astrocytes: a novel mechanism of glutamate release. J Neurosci 23(9):3588-96. **Part 4**

**Roles of Glial Cells in Neurodegeneration** 

254 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

Ye ZC, Wyeth MS, Baltan-Tekkok S, Ransom BR. 2003. Functional hemichannels in

**11** 

*1Colombia 2Argentina* 

**Role of Astrocytes in** 

**Neurodegenerative Diseases** 

*1Departamento de Nutrición y Bioquímica, Facultad de Ciencias,* 

*2Instituto de Investigaciones Cardiológicas, Prof. Dr. Alberto Taquini,* 

The past several decades have given rise to many important discoveries and novel insights into the role of astrocytes in normal brain function and disease, firmly establishing concepts that describe the dynamic and reciprocal signaling networks between astrocytes, neurons

Brain aging, overt any neurodegenerative state, leads to inflammation, oxidative stress and cell death. Neurons are more susceptible to injury than astrocytes, as they have fewer antioxidant mechanisms and are therefore prone to excitotoxicity (Swanson *et al.,* 2004). Both normally and with aging, astrocytes support neurons by providing antioxidant protection, substrates for neuronal metabolism via neurovascular coupling, and glutamate clearance. Although astrocytes are generally more resilient than neurons, severe damage also results in astrocyte dysfunction, leading to increased neuronal death (Nedergaard & Dirnagl, 2005). Therefore, many recent efforts have focused on the astrocyte-neuron interaction and how astrocyte function can be improved to enhance neuronal support and survival (Swanson *et al.,* 2004). A growing body of data demonstrates that astrocytes play a multifaceted and complex role in the response to neuropathologies, including neurodegenerative, as they have potential to both enhance neuronal survival and regeneration and contribute to further injury (Sofroniew, 2000, 2009; Sofroniew & Vinters, 2010). Because of the diverse nature and complex biology of these cells, and the limited number of studies to date, their role in

It is likely that diminished astrocytes function throughout the neurodegenerative process is a prominent determinant of both neuronal survival as well as survival of the entire organism (Shibata & Kobayashi, 2008). In this chapter we provide a brief overview of the pathophysiological events underlying brain aging, and in neurodegenerative diseases, and discusses how these events affect astrocytes response to these chronic neuropathologies, such as Alzheimer's AD and Parkinson´s PD diseases, Amyotrophic Lateral Syndrome

**1. Introduction** 

and other cell types.

neurodegeneration deserves further study.

(ALS), and Multiple Sclerosis (MS).

George E. Barreto1, Janneth Gonzalez1, Francisco Capani2 and Ludis Morales1

*Pontificia Universidad Javeriana, Bogotá D.C.,* 

*CONICET-UBA Buenos Aires,* 

### **Role of Astrocytes in Neurodegenerative Diseases**

George E. Barreto1, Janneth Gonzalez1, Francisco Capani2 and Ludis Morales1 *1Departamento de Nutrición y Bioquímica, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá D.C., 2Instituto de Investigaciones Cardiológicas, Prof. Dr. Alberto Taquini, CONICET-UBA Buenos Aires, 1Colombia 2Argentina* 

#### **1. Introduction**

The past several decades have given rise to many important discoveries and novel insights into the role of astrocytes in normal brain function and disease, firmly establishing concepts that describe the dynamic and reciprocal signaling networks between astrocytes, neurons and other cell types.

Brain aging, overt any neurodegenerative state, leads to inflammation, oxidative stress and cell death. Neurons are more susceptible to injury than astrocytes, as they have fewer antioxidant mechanisms and are therefore prone to excitotoxicity (Swanson *et al.,* 2004). Both normally and with aging, astrocytes support neurons by providing antioxidant protection, substrates for neuronal metabolism via neurovascular coupling, and glutamate clearance. Although astrocytes are generally more resilient than neurons, severe damage also results in astrocyte dysfunction, leading to increased neuronal death (Nedergaard & Dirnagl, 2005). Therefore, many recent efforts have focused on the astrocyte-neuron interaction and how astrocyte function can be improved to enhance neuronal support and survival (Swanson *et al.,* 2004). A growing body of data demonstrates that astrocytes play a multifaceted and complex role in the response to neuropathologies, including neurodegenerative, as they have potential to both enhance neuronal survival and regeneration and contribute to further injury (Sofroniew, 2000, 2009; Sofroniew & Vinters, 2010). Because of the diverse nature and complex biology of these cells, and the limited number of studies to date, their role in neurodegeneration deserves further study.

It is likely that diminished astrocytes function throughout the neurodegenerative process is a prominent determinant of both neuronal survival as well as survival of the entire organism (Shibata & Kobayashi, 2008). In this chapter we provide a brief overview of the pathophysiological events underlying brain aging, and in neurodegenerative diseases, and discusses how these events affect astrocytes response to these chronic neuropathologies, such as Alzheimer's AD and Parkinson´s PD diseases, Amyotrophic Lateral Syndrome (ALS), and Multiple Sclerosis (MS).

Role of Astrocytes in Neurodegenerative Diseases 259

**Glutamate**

**Neuron**

**Pyruvate lactate**

**K+**

**GS**

**Glutamine**

**Astrocyte**

expressed by neurons. Scale bar, 50 µm

 **GSH ascorbine**

Fig. 1. Mechanisms of astrocyte support of neurons in the normal brain. Antioxidant defence includes release of glutathione and ascorbate. Regulation of extracellular levels of ions and neurotransmitters, especially K+ and glutamate, strongly influence neuronal excitability. Elevated extracellular K+ triggers astrocyte glycolysis and enhances lactate and pyruvate release which support neuronal metabolism. Sodium dependent glutamate uptake by astrocytes activates the Na+/K+ ATPase, stimulating glycolytic activity and production of lactate. Astrocytes and neurons are also coupled by the glutamate-glutamine cycle.

Astrocytes take up glutamate, convert it to glutamine, release glutamine to the extracellular space where it is taken up by neurons and used to synthesize glutamate to replenishment the neurotransmitter pool. Any deregulation of these mechanisms, as a common situation in

Fig. 2. Astrocytic HO-1 expression in corpus callosum. Immunostaining for HO-1 and NeuN (neuronal marker) was carried out in free floating brain sections of 3 months-old naïve male mice. Morphologically, HO-1 is expressed in shaped-like astrocytes, and does not seem to be

some neurodegenerative diseases, will likely influence neuronal survival

 **HO1 NeuN Merge** 

#### **2. Astrocytes function in the brain**

Astrocytes are the most common cell type in mammalian brain. Glial fibrillary acidic protein (GFAP) and vimentin (Vim) constitute intermediate filaments (known also as nanofilaments) as part of the cytoskeleton in astrocytes. Reactive gliosis is a response of astrocytes to a variety of brain insults that is characterized by hypertrophy of the cell bodies and processes, altered gene expression, increase in the expression of GFAP, Vim and the calcium binding protein S100β (Ridet *et al.,* 1997), and proliferation that may likely occur in some neurodegenerative diseases (Sofroniew, 2009; Sofroniew & Vinters, 2010). In contrast, because reactive astrocytes are ubiquitous in aged central nervous system (CNS) tissue, they are often regarded as uniformly harmful, provoking inflammation, releasing cytotoxins and chemokines that serve no purpose but to inhibit axonal regeneration and increase damage. The wide range of activities that astrocytes can exhibit *in vitro* contributes to uncertainty over whether these cells exert beneficial or detrimental effects after CNS degeneration. For example, potential protective effects could be provided by glutamate uptake and neurotrophin release, while potential detrimental effects might be caused by the release of inflammatory cytokines and cytotoxic radicals. Little information has been available on the roles played by reactive astrocytes in the response to experimental models of neurodegenerative diseases *in vivo.* For instance, aged astrocytes exhibit an elevated content of GFAP and of S100β (Barreto *et al.,* 2009; Nichols, 1999). Use of oligonucleotide arrays has yielded the first profile of gene expression from the aging brain of mice and evidence that aging seems to be associated with an inflammatory response and oxidative stress both in neocortex, hippocampus and in cerebellum (Lee *et al.,* 2000; Zeier *et al.,* 2011), with parallels to human neurodegenerative disorders. GFAP is also one of the genes that undergoes a twofold increase in expression. Thus, the GFAP increases of the aged astrocytes may be the result of a response to the inflammatory and oxidative state of the aging brain. Indeed, better comprehension of the features that distinguish a normal, "healthy" old brain from a brain that is at an early stage of a neurodegenerative disease is a key aspect in developing treatments.

It is interesting to note that one of the characteristics of astrocytes in the aging brain – the number of astrocytes – is increased by ~20% (Peinado *et al.,* 1998; Pilegaard & Ladefoged, 1996; Rozovsky *et al.,* 1998; Salminen *et al.,* 2011). This response has been compared with reactive gliosis in response to injured or damaged neurons during aging. However, an alternative explanation is that increased number of astrocytes in the aging brain is required to provide the same level of neuroprotection that is present in the brain of a young animal.

One hallmark of the cellular response to brain aging, and in neurodegenerative states, is a rapid, dramatic increase in damaging free radicals, including nitric oxide (NO), superoxide, and peroxynitrite (Shibata & Kobayashi, 2008). On the other hand, astrocytes produce the beneficial antioxidants glutathione, superoxide dismutases (SODs 1, 2 and 3), and ascorbate (Figure 1, Anderson & Swanson, 2000; Dringen, 2000; Dringen *et al.,* 2000; Lindenau *et al.,* 2000; Sims *et al.,* 2004). Interestingly, neurons cocultured with astrocyte exhibit higher levels of glutathione compared with neurons cultured alone (Giordano *et al.,* 2009), suggesting that astrocytes provide additional antioxidant defense to neurons (Slemmer *et al.,* 2008). Similarly, astrocytes upregulate HO-1 (heme-oxygenase 1, Figure 2), a 32 kDa stress protein that degrades heme to biliverdin, free iron and carbon monoxide. Although the upregulation of this enzyme has been previously reported to confer neuroprotection following various brain insults (Beschorner *et al.,* 2000; Chen *et al.,* 2000; Espada *et al.,* 2010;

Astrocytes are the most common cell type in mammalian brain. Glial fibrillary acidic protein (GFAP) and vimentin (Vim) constitute intermediate filaments (known also as nanofilaments) as part of the cytoskeleton in astrocytes. Reactive gliosis is a response of astrocytes to a variety of brain insults that is characterized by hypertrophy of the cell bodies and processes, altered gene expression, increase in the expression of GFAP, Vim and the calcium binding protein S100β (Ridet *et al.,* 1997), and proliferation that may likely occur in some neurodegenerative diseases (Sofroniew, 2009; Sofroniew & Vinters, 2010). In contrast, because reactive astrocytes are ubiquitous in aged central nervous system (CNS) tissue, they are often regarded as uniformly harmful, provoking inflammation, releasing cytotoxins and chemokines that serve no purpose but to inhibit axonal regeneration and increase damage. The wide range of activities that astrocytes can exhibit *in vitro* contributes to uncertainty over whether these cells exert beneficial or detrimental effects after CNS degeneration. For example, potential protective effects could be provided by glutamate uptake and neurotrophin release, while potential detrimental effects might be caused by the release of inflammatory cytokines and cytotoxic radicals. Little information has been available on the roles played by reactive astrocytes in the response to experimental models of neurodegenerative diseases *in vivo.* For instance, aged astrocytes exhibit an elevated content of GFAP and of S100β (Barreto *et al.,* 2009; Nichols, 1999). Use of oligonucleotide arrays has yielded the first profile of gene expression from the aging brain of mice and evidence that aging seems to be associated with an inflammatory response and oxidative stress both in neocortex, hippocampus and in cerebellum (Lee *et al.,* 2000; Zeier *et al.,* 2011), with parallels to human neurodegenerative disorders. GFAP is also one of the genes that undergoes a twofold increase in expression. Thus, the GFAP increases of the aged astrocytes may be the result of a response to the inflammatory and oxidative state of the aging brain. Indeed, better comprehension of the features that distinguish a normal, "healthy" old brain from a brain that is at an early stage of a neurodegenerative disease is a key aspect in developing

It is interesting to note that one of the characteristics of astrocytes in the aging brain – the number of astrocytes – is increased by ~20% (Peinado *et al.,* 1998; Pilegaard & Ladefoged, 1996; Rozovsky *et al.,* 1998; Salminen *et al.,* 2011). This response has been compared with reactive gliosis in response to injured or damaged neurons during aging. However, an alternative explanation is that increased number of astrocytes in the aging brain is required to provide the same level of neuroprotection that is present in the brain of a young animal. One hallmark of the cellular response to brain aging, and in neurodegenerative states, is a rapid, dramatic increase in damaging free radicals, including nitric oxide (NO), superoxide, and peroxynitrite (Shibata & Kobayashi, 2008). On the other hand, astrocytes produce the beneficial antioxidants glutathione, superoxide dismutases (SODs 1, 2 and 3), and ascorbate (Figure 1, Anderson & Swanson, 2000; Dringen, 2000; Dringen *et al.,* 2000; Lindenau *et al.,* 2000; Sims *et al.,* 2004). Interestingly, neurons cocultured with astrocyte exhibit higher levels of glutathione compared with neurons cultured alone (Giordano *et al.,* 2009), suggesting that astrocytes provide additional antioxidant defense to neurons (Slemmer *et al.,* 2008). Similarly, astrocytes upregulate HO-1 (heme-oxygenase 1, Figure 2), a 32 kDa stress protein that degrades heme to biliverdin, free iron and carbon monoxide. Although the upregulation of this enzyme has been previously reported to confer neuroprotection following various brain insults (Beschorner *et al.,* 2000; Chen *et al.,* 2000; Espada *et al.,* 2010;

**2. Astrocytes function in the brain** 

treatments.

Fig. 1. Mechanisms of astrocyte support of neurons in the normal brain. Antioxidant defence includes release of glutathione and ascorbate. Regulation of extracellular levels of ions and neurotransmitters, especially K+ and glutamate, strongly influence neuronal excitability. Elevated extracellular K+ triggers astrocyte glycolysis and enhances lactate and pyruvate release which support neuronal metabolism. Sodium dependent glutamate uptake by astrocytes activates the Na+/K+ ATPase, stimulating glycolytic activity and production of lactate. Astrocytes and neurons are also coupled by the glutamate-glutamine cycle. Astrocytes take up glutamate, convert it to glutamine, release glutamine to the extracellular space where it is taken up by neurons and used to synthesize glutamate to replenishment the neurotransmitter pool. Any deregulation of these mechanisms, as a common situation in some neurodegenerative diseases, will likely influence neuronal survival

Fig. 2. Astrocytic HO-1 expression in corpus callosum. Immunostaining for HO-1 and NeuN (neuronal marker) was carried out in free floating brain sections of 3 months-old naïve male mice. Morphologically, HO-1 is expressed in shaped-like astrocytes, and does not seem to be expressed by neurons. Scale bar, 50 µm

Role of Astrocytes in Neurodegenerative Diseases 261

Nevertheless, much of the pial circulation is in contact with the glia-limitans, a de-facto extension of astrocytic processes (Kontos *et al.,* 1971; Xu *et al.,* 2004). This domain organization has been proposed as being the key linking element of the neuronal– (astrocyte)–vascular unit (Volterra & Meldolesi, 2005). For example, working with neocortical slices, Zonta et al. (Zonta *et al.,* 2003) demonstrated that electrical stimulation of neuronal processes raises intracellular Ca2+ levels in astrocytic endfeet and leads to a slowly developing dilatation of local intracerebral arterioles. Additionally, electrical stimulation of individual astrocytes had the same effect. Since this initial report, several investigators observed a vascular response in conjunction with an elevation of intracellular Ca2+ levels in astrocytic endfeet. However, these studies reported inconsistent vascular responses ranging from vasorelaxation to vasodilatation or the combination of both (Gordon *et al.,* 2007; Iadecola & Nedergaard, 2007). Mediators implicated in this mechanism are vasoactive metabolites of the cyclooxygenase or cytochrome P450 ω-hydroxylase pathways. All of these studies were performed in brain slices in which the vessels are lacking in intraluminal pressure. This might account for disparate results. In vivo analysis with two-photon laser scanning microscopy revealed that increases of astrocytic Ca2+ by photolysis of caged Ca2+ evoked a vasodilatation of cortical arterioles (Takano *et al.,* 2006). This interaction between the vessel and the endfeet appeared to be mediated by metabolites of the COX-1 pathway, because inhibitors of nitric oxide synthetase (NOS), COX-2, p450 epoxygenases, and adenosine receptor antagonists had no effect. These and other studies strongly implicate a role for astrocytes in cerebral blood flow regulation during neuronal activation (Haydon &

It is important to point that some, if not all, of these astrocytic functions may likely be altered or reduced in neurodegenerative states (Rossi & Volterra, 2009). The role of astrocytes in various neurodegenerative diseases will briefly be discussed more thoroughly below, specifically looking at their involvement during the pathologic processes of Alzheimer´s and Parkinson diseases, Amyotrophic Lateral Syndrome (ALS) and Multiple

Neurodegenerative diseases represent a heterogeneous group of disorders affecting the nervous system. In most instances, they affect adults, their causes are unknown, and progression is relentless. Some are genetic, but most are sporadic. They involve all parts of the nervous system, although the cerebral cortex and the basal ganglia are the most frequent loci of pathology. The historical classification of neurodegenerative diseases, based on clinical and pathological characteristics, is imperfect. New classifications are rather based on molecular determinants. Contrary to common belief, it is now recognized that neurodegenerative disorders are multisystemic, even if specific neuronal pathways are more affected than others. The death of astrocytes and specific types of neurons in neurodegenerative diseases is provoked, not by a single pathogenic factor, but rather by a

cascade of multiple deleterious molecular and cellular events as described earlier.

Mitochondria are central neuronal organelles that play a vital role in neuronal life and death. Both mitochondrial dysfunction and proper function are essential components in neurodegeneration. Further elucidation of the mechanisms of interaction between

**3. Astrocytes dysfunction in neurodegenerative diseases** 

**3.1 Oxidative stress and neurodegeneration** 

Carmignoto, 2006).

Sclerosis.

Imuta *et al.,* 2007; Ku *et al.,* 2006; Le *et al.,* 1999; Takeda *et al.,* 2000), its overproduction in astrocytes may contribute to iron overload and mitochondrial insufciency, characteristic of some neurodegenerative disorders (Fernandez-Checa *et al.,* 2010; Serviddio *et al.,* 2011). HO-1 is expressed by approximately 86% (Schipper *et al.,* 1995) and 77.1% (Schipper *et al.,* 1998) of GFAP-positive astrocytes in AD and PD, respectively, suggesting a possible role in the pathogenesis of these neurodegenerative diseases.

Control of energy metabolism is also controlled by astrocytes in the CNS. When astrocytes take up extracellular glutamate as a result of neuronal activity, the Na+/ K+-ATPase and AMPA signaling trigger astrocyte uptake of glucose from the blood, as astrocytic endfeet contact capillaries (Caesar *et al.,* 2008; Magistretti, 2006). The glucose is then made into lactate, a substrate for neuronal energy, to further "fuel" active neurons (Magistretti & Pellerin, 1999; Figure 1). As mentioned above, astrocytes produce glutathione. In addition to its antioxidant properties, glutathione is the enzyme needed for the conversion of methylglyoxal, a toxic by-product of metabolism, into D-lactate by glyoxalase 1 (Cliffe & Waley, 1961). Although the role of astrocyte metabolism is relatively well-established in normal tissues, the role of astrocyte metabolism maintenance with aging and in neurodegenerative diseases is less clear (Bartnik-Olson *et al.,* 2010; Bentzer *et al.,* 2000; Floyd & Lyeth, 2007).

Astrocytes are also key players in the production and regulation of neurotransmitters, antioxidant production, potassium uptake, energy metabolism and neurovascular coupling in the CNS. Notably, astrocytes make glutamine, the precursor for the neurotransmitters glutamate and GABA, from glucose (Zou *et al.,* 2010). In addition to providing the precursors for neurotransmitters, one important role of astrocytes in the normal brain is to take up glutamate using the glutamate transporters GLAST and GLT-1 (Anderson & Swanson, 2000; Romera *et al.,* 2004; Schousboe & Waagepetersen, 2006), as excess glutamate leads to cell death via excitotoxicity (Tilleux & Hermans, 2007).

Astrocytes regulate neuronal activation by extracellular potassium uptake, and proper maintenance of ion gradients, such as potassium, as an important mechanism for regulating cell volume in both normal and pathological conditions (Jayakumar & Norenberg, 2010; Lambert & Oberwinkler, 2005; Lang *et al.,* 1998; Obara *et al.,* 2008). Indeed, astrocytes upregulate glucose transporters in order to provide energy to dying neuronal cells (Floyd & Lyeth, 2007; Scafidi *et al.,* 2009; Yi & Hazell, 2006,) suggesting that astrocytes are necessary for improvement in chronic neurodegenerative diseases energy metabolism. In summary, astrocytes are important producers of antioxidants in the normal CNS, and astrocytic production of these molecules after brain injury may enhance neuronal survival and protect astrocyte function.

Astrocytes are critical in the development and/or maintenance of blood-brain barrier characteristics (Gordon *et al.,* 2007; Koehler *et al.,* 2009). Astrocytes are arranged in nonoverlapping spatial domains (Bushong *et al.,* 2002; Halassa *et al.,* 2007), but coupled to each other in a syncytial network (Haydon & Carmignoto, 2006). Since one astrocyte maintains contacts with approximately 160,000 synapses (Bushong *et al.,* 2002), this cell population is well positioned to integrate neuronal activity and link neuronal activity to the vascular network (Ransom *et al.,* 2003).

Astrocytes terminal processes are also known as "endfeet" cover 99% of the abluminal vascular surface of capillaries, intracerebral arterioles, and venules (Simard *et al.,* 2003). The extent of contact between endfeet and penetrating and pial arterioles remains unclear. Pial arterioles and arteries lying free in the subarachnoid space are not covered (Jones, 1970).

Imuta *et al.,* 2007; Ku *et al.,* 2006; Le *et al.,* 1999; Takeda *et al.,* 2000), its overproduction in astrocytes may contribute to iron overload and mitochondrial insufciency, characteristic of some neurodegenerative disorders (Fernandez-Checa *et al.,* 2010; Serviddio *et al.,* 2011). HO-1 is expressed by approximately 86% (Schipper *et al.,* 1995) and 77.1% (Schipper *et al.,* 1998) of GFAP-positive astrocytes in AD and PD, respectively, suggesting a possible role in the

Control of energy metabolism is also controlled by astrocytes in the CNS. When astrocytes take up extracellular glutamate as a result of neuronal activity, the Na+/ K+-ATPase and AMPA signaling trigger astrocyte uptake of glucose from the blood, as astrocytic endfeet contact capillaries (Caesar *et al.,* 2008; Magistretti, 2006). The glucose is then made into lactate, a substrate for neuronal energy, to further "fuel" active neurons (Magistretti & Pellerin, 1999; Figure 1). As mentioned above, astrocytes produce glutathione. In addition to its antioxidant properties, glutathione is the enzyme needed for the conversion of methylglyoxal, a toxic by-product of metabolism, into D-lactate by glyoxalase 1 (Cliffe & Waley, 1961). Although the role of astrocyte metabolism is relatively well-established in normal tissues, the role of astrocyte metabolism maintenance with aging and in neurodegenerative diseases is less clear (Bartnik-Olson *et al.,* 2010; Bentzer *et al.,* 2000; Floyd

Astrocytes are also key players in the production and regulation of neurotransmitters, antioxidant production, potassium uptake, energy metabolism and neurovascular coupling in the CNS. Notably, astrocytes make glutamine, the precursor for the neurotransmitters glutamate and GABA, from glucose (Zou *et al.,* 2010). In addition to providing the precursors for neurotransmitters, one important role of astrocytes in the normal brain is to take up glutamate using the glutamate transporters GLAST and GLT-1 (Anderson & Swanson, 2000; Romera *et al.,* 2004; Schousboe & Waagepetersen, 2006), as excess glutamate

Astrocytes regulate neuronal activation by extracellular potassium uptake, and proper maintenance of ion gradients, such as potassium, as an important mechanism for regulating cell volume in both normal and pathological conditions (Jayakumar & Norenberg, 2010; Lambert & Oberwinkler, 2005; Lang *et al.,* 1998; Obara *et al.,* 2008). Indeed, astrocytes upregulate glucose transporters in order to provide energy to dying neuronal cells (Floyd & Lyeth, 2007; Scafidi *et al.,* 2009; Yi & Hazell, 2006,) suggesting that astrocytes are necessary for improvement in chronic neurodegenerative diseases energy metabolism. In summary, astrocytes are important producers of antioxidants in the normal CNS, and astrocytic production of these molecules after brain injury may enhance neuronal survival and protect

Astrocytes are critical in the development and/or maintenance of blood-brain barrier characteristics (Gordon *et al.,* 2007; Koehler *et al.,* 2009). Astrocytes are arranged in nonoverlapping spatial domains (Bushong *et al.,* 2002; Halassa *et al.,* 2007), but coupled to each other in a syncytial network (Haydon & Carmignoto, 2006). Since one astrocyte maintains contacts with approximately 160,000 synapses (Bushong *et al.,* 2002), this cell population is well positioned to integrate neuronal activity and link neuronal activity to the vascular

Astrocytes terminal processes are also known as "endfeet" cover 99% of the abluminal vascular surface of capillaries, intracerebral arterioles, and venules (Simard *et al.,* 2003). The extent of contact between endfeet and penetrating and pial arterioles remains unclear. Pial arterioles and arteries lying free in the subarachnoid space are not covered (Jones, 1970).

pathogenesis of these neurodegenerative diseases.

leads to cell death via excitotoxicity (Tilleux & Hermans, 2007).

& Lyeth, 2007).

astrocyte function.

network (Ransom *et al.,* 2003).

Nevertheless, much of the pial circulation is in contact with the glia-limitans, a de-facto extension of astrocytic processes (Kontos *et al.,* 1971; Xu *et al.,* 2004). This domain organization has been proposed as being the key linking element of the neuronal– (astrocyte)–vascular unit (Volterra & Meldolesi, 2005). For example, working with neocortical slices, Zonta et al. (Zonta *et al.,* 2003) demonstrated that electrical stimulation of neuronal processes raises intracellular Ca2+ levels in astrocytic endfeet and leads to a slowly developing dilatation of local intracerebral arterioles. Additionally, electrical stimulation of individual astrocytes had the same effect. Since this initial report, several investigators observed a vascular response in conjunction with an elevation of intracellular Ca2+ levels in astrocytic endfeet. However, these studies reported inconsistent vascular responses ranging from vasorelaxation to vasodilatation or the combination of both (Gordon *et al.,* 2007; Iadecola & Nedergaard, 2007). Mediators implicated in this mechanism are vasoactive metabolites of the cyclooxygenase or cytochrome P450 ω-hydroxylase pathways. All of these studies were performed in brain slices in which the vessels are lacking in intraluminal pressure. This might account for disparate results. In vivo analysis with two-photon laser scanning microscopy revealed that increases of astrocytic Ca2+ by photolysis of caged Ca2+ evoked a vasodilatation of cortical arterioles (Takano *et al.,* 2006). This interaction between the vessel and the endfeet appeared to be mediated by metabolites of the COX-1 pathway,

because inhibitors of nitric oxide synthetase (NOS), COX-2, p450 epoxygenases, and adenosine receptor antagonists had no effect. These and other studies strongly implicate a role for astrocytes in cerebral blood flow regulation during neuronal activation (Haydon & Carmignoto, 2006).

It is important to point that some, if not all, of these astrocytic functions may likely be altered or reduced in neurodegenerative states (Rossi & Volterra, 2009). The role of astrocytes in various neurodegenerative diseases will briefly be discussed more thoroughly below, specifically looking at their involvement during the pathologic processes of Alzheimer´s and Parkinson diseases, Amyotrophic Lateral Syndrome (ALS) and Multiple Sclerosis.

#### **3. Astrocytes dysfunction in neurodegenerative diseases**

Neurodegenerative diseases represent a heterogeneous group of disorders affecting the nervous system. In most instances, they affect adults, their causes are unknown, and progression is relentless. Some are genetic, but most are sporadic. They involve all parts of the nervous system, although the cerebral cortex and the basal ganglia are the most frequent loci of pathology. The historical classification of neurodegenerative diseases, based on clinical and pathological characteristics, is imperfect. New classifications are rather based on molecular determinants. Contrary to common belief, it is now recognized that neurodegenerative disorders are multisystemic, even if specific neuronal pathways are more affected than others. The death of astrocytes and specific types of neurons in neurodegenerative diseases is provoked, not by a single pathogenic factor, but rather by a cascade of multiple deleterious molecular and cellular events as described earlier.

#### **3.1 Oxidative stress and neurodegeneration**

Mitochondria are central neuronal organelles that play a vital role in neuronal life and death. Both mitochondrial dysfunction and proper function are essential components in neurodegeneration. Further elucidation of the mechanisms of interaction between

Role of Astrocytes in Neurodegenerative Diseases 263

molecules such as glutamate, produced during synaptic transmission through neurons (Hertz & Zielke, 2004). This is, perhaps, the most common astrocytic dysfunction that likely

Astrocytes react to various neurodegenerative insults rapidly, leading to vigorous astrogliosis. This reactive gliosis is associated with alteration in morphology and structure of activated astrocytes along with its functional characteristics (Eddleston & Mucke, 1993). The astrocytic processes construct a bushy network surrounding the injury site, thus secluding the affected part from the rest of the CNS area. Subsequently, astrogliosis has been implicated in the pathogenesis of a variety of chronic neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease, Amyotrophic Lateral Syndrome (ALS ), acute traumatic brain injury, stroke, and neuroinflammatory brain diseases (Axelsson *et al.,* 2011; Ciesielska *et al.,* 2009; Garcia-Matas *et al.,* 2010; Heales *et al.,* 2004; Li *et al.,* 2011;

AD is characterized clinically by cognitive loss in two or more domains, including memory, language, calculations, orientation and judgment; the loss must be of sufficient severity to cause social or occupational disability. These clinical features are the result of neuronal death and dysfunction in the cerebral cortex, entorhinal area, hippocampus, ventral striatum and basal forebrain, eventually resulting in severe dementia. Pathologically, the two hallmark findings of the disorder are neurofibrillary tangles and amyloid plaques (Veerhuis, 2011). Senile plaques, a pathologic hallmark of Alzheimer's disease, are associated with GFAPpositive activated astrocytes (Nagele *et al.,* 2004). It is reported that in various neuropathological states, the increased GFAP expression corresponds to the severity of astroglial activation (Axelsson *et al.,* 2011; Kashon *et al.,* 2004; Notturno *et al.,* 2009; Pelinka *et* 

Concerning astrocytes, recent findings suggest that they play a role in the clearance of the Aβ- peptide and thus in preventing plaque formation (Li *et al.,* 2011). Similarly, this peptide decreases glutamate uptake in cultured astrocytes, thus increasing oxidative stress and and activation of mitogen-activated protein kinase cascades (Agostinho *et al.,* 2010; Matos *et al.,* 2008). High levels of pro-inflammatory cytokines such as interleukin 1β, interleukin 6 and TNFα, mostly produced by reactive astrocytes, are detected in the brain of AD subjects, so the consequences of this phenomenon are unclear, also because pro-inflammatory cytokines

A previous study indicated that activated astrocytes were closely associated with amyloid plaques in the molecular layer of the cerebral cortex (Wisniewski & Wegiel, 1991). Astrocytes might be activated by human amyloid-β (Aβ) (DeWitt *et al.,* 1998), indicating a correlation between this protein and subsequent alterations in astrocyte function. Astrocytes also accumulate neuron-derived amyloid material resulting from local neurodegeneration. Once substantial accumulation of this debris occurs, the astrocytes themselves might undergo cell death, resulting in the formation of GFAP+ amyloid plaques (Nagele *et al.,* 2004). *In vitro* analyses also indicate that treatment of astrocytes with Aβ results in an increase in calcium-wave signaling between these cells (Haughey & Mattson, 2003). In cells expressing the familial AD presenilin 1 (*PSEN1*) mutation, calcium oscillations in astrocytes were found to occur at lower ATP and glutamate concentrations than in wild-type astrocytes (Johnston *et al.,* 2006). These data support a model in which calcium signaling between astrocytes is altered by the disease process, which might, in ways that are not fully

occurs in some neurodegenerative states.

Simpson *et al.,* 2010; Sofroniew, 2000).

*al.,* 2004; Simpson *et al.,* 2010; Toft-Hansen *et al.,* 2011).

have varied effects depending on the biological context (Veerhuis, 2011).

understood, contribute to dysfunction or death of neurons.

**3.2 Alzheimer´s disease** 

mitochondria and neuronal death will allow better description of the pathogenesis of neurodegenerative diseases and provide potential targets for therapeutic intervention.

One of the hallmarks of various neurodegenerative and neuroinflammatory disorders is oxidative stress-induced CNS damage. Similarly, the natural aging process per se is associated with increased oxidative stress (Figure 3). Such oxidative stress can damage lipids, proteins and nucleic acids of cells and power-house mitochondria causing cell death in assorted cell types including astrocytes and neurons. However, astrocytes having high levels of anti-oxidant enzymes (glutathione peroxidase, catalase, glutathione reductase, and superoxide dismutase) and antioxidants (glutathione and ascorbic acid) try to absorb reactive oxygen species (O2 =, O2 −, and OH.) and reactive nitrogen species (NO, ONOO−), maintain redox homeostasis and defend the insulted CNS (Chen & Swanson, 2003; Dringen & Hirrlinger, 2003; Wilson, 1997). In addition, astrocytes also scavenge detrimental

molecules such as glutamate, produced during synaptic transmission through neurons (Hertz & Zielke, 2004). This is, perhaps, the most common astrocytic dysfunction that likely occurs in some neurodegenerative states.

Astrocytes react to various neurodegenerative insults rapidly, leading to vigorous astrogliosis. This reactive gliosis is associated with alteration in morphology and structure of activated astrocytes along with its functional characteristics (Eddleston & Mucke, 1993). The astrocytic processes construct a bushy network surrounding the injury site, thus secluding the affected part from the rest of the CNS area. Subsequently, astrogliosis has been implicated in the pathogenesis of a variety of chronic neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease, Amyotrophic Lateral Syndrome (ALS ), acute traumatic brain injury, stroke, and neuroinflammatory brain diseases (Axelsson *et al.,* 2011; Ciesielska *et al.,* 2009; Garcia-Matas *et al.,* 2010; Heales *et al.,* 2004; Li *et al.,* 2011; Simpson *et al.,* 2010; Sofroniew, 2000).

#### **3.2 Alzheimer´s disease**

262 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

mitochondria and neuronal death will allow better description of the pathogenesis of neurodegenerative diseases and provide potential targets for therapeutic intervention. One of the hallmarks of various neurodegenerative and neuroinflammatory disorders is oxidative stress-induced CNS damage. Similarly, the natural aging process per se is associated with increased oxidative stress (Figure 3). Such oxidative stress can damage lipids, proteins and nucleic acids of cells and power-house mitochondria causing cell death in assorted cell types including astrocytes and neurons. However, astrocytes having high levels of anti-oxidant enzymes (glutathione peroxidase, catalase, glutathione reductase, and superoxide dismutase) and antioxidants (glutathione and ascorbic acid) try to absorb reactive oxygen species (O2 =, O2 −, and OH.) and reactive nitrogen species (NO, ONOO−), maintain redox homeostasis and defend the insulted CNS (Chen & Swanson, 2003; Dringen & Hirrlinger, 2003; Wilson, 1997). In addition, astrocytes also scavenge detrimental

Young Aged

Fig. 3. Increased oxidative stress production in the normal aged brain. 3- or 18-months naïve

4-Hydroxynonenal (4-HNE), a marker of lipid peroxidation. Mean Fluorescence intensity assessed by ImageJ program showed that aged animals, overt any pathological condition, had an increased expression of 4-HNE in cortical layers II-III, compared to young mice. 3 sections/animal for 4 animals were analyzed in each condition. Data are represented as

old male mice were sacrificed and 40 µm brain sections were stained for

**Young Aged**

250

200

150

Mean Flourescence Intensity

100

50

0

Means ± SEM. Scale bar, 100 µm

AD is characterized clinically by cognitive loss in two or more domains, including memory, language, calculations, orientation and judgment; the loss must be of sufficient severity to cause social or occupational disability. These clinical features are the result of neuronal death and dysfunction in the cerebral cortex, entorhinal area, hippocampus, ventral striatum and basal forebrain, eventually resulting in severe dementia. Pathologically, the two hallmark findings of the disorder are neurofibrillary tangles and amyloid plaques (Veerhuis, 2011).

Senile plaques, a pathologic hallmark of Alzheimer's disease, are associated with GFAPpositive activated astrocytes (Nagele *et al.,* 2004). It is reported that in various neuropathological states, the increased GFAP expression corresponds to the severity of astroglial activation (Axelsson *et al.,* 2011; Kashon *et al.,* 2004; Notturno *et al.,* 2009; Pelinka *et al.,* 2004; Simpson *et al.,* 2010; Toft-Hansen *et al.,* 2011).

Concerning astrocytes, recent findings suggest that they play a role in the clearance of the Aβ- peptide and thus in preventing plaque formation (Li *et al.,* 2011). Similarly, this peptide decreases glutamate uptake in cultured astrocytes, thus increasing oxidative stress and and activation of mitogen-activated protein kinase cascades (Agostinho *et al.,* 2010; Matos *et al.,* 2008). High levels of pro-inflammatory cytokines such as interleukin 1β, interleukin 6 and TNFα, mostly produced by reactive astrocytes, are detected in the brain of AD subjects, so the consequences of this phenomenon are unclear, also because pro-inflammatory cytokines have varied effects depending on the biological context (Veerhuis, 2011).

A previous study indicated that activated astrocytes were closely associated with amyloid plaques in the molecular layer of the cerebral cortex (Wisniewski & Wegiel, 1991). Astrocytes might be activated by human amyloid-β (Aβ) (DeWitt *et al.,* 1998), indicating a correlation between this protein and subsequent alterations in astrocyte function. Astrocytes also accumulate neuron-derived amyloid material resulting from local neurodegeneration. Once substantial accumulation of this debris occurs, the astrocytes themselves might undergo cell death, resulting in the formation of GFAP+ amyloid plaques (Nagele *et al.,* 2004). *In vitro* analyses also indicate that treatment of astrocytes with Aβ results in an increase in calcium-wave signaling between these cells (Haughey & Mattson, 2003). In cells expressing the familial AD presenilin 1 (*PSEN1*) mutation, calcium oscillations in astrocytes were found to occur at lower ATP and glutamate concentrations than in wild-type astrocytes (Johnston *et al.,* 2006). These data support a model in which calcium signaling between astrocytes is altered by the disease process, which might, in ways that are not fully understood, contribute to dysfunction or death of neurons.

Role of Astrocytes in Neurodegenerative Diseases 265

muscle atrophy. Patients eventually experience respiratory failure, usually within three to five years from diagnosis. However, the onset of ALS may be subtle and early symptoms are

Common to familial and sporadic ALS is the loss of the astrocyte glutamate transporter EAAT2. Studies of the EAAT2 transporter in tissue from individuals with sporadic ALS showed a marked loss of up to 95% of astroglial EAAT2 protein expression and activity in affected areas of the CNS (Bristol & Rothstein, 1996). A clue to a possible mechanism for EAAT2 reduction or dysfunction was provided by the finding of aberrant *EAAT2* RNA species, which has been implicated in multiple neurodegenerative diseases. The production of truncated EAAT2 protein results in reduced function, and the retention of normal EAAT2 protein within the cytoplasm (Lin *et al.,* 1998). The significance of these aberrant *EAAT2* RNA species continues to be debated, however, as they have also been found in some

In both human tissue and transgenic models of ALS, there is abundant evidence that astroglial abnormalities and physiological dysfunction precede clinical disease. These changes include reactive astrocytosis that can be seen many months before motor neuron degeneration (G85R) (Bruijn *et al.,* 1997), and loss of glutamate transport and GLT1 protein expression before the onset of clinical disease or overt motor neuron degeneration (Howland *et al.,* 2002). Similarly, increased astrocytes activation and expression of immune/inflammatory markers are hallmark of this pathology (Chiu *et al.,* 2008; Chiu *et al.,* 2009). Is the reduction in GLT1 protein in astrocytes significant? Guo and colleagues addressed this question by overexpressing the EAAT2 protein in astrocytes in the mSOD1 mouse model, and demonstrated an increase in motor neuron survival and a delay in disease onset; similar outcomes are seen with drugs that increase GLT1 expression (Guo *et al.,* 2003). This evidence indicates that EAAT2 expressed in astrocytes - and probably also glutamate- influences the timing of disease onset and motor neuron survival (Guo *et al.,* 2003). Other changes associated with ALS include increased expression of various proteins in astrocytes, including inducible nitric oxide synthase (iNOS), the copper chaperone CCS, and metallothioneins. Pathologically, early cytosolic proteinaceous aggregates have been found in spinal cord astrocytes from the entire mSOD1 mouse lines examined to date (Patel

Multiple Sclerosis is a chronic inflammatory demyelinating disease of the central nervous system in which glial cells play a prominent role. In murine experimental autoimmune encephalomyelitis (EAE), an established animal model of multiple sclerosis, astrocyte hypertrophy coincided with manifestation of axonal damage (Wang *et al.,* 2005). Astrocytes in multiple sclerosis plaques produce IL-6 (Okuda *et al.,* 1998), lack β-2 adrenergic receptors, and potentially serve as antigen-presenting cells (Zeinstra *et al.,* 2000b), thus facilitating Tcell invasion and activation. Repeated exposure of these astrocytes to inflammatory cytokines triggers unregulated inflammatory responses and increased noradrenalin levels, leading to focal areas of myelin and axonal damage (De Keyser *et al.,* 1999; Zeinstra *et al.,*

Concerning the immune system, class II MHC expressing astrocytes have been shown to process and present antigens and activate both naïve and memory T cells (Nikcevich *et al.,* 1997; Soos *et al.,* 1998). In contrast, other investigators have shown that class II MHC expressing astrocytes are not capable of stimulating T-cell proliferation and instead induce

frequently overlooked.

& Maragakis, 2002).

2000a).

**3.5 Multiple Sclerosis** 

normal controls (Flowers *et al.,* 2001; Meyer *et al.,* 1999).

Either prooxidant agents or amyloid beta peptide did not cause deleterious effects in the astrocytes, but the combined treatment let to oxidative stress and apoptosis in vitro and inflammation and degenerative traits in vivo. Therefore, a reduced oxidative stress defense capacity in frail aged astrocytes may contribute to neuron death by failure of astrocyte support. To preserve astrocyte function and reduce oxidative stress in old age is a new goal against AD (Aliev *et al.,* 2009a; Aliev *et al.,* 2009b; Garcia-Matas *et al.,* 2010).

#### **3.3 Parkinson´s disease**

PD is the second most prevalent neurodegenerative disease, after AD. PD is estimated to affect about 1 million Americans, or about 1% of the population over 60 years of age. PD is caused by the disruption of dopaminergic neurotransmission in the basal ganglia. On pathological examination, the numbers of dopaminergic neurons in the substantia nigra are markedly reduced, and Lewy bodies (cytoplasmic inclusions) are present in the residual dopaminergic neurons (Nutt & Wooten, 2005). The focus has always been on the loss of these dopamanergic neurons and subsequent depletion of dopamine, but a role for nonneuronal cells in producing neuropathological or neuroprotective functions in PD is becoming increasingly recognized.

The studies that have been carried out to date appear to support a neuroprotective role for astrocytes in PD. From pathological examinations, an increase in the number of astrocytes as well as in GFAP expression is observed in PD, (Ciesielska *et al.,* 2009; Muramatsu *et al.,* 2003), as with other neurodegenerative disorders. The pathological evidence indirectly indicates that antioxidant pathways might contribute to this neuroprotective effect, because in control brains the density of glutathione-peroxidase-positive cells was higher in the vicinity of the dopaminergic cell groups known to be resistant to the pathological process of PD. The increase in glutathione-peroxidase-containing cells was inversely correlated with the severity of dopaminergic cell loss in the respective cell groups in patients with PD. The quantity of glutathione-peroxidase-containing cells, therefore, might be critical for a protective effect against oxidative stress (Damier *et al.,* 1993). Conversely, the presence of synuclein-positive astrocytes in pathological samples has been shown to correlate with nigral neuronal cell death (Wakabayashi *et al.,* 2000).

Nitric oxide production and glutathione depletion also appear as consistent features in human PD. The release of glutathione represents another pathway by which astrocytes might be neuroprotective in PD models. Glutathione production appears to be increased by exposure of astrocytes to nitric oxide, and the increase in glutathione release by astrocytes might increase its availability to neurons, thereby making them less susceptible to reactive nitrogen species. This pattern is consistent with the data in PD patients, in whom glutathione-containing cells are in regions with preserved dopaminergic neurons (Heales *et al.,* 2004).

Evidence regarding regulation of glutamate transporter expression and function in PD has been somewhat mixed, with downregulation of glutamate transporters being reported in some studies and upregulation being reported in others. The differences in these studies might be related to the methods by which the lesions were induced (Maragakis & Rothstein, 2004).

#### **3.4 Amyotrophic Lateral Syndrome (ALS)**

Amyotrophic Lateral Syndrome is an inexorably progressive motor neuron disease, in which both the upper motor neurons and the lower motor neurons degenerate leading to

Either prooxidant agents or amyloid beta peptide did not cause deleterious effects in the astrocytes, but the combined treatment let to oxidative stress and apoptosis in vitro and inflammation and degenerative traits in vivo. Therefore, a reduced oxidative stress defense capacity in frail aged astrocytes may contribute to neuron death by failure of astrocyte support. To preserve astrocyte function and reduce oxidative stress in old age is a new goal

PD is the second most prevalent neurodegenerative disease, after AD. PD is estimated to affect about 1 million Americans, or about 1% of the population over 60 years of age. PD is caused by the disruption of dopaminergic neurotransmission in the basal ganglia. On pathological examination, the numbers of dopaminergic neurons in the substantia nigra are markedly reduced, and Lewy bodies (cytoplasmic inclusions) are present in the residual dopaminergic neurons (Nutt & Wooten, 2005). The focus has always been on the loss of these dopamanergic neurons and subsequent depletion of dopamine, but a role for nonneuronal cells in producing neuropathological or neuroprotective functions in PD is

The studies that have been carried out to date appear to support a neuroprotective role for astrocytes in PD. From pathological examinations, an increase in the number of astrocytes as well as in GFAP expression is observed in PD, (Ciesielska *et al.,* 2009; Muramatsu *et al.,* 2003), as with other neurodegenerative disorders. The pathological evidence indirectly indicates that antioxidant pathways might contribute to this neuroprotective effect, because in control brains the density of glutathione-peroxidase-positive cells was higher in the vicinity of the dopaminergic cell groups known to be resistant to the pathological process of PD. The increase in glutathione-peroxidase-containing cells was inversely correlated with the severity of dopaminergic cell loss in the respective cell groups in patients with PD. The quantity of glutathione-peroxidase-containing cells, therefore, might be critical for a protective effect against oxidative stress (Damier *et al.,* 1993). Conversely, the presence of synuclein-positive astrocytes in pathological samples has been shown to correlate with

Nitric oxide production and glutathione depletion also appear as consistent features in human PD. The release of glutathione represents another pathway by which astrocytes might be neuroprotective in PD models. Glutathione production appears to be increased by exposure of astrocytes to nitric oxide, and the increase in glutathione release by astrocytes might increase its availability to neurons, thereby making them less susceptible to reactive nitrogen species. This pattern is consistent with the data in PD patients, in whom glutathione-containing cells are in regions with preserved dopaminergic neurons (Heales *et* 

Evidence regarding regulation of glutamate transporter expression and function in PD has been somewhat mixed, with downregulation of glutamate transporters being reported in some studies and upregulation being reported in others. The differences in these studies might be related to the methods by which the lesions were induced (Maragakis & Rothstein, 2004).

Amyotrophic Lateral Syndrome is an inexorably progressive motor neuron disease, in which both the upper motor neurons and the lower motor neurons degenerate leading to

against AD (Aliev *et al.,* 2009a; Aliev *et al.,* 2009b; Garcia-Matas *et al.,* 2010).

**3.3 Parkinson´s disease** 

becoming increasingly recognized.

*al.,* 2004).

nigral neuronal cell death (Wakabayashi *et al.,* 2000).

**3.4 Amyotrophic Lateral Syndrome (ALS)** 

muscle atrophy. Patients eventually experience respiratory failure, usually within three to five years from diagnosis. However, the onset of ALS may be subtle and early symptoms are frequently overlooked.

Common to familial and sporadic ALS is the loss of the astrocyte glutamate transporter EAAT2. Studies of the EAAT2 transporter in tissue from individuals with sporadic ALS showed a marked loss of up to 95% of astroglial EAAT2 protein expression and activity in affected areas of the CNS (Bristol & Rothstein, 1996). A clue to a possible mechanism for EAAT2 reduction or dysfunction was provided by the finding of aberrant *EAAT2* RNA species, which has been implicated in multiple neurodegenerative diseases. The production of truncated EAAT2 protein results in reduced function, and the retention of normal EAAT2 protein within the cytoplasm (Lin *et al.,* 1998). The significance of these aberrant *EAAT2* RNA species continues to be debated, however, as they have also been found in some normal controls (Flowers *et al.,* 2001; Meyer *et al.,* 1999).

In both human tissue and transgenic models of ALS, there is abundant evidence that astroglial abnormalities and physiological dysfunction precede clinical disease. These changes include reactive astrocytosis that can be seen many months before motor neuron degeneration (G85R) (Bruijn *et al.,* 1997), and loss of glutamate transport and GLT1 protein expression before the onset of clinical disease or overt motor neuron degeneration (Howland *et al.,* 2002). Similarly, increased astrocytes activation and expression of immune/inflammatory markers are hallmark of this pathology (Chiu *et al.,* 2008; Chiu *et al.,* 2009). Is the reduction in GLT1 protein in astrocytes significant? Guo and colleagues addressed this question by overexpressing the EAAT2 protein in astrocytes in the mSOD1 mouse model, and demonstrated an increase in motor neuron survival and a delay in disease onset; similar outcomes are seen with drugs that increase GLT1 expression (Guo *et al.,* 2003). This evidence indicates that EAAT2 expressed in astrocytes - and probably also glutamate- influences the timing of disease onset and motor neuron survival (Guo *et al.,* 2003). Other changes associated with ALS include increased expression of various proteins in astrocytes, including inducible nitric oxide synthase (iNOS), the copper chaperone CCS, and metallothioneins. Pathologically, early cytosolic proteinaceous aggregates have been found in spinal cord astrocytes from the entire mSOD1 mouse lines examined to date (Patel & Maragakis, 2002).

#### **3.5 Multiple Sclerosis**

Multiple Sclerosis is a chronic inflammatory demyelinating disease of the central nervous system in which glial cells play a prominent role. In murine experimental autoimmune encephalomyelitis (EAE), an established animal model of multiple sclerosis, astrocyte hypertrophy coincided with manifestation of axonal damage (Wang *et al.,* 2005). Astrocytes in multiple sclerosis plaques produce IL-6 (Okuda *et al.,* 1998), lack β-2 adrenergic receptors, and potentially serve as antigen-presenting cells (Zeinstra *et al.,* 2000b), thus facilitating Tcell invasion and activation. Repeated exposure of these astrocytes to inflammatory cytokines triggers unregulated inflammatory responses and increased noradrenalin levels, leading to focal areas of myelin and axonal damage (De Keyser *et al.,* 1999; Zeinstra *et al.,* 2000a).

Concerning the immune system, class II MHC expressing astrocytes have been shown to process and present antigens and activate both naïve and memory T cells (Nikcevich *et al.,* 1997; Soos *et al.,* 1998). In contrast, other investigators have shown that class II MHC expressing astrocytes are not capable of stimulating T-cell proliferation and instead induce

Role of Astrocytes in Neurodegenerative Diseases 267

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microglial phagocytosis of senile plaque cores of Alzheimer's disease. *Exp Neurol*

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#### **4. Conclusions**

Astrocytes play a critical role in normal function of the mammalian nervous system. Astrocytes regulate K+ buffering, glutamate clearance, brain antioxidant defense, close metabolic coupling with neurons, and modulation of neuronal excitability. In numerous pathological states, such as AD, PD, ALS and ME, astrocytes are involved in both exacerbation of damage and neuroprotective mechanisms. As discussed in this chapter, they support neurons in many ways, all of which are essential for repair and regeneration. Disturbances in astrocytic functions are implicated in neurodegenerative diseases pathogenesis, therefore, modulation of astrocyte functioning may prove to be an efficient therapeutic strategy in many chronic CNS disorders.

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**12** 

*Russia* 

**Alteration of Neuron-Glia** 

Alla B. Salmina et al.\*

**Interactions in Neurodegeneration:** 

**Molecular Biomarkers and Therapeutic Strategy** 

Accumulating evidence suggests that alterations of neuron-astroglia interactions are associated with development of neurodegenerative diseases (Barres, 2008; Ricci et al., 2009; Verkhratsky et al., 2010). Astrocytes contribute to a variety of functions of neurons, including synapse formation and plasticity, energetic and redox metabolism, and synaptic homeostasis of neurotransmitters and ions (Bolton & Eroglu, 2009; Nimmerjahn, 2009; Salmina, 2009; Verkhratsky, 2010; Wang & Bordey, 2008). It is well known that astrocytes play important role in supporting energy production in neurons. Astrocytes produce lactate which is actively taken up by active neurons. They utilize lactate as an alternative energy fuel. In turn, neuronal activation results in glutamate-stimulated glycolysis in astroglial cells. Thus, neuron-astrocyte metabolic coupling provides tight interactions between the activated neuronal cells consuming lactate and lactate-producing astrocytes. Remodeling of astrocytes is required for adequate synapse turnover in the brain, and astrocytes or the closely related radial glial cells possess all the attributes of a neural stem cells, thereby playing a key role in

Functional relationship between neurons, glial cells, and vascular cells within so-called neurovascular unit is very important in the context of pathogenesis of neurodegenerative disorders. A major function of the neurovascular unit is to regulate the transport and diffusion properties of brain capillary endothelial cells that compose the brain-blood barrier. Astrocytes exhibit anatomic relationships with cerebral arterioles and neurons. In the brain parenchyma, the extensive ensheatment of cerebral arterioles by astrocytic end-feet far exceeds any direct neural contacts with those perfusion-regulating microvessels. That unique arrangement permits astrocytes to transduce signals arising from activated neurons and to transmit that information to the cerebral microcirculation. Alteration of these processes may play a particularly significantrole in the pathogenesis of neurodegenerative diseases. The early and mid-term stages of neurodegenerative processes are associated with generalised atrophy of astroglia, whereas the later stages are characterized with an

Marina M. Petrova, Tatyana E. Taranushenko, Semen V. Prokopenko, Natalia A. Malinovskaya, Olesya

neurogenesis (Steindler & Laywell, 2003; Theodosis et al., 2006).

S. Okuneva, Alyona I. Inzhutova, Andrei V. Morgun, Alexander A. Fursov *Krasnoyarsk State Medical University named after Prof. V.F.Voino-Yasenetsky; Russia* 

**1. Introduction** 

 \*

*Krasnoyarsk State Medical University named after Prof. V.F.Voino-Yasenetsky* 


## **Alteration of Neuron-Glia Interactions in Neurodegeneration: Molecular Biomarkers and Therapeutic Strategy**

Alla B. Salmina et al.\*

*Krasnoyarsk State Medical University named after Prof. V.F.Voino-Yasenetsky Russia* 

#### **1. Introduction**

272 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

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Astrocyte-associated axonal damage in pre-onset stages of experimental

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expression in the hippocampus: regionally specific effects of aging and caloric

Carmignoto, G. (2003). Neuron-to-astrocyte signaling is central to the dynamic

synthetase down-regulation reduces astrocyte protection against glutamate

Accumulating evidence suggests that alterations of neuron-astroglia interactions are associated with development of neurodegenerative diseases (Barres, 2008; Ricci et al., 2009; Verkhratsky et al., 2010). Astrocytes contribute to a variety of functions of neurons, including synapse formation and plasticity, energetic and redox metabolism, and synaptic homeostasis of neurotransmitters and ions (Bolton & Eroglu, 2009; Nimmerjahn, 2009; Salmina, 2009; Verkhratsky, 2010; Wang & Bordey, 2008). It is well known that astrocytes play important role in supporting energy production in neurons. Astrocytes produce lactate which is actively taken up by active neurons. They utilize lactate as an alternative energy fuel. In turn, neuronal activation results in glutamate-stimulated glycolysis in astroglial cells. Thus, neuron-astrocyte metabolic coupling provides tight interactions between the activated neuronal cells consuming lactate and lactate-producing astrocytes. Remodeling of astrocytes is required for adequate synapse turnover in the brain, and astrocytes or the closely related radial glial cells possess all the attributes of a neural stem cells, thereby playing a key role in neurogenesis (Steindler & Laywell, 2003; Theodosis et al., 2006).

Functional relationship between neurons, glial cells, and vascular cells within so-called neurovascular unit is very important in the context of pathogenesis of neurodegenerative disorders. A major function of the neurovascular unit is to regulate the transport and diffusion properties of brain capillary endothelial cells that compose the brain-blood barrier. Astrocytes exhibit anatomic relationships with cerebral arterioles and neurons. In the brain parenchyma, the extensive ensheatment of cerebral arterioles by astrocytic end-feet far exceeds any direct neural contacts with those perfusion-regulating microvessels. That unique arrangement permits astrocytes to transduce signals arising from activated neurons and to transmit that information to the cerebral microcirculation. Alteration of these processes may play a particularly significantrole in the pathogenesis of neurodegenerative diseases. The early and mid-term stages of neurodegenerative processes are associated with generalised atrophy of astroglia, whereas the later stages are characterized with an

<sup>\*</sup> Marina M. Petrova, Tatyana E. Taranushenko, Semen V. Prokopenko, Natalia A. Malinovskaya, Olesya S. Okuneva, Alyona I. Inzhutova, Andrei V. Morgun, Alexander A. Fursov

*Krasnoyarsk State Medical University named after Prof. V.F.Voino-Yasenetsky; Russia* 

Alteration of Neuron-Glia Interactions in Neurodegeneration:

Molecular Biomarkers and Therapeutic Strategy 275

phosphoryls and NAD+. In cytosol, NAD+ and NADH mediate glycolysis acting as cofactors for rate-limiting glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase, participate in lactate-pyruvate conversions, and affect mitochondrial oxidative phosphorylation. Also, NAD+ serves as a substrate for NAD+-converting enzymes (ADP-

Synthesis of NAD+ *in vivo* and *de novo* is possible by means of 4 pathways: 1) from nicotinic acid (niacin); 2) from nicotinamide (vitamin PP); 3) from tryptophan; 4) from aspartate (in plants only). Recently, a biosynthesis pathway which uses nicotinamide riboside as a precursor has been described. For the synthesis of NAD+ from L-tryptophan, the indole ring should undergo transformation into the pyridine ring, and finally quinolinate is formed. Quinolinic acid undergoes further transformations resulting in NAD+ formation. Indoleamine 2,3-dioxygenase (expressed in various tissues) and tryptophan 2,3-dioxygenase (expressed in liver) catalyze the formation of N-formylkynurenine in the reaction representing the first rate-limiting step in this pathway. Indoleamine 2,3-dioxygenase is a unique enzyme in that it can utilize as the substrate, in place of oxygen, the superoxide anion radical, thus acting as a radical scavenger. The second rate-limiting step in this pathway is represented by the reaction catalyzing by quinolinate phosphoribosyltransferase responsible for the conversion of quinolinic acid and 5-phospho-α-D-ribose 1-diphosphate

Enzymes responsible for producing neuroactive compounds in the kynurenine pathway are preferentially localized in astrocytes and microglia. Astrocytes are well equipped with enzymes for kynurenine metabolism, i.e indoleamine 2,3-dioxygenase is highly expressed in these cells (Suh et al., 2007), thus raising the possibility that generation of the neuroactive compounds may play significant role in neuron-glial cell interactions (Ying, 2006). Interestingly, tryptophan and kynurenine stimulate expression of nerve growth factor in astrocytes (Dong-Ruyl et al., 1998), while interferon-gamma hyperactivates indoleamine 2,3 dioxygenase resulting in elevated production of kynunerines (i.e. quinolinic and picolinic acids) and stimulation of iNOS in human aged brain and vascular cognitive impairment (Oxenkrug, 2007). In general, kynurenine pathway is up-regulated in Alzheimer's disease brain, i.e. in hippocampus, where indoleamine 2,3-dioxygenase and quinolinic acid immunoreactivity was detected in astrocytes, microglia and neurons, with highest expression in glial cells in the perimeter of senile plaques. Quinolinic acid immunoreactivity was also present in granular deposits within the neuronal soma of cortex, and looks to label neurofibrillary tangles (Guillemin et al., 2005). Dysfunction of quinolinate metabolism in the human brain has been postulated to be involved in the pathogenesis of Alzheimer's disease ("quinolinate hypothesis") (Fukuoka et al., 2002). At the same time, it was suggested that astrocytes alone are neuroprotective by minimizing quinolinic acid production and maximizing synthesis of kynurenic acid (due to absence of kynurenine hydroxylase), but in the presence of macrophages and/or microglia, astrocytes become indirectly neurotoxic by the production of high concentrations of kynurenine that can be secondary metabolized by

Mononucleotide adenylyltransferase (NMNAT) is a central enzyme in NAD+ biosynthesis, catalyzing the condensation of nicotinamide mononucleotide or nicotinic acid mononucleotide with the AMP moiety of ATP to form NAD+. NMNAT-1 has nuclear localization, and was proposed to have functional relations with poly (ADP-ribosyl) polymerase (PARP) in prevention of NAD+ depletion during PARP over-activation. NMNAT-2 isoform has cytoplasmic localization, and is very prone to oxidation due to

ribosyltransferases, poly(ADP-ribosyl)polymerase and ADP-ribosyl cyclase).

to nicotinate mononucleotide, pyrophosphate and CO2 (Magni et al., 2004).

neighboring cells to quinolinic acid (Guillemin et al., 2001).

astrogliosis and microglial activation linked to neuropathological lesions such as senile plaques (Rodriguez & Verkhratsky, 2010).

Acute neurodegeneration is encountered during and following stroke, transient cardiac arrest, brain trauma, insulin-induced hypoglycemia and status epilepticus. All these severe clinical conditions are characterized by neuronal calcium overload, aberrant cell signaling, generation of free radicals and elevation of cellular free fatty acids, conditions that favor activation of the mitochondrial permeability transition pore (mtPTP) (Friberg & Wieloch, 2002).

Pathological cascade leading to clinical manifestations of chronic neurodegeneration (i.e. Parkinson's disease, Alzheimer's disease, Hungtington's disease) includes progressive loss of functional synapses, irreversible damage and loss of neurons, neurotoxicity, and excessive activation of astroglial (reactive astrogliosis) and microglial (neuroinflammation) cells. Neurodegeneration is associated with axonal and synapse degeneration which is triggered by mechanical, metabolic, infectious, toxic, hereditary and inflammatory stimuli. Several signaling pathways are implicated in axonal and synapse degeneration, but identification of an integrative mechanism for these self-destructive processes has remained elusive. Also, neurodegenerative events are known to be associated with alterations in cellcell interactions, gene expression, dynamics of neuronal networks, development of oxidative stress, accumulation of lipid and protein oxidation products, production of fatty acids metabolites with biological activity, mitochondrial dysfunction, impairment of multiple signaling pathways, activation of programmed cell death (Salmina, 2009).

In general, the link between the character of astroglial activation and neuronal damage or repair in neurodegeneration is well established (Theodosis et al., 2008). The current conception includes impairment of astroglia-assisted synapse formation and plasticity, synapse elimination, neurogenesis, function of neural circuits, and functioning of the bloodbrain barrier; dysregulation of gliovascular control and cerebral blood flow, alterations of neuronal metabolism, astrocyte-dependent augmentation of oxidative stress due to impaired antioxidant activity, stimulation of neuroinflammatory response, potentiation of excitotoxic insult, mitochondrial and glycolytic failure, impairment of glial calcium homeostasis, pathology of neurovascular unit, reactive astrogliosis accompanied by scar formation and initiation of brain repair (Bambrick et al., 2004; Barres, 2008; Buffo et al., 2008; L'Episcopo et al., 2010; Ricci, et al., 2009; Sofroniew, 2009; Stevens, 2008; Verkhratsky, et al., 2010; Verkhratsky et al., 1998).

Recent achievements in deciphering cell and molecular mechanisms of acute and chronic neurodegeneration suggest new prospective biomarkers and therapeutic targets for modulation of neuron-glia interactions. In this chapter, we will focus on several aspects of metabolism of nicotinamide adenine dinucleotide (NAD+) in neurons and astrocytes as a critical factor in neurodegeneration-associated cell damage.

#### **2. Neuronal and glial NAD<sup>+</sup> -generating and NAD<sup>+</sup> -converting enzymes in neurodegeneration**

Few decades ago, the actions of NAD+ have been extended from being an oxidoreductase cofactor for single enzymatic activities to acting as substrate for a wide range of proteins. These include NAD+-converting enzymes, and transcription factors that affect a large array of cellular functions. Through these effects, NAD+ provides a direct link between the cellular redox status and the control of signaling and transcriptional events (Houtkooper et al., 2010). Cellular bioenergetic homeostasis requires production and delivery of energy-rich

astrogliosis and microglial activation linked to neuropathological lesions such as senile

Acute neurodegeneration is encountered during and following stroke, transient cardiac arrest, brain trauma, insulin-induced hypoglycemia and status epilepticus. All these severe clinical conditions are characterized by neuronal calcium overload, aberrant cell signaling, generation of free radicals and elevation of cellular free fatty acids, conditions that favor activation of the mitochondrial permeability transition pore (mtPTP) (Friberg & Wieloch,

Pathological cascade leading to clinical manifestations of chronic neurodegeneration (i.e. Parkinson's disease, Alzheimer's disease, Hungtington's disease) includes progressive loss of functional synapses, irreversible damage and loss of neurons, neurotoxicity, and excessive activation of astroglial (reactive astrogliosis) and microglial (neuroinflammation) cells. Neurodegeneration is associated with axonal and synapse degeneration which is triggered by mechanical, metabolic, infectious, toxic, hereditary and inflammatory stimuli. Several signaling pathways are implicated in axonal and synapse degeneration, but identification of an integrative mechanism for these self-destructive processes has remained elusive. Also, neurodegenerative events are known to be associated with alterations in cellcell interactions, gene expression, dynamics of neuronal networks, development of oxidative stress, accumulation of lipid and protein oxidation products, production of fatty acids metabolites with biological activity, mitochondrial dysfunction, impairment of multiple

In general, the link between the character of astroglial activation and neuronal damage or repair in neurodegeneration is well established (Theodosis et al., 2008). The current conception includes impairment of astroglia-assisted synapse formation and plasticity, synapse elimination, neurogenesis, function of neural circuits, and functioning of the bloodbrain barrier; dysregulation of gliovascular control and cerebral blood flow, alterations of neuronal metabolism, astrocyte-dependent augmentation of oxidative stress due to impaired antioxidant activity, stimulation of neuroinflammatory response, potentiation of excitotoxic insult, mitochondrial and glycolytic failure, impairment of glial calcium homeostasis, pathology of neurovascular unit, reactive astrogliosis accompanied by scar formation and initiation of brain repair (Bambrick et al., 2004; Barres, 2008; Buffo et al., 2008; L'Episcopo et al., 2010; Ricci, et al., 2009; Sofroniew, 2009; Stevens, 2008; Verkhratsky, et al.,

Recent achievements in deciphering cell and molecular mechanisms of acute and chronic neurodegeneration suggest new prospective biomarkers and therapeutic targets for modulation of neuron-glia interactions. In this chapter, we will focus on several aspects of metabolism of nicotinamide adenine dinucleotide (NAD+) in neurons and astrocytes as a

**-generating and NAD<sup>+</sup>**

Few decades ago, the actions of NAD+ have been extended from being an oxidoreductase cofactor for single enzymatic activities to acting as substrate for a wide range of proteins. These include NAD+-converting enzymes, and transcription factors that affect a large array of cellular functions. Through these effects, NAD+ provides a direct link between the cellular redox status and the control of signaling and transcriptional events (Houtkooper et al., 2010). Cellular bioenergetic homeostasis requires production and delivery of energy-rich

**-converting enzymes in** 

signaling pathways, activation of programmed cell death (Salmina, 2009).

critical factor in neurodegeneration-associated cell damage.

plaques (Rodriguez & Verkhratsky, 2010).

2010; Verkhratsky et al., 1998).

**2. Neuronal and glial NAD<sup>+</sup>**

**neurodegeneration** 

2002).

phosphoryls and NAD+. In cytosol, NAD+ and NADH mediate glycolysis acting as cofactors for rate-limiting glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase, participate in lactate-pyruvate conversions, and affect mitochondrial oxidative phosphorylation. Also, NAD+ serves as a substrate for NAD+-converting enzymes (ADPribosyltransferases, poly(ADP-ribosyl)polymerase and ADP-ribosyl cyclase).

Synthesis of NAD+ *in vivo* and *de novo* is possible by means of 4 pathways: 1) from nicotinic acid (niacin); 2) from nicotinamide (vitamin PP); 3) from tryptophan; 4) from aspartate (in plants only). Recently, a biosynthesis pathway which uses nicotinamide riboside as a precursor has been described. For the synthesis of NAD+ from L-tryptophan, the indole ring should undergo transformation into the pyridine ring, and finally quinolinate is formed. Quinolinic acid undergoes further transformations resulting in NAD+ formation. Indoleamine 2,3-dioxygenase (expressed in various tissues) and tryptophan 2,3-dioxygenase (expressed in liver) catalyze the formation of N-formylkynurenine in the reaction representing the first rate-limiting step in this pathway. Indoleamine 2,3-dioxygenase is a unique enzyme in that it can utilize as the substrate, in place of oxygen, the superoxide anion radical, thus acting as a radical scavenger. The second rate-limiting step in this pathway is represented by the reaction catalyzing by quinolinate phosphoribosyltransferase responsible for the conversion of quinolinic acid and 5-phospho-α-D-ribose 1-diphosphate to nicotinate mononucleotide, pyrophosphate and CO2 (Magni et al., 2004).

Enzymes responsible for producing neuroactive compounds in the kynurenine pathway are preferentially localized in astrocytes and microglia. Astrocytes are well equipped with enzymes for kynurenine metabolism, i.e indoleamine 2,3-dioxygenase is highly expressed in these cells (Suh et al., 2007), thus raising the possibility that generation of the neuroactive compounds may play significant role in neuron-glial cell interactions (Ying, 2006). Interestingly, tryptophan and kynurenine stimulate expression of nerve growth factor in astrocytes (Dong-Ruyl et al., 1998), while interferon-gamma hyperactivates indoleamine 2,3 dioxygenase resulting in elevated production of kynunerines (i.e. quinolinic and picolinic acids) and stimulation of iNOS in human aged brain and vascular cognitive impairment (Oxenkrug, 2007). In general, kynurenine pathway is up-regulated in Alzheimer's disease brain, i.e. in hippocampus, where indoleamine 2,3-dioxygenase and quinolinic acid immunoreactivity was detected in astrocytes, microglia and neurons, with highest expression in glial cells in the perimeter of senile plaques. Quinolinic acid immunoreactivity was also present in granular deposits within the neuronal soma of cortex, and looks to label neurofibrillary tangles (Guillemin et al., 2005). Dysfunction of quinolinate metabolism in the human brain has been postulated to be involved in the pathogenesis of Alzheimer's disease ("quinolinate hypothesis") (Fukuoka et al., 2002). At the same time, it was suggested that astrocytes alone are neuroprotective by minimizing quinolinic acid production and maximizing synthesis of kynurenic acid (due to absence of kynurenine hydroxylase), but in the presence of macrophages and/or microglia, astrocytes become indirectly neurotoxic by the production of high concentrations of kynurenine that can be secondary metabolized by neighboring cells to quinolinic acid (Guillemin et al., 2001).

Mononucleotide adenylyltransferase (NMNAT) is a central enzyme in NAD+ biosynthesis, catalyzing the condensation of nicotinamide mononucleotide or nicotinic acid mononucleotide with the AMP moiety of ATP to form NAD+. NMNAT-1 has nuclear localization, and was proposed to have functional relations with poly (ADP-ribosyl) polymerase (PARP) in prevention of NAD+ depletion during PARP over-activation. NMNAT-2 isoform has cytoplasmic localization, and is very prone to oxidation due to

Alteration of Neuron-Glia Interactions in Neurodegeneration:

NAD+ biosynthesis (Grant & Kapoor, 1998).

appears to be the enzyme itself (Ziegler, 2000).

Molecular Biomarkers and Therapeutic Strategy 277

however, the pathway for NAD+ regeneration from nicotinic acid is a preferred route for

Among all the NAD+-converting enzymes, poly(ADP-ribosyl) polymerase (PARP) and ADP-ribosyl cyclase attract the main attention in terms of neurodegenerative disorders (Kauppinen & Swanson, 2007). Poly(ADP-ribosyl) polymerase functions as DNA damage sensor and signaling molecule binding to single- and double-stranded DNA breaks. Upon binding to damaged DNA PARP forms homodimers and catalyzes the cleavage of NAD+ into ADP-ribose and nicotinamide. ADP-ribose is then used to synthesize the branched polymer attached to nuclear (or mitochondrial) acceptor proteins. Variety of acceptor proteins has been described (histones, DNA repair enzymes, topoisomerases, transcription factors, DNA-dependent protein kinase, lamin B, p53), but the most efficient acceptor

There is a growing number of evidences on involvement of PARP and PARP-mediated depletion of intracellular NAD+ in the acute and chronic injury of cells (ischemia/reperfusion, endothelial dysfunction, genotoxicity, inflammation, traumatic injury) (Oliver et al., 1999). In respect to the CNS, NAD+ depletion and mitochondrial permeability transition were shown to be sequential and necessary steps in PARP-1 overactivation-dependent cell death in astrocytes (Alano et al., 2004). Increased poly(ADPribosylation) of nuclear proteins was demonstrated in neurons in Alzheimer's disease (Love et al., 1999). Intra-mitochondrial PARP contributes to NAD+ depletion and cell death induced by oxidative stress in neurons (Du et al., 2003). Increased poly ADP-ribosylation of nuclear proteins in Alzheimer's disease has been detected, and double immunolabelling for poly(ADP-ribose) and markers of neuronal, astrocytic and microglial differentiation showed many of the cells containing poly(ADP-ribose) to be neurons, while few of the cells were astrocytes, and no poly(ADP-ribose) accumulation was found in microglia (Kauppinen & Swanson, 2007; Love, et al., 1999). Moreover, it was shown that β-amyloid affected cholinergic receptor-mediated signal transduction to PARP, probably, through free radical evoked inhibition of inositol-3-phosphate formation in the hippocampal cells (Adamczyk et al., 2005). Glutamate neurotransmission involving NMDA receptors and neuronal nitric oxide synthase activity in part mediates neuronal DNA strand breaks and PARP activation. These events are especially important for neurons since astrocytes able to maintain higher levels of NAD+ comparing with neurons (Pieper et al., 2000), and much higher concentrations of oxidants are required for killing astrocytes (Ying et al., 2002). It should be taken into the consideration that excessive PARP activation leads to impairment of glycolysis in affected cells, thereby impaired glycolytic flux is involved into PARP-mediated neuronal and astroglial cell death. Since astrocyte-produced lactate is a major endogenous energy substrate used by neurons in brain, NAD+ depletion caused by excessive PARP activation in neurons would result in alteration of lactate-pyruvate conversion thus affecting the efficacy of oxidative metabolism in neurons and astrocyte-neuronal lactate shuttle mechanism. In addition, neurological metabolic coupling implies subcellular compartmentation of pyruvate and monocarboxylate recycling through the plasma membrane of both neurons and glial cells, subcellular compartmentation of pyruvate allows neurons and astrocytes to select between glucose and lactate as alternative substrates depending of the concentrations and the operation of a redox switch (Cerdan et al., 2006). Pyruvate compartmentation results in effective transcellular coupling between the cytosolic NAD+/NADH redox states of neuronal and glial cells, therefore, impairment of this

presence of nine cysteines versus four cysteines present in NMNAT-1. NMNAT-3 presents in cytoplasm and mitochondria, has much lower enzymatic activity comparing with NMNAT-1 and NMNAT-2 (Raffaelli et al., 2002). Extracelullar nucleotides (*e.g.* NAD+ and NMN) undergo extracellular degradation resulting in the formation of permeable precursors which are further converted to NAD+ in mitochondria due to activity of NMNAT3 localized to the mitochondrial matrix (Nikiforov et al., 2011). Interestingly enough, in genomewide screen for late-onset Alzheimer's disease, SNP of the NMNAT-3 gene was found, thus suggesting involvement of NAD+ synthesizing pathways in pathogenesis of this neurodegenerative disorder (Liu et al., 2007)].

Recently, a role for mitochondrial permeability transition, and mitochondrial dysfunction, in development of axonal degeneration has been proposed. Axonal degeneration has been shown to be regulated by proapoptotic proteins (i.e. caspases 3 and 6) and/or NAD+ sensitive pathways (Schoenmann et al., 2010). Since these degenerative processes can cause permanent loss of function, they represent a focus for neuroprotective strategies (Barrientos et al., 2011). Functioning of NMNAT as a chaperone acting through a proteasome-mediated pathway was found (Zhai et al., 2008), thus suggesting novel aspects in regulation of NAD+ homeostasis under the conditions of cellular stress. Overexpression of NMNAT in the mitochondrial matrix resulted in suppression of axonal degeneration seen in neurodenegeration (Sasaki & Milbrandt, 2010; Sasaki et al., 2009; Yahata et al., 2009). In amyloid-treated cells, NMNAT-sensitive program is uniquely involved in axonal, but not cell body, degeneration (Vohra et al., 2010). Axonal degeneration can be slowed by the addition of extracellular NAD+ (Billington et al., 2008).

Nicotinamide N-methyltransferase (NNMT) methylates pyridines, in particular nicotinamide, to N-methyl nicotinamide which is further used for synthesis of NAD(P) and NAD(P)H. Increased activity of NNMT leads to cellular nicotinamide deficiency. It was demonstrated that elevated levels of NNMT result in reduced Complex I activity in idiopathic Parkinson's disease (IPD) in two ways: (1) reduction in the levels of nicotinamide available for nicotinamide adenine dinucleotide synthesis; and (2) increased methylation of compounds such as tetrahydroisoquinolines and β-carbolines, which are potent Complex I inhibitors. Expression of NNMT is increased in Parkinson's disease which may ultimately lead to neurodegeneration via a reduction in Complex I activity (Parsons et al., 2003). A.C.Williams has proposed that elevated activity of NNMT may be responsible for dopaminergic toxicity of N-methylated pyridines (i.e. MPP+) and for depletion of NAD+ in the cells (Williams et al., 2005).

Apart from the pathways of NAD+ synthesis *de novo*, there are some reactions for the regeneration of NAD+ from molecules formed during its functioning and catabolism (reduced pyridine nucleotides (NADH, ADP-ribose, nicotinamide, NAAD+). It should be noted that re-synthesis of NAD+ from ADP-ribose and nicotinamide requires as many as four molecules of ATP, therefore being energetically unfavorable (Di Lisa & Ziegler, 2001). Regeneration of NAD+ from NADH may be achieved through the activity of following enzymes and processes: 1) specific NADH flavin dehydrogenase acting in the respiratory chain; 2) transhydrogenase of the outer mitochondrial membrane; 3) specific NADH oxidases; 4) malate-aspartate shuttle. Regeneration of NAD+ is very important not only for the economic using of cellular pool of pyridine nucleotides, but also for their effective intracellular redistribution at the appropriate moment under physiological and pathological conditions. Murine glial cells have been shown to synthesize NAD+ from quinolinic acid,

presence of nine cysteines versus four cysteines present in NMNAT-1. NMNAT-3 presents in cytoplasm and mitochondria, has much lower enzymatic activity comparing with NMNAT-1 and NMNAT-2 (Raffaelli et al., 2002). Extracelullar nucleotides (*e.g.* NAD+ and NMN) undergo extracellular degradation resulting in the formation of permeable precursors which are further converted to NAD+ in mitochondria due to activity of NMNAT3 localized to the mitochondrial matrix (Nikiforov et al., 2011). Interestingly enough, in genomewide screen for late-onset Alzheimer's disease, SNP of the NMNAT-3 gene was found, thus suggesting involvement of NAD+ synthesizing pathways in

Recently, a role for mitochondrial permeability transition, and mitochondrial dysfunction, in development of axonal degeneration has been proposed. Axonal degeneration has been shown to be regulated by proapoptotic proteins (i.e. caspases 3 and 6) and/or NAD+ sensitive pathways (Schoenmann et al., 2010). Since these degenerative processes can cause permanent loss of function, they represent a focus for neuroprotective strategies (Barrientos et al., 2011). Functioning of NMNAT as a chaperone acting through a proteasome-mediated pathway was found (Zhai et al., 2008), thus suggesting novel aspects in regulation of NAD+ homeostasis under the conditions of cellular stress. Overexpression of NMNAT in the mitochondrial matrix resulted in suppression of axonal degeneration seen in neurodenegeration (Sasaki & Milbrandt, 2010; Sasaki et al., 2009; Yahata et al., 2009). In amyloid-treated cells, NMNAT-sensitive program is uniquely involved in axonal, but not cell body, degeneration (Vohra et al., 2010). Axonal degeneration can be slowed by the

Nicotinamide N-methyltransferase (NNMT) methylates pyridines, in particular nicotinamide, to N-methyl nicotinamide which is further used for synthesis of NAD(P) and NAD(P)H. Increased activity of NNMT leads to cellular nicotinamide deficiency. It was demonstrated that elevated levels of NNMT result in reduced Complex I activity in idiopathic Parkinson's disease (IPD) in two ways: (1) reduction in the levels of nicotinamide available for nicotinamide adenine dinucleotide synthesis; and (2) increased methylation of compounds such as tetrahydroisoquinolines and β-carbolines, which are potent Complex I inhibitors. Expression of NNMT is increased in Parkinson's disease which may ultimately lead to neurodegeneration via a reduction in Complex I activity (Parsons et al., 2003). A.C.Williams has proposed that elevated activity of NNMT may be responsible for dopaminergic toxicity of N-methylated pyridines (i.e. MPP+) and for depletion of NAD+ in the cells (Williams et

Apart from the pathways of NAD+ synthesis *de novo*, there are some reactions for the regeneration of NAD+ from molecules formed during its functioning and catabolism (reduced pyridine nucleotides (NADH, ADP-ribose, nicotinamide, NAAD+). It should be noted that re-synthesis of NAD+ from ADP-ribose and nicotinamide requires as many as four molecules of ATP, therefore being energetically unfavorable (Di Lisa & Ziegler, 2001). Regeneration of NAD+ from NADH may be achieved through the activity of following enzymes and processes: 1) specific NADH flavin dehydrogenase acting in the respiratory chain; 2) transhydrogenase of the outer mitochondrial membrane; 3) specific NADH oxidases; 4) malate-aspartate shuttle. Regeneration of NAD+ is very important not only for the economic using of cellular pool of pyridine nucleotides, but also for their effective intracellular redistribution at the appropriate moment under physiological and pathological conditions. Murine glial cells have been shown to synthesize NAD+ from quinolinic acid,

pathogenesis of this neurodegenerative disorder (Liu et al., 2007)].

addition of extracellular NAD+ (Billington et al., 2008).

al., 2005).

however, the pathway for NAD+ regeneration from nicotinic acid is a preferred route for NAD+ biosynthesis (Grant & Kapoor, 1998).

Among all the NAD+-converting enzymes, poly(ADP-ribosyl) polymerase (PARP) and ADP-ribosyl cyclase attract the main attention in terms of neurodegenerative disorders (Kauppinen & Swanson, 2007). Poly(ADP-ribosyl) polymerase functions as DNA damage sensor and signaling molecule binding to single- and double-stranded DNA breaks. Upon binding to damaged DNA PARP forms homodimers and catalyzes the cleavage of NAD+ into ADP-ribose and nicotinamide. ADP-ribose is then used to synthesize the branched polymer attached to nuclear (or mitochondrial) acceptor proteins. Variety of acceptor proteins has been described (histones, DNA repair enzymes, topoisomerases, transcription factors, DNA-dependent protein kinase, lamin B, p53), but the most efficient acceptor appears to be the enzyme itself (Ziegler, 2000).

There is a growing number of evidences on involvement of PARP and PARP-mediated depletion of intracellular NAD+ in the acute and chronic injury of cells (ischemia/reperfusion, endothelial dysfunction, genotoxicity, inflammation, traumatic injury) (Oliver et al., 1999). In respect to the CNS, NAD+ depletion and mitochondrial permeability transition were shown to be sequential and necessary steps in PARP-1 overactivation-dependent cell death in astrocytes (Alano et al., 2004). Increased poly(ADPribosylation) of nuclear proteins was demonstrated in neurons in Alzheimer's disease (Love et al., 1999). Intra-mitochondrial PARP contributes to NAD+ depletion and cell death induced by oxidative stress in neurons (Du et al., 2003). Increased poly ADP-ribosylation of nuclear proteins in Alzheimer's disease has been detected, and double immunolabelling for poly(ADP-ribose) and markers of neuronal, astrocytic and microglial differentiation showed many of the cells containing poly(ADP-ribose) to be neurons, while few of the cells were astrocytes, and no poly(ADP-ribose) accumulation was found in microglia (Kauppinen & Swanson, 2007; Love, et al., 1999). Moreover, it was shown that β-amyloid affected cholinergic receptor-mediated signal transduction to PARP, probably, through free radical evoked inhibition of inositol-3-phosphate formation in the hippocampal cells (Adamczyk et al., 2005). Glutamate neurotransmission involving NMDA receptors and neuronal nitric oxide synthase activity in part mediates neuronal DNA strand breaks and PARP activation. These events are especially important for neurons since astrocytes able to maintain higher levels of NAD+ comparing with neurons (Pieper et al., 2000), and much higher concentrations of oxidants are required for killing astrocytes (Ying et al., 2002). It should be taken into the consideration that excessive PARP activation leads to impairment of glycolysis in affected cells, thereby impaired glycolytic flux is involved into PARP-mediated neuronal and astroglial cell death. Since astrocyte-produced lactate is a major endogenous energy substrate used by neurons in brain, NAD+ depletion caused by excessive PARP activation in neurons would result in alteration of lactate-pyruvate conversion thus affecting the efficacy of oxidative metabolism in neurons and astrocyte-neuronal lactate shuttle mechanism. In addition, neurological metabolic coupling implies subcellular compartmentation of pyruvate and monocarboxylate recycling through the plasma membrane of both neurons and glial cells, subcellular compartmentation of pyruvate allows neurons and astrocytes to select between glucose and lactate as alternative substrates depending of the concentrations and the operation of a redox switch (Cerdan et al., 2006). Pyruvate compartmentation results in effective transcellular coupling between the cytosolic NAD+/NADH redox states of neuronal and glial cells, therefore, impairment of this

Alteration of Neuron-Glia Interactions in Neurodegeneration:

or by NAD+ (Togashi et al., 2006).

observed ryanodine receptor dysfunction.

glutathione (Giaume et al., 2010).

mammalian cells.

Molecular Biomarkers and Therapeutic Strategy 279

cells. SIRT expressed in neuronal and astroglial cells requires NAD+ as an essential cofactor for their deacetylase activity, thus providing direct link between the metabolic and transcriptional response (Cohen et al., 2009; Kwon & Ott, 2008), while TRPM2 channels are expressed predominantly in neurons and microglia and are activated by cyclic ADP-ribose

It is known that astrocytic response to neuronal activity can be most readily detected by observing changes in the intracellular Ca2+ concentrations mediated via calcium flux through the plasma membrane calcium channels or calcium release from intracellular stores. Products of enzymatic activity of CD38 – cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP+) – work as potent Ca2+ mobilizing second messengers acting at ryanodine receptors (Higashida, et al., 2001a). Immunocytochemical studies revealed association of altered Ca2+ regulation in astrocytes (i.e. calcineurin upregulation) with their activation in aging or Alzheimer's disease models (Norris et al., 2005). Functional ryanodine receptors are required for astrocyte migration that is important component of regenerative process in the brain (Matyash et al., 2002). Therefore, expression and functional activity of CD38 in astrocytes and/or neurons, and ectocellular action of cADPR and NAADP+ on astrocytes resulting in Ca2+ signaling (Heidemann et al., 2005; Pawlikowska et al., 1996) would have physiological and pathophysiological meaning as a mechanism of Ca2+ signaling involved in neuron-astroglia cell interactions. Alterations in ryanodine receptor binding and function are very early events in the pathogenesis of Alzheimer's disease (Kelliher et al., 1999) while Aβ increases ryanodine receptors expression and function in cortical neurons (Supnet et al., 2006). Taking into account importance of neuronal calcium mishandling in the development of Alzheimer's disease (Verkhratsky, et al., 1998), one can suggest involvement of cADPR-associated signaling pathways in

Figure 1 summarizes data on NAD+-generating and NAD+-converting pathways in

Glial cells can communicate with each other by means of Ca2+ waves, and any perturbation of astrocytic intracellular concentration can propagate to other adjacent astrocytes through gap junction formed by connexins (Cx). Astroglial calcium signaling can be linked to synaptic transduction between neurons and neuronal-astroglial metabolic coupling (Allegrini et al., 2009). Cx43-formed gap junctions extensively couple neurons with glia (Nagy et al., 2004), and astrocytes represent the largest gap junction-coupled cellular network within the brain (Nakase & Naus, 2004). In the adult brain, Cx43 levels vary according to the developmental stage and brain region: Cx43 is expressed from early in development and further its expression increases.Cx43 is believed to be mediator of intercellular communication and operator between processes originating from a single astrocyte. In astrocytes, connexons are activated at metabolic inhibition, pro-inflammatory microenvironment, brain injury. Cx43 contributes to paracrine pathways in astroglial cells by regulating Ca2+ waves and uptake and release of glutamate, ATP, glucose, and

Astroglial cells express connexin-based gap junction channels and hemichannels that allow passage of molecules between the cytoplasm and extracellular cells or between the cells

**3. Astroglial CD38 and Cx43 in neuron-glia metabolic coupling** 

mechanism due to PARP hyperactivation in neurons could directly affect restoring the basal redox state in astrocytes.

Another class of enzymes utilizing NAD+ is represented by the CD38 family (EC 3.2.2.5, EC 3.2.2.6). Two ligands of CD38 – the substrate ligand NAD+ acting either extracellularly or intracellularly, and the non-substrate ligand CD31 expressed in endothelial cells – have important functions in brain cells under (patho)physiological conditions (Higashida et al., 2001a; Higashida et al., 2007; Salmina et al., 2010a; Salmina et al., 2006b). CD38 possesses the capability to catalyze different reactions, such as the hydrolysis of NAD+ and cADPR to ADP-ribose, and the cyclization of NAD+ and nicotinamine guanine dinucleotide (NGD+) to cADPR and cGDPR, respectively. The physiological meaning of the latter reaction is still unclear, but biological activity of cADPR is well defined in many cell types (Deaglio et al., 2008; Malavasi et al., 2008a). ADP-ribosyl cyclase attributable to CD38 was detected in the central nervous system where its activity and expression showed developmental changes. ADP-ribosyl cyclase synthesizes Ca2+ mobilizing messengers by cyclizing NAD+ to produce cyclic ADP-ribose (cADPR) acting through activation/modulation of ryanodine receptor channels involving FKBP12.6. In addition, cADPR was also shown to affect some potassium currents and thereby could be involved in synaptic activity. In murine brain, CD38 was found in both neurons and glial cells, showed predominant intracellular location, and was enriched in neuronal perikarya. In human brain, CD38 immunoreactivity was demonstrated in neurons, astrocytes, and microglial cells. In rat astrocytes, ADP-ribosyl cyclase has been reported to have both intracellular and extracellular actions. Co-culture of astrocytes with neurons resulted in significantly increased expression of astrocytic CD38 both on the plasma membrane and cytosol, and this effect was attributed to neuron-released glutamate action on astrocytes (Bruzzone et al., 2004). It is known that astrocytic response to neuronal activity can be most readily detected by observing changes in the intracellular Ca2+ concentrations mediated via calcium flux trough the plasma membrane calcium channels or calcium release from intracellular stores. Subtype-specific coupling with ADP-ribosyl cyclase of various neurotransmitter receptors confirms the involvement of this enzyme in signal transduction in neuronal and glial cells. The expression of CD38 is regulated by various substances (cytokines, retinoic acid), while enzymatic activity of ADP-ribosyl cyclase/CD38 is controlled by the structure of the catalytic center, the integrity of the sulfhydryl cysteine residues in this center, the intracellular levels of ATP and NADH, intracellular localization of the enzyme (plasma membrane, mitochondrial membrane, nuclear membrane, and cytosol), conformational plasticity, ligands (NAD+, CD31), and capacity to form dimers in a membrane for effective transport of reaction product.

In the cells of the CNS, ADP-ribosyl cyclase is expressed in different cell compartments (the nucleus, cytosol, and mitochondria), including the plasma membrane; however, the mechanisms that control translocation of the enzyme molecules, role of intracellular localization in the realization of enzymatic activity, and the mechanisms of directed transport of the enzyme to different cell compartments are unclear (Higashida et al., 2001; Salmina et al., 2008). Proposal exists that CD38 is a regulator of cellular NAD+ levels under physiological conditions, while PARP is the key factor determining intracellular NAD+ levels when significant DNA damage occurs (Ying et al., 2005).

A key role of CD38 in regulation of NAD+ homeostasis in cells has been suggested (Aksoy et al., 2006). Thereby, CD38 may contribute to regulation of activity of SIRT proteins or TRPM (transient receptor potential) channels. SIRT1 promotes survival and stress tolerance in brain

mechanism due to PARP hyperactivation in neurons could directly affect restoring the basal

Another class of enzymes utilizing NAD+ is represented by the CD38 family (EC 3.2.2.5, EC 3.2.2.6). Two ligands of CD38 – the substrate ligand NAD+ acting either extracellularly or intracellularly, and the non-substrate ligand CD31 expressed in endothelial cells – have important functions in brain cells under (patho)physiological conditions (Higashida et al., 2001a; Higashida et al., 2007; Salmina et al., 2010a; Salmina et al., 2006b). CD38 possesses the capability to catalyze different reactions, such as the hydrolysis of NAD+ and cADPR to ADP-ribose, and the cyclization of NAD+ and nicotinamine guanine dinucleotide (NGD+) to cADPR and cGDPR, respectively. The physiological meaning of the latter reaction is still unclear, but biological activity of cADPR is well defined in many cell types (Deaglio et al., 2008; Malavasi et al., 2008a). ADP-ribosyl cyclase attributable to CD38 was detected in the central nervous system where its activity and expression showed developmental changes. ADP-ribosyl cyclase synthesizes Ca2+ mobilizing messengers by cyclizing NAD+ to produce cyclic ADP-ribose (cADPR) acting through activation/modulation of ryanodine receptor channels involving FKBP12.6. In addition, cADPR was also shown to affect some potassium currents and thereby could be involved in synaptic activity. In murine brain, CD38 was found in both neurons and glial cells, showed predominant intracellular location, and was enriched in neuronal perikarya. In human brain, CD38 immunoreactivity was demonstrated in neurons, astrocytes, and microglial cells. In rat astrocytes, ADP-ribosyl cyclase has been reported to have both intracellular and extracellular actions. Co-culture of astrocytes with neurons resulted in significantly increased expression of astrocytic CD38 both on the plasma membrane and cytosol, and this effect was attributed to neuron-released glutamate action on astrocytes (Bruzzone et al., 2004). It is known that astrocytic response to neuronal activity can be most readily detected by observing changes in the intracellular Ca2+ concentrations mediated via calcium flux trough the plasma membrane calcium channels or calcium release from intracellular stores. Subtype-specific coupling with ADP-ribosyl cyclase of various neurotransmitter receptors confirms the involvement of this enzyme in signal transduction in neuronal and glial cells. The expression of CD38 is regulated by various substances (cytokines, retinoic acid), while enzymatic activity of ADP-ribosyl cyclase/CD38 is controlled by the structure of the catalytic center, the integrity of the sulfhydryl cysteine residues in this center, the intracellular levels of ATP and NADH, intracellular localization of the enzyme (plasma membrane, mitochondrial membrane, nuclear membrane, and cytosol), conformational plasticity, ligands (NAD+, CD31), and capacity to form dimers in a

In the cells of the CNS, ADP-ribosyl cyclase is expressed in different cell compartments (the nucleus, cytosol, and mitochondria), including the plasma membrane; however, the mechanisms that control translocation of the enzyme molecules, role of intracellular localization in the realization of enzymatic activity, and the mechanisms of directed transport of the enzyme to different cell compartments are unclear (Higashida et al., 2001; Salmina et al., 2008). Proposal exists that CD38 is a regulator of cellular NAD+ levels under physiological conditions, while PARP is the key factor determining intracellular NAD+

A key role of CD38 in regulation of NAD+ homeostasis in cells has been suggested (Aksoy et al., 2006). Thereby, CD38 may contribute to regulation of activity of SIRT proteins or TRPM (transient receptor potential) channels. SIRT1 promotes survival and stress tolerance in brain

redox state in astrocytes.

membrane for effective transport of reaction product.

levels when significant DNA damage occurs (Ying et al., 2005).

cells. SIRT expressed in neuronal and astroglial cells requires NAD+ as an essential cofactor for their deacetylase activity, thus providing direct link between the metabolic and transcriptional response (Cohen et al., 2009; Kwon & Ott, 2008), while TRPM2 channels are expressed predominantly in neurons and microglia and are activated by cyclic ADP-ribose or by NAD+ (Togashi et al., 2006).

It is known that astrocytic response to neuronal activity can be most readily detected by observing changes in the intracellular Ca2+ concentrations mediated via calcium flux through the plasma membrane calcium channels or calcium release from intracellular stores. Products of enzymatic activity of CD38 – cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP+) – work as potent Ca2+ mobilizing second messengers acting at ryanodine receptors (Higashida, et al., 2001a). Immunocytochemical studies revealed association of altered Ca2+ regulation in astrocytes (i.e. calcineurin upregulation) with their activation in aging or Alzheimer's disease models (Norris et al., 2005). Functional ryanodine receptors are required for astrocyte migration that is important component of regenerative process in the brain (Matyash et al., 2002). Therefore, expression and functional activity of CD38 in astrocytes and/or neurons, and ectocellular action of cADPR and NAADP+ on astrocytes resulting in Ca2+ signaling (Heidemann et al., 2005; Pawlikowska et al., 1996) would have physiological and pathophysiological meaning as a mechanism of Ca2+ signaling involved in neuron-astroglia cell interactions. Alterations in ryanodine receptor binding and function are very early events in the pathogenesis of Alzheimer's disease (Kelliher et al., 1999) while Aβ increases ryanodine receptors expression and function in cortical neurons (Supnet et al., 2006). Taking into account importance of neuronal calcium mishandling in the development of Alzheimer's disease (Verkhratsky, et al., 1998), one can suggest involvement of cADPR-associated signaling pathways in observed ryanodine receptor dysfunction.

Figure 1 summarizes data on NAD+-generating and NAD+-converting pathways in mammalian cells.

#### **3. Astroglial CD38 and Cx43 in neuron-glia metabolic coupling**

Glial cells can communicate with each other by means of Ca2+ waves, and any perturbation of astrocytic intracellular concentration can propagate to other adjacent astrocytes through gap junction formed by connexins (Cx). Astroglial calcium signaling can be linked to synaptic transduction between neurons and neuronal-astroglial metabolic coupling (Allegrini et al., 2009). Cx43-formed gap junctions extensively couple neurons with glia (Nagy et al., 2004), and astrocytes represent the largest gap junction-coupled cellular network within the brain (Nakase & Naus, 2004). In the adult brain, Cx43 levels vary according to the developmental stage and brain region: Cx43 is expressed from early in development and further its expression increases.Cx43 is believed to be mediator of intercellular communication and operator between processes originating from a single astrocyte. In astrocytes, connexons are activated at metabolic inhibition, pro-inflammatory microenvironment, brain injury. Cx43 contributes to paracrine pathways in astroglial cells by regulating Ca2+ waves and uptake and release of glutamate, ATP, glucose, and glutathione (Giaume et al., 2010).

Astroglial cells express connexin-based gap junction channels and hemichannels that allow passage of molecules between the cytoplasm and extracellular cells or between the cells

Alteration of Neuron-Glia Interactions in Neurodegeneration:

neuronal signals and local blood flow (neurovascular coupling).

toxic levels (Salmina et al., 2009a).

Molecular Biomarkers and Therapeutic Strategy 281

Two primary hypotheses of gap junction coupling in the CNS are the following: (1) generalized coupling occurs between neurons and glia, with some connexins expressed in both neurons and glia, and (2) intercellular junctional coupling is restricted to specific coupling partners, with different connexins expressed in each cell type. The most important question is a role of connexon in mediating communication between different cell types in the brain (Rash et al., 2001). It is well-known that intercellular calcium signaling between different types of glial cells (astrocytes and oligodendocytes) is mediated by Cx43 (Parys et al., 2010). Several connexin proteins have been identified at gap junctions between neuronal and astroglial cells. Moreover, expression of Cx43 but at much lower levels comparing to astrocytes has been detected in oligodendrocytes, Schwann cells and neurons (Nagy, et al., 2004). However, astrocytes are still considered as connexin-dependent signaling cells (Saez, 2008). Expression of Cx43 is closely related to the role of astrocytes in coordinating the

In the CNS, expression and activity of Cx43 are modulated by cytokines, NO. Recently, novel protein interacting with Cx43 in astrocytes has been identified – brain-derived integrating factor-1 (BDIF-1) which is probably involved in regulating of astroglial activity (Ito et al., 2011). Pharmacological modulation of Cx43 can be achieved by various compounds (Dhein et al., 2002). T. Nakase et al. (Nakase & Naus, 2004) reported that the activation of astrocytes associated with an increase in the expression and activity of connexin 43 protects neurons from ischemia-induced damage. It should be noted that Cx43 can provide NAD+ to the ectodomain of CD38. In fibroblasts, Cx43 hemichannels mediate release of NAD+ to the extracellular medium, presumably allowing cytoplasmic NAD+ to reach the active site of ADP-ribosyl cyclase/CD38 located at the extracellular part of plasma membrane (Franco et al., 2001). The same mechanism could work in astrocytes (Verderio et al., 2001) since glutamate-induced CD38 overexpression in astrocytes was observed in the model of neuronal-astroglial cell coupling. Functional cross-talk between Cx43 hemichannels and CD38 may be triggered by elevation of intracellular Ca2+ concentrations or by binding of a ligand to Cx43 or CD38. De Flora et al. (De Flora et al., 2004) suggested that extra phosphorylation of Cx43 induced by Ca2+ rise decreases the open-state probability of connexons, thereby downregulating NAD+ transport across them and resulting in lower access of NAD+ to the active site of CD38 and decreasing cADPR generation and limiting of Ca2+ levels inside the cell. Thus, autocrine and paracrine Ca2+ signaling is mediated by Cx43- CD38 system. Whether or not such system is operating in astrocytes in the context of neuronal-astroglial coupling remains to be elucidated. This mechanism is very important, because it helps to prevent NAD+ loss from cells, depletion of intracellular NAD+ due to elevated CD38 activity, and cADPR overproduction leading to calcium accumulation to

CD38 and Cx43 also may serve as transporters of nucleotides (NAD(H), cADPR, and NAADP+) into the cell from the extracellular space (Billington, et al., 2008), thus contributing to maintaining NAD+ homeostasis within the cell. NAD+ itself can enter

What are the possible roles for Cx43 hemichannes in neurodegenerative processes? Elevated expression of Cx43 in reactive astrocytes may reflect: 1) extensive buffering of neurotoxic substances by activated astroglial cells required for protecting the neurons from cell death (i.e. inhibition of gap junctions with octanol abolishes the ability of Aβ to enhance the velocity and extent of propagation of astroglial calcium waves (Orellana et al., 2009));

astrocytes via gap junction-mediated pathway (Ying, et al., 2005).

Fig. 1. Metabolism of NAD+ in mammalian cells. Abbreviations used: NAPRT – nicotinic acid phosphoribosyltransferase, NAMPT – nicotinamide phosphoribosyltransferase, NRK – nicotinamide riboside kinase, NaMN – nicotinic acid mononucleotide, NMN – nicotinamide mononucleotide, QPRT – quinolinate phosphoribosyltransferase, NMNAT – nicotinamide mononucleotide adenylyltransferase, NAAD – nicotinic acid adenine dinucleotide, NAD+ - nicotinamide dinucleotide

(Contreras et al., 2004). Synapse-glial interactions in the developing, adult and injured brain are very important for brain plasticity. Distinct phases of synapse development depend on assistance from glial cells (Pfrieger, 2010). Astrocytic hemichannels consisted of connexin 43 mediate release of glutamate and other amino acids (Ye et al., 2003). Gap junction channels allow the coordination of intrinsic or elicited metabolic and/or electrical responses of cells in a heterogenous population, and regulate poroliferative activity of astroglial cells. Astrocytes with a radial glia–like morphology in the subgranular zone of the dentate gyrus are considered as stem cells which give rise to neurons within different regions of the adult brain. Radial glia-like cells express GFAP, nestin, and Cx43. The latter contribute to controlling proliferation and differentiation of these cells since no other mechanisms (i.e. glutamatergic) have been detected in radial glia-like cells (Kunze, 2009).

**Indoleamine- 2,3 dioxygenase**

**CONVERSION OF NAD+ SYNTHESIS OF NAD+**

**L-Formylkynurenine**

**L-Kynurenine**

**Quinolinic acid**

 **Poly(ADP) ribosyl polymerase**

adenine dinucleotide, NAD+ - nicotinamide dinucleotide

glutamatergic) have been detected in radial glia-like cells (Kunze, 2009).

**L-Tryptophan Nicotinic acid Nicotinamide** 

**NAPRT NAMPT NRK**

**NAAD**

 **Sirtuin deacetylase**

**NaMN NMN**

**QPRT NMNAT**

**ADP-ribosyl cyclase**

 **NAD+ synthase**

**NAD+**

**Mono(ADP) ribosyl transferase**

Fig. 1. Metabolism of NAD+ in mammalian cells. Abbreviations used: NAPRT – nicotinic acid phosphoribosyltransferase, NAMPT – nicotinamide phosphoribosyltransferase, NRK – nicotinamide riboside kinase, NaMN – nicotinic acid mononucleotide,

NMN – nicotinamide mononucleotide, QPRT – quinolinate phosphoribosyltransferase, NMNAT – nicotinamide mononucleotide adenylyltransferase, NAAD – nicotinic acid

(Contreras et al., 2004). Synapse-glial interactions in the developing, adult and injured brain are very important for brain plasticity. Distinct phases of synapse development depend on assistance from glial cells (Pfrieger, 2010). Astrocytic hemichannels consisted of connexin 43 mediate release of glutamate and other amino acids (Ye et al., 2003). Gap junction channels allow the coordination of intrinsic or elicited metabolic and/or electrical responses of cells in a heterogenous population, and regulate poroliferative activity of astroglial cells. Astrocytes with a radial glia–like morphology in the subgranular zone of the dentate gyrus are considered as stem cells which give rise to neurons within different regions of the adult brain. Radial glia-like cells express GFAP, nestin, and Cx43. The latter contribute to controlling proliferation and differentiation of these cells since no other mechanisms (i.e.

**Nicotin amide riboside** Two primary hypotheses of gap junction coupling in the CNS are the following: (1) generalized coupling occurs between neurons and glia, with some connexins expressed in both neurons and glia, and (2) intercellular junctional coupling is restricted to specific coupling partners, with different connexins expressed in each cell type. The most important question is a role of connexon in mediating communication between different cell types in the brain (Rash et al., 2001). It is well-known that intercellular calcium signaling between different types of glial cells (astrocytes and oligodendocytes) is mediated by Cx43 (Parys et al., 2010). Several connexin proteins have been identified at gap junctions between neuronal and astroglial cells. Moreover, expression of Cx43 but at much lower levels comparing to astrocytes has been detected in oligodendrocytes, Schwann cells and neurons (Nagy, et al., 2004). However, astrocytes are still considered as connexin-dependent signaling cells (Saez, 2008). Expression of Cx43 is closely related to the role of astrocytes in coordinating the neuronal signals and local blood flow (neurovascular coupling).

In the CNS, expression and activity of Cx43 are modulated by cytokines, NO. Recently, novel protein interacting with Cx43 in astrocytes has been identified – brain-derived integrating factor-1 (BDIF-1) which is probably involved in regulating of astroglial activity (Ito et al., 2011). Pharmacological modulation of Cx43 can be achieved by various compounds (Dhein et al., 2002). T. Nakase et al. (Nakase & Naus, 2004) reported that the activation of astrocytes associated with an increase in the expression and activity of connexin 43 protects neurons from ischemia-induced damage. It should be noted that Cx43 can provide NAD+ to the ectodomain of CD38. In fibroblasts, Cx43 hemichannels mediate release of NAD+ to the extracellular medium, presumably allowing cytoplasmic NAD+ to reach the active site of ADP-ribosyl cyclase/CD38 located at the extracellular part of plasma membrane (Franco et al., 2001). The same mechanism could work in astrocytes (Verderio et al., 2001) since glutamate-induced CD38 overexpression in astrocytes was observed in the model of neuronal-astroglial cell coupling. Functional cross-talk between Cx43 hemichannels and CD38 may be triggered by elevation of intracellular Ca2+ concentrations or by binding of a ligand to Cx43 or CD38. De Flora et al. (De Flora et al., 2004) suggested that extra phosphorylation of Cx43 induced by Ca2+ rise decreases the open-state probability of connexons, thereby downregulating NAD+ transport across them and resulting in lower access of NAD+ to the active site of CD38 and decreasing cADPR generation and limiting of Ca2+ levels inside the cell. Thus, autocrine and paracrine Ca2+ signaling is mediated by Cx43- CD38 system. Whether or not such system is operating in astrocytes in the context of neuronal-astroglial coupling remains to be elucidated. This mechanism is very important, because it helps to prevent NAD+ loss from cells, depletion of intracellular NAD+ due to elevated CD38 activity, and cADPR overproduction leading to calcium accumulation to toxic levels (Salmina et al., 2009a).

CD38 and Cx43 also may serve as transporters of nucleotides (NAD(H), cADPR, and NAADP+) into the cell from the extracellular space (Billington, et al., 2008), thus contributing to maintaining NAD+ homeostasis within the cell. NAD+ itself can enter astrocytes via gap junction-mediated pathway (Ying, et al., 2005).

What are the possible roles for Cx43 hemichannes in neurodegenerative processes? Elevated expression of Cx43 in reactive astrocytes may reflect: 1) extensive buffering of neurotoxic substances by activated astroglial cells required for protecting the neurons from cell death (i.e. inhibition of gap junctions with octanol abolishes the ability of Aβ to enhance the velocity and extent of propagation of astroglial calcium waves (Orellana et al., 2009));

Alteration of Neuron-Glia Interactions in Neurodegeneration:

mechanisms.

Molecular Biomarkers and Therapeutic Strategy 283

intracellular stores. Thereby, amplification of initial rise in intracellular Ca2+ levels might be important in cell damage. It was reported that mitochondrial dysfucntion leads to postponed changes in CD38 expression (Mills et al., 1999). Thus, accumulating data suggest that ADP-ribosyl cyclase may affect mitochondrial functioning through various

Mitochondrial localization of Cx43 has been shown in cardiomyocytes, and the role for Cx43 as regulator of mitochondrial potassium uptake (Miro-Casas et al., 2009) or mitochondrial respiration (Boengler et al., 2008a) has been suggested. Stimulatory effect of Cx43 on mitochondrial KATP channels resulting in cytoprotectiion has been shown in cardiomyocytes (Rottlaender et al., 2010). It is intriguing to speculate that mitochondrial Cx43 contributes to regulation of respiration and potassium uptake in astroglial cells, thus providing adaptation of mitochondrial activity to altered microenvironment in activated astrocytes in the site of brain injury. It was proposed that Cx43 may directly regulate respiratory chain complex I activity and mitochondrial oxygen consumption: decrease in

Mitochondrial localization of CD38 is well documented. I. Balan et al. (Balan et al., 2010) detected NAD+- glycohydrolase activity in isolated synaptosomes and also in intact brain mitochondria, confirming localization of CD38 also in outer mitochondrial membranes. Interestingly, the NAD+- glycohydrolase activity appeared to be much higher in nonsynaptic mitochondria compared with mitochondria isolated from synaptosomes. Taken together, these data suggest that NAD+ depletion can occur more rapidly in astrocytes following ischemic insult, compromising the ability of astrocytes to support neuronal functions. Interestingly, the NAD+ catabolic activity is higher in brain regions that are vulnerable to ischemic insult, furthermore, the CD38 NAD+ glycohydrolase activity is significantly increased in postischemic tissue, and the immunohistochemistry shows overexpression of this enzyme preferentially in neuroglial cells (Kristian et al., 2011; Salmina, et al., 2008; Salmina, et al., 2009a). Activation of CD38 can lead to rapid and almost complete tissue NAD+ depletion (Balan, et al., 2010). A prolonged MPT results not only in dissipation of the mitochondrial electrochemical hydrogen ion gradient and swelling of mitochondria but also depletion of pyridine nucleotides from the matrix (Di Lisa & Ziegler, 2001), however, significant loss of matrix pyridine nucleotides can lead to inhibition of mitochondrial respiration but without irreversible damage to the respiratory complexes or mitochondrial membranes (Kristian, et al., 2011). It was proposed, that once the cellular CD38 enzymatic pool is saturated, cytosolic NAD+ concentrations rise to a level that permits efflux into extracellular space where NAD+ becomes to be the substrate for surface-

mitochondrial Cx43 levels reduced complex I activity (Boengler et al., 2008b).

expressed CD38 acting as autocrine or paracrine regulator of Ca2+ signaling.

Neurodegeneration is associated with altered energy metabolism in the brain; accumulation of glycolytic enzymes, such as enolase and glyceraldehyde 3-phosphate dehydrogenase, decrease in expression of voltage-dependent anion-selective channel protein-1 (VDAC-1), and decrease in expression of subunits of multiprotein enzyme complex NADH:ubiquinone oxidoreductase (complex I of the mitochondrial electron transport chain) have been registered in Alzheimer's disease (Butterfield et al., 2003)]. It is well known that astrocytes play important roles in supporting energy production in neurons. According to astrocyteneuron lactate shuttle hypothesis, lactate is produced in an activity-dependent and glutamate-mediated manner by astrocytes and is then transferred to and used by active neurons (Pellerin et al., 1998). Neuronal activation results in uptake of glutamate by astrocytes leading to activation of glutamine synthetase and Na+-K+-ATPase, followed by

2) propagating Ca2+ waves between communicating astrocytes and/or individual processes of a single cell; 3) active proliferation and migration of Cx43-immunopositive progenitor cells to the site of neurodegeneration; 4) activation of autocrine and paracrine signaling in astrocytes and adjacent neurons; 5) action of stimuli (prooxidant and proinflammatory) able to increase Cx43 expression. In neurodegenerative diseases and ischemia, reactive astrocytes have increased levels of Cx43, and changes in Cx43 expression are dependent on the proximity of the reactive astrocytes to the focus of neurodegeneration. In the site of injury, reactive astrocytes can lose their non-overlapping domains (Giaume, et al., 2010). Functional hemichannels that have a role in glutamate homeostasis may significantly contribute to astrocyte-mediated regulation of neuronal activity. It is interesting that in the Alzheimer's type of neurodegeneration, elevated expression of Cx43 has been detected at β-amyloid plaques due to reactive astrogliosis (Mei et al., 2010). It was suggested that in Alzheimer's disease, increased Cx43 expression might represent an attempt to maintain tissue homeostasis by augmented intercellular communication via gap junction formation between astrocytic processes that invest senile plaques. In addition, one can propose that elevated expression of Cx43 hemichannels could result in massive release of glutamate from astrocytes into the extracellular space resulting in excitotoxic injury of neurons. Connexin 43 regulates astrocytic migration and proliferation in response to injury. Reactive astrocytes display up-regulation of the gap junction protein Cx43, and astroglial cells with depleted expression of Cx43 show diminished ability to migrate and to proliferate in the wound area of the brain (Homkajorn et al., 2010). Drebrin as an actin binding protein whose level is greatly decreased in brains of Alzheimer's patients was found to be a binding partner of the Cx43 COOH-terminal domain in astrocytes. In experimental model, depletion of drebrin in cells results in impaired cell-cell coupling, internalization of gap junctions, and targeting of Cx43 to a degradative pathway (Butkevich et al., 2004)]. It was suggested that increased Cx43 expression in a close vicinity to amyloid plaques might represent aberrant induction of Cx43 expression stimulated by excessive degradative pathway. In support of this hypothesis, increased expression of Cx43 was found to be induced by β-amyloid precursor protein (Hallaq & Killick, 2006)], thus suggesting direct effect of amyloid deposites on the mechanism of glutamate release from astroglial cells and neuron-astrocyte communication.

Recently, changes in expression and activity of Cx43 have been registered in rotenoneinduced model of Parkinson's disease: enhancement of Cx43 protein levels in cells treated with rotenone (mitochondrial complex I inhibitor) resulted in increased efficacy of gap junctional intercellular communication (Kawasaki et al., 2009). However, some authors registered inhibited expression of astrocytic Cx43 and gap junction permeability in astrocytes in rotenone models of Parkinson's disease. Moreover, rotenone-induced dysfunction of astrocytic Cx43 can be reversed by opening mitochondrial ATP-sensitive potassium channels (iptakalim and diazoxide) resulting in prevention of astrocyte apoptosis (Zhang et al., 2010). Thus, we believe that Cx43-CD38 functional coupling in astrocytes significantly contribute to controlling energy homeostasis in astroglial and neuronal cells.

Opening of the mitochondrial permeability transition pore is known to play a role in cell death. Its opening has been shown to cause activation of NAD+ glycohydrolase located in the outer mitochondrial membrane following by NAD+ hydrolysis in cardiomyocytes at postischemic reperfusion (Di Lisa et al., 2001). Since mitochondrial NAD+ glycohydrolase has been identified as ADP-ribosyl cyclase (Ziegler et al., 1997), NAD+ released from mitochondrial matrix could be transformed into cADPR which promoting Ca2+ release from

2) propagating Ca2+ waves between communicating astrocytes and/or individual processes of a single cell; 3) active proliferation and migration of Cx43-immunopositive progenitor cells to the site of neurodegeneration; 4) activation of autocrine and paracrine signaling in astrocytes and adjacent neurons; 5) action of stimuli (prooxidant and proinflammatory) able to increase Cx43 expression. In neurodegenerative diseases and ischemia, reactive astrocytes have increased levels of Cx43, and changes in Cx43 expression are dependent on the proximity of the reactive astrocytes to the focus of neurodegeneration. In the site of injury, reactive astrocytes can lose their non-overlapping domains (Giaume, et al., 2010). Functional hemichannels that have a role in glutamate homeostasis may significantly contribute to astrocyte-mediated regulation of neuronal activity. It is interesting that in the Alzheimer's type of neurodegeneration, elevated expression of Cx43 has been detected at β-amyloid plaques due to reactive astrogliosis (Mei et al., 2010). It was suggested that in Alzheimer's disease, increased Cx43 expression might represent an attempt to maintain tissue homeostasis by augmented intercellular communication via gap junction formation between astrocytic processes that invest senile plaques. In addition, one can propose that elevated expression of Cx43 hemichannels could result in massive release of glutamate from astrocytes into the extracellular space resulting in excitotoxic injury of neurons. Connexin 43 regulates astrocytic migration and proliferation in response to injury. Reactive astrocytes display up-regulation of the gap junction protein Cx43, and astroglial cells with depleted expression of Cx43 show diminished ability to migrate and to proliferate in the wound area of the brain (Homkajorn et al., 2010). Drebrin as an actin binding protein whose level is greatly decreased in brains of Alzheimer's patients was found to be a binding partner of the Cx43 COOH-terminal domain in astrocytes. In experimental model, depletion of drebrin in cells results in impaired cell-cell coupling, internalization of gap junctions, and targeting of Cx43 to a degradative pathway (Butkevich et al., 2004)]. It was suggested that increased Cx43 expression in a close vicinity to amyloid plaques might represent aberrant induction of Cx43 expression stimulated by excessive degradative pathway. In support of this hypothesis, increased expression of Cx43 was found to be induced by β-amyloid precursor protein (Hallaq & Killick, 2006)], thus suggesting direct effect of amyloid deposites on the mechanism of glutamate release from astroglial cells and

Recently, changes in expression and activity of Cx43 have been registered in rotenoneinduced model of Parkinson's disease: enhancement of Cx43 protein levels in cells treated with rotenone (mitochondrial complex I inhibitor) resulted in increased efficacy of gap junctional intercellular communication (Kawasaki et al., 2009). However, some authors registered inhibited expression of astrocytic Cx43 and gap junction permeability in astrocytes in rotenone models of Parkinson's disease. Moreover, rotenone-induced dysfunction of astrocytic Cx43 can be reversed by opening mitochondrial ATP-sensitive potassium channels (iptakalim and diazoxide) resulting in prevention of astrocyte apoptosis (Zhang et al., 2010). Thus, we believe that Cx43-CD38 functional coupling in astrocytes significantly contribute to controlling energy homeostasis in astroglial and neuronal cells. Opening of the mitochondrial permeability transition pore is known to play a role in cell death. Its opening has been shown to cause activation of NAD+ glycohydrolase located in the outer mitochondrial membrane following by NAD+ hydrolysis in cardiomyocytes at postischemic reperfusion (Di Lisa et al., 2001). Since mitochondrial NAD+ glycohydrolase has been identified as ADP-ribosyl cyclase (Ziegler et al., 1997), NAD+ released from mitochondrial matrix could be transformed into cADPR which promoting Ca2+ release from

neuron-astrocyte communication.

intracellular stores. Thereby, amplification of initial rise in intracellular Ca2+ levels might be important in cell damage. It was reported that mitochondrial dysfucntion leads to postponed changes in CD38 expression (Mills et al., 1999). Thus, accumulating data suggest that ADP-ribosyl cyclase may affect mitochondrial functioning through various mechanisms.

Mitochondrial localization of Cx43 has been shown in cardiomyocytes, and the role for Cx43 as regulator of mitochondrial potassium uptake (Miro-Casas et al., 2009) or mitochondrial respiration (Boengler et al., 2008a) has been suggested. Stimulatory effect of Cx43 on mitochondrial KATP channels resulting in cytoprotectiion has been shown in cardiomyocytes (Rottlaender et al., 2010). It is intriguing to speculate that mitochondrial Cx43 contributes to regulation of respiration and potassium uptake in astroglial cells, thus providing adaptation of mitochondrial activity to altered microenvironment in activated astrocytes in the site of brain injury. It was proposed that Cx43 may directly regulate respiratory chain complex I activity and mitochondrial oxygen consumption: decrease in mitochondrial Cx43 levels reduced complex I activity (Boengler et al., 2008b).

Mitochondrial localization of CD38 is well documented. I. Balan et al. (Balan et al., 2010) detected NAD+- glycohydrolase activity in isolated synaptosomes and also in intact brain mitochondria, confirming localization of CD38 also in outer mitochondrial membranes. Interestingly, the NAD+- glycohydrolase activity appeared to be much higher in nonsynaptic mitochondria compared with mitochondria isolated from synaptosomes. Taken together, these data suggest that NAD+ depletion can occur more rapidly in astrocytes following ischemic insult, compromising the ability of astrocytes to support neuronal functions. Interestingly, the NAD+ catabolic activity is higher in brain regions that are vulnerable to ischemic insult, furthermore, the CD38 NAD+ glycohydrolase activity is significantly increased in postischemic tissue, and the immunohistochemistry shows overexpression of this enzyme preferentially in neuroglial cells (Kristian et al., 2011; Salmina, et al., 2008; Salmina, et al., 2009a). Activation of CD38 can lead to rapid and almost complete tissue NAD+ depletion (Balan, et al., 2010). A prolonged MPT results not only in dissipation of the mitochondrial electrochemical hydrogen ion gradient and swelling of mitochondria but also depletion of pyridine nucleotides from the matrix (Di Lisa & Ziegler, 2001), however, significant loss of matrix pyridine nucleotides can lead to inhibition of mitochondrial respiration but without irreversible damage to the respiratory complexes or mitochondrial membranes (Kristian, et al., 2011). It was proposed, that once the cellular CD38 enzymatic pool is saturated, cytosolic NAD+ concentrations rise to a level that permits efflux into extracellular space where NAD+ becomes to be the substrate for surfaceexpressed CD38 acting as autocrine or paracrine regulator of Ca2+ signaling.

Neurodegeneration is associated with altered energy metabolism in the brain; accumulation of glycolytic enzymes, such as enolase and glyceraldehyde 3-phosphate dehydrogenase, decrease in expression of voltage-dependent anion-selective channel protein-1 (VDAC-1), and decrease in expression of subunits of multiprotein enzyme complex NADH:ubiquinone oxidoreductase (complex I of the mitochondrial electron transport chain) have been registered in Alzheimer's disease (Butterfield et al., 2003)]. It is well known that astrocytes play important roles in supporting energy production in neurons. According to astrocyteneuron lactate shuttle hypothesis, lactate is produced in an activity-dependent and glutamate-mediated manner by astrocytes and is then transferred to and used by active neurons (Pellerin et al., 1998). Neuronal activation results in uptake of glutamate by astrocytes leading to activation of glutamine synthetase and Na+-K+-ATPase, followed by

Alteration of Neuron-Glia Interactions in Neurodegeneration:

2008).

neurons.

al., 2000).

with ischemia-induced neurodegeneration (Salmina, et al., 2006a).

i.e. by homocysteine (Boldyrev, 2010) in neurodegenerative processes.

Molecular Biomarkers and Therapeutic Strategy 285

brain significantly improved clinical manifestations of neurological dysfunction associated

In patients with ischemic stroke, elevated expression of CD38 in peripheral blood leukocytes corresponds to formation of membrane-derived microparticles and progression of endothelial dysfunction due to CD38-CD31 interactions (Inzhutova et al., 2008; Salmina et al., 2010b). It is well known that astrocytes play important role in the formation, extent and configuration of the junctional complexes in the brain endothelium in a manner that astrocyte-induced enhanced tight junction communication is associated with the reduction of gap junctions. A major function of the neurovascular unit is to regulate the transport and diffusion properties of brain capillary endothelial cells that compose the brain-blood barrier (Banerjee & Bhat, 2007). Astrocytes exhibit anatomic relationships with cerebral arterioles and neurons. In the brain parenchyma, the extensive ensheatment of cerebral arterioles by astrocytic end-feet far exceeds any direct neural contacts with those perfusion-regulating microvessels. That unique arrangement permits astrocytes to transduce signals arising from activated neurons and to transmitthat information to the cerebral microcirculation (Xu et al.,

Coupling of NMDA receptors to ADP-ribosyl cyclase/CD38 in neuronal and glial cells, involvement of CD38 in neuronal-glial (Higashida et al., 2007a; Salmina, et al., 2009a) and leukocyte-endothelial interactions (Deaglio, et al., 2008; Inzhutova, et al., 2008; Malavasi et al., 2008) suggest new approach to treat endothelial dysfunction caused by various stimuli,

As we mentioned above, in acute neurodegeneration, CD38+-expressing cells were predominantly represented by GFAP+/Cx43+ cells of astroglial origin. We found that in neurons, elevated CD38 expression resulted in intracellular NAD+ depletion and cell death, while in astrocytes high levels of CD38 expression relate to increased resistance to the action of apoptogenic stimuli, development of reactive gliosis, and changes in their glycolytic activity. Mitochondrial ADP-ribosyl cyclase activity was mainly induced by ischemic stimuli. Our data well fit the previous observations that intracellular NAD+ levels regulate astroglial response to neuronal activation, NAD+ released from astrocytes regulate apoptosis of neurons in postischemic period, and astrocytes are more resistant to hypoxia than

In the developing brain, direct correlation between the CD38 expression and apoptosis development in the given cell populations has been registered. We found stimulating effects of agonists of mGluRI, mGluRIII, suppressive effects of agonists of NMDAR on CD38 activity in premature injured brain cells. However, in the adult brain, reverse correlation between CD38 expression and apoptosis progression was observed. Under the pathophysiological conditions, development of cell death and ability of brain cells to maintain the levels of intracellular NAD+ are determined by hypoxia/ischemia-induced disturbance in the dynamics of ADP-ribosyl cyclase activity in the brain cells. These data are in agreement with our report on the contribution of CD38 overexpression to development of plasma membrane blebbing in neuroblastoma x glioma NG108-15 hybrid cells (Egorova et

In the experimental model of Parkinson's disease, we found that loss of dopaminergic neurons via apoptosis was associated with elevation of expression and activity of ADP-ribosyl cyclase/CD38 in remaining tyrosine hydroxylase-immunopositive cells. Immunochemical studies with anti-CD38 antibodies indicated accumulation of CD38 antigen in the neurofibrillary tangles that occur in neuronal perikarya and proximal

activation of anaerobic glycolysis in astrocytes, and release of lactate supporting the activityrelated energy required for neurons (Magistretti, 2000)]. In general, astrocytes metabolize glucose mainly to lactate and release it into the extracellular medium, while neurons appear to have a kinetic preference for oxidizing lactate imported from the external medium over pyruvate/lactate produced in neurons by glycolysis (Itoh et al., 2003). However, neurons die more intensively than astrocytes even though the apparent dysfunction takes place in astrocytes. Glycolysis is high in astrocytes, so they need a way to maintain the adequate levels of NAD+. Probably, astrocytes can sustain themselves adequately with glycolytic metabolism, not significantly affected by mitochondrial dysfunction, thereby being more resistant to oxidative stress and NAD+ depletion.

#### **4. CD38 expression in acute and chronic neurodegeneration**

Very few data are available on CD38 expression in neurodegeneration. In different experimental models of acute and chronic neurodegeneration in rats (focal brain ischemia, rotenone model of Parkinson's disease, and perinatal hypoxic-ischemic brain injury), we found elevated expression of CD38 in neurons and astroglial cells in the acute period of brain injury (Salmina, et al., 2008; Salmina, et al., 2009a; Salmina et al., 2006a).

In the model of perinatal hypoxic-ischemic brain damage, we found that changes in CD38 expression and ADP-ribosyl cyclase activity in neuronal and glial cells attribute to alterations in intracellular NAD+ level as well as to susceptibility of the cells to the action of apoptogenic stimuli; acute period of perinatal hypoxic/ischemic brain injury is characterized by reactive astrogliosis and elevation of CD38 expression, changes in CD38 and Cx43 expression in astrocytes serving as markers of neuron-glial interactions in perinatal CNS injury; ADP-ribosyl cyclase activity in neurons in response to stimulation of NMDA is changed after perinatal hypoxic-ischemic brain injury. It has been demonstrated that, in the immature brain, the impairment of intracellular calcium homeostasis is the leading mechanism of perinatal damage to both neuronal and glial cells (Vannucci et al., 2001). Based on our data, we can conclude that the mechanisms of maintenance of intracellular calcium homeostasis, which are under the control of ADPribosyl cyclase and Cx43, play a special role in responses of neurons and glia to hypoxic– ischemic damage.

We demonstrated that most of the cells that expressed Cx43 were CD38-immunopositive in acute neurodegeneration. Interestingly, in the 10-day-old rats subjected to cerebral damage, the number of Cx43 and CD38 coexpressing cells was several times higher as compared to GFAP and CD38 coexpressing cells, whereas the fractions of cells that expressed both GFAP and CD38 were similar in the control and experimental groups. These data suggest that other types of Cx43-containing cells, such as microglia, substantially contributed to the total elevation in CD38 expression after perinatal brain damage. Taking into account the data on the functional coupling of CD38 ADP-ribosyl cyclase activity and connexin 43, we studied the effects of a connexin blocker, glycyrrhetinic acid (GRA), on ADP-ribosyl cyclase activity in astrocytes isolated from brain tissue of control and experimental animals. We found that a 30-minute incubation of astrocytes with 5μM GRA resulted in decrease in ADP-rybosyl cyclase activity in these cells (Salmina, et al., 2009a).

In the model of focal brain ischemia in adult animals, we confirmed that CD38 could be considered as a marker of neuron-glia interactions disturbances caused by acute ischemic injury, and that modulation of ADP-ribosyl cyclase/CD38 expression and activity in the

activation of anaerobic glycolysis in astrocytes, and release of lactate supporting the activityrelated energy required for neurons (Magistretti, 2000)]. In general, astrocytes metabolize glucose mainly to lactate and release it into the extracellular medium, while neurons appear to have a kinetic preference for oxidizing lactate imported from the external medium over pyruvate/lactate produced in neurons by glycolysis (Itoh et al., 2003). However, neurons die more intensively than astrocytes even though the apparent dysfunction takes place in astrocytes. Glycolysis is high in astrocytes, so they need a way to maintain the adequate levels of NAD+. Probably, astrocytes can sustain themselves adequately with glycolytic metabolism, not significantly affected by mitochondrial dysfunction, thereby being more

Very few data are available on CD38 expression in neurodegeneration. In different experimental models of acute and chronic neurodegeneration in rats (focal brain ischemia, rotenone model of Parkinson's disease, and perinatal hypoxic-ischemic brain injury), we found elevated expression of CD38 in neurons and astroglial cells in the acute period of

In the model of perinatal hypoxic-ischemic brain damage, we found that changes in CD38 expression and ADP-ribosyl cyclase activity in neuronal and glial cells attribute to alterations in intracellular NAD+ level as well as to susceptibility of the cells to the action of apoptogenic stimuli; acute period of perinatal hypoxic/ischemic brain injury is characterized by reactive astrogliosis and elevation of CD38 expression, changes in CD38 and Cx43 expression in astrocytes serving as markers of neuron-glial interactions in perinatal CNS injury; ADP-ribosyl cyclase activity in neurons in response to stimulation of NMDA is changed after perinatal hypoxic-ischemic brain injury. It has been demonstrated that, in the immature brain, the impairment of intracellular calcium homeostasis is the leading mechanism of perinatal damage to both neuronal and glial cells (Vannucci et al., 2001). Based on our data, we can conclude that the mechanisms of maintenance of intracellular calcium homeostasis, which are under the control of ADPribosyl cyclase and Cx43, play a special role in responses of neurons and glia to hypoxic–

We demonstrated that most of the cells that expressed Cx43 were CD38-immunopositive in acute neurodegeneration. Interestingly, in the 10-day-old rats subjected to cerebral damage, the number of Cx43 and CD38 coexpressing cells was several times higher as compared to GFAP and CD38 coexpressing cells, whereas the fractions of cells that expressed both GFAP and CD38 were similar in the control and experimental groups. These data suggest that other types of Cx43-containing cells, such as microglia, substantially contributed to the total elevation in CD38 expression after perinatal brain damage. Taking into account the data on the functional coupling of CD38 ADP-ribosyl cyclase activity and connexin 43, we studied the effects of a connexin blocker, glycyrrhetinic acid (GRA), on ADP-ribosyl cyclase activity in astrocytes isolated from brain tissue of control and experimental animals. We found that a 30-minute incubation of astrocytes with 5μM GRA resulted in decrease in ADP-rybosyl

In the model of focal brain ischemia in adult animals, we confirmed that CD38 could be considered as a marker of neuron-glia interactions disturbances caused by acute ischemic injury, and that modulation of ADP-ribosyl cyclase/CD38 expression and activity in the

resistant to oxidative stress and NAD+ depletion.

cyclase activity in these cells (Salmina, et al., 2009a).

ischemic damage.

**4. CD38 expression in acute and chronic neurodegeneration** 

brain injury (Salmina, et al., 2008; Salmina, et al., 2009a; Salmina et al., 2006a).

brain significantly improved clinical manifestations of neurological dysfunction associated with ischemia-induced neurodegeneration (Salmina, et al., 2006a).

In patients with ischemic stroke, elevated expression of CD38 in peripheral blood leukocytes corresponds to formation of membrane-derived microparticles and progression of endothelial dysfunction due to CD38-CD31 interactions (Inzhutova et al., 2008; Salmina et al., 2010b). It is well known that astrocytes play important role in the formation, extent and configuration of the junctional complexes in the brain endothelium in a manner that astrocyte-induced enhanced tight junction communication is associated with the reduction of gap junctions. A major function of the neurovascular unit is to regulate the transport and diffusion properties of brain capillary endothelial cells that compose the brain-blood barrier (Banerjee & Bhat, 2007). Astrocytes exhibit anatomic relationships with cerebral arterioles and neurons. In the brain parenchyma, the extensive ensheatment of cerebral arterioles by astrocytic end-feet far exceeds any direct neural contacts with those perfusion-regulating microvessels. That unique arrangement permits astrocytes to transduce signals arising from activated neurons and to transmitthat information to the cerebral microcirculation (Xu et al., 2008).

Coupling of NMDA receptors to ADP-ribosyl cyclase/CD38 in neuronal and glial cells, involvement of CD38 in neuronal-glial (Higashida et al., 2007a; Salmina, et al., 2009a) and leukocyte-endothelial interactions (Deaglio, et al., 2008; Inzhutova, et al., 2008; Malavasi et al., 2008) suggest new approach to treat endothelial dysfunction caused by various stimuli, i.e. by homocysteine (Boldyrev, 2010) in neurodegenerative processes.

As we mentioned above, in acute neurodegeneration, CD38+-expressing cells were predominantly represented by GFAP+/Cx43+ cells of astroglial origin. We found that in neurons, elevated CD38 expression resulted in intracellular NAD+ depletion and cell death, while in astrocytes high levels of CD38 expression relate to increased resistance to the action of apoptogenic stimuli, development of reactive gliosis, and changes in their glycolytic activity. Mitochondrial ADP-ribosyl cyclase activity was mainly induced by ischemic stimuli. Our data well fit the previous observations that intracellular NAD+ levels regulate astroglial response to neuronal activation, NAD+ released from astrocytes regulate apoptosis of neurons in postischemic period, and astrocytes are more resistant to hypoxia than neurons.

In the developing brain, direct correlation between the CD38 expression and apoptosis development in the given cell populations has been registered. We found stimulating effects of agonists of mGluRI, mGluRIII, suppressive effects of agonists of NMDAR on CD38 activity in premature injured brain cells. However, in the adult brain, reverse correlation between CD38 expression and apoptosis progression was observed. Under the pathophysiological conditions, development of cell death and ability of brain cells to maintain the levels of intracellular NAD+ are determined by hypoxia/ischemia-induced disturbance in the dynamics of ADP-ribosyl cyclase activity in the brain cells. These data are in agreement with our report on the contribution of CD38 overexpression to development of plasma membrane blebbing in neuroblastoma x glioma NG108-15 hybrid cells (Egorova et al., 2000).

In the experimental model of Parkinson's disease, we found that loss of dopaminergic neurons via apoptosis was associated with elevation of expression and activity of ADP-ribosyl cyclase/CD38 in remaining tyrosine hydroxylase-immunopositive cells. Immunochemical studies with anti-CD38 antibodies indicated accumulation of CD38 antigen in the neurofibrillary tangles that occur in neuronal perikarya and proximal

Alteration of Neuron-Glia Interactions in Neurodegeneration:

concentrations and metabolism.

Molecular Biomarkers and Therapeutic Strategy 287

the therapeutic potential of sirtuins (NAD+-dependent histone deacetylases consuming NAD+) for Alzheimer's disease (Anekonda & Reddy, 2006). Inhibitors of kynurenine 3-hydroxylase can reduce the production of neurotoxic metabolites (Khan et al., 2007). It was reported that decreases in β-amyloid content in the brain can be achieved by governing cellular sirtuin activity (Qin et al., 2006). Improvement of cognitive functions was detected after treatment with oral stabilized NADH (Demarin et al., 2004), probably, due to ability of NADH to enter into astrocyets and block PARP-mediated astrocyte death (Zhu et al., 2005). However, efficacy of NADH to correction of cognitive dysfunction in dementia remains to be ambiguos (Rainer et al., 2000). Recently, CD38 was suggested as a new target for dementias including Alzheimer's disease (Chini et al., 2007). It is interesting enough that NAD+ (and, probably, NADH) can be transported across the plasma membranes of astrocytes through connexin hemichannels (Verderio, et al., 2001) or purinergic receptors (Lu et al., 2007), thus suggesting new approach to manipulating its intracellular

Overexpression of NMNAT results in suppression of Wallerian degeneration in neurons, however, in Cd38-/- cells with higher levels of intracellular NAD+ no difference in the axon degeneration patterns were registered. In general, increased NAD+ synthesis is responsible for axonal protection (Yan et al., 2010). In vitro NNMT expression significantly decreased cell death which correlated with increased intracellular ATP content, ATP: ADP ratio, Complex I activity and a reduction in the degradation of the NDUFS3 subunit of Complex I. These effects were replicated by incubation of cells with 1-methylnicotinamide. In the context of pathogenesis of Parkinson's disease, it is important that both NNMT expression and 1-methylnicotinamide protected SH-SY5Y cells from the toxicity of the Complex I inhibitors MPP+ and rotenone by reversing their effects upon ATP synthesis, ATP:ADP ratio, Complex I activity and the NDUFS3 subunit (Parsons et al., 2011). Overexpression of SIRT1 or its activation in neuronal and glial cells with resveratrol has been shown to protect the brain tissue from degeneration in Alzheimer's disease and Huntington's disease, and calorie restriction able to modulate SIRT activity is neuroprotective against Parkinson's disease and Alzheimer's disease (Outeiro et al., 2008). Modulation of TRPM channels which are abundantly expressed in the brain has neuroprotective activity in Parkinson's disease and Alzheimer's disease (Yamamoto et al., 2007). Protection of neurons from glutamate and β-amyloid toxicity was achieved by preloading neurons with creatine (Brewer & Wallimann, 2000) or by pyruvate (Massieu et al., 2001). The latter as well as another tricarboxilic acid cycle substrate – α-ketoglutarate - were also potent in preventing death of neurons and

Experimental pharmacological intervention in the cADPR-signaling pathway are usually restricted to two targets, the cADPR-binding protein and the ADP-ribosyl cyclase (Guse, 2000). The agents used for such properties are: a) cADP-ribose and its analogues; b) modulators of ryanodine receptors activity such as caffeine, ryanodine, procaine, ruthenium red; c) ligands of FKBP. The usefulness of cADP-ribose as a pharmacological tool is limited

Among all inhibitors of NAD+-consuming enzymes, nicotinamide attracts the biggest interest. Nicotinamide has several cellular functions in CNS and serves as an anxiolytic, increases brain choline concentrations and is endogenous ligand of benzodiazepine receptors (Maiese & Chong, 2003). Neuroprotective action of nicotinamide has been reported in neurons at oxidative stress even it may be attributed to changes in glycolysis, apoptotic machinery, MAP kinase activity etc. rather than inhibition of NAD+

by its rapid hydrolysis, therefore various cADPR analogues have been synthesized.

astrocytes caused by intracellular NAD+ depletion (Ying, et al., 2002).

dendrites in Alzheimer's disease (Otsuka et al., 1994). Literature data suggest that in Alzheimer's disease, accumulation of CD38 antigen in the neurofibrillary tangles, an association of altered Ca2+ regulation in astrocytes, alterations in ryanodine receptors binding and functions are detectable.

Dramatic rise in CD38 messenger RNA levels in IL-1β-activated astrocytes was reported in HIV-associated neurodegeneration and dementia: in astrocytes, pre-treatment with the cADPR-specific antagonist 8-Br-cADPR and CD38 siRNA transfection returned elevated [Ca2+]i to baseline, thus confirming a CD38-cADPR specific response. These data have broader implications in other inflammatory diseases involving astrocyte activation and CD38 dysregulation (Banerjee et al., 2008).

We suggest that possible causes of elevation of CD38 expression in brain cells are the following: 1) changes in NAD+ bioavailability (release from mitochondria into cytosol, cell death, connexin Cx43 activation); 2) redistribution of the enzyme in the cells; 3) cytokinedependent (IL, TNF) changes in expression of gene encoding for CD38 in the sites of brain injury of neurodegeneration; 4) action of neuro- and gliotransmitters. Since CD38 expression in astrocytes is stimulated by glutamate release from neurons, we can suggest that CD38 is a marker of altered neuron-astrocyte interactions under the conditions of excitotoxic insult. Also, since mitochondrial complex I dysfunction causes elevation of CD38 expression, we suggest that CD38 is a marker of mitochondrial dysfunction in the context of neuron-glia metabolic coupling. Activity of Cx43 prevents NAD+ depletion in CD38-overexpressing astrocytes and provides enough NAD+ for glycolysis, thus making astrocytes more resistant to the action of stimuli causing neurodegeneration.

Therefore, we propose that expression of CD38 in neuronal and glial cells: 1) is regulated under (patho)physiological conditions; 2) is associated with various signal transduction pathways (i.e. GluR in neurons and Cx43 in astrocytes) whose activity is important for molecular pathogenesis of neurodegeneration; 3) reflects – specifically or non-specifically – mitochondrial dysfunction; 4) could be considered as a target for pharmacological correction of neurodegeneration. Deciphering of CD38-associated molecules/events in neuronal and glial populations would give us new biomarkers for diagnostics of neurodegeneration, while pharmacological manipulation of ADP-ribosyl cyclase activity in brain cells would provide new therapeutic opportunities for the treatment of neurodegenerative disorders.

#### **5. Modulation of NAD<sup>+</sup> metabolism in glial cells as therapeutic approach in neurodegeneration**

Manipulating the neuron-glial cell interactions associated with changes in NAD+ levels represent one of the promising approaches to treatment of Alzheimer's disease (Braidy et al., 2008; Henricksen & Federoff, 2004). Pharmacological manipulation may be targeted to the modulation of intracellular NAD+ metabolism with substances affecting activity of NAD+ synthesizing and NAD+-converting enzymes, modulators of NAD+-dependent enzymes (i.e. sirtuins, glycolytic enzymes), regulators of tryptophan kynurenine metabolism, substrates of NAD+ synthetic pathways, ligands and regulators of CD38 expression and activity, modulators of NAD+ and cyclic ADP-ribose transport across the membrane (i.e. Cx43).

Metabolism of NAD+ could be efficiently regulated by inhibitors of mono(ADP-ribosyl) transferase and poly-ADP-ribosyl polymerase activities. Pharmacological interventions aimed at inhibiting PARP activity have been shown to be efficient in prevention of PARPmediated death of neurons and astrocytes (Ying, et al., 2002). Several studies have suggested

dendrites in Alzheimer's disease (Otsuka et al., 1994). Literature data suggest that in Alzheimer's disease, accumulation of CD38 antigen in the neurofibrillary tangles, an association of altered Ca2+ regulation in astrocytes, alterations in ryanodine receptors

Dramatic rise in CD38 messenger RNA levels in IL-1β-activated astrocytes was reported in HIV-associated neurodegeneration and dementia: in astrocytes, pre-treatment with the cADPR-specific antagonist 8-Br-cADPR and CD38 siRNA transfection returned elevated [Ca2+]i to baseline, thus confirming a CD38-cADPR specific response. These data have broader implications in other inflammatory diseases involving astrocyte activation and

We suggest that possible causes of elevation of CD38 expression in brain cells are the following: 1) changes in NAD+ bioavailability (release from mitochondria into cytosol, cell death, connexin Cx43 activation); 2) redistribution of the enzyme in the cells; 3) cytokinedependent (IL, TNF) changes in expression of gene encoding for CD38 in the sites of brain injury of neurodegeneration; 4) action of neuro- and gliotransmitters. Since CD38 expression in astrocytes is stimulated by glutamate release from neurons, we can suggest that CD38 is a marker of altered neuron-astrocyte interactions under the conditions of excitotoxic insult. Also, since mitochondrial complex I dysfunction causes elevation of CD38 expression, we suggest that CD38 is a marker of mitochondrial dysfunction in the context of neuron-glia metabolic coupling. Activity of Cx43 prevents NAD+ depletion in CD38-overexpressing astrocytes and provides enough NAD+ for glycolysis, thus making astrocytes more resistant

Therefore, we propose that expression of CD38 in neuronal and glial cells: 1) is regulated under (patho)physiological conditions; 2) is associated with various signal transduction pathways (i.e. GluR in neurons and Cx43 in astrocytes) whose activity is important for molecular pathogenesis of neurodegeneration; 3) reflects – specifically or non-specifically – mitochondrial dysfunction; 4) could be considered as a target for pharmacological correction of neurodegeneration. Deciphering of CD38-associated molecules/events in neuronal and glial populations would give us new biomarkers for diagnostics of neurodegeneration, while pharmacological manipulation of ADP-ribosyl cyclase activity in brain cells would provide new therapeutic opportunities for the treatment of neurodegenerative disorders.

Manipulating the neuron-glial cell interactions associated with changes in NAD+ levels represent one of the promising approaches to treatment of Alzheimer's disease (Braidy et al., 2008; Henricksen & Federoff, 2004). Pharmacological manipulation may be targeted to the modulation of intracellular NAD+ metabolism with substances affecting activity of NAD+ synthesizing and NAD+-converting enzymes, modulators of NAD+-dependent enzymes (i.e. sirtuins, glycolytic enzymes), regulators of tryptophan kynurenine metabolism, substrates of NAD+ synthetic pathways, ligands and regulators of CD38 expression and activity, modulators of NAD+ and cyclic ADP-ribose transport across the membrane (i.e. Cx43). Metabolism of NAD+ could be efficiently regulated by inhibitors of mono(ADP-ribosyl) transferase and poly-ADP-ribosyl polymerase activities. Pharmacological interventions aimed at inhibiting PARP activity have been shown to be efficient in prevention of PARPmediated death of neurons and astrocytes (Ying, et al., 2002). Several studies have suggested

 **metabolism in glial cells as therapeutic approach in** 

binding and functions are detectable.

CD38 dysregulation (Banerjee et al., 2008).

to the action of stimuli causing neurodegeneration.

**5. Modulation of NAD<sup>+</sup>**

**neurodegeneration** 

the therapeutic potential of sirtuins (NAD+-dependent histone deacetylases consuming NAD+) for Alzheimer's disease (Anekonda & Reddy, 2006). Inhibitors of kynurenine 3-hydroxylase can reduce the production of neurotoxic metabolites (Khan et al., 2007). It was reported that decreases in β-amyloid content in the brain can be achieved by governing cellular sirtuin activity (Qin et al., 2006). Improvement of cognitive functions was detected after treatment with oral stabilized NADH (Demarin et al., 2004), probably, due to ability of NADH to enter into astrocyets and block PARP-mediated astrocyte death (Zhu et al., 2005). However, efficacy of NADH to correction of cognitive dysfunction in dementia remains to be ambiguos (Rainer et al., 2000). Recently, CD38 was suggested as a new target for dementias including Alzheimer's disease (Chini et al., 2007). It is interesting enough that NAD+ (and, probably, NADH) can be transported across the plasma membranes of astrocytes through connexin hemichannels (Verderio, et al., 2001) or purinergic receptors (Lu et al., 2007), thus suggesting new approach to manipulating its intracellular concentrations and metabolism.

Overexpression of NMNAT results in suppression of Wallerian degeneration in neurons, however, in Cd38-/- cells with higher levels of intracellular NAD+ no difference in the axon degeneration patterns were registered. In general, increased NAD+ synthesis is responsible for axonal protection (Yan et al., 2010). In vitro NNMT expression significantly decreased cell death which correlated with increased intracellular ATP content, ATP: ADP ratio, Complex I activity and a reduction in the degradation of the NDUFS3 subunit of Complex I. These effects were replicated by incubation of cells with 1-methylnicotinamide. In the context of pathogenesis of Parkinson's disease, it is important that both NNMT expression and 1-methylnicotinamide protected SH-SY5Y cells from the toxicity of the Complex I inhibitors MPP+ and rotenone by reversing their effects upon ATP synthesis, ATP:ADP ratio, Complex I activity and the NDUFS3 subunit (Parsons et al., 2011). Overexpression of SIRT1 or its activation in neuronal and glial cells with resveratrol has been shown to protect the brain tissue from degeneration in Alzheimer's disease and Huntington's disease, and calorie restriction able to modulate SIRT activity is neuroprotective against Parkinson's disease and Alzheimer's disease (Outeiro et al., 2008). Modulation of TRPM channels which are abundantly expressed in the brain has neuroprotective activity in Parkinson's disease and Alzheimer's disease (Yamamoto et al., 2007). Protection of neurons from glutamate and β-amyloid toxicity was achieved by preloading neurons with creatine (Brewer & Wallimann, 2000) or by pyruvate (Massieu et al., 2001). The latter as well as another tricarboxilic acid cycle substrate – α-ketoglutarate - were also potent in preventing death of neurons and astrocytes caused by intracellular NAD+ depletion (Ying, et al., 2002).

Experimental pharmacological intervention in the cADPR-signaling pathway are usually restricted to two targets, the cADPR-binding protein and the ADP-ribosyl cyclase (Guse, 2000). The agents used for such properties are: a) cADP-ribose and its analogues; b) modulators of ryanodine receptors activity such as caffeine, ryanodine, procaine, ruthenium red; c) ligands of FKBP. The usefulness of cADP-ribose as a pharmacological tool is limited by its rapid hydrolysis, therefore various cADPR analogues have been synthesized.

Among all inhibitors of NAD+-consuming enzymes, nicotinamide attracts the biggest interest. Nicotinamide has several cellular functions in CNS and serves as an anxiolytic, increases brain choline concentrations and is endogenous ligand of benzodiazepine receptors (Maiese & Chong, 2003). Neuroprotective action of nicotinamide has been reported in neurons at oxidative stress even it may be attributed to changes in glycolysis, apoptotic machinery, MAP kinase activity etc. rather than inhibition of NAD+

Alteration of Neuron-Glia Interactions in Neurodegeneration:

mechanisms in neurons and astrocytes?

mitochondrial dysfunction in neurodegeneration?

in axonal degeneration and neuronal repair?

addressed:

**7. References** 

Molecular Biomarkers and Therapeutic Strategy 289

1. Which glial- and neuronal-derived factors can affect NAD+ metabolism in acute and chronic neurodegeneration? What is an integrative scheme for NAD+ homeostatic

2. What is the role for CD38-Cx43 interactions in initiation and progression of

3. Which molecular mechanisms coupled to NAD+ homeostasis in brain cells are involved

4. Which new biomarkers could be developed for early diagnostics of astroglial dysfunction in neurodegeneration? Which molecular targets in neurons and glial cells

Adamczyk, A., Jesko, H. & Strosznajder, R.P. (2005) Alzheimer's disease related peptides

Aksoy, P., White, T.A., Thompson, M. & Chini, E.N. (2006) Regulation of intracellular levels

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affected cholinergic receptor mediated poly(ADP-ribose) polymerase activity in the

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**6. Concluding remarks and outstanding questions for further investigation**  So far, several hypotheses have been suggested to explain pathogenesis of acute and chronic neurodegenration. Almost all of the proposed mechanisms include processes considered as gliopathy (Maragakis & Rothstein, 2006; Verkhratsky, 2010). Different molecules mark gliopathological changes, and CD38 expressed in astroglial cells should be considered as one of the markers of neuron-astrocyte metabolic coupling. Neuron-glia communication is responsible for establishment of vicious circles in the pathogenesis of neurodegeneration, therefore deciphering new molecular mechanisms of intercellular communication will provide us with new diagnostic and therapeutic strategies. To achieve this goal in the context of NAD+-controlled neuronal-astroglial coupling, the following questions should be

glycohydrolases. Age-dependent susceptibility of glial cells to the action of nicotinamide analogs has been reported (Krum, 1995). 6-aminonicotinamide as niacin antagonist produces neurotoxic activity by inducing inflammatory response of astroglial and microglial cells (Penkowa et al., 2003). But using of inhibitors of NAD+ hydrolysis may have even unfavorable results: partial inhibition of poly-ADP-ribosylation with 5-iodo-6-amino-1,2 benzopyrone preserves NAD+ and improves functional outcome after traumatic brain injury, whereas more complete inhibition impairs spatial memory acquisition independent of injury (Satchell et al., 2003). Therefore, cytoprotection with inhibitors of NAD+-consuming enzymes might be concentration specific. We found that nicotinamide (500 mg/kg) reduced expression of CD38 in the brain cortex in the model of ischemia-induced acute neurodegeneration in adult rats in vivo and potentiate neurological dysfunction caused by ischemia, thereby further confirming ambiguity of nicotinamide action on neuronal and glial cells.

Retinoic acid (RA) is a potent inducer of CD38 in peripheral blood cells, and recently it was suggested that this compound can be used to 'rescue' cells exhibiting low CD38 synthesis and hence might be a novel therapeutic strategy in treatment of autism associated with impaired CD38 expression in neurosecretory cells (Ebstein et al., 2011). Cultured astrocytes express the key enzyme mRNAs of retinoic acid biosynthesis and actively produce retinoic acid acting at RA receptors (RAR). Synthesis of retinoic acid in astrocytes is provided by retinal dehydrogenase and alcohol dehydrogenase (Wagner et al., 2002). It was shown that blockage of retinoic acid signaling by the pan-RAR antagonist prevented glia-induced neuron formation by noncommitted stem cells, thus suggesting a role for retinoic acid in astroglia-induced neuronal differentiation (Kornyei et al., 2007). Retinoids control expression of wide spectrum of genes in neuronal and glial cells (Lane & Bailey, 2005).

We tested effect of all-trans retinoic acid in vivo (20 mg/kg with ethanol to suppress endogenous synthesis of retinoic acid) in rats with experimental model of perinatal ischemic-hypoxic acute neurodegeneration. We found that suppression of endogenous synthesis of retinoic acid with ethanol reduced expression of CD38 in the cortex, while retinoic acid itself partially restored the level of CD38 (Salmina et al., 2009b).

Retinoid and retinoid-associated signaling plays an essential role in normal neurodevelopment and appears to remain active in the adult CNS. Molecular factors involved in RA-mediated responses become up-regulated in the adult CNS as a consequence of injury or degeneration. Our data and recent findings of R. Ebstein et al. (Ebstein, et al., 2011) suggest that intervention that modulates RA-regulated CD38 may have therapeutic potential in CNS disorders. It is interesting that a prolonged regime of vitamin A deprivation in adult rats has been shown to cause a deposition of β-amyloid peptide in the forebrain, RA could regulate the expression of the tau protein, and in particular the level of phosphorylated forms of tau, as suggested by *in vitro* observations, vitamin A, as well as β-carotene and coenzyme Q10, have also been shown to dose-dependently inhibit the formation of α-synuclein fibrils *in vitro,* RA reduces the effect of β-amyloid, and thus inhibits the neurotoxic effect of activated microglia, by suppressing the production of these cytotoxic molecules (Malaspina & Michael Titus, 2008). Retinoid receptors are involved in the regulation of brain functions, and retinoic acid signaling defects may contribute to pathologies such as Parkinson's disease (Krezel et al., 1998). Whether or not these events are associated with NAD+-converting activity of CD38 in neuronal and astroglial cells remains to be elucidated.

#### **6. Concluding remarks and outstanding questions for further investigation**

So far, several hypotheses have been suggested to explain pathogenesis of acute and chronic neurodegenration. Almost all of the proposed mechanisms include processes considered as gliopathy (Maragakis & Rothstein, 2006; Verkhratsky, 2010). Different molecules mark gliopathological changes, and CD38 expressed in astroglial cells should be considered as one of the markers of neuron-astrocyte metabolic coupling. Neuron-glia communication is responsible for establishment of vicious circles in the pathogenesis of neurodegeneration, therefore deciphering new molecular mechanisms of intercellular communication will provide us with new diagnostic and therapeutic strategies. To achieve this goal in the context of NAD+-controlled neuronal-astroglial coupling, the following questions should be addressed:


#### **7. References**

288 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

glycohydrolases. Age-dependent susceptibility of glial cells to the action of nicotinamide analogs has been reported (Krum, 1995). 6-aminonicotinamide as niacin antagonist produces neurotoxic activity by inducing inflammatory response of astroglial and microglial cells (Penkowa et al., 2003). But using of inhibitors of NAD+ hydrolysis may have even unfavorable results: partial inhibition of poly-ADP-ribosylation with 5-iodo-6-amino-1,2 benzopyrone preserves NAD+ and improves functional outcome after traumatic brain injury, whereas more complete inhibition impairs spatial memory acquisition independent of injury (Satchell et al., 2003). Therefore, cytoprotection with inhibitors of NAD+-consuming enzymes might be concentration specific. We found that nicotinamide (500 mg/kg) reduced expression of CD38 in the brain cortex in the model of ischemia-induced acute neurodegeneration in adult rats in vivo and potentiate neurological dysfunction caused by ischemia, thereby further confirming ambiguity of nicotinamide action on neuronal and

Retinoic acid (RA) is a potent inducer of CD38 in peripheral blood cells, and recently it was suggested that this compound can be used to 'rescue' cells exhibiting low CD38 synthesis and hence might be a novel therapeutic strategy in treatment of autism associated with impaired CD38 expression in neurosecretory cells (Ebstein et al., 2011). Cultured astrocytes express the key enzyme mRNAs of retinoic acid biosynthesis and actively produce retinoic acid acting at RA receptors (RAR). Synthesis of retinoic acid in astrocytes is provided by retinal dehydrogenase and alcohol dehydrogenase (Wagner et al., 2002). It was shown that blockage of retinoic acid signaling by the pan-RAR antagonist prevented glia-induced neuron formation by noncommitted stem cells, thus suggesting a role for retinoic acid in astroglia-induced neuronal differentiation (Kornyei et al., 2007). Retinoids control expression of wide spectrum of genes in neuronal and glial cells (Lane &

We tested effect of all-trans retinoic acid in vivo (20 mg/kg with ethanol to suppress endogenous synthesis of retinoic acid) in rats with experimental model of perinatal ischemic-hypoxic acute neurodegeneration. We found that suppression of endogenous synthesis of retinoic acid with ethanol reduced expression of CD38 in the cortex, while

Retinoid and retinoid-associated signaling plays an essential role in normal neurodevelopment and appears to remain active in the adult CNS. Molecular factors involved in RA-mediated responses become up-regulated in the adult CNS as a consequence of injury or degeneration. Our data and recent findings of R. Ebstein et al. (Ebstein, et al., 2011) suggest that intervention that modulates RA-regulated CD38 may have therapeutic potential in CNS disorders. It is interesting that a prolonged regime of vitamin A deprivation in adult rats has been shown to cause a deposition of β-amyloid peptide in the forebrain, RA could regulate the expression of the tau protein, and in particular the level of phosphorylated forms of tau, as suggested by *in vitro* observations, vitamin A, as well as β-carotene and coenzyme Q10, have also been shown to dose-dependently inhibit the formation of α-synuclein fibrils *in vitro,* RA reduces the effect of β-amyloid, and thus inhibits the neurotoxic effect of activated microglia, by suppressing the production of these cytotoxic molecules (Malaspina & Michael Titus, 2008). Retinoid receptors are involved in the regulation of brain functions, and retinoic acid signaling defects may contribute to pathologies such as Parkinson's disease (Krezel et al., 1998). Whether or not these events are associated with NAD+-converting activity of CD38 in neuronal and astroglial cells remains

retinoic acid itself partially restored the level of CD38 (Salmina et al., 2009b).

glial cells.

Bailey, 2005).

to be elucidated.


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**13** 

*Barcelona Spain* 

**Microglia, Calcification and** 

**Neurodegenerative Diseases** 

Jose M. Vidal-Taboada, Nicole Mahy and Manuel J. Rodríguez

*Unitat de Bioquímica i Biologia Molecular, Facultat de Medicina, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED),* 

Neurodegeneration is a complex process involving different cell types and neurotransmitters. A common characteristic of neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis, Huntington's disease (HD) and Amyotrophic Lateral Sclerosis (ALS) is the occurrence of a neuroinammatory reaction in which cellular processes involving glial cells (mainly microglia and astrocytes) and T cells are activated in response to neuronal death. This inammatory reaction has recently received attention as an unexpected potential target for the treatment of these

Microglial cells have a mesenchymal origin, invade the central nervous system (CNS) prenatally (Chan et al., 2007b) and are the resident macrophages in the CNS (Ransohoff & Perry, 2009). They comprise approximately 10-20% of adult glia and serve as the CNS innate immune system. In neurodegenerative diseases, microglia is activated by misfolded proteins. In the case of AD, amyloid-β (Aβ) peptides accumulate extracellularly and activate the microglia locally. In the case of PD, ALS and HD, the misfolded proteins accumulate intracellularly but are still associated with activation of the microglia (Perry et al., 2010). Reactive microglia in the substantia nigra and striatum of PD brains have been described, and increased levels of proinflammatory cytokines and inducible nitric oxide synthase have been detected in these brain regions, providing evidence of a local inflammatory reaction (Hirsch & Hunot, 2009). The injection of lipopolysaccharide (a potent microglia activator) into the substantia nigra produces microglial activation and the death of dopaminergic cells. These findings support the hypothesis that microglial activation and neuroinflammation

Astrocytes are ectodermal cells, and they are probably about ten times as numerous as neurons. Astroglial cells were initially believed to be passive support cells providing trophic support for surrounding neurons (Sofroniew & Vinters, 2010), maintaining extracellular ion homeostasis and capturing excess extracellular neurotransmitters such as glutamate, which is considered particularly important given its involvement in excitotoxicity. However, recent studies have implicated astrocytes in many complex CNS functions, such as physical

**1. Introduction** 

diseases.

**1.1 Neurodegeneration involve different cell types** 

contribute to PD pathogenesis (Herrera et al., 2000).

Ziegler, M., Jorcke, D. & Schweiger, M. (1997) Identification of bovine liver mitochondrial NAD+ glycohydrolase as ADP-ribosyl cyclase. *Biochemical Journal*. Vol. 326, No. Pt 2, pp. 401-405

## **Microglia, Calcification and Neurodegenerative Diseases**

Jose M. Vidal-Taboada, Nicole Mahy and Manuel J. Rodríguez *Unitat de Bioquímica i Biologia Molecular, Facultat de Medicina, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Barcelona* 

*Spain* 

#### **1. Introduction**

300 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

Ziegler, M., Jorcke, D. & Schweiger, M. (1997) Identification of bovine liver mitochondrial

2, pp. 401-405

NAD+ glycohydrolase as ADP-ribosyl cyclase. *Biochemical Journal*. Vol. 326, No. Pt

#### **1.1 Neurodegeneration involve different cell types**

Neurodegeneration is a complex process involving different cell types and neurotransmitters. A common characteristic of neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis, Huntington's disease (HD) and Amyotrophic Lateral Sclerosis (ALS) is the occurrence of a neuroinammatory reaction in which cellular processes involving glial cells (mainly microglia and astrocytes) and T cells are activated in response to neuronal death. This inammatory reaction has recently received attention as an unexpected potential target for the treatment of these diseases.

Microglial cells have a mesenchymal origin, invade the central nervous system (CNS) prenatally (Chan et al., 2007b) and are the resident macrophages in the CNS (Ransohoff & Perry, 2009). They comprise approximately 10-20% of adult glia and serve as the CNS innate immune system. In neurodegenerative diseases, microglia is activated by misfolded proteins. In the case of AD, amyloid-β (Aβ) peptides accumulate extracellularly and activate the microglia locally. In the case of PD, ALS and HD, the misfolded proteins accumulate intracellularly but are still associated with activation of the microglia (Perry et al., 2010). Reactive microglia in the substantia nigra and striatum of PD brains have been described, and increased levels of proinflammatory cytokines and inducible nitric oxide synthase have been detected in these brain regions, providing evidence of a local inflammatory reaction (Hirsch & Hunot, 2009). The injection of lipopolysaccharide (a potent microglia activator) into the substantia nigra produces microglial activation and the death of dopaminergic cells. These findings support the hypothesis that microglial activation and neuroinflammation contribute to PD pathogenesis (Herrera et al., 2000).

Astrocytes are ectodermal cells, and they are probably about ten times as numerous as neurons. Astroglial cells were initially believed to be passive support cells providing trophic support for surrounding neurons (Sofroniew & Vinters, 2010), maintaining extracellular ion homeostasis and capturing excess extracellular neurotransmitters such as glutamate, which is considered particularly important given its involvement in excitotoxicity. However, recent studies have implicated astrocytes in many complex CNS functions, such as physical

Microglia, Calcification and Neurodegenerative Diseases 303

inuenced by surrounding astrocytes and inammatory T-cell subsets, which can affect

All neurological disorders lead to activation of the microglia. Thus, microglial reaction represents the main mediator of the inflammatory process in neurodegenerative diseases, and microgliosis is directly related to the physiopathology of these. For example, microgliosis is associated with atypical and insoluble components caused by irregular protein folding and degradation pathways, altered subcellular localization, and the abnormal interactions with other cellular proteins found in AD, PD HD, Down syndrome and normal aging. Microgliosis is also associated with the formation of extracellular ionic precipitates, such as hydroxyapatites, which are frequently observed within the CNS areas involved in the disease (Rodriguez et al., 2009a, Saura et al., 1995), and is also present in encephalopathies caused by prions. This innate immune response is currently considered to be a potential pathogenic factor, since microglial reaction may engender neurodegenerative events, including amyloid-beta plaque formation, dystrophic neurite growth, and excessive

In the healthy CNS, ramified resting microglia are active cells since they permanently scan their microenvironment (Wake et al., 2009). In response to any CNS injury or immunological stimuli, the microglia rapidly evolve from a surveillance state towards a more reactive one, through important phenotypical changes in response to activation signals released by the tissue (Schwartz et al., 2006). Microglia undergo a dramatic morphological transformation into amoeboid form and express an upregulated catalogue of molecules, such as CD14, major histocompatibility complex (MHC) molecules, chemokine receptors, CD11c, integrins, neurotrophins, and several other markers (Kettenmann et al., 2011). As such, reactive microglia can perform functions essential to neuron survival, such as phagocytosis to clear toxic and cellular debris, and innate immunity. Also involved in the release of trophic and anti-inflammatory factors, microglia facilitate repair through the guided migration of stem

In contrast, once microglia become overactivated they can produce detrimental effects through excessive production of a large array of cytotoxic factors, such as NO, TNF-α, reactive oxygen species, and pro-inflammatory cytokines (Lull & Block, 2010, Milligan & Watkins, 2009). Currently, the conditions that determine whether microglial reaction will be detrimental or beneficial to neuronal survival are poorly understood. However, it is becoming more widely accepted that although microglial activation is necessary and crucial for host defense and neuron survival, microglial overactivation leads to deleterious

Since every single microglial cell generates its own response to damage according to the nature and intensity of the signals released by the injured tissue, microglial cells do not constitute a homogenous cell population, but instead present a range of different phenotypes closely related to the evolution of the lesion process. In addition, some microglial cells become increasingly dysfunctional as they age, and may participate directly in the development of neurodegeneration (Block et al., 2007, Stoll et al., 2002). Microglia adopts a phenotype that mostly exacerbates tissue injury or promotes brain repair. Microglia can thus present two phenotypes, one of which is deleterious (also called M1 microglial phenotype) and the other benign (M2 microglial phenotype), depending on their intrinsic properties, interaction with the cellular microenvironment, and presence of

their phagocytic capacity and antigen-presenting cell properties.

**1.3 Microglial reaction: Two sides of the same coin** 

cells to the site of inflammation and injury.

tau phosphorylation.

consequences.

structuring of the brain (they are the main cells involved in cholesterol synthesis), active control of synaptogenesis and plasticity (Graeber, 2010), regulation of blood flow and promotion of myelination (Halliday & Stevens, 2011). Astroglial activation is characterized by an increase in expression of intermediate lament glial brillary acidic protein (GFAP) and the gene aldehyde dehydrogenase 1 family member L1 (ALDH1L1). Astrocytes are not immune cells per se, but they can, under specic conditions, contribute to the immune response (Farina et al., 2007).

Several other cell types have been associated with neurodegeneration and neuroinammation, such as T-cells, oligodendrocytes and ependymal and subependymal cells (Philips & Robberecht, 2011). Once inltrated in the CNS, T-cell subpopulations modulate the neuroinammatory reaction differently, depending on stage of disease progression (Beers et al., 2011, Philips & Robberecht, 2011). Oligodendrocyte cells are widely distributed in the adult nervous system and their precursors have been reported to differentiate into astrocytes and even neurons in specic conditions (Rivers et al., 2008); however, their role in neuroinammation is still poorly understood, as is that of ependymal and subependymal (Chi et al., 2006).

In addition to the death of specific neuronal populations, there are many other parallels between different neurodegenerative disorders. These include alteration of a diversity of neurotransmitters and intracellular signals, in particular of glutamate and calcium, which play key roles in excitotoxicity. Thus, alterations in cellular and molecular steady states give rise to changes that cannot be counteracted by the tissue, and lead to chronic and progressive neurodegenerative processes from which a return to normality is impossible. As advances in research are made, more similarities between these neurodegenerative diseases have been found on many different levels, from molecular to tissular.

#### **1.2 Inflammation and neurodegeneration**

The term neuroinflammation describes endogenous CNS tissue response to injury. Classically known as reactive gliosis, neuroinflammation refers to the aggressive response of glia to activating stimuli, analogous to the response of activated immune cells in peripheral tissues. Neuroinflammation has been associated with chronic CNS diseases such as multiple sclerosis, which is an unequivocal example of an inflammatory CNS disease. Other neurodegenerative diseases such as AD, ALS, PD, and HD lack the prominent infiltrates of blood-derived mononuclear cells that characterize autoimmune diseases. However, many substances involved in the promotion of inflammatory processes are present in the CNS of patients with such neurodegenerative diseases (Block et al., 2007).

Microglia are transformed and activated by a range of signals, such as neuronal death, mechanical injury and toxins (Block et al., 2007, Streit et al., 2004), and once activated they form the rst line of defense against infection or injury to the CNS (Schwartz et al., 2006). Activated microglia acquire an amoeboid phenotype morphology similar to macrophages expressing the same markers, such as MHCI, MHCII, Iba1 and GLUT5 (Halliday & Stevens, 2011), and secrete proinammatory molecules such as tumor necrosis factor-alpha (TNF-α), interferon γ, and interleukin 1β; they also upregulate oxidant molecules such as nitric oxide (NO) and O2, which can protect against pathogens. This proinammatory reaction eliminates hazards and repairs any damage. Microglia also release anti-inammatory and trophic factors such as insulin-like growth factor 1 (IGF-1), interleukin 4, and interleukin 10, contributing to the repair and limitation of the inammatory process (Block et al., 2007, Stoll et al., 2002). The proinammatory or anti-inammatory responses of the microglia are

structuring of the brain (they are the main cells involved in cholesterol synthesis), active control of synaptogenesis and plasticity (Graeber, 2010), regulation of blood flow and promotion of myelination (Halliday & Stevens, 2011). Astroglial activation is characterized by an increase in expression of intermediate lament glial brillary acidic protein (GFAP) and the gene aldehyde dehydrogenase 1 family member L1 (ALDH1L1). Astrocytes are not immune cells per se, but they can, under specic conditions, contribute to the immune

Several other cell types have been associated with neurodegeneration and neuroinammation, such as T-cells, oligodendrocytes and ependymal and subependymal cells (Philips & Robberecht, 2011). Once inltrated in the CNS, T-cell subpopulations modulate the neuroinammatory reaction differently, depending on stage of disease progression (Beers et al., 2011, Philips & Robberecht, 2011). Oligodendrocyte cells are widely distributed in the adult nervous system and their precursors have been reported to differentiate into astrocytes and even neurons in specic conditions (Rivers et al., 2008); however, their role in neuroinammation is still poorly understood, as is that of ependymal

In addition to the death of specific neuronal populations, there are many other parallels between different neurodegenerative disorders. These include alteration of a diversity of neurotransmitters and intracellular signals, in particular of glutamate and calcium, which play key roles in excitotoxicity. Thus, alterations in cellular and molecular steady states give rise to changes that cannot be counteracted by the tissue, and lead to chronic and progressive neurodegenerative processes from which a return to normality is impossible. As advances in research are made, more similarities between these neurodegenerative diseases

The term neuroinflammation describes endogenous CNS tissue response to injury. Classically known as reactive gliosis, neuroinflammation refers to the aggressive response of glia to activating stimuli, analogous to the response of activated immune cells in peripheral tissues. Neuroinflammation has been associated with chronic CNS diseases such as multiple sclerosis, which is an unequivocal example of an inflammatory CNS disease. Other neurodegenerative diseases such as AD, ALS, PD, and HD lack the prominent infiltrates of blood-derived mononuclear cells that characterize autoimmune diseases. However, many substances involved in the promotion of inflammatory processes are present in the CNS of

Microglia are transformed and activated by a range of signals, such as neuronal death, mechanical injury and toxins (Block et al., 2007, Streit et al., 2004), and once activated they form the rst line of defense against infection or injury to the CNS (Schwartz et al., 2006). Activated microglia acquire an amoeboid phenotype morphology similar to macrophages expressing the same markers, such as MHCI, MHCII, Iba1 and GLUT5 (Halliday & Stevens, 2011), and secrete proinammatory molecules such as tumor necrosis factor-alpha (TNF-α), interferon γ, and interleukin 1β; they also upregulate oxidant molecules such as nitric oxide (NO) and O2, which can protect against pathogens. This proinammatory reaction eliminates hazards and repairs any damage. Microglia also release anti-inammatory and trophic factors such as insulin-like growth factor 1 (IGF-1), interleukin 4, and interleukin 10, contributing to the repair and limitation of the inammatory process (Block et al., 2007, Stoll et al., 2002). The proinammatory or anti-inammatory responses of the microglia are

have been found on many different levels, from molecular to tissular.

patients with such neurodegenerative diseases (Block et al., 2007).

response (Farina et al., 2007).

and subependymal (Chi et al., 2006).

**1.2 Inflammation and neurodegeneration** 

inuenced by surrounding astrocytes and inammatory T-cell subsets, which can affect their phagocytic capacity and antigen-presenting cell properties.

All neurological disorders lead to activation of the microglia. Thus, microglial reaction represents the main mediator of the inflammatory process in neurodegenerative diseases, and microgliosis is directly related to the physiopathology of these. For example, microgliosis is associated with atypical and insoluble components caused by irregular protein folding and degradation pathways, altered subcellular localization, and the abnormal interactions with other cellular proteins found in AD, PD HD, Down syndrome and normal aging. Microgliosis is also associated with the formation of extracellular ionic precipitates, such as hydroxyapatites, which are frequently observed within the CNS areas involved in the disease (Rodriguez et al., 2009a, Saura et al., 1995), and is also present in encephalopathies caused by prions. This innate immune response is currently considered to be a potential pathogenic factor, since microglial reaction may engender neurodegenerative events, including amyloid-beta plaque formation, dystrophic neurite growth, and excessive tau phosphorylation.

#### **1.3 Microglial reaction: Two sides of the same coin**

In the healthy CNS, ramified resting microglia are active cells since they permanently scan their microenvironment (Wake et al., 2009). In response to any CNS injury or immunological stimuli, the microglia rapidly evolve from a surveillance state towards a more reactive one, through important phenotypical changes in response to activation signals released by the tissue (Schwartz et al., 2006). Microglia undergo a dramatic morphological transformation into amoeboid form and express an upregulated catalogue of molecules, such as CD14, major histocompatibility complex (MHC) molecules, chemokine receptors, CD11c, integrins, neurotrophins, and several other markers (Kettenmann et al., 2011). As such, reactive microglia can perform functions essential to neuron survival, such as phagocytosis to clear toxic and cellular debris, and innate immunity. Also involved in the release of trophic and anti-inflammatory factors, microglia facilitate repair through the guided migration of stem cells to the site of inflammation and injury.

In contrast, once microglia become overactivated they can produce detrimental effects through excessive production of a large array of cytotoxic factors, such as NO, TNF-α, reactive oxygen species, and pro-inflammatory cytokines (Lull & Block, 2010, Milligan & Watkins, 2009). Currently, the conditions that determine whether microglial reaction will be detrimental or beneficial to neuronal survival are poorly understood. However, it is becoming more widely accepted that although microglial activation is necessary and crucial for host defense and neuron survival, microglial overactivation leads to deleterious consequences.

Since every single microglial cell generates its own response to damage according to the nature and intensity of the signals released by the injured tissue, microglial cells do not constitute a homogenous cell population, but instead present a range of different phenotypes closely related to the evolution of the lesion process. In addition, some microglial cells become increasingly dysfunctional as they age, and may participate directly in the development of neurodegeneration (Block et al., 2007, Stoll et al., 2002). Microglia adopts a phenotype that mostly exacerbates tissue injury or promotes brain repair. Microglia can thus present two phenotypes, one of which is deleterious (also called M1 microglial phenotype) and the other benign (M2 microglial phenotype), depending on their intrinsic properties, interaction with the cellular microenvironment, and presence of

Microglia, Calcification and Neurodegenerative Diseases 305

α

**S100β, TNF- , Ado, Tau, ATP, Glu, GABA, GDNF**

 **↓Glc, ↓O , ↑Glu, ↑[Ca ] , ROS, Aβ 2+ 2** *i*

Fig. 1. Signalling systems in the microglia-astrocyte-neuron cross-talk. Astrocytes and microglia monitorize neuronal activity by sensing neurotransmitter release. In the same way, microglial cells have receptors to molecules released by astrocytes. Microglia integrate all these signals and release molecules that may modulate neuronal and astroglial activity. During neurodegeneration, changes in physiological parameters may trigger neuronal injury and/or microglial activation (molecules inside a red square) that modify those signal transduction systems. Aβ, amyloid beta; Ach, acetylcholine; Ado, adenosine; BDNF, Brainderived neurotrophic factor; GABA, γ-aminobutyric acid; GDNF, Glial cell-derived

neurotrophic factor; Glc, glucose; Glu, glutamate; 5-HT, serotonin; VIP, Vasoactive intestinal

reactive oxygen species or TNF-α, also modify the RAGE response to S100β (Edwards & Robinson, 2006) in a microglia-astroglia cross-talk that integrates these signaling systems. In contrast, at high concentrations S100β binds the RAGE, which may mediate microglial activation during the course of brain damage (Bianchi et al., 2010). An increased release of S100β during neurodegeneration (Li et al., 2011) will enhance inflammatory cytokine production and potentiate the switch of microglia to chronic cytotoxic activity, feeding the

peptide; IFNγ, interferon gamma; ROS, reactive oxygen species; Tau, taurine

**Neuron**

**TNF- , NO, IL-1β, IGF-1** α

**ATP, Glu, GABA, ACh, dopamine, adrenaline, 5-HT, VIP, BDNF**

**Glu, IFN , ROS, Aβ, -synuclein** γ α

α

**S100β TNF-Ado ATP Glu CCL2 CXCL10**

**Astrocyte**

neurotoxic process and leading to neurodegeneration.

α

**TNF- NO IL-1β IGF-1**

**Microglial cell**

pathogenic factors (Halliday & Stevens, 2011, Henkel et al., 2009). Therefore, controlling microglial cell activation and the acquisition of positive or negative phenotypes is of major therapeutic interest in all CNS disorders related to neuroinflammation.

#### **1.4 Astrocyte-microglia interactions: Who's the bad guy after all?**

The classical view of astroglia, as simply presenting non-excitable support to neurons, has changed radically in recent years. Astrocytes are now seen as elements that generate various local signals, including glutamate, to communicate with neurons and that influence the tissue outcome during neurodegeneration (Allaman et al., 2011). There is increasing evidence in support of this active role of astrocytes, suggesting that atypical astrocyte activation or astroglial dysfunction constitute maladaptive responses to brain injury that may feed the ongoing pathologic process during neurodegeneration. For example, astrocyte dysfunction is a key factor in the pathogenesis of human neurological disorders (Seifert et al., 2006), and in the cognitive impairment of aged rats (Andrés et al., 2000). Furthermore, glutamate-induced chronic lesion in rat brain not only presents a lack of astrogliosis but also long term atrophy of astrocytes, suggesting a maladaptive response that may be a cause of the on-going pathologic process (Rodriguez et al., 2009a).

Moreover, astrocytes influence microglial behaviour (figure 1). For instance, astrocytes play a critical role in the activation of microglia under infectious conditions (Ovanesov et al., 2008). In addition, astroglial chemokines are involved in microglia/macrophage activation in multiple sclerosis with MCP-1/CCL2 and IP-10/CXCL10 directing reactive gliosis (Tanuma et al., 2006). Therefore, it is reasonable to assume that astrocytic activity can be influenced by microglial activation. Although a clear account of this dynamic relationship has yet to be proposed, the astrocyte-microglia interplay may determine the phenotype that microglial cells adopt during neurodegeneration.

Some findings have implicated astrocytes in chronic microgliosis, with a transition from an initial neuroprotective activity to a later cytotoxic one. TNF-α secretion is crucial for rapid autocrine microglial activation with both neuroprotective and cytotoxic effects, a process that is also fed by TNF-α released by reactive astrocytes (Suzumura et al., 2006). TNF-α actions leading to neuronal death or survival are dose dependent (Bernardino et al., 2008), since it can activate two specific receptors: TNFR1, with an intracellular death domain, and TNFR2, mainly involved in neuroprotection (Fontaine et al., 2002). Consequently, low concentrations of TNF-α would initially induce TNFR2-mediated neuroprotection, whereas a subsequent high concentration of TNF-α would be able to activate TNFR1 both in astrocytes and microglia and contribute to cell injury through the death domain of the receptor.

Astroglial S100β is another of the factors that control microglial activity. Astrocytes release S100β constitutively (Van Eldik & Wainwright, 2003) and increase this release upon stimulation by several factors, including TNF-α (Edwards & Robinson, 2006). Under normal conditions, released S100β acts as a neurotrophic factor, countering the stimulatory effect of neurotoxins on (Reali et al., 2005) and stimulating astrocyte glutamate uptake (Tramontina et al., 2006). Released S100β modifies astrocytic, neuronal and microglial activities, depending on the extracellular concentration of the former and the expression of the specific receptor for advanced glycation end-products (RAGE). At micromolar concentrations, S100β upregulates IL-1β and TNF-α expression in activated microglia via RAGE, with the requirement of concurrent activation of NF-κB and AP-1 transcription factors (Bianchi et al., 2010). Furthermore, factors that modulate microglial reactivity, such as Ca2+ concentration,

pathogenic factors (Halliday & Stevens, 2011, Henkel et al., 2009). Therefore, controlling microglial cell activation and the acquisition of positive or negative phenotypes is of major

The classical view of astroglia, as simply presenting non-excitable support to neurons, has changed radically in recent years. Astrocytes are now seen as elements that generate various local signals, including glutamate, to communicate with neurons and that influence the tissue outcome during neurodegeneration (Allaman et al., 2011). There is increasing evidence in support of this active role of astrocytes, suggesting that atypical astrocyte activation or astroglial dysfunction constitute maladaptive responses to brain injury that may feed the ongoing pathologic process during neurodegeneration. For example, astrocyte dysfunction is a key factor in the pathogenesis of human neurological disorders (Seifert et al., 2006), and in the cognitive impairment of aged rats (Andrés et al., 2000). Furthermore, glutamate-induced chronic lesion in rat brain not only presents a lack of astrogliosis but also long term atrophy of astrocytes, suggesting a maladaptive response that may be a cause of the on-going

Moreover, astrocytes influence microglial behaviour (figure 1). For instance, astrocytes play a critical role in the activation of microglia under infectious conditions (Ovanesov et al., 2008). In addition, astroglial chemokines are involved in microglia/macrophage activation in multiple sclerosis with MCP-1/CCL2 and IP-10/CXCL10 directing reactive gliosis (Tanuma et al., 2006). Therefore, it is reasonable to assume that astrocytic activity can be influenced by microglial activation. Although a clear account of this dynamic relationship has yet to be proposed, the astrocyte-microglia interplay may determine the phenotype that

Some findings have implicated astrocytes in chronic microgliosis, with a transition from an initial neuroprotective activity to a later cytotoxic one. TNF-α secretion is crucial for rapid autocrine microglial activation with both neuroprotective and cytotoxic effects, a process that is also fed by TNF-α released by reactive astrocytes (Suzumura et al., 2006). TNF-α actions leading to neuronal death or survival are dose dependent (Bernardino et al., 2008), since it can activate two specific receptors: TNFR1, with an intracellular death domain, and TNFR2, mainly involved in neuroprotection (Fontaine et al., 2002). Consequently, low concentrations of TNF-α would initially induce TNFR2-mediated neuroprotection, whereas a subsequent high concentration of TNF-α would be able to activate TNFR1 both in astrocytes and microglia and contribute to cell injury through the death domain of the

Astroglial S100β is another of the factors that control microglial activity. Astrocytes release S100β constitutively (Van Eldik & Wainwright, 2003) and increase this release upon stimulation by several factors, including TNF-α (Edwards & Robinson, 2006). Under normal conditions, released S100β acts as a neurotrophic factor, countering the stimulatory effect of neurotoxins on (Reali et al., 2005) and stimulating astrocyte glutamate uptake (Tramontina et al., 2006). Released S100β modifies astrocytic, neuronal and microglial activities, depending on the extracellular concentration of the former and the expression of the specific receptor for advanced glycation end-products (RAGE). At micromolar concentrations, S100β upregulates IL-1β and TNF-α expression in activated microglia via RAGE, with the requirement of concurrent activation of NF-κB and AP-1 transcription factors (Bianchi et al., 2010). Furthermore, factors that modulate microglial reactivity, such as Ca2+ concentration,

therapeutic interest in all CNS disorders related to neuroinflammation.

**1.4 Astrocyte-microglia interactions: Who's the bad guy after all?** 

pathologic process (Rodriguez et al., 2009a).

microglial cells adopt during neurodegeneration.

receptor.

Fig. 1. Signalling systems in the microglia-astrocyte-neuron cross-talk. Astrocytes and microglia monitorize neuronal activity by sensing neurotransmitter release. In the same way, microglial cells have receptors to molecules released by astrocytes. Microglia integrate all these signals and release molecules that may modulate neuronal and astroglial activity. During neurodegeneration, changes in physiological parameters may trigger neuronal injury and/or microglial activation (molecules inside a red square) that modify those signal transduction systems. Aβ, amyloid beta; Ach, acetylcholine; Ado, adenosine; BDNF, Brainderived neurotrophic factor; GABA, γ-aminobutyric acid; GDNF, Glial cell-derived neurotrophic factor; Glc, glucose; Glu, glutamate; 5-HT, serotonin; VIP, Vasoactive intestinal peptide; IFNγ, interferon gamma; ROS, reactive oxygen species; Tau, taurine

reactive oxygen species or TNF-α, also modify the RAGE response to S100β (Edwards & Robinson, 2006) in a microglia-astroglia cross-talk that integrates these signaling systems. In contrast, at high concentrations S100β binds the RAGE, which may mediate microglial activation during the course of brain damage (Bianchi et al., 2010). An increased release of S100β during neurodegeneration (Li et al., 2011) will enhance inflammatory cytokine production and potentiate the switch of microglia to chronic cytotoxic activity, feeding the neurotoxic process and leading to neurodegeneration.

Microglia, Calcification and Neurodegenerative Diseases 307

excitotoxic lesions in basal forebrain can modify long-term cortical adaptative responses, and may modulate the expression of glutamate receptor. Some of these effects, such as the decrease in brain-derived neurotrophic factor and the increase in c-fos expression, also

Given these toxic effects, adaptations that act to control glutamatergic neurotransmission and Ca2+ movements in the cell may be potentially protective. Over time, these defenses are developed to act at any time during an excitotoxic event, involve different cellular types such as neurons, astrocytes and microglia, and deal with the cellular and molecular mechanisms of glutamatergic neurotransmission. These mechanisms include defenses that: a) decrease neuronal excitability, b) decrease glutamate accumulation in the synapse, c) limit Ca2+ mobilization in the postsynaptic neuron and protect against calcium-dependent degenerative effects, and d) enhance neuronal energy (Rodriguez et al., 2009b, Sapolsky,

If the compensatory mechanisms are not sufficiently effective, the initial acute neuronal injury due to an increase in [Ca2+]i leads, with time, to a chronic lesion. This secondary excitotoxicity appears in neurons after the massive entrance of Ca2+ and Na+ through ionotropic glutamate receptors, an entrance that is supplemented by Ca2+ release from the endoplamic reticulum following activation of mGluRs. As a result, an excessive [Ca2+]i increment occurs (Verkhratsky, 2007), which activates the mechanisms triggering neuronal death. Ca2+ extrusion and buffering are activated when [Ca2+]i increases (Mattson & Chan,

Calcium homeostasis disturbances are present in all neurodegenerative disorders (Mattson, 2006). Dysregulation of Ca2+ homeostasis alters the rapid and coherent activation of neurons, and is therefore ultimately responsible for many aspects of brain dysfunction and CNS diseases. For example, an increased rate of Ca2+-mediated apoptosis may cause neuronal death in the penumbra of cerebral ischemia, or may underlie the etiology of chronic neurodegenerative disorders such as PD and AD. Calcium precipitation that coincides with microglial activation, amyloid deposits and other ion accumulations in AD

Regulation of intraneuronal Ca2+ movements is a key element to ensure adequate cellular response at all times and produce a physiological effect. Ca2+ participates in most cellular functions, including membrane excitability and secretion, energy production, synaptic transmission, gene regulation and plasticity, cell proliferation and cell death (Arundine & Tymianski, 2003). As a consequence, cell function requires tight control of Ca2+ homeostasis between extra- and intracellular compartments at all times, including the production of sufficient energy from glycolysis to maintain different gradient concentrations such as a 1/10,000 inside-outside cell concentration (Verkhratsky, 2005). Most calcium within cells is sequestered in the mitochondria and the endoplasmic reticulum. Intracellular free calcium concentrations fluctuate widely, from roughly 100 nM to over 1 μM, due to release from cellular stores or influx from extracellular fluid. These fluctuations are integral to the role of calcium, and unless compensated for by some other mechanism, any dysregulation will have severe cellular consequences, the effects of which will be propagated to the surrounding cells, namely, neurons and glial cells (Beck et al., 2004). For example, activation of glutamate receptors by excitatory signals leads to a massive increase in cytoplasm Ca2+ levels, which in turn activates a cascade of events to produce a neuronal response. A return

may thus be a key element of the neurodegenerative process (Ramonet et al., 2006).

**2.2 Neurodegeneration as a result of disturbances in calcium homeostasis** 

2003), with high expenditure of energy through Ca2+-ATPases. (Figure 4).

reflect the molecular alterations present in AD.

2001).

Thus, CNS neurodegeneration involves chronic microgliosis with a putative transition from an initial neuroprotective activity to a later cytotoxic one, with S100β as a key modulator of microglial transition towards a cytotoxic response. This suggests a role for astrocytes in promoting the cytotoxic microglial phenotype through secretion of TNF-α, S100β and other signals (Donato et al., 2009, Suzumura et al., 2006). In this scenario, the interplay between trophic, neuroprotective, inflammatory and cytotoxic functions of both cell types during brain injury will determine the evolution of the neurodegenerative process. Precise control of these processes thus requires a dynamic view of their interactions so as to allow effective development of approaches to neuroprotection.

#### **2. CNS calcification and neurodegeneration**

#### **2.1 Enduring effects of excessive synaptic glutamate**

Glutamate accounts for most of the excitatory synaptic activity in the CNS and has been implicated in learning, memory, synaptic plasticity and neurotrophic activity processes. Glutamate receptors have been classified into three groups: two ionotropic groups -Nmethyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-kainate receptors-; and one group of metabotropic receptors, which are coupled to G proteins. Although non-NMDA receptors are not initially permeable to Ca2+, glutamate release in the synaptic cleft increases post-synaptic and glial membrane permeability, leading to a transient increase in intracellular Ca2+ concentration ([Ca2+]i) (Obrenovitch et al., 2000).

Excessive activation of glutamate receptors can trigger neuronal death through a process characterized by chronic glutamate release and the consequent [Ca2+]i dys-homeostasis in neurons and astrocytes (Arundine & Tymianski, 2004) and formation of [Ca2+]i precipitates, the size and number of which depends on the CNS area involved and CNS maturation (Bernal et al., 2000b, Rodríguez et al., 2001). This process, defined as excitotoxicity, also involves cellular influxes of Na+ and Cl and efflux of K+, with ensuing cell swelling (Chen et al., 1998, Katayama et al., 1995). Because of the complexity and diversity of the processes taking place at the glutamatergic synapse, any disturbance at the pre-synaptic, postsynaptic, or astroglial level may trigger a chronic excitotoxic process. For example, ALS presents a loss of selectivity of ionotropic receptors (Obrenovitch et al., 2000) and deficiencies in glial re-uptake of glutamate (Liévens et al., 2000). These glutamate-related dysfunctions contribute to explaining phenomena such as the aging-associated hypoactivity of NMDA receptors observed in AD (Olney et al., 1997), and the specific AMPA-receptor increment detected in the hippocampus of aged, cognitive-impaired rats (Le Jeune et al., 1996).

One of the consequences of excitotoxic-induced neuronal loss is the alteration of other neurotransmitter systems and neuromodulators. For example, long-term ibotenic-induced lesion in the basal forebrain of rat leads to a loss of cholinergic afferences and to decreased extracellular noradrenaline, glutamate, and taurine (Boatell et al., 1995). This cortical reduction in glutamatergic transmission presents a temporal pattern which, together with the development of Ca2+ precipitates and a decrease in the cholinergic and noradrenergic functions (Saura et al., 1995), mimics the neurochemical modifications described in AD. Similarly, one year after acute lesion, the cortical and hippocampal decrease in brainderived neurotrophic factor, fibroblastic growth factor and glucocorticoid receptor, and the increase in c-fos expression in the septal area were still significant (Boatell et al., 1992). Thus,

Thus, CNS neurodegeneration involves chronic microgliosis with a putative transition from an initial neuroprotective activity to a later cytotoxic one, with S100β as a key modulator of microglial transition towards a cytotoxic response. This suggests a role for astrocytes in promoting the cytotoxic microglial phenotype through secretion of TNF-α, S100β and other signals (Donato et al., 2009, Suzumura et al., 2006). In this scenario, the interplay between trophic, neuroprotective, inflammatory and cytotoxic functions of both cell types during brain injury will determine the evolution of the neurodegenerative process. Precise control of these processes thus requires a dynamic view of their interactions so as to allow effective

Glutamate accounts for most of the excitatory synaptic activity in the CNS and has been implicated in learning, memory, synaptic plasticity and neurotrophic activity processes. Glutamate receptors have been classified into three groups: two ionotropic groups -Nmethyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-kainate receptors-; and one group of metabotropic receptors, which are coupled to G proteins. Although non-NMDA receptors are not initially permeable to Ca2+, glutamate release in the synaptic cleft increases post-synaptic and glial membrane permeability, leading to a transient increase in intracellular Ca2+ concentration ([Ca2+]i) (Obrenovitch et al.,

Excessive activation of glutamate receptors can trigger neuronal death through a process characterized by chronic glutamate release and the consequent [Ca2+]i dys-homeostasis in neurons and astrocytes (Arundine & Tymianski, 2004) and formation of [Ca2+]i precipitates, the size and number of which depends on the CNS area involved and CNS maturation (Bernal et al., 2000b, Rodríguez et al., 2001). This process, defined as excitotoxicity, also

al., 1998, Katayama et al., 1995). Because of the complexity and diversity of the processes taking place at the glutamatergic synapse, any disturbance at the pre-synaptic, postsynaptic, or astroglial level may trigger a chronic excitotoxic process. For example, ALS presents a loss of selectivity of ionotropic receptors (Obrenovitch et al., 2000) and deficiencies in glial re-uptake of glutamate (Liévens et al., 2000). These glutamate-related dysfunctions contribute to explaining phenomena such as the aging-associated hypoactivity of NMDA receptors observed in AD (Olney et al., 1997), and the specific AMPA-receptor increment detected in the hippocampus of aged, cognitive-impaired rats (Le Jeune et al.,

One of the consequences of excitotoxic-induced neuronal loss is the alteration of other neurotransmitter systems and neuromodulators. For example, long-term ibotenic-induced lesion in the basal forebrain of rat leads to a loss of cholinergic afferences and to decreased extracellular noradrenaline, glutamate, and taurine (Boatell et al., 1995). This cortical reduction in glutamatergic transmission presents a temporal pattern which, together with the development of Ca2+ precipitates and a decrease in the cholinergic and noradrenergic functions (Saura et al., 1995), mimics the neurochemical modifications described in AD. Similarly, one year after acute lesion, the cortical and hippocampal decrease in brainderived neurotrophic factor, fibroblastic growth factor and glucocorticoid receptor, and the increase in c-fos expression in the septal area were still significant (Boatell et al., 1992). Thus,

and efflux of K+, with ensuing cell swelling (Chen et

development of approaches to neuroprotection.

involves cellular influxes of Na+ and Cl-

2000).

1996).

**2. CNS calcification and neurodegeneration** 

**2.1 Enduring effects of excessive synaptic glutamate** 

excitotoxic lesions in basal forebrain can modify long-term cortical adaptative responses, and may modulate the expression of glutamate receptor. Some of these effects, such as the decrease in brain-derived neurotrophic factor and the increase in c-fos expression, also reflect the molecular alterations present in AD.

Given these toxic effects, adaptations that act to control glutamatergic neurotransmission and Ca2+ movements in the cell may be potentially protective. Over time, these defenses are developed to act at any time during an excitotoxic event, involve different cellular types such as neurons, astrocytes and microglia, and deal with the cellular and molecular mechanisms of glutamatergic neurotransmission. These mechanisms include defenses that: a) decrease neuronal excitability, b) decrease glutamate accumulation in the synapse, c) limit Ca2+ mobilization in the postsynaptic neuron and protect against calcium-dependent degenerative effects, and d) enhance neuronal energy (Rodriguez et al., 2009b, Sapolsky, 2001).

If the compensatory mechanisms are not sufficiently effective, the initial acute neuronal injury due to an increase in [Ca2+]i leads, with time, to a chronic lesion. This secondary excitotoxicity appears in neurons after the massive entrance of Ca2+ and Na+ through ionotropic glutamate receptors, an entrance that is supplemented by Ca2+ release from the endoplamic reticulum following activation of mGluRs. As a result, an excessive [Ca2+]i increment occurs (Verkhratsky, 2007), which activates the mechanisms triggering neuronal death. Ca2+ extrusion and buffering are activated when [Ca2+]i increases (Mattson & Chan, 2003), with high expenditure of energy through Ca2+-ATPases. (Figure 4).

Calcium homeostasis disturbances are present in all neurodegenerative disorders (Mattson, 2006). Dysregulation of Ca2+ homeostasis alters the rapid and coherent activation of neurons, and is therefore ultimately responsible for many aspects of brain dysfunction and CNS diseases. For example, an increased rate of Ca2+-mediated apoptosis may cause neuronal death in the penumbra of cerebral ischemia, or may underlie the etiology of chronic neurodegenerative disorders such as PD and AD. Calcium precipitation that coincides with microglial activation, amyloid deposits and other ion accumulations in AD may thus be a key element of the neurodegenerative process (Ramonet et al., 2006).

#### **2.2 Neurodegeneration as a result of disturbances in calcium homeostasis**

Regulation of intraneuronal Ca2+ movements is a key element to ensure adequate cellular response at all times and produce a physiological effect. Ca2+ participates in most cellular functions, including membrane excitability and secretion, energy production, synaptic transmission, gene regulation and plasticity, cell proliferation and cell death (Arundine & Tymianski, 2003). As a consequence, cell function requires tight control of Ca2+ homeostasis between extra- and intracellular compartments at all times, including the production of sufficient energy from glycolysis to maintain different gradient concentrations such as a 1/10,000 inside-outside cell concentration (Verkhratsky, 2005). Most calcium within cells is sequestered in the mitochondria and the endoplasmic reticulum. Intracellular free calcium concentrations fluctuate widely, from roughly 100 nM to over 1 μM, due to release from cellular stores or influx from extracellular fluid. These fluctuations are integral to the role of calcium, and unless compensated for by some other mechanism, any dysregulation will have severe cellular consequences, the effects of which will be propagated to the surrounding cells, namely, neurons and glial cells (Beck et al., 2004). For example, activation of glutamate receptors by excitatory signals leads to a massive increase in cytoplasm Ca2+ levels, which in turn activates a cascade of events to produce a neuronal response. A return

Microglia, Calcification and Neurodegenerative Diseases 309

regulating neuroinflammation, as has been recently reported (Haider et al., 2011). In all of these diseases, dysregulation of Ca2+ homeostasis has been considered a pathophysiological factor linked to neuronal degeneration, and the formation of intracellular Ca2+ deposits with different characteristics as regards size and distribution - reflecting differential CNS area vulnerability - has frequently been reported (Hashimoto et al., 2003, Ramonet et al.,

In immature human CNS, Ca2+-mediated excitotoxicity is associated with a calcification process that directly correlates with neuronal loss and the extent of injury. Revealed by Alizarin red staining and appearing in TEM and X-ray microanalyses of animal neurodegeneration models, small and large intracellular Ca2+ precipitates indicate the formation of a paracrystalline structure of hydroxyapatites localized within neurons and

Glutamate analog microinjection in rat CNS leads to an intracellular Ca2+ precipitation similar to brain calcification in humans (Ramonet et al., 2006, 2002). As these Ca2+ deposits can be observed in several areas of rat brain after microinjection of different excitotoxins (Bernal et al., 2000b, Rodriguez et al., 2000, Saura et al., 1995), their formation does not depend on the glutamate receptor subtype initially stimulated. However, their size, number and distribution vary with both the activated receptor and the CNS area. For example, sensitivity to AMPA-induced calcification decreased from the globus pallidus, cerebral cortex, hippocampus, medial septum, to retina (Rodriguez et al., 2000). Moreover, in medial septum, the degeneration associated with microinjection of ibotenic and quisqualic acids was characterized by significant atrophy and no calcification (Mahy et al., 1996, Saura et al., 1995). In similar conditions, AMPA microinjection resulted in similar atrophy and Ca2+

Ca2+ deposits do not occur in all cells that degenerate in response to excitotoxins. For example, in the basal forebrain and medial septum, the calcification observed in GABAergic cells was not detected in cholinergic neurons. The former, together with astrocytes, seem to participate actively in the calcification process (Mahy et al., 1999). Differences in the neuronal phenotype of Ca2+ buffering and extrusion systems, specific energy needs, expression of the glutamate subtype receptor and different astroglial populations, should explain this variability. The ultrastructural study of tissue affected by excitotoxicity has also contributed to our understanding of calcification. Ca2+ deposits within hypertrophied astrocytes have been characterized in the basal forebrain and hippocampus which ranged from 0.5 to 10 μm in diameter and were formed by numerous, small, needle-shaped crystals associated with cellular organelles, such as microtubules, cisternae, vesicles or mitochondria, with no signs of neurodegeneration (figure 2). Larger inclusions were surrounded by reactive microglia, a finding that was also observed in tissue after specific localization by in vitro autoradiography (Bernal et al., 2000b, Petegnief et al., 1999). X-ray microanalysis has shown an electron-diffraction ring pattern characteristic of a crystalline structure similar to apatites (Kim, 1995), and a Ca/P ratio of 1.3±0.2 of cytoplasmic deposits (Figure 2). This ratio, lower than the theoretical apatite value of 1.67, is also typical of biological crystals, which do not present an ideal organization (Rodriguez et al., 2000). As biological hydroxyapatites, these deposits are similar to those observed in several human

2006).

**2.3 CNS calcification** 

astrocytes (figure 2).

deposits at the injection site (Rodriguez et al., 2009a).

peripheral nervous system tissues (Kodaka et al., 1994).

to basal activity requires a considerable expenditure of energy to bring Ca2+ back to initial levels. Any failure in these multiple, coordinated steps will alter neuronal signalling and interfere with neuronal network functions (Rodriguez et al., 2009b).

Reduction of [Ca2+]i involves a high mitochondrial intake of Ca2+ that may lead to loss of the mitochondrial membrane potential and the production of reactive oxygen species, thereby decreasing cellular respiratory capacity and ATP formation from ADP and Pi (Chan et al., 2007a). As a result, there is an acceleration of anaerobic glycolysis with a net lactate production that contributes to tissue acidification and progression of damage. Disturbances in Ca2+ homeostasis in astrocytes reduce neuronal support, in particular through alteration of the glutamate/glutamine cycle and a reduction of glucose delivery to neurons (Pellerin et al., 2007, Ramonet et al., 2004) (Figure 4). Consequently, astrocyte dysfunction can lead to increased synaptic glutamate levels and glutamate receptor overactivation, combined with reduced neuronal energy, resulting in neuronal damage. Under these conditions of high Ca2+ and Pi and low ATP, formation of hydroxyapatite precipitates to reduce Ca2+ cytoplasm activity at low energy costs can occur in neurons and astrocytes (Rodriguez et al., 2000). This new step in calcium homeostasis temporarily helps the cell to resist excessive stimulatory signals and return to basal activity by dissolving paracrystal elements. However, in most cases, the hydroxyapatite crystals show progressive growth and participate in cell death and CNS damage.

Depending on the importance of the damage, microglial activation may take place, initially expressing neuroprotective signals to help avoid further neuronal death, but then changing progressively to an inflammatory phenotype (Graeber, 2010). Glycolysis, highly stimulated in microglia to ensure and maintain their activated stage, reduces neuronal glucose and oxygen availability. Consequently, microglial participation in neuronal death includes not only neuroinflammation, but also reduction in neuronal energy availability (Allaman et al., 2011). If, as asserted by Gyuri Buzsaki in Rhythms of the Brain (Buzsáki, 2006), "Brains are foretelling devices and their predictive powers emerge from the various rhythms they perpetually generate", any significant alteration of the neuronal rhythms caused by an abnormally high Ca2+ concentration in neurons or glia should alter the brain's ability to pause, adapt and learn, and can lead to disease. This is the case of neurodegenerative diseases, which exhibit diverse clinical and neuropathological phenotypes but share the common feature of progressively reduced cell function and survival within the nervous system, leading to neurological disability and often death. As such, several different CNS disorders can be induced following the same injury, due to the multi-directional interactions between the neurons, glial cells, extracellular matrix, endothelia and host immune cells that regulate tissue homeostasis and orchestrate neuroinflammation and degeneration. Furthermore, the characteristics of each neuronal population and network, the different gliopathic changes occurring between CNS areas, the various microglia phenotypes and the abundance and distribution of glutamate receptor subtypes and of Ca2+-binding proteins, all participate directly in the properties of the neurodegenerative parameters (Graeber & Streit, 2010, Rodriguez et al., 2004, Rodriguez et al., 2009a) that will determine the dynamics and progression of the disease in the specific affected areas. For example, PD and AD are both regarded as diseases that are initiated by neuronal death to which the immune system responds, as evidenced by astroglial and microglial activation with pathogenic consequences (Agostinho et al., 2010, Halliday & Stevens, 2011). Similarly, multiple sclerosis is typically considered a neuroinflammatory disorder, but one in which neuronal injury plays an active role in regulating neuroinflammation, as has been recently reported (Haider et al., 2011). In all of these diseases, dysregulation of Ca2+ homeostasis has been considered a pathophysiological factor linked to neuronal degeneration, and the formation of intracellular Ca2+ deposits with different characteristics as regards size and distribution - reflecting differential CNS area vulnerability - has frequently been reported (Hashimoto et al., 2003, Ramonet et al., 2006).

#### **2.3 CNS calcification**

308 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

to basal activity requires a considerable expenditure of energy to bring Ca2+ back to initial levels. Any failure in these multiple, coordinated steps will alter neuronal signalling and

Reduction of [Ca2+]i involves a high mitochondrial intake of Ca2+ that may lead to loss of the mitochondrial membrane potential and the production of reactive oxygen species, thereby decreasing cellular respiratory capacity and ATP formation from ADP and Pi (Chan et al., 2007a). As a result, there is an acceleration of anaerobic glycolysis with a net lactate production that contributes to tissue acidification and progression of damage. Disturbances in Ca2+ homeostasis in astrocytes reduce neuronal support, in particular through alteration of the glutamate/glutamine cycle and a reduction of glucose delivery to neurons (Pellerin et al., 2007, Ramonet et al., 2004) (Figure 4). Consequently, astrocyte dysfunction can lead to increased synaptic glutamate levels and glutamate receptor overactivation, combined with reduced neuronal energy, resulting in neuronal damage. Under these conditions of high Ca2+ and Pi and low ATP, formation of hydroxyapatite precipitates to reduce Ca2+ cytoplasm activity at low energy costs can occur in neurons and astrocytes (Rodriguez et al., 2000). This new step in calcium homeostasis temporarily helps the cell to resist excessive stimulatory signals and return to basal activity by dissolving paracrystal elements. However, in most cases, the hydroxyapatite crystals show progressive growth and

Depending on the importance of the damage, microglial activation may take place, initially expressing neuroprotective signals to help avoid further neuronal death, but then changing progressively to an inflammatory phenotype (Graeber, 2010). Glycolysis, highly stimulated in microglia to ensure and maintain their activated stage, reduces neuronal glucose and oxygen availability. Consequently, microglial participation in neuronal death includes not only neuroinflammation, but also reduction in neuronal energy availability (Allaman et al., 2011). If, as asserted by Gyuri Buzsaki in Rhythms of the Brain (Buzsáki, 2006), "Brains are foretelling devices and their predictive powers emerge from the various rhythms they perpetually generate", any significant alteration of the neuronal rhythms caused by an abnormally high Ca2+ concentration in neurons or glia should alter the brain's ability to pause, adapt and learn, and can lead to disease. This is the case of neurodegenerative diseases, which exhibit diverse clinical and neuropathological phenotypes but share the common feature of progressively reduced cell function and survival within the nervous system, leading to neurological disability and often death. As such, several different CNS disorders can be induced following the same injury, due to the multi-directional interactions between the neurons, glial cells, extracellular matrix, endothelia and host immune cells that regulate tissue homeostasis and orchestrate neuroinflammation and degeneration. Furthermore, the characteristics of each neuronal population and network, the different gliopathic changes occurring between CNS areas, the various microglia phenotypes and the abundance and distribution of glutamate receptor subtypes and of Ca2+-binding proteins, all participate directly in the properties of the neurodegenerative parameters (Graeber & Streit, 2010, Rodriguez et al., 2004, Rodriguez et al., 2009a) that will determine the dynamics and progression of the disease in the specific affected areas. For example, PD and AD are both regarded as diseases that are initiated by neuronal death to which the immune system responds, as evidenced by astroglial and microglial activation with pathogenic consequences (Agostinho et al., 2010, Halliday & Stevens, 2011). Similarly, multiple sclerosis is typically considered a neuroinflammatory disorder, but one in which neuronal injury plays an active role in

interfere with neuronal network functions (Rodriguez et al., 2009b).

participate in cell death and CNS damage.

In immature human CNS, Ca2+-mediated excitotoxicity is associated with a calcification process that directly correlates with neuronal loss and the extent of injury. Revealed by Alizarin red staining and appearing in TEM and X-ray microanalyses of animal neurodegeneration models, small and large intracellular Ca2+ precipitates indicate the formation of a paracrystalline structure of hydroxyapatites localized within neurons and astrocytes (figure 2).

Glutamate analog microinjection in rat CNS leads to an intracellular Ca2+ precipitation similar to brain calcification in humans (Ramonet et al., 2006, 2002). As these Ca2+ deposits can be observed in several areas of rat brain after microinjection of different excitotoxins (Bernal et al., 2000b, Rodriguez et al., 2000, Saura et al., 1995), their formation does not depend on the glutamate receptor subtype initially stimulated. However, their size, number and distribution vary with both the activated receptor and the CNS area. For example, sensitivity to AMPA-induced calcification decreased from the globus pallidus, cerebral cortex, hippocampus, medial septum, to retina (Rodriguez et al., 2000). Moreover, in medial septum, the degeneration associated with microinjection of ibotenic and quisqualic acids was characterized by significant atrophy and no calcification (Mahy et al., 1996, Saura et al., 1995). In similar conditions, AMPA microinjection resulted in similar atrophy and Ca2+ deposits at the injection site (Rodriguez et al., 2009a).

Ca2+ deposits do not occur in all cells that degenerate in response to excitotoxins. For example, in the basal forebrain and medial septum, the calcification observed in GABAergic cells was not detected in cholinergic neurons. The former, together with astrocytes, seem to participate actively in the calcification process (Mahy et al., 1999). Differences in the neuronal phenotype of Ca2+ buffering and extrusion systems, specific energy needs, expression of the glutamate subtype receptor and different astroglial populations, should explain this variability. The ultrastructural study of tissue affected by excitotoxicity has also contributed to our understanding of calcification. Ca2+ deposits within hypertrophied astrocytes have been characterized in the basal forebrain and hippocampus which ranged from 0.5 to 10 μm in diameter and were formed by numerous, small, needle-shaped crystals associated with cellular organelles, such as microtubules, cisternae, vesicles or mitochondria, with no signs of neurodegeneration (figure 2). Larger inclusions were surrounded by reactive microglia, a finding that was also observed in tissue after specific localization by in vitro autoradiography (Bernal et al., 2000b, Petegnief et al., 1999). X-ray microanalysis has shown an electron-diffraction ring pattern characteristic of a crystalline structure similar to apatites (Kim, 1995), and a Ca/P ratio of 1.3±0.2 of cytoplasmic deposits (Figure 2). This ratio, lower than the theoretical apatite value of 1.67, is also typical of biological crystals, which do not present an ideal organization (Rodriguez et al., 2000). As biological hydroxyapatites, these deposits are similar to those observed in several human peripheral nervous system tissues (Kodaka et al., 1994).

Microglia, Calcification and Neurodegenerative Diseases 311

Experimental models of bone formation (i.e. hydroxyapatite formation in vitro) (Andre-Frei et al., 2000) have shown that rather than Ca2+, a minimal amount of phosphorus, as inorganic phosphate, is crucial for crystal nucleation in a collagen matrix. Similarly, organic phosphate residues of the phosphoproteins also play a direct and significant role in the process of in vitro nucleation of apatite by bone collagen, whereas collagen itself does not promote the precipitation of Ca2+ or phosphate (Andre-Frei et al., 2000). Therefore, excitotoxicity-induced calcification in the rat brain depends on an increase in intracellular inorganic phosphate (i.e. ATP depletion) and, most importantly, on the degree of protein phosphorylation. Thus, the Ca2+-binding-protein-dependent kinases and activity of the

In aqueous solutions, hydroxyapatite crystallization takes place in two sequential steps (Barat et al., 2011): in the first, crystal nucleation occurs spontaneously with subsequent growth to some nanometers, when phosphate and Ca2+ ions reach a certain concentration; in the second step, an accretion process of these nanocrystals on a proteinic net takes place until reaching a maximum size of 20 micrometers. While the first process facilitates resolubilization of the crystal, the second produces a stable precipitate and requires a catalytic agent. These two mechanisms may help explain the size differences we found between several areas of the CNS. For example, the large insoluble Ca2+ precipitates (mean size 20 μm) found after AMPA microinjection in globus pallidus (Petegnief et al., 1999) fit well with the second step theory, whereas the small deposits (mean size lower than 3 μm) obtained in hippocampus after the same procedure (Rodriguez et al., 2004) may reflect the lack of a catalytic agent for accretion, or an equilibrium between crystal formation and solubilization. Furthermore, blockade of glial glutamate uptake in rat striatum (Liévens et al., 2000) produced a spherical lesion with a central necrotic core surrounded by a penumbra zone similar to that caused by focal ischemia. Three days after treatment, an astroglial reaction and small Ca2+ deposits (mean diameter < 1 μm) were observed in the penumbra area. Eleven days later, these deposits had disappeared, the penumbra zone had recovered from injury and the necrotic area was partially repaired (Liévens et al., 2000). In this scenario, compensatory mechanisms helped normalize Ca2+ homeostasis and avoid further neuronal death. The tissue recovered the ability to use extrusion mechanisms, and re-solubilization of

When Ca2+ deposits are localized extracellularly due to cell death, a microglial reaction is activated for their phagocytic removal. This microglial reaction also participates in the neuronal death seen in chronic neurodegenerative processes, but is dissociated from astrogliosis. In some animal models of neurodegeneration, a recovery has been observed associated with the disappearance of Ca2+ deposits. In other excitotoxic rodent models, the on-going neurodegenerative process increased with time and the Ca2+ deposits remained

In the neonate mammalian brain, considered more resistant to hypoxia-ischemia than adult CNS, dysregulation of Ca2+ homeostasis together with lactate acidosis are considered the main factors causing neuronal death. As premature-neonates are more resistant to hypoxiaischemia than term neonates, we studied the relationship between differences in human brain vulnerability to hypoxia-ischemia during the perinatal period and brain calcification in the basal ganglia, cerebral cortex, and hippocampus (Rodríguez et al., 2001). The number

neurotrophic factor ultimately determine calcification.

Ca2+ precipitates took place.

present, associated with microglial reaction.

**2.4 The calcification process and ageing** 

Fig. 2. Characterization of calcium deposits induced by ibotenic acid in the rat brain. Microphotographs of a) Nissl stained section of a rat hippocampus 15 days after the injection and b) Alizarin red stained section of the same hippocampus showing calcium deposits associated with the lesion. c-d) calcium deposits showed different sizes in the rat globus pallidus 2 months after injection. e) Isolectin B4 histochemistry (brown staining) counterstained with alizarin red showing the microglial reaction (arrowhead) associated with calcium deposits. f) Hypertrophic astrocyte with an intracytoplasmic calcium deposit by TEM. g) Detail of the ultrastructure of calcium deposit within an astrocyte. Note the normal appearance of the surrounding mitochondria (arrowhead). X-ray image h) and spectrum analysis i) of one calcium deposit in a non-osmificated sample with a calculated Ca/P ratio of 1.3. False colour X-ray image mapping j) and distribution plots k) of Ca and P of the same deposit. Bars; a-b, 1 mm; c, 100 μm; d-e, 20 μm; f, 0.6 μm, g, 0.2 μm; h, 10 μm

Fig. 2. Characterization of calcium deposits induced by ibotenic acid in the rat brain. Microphotographs of a) Nissl stained section of a rat hippocampus 15 days after the injection and b) Alizarin red stained section of the same hippocampus showing calcium deposits associated with the lesion. c-d) calcium deposits showed different sizes in the rat globus pallidus 2 months after injection. e) Isolectin B4 histochemistry (brown staining) counterstained with alizarin red showing the microglial reaction (arrowhead) associated with calcium deposits. f) Hypertrophic astrocyte with an intracytoplasmic calcium deposit by TEM. g) Detail of the ultrastructure of calcium deposit within an astrocyte. Note the normal appearance of the surrounding mitochondria (arrowhead). X-ray image h) and spectrum analysis i) of one calcium deposit in a non-osmificated sample with a calculated Ca/P ratio of 1.3. False colour X-ray image mapping j) and distribution plots k) of Ca and P of the same deposit. Bars; a-b, 1 mm; c, 100 μm; d-e, 20 μm; f, 0.6 μm, g, 0.2 μm; h, 10 μm

Experimental models of bone formation (i.e. hydroxyapatite formation in vitro) (Andre-Frei et al., 2000) have shown that rather than Ca2+, a minimal amount of phosphorus, as inorganic phosphate, is crucial for crystal nucleation in a collagen matrix. Similarly, organic phosphate residues of the phosphoproteins also play a direct and significant role in the process of in vitro nucleation of apatite by bone collagen, whereas collagen itself does not promote the precipitation of Ca2+ or phosphate (Andre-Frei et al., 2000). Therefore, excitotoxicity-induced calcification in the rat brain depends on an increase in intracellular inorganic phosphate (i.e. ATP depletion) and, most importantly, on the degree of protein phosphorylation. Thus, the Ca2+-binding-protein-dependent kinases and activity of the neurotrophic factor ultimately determine calcification.

In aqueous solutions, hydroxyapatite crystallization takes place in two sequential steps (Barat et al., 2011): in the first, crystal nucleation occurs spontaneously with subsequent growth to some nanometers, when phosphate and Ca2+ ions reach a certain concentration; in the second step, an accretion process of these nanocrystals on a proteinic net takes place until reaching a maximum size of 20 micrometers. While the first process facilitates resolubilization of the crystal, the second produces a stable precipitate and requires a catalytic agent. These two mechanisms may help explain the size differences we found between several areas of the CNS. For example, the large insoluble Ca2+ precipitates (mean size 20 μm) found after AMPA microinjection in globus pallidus (Petegnief et al., 1999) fit well with the second step theory, whereas the small deposits (mean size lower than 3 μm) obtained in hippocampus after the same procedure (Rodriguez et al., 2004) may reflect the lack of a catalytic agent for accretion, or an equilibrium between crystal formation and solubilization. Furthermore, blockade of glial glutamate uptake in rat striatum (Liévens et al., 2000) produced a spherical lesion with a central necrotic core surrounded by a penumbra zone similar to that caused by focal ischemia. Three days after treatment, an astroglial reaction and small Ca2+ deposits (mean diameter < 1 μm) were observed in the penumbra area. Eleven days later, these deposits had disappeared, the penumbra zone had recovered from injury and the necrotic area was partially repaired (Liévens et al., 2000). In this scenario, compensatory mechanisms helped normalize Ca2+ homeostasis and avoid further neuronal death. The tissue recovered the ability to use extrusion mechanisms, and re-solubilization of Ca2+ precipitates took place.

When Ca2+ deposits are localized extracellularly due to cell death, a microglial reaction is activated for their phagocytic removal. This microglial reaction also participates in the neuronal death seen in chronic neurodegenerative processes, but is dissociated from astrogliosis. In some animal models of neurodegeneration, a recovery has been observed associated with the disappearance of Ca2+ deposits. In other excitotoxic rodent models, the on-going neurodegenerative process increased with time and the Ca2+ deposits remained present, associated with microglial reaction.

#### **2.4 The calcification process and ageing**

In the neonate mammalian brain, considered more resistant to hypoxia-ischemia than adult CNS, dysregulation of Ca2+ homeostasis together with lactate acidosis are considered the main factors causing neuronal death. As premature-neonates are more resistant to hypoxiaischemia than term neonates, we studied the relationship between differences in human brain vulnerability to hypoxia-ischemia during the perinatal period and brain calcification in the basal ganglia, cerebral cortex, and hippocampus (Rodríguez et al., 2001). The number

Microglia, Calcification and Neurodegenerative Diseases 313

Fig. 3. Calcification depends on the brain area but also on the glutamate receptor involved. a) AMPA induces small calcium deposits in the rat hippocampus, affecting mainly the CA1 radiatum and lacunosum moleculare subfields. b) AMPA microinjection in the globus pallidus induces larger calcium deposits. c) NMDA microinjection in the hippocampus induces the formation of large calcium deposits located mainly in the pyramidal CA1 and granual dentate gyrus. d) The plots show comparison of the AMPA dose-response study in the hippocampus and the globus pallidus. e-f) Correlation plots of the hypoxia ischemiainduced calcification in the basal ganglia e), cerebral cortex f) and hippocampus g) of premature and term neonates. Calcification was calculated in a representative area (1 mm2) and the lifespan corresponds to the time of injury in days. k, days to reach half of the

Thus, the correlation between the calcification process, neuronal loss and the extent of CNS injury disappears with aging, but differences in CNS area vulnerability to calcification are maintained. The components that underlie the specific vulnerability of each brain area are thus already expressed in human neonates. The permanent area differences are associated with significant variations in the response to specific Ca2+ channel blockers such as nimodipine and TMB-8 (Bernal et al., 2009, Petegnief et al., 2004), and illustrate the functional diversity of each area and the difficulty encountered in ensuring the efficacy of

maximal calcified area. Bars, 300 μm

and size of the observed non-arteriosclerotic calcifications were area-specific and increased in term neonates (Figure 3). The basal ganglia presented the highest degree of calcification and the hippocampus the lowest, mainly in the CA1 subfield. In all cases, neuronal damage was associated with astroglial reaction and Ca2+ precipitates, with microglial reaction absent in the hippocampus. These data are consistent with those obtained following long-term excitotoxic lesions in adult rat brain and support the involvement of excitotoxic processes in hypoxia-ischemia damage.

A comparison between lifespan and degree of calcification (Figure 3) demonstrated that in all cases, highest calcification occurred within two months of hypoxia-ischemia, and that semi-calcification time was very short (less than 10 days). Independent of subjective measurements, this last parameter suggests that calcification depends on the degree of brain differentiation and initial cerebral injury, but not on the time-course of the lesion. Moreover, the mechanisms leading to Ca2+ precipitation seem to be similar for all brain areas. If this is true, neurons of each CNS structure degenerate through a common mechanism, which is linked to disturbances in Ca2+ homeostasis. As each area of the brain participates in specific physiological functions, the resultant pathology will depend on the specific neuronal death of the area affected.

Aging increases neuronal vulnerability to toxic compounds, including drugs that impair energy metabolism and induce secondary excitotoxic processes (Brouillet et al., 1993). However, a decreased susceptibility of aged rats to excitotoxins such as quinolinic or kainic acids has been reported (Kesslak et al., 1995). AMPA-induced Ca2+ deposits in rat hippocampus are age-dependent, since young rats (3 months old) present greater areas of calcification than middle-aged ones (15 months old) (Bernal et al., 2000a). In this study, glial reaction, γ-aminobutiric acid (GABA)-uptake activity and immunostaining of Ca2+ binding proteins showed the same response. Therefore, the vulnerability of hippocampal neurons to AMPA-induced neurodegeneration decreases with age between 3 and 15 months. Similar results have been found in other brain areas, such as the striatum and the nucleus basalis magnocellularis. This reduced vulnerability may be related to several factors: for example, age-associated variations in the relative abundance of glutamate receptors and pre-synaptic alterations of glutamate release may explain, at least in part, an increased resistance to excitotoxicity in the hippocampus (Mullany et al., 1996, Nicolle et al., 1996).

This effect is compatible with the increased vulnerability to excitotoxicity observed in the oldest animals (Brouillet et al., 1993), since some of the factors responsible for injury resistance may follow a biphasic pattern, with a progressive increase until reaching maturity followed by a subsequent decrease (Coleman et al., 1990). Many authors have also described biphasic variations in several parameters during aging, with an opposite tendency before and after middle age (Villa et al., 1994). We observed a biphasic variation in monoamine oxidase B (MAO-B) during aging in most human brain areas: up to the age of 50-60 years old, MAO-B levels remain constant, but start to increase thereafter (Saura et al., 1997). This finding may be due to the presence of MAO-B rich reactive astrocytes in response to neuronal degeneration. A similar increase in plaque-associated astrocytes has been found in patients with AD (Saura et al., 1994). As MAO-B activity is associated with reactive oxygen species production, astrocytes may contribute to the age-associated decline of neurological functions. The evidence that an increase in AMPA receptor correlates negatively with MAO-B in age-associated learning-impaired rats also suggests that a gliopathic reaction may be involved in neuronal dysfunction (Andrés et al., 2000).

and size of the observed non-arteriosclerotic calcifications were area-specific and increased in term neonates (Figure 3). The basal ganglia presented the highest degree of calcification and the hippocampus the lowest, mainly in the CA1 subfield. In all cases, neuronal damage was associated with astroglial reaction and Ca2+ precipitates, with microglial reaction absent in the hippocampus. These data are consistent with those obtained following long-term excitotoxic lesions in adult rat brain and support the involvement of excitotoxic processes in

A comparison between lifespan and degree of calcification (Figure 3) demonstrated that in all cases, highest calcification occurred within two months of hypoxia-ischemia, and that semi-calcification time was very short (less than 10 days). Independent of subjective measurements, this last parameter suggests that calcification depends on the degree of brain differentiation and initial cerebral injury, but not on the time-course of the lesion. Moreover, the mechanisms leading to Ca2+ precipitation seem to be similar for all brain areas. If this is true, neurons of each CNS structure degenerate through a common mechanism, which is linked to disturbances in Ca2+ homeostasis. As each area of the brain participates in specific physiological functions, the resultant pathology will depend on the specific neuronal death

Aging increases neuronal vulnerability to toxic compounds, including drugs that impair energy metabolism and induce secondary excitotoxic processes (Brouillet et al., 1993). However, a decreased susceptibility of aged rats to excitotoxins such as quinolinic or kainic acids has been reported (Kesslak et al., 1995). AMPA-induced Ca2+ deposits in rat hippocampus are age-dependent, since young rats (3 months old) present greater areas of calcification than middle-aged ones (15 months old) (Bernal et al., 2000a). In this study, glial reaction, γ-aminobutiric acid (GABA)-uptake activity and immunostaining of Ca2+ binding proteins showed the same response. Therefore, the vulnerability of hippocampal neurons to AMPA-induced neurodegeneration decreases with age between 3 and 15 months. Similar results have been found in other brain areas, such as the striatum and the nucleus basalis magnocellularis. This reduced vulnerability may be related to several factors: for example, age-associated variations in the relative abundance of glutamate receptors and pre-synaptic alterations of glutamate release may explain, at least in part, an increased resistance to

This effect is compatible with the increased vulnerability to excitotoxicity observed in the oldest animals (Brouillet et al., 1993), since some of the factors responsible for injury resistance may follow a biphasic pattern, with a progressive increase until reaching maturity followed by a subsequent decrease (Coleman et al., 1990). Many authors have also described biphasic variations in several parameters during aging, with an opposite tendency before and after middle age (Villa et al., 1994). We observed a biphasic variation in monoamine oxidase B (MAO-B) during aging in most human brain areas: up to the age of 50-60 years old, MAO-B levels remain constant, but start to increase thereafter (Saura et al., 1997). This finding may be due to the presence of MAO-B rich reactive astrocytes in response to neuronal degeneration. A similar increase in plaque-associated astrocytes has been found in patients with AD (Saura et al., 1994). As MAO-B activity is associated with reactive oxygen species production, astrocytes may contribute to the age-associated decline of neurological functions. The evidence that an increase in AMPA receptor correlates negatively with MAO-B in age-associated learning-impaired rats also suggests that a gliopathic reaction may be

excitotoxicity in the hippocampus (Mullany et al., 1996, Nicolle et al., 1996).

involved in neuronal dysfunction (Andrés et al., 2000).

hypoxia-ischemia damage.

of the area affected.

Fig. 3. Calcification depends on the brain area but also on the glutamate receptor involved. a) AMPA induces small calcium deposits in the rat hippocampus, affecting mainly the CA1 radiatum and lacunosum moleculare subfields. b) AMPA microinjection in the globus pallidus induces larger calcium deposits. c) NMDA microinjection in the hippocampus induces the formation of large calcium deposits located mainly in the pyramidal CA1 and granual dentate gyrus. d) The plots show comparison of the AMPA dose-response study in the hippocampus and the globus pallidus. e-f) Correlation plots of the hypoxia ischemiainduced calcification in the basal ganglia e), cerebral cortex f) and hippocampus g) of premature and term neonates. Calcification was calculated in a representative area (1 mm2) and the lifespan corresponds to the time of injury in days. k, days to reach half of the maximal calcified area. Bars, 300 μm

Thus, the correlation between the calcification process, neuronal loss and the extent of CNS injury disappears with aging, but differences in CNS area vulnerability to calcification are maintained. The components that underlie the specific vulnerability of each brain area are thus already expressed in human neonates. The permanent area differences are associated with significant variations in the response to specific Ca2+ channel blockers such as nimodipine and TMB-8 (Bernal et al., 2009, Petegnief et al., 2004), and illustrate the functional diversity of each area and the difficulty encountered in ensuring the efficacy of

Microglia, Calcification and Neurodegenerative Diseases 315

reduction associated with increased lactate concentration facilitates solubility of Ca2+ and the formation of H2PO4-, HPO42- and PO43- ions from inorganic phosphate (Rodriguez et al.,

affinity, apatite nucleation may occur, with the subsequent growth of crystalline formations together with neurodegeneration. In this case, calcification of each lesioned area will also depend on phosphate availability and the differential capacity of glial cells to release lactate

Wide variations have been described in the extent of calcification in pathological cases (Ramonet et al., 2002, Rodríguez et al., 2001) and animal species (Ramonet et al., 2006) However, the homogeneous morphology of these deposits suggests common synaptic processes (Ramonet et al., 2006) where variability depends on cellular type (astrocyte or neuron), glutamatergic activity, and energy availability (Figure 4). These factors modify Ca2+ homeostasis and may trigger cellular calcification through a common mechanism (Ramonet et al., 2006). Thus, hydroxyapatite formation, with the subsequent reduction of free Ca2+ ions, may take place as an alternative homeostatic step to reduce excitotoxicity (Ramonet et al., 2006, Rodriguez et al., 2000), and a number of findings lend support to this interpretation. For example, it has been observed that mitochondria close to Ca2+ deposits appear normal at electron microscopy level (Mahy et al., 1999, Rodriguez et al., 2000), despite

Fig. 4. Excitotoxicity modifies cell calcium homeostasis in the brain. Drawing of the excitotoxic process induced by glutamate, with the intercellular precipitation of calcium as part of the calcium homeostasis. The metabolic pathway of lactate with the communication between endothelial, astroglial, microglial and neuronal compartments is included in the


2-, PO4 3-

2000) and phosphorylated proteins. Because of the very high Ca2+ / H2PO4

during degeneration.

diagram

such types of treatment. Similar results concerning differences between calcium precipitates and brain area susceptibility have been observed in congenital toxoplasmosis (Safadi et al., 2003, Surendrababu et al., 2006). Cerebral calcification has been described in 65% of these patients, with calcified foci distributed predominantly in the cortex in the form of tiny flecks, and as linear streaks in the basal ganglia.

The calcification process can thus be considered a new stage in cytoplasmic calcium homeostasis taking place in a diversity of CNS injuries to reduce calcium signalling at no energy cost. When located extracellularly due to cell-death, these precipitates activate a permanent microglial reaction aimed at their removal but which rapidly turns into chronic damage and aggravation of neurodegeneration.

#### **2.5 Uncoupling of retaliatory systems and energy availability**

The balance between retaliatory system actions and energy metabolism constitutes a fine equilibrium in physiological conditions, but it can be disrupted by glutamate-mediated neuronal injury to then participate in the evoked neurodegenerative process. For example, AMPA-microinjection in medial septum induces a progressive cholinergic and GABAergic loss associated with a long-term decline of the hippocampal functions and decreased glutamatergic activity (Rodriguez et al., 2005). Other effects of this lesion imply modifications of adenosine and taurine transmissions, glutamate recycling and glucose metabolism (Ramonet et al., 2004, Rodriguez et al., 2005). Over time, adenosine replaces GABA functions to avoid further excitotoxic damage when cholinergic and GABAergic processes are compromised.

Long-term septal lesion-induced neuronal loss in the hippocampus is apoptotic, with enhancement of neuronal glycolysis. Together with the cleavage of caspase 3, a glutamateglutamine cycle displacement towards glutamine production reduces glutamate synthesis (Ramonet et al., 2004). In addition, synaptic glutamine is decreased, probably through expulsion to vessels, where it exerts a vasodilatory effect through NO synthesis inhibition (Mates et al., 2002). In this scenario, the reduction in glutamate signaling and increased neuronal energy metabolism both reflect a neurodegenerative process with deficient adaptation of the retaliatory systems and a chronic energy requirement to execute the apoptotic program.

This chronic energy requirement induces mitochondrial damage, in turn leading to acidosis in cells and the extracellular space (Hertz, 2008). Mitochondrial damage forces the cell to shift from an aerobic to an anaerobic metabolism, and as a result lactate is produced with the formation of two ATPs and the release of two protons. After trauma and ischemia, extracellular lactate increases dramatically and pH decreases. To ensure neuronal viability during and even after human hypoxia, glial glucose is only oxidized to lactate, which is rapidly transported into neurons for its complete oxidation (Sibson et al., 1998). In parallel, a Ca2+ influx causes rapid cytoplasmic acidification (Verkhratsky, 2007) through: a) activation of membrane Na+/H+ exchanger to restore the Na+ gradient, and b) the Ca2+-dependent displacement of protons bound to cytoplasmic anions (Arundine & Tymianski, 2004). Furthermore, H+ also appears during some chemical reactions, such as phospholipid hydrolysis.

#### **2.6 Functional relevance of the calcification process**

The massive astroglial production of lactate to help compensate for the neuronal energy depletion caused by excitotoxicity is a key factor in brain calcification (Figure 4). pH

such types of treatment. Similar results concerning differences between calcium precipitates and brain area susceptibility have been observed in congenital toxoplasmosis (Safadi et al., 2003, Surendrababu et al., 2006). Cerebral calcification has been described in 65% of these patients, with calcified foci distributed predominantly in the cortex in the form of tiny

The calcification process can thus be considered a new stage in cytoplasmic calcium homeostasis taking place in a diversity of CNS injuries to reduce calcium signalling at no energy cost. When located extracellularly due to cell-death, these precipitates activate a permanent microglial reaction aimed at their removal but which rapidly turns into chronic

The balance between retaliatory system actions and energy metabolism constitutes a fine equilibrium in physiological conditions, but it can be disrupted by glutamate-mediated neuronal injury to then participate in the evoked neurodegenerative process. For example, AMPA-microinjection in medial septum induces a progressive cholinergic and GABAergic loss associated with a long-term decline of the hippocampal functions and decreased glutamatergic activity (Rodriguez et al., 2005). Other effects of this lesion imply modifications of adenosine and taurine transmissions, glutamate recycling and glucose metabolism (Ramonet et al., 2004, Rodriguez et al., 2005). Over time, adenosine replaces GABA functions to avoid further excitotoxic damage when cholinergic and GABAergic

Long-term septal lesion-induced neuronal loss in the hippocampus is apoptotic, with enhancement of neuronal glycolysis. Together with the cleavage of caspase 3, a glutamateglutamine cycle displacement towards glutamine production reduces glutamate synthesis (Ramonet et al., 2004). In addition, synaptic glutamine is decreased, probably through expulsion to vessels, where it exerts a vasodilatory effect through NO synthesis inhibition (Mates et al., 2002). In this scenario, the reduction in glutamate signaling and increased neuronal energy metabolism both reflect a neurodegenerative process with deficient adaptation of the retaliatory systems and a chronic energy requirement to execute the

This chronic energy requirement induces mitochondrial damage, in turn leading to acidosis in cells and the extracellular space (Hertz, 2008). Mitochondrial damage forces the cell to shift from an aerobic to an anaerobic metabolism, and as a result lactate is produced with the formation of two ATPs and the release of two protons. After trauma and ischemia, extracellular lactate increases dramatically and pH decreases. To ensure neuronal viability during and even after human hypoxia, glial glucose is only oxidized to lactate, which is rapidly transported into neurons for its complete oxidation (Sibson et al., 1998). In parallel, a Ca2+ influx causes rapid cytoplasmic acidification (Verkhratsky, 2007) through: a) activation of membrane Na+/H+ exchanger to restore the Na+ gradient, and b) the Ca2+-dependent displacement of protons bound to cytoplasmic anions (Arundine & Tymianski, 2004). Furthermore, H+ also appears during some chemical reactions, such as phospholipid

The massive astroglial production of lactate to help compensate for the neuronal energy depletion caused by excitotoxicity is a key factor in brain calcification (Figure 4). pH

flecks, and as linear streaks in the basal ganglia.

damage and aggravation of neurodegeneration.

processes are compromised.

apoptotic program.

hydrolysis.

**2.5 Uncoupling of retaliatory systems and energy availability** 

**2.6 Functional relevance of the calcification process** 

reduction associated with increased lactate concentration facilitates solubility of Ca2+ and the formation of H2PO4-, HPO42- and PO43- ions from inorganic phosphate (Rodriguez et al., 2000) and phosphorylated proteins. Because of the very high Ca2+ / H2PO4 -, HPO4 2-, PO4 3 affinity, apatite nucleation may occur, with the subsequent growth of crystalline formations together with neurodegeneration. In this case, calcification of each lesioned area will also depend on phosphate availability and the differential capacity of glial cells to release lactate during degeneration.

Wide variations have been described in the extent of calcification in pathological cases (Ramonet et al., 2002, Rodríguez et al., 2001) and animal species (Ramonet et al., 2006) However, the homogeneous morphology of these deposits suggests common synaptic processes (Ramonet et al., 2006) where variability depends on cellular type (astrocyte or neuron), glutamatergic activity, and energy availability (Figure 4). These factors modify Ca2+ homeostasis and may trigger cellular calcification through a common mechanism (Ramonet et al., 2006). Thus, hydroxyapatite formation, with the subsequent reduction of free Ca2+ ions, may take place as an alternative homeostatic step to reduce excitotoxicity (Ramonet et al., 2006, Rodriguez et al., 2000), and a number of findings lend support to this interpretation. For example, it has been observed that mitochondria close to Ca2+ deposits appear normal at electron microscopy level (Mahy et al., 1999, Rodriguez et al., 2000), despite

Fig. 4. Excitotoxicity modifies cell calcium homeostasis in the brain. Drawing of the excitotoxic process induced by glutamate, with the intercellular precipitation of calcium as part of the calcium homeostasis. The metabolic pathway of lactate with the communication between endothelial, astroglial, microglial and neuronal compartments is included in the diagram

Microglia, Calcification and Neurodegenerative Diseases 317

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the fact that mitochondrial dysfunction constitutes a primary event in NMDA-induced degeneration (Schinder et al., 1996). This hypothesis is also consistent with the finding that neurons undergoing prolonged stimulation of NMDA receptors can survive in the presence of [Ca2+]i chelators. Very high levels of cytoplasmic Ca2+ are not necessarily neurotoxic, and an effective uptake of this element into mitochondria is required to trigger NMDA-receptorstimulated neuronal death (Stout et al., 1998). Moreover, in rat globus pallidus, an AMPAdose-response study has shown a dose-dependent increase in calcification, which was not accompanied by an increase in astrogliosis (Petegnief et al., 1999). In the hippocampus, AMPA induced a calcified area larger than the injured area. In this same structure, the selective adenosine-A2a-receptor antagonist 8-(3-chlorostyryl)-caffeine increased NMDAinduced neuronal loss while calcification was decreased (Robledo et al., 1999). All these data indicate that Ca2+ precipitation does not necessarily reflect neuronal death and that, as proposed for retinal excitotoxic damage (Chen et al., 1999), in addition to Ca2+ other factors such as Na+ and Cl- influx, cell swelling and acidosis induce excitotoxic neuronal damage.

#### **3. Conclusions**

Neurodegenerative disorders are characterized by the appearance of distinct neurodegenerative parameters that determine the induction of a chronic process with underlying glutamate-mediated excitotoxicity and Ca2+ dys-homeostasis. At tissue level, the pathogenesis of each disorder depends on the neuronal type involved, synaptic density, glial interactions, and vicinity of vascularization. For each neuron, astrocyte and microglial type, the group of glutamate and cytokine receptors, the Ca2+ binding protein content, protein phosphorylation levels, and all elements that participate in energy needs and glucose availability will constitute the factors involved in the appearance of the lesion. In this scenario, CNS calcification can be considered one of the few common mechanisms already available at an early age to help buffer disturbances in Ca2+ homeostasis at no energy cost. Over time, CNS maturation includes a massive increase in synaptic connections, the organization of inhibitory systems and greater cellular complexity. As it becomes more sophisticated, the CNS relies on a greater diversity of mechanisms to prevent CNS injury. This would explain why calcification is observed in neurodegenerative diseases, but does not correlate with CNS damage.

#### **4. Acknowledgements**

This research was supported by grants SAF2008-01902 and IPT-010000-2010-35 from the Ministerio de Ciencia e Innovación, and by grant 2009SGR1380 from the Generalitat de Catalunya, Spain.

#### **5. References**


the fact that mitochondrial dysfunction constitutes a primary event in NMDA-induced degeneration (Schinder et al., 1996). This hypothesis is also consistent with the finding that neurons undergoing prolonged stimulation of NMDA receptors can survive in the presence of [Ca2+]i chelators. Very high levels of cytoplasmic Ca2+ are not necessarily neurotoxic, and an effective uptake of this element into mitochondria is required to trigger NMDA-receptorstimulated neuronal death (Stout et al., 1998). Moreover, in rat globus pallidus, an AMPAdose-response study has shown a dose-dependent increase in calcification, which was not accompanied by an increase in astrogliosis (Petegnief et al., 1999). In the hippocampus, AMPA induced a calcified area larger than the injured area. In this same structure, the selective adenosine-A2a-receptor antagonist 8-(3-chlorostyryl)-caffeine increased NMDAinduced neuronal loss while calcification was decreased (Robledo et al., 1999). All these data indicate that Ca2+ precipitation does not necessarily reflect neuronal death and that, as proposed for retinal excitotoxic damage (Chen et al., 1999), in addition to Ca2+ other factors such as Na+ and Cl- influx, cell swelling and acidosis induce excitotoxic neuronal damage.

Neurodegenerative disorders are characterized by the appearance of distinct neurodegenerative parameters that determine the induction of a chronic process with underlying glutamate-mediated excitotoxicity and Ca2+ dys-homeostasis. At tissue level, the pathogenesis of each disorder depends on the neuronal type involved, synaptic density, glial interactions, and vicinity of vascularization. For each neuron, astrocyte and microglial type, the group of glutamate and cytokine receptors, the Ca2+ binding protein content, protein phosphorylation levels, and all elements that participate in energy needs and glucose availability will constitute the factors involved in the appearance of the lesion. In this scenario, CNS calcification can be considered one of the few common mechanisms already available at an early age to help buffer disturbances in Ca2+ homeostasis at no energy cost. Over time, CNS maturation includes a massive increase in synaptic connections, the organization of inhibitory systems and greater cellular complexity. As it becomes more sophisticated, the CNS relies on a greater diversity of mechanisms to prevent CNS injury. This would explain why calcification is observed in neurodegenerative diseases,

This research was supported by grants SAF2008-01902 and IPT-010000-2010-35 from the Ministerio de Ciencia e Innovación, and by grant 2009SGR1380 from the Generalitat de

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**3. Conclusions** 

but does not correlate with CNS damage.

78, ISSN 1873-4286

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**4. Acknowledgements** 

Catalunya, Spain.

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**14** 

*Ireland* 

*2University College, Cork,* 

**Analysis of the Impact of CD200 on Neurodegenerative Diseases** 

Anne-Marie Miller1, Brian F. Deighan1, Eric Downer1, Anthony Lyons1

Neuroinflammation, accompanied by neuronal loss and dysfunction, is a characteristic of neurodegenerative disorders like Alzheimer's disease (AD) and Parkinson's disease (PD). It is well documented that inappropriate activation of glia is the primary cause of neuroinflammation (Masocha, 2009), but their role in the pathogenesis of neurodegenerative diseases is not known. However it is certainly the case that dying neurons act to stimulate glia since they release alarmins which activate pathogen recognition receptors (PRR) and therefore the possibility exists that activation of glia especially microglia, may be a consequence, rather than a cause, of neurodegenerative processes which characterize diseases like AD and PD. Understanding microglial function remains a major goal since it is widely believed that modulating glial function will provide a possible strategy for limiting the progression of neurodegenerative diseases. Consequently it is imperative to increase our understanding of the factors which control microglial function and the mechanisms by

Secreted factors including neurotrophins and growth factors like transforming growth factor (TGF)-β, as well as anti-inflammatory cytokines, impact on microglial activation and help to maintain these cells in a relatively quiescent state. Similarly, the interaction of microglia with other cells affects their activation state. However the recognition that macrophages, the peripheral cells which are derived from the same myeloid precursors as microglia, adopt different activation states has led to the acknowledgement that microglia can also adopt different activation states (Gordon, 2003). As the primary immune cells in the brain, microglia express PRR and therefore pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) interact with these receptors and trigger the innate immune response (Blasko et al., 2004, Koenigsknecht and Landreth, 2004). Microglia, like macrophages, are activated by the secreted proinflammatory cytokine, interferon-γ (IFNγ) inducing classical activation, and by the anti-inflammatory cytokines interleukin (IL)-4 and IL-13 to induce the alternative activation state (Gordon, 2003). Humoral activation of microglia, involving the complement system has also been described

**1. Introduction** 

which expression of these factors are controlled.

**2. Microglia adopt different activation states** 

Petra Henrich-Noack1, Yvonne Nolan2 and Marina A. Lynch1

*1Trinity College Institute of Neuroscience, Trinity College Dublin* 


## **Analysis of the Impact of CD200 on Neurodegenerative Diseases**

Anne-Marie Miller1, Brian F. Deighan1, Eric Downer1, Anthony Lyons1 Petra Henrich-Noack1, Yvonne Nolan2 and Marina A. Lynch1 *1Trinity College Institute of Neuroscience, Trinity College Dublin 2University College, Cork, Ireland* 

#### **1. Introduction**

322 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

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directly monitor the functional state of synapses in vivo and determine the fate of

Neuroinflammation, accompanied by neuronal loss and dysfunction, is a characteristic of neurodegenerative disorders like Alzheimer's disease (AD) and Parkinson's disease (PD). It is well documented that inappropriate activation of glia is the primary cause of neuroinflammation (Masocha, 2009), but their role in the pathogenesis of neurodegenerative diseases is not known. However it is certainly the case that dying neurons act to stimulate glia since they release alarmins which activate pathogen recognition receptors (PRR) and therefore the possibility exists that activation of glia especially microglia, may be a consequence, rather than a cause, of neurodegenerative processes which characterize diseases like AD and PD. Understanding microglial function remains a major goal since it is widely believed that modulating glial function will provide a possible strategy for limiting the progression of neurodegenerative diseases. Consequently it is imperative to increase our understanding of the factors which control microglial function and the mechanisms by which expression of these factors are controlled.

#### **2. Microglia adopt different activation states**

Secreted factors including neurotrophins and growth factors like transforming growth factor (TGF)-β, as well as anti-inflammatory cytokines, impact on microglial activation and help to maintain these cells in a relatively quiescent state. Similarly, the interaction of microglia with other cells affects their activation state. However the recognition that macrophages, the peripheral cells which are derived from the same myeloid precursors as microglia, adopt different activation states has led to the acknowledgement that microglia can also adopt different activation states (Gordon, 2003). As the primary immune cells in the brain, microglia express PRR and therefore pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) interact with these receptors and trigger the innate immune response (Blasko et al., 2004, Koenigsknecht and Landreth, 2004). Microglia, like macrophages, are activated by the secreted proinflammatory cytokine, interferon-γ (IFNγ) inducing classical activation, and by the anti-inflammatory cytokines interleukin (IL)-4 and IL-13 to induce the alternative activation state (Gordon, 2003). Humoral activation of microglia, involving the complement system has also been described

Analysis of the Impact of CD200 on Neurodegenerative Diseases 325

as a consequence of its interaction with plexin B1 (Chabbert-de Ponnat et al., 2005). However, in complete contrast to these findings, a Sema4D fusion protein has been reported to increase NO production in microglia and this was abolished in cells prepared from plexin B1-deficient mice (Okuno et al., 2010). The possible role of SEMA4D as a regulator of

Interest in understanding the roles of the NIRegs identified above has been increasing in the past few years and, to date, most emphasis has been placed on evaluating the role of the interaction between CD200 and its receptor on microglial activation. This interaction is recognized as a potent immune suppressor and therefore it is predicted that reduced inhibitory input from CD200 results in dysregulation of microglial function and the risk of

CD200, previously known as OX2, is a 41-47 kDa type-1 cell surface glycoprotein with two immunoglobulin domains arranged in a typical V-/C2 set (Clark et al., 1985). The family of IgSF glycoproteins to which CD200 belongs includes neural cell adhesion molecule (NCAM), Thy-1 and L1, which are expressed on both lymphoid tissue and also neuronal tissue; CD200 was originally identified in the thymus and brain (Barclay, 1981) and thereafter in several tissues, and cells including neurons, T cells and astrocytes (Webb and Barclay, 1984, Preston et al., 1997, Wright et al., 2000). Expression of CD200 on vascular endothelium has been described with evidence of more intense staining on veins and venules rather than arteries, although staining in arteries was increased following injection with LPS. Distribution of CD200 in capillaries appears to be tissue-dependent and varies with the type of capillary; thus intense immunoreactivity is observed in continuous endothelia (both fenestrated and non-fenestrated) compared with relatively lower expression on discontinuous endothelia. Interestingly it has been shown that an anti-CD200 antibody blocked the adhesion of T cells to endothelial cells but did not affect the adhesion of macrophages; thus it was suggested that, whereas the primary role of the interaction between CD200 and its receptor may be to reduce activity of macrophages, a second role may be to modulate adhesion and migration of T cells into tissues (Ko et al., 2009). CD200 expression has also been examined on endothelial cells in the brain and it has been reported that expression in the hippocampus was evident only on the luminal surface of endothelial cells that made up the blood brain barrier (BBB), whereas in the area postrema, which lacks a BBB, clear

staining was observed on the luminal and abluminal surfaces (Ko et al., 2009).

CD200 expression in brain tissue was found to be widespread with stronger staining in grey matter compared with white matter (Webb and Barclay, 1984). Immunostaining has been reported in the spinal cord, cerebellum and striatum, as well as the hippocampus and parietal cortex, and the evidence suggested that while it was expressed on the cell membrane in most brain areas, there was evidence of CD200 staining in the cytosol in hippocampal neurons. In the spinal cord, axons were CD200-positive whereas myelin did

CD200 receptor (CD200R), CD200's cognate receptor is also a glycoprotein and, like the ligand, it contains two IgSF domains in a V/C2 set arrangement and cysteine residues in their V-like domains. To date, 5 CD200R family members (R1-R5) have been identified in mice (Gorczynski et al., 2008). The most studied receptor, CD200R1, is expressed primarily

microglial function requires further examination.

inappropriate cellular activation and tissue damage.

**3.1 Expression of CD200 and its receptor** 

not stain for CD200 (Koning et al., 2009).

**3. CD200 and CD200R** 

(Griffiths et al., 2010). In addition to the modulatory effect of secreted factors like pro- and anti-inflammatory cytokines and factors like TGFβ, a deactivation/suppression state of microglia has been described, and this state is controlled by neuroimmunoregulatory proteins (NIRegs).

#### **2.1 Neuroimmunoregulatory proteins modulate microglial activation**

NIRegs act on specific receptors expressed on microglia and ensure that cell activation is checked. These NIRegs include CD200, CD22, CD47, semaphorin and fractalkine which interact with CD200R, CD45, SIRPα, plexin B1 or CD72, and fractalkine receptor respectively. In most of these cases, expression of the receptors is relatively restricted to cells of the myeloid lineage, whereas expression of the ligands is more widespread.

CD47 is a membrane glycoprotein and a member of the immunoglobulin superfamily. It is expressed on neurons and endothelial cells and its expression on macrophages has also been reported (Reinhold et al., 1995). CD47 is a 'don't eat me' signal and circulating cells lacking CD47 are rapidly cleared. Activation of SIRPα by CD47 leads to activation of an inhibitory signal as a consequence of the interaction between tyrosine phosphatases SHP-1 and SHP2 with cytoplasmic tyrosine-linked inhibition motifs (Hatherley et al., 2009). SIRPα, and another receptor for CD47, thrombospondin, are expressed on microglia, although SIRPα is also expressed on neurons (Brown and Frazier, 2001; Lamy et al., 2007).

CD45 is expressed on microglia, albeit at low levels when cells are unstimulated, contrasting with the higher expression on macrophages. It is a transmembrane protein tyrosine phosphatase which has been identified as a negative regulator of microglial activation (Tan et al., 2000). It has been known for 20 years that CD22 is a ligand for CD45 (Stamenkovic et al., 1991), but the fact that CD22 is expressed on neurons, and also released from neurons, has been established only relatively recently (Mott et al., 2004). These authors identified a role for CD22 in modulating tumour necrosis factor (TNF)-α release from microglia.

Fractalkine (also known as CX3CL1) is the only member of the CX3C subfamily of chemokines (Bazan et al., 1997). In the brain, it is expressed mainly on neurons (Harrison et al., 1998, Maciejewski-Lenoir et al., 1999), whereas its receptor is expressed chiefly on microglial cells (Harrison et al., 1998). However this expression pattern is probably not exclusive with evidence indicating that the ligand is expressed on glia (Maciejewski-Lenoir et al., 1999) and the receptor is expressed on neurons (Hughes et al., 2002). The engagement of fractalkine with its receptor decreases microglial activation and inhibits lipopolysaccharide (LPS) induced proinflammatory cytokine production (Zujovic et al., 2000; Lyons et al., 2009a). Evidence from this laboratory suggested that fractalkine expression was decreased in hippocampal tissue prepared from aged rats in which microglial activation is upregulated, and that the combination of these changes was coupled with a deficit in neuronal plasticity (Lyons et al., 2009a).

Although they were originally identified because of their importance as axon guidance molecules, an immunoregulatory role for some semaphorins has been described (Suzuki et al., 2008). SEMA4D (also referred to as CD100), a transmembrane protein which belongs to class 4 group of the semaphorin family, has been the focus of the studies designed to understand this immunomodulatory role. It is expressed on neurons, though not on microglia (Hirsch et al., 1999), whereas the 2 major receptors for SEMA4D, plexin B1 and CD72 are expressed on microglia (Toguchi et al., 2009). Soluble Sema4D inhibits LPSinduced microglial activation as assessed by a change in cell morphology, nitric oxide (NO) production and cell migration (Toguchi et al., 2009). It also prevents migration of monocytes as a consequence of its interaction with plexin B1 (Chabbert-de Ponnat et al., 2005). However, in complete contrast to these findings, a Sema4D fusion protein has been reported to increase NO production in microglia and this was abolished in cells prepared from plexin B1-deficient mice (Okuno et al., 2010). The possible role of SEMA4D as a regulator of microglial function requires further examination.

#### **3. CD200 and CD200R**

324 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

(Griffiths et al., 2010). In addition to the modulatory effect of secreted factors like pro- and anti-inflammatory cytokines and factors like TGFβ, a deactivation/suppression state of microglia has been described, and this state is controlled by neuroimmunoregulatory

NIRegs act on specific receptors expressed on microglia and ensure that cell activation is checked. These NIRegs include CD200, CD22, CD47, semaphorin and fractalkine which interact with CD200R, CD45, SIRPα, plexin B1 or CD72, and fractalkine receptor respectively. In most of these cases, expression of the receptors is relatively restricted to cells

CD47 is a membrane glycoprotein and a member of the immunoglobulin superfamily. It is expressed on neurons and endothelial cells and its expression on macrophages has also been reported (Reinhold et al., 1995). CD47 is a 'don't eat me' signal and circulating cells lacking CD47 are rapidly cleared. Activation of SIRPα by CD47 leads to activation of an inhibitory signal as a consequence of the interaction between tyrosine phosphatases SHP-1 and SHP2 with cytoplasmic tyrosine-linked inhibition motifs (Hatherley et al., 2009). SIRPα, and another receptor for CD47, thrombospondin, are expressed on microglia, although SIRPα is

CD45 is expressed on microglia, albeit at low levels when cells are unstimulated, contrasting with the higher expression on macrophages. It is a transmembrane protein tyrosine phosphatase which has been identified as a negative regulator of microglial activation (Tan et al., 2000). It has been known for 20 years that CD22 is a ligand for CD45 (Stamenkovic et al., 1991), but the fact that CD22 is expressed on neurons, and also released from neurons, has been established only relatively recently (Mott et al., 2004). These authors identified a

Fractalkine (also known as CX3CL1) is the only member of the CX3C subfamily of chemokines (Bazan et al., 1997). In the brain, it is expressed mainly on neurons (Harrison et al., 1998, Maciejewski-Lenoir et al., 1999), whereas its receptor is expressed chiefly on microglial cells (Harrison et al., 1998). However this expression pattern is probably not exclusive with evidence indicating that the ligand is expressed on glia (Maciejewski-Lenoir et al., 1999) and the receptor is expressed on neurons (Hughes et al., 2002). The engagement of fractalkine with its receptor decreases microglial activation and inhibits lipopolysaccharide (LPS) induced proinflammatory cytokine production (Zujovic et al., 2000; Lyons et al., 2009a). Evidence from this laboratory suggested that fractalkine expression was decreased in hippocampal tissue prepared from aged rats in which microglial activation is upregulated, and that the combination of these changes was coupled with a deficit in neuronal plasticity

Although they were originally identified because of their importance as axon guidance molecules, an immunoregulatory role for some semaphorins has been described (Suzuki et al., 2008). SEMA4D (also referred to as CD100), a transmembrane protein which belongs to class 4 group of the semaphorin family, has been the focus of the studies designed to understand this immunomodulatory role. It is expressed on neurons, though not on microglia (Hirsch et al., 1999), whereas the 2 major receptors for SEMA4D, plexin B1 and CD72 are expressed on microglia (Toguchi et al., 2009). Soluble Sema4D inhibits LPSinduced microglial activation as assessed by a change in cell morphology, nitric oxide (NO) production and cell migration (Toguchi et al., 2009). It also prevents migration of monocytes

role for CD22 in modulating tumour necrosis factor (TNF)-α release from microglia.

**2.1 Neuroimmunoregulatory proteins modulate microglial activation** 

of the myeloid lineage, whereas expression of the ligands is more widespread.

also expressed on neurons (Brown and Frazier, 2001; Lamy et al., 2007).

proteins (NIRegs).

(Lyons et al., 2009a).

#### **3.1 Expression of CD200 and its receptor**

Interest in understanding the roles of the NIRegs identified above has been increasing in the past few years and, to date, most emphasis has been placed on evaluating the role of the interaction between CD200 and its receptor on microglial activation. This interaction is recognized as a potent immune suppressor and therefore it is predicted that reduced inhibitory input from CD200 results in dysregulation of microglial function and the risk of inappropriate cellular activation and tissue damage.

CD200, previously known as OX2, is a 41-47 kDa type-1 cell surface glycoprotein with two immunoglobulin domains arranged in a typical V-/C2 set (Clark et al., 1985). The family of IgSF glycoproteins to which CD200 belongs includes neural cell adhesion molecule (NCAM), Thy-1 and L1, which are expressed on both lymphoid tissue and also neuronal tissue; CD200 was originally identified in the thymus and brain (Barclay, 1981) and thereafter in several tissues, and cells including neurons, T cells and astrocytes (Webb and Barclay, 1984, Preston et al., 1997, Wright et al., 2000). Expression of CD200 on vascular endothelium has been described with evidence of more intense staining on veins and venules rather than arteries, although staining in arteries was increased following injection with LPS. Distribution of CD200 in capillaries appears to be tissue-dependent and varies with the type of capillary; thus intense immunoreactivity is observed in continuous endothelia (both fenestrated and non-fenestrated) compared with relatively lower expression on discontinuous endothelia. Interestingly it has been shown that an anti-CD200 antibody blocked the adhesion of T cells to endothelial cells but did not affect the adhesion of macrophages; thus it was suggested that, whereas the primary role of the interaction between CD200 and its receptor may be to reduce activity of macrophages, a second role may be to modulate adhesion and migration of T cells into tissues (Ko et al., 2009). CD200 expression has also been examined on endothelial cells in the brain and it has been reported that expression in the hippocampus was evident only on the luminal surface of endothelial cells that made up the blood brain barrier (BBB), whereas in the area postrema, which lacks a BBB, clear staining was observed on the luminal and abluminal surfaces (Ko et al., 2009).

CD200 expression in brain tissue was found to be widespread with stronger staining in grey matter compared with white matter (Webb and Barclay, 1984). Immunostaining has been reported in the spinal cord, cerebellum and striatum, as well as the hippocampus and parietal cortex, and the evidence suggested that while it was expressed on the cell membrane in most brain areas, there was evidence of CD200 staining in the cytosol in hippocampal neurons. In the spinal cord, axons were CD200-positive whereas myelin did not stain for CD200 (Koning et al., 2009).

CD200 receptor (CD200R), CD200's cognate receptor is also a glycoprotein and, like the ligand, it contains two IgSF domains in a V/C2 set arrangement and cysteine residues in their V-like domains. To date, 5 CD200R family members (R1-R5) have been identified in mice (Gorczynski et al., 2008). The most studied receptor, CD200R1, is expressed primarily

Analysis of the Impact of CD200 on Neurodegenerative Diseases 327

**CD200R**

**CD200R**

**pY**

Fig. 1. CD200-induced signalling downregulates glial production of inflammatory cytokines. CD200 is expressed on several cell types including neurons and endothelial cells whereas expression of CD200R is relatively restricted to cells of the myeloid lineage. CD200 has a short cytosolic domain with no signalling capability whereas the signalling motif in the cytosolic domain of CD200R contains 3 tyrosine residues which, when phosphorylated, recruits Dok 1and Dok 2 which leads to activation of SHIP and RasGAP respectively, the latter of which leads to inhibition of MAP kinases thereby permitting increased production

and a defect in the organization of the mesenteric lymph nodes was described (Barclay et al., 2002). The findings of these studies indicated that CD200R activation provides a mechanism for negatively modulating cell responses and controlling responses of cells to immunological stimuli. An increase in the activation state of microglia was also reported with evidence of increased expression of CD11b and CD45, and the response of microglia to trauma is markedly enhanced in CD200-deficient mice where activated microglia cluster around the lesion area (Hoek et al., 2000). The clustering of activated macrophages or microglia in

**pY**

**p**

**GAP Ras MAP kinase**

**p Dok**

**SHIP**

**CD200**

**Microglia Macrophage**

**Neuron Other cells**

**19aa CD200**

**67aa**

of inflammatory cytokines

**CD200R**

 **Release of Immunomodulatory molecules**

**eg Cytokines**

on myeloid lineage cells such as microglia and macrophages (Meuth et al., 2008, Masocha, 2009) and also monocytes, granulocytes and dendritic cells (DC) (Wright et al., 2000, Wright et al., 2003). More recent flow cytometry data suggest that CD200R is also expressed on natural killer cells and B cells, as well as on CD4+ T cells which had been reported previously (Wright et al., 2003, Rijkers et al., 2008). It was suggested that CD200 is the natural ligand for only CD200R1 (Wright et al., 2003) although others suggest that this may not be the case (Gorczynski et al., 2004).

#### **3.2 The signaling events induced by CD200R activation**

Most inhibitory receptors contain immunoreceptor tyrosine-based inhibitory motifs (ITIM) which enables cell signalling through recruitment of Src homology 2 domain containing phosphatases (SHP), or SHIP, which is an inositol phosphataseSH2-containing inositol phosphatase (SHIP). This is not the case with CD200R; instead, CD200R has a long cytoplasmic tail of 67 amino acids (Figure 1).

This longer cytoplasmic domain on CD200R contrasts with the short intracellular domain of CD200, which contains 19 amino acids and no signalling motifs (Barclay et al., 2002). The cytoplasmic tail of CD200R includes an NPXY signalling motif which interacts with the phosphotyrosine-binding (PTB) domains present in several signalling adaptor molecules (Wright et al., 2000). The NPXY signalling motif contains 3 tyrosine residues, which are phosphorylated following the interaction between CD200 and CD200R (Wright *et al*., 2000: Snelgrove *et al.,* 2008). This initiates a signaling cascade, which involves recruitment and phosphorylation of adaptor proteins, downstream of tyrosine kinase (Dok) 1 and Dok 2 and the subsequent binding to RasGAP and SHIP (Mihrshahi et al., 2009); the downstream events include inhibition of the Ras/mitogen-activated protein kinase (MAPK) pathway (Zhang et al., 2004). Ultimately this results in a decrease in release of inflammatory cytokines. Thus CD200R agonists inhibited IFNγ-induced release of TNFα from peritoneal macrophages, although no effect on LPS-induced release was observed (Jenmalm et al., 2006). These agonists also increased IFNγ-induced and IL-17-induced release of IL-6, although production of monocyte chemoattractant protein-1 (MCP-1) was unaffected. Tetanus toxin-induced production of IL-5 and IL-13, but not other cytokines, was inhibited by CD200R agonists (Jenmalm et al., 2006). The effects of these agonists were cell-specific; activation of DC by several stimuli, including LPS and inflammatory cytokines, increased numerous markers of cell activation and resulted in release of many cytokines but these changes were resistant to modulation by CD200R agonists.

Recent evidence suggests that Dok 1 negatively regulates Dok 2-induced signalling (Mihrshahi and Brown, 2010) and that the negative regulation induced by CD200R activation is mediated by sequential activation of Dok 2 and RasGAP (Mihrshahi et al., 2009).

#### **3.3 Characteristics of CD200-deficient mice**

Deletion of the CD200 gene in mice provided a significant insight into the role of CD200 with the important observation that susceptibility of these mice to autoimmune diseases was markedly increased, with evidence of upregulated inflammatory responses (Hoek et al., 2000). The population of macrophages was increased in these animals and there was evidence of an enhanced activation state, even under resting conditions (Hoek et al., 2000). Specifically, macrophage numbers in the spleen and mesenteric lymph nodes were increased

on myeloid lineage cells such as microglia and macrophages (Meuth et al., 2008, Masocha, 2009) and also monocytes, granulocytes and dendritic cells (DC) (Wright et al., 2000, Wright et al., 2003). More recent flow cytometry data suggest that CD200R is also expressed on natural killer cells and B cells, as well as on CD4+ T cells which had been reported previously (Wright et al., 2003, Rijkers et al., 2008). It was suggested that CD200 is the natural ligand for only CD200R1 (Wright et al., 2003) although others suggest that this may

Most inhibitory receptors contain immunoreceptor tyrosine-based inhibitory motifs (ITIM) which enables cell signalling through recruitment of Src homology 2 domain containing phosphatases (SHP), or SHIP, which is an inositol phosphataseSH2-containing inositol phosphatase (SHIP). This is not the case with CD200R; instead, CD200R has a long

This longer cytoplasmic domain on CD200R contrasts with the short intracellular domain of CD200, which contains 19 amino acids and no signalling motifs (Barclay et al., 2002). The cytoplasmic tail of CD200R includes an NPXY signalling motif which interacts with the phosphotyrosine-binding (PTB) domains present in several signalling adaptor molecules (Wright et al., 2000). The NPXY signalling motif contains 3 tyrosine residues, which are phosphorylated following the interaction between CD200 and CD200R (Wright *et al*., 2000: Snelgrove *et al.,* 2008). This initiates a signaling cascade, which involves recruitment and phosphorylation of adaptor proteins, downstream of tyrosine kinase (Dok) 1 and Dok 2 and the subsequent binding to RasGAP and SHIP (Mihrshahi et al., 2009); the downstream events include inhibition of the Ras/mitogen-activated protein kinase (MAPK) pathway (Zhang et al., 2004). Ultimately this results in a decrease in release of inflammatory cytokines. Thus CD200R agonists inhibited IFNγ-induced release of TNFα from peritoneal macrophages, although no effect on LPS-induced release was observed (Jenmalm et al., 2006). These agonists also increased IFNγ-induced and IL-17-induced release of IL-6, although production of monocyte chemoattractant protein-1 (MCP-1) was unaffected. Tetanus toxin-induced production of IL-5 and IL-13, but not other cytokines, was inhibited by CD200R agonists (Jenmalm et al., 2006). The effects of these agonists were cell-specific; activation of DC by several stimuli, including LPS and inflammatory cytokines, increased numerous markers of cell activation and resulted in release of many cytokines but these

Recent evidence suggests that Dok 1 negatively regulates Dok 2-induced signalling (Mihrshahi and Brown, 2010) and that the negative regulation induced by CD200R activation is mediated by sequential activation of Dok 2 and RasGAP (Mihrshahi et al.,

Deletion of the CD200 gene in mice provided a significant insight into the role of CD200 with the important observation that susceptibility of these mice to autoimmune diseases was markedly increased, with evidence of upregulated inflammatory responses (Hoek et al., 2000). The population of macrophages was increased in these animals and there was evidence of an enhanced activation state, even under resting conditions (Hoek et al., 2000). Specifically, macrophage numbers in the spleen and mesenteric lymph nodes were increased

not be the case (Gorczynski et al., 2004).

cytoplasmic tail of 67 amino acids (Figure 1).

**3.2 The signaling events induced by CD200R activation** 

changes were resistant to modulation by CD200R agonists.

**3.3 Characteristics of CD200-deficient mice** 

2009).

Fig. 1. CD200-induced signalling downregulates glial production of inflammatory cytokines. CD200 is expressed on several cell types including neurons and endothelial cells whereas expression of CD200R is relatively restricted to cells of the myeloid lineage. CD200 has a short cytosolic domain with no signalling capability whereas the signalling motif in the cytosolic domain of CD200R contains 3 tyrosine residues which, when phosphorylated, recruits Dok 1and Dok 2 which leads to activation of SHIP and RasGAP respectively, the latter of which leads to inhibition of MAP kinases thereby permitting increased production of inflammatory cytokines

and a defect in the organization of the mesenteric lymph nodes was described (Barclay et al., 2002). The findings of these studies indicated that CD200R activation provides a mechanism for negatively modulating cell responses and controlling responses of cells to immunological stimuli. An increase in the activation state of microglia was also reported with evidence of increased expression of CD11b and CD45, and the response of microglia to trauma is markedly enhanced in CD200-deficient mice where activated microglia cluster around the lesion area (Hoek et al., 2000). The clustering of activated macrophages or microglia in

Analysis of the Impact of CD200 on Neurodegenerative Diseases 329

Fig. 2. Endothelial cells, which express CD200, modulate LPS-induced IL-1β production from glia in a manner which resembles the effect of neurons. a,b. Neurons (a) and endothelial cells (b; bEnd.3) express CD200. Mixed glia were incubated in the presence or absence of LPS (100ng/ml), and either neurons (1:2) or endothelial cells (bEnd.3; 1:8) were added. c,d. LPS significantly increased supernatant concentration of IL-1β (\*\*\*p < 0.001; ANOVA) and this was significantly attenuated when mixed glia were co-cultured with either neurons or

and CD11b, are increased in hippocampal and cortical tissue with age and these changes are accompanied by increased expression of inflammatory cytokines (Lynch, 2010). Evidence from this laboratory indicates that CD200 expression is decreased in hippocampal tissue prepared from aged, compared with young, rats. We have proposed that this significantly contributes to the age-related increase in microglial activation (Lyons et al., 2007a) and consequently the age-related decrease in synaptic plasticity, typified by the deficit in longterm potentiation (LTP). Recent evidence has revealed that intracerebroventricular injection of CD200Fc attenuated the age-related deficit in LTP (Cox et al., unpublished). Interestingly, amyloid-β (Aβ), which has been shown to decrease LTP (Lyons et al., 2007a, Lyons et al., 2007b) is associated with increased microglial activation as demonstrated by increased expression of the cell surface markers of microglial activation, MHCII (Lyons et al., 2007a, Lyons et al., 2007b), ICAM and CD86 (Clarke et al., 2007), increased production of inflammatory cytokines, IFNγ and IL-1β (Minogue et al., 2007) and increased production of chemokines MCP-1 and IP-10 (Clarke et al., 2007). Significantly Aβ also decreases CD200 expression *in vitro* while expression of CD200 is also decreased in hippocampal tissue prepared from rats which received an intracerebroventricular injection of Aβ (Lyons et al.,

Apoptosis is an ongoing process which is necessary to permit natural cell turnover. It is important to ensure that this occurs without production of inflammatory cytokines which can negatively impact on cells in the microenvironment; a key factor in ensuring maintenance of this steady state is the expression of immunoregulatory signals. Like other peripheral cells, apoptosis of DC occurs on an ongoing basis and, experimentally, apoptosis can be induced by growth factor deprivaton. Recent data have indicated that up to 75% of apoptotic CD11c+ cells express CD200, whereas about one third of non-apoptotic CD11c+ cells express CD200; the evidence indicates that expression of CD200 is p53- and caspase-

endothelial cells (+++p < 0.001; ANOVA)

**4.3 CD200 is a protective molecule during apoptosis** 

2007a).

tissues of CD200-deficient mice has suggested that CD200-CD200R interaction may not simply provide a mechanism by which these myeloid cells are maintained in a relatively quiescent state, but that this interaction may play a key role in controlling migration of cells (Nathan and Muller, 2001). Interestingly, one of the earliest papers on the actions of CD200 suggested that it was expressed on immature (as well as mature) neurons and that it may be involved in migration of these neurons during development of the CNS (Webb and Barclay, 1984).

Symptoms in several models of neurodegenerative and/or neuroinflammatory disease, or the responses to certain infections, or the effects of injury to neurons (detailed in Section 4 below) have been examined in CD200-deficient mice. The evidence consistently shows, across these experiments, that the symptoms are worse, the mortality rate is higher and activation of microglial cells or macrophages is more profound in CD200-deficient, compared with wildtype, mice. Thus CD200-deficient mice exhibit increased sensitivity to infections like influenza where evidence of greater macrophage activity was linked with prolonged symptoms and increased mortality (Snelgrove et al., 2008) and to *Toxoplasma gondii* where the increased macrophage infiltration, accompanied by increased activation of these cells and also microglia, was associated with poorer survival rates (Deckert et al., 2006). In a striking parallel with microglia from CD200-deficient mice, microglia prepared from mice lacking either Dok 1 or Dok 2 also respond more profoundly to LPS than cells from wildtype mice (Shinohara et al., 2005).

#### **4. CD200 functions as a neuroimmunoregulatory protein**

#### **4.1 CD200-CD200R interaction maintains microglia in a quiescent state**

The findings of several experiments indicate that the interaction between CD200 and CD200R maintains microglia or macrophages in a quiescent state whereas the absence of CD200 is linked with evidence of cell activation and inflammatory changes. Evidence from this laboratory has revealed that co-culture of neurons with mixed glia inhibited LPSinduced increases in release of IL-1β, IL-6 and TNFα. The effect of neurons was blocked when the incubation was carried out in the presence of a blocking anti-CD200 antibody (Lyons et al., 2009b) pinpointing a role for CD200 in modulating cytokine release. Similarly, the Aβ-induced release of IL-1β, IL-6 and TNFα from mixed glia is inhibited when cells are co-cultured with neurons and this effect of neurons is also inhibited by the presence of a blocking anti-CD200 antibody (Lyons et al., 2007a).

One factor which increases CD200 expression is IL-4 and, interestingly, a marked reduction in CD200 expression has been reported on neurons prepared from IL-4-deficient mice (Lyons et al., 2009b). Predictably, therefore, co-incubation of mixed glia with neurons prepared from IL-4-deficient mice did not attenuate Aβ-induced production of inflammatory cytokines (Lyons et al., 2009b), contrasting with the effect of neurons prepared from wildtype mice. As highlighted above, endothelial cells express CD200 and, like neurons, incubation of LPS-treated mixed glia with endothelial cells inhibits the LPSinduced release of IL-1β from mixed glia (Figure 2).

#### **4.2 The age-related increase in microglial activation is associated with decreased CD200 expression**

It has been recognized for several years that microglial activation is increased in the brain with age; the evidence suggests that expression of markers of activation, for example MHCII

tissues of CD200-deficient mice has suggested that CD200-CD200R interaction may not simply provide a mechanism by which these myeloid cells are maintained in a relatively quiescent state, but that this interaction may play a key role in controlling migration of cells (Nathan and Muller, 2001). Interestingly, one of the earliest papers on the actions of CD200 suggested that it was expressed on immature (as well as mature) neurons and that it may be involved in migration of these neurons during development of the CNS (Webb and Barclay,

Symptoms in several models of neurodegenerative and/or neuroinflammatory disease, or the responses to certain infections, or the effects of injury to neurons (detailed in Section 4 below) have been examined in CD200-deficient mice. The evidence consistently shows, across these experiments, that the symptoms are worse, the mortality rate is higher and activation of microglial cells or macrophages is more profound in CD200-deficient, compared with wildtype, mice. Thus CD200-deficient mice exhibit increased sensitivity to infections like influenza where evidence of greater macrophage activity was linked with prolonged symptoms and increased mortality (Snelgrove et al., 2008) and to *Toxoplasma gondii* where the increased macrophage infiltration, accompanied by increased activation of these cells and also microglia, was associated with poorer survival rates (Deckert et al., 2006). In a striking parallel with microglia from CD200-deficient mice, microglia prepared from mice lacking either Dok 1 or Dok 2 also respond more profoundly to LPS than cells

1984).

from wildtype mice (Shinohara et al., 2005).

blocking anti-CD200 antibody (Lyons et al., 2007a).

induced release of IL-1β from mixed glia (Figure 2).

**CD200 expression** 

**4. CD200 functions as a neuroimmunoregulatory protein** 

**4.1 CD200-CD200R interaction maintains microglia in a quiescent state** 

The findings of several experiments indicate that the interaction between CD200 and CD200R maintains microglia or macrophages in a quiescent state whereas the absence of CD200 is linked with evidence of cell activation and inflammatory changes. Evidence from this laboratory has revealed that co-culture of neurons with mixed glia inhibited LPSinduced increases in release of IL-1β, IL-6 and TNFα. The effect of neurons was blocked when the incubation was carried out in the presence of a blocking anti-CD200 antibody (Lyons et al., 2009b) pinpointing a role for CD200 in modulating cytokine release. Similarly, the Aβ-induced release of IL-1β, IL-6 and TNFα from mixed glia is inhibited when cells are co-cultured with neurons and this effect of neurons is also inhibited by the presence of a

One factor which increases CD200 expression is IL-4 and, interestingly, a marked reduction in CD200 expression has been reported on neurons prepared from IL-4-deficient mice (Lyons et al., 2009b). Predictably, therefore, co-incubation of mixed glia with neurons prepared from IL-4-deficient mice did not attenuate Aβ-induced production of inflammatory cytokines (Lyons et al., 2009b), contrasting with the effect of neurons prepared from wildtype mice. As highlighted above, endothelial cells express CD200 and, like neurons, incubation of LPS-treated mixed glia with endothelial cells inhibits the LPS-

**4.2 The age-related increase in microglial activation is associated with decreased** 

It has been recognized for several years that microglial activation is increased in the brain with age; the evidence suggests that expression of markers of activation, for example MHCII

Fig. 2. Endothelial cells, which express CD200, modulate LPS-induced IL-1β production from glia in a manner which resembles the effect of neurons. a,b. Neurons (a) and endothelial cells (b; bEnd.3) express CD200. Mixed glia were incubated in the presence or absence of LPS (100ng/ml), and either neurons (1:2) or endothelial cells (bEnd.3; 1:8) were added. c,d. LPS significantly increased supernatant concentration of IL-1β (\*\*\*p < 0.001; ANOVA) and this was significantly attenuated when mixed glia were co-cultured with either neurons or endothelial cells (+++p < 0.001; ANOVA)

and CD11b, are increased in hippocampal and cortical tissue with age and these changes are accompanied by increased expression of inflammatory cytokines (Lynch, 2010). Evidence from this laboratory indicates that CD200 expression is decreased in hippocampal tissue prepared from aged, compared with young, rats. We have proposed that this significantly contributes to the age-related increase in microglial activation (Lyons et al., 2007a) and consequently the age-related decrease in synaptic plasticity, typified by the deficit in longterm potentiation (LTP). Recent evidence has revealed that intracerebroventricular injection of CD200Fc attenuated the age-related deficit in LTP (Cox et al., unpublished). Interestingly, amyloid-β (Aβ), which has been shown to decrease LTP (Lyons et al., 2007a, Lyons et al., 2007b) is associated with increased microglial activation as demonstrated by increased expression of the cell surface markers of microglial activation, MHCII (Lyons et al., 2007a, Lyons et al., 2007b), ICAM and CD86 (Clarke et al., 2007), increased production of inflammatory cytokines, IFNγ and IL-1β (Minogue et al., 2007) and increased production of chemokines MCP-1 and IP-10 (Clarke et al., 2007). Significantly Aβ also decreases CD200 expression *in vitro* while expression of CD200 is also decreased in hippocampal tissue prepared from rats which received an intracerebroventricular injection of Aβ (Lyons et al., 2007a).

#### **4.3 CD200 is a protective molecule during apoptosis**

Apoptosis is an ongoing process which is necessary to permit natural cell turnover. It is important to ensure that this occurs without production of inflammatory cytokines which can negatively impact on cells in the microenvironment; a key factor in ensuring maintenance of this steady state is the expression of immunoregulatory signals. Like other peripheral cells, apoptosis of DC occurs on an ongoing basis and, experimentally, apoptosis can be induced by growth factor deprivaton. Recent data have indicated that up to 75% of apoptotic CD11c+ cells express CD200, whereas about one third of non-apoptotic CD11c+ cells express CD200; the evidence indicates that expression of CD200 is p53- and caspase-

Analysis of the Impact of CD200 on Neurodegenerative Diseases 331

exaggerated symptoms in models of autoimmune diseases (Feuer, 2007). The majority of studies which have examined the role of CD200 as a negative regulator of myeloid cells have focussed on three autoimmune disease models, collagen-induced arthritis (CIA), a model for rheumatoid arthritis, experimental autoimmune uveoretinitis (EAU), a murine model for uveitis and myelin oligodendrocyte glycoprotein (MOG)-induced experimental

Rheumatoid arthritis is a classical inflammatory disease of the joints, typified by infiltrates of inflammatory cells. The most widely-used model is CIA and the evidence indicates that the symptoms of the disease, including inflammation and joint pathology was much more severe in CD200-/- mice, compared with their wildtype counterparts (Hoek et al., 2000). In contrast, treatment of mice with recombinant CD200 at 3-day intervals, concomitant with collagen immunization, markedly reduced symptoms; this included a reduction in infiltration of inflammatory cells and reduced bone erosion (Melnyk et al., 2011). A similar reduction in the severity of the disease, pathology and production of inflammatory mediators was observed when mice were treated with CD200Fc (Simelyte et al., 2008). These findings suggest that an agonistic antibody to CD200R, as substitute for the CD200-CD200R interaction, might be a useful therapeutic strategy in CIA. Predictably, CD200Fc, an immunoadhensin, produced by fusing the extracellular domains of CD200 to a murine IgG2a Fc construct, decreases TNFα and IFNγ production following collagen injection and

EAU, which is induced by immunization with interphotoreceptor retinoid-binding protein, is characterized by destruction of the neuroretina and photoreceptors, and the evidence indicates that this is T cell mediated; the symptoms include leukocyte infiltration of the vitreous and retina, vasculitis and ultimately photoreceptor and ganglion cell death. Symptoms become evident more quickly and are more profound in CD200-/- mice, compared with wildtype animals with significant additional infiltration of CD45+ CD11b+ cells and evidence of photoreceptor death, coupled with increased expression of nitric oxide synthase (NOS)-2 (Broderick et al., 2002). These findings were replicated subsequently and extended to show that the progression of the disease was suppressed by an agonist CD200R antibody (Copland et al., 2007). The modulatory role for CD200 in EAU was also identified in a rat model and, in this case, the evidence indicated that blocking CD200-CD200R interaction by an antibody accelerated the onset and severity of symptoms (Banerjee and Dick, 2004). Experimentally-induced glaucoma, caused by injecting hypertonic saline into the superior episcleral aqueous drainage vein, is another inflammatory and degenerative condition of the eye which is associated with a time-related increase in microglial activation; like EAU a role for CD200-CD200R has been implicated by the finding that the microglial activation is coupled with a decrease in CD200 and evidence of retinal ganglion cell death

Multiple sclerosis is a chronic, progressive, disabling autoimmune disease. The generallyaccepted view is that the disease is caused by uncontrolled inflammatory T cell responses to

autoimmune encephalomyelitis (EAE), a model of multiple sclerosis.

halted the progression of symptoms of CIA (Gorczynski et al., 2002).

**5.2 CD200-CD200R interaction and Multiple Sclerosis** 

**5.1.1 CD200-CD200R interaction in CIA** 

**5.1.2 CD200-CD200R interaction in EAU** 

(Taylor et al., 2011).

dependent. Similarly γ-irradiation, which induces apoptosis in C1498 leukemia cells, is associated with increased expression of CD200 (Rosenblum et al., 2004).

It has been proposed that CD200 also plays a role in tolerance. This has been demonstrated in a model of contact hypersensitivity which is induced by 2,4-dinitro-fluorobenzene. In this model, the inflammatory changes which typify contact hypersensitivity are attenuated by prior exposure to low dose ultraviolet light (UVB) and it has been proposed that UVBinduced apoptosis of epidermal DCs is the key to this tolerance. Significantly, and consistent with the findings obtained in vitro, this is dependent on CD200, since tolerance was absent when this experiment was conducted in CD200-deficient mice. The data suggest that CD200- CD200R interaction may be a key event in ensuring that inflammatory changes do not accompany steady-state ongoing apoptosis (Rosenblum et al., 2004). Interestingly several studies have highlighted a role for CD200 in tolerance following transplants (Clark et al., 2008, Gorczynski et al., 2009)

#### **5. The importance of the interaction between CD200 and CD200R in modulating inflammation**

CD200-CD200R interaction provides a regulatory signal to macrophages (Broderick *et al.,* 2002) and consequently macrophage numbers in the spleen are increased in CD200-/- mice compared with wildtype mice, while CD200-/- mice also have enlarged lymph nodes (Hoek et al., 2000). A similar regulatory signal modulates microglia and therefore the absence of CD200 is associated with microglial activation. Thus cells prepared from CD200-/- mice exhibited an activated phenotype, and had less ramified morphology and shorter processes, as well as increased expression of cell surface markers, CD11b and CD45, which are indicative of activation (Hoek et al., 2000). Microglia from CD200-/- animals also appeared to form aggregates, which occurs in neuroinflammatory and neurodegenerative, but not under normal, conditions (Hoek et al., 2000). Predictably, cells prepared from CD200-/- mice exhibited a greater response to stimuli including LPS and Aβ (Lyons et al., 2007a). These data indicate that disruption of this interaction between CD200-CD200R results in dysregulation of macrophages and microglia, with cells shifting to a more tonically active state (Hoek *et al.*, 2000).

Evidence from experimental conditions associated with inflammatory changes and microglial activation, adds support to the finding that CD200-CD200R interaction is an important regulator of neuroimmune function. For example, *Toxoplasma gondii*-induced encephalitis is characterized by lymphocytic infiltrates and microglial activation and it has been reported that infection induced a more profound microglial proliferation and greater expression of markers of activation in CD200-deficient, compared with wildtype, mice (Deckert et al., 2006). In addition, nerve injury is associated with microglial activation and it has been reported that facial nerve transaction induced a greater degree of microglial activation in CD200-deficient, compared with wildtype, mice (Hoek et al., 2000). Similarly the neurodegenerative changes that occurred following sciatic nerve crush was associated with a profound loss of CD200 and evidence of macrophage activation (Chang et al., 2011).

#### **5.1 CD200-CD200R interaction in inflammatory diseases and models of disease**

One of the most clearcut consequences of the loss of the interaction between CD200 and CD200R is the development of inflammatory changes (Masocha 2009), and therefore, as described above, CD200-/- mice are more susceptible to inflammatory stimuli and exhibit exaggerated symptoms in models of autoimmune diseases (Feuer, 2007). The majority of studies which have examined the role of CD200 as a negative regulator of myeloid cells have focussed on three autoimmune disease models, collagen-induced arthritis (CIA), a model for rheumatoid arthritis, experimental autoimmune uveoretinitis (EAU), a murine model for uveitis and myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis.

#### **5.1.1 CD200-CD200R interaction in CIA**

330 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

dependent. Similarly γ-irradiation, which induces apoptosis in C1498 leukemia cells, is

It has been proposed that CD200 also plays a role in tolerance. This has been demonstrated in a model of contact hypersensitivity which is induced by 2,4-dinitro-fluorobenzene. In this model, the inflammatory changes which typify contact hypersensitivity are attenuated by prior exposure to low dose ultraviolet light (UVB) and it has been proposed that UVBinduced apoptosis of epidermal DCs is the key to this tolerance. Significantly, and consistent with the findings obtained in vitro, this is dependent on CD200, since tolerance was absent when this experiment was conducted in CD200-deficient mice. The data suggest that CD200- CD200R interaction may be a key event in ensuring that inflammatory changes do not accompany steady-state ongoing apoptosis (Rosenblum et al., 2004). Interestingly several studies have highlighted a role for CD200 in tolerance following transplants (Clark et al.,

associated with increased expression of CD200 (Rosenblum et al., 2004).

**5. The importance of the interaction between CD200 and CD200R in** 

CD200-CD200R interaction provides a regulatory signal to macrophages (Broderick *et al.,* 2002) and consequently macrophage numbers in the spleen are increased in CD200-/- mice compared with wildtype mice, while CD200-/- mice also have enlarged lymph nodes (Hoek et al., 2000). A similar regulatory signal modulates microglia and therefore the absence of CD200 is associated with microglial activation. Thus cells prepared from CD200-/- mice exhibited an activated phenotype, and had less ramified morphology and shorter processes, as well as increased expression of cell surface markers, CD11b and CD45, which are indicative of activation (Hoek et al., 2000). Microglia from CD200-/- animals also appeared to form aggregates, which occurs in neuroinflammatory and neurodegenerative, but not under normal, conditions (Hoek et al., 2000). Predictably, cells prepared from CD200-/- mice exhibited a greater response to stimuli including LPS and Aβ (Lyons et al., 2007a). These data indicate that disruption of this interaction between CD200-CD200R results in dysregulation of macrophages and microglia, with cells shifting to a more tonically active

Evidence from experimental conditions associated with inflammatory changes and microglial activation, adds support to the finding that CD200-CD200R interaction is an important regulator of neuroimmune function. For example, *Toxoplasma gondii*-induced encephalitis is characterized by lymphocytic infiltrates and microglial activation and it has been reported that infection induced a more profound microglial proliferation and greater expression of markers of activation in CD200-deficient, compared with wildtype, mice (Deckert et al., 2006). In addition, nerve injury is associated with microglial activation and it has been reported that facial nerve transaction induced a greater degree of microglial activation in CD200-deficient, compared with wildtype, mice (Hoek et al., 2000). Similarly the neurodegenerative changes that occurred following sciatic nerve crush was associated with a profound loss of CD200 and evidence of macrophage activation (Chang et al., 2011).

**5.1 CD200-CD200R interaction in inflammatory diseases and models of disease**  One of the most clearcut consequences of the loss of the interaction between CD200 and CD200R is the development of inflammatory changes (Masocha 2009), and therefore, as described above, CD200-/- mice are more susceptible to inflammatory stimuli and exhibit

2008, Gorczynski et al., 2009)

**modulating inflammation** 

state (Hoek *et al.*, 2000).

Rheumatoid arthritis is a classical inflammatory disease of the joints, typified by infiltrates of inflammatory cells. The most widely-used model is CIA and the evidence indicates that the symptoms of the disease, including inflammation and joint pathology was much more severe in CD200-/- mice, compared with their wildtype counterparts (Hoek et al., 2000). In contrast, treatment of mice with recombinant CD200 at 3-day intervals, concomitant with collagen immunization, markedly reduced symptoms; this included a reduction in infiltration of inflammatory cells and reduced bone erosion (Melnyk et al., 2011). A similar reduction in the severity of the disease, pathology and production of inflammatory mediators was observed when mice were treated with CD200Fc (Simelyte et al., 2008). These findings suggest that an agonistic antibody to CD200R, as substitute for the CD200-CD200R interaction, might be a useful therapeutic strategy in CIA. Predictably, CD200Fc, an immunoadhensin, produced by fusing the extracellular domains of CD200 to a murine IgG2a Fc construct, decreases TNFα and IFNγ production following collagen injection and halted the progression of symptoms of CIA (Gorczynski et al., 2002).

#### **5.1.2 CD200-CD200R interaction in EAU**

EAU, which is induced by immunization with interphotoreceptor retinoid-binding protein, is characterized by destruction of the neuroretina and photoreceptors, and the evidence indicates that this is T cell mediated; the symptoms include leukocyte infiltration of the vitreous and retina, vasculitis and ultimately photoreceptor and ganglion cell death. Symptoms become evident more quickly and are more profound in CD200-/- mice, compared with wildtype animals with significant additional infiltration of CD45+ CD11b+ cells and evidence of photoreceptor death, coupled with increased expression of nitric oxide synthase (NOS)-2 (Broderick et al., 2002). These findings were replicated subsequently and extended to show that the progression of the disease was suppressed by an agonist CD200R antibody (Copland et al., 2007). The modulatory role for CD200 in EAU was also identified in a rat model and, in this case, the evidence indicated that blocking CD200-CD200R interaction by an antibody accelerated the onset and severity of symptoms (Banerjee and Dick, 2004). Experimentally-induced glaucoma, caused by injecting hypertonic saline into the superior episcleral aqueous drainage vein, is another inflammatory and degenerative condition of the eye which is associated with a time-related increase in microglial activation; like EAU a role for CD200-CD200R has been implicated by the finding that the microglial activation is coupled with a decrease in CD200 and evidence of retinal ganglion cell death (Taylor et al., 2011).

#### **5.2 CD200-CD200R interaction and Multiple Sclerosis**

Multiple sclerosis is a chronic, progressive, disabling autoimmune disease. The generallyaccepted view is that the disease is caused by uncontrolled inflammatory T cell responses to

Analysis of the Impact of CD200 on Neurodegenerative Diseases 333

Fig. 3. Immunization with MOG increases microglial activation and decreases CD200 expression. Immunization of mice with MOG induced clinical signs which became evident after 7 days. This was accompanied, in the spinal cord, by a time-related increase in CD40 mRNA, which was significant 10 and 21 days post-immunization (\*p < 0.05; ANOVA; Figure 3b) and a significant decrease in CD200 mRNA (\*\*p < 0.01; ANOVA; Figure 3c). A significant inverse correlation between CD200 mRNA and CD40 mRNA is demonstrated

(p = 0.0017; Figure 3d). IL-4 mRNA expression was significantly decreased in tissue

Fig. 4. The loss of IL-4 leads to more profound clinical symptoms of EAE. The symptoms of EAE developed more rapidly in IL-4-deficient mice compared with wildtype mice (Figure 3a). CD200 mRNA (Figure 3b) and CD200 protein (Figure 3c) were both decreased in tissue prepared from mice with EAE at the end of the 21-day experiment (+p < 0.05; +++p < 0.001; ANOVA; Figure 3b,c) and a decrease in both was identified in tissue prepared from IL-4-

deficient, compared with wildtype, mice (\*p < 0.05; ANOVA; Figure 3b,c)

prepared from animals with EAE (\*p< 0.05; ANOVA; Figure 3e)

self antigens (myelin) in the brain and spinal cord. This results in a cascade of events which triggers inflammation, as a consequence of microglial and macrophage activation, and is followed by demyelination and degeneration of axons. One of the characteristics of this disease is the presence of inflammatory plaques, which are detected by magnetic resonance imaging (MRI), and post mortem examination has established that the brain lesions are associated with the presence of inflammatory cells (Frohman et al., 2006).

#### **5.2.1 What factors contribute to the pathogenesis of EAE?**

A great deal of progress in understanding the mechanisms which precipitate the disease has been made by examining changes which trigger disease symptoms in the widely-used animal model of multiple sclerosis, EAE. EAE is induced by stimulating an immune response directed against CNS antigens, such as myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG). EAE can be induced by immunisation with these myelin antigens in adjuvant or by adoptive transfer of myelinspecific T cells, both of which result in inflammatory infiltrates into the CNS and demyelination (Stromnes and Goverman, 2006).

Like multiple sclerosis, EAE is characterized by infiltration of macrophages and CD4+ T cells into the CNS, accompanied by microglial activation which, together, are responsible for the temporary paralysis that typifies the disease. The symptoms have been shown to be exaggerated, and the onset of the symptoms is hastened, when CD200-CD200R interaction is attenuated (Hoek et al., 2000, Wright et al., 2000, Meuth et al., 2008). Specifically, initial symptoms following MOG injection appeared 2-3 days earlier in CD200-/- mice than in wildtype mice, and expression of iNOS and CD68 were markedly increased in spinal cord of these mice 7 days after immunization (Hoek et al., 2000), while more severe symptoms were also observed (Wright et al., 2000). Consistently, an anti-CD200R antibody increased the severity of the symptoms and, in parallel, increased T cell infiltration and macrophage numbers in the spinal cord of MOG-treated mice (Meuth et al., 2008). Furthermore, the Wlds mouse, which exhibits unique protection against neurodegenerative conditions, including EAE, overexpresses CD200 (Chitnis *et al*., 2007). Interestingly, CD200 expression was reported to be decreased, in parallel with another NIReg, CD47, in laser-dissected active lesions of the post mortem brain of individuals with multiple sclerosis, although CD200R expression was unchanged and there was also no evidence of a change in SIRPα expression (Koning et al., 2007). However more recent studies revealed that CD200 was expressed on astrocytes associated with lesions in multiple sclerosis (Koning et al., 2009).

#### **5.2.2 The development of EAE is associated with altered CD200 expression**

MOG-induced EAE is typified by the development of clinical signs which appear initially at about day 7 post-immunization; the well-defined changes progress from the initial flaccid paralysis manifest by a limp tail and developing to paralysis in hindlimbs and ultimately the forelimbs (Stromnes and Goverman, 2006). We have investigated the accompanying changes induced in microglial activation and CD200 in the spinal cord following immunization (Figure 3). CD40 mRNA, which is indicative of microglial activation, increased after immunization whereas CD200 mRNA expression decreased and a significant inverse relationship between the 2 measures was observed. IL-4, which modulates CD200 expression, was decreased at the end of the experiment, paralleling the change in CD200. Similar data were obtained in the hippocampus (not shown).

self antigens (myelin) in the brain and spinal cord. This results in a cascade of events which triggers inflammation, as a consequence of microglial and macrophage activation, and is followed by demyelination and degeneration of axons. One of the characteristics of this disease is the presence of inflammatory plaques, which are detected by magnetic resonance imaging (MRI), and post mortem examination has established that the brain lesions are

A great deal of progress in understanding the mechanisms which precipitate the disease has been made by examining changes which trigger disease symptoms in the widely-used animal model of multiple sclerosis, EAE. EAE is induced by stimulating an immune response directed against CNS antigens, such as myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG). EAE can be induced by immunisation with these myelin antigens in adjuvant or by adoptive transfer of myelinspecific T cells, both of which result in inflammatory infiltrates into the CNS and

Like multiple sclerosis, EAE is characterized by infiltration of macrophages and CD4+ T cells into the CNS, accompanied by microglial activation which, together, are responsible for the temporary paralysis that typifies the disease. The symptoms have been shown to be exaggerated, and the onset of the symptoms is hastened, when CD200-CD200R interaction is attenuated (Hoek et al., 2000, Wright et al., 2000, Meuth et al., 2008). Specifically, initial symptoms following MOG injection appeared 2-3 days earlier in CD200-/- mice than in wildtype mice, and expression of iNOS and CD68 were markedly increased in spinal cord of these mice 7 days after immunization (Hoek et al., 2000), while more severe symptoms were also observed (Wright et al., 2000). Consistently, an anti-CD200R antibody increased the severity of the symptoms and, in parallel, increased T cell infiltration and macrophage numbers in the spinal cord of MOG-treated mice (Meuth et al., 2008). Furthermore, the Wlds mouse, which exhibits unique protection against neurodegenerative conditions, including EAE, overexpresses CD200 (Chitnis *et al*., 2007). Interestingly, CD200 expression was reported to be decreased, in parallel with another NIReg, CD47, in laser-dissected active lesions of the post mortem brain of individuals with multiple sclerosis, although CD200R expression was unchanged and there was also no evidence of a change in SIRPα expression (Koning et al., 2007). However more recent studies revealed that CD200 was expressed on astrocytes associated with lesions in

**5.2.2 The development of EAE is associated with altered CD200 expression** 

Similar data were obtained in the hippocampus (not shown).

MOG-induced EAE is typified by the development of clinical signs which appear initially at about day 7 post-immunization; the well-defined changes progress from the initial flaccid paralysis manifest by a limp tail and developing to paralysis in hindlimbs and ultimately the forelimbs (Stromnes and Goverman, 2006). We have investigated the accompanying changes induced in microglial activation and CD200 in the spinal cord following immunization (Figure 3). CD40 mRNA, which is indicative of microglial activation, increased after immunization whereas CD200 mRNA expression decreased and a significant inverse relationship between the 2 measures was observed. IL-4, which modulates CD200 expression, was decreased at the end of the experiment, paralleling the change in CD200.

associated with the presence of inflammatory cells (Frohman et al., 2006).

**5.2.1 What factors contribute to the pathogenesis of EAE?** 

demyelination (Stromnes and Goverman, 2006).

multiple sclerosis (Koning et al., 2009).

Fig. 3. Immunization with MOG increases microglial activation and decreases CD200 expression. Immunization of mice with MOG induced clinical signs which became evident after 7 days. This was accompanied, in the spinal cord, by a time-related increase in CD40 mRNA, which was significant 10 and 21 days post-immunization (\*p < 0.05; ANOVA; Figure 3b) and a significant decrease in CD200 mRNA (\*\*p < 0.01; ANOVA; Figure 3c). A significant inverse correlation between CD200 mRNA and CD40 mRNA is demonstrated (p = 0.0017; Figure 3d). IL-4 mRNA expression was significantly decreased in tissue prepared from animals with EAE (\*p< 0.05; ANOVA; Figure 3e)

Fig. 4. The loss of IL-4 leads to more profound clinical symptoms of EAE. The symptoms of EAE developed more rapidly in IL-4-deficient mice compared with wildtype mice (Figure 3a). CD200 mRNA (Figure 3b) and CD200 protein (Figure 3c) were both decreased in tissue prepared from mice with EAE at the end of the 21-day experiment (+p < 0.05; +++p < 0.001; ANOVA; Figure 3b,c) and a decrease in both was identified in tissue prepared from IL-4 deficient, compared with wildtype, mice (\*p < 0.05; ANOVA; Figure 3b,c)

Analysis of the Impact of CD200 on Neurodegenerative Diseases 335

progression of the diseases. Interestingly, the protective effects in AD may be confined to particular subpopulations. Recent retrospective studies indicated that statin therapy

It is unclear whether microglial activation directly contributes to neuronal loss in PD (or indeed AD) but post mortem examination has established that activated microglia are clustered at high density in the SN (McGeer et al., 1988), the most vulnerable area of the brain in PD due to the low intracellular glutathione concentration and high iron level within nigrostriatal dopaminergic neurons (Sian et al., 1994). Indeed dopaminergic neurons are especially vulnerable to LPS-induced neurodegeneration (Smidt, 2009). Interestingly the number of MHCII-positive cells in this area increases in parallel with neuronal loss (Imamura *et al.* 2003). Similarly, an inverse relationship between 11C-(R)- PK11195 binding (which is indicative of microglial activation) in the midbrain and binding of [11C]CFT to the dopamine transporter (which reflects the viability of presynaptic dopaminergic neurons) in the putamen has been described. It has been reported that the combination of binding of these 2 tracers positively correlates with motor deficits in early PD (Ouchi *et al.* 2009). The correlative changes suggest a role for microglial activation in the pathogenesis of PD but do not address the question whether microglial activation plays an explicit role in dopaminergic cell death; animal models have been used to explore this. Environmental factors including pesticides have been implicated in the aetiology of PD and therefore experimental models of the disease include those in which animals are treated with rotenone or paraquat (Cicchetti et al., 2009); the loss of dopaminergic neurons in these models appears to be mediated by microglia since the superoxide which is considered to be pivotal to inducing cell damage was generated from microglia (Gao et al., 2002, Wu et al., 2005). Another animal model of PD involves prenatal exposure to LPS, which ultimately causes protracted inflammation and loss of dopaminergic neurons which progresses with subsequent insults; data from this model suggests that the priming of microglia is responsible for the ongoing degeneration and has led to the development of the 'multiple hit' hypothesis (Ling *et al.* 2006). An important tenet of this theory is that prolonged inflammation, rather than an acute inflammatory response, is responsible for the progressive neuronal loss (Park *et al.* 2009, Long-Smith et al. 2009). Interestingly, an age-related increase in microglialactivation in the SN has been reported (Beach et al., 2007) and the suggestion is that this

The most commonly-used models of PD which lead to neurodegeneration of dopaminergic neurons and induce Parkinson-like symptoms involve injection of rotenone or 6-hydroxydopamine (6-OHDA). It has recently been reported that rotenone+ironinduced dopaminergic neurotoxicity is mediated by microglia and that the toxicity is enhanced by a CD200R blocking antibody (Wang et al., 2011). The evidence indicated that microglia were the source of superoxide, that production was enhanced by the antibody and that inhibiting CD200R activation in microglia has detrimental effects on neuronal function. In addition to the Parkinson-like symptoms, injection of 6-OHDA also induces marked microglial activation (Long-Smith et al., 2009). These findings and the observations of other groups over many years (Chen et al., 1998, Le et al., 2001, Liu and Hong, 2003, Kim and Joh, 2006, Purisai et al., 2007) have provided significant support for the thesis that activated microglia play an important part in the onset and/or progression

reduced the risk of developing PD and AD.

'priming' may contribute to development of the disease.

of PD.

#### **5.2.3 IL-4 modulates CD200 expression and the course of EAE**

Because the evidence indicates a close parallel between IL-4 and CD200, and because CD200 appears to be linked with the increase in microglial activation which contributes to the inflammatory changes in EAE, the onset and severity of clinical symptoms were compared following MOG immunization in wildtype and IL-4-/- mice. The evidence indicates that loss of IL-4 exacerbates the clinical symptoms (Figure 4a) and CD200 mRNA expression (Figure 4b), as well as CD200 protein (Figure 4c) was decreased in spinal cord prepared from IL-4-/ mice, compared with wildtype mice. Moreover, the decrease in both measures was significantly greater in mice with EAE compared with controls.

#### **5.2.4 CD200R ligation on specific T cell subtypes may contribute to the development of EAE**

A comprehensive analysis of the expression of CD200R on cells and tissue prepared from mouse and humans revealed that expression levels were greatest on macrophages, mast cells and dendritic cells, and in lymph nodes, spleen, bone marrow and, to a lesser extent, in lung and liver (Wright et al., 2003). However the receptor was also found on polarized Th2 cells, whereas expression on polarized Th1 cells was markedly less; this differential expression on T cells was observed in mice and human cells (Wright et al., 2003). Subsequent analysis indicated that CD200R was expressed to a greater extent on CD4+ effector memory cells compared with central memory cells and naïve cells. Similarly CD8+ central memory cells had higher expression than naïve cells (Rijkers et al., 2008). Clearly these findings suggest that CD200R ligation can modulate T cell function in addition to myeloid cell function; this may contribute to the exaggerated symptoms in autoimmune diseases, for example EAE.

#### **6. Evidence of a role for CD200-CD200R interaction in other neurodegenerative diseases**

#### **6.1 Parkinson's Disease**

PD is the most common movement disorder and the second most common neurodegenerative disease. It shares some characteristics with AD. Both are, at least to some extent, age-related disorders, characterized by neuroinflammatory changes accompanied by increased expression of inflammatory cytokines. PD is a chronic and progressive disorder, resulting in the selective loss of dopaminergic neurons within the substantia nigra (SN) of the midbrain. As the disease progresses there is gradual circuitry degeneration and neuronal loss within the nigrostriatal pathway, producing cognitive and psychiatric symptoms, as well as disturbances in movement (Braak et al, 2003). Cytoplasmic accumulations of insoluble proteins are likely to significantly contribute to the neuronal loss apparent in both AD and PD. Cognitive dysfunction is particularly marked in AD but there is also evidence of deterioration in cognition in PD. It has been suggested that the microglial activation which is prevalent in hippocampus and parahippocampal regions, coupled with the decrease in hippocampal volume (Laakso et al., 1996) and associated neuronal loss in the limbic areas (Emre, 2003a, b) may account for the cognitive dysfunction in PD. Although clinical trials have failed to show that NSAIDs are effective in treating AD or PD, epidemiological studies have suggested that chronic treatment with NSAIDs reduces the risk of both diseases suggesting that inflammatory changes may contribute to the

Because the evidence indicates a close parallel between IL-4 and CD200, and because CD200 appears to be linked with the increase in microglial activation which contributes to the inflammatory changes in EAE, the onset and severity of clinical symptoms were compared following MOG immunization in wildtype and IL-4-/- mice. The evidence indicates that loss of IL-4 exacerbates the clinical symptoms (Figure 4a) and CD200 mRNA expression (Figure 4b), as well as CD200 protein (Figure 4c) was decreased in spinal cord prepared from IL-4-/ mice, compared with wildtype mice. Moreover, the decrease in both measures was

**5.2.4 CD200R ligation on specific T cell subtypes may contribute to the development** 

A comprehensive analysis of the expression of CD200R on cells and tissue prepared from mouse and humans revealed that expression levels were greatest on macrophages, mast cells and dendritic cells, and in lymph nodes, spleen, bone marrow and, to a lesser extent, in lung and liver (Wright et al., 2003). However the receptor was also found on polarized Th2 cells, whereas expression on polarized Th1 cells was markedly less; this differential expression on T cells was observed in mice and human cells (Wright et al., 2003). Subsequent analysis indicated that CD200R was expressed to a greater extent on CD4+ effector memory cells compared with central memory cells and naïve cells. Similarly CD8+ central memory cells had higher expression than naïve cells (Rijkers et al., 2008). Clearly these findings suggest that CD200R ligation can modulate T cell function in addition to myeloid cell function; this may contribute to the exaggerated symptoms in autoimmune

PD is the most common movement disorder and the second most common neurodegenerative disease. It shares some characteristics with AD. Both are, at least to some extent, age-related disorders, characterized by neuroinflammatory changes accompanied by increased expression of inflammatory cytokines. PD is a chronic and progressive disorder, resulting in the selective loss of dopaminergic neurons within the substantia nigra (SN) of the midbrain. As the disease progresses there is gradual circuitry degeneration and neuronal loss within the nigrostriatal pathway, producing cognitive and psychiatric symptoms, as well as disturbances in movement (Braak et al, 2003). Cytoplasmic accumulations of insoluble proteins are likely to significantly contribute to the neuronal loss apparent in both AD and PD. Cognitive dysfunction is particularly marked in AD but there is also evidence of deterioration in cognition in PD. It has been suggested that the microglial activation which is prevalent in hippocampus and parahippocampal regions, coupled with the decrease in hippocampal volume (Laakso et al., 1996) and associated neuronal loss in the limbic areas (Emre, 2003a, b) may account for the cognitive dysfunction in PD. Although clinical trials have failed to show that NSAIDs are effective in treating AD or PD, epidemiological studies have suggested that chronic treatment with NSAIDs reduces the risk of both diseases suggesting that inflammatory changes may contribute to the

**5.2.3 IL-4 modulates CD200 expression and the course of EAE** 

significantly greater in mice with EAE compared with controls.

**6. Evidence of a role for CD200-CD200R interaction in other** 

**of EAE** 

diseases, for example EAE.

**6.1 Parkinson's Disease** 

**neurodegenerative diseases** 

progression of the diseases. Interestingly, the protective effects in AD may be confined to particular subpopulations. Recent retrospective studies indicated that statin therapy reduced the risk of developing PD and AD.

It is unclear whether microglial activation directly contributes to neuronal loss in PD (or indeed AD) but post mortem examination has established that activated microglia are clustered at high density in the SN (McGeer et al., 1988), the most vulnerable area of the brain in PD due to the low intracellular glutathione concentration and high iron level within nigrostriatal dopaminergic neurons (Sian et al., 1994). Indeed dopaminergic neurons are especially vulnerable to LPS-induced neurodegeneration (Smidt, 2009). Interestingly the number of MHCII-positive cells in this area increases in parallel with neuronal loss (Imamura *et al.* 2003). Similarly, an inverse relationship between 11C-(R)- PK11195 binding (which is indicative of microglial activation) in the midbrain and binding of [11C]CFT to the dopamine transporter (which reflects the viability of presynaptic dopaminergic neurons) in the putamen has been described. It has been reported that the combination of binding of these 2 tracers positively correlates with motor deficits in early PD (Ouchi *et al.* 2009). The correlative changes suggest a role for microglial activation in the pathogenesis of PD but do not address the question whether microglial activation plays an explicit role in dopaminergic cell death; animal models have been used to explore this. Environmental factors including pesticides have been implicated in the aetiology of PD and therefore experimental models of the disease include those in which animals are treated with rotenone or paraquat (Cicchetti et al., 2009); the loss of dopaminergic neurons in these models appears to be mediated by microglia since the superoxide which is considered to be pivotal to inducing cell damage was generated from microglia (Gao et al., 2002, Wu et al., 2005). Another animal model of PD involves prenatal exposure to LPS, which ultimately causes protracted inflammation and loss of dopaminergic neurons which progresses with subsequent insults; data from this model suggests that the priming of microglia is responsible for the ongoing degeneration and has led to the development of the 'multiple hit' hypothesis (Ling *et al.* 2006). An important tenet of this theory is that prolonged inflammation, rather than an acute inflammatory response, is responsible for the progressive neuronal loss (Park *et al.* 2009, Long-Smith et al. 2009). Interestingly, an age-related increase in microglialactivation in the SN has been reported (Beach et al., 2007) and the suggestion is that this 'priming' may contribute to development of the disease.

The most commonly-used models of PD which lead to neurodegeneration of dopaminergic neurons and induce Parkinson-like symptoms involve injection of rotenone or 6-hydroxydopamine (6-OHDA). It has recently been reported that rotenone+ironinduced dopaminergic neurotoxicity is mediated by microglia and that the toxicity is enhanced by a CD200R blocking antibody (Wang et al., 2011). The evidence indicated that microglia were the source of superoxide, that production was enhanced by the antibody and that inhibiting CD200R activation in microglia has detrimental effects on neuronal function. In addition to the Parkinson-like symptoms, injection of 6-OHDA also induces marked microglial activation (Long-Smith et al., 2009). These findings and the observations of other groups over many years (Chen et al., 1998, Le et al., 2001, Liu and Hong, 2003, Kim and Joh, 2006, Purisai et al., 2007) have provided significant support for the thesis that activated microglia play an important part in the onset and/or progression of PD.

Analysis of the Impact of CD200 on Neurodegenerative Diseases 337

Fig. 5. 6-OHDA injection leads to dopaminergic cell loss and a marked reduction in CD200 immunoreactivity, coupled with microglial activation. Rats were anaesthetized with equal amounts of xylazine hydrochloride and ketamine hydrochloride (0.2ml/100g body weight; 1.5 ml of each compound dissolved in 7 ml PBS). Rats received a single injection of 6-OHDA (2μg/μl) into the medial forebrain bundle (AP –2.2 mm, ML + 1.5 mm from bregma; depth 7.8 mm). Rats were killed 10 days later. a-d: CD200 immunoreactivity (green) and tyrosine hydroxylase (TH) immunoreactivity was evident in contralateral and ipsilateral SN and there was clear evidence of co-localization indicating the presence of CD200 on dopaminergic neurons. There was a marked decrease in TH immunoreactivity in sections prepared from the ipsilateral SN of rats which received 6-OHDA, indicative of substantial dopaminergic cell loss but no changes were observed in the other treatment groups. TH loss was

accompanied by a loss in CD200 immunoreactivity. e-h: TH expression (green) was similar in the contralateral SN obtained from sham- or 6-OHDA-treated rats and in the ipsilateral side of sham-treated rats. In contrast, a marked change in morphology indicative of dopaminergic cell loss was observed in sections prepared from the ipsilateral SN of rats which received 6-OHDA. Microglial activation was assessed by evaluating OX42

immunoreactivity (red staining); marked staining was observed in sections prepared from

Ischaemia induces a profound disturbance in homeostasis and significant pathology in the brain. Among the earliest changes is infiltration of neutrophils into the brain parenchyma and the evidence indicates that, in the endothelin model of stroke, these cells accumulated in the core of the lesion 2 weeks after injection and correlated with the infarct volume (Weston

the ipsilateral SN of rats which received 6-OHDA but in none of the other groups

**6.3 Stroke** 

In an effort to further address this question, and specifically to evaluate whether CD200 may play a role in modulating microglial activation which accompanies the loss of dopaminergic neurons following 6-OHDA injection, we examined the expression of CD200 in the ipsilateral and contralateral SN of rats following unilateral injection of 6-OHDA into the medial forebrain bundle. Immunocytochemical analysis of sections prepared from these animals revealed that there was marked dopaminergic cell loss in the side of the brain in which the 6-OHDA injection was made, as shown by decreased expression of tyrosine hydroxylase (Figure 5d), but that there was no evidence of cell loss on the contralateral side (Figure 5c). No cell loss was evident on either the ipsilateral or contralateral side of shamtreated rats Figure 5a,b). The data show that the marked 6-OHDA-induced dopaminergic cell loss in the ipsilateral SN was coupled with a marked decrease in CD200 expression (Figure 5d) whereas there was no discernible loss in the contralateral side of 6-OHDAinjected animals (Figure 5c) or the ipsilateral or contralateral side of sham-treated rats (Figure 5a,b). The loss of dopaminergic neurons was also associated with marked microglial activation as indicated by increased OX42 staining (red; Figure 5h); this is consistent with previous evidence indicating that loss of CD200 is linked with microglial activation (Lyons et al., 2007a). There was no evidence of microglial staining in sections prepared from the contralateral side of 6-OHDA-injected animals (Figure 5g) or the ipsilateral or contralateral side of sham-treated rats (Figure 5e,f).

#### **6.2 Alzheimer's Disease**

Despite an enormous effort, the molecular/cellular events which trigger AD remain unknown. It is undoubtedly the case that neuroinflammatory changes characterize the disease with evidence of profound microglial activation and, specifically, activated microglia and astrocytes clustered around Aβ-containing plaques (Xiang et al., 2006) and blood vessels (McGeer and McGeer, 2003) where amyloid deposits are also observed. As indicated above, several reports suggest that NSAID treatment reduces the risk of developing AD (McGeer and McGeer, 1999, Szekely et al., 2008, Vlad et al., 2008, Breitner et al., 2009) but NSAIDs are of little value in treating the disease. One possible explanation for this might be that inflammatory changes occur very early in the disease, prior to development of symptoms, and that preventing or delaying inflammation is beneficial because it is factor which contributes to the later neurodegenerative changes. A corollary to this previously-rehearsed proposal is that anti-inflammatory agents will not be beneficial once neurodegenerative changes are advanced. In support of this view, it has been consistently shown that inflammatory cytokines like IL-1β, IL-6 and TNFα negatively impact on neuronal and synaptic function (Lynch, 2010), and that these cytokines can contribute to neuronal cell death (Thornton et al., 2008, Long-Smith et al., 2010). Since activated glia, particularly microglia, are responsible for releasing these cytokines, it could be argued that targeting these cells might be a reasonable strategy for the treatment of AD, at least in its very early stages. This argument has been advanced by Walker and colleagues, who reported that CD200 expression was decreased in brains of individuals with AD. Thus sections prepared from inferior temporal gyrus of non-demented individuals exhibited colocalization of CD200 with NeuN but a marked loss of CD200 immunoreactivity was observed in sections prepared from post-mortem brains of AD patients. An AD-associated decrease in CD200R was also observed. Furthermore, the evidence suggested that the plaque density, and also the neurofibrillary tangle score, was inversely related to CD200R expression (Walker et al., 2009).

In an effort to further address this question, and specifically to evaluate whether CD200 may play a role in modulating microglial activation which accompanies the loss of dopaminergic neurons following 6-OHDA injection, we examined the expression of CD200 in the ipsilateral and contralateral SN of rats following unilateral injection of 6-OHDA into the medial forebrain bundle. Immunocytochemical analysis of sections prepared from these animals revealed that there was marked dopaminergic cell loss in the side of the brain in which the 6-OHDA injection was made, as shown by decreased expression of tyrosine hydroxylase (Figure 5d), but that there was no evidence of cell loss on the contralateral side (Figure 5c). No cell loss was evident on either the ipsilateral or contralateral side of shamtreated rats Figure 5a,b). The data show that the marked 6-OHDA-induced dopaminergic cell loss in the ipsilateral SN was coupled with a marked decrease in CD200 expression (Figure 5d) whereas there was no discernible loss in the contralateral side of 6-OHDAinjected animals (Figure 5c) or the ipsilateral or contralateral side of sham-treated rats (Figure 5a,b). The loss of dopaminergic neurons was also associated with marked microglial activation as indicated by increased OX42 staining (red; Figure 5h); this is consistent with previous evidence indicating that loss of CD200 is linked with microglial activation (Lyons et al., 2007a). There was no evidence of microglial staining in sections prepared from the contralateral side of 6-OHDA-injected animals (Figure 5g) or the ipsilateral or contralateral

Despite an enormous effort, the molecular/cellular events which trigger AD remain unknown. It is undoubtedly the case that neuroinflammatory changes characterize the disease with evidence of profound microglial activation and, specifically, activated microglia and astrocytes clustered around Aβ-containing plaques (Xiang et al., 2006) and blood vessels (McGeer and McGeer, 2003) where amyloid deposits are also observed. As indicated above, several reports suggest that NSAID treatment reduces the risk of developing AD (McGeer and McGeer, 1999, Szekely et al., 2008, Vlad et al., 2008, Breitner et al., 2009) but NSAIDs are of little value in treating the disease. One possible explanation for this might be that inflammatory changes occur very early in the disease, prior to development of symptoms, and that preventing or delaying inflammation is beneficial because it is factor which contributes to the later neurodegenerative changes. A corollary to this previously-rehearsed proposal is that anti-inflammatory agents will not be beneficial once neurodegenerative changes are advanced. In support of this view, it has been consistently shown that inflammatory cytokines like IL-1β, IL-6 and TNFα negatively impact on neuronal and synaptic function (Lynch, 2010), and that these cytokines can contribute to neuronal cell death (Thornton et al., 2008, Long-Smith et al., 2010). Since activated glia, particularly microglia, are responsible for releasing these cytokines, it could be argued that targeting these cells might be a reasonable strategy for the treatment of AD, at least in its very early stages. This argument has been advanced by Walker and colleagues, who reported that CD200 expression was decreased in brains of individuals with AD. Thus sections prepared from inferior temporal gyrus of non-demented individuals exhibited colocalization of CD200 with NeuN but a marked loss of CD200 immunoreactivity was observed in sections prepared from post-mortem brains of AD patients. An AD-associated decrease in CD200R was also observed. Furthermore, the evidence suggested that the plaque density, and also the neurofibrillary tangle score, was inversely related to CD200R

side of sham-treated rats (Figure 5e,f).

**6.2 Alzheimer's Disease** 

expression (Walker et al., 2009).

Fig. 5. 6-OHDA injection leads to dopaminergic cell loss and a marked reduction in CD200 immunoreactivity, coupled with microglial activation. Rats were anaesthetized with equal amounts of xylazine hydrochloride and ketamine hydrochloride (0.2ml/100g body weight; 1.5 ml of each compound dissolved in 7 ml PBS). Rats received a single injection of 6-OHDA (2μg/μl) into the medial forebrain bundle (AP –2.2 mm, ML + 1.5 mm from bregma; depth 7.8 mm). Rats were killed 10 days later. a-d: CD200 immunoreactivity (green) and tyrosine hydroxylase (TH) immunoreactivity was evident in contralateral and ipsilateral SN and there was clear evidence of co-localization indicating the presence of CD200 on dopaminergic neurons. There was a marked decrease in TH immunoreactivity in sections prepared from the ipsilateral SN of rats which received 6-OHDA, indicative of substantial dopaminergic cell loss but no changes were observed in the other treatment groups. TH loss was accompanied by a loss in CD200 immunoreactivity. e-h: TH expression (green) was similar in the contralateral SN obtained from sham- or 6-OHDA-treated rats and in the ipsilateral side of sham-treated rats. In contrast, a marked change in morphology indicative of dopaminergic cell loss was observed in sections prepared from the ipsilateral SN of rats which received 6-OHDA. Microglial activation was assessed by evaluating OX42 immunoreactivity (red staining); marked staining was observed in sections prepared from the ipsilateral SN of rats which received 6-OHDA but in none of the other groups

#### **6.3 Stroke**

Ischaemia induces a profound disturbance in homeostasis and significant pathology in the brain. Among the earliest changes is infiltration of neutrophils into the brain parenchyma and the evidence indicates that, in the endothelin model of stroke, these cells accumulated in the core of the lesion 2 weeks after injection and correlated with the infarct volume (Weston

Analysis of the Impact of CD200 on Neurodegenerative Diseases 339

reduced the associated neurodegenerative changes (Relton and Rothwell, 1992). However, more recent findings have indicated that the reduction in synaptic responses following ischaemia, and the decrease in LTP, were partially reversed following intra-arterial injection of microglia into rats (Hayashi et al., 2006). Thus microglia exert both a positive and

We investigated microglial activation 7 days after endothelin-1 injection and demonstrate that markers of activation were increased while CD200 was decreased. Thus staining of the tissue revealed that there was marked cell loss (Figure 6a) and extensive OX6 staining (Figure 6b), with particularly marked staining in the striatum. PCR analysis revealed that there was a significant increase in expression of MHCII mRNA (Figure 6c) as well as another marker of microglial activation, CD40 mRNA (Figure 6e), in striatal tissue prepared from endothelin 1-treated rats compared with controls (\*p < 0.05). CD200 mRNA was markedly decreased in striatal tissue prepared from endothelin-1-injected animals and, interestingly, there was a significant inverse relationship between these CD200 mRNA and CD40 mRNA (p = 0.0039; Figure 6f). These findings indicate that there is a persistent increase in microglial activation following ischaemia which has been reported previously (Denes et al., 2010). The underlying cause of this increase has not been fully explained. The present results suggest that the decrease in CD200, which might be anticipated to accompany the loss of neurons,

In the past decade or so, it has become clear that microglia are maintained in a non-activated state by soluble factors including growth factors and anti-inflammatory cytokines, as well as cell-cell interactions. Among the ligand-receptor pairs which play a key role in modulating microglial activation is CD200-CD200R and the evidence indicates that when CD200R activation is disrupted, for example in CD200-deficient mice, the result is activation of microglia and macrophages, accompanied by inflammatory changes, and exacerbation of changes in models of autoimmune disease. The experimental evidence certainly suggests that targeting the interaction between CD200 and its receptor is a powerful weapon in attenuating inflammation and there is a growing body of evidence suggesting that disruption of the interaction, in combination with microglial and/or macrophage activation occurs in

> **Decreased CD200**

> > **Microglial activation**

**Decreased CD200Ractivated signaling**

**Neuronal loss**

**Increased secretion of inflammatory mediators**

Fig. 7. Proposed role for CD200-CD200R interaction in neurodegenerative changes

negative impact.

may be a contributory factor.

**6.4 Conclusions** 

 **Age**

 **Neurodegenerative diseases Neuroinflammatory diseases**

Fig. 6. CD40 mRNA is increased and CD200 mRNA is decreased in striatum following endothelin injection. Male Wistar rats (3 months; 270-350g; BioResources Unit, Trinity College, Dublin, Ireland) were anaesthetized with isofluorane (5% in O2), placed in a stereotaxic frame and injected with endothelin-1 (ET-1; 600pmol; 3 µl), delivered through a drill hole (0.52 cm lateral to midline, 0.05 cm posterior to bregma; depth 0.05cm from the base of the skull ) (Moyanova et al., 2003). Animals were killed and brain tissue harvested 7 days after injection. Cryostat sections were prepared from part of the brain and striatum was taken from the remaining brain tissue to analyse expression of CD40 and CD200 mRNA. Endothelin induced significant neuronal loss (Figure 1a) and microglial activation, as assessed by OX6 staining, particularly in striatum (Figure 1b). Analysis of striatal tissue indicated that MHCII mRNA and CD40 mRNA were significantly increased in tissue prepared from endothelin-1-injected animals (\*p < 0.05; student's t test for independent means; Figure 1c,d) whereas CD200 mRNA was significantly decreased (\*\*p < 0.01; student's t test for independent means; Figure 1e); a significant inverse relationship between CD40 mRNA and CD200 mRNA was observed (p = 0.0039; Figure 1f)

et al., 2007). Neutrophils have also been shown to contribute to the increase in BBB permeability following stroke (McColl et al., 2007). Neutrophils release inflammatory cytokines, chemokines and reactive oxygen species, all of which contribute to the pathology, and also recruitment of immune cells (Denes et al., 2010). However microglia can also act similarly and the evidence has indicated that microglial activation is increased following ischaemia but the effect of this activation remains unclear. On the one hand, these cells may contribute to the cell damage because of their ability to secrete inflammatory molecules but their reparative role has also been clearly identified (Denes et al., 2010). It has been known for almost 2 decades that release of the inflammatory cytokine, IL-1β, was increased following ischaemia and that the endogenous antagonist, IL-1 receptor antagonist (IL-1ra) reduced the associated neurodegenerative changes (Relton and Rothwell, 1992). However, more recent findings have indicated that the reduction in synaptic responses following ischaemia, and the decrease in LTP, were partially reversed following intra-arterial injection of microglia into rats (Hayashi et al., 2006). Thus microglia exert both a positive and negative impact.

We investigated microglial activation 7 days after endothelin-1 injection and demonstrate that markers of activation were increased while CD200 was decreased. Thus staining of the tissue revealed that there was marked cell loss (Figure 6a) and extensive OX6 staining (Figure 6b), with particularly marked staining in the striatum. PCR analysis revealed that there was a significant increase in expression of MHCII mRNA (Figure 6c) as well as another marker of microglial activation, CD40 mRNA (Figure 6e), in striatal tissue prepared from endothelin 1-treated rats compared with controls (\*p < 0.05). CD200 mRNA was markedly decreased in striatal tissue prepared from endothelin-1-injected animals and, interestingly, there was a significant inverse relationship between these CD200 mRNA and CD40 mRNA (p = 0.0039; Figure 6f). These findings indicate that there is a persistent increase in microglial activation following ischaemia which has been reported previously (Denes et al., 2010). The underlying cause of this increase has not been fully explained. The present results suggest that the decrease in CD200, which might be anticipated to accompany the loss of neurons, may be a contributory factor.

#### **6.4 Conclusions**

338 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

Fig. 6. CD40 mRNA is increased and CD200 mRNA is decreased in striatum following endothelin injection. Male Wistar rats (3 months; 270-350g; BioResources Unit, Trinity College, Dublin, Ireland) were anaesthetized with isofluorane (5% in O2), placed in a stereotaxic frame and injected with endothelin-1 (ET-1; 600pmol; 3 µl), delivered through a drill hole (0.52 cm lateral to midline, 0.05 cm posterior to bregma; depth 0.05cm from the base of the skull ) (Moyanova et al., 2003). Animals were killed and brain tissue harvested 7 days after injection. Cryostat sections were prepared from part of the brain and striatum was taken from the remaining brain tissue to analyse expression of CD40 and CD200 mRNA. Endothelin induced significant neuronal loss (Figure 1a) and microglial activation, as assessed by OX6 staining, particularly in striatum (Figure 1b). Analysis of striatal tissue indicated that MHCII mRNA and CD40 mRNA were significantly increased in tissue prepared from endothelin-1-injected animals (\*p < 0.05; student's t test for independent means; Figure 1c,d) whereas CD200 mRNA was significantly decreased (\*\*p < 0.01;

student's t test for independent means; Figure 1e); a significant inverse relationship between

et al., 2007). Neutrophils have also been shown to contribute to the increase in BBB permeability following stroke (McColl et al., 2007). Neutrophils release inflammatory cytokines, chemokines and reactive oxygen species, all of which contribute to the pathology, and also recruitment of immune cells (Denes et al., 2010). However microglia can also act similarly and the evidence has indicated that microglial activation is increased following ischaemia but the effect of this activation remains unclear. On the one hand, these cells may contribute to the cell damage because of their ability to secrete inflammatory molecules but their reparative role has also been clearly identified (Denes et al., 2010). It has been known for almost 2 decades that release of the inflammatory cytokine, IL-1β, was increased following ischaemia and that the endogenous antagonist, IL-1 receptor antagonist (IL-1ra)

CD40 mRNA and CD200 mRNA was observed (p = 0.0039; Figure 1f)

In the past decade or so, it has become clear that microglia are maintained in a non-activated state by soluble factors including growth factors and anti-inflammatory cytokines, as well as cell-cell interactions. Among the ligand-receptor pairs which play a key role in modulating microglial activation is CD200-CD200R and the evidence indicates that when CD200R activation is disrupted, for example in CD200-deficient mice, the result is activation of microglia and macrophages, accompanied by inflammatory changes, and exacerbation of changes in models of autoimmune disease. The experimental evidence certainly suggests that targeting the interaction between CD200 and its receptor is a powerful weapon in attenuating inflammation and there is a growing body of evidence suggesting that disruption of the interaction, in combination with microglial and/or macrophage activation occurs in

Fig. 7. Proposed role for CD200-CD200R interaction in neurodegenerative changes

Analysis of the Impact of CD200 on Neurodegenerative Diseases 341

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models of neurodegenerative diseases. Figure 7 presents a schematic diagram which suggests that CD200R activation plays a pivotal role in modulating microglial activation. It is proposed that the secretion of immunomodulatory molecules from activated microglia contributes to the development of neurodegenerative changes which characterize neurodegenerative and neuroinflammatory diseases, and which also occur with age, these changes are inextricably linked with neuronal loss and consequently CD200 expression is decreased resulting in a decrease in signalling through CD200R, completing the continuing cycle of events.

#### **7. Acknowledgements**

Funding was obtained from Science Foundation Ireland and the Health Research Board (Ireland).

#### **8. References**


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**Part 5** 

**Hormonal Control and** 

**Metabolism in Neurodegeneration** 

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## **Part 5**

**Hormonal Control and Metabolism in Neurodegeneration** 

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**15** 

*Russia* 

**Hormonal Signaling Systems** 

Alexander Shpakov, Oksana Chistyakova,

Kira Derkach and Vera Bondareva

**of the Brain in Diabetes Mellitus** 

*Sechenov Institute of Evolutionary Physiology and Biochemistry* 

Diabetes mellitus (DM) is nowadays a major global health problem affecting more than 200 million people worldwide. It is one of the most severe metabolic disorders in humans characterized by hyperglycemia due to a relative or an absolute lack of insulin or the action of insulin on its target tissue or both. Many neurodegenerative disorders, such as diabetic encephalopathy and Alzheimer's disease (AD), are associated with the type 1, insulindependent, and the type 2, non-insulin-dependent, diabetes mellitus (DM1 and DM2). Manifestations of these disorders in diabetic patients include alterations in neurotransmission, electrophysiological abnormalities, structural changes and cognitive deficit (Biessels et al., 2001). In the recent time attention to the neurological consequences of

Many approaches and tools have been used to study etiology and pathogenesis of DM and DM-associated neurodegenerative disorders, and their diagnostics and treatment. The most perspective approaches are based on a combined use of the methods of biochemistry, molecular biology and physiology, they include clinical investigations of diabetic patients and the experimental models of DM and their complications, such as the model of DM1 induced by streptozotocin (STZ) treatment of young or adult rodents, the neonatal model of DM2 induced by the STZ treatment of newborn rats, and also the models of spontaneous DM and nutritional background causing DM2, as well as the models produced by transgenic manipulations or gene knockout techniques are all successfully used to study the

A severe hyperglycemia in DM1, mild hyperglycemia typical of DM2, and recurrent hypoglycemia induced by inadequate insulin therapy are the major factors responsible for the development of CNS complications in DM. The brain is mainly a glucose-dependent organ, which can be damaged by hyper- as well as by hypoglycemia (Scheen, 2010). Being a major problem in clinical practice, hypoglycemia unawareness is associated with an increased risk of coma. Note that low blood glucose level induces negative mood states, primarily self-reported "nervousness" (Boyle & Zrebiec, 2007). Moreover, patients with a history of severe hypoglycemia show a much higher level of anxiety compared to other DM patients (Wredling, 1992). The prolonged influence of mild hypoglycemia on the brain leads to deregulation of many processes in CNS, which underlines the importance of scrupulously avoiding even mild hypoglycemic episodes in patients with DM. Hypoglycemia induces

molecular, cellular and morphological changes in diabetic brain (Shafrir, 2010).

**1. Introduction** 

DM in the CNS has increased considerably.

## **Hormonal Signaling Systems of the Brain in Diabetes Mellitus**

Alexander Shpakov, Oksana Chistyakova, Kira Derkach and Vera Bondareva *Sechenov Institute of Evolutionary Physiology and Biochemistry Russia* 

#### **1. Introduction**

Diabetes mellitus (DM) is nowadays a major global health problem affecting more than 200 million people worldwide. It is one of the most severe metabolic disorders in humans characterized by hyperglycemia due to a relative or an absolute lack of insulin or the action of insulin on its target tissue or both. Many neurodegenerative disorders, such as diabetic encephalopathy and Alzheimer's disease (AD), are associated with the type 1, insulindependent, and the type 2, non-insulin-dependent, diabetes mellitus (DM1 and DM2). Manifestations of these disorders in diabetic patients include alterations in neurotransmission, electrophysiological abnormalities, structural changes and cognitive deficit (Biessels et al., 2001). In the recent time attention to the neurological consequences of DM in the CNS has increased considerably.

Many approaches and tools have been used to study etiology and pathogenesis of DM and DM-associated neurodegenerative disorders, and their diagnostics and treatment. The most perspective approaches are based on a combined use of the methods of biochemistry, molecular biology and physiology, they include clinical investigations of diabetic patients and the experimental models of DM and their complications, such as the model of DM1 induced by streptozotocin (STZ) treatment of young or adult rodents, the neonatal model of DM2 induced by the STZ treatment of newborn rats, and also the models of spontaneous DM and nutritional background causing DM2, as well as the models produced by transgenic manipulations or gene knockout techniques are all successfully used to study the molecular, cellular and morphological changes in diabetic brain (Shafrir, 2010).

A severe hyperglycemia in DM1, mild hyperglycemia typical of DM2, and recurrent hypoglycemia induced by inadequate insulin therapy are the major factors responsible for the development of CNS complications in DM. The brain is mainly a glucose-dependent organ, which can be damaged by hyper- as well as by hypoglycemia (Scheen, 2010). Being a major problem in clinical practice, hypoglycemia unawareness is associated with an increased risk of coma. Note that low blood glucose level induces negative mood states, primarily self-reported "nervousness" (Boyle & Zrebiec, 2007). Moreover, patients with a history of severe hypoglycemia show a much higher level of anxiety compared to other DM patients (Wredling, 1992). The prolonged influence of mild hypoglycemia on the brain leads to deregulation of many processes in CNS, which underlines the importance of scrupulously avoiding even mild hypoglycemic episodes in patients with DM. Hypoglycemia induces

Hormonal Signaling Systems of the Brain in Diabetes Mellitus 351

the blood-brain barrier (BBB) and binds to brain insulin receptors (IRs), which leads to the triggering of their intrinsic tyrosine kinase activity and, as a result, to tyrosine phosphorylation and activation of IRS proteins (Boura-Halfon & Zick, 2009). Phosphorylated IRS proteins then activate p110/p85 heterodimeric PI 3-kinase, protein phosphotyrosine phosphatase and adaptor Shc/GRB2 dimer complex, which triggers the intracellular signaling cascades controlling the gene expression and, thus, regulating growth,

Fig. 1. Critical nodes in the insulin/IGF-1 and leptin signaling systems. The signal components of the systems whose expression and functional activity are significantly changed in DM are underlined. These changes are brain area-specific, they depend on the type of human DM, its severity and duration, DM-induced complications, and on the model of experimental DM. Abbreviations: IRS, insulin receptor substrate proteins; GRB2, growthfactor-receptor-bound protein-2; mSOS, mammalian *son of sevenless* nucleotide exchange factor; Ras, small G protein of Ras family; c-Raf, cytoplasmic serine/threonine-specific protein kinase Raf; MEK, mitogene-activated protein kinase; ERK1/2, extracellular signalregulated kinases 1 and 2; p85/p110 PI 3K, heterodimeric p85/p110 phosphatidylinositol 3 kinase; PTEN, phosphatase and tensin homologue; PDK1, phosphoinositide-dependent kinase 1; PKC, protein kinase C; AKT, protein kinase B; mTOR, mammalian target of

rapamycin; GSK3, glycogen synthase kinase 3; FoxO1, forkhead box O1 protein; JAK2, Janus kinase-2; STAT3, signal transducer and activator of transcription of the type 3; PIP2 and PIP3,

phosphatidylinositol 3,4-diphosphate and phosphatidylinositol 3,4,5-triphosphate,

respectively

progressive reduction in cerebral glycogen and glucose, which is due to an increase in gene expression of GLUT3, the glucose transporter rather abundant in the brain (Antony et al., 2010b). Alteration of expression of GLUT3 in the cerebral cortex in hypoglycemia is the evidence for impairment of neuronal glucose transport during glucose deprivation. The impaired transport and utilization of neuronal glucose in hypoglycemia is likely to be an important factor contributing to an increase of neuronal vulnerability. The disturbances of neuronal glucose transport and metabolism in hyperglycemia are similar to those in hypoglycemia and also induce neuronal damages and CNS disorders. For example, chronic diabetic encephalopathy leading to cognitive dysfunctions and dementia may be the result of recurrent hypoglycaemia and/or chronic hyperglycaemia, both inducing cerebral vascular damages (Scheen, 2010).

A new view of the nature and pathogenesis of DM-induced cerebral complications shared by many specialists nowadays has been prompted by the results of study of functional activity of hormonal signaling systems regulated by insulin, insulin-like growth factor-1 (IGF-1), leptin, biogenic amines, purines, glutamate, and peptide hormones controlling the fundamental processes in the neuronal and glial cells. The data were obtained showing that the alterations and abnormalities of hormonal signaling systems regulated by these hormones and the changes in expression of hormones and signal proteins, the components of these systems, induce disturbances of growth, differentiation, metabolism and apoptosis in neuronal cells and contribute to triggering and development of neurodegenerative processes in the diabetic brain. The present review is devoted to the achievements in the study of the functional state of hormone-sensitive signaling systems of the brain in human and experimental DM, to the alterations and abnormalities in these systems, and to the search of new approaches in the therapy of cerebral complications of DM based on restoration of normal functioning of some signaling systems and overall integrative signaling network in the diabetic brain.

#### **2. Insulin, insulin-like growth factor-1 and leptin in the diabetic brain**

Polypeptide hormones insulin, IGF-1 and leptin, the principal players responsible for pathogenesis of DM and its central and peripheral complications, are to a large extent affected in the diabetic brain. The abnormalities in numerous signaling pathways regulated by insulin, IGF-1 and leptin lead to disturbances of the biochemical and physiological functions of the neuronal and glial cells. It was shown by many investigators that the level of these hormones in the brain is decreased in DM, and the signaling pathways regulated by insulin, IGF-1 and leptin and involving a large number of effector proteins, such as insulin receptor substrate (IRS) proteins, phosphatidylinositol 3-kinase (PI 3-kinase), protein phosphotyrosine phosphatases, AKT kinase, ERK1/ERK2 kinases and glycogen synthase kinase 3β (GSK3β), are impaired (Fig. 1). Therefore, the treatment of diabetic patients with insulin, IGF-1 and leptin, and the restoration of activity of the signaling pathways they regulate are a reliable approach in the therapy of central and neuroendocrine dysfunctions in DM.

#### **2.1 Insulin and insulin-like growth factor-1**

Insulin and IGF-1 are genetically related polypeptides with similar three-dimensional and primary structures. Insulin is synthesized predominantly in pancreatic β-cells, while IGF-1 is synthesized primarily in the liver and also in the brain. Peripheral insulin penetrates

progressive reduction in cerebral glycogen and glucose, which is due to an increase in gene expression of GLUT3, the glucose transporter rather abundant in the brain (Antony et al., 2010b). Alteration of expression of GLUT3 in the cerebral cortex in hypoglycemia is the evidence for impairment of neuronal glucose transport during glucose deprivation. The impaired transport and utilization of neuronal glucose in hypoglycemia is likely to be an important factor contributing to an increase of neuronal vulnerability. The disturbances of neuronal glucose transport and metabolism in hyperglycemia are similar to those in hypoglycemia and also induce neuronal damages and CNS disorders. For example, chronic diabetic encephalopathy leading to cognitive dysfunctions and dementia may be the result of recurrent hypoglycaemia and/or chronic hyperglycaemia, both inducing cerebral

A new view of the nature and pathogenesis of DM-induced cerebral complications shared by many specialists nowadays has been prompted by the results of study of functional activity of hormonal signaling systems regulated by insulin, insulin-like growth factor-1 (IGF-1), leptin, biogenic amines, purines, glutamate, and peptide hormones controlling the fundamental processes in the neuronal and glial cells. The data were obtained showing that the alterations and abnormalities of hormonal signaling systems regulated by these hormones and the changes in expression of hormones and signal proteins, the components of these systems, induce disturbances of growth, differentiation, metabolism and apoptosis in neuronal cells and contribute to triggering and development of neurodegenerative processes in the diabetic brain. The present review is devoted to the achievements in the study of the functional state of hormone-sensitive signaling systems of the brain in human and experimental DM, to the alterations and abnormalities in these systems, and to the search of new approaches in the therapy of cerebral complications of DM based on restoration of normal functioning of some signaling systems and overall integrative

**2. Insulin, insulin-like growth factor-1 and leptin in the diabetic brain** 

Polypeptide hormones insulin, IGF-1 and leptin, the principal players responsible for pathogenesis of DM and its central and peripheral complications, are to a large extent affected in the diabetic brain. The abnormalities in numerous signaling pathways regulated by insulin, IGF-1 and leptin lead to disturbances of the biochemical and physiological functions of the neuronal and glial cells. It was shown by many investigators that the level of these hormones in the brain is decreased in DM, and the signaling pathways regulated by insulin, IGF-1 and leptin and involving a large number of effector proteins, such as insulin receptor substrate (IRS) proteins, phosphatidylinositol 3-kinase (PI 3-kinase), protein phosphotyrosine phosphatases, AKT kinase, ERK1/ERK2 kinases and glycogen synthase kinase 3β (GSK3β), are impaired (Fig. 1). Therefore, the treatment of diabetic patients with insulin, IGF-1 and leptin, and the restoration of activity of the signaling pathways they regulate are a reliable approach in the therapy of central and neuroendocrine dysfunctions

Insulin and IGF-1 are genetically related polypeptides with similar three-dimensional and primary structures. Insulin is synthesized predominantly in pancreatic β-cells, while IGF-1 is synthesized primarily in the liver and also in the brain. Peripheral insulin penetrates

vascular damages (Scheen, 2010).

signaling network in the diabetic brain.

**2.1 Insulin and insulin-like growth factor-1** 

in DM.

the blood-brain barrier (BBB) and binds to brain insulin receptors (IRs), which leads to the triggering of their intrinsic tyrosine kinase activity and, as a result, to tyrosine phosphorylation and activation of IRS proteins (Boura-Halfon & Zick, 2009). Phosphorylated IRS proteins then activate p110/p85 heterodimeric PI 3-kinase, protein phosphotyrosine phosphatase and adaptor Shc/GRB2 dimer complex, which triggers the intracellular signaling cascades controlling the gene expression and, thus, regulating growth,

Fig. 1. Critical nodes in the insulin/IGF-1 and leptin signaling systems. The signal components of the systems whose expression and functional activity are significantly changed in DM are underlined. These changes are brain area-specific, they depend on the type of human DM, its severity and duration, DM-induced complications, and on the model of experimental DM. Abbreviations: IRS, insulin receptor substrate proteins; GRB2, growthfactor-receptor-bound protein-2; mSOS, mammalian *son of sevenless* nucleotide exchange factor; Ras, small G protein of Ras family; c-Raf, cytoplasmic serine/threonine-specific protein kinase Raf; MEK, mitogene-activated protein kinase; ERK1/2, extracellular signalregulated kinases 1 and 2; p85/p110 PI 3K, heterodimeric p85/p110 phosphatidylinositol 3 kinase; PTEN, phosphatase and tensin homologue; PDK1, phosphoinositide-dependent kinase 1; PKC, protein kinase C; AKT, protein kinase B; mTOR, mammalian target of rapamycin; GSK3, glycogen synthase kinase 3; FoxO1, forkhead box O1 protein; JAK2, Janus kinase-2; STAT3, signal transducer and activator of transcription of the type 3; PIP2 and PIP3, phosphatidylinositol 3,4-diphosphate and phosphatidylinositol 3,4,5-triphosphate, respectively

Hormonal Signaling Systems of the Brain in Diabetes Mellitus 353

significantly, but began to decline markedly in prolonged DM2 and in long-term hyperglycemia (Clauson et al., 1998). It indicates the temporal dynamics of a decrease of IGF-1 and the impairments of its signaling in the diabetic brain, correlating with an increase

Central administration of insulin and IGF-1 restores to a great extent the function of the CNS, being in some cases the most effective co-administration of insulin and IGF-1, the latter refers mostly to the cases of much lower concentrations. It is shown that in DM1, in the case of insulin deficit, a concomitant decrease of insulin and IGF-1 levels in the brain leads to atrophy of some brain areas inducing impaired learning and memory. A combined infusion for 12 weeks of insulin and IGF-1 into the brain lateral ventricles of STZ rats prevents a decrease of the brain weight, and leads to normalization of the level of DNA and the content of proteins associated with neurons and glial cells, whose level and activity are significantly decreased in the diabetic brain. As a result, the brain DNA loss in DM1 is prevented (Serbedzija et al., 2009). The administration of IGF-1 to STZ rats prevents irrespective of the severity of hyperglycemia IGF-1 reduction in the brain and the DMassociated cognitive disturbances (Lupien et al., 2003). Anti-IGF-1 antibody infused into the lateral ventricles led, on the contrary, to deterioration of learning and memory functions of diabetic as well as non-diabetic rats. Quite often DM and its complications in human are associated with the changes in IGF-1 binding proteins, which contribute to the concentration of peripheral and central IGF-1 (Busiguina et al., 2000). The alterations of the content of these proteins are responsible for a decline in memory and for many DM-associated

neurodegenerative disorders, such as AD and vascular dementia (Zhu et al., 2005).

The second component of insulin/IGF-1 signaling is IR or IGF-1 receptor. According to some reports, mice with a neuron-specific disruption of the IR gene increased food intake and diet-sensitive obesity with an increase in body fat, mild insulin resistance, elevated plasma insulin and leptin levels, and hypertriglyceridemia typical of DM2 (Bruning et al., 2000). These mutant mice also exhibited impaired spermatogenesis and ovarian follicle maturation due to deregulation of luteinizing hormone-releasing factor secretion caused by attenuation of insulin signaling in the hypothalamus. The restoration of IRs in the brain of these mice maintained energy homeostasis, improved functions of the CNS and prevented DM (Okamoto et al., 2004). The expression of IRs in the brain of mice lacking the genes encoding IR and the glucose transporter GLUT4 also improved their survival, but did not completely eliminate the symptoms of DM2 due to dysfunction of GLUT4 (H.V. Lin & Accili, 2011). The study of expression of IRs and IGF-1 receptors in the frontal cortices of 8 month-old diabetic rats with spontaneous onset of DM1 and DM2 showed that the IR expression was decreased in DM1 only, whereas IGF-1 receptor expression was decreased in both models (Z.G. Li et al., 2007). The disruption of IR expression in discrete hypothalamic nuclei led to hyperphagia and increased fat mass, which was a result of disturbances of regulation of hepatic glucose production by central insulin (Obici et al., 2002). The mice lacking the brain IR had severe hypoleptinemia as well as more severe hyperinsulinemia and hyperglycemia than the mice lacking the receptor in the peripheral tissues, which demonstrates the major role of central insulin in regulating white adipose tissue mass and glucose metabolism in the liver (Koch et al., 2008). Both neuron-specific IR knockout (NIRKO) mice and the rats with spontaneous DM exhibited a complete loss of insulinmediated activation of PI 3-kinase and inhibition of neuronal apoptosis, and had markedly reduced phosphorylation of AKT kinase and GSK3β, leading to substantially increased phosphorylation of the microtubule-associated Tau protein at sites associated with

of neurological disorders in prolonged uncontrolled DM2.

differentiation and the other processes in neuronal cells. The activation of PI 3-kinase leads to phosphorylation and activation of AKT kinase that regulates the metabolism and cell survival via numerous downstream proteins in the peripheral insulin-sensitive tissues as well as in the CNS, primarily in hypothalamic neurons (Iskandar et al., 2010). AKT kinase partly facilitates signal transduction via phosphorylation and cytoplasmic sequestering of forkhead-box protein O1, a negative regulator of insulin signaling, whose nuclear translocation is associated with obesity and hyperphagia (Kitamura et al., 2006). The same signaling network is regulated by IGF-1 that specifically binds with cognate IGF-1 receptor demonstrating a close structural homology and sequence identity with IR and also possessing the tyrosine kinase activity and triggerring IRS-dependent signaling pathways.

Both IRs and IGF-1 receptors are widely expressed in the brain and are localized preferably in neuron rich structures in many brain areas, such as the granule cell layers of the olfactory bulb, hippocampal formation and cerebral cortex. The fact that these receptors are localized in the brain accounts for the role of insulin and IGF-1 in CNS functioning. Since the main function of insulin is to regulate glucose homeostasis, central insulin and brain IRs specifically recognizing the hormone modulate the energy, glucose and fat homeostasis in the brain, being involved, in addition, in the regulation of metabolism in the peripheral tissues. However, in the brain insulin performs some other functions specific of the CNS. Interacting with the other regulatory peptides and neurotransmitters, central insulin participates in controlling the feeding behavior, learning and memory, and is involved in the intercellular communication within brain structures, the hypothalamus and the limbic system in particular (Gerozissis, 2008). IGF-1 is involved in neuronal development, stimulates neurogenesis and synaptogenesis, facilitates oligodendrocyte development, promotes neuron and oligodendrocyte survival, and stimulates myelination. All this speaks about a very important role it has in preserving the integrity of neuronal cells and in protecting the brain structures from damages and injury (D'Ercole et al., 2002).

The alterations of proteins, the components of brain insulin- and IGF-1-regulated signaling cascades, typical of DM and pre-diabetic states, are the causes of the DM-associated neurodegenerative diseases. It should be emphasized that the abnormalities in brain insulin/IGF-1 signaling can be provoked by DM, being a result of the systemic changes of integral signaling network in the diabetic brain, and, on the other hand, the disturbances of the functioning of insulin/IGF-1 signaling systems of the brain induced by neurodegenerative disorders can also lead to DM. In the latter case we can talk about the central genesis of DM.

The initial component of insulin/IGF-1-regulated cascades is a hormonal molecule, insulin or IGF-1, whose brain concentrations are significantly reduced in DM (Gelling et al., 2006). A significant decrease of the IGF-1 level was found in the cerebellum of insulin-deficient rats with STZ-induced DM with poorly controlled glycemia, whereas there were no changes in cerebellar IGF-1 mRNA level, which indicates the abnormalities of hormone processing and secretion in the diabetic brain (Busiguina et al., 1996). The appropriate glycemic control with insulin completely restored IGF-1 concentration in the cerebellum (D'Ercole et al., 2002). Since IGF-1, the same as insulin, crosses the BBB, a decrease of serum IGF-1 in human DM1 and STZ-induced DM also contributes to brain IGF-1 deficit leading to attenuation of IGF-1 signaling (Busiguina et al., 2000). The children with DM1 had a 50% decrease of peripheral IGF-1 level compared with control group, and in diabetic children with poor glucose control it was decreased even more compared with moderate metabolic control. In the patients with DM2 the peripheral level of IGF-1 at the early stages of the disease did not change

differentiation and the other processes in neuronal cells. The activation of PI 3-kinase leads to phosphorylation and activation of AKT kinase that regulates the metabolism and cell survival via numerous downstream proteins in the peripheral insulin-sensitive tissues as well as in the CNS, primarily in hypothalamic neurons (Iskandar et al., 2010). AKT kinase partly facilitates signal transduction via phosphorylation and cytoplasmic sequestering of forkhead-box protein O1, a negative regulator of insulin signaling, whose nuclear translocation is associated with obesity and hyperphagia (Kitamura et al., 2006). The same signaling network is regulated by IGF-1 that specifically binds with cognate IGF-1 receptor demonstrating a close structural homology and sequence identity with IR and also possessing the tyrosine kinase activity and triggerring IRS-dependent signaling pathways. Both IRs and IGF-1 receptors are widely expressed in the brain and are localized preferably in neuron rich structures in many brain areas, such as the granule cell layers of the olfactory bulb, hippocampal formation and cerebral cortex. The fact that these receptors are localized in the brain accounts for the role of insulin and IGF-1 in CNS functioning. Since the main function of insulin is to regulate glucose homeostasis, central insulin and brain IRs specifically recognizing the hormone modulate the energy, glucose and fat homeostasis in the brain, being involved, in addition, in the regulation of metabolism in the peripheral tissues. However, in the brain insulin performs some other functions specific of the CNS. Interacting with the other regulatory peptides and neurotransmitters, central insulin participates in controlling the feeding behavior, learning and memory, and is involved in the intercellular communication within brain structures, the hypothalamus and the limbic system in particular (Gerozissis, 2008). IGF-1 is involved in neuronal development, stimulates neurogenesis and synaptogenesis, facilitates oligodendrocyte development, promotes neuron and oligodendrocyte survival, and stimulates myelination. All this speaks about a very important role it has in preserving the integrity of neuronal cells and in

protecting the brain structures from damages and injury (D'Ercole et al., 2002).

central genesis of DM.

The alterations of proteins, the components of brain insulin- and IGF-1-regulated signaling cascades, typical of DM and pre-diabetic states, are the causes of the DM-associated neurodegenerative diseases. It should be emphasized that the abnormalities in brain insulin/IGF-1 signaling can be provoked by DM, being a result of the systemic changes of integral signaling network in the diabetic brain, and, on the other hand, the disturbances of the functioning of insulin/IGF-1 signaling systems of the brain induced by neurodegenerative disorders can also lead to DM. In the latter case we can talk about the

The initial component of insulin/IGF-1-regulated cascades is a hormonal molecule, insulin or IGF-1, whose brain concentrations are significantly reduced in DM (Gelling et al., 2006). A significant decrease of the IGF-1 level was found in the cerebellum of insulin-deficient rats with STZ-induced DM with poorly controlled glycemia, whereas there were no changes in cerebellar IGF-1 mRNA level, which indicates the abnormalities of hormone processing and secretion in the diabetic brain (Busiguina et al., 1996). The appropriate glycemic control with insulin completely restored IGF-1 concentration in the cerebellum (D'Ercole et al., 2002). Since IGF-1, the same as insulin, crosses the BBB, a decrease of serum IGF-1 in human DM1 and STZ-induced DM also contributes to brain IGF-1 deficit leading to attenuation of IGF-1 signaling (Busiguina et al., 2000). The children with DM1 had a 50% decrease of peripheral IGF-1 level compared with control group, and in diabetic children with poor glucose control it was decreased even more compared with moderate metabolic control. In the patients with DM2 the peripheral level of IGF-1 at the early stages of the disease did not change significantly, but began to decline markedly in prolonged DM2 and in long-term hyperglycemia (Clauson et al., 1998). It indicates the temporal dynamics of a decrease of IGF-1 and the impairments of its signaling in the diabetic brain, correlating with an increase of neurological disorders in prolonged uncontrolled DM2.

Central administration of insulin and IGF-1 restores to a great extent the function of the CNS, being in some cases the most effective co-administration of insulin and IGF-1, the latter refers mostly to the cases of much lower concentrations. It is shown that in DM1, in the case of insulin deficit, a concomitant decrease of insulin and IGF-1 levels in the brain leads to atrophy of some brain areas inducing impaired learning and memory. A combined infusion for 12 weeks of insulin and IGF-1 into the brain lateral ventricles of STZ rats prevents a decrease of the brain weight, and leads to normalization of the level of DNA and the content of proteins associated with neurons and glial cells, whose level and activity are significantly decreased in the diabetic brain. As a result, the brain DNA loss in DM1 is prevented (Serbedzija et al., 2009). The administration of IGF-1 to STZ rats prevents irrespective of the severity of hyperglycemia IGF-1 reduction in the brain and the DMassociated cognitive disturbances (Lupien et al., 2003). Anti-IGF-1 antibody infused into the lateral ventricles led, on the contrary, to deterioration of learning and memory functions of diabetic as well as non-diabetic rats. Quite often DM and its complications in human are associated with the changes in IGF-1 binding proteins, which contribute to the concentration of peripheral and central IGF-1 (Busiguina et al., 2000). The alterations of the content of these proteins are responsible for a decline in memory and for many DM-associated neurodegenerative disorders, such as AD and vascular dementia (Zhu et al., 2005).

The second component of insulin/IGF-1 signaling is IR or IGF-1 receptor. According to some reports, mice with a neuron-specific disruption of the IR gene increased food intake and diet-sensitive obesity with an increase in body fat, mild insulin resistance, elevated plasma insulin and leptin levels, and hypertriglyceridemia typical of DM2 (Bruning et al., 2000). These mutant mice also exhibited impaired spermatogenesis and ovarian follicle maturation due to deregulation of luteinizing hormone-releasing factor secretion caused by attenuation of insulin signaling in the hypothalamus. The restoration of IRs in the brain of these mice maintained energy homeostasis, improved functions of the CNS and prevented DM (Okamoto et al., 2004). The expression of IRs in the brain of mice lacking the genes encoding IR and the glucose transporter GLUT4 also improved their survival, but did not completely eliminate the symptoms of DM2 due to dysfunction of GLUT4 (H.V. Lin & Accili, 2011). The study of expression of IRs and IGF-1 receptors in the frontal cortices of 8 month-old diabetic rats with spontaneous onset of DM1 and DM2 showed that the IR expression was decreased in DM1 only, whereas IGF-1 receptor expression was decreased in both models (Z.G. Li et al., 2007). The disruption of IR expression in discrete hypothalamic nuclei led to hyperphagia and increased fat mass, which was a result of disturbances of regulation of hepatic glucose production by central insulin (Obici et al., 2002). The mice lacking the brain IR had severe hypoleptinemia as well as more severe hyperinsulinemia and hyperglycemia than the mice lacking the receptor in the peripheral tissues, which demonstrates the major role of central insulin in regulating white adipose tissue mass and glucose metabolism in the liver (Koch et al., 2008). Both neuron-specific IR knockout (NIRKO) mice and the rats with spontaneous DM exhibited a complete loss of insulinmediated activation of PI 3-kinase and inhibition of neuronal apoptosis, and had markedly reduced phosphorylation of AKT kinase and GSK3β, leading to substantially increased phosphorylation of the microtubule-associated Tau protein at sites associated with

Hormonal Signaling Systems of the Brain in Diabetes Mellitus 355

likely to account for a majority of cases of a significant increase in Tau phosphorylation caused by STZ treatment. The decreased PP2A activity and Tau hyperphosphorylation on the background of insulin deficiency may increase the susceptibility of the diabetic brain to insults associated with AD, thereby contributing to the relationship between DM and

To study the role of PI 3-kinase in the diabetic brain, it was shown by making i.c.v. infusion of LY294002, a specific inhibitor of the enzyme, into the 3rd cerebral ventricle of STZ rats that the inhibition of PI 3-kinase activity and downstream effector AKT kinase in this case leads to attenuation of the glycemic response to systemic insulin treatment (Gelling et al., 2006). The glucose-lowering effect of insulin in STZ rats after adenovirus delivery of *Irs-2* gene into the hypothalamic arcuate nucleus was increased 2-fold compared to diabetic rats receiving a control adenovirus. The same results were obtained after injection of adenovirus encoding a constitutively active AKT kinase. These findings indicate that the response to adenovirus encoding IRS-2 involves signal transduction via PI 3-kinase and AKT kinase, and the increased hypothalamic signaling either upstream or downstream of PI 3-kinase is sufficient to enhance insulin-induced glucose lowering in diabetic rats (Gelling et al., 2006). Hence, being the most insulin-responsive brain area, the hypothalamus contributes to whole-body

The prime function of the other mechanism of neuroprotective action of insulin and IGF-1 realized via PI 3-kinase is to control the oxidative stress and susceptibility of the brain endothelium, the important contributing factors in the development of CNS disorders in DM (Okouchi et al., 2006). It was found that chronic hyperglycemia exacerbated apoptosis of human brain endothelial cells in accordance with exaggerated cytosolic and mitochondrial glutathione and protein-thiol redox imbalance. Insulin activates the PI 3-kinase/AKT kinase/mTOR kinase cascade, increases serine phosphorylation and nuclear translocation of nuclear NF-E2-related factor 2 (Nrf2), and enhances the expression of catalytic subunit of Nrf2-dependent glutamate-L-cysteine ligase, a heterodimeric enzyme participating in glutathione metabolism, and, hence, attenuates hyperglycemia-induced apoptosis via the restored cytosolic and mitochondrial redox balance. Inhibitors of IR tyrosine kinase, PI 3-kinase, AKT kinase and mTOR kinase abrogate insulin-induced Nrf2-mediated glutamate-L-cysteine ligase expression, redox balance, and the survival of human brain endothelial cells (Okouchi et al., 2006). Insulin-regulated PI 3-kinase-dependent pathways are involved in the prevention of endoplasmic reticulum stress that contributes to DM and neurodegenerative disorders (Hosoi et al., 2007). It was found that PI 3-kinase regulates the expression of CHOP protein, an endoplasmic reticulum stress-induced transcription factor

The important role in regulation of insulin level in the diabetic brain belongs to the insulindegrading enzyme (IDE). In addition to insulin, it also degrades β-amyloid peptide. Thus, in the case of hyperinsulinemia in DM2, insulin competes with β-amyloid peptide for IDE and this leads to an increase in β-amyloid peptide concentration and provokes neurodegenerative processes and the development of AD (Qiu & Folstein, 2006). The genetic studies indicate that *IDE* gene variations are associated with the clinical symptoms of AD as well as with the risk of DM2. In DM1 it was shown that the activity of IDE and the level of mRNA encoding IDE were significantly decreased in the temporal cortex of STZ rats. Since the activity of two other β-amyloid peptide-degrading enzymes, neprilysin and endothelinconverting enzyme 1, was also decreased though to a different extent in the brain of diabetic rats, the level of the β-amyloid peptide 1–40 was markedly elevated, which induced DM-

heightened susceptibility to AD (Clodfelder-Miller et al., 2006).

glucose homeostasis via IRS–PI 3-kinase signaling.

involved in control of neuronal cell survival.

neurodegenerative diseases (Z.G. Li et al., 2007; Koch et al., 2008). This is one of the molecular mechanisms responsible for the altered insulin signaling and insulin resistance in the brain to be predisposed for the development of neurodegeneration, creating a clinical link between DM2 and AD and other CNS dysfunctions (Schubert et al., 2004).

The third component of insulin/IGF-1 signaling is IRS proteins. They have a key role in linking IR and IGF-1 receptor to the intracellular signaling cascades and in coordinating signals from these receptors with those generated by other neurotransmitters, peptide hormones, pro-inflammatory cytokines and nutrients. The alterations of the IRS protein functions are responsible for the failure of insulin/IGF-1 signaling not only in the peripheral tissues, but also in neuronal cells, they induce insulin resistance and, finally, cause DM and neurodegenerative diseases associated with it (Lee & White, 2004). The deletion of gene encoding IRS-2 protein leads to the weakening of hypothalamic insulin signaling and increases both food intake and hepatic glucose production (X. Lin et al., 2004). Conversely, over expression of IRS-2 in the mediobasal hypothalamus was found to significantly enhance the glycemic response to systemic insulin treatment in STZ rats (Gelling et al., 2006). It was shown that in *Irs2* gene knockout mice the embryonic brain size is 55% of that in normal animals due to the reduced neuronal proliferation in the course of development, indicating IRS-2 to be involved in the brain growth. It seems likely that IRS-2 are involved in neuroprotective effects of insulin and IGF-1, because in the hippocampus of old *Irs2* knockout mice there are formed neurofibrillary tangles containing phosphorylated Tau protein, a hallmark of neurodegenerative processes (Schubert et al., 2003). No direct evidence for IRS-2 being involved in human brain growth and differentiation is available, but breaks at the distal end of human chromosome 13 (13q) near the *Irs2* gene between micro satellites D13S285 and D13S1295 are frequently associated with microcephaly, while very distal deletions between D13S274 and D13S1311 with microcephaly and neural tube defects, suggesting a possible contribution of partial *Irs2* deficiency to microcephaly (J. Luo et al., 2000). Based on these data, the conclusion was made that the regulation of activity of IRS-1 and IRS-2 controlling the growth, metabolism and survival of neuronal cells is a new strategy aimed at prevention or cure of DM and its CNS complications. However, according to the recently obtained data, the deletion of gene encoding IRS-2 improves the functioning of the brain of mutant mice, because IRS-2 act as negative regulators of memory formation by restricting dendritic spine generation (Irvine et al., 2011). The above may be due to the fact that various groups of scientists are engaged in the study of mutant lines of animals with a large number of alterations of insulin/IGF-1 signaling, and these alterations induce different changes in the brain signaling network. With this in mind, it is clear why the functions of IRS-2 can be redistributed among the other types of IRS proteins or described as depending on the activity of upstream or downstream signal proteins interacting with IRS-2. The downstream components of insulin/IGF-1 signaling, such as PI 3-kinase, AKT kinase and protein phosphotyrosine phosphatase 2A (PP2A) are also changed in DM and greatly contribute in etiology and pathogenesis of DM-induced neurodegenerative diseases. The main molecular mechanism in this case is a rapid and significant increase of phosphorylation of Tau protein (Clodfelder-Miller et al., 2006). The hyperphosphorylation of Tau was detected in the mouse cerebral cortex and hippocampus within 3 days after STZ treatment and can be rapidly reversed by peripheral insulin administration. The increase of Tau phosphorylation in the brain in DM partly depends on the fact that the activity of PP2A, the major protein phosphatase acting on Tau, was decreased by 44% in the cerebral cortex and by 55% in the hippocampus. This indicates that a significant decrease in PP2A activity is

neurodegenerative diseases (Z.G. Li et al., 2007; Koch et al., 2008). This is one of the molecular mechanisms responsible for the altered insulin signaling and insulin resistance in the brain to be predisposed for the development of neurodegeneration, creating a clinical

The third component of insulin/IGF-1 signaling is IRS proteins. They have a key role in linking IR and IGF-1 receptor to the intracellular signaling cascades and in coordinating signals from these receptors with those generated by other neurotransmitters, peptide hormones, pro-inflammatory cytokines and nutrients. The alterations of the IRS protein functions are responsible for the failure of insulin/IGF-1 signaling not only in the peripheral tissues, but also in neuronal cells, they induce insulin resistance and, finally, cause DM and neurodegenerative diseases associated with it (Lee & White, 2004). The deletion of gene encoding IRS-2 protein leads to the weakening of hypothalamic insulin signaling and increases both food intake and hepatic glucose production (X. Lin et al., 2004). Conversely, over expression of IRS-2 in the mediobasal hypothalamus was found to significantly enhance the glycemic response to systemic insulin treatment in STZ rats (Gelling et al., 2006). It was shown that in *Irs2* gene knockout mice the embryonic brain size is 55% of that in normal animals due to the reduced neuronal proliferation in the course of development, indicating IRS-2 to be involved in the brain growth. It seems likely that IRS-2 are involved in neuroprotective effects of insulin and IGF-1, because in the hippocampus of old *Irs2* knockout mice there are formed neurofibrillary tangles containing phosphorylated Tau protein, a hallmark of neurodegenerative processes (Schubert et al., 2003). No direct evidence for IRS-2 being involved in human brain growth and differentiation is available, but breaks at the distal end of human chromosome 13 (13q) near the *Irs2* gene between micro satellites D13S285 and D13S1295 are frequently associated with microcephaly, while very distal deletions between D13S274 and D13S1311 with microcephaly and neural tube defects, suggesting a possible contribution of partial *Irs2* deficiency to microcephaly (J. Luo et al., 2000). Based on these data, the conclusion was made that the regulation of activity of IRS-1 and IRS-2 controlling the growth, metabolism and survival of neuronal cells is a new strategy aimed at prevention or cure of DM and its CNS complications. However, according to the recently obtained data, the deletion of gene encoding IRS-2 improves the functioning of the brain of mutant mice, because IRS-2 act as negative regulators of memory formation by restricting dendritic spine generation (Irvine et al., 2011). The above may be due to the fact that various groups of scientists are engaged in the study of mutant lines of animals with a large number of alterations of insulin/IGF-1 signaling, and these alterations induce different changes in the brain signaling network. With this in mind, it is clear why the functions of IRS-2 can be redistributed among the other types of IRS proteins or described as depending on the activity of upstream or downstream signal proteins interacting with IRS-2. The downstream components of insulin/IGF-1 signaling, such as PI 3-kinase, AKT kinase and protein phosphotyrosine phosphatase 2A (PP2A) are also changed in DM and greatly contribute in etiology and pathogenesis of DM-induced neurodegenerative diseases. The main molecular mechanism in this case is a rapid and significant increase of phosphorylation of Tau protein (Clodfelder-Miller et al., 2006). The hyperphosphorylation of Tau was detected in the mouse cerebral cortex and hippocampus within 3 days after STZ treatment and can be rapidly reversed by peripheral insulin administration. The increase of Tau phosphorylation in the brain in DM partly depends on the fact that the activity of PP2A, the major protein phosphatase acting on Tau, was decreased by 44% in the cerebral cortex and by 55% in the hippocampus. This indicates that a significant decrease in PP2A activity is

link between DM2 and AD and other CNS dysfunctions (Schubert et al., 2004).

likely to account for a majority of cases of a significant increase in Tau phosphorylation caused by STZ treatment. The decreased PP2A activity and Tau hyperphosphorylation on the background of insulin deficiency may increase the susceptibility of the diabetic brain to insults associated with AD, thereby contributing to the relationship between DM and heightened susceptibility to AD (Clodfelder-Miller et al., 2006).

To study the role of PI 3-kinase in the diabetic brain, it was shown by making i.c.v. infusion of LY294002, a specific inhibitor of the enzyme, into the 3rd cerebral ventricle of STZ rats that the inhibition of PI 3-kinase activity and downstream effector AKT kinase in this case leads to attenuation of the glycemic response to systemic insulin treatment (Gelling et al., 2006). The glucose-lowering effect of insulin in STZ rats after adenovirus delivery of *Irs-2* gene into the hypothalamic arcuate nucleus was increased 2-fold compared to diabetic rats receiving a control adenovirus. The same results were obtained after injection of adenovirus encoding a constitutively active AKT kinase. These findings indicate that the response to adenovirus encoding IRS-2 involves signal transduction via PI 3-kinase and AKT kinase, and the increased hypothalamic signaling either upstream or downstream of PI 3-kinase is sufficient to enhance insulin-induced glucose lowering in diabetic rats (Gelling et al., 2006). Hence, being the most insulin-responsive brain area, the hypothalamus contributes to whole-body glucose homeostasis via IRS–PI 3-kinase signaling.

The prime function of the other mechanism of neuroprotective action of insulin and IGF-1 realized via PI 3-kinase is to control the oxidative stress and susceptibility of the brain endothelium, the important contributing factors in the development of CNS disorders in DM (Okouchi et al., 2006). It was found that chronic hyperglycemia exacerbated apoptosis of human brain endothelial cells in accordance with exaggerated cytosolic and mitochondrial glutathione and protein-thiol redox imbalance. Insulin activates the PI 3-kinase/AKT kinase/mTOR kinase cascade, increases serine phosphorylation and nuclear translocation of nuclear NF-E2-related factor 2 (Nrf2), and enhances the expression of catalytic subunit of Nrf2-dependent glutamate-L-cysteine ligase, a heterodimeric enzyme participating in glutathione metabolism, and, hence, attenuates hyperglycemia-induced apoptosis via the restored cytosolic and mitochondrial redox balance. Inhibitors of IR tyrosine kinase, PI 3-kinase, AKT kinase and mTOR kinase abrogate insulin-induced Nrf2-mediated glutamate-L-cysteine ligase expression, redox balance, and the survival of human brain endothelial cells (Okouchi et al., 2006). Insulin-regulated PI 3-kinase-dependent pathways are involved in the prevention of endoplasmic reticulum stress that contributes to DM and neurodegenerative disorders (Hosoi et al., 2007). It was found that PI 3-kinase regulates the expression of CHOP protein, an endoplasmic reticulum stress-induced transcription factor involved in control of neuronal cell survival.

The important role in regulation of insulin level in the diabetic brain belongs to the insulindegrading enzyme (IDE). In addition to insulin, it also degrades β-amyloid peptide. Thus, in the case of hyperinsulinemia in DM2, insulin competes with β-amyloid peptide for IDE and this leads to an increase in β-amyloid peptide concentration and provokes neurodegenerative processes and the development of AD (Qiu & Folstein, 2006). The genetic studies indicate that *IDE* gene variations are associated with the clinical symptoms of AD as well as with the risk of DM2. In DM1 it was shown that the activity of IDE and the level of mRNA encoding IDE were significantly decreased in the temporal cortex of STZ rats. Since the activity of two other β-amyloid peptide-degrading enzymes, neprilysin and endothelinconverting enzyme 1, was also decreased though to a different extent in the brain of diabetic rats, the level of the β-amyloid peptide 1–40 was markedly elevated, which induced DM-

Hormonal Signaling Systems of the Brain in Diabetes Mellitus 357

of insensitivity of the hippocampus to the hormone. Since the deficiency in hippocampal plasticity in diabetic patients and STZ-treated animals is independent of insulin level, it can be assumed that the cause for these abnormalities is the concert functioning of leptin and insulin signaling systems and their ability to modulate other neuronal systems regulated by γ-aminobutyric acid (GABA), DA and melanocortin (Van der Heide et al., 2005). A close interrelation between the signaling pathways controlled by leptin and the dopaminergic and peptidergic signaling systems is supported by the following data obtained with experimental DM and obesity. The leptin deficiency in the obese mice lacking leptin (*Lepob/ob* mice) led to a decrease in the content of somatodendritic vesicular DA and the amount of DA to be released (Roseberry et al., 2007). One possible cause is related to a decrease of the number of functionally active DA transporters controlling the synaptic level of DA. I.c.v. and parenchymal hypothalamic administration of leptin into MKR mice, a model of nonobese DM2, lacking IGF-1 receptor and having hyperglycemia, hyperinsulinemia, and hyperlipidemia, significantly increased the rate of disappearance of glucose. These effects were mediated by brain MCRs, as central administration of SHU9119, the antagonist of MCRs of the types 3 and 4 (MC3R and MC4R), blocked the ability of hypothalamic leptin to increase skeletal muscle glucose metabolism, glucose uptake and fat oxidation, while in the presence of the agonists of the receptors the anti-diabetic effects of leptin were retained and intensified even more (Toda et al., 2009). The involvement of hypothalamic signaling systems regulated by neurotransmitters in the regulatory effects of central leptin on the energy balance and peripheral glucose homeostasis is supported by the results of the study of non-obese diabetic MKR mice, where i.c.v. administration of leptin dramatically improved insulin sensitivity both via the hypothalamus and direct contact with the

Studying the action of i.c.v. administered leptin on metabolic imbalance caused by experimental DM1 it was found that leptin normalizes the glucose homeostasis and ameliorates the functioning of CNS in STZ-treated rodents (Kojima et al., 2009; Wang et al., 2010). I.c.v. infusion of leptin reversed lethality and greatly improved hyperglycemia, hyperglucagonemia, hyperketonemia, and polyuria in STZ mice. The leptin therapy improved the expression of the metabolically relevant hypothalamic neuropeptides proopiomelanocortin (POMC) and NPY, and also the expression of AgRP in the brain of diabetic mice and restored their signaling cascades impaired in DM1. For the effects of leptin to be long-term, the technique of i.c.v. administration of recombinant adenoassociated virus vector (*rAAV*) encoding leptin gene (*rAAV-lep*) was developed and used in adult STZ-treated mice. The injection of *rAAV-lep* gene markedly increased the level of hypothalamic leptin, rescued the STZ mice from early mortality, gradually decreased hyperphagia to normalize food intake by the 20th week, and maintained body weight within significantly lower than the control range. The blood levels of glucose in these mice started to recede dramatically by the 2nd-3rd week to normalize by the 8th week, and euglycemia was sustained during 52 weeks of experiment. *rAAV-lep* gene injected mice did not exhibit any discernible untoward behavioral changes, nor diabetic complications (Kojima et al., 2009). The addition of low-dose insulin to the leptin therapy provides physiological insulin level for the peripheral targets of STZ rats and leptin in this case suppresses the hyperglucagonemia, avoiding high doses of insulin required to decrease the elevated glucagon level (Wang et al., 2010). Thus, leptin administration has multiple short- and longterm advantages over insulin monotherapy of DM1, and the combined application of leptin and insulin can be recommended for the treatment of human DM1. A high efficiency of the

peripheral tissues (X. Li et al., 2011).

associated AD and other abnormalities of CNS (Y. Liu et al., 2011). The other authors reported a significant reduction of IDE expression in the brain of STZ mice after 9 weeks of hyperglycemia (Jolivalt et al., 2008). The treatment with insulin partially restored phosphorylation of IR and downstream components of insulin signaling system and led to restoration of IDE activity. Based on these data the conclusion was made that in both types of DM the level of β-amyloid peptides was increased, although the molecular mechanisms and the role of IDE in this case may be different.

#### **2.2 Leptin**

Leptin, the product of the *ob* gene, is mainly secreted by peripheral adipocytes, it regulates energy metabolism and body weight. Leptin deficiency in rodents and humans leads to severe obesity. Leptin penetrates into the brain through the BBB as a result of receptormediated endocytosis, binds to the leptin receptors located on neurons in the hypothalamus, where the density of receptors is high, and in some extrahypothalamic regions including the cortex, thalamus, cerebellum, choroid plexus and olfactory bulb (Mutze et al., 2006, Marino et al., 2011). The leptin receptor belonging to the cytokine family receptors has several isoforms, but only the full-length isoform generates an intracellular signal. Activated leptin receptors trigger the stimulation of JAK2 tyrosine kinase that phosphorylates the intracellular domain of the receptor to create a binding site for IRS proteins activating PI 3 kinase and the MEK/ERK signaling pathway (Hegyi et al, 2004). JAK2 kinase also activates the transcription factor STAT3, and the JAK/STAT pathway plays the major role in leptin signaling via the membrane receptors (Mutze et al., 2006).

Central leptin interacts with the hypothalamic nuclei and regulates energy expenditure and food intake through production of agouti-related protein (AgRP), the antagonist of melanocotin receptors (MCRs), and neuropeptide Y (NPY), and α-melanocyte-stimulating hormone (α-MSH) (M.W. Schwartz et al. 2000; Signore et al., 2008). Leptin, like insulin, is involved in the control of the excitability of hypothalamic neurons, modulates the synaptic plasticity and promotes the learning and cognition. Leptin facilitates the presynaptic transmitter release and postsynaptic sensitivity to the transmitters in the hippocampal neurons and regulates hippocampal synaptic plasticity and neuronal development. The rodents with dysfunction of leptin signaling display impaired hippocampal synaptic plasticity, and the application of leptin restores the functions of hippocampus (X.L. Li et al, 2002). In neuronal cells leptin activates JAK/STAT, MEK/ERK and PI 3-kinase signaling pathways and functions as the antiapoptotic factor regulating cell survival. The central effects of leptin are mainly mediated via PI 3-kinase and AKT kinase (Morton et al., 2005). Leptin also serves as neurotrophic factor, because it reverses the loss of dopaminergic neurons and dopamine (DA)-mediated behavior induced by the toxin destroying these neurons (Weng et al., 2007). Therefore, leptin not only protects the rescuing dopaminergic neurons from toxicity, but also preserves the DA-regulated signaling network in neurodegenerative diseases, which might prove useful in the treatment of DM-associated neurodegenerative diseases.

Some time ago in the CA1 hippocampal region of leptin receptor-deficient rodents (Zucker *fa/fa* rats and *db/db* mice) the impairments of hippocampal long-term potentiation (LTP) and long-term depression (LTD) were detected (X.L. Li et al., 2002). The animals showed deficiencies in neuronal and behavioral plasticity and, as demonstrated by the impairment of spatial memory in the Morris water-maze test, had memory deficit due, at least in part, to a deficiency in leptin receptors. The leptin administration gave no results probably because

associated AD and other abnormalities of CNS (Y. Liu et al., 2011). The other authors reported a significant reduction of IDE expression in the brain of STZ mice after 9 weeks of hyperglycemia (Jolivalt et al., 2008). The treatment with insulin partially restored phosphorylation of IR and downstream components of insulin signaling system and led to restoration of IDE activity. Based on these data the conclusion was made that in both types of DM the level of β-amyloid peptides was increased, although the molecular mechanisms

Leptin, the product of the *ob* gene, is mainly secreted by peripheral adipocytes, it regulates energy metabolism and body weight. Leptin deficiency in rodents and humans leads to severe obesity. Leptin penetrates into the brain through the BBB as a result of receptormediated endocytosis, binds to the leptin receptors located on neurons in the hypothalamus, where the density of receptors is high, and in some extrahypothalamic regions including the cortex, thalamus, cerebellum, choroid plexus and olfactory bulb (Mutze et al., 2006, Marino et al., 2011). The leptin receptor belonging to the cytokine family receptors has several isoforms, but only the full-length isoform generates an intracellular signal. Activated leptin receptors trigger the stimulation of JAK2 tyrosine kinase that phosphorylates the intracellular domain of the receptor to create a binding site for IRS proteins activating PI 3 kinase and the MEK/ERK signaling pathway (Hegyi et al, 2004). JAK2 kinase also activates the transcription factor STAT3, and the JAK/STAT pathway plays the major role in leptin

Central leptin interacts with the hypothalamic nuclei and regulates energy expenditure and food intake through production of agouti-related protein (AgRP), the antagonist of melanocotin receptors (MCRs), and neuropeptide Y (NPY), and α-melanocyte-stimulating hormone (α-MSH) (M.W. Schwartz et al. 2000; Signore et al., 2008). Leptin, like insulin, is involved in the control of the excitability of hypothalamic neurons, modulates the synaptic plasticity and promotes the learning and cognition. Leptin facilitates the presynaptic transmitter release and postsynaptic sensitivity to the transmitters in the hippocampal neurons and regulates hippocampal synaptic plasticity and neuronal development. The rodents with dysfunction of leptin signaling display impaired hippocampal synaptic plasticity, and the application of leptin restores the functions of hippocampus (X.L. Li et al, 2002). In neuronal cells leptin activates JAK/STAT, MEK/ERK and PI 3-kinase signaling pathways and functions as the antiapoptotic factor regulating cell survival. The central effects of leptin are mainly mediated via PI 3-kinase and AKT kinase (Morton et al., 2005). Leptin also serves as neurotrophic factor, because it reverses the loss of dopaminergic neurons and dopamine (DA)-mediated behavior induced by the toxin destroying these neurons (Weng et al., 2007). Therefore, leptin not only protects the rescuing dopaminergic neurons from toxicity, but also preserves the DA-regulated signaling network in neurodegenerative diseases, which might prove useful in the treatment of DM-associated

Some time ago in the CA1 hippocampal region of leptin receptor-deficient rodents (Zucker *fa/fa* rats and *db/db* mice) the impairments of hippocampal long-term potentiation (LTP) and long-term depression (LTD) were detected (X.L. Li et al., 2002). The animals showed deficiencies in neuronal and behavioral plasticity and, as demonstrated by the impairment of spatial memory in the Morris water-maze test, had memory deficit due, at least in part, to a deficiency in leptin receptors. The leptin administration gave no results probably because

and the role of IDE in this case may be different.

signaling via the membrane receptors (Mutze et al., 2006).

neurodegenerative diseases.

**2.2 Leptin** 

of insensitivity of the hippocampus to the hormone. Since the deficiency in hippocampal plasticity in diabetic patients and STZ-treated animals is independent of insulin level, it can be assumed that the cause for these abnormalities is the concert functioning of leptin and insulin signaling systems and their ability to modulate other neuronal systems regulated by γ-aminobutyric acid (GABA), DA and melanocortin (Van der Heide et al., 2005). A close interrelation between the signaling pathways controlled by leptin and the dopaminergic and peptidergic signaling systems is supported by the following data obtained with experimental DM and obesity. The leptin deficiency in the obese mice lacking leptin (*Lepob/ob* mice) led to a decrease in the content of somatodendritic vesicular DA and the amount of DA to be released (Roseberry et al., 2007). One possible cause is related to a decrease of the number of functionally active DA transporters controlling the synaptic level of DA. I.c.v. and parenchymal hypothalamic administration of leptin into MKR mice, a model of nonobese DM2, lacking IGF-1 receptor and having hyperglycemia, hyperinsulinemia, and hyperlipidemia, significantly increased the rate of disappearance of glucose. These effects were mediated by brain MCRs, as central administration of SHU9119, the antagonist of MCRs of the types 3 and 4 (MC3R and MC4R), blocked the ability of hypothalamic leptin to increase skeletal muscle glucose metabolism, glucose uptake and fat oxidation, while in the presence of the agonists of the receptors the anti-diabetic effects of leptin were retained and intensified even more (Toda et al., 2009). The involvement of hypothalamic signaling systems regulated by neurotransmitters in the regulatory effects of central leptin on the energy balance and peripheral glucose homeostasis is supported by the results of the study

of non-obese diabetic MKR mice, where i.c.v. administration of leptin dramatically improved insulin sensitivity both via the hypothalamus and direct contact with the peripheral tissues (X. Li et al., 2011).

Studying the action of i.c.v. administered leptin on metabolic imbalance caused by experimental DM1 it was found that leptin normalizes the glucose homeostasis and ameliorates the functioning of CNS in STZ-treated rodents (Kojima et al., 2009; Wang et al., 2010). I.c.v. infusion of leptin reversed lethality and greatly improved hyperglycemia, hyperglucagonemia, hyperketonemia, and polyuria in STZ mice. The leptin therapy improved the expression of the metabolically relevant hypothalamic neuropeptides proopiomelanocortin (POMC) and NPY, and also the expression of AgRP in the brain of diabetic mice and restored their signaling cascades impaired in DM1. For the effects of leptin to be long-term, the technique of i.c.v. administration of recombinant adenoassociated virus vector (*rAAV*) encoding leptin gene (*rAAV-lep*) was developed and used in adult STZ-treated mice. The injection of *rAAV-lep* gene markedly increased the level of hypothalamic leptin, rescued the STZ mice from early mortality, gradually decreased hyperphagia to normalize food intake by the 20th week, and maintained body weight within significantly lower than the control range. The blood levels of glucose in these mice started to recede dramatically by the 2nd-3rd week to normalize by the 8th week, and euglycemia was sustained during 52 weeks of experiment. *rAAV-lep* gene injected mice did not exhibit any discernible untoward behavioral changes, nor diabetic complications (Kojima et al., 2009).

The addition of low-dose insulin to the leptin therapy provides physiological insulin level for the peripheral targets of STZ rats and leptin in this case suppresses the hyperglucagonemia, avoiding high doses of insulin required to decrease the elevated glucagon level (Wang et al., 2010). Thus, leptin administration has multiple short- and longterm advantages over insulin monotherapy of DM1, and the combined application of leptin and insulin can be recommended for the treatment of human DM1. A high efficiency of the

Hormonal Signaling Systems of the Brain in Diabetes Mellitus 359

Fig. 2. Gs-, Gi/o- and Gq-coupled signaling pathways including the receptors of the serpentine type regulated by biogenic amines, glutamate, acetylcholine and peptide hormones. The signal components whose activity and expression are significantly altered in DM are underlined. Abbreviations: NPY, neuropeptide Y; GLP-1, glucagon-like peptide-1; D1,2DARs, dopamine receptors of the types 1 and 2; 5-HT1,2,6Rs, 5-hydroxytryptamine receptors of the types 1, 2 and 6; MC3,4Rs, melanocortin receptors of the types 3 and 4; mGlu1,5Rs, metabotropic glutamate receptors of the types 1 and 5; m1,3-MAChRs, muscarinic

acetylcholine receptors of the types 1 and 3; αs,i/o,qβγ, heterotrimeric Gs-, Gi/o- and Gqproteins; PKA, protein kinase A; CREB, cAMP response element-binding; PLC, phosphoinositide-specific phospholipase C; PKC, protein kinase C; cAMP, 3',5'-cyclic adenosine monophosphate; DAG, diacylglycerol; Ins(1,4,5)P3, phosphatidylinositol 1,4,5-

The treatment with bromocriptine can reverse the metabolic abnormalities in humans with DM2 and obesity and in obese experimental animals. Using 22 obese patients with DM2 it was found that bromocriptine significantly reduces both glycosylated hemoglobin level and fasting and postprandial plasma glucose concentrations, it decreases the mean plasma glucose concentration during oral glucose tolerance test, which indicates the improvement in glucose tolerance (Pijl et al., 2000). There are also reports that administration of Cycloset (bromocriptine mesylate) either as monotherapy or adjunctive therapy to sulfonylurea or insulin markedly reduces glycosylated hemoglobin, plasma triglycerides and free fatty acid levels (Scranton et al., 2007). The effects of once-daily morning Cycloset therapy on glycemic

triphosphate

combined action of insulin and leptin suggests that the brain signaling systems sensitive to these hormones have the common components enabling their interaction which takes place in the hypothalamus or the other brain areas sensitive to insulin and leptin. This view finds support in the fact that leptin directly governs glucose homeostasis via activation of leptin receptors in neurons within the hypothalamic arcuate nucleus enriched by IRs (Huo et al., 2009). Summing up, the brain is a critical site for mediating leptin metabolic-improving actions in DM and the action of central leptin is in concert with the action of insulin and, probably of IGF-1.

#### **3. Neurotransmitter signaling systems in the diabetic brain**

The various neurotransmitter systems, including dopaminergic, serotonergic, cholinergic, glutamatergic, and GABAergic, undergo a significant change in DM (Jackson & Paulose, 1999; Gireesh et al., 2008; Antony et al., 2010a; Anu et al., 2010; T.P. Kumar et al., 2010) (Fig. 2). The well-coordinated activation and inhibition of different neurotransmitter systems in normal brain are disrupted in DM-associated hyper- and hypoglycemia and in the case of insulin and leptin deficit. The synergistic effect of alterations of neurotransmitter receptors leads to neurodegenerative changes in different brain areas and to the development of CNS disorders and dysfunctions.

#### **3.1 Dopamine signaling**

DA is the predominant catecholamine neurotransmitter in the brain of mammals, where it controls a variety of functions including locomotor activity, cognition, emotion, positive reinforcement, food intake, and endocrine regulation. DA also plays multiple roles in the periphery as a modulator of cardiovascular function, catecholamine release, hormone secretion, vascular tone, and gastrointestinal motility. The results obtained with diabetic animals and the clinical study of patients with DM2 showed that reduced dopaminergic activity in the brain is involved in the pathogenesis of DM2 and metabolic syndrome and is responsible for DM-induced changes in the CNS (Pijl & Edo, 2002).

The treatment of diabetic patients with selective ligands of dopamine receptors (DARs) is a promising approach to improve the functions of CNS in DM. In the recent years a selective D2-DAR agonist bromocriptine, an ergot derivative, has been widely used in the treatment of DM, especially DM2, and obesity. Bromocriptine acts on a central target in the brain, mainly in hypothalamus, and reduces ventromedial, arcuate and paraventricular hypothalamic drive for increased hepatic glucose production, lipid synthesis and mobilization, and insulin resistance, which decreases the risk of damage of neuronal cells and the cardiovascular system in patients with DM2 (Scranton et al., 2007). It is very important that bromocriptine reduces fasting and postprandial glucose without increasing insulin level and its therapeutic effects are not associated with weight gain or hypoglycemia. The main mechanism of action of bromocriptine is based on its ability to bind with D2-DAR coupled with the adenylyl cyclase (AC) via Gi protein, which provides the utility in resetting hypothalamic circadian organization of monoamine neuronal activities in patients with DM2. The other mechanisms include the influence of bromocriptine on signaling pathways regulated by α-adrenergic ligands and prolactin, as well as its inhibitory effect on serotonin (5-hydroxytryptamine, 5-HT) turnover in the CNS, and may also be involved in glucoselowering effects of bromocriptine (Kerr et al., 2010).

combined action of insulin and leptin suggests that the brain signaling systems sensitive to these hormones have the common components enabling their interaction which takes place in the hypothalamus or the other brain areas sensitive to insulin and leptin. This view finds support in the fact that leptin directly governs glucose homeostasis via activation of leptin receptors in neurons within the hypothalamic arcuate nucleus enriched by IRs (Huo et al., 2009). Summing up, the brain is a critical site for mediating leptin metabolic-improving actions in DM and the action of central leptin is in concert with the action of insulin and,

The various neurotransmitter systems, including dopaminergic, serotonergic, cholinergic, glutamatergic, and GABAergic, undergo a significant change in DM (Jackson & Paulose, 1999; Gireesh et al., 2008; Antony et al., 2010a; Anu et al., 2010; T.P. Kumar et al., 2010) (Fig. 2). The well-coordinated activation and inhibition of different neurotransmitter systems in normal brain are disrupted in DM-associated hyper- and hypoglycemia and in the case of insulin and leptin deficit. The synergistic effect of alterations of neurotransmitter receptors leads to neurodegenerative changes in different brain areas and to the development of CNS

DA is the predominant catecholamine neurotransmitter in the brain of mammals, where it controls a variety of functions including locomotor activity, cognition, emotion, positive reinforcement, food intake, and endocrine regulation. DA also plays multiple roles in the periphery as a modulator of cardiovascular function, catecholamine release, hormone secretion, vascular tone, and gastrointestinal motility. The results obtained with diabetic animals and the clinical study of patients with DM2 showed that reduced dopaminergic activity in the brain is involved in the pathogenesis of DM2 and metabolic syndrome and is

The treatment of diabetic patients with selective ligands of dopamine receptors (DARs) is a promising approach to improve the functions of CNS in DM. In the recent years a selective D2-DAR agonist bromocriptine, an ergot derivative, has been widely used in the treatment of DM, especially DM2, and obesity. Bromocriptine acts on a central target in the brain, mainly in hypothalamus, and reduces ventromedial, arcuate and paraventricular hypothalamic drive for increased hepatic glucose production, lipid synthesis and mobilization, and insulin resistance, which decreases the risk of damage of neuronal cells and the cardiovascular system in patients with DM2 (Scranton et al., 2007). It is very important that bromocriptine reduces fasting and postprandial glucose without increasing insulin level and its therapeutic effects are not associated with weight gain or hypoglycemia. The main mechanism of action of bromocriptine is based on its ability to bind with D2-DAR coupled with the adenylyl cyclase (AC) via Gi protein, which provides the utility in resetting hypothalamic circadian organization of monoamine neuronal activities in patients with DM2. The other mechanisms include the influence of bromocriptine on signaling pathways regulated by α-adrenergic ligands and prolactin, as well as its inhibitory effect on serotonin (5-hydroxytryptamine, 5-HT) turnover in the CNS, and may also be involved in glucose-

**3. Neurotransmitter signaling systems in the diabetic brain** 

responsible for DM-induced changes in the CNS (Pijl & Edo, 2002).

lowering effects of bromocriptine (Kerr et al., 2010).

probably of IGF-1.

disorders and dysfunctions.

**3.1 Dopamine signaling** 

Fig. 2. Gs-, Gi/o- and Gq-coupled signaling pathways including the receptors of the serpentine type regulated by biogenic amines, glutamate, acetylcholine and peptide hormones. The signal components whose activity and expression are significantly altered in DM are underlined. Abbreviations: NPY, neuropeptide Y; GLP-1, glucagon-like peptide-1; D1,2DARs, dopamine receptors of the types 1 and 2; 5-HT1,2,6Rs, 5-hydroxytryptamine receptors of the types 1, 2 and 6; MC3,4Rs, melanocortin receptors of the types 3 and 4; mGlu1,5Rs, metabotropic glutamate receptors of the types 1 and 5; m1,3-MAChRs, muscarinic acetylcholine receptors of the types 1 and 3; αs,i/o,qβγ, heterotrimeric Gs-, Gi/o- and Gqproteins; PKA, protein kinase A; CREB, cAMP response element-binding; PLC, phosphoinositide-specific phospholipase C; PKC, protein kinase C; cAMP, 3',5'-cyclic adenosine monophosphate; DAG, diacylglycerol; Ins(1,4,5)P3, phosphatidylinositol 1,4,5 triphosphate

The treatment with bromocriptine can reverse the metabolic abnormalities in humans with DM2 and obesity and in obese experimental animals. Using 22 obese patients with DM2 it was found that bromocriptine significantly reduces both glycosylated hemoglobin level and fasting and postprandial plasma glucose concentrations, it decreases the mean plasma glucose concentration during oral glucose tolerance test, which indicates the improvement in glucose tolerance (Pijl et al., 2000). There are also reports that administration of Cycloset (bromocriptine mesylate) either as monotherapy or adjunctive therapy to sulfonylurea or insulin markedly reduces glycosylated hemoglobin, plasma triglycerides and free fatty acid levels (Scranton et al., 2007). The effects of once-daily morning Cycloset therapy on glycemic

Hormonal Signaling Systems of the Brain in Diabetes Mellitus 361

reverse the DM-induced changes of D2-DAR functions (Shankar et al., 2007). The hypothalamus and brainstem are two parts of the brain very important for monitoring the glucose status and the regulation of feeding. The hypothalamus, in addition, controls the release of pituitary hormones having a key role in regulation of the CNS and the periphery. These data indicate that the activity of the dopaminergic system in different areas of the diabetic brain either increases or decreases, which must be taken into consideration in clinic

The alteration of DA-regulated signaling cascades in DM is associated with their downstream components, such as the transcription factor CREB playing a pivotal role in DAR-mediated nuclear signaling and neuroplasticity (Finkbeiner, 2000) and D1-DARcoupled PLC involved in the neuromodulation of hippocampal LTD (J. Liu et al., 2009). It was found that STZ-induced DM produces a significant attenuation of functional activity of CREB and PLC in the cerebral cortex and cerebellum of diabetic rats and these alterations are largely eliminated by the treatment with insulin and curcumin (T.P. Kumar et al., 2010). We showed that in the brain of rats with STZ-induced DM1, duration one month, as well as with neonatal model of DM2, duration 3 to 6 months, the sensitivity of AC to regulatory action of bromocriptine was decreased (Shpakov et al., 2006, 2007a). The inhibitory effect of bromocriptine on forskolin-stimulated AC activity and its stimulating effect on GppNHp binding of Gi proteins in synaptosomal membranes of diabetic rats were significantly decreased, predominantly in DM1. As the binding characteristics of DARs and the catalytic activity of AC did not change essentially, a suggestion was made that the impairment of bromocriptine-induced signaling in the diabetic brain was due to the reduced function of Gi proteins (Shpakov et al., 2007b). This view finds support in the fact that the regulatory effects of somatostatin and 5-HT1R agonists acting, like bromocryptine, on AC via Gi protein-coupled receptors were decreased in the brain of diabetic rats (Shpakov et al., 2007a). The attenuation of D2 agonist-induced suppression of appetite in STZ rats (Kuo, 2006) is also likely to be the result of reduction of Gi protein activity in the diabetic brain. Another cause why the activity of dopaminergic system in the diabetic brain is decreased is the reduction in DA uptake and the DA transporter (DAT) expression that depend on the activity of PI 3-kinase and AKT kinase (Garcia et al., 2005). The uptake by DAT is the primary pathway for the clearance of extracellular DA and hence for regulating the magnitude and duration of dopaminergic signaling. Insulin activates PI 3-kinase and AKT kinase, increases DA uptake and blocks the amphetamine-induced DAT intracellular accumulation leading to a decrease of the number of active transporters. In DM1, which is characterized by hypoinsulinemia, the available cell surface DATs are reduced, and this leads to decrease of synaptic DA level. As a result, the DM-induced alterations in DA uptake and transport induce attenuation of synaptic DA signaling. Actually, the impairment of DA uptake and transport systems in the hippocampus of both STZ and spontaneously diabetic *WBN/Kob* rats leads to a significant decrease in the basal level of DA (Yamato et al., 2004).

The brain serotonergic system regulates several behaviors (e.g., feeding, locomotion, reproduction, sleep, pain, aggression and stress responses) as well as some autonomic functions (e.g., thermogenesis, cardiovascular control, circadian rhythm and pancreatic function). The changes of serotonergic transmission in the diabetic brain provoke disturbances in neuronal processing and the altered plasticity of neurotransmission, and play an important role in DM-induced behavioral abnormalities. This is due first of all to the

practice for successful management of DM and its cerebral complications.

**3.2 Serotonin signaling** 

control and plasma lipids are demonstrable throughout the diurnal portion of the day (7 a.m. to 7 p.m.) across postprandial time points. Recently it was shown that the bromocriptine therapy of 4328 patients with DM2 during 6–24 weeks leads to a significant decrease of glycosylated hemoglobin and plasma glucose levels (Kerr et al., 2010).

Bromocriptine improved the functional state of obese glucose-intolerant Syrian hamsters, inducing a decrease in their insulin resistance and markedly lowering the plasma levels of insulin and free fatty acids (S. Luo et al., 2000). These anti-diabetic effects of bromocriptine are associated with its influence on the daily rhythms of metabolic hormones and daily monoamine profiles within the hypothalamic suprachiasmatic nuclei that modulate circadian neuroendocrine activities and, thus, regulate metabolism of seasonal animals. The bromocriptine significantly reduced DA turnover during the light period and shifted daily peaks of the content of 5-HT and 5-hydroxy-indoleacetic acid (5-HIAA), the main metabolite of 5-HT, by 12 h from the light to the dark period of the day within the hypothalamic suprachiasmatic nuclei, it also increased extracellular 5-HIAA in the brain of diabetic hamsters during the dark phase toward levels observed in normal glucose-tolerant animals.

Using animal models it was found that a combined administration of agonists of D1- and D2- DARs is a successful approach for decreasing appetite in both STZ rats and ob/ob mice (Bina & Cincotta, 2000; Kuo, 2006). The anorectic response induced by D1/D2 agonists is due to their antagonistic action on hypothalamic neurons containing NPY, the most potent appetite transducer in the CNS, and on NPY-dependent signaling. In DM the NPY system is up-regulated due to increased expression of both NPY and its receptor and to enhanced release of NPY. The co-administration of D1/D2 agonists normalized the elevated NPY content and hyperphagic effect observed in STZ rats and *ob/ob* mice (Bina & Cincotta, 2000; Kuo, 2006). However, the response of D1/D2 agonist-induced appetite suppression was attenuated in diabetic rats compared to normal animals, which can be ascribed both to a decreased inhibitory action of central dopaminergic system and to enhanced activity of hypothalamic NPY neurons in DM. The insulin treatment in DM normalized the response to D1/D2 agonists owing to the restoration of NPY content in the hypothalamus and DA signaling.

The reduction of activity of the brain dopaminergic system in DM is mainly due to changes of the initial stages of DA-induced signal transduction which involves DARs, Gi or Gs proteins and effectors, AC and phospholipase C (PLC), generating second messengers. In many brain regions the activity of DARs and signal proteins coupled to them has DARspecific differential alterations. The studies in this area are mostly devoted to the functional state of DARs in DM. In the early 1980s it was found that the binding of [3H]-spiperone, antagonist of D2-DAR, to striatal membranes is significantly increased in rats with DM induced by alloxan or STZ treatment, and insulin therapy leads to normalization of functioning of central dopaminergic system (Lozovsky et al., 1981). Recently it was shown that the expression of D1- and D2-DARs and total DAR binding (Bmax) are increased in the cerebral cortex of STZ rats (T.P. Kumar et al., 2010). In the cerebellum D1-DAR was down regulated and D2-DAR up regulated, a total number of DARs being however decreased. The treatment with insulin or curcumin, an active component in rhizome of *Curcuma longa*, reduced DM-induced alteration of D1- and D2-DARs in the cerebral cortex and increased D1- DAR expression in the cerebellum to near control, thereby improving the cognitive and emotional functions associated with these regions. In the hypothalamus and brainstem of STZ rats a significant decrease in the DA content and the number of D2-DARs, and an increase in affinity of the latter were found, and the insulin therapy did not completely

control and plasma lipids are demonstrable throughout the diurnal portion of the day (7 a.m. to 7 p.m.) across postprandial time points. Recently it was shown that the bromocriptine therapy of 4328 patients with DM2 during 6–24 weeks leads to a significant

Bromocriptine improved the functional state of obese glucose-intolerant Syrian hamsters, inducing a decrease in their insulin resistance and markedly lowering the plasma levels of insulin and free fatty acids (S. Luo et al., 2000). These anti-diabetic effects of bromocriptine are associated with its influence on the daily rhythms of metabolic hormones and daily monoamine profiles within the hypothalamic suprachiasmatic nuclei that modulate circadian neuroendocrine activities and, thus, regulate metabolism of seasonal animals. The bromocriptine significantly reduced DA turnover during the light period and shifted daily peaks of the content of 5-HT and 5-hydroxy-indoleacetic acid (5-HIAA), the main metabolite of 5-HT, by 12 h from the light to the dark period of the day within the hypothalamic suprachiasmatic nuclei, it also increased extracellular 5-HIAA in the brain of diabetic hamsters during the dark phase toward levels observed in normal glucose-tolerant animals. Using animal models it was found that a combined administration of agonists of D1- and D2- DARs is a successful approach for decreasing appetite in both STZ rats and ob/ob mice (Bina & Cincotta, 2000; Kuo, 2006). The anorectic response induced by D1/D2 agonists is due to their antagonistic action on hypothalamic neurons containing NPY, the most potent appetite transducer in the CNS, and on NPY-dependent signaling. In DM the NPY system is up-regulated due to increased expression of both NPY and its receptor and to enhanced release of NPY. The co-administration of D1/D2 agonists normalized the elevated NPY content and hyperphagic effect observed in STZ rats and *ob/ob* mice (Bina & Cincotta, 2000; Kuo, 2006). However, the response of D1/D2 agonist-induced appetite suppression was attenuated in diabetic rats compared to normal animals, which can be ascribed both to a decreased inhibitory action of central dopaminergic system and to enhanced activity of hypothalamic NPY neurons in DM. The insulin treatment in DM normalized the response to D1/D2 agonists owing to the restoration of NPY content in the hypothalamus and DA

The reduction of activity of the brain dopaminergic system in DM is mainly due to changes of the initial stages of DA-induced signal transduction which involves DARs, Gi or Gs proteins and effectors, AC and phospholipase C (PLC), generating second messengers. In many brain regions the activity of DARs and signal proteins coupled to them has DARspecific differential alterations. The studies in this area are mostly devoted to the functional state of DARs in DM. In the early 1980s it was found that the binding of [3H]-spiperone, antagonist of D2-DAR, to striatal membranes is significantly increased in rats with DM induced by alloxan or STZ treatment, and insulin therapy leads to normalization of functioning of central dopaminergic system (Lozovsky et al., 1981). Recently it was shown that the expression of D1- and D2-DARs and total DAR binding (Bmax) are increased in the cerebral cortex of STZ rats (T.P. Kumar et al., 2010). In the cerebellum D1-DAR was down regulated and D2-DAR up regulated, a total number of DARs being however decreased. The treatment with insulin or curcumin, an active component in rhizome of *Curcuma longa*, reduced DM-induced alteration of D1- and D2-DARs in the cerebral cortex and increased D1- DAR expression in the cerebellum to near control, thereby improving the cognitive and emotional functions associated with these regions. In the hypothalamus and brainstem of STZ rats a significant decrease in the DA content and the number of D2-DARs, and an increase in affinity of the latter were found, and the insulin therapy did not completely

decrease of glycosylated hemoglobin and plasma glucose levels (Kerr et al., 2010).

signaling.

reverse the DM-induced changes of D2-DAR functions (Shankar et al., 2007). The hypothalamus and brainstem are two parts of the brain very important for monitoring the glucose status and the regulation of feeding. The hypothalamus, in addition, controls the release of pituitary hormones having a key role in regulation of the CNS and the periphery. These data indicate that the activity of the dopaminergic system in different areas of the diabetic brain either increases or decreases, which must be taken into consideration in clinic practice for successful management of DM and its cerebral complications.

The alteration of DA-regulated signaling cascades in DM is associated with their downstream components, such as the transcription factor CREB playing a pivotal role in DAR-mediated nuclear signaling and neuroplasticity (Finkbeiner, 2000) and D1-DARcoupled PLC involved in the neuromodulation of hippocampal LTD (J. Liu et al., 2009). It was found that STZ-induced DM produces a significant attenuation of functional activity of CREB and PLC in the cerebral cortex and cerebellum of diabetic rats and these alterations are largely eliminated by the treatment with insulin and curcumin (T.P. Kumar et al., 2010).

We showed that in the brain of rats with STZ-induced DM1, duration one month, as well as with neonatal model of DM2, duration 3 to 6 months, the sensitivity of AC to regulatory action of bromocriptine was decreased (Shpakov et al., 2006, 2007a). The inhibitory effect of bromocriptine on forskolin-stimulated AC activity and its stimulating effect on GppNHp binding of Gi proteins in synaptosomal membranes of diabetic rats were significantly decreased, predominantly in DM1. As the binding characteristics of DARs and the catalytic activity of AC did not change essentially, a suggestion was made that the impairment of bromocriptine-induced signaling in the diabetic brain was due to the reduced function of Gi proteins (Shpakov et al., 2007b). This view finds support in the fact that the regulatory effects of somatostatin and 5-HT1R agonists acting, like bromocryptine, on AC via Gi protein-coupled receptors were decreased in the brain of diabetic rats (Shpakov et al., 2007a). The attenuation of D2 agonist-induced suppression of appetite in STZ rats (Kuo, 2006) is also likely to be the result of reduction of Gi protein activity in the diabetic brain.

Another cause why the activity of dopaminergic system in the diabetic brain is decreased is the reduction in DA uptake and the DA transporter (DAT) expression that depend on the activity of PI 3-kinase and AKT kinase (Garcia et al., 2005). The uptake by DAT is the primary pathway for the clearance of extracellular DA and hence for regulating the magnitude and duration of dopaminergic signaling. Insulin activates PI 3-kinase and AKT kinase, increases DA uptake and blocks the amphetamine-induced DAT intracellular accumulation leading to a decrease of the number of active transporters. In DM1, which is characterized by hypoinsulinemia, the available cell surface DATs are reduced, and this leads to decrease of synaptic DA level. As a result, the DM-induced alterations in DA uptake and transport induce attenuation of synaptic DA signaling. Actually, the impairment of DA uptake and transport systems in the hippocampus of both STZ and spontaneously diabetic *WBN/Kob* rats leads to a significant decrease in the basal level of DA (Yamato et al., 2004).

#### **3.2 Serotonin signaling**

The brain serotonergic system regulates several behaviors (e.g., feeding, locomotion, reproduction, sleep, pain, aggression and stress responses) as well as some autonomic functions (e.g., thermogenesis, cardiovascular control, circadian rhythm and pancreatic function). The changes of serotonergic transmission in the diabetic brain provoke disturbances in neuronal processing and the altered plasticity of neurotransmission, and play an important role in DM-induced behavioral abnormalities. This is due first of all to the

Hormonal Signaling Systems of the Brain in Diabetes Mellitus 363

hyperglycemic rats with alloxan DM and partially restored 5-HT-regulated functions of the CNS (Bhattacharya & Saraswati, 1991). It indicates the importance of the appropriate

With a decrease of concentration of 5-HT and 5-HIAA in the diabetic brain the number of different types of 5-HTRs and their affinity to available 5-HT increases inducing alteration of 5-HT neurotransmission. Thus, in the frontal cortex of STZ rats the density of 5-HT2AR, coupled to PLC via Gq proteins, was significantly higher than in control group of animals (Sandrini et al., 1997). An increase in affinity of 5-HT2ARs in the cerebral cortex without any change in the number of receptors, and a significant increase in Bmax for these receptors in the brainstem with a decrease in affinity during STZ-induced DM were also shown (Jackson & Paulose, 1999). The alterations of 5-HT2AR in the cerebral cortex and brainstem are a compensatory mechanism responsible for a decrease of 5-HT level in these brain areas in DM. All these parameters returned to normal level by insulin therapy. It seems likely that up-regulation of the 5-HT2AR may have a role in the regulation of insulin secretion from pancreatic islets. As is known, the increased activity of 5-HT2AR in the cerebral cortex and brainstem can increase the sympathetic nerve discharge, thereby increasing the levels of circulating norepinephrine and epinephrine, which leads to inhibition of insulin release from the pancreas. In addition to insulin regulation, an increase in affinity and the number

glycemic control for restoration of 5-HT metabolism in the diabetic brain.

of 5-HT2ARs has a role in pathogenesis of depression and cognitive deficit in DM.

drug, and to improve the clinical as well as cognitive and emotional variables.

Dysfunctions of the serotonergic system of the brain can be the result of DM, but on the other hand, they can be the cause of DM. The attenuation of 5-HT signaling in the brain

In our view, being a compensatory response of the brain to lower levels of 5-HT and its precursors, the increase of the number of 5-HTRs is also a reaction to the weakening of signal transduction through these receptors. The latter may be associated with a decreased expression or the functions of signal proteins, the components of 5-HT-regulated signaling pathways. It was shown that one week after STZ treatment the flat body posture induced by 5-HT1AR agonist 8-hydroxy-2-(dipropylamino)tetralin hydrobromide (8-OH-DPAT) and head twitching induced by 5-HT2AR agonist 2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) were markedly reduced in the diabetic rats compared with control animals, which indicates that STZ-induced DM profoundly affects the sensitivity to drugs acting at 5-HT1A- and 5-HT2ARs (J.X. Li & France, 2008). Insulin treatment during one week restored 8-OH-DPAT and DOI-induced behavioral effects. We found no alteration of the sensitivity of AC signaling system in the brain of STZ rats to selective agonists of 5-HT6R coupled with Gs proteins, while the sensitivity of this system to agonists of 5-HT1AR and 5- HT1BR coupled with Gi proteins was significantly decreased (Shpakov et al., 2007a). We consider the weakening of 5-HT1R-mediated signaling to be associated with decreased expression and activity of Gi proteins because, as mentioned above, a decrease in activity of the other Gi protein-coupled cascades regulated by somatostatin and DA was also detected in the brain in DM. Note that in the diabetic brain the signaling pathways involving Gs proteins were either unchanged or changed very little (Shpakov et al., 2007b). The impairment of response of the diabetic brain to 5-HT was made evident in the recent clinic study where citalopram, a selective 5-HT reuptake inhibitor, was used in the treatment of patients with DM2. It was shown that citalopram is less effective in diabetic patients compared with healthy individuals (Trento et al., 2010). The appropriate control of glucose and insulin plasma level in patients with DM2 makes it possible to increase the efficiency of citalopram treatment and the response of the hypothalamic-pituitary-adrenal axis to this

alteration of the brain sensitivity to 5-HT, which depends on the functioning of 5-HTregulated signaling pathways and the disturbances in the biochemical conversion, reuptake and transport of 5-HT and its metabolites. These changes cause a distorted response of neuronal cells and the CNS as a whole to 5-HT and its analogs, as well as to the drugs that increase the level of central 5-HT.

Selective 5-HT reuptake inhibitors are widely used in the pharmacological treatment of depression typical of both DM1 and DM2 and have a significant effect on the course and outcome of this medical illness (Lustman & Clouse, 2005). The 5-HT reuptake inhibitors contribute to lowering the level of hyperglycemia, decrease the rate of hemoglobin glycosylation, improve metabolic control through their positive effect on weight loss, thereby improving insulin resistance, and restore cognitive functions impaired in DM (Van Tilburg et al., 2001). It was shown that the treatment of 60 patients with depression associated with DM1 and DM2 by fluoxetine, selective 5-HT reuptake inhibitor, significantly reduces depressive symptoms and increases the sensitivity of the brain and the peripheral tissues to insulin (Lustman et al., 2000). Consequently, the approach leading to an increase of the brain 5-HT level and, thus, improving 5-HTR signaling in the CNS is a successful strategy to treat DM (Zhou et al., 2007).

In the late 1970s, it was shown that STZ-induced DM and hyperglycemia have a significant influence on the brain tryptophan (Trp) and 5-HT metabolism (MacKenzie & Trulson, 1978). The content of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA), the main metabolite of 5-HT, as well as 5-HT turnover (5-HIAA/5-HT) is decreased in different brain areas of STZ rats with long-term hyperglycemia and in the hippocampus of spontaneously diabetic *WBN/Kob* rats (Sandirini et al., 1997; Jackson & Paulose, 1999; Yamato et al., 2004). A decrease in 5-HT level is due to the decreased uptake of Trp, the precursor of 5-HT, by the brain (Mackenzie & Trulson, 1978). An increase in the level of insulin can result in decreased plasma concentrations of large neutral amino acids (phenylalanine, valine, leucine, isoleucine, tyrosine) competing with Trp for uptake by the brain, which accounts for a low availability of plasma Trp. The other cause of a decrease of the biosynthesis of 5-HT is a long-lasting inhibition of the rate-limiting enzyme tryptophan-5-hydroxylase 2 (Herrera et al., 2005). It was shown that the Trp level and the free/total Trp ratio in the plasma and in the brain of children and adolescents with DM1 and in women with DM2 were also significantly decreased (Manjarrez-Gutierrez et al., 2009). Free fraction and free fraction/total Trp ratio were also decreased in adolescents with metabolic syndrome, although to a small extent (Herrera-Marquez et al., 2011). In the case of diabetic adolescents two groups of patients, with and without depression, were studied and it was shown that diabetic patients with depression had a lower level of Trp compared with diabetic adolescents without depression (Manjarrez-Gutierrez et al., 2009). Diabetic patients with depression had the most expressed hypoinsulinemia and more extended episodes of hyperglycemia than patients without depression. These results indicate that the degree of disturbances of brain serotonergic activity is likely to correlate with the degree of metabolic disturbances induced by DM1.

Hypoglycemia caused by fasting or by treatment of diabetic patients with peripheral insulin, like hyperglycemia associated with STZ DM, leads to disturbances in serotoninergic system of the brain (Das, 2010). Hypoglycemia increases turnover of 5-HT and decreases the level of 5-HT precursor 5-HIAA in both ventromedial and lateral hypothalamic areas, which induces a decrease of central 5-HT concentration (Shimizu & Bray, 1990). At the same time, i.c.v. administered insulin at doses 50 and 100 μUnits, which induced minimal hypoglycemia, increased 5-HT concentration in the midbrain and ponsmedulla oblongata of

alteration of the brain sensitivity to 5-HT, which depends on the functioning of 5-HTregulated signaling pathways and the disturbances in the biochemical conversion, reuptake and transport of 5-HT and its metabolites. These changes cause a distorted response of neuronal cells and the CNS as a whole to 5-HT and its analogs, as well as to the drugs that

Selective 5-HT reuptake inhibitors are widely used in the pharmacological treatment of depression typical of both DM1 and DM2 and have a significant effect on the course and outcome of this medical illness (Lustman & Clouse, 2005). The 5-HT reuptake inhibitors contribute to lowering the level of hyperglycemia, decrease the rate of hemoglobin glycosylation, improve metabolic control through their positive effect on weight loss, thereby improving insulin resistance, and restore cognitive functions impaired in DM (Van Tilburg et al., 2001). It was shown that the treatment of 60 patients with depression associated with DM1 and DM2 by fluoxetine, selective 5-HT reuptake inhibitor, significantly reduces depressive symptoms and increases the sensitivity of the brain and the peripheral tissues to insulin (Lustman et al., 2000). Consequently, the approach leading to an increase of the brain 5-HT level and, thus, improving 5-HTR signaling in the CNS is a successful

In the late 1970s, it was shown that STZ-induced DM and hyperglycemia have a significant influence on the brain tryptophan (Trp) and 5-HT metabolism (MacKenzie & Trulson, 1978). The content of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA), the main metabolite of 5-HT, as well as 5-HT turnover (5-HIAA/5-HT) is decreased in different brain areas of STZ rats with long-term hyperglycemia and in the hippocampus of spontaneously diabetic *WBN/Kob* rats (Sandirini et al., 1997; Jackson & Paulose, 1999; Yamato et al., 2004). A decrease in 5-HT level is due to the decreased uptake of Trp, the precursor of 5-HT, by the brain (Mackenzie & Trulson, 1978). An increase in the level of insulin can result in decreased plasma concentrations of large neutral amino acids (phenylalanine, valine, leucine, isoleucine, tyrosine) competing with Trp for uptake by the brain, which accounts for a low availability of plasma Trp. The other cause of a decrease of the biosynthesis of 5-HT is a long-lasting inhibition of the rate-limiting enzyme tryptophan-5-hydroxylase 2 (Herrera et al., 2005). It was shown that the Trp level and the free/total Trp ratio in the plasma and in the brain of children and adolescents with DM1 and in women with DM2 were also significantly decreased (Manjarrez-Gutierrez et al., 2009). Free fraction and free fraction/total Trp ratio were also decreased in adolescents with metabolic syndrome, although to a small extent (Herrera-Marquez et al., 2011). In the case of diabetic adolescents two groups of patients, with and without depression, were studied and it was shown that diabetic patients with depression had a lower level of Trp compared with diabetic adolescents without depression (Manjarrez-Gutierrez et al., 2009). Diabetic patients with depression had the most expressed hypoinsulinemia and more extended episodes of hyperglycemia than patients without depression. These results indicate that the degree of disturbances of brain serotonergic activity is likely to correlate with the degree of metabolic disturbances induced by DM1. Hypoglycemia caused by fasting or by treatment of diabetic patients with peripheral insulin, like hyperglycemia associated with STZ DM, leads to disturbances in serotoninergic system of the brain (Das, 2010). Hypoglycemia increases turnover of 5-HT and decreases the level of 5-HT precursor 5-HIAA in both ventromedial and lateral hypothalamic areas, which induces a decrease of central 5-HT concentration (Shimizu & Bray, 1990). At the same time, i.c.v. administered insulin at doses 50 and 100 μUnits, which induced minimal hypoglycemia, increased 5-HT concentration in the midbrain and ponsmedulla oblongata of

increase the level of central 5-HT.

strategy to treat DM (Zhou et al., 2007).

hyperglycemic rats with alloxan DM and partially restored 5-HT-regulated functions of the CNS (Bhattacharya & Saraswati, 1991). It indicates the importance of the appropriate glycemic control for restoration of 5-HT metabolism in the diabetic brain.

With a decrease of concentration of 5-HT and 5-HIAA in the diabetic brain the number of different types of 5-HTRs and their affinity to available 5-HT increases inducing alteration of 5-HT neurotransmission. Thus, in the frontal cortex of STZ rats the density of 5-HT2AR, coupled to PLC via Gq proteins, was significantly higher than in control group of animals (Sandrini et al., 1997). An increase in affinity of 5-HT2ARs in the cerebral cortex without any change in the number of receptors, and a significant increase in Bmax for these receptors in the brainstem with a decrease in affinity during STZ-induced DM were also shown (Jackson & Paulose, 1999). The alterations of 5-HT2AR in the cerebral cortex and brainstem are a compensatory mechanism responsible for a decrease of 5-HT level in these brain areas in DM. All these parameters returned to normal level by insulin therapy. It seems likely that up-regulation of the 5-HT2AR may have a role in the regulation of insulin secretion from pancreatic islets. As is known, the increased activity of 5-HT2AR in the cerebral cortex and brainstem can increase the sympathetic nerve discharge, thereby increasing the levels of circulating norepinephrine and epinephrine, which leads to inhibition of insulin release from the pancreas. In addition to insulin regulation, an increase in affinity and the number of 5-HT2ARs has a role in pathogenesis of depression and cognitive deficit in DM.

In our view, being a compensatory response of the brain to lower levels of 5-HT and its precursors, the increase of the number of 5-HTRs is also a reaction to the weakening of signal transduction through these receptors. The latter may be associated with a decreased expression or the functions of signal proteins, the components of 5-HT-regulated signaling pathways. It was shown that one week after STZ treatment the flat body posture induced by 5-HT1AR agonist 8-hydroxy-2-(dipropylamino)tetralin hydrobromide (8-OH-DPAT) and head twitching induced by 5-HT2AR agonist 2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) were markedly reduced in the diabetic rats compared with control animals, which indicates that STZ-induced DM profoundly affects the sensitivity to drugs acting at 5-HT1A- and 5-HT2ARs (J.X. Li & France, 2008). Insulin treatment during one week restored 8-OH-DPAT and DOI-induced behavioral effects. We found no alteration of the sensitivity of AC signaling system in the brain of STZ rats to selective agonists of 5-HT6R coupled with Gs proteins, while the sensitivity of this system to agonists of 5-HT1AR and 5- HT1BR coupled with Gi proteins was significantly decreased (Shpakov et al., 2007a). We consider the weakening of 5-HT1R-mediated signaling to be associated with decreased expression and activity of Gi proteins because, as mentioned above, a decrease in activity of the other Gi protein-coupled cascades regulated by somatostatin and DA was also detected in the brain in DM. Note that in the diabetic brain the signaling pathways involving Gs proteins were either unchanged or changed very little (Shpakov et al., 2007b). The impairment of response of the diabetic brain to 5-HT was made evident in the recent clinic study where citalopram, a selective 5-HT reuptake inhibitor, was used in the treatment of patients with DM2. It was shown that citalopram is less effective in diabetic patients compared with healthy individuals (Trento et al., 2010). The appropriate control of glucose and insulin plasma level in patients with DM2 makes it possible to increase the efficiency of citalopram treatment and the response of the hypothalamic-pituitary-adrenal axis to this drug, and to improve the clinical as well as cognitive and emotional variables.

Dysfunctions of the serotonergic system of the brain can be the result of DM, but on the other hand, they can be the cause of DM. The attenuation of 5-HT signaling in the brain

Hormonal Signaling Systems of the Brain in Diabetes Mellitus 365

GluRs are significantly increased, which is the main cause of neurodegenerative changes in DM (N. Li et al., 1999; Tomiyama et al., 2005; Joseph et al., 2008; Anu et al., 2010) (Fig. 3).

Fig. 3. Signaling pathways responsible for glutamate toxicity

transmission at the later stages of DM (Baptista et al., 2011).

Abbreviations: mGluRs, metabotropic glutamate receptors; NMDARs and AMPARs,

LTP and LTD, long-term potentiation and long-term depression, respectively.

ionotropic glutamate receptors of NMDA and AMPA types; αqβγ, heterotrimeric Gq-protein;

The synaptic level of glutamate in the brain depends on the high-affinity glutamate transporter GLAST, the major component of synaptic glutamate reuptake system, that plays an important role in the termination of glutamatergic neurotransmission and prevention of excitotoxicity, it also depends on the activity of GluRs regulating synaptic glutamate release (Danbolt, 2001). In nerve terminals specific vesicular transporters GluT1-3 allow incorporation of glutamate into synaptic vesicles. These transporters have an essential role in glutamate recycling and homeostasis in the CNS and the abnormalities of this functioning are responsible for development of neurological disorders (Benarroch, 2010). Synaptic release of endogenous glutamate is mediated with the voltage-dependent N-, L- and P/Qtype Ca2+ channels controlling the entry of Ca2+ into nerve terminals. In the diabetic brain the content of glutamate transporters and the α1A subunit of P/Q type Ca2+ channels are changed. In the cerebellum of STZ rats the expression of the glutamate transporter GLAST gene was decreased, which indicates a decrease of glutamate reuptake (Anu et al., 2010). In the hippocampus a decrease of the level of glutamate transporters was transient, being evident mainly at the early stages of DM. This suggests that after the initial stress induced by DM the hippocampus was somehow able to respond to DM-induced stress, and after two weeks of DM the level of glutamate transporters recovered so that the values remained under control longer. After eight weeks of DM, the levels of glutamate transporters and P/Q-type Ca2+ channels did not change but the basal release of glutamate was significantly increased in hippocampal synaptosomes, which may underlie alterations in synaptic

induces hyperphagia and other disturbances of feeding behavior, which, in turn, leads to the obesity and DM2 (Heisler et al., 2002). The cause of this is in that the central 5-HT activates, via 5-HT2CRs expressed on POMC neurons, signaling pathways regulated by melanocortin and its analogs via MC4R/MC3R located on the same neurons in the arcuate nucleus of the hypothalamus (Zhou et al., 2007; Nonogaki et al., 2008). It follows, these neurons are a potential target for 5-HT2CR agonists because they receive direct input from 5-HT dorsal raphe nucleus neurons and project to the regions associated with energy regulation. 5- HT2CR agonists significantly improved glucose tolerance and reduced plasma insulin in animals with obesity and DM2. 5-HT2CR agonist-induced improvements in glucose homeostasis occurred at concentrations of agonist that had no effect on feeding behavior, energy expenditure, locomotor activity, body weight, and fat mass (Zhou et al., 2007). These data are supported by the results of genetic studies. It was revealed in the murine knockout studies that only deletion of the gene encoding the 5-HT2C receptor produces insulin resistance and DM2 with antecedent hyperphagia and obesity, which demonstrates that 5- HT2CRs are critical for energy homeostasis (Bonasera & Tecott, 2000). It was found that three loci of single nucleotide substitution (G → A at -995, C → T at -759, G → C at -697) and (GT)n dinucleotide repeat polymorphism in the upstream region (promoter) of the 5-HT2CR gene are involved in the development of obesity and DM2 in human (Yuan et al., 2000). The haplotypes containing the nucleotide substitutions are associated with higher transcription levels of the gene and thereby with resistance to obesity and DM2.

#### **3.3 Glutamate signaling**

Glutamate is the major excitatory neurotransmitter in the CNS. It exerts action via ionotropic glutamate receptors (iGluRs) – AMPA and NMDA receptors, and via metabotropic glutamate receptors (mGluRs). mGluRs are predominantly found in pre- and post-synaptic neurons in synapses of the hippocampus, cerebellum and cerebral cortex but are also present in other parts of the brain and in the peripheral tissues. mGluR subtypes are critical in gating the plasticity and memory formation. mGluRs interact with iGluRs, ion channels and membrane-associated enzymes, the generators of second messengers, that modulate the cellular response involved in the processes of differentiation and degeneration of neuronal cells. The activation of mGlu1R and mGlu5R, belonging to group I of mGluRs, enhances phosphoinositide hydrolysis and mobilization of intracellular Ca2+ due to stimulation of PLC, induces the activation of Na+ and K+ channels, modulates voltagedependent Ca2+ channels and inhibits glutamate release, all this being of great importance in the regulation of cascades of biochemical reactions resulting in death of neuronal cells (N.E. Schwartz & Alford, 2000). The iGluRs are ligand-gated nonselective cation channels allowing the flow of K+, Na+ and Ca2+ in response to glutamate binding. These receptors, like mGluRs, have influence on synaptic plasticity and are of prime importance in excitotoxicity. An increase or a decrease of the number of iGluRs on post-synaptic neurons leads to LTP or LTD of neuronal cell, respectively. The activation of NMDA receptors in post-synaptic neurons increases Ca2+ influx, leading to phospholipase A2-mediated arachidonic acid release and neuronal injury by inhibiting the Na+-channels.

Glutamate is essential for synaptic communication in the CNS, but inadequate increase of extracellular glutamate and excessive activation of GluRs causes toxicity in the brain leading to neurodegenerative disorders (Trudeau et al., 2004). Excessive glutamate over-activates the cognate receptors, specifically NMDA receptors, which gives the influx of high level of Ca2+ in the post-synaptic cell. In the diabetic brain the glutamate level and the number of

induces hyperphagia and other disturbances of feeding behavior, which, in turn, leads to the obesity and DM2 (Heisler et al., 2002). The cause of this is in that the central 5-HT activates, via 5-HT2CRs expressed on POMC neurons, signaling pathways regulated by melanocortin and its analogs via MC4R/MC3R located on the same neurons in the arcuate nucleus of the hypothalamus (Zhou et al., 2007; Nonogaki et al., 2008). It follows, these neurons are a potential target for 5-HT2CR agonists because they receive direct input from 5-HT dorsal raphe nucleus neurons and project to the regions associated with energy regulation. 5- HT2CR agonists significantly improved glucose tolerance and reduced plasma insulin in animals with obesity and DM2. 5-HT2CR agonist-induced improvements in glucose homeostasis occurred at concentrations of agonist that had no effect on feeding behavior, energy expenditure, locomotor activity, body weight, and fat mass (Zhou et al., 2007). These data are supported by the results of genetic studies. It was revealed in the murine knockout studies that only deletion of the gene encoding the 5-HT2C receptor produces insulin resistance and DM2 with antecedent hyperphagia and obesity, which demonstrates that 5- HT2CRs are critical for energy homeostasis (Bonasera & Tecott, 2000). It was found that three loci of single nucleotide substitution (G → A at -995, C → T at -759, G → C at -697) and (GT)n dinucleotide repeat polymorphism in the upstream region (promoter) of the 5-HT2CR gene are involved in the development of obesity and DM2 in human (Yuan et al., 2000). The haplotypes containing the nucleotide substitutions are associated with higher transcription

Glutamate is the major excitatory neurotransmitter in the CNS. It exerts action via ionotropic glutamate receptors (iGluRs) – AMPA and NMDA receptors, and via metabotropic glutamate receptors (mGluRs). mGluRs are predominantly found in pre- and post-synaptic neurons in synapses of the hippocampus, cerebellum and cerebral cortex but are also present in other parts of the brain and in the peripheral tissues. mGluR subtypes are critical in gating the plasticity and memory formation. mGluRs interact with iGluRs, ion channels and membrane-associated enzymes, the generators of second messengers, that modulate the cellular response involved in the processes of differentiation and degeneration of neuronal cells. The activation of mGlu1R and mGlu5R, belonging to group I of mGluRs, enhances phosphoinositide hydrolysis and mobilization of intracellular Ca2+ due to stimulation of PLC, induces the activation of Na+ and K+ channels, modulates voltagedependent Ca2+ channels and inhibits glutamate release, all this being of great importance in the regulation of cascades of biochemical reactions resulting in death of neuronal cells (N.E. Schwartz & Alford, 2000). The iGluRs are ligand-gated nonselective cation channels allowing the flow of K+, Na+ and Ca2+ in response to glutamate binding. These receptors, like mGluRs, have influence on synaptic plasticity and are of prime importance in excitotoxicity. An increase or a decrease of the number of iGluRs on post-synaptic neurons leads to LTP or LTD of neuronal cell, respectively. The activation of NMDA receptors in post-synaptic neurons increases Ca2+ influx, leading to phospholipase A2-mediated

levels of the gene and thereby with resistance to obesity and DM2.

arachidonic acid release and neuronal injury by inhibiting the Na+-channels.

Glutamate is essential for synaptic communication in the CNS, but inadequate increase of extracellular glutamate and excessive activation of GluRs causes toxicity in the brain leading to neurodegenerative disorders (Trudeau et al., 2004). Excessive glutamate over-activates the cognate receptors, specifically NMDA receptors, which gives the influx of high level of Ca2+ in the post-synaptic cell. In the diabetic brain the glutamate level and the number of

**3.3 Glutamate signaling** 

GluRs are significantly increased, which is the main cause of neurodegenerative changes in DM (N. Li et al., 1999; Tomiyama et al., 2005; Joseph et al., 2008; Anu et al., 2010) (Fig. 3).

Fig. 3. Signaling pathways responsible for glutamate toxicity

Abbreviations: mGluRs, metabotropic glutamate receptors; NMDARs and AMPARs, ionotropic glutamate receptors of NMDA and AMPA types; αqβγ, heterotrimeric Gq-protein; LTP and LTD, long-term potentiation and long-term depression, respectively.

The synaptic level of glutamate in the brain depends on the high-affinity glutamate transporter GLAST, the major component of synaptic glutamate reuptake system, that plays an important role in the termination of glutamatergic neurotransmission and prevention of excitotoxicity, it also depends on the activity of GluRs regulating synaptic glutamate release (Danbolt, 2001). In nerve terminals specific vesicular transporters GluT1-3 allow incorporation of glutamate into synaptic vesicles. These transporters have an essential role in glutamate recycling and homeostasis in the CNS and the abnormalities of this functioning are responsible for development of neurological disorders (Benarroch, 2010). Synaptic release of endogenous glutamate is mediated with the voltage-dependent N-, L- and P/Qtype Ca2+ channels controlling the entry of Ca2+ into nerve terminals. In the diabetic brain the content of glutamate transporters and the α1A subunit of P/Q type Ca2+ channels are changed. In the cerebellum of STZ rats the expression of the glutamate transporter GLAST gene was decreased, which indicates a decrease of glutamate reuptake (Anu et al., 2010). In the hippocampus a decrease of the level of glutamate transporters was transient, being evident mainly at the early stages of DM. This suggests that after the initial stress induced by DM the hippocampus was somehow able to respond to DM-induced stress, and after two weeks of DM the level of glutamate transporters recovered so that the values remained under control longer. After eight weeks of DM, the levels of glutamate transporters and P/Q-type Ca2+ channels did not change but the basal release of glutamate was significantly increased in hippocampal synaptosomes, which may underlie alterations in synaptic transmission at the later stages of DM (Baptista et al., 2011).

Hormonal Signaling Systems of the Brain in Diabetes Mellitus 367

GABAA receptors, which open membrane chloride channels and stabilize the membrane potential below firing threshold, and GABAB receptors, which act via G proteins to reduce transmitter release from presynaptic terminals. The inhibitory GABA-releasing interneurons mediate the function of excitatory glutamatergic neurons in the brain regions, which contributes significantly to the control of glutamate content in brain regions and prevents glutamate toxicity induced in the brain of hypo- and hyperglycemic diabetic rats. Disruption of GABAergic inhibition induces seizures leading to neuronal damage and, therefore, the pathophysiology of many seizure disorders is the result of alteration of GABA receptor

It was shown that the synaptic level of GABA and its release in the diabetic brain are slightly changed or remain unchanged. The extracellular basal level of GABA at dentate gyrus of STZ rats 12 weeks after the induction of DM showed no changes (Reisi et al., 2009). The content of vesicular GABA transporter was significantly decreased in hippocampal synaptosomal membranes in two week DM, although only minor changes in the release of GABA and in the loading capacity of GABA transporters were found (Baptista et al., 2011). This indicates that the alterations of GABA signaling, typical of the diabetic brain, are due to the changes in the level and functional activity of GABA receptors and down-stream signal

Actually, the GABA binding and the gene expression of the subunits of GABAAα1 and GABAB receptors were decreased in the cerebral cortex of diabetic rats compared to control animals. In the diabetic hypoglycemic rats having two episodes of insulin-induced hypoglycemia in the course of 10 days GABA binding and expression of GABA receptor subunits were reduced to a greater extent in comparison with diabetic hyper/euglycemic animals. This is the evidence that hypoglycemia amplifies the adverse effects of hyperglycemia on GABAergic system, and the impairments of functions of GABAergic neurons in the diabetic cerebral cortex are intensified in hypoglycemia. The expression of glutamate decarboxylase, the rate-limiting enzyme of GABA synthesis, which is used as a marker of GABAergic activity, was also significantly down regulated in DM and hypoglycemia exacerbated the altered expression (Antony et al., 2010a). The same picture is found in the cerebellum, where GABA receptors are involved in control of coordination and motor learning and, like in the cerebral cortex, play a critical role in neuronal excitability and modulation of synaptic neurotransmission (Luján, 2007). In the cerebellum of STZ rats with hyperglycemia the gene expression of GABAAα1 subunit and glutamate decarboxylase was decreased and these molecular alterations were exacerbated by recurrent hypoglycemia (Sherin et al., 2010). The gene expression of CREB, a stimulus-inducible transcription activator implicated in the activation of protein synthesis required for long-term memory and seizure formation, was significantly down regulated in DM and recurrent hypoglycemia. Since CREB up-regulates endogenous GABAAα1 transcription, the decreased expression of CREB in the cerebellum of hypoglycemic and hyperglycemic rats led to the attenuation of GABAergic system and, as a result, to excitotoxic damage of neuronal cells (Sherin et al., 2010). It follows that hypo- and hyperglycemia in DM both decrease GABAergic neuroprotective function in the cerebral cortex and cerebellum, which accounts

for increased vulnerability of these brain areas to subsequent neuronal damage.

In the brain acetylcholine functions either as a neuromodulator, or as a neutotransmitter, activating via metabotropic muscarinic acetylcholine receptors (MAChRs) a multitude of

function (Antony et al., 2010a).

**3.5 Acetylcholine signaling** 

components of GABA-regulated intracellular cascades.

In the cerebral synaptosomes from STZ mice the K+- and 4-aminopyridine-evoked Ca2+ dependent glutamate release was significantly increased. The treatment of synaptosomes with a combination of ω-agatoxin IVA (a P-type Ca2+ channel blocker) and ω-conotoxin GVIA (an N-type Ca2+ channel blocker) completely inhibited K+- or 4-aminopyridineinduced increase in glutamate release and prevented glutamate toxicity typical of the diabetic brain (Satoh & Takahashi, 2008). It means that STZ-induced DM enhanced a depolarization-evoked Ca2+-dependent glutamate release in cerebral synaptosomes by stimulating Ca2+ entry through both P- and N-type Ca2+ channels. It was also shown that voltage-dependent Ca2+ currents through N-, P- and L-type Ca2+ channels were enhanced in dorsal root ganglion neurons of STZ rats and Bio Bred/Worchester diabetic rats, which directly mediated the increase of glutamate exocytosis and induced DM-associated excitotoxicity (Voitenko et al., 2000; Hall et al., 2001). These data allow the selective blockers of the Ca2+ channels to be considered possible drugs for the treatment of diabetic patients with neuronal disorders associated with an increased level of synaptic glutamate.

In the cerebral cortex and cerebellum of STZ rats and hypoglycemic diabetic rats the expression of NR1 and NR2B receptor subunits and mGlu5R genes and the number of the receptors were increased (Joseph et al., 2008). The activity of mGlu5R was increased, which led to stimulation of the activity of PLC coupled with mGlu5R via Gq protein and to an increase of the content of intracellular inositol 1,4,5-triphosphate receptors interacting with the second messenger phosphatidyl inositol 1,4,5-triphosphate generated by PLC. The increase of activity of NMDA receptors and the mGlu5R-associated stimulation of PLC activity mediated Ca2+ overload in cells causing neuronal cell damage and neurodegeneration in the diabetic brain, affecting as it did the motor learning and memory ability (Anu et al., 2010). In the dorsal horn of the lumbar spinal cord of STZ rats the levels of mRNAs coding several AMPA receptor subunits (GluR1, GluR2, and GluR3), NMDA receptor subunits (NR2A and NR2B), as well as mGlu1R and mGlu5R were also up regulated (Tomiyama et al., 2005). In the deep dorsal horn of STZ rats the level of NMDA receptors with high affinity for glutamate, namely NR1/NR2A or NR1/NR2B receptors, was the highest. Also increased was the number of NMDA and AMPA receptors in the gray matter of the spinal cord of the *ob/ob* mice responsible for pain, sensory perception and muscle control (N. Li et al., 1999). Thus, the elevated level of specific GluRs/GluR subunits in the spinal cord is a precondition for the pathogenesis of sensory impairment leading to diabetic neuropathy in DM. The use of GluRs antagonists decreasing enhanced activity of these receptors in the diabetic brain significantly ameliorated hyperalgesia and allodynia in experimental DM1 (Malcangio & Tomlinson, 1998; Calcutt & Chaplan, 1997), which suggests that increased excitatory tone in the spinal cord plays an important role in the development of diabetic neuropathy. It should be pointed out that NR2B-selective antagonists are effective in suppressing hyperalgesia in STZ rats with neuropathic pain at doses devoid of negative side effects, which indicates their suitability for control of sensory symptoms induced by DM (Tomiyama et al., 2005). It is worth mentioning that some antagonists of GluRs, e.g. the NMDA receptor antagonists dextromethorphan and amantadine, are used in clinical practice in the treatment of diabetic patients and markedly ameliorate the neuropathic pain in some patients (Nelson et al., 1997; Amin & Sturrock, 2003).

#### **3.4 GABA signaling**

GABAergic inhibitory function in the cerebral cortex is of great importance in the regulation of excitability and responsiveness of cortical neurons. GABA inhibition is mediated both by

In the cerebral synaptosomes from STZ mice the K+- and 4-aminopyridine-evoked Ca2+ dependent glutamate release was significantly increased. The treatment of synaptosomes with a combination of ω-agatoxin IVA (a P-type Ca2+ channel blocker) and ω-conotoxin GVIA (an N-type Ca2+ channel blocker) completely inhibited K+- or 4-aminopyridineinduced increase in glutamate release and prevented glutamate toxicity typical of the diabetic brain (Satoh & Takahashi, 2008). It means that STZ-induced DM enhanced a depolarization-evoked Ca2+-dependent glutamate release in cerebral synaptosomes by stimulating Ca2+ entry through both P- and N-type Ca2+ channels. It was also shown that voltage-dependent Ca2+ currents through N-, P- and L-type Ca2+ channels were enhanced in dorsal root ganglion neurons of STZ rats and Bio Bred/Worchester diabetic rats, which directly mediated the increase of glutamate exocytosis and induced DM-associated excitotoxicity (Voitenko et al., 2000; Hall et al., 2001). These data allow the selective blockers of the Ca2+ channels to be considered possible drugs for the treatment of diabetic patients

with neuronal disorders associated with an increased level of synaptic glutamate.

neuropathic pain in some patients (Nelson et al., 1997; Amin & Sturrock, 2003).

GABAergic inhibitory function in the cerebral cortex is of great importance in the regulation of excitability and responsiveness of cortical neurons. GABA inhibition is mediated both by

**3.4 GABA signaling** 

In the cerebral cortex and cerebellum of STZ rats and hypoglycemic diabetic rats the expression of NR1 and NR2B receptor subunits and mGlu5R genes and the number of the receptors were increased (Joseph et al., 2008). The activity of mGlu5R was increased, which led to stimulation of the activity of PLC coupled with mGlu5R via Gq protein and to an increase of the content of intracellular inositol 1,4,5-triphosphate receptors interacting with the second messenger phosphatidyl inositol 1,4,5-triphosphate generated by PLC. The increase of activity of NMDA receptors and the mGlu5R-associated stimulation of PLC activity mediated Ca2+ overload in cells causing neuronal cell damage and neurodegeneration in the diabetic brain, affecting as it did the motor learning and memory ability (Anu et al., 2010). In the dorsal horn of the lumbar spinal cord of STZ rats the levels of mRNAs coding several AMPA receptor subunits (GluR1, GluR2, and GluR3), NMDA receptor subunits (NR2A and NR2B), as well as mGlu1R and mGlu5R were also up regulated (Tomiyama et al., 2005). In the deep dorsal horn of STZ rats the level of NMDA receptors with high affinity for glutamate, namely NR1/NR2A or NR1/NR2B receptors, was the highest. Also increased was the number of NMDA and AMPA receptors in the gray matter of the spinal cord of the *ob/ob* mice responsible for pain, sensory perception and muscle control (N. Li et al., 1999). Thus, the elevated level of specific GluRs/GluR subunits in the spinal cord is a precondition for the pathogenesis of sensory impairment leading to diabetic neuropathy in DM. The use of GluRs antagonists decreasing enhanced activity of these receptors in the diabetic brain significantly ameliorated hyperalgesia and allodynia in experimental DM1 (Malcangio & Tomlinson, 1998; Calcutt & Chaplan, 1997), which suggests that increased excitatory tone in the spinal cord plays an important role in the development of diabetic neuropathy. It should be pointed out that NR2B-selective antagonists are effective in suppressing hyperalgesia in STZ rats with neuropathic pain at doses devoid of negative side effects, which indicates their suitability for control of sensory symptoms induced by DM (Tomiyama et al., 2005). It is worth mentioning that some antagonists of GluRs, e.g. the NMDA receptor antagonists dextromethorphan and amantadine, are used in clinical practice in the treatment of diabetic patients and markedly ameliorate the GABAA receptors, which open membrane chloride channels and stabilize the membrane potential below firing threshold, and GABAB receptors, which act via G proteins to reduce transmitter release from presynaptic terminals. The inhibitory GABA-releasing interneurons mediate the function of excitatory glutamatergic neurons in the brain regions, which contributes significantly to the control of glutamate content in brain regions and prevents glutamate toxicity induced in the brain of hypo- and hyperglycemic diabetic rats. Disruption of GABAergic inhibition induces seizures leading to neuronal damage and, therefore, the pathophysiology of many seizure disorders is the result of alteration of GABA receptor function (Antony et al., 2010a).

It was shown that the synaptic level of GABA and its release in the diabetic brain are slightly changed or remain unchanged. The extracellular basal level of GABA at dentate gyrus of STZ rats 12 weeks after the induction of DM showed no changes (Reisi et al., 2009). The content of vesicular GABA transporter was significantly decreased in hippocampal synaptosomal membranes in two week DM, although only minor changes in the release of GABA and in the loading capacity of GABA transporters were found (Baptista et al., 2011). This indicates that the alterations of GABA signaling, typical of the diabetic brain, are due to the changes in the level and functional activity of GABA receptors and down-stream signal components of GABA-regulated intracellular cascades.

Actually, the GABA binding and the gene expression of the subunits of GABAAα1 and GABAB receptors were decreased in the cerebral cortex of diabetic rats compared to control animals. In the diabetic hypoglycemic rats having two episodes of insulin-induced hypoglycemia in the course of 10 days GABA binding and expression of GABA receptor subunits were reduced to a greater extent in comparison with diabetic hyper/euglycemic animals. This is the evidence that hypoglycemia amplifies the adverse effects of hyperglycemia on GABAergic system, and the impairments of functions of GABAergic neurons in the diabetic cerebral cortex are intensified in hypoglycemia. The expression of glutamate decarboxylase, the rate-limiting enzyme of GABA synthesis, which is used as a marker of GABAergic activity, was also significantly down regulated in DM and hypoglycemia exacerbated the altered expression (Antony et al., 2010a). The same picture is found in the cerebellum, where GABA receptors are involved in control of coordination and motor learning and, like in the cerebral cortex, play a critical role in neuronal excitability and modulation of synaptic neurotransmission (Luján, 2007). In the cerebellum of STZ rats with hyperglycemia the gene expression of GABAAα1 subunit and glutamate decarboxylase was decreased and these molecular alterations were exacerbated by recurrent hypoglycemia (Sherin et al., 2010). The gene expression of CREB, a stimulus-inducible transcription activator implicated in the activation of protein synthesis required for long-term memory and seizure formation, was significantly down regulated in DM and recurrent hypoglycemia. Since CREB up-regulates endogenous GABAAα1 transcription, the decreased expression of CREB in the cerebellum of hypoglycemic and hyperglycemic rats led to the attenuation of GABAergic system and, as a result, to excitotoxic damage of neuronal cells (Sherin et al., 2010). It follows that hypo- and hyperglycemia in DM both decrease GABAergic neuroprotective function in the cerebral cortex and cerebellum, which accounts for increased vulnerability of these brain areas to subsequent neuronal damage.

#### **3.5 Acetylcholine signaling**

In the brain acetylcholine functions either as a neuromodulator, or as a neutotransmitter, activating via metabotropic muscarinic acetylcholine receptors (MAChRs) a multitude of

Hormonal Signaling Systems of the Brain in Diabetes Mellitus 369

against obesity-associated glucose intolerance, insulin resistance, hyperinsulinemia, and hyperglycemia triggered by a high-fat diet, chemical disruption of hypothalamic neurons by gold-thioglucose, and genetic disruption of the leptin gene. These data favor the fact that the m3-MAChR and other subtypes of MAChRs can represent a potential pharmacologic target

Along with insulin, some substances, vitamin D3 and curcumin in particular, which differ in the chemical nature and the mechanism of action are also capable of restoring the functions of cholinergic system in the diabetic brain. Vitamin D3, as well as insulin, markedly recovers the altered gene expression of m1- and m3-MAChRs in the cerebral cortex and cerebellum of STZ rats and binding parameters of these receptors to near control (P.T. Kumar et al., 2011). Vitamin D3-induced improvement of the cholinergic system and glucose homeostasis in the diabetic brain is due to the influence of vitamin D3 on activity of pancreatic m3-MAChR followed by enhanced synthesis and secretion of insulin and reduction of the neuronal disorders in DM (P.T. Kumar et al., 2011). It was found, in addition, that vitamin D3 restores the disrupted expression of IR in the cerebral cortex of diabetic rats. Curcumin possesses powerful anti-diabetic properties and has the ability to modulate MAChRs thereby

ameliorating the impaired cognitive functions in DM (Peeyush Kumar et al., 2011).

learning and memory.

**4.1 Melanocortin signaling** 

**4. Peptide hormones in the diabetic brain** 

Ionotropic nicotine acetylcholine receptors are also involved in the pathogenesis of neurodegenerative processes in DM. Note that the stimulation of nicotinic acetylcholine receptors and MAChRs provokes opposing physiological and behavioral responses, which is due to the existence of multiple nicotinic and muscarinic receptor subtypes and their different anatomical distributions in the CNS. For example, nicotine administration inhibits food intake, increases metabolic rate, and leads to reduced adiposity (M.D. Li et al., 2003), while the activation of m3-MAChRs induces hyperphagia and obesity (Gautam et al., 2008). α7-Nicotinic receptors highly expressed in the course of brain development are implicated in memory, attention and information processing (Picciotto et al., 2000). In the cortex of STZ rats the expression of α7-nicotinic receptors was markedly increased. The receptors significantly influenced the activity within the cortex circuitry, and DM-associated deregulation of this activity could contribute to disorders involving the cerebral cortex (Peeyush Kumar et al., 2011). Alongside with the increase in α7-nicotinic receptors expression, in the cerebral cortex of diabetic rats were revealed the increased acetylcholine esterase and the decreased choline acetyl transferase mRNA levels, which indicates fast acetylcholine degradation and a subsequent down stimulation of acetylcholine receptors causing undesirable effects on cognitive functions. These changes in the expression of acetylcholine esterase and choline acetyl transferase in DM led to a reduction of cholinergic neurotransmission efficiency due to a decrease in acetylcholine levels in the synaptic cleft, thus contributing to progressive cognitive impairment and other neurological dysfunctions in DM. Insulin therapy and curcumin substantially regularize the increased expression of acetylcholine esterase and choline acetyl transferase, and significantly revert up-regulation of α7-nicotinic receptor in the cortex of STZ rats improving the cognitive functions, such as

The DM2 and obesity of humans and animals are strongly associated with variations in a gene encoding MC4R coupled with AC via Gs proteins (Farooqi et al., 2003) (Fig. 2). MC4R

for the treatment of DM, obesity and associated neurological disorders.

signaling pathways important for modulating neuronal excitability, synaptic plasticity and feedback regulation of acetylcholine release and, thus, controls the functional, behavioral and pathological states of the CNS (Dani, 2001). Acetylcholine also activates ionotropic nicotinic acetylcholine receptors that form ligand-gated ion channels in the plasma membranes of the neurons and on the postsynaptic side of the neuromuscular junction. The activation of nicotinic receptors in the CNS induces depolarization of the plasma membrane, culminating in an excitatory postsynaptic potential in neuron, the activation of voltagegated ion channels and the increase of calcium permeability. The changes in the number and activity of the metabotropic and ionotropic acetylcholine receptors have been implicated in the pathophysiology of many diseases of the CNS, including cognitive impairment.

It was shown that in the cerebral cortex, hypothalamus and brainstem of STZ rats the number of Gq-coupled m1-MAChRs and the expression of genes encoding m1-MAChR were decreased with an increase in affinity of the receptor to agonists, and the binding parameters of the m1- MAChR were reversed to near control by the treatment with insulin (Gireesh et al., 2008; Peeyush Kumar et al., 2011). In the cerebral cortex of the diabetic and control rats with insulininduced long-term hypoglycemia the maximal binding of m1-MAChRs and their expression were reduced to a greater extent compared with diabetic animals with hyperglycemia (Sherin et al., 2011). At the same time, in the cerebellum and corpus striatum of both diabetic rats and hypoglycemic diabetic and control rats the binding parameters and gene expression of m1-MAChRs was, on the contrary, increased (Antony et al., 2010b). This indicates that the alterations in the initial steps of m1-MAChR signaling in the diabetic brain are area-specific.

The STZ-induced DM and insulin-induced hypoglycemia both lead to a significant increase of the binding of another Gq-coupled m3-MAChR in the cerebral cortex and cerebellum but the extent of changes induced by hypoglycemia was significantly higher compared to DM, which indicates the detrimental effect of recurrent hypoglycemia on cholinergic system in the brain (Antony et al., 2010b; Peeyush Kumar et al., 2011; Sherin et al., 2011). This allows a conclusion that the imbalance in glucose homeostasis affects acetylcholine metabolism and cholinergic muscarinic neurotransmission in the brain, and changes the expression and function of cholinergic receptors. The study of 7-week- and 90-week-old STZ rats showed that in the brainstem of both groups of animals the number of m1-MAChRs was significantly decreased whereas the number of m3-MAChRs greatly increased compared to their respective controls, and the insulin treatment reversed the binding parameters of m1- and m3-MAChRs to near control level (Balakrishnan et al., 2009). In the cerebral cortex of 7 week-old STZ rats the number of m1-MAChRs decreased by 28 %, while the number of m3- MAChRs increased by 30 %. In the cerebral cortex of 90-week-old diabetic rats the number of m1- and m3-MAChRs increased by 43 and 23 %, respectively, and the level of acetylcholine was significantly increased compared to control (Savitha et al., 2010). These alterations of m1- and m3-MAChR expression correlate with cholinergic hypofunction in short-term and prolonged STZ-induced DM. It should be noted that m1- and m3-MAChRs are abundantly expressed in the brain regions involved in cognition, including the cerebral cortex, hippocampus and striatum (Porter et al., 2002).

As a rule, most animal models of obesity and hyperinsulinemia are associated with increased vagal cholinergic activity that is strongly associated with the m3-MAChR expressed in the brain and the peripheral tissues (Gautam et al., 2008). The absence of m3- MAChR protects the animals against experimentally or genetically induced obesity and obesity-associated metabolic deficit and greatly ameliorates the impairments in glucose homeostasis and insulin sensitivity. The m3-MAChR-deficient mice are largely protected

signaling pathways important for modulating neuronal excitability, synaptic plasticity and feedback regulation of acetylcholine release and, thus, controls the functional, behavioral and pathological states of the CNS (Dani, 2001). Acetylcholine also activates ionotropic nicotinic acetylcholine receptors that form ligand-gated ion channels in the plasma membranes of the neurons and on the postsynaptic side of the neuromuscular junction. The activation of nicotinic receptors in the CNS induces depolarization of the plasma membrane, culminating in an excitatory postsynaptic potential in neuron, the activation of voltagegated ion channels and the increase of calcium permeability. The changes in the number and activity of the metabotropic and ionotropic acetylcholine receptors have been implicated in

the pathophysiology of many diseases of the CNS, including cognitive impairment.

cortex, hippocampus and striatum (Porter et al., 2002).

It was shown that in the cerebral cortex, hypothalamus and brainstem of STZ rats the number of Gq-coupled m1-MAChRs and the expression of genes encoding m1-MAChR were decreased with an increase in affinity of the receptor to agonists, and the binding parameters of the m1- MAChR were reversed to near control by the treatment with insulin (Gireesh et al., 2008; Peeyush Kumar et al., 2011). In the cerebral cortex of the diabetic and control rats with insulininduced long-term hypoglycemia the maximal binding of m1-MAChRs and their expression were reduced to a greater extent compared with diabetic animals with hyperglycemia (Sherin et al., 2011). At the same time, in the cerebellum and corpus striatum of both diabetic rats and hypoglycemic diabetic and control rats the binding parameters and gene expression of m1-MAChRs was, on the contrary, increased (Antony et al., 2010b). This indicates that the alterations in the initial steps of m1-MAChR signaling in the diabetic brain are area-specific. The STZ-induced DM and insulin-induced hypoglycemia both lead to a significant increase of the binding of another Gq-coupled m3-MAChR in the cerebral cortex and cerebellum but the extent of changes induced by hypoglycemia was significantly higher compared to DM, which indicates the detrimental effect of recurrent hypoglycemia on cholinergic system in the brain (Antony et al., 2010b; Peeyush Kumar et al., 2011; Sherin et al., 2011). This allows a conclusion that the imbalance in glucose homeostasis affects acetylcholine metabolism and cholinergic muscarinic neurotransmission in the brain, and changes the expression and function of cholinergic receptors. The study of 7-week- and 90-week-old STZ rats showed that in the brainstem of both groups of animals the number of m1-MAChRs was significantly decreased whereas the number of m3-MAChRs greatly increased compared to their respective controls, and the insulin treatment reversed the binding parameters of m1- and m3-MAChRs to near control level (Balakrishnan et al., 2009). In the cerebral cortex of 7 week-old STZ rats the number of m1-MAChRs decreased by 28 %, while the number of m3- MAChRs increased by 30 %. In the cerebral cortex of 90-week-old diabetic rats the number of m1- and m3-MAChRs increased by 43 and 23 %, respectively, and the level of acetylcholine was significantly increased compared to control (Savitha et al., 2010). These alterations of m1- and m3-MAChR expression correlate with cholinergic hypofunction in short-term and prolonged STZ-induced DM. It should be noted that m1- and m3-MAChRs are abundantly expressed in the brain regions involved in cognition, including the cerebral

As a rule, most animal models of obesity and hyperinsulinemia are associated with increased vagal cholinergic activity that is strongly associated with the m3-MAChR expressed in the brain and the peripheral tissues (Gautam et al., 2008). The absence of m3- MAChR protects the animals against experimentally or genetically induced obesity and obesity-associated metabolic deficit and greatly ameliorates the impairments in glucose homeostasis and insulin sensitivity. The m3-MAChR-deficient mice are largely protected against obesity-associated glucose intolerance, insulin resistance, hyperinsulinemia, and hyperglycemia triggered by a high-fat diet, chemical disruption of hypothalamic neurons by gold-thioglucose, and genetic disruption of the leptin gene. These data favor the fact that the m3-MAChR and other subtypes of MAChRs can represent a potential pharmacologic target for the treatment of DM, obesity and associated neurological disorders.

Along with insulin, some substances, vitamin D3 and curcumin in particular, which differ in the chemical nature and the mechanism of action are also capable of restoring the functions of cholinergic system in the diabetic brain. Vitamin D3, as well as insulin, markedly recovers the altered gene expression of m1- and m3-MAChRs in the cerebral cortex and cerebellum of STZ rats and binding parameters of these receptors to near control (P.T. Kumar et al., 2011). Vitamin D3-induced improvement of the cholinergic system and glucose homeostasis in the diabetic brain is due to the influence of vitamin D3 on activity of pancreatic m3-MAChR followed by enhanced synthesis and secretion of insulin and reduction of the neuronal disorders in DM (P.T. Kumar et al., 2011). It was found, in addition, that vitamin D3 restores the disrupted expression of IR in the cerebral cortex of diabetic rats. Curcumin possesses powerful anti-diabetic properties and has the ability to modulate MAChRs thereby ameliorating the impaired cognitive functions in DM (Peeyush Kumar et al., 2011).

Ionotropic nicotine acetylcholine receptors are also involved in the pathogenesis of neurodegenerative processes in DM. Note that the stimulation of nicotinic acetylcholine receptors and MAChRs provokes opposing physiological and behavioral responses, which is due to the existence of multiple nicotinic and muscarinic receptor subtypes and their different anatomical distributions in the CNS. For example, nicotine administration inhibits food intake, increases metabolic rate, and leads to reduced adiposity (M.D. Li et al., 2003), while the activation of m3-MAChRs induces hyperphagia and obesity (Gautam et al., 2008).

α7-Nicotinic receptors highly expressed in the course of brain development are implicated in memory, attention and information processing (Picciotto et al., 2000). In the cortex of STZ rats the expression of α7-nicotinic receptors was markedly increased. The receptors significantly influenced the activity within the cortex circuitry, and DM-associated deregulation of this activity could contribute to disorders involving the cerebral cortex (Peeyush Kumar et al., 2011). Alongside with the increase in α7-nicotinic receptors expression, in the cerebral cortex of diabetic rats were revealed the increased acetylcholine esterase and the decreased choline acetyl transferase mRNA levels, which indicates fast acetylcholine degradation and a subsequent down stimulation of acetylcholine receptors causing undesirable effects on cognitive functions. These changes in the expression of acetylcholine esterase and choline acetyl transferase in DM led to a reduction of cholinergic neurotransmission efficiency due to a decrease in acetylcholine levels in the synaptic cleft, thus contributing to progressive cognitive impairment and other neurological dysfunctions in DM. Insulin therapy and curcumin substantially regularize the increased expression of acetylcholine esterase and choline acetyl transferase, and significantly revert up-regulation of α7-nicotinic receptor in the cortex of STZ rats improving the cognitive functions, such as learning and memory.

#### **4. Peptide hormones in the diabetic brain**

#### **4.1 Melanocortin signaling**

The DM2 and obesity of humans and animals are strongly associated with variations in a gene encoding MC4R coupled with AC via Gs proteins (Farooqi et al., 2003) (Fig. 2). MC4R

Hormonal Signaling Systems of the Brain in Diabetes Mellitus 371

acted as partial agonists and decreased the level of cAMP in cell cultures. In rats injected with peptide corresponding to the N-terminal domain of MC4R, like in the case of blockade of hypothalamic MCRs, the food intake, body weight, plasma insulin and triglycerides levels increased significantly (Hofbauer et al., 2008). Antibodies against peptide derived from the first loop of MC3R amplified AC stimulating effect of α-MSH; contrary to this, antibodies against the peptide derivatives of the third loop of the same receptor reduced the effect of hormone, acting as non-competitive antagonist. In rats injected with peptide derived from the third loop of MC3R, the body weight and blood pressure were increased and motor activity was decreased. In plasma the levels of triglycerides, insulin and leptin were significantly increased compared with control. At the same time, the rats injected with peptide derived from the first loop had no changes of physiological and biochemical parameters (Peter et al., 2010). These data indicate that peptides derived from the MCRs and the antibodies to them directly influence melanocortin signaling pathways and cause changes in brain signaling, their action being receptor- and site-specific, i.e. depends on the antigenic determinants they correspond to, and can either inhibit or enhance signal transduction via the cognate receptor. This is in good agreement with the results obtained with other peptides, the derivatives of extracellular and intracellular regions of G proteincoupled receptors (Shpakov, 2011). Thus, peptides derived from the extracellular loops of MCRs and the other receptors involved in the functioning of the brain are a promising tool in the study of pathogenesis of DM and its CNS complications and give a perspective approach to develop new models of DM and obesity based on antibody-induced deregulation of the central signaling network controlled by hormones of different nature.

NPY, a 36-amino acid peptide, stimulates feeding and decreases energy expenditure. NPY, one of the most abundant brain peptides in the paraventricular and arcuate nuclei and in the other regions of the hypothalamus is implicated in regulation of the feeding behavior, energy balance, and pituitary secretion. Disruptions in NPY signaling due to high or low abundance of NPY and cognate receptors deregulate the homeostatic milieu to promote hyperinsulinemia, hyperglycemia, fat accrual, and overt DM. In STZ rats the activity of hypothalamic NPY neurons was significantly increased, and induced marked hyperphagia (Sindelar et al., 2002; Kuo et al., 2006). STZ rats between 3 and 14 weeks after induction of DM1 had a significant increase (35–200 %) of NPY concentration in the paraventricular and the ventromedial nuclei and lateral hypothalamic area of hypothalamus, the major appetiteregulating areas sensitive to hyperphagic and polydipsic action of NPY. The concentration of NPY was also increased in the arcuate nucleus and medial preoptic area, the regions involved in modulating hormone secretion. A significant increase of NPY level was found in the hypothalamic sites of diabetic rats 6 months after STZ treatment, and insulin therapy for 3 months completely prevented the STZ-induced increments in NPY levels in all

In the rats with DM2 the level of NPY and the activity of arcuate nucleus NPY neurons were also increased, which led to hyperphagia and obesity, and may have contributed to hyperinsulinemia and altered pituitary secretion, and the insulin treatment returned the activity of NPY system (Maekawa et al., 2006). The level of mRNA encoding NPY was increased in cells of the arcuate nucleus of young 11-week-old Goto-Kakizaki rats having hyperphagia associated with leptin resistance. Following i.c.v. injection of the NPY-Y1

**4.2 Neuropeptide Y signaling** 

hypothalamic sites (Sahu et al., 1990).

expression is restricted primarily to the brain, where it is widely expressed. MC4R agonists α-MSH, a product of POMC, and melanotan II promote a negative energy balance by decreasing the food intake and increasing the CNS activity and energy expenditure, whereas hypothalamic AgRP, MC4R antagonist, on the contrary, increases food intake (Balthasar et al., 2005). MC4R pathways also regulate glucose metabolism and insulin sensitivity (Fan et al., 2000; Obici et al., 2001; Nogueiras et al., 2007). Central injection of the MC4R agonist reduces insulin secretion, while administration of the MC4R antagonist increases serum insulin levels. Furthermore, elevated plasma insulin level was detected in the young lean MC4R knockout mice, and impaired insulin tolerance before the onset of detectable hyperphagia or obesity (Fan et al., 2000; Haskell-Luevano et al., 2009). The mice with functionally inactive MC4R had obesity strikingly reminiscent of the agouti syndrome, which indicates that the disturbances in MC4R signaling pathways were the primary cause of the agouti obesity. The available data indicate that hypothalamic melanocortin system controls adiposity levels rapidly and perhaps more efficiently than the other CNS signaling pathways (Nogueiras et al., 2007). It should be emphasized that the hypothalamic melanocortin system is regulated by leptin. It must be really so because the conditions associated with low leptin levels, such as fasting or genetic leptin deficiency, provide for decreased hypothalamic POMC mRNA level as well as increased expression of AgRP (Havel et al., 2000). Leptin infusion is followed by an increase in POMC mRNA level as well as in MC4R mRNA level and inhibits the production of AgRP (Gout et al., 2008).

Despite the lack of data on the relationship between neurodegenerative diseases and the alterations of the hypothalamic melanocotrin system in obesity and DM, a suggestion was made that a decreased activity of this system and increased expression of AgRP are the prime causes of neurodegenerative processes in the diabetic brain. As is known, MC4Rmediated improvement of cognitive functions involves neuroprotective action, regenerative trophic effects, promotion of adaptive plasticity, and suppression of damage pathways triggered by apoptotic and inflammatory factors (Tatro, 2006). The treatment with Nle4,D-Phe7-MSH, a selective MC4R agonist, reduced postischemic tissue injury and improved the recovery of behavioral functions even when the treatment began as late as 9 hours after ischemia. The neuroprotective effect of Nle4,D-Phe7-MSH was prevented by MC4R antagonists (Giuliani et al., 2006). The treatment blocked the ischemia-induced impairment of spatial learning and memory for at least 12 days due to the MC4R-mediated reduction of death of hippocampal cells. Because a very high dose of MC4R agonists actually enhanced learning, it was assumed that their effect is likely to have involved neurotrophic action of melanocortin, including promotion of neurite sprouting and functional recovery from nerve injury. The regulatory effects of α-MSH and selective MC4R agonists on neuronal plasticity and survival could be mediated by their influence on neuronal signaling pathways regulated by other neurotransmitters. It was shown that MC4R activation by agonists exerts the inhibitory effect on hypothalamic neurons through inhibition of neuronal firing rate and facilitation of GABA transmission (Nargund et al., 2006). This suggests the central melanocortin system to be responsible for a large number of neurodegenerative processes in the CNS previously associated with the other signaling systems of the brain.

Studying the activity of antibodies against extracellular loops of MC3R and MC4R strong evidence was obtained for the involvement of central melanocortin system in DM and obesity. Hofbauer and coworkers immunized the rats with peptides corresponding to the Nterminal extracellular domain MC4R and to the first and third extracellular loops of MC3R (Hofbauer et al., 2008; Peter et al., 2010). The antibodies to the N-terminal domain of MC4R

expression is restricted primarily to the brain, where it is widely expressed. MC4R agonists α-MSH, a product of POMC, and melanotan II promote a negative energy balance by decreasing the food intake and increasing the CNS activity and energy expenditure, whereas hypothalamic AgRP, MC4R antagonist, on the contrary, increases food intake (Balthasar et al., 2005). MC4R pathways also regulate glucose metabolism and insulin sensitivity (Fan et al., 2000; Obici et al., 2001; Nogueiras et al., 2007). Central injection of the MC4R agonist reduces insulin secretion, while administration of the MC4R antagonist increases serum insulin levels. Furthermore, elevated plasma insulin level was detected in the young lean MC4R knockout mice, and impaired insulin tolerance before the onset of detectable hyperphagia or obesity (Fan et al., 2000; Haskell-Luevano et al., 2009). The mice with functionally inactive MC4R had obesity strikingly reminiscent of the agouti syndrome, which indicates that the disturbances in MC4R signaling pathways were the primary cause of the agouti obesity. The available data indicate that hypothalamic melanocortin system controls adiposity levels rapidly and perhaps more efficiently than the other CNS signaling pathways (Nogueiras et al., 2007). It should be emphasized that the hypothalamic melanocortin system is regulated by leptin. It must be really so because the conditions associated with low leptin levels, such as fasting or genetic leptin deficiency, provide for decreased hypothalamic POMC mRNA level as well as increased expression of AgRP (Havel et al., 2000). Leptin infusion is followed by an increase in POMC mRNA level as well

as in MC4R mRNA level and inhibits the production of AgRP (Gout et al., 2008).

the CNS previously associated with the other signaling systems of the brain.

Studying the activity of antibodies against extracellular loops of MC3R and MC4R strong evidence was obtained for the involvement of central melanocortin system in DM and obesity. Hofbauer and coworkers immunized the rats with peptides corresponding to the Nterminal extracellular domain MC4R and to the first and third extracellular loops of MC3R (Hofbauer et al., 2008; Peter et al., 2010). The antibodies to the N-terminal domain of MC4R

Despite the lack of data on the relationship between neurodegenerative diseases and the alterations of the hypothalamic melanocotrin system in obesity and DM, a suggestion was made that a decreased activity of this system and increased expression of AgRP are the prime causes of neurodegenerative processes in the diabetic brain. As is known, MC4Rmediated improvement of cognitive functions involves neuroprotective action, regenerative trophic effects, promotion of adaptive plasticity, and suppression of damage pathways triggered by apoptotic and inflammatory factors (Tatro, 2006). The treatment with Nle4,D-Phe7-MSH, a selective MC4R agonist, reduced postischemic tissue injury and improved the recovery of behavioral functions even when the treatment began as late as 9 hours after ischemia. The neuroprotective effect of Nle4,D-Phe7-MSH was prevented by MC4R antagonists (Giuliani et al., 2006). The treatment blocked the ischemia-induced impairment of spatial learning and memory for at least 12 days due to the MC4R-mediated reduction of death of hippocampal cells. Because a very high dose of MC4R agonists actually enhanced learning, it was assumed that their effect is likely to have involved neurotrophic action of melanocortin, including promotion of neurite sprouting and functional recovery from nerve injury. The regulatory effects of α-MSH and selective MC4R agonists on neuronal plasticity and survival could be mediated by their influence on neuronal signaling pathways regulated by other neurotransmitters. It was shown that MC4R activation by agonists exerts the inhibitory effect on hypothalamic neurons through inhibition of neuronal firing rate and facilitation of GABA transmission (Nargund et al., 2006). This suggests the central melanocortin system to be responsible for a large number of neurodegenerative processes in

acted as partial agonists and decreased the level of cAMP in cell cultures. In rats injected with peptide corresponding to the N-terminal domain of MC4R, like in the case of blockade of hypothalamic MCRs, the food intake, body weight, plasma insulin and triglycerides levels increased significantly (Hofbauer et al., 2008). Antibodies against peptide derived from the first loop of MC3R amplified AC stimulating effect of α-MSH; contrary to this, antibodies against the peptide derivatives of the third loop of the same receptor reduced the effect of hormone, acting as non-competitive antagonist. In rats injected with peptide derived from the third loop of MC3R, the body weight and blood pressure were increased and motor activity was decreased. In plasma the levels of triglycerides, insulin and leptin were significantly increased compared with control. At the same time, the rats injected with peptide derived from the first loop had no changes of physiological and biochemical parameters (Peter et al., 2010). These data indicate that peptides derived from the MCRs and the antibodies to them directly influence melanocortin signaling pathways and cause changes in brain signaling, their action being receptor- and site-specific, i.e. depends on the antigenic determinants they correspond to, and can either inhibit or enhance signal transduction via the cognate receptor. This is in good agreement with the results obtained with other peptides, the derivatives of extracellular and intracellular regions of G proteincoupled receptors (Shpakov, 2011). Thus, peptides derived from the extracellular loops of MCRs and the other receptors involved in the functioning of the brain are a promising tool in the study of pathogenesis of DM and its CNS complications and give a perspective approach to develop new models of DM and obesity based on antibody-induced deregulation of the central signaling network controlled by hormones of different nature.

#### **4.2 Neuropeptide Y signaling**

NPY, a 36-amino acid peptide, stimulates feeding and decreases energy expenditure. NPY, one of the most abundant brain peptides in the paraventricular and arcuate nuclei and in the other regions of the hypothalamus is implicated in regulation of the feeding behavior, energy balance, and pituitary secretion. Disruptions in NPY signaling due to high or low abundance of NPY and cognate receptors deregulate the homeostatic milieu to promote hyperinsulinemia, hyperglycemia, fat accrual, and overt DM. In STZ rats the activity of hypothalamic NPY neurons was significantly increased, and induced marked hyperphagia (Sindelar et al., 2002; Kuo et al., 2006). STZ rats between 3 and 14 weeks after induction of DM1 had a significant increase (35–200 %) of NPY concentration in the paraventricular and the ventromedial nuclei and lateral hypothalamic area of hypothalamus, the major appetiteregulating areas sensitive to hyperphagic and polydipsic action of NPY. The concentration of NPY was also increased in the arcuate nucleus and medial preoptic area, the regions involved in modulating hormone secretion. A significant increase of NPY level was found in the hypothalamic sites of diabetic rats 6 months after STZ treatment, and insulin therapy for 3 months completely prevented the STZ-induced increments in NPY levels in all hypothalamic sites (Sahu et al., 1990).

In the rats with DM2 the level of NPY and the activity of arcuate nucleus NPY neurons were also increased, which led to hyperphagia and obesity, and may have contributed to hyperinsulinemia and altered pituitary secretion, and the insulin treatment returned the activity of NPY system (Maekawa et al., 2006). The level of mRNA encoding NPY was increased in cells of the arcuate nucleus of young 11-week-old Goto-Kakizaki rats having hyperphagia associated with leptin resistance. Following i.c.v. injection of the NPY-Y1

Hormonal Signaling Systems of the Brain in Diabetes Mellitus 373

GLP-1 analogs show promise in the treatment of neurodegenerative diseases induced by DM, because they cross the BBB and increase neuroneogenesis. The GLP-1 analogs, such as GLP-1 with the substitution of Ala82-aminobutyric acid, with the increased stability to dipeptidyl peptidase IV elicit the insulinotropic activity and improve the central and peripheral symptoms of DM2 (Green & Flatt, 2007). The dipeptidyl peptidase-stable analogs of GLP-1 stimulate AC activity in neuronal cells and the AC stimulating effect correlates

The data presented in this review suggest that alterations and disturbances occurring in a majority of hormonal signaling systems in the diabetic brain are responsible for the functioning of the CNS, the central regulation of peripheral functions as well as for memory, cognitive processes, emotion, and social behavior. These alterations leading to the DMassociated CNS disorders and centrally induced diseases of the peripheral systems are likely

The first mechanism is associated with the appearance of damages in one of the signaling systems that may be caused by alterations in the expression or functional activity of sensory, adaptor or effector protein, a component of this system, and also by a deficit or, on the contrary, an excess of hormonal or hormone-like molecules that specifically regulate the system. The damages may be a result of hyperactivation, weakening or modification of the functions of signal protein due to mutations in the translated region of the gene encoding this protein or in the untranslated regions responsible for gene transcription, or else be induced by gene polymorphism in human DM. The other causes are the gene knockout and the mutations leading to gain, loss or modification of the function of signal proteins in experimental models of DM. The changes in concentration and availability of signal molecules can be ascribed to abnormalities in the systems responsible for their synthesis, transport and degradation. In the case of insulin and IGF-1 that penetrate the BBB, a decrease or increase of their level in plasma induces the corresponding alterations of insulin and IGF-1 levels in the brain, which directly affects the functioning of the signaling pathways regulated by these hormones. DM1 gives rise to peripheral hypoinsulinemia which leads to insulin deficiency in the brain, and DM2 to moderate hyperinsulinemia which leads to an increase of central insulin concentration. The abnormalities in one single signaling system influence the activity of the other signaling cascades coupled with and depending on it and induce changes in their functional activity which is a compensatory response of the brain to the primary local dysfunction of hormonal signaling. If the abnormalities are not eliminated, then the changes of brain signaling will amplify and cause deregulation of a comprehensive neuronal signaling network, which resembles "a domino effect". As a result, the disturbances are systemic and irreversible; they have influence on the signal transduction pathways regulated by insulin, IGF-1, leptin, biogenic amines,

The second mechanism is based on the systemic response of the hormonal signaling systems in the brain to significant and prolonged changes of cerebral glucose homeostasis, the state of recurrent hypoglycemia and severe long-term hyperglycemia. This causes alterations in the energy balance in the neuronal and glial cells, inducing different compensatory changes in the signal network to allow maintaining the activity of the brain in the case of inadequate glucose concentrations. The short-term fluctuations in cerebral glucose level cause

with their neuroprotective properties.

to develop via several mechanisms.

glutamate, and neuropeptides.

**5. Conclusion** 

receptor antagonist 1229U91, the amount of food intake in Goto-Kakizaki rats was indistinguishable from that in Wistar rats, thus eliminating hyperphagia. Note that in NPYdeficient diabetic mice the mean daily food intake did not change, while in wild diabetic mice it increased two-fold. Alongside, in NPY-deficient mice the level of mRNA encoding POMC was decreased by as little as 11%, but in wild diabetic mice by 65%. Proceeding from these results, the conclusion was made that NPY is required both for an increase of food intake and for a decrease of POMC gene expression in DM (Sindelar et al., 2002).

The NPY signaling system is tightly associated with dopaminergic, melanocortin and leptin systems of the brain. The increased content of hypothalamic NPY plays a major role in attenuating the anorectic response of D1/D2-DARs agonists in STZ rats (Bina, Cincotta, 2000; Kuo, 2006). Leptin directly restrains the release of NPY and cohorts from the hypothalamic NPY neuronal network, and the complete absence of leptin or hypothalamic leptin receptors induces up-regulation of NPY signaling, which promotes unabated hyperphagia and fat storage (Kalra, 2008). The NPY and melanocortin signaling systems in the arcuate nucleus, where NPY and α-MSH are expressed, act in concert but have opposite functions. Hypothalamic NPY pathways favor anabolic processes and increase the food intake, whereas POMC neurons do the reverse. As a result, in hypothalamus signaling systems both form a complex network integrating hormonal (e.g., insulin and leptin) and metabolic (e.g., glucose) signals of energy homeostasis and initiating the adaptive responses of the diabetic brain (Fioramonti et al., 2007).

#### **4.3 Glucagon-like peptide-1 signaling**

Glucagon-like peptide-1 (GLP-1), a 30-amino-acid peptide hormone, is responsible for modulating blood glucose concentrations by stimulating glucose-dependent insulin secretion and by activating β-cell proliferation. GLP-1 is effective in restoring first-phase insulin response and lowering hyperglycemia in DM2 (Doyle & Egan, 2007). GLP1 also functions in the brain as a neurotransmitter, has the growth factor-like properties and protects neurons from neurotoxic influence, controlling learning behavior, memory and synaptic plasticity (Hamilton & Holscher, 2009; Hamilton et al., 2011). The action of GLP-1 is realized via GLP-1 receptors that in the brain affect neuronal activity through regulation of intracellular cAMP-dependent pathways, modulation of Ca2+ channels, activation of ERK1/ERK2 kinases and other second messenger systems involved in transmitter vesicle release (Gilman et al., 2003) (Fig. 2).

GLP-1 receptor agonists, exendin-4 and Liraglutide, like the inhibitors of GLP-1 degradation (dipeptidylpeptidase IV inhibitors), have been approved for treatment of DM2 (Lovshin & Drucker, 2009; Holst et al., 2011). Note that Liraglutide, analog of human GLP-1 with prolonged half life having a fatty acid palmitoyl group conjugated to the side-chain of Lys26 and an Arg34Ser substitution, is now widely used in DM2 therapy (Lovshin, Drucker, 2009). Exendin-4 and Liraglutide injected subcutaneously for 4, 6, or 10 weeks once daily in *ob/ob*, *db/db* and high-fat-diet-fed mice enhanced proliferation rate of progenitor cells by 100–150 % and stimulated differentiation into neurons in the dentate gyrus (Hamilton et al., 2011). The GLP-1 receptor antagonist exendin(9–36) significantly reduced progenitor cell proliferation in these mice. Exendin-4 and Liraglutide enhanced LTP in the brain and once-daily injection of the GLP-1 analog with Ala8Val substitution enhanced LTP in the brain and reduced the number of amyloid dense-core plaques in mice with insulin resistance and in patients with DM-associated obesity and AD (McClean et al., 2010). These results demonstrate that the GLP-1 analogs show promise in the treatment of neurodegenerative diseases induced by DM, because they cross the BBB and increase neuroneogenesis. The GLP-1 analogs, such as GLP-1 with the substitution of Ala82-aminobutyric acid, with the increased stability to dipeptidyl peptidase IV elicit the insulinotropic activity and improve the central and peripheral symptoms of DM2 (Green & Flatt, 2007). The dipeptidyl peptidase-stable analogs of GLP-1 stimulate AC activity in neuronal cells and the AC stimulating effect correlates with their neuroprotective properties.

#### **5. Conclusion**

372 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

receptor antagonist 1229U91, the amount of food intake in Goto-Kakizaki rats was indistinguishable from that in Wistar rats, thus eliminating hyperphagia. Note that in NPYdeficient diabetic mice the mean daily food intake did not change, while in wild diabetic mice it increased two-fold. Alongside, in NPY-deficient mice the level of mRNA encoding POMC was decreased by as little as 11%, but in wild diabetic mice by 65%. Proceeding from these results, the conclusion was made that NPY is required both for an increase of food

The NPY signaling system is tightly associated with dopaminergic, melanocortin and leptin systems of the brain. The increased content of hypothalamic NPY plays a major role in attenuating the anorectic response of D1/D2-DARs agonists in STZ rats (Bina, Cincotta, 2000; Kuo, 2006). Leptin directly restrains the release of NPY and cohorts from the hypothalamic NPY neuronal network, and the complete absence of leptin or hypothalamic leptin receptors induces up-regulation of NPY signaling, which promotes unabated hyperphagia and fat storage (Kalra, 2008). The NPY and melanocortin signaling systems in the arcuate nucleus, where NPY and α-MSH are expressed, act in concert but have opposite functions. Hypothalamic NPY pathways favor anabolic processes and increase the food intake, whereas POMC neurons do the reverse. As a result, in hypothalamus signaling systems both form a complex network integrating hormonal (e.g., insulin and leptin) and metabolic (e.g., glucose) signals of energy homeostasis and initiating the adaptive responses of the diabetic

Glucagon-like peptide-1 (GLP-1), a 30-amino-acid peptide hormone, is responsible for modulating blood glucose concentrations by stimulating glucose-dependent insulin secretion and by activating β-cell proliferation. GLP-1 is effective in restoring first-phase insulin response and lowering hyperglycemia in DM2 (Doyle & Egan, 2007). GLP1 also functions in the brain as a neurotransmitter, has the growth factor-like properties and protects neurons from neurotoxic influence, controlling learning behavior, memory and synaptic plasticity (Hamilton & Holscher, 2009; Hamilton et al., 2011). The action of GLP-1 is realized via GLP-1 receptors that in the brain affect neuronal activity through regulation of intracellular cAMP-dependent pathways, modulation of Ca2+ channels, activation of ERK1/ERK2 kinases and other second messenger systems involved in transmitter vesicle

GLP-1 receptor agonists, exendin-4 and Liraglutide, like the inhibitors of GLP-1 degradation (dipeptidylpeptidase IV inhibitors), have been approved for treatment of DM2 (Lovshin & Drucker, 2009; Holst et al., 2011). Note that Liraglutide, analog of human GLP-1 with prolonged half life having a fatty acid palmitoyl group conjugated to the side-chain of Lys26 and an Arg34Ser substitution, is now widely used in DM2 therapy (Lovshin, Drucker, 2009). Exendin-4 and Liraglutide injected subcutaneously for 4, 6, or 10 weeks once daily in *ob/ob*, *db/db* and high-fat-diet-fed mice enhanced proliferation rate of progenitor cells by 100–150 % and stimulated differentiation into neurons in the dentate gyrus (Hamilton et al., 2011). The GLP-1 receptor antagonist exendin(9–36) significantly reduced progenitor cell proliferation in these mice. Exendin-4 and Liraglutide enhanced LTP in the brain and once-daily injection of the GLP-1 analog with Ala8Val substitution enhanced LTP in the brain and reduced the number of amyloid dense-core plaques in mice with insulin resistance and in patients with DM-associated obesity and AD (McClean et al., 2010). These results demonstrate that the

intake and for a decrease of POMC gene expression in DM (Sindelar et al., 2002).

brain (Fioramonti et al., 2007).

**4.3 Glucagon-like peptide-1 signaling** 

release (Gilman et al., 2003) (Fig. 2).

The data presented in this review suggest that alterations and disturbances occurring in a majority of hormonal signaling systems in the diabetic brain are responsible for the functioning of the CNS, the central regulation of peripheral functions as well as for memory, cognitive processes, emotion, and social behavior. These alterations leading to the DMassociated CNS disorders and centrally induced diseases of the peripheral systems are likely to develop via several mechanisms.

The first mechanism is associated with the appearance of damages in one of the signaling systems that may be caused by alterations in the expression or functional activity of sensory, adaptor or effector protein, a component of this system, and also by a deficit or, on the contrary, an excess of hormonal or hormone-like molecules that specifically regulate the system. The damages may be a result of hyperactivation, weakening or modification of the functions of signal protein due to mutations in the translated region of the gene encoding this protein or in the untranslated regions responsible for gene transcription, or else be induced by gene polymorphism in human DM. The other causes are the gene knockout and the mutations leading to gain, loss or modification of the function of signal proteins in experimental models of DM. The changes in concentration and availability of signal molecules can be ascribed to abnormalities in the systems responsible for their synthesis, transport and degradation. In the case of insulin and IGF-1 that penetrate the BBB, a decrease or increase of their level in plasma induces the corresponding alterations of insulin and IGF-1 levels in the brain, which directly affects the functioning of the signaling pathways regulated by these hormones. DM1 gives rise to peripheral hypoinsulinemia which leads to insulin deficiency in the brain, and DM2 to moderate hyperinsulinemia which leads to an increase of central insulin concentration. The abnormalities in one single signaling system influence the activity of the other signaling cascades coupled with and depending on it and induce changes in their functional activity which is a compensatory response of the brain to the primary local dysfunction of hormonal signaling. If the abnormalities are not eliminated, then the changes of brain signaling will amplify and cause deregulation of a comprehensive neuronal signaling network, which resembles "a domino effect". As a result, the disturbances are systemic and irreversible; they have influence on the signal transduction pathways regulated by insulin, IGF-1, leptin, biogenic amines, glutamate, and neuropeptides.

The second mechanism is based on the systemic response of the hormonal signaling systems in the brain to significant and prolonged changes of cerebral glucose homeostasis, the state of recurrent hypoglycemia and severe long-term hyperglycemia. This causes alterations in the energy balance in the neuronal and glial cells, inducing different compensatory changes in the signal network to allow maintaining the activity of the brain in the case of inadequate glucose concentrations. The short-term fluctuations in cerebral glucose level cause

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temporary changes in brain signaling, they are reversible and do not significantly affect the physiological functions of the brain, but the long-term alterations of the level and its large amplitude provoke dramatic and irreversible changes and cause the neurodegenerative disorders. For example, a prolonged and untreated DM1 with markedly expressed hyperglycemia as well as DM1 with intensive therapy using high doses of insulin and inadequate control of glucose plasma level, leading to recurrent hypoglycemia, are the major factors causing abnormalities in several signaling systems in parallel including the glutamatergic system responsible for development of glutamate excitotoxicity and CNS disorders.

Until recently, it was generally accepted that abnormalities and alterations in the neurotransmitter systems of the brain and the associated neurodegenerative disorders are the complications of DM and their role in the etiology of this disease is not very important. In the last few years, however, the conception of central genesis of DM has been significantly extended (Cole et al., 2007; de la Monte, 2009). According to this conception, there are cases when the abnormalities in the hormonal signaling systems of the brain will trigger the mechanism leading to insulin resistance or insulin deficiency and, as a result, to the development of DM and its central and peripheral complications. The following factors contribute to DM, a dysfunction in the leptin and the melanocortin systems (leptin and melanocortin model of DM2), and alterations in the 5-HT2CR-coupled serotonergic and the D2R-coupled dopaminergic systems (Bonasera & Tecott, 2000; Heisler et al., 2002; Zhou et al., 2007; Hofbauer et al., 2008; Toda et al., 2009; Peter et al., 2010). In the years to come, this list will, no doubt, be extended with the results of study of the forms of DM with central genesis. Some neurodegenerative diseases are considered to be pre-diabetes or specific forms of earlier DM, e.g. AD is referred to as the third type of DM (de la Monte, 2009).

The etiology of DM should be studied in order to find the most optimal strategy for adequate therapy and clinical management of DM and its CNS complications. The neuronal abnormalities precede DM as its causal factors; therefore it seems appropriate to eliminate the changes in the central signaling systems responsible for these abnormalities, and then to use the effective treatment of DM without high doses of insulin causing dangerous hypoglycemic episodes. A high efficiency has been shown in the case of combined use of insulin and IGF-1 and the drugs that improve the function of dopaminergic, serotonergic, melanocortin, GABAergic and glutamatergic systems. The approaches based on restoration of the functioning of a comprehensive signaling network of the brain are a new avenue of the treatment of DM of both central and peripheral genesis. This will allow avoiding many side effects of insulin monotherapy negatively affecting the CNS in diabetic patients.

#### **6. Acknowledgment**

This work was supported by Grant No. 09-04-00746 from the Russian Foundation of Basic Research and Program of the Russian Academy of Sciences "Fundamental Sciences – Medicine" (2009–2011). We express our thanks to *Inga Menina* for linguistic assistance.

#### **7. References**

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Until recently, it was generally accepted that abnormalities and alterations in the neurotransmitter systems of the brain and the associated neurodegenerative disorders are the complications of DM and their role in the etiology of this disease is not very important. In the last few years, however, the conception of central genesis of DM has been significantly extended (Cole et al., 2007; de la Monte, 2009). According to this conception, there are cases when the abnormalities in the hormonal signaling systems of the brain will trigger the mechanism leading to insulin resistance or insulin deficiency and, as a result, to the development of DM and its central and peripheral complications. The following factors contribute to DM, a dysfunction in the leptin and the melanocortin systems (leptin and melanocortin model of DM2), and alterations in the 5-HT2CR-coupled serotonergic and the D2R-coupled dopaminergic systems (Bonasera & Tecott, 2000; Heisler et al., 2002; Zhou et al., 2007; Hofbauer et al., 2008; Toda et al., 2009; Peter et al., 2010). In the years to come, this list will, no doubt, be extended with the results of study of the forms of DM with central genesis. Some neurodegenerative diseases are considered to be pre-diabetes or specific forms of earlier DM, e.g. AD is referred to as the third type of DM (de la Monte, 2009). The etiology of DM should be studied in order to find the most optimal strategy for adequate therapy and clinical management of DM and its CNS complications. The neuronal abnormalities precede DM as its causal factors; therefore it seems appropriate to eliminate the changes in the central signaling systems responsible for these abnormalities, and then to use the effective treatment of DM without high doses of insulin causing dangerous hypoglycemic episodes. A high efficiency has been shown in the case of combined use of insulin and IGF-1 and the drugs that improve the function of dopaminergic, serotonergic, melanocortin, GABAergic and glutamatergic systems. The approaches based on restoration of the functioning of a comprehensive signaling network of the brain are a new avenue of the treatment of DM of both central and peripheral genesis. This will allow avoiding many

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This work was supported by Grant No. 09-04-00746 from the Russian Foundation of Basic Research and Program of the Russian Academy of Sciences "Fundamental Sciences – Medicine" (2009–2011). We express our thanks to *Inga Menina* for linguistic assistance.

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**16** 

*United States of America* 

**Can VEGF-B Be Used to Treat Neurodegenerative Diseases?** 

Xuri Li, Anil Kumar, Chunsik Lee, Zhongshu Tang, Yang Li, Pachiappan Arjunan, Xu Hou and Fan Zhang

*National Eye Institute, National Institutes of Health, Rockville, Maryland* 

Studies on vascular endothelial growth factor B (VEGF-B) during the past decade or so have shown that VEGF-B appears to be a mysterious molecule with obscure, if not controversial, functions. When VEGF-B was initially discovered (Grimmond et al., 1996; Olofsson et al., 1996a), it was naturally believed to be an angiogenic factor, due to its high sequence homology and similar receptor binding pattern to VEGF, the prototypic angiogenic molecule. Much of our research effort was focused on this speculated angiogenic activity of VEGF-B for a long time. However, studies into this aspect, most of the time, turned out to be disappointing because of the negative findings. Unlike VEGF-A, VEGF-B did not seem to play a significant role in inducing blood vessel growth or vascular permeability, etc (Li et al., 2009). In addition, VEGF-B deficiency in mice did not seem to matter greatly, since VEGF-Bnull mice appeared largely healthy (Aase et al., 2001; Bellomo et al., 2000; Louzier et al., 2003; Reichelt et al., 2003), in contrast to the early embryonic lethality of VEGF-A null mice (Carmeliet et al., 1996; Ferrara et al., 1996). Based on the negative findings, we had once suspected that VEGF-B might be a redundant molecule. In recent years, VEGF-B has been shown to be a potent neuroprotective factor and an apoptosis inhibitor (Li et al., 2009; Li et al., 2008b; Poesen et al., 2008; Sun et al., 2004; Sun et al., 2006), opening up a new research

Thus far, there are five members within the VEGF family, VEGF-A, VEGF-B, PlGF, VEGF-C and VEGF-D (Li and Eriksson, 2001; Lohela et al., 2009). As a prototypic angiogenic factor, VEGF-A has a potent and "universal" angiogenic effect under most physiological and pathological conditions (Carmeliet & Jain, 2000; Ferrara & Kerbel, 2005; Folkman, 2007). The placenta growth factor (PlGF) is required for pathological angiogenesis (Luttun et al., 2002). However, when PlGF-1 is produced in the same population of cells with VEGF-A, it can also act as a natural antagonist of VEGF-A (Cao, 2009; Eriksson et al., 2002). VEGF-C and VEGF-D are important players in lymphangiogenesis (Alitalo et al., 2005; Lohela et al., 2009). Remarkably, the biological function of VEGF-B has remained less studied. VEGF-B displays a high degree of sequence homology to VEGF-A and PlGF, and also binds to the tyrosine kinase VEGF receptor-1 (VEGFR-1) and neuropilin-1 (NP-1), like VEGF-A and PlGF (Olofsson et al., 1998; Olofsson et al., 1996a). VEGF-B is abundantly expressed in most tissues and organs (Aase et al., 1999; Li et al., 2001; Olofsson et al., 1996a). However, VEGF-B under most conditions appeared to be "redundant" or "inert" with no obvious function. The

**1. Introduction** 

avenue in VEGF-B biology.


### **Can VEGF-B Be Used to Treat Neurodegenerative Diseases?**

Xuri Li, Anil Kumar, Chunsik Lee, Zhongshu Tang, Yang Li, Pachiappan Arjunan, Xu Hou and Fan Zhang *National Eye Institute, National Institutes of Health, Rockville, Maryland United States of America* 

#### **1. Introduction**

386 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

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Studies on vascular endothelial growth factor B (VEGF-B) during the past decade or so have shown that VEGF-B appears to be a mysterious molecule with obscure, if not controversial, functions. When VEGF-B was initially discovered (Grimmond et al., 1996; Olofsson et al., 1996a), it was naturally believed to be an angiogenic factor, due to its high sequence homology and similar receptor binding pattern to VEGF, the prototypic angiogenic molecule. Much of our research effort was focused on this speculated angiogenic activity of VEGF-B for a long time. However, studies into this aspect, most of the time, turned out to be disappointing because of the negative findings. Unlike VEGF-A, VEGF-B did not seem to play a significant role in inducing blood vessel growth or vascular permeability, etc (Li et al., 2009). In addition, VEGF-B deficiency in mice did not seem to matter greatly, since VEGF-Bnull mice appeared largely healthy (Aase et al., 2001; Bellomo et al., 2000; Louzier et al., 2003; Reichelt et al., 2003), in contrast to the early embryonic lethality of VEGF-A null mice (Carmeliet et al., 1996; Ferrara et al., 1996). Based on the negative findings, we had once suspected that VEGF-B might be a redundant molecule. In recent years, VEGF-B has been shown to be a potent neuroprotective factor and an apoptosis inhibitor (Li et al., 2009; Li et al., 2008b; Poesen et al., 2008; Sun et al., 2004; Sun et al., 2006), opening up a new research avenue in VEGF-B biology.

Thus far, there are five members within the VEGF family, VEGF-A, VEGF-B, PlGF, VEGF-C and VEGF-D (Li and Eriksson, 2001; Lohela et al., 2009). As a prototypic angiogenic factor, VEGF-A has a potent and "universal" angiogenic effect under most physiological and pathological conditions (Carmeliet & Jain, 2000; Ferrara & Kerbel, 2005; Folkman, 2007). The placenta growth factor (PlGF) is required for pathological angiogenesis (Luttun et al., 2002). However, when PlGF-1 is produced in the same population of cells with VEGF-A, it can also act as a natural antagonist of VEGF-A (Cao, 2009; Eriksson et al., 2002). VEGF-C and VEGF-D are important players in lymphangiogenesis (Alitalo et al., 2005; Lohela et al., 2009). Remarkably, the biological function of VEGF-B has remained less studied. VEGF-B displays a high degree of sequence homology to VEGF-A and PlGF, and also binds to the tyrosine kinase VEGF receptor-1 (VEGFR-1) and neuropilin-1 (NP-1), like VEGF-A and PlGF (Olofsson et al., 1998; Olofsson et al., 1996a). VEGF-B is abundantly expressed in most tissues and organs (Aase et al., 1999; Li et al., 2001; Olofsson et al., 1996a). However, VEGF-B under most conditions appeared to be "redundant" or "inert" with no obvious function. The

Can VEGF-B Be Used to Treat Neurodegenerative Diseases? 389

neurons (Li et al., 2008b), and motor neurons in the spinal cord (Poesen et al., 2008). *In vitro,* VEGF-B protein treatment dose-dependently increased the survival of cultured primary brain cortex neurons (Li et al., 2008b; Sun et al., 2004). *In vivo,* VEGF-B treatment inhibited apoptosis of brain cortical neurons and reduced stroke volume in a middle cerebral artery ligation-induced brain stroke model (Li et al., 2008b). In the retina, we have shown that VEGF-B treatment protected different types of retinal neurons from apoptosis under different pathological conditions. In an optic nerve crush injury model, VEGF-B treatment increased the survival of retinal ganglion cells. In a NMDA-induced retinal neuron apoptosis model, VEGF-B treatment protected retinal neurons in the ganglion cell layer, inner nuclear layer, and outer nuclear layer (Li et al., 2008b). Moreover, Poesen, K *et al* recently showed that VEGF-B treatment protected cultured primary motor neurons from apoptosis (Poesen et al., 2008). Indeed, the neuroprotective effect of VEGF-B was further confirmed using mice in which VEGF-B was genetically deleted. VEGF-B deficiency led to more severe strokes in an experimental stroke model, and exacerbated retinal ganglion cell death in an optic nerve crush injury model (Li et al., 2008b). Moreover, VEGF-B deficient mice developed a more severe form of motor neuron degeneration when intercrossed with the mutant SOD1 mice, whereas VEGF-B intracerebroventricular injection prolonged the survival of mutant SOD1 rats (Poesen et al., 2008). Taken together, both *in vitro* data derived from cultured neurons and *in vivo* work obtained using different animal models showed

that VEGF-B is a critical survival factor for different types of neurons (Fig. 1).

VEGF-B and its receptors are expressed by different types of vascular cells (Aase et al., 1999; Li et al., 2008a; Zhang et al., 2009). We recently found that VEGF-B is a potent survival factor for multiple types of vascular cells, including vascular endothelial cells (EC), pericytes (PC), and smooth muscle cells (SMC) (Li et al., 2009; Zhang et al., 2009). *In vitro,* in both cultured primary vascular cells and established vascular cell lines, VEGF-B treatment increased the survival of not only ECs, but also that of PCs and SMCs (Zhang et al., 2009). In contrast, VEGF-B inhibition by shRNA treatment led to apoptosis in the ECs and PCs. Moreover, increased apoptosis was found in VEGF-B deficient ECs and SMCs isolated from VEGF-B null mice, when the cells were cultured in serum-free medium or under H2O2-induced oxidative stress (Zhang et al., 2009). *In vivo*, VEGF-B deficiency led to poorer blood vessel survival in the cornea after withdrawal of the implanted growth factors, fewer surviving hyaloid vessels in postnatal mouse eyes, and greater oxygen-induced retinal blood vessel degeneration in neonatal mice (Zhang et al., 2009). Thus, both gain- and loss-of-function analyses showed that VEGF-B is required for the survival of multiple types of vascular cells,

The human brain weighs only about 2% of the total body weight. However, it consumes about 20% of the total energy produced in the body, demonstrating the importance of energy metabolism to the neural systems. Indeed, numerous reports have shown that energy deficit is involved in various neurodegenerative disorders, such as Alzheimer's disease (AD) (Beal, 2007), Huntington's Disease (HD) (Browne and Beal, 2004), Parkinson's disease (PD) (Elstner et al., 2011) and Amyotrophic lateral sclerosis (ALS) (D'Alessandro et

**3. VEGF-B is a vascular survival factor** 

especially, under pathological conditions (Fig. 1).

**4. VEGF-B promotes energy metabolism** 

*in vivo* role of VEGF-B therefore remained enigmatic for a long time. In this review, we summarize the recent advances on VEGF-B biology, with a particular interest in its neuroprotective/survival effect on neuronal and vascular cells (Claesson-Welsh, 2008; Karpanen et al., 2008; Lahteenvuo et al., 2009; Li et al., 2008a; Li et al., 2008b; Poesen et al., 2008; Zhang et al., 2009), and further discuss the therapeutic potential of VEGF-B in treating different types of neurodegenerative diseases.

#### **2. VEGF-B is a neuronal protective factor**

VEGF-B is highly expressed in different types of neural tissues, such as the brain (Li et al., 2001; Sun et al., 2004), retina (Li et al., 2008b), spinal cord (Poesen et al., 2008), etc.

Fig. 1. Pleiotropic protective/survival effect of VEGF-B on multiple cell types. Both *in vitro* data derived from cultured neurons and *in vivo* work using different types of animal models have shown that VEGF-B is a critical protective/survival factor for different types of neurons, including cortical, retinal, and spinal cord motor neurons. In addition, VEGF-B is also a potent protective/survival factor for different types of vascular cells, including vascular endothelial cells, smooth muscle cells and pericytes. Moreover, VEGF-B has also been reported to be a protective factor for cardiac myocytes

We and others have shown that VEGF-B is a potent protective/survival factor for different types of neurons, including brain cortical neurons (Li et al., 2008b; Sun et al., 2004), retinal

*in vivo* role of VEGF-B therefore remained enigmatic for a long time. In this review, we summarize the recent advances on VEGF-B biology, with a particular interest in its neuroprotective/survival effect on neuronal and vascular cells (Claesson-Welsh, 2008; Karpanen et al., 2008; Lahteenvuo et al., 2009; Li et al., 2008a; Li et al., 2008b; Poesen et al., 2008; Zhang et al., 2009), and further discuss the therapeutic potential of VEGF-B in treating

VEGF-B is highly expressed in different types of neural tissues, such as the brain (Li et al.,

Fig. 1. Pleiotropic protective/survival effect of VEGF-B on multiple cell types. Both *in vitro* data derived from cultured neurons and *in vivo* work using different types of animal models

We and others have shown that VEGF-B is a potent protective/survival factor for different types of neurons, including brain cortical neurons (Li et al., 2008b; Sun et al., 2004), retinal

have shown that VEGF-B is a critical protective/survival factor for different types of neurons, including cortical, retinal, and spinal cord motor neurons. In addition, VEGF-B is also a potent protective/survival factor for different types of vascular cells, including vascular endothelial cells, smooth muscle cells and pericytes. Moreover, VEGF-B has also

been reported to be a protective factor for cardiac myocytes

2001; Sun et al., 2004), retina (Li et al., 2008b), spinal cord (Poesen et al., 2008), etc.

different types of neurodegenerative diseases.

**2. VEGF-B is a neuronal protective factor** 

neurons (Li et al., 2008b), and motor neurons in the spinal cord (Poesen et al., 2008). *In vitro,* VEGF-B protein treatment dose-dependently increased the survival of cultured primary brain cortex neurons (Li et al., 2008b; Sun et al., 2004). *In vivo,* VEGF-B treatment inhibited apoptosis of brain cortical neurons and reduced stroke volume in a middle cerebral artery ligation-induced brain stroke model (Li et al., 2008b). In the retina, we have shown that VEGF-B treatment protected different types of retinal neurons from apoptosis under different pathological conditions. In an optic nerve crush injury model, VEGF-B treatment increased the survival of retinal ganglion cells. In a NMDA-induced retinal neuron apoptosis model, VEGF-B treatment protected retinal neurons in the ganglion cell layer, inner nuclear layer, and outer nuclear layer (Li et al., 2008b). Moreover, Poesen, K *et al* recently showed that VEGF-B treatment protected cultured primary motor neurons from apoptosis (Poesen et al., 2008). Indeed, the neuroprotective effect of VEGF-B was further confirmed using mice in which VEGF-B was genetically deleted. VEGF-B deficiency led to more severe strokes in an experimental stroke model, and exacerbated retinal ganglion cell death in an optic nerve crush injury model (Li et al., 2008b). Moreover, VEGF-B deficient mice developed a more severe form of motor neuron degeneration when intercrossed with the mutant SOD1 mice, whereas VEGF-B intracerebroventricular injection prolonged the survival of mutant SOD1 rats (Poesen et al., 2008). Taken together, both *in vitro* data derived from cultured neurons and *in vivo* work obtained using different animal models showed that VEGF-B is a critical survival factor for different types of neurons (Fig. 1).

#### **3. VEGF-B is a vascular survival factor**

VEGF-B and its receptors are expressed by different types of vascular cells (Aase et al., 1999; Li et al., 2008a; Zhang et al., 2009). We recently found that VEGF-B is a potent survival factor for multiple types of vascular cells, including vascular endothelial cells (EC), pericytes (PC), and smooth muscle cells (SMC) (Li et al., 2009; Zhang et al., 2009). *In vitro,* in both cultured primary vascular cells and established vascular cell lines, VEGF-B treatment increased the survival of not only ECs, but also that of PCs and SMCs (Zhang et al., 2009). In contrast, VEGF-B inhibition by shRNA treatment led to apoptosis in the ECs and PCs. Moreover, increased apoptosis was found in VEGF-B deficient ECs and SMCs isolated from VEGF-B null mice, when the cells were cultured in serum-free medium or under H2O2-induced oxidative stress (Zhang et al., 2009). *In vivo*, VEGF-B deficiency led to poorer blood vessel survival in the cornea after withdrawal of the implanted growth factors, fewer surviving hyaloid vessels in postnatal mouse eyes, and greater oxygen-induced retinal blood vessel degeneration in neonatal mice (Zhang et al., 2009). Thus, both gain- and loss-of-function analyses showed that VEGF-B is required for the survival of multiple types of vascular cells, especially, under pathological conditions (Fig. 1).

#### **4. VEGF-B promotes energy metabolism**

The human brain weighs only about 2% of the total body weight. However, it consumes about 20% of the total energy produced in the body, demonstrating the importance of energy metabolism to the neural systems. Indeed, numerous reports have shown that energy deficit is involved in various neurodegenerative disorders, such as Alzheimer's disease (AD) (Beal, 2007), Huntington's Disease (HD) (Browne and Beal, 2004), Parkinson's disease (PD) (Elstner et al., 2011) and Amyotrophic lateral sclerosis (ALS) (D'Alessandro et

Can VEGF-B Be Used to Treat Neurodegenerative Diseases? 391

proliferative retinopathy (Reichelt et al., 2003) or blood vessel remodeling in pulmonary

Transgenic expression of all the other VEGF family members, such as VEGF-A (Detmar et al., 1998; Larcher et al., 1998; Xia et al., 2003), PlGF (Odorisio et al., 2002), VEGF-C (Jeltsch et al., 1997), VEGF-D (Karkkainen et al., 2009) or VEGF-E (Kiba et al., 2003) induced either angiogenesis or lymphangiogenesis. VEGF-B is the only member of the VEGF family, transgenic overexpression of which in different organs did not induce angiogenesis or lymphangiogenesis (Karpanen et al., 2008; Mould et al., 2005). VEGF-B overexpression in cardiac myocytes under the alpha-myosin heavy chain promoter did not induce angiogenesis in the heart (Karpanen et al., 2008). Instead, blood vessel density was decreased in the hearts overexpressing VEGF-B (Karpanen et al., 2008). In addition, VEGF-B

Fig. 2. VEGF-B does not induce blood vessel permeability and is minimally angiogenic. Adenoviral gene transfer of the other VEGF family members, such as VEGF-A, VEGF-C and VEGF-D, into rabbit hindlimb skeletal muscles induced strong angiogenesis, vascular permeability, or lymphangiogenesis (Rissanen et al., 2003). VEGF-B adenoviral gene transfer, however, did not induce angiogenesis or lymphangiogenesis in the same model system (Rissanen et al., 2003). Similarly, adenoviral gene transfer of VEGF-A and VEGF-D to rabbit carotid arteries induced robust adventitial angiogenesis, whereas VEGF-B adenoviral gene transfer failed to do so (Bhardwaj et al., 2003, 2005). Another study also showed that VEGF-B167 gene delivery to the mouse skin or ischemic limb did not induce blood vessel

hypertension (Louzier et al., 2003).

growth (Li et al., 2008a)

al., 2011). In addition, dysregulation of lipid pathways has been implicated in AD (Di Paolo and Kim, 2011). These findings thus warrant investigating and developing therapeutic reagents that can regulate neuronal bioenergetic pathways. Recently, VEGF-B has been shown to be involved in energy metabolism, where it facilitates fatty acid uptake from circulation and transfer to metabolically active tissues (Hagberg et al., 2010). We have also seen that VEGF-B upregulated the expression of a number of key enzymes that are involved in lipid and glucose metabolism in cultured cells (our own unpublished data). Based on the above findings, VEGF-B might be an important molecule that could be used to regulate neuronal bioenergetic pathways. Further studies are needed to verify this.

#### **5. VEGF-B does not induce blood vessel permeability**

It is known that all the other VEGF family members, VEGF-A (Dvorak et al., 1995), PlGF (Carmeliet et al., 2001), VEGF-C (Joukov et al., 1997), VEGF-D (Rissanen et al., 2003) and VEGF-E (Ogawa et al., 1998), induce blood vessel permeability. However, numerous studies using different models and approaches, such as VEGF-B deficient and transgenic mice, recombinant protein or gene transfer, have shown that VEGF-B does not affect blood vessel permeability (Aase et al., 2001; Mould et al., 2005; Reichelt et al., 2003) (Fig. 2). Intradermal injection of VEGF-A165, VEGF-A121, and VEGF-C in mice ears increased vascular permeability, while VEGF-B administration had no such effect (Brkovic & Sirois, 2007). VEGF-B167 recombinant protein injection into mouse brain or eye did not induce blood vessel permeability (Li et al., 2008b). In preserved lung grafts, VEGF-A and VEGF-C, but not VEGF-B mediate increased vascular permeability (Abraham et al., 2002). Indeed, when overexpressed in the lung by adenoviral gene transfer, VEGF-B had no effect on blood vessel permeability (Louzier et al., 2003). Adenoviruses expressing VEGF-A and VEGF-D delivered into rabbit hind limb skeletal muscles induced vascular permeability, while adenovirus encoding VEGF-B did not affect blood vessel permeability when administered into skeletal muscles (Rissanen et al., 2003). Thus, data derived from different model systems showed that VEGF-B is the only member of the VEGF family that does not have a significant role in inducing blood vessel permeability

#### **6. Minimum side effect of VEGF-B and its negligible role in angiogenesis**

Due to its high sequence homology and similar receptor binding patterns to VEGF-A (Li and Eriksson, 2001; Nash et al., 2006), VEGF-B was initially believed to be an angiogenic factor. However, studies along this line using VEGF-B deficient and transgenic mice and gene transfer approaches have, most of the time, led to negative findings (Fig. 2).

VEGF-A or VEGF-C deficiency caused embryonic lethality in mice (Carmeliet et al., 1996; Ferrara et al., 1996; Karkkainen et al., 2004). VEGF-B deficient mice, however, are largely healthy with normal physiological angiogenesis (Aase et al., 2001; Bellomo et al., 2000; Louzier et al., 2003; Reichelt et al., 2003). PlGF deficient mice display impaired pathological angiogenesis (Carmeliet et al., 2001; Luttun et al., 2002). VEGF-B deficiency, however, does not affect pathological angiogenesis in most organs studied, such as the wounded skin, hypoxic lung, ischemic retina and limb (Li et al., 2008a). Even though one study reported a role of VEGF-B in pathological (inflammatory) angiogenesis using arthritis models (Mould et al., 2003), we did not observe such an effect in our study (unpublished observation). In contrast to VEGF-A and PlGF, VEGF-B is not required for neovessel formation in

al., 2011). In addition, dysregulation of lipid pathways has been implicated in AD (Di Paolo and Kim, 2011). These findings thus warrant investigating and developing therapeutic reagents that can regulate neuronal bioenergetic pathways. Recently, VEGF-B has been shown to be involved in energy metabolism, where it facilitates fatty acid uptake from circulation and transfer to metabolically active tissues (Hagberg et al., 2010). We have also seen that VEGF-B upregulated the expression of a number of key enzymes that are involved in lipid and glucose metabolism in cultured cells (our own unpublished data). Based on the above findings, VEGF-B might be an important molecule that could be used to regulate

It is known that all the other VEGF family members, VEGF-A (Dvorak et al., 1995), PlGF (Carmeliet et al., 2001), VEGF-C (Joukov et al., 1997), VEGF-D (Rissanen et al., 2003) and VEGF-E (Ogawa et al., 1998), induce blood vessel permeability. However, numerous studies using different models and approaches, such as VEGF-B deficient and transgenic mice, recombinant protein or gene transfer, have shown that VEGF-B does not affect blood vessel permeability (Aase et al., 2001; Mould et al., 2005; Reichelt et al., 2003) (Fig. 2). Intradermal injection of VEGF-A165, VEGF-A121, and VEGF-C in mice ears increased vascular permeability, while VEGF-B administration had no such effect (Brkovic & Sirois, 2007). VEGF-B167 recombinant protein injection into mouse brain or eye did not induce blood vessel permeability (Li et al., 2008b). In preserved lung grafts, VEGF-A and VEGF-C, but not VEGF-B mediate increased vascular permeability (Abraham et al., 2002). Indeed, when overexpressed in the lung by adenoviral gene transfer, VEGF-B had no effect on blood vessel permeability (Louzier et al., 2003). Adenoviruses expressing VEGF-A and VEGF-D delivered into rabbit hind limb skeletal muscles induced vascular permeability, while adenovirus encoding VEGF-B did not affect blood vessel permeability when administered into skeletal muscles (Rissanen et al., 2003). Thus, data derived from different model systems showed that VEGF-B is the only member of the VEGF family that does not have a

**6. Minimum side effect of VEGF-B and its negligible role in angiogenesis** 

transfer approaches have, most of the time, led to negative findings (Fig. 2).

Due to its high sequence homology and similar receptor binding patterns to VEGF-A (Li and Eriksson, 2001; Nash et al., 2006), VEGF-B was initially believed to be an angiogenic factor. However, studies along this line using VEGF-B deficient and transgenic mice and gene

VEGF-A or VEGF-C deficiency caused embryonic lethality in mice (Carmeliet et al., 1996; Ferrara et al., 1996; Karkkainen et al., 2004). VEGF-B deficient mice, however, are largely healthy with normal physiological angiogenesis (Aase et al., 2001; Bellomo et al., 2000; Louzier et al., 2003; Reichelt et al., 2003). PlGF deficient mice display impaired pathological angiogenesis (Carmeliet et al., 2001; Luttun et al., 2002). VEGF-B deficiency, however, does not affect pathological angiogenesis in most organs studied, such as the wounded skin, hypoxic lung, ischemic retina and limb (Li et al., 2008a). Even though one study reported a role of VEGF-B in pathological (inflammatory) angiogenesis using arthritis models (Mould et al., 2003), we did not observe such an effect in our study (unpublished observation). In contrast to VEGF-A and PlGF, VEGF-B is not required for neovessel formation in

neuronal bioenergetic pathways. Further studies are needed to verify this.

**5. VEGF-B does not induce blood vessel permeability** 

significant role in inducing blood vessel permeability

proliferative retinopathy (Reichelt et al., 2003) or blood vessel remodeling in pulmonary hypertension (Louzier et al., 2003).

Transgenic expression of all the other VEGF family members, such as VEGF-A (Detmar et al., 1998; Larcher et al., 1998; Xia et al., 2003), PlGF (Odorisio et al., 2002), VEGF-C (Jeltsch et al., 1997), VEGF-D (Karkkainen et al., 2009) or VEGF-E (Kiba et al., 2003) induced either angiogenesis or lymphangiogenesis. VEGF-B is the only member of the VEGF family, transgenic overexpression of which in different organs did not induce angiogenesis or lymphangiogenesis (Karpanen et al., 2008; Mould et al., 2005). VEGF-B overexpression in cardiac myocytes under the alpha-myosin heavy chain promoter did not induce angiogenesis in the heart (Karpanen et al., 2008). Instead, blood vessel density was decreased in the hearts overexpressing VEGF-B (Karpanen et al., 2008). In addition, VEGF-B

Fig. 2. VEGF-B does not induce blood vessel permeability and is minimally angiogenic. Adenoviral gene transfer of the other VEGF family members, such as VEGF-A, VEGF-C and VEGF-D, into rabbit hindlimb skeletal muscles induced strong angiogenesis, vascular permeability, or lymphangiogenesis (Rissanen et al., 2003). VEGF-B adenoviral gene transfer, however, did not induce angiogenesis or lymphangiogenesis in the same model system (Rissanen et al., 2003). Similarly, adenoviral gene transfer of VEGF-A and VEGF-D to rabbit carotid arteries induced robust adventitial angiogenesis, whereas VEGF-B adenoviral gene transfer failed to do so (Bhardwaj et al., 2003, 2005). Another study also showed that VEGF-B167 gene delivery to the mouse skin or ischemic limb did not induce blood vessel growth (Li et al., 2008a)

Can VEGF-B Be Used to Treat Neurodegenerative Diseases? 393

and neurofibrillary tangles of hyperphosphorylated tau protein accumulate intracellularly in the brain, leading to the degeneration of synapses and neurons, and eventually the loss of memory and cognitive ability (Mattson, 2004). Both genetic and environmental factors contribute to the development of AD. Several drugs are currently available for AD treatment, such as tacrine, donepezil, rivastigmine tartrate and galantamine hydrobromide. These drugs can sometimes relieve the symptoms of early stage AD patients. However, they cannot stop or reverse the progression of the illness, and the effects of these drugs are often inconsistent and diminished over time. Therefore, more effective treatments are still needed. Many new reagents have been tested in preclinical or clinical studies, such as intravenous immunoglobulin (Relkin et al., 2008), γ-secretase inhibitors (Siemers et al., 2006; Wilcock et al., 2008), blockers of the receptor for advanced glycation end product (Chen et al., 2007), Dimebon (Doody et al., 2008), etc. However, their therapeutic efficacies are yet to be proven. It is noteworthy that in recent years, AD has been considered more as a vascular, rather than a neural disease based on clinical imaging, epidemiological, pharmacotherapy and histopathological evidence (Chow et al., 2007; de la Torre, 2002; de la Torre, 2004; Kalaria and Hedera, 1995). Indeed, vascular degeneration has been observed in different experimental Alzheimer's disease models (Girouard and Iadecola, 2006; Wu et al.,

(Hsu et al., 2007). In addition, it has been known that the functional relationships among neuronal, glial, and vascular cells within the so-called neurovascular unit is compromised in Alzheimer's disease (Salmina, 2009). Thus, mounting evidence indicates that vascular abnormalities, such as capillary degeneration, are important factors that can initiate Alzheimer's disease. Due to the potent survival effect of VEGF-B on both neuronal and vascular cells, VEGF-B may have a therapeutic potential in the prevention and

Parkinson's Disease (PD) is the second most prevalent neurodegenerative disease following AD. PD is characterized by the age-related progressive loss of dopaminergic neurotransmission in the basal ganglia (Nutt and Wooten, 2005). The etiology of PD is complicated and involves multiple factors and mechanisms. PD patients suffer from severe motor symptoms, including uncontrollable resting tremor, bradykinesia, rigidity and postural imbalance. Current treatment for PD can only attenuate the symptoms. There is no effective drug that can stop the neuronal death in PD patients. Levodopa, in combination with a peripheral dopa decarboxylase inhibitor, is the most effective therapy thus far (Lees et al., 2009). However, levodopa motor and nonmotor complications are challenging issues to overcome clinically (Jankovic, 2005). Dopamine agonists and monoamine oxidase-B inhibitors can reduce the symptoms either as a monotherapy or in combination with levodopa (Jankovic, 2006). However, even though the symptoms may be controlled after the administration of these drugs, at least following the initial treatment, the death of the

Neuroprotection is at the forefront of PD research, and many neuroprotective reagents have been investigated (Bonuccelli and Del Dotto, 2006; Djaldetti and Melamed, 2002). The glial cell derived neurotrophic factor (GDNF) has been shown to enhance the survival of midbrain dopaminergic neurons *in vitro* and rescued degenerating neurons *in vivo* (Love et al., 2005). However, a multicenter clinical trial showed no clinical benefit (Lang et al., 2006), and GDNF antibody development was observed in some PD patients (Sherer et al., 2006).

treatment of Alzheimer's disease. Further studies are needed to verify this.

dopaminergic neurons persists and the disease continues to progress.

2005),

**7.2 Parkinson's Disease** 

transgenic expression in endothelial cells under Tie2 promoter did not induce angiogenesis in different types of organs (liver, heart, kidney, etc) (Mould et al., 2005), and VEGF-B transgenic expression in the skin under keratin-14 promoter only marginal potentiated angiogenesis (Karpanen et al., 2008).

Studies using VEGF-B protein treatment also showed a minimum side effect of VEGF-B and a negligible role in angiogenesis. VEGF-B167 recombinant protein injection into adult mouse eyes at a dose effective for retinal neuron survival did not induce ocular angiogenesis (Li et al., 2008b). Poesen, K.*, et al* has also shown that intracerebroventricular injection of the VEGF-B186 recombinant protein did not cause any blood vessel growth or blood-brain barrier leakage (Poesen et al., 2008).

VEGF-B is most abundantly expressed in the heart (Li et al., 2001; Olofsson et al., 1996a). Using a cardiac ischemia model, we found that VEGF-B has a restricted role in the revascularization of ischemic myocardium (Claesson-Welsh, 2008; Li et al., 2008a). Indeed, this observation was also reported by another study demonstrating that in pigs and rabbits, VEGF-B186 gene transfer induced myocardium-specific angiogenesis and arteriogenesis (Lahteenvuo et al., 2009). Thus, ours and others' work have shown that in most organs, VEGF-B is dispensable for blood vessel growth in development, normal physiology, and many pathological conditions but with a selective angiogenic activity in the ischemic heart. Taken together, compared with the other VEGF family members, VEGF-B appears to have a unique safety profile that is highly desirable as a potential therapeutic reagent to treat human diseases.

#### **7. Therapeutic potential of VEGF-B in treating neurodegenerative diseases**

Currently, for most neurodegenerative diseases, there are no effective treatments. Although novel remedies such as gene or cell therapies are being explored intensively, few have proved to be clinically beneficial. Neurodegenerative diseases often involve complex multietiological aspects. Neuronal apoptosis is a central characteristic of neurodegenerative diseases. In addition, blood vessel degeneration in the relevant neural system is often seen in many of the neurodegenerative disorders. Therefore, therapeutic reagents targeting one pathway only will most likely not be sufficient to cure the disease. Reagents that can improve multiple pathological aspects are more desirable. Based on our recent findings that VEGF-B is a potent protective/survival factor for both the neuronal and vascular systems, which are two critical components in most neurodegenerative disorders, we hypothesize that VEGF-B may have therapeutic implications in treating various types of neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) stroke, retinitis pigmentosa (RP), glaucoma, diabetic retinopathy (DR) and atrophic age-related macular degeneration (AMD). Below, we discuss the therapeutic potential of VEGF-B in relation to these pathologies.

#### **7.1 Alzheimer's Disease**

Alzheimer's Disease (AD) is a major contributor to dementia in the elderly, and affects about 2% of the population in developed countries. The total number of AD patients is estimated to increase significantly in the near future due to the growing aging population (Mattson, 2004). In AD patients, plaques containing the beta-amyloid protein deposit extracellularly,

transgenic expression in endothelial cells under Tie2 promoter did not induce angiogenesis in different types of organs (liver, heart, kidney, etc) (Mould et al., 2005), and VEGF-B transgenic expression in the skin under keratin-14 promoter only marginal potentiated

Studies using VEGF-B protein treatment also showed a minimum side effect of VEGF-B and a negligible role in angiogenesis. VEGF-B167 recombinant protein injection into adult mouse eyes at a dose effective for retinal neuron survival did not induce ocular angiogenesis (Li et al., 2008b). Poesen, K.*, et al* has also shown that intracerebroventricular injection of the VEGF-B186 recombinant protein did not cause any blood vessel growth or blood-brain

VEGF-B is most abundantly expressed in the heart (Li et al., 2001; Olofsson et al., 1996a). Using a cardiac ischemia model, we found that VEGF-B has a restricted role in the revascularization of ischemic myocardium (Claesson-Welsh, 2008; Li et al., 2008a). Indeed, this observation was also reported by another study demonstrating that in pigs and rabbits, VEGF-B186 gene transfer induced myocardium-specific angiogenesis and arteriogenesis (Lahteenvuo et al., 2009). Thus, ours and others' work have shown that in most organs, VEGF-B is dispensable for blood vessel growth in development, normal physiology, and many pathological conditions but with a selective angiogenic activity in the ischemic heart. Taken together, compared with the other VEGF family members, VEGF-B appears to have a unique safety profile that is highly desirable as a potential therapeutic reagent to treat

**7. Therapeutic potential of VEGF-B in treating neurodegenerative diseases**  Currently, for most neurodegenerative diseases, there are no effective treatments. Although novel remedies such as gene or cell therapies are being explored intensively, few have proved to be clinically beneficial. Neurodegenerative diseases often involve complex multietiological aspects. Neuronal apoptosis is a central characteristic of neurodegenerative diseases. In addition, blood vessel degeneration in the relevant neural system is often seen in many of the neurodegenerative disorders. Therefore, therapeutic reagents targeting one pathway only will most likely not be sufficient to cure the disease. Reagents that can improve multiple pathological aspects are more desirable. Based on our recent findings that VEGF-B is a potent protective/survival factor for both the neuronal and vascular systems, which are two critical components in most neurodegenerative disorders, we hypothesize that VEGF-B may have therapeutic implications in treating various types of neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) stroke, retinitis pigmentosa (RP), glaucoma, diabetic retinopathy (DR) and atrophic age-related macular degeneration (AMD). Below, we discuss the therapeutic potential of VEGF-B in relation to these

Alzheimer's Disease (AD) is a major contributor to dementia in the elderly, and affects about 2% of the population in developed countries. The total number of AD patients is estimated to increase significantly in the near future due to the growing aging population (Mattson, 2004). In AD patients, plaques containing the beta-amyloid protein deposit extracellularly,

angiogenesis (Karpanen et al., 2008).

barrier leakage (Poesen et al., 2008).

human diseases.

pathologies.

**7.1 Alzheimer's Disease** 

and neurofibrillary tangles of hyperphosphorylated tau protein accumulate intracellularly in the brain, leading to the degeneration of synapses and neurons, and eventually the loss of memory and cognitive ability (Mattson, 2004). Both genetic and environmental factors contribute to the development of AD. Several drugs are currently available for AD treatment, such as tacrine, donepezil, rivastigmine tartrate and galantamine hydrobromide. These drugs can sometimes relieve the symptoms of early stage AD patients. However, they cannot stop or reverse the progression of the illness, and the effects of these drugs are often inconsistent and diminished over time. Therefore, more effective treatments are still needed. Many new reagents have been tested in preclinical or clinical studies, such as intravenous immunoglobulin (Relkin et al., 2008), γ-secretase inhibitors (Siemers et al., 2006; Wilcock et al., 2008), blockers of the receptor for advanced glycation end product (Chen et al., 2007), Dimebon (Doody et al., 2008), etc. However, their therapeutic efficacies are yet to be proven. It is noteworthy that in recent years, AD has been considered more as a vascular, rather than a neural disease based on clinical imaging, epidemiological, pharmacotherapy and histopathological evidence (Chow et al., 2007; de la Torre, 2002; de la Torre, 2004; Kalaria and Hedera, 1995). Indeed, vascular degeneration has been observed in different experimental Alzheimer's disease models (Girouard and Iadecola, 2006; Wu et al., 2005), (Hsu et al., 2007). In addition, it has been known that the functional relationships among neuronal, glial, and vascular cells within the so-called neurovascular unit is compromised in Alzheimer's disease (Salmina, 2009). Thus, mounting evidence indicates that vascular abnormalities, such as capillary degeneration, are important factors that can initiate Alzheimer's disease. Due to the potent survival effect of VEGF-B on both neuronal and vascular cells, VEGF-B may have a therapeutic potential in the prevention and treatment of Alzheimer's disease. Further studies are needed to verify this.

#### **7.2 Parkinson's Disease**

Parkinson's Disease (PD) is the second most prevalent neurodegenerative disease following AD. PD is characterized by the age-related progressive loss of dopaminergic neurotransmission in the basal ganglia (Nutt and Wooten, 2005). The etiology of PD is complicated and involves multiple factors and mechanisms. PD patients suffer from severe motor symptoms, including uncontrollable resting tremor, bradykinesia, rigidity and postural imbalance. Current treatment for PD can only attenuate the symptoms. There is no effective drug that can stop the neuronal death in PD patients. Levodopa, in combination with a peripheral dopa decarboxylase inhibitor, is the most effective therapy thus far (Lees et al., 2009). However, levodopa motor and nonmotor complications are challenging issues to overcome clinically (Jankovic, 2005). Dopamine agonists and monoamine oxidase-B inhibitors can reduce the symptoms either as a monotherapy or in combination with levodopa (Jankovic, 2006). However, even though the symptoms may be controlled after the administration of these drugs, at least following the initial treatment, the death of the dopaminergic neurons persists and the disease continues to progress.

Neuroprotection is at the forefront of PD research, and many neuroprotective reagents have been investigated (Bonuccelli and Del Dotto, 2006; Djaldetti and Melamed, 2002). The glial cell derived neurotrophic factor (GDNF) has been shown to enhance the survival of midbrain dopaminergic neurons *in vitro* and rescued degenerating neurons *in vivo* (Love et al., 2005). However, a multicenter clinical trial showed no clinical benefit (Lang et al., 2006), and GDNF antibody development was observed in some PD patients (Sherer et al., 2006).

Can VEGF-B Be Used to Treat Neurodegenerative Diseases? 395

stroke patients despite extensive effort on identifying better interventions. Since early 1990s, neuroprotection as a potential therapeutic strategy for stroke treatment has received much attention (Ginsberg, 2008). During the past decade or so, about 160 clinical trials on neuroprotection for ischemic stroke treatment have been conducted (Ginsberg, 2008). However, no effective neuroprotective drug has been identified. The potential therapeutic value of VEGF-B for stroke treatment has been supported by several studies. It has been shown that VEGF-B is a potent survival factor for cortical neurons. VEGF-B deficiency in mouse increased stroke volume by about 40% in an experimental stroke model, and led to more severe neurologic impairment (Sun et al., 2004). Indeed, VEGF-B protein treatment protected cultured cerebral cortical neurons from hypoxic injury, demonstrating a direct survival effect of VEGF-B on neurons (Sun et al., 2004). Furthermore, intraventricular administration of VEGF-B decreased stroke volume (Li et al., 2008b) and restored neurogenesis to normal level in VEGF-B deficient mice (Sun et al., 2006). Mechanistically, we have shown that VEGF-B exerts its neuronal survival effect by inhibiting the expression of many proapoptotic BH3-only protein genes (Li et al., 2008b). In summary, both *in vitro* and *in vivo* data from several groups have suggested a therapeutic potential of VEGF-B in stroke

Huntington's Disease (HD) is a hereditary autosomal dominant neurodegenerative disorder characterized by the selective degeneration of striatal projection neurons that are responsible for choreic movements, resulting in progressive movement disorder, cognitive decline and psychiatric disturbances. Over the course of HD, the mutated huntingtin protein leads to intracellular dysfunctions and neuronal death in the striatum, selected layers of the cerebral cortex, as well as other brain regions (Gil and Rego, 2008). Currently, no effective therapy exists for HD. Pharmacological treatment may ameliorate hyperkinesis and psychiatric symptoms, but neuropsychological deficits and dementia remain untreatable. The apoptotic cascade is believed to be a possible cause of neurodegeneration in HD (Pattison et al., 2006). The therapeutic potential of some neuroprotective reagents in HD treatment, such as GDNF, coenzyme Q10, minocycline and unsaturated fatty acids, has been investigated (Alberch et al., 2002; Bonelli and Hofmann, 2007). Since VEGF-B is a potent apoptosis inhibitor (Li et al., 2008b), it will be interesting to test whether VEGF-B could slow

Retinal degenerative diseases are a group of disorders involving degeneration of the retina. Progressive loss of retinal neurons is a common characteristic of such disorders and the major reason for vision impair or loss. Further, blood vessel deterioration is often seen in many of the retinal degenerative diseases. Unfortunately, thus far, there is no efficacious

Retinitis pigmentosa (RP) is a heterogeneous retinal dystrophy characterized by the progressive loss of photoreceptors and subsequent degeneration of retinal pigmented epithelial (RPE) cells (Hartong et al., 2006). RP is the leading cause of blindness in inherited

treatment and warrant further studies to investigate into this.

down, if not stop, neuronal degeneration in HD.

treatment for most of the retinal degenerative diseases.

**7.6 Retinal degenerative diseases** 

**7.6.1 Retinitis pigmentosa (RP)** 

**7.5 Huntington's Disease** 

Indeed, in a rat α-synuclein PD model, overexpression of GDNF failed to exert effective neuroprotection (Decressac et al., 2011). The vascular endothelial growth factor–A (VEGF-A) has been shown to induce neuroprotection in a PD model of the 6-hydroxydopamine (6- OHDA) lesioned rats (Yasuhara et al., 2004). However unwarranted side effect of VEGF-A proved to be detrimental to the brain, since VEGF-A also induced edema and undesired angiogenesis in the brain (Yasuhara et al., 2005). In addition, it has also been reported that VEGF-A induces astrogliosis, microgliosis and disrupts the blood-brain barrier (Rite et al., 2007). Thus, new and better neuroprotective reagents are still needed.

Apart from neuronal death, normal contact between nigral neurons and capillaries is often impaired in the brains of PD patients. Capillary basement membrane thickening and collagen accumulation are often seen in PD patients, suggesting that capillary dysfunction may play an important role in PD development (Farkas et al., 2000; Faucheux et al., 1999). Indeed, it is believed that markers of cerebrovascular disease may predict the development of different types of dementia, including PD (Staekenborg et al., 2009). Recent work has shown that VEGF-B expression was upregulated by neurodegenerative challenges in the midbrain, and exogenous application of VEGF-B has a neuroprotective effect in a culture model of PD (Falk et al., 2009). In another study, VEGF-B186 was used to test its neuroprotective effect in a PD model since it is more diffusable and hardly binds to extracellular matrix than VEGF-B167 (Olofsson et al., 1996b; Poesen et al., 2008). In this study, a single dose of VEGF-B186 (3µg/rat) rescued dopaminergic neurons from death in the caudal sub region of substantia nigra in rats (Falk et al., 2011). Thus, as a potent neuronal and vascular protective factor, VEGF-B may have therapeutic implications in PD treatment. Future investigations are needed to investigate into this.

#### **7.3 Amyotrophic lateral sclerosis**

Amyotrophic lateral sclerosis (ALS) is a devastating adult-onset neurodegenerative disorder characterized by progressive loss of motoneurons in the primary motor cortex, corticospinal tracts, brainstem and spinal cord, leading to muscular paralysis and eventually death (Wijesekera and Leigh, 2009). The pathogenesis of familial ALS is unclear. Sporadic ALS is believed to be related to superoxide dismutase (SOD) 1 mutation in about 20-30% of the patients (Yamamoto et al., 2008). Although many drug candidates have been tested, such as antioxidants, neurotrophic factors, anti-apoptotic, anti-inflammatory and anti-aggregation reagents, the only drug currently available for ALS patients is Riluzole, a glutamate antagonist (Traynor et al., 2006; Yamamoto et al., 2008). Recently, it is believed that vascular defect may be a critical contributor to the pathogenesis of ALS. In the amyotrophic lateral sclerosis-linked SOD1 mutant mice, vascular endothelial damage accumulates before motor neuron degeneration and plays a central role in ALS initiation (Segura et al., 2009). The therapeutic promise of VEGF-B in ALS treatment has been shown by Poesen et al. VEGF-B186 protected cultured primary motor neurons against degeneration (Poesen et al., 2008). *In vivo*, VEGF-B treatment protected motor neurons from degeneration in several experimental ALS models (Poesen et al., 2008). In the future, it will be exciting to see whether this effect of VEGF-B holds true in ALS patients.

#### **7.4 Stroke**

Ischemic stroke due to sudden loss of blood supply in the brain is a leading cause of morbidity and mortality in the United States. Currently, there is no satisfying therapy for stroke patients despite extensive effort on identifying better interventions. Since early 1990s, neuroprotection as a potential therapeutic strategy for stroke treatment has received much attention (Ginsberg, 2008). During the past decade or so, about 160 clinical trials on neuroprotection for ischemic stroke treatment have been conducted (Ginsberg, 2008). However, no effective neuroprotective drug has been identified. The potential therapeutic value of VEGF-B for stroke treatment has been supported by several studies. It has been shown that VEGF-B is a potent survival factor for cortical neurons. VEGF-B deficiency in mouse increased stroke volume by about 40% in an experimental stroke model, and led to more severe neurologic impairment (Sun et al., 2004). Indeed, VEGF-B protein treatment protected cultured cerebral cortical neurons from hypoxic injury, demonstrating a direct survival effect of VEGF-B on neurons (Sun et al., 2004). Furthermore, intraventricular administration of VEGF-B decreased stroke volume (Li et al., 2008b) and restored neurogenesis to normal level in VEGF-B deficient mice (Sun et al., 2006). Mechanistically, we have shown that VEGF-B exerts its neuronal survival effect by inhibiting the expression of many proapoptotic BH3-only protein genes (Li et al., 2008b). In summary, both *in vitro* and *in vivo* data from several groups have suggested a therapeutic potential of VEGF-B in stroke treatment and warrant further studies to investigate into this.

#### **7.5 Huntington's Disease**

394 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

Indeed, in a rat α-synuclein PD model, overexpression of GDNF failed to exert effective neuroprotection (Decressac et al., 2011). The vascular endothelial growth factor–A (VEGF-A) has been shown to induce neuroprotection in a PD model of the 6-hydroxydopamine (6- OHDA) lesioned rats (Yasuhara et al., 2004). However unwarranted side effect of VEGF-A proved to be detrimental to the brain, since VEGF-A also induced edema and undesired angiogenesis in the brain (Yasuhara et al., 2005). In addition, it has also been reported that VEGF-A induces astrogliosis, microgliosis and disrupts the blood-brain barrier (Rite et al.,

Apart from neuronal death, normal contact between nigral neurons and capillaries is often impaired in the brains of PD patients. Capillary basement membrane thickening and collagen accumulation are often seen in PD patients, suggesting that capillary dysfunction may play an important role in PD development (Farkas et al., 2000; Faucheux et al., 1999). Indeed, it is believed that markers of cerebrovascular disease may predict the development of different types of dementia, including PD (Staekenborg et al., 2009). Recent work has shown that VEGF-B expression was upregulated by neurodegenerative challenges in the midbrain, and exogenous application of VEGF-B has a neuroprotective effect in a culture model of PD (Falk et al., 2009). In another study, VEGF-B186 was used to test its neuroprotective effect in a PD model since it is more diffusable and hardly binds to extracellular matrix than VEGF-B167 (Olofsson et al., 1996b; Poesen et al., 2008). In this study, a single dose of VEGF-B186 (3µg/rat) rescued dopaminergic neurons from death in the caudal sub region of substantia nigra in rats (Falk et al., 2011). Thus, as a potent neuronal and vascular protective factor, VEGF-B may have therapeutic implications in PD treatment.

Amyotrophic lateral sclerosis (ALS) is a devastating adult-onset neurodegenerative disorder characterized by progressive loss of motoneurons in the primary motor cortex, corticospinal tracts, brainstem and spinal cord, leading to muscular paralysis and eventually death (Wijesekera and Leigh, 2009). The pathogenesis of familial ALS is unclear. Sporadic ALS is believed to be related to superoxide dismutase (SOD) 1 mutation in about 20-30% of the patients (Yamamoto et al., 2008). Although many drug candidates have been tested, such as antioxidants, neurotrophic factors, anti-apoptotic, anti-inflammatory and anti-aggregation reagents, the only drug currently available for ALS patients is Riluzole, a glutamate antagonist (Traynor et al., 2006; Yamamoto et al., 2008). Recently, it is believed that vascular defect may be a critical contributor to the pathogenesis of ALS. In the amyotrophic lateral sclerosis-linked SOD1 mutant mice, vascular endothelial damage accumulates before motor neuron degeneration and plays a central role in ALS initiation (Segura et al., 2009). The therapeutic promise of VEGF-B in ALS treatment has been shown by Poesen et al. VEGF-B186 protected cultured primary motor neurons against degeneration (Poesen et al., 2008). *In vivo*, VEGF-B treatment protected motor neurons from degeneration in several experimental ALS models (Poesen et al., 2008). In the future, it will be exciting to see whether this effect of

Ischemic stroke due to sudden loss of blood supply in the brain is a leading cause of morbidity and mortality in the United States. Currently, there is no satisfying therapy for

2007). Thus, new and better neuroprotective reagents are still needed.

Future investigations are needed to investigate into this.

**7.3 Amyotrophic lateral sclerosis** 

VEGF-B holds true in ALS patients.

**7.4 Stroke** 

Huntington's Disease (HD) is a hereditary autosomal dominant neurodegenerative disorder characterized by the selective degeneration of striatal projection neurons that are responsible for choreic movements, resulting in progressive movement disorder, cognitive decline and psychiatric disturbances. Over the course of HD, the mutated huntingtin protein leads to intracellular dysfunctions and neuronal death in the striatum, selected layers of the cerebral cortex, as well as other brain regions (Gil and Rego, 2008). Currently, no effective therapy exists for HD. Pharmacological treatment may ameliorate hyperkinesis and psychiatric symptoms, but neuropsychological deficits and dementia remain untreatable. The apoptotic cascade is believed to be a possible cause of neurodegeneration in HD (Pattison et al., 2006). The therapeutic potential of some neuroprotective reagents in HD treatment, such as GDNF, coenzyme Q10, minocycline and unsaturated fatty acids, has been investigated (Alberch et al., 2002; Bonelli and Hofmann, 2007). Since VEGF-B is a potent apoptosis inhibitor (Li et al., 2008b), it will be interesting to test whether VEGF-B could slow down, if not stop, neuronal degeneration in HD.

#### **7.6 Retinal degenerative diseases**

Retinal degenerative diseases are a group of disorders involving degeneration of the retina. Progressive loss of retinal neurons is a common characteristic of such disorders and the major reason for vision impair or loss. Further, blood vessel deterioration is often seen in many of the retinal degenerative diseases. Unfortunately, thus far, there is no efficacious treatment for most of the retinal degenerative diseases.

#### **7.6.1 Retinitis pigmentosa (RP)**

Retinitis pigmentosa (RP) is a heterogeneous retinal dystrophy characterized by the progressive loss of photoreceptors and subsequent degeneration of retinal pigmented epithelial (RPE) cells (Hartong et al., 2006). RP is the leading cause of blindness in inherited

Can VEGF-B Be Used to Treat Neurodegenerative Diseases? 397

a microvascular disease. However, it is now considered to be also a neurodegenerative disease involving functional and structural defects of different types of neurons in the retina (Imai et al., 2009). Indeed, neuronal apoptosis has been found to be an early event in a rat model of diabetes (Barber et al., 1998). Four months after the onset of diabetes, there were only about 50% of total neurons left in the retinae of the rats (Barber et al., 1998), and the number of retinal ganglion cells (RGC) and the thickness of the inner retina layer were significantly reduced (Barber et al., 1998). In diabetic patients, increased apoptosis was also observed in the retina (Imai et al., 2009). Moreover, significant nerve fibre loss in the superior segment of the retina was observed in type 1 diabetic patients, suggesting RGC loss (Kern and Barber, 2008; Lopes de Faria et al., 2002). In addition, thinning of the inner retinal layer was observed in early stage of type 1 diabetic patients (van Dijk et al., 2009). It is reported that the mitochondria- and caspase-dependent cell-death pathways are involved in the neuronal degeneration in diabetic retinopathy (Oshitari et al., 2008). The potential role of VEGF-B in diabetic retinopathy has not been investigated thus far. However, given that VEGF-B is a potent apoptosis inhibitor and has a strong protective effect on both retinal ganglion cells and different types of vascular cells, it is reasonable to speculate that VEGF-B could be used to rescue the chronic retinal degeneration in DR. However, further

Age-related macular degeneration (AMD) is the most common cause of blindness in developed countries. Atrophic (dry) AMD is a late-onset, multifactorial, slowly progressing retinal neurodegenerative disease caused by the degeneration of retinal pigment epithelium (RPE) that lies beneath the photoreceptor cells in the retina. Although RPE is a central element in the pathogenesis of age-related macular degeneration, RPE dysfunction results in the secondary death of macular rods and cones due to abnormal metabolic support from the RPE, eventually leading to irreversible vision loss (de Jong, 2006). Drusen formation, oxidative stress, accumulation of lipofuscin, local inflammation and reactive gliosis are believed to be involved in the pathogenesis of atrophic AMD (Petrukhin, 2007). Currently, there is no effective treatment for atrophic AMD. There are reports showing that antioxidants supplement can provide protection against age-related macular degeneration. A high dietary intake of beta carotene, vitamin C, vitamin E, and zinc may reduce the risk of AMD in elderly people substantially (Johnson, 2009; van Leeuwen et al., 2005). Compared with the other types of retinal degenerative diseases, neuroprotection as a potential therapeutic strategy has been less studied in atrophic AMD. Our recent findings showed that VEGF-B is a potent apoptosis inhibitor. Moreover, the anti-apoptotic property of VEGF-B is likely a general effect on many different types of cells, including RPE cells (Li et al., 2009; Li et al., 2008b; Zhang et al., 2009). VEGF-B therefore might potentially be used to

In summary, despite the complex etiology of different types of neurodegenerative diseases, one common characteristic of them is the apoptotic neuronal death. In addition, degeneration of the blood vessels is often seen in many of the neurodegenerative diseases. Thus, combination therapy acting on both aspects is highly desirable. We and others have

investigation and research into this aspect are still needed.

enhance RPE survival for AMD treatment.

**8. Conclusion** 

**7.6.4 Atrophic AMD** 

retinal degeneration-associated diseases world-wide. The first symptom of RP is often night blindness, followed by the gradual loss of peripheral visual field, and ultimately blindness. Apart from the photoreceptor dystrophy, retinal arterioles are attenuated in RP, leading to poor oxygenation of rods and cones and increased apoptosis in the neural retina. It is known that about 45 genes/loci are involved in this pathology. Due to the large number of genes and mutations implicated, correcting the defective genes/mutations represents an overwhelming challenge. The current available therapies are vitamin supplement and sunlight protection, which can only slow down the degenerative process (Hamel, 2006). There is no treatment that can stop the progress of the disease or restore vision in RP patients. Since VEGF-B can protect both neuronal and vascular cells from apoptosis, VEGF-B administration may preserve both the photoreceptors and blood vessels in RP. Future studies are needed to verify this.

#### **7.6.2 Glaucoma**

Glaucoma is the most prevalent form of adult optic neuropathies affecting approximately 2% of the population over the age of 40 (Levin, 2005; Marcic et al., 2003). Glaucoma is characterized by the increased apoptosis of retinal ganglion cells, loss of optic nerve bers, and, if uncontrolled, impair or loss of vision (Weinreb, 2005). Apoptosis of retinal ganglion cells is believed to be an early event in glaucoma (Cheung et al., 2008). The number of glaucoma patients is significantly increasing because of the growing ageing population and other factors (Morley and Murdoch, 2006). Currently, there is no general treatment effective for all glaucoma patients. Recent years have seen increasing evidence showing that glaucoma is, to a large extent, a neurodegenerative disease similar to other neurodegenerative disorders in the central nervous system, such as Alzheimer's disease (Cheung et al., 2008). Traditionally, lowering the intraocular pressure (IOP) has been a major therapeutic goal in glaucoma treatment. However, such therapeutic approaches have not been effective in preventing many patients from progressive vision loss. Thus, the fact that retinal ganglion cells (RGC) continue to die in some glaucoma patients with normal or even lower IOP has changed the research focus to neuroprotection for glaucoma treatment in recent years. Therefore, neuroprotective reagents used to treat other neurodegenerative diseases have been under considerable investigation for glaucoma treatment, and neuroprotection in glaucoma treatment has gained more and more attention. However, the number of effective neuroprotective reagents is limited. We have recently revealed that VEGF-B is expressed in normal retinal ganglion cells (Li et al., 2008b). Importantly, the expression of VEGF-B is up-regulated after optic nerve crush injury in the retina (Li et al., 2008b), suggesting a role of VEGF-B in retinal ganglion cell function. Indeed, VEGF-B inhibits the expression of many apoptotic genes in the retina and protected retinal ganglion cells from axotomy-induced apoptosis (Li et al., 2008b). These data have provided evidence that VEGF-B may be a promising drug candidate for glaucoma treatment as a neuroprotective factor. Further studies are warranted to investigate this.

#### **7.6.3 Diabetic retinopathy**

Diabetic retinopathy (DR) is a common complication of diabetes. About 50-75% of diabetic patients develop DR. In the United States, DR is the leading cause of legal blindness in the 20 to 74 year-old population (Imai et al., 2009). Conventionally, DR is believed mainly to be a microvascular disease. However, it is now considered to be also a neurodegenerative disease involving functional and structural defects of different types of neurons in the retina (Imai et al., 2009). Indeed, neuronal apoptosis has been found to be an early event in a rat model of diabetes (Barber et al., 1998). Four months after the onset of diabetes, there were only about 50% of total neurons left in the retinae of the rats (Barber et al., 1998), and the number of retinal ganglion cells (RGC) and the thickness of the inner retina layer were significantly reduced (Barber et al., 1998). In diabetic patients, increased apoptosis was also observed in the retina (Imai et al., 2009). Moreover, significant nerve fibre loss in the superior segment of the retina was observed in type 1 diabetic patients, suggesting RGC loss (Kern and Barber, 2008; Lopes de Faria et al., 2002). In addition, thinning of the inner retinal layer was observed in early stage of type 1 diabetic patients (van Dijk et al., 2009). It is reported that the mitochondria- and caspase-dependent cell-death pathways are involved in the neuronal degeneration in diabetic retinopathy (Oshitari et al., 2008). The potential role of VEGF-B in diabetic retinopathy has not been investigated thus far. However, given that VEGF-B is a potent apoptosis inhibitor and has a strong protective effect on both retinal ganglion cells and different types of vascular cells, it is reasonable to speculate that VEGF-B could be used to rescue the chronic retinal degeneration in DR. However, further investigation and research into this aspect are still needed.

#### **7.6.4 Atrophic AMD**

396 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

retinal degeneration-associated diseases world-wide. The first symptom of RP is often night blindness, followed by the gradual loss of peripheral visual field, and ultimately blindness. Apart from the photoreceptor dystrophy, retinal arterioles are attenuated in RP, leading to poor oxygenation of rods and cones and increased apoptosis in the neural retina. It is known that about 45 genes/loci are involved in this pathology. Due to the large number of genes and mutations implicated, correcting the defective genes/mutations represents an overwhelming challenge. The current available therapies are vitamin supplement and sunlight protection, which can only slow down the degenerative process (Hamel, 2006). There is no treatment that can stop the progress of the disease or restore vision in RP patients. Since VEGF-B can protect both neuronal and vascular cells from apoptosis, VEGF-B administration may preserve both the photoreceptors and blood vessels in RP. Future

Glaucoma is the most prevalent form of adult optic neuropathies affecting approximately 2% of the population over the age of 40 (Levin, 2005; Marcic et al., 2003). Glaucoma is characterized by the increased apoptosis of retinal ganglion cells, loss of optic nerve bers, and, if uncontrolled, impair or loss of vision (Weinreb, 2005). Apoptosis of retinal ganglion cells is believed to be an early event in glaucoma (Cheung et al., 2008). The number of glaucoma patients is significantly increasing because of the growing ageing population and other factors (Morley and Murdoch, 2006). Currently, there is no general treatment effective for all glaucoma patients. Recent years have seen increasing evidence showing that glaucoma is, to a large extent, a neurodegenerative disease similar to other neurodegenerative disorders in the central nervous system, such as Alzheimer's disease (Cheung et al., 2008). Traditionally, lowering the intraocular pressure (IOP) has been a major therapeutic goal in glaucoma treatment. However, such therapeutic approaches have not been effective in preventing many patients from progressive vision loss. Thus, the fact that retinal ganglion cells (RGC) continue to die in some glaucoma patients with normal or even lower IOP has changed the research focus to neuroprotection for glaucoma treatment in recent years. Therefore, neuroprotective reagents used to treat other neurodegenerative diseases have been under considerable investigation for glaucoma treatment, and neuroprotection in glaucoma treatment has gained more and more attention. However, the number of effective neuroprotective reagents is limited. We have recently revealed that VEGF-B is expressed in normal retinal ganglion cells (Li et al., 2008b). Importantly, the expression of VEGF-B is up-regulated after optic nerve crush injury in the retina (Li et al., 2008b), suggesting a role of VEGF-B in retinal ganglion cell function. Indeed, VEGF-B inhibits the expression of many apoptotic genes in the retina and protected retinal ganglion cells from axotomy-induced apoptosis (Li et al., 2008b). These data have provided evidence that VEGF-B may be a promising drug candidate for glaucoma treatment as a

neuroprotective factor. Further studies are warranted to investigate this.

Diabetic retinopathy (DR) is a common complication of diabetes. About 50-75% of diabetic patients develop DR. In the United States, DR is the leading cause of legal blindness in the 20 to 74 year-old population (Imai et al., 2009). Conventionally, DR is believed mainly to be

studies are needed to verify this.

**7.6.3 Diabetic retinopathy** 

**7.6.2 Glaucoma** 

Age-related macular degeneration (AMD) is the most common cause of blindness in developed countries. Atrophic (dry) AMD is a late-onset, multifactorial, slowly progressing retinal neurodegenerative disease caused by the degeneration of retinal pigment epithelium (RPE) that lies beneath the photoreceptor cells in the retina. Although RPE is a central element in the pathogenesis of age-related macular degeneration, RPE dysfunction results in the secondary death of macular rods and cones due to abnormal metabolic support from the RPE, eventually leading to irreversible vision loss (de Jong, 2006). Drusen formation, oxidative stress, accumulation of lipofuscin, local inflammation and reactive gliosis are believed to be involved in the pathogenesis of atrophic AMD (Petrukhin, 2007). Currently, there is no effective treatment for atrophic AMD. There are reports showing that antioxidants supplement can provide protection against age-related macular degeneration. A high dietary intake of beta carotene, vitamin C, vitamin E, and zinc may reduce the risk of AMD in elderly people substantially (Johnson, 2009; van Leeuwen et al., 2005). Compared with the other types of retinal degenerative diseases, neuroprotection as a potential therapeutic strategy has been less studied in atrophic AMD. Our recent findings showed that VEGF-B is a potent apoptosis inhibitor. Moreover, the anti-apoptotic property of VEGF-B is likely a general effect on many different types of cells, including RPE cells (Li et al., 2009; Li et al., 2008b; Zhang et al., 2009). VEGF-B therefore might potentially be used to enhance RPE survival for AMD treatment.

#### **8. Conclusion**

In summary, despite the complex etiology of different types of neurodegenerative diseases, one common characteristic of them is the apoptotic neuronal death. In addition, degeneration of the blood vessels is often seen in many of the neurodegenerative diseases. Thus, combination therapy acting on both aspects is highly desirable. We and others have

Can VEGF-B Be Used to Treat Neurodegenerative Diseases? 399

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#### **9. Acknowledgment**

This research was supported in part by the Macular Degeneration Research, a program of the American Health Assistance Foundation, and the Intramural Research Program of the NIH, National Eye Institute.

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**17** 

*France* 

**Power of a Metabonomic Approach to** 

*1Toulouse Nationale Veterinary School, Alimentation & Botanics, Toulouse* 

*2INRA - Mét@risk Unit, AgroParisTech, Paris 3UR AFPA, INRA UC340, Nancy University, Faculty of Sciences & Technologies, Nancy 4UMR 1331 ToxAlim INRA INP, Toulouse* 

**Investigate an Unknown Nervous Disease** 

Céline Domange1, Alain Paris2, Henri Schroeder3 and Nathalie Priymenko1,4

The field of neurological disorders becomes now one of the most important investigation areas in clinical medicine, whatever the toxicological, genetic, degenerative or environmental aetiology they have. Because it involves the main complex organ as target tissue, because also of the intrinsic specificity of the biological network of the nervous system, or the technical difficulty to access such a composite organ, the nervous diseases remain particularly difficult to study. Certainly, the rapid development of transgenic animal models of neurological diseases and the expanding growth of imaging techniques to functionally and non-invasively access some specific brain regions constitute a favourable situation to study the basis and the progression of some nervous diseases. However, the use of such transgenic animals or spontaneous animal models needs that the clinical symptoms are reproducible and that a *prior* knowledge of the aetiopathology of these diseases may exist. These latter conditions are not always available, especially concerning toxicology. In this case, how can both pathophysiology and therapies be investigated? Indeed, classically, when considering a toxicological approach, clinical signs, similar to those ascribed on the target species, need to be reproduced on the animal model. But how to do with disease displaying no known aetiology or with an animal model, on which it is impossible to reproduce, at least partially, some clinical signs of the target species? Furthermore, because of evident ethical reasons added to practical ones, some neurological disorders in humans or in large animals remain scarcely explored. "Omics" approaches seem to be a good alternative in the clinical medical research, enabling to take advantage of the global living system and, simultaneously of the control of the toxicological factor. To illustrate such an original approach, a neurological horse disease, Australian stringhalt, which has been described for several centuries, but for which aetiology is still only partially known, was reassessed using metabonomics in combination with other classical techniques. This has led to show how powerful this method may stand for in clinical medical research and,

**1. Introduction** 

particularly in neurological studies.

Zhang, F.; Tang, Z.; Hou, X.; Lennartsson, J.; Li, Y.; Koch, A.W.; Scotney, P.; Lee, C.; Arjunan, P.; Dong, L.; Kumar, A.; Rissanen, T.T.; Wang, B.; Nagai, N.; Fons, P.; Fariss, R.; Zhang, Y.; Wawrousek, E.; Tansey, G.; Raber, J.; Fong, G.H.; Ding, H.; Greenberg, D.A.; Becker, K.G.; Herbert, J.M.; Nash, A.; Yla-Herttuala, S.; Cao, Y.; Watts, R.J. & Li, X. (2009). VEGF-B is dispensable for blood vessel growth but critical for their survival, and VEGF-B targeting inhibits pathological angiogenesis. *Proc Natl Acad Sci U S A*. 106:6152-6157.

### **Power of a Metabonomic Approach to Investigate an Unknown Nervous Disease**

Céline Domange1, Alain Paris2, Henri Schroeder3 and Nathalie Priymenko1,4

*1Toulouse Nationale Veterinary School, Alimentation & Botanics, Toulouse 2INRA - Mét@risk Unit, AgroParisTech, Paris 3UR AFPA, INRA UC340, Nancy University, Faculty of Sciences & Technologies, Nancy 4UMR 1331 ToxAlim INRA INP, Toulouse France* 

#### **1. Introduction**

406 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

Zhang, F.; Tang, Z.; Hou, X.; Lennartsson, J.; Li, Y.; Koch, A.W.; Scotney, P.; Lee, C.;

*Proc Natl Acad Sci U S A*. 106:6152-6157.

Arjunan, P.; Dong, L.; Kumar, A.; Rissanen, T.T.; Wang, B.; Nagai, N.; Fons, P.; Fariss, R.; Zhang, Y.; Wawrousek, E.; Tansey, G.; Raber, J.; Fong, G.H.; Ding, H.; Greenberg, D.A.; Becker, K.G.; Herbert, J.M.; Nash, A.; Yla-Herttuala, S.; Cao, Y.; Watts, R.J. & Li, X. (2009). VEGF-B is dispensable for blood vessel growth but critical for their survival, and VEGF-B targeting inhibits pathological angiogenesis.

> The field of neurological disorders becomes now one of the most important investigation areas in clinical medicine, whatever the toxicological, genetic, degenerative or environmental aetiology they have. Because it involves the main complex organ as target tissue, because also of the intrinsic specificity of the biological network of the nervous system, or the technical difficulty to access such a composite organ, the nervous diseases remain particularly difficult to study. Certainly, the rapid development of transgenic animal models of neurological diseases and the expanding growth of imaging techniques to functionally and non-invasively access some specific brain regions constitute a favourable situation to study the basis and the progression of some nervous diseases. However, the use of such transgenic animals or spontaneous animal models needs that the clinical symptoms are reproducible and that a *prior* knowledge of the aetiopathology of these diseases may exist. These latter conditions are not always available, especially concerning toxicology. In this case, how can both pathophysiology and therapies be investigated? Indeed, classically, when considering a toxicological approach, clinical signs, similar to those ascribed on the target species, need to be reproduced on the animal model. But how to do with disease displaying no known aetiology or with an animal model, on which it is impossible to reproduce, at least partially, some clinical signs of the target species? Furthermore, because of evident ethical reasons added to practical ones, some neurological disorders in humans or in large animals remain scarcely explored. "Omics" approaches seem to be a good alternative in the clinical medical research, enabling to take advantage of the global living system and, simultaneously of the control of the toxicological factor. To illustrate such an original approach, a neurological horse disease, Australian stringhalt, which has been described for several centuries, but for which aetiology is still only partially known, was reassessed using metabonomics in combination with other classical techniques. This has led to show how powerful this method may stand for in clinical medical research and, particularly in neurological studies.

Power of a Metabonomic Approach to Investigate an Unknown Nervous Disease 409

spatial or the temporal resolution according to what it has to be focussed on. Among these different techniques, anatomic or functional imaging techniques have to be distinguished. The first ones, tomodensitometry and magnetic resonance imaging or MRI (Griffith et al., 2007) provide highly detailed anatomic information. Their ability to give an access to *in vivo* biological information acquired non-invasively or to define a seemingly normal body composition and its perturbation in response to a pharmacological or a pathological event may facilitate exploration of nervous diseases (Frisoni and Filippi, 2005; Griffith et al., 2007; Tartaglia and Arnold, 2006). In parallel with the description of novel biomarkers coming from transgenic animal models developed for studying neurodegenerative diseases and more efficient therapies, the use of MRI and magnetic resonance spectroscopy (MRS) provide new information for *in vivo* neurochemistry, such as neuronal apoptosis, osmoregulation, energy metabolism, membrane function or signalling disruptions (Choi et al., 2007; Ross and Sachdev, 2004; Ross and Bluml, 2001). Most of clinical researches are based on the metabolites that are detectable using proton spectroscopy (Figure 1), which can

Creatine Creatine

Taurine

*Scyllo*-inositol

Choline

Fig. 1. 600.13 MHz 1H NMR spectra from aqueous extracts of brain in mouse (control

Nuclear magnetic resonance (or NMR) methods (MRI or MRS) can be successfully used to reveal neurological markers like N-acetyl-aspartate (a neuronal and axonal marker associated with neuronal viability), *myo*-inositol (a cerebral osmolyte and an astrocytic marker), glutamate and glutamine (the first is a major excitatory neurotransmitter and the second can restore it), creatine (which plays a crucial role in ATP biosynthesis in astrocytes),

PCholineGlyceroPcholine

GABA

Glutathione reduced

4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

Glutathione reduced

Gln

GABA

Glu/Gln

Glu

Citrate

Alanine

GABA

Acetate

Lactate

quantify them in localized volumes in brain.

**1H NMR spectrum Control mouse** 

Lactate

Inosine

Glutathione reduced

animal) (from Domange, 2008)

*Myo*-inositol

Choline

*Myo*-inositol

Glu/Gln

Alanine

*Myo*-inositol

#### **2. Current neurological investigations: Advantages and limits of routinely used approaches and techniques**

#### **2.1 Limits of classical studies**

Up to now, the neurological investigations tended to reproduce a human disease using a convenient animal model. However, they laboured to give results. In fact, it may appear surprising to recreate all the metabolic complexity prevailing in the genesis of a given disease and, to work on it, before having any knowledge of the specifically involved metabolic pathways specifically involved. Before considering an animal model as a convenient model of a human disease, it seems more consistent to record and describe all the putative impacts of a controlled *stimulus* on a living system without any *a priori* hypothesis because of our ignorance of the inherent metabolic disruptions involved. Indeed, this may help to efficiently tackle a neurological disease.

#### **2.2 Behavioural approaches**

The use of animal models of human diseases, on which some behavioural tests are carried out, is fundamental to investigate nervous disorders. The field of psychopharmacology or behavioural pharmacology enables to test and to measure effects of drugs on behaviour. The toxicological studies test the short- or long-term exposure, the acute intoxication or the chronic effects following administration of subclinical doses and the associated effects of chemical compounds or contaminants. Each behavioural manifestation in animal model tends to reflect a specific human behavioural alteration or cognitive effect like depression, anxiety, fear or schizophrenia. This may be susceptible to reveal a disruption in some mean way of neurological transduction involving, for example, dopamine, acetylcholine, amphetamine or catecholamine's impairments. However, this approach has some limits. In case of the lack of any behavioural manifestation in animals, the conclusion isn't that there is a lack of effect but only that there is an impossibility to give an interpretation of this lack of effect because of an inadequate "observation window" as in delayed toxicity of some contaminants for example. Moreover, the putative link between a visible behavioural impairment and a putative mechanistic explanation requires going back to the cerebral metabolism to translate the observed behavioural variance and to confirm the pertinence of metabolic pathways specifically involved. Most of the time, such behavioural approaches are hardly self-sufficient; they need to be completed by other studies such as metabolic, histological, anatomical or immunologic ones.

#### **2.3 Imaging techniques**

A wide range of imaging techniques provides powerful tools for studying tumours (Cooper et al., 2011), congenital diseases (Toga et al., 2006), metabolic and infectious diseases (Kastrup et al., 2005), development of organisms (Davis et al., 2011) and for realizing preclinical or clinical studies, or for measuring a treatment effect (Song et al., 2011). These techniques can also be used in neurotoxicology (Pogge and Slikker, 2004) or for exploring neurodegenerative or psychiatric disorders (Masdeu, 2011; Stoessl, 2011). The choice of one of these techniques is made according to some awaited answers to a specific anatomical, metabolic or functional information question, some of imaging techniques being able to perform several specific assessments. They are well adapted to describe functions in the frame of non-invasive *in vivo* studies, some being planned with a longitudinal follow-up. Concerning some specific tissues analyses, some compromises have to be done between the

Up to now, the neurological investigations tended to reproduce a human disease using a convenient animal model. However, they laboured to give results. In fact, it may appear surprising to recreate all the metabolic complexity prevailing in the genesis of a given disease and, to work on it, before having any knowledge of the specifically involved metabolic pathways specifically involved. Before considering an animal model as a convenient model of a human disease, it seems more consistent to record and describe all the putative impacts of a controlled *stimulus* on a living system without any *a priori* hypothesis because of our ignorance of the inherent metabolic disruptions involved. Indeed,

The use of animal models of human diseases, on which some behavioural tests are carried out, is fundamental to investigate nervous disorders. The field of psychopharmacology or behavioural pharmacology enables to test and to measure effects of drugs on behaviour. The toxicological studies test the short- or long-term exposure, the acute intoxication or the chronic effects following administration of subclinical doses and the associated effects of chemical compounds or contaminants. Each behavioural manifestation in animal model tends to reflect a specific human behavioural alteration or cognitive effect like depression, anxiety, fear or schizophrenia. This may be susceptible to reveal a disruption in some mean way of neurological transduction involving, for example, dopamine, acetylcholine, amphetamine or catecholamine's impairments. However, this approach has some limits. In case of the lack of any behavioural manifestation in animals, the conclusion isn't that there is a lack of effect but only that there is an impossibility to give an interpretation of this lack of effect because of an inadequate "observation window" as in delayed toxicity of some contaminants for example. Moreover, the putative link between a visible behavioural impairment and a putative mechanistic explanation requires going back to the cerebral metabolism to translate the observed behavioural variance and to confirm the pertinence of metabolic pathways specifically involved. Most of the time, such behavioural approaches are hardly self-sufficient; they need to be completed by other studies such as metabolic,

A wide range of imaging techniques provides powerful tools for studying tumours (Cooper et al., 2011), congenital diseases (Toga et al., 2006), metabolic and infectious diseases (Kastrup et al., 2005), development of organisms (Davis et al., 2011) and for realizing preclinical or clinical studies, or for measuring a treatment effect (Song et al., 2011). These techniques can also be used in neurotoxicology (Pogge and Slikker, 2004) or for exploring neurodegenerative or psychiatric disorders (Masdeu, 2011; Stoessl, 2011). The choice of one of these techniques is made according to some awaited answers to a specific anatomical, metabolic or functional information question, some of imaging techniques being able to perform several specific assessments. They are well adapted to describe functions in the frame of non-invasive *in vivo* studies, some being planned with a longitudinal follow-up. Concerning some specific tissues analyses, some compromises have to be done between the

**2. Current neurological investigations: Advantages and limits of routinely** 

**used approaches and techniques** 

this may help to efficiently tackle a neurological disease.

histological, anatomical or immunologic ones.

**2.3 Imaging techniques** 

**2.1 Limits of classical studies** 

**2.2 Behavioural approaches** 

spatial or the temporal resolution according to what it has to be focussed on. Among these different techniques, anatomic or functional imaging techniques have to be distinguished. The first ones, tomodensitometry and magnetic resonance imaging or MRI (Griffith et al., 2007) provide highly detailed anatomic information. Their ability to give an access to *in vivo* biological information acquired non-invasively or to define a seemingly normal body composition and its perturbation in response to a pharmacological or a pathological event may facilitate exploration of nervous diseases (Frisoni and Filippi, 2005; Griffith et al., 2007; Tartaglia and Arnold, 2006). In parallel with the description of novel biomarkers coming from transgenic animal models developed for studying neurodegenerative diseases and more efficient therapies, the use of MRI and magnetic resonance spectroscopy (MRS) provide new information for *in vivo* neurochemistry, such as neuronal apoptosis, osmoregulation, energy metabolism, membrane function or signalling disruptions (Choi et al., 2007; Ross and Sachdev, 2004; Ross and Bluml, 2001). Most of clinical researches are based on the metabolites that are detectable using proton spectroscopy (Figure 1), which can quantify them in localized volumes in brain.

Fig. 1. 600.13 MHz 1H NMR spectra from aqueous extracts of brain in mouse (control animal) (from Domange, 2008)

Nuclear magnetic resonance (or NMR) methods (MRI or MRS) can be successfully used to reveal neurological markers like N-acetyl-aspartate (a neuronal and axonal marker associated with neuronal viability), *myo*-inositol (a cerebral osmolyte and an astrocytic marker), glutamate and glutamine (the first is a major excitatory neurotransmitter and the second can restore it), creatine (which plays a crucial role in ATP biosynthesis in astrocytes),

Power of a Metabonomic Approach to Investigate an Unknown Nervous Disease 411

target they have to reach (gene, enzymatic mechanism, biomarker). The full range of metabolites synthesized by a given biological system corresponds to its metabolome. In the same way, the full range of genes is contained in the term genome, the mRNA and the proteins ones, respectively, in the terms transcriptome and proteome (Figure 2). All these systems can be defined according to the level of biological organization, *i.e.* organism, organ, tissue, and cell. Related to these biological levels, omics-based approaches, mainly genomics, transcriptomics, proteomics (Colucci-D'Amato et al., 2011), lipidomics (Li et al., 2007), and metabolomics are terms standing for various global molecular-oriented approaches to better understand the underlying mechanisms, as the physiological regulations and the networks involved on all levels of gene products (mRNA, proteins, metabolites) in their respective systems and, if possible, between different sub-networks. Indeed, the observable property of organisms, *i.e.* their phenotype, is issued from genotype submitted to the concomitantly interactive action of the environment. Most of the time, the association between some of these approaches can be beneficial, enabling to understand the temporal progression of a pathophysiological state or the functioning of metabolic networks (Fiehn, 2001). Interest of these methods is to apply a controlled disruption to a biological system, whatever its nature (genetic, toxicological, pathophysiological, dietary), under some

Genome

Transcriptome

Proteome

Omics

Clinic Function

Subclinic approaches

Clinical biomarker

Subclinical biomarker

Metabolome

Fig. 2. "Omics" approaches and their different levels of biological living systems investigation

Phenotype

Genotype

DNA

mRNA

Proteins

Metabolites

choline (its increased concentration theoretically means an alteration of myelin) and gammaamino butyric acid (a main inhibitory neurotransmitter) (Martin, 2007). But MRS can also record intrinsic containing metabolites containing other atoms, as phosphorus, sodium, potassium, carbon, nitrogen and fluorine (this last atom often being a constituent of many drugs). Among the functional imaging techniques, positron emission tomography (PET) is a three-dimensional diagnostic imaging technology used in nuclear medicine that measures physiological function by looking at various functions of the body. It is a non-invasive diagnostic imaging tool enabling to follow some chemical neurotransmitters like dopamine in Parkinson's disease. Whatever the technique used to cover a specific neurological question, most of the time, it often requires laboratory animal and more particularly animal models of given human diseases.

#### **2.4 Laboratory animals model contribution**

The use of animal models in clinical research is crucial. As models, they usually display the same features and clinical signs as those observed in humans. So, they enable to establish some comparisons and extrapolations with the human physiology, to give access putative metabolic mechanisms involved in the progression of the disease and, hence, to identify biomarkers. A wide range of animal models has been used according to their origin. Animal models can be spontaneous, namely "mutant". Therefore, identification and characterization of novel laboratory animal lines carrying an interesting mutation combined with genotype-driven approaches are useful approaches to investigate some specific mechanistically-related molecules, to give new information about the function of the mammalian nervous system (Banks et al., 2011) or to study how genetic, environmental, toxicological or dietary factors can explain aetiology of a given disease. Animal models can also be created, using surgery, pharmacology or genetic interventions. These models are used to identify aetiological markers of disease or drug target and to test some new therapeutic drugs. The first cases have traditionally been induced by neurotoxins, acting selectively on neurons affected by human diseases. They are particularly useful for the study of the pathogenetic mechanism or to test new therapies for human neurological disorders (psychiatric or motor disorders) like obsessive-compulsive disorder or Parkinson's disease (Nowak et al., 2011). In parallel, the knockout technique, in which a gene is made inoperative leading to animals deficient in one specific gene, enables to evaluate the effects of the depletion of one protein in all the series of biological reactions within an organism and the putative followed consequences (Berman et al., 2011). More recently, the use of transgenic animals, constructed by inserting a human gene downstream into promoter, followed by microinjection in animal, ensures to indicate whether an over- or under-expression of a gene in one or several tissues should be susceptible to promote the pathogenesis and the development of a disease (Liu et al., 2011). The common point of all these animal models is the necessity of having some preliminary knowledge concerning a disease or the deleterious effect of a given xenobiotic. However, this information is not always available. Therefore, researchers have apace become aware of the necessity to access a wider range of knowledge in a living system and not only a specific molecular entity.

#### **3. "Omics" approaches and their interest in clinical research**

#### **3.1 "Omics" approaches presentation**

Similarly to imaging techniques, omics-based approaches appeared to be used according to the biological pool they consider (genes, proteins, lipids, metabolites) and the nature of

choline (its increased concentration theoretically means an alteration of myelin) and gammaamino butyric acid (a main inhibitory neurotransmitter) (Martin, 2007). But MRS can also record intrinsic containing metabolites containing other atoms, as phosphorus, sodium, potassium, carbon, nitrogen and fluorine (this last atom often being a constituent of many drugs). Among the functional imaging techniques, positron emission tomography (PET) is a three-dimensional diagnostic imaging technology used in nuclear medicine that measures physiological function by looking at various functions of the body. It is a non-invasive diagnostic imaging tool enabling to follow some chemical neurotransmitters like dopamine in Parkinson's disease. Whatever the technique used to cover a specific neurological question, most of the time, it often requires laboratory animal and more particularly animal

The use of animal models in clinical research is crucial. As models, they usually display the same features and clinical signs as those observed in humans. So, they enable to establish some comparisons and extrapolations with the human physiology, to give access putative metabolic mechanisms involved in the progression of the disease and, hence, to identify biomarkers. A wide range of animal models has been used according to their origin. Animal models can be spontaneous, namely "mutant". Therefore, identification and characterization of novel laboratory animal lines carrying an interesting mutation combined with genotype-driven approaches are useful approaches to investigate some specific mechanistically-related molecules, to give new information about the function of the mammalian nervous system (Banks et al., 2011) or to study how genetic, environmental, toxicological or dietary factors can explain aetiology of a given disease. Animal models can also be created, using surgery, pharmacology or genetic interventions. These models are used to identify aetiological markers of disease or drug target and to test some new therapeutic drugs. The first cases have traditionally been induced by neurotoxins, acting selectively on neurons affected by human diseases. They are particularly useful for the study of the pathogenetic mechanism or to test new therapies for human neurological disorders (psychiatric or motor disorders) like obsessive-compulsive disorder or Parkinson's disease (Nowak et al., 2011). In parallel, the knockout technique, in which a gene is made inoperative leading to animals deficient in one specific gene, enables to evaluate the effects of the depletion of one protein in all the series of biological reactions within an organism and the putative followed consequences (Berman et al., 2011). More recently, the use of transgenic animals, constructed by inserting a human gene downstream into promoter, followed by microinjection in animal, ensures to indicate whether an over- or under-expression of a gene in one or several tissues should be susceptible to promote the pathogenesis and the development of a disease (Liu et al., 2011). The common point of all these animal models is the necessity of having some preliminary knowledge concerning a disease or the deleterious effect of a given xenobiotic. However, this information is not always available. Therefore, researchers have apace become aware of the necessity to access a wider range of knowledge in a living system and not only a specific molecular entity.

**3. "Omics" approaches and their interest in clinical research** 

Similarly to imaging techniques, omics-based approaches appeared to be used according to the biological pool they consider (genes, proteins, lipids, metabolites) and the nature of

**3.1 "Omics" approaches presentation** 

models of given human diseases.

**2.4 Laboratory animals model contribution** 

target they have to reach (gene, enzymatic mechanism, biomarker). The full range of metabolites synthesized by a given biological system corresponds to its metabolome. In the same way, the full range of genes is contained in the term genome, the mRNA and the proteins ones, respectively, in the terms transcriptome and proteome (Figure 2). All these systems can be defined according to the level of biological organization, *i.e.* organism, organ, tissue, and cell. Related to these biological levels, omics-based approaches, mainly genomics, transcriptomics, proteomics (Colucci-D'Amato et al., 2011), lipidomics (Li et al., 2007), and metabolomics are terms standing for various global molecular-oriented approaches to better understand the underlying mechanisms, as the physiological regulations and the networks involved on all levels of gene products (mRNA, proteins, metabolites) in their respective systems and, if possible, between different sub-networks. Indeed, the observable property of organisms, *i.e.* their phenotype, is issued from genotype submitted to the concomitantly interactive action of the environment. Most of the time, the association between some of these approaches can be beneficial, enabling to understand the temporal progression of a pathophysiological state or the functioning of metabolic networks (Fiehn, 2001). Interest of these methods is to apply a controlled disruption to a biological system, whatever its nature (genetic, toxicological, pathophysiological, dietary), under some

Fig. 2. "Omics" approaches and their different levels of biological living systems investigation

Genotype

Power of a Metabonomic Approach to Investigate an Unknown Nervous Disease 413

 ionic current measured at a specific mass to charge ratio in MS. Datasets are then treated using sophisticated statistical tools, *i.e.* multivariate or multidimensional statistical analysis tools, to access the most suitable model able to discriminate the different groups of samples according to the studied factors and to reveal main variables explaining this segregation. These variables can be considered at this step as many putative biomarkers, which need to be fully characterized by convenient structural identification methods. Finally, a detailed map of regulation and interaction between identified metabolites, their disruptions and the putative explanation of the pathophysiological state according to all involved factors may be suggested. Among analytical techniques mainly used in metabolomics, MS spectroscopy coupled to liquid (LC-MS) or gas chromatography (GC-MS) and NMR spectroscopy are the most appropriate ones concerning analysis of biofluids or liquid samples, whereas high resolution magic angle spinning (HR-MAS) NMR and MRS are adapted to solid samples

As previously mentioned above, the major constraint the "omics" approaches have to answer to, is to give access to a global pool of information belonging to the living system without focusing on a specific molecular entity. Indeed, one of the main assets of these approaches is the property of data integration necessary to render it as functionally understandable as possible. These features can be revealed through three complementary characteristics, namely *i)* the global nature of living systems underlined by homeostasis, *ii)* the multifactorial nature of diseases with both intrinsic and extrinsic factors, and *iii)* the ability to access multiple biological levels in living systems, and then to compile them to reveal one of the most realistic progressions of a disruption within a complex organism. Let's go into details of these three points. i) Contrary to classical biochemical approaches, which are set out to study only a single or few metabolites or metabolic reactions at the same time, metabolomics provides quantitative data on a wide range of known and unknown metabolites. It enables to visualize an overall pattern comprehensively linked to a set of interactions between metabolites or metabolic pathways and, hence, to an intrinsic homeostasis defined in these specific conditions (Kaddurah-Daouk et al., 2008). Indeed, whatever the stress applied on living systems, some allostatic changes, defined as an adaptive process, lead to short-term corrective changes of the different relevant regulatory systems to maintain a metabolic homeostasis. Concept of homeostasis is fundamental in biology and in clinical medicine to understand pathophysiological processes. The current clinical medicine tends now to come back to a more global view and, at the same time, on a more individualized approach of every patient because each of them differently answers to the environment according to their own homeostatic specificity. Clinical and subclinical signs give personalized information for every subject and, hence, physiological "means" used to adapt for everybody the set of parameters of homeostatic control in response to a disruptive stimulus and so to avoid falling down in a pathological state. The understanding of overall adaptation requires a good knowledge of metabolic pathways and related biochemical networks involved in the efficient control of homeostasis. For example, the knowledge of the glucose metabolism and the different ways by which homeostatic control of the circulating glucose concentration is achieved is crucial in the investigation of the Type 2 diabetes (Fiehn et al., 2010). ii) From global approaches can emerge a more accurate understanding of a given disease considering it is not only a single functional entity which is

like tissue or to achieve *in vivo* studies, respectively.

**3.2 An integrated and functional approach** 

specific conditions (most of the time, the investigation is focused on an animal model), and to consider the subclinical consequences of such a disruptive perturbation. Therefore, "omics" approaches are widely used in biomedical research to make easier the understanding of disease mechanisms and to give access target tissues, to make easier the identification of biomarkers useful for therapeutic and diagnostic development and to predict clinical responses to treatments. The large amount of acquired data is as much an advantage as a hindrance. Indeed, the challenging subtlety is that all this information needs to be explored without any *a priori* hypothesis but by extracting only the interesting data. This fact partially explains why the real capacity of "omics" technologies stays in some instances rather limited because of the necessary requirement of some specific bioinformatics tools to efficiently mine multidimensional data but also the requirement of some specific analytical database to identify the candidate biomarkers at the gene, mRNA, enzyme, protein, or metabolite level. Moreover, transcriptomic studies require high-cost technologies and so, are less used than proteomic ones, which are based on two-dimensional gel-electrophoresis, which is cheaper and can be more easily used in many laboratories. However, analytical techniques related to the detection of large arrays of metabolites seem more robust, the resulting information being often easier to interpret because of the lower number of molecular entities, even though a rigorous identification of new metabolites still remains particularly fussy. According to the aim of studies and considering an increasing level of complexity of the analytical strategy used, investigation of metabolites may require either a metabolic profiling approach, which is focused on a small number of known metabolites (targeted metabolic profiling), or metabolomics including investigation of several classes of compounds (open metabolic profiling) or functional genomics, also called metabolic fingerprinting or metabonomics. This latter one is based on classification of samples according to their biological relevance to the studied disruption event and on identification of the fully informative markers detected at the statistical and functional sides and measured within the analyzed biological matrices.

Therefore, among these omics-based approaches, metabonomics stands for one of the most used holistic methods. Its emergence and its development mainly come from pioneering works of Pr J. Nicholson from the Imperial College of London. Because metabonomics enables to identify and quantify simultaneously low molecular weight compounds (metabolites) using spectroscopic methods such as nuclear magnetic resonance (NMR) or mass spectroscopy (MS), it gives access to a molecular level and may define the quantitative measurement of multiparametric metabolic responses of living system to pathophysiological stimuli. This can bring to the determination of some comprehensive metabolic signatures of biological matrices (Nicholson et al., 1999; Robertson, 2005). Metabonomics approach can be divided into successive steps. After the crucial step concerning the development of the experimental design, the choice of the animal model, the choice of the instrumentation used to quantitatively generate the metabolic information, the choice of samples of interest to be collected during the animal or the human experiment (biofluids such as plasma, urine, cerebral spinal fluid, saliva or faeces, tissues or organs), these biological samples are treated using appropriate analytical techniques. These latter ones enable to establish a metabolic fingerprint through the spectrum recording for every sample. All these fingerprints are summed up into datasets, in which metabolic information is subdivided and identified through coding variables. Each of them stands for either an integration bucket in NMR spectra corresponding to a defined chemical shift, or a relative or an absolute intensity of the

 ionic current measured at a specific mass to charge ratio in MS. Datasets are then treated using sophisticated statistical tools, *i.e.* multivariate or multidimensional statistical analysis tools, to access the most suitable model able to discriminate the different groups of samples according to the studied factors and to reveal main variables explaining this segregation. These variables can be considered at this step as many putative biomarkers, which need to be fully characterized by convenient structural identification methods. Finally, a detailed map of regulation and interaction between identified metabolites, their disruptions and the putative explanation of the pathophysiological state according to all involved factors may be suggested. Among analytical techniques mainly used in metabolomics, MS spectroscopy coupled to liquid (LC-MS) or gas chromatography (GC-MS) and NMR spectroscopy are the most appropriate ones concerning analysis of biofluids or liquid samples, whereas high resolution magic angle spinning (HR-MAS) NMR and MRS are adapted to solid samples like tissue or to achieve *in vivo* studies, respectively.

#### **3.2 An integrated and functional approach**

412 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

specific conditions (most of the time, the investigation is focused on an animal model), and to consider the subclinical consequences of such a disruptive perturbation. Therefore, "omics" approaches are widely used in biomedical research to make easier the understanding of disease mechanisms and to give access target tissues, to make easier the identification of biomarkers useful for therapeutic and diagnostic development and to predict clinical responses to treatments. The large amount of acquired data is as much an advantage as a hindrance. Indeed, the challenging subtlety is that all this information needs to be explored without any *a priori* hypothesis but by extracting only the interesting data. This fact partially explains why the real capacity of "omics" technologies stays in some instances rather limited because of the necessary requirement of some specific bioinformatics tools to efficiently mine multidimensional data but also the requirement of some specific analytical database to identify the candidate biomarkers at the gene, mRNA, enzyme, protein, or metabolite level. Moreover, transcriptomic studies require high-cost technologies and so, are less used than proteomic ones, which are based on two-dimensional gel-electrophoresis, which is cheaper and can be more easily used in many laboratories. However, analytical techniques related to the detection of large arrays of metabolites seem more robust, the resulting information being often easier to interpret because of the lower number of molecular entities, even though a rigorous identification of new metabolites still remains particularly fussy. According to the aim of studies and considering an increasing level of complexity of the analytical strategy used, investigation of metabolites may require either a metabolic profiling approach, which is focused on a small number of known metabolites (targeted metabolic profiling), or metabolomics including investigation of several classes of compounds (open metabolic profiling) or functional genomics, also called metabolic fingerprinting or metabonomics. This latter one is based on classification of samples according to their biological relevance to the studied disruption event and on identification of the fully informative markers detected at the statistical and functional sides

Therefore, among these omics-based approaches, metabonomics stands for one of the most used holistic methods. Its emergence and its development mainly come from pioneering works of Pr J. Nicholson from the Imperial College of London. Because metabonomics enables to identify and quantify simultaneously low molecular weight compounds (metabolites) using spectroscopic methods such as nuclear magnetic resonance (NMR) or mass spectroscopy (MS), it gives access to a molecular level and may define the quantitative measurement of multiparametric metabolic responses of living system to pathophysiological stimuli. This can bring to the determination of some comprehensive metabolic signatures of biological matrices (Nicholson et al., 1999; Robertson, 2005). Metabonomics approach can be divided into successive steps. After the crucial step concerning the development of the experimental design, the choice of the animal model, the choice of the instrumentation used to quantitatively generate the metabolic information, the choice of samples of interest to be collected during the animal or the human experiment (biofluids such as plasma, urine, cerebral spinal fluid, saliva or faeces, tissues or organs), these biological samples are treated using appropriate analytical techniques. These latter ones enable to establish a metabolic fingerprint through the spectrum recording for every sample. All these fingerprints are summed up into datasets, in which metabolic information is subdivided and identified through coding variables. Each of them stands for either an integration bucket in NMR spectra corresponding to a defined chemical shift, or a relative or an absolute intensity of the

and measured within the analyzed biological matrices.

As previously mentioned above, the major constraint the "omics" approaches have to answer to, is to give access to a global pool of information belonging to the living system without focusing on a specific molecular entity. Indeed, one of the main assets of these approaches is the property of data integration necessary to render it as functionally understandable as possible. These features can be revealed through three complementary characteristics, namely *i)* the global nature of living systems underlined by homeostasis, *ii)* the multifactorial nature of diseases with both intrinsic and extrinsic factors, and *iii)* the ability to access multiple biological levels in living systems, and then to compile them to reveal one of the most realistic progressions of a disruption within a complex organism. Let's go into details of these three points. i) Contrary to classical biochemical approaches, which are set out to study only a single or few metabolites or metabolic reactions at the same time, metabolomics provides quantitative data on a wide range of known and unknown metabolites. It enables to visualize an overall pattern comprehensively linked to a set of interactions between metabolites or metabolic pathways and, hence, to an intrinsic homeostasis defined in these specific conditions (Kaddurah-Daouk et al., 2008). Indeed, whatever the stress applied on living systems, some allostatic changes, defined as an adaptive process, lead to short-term corrective changes of the different relevant regulatory systems to maintain a metabolic homeostasis. Concept of homeostasis is fundamental in biology and in clinical medicine to understand pathophysiological processes. The current clinical medicine tends now to come back to a more global view and, at the same time, on a more individualized approach of every patient because each of them differently answers to the environment according to their own homeostatic specificity. Clinical and subclinical signs give personalized information for every subject and, hence, physiological "means" used to adapt for everybody the set of parameters of homeostatic control in response to a disruptive stimulus and so to avoid falling down in a pathological state. The understanding of overall adaptation requires a good knowledge of metabolic pathways and related biochemical networks involved in the efficient control of homeostasis. For example, the knowledge of the glucose metabolism and the different ways by which homeostatic control of the circulating glucose concentration is achieved is crucial in the investigation of the Type 2 diabetes (Fiehn et al., 2010). ii) From global approaches can emerge a more accurate understanding of a given disease considering it is not only a single functional entity which is

Power of a Metabonomic Approach to Investigate an Unknown Nervous Disease 415

urine metabolomic analysis is not prone to reflect the real state of the subject, contrary to data coming from other organs like liver and kidney. Nevertheless, some first encouraging studies on neurological disorders performed using metabolomics have confirmed the interest of application of this approach in the field of neuroscience (Griffin and Salek, 2007). Analysis of blood or urine gives access to putative cerebral disruptions and can help to successfully reveal some biomarkers, as in the case of the manganese neurotoxicity, which is a significant public health concern (Dorman et al., 2008). So, because it reflects the presence of both extrinsic and intrinsic disruptive factors, metabonomics can define accurate biomarkers in neurology. Moreover, some specific metabolic pathways or some biological disruptions can be particularly interesting to study, because of their central or ubiquitous role in many pathological states. One example is the oxidative stress, leading to neuronal death, a mechanism that is found in early stages but also in secondary manifestations of many neurodegenerative states like Alzheimer's, Parkinson's and Huntington's diseases, amyotrophic lateral sclerosis, and neuroinflammatory disorders (Sayre et al., 2008). Because

better understanding of some metabolic pathways like the biosynthesis of the amino acid L-serine can be interesting to investigate (Tabatabaie et al., 2010). Metabolic profiles acquired on human or animal biofluids like urine, cerebrospinal fluid (Lutz et al., 2007b), plasma, serum or tissue extracts, using either NMR or MS techniques, can give some precious information concerning neurological disorders (Sinclair et al., 2009). For example, ultra performance liquid chromatography/mass spectroscopy (UPLC/MS) metabolic profiles from serum collected on cerebral infarction patients have been analyzed using a metabonomic approach (Jiang et al., 2011). Quantitative analysis of human cerebrospinal fluid using NMR spectroscopy has been performed in multiple sclerosis (Lutz et al., 2007a), to identify biomarkers in the early stages of the amyotrophic lateral sclerosis (Blasco et al., 2010). Plasmatic metabolic disruptions between healthy and old persons with Alzheimer's disease were investigated using UPLC/MS-based metabonomic approach (Li et al., 2010). CRND8 transgenic mouse, model of this disease, enabled to analyze brain extracts using 1H NMR spectroscopy (Salek et al., 2010). The interest of brain extracts coming from an animal model has been also illustrated to investigate epilepsy, for which the pharmacologically-induced animal model was obtained using pentylenetetrazole, a drug that induces seizures (Carmody and Brennan, 2010). Plasma from an experimental animal model of the spinal cord injury (Blasco et al., 2010) has been analyzed by 1H NMR to get fingerprint profiles of this pathology (Jiang et al., 2010). Other cerebral alterations like brain tumors (Tate et al., 1996; Tate et al., 1998), schizophrenia and meningitis (Holmes et al., 2006;

Beyond the use of a unique "omics" approach, it seems that it is all the more interesting and powerful to call for several complementary approaches and to tend to integrate sogenerated data to yield a more comprehensive understanding of many diseases. In this way, Caudle et al. have used "omics" to characterize and identify some biomarkers of Parkinson's disease (Caudle et al., 2010). As an example, the following part illustrates the power of such a use, in a rodent model, of a neuro-intoxication caused in the horse by a plant, *Hypochoeris radicata* (L.). Indeed, because of the lack of knowledge about a neurological disease described only in the horse, we have tempted to use a laboratory animal model of this intoxication by applying metabonomics combined to imaging or behavioural experiments to

of the pivotal role of a metabolite in many biological functions, a

Lutz et al., 2007a) have also been investigated.

reveal, in brain, candidate biomarkers of this pathology.

concerned, that is not only the consequence of a single causative explanation with a single mechanism involved in a single cell type in a given condition. Indeed, most of the pathophysiological disorders are not unique functional events but are resulting from complex interactions. These latter ones involve different concomitant actions in different biological compartments leading to different disruptions, which can be categorized according to the environmental conditions encountered and the inherent variability of subjects. Becoming aware of the importance of the environment and, more particularly, of the multifactorial nature of most of the disruptive events displayed by a living system is among the first pillars of the concept of global approach used in biological research in clinical medicine or in toxicology. iii) Finally, as a microscope could do it, omics-based approaches enable to focus on a specific level of a living system depending on the available analytical techniques used to generate data, but also to statistically integrate data coming from complementary fingerprinting techniques by using canonical analyses.

#### **3.3 A metabolomic-based approach to reveal subclinical metabolic disruptions: A powerful tool in investigation of biomarkers**

Besides the ability to define and to understand the aetiology of a disease, the discovery of novel biomarkers stands for a fundamental step to characterize and to manage it, especially to spot the homeostatic break down before appearance of the first clinical signs. Biomarkers, which are relevant indicators of disrupted biological processes in a given pathophysiological context, have to disclose features of disease (Moore et al., 2007; Nicholson and Lindon, 2008). The metabonomic approach is particularly interesting to explore subclinical disruptions of a living system before the outset of manifest clinical signs, and to identify biomarkers of disease risk and, if possible, to initiate prevention like in cancer (Roberts et al., 2011), diabetes (Wang et al., 2011), or nervous system illnesses (Kaddurah-Daouk and Krishnan, 2009; Nicholson and Lindon, 2008; Quinones and Kaddurah-Daouk, 2009). The identification of the metabolites requires the use of up-to-date structural databases of metabolites and metabolic pathway resources (Kouskoumvekaki and Panagiotou, 2011). As it has been previously mentioned, the use of complementary approaches stands for a wise way search of biomarkers displayed at different levels, namely biochemical, neuroanatomical, metabolic, genetic and neuropsychological ones, as it can be reported in the case of Alzheimer's disease investigation (Wattamwar and Mathuranath, 2010).

#### **3.4 Examples of "omics" approaches in neurological investigation area**

Use of metabolomics in neurological studies has been reported in many reviews (Choi et al., 2003; Rudkin and Arnold, 1999). It has been applied to a variety of biological samples for a better understanding of pathogenesis. This approach, because of its integrated and functional nature, stands for a powerful tool to study normal or pathological living systems, especially in central nervous system disorders through the use of specific animal models (Pears et al., 2005). Thus, it allows the identification of biomarkers of such diseases, but also of illness progression or response to therapy. In the drug discovery process, metabolomics brings some biochemical information about drug candidates, their mechanism of action and their therapeutic potential. In the field of neurosciences, the use of metabolomic approach can generate some questionings. Contrary to other organs in mammals, brain is isolated from the rest of organism by the blood-brain barrier, with consequences on the passage of some metabolites. Therefore, a metabolic fingerprint of brain predicted from a blood or

concerned, that is not only the consequence of a single causative explanation with a single mechanism involved in a single cell type in a given condition. Indeed, most of the pathophysiological disorders are not unique functional events but are resulting from complex interactions. These latter ones involve different concomitant actions in different biological compartments leading to different disruptions, which can be categorized according to the environmental conditions encountered and the inherent variability of subjects. Becoming aware of the importance of the environment and, more particularly, of the multifactorial nature of most of the disruptive events displayed by a living system is among the first pillars of the concept of global approach used in biological research in clinical medicine or in toxicology. iii) Finally, as a microscope could do it, omics-based approaches enable to focus on a specific level of a living system depending on the available analytical techniques used to generate data, but also to statistically integrate data coming

from complementary fingerprinting techniques by using canonical analyses.

**A powerful tool in investigation of biomarkers** 

**3.3 A metabolomic-based approach to reveal subclinical metabolic disruptions:** 

the case of Alzheimer's disease investigation (Wattamwar and Mathuranath, 2010).

Use of metabolomics in neurological studies has been reported in many reviews (Choi et al., 2003; Rudkin and Arnold, 1999). It has been applied to a variety of biological samples for a better understanding of pathogenesis. This approach, because of its integrated and functional nature, stands for a powerful tool to study normal or pathological living systems, especially in central nervous system disorders through the use of specific animal models (Pears et al., 2005). Thus, it allows the identification of biomarkers of such diseases, but also of illness progression or response to therapy. In the drug discovery process, metabolomics brings some biochemical information about drug candidates, their mechanism of action and their therapeutic potential. In the field of neurosciences, the use of metabolomic approach can generate some questionings. Contrary to other organs in mammals, brain is isolated from the rest of organism by the blood-brain barrier, with consequences on the passage of some metabolites. Therefore, a metabolic fingerprint of brain predicted from a blood or

**3.4 Examples of "omics" approaches in neurological investigation area** 

Besides the ability to define and to understand the aetiology of a disease, the discovery of novel biomarkers stands for a fundamental step to characterize and to manage it, especially to spot the homeostatic break down before appearance of the first clinical signs. Biomarkers, which are relevant indicators of disrupted biological processes in a given pathophysiological context, have to disclose features of disease (Moore et al., 2007; Nicholson and Lindon, 2008). The metabonomic approach is particularly interesting to explore subclinical disruptions of a living system before the outset of manifest clinical signs, and to identify biomarkers of disease risk and, if possible, to initiate prevention like in cancer (Roberts et al., 2011), diabetes (Wang et al., 2011), or nervous system illnesses (Kaddurah-Daouk and Krishnan, 2009; Nicholson and Lindon, 2008; Quinones and Kaddurah-Daouk, 2009). The identification of the metabolites requires the use of up-to-date structural databases of metabolites and metabolic pathway resources (Kouskoumvekaki and Panagiotou, 2011). As it has been previously mentioned, the use of complementary approaches stands for a wise way search of biomarkers displayed at different levels, namely biochemical, neuroanatomical, metabolic, genetic and neuropsychological ones, as it can be reported in urine metabolomic analysis is not prone to reflect the real state of the subject, contrary to data coming from other organs like liver and kidney. Nevertheless, some first encouraging studies on neurological disorders performed using metabolomics have confirmed the interest of application of this approach in the field of neuroscience (Griffin and Salek, 2007). Analysis of blood or urine gives access to putative cerebral disruptions and can help to successfully reveal some biomarkers, as in the case of the manganese neurotoxicity, which is a significant public health concern (Dorman et al., 2008). So, because it reflects the presence of both extrinsic and intrinsic disruptive factors, metabonomics can define accurate biomarkers in neurology. Moreover, some specific metabolic pathways or some biological disruptions can be particularly interesting to study, because of their central or ubiquitous role in many pathological states. One example is the oxidative stress, leading to neuronal death, a mechanism that is found in early stages but also in secondary manifestations of many neurodegenerative states like Alzheimer's, Parkinson's and Huntington's diseases, amyotrophic lateral sclerosis, and neuroinflammatory disorders (Sayre et al., 2008). Because of the pivotal role of a metabolite in many biological functions, a

better understanding of some metabolic pathways like the biosynthesis of the amino acid L-serine can be interesting to investigate (Tabatabaie et al., 2010). Metabolic profiles acquired on human or animal biofluids like urine, cerebrospinal fluid (Lutz et al., 2007b), plasma, serum or tissue extracts, using either NMR or MS techniques, can give some precious information concerning neurological disorders (Sinclair et al., 2009). For example, ultra performance liquid chromatography/mass spectroscopy (UPLC/MS) metabolic profiles from serum collected on cerebral infarction patients have been analyzed using a metabonomic approach (Jiang et al., 2011). Quantitative analysis of human cerebrospinal fluid using NMR spectroscopy has been performed in multiple sclerosis (Lutz et al., 2007a), to identify biomarkers in the early stages of the amyotrophic lateral sclerosis (Blasco et al., 2010). Plasmatic metabolic disruptions between healthy and old persons with Alzheimer's disease were investigated using UPLC/MS-based metabonomic approach (Li et al., 2010). CRND8 transgenic mouse, model of this disease, enabled to analyze brain extracts using 1H NMR spectroscopy (Salek et al., 2010). The interest of brain extracts coming from an animal model has been also illustrated to investigate epilepsy, for which the pharmacologically-induced animal model was obtained using pentylenetetrazole, a drug that induces seizures (Carmody and Brennan, 2010). Plasma from an experimental animal model of the spinal cord injury (Blasco et al., 2010) has been analyzed by 1H NMR to get fingerprint profiles of this pathology (Jiang et al., 2010). Other cerebral alterations like brain tumors (Tate et al., 1996; Tate et al., 1998), schizophrenia and meningitis (Holmes et al., 2006; Lutz et al., 2007a) have also been investigated.

Beyond the use of a unique "omics" approach, it seems that it is all the more interesting and powerful to call for several complementary approaches and to tend to integrate sogenerated data to yield a more comprehensive understanding of many diseases. In this way, Caudle et al. have used "omics" to characterize and identify some biomarkers of Parkinson's disease (Caudle et al., 2010). As an example, the following part illustrates the power of such a use, in a rodent model, of a neuro-intoxication caused in the horse by a plant, *Hypochoeris radicata* (L.). Indeed, because of the lack of knowledge about a neurological disease described only in the horse, we have tempted to use a laboratory animal model of this intoxication by applying metabonomics combined to imaging or behavioural experiments to reveal, in brain, candidate biomarkers of this pathology.

Power of a Metabonomic Approach to Investigate an Unknown Nervous Disease 417

Since this time, several other outbreaks had been reported in many countries such as New Zealand (Cahill et al., 1985; Cahill et al., 1986; Cahill and Goulden, 1992), Chile (Araya et al., 1998), United States (Gay et al., 1993; Huntington et al., 1989; Robertson-Smith et al., 1985; Slocombe et al., 1992), Italy (Torre, 2005), Brasil (Araujo), more recently in France (Domange et al., 2010; Gouy et al., 2005) and were suspected in Japan (Takahashi et al., 2002). According to most of the authors, a plant of the Asteraceae family (formerly Compositeae family), *Hypochoeris radicata* L. also named cat's ear, flatweed or capeweed was suspected to be responsible for this disease (Araujo et al., 2008; Gardner et al., 2005; Gay et al., 1993; Gouy et al., 2005). This rosette-forming herb with a yellow terminal flower has a deep taproot, giving it resistance to drought. This explains a growth achieved preferentially on poorquality pastures after a prolonged dry period, mainly in late summer and early autumn. Such climatic conditions, associated with the aggressiveness and the dominance of *Hypochoeris radicata* L. on other species, enable it to colonize pastures and to become the major plant available as herbivore feeding. These favouring factors, in aggravation for many years because of the global change in climatic conditions, appeared particularly marked in 2003 in France, after a blistering and dry summer, leading to an epizooty with a few tens of recorded intoxicated horses (Domange et al., 2010; Gouy et al., 2005). These latter's showed a wide range of symptoms but mainly dominated by several severity degrees from grade I to grade V, (Huntington et al., 1989) with an involuntary exaggerated hyperflexion of hind limbs and a delayed extension of hocks during forward movement. A marked atrophy of the hind limbs musculature, especially in the distal muscles, is often associated with this gait in the most affected animals. Most of the time, this amyotrophy is related to neurological lesions of the hind limbs with a proximal-to-distal gradient in the intensity, *i.e.* a loss of fibres, a decrease of the number of large myelinated nerve fibres, in agreement with the supposed pathogenesis described as a distal axonopathy (Cahill et al., 1986; Domange et al., 2010). However, in spite of these rare epidemiological and pathological data, the link between this horse disease and the toxicity of *Hypochoeris radicata* (HR) has been poorly investigated in spite of a recent study, which tended to reproduce the disease on animals after a 50-day HR treatment (9.8 kg HR/animal/day) (Araujo et al., 2008). The lack of investigation of such a disease is further partially explained by the critical approach of the nervous system, especially the peripheral nervous system and by the only target species. Besides, we need to consider ethical and financial issues. Moreover, as Araujo and colleagues underlined, the plant material is susceptible to differ in toxicity depending on

several factors, one being the geographical location (Araujo et al., 2008).

Most of the time, investigating a disease often requires a convenient laboratory animal model enabling to reproduce clinical symptoms, to access pharmacological data, to reveal some biomarkers of the disease and, in the best cases, to suggest some therapeutic treatments. Because of the nervous nature of Australian stringhalt, the fact that this illness was only described in target species, and the difficulties to link the supposed plant (more particularly if a specific secondary metabolite present in the plant would be involved) to the pathogenesis, the assessment of such an induced intoxication using a "classical" neurological approach seemed not efficient enough to reveal valuable biomarkers. Data obtained until recently remained too scarce. The "omics" approach, more particularly metabonomics, appears to be the most suitable mean to obtain some pertinent information about the target organs and candidate metabolic biomarkers by using an *a priori*

**4.2 Concept of orthology and interest in metabonomics** 

#### **4. Example of a metabonomic approach of a neurological horse disease, the Australian stringhalt or how to address a toxicological issue on a seemingly non-target species without referring to a known toxic molecule**

#### **4.1 Problem for studying such an animal disease**

Australian stringhalt is the name of a horse disease described since the middle of the 19th century in Australia (Robertson-Smith et al., 1985). It is defined as a syndrome characterized by an abnormal gait and an involuntary hyperflexion of both hind limbs during movement (Figure 3).

Fig. 3. Horses displaying clinical signs of Australian stringhalt (grade IV on the left, grade V on the right) (from (Collignon, 2007))

**4. Example of a metabonomic approach of a neurological horse disease, the Australian stringhalt or how to address a toxicological issue on a seemingly** 

Australian stringhalt is the name of a horse disease described since the middle of the 19th century in Australia (Robertson-Smith et al., 1985). It is defined as a syndrome characterized by an abnormal gait and an involuntary hyperflexion of both hind limbs during movement

Fig. 3. Horses displaying clinical signs of Australian stringhalt (grade IV on the left, grade V

on the right) (from (Collignon, 2007))

**non-target species without referring to a known toxic molecule** 

**4.1 Problem for studying such an animal disease** 

(Figure 3).

Since this time, several other outbreaks had been reported in many countries such as New Zealand (Cahill et al., 1985; Cahill et al., 1986; Cahill and Goulden, 1992), Chile (Araya et al., 1998), United States (Gay et al., 1993; Huntington et al., 1989; Robertson-Smith et al., 1985; Slocombe et al., 1992), Italy (Torre, 2005), Brasil (Araujo), more recently in France (Domange et al., 2010; Gouy et al., 2005) and were suspected in Japan (Takahashi et al., 2002). According to most of the authors, a plant of the Asteraceae family (formerly Compositeae family), *Hypochoeris radicata* L. also named cat's ear, flatweed or capeweed was suspected to be responsible for this disease (Araujo et al., 2008; Gardner et al., 2005; Gay et al., 1993; Gouy et al., 2005). This rosette-forming herb with a yellow terminal flower has a deep taproot, giving it resistance to drought. This explains a growth achieved preferentially on poorquality pastures after a prolonged dry period, mainly in late summer and early autumn. Such climatic conditions, associated with the aggressiveness and the dominance of *Hypochoeris radicata* L. on other species, enable it to colonize pastures and to become the major plant available as herbivore feeding. These favouring factors, in aggravation for many years because of the global change in climatic conditions, appeared particularly marked in 2003 in France, after a blistering and dry summer, leading to an epizooty with a few tens of recorded intoxicated horses (Domange et al., 2010; Gouy et al., 2005). These latter's showed a wide range of symptoms but mainly dominated by several severity degrees from grade I to grade V, (Huntington et al., 1989) with an involuntary exaggerated hyperflexion of hind limbs and a delayed extension of hocks during forward movement. A marked atrophy of the hind limbs musculature, especially in the distal muscles, is often associated with this gait in the most affected animals. Most of the time, this amyotrophy is related to neurological lesions of the hind limbs with a proximal-to-distal gradient in the intensity, *i.e.* a loss of fibres, a decrease of the number of large myelinated nerve fibres, in agreement with the supposed pathogenesis described as a distal axonopathy (Cahill et al., 1986; Domange et al., 2010). However, in spite of these rare epidemiological and pathological data, the link between this horse disease and the toxicity of *Hypochoeris radicata* (HR) has been poorly investigated in spite of a recent study, which tended to reproduce the disease on animals after a 50-day HR treatment (9.8 kg HR/animal/day) (Araujo et al., 2008). The lack of investigation of such a disease is further partially explained by the critical approach of the nervous system, especially the peripheral nervous system and by the only target species. Besides, we need to consider ethical and financial issues. Moreover, as Araujo and colleagues underlined, the plant material is susceptible to differ in toxicity depending on several factors, one being the geographical location (Araujo et al., 2008).

#### **4.2 Concept of orthology and interest in metabonomics**

Most of the time, investigating a disease often requires a convenient laboratory animal model enabling to reproduce clinical symptoms, to access pharmacological data, to reveal some biomarkers of the disease and, in the best cases, to suggest some therapeutic treatments. Because of the nervous nature of Australian stringhalt, the fact that this illness was only described in target species, and the difficulties to link the supposed plant (more particularly if a specific secondary metabolite present in the plant would be involved) to the pathogenesis, the assessment of such an induced intoxication using a "classical" neurological approach seemed not efficient enough to reveal valuable biomarkers. Data obtained until recently remained too scarce. The "omics" approach, more particularly metabonomics, appears to be the most suitable mean to obtain some pertinent information about the target organs and candidate metabolic biomarkers by using an *a priori*

Power of a Metabonomic Approach to Investigate an Unknown Nervous Disease 419

Fig. 4. LDA performed on 150 metabolic variables selected from fingerprints obtained by 1H NMR performed at 600.13 MHz on 332 urinary samples (from Domange et al., 2008). The dummy variable selected is the « group » factor. A 61.5% amount of the total metabolic information is projected on the factorial plan LD1 x LD2. Arrows stand for metabolic trajectories throughout the study followed by every group fed either a control or a 3 or 9% HR diet. Barycenters give the dates of urine collection and correspond to the duration of HR

Fig. 5. LDA performed on 20 variables filtered from 600.13 MHz 1H NMR data of brain aqueous extracts from male and female mice according to groups and in agreement with the two first components (from Domange, 2008). Barycenters give the date of brain collection and correspond to the duration of HR intoxication (d8, day 8; d15, day 15; d21, day 21)

intoxication (d8, day 8; d15, day 15; d21, day 21)

"metabolically competent" animal model. The orthologous hypothesis considered in the case of an induction of a metabolic disruption in a rodent animal model of another animal species, here horse species, is crucial in characterizing a set of candidate metabolic biomarkers. Even though clinical signs may strikingly differ between the two species, some metabolic similarities may exist between their metabolic networks, particularly in their ability to be similarly disrupted by one or few toxicants. Among these latter's, plant secondary metabolites, for which nothing is known at the chemical and pharmacological sides, can be studied.

#### **4.3 Use of complementary approaches: <sup>1</sup> H NMR-based metabonomics, MRI and behavioural tests**

#### **4.3.1 Metabolic fingerprints on biofluids and tissue extracts**

Using the orthologous metabolic disruption assumption existing between two species, horse and mouse in the present case, metabonomics was used to investigate at the metabolic side this orphan neurological disease, Australian stringhalt. The purpose was to combine it with MRI as published elsewhere (Griffith et al., 2007) and with behavioural tests to improve the functional understanding of the metabolic data. Based on the orthologous hypothesis previously mentioned, the mouse was chosen as a "metabolically competent" laboratory animal model of horse intoxicated by HR, even though this rodent model of exposure to HR does not display any observable clinical sign. In a first time, metabonomic studies using male and female C57BL/6J mice fed for 21 days a diet containing 3 or 9% HR had been performed (Domange et al., 2008). 1H NMR spectroscopy analyses have been done on weekly collected urine samples but also on tissue extracts prepared from liver and brain tissues collected at 0, 8, 15 and 21 days of treatment, after sacrifice of a subpopulation of the animals included in the experimental design. Urine and liver analyses were performed to detect the putative systemic disruption after the HR ingestion, and the brain analysis to access the nervous system disruption. All 1H NMR spectra were acquired at 300 K on a Bruker DRX-600 Avance NMR spectrometer operating at 600.13 MHz for 1H resonance frequency, using a cryoprobe and the 1D "Improved Watergate" sequence for suppression of water resonance. Multidimensional statistical analyses of fingerprint data were achieved on log-transformed variables. After removing redundant variables, linear discriminant analyses and partial least-squares regression-based discriminant analyses (PLS-DA) were performed on NMR data to maximize the groups' separation on a factorial map. Projection of these groups on every factorial axis enables to associate canonical 1H NMR variables to the axis construction revealing thus the respective influence of the different factors of interest (gender, intoxication duration, toxicant dose). Therefore, the main part of the metabolic information related to urine 1H NMR data and enabling the discrimination between the different groups of animals can be summed up in a factorial map (Figure 4). On this map, appears the temporal evolution between day 0 and day 21 of the metabolism of animals. This latter depends significantly on the gender of mice, through the 1st axis (this factor contains the main part of the variance explained by the statistical model used) and on the diet factor, through the second axis, covering from the bottom part of the factorial map diets without HR (named "control") to the middle part, diets with 3% HR (named "3%HR"), then to the top part, diets with 9% HR (named "9%HR"). By searching the first variables involved in the second axis construction, we reached the main metabolites, the concentration of which was influenced by a HR-induced metabolic disruption (Domange et al., 2008).

"metabolically competent" animal model. The orthologous hypothesis considered in the case of an induction of a metabolic disruption in a rodent animal model of another animal species, here horse species, is crucial in characterizing a set of candidate metabolic biomarkers. Even though clinical signs may strikingly differ between the two species, some metabolic similarities may exist between their metabolic networks, particularly in their ability to be similarly disrupted by one or few toxicants. Among these latter's, plant secondary metabolites, for which nothing is known at the chemical and pharmacological

Using the orthologous metabolic disruption assumption existing between two species, horse and mouse in the present case, metabonomics was used to investigate at the metabolic side this orphan neurological disease, Australian stringhalt. The purpose was to combine it with MRI as published elsewhere (Griffith et al., 2007) and with behavioural tests to improve the functional understanding of the metabolic data. Based on the orthologous hypothesis previously mentioned, the mouse was chosen as a "metabolically competent" laboratory animal model of horse intoxicated by HR, even though this rodent model of exposure to HR does not display any observable clinical sign. In a first time, metabonomic studies using male and female C57BL/6J mice fed for 21 days a diet containing 3 or 9% HR had been performed (Domange et al., 2008). 1H NMR spectroscopy analyses have been done on weekly collected urine samples but also on tissue extracts prepared from liver and brain tissues collected at 0, 8, 15 and 21 days of treatment, after sacrifice of a subpopulation of the animals included in the experimental design. Urine and liver analyses were performed to detect the putative systemic disruption after the HR ingestion, and the brain analysis to access the nervous system disruption. All 1H NMR spectra were acquired at 300 K on a Bruker DRX-600 Avance NMR spectrometer operating at 600.13 MHz for 1H resonance frequency, using a cryoprobe and the 1D "Improved Watergate" sequence for suppression of water resonance. Multidimensional statistical analyses of fingerprint data were achieved on log-transformed variables. After removing redundant variables, linear discriminant analyses and partial least-squares regression-based discriminant analyses (PLS-DA) were performed on NMR data to maximize the groups' separation on a factorial map. Projection of these groups on every factorial axis enables to associate canonical 1H NMR variables to the axis construction revealing thus the respective influence of the different factors of interest (gender, intoxication duration, toxicant dose). Therefore, the main part of the metabolic information related to urine 1H NMR data and enabling the discrimination between the different groups of animals can be summed up in a factorial map (Figure 4). On this map, appears the temporal evolution between day 0 and day 21 of the metabolism of animals. This latter depends significantly on the gender of mice, through the 1st axis (this factor contains the main part of the variance explained by the statistical model used) and on the diet factor, through the second axis, covering from the bottom part of the factorial map diets without HR (named "control") to the middle part, diets with 3% HR (named "3%HR"), then to the top part, diets with 9% HR (named "9%HR"). By searching the first variables involved in the second axis construction, we reached the main metabolites, the concentration of which was influenced by a HR-induced metabolic disruption (Domange et

**H NMR-based metabonomics, MRI and** 

sides, can be studied.

**behavioural tests** 

al., 2008).

**4.3 Use of complementary approaches: <sup>1</sup>**

**4.3.1 Metabolic fingerprints on biofluids and tissue extracts** 

Fig. 4. LDA performed on 150 metabolic variables selected from fingerprints obtained by 1H NMR performed at 600.13 MHz on 332 urinary samples (from Domange et al., 2008). The dummy variable selected is the « group » factor. A 61.5% amount of the total metabolic information is projected on the factorial plan LD1 x LD2. Arrows stand for metabolic trajectories throughout the study followed by every group fed either a control or a 3 or 9% HR diet. Barycenters give the dates of urine collection and correspond to the duration of HR intoxication (d8, day 8; d15, day 15; d21, day 21)

Fig. 5. LDA performed on 20 variables filtered from 600.13 MHz 1H NMR data of brain aqueous extracts from male and female mice according to groups and in agreement with the two first components (from Domange, 2008). Barycenters give the date of brain collection and correspond to the duration of HR intoxication (d8, day 8; d15, day 15; d21, day 21)

Power of a Metabonomic Approach to Investigate an Unknown Nervous Disease 421

Fig. 7. 600.13 MHz 1H NMR spectra from aqueous extracts of brain in 9% HR-treated mouse

and control mouse (from Domange, 2008)

In a same way, the main part of metabolic information contained in 1H NMR data characterizing liver and brain hydrosoluble extracts and enabling the discrimination between the different groups of animals during the experiment could be summed up into a more complex factorial map (Figure 5). Firstly, is displayed the temporal evolution of the brain metabolism of mice orally exposed or not to HR, which holds almost all the part of the variance explained by the statistical model with, respectively from the right to the left side, a projection of the cerebral metabolisms of control animals, then the 3%HR-treated mice, and finally, the 9%HR-treated ones (Domange, 2008). The factor "time" is clearly revealed through every HR treatment with, respectively, from the right to the left side, an emphasis of the disrupted metabolism in a given direction all along the experiment duration. Given the fact that the two matrices of 1H NMR fingerprinting data obtained on hydrosoluble brain and liver extracts were issued from the same individuals, a global correlation using a canonical analysis (PLS2 here) have been performed between them. A significant correlation between the two first PLS2 components has been revealed (Figure 6), in which, the gender factor is orthogonally projected to the diet one. Concerning the projection of variables involved in the variance calculation, *i.e.* the information explaining this construction, on the same plot, we can show that liver and brain 1H NMR fingerprint data display close

Fig. 6. Resulting biplot performed on the two first PLS2 components calculated between the hydrosoluble liver and brain extracts (from (Domange et al., 2008). Only the projection of the variables with contribution is above 0.5 is displayed (in grey for brain variables, in pink for the liver ones). Most of the variables containing the variance explained by the statistical model is spread according to the gender factor for the liver and according to the diet concerning the brain. The brain variable named B3.34 (arrow numbered 1) and the corresponding liver variable named L3.34 (arrow numbered 2) stand for the *scyllo-*inositol, detected at δ = 3.34 ppm. The brain variable named B3.60 (arrow numbered 3) and the corresponding liver variable named L3.60 (arrow numbered 4) stand for the *myo*-inositol detected at δ = 3.60 ppm

In a same way, the main part of metabolic information contained in 1H NMR data characterizing liver and brain hydrosoluble extracts and enabling the discrimination between the different groups of animals during the experiment could be summed up into a more complex factorial map (Figure 5). Firstly, is displayed the temporal evolution of the brain metabolism of mice orally exposed or not to HR, which holds almost all the part of the variance explained by the statistical model with, respectively from the right to the left side, a projection of the cerebral metabolisms of control animals, then the 3%HR-treated mice, and finally, the 9%HR-treated ones (Domange, 2008). The factor "time" is clearly revealed through every HR treatment with, respectively, from the right to the left side, an emphasis of the disrupted metabolism in a given direction all along the experiment duration. Given the fact that the two matrices of 1H NMR fingerprinting data obtained on hydrosoluble brain and liver extracts were issued from the same individuals, a global correlation using a canonical analysis (PLS2 here) have been performed between them. A significant correlation between the two first PLS2 components has been revealed (Figure 6), in which, the gender factor is orthogonally projected to the diet one. Concerning the projection of variables involved in the variance calculation, *i.e.* the information explaining this construction, on the same plot, we can show that liver and brain 1H NMR fingerprint data display close

Fig. 6. Resulting biplot performed on the two first PLS2 components calculated between the hydrosoluble liver and brain extracts (from (Domange et al., 2008). Only the projection of the variables with contribution is above 0.5 is displayed (in grey for brain variables, in pink for the liver ones). Most of the variables containing the variance explained by the statistical model is spread according to the gender factor for the liver and according to the diet concerning the brain. The brain variable named B3.34 (arrow numbered 1) and the

corresponding liver variable named L3.34 (arrow numbered 2) stand for the *scyllo-*inositol, detected at δ = 3.34 ppm. The brain variable named B3.60 (arrow numbered 3) and the corresponding liver variable named L3.60 (arrow numbered 4) stand for the *myo*-inositol

detected at δ = 3.60 ppm

Fig. 7. 600.13 MHz 1H NMR spectra from aqueous extracts of brain in 9% HR-treated mouse and control mouse (from Domange, 2008)

Power of a Metabonomic Approach to Investigate an Unknown Nervous Disease 423

one of the interests of metabonomic approach is to combine data generated by different techniques to get more powerful biomarkers, a PLS2-based canonical regression between the set of brain metabolites issued by *in vivo* MRI and the 1H NMR fingerprints of hydrosoluble brain extracts performed on the same animals has been obtained after correction of the two data sets by an OSC-PLS-driven correction procedure. The canonical analysis obtained by the PLS2 analysis between these two corrected data sets showed that the 1H NMR variable called B3.34, namely *scyllo*-inositol, was projected in the region where scores of HR-treated animals were also projected (Figure 9.c). Moreover, the relative contents of *N*-acetyl-aspartate (NAA), lactate and choline were increased (*p* < 10-5, *p* = 0.02 and *p* = 0.03, respectively) whereas the glutamine one was decreased (*p* = 0.04) in response to the 9% HR treatment. MRI studies were also conducted in poisoned living mice and corroborated the abnormal higher presence of *scyllo*-inositol in the thalamus of poisoned animals. Even this result was unable to explain the exact pathophysiological mechanism involved and the outset of the illness, it confirmed that *scyllo*-inositol was a biomarker of interest in the central nervous system, particularly when it is related to some brain metabolic disturbances (Griffith et al., 2007; Jenkins et al., 1993; Viola et al., 2004). The increase in NAA, which has been previously revealed following MRI and 1H NMR spectroscopy of hydrosoluble brain extracts was suggested to be linked to the enhanced locomotor activity observed in 9% HR-treated mice. Besides, NAA has been reported in epileptic seizures cases

This accumulation of NAA has also been shown in a rat model of the Canavan's disease, suggesting that NAA increase in brain should be linked to neuroexcitation and

The two previous exploratory studies led us to consider in more details the role of inositols in the development of the Australian stringhalt. The location of such metabolic disturbances, the current knowledge of the metabolism and the pathways involved, such as neurotransmission, signalling system and regulation of many cellular functions, depending on the balance between *scyllo* and *myo* inositol needed to be rounded out by a complementary functional assessment as a large extent behavioural testing of HR-treated animals can provide it. Indeed, the administration of inositol (*myo*-inositol) is used as a therapeutic molecule in depression (Einat et al., 1999), panic disorder, obsessive-compulsive disorder (Cohen et al., 1997; Levine, 1997). It partially explains an enhanced locomotion (Kofman et al., 1998) and may be linked to a putative anxiolytic effect (Kofman et al., 2000) with possible involvement of serotoninergic (5-HT2) receptors (Einat et al., 2001). Therefore, to investigate the functional consequences of such previous disrupted metabolic events, various behavioural aspects of C57BL/6J mice orally exposed to 9% HR for 3 weeks were performed in parallel with the 1H NMR metabolomic exploration of the brain. Several behavioural tests related to locomotor activity (open-field test), motor coordination (Locotronic® apparatus, Wespoc test), learning and memory [Y maze, (Hughes, 2004), Figure 10.a and Morris water maze], anxiety [elevated plus maze, (Rodgers and Johnson, 1995), hole board (do-Rego et al., 2006; Takeda et al., 1998), Figure 10.b], and depression forced swimming test or test of Porsolt (Porsolt et al., 1977; Porsolt et al., 1979), social interaction (resident/intruder model), and addiction (place preference test) were carried out (Domange

(Akimitsu et al., 2000).

**4.3.3 Behavioural testing** 

et al., submitted).

neurodegeneration (Kitada et al., 2000).

information. Among the main variables involved in the segregation of the groups of animals, *i.e.* which are related to the HR-treatment factor, and whatever the biological matrix analysed, the first identified variables correspond to the chemical shifts of the *scyllo*inositol (δ = 3.35 or 3.36 ppm in urine and in liver extract fingerprints, δ = 3.34 ppm in cerebral and liver fingerprints), which are positively correlated to HR-treatment, when the *myo*-inositol ones (δ = 3.60 ppm) are negatively correlated to HR-treatment (Figures 7 and 8). Moreover, the comparison between 1H-NMR metabolic fingerprints in control and HR-fed laboratory animals revealed a dose-dependent increase of the ratio *scyllo*-inositol/*myo*inositol in urine, plasma, and hydrosoluble extracts of liver and brain of the HR-treated animals, enabling us to reveal some putative candidate metabolic biomarker(s) even though no aetiological factor was characterized, and no requirement of the target species was performed in this toxicological exploration.

Fig. 8. Loading plot from O-PLS models performed from the aqueous extracts of brain in 9% HR-treated mice

#### **4.3.2 Magnetic resonance imaging**

To get access *in situ* to some potent cerebral metabolic changes thanks to a second spectroscopic technique, 1H NMR localized spectroscopy, six male mice given a 9% HR diet and six control mice were used for *in vivo* metabolite quantification. All experiments were performed at 9.4 T on a Bruker Avance DRX 400 microimaging system with a wide-bore vertical magnet and a Micro 2.5 gradient system (Bruker Ettlingen, Germany). Because a preliminary experiment performed on a spectroscopic volume of interest (VOI) positioned in the cortex of mice was inconclusive, spectra have been performed from 1H NMR data acquired by *in vivo* MRI using a VOI positioned in the thalamus of control and 9% HR-treated male mice (Figure 9.a). In this region, only the 9% HR-treated mice displayed a significant although minor signal found at δ = 3.34 ppm corresponding to *scyllo*-inositol (Figure 9.b). A one-way ANOVA performed for every other identified variables quantified at the same time from raw integrated spectra coming from *in vivo* MRI enables us to give significant results only for *scyllo*-inositol (p = 0.0013). As it could be described above, and as one of the interests of metabonomic approach is to combine data generated by different techniques to get more powerful biomarkers, a PLS2-based canonical regression between the set of brain metabolites issued by *in vivo* MRI and the 1H NMR fingerprints of hydrosoluble brain extracts performed on the same animals has been obtained after correction of the two data sets by an OSC-PLS-driven correction procedure. The canonical analysis obtained by the PLS2 analysis between these two corrected data sets showed that the 1H NMR variable called B3.34, namely *scyllo*-inositol, was projected in the region where scores of HR-treated animals were also projected (Figure 9.c). Moreover, the relative contents of *N*-acetyl-aspartate (NAA), lactate and choline were increased (*p* < 10-5, *p* = 0.02 and *p* = 0.03, respectively) whereas the glutamine one was decreased (*p* = 0.04) in response to the 9% HR treatment. MRI studies were also conducted in poisoned living mice and corroborated the abnormal higher presence of *scyllo*-inositol in the thalamus of poisoned animals. Even this result was unable to explain the exact pathophysiological mechanism involved and the outset of the illness, it confirmed that *scyllo*-inositol was a biomarker of interest in the central nervous system, particularly when it is related to some brain metabolic disturbances (Griffith et al., 2007; Jenkins et al., 1993; Viola et al., 2004). The increase in NAA, which has been previously revealed following MRI and 1H NMR spectroscopy of hydrosoluble brain extracts was suggested to be linked to the enhanced locomotor activity observed in 9% HR-treated mice. Besides, NAA has been reported in epileptic seizures cases (Akimitsu et al., 2000).

This accumulation of NAA has also been shown in a rat model of the Canavan's disease, suggesting that NAA increase in brain should be linked to neuroexcitation and neurodegeneration (Kitada et al., 2000).

#### **4.3.3 Behavioural testing**

422 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

information. Among the main variables involved in the segregation of the groups of animals, *i.e.* which are related to the HR-treatment factor, and whatever the biological matrix analysed, the first identified variables correspond to the chemical shifts of the *scyllo*inositol (δ = 3.35 or 3.36 ppm in urine and in liver extract fingerprints, δ = 3.34 ppm in cerebral and liver fingerprints), which are positively correlated to HR-treatment, when the *myo*-inositol ones (δ = 3.60 ppm) are negatively correlated to HR-treatment (Figures 7 and 8). Moreover, the comparison between 1H-NMR metabolic fingerprints in control and HR-fed laboratory animals revealed a dose-dependent increase of the ratio *scyllo*-inositol/*myo*inositol in urine, plasma, and hydrosoluble extracts of liver and brain of the HR-treated animals, enabling us to reveal some putative candidate metabolic biomarker(s) even though no aetiological factor was characterized, and no requirement of the target species was

Fig. 8. Loading plot from O-PLS models performed from the aqueous extracts of brain in 9%

To get access *in situ* to some potent cerebral metabolic changes thanks to a second spectroscopic technique, 1H NMR localized spectroscopy, six male mice given a 9% HR diet and six control mice were used for *in vivo* metabolite quantification. All experiments were performed at 9.4 T on a Bruker Avance DRX 400 microimaging system with a wide-bore vertical magnet and a Micro 2.5 gradient system (Bruker Ettlingen, Germany). Because a preliminary experiment performed on a spectroscopic volume of interest (VOI) positioned in the cortex of mice was inconclusive, spectra have been performed from 1H NMR data acquired by *in vivo* MRI using a VOI positioned in the thalamus of control and 9% HR-treated male mice (Figure 9.a). In this region, only the 9% HR-treated mice displayed a significant although minor signal found at δ = 3.34 ppm corresponding to *scyllo*-inositol (Figure 9.b). A one-way ANOVA performed for every other identified variables quantified at the same time from raw integrated spectra coming from *in vivo* MRI enables us to give significant results only for *scyllo*-inositol (p = 0.0013). As it could be described above, and as

performed in this toxicological exploration.

HR-treated mice

**4.3.2 Magnetic resonance imaging** 

The two previous exploratory studies led us to consider in more details the role of inositols in the development of the Australian stringhalt. The location of such metabolic disturbances, the current knowledge of the metabolism and the pathways involved, such as neurotransmission, signalling system and regulation of many cellular functions, depending on the balance between *scyllo* and *myo* inositol needed to be rounded out by a complementary functional assessment as a large extent behavioural testing of HR-treated animals can provide it. Indeed, the administration of inositol (*myo*-inositol) is used as a therapeutic molecule in depression (Einat et al., 1999), panic disorder, obsessive-compulsive disorder (Cohen et al., 1997; Levine, 1997). It partially explains an enhanced locomotion (Kofman et al., 1998) and may be linked to a putative anxiolytic effect (Kofman et al., 2000) with possible involvement of serotoninergic (5-HT2) receptors (Einat et al., 2001). Therefore, to investigate the functional consequences of such previous disrupted metabolic events, various behavioural aspects of C57BL/6J mice orally exposed to 9% HR for 3 weeks were performed in parallel with the 1H NMR metabolomic exploration of the brain. Several behavioural tests related to locomotor activity (open-field test), motor coordination (Locotronic® apparatus, Wespoc test), learning and memory [Y maze, (Hughes, 2004), Figure 10.a and Morris water maze], anxiety [elevated plus maze, (Rodgers and Johnson, 1995), hole board (do-Rego et al., 2006; Takeda et al., 1998), Figure 10.b], and depression forced swimming test or test of Porsolt (Porsolt et al., 1977; Porsolt et al., 1979), social interaction (resident/intruder model), and addiction (place preference test) were carried out (Domange et al., submitted).

Power of a Metabonomic Approach to Investigate an Unknown Nervous Disease 425

visualize a depression state (from Domange et al., submitted)

stringhalt in a seemingly metabolically orthologous murine species.

**5. Conclusion** 

 (a) (b) (c) Fig. 10. Examples of behavioural tests used in mice: a) Y-maze or Y-maze spontaneous alternation that estimates the immediate working memory performance. b) Hole board test that evaluates the exploratory rate and the anxiety level. c) Forced swimming test defined to

Although the lack of motor coordination impairment is commonly observed in the sick horses, 9% HR-treated mice displayed a motor hyperactivity, which is reflected by the decrease of immobility time in the forced swimming test, and the increased numbers of head dipping in the hole board test, of arms visited in Y-maze and of the number of entries in the upper quarter of the maze in the Morris water maze (Domange et al., submitted). This increased activity of treated mice, which is clearly observable at the end of tests, could be linked to a decrease in the resignation state or an enhanced motivation. Moreover, the 9% HR-contaminated mice seem to be addicted to the plant as indicated by results obtained in the place preference test. A regularized canonical analyses performed using mixOmics, an R package (Le Cao et al., 2009) to establish a canonical link between the two multidimensional data sets, *i.e.* the one containing the 1H NMR fingerprints of hydrosoluble brain extracts and the one corresponding to the behavioural data set, which comprises nearly 100 variables, has revealed a clear relationship between some behavioural impairment variables (the motor hyperactivity and the addiction for the plant) and the main metabolic disruptions, *i.e.* the increase in *scyllo*-inositol in the brain of HR-treated mice and the relative decrease in *myo*-inositol. These results underlie the interest of such a dual and combined approach to characterize the functional end-points of a pathophysiological model of the horse Australian

In this chapter, we underlined the interest of "omics" approaches and their recent introduction in the field of neuro-toxicological research. Indeed, metabonomics can especially be considered as a potentially powerful mean to explore the subclinical disruptions of an organism before the outset of clinical signs, and would particularly be useful in discovery markers of disease risk. This approach would help to prevent some risks in spite of the difficulty to detect some minor metabolites or molecules in tiny doses or mixtures, with the ability to access and explore some isolated and intricate tissues (like brain) *via* the general metabolism (urine, plasma) and to link statistically these subclinical metabolic changes with complementary data coming from other phenotyping approaches and across multiple physiological levels. Besides, these combined techniques have been

Fig. 9. a) MRI performed at 9.4 T on a Bruker Avance DRX 400 microimaging system positioned in the thalamus region with an *in vivo* parallel metabolite quantification using 1H NMR localized spectroscopy (VOI, 12 mm3). b) Spectrum comparison between the sum of six 1H NMR spectra acquired on control male mice and the sum of six 1H NMR spectra acquired on 9%HR-treated male mice with the presence of *scyllo*-inositol (chemical shift detected at δ = 3.34 ppm). c) PLS2 between MRI quantitative data and 1H NMR data. A loading projection is given for metabonomic variables having a norm above 0.5 (pale blue circle) or above 0.75 (pale green circle). The purple and the dark-blue ellipses, respectively, correspond to the scores of control and 9% HR-treated mice. Among the main MRI loadings having a positive correlation with HR treatment are *scyllo*-inositol (*s*-Ins), *N*-acetyl-aspartate, lactate and choline. For MRI variables having a negative correlation with HR treatment are glutamate (Glu.2, second chemical shift) and glutamine (Gln.2, second chemical shift). Uninformative MRI loadings: *myo*-inositol (*m*-Ins), glutamate, first chemical shift (Glu.1), glutamine, first chemical shift (Gln.1), GABA, taurine and unknown 1 are projected in the centre of the biplot (from Domange et al., 2008)

Fig. 10. Examples of behavioural tests used in mice: a) Y-maze or Y-maze spontaneous alternation that estimates the immediate working memory performance. b) Hole board test that evaluates the exploratory rate and the anxiety level. c) Forced swimming test defined to visualize a depression state (from Domange et al., submitted)

Although the lack of motor coordination impairment is commonly observed in the sick horses, 9% HR-treated mice displayed a motor hyperactivity, which is reflected by the decrease of immobility time in the forced swimming test, and the increased numbers of head dipping in the hole board test, of arms visited in Y-maze and of the number of entries in the upper quarter of the maze in the Morris water maze (Domange et al., submitted). This increased activity of treated mice, which is clearly observable at the end of tests, could be linked to a decrease in the resignation state or an enhanced motivation. Moreover, the 9% HR-contaminated mice seem to be addicted to the plant as indicated by results obtained in the place preference test. A regularized canonical analyses performed using mixOmics, an R package (Le Cao et al., 2009) to establish a canonical link between the two multidimensional data sets, *i.e.* the one containing the 1H NMR fingerprints of hydrosoluble brain extracts and the one corresponding to the behavioural data set, which comprises nearly 100 variables, has revealed a clear relationship between some behavioural impairment variables (the motor hyperactivity and the addiction for the plant) and the main metabolic disruptions, *i.e.* the increase in *scyllo*-inositol in the brain of HR-treated mice and the relative decrease in *myo*-inositol. These results underlie the interest of such a dual and combined approach to characterize the functional end-points of a pathophysiological model of the horse Australian stringhalt in a seemingly metabolically orthologous murine species.

#### **5. Conclusion**

424 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

Fig. 9. a) MRI performed at 9.4 T on a Bruker Avance DRX 400 microimaging system positioned in the thalamus region with an *in vivo* parallel metabolite quantification using 1H NMR localized spectroscopy (VOI, 12 mm3). b) Spectrum comparison between the sum of six 1H NMR spectra acquired on control male mice and the sum of six 1H NMR spectra acquired on 9%HR-treated male mice with the presence of *scyllo*-inositol (chemical shift detected at δ = 3.34 ppm). c) PLS2 between MRI quantitative data and 1H NMR data. A loading projection is given for metabonomic variables having a norm above 0.5 (pale blue circle) or above 0.75 (pale green circle). The purple and the dark-blue ellipses, respectively, correspond to the scores of control and 9% HR-treated mice. Among the main MRI loadings having a positive correlation with HR treatment are *scyllo*-inositol (*s*-Ins), *N*-acetyl-aspartate, lactate and choline. For MRI variables having a negative correlation with HR treatment are glutamate (Glu.2, second chemical shift) and glutamine (Gln.2, second chemical shift). Uninformative MRI loadings: *myo*-inositol (*m*-Ins), glutamate, first chemical shift (Glu.1), glutamine, first chemical shift (Gln.1), GABA, taurine and unknown 1 are projected in the

centre of the biplot (from Domange et al., 2008)

In this chapter, we underlined the interest of "omics" approaches and their recent introduction in the field of neuro-toxicological research. Indeed, metabonomics can especially be considered as a potentially powerful mean to explore the subclinical disruptions of an organism before the outset of clinical signs, and would particularly be useful in discovery markers of disease risk. This approach would help to prevent some risks in spite of the difficulty to detect some minor metabolites or molecules in tiny doses or mixtures, with the ability to access and explore some isolated and intricate tissues (like brain) *via* the general metabolism (urine, plasma) and to link statistically these subclinical metabolic changes with complementary data coming from other phenotyping approaches and across multiple physiological levels. Besides, these combined techniques have been

Power of a Metabonomic Approach to Investigate an Unknown Nervous Disease 427

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#### **6. Acknowledgment**

We gratefully acknowledge the excellent technical support and the contribution in the animal experimentation of Florence Blas Y Estrada and Raymond Gazel (INRA Toulouse, INP, UMR1331 ToxAlim) during metabonomic studies, Amidou Traoré, Guy Biélicki and Cécile Keller (INRA Clermont-Ferrand/Theix, QuaPA STIM, F-63122 St Genès Champanelle) during MRI experiments and Nicolas Violle and Julie Peiffer (UR AFPA, INRA UC340, Nancy University) during behavioural tests.

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**Part 6** 

**Prevention, Protection and** 

**Monitoring of Neurodegeneration** 


## **Part 6**

**Prevention, Protection and Monitoring of Neurodegeneration** 

432 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

Wang, T.J., Larson, M.G., Vasan, R.S., Cheng, S., Rhee, E.P., McCabe, E., Lewis, G.D., Fox,

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**18** 

*Israel* 

**Extract of** *Achillea fragrantissima*

**Oxidative-Stress-Induced Cell Death** 

Hilla Erlank1, Miriam Rindner1, Rivka Ofir2 and Elie Beit-Yannai3

*2Dead Sea & Arava Science Center and Department of Microbiology & Immunology* 

Oxidative damage plays a pivotal role in the initiation and progress of many human diseases and also in the development of acute and chronic pathological conditions in brain tissue (Halliwell, 2006; Hyslop et al., 1995; Ischiropoulos & Beckman, 2003; Minghetti, 2005). Compared with other tissues, the brain is particularly vulnerable to oxidative damage due to its high rate of oxygen utilization and high contents of oxidizable polyunsaturated fatty acids (Floyd, 1999; Sastry, 1985). In addition, certain regions of the brain are highly enriched in iron, a metal that is catalytically involved in the production of damaging reactive oxygen species (ROS) (Hallgren & Sourander, 1958). Although ROS are critical intracellular signaling messengers (Schrecka & Baeuerlea, 1991), excess of free radicals may lead to peroxidative impairment of membrane lipids and, consequently, to disruption of neuronal functions, and apoptosis. Among the ROS that are responsible for oxidative stress, H2O2 is thought to be the major precursor of highly reactive free radicals, and is regarded as a key factor in both neuronal (Vaudry et al., 2002) and astroglial cell death (Ferrero-Gutierrez et al., 2008). H2O2 is normally produced in reactions predominantly catalyzed by superoxide dismutase (SOD) and monoaminoxidases (MAO) A and B in the brain (Almeida et al., 2006; Duarte et al., 2007). As with both Ca2+ and NO, H2O2 appears to play contradictory roles, in that it is potentially toxic at high concentrations, even though it is a central signaling compound at low concentrations (Miura et al., 2002). Brain cells have the capacity to produce peroxides, particularly H2O2, in large amounts (Dringen et al., 2005). Excess of H2O2 accumulates during brain injuries and neurodegenerative diseases, and can cross cell membranes to elicit its biological effects intracellularly (Bienert et al., 2006). Although H2O2 is generally poorly reactive, it forms highly toxic hydroxyl radicals, which may damage all

**1. Introduction** 

Anat Elmann1, Alona Telerman1, Sharon Mordechay1,

*3Department of Clinical Pharmacology, Faculty of Health Sciences,* 

**Downregulates ROS Production** 

**and Protects Astrocytes from** 

*1Department of Food Science, Volcani Center, Agricultural Research Organization, Bet Dagan,* 

*Ben-Gurion University of the Negev, Beer-Sheva,* 

*Ben-Gurion University of the Negev, Beer-Sheva,* 

### **Extract of** *Achillea fragrantissima* **Downregulates ROS Production and Protects Astrocytes from Oxidative-Stress-Induced Cell Death**

Anat Elmann1, Alona Telerman1, Sharon Mordechay1, Hilla Erlank1, Miriam Rindner1, Rivka Ofir2 and Elie Beit-Yannai3 *1Department of Food Science, Volcani Center, Agricultural Research Organization, Bet Dagan, 2Dead Sea & Arava Science Center and Department of Microbiology & Immunology Ben-Gurion University of the Negev, Beer-Sheva, 3Department of Clinical Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel* 

#### **1. Introduction**

Oxidative damage plays a pivotal role in the initiation and progress of many human diseases and also in the development of acute and chronic pathological conditions in brain tissue (Halliwell, 2006; Hyslop et al., 1995; Ischiropoulos & Beckman, 2003; Minghetti, 2005). Compared with other tissues, the brain is particularly vulnerable to oxidative damage due to its high rate of oxygen utilization and high contents of oxidizable polyunsaturated fatty acids (Floyd, 1999; Sastry, 1985). In addition, certain regions of the brain are highly enriched in iron, a metal that is catalytically involved in the production of damaging reactive oxygen species (ROS) (Hallgren & Sourander, 1958). Although ROS are critical intracellular signaling messengers (Schrecka & Baeuerlea, 1991), excess of free radicals may lead to peroxidative impairment of membrane lipids and, consequently, to disruption of neuronal functions, and apoptosis. Among the ROS that are responsible for oxidative stress, H2O2 is thought to be the major precursor of highly reactive free radicals, and is regarded as a key factor in both neuronal (Vaudry et al., 2002) and astroglial cell death (Ferrero-Gutierrez et al., 2008). H2O2 is normally produced in reactions predominantly catalyzed by superoxide dismutase (SOD) and monoaminoxidases (MAO) A and B in the brain (Almeida et al., 2006; Duarte et al., 2007). As with both Ca2+ and NO, H2O2 appears to play contradictory roles, in that it is potentially toxic at high concentrations, even though it is a central signaling compound at low concentrations (Miura et al., 2002). Brain cells have the capacity to produce peroxides, particularly H2O2, in large amounts (Dringen et al., 2005). Excess of H2O2 accumulates during brain injuries and neurodegenerative diseases, and can cross cell membranes to elicit its biological effects intracellularly (Bienert et al., 2006). Although H2O2 is generally poorly reactive, it forms highly toxic hydroxyl radicals, which may damage all

Extract of *Achillea fragrantissima* Downregulates ROS Production and Protects Astrocytes from Oxidative-Stress-Induced Cell Death

activities of this plant extract.

**2. Materials and methods** 

Co. (St Louis, MO, USA).

**2.2 Preparation of** *Af* **Extracts** 

**2.1 Reagents** 

1987), and for the treatment of gastrointestinal disturbances (Segal et al., 1987). The ingredient responsible for the anti-spasmolytic activity was found to be a flavone aglycone named cirsiliol (5,3',4'-trihydroxy-6,7-dimethoxyflavone) that was shown to antagonize the spasmodic effects, inhibit Ca2+ influx and stimulate Ca2+ release from intracellular stores (Mustafa et al., 1992). In addition, the hydro-alcoholic extract of *Af* was shown to have a remarkable antiviral activity against poliomyelitis-1 virus (Soltan & Zaki, 2009). However, the effects of *Af* in the context of brain injuries and neurodegenerative diseases, have not been studied to date. In a recent study we have found that the ethanolic extract of *Achillea fragrantissima* inhibited lipopolysaccharide (LPS) –induced nitric oxide (NO) production by activated primary microglial cells. This extract also inhibited LPS - elicited expression of the pro-inflammatory cytokines interleukin1β (IL-1β) and tumor necrosis factor-α (TNFα), as well as expression of the proinflammatory enzymes, cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS) by these cells (in preparation). Since oxidative stress has become accepted as a suitable target for early therapeutic intervention in brain injuries and neurodegenerative diseases, the present study addressed the astroprotective and antioxidant

Dulbecco's modified Eagle's medium (DMEM), Leibovitz-15 medium, glutamine, antibiotics (10,000 IU/ml penicillin and 10,000 μg/ml streptomycin), soybean trypsin inhibitor, fetal bovine serum (FBS) and Dulbecco's phosphate buffered saline (PBS) (without calcium and magnesium) were purchased from Biological Industries (Beit Haemek, Israel); dimethyl sulfoxide (DMSO) was obtained from Applichem (Darmstadt, Germany); Hydrogen peroxide was obtained from MP Biomedicals (Ohio, USA); 2,2'-Azobis(amidinopropane) (ABAP) was obtained from Wako chemicals (Richmond, VA), and other chemicals including ZnCl2 and 2'7'-dichlorofluorescein diacetate (DCF) were purchased from Sigma Chemical

The plant was collected in the Arava Valley and authenticated. The voucher specimens have been kept in as part of the Arava Rift Valley Plant Collection; VPC (Dead Sea & Arava Science Center, Central Arava Branch, Israel, http://www.deadseaarava-rd.co.il/) under the accession code AVPC0040. Freshly collected plants were dried at 40 °C for three days and extracted in ethanol (96%). The liquid phase was then evaporated off, and the dry material was dissolved in DMSO to a concentration of 100 mg/ml to produce the *Af* extract.

The ethanolic extract of *Af* was subjected to HPLC chromatography. Separation was made using reverse phase column (Betasil C-18, 5 μm, 250 × 0.46 mm; Thermo-Hypersil, UK) by gradient elution with water-acetic acid (97 : 3 V/V) and methanol as described previously

The ethanolic extract of *Af* was subjected to MS/MS (Fig. 2). The mass spectra were performed on a liquid chromatography–mass spectrometry (LC–MS) Agilent 1100LC series

(Chen et al., 2010), and detection at 360 nm (Blue line) and 280 nm (Red line) (Fig. 1).

**2.3 High performance liquid chromatography (HPLC) conditions** 

**2.4 Liquid chromatography–mass spectrometry (LC–MS) conditions** 

437

the major classes of biological macromolecules in the cell, through iron- or copper ionmediated oxidation of lipids, proteins, and nucleic acids. This capability can partly account for H2O2-mediated neuronal and glial cell death. H2O2 also induces differential protein activation, which indicates varied biological effects of this molecule. In the mammalian central nervous system (CNS), the transition metal zinc is an endogenous molecule that is localized exclusively to the synaptic vesicles of glutamatergic neurons and that has a special role in modulating synaptic transmission. Chelatable zinc is released into the synaptic cleft with the neurotransmitter during neuronal execution (Assaf & Chung, 1984), and under normal circumstances the robust release of zinc is transient and is efficiently cleared from the synaptic cleft to ensure the performance of successive stimuli. However, in pathological conditions, zinc dyshomeostasis, with consequently elevated levels of extracellular zinc has been recognized as an important factor in the resulting neuropathology (Choi & Koh, 1998; Cote et al., 2005; Li et al., 2009). In neurotransmission, the amount of zinc in the synaptic cleft is in the 10- to 30-μM range, but in pathological conditions that involve sustained neuronal depolarization, e.g., ischemia, stroke, or traumatic brain injury, the levels of extracellular zinc can increase to 100- to 400-μM, at which it can contribute to the resulting neuropathology (Frederickson et al., 2005; Li et al., 2001). *In vivo* and *in vitro* studies showed that, at concentrations that can be reached in the mammalian CNS during excitotoxic episodes, injuries or diseases, zinc is toxic to both neurons and astrocytes (Bishop et al., 2007; Hwang et al., 2008; Kim et al., 1999a; Kim et al., 1999b; Koh et al., 1996; Ryu et al., 2002; Sheline et al., 2000; Stork & Li, 2009). Zinc induces oxidative stress and ROS production, which contribute to both glial cell death (Ryu et al., 2002) and neuronal cell death (Kim et al. 1999a; Kim et al. 1999b). Zinc decreased the GSH content of primary cultures of astrocytes (Kim et al., 2003; Ryu et al., 2002), increased their GSSG content (Kim et al., 2003) and inhibited glutathione reductase activity in these cells (Bishop et al., 2007); furthermore, it slowed the clearance of exogenous H2O2 by astrocytes, and promoted intracellular production of ROS (Bishop et al., 2007). Thus, ROS generation, glutathione depletion and mitochondrial dysfunction may be key factors in ZnCl2–induced glial toxicity (Ryu et al., 2002). Astrocytes are the most abundant glial cell type in the brain. They play important roles in maintenance of homeostasis, in provision of metabolic substrates for neurons, and also in coupling cerebral blood flow to neuronal activity. They are prominent in protecting neurons against oxidative stress and cell death, and in providing trophic supports such as the glial cell-line-derived neurotrophic factor (GDNF) (Sandhu et al., 2009). There is evidence that dysfunctional astrocytes can enhance neuronal degeneration by diminishing secretion of trophic factors (Takuma et al., 2004). The study of astrocytes is particularly important, in light of the co- existence of apoptotic death of neurons and astrocytes in damaged brains affected by ischemia and neurodegenerative diseases. Despite their high antioxidative activities, astrocytes exhibit a high degree of vulnerability, and are not resistant to the effects of ROS. They respond to substantial or sustained oxidative stress with increased intracellular Ca2+, loss of mitochondrial potential, and decreased oxidative phosphorylation (Robb et al., 1999). Since astrocytes determine the brain's vulnerability to oxidative injury, and form a tight functional unit with neurons, once astrocyte energy metabolism and antioxidant capacity are impaired, astrocytic death may critically impair neuronal survival (Feeney et al., 2008; Lu et al., 2008). Thus, protection of astrocytes from oxidative insult appears essential to brain function maintenance. Many herb and plant extracts are used as folk medicines for various kinds of diseases and organ dysfunctions. *Achillea fragrantissima* (*Af;* Asteraceae) is a desert plant that for many years has been used as a hypoglycemic medicinal plant in traditional medicine in the Arabian region (Yaniv et al.,

1987), and for the treatment of gastrointestinal disturbances (Segal et al., 1987). The ingredient responsible for the anti-spasmolytic activity was found to be a flavone aglycone named cirsiliol (5,3',4'-trihydroxy-6,7-dimethoxyflavone) that was shown to antagonize the spasmodic effects, inhibit Ca2+ influx and stimulate Ca2+ release from intracellular stores (Mustafa et al., 1992). In addition, the hydro-alcoholic extract of *Af* was shown to have a remarkable antiviral activity against poliomyelitis-1 virus (Soltan & Zaki, 2009). However, the effects of *Af* in the context of brain injuries and neurodegenerative diseases, have not been studied to date. In a recent study we have found that the ethanolic extract of *Achillea fragrantissima* inhibited lipopolysaccharide (LPS) –induced nitric oxide (NO) production by activated primary microglial cells. This extract also inhibited LPS - elicited expression of the pro-inflammatory cytokines interleukin1β (IL-1β) and tumor necrosis factor-α (TNFα), as well as expression of the proinflammatory enzymes, cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS) by these cells (in preparation). Since oxidative stress has become accepted as a suitable target for early therapeutic intervention in brain injuries and neurodegenerative diseases, the present study addressed the astroprotective and antioxidant activities of this plant extract.

#### **2. Materials and methods**

#### **2.1 Reagents**

Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring 436

the major classes of biological macromolecules in the cell, through iron- or copper ionmediated oxidation of lipids, proteins, and nucleic acids. This capability can partly account for H2O2-mediated neuronal and glial cell death. H2O2 also induces differential protein activation, which indicates varied biological effects of this molecule. In the mammalian central nervous system (CNS), the transition metal zinc is an endogenous molecule that is localized exclusively to the synaptic vesicles of glutamatergic neurons and that has a special role in modulating synaptic transmission. Chelatable zinc is released into the synaptic cleft with the neurotransmitter during neuronal execution (Assaf & Chung, 1984), and under normal circumstances the robust release of zinc is transient and is efficiently cleared from the synaptic cleft to ensure the performance of successive stimuli. However, in pathological conditions, zinc dyshomeostasis, with consequently elevated levels of extracellular zinc has been recognized as an important factor in the resulting neuropathology (Choi & Koh, 1998; Cote et al., 2005; Li et al., 2009). In neurotransmission, the amount of zinc in the synaptic cleft is in the 10- to 30-μM range, but in pathological conditions that involve sustained neuronal depolarization, e.g., ischemia, stroke, or traumatic brain injury, the levels of extracellular zinc can increase to 100- to 400-μM, at which it can contribute to the resulting neuropathology (Frederickson et al., 2005; Li et al., 2001). *In vivo* and *in vitro* studies showed that, at concentrations that can be reached in the mammalian CNS during excitotoxic episodes, injuries or diseases, zinc is toxic to both neurons and astrocytes (Bishop et al., 2007; Hwang et al., 2008; Kim et al., 1999a; Kim et al., 1999b; Koh et al., 1996; Ryu et al., 2002; Sheline et al., 2000; Stork & Li, 2009). Zinc induces oxidative stress and ROS production, which contribute to both glial cell death (Ryu et al., 2002) and neuronal cell death (Kim et al. 1999a; Kim et al. 1999b). Zinc decreased the GSH content of primary cultures of astrocytes (Kim et al., 2003; Ryu et al., 2002), increased their GSSG content (Kim et al., 2003) and inhibited glutathione reductase activity in these cells (Bishop et al., 2007); furthermore, it slowed the clearance of exogenous H2O2 by astrocytes, and promoted intracellular production of ROS (Bishop et al., 2007). Thus, ROS generation, glutathione depletion and mitochondrial dysfunction may be key factors in ZnCl2–induced glial toxicity (Ryu et al., 2002). Astrocytes are the most abundant glial cell type in the brain. They play important roles in maintenance of homeostasis, in provision of metabolic substrates for neurons, and also in coupling cerebral blood flow to neuronal activity. They are prominent in protecting neurons against oxidative stress and cell death, and in providing trophic supports such as the glial cell-line-derived neurotrophic factor (GDNF) (Sandhu et al., 2009). There is evidence that dysfunctional astrocytes can enhance neuronal degeneration by diminishing secretion of trophic factors (Takuma et al., 2004). The study of astrocytes is particularly important, in light of the co- existence of apoptotic death of neurons and astrocytes in damaged brains affected by ischemia and neurodegenerative diseases. Despite their high antioxidative activities, astrocytes exhibit a high degree of vulnerability, and are not resistant to the effects of ROS. They respond to substantial or sustained oxidative stress with increased intracellular Ca2+, loss of mitochondrial potential, and decreased oxidative phosphorylation (Robb et al., 1999). Since astrocytes determine the brain's vulnerability to oxidative injury, and form a tight functional unit with neurons, once astrocyte energy metabolism and antioxidant capacity are impaired, astrocytic death may critically impair neuronal survival (Feeney et al., 2008; Lu et al., 2008). Thus, protection of astrocytes from oxidative insult appears essential to brain function maintenance. Many herb and plant extracts are used as folk medicines for various kinds of diseases and organ dysfunctions. *Achillea fragrantissima* (*Af;* Asteraceae) is a desert plant that for many years has been used as a hypoglycemic medicinal plant in traditional medicine in the Arabian region (Yaniv et al.,

Dulbecco's modified Eagle's medium (DMEM), Leibovitz-15 medium, glutamine, antibiotics (10,000 IU/ml penicillin and 10,000 μg/ml streptomycin), soybean trypsin inhibitor, fetal bovine serum (FBS) and Dulbecco's phosphate buffered saline (PBS) (without calcium and magnesium) were purchased from Biological Industries (Beit Haemek, Israel); dimethyl sulfoxide (DMSO) was obtained from Applichem (Darmstadt, Germany); Hydrogen peroxide was obtained from MP Biomedicals (Ohio, USA); 2,2'-Azobis(amidinopropane) (ABAP) was obtained from Wako chemicals (Richmond, VA), and other chemicals including ZnCl2 and 2'7'-dichlorofluorescein diacetate (DCF) were purchased from Sigma Chemical Co. (St Louis, MO, USA).

#### **2.2 Preparation of** *Af* **Extracts**

The plant was collected in the Arava Valley and authenticated. The voucher specimens have been kept in as part of the Arava Rift Valley Plant Collection; VPC (Dead Sea & Arava Science Center, Central Arava Branch, Israel, http://www.deadseaarava-rd.co.il/) under the accession code AVPC0040. Freshly collected plants were dried at 40 °C for three days and extracted in ethanol (96%). The liquid phase was then evaporated off, and the dry material was dissolved in DMSO to a concentration of 100 mg/ml to produce the *Af* extract.

#### **2.3 High performance liquid chromatography (HPLC) conditions**

The ethanolic extract of *Af* was subjected to HPLC chromatography. Separation was made using reverse phase column (Betasil C-18, 5 μm, 250 × 0.46 mm; Thermo-Hypersil, UK) by gradient elution with water-acetic acid (97 : 3 V/V) and methanol as described previously (Chen et al., 2010), and detection at 360 nm (Blue line) and 280 nm (Red line) (Fig. 1).

#### **2.4 Liquid chromatography–mass spectrometry (LC–MS) conditions**

The ethanolic extract of *Af* was subjected to MS/MS (Fig. 2). The mass spectra were performed on a liquid chromatography–mass spectrometry (LC–MS) Agilent 1100LC series

437

Extract of *Achillea fragrantissima* Downregulates ROS Production and Protects Astrocytes from Oxidative-Stress-Induced Cell Death

a fragmentor voltage of 0.4V and capillary voltage of 4.5kV.

**2.5 Preparation of primary glial cell cultures** 

penicillin, and 100 μg/ml streptomycin.

**2.6 Treatment of astrocytes** 

**2.7 Determination of cell viability** 

cells into the incubation medium.

**2.8 Evaluation of intracellular ROS production** 

[M+H]+ aglycon.

Organization.

were optimized as follows: API electron spray interface, positive mode polarity, a drying gas flow of 10L/min, an nebulizer gas pressure of 60psi,a drying gas temperature of 300°C,

Four main peaks were identified by ESI-MS: Compound 1, (C27H34O14Na), r.t. 50.1, m/z 605 [M+Na+146+146], m/z 582 [M+H+146+146]+, ; suggested as epicatechin-rhamnoside. Compound 2, (C28H33O13), r.t. 48.8- m/z 577 [M+H+146+146]+; suggested as Acacetin rhamnoside. Compound 3, (C22H22O10Na ), r.t. 47.4- m/z 469 [M+Na+162]+, , m/z 447 [M+H+162]+,; suggested as Acacetin-glucoside, m/z 285 [M+H]+ aglycon. Compound 4, (C24H25O12), r.t. 46.6- m/z 465 [M+H+162]+, suggested as Quercetin-glucoside, m/z 303

Cultures of primary rat glial cells were prepared from cerebral cortices of 1- to 2-day-old neonatal Wistar rats. Briefly, meninges and blood vessels were carefully removed from cerebral cortices kept in Leibovitz-15 medium; brain tissues were dissociated by trypsinization with 0.5% trypsin (10 min, 37 °C, 5% CO2); and cells were washed first with DMEM containing soybean trypsin inhibitor (100 μg/ml) and 10% FBS and then with DMEM containing 10% FBS. Cells were seeded in tissue culture flasks pre-coated with poly-D-lysine (20 μg/ml in 0.1 M borate buffer pH 8.4) and incubated at 37 °C in humidified air with 5% CO2. The medium was changed on the second day and every second day thereafter. At the time of primary cell confluence (day 10), microglial and progenitor cells were discarded by shaking (180 RPM, 37 °C) the flasks on a horizontal shaking platform. Astrocytes were then replated on 24-well poly-D-lysine-coated plastic plates, at a density of 1×105/well, in DMEM (without phenol red) containing 2% FBS, 2 mM glutamine, 100 U/ml

The research was conducted in accordance with the internationally accepted principles for laboratory animal use and care, as found in the US guidelines, and was approved by the Institutional Animal Care and Use Committee of The Volcani Center, Agricultural Research

Twenty four hours after plating, the original medium in which the cells were grown was aspirated off, and fresh medium was added to the cells. Dilutions of plant extracts first in DMSO and then in the growth medium were made freshly from stock solution just prior to each experiment and were used immediately. The final concentration of DMSO in the medium was 0.2%. Dilutions of H2O2 in the growth medium were made freshly from a 30%

Cell viability was determined using a commercial colorimetric assay (Roche Applied Science, Germany) according to the manufacturer's instructions. This assay is based on the measurement of lactate dehydrogenase (LDH) activity released from the cytosol of damaged

Intracellular ROS production was detected using the non-fluorescent cell permeating compound, 2'7'-dichlorofluorescein diacetate (DCF-DA). DCF-DA is hydrolyzed by

stock solution immediately prior to each experiment and were used immediately.

439

(Wald- bronn, Germany) and Bruker Esquire 3000plus MS (Bremen, Germany) instrument, operated in the electrospray ionization (ESI) in a positive ion mode. A reverse phase column (BetasilC-18,5 mm, 250 mm x 0.46 mm, Thermo-Hypersil,UK) was used. The MS conditions

Fig. 1. HPLC analysis of the ethanolic extract of *Af*

Fig. 2. Liquid chromatography–mass spectrometry (LC–MS) analysis of the ethanolic extract of *Af* 

(Wald- bronn, Germany) and Bruker Esquire 3000plus MS (Bremen, Germany) instrument, operated in the electrospray ionization (ESI) in a positive ion mode. A reverse phase column (BetasilC-18,5 mm, 250 mm x 0.46 mm, Thermo-Hypersil,UK) was used. The MS conditions

Fig. 1. HPLC analysis of the ethanolic extract of *Af*

of *Af* 

 **35 40 45 50 55** 

Fig. 2. Liquid chromatography–mass spectrometry (LC–MS) analysis of the ethanolic extract

were optimized as follows: API electron spray interface, positive mode polarity, a drying gas flow of 10L/min, an nebulizer gas pressure of 60psi,a drying gas temperature of 300°C, a fragmentor voltage of 0.4V and capillary voltage of 4.5kV.

Four main peaks were identified by ESI-MS: Compound 1, (C27H34O14Na), r.t. 50.1, m/z 605 [M+Na+146+146], m/z 582 [M+H+146+146]+, ; suggested as epicatechin-rhamnoside. Compound 2, (C28H33O13), r.t. 48.8- m/z 577 [M+H+146+146]+; suggested as Acacetin rhamnoside. Compound 3, (C22H22O10Na ), r.t. 47.4- m/z 469 [M+Na+162]+, , m/z 447 [M+H+162]+,; suggested as Acacetin-glucoside, m/z 285 [M+H]+ aglycon. Compound 4, (C24H25O12), r.t. 46.6- m/z 465 [M+H+162]+, suggested as Quercetin-glucoside, m/z 303 [M+H]+ aglycon.

#### **2.5 Preparation of primary glial cell cultures**

Cultures of primary rat glial cells were prepared from cerebral cortices of 1- to 2-day-old neonatal Wistar rats. Briefly, meninges and blood vessels were carefully removed from cerebral cortices kept in Leibovitz-15 medium; brain tissues were dissociated by trypsinization with 0.5% trypsin (10 min, 37 °C, 5% CO2); and cells were washed first with DMEM containing soybean trypsin inhibitor (100 μg/ml) and 10% FBS and then with DMEM containing 10% FBS. Cells were seeded in tissue culture flasks pre-coated with poly-D-lysine (20 μg/ml in 0.1 M borate buffer pH 8.4) and incubated at 37 °C in humidified air with 5% CO2. The medium was changed on the second day and every second day thereafter. At the time of primary cell confluence (day 10), microglial and progenitor cells were discarded by shaking (180 RPM, 37 °C) the flasks on a horizontal shaking platform. Astrocytes were then replated on 24-well poly-D-lysine-coated plastic plates, at a density of 1×105/well, in DMEM (without phenol red) containing 2% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin.

The research was conducted in accordance with the internationally accepted principles for laboratory animal use and care, as found in the US guidelines, and was approved by the Institutional Animal Care and Use Committee of The Volcani Center, Agricultural Research Organization.

#### **2.6 Treatment of astrocytes**

Twenty four hours after plating, the original medium in which the cells were grown was aspirated off, and fresh medium was added to the cells. Dilutions of plant extracts first in DMSO and then in the growth medium were made freshly from stock solution just prior to each experiment and were used immediately. The final concentration of DMSO in the medium was 0.2%. Dilutions of H2O2 in the growth medium were made freshly from a 30% stock solution immediately prior to each experiment and were used immediately.

#### **2.7 Determination of cell viability**

Cell viability was determined using a commercial colorimetric assay (Roche Applied Science, Germany) according to the manufacturer's instructions. This assay is based on the measurement of lactate dehydrogenase (LDH) activity released from the cytosol of damaged cells into the incubation medium.

#### **2.8 Evaluation of intracellular ROS production**

Intracellular ROS production was detected using the non-fluorescent cell permeating compound, 2'7'-dichlorofluorescein diacetate (DCF-DA). DCF-DA is hydrolyzed by

Extract of *Achillea fragrantissima* Downregulates ROS Production and Protects Astrocytes from Oxidative-Stress-Induced Cell Death

of the *Af* extract in the absence of H2O2 (Fig. 3).

**0**

**20**

**40**

**60**

**Cytotoxicity**

extract of *Af* 

with H2O2 alone.

 **(%)** 

**80**

**100**

**120**

addition of this compound to cultured primary astrocytes. Exposure of normal primary astrocytes with H2O2 resulted in a time and concentration dependent astrocytic cell death 20 h later (data not shown). To find out whether the *Af* extract has a protective effect and to determine the optimal concentration of the extract needed for such an effect, astrocytes were pre-incubated with different concentrations of *Af* extract. H2O2 was then added, and cytotoxicity was determined after 20 h. Our results showed that the *Af* extract exerted a protective action against H2O2 -induced cell death in a dose-dependent manner (Fig. 3). No significant changes were observed in the viability of cells treated with similar concentrations

**0 50 100 150 200**

Fig. 3. Protection from H2O2-induced astrocytic cell death by different concentrations of the

Astrocytes were treated with different concentrations of *Af* extract. H2O2 (200 μM) was added 2 h after the addition of *Af* extract. Cell death was determined 20 h later. Each point represents the means ± SEM of five experiments (*n = 20*). \*\**p*<0.001 compared to cells treated

In order to gain more insight into the mechanisms by which the *Af* extract might exert its protective effects, and to determine whether this extract could inhibit ROS production induced by H2O2 and ZnCl2, we assessed the intracellular generation of ROS by these toxic molecules, and tested whether treatment of astrocytes with the *Af* extract affected intracellular ROS levels. For the study of preventive effects against intracellular ROS formation the cells were preloaded with the ROS indicator DCF-DA, and were pretreated with various concentrations of *Af* extract before the application of H2O2 or ZnCl2 stress, and ROS formation was determined by reading fluorescence every hour for 4 h. As can be seen in Fig. 4A, H2O2 induced ROS production in astrocytes, with the maximum levels produced after 1 h. Pretreatment of astrocytes with the *Af* extract inhibited the H2O2-induced elevation of the levels of intracellular ROS in a dose-dependent manner (Fig. 4B). We also found that treatment with ZnCl2 increased ROS generation in astrocytes, and that, similarly to the effect

**3.2** *Af* **extract inhibits H2O2- and ZnCl2-induced ROS generation** 

**\*\***

**Af extract ( µg/mL)**

*Af* **(µg/mL)**

**\*\***

441

**With H2O2 W/O H2O2**

**With H2O2W/O H2O2**

**\*\***

intracellular esterases and then oxidized by ROS to a fluorescent compound 2'-7'-DCF. Astrocytes were plated onto 24 wells plates (300,000 cells/well) and treated with DCF-DA (20 µM) for 30 min at 370C. Following incubation with DCF, cultures were rinsed twice with PBS and then re-suspended (1) For measurement of H2O2-induced ROS: in DMEM containing 10% FBS, 8 mM HEPES, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (2) For measurement of ZnCl2 - induced ROS: in a defined buffer containing 116 mM NaCl, 1.8 mM CaCl2, 0.8 mM MgSO4, 5.4 mM KCl, 1 mM NaH2PO4, 14.7 mM NaHCO3, and 10 mM HEPES, pH, 7.4. The fluorescence was measured in a plate reader with excitation at 485 nm and emission at 520 nm.

#### **2.9 Cellular antioxidant activity of** *Af* **extract**

Peroxyl radicals are generated by thermolysis of 2,2'-Azobis(amidinopropane) (ABAP) at physiological temperature. ABAP decomposes at approximately 1.36x10-6s-1 at 37°C, producing at most 1x1012 radicals/ml/s (Bowry & Stocker, 1993; Niki et al., 1986; Thomas et al., 1997). Astrocytes were plated onto 24 wells plates (300,000 cells/well) and were incubated for 1 hr with *Af* extract. Then astrocytes were preloaded with DCF-DA for 30 min, washed, and ABAP (0.6 mM final concentration) was then added. The fluorescence, which indicates ROS levels, was measured in a plate reader with excitation at 485 nm and emission at 520 nm.

#### **2.10 Differential pulse voltammetry analysis**

Ethanolic extracts were obtained by dissolving 1 g of dry plant powder in 10 ml of ethanol overnight at room temperature. Before performing the differential pulse voltammetry (DPV) analysis, tetrabutylammonium perchlorate was added to the ethanolic extract to final concentration of 1% and the total reducing capacity of the *Af* extracts was analyzed, as described before (Butera et al., 2002). Briefly, the plant extract was placed in a cyclic voltammeter cell equipped with a working electrode (3.2 mm in diameters, glassy carbon), a reference electrode (Ag/AgCl), and an auxiliary electrode (platinum wire). The DPV potential was conducted at a scan rate of 40 mV/s, pulse amplitude 50 mV, sample width 17 ms, pulse width 50 ms, pulse period 200 ms. An electrochemical working station (CH Instruments Inc., 610B, Austin, TX, USA) was used. The output of the DPV experiments was a potential-current curve (Kohen et al., 1999).

#### **2.11 Data analysis**

Statistical analyses were performed with one-way ANOVA followed by Tukey-Kramer multiple comparison tests using Graph Pad InStat 3 for windows (GraphPad Software, San Diego, CA, USA).

#### **3. Results**

#### **3.1 Protection by the** *Af* **extract of astrocytes from H2O2 -induced cell death**

H2O2 exposure is used as a model of ischemia reperfusion. The concentration of H2O2 used in our experiments (175-200 microM) resembles the concentration reported by Hyslop *et al* to be the concentration of H2O2 that appears in the rat striatum under ischemic conditions (Hyslop et al., 1995). In order to characterize the astroprotective potential of the *Af* extract against H2O2 -induced oxidative stress, we have assessed changes in intracellular ROS production and in cell viability, using a model in which oxidative stress was induced by the

intracellular esterases and then oxidized by ROS to a fluorescent compound 2'-7'-DCF. Astrocytes were plated onto 24 wells plates (300,000 cells/well) and treated with DCF-DA (20 µM) for 30 min at 370C. Following incubation with DCF, cultures were rinsed twice with PBS and then re-suspended (1) For measurement of H2O2-induced ROS: in DMEM containing 10% FBS, 8 mM HEPES, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (2) For measurement of ZnCl2 - induced ROS: in a defined buffer containing 116 mM NaCl, 1.8 mM CaCl2, 0.8 mM MgSO4, 5.4 mM KCl, 1 mM NaH2PO4, 14.7 mM NaHCO3, and 10 mM HEPES, pH, 7.4. The fluorescence was measured in a plate reader with

Peroxyl radicals are generated by thermolysis of 2,2'-Azobis(amidinopropane) (ABAP) at physiological temperature. ABAP decomposes at approximately 1.36x10-6s-1 at 37°C, producing at most 1x1012 radicals/ml/s (Bowry & Stocker, 1993; Niki et al., 1986; Thomas et al., 1997). Astrocytes were plated onto 24 wells plates (300,000 cells/well) and were incubated for 1 hr with *Af* extract. Then astrocytes were preloaded with DCF-DA for 30 min, washed, and ABAP (0.6 mM final concentration) was then added. The fluorescence, which indicates ROS levels, was measured in a plate reader with excitation at 485 nm and emission

Ethanolic extracts were obtained by dissolving 1 g of dry plant powder in 10 ml of ethanol overnight at room temperature. Before performing the differential pulse voltammetry (DPV) analysis, tetrabutylammonium perchlorate was added to the ethanolic extract to final concentration of 1% and the total reducing capacity of the *Af* extracts was analyzed, as described before (Butera et al., 2002). Briefly, the plant extract was placed in a cyclic voltammeter cell equipped with a working electrode (3.2 mm in diameters, glassy carbon), a reference electrode (Ag/AgCl), and an auxiliary electrode (platinum wire). The DPV potential was conducted at a scan rate of 40 mV/s, pulse amplitude 50 mV, sample width 17 ms, pulse width 50 ms, pulse period 200 ms. An electrochemical working station (CH Instruments Inc., 610B, Austin, TX, USA) was used. The output of the DPV experiments was

Statistical analyses were performed with one-way ANOVA followed by Tukey-Kramer multiple comparison tests using Graph Pad InStat 3 for windows (GraphPad Software, San

H2O2 exposure is used as a model of ischemia reperfusion. The concentration of H2O2 used in our experiments (175-200 microM) resembles the concentration reported by Hyslop *et al* to be the concentration of H2O2 that appears in the rat striatum under ischemic conditions (Hyslop et al., 1995). In order to characterize the astroprotective potential of the *Af* extract against H2O2 -induced oxidative stress, we have assessed changes in intracellular ROS production and in cell viability, using a model in which oxidative stress was induced by the

**3.1 Protection by the** *Af* **extract of astrocytes from H2O2 -induced cell death** 

excitation at 485 nm and emission at 520 nm.

**2.9 Cellular antioxidant activity of** *Af* **extract** 

**2.10 Differential pulse voltammetry analysis** 

a potential-current curve (Kohen et al., 1999).

at 520 nm.

**2.11 Data analysis** 

Diego, CA, USA).

**3. Results** 

addition of this compound to cultured primary astrocytes. Exposure of normal primary astrocytes with H2O2 resulted in a time and concentration dependent astrocytic cell death 20 h later (data not shown). To find out whether the *Af* extract has a protective effect and to determine the optimal concentration of the extract needed for such an effect, astrocytes were pre-incubated with different concentrations of *Af* extract. H2O2 was then added, and cytotoxicity was determined after 20 h. Our results showed that the *Af* extract exerted a protective action against H2O2 -induced cell death in a dose-dependent manner (Fig. 3). No significant changes were observed in the viability of cells treated with similar concentrations of the *Af* extract in the absence of H2O2 (Fig. 3).

Fig. 3. Protection from H2O2-induced astrocytic cell death by different concentrations of the extract of *Af* 

Astrocytes were treated with different concentrations of *Af* extract. H2O2 (200 μM) was added 2 h after the addition of *Af* extract. Cell death was determined 20 h later. Each point represents the means ± SEM of five experiments (*n = 20*). \*\**p*<0.001 compared to cells treated with H2O2 alone.

#### **3.2** *Af* **extract inhibits H2O2- and ZnCl2-induced ROS generation**

In order to gain more insight into the mechanisms by which the *Af* extract might exert its protective effects, and to determine whether this extract could inhibit ROS production induced by H2O2 and ZnCl2, we assessed the intracellular generation of ROS by these toxic molecules, and tested whether treatment of astrocytes with the *Af* extract affected intracellular ROS levels. For the study of preventive effects against intracellular ROS formation the cells were preloaded with the ROS indicator DCF-DA, and were pretreated with various concentrations of *Af* extract before the application of H2O2 or ZnCl2 stress, and ROS formation was determined by reading fluorescence every hour for 4 h. As can be seen in Fig. 4A, H2O2 induced ROS production in astrocytes, with the maximum levels produced after 1 h. Pretreatment of astrocytes with the *Af* extract inhibited the H2O2-induced elevation of the levels of intracellular ROS in a dose-dependent manner (Fig. 4B). We also found that treatment with ZnCl2 increased ROS generation in astrocytes, and that, similarly to the effect

Extract of *Achillea fragrantissima* Downregulates ROS Production and Protects Astrocytes from Oxidative-Stress-Induced Cell Death

> **012345 Time (hr)**

Fig. 5. Zinc induces ROS generation, and the *Af* extract attenuates ROS production following

Astrocytes were preloaded with DCF-DA for 30 min and washed with PBS. They were then pre-incubated for 2 h with various concentrations of *Af* extract, after which, ZnCl2 (50 μM) was added and the resulting fluorescence signal was measured at the indicated time points. Each point represents the mean ± SEM (*n* = 7). *p*<0.01 when ROS production following treatment with ZnCl2+*Af* extract was compared to cells treated with ZnCl2 alone at each of

**3.3** *Af* **extract reduces 2,2'-azobis(amidinopropane) (ABAP)-mediated peroxyl radicals** 

In addition to H2O2, various other species, such as peroxynitrite (ONOO-), nitric oxide (NO.

**3.4 Differential pulse voltammetry (DPV) analysis of** *the* **antioxidant capacity of** *Af*

Extract antioxidant capacity was evaluated by differential pulse voltammetry approach (DPV). Voltammetric techniques of analysis are increasingly being used for the determination of many substances of pharmaceutical importance (Zapata-Urzua et al., 2010) as well as of fruit extracts (Butera et al., 2002). These techniques are based on the measurement of current that results from oxidation or reduction at an electrode surface following an applied potential

and peroxyl radicals have been found to oxidize DCFH to DCF in cell culture (Wang & Joseph, 1999), therefore we have used the cellular antioxidant activity assay to measure the ability of compounds present in the *Af* extract to prevent formation of DCF by ABAPgenerated peroxyl radicals (Wolfe & Liu, 2007). The kinetics of DCFH oxidation in astrocytes by peroxyl radicals generated from ABAP is shown in Fig. 6A, where it can be seen that ABAP generated radicals in a time-dependent manner, and that treatment of cells with *Af* extract moderated this induction. Fig. 6B shows that the increase in ROS–induced fluorescence was inhibited by *Af* extract in a dose-dependent manner. This indicates that compounds present in the *Af* extract entered the cells and acted as efficient intracellular hydroperoxyl

treatment of astrocytes with zinc

the equivalent time points

**levels in astrocytes** 

radical scavengers.

**extract** 

**ROS production (FU)**

443

)

**W/O ZnCl2**

**ZnCl2** *Af*

**ZnCl2** *Af*

**ZnCl2** *Af*

**ZnCl2**

**ZnCl2 + Af (12.5 µg/mL)**

**ZnCl2 + Af (25 µg/mL)**

**ZnCl2 + Af (50 µg/mL)**

**ZnCl2**

**ZnCl2**

of the *Af* extract on H2O2-induced ROS, this extract greatly attenuated ZnCl2-induced ROS generation (Fig. 5).

Fig. 4. The *Af* extract attenuates H2O2-induced ROS production in astrocytes

Astrocytes were preloaded with the redox - sensitive DCF-DA for 30 min and washed with PBS. Preloaded astrocytes were then pre-incubated for 2 h with various concentrations of *Af* extract. H2O2 (175 μM) was added to the culture and the fluorescence intensity representing ROS production was measured. (A) Pre-incubation with 100 μg/ml *Af* extract and measurements at the indicated time points (B) Pre-incubation with various concentrations of *Af* extract and measurements after 1 h. Each point represents the mean ± SEM of two experiments (*n=7*). \*\**p*<0.001 when ROS production following treatment with H2O2+*Af* extract was compared to cells treated with H2O2 alone at each of the equivalent time points.

of the *Af* extract on H2O2-induced ROS, this extract greatly attenuated ZnCl2-induced ROS

**H2O2**

**H2O2 + Af extract**

*Af*

**012345 Time (hr)**

(A)

**0 13 25 38 50 63 75 88 100 113** *Af* **extract (µg/mL)**

**\*\* \*\***

(B)

Astrocytes were preloaded with the redox - sensitive DCF-DA for 30 min and washed with PBS. Preloaded astrocytes were then pre-incubated for 2 h with various concentrations of *Af* extract. H2O2 (175 μM) was added to the culture and the fluorescence intensity representing ROS production was measured. (A) Pre-incubation with 100 μg/ml *Af* extract and measurements at the indicated time points (B) Pre-incubation with various concentrations of *Af* extract and measurements after 1 h. Each point represents the mean ± SEM of two experiments (*n=7*). \*\**p*<0.001 when ROS production following treatment with H2O2+*Af* extract was compared to cells treated with H2O2 alone at each of the equivalent time points.

**\*\***

Fig. 4. The *Af* extract attenuates H2O2-induced ROS production in astrocytes

**\*\***

**\*\* \*\* \*\***

generation (Fig. 5).

**0**

**20**

**40**

**60**

**ROS production (%)**

**80**

**100**

**120**

**ROS production (%)**

Fig. 5. Zinc induces ROS generation, and the *Af* extract attenuates ROS production following treatment of astrocytes with zinc

Astrocytes were preloaded with DCF-DA for 30 min and washed with PBS. They were then pre-incubated for 2 h with various concentrations of *Af* extract, after which, ZnCl2 (50 μM) was added and the resulting fluorescence signal was measured at the indicated time points. Each point represents the mean ± SEM (*n* = 7). *p*<0.01 when ROS production following treatment with ZnCl2+*Af* extract was compared to cells treated with ZnCl2 alone at each of the equivalent time points

#### **3.3** *Af* **extract reduces 2,2'-azobis(amidinopropane) (ABAP)-mediated peroxyl radicals levels in astrocytes**

In addition to H2O2, various other species, such as peroxynitrite (ONOO-), nitric oxide (NO. ) and peroxyl radicals have been found to oxidize DCFH to DCF in cell culture (Wang & Joseph, 1999), therefore we have used the cellular antioxidant activity assay to measure the ability of compounds present in the *Af* extract to prevent formation of DCF by ABAPgenerated peroxyl radicals (Wolfe & Liu, 2007). The kinetics of DCFH oxidation in astrocytes by peroxyl radicals generated from ABAP is shown in Fig. 6A, where it can be seen that ABAP generated radicals in a time-dependent manner, and that treatment of cells with *Af* extract moderated this induction. Fig. 6B shows that the increase in ROS–induced fluorescence was inhibited by *Af* extract in a dose-dependent manner. This indicates that compounds present in the *Af* extract entered the cells and acted as efficient intracellular hydroperoxyl radical scavengers.

#### **3.4 Differential pulse voltammetry (DPV) analysis of** *the* **antioxidant capacity of** *Af* **extract**

Extract antioxidant capacity was evaluated by differential pulse voltammetry approach (DPV). Voltammetric techniques of analysis are increasingly being used for the determination of many substances of pharmaceutical importance (Zapata-Urzua et al., 2010) as well as of fruit extracts (Butera et al., 2002). These techniques are based on the measurement of current that results from oxidation or reduction at an electrode surface following an applied potential

Extract of *Achillea fragrantissima* Downregulates ROS Production and Protects Astrocytes from Oxidative-Stress-Induced Cell Death

presented at Table 1.

that 50 mV apart (Smyth & Woolfson, 1987). In the present study we have used the DPV approach to analyze the total reducing capacity of the ethanolic *Af* extract. On the potentialcurrent curve generated by DPV, the values of the potential are a characteristic of the antioxidant material and the values of the current are proportional to the amounts of the corresponding antioxidant. Analysis of the *Af* extract by DPV revealed two anodic waves that are caused by two major reducing groups of low-molecular-weight antioxidants, representing the total antioxidants in the extract (Fig. 7). The anodic wave potentials and their corresponding anodic currents, representing the amount of each antioxidant, are

b

Differential pulse voltammetry (DPV) was conducted from E = 0.0 V to final E = 2.0 V at a scan rate of 40 mV/s, pulse amplitude 50 mV, sample width 17 ms, pulse width 50 ms, pulse period 200 ms. Extracts were prepared in duplicate, and each sample was traced three times.

The main findings of the present study were that an ethanolic extract of the desert plant *Af* could protect primary cultures of rat brain astrocytes from H2O2 -induced cell death, and reduced the levels of intracellular ROS produced after treatment with H2O2, ZnCl2 or ABAP. This protective effect of *Af* and the reduction in ROS levels might be mediated by its antioxidant activities (as was demonstrated by the DPV experiments) or by modulation of

**Potential (V ± SD)** 0.625±0.003 1.039±0.024 **Current (µA ± SD )** 3.233±0.251 7.027±0.063

**Anodic wave a Anodic wave b** 

Fig. 7. Representative differential pulse voltammogram of the *Af* extract

Table 1. Anodic potentials and currents of the ethanolic extracts of *Af*

**a** - first anodic wave; **b** - second anodic wave.

**4. Discussion** 

a

445

Fig. 6. Peroxyl radical - induced oxidation of DCFH to DCF in primary astrocytes, and the inhibition of oxidation by *Af* extract

Astrocytes were incubated for 1 h with *Af* extract. They were then preloaded with DCF-DA for 30 min and washed with PBS, after which, 0.6 mM ABAP was added and ROS levels were measured at the indicated time points. Each point represents mean ± SEM of two experiments (*n* = 7). **A**. *Af* extract at 25 μg/ml. **B**. ROS production was measured 20 h after the addition of ABAP *\*p*<0.01, \**\*p*<0.001 compared to cells treated with ABAP only at the equivalent time points.

difference. The DPV technique has excellent resolving power, and is able to differentiate between peaks due to different electroactive species in the same solution which are no more

**ABAP only**

**ABAP + Af extract**

**+** *Af* **extract**

**0 3 6 9 12 15 18 21**

**0 13 25 38 50 63 75 88 100 113**

**\*\* \* \***

**\* \***

**ABAP+** *Af* **extract (µg/mL)**

(B) Fig. 6. Peroxyl radical - induced oxidation of DCFH to DCF in primary astrocytes, and the

Astrocytes were incubated for 1 h with *Af* extract. They were then preloaded with DCF-DA for 30 min and washed with PBS, after which, 0.6 mM ABAP was added and ROS levels were measured at the indicated time points. Each point represents mean ± SEM of two experiments (*n* = 7). **A**. *Af* extract at 25 μg/ml. **B**. ROS production was measured 20 h after the addition of ABAP *\*p*<0.01, \**\*p*<0.001 compared to cells treated with ABAP only at the

difference. The DPV technique has excellent resolving power, and is able to differentiate between peaks due to different electroactive species in the same solution which are no more

**\***

(A)

**0 3 6 9 12 15 18 21 Time (hr)**

**Time (hr)**

**\*\***

**\*\***

**0**

**0**

inhibition of oxidation by *Af* extract

equivalent time points.

**20**

**40**

**60**

**ROS production (%)**

**80**

**\***

**\*\***

**100**

**120**

**2000**

**4000**

**6000**

**ROS production (FU)**

**ROS production (FU)**

**8000**

**10000**

**12000**

**12000**

**10000**

 **8000**

 **6000**

 **4000**

 **2000**

 **0**

that 50 mV apart (Smyth & Woolfson, 1987). In the present study we have used the DPV approach to analyze the total reducing capacity of the ethanolic *Af* extract. On the potentialcurrent curve generated by DPV, the values of the potential are a characteristic of the antioxidant material and the values of the current are proportional to the amounts of the corresponding antioxidant. Analysis of the *Af* extract by DPV revealed two anodic waves that are caused by two major reducing groups of low-molecular-weight antioxidants, representing the total antioxidants in the extract (Fig. 7). The anodic wave potentials and their corresponding anodic currents, representing the amount of each antioxidant, are presented at Table 1.

Fig. 7. Representative differential pulse voltammogram of the *Af* extract

Differential pulse voltammetry (DPV) was conducted from E = 0.0 V to final E = 2.0 V at a scan rate of 40 mV/s, pulse amplitude 50 mV, sample width 17 ms, pulse width 50 ms, pulse period 200 ms. Extracts were prepared in duplicate, and each sample was traced three times. **a** - first anodic wave; **b** - second anodic wave.


Table 1. Anodic potentials and currents of the ethanolic extracts of *Af*

#### **4. Discussion**

The main findings of the present study were that an ethanolic extract of the desert plant *Af* could protect primary cultures of rat brain astrocytes from H2O2 -induced cell death, and reduced the levels of intracellular ROS produced after treatment with H2O2, ZnCl2 or ABAP. This protective effect of *Af* and the reduction in ROS levels might be mediated by its antioxidant activities (as was demonstrated by the DPV experiments) or by modulation of

Extract of *Achillea fragrantissima* Downregulates ROS Production and Protects Astrocytes from Oxidative-Stress-Induced Cell Death

cope with stresses that develop during pathological conditions.

temperatures and extreme pH values.

death form part of the pathophysiology.

Israel Science Foundation (grant No. 600/08).

1, pp.18-26, ISSN 0189-6016

*Ciências,* Vol.78, No. 3, pp.505-514, ISSN 0001-3765

*NeuroToxicology,* Vol.27, No. 2, pp.158-163, ISSN 0161-813X

et al., 2008).

**5. Conclusions** 

**6. Acknowledgments** 

**7. References** 

aorta, along with the attenuation of hyperlipidemia (Kamada et al., 2005; Terao, 1999; Terao

Two other compounds in the *Af* extract were also identified by LC-MS: acacetin 7-o-rhamnoside, which was also identified in the aerial parts of several plants (El-Wakil, 2007; Sharaf et al., 1997), and acacetin 7-o-glucoside, which was also found in the antiinflammatory extract of Mcfadyena unguis-cati L. (Aboutabl et al., 2008). All four compounds identified by LC-MS analysis as major peaks in *Af* extract, namely epicatechinrhamnoside, Acacetin rhamnoside, Acacetin-glucoside, and Quercetin-glucoside, are stable compounds, that under our experimental conditions (ethanol extraction, resolubilization in DMSO, and tissue culture experiments at 37°C and neutral pH) would not react chemically with each other. Chemical interactions between these compounds might occur under high

Several studies have revealed that some herbal medications and antioxidants show promise in prevention of neurodegenerative diseases (Iriti et al., 2010). Substances that can restrict and/or protect brain cells from oxidative stress show promise as potential tools in the therapy of various brain injuries and neurodegenerative diseases. Desert plants survive various stress conditions, including oxidative stress., therefore it is reasonable to suppose that various endogenous molecules present in these plants might also assist animal cells to

In light of their antioxidant and astroprotective properties, we suggest that *Af* extracts might serve as a new source of beneficial phytochemicals, and should be further evaluated for nutraceutical development as polyvalent cocktails for prevention or treatment of various brain injuries and neurodegenerative diseases, in which oxidative stress and astrocytic cell

This work was supported by the Chief Scientist of the Ministry of Science, Israel, and by The

Aboutabl, E.A., Hashem F.A., Sleem A.A., & Maamoon A.A. (2008). Flavonoids, anti-

Almeida, A.M., Bertoncini C.R., Borecky J., Souza-Pinto N.C., & Vercesi A.E. (2006).

Altiok, N., Ersoz M., Karpuz V., & Koyuturk M. (2006). *Ginkgo biloba* extract regulates

Arts, I.C., Sesink A.L., Faassen-Peters M., & Hollman P.C. (2004). The type of sugar moiety is

inflammatory activity and cytotoxicity of *Macfadyena Unguis-Cati* L. *The African Journal of Traditional, Complementary and Alternative medicines (AJTCAM),* Vol.5, No.

Mitochondrial DNA damage associated with lipid peroxidation of the mitochondrial membrane induced by Fe2+-citrate. *Anais da Academia Brasileira de* 

differentially the cell death induced by hydrogen peroxide and simvastatin.

a major determinant of the small intestinal uptake and subsequent biliary excretion

447

signals and processes induced by H2O2 and ZnCl2. For example, it has been found, that H2O2 induced the phosphorylation of ERK1/2, AKT/protein kinase B and ATF-2 in C6 glioma cells (Altiok et al., 2006). It also has been demonstrated that cell death caused by zinc was accompanied by membrane translocation of protein kinase C-alpha (PKC-α), phosphorylation of extracellular signal-regulated kinase (ERK), and activation of group IV calcium-dependent cytosolic phospholipase A2 (cPLA2) (Chang et al., 2010; Liao et al., 2011). It was also reported that Zn2+ bound to and inhibited glutathione reductase and peroxidase, the major enzymes responsible for glutathione (GSH) metabolism and cellular antioxidative defense mechanisms (Mize & Langdon, 1962; Splittgerber & Tappel, 1979).

Hydrogen peroxide also decreased astrocyte membrane fluidity, induced cytoskeletal reorganization, decreased the activities of the antioxidant enzymes catalase and superoxide dismutase (SOD) (Naval et al., 2007), and increased formation of cytonemes and cell-to-cell tunneling nanotube (TNT)-like connections (Zhu et al., 2005). Thus, the *Af* extract might interfere with any or all of the described processes, and enhance the resistance of astrocytes to ZnCl2 and H2O2 toxicity, and to oxidative stress. Moreover, defense of glial cells against oxidative damage would be essential for maintaining brain functions.

There are two opportunities for compounds present in *Af* extract to elicit their antioxidant effects in our model: they can act at the cell membrane and break peroxyl radical chain reactions at the cell surface; or they can be taken up by the cell and react intracellularly with ROS. Therefore, the efficiency of cellular uptake and/or membrane binding, combined with the radical-scavenging activity dictates the efficacy of the tested compounds. In order to discriminate between these possibilities, astrocytes were pre-incubated with ABAP, which generates ROS intracellularly. According to our results, which show that *Af* extract inhibited intracellular ROS levels, in addition to other possible activities, compounds present in *Af* extract could enter the cells and react with ROS intracellularly.

Because many low-molecular-weight antioxidants might contribute to the cellular antioxidant defense properties, we analyzed the total antioxidant content of the *Af* extract by the DPV method, which enabled us to demonstrate the presence of two reducing equivalents in the *Af* extract. The advantages of DPV over other voltammetric techniques include excellent sensitivity with a very wide useful linear concentration range for organic species (10–6 to 10–3 M), short analysis times, simultaneous determination of several analytes, and ease of generating a variety of potential waveforms and measuring small currents.

Our LC-MS analysis identified quercetin-glucoside as one of the major peaks in the *Af* extract. Quercetin glycosides are widely consumed flavonoids that are found in many fruits and vegetables, e.g., onion, and, like other flavonoids, offer a wide range of potential health benefits, including prevention of atherosclerosis and cardiovascular diseases (Peluso, 2006; Terao et al., 2008). In recent years, intestinal absorption and metabolism of quercetin glucosides have been extensively investigated with regard to their bioavailability (Spencer et al., 2004; Walle, 2004). Quercetin glucosides are well absorbed by the small intestine because the presence of a glucose moiety significantly enhances absorption (Arts et al. 2004; Boyer et al., 2005; Hollman & Arts, 2000). In the process of intestinal absorption quercetinglucosides are subjected to hydrolysis and subsequent conversion into conjugated glucuronides and/or sulfates (Murota & Terao, 2003). A variety of metabolites circulating in the blood-stream were identified (Day et al., 2001; Mullen et al., 2002), and some of them were found to possess a substantial antioxidant activity (da Silva et al., 1998; Manach et al., 1998). It was suggested that metabolites of quercetin glucosides accumulate in the aorta - a target site for its anti-atherosclerotic effect, and attenuate lipid peroxidation that occur in the

aorta, along with the attenuation of hyperlipidemia (Kamada et al., 2005; Terao, 1999; Terao et al., 2008).

Two other compounds in the *Af* extract were also identified by LC-MS: acacetin 7-o-rhamnoside, which was also identified in the aerial parts of several plants (El-Wakil, 2007; Sharaf et al., 1997), and acacetin 7-o-glucoside, which was also found in the antiinflammatory extract of Mcfadyena unguis-cati L. (Aboutabl et al., 2008). All four compounds identified by LC-MS analysis as major peaks in *Af* extract, namely epicatechinrhamnoside, Acacetin rhamnoside, Acacetin-glucoside, and Quercetin-glucoside, are stable compounds, that under our experimental conditions (ethanol extraction, resolubilization in DMSO, and tissue culture experiments at 37°C and neutral pH) would not react chemically with each other. Chemical interactions between these compounds might occur under high temperatures and extreme pH values.

Several studies have revealed that some herbal medications and antioxidants show promise in prevention of neurodegenerative diseases (Iriti et al., 2010). Substances that can restrict and/or protect brain cells from oxidative stress show promise as potential tools in the therapy of various brain injuries and neurodegenerative diseases. Desert plants survive various stress conditions, including oxidative stress., therefore it is reasonable to suppose that various endogenous molecules present in these plants might also assist animal cells to cope with stresses that develop during pathological conditions.

#### **5. Conclusions**

Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring 446

signals and processes induced by H2O2 and ZnCl2. For example, it has been found, that H2O2 induced the phosphorylation of ERK1/2, AKT/protein kinase B and ATF-2 in C6 glioma cells (Altiok et al., 2006). It also has been demonstrated that cell death caused by zinc was accompanied by membrane translocation of protein kinase C-alpha (PKC-α), phosphorylation of extracellular signal-regulated kinase (ERK), and activation of group IV calcium-dependent cytosolic phospholipase A2 (cPLA2) (Chang et al., 2010; Liao et al., 2011). It was also reported that Zn2+ bound to and inhibited glutathione reductase and peroxidase, the major enzymes responsible for glutathione (GSH) metabolism and cellular antioxidative

Hydrogen peroxide also decreased astrocyte membrane fluidity, induced cytoskeletal reorganization, decreased the activities of the antioxidant enzymes catalase and superoxide dismutase (SOD) (Naval et al., 2007), and increased formation of cytonemes and cell-to-cell tunneling nanotube (TNT)-like connections (Zhu et al., 2005). Thus, the *Af* extract might interfere with any or all of the described processes, and enhance the resistance of astrocytes to ZnCl2 and H2O2 toxicity, and to oxidative stress. Moreover, defense of glial cells against

There are two opportunities for compounds present in *Af* extract to elicit their antioxidant effects in our model: they can act at the cell membrane and break peroxyl radical chain reactions at the cell surface; or they can be taken up by the cell and react intracellularly with ROS. Therefore, the efficiency of cellular uptake and/or membrane binding, combined with the radical-scavenging activity dictates the efficacy of the tested compounds. In order to discriminate between these possibilities, astrocytes were pre-incubated with ABAP, which generates ROS intracellularly. According to our results, which show that *Af* extract inhibited intracellular ROS levels, in addition to other possible activities, compounds present in *Af*

Because many low-molecular-weight antioxidants might contribute to the cellular antioxidant defense properties, we analyzed the total antioxidant content of the *Af* extract by the DPV method, which enabled us to demonstrate the presence of two reducing equivalents in the *Af* extract. The advantages of DPV over other voltammetric techniques include excellent sensitivity with a very wide useful linear concentration range for organic species (10–6 to 10–3 M), short analysis times, simultaneous determination of several analytes, and ease of generating a variety of potential waveforms and measuring small currents. Our LC-MS analysis identified quercetin-glucoside as one of the major peaks in the *Af* extract. Quercetin glycosides are widely consumed flavonoids that are found in many fruits and vegetables, e.g., onion, and, like other flavonoids, offer a wide range of potential health benefits, including prevention of atherosclerosis and cardiovascular diseases (Peluso, 2006; Terao et al., 2008). In recent years, intestinal absorption and metabolism of quercetin glucosides have been extensively investigated with regard to their bioavailability (Spencer et al., 2004; Walle, 2004). Quercetin glucosides are well absorbed by the small intestine because the presence of a glucose moiety significantly enhances absorption (Arts et al. 2004; Boyer et al., 2005; Hollman & Arts, 2000). In the process of intestinal absorption quercetinglucosides are subjected to hydrolysis and subsequent conversion into conjugated glucuronides and/or sulfates (Murota & Terao, 2003). A variety of metabolites circulating in the blood-stream were identified (Day et al., 2001; Mullen et al., 2002), and some of them were found to possess a substantial antioxidant activity (da Silva et al., 1998; Manach et al., 1998). It was suggested that metabolites of quercetin glucosides accumulate in the aorta - a target site for its anti-atherosclerotic effect, and attenuate lipid peroxidation that occur in the

defense mechanisms (Mize & Langdon, 1962; Splittgerber & Tappel, 1979).

oxidative damage would be essential for maintaining brain functions.

extract could enter the cells and react with ROS intracellularly.

In light of their antioxidant and astroprotective properties, we suggest that *Af* extracts might serve as a new source of beneficial phytochemicals, and should be further evaluated for nutraceutical development as polyvalent cocktails for prevention or treatment of various brain injuries and neurodegenerative diseases, in which oxidative stress and astrocytic cell death form part of the pathophysiology.

#### **6. Acknowledgments**

This work was supported by the Chief Scientist of the Ministry of Science, Israel, and by The Israel Science Foundation (grant No. 600/08).

#### **7. References**


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**19** 

*Turkey* 

**Quantification of Volumetric Changes of** 

*1Dept. of Anatomy, Erciyes University School of Medicine, Kayseri, 2Dept. of Radiology, Ankara Training and Research Hospital, Ankara,* 

*3Dept. of Radiology, Adnan Menderes University School of Medicine, Aydn, 4Dept. of Neurosurgery, Adnan Menderes University School of Medicine, Aydn,* 

Niyazi Acer1, Ahmet Tuncay Turgut2, Yelda Özsunar3 and Mehmet Turgut4

**Brain in Neurodegenerative Diseases Using** 

**Magnetic Resonance Imaging and Stereology** 

In this chapter, we review the different magnetic resonance imaging (MRI)-based methods used to quantify whole and subcortical brain structures volume, and discuss the relevance of the brain atrophy in different neurodegenerative disseases. Although there are a lot of studies for multiple sclerosis (MS) and dementia of Alzheimer's type (AD) for the brain atrophy using different methods, the optimal method for quantifying atrophy has not been

In recent years, computed tomography (CT) scanning has been replaced with MRI scanning due to its enhanced soft-tissue resolution, especially for cerebrospinal fluid (CSF)-filled spaces, such as ventricular enlargement in patients with AD. Thus, a transition has occurred from CT to MRI in longitudinal studies investigating the human brain. As a result of development of new neuroimaging methods in clinical practice, volumetric methods started to be more sophisticated depending on various imaging methods (Lim et al., 2000). There are numerous reasons for the aforementioned transition; first of all, unlike CT, MRI has no inherent radiation effect, and secondly, CT underestimates cortical sulcal volume relative to MRI due to poorer resolution and spectral shift artifact on CT (Lim et al., 2000)*.* Due to higher contrast resolution, MRI can better characterize the brain morphology including the size, tissue composition such as gray (or grey) matter and white matter, and shape of different cortical or subcortical neuroanatomic structures (Lim et al., 2000). Nowadays, it is possible to use MRI to visualize and quantify the directional coherence of white matter fibers, called diffusion tensor imaging (DTI), for investigation of connectivity and disconnectivity between different brain regions (Basser et al., 1994). Additionally, MRI equipments are also used to provide functional brain responses with functional MRI (fMRI) and perfusion MRI as in some nuclear medicine neuroimaging methods such as positron emission tomography (PET) and single photon emission computed tomography (SPECT). These methods can provide pathognomonic data of certain structural lesions in AD, as they can demonstrate neuronal activity or receptor characteristics (Small, 2002). High field MRI

**1. Introduction** 

established to date.


### **Quantification of Volumetric Changes of Brain in Neurodegenerative Diseases Using Magnetic Resonance Imaging and Stereology**

Niyazi Acer1, Ahmet Tuncay Turgut2, Yelda Özsunar3 and Mehmet Turgut4

*1Dept. of Anatomy, Erciyes University School of Medicine, Kayseri, 2Dept. of Radiology, Ankara Training and Research Hospital, Ankara, 3Dept. of Radiology, Adnan Menderes University School of Medicine, Aydn, 4Dept. of Neurosurgery, Adnan Menderes University School of Medicine, Aydn, Turkey* 

#### **1. Introduction**

Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring 452

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Walle, T. (2004). Absorption and metabolism of flavonoids. *Free Radical Biology and Medicine,* 

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Simultaneous voltammetric determination of levodopa, carbidopa and benserazide in pharmaceuticals using multivariate calibration. *Talanta (Oxford),* Vol.82, No. 3,

membrane and cytoskeleton properties and increases intercellular connections in

In this chapter, we review the different magnetic resonance imaging (MRI)-based methods used to quantify whole and subcortical brain structures volume, and discuss the relevance of the brain atrophy in different neurodegenerative disseases. Although there are a lot of studies for multiple sclerosis (MS) and dementia of Alzheimer's type (AD) for the brain atrophy using different methods, the optimal method for quantifying atrophy has not been established to date.

In recent years, computed tomography (CT) scanning has been replaced with MRI scanning due to its enhanced soft-tissue resolution, especially for cerebrospinal fluid (CSF)-filled spaces, such as ventricular enlargement in patients with AD. Thus, a transition has occurred from CT to MRI in longitudinal studies investigating the human brain. As a result of development of new neuroimaging methods in clinical practice, volumetric methods started to be more sophisticated depending on various imaging methods (Lim et al., 2000). There are numerous reasons for the aforementioned transition; first of all, unlike CT, MRI has no inherent radiation effect, and secondly, CT underestimates cortical sulcal volume relative to MRI due to poorer resolution and spectral shift artifact on CT (Lim et al., 2000)*.* Due to higher contrast resolution, MRI can better characterize the brain morphology including the size, tissue composition such as gray (or grey) matter and white matter, and shape of different cortical or subcortical neuroanatomic structures (Lim et al., 2000). Nowadays, it is possible to use MRI to visualize and quantify the directional coherence of white matter fibers, called diffusion tensor imaging (DTI), for investigation of connectivity and disconnectivity between different brain regions (Basser et al., 1994). Additionally, MRI equipments are also used to provide functional brain responses with functional MRI (fMRI) and perfusion MRI as in some nuclear medicine neuroimaging methods such as positron emission tomography (PET) and single photon emission computed tomography (SPECT). These methods can provide pathognomonic data of certain structural lesions in AD, as they can demonstrate neuronal activity or receptor characteristics (Small, 2002). High field MRI

Quantification of Volumetric Changes of Brain in

**2.4 Conventional magnetic resonance imaging (MRI)** 

**2.5 Functional magnetic resonance imaging (fMRI)** 

detected (Small, 2002).

al., 1997; Ewers et al., 2011).

**2.7 Magnetic resonance spectroscopy (MRS)** 

**2.6 Diffusion tensor imaging (DTI)** 

Neurodegenerative Diseases Using Magnetic Resonance Imaging and Stereology 455

The MRI scanner detects the radiofrequency energy emitted and energy level changes represent different brain structures. Typically, T1-weighted images differentiate gray and white matter, while T2-weighted images delineate white matter hyperintensities. It is reported that spatial resolution for MRI is 1 to 2 mm, less than that of CT (Small, 2002). Fortunately, patients can have multiple MRI scans because it does not involve ionizing radiation. In MRI study, the object is placed in a high field strength magnetic field varying from 0.5 to 3 Tesla (T). Technically, different relaxation times, in addition to proton density, are measured and further manipulations by using various pulse sequences are possible. Today, various MRI techniques such as fast spin echo, high performance gradients, echo planar and diffusion weighted imaging are available for clinical use, in addition to MRI contrast agents, CSF velocity analysis, and interventional MRI (Bradley & Bydder, 1997). Recently, some technical improvements regarding the acquisition and processing of structural data have provided vivid visual representations of the external surface and internal structures of the human brain (Lim et al., 2000). Clinically, the progressive neuronal loss leading to atrophy in

With recent developments in MRI techniques, it is also possible to measure brain activity and tissue signal changes, reflecting local changes in oxygenation of haemoglobin, which depend on regional blood perfusion. Technical point of view, the signal intensity of deoxygenated hemoglobin differs from that of oxygenated hemoglobin (Belliveau et al., 1992). This MRI method also called BOLD technique. As a rule, the brain tissue during brain activity does not use this excess oxygen, causing high concentration of oxygenated blood, greater levels of magnetic field homogeneity and higher MRI signal intensity (Wagner et al., 1998). Thus, brain regions receiving greater blood flow during brain activity produce a stronger MRI signal than do other regions and areas of relative brain activity can be easily

DTI provides detailed information concerning the anatomy of white matter structure in the central nervous system. With use of DTI, visualizations of projections of axonal fibers, i.e. neuronal connectivity, is possible by quantitative evaluation on the anisotropy of water diffusion, local fiber orientation and integrity of white matter tracks (Jones et al., 1999). Technically, DTI visualizes diffusional anisotropy within each voxel as three-dimensional projections of axonal fibers. In patients with AD and other neuropsychiatric disorders, the degree of neuronal connectivity loss is a useful marker in the progression of the disease (Buchsbaum et al., 1998; Ewers et al., 2011). Moreover, recent studies revealed the presence of loss of myelin and axons in patients with AD, particularly periventricular areas (Hanyu et

From the technical view, the magnetic resonance spectrum display according to frequency shows different chemical forms of the element such as characteristic peaks, thus reflecting tissue metabolite concentrations (Weiner, 1987; Bothwell & Griffin 2011). As a noninvasive study, MRS provides quantitative regional biochemical and physiologic features of the

neurodegenerative disease increases the value of MRI (Loewe et al., 2002).

has started to further depict the regional atrophy patterns AD and other neurodegenerative disorders.

In this chapter, we aim to overview the challenging and exciting radiological methods used for the diagnosis of various neurodegenerative diseases. Specifically, we focus on the neuroimaging techniques in the first part of the chapter, their clinical applications in the second part, and methods for volume estimation including stereological techniques on the last part of the text.

#### **2. Neuroimaging techniques**

Primary neuroimaging techniques that are widely used clinically are CT, PET, SPECT, conventional MRI, fMRI, magnetic resonance spectroscopy (MRS), and tractography or DTI. These techniques enlighten different aspects of brain structures or functions (Frey et al., 1999; Small, 2002). An overview of the neuroimaging techniques is following:

#### **2.1 Computed tomography (CT)**

CT is the first imaging modality to provide *in vivo* evidence of the brain atrophy in different neurodegenerative diseases. CT images are generated by passing an x–ray beam through the skull or other object (e.g. spine and vertebral column) and it measures the attenuation of an x-ray beam through different body tissues e.g. brain, bone and CSF. Therefore, tissue's appearance will vary according to degree of its attenuation (Frey et al., 1999). The degree of attenuation can be measured numerically as a tissue density number for each voxel (volume element) and then these numbers can be converted to gray scale values and presented visually as pixels (Frey et al., 1999). Among different body tissues, the bone has the highest attenuation and appears white on CT images (Small, 2002). On the other hand, CT study has some limitations such as radiation hazards, inability to differentiate gray and white matter due to low contrast resolution and visualization of the posterior fossa structures, particularly brain stem and cerebellum. Despite to these limitations, quantitative CT still can demonstrate the presence of greater brain atrophy and ventricular dilatation in patients with AD compared with controls (Creasey et al., 1986).

#### **2.2 Single photon emission computed tomography (SPECT)**

In SPECT, the scanner determines the site of the photon source following adminstration of an unstable isotope or inhaled/injected tracer and thus an image reflecting cerebral blood flow or receptor distribution is produced (Schuckit et al., 1992). Unfortunately, its spatial resolution is not high enough for imaging deep structures and determination of the source of single photon emitters is difficult (Small, 2002).

#### **2.3 Positron emission tomography (PET)**

A PET scanner determines the line along which the photons travel, by recording the simultaneous arrival of two different photons at different detectors, and an image is then constructed from information received by the scanner. Importantly, PET study demonstrates receptor characteristics like density and affinity following injection of receptor ligands labeled with nuclides, in addition to cerebral blood flow. In patients with AD, PET studies using fluorodeoxyglucose have revealed characteristic alterations in cerebral blood flow and metabolism in the parietal, temporal and prefrontal cortices (Mazziotta et al., 1992).

#### **2.4 Conventional magnetic resonance imaging (MRI)**

454 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

has started to further depict the regional atrophy patterns AD and other neurodegenerative

In this chapter, we aim to overview the challenging and exciting radiological methods used for the diagnosis of various neurodegenerative diseases. Specifically, we focus on the neuroimaging techniques in the first part of the chapter, their clinical applications in the second part, and methods for volume estimation including stereological techniques on the

Primary neuroimaging techniques that are widely used clinically are CT, PET, SPECT, conventional MRI, fMRI, magnetic resonance spectroscopy (MRS), and tractography or DTI. These techniques enlighten different aspects of brain structures or functions (Frey et al.,

CT is the first imaging modality to provide *in vivo* evidence of the brain atrophy in different neurodegenerative diseases. CT images are generated by passing an x–ray beam through the skull or other object (e.g. spine and vertebral column) and it measures the attenuation of an x-ray beam through different body tissues e.g. brain, bone and CSF. Therefore, tissue's appearance will vary according to degree of its attenuation (Frey et al., 1999). The degree of attenuation can be measured numerically as a tissue density number for each voxel (volume element) and then these numbers can be converted to gray scale values and presented visually as pixels (Frey et al., 1999). Among different body tissues, the bone has the highest attenuation and appears white on CT images (Small, 2002). On the other hand, CT study has some limitations such as radiation hazards, inability to differentiate gray and white matter due to low contrast resolution and visualization of the posterior fossa structures, particularly brain stem and cerebellum. Despite to these limitations, quantitative CT still can demonstrate the presence of greater brain atrophy and ventricular dilatation in patients with AD

In SPECT, the scanner determines the site of the photon source following adminstration of an unstable isotope or inhaled/injected tracer and thus an image reflecting cerebral blood flow or receptor distribution is produced (Schuckit et al., 1992). Unfortunately, its spatial resolution is not high enough for imaging deep structures and determination of the source

A PET scanner determines the line along which the photons travel, by recording the simultaneous arrival of two different photons at different detectors, and an image is then constructed from information received by the scanner. Importantly, PET study demonstrates receptor characteristics like density and affinity following injection of receptor ligands labeled with nuclides, in addition to cerebral blood flow. In patients with AD, PET studies using fluorodeoxyglucose have revealed characteristic alterations in cerebral blood flow and

metabolism in the parietal, temporal and prefrontal cortices (Mazziotta et al., 1992).

1999; Small, 2002). An overview of the neuroimaging techniques is following:

disorders.

last part of the text.

**2. Neuroimaging techniques** 

**2.1 Computed tomography (CT)** 

compared with controls (Creasey et al., 1986).

of single photon emitters is difficult (Small, 2002).

**2.3 Positron emission tomography (PET)** 

**2.2 Single photon emission computed tomography (SPECT)** 

The MRI scanner detects the radiofrequency energy emitted and energy level changes represent different brain structures. Typically, T1-weighted images differentiate gray and white matter, while T2-weighted images delineate white matter hyperintensities. It is reported that spatial resolution for MRI is 1 to 2 mm, less than that of CT (Small, 2002). Fortunately, patients can have multiple MRI scans because it does not involve ionizing radiation. In MRI study, the object is placed in a high field strength magnetic field varying from 0.5 to 3 Tesla (T). Technically, different relaxation times, in addition to proton density, are measured and further manipulations by using various pulse sequences are possible. Today, various MRI techniques such as fast spin echo, high performance gradients, echo planar and diffusion weighted imaging are available for clinical use, in addition to MRI contrast agents, CSF velocity analysis, and interventional MRI (Bradley & Bydder, 1997). Recently, some technical improvements regarding the acquisition and processing of structural data have provided vivid visual representations of the external surface and internal structures of the human brain (Lim et al., 2000). Clinically, the progressive neuronal loss leading to atrophy in neurodegenerative disease increases the value of MRI (Loewe et al., 2002).

#### **2.5 Functional magnetic resonance imaging (fMRI)**

With recent developments in MRI techniques, it is also possible to measure brain activity and tissue signal changes, reflecting local changes in oxygenation of haemoglobin, which depend on regional blood perfusion. Technical point of view, the signal intensity of deoxygenated hemoglobin differs from that of oxygenated hemoglobin (Belliveau et al., 1992). This MRI method also called BOLD technique. As a rule, the brain tissue during brain activity does not use this excess oxygen, causing high concentration of oxygenated blood, greater levels of magnetic field homogeneity and higher MRI signal intensity (Wagner et al., 1998). Thus, brain regions receiving greater blood flow during brain activity produce a stronger MRI signal than do other regions and areas of relative brain activity can be easily detected (Small, 2002).

#### **2.6 Diffusion tensor imaging (DTI)**

DTI provides detailed information concerning the anatomy of white matter structure in the central nervous system. With use of DTI, visualizations of projections of axonal fibers, i.e. neuronal connectivity, is possible by quantitative evaluation on the anisotropy of water diffusion, local fiber orientation and integrity of white matter tracks (Jones et al., 1999). Technically, DTI visualizes diffusional anisotropy within each voxel as three-dimensional projections of axonal fibers. In patients with AD and other neuropsychiatric disorders, the degree of neuronal connectivity loss is a useful marker in the progression of the disease (Buchsbaum et al., 1998; Ewers et al., 2011). Moreover, recent studies revealed the presence of loss of myelin and axons in patients with AD, particularly periventricular areas (Hanyu et al., 1997; Ewers et al., 2011).

#### **2.7 Magnetic resonance spectroscopy (MRS)**

From the technical view, the magnetic resonance spectrum display according to frequency shows different chemical forms of the element such as characteristic peaks, thus reflecting tissue metabolite concentrations (Weiner, 1987; Bothwell & Griffin 2011). As a noninvasive study, MRS provides quantitative regional biochemical and physiologic features of the

Quantification of Volumetric Changes of Brain in

pixel in the image matrix represents 1 mm2 (Lim et al., 2000).

and deep gray matter nuclei (Keller & Roberts, 2009)*.*

associated with white matter loss and gliosis (Kern & Behl, 2009).

withdrawal, and hallucinations (Mohs & Haroutunian, 2002)*.*

**3. Clinical applications 3.1 Dementia syndromes** 

**3.1.1 Alzheimer disease (AD)** 

Neurodegenerative Diseases Using Magnetic Resonance Imaging and Stereology 457

It is important to know that 2D image has a limitation so that only selected slices imaged and therefore a comparison across subjects is difficult, although it may be possible by orienting each slice acquisition relative to a specific anatomic plane. On the other hand, three-dimensional (3D) volume acquisition protocols include the entire brain and they are widely used in psychiatric neuroimaging. Using T1-weighted MRI sections with a good gray/white matter differentiation, the entire brain with 1.5 mm or thinner slices are obtained in 10 minutes or less. As a rule, an in-plane resolution of 1 mm means that each

Quantitative investigations of the brain using MRI may reveal important information about the function and organisation of the brain being studied, recently. MRI has become the method of choice for the examination of macroscopic neuroanatomy in vivo due to excellent levels of image resolution and between tissue contrasts. Estimation of brain compartment volume needs high resolution MRIs for the delineation of anatomical boundaries. With the use of higher magnetic field strength, a better image quality with can be obtained using thinner slices and shorter imaging time. For this reason, many researchers frequently use MRI scanners which are either with 1.5 T or 3 T systems (Fig. 2). Although 3 T systems offer increased resolution of between-tissue contrast (i.e. increased visualisation of the borders between gray matter, white matter and CSF), MRI scans on 1.5 T systems are sufficient for the quantification of relatively small brain structures, such as the hippocampus, amygdala,

A number of studies reported that patients with MS have smaller volumes of the parenchyma than in age-matched control subjects (Bermel et al., 2003; Sanfilipo et al., 2005, 2006). The first method used for the estimation of brain atrophy is linear measurement of ventricles or other brain structural dimensions (Smith et al., 2002). In general, MRI studies reveal some differences in the volume of the brain structures in certain neurodegenerative diseases, an inhomogeneous group of neurological diseases with unknown etiology, such as demantia. In such diseases, multiple systems or one system or one group of nuclei may partly or totally be involved (Loewe et al., 2002). Basically, there are two principal pathological processes which determine imaging findings: neuronal or white matter loss and deposition of different compounds. The loss of neurons leads to progressive atrophy

Nowadays, dementia is a well-known illness with a high incidence in the aged population. Clinically, there are a number of neurodegenerative diseases causing dementia, including AD, dementia with Lewy bodies, and frontotemporal dementia. Furthermore, dementia picture is also present in some neurodegenerative illnesses including Creutzfeldt–Jakob disease, Huntington's disease, progressive supranuclear palsy, multiple system atrophy, amyotrophic lateral sclerosis, and Parkinson's disease (Loewe et al., 2002; Vitali et al., 2008).

AD is well-known progressive neurodegenerative pathology, accounting for around 60% of all cases dementia. Clinically, patients with AD have serious cognitive findings related with memory, language, such as confusion, poor judgment, language disturbance, agitation,

tissue. To determine *N*-acetylaspartate (NAA) content of hippocampus in patients with AD, some authors used proton MRS (1H MRS) and volumetric MRI (Weiner, 1987; Schuff et al. 1997).

#### **2.8 Improvements in magnetic resonance imaging 2.8.1 Mechanisms of tissue contrast: Pulse sequences**

By varying elements of the image acquisition sequence of MRI, it is possible to manipulate the amount of contrast between various tissues. It is well-known that hydrogen atoms are the most important element of the tissue and MRI device demonstrate signals related with free water. Based on proton density and relaxation time of any tissue, different structures will appear in an acquired image*.* T1 means the time taken for excited nuclei to return to equilibrium, while T2 is an xponential time constant related with the time for the excited nuclei to lose signal (Lim et al., 2000). Technical view of point, the time between radiofrequency pulses (TR) and the amount of time after the pulse called echo time (TE) are important parameters; a long TR and a long TE give T2-weighted image, while a short TR and a short TE gives T1-weighted image. Although T1-weighted spin-echo and inversion recovery sequences have poor definition of CSF/skull margins for reliably measuring intracranial volume, they are used for morphometric studies because they provide good white-gray contrast (Lim et al., 2000)*.*

Sources of contrast other than that based on manipulation of T1, T2, fluid-attenuated inversion recovery (FLAIR) and proton density are used to obtain further information. T1 weighted images are superior to T2-weighted images for the evaluation of atrophy, because T2-weighted ones overestimate the dimensions of ventricles and sulci (Kucharczyk & Henkelman, 1994). On the other hand, T1-weighted imaging gives a clear distinction between grey matter, white matter and CSF; therefore, they are used for quantitative MRI studies of brain morphology, particularly of individual brain structures (Keller & Roberts, 2009) (Fig. 1)*.*

Fast spin echo T2 sequences has been usually used in brain imaging due to their short acquisition time and increased robustness to motion artifacts. In imaging of neurodegenerative disorders like Parkinson-like syndromes, however, gradient echo T2 weighted spin-echo sequences are preferred because they increase the sensitivity for paramagnetic materials (ferritin, melanin etc.). Also, proton-density or FLAIR sequences identify gliosis owing to result of progressive neuronal loss (Loewe et al., 2002). Therefore, T2-weighted imaging may be used for determination of intracranial volume as the increased signal intensity of CSF provides better determination of CSF and the parenchyma of the brain (Keller & Roberts, 2009)*.* Therefore, the type of MRI sequence used is important for volume estimation.

#### **2.8.2 Two-dimensional multi-slice and three-dimensional imaging**

Two-dimensional (2D) images are obtained in axial, sagittal and coronal planes (Fig. 1). Image orientation, giving a different view of the brain with optimal visualization of different structures, is described according to radiofrequency pulse excitations and the magnetic gradients in three orthogonal axes. Basically, a mid-sagittal section provides an image of the corpus callosum and the prefrontal cortex, coronal section gives an image of the limbic structures including hippocampus, and axial section gives an image of basal ganglia structures and the lateral ventricular system.

It is important to know that 2D image has a limitation so that only selected slices imaged and therefore a comparison across subjects is difficult, although it may be possible by orienting each slice acquisition relative to a specific anatomic plane. On the other hand, three-dimensional (3D) volume acquisition protocols include the entire brain and they are widely used in psychiatric neuroimaging. Using T1-weighted MRI sections with a good gray/white matter differentiation, the entire brain with 1.5 mm or thinner slices are obtained in 10 minutes or less. As a rule, an in-plane resolution of 1 mm means that each pixel in the image matrix represents 1 mm2 (Lim et al., 2000).

Quantitative investigations of the brain using MRI may reveal important information about the function and organisation of the brain being studied, recently. MRI has become the method of choice for the examination of macroscopic neuroanatomy in vivo due to excellent levels of image resolution and between tissue contrasts. Estimation of brain compartment volume needs high resolution MRIs for the delineation of anatomical boundaries. With the use of higher magnetic field strength, a better image quality with can be obtained using thinner slices and shorter imaging time. For this reason, many researchers frequently use MRI scanners which are either with 1.5 T or 3 T systems (Fig. 2). Although 3 T systems offer increased resolution of between-tissue contrast (i.e. increased visualisation of the borders between gray matter, white matter and CSF), MRI scans on 1.5 T systems are sufficient for the quantification of relatively small brain structures, such as the hippocampus, amygdala, and deep gray matter nuclei (Keller & Roberts, 2009)*.*

#### **3. Clinical applications**

456 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

tissue. To determine *N*-acetylaspartate (NAA) content of hippocampus in patients with AD, some authors used proton MRS (1H MRS) and volumetric MRI (Weiner, 1987; Schuff et al.

By varying elements of the image acquisition sequence of MRI, it is possible to manipulate the amount of contrast between various tissues. It is well-known that hydrogen atoms are the most important element of the tissue and MRI device demonstrate signals related with free water. Based on proton density and relaxation time of any tissue, different structures will appear in an acquired image*.* T1 means the time taken for excited nuclei to return to equilibrium, while T2 is an xponential time constant related with the time for the excited nuclei to lose signal (Lim et al., 2000). Technical view of point, the time between radiofrequency pulses (TR) and the amount of time after the pulse called echo time (TE) are important parameters; a long TR and a long TE give T2-weighted image, while a short TR and a short TE gives T1-weighted image. Although T1-weighted spin-echo and inversion recovery sequences have poor definition of CSF/skull margins for reliably measuring intracranial volume, they are used for morphometric studies because they provide good

Sources of contrast other than that based on manipulation of T1, T2, fluid-attenuated inversion recovery (FLAIR) and proton density are used to obtain further information. T1 weighted images are superior to T2-weighted images for the evaluation of atrophy, because T2-weighted ones overestimate the dimensions of ventricles and sulci (Kucharczyk & Henkelman, 1994). On the other hand, T1-weighted imaging gives a clear distinction between grey matter, white matter and CSF; therefore, they are used for quantitative MRI studies of brain morphology, particularly of individual brain structures (Keller & Roberts,

Fast spin echo T2 sequences has been usually used in brain imaging due to their short acquisition time and increased robustness to motion artifacts. In imaging of neurodegenerative disorders like Parkinson-like syndromes, however, gradient echo T2 weighted spin-echo sequences are preferred because they increase the sensitivity for paramagnetic materials (ferritin, melanin etc.). Also, proton-density or FLAIR sequences identify gliosis owing to result of progressive neuronal loss (Loewe et al., 2002). Therefore, T2-weighted imaging may be used for determination of intracranial volume as the increased signal intensity of CSF provides better determination of CSF and the parenchyma of the brain (Keller & Roberts, 2009)*.* Therefore, the type of MRI sequence used is important for

Two-dimensional (2D) images are obtained in axial, sagittal and coronal planes (Fig. 1). Image orientation, giving a different view of the brain with optimal visualization of different structures, is described according to radiofrequency pulse excitations and the magnetic gradients in three orthogonal axes. Basically, a mid-sagittal section provides an image of the corpus callosum and the prefrontal cortex, coronal section gives an image of the limbic structures including hippocampus, and axial section gives an image of basal ganglia

**2.8.2 Two-dimensional multi-slice and three-dimensional imaging** 

structures and the lateral ventricular system.

**2.8 Improvements in magnetic resonance imaging 2.8.1 Mechanisms of tissue contrast: Pulse sequences** 

white-gray contrast (Lim et al., 2000)*.*

2009) (Fig. 1)*.*

volume estimation.

1997).

#### **3.1 Dementia syndromes**

A number of studies reported that patients with MS have smaller volumes of the parenchyma than in age-matched control subjects (Bermel et al., 2003; Sanfilipo et al., 2005, 2006). The first method used for the estimation of brain atrophy is linear measurement of ventricles or other brain structural dimensions (Smith et al., 2002). In general, MRI studies reveal some differences in the volume of the brain structures in certain neurodegenerative diseases, an inhomogeneous group of neurological diseases with unknown etiology, such as demantia. In such diseases, multiple systems or one system or one group of nuclei may partly or totally be involved (Loewe et al., 2002). Basically, there are two principal pathological processes which determine imaging findings: neuronal or white matter loss and deposition of different compounds. The loss of neurons leads to progressive atrophy associated with white matter loss and gliosis (Kern & Behl, 2009).

Nowadays, dementia is a well-known illness with a high incidence in the aged population. Clinically, there are a number of neurodegenerative diseases causing dementia, including AD, dementia with Lewy bodies, and frontotemporal dementia. Furthermore, dementia picture is also present in some neurodegenerative illnesses including Creutzfeldt–Jakob disease, Huntington's disease, progressive supranuclear palsy, multiple system atrophy, amyotrophic lateral sclerosis, and Parkinson's disease (Loewe et al., 2002; Vitali et al., 2008).

#### **3.1.1 Alzheimer disease (AD)**

AD is well-known progressive neurodegenerative pathology, accounting for around 60% of all cases dementia. Clinically, patients with AD have serious cognitive findings related with memory, language, such as confusion, poor judgment, language disturbance, agitation, withdrawal, and hallucinations (Mohs & Haroutunian, 2002)*.*

Quantification of Volumetric Changes of Brain in

Neurodegenerative Diseases Using Magnetic Resonance Imaging and Stereology 459

Gross examination of the brain in patients with AD demonstrated an obvious atrophy, widening of the sulci, and erosion of the gyri. Histologically, the atrophy of the cortex is associated with significant reductions in the numbers of neurons. Macroscopically, the weight of the brain is decreased compared to normal controls (Masliah et al., 1991). In a previous study using unbiased stereologic sampling techniques, about 50% loss in neurons of the superior temporal gyrus has been reported (Gomez-Isla et al., 1996, 1997). In another study, 40-46% loss of large neurons in the frontal and temporal cortices of specimens has been reported in patients with AD (Terry et al., 1981). In fact, neuronal loss and degeneration are not restricted to the cortex; it may be observed in subcortical nuclei such as the locus ceruleus, raphe aminergic nuclei (Zweig et al., 1988; Chan-Palay & Asan, 1989), and the nucleus basalis of Meynert (Whitehouse et al., 1982). In such cases, synaptic markers such as synaptophysin are significantly reduced in the cerebral cortex, especially the frontal

and parietal cortices and in the hippocampus, with increasing age (Nagy et al., 1995).

and ERC volumes in patients with AD (Pearlson et al., 1992; Obrien, 2007).

pathology (Neary et al., 1998; McKhann, 2001; Ratnavalli, 2002).

technologies (Mohs & Haroutunian, 2002).

**3.1.2 Frontotemporal demantia (FTD)** 

**3.1.3 Dementia with Lewy bodies (DLB)** 

Radiologically, MRI provides understanding of disease progression in AD and other dementias. Recently, it has been reported that patients with AD have atrophy in parietal lobes, medial temporal lobe and hippocampus on MRI (Loewe et al., 2002, Vitali et al., 2008). The parietal lobe atrophy is observed on axial or coronal T1-weighted or FLAIR sequences with thinning of the posterior part of the body of the corpus callosum on T1-weighted sagittal sequences (Yamauchi et al., 2000). In some studies, decreased hippocampal and entorhinal cortex (ERC) volumes in patients with AD were noted (Appel et al., 2009). Hippocampal atrophy is observed with thin coronal T1-weighted or FLAIR tomographic slices through the medial temporal lobes (Teipel et al., 2003). A lot of quantitative MRI studies indicate that white matter hyperintensities correlate with neuropsychological functioning in both healthy elderly persons and demented patients (Boone et al., 1992; Lopez et al., 1992). Other studies indicate loss of cerebral gray matter (Rusinek et al., 1991), hippocampal and parahippocampal atrophy (Kesslak et al., 1991), and lower left amygdala

Recently, a longitudinal study demonstrated that most common neuropathologic findings in elderly patients are neuritic plaques and neurofibrillary tangles (Mohs &Haroutunian, 2002). The presence of these findings before clinical AD diagnosis suggests that *in vivo* methods that directly image these pathognomonic lesions would be useful presymptomatic detection

Frontotemporal demantia (FTD) is as common a cause of dementia. In particular, volumes of some regions of the frontal lobe (the ventromedial and posterior orbital regions of the frontal lobe), the cingulate cortex, and the insula are reduced in patients with the FTD, compared with those of both AD patients and age-matched controls. This feature differentiates this illness from AD as these areas are relatively spared in the latter disease (Rosen et al., 2002). In patients with the semantic variant of FTD, there is a relative preservation of frontal lobe volumes but marked loss of volumes in the temporal lobes (Rosen et al,. 2005, 2006). In clinical practice, FTD includes a group of neurodegenerative diseases characterized by focal atrophy of frontal and anterior temporal lobes and non-AD

Dementia with Lewy bodies results a diffuse, irreversible and destructive atrophy (Seppi & Schocke, 2005). Measurement of brain volume to predict atrophy using MRI may be used as

Fig. 1. T1-weighted MRI scans acquired in coronal (left), axial (center) and sagittal (right) planes with 3 T. All images were acquired with a field of view of 25 cm and 256 x 256 matrix, 1-mm slice thickness. Image was acquired using a turbo field echo sequence, gated to achieve an effective TR of >8 ms and TE of 4 ms. Left: Coronal image passing through lateral ventricles and temporal lobes. Center: Axial image passing through the lateral ventricles and basal ganglia. Right: Mid-sagittal image highlighting the corpus callosum, brain stem and cerebellum

Fig. 2. T1-weighted axial MRIs acquired with 1.5 T (left) and 3T (right) MRI scanners. 3T MRI was acquired with a field of view of 25 cm and 256 x 256 matrix, 1-mm slice thickness. Image was acquired using a turbo field echo sequence, gated to achieve an effective TR of >8 ms and TE of 4 ms. 1.5T MRI was acquired with field of view of 24 cm, 1.5 mm slice thickness. Image was acquired using a spoiled gradient recalled acquisition sequence, gated to achieve an effective TR of >35 ms and TE of 15 ms

Fig. 1. T1-weighted MRI scans acquired in coronal (left), axial (center) and sagittal (right) planes with 3 T. All images were acquired with a field of view of 25 cm and 256 x 256 matrix, 1-mm slice thickness. Image was acquired using a turbo field echo sequence, gated to achieve an effective TR of >8 ms and TE of 4 ms. Left: Coronal image passing through lateral ventricles and temporal lobes. Center: Axial image passing through the lateral ventricles and basal ganglia. Right: Mid-sagittal image highlighting the corpus callosum,

Fig. 2. T1-weighted axial MRIs acquired with 1.5 T (left) and 3T (right) MRI scanners. 3T MRI was acquired with a field of view of 25 cm and 256 x 256 matrix, 1-mm slice thickness. Image was acquired using a turbo field echo sequence, gated to achieve an effective TR of >8

thickness. Image was acquired using a spoiled gradient recalled acquisition sequence, gated

ms and TE of 4 ms. 1.5T MRI was acquired with field of view of 24 cm, 1.5 mm slice

to achieve an effective TR of >35 ms and TE of 15 ms

brain stem and cerebellum

Gross examination of the brain in patients with AD demonstrated an obvious atrophy, widening of the sulci, and erosion of the gyri. Histologically, the atrophy of the cortex is associated with significant reductions in the numbers of neurons. Macroscopically, the weight of the brain is decreased compared to normal controls (Masliah et al., 1991). In a previous study using unbiased stereologic sampling techniques, about 50% loss in neurons of the superior temporal gyrus has been reported (Gomez-Isla et al., 1996, 1997). In another study, 40-46% loss of large neurons in the frontal and temporal cortices of specimens has been reported in patients with AD (Terry et al., 1981). In fact, neuronal loss and degeneration are not restricted to the cortex; it may be observed in subcortical nuclei such as the locus ceruleus, raphe aminergic nuclei (Zweig et al., 1988; Chan-Palay & Asan, 1989), and the nucleus basalis of Meynert (Whitehouse et al., 1982). In such cases, synaptic markers such as synaptophysin are significantly reduced in the cerebral cortex, especially the frontal and parietal cortices and in the hippocampus, with increasing age (Nagy et al., 1995).

Radiologically, MRI provides understanding of disease progression in AD and other dementias. Recently, it has been reported that patients with AD have atrophy in parietal lobes, medial temporal lobe and hippocampus on MRI (Loewe et al., 2002, Vitali et al., 2008). The parietal lobe atrophy is observed on axial or coronal T1-weighted or FLAIR sequences with thinning of the posterior part of the body of the corpus callosum on T1-weighted sagittal sequences (Yamauchi et al., 2000). In some studies, decreased hippocampal and entorhinal cortex (ERC) volumes in patients with AD were noted (Appel et al., 2009). Hippocampal atrophy is observed with thin coronal T1-weighted or FLAIR tomographic slices through the medial temporal lobes (Teipel et al., 2003). A lot of quantitative MRI studies indicate that white matter hyperintensities correlate with neuropsychological functioning in both healthy elderly persons and demented patients (Boone et al., 1992; Lopez et al., 1992). Other studies indicate loss of cerebral gray matter (Rusinek et al., 1991), hippocampal and parahippocampal atrophy (Kesslak et al., 1991), and lower left amygdala and ERC volumes in patients with AD (Pearlson et al., 1992; Obrien, 2007).

Recently, a longitudinal study demonstrated that most common neuropathologic findings in elderly patients are neuritic plaques and neurofibrillary tangles (Mohs &Haroutunian, 2002). The presence of these findings before clinical AD diagnosis suggests that *in vivo* methods that directly image these pathognomonic lesions would be useful presymptomatic detection technologies (Mohs & Haroutunian, 2002).

#### **3.1.2 Frontotemporal demantia (FTD)**

Frontotemporal demantia (FTD) is as common a cause of dementia. In particular, volumes of some regions of the frontal lobe (the ventromedial and posterior orbital regions of the frontal lobe), the cingulate cortex, and the insula are reduced in patients with the FTD, compared with those of both AD patients and age-matched controls. This feature differentiates this illness from AD as these areas are relatively spared in the latter disease (Rosen et al., 2002). In patients with the semantic variant of FTD, there is a relative preservation of frontal lobe volumes but marked loss of volumes in the temporal lobes (Rosen et al,. 2005, 2006). In clinical practice, FTD includes a group of neurodegenerative diseases characterized by focal atrophy of frontal and anterior temporal lobes and non-AD pathology (Neary et al., 1998; McKhann, 2001; Ratnavalli, 2002).

#### **3.1.3 Dementia with Lewy bodies (DLB)**

Dementia with Lewy bodies results a diffuse, irreversible and destructive atrophy (Seppi & Schocke, 2005). Measurement of brain volume to predict atrophy using MRI may be used as

Quantification of Volumetric Changes of Brain in

time consuming method (Flippi et al., 1998).

are possible (Keller & Roberts, 2009).

MRI sections (Keller & Roberts, 2009).

a single sequence.

Neurodegenerative Diseases Using Magnetic Resonance Imaging and Stereology 461

easier. Depending on the compartment of interest, tissue contrast can be chosen such as CSF/parenchyma or gray/white matter (Grassiot et al., 2009). There are many different segmentation methods for estimating brain volume using manual or automated techniques. Flippi et al. (1998) used manual technique for whole brain volume with MS. Although the manual tracing of brain structure allows brain volume to be estimated, this technique is a

Semi-automated techniques, quicker and more reproducible, use various algorithms of brain segmentation from 3D volume (Horsfield et al., 2003). For both semi-automated and automated methods, however, manual defining of brain structures is necessary. In semiautomated methods, manual marking of some anatomical landmarks and an automatic segmentation of the region of interest (ROI) are required, while automated methods are completely user-independent in the determination of various parameters such as brain size and shape. Importantly, experienced raters with detailed knowledge of neuroanatomy are necessary for manual techniques and correct estimations related to the neuroanatomical ROI

Automated and semi-automated methods for segmentation and quantification of the brain are used in most studies. More recently, various image analysis tools have been developed, including both automated and semi-automated algorithms, relying on either raw or normalized brain volume assessments (Pelletier et al., 2004). Several previous studies have described automatic segmentation methods using MRIs. Calmon & Roberts (2000) reported a segmentation method for the lateral ventricles on coronal MR images. Stokking et al. (2000) described the development of a morphology-based brain segmentation method for fully automatic segmentation of the brain using T1-weighted MRI data. Webb et al. (1999) reported a method of automatic detection of the hippocampus with atrophy. These methods each segmented one target object on each MRI obtained by different sequences. Therefore, these methods could not segment two or more objects simultaneously on MRIs obtained by

There are two primary methods for manual quantification of brain compartment volume from MRIs, namely stereology in conjunction with point counting and planimetric methods or manual tracing (Acer et al., 2007; Keller & Roberts, 2009). Authors used manual tracing of brain boundaries from MRI scans using various softwares such as Analyze (Biomedical Imaging Resource, Mayo Foundation), BRAINS (Iowa Mental Health Clinical Research Center 2008), and FreeSurfer (Dale et al., 1999; Fischl et al., 1999; Fischl et al., 2002). Manual techniques such as planimetry or tracing methods require the investigator to delineate a brain region based on reliable anatomical landmarks, whilst the software package provides information on volume. Tracing methods require the investigator to trace the brain ROI using a mouse driven cursor throughout a defined number of MRI sections (Keller & Roberts, 2009). The cut surface areas, determined by pixel counting within the traced region, are summed and multiplied by the distance between the consecutive sections traced to estimate the total volume. Although tracing methods represent the most commonly used tool to estimate brain structure volume on MRIs, there are some drawbacks associated this technique (Geuze et al., 2005). Firstly, the time taken to perform manual tracing or manual segmentation methods is significantly longer than stereological point counting methods (Acer et al., 2007, 2008; Keller & Roberts, 2009). Secondly, tracing and manual segmentation methods suffer from the risk of "hand wobble" during the delineation of ROI boundaries on

The measurement of rates of change requires volume quantification. In general, manual or semi-automated methods have been employed for volume quantification on structural brain

a predicter for outcome in different neurodegenerative diseases such as AD. There are a lot of biologic factors influencing cerebral volume measurement such as inflammation and edema, cerebrovascular disease, chronic alcoholism and normal aging (Ron et al., 1982; Molyneux et al., 2000).

#### **3.2 Multiple Sclerosis (MS)**

In cases with MS, various measurement techniques revealed atrophy of brain and spinal cord, axonal loss, and Wallerian degeneration (Sharma et al., 2004). Recent studies show that the MS is a destructive disease process and whole-brain atrophy is a valuable marker for the progression of the disease (Sharma et al., 2004).

#### **3.3 Medial temporal lobe epilepsy (MTLE)**

In patients with medial temporal lobe epilepsy (MTLE), the atrophy of the hippocampus is often observed on routine MRI. Recently, it has been reported that automatic morphometry can be used as a clinical tool to provide a quantifiable estimation of of hippocampal atrophy in patients with MTLE (Bonilha et al., 2009). Most recently, Henry et al. (2011) suggested that ultrahigh-field-strength MRI revealed prominent atrophy of Ammon horn in patients with MTLE and hippocampal sclerosis.

#### **3.4 Ageing**

With increasing age, there are some volumetric changes in the gray matter structures of the temporal lobe, amygdale, and hippocampus, a critical structure for memory in AD, but they are heterogenous, with some regions showing more atrophic changes than others (O'Sullivan, 2009). Recently, it has been reported that it is possible to differentiate ageing from AD with 87% accuracy (Likeman et al., 2005; O'Sullivan, 2009). Some volumetric studies demonstrated that changes in white matter regions provide an early and accurate diagnosis (Davatzikos et al., 2008).

#### **4. Methods for volume estimation**

#### **4.1 Manual, automated and semiautomated methods for volume estimation**

Use of imaging methods for quantitive volume estimation such as manual, semi-automated and automated methods can provide the capability to reliably detect and identify general and specific structural abnormalities of the brain. Use of these methods can aid to diagnose some specific neurological diseases and facilitate monitoring of the progression of the disease. Quantative measures of the brain atrophy can be clinically relevant and much work has been carried out to establish diagnosis of AD (Furlong, 2008).

At present, a number of manual, semi-automated and automated methods based on conventional MRI are available for measuring whole or regional brain volume. Ideally, the technique for measuring tissue volume should be reproducible, sensitive to subtle modifications, practical, fast and correct. Theoretically, many factors may affect the quantification of brain atrophy using segmentation methods, such as the pulse sequence and the resolution parameters chosen for the acquisition (Horsfield et al., 2003; Sharma et al., 2004).

One of these most important factors is slice thickness. The use of thin slice helps to reduce the partial volume effect and consequently permits a better estimation of tissue volumes. Moreover, high contrast makes segmentation between the different cerebral compartments

a predicter for outcome in different neurodegenerative diseases such as AD. There are a lot of biologic factors influencing cerebral volume measurement such as inflammation and edema, cerebrovascular disease, chronic alcoholism and normal aging (Ron et al., 1982;

In cases with MS, various measurement techniques revealed atrophy of brain and spinal cord, axonal loss, and Wallerian degeneration (Sharma et al., 2004). Recent studies show that the MS is a destructive disease process and whole-brain atrophy is a valuable marker for the

In patients with medial temporal lobe epilepsy (MTLE), the atrophy of the hippocampus is often observed on routine MRI. Recently, it has been reported that automatic morphometry can be used as a clinical tool to provide a quantifiable estimation of of hippocampal atrophy in patients with MTLE (Bonilha et al., 2009). Most recently, Henry et al. (2011) suggested that ultrahigh-field-strength MRI revealed prominent atrophy of Ammon horn in patients

With increasing age, there are some volumetric changes in the gray matter structures of the temporal lobe, amygdale, and hippocampus, a critical structure for memory in AD, but they are heterogenous, with some regions showing more atrophic changes than others (O'Sullivan, 2009). Recently, it has been reported that it is possible to differentiate ageing from AD with 87% accuracy (Likeman et al., 2005; O'Sullivan, 2009). Some volumetric studies demonstrated that changes in white matter regions provide an early and accurate

Use of imaging methods for quantitive volume estimation such as manual, semi-automated and automated methods can provide the capability to reliably detect and identify general and specific structural abnormalities of the brain. Use of these methods can aid to diagnose some specific neurological diseases and facilitate monitoring of the progression of the disease. Quantative measures of the brain atrophy can be clinically relevant and much work

At present, a number of manual, semi-automated and automated methods based on conventional MRI are available for measuring whole or regional brain volume. Ideally, the technique for measuring tissue volume should be reproducible, sensitive to subtle modifications, practical, fast and correct. Theoretically, many factors may affect the quantification of brain atrophy using segmentation methods, such as the pulse sequence and the resolution parameters chosen for the acquisition (Horsfield et al., 2003; Sharma et al.,

One of these most important factors is slice thickness. The use of thin slice helps to reduce the partial volume effect and consequently permits a better estimation of tissue volumes. Moreover, high contrast makes segmentation between the different cerebral compartments

**4.1 Manual, automated and semiautomated methods for volume estimation** 

has been carried out to establish diagnosis of AD (Furlong, 2008).

Molyneux et al., 2000).

**3.4 Ageing** 

2004).

**3.2 Multiple Sclerosis (MS)** 

progression of the disease (Sharma et al., 2004).

**3.3 Medial temporal lobe epilepsy (MTLE)** 

with MTLE and hippocampal sclerosis.

diagnosis (Davatzikos et al., 2008).

**4. Methods for volume estimation** 

easier. Depending on the compartment of interest, tissue contrast can be chosen such as CSF/parenchyma or gray/white matter (Grassiot et al., 2009). There are many different segmentation methods for estimating brain volume using manual or automated techniques. Flippi et al. (1998) used manual technique for whole brain volume with MS. Although the manual tracing of brain structure allows brain volume to be estimated, this technique is a time consuming method (Flippi et al., 1998).

Semi-automated techniques, quicker and more reproducible, use various algorithms of brain segmentation from 3D volume (Horsfield et al., 2003). For both semi-automated and automated methods, however, manual defining of brain structures is necessary. In semiautomated methods, manual marking of some anatomical landmarks and an automatic segmentation of the region of interest (ROI) are required, while automated methods are completely user-independent in the determination of various parameters such as brain size and shape. Importantly, experienced raters with detailed knowledge of neuroanatomy are necessary for manual techniques and correct estimations related to the neuroanatomical ROI are possible (Keller & Roberts, 2009).

Automated and semi-automated methods for segmentation and quantification of the brain are used in most studies. More recently, various image analysis tools have been developed, including both automated and semi-automated algorithms, relying on either raw or normalized brain volume assessments (Pelletier et al., 2004). Several previous studies have described automatic segmentation methods using MRIs. Calmon & Roberts (2000) reported a segmentation method for the lateral ventricles on coronal MR images. Stokking et al. (2000) described the development of a morphology-based brain segmentation method for fully automatic segmentation of the brain using T1-weighted MRI data. Webb et al. (1999) reported a method of automatic detection of the hippocampus with atrophy. These methods each segmented one target object on each MRI obtained by different sequences. Therefore, these methods could not segment two or more objects simultaneously on MRIs obtained by a single sequence.

There are two primary methods for manual quantification of brain compartment volume from MRIs, namely stereology in conjunction with point counting and planimetric methods or manual tracing (Acer et al., 2007; Keller & Roberts, 2009). Authors used manual tracing of brain boundaries from MRI scans using various softwares such as Analyze (Biomedical Imaging Resource, Mayo Foundation), BRAINS (Iowa Mental Health Clinical Research Center 2008), and FreeSurfer (Dale et al., 1999; Fischl et al., 1999; Fischl et al., 2002). Manual techniques such as planimetry or tracing methods require the investigator to delineate a brain region based on reliable anatomical landmarks, whilst the software package provides information on volume. Tracing methods require the investigator to trace the brain ROI using a mouse driven cursor throughout a defined number of MRI sections (Keller & Roberts, 2009). The cut surface areas, determined by pixel counting within the traced region, are summed and multiplied by the distance between the consecutive sections traced to estimate the total volume. Although tracing methods represent the most commonly used tool to estimate brain structure volume on MRIs, there are some drawbacks associated this technique (Geuze et al., 2005). Firstly, the time taken to perform manual tracing or manual segmentation methods is significantly longer than stereological point counting methods (Acer et al., 2007, 2008; Keller & Roberts, 2009). Secondly, tracing and manual segmentation methods suffer from the risk of "hand wobble" during the delineation of ROI boundaries on MRI sections (Keller & Roberts, 2009).

The measurement of rates of change requires volume quantification. In general, manual or semi-automated methods have been employed for volume quantification on structural brain

Quantification of Volumetric Changes of Brain in

Neurodegenerative Diseases Using Magnetic Resonance Imaging and Stereology 463

Cortical Region Masking

Morfological Image Processing

Fuzzy C Means Clustering

Boundery Analysis

Calculation of Surface Area and Cortical Volume

Fig. 4. Segmentation called the Fuzzy C. Left: Original T1-weighted image. Center: Masking and removal of artifacts the outer contour of the brain. Right: Contour of segmented image

The Cavalieri method in combination with point counting requires beginning from a uniform random starting within the sectioning interval, a structure of interest is exhaustively sectioned with a series of parallel plane probes a constant distant apart. An unbiased estimate of volume is obtained by multiplying the total area of all sections through the

Fig. 3. Brain image segmentation blocks (Nakamura & Fisher, 2009)

is outlined

**4.4.1 Point-counting method** 

structure by sectioning interval *t* as following:

MRIs, but they are generally severely limited in practicality and reliability. For example, a high-resolution 3D brain MRI data set can contain more than 100 slices to cover an entire brain (Keller & Roberts, 2009). Manually delineating tissue boundaries for volumetric measurement can be a tedious and demanding process because of the presence of the extremely complex convoluted structures of the brain. Manual tracing is also well-known to be associated with large subjective variability and low reproducibility. As a result, methods with better reproducibility and higher precision are required for measuring subtle neuroanatomic changes and these methods are likely to be based on computerized approaches. FreeSurfer is freely available on the World Wide Web (www or commonly known as the Web), it has been widely used in the neuroimaging field. At present, fully automated methods are most often used. Fully automated or semi-automated methods can be applied to a specific ROI (such as the thalamus or the hippocampus) to obtain a regional brain volume (Houtchens et al., 2007).

#### **4.2 Brain segmentation**

Brain tissue segmentation of MRIs means to specify the tissue type for each pixel or voxel in a 2D or 3D data set, respectively, on the basis of information available from both MRIs and the prior knowledge of the brain. It is an important preprocessing step in many medical research and clinical applications, such as quantification of tissue volume, visualization and analysis of anatomical structures, multimodality fusion and registration, functional brain mapping, detection of pathology, surgical planning, surgical navigation, and brain substructure segmentation (Suri et al., 2002). So far, various segmentation techniques such as Gaussian mixture models (Ashburner & Friston, 2005), discriminant analysis (Amato et al., 2003), k-nearest neighbor classification (Mohamed et al., 1999), and fuzzy c-means clustering (Pham & Prince, 1999; Suckling et al., 1999; Ahmed et al., 2002; Zhou & Bai, 2007) were used to determine gray and white matter volume. Most recently, it has been reported that "fuzzy" cluster or classifier approaches were found to have a high reliability, accuracy, and validity (Herndon, 1998).

#### **4.3 Image processing and segmentation**

Today, medical image processing and segmentation are used to improve the quality of diagnosis. We can calculate the cortical volume and surface area using the Fuzzy C-Means algorithm as a semi-automated segmentation method as described in Figure 3.

Firstly, T1-weighted MRIs are normalized using registration algorithms. Following normalization process, we obtain brain contour to calculate volume and surface area of the brain using image working algorithms. Images of brain are cleared brain contour using morphological image processing. This method involves two major steps and final segmented images result from separation of parenchyma for brain volume and surrounding line for cortical surface area of the brain (Tosun et al., 2004; Ueda et al., 2009; Brouwer et al., 2010; Lui et al., 2010) (Fig. 4).

#### **4.4 Stereological approaches**

In general, stereological methods provide quantitative data on 3D structures using 2D images. Stereological methods have been widely applied on MRIs to estimate geometric variables, such as volume and surface area, and various internal brain compartments. The volume of internal brain structure can be obtained using the Cavalieri principle of stereologic approaches.

MRIs, but they are generally severely limited in practicality and reliability. For example, a high-resolution 3D brain MRI data set can contain more than 100 slices to cover an entire brain (Keller & Roberts, 2009). Manually delineating tissue boundaries for volumetric measurement can be a tedious and demanding process because of the presence of the extremely complex convoluted structures of the brain. Manual tracing is also well-known to be associated with large subjective variability and low reproducibility. As a result, methods with better reproducibility and higher precision are required for measuring subtle neuroanatomic changes and these methods are likely to be based on computerized approaches. FreeSurfer is freely available on the World Wide Web (www or commonly known as the Web), it has been widely used in the neuroimaging field. At present, fully automated methods are most often used. Fully automated or semi-automated methods can be applied to a specific ROI (such as the thalamus or the hippocampus) to obtain a regional brain

Brain tissue segmentation of MRIs means to specify the tissue type for each pixel or voxel in a 2D or 3D data set, respectively, on the basis of information available from both MRIs and the prior knowledge of the brain. It is an important preprocessing step in many medical research and clinical applications, such as quantification of tissue volume, visualization and analysis of anatomical structures, multimodality fusion and registration, functional brain mapping, detection of pathology, surgical planning, surgical navigation, and brain substructure segmentation (Suri et al., 2002). So far, various segmentation techniques such as Gaussian mixture models (Ashburner & Friston, 2005), discriminant analysis (Amato et al., 2003), k-nearest neighbor classification (Mohamed et al., 1999), and fuzzy c-means clustering (Pham & Prince, 1999; Suckling et al., 1999; Ahmed et al., 2002; Zhou & Bai, 2007) were used to determine gray and white matter volume. Most recently, it has been reported that "fuzzy" cluster or classifier approaches were found to have a high reliability, accuracy,

Today, medical image processing and segmentation are used to improve the quality of diagnosis. We can calculate the cortical volume and surface area using the Fuzzy C-Means

Firstly, T1-weighted MRIs are normalized using registration algorithms. Following normalization process, we obtain brain contour to calculate volume and surface area of the brain using image working algorithms. Images of brain are cleared brain contour using morphological image processing. This method involves two major steps and final segmented images result from separation of parenchyma for brain volume and surrounding line for cortical surface area of the brain (Tosun et al., 2004; Ueda et al., 2009; Brouwer et al.,

In general, stereological methods provide quantitative data on 3D structures using 2D images. Stereological methods have been widely applied on MRIs to estimate geometric variables, such as volume and surface area, and various internal brain compartments. The volume of internal brain structure can be obtained using the Cavalieri principle of

algorithm as a semi-automated segmentation method as described in Figure 3.

volume (Houtchens et al., 2007).

**4.2 Brain segmentation** 

and validity (Herndon, 1998).

2010; Lui et al., 2010) (Fig. 4).

**4.4 Stereological approaches** 

stereologic approaches.

**4.3 Image processing and segmentation** 

Fig. 3. Brain image segmentation blocks (Nakamura & Fisher, 2009)

Fig. 4. Segmentation called the Fuzzy C. Left: Original T1-weighted image. Center: Masking and removal of artifacts the outer contour of the brain. Right: Contour of segmented image is outlined

#### **4.4.1 Point-counting method**

The Cavalieri method in combination with point counting requires beginning from a uniform random starting within the sectioning interval, a structure of interest is exhaustively sectioned with a series of parallel plane probes a constant distant apart. An unbiased estimate of volume is obtained by multiplying the total area of all sections through the structure by sectioning interval *t* as following:

$$testV = \mathbf{t} \times \left(a\_1 + a\_2 + \ldots + a\_n\right) \tag{1}$$

Quantification of Volumetric Changes of Brain in

Roberts, 2009).

technique

Neurodegenerative Diseases Using Magnetic Resonance Imaging and Stereology 465

method gives a CE of estimation for each volume assessment. Thus, an investigator may easily observe the potential variability in any given volume measurement. It may cause some problems in accuracy and hence interpretation in the presence of high CE for these measurements. If too few slices or too few points are taken for volume estimation, it is possible to encounter with such problems. The investigator is eligible to change the spacing of points in the grid or the number of slices available in any CT or MR study to provide a reasonable CE value. More importantly, an appropriate grid size and the number of slices required for volume estimation of an object is crucial at the beginning, obviating the need to

In the stereological method, continuous investigator computer interaction is necessary because all points intersecting the cerebral hemispheres should be removed or marked on consecutive MRI sections. For the reliable measurement of each brain structure using stereology in conjunction with point counting, the stereological parameters like grid size and slice gap should be optimized by counting at least 200 points per structure. In a previous study investigating the cerebral hemispheres, a grid size of 15 and slice gap of every 15 sections results in approximately 200 points being counted per hemisphere on frequently acquired 3D T1-weighted images (Mackay et al., 1998; Cowell et al., 2007), and it achieves a CE lower than the optimal 5% (Roberts et al., 2000). It has been reported that stereological volume estimation of a cerebral hemisphere using the Windows based software packages (EASYMEASURE and MEASURE) takes approximately 10 minutes (Keller &

Stereological point counting method involves the random placement of a grid with sufficient resolution in 2D or 3D over the structure of interest and counting the points overlying the ROI. For this method, the requirements are a grid encompassing the region or structure completely, the structure placed with a grid randomly, and an adequate number of points counted on an adequate number of slices. Thus, the stereological point counting approach is very efficient and statistically sound, in addition to providing a CE of the

According to point-counting technique, a square grid of test points is positioned on each

2

∑

2

*V cm*

7.8

⎡ ⎤ <sup>×</sup> =× × = ⎢ ⎥ <sup>⎣</sup> <sup>⎦</sup>

*su d V pc t <sup>p</sup> sl*

⎡ ⎤ <sup>×</sup> =× × ⎢ ⎥ ⎣ ⎦

8 0.8 1.6 796 856.90

In the Cavalieri method in combination with point-counting technique using MRI sections,

Section number 1 2 3 4 5 6 7 8 Total Point Counts Point number 42 72 122 132 129 116 115 68 796

Table 1. Relationship between numbers of section and counts of point in point-counting

3

(3)

measurement of the volume of the structure of interest.

**4.4.2 Worked example for point-counting technique** 

T = 1.6cm, d = 0.8cm, SU=8 cm, SL=7.8 cm, ∑P = 796

MRI section, and all points hitting the cerebrum are counted (Fig. 5).

relationship between numbers of section and counts is given in Table 1.

( )

calculate the CE value for repeated sessions (Sahin et al., 2003; Acer et al., 2008).

where *a1,a2…an* show the section areas and *t* is the sectioning interval (Roberts et al., 2000; García-Fiñana et al., 2003).

The point counting method involves overlying each MRI with a regular grid of test points. After each superimposition, the number of test points hitting the structure of interest is counted on each section and we can estimate volume following formula:

$$testV = t \times \left(\frac{a}{p}\right) \times \left(p\_1 + p\_2 + \ldots + p\_n\right) \tag{2}$$

where *p1, p2,…..pn* show point counts and *a/p* represent the area associated with each test point. To avoid bias, the position of the test system should be uniform randomly (Roberts et al., 2000; García-Fiñana et al., 2003).

In any case, the following formula (Eq.3) can be used for volume estimation from MRIs of the brain (Şahin & Ergür, 2006; Acer et al., 2008):

$$V(pc) = t \times \left[\frac{su \times d}{sl}\right]^2 \times \sum p \tag{3}$$

where '*t*' is the section thickness, '*su*' the scale unit of the printed film, '*d*' the distance between the test points of the grid, '*sl*' the measured length of the scale printed on the film and '*Σp'* is the total number of points hitting the sectioned cut surface areas of the related structures such as the cerebrum.

From stereological point of view, planimetry and point-counting are two different methods for estimating volume based on the Cavalieri principle. The Cavalieri principle may be used for estimating the volume of brain and substructures such as hypocampus, amygdale and thalamus. Therefore, a random beginning is necessary and the object is cut into slices of a known and fixed thickness. The volume is estimated by multiplying the distance between the slices by the total cut area of the structures are under investigation. The cut area of the structures may be estimated by point-counting or planimetry. Nevertheless, the Cavalieri principle in combination with point-counting is ideal for estimating total volumes of various brain and any compartments. Keller et al. (2002, 2007, 2008) and Acer et al. (2008, 2010) have previously applied this technique to obtain volume estimations of various brain structures such as Broca's area, hippocampus, ventricles, cerebral hemisphere, and cerebellum. In these studies, a set of parallel and equidistant MRIs of the brain is randomly selected, and the ROI is directly estimated on each image by randomly superimposing a grid of points, and subsequently, counting the number of points that fall within the ROI (Keller et al., 2002, 2007, 2008; Acer et al., 2008, 2010).

Stereology in combination with point counting has an advantage related with the time taken to estimate volume of brain structures on MRIs. Compared with manual tracing or segmentation methods, this technique is much more time efficient. Another advantage of stereology in combination with point counting is the prediction of the coefficient of error (CE) so that it may be used to identify the optimal parameters of sampling needed to achieve a given precision such as we need the number of MRI sections and the density of the point grid.

Importantly, the stereological approach provides an opportunity for the investigator making appropriate changes on their sampling or estimating procedures. Therefore, the Cavalieri

where *a1,a2…an* show the section areas and *t* is the sectioning interval (Roberts et al., 2000;

The point counting method involves overlying each MRI with a regular grid of test points. After each superimposition, the number of test points hitting the structure of interest is

> *<sup>a</sup> estV t pp p p* ⎛ ⎞

⎝ ⎠

=× × + + + ⎜ ⎟

where *p1, p2,…..pn* show point counts and *a/p* represent the area associated with each test point. To avoid bias, the position of the test system should be uniform randomly (Roberts et

In any case, the following formula (Eq.3) can be used for volume estimation from MRIs of

( ) *su d V pc t <sup>p</sup> sl* ⎡ ⎤ <sup>×</sup> =× × ⎢ ⎥

where '*t*' is the section thickness, '*su*' the scale unit of the printed film, '*d*' the distance between the test points of the grid, '*sl*' the measured length of the scale printed on the film and '*Σp'* is the total number of points hitting the sectioned cut surface areas of the related

From stereological point of view, planimetry and point-counting are two different methods for estimating volume based on the Cavalieri principle. The Cavalieri principle may be used for estimating the volume of brain and substructures such as hypocampus, amygdale and thalamus. Therefore, a random beginning is necessary and the object is cut into slices of a known and fixed thickness. The volume is estimated by multiplying the distance between the slices by the total cut area of the structures are under investigation. The cut area of the structures may be estimated by point-counting or planimetry. Nevertheless, the Cavalieri principle in combination with point-counting is ideal for estimating total volumes of various brain and any compartments. Keller et al. (2002, 2007, 2008) and Acer et al. (2008, 2010) have previously applied this technique to obtain volume estimations of various brain structures such as Broca's area, hippocampus, ventricles, cerebral hemisphere, and cerebellum. In these studies, a set of parallel and equidistant MRIs of the brain is randomly selected, and the ROI is directly estimated on each image by randomly superimposing a grid of points, and subsequently, counting the number of points that fall within the ROI (Keller et al., 2002,

Stereology in combination with point counting has an advantage related with the time taken to estimate volume of brain structures on MRIs. Compared with manual tracing or segmentation methods, this technique is much more time efficient. Another advantage of stereology in combination with point counting is the prediction of the coefficient of error (CE) so that it may be used to identify the optimal parameters of sampling needed to achieve a given precision such as we need the number of MRI sections and the density of the

Importantly, the stereological approach provides an opportunity for the investigator making appropriate changes on their sampling or estimating procedures. Therefore, the Cavalieri

( ) 1 2 .. *<sup>n</sup>*

2

counted on each section and we can estimate volume following formula:

García-Fiñana et al., 2003).

al., 2000; García-Fiñana et al., 2003).

structures such as the cerebrum.

2007, 2008; Acer et al., 2008, 2010).

point grid.

the brain (Şahin & Ergür, 2006; Acer et al., 2008):

*estV t a a a* =× + + + ( 1 2 .. *<sup>n</sup>* ) (1)

(2)

<sup>⎣</sup> <sup>⎦</sup> ∑ (3)

method gives a CE of estimation for each volume assessment. Thus, an investigator may easily observe the potential variability in any given volume measurement. It may cause some problems in accuracy and hence interpretation in the presence of high CE for these measurements. If too few slices or too few points are taken for volume estimation, it is possible to encounter with such problems. The investigator is eligible to change the spacing of points in the grid or the number of slices available in any CT or MR study to provide a reasonable CE value. More importantly, an appropriate grid size and the number of slices required for volume estimation of an object is crucial at the beginning, obviating the need to calculate the CE value for repeated sessions (Sahin et al., 2003; Acer et al., 2008).

In the stereological method, continuous investigator computer interaction is necessary because all points intersecting the cerebral hemispheres should be removed or marked on consecutive MRI sections. For the reliable measurement of each brain structure using stereology in conjunction with point counting, the stereological parameters like grid size and slice gap should be optimized by counting at least 200 points per structure. In a previous study investigating the cerebral hemispheres, a grid size of 15 and slice gap of every 15 sections results in approximately 200 points being counted per hemisphere on frequently acquired 3D T1-weighted images (Mackay et al., 1998; Cowell et al., 2007), and it achieves a CE lower than the optimal 5% (Roberts et al., 2000). It has been reported that stereological volume estimation of a cerebral hemisphere using the Windows based software packages (EASYMEASURE and MEASURE) takes approximately 10 minutes (Keller & Roberts, 2009).

Stereological point counting method involves the random placement of a grid with sufficient resolution in 2D or 3D over the structure of interest and counting the points overlying the ROI. For this method, the requirements are a grid encompassing the region or structure completely, the structure placed with a grid randomly, and an adequate number of points counted on an adequate number of slices. Thus, the stereological point counting approach is very efficient and statistically sound, in addition to providing a CE of the measurement of the volume of the structure of interest.

#### **4.4.2 Worked example for point-counting technique**

According to point-counting technique, a square grid of test points is positioned on each MRI section, and all points hitting the cerebrum are counted (Fig. 5). T = 1.6cm, d = 0.8cm, SU=8 cm, SL=7.8 cm, ∑P = 796

$$\begin{aligned} V(pc) &= t \times \left[ \frac{su \times d}{sl} \right]^2 \times \sum p \\ V &= 1.6 \times \left[ \frac{8 \times 0.8}{7.8} \right]^2 \times 796 = 856.90 cm^3 \end{aligned} \tag{3}$$

In the Cavalieri method in combination with point-counting technique using MRI sections, relationship between numbers of section and counts is given in Table 1.


Table 1. Relationship between numbers of section and counts of point in point-counting technique

Quantification of Volumetric Changes of Brain in

Section (i) Pi Pi

Table 2. Calculation of the constants *C*0, *C*1, *C*2, *C*4

The coefficient α(*q*) has the following expression (Eq.8):

α

*a*

var( ) 9.2

=

*T T T*

*Q Q*

We apply Eq. (7) with α = 0.53.

Therefore, the estimate of Var ( ˆ

We predict the value of CE;

The smoothness constant can be estimated from Eq. (7) as follows:

Neurodegenerative Diseases Using Magnetic Resonance Imaging and Stereology 467

1 42 1764 3024 5124 5418 2 72 5184 8784 9504 8352 3 122 14884 16104 15738 14030 4 132 17424 17028 15312 8976 5 129 16641 14964 14835 0 6 116 13456 13340 7888 0 7 115 13225 7820 0 0 8 68 4624 0 0 0 87202 81064 68401 36776 Total 796 C0 C1 C2 C4

> ( ) ( ) 0 24 0 12

(7)

*C CC*

1 1 3 4 max 0, log 2log 2 34 2

1 3 87202 4 68401 36776 1 0, log 0.53

( ) ( ) [ ] 2 2 2 1

 π

0,1

2

(9)

Γ+ + <sup>=</sup> <sup>∈</sup> − (8)

<sup>⎧</sup> ⎡ ⎤ − + <sup>⎫</sup> <sup>⎪</sup> <sup>⎪</sup> <sup>=</sup> <sup>⎨</sup> ⎢ ⎥ <sup>−</sup> <sup>⎬</sup> − + ⎩ ⎭ <sup>⎪</sup> ⎣ ⎦ <sup>⎪</sup>

*<sup>q</sup> C CC*

2log 2 3 87202 4 81064 68401 2 *<sup>q</sup>* ⎧ ⎫ ⎪ ⎪ ⎡ ⎤ × −× + <sup>=</sup> ⎨ ⎬ <sup>−</sup> <sup>=</sup> ⎢ ⎥ ⎪ ⎪ ⎣ ⎦ × −× + ⎩ ⎭

( ) ( ) ( ) ( )

π

2 2 2 2 cos

2 12 *<sup>q</sup> <sup>q</sup> qq q q q* ζ

where Γ and ζ denote the gamma function and the Riemann Zeta function, respectively.

ζ

τ

0 12

var( ) ( )(3 4 )

*Q aq C C C T*

= −+

2.06 0.06 (2.06) (2.06)cos(1.66) (0.53) 0.018 (2 ) (1 2 )

2

<sup>Γ</sup> <sup>=</sup> <sup>=</sup> <sup>−</sup>

*QT* ) obtained via Eq. (5) is:

var( ) 0.018 (3 87202 4 81064 68401) (1.6)

*CE Q*( ) 9.2 /856.9 0.0103 1.03% *<sup>T</sup>* = = = In our studies, we calculate the CE values as predictive using the R program. First, by using the statistical package R, codes are developed to calculate the contribution to the predictive

= × × −× + ×

<sup>+</sup> <sup>−</sup>

2 Pi.Pi+1 Pi.Pi+2 Pi.Pi+4

Fig. 5. Acoronal MRI series with a point-counting on it for the estimation of the cerebral volume from first to last section (T=1.6 cm)

#### **4.4.3 Error prediction for point counting technique**

The error predictors given below originate from the recent literature (García-Fiñana & Cruz-Orive, 2000; Garcia Finana, 2006; Garcia Finana et al., 2009). In particular, the estimation of volume and variance of the volume estimate for the cerebral volume are calculated as follows.

An unbiased estimator of *Q* can be constructed from a sample of equidistant observations of *f*, with a distance *T* apart, as follows:

$$\hat{Q}\_T = T \sum\_{k \in \mathbb{Z}} f\left(\mathbf{x}\_0 + kT\right) = T\left(f\_1 + f\_2 + \dots f\_n\right) \tag{4}$$

where *x*0 is a uniform random variable in the interval [0,*T*) and {*f*1, *f*2, … , *fn*} is the set of equidistant observations of *f* at the sampling points which lie in [*a*, *b*]. In many applications, *Q* represents the volume of a structure and *f*(*x*) is the area of the intersection between the structure and a plane that is perpendicular to a given sampling axis at the point of abscissa *x* (García-Fiñana & Cruz-Orive, 2000; Garcia Finana, 2006; Garcia Finana et al., 2009).

This data sample represents the area of cerebrum in cm2 on a total of 8 MRI sections a distance *T* = 1.6 cm apart (Table 1).

To estimate Var ( ˆ *QT* ) via Eq. (5) we have to calculate first α(*q*), *C*0, *C*1, *C*2 and *C*4 (Table 2).

$$\text{var}\left(\hat{Q}\_{T}\right) = \alpha\left(q\right)\left(\mathfrak{K}\_{0} - 4\mathfrak{C}\_{1} + \mathfrak{C}\_{2}\right)T^{2} \qquad q \in \left[0, 1\right] \tag{5}$$

From Eq. (6), we have:

$$\mathbf{C}\_{k}\sum\_{i=1}^{n-k} f\_{i} f\_{i+k} \quad \quad k=1,\ 2,\ \dots \ n-1 \tag{6}$$

Equation (5) is an extended version of the variance estimator given in (García-Fiñana & Cruz-Orive, 2004).


Table 2. Calculation of the constants *C*0, *C*1, *C*2, *C*4

The smoothness constant can be estimated from Eq. (7) as follows:

$$q = \max\left\{0, \frac{1}{2\log 2} \log \left[\frac{\left(3\mathbf{C}\_0 - 4\mathbf{C}\_2 + \mathbf{C}\_4\right)}{\left(3\mathbf{C}\_0 - 4\mathbf{C}\_1 + \mathbf{C}\_2\right)}\right] - \frac{1}{2}\right\} \tag{7}$$

$$q = \left\{ 0, \frac{1}{2\log 2} \log \left[ \frac{3 \times 87202 - 4 \times 68401 + 36776}{3 \times 87202 - 4 \times 81064 + 68401} \right] - \frac{1}{2} \right\} = 0.533$$

We apply Eq. (7) with α = 0.53.

466 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

Fig. 5. Acoronal MRI series with a point-counting on it for the estimation of the cerebral

The error predictors given below originate from the recent literature (García-Fiñana & Cruz-Orive, 2000; Garcia Finana, 2006; Garcia Finana et al., 2009). In particular, the estimation of volume and variance of the volume estimate for the cerebral volume are calculated as

An unbiased estimator of *Q* can be constructed from a sample of equidistant observations of

( 0 1 ) ( <sup>2</sup> ) <sup>ˆ</sup> ... *T n*

where *x*0 is a uniform random variable in the interval [0,*T*) and {*f*1, *f*2, … , *fn*} is the set of equidistant observations of *f* at the sampling points which lie in [*a*, *b*]. In many applications, *Q* represents the volume of a structure and *f*(*x*) is the area of the intersection between the structure and a plane that is perpendicular to a given sampling axis at the point of abscissa *x*

This data sample represents the area of cerebrum in cm2 on a total of 8 MRI sections a

( ) ( )( ) [ ] <sup>2</sup> 0 12 <sup>ˆ</sup> var 3 4 0,1 *Q q C C CT q <sup>T</sup>* = −+ ∈

*C ff k n*

Equation (5) is an extended version of the variance estimator given in (García-Fiñana &

+

*QT* ) via Eq. (5) we have to calculate first α(*q*), *C*0, *C*1, *C*2 and *C*4 (Table 2).

, 1, 2, ... - 1

= + = ++ ∑ (4)

∑ <sup>=</sup> (6)

(5)

*Q T f x kT T ff f*

(García-Fiñana & Cruz-Orive, 2000; Garcia Finana, 2006; Garcia Finana et al., 2009).

*k Z*

∈

α

1

=

*n k k iik i*

−

volume from first to last section (T=1.6 cm)

*f*, with a distance *T* apart, as follows:

distance *T* = 1.6 cm apart (Table 1).

To estimate Var ( ˆ

From Eq. (6), we have:

Cruz-Orive, 2004).

follows.

**4.4.3 Error prediction for point counting technique** 

The coefficient α(*q*) has the following expression (Eq.8):

$$\alpha(q) = \frac{\Gamma\left(2q+2\right)\zeta\left(2q+2\right)\cos\left(\pi q\right)}{\left(2\pi\right)^{2q+2}\left(1-2^{2q-1}\right)} \quad q \in \left[0,1\right] \tag{8}$$

where Γ and ζ denote the gamma function and the Riemann Zeta function, respectively.

$$a(0.53) = \frac{\Gamma(2.06)\zeta(2.06)\cos(1.66)}{(2\pi)^{2.06}(1-2^{0.06})} = 0.018$$

Therefore, the estimate of Var ( ˆ *QT* ) obtained via Eq. (5) is:

$$\begin{aligned} \text{var}(Q\_T) &= a(q)(3\mathcal{C}\_0 - 4\mathcal{C}\_1 + \mathcal{C}\_2)T^2 \\ \text{var}(Q\_T) &= 0.018 \times (3 \times 87202 - 4 \times 81064 + 68401) \times (1.6)^2 \\ \text{var}(Q\_T) &= 9.2 \end{aligned} \tag{9}$$

We predict the value of CE;

$$CE(Q\_T) = \sqrt{9.2 \times 856.9} = 0.0103 = 1.03\%$$

In our studies, we calculate the CE values as predictive using the R program. First, by using the statistical package R, codes are developed to calculate the contribution to the predictive

Quantification of Volumetric Changes of Brain in

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### **5. Conclusion**

In conlusion, MRI may help to specify the cause of the disease such as the brain atrophy, if a kind of neurodegenerative dissease is present. Unfortunately, however, conventional MRI study not give subsutructural detailed information about cellular and molecular organisation of the brain tissue. On the other hand, it is also possible to define the etiologies of the pathologies using new functional MRI methods, such as diffusion weighted imaging and MRS. Future advances in functional and anatomic neuroimaging techniques provide further insights into certain neurodegenerative disseases of the brain. A combination of different neuroimaging techniques and atrophy correction through MRI, PET and SPECT superimposition may demonstrate functional and morphological features of the brain tissue. In similar to MRI, PET and SPECT are useful in the diagnosis of some neurodegenerative diseases.

By using MRI, the estimation of the brain volume is a well-known entity for the determination of the brain atrophy. Several different methods such as segmentation techniques are available for the estimation of the brain volume, but there are only a few stereological studies using point-counting and planimetric methods. Recently, the Cavalieri principle in combination with point-counting has become popular in the understanding of the pathologies of brain morphology. There is no doubt that determination of the brain atrophy using MRI will be useful in understanding of neurodegenerative diseases, monitoring of disease progression, and treatment of such patients. In conclusion, the Cavalieri principle in combination with point-counting is an ideal method for the estimation of total volume of the brain or any of its compartment for the diagnosis of atrophic neurodegenerative diseases.

Nevertheless, further combinations of new imaging techniques with different methods for volume estimation using a combination of different neuroimaging techniques are needed for early diagnosis and monitorization of course of the disease. It is important to know that these techniques for volume estimation must be reproducible and reliable. The greater accuracy of imaging methods in detection of early neurodegenerative disseases will result in early optimal treatment to delay further cognitive decline.

#### **6. References**


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In conlusion, MRI may help to specify the cause of the disease such as the brain atrophy, if a kind of neurodegenerative dissease is present. Unfortunately, however, conventional MRI study not give subsutructural detailed information about cellular and molecular organisation of the brain tissue. On the other hand, it is also possible to define the etiologies of the pathologies using new functional MRI methods, such as diffusion weighted imaging and MRS. Future advances in functional and anatomic neuroimaging techniques provide further insights into certain neurodegenerative disseases of the brain. A combination of different neuroimaging techniques and atrophy correction through MRI, PET and SPECT superimposition may demonstrate functional and morphological features of the brain tissue. In similar to MRI, PET and SPECT are useful in the diagnosis of some neurodegenerative

By using MRI, the estimation of the brain volume is a well-known entity for the determination of the brain atrophy. Several different methods such as segmentation techniques are available for the estimation of the brain volume, but there are only a few stereological studies using point-counting and planimetric methods. Recently, the Cavalieri principle in combination with point-counting has become popular in the understanding of the pathologies of brain morphology. There is no doubt that determination of the brain atrophy using MRI will be useful in understanding of neurodegenerative diseases, monitoring of disease progression, and treatment of such patients. In conclusion, the Cavalieri principle in combination with point-counting is an ideal method for the estimation of total volume of the brain or any of its compartment for the diagnosis of atrophic

Nevertheless, further combinations of new imaging techniques with different methods for volume estimation using a combination of different neuroimaging techniques are needed for early diagnosis and monitorization of course of the disease. It is important to know that these techniques for volume estimation must be reproducible and reliable. The greater accuracy of imaging methods in detection of early neurodegenerative disseases will result in

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**5. Conclusion** 

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**20** 

*USA* 

**Acid-Sensing Ion Channels in Neurodegenerative Diseases:** 

**Potential Therapeutic Target** 

*Morehouse School of Medicine, Atlanta, Georgia;* 

*1Department of Basic Medical Science,* 

*2Department of Neurobiology,* 

Chu Xiang-Ping1, Wang John Q.1 and Xiong Zhi-Gang2

Under pathological conditions such as tissue inflammation, ischemic stroke, traumatic brain injury, and epileptic seizure, accumulations of lactic acid due to enhanced anaerobic glucose metabolism and the release of proton from ATP hydrolysis result in significant reduction of tissue pH, a condition termed acidosis. Acidosis can activate a distinct family of ion channels: acid-sensing ion channels (ASICs) (Waldmann et al., 1997b), which are heavily expressed in the peripheral sensory and central neurons (Waldmann & Lazdunski, 1998; Krishtal, 2003; Wemmie et al., 2006; Lingueglia, 2007; Xiong et al., 2006, 2007, 2008; Sluka et al., 2009). ASICs belong to the amiloride-sensitive degenerin/epithelial Na+ channel (DEG/ENaC) superfamily (Kellenberger & Schild, 2002). Four genes (*ACCN1 - 4*) encoding at least six ASIC subunits have been cloned. Each subunit has two transmembrane domains with a large extracellular loop and short intracellular N- and C-termini (Waldmann et al., 1997b). Functional ASICs are trimeric complexes of these subunits (Jasti et al., 2007; Gonzales et al., 2009) and most of these subunits can form homomeric and/or heteromeric channels (Benson et al., 2002; Baron et al., 2002, 2008; Wemmie et al., 2002, 2003; Askwith et al., 2004; Chu et al., 2004, 2006; Xiong et al., 2004; Zha et al., 2006; Sherwood et al., 2011). ASICs are enriched in brain neurons (Alvarez de la Rosa et al., 2003; Wemmie et al., 2003; Xiong et al., 2004; Sherwood et al., 2011), where at least three (ASIC1a, ASIC2a and ASIC2b) of the seven subunits can be found. ASIC1a is the dominant subunit in brain and homomeric ASIC1a and heteromeric ASIC1a/2b channels are permeable to both Na+ and Ca2+ ions (Waldmann et al., 1997b; Yermolaieva et al., 2004; Zha et al., 2006; Sherwood et al., 2011). ASICs are inhibited by the diuretic amiloride, a non-specific ASIC blocker (Waldmann et al., 1997b). The tarantula toxin psalmotoxin 1 (PcTX1) blocks the homomeric ASIC1a (Escoubas et al., 2000) and heteromeric ASIC1a/2b (Sherwood et al., 2011) channels. The roles of ASICs in a variety of neurologic conditions are still under active investigation. ASIC1a channels localize at synapse and contribute to synaptic plasticity, learning/memory, and fear conditioning (Wemmie et al., 2002, 2003, 2004). Activation of Ca2+-permeable homomeric ASIC1a and heteromeric ASIC1a/2b channels is involved in acidosis-mediated ischemic

**1. Introduction** 

*University of Missouri-Kansas City, Kansas City, Missouri;* 


## **Acid-Sensing Ion Channels in Neurodegenerative Diseases: Potential Therapeutic Target**

Chu Xiang-Ping1, Wang John Q.1 and Xiong Zhi-Gang2 *1Department of Basic Medical Science, University of Missouri-Kansas City, Kansas City, Missouri; 2Department of Neurobiology, Morehouse School of Medicine, Atlanta, Georgia; USA* 

#### **1. Introduction**

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Under pathological conditions such as tissue inflammation, ischemic stroke, traumatic brain injury, and epileptic seizure, accumulations of lactic acid due to enhanced anaerobic glucose metabolism and the release of proton from ATP hydrolysis result in significant reduction of tissue pH, a condition termed acidosis. Acidosis can activate a distinct family of ion channels: acid-sensing ion channels (ASICs) (Waldmann et al., 1997b), which are heavily expressed in the peripheral sensory and central neurons (Waldmann & Lazdunski, 1998; Krishtal, 2003; Wemmie et al., 2006; Lingueglia, 2007; Xiong et al., 2006, 2007, 2008; Sluka et al., 2009). ASICs belong to the amiloride-sensitive degenerin/epithelial Na+ channel (DEG/ENaC) superfamily (Kellenberger & Schild, 2002). Four genes (*ACCN1 - 4*) encoding at least six ASIC subunits have been cloned. Each subunit has two transmembrane domains with a large extracellular loop and short intracellular N- and C-termini (Waldmann et al., 1997b). Functional ASICs are trimeric complexes of these subunits (Jasti et al., 2007; Gonzales et al., 2009) and most of these subunits can form homomeric and/or heteromeric channels (Benson et al., 2002; Baron et al., 2002, 2008; Wemmie et al., 2002, 2003; Askwith et al., 2004; Chu et al., 2004, 2006; Xiong et al., 2004; Zha et al., 2006; Sherwood et al., 2011). ASICs are enriched in brain neurons (Alvarez de la Rosa et al., 2003; Wemmie et al., 2003; Xiong et al., 2004; Sherwood et al., 2011), where at least three (ASIC1a, ASIC2a and ASIC2b) of the seven subunits can be found. ASIC1a is the dominant subunit in brain and homomeric ASIC1a and heteromeric ASIC1a/2b channels are permeable to both Na+ and Ca2+ ions (Waldmann et al., 1997b; Yermolaieva et al., 2004; Zha et al., 2006; Sherwood et al., 2011). ASICs are inhibited by the diuretic amiloride, a non-specific ASIC blocker (Waldmann et al., 1997b). The tarantula toxin psalmotoxin 1 (PcTX1) blocks the homomeric ASIC1a (Escoubas et al., 2000) and heteromeric ASIC1a/2b (Sherwood et al., 2011) channels. The roles of ASICs in a variety of neurologic conditions are still under active investigation. ASIC1a channels localize at synapse and contribute to synaptic plasticity, learning/memory, and fear conditioning (Wemmie et al., 2002, 2003, 2004). Activation of Ca2+-permeable homomeric ASIC1a and heteromeric ASIC1a/2b channels is involved in acidosis-mediated ischemic

Acid-Sensing Ion Channels in Neurodegenerative Diseases: Potential Therapeutic Target 479

**Relative**

**0.0**

**D**

**0.2**

**0.4**

**0.6**

**0.8**

**1.0**

**Amplitude**

**B**

**pH**

**5678 4 3**

**-1.5**

**-0.5**

**-1.0**

**-2.0 -2.5 -3.0 -3.5**

**0.5**

**nA**

**-80 -60 -40 -20 20 40 60 80 0**

**n = 6**

**mV**

**n = 7**

**5.0 4.0**

**C**

**-80 -60**

**-40**

**mV mV mV**

**mV**

**mV**

**mV mV**

specific inhibitor of ASIC1a.

**2 Sec**

**pH 6.0**

**2 Sec**

activation of ASIC currentsin in MSNs. (B) Dose-response curve for activation of the currents by pH drops. The pH50 value is 6.25 and the Hill coeficient is 0.94. (C) The I-V relationship of acid-activated currents with different holding levels by decreasing the pH from 7.4 to 6.0 in MSNs. (D) The I-V curve. The extrapolated reversal potential is close to 60

Fig. 2. Electrophysiological properties of ASICs in cultured mouse MSNs. (A) pH-dependent

ASIC1a and ASIC1b proteins are identical, there are significant differences in the sequence for the first one third (about 172 amino acids) of the protein beginning at the N terminal; this sequence includes the intracellular N-terminus, the first transmembrane domain, and the proximal part of the ectodomain (Chen et al., 1998; Bassler et al., 2001); (2), the expression of ASIC1b in the nervous system is limited to peripheral sensory neurons, while ASIC1a is also expressed in the CNS; (3), rodent ASIC1b is impermeable to Ca2+ while ASIC1a channels have significant Ca2+ permeability; Interestingly, a recent study has shown that human ASIC1b channels are permeable to Ca2+ (Hoagland et al., 2010); (4), the threshold for activation of ASIC1b current is lower than ASIC1a (~6.5 for ASIC1b and ~7.0 for ASIC1a) and it has lower pH50 (5.9); (5), ASIC1b is potentiated by PcTx1(Chen et al., 2006), which is a

**2.0 nA**

mV, which is close to the sodium equilibrium potential

**2.0 nA**

**7.4 7.4 pH drop**

**6.0**

**6.5**

**7.0**

**A**

brain injury (Xiong et al., 2004; Pignataro et al., 2007; Sherwood et al., 2011). Moreover, ASIC1a channels play critical roles in neurodegenerative diseases such as multiple sclerosis (Friese et al., 2007; Vergo et al., 2011), Parkinson's (Arias et al., 2008) and Huntington's (Wong et al., 2008) disease and in seizures (Chang et al., 2007; Ziemann et al., 2008) and depression (Coryell et al., 2009). Thus, controlling their activation might ameliorate acidosismediated CNS disorders (Xiong et al., 2008). This chapter provides an overview of recent advance in electrophysiological properties as well as pharmacological profiles of ASICs, and their roles in neurodegenerative disorders.

#### **2. Electrophysiological and pharmacological properties of ASICs**

#### **2.1 Electrophysiological properties of ASICs**

The electrophysiological properties and pharmacological profiles of ASICs have been extensively explored in heterologous expression systems (Chu et al., 2004; Hesselager et al., 2004) and in neurons from different brain regions, such as cortex (Varming, 1998; Xiong et al., 2004; Chu et al., 2004, 2006), hippocampus (Baron et al., 2002; Askwith et al., 2004), striatum (Jiang et al., 2009), cerebellum (Allen & Attwell, 2002), retinal ganglion (Lilley et al., 2004), and spinal cord (Wu et al., 2004; Baron et al., 2008). Fig. 1 shows typical ASIC current mediated by homomeric ASIC1a, 1b, 2a, or 3 channels expressed in CHO cells.

Fig. 1. Acid-triggered inward currents in CHO cells expressing indicated ASIC subunits

Homomeric ASIC1a channels have a pH for half-maximal activation (pH50) between 6.2 and 6.8 (Babini et al., 2002; Benson et al., 2002; Chu et al., 2002; Jiang et al., 2009). Although the precise configuration of ASICs in native neurons is not clear, homomeric ASIC1a and heteromeric ASIC1a/2 channels are the major components in brain neurons (Wemmie et al., 2002; Askwith et al., 2004; Xiong et al., 2004; Jiang et al., 2009; Sherwood et al., 2011). For example, our recent studies have shown that rapid drops in extracellular pH from 7.4 to lower levels (e.g., 6.5, 6.0, 5.0 and 4.0) induced transient inward currents in cultured medium spiny neurons (MSNs) of the mouse striatum (Fig. 2A) (Jiang et al., 2009). The doseresponse curve for activation of ASICs revealed a pH50 value of 6.25 (Fig. 2B). This pH50 value of ASICs in MSNs is comparable to that of homomeric ASIC1a channels (Walmann et al., 1997). The ASIC currents in MSNs had a linear I-V relationship with a reversal potential close to +60 mV (Fig. 2C, D), indicating that ASICs in MSNs are Na+-selective.

In contrast to homomeric ASIC1a channels, the following properties distinguish rodent ASIC1b from ASIC1a: (1), although the amino acid sequence of approximately 2/3 of the

brain injury (Xiong et al., 2004; Pignataro et al., 2007; Sherwood et al., 2011). Moreover, ASIC1a channels play critical roles in neurodegenerative diseases such as multiple sclerosis (Friese et al., 2007; Vergo et al., 2011), Parkinson's (Arias et al., 2008) and Huntington's (Wong et al., 2008) disease and in seizures (Chang et al., 2007; Ziemann et al., 2008) and depression (Coryell et al., 2009). Thus, controlling their activation might ameliorate acidosismediated CNS disorders (Xiong et al., 2008). This chapter provides an overview of recent advance in electrophysiological properties as well as pharmacological profiles of ASICs, and

The electrophysiological properties and pharmacological profiles of ASICs have been extensively explored in heterologous expression systems (Chu et al., 2004; Hesselager et al., 2004) and in neurons from different brain regions, such as cortex (Varming, 1998; Xiong et al., 2004; Chu et al., 2004, 2006), hippocampus (Baron et al., 2002; Askwith et al., 2004), striatum (Jiang et al., 2009), cerebellum (Allen & Attwell, 2002), retinal ganglion (Lilley et al., 2004), and spinal cord (Wu et al., 2004; Baron et al., 2008). Fig. 1 shows typical ASIC current

**0.3 nA**

**3 sec 3 sec 3 sec**

**ASIC2a**

**ASIC3**

**1.0 nA**

**pH 6.0 pH 5.0 pH 4.0**

**2. Electrophysiological and pharmacological properties of ASICs** 

mediated by homomeric ASIC1a, 1b, 2a, or 3 channels expressed in CHO cells.

Fig. 1. Acid-triggered inward currents in CHO cells expressing indicated ASIC subunits

close to +60 mV (Fig. 2C, D), indicating that ASICs in MSNs are Na+-selective.

Homomeric ASIC1a channels have a pH for half-maximal activation (pH50) between 6.2 and 6.8 (Babini et al., 2002; Benson et al., 2002; Chu et al., 2002; Jiang et al., 2009). Although the precise configuration of ASICs in native neurons is not clear, homomeric ASIC1a and heteromeric ASIC1a/2 channels are the major components in brain neurons (Wemmie et al., 2002; Askwith et al., 2004; Xiong et al., 2004; Jiang et al., 2009; Sherwood et al., 2011). For example, our recent studies have shown that rapid drops in extracellular pH from 7.4 to lower levels (e.g., 6.5, 6.0, 5.0 and 4.0) induced transient inward currents in cultured medium spiny neurons (MSNs) of the mouse striatum (Fig. 2A) (Jiang et al., 2009). The doseresponse curve for activation of ASICs revealed a pH50 value of 6.25 (Fig. 2B). This pH50 value of ASICs in MSNs is comparable to that of homomeric ASIC1a channels (Walmann et al., 1997). The ASIC currents in MSNs had a linear I-V relationship with a reversal potential

In contrast to homomeric ASIC1a channels, the following properties distinguish rodent ASIC1b from ASIC1a: (1), although the amino acid sequence of approximately 2/3 of the

**ASIC1b**

**1.0 nA**

their roles in neurodegenerative disorders.

**pH 6.0**

**ASIC1a**

**0.5 nA**

**3 sec**

**2.1 Electrophysiological properties of ASICs** 

Fig. 2. Electrophysiological properties of ASICs in cultured mouse MSNs. (A) pH-dependent activation of ASIC currentsin in MSNs. (B) Dose-response curve for activation of the currents by pH drops. The pH50 value is 6.25 and the Hill coeficient is 0.94. (C) The I-V relationship of acid-activated currents with different holding levels by decreasing the pH from 7.4 to 6.0 in MSNs. (D) The I-V curve. The extrapolated reversal potential is close to 60 mV, which is close to the sodium equilibrium potential

ASIC1a and ASIC1b proteins are identical, there are significant differences in the sequence for the first one third (about 172 amino acids) of the protein beginning at the N terminal; this sequence includes the intracellular N-terminus, the first transmembrane domain, and the proximal part of the ectodomain (Chen et al., 1998; Bassler et al., 2001); (2), the expression of ASIC1b in the nervous system is limited to peripheral sensory neurons, while ASIC1a is also expressed in the CNS; (3), rodent ASIC1b is impermeable to Ca2+ while ASIC1a channels have significant Ca2+ permeability; Interestingly, a recent study has shown that human ASIC1b channels are permeable to Ca2+ (Hoagland et al., 2010); (4), the threshold for activation of ASIC1b current is lower than ASIC1a (~6.5 for ASIC1b and ~7.0 for ASIC1a) and it has lower pH50 (5.9); (5), ASIC1b is potentiated by PcTx1(Chen et al., 2006), which is a specific inhibitor of ASIC1a.

Acid-Sensing Ion Channels in Neurodegenerative Diseases: Potential Therapeutic Target 481

sensory system (Ugawa et al., 2002; Sluka et al., 2003; Jones et al., 2004; Dube et al., 2005), and acidosis-mediated injury of CNS neurons (Xiong et al., 2004; Yermolaieva et al., 2004). However, because of its nonspecificity for other ion channels (e.g., ENaC and T-type Ca2+ channels) and ion exchange systems (e.g., Na+/H+ and Na+/Ca2+ exchanger), it is less likely that amiloride will be used as a future neuroprotective agent in human subjects. It is worth mentioning that the normal activity of Na+/Ca2+ exchanger, for example, is critical for maintaining the cellular Ca2+ homeostasis and the survival of neurons against delayed calcium deregulation caused by glutamate receptor activation (Bano et al., 2005). Inhibition of Na+/Ca2+ exchange by amiloride may therefore compromise normal neuronal Ca2+

**4 Sec**

**1.0 nA**

**0.1 1 10 100 1000**

**Amiloride Concentration (µM)**

Fig. 3. Dose-dependent blockade of ASIC currents in cultured MSNs by amiloride, a nonspecific ASIC blocker. (A) Amiloride dose-dependently inhibits the ASIC currents activated by pH 6.0. (B) Dose-inhibition curve of the acid-induced currents by amiloride. The IC50 of

**Control 10030103 Wash**

**Amiloride ( M) µ**

handling, thus potentiating the glutamate toxicity (Bano et al., 2005).

**A**

**pH 6.0**

**0.0**

**0.2**

**0.4**

**0.6**

**Normalized Amplitude**

amiloride is 13.6 μM

**B**

**0.8**

**1.0**

Homomeric ASIC2a channels are relatively insensitive to proton, with a pH50 of 4.4 (Price et al., 1996; Waldmann et al., 1996; Lingueglia et al., 1997). However, ASIC2a subunits can associate with ASIC1a to form heteromeric channels in brain (Askwith et al., 2004; Chu et al., 2004, 2006; Xiong et al., 2004; Jiang et al., 2009). Different from homomeric ASIC2a subunits, homomeric ASIC2b subunits do not form functional channels by themselves, but can associate with other ASIC subunits to form heteromultimeric channels (Lingueglia et al., 1997; Hesselager et al., 2004; Sherwood et al., 2011). For example, ASIC2b can be associated with ASIC1a to form functional channels and contribute to acidosis-induced neuronal injury (Sherwood et al., 2011).

ASIC3, like ASIC1b (Chen et al., 1998), is expressed primarily in peripheral sensory neurons (Waldmann et al., 1997a; Babinski et al., 1999; Wu et al., 2004; Lingueglia, 2007; Lin et al., 2008). In contrast to other subunits of ASICs, homomeric ASIC3 channels can respond to a large drop of extracellular pH with a transient inactivating current followed by a sustained component (Waldmann et al., 1997a; Sanilas et al., 2009) (Fig. 1). The transient currents are highly sensitive to protons, with a pH50 of around 6.5 (Waldmann et al., 1997a; Hesselager et al., 2004). Electrophysiological studies have shown that ASIC3 subunits function as homomeric or heteromeric channels in sensory neurons (Sutherland et al., 2001; Benson et al., 2002; Deval et al., 2004, 2008; Lin et al., 2008; Hattori et al., 2009). They can sense extracellular acidification occurring in physiological and/or pathological processes, such as cutaneous touch, pain perception, inflammation and ischemia (Benson et al., 1999; Immke & McCleskey, 2001; Price et al., 2001; Sutherland et al., 2001; Mamet et al., 2003; Molliver et al., 2005; Sluka et al., 2007; Ikeuchi et al., 2009). For example, ASIC3 channels expressed in cardiac sensory neurons can respond to myocardial ischemia (Benson et al., 1999; Sutherland et al., 2001; Yagi et al., 2006). Further, cutaneous sensory neurons from rats display large ASIC3-like currents when stimulated by moderate acidosis (Deval et al., 2008). Consequently, it is generally accepted that ASIC3 is a sensor of moderate acidosis during ischemia and inflammatory pain in sensory neurons (Lingueglia, 2007).

ASIC4 subunits are expressed in pituitary gland. Similar to ASIC2b, they do not seem to form functional homomeric channels (Aropian et al., 2000; Grunder et al., 2000).

#### **2.2 Pharmacological profiles of ASICs**

#### **2.2.1 Amiloride**

Amiloride, the potassium-sparing diuretic agent, is a commonly used nonspecific blocker for ASICs. It inhibits the ASIC current and acid-induced increase in intracellular Ca2+ ([Ca2+]i) with an IC50 of 10–60 μM (Waldmann et al., 1997b; de Weille et al., 1998; Chen et al., 1998; Benson et al., 1999; Chu et al., 2002; Wu et al., 2004; Xiong et al., 2004; Yermolaieva et al., 2004; Jiang et al., 2009). For example, our recent study has shown that amiloride dosedependently inhibited the ASIC currents in MSNs with an IC50 of 13.6 μM (Fig. 3) (Jiang et al., 2009). Unlike the currents mediated by other homomeric ASICs, however, the sustained current mediated by homomeric ASIC3 channels is insensitive to amiloride (Waldmann et al., 1997b; Benson et al., 1999; Yagi et al., 2006). Based on the studies of ENaC, it is believed that amiloride inhibits ASICs by a direct blockade of the channel (Schild et al., 1997; Adams et al., 1999). The pre-TM II region of the channel is critical for the effect of amiloride. Mutation of Gly-430 in this region, for example, dramatically changed the sensitivity of ASIC2a current to amiloride (Champigny et al., 1998). Consistent with its inhibition on the ASIC current, amiloride has been shown to suppress acid-induced pain in peripheral

Homomeric ASIC2a channels are relatively insensitive to proton, with a pH50 of 4.4 (Price et al., 1996; Waldmann et al., 1996; Lingueglia et al., 1997). However, ASIC2a subunits can associate with ASIC1a to form heteromeric channels in brain (Askwith et al., 2004; Chu et al., 2004, 2006; Xiong et al., 2004; Jiang et al., 2009). Different from homomeric ASIC2a subunits, homomeric ASIC2b subunits do not form functional channels by themselves, but can associate with other ASIC subunits to form heteromultimeric channels (Lingueglia et al., 1997; Hesselager et al., 2004; Sherwood et al., 2011). For example, ASIC2b can be associated with ASIC1a to form functional channels and contribute to acidosis-induced neuronal injury

ASIC3, like ASIC1b (Chen et al., 1998), is expressed primarily in peripheral sensory neurons (Waldmann et al., 1997a; Babinski et al., 1999; Wu et al., 2004; Lingueglia, 2007; Lin et al., 2008). In contrast to other subunits of ASICs, homomeric ASIC3 channels can respond to a large drop of extracellular pH with a transient inactivating current followed by a sustained component (Waldmann et al., 1997a; Sanilas et al., 2009) (Fig. 1). The transient currents are highly sensitive to protons, with a pH50 of around 6.5 (Waldmann et al., 1997a; Hesselager et al., 2004). Electrophysiological studies have shown that ASIC3 subunits function as homomeric or heteromeric channels in sensory neurons (Sutherland et al., 2001; Benson et al., 2002; Deval et al., 2004, 2008; Lin et al., 2008; Hattori et al., 2009). They can sense extracellular acidification occurring in physiological and/or pathological processes, such as cutaneous touch, pain perception, inflammation and ischemia (Benson et al., 1999; Immke & McCleskey, 2001; Price et al., 2001; Sutherland et al., 2001; Mamet et al., 2003; Molliver et al., 2005; Sluka et al., 2007; Ikeuchi et al., 2009). For example, ASIC3 channels expressed in cardiac sensory neurons can respond to myocardial ischemia (Benson et al., 1999; Sutherland et al., 2001; Yagi et al., 2006). Further, cutaneous sensory neurons from rats display large ASIC3-like currents when stimulated by moderate acidosis (Deval et al., 2008). Consequently, it is generally accepted that ASIC3 is a sensor of moderate acidosis during

ASIC4 subunits are expressed in pituitary gland. Similar to ASIC2b, they do not seem to

Amiloride, the potassium-sparing diuretic agent, is a commonly used nonspecific blocker for ASICs. It inhibits the ASIC current and acid-induced increase in intracellular Ca2+ ([Ca2+]i) with an IC50 of 10–60 μM (Waldmann et al., 1997b; de Weille et al., 1998; Chen et al., 1998; Benson et al., 1999; Chu et al., 2002; Wu et al., 2004; Xiong et al., 2004; Yermolaieva et al., 2004; Jiang et al., 2009). For example, our recent study has shown that amiloride dosedependently inhibited the ASIC currents in MSNs with an IC50 of 13.6 μM (Fig. 3) (Jiang et al., 2009). Unlike the currents mediated by other homomeric ASICs, however, the sustained current mediated by homomeric ASIC3 channels is insensitive to amiloride (Waldmann et al., 1997b; Benson et al., 1999; Yagi et al., 2006). Based on the studies of ENaC, it is believed that amiloride inhibits ASICs by a direct blockade of the channel (Schild et al., 1997; Adams et al., 1999). The pre-TM II region of the channel is critical for the effect of amiloride. Mutation of Gly-430 in this region, for example, dramatically changed the sensitivity of ASIC2a current to amiloride (Champigny et al., 1998). Consistent with its inhibition on the ASIC current, amiloride has been shown to suppress acid-induced pain in peripheral

ischemia and inflammatory pain in sensory neurons (Lingueglia, 2007).

**2.2 Pharmacological profiles of ASICs** 

**2.2.1 Amiloride** 

form functional homomeric channels (Aropian et al., 2000; Grunder et al., 2000).

(Sherwood et al., 2011).

sensory system (Ugawa et al., 2002; Sluka et al., 2003; Jones et al., 2004; Dube et al., 2005), and acidosis-mediated injury of CNS neurons (Xiong et al., 2004; Yermolaieva et al., 2004). However, because of its nonspecificity for other ion channels (e.g., ENaC and T-type Ca2+ channels) and ion exchange systems (e.g., Na+/H+ and Na+/Ca2+ exchanger), it is less likely that amiloride will be used as a future neuroprotective agent in human subjects. It is worth mentioning that the normal activity of Na+/Ca2+ exchanger, for example, is critical for maintaining the cellular Ca2+ homeostasis and the survival of neurons against delayed calcium deregulation caused by glutamate receptor activation (Bano et al., 2005). Inhibition of Na+/Ca2+ exchange by amiloride may therefore compromise normal neuronal Ca2+ handling, thus potentiating the glutamate toxicity (Bano et al., 2005).

Fig. 3. Dose-dependent blockade of ASIC currents in cultured MSNs by amiloride, a nonspecific ASIC blocker. (A) Amiloride dose-dependently inhibits the ASIC currents activated by pH 6.0. (B) Dose-inhibition curve of the acid-induced currents by amiloride. The IC50 of amiloride is 13.6 μM

Acid-Sensing Ion Channels in Neurodegenerative Diseases: Potential Therapeutic Target 483

AGs (streptomycin, neomycin and gentamicin) are a group of antibiotics that have been shown to block Ca2+ channels (Zhou and Zhao, 2002), excitatory amino acid receptors (Pérez et al., 1991), and transient-receptor-potential V1 channels (Raisinghani and Premkumar, 2005). Recently, Garza et al determined the effect of AGs on proton-gated ionic currents in DRG neurons of the rat, and in human embryonic kidney (HEK)-293 cells (Garza et al., 2010). In DRG neurons, streptomycin and neomycin produced a significant, reversible reduction in the amplitude of proton-gated currents in a concentration-dependent manner. In addition, they slowed desensitization rates of ASIC currents. Gentamicin also showed a significant reversible action on the ASIC currents. In HEK-293 cells, streptomycin produced a significant reduction in the amplitude of the proton-gated current, whereas neomycin and gentamicin had no significant effect. These results indicate that ASICs are molecular targets for AGs, which may explain, in part, their effects on excitable cells. Moreover, AGs might potentially represent a novel class of molecules with high affinity, specificity, and selectivity

Diarylamidines have been widely used for the treatment of protozoan diseases such as trypanosomiasis and leishmaniasis since 1930s (Baraldi et al., 2004; Mishra et al., 2007). Recently, Chen and colleges found that four members of the diarylamidines, 4', 6 diamidino-2-phenylindole, diminazene, hydroxystilbamidine and pentamidine strongly inhibit ASIC currents in hippocampal neurons with IC50 of 2.8, 0.3, 1.5 and 38 µM, respectively. The inhibitory concentration is much lower than amiloride. Sub-maximal concentrations of diminazene also potently accelerate desensitization of ASIC currents in hippocampal neurons. Diminazene blocks ASIC1a, -1b, -2a, and -3 currents expressed in CHO cells with a rank order of potency 1b > 3 > 2a > or = 1a. This study indicates that diarylamidines represent a novel class of non-amiloride ASIC blockers and suggests that diarylamidines as small molecules may be developed as therapeutic agents in the treatment

**3. Activation of ASICs induces membrane depolarization and increases** 

Since all ASICs are Na+-selective channels which have a reversal potential near Na+ equilibrium potential (+60 mV), activation of ASICs at normal resting potentials produces exclusively inward currents which result in membrane depolarization and the excitation of neurons (Baron et al., 2002; Wu et al., 2004; Jiang et al., 2009). For example, our recent study has shown that a minor drop in extracellular pH from 7.4 to 6.8 induces significant membrane depolarization, which accompanies trains of action potentials (Fig. 4) (Jiang et al., 2009). This acid-induced membrane depolarization is significantly attenuated by either amiloride or PcTX1 (Fig. 4). Tetrodotoxin, a voltage-gated Na+ channel blocker, has little effect on the membrane depolarization but completely diminished the action potentials triggered by a drop in pH from 7.4 to 6.8. For homomeric ASIC1a channels, acid activation induces Ca2+ entry directly through these channels (Walmann et al., 1997b; Chu et al., 2002; Xiong et al., 2004; Yermolaieva et al., 2004). In addition, the ASIC-mediated membrane depolarization may facilitate the activation of voltage-gated Ca2+ channels and NMDA receptor-gated channels (Wemmie et al., 2002; Zha et al., 2006), further promoting neuronal

**2.2.6 Aminoglycosides (AGs)** 

for different ASIC subunits.

of ASIC-involved diseases (Chen et al., 2010).

**intracellular Ca2+ in brain neurons** 

**2.2.7 Diarylamidines** 

#### **2.2.2 A-317567**

A-317567, a small molecule structurally unrelated to amiloride, is another nonselective ASIC blocker (Dube et al., 2005). It inhibits the ASIC1a, ASIC2a, and ASIC3-like currents with an IC50 of 2–30 μM. Unlike amiloride, which has no effect on the slow component of the ASIC3 current, A-317567 blocks both the fast and the sustained ASIC3 currents. Also different from amiloride, A-317567 does not show diuresis or natriuresis activity (Dube et al., 2005), suggesting that it is more specific for ASICs than amiloride. Its inhibition of sustained ASIC3 current suggests that it might be potent in reducing acidosis-mediated chronic pain. Indeed, A-317567 has been shown to be effective in suppressing the pain in a rat model of thermal hyperalgesia at a dose tenfold lower than amiloride (Dube et al., 2005).

#### **2.2.3 PcTX1**

Being a peptide toxin isolated from venom of the South American tarantula *Psalmopoeus cambridgei*, PcTX1 is a potent and specific inhibitor for homomeric ASIC1a channels (Escoubas et al., 2000). This toxin contains 40 amino acids cross-linked by three disulfide bridges. In heterologous expression systems, PcTX1 specifically inhibits the acid-activated current mediated by homomeric ASIC1a subunits with an IC50 of 1 nM (Escoubas et al., 2000). At concentrations that effectively inhibit the ASIC1a current, it has no effect on the currents mediated by other configurations of ASICs (Escoubas et al., 2000), or known voltage-gated Na+, K+, Ca2+ channels as well as several ligand-gated ion channels (Xiong et al., 2004). Unlike amiloride, which directly blocks the ASICs, PcTX1 acts as a gating modifier. It shifts the channel from its resting state toward the inactivated state by increasing its apparent affinity for protons (Chen et al., 2005). Recently, PcTX1 has also been shown to suppress heteromeric ASIC1a/2b channels (Sherwood et al., 2011).

#### **2.2.4 APETx2**

Being a peptide toxin isolated from sea anemone *Anthopleura elegantissima*, APETx2 is a potent and selective inhibitor for homomeric ASIC3 and ASIC3 containing channels (Diochot et al., 2004). The toxin contains 42 amino acids, also cross-linked by three disulfide bridges. It reduces transient peak acid-evoked currents mediated by homomeric ASIC3 channels (Diochot et al., 2004). In contrast to the peak ASIC3 current, the sustained component of the ASIC3 current is insensitive to APETx2. In addition to homomeric ASIC3 channels (IC50 = 63 nM for rat and 175 nM for human), APETx2 inhibits heteromeric ASIC3/1a (IC50 = 2 μM), ASIC3/1b (IC50 = 900 nM), and ASIC3/2b (IC50 = 117 nM). Homomeric ASIC1a, ASIC1b, ASIC2a, and heteromeric ASIC3/2a channels, on the other hand, are not sensitive to APETx2 (Diochot et al., 2004).

#### **2.2.5 Nonsteroid anti-inflammatory drugs (NSAIDs)**

NSAIDs are the most commonly used anti-inflammatory and analgesic agents. They inhibit the synthesis of prostaglandins (PGs), a main tissue inflammatory substance. A recent study demonstrated that NSAIDs also inhibit the activity of ASICs at their therapeutic doses for analgesic effects (Voilley et al., 2001). Ibuprofen and flurbiprofen, for example, inhibit ASIC1a containing channels with an IC50 of 350 μM. Aspirin and salicylate inhibit ASIC3 containing channels with an IC50 of 260 μM, whereas diclofenac inhibits the same channels with an IC50 of 92 μM. In addition to a direct inhibition of the ASIC activity, NSAIDs also prevent inflammation-induced increase of ASIC expression in sensory neurons (Voilley et al., 2001).

#### **2.2.6 Aminoglycosides (AGs)**

482 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

A-317567, a small molecule structurally unrelated to amiloride, is another nonselective ASIC blocker (Dube et al., 2005). It inhibits the ASIC1a, ASIC2a, and ASIC3-like currents with an IC50 of 2–30 μM. Unlike amiloride, which has no effect on the slow component of the ASIC3 current, A-317567 blocks both the fast and the sustained ASIC3 currents. Also different from amiloride, A-317567 does not show diuresis or natriuresis activity (Dube et al., 2005), suggesting that it is more specific for ASICs than amiloride. Its inhibition of sustained ASIC3 current suggests that it might be potent in reducing acidosis-mediated chronic pain. Indeed, A-317567 has been shown to be effective in suppressing the pain in a rat model of thermal

Being a peptide toxin isolated from venom of the South American tarantula *Psalmopoeus cambridgei*, PcTX1 is a potent and specific inhibitor for homomeric ASIC1a channels (Escoubas et al., 2000). This toxin contains 40 amino acids cross-linked by three disulfide bridges. In heterologous expression systems, PcTX1 specifically inhibits the acid-activated current mediated by homomeric ASIC1a subunits with an IC50 of 1 nM (Escoubas et al., 2000). At concentrations that effectively inhibit the ASIC1a current, it has no effect on the currents mediated by other configurations of ASICs (Escoubas et al., 2000), or known voltage-gated Na+, K+, Ca2+ channels as well as several ligand-gated ion channels (Xiong et al., 2004). Unlike amiloride, which directly blocks the ASICs, PcTX1 acts as a gating modifier. It shifts the channel from its resting state toward the inactivated state by increasing its apparent affinity for protons (Chen et al., 2005). Recently, PcTX1 has also been

Being a peptide toxin isolated from sea anemone *Anthopleura elegantissima*, APETx2 is a potent and selective inhibitor for homomeric ASIC3 and ASIC3 containing channels (Diochot et al., 2004). The toxin contains 42 amino acids, also cross-linked by three disulfide bridges. It reduces transient peak acid-evoked currents mediated by homomeric ASIC3 channels (Diochot et al., 2004). In contrast to the peak ASIC3 current, the sustained component of the ASIC3 current is insensitive to APETx2. In addition to homomeric ASIC3 channels (IC50 = 63 nM for rat and 175 nM for human), APETx2 inhibits heteromeric ASIC3/1a (IC50 = 2 μM), ASIC3/1b (IC50 = 900 nM), and ASIC3/2b (IC50 = 117 nM). Homomeric ASIC1a, ASIC1b, ASIC2a, and heteromeric ASIC3/2a channels, on the other

NSAIDs are the most commonly used anti-inflammatory and analgesic agents. They inhibit the synthesis of prostaglandins (PGs), a main tissue inflammatory substance. A recent study demonstrated that NSAIDs also inhibit the activity of ASICs at their therapeutic doses for analgesic effects (Voilley et al., 2001). Ibuprofen and flurbiprofen, for example, inhibit ASIC1a containing channels with an IC50 of 350 μM. Aspirin and salicylate inhibit ASIC3 containing channels with an IC50 of 260 μM, whereas diclofenac inhibits the same channels with an IC50 of 92 μM. In addition to a direct inhibition of the ASIC activity, NSAIDs also prevent inflammation-induced increase of ASIC expression in sensory neurons (Voilley et

hyperalgesia at a dose tenfold lower than amiloride (Dube et al., 2005).

shown to suppress heteromeric ASIC1a/2b channels (Sherwood et al., 2011).

hand, are not sensitive to APETx2 (Diochot et al., 2004).

**2.2.5 Nonsteroid anti-inflammatory drugs (NSAIDs)** 

**2.2.2 A-317567** 

**2.2.3 PcTX1** 

**2.2.4 APETx2** 

al., 2001).

AGs (streptomycin, neomycin and gentamicin) are a group of antibiotics that have been shown to block Ca2+ channels (Zhou and Zhao, 2002), excitatory amino acid receptors (Pérez et al., 1991), and transient-receptor-potential V1 channels (Raisinghani and Premkumar, 2005). Recently, Garza et al determined the effect of AGs on proton-gated ionic currents in DRG neurons of the rat, and in human embryonic kidney (HEK)-293 cells (Garza et al., 2010). In DRG neurons, streptomycin and neomycin produced a significant, reversible reduction in the amplitude of proton-gated currents in a concentration-dependent manner. In addition, they slowed desensitization rates of ASIC currents. Gentamicin also showed a significant reversible action on the ASIC currents. In HEK-293 cells, streptomycin produced a significant reduction in the amplitude of the proton-gated current, whereas neomycin and gentamicin had no significant effect. These results indicate that ASICs are molecular targets for AGs, which may explain, in part, their effects on excitable cells. Moreover, AGs might potentially represent a novel class of molecules with high affinity, specificity, and selectivity for different ASIC subunits.

#### **2.2.7 Diarylamidines**

Diarylamidines have been widely used for the treatment of protozoan diseases such as trypanosomiasis and leishmaniasis since 1930s (Baraldi et al., 2004; Mishra et al., 2007). Recently, Chen and colleges found that four members of the diarylamidines, 4', 6 diamidino-2-phenylindole, diminazene, hydroxystilbamidine and pentamidine strongly inhibit ASIC currents in hippocampal neurons with IC50 of 2.8, 0.3, 1.5 and 38 µM, respectively. The inhibitory concentration is much lower than amiloride. Sub-maximal concentrations of diminazene also potently accelerate desensitization of ASIC currents in hippocampal neurons. Diminazene blocks ASIC1a, -1b, -2a, and -3 currents expressed in CHO cells with a rank order of potency 1b > 3 > 2a > or = 1a. This study indicates that diarylamidines represent a novel class of non-amiloride ASIC blockers and suggests that diarylamidines as small molecules may be developed as therapeutic agents in the treatment of ASIC-involved diseases (Chen et al., 2010).

#### **3. Activation of ASICs induces membrane depolarization and increases intracellular Ca2+ in brain neurons**

Since all ASICs are Na+-selective channels which have a reversal potential near Na+ equilibrium potential (+60 mV), activation of ASICs at normal resting potentials produces exclusively inward currents which result in membrane depolarization and the excitation of neurons (Baron et al., 2002; Wu et al., 2004; Jiang et al., 2009). For example, our recent study has shown that a minor drop in extracellular pH from 7.4 to 6.8 induces significant membrane depolarization, which accompanies trains of action potentials (Fig. 4) (Jiang et al., 2009). This acid-induced membrane depolarization is significantly attenuated by either amiloride or PcTX1 (Fig. 4). Tetrodotoxin, a voltage-gated Na+ channel blocker, has little effect on the membrane depolarization but completely diminished the action potentials triggered by a drop in pH from 7.4 to 6.8. For homomeric ASIC1a channels, acid activation induces Ca2+ entry directly through these channels (Walmann et al., 1997b; Chu et al., 2002; Xiong et al., 2004; Yermolaieva et al., 2004). In addition, the ASIC-mediated membrane depolarization may facilitate the activation of voltage-gated Ca2+ channels and NMDA receptor-gated channels (Wemmie et al., 2002; Zha et al., 2006), further promoting neuronal

Acid-Sensing Ion Channels in Neurodegenerative Diseases: Potential Therapeutic Target 485

potentiation of excitatory postsynaptic potentials (EPSP) in hippocampal slices from wildtype mice. However, the potentiation of EPSP decays rapidly to the baseline in slices from ASIC1a null mice. Further studies showed that the NMDA receptor antagonist D-2-Amino-5-phosphonovalerate inhibits EPSP summation in slices from wild-type but not ASIC1aknockout mice, suggesting that the loss of ASIC1a impaired NMDA-receptor function. ASIC1a disruption does not impair presynaptic vesicle release, as evidenced by normal single evoked EPSPs and paired-pulse facilitation. Interestingly, a later study by Cho and Askwith demonstrated that the presynaptic release probability is increased in cultured hippocampal neurons from the ASIC1 knockout mice (Cho & Askwith, 2008). Although localizations of ASICs at neuronal cell body and postsynaptic sites have been clearly demonstrated (Wemmie et al., 2002; Zha et al., 2006), it remains to be determined whether

ASIC1a is enriched in key structures of fear circuit (e.g. amygdala) (Wemmie et al., 2003). Thus, ASIC1a may influence fear responses. Indeed, Wemmie and colleagues demonstrated that ASIC1-null mice display significant deficits in cue and context fear conditioning (Wemmie et al., 2003). The loss of ASIC1a also reduces unconditioned fear in the open field test, during acoustic startle, and in response to predator odor (Coryell et al., 2007). Overexpressing ASIC1a, on the other hand, increases fear conditioning (Wemmie et al.,

Further studies by Wemmie's group suggest that activation of ASIC1a in brain chemosensors contributes to CO2 induced fear-related behavior (Ziemann et al., 2009). It has long been known that breathing CO2 triggers panic attacks in patients with panic disorder, and that these patients show an increased sensitivity to CO2 inhalation (Papp et al., 1993). In addition, patients with increasing hypercarbia due to respiratory failure become extremely anxious. How can CO2 inhalation contribute to fear behavior and related panic disorders? Wemmie and colleagues have provided evidence that ASIC1a channels are involved (Ziemann et al., 2009). They showed that inhaled CO2 triggers a drop in brain pH and induces fear behavior in mice. Eliminating or inhibiting ASIC1a significantly limits this activity. Overexpressing ASIC1a in the amygdala rescues the CO2-induced fear deficit in ASIC1a null mice. Buffering brain pH, on the other hand, attenuates fear behavior, whereas lowering pH in the amygdale reproduces the effect of CO2. These studies provide a novel molecular mechanism underlying CO2-induced intense fear and related anxiety/panic disorders and define the amygdala as an important chemosensor that detects

hypercarbia/acidosis and initiates behavioral responses (Ziemann et al., 2009).

pH variations in the retina are involved in the fine-tuning of visual perception. Expression of ASICs in the retina suggests that they might play a role (Lilley et al., 2004). One study by Ettaiche suggested that ASIC2 is important for retinal function and likely protects against light-induced retinal degeneration. They showed that both photoreceptors and neurons of the mouse retina express ASIC2a and ASIC2b. Inactivation of the ASIC2 gene in mice leads to an increased rod electroretinogram of a- and b-waves, indicating an enhanced gain of visual transduction. ASIC2 knockout mice also show more sensitivity to light-induced retinal degeneration. Thus, ASIC2 is likely a negative modulator of rod phototransduction,

ASICs are also expressed at presynaptic sites.

**4.2 ASIC1a channels in fear-related behavior** 

**4.3 ASICs and retinal integrity** 

2004), but not unconditioned fear responses (Coryell et al., 2008).

excitation and [Ca2+]i accumulation. The Ca2+-permeability of ASICs in CNS neurons has been characterized using fluorescent Ca2+ imaging and ion-substitution protocols (Xiong et al., 2004; Yermolaieva et al., 2004). In mouse cortical, striatal and hippocampal neurons, activation of ASICs by decreasing in extracellular pH induces increases in [Ca2+]i. This acidinduced increase in [Ca2+]i could be recorded in the presence of a cocktail blocking other voltage-gated and ligand-gated Ca2+ channels (Xiong et al., 2004; Jiang et al., 2009), indicating Ca2+ entry directly through ASICs. The acid-induced increase in [Ca2+]i is eliminated by specific and non-specific ASIC1a blockade, or by ASIC1 gene knockout (Xiong et al., 2004; Yermolaieva et al., 2004; Jiang et al., 2009). Consistent with the finding of fluorescent imaging, acid-activated inward current is activated when extracellular solution contains Ca2+ as the only conducting cation (Xiong et al., 2004). Thus, homomeric ASIC1a channels constitute an additional and important Ca2+ entry pathway for neurons.

Fig. 4. pH drop triggered membrane depolarization and action potentials by activation of ASICs in cultured MSNs. Membrane depolarization by a drop in pH from 7.4 to 6.8 subsequently triggered trains of action potentials. The membrane depolarization was inhibited by amiloride (A) and PcTX1 (B)

#### **4. Physiological implications of ASICs in the CNS**

#### **4.1 ASIC1a channels in synaptic plasticity, learning and memory**

A change in pH at the synaptic cleft following synaptic release may render ASICs the opportunity to regulate synaptic transmission. The findings that ASICs are present at synaptic sites and can interact with postsynaptic density protein 95 as well as C kinase 1 interacting proteins (Hruska-Hageman et al., 2002; Wemmie et al., 2002; Zha et al., 2006, 2009) support this notion. Indeed, studies by Wemmie and coworkers have demonstrated that ASIC1a activation is involved in synaptic plasticity, learning and memory (Wemmie et al., 2002). They demonstrated that high frequency stimulation produces long-lasting potentiation of excitatory postsynaptic potentials (EPSP) in hippocampal slices from wildtype mice. However, the potentiation of EPSP decays rapidly to the baseline in slices from ASIC1a null mice. Further studies showed that the NMDA receptor antagonist D-2-Amino-5-phosphonovalerate inhibits EPSP summation in slices from wild-type but not ASIC1aknockout mice, suggesting that the loss of ASIC1a impaired NMDA-receptor function. ASIC1a disruption does not impair presynaptic vesicle release, as evidenced by normal single evoked EPSPs and paired-pulse facilitation. Interestingly, a later study by Cho and Askwith demonstrated that the presynaptic release probability is increased in cultured hippocampal neurons from the ASIC1 knockout mice (Cho & Askwith, 2008). Although localizations of ASICs at neuronal cell body and postsynaptic sites have been clearly demonstrated (Wemmie et al., 2002; Zha et al., 2006), it remains to be determined whether ASICs are also expressed at presynaptic sites.

#### **4.2 ASIC1a channels in fear-related behavior**

484 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

excitation and [Ca2+]i accumulation. The Ca2+-permeability of ASICs in CNS neurons has been characterized using fluorescent Ca2+ imaging and ion-substitution protocols (Xiong et al., 2004; Yermolaieva et al., 2004). In mouse cortical, striatal and hippocampal neurons, activation of ASICs by decreasing in extracellular pH induces increases in [Ca2+]i. This acidinduced increase in [Ca2+]i could be recorded in the presence of a cocktail blocking other voltage-gated and ligand-gated Ca2+ channels (Xiong et al., 2004; Jiang et al., 2009), indicating Ca2+ entry directly through ASICs. The acid-induced increase in [Ca2+]i is eliminated by specific and non-specific ASIC1a blockade, or by ASIC1 gene knockout (Xiong et al., 2004; Yermolaieva et al., 2004; Jiang et al., 2009). Consistent with the finding of fluorescent imaging, acid-activated inward current is activated when extracellular solution contains Ca2+ as the only conducting cation (Xiong et al., 2004). Thus, homomeric ASIC1a

channels constitute an additional and important Ca2+ entry pathway for neurons.

**mV Control Amiloride Wash**

**mV PcTX1 Wash**

Fig. 4. pH drop triggered membrane depolarization and action potentials by activation of ASICs in cultured MSNs. Membrane depolarization by a drop in pH from 7.4 to 6.8 subsequently triggered trains of action potentials. The membrane depolarization was

A change in pH at the synaptic cleft following synaptic release may render ASICs the opportunity to regulate synaptic transmission. The findings that ASICs are present at synaptic sites and can interact with postsynaptic density protein 95 as well as C kinase 1 interacting proteins (Hruska-Hageman et al., 2002; Wemmie et al., 2002; Zha et al., 2006, 2009) support this notion. Indeed, studies by Wemmie and coworkers have demonstrated that ASIC1a activation is involved in synaptic plasticity, learning and memory (Wemmie et al., 2002). They demonstrated that high frequency stimulation produces long-lasting

**-80 -60 -40 -20 0 20**

**B**

**Membrane Potential**

**A**

**Membrane Potential**

**pH 6.8**

**pH 6.8 -80 -60 -40 -20 0 20**

**7 Sec**

**Control**

**7 Sec**

inhibited by amiloride (A) and PcTX1 (B)

**4. Physiological implications of ASICs in the CNS** 

**4.1 ASIC1a channels in synaptic plasticity, learning and memory** 

ASIC1a is enriched in key structures of fear circuit (e.g. amygdala) (Wemmie et al., 2003). Thus, ASIC1a may influence fear responses. Indeed, Wemmie and colleagues demonstrated that ASIC1-null mice display significant deficits in cue and context fear conditioning (Wemmie et al., 2003). The loss of ASIC1a also reduces unconditioned fear in the open field test, during acoustic startle, and in response to predator odor (Coryell et al., 2007). Overexpressing ASIC1a, on the other hand, increases fear conditioning (Wemmie et al., 2004), but not unconditioned fear responses (Coryell et al., 2008).

Further studies by Wemmie's group suggest that activation of ASIC1a in brain chemosensors contributes to CO2 induced fear-related behavior (Ziemann et al., 2009). It has long been known that breathing CO2 triggers panic attacks in patients with panic disorder, and that these patients show an increased sensitivity to CO2 inhalation (Papp et al., 1993). In addition, patients with increasing hypercarbia due to respiratory failure become extremely anxious. How can CO2 inhalation contribute to fear behavior and related panic disorders? Wemmie and colleagues have provided evidence that ASIC1a channels are involved (Ziemann et al., 2009). They showed that inhaled CO2 triggers a drop in brain pH and induces fear behavior in mice. Eliminating or inhibiting ASIC1a significantly limits this activity. Overexpressing ASIC1a in the amygdala rescues the CO2-induced fear deficit in ASIC1a null mice. Buffering brain pH, on the other hand, attenuates fear behavior, whereas lowering pH in the amygdale reproduces the effect of CO2. These studies provide a novel molecular mechanism underlying CO2-induced intense fear and related anxiety/panic disorders and define the amygdala as an important chemosensor that detects hypercarbia/acidosis and initiates behavioral responses (Ziemann et al., 2009).

#### **4.3 ASICs and retinal integrity**

pH variations in the retina are involved in the fine-tuning of visual perception. Expression of ASICs in the retina suggests that they might play a role (Lilley et al., 2004). One study by Ettaiche suggested that ASIC2 is important for retinal function and likely protects against light-induced retinal degeneration. They showed that both photoreceptors and neurons of the mouse retina express ASIC2a and ASIC2b. Inactivation of the ASIC2 gene in mice leads to an increased rod electroretinogram of a- and b-waves, indicating an enhanced gain of visual transduction. ASIC2 knockout mice also show more sensitivity to light-induced retinal degeneration. Thus, ASIC2 is likely a negative modulator of rod phototransduction,

Acid-Sensing Ion Channels in Neurodegenerative Diseases: Potential Therapeutic Target 487

More recently, Vergo et al., from the same group studied acute and chronic EAE and multiple sclerosis spinal cord and optic nerve tissues to examine the distribution of ASIC1 and its relationship with neuronal and glial damage (Vergo et al., 2011). They found that ASIC1 was upregulated in axons and oligodendrocytes within lesions from mice with acute EAE and from patients with active multiple sclerosis. The expression of ASIC1 was associated with axonal damage as indicated by co-localization with the axonal injury marker beta amyloid precursor protein. Moreover, blocking ASIC1 with amiloride protected both myelin and neurons from damage in the acute model, and when given either at disease onset or, more clinically relevant, at first relapse, ameliorated disability in mice with chronic-relapsing EAE. Together these findings suggest that blockade of ASIC1 has the potential to provide both neuro- and myelo-protective benefits in multiple sclerosis (Vergo

PD is characterized by motor impairments and a loss of dopaminergic neurons in the substantia nigra (SNc) (Dauer & Przedborski, 2003). However, the mechanism of neuronal injury is not entirely clear. Previous studies have shown that the vulnerable neurons in this region also express ASIC1a (Wemmie et al., 2003; Pidoplichko & Dani, 2006). Given that PD, like ischemia, is associated with cerebral lactic acidosis, Arias et al tested the effect of ASIC blockade in a mouse model of PD induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment (Arias et al., 2008). As expected, amiloride was found to protect SNc neurons from MPTP-induced degeneration, and to preserve dopaminergic cell bodies in the SNc. Administration of PcTX venom resulted in a modest effect, attenuating the deficits in striatal DAT binding and dopamine. These findings suggest a potential role for ASICs in the

HD is a fatal neurodegenerative disorder. Energy metabolism deficit and acidosis have been observed in both *in vitro* and *in vivo* models of HD as well as in the brains of HD patients (Wong et al., 2008). To examine the potential involvement of ASICs in the pathology of HD, Wong et al tested effect of amiloride derivative benzamil both *in vitro* and *in vivo* (Wong et al., 2008). They showed that benzamil markedly reduced the huntingtin-polyglutamine (httpolyQ) aggregation in an inducible cellular system. In addition, the effect of benzamil was recapitulated in the R6/2 animal model of HD. Further experimentation showed that benzamil alleviated the inhibition of ubiquitin-proteasome system (UPS) activity, resulting in enhanced degradation of soluble htt-polyQ specifically in its pathological range. Blocking the expression of ASIC1a with siRNA also enhanced UPS activity, resulting in decreased httpolyQ aggregation in the striatum of R6/2 mice. Thus, targeting ASIC1a might be an

Based on ASIC1a channels in synaptic plasticity and learning/memory, a recent preliminary study has suggested that a reduced function of ASIC1a channels may contribute to the learning and memory deficit associated with AD (Maysami et al., 2009). In this study, Maysami et al showed that acid-activated currents in mouse cortical neurons and in CHO cells expressing ASIC1a are inhibited by nanomolar concentrations of amyloid beta peptide,

alternative approach to combat HD and other polyQ-related disorders.

et al., 2011).

**5.2 ASICs and Parkinson's disease (PD)** 

pathogenesis of Parkinson's disease.

**5.3 ASICs and Huntington's disease (HD)** 

**5.4 ASIC1a and Alzheimer's disease (AD)** 

and that functional ASIC2 channels are beneficial for the maintenance of retinal integrity (Ettaiche et al., 2004). However, since homomeric ASIC2a channels have an extremely lowsensitivity to protons (i.e. pH50 of 4.4), it is not clear whether active channel activity is required for this role.

Further studies by Ettaiche and colleagues also suggested an involvement of ASIC1a in retinal physiology (Ettaiche et al., 2006). In situ hybridization and immunohistochemistry detected the expression of ASIC1a in the outer and inner nuclear layers (cone photoreceptors, horizontal cells, some amacrine and bipolar cells) and in the ganglion cell layer. ASIC1a knockdown by antisense oligonucleotides and ASIC1a blockade by relatively specific inhibitor PcTX1 decreased the photopic a- and b-waves and oscillatory potentials. This finding suggests that ASIC1a is involved in normal retinal activity. Interestingly, a recent study by Render and colleagues did not detect any remarkable morphological changes in cone photoreceptors in ASIC1a-/- mice, at least at 5 or 22-27 weeks of age (Render et al., 2010). Thus, the exact role of this subunit in retinal integrity and/or function remains to be determined.

In addition to ASIC1a and ASIC2, a potential role of ASIC3 in retinal function and survival has been reported (Ettaiche et al., 2009). Ettaiche and colleagues demonstrated the presence of ASIC3 in the rod inner segment of photoreceptors, in horizontal and some amacrine cells. ASIC3 is also detected in retinal ganglion cells (RGCs) but contributes little to ASIC currents recorded in cultured RGCs. At 2 - 3 months, knockout mice experienced a moderate enhancement of scotopic electroretinogram a-wave amplitude and a concomitant increase of b-wave amplitude without alteration of retinal structure. Older (8-month-old) mice had large reductions in scotopic a- and b-waves, respectively, and reductions in oscillatory potential amplitudes associated with complete disorganization of the retina and degenerating rod inner segments. At 8 and 12 months of age, GFAP and TUNEL staining revealed an up-regulation of GFAP expression in Müller cells and the presence of apoptotic cells in the inner and outer retina (Ettaiche et al., 2009). Thus, ASIC3 also appears to be required for the maintenance of retina integrity.

#### **5. ASICs in neurodegenerative diseases**

#### **5.1 ASIC1 channels and multiple sclerosis**

Multiple sclerosis is a neuroinflammatory disease associated with axonal degeneration. Although inflammation and demyelination are the primary features of CNS lesions, axonal degeneration correlates best with clinical deficits in individuals with this disease. It has been suggested that the inflammatory insult leads to axonal degeneration by causing neuronal mitochondrial dysfunction, energy failure and alteration of ion exchange mechanisms (Waxman, 2006). Since excessive accumulation of Na+ and Ca2+ ions is associated with axonal degeneration (Stys & LoPachin, 1998), Friese et al determined whether ASIC1a activation, which is known to cause accumulation of Na+ and Ca2+ ions, contributes to such process in inflammatory lesions of the CNS (Friese et al., 2007). They showed that in an experimental model of autoimmune encephalomyelitis (EAE), ASIC1 null mice exhibit a significantly reduced clinical deficit and axonal degeneration as compared to wild-type mice. Further, pH measurements in the spinal cord of EAE mice display tissue acidosis sufficient to open ASIC1. The ASIC1 gene disruption also shows protective effect in nerve explants in vitro. ASIC blockade by amiloride is equally neuroprotective in nerve explants and in EAE. Thus, ASIC1a may be a potential target for axon degeneration associated with multiple sclerosis.

More recently, Vergo et al., from the same group studied acute and chronic EAE and multiple sclerosis spinal cord and optic nerve tissues to examine the distribution of ASIC1 and its relationship with neuronal and glial damage (Vergo et al., 2011). They found that ASIC1 was upregulated in axons and oligodendrocytes within lesions from mice with acute EAE and from patients with active multiple sclerosis. The expression of ASIC1 was associated with axonal damage as indicated by co-localization with the axonal injury marker beta amyloid precursor protein. Moreover, blocking ASIC1 with amiloride protected both myelin and neurons from damage in the acute model, and when given either at disease onset or, more clinically relevant, at first relapse, ameliorated disability in mice with chronic-relapsing EAE. Together these findings suggest that blockade of ASIC1 has the potential to provide both neuro- and myelo-protective benefits in multiple sclerosis (Vergo et al., 2011).

#### **5.2 ASICs and Parkinson's disease (PD)**

486 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

and that functional ASIC2 channels are beneficial for the maintenance of retinal integrity (Ettaiche et al., 2004). However, since homomeric ASIC2a channels have an extremely lowsensitivity to protons (i.e. pH50 of 4.4), it is not clear whether active channel activity is

Further studies by Ettaiche and colleagues also suggested an involvement of ASIC1a in retinal physiology (Ettaiche et al., 2006). In situ hybridization and immunohistochemistry detected the expression of ASIC1a in the outer and inner nuclear layers (cone photoreceptors, horizontal cells, some amacrine and bipolar cells) and in the ganglion cell layer. ASIC1a knockdown by antisense oligonucleotides and ASIC1a blockade by relatively specific inhibitor PcTX1 decreased the photopic a- and b-waves and oscillatory potentials. This finding suggests that ASIC1a is involved in normal retinal activity. Interestingly, a recent study by Render and colleagues did not detect any remarkable morphological changes in cone photoreceptors in ASIC1a-/- mice, at least at 5 or 22-27 weeks of age (Render et al., 2010). Thus, the exact role of this subunit in retinal integrity and/or function

In addition to ASIC1a and ASIC2, a potential role of ASIC3 in retinal function and survival has been reported (Ettaiche et al., 2009). Ettaiche and colleagues demonstrated the presence of ASIC3 in the rod inner segment of photoreceptors, in horizontal and some amacrine cells. ASIC3 is also detected in retinal ganglion cells (RGCs) but contributes little to ASIC currents recorded in cultured RGCs. At 2 - 3 months, knockout mice experienced a moderate enhancement of scotopic electroretinogram a-wave amplitude and a concomitant increase of b-wave amplitude without alteration of retinal structure. Older (8-month-old) mice had large reductions in scotopic a- and b-waves, respectively, and reductions in oscillatory potential amplitudes associated with complete disorganization of the retina and degenerating rod inner segments. At 8 and 12 months of age, GFAP and TUNEL staining revealed an up-regulation of GFAP expression in Müller cells and the presence of apoptotic cells in the inner and outer retina (Ettaiche et al., 2009). Thus, ASIC3 also appears to be

Multiple sclerosis is a neuroinflammatory disease associated with axonal degeneration. Although inflammation and demyelination are the primary features of CNS lesions, axonal degeneration correlates best with clinical deficits in individuals with this disease. It has been suggested that the inflammatory insult leads to axonal degeneration by causing neuronal mitochondrial dysfunction, energy failure and alteration of ion exchange mechanisms (Waxman, 2006). Since excessive accumulation of Na+ and Ca2+ ions is associated with axonal degeneration (Stys & LoPachin, 1998), Friese et al determined whether ASIC1a activation, which is known to cause accumulation of Na+ and Ca2+ ions, contributes to such process in inflammatory lesions of the CNS (Friese et al., 2007). They showed that in an experimental model of autoimmune encephalomyelitis (EAE), ASIC1 null mice exhibit a significantly reduced clinical deficit and axonal degeneration as compared to wild-type mice. Further, pH measurements in the spinal cord of EAE mice display tissue acidosis sufficient to open ASIC1. The ASIC1 gene disruption also shows protective effect in nerve explants in vitro. ASIC blockade by amiloride is equally neuroprotective in nerve explants and in EAE. Thus, ASIC1a may be a potential target for axon degeneration associated with

required for this role.

remains to be determined.

multiple sclerosis.

required for the maintenance of retina integrity.

**5. ASICs in neurodegenerative diseases 5.1 ASIC1 channels and multiple sclerosis** 

PD is characterized by motor impairments and a loss of dopaminergic neurons in the substantia nigra (SNc) (Dauer & Przedborski, 2003). However, the mechanism of neuronal injury is not entirely clear. Previous studies have shown that the vulnerable neurons in this region also express ASIC1a (Wemmie et al., 2003; Pidoplichko & Dani, 2006). Given that PD, like ischemia, is associated with cerebral lactic acidosis, Arias et al tested the effect of ASIC blockade in a mouse model of PD induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment (Arias et al., 2008). As expected, amiloride was found to protect SNc neurons from MPTP-induced degeneration, and to preserve dopaminergic cell bodies in the SNc. Administration of PcTX venom resulted in a modest effect, attenuating the deficits in striatal DAT binding and dopamine. These findings suggest a potential role for ASICs in the pathogenesis of Parkinson's disease.

#### **5.3 ASICs and Huntington's disease (HD)**

HD is a fatal neurodegenerative disorder. Energy metabolism deficit and acidosis have been observed in both *in vitro* and *in vivo* models of HD as well as in the brains of HD patients (Wong et al., 2008). To examine the potential involvement of ASICs in the pathology of HD, Wong et al tested effect of amiloride derivative benzamil both *in vitro* and *in vivo* (Wong et al., 2008). They showed that benzamil markedly reduced the huntingtin-polyglutamine (httpolyQ) aggregation in an inducible cellular system. In addition, the effect of benzamil was recapitulated in the R6/2 animal model of HD. Further experimentation showed that benzamil alleviated the inhibition of ubiquitin-proteasome system (UPS) activity, resulting in enhanced degradation of soluble htt-polyQ specifically in its pathological range. Blocking the expression of ASIC1a with siRNA also enhanced UPS activity, resulting in decreased httpolyQ aggregation in the striatum of R6/2 mice. Thus, targeting ASIC1a might be an alternative approach to combat HD and other polyQ-related disorders.

#### **5.4 ASIC1a and Alzheimer's disease (AD)**

Based on ASIC1a channels in synaptic plasticity and learning/memory, a recent preliminary study has suggested that a reduced function of ASIC1a channels may contribute to the learning and memory deficit associated with AD (Maysami et al., 2009). In this study, Maysami et al showed that acid-activated currents in mouse cortical neurons and in CHO cells expressing ASIC1a are inhibited by nanomolar concentrations of amyloid beta peptide,

Acid-Sensing Ion Channels in Neurodegenerative Diseases: Potential Therapeutic Target 489

Although the studies from ASIC1a and ASIC3 knockout mice indicated that ASICs contribute to neuropsychiatric disorders such as depression and anxiety, whether these neurological conditions are associated with significant change in local or global pH in the

During neurological conditions such as brain ischemia, increased anaerobic glycolysis due to reduced oxygen supply leads to lactic acid accumulation (Rehncrona, 1985). Accumulation of lactic acid, alone with increased H+ release from ATP hydrolysis, causes a decrease in pH, resulting in brain acidosis. During brain ischemia, for example, extracellular

Acidosis has long been known to play an important role in ischemic brain injury (Tombaugh & Sapolsky, 1993; Siesjo, et al., 1996), and a direct correlation of brain acidosis with infarct size has been described (Siesjo, 1988). However, the exact mechanism underlying acidosismediated neuronal injury remained uncertain. Severe acidosis may cause non-selective denaturation of proteins and nucleic acids (Kalimo et al., 1981); trigger cell swelling through

osmolysis (Kimelberg et al., 1990); hinder postischemic metabolic recovery by inhibiting mitochondrial energy metabolism and impairing postischemic blood flow via vascular edema (Hillered et al., 1985). The stimulation of pathologic free radical formation by acidosis has also been described (Rehncrona et al., 1989). At the neurotransmitter level, profound acidosis inhibits astrocytic glutamate uptake, which may contribute to excitatory neuronal injury (Swanson et al., 1995). Marked acidosis, with tissue pH<5.5, may influence neuronal

The widespread expression of ASIC1a in the brain, its activation by pH drops to the level commonly seen during ischemia, and its demonstrated role in intracellular Ca2+ accumulation suggested a potential involvement of these channels in the pathology of brain injury. Indeed, a number of recent studies have demonstrated an important role for ASIC1a activation in acidosis-mediated neuronal injury (Xiong et al., 2004; Yermolaieva et al., 2004; Gao et al., 2005; Pignataro nt al., 2007; Sherwood et al., 2009, 2011; Gu et al., 2010; Jetti et al., 2010; Li et al., 2010; Mari et al., 2010). In cultured mouse and human cortical neurons, for example, activation of ASICs by acid incubation induced glutamate receptor-independent neuronal injury inhibited by specific ASIC1a blockade, and/or by ASIC1 gene knockout (Xiong et al., 2004; Li et al., 2010). In rodent models of brain ischemia, intracerebroventricular injection of ASIC1a blocker/inhibitor reduced the infarct volume from transient or permanent focal ischemia by up to 60%(Xiong et al., 2004; Pignataro et al., 2007). Similarly, ASIC1 gene knockout produced significant neuroprotection in mice (Xiong et al., 2004). The protection by ASIC1a blockade had a time window of efficacy of up to 5 hours, and the

More recently, Sherwood et al., found that ASIC2b subunit can form functional channels with ASIC1a in cultured hippocampal neurons, and that the heteromeric ASIC1a/2b channels are calcium-permeable (Sherwood et al., 2011). Further, activation of heteromeric ASIC1a/2b channels contributes to acidosis-induced neuronal death. These data indicate that ASIC2, like ASIC1a, plays a role in acidosis-induced neuronal death and implicate the ASIC1a/2b subtype as a novel pharmacological target to prevent neuronal injury after

exchangers, which leads to cellular edema and

CNS remains to be determined.

stimulation of Na+/H+ and Cl-/HCO3-

**5.7 ASICs in acidosis-mediated ischemic neuronal injury** 

pH falls to 6.5 or lowers (Rehncrona, 1985; Nedergaard et al., 1991).

vulnerability indirectly by damaging glial cells (Giffard et al., 1990).

protection persists for at least 7 days (Pignataro nt al., 2007).

stroke (Sherwood et al., 2011).

a critical player for the pathology of AD. In addition to a reduction of current amplitude, amyloid beta peptide also slows down the activation of the channels. Thus, restoring the activity of ASIC1a channels could be a new intervention for AD.

#### **5.5 ASICs in depression-related behavior**

Depression disorders are a highly prevalent condition among adults in general population but the molecular pathways underlying depression are poorly understood. Recent studies by Coryell and colleagues have linked ASIC function to depression-related behavior (Coryell et al., 2009). They demonstrated that genetically disrupting ASIC1a in mice produced antidepressant-like effects in the forced swim test, the tail suspension test, and following unpredictable mild stress. Pharmacologically inhibiting ASIC1a also had antidepressant-like effects. The effects of ASIC1a disruption in the forced swim test were independent and additive to those of several commonly used antidepressants. Restoring ASIC1a to the amygdale of ASIC1a null mice reversed the forced swim test effects. The mechanism underlying the involvement of ASIC1a in depression-related behavior is not clear. It is likely that brain-derived neurotrophic factor (BDNF) is involved since both ASIC1a disruption and inhibition interfere with the ability of stress to reduce BDNF in the hippocampus. Thus, antagonists of ASIC1a channels may have potential for combating human depression.

#### **5.6 ASICs and anxiety disorders**

Anxiety disorders are debilitating neuropsychiatric disorders. Current treatments for anxiety disorders include pharmacological agents such as benzodiazepines and selective serotonin reuptake inhibitors. These agents, while effective in many patients, can induce a variety of side effects. Thus, it is necessary to develop a new generation of effective and better-tolerated anxiolytic agents. In this regard, Dwyer et al have shown that ASIC1a inhibitors have an effect in preclinical rodent models of autonomic and behavioral parameters of anxiety (Dwyer et al., 2009). In the stress-induced hyperthermia model, acute administration of ASIC inhibitors PcTX1, A-317567, and amiloride prevented stress-induced elevations in core body temperature. In the four-plate test, acute treatment with PcTX1 and A-317567 produced dose-dependent increases in the number of punished crossings. Further experiment showed that infusion of A-317567 into the amygdala significantly elevated the extracellular levels of GABA, but not glutamate, in this brain region. These findings suggest that ASIC inhibition has anxiolytic-like effects in some behavioral models and that GABAergic mechanisms are involved in the effects.

A recent study also suggests an involvement of ASIC3 in anxiety-like behavior (Wu et al., 2010). Although it is widely accepted that ASIC3 is predominately distributed in the peripheral nervous system, its expression has been found in rat hypothalamus (Meng et al., 2009). Study by Wu and colleagues also reported the expression of ASIC3 in the sensory mesencephalic trigeminal nucleus of mouse brain (Wu et al., 2010). However, whether ASIC3 plays any functional role in the brain was unclear. Wu et al showed that, in anxiety behavior tasks, ASIC3 null mice spent more time in the open arms of an elevated plus maze than did their wild-type littermates. ASIC3 null mice also displayed less aggressiveness toward intruders but more stereotypic repetitive behaviors during resident-intruder testing than did wild-type littermates. Therefore, loss of ASIC3 produces behavioral changes in anxiety and aggression in mice, which suggests that ASIC3-dependent sensory activities might be related to the central process of emotion modulation (Wu et al., 2010).

Although the studies from ASIC1a and ASIC3 knockout mice indicated that ASICs contribute to neuropsychiatric disorders such as depression and anxiety, whether these neurological conditions are associated with significant change in local or global pH in the CNS remains to be determined.

#### **5.7 ASICs in acidosis-mediated ischemic neuronal injury**

488 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

a critical player for the pathology of AD. In addition to a reduction of current amplitude, amyloid beta peptide also slows down the activation of the channels. Thus, restoring the

Depression disorders are a highly prevalent condition among adults in general population but the molecular pathways underlying depression are poorly understood. Recent studies by Coryell and colleagues have linked ASIC function to depression-related behavior (Coryell et al., 2009). They demonstrated that genetically disrupting ASIC1a in mice produced antidepressant-like effects in the forced swim test, the tail suspension test, and following unpredictable mild stress. Pharmacologically inhibiting ASIC1a also had antidepressant-like effects. The effects of ASIC1a disruption in the forced swim test were independent and additive to those of several commonly used antidepressants. Restoring ASIC1a to the amygdale of ASIC1a null mice reversed the forced swim test effects. The mechanism underlying the involvement of ASIC1a in depression-related behavior is not clear. It is likely that brain-derived neurotrophic factor (BDNF) is involved since both ASIC1a disruption and inhibition interfere with the ability of stress to reduce BDNF in the hippocampus. Thus, antagonists of ASIC1a channels may have potential for combating

Anxiety disorders are debilitating neuropsychiatric disorders. Current treatments for anxiety disorders include pharmacological agents such as benzodiazepines and selective serotonin reuptake inhibitors. These agents, while effective in many patients, can induce a variety of side effects. Thus, it is necessary to develop a new generation of effective and better-tolerated anxiolytic agents. In this regard, Dwyer et al have shown that ASIC1a inhibitors have an effect in preclinical rodent models of autonomic and behavioral parameters of anxiety (Dwyer et al., 2009). In the stress-induced hyperthermia model, acute administration of ASIC inhibitors PcTX1, A-317567, and amiloride prevented stress-induced elevations in core body temperature. In the four-plate test, acute treatment with PcTX1 and A-317567 produced dose-dependent increases in the number of punished crossings. Further experiment showed that infusion of A-317567 into the amygdala significantly elevated the extracellular levels of GABA, but not glutamate, in this brain region. These findings suggest that ASIC inhibition has anxiolytic-like effects in some behavioral models and that

A recent study also suggests an involvement of ASIC3 in anxiety-like behavior (Wu et al., 2010). Although it is widely accepted that ASIC3 is predominately distributed in the peripheral nervous system, its expression has been found in rat hypothalamus (Meng et al., 2009). Study by Wu and colleagues also reported the expression of ASIC3 in the sensory mesencephalic trigeminal nucleus of mouse brain (Wu et al., 2010). However, whether ASIC3 plays any functional role in the brain was unclear. Wu et al showed that, in anxiety behavior tasks, ASIC3 null mice spent more time in the open arms of an elevated plus maze than did their wild-type littermates. ASIC3 null mice also displayed less aggressiveness toward intruders but more stereotypic repetitive behaviors during resident-intruder testing than did wild-type littermates. Therefore, loss of ASIC3 produces behavioral changes in anxiety and aggression in mice, which suggests that ASIC3-dependent sensory activities

might be related to the central process of emotion modulation (Wu et al., 2010).

activity of ASIC1a channels could be a new intervention for AD.

**5.5 ASICs in depression-related behavior** 

human depression.

**5.6 ASICs and anxiety disorders** 

GABAergic mechanisms are involved in the effects.

During neurological conditions such as brain ischemia, increased anaerobic glycolysis due to reduced oxygen supply leads to lactic acid accumulation (Rehncrona, 1985). Accumulation of lactic acid, alone with increased H+ release from ATP hydrolysis, causes a decrease in pH, resulting in brain acidosis. During brain ischemia, for example, extracellular pH falls to 6.5 or lowers (Rehncrona, 1985; Nedergaard et al., 1991).

Acidosis has long been known to play an important role in ischemic brain injury (Tombaugh & Sapolsky, 1993; Siesjo, et al., 1996), and a direct correlation of brain acidosis with infarct size has been described (Siesjo, 1988). However, the exact mechanism underlying acidosismediated neuronal injury remained uncertain. Severe acidosis may cause non-selective denaturation of proteins and nucleic acids (Kalimo et al., 1981); trigger cell swelling through stimulation of Na+/H+ and Cl- /HCO3 exchangers, which leads to cellular edema and osmolysis (Kimelberg et al., 1990); hinder postischemic metabolic recovery by inhibiting mitochondrial energy metabolism and impairing postischemic blood flow via vascular edema (Hillered et al., 1985). The stimulation of pathologic free radical formation by acidosis has also been described (Rehncrona et al., 1989). At the neurotransmitter level, profound acidosis inhibits astrocytic glutamate uptake, which may contribute to excitatory neuronal injury (Swanson et al., 1995). Marked acidosis, with tissue pH<5.5, may influence neuronal vulnerability indirectly by damaging glial cells (Giffard et al., 1990).

The widespread expression of ASIC1a in the brain, its activation by pH drops to the level commonly seen during ischemia, and its demonstrated role in intracellular Ca2+ accumulation suggested a potential involvement of these channels in the pathology of brain injury. Indeed, a number of recent studies have demonstrated an important role for ASIC1a activation in acidosis-mediated neuronal injury (Xiong et al., 2004; Yermolaieva et al., 2004; Gao et al., 2005; Pignataro nt al., 2007; Sherwood et al., 2009, 2011; Gu et al., 2010; Jetti et al., 2010; Li et al., 2010; Mari et al., 2010). In cultured mouse and human cortical neurons, for example, activation of ASICs by acid incubation induced glutamate receptor-independent neuronal injury inhibited by specific ASIC1a blockade, and/or by ASIC1 gene knockout (Xiong et al., 2004; Li et al., 2010). In rodent models of brain ischemia, intracerebroventricular injection of ASIC1a blocker/inhibitor reduced the infarct volume from transient or permanent focal ischemia by up to 60%(Xiong et al., 2004; Pignataro et al., 2007). Similarly, ASIC1 gene knockout produced significant neuroprotection in mice (Xiong et al., 2004). The protection by ASIC1a blockade had a time window of efficacy of up to 5 hours, and the protection persists for at least 7 days (Pignataro nt al., 2007).

More recently, Sherwood et al., found that ASIC2b subunit can form functional channels with ASIC1a in cultured hippocampal neurons, and that the heteromeric ASIC1a/2b channels are calcium-permeable (Sherwood et al., 2011). Further, activation of heteromeric ASIC1a/2b channels contributes to acidosis-induced neuronal death. These data indicate that ASIC2, like ASIC1a, plays a role in acidosis-induced neuronal death and implicate the ASIC1a/2b subtype as a novel pharmacological target to prevent neuronal injury after stroke (Sherwood et al., 2011).

Acid-Sensing Ion Channels in Neurodegenerative Diseases: Potential Therapeutic Target 491

ASIC activation in inhibitory interneurons and facilitate GABAergic transmission, resulting

The inconsistent data on the role of ASICs in epileptic seizures may result from the use of different epilepsy models. The different ages of animals used may also contribute to the inconsistency since expression and function of ASICs in CNS neurons undergo dramatic developmental changes (Li et al., 2010). In addition, the finding that hippocampal interneurons are highly diverse with dramatically different expression level of ASICs (Weng

ASICs represent new biological components in peripheral sensory and CNS neurons. Increasing evidence indicates the involvement of these channels in both physiological and pathological processes of CNS (Grunder & Chen, 2010). Therefore, targeting these channels may provide novel and effective therapeutic interventions for a number of CNS diseases. In addition to establishing ASIC-specific small molecule antagonists that can easily pass through the blood brain barrier, alternative strategies may consider targeting endogenous modulators that are known to influence the expression and/or activity of these channels.

Adams, C.M.; Snyder, P.M. & Welsh, M.J. (1999). Paradoxical Stimulation of a DEG/ENaC

Akopian, A.N.; Chen, C.C.; Ding, Y. Cesare, P. & Wood, J.N. (2000). A New Member of the

Ali, A.; Ahmad, F.J.; Pillai, K.K. & Vohora, D. (2004). Evidence of the Antiepileptic Potential

and Behavior. *Epilepsy & Behavior,* Vol. 5, No. 3, pp. 322-328, ISSN 1525-5050 Ali, A.; Pillai, K.P.; Ahmad, F.J.; Dua, Y. & Vohora, D. (2006). Anticonvulsant Effect of

Allen, N.J. & Attwell, D. (2002). Modulation of ASIC Channels in Rat Crebellar Purkinje

Alvarez de la Rosa, D.; Canessa, C.M.; Fyfe, G.K. & Zhang, P. (2000). Structure and

Arias, R.L.; Sung, M.L.; Vasylyev, D.; Zhang, M.Y.; Albinson, K.; Kubek, K.; Kagan, N.;

Askwith, C.C.; Wemmie, J.A.; Price, M.P.; Rokhlina, T. & Welsh, M.J. (2004). Acid-Sensing

*Reports,* Vol. 58, No.2, pp. 242-245, ISSN 1734-1140

Vol. 62, No.1, pp.573-594, ISSN 0066-4278

Channel by Amiloride. *Journal of Biological Chemistry*, Vol. 274, No. 22, pp.15500-

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of Amiloride With Neuropharmacological Benefits in Rodent Models of Epilepsy

Amiloride in Pentetrazole-Induced Status Epilepticus in Mice. *Pharmacological* 

Nurons by Ichemia-Rlated Sgnals. *Journal of Phsiology,* Vol. 543, No. 2, pp. 521 – 529,

Regulation of Amiloride Sensitive Sodium Channels. *Annual Review of Physiology,* 

Beyer, C.; Lin, Q.; Dwyer, J.M.; Zaleska, M.M.; Bowlby, M.R.; Dunlop, J. & Monaghan, M. (2008). Amiloride is Neuroprotective in an MPTP Model of Parkinson's Disease. *Neurobiology of Disease,* Vol. 31, No. 3, pp. 334-341, ISSN 0969-

Ion Channel 2 (ASIC2) Modulates ASIC1 H+-Activated Currents in Hippocampal

in seizure termination.

**6. Conclusion** 

**7. References** 

15504, ISSN 0021-9258

0959-4956

ISSN 0022-3751

9961

et al., 2010) adds additional complexity to this subject.

Since activation of NMDA receptors and subsequent Ca2+ toxicity have been known to play an important role in ischemic brain injury, the outcome of co-application of both antagonists has also been investigated. Compared to ASIC1a or NMDA blockade alone, co-application of NMDA and ASIC antagonists produced additional neuroprotection, and the presence of ASIC1a blockade prolonged the time window of effectiveness of NMDA blockade (Pignataro nt al., 2007). Thus, ASIC1a represents a novel pharmacological target for ischemic brain injury.

In contrast to ASIC1a, a study by Johnson and colleagues suggests that an increased ASIC2a expression could provide protection against ischemic injury (Johnson et al., 2001). They showed an increased ASIC2a expression in neurons that survived global ischemia. This may be explained by the possibility that increased ASIC2a expression favors the formation of heteromeric ASIC1a/ASIC2a channels with reduced acid-sensitivity and no Ca2+ permeability.

#### **5.8 ASIC activation and epileptic seizure activity**

A significant drop of brain pH during intense neuronal excitation or seizure activity (Urbanics et al., 1978; Somjen et al., 1984; Simon et al., 1985, 1987; Chesler & Chan, 1988; Chesler & Kaila, 1992) suggests that ASIC activation might occur and activated ASICs then play a role in the generation/maintenance of epileptic seizures. However, the exact role of ASIC activation in seizure generation, propagation, and termination seems controversial.

Babinski and colleagues first reported a change of ASIC1a and ASIC2b expression in the hippocampal area following pilocarpine-induced epilepticus (Biagini et al., 2001), suggesting that the channels containing ASIC1a and ASIC2b subunits might play a role in the pathology of epilepsy.

Later on, a number of studies showed that amiloride, a commonly used non-selective ASIC blocker, has an anticonvulsant property *in vivo* in pilocarpine and pentylenetetrazole models of seizures (Ali et al., 2004, 2006; N'Gouemo, 2008), suggesting that ASIC activation might be proconvulsant. However, since amiloride also inhibits a number of other channels and ion exchange systems, these findings do not define ASICs as a specific target for amiloride to achieve its anti-epileptic action.

Using a number of *in vitro* epilepsy models, a preliminary study by Chang et al provided additional evidence that ASIC1a activation might be proconvulsant (Chang et al., 2007). In a cell culture model of epilepsy, brief withdrawal of the NMDA antagonist kynurenic acid induces a dramatic increase in the firing of action potentials, in addition to a sustained membrane depolarization. ASIC blockade by amiloride and the selective ASIC1a blocker PcTX1 significantly inhibited the increase of neuronal firing and the sustained membrane depolarization. In hippocampal slices, high frequency electrical stimulation or removal of extracellular Mg2+ triggers spontaneous seizure-like bursting. Bath perfusion of amiloride and PcTX1 decreased the amplitude and the frequency of these seizure-like bursting activities. Similarly, slices prepared from the brains of ASIC1a knockout mice demonstrated a reduced sensitivity to low extracellular Mg2+-induced or stimulation-evoked seizure activities (Chang et al., 2007).

In contrast, studies by Ziemann and colleagues, performed largely *in vivo*, have suggested that activation of ASIC1a channels is involved in the termination of epileptic seizure activity (Ziemann et al., 2008). An interesting finding by Ziemann and colleagues was that the level of ASIC1a expression is higher in GABAergic interneurons than in excitatory neurons (Ziemann et al., 2008). Therefore, acidosis generated during seizures might produce more ASIC activation in inhibitory interneurons and facilitate GABAergic transmission, resulting in seizure termination.

The inconsistent data on the role of ASICs in epileptic seizures may result from the use of different epilepsy models. The different ages of animals used may also contribute to the inconsistency since expression and function of ASICs in CNS neurons undergo dramatic developmental changes (Li et al., 2010). In addition, the finding that hippocampal interneurons are highly diverse with dramatically different expression level of ASICs (Weng et al., 2010) adds additional complexity to this subject.

#### **6. Conclusion**

490 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

Since activation of NMDA receptors and subsequent Ca2+ toxicity have been known to play an important role in ischemic brain injury, the outcome of co-application of both antagonists has also been investigated. Compared to ASIC1a or NMDA blockade alone, co-application of NMDA and ASIC antagonists produced additional neuroprotection, and the presence of ASIC1a blockade prolonged the time window of effectiveness of NMDA blockade (Pignataro nt al., 2007). Thus, ASIC1a represents a novel pharmacological target for ischemic

In contrast to ASIC1a, a study by Johnson and colleagues suggests that an increased ASIC2a expression could provide protection against ischemic injury (Johnson et al., 2001). They showed an increased ASIC2a expression in neurons that survived global ischemia. This may be explained by the possibility that increased ASIC2a expression favors the formation of heteromeric ASIC1a/ASIC2a channels with reduced acid-sensitivity and no Ca2+

A significant drop of brain pH during intense neuronal excitation or seizure activity (Urbanics et al., 1978; Somjen et al., 1984; Simon et al., 1985, 1987; Chesler & Chan, 1988; Chesler & Kaila, 1992) suggests that ASIC activation might occur and activated ASICs then play a role in the generation/maintenance of epileptic seizures. However, the exact role of ASIC activation in seizure generation, propagation, and termination seems controversial. Babinski and colleagues first reported a change of ASIC1a and ASIC2b expression in the hippocampal area following pilocarpine-induced epilepticus (Biagini et al., 2001), suggesting that the channels containing ASIC1a and ASIC2b subunits might play a role in

Later on, a number of studies showed that amiloride, a commonly used non-selective ASIC blocker, has an anticonvulsant property *in vivo* in pilocarpine and pentylenetetrazole models of seizures (Ali et al., 2004, 2006; N'Gouemo, 2008), suggesting that ASIC activation might be proconvulsant. However, since amiloride also inhibits a number of other channels and ion exchange systems, these findings do not define ASICs as a specific target for amiloride to

Using a number of *in vitro* epilepsy models, a preliminary study by Chang et al provided additional evidence that ASIC1a activation might be proconvulsant (Chang et al., 2007). In a cell culture model of epilepsy, brief withdrawal of the NMDA antagonist kynurenic acid induces a dramatic increase in the firing of action potentials, in addition to a sustained membrane depolarization. ASIC blockade by amiloride and the selective ASIC1a blocker PcTX1 significantly inhibited the increase of neuronal firing and the sustained membrane depolarization. In hippocampal slices, high frequency electrical stimulation or removal of extracellular Mg2+ triggers spontaneous seizure-like bursting. Bath perfusion of amiloride and PcTX1 decreased the amplitude and the frequency of these seizure-like bursting activities. Similarly, slices prepared from the brains of ASIC1a knockout mice demonstrated a reduced sensitivity to low extracellular Mg2+-induced or stimulation-evoked seizure

In contrast, studies by Ziemann and colleagues, performed largely *in vivo*, have suggested that activation of ASIC1a channels is involved in the termination of epileptic seizure activity (Ziemann et al., 2008). An interesting finding by Ziemann and colleagues was that the level of ASIC1a expression is higher in GABAergic interneurons than in excitatory neurons (Ziemann et al., 2008). Therefore, acidosis generated during seizures might produce more

brain injury.

permeability.

the pathology of epilepsy.

achieve its anti-epileptic action.

activities (Chang et al., 2007).

**5.8 ASIC activation and epileptic seizure activity** 

ASICs represent new biological components in peripheral sensory and CNS neurons. Increasing evidence indicates the involvement of these channels in both physiological and pathological processes of CNS (Grunder & Chen, 2010). Therefore, targeting these channels may provide novel and effective therapeutic interventions for a number of CNS diseases. In addition to establishing ASIC-specific small molecule antagonists that can easily pass through the blood brain barrier, alternative strategies may consider targeting endogenous modulators that are known to influence the expression and/or activity of these channels.

#### **7. References**


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**21** 

*Slovenia* 

**Genome Profiling and Potential** 

Luca Lovrečić, Aleš Maver and Borut Peterlin

**Biomarkers in Neurodegenerative Disorders** 

Neurodegenerative disorders (NDG) are incurable, progressive and debilitating conditions resulting from progressive degeneration and death of nerve cells. They are among the most serious health problems faced by modern society. Most of these disorders become more common with advancing age, including Alzheimer's disease and Parkinson's disease. The burden of these neurodegenerative diseases is growing inexorably as the population ages, with incalculable economic and human costs. According to the Global Burden of Disease Study, a collaborative study of the World Health Organization, the World Bank and the Harvard School of Public Health, dementia and other neurodegenerative diseases will be the eighth cause of disease burden for developed regions in 2020 [1, 2]. Also, according to the WHO, neurodegenerative diseases will become the world's second leading cause of death by 2050, overtaking cancer [2]. True, such estimates and predictions need to be taken with caution, but they definitely confirm that neurodegenerative diseases are of an increasing

Most NDG diseases are characterized by the aggregation of intracellular proteins. Majority of neurodegenerative disorders occur sporadically and are believed to arise through interactions between genetic and environmental factors. Only a small minority belong to familial forms where certain disease occurs due to a mutation of the gene coding for the

We differentiate many types of NDG disease, but the lines that separate one from another are often unclear. For instance, symptoms such as motor impairment and dementia may occur in many different types of NDG disease. Motor impairment similar to that seen in Parkinson's disease is not enough to rule out other diagnoses, especially when both motor and cognitive impairment are present. At the time being, there is no such diagnostic test that can clearly indicate the presence, absence, or category of a NDG disease. Individual diagnosis is based on clinical evaluation of the symptoms, with the exception of monogenic NDG diseases, such as Huntington's disease (HD). HD is a single gene disorder and cause is

Definitive diagnosis of certain NDG diseases still relies on neuropathological evaluation. But it has been demonstrated that brain pathology can show marked overlap among the syndromes of age-related cognitive and motor impairment [4]. Also, previous research reports have shown that pathological markers do not always correlate optimally with clinical findings. Some individuals with extensive neuropathology may retain relatively

**1. Introduction** 

public concern.

abnormally aggregating protein.

invariably trinucleotide expansion mutation [3].

*Clinical Institute of Medical Genetics, University Medical Center Ljubljana,* 


### **Genome Profiling and Potential Biomarkers in Neurodegenerative Disorders**

Luca Lovrečić, Aleš Maver and Borut Peterlin *Clinical Institute of Medical Genetics, University Medical Center Ljubljana, Slovenia* 

#### **1. Introduction**

502 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

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& Wemmie, J.A. (2008). Seizure Termination by Acidosis Depends on ASIC1a.

Neurodegenerative disorders (NDG) are incurable, progressive and debilitating conditions resulting from progressive degeneration and death of nerve cells. They are among the most serious health problems faced by modern society. Most of these disorders become more common with advancing age, including Alzheimer's disease and Parkinson's disease. The burden of these neurodegenerative diseases is growing inexorably as the population ages, with incalculable economic and human costs. According to the Global Burden of Disease Study, a collaborative study of the World Health Organization, the World Bank and the Harvard School of Public Health, dementia and other neurodegenerative diseases will be the eighth cause of disease burden for developed regions in 2020 [1, 2]. Also, according to the WHO, neurodegenerative diseases will become the world's second leading cause of death by 2050, overtaking cancer [2]. True, such estimates and predictions need to be taken with caution, but they definitely confirm that neurodegenerative diseases are of an increasing public concern.

Most NDG diseases are characterized by the aggregation of intracellular proteins. Majority of neurodegenerative disorders occur sporadically and are believed to arise through interactions between genetic and environmental factors. Only a small minority belong to familial forms where certain disease occurs due to a mutation of the gene coding for the abnormally aggregating protein.

We differentiate many types of NDG disease, but the lines that separate one from another are often unclear. For instance, symptoms such as motor impairment and dementia may occur in many different types of NDG disease. Motor impairment similar to that seen in Parkinson's disease is not enough to rule out other diagnoses, especially when both motor and cognitive impairment are present. At the time being, there is no such diagnostic test that can clearly indicate the presence, absence, or category of a NDG disease. Individual diagnosis is based on clinical evaluation of the symptoms, with the exception of monogenic NDG diseases, such as Huntington's disease (HD). HD is a single gene disorder and cause is invariably trinucleotide expansion mutation [3].

Definitive diagnosis of certain NDG diseases still relies on neuropathological evaluation. But it has been demonstrated that brain pathology can show marked overlap among the syndromes of age-related cognitive and motor impairment [4]. Also, previous research reports have shown that pathological markers do not always correlate optimally with clinical findings. Some individuals with extensive neuropathology may retain relatively

Genome Profiling and Potential Biomarkers in Neurodegenerative Disorders 505

pathogenesis and development of diagnostic approaches, including disease/process

Huntington disease is a late onset, single gene disorder and its cause is invariably trinucleotide expansion mutation, known for almost 2 decades [3]. Clinical characteristics of the disease include progressive motor impairment, cognitive decline and various psychiatric symptoms with the typical age of onset in the third to fifth decade. The disease is fatal after 15-20 years of progressive neurodegeneration [12]. So far, no effective treatment has been available to cure the disease or to slow its progression. Hyperkinesias and psychiatric symptoms may respond well to pharmacotherapy, but neuropsychological deficits and dementia remain untreatable [13]. We are unable to predict the age at onset and to follow the disease progression over short time periods due to the unsensitivity of rating scales. Even more, no useful measures to follow response to symptomatic treatment over short time periods are known. In addition, in the presymptomatic period when preventive treatment and slowing of neurodegeneration might be most effective, we have no measures/markers

Although the responsible gene and mutation were already identified and characterized in 1993, the function of normal huntingtin and the mutation mechanism that leads to neurodegeneration are still not clear. Basic research has demonstrated that the pathogenesis of HD involves recruitment of multiple biochemical pathways like protein degradation, apoptosis, accumulation of misfolded mutated proteins, intracelular signaling, oxidative

Dementia, common symptom of all three already mentioned neurodegenerative diseases is also a common symptom in individuals with Down syndrome (DS). Most of individuals with DS after about age of 30 have the characteristic plaques and neurofibrillary tangles, associated with AD. As in general population, the prevalence of AD in people with DS increases significantly with age. On the other hand, age-related cognitive decline and dementia in people with DS occurs 30–40 years earlier than in the general population, reaching almost 40% in the 50s [16]. Life expectancy of people with DS continues to increase

Research in the field of biomarkers is a rapidly growing and developing area in medicine. Everyday advances in genomic, proteomic, metabolomic and epigenomic knowledge and technologies have made their way also in the neuroscientific research area. Biomarkers are very important indicators of normal and abnormal biological processes. By definition, biological marker or biomarker is a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention [17]. Despite the fact that enormous effort and extensive research have been concentrated on this area, there is still a major lack of biomarkers for diagnosis, progression monitoring, response to treatment evaluation, etc. in neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD)

stress, mitochondrial involvement and in the last years also transcription [14, 15].

**2.2 Huntington disease – A model of genetic neurodegenerative disorder** 

specific biomarkers.

to monitor those responses and benefits.

**2.3 Dementia and Down syndrome** 

**2.4 Biomarkers** 

and Huntington's disease (HD).

and therefore, dementia is becoming an important issue.

intact neurological function while others with less extensive pathology may be significantly impaired [5, 6]. The neuropathological findings may be the response to other antecedent disease processes and are not necessarily the cause of the underlying disease at the early disease stages. Later, as disease progresses, they probably contribute to disease progression in a positive feedback loop.

Analysis of whole genome transcriptome in brain might give us insights into the disturbed pathways and processes involved in disease onset and progression. Many different mechanisms have been proposed to be dysregulated in NDG diseases. We collected all reported studies to date on brain transcriptome in Parkinson's disease, Alzheimer disease, Huntington disease and Down syndrome and performed an integrated meta-analysis.

#### **2. Background**

#### **2.1 Common neurodegenerative disorders – Alzheimer and Parkinson disease**

Two most common neurodegenerative diseases, Parkinson's disease (PD) and Alzheimer disease(AD) are believed to be heterogeneous based on the causes - combination of genetic and environmental factors, vast variety in the age at onset, variability in leading symptoms and presenting clinical manifestations, disease progression and responses to different therapies employed. Definitive diagnosis of both, AD and PD still relies on a 'gold standard' post mortem neuropathological evaluation, although a number of clinical and neuropsychological tests are often employed when making a clinical diagnosis. AD is detected with approximately 85–90% accuracy and PD with approximaly 75% accuracy. The pathogenesis of both AD and PD are complex and still remain unexplained in worldwide research community.

It has been recently estimated [7] that 24 million people have dementia worldwide and majority is attributable to AD. The authors emphasized the urgency of better understanding of pathophysiology of the disease in order to improve development of disease-modifying treatment. Due to the age-dependent incidence rate of AD and due to the population ageing, it is foreseen that more than 80 million people will have AD by 2040 [8]. It is a progressive neurologic disease affecting particularly cortical and hippocampal neurons, leading to their irreversible loss [9]. Major clinical signs and symptoms are progressive impairment in memory, judgment, decision making, orientation to physical surroundings, and language. The key pathological characteristics are neuronal loss, β amyloid containing extracellular senile plaques, and neurofibrillary tangles, which are composed of a hyperphosphorylated form of the microtubular protein tau.

PD is the second most prevalent NDG disease after AD. According to available data of European Parkinson's Disease Association (EPDA), there are 6.3 million people with PD worldwide. Prevalence is age-dependent - there are approximately 0.5 to 1 percent of individuals with PD in the age group 65 to 69 years, and 1 to 3 percent of individuals with PD in the group of people older than 80 years [10]. Typical clinical sign is parkinsonism resting tremor, bradykinesia, rigidity, and postural instability. Neuropathological characteristics are the loss of neurons in the substantia nigra and the presence of neuronal inclusions termed Lewy bodies and Lewy neurites whose main component is aggregated and phosphorylated alpha-synuclein [11].

Important futuristic challenge in the management AD and PD remains the establishment of early diagnosis or even identification of individuals prior to the onset of dementia in AD or resting tremor in PD. This implicates advancement in understanding disease pathogenesis and development of diagnostic approaches, including disease/process specific biomarkers.

#### **2.2 Huntington disease – A model of genetic neurodegenerative disorder**

Huntington disease is a late onset, single gene disorder and its cause is invariably trinucleotide expansion mutation, known for almost 2 decades [3]. Clinical characteristics of the disease include progressive motor impairment, cognitive decline and various psychiatric symptoms with the typical age of onset in the third to fifth decade. The disease is fatal after 15-20 years of progressive neurodegeneration [12]. So far, no effective treatment has been available to cure the disease or to slow its progression. Hyperkinesias and psychiatric symptoms may respond well to pharmacotherapy, but neuropsychological deficits and dementia remain untreatable [13]. We are unable to predict the age at onset and to follow the disease progression over short time periods due to the unsensitivity of rating scales. Even more, no useful measures to follow response to symptomatic treatment over short time periods are known. In addition, in the presymptomatic period when preventive treatment and slowing of neurodegeneration might be most effective, we have no measures/markers to monitor those responses and benefits.

Although the responsible gene and mutation were already identified and characterized in 1993, the function of normal huntingtin and the mutation mechanism that leads to neurodegeneration are still not clear. Basic research has demonstrated that the pathogenesis of HD involves recruitment of multiple biochemical pathways like protein degradation, apoptosis, accumulation of misfolded mutated proteins, intracelular signaling, oxidative stress, mitochondrial involvement and in the last years also transcription [14, 15].

#### **2.3 Dementia and Down syndrome**

Dementia, common symptom of all three already mentioned neurodegenerative diseases is also a common symptom in individuals with Down syndrome (DS). Most of individuals with DS after about age of 30 have the characteristic plaques and neurofibrillary tangles, associated with AD. As in general population, the prevalence of AD in people with DS increases significantly with age. On the other hand, age-related cognitive decline and dementia in people with DS occurs 30–40 years earlier than in the general population, reaching almost 40% in the 50s [16]. Life expectancy of people with DS continues to increase and therefore, dementia is becoming an important issue.

#### **2.4 Biomarkers**

504 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

intact neurological function while others with less extensive pathology may be significantly impaired [5, 6]. The neuropathological findings may be the response to other antecedent disease processes and are not necessarily the cause of the underlying disease at the early disease stages. Later, as disease progresses, they probably contribute to disease progression

Analysis of whole genome transcriptome in brain might give us insights into the disturbed pathways and processes involved in disease onset and progression. Many different mechanisms have been proposed to be dysregulated in NDG diseases. We collected all reported studies to date on brain transcriptome in Parkinson's disease, Alzheimer disease, Huntington disease and Down syndrome and performed an integrated meta-analysis.

**2.1 Common neurodegenerative disorders – Alzheimer and Parkinson disease** 

Two most common neurodegenerative diseases, Parkinson's disease (PD) and Alzheimer disease(AD) are believed to be heterogeneous based on the causes - combination of genetic and environmental factors, vast variety in the age at onset, variability in leading symptoms and presenting clinical manifestations, disease progression and responses to different therapies employed. Definitive diagnosis of both, AD and PD still relies on a 'gold standard' post mortem neuropathological evaluation, although a number of clinical and neuropsychological tests are often employed when making a clinical diagnosis. AD is detected with approximately 85–90% accuracy and PD with approximaly 75% accuracy. The pathogenesis of both AD and PD are complex and still remain unexplained in worldwide

It has been recently estimated [7] that 24 million people have dementia worldwide and majority is attributable to AD. The authors emphasized the urgency of better understanding of pathophysiology of the disease in order to improve development of disease-modifying treatment. Due to the age-dependent incidence rate of AD and due to the population ageing, it is foreseen that more than 80 million people will have AD by 2040 [8]. It is a progressive neurologic disease affecting particularly cortical and hippocampal neurons, leading to their irreversible loss [9]. Major clinical signs and symptoms are progressive impairment in memory, judgment, decision making, orientation to physical surroundings, and language. The key pathological characteristics are neuronal loss, β amyloid containing extracellular senile plaques, and neurofibrillary tangles, which are composed of a hyperphosphorylated

PD is the second most prevalent NDG disease after AD. According to available data of European Parkinson's Disease Association (EPDA), there are 6.3 million people with PD worldwide. Prevalence is age-dependent - there are approximately 0.5 to 1 percent of individuals with PD in the age group 65 to 69 years, and 1 to 3 percent of individuals with PD in the group of people older than 80 years [10]. Typical clinical sign is parkinsonism resting tremor, bradykinesia, rigidity, and postural instability. Neuropathological characteristics are the loss of neurons in the substantia nigra and the presence of neuronal inclusions termed Lewy bodies and Lewy neurites whose main component is aggregated

Important futuristic challenge in the management AD and PD remains the establishment of early diagnosis or even identification of individuals prior to the onset of dementia in AD or resting tremor in PD. This implicates advancement in understanding disease

in a positive feedback loop.

**2. Background** 

research community.

form of the microtubular protein tau.

and phosphorylated alpha-synuclein [11].

Research in the field of biomarkers is a rapidly growing and developing area in medicine. Everyday advances in genomic, proteomic, metabolomic and epigenomic knowledge and technologies have made their way also in the neuroscientific research area. Biomarkers are very important indicators of normal and abnormal biological processes. By definition, biological marker or biomarker is a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention [17]. Despite the fact that enormous effort and extensive research have been concentrated on this area, there is still a major lack of biomarkers for diagnosis, progression monitoring, response to treatment evaluation, etc. in neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD).

Genome Profiling and Potential Biomarkers in Neurodegenerative Disorders 507

As we were primarily interested in the studies with microarray experimental results accessible from biological repositories, we then searched Gene Expression Omnibus (GEO) repository (*http://www.ncbi.nlm.nih.gov/geo/*), ArrayExpress database (*http://www.ebi.ac.uk/arrayexpress/*) and Stanford Microarray database (*http://smd.stanford.edu*) for studies with data available in the raw or processed form. As most of the gene expression profiling experiments were performed on Affymetrix platform and to avoid difficulties due to different probe annotations utilized by different microarray manufacturers, only results from experiments performed on the Affymetrix U133 platform were included to facilitate further steps in probe level meta-analysis of microarray data. The detailed information on datasets included

All the integration and statistical steps described were performed in R statistical environment version 2.13.1 (http://cran.r-project.org), using Bioconductor version 2.8 packages (available at http://bioconductor.org) [19]. Raw data from all microarray experiments listed in Table 1 was obtained directly from Gene Expression Omnibus (GEO) repository (http://www.ncbi.nlm.nih.gov/geo/) utilizing the GEOquery package for R [20, 21]. Before the meta-analysis of data from selected studies was performed, all the datasets obtained in such manner were inspected for significant inter-array differences in distribution of probe intensities. For this reason, raw datasets were initially examined using arrayQualityMetrics package and where necessary the straightforward quantile normalization functions in the affyPLM package was utilized [30, 31]. Non-specific intensity and interquartile variation filters were applied using methods in genefilter package [19]. Log2 transformations were applied where discrepancies in data reporting format were

Data collections for each individual neurodegenerative disease were then merged using probeset annotations as the common denominator. Using this approach we avoided potential statistical issues originating from averaging probe intensity values to obtain a single mean intensity value for each gene, possibly disregarding distinct expression of

These steps resulted in generation of 4 separate data matrices, each carrying data for a single disease, originating from multiple studies – Alzheimer disease (AD), Down syndrome (DS),

Summarized differential expression of genes in each merged dataset was calculated using meta-analysis algorithms incorporated in the RankProd package for R [32]. RankProd uses a non-parametric statistical algorithm that facilitates detection of genes that are consistently highly ranked across microarray datasets originating from various microarray experiments in various studies perfomed on the same condition (ie. disease). As this approach is based on rank statistics in contrast to approaches requiring analyzing absolute intensity values, it allows for inclusion of data originating from different laboratories, differing platforms and

For analyses of such multi-study data, RPadvance function was utilized in our analyses, with origin parameter set to account for data originating from number of different sources corresponding to the number of different originating study [32]. Here it is important to

**3.2 Microarray data pre-processing and preparation for meta-analysis** 

in the analyses may be observed in Table 1.

different transcripts from the same gene.

Huntington disease (HD) and Parkinson disease (PD) datasets.

potentially studies performed under differing conditions [32].

observed.

**3.3 Meta-analysis** 

Biomarkers have many valuable applications, such as identification of major neuropathological processes in specific disease, disease detection and monitoring of health status, early efficacy and safety evaluations in *in vitro* studies in tissue samples, *in vivo* studies in animal models, and early-phase clinical trials. They are invaluable as a diagnostic tool for identification of patients with a disease or abnormal condition, as a tool in staging the disease or classification of the extent of disease, as an indicator of disease prognosis and in predicting and monitoring of a clinical response to treatment.

Biomarkers are of extreme relevance in chronic NDG diseases - there are no cures for these diseases, as neurons of the central nervous system cannot regenerate on their own after cell death or damage. Tremendous efforts have been made in recent years to identify the neuropathological, biochemical, and genetic biomarkers of these diseases aiming to establish the diagnosis in earlier stages, to survey the rate of progression, or response to treatment. Currently, the neuropathologic diagnosis is a gold standard, but it can only be made in the form of an autopsy after the patient's death. On the other hand, biomarkers may improve the early diagnosis at a stage when disease-modifying therapies are likely to be most effective, the monitoring of disease progression and the efficacy of any therapeutic intervention [18].

#### **2.5 Brain transcriptome in neurodegenerative disorders**

Many different research groups have tried to solve the neuropathophysiological puzzle in PD, AD, HD and DS. Human brain has been extensivelly studied using many approaches, in the last decade also variety of »omic« technologies. Whole-genome gene expression studies in brain of each of four diseases individually have shown changes in transcription of number of genes when compared to normal human brain.

We investigated, reviewed and collected data from all reported studies to date on brain transcriptome in Parkinson's disease, Alzheimer disease, Huntington disease and Down syndrome and performed integrated meta-analysis.

#### **3. Methods**

In an attempt to present the alterations consistently reported by studies of brain transcriptome in neurodegenerative diseases, we initially searched for such reports in literature databases, then obtained raw and processed experimental data from microarray data repositories, after which we performed probe level meta-analyses of datasets originating from various studies. In addition, to reveal possible commonalities and shared pathways across various neurodegenerative diseases, we inspected the similarities and differences in gene expression dysregularities occurring in these conditions.

#### **3.1 Study inclusion**

Initially, we have searched Medline database (http://www.ncbi.nlm.nih.gov/pubmed) for reports from studies of interest using the search string (transcriptom\* OR microarray OR profiling OR Affymetrix OR Agilent OR Illumina OR array) AND (Parkinson's disease OR Parkinsons disease OR Parkinson disease AND Alzheimer's disease OR Alzheimers disease OR Alzheimer disease OR dementia OR Down's syndrome OR Downs syndrome OR Down syndrome OR trisomy 21 OR Huntington's disease OR Huntingtons disease OR Huntington disease) to obtain the complete list of studies reporting results relating to transcriptional alterations in brain tissues affected by neurodegenerative processes.

As we were primarily interested in the studies with microarray experimental results accessible from biological repositories, we then searched Gene Expression Omnibus (GEO) repository (*http://www.ncbi.nlm.nih.gov/geo/*), ArrayExpress database (*http://www.ebi.ac.uk/arrayexpress/*) and Stanford Microarray database (*http://smd.stanford.edu*) for studies with data available in the raw or processed form. As most of the gene expression profiling experiments were performed on Affymetrix platform and to avoid difficulties due to different probe annotations utilized by different microarray manufacturers, only results from experiments performed on the Affymetrix U133 platform were included to facilitate further steps in probe level meta-analysis of microarray data. The detailed information on datasets included in the analyses may be observed in Table 1.

#### **3.2 Microarray data pre-processing and preparation for meta-analysis**

All the integration and statistical steps described were performed in R statistical environment version 2.13.1 (http://cran.r-project.org), using Bioconductor version 2.8 packages (available at http://bioconductor.org) [19]. Raw data from all microarray experiments listed in Table 1 was obtained directly from Gene Expression Omnibus (GEO) repository (http://www.ncbi.nlm.nih.gov/geo/) utilizing the GEOquery package for R [20, 21].

Before the meta-analysis of data from selected studies was performed, all the datasets obtained in such manner were inspected for significant inter-array differences in distribution of probe intensities. For this reason, raw datasets were initially examined using arrayQualityMetrics package and where necessary the straightforward quantile normalization functions in the affyPLM package was utilized [30, 31]. Non-specific intensity and interquartile variation filters were applied using methods in genefilter package [19]. Log2 transformations were applied where discrepancies in data reporting format were observed.

Data collections for each individual neurodegenerative disease were then merged using probeset annotations as the common denominator. Using this approach we avoided potential statistical issues originating from averaging probe intensity values to obtain a single mean intensity value for each gene, possibly disregarding distinct expression of different transcripts from the same gene.

These steps resulted in generation of 4 separate data matrices, each carrying data for a single disease, originating from multiple studies – Alzheimer disease (AD), Down syndrome (DS), Huntington disease (HD) and Parkinson disease (PD) datasets.

#### **3.3 Meta-analysis**

506 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

Biomarkers have many valuable applications, such as identification of major neuropathological processes in specific disease, disease detection and monitoring of health status, early efficacy and safety evaluations in *in vitro* studies in tissue samples, *in vivo* studies in animal models, and early-phase clinical trials. They are invaluable as a diagnostic tool for identification of patients with a disease or abnormal condition, as a tool in staging the disease or classification of the extent of disease, as an indicator of disease prognosis and

Biomarkers are of extreme relevance in chronic NDG diseases - there are no cures for these diseases, as neurons of the central nervous system cannot regenerate on their own after cell death or damage. Tremendous efforts have been made in recent years to identify the neuropathological, biochemical, and genetic biomarkers of these diseases aiming to establish the diagnosis in earlier stages, to survey the rate of progression, or response to treatment. Currently, the neuropathologic diagnosis is a gold standard, but it can only be made in the form of an autopsy after the patient's death. On the other hand, biomarkers may improve the early diagnosis at a stage when disease-modifying therapies are likely to be most effective, the monitoring of disease progression and the efficacy of any therapeutic

Many different research groups have tried to solve the neuropathophysiological puzzle in PD, AD, HD and DS. Human brain has been extensivelly studied using many approaches, in the last decade also variety of »omic« technologies. Whole-genome gene expression studies in brain of each of four diseases individually have shown changes in transcription of

We investigated, reviewed and collected data from all reported studies to date on brain transcriptome in Parkinson's disease, Alzheimer disease, Huntington disease and Down

In an attempt to present the alterations consistently reported by studies of brain transcriptome in neurodegenerative diseases, we initially searched for such reports in literature databases, then obtained raw and processed experimental data from microarray data repositories, after which we performed probe level meta-analyses of datasets originating from various studies. In addition, to reveal possible commonalities and shared pathways across various neurodegenerative diseases, we inspected the similarities and

Initially, we have searched Medline database (http://www.ncbi.nlm.nih.gov/pubmed) for reports from studies of interest using the search string (transcriptom\* OR microarray OR profiling OR Affymetrix OR Agilent OR Illumina OR array) AND (Parkinson's disease OR Parkinsons disease OR Parkinson disease AND Alzheimer's disease OR Alzheimers disease OR Alzheimer disease OR dementia OR Down's syndrome OR Downs syndrome OR Down syndrome OR trisomy 21 OR Huntington's disease OR Huntingtons disease OR Huntington disease) to obtain the complete list of studies reporting results relating to transcriptional

differences in gene expression dysregularities occurring in these conditions.

alterations in brain tissues affected by neurodegenerative processes.

in predicting and monitoring of a clinical response to treatment.

**2.5 Brain transcriptome in neurodegenerative disorders** 

number of genes when compared to normal human brain.

syndrome and performed integrated meta-analysis.

intervention [18].

**3. Methods**

**3.1 Study inclusion** 

Summarized differential expression of genes in each merged dataset was calculated using meta-analysis algorithms incorporated in the RankProd package for R [32]. RankProd uses a non-parametric statistical algorithm that facilitates detection of genes that are consistently highly ranked across microarray datasets originating from various microarray experiments in various studies perfomed on the same condition (ie. disease). As this approach is based on rank statistics in contrast to approaches requiring analyzing absolute intensity values, it allows for inclusion of data originating from different laboratories, differing platforms and potentially studies performed under differing conditions [32].

For analyses of such multi-study data, RPadvance function was utilized in our analyses, with origin parameter set to account for data originating from number of different sources corresponding to the number of different originating study [32]. Here it is important to

Genome Profiling and Potential Biomarkers in Neurodegenerative Disorders 509

stress that we have faced the issue of multiple studies simultaneously reporting differential expression in several different anatomical brain parts. As we wanted to facilitate the discovery of differentially expressed genes in diseased tissue in comparison to control samples, we set the origin parameter to take into account these considerations and regard such data as originating from different sources, thereby avoiding comparisons of gene expression between different brain regions rather than between affected and unaffected samples. Afterwards, P-values and q-values were obtained by performing 100 permutation cycles of complete originating datasets. An arbitrary P-value cut-off for significance of

**3.4 Investigating intersections between datasets and gene set enrichment analyses**  Resulting ordered lists of differentially expressed probesets were subsequently investigated for overlap between AD, DS, HD and PD datasets. Top 1000 genes from each dataset were used and intersections between combinations of two, three and four datasets were obtained. Venn diagrams in the results section were produced using Venny utility available at *http://bioinfogp.cnb.csic.es/tools/venny/index.html*. Furthermore, to gain insight in functional properties of genes in the intersections, gene set enrichment analyses (GSEA) were performed, utilizing GOstats package for R and investigating significant (uncorrected p<0.05) over- or underrepresentation of GeneOntology (GO) and KEGG terms annotating genes occurring in the intersections [33-36]. Additionally, DAVID tool (http://david.abcc.ncifcrf.gov/) was used to reveal the functional annotation clusters related to intersecting genes [37]. Required annotation conversions were performed using the hgu133plus.db package from Bioconductor annotation package collection and using biomaRt package for R in combination with Ensembl

Alltogether, our data collection comprised of data from 9 whole-genome expression studies, performed on samples from 4 neurodegenerative conditions (AD, DS, HD and PD). Collectively, 200, 33, 201, and 186 microarray analysed samples were included in the investigations of AD, DS, HD and PD, respectively, which accounted for 620 separate experiments included overall. A slight predominance of experiments performed on case tissues was noted in most of the experiements with summary case:control ratio amounting

Separate analyses of datasets for each NDG disorder have revealed significant perturbances in expression profiles of several genes. When arbitrary permutation p-value cut-off was set at 0.05 for upregulated genes, 5701 probesets attained significance in the AD dataset, 3291 in DS dataset, 4174 in the HD dataset and 3043 in the PD dataset. In the downregulated gene group the p<0.05 significance was reached for 5496 probesets in the AD dataset, 2983 probesets in the DS dataset, 4079 in the HD dataset and 3410 in the PD dataset. A detailed view of the distribution of significance values of the top 10,000 ordered differentially

The resulting numbers of significant results are inflated by the effect of multiple testing and therefore the q- values were also estimated as described in the article by Breitling et al [40]. The numbers of upregulated probesets with estimated q-values below 0.05 were 3775 for AD, 1496 for DS, 3182 for HD and 1894 for PD datasets. The numbers of downregulated probesets meeting this criterion were 3624 in AD, 652 in DS, 3065 in HD and 2541 probesets

differential gene expression was then set at P<0.05.

Biomart service (http://www.biomart.org/) [38, 39].

to 1,2:1 (339 affected tissues and 281 unaffected tissues included).

expressed genes may be observed in Figure 1 for each of the NDG disorders.

**4. Results**

in the PD dataset.


\* According to data obtained from the GEO site

† The dataset included some microarray experiments not related to the scope of this study and those were omitted from the analyses

‡ The study related to listed GEO entry was not yet published

Table 1. Detailed information on studies included in meta-analysis

stress that we have faced the issue of multiple studies simultaneously reporting differential expression in several different anatomical brain parts. As we wanted to facilitate the discovery of differentially expressed genes in diseased tissue in comparison to control samples, we set the origin parameter to take into account these considerations and regard such data as originating from different sources, thereby avoiding comparisons of gene expression between different brain regions rather than between affected and unaffected samples. Afterwards, P-values and q-values were obtained by performing 100 permutation cycles of complete originating datasets. An arbitrary P-value cut-off for significance of differential gene expression was then set at P<0.05.

#### **3.4 Investigating intersections between datasets and gene set enrichment analyses**

Resulting ordered lists of differentially expressed probesets were subsequently investigated for overlap between AD, DS, HD and PD datasets. Top 1000 genes from each dataset were used and intersections between combinations of two, three and four datasets were obtained. Venn diagrams in the results section were produced using Venny utility available at *http://bioinfogp.cnb.csic.es/tools/venny/index.html*. Furthermore, to gain insight in functional properties of genes in the intersections, gene set enrichment analyses (GSEA) were performed, utilizing GOstats package for R and investigating significant (uncorrected p<0.05) over- or underrepresentation of GeneOntology (GO) and KEGG terms annotating genes occurring in the intersections [33-36]. Additionally, DAVID tool (http://david.abcc.ncifcrf.gov/) was used to reveal the functional annotation clusters related to intersecting genes [37]. Required annotation conversions were performed using the hgu133plus.db package from Bioconductor annotation package collection and using biomaRt package for R in combination with Ensembl Biomart service (http://www.biomart.org/) [38, 39].

#### **4. Results**

508 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

probesets\* Affected

tissue

Platform Number

Affymetrix HG-U133Plus2

Affymetrix HG-U133A

Affymetrix HG-U133Plus2

Affymetrix HG-U133Plus2

Affymetrix HG-U133A and

22,283 and 22,645

22,283 and 22,645

† The dataset included some microarray experiments not related to the scope of this study and those

29 and 29

Affymetrix HG-U133B

Affymetrix HG-U133Plus2

Affymetrix HG-U133A and

Affymetrix HG-U133B

Affymetrix HG-U133A

Affymetrix HG-U133A

‡ The study related to listed GEO entry was not yet published

Table 1. Detailed information on studies included in meta-analysis

of

GEO Accession Disease name

GSE5281 Alzheimer's disease

GSE1297 Alzheimer's disease

†GSE16759 Alzheimer's disease

†GSE7307 Parkinson's disease

GSE8397 Parkinson's disease

GSE7621 Parkinson's disease

GSE3790 Huntington'

†GSE1397 Down

GSE5390 Down

\*

s disease

syndrome

Syndrome

were omitted from the analyses

According to data obtained from the GEO site

Number of array experiments

54,675 87 74 Entorhinal

Unaffected tissue

22,283 22 9 Hippocampus [23]

54,675 4 4 Parietal lobe

18 and 18

54,675 16 9 Substantia

22,283 9 9 Cerebrum

22,283 7 8 Dorsolateral

114 87 Cerebellum

54,675 22 45 Caudate

Tissue Ref

[22]

[24]

NA‡

[25]

[26]

[27]

[28]

[29]

cortex Hippocampus Medial temporal gyrus Posterior cingulate cortex Primary visual cortex Superior frontal gyrus

tissue

Gloubus pallidum Putamen Substantia nigra Subthalamic nucleus Thalamus lateral nuclei Thalamus subthalamic nucleus

Substantia nigra Frontal cortex

nigra

Frontal cortex Caudate nucleus

Cerebellum Astrocyte samples

prefrontal cortex

Alltogether, our data collection comprised of data from 9 whole-genome expression studies, performed on samples from 4 neurodegenerative conditions (AD, DS, HD and PD). Collectively, 200, 33, 201, and 186 microarray analysed samples were included in the investigations of AD, DS, HD and PD, respectively, which accounted for 620 separate experiments included overall. A slight predominance of experiments performed on case tissues was noted in most of the experiements with summary case:control ratio amounting to 1,2:1 (339 affected tissues and 281 unaffected tissues included).

Separate analyses of datasets for each NDG disorder have revealed significant perturbances in expression profiles of several genes. When arbitrary permutation p-value cut-off was set at 0.05 for upregulated genes, 5701 probesets attained significance in the AD dataset, 3291 in DS dataset, 4174 in the HD dataset and 3043 in the PD dataset. In the downregulated gene group the p<0.05 significance was reached for 5496 probesets in the AD dataset, 2983 probesets in the DS dataset, 4079 in the HD dataset and 3410 in the PD dataset. A detailed view of the distribution of significance values of the top 10,000 ordered differentially expressed genes may be observed in Figure 1 for each of the NDG disorders.

The resulting numbers of significant results are inflated by the effect of multiple testing and therefore the q- values were also estimated as described in the article by Breitling et al [40]. The numbers of upregulated probesets with estimated q-values below 0.05 were 3775 for AD, 1496 for DS, 3182 for HD and 1894 for PD datasets. The numbers of downregulated probesets meeting this criterion were 3624 in AD, 652 in DS, 3065 in HD and 2541 probesets in the PD dataset.

Genome Profiling and Potential Biomarkers in Neurodegenerative Disorders 511

We have also investigated the extent of similarity of GSEA profiles across four diseases. Top 200 enriched GO terms were inspected in each neurodegenerative disorder and compared for matching terms in pair with other three disorders. Greatest similarity was observed between GSEA terms annotating downregulated genes in all four disorders, which may be observed in more detail in Figure 4. As previously observed for overlapping genes, greatest overlap was observed between PD and HD GO profiles in the upregulated (40.0% overlap)

Fig. 2. Number of probesets overlapping between four sets of top 1000 DE upregulated (2a)

Please note the abbreviations: Alzheimer disease (AD), Down syndrome (DS), Huntington

Fig. 3. a) Pairwise overlaps between lists of top DE upregulated (in red) and downregulated genes (in blue). Color intensiy of each square is proportional to size of overlap between a pair of DE gene lists. b) Pairwise overlaps between lists of top DE upregulated (in red) and downregulated genes (in blue). Color intensiy of each square is proportional to the value of

and downregulated sets (59.5% overlap).

and of top 1000 DE downregulated (2b) genes

–logp value obtained by performing hypergeometric test

disease (HD) and Parkinson disease (PD).

Fig. 1. Distribution of significance estimations for differential expression in 4 neurodegenerative disorders

An extent of global perturbation of the transciptome may be compared, with AD displaying the greatest extent of differentially expressed genes (blue line) and DS displaying the lowest extent, especially in the case of genes displaying downregulation.

#### **4.1 Common patterns of differential expression in neurodegenerative disorders**

Comparisons of comformity between profiles of transcriptome perturbations in four neurodegenerative diseases was initially performed by inspecting lists of top 1000 DE (differentially expressed) probesets for each condition and subsequently obtaining probesets (and genes) found to be differentially expressed simultaneously in several conditions.

The numbers of overlapping probesets may be observed in Figure 2. The largest overlap was observed between between the PD and HD lists, with altogether 338 (33.8%) upregulated and 267 (26.7%) downregulated genes differentially expressed in both conditions. Detailed overview of the extent of overlap between pairs of top DE gene list may be observed in Figure 3. A notable number of probesets was DE in all four conditions: 44 upregulated and 16 downregulated as presented in Figure 2a and 2b.

#### **4.2 Comparative functional analyses of differential expression profile in neurodegenerative diseases**

Calculations of gene set enrichment profile of upregulated and downregulated sets of genes presented here, were performed using hypergeometric test in the GOstats package. The profiles of DE genes were first calculated for each disorder separately, and afterwards every intersection between combinations of four sets of DE genes was evaluated.

Results of interests from separate GSEA analyses are presented in Table2(a-d) for top 1000 downregulated DE gene sets (the data for upregulated GSEA are not shown). Several GO biological process annotations appeared in all of the four analyses, most notably terms related to synaptic transmission and to cognitive processes.

Fig. 1. Distribution of significance estimations for differential expression in 4

extent, especially in the case of genes displaying downregulation.

16 downregulated as presented in Figure 2a and 2b.

An extent of global perturbation of the transciptome may be compared, with AD displaying the greatest extent of differentially expressed genes (blue line) and DS displaying the lowest

The numbers of overlapping probesets may be observed in Figure 2. The largest overlap was observed between between the PD and HD lists, with altogether 338 (33.8%) upregulated and 267 (26.7%) downregulated genes differentially expressed in both conditions. Detailed overview of the extent of overlap between pairs of top DE gene list may be observed in Figure 3. A notable number of probesets was DE in all four conditions: 44 upregulated and

Calculations of gene set enrichment profile of upregulated and downregulated sets of genes presented here, were performed using hypergeometric test in the GOstats package. The profiles of DE genes were first calculated for each disorder separately, and afterwards every

Results of interests from separate GSEA analyses are presented in Table2(a-d) for top 1000 downregulated DE gene sets (the data for upregulated GSEA are not shown). Several GO biological process annotations appeared in all of the four analyses, most notably terms

**4.1 Common patterns of differential expression in neurodegenerative disorders**  Comparisons of comformity between profiles of transcriptome perturbations in four neurodegenerative diseases was initially performed by inspecting lists of top 1000 DE (differentially expressed) probesets for each condition and subsequently obtaining probesets (and genes) found to be differentially expressed simultaneously in several

**4.2 Comparative functional analyses of differential expression profile in** 

intersection between combinations of four sets of DE genes was evaluated.

related to synaptic transmission and to cognitive processes.

neurodegenerative disorders

**neurodegenerative diseases** 

conditions.

We have also investigated the extent of similarity of GSEA profiles across four diseases. Top 200 enriched GO terms were inspected in each neurodegenerative disorder and compared for matching terms in pair with other three disorders. Greatest similarity was observed between GSEA terms annotating downregulated genes in all four disorders, which may be observed in more detail in Figure 4. As previously observed for overlapping genes, greatest overlap was observed between PD and HD GO profiles in the upregulated (40.0% overlap) and downregulated sets (59.5% overlap).

Fig. 2. Number of probesets overlapping between four sets of top 1000 DE upregulated (2a) and of top 1000 DE downregulated (2b) genes

Please note the abbreviations: Alzheimer disease (AD), Down syndrome (DS), Huntington disease (HD) and Parkinson disease (PD).

Fig. 3. a) Pairwise overlaps between lists of top DE upregulated (in red) and downregulated genes (in blue). Color intensiy of each square is proportional to size of overlap between a pair of DE gene lists. b) Pairwise overlaps between lists of top DE upregulated (in red) and downregulated genes (in blue). Color intensiy of each square is proportional to the value of –logp value obtained by performing hypergeometric test

Genome Profiling and Potential Biomarkers in Neurodegenerative Disorders 513

Term

GO:0009144 1,47E-12 55 purine nucleoside triphosphate metabolic process

GO:0072521 9,91E-11 69 purine-containing compound metabolic process

GO:0015980 9,19E-10 39 energy derivation by oxidation of organic compounds Table 2c Parkinson's disease (downregulated genes). GOBPID stands for GeneOntology

Term

GO:0007186 1,76E-05 41 G-protein coupled receptor protein signaling pathway

particle remodeling

GO:0003001 4,34E-05 24 generation of a signal involved in cell-cell signaling GO:0010903 5,15E-05 3 negative regulation of very-low-density lipoprotein

GO:0048667 7,44E-05 35 cell morphogenesis involved in neuron differentiation

Table 2d Down's syndrome (downregulated genes). GOBPID stands for GeneOntology

GO:0006753 2,95E-10 73 nucleoside phosphate metabolic process

GOBPID Accession

biological process ID

biological process ID

GOBPID Accession P-value Count

of genes annotated

GO:0007399 8,20E-11 115 nervous system development GO:0001505 8,41E-11 24 regulation of neurotransmitter levels

GO:0007269 1,05E-10 20 neurotransmitter secretion

GO:0009117 2,95E-10 73 nucleotide metabolic process

GO:0007267 3,23E-10 82 cell-cell signaling

of genes annotated

GO:0007267 2,83E-08 65 cell-cell signaling

GO:0022008 3,01E-06 58 neurogenesis

GO:0048856 6,65E-11 173 anatomical structure development

GO:0050877 6,86E-07 69 neurological system process GO:0050789 1,60E-06 318 regulation of biological process

GO:0051716 2,70E-05 194 cellular response to stimulus

GO:0007268 7,44E-05 35 synaptic transmission

GO:0007165 9,36E-05 165 signal transduction GO:0048666 1,73E-04 41 neuron development

GO:0030182 3,71E-06 53 neuron differentiation GO:0007399 4,91E-06 82 nervous system development GO:0048839 8,26E-06 14 inner ear development GO:0009887 1,69E-05 42 organ morphogenesis

P-value Count

GO:0007268 4,16E-17 68 synaptic transmission GO:0051234 1,99E-16 229 establishment of localization GO:0019226 1,34E-15 70 transmission of nerve impulse GO:0035637 1,34E-15 70 multicellular organismal signaling GO:0006836 9,40E-14 29 neurotransmitter transport GO:0009259 8,03E-13 58 ribonucleotide metabolic process


Table 2a Alzheimer disease (downregulated genes). GOBPID stands for GeneOntology biological process ID


Table 2b Huntington's disease (downregulated genes). GOBPID stands for GeneOntology biological process ID

GOBPID Accession

biological process ID

biological process ID

GOBPID Accession P-value Count

of genes annotated

GO:0007268 1,01E-10 61 synaptic transmission

GO:0019226 1,49E-09 63 transmission of nerve impulse GO:0035637 1,49E-09 63 multicellular organismal signaling GO:0044282 2,61E-07 68 small molecule catabolic process

GO:0019752 9,12E-07 70 carboxylic acid metabolic process

GO:0007017 9,55E-06 35 microtubule-based process

GO:0007611 1,65E-05 18 learning or memory

P-value Count

GO:0007610 1,09E-12 46 behavior GO:0050890 5,29E-12 25 cognition

GO:0006811 9,51E-10 73 ion transport

GO:0032940 9,52E-09 50 secretion by cell

GO:0022008 3,13E-08 67 neurogenesis

Term

GO:0051443 5,09E-07 17 positive regulation of ubiquitin-protein ligase activity

GO:0030330 4,23E-05 16 DNA damage response, signal transduction by p53 class mediator

Table 2a Alzheimer disease (downregulated genes). GOBPID stands for GeneOntology

Term

GO:0009144 1,82E-06 46 purine nucleoside triphosphate metabolic process GO:0051438 5,03E-06 17 regulation of ubiquitin-protein ligase activity

GO:0031398 4,70E-05 17 positive regulation of protein ubiquitination

of genes annotated

GO:0019226 2,02E-35 93 transmission of nerve impulse

GO:0007399 8,47E-15 113 nervous system development

GO:0001505 1,00E-09 21 regulation of neurotransmitter levels GO:0031175 3,23E-09 52 neuron projection development

GO:0048667 1,66E-08 46 cell morphogenesis involved in neuron differentiation

Table 2b Huntington's disease (downregulated genes). GOBPID stands for GeneOntology

GO:0007268 3,47E-37 90 synaptic transmission

GO:0007267 5,12E-29 110 cell-cell signaling

GO:0007611 6,38E-13 25 learning or memory

GO:0048666 2,05E-10 59 neuron development GO:0006836 2,24E-10 23 neurotransmitter transport




Table 2d Down's syndrome (downregulated genes). GOBPID stands for GeneOntology biological process ID

Genome Profiling and Potential Biomarkers in Neurodegenerative Disorders 515

also the profile overlap between AD and PD, which present as clinically somewhat distinct entities. Recently however, it has been becoming progressively more obvious that the two disorders share not only a significant proportion of clinical elements (movement disorder, cognitive decline, mood and psychiatric disorders) but also share common pathophysiological pathways [42]. These results potentially suggest that clinical distinction between disease entities may not be perfect projection of actual processes at cellular and molecular level. Additionally, in contrast to expectation, however, the lowest overlap was observed between samples from patients with DS and AD, especially as these conditions have been known to share NDG pathways related to amyloid beta deposition in neurons. Reasons for lower extent of overlap may be found in significant differences in the age of patients from whom the brain samples were obtained for studies of DS in comparison with AD. Additionally, it is important that in most instances, a complete triplication of genes located on chromosome 21 may dominate genes commonly dysregulated in DS and AD [29]. Also, the number of brain tissue samples samples profiled in microarray experiments was by far the lowest among other types of NDG diseases investigated in our survey. Therefore, before final answer regarding this finding is obtained, more studies investigating

Several GO categories appeared to be consistently singled out in GSEA analyses of separate and overlapping genes DE in NDG disorders. Interestingly several terms were related to processes previously associated with neuron degeneration [42], most prominently GO terms: synaptic transmission (GO:0007268), neurogenesis (GO:0022008) and terms related to higher cognitive processes (GO:0007611). Dysfunctional synaptic transmission (as in glutamate exitotoxicity) and defects in neurogenesis have been previously repeatedly shown to be related to various NDG diseases [42-44]. It is interesting that although disturbances in neuroinflammatory mechanisms have been proposed as a possible causative factor in a number of NDG diseases, our analysis of intersecting genes dysregulated in brain samples of these conditions did not single out a particular common inflammatory pathogenetic pathway. This notion may be interpreted in the light of previously recognized differences in complement-activating immunogenic activity of plaques in different NDG diseases, resulting in absence of commonly overlapping inflammatory genes and GO terms [42]. When we investigated the compatibility of functional profiles between four NDG diseases, we have found greatest overlaps between sets of GO terms annotating genes characterized by downregulation in NDG diseases, where an overlap greater than 40% was observed in all of the pairwise comparisons of the sets of top 200 enriched GO terms. Again, the greatest functional conformance was noted between top downregulated genes in HD and PD as well as AD and PD dataset pairs. Notable overlap was also observed in the functional profiles of upregulated genes, where we noted good functional conformity between DS and HD

It is important to stress that genome-wide expression studies included in this survey are inherently burdened by important statistical issues that predominantly originate from the issue of testing a large number of variables on a relatively small population of biological replicates (ie. study subjects) [45]. For this reason we attempted to gain a more complete account of biological alterations in neurodegenerative diseases by merging data from several different studies investigating transcriptional changes in brain samples of distinct neurological conditions (AD, DS, HD and PD) [46]. This increased the number of biological replicates considerably, allowing for potentially more reliable calling of DE genes in these conditions. There are, however, important downsides to this approach: the studies included

transcriptional alterations in DS brain samples must be performed.

datasets in addition to HD-PD and AD-PD functional overlaps.

Fig. 4. Pairwise comparison of GO terms between pairs of datasets representing four neurodegenerative diseases. Percentages were calculated by dividing the number of GO terms overlapping by the number of all GO terms included in the overlapping analysis (N=200). GO terms annotating upregulated genes are presented in shades of red color and those annotating downregulated genes in blue

#### **5. Conclusion**

We have shown that whole-genome transcription analysis might be useful for identification and clarification of pathophysiological mechanisms in neurodegenerative diseases. We have used innovative approach of comparing and integrating experiment results from different NDG diseases and provided new important insights into the common NDG processes. Elucidation of these mechanisms holds important potential for future prediction and development of new useful treatments as well as for identification of biomarkers of neurodegeneration.

When comparisons of intersections between groups of top DE genes were performed, the greatest overlap was found between DE genes in brain samples of patients with HD and PD, which is possibly in accordance with their primary manifestation in movement disturbances related to function of basal ganglia. On the other hand, this similarity is surprising, as the known etiological agents in HD and PD differ significantly, one disorder being a consequence of monogenic disruption and other being a complex disorder with heterogeneous combination of genetic and environmental factors [41]. Surprisingly high is

Fig. 4. Pairwise comparison of GO terms between pairs of datasets representing four neurodegenerative diseases. Percentages were calculated by dividing the number of GO terms overlapping by the number of all GO terms included in the overlapping analysis (N=200). GO terms annotating upregulated genes are presented in shades of red color and

We have shown that whole-genome transcription analysis might be useful for identification and clarification of pathophysiological mechanisms in neurodegenerative diseases. We have used innovative approach of comparing and integrating experiment results from different NDG diseases and provided new important insights into the common NDG processes. Elucidation of these mechanisms holds important potential for future prediction and development of new useful treatments as well as for identification of biomarkers of

When comparisons of intersections between groups of top DE genes were performed, the greatest overlap was found between DE genes in brain samples of patients with HD and PD, which is possibly in accordance with their primary manifestation in movement disturbances related to function of basal ganglia. On the other hand, this similarity is surprising, as the known etiological agents in HD and PD differ significantly, one disorder being a consequence of monogenic disruption and other being a complex disorder with heterogeneous combination of genetic and environmental factors [41]. Surprisingly high is

those annotating downregulated genes in blue

**5. Conclusion** 

neurodegeneration.

also the profile overlap between AD and PD, which present as clinically somewhat distinct entities. Recently however, it has been becoming progressively more obvious that the two disorders share not only a significant proportion of clinical elements (movement disorder, cognitive decline, mood and psychiatric disorders) but also share common pathophysiological pathways [42]. These results potentially suggest that clinical distinction between disease entities may not be perfect projection of actual processes at cellular and molecular level. Additionally, in contrast to expectation, however, the lowest overlap was observed between samples from patients with DS and AD, especially as these conditions have been known to share NDG pathways related to amyloid beta deposition in neurons. Reasons for lower extent of overlap may be found in significant differences in the age of patients from whom the brain samples were obtained for studies of DS in comparison with AD. Additionally, it is important that in most instances, a complete triplication of genes located on chromosome 21 may dominate genes commonly dysregulated in DS and AD [29]. Also, the number of brain tissue samples samples profiled in microarray experiments was by far the lowest among other types of NDG diseases investigated in our survey. Therefore, before final answer regarding this finding is obtained, more studies investigating transcriptional alterations in DS brain samples must be performed.

Several GO categories appeared to be consistently singled out in GSEA analyses of separate and overlapping genes DE in NDG disorders. Interestingly several terms were related to processes previously associated with neuron degeneration [42], most prominently GO terms: synaptic transmission (GO:0007268), neurogenesis (GO:0022008) and terms related to higher cognitive processes (GO:0007611). Dysfunctional synaptic transmission (as in glutamate exitotoxicity) and defects in neurogenesis have been previously repeatedly shown to be related to various NDG diseases [42-44]. It is interesting that although disturbances in neuroinflammatory mechanisms have been proposed as a possible causative factor in a number of NDG diseases, our analysis of intersecting genes dysregulated in brain samples of these conditions did not single out a particular common inflammatory pathogenetic pathway. This notion may be interpreted in the light of previously recognized differences in complement-activating immunogenic activity of plaques in different NDG diseases, resulting in absence of commonly overlapping inflammatory genes and GO terms [42].

When we investigated the compatibility of functional profiles between four NDG diseases, we have found greatest overlaps between sets of GO terms annotating genes characterized by downregulation in NDG diseases, where an overlap greater than 40% was observed in all of the pairwise comparisons of the sets of top 200 enriched GO terms. Again, the greatest functional conformance was noted between top downregulated genes in HD and PD as well as AD and PD dataset pairs. Notable overlap was also observed in the functional profiles of upregulated genes, where we noted good functional conformity between DS and HD datasets in addition to HD-PD and AD-PD functional overlaps.

It is important to stress that genome-wide expression studies included in this survey are inherently burdened by important statistical issues that predominantly originate from the issue of testing a large number of variables on a relatively small population of biological replicates (ie. study subjects) [45]. For this reason we attempted to gain a more complete account of biological alterations in neurodegenerative diseases by merging data from several different studies investigating transcriptional changes in brain samples of distinct neurological conditions (AD, DS, HD and PD) [46]. This increased the number of biological replicates considerably, allowing for potentially more reliable calling of DE genes in these conditions. There are, however, important downsides to this approach: the studies included

Genome Profiling and Potential Biomarkers in Neurodegenerative Disorders 517

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#### **6. References**


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**22** 

*Mexico* 

**Immunization with Neural-Derived** 

There is a nosological dilemma when it comes to classifying what comprises a neurodegenerative disease (NDD). Degeneration – purely speaking – is to go from a higher to a lower level of functioning; it is deterioration from normalcy. Neurons are the functional elements of the nervous system. Then degeneration of the nervous system consists of a decrease or loss in the function of neurons. Not necessarily an atrophy, which consists of the death of a particular population of neurons. Clinically, NDD are comprised of progressive dementias, progressive ataxias, disorders in posture and movement, muscle weakness, and progressive blindness. The common characteristic in all of these pathologies is their chronicity. Each and every one of the aforementioned diseases consists of a chronic progression towards the loss of a particular function. However, this definition does not include a limit on temporality. Nosologically speaking neurodegeneration could include several other pathologies from an acute time frame. NDD can further be divided into an acute and chronic classification. Chronic diseases such as: amyotrophic lateral sclerosis (ALS), Alzheimer disease (AD) and Parkinson disease (PD) were the common conception of NDD. The latter was sustained until acute traumatic injuries to the central nervous system (CNS) were found to cause generalized inflammation and other phenomena that lead to degeneration. Examples of CNS injury that cause this secondary degeneration are: global or focal cerebral ischemia (stroke), spinal cord injury (SCI), and traumatic brain injury (TBI). The similarities in neurodegenerative processes between these and chronic NDD allows us to classify them within acute NDD. Neurodegeneration previously consisted of progressive atrophic disorders but has now expanded into the study of all pathophysiological processes that deteriorate the CNS. As a whole, NDD are the cause of many deaths around the world. In the US, stroke, traumatic injuries (such as: SCI and TBI), AD, and PD are within the top 15 causes of mortality, averaging 350,000 deaths per year (Xu et al., 2007). Although NDD have an elevated mortality their greatest impact is on morbidity, affecting 50 million Americans each year and generating a large amount of federal spending (Brown et al., 2005). Every year \$144 billion USD are spent on AD alone, and that is excluding the spending required for the other 600 neurological disorders that have been described (Alzheimer's Association, 2010; Meek et al., 1998). The elevated prevalence and incidence require a large initiative to research the hallmarks of these diseases. Until now, our understanding of NDD is quite

**1. Introduction** 

**Peptides as a Potential Therapy** 

**in Neurodegenerative Diseases** 

Humberto Mestre and Antonio Ibarra

*Universidad Anahuac Mexico Norte* 


### **Immunization with Neural-Derived Peptides as a Potential Therapy in Neurodegenerative Diseases**

Humberto Mestre and Antonio Ibarra *Universidad Anahuac Mexico Norte Mexico* 

#### **1. Introduction**

518 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

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8.

There is a nosological dilemma when it comes to classifying what comprises a neurodegenerative disease (NDD). Degeneration – purely speaking – is to go from a higher to a lower level of functioning; it is deterioration from normalcy. Neurons are the functional elements of the nervous system. Then degeneration of the nervous system consists of a decrease or loss in the function of neurons. Not necessarily an atrophy, which consists of the death of a particular population of neurons. Clinically, NDD are comprised of progressive dementias, progressive ataxias, disorders in posture and movement, muscle weakness, and progressive blindness. The common characteristic in all of these pathologies is their chronicity. Each and every one of the aforementioned diseases consists of a chronic progression towards the loss of a particular function. However, this definition does not include a limit on temporality. Nosologically speaking neurodegeneration could include several other pathologies from an acute time frame. NDD can further be divided into an acute and chronic classification. Chronic diseases such as: amyotrophic lateral sclerosis (ALS), Alzheimer disease (AD) and Parkinson disease (PD) were the common conception of NDD. The latter was sustained until acute traumatic injuries to the central nervous system (CNS) were found to cause generalized inflammation and other phenomena that lead to degeneration. Examples of CNS injury that cause this secondary degeneration are: global or focal cerebral ischemia (stroke), spinal cord injury (SCI), and traumatic brain injury (TBI). The similarities in neurodegenerative processes between these and chronic NDD allows us to classify them within acute NDD. Neurodegeneration previously consisted of progressive atrophic disorders but has now expanded into the study of all pathophysiological processes that deteriorate the CNS. As a whole, NDD are the cause of many deaths around the world. In the US, stroke, traumatic injuries (such as: SCI and TBI), AD, and PD are within the top 15 causes of mortality, averaging 350,000 deaths per year (Xu et al., 2007). Although NDD have an elevated mortality their greatest impact is on morbidity, affecting 50 million Americans each year and generating a large amount of federal spending (Brown et al., 2005). Every year \$144 billion USD are spent on AD alone, and that is excluding the spending required for the other 600 neurological disorders that have been described (Alzheimer's Association, 2010; Meek et al., 1998). The elevated prevalence and incidence require a large initiative to research the hallmarks of these diseases. Until now, our understanding of NDD is quite

Immunization with Neural-Derived Peptides as a

described in detail throughout this chapter.

**2. Role of immune cells and their potential therapeutic effect** 

Potential Therapy in Neurodegenerative Diseases 521

model of MS). Therefore, a different way of eliciting PA had to be obtained in order to prevent this complication. Studies suggested that immunizing with a weaker version of the self-antigen could solve the problem, these type of antigens became known as altered peptide ligands (APL). Vaccinating with APL would generate PA without degenerative autoimmunity. In the study of NDD, APL were derived from neural constituents and were therefore coined under the term neural-derived peptides (NDP). The success in the development of these immunomodulatory peptides has inspired a lot of research into their possible therapeutic applications in both chronic and acute NDD. These applications will be

The CNS has long been considered to be an immunologically privileged location. The bloodbrain barrier (BBB) was thought to maintain blood-borne cells of both the innate and adaptive immune system out of the CNS. This hypothesis assumed that microglia were the only innate immune cells of the CNS. During damage, microglia became activated and functioned as destructive inflammatory cells indistinguishable from infiltrating macrophages. Immune cells were thought to contribute to the increase in tissue damage during CNS disease (Bethea et al., 1998; Blight, 1992; Dusart et al., 1994; Popovich et al., 1997). The idea was supported by the following: i) CNS trauma activates T lymphocytes against neural constituents, and ii) the passive transfer of myelin autoreactive T cells caused EAE in previously healthy rats (Popovich et al., 1996). The notion was sustained in such a way that the complete inhibition of these responses was proposed as a potential therapeutic intervention, and remains to this day as the predominant clinical approach (Lopez-Vales et al., 2005; Popovich et al., 1999). However, it is now clear that these cells have a pivotal role in CNS repair (Hammarberg et al., 2000; Hashimoto et al., 2007; Hendrix & Nitsch, 2007; Moalem et al., 1999; Rapalino et al., 1998; Turrin & Rivest, 2006; Yin et al., 2003). In the healthy CNS the microglia is in a resting state where its morphology consists of a small cell soma and numerous branching processes, known as resting/ramified state. The ramifications are dynamic structures that enable the cell to sample and monitor its microenvironment (Nimmerjahn et al., 2005; Raivich, 2005). Resting microglia express CD45 (leukocyte common antigen), CD14, and CD11b/CD18 (Kreutzberg, 1996). Under duress, microglial expression patterns are modified from a monitoring role to one of protection and repair. Microglia begin to express key surface receptors such as: CD1, lymphocyte functionassociated antigen 1 (LFA-1), intracellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1). Besides changing their surface receptor repertoire they begin to secrete: inflammatory cytokines such as TNFα and interleukins IL-1β and IL-6, chemokines like macrophage inflammatory protein (MIP-1α), monocyte chemoattractant protein (MCP-1), and interferon inducible protein 10 (IP-10). This change in microenvironment changes the resting/ramified state of the microglia into an amoeboid/phagocytic state. The activated state of microglia has beneficial functions during NDD such as: scavenging neurotoxins, removing cellular debris, and the secretion of trophic factors that promote neuronal survival (Frank-Cannon et al., 2009). During CNS injury, if microglia come in contact with products of the adaptive immune response such as interferon gamma (IFN-γ) and IL-4 it will acquire a phenotype that has antigen presenting cell (APC)-like qualities. This phenotype expresses major histocompatibility complex II (MHC-II) and B 7.2 receptors, giving it the ability to interact with elements of the adaptive

complex but there is still a lot to uncover. Research is normally directed towards the NDD with the most impact on society such as: ALS, AD and PD. Due to the increased availability of information on the previous diseases this chapter will only discuss these diseases within the chronic NDD section. In order to find treatment opportunities for each one of these diseases we must first understand the basic pathophysiology. ALS is a progressive degeneration of upper and lower motor neurons in the brain and spinal cord. This atrophy eliminates the brain's control over muscle movements and causes them to weaken and become paralyzed. Progressive muscular paralysis causes the inability to move, swallow, and eventually, breathe (Angelov et al., 2003). AD is a progressive disorder characterized by memory loss and severe cognitive decline. This degeneration is caused by excessive accumulations of extracellular amyloid beta peptide, which forms plaques in the hippocampus and cerebral cortex, leading to neuronal death (Frenkel et al., 2005; Butovsky et al., 2006). PD is a chronic progressive disease characterized by motor symptoms (tremor, rigidity and bradykinesia) and nonmotor symptoms (e.g. autonomic, mood and cognitive). These clinical hallmarks are attributed to the degeneration of nigrostraital dopaminergic neurons and other structures in the brainstem, cortex, and subcortex (Laurie et al., 2007). Multiple sclerosis (MS) is an inflammatory autoimmune CNS demyelinating disease that is thought to be perpetrated by myelin-reactive lymphocytes. Demyelination of the CNS causes the loss of function of the affected tract (Stuve et al., 2006). MS is considered an autoimmune disease and not a NDD because there is no direct neuronal death only demyelination. The nosology of NDD excludes MS from our study but it still shares very similar immune pathophysiology and most of the therapies mentioned are derived or designed for use in MS. The inflammatory component of acute injury to the CNS provided new insight into the autoimmune response propagated after a CNS insult. These findings gave immune cells a crucial role in the protection and regeneration of the injured CNS, as well as a role in chronic progressive NDD. Further insight into the immunological component of neurodegenerative diseases provides us with new mechanisms where we are able to intervene in order to resolve these disorders. One of these mechanisms is protective autoimmunity (PA). PA is a new concept where autoreactive mechanisms are being modulated in order to promote neuroprotection. Dr. Michal Schwartz from the Weizmann Institute of Science in Israel originally conceived this concept. Infiltration of immune cells after CNS injury was traditionally regarded as pathological. This view was based on the fact that immune cell-infiltration has been exclusively identified with inflammation, and that inflammation is generally harmful to the injured CNS. However, recent studies indicate that a well-controlled innate and adaptive immune response is essential for the repair of the injured tissue. These results brought about research into immunomodulatory therapies in several NDD. In acute NDD and MS, recent findings have suggested that the inflammatory response is strongly modulated by an autoimmune reaction directed against neural constituents, specifically against myelin basic protein (MBP), one of the most abundant and immunogenic proteins in the CNS (Butovsky et al., 2001; Ibarra et al., 2003; Popovich et al., 1996; Sospedra & Martin, 2005). Dr. Schwartz started to modulate the action of myelinspecific autoreactive lymphocytes by immunizing with MBP. This strategy improved tissue preservation, neuronal survival and motor recovery after acute SCI (Hauben et al., 2000a; Hauben et al., 2000b). PA also proved to be a T cell-dependent response that is genetically determined (Kipnis et al., 2001) and triggered as a physiological response to CNS trauma (Yoles et al., 2001). However, immunizing animals with self-antigens (i.e. MBP) induced an autoimmune disease known as experimental autoimmune encephalomyelitis (EAE, animal

complex but there is still a lot to uncover. Research is normally directed towards the NDD with the most impact on society such as: ALS, AD and PD. Due to the increased availability of information on the previous diseases this chapter will only discuss these diseases within the chronic NDD section. In order to find treatment opportunities for each one of these diseases we must first understand the basic pathophysiology. ALS is a progressive degeneration of upper and lower motor neurons in the brain and spinal cord. This atrophy eliminates the brain's control over muscle movements and causes them to weaken and become paralyzed. Progressive muscular paralysis causes the inability to move, swallow, and eventually, breathe (Angelov et al., 2003). AD is a progressive disorder characterized by memory loss and severe cognitive decline. This degeneration is caused by excessive accumulations of extracellular amyloid beta peptide, which forms plaques in the hippocampus and cerebral cortex, leading to neuronal death (Frenkel et al., 2005; Butovsky et al., 2006). PD is a chronic progressive disease characterized by motor symptoms (tremor, rigidity and bradykinesia) and nonmotor symptoms (e.g. autonomic, mood and cognitive). These clinical hallmarks are attributed to the degeneration of nigrostraital dopaminergic neurons and other structures in the brainstem, cortex, and subcortex (Laurie et al., 2007). Multiple sclerosis (MS) is an inflammatory autoimmune CNS demyelinating disease that is thought to be perpetrated by myelin-reactive lymphocytes. Demyelination of the CNS causes the loss of function of the affected tract (Stuve et al., 2006). MS is considered an autoimmune disease and not a NDD because there is no direct neuronal death only demyelination. The nosology of NDD excludes MS from our study but it still shares very similar immune pathophysiology and most of the therapies mentioned are derived or designed for use in MS. The inflammatory component of acute injury to the CNS provided new insight into the autoimmune response propagated after a CNS insult. These findings gave immune cells a crucial role in the protection and regeneration of the injured CNS, as well as a role in chronic progressive NDD. Further insight into the immunological component of neurodegenerative diseases provides us with new mechanisms where we are able to intervene in order to resolve these disorders. One of these mechanisms is protective autoimmunity (PA). PA is a new concept where autoreactive mechanisms are being modulated in order to promote neuroprotection. Dr. Michal Schwartz from the Weizmann Institute of Science in Israel originally conceived this concept. Infiltration of immune cells after CNS injury was traditionally regarded as pathological. This view was based on the fact that immune cell-infiltration has been exclusively identified with inflammation, and that inflammation is generally harmful to the injured CNS. However, recent studies indicate that a well-controlled innate and adaptive immune response is essential for the repair of the injured tissue. These results brought about research into immunomodulatory therapies in several NDD. In acute NDD and MS, recent findings have suggested that the inflammatory response is strongly modulated by an autoimmune reaction directed against neural constituents, specifically against myelin basic protein (MBP), one of the most abundant and immunogenic proteins in the CNS (Butovsky et al., 2001; Ibarra et al., 2003; Popovich et al., 1996; Sospedra & Martin, 2005). Dr. Schwartz started to modulate the action of myelinspecific autoreactive lymphocytes by immunizing with MBP. This strategy improved tissue preservation, neuronal survival and motor recovery after acute SCI (Hauben et al., 2000a; Hauben et al., 2000b). PA also proved to be a T cell-dependent response that is genetically determined (Kipnis et al., 2001) and triggered as a physiological response to CNS trauma (Yoles et al., 2001). However, immunizing animals with self-antigens (i.e. MBP) induced an autoimmune disease known as experimental autoimmune encephalomyelitis (EAE, animal model of MS). Therefore, a different way of eliciting PA had to be obtained in order to prevent this complication. Studies suggested that immunizing with a weaker version of the self-antigen could solve the problem, these type of antigens became known as altered peptide ligands (APL). Vaccinating with APL would generate PA without degenerative autoimmunity. In the study of NDD, APL were derived from neural constituents and were therefore coined under the term neural-derived peptides (NDP). The success in the development of these immunomodulatory peptides has inspired a lot of research into their possible therapeutic applications in both chronic and acute NDD. These applications will be described in detail throughout this chapter.

#### **2. Role of immune cells and their potential therapeutic effect**

The CNS has long been considered to be an immunologically privileged location. The bloodbrain barrier (BBB) was thought to maintain blood-borne cells of both the innate and adaptive immune system out of the CNS. This hypothesis assumed that microglia were the only innate immune cells of the CNS. During damage, microglia became activated and functioned as destructive inflammatory cells indistinguishable from infiltrating macrophages. Immune cells were thought to contribute to the increase in tissue damage during CNS disease (Bethea et al., 1998; Blight, 1992; Dusart et al., 1994; Popovich et al., 1997). The idea was supported by the following: i) CNS trauma activates T lymphocytes against neural constituents, and ii) the passive transfer of myelin autoreactive T cells caused EAE in previously healthy rats (Popovich et al., 1996). The notion was sustained in such a way that the complete inhibition of these responses was proposed as a potential therapeutic intervention, and remains to this day as the predominant clinical approach (Lopez-Vales et al., 2005; Popovich et al., 1999). However, it is now clear that these cells have a pivotal role in CNS repair (Hammarberg et al., 2000; Hashimoto et al., 2007; Hendrix & Nitsch, 2007; Moalem et al., 1999; Rapalino et al., 1998; Turrin & Rivest, 2006; Yin et al., 2003). In the healthy CNS the microglia is in a resting state where its morphology consists of a small cell soma and numerous branching processes, known as resting/ramified state. The ramifications are dynamic structures that enable the cell to sample and monitor its microenvironment (Nimmerjahn et al., 2005; Raivich, 2005). Resting microglia express CD45 (leukocyte common antigen), CD14, and CD11b/CD18 (Kreutzberg, 1996). Under duress, microglial expression patterns are modified from a monitoring role to one of protection and repair. Microglia begin to express key surface receptors such as: CD1, lymphocyte functionassociated antigen 1 (LFA-1), intracellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1). Besides changing their surface receptor repertoire they begin to secrete: inflammatory cytokines such as TNFα and interleukins IL-1β and IL-6, chemokines like macrophage inflammatory protein (MIP-1α), monocyte chemoattractant protein (MCP-1), and interferon inducible protein 10 (IP-10). This change in microenvironment changes the resting/ramified state of the microglia into an amoeboid/phagocytic state. The activated state of microglia has beneficial functions during NDD such as: scavenging neurotoxins, removing cellular debris, and the secretion of trophic factors that promote neuronal survival (Frank-Cannon et al., 2009). During CNS injury, if microglia come in contact with products of the adaptive immune response such as interferon gamma (IFN-γ) and IL-4 it will acquire a phenotype that has antigen presenting cell (APC)-like qualities. This phenotype expresses major histocompatibility complex II (MHC-II) and B 7.2 receptors, giving it the ability to interact with elements of the adaptive

Immunization with Neural-Derived Peptides as a

response.

during CNS injury.

Potential Therapy in Neurodegenerative Diseases 523

neuroregeneration such as: neurotrophins (NT), nerve growth factor (NGF), and insulin-like growth factor 1 (IGF-1). The early arrival of T cells due to immunization with NDP regulates the response so that we can obtain the benefits and not the detriments of the immune

Immunomodulation is an idea from the past that looks more promising than ever. It is a change in the body's normal physiological immune response to a specific antigen. This modulation changes the way the immune system would normally respond to an event and replaces it with an alternate desired response. The modification of immune responses is different from agents that suppress the immune response (such as corticosteroids). Immunomodulation has already become a reality. For example, IFN-γ is used in patients with chronic granulomatous disease (Farhoudi et al., 2003), IFN-β is used in patients with multiple sclerosis (Kumpfel et al., 2007), and IL-2 in patients with AIDS and metastatic melanoma (Davey et al., 1997; Terando et al., 2003). Aside from this, numerous vaccines use adjuvants to achieve the desired immune response (Partidos et al., 2004; Petrovsky & Aguilar, 2004). Modulation of the immune response as a therapeutic strategy is a promising alternative for several diseases. PA allows us to speculate that it is better to modulate the immune response rather than eliminating it. In chronic NDD, patients require a competent immune response to fend off pathogens and evade complications due to infections. The ablation of the immune response is usually done with steroids or immunosuppressants, which severely affect the patient's ability to initiate an adequate immune response. In the acute form of NDD the immune system is vital in the return to homeostasis. Immune cells extract cellular debris, reestablish blood flow, secrete neurotrophic factors and eliminate pathogens. All these beneficial effects are lost when the immune response is inhibited using immunosuppressant therapy. Accordingly, it seems only logical that the immune response is essential in NDD. In line with this, it is realistic to envision that the harmful effects exerted by immune cells could be reverted or changed to promote beneficial actions. In order to achieve this goal, it is crucial to avoid or at least diminish the activation of microglial cells by means of the classic pathway (destructive phenotype). For this purpose, an earlier and larger arrival of T cells to the site of injury should be promoted. The opportune and adequate arrival of these cells will favor the activation of microglia under the bases of a protective phenotype (Shaked et al., 2004). A simple way of making this possible is by immunizing with the same antigen that induces the autoreactive response: neural antigens. With this approach, an important number of microglial cells will acquire the protective phenotype and will then release molecules that instead of increasing damage will promote neuroprotection. Thus, we will obtain the benefits and not the detriments of this immune response. The present strategy proposes the modulation of the immunological response by boosting an autoreactive reaction. This could be a bit conflicting for general understanding since it is common to associate autoimmunity with disease. However, at present, it is very clear that autoimmunity is a physiological phenomenon perfectly compatible with homeostasis (Schwartz & Cohen, 2000). Furthermore, autoimmunity has been proposed as a useful and beneficial event (Hauben et al., 2005). Therefore, PA is a protective strategy where autoimmunity is the main player in providing beneficial effects

**3. Modulation of the immune response using neural-derived peptides** 

immune response. As an APC, microglia can hold dialogue with T cells and are capable of releasing neurotrophic factors (BDNF, NT-3, NGF) and scavenging toxic neurotransmitters and reactive oxygen species (ROS) that endanger the tissue (Li et al., 2007; Schwartz et al., 2003). However, the chronic and uncontrolled activation of microglia increases the permeability of the BBB and elevates the amount of infiltrating blood-borne immune cells (Schmid et al., 2009). This promotes the activation of microglial cells into a destructive phenotype characterized by the production of high levels of nitric oxide (NO, a potent free radical), as well as TNFα, and cyclooxygenase 2 (COX2) (Franciosi et al., 2005; Lee et al., 2007; Shaked et al., 2004). In this phenotype microglia express low amounts of MHC-II and are thus incapable of communicating with the adaptive immune system, an important condition to promote neuroprotection (Schwartz et al., 2003; Shaked et al., 2004). In addition, T lymphocytes are recruited in small amounts and very late. The lack of T cell-mediated activation of microglia results in an uncoordinated release of additional pro-inflammatory cytokines, exacerbating the damage (Bethea et al., 1999; Lopez-Vales et al., 2006; Pan et al., 2003; Resnick et al., 1998; Schwartz et al., 2003; Vanegas & Schaible, 2001). The best way to elicit a T cell-mediated activation of microglial cells is through neural autoreactive T cells. This assures that T cells arrive to the CNS and activate microglia into their protective phenotype propagating the beneficial effects mentioned above (Figure 1). PA has proven to yield clinical improvements in the treatment of several NDD.

Fig. 1. T cell recruitment into the injured CNS

*Left panel:* An uncontrolled response where T cells are recruited very late allows the activation of microglia into a destructive phenotype. This is characterized by the release of nitric oxide (NO) and proinflammatory molecules like tumor necrosis factor alfa (TNF-α) and cyclooxygenase-2 (COX2). Under these circumstances, T cells intensify the inflammatory response and exacerbate neurodegeneration. *Right panel:* When the autoreactive response is elicited by immunizing with NDP there is an earlier and larger arrival of T cells. With this approach, microglial cells undergo a T cell-mediated activation into a protective phenotype. This regulated activation releases molecules that promote neuroprotection and

immune response. As an APC, microglia can hold dialogue with T cells and are capable of releasing neurotrophic factors (BDNF, NT-3, NGF) and scavenging toxic neurotransmitters and reactive oxygen species (ROS) that endanger the tissue (Li et al., 2007; Schwartz et al., 2003). However, the chronic and uncontrolled activation of microglia increases the permeability of the BBB and elevates the amount of infiltrating blood-borne immune cells (Schmid et al., 2009). This promotes the activation of microglial cells into a destructive phenotype characterized by the production of high levels of nitric oxide (NO, a potent free radical), as well as TNFα, and cyclooxygenase 2 (COX2) (Franciosi et al., 2005; Lee et al., 2007; Shaked et al., 2004). In this phenotype microglia express low amounts of MHC-II and are thus incapable of communicating with the adaptive immune system, an important condition to promote neuroprotection (Schwartz et al., 2003; Shaked et al., 2004). In addition, T lymphocytes are recruited in small amounts and very late. The lack of T cell-mediated activation of microglia results in an uncoordinated release of additional pro-inflammatory cytokines, exacerbating the damage (Bethea et al., 1999; Lopez-Vales et al., 2006; Pan et al., 2003; Resnick et al., 1998; Schwartz et al., 2003; Vanegas & Schaible, 2001). The best way to elicit a T cell-mediated activation of microglial cells is through neural autoreactive T cells. This assures that T cells arrive to the CNS and activate microglia into their protective phenotype propagating the beneficial effects mentioned above (Figure 1). PA has proven to

*Left panel:* An uncontrolled response where T cells are recruited very late allows the activation of microglia into a destructive phenotype. This is characterized by the release of nitric oxide (NO) and proinflammatory molecules like tumor necrosis factor alfa (TNF-α) and cyclooxygenase-2 (COX2). Under these circumstances, T cells intensify the inflammatory response and exacerbate neurodegeneration. *Right panel:* When the autoreactive response is elicited by immunizing with NDP there is an earlier and larger arrival of T cells. With this approach, microglial cells undergo a T cell-mediated activation into a protective phenotype. This regulated activation releases molecules that promote neuroprotection and

yield clinical improvements in the treatment of several NDD.

Fig. 1. T cell recruitment into the injured CNS

neuroregeneration such as: neurotrophins (NT), nerve growth factor (NGF), and insulin-like growth factor 1 (IGF-1). The early arrival of T cells due to immunization with NDP regulates the response so that we can obtain the benefits and not the detriments of the immune response.

#### **3. Modulation of the immune response using neural-derived peptides**

Immunomodulation is an idea from the past that looks more promising than ever. It is a change in the body's normal physiological immune response to a specific antigen. This modulation changes the way the immune system would normally respond to an event and replaces it with an alternate desired response. The modification of immune responses is different from agents that suppress the immune response (such as corticosteroids). Immunomodulation has already become a reality. For example, IFN-γ is used in patients with chronic granulomatous disease (Farhoudi et al., 2003), IFN-β is used in patients with multiple sclerosis (Kumpfel et al., 2007), and IL-2 in patients with AIDS and metastatic melanoma (Davey et al., 1997; Terando et al., 2003). Aside from this, numerous vaccines use adjuvants to achieve the desired immune response (Partidos et al., 2004; Petrovsky & Aguilar, 2004). Modulation of the immune response as a therapeutic strategy is a promising alternative for several diseases. PA allows us to speculate that it is better to modulate the immune response rather than eliminating it. In chronic NDD, patients require a competent immune response to fend off pathogens and evade complications due to infections. The ablation of the immune response is usually done with steroids or immunosuppressants, which severely affect the patient's ability to initiate an adequate immune response. In the acute form of NDD the immune system is vital in the return to homeostasis. Immune cells extract cellular debris, reestablish blood flow, secrete neurotrophic factors and eliminate pathogens. All these beneficial effects are lost when the immune response is inhibited using immunosuppressant therapy. Accordingly, it seems only logical that the immune response is essential in NDD. In line with this, it is realistic to envision that the harmful effects exerted by immune cells could be reverted or changed to promote beneficial actions. In order to achieve this goal, it is crucial to avoid or at least diminish the activation of microglial cells by means of the classic pathway (destructive phenotype). For this purpose, an earlier and larger arrival of T cells to the site of injury should be promoted. The opportune and adequate arrival of these cells will favor the activation of microglia under the bases of a protective phenotype (Shaked et al., 2004). A simple way of making this possible is by immunizing with the same antigen that induces the autoreactive response: neural antigens. With this approach, an important number of microglial cells will acquire the protective phenotype and will then release molecules that instead of increasing damage will promote neuroprotection. Thus, we will obtain the benefits and not the detriments of this immune response. The present strategy proposes the modulation of the immunological response by boosting an autoreactive reaction. This could be a bit conflicting for general understanding since it is common to associate autoimmunity with disease. However, at present, it is very clear that autoimmunity is a physiological phenomenon perfectly compatible with homeostasis (Schwartz & Cohen, 2000). Furthermore, autoimmunity has been proposed as a useful and beneficial event (Hauben et al., 2005). Therefore, PA is a protective strategy where autoimmunity is the main player in providing beneficial effects during CNS injury.

Immunization with Neural-Derived Peptides as a

Potential Therapy in Neurodegenerative Diseases 525

Fig. 2. Immunization with NDP causes repair and protection of the injured CNS Immunizing with NDP causes a peculiar adaptive immune response. The similarity of NDP with neural peptides (NP) causes T cell activation to deviate towards a Th2 phenotype. These NDPreactive T cells are released into systemic blood flow where they can hone towards the site of injury. Once these autoreactive T cells infiltrate into the CNS, they come in contact with glial cells and activate microglia into a neuroprotective phenotype. Activated microglia function as antigen presenting cells (APC) and present NP to anti-NDP Th2 cells producing anti-inflammatory cytokines like interleukin- 4 and -10 (IL-4, IL-10) and transforming growth factor beta (TGF-β). This T cell-mediated anti-inflammatory effect further

ameliorates the degenerative phenomena developed after CNS insult. These cells have also

been shown to produce neurotrophic factors implicated in neuroregeneration like

neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF)

#### **4. Modulation of protective autoimmunity with no risk of autoimmune disease**

As it was mentioned before, the possibility of inducing an autoimmune disease after vaccination with neural constituents is perhaps the main complication of this therapy. In order to solve this issue, immunizations are done with NDP. NDP are analogs of immunogenic epitopes with one (or a few) substitution(s) at specific amino acid positions of neural peptides (NP). The variation between the amino acid sequence is essential for contact with the T cell receptor (TCR) during antigen processing. This variation allows them to compete for TCR binding and to interfere with the necessary sequence of events required for T cell activation. The interference caused by NDP in TCR antigen recognition could affect T cell differentiation or induce a state of anergy (Nel & Slaughter, 2002). The specificity and avidity of the TCR with its ligand is determined by the primary sequence of the antigenic peptide. That particular sequence affects its binding to the complementary-determining regions of the TCR and the peptide-binding groove of the HLA molecule (Garboczi et al., 1996). A small variation in amino acid sequence can alter its ability to interact with either the MHC-II or TCR receptor molecule. This competition thereby converts an agonist peptide into a partial agonist or even an antagonist (Jameson & Bevan, 1995). Agonist peptides engage in high-affinity interactions with the TCR and induce a robust T cell response; whereas partial agonists or antagonists engage in lower affinity interactions that lead to altered or inhibitory responses (Jameson & Bevan, 1995; Kersh & Allen, 1996). Stimulation of naïve CD4+ T cells with an agonist peptide induces sufficient assembly of signaling complexes to allow activation of the IL-2 promoter and support a Th1 differentiation pathway. In contrast, the signals generated by APL activation are generally insufficient to induce IL-2 synthesis and therefore will not cause activation. That lack of IL-2 production might induce an anergic state or a skewing of the Th1/Th2 differentiation (Nel & Slaughter, 2002). Some APL are already being explored for neurological diseases (Figure 2). These peptides are derived from MBP-encephalitogenic epitopes. A group of them (G91, A96 and A91) have already been tested in animal models (Hauben et al., 2001). Importantly, immunized animals did not present clinical signs of EAE. A91 is a peptide derived from MBP (sequence 87-99), where the lysine residue at position 91 is replaced for alanine. This NDP cross-reacts with the original encephalitogenic epitope of MBP but it activates weak self-reactive T cells thus inducing autoimmunity without developing EAE. Immunizing with A91 inhibits EAE but neither causes anergy nor clonal deletion (Gaur et al., 1997). During antigenic presentation, A91 works as a partial agonist that instead of inducing a Th1 response promotes a Th2 differentiation pathway. This preference for the Th2 phenotype may be responsible for the elimination of the Th1-dependent response observed in EAE. Studies also indicate that post-injury injection of bone marrow-derived dendritic cells pulsed with A91, induce the same significant beneficial effects (Hauben et al., 2003). This indicates that the APC properties of the dendritic cell are enough to activate anti-A91 CD4+ T cells that are responsible for the elevated neuroprotection. To further support the use of immunomodulatory NDP, our laboratory examined the effects of combining immunizations with A91 and methylprednisolone (MP). The use of corticosteroids, such as MP, is the only therapeutic agent currently available for the treatment of a variety of NDD, primarily CNS trauma. In our study, a high dose of MP was administered together with an A91 immunization after SCI. As expected, MP eliminated the beneficial effects of A91. Nevertheless, when vaccination with A91 was delayed for 48 h after injury, there was no interference with its effect by the anti-inflammatory action of MP injected immediately after

**4. Modulation of protective autoimmunity with no risk of autoimmune disease**  As it was mentioned before, the possibility of inducing an autoimmune disease after vaccination with neural constituents is perhaps the main complication of this therapy. In order to solve this issue, immunizations are done with NDP. NDP are analogs of immunogenic epitopes with one (or a few) substitution(s) at specific amino acid positions of neural peptides (NP). The variation between the amino acid sequence is essential for contact with the T cell receptor (TCR) during antigen processing. This variation allows them to compete for TCR binding and to interfere with the necessary sequence of events required for T cell activation. The interference caused by NDP in TCR antigen recognition could affect T cell differentiation or induce a state of anergy (Nel & Slaughter, 2002). The specificity and avidity of the TCR with its ligand is determined by the primary sequence of the antigenic peptide. That particular sequence affects its binding to the complementary-determining regions of the TCR and the peptide-binding groove of the HLA molecule (Garboczi et al., 1996). A small variation in amino acid sequence can alter its ability to interact with either the MHC-II or TCR receptor molecule. This competition thereby converts an agonist peptide into a partial agonist or even an antagonist (Jameson & Bevan, 1995). Agonist peptides engage in high-affinity interactions with the TCR and induce a robust T cell response; whereas partial agonists or antagonists engage in lower affinity interactions that lead to altered or inhibitory responses (Jameson & Bevan, 1995; Kersh & Allen, 1996). Stimulation of naïve CD4+ T cells with an agonist peptide induces sufficient assembly of signaling complexes to allow activation of the IL-2 promoter and support a Th1 differentiation pathway. In contrast, the signals generated by APL activation are generally insufficient to induce IL-2 synthesis and therefore will not cause activation. That lack of IL-2 production might induce an anergic state or a skewing of the Th1/Th2 differentiation (Nel & Slaughter, 2002). Some APL are already being explored for neurological diseases (Figure 2). These peptides are derived from MBP-encephalitogenic epitopes. A group of them (G91, A96 and A91) have already been tested in animal models (Hauben et al., 2001). Importantly, immunized animals did not present clinical signs of EAE. A91 is a peptide derived from MBP (sequence 87-99), where the lysine residue at position 91 is replaced for alanine. This NDP cross-reacts with the original encephalitogenic epitope of MBP but it activates weak self-reactive T cells thus inducing autoimmunity without developing EAE. Immunizing with A91 inhibits EAE but neither causes anergy nor clonal deletion (Gaur et al., 1997). During antigenic presentation, A91 works as a partial agonist that instead of inducing a Th1 response promotes a Th2 differentiation pathway. This preference for the Th2 phenotype may be responsible for the elimination of the Th1-dependent response observed in EAE. Studies also indicate that post-injury injection of bone marrow-derived dendritic cells pulsed with A91, induce the same significant beneficial effects (Hauben et al., 2003). This indicates that the APC properties of the dendritic cell are enough to activate anti-A91 CD4+ T cells that are responsible for the elevated neuroprotection. To further support the use of immunomodulatory NDP, our laboratory examined the effects of combining immunizations with A91 and methylprednisolone (MP). The use of corticosteroids, such as MP, is the only therapeutic agent currently available for the treatment of a variety of NDD, primarily CNS trauma. In our study, a high dose of MP was administered together with an A91 immunization after SCI. As expected, MP eliminated the beneficial effects of A91. Nevertheless, when vaccination with A91 was delayed for 48 h after injury, there was no interference with its effect by the anti-inflammatory action of MP injected immediately after

Fig. 2. Immunization with NDP causes repair and protection of the injured CNS Immunizing with NDP causes a peculiar adaptive immune response. The similarity of NDP with neural peptides (NP) causes T cell activation to deviate towards a Th2 phenotype. These NDPreactive T cells are released into systemic blood flow where they can hone towards the site of injury. Once these autoreactive T cells infiltrate into the CNS, they come in contact with glial cells and activate microglia into a neuroprotective phenotype. Activated microglia function as antigen presenting cells (APC) and present NP to anti-NDP Th2 cells producing anti-inflammatory cytokines like interleukin- 4 and -10 (IL-4, IL-10) and transforming growth factor beta (TGF-β). This T cell-mediated anti-inflammatory effect further ameliorates the degenerative phenomena developed after CNS insult. These cells have also been shown to produce neurotrophic factors implicated in neuroregeneration like neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF)

Immunization with Neural-Derived Peptides as a

have been conducted on the use of NDP in NDD.

**5.1 Chronic neurodegenerative diseases 5.1.1 Amyotrophic lateral sclerosis** 

Potential Therapy in Neurodegenerative Diseases 527

(Ziemssen, 2002, 2005). This Th1-mediated effect also induces astrocyte and neuronal production of these neurotrophic factors through a bystander effect (Aharoni et al., 2005). However, the effect of Cop-1 is not only mediated by its direct effect on CD4+ lymphocytes but also of its effect on APC, especially dendritic cells (DC). A recent study demonstrated that Cop-1 induced a Th2 response by modulating the APC function of DC. They demonstrated that DC exposed to Cop-1 during maturation had an impaired capacity of secreting IL-17p70 (the main Th1-polarizing cytokine). This effect resulted in the induction of a population with an increased frequency of effector Th2 cells that secreted IL-4 (Sanna et al., 2006; Vieira et al., 2003). Although the main components of NDP-induced PA are superficially understood, more research initiatives should be taken to better understand the therapeutic potential of these peptides. Most of the studies published use Cop-1 as the NDP, but the use of alternate peptide sequences such as A91 must be better understood. Nonetheless, there should be a constant effort to develop shorter, cheaper and more efficacious peptide sequences so that the true potential of NDP can be unlocked. Few studies

There have been many attempts to halt the progression of ALS by blocking different mediators of cytotoxicity (Ludolph et al., 2000). Because not all ALS patients have the defective SOD1 gene, motor neuron death is taken as the hallmark of disease because it is common to all cases of ALS. The animal model of ALS is acute peripheral nerve axotomy (Liu & Martin, 2001; Martin et al., 2000). The only drug currently used to slow down the progression of ALS is riluzole. Riluzole blocks the release of the excitatory neurotransmitter glutamate that can be toxic in elevated concentrations and is fundamental to ALS pathophysiology (Doble & Kennel, 2000; Meininger et al., 2000). In this study conducted by Angelov *et al.*, mice treated with Cop-1 (using a different regimen than MS) show more motor neuron survival in the acute and chronic phases of ALS (Angelov et al., 2003). In the study, mice were subjected to a unilateral facial nerve axotomy. They were then immunized with Cop-1 and assessed. The results showed that vaccination with Cop-1 protected against motor neuron death induced after facial nerve axotomy. Transection of the facial nerve in the adult mouse is known to cause an easily visible late degeneration of axotomized motor neurons (Sendtner et al., 1996). Eight weeks after axotomy, mice immunized with Cop-1 had significantly larger numbers of motor neurons compared to PBS-immunized controls. Studies also indicated that immunization with Cop-1 preserved the activity of axotomized motor neurons. The study concluded that there was an elevated preservation of facial nerve motor neurons but the next step was to confirm that these were still functional. Using biometrical analysis of the mice's whisking patterns they found that Cop-1-treated animals exhibited significantly better facial nerve functionality than controls. The previous results demonstrated that Cop-1-immunized ALS mice benefited from improved motor neuron survival and the preservation of their function after facial nerve axotomy. A mice strain that expresses human mutant SOD1 gene develops a motor disease that closely resembles human ALS. The loss of motor function eventually causes death because of the lack of muscular respiratory control. Angelov *et al.* concluded that treatment with Cop-1 immunizations resulted in an increased survival of the ALS mice. Immunizations with Cop-1 proved to be an adequate and efficacious therapy in an animal model of ALS. A small phase II study was held in human patients with ALS that finished with inconclusive results.

SCI (Ibarra et al., 2004). This finding suggests that vaccination with A91 is neuroprotective even if administered 48 h after injury, and that the effect of MP over the immune system is transient and does not interfere with later therapy even if that treatment is immune related. These results offer another interesting benefit of NDP-induced PA, and that is the clinical plausibility of these therapies. In the clinical setting, CNS trauma and pathology is diagnosed long after the moment of incidence. NDP-induced PA is functional even when administered 48 h after the development of NDD and works as an adjuvant in traditional clinical treatment protocols (MP administration post-CNS trauma). It appears that the beneficial effect of the vaccination with A91 will not necessarily be neutralized by concomitant treatment with MP. It is worth mentioning that one of the most prevailing adverse effects observed after NDP immunization is immediate-type hypersensitivity reactions. This undesirable effect is generally associated with the immune deviation toward Th2 phenotype. These observations should stimulate further research into which patients are most likely to benefit from this therapy. Taking into consideration all of the data, therapeutic vaccination with NDP appears to be a promising strategy that could be adapted for treatment in several NDD.

#### **5. Effect of immunizing with neural-derived peptides**

In the study of neuroprotection, the term autoreactivity is immediately associated with increased cell death, inhibition of neuroprotective mechanisms and a worse clinical outcome after CNS injury. However, our understanding of the immune system's role in the pathological CNS has changed drastically in the last couple of years. The old school of thought indicated that the immune response was responsible for the exacerbation of neurodestructive phenomenon, so the first line of defense was immunosuppression. The recent findings of PA suggested that the immune response was not only needed after an insult to the CNS but it also had a beneficial neuroprotective role in most NDD. This radical change in information forces us to reevaluate the existing treatment protocols for all NDD. If PA is present in a number of CNS diseases then the use of NDP immunizations is a reasonable treatment option. The use of NDP-induced PA results in the generation of a prevalent Th2 phenotype. These cell types have shown to have the most overwhelming neuroprotective effect in the CNS. The influential roles that these cells have on the outcome of disease have made them the goal of therapy development. The increase in Th2-inducing interventions has been studied in ALS, AD, PD, SCI, TBI, and stroke; it has even been proposed as a treatment for neurodevelopmental disorders such as Rett syndrome (Ben-Zeev et al., 2011). There are many different approaches to the induction of autoreactive Th2 lymphocytes some of these are: glatiramer acetate (GA, Coplymer-1, Cop-1, Copaxone), A91, poly-YE, p472 (Nogo-A-derived peptide). However, the only FDA-approved use of NDPinduced PA is GA under the brand name Copaxone for the treatment of MS. GA, also known as Cop-1, is the most studied of all APL-based therapies. Cop-1 is a synthetic polypeptide consisting of the amino acids tyrosine, glutamate, alanine and lysine that shows cross-reactivity with MBP (Schori et al., 2001; Kipnis & Schwartz, 2002). While the exact mechanism of Cop-1 is still not clearly elucidated, there is reason to believe that it induces Th2 differentiation, which later goes on to mediate neuroprotection (Aharoni et al., 2003; Aharoni et al., 2000). Although Th2 induction is the primary effect, immunization with Cop-1 also results in a Th1 cell deviation. This effect may seem paradoxical in nature but these pro-inflammatory Th1 cells are responsible for a sustained release of BDNF, NT-3, and NT-4

SCI (Ibarra et al., 2004). This finding suggests that vaccination with A91 is neuroprotective even if administered 48 h after injury, and that the effect of MP over the immune system is transient and does not interfere with later therapy even if that treatment is immune related. These results offer another interesting benefit of NDP-induced PA, and that is the clinical plausibility of these therapies. In the clinical setting, CNS trauma and pathology is diagnosed long after the moment of incidence. NDP-induced PA is functional even when administered 48 h after the development of NDD and works as an adjuvant in traditional clinical treatment protocols (MP administration post-CNS trauma). It appears that the beneficial effect of the vaccination with A91 will not necessarily be neutralized by concomitant treatment with MP. It is worth mentioning that one of the most prevailing adverse effects observed after NDP immunization is immediate-type hypersensitivity reactions. This undesirable effect is generally associated with the immune deviation toward Th2 phenotype. These observations should stimulate further research into which patients are most likely to benefit from this therapy. Taking into consideration all of the data, therapeutic vaccination with NDP appears to be a promising strategy that could be adapted

In the study of neuroprotection, the term autoreactivity is immediately associated with increased cell death, inhibition of neuroprotective mechanisms and a worse clinical outcome after CNS injury. However, our understanding of the immune system's role in the pathological CNS has changed drastically in the last couple of years. The old school of thought indicated that the immune response was responsible for the exacerbation of neurodestructive phenomenon, so the first line of defense was immunosuppression. The recent findings of PA suggested that the immune response was not only needed after an insult to the CNS but it also had a beneficial neuroprotective role in most NDD. This radical change in information forces us to reevaluate the existing treatment protocols for all NDD. If PA is present in a number of CNS diseases then the use of NDP immunizations is a reasonable treatment option. The use of NDP-induced PA results in the generation of a prevalent Th2 phenotype. These cell types have shown to have the most overwhelming neuroprotective effect in the CNS. The influential roles that these cells have on the outcome of disease have made them the goal of therapy development. The increase in Th2-inducing interventions has been studied in ALS, AD, PD, SCI, TBI, and stroke; it has even been proposed as a treatment for neurodevelopmental disorders such as Rett syndrome (Ben-Zeev et al., 2011). There are many different approaches to the induction of autoreactive Th2 lymphocytes some of these are: glatiramer acetate (GA, Coplymer-1, Cop-1, Copaxone), A91, poly-YE, p472 (Nogo-A-derived peptide). However, the only FDA-approved use of NDPinduced PA is GA under the brand name Copaxone for the treatment of MS. GA, also known as Cop-1, is the most studied of all APL-based therapies. Cop-1 is a synthetic polypeptide consisting of the amino acids tyrosine, glutamate, alanine and lysine that shows cross-reactivity with MBP (Schori et al., 2001; Kipnis & Schwartz, 2002). While the exact mechanism of Cop-1 is still not clearly elucidated, there is reason to believe that it induces Th2 differentiation, which later goes on to mediate neuroprotection (Aharoni et al., 2003; Aharoni et al., 2000). Although Th2 induction is the primary effect, immunization with Cop-1 also results in a Th1 cell deviation. This effect may seem paradoxical in nature but these pro-inflammatory Th1 cells are responsible for a sustained release of BDNF, NT-3, and NT-4

for treatment in several NDD.

**5. Effect of immunizing with neural-derived peptides** 

(Ziemssen, 2002, 2005). This Th1-mediated effect also induces astrocyte and neuronal production of these neurotrophic factors through a bystander effect (Aharoni et al., 2005). However, the effect of Cop-1 is not only mediated by its direct effect on CD4+ lymphocytes but also of its effect on APC, especially dendritic cells (DC). A recent study demonstrated that Cop-1 induced a Th2 response by modulating the APC function of DC. They demonstrated that DC exposed to Cop-1 during maturation had an impaired capacity of secreting IL-17p70 (the main Th1-polarizing cytokine). This effect resulted in the induction of a population with an increased frequency of effector Th2 cells that secreted IL-4 (Sanna et al., 2006; Vieira et al., 2003). Although the main components of NDP-induced PA are superficially understood, more research initiatives should be taken to better understand the therapeutic potential of these peptides. Most of the studies published use Cop-1 as the NDP, but the use of alternate peptide sequences such as A91 must be better understood. Nonetheless, there should be a constant effort to develop shorter, cheaper and more efficacious peptide sequences so that the true potential of NDP can be unlocked. Few studies have been conducted on the use of NDP in NDD.

#### **5.1 Chronic neurodegenerative diseases 5.1.1 Amyotrophic lateral sclerosis**

There have been many attempts to halt the progression of ALS by blocking different mediators of cytotoxicity (Ludolph et al., 2000). Because not all ALS patients have the defective SOD1 gene, motor neuron death is taken as the hallmark of disease because it is common to all cases of ALS. The animal model of ALS is acute peripheral nerve axotomy (Liu & Martin, 2001; Martin et al., 2000). The only drug currently used to slow down the progression of ALS is riluzole. Riluzole blocks the release of the excitatory neurotransmitter glutamate that can be toxic in elevated concentrations and is fundamental to ALS pathophysiology (Doble & Kennel, 2000; Meininger et al., 2000). In this study conducted by Angelov *et al.*, mice treated with Cop-1 (using a different regimen than MS) show more motor neuron survival in the acute and chronic phases of ALS (Angelov et al., 2003). In the study, mice were subjected to a unilateral facial nerve axotomy. They were then immunized with Cop-1 and assessed. The results showed that vaccination with Cop-1 protected against motor neuron death induced after facial nerve axotomy. Transection of the facial nerve in the adult mouse is known to cause an easily visible late degeneration of axotomized motor neurons (Sendtner et al., 1996). Eight weeks after axotomy, mice immunized with Cop-1 had significantly larger numbers of motor neurons compared to PBS-immunized controls. Studies also indicated that immunization with Cop-1 preserved the activity of axotomized motor neurons. The study concluded that there was an elevated preservation of facial nerve motor neurons but the next step was to confirm that these were still functional. Using biometrical analysis of the mice's whisking patterns they found that Cop-1-treated animals exhibited significantly better facial nerve functionality than controls. The previous results demonstrated that Cop-1-immunized ALS mice benefited from improved motor neuron survival and the preservation of their function after facial nerve axotomy. A mice strain that expresses human mutant SOD1 gene develops a motor disease that closely resembles human ALS. The loss of motor function eventually causes death because of the lack of muscular respiratory control. Angelov *et al.* concluded that treatment with Cop-1 immunizations resulted in an increased survival of the ALS mice. Immunizations with Cop-1 proved to be an adequate and efficacious therapy in an animal model of ALS. A small phase II study was held in human patients with ALS that finished with inconclusive results.

Immunization with Neural-Derived Peptides as a

system and not just the evaluation through substitution studies.

Immunization with NDP has also proved to be beneficial in cases of focal and global cerebral ischemia. There have been several studies of oral and nasal tolerization with neural constituents (Becker et al., 1997; Frenkel et al., 2003); however, only a few have resorted to NDP. There are primarily two studies that analyze the effects of this Th2-induced response after middle cerebral artery occlusion. The first by Ziv et al. used poly-YE, a high molecular weight (22 to 45 kDa) copolymer that was shown to exert modulatory effects on the immune system (Cady et al., 2000; Vidovic & Matzinger, 1988). This peptide demonstrated abilities to downregulate regulatory T cell functions and allows effector T cell activation. The study showed that a single immunization with poly-YE produced long-lasting clinical and behavioral benefits, along with neuroprotection and increased neurogenesis, starting from the subacute phase. They also found that poly-YE was beneficial even when administered 24 hours after occlusion. The effects of poly-YE immunization were long lasting as animals showed less residual impairment against controls even after 6 weeks. Histological analysis indicated that poly-YE attenuated cell loss in the hippocampus where PBS-treated rats showed large numbers of necrotic cells. The reduction in cell necrosis induced by poly-YE was so dramatic that the ipsilateral and contralateral sides were indistinguishable.

**5.2 Acute neurodegenerative diseases** 

**5.2.1 Cerebral ischemia** 

Potential Therapy in Neurodegenerative Diseases 529

Cop-1-reactive T cells exhibited a much smaller reduction in the number of SNpc dopaminergic neurons. For the functional analysis of dopaminergic circuits they quantified tyrosine hydroxylase (TH) density. The loss of TH density was significantly less in Cop-1 immunized mice than in controls. Unfortunately, even in Cop-1 immunized mice, the loss of TH density was up to 72%. However, the conclusion was that Cop-1-reactive T cell passive immunization protected neuronal dopamine metabolism as well as structural neuronal elements and its projections. Complementary analysis stated that transferred lymphocytes were readily observed both in ventral midbrains and striata of MPTP mice. The study was also interested in evaluating microglial activation due to the fact that these cells are considered to be pathological in this NDD. To assess microglial activation they analyzed the Mac-1 gene using real time RT-PCR. Results showed that Cop-1 splenocytes are capable of attenuating MPTP-induced microglial reactions and in turn limiting their neurodestructive processes. In accordance to previously demonstrated concepts, the beneficial effects of Cop-1 immunizations were T cell-dependent. Treatment with NDP also increased the expression of the neurotrophic factor GDNF. All results demonstrate the beneficial effects of immunizing with Cop-1 in PD (Benner et al., 2004). A similar study by Laurie et al. corroborated the results observed by Benner and co-workers. Although similar results were obtained, the latter was able to recollect new data. The study concluded that anti-Cop-1 CD4+ T cell transfer into MPTP intoxicated mice exerted its reparative effects in a dose dependent manner. Also this study attributed the neuroprotection to a particular subset of T lymphocytes, CD4+ T cells. This further implicated T helper cells as the main player in PA. In order to support that PA is T cell-dependent, authors' transplanted Cop-1 specific antibodies to MPTP intoxicated mice to see if this conferred neuroprotection, as expected the effects of Cop-1 are CD4+ T cell-dependent (Laurie et al., 2006). This study reiterates the outstanding potential that NDP-induced PA holds in the outcome of PD. Nonetheless, this topic deserves more investigation as to identify the effect of a normal functional immune

Most patients demonstrated adverse reactions at the site of immunization and elevated lymphocyte proliferation. Although the results showed promise, efforts must be taken to increase the sample size and scrutinize the possible mechanisms through which Cop-1 exerts its protective effects (Gordon et al., 2006). These small but conclusive examples of NDP-induced PA in ALS provide us with enough proof to understand the possible therapeutic advantages. The study of Cop-1 in ALS is still in its beginning and should therefore be a priority in the coming years for NDD researchers. The maximal benefits of PA in ALS have not yet been achieved.

#### **5.1.2 Alzheimer disease**

Previous studies proved that immunotherapy in AD via amyloid beta (Aβ) antibodies reduced the levels of Aβ plaques in transgenic mice. However, a human trial with Aβ antibodies caused severe adverse reactions in the form of meningoencephalitis (Nicoll et al., 2003; Orgogozo et al., 2003). A study done by Frenkel et al. postulated that meningoencephalitis was very similar to EAE. They decided to test if amyloid precursor protein-transgenic (APP-Tg) mice were more susceptible to develop EAE. They concluded that EAE lowered the levels of Aβ in APP-Tg mice using antibody-independent mechanisms. As a follow-up they decided to see if they could achieve the low Aβ levels without causing EAE. GA or Cop-1 was an FDA-approved treatment for relapsing-remitting MS and was known to cause an autoreactive response without developing EAE. They were able to reproduce the amyloid load achieved in EAE using immunization with GA (Frenkel et al., 2005). Butovsky et al performed a more directed study, towards the analysis of PA in AD. This work found that Aβ activated microglia supports neurogenesis when stimulated by IL-4. This means that a Th2 phenotype will result in the overexpression of IL-4 and increased neurogenesis after microglial activation with Aβ. Vaccination with autoreactive T cells besides aiding in neurogenesis helped in the elimination of the Aβ plaque in APP-Tg mice. The increase in neurogenesis and the removal of the Aβ plaques resulted in the counteraction of the cognitive decline normally seen in AD (Butovsky et al., 2006). The vaccination with NDP has proven to be of paramount importance in the treatment of yet another NDD. This data is also an indicator of the urgency with which these therapies should be developed, standardized, and translated into clinical trials where they can bear fruits to human disease.

#### **5.1.3 Parkinson disease**

Immunological studies in PD are controversial. The animal model is 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication (Benner et al., 2004; Laurie et al., 2006). This intoxication depletes dopaminergic neurons in the substantia nigra pars compacta (SNpc), simulating PD. The complication arises because MPTP toxicity also destroys the animal's immune system, causing significant changes in spleen size and diminished numbers of CD3+ T cells 7 days after intoxication (Benner et al., 2004). The alterations in normal immune response impede the researcher's ability to analyze the role of the immune system in PD. However, researchers bypass this complication by cell subset replacements. The use of NDP in PD has been briefly evaluated by several studies from the same laboratory. All studies use the MPTP toxicity model of PD and use adoptive transfer of T cells from Cop-1-immunized mice. In the first study of Benner et al. Cop-1-immunity was found to confer dopaminergic neuroprotection after MPTP intoxication. Animals that received the adoptive transfer of

Most patients demonstrated adverse reactions at the site of immunization and elevated lymphocyte proliferation. Although the results showed promise, efforts must be taken to increase the sample size and scrutinize the possible mechanisms through which Cop-1 exerts its protective effects (Gordon et al., 2006). These small but conclusive examples of NDP-induced PA in ALS provide us with enough proof to understand the possible therapeutic advantages. The study of Cop-1 in ALS is still in its beginning and should therefore be a priority in the coming years for NDD researchers. The maximal benefits of PA

Previous studies proved that immunotherapy in AD via amyloid beta (Aβ) antibodies reduced the levels of Aβ plaques in transgenic mice. However, a human trial with Aβ antibodies caused severe adverse reactions in the form of meningoencephalitis (Nicoll et al., 2003; Orgogozo et al., 2003). A study done by Frenkel et al. postulated that meningoencephalitis was very similar to EAE. They decided to test if amyloid precursor protein-transgenic (APP-Tg) mice were more susceptible to develop EAE. They concluded that EAE lowered the levels of Aβ in APP-Tg mice using antibody-independent mechanisms. As a follow-up they decided to see if they could achieve the low Aβ levels without causing EAE. GA or Cop-1 was an FDA-approved treatment for relapsing-remitting MS and was known to cause an autoreactive response without developing EAE. They were able to reproduce the amyloid load achieved in EAE using immunization with GA (Frenkel et al., 2005). Butovsky et al performed a more directed study, towards the analysis of PA in AD. This work found that Aβ activated microglia supports neurogenesis when stimulated by IL-4. This means that a Th2 phenotype will result in the overexpression of IL-4 and increased neurogenesis after microglial activation with Aβ. Vaccination with autoreactive T cells besides aiding in neurogenesis helped in the elimination of the Aβ plaque in APP-Tg mice. The increase in neurogenesis and the removal of the Aβ plaques resulted in the counteraction of the cognitive decline normally seen in AD (Butovsky et al., 2006). The vaccination with NDP has proven to be of paramount importance in the treatment of yet another NDD. This data is also an indicator of the urgency with which these therapies should be developed, standardized, and translated into clinical trials where they can bear

Immunological studies in PD are controversial. The animal model is 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication (Benner et al., 2004; Laurie et al., 2006). This intoxication depletes dopaminergic neurons in the substantia nigra pars compacta (SNpc), simulating PD. The complication arises because MPTP toxicity also destroys the animal's immune system, causing significant changes in spleen size and diminished numbers of CD3+ T cells 7 days after intoxication (Benner et al., 2004). The alterations in normal immune response impede the researcher's ability to analyze the role of the immune system in PD. However, researchers bypass this complication by cell subset replacements. The use of NDP in PD has been briefly evaluated by several studies from the same laboratory. All studies use the MPTP toxicity model of PD and use adoptive transfer of T cells from Cop-1-immunized mice. In the first study of Benner et al. Cop-1-immunity was found to confer dopaminergic neuroprotection after MPTP intoxication. Animals that received the adoptive transfer of

in ALS have not yet been achieved.

**5.1.2 Alzheimer disease** 

fruits to human disease.

**5.1.3 Parkinson disease** 

Cop-1-reactive T cells exhibited a much smaller reduction in the number of SNpc dopaminergic neurons. For the functional analysis of dopaminergic circuits they quantified tyrosine hydroxylase (TH) density. The loss of TH density was significantly less in Cop-1 immunized mice than in controls. Unfortunately, even in Cop-1 immunized mice, the loss of TH density was up to 72%. However, the conclusion was that Cop-1-reactive T cell passive immunization protected neuronal dopamine metabolism as well as structural neuronal elements and its projections. Complementary analysis stated that transferred lymphocytes were readily observed both in ventral midbrains and striata of MPTP mice. The study was also interested in evaluating microglial activation due to the fact that these cells are considered to be pathological in this NDD. To assess microglial activation they analyzed the Mac-1 gene using real time RT-PCR. Results showed that Cop-1 splenocytes are capable of attenuating MPTP-induced microglial reactions and in turn limiting their neurodestructive processes. In accordance to previously demonstrated concepts, the beneficial effects of Cop-1 immunizations were T cell-dependent. Treatment with NDP also increased the expression of the neurotrophic factor GDNF. All results demonstrate the beneficial effects of immunizing with Cop-1 in PD (Benner et al., 2004). A similar study by Laurie et al. corroborated the results observed by Benner and co-workers. Although similar results were obtained, the latter was able to recollect new data. The study concluded that anti-Cop-1 CD4+ T cell transfer into MPTP intoxicated mice exerted its reparative effects in a dose dependent manner. Also this study attributed the neuroprotection to a particular subset of T lymphocytes, CD4+ T cells. This further implicated T helper cells as the main player in PA. In order to support that PA is T cell-dependent, authors' transplanted Cop-1 specific antibodies to MPTP intoxicated mice to see if this conferred neuroprotection, as expected the effects of Cop-1 are CD4+ T cell-dependent (Laurie et al., 2006). This study reiterates the outstanding potential that NDP-induced PA holds in the outcome of PD. Nonetheless, this topic deserves more investigation as to identify the effect of a normal functional immune system and not just the evaluation through substitution studies.

### **5.2 Acute neurodegenerative diseases**

#### **5.2.1 Cerebral ischemia**

Immunization with NDP has also proved to be beneficial in cases of focal and global cerebral ischemia. There have been several studies of oral and nasal tolerization with neural constituents (Becker et al., 1997; Frenkel et al., 2003); however, only a few have resorted to NDP. There are primarily two studies that analyze the effects of this Th2-induced response after middle cerebral artery occlusion. The first by Ziv et al. used poly-YE, a high molecular weight (22 to 45 kDa) copolymer that was shown to exert modulatory effects on the immune system (Cady et al., 2000; Vidovic & Matzinger, 1988). This peptide demonstrated abilities to downregulate regulatory T cell functions and allows effector T cell activation. The study showed that a single immunization with poly-YE produced long-lasting clinical and behavioral benefits, along with neuroprotection and increased neurogenesis, starting from the subacute phase. They also found that poly-YE was beneficial even when administered 24 hours after occlusion. The effects of poly-YE immunization were long lasting as animals showed less residual impairment against controls even after 6 weeks. Histological analysis indicated that poly-YE attenuated cell loss in the hippocampus where PBS-treated rats showed large numbers of necrotic cells. The reduction in cell necrosis induced by poly-YE was so dramatic that the ipsilateral and contralateral sides were indistinguishable.

Immunization with Neural-Derived Peptides as a

Potential Therapy in Neurodegenerative Diseases 531

secondary phase of damage after trauma is NO. When NO is produced in an unregulated fashion it can react with other free radicals such as superoxide anion and produce peroxynitrite a powerful neurotoxic substance. We determined that the decrease in lipid peroxidation was caused by an inhibition in the synthesis of NO after immunization with NDP after SCI (unpublished data). Our results supported our hypothesis and allowed us to corroborate the data with expression analysis. We used real time RT-PCR to also demonstrate a reduction in the expression of the enzyme implicated in post-injury synthesis of NO, the inducible form of nitric oxide synthase (iNOS) (unpublished data). By determining that A91 reactive T cells also secrete NT-3 and IL-4 after SCI, making them a Th2 phenotype, we further substantiate the PA hypothesis. Immunizing with NDP deviates the Th response down a Th2 pathway increasing the synthesis of molecules such as IL-4 and IL-10 and secretion of neurotrophic factors like NT-3. Finally, we have found that the severity of injury would determine the strength and the effect of the PA response (unpublished data). This new data adds more factors into the induction of an autoreactive response. Our study noticed that animals that sustained a non-complete injury to the spinal cord had an increased recovery when immunized with A91. These autoreactive T cells also secreted BDNF and had greater recognition for A91 in vitro. On the other hand, animals that sustained complete or severe SCI did not recover even after A91-immunization. Unexpectedly, these animals did not even possess a clonal response to A91, meaning they were not even able to recognize the antigen in vivo, even with an adjuvant. This indicates that animals that sustained a severe or complete injury to the spinal cord are severely immunosuppressed and may therefore not engage a true PA response (unpublished data). This data that has just surfaced indicates that the neuroimmunological components of CNS disease require much more research in order to elucidate this unknown mechanisms. Even further, we must continue to delve into this immunosuppression caused by severe injury. The study of the body's physiology under duress shows us some of the mechanisms it possesses that could help in regenerating the CNS during disease. Immunization with NDP has proven to be an excellent therapeutical intervention in SCI and several other NDD,

providing it with reasonable necessity to continue research on the topic.

**6. Improving the beneficial effect of protective autoimmunity** 

Even though the positive effect of immunizing with NDP has rendered significant results, it is possible to potentiate this effect. The improvement of this strategy would yield a better functional recovery and, thereby, a better quality of life for NDD-affected individuals. It is clear that several damaging mechanisms take place during the acute phase of injury. Unfortunately, NDP-induced PA develops after a few days of immunization. Before PA sets in, the neural tissue is unprotected; therefore, the best approach is a combination of neuroprotective strategies. A therapeutic intervention tailored to each specific time point of injury pathophysiology. This approach will ameliorate one or more of the destructive events and may improve the functional outcome even more than PA alone. Excessive production of ROS from the beginning of CNS injury causes lipid peroxidation LP (Hall, 1994). Peroxidation of membrane lipids affects the integrity of the cell membrane and is the most damaging mechanism. The unregulated synthesis of free radicals offers a potential intervention route for the treatment of NDD. An example of this is the use of glutathione monoethyl ester (GSHE). This cell-permeant derivative of glutathione (GSH) is an

Immunization with poly-YE had a significant neuroprotective effect after stroke, but authors' also wanted to evaluate its neuroregenerative properties. They found that poly-YE promotes neurogenesis after stroke as they saw an overall increase in the number of newly formed neurons in the dentate gyri of treated animals. The results presented in this study showed that the administrations of poly-YE as late as 24 hours after the induction of ischemic stroke greatly improved subsequent recovery. It had a positive effect on the neurological outcome of stroke, delayed degeneration, and enhanced the repair of damaged structures. Also, the therapeutic window (24 hours) seemed to be significantly wider than most of the current candidate therapies for stroke, giving it much more clinically translational value (Ziv et al., 2007). A separate study in our laboratory examined the effect of Cop-1 immunizations on the outcome of ischemic stroke, using the middle cerebral artery occlusion model. Results suggested that Cop-1 significantly improved the neurological outcome of animals after stroke. Histolopathological assessment also demonstrated a decrease in infarct size and infarct volume in Cop-1-treated animals (Ibarra et al., 2007). The results of both studies do not necessarily elucidate the mechanisms through which NDPinduced PA exerts its protective effects in focal cerebral ischemia but they provide evidence of its neuroprotective, and even neuroregenerative, properties. These studies provide NDPinduced PA with another consequential benefit, and that is the wide therapeutic window. Immunizations with NDP in the treatment of stroke require exhaustive research before they reach clinical trial potential but these preliminary results are an enormous step closer.

#### **5.2.2 Traumatic CNS injury**

Traumatic CNS injury can be broken down into two compartments: TBI and SCI. A study by Kipnis et al. found that immunizing with Cop-1 after traumatic brain injury had a better outcome on neurological and histological evaluations after injury (Kipnis et al., 2003). TBI triggers self-destructive processes, like other injuries to the CNS. Kipnis et al. studied mice with closed head injury and determined that the immune system plays a key role in the spontaneous recovery. The trauma-induced deficit was reduced, both functionally and anatomically, by post-traumatic vaccination with Cop-1. Several studies have been published on the use of NDP in SCI. Hauben et al. used immunization with a variety of myelin-associated peptides, including those derived from Nogo-A, can be used to evoke a T cell-mediated response that promotes recovery. They show that neuronal degeneration after incomplete spinal cord contusion in rats was substantially reduced, and hence recovery was significantly promoted, by posttraumatic immunization with Nogo-A-derived, p472 (Hauben et al., 2001). Our laboratory has also demonstrated the beneficial effect of immunizing with NDP (A91) on motor recovery and neuronal survival after SCI (Martiñon et al., 2007). Furthermore, we have determined some of the mechanisms of action of NDPinduced PA. In a recent study we found that immunization with Cop-1 and A91 exerted its neuroprotective effect through the inhibition of lipid peroxidation (LP). Animals were immunized with A91 seven days before injury. With the aim of inducing the functional elimination of CNS-specific T cells, animals were tolerized against SC-protein extract and thereafter subjected to a SCI. The lipid-soluble fluorescent products were used as an index of LP and were assessed after injury. Immunization with NDP reduced LP after SCI. Functional elimination of CNS-specific T cells avoided the beneficial effect induced by PA (Ibarra et al., 2010). A consequential study hypothesized that LP was caused by an unregulated production of ROS seen after CNS injury. The main ROS produced during the

Immunization with poly-YE had a significant neuroprotective effect after stroke, but authors' also wanted to evaluate its neuroregenerative properties. They found that poly-YE promotes neurogenesis after stroke as they saw an overall increase in the number of newly formed neurons in the dentate gyri of treated animals. The results presented in this study showed that the administrations of poly-YE as late as 24 hours after the induction of ischemic stroke greatly improved subsequent recovery. It had a positive effect on the neurological outcome of stroke, delayed degeneration, and enhanced the repair of damaged structures. Also, the therapeutic window (24 hours) seemed to be significantly wider than most of the current candidate therapies for stroke, giving it much more clinically translational value (Ziv et al., 2007). A separate study in our laboratory examined the effect of Cop-1 immunizations on the outcome of ischemic stroke, using the middle cerebral artery occlusion model. Results suggested that Cop-1 significantly improved the neurological outcome of animals after stroke. Histolopathological assessment also demonstrated a decrease in infarct size and infarct volume in Cop-1-treated animals (Ibarra et al., 2007). The results of both studies do not necessarily elucidate the mechanisms through which NDPinduced PA exerts its protective effects in focal cerebral ischemia but they provide evidence of its neuroprotective, and even neuroregenerative, properties. These studies provide NDPinduced PA with another consequential benefit, and that is the wide therapeutic window. Immunizations with NDP in the treatment of stroke require exhaustive research before they reach clinical trial potential but these preliminary results are an enormous step closer.

Traumatic CNS injury can be broken down into two compartments: TBI and SCI. A study by Kipnis et al. found that immunizing with Cop-1 after traumatic brain injury had a better outcome on neurological and histological evaluations after injury (Kipnis et al., 2003). TBI triggers self-destructive processes, like other injuries to the CNS. Kipnis et al. studied mice with closed head injury and determined that the immune system plays a key role in the spontaneous recovery. The trauma-induced deficit was reduced, both functionally and anatomically, by post-traumatic vaccination with Cop-1. Several studies have been published on the use of NDP in SCI. Hauben et al. used immunization with a variety of myelin-associated peptides, including those derived from Nogo-A, can be used to evoke a T cell-mediated response that promotes recovery. They show that neuronal degeneration after incomplete spinal cord contusion in rats was substantially reduced, and hence recovery was significantly promoted, by posttraumatic immunization with Nogo-A-derived, p472 (Hauben et al., 2001). Our laboratory has also demonstrated the beneficial effect of immunizing with NDP (A91) on motor recovery and neuronal survival after SCI (Martiñon et al., 2007). Furthermore, we have determined some of the mechanisms of action of NDPinduced PA. In a recent study we found that immunization with Cop-1 and A91 exerted its neuroprotective effect through the inhibition of lipid peroxidation (LP). Animals were immunized with A91 seven days before injury. With the aim of inducing the functional elimination of CNS-specific T cells, animals were tolerized against SC-protein extract and thereafter subjected to a SCI. The lipid-soluble fluorescent products were used as an index of LP and were assessed after injury. Immunization with NDP reduced LP after SCI. Functional elimination of CNS-specific T cells avoided the beneficial effect induced by PA (Ibarra et al., 2010). A consequential study hypothesized that LP was caused by an unregulated production of ROS seen after CNS injury. The main ROS produced during the

**5.2.2 Traumatic CNS injury** 

secondary phase of damage after trauma is NO. When NO is produced in an unregulated fashion it can react with other free radicals such as superoxide anion and produce peroxynitrite a powerful neurotoxic substance. We determined that the decrease in lipid peroxidation was caused by an inhibition in the synthesis of NO after immunization with NDP after SCI (unpublished data). Our results supported our hypothesis and allowed us to corroborate the data with expression analysis. We used real time RT-PCR to also demonstrate a reduction in the expression of the enzyme implicated in post-injury synthesis of NO, the inducible form of nitric oxide synthase (iNOS) (unpublished data). By determining that A91 reactive T cells also secrete NT-3 and IL-4 after SCI, making them a Th2 phenotype, we further substantiate the PA hypothesis. Immunizing with NDP deviates the Th response down a Th2 pathway increasing the synthesis of molecules such as IL-4 and IL-10 and secretion of neurotrophic factors like NT-3. Finally, we have found that the severity of injury would determine the strength and the effect of the PA response (unpublished data). This new data adds more factors into the induction of an autoreactive response. Our study noticed that animals that sustained a non-complete injury to the spinal cord had an increased recovery when immunized with A91. These autoreactive T cells also secreted BDNF and had greater recognition for A91 in vitro. On the other hand, animals that sustained complete or severe SCI did not recover even after A91-immunization. Unexpectedly, these animals did not even possess a clonal response to A91, meaning they were not even able to recognize the antigen in vivo, even with an adjuvant. This indicates that animals that sustained a severe or complete injury to the spinal cord are severely immunosuppressed and may therefore not engage a true PA response (unpublished data). This data that has just surfaced indicates that the neuroimmunological components of CNS disease require much more research in order to elucidate this unknown mechanisms. Even further, we must continue to delve into this immunosuppression caused by severe injury. The study of the body's physiology under duress shows us some of the mechanisms it possesses that could help in regenerating the CNS during disease. Immunization with NDP has proven to be an excellent therapeutical intervention in SCI and several other NDD, providing it with reasonable necessity to continue research on the topic.

#### **6. Improving the beneficial effect of protective autoimmunity**

Even though the positive effect of immunizing with NDP has rendered significant results, it is possible to potentiate this effect. The improvement of this strategy would yield a better functional recovery and, thereby, a better quality of life for NDD-affected individuals. It is clear that several damaging mechanisms take place during the acute phase of injury. Unfortunately, NDP-induced PA develops after a few days of immunization. Before PA sets in, the neural tissue is unprotected; therefore, the best approach is a combination of neuroprotective strategies. A therapeutic intervention tailored to each specific time point of injury pathophysiology. This approach will ameliorate one or more of the destructive events and may improve the functional outcome even more than PA alone. Excessive production of ROS from the beginning of CNS injury causes lipid peroxidation LP (Hall, 1994). Peroxidation of membrane lipids affects the integrity of the cell membrane and is the most damaging mechanism. The unregulated synthesis of free radicals offers a potential intervention route for the treatment of NDD. An example of this is the use of glutathione monoethyl ester (GSHE). This cell-permeant derivative of glutathione (GSH) is an

Immunization with Neural-Derived Peptides as a

**8. References** 

Potential Therapy in Neurodegenerative Diseases 533

mechanisms in order to make this therapeutic intervention efficacious and safe. The ultimate

Aharoni, R.; Eilam, R.; Domev, H.; Labunskay, G.; Sela, M. & Arnon, R. (2005). The

Aharoni, R.; Kayhan, B.; Eilam, R.; Sela, M. & Arnon, R. (2003). Glatiramer acetate-specific T

*America,* Vol.100, No.24, (November 2003), pp. 14157-14162, ISSN 0027-8424 Aharoni, R.; Teitelbaum, D.; Leitner, O.; Meshorer, A.; Sela, M. & Arnon, R. (2000). Specific

Alzheimer's Association. (2010). Alzheimer's disease facts and figures. *Alzheimer's &* 

Angelov, D.N.; Waibel, S.; Guntinas-Lichius, O.; Lenzen, M.; Neiss, W.F.; Tomov, T.L.; Yoles,

Becker, K.J.; McCarron, R.M.; Ruetzler, C.; Laban, O.; Sternberg, E.; Flanders, K.C. &

Ben-Zeev, B.; Aharoni, R.; Nissenkorn, A. & Arnon, R. (2011). Glatiramer acetate (GA,

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*Hypotheses,* Vol.76, No.2, (February 2011), pp. 190-193, ISSN 1532-2777 Benner, E.J.; Mosley, R.L.; Destache, C.J.; Lewis, T.B.; Jackson-Lewis, V.; Gorantla, S.;

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*dementia,* Vol.6, No.2, (March 2010), pp. 158-194. ISSN 1552-5279

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immunomodulator glatiramer acetate augments the expression of neurotrophic factors in brains of experimental autoimmune encephalomyelitis mice. *Proceedings of the National Academy of Sciences of the United States of America,* Vol.102*,* No.52,

cells in the brain express T helper 2/3 cytokines and brain-derived neurotrophic factor in situ. *Proceedings of the National Academy of Sciences of the United States of* 

Th2 cells accumulate in the central nervous system of mice protected against experimental autoimmune encephalomyelitis by copolymer 1. *Proceedings of the National Academy of Sciences of the United States of America,* Vol.97, No.21, (October

E.; Kipnis, J.; Schori, H.; Reuter, A.; Ludolph, A. & Schwartz, M. (2003). Therapeutic vaccine for acute and chronic motor neuron diseases: implications for amyotrophic lateral sclerosis. *Proceedings of the National Academy of Sciences of the United States of* 

Hallenbeck, J.M. (1997). Immunologic tolerance to myelin basic protein decreases stroke size after transient focal cerebral ischemia. *Proceedings of the National Academy of Sciences of the United States of America,* Vol.94*,* No.20, (September 1997), pp. 10873-

Copolymer-1) an hypothetical treatment option for Rett syndrome. *Medical* 

Nemachek, C.; Green, S.R.; Przedborski, S. & Gendelman, H.E. (2004). Therapeutic immunization protects dopaminergic neurons in a mouse model of Parkinson's disease. *Proceedings of the National Academy of Sciences of the United States of America,* 

Traumatic spinal cord injury induces nuclear factor-kappaB activation. *The Journal* 

Green, J. & Dietrich, W.D. (1999). Systemically administered interleukin-10 reduces tumor necrosis factor-alpha production and significantly improves functional recovery following traumatic spinal cord injury in rats. *Journal of Neurotrauma,* 

goal is to help the suffering and the complications of human disease.

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10878, ISSN 0027-8424

antioxidant that limits the effect of ROS on the bi-lipid membrane. GSH has shown neuroprotective properties after SCI (Guizar-Sahagun et al., 2005; Santoscoy et al., 2002). Aside from this effect, GSH supports the proliferation, growth, and differentiation of immune cells. Moreover, GSH is actually required for many specific T cell functions, including DNA replication and IL-2 synthesis (Kidd., 1997). The amount of GSH determines the magnitude of the immunological response (Droge et al., 1994) as well as its depletion inhibits normal function (Kidd, 1997). According to the data presented above, the addition of GSHE to NDP immunizations could significantly improve neuroprotection. The antioxidant properties of GSH will cover the overproduction of ROS from the beginning of injury while it could also assist in inducing a better PA response. A previous work carried out in our laboratory, examined the effect of this combination and demonstrated that the addition of GSHE to NDP immunizations induced earlier and better motor recovery after SCI compared to immunizations alone (Martinon et al., 2007). This effect was observed in animals subjected to either a contusive or a compressive SCI. The substantial improvement observed in treated animals allowed them to attain weight-supported plantar steps. This recovery is of great relevance when translating this treatment into a clinical setting. Motor improvement significantly correlated with increased axonal myelination as well as a marked survival of rubrospinal neurons. Besides finding adjuvant therapies for NDP-induced PA we wanted to see if multiple immunizations would increase the beneficial effect. We examined the effect of double immunizations and their effect on PA. Contrary to our expectations, double immunizations abolished the neuroprotective effect of single dose NDP-induced PA. The findings support the notion that the second immunization after SCI has a negative effect on PA. Rather than strengthening the protective effect, it eliminated it. This phenomenon was probably secondary to anergy since double immunization did not induce cell death (Martinon et al., 2007). According to the present data, the use of NDP and GSHE in SCI is a promising strategy. Further studies are necessary in order to establish the efficacy of this therapy and its potential applications into other NDD. Another attempt of synergistic therapeutic interventions is the use of GA with IFN-β-1a in MS (Lublin & Reingold, 2001). The development of adjuvant and synergistic therapies will aid in the optimization of NDP-induced PA allowing us to tackle the pathophysiology of several NDD.

#### **7. Conclusion**

The concept of PA revolutionized the way we saw the immune system in several different diseases. We figured out that it was more important to modulate the response than to eliminate it. With the logarithmic explosion in knowledge we must now hold these conclusions. The use of NDP and their effect on the immune response have proven to be helpful in several different pathologies, particularly in NDD. Using the information that we have recollected across the years, the mechanisms through which NDP-induced PA exerts its effects is everyday less obscure. Unfortunately, due to hypersensitivity reactions and heterogeneous responses among patients NDP have not been taken to their maximum potential. Unfortunately, PA is developed under the bases that the immune system is healthy and will function normally following an insult to the CNS. However, MS is an autoimmune disease, a case where the immune system is fatally skewed. This paradox forces us to adopt a revolutionary idea such as PA and apply it to NDD. The application of NDP-induced PA to the field of NDD can yield insurmountable results and therefore we urge the scientific community to aid in continuing to shed light on these once obscure mechanisms in order to make this therapeutic intervention efficacious and safe. The ultimate goal is to help the suffering and the complications of human disease.

#### **8. References**

532 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

antioxidant that limits the effect of ROS on the bi-lipid membrane. GSH has shown neuroprotective properties after SCI (Guizar-Sahagun et al., 2005; Santoscoy et al., 2002). Aside from this effect, GSH supports the proliferation, growth, and differentiation of immune cells. Moreover, GSH is actually required for many specific T cell functions, including DNA replication and IL-2 synthesis (Kidd., 1997). The amount of GSH determines the magnitude of the immunological response (Droge et al., 1994) as well as its depletion inhibits normal function (Kidd, 1997). According to the data presented above, the addition of GSHE to NDP immunizations could significantly improve neuroprotection. The antioxidant properties of GSH will cover the overproduction of ROS from the beginning of injury while it could also assist in inducing a better PA response. A previous work carried out in our laboratory, examined the effect of this combination and demonstrated that the addition of GSHE to NDP immunizations induced earlier and better motor recovery after SCI compared to immunizations alone (Martinon et al., 2007). This effect was observed in animals subjected to either a contusive or a compressive SCI. The substantial improvement observed in treated animals allowed them to attain weight-supported plantar steps. This recovery is of great relevance when translating this treatment into a clinical setting. Motor improvement significantly correlated with increased axonal myelination as well as a marked survival of rubrospinal neurons. Besides finding adjuvant therapies for NDP-induced PA we wanted to see if multiple immunizations would increase the beneficial effect. We examined the effect of double immunizations and their effect on PA. Contrary to our expectations, double immunizations abolished the neuroprotective effect of single dose NDP-induced PA. The findings support the notion that the second immunization after SCI has a negative effect on PA. Rather than strengthening the protective effect, it eliminated it. This phenomenon was probably secondary to anergy since double immunization did not induce cell death (Martinon et al., 2007). According to the present data, the use of NDP and GSHE in SCI is a promising strategy. Further studies are necessary in order to establish the efficacy of this therapy and its potential applications into other NDD. Another attempt of synergistic therapeutic interventions is the use of GA with IFN-β-1a in MS (Lublin & Reingold, 2001). The development of adjuvant and synergistic therapies will aid in the optimization of NDP-induced PA allowing us to tackle the pathophysiology of several NDD.

The concept of PA revolutionized the way we saw the immune system in several different diseases. We figured out that it was more important to modulate the response than to eliminate it. With the logarithmic explosion in knowledge we must now hold these conclusions. The use of NDP and their effect on the immune response have proven to be helpful in several different pathologies, particularly in NDD. Using the information that we have recollected across the years, the mechanisms through which NDP-induced PA exerts its effects is everyday less obscure. Unfortunately, due to hypersensitivity reactions and heterogeneous responses among patients NDP have not been taken to their maximum potential. Unfortunately, PA is developed under the bases that the immune system is healthy and will function normally following an insult to the CNS. However, MS is an autoimmune disease, a case where the immune system is fatally skewed. This paradox forces us to adopt a revolutionary idea such as PA and apply it to NDD. The application of NDP-induced PA to the field of NDD can yield insurmountable results and therefore we urge the scientific community to aid in continuing to shed light on these once obscure

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**23** 

*Spain* 

Paul Bustamante1,2,

*1CEIT University of Navarra 2Tecnun University of Navarra* 

Gonzalo Solas1 and Karol Grandez1

**Neurodegenerative Disease Monitoring** 

**Using a Portable Wireless Sensor Device** 

Neurodegenerative diseases are characterized by progressive loss of neurons in the central nervous system. The disorders are clinically well-defined as a disease-related dementia, Alzheimer's disease the most typical case, or as a movement disorder, Parkinson's disease (PD). The risk of developing these diseases increases significantly with age: Parkinson's disease affects 1% of the population over 65 years of age, rising to 2% for those over 80

Parkinson's disease is a common neurodegenerative disorder that often impairs motor skills and speech of the patient. PD is characterized by muscle rigidity, tremor, slowing of physical movement (bradykinesia) and in extreme cases, loss of physical movement (akinesia). In particular, PD is due to a loss of dopaminergic neurons (related to the neurotransmitter dopamine), and subcortical neurons in the brain. Replacement therapy with dopaminergic drugs (levodopa, pramipexole) effectively reverses all the symptoms and signs of the disease. After a changeable period of time, however, this excellent initial response to dopaminergic treatment is complicated by the appearance of disorders known as motor response complications (MRC). These complications are divided into two main categories: (i) fluctuations in motor response and (ii) the emergence of abnormal involuntary

Generally, motor fluctuations appear first as a shortening of the initially soft and lasting dopaminergic response. For patients with advanced PD, a few hours after the administration of medication the patient begins to notice the reappearance of signs and symptoms of the disease. This is known as "end of dose deterioration" or "wearing off". This may happen several times a day, so the patient can actually spend several hours per day in an "off" state. During the short visit with the neurologist, the patient may appear to be well and thus the neurologist misses the symptoms related to wearing off. As a result, changes in the recent drug treatment availability do not take place in time. It is now well known that early treatment of wearing-off fluctuations delay the onset of more severe complications in the future, as well as the appearance of LID. Therefore any strategy that can detect early changes associated with wearing off would provide a valuable clinical tool that would allow

movements known as levodopa-induced dyskinesias (LID) (Konitsiotis, 2005).

**1. Introduction** 

early treatment interventions.

years.


### **Neurodegenerative Disease Monitoring Using a Portable Wireless Sensor Device**

Paul Bustamante1,2, Gonzalo Solas1 and Karol Grandez1 *1CEIT University of Navarra 2Tecnun University of Navarra Spain* 

#### **1. Introduction**

540 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

Terando, A.; Sabel, M.S. & Sondak, V.K. (2003). Melanoma: adjuvant therapy and other

Turrin, N.P. & Rivest, S. (2006). Molecular and cellular immune mediators of

Vanegas, H. & Schaible, H.G. (2001). Prostaglandins and cyclooxygenases [correction of

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Vieira, P.L.; Heystek, H.C.; Wormmeester, J.; Wierenga, E.A. & Kapsenberg, M.L. (2003).

Yin, Y.; Cui, Q.; Li, Y.; Irwin, N.; Fischer, D.; Harvey, A.R. & Benowitz, L.I. (2003).

Ziemssen, T.; Kumpfel, T.; Klinkert, W.E.; Neuhaus, O. & Hohlfeld, R. (2002). Glatiramer

Ziemssen, T.; Kumpfel, T.; Schneider, H.; Klinkert, W.E.; Neuhaus, O. & Hohlfeld, R. (2005).

*Neuroscience*, Vol.23, No.6, (March 2003), pp. 2284-2293, ISSN 0270-6474 Yoles, E.; Hauben, E.; Palgi, O.; Agranov, E.; Gothilf, A.; Cohen, A.; Kuchroo, V.; Cohen, I.R.;

*Immunology*, Vol.170, No.9, (May 2003), pp. 4483-4488, ISSN 0022-1767 Xu, J.Q.; Kochanek, K.D.; Murphy, S.L. & Tejada-Vera, B. (2007). *Deaths: Final data for 2007*.

Hyattsville, MD: National Center for Health Statistics. 2010.

11, (November 2002), pp. 2381-2391, ISSN 0006-8950

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2001), pp. 327-363, ISSN 0301-0082

3740-3748, ISSN 0270-6474

ISSN 0039-2499

242, ISSN 0893-7648

0836

treatment options. *Current Treatment Options in Oncology*, Vol.4, No.3, (June 2003),

neuroprotection. *Molecular Neurobiology*, Vol.34, No.3, (December 2007), pp. 221-

cycloxygenases] in the spinal cord. *Progress in Neurobiology*, Vol.64, No.4, (July

self-tolerance. *Nature*, Vol.336, No.6196, (November 1988), pp. 222-225, ISSN 0028-

Glatiramer acetate (copolymer-1, copaxone) promotes Th2 cell development and increased IL-10 production through modulation of dendritic cells. *Journal of* 

Macrophage-derived factors stimulate optic nerve regeneration. *The Journal of* 

Weiner, H. & Schwartz, M. (2001). Protective autoimmunity is a physiological response to CNS trauma. *The Journal of Neuroscience*, Vol.21, No.11, (June 2001), pp.

acetate-specific T-helper 1- and 2-type cell lines produce BDNF: implications for multiple sclerosis therapy. Brain-derived neurotrophic factor. *Brain,* Vol.125, No.Pt

Secretion of brain-derived neurotrophic factor by glatiramer acetate-reactive Thelper cell lines: Implications for multiple sclerosis therapy. *Journal of the Neurological Sciences*, Vol.233, No.1-2, (June 2005), pp. 109-112, ISSN 0022-510X Ziv, Y.; Finkelstein, A.; Geffen, Y.; Kipnis, J.; Smirnov, I.; Shpilman, S.; Vertkin, I.; Kimron,

M.; Lange, A.; Hecht, T.; Reyman, K.G.; Marder, J.B.; Schwartz, M. & Yoles, E. (2007). A novel immune-based therapy for stroke induces neuroprotection and supports neurogenesis. *Stroke*, Vol.38, No.2 Suppl, (February 2007), pp. 774-782, Neurodegenerative diseases are characterized by progressive loss of neurons in the central nervous system. The disorders are clinically well-defined as a disease-related dementia, Alzheimer's disease the most typical case, or as a movement disorder, Parkinson's disease (PD). The risk of developing these diseases increases significantly with age: Parkinson's disease affects 1% of the population over 65 years of age, rising to 2% for those over 80 years.

Parkinson's disease is a common neurodegenerative disorder that often impairs motor skills and speech of the patient. PD is characterized by muscle rigidity, tremor, slowing of physical movement (bradykinesia) and in extreme cases, loss of physical movement (akinesia). In particular, PD is due to a loss of dopaminergic neurons (related to the neurotransmitter dopamine), and subcortical neurons in the brain. Replacement therapy with dopaminergic drugs (levodopa, pramipexole) effectively reverses all the symptoms and signs of the disease. After a changeable period of time, however, this excellent initial response to dopaminergic treatment is complicated by the appearance of disorders known as motor response complications (MRC). These complications are divided into two main categories: (i) fluctuations in motor response and (ii) the emergence of abnormal involuntary movements known as levodopa-induced dyskinesias (LID) (Konitsiotis, 2005).

Generally, motor fluctuations appear first as a shortening of the initially soft and lasting dopaminergic response. For patients with advanced PD, a few hours after the administration of medication the patient begins to notice the reappearance of signs and symptoms of the disease. This is known as "end of dose deterioration" or "wearing off". This may happen several times a day, so the patient can actually spend several hours per day in an "off" state. During the short visit with the neurologist, the patient may appear to be well and thus the neurologist misses the symptoms related to wearing off. As a result, changes in the recent drug treatment availability do not take place in time. It is now well known that early treatment of wearing-off fluctuations delay the onset of more severe complications in the future, as well as the appearance of LID. Therefore any strategy that can detect early changes associated with wearing off would provide a valuable clinical tool that would allow early treatment interventions.

Neurodegenerative Disease Monitoring Using a Portable Wireless Sensor Device 543

interior environment that responds and interacts with its external environment, but is somehow "independent." The monitoring of the human body using a wireless sensor network can be achieved by attaching the sensors to the body (or even implanted in the

The wireless sensor networks are formed by a group of sensor nodes with certain capacity for sensing environment variables and transmitting them wirelessly. These nodes allow forming ad-hoc networks without an established physical infrastructure or a centralized management. These kinds of networks are known for being easy to deploy and for being

The majority of the researches carried out in the field of wireless sensor networks are focused on the network architecture, as well as on the communication protocols within the network. But few advances have been made in the development of novel sensor node architectures. The efforts are focused on the miniaturization of the nodes and the reduction

The objective of the work described in this chapter is to develop a single device which could be used in several application fields, due to its capability of being able to acquire signals coming from different types of sensors. Apart from that, the treatment of the data can be carried out in multiple ways, as the device is equipped with an SD card, a RF transceiver (IEEE 802.15.4 specification compliant) and a USB connector, for communication as well as

In order to test its versatility, an application field has been chosen and several tests have been carried out related to that field. More concretely, the application field that has been selected the validity of the objectives proposed in this work has been e-Health, and thus, a

The study of the state of the art shows that the devices and methods developed so far for the testing activities in patients affected by PD and ALS lack the most important characteristics

• **Accuracy**: the data provided by the device show exact values for the parameters the doctors are interested in. They are not based on subjective appreciation of the

• **Ease of use**: both for the clinicians and for the patients. The patient can carry out the tests without having to move from their own homes. And the data is stored in a PC, which offers the possibility of sending it to the hospital via Internet, for the doctors to

• **Frequency**: the ease of use of the system makes it possible to carry out more frequent tests, so the tracking of the variations of the motor functions of the patients is more

• **Versatility**: using the devices presented in this article, several different tests can be performed, and in each test, several parameters can be measured. For example, for the finger tapping case, both the speed and the regularity (periodicity) can be obtained, which enriches the results of the test and enhances the analysis and the conclusions

In order to comply with this characteristics or requirements, in this work we describe the

system developed, which is based on the architecture shown in Fig. 1.

tissues).

auto configurable.

for charging functions.

**3. System architecture** 

analyze the results.

obtained with it.

accurate.

of the device described in this article:

of the energy consumption (Anastasi et al., 2009).

continuous monitoring system has been developed.

performance of the tests carried out by the patients.

The quantitative assessment of the human body and motor movement disorders has been a topic of great interest for decades. Advanced equipment has been used to study various pathologies of the motor performance of the human body. However, sophisticated equipment alone is not a guarantee for success in the detection and analysis of motor disorders. In many situations, deficiencies in motor performance are not always frequent and motor disorders can occur only in very specific situations that are difficult to imitate or reproduce in a laboratory. The underlying testing and monitoring processes have not experienced the innovation and advancement required to fulfil the needs that such detection and analysis present.

Amyotrophic lateral sclerosis (ALS), often referred to as "Lou Gehrig's Disease," is a progressive neurodegenerative disease that affects nerve cells in the brain and the spinal cord. Motor neurons reach the spinal cord from the brain, and from the spinal cord to the muscles throughout the body. The progressive degeneration of the motor neurons in ALS causes these motor neurons to die and when this happens the ability of the brain to initiate and control muscle movement is lost. With voluntary muscle action progressively affected, patients in the later stages of the disease may become totally paralyzed.

The cases for Parkinson disease and ALS are expected to double worldwide by the year 2020 (Von Campenhausen et al., 2005). Proper medical care of these patients is becoming increasingly complex and expensive. Lengthy hospital stays for monitoring and adjustment of the patients' treatment and the problems related with it, contribute to cost increase and morbidity due to the hospitalization itself. But there is a clinical deficit of objective data on which neurologists can base the assessment and care of patients with chronic neurologicallybased movement disorders.

#### **2. Patient monitoring**

The patient monitoring is a technique that has become popular in recent years in the field of research, and soon the number of actual implementations in clinics and hospitals will begin to increase. Monitoring of patients is not new, in fact, today there are many hospitals that supply devices (thermometers, gauges, pulse and blood pressure, pulse oximetry, electrocardiogram, electro-devices, etc). The disadvantage of these devices is their large size and weight, and the little mobility they offer. The key innovation lies in one word: continuous monitoring. It consists of a series of devices and techniques designed to monitor, continuously and for a period of time established by the specialists, the physiological parameters of the patient. The specific values and the time evolution of these parameters allow a more precise analysis of the evolution of the disease, and therefore more effective treatment.

There are two main factors that have contributed to the rise of this technology. On one hand, the development of new physiological sensors that allow the measurement of more and more parameters related to the human body. Advances in biological, chemical, electrical and mechanical sensor technologies have led to their wider use as wearable sensors or implants. Improvements in the manufacture of sensors and techniques for nano-engineering, along with parallel advances in technology of microelectromechanical systems (MEMS) offer the potential for implantable or attachable sensors getting smaller.

On the other hand, the popularization of wireless sensor networks (WSN) and the recent advances in their use as body sensor networks (BSN), has been another key development for recent continuous monitoring of patients (Yick et al., 2008). The human body is a complex interior environment that responds and interacts with its external environment, but is somehow "independent." The monitoring of the human body using a wireless sensor network can be achieved by attaching the sensors to the body (or even implanted in the tissues).

The wireless sensor networks are formed by a group of sensor nodes with certain capacity for sensing environment variables and transmitting them wirelessly. These nodes allow forming ad-hoc networks without an established physical infrastructure or a centralized management. These kinds of networks are known for being easy to deploy and for being auto configurable.

The majority of the researches carried out in the field of wireless sensor networks are focused on the network architecture, as well as on the communication protocols within the network. But few advances have been made in the development of novel sensor node architectures. The efforts are focused on the miniaturization of the nodes and the reduction of the energy consumption (Anastasi et al., 2009).

The objective of the work described in this chapter is to develop a single device which could be used in several application fields, due to its capability of being able to acquire signals coming from different types of sensors. Apart from that, the treatment of the data can be carried out in multiple ways, as the device is equipped with an SD card, a RF transceiver (IEEE 802.15.4 specification compliant) and a USB connector, for communication as well as for charging functions.

In order to test its versatility, an application field has been chosen and several tests have been carried out related to that field. More concretely, the application field that has been selected the validity of the objectives proposed in this work has been e-Health, and thus, a continuous monitoring system has been developed.

#### **3. System architecture**

542 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

The quantitative assessment of the human body and motor movement disorders has been a topic of great interest for decades. Advanced equipment has been used to study various pathologies of the motor performance of the human body. However, sophisticated equipment alone is not a guarantee for success in the detection and analysis of motor disorders. In many situations, deficiencies in motor performance are not always frequent and motor disorders can occur only in very specific situations that are difficult to imitate or reproduce in a laboratory. The underlying testing and monitoring processes have not experienced the innovation and advancement required to fulfil the needs that such detection

Amyotrophic lateral sclerosis (ALS), often referred to as "Lou Gehrig's Disease," is a progressive neurodegenerative disease that affects nerve cells in the brain and the spinal cord. Motor neurons reach the spinal cord from the brain, and from the spinal cord to the muscles throughout the body. The progressive degeneration of the motor neurons in ALS causes these motor neurons to die and when this happens the ability of the brain to initiate and control muscle movement is lost. With voluntary muscle action progressively affected,

The cases for Parkinson disease and ALS are expected to double worldwide by the year 2020 (Von Campenhausen et al., 2005). Proper medical care of these patients is becoming increasingly complex and expensive. Lengthy hospital stays for monitoring and adjustment of the patients' treatment and the problems related with it, contribute to cost increase and morbidity due to the hospitalization itself. But there is a clinical deficit of objective data on which neurologists can base the assessment and care of patients with chronic neurologically-

The patient monitoring is a technique that has become popular in recent years in the field of research, and soon the number of actual implementations in clinics and hospitals will begin to increase. Monitoring of patients is not new, in fact, today there are many hospitals that supply devices (thermometers, gauges, pulse and blood pressure, pulse oximetry, electrocardiogram, electro-devices, etc). The disadvantage of these devices is their large size and weight, and the little mobility they offer. The key innovation lies in one word: continuous monitoring. It consists of a series of devices and techniques designed to monitor, continuously and for a period of time established by the specialists, the physiological parameters of the patient. The specific values and the time evolution of these parameters allow a more precise analysis of the evolution of the disease, and therefore more effective

There are two main factors that have contributed to the rise of this technology. On one hand, the development of new physiological sensors that allow the measurement of more and more parameters related to the human body. Advances in biological, chemical, electrical and mechanical sensor technologies have led to their wider use as wearable sensors or implants. Improvements in the manufacture of sensors and techniques for nano-engineering, along with parallel advances in technology of microelectromechanical systems (MEMS) offer the

On the other hand, the popularization of wireless sensor networks (WSN) and the recent advances in their use as body sensor networks (BSN), has been another key development for recent continuous monitoring of patients (Yick et al., 2008). The human body is a complex

potential for implantable or attachable sensors getting smaller.

patients in the later stages of the disease may become totally paralyzed.

and analysis present.

based movement disorders.

**2. Patient monitoring** 

treatment.

The study of the state of the art shows that the devices and methods developed so far for the testing activities in patients affected by PD and ALS lack the most important characteristics of the device described in this article:


In order to comply with this characteristics or requirements, in this work we describe the system developed, which is based on the architecture shown in Fig. 1.

Neurodegenerative Disease Monitoring Using a Portable Wireless Sensor Device 545

The sensors used in this work have been Force Sensitive Resistors (FSR). A force-sensitive resistor (alternatively called a force-sensing resistor) has a variable resistance as a function of applied pressure. In this sense, the term "force-sensitive" is misleading – a more appropriate one would be "pressure-sensitive", since the sensor's output is dependent on

The sensors used in this work are manufactured by Tekscan, and are constructed of two layers of substrate film. On each layer, a conductive material (silver) is applied, followed by a layer of pressure-sensitive ink (Vecchi et al., 2000). Adhesive is then used to laminate the two layers of substrate together to form the force sensor. The active sensing area is defined by the silver circle on top of the pressure-sensitive ink. Silver extends from the sensing area to the connectors at the other end of the sensor, forming the conductive leads. Fig. 2 shows a

After choosing Force Sensitive Resistors (FSR) as transducers, both a sensorized glove and an insole have been designed, and then used to carry out several tests related to Parkinson

The design of the device was related to its main functionality explained above. Measurements obtained from sensors are transmitted through wires to an IDC connector located at one edge of the device. This connector allows these inputs to be connected to A/D channels extended from the CPU. An interface stage is needed for each input due to sensors,

The architecture of the approach presented in this work is shown in Fig. 3. The CPU of the portable wireless device is the 18LF4550, a Microchip PIC18 Microcontroller with nanoWatt technology. It is an 8-bit System On-chip mainly featured by USB and SPI communication interfaces; it has a maximum number of 13 input A/D channels; each with a 10-bit resolution. It is also characterized for its low power consumption in deep-sleep mode, ideal to work as sensor node in monitoring applications. Also, this CPU has an RTC (Real Timer Clock), ideal for use in applications where is necessary to store the data

Analogical input signals attached to the IDC connector are converted into digital values which are put in an established frame structure according to a particular protocol. The frame

the area on the sensor's surface to which force is applied.

Disease (PD) and Amyotrophic lateral sclerosis (ALS).

arranged is ready to be transmitted via USB or RF.

done basically with some operational amplifiers and passive filters.

**4.1 FSR sensors** 

picture of the Tekscan FSR sensor.

Fig. 2. Tekscan FSR sensor

**4.2 Hardware architecture** 

sample time.

Fig. 1. System architecture

The whole system architecture is composed of four main building blocks:


#### **4. Device architecture**

The main aim of the system is to gather data from any kind of sensor in order to store those data in an SD card or transmit them to Base Central Unit (BCU), connected to PC through USB connection. The SD card gives the system the possibility of having longer recording periods which allows the device to be used at a further distance from the BCU.

The data gathered and stored in the SD by the system is downloaded to a PC, by USB connection or by the radio transceiver, which operates in the 2.4GHz ISM band, using the BCU.

#### **4.1 FSR sensors**

544 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

The whole system architecture is composed of four main building blocks:

saving space with another chip converter.

Windows with Visual C++ environment.

it is converted in voltage, in order to be acquired by the A/D converter.

• **Sensorized glove**: this glove is equipped with five sensors, which are attached to it. Each sensor is a FSR sensor and is connected to the circuit with a simple interface, done with a division resistor. When the user presses the FSR sensor, its resistance varies, and

• **Sensorized insole**: each insole is designed with five FSR sensors, in order to measure the area where the patient puts more pressure and to analyse the way he/she walks. • **Hardware device**: this is the main development of the present research work. It consists of a tiny electronic circuit, based on a low cost and low energy microprocessor (PIC), protected by a case specifically designed for it. Its main functions are the acquisition and processing of the signals coming from the sensors, and transmitting them to the PC via the USB connection. The selected microprocessor has an 8 channel 10 bit A/D converter and an USB interface, which can be easily programmed and this allows

• **PC application**: the fourth component of the system architecture is in charge of receiving the data sent by the hardware device via the USB connection, storing and visualizing them, using a graphical user interface. This application was done in

The main aim of the system is to gather data from any kind of sensor in order to store those data in an SD card or transmit them to Base Central Unit (BCU), connected to PC through USB connection. The SD card gives the system the possibility of having longer recording

The data gathered and stored in the SD by the system is downloaded to a PC, by USB connection or by the radio transceiver, which operates in the 2.4GHz ISM band, using the

periods which allows the device to be used at a further distance from the BCU.

Fig. 1. System architecture

**4. Device architecture** 

BCU.

The sensors used in this work have been Force Sensitive Resistors (FSR). A force-sensitive resistor (alternatively called a force-sensing resistor) has a variable resistance as a function of applied pressure. In this sense, the term "force-sensitive" is misleading – a more appropriate one would be "pressure-sensitive", since the sensor's output is dependent on the area on the sensor's surface to which force is applied.

The sensors used in this work are manufactured by Tekscan, and are constructed of two layers of substrate film. On each layer, a conductive material (silver) is applied, followed by a layer of pressure-sensitive ink (Vecchi et al., 2000). Adhesive is then used to laminate the two layers of substrate together to form the force sensor. The active sensing area is defined by the silver circle on top of the pressure-sensitive ink. Silver extends from the sensing area to the connectors at the other end of the sensor, forming the conductive leads. Fig. 2 shows a picture of the Tekscan FSR sensor.

Fig. 2. Tekscan FSR sensor

After choosing Force Sensitive Resistors (FSR) as transducers, both a sensorized glove and an insole have been designed, and then used to carry out several tests related to Parkinson Disease (PD) and Amyotrophic lateral sclerosis (ALS).

#### **4.2 Hardware architecture**

The design of the device was related to its main functionality explained above. Measurements obtained from sensors are transmitted through wires to an IDC connector located at one edge of the device. This connector allows these inputs to be connected to A/D channels extended from the CPU. An interface stage is needed for each input due to sensors, done basically with some operational amplifiers and passive filters.

The architecture of the approach presented in this work is shown in Fig. 3. The CPU of the portable wireless device is the 18LF4550, a Microchip PIC18 Microcontroller with nanoWatt technology. It is an 8-bit System On-chip mainly featured by USB and SPI communication interfaces; it has a maximum number of 13 input A/D channels; each with a 10-bit resolution. It is also characterized for its low power consumption in deep-sleep mode, ideal to work as sensor node in monitoring applications. Also, this CPU has an RTC (Real Timer Clock), ideal for use in applications where is necessary to store the data sample time.

Analogical input signals attached to the IDC connector are converted into digital values which are put in an established frame structure according to a particular protocol. The frame arranged is ready to be transmitted via USB or RF.

Neurodegenerative Disease Monitoring Using a Portable Wireless Sensor Device 547

The developed software to be embedded into the device has a modular scheme. This design

The whole software structure is divided in 4 layers as depicted in Fig. 5. The layers are

• **Physical level**: is the lowest level and it depends on the hardware directly. The modules present in this level correspond to the physical modules of the node; these are the force sensors, accelerometer (not mounted), the USB port, the mini-SD slot

(optional) and the RF module, which is controlled by the CPU using the SPI bus. • **Controller level**: the functions developed in this level permit the application level to invoke controller functions. The ADC module converts analogical signals from the force sensors to digital, and the SPI allows communications of the CPU with the accelerometer and the mini-SD card and the RF chip. In this layer the set of USB and

separated by dotted lines and a short description for each one is given below:

RTC (Real Time Clock) functions are also included.

allows the software to be independent from the platform and also gives flexibility.

Fig. 4. PCB layout and sensor interface

Fig. 5. Embedded software architecture

**4.3 Embedded software** 

Fig. 3. Wireless device architecture

The USB device module is a mini-USB 2.0 compliant allowing fast transmission of data. It allows also charging the battery using a standard chip with a LED to monitor the charging action.

The device has also 3 more LEDs, whose main functionality is to indicate states in the program or can be programmed for different functions.

The device is designed also to use a 3-axis accelerometer in case it was needed; it also offers the option to record data in a micro-SD card placed in the bottom side. In order to increase the time access to the SD card, which means saving power, a proprietary system files access was implemented, based on the standard FAT32.

The wireless device has a radio frequency chip, the MRF24J40, from Microchip, which works in the 2.4GHz and has a SPI interface to communicate with the CPU. This chip was mainly chosen due to its IEEE 802.15.4 specification compliant (Hardware CSMA-CA mechanism, Automatic ACK response and support RSSI/LQI), additionally it has a hardware security engine and offers a low power consumption: 2uA in sleep mode, 22mA in TX mode (at +0dBm) and 18mA in RX mode.

The distribution of the device's components is illustrated in Fig. 4, where the left side shows the layout of the PCB and the distribution of the chips on the device. On the right side the connection procedure of the device with different kinds of sensors through a sensor interface is shown. This connection is possible by using the IDC connector, which includes pins for a VCC signal, the GND and 10 signals which are directed to the A/D converter. That was done due the fact that the main functionality of this device is to be a multifunctional wireless device.

Fig. 4. PCB layout and sensor interface

#### **4.3 Embedded software**

546 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

Battery

S1 S2 Sn

. . .

The USB device module is a mini-USB 2.0 compliant allowing fast transmission of data. It allows also charging the battery using a standard chip with a LED to monitor the charging

The device has also 3 more LEDs, whose main functionality is to indicate states in the

The device is designed also to use a 3-axis accelerometer in case it was needed; it also offers the option to record data in a micro-SD card placed in the bottom side. In order to increase the time access to the SD card, which means saving power, a proprietary system files access

The wireless device has a radio frequency chip, the MRF24J40, from Microchip, which works in the 2.4GHz and has a SPI interface to communicate with the CPU. This chip was mainly chosen due to its IEEE 802.15.4 specification compliant (Hardware CSMA-CA mechanism, Automatic ACK response and support RSSI/LQI), additionally it has a hardware security engine and offers a low power consumption: 2uA in sleep mode, 22mA in TX mode (at

The distribution of the device's components is illustrated in Fig. 4, where the left side shows the layout of the PCB and the distribution of the chips on the device. On the right side the connection procedure of the device with different kinds of sensors through a sensor interface is shown. This connection is possible by using the IDC connector, which includes pins for a VCC signal, the GND and 10 signals which are directed to the A/D converter. That was done due the fact that the main functionality of this device is to be a

USB

Fig. 3. Wireless device architecture

+0dBm) and 18mA in RX mode.

multifunctional wireless device.

program or can be programmed for different functions.

was implemented, based on the standard FAT32.

action.

Charger

RF µC

SPI

A/D Acc

SD

The developed software to be embedded into the device has a modular scheme. This design allows the software to be independent from the platform and also gives flexibility.

Fig. 5. Embedded software architecture

The whole software structure is divided in 4 layers as depicted in Fig. 5. The layers are separated by dotted lines and a short description for each one is given below:


Neurodegenerative Disease Monitoring Using a Portable Wireless Sensor Device 549

brain, the motor strip rostral to the central sulcus is the most important, and the functioning of this area is reflected directly in the FTT. As well as direct motor effects, the speed, coordination, and pacing requirements of finger tapping can be affected by levels of alertness, impaired ability to focus attention, or slowing of responses. Tapping frequency can distinguish patients with motor dysfunctions of cerebella, basal ganglia, and cerebral origins

At the onset of ALS the symptoms may be so slight that they are frequently overlooked. With regard to the appearance of symptoms and the progression of the illness, the course of the disease may include muscle weakness. Muscle weakness is a hallmark initial sign in ALS, occurring in approximately 60% of patients. The hands and feet may be affected first, causing difficulty in lifting, walking or using the hands for the activities of daily living such

ALS is a very difficult disease to diagnose. To date, there is no one test or procedure to ultimately establish the diagnosis of ALS. Methods for the evaluation of strength in people with ALS include a clinical neurological exam, manual muscle testing (MMT) (Aitkens et al., 1989), and rating scales. These methods are subjective and lack sensitivity to detect small changes. The purpose of the Hand Grip Strength Test is to measure the maximum isometric

The devices and methods used so far for the proposed tests have not had any significant improvement or innovation for many years. Traditional ways of performing the tests are

For the finger tapping test, several methods have been proposed and used. The standard method consists of asking the patients to start with the finger tapping process and an examiner using a stopwatch to keep track of the 10-second trial interval. Electronic devices which are based on the same testing methodology have been marketed. The electronic device has an internal timer that starts on the first tap and stops counting taps when the 10 seconds have elapsed. The use of automatic timing is intended to increase the accuracy of

Other devices used, which can be found in the literature, include precision image-based motion analyzer and passive marker-based movement analyzer (Jobbágy et al., 2005); the Halstead-Reitan finger tapping test (HRFTT), developed and manufactured by Reitan Neuropsychological Laboratory, which uses an electronic counter and a tapping key; finger tapping devices containing pressure sensors (Soichiro et al., 2004); systems consisting of

In the case of the hand-grip strength measurement, the innovations carried out in recent years have been even poorer. The most usual way to carry out this specific test is by using hand-grip and pinch-force dynamometers, which offer very poor information about the way the hand grabs objects. Electromyography has been also used in some studies (Long, 1970). In this work we have used our wireless device to carry out both of the tests. It is integrated in a system consisting of the mentioned device, a sensorized glove (see Fig. 7) which is worn by the patient, and a PC or base station, which is in charge of receiving the data sent by the

As mentioned before, the sensors used in this test have been Force Sensitive Resistors (FSR). The approach followed in this research work has been the one of attaching several sensors to a glove. This design allows complying with one of the key characteristics identified in the system architecture section: versatility. We consider that this design is more versatile in order to allow carrying out different type of tests and obtaining a wide range of results.

accelerometers and touch sensor (Yokoe et a., 2009) (Okuno et al., 2007).

device, and visualizing them graphically in order to be analyzed by the doctors.

from normal subjects.

still used.

testing (McDermid, 2000).

as dressing, washing and buttoning clothes.

strength of the hand and forearm muscles.


#### **4.4 Data frame**

The structure of the data frame, which is sent by RF or USB, is composed of a header which contains the ID of the device, followed by 4 bytes, indicating the measurement time, and a byte which indicates the length of the data.

Fig. 6 shows the data frame enclosed information of the measurements taken; the first two bytes give information about the frequency of sampling and the next byte gives the number of sensors measured. According to this last parameter, the rest of bytes corresponding to each sensor in groups of two bytes due to the 10-bit conversion configuration of the A/D converter.

Fig. 6. Data frame

#### **5. Device test in ALS disease**

One of the ways of overcoming the lack of data in ALS disease is to develop new easy-to-use testing devices, which can be left in the patient's own home and used to carry out periodic tests without having to go to hospital to do so. The comfortable testing processes and devices make the patients more willing to wear them outside the home, and this leads to a wider amount of data available for the doctors.

Two of the more widely used tests with neurodegenerative disease patients are the Finger Tapping Test (FTT) (Jobbágy et al., 2005) and the Hand-grip Strength Test (Long, 1970). In the case of the FTT, the patient is asked to tap two of the fingers of one hand as quick as possible, and the main parameter measured by the doctors is the tapping frequency. On the other hand, in the hand grip strength test the measured parameter is the force the patient is capable of apply when grabbing an object.

The Finger Tapping Test (FTT), originally developed as part of the Halstead Reitan Battery (HRB) of neuropsychological tests, is a simple measure of motor speed and motor control and is used in neuropsychology as a sensitive test for brain damage (Christianson & Leathem, 2004). Although motor functioning in humans is controlled by many areas of the

• **Interface level**: this layer is the interface between controller and application; it contains the main functions that the device performs during its duty cycle. These functions range from reading ADC channels or communicating through the SPI interface, to sending and receiving data from the USB, the SD card and the RF chip. Interruption routines are

• **Application level**: this is the top level layer and executes related actions according to

The structure of the data frame, which is sent by RF or USB, is composed of a header which contains the ID of the device, followed by 4 bytes, indicating the measurement time, and a

Fig. 6 shows the data frame enclosed information of the measurements taken; the first two bytes give information about the frequency of sampling and the next byte gives the number of sensors measured. According to this last parameter, the rest of bytes corresponding to each sensor in groups of two bytes due to the 10-bit conversion configuration of the A/D

One of the ways of overcoming the lack of data in ALS disease is to develop new easy-to-use testing devices, which can be left in the patient's own home and used to carry out periodic tests without having to go to hospital to do so. The comfortable testing processes and devices make the patients more willing to wear them outside the home, and this leads to a

Two of the more widely used tests with neurodegenerative disease patients are the Finger Tapping Test (FTT) (Jobbágy et al., 2005) and the Hand-grip Strength Test (Long, 1970). In the case of the FTT, the patient is asked to tap two of the fingers of one hand as quick as possible, and the main parameter measured by the doctors is the tapping frequency. On the other hand, in the hand grip strength test the measured parameter is the force the patient is

The Finger Tapping Test (FTT), originally developed as part of the Halstead Reitan Battery (HRB) of neuropsychological tests, is a simple measure of motor speed and motor control and is used in neuropsychology as a sensitive test for brain damage (Christianson & Leathem, 2004). Although motor functioning in humans is controlled by many areas of the

received interruptions (external switches or internal interruptions).

also developed in this layer.

byte which indicates the length of the data.

**4.4 Data frame** 

converter.

Fig. 6. Data frame

**5. Device test in ALS disease** 

wider amount of data available for the doctors.

capable of apply when grabbing an object.

brain, the motor strip rostral to the central sulcus is the most important, and the functioning of this area is reflected directly in the FTT. As well as direct motor effects, the speed, coordination, and pacing requirements of finger tapping can be affected by levels of alertness, impaired ability to focus attention, or slowing of responses. Tapping frequency can distinguish patients with motor dysfunctions of cerebella, basal ganglia, and cerebral origins from normal subjects.

At the onset of ALS the symptoms may be so slight that they are frequently overlooked. With regard to the appearance of symptoms and the progression of the illness, the course of the disease may include muscle weakness. Muscle weakness is a hallmark initial sign in ALS, occurring in approximately 60% of patients. The hands and feet may be affected first, causing difficulty in lifting, walking or using the hands for the activities of daily living such as dressing, washing and buttoning clothes.

ALS is a very difficult disease to diagnose. To date, there is no one test or procedure to ultimately establish the diagnosis of ALS. Methods for the evaluation of strength in people with ALS include a clinical neurological exam, manual muscle testing (MMT) (Aitkens et al., 1989), and rating scales. These methods are subjective and lack sensitivity to detect small changes. The purpose of the Hand Grip Strength Test is to measure the maximum isometric strength of the hand and forearm muscles.

The devices and methods used so far for the proposed tests have not had any significant improvement or innovation for many years. Traditional ways of performing the tests are still used.

For the finger tapping test, several methods have been proposed and used. The standard method consists of asking the patients to start with the finger tapping process and an examiner using a stopwatch to keep track of the 10-second trial interval. Electronic devices which are based on the same testing methodology have been marketed. The electronic device has an internal timer that starts on the first tap and stops counting taps when the 10 seconds have elapsed. The use of automatic timing is intended to increase the accuracy of testing (McDermid, 2000).

Other devices used, which can be found in the literature, include precision image-based motion analyzer and passive marker-based movement analyzer (Jobbágy et al., 2005); the Halstead-Reitan finger tapping test (HRFTT), developed and manufactured by Reitan Neuropsychological Laboratory, which uses an electronic counter and a tapping key; finger tapping devices containing pressure sensors (Soichiro et al., 2004); systems consisting of accelerometers and touch sensor (Yokoe et a., 2009) (Okuno et al., 2007).

In the case of the hand-grip strength measurement, the innovations carried out in recent years have been even poorer. The most usual way to carry out this specific test is by using hand-grip and pinch-force dynamometers, which offer very poor information about the way the hand grabs objects. Electromyography has been also used in some studies (Long, 1970).

In this work we have used our wireless device to carry out both of the tests. It is integrated in a system consisting of the mentioned device, a sensorized glove (see Fig. 7) which is worn by the patient, and a PC or base station, which is in charge of receiving the data sent by the device, and visualizing them graphically in order to be analyzed by the doctors.

As mentioned before, the sensors used in this test have been Force Sensitive Resistors (FSR). The approach followed in this research work has been the one of attaching several sensors to a glove. This design allows complying with one of the key characteristics identified in the system architecture section: versatility. We consider that this design is more versatile in order to allow carrying out different type of tests and obtaining a wide range of results.

Neurodegenerative Disease Monitoring Using a Portable Wireless Sensor Device 551

• **Start/Stop**: this button allows the exact moment in which the test starts and ends to be controlled. When the test starts, a new process is created in the application, which is constantly controlling the USB communications with the device, and passing the

• **Zoom**: the zooming tool enables the signals corresponding to the force applied by the patient's each of the fingers to be visualised more accurately. It is also possible to analyze only one finger in the application or to compare with other tests carried out

• **Log**: the application enables a registry or log with the messages corresponding to the events that appear during the testing process (communication states) to be visualised. • **Files**: the application allows the data in files with ".csv" format to be saved, in order to edit and analyze later in a PC program such as Excel. Also, in the new version of the program, it is possible to save in a Matlab binary format, as some clinicians have

• **Options**: in this option, the user can configure the device, by changing the sample frequency, the date of the device in order to maintain well synchronized, etc.

Fig. 9 shows a screen capture of the PC application, where a hand-grip force test is being carried out. As it can be seen in that figure, the force signal corresponding to each of the fingers is plotted using a different colour. That way the analysis of the graph is easier for the clinicians, where they can see for example that the patient has more force with one finger.

gathered data to the GUI window.

experience with that mathematical tool.

Fig. 9. PC application showing hand-grip force test results

previously.

**5.2 Hand-grip results** 

Fig. 7. Sensorized Glove attached to the wireless device

#### **5.1 PC application**

In order to gather the data and to be analyzed, a PC application was designed. It has been developed in Visual C++ using the Object Oriented Programming methodology (OOP), which is based in classes. The architecture is shown in Fig. 8. There are five blocks; the most important ones are the USB process and the graphical routines.

Fig. 8. PC application diagram

The data obtained by the hardware device after gathering and processing the signals coming from the FSR sensor, are sent via the USB connection to a PC, where an application is running. This application receives the data and visualizes and stores them.

Due the fact that the data rate of the device is low (less than 1KBps), the HID protocol has been implemented in the Sensor Device, providing the PC application an easier method of gathering the data, because most operating systems recognize standard USB HID devices, like keyboards and mice, without needing a special driver. In this way, the software can run in any compatible PC with Windows XP Operating System installed.

The application has some functionality that makes it easier for the doctors to analyze the data gathered by the hardware device. These functionalities are:


#### **5.2 Hand-grip results**

550 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

In order to gather the data and to be analyzed, a PC application was designed. It has been developed in Visual C++ using the Object Oriented Programming methodology (OOP), which is based in classes. The architecture is shown in Fig. 8. There are five blocks; the most

The data obtained by the hardware device after gathering and processing the signals coming from the FSR sensor, are sent via the USB connection to a PC, where an application is

Due the fact that the data rate of the device is low (less than 1KBps), the HID protocol has been implemented in the Sensor Device, providing the PC application an easier method of gathering the data, because most operating systems recognize standard USB HID devices, like keyboards and mice, without needing a special driver. In this way, the software can run

The application has some functionality that makes it easier for the doctors to analyze the

**Log Window**

**USB Process**

Fig. 7. Sensorized Glove attached to the wireless device

important ones are the USB process and the graphical routines.

**Graph Window**

running. This application receives the data and visualizes and stores them.

in any compatible PC with Windows XP Operating System installed.

data gathered by the hardware device. These functionalities are:

**Application**

**5.1 PC application** 

**Data Disk**

Fig. 8. PC application diagram

Fig. 9 shows a screen capture of the PC application, where a hand-grip force test is being carried out. As it can be seen in that figure, the force signal corresponding to each of the fingers is plotted using a different colour. That way the analysis of the graph is easier for the clinicians, where they can see for example that the patient has more force with one finger.

Fig. 9. PC application showing hand-grip force test results

Neurodegenerative Disease Monitoring Using a Portable Wireless Sensor Device 553

Recent advances on gait analysis of PD patients include portable digital monitoring systems. These systems allow gathering data by the patient themselves, wearing sensors at home and outside home. The developments performed to date are based on tiny electronic circuits which gather and transmit data coming from sensors, mainly accelerometers (Kauw-A-Tjoe

Combining the advantages of both approaches used till date (sensorized ground platforms and portable monitoring devices) a gait monitoring system has been developed, using our wireless sensor device. For the approach presented in this test, Force Sensitive Resistors (FSR) sensors have also been selected. Regarding the location of the sensors on the insole, several medical considerations have to be taken into account. As shown in Fig. 11, the most interesting zones to place the sensors are three: the plantar area, the heel and one in the middle. These zones are the ones in which most of the force is applied and, thus, the zones

et al., 2007).

from which more information can be obtained.

Fig. 11. Insole with the FSR sensors and wireless sensor device

each of them carried out three repetitions, in order to avoid random results.

The test methodology carried out consists of several tests performed on patients affected by PD and on healthy individuals. Two people from each group participated in the tests, and

**6.1 Gait analysis results** 

#### **5.3 Finger tapping results**

The main innovation of this system is that it can measure both the frequency of the tapping and the force the patient applies when carrying out the test. Fig. 10 shows a screen capture of the PC application used to visualize the results in real-time, in which an ongoing finger tapping test can be seen.

Fig. 10. PC application showing Finger-Tapping test results

Another key point of this finger-tapping test system compared to the existing ones is that the test can be performed using any of the five fingers of the hand. That way, two kinds of finger tapping tests can be carried out: one in which the fingers the patient uses most are involved in the testing process, and another one in which the patient uses the fingers that he or she is less likely to use.

#### **6. Device's test in gait analysis**

One of the ways of measuring and quantifying the movement disorders is performing gait analysis. Although several techniques and methods have been developed and used for years, all of them are based on hospitalizing patients and using in-hospital equipment.

Several interviews and meetings held with experts in neurology show that the most common way to carry out the gait analysis is by using sensorized ground platforms, as well as video cameras, in order to capture movement, where the two main disadvantages of these methods are the limited, and short period of time over which the patient can be monitored; and the fact of the monitoring process being carried out in a controlled environment, in which the patient may feel safe.

The main innovation of this system is that it can measure both the frequency of the tapping and the force the patient applies when carrying out the test. Fig. 10 shows a screen capture of the PC application used to visualize the results in real-time, in which an ongoing finger

Another key point of this finger-tapping test system compared to the existing ones is that the test can be performed using any of the five fingers of the hand. That way, two kinds of finger tapping tests can be carried out: one in which the fingers the patient uses most are involved in the testing process, and another one in which the patient uses the fingers that he

One of the ways of measuring and quantifying the movement disorders is performing gait analysis. Although several techniques and methods have been developed and used for years, all of them are based on hospitalizing patients and using in-hospital equipment. Several interviews and meetings held with experts in neurology show that the most common way to carry out the gait analysis is by using sensorized ground platforms, as well as video cameras, in order to capture movement, where the two main disadvantages of these methods are the limited, and short period of time over which the patient can be monitored; and the fact of the monitoring process being carried out in a controlled

**5.3 Finger tapping results** 

tapping test can be seen.

or she is less likely to use.

**6. Device's test in gait analysis** 

environment, in which the patient may feel safe.

Fig. 10. PC application showing Finger-Tapping test results

Recent advances on gait analysis of PD patients include portable digital monitoring systems. These systems allow gathering data by the patient themselves, wearing sensors at home and outside home. The developments performed to date are based on tiny electronic circuits which gather and transmit data coming from sensors, mainly accelerometers (Kauw-A-Tjoe et al., 2007).

Combining the advantages of both approaches used till date (sensorized ground platforms and portable monitoring devices) a gait monitoring system has been developed, using our wireless sensor device. For the approach presented in this test, Force Sensitive Resistors (FSR) sensors have also been selected. Regarding the location of the sensors on the insole, several medical considerations have to be taken into account. As shown in Fig. 11, the most interesting zones to place the sensors are three: the plantar area, the heel and one in the middle. These zones are the ones in which most of the force is applied and, thus, the zones from which more information can be obtained.

Fig. 11. Insole with the FSR sensors and wireless sensor device

#### **6.1 Gait analysis results**

The test methodology carried out consists of several tests performed on patients affected by PD and on healthy individuals. Two people from each group participated in the tests, and each of them carried out three repetitions, in order to avoid random results.

Neurodegenerative Disease Monitoring Using a Portable Wireless Sensor Device 555

Fig. 13. Signal of a Parkinsonian patient

Fig. 14. FFT Signal of a Parkinsonian patient

The parameters to be measured are the amplitude of the signal of each sensor (i.e., the force of the step) and the frequency of the signal, which gives an idea of the cadence of the gait. Table 1 shows the results obtained, where it can be seen that parkinsonian people has more frequency in their steps than healthy people. Fig. 11 shows the results for 2 sensors, gathered on the gait of a healthy person. The signal with the greater amplitude corresponds to a sensor located in the heel and the other one to the plantar area.


Table 1. Results of the test in patients

A delay can be noted between the two signal in Fig. 12. This is due to the nature of the step in a normal gait. Another difference lies in the amplitude of the signals and this is because most of the weight rests on the heel. On the other hand, Fig. 13 shows the analogous results for a Parkinsonian individual.

Fig. 12. Signal of a non-Parkinsonian individual over a temporal axis

Fig. 13. Signal of a Parkinsonian patient

The parameters to be measured are the amplitude of the signal of each sensor (i.e., the force of the step) and the frequency of the signal, which gives an idea of the cadence of the gait. Table 1 shows the results obtained, where it can be seen that parkinsonian people has more frequency in their steps than healthy people. Fig. 11 shows the results for 2 sensors, gathered on the gait of a healthy person. The signal with the greater amplitude corresponds

Results

1 2.64 0.88

2 2.42 0.82

1 1.64 1.76

2 1.76 1.85

A delay can be noted between the two signal in Fig. 12. This is due to the nature of the step in a normal gait. Another difference lies in the amplitude of the signals and this is because most of the weight rests on the heel. On the other hand, Fig. 13 shows the analogous results

Amplitude(V) Central frequency (Hz.)

to a sensor located in the heel and the other one to the plantar area.

Fig. 12. Signal of a non-Parkinsonian individual over a temporal axis

Samples

Non-Parkinsonian

Table 1. Results of the test in patients

Parkinsonian

for a Parkinsonian individual.

Fig. 14. FFT Signal of a Parkinsonian patient

Neurodegenerative Disease Monitoring Using a Portable Wireless Sensor Device 557

Aitkens, S., Lord, J., Bernauer, E., Fowler, W.M. Jr, Lieberman, J.S., Berck, P. (1989).

Christianson, M. K., Leathem, J. M. (2004). Development and Standardisation of the

Jobbágy Á., Harcos, P., Karoly, R., & Fazekas, G. (2005). Analysis of finger-tapping

Kauw-A-Tjoe, R.G., Thalen, J., Marin-Perianu, M., & Havinga, P. (2007). SensorShoe: Mobile

Konitsiotis, S. (2005). Novel pharmacological strategies for motor complications in

Long, C. (1970). Intrinsic-extrinsic muscle control of hand in power grip and precision

McDermid, R. (2000). A comparison of alternative devices of the finger tapping test. *Archives* 

Okuno, R., Yokoe, M., Fukawa, K., Sakoda, S., & Akazawa, K. (2007). Measurement system

Soichiro, M., Hisayoshi, O., Akira, K., Hironori, S., & Ko, K. (2004). Quantitative analysis of

Vecchi, F., Freschi, C., Micera, S., Sabatini, A. M., & Dario, P. (2000). Experimental

Von Campenhausen, S., Bornschein, B., Wick, R., Bötzel, K., Sampaio, C., Poewe, W., Oertel,

Yick, J., Mukherjee, B., & Ghosal, D. Wireless sensor network survey. *Computer Networks (Elsevier).* Vol. 52, No. 12, (August 2008), pp. (2292-2330), ISSN 1389-1286.

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wireless sensor networks: A survey. *Ad Hoc Networks.* Vol. 7, No. 3, (May 2009), pp.

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handling - an electromyographic study. *Journal of bone and joint surgery - American* 

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cerebellar ataxia with a finger tapping device containing a pressure sensor.

evaluation of two commercial force sensors for applications in biomechanics and motor control, *Proceedings of the 5th IFESS Annual Conference*, ISBN 4-9980783-1-3,

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**9. References** 

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Aalborg (Denmark), June 2000.

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As Fig. 13 shows, there is no delay between signals which suggests that this is due to the typical short steps of a Pakinsonian patient. Two more interesting conclusions are that the amplitude of these signals is lower than in the previous case, and the frequency is greater, around twice as much. This can be seen in Fig. 14, where the FFT of the Parkinsonian patient's signal is plotted.

#### **7. Conclusion**

The design of a tiny wireless sensor node platform has been carried out in this work. This device is mainly featured with its multifunctional functionality which has been proven in this paper on e-Health applications, specifically on tests related to patients affected by neurodegenerative diseases.

The presented work is based on the development of two specific tests for the treatment and analysis of Parkinson Disease (PD) and Amyotrophic Lateral Sclerosis (ALS). For each, the device has a different sensorized platform according to the nature of the performed test. Collected data from sensors can be either transmitted online through RF or downloaded via USB to a PC, or just stored in a card memory for a further download and analysis of data. A sensorized glove allows two tests to be carried out, mostly used on ALS patients; those are the hand-grip and the finger tapping tests. In the same way an insole with sensors located strategically is used to carry out a gait analysis which is one of the ways of measuring the movement disorders in parkinsonian people.

Results from both kinds of tests can be visualized and analyzed with the PC application developed in this work which also proves the versatility of the whole designed system. This application provides useful tools for the analysis of results; it was designed taking into account clinicians feedback as part of the work under the scope of the PERFORM project, acting as an interface between the clinician and the system.

The obtained results show and prove the viability and value of the multifunctional characteristics of the designed device. Additionally, by using the several tools provided by the PC application tools, important parameters can be obtained such as the frequency of a signal through the implemented FFT calculation function, the correlation among sensor signals in terms of phase and magnitude, the customization in the selection of specific signals and the zoom tool for a better appreciation of data.

These functionalities of the PC application allow clinician to obtain valuable conclusions like the stability of the gait (from the harmonics of the signals), the relation between air and ground time of the step (in PD analysis), the finger tapping frequency, the relation between the force applied by the different fingers, or the recording of the periods of time in which the patient is in "on" or "off" state.

Future work, which remains to be done is to focus on the accelerometer not mounted in this work. This component will provide relevant information for the gait analysis mainly helping to determine orientation and acceleration parameters of the patient.

#### **8. Acknowledgement**

This work is partly funded by the ICT programme of the European Commission (PERFORM Project: FP7-ICT-2007-1-215952)

#### **9. References**

556 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

As Fig. 13 shows, there is no delay between signals which suggests that this is due to the typical short steps of a Pakinsonian patient. Two more interesting conclusions are that the amplitude of these signals is lower than in the previous case, and the frequency is greater, around twice as much. This can be seen in Fig. 14, where the FFT of the Parkinsonian

The design of a tiny wireless sensor node platform has been carried out in this work. This device is mainly featured with its multifunctional functionality which has been proven in this paper on e-Health applications, specifically on tests related to patients affected by

The presented work is based on the development of two specific tests for the treatment and analysis of Parkinson Disease (PD) and Amyotrophic Lateral Sclerosis (ALS). For each, the device has a different sensorized platform according to the nature of the performed test. Collected data from sensors can be either transmitted online through RF or downloaded via USB to a PC, or just stored in a card memory for a further download and analysis of data. A sensorized glove allows two tests to be carried out, mostly used on ALS patients; those are the hand-grip and the finger tapping tests. In the same way an insole with sensors located strategically is used to carry out a gait analysis which is one of the ways of measuring the

Results from both kinds of tests can be visualized and analyzed with the PC application developed in this work which also proves the versatility of the whole designed system. This application provides useful tools for the analysis of results; it was designed taking into account clinicians feedback as part of the work under the scope of the PERFORM project,

The obtained results show and prove the viability and value of the multifunctional characteristics of the designed device. Additionally, by using the several tools provided by the PC application tools, important parameters can be obtained such as the frequency of a signal through the implemented FFT calculation function, the correlation among sensor signals in terms of phase and magnitude, the customization in the selection of specific

These functionalities of the PC application allow clinician to obtain valuable conclusions like the stability of the gait (from the harmonics of the signals), the relation between air and ground time of the step (in PD analysis), the finger tapping frequency, the relation between the force applied by the different fingers, or the recording of the periods of time in which the

Future work, which remains to be done is to focus on the accelerometer not mounted in this work. This component will provide relevant information for the gait analysis mainly

This work is partly funded by the ICT programme of the European Commission (PERFORM

helping to determine orientation and acceleration parameters of the patient.

patient's signal is plotted.

neurodegenerative diseases.

patient is in "on" or "off" state.

**8. Acknowledgement** 

Project: FP7-ICT-2007-1-215952)

movement disorders in parkinsonian people.

acting as an interface between the clinician and the system.

signals and the zoom tool for a better appreciation of data.

**7. Conclusion** 


Yokoe, M., Okuno, R., Hamasakib, T., Kurachic, Y., Akazawaf, K., & Sakoda, S. (2009). Opening velocity, a novel parameter, for finger tapping test in patients with Parkinson's disease. *Parkinsonism Related Disorders*, Vol. 15, No. 6, (July 2009), pp. (440-444), ISSN 1353-8020

Yokoe, M., Okuno, R., Hamasakib, T., Kurachic, Y., Akazawaf, K., & Sakoda, S. (2009).

(440-444), ISSN 1353-8020

Opening velocity, a novel parameter, for finger tapping test in patients with Parkinson's disease. *Parkinsonism Related Disorders*, Vol. 15, No. 6, (July 2009), pp.

### *Edited by Raymond Chuen-Chung Chang*

Neurodegenerative Diseases - Processes, Prevention, Protection and Monitoring focuses on biological mechanisms, prevention, neuroprotection and even monitoring of disease progression. This book emphasizes the general biological processes of neurodegeneration in different neurodegenerative diseases. Although the primary etiology for different neurodegenerative diseases is different, there is a high level of similarity in the disease processes. The first three sections introduce how toxic proteins, intracellular calcium and oxidative stress affect different biological signaling pathways or molecular machineries to inform neurons to undergo degeneration. A section discusses how neighboring glial cells modulate or promote neurodegeneration. In the next section an evaluation is given of how hormonal and metabolic control modulate disease progression, which is followed by a section exploring some preventive methods using natural products and new pharmacological targets. We also explore how medical devices facilitate patient monitoring. This book is suitable for different readers: college students can use it as a textbook; researchers in academic institutions and pharmaceutical companies can take it as updated research information; health care professionals can take it as a reference book, even patients' families, relatives and friends can take it as a good basis to understand neurodegenerative diseases.

> ISBN 978-953-307-485-6 ISBN 978-953-51-4391-8

Neurodegenerative Diseases - Processes, Prevention, Protection and Monitoring

Neurodegenerative Diseases

Processes, Prevention, Protection

and Monitoring

*Edited by Raymond Chuen-Chung Chang*

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