**Oxidative Changes and Possible Effects of Polymorphism of Antioxidant Enzymes in Neurodegenerative Disease**

Eva Babusikova, Andrea Evinova, Jozef Hatok, Dusan Dobrota and Jana Jurecekova

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54619

#### **1. Introduction**

[61] Velasques B, Machado S, Portella CE, Silva JG, Basile LF, Cagy M, et al. Electrophy‐ siological analysis of a sensorimotor integration task. Neurosci Lett. 2007 Oct;426(3):

[62] Wittstock M. [Neurophysiological analysis of interhemispheric motor tracts in neuro‐ degenerative diseases]. Fortschr Neurol Psychiatr. 2009 Aug;77 Suppl 1:S42-4.

[63] Braak H, Ghebremedhin E, Rüb U, Bratzke H, Del Tredici K. Stages in the develop‐ ment of Parkinson's disease-related pathology. Cell Tissue Res. 2004 Oct;318(1):

[64] Nojszewska M, Pilczuk B, Zakrzewska-Pniewska B, Rowińska-Marcińska K. The au‐ ditory system involvement in Parkinson disease: electrophysiological and neuropsy‐

[65] Kamei S. [Electroencephalogram and event-related potential analyses in Parkinson

[66] Beste C, Saft C, Andrich J, Gold R, Falkenstein M. Response inhibition in Hunting‐ ton's disease-a study using ERPs and sLORETA. Neuropsychologia. 2008 Apr;46(5):

[67] Orth M, Schippling S, Schneider SA, Bhatia KP, Talelli P, Tabrizi SJ, et al. Abnormal motor cortex plasticity in premanifest and very early manifest Huntington disease. J

[68] Höhn S, Dallérac G, Faure A, Urbach YK, Nguyen HP, Riess O, et al. Behavioral and in vivo electrophysiological evidence for presymptomatic alteration of prefrontos‐ triatal processing in the transgenic rat model for huntington disease. J Neurosci. 2011

[69] Cooper DB, Ales G, Lange C, Clement P. Atypical onset of symptoms in Huntington disease: severe cognitive decline preceding chorea or other motor manifestations.

[70] Ellenberger C, Petro DJ, Ziegler SB. The visually evoked potential in Huntington dis‐

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420 Neurodegenerative Diseases

121-34.

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Frequency of neurodegenerative diseases increase significantly with the age. In the present, average age is increasing and the number of people over 60 years increases as well. Ageing is a physiological process; however it seems to be linked with an increasing risk of origin and development of several diseases including neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. Exact mechanisms of ageing are still unclear but experimental evidences support a hypothesis that ageing changes are consequen‐ ces of increasing oxidative damage of organs, tissues, cells and all biomolecules. Oxidative damage is elevated when production of reactive oxygen species is increased compared to the physiological condition or a defence ability of organism against attacks of reactive oxygen species is decreased. Oxidation of specific proteins could play a key role in age associated damage. A relationship between protein aggregation, oxidative stress and neurodegeneration remains unclear although neurodegenerative diseases are connected with an origin of protein deposits. It assumes that protein oxidation and generation of protein aggregates generate a base for a loss of cell function and a reduced ability aged organisms to resist to physiological stress. Accumulation of modified proteins, disturbance of ion homeostasis, lipid and DNA modifications, and impairment of energy production are some of the crucial mechanisms linking ageing to neurodegeneration. In addition mitochondrial dysfunction plays a key role in neurology. Damage of mitochondrial electron transport may be an important factor in the pathogenesis of neurodegenerative diseases, such as Alzheimer's, Parkinson's, and Hunting‐ ton's diseases.

© 2013 Babusikova et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Oxygen is vital for all aerobic organisms and reactive oxygen species (ROS) are formed in cells as a consequence of aerobic metabolism. Moreover mitochondrial respiration is associated with inevitable electron leak, resulting in a non-stop production of reactive oxygen species, such as superoxide anion radical, hydrogen peroxide and hydrogen radical. Universal nature of reactive oxygen species is underlined by the presence of superoxide dismutase in all aerobic organisms. Genes involved in detoxification of reactive oxygen species are highly conserved among eukaryotes and their deficiency could be limit of several diseases and life span. Oxidative stress is a unique pathophysiological condition resulting from the disrupted balance between oxidants and antioxidants. Increased level of reactive oxygen species may cause oxidative damage of all four biomolecules: nucleic acids, proteins, lipids, saccharides. A progressive grow of oxidative damage is the result of increasing production of reactive oxygen species and this damage may contribute to the origin and development of several diseases including neurodegenerative diseases, but on the other hand oxidative damage can be the consequence of them as well (fig. 1). Cells possess defence systems: enzymatic and nonenzymatic against ROS. The most important enzymatic antioxidants are: superoxide dismu‐ tase, catalase and glutathione peroxidase but many others enzymes have antioxidant potential.

generate in cells under normal physiological conditions in different metabolic pathways and

Oxidative Changes and Possible Effects of Polymorphism of Antioxidant Enzymes in Neurodegenerative Disease

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423

**Figure 2. The main endogenous sources of reactive oxygen species (ROS) in cell.** Organelles such as mitochon‐ dria, peroxisomes, phagocytes and endoplasmic reticulum and enzymes produce ROS during a pursuance their physio‐

Four from the endogenous sources (mitochondria, phagocytes, peroxisomes, and cyto‐ chrome P450 enzymes) are responsible for origin of the majority of oxidants produced by cells [5]. The main endogenous sources of reactive oxygen species are mitochondria which produce reactive oxygen species continuously. Mitochondria serve mainly as pro‐ ducers of energy. In normal aerobic respiration mitochondria utilize oxygen that is re‐ duced by serial steps whereby is produced water. Mitochondria are the major producer of reactive oxygen species via incomplete reduction of oxygen by electrons leaked out of the respiratory chain in the animal and human cells. Mitochondrial oxidative damage can lead to the release of greater amount of reactive oxygen species and cause increased oxi‐ dative damage of mitochondrial, cytoplasmic and nuclear components what subsequently may lead to dysfunctional mitochondria. Damage of mitochondrial electron transport

Phagocyting cells are another important endogenous source of oxidants. The main function of phagocytosis is the defence of host organisms against pathogens. Neutrophils and another

oxide (•NO), hydrogen peroxide (H2O2), hypochlorous acid (HClO) [129]. Chronic virus, bacterial or parasite infection results in chronic increased phagocyting activity and finally chronic inflammation, which is a main risk factor for development of several diseases [5], and

•-), nitric

phagocytes attack pathogens by mixture of reactive oxygen species: singlet oxygen (O2

logic roles in cell and during metabolism of several components.

raising oxidative damage.

may be an important factor in the pathogenesis of many diseases.

cell compartments (fig. 2).

**Figure 1. Reactive oxygen species in the development of disease.** As a consequence of imbalance between reac‐ tive oxygen species (ROS) and antioxidant mechanisms (AOM) on the side of reactive oxygen species, oxidative stress is increasing. Increased level of reactive oxygen species causes increased oxidative damage of biomolecules, an accu‐ mulation of damage, and the development of many diseases.

### **2. Oxidative damage**

Reactive oxygen species (ROS) are necessary for human life. The main characteristics of ROS are their high effectiveness in a small concentration and their extremely highly reactivity. ROS are oxidants which can be produced endogenously and exogenously. Potential harmful ROS generate in cells under normal physiological conditions in different metabolic pathways and cell compartments (fig. 2).

Oxygen is vital for all aerobic organisms and reactive oxygen species (ROS) are formed in cells as a consequence of aerobic metabolism. Moreover mitochondrial respiration is associated with inevitable electron leak, resulting in a non-stop production of reactive oxygen species, such as superoxide anion radical, hydrogen peroxide and hydrogen radical. Universal nature of reactive oxygen species is underlined by the presence of superoxide dismutase in all aerobic organisms. Genes involved in detoxification of reactive oxygen species are highly conserved among eukaryotes and their deficiency could be limit of several diseases and life span. Oxidative stress is a unique pathophysiological condition resulting from the disrupted balance between oxidants and antioxidants. Increased level of reactive oxygen species may cause oxidative damage of all four biomolecules: nucleic acids, proteins, lipids, saccharides. A progressive grow of oxidative damage is the result of increasing production of reactive oxygen species and this damage may contribute to the origin and development of several diseases including neurodegenerative diseases, but on the other hand oxidative damage can be the consequence of them as well (fig. 1). Cells possess defence systems: enzymatic and nonenzymatic against ROS. The most important enzymatic antioxidants are: superoxide dismu‐ tase, catalase and glutathione peroxidase but many others enzymes have antioxidant potential.

**Figure 1. Reactive oxygen species in the development of disease.** As a consequence of imbalance between reac‐ tive oxygen species (ROS) and antioxidant mechanisms (AOM) on the side of reactive oxygen species, oxidative stress is increasing. Increased level of reactive oxygen species causes increased oxidative damage of biomolecules, an accu‐

Reactive oxygen species (ROS) are necessary for human life. The main characteristics of ROS are their high effectiveness in a small concentration and their extremely highly reactivity. ROS are oxidants which can be produced endogenously and exogenously. Potential harmful ROS

mulation of damage, and the development of many diseases.

**2. Oxidative damage**

422 Neurodegenerative Diseases

**Figure 2. The main endogenous sources of reactive oxygen species (ROS) in cell.** Organelles such as mitochon‐ dria, peroxisomes, phagocytes and endoplasmic reticulum and enzymes produce ROS during a pursuance their physio‐ logic roles in cell and during metabolism of several components.

Four from the endogenous sources (mitochondria, phagocytes, peroxisomes, and cyto‐ chrome P450 enzymes) are responsible for origin of the majority of oxidants produced by cells [5]. The main endogenous sources of reactive oxygen species are mitochondria which produce reactive oxygen species continuously. Mitochondria serve mainly as pro‐ ducers of energy. In normal aerobic respiration mitochondria utilize oxygen that is re‐ duced by serial steps whereby is produced water. Mitochondria are the major producer of reactive oxygen species via incomplete reduction of oxygen by electrons leaked out of the respiratory chain in the animal and human cells. Mitochondrial oxidative damage can lead to the release of greater amount of reactive oxygen species and cause increased oxi‐ dative damage of mitochondrial, cytoplasmic and nuclear components what subsequently may lead to dysfunctional mitochondria. Damage of mitochondrial electron transport may be an important factor in the pathogenesis of many diseases.

Phagocyting cells are another important endogenous source of oxidants. The main function of phagocytosis is the defence of host organisms against pathogens. Neutrophils and another phagocytes attack pathogens by mixture of reactive oxygen species: singlet oxygen (O2 •-), nitric oxide (•NO), hydrogen peroxide (H2O2), hypochlorous acid (HClO) [129]. Chronic virus, bacterial or parasite infection results in chronic increased phagocyting activity and finally chronic inflammation, which is a main risk factor for development of several diseases [5], and raising oxidative damage.

Peroxisomes are organelles from the microbody family and are present in almost all eu‐ karyotic cells. They participate in the β-oxidation of fatty acids and in the metabolism of many others metabolites. Certain enzymes within peroxisome, by using molecular oxy‐ gen, remove hydrogen atoms from specific organic substrates, in an oxidative reaction, producing hydrogen peroxide. Hydrogen peroxide is degraded by catalase, another en‐ zyme in peroxisome. Peroxisomes contain also xanthine oxidase which produces singlet oxygen and hydrogen peroxide.

**•** Brain has a high content of unsaturated fatty acid (especially 20:4 and 22:6 fatty acids).

**•** In the brain is high concentration of iron and ascorbate (key elements responsible for

Oxidative Changes and Possible Effects of Polymorphism of Antioxidant Enzymes in Neurodegenerative Disease

http://dx.doi.org/10.5772/54619

425

**Figure 3. Markers of oxidative damage.** Proteins, lipids and DNA are main targets of reactive oxygen species (ROS). These molecules generate a spectrum of molecules in the condition of oxidative stress which may be estimated for example in plasma, serum, bronchoalveolar lavage fluid, tissues and in exhalated breath condensate and reflect a dan‐

Exclusive using of glucose as a source of energy by brain is responsible for high oxygen concentrations which are necessary for normal brain function. A predominant quantity of reactive oxygen species (90-95%) is generated during aerobic metabolism as a by-product in an electron transport chain of mitochondria. Mitochondrial dysfunction plays a key role in neurology [8]. A decline in respiratory chain Complex I and Complex IV activity is associated with ageing [38]. Damage of mitochondrial electron transport may be an important factor in the pathogenesis of neurodegenerative. Increased oxidative stress in consequence of unpro‐ portional ROS production is considered a main feature in the pathogenesis of neurodegener‐ ative diseases [16, 141]. Apoptosis is an important mechanism of neuronal loss in age-related neurodegenerative diseases [38, 141]. Neuronal apoptosis in age-associated neurodegenera‐ tive disorders can be triggered by oxidative damage of proteins, lipids and DNA, metabolic compromise resulting from impaired glucose metabolism and mitochondrial dysfunction, and over activation of glutamate receptors resulting in disruption of neuronal calcium homeostasis. Several different kind of oxidative protein and lipid damages were observed in brain during ageing as well as increased generation of reactive oxygen species [59, 138, 144, 153, 154]. Increasing protein oxidation and lipid peroxidation can participate on the age-related brain

ger of ROS for a subject in consequence of origin and development of several diseases.

**•** Brain has high oxygen consumption (20% of using oxygen is consumed by brain).

membrane lipid peroxidation).

**•** Brain is to poor of antioxidants and defence mechanisms.

Microsomal cytochrome P450 enzymes are a very large and diverse superfamily of hemopro‐ teins identified from all lineages of life including humans, mammals, birds, fish, plants, bacteria. They form one of the primary defence system against xenobiotic compounds usually plant origin. Human cytochrome P450 enzymes are primarily membrane-associated proteins, located in the inner mitochondrial membrane or in the endoplasmic reticulum of cells. They modify thousands of endogenous and exogenous compounds by univalent oxidation or reduction. Induction of these enzymes protects before acute oxidative effects of foreign compounds or chemicals but also results in production of oxidants.

Although cells possess complex net of antioxidant defence, defence is not completely effective. Small fractions of pro-oxidants escape from elimination and cause molecular damage. Some of these damages are irreversible therefore they are accumulated in time and they make base of functional decline associated with age. Disruption of a balance between the level of reactive species generated during normal cellular metabolism and the level of endogenous antioxidant, either due to increased generation of ROS or decreased level of antioxidants, leads to oxidative damage and to several pathological conditions, including accelerated ageing and neurodege‐ nerative disorders.

Exact mechanisms of reasons for origin of some neurodegenerative diseases are still un‐ clear but experimental evidences support a hypothesis that ageing is a major risk and ageing changes are consequences of increasing oxidative damage. One of the basic prob‐ lems is the analysis of mechanisms that are base of damage. ROS are effective in a very small concentrations and their half-life is very short. We still do not have sensitive in‐ struments for measurement ROS (concentration and localisation) in living systems. Usual‐ ly effects of ROS are determined indirect, by several markers of oxidative damage (fig. 3). Both localisation and kind of damage are necessary for understanding of neurodegen‐ eration. Oxidative damage may by the most important contribution to ageing and age-re‐ lated diseases. Literature is full of controversy results. Oxidative modification of proteins, saccharides, nucleic acid (nuclear and mitochondrial) and lipid peroxidation were ob‐ served in different tissues, cells, compartments, including mitochondria with advancing of age. There is no detail information how the higher availability of reactive oxygen spe‐ cies could be translated to an accumulation of oxidized biomolecules so far.

An accumulation of oxidized proteins, disturbance of ion homeostasis, modifications of lipids, saccharides, proteins and nucleic acids, and impairment of energy production are some of the crucial mechanisms linking ageing to neurodegeneration. Brain is particularly sensitive to reactive oxygen species attack and to oxidative damage consequence of several factors:


Peroxisomes are organelles from the microbody family and are present in almost all eu‐ karyotic cells. They participate in the β-oxidation of fatty acids and in the metabolism of many others metabolites. Certain enzymes within peroxisome, by using molecular oxy‐ gen, remove hydrogen atoms from specific organic substrates, in an oxidative reaction, producing hydrogen peroxide. Hydrogen peroxide is degraded by catalase, another en‐ zyme in peroxisome. Peroxisomes contain also xanthine oxidase which produces singlet

Microsomal cytochrome P450 enzymes are a very large and diverse superfamily of hemopro‐ teins identified from all lineages of life including humans, mammals, birds, fish, plants, bacteria. They form one of the primary defence system against xenobiotic compounds usually plant origin. Human cytochrome P450 enzymes are primarily membrane-associated proteins, located in the inner mitochondrial membrane or in the endoplasmic reticulum of cells. They modify thousands of endogenous and exogenous compounds by univalent oxidation or reduction. Induction of these enzymes protects before acute oxidative effects of foreign

Although cells possess complex net of antioxidant defence, defence is not completely effective. Small fractions of pro-oxidants escape from elimination and cause molecular damage. Some of these damages are irreversible therefore they are accumulated in time and they make base of functional decline associated with age. Disruption of a balance between the level of reactive species generated during normal cellular metabolism and the level of endogenous antioxidant, either due to increased generation of ROS or decreased level of antioxidants, leads to oxidative damage and to several pathological conditions, including accelerated ageing and neurodege‐

Exact mechanisms of reasons for origin of some neurodegenerative diseases are still un‐ clear but experimental evidences support a hypothesis that ageing is a major risk and ageing changes are consequences of increasing oxidative damage. One of the basic prob‐ lems is the analysis of mechanisms that are base of damage. ROS are effective in a very small concentrations and their half-life is very short. We still do not have sensitive in‐ struments for measurement ROS (concentration and localisation) in living systems. Usual‐ ly effects of ROS are determined indirect, by several markers of oxidative damage (fig. 3). Both localisation and kind of damage are necessary for understanding of neurodegen‐ eration. Oxidative damage may by the most important contribution to ageing and age-re‐ lated diseases. Literature is full of controversy results. Oxidative modification of proteins, saccharides, nucleic acid (nuclear and mitochondrial) and lipid peroxidation were ob‐ served in different tissues, cells, compartments, including mitochondria with advancing of age. There is no detail information how the higher availability of reactive oxygen spe‐

cies could be translated to an accumulation of oxidized biomolecules so far.

An accumulation of oxidized proteins, disturbance of ion homeostasis, modifications of lipids, saccharides, proteins and nucleic acids, and impairment of energy production are some of the crucial mechanisms linking ageing to neurodegeneration. Brain is particularly sensitive to reactive oxygen species attack and to oxidative damage consequence of several factors:

compounds or chemicals but also results in production of oxidants.

oxygen and hydrogen peroxide.

424 Neurodegenerative Diseases

nerative disorders.

**Figure 3. Markers of oxidative damage.** Proteins, lipids and DNA are main targets of reactive oxygen species (ROS). These molecules generate a spectrum of molecules in the condition of oxidative stress which may be estimated for example in plasma, serum, bronchoalveolar lavage fluid, tissues and in exhalated breath condensate and reflect a dan‐ ger of ROS for a subject in consequence of origin and development of several diseases.

Exclusive using of glucose as a source of energy by brain is responsible for high oxygen concentrations which are necessary for normal brain function. A predominant quantity of reactive oxygen species (90-95%) is generated during aerobic metabolism as a by-product in an electron transport chain of mitochondria. Mitochondrial dysfunction plays a key role in neurology [8]. A decline in respiratory chain Complex I and Complex IV activity is associated with ageing [38]. Damage of mitochondrial electron transport may be an important factor in the pathogenesis of neurodegenerative. Increased oxidative stress in consequence of unpro‐ portional ROS production is considered a main feature in the pathogenesis of neurodegener‐ ative diseases [16, 141]. Apoptosis is an important mechanism of neuronal loss in age-related neurodegenerative diseases [38, 141]. Neuronal apoptosis in age-associated neurodegenera‐ tive disorders can be triggered by oxidative damage of proteins, lipids and DNA, metabolic compromise resulting from impaired glucose metabolism and mitochondrial dysfunction, and over activation of glutamate receptors resulting in disruption of neuronal calcium homeostasis. Several different kind of oxidative protein and lipid damages were observed in brain during ageing as well as increased generation of reactive oxygen species [59, 138, 144, 153, 154]. Increasing protein oxidation and lipid peroxidation can participate on the age-related brain cell dysfunction. There are many studies demonstrated elevated concentration of different ROS and decreased antioxidant status during ageing and in neurodegenerative disorders but majority of them are determined on animal models or cell lines. We are still limited in human studies especially in the case of neurodegenerative disorders; and ethnicity, environment and life style may be responsible for controversial results.

**•** an increased sensitivity of damaged proteins to become oxidized as a consequence of

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427

**•** a decreased levels or activities of the proteasome or proteases which degrade oxidized

Lipid peroxidation is an example of oxidative damage of cell membranes, lipoproteins and other lipid containing structures. This oxidative damage is degenerative process, which affects cellular membranes under conditions of oxidative stress [136]. Membranes are particularly sensitive to oxidative damage because they are rich on two or more carbon-carbon double bonds -C=C- [148]. During lipid peroxidation, polyunsaturated higher fatty acids are damaged in auto-catalytic, uncontrolled process which result is production of hydroperoxides of membrane lipids and wide spectrum of secondary metabolites including different aldehydes. Aldehyde products of lipoperoxidation may interact with mitochondrial membrane lipids and can change physiochemical state of membrane [150]. The major components of biological membranes are lipids and proteins. The amount of proteins is increasing with the number of functions that are on membranes performed. Lipid peroxidation can therefore cause damage of membrane proteins as well as lipids. Lipoproteins are also target of oxidative damage. Lipid hydroperoxides are primary products of lipid peroxidation. Dissociation of hydroperoxides

transcriptional and translational errors [1],

is important from toxicological point of view for two reasons:

**•** non-radical products are produced which can be also biologically active.

with thiol group of cysteine and with imidazol group of histidine [16].

It was found, that lipid peroxidation produces: unsaturated aldehydes, malondialdehyde, 4 hydroxy-2-nonenal (HNE) and other products that are cytotoxic and mutagenic and can damaged other biomolecules [91]. Malondialdehyde arises largely from peroxidation of polyunsaturated fatty acids with more than two double bonds and it can also arise enzymat‐ ically during eicosanoid metabolism. 4-hydroxy-2-nonenal has toxic property like cell growth inhibition, genotoxicity, chemotaxic activity and able ability to modify lipoproteins and promote atherosclerosis [51]. It is able react with nucleofil components largely with metabolites and proteins contained thiol groups [50]. Effect of HNE is depending on its concentration. Lipid peroxidation can be overly destructive process in living system. It damages biological membrane thereby are changed their biophysiological properties. Aldehyde products of lipoperoxidation reacted with mitochondria membrane lipids and can change physiochemical state of membrane [28]. Peroxidation of membrane phospholipids is accompanied by change structural and functional characteristics of membranes. Lipid peroxidation impacts also function of proteins that are component of biological membranes. Consequence of near physiological junction lipids and proteins can lead oxidative damage of mitochondrial proteins to form proteins cross linkage, protein degradation or to lose of their function [166]. Oxidative damage can be established into proteins with reaction with aldehydes arisen during lipid peroxidation. For example HNE or malondialdehyde can react with ε-amino group of lysine,

**•** new radicals are generated and ramify radical reactions,

proteins [24].

**2.2. Lipid peroxidation**

#### **2.1. Protein oxidation**

One of the important targets of oxidative damage can be proteins which play elementary roles such as biological accelerators, gene regulators, receptors, transport proteins and structural components of cells. Oxidative modification of proteins by reactive oxygen species or by other reactive molecules (e.g. products of lipid peroxidation) is implicated in aetiology or develop‐ ment of many diseases and it can also contribute to secondary damage of other molecules. Damage of DNA repair enzymes could raise levels of DNA oxidative damage and increase of mutation frequency. DNA polymerase damage might result in decreasing of fidelity in replicating DNA. Endogenous proteins are very sensitive to free radical modification as by byproducts of normal metabolic processes or after exposition to oxidative stress *in vivo* or *in vitro* [48, 64, 86]. ROS-associated protein modification can lead to loss of biological functions and to the change of protein forms. Modified proteins have increased sensitivity to intracellular proteolysis [176] and they are quickly degraded by endogenous proteases, particularly by multi-catalytic system [45]. Reactive oxygen species can react directly with proteins or they can react with molecules such as saccharides and lipids forming reactive products, which consecutively attack proteins. Reactions are often influenced by redox cycle of metal cations, particularly by iron and copper. Proteins can go through many covalent changes after exposing to oxidants [98]. Some of this alternations result from direct attacks of ROS on protein molecule; meanwhile another changes are produced indirectly [110]. Protein oxidation can lead to the amino acid side chain residues oxidation. All protein amino acid residues are sensitive to oxidative damage by hydroxyl radical that is generated by radiation but no all products generated during oxidation of some amino acid residues were absolutely characterised. However, tyrosine, phenylalanine, tryptophan, histidine, methionine and cysteine are preferred target of •OH attack [38]. In consequence of •OH attack of side chains in presence of oxygen can form hydroperoxides, alcohols and carbonyl compounds [39]. Proteins can contain after ROS attack new functional groups (hydroxyl and carbonyl groups) [22]. Protein oxidation can lead to the breaking of peptide bounds (formation of products with lower molecular weight) and to the formation of protein-protein cross-links (formation of products with higher molecular weight) and to the protein netting. These changes can result in different secondary effects including protein fragmentation, aggregation and unfolding. These processes are ordinarily connected with loss or change of protein activity and function [11, 102]. Increased oxidative damage of proteins result in:


#### **2.2. Lipid peroxidation**

cell dysfunction. There are many studies demonstrated elevated concentration of different ROS and decreased antioxidant status during ageing and in neurodegenerative disorders but majority of them are determined on animal models or cell lines. We are still limited in human studies especially in the case of neurodegenerative disorders; and ethnicity, environment and

One of the important targets of oxidative damage can be proteins which play elementary roles such as biological accelerators, gene regulators, receptors, transport proteins and structural components of cells. Oxidative modification of proteins by reactive oxygen species or by other reactive molecules (e.g. products of lipid peroxidation) is implicated in aetiology or develop‐ ment of many diseases and it can also contribute to secondary damage of other molecules. Damage of DNA repair enzymes could raise levels of DNA oxidative damage and increase of mutation frequency. DNA polymerase damage might result in decreasing of fidelity in replicating DNA. Endogenous proteins are very sensitive to free radical modification as by byproducts of normal metabolic processes or after exposition to oxidative stress *in vivo* or *in vitro* [48, 64, 86]. ROS-associated protein modification can lead to loss of biological functions and to the change of protein forms. Modified proteins have increased sensitivity to intracellular proteolysis [176] and they are quickly degraded by endogenous proteases, particularly by multi-catalytic system [45]. Reactive oxygen species can react directly with proteins or they can react with molecules such as saccharides and lipids forming reactive products, which consecutively attack proteins. Reactions are often influenced by redox cycle of metal cations, particularly by iron and copper. Proteins can go through many covalent changes after exposing to oxidants [98]. Some of this alternations result from direct attacks of ROS on protein molecule; meanwhile another changes are produced indirectly [110]. Protein oxidation can lead to the amino acid side chain residues oxidation. All protein amino acid residues are sensitive to oxidative damage by hydroxyl radical that is generated by radiation but no all products generated during oxidation of some amino acid residues were absolutely characterised. However, tyrosine, phenylalanine, tryptophan, histidine, methionine and cysteine are preferred target of •OH attack [38]. In consequence of •OH attack of side chains in presence of oxygen can form hydroperoxides, alcohols and carbonyl compounds [39]. Proteins can contain after ROS attack new functional groups (hydroxyl and carbonyl groups) [22]. Protein oxidation can lead to the breaking of peptide bounds (formation of products with lower molecular weight) and to the formation of protein-protein cross-links (formation of products with higher molecular weight) and to the protein netting. These changes can result in different secondary effects including protein fragmentation, aggregation and unfolding. These processes are ordinarily connected with loss or change of protein activity and function [11, 102]. Increased

life style may be responsible for controversial results.

**2.1. Protein oxidation**

426 Neurodegenerative Diseases

oxidative damage of proteins result in:

**•** an increased production of reactive oxygen species [77],

**•** a decreased capacity to scavenge reactive oxygen species,

Lipid peroxidation is an example of oxidative damage of cell membranes, lipoproteins and other lipid containing structures. This oxidative damage is degenerative process, which affects cellular membranes under conditions of oxidative stress [136]. Membranes are particularly sensitive to oxidative damage because they are rich on two or more carbon-carbon double bonds -C=C- [148]. During lipid peroxidation, polyunsaturated higher fatty acids are damaged in auto-catalytic, uncontrolled process which result is production of hydroperoxides of membrane lipids and wide spectrum of secondary metabolites including different aldehydes. Aldehyde products of lipoperoxidation may interact with mitochondrial membrane lipids and can change physiochemical state of membrane [150]. The major components of biological membranes are lipids and proteins. The amount of proteins is increasing with the number of functions that are on membranes performed. Lipid peroxidation can therefore cause damage of membrane proteins as well as lipids. Lipoproteins are also target of oxidative damage. Lipid hydroperoxides are primary products of lipid peroxidation. Dissociation of hydroperoxides is important from toxicological point of view for two reasons:


It was found, that lipid peroxidation produces: unsaturated aldehydes, malondialdehyde, 4 hydroxy-2-nonenal (HNE) and other products that are cytotoxic and mutagenic and can damaged other biomolecules [91]. Malondialdehyde arises largely from peroxidation of polyunsaturated fatty acids with more than two double bonds and it can also arise enzymat‐ ically during eicosanoid metabolism. 4-hydroxy-2-nonenal has toxic property like cell growth inhibition, genotoxicity, chemotaxic activity and able ability to modify lipoproteins and promote atherosclerosis [51]. It is able react with nucleofil components largely with metabolites and proteins contained thiol groups [50]. Effect of HNE is depending on its concentration. Lipid peroxidation can be overly destructive process in living system. It damages biological membrane thereby are changed their biophysiological properties. Aldehyde products of lipoperoxidation reacted with mitochondria membrane lipids and can change physiochemical state of membrane [28]. Peroxidation of membrane phospholipids is accompanied by change structural and functional characteristics of membranes. Lipid peroxidation impacts also function of proteins that are component of biological membranes. Consequence of near physiological junction lipids and proteins can lead oxidative damage of mitochondrial proteins to form proteins cross linkage, protein degradation or to lose of their function [166]. Oxidative damage can be established into proteins with reaction with aldehydes arisen during lipid peroxidation. For example HNE or malondialdehyde can react with ε-amino group of lysine, with thiol group of cysteine and with imidazol group of histidine [16].

#### **2.3. Oxidative damage of DNA**

Nucleic acids are particularly sensitive to oxidative damage. There is increasing evidence that ROS are involved in the development of cancer, not only by direct effects on DNA but also by affecting signal transduction, cell proliferation, cell death and intracellular communication. ROS can damage DNA by direct chemical attack of purine and pyrimidine bases and deoxy‐ ribose sugars and also by indirect mechanisms. There is a known more than 20 product of oxidative damage of the nucleic acids.

The most important enzymatic antioxidants are: superoxide dismutase, catalase and gluta‐ thione peroxidase. Superoxide dismutase (SOD, EC 1.15.1.1) is universal enzymatic antioxi‐ dant. This enzyme is extremely efficient and catalyses the neutralization of superoxide anion to oxygen and hydrogen peroxide. There are three major families of superoxide dismutase, depending on the metal cofactor: Cu/Zn (which binds both copper and zinc), Fe and Mn types (which bind either iron or manganese), and the Ni type, which binds nickel. In humans three form of SOD are present: cytoplasmic Cu/Zn-SOD (SOD1), mitochondrial Mn-SOD (SOD2), and extracellular Cu/Zn-SOD (ECSOD, SOD3). Catalase (CAT, EC 1.11.1.6) is a common antioxidant enzyme responsible for controlling hydrogen peroxide concentrations in cells. Catalase as an intracellular antioxidant enzyme catalyzes the decomposition of two molecules of hydrogen peroxide into one molecule of oxygen and two of water and its activity is genetically determined. Glutathione peroxidases (GPXs, EC 1.11.1.9) are family of enzymes ubiquitously distributed which have peroxidase activity whose main biological role is to protect the organism from oxidative damage. Glutathione peroxidases reduce hydrogen peroxide to water and reduced glutathione and lipid hydroperoxides to their corresponding alcohols, water and reduced glutathione. Four types of GPXs have been identified: cellular GPX, gastrointestinal GPX, extracellular GPX, and phospholipid hydroperoxide GPX [161]. Other an essential part of defence mechanism is a super-family of enzymes called glutathione transferases (GSTs, EC 2.5.1.18). These enzymes are involved in the cellular detoxification of various electrophilic xenobiotic substances such as chemical carcinogens, environmental pollutants, drugs and antitumor agents. These enzymes also inactivate endogenous α,βunsaturated aldehydes, quinone, epoxides, and hydroperoxides formed as secondary metab‐ olites during oxidative damage. GSTs may reduce reactive oxygen species to less reactive metabolites and protect organism against consequences of lipid peroxidation. Heme oxygen‐ ase (heat shock protein 32, HO; EC 1.14.99.3) plays an important role in organism defence to oxidative stress [114] and inflammation [111]. There are known three isoforms of HO: HO-1, HO-2, and HO-3. HO-1 is activated by a lot of inflammatory mediators, reactive oxygen species and by other stimuli [109, 135]. Upregulation of HO-1 is accepted as a sensitive marker of oxidative stress. Oxidative modified DNA can be repaired by several enzymes such as glycosylases: 8-oxoguanine-DNA-glysocylase (OGG1, EC 4.2.99.18), Nei-like protein 1 and 2 (NEIL, EC 4.2.99.18); Apurinic/apyrimidinic endonuclease 1 (APE 1, EC 4.2.99.18), X ray repair cross-complementing group 1 (XRCC 1, EC 4.2.99.18) and poly(ADP-ribose) polymerase-1 (PARP 1, EC 2.4.2.30). OGG1 removes 8-oxoguanine paired with a cytosine. Human *OGG1* gene consists of eight exons which can be alternatively spliced to produce different isoforms. The most abundant mRNAs of OGG1 are type 1a and 2a. These two isoforms are ubiquitously

Oxidative Changes and Possible Effects of Polymorphism of Antioxidant Enzymes in Neurodegenerative Disease

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429

Ageing is the main risk factor of neurodegenerative disorders. Approximately 5% of people in age 65 years have Alzheimer`s disease (AD) and the prevalence of this disease increases with increasing age from 19% to 30% after 75 years of age. Overall, 90-95% of Alzheimer`s

expressed in human tissues.

**4. Alzheimer`s disease**

Superoxide, nitric oxide or hydrogen peroxide at physiologically relevant levels do not react with any of DNA or RNA bases or with the ribose or deoxyribose sugars at significant rates. Particularly hydroxyl radical is known to cause chemical modifications of DNA through the formation of one strand and two strands breaks and cross linkages with other molecules. Different saccharides radicals of DNA can arise by abstraction of a hydrogen atom from 2`deoxyribose because all positions in saccharides are susceptible to oxidative damage. Hydroxyl radical reacts with aromatic rings therefore also nitrogen bases of nucleic acids are modified. C5-C6 double bonds of pyrimidines and carbon atoms C4, C5, and C8 of purines are the most sensitive position to oxidative effect of hydroxyl radical and hydroxyl radical abstract electron and no hydrogen atom [169]. In consequence of ROS attack to nuclear proteins are generated protein radicals and radicals of base react under formation of DNA-protein crosslinking.

Mitochondrial DNA (mtDNA) is excessively sensitive on an oxidative damage because mtDNA is situated near inner mitochondrial membrane, where are formed ROS. Mitochon‐ drial DNA is small and is not protected by histones like nuclear DNA. Mitochondria are able repair an oxidative damage of mtDNA and that base excise repair pathway plays a dominant role in mtDNA repair [36]. Damage of mtDNA can be potentially more important like damage of nuclear DNA because all mitochondrial genome code genes which are expressed whereas nuclear DNA includes great number of untranscribed sequences [166]. Linnane and coworkers [101] assume that accumulation of somatic mutations mtDNA is the main origin of human ageing and degenerative diseases.

### **3. Antioxidant defence**

In consequence, imbalance between pro-oxidant and antioxidant in favour of pro-oxidants and their harmful effects, oxidative stress is increased. Cells possess antioxidant defence systems: enzymatic and non-enzymatic [15]. Antioxidants can work at various levels:


The most important enzymatic antioxidants are: superoxide dismutase, catalase and gluta‐ thione peroxidase. Superoxide dismutase (SOD, EC 1.15.1.1) is universal enzymatic antioxi‐ dant. This enzyme is extremely efficient and catalyses the neutralization of superoxide anion to oxygen and hydrogen peroxide. There are three major families of superoxide dismutase, depending on the metal cofactor: Cu/Zn (which binds both copper and zinc), Fe and Mn types (which bind either iron or manganese), and the Ni type, which binds nickel. In humans three form of SOD are present: cytoplasmic Cu/Zn-SOD (SOD1), mitochondrial Mn-SOD (SOD2), and extracellular Cu/Zn-SOD (ECSOD, SOD3). Catalase (CAT, EC 1.11.1.6) is a common antioxidant enzyme responsible for controlling hydrogen peroxide concentrations in cells. Catalase as an intracellular antioxidant enzyme catalyzes the decomposition of two molecules of hydrogen peroxide into one molecule of oxygen and two of water and its activity is genetically determined. Glutathione peroxidases (GPXs, EC 1.11.1.9) are family of enzymes ubiquitously distributed which have peroxidase activity whose main biological role is to protect the organism from oxidative damage. Glutathione peroxidases reduce hydrogen peroxide to water and reduced glutathione and lipid hydroperoxides to their corresponding alcohols, water and reduced glutathione. Four types of GPXs have been identified: cellular GPX, gastrointestinal GPX, extracellular GPX, and phospholipid hydroperoxide GPX [161]. Other an essential part of defence mechanism is a super-family of enzymes called glutathione transferases (GSTs, EC 2.5.1.18). These enzymes are involved in the cellular detoxification of various electrophilic xenobiotic substances such as chemical carcinogens, environmental pollutants, drugs and antitumor agents. These enzymes also inactivate endogenous α,βunsaturated aldehydes, quinone, epoxides, and hydroperoxides formed as secondary metab‐ olites during oxidative damage. GSTs may reduce reactive oxygen species to less reactive metabolites and protect organism against consequences of lipid peroxidation. Heme oxygen‐ ase (heat shock protein 32, HO; EC 1.14.99.3) plays an important role in organism defence to oxidative stress [114] and inflammation [111]. There are known three isoforms of HO: HO-1, HO-2, and HO-3. HO-1 is activated by a lot of inflammatory mediators, reactive oxygen species and by other stimuli [109, 135]. Upregulation of HO-1 is accepted as a sensitive marker of oxidative stress. Oxidative modified DNA can be repaired by several enzymes such as glycosylases: 8-oxoguanine-DNA-glysocylase (OGG1, EC 4.2.99.18), Nei-like protein 1 and 2 (NEIL, EC 4.2.99.18); Apurinic/apyrimidinic endonuclease 1 (APE 1, EC 4.2.99.18), X ray repair cross-complementing group 1 (XRCC 1, EC 4.2.99.18) and poly(ADP-ribose) polymerase-1 (PARP 1, EC 2.4.2.30). OGG1 removes 8-oxoguanine paired with a cytosine. Human *OGG1* gene consists of eight exons which can be alternatively spliced to produce different isoforms. The most abundant mRNAs of OGG1 are type 1a and 2a. These two isoforms are ubiquitously expressed in human tissues.

#### **4. Alzheimer`s disease**

**2.3. Oxidative damage of DNA**

428 Neurodegenerative Diseases

oxidative damage of the nucleic acids.

human ageing and degenerative diseases.

**3. Antioxidant defence**

reactive ROS,

**•** removing of oxidised molecules.

linking.

Nucleic acids are particularly sensitive to oxidative damage. There is increasing evidence that ROS are involved in the development of cancer, not only by direct effects on DNA but also by affecting signal transduction, cell proliferation, cell death and intracellular communication. ROS can damage DNA by direct chemical attack of purine and pyrimidine bases and deoxy‐ ribose sugars and also by indirect mechanisms. There is a known more than 20 product of

Superoxide, nitric oxide or hydrogen peroxide at physiologically relevant levels do not react with any of DNA or RNA bases or with the ribose or deoxyribose sugars at significant rates. Particularly hydroxyl radical is known to cause chemical modifications of DNA through the formation of one strand and two strands breaks and cross linkages with other molecules. Different saccharides radicals of DNA can arise by abstraction of a hydrogen atom from 2`deoxyribose because all positions in saccharides are susceptible to oxidative damage. Hydroxyl radical reacts with aromatic rings therefore also nitrogen bases of nucleic acids are modified. C5-C6 double bonds of pyrimidines and carbon atoms C4, C5, and C8 of purines are the most sensitive position to oxidative effect of hydroxyl radical and hydroxyl radical abstract electron and no hydrogen atom [169]. In consequence of ROS attack to nuclear proteins are generated protein radicals and radicals of base react under formation of DNA-protein cross-

Mitochondrial DNA (mtDNA) is excessively sensitive on an oxidative damage because mtDNA is situated near inner mitochondrial membrane, where are formed ROS. Mitochon‐ drial DNA is small and is not protected by histones like nuclear DNA. Mitochondria are able repair an oxidative damage of mtDNA and that base excise repair pathway plays a dominant role in mtDNA repair [36]. Damage of mtDNA can be potentially more important like damage of nuclear DNA because all mitochondrial genome code genes which are expressed whereas nuclear DNA includes great number of untranscribed sequences [166]. Linnane and coworkers [101] assume that accumulation of somatic mutations mtDNA is the main origin of

In consequence, imbalance between pro-oxidant and antioxidant in favour of pro-oxidants and their harmful effects, oxidative stress is increased. Cells possess antioxidant defence systems:

**•** elimination of reactive oxygen species by conversion to un-radical molecules or to less

enzymatic and non-enzymatic [15]. Antioxidants can work at various levels:

**•** protection the organism from the formation of reactive oxygen species,

**•** reparation of damaged molecules and cell structures,

Ageing is the main risk factor of neurodegenerative disorders. Approximately 5% of people in age 65 years have Alzheimer`s disease (AD) and the prevalence of this disease increases with increasing age from 19% to 30% after 75 years of age. Overall, 90-95% of Alzheimer`s disease represents a sporadic form and 5-10% represents familiar form. Alzheimer`s disease is neurodegenerative disorder characterised by cognitive failures, impairment of memory and by dramatic changes in behaviour. AD symptoms may include:

tracellular and intracellular region of brain. Extracellular deposits or senile amyloid plaques occur the most frequently in neocortex. Primary they are consisting of 4 kDa, 40-42 amino acid polypeptide chain called amyloid β peptide (Aβ) [66]. Intracellular deposits represent neurofibrillar tangles which are generated from filaments of microtubullar hyperphosphory‐ lated tau protein [4, 70, 99]. Amyloid plaques are example of a specific damage that is char‐ acteristic for AD while neurofibrillar tangles are present in different neurodegenerative pathological situations [134]. Created aggregates are involved in a process which leads to progressive degeneration and to neuron death. In the past decade, a significant body of evi‐ dence has pointed the attention to the amyloid processing of amyloid precurcor protein -

Oxidative Changes and Possible Effects of Polymorphism of Antioxidant Enzymes in Neurodegenerative Disease

http://dx.doi.org/10.5772/54619

431

Pathogenesis of AD is complex and involves many molecular, cellular, biochemical and physiological pathologies [9]. Alzheimer`s disease is a characteristic process with identifiable clinical state which are in a continuity with normal ageing process. It is a multifactorial disease and genetic as well as environmental factors are included in its pathogenesis. Whereas majority of AD is sporadic 5% is caused by mutations (familiar AD). There was observed a large loss of synapses and a neuronal death in a part of brain which is crucial for cognitive function including cerebral cortex, entorhinal cartex and hippocampus. Senile plaques created by

Reactive oxygen species probably play an important role in the generation of amyloid plaques, the development of neurofibrillary tangles and the neurodegenerative process itself. Agerelated accumulation of reactive oxygen species results in damage to nuclear and mitochon‐ drial DNA, lipids (lipid peroxidation), proteins (protein oxidation), and sugars (advanced glycosylation end products). Oxidative damage caused by reactive oxygen species can account for the vastly heterogeneous nature of Alzheimer`s disease. Several different kind of oxidative protein and lipid damages were observed in brain during ageing as well as increased gener‐ ation of reactive oxygen species [1, 24, 136, 147, 150]. We have found increased lipid peroxi‐ dation, accompanied by accumulation of conjugates of lipid peroxidation products with proteins, formation of dityrosines, loss of sulfhydryl groups and change in ANS (fluorescent probe 1-anilino-8-naphthalenesulfonate) in the ageing rat brain [7]. Keller et al. [91] observed that 4-hydroxy-2-nonenal damage a glutamate transport in synaptosome and mitochondrial function in brain. Increasing lipid peroxidation can participate on the age-related brain cell dysfunction. Recently, several reports have suggested that mitochondrial abnormalities and oxidative stress play a role in sporadic Alzheimer`s disease [26, 89, 132, 158, 181]. In brain tissue from Alzheimer`s disease patients, there are increased levels of markers of oxidative stress, including oxidized proteins, membrane lipids, and DNA [121, 127, 147, 148]. Oxidative modification of biomolecules is a marking process for the targeting of proteases. In the process of ageing there is a marked decrease in protease activity, damaged molecules can be cumulated with age and it may contribute to the age-related neurodegenerative diseases, including

"amyloid cascade" (fig. 4). This event is the major causative factor in AD.

deposits of amyloid fibres were localized in the brain.

**4.1. Oxidative damage and Alzheimer`s disease**

Alzheimer`s disease.


Although the cause or causes of Alzheimer's disease are not yet known, most experts agree that AD, like other common chronic conditions, probably develops as a result of multiple factors rather than a single cause.

**Figure 4. Processing of amyloid precursor protein (APP).** Non-amyloid pathway of APP starts by α-secretase cleav‐ age and continues by γ-secretase. Non toxic a soluble fragment of amyloid precursor protein (sAPPα), a small peptide (p3) and an amyloid intracellular domain (ACID) are produced. Amyloid pathway starts with β–secretase cleavage and after that it continues by γ–secretase. A soluble fragment of amyloid precursor protein (sAPPβ), a toxic amyloid β pep‐ tide (Aβ) and an amyloid intracellular domain are generated. Amyloid β peptide can be degradated or accumulated and therefore can be responsible for generation of amyloid plaques.

For Alzheimer`s disease many neurochemical and pathological changes are characteristic such as gliosis, tissue atrophy caused by loss of synapses which is the most striking in fron‐ tal and temporal parts of brain cortex and by formation of two main protein clusters in ex‐ tracellular and intracellular region of brain. Extracellular deposits or senile amyloid plaques occur the most frequently in neocortex. Primary they are consisting of 4 kDa, 40-42 amino acid polypeptide chain called amyloid β peptide (Aβ) [66]. Intracellular deposits represent neurofibrillar tangles which are generated from filaments of microtubullar hyperphosphory‐ lated tau protein [4, 70, 99]. Amyloid plaques are example of a specific damage that is char‐ acteristic for AD while neurofibrillar tangles are present in different neurodegenerative pathological situations [134]. Created aggregates are involved in a process which leads to progressive degeneration and to neuron death. In the past decade, a significant body of evi‐ dence has pointed the attention to the amyloid processing of amyloid precurcor protein - "amyloid cascade" (fig. 4). This event is the major causative factor in AD.

Pathogenesis of AD is complex and involves many molecular, cellular, biochemical and physiological pathologies [9]. Alzheimer`s disease is a characteristic process with identifiable clinical state which are in a continuity with normal ageing process. It is a multifactorial disease and genetic as well as environmental factors are included in its pathogenesis. Whereas majority of AD is sporadic 5% is caused by mutations (familiar AD). There was observed a large loss of synapses and a neuronal death in a part of brain which is crucial for cognitive function including cerebral cortex, entorhinal cartex and hippocampus. Senile plaques created by deposits of amyloid fibres were localized in the brain.

#### **4.1. Oxidative damage and Alzheimer`s disease**

disease represents a sporadic form and 5-10% represents familiar form. Alzheimer`s disease is neurodegenerative disorder characterised by cognitive failures, impairment of memory and

Although the cause or causes of Alzheimer's disease are not yet known, most experts agree that AD, like other common chronic conditions, probably develops as a result of multiple

**Figure 4. Processing of amyloid precursor protein (APP).** Non-amyloid pathway of APP starts by α-secretase cleav‐ age and continues by γ-secretase. Non toxic a soluble fragment of amyloid precursor protein (sAPPα), a small peptide (p3) and an amyloid intracellular domain (ACID) are produced. Amyloid pathway starts with β–secretase cleavage and after that it continues by γ–secretase. A soluble fragment of amyloid precursor protein (sAPPβ), a toxic amyloid β pep‐ tide (Aβ) and an amyloid intracellular domain are generated. Amyloid β peptide can be degradated or accumulated

For Alzheimer`s disease many neurochemical and pathological changes are characteristic such as gliosis, tissue atrophy caused by loss of synapses which is the most striking in fron‐ tal and temporal parts of brain cortex and by formation of two main protein clusters in ex‐

and therefore can be responsible for generation of amyloid plaques.

**•** difficulty in finding the right words or understanding what people are saying,

by dramatic changes in behaviour. AD symptoms may include:

**•** difficulty in performing previously routine tasks, and activities,

**•** loss of memory,

430 Neurodegenerative Diseases

**•** problems with language,

**•** personality and mood changes.

factors rather than a single cause.

Reactive oxygen species probably play an important role in the generation of amyloid plaques, the development of neurofibrillary tangles and the neurodegenerative process itself. Agerelated accumulation of reactive oxygen species results in damage to nuclear and mitochon‐ drial DNA, lipids (lipid peroxidation), proteins (protein oxidation), and sugars (advanced glycosylation end products). Oxidative damage caused by reactive oxygen species can account for the vastly heterogeneous nature of Alzheimer`s disease. Several different kind of oxidative protein and lipid damages were observed in brain during ageing as well as increased gener‐ ation of reactive oxygen species [1, 24, 136, 147, 150]. We have found increased lipid peroxi‐ dation, accompanied by accumulation of conjugates of lipid peroxidation products with proteins, formation of dityrosines, loss of sulfhydryl groups and change in ANS (fluorescent probe 1-anilino-8-naphthalenesulfonate) in the ageing rat brain [7]. Keller et al. [91] observed that 4-hydroxy-2-nonenal damage a glutamate transport in synaptosome and mitochondrial function in brain. Increasing lipid peroxidation can participate on the age-related brain cell dysfunction. Recently, several reports have suggested that mitochondrial abnormalities and oxidative stress play a role in sporadic Alzheimer`s disease [26, 89, 132, 158, 181]. In brain tissue from Alzheimer`s disease patients, there are increased levels of markers of oxidative stress, including oxidized proteins, membrane lipids, and DNA [121, 127, 147, 148]. Oxidative modification of biomolecules is a marking process for the targeting of proteases. In the process of ageing there is a marked decrease in protease activity, damaged molecules can be cumulated with age and it may contribute to the age-related neurodegenerative diseases, including Alzheimer`s disease.

Brain and cerebral blood vessel deposits of amyloid β peptide are the main signs of Alzheimer`s disease. Experimental and clinical studies showed an causal relationship between an accu‐ mulation of amyloid β peptide and origin of Alzheimer`s disease [25, 63, 73]. An abnormal production of amyloid β peptide or disturbed amyloid β peptide degradation can cause a pathological accumulation of amyloid β peptide and subsequent production of amyloid plaques [182]. It is suggested hypothesis that amyloid β peptide cause neuronal damage and cognitive failure via the generation of reactive oxygen species, mitochondrial oxidative damage, synaptic failure, and by inflammation changing in the brains of Alzheimer`s disease patients [133, 140, 158, 160]. Several *in vitro* studies have shown that synthetic amyloid β peptide facilitates the production of reactive oxygen species [12, 78, 132]. It was observed increased levels of soluble amyloid precursor protein in plasma and cerebrospinal fluid with advancing age [88, 106]. Increased level of soluble amyloid precursor protein may be a sorce of amyloid β peptide in the brain and vessels. Pluta et al. [126] demonstrated a transit of amyloid β peptide through the blood brain barrier. Expression of amyloid precursor protein, α-secretase, β-secretase, enothelin-converting enzyme, neprilysin as well as insulin-degrading enzyme was demonstrated at the brain barrier system [35]. It is possible that 80% of amyloid plaques in transgenic models Alzheimer`s disease [44] and 90% of human amyloid plaques is in a contact with capillaries [87]. Transit of amyloid β peptide or fragments of amyloid precursor protein from blood to the endothelial cells and brain parenchyma can cause changes in the vascular elasticity [127, 162] or can have direct pathological implications in the brain tissue. Changes in the brain blood vessels after brain attack or during the ageing process can cooperate in the pathogenesis and development of Alzheimer`s disease. Beta-secretase (BACE1) expression and its mediated β-site amyloid precursor protein cleavage activity appear to be tightly coupled to mitochondrial function. Beta-secretase upregulation may be a common consequence in various potential Alzheimer`s disease-related etiological conditions that involve energy insufficiency and/or oxidative stress [174]. *Post mortem* analysis of Alzheimer`s disease patients showed increased level of β-secretase [100, 163] and it can be responsible for an accumulation of amyloid plaques [179] and amyloid cascade. Reactive oxygen species that are generated due to ageing can activate β-secretase and facilitate the cleavage of amyloid precursor protein. Amyloid β peptide can be toxic for organism not only for its accumulation and amyloid plaques generation but also for possibility to increase oxidative stress and oxidative damage, to inhibit complexes of respiratory chain in mitochon‐ dria, and to inhibit enzymes of Krebs cycle [117, 132, 133, 147, 148]. However clear and exact mechanisms interface between amyloid β peptide and increased oxidative damage are unknown. Oxidative damage of amyloid degrading enzymes can be result of pathological changes of Alzheimer`s disease as well as a contributing factor to amyloid β peptide accumu‐ lation and generation of amyloid plaques. During ageing we observed protein and lipid oxidative damage that was caused by increased production of reactive oxygen species and insufficient or inadequate antioxidant defence. Age-related increasing production of reactive oxygen species could be one of a key factor leading to the age-related diseases, including neurodegenerative disease. Elevated oxidative damage and accumulation of amyloid deposits can have repercussion on deepened oxidative damage, preference of amyloid processing of amyloid precursor protein and development of Alzheimer`s disease.

**4.2. Alzheimer`s disease and polymorphism in antioxidant enzymes**

be associated with development of AD [173].

AD patients.

Oxidative damage is one of the mechanisms which results in stimulation of the amyloid pathway of APP processing therefore genes of antioxidant enzymes could present another group of candidate genes. Superoxide dismutase represents the most important part of an active antioxidant defence. The genes encoding SOD1, SOD2, SOD3 are located in different chromosomes and in all of them polymorphisms have been described. *SOD1* is encoded on 21q22.1, *SOD2* on 6q25.3, and *SOD3* on 4p16.3-q21. Regulation of *SOD* genes plays a crucial role in balancing the reactive oxygen species concentration. In *SOD1* has been observed substitution of A to C at the non-coding position 35. This polymorphism influence SOD1 activity [56]. Substitution T to C at position 24, resulting in a valine to alanine substitution at amino acid 16 has been identified in *SOD2*. In *SOD3* gene has been identified three single nucleotide polymorphism: alanine to threonine substitution at amino acid 40, phenylalanine to cysteine at amino acid 131, and finally the most studied polymorphism which represents substitution of arginine to glycine at amino acid 213. It has been observed no linkage between AD polymorphism in *SOD1* [37, 105]. It was observed that three polymorphisms in *SOD2* can

Oxidative Changes and Possible Effects of Polymorphism of Antioxidant Enzymes in Neurodegenerative Disease

http://dx.doi.org/10.5772/54619

433

Catalase is a common antioxidant enzyme responsible for controlling hydrogen peroxide concentrations in cells. The catalase gene is located on chromosome 11p13. There are known different polymorphisms of this enzyme in coding regions [67, 93] and in non-coding regions as well [27, 58, 68, 93, 164, 180]. A common polymorphism in the promoter region of the catalase gene consists of a C to T substitution at position -262 in the 5' region [58], which is thought to result in reduced activity. Catalase gene polymorphism does not confirm a protective role in

Glutathione transferases have historically also been called glutathione-S-transferases, and it is this latter name that gives rise to the widely used abbrevation, GST. Three major families of proteins the cytosolic, mitochondrial and microsomal (membrane-associated proteins in eicosanoids and glutathione metabolism, MAPEG) are known. In some organisms expression of *GSTs* are upregulated by exposure to prooxidants [6, 41, 167]. Seven classes of cytosolic glutathione transferases are recognising in mammals (Alpha, Mu, Pi, Sigma, Theta, Omega, and Zeta) [76]. At least 16 cytosolic GST subunits exist in human and display polymorphisms, and this is probably to contribute to interindividual differences in responses to diseases and xenobiotics. *GSTM1* is one of the genes encoding the Mu class of enzymes. Gene for GSTM1 has been mapped to glutathione transferase mu gene cluster on chromosome 1p13.3. Three polymorphisms of *GSTM1* have been identified: a substitution (*GSTM1A* and *GSTM1B*) and a deletion [131, 175]. The alleles of the substitution variant differ by C to G transition at base position 534, resulting in a lysine to asparagine substitution at amino acid 172 [34, 75]. There is no evidence to date that *GSTM1A* and *GSTM1B* alleles are functionally different from one another; thus these alleles are typically categorized together as a single functional phenotype. Other polymorphism is a deletion – *GSTM1* null variant that results in a lack of functional gene product and null genotype of GSTM1 was shown as a risk factor in Italian AD patients. The *GSTT1* gene is located at 22q11.2. Absence of both alleles for this gene represents null variant analogous to *GSTM1*. Deletion of whole gene results in the lack of enzymatic activity [152].

#### **4.2. Alzheimer`s disease and polymorphism in antioxidant enzymes**

Brain and cerebral blood vessel deposits of amyloid β peptide are the main signs of Alzheimer`s disease. Experimental and clinical studies showed an causal relationship between an accu‐ mulation of amyloid β peptide and origin of Alzheimer`s disease [25, 63, 73]. An abnormal production of amyloid β peptide or disturbed amyloid β peptide degradation can cause a pathological accumulation of amyloid β peptide and subsequent production of amyloid plaques [182]. It is suggested hypothesis that amyloid β peptide cause neuronal damage and cognitive failure via the generation of reactive oxygen species, mitochondrial oxidative damage, synaptic failure, and by inflammation changing in the brains of Alzheimer`s disease patients [133, 140, 158, 160]. Several *in vitro* studies have shown that synthetic amyloid β peptide facilitates the production of reactive oxygen species [12, 78, 132]. It was observed increased levels of soluble amyloid precursor protein in plasma and cerebrospinal fluid with advancing age [88, 106]. Increased level of soluble amyloid precursor protein may be a sorce of amyloid β peptide in the brain and vessels. Pluta et al. [126] demonstrated a transit of amyloid β peptide through the blood brain barrier. Expression of amyloid precursor protein, α-secretase, β-secretase, enothelin-converting enzyme, neprilysin as well as insulin-degrading enzyme was demonstrated at the brain barrier system [35]. It is possible that 80% of amyloid plaques in transgenic models Alzheimer`s disease [44] and 90% of human amyloid plaques is in a contact with capillaries [87]. Transit of amyloid β peptide or fragments of amyloid precursor protein from blood to the endothelial cells and brain parenchyma can cause changes in the vascular elasticity [127, 162] or can have direct pathological implications in the brain tissue. Changes in the brain blood vessels after brain attack or during the ageing process can cooperate in the pathogenesis and development of Alzheimer`s disease. Beta-secretase (BACE1) expression and its mediated β-site amyloid precursor protein cleavage activity appear to be tightly coupled to mitochondrial function. Beta-secretase upregulation may be a common consequence in various potential Alzheimer`s disease-related etiological conditions that involve energy insufficiency and/or oxidative stress [174]. *Post mortem* analysis of Alzheimer`s disease patients showed increased level of β-secretase [100, 163] and it can be responsible for an accumulation of amyloid plaques [179] and amyloid cascade. Reactive oxygen species that are generated due to ageing can activate β-secretase and facilitate the cleavage of amyloid precursor protein. Amyloid β peptide can be toxic for organism not only for its accumulation and amyloid plaques generation but also for possibility to increase oxidative stress and oxidative damage, to inhibit complexes of respiratory chain in mitochon‐ dria, and to inhibit enzymes of Krebs cycle [117, 132, 133, 147, 148]. However clear and exact mechanisms interface between amyloid β peptide and increased oxidative damage are unknown. Oxidative damage of amyloid degrading enzymes can be result of pathological changes of Alzheimer`s disease as well as a contributing factor to amyloid β peptide accumu‐ lation and generation of amyloid plaques. During ageing we observed protein and lipid oxidative damage that was caused by increased production of reactive oxygen species and insufficient or inadequate antioxidant defence. Age-related increasing production of reactive oxygen species could be one of a key factor leading to the age-related diseases, including neurodegenerative disease. Elevated oxidative damage and accumulation of amyloid deposits can have repercussion on deepened oxidative damage, preference of amyloid processing of

432 Neurodegenerative Diseases

amyloid precursor protein and development of Alzheimer`s disease.

Oxidative damage is one of the mechanisms which results in stimulation of the amyloid pathway of APP processing therefore genes of antioxidant enzymes could present another group of candidate genes. Superoxide dismutase represents the most important part of an active antioxidant defence. The genes encoding SOD1, SOD2, SOD3 are located in different chromosomes and in all of them polymorphisms have been described. *SOD1* is encoded on 21q22.1, *SOD2* on 6q25.3, and *SOD3* on 4p16.3-q21. Regulation of *SOD* genes plays a crucial role in balancing the reactive oxygen species concentration. In *SOD1* has been observed substitution of A to C at the non-coding position 35. This polymorphism influence SOD1 activity [56]. Substitution T to C at position 24, resulting in a valine to alanine substitution at amino acid 16 has been identified in *SOD2*. In *SOD3* gene has been identified three single nucleotide polymorphism: alanine to threonine substitution at amino acid 40, phenylalanine to cysteine at amino acid 131, and finally the most studied polymorphism which represents substitution of arginine to glycine at amino acid 213. It has been observed no linkage between AD polymorphism in *SOD1* [37, 105]. It was observed that three polymorphisms in *SOD2* can be associated with development of AD [173].

Catalase is a common antioxidant enzyme responsible for controlling hydrogen peroxide concentrations in cells. The catalase gene is located on chromosome 11p13. There are known different polymorphisms of this enzyme in coding regions [67, 93] and in non-coding regions as well [27, 58, 68, 93, 164, 180]. A common polymorphism in the promoter region of the catalase gene consists of a C to T substitution at position -262 in the 5' region [58], which is thought to result in reduced activity. Catalase gene polymorphism does not confirm a protective role in AD patients.

Glutathione transferases have historically also been called glutathione-S-transferases, and it is this latter name that gives rise to the widely used abbrevation, GST. Three major families of proteins the cytosolic, mitochondrial and microsomal (membrane-associated proteins in eicosanoids and glutathione metabolism, MAPEG) are known. In some organisms expression of *GSTs* are upregulated by exposure to prooxidants [6, 41, 167]. Seven classes of cytosolic glutathione transferases are recognising in mammals (Alpha, Mu, Pi, Sigma, Theta, Omega, and Zeta) [76]. At least 16 cytosolic GST subunits exist in human and display polymorphisms, and this is probably to contribute to interindividual differences in responses to diseases and xenobiotics. *GSTM1* is one of the genes encoding the Mu class of enzymes. Gene for GSTM1 has been mapped to glutathione transferase mu gene cluster on chromosome 1p13.3. Three polymorphisms of *GSTM1* have been identified: a substitution (*GSTM1A* and *GSTM1B*) and a deletion [131, 175]. The alleles of the substitution variant differ by C to G transition at base position 534, resulting in a lysine to asparagine substitution at amino acid 172 [34, 75]. There is no evidence to date that *GSTM1A* and *GSTM1B* alleles are functionally different from one another; thus these alleles are typically categorized together as a single functional phenotype. Other polymorphism is a deletion – *GSTM1* null variant that results in a lack of functional gene product and null genotype of GSTM1 was shown as a risk factor in Italian AD patients. The *GSTT1* gene is located at 22q11.2. Absence of both alleles for this gene represents null variant analogous to *GSTM1*. Deletion of whole gene results in the lack of enzymatic activity [152]. Gene for GSTP1 is one of the most intensive studying genes of glutathione transferase family and has been mapped on chromosome 11q13 and comprising nine exons. There are known two polymorphisms of *GSTP1*: substitution of isoleucine to valine at amino acid 105 and alanine to valine at amino acid 114, demonstrating different catalytic efficiencies due to changes in the active site [3]. Polymorphism in *GSTP1* may represent risk factor for AD and with advancing age [125, 151]. Presence of gene for GSTM1 and GSTT1 could be a protective factor [125, 157].

hOGG1, APE1, DNA polymerase β (pol β) and DNA ligase IIIα [115]. XRCC1 is recruited to the site of repair till the last stage of ligation, regulating and coordinating the whole process. More than 60 single nucleotide polymorphisms (SNPs) XRCC1 polymorphisms are known but the most common polymorphisms in *XRCC1* gene are: Arg149Trp, Arg280His, and Arg399Gln, and are associated with decreased function of XRCC1. It is unlikely that the XRCC1 Arg194Trp polymorphism plays a major role in the pathogenesis of late-onset AD in elderly Han Chinese [130] but positive association was found in Turkish population [46]. Arg280His, and

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Parkinson´s disease (PD) was firstly described by James Parkinson in 1817. It is the second most common neurodegenerative disease which affects approximately 1% of the human population aged 65 and more [80]. PD is slowly progressed disorder characterized by selective degeneration and loss of dopaminergic neurons in the *substantia nigra* (SN) *pars compacta* region of the midbrain, as well as with the appearance of intracytoplasmic inclusions known as Lewy bodies [30]. Among clinical symptoms of the disease belong rigidity, resting tremor, bradyki‐ nesia and postural imbalance [139]. Beside motor dysfunctions, as the major clinical features, non-motor symptoms, such as sleep disturbances, dementia and depression may be present

Familiar PD represents only 5-10% of the total cases of PD. Recently, several gene mutations have been linked with rare familiar forms of PD (e.g. α-synuclein, parkin, nuclear receptorrelated 1). The most important in early-onset familiar PD is the parkin gene [40]. So far no genetic changes are definitely connected with sporadic or idiopathic form of the disease. However, the prevalent form of PD appears to be multifactorial and a combination of envi‐ ronmental and genetic factors, together with ageing, may contribute to the development of the disease. Biochemical abnormalities that have been detected in sporadic PD include: oxidative stress [108, 124, 168], mitochondrial and proteasomal dysfunction [116, 142] and glutathione

Although the primary cause of PD is still unknown, oxidative stress together with mitochon‐ drial dysfunction are thought to be significantly implicated in the neurodegenerative process. Excessive formation of reactive oxygen and nitrogen species in PD may damage cellular components such as lipids, proteins and DNA. Increased lipid peroxidation, measured by increased amounts of malondialdehyde [42] and 4-hydroxynonenal, as well as increase in the extent of protein oxidation and elevated concentration of 8-hydroxy-2´-deoxyguanosine, a product of DNA oxidation [84], in SN provides direct evidence of oxidative damage [43].

Since majority of all oxygen is used in mitochondria, electron transport chain is major source of free radicals. Recent research confirmed that about 1-2% of total molecular oxy‐ gen is converted into reactive oxygen species [23]. Post-mortem studies on patients with

Arg399Gln were not associated with AD [115].

**5. Parkinson´s disease**

as well [177].

depletion [10, 177].

**5.1. Oxidative damage and Parkinson´s disease**

The *GPX1* gene is located in the 3p21 locus; the pro198leu polymorphism involves a change of thymine (T) for cytosine (C), which leads to the substitution of leucine (leu) for proline (pro), whose recessive allele leu has been linked to 70% reduction of enzyme activity. The leucine allele of *GPX1* may be a possible risk factor [118].

Subjects carrying the *HO-1* (-413) TT genotype might show a higher AD risk due to their genetic inability to induce a more effective HO-1 protective response [83]. Authors showed that an AD risk effect of *HO-1* (−413) TT genotype is only apparent in the presence of liver X receptor (LXR) LXR-β (intron 2) TT, LXR-β (intron 5) AA, or LXR-β (intron 7) TT genotypes. No genetic association between AD and polymorphisms of heme oxygenase 1 and 2 were observed in a Japanese population [143]. A (GT)n repeat in the human HO-1 gene promoter region is highly polymorphic, although no particular alleles are associated with AD or PD [92].

The DNA base excision repair (BER) pathway is the major pathway responsible for removing oxidative DNA damage caused by oxidative reagents and alkylation and thus protects cells against the toxic effects of endogenous and exogenous agents and this pathway is of particular importance in postmitotic tissues such as brain. The first step in the BER pathway is recognition and removal of the damaged base by a DNA glycosylase. A variety of glycosylases have evolved to recognize oxidized bases, which are commonly formed by reactive oxygen species generated during cellular metabolism. One of them is 8-oxoguanine-DNA-glysocylase (OGG1). The most common polymorphism of *OGG1* is in this gene substitution of serine (Ser) for cysteine (Cys) at codon 326 in exon 7. The variant homozygote is associated with reduced enzymatic activity [94]. Allele and genotype frequencies of *OGG1* were equally distributed between AD patients and healthy subjects [32, 115]. The Arg46Gln polymorphism of *OGG1* is also not associated with the pathogenesis of AD [47]. Mao et al. [104] identified in AD patients deletion in *OGG1* and their results suggest that defects in OGG1 may be important in the pathogenesis of AD in a significant fraction of AD.

Apurinic/apyrimidinic endonuclease 1 (APE1) is an enzyme involved in BER pathway. Its main role in the repair of damaged or mismatched nucleotides in DNA is to create a nick in the phosphodiester backbone of the AP site created when DNA glycosylase removes the damaged base. There are four types of AP endonucleases that have been classified according to their sites of incision. Genetic polymorphism has been identified in *APE1* gene and associ‐ ated with cancer risk. One of the most studied polymorphism is Asp148Glu. No association was found in AD patients [115].

X-ray repair cross-complementing-1 (XRCC) is a protein, which plays a coordinating role for consecutive stages of the BER system and interacts with several proteins of BER including hOGG1, APE1, DNA polymerase β (pol β) and DNA ligase IIIα [115]. XRCC1 is recruited to the site of repair till the last stage of ligation, regulating and coordinating the whole process. More than 60 single nucleotide polymorphisms (SNPs) XRCC1 polymorphisms are known but the most common polymorphisms in *XRCC1* gene are: Arg149Trp, Arg280His, and Arg399Gln, and are associated with decreased function of XRCC1. It is unlikely that the XRCC1 Arg194Trp polymorphism plays a major role in the pathogenesis of late-onset AD in elderly Han Chinese [130] but positive association was found in Turkish population [46]. Arg280His, and Arg399Gln were not associated with AD [115].

### **5. Parkinson´s disease**

Gene for GSTP1 is one of the most intensive studying genes of glutathione transferase family and has been mapped on chromosome 11q13 and comprising nine exons. There are known two polymorphisms of *GSTP1*: substitution of isoleucine to valine at amino acid 105 and alanine to valine at amino acid 114, demonstrating different catalytic efficiencies due to changes in the active site [3]. Polymorphism in *GSTP1* may represent risk factor for AD and with advancing age [125, 151]. Presence of gene for GSTM1 and GSTT1 could be a protective

The *GPX1* gene is located in the 3p21 locus; the pro198leu polymorphism involves a change of thymine (T) for cytosine (C), which leads to the substitution of leucine (leu) for proline (pro), whose recessive allele leu has been linked to 70% reduction of enzyme activity. The leucine

Subjects carrying the *HO-1* (-413) TT genotype might show a higher AD risk due to their genetic inability to induce a more effective HO-1 protective response [83]. Authors showed that an AD risk effect of *HO-1* (−413) TT genotype is only apparent in the presence of liver X receptor (LXR) LXR-β (intron 2) TT, LXR-β (intron 5) AA, or LXR-β (intron 7) TT genotypes. No genetic association between AD and polymorphisms of heme oxygenase 1 and 2 were observed in a Japanese population [143]. A (GT)n repeat in the human HO-1 gene promoter region is highly

The DNA base excision repair (BER) pathway is the major pathway responsible for removing oxidative DNA damage caused by oxidative reagents and alkylation and thus protects cells against the toxic effects of endogenous and exogenous agents and this pathway is of particular importance in postmitotic tissues such as brain. The first step in the BER pathway is recognition and removal of the damaged base by a DNA glycosylase. A variety of glycosylases have evolved to recognize oxidized bases, which are commonly formed by reactive oxygen species generated during cellular metabolism. One of them is 8-oxoguanine-DNA-glysocylase (OGG1). The most common polymorphism of *OGG1* is in this gene substitution of serine (Ser) for cysteine (Cys) at codon 326 in exon 7. The variant homozygote is associated with reduced enzymatic activity [94]. Allele and genotype frequencies of *OGG1* were equally distributed between AD patients and healthy subjects [32, 115]. The Arg46Gln polymorphism of *OGG1* is also not associated with the pathogenesis of AD [47]. Mao et al. [104] identified in AD patients deletion in *OGG1* and their results suggest that defects in OGG1 may be important in the

Apurinic/apyrimidinic endonuclease 1 (APE1) is an enzyme involved in BER pathway. Its main role in the repair of damaged or mismatched nucleotides in DNA is to create a nick in the phosphodiester backbone of the AP site created when DNA glycosylase removes the damaged base. There are four types of AP endonucleases that have been classified according to their sites of incision. Genetic polymorphism has been identified in *APE1* gene and associ‐ ated with cancer risk. One of the most studied polymorphism is Asp148Glu. No association

X-ray repair cross-complementing-1 (XRCC) is a protein, which plays a coordinating role for consecutive stages of the BER system and interacts with several proteins of BER including

polymorphic, although no particular alleles are associated with AD or PD [92].

factor [125, 157].

434 Neurodegenerative Diseases

allele of *GPX1* may be a possible risk factor [118].

pathogenesis of AD in a significant fraction of AD.

was found in AD patients [115].

Parkinson´s disease (PD) was firstly described by James Parkinson in 1817. It is the second most common neurodegenerative disease which affects approximately 1% of the human population aged 65 and more [80]. PD is slowly progressed disorder characterized by selective degeneration and loss of dopaminergic neurons in the *substantia nigra* (SN) *pars compacta* region of the midbrain, as well as with the appearance of intracytoplasmic inclusions known as Lewy bodies [30]. Among clinical symptoms of the disease belong rigidity, resting tremor, bradyki‐ nesia and postural imbalance [139]. Beside motor dysfunctions, as the major clinical features, non-motor symptoms, such as sleep disturbances, dementia and depression may be present as well [177].

Familiar PD represents only 5-10% of the total cases of PD. Recently, several gene mutations have been linked with rare familiar forms of PD (e.g. α-synuclein, parkin, nuclear receptorrelated 1). The most important in early-onset familiar PD is the parkin gene [40]. So far no genetic changes are definitely connected with sporadic or idiopathic form of the disease. However, the prevalent form of PD appears to be multifactorial and a combination of envi‐ ronmental and genetic factors, together with ageing, may contribute to the development of the disease. Biochemical abnormalities that have been detected in sporadic PD include: oxidative stress [108, 124, 168], mitochondrial and proteasomal dysfunction [116, 142] and glutathione depletion [10, 177].

#### **5.1. Oxidative damage and Parkinson´s disease**

Although the primary cause of PD is still unknown, oxidative stress together with mitochon‐ drial dysfunction are thought to be significantly implicated in the neurodegenerative process. Excessive formation of reactive oxygen and nitrogen species in PD may damage cellular components such as lipids, proteins and DNA. Increased lipid peroxidation, measured by increased amounts of malondialdehyde [42] and 4-hydroxynonenal, as well as increase in the extent of protein oxidation and elevated concentration of 8-hydroxy-2´-deoxyguanosine, a product of DNA oxidation [84], in SN provides direct evidence of oxidative damage [43].

Since majority of all oxygen is used in mitochondria, electron transport chain is major source of free radicals. Recent research confirmed that about 1-2% of total molecular oxy‐ gen is converted into reactive oxygen species [23]. Post-mortem studies on patients with PD have revealed a specific decrease in the activity of NADPH dehydrogenase (complex I). The alterations in complex I activity were not detected in other regions of the brain, as well as in other neurodegenerative diseases [142]. Complex I deficiency could contribute to neurodegeneration in PD not only due to decreased ATP synthesis but also increased ROS production [11, 137]. Van der Walt et al. [165] also published that the 10398G poly‐ morphism within NADH dehydrogenase 3 may provide significant protection against de‐ veloping PD in Caucasian populations.

frequency in the group of PD patients [57, 172]. Observed results might be explained by ethnic

Oxidative Changes and Possible Effects of Polymorphism of Antioxidant Enzymes in Neurodegenerative Disease

http://dx.doi.org/10.5772/54619

437

Little is known about the association of catalase polymorphisms and PD. However, Parboo‐

Glutathione transferases (GST) are the most studied phase II detoxification enzymes. Activity of GST was observed to be reduced in substantia nigra of PD [145]. Studies of *GST* polymor‐ phisms and PD yielded mixed results. Several studies confirmed positive association between *GSTM1* and *GSTT1* null genotypes and higher risk of PD [120, 157]. On the other hand, other studies found no association [90, 171]. Similarly, *GSTP1* was detected to have only a minor role in PD [90,, 171]. However, Wahner et al. [90] noted a 32% risk reduction among Caucasian PD

Markedly elevated expression of heme oxygenase 1 was observed in PD and it has been shown that its overexpression may protect neurons against oxidative stress-induced toxicity [81]. However, Funke et al. [61] found no association of (GT)n fragment length polymorphism in

Since increased 8-oxo-guanine levels have been observed in PD patients, still more attention is paid to analysis of genetic polymorphisms of oxoguanine DNA glycosylase (OGG1), which removes oxidized guanine from DNA. Coppede et al. [33] published that *hOGG1* Ser326Cys

Huntington`s disease (HD) is an autosomal dominant inherited neurodegenerative disorder of the central nervous system. Worldwide prevalence of HD is 5 to 8 per 100,000 people with no gender preponderance [74]. HD is characterized by cognitive and memory dysfunctions, weight loss, and choreiform movements. It is caused by an expansion of a polymorphic three nucleotide repeat sequence CAG in the exon 1 of the gene coding for the protein huntingtin (htt) on chromosome 4 (4p16.3) [82]. Wild type htt may exert a variety of intracellular functions such as: protein trafficking, vesicle transport and anchoring to the cytoskeleton, clathrinemediated endocytosis, postsynaptic signaling, transcriptional regulation, and anti-apoptotic function [65]. For instance, htt is involved in fast axonal transport, enhancing vesicular transport of brain-derived neurotrophic factor along microtubules [62, 159]. The protein htt consists of a series of CAG repeats coding for glutamine residues (polyQ) followed by two short stretches of prolines. Normally, the number of the polyglutamine repeats is 10-29 (median, 18). By contrast, HD patients have expanded numbers of CAG repeats, from 39 to 121 (median, 44). Expanded repeats cause a conformational change in the htt promoting the formation of intracellular aggregates, mainly in medium spiny neurons where the expression of huntingtin is elevated. The number of CAG repeats is inversely correlated with the age of disease onset, and disease progression is rapid in patients with more CAG expansion [96]. In brain, the most remarkable changes are found within the striatum, there is a gradual atrophy

singh et al. [113] observed no connection between mutations of catalase gene and PD.

patients carrying at least one *GSTO1* and *GSTO2* variant allele.

the promoter region, as well as three other coding SNPs with PD.

polymorphism is not associated with sporadic form of PD.

**6. Huntington`s disease**

of the caudate nucleus and putamen [170].

differences in genotypes frequencies.

Alternatively, ROS may arise during metabolism of dopamine. Autooxidation of dopamine and its polymerization into neuromelanin produces electrophilic semiquinones and quinones, which can contribute to ROS production, especially superoxide anion radicals [60]. It is also known that dopamine is able to coordinate iron and generate Fe2+, providing an important source of hydroxyl and superoxide radical production [13]. In generally, elevated iron levels were observed in *substantia nigra* in PD [149]. A major source of increased iron levels during PD is microglial activation which induces iron release and free radical production [123]. Free iron may promote already mentioned autooxidation of dopamine [14]. Oxidative stress induced by elevated levels of free iron also appears to promote α-synuclein (a prominent component of Lewy bodies) aggregation, the major histopathological hallmark of PD [75].

Important role in protection of dopaminergic neurons against oxidative stress plays the antioxidant molecule glutathione (GSH). GSH removes H2O2, which is produced during cellular metabolism. Perry et al. [121] firstly reported decreased levels of total GSH in autopsied brains from PD patients. Total GSH is reported to be decreased by 40-50% specifically in nigral dopaminergic neurons [119]. GSH depletion also appears to correlate with the severity of the disease and is the earliest known marker of oxidative stress and indicator of degeneration of nigral neurons [85]. On the other hand, Mythri et al. [108] observed 3-5 fold increase in total GSH levels in all the non-SN regions of tested PD brains compared to control samples. On the contrary, they found no significant changes in the levels of protein carbonyls, as markers of protein oxidation, or nitrosative stress markers. According to these results, they expected that specific signals from the degenerating dopaminergic SN neurons might induce elevated levels of GSH and such prevent oxidative damage [108]. Beside GSH, other antioxidant activities are altered in the SN. The levels of antioxidant enzymes, such as superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase, were found to be changed in SN [168].

#### **5.2. Parkinson´s disease and polymorphism in antioxidant enzymes**

Current research shows that ROS and oxidative damage are part of pathological processes during PD, but it remains to be determined whether this is a primary event or a consequence of other cellular dysfunctions. Analysis of polymorphisms of detoxifying enzymes may help to clarify whether PD may be caused by a genetic predisposition to oxidative stress.

Among antioxidant enzymes, superoxide dismutase (SOD) represents the first line of defence. From three different SOD isoenzymes, SOD2 appears to be the most relevant in PD because of its mitochondrial localization. Several research groups confirmed that *SOD2* (Ala9Val) polymorphism is not significantly associated with PD in Caucasian population [52, 69, 146]. On the other hand, some studies, especially on Asian population, found higher Ala allele frequency in the group of PD patients [57, 172]. Observed results might be explained by ethnic differences in genotypes frequencies.

Little is known about the association of catalase polymorphisms and PD. However, Parboo‐ singh et al. [113] observed no connection between mutations of catalase gene and PD.

Glutathione transferases (GST) are the most studied phase II detoxification enzymes. Activity of GST was observed to be reduced in substantia nigra of PD [145]. Studies of *GST* polymor‐ phisms and PD yielded mixed results. Several studies confirmed positive association between *GSTM1* and *GSTT1* null genotypes and higher risk of PD [120, 157]. On the other hand, other studies found no association [90, 171]. Similarly, *GSTP1* was detected to have only a minor role in PD [90,, 171]. However, Wahner et al. [90] noted a 32% risk reduction among Caucasian PD patients carrying at least one *GSTO1* and *GSTO2* variant allele.

Markedly elevated expression of heme oxygenase 1 was observed in PD and it has been shown that its overexpression may protect neurons against oxidative stress-induced toxicity [81]. However, Funke et al. [61] found no association of (GT)n fragment length polymorphism in the promoter region, as well as three other coding SNPs with PD.

Since increased 8-oxo-guanine levels have been observed in PD patients, still more attention is paid to analysis of genetic polymorphisms of oxoguanine DNA glycosylase (OGG1), which removes oxidized guanine from DNA. Coppede et al. [33] published that *hOGG1* Ser326Cys polymorphism is not associated with sporadic form of PD.

### **6. Huntington`s disease**

PD have revealed a specific decrease in the activity of NADPH dehydrogenase (complex I). The alterations in complex I activity were not detected in other regions of the brain, as well as in other neurodegenerative diseases [142]. Complex I deficiency could contribute to neurodegeneration in PD not only due to decreased ATP synthesis but also increased ROS production [11, 137]. Van der Walt et al. [165] also published that the 10398G poly‐ morphism within NADH dehydrogenase 3 may provide significant protection against de‐

Alternatively, ROS may arise during metabolism of dopamine. Autooxidation of dopamine and its polymerization into neuromelanin produces electrophilic semiquinones and quinones, which can contribute to ROS production, especially superoxide anion radicals [60]. It is also known that dopamine is able to coordinate iron and generate Fe2+, providing an important source of hydroxyl and superoxide radical production [13]. In generally, elevated iron levels were observed in *substantia nigra* in PD [149]. A major source of increased iron levels during PD is microglial activation which induces iron release and free radical production [123]. Free iron may promote already mentioned autooxidation of dopamine [14]. Oxidative stress induced by elevated levels of free iron also appears to promote α-synuclein (a prominent component of Lewy bodies) aggregation, the major histopathological hallmark of PD [75]. Important role in protection of dopaminergic neurons against oxidative stress plays the antioxidant molecule glutathione (GSH). GSH removes H2O2, which is produced during cellular metabolism. Perry et al. [121] firstly reported decreased levels of total GSH in autopsied brains from PD patients. Total GSH is reported to be decreased by 40-50% specifically in nigral dopaminergic neurons [119]. GSH depletion also appears to correlate with the severity of the disease and is the earliest known marker of oxidative stress and indicator of degeneration of nigral neurons [85]. On the other hand, Mythri et al. [108] observed 3-5 fold increase in total GSH levels in all the non-SN regions of tested PD brains compared to control samples. On the contrary, they found no significant changes in the levels of protein carbonyls, as markers of protein oxidation, or nitrosative stress markers. According to these results, they expected that specific signals from the degenerating dopaminergic SN neurons might induce elevated levels of GSH and such prevent oxidative damage [108]. Beside GSH, other antioxidant activities are altered in the SN. The levels of antioxidant enzymes, such as superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase, were found to be changed in SN [168].

**5.2. Parkinson´s disease and polymorphism in antioxidant enzymes**

Current research shows that ROS and oxidative damage are part of pathological processes during PD, but it remains to be determined whether this is a primary event or a consequence of other cellular dysfunctions. Analysis of polymorphisms of detoxifying enzymes may help

Among antioxidant enzymes, superoxide dismutase (SOD) represents the first line of defence. From three different SOD isoenzymes, SOD2 appears to be the most relevant in PD because of its mitochondrial localization. Several research groups confirmed that *SOD2* (Ala9Val) polymorphism is not significantly associated with PD in Caucasian population [52, 69, 146]. On the other hand, some studies, especially on Asian population, found higher Ala allele

to clarify whether PD may be caused by a genetic predisposition to oxidative stress.

veloping PD in Caucasian populations.

436 Neurodegenerative Diseases

Huntington`s disease (HD) is an autosomal dominant inherited neurodegenerative disorder of the central nervous system. Worldwide prevalence of HD is 5 to 8 per 100,000 people with no gender preponderance [74]. HD is characterized by cognitive and memory dysfunctions, weight loss, and choreiform movements. It is caused by an expansion of a polymorphic three nucleotide repeat sequence CAG in the exon 1 of the gene coding for the protein huntingtin (htt) on chromosome 4 (4p16.3) [82]. Wild type htt may exert a variety of intracellular functions such as: protein trafficking, vesicle transport and anchoring to the cytoskeleton, clathrinemediated endocytosis, postsynaptic signaling, transcriptional regulation, and anti-apoptotic function [65]. For instance, htt is involved in fast axonal transport, enhancing vesicular transport of brain-derived neurotrophic factor along microtubules [62, 159]. The protein htt consists of a series of CAG repeats coding for glutamine residues (polyQ) followed by two short stretches of prolines. Normally, the number of the polyglutamine repeats is 10-29 (median, 18). By contrast, HD patients have expanded numbers of CAG repeats, from 39 to 121 (median, 44). Expanded repeats cause a conformational change in the htt promoting the formation of intracellular aggregates, mainly in medium spiny neurons where the expression of huntingtin is elevated. The number of CAG repeats is inversely correlated with the age of disease onset, and disease progression is rapid in patients with more CAG expansion [96]. In brain, the most remarkable changes are found within the striatum, there is a gradual atrophy of the caudate nucleus and putamen [170].

#### **6.1. Oxidative damage and Huntington`s disease**

The generation of reactive oxygen species and the consequent oxidative stress is thought to play a pivotal role in the neurodegeneration observed in HD [53, 71] (Ferrante, Grunewald). Several lines of evidence suggest that not only increased oxidative stress, but also protein metabolism impairment, mitochondrial dysfunction, and they interplay contribute to neuronal dysfunction in HD [18, 19, 112, 128].

glutamate aminopeptidase activities were significantly reduced in HD patients. These changes

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Besides CAG repetition in huntingtin gene, there are 2 genetic polymorphisms in the fulllength htt gene. One of them, named CCG polymorphism is located in the first proline-rich fragment, second one is delta 2642 glutamic acid polymorphism. Previous studies have shown that alleles with 7 or 10 repeats are predominant in CGG polymorphism, but the distribution from among ethnics is variable. In western population is strong association between the 7 repeat CCG allele and HD [156]. On contrary, in Japanese population has been shown association with the 10 repeat CCG allele and HD [107]. Latest study performed on Chinese population compared the clinical features between the 7 and 10 repeats CCG alleles, but did

It was proposed a model that somatic expansion of CAG repeats in HD cells might contribute to disease onset and progression of HD and is mediated by the DNA repair oxoguanine glycosylase 1 (OGG1) enzyme [95]. This enzyme removes 8-oxoguanine from the DNA. Study of the *hOGG1* Ser326Cys polymorphism performed on 91 patients showed that bearers of mutant Cys allele appear to have an increased number of the CAG repeats of the expanded HD allele. Since this is the first evidence of an association between the hOGG1 genotype and both CAG repeat length and age at onset of the HD, for confirmation, further studies are

Population ages therefore neurodegenerative diseases are in a centre of interest. Several studies showed that missing antioxidant genes may have negative effect on central nervous system and may represent a risk for development of neurodegenerative diseases. Unfortunately majority of results are from animals and cell tissue studies. Animal studies, in vitro and ex vivo studies are full of positive effects of single antioxidant gene overexpression such as superoxide dismutase, catalase and antioxidant therapy may represent promising treatment. Molecular and genetic analyses represent a new potential for neurodegenerative diseases studying. The role of gene polymorphisms and many others gene polymorphisms as risk factors for the occurrence of neurodegenerative diseases are still controversial. Moreover impact of gene polymorphisms can depend on several different factors, especially for neuro‐ degenerative diseases, such as ethnicity, social environment, life style. We need new studies for clear determination of antioxidant gene polymorphisms. Moreover multiple genotype analyses are necessary as well because a single gene polymorphism can be without relationship to increased risk of neurodegenerative disorders but the combination of gene polymorphisms

may be related to the progression of the disease [49].

**6.2. Polymorphisms and Huntington`s disease**

not find any statistical significant results [178].

may have significant effect, positive or negative.

required [31].

**7. Conclusions**

DNA fragmentation is significantly increased in human HD patients and correlates with the length of CAG repeats [20]. The oxidation of DNA leads to the formation of the metabolite 8 hydroxy-2`-deoxyguanosine (OHdG) which is a direct result of free radical activity [128]. Significant increases of OHdG levels, coming from nuclear DNA, occur in the caudate nucleus and parietal cortex in postmortem tissue of HD patients [18, 128]. In addition, increased oxidative damage to DNA is present in serum from HD patients [29, 79. Moreover, the improvement of elevated OHdG by creatine treatment suggests OHdG as a promising peripheral biomarker [79]. These findings are in consensus with the elevated levels of OHdG that occur in other neurodegenerative diseases in which oxidative stress is implicated as a pivotal pathogenic mechanism [55]. Nevertheless, some research groups have not confirmed changes in OHdG levels in HD patients [2].

Elevated levels of malondialdehyde (MDA), a marker of lipid peroxidation, have been documented in HD brain [72]. Elevated levels of MDA have also been shown in the peripheral blood of HD patients, and preliminary results suggest that the levels of lipid peroxidation (MDA level) appear to be correlated to disease severity [29, 155]. Reduced activities of erythrocyte glutathione peroxidase and Cu/Zn-superoxid dismutase in HD patients implicate that the defense mechanism is impaired in peripheral blood cells of HD. Because of ubiquitous expression of huntingtin, the peripheral abnormalities may reflect the same consequences to mutant huntingtin in the brain [29]. An abnormal accumulation of lipofuscin, product of unsaturated fatty acid lipid peroxidation, has been proven in HD patients [17]. Supplemental markers of oxidative damage, as for example inducible form of heme oxygenase, 3-nitrotyro‐ sine, and above mentioned malondialdehyde, are elevated in human HD striatum and cortex compared with age-matched control brain specimens [19, 54].

The primary source of reactive oxygen species in neurons is mitochondria. Mitochondrial dysfunction in HD is closely associated with oxidative stress. In was first reflected that here was an energetic impairment in HD, because HD patients exhibit profound weight loss despite continual caloric intake [20, 21]. Lowered glucose utilization in striatum of HD patients early stages prior to pronounced striatal atrophy [97]. Mitochondrial functional abnormalities were observed, such is a defect in succinate dehydrogenase in the caudate of postmortem HD brains. Subsequent studies confirmed that there was a significant decrease in complex II activity in the caudate nucleus, in complex III activity in the caudate and putamen, and of complex IV in the putamen of HD brains [18, 72, 103].

Plasma lipid peroxide and lactate concentrations as indicators of oxidative stress and mito‐ chondrial dysfunction, were significantly elevated in HD patients. On contrary, aspartate and glutamate aminopeptidase activities were significantly reduced in HD patients. These changes may be related to the progression of the disease [49].

#### **6.2. Polymorphisms and Huntington`s disease**

Besides CAG repetition in huntingtin gene, there are 2 genetic polymorphisms in the fulllength htt gene. One of them, named CCG polymorphism is located in the first proline-rich fragment, second one is delta 2642 glutamic acid polymorphism. Previous studies have shown that alleles with 7 or 10 repeats are predominant in CGG polymorphism, but the distribution from among ethnics is variable. In western population is strong association between the 7 repeat CCG allele and HD [156]. On contrary, in Japanese population has been shown association with the 10 repeat CCG allele and HD [107]. Latest study performed on Chinese population compared the clinical features between the 7 and 10 repeats CCG alleles, but did not find any statistical significant results [178].

It was proposed a model that somatic expansion of CAG repeats in HD cells might contribute to disease onset and progression of HD and is mediated by the DNA repair oxoguanine glycosylase 1 (OGG1) enzyme [95]. This enzyme removes 8-oxoguanine from the DNA. Study of the *hOGG1* Ser326Cys polymorphism performed on 91 patients showed that bearers of mutant Cys allele appear to have an increased number of the CAG repeats of the expanded HD allele. Since this is the first evidence of an association between the hOGG1 genotype and both CAG repeat length and age at onset of the HD, for confirmation, further studies are required [31].

### **7. Conclusions**

**6.1. Oxidative damage and Huntington`s disease**

dysfunction in HD [18, 19, 112, 128].

438 Neurodegenerative Diseases

changes in OHdG levels in HD patients [2].

the putamen of HD brains [18, 72, 103].

compared with age-matched control brain specimens [19, 54].

The generation of reactive oxygen species and the consequent oxidative stress is thought to play a pivotal role in the neurodegeneration observed in HD [53, 71] (Ferrante, Grunewald). Several lines of evidence suggest that not only increased oxidative stress, but also protein metabolism impairment, mitochondrial dysfunction, and they interplay contribute to neuronal

DNA fragmentation is significantly increased in human HD patients and correlates with the length of CAG repeats [20]. The oxidation of DNA leads to the formation of the metabolite 8 hydroxy-2`-deoxyguanosine (OHdG) which is a direct result of free radical activity [128]. Significant increases of OHdG levels, coming from nuclear DNA, occur in the caudate nucleus and parietal cortex in postmortem tissue of HD patients [18, 128]. In addition, increased oxidative damage to DNA is present in serum from HD patients [29, 79. Moreover, the improvement of elevated OHdG by creatine treatment suggests OHdG as a promising peripheral biomarker [79]. These findings are in consensus with the elevated levels of OHdG that occur in other neurodegenerative diseases in which oxidative stress is implicated as a pivotal pathogenic mechanism [55]. Nevertheless, some research groups have not confirmed

Elevated levels of malondialdehyde (MDA), a marker of lipid peroxidation, have been documented in HD brain [72]. Elevated levels of MDA have also been shown in the peripheral blood of HD patients, and preliminary results suggest that the levels of lipid peroxidation (MDA level) appear to be correlated to disease severity [29, 155]. Reduced activities of erythrocyte glutathione peroxidase and Cu/Zn-superoxid dismutase in HD patients implicate that the defense mechanism is impaired in peripheral blood cells of HD. Because of ubiquitous expression of huntingtin, the peripheral abnormalities may reflect the same consequences to mutant huntingtin in the brain [29]. An abnormal accumulation of lipofuscin, product of unsaturated fatty acid lipid peroxidation, has been proven in HD patients [17]. Supplemental markers of oxidative damage, as for example inducible form of heme oxygenase, 3-nitrotyro‐ sine, and above mentioned malondialdehyde, are elevated in human HD striatum and cortex

The primary source of reactive oxygen species in neurons is mitochondria. Mitochondrial dysfunction in HD is closely associated with oxidative stress. In was first reflected that here was an energetic impairment in HD, because HD patients exhibit profound weight loss despite continual caloric intake [20, 21]. Lowered glucose utilization in striatum of HD patients early stages prior to pronounced striatal atrophy [97]. Mitochondrial functional abnormalities were observed, such is a defect in succinate dehydrogenase in the caudate of postmortem HD brains. Subsequent studies confirmed that there was a significant decrease in complex II activity in the caudate nucleus, in complex III activity in the caudate and putamen, and of complex IV in

Plasma lipid peroxide and lactate concentrations as indicators of oxidative stress and mito‐ chondrial dysfunction, were significantly elevated in HD patients. On contrary, aspartate and Population ages therefore neurodegenerative diseases are in a centre of interest. Several studies showed that missing antioxidant genes may have negative effect on central nervous system and may represent a risk for development of neurodegenerative diseases. Unfortunately majority of results are from animals and cell tissue studies. Animal studies, in vitro and ex vivo studies are full of positive effects of single antioxidant gene overexpression such as superoxide dismutase, catalase and antioxidant therapy may represent promising treatment. Molecular and genetic analyses represent a new potential for neurodegenerative diseases studying. The role of gene polymorphisms and many others gene polymorphisms as risk factors for the occurrence of neurodegenerative diseases are still controversial. Moreover impact of gene polymorphisms can depend on several different factors, especially for neuro‐ degenerative diseases, such as ethnicity, social environment, life style. We need new studies for clear determination of antioxidant gene polymorphisms. Moreover multiple genotype analyses are necessary as well because a single gene polymorphism can be without relationship to increased risk of neurodegenerative disorders but the combination of gene polymorphisms may have significant effect, positive or negative.

### **Acknowledgements**

Project was supported by grants VEGA 1/0071/11 and Ministry of Health 2007/57- UK-17.

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### **Author details**

Eva Babusikova, Andrea Evinova, Jozef Hatok, Dusan Dobrota and Jana Jurecekova

Comenius University in Bratislava, Jessenius Faculty of Medicine in Martin, Department of Medical Biochemistry, Martin, Slovakia

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**Chapter 19**

**Intermediate Filaments in Neurodegenerative Diseases**

In adult central (CNS) and peripheral nervous system (PNS), intermediate filaments (IFs) are the most abundant cytoskeletal components [1]. Neurons express differentially several IF pro‐ teins depending on their developing stage or their localization in the nervous system. In CNS, IFs are principally composed of the neurofilament (NF) triplet proteins (called NFL (light, 68 kDa), NFM (medium, 160 kDa) and NFH (heavy, 205 kDa); type IV) and α-internexin (66 kDa; type IV), while in the PNS, NFs are made up of NFL, NFM, NFH and peripherin (57 kDa; type III) [2, 3]. Neurons may also express other IF proteins, including nestin (200 kDa; type IV), vimentin (57 kDa, type III) syncoilin isoforms (Sync1 (64 kDa), Sync2 (64 kDa); type III) and synemin isoforms (Low synemin (41 kDa), Middle or beta synemin (150 kDa), High or alpha synemin (180 kDa); type IV). While present in perikarya and dendrites, IFs are particularly abundant in large myelinated axons, where they are essential for axon radial growth during development and axon caliber maintenance [4]. Consequently they are crucial to optimize the conduction velocity of the nerve impulse. They also contribute to the dynamic properties of the axonal cytoskeleton during neuronal differentiation, axon outgrowth, regeneration and guidance [5]. The IF proteins share a common tripartite structure with a globular head, a central α-helical rod domain and variable tail domains that differ in length and amino acids compo‐ sition. The central rod domain of approximately 310 amino acids contains four α-helical re‐ gions and is involved in the assembly of 10 nm filaments [6]. NFM and NFH subunits differ from other neuronal IF proteins by their long tail domains containing numerous repeats of

An increasing body of evidence supports the view that the most common mechanism of chronic neurodegenerative disorders involves abnormal protein production, processing or misfolding and subsequent accumulation in nervous system. Alterations in the metabolism and/or organization of neuronal IFs are frequently associated, directly or indirectly, with var‐ ious neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Charcot-Mar‐

and reproduction in any medium, provided the original work is properly cited.

© 2013 Perrot and Eyer; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

Rodolphe Perrot and Joel Eyer

Lys-Ser-Pro (KSP) phosphorylation sites [4].

http://dx.doi.org/10.5772/54676

**1. Introduction**

Additional information is available at the end of the chapter

### **Intermediate Filaments in Neurodegenerative Diseases**

Rodolphe Perrot and Joel Eyer

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54676

### **1. Introduction**

In adult central (CNS) and peripheral nervous system (PNS), intermediate filaments (IFs) are the most abundant cytoskeletal components [1]. Neurons express differentially several IF pro‐ teins depending on their developing stage or their localization in the nervous system. In CNS, IFs are principally composed of the neurofilament (NF) triplet proteins (called NFL (light, 68 kDa), NFM (medium, 160 kDa) and NFH (heavy, 205 kDa); type IV) and α-internexin (66 kDa; type IV), while in the PNS, NFs are made up of NFL, NFM, NFH and peripherin (57 kDa; type III) [2, 3]. Neurons may also express other IF proteins, including nestin (200 kDa; type IV), vimentin (57 kDa, type III) syncoilin isoforms (Sync1 (64 kDa), Sync2 (64 kDa); type III) and synemin isoforms (Low synemin (41 kDa), Middle or beta synemin (150 kDa), High or alpha synemin (180 kDa); type IV). While present in perikarya and dendrites, IFs are particularly abundant in large myelinated axons, where they are essential for axon radial growth during development and axon caliber maintenance [4]. Consequently they are crucial to optimize the conduction velocity of the nerve impulse. They also contribute to the dynamic properties of the axonal cytoskeleton during neuronal differentiation, axon outgrowth, regeneration and guidance [5]. The IF proteins share a common tripartite structure with a globular head, a central α-helical rod domain and variable tail domains that differ in length and amino acids compo‐ sition. The central rod domain of approximately 310 amino acids contains four α-helical re‐ gions and is involved in the assembly of 10 nm filaments [6]. NFM and NFH subunits differ from other neuronal IF proteins by their long tail domains containing numerous repeats of Lys-Ser-Pro (KSP) phosphorylation sites [4].

An increasing body of evidence supports the view that the most common mechanism of chronic neurodegenerative disorders involves abnormal protein production, processing or misfolding and subsequent accumulation in nervous system. Alterations in the metabolism and/or organization of neuronal IFs are frequently associated, directly or indirectly, with var‐ ious neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Charcot-Mar‐

© 2013 Perrot and Eyer; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ie-Tooth (CMT) disease, giant axonal neuropathy ( GAN), neuronal intermediate filament inclusion disease (NIFID), Parkinson disease (PD), diabetic neuropathy, dementia with Lewy bodies and spinal muscular atrophy [7]. While IF abnormalities in neurodegenerative disor‐ ders could simply reflect a pathological consequence of neuronal dysfunction, recent studies using transgenic mouse models suggested that IF disorganization itself can also produce del‐ eterious effects and therefore could contribute to the neurodegeneration process. Glial IF, and more particularly GFAP in astrocytes, is also the target of mutations leading to neurodege‐ nerative diseases. Astrocytes express various IF proteins, including nestin, vimentin and syn‐ emin, but GFAP is the most abundant. GFAP is a type III IF protein existing under different spliced forms. The relative abundance of these GFAP transcripts is variable and can be de‐ pendent upon astrocyte location or pathological states [8]. *GFAP* mutations lead to accumu‐ lations of GFAP protein and cause Alexander disease (AXD), a rare leukodystrophy. Here, we tempted to cover the current knowledge related to neuronal and glial IF involvement in human neurodegenerative diseases (Table 1).

**Disease Mutations in IF genes**

**PD** A point mutation in

**AXD** ≈ 100 mutations in the *GFAP* gene were identified in AXD patients. ≈ 95% of AXD cases are due to GFAP mutation.

**Table 1.** IFs in neurodegenerative diseases.

**2.1. Amyotrophic lateral sclerosis**

the *NEFM* gene was reported in only one case of PD with early onset.

**Accumulation of IFs Possible causes of IF**


inclusions containing all type IV neuronal IFs, and especially α-internexin.

Cytoplasmic inclusion bodies (Lewy bodies) composed of α-synuclein, NF proteins, ubiquitin and proteasome subunits. Inappropriate phosphorylation and proteolysis of NFs occur in Lewy bodies.

Presence of protein aggregates (Rosenthal fibers) composed of GFAP, αB-crystallin, HSP27 and ubiquitin within the cytoplasm of astrocytes throughout

the CNS.

**NIFID** No Neuronal cytoplasmic

**accumulation**

to the formation of distal axonal swelling with packed NFs. - Disturbed cytoskeleton regulation and modulation.


aggregation of GFAP. - Some mutations could impair interaction of GFAP with partners which normally prevent its assembly, resulting in the accumulation of GFAP polymers. - Insufficient amount of plectin seems promote GFAP aggregates.

**2. Neuronal intermediate filaments and neurodegenerative diseases**

ALS, also referred to as Lou Gehrig's disease, is a neurodegenerative disease which, by affect‐ ing the motor neurons in the motor cortex, brain stem and spinal cord, causes progressive physical impairment, together with worsening limitations in the functions of breathing, swal‐ lowing and communication. The disease has an incidence rate of 1-2 per 100,000, with a higher occurrence in men than in women. There is no cure and death usually occurs within 3 to 5

Not determined. Not determined. Abnormal

Intermediate Filaments in Neurodegenerative Diseases

Not determined. Data suggest no direct

**Possible roles of IF in disease pathogenesis**

http://dx.doi.org/10.5772/54676

459

accumulation of IFs may only be a secondary phenomenon.

implication of IFs in pathogenesis of PD.

The specific aspects of astrocytes functions that are compromised by the mutations of GFAP have not yet been discovered, but inhibited proteasome activity and activated stress pathways seemed to be important consequences of GFAP accumulation.



**Table 1.** IFs in neurodegenerative diseases.

ie-Tooth (CMT) disease, giant axonal neuropathy ( GAN), neuronal intermediate filament inclusion disease (NIFID), Parkinson disease (PD), diabetic neuropathy, dementia with Lewy bodies and spinal muscular atrophy [7]. While IF abnormalities in neurodegenerative disor‐ ders could simply reflect a pathological consequence of neuronal dysfunction, recent studies using transgenic mouse models suggested that IF disorganization itself can also produce del‐ eterious effects and therefore could contribute to the neurodegeneration process. Glial IF, and more particularly GFAP in astrocytes, is also the target of mutations leading to neurodege‐ nerative diseases. Astrocytes express various IF proteins, including nestin, vimentin and syn‐ emin, but GFAP is the most abundant. GFAP is a type III IF protein existing under different spliced forms. The relative abundance of these GFAP transcripts is variable and can be de‐ pendent upon astrocyte location or pathological states [8]. *GFAP* mutations lead to accumu‐ lations of GFAP protein and cause Alexander disease (AXD), a rare leukodystrophy. Here, we tempted to cover the current knowledge related to neuronal and glial IF involvement in human

**Accumulation of IFs Possible causes of IF**

Accumulation of peripherin and extensively

spheroids.



with abnormally packed

NFs.

cell body.

NFs.

**GAN** No - Enlarged axons filled

phosphorylated NFs in the perikaryon of motor neurons and in axonal

**accumulation**

motors.

mutant NFs.


proteins modifications.


phosphorylation of NFs and/or alteration of the molecular





**Possible roles of IF in disease pathogenesis**

animals.

body. - Alteration of

and dynamic.


chelators or

Paradoxically, perikaryal NF aggregates appeared protective in mouse models of ALS, slowing disease progression in these


phosphorylation sink. - Removal of NFs from the axonal compartment could enhance axonal transport.


mitochondrial morphology

neurodegenerative diseases (Table 1).

**Disease Mutations in IF genes**

458 Neurodegenerative Diseases

**ALS** - Only 3 variants

gene. - Peripherin mutations identified in 3 sporadic ALS patients.

**CMT** 20 mutations in the *NEFL* gene have been linked to CMT2E and CMT1F (represent ≈ 2% of CMT cases).

identified in *NEFH*

### **2. Neuronal intermediate filaments and neurodegenerative diseases**

#### **2.1. Amyotrophic lateral sclerosis**

ALS, also referred to as Lou Gehrig's disease, is a neurodegenerative disease which, by affect‐ ing the motor neurons in the motor cortex, brain stem and spinal cord, causes progressive physical impairment, together with worsening limitations in the functions of breathing, swal‐ lowing and communication. The disease has an incidence rate of 1-2 per 100,000, with a higher occurrence in men than in women. There is no cure and death usually occurs within 3 to 5 years from symptom onset. Only 10% of cases are inherited in an autosomal dominant pattern with the remaining 90% sporadic. 20% of all the familial cases are due to mutations in Cu/Zn superoxide dismutase 1 (SOD1), the most abundant cytosolic enzyme.

to reduce pathological accumulation of phosphorylated NFs. Pin1 associates with phosphory‐ lated NFH in neurons and is found in aggregates in spinal cord from ALS patients [33]. Its inhibition by inhibitor or down-regulating Pin1 levels reduces glutamate-induced perikaryal accumulation of phosphorylated NFH. Finally, riluzole protects against glutamate-induced slowing of NF axonal transport by decreasing perikaryal NF side-arm phosphorylation [34], probably via the inhibition of ERK and p38 activities, two NF kinases activated in ALS.

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Alterations of the anterograde or retrograde molecular motors may also be responsible for aggregation of IFs. Mutation of dynein or p150glued [35], overexpression of dynamitin [36] and absence of kinesin heavy chain isoform 5A (KIF5A) [37] induce NF accumulations in mice. Recent studies suggest that inhibition of retrograde transport is more susceptible to cause accumulation of NFs than inhibition of anterograde transport. The inhibition of dynein by increasing the level of dynamitin induces aberrant focal accumulation of NFs within axonal neurites whereas inhibition of kinesin inhibits anterograde transport but does not induce sim‐ ilar focal aggregations [38]. Similarly, the neuron-specific expression of Bicaudal D2 N-termi‐ nus (BICD2-N), a motor-adaptor protein, impairs dynein-dynactin function, causing the appearance of giant NF swellings in the proximal axons [39]. However these mice did not

Modification in NF stoichiometry was also proposed to induce accumulation of NFs. Sin‐ gly overexpressing any of the NF subunit in transgenic mice led to prominent motor neuropathy characterized by the presence of abnormal NF accumulations resembling those found in ALS [40-42]. Remarkably, the motor neuron disease caused by excess hu‐ man NFH (hNFH) can be rescued by restoring a correct stoichiometry of NF subunits via the overexpression of hNFL in a dosage-dependent fashion [43]. Overexpression of peripherin in mice also provokes the formation of cytoplasmic protein aggregates and the subsequent selective loss of motor neurons during ageing [44, 45]. This loss is pre‐ ceded by axonal transport defects and formation of axonal spheroids [46]. Because NFL mRNA levels are reduced in cases of ALS, Beaulieu et al. [45] generated double trans‐ genic mice overexpressing peripherin and deficient for NFL (Per;NFL-/- mice). Here, the onset of peripherin-mediated disease is accelerated by the deficiency of NFL. Without NFL, peripherin interacts with NFM and NFH to form disorganized IF structures. This could explain why the number of IF inclusion bodies is increased in Per;NFL-/- mice, leading to an earlier neuronal death and to defects of fast axonal transport in cultured Per;NFL-/- neurons [47]. In contrast, peripherin toxicity can be attenuated by coexpres‐ sion of NFL or NFH [48, 49], illustrating once again the importance of IF protein stoichi‐ ometry. NFH overexpression shifted the intracellular localization of inclusion bodies from the axonal to the perikaryal compartment of motor neurons, suggesting that the toxicity of peripherin inclusions may be related to their axonal localization, possibly by altering the axonal transport. However, it should be noted that peripherin is not a key contributing factor to the neuronal death in disease caused by SOD1 mutations because absence or overexpression of peripherin in SOD1G37R mice do not affect the onset and

develop signs of motor neuron degeneration and motor abnormalities.

progression of motor neuron disease [50].

One common pathological finding of both sporadic and familial ALS is the accumulation of NFs and/or peripherin in the perikaryon of motor neurons and in axonal spheroids [9]. Because of the presence of IF proteins in these aggregates, several studies have searched for mutations in genes coding for NF proteins and peripherin. The discovery of a small number of NF gene variants in ALS patients suggested the involvement of NFs in the pathogenesis of the disease. Indeed, codon deletions or insertions in the KSP repeat motifs of NFH have been identified in patients with sporadic ALS [10-12]. However, two others studies failed to identify such var‐ iants in the NF genes linked to sporadic and familial ALS [13, 14], suggesting that mutations in the NF genes are not a systematic common cause of ALS but could be a risk factor for sporadic ALS. Peripherin mutations have also been identified in three sporadic ALS patients [15-17], including a frameshift mutation in the *PRPH* gene able to disrupt the NF network assembly *in vitro*, reinforcing the view that NF disorganization may contribute to pathogenesis. These results suggest that peripherin mutations may be responsible for a small percentage of ALS cases. Two peripherin isoforms have been linked to ALS: aggregate-inducing Per28 is upre‐ gulated in patients with ALS, at both the mRNA and protein levels, and an antibody specific for Per28 stained the filamentous inclusions [18]. The Per61 splice variant is neurotoxic and has been observed in ALS mouse models and human patients [19]. These observations raise the possibility that missplicing of peripherin could lead to disease. It is also of interest to note the presence of high NFL and NFH levels and auto-antibodies against NFL in cerebrospinal fluid of ALS patients [20-22]. Furthermore, plasma NFH levels closely reflect later stages of disease progression and therapeutic response in a mouse model of ALS [23]. In the same way, a significant relation exists between cerebrospinal fluid NFL levels and disease progression in ALS patients [24]. Accordingly, it seems that NFs levels may be valuable biomarkers of later disease progression in ALS.

NFs found in perikaryal aggregates are extensively phosphorylated, a process that occurs normally only within the axon [25]. The mechanisms governing the formation of IF aggregates are still not clearly established but defects of axonal transport or abnormal stoichiometry of IF proteins could be involved. Perturbations of the axonal transport of NFs and organelles are one of the earliest pathological changes seen in several transgenic mouse models of ALS [26-29]. The premature phosphorylation of NF tail-domains in motor neurons cell bodies could directly mediate their accumulation in this region. Glutamate excitotoxicity, another patho‐ genic process in ALS, may induce this abnormal phosphorylation of NFs. Treatment of primary neurons with glutamate activates members of the mitogen-activated protein kinase family which phosphorylate NFs with ensuing slowing of their axonal transport [30]. In addition, glutamate leads to caspase cleavage and activation of protein kinase N1 (PKN1), a NF headrod domain kinase [31]. This cleaved form of PKN1 disrupts NF organization and axonal transport. Excitotoxicity mediated by non-N-methyl-D-aspartic acid (NMDA) receptor is also associated with the aberrant colocalization of phosphorylated and dephosphorylated NF pro‐ teins [32]. Inhibition of Pin1, a prolyl isomerase, was suggested as a possible therapeutic target to reduce pathological accumulation of phosphorylated NFs. Pin1 associates with phosphory‐ lated NFH in neurons and is found in aggregates in spinal cord from ALS patients [33]. Its inhibition by inhibitor or down-regulating Pin1 levels reduces glutamate-induced perikaryal accumulation of phosphorylated NFH. Finally, riluzole protects against glutamate-induced slowing of NF axonal transport by decreasing perikaryal NF side-arm phosphorylation [34], probably via the inhibition of ERK and p38 activities, two NF kinases activated in ALS.

years from symptom onset. Only 10% of cases are inherited in an autosomal dominant pattern with the remaining 90% sporadic. 20% of all the familial cases are due to mutations in Cu/Zn

One common pathological finding of both sporadic and familial ALS is the accumulation of NFs and/or peripherin in the perikaryon of motor neurons and in axonal spheroids [9]. Because of the presence of IF proteins in these aggregates, several studies have searched for mutations in genes coding for NF proteins and peripherin. The discovery of a small number of NF gene variants in ALS patients suggested the involvement of NFs in the pathogenesis of the disease. Indeed, codon deletions or insertions in the KSP repeat motifs of NFH have been identified in patients with sporadic ALS [10-12]. However, two others studies failed to identify such var‐ iants in the NF genes linked to sporadic and familial ALS [13, 14], suggesting that mutations in the NF genes are not a systematic common cause of ALS but could be a risk factor for sporadic ALS. Peripherin mutations have also been identified in three sporadic ALS patients [15-17], including a frameshift mutation in the *PRPH* gene able to disrupt the NF network assembly *in vitro*, reinforcing the view that NF disorganization may contribute to pathogenesis. These results suggest that peripherin mutations may be responsible for a small percentage of ALS cases. Two peripherin isoforms have been linked to ALS: aggregate-inducing Per28 is upre‐ gulated in patients with ALS, at both the mRNA and protein levels, and an antibody specific for Per28 stained the filamentous inclusions [18]. The Per61 splice variant is neurotoxic and has been observed in ALS mouse models and human patients [19]. These observations raise the possibility that missplicing of peripherin could lead to disease. It is also of interest to note the presence of high NFL and NFH levels and auto-antibodies against NFL in cerebrospinal fluid of ALS patients [20-22]. Furthermore, plasma NFH levels closely reflect later stages of disease progression and therapeutic response in a mouse model of ALS [23]. In the same way, a significant relation exists between cerebrospinal fluid NFL levels and disease progression in ALS patients [24]. Accordingly, it seems that NFs levels may be valuable biomarkers of later

NFs found in perikaryal aggregates are extensively phosphorylated, a process that occurs normally only within the axon [25]. The mechanisms governing the formation of IF aggregates are still not clearly established but defects of axonal transport or abnormal stoichiometry of IF proteins could be involved. Perturbations of the axonal transport of NFs and organelles are one of the earliest pathological changes seen in several transgenic mouse models of ALS [26-29]. The premature phosphorylation of NF tail-domains in motor neurons cell bodies could directly mediate their accumulation in this region. Glutamate excitotoxicity, another patho‐ genic process in ALS, may induce this abnormal phosphorylation of NFs. Treatment of primary neurons with glutamate activates members of the mitogen-activated protein kinase family which phosphorylate NFs with ensuing slowing of their axonal transport [30]. In addition, glutamate leads to caspase cleavage and activation of protein kinase N1 (PKN1), a NF headrod domain kinase [31]. This cleaved form of PKN1 disrupts NF organization and axonal transport. Excitotoxicity mediated by non-N-methyl-D-aspartic acid (NMDA) receptor is also associated with the aberrant colocalization of phosphorylated and dephosphorylated NF pro‐ teins [32]. Inhibition of Pin1, a prolyl isomerase, was suggested as a possible therapeutic target

superoxide dismutase 1 (SOD1), the most abundant cytosolic enzyme.

disease progression in ALS.

460 Neurodegenerative Diseases

Alterations of the anterograde or retrograde molecular motors may also be responsible for aggregation of IFs. Mutation of dynein or p150glued [35], overexpression of dynamitin [36] and absence of kinesin heavy chain isoform 5A (KIF5A) [37] induce NF accumulations in mice. Recent studies suggest that inhibition of retrograde transport is more susceptible to cause accumulation of NFs than inhibition of anterograde transport. The inhibition of dynein by increasing the level of dynamitin induces aberrant focal accumulation of NFs within axonal neurites whereas inhibition of kinesin inhibits anterograde transport but does not induce sim‐ ilar focal aggregations [38]. Similarly, the neuron-specific expression of Bicaudal D2 N-termi‐ nus (BICD2-N), a motor-adaptor protein, impairs dynein-dynactin function, causing the appearance of giant NF swellings in the proximal axons [39]. However these mice did not develop signs of motor neuron degeneration and motor abnormalities.

Modification in NF stoichiometry was also proposed to induce accumulation of NFs. Sin‐ gly overexpressing any of the NF subunit in transgenic mice led to prominent motor neuropathy characterized by the presence of abnormal NF accumulations resembling those found in ALS [40-42]. Remarkably, the motor neuron disease caused by excess hu‐ man NFH (hNFH) can be rescued by restoring a correct stoichiometry of NF subunits via the overexpression of hNFL in a dosage-dependent fashion [43]. Overexpression of peripherin in mice also provokes the formation of cytoplasmic protein aggregates and the subsequent selective loss of motor neurons during ageing [44, 45]. This loss is pre‐ ceded by axonal transport defects and formation of axonal spheroids [46]. Because NFL mRNA levels are reduced in cases of ALS, Beaulieu et al. [45] generated double trans‐ genic mice overexpressing peripherin and deficient for NFL (Per;NFL-/- mice). Here, the onset of peripherin-mediated disease is accelerated by the deficiency of NFL. Without NFL, peripherin interacts with NFM and NFH to form disorganized IF structures. This could explain why the number of IF inclusion bodies is increased in Per;NFL-/- mice, leading to an earlier neuronal death and to defects of fast axonal transport in cultured Per;NFL-/- neurons [47]. In contrast, peripherin toxicity can be attenuated by coexpres‐ sion of NFL or NFH [48, 49], illustrating once again the importance of IF protein stoichi‐ ometry. NFH overexpression shifted the intracellular localization of inclusion bodies from the axonal to the perikaryal compartment of motor neurons, suggesting that the toxicity of peripherin inclusions may be related to their axonal localization, possibly by altering the axonal transport. However, it should be noted that peripherin is not a key contributing factor to the neuronal death in disease caused by SOD1 mutations because absence or overexpression of peripherin in SOD1G37R mice do not affect the onset and progression of motor neuron disease [50].

Changes in stoichiometry were reported in ALS motor neurons as the levels of NFL, α-inter‐ nexin and peripherin mRNA are decreased, while in familial ALS the levels of peripherin mRNA appear to be abnormally elevated [51-53]. This suggests a change in the stoichiometry of cytoskeletal protein expression which could be conducive to the formation of neurofila‐ mentous aggregates in ALS. This decrease of IF mRNA could be due in part to modification in their stability. Several NFL mRNA binding proteins have been identified in human, includ‐ ing 14-3-3 proteins [54], TAR (trans-active regulatory) DNA-binding protein (TDP43) [55], both mutant and wild-type SOD1 [56] and Rho guanine nucleotide exchange factor (RGNEF) [57]. These proteins are incorporated in ALS intraneuronal aggregates and affect the stability of NFL mRNA. Mice expressing human TDP-43 displayed reduced NF mRNAs and proteins contents, inducing a decrease of caliber of their motor axons [58]. The involvement of TDP-43 in ALS pathogenesis was reinforced by the recent discovery of several mutant forms of this protein in familial and sporadic ALS [59]. Motor neurons from mice expressing such mutated TDP-43 displayed peripherin and NFs (NFM and NFH) aggregates, concomitant with a down‐ regulation of NFL and an overexpression of peripherin [60]. In addition, they detected in these mice the presence of abnormal splicing variants of peripherin, such as Per61, that can contrib‐ ute to formation of IF aggregates. RGNEF is another RNA binding protein that acts as an NFL mRNA stability factor via 3′ untranslated region destabilization and reduces NFL protein lev‐ els when overexpressed in a stable cell line. Furthermore, RGNEF cytoplasmic inclusions were detected in ALS spinal motor neurons that colocalized with ubiquitin, p62/sequestosome-1, and TDP-43 [61]. These observations provide a possible mechanism for NF aggregate forma‐ tion together with a link between ALS and Rho signaling pathways.

NFH, α-internexin and peripherin, and induced the accumulation of mitochondria and vesicle-like structures, suggesting a disruption of the axonal transport. Moreover, the se‐ verity of this axonopathy correlated with the phenotype of the glial cells, with a signifi‐ cant increase being induced by a glial feeder layer expressing mutant SOD1 or that was

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To further determine whether NFs are directly involved in SOD1-mediated disease, mice ex‐ pressing mutant SOD1 were mated with transgenic mice deficient for axonal NFs. The with‐ drawal of NFs from the axonal compartment and their perikaryal accumulation induced by the expression of NFH-β-galactosidase fusion protein conferred no beneficial effect to SOD1G37R mice [68], indicating that axonal NFs are not necessary for SOD1-mediated disease. This was also observed in SOD1G85R mice deprived of NFL, but the absence of axonal NFs in these animals prolongs their life span by approximately 15% [69]. Surprisingly, overexpression of mouse NFL or mouse NFH in SOD1G93A mice [70], and overexpression of hNFH in SOD1G37R mice [71], also increase their life span by respectively 15% and 65%. This suggests a protective effect of NF perikaryal accumulation in motor neuron disease caused by mutant SOD1. While the mechanism of protection is unclear, it seems that perikaryal accumulation of NFs rather than their axonal deficiency is responsible for slowing disease in these models. Indeed, the formation of large perikaryal aggregates and a massive depletion of axonal NFs due to the expression of the human NFH43 allele cause more positive effects than human NFH44 allele which induces smaller aggregates and more axonal NFs [71]. Moreover, the dis‐ ruption of one allele for each NF gene induces a 40% decrease of axonal NF proteins content and an important axonal atrophy without perikaryal accumulation of NFs in SOD1G37R mice, but it does not extend their life span nor does it alleviate the loss of motor axons [72]. Several hypotheses were proposed to explain this protective effect of perikaryal aggregates in SOD1 mediated disease. Through their multiple calcium-binding sites NFs may act as calcium che‐ lators. Supporting this hypothesis, a significant neuroprotection was obtained by overexpressing the calcium-binding protein calbindin-D28k in cultured motor neurons [73]. It was also proposed that perikaryal accumulations of NFs in motor neurons may alleviate ALS pathogenesis by acting as a phosphorylation sink for cyclin-dependent kinase 5 dysre‐ gulation induced by mutant SOD1, thereby reducing the excessive phosphorylation of tau and other neuronal substrates [72]. This was supported by the fact that NF accumulations contain hyperphosphorylated NFM and NFH subunits in ALS patients [25] and in SOD1 mutant mice [74]. However, removal of NFM and NFH sidearms led to a delay of disease in SOD1 mutant mice rather than the acceleration predicted by a kinase dysregulation model [75], indicating that perikaryal phosphorylation of NFs is not an essential contributor to reduced toxicity of SOD1 mutants and that abnormal phosphorylation of NF proteins may be a detrimental factor. Alternatively, axonal removal of NFs could enhance axonal transport, which is impaired in

Finally, it was shown that NFs are involved in the localization of NMDA receptors in the neuronal plasma membrane by interacting with the NMDA NR1 subunit [76]. Thus, accumu‐ lation of NFs could interfere with glutamate receptor function and prevent glutamate excito‐ toxicity. However, NF aggregate-bearing neurons demonstrate increased intracellular calcium

pre-aged prior to plating the motor neurons [67].

SOD1 mice, by providing a more flexible axoplasm.

Neuronal IF abnormalities in ALS may also occur as a result of post-translational protein modifications. Indeed, advanced glycation endproducts were detected in NF aggregates of motor neurons in familial and sporadic ALS [62]. *O*-glycosylation of NFM is strongly decreased in spinal cord of different models for ALS, whereas phosphorylation is increased relative to total NFM [63, 64], suggesting competition of the binding sites of these two modifications and a potential mechanism for the formation of NF protein accumulations in ALS. Interestingly, inhibition of *O*-GlcNAcase (OGA), the enzyme catalyzing removal of *O*-GlcNAc, increased levels of *O*-GlcNAc modified NFM in spinal cords of control mice, but not in mutant SOD1 mice. Moreover, phosphorylation state of NFM appeared unchanged in these mutant mice [64]. The authors speculate that this lack of difference in NFM phosphorylation in mutant SOD1 mice may arise from the aggregation of hyperphosphorylated NFs, which may prevent de‐ phosphorylation and subsequent *O*-GlcNAc modification. It was also showed that SOD1 can catalyze nitration of tyrosines by peroxynitrite in the rod and head domains of NFL [65]. However, no significant changes were detected in the nitration of NFL isolated from cervical spinal cord tissue of sporadic ALS cases [66].

Finally, it seems that non-neuronal cells could be directly involved in the formation of cytoskeletal aggregates within proximal axon from motor neurons. Indeed, cultured mouse spinal motor neurons in contact with non-neuronal cells displayed swellings that were morphologically and neurochemically comparable to axonal spheroids that develop *in vivo* in ALS transgenic mouse models [67]. These swellings contained NFL, NFM, NFH, α-internexin and peripherin, and induced the accumulation of mitochondria and vesicle-like structures, suggesting a disruption of the axonal transport. Moreover, the se‐ verity of this axonopathy correlated with the phenotype of the glial cells, with a signifi‐ cant increase being induced by a glial feeder layer expressing mutant SOD1 or that was pre-aged prior to plating the motor neurons [67].

Changes in stoichiometry were reported in ALS motor neurons as the levels of NFL, α-inter‐ nexin and peripherin mRNA are decreased, while in familial ALS the levels of peripherin mRNA appear to be abnormally elevated [51-53]. This suggests a change in the stoichiometry of cytoskeletal protein expression which could be conducive to the formation of neurofila‐ mentous aggregates in ALS. This decrease of IF mRNA could be due in part to modification in their stability. Several NFL mRNA binding proteins have been identified in human, includ‐ ing 14-3-3 proteins [54], TAR (trans-active regulatory) DNA-binding protein (TDP43) [55], both mutant and wild-type SOD1 [56] and Rho guanine nucleotide exchange factor (RGNEF) [57]. These proteins are incorporated in ALS intraneuronal aggregates and affect the stability of NFL mRNA. Mice expressing human TDP-43 displayed reduced NF mRNAs and proteins contents, inducing a decrease of caliber of their motor axons [58]. The involvement of TDP-43 in ALS pathogenesis was reinforced by the recent discovery of several mutant forms of this protein in familial and sporadic ALS [59]. Motor neurons from mice expressing such mutated TDP-43 displayed peripherin and NFs (NFM and NFH) aggregates, concomitant with a down‐ regulation of NFL and an overexpression of peripherin [60]. In addition, they detected in these mice the presence of abnormal splicing variants of peripherin, such as Per61, that can contrib‐ ute to formation of IF aggregates. RGNEF is another RNA binding protein that acts as an NFL mRNA stability factor via 3′ untranslated region destabilization and reduces NFL protein lev‐ els when overexpressed in a stable cell line. Furthermore, RGNEF cytoplasmic inclusions were detected in ALS spinal motor neurons that colocalized with ubiquitin, p62/sequestosome-1, and TDP-43 [61]. These observations provide a possible mechanism for NF aggregate forma‐

tion together with a link between ALS and Rho signaling pathways.

spinal cord tissue of sporadic ALS cases [66].

462 Neurodegenerative Diseases

Neuronal IF abnormalities in ALS may also occur as a result of post-translational protein modifications. Indeed, advanced glycation endproducts were detected in NF aggregates of motor neurons in familial and sporadic ALS [62]. *O*-glycosylation of NFM is strongly decreased in spinal cord of different models for ALS, whereas phosphorylation is increased relative to total NFM [63, 64], suggesting competition of the binding sites of these two modifications and a potential mechanism for the formation of NF protein accumulations in ALS. Interestingly, inhibition of *O*-GlcNAcase (OGA), the enzyme catalyzing removal of *O*-GlcNAc, increased levels of *O*-GlcNAc modified NFM in spinal cords of control mice, but not in mutant SOD1 mice. Moreover, phosphorylation state of NFM appeared unchanged in these mutant mice [64]. The authors speculate that this lack of difference in NFM phosphorylation in mutant SOD1 mice may arise from the aggregation of hyperphosphorylated NFs, which may prevent de‐ phosphorylation and subsequent *O*-GlcNAc modification. It was also showed that SOD1 can catalyze nitration of tyrosines by peroxynitrite in the rod and head domains of NFL [65]. However, no significant changes were detected in the nitration of NFL isolated from cervical

Finally, it seems that non-neuronal cells could be directly involved in the formation of cytoskeletal aggregates within proximal axon from motor neurons. Indeed, cultured mouse spinal motor neurons in contact with non-neuronal cells displayed swellings that were morphologically and neurochemically comparable to axonal spheroids that develop *in vivo* in ALS transgenic mouse models [67]. These swellings contained NFL, NFM, To further determine whether NFs are directly involved in SOD1-mediated disease, mice ex‐ pressing mutant SOD1 were mated with transgenic mice deficient for axonal NFs. The with‐ drawal of NFs from the axonal compartment and their perikaryal accumulation induced by the expression of NFH-β-galactosidase fusion protein conferred no beneficial effect to SOD1G37R mice [68], indicating that axonal NFs are not necessary for SOD1-mediated disease. This was also observed in SOD1G85R mice deprived of NFL, but the absence of axonal NFs in these animals prolongs their life span by approximately 15% [69]. Surprisingly, overexpression of mouse NFL or mouse NFH in SOD1G93A mice [70], and overexpression of hNFH in SOD1G37R mice [71], also increase their life span by respectively 15% and 65%. This suggests a protective effect of NF perikaryal accumulation in motor neuron disease caused by mutant SOD1. While the mechanism of protection is unclear, it seems that perikaryal accumulation of NFs rather than their axonal deficiency is responsible for slowing disease in these models. Indeed, the formation of large perikaryal aggregates and a massive depletion of axonal NFs due to the expression of the human NFH43 allele cause more positive effects than human NFH44 allele which induces smaller aggregates and more axonal NFs [71]. Moreover, the dis‐ ruption of one allele for each NF gene induces a 40% decrease of axonal NF proteins content and an important axonal atrophy without perikaryal accumulation of NFs in SOD1G37R mice, but it does not extend their life span nor does it alleviate the loss of motor axons [72]. Several hypotheses were proposed to explain this protective effect of perikaryal aggregates in SOD1 mediated disease. Through their multiple calcium-binding sites NFs may act as calcium che‐ lators. Supporting this hypothesis, a significant neuroprotection was obtained by overexpressing the calcium-binding protein calbindin-D28k in cultured motor neurons [73]. It was also proposed that perikaryal accumulations of NFs in motor neurons may alleviate ALS pathogenesis by acting as a phosphorylation sink for cyclin-dependent kinase 5 dysre‐ gulation induced by mutant SOD1, thereby reducing the excessive phosphorylation of tau and other neuronal substrates [72]. This was supported by the fact that NF accumulations contain hyperphosphorylated NFM and NFH subunits in ALS patients [25] and in SOD1 mutant mice [74]. However, removal of NFM and NFH sidearms led to a delay of disease in SOD1 mutant mice rather than the acceleration predicted by a kinase dysregulation model [75], indicating that perikaryal phosphorylation of NFs is not an essential contributor to reduced toxicity of SOD1 mutants and that abnormal phosphorylation of NF proteins may be a detrimental factor. Alternatively, axonal removal of NFs could enhance axonal transport, which is impaired in SOD1 mice, by providing a more flexible axoplasm.

Finally, it was shown that NFs are involved in the localization of NMDA receptors in the neuronal plasma membrane by interacting with the NMDA NR1 subunit [76]. Thus, accumu‐ lation of NFs could interfere with glutamate receptor function and prevent glutamate excito‐ toxicity. However, NF aggregate-bearing neurons demonstrate increased intracellular calcium levels and enhanced cell death in response to NMDA receptor activation without increased NMDA receptor expression. These results suggest that the presence of NF aggregates renders motor neurons more susceptible to NMDA-mediated excitotoxicity [77].

tation revealed a mixed axonal and demyelinating neuropathy [85], emphasizing the complexity of genotype-phenotype correlations in CMT. Finally, the P22T mutation was detected in unrelated Japanese patients with CMT disease [86]. The formation of NF ag‐ gregates in patients expressing NFLP22S and NFLP22T mutant proteins could be explain by the ability of these mutations to abolish the phosphorylation of the adjacent Thr21 by cyclin-dependent kinase 5, which normally inhibits filament assembly [87]. The phos‐ phorylation of NFL head domain by PKA alleviated aggregates in cortical neurons, pro‐ viding a potential therapeutic approach to dissociate NF aggregates in CMT disease [87].

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The screening of 323 patients with CMT or related peripheral neuropathies allowed the identification of six disease-associated missense mutations and one 3-bp in-frame deletion in the *NEFL* gene [88]. Other mutations were also detected in Korean CMT patients [89], in a German family [90], and four mutations in the head and rod domains of NFL, includ‐ ing a L268P substitution and a del322Cys\_326 Asn deletion, were identified by the screen‐ ing of 177 patients [91]. Most of these mutated proteins (except E7K and D469N) form aggregates, and thus could alter the axonal transport following their abnormal aggrega‐ tion in cell bodies and axons. A duplication-insertion mutation of NFL in a patient with CMT was also reported [92], which probably provoked neuronal degeneration through both aggregation and destabilization of the IF network. Finally, new mutations in the *NEFL* gene were identified following the screening of 223 Japanese CMT patients [93]. Four heterozygous missense mutations (P8L, E90K, N98S and E396K) were detected in five unrelated patients as well as a homozygous nonsense mutation (E140Stop) in one pa‐ tient. All these patients displayed moderate delayed nerve conduction velocities, possibly caused by a loss of large diameter fibers. This study suggested that nonsense *NEFL* muta‐ tions probably cause a recessive phenotype, while missense mutations cause a dominant phenotype [93]. The majority of NFL mutations are linked to axonal forms of CMT but their implication in demyelinating CMT cannot be excluded since nerves from patients ex‐ pressing NFLL268P or NFLE90K showed evidence of Schwann cell abnormalities [88, 91].

The first mouse model of the CMT2E disease expressed the hNFLP22S mutant protein specifi‐ cally in the nervous system and recapitulate many of the overt phenotypes observed in CMT2E patients, including aberrant hind limb posture, motor deficits, hypertrophy of muscle fibres and loss of muscle innervation without neuronal loss [94]. To address whether CMT2E disease is potentially reversible, this mouse model was based on the tetracycline-responsive gene sys‐ tem that allows the suppression of mutant hNFLP22S expression in mature neurons through administration of doxycycline. Remarkably, a 3-month treatment of these mice with doxycy‐ cline after disease onset efficiently down-regulated expression of hNFLP22S and reversed the neurological phenotype [94], providing hope that future therapeutic strategies might not only stop progress of CMT2E disease but also reverse it. A novel line of CMT2E mice that consti‐ tutively express hNFLE397K was recently generated [95]. As with the hNFLP22S mice, these mice developed as early as 4 months signs consistent with CMT2E patients, such as aberrant hind limb posture, digit deformities, reduced locomotor activity and reduced motor nerve conduc‐ tion velocities. However, some aspects differed between the two lines of CMT2E mice. Indeed, hNFLE397K mice showed no significant denervation and their muscles were atrophied. More‐

#### **2.2. Charcot-Marie-Tooth disease**

CMT represents a heterogeneous group of inherited peripheral neuropathies affecting both motor and sensory neurons to the muscles. CMT is the most common inherited disorder of the PNS, with approximately 1 per 2,500 people affected. Patients slowly lose function of their feet/ legs and hand/arms as nerves to the extremities degenerate. First signs typically appear in the first or second decade of life, although it may be detected in infancy. This disease shows a high degree of heterogeneity, both in the clinical presentation and at the genetic level. CMT was originally subclassified into CMT1 and CMT2 on the basis of electrophysiological properties and histopathology. CMT1 is a demyelinating disease with reduced nerve conduction velocity whereas CMT2 is an axonal neuropathy with relatively normal nerve conduction velocity. CMT patients show a high degree of heterogeneity, due to mutations in multiple genes. This led to the distinction of other subtypes of CMT, including CMT3 (or Dejerine-Sottas disease, a particularly severe demyelinating form of CMT), CMT4 (autosomal recessive form of de‐ myelinating CMT) and CMTX (X-linked form of CMT with both demyelinating and axonal features). Moreover, each type of CMT has several subtypes.

Vogel et al. [78] reported the presence of NF accumulations in CMT. Evidence for the involve‐ ment of IFs in the pathogenesis of CMT was provided by the identification of 20 mutations of the *NEFL* gene on chromosome 8 in patients with CMT1F and CMT2E. Mutations in *NEFL* gene are responsible for approximately 2% of CMT cases and a high percentage of CMT2 cases. These mutations are located throughout the three functional domains of this protein (head, rod and tail) and consist of substitutions, deletions and frame-shift mutations. Co-expression of most NFL mutants with wild-type NFM or NFH subunits disrupted the NF cytoskeleton *in vitro*, resulting in the formation of aggregates within the cell body [79, 80]. The first two CMTassociated *NEFL* mutations, NFLP8R and NFLQ333P, were identified in respectively a Belgian and a Russian family. In addition to disturb the assembly of NFs, these mutations affect the axonal transport of wild-type and mutant NFs, but also the transport of mitochondria and human amyloid β protein precursor, resulting in alterations of retrograde axonal transport, fragmen‐ tation of the Golgi apparatus and increased neuritic degeneration [79, 80]. The effect of these mutant proteins on filament assembly was dominant, since wild-type NFL could not rescue the assembly defect. Filament formation was also abolished in SW13 cells by the rod domain A148V mutation [81]. These data provide possible mechanisms by which these mutants could be involved in axonal degeneration and CMT pathogenesis.

The Pro-22 residue of NFL is also the target of several mutations: P22R, P22S and P22T. The P22R mutation, identified in a Korean family, is associated with demyelinating neu‐ ropathy features of CMT1F [82]. The P22S substitution was first described in a Sloven‐ ian CMT2 family [83], then in an Italian family developing a primary axonopathy characterized by giant axons with swellings composed essentially of aggregated NFs [84]. Interestingly, clinical and electrophysiological studies from patients with P22S mu‐ tation revealed a mixed axonal and demyelinating neuropathy [85], emphasizing the complexity of genotype-phenotype correlations in CMT. Finally, the P22T mutation was detected in unrelated Japanese patients with CMT disease [86]. The formation of NF ag‐ gregates in patients expressing NFLP22S and NFLP22T mutant proteins could be explain by the ability of these mutations to abolish the phosphorylation of the adjacent Thr21 by cyclin-dependent kinase 5, which normally inhibits filament assembly [87]. The phos‐ phorylation of NFL head domain by PKA alleviated aggregates in cortical neurons, pro‐ viding a potential therapeutic approach to dissociate NF aggregates in CMT disease [87].

levels and enhanced cell death in response to NMDA receptor activation without increased NMDA receptor expression. These results suggest that the presence of NF aggregates renders

CMT represents a heterogeneous group of inherited peripheral neuropathies affecting both motor and sensory neurons to the muscles. CMT is the most common inherited disorder of the PNS, with approximately 1 per 2,500 people affected. Patients slowly lose function of their feet/ legs and hand/arms as nerves to the extremities degenerate. First signs typically appear in the first or second decade of life, although it may be detected in infancy. This disease shows a high degree of heterogeneity, both in the clinical presentation and at the genetic level. CMT was originally subclassified into CMT1 and CMT2 on the basis of electrophysiological properties and histopathology. CMT1 is a demyelinating disease with reduced nerve conduction velocity whereas CMT2 is an axonal neuropathy with relatively normal nerve conduction velocity. CMT patients show a high degree of heterogeneity, due to mutations in multiple genes. This led to the distinction of other subtypes of CMT, including CMT3 (or Dejerine-Sottas disease, a particularly severe demyelinating form of CMT), CMT4 (autosomal recessive form of de‐ myelinating CMT) and CMTX (X-linked form of CMT with both demyelinating and axonal

Vogel et al. [78] reported the presence of NF accumulations in CMT. Evidence for the involve‐ ment of IFs in the pathogenesis of CMT was provided by the identification of 20 mutations of the *NEFL* gene on chromosome 8 in patients with CMT1F and CMT2E. Mutations in *NEFL* gene are responsible for approximately 2% of CMT cases and a high percentage of CMT2 cases. These mutations are located throughout the three functional domains of this protein (head, rod and tail) and consist of substitutions, deletions and frame-shift mutations. Co-expression of most NFL mutants with wild-type NFM or NFH subunits disrupted the NF cytoskeleton *in vitro*, resulting in the formation of aggregates within the cell body [79, 80]. The first two CMTassociated *NEFL* mutations, NFLP8R and NFLQ333P, were identified in respectively a Belgian and a Russian family. In addition to disturb the assembly of NFs, these mutations affect the axonal transport of wild-type and mutant NFs, but also the transport of mitochondria and human amyloid β protein precursor, resulting in alterations of retrograde axonal transport, fragmen‐ tation of the Golgi apparatus and increased neuritic degeneration [79, 80]. The effect of these mutant proteins on filament assembly was dominant, since wild-type NFL could not rescue the assembly defect. Filament formation was also abolished in SW13 cells by the rod domain A148V mutation [81]. These data provide possible mechanisms by which these mutants could

The Pro-22 residue of NFL is also the target of several mutations: P22R, P22S and P22T. The P22R mutation, identified in a Korean family, is associated with demyelinating neu‐ ropathy features of CMT1F [82]. The P22S substitution was first described in a Sloven‐ ian CMT2 family [83], then in an Italian family developing a primary axonopathy characterized by giant axons with swellings composed essentially of aggregated NFs [84]. Interestingly, clinical and electrophysiological studies from patients with P22S mu‐

motor neurons more susceptible to NMDA-mediated excitotoxicity [77].

features). Moreover, each type of CMT has several subtypes.

be involved in axonal degeneration and CMT pathogenesis.

**2.2. Charcot-Marie-Tooth disease**

464 Neurodegenerative Diseases

The screening of 323 patients with CMT or related peripheral neuropathies allowed the identification of six disease-associated missense mutations and one 3-bp in-frame deletion in the *NEFL* gene [88]. Other mutations were also detected in Korean CMT patients [89], in a German family [90], and four mutations in the head and rod domains of NFL, includ‐ ing a L268P substitution and a del322Cys\_326 Asn deletion, were identified by the screen‐ ing of 177 patients [91]. Most of these mutated proteins (except E7K and D469N) form aggregates, and thus could alter the axonal transport following their abnormal aggrega‐ tion in cell bodies and axons. A duplication-insertion mutation of NFL in a patient with CMT was also reported [92], which probably provoked neuronal degeneration through both aggregation and destabilization of the IF network. Finally, new mutations in the *NEFL* gene were identified following the screening of 223 Japanese CMT patients [93]. Four heterozygous missense mutations (P8L, E90K, N98S and E396K) were detected in five unrelated patients as well as a homozygous nonsense mutation (E140Stop) in one pa‐ tient. All these patients displayed moderate delayed nerve conduction velocities, possibly caused by a loss of large diameter fibers. This study suggested that nonsense *NEFL* muta‐ tions probably cause a recessive phenotype, while missense mutations cause a dominant phenotype [93]. The majority of NFL mutations are linked to axonal forms of CMT but their implication in demyelinating CMT cannot be excluded since nerves from patients ex‐ pressing NFLL268P or NFLE90K showed evidence of Schwann cell abnormalities [88, 91].

The first mouse model of the CMT2E disease expressed the hNFLP22S mutant protein specifi‐ cally in the nervous system and recapitulate many of the overt phenotypes observed in CMT2E patients, including aberrant hind limb posture, motor deficits, hypertrophy of muscle fibres and loss of muscle innervation without neuronal loss [94]. To address whether CMT2E disease is potentially reversible, this mouse model was based on the tetracycline-responsive gene sys‐ tem that allows the suppression of mutant hNFLP22S expression in mature neurons through administration of doxycycline. Remarkably, a 3-month treatment of these mice with doxycy‐ cline after disease onset efficiently down-regulated expression of hNFLP22S and reversed the neurological phenotype [94], providing hope that future therapeutic strategies might not only stop progress of CMT2E disease but also reverse it. A novel line of CMT2E mice that consti‐ tutively express hNFLE397K was recently generated [95]. As with the hNFLP22S mice, these mice developed as early as 4 months signs consistent with CMT2E patients, such as aberrant hind limb posture, digit deformities, reduced locomotor activity and reduced motor nerve conduc‐ tion velocities. However, some aspects differed between the two lines of CMT2E mice. Indeed, hNFLE397K mice showed no significant denervation and their muscles were atrophied. More‐ over, they showed only relatively mild signs of nerve pathology, including ectopic accumu‐ lations of phosphorylated NFs in motor neuron cell bodies, NF disorganization in motor and sensory roots, and reduced axonal caliber [95]. The divergence in cellular pathology between the two animal models may suggests that overt CMT2E phenotypes may arise through dif‐ ferent cellular mechanisms.

kelch proteins are organizers of the cytoskeletal network and closely linked to the ubiquitin degradation pathway. More than 45 distinct mutations of the gigaxonin have been identified to date along the entire *GAN* gene in patients. By revealing a high instability of gigaxonin in multiple lymphoblasts cell lines from unrelated patients, Cleveland et al. [106] showed that

Intermediate Filaments in Neurodegenerative Diseases

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467

The major cytopathological hallmark of GAN is the presence of distal enlarged axons, also called giant axons, filled with abnormally packed IFs associated with a reduced number of MTs [107]. In contrast, axonal segments proximal to the swellings exhibit a reduced number of NFs [108]. Disorganization and accumulation of other types of IFs are also found in skin fibroblasts, Schwann cells and muscle fibers [109-111], suggesting a critical role of gigaxonin in maintaining cytoskeletal architecture. A decreased inter-NF distance was observed in sural nerve axons of a GAN patient and, more surprisingly, the mean diameter of NFs was increased (12.4 nm in GAN compared with 10.1 nm in controls) [112]. Although the mechanism leading to the distal axonal accumulation of NFs is still unclear, an acceleration of their axonal transport was observed in optic nerve from experimentally induced GAN rat model, concomitant with a proximal decreased content of NFs and their distal accumulation [113]. The authors proposed that acceleration of NF transport in the presence of a normal rate of NF protein synthesis and insertion into transport system would lead to the formation of distal axonal swellings with

In order to determine how loss of gigaxonin's function leads to GAN, mice deleted in exons 3-5 of the *GAN* gene (GANΔ3-5 mice) were produced [114]. These mice develop strong motor deficits as early as 6 months of age, including reduction of spontaneous movement, bizarre limb posture and overall weakness. However, they displayed normal life span and fertility, and giant axons were never seen. Nevertheless these mice exhibited enlarged axons with densely packed NF, leading to the segregation of axonal organelles, a feature characteristic of human GAN pathology. This was accompanied by an axonal loss at the age of 9-12 months. However, it should be noted that some null mice showed no overt neurological phenotypes, suggesting that some genetic modifiers may exist [115]. Another mouse model with deletion of exon 1 of the *GAN* gene was generated [116] which exhibited no overt phenotype over 15 months in contrast to GANΔ3-5 mice. Nevertheless, they developed aggregates composed of non-phosphorylated NFH and α-internexin in cerebral cortex and thalamus. Small aggregates of NFL and peripherin also formed in cell bodies of dorsal root ganglion neurons. Moreover, increased levels of neuronal IF proteins were detected in various regions of the nervous system, confirming the importance of gigaxonin in modulating the levels and organization of IF pro‐ teins. Given the very different phenotypes between these two GAN models, Ganay et al. [117] conducted a behavioral analysis over a 72-week period in their own GANΔ3-5 mice as well as in GANΔ3-5 mice developed by Ding et al [114]. Analysis performed on their own model re‐ vealed difference depending on the genetic background. Indeed, a mild but persistent motor impairment was reported in the 129/SvJ genetic background, while C57BL/6 animals displayed rather a deterioration of sensory functions. Despite the modest phenotypic manifestation and no pronounced signs of neurodegeneration, these mice exhibited severe cytoskeletal altera‐ tions, including an increase in the diameter of NFs, an overt impairment in their orientation

GAN is caused by a loss of function of gigaxonin.

packed NFs.

Mutations of myotubularin-related protein 2 (MTMR2) (CMT4B), heat-shock protein B1 (HSPB1) (CMT2F) or HSPB8 (CMT2L) can also cause NFL aggregation [96-99], indicating that mutation of NFs is not the only mechanism inducing their accumulation in CMT. Co-expres‐ sion of Wt HSPB1 with P8R or Q333P CMT mutant NFL reduced their aggregation, induced reversal of mutant NFL aggregates and decreased mutant NFL-induced loss of motor neuron viability [100]. On the opposite, mutant HSPB1 was found to have an inhibitory effect on the assembly of NFL in transfected cells. Zhai et al. [100] showed that deletion of NFL markedly reduces degeneration and loss of motor neurons induced by mutant HSPB1. Finally, mice expressing mutant HSPB1 throughout the nervous system showed axonal pathology in spinal cord and peripheral nerve that was age-dependent, with evidence of impaired NF cytoskele‐ ton, associated with organelle accumulation. These data suggest that overexpression of mutant HSPB1 in neurons is sufficient to cause pathological changes in mice that are seen in patients with CMT. Mutant MTMR2 also induces abnormal NFL assembly in transfected cells [98] and mice lacking MTMR2 develop a CMT-like neuropathy, including several characteristics of dysmyelination [101]. A similar phenotype was observed following Schwann cell-specific *MTMR2* inactivation, whereas neuron-specific inactivation did not provoke myelin outfold‐ ings nor axonal defects, suggesting that loss of MTMR2 in Schwann cells, but not in motor neurons, is both sufficient and necessary to cause CMT4B neuropathy [102]. In addition to disrupt the NF network, recent studies showed that expression of NFLP8R or NFLQ333P in cul‐ tured motor neurons caused the rounding of mitochondria and decreased their rate of fusion concomitant with increased motility [103, 104], indicating an important function of NFs in mitochondrial dynamics. Cotransfection of HSPB1 helped to maintain normal NF network, axonal caliber and mitochondrial morphology. On the other hand, the cotransfection of HSPA1 was effective in neurons expressing NFLQ333P, but not NFLP8R, suggesting that chaperone-based therapies have potential for the treatment of CMT2E but their efficacy would depend on the profile of HSPs induced and the type of *NEFL* mutation.

#### **2.3. Giant axonal neuropathy**

GAN is a rare progressive neurodegenerative disorder with early onset affecting both PNS and CNS. Phenotypic variability has been reported but typical clinical features include distal limb weakness, areflexia and a marked gait disturbance. The motor deficits encompass amyo‐ trophy, muscle weakness and evolve with skeletal deformations and loss of ambulation by the adolescence. As the disorder progresses, CNS involvement includes electroencephalographic abnormalities, mental retardation, speech defect, seizures and defective upper motor neuron function. GAN is caused by mutations in the *GAN* gene encoding the ubiquitously expressed protein gigaxonin. Gigaxonin belongs to a protein family that is characterized by an N-terminal BTB (broad-complex, Tramtrack, and Bric a brac) domain and six kelch repeats [105]. BTB/ kelch proteins are organizers of the cytoskeletal network and closely linked to the ubiquitin degradation pathway. More than 45 distinct mutations of the gigaxonin have been identified to date along the entire *GAN* gene in patients. By revealing a high instability of gigaxonin in multiple lymphoblasts cell lines from unrelated patients, Cleveland et al. [106] showed that GAN is caused by a loss of function of gigaxonin.

over, they showed only relatively mild signs of nerve pathology, including ectopic accumu‐ lations of phosphorylated NFs in motor neuron cell bodies, NF disorganization in motor and sensory roots, and reduced axonal caliber [95]. The divergence in cellular pathology between the two animal models may suggests that overt CMT2E phenotypes may arise through dif‐

Mutations of myotubularin-related protein 2 (MTMR2) (CMT4B), heat-shock protein B1 (HSPB1) (CMT2F) or HSPB8 (CMT2L) can also cause NFL aggregation [96-99], indicating that mutation of NFs is not the only mechanism inducing their accumulation in CMT. Co-expres‐ sion of Wt HSPB1 with P8R or Q333P CMT mutant NFL reduced their aggregation, induced reversal of mutant NFL aggregates and decreased mutant NFL-induced loss of motor neuron viability [100]. On the opposite, mutant HSPB1 was found to have an inhibitory effect on the assembly of NFL in transfected cells. Zhai et al. [100] showed that deletion of NFL markedly reduces degeneration and loss of motor neurons induced by mutant HSPB1. Finally, mice expressing mutant HSPB1 throughout the nervous system showed axonal pathology in spinal cord and peripheral nerve that was age-dependent, with evidence of impaired NF cytoskele‐ ton, associated with organelle accumulation. These data suggest that overexpression of mutant HSPB1 in neurons is sufficient to cause pathological changes in mice that are seen in patients with CMT. Mutant MTMR2 also induces abnormal NFL assembly in transfected cells [98] and mice lacking MTMR2 develop a CMT-like neuropathy, including several characteristics of dysmyelination [101]. A similar phenotype was observed following Schwann cell-specific *MTMR2* inactivation, whereas neuron-specific inactivation did not provoke myelin outfold‐ ings nor axonal defects, suggesting that loss of MTMR2 in Schwann cells, but not in motor neurons, is both sufficient and necessary to cause CMT4B neuropathy [102]. In addition to disrupt the NF network, recent studies showed that expression of NFLP8R or NFLQ333P in cul‐ tured motor neurons caused the rounding of mitochondria and decreased their rate of fusion concomitant with increased motility [103, 104], indicating an important function of NFs in mitochondrial dynamics. Cotransfection of HSPB1 helped to maintain normal NF network, axonal caliber and mitochondrial morphology. On the other hand, the cotransfection of HSPA1 was effective in neurons expressing NFLQ333P, but not NFLP8R, suggesting that chaperone-based therapies have potential for the treatment of CMT2E but their efficacy would depend on the

GAN is a rare progressive neurodegenerative disorder with early onset affecting both PNS and CNS. Phenotypic variability has been reported but typical clinical features include distal limb weakness, areflexia and a marked gait disturbance. The motor deficits encompass amyo‐ trophy, muscle weakness and evolve with skeletal deformations and loss of ambulation by the adolescence. As the disorder progresses, CNS involvement includes electroencephalographic abnormalities, mental retardation, speech defect, seizures and defective upper motor neuron function. GAN is caused by mutations in the *GAN* gene encoding the ubiquitously expressed protein gigaxonin. Gigaxonin belongs to a protein family that is characterized by an N-terminal BTB (broad-complex, Tramtrack, and Bric a brac) domain and six kelch repeats [105]. BTB/

ferent cellular mechanisms.

466 Neurodegenerative Diseases

profile of HSPs induced and the type of *NEFL* mutation.

**2.3. Giant axonal neuropathy**

The major cytopathological hallmark of GAN is the presence of distal enlarged axons, also called giant axons, filled with abnormally packed IFs associated with a reduced number of MTs [107]. In contrast, axonal segments proximal to the swellings exhibit a reduced number of NFs [108]. Disorganization and accumulation of other types of IFs are also found in skin fibroblasts, Schwann cells and muscle fibers [109-111], suggesting a critical role of gigaxonin in maintaining cytoskeletal architecture. A decreased inter-NF distance was observed in sural nerve axons of a GAN patient and, more surprisingly, the mean diameter of NFs was increased (12.4 nm in GAN compared with 10.1 nm in controls) [112]. Although the mechanism leading to the distal axonal accumulation of NFs is still unclear, an acceleration of their axonal transport was observed in optic nerve from experimentally induced GAN rat model, concomitant with a proximal decreased content of NFs and their distal accumulation [113]. The authors proposed that acceleration of NF transport in the presence of a normal rate of NF protein synthesis and insertion into transport system would lead to the formation of distal axonal swellings with packed NFs.

In order to determine how loss of gigaxonin's function leads to GAN, mice deleted in exons 3-5 of the *GAN* gene (GANΔ3-5 mice) were produced [114]. These mice develop strong motor deficits as early as 6 months of age, including reduction of spontaneous movement, bizarre limb posture and overall weakness. However, they displayed normal life span and fertility, and giant axons were never seen. Nevertheless these mice exhibited enlarged axons with densely packed NF, leading to the segregation of axonal organelles, a feature characteristic of human GAN pathology. This was accompanied by an axonal loss at the age of 9-12 months. However, it should be noted that some null mice showed no overt neurological phenotypes, suggesting that some genetic modifiers may exist [115]. Another mouse model with deletion of exon 1 of the *GAN* gene was generated [116] which exhibited no overt phenotype over 15 months in contrast to GANΔ3-5 mice. Nevertheless, they developed aggregates composed of non-phosphorylated NFH and α-internexin in cerebral cortex and thalamus. Small aggregates of NFL and peripherin also formed in cell bodies of dorsal root ganglion neurons. Moreover, increased levels of neuronal IF proteins were detected in various regions of the nervous system, confirming the importance of gigaxonin in modulating the levels and organization of IF pro‐ teins. Given the very different phenotypes between these two GAN models, Ganay et al. [117] conducted a behavioral analysis over a 72-week period in their own GANΔ3-5 mice as well as in GANΔ3-5 mice developed by Ding et al [114]. Analysis performed on their own model re‐ vealed difference depending on the genetic background. Indeed, a mild but persistent motor impairment was reported in the 129/SvJ genetic background, while C57BL/6 animals displayed rather a deterioration of sensory functions. Despite the modest phenotypic manifestation and no pronounced signs of neurodegeneration, these mice exhibited severe cytoskeletal altera‐ tions, including an increase in the diameter of NFs, an overt impairment in their orientation and a strikingly increased abundance of the three NF subunits. Finally, they tested motor deficits in GANΔ3-5 mice produced by Ding et al [114] and detected no clinical signs within the first year. This is consistent with a mild progression of the disease in mice and suggests that the three existing models probably display a phenotype of similar intensity. Altogether, these data shown that the absence of gigaxonin results in a milder version of the GAN disease in mice at the behavioral level, associated with a severe disorganization of the NF network that recapitulates what is observed in patients [117].

and personality changes, which can be associated to memory loss, cognitive impairment, lan‐ guage impairment, hyperreflexia and motor weakness. Neuropathologically, NIFID is char‐ acterized by widespread degeneration of the frontal and temporal lobes. The cytopathological characteristics consist of neuronal loss, gliosis, swollen neurons and presence of large inclu‐ sions in the cell body of many neurons that are immunoreactive for all of the class IV neuronal IFs and especially in α-internexin [125, 126]. These inclusions of α-internexin but negative for tau or synuclein distinguish NIFID from other disease that involve IF inclusions, such as syn‐ ucleinopathies (e.g., PD), tauopathies (e.g., AD and frontotemporal dementia), and motor neuron disease. Although α-internexin has been observed in neuronal inclusions in other neu‐ rodegenerative disorders, it is generally a relatively minor component. This raises the question whether α-internexin-positive neuronal inclusions in NIFID reflect any selective neuronal dysfunction, and as such if they are associated with some specific clinical symptoms. Genetic screening revealed no pathogenic variants for all type IV neuronal IFs, SOD1, NUDEL and gigaxonin [127, 128]. To date, no genetic mutations leading to NIFID have been described.

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469

Interestingly, the number of IFs aggregates is high in areas with reduced neuronal loss, and low in sites of intense neuronal degeneration. Cairns et al. [125] proposed that the formation of these inclusions is an early event in the pathogenesis of NIFID, and these aggregates are then released and degraded into the extracellular space following degen‐ eration of the neuron. The mechanism of IF aggregation and the role they play in neuro‐ nal dysfunction and cell death are still unclear. Although immunoreactivity for IFs was initially described as the defining pathological feature of NIFID, not all the inclusions in NIFID are IF-positive. It now appears that aggregates of FUS (fused in sarcoma) protein, is a more consistent feature of NIFID. Indeed, intracellular accumulations of FUS are more often encountered than IF inclusions and all neurons that contained abnormal IF aggregates also contained FUS inclusions [129]. It should also be noted that clusters of FUS-immunoreactive inclusions are larger than those revealed by NFH or α-internexin [130]. The authors interpreted this finding as suggesting that FUS plays a more central role in the pathogenesis of NIFID and that the abnormal accumulation of IFs is likely a secondary phenomenon. It now remains to determine the exact implication of FUS in

Diabetes is the leading cause of peripheral neuropathy worldwide. About 60 to 70 percent of people with diabetes have some form of neuropathy. People with diabetes can develop nerve problems at any time, but risk rises with age and longer duration of diabetes. Diabetic neuro‐ pathies are complex, heterogeneous disorders that affect dorsal root ganglia and sensory axons more so than motor fibers. Nerve damage is likely due to a combination of factors, including metabolic factors (e.g., high blood glucose, abnormal blood fat levels), neurovascular factors leading to damage to the blood vessels, autoimmune factors, lifestyle factors and inherited traits that increase susceptibility to nerve disease. Although its pathogenesis has not been fully elucidated, diabetic neuropathy is characterized by slower conduction velocity, impairment of axonal transport, axonal atrophy and reduced capacity for nerve regeneration. All these

the pathogenesis of NIFID.

**2.5. Diabetic neuropathy**

Gigaxonin was shown to be a direct key player in the Ubiquitin Proteasome System (UPS). Indeed, BTB-containing proteins, including gigaxonin have been found to be the substrate adaptors of Cul3-dependant E3 ubiquitin ligases, interacting with Cul3 and the substrates through the BTB and the C-terminal domains, respectively [118-120]. Gigaxo‐ nin was shown to promote the ubiquitin-mediated degradation of its three known sub‐ strates, the microtubule-associated protein 1B (MAP1B) [121], tubulin folding cofactor B (TBCB) [122] and MAP1S (also called MAP8) [114]. Disease associated gigaxonin muta‐ tions perturb its association with these partners while gigaxonin ablation results in their accumulation [114, 122, 123]. This raised the possibility that IF accumulation in GAN re‐ sults from a MT reorganization/destabilization. However, it is intriguing to note that these proteins have opposite effects on MT network: MAP1B is a MT-stabilizing phosphopro‐ tein, whereas overexpression of TBCB depolymerizes MTs. Using primary fibroblasts de‐ rived from skin biopsies of multiple GAN patients with aberrant aggregates of vimentin, Cleveland et al. [106] demonstrated that vimentin aggregation is greatly enhanced in con‐ ditions driving quiescence and is not caused by an abnormal accumulation of the tubulin chaperone TBCB and its effect on MT stability. Moreover, the prolonged depletion of the MT network did not induce GAN-like aggregates of vimentin in normal fibroblasts. These results indicated that the generalized disorganization of IFs in GAN patients may not in‐ volve TBCB-mediated MT disassembly and must be regulated by a yet unidentified mech‐ anism [106]. Recently, proteomic analysis performed in fibroblasts from four GAN patients provided new insights into disease mechanisms [124]. Although the major role of gigaxonin is reported to be degradation of cytoskeleton-associated proteins, the amount of 76 structural cytoskeletal proteins was unaltered. However, several proteins linked to reg‐ ulation of the cytoskeleton network were found to be upregulated or downregulated. The authors speculated that in GAN, dysregulation of the cytoskeletal network is responsible for formation of aggregates of IFs. In the case of fibroblasts, disturbed cytoskeletal regula‐ tion could lead to a hyperphosphorylation state of vimentin that results in massive depo‐ lymerization of vimentin filaments and finally in collapse of the vimentin network. The unpolymerized filaments are collected in the aggresome near the nucleus where they form the typical aggregates [124].

#### **2.4. Neuronal intermediate filament inclusion disease**

NIFID is a recently described uncommon neurological disorder of early onset with a hetero‐ geneous clinical phenotype, including sporadic fronto-temporal dementia associated with a pyramidal and/or extrapyramidal movement disorder. The symptoms comprise behavioural and personality changes, which can be associated to memory loss, cognitive impairment, lan‐ guage impairment, hyperreflexia and motor weakness. Neuropathologically, NIFID is char‐ acterized by widespread degeneration of the frontal and temporal lobes. The cytopathological characteristics consist of neuronal loss, gliosis, swollen neurons and presence of large inclu‐ sions in the cell body of many neurons that are immunoreactive for all of the class IV neuronal IFs and especially in α-internexin [125, 126]. These inclusions of α-internexin but negative for tau or synuclein distinguish NIFID from other disease that involve IF inclusions, such as syn‐ ucleinopathies (e.g., PD), tauopathies (e.g., AD and frontotemporal dementia), and motor neuron disease. Although α-internexin has been observed in neuronal inclusions in other neu‐ rodegenerative disorders, it is generally a relatively minor component. This raises the question whether α-internexin-positive neuronal inclusions in NIFID reflect any selective neuronal dysfunction, and as such if they are associated with some specific clinical symptoms. Genetic screening revealed no pathogenic variants for all type IV neuronal IFs, SOD1, NUDEL and gigaxonin [127, 128]. To date, no genetic mutations leading to NIFID have been described.

Interestingly, the number of IFs aggregates is high in areas with reduced neuronal loss, and low in sites of intense neuronal degeneration. Cairns et al. [125] proposed that the formation of these inclusions is an early event in the pathogenesis of NIFID, and these aggregates are then released and degraded into the extracellular space following degen‐ eration of the neuron. The mechanism of IF aggregation and the role they play in neuro‐ nal dysfunction and cell death are still unclear. Although immunoreactivity for IFs was initially described as the defining pathological feature of NIFID, not all the inclusions in NIFID are IF-positive. It now appears that aggregates of FUS (fused in sarcoma) protein, is a more consistent feature of NIFID. Indeed, intracellular accumulations of FUS are more often encountered than IF inclusions and all neurons that contained abnormal IF aggregates also contained FUS inclusions [129]. It should also be noted that clusters of FUS-immunoreactive inclusions are larger than those revealed by NFH or α-internexin [130]. The authors interpreted this finding as suggesting that FUS plays a more central role in the pathogenesis of NIFID and that the abnormal accumulation of IFs is likely a secondary phenomenon. It now remains to determine the exact implication of FUS in the pathogenesis of NIFID.

#### **2.5. Diabetic neuropathy**

and a strikingly increased abundance of the three NF subunits. Finally, they tested motor deficits in GANΔ3-5 mice produced by Ding et al [114] and detected no clinical signs within the first year. This is consistent with a mild progression of the disease in mice and suggests that the three existing models probably display a phenotype of similar intensity. Altogether, these data shown that the absence of gigaxonin results in a milder version of the GAN disease in mice at the behavioral level, associated with a severe disorganization of the NF network that

Gigaxonin was shown to be a direct key player in the Ubiquitin Proteasome System (UPS). Indeed, BTB-containing proteins, including gigaxonin have been found to be the substrate adaptors of Cul3-dependant E3 ubiquitin ligases, interacting with Cul3 and the substrates through the BTB and the C-terminal domains, respectively [118-120]. Gigaxo‐ nin was shown to promote the ubiquitin-mediated degradation of its three known sub‐ strates, the microtubule-associated protein 1B (MAP1B) [121], tubulin folding cofactor B (TBCB) [122] and MAP1S (also called MAP8) [114]. Disease associated gigaxonin muta‐ tions perturb its association with these partners while gigaxonin ablation results in their accumulation [114, 122, 123]. This raised the possibility that IF accumulation in GAN re‐ sults from a MT reorganization/destabilization. However, it is intriguing to note that these proteins have opposite effects on MT network: MAP1B is a MT-stabilizing phosphopro‐ tein, whereas overexpression of TBCB depolymerizes MTs. Using primary fibroblasts de‐ rived from skin biopsies of multiple GAN patients with aberrant aggregates of vimentin, Cleveland et al. [106] demonstrated that vimentin aggregation is greatly enhanced in con‐ ditions driving quiescence and is not caused by an abnormal accumulation of the tubulin chaperone TBCB and its effect on MT stability. Moreover, the prolonged depletion of the MT network did not induce GAN-like aggregates of vimentin in normal fibroblasts. These results indicated that the generalized disorganization of IFs in GAN patients may not in‐ volve TBCB-mediated MT disassembly and must be regulated by a yet unidentified mech‐ anism [106]. Recently, proteomic analysis performed in fibroblasts from four GAN patients provided new insights into disease mechanisms [124]. Although the major role of gigaxonin is reported to be degradation of cytoskeleton-associated proteins, the amount of 76 structural cytoskeletal proteins was unaltered. However, several proteins linked to reg‐ ulation of the cytoskeleton network were found to be upregulated or downregulated. The authors speculated that in GAN, dysregulation of the cytoskeletal network is responsible for formation of aggregates of IFs. In the case of fibroblasts, disturbed cytoskeletal regula‐ tion could lead to a hyperphosphorylation state of vimentin that results in massive depo‐ lymerization of vimentin filaments and finally in collapse of the vimentin network. The unpolymerized filaments are collected in the aggresome near the nucleus where they

recapitulates what is observed in patients [117].

468 Neurodegenerative Diseases

form the typical aggregates [124].

**2.4. Neuronal intermediate filament inclusion disease**

NIFID is a recently described uncommon neurological disorder of early onset with a hetero‐ geneous clinical phenotype, including sporadic fronto-temporal dementia associated with a pyramidal and/or extrapyramidal movement disorder. The symptoms comprise behavioural Diabetes is the leading cause of peripheral neuropathy worldwide. About 60 to 70 percent of people with diabetes have some form of neuropathy. People with diabetes can develop nerve problems at any time, but risk rises with age and longer duration of diabetes. Diabetic neuro‐ pathies are complex, heterogeneous disorders that affect dorsal root ganglia and sensory axons more so than motor fibers. Nerve damage is likely due to a combination of factors, including metabolic factors (e.g., high blood glucose, abnormal blood fat levels), neurovascular factors leading to damage to the blood vessels, autoimmune factors, lifestyle factors and inherited traits that increase susceptibility to nerve disease. Although its pathogenesis has not been fully elucidated, diabetic neuropathy is characterized by slower conduction velocity, impairment of axonal transport, axonal atrophy and reduced capacity for nerve regeneration. All these features of nerve function depend on the integrity of the axonal cytoskeleton and particularly on NFs. In agreement with this, multiple abnormalities of NF biology were identified in models of diabetes. An impairment of the axonal transport of NFs, actin and tubulin concomitant with a proximal increase and a distal decrease of axonal calibers were observed in rats with strep‐ tozotocin-induced diabetes and in BioBreeding rats (a model of spontaneous type I diabetes) [131, 132]. The distal axonal atrophy is accompanied by a concomitant NF loss in this region [133], and accumulations of highly phosphorylated NF epitopes are present in proximal axonal segments of dorsal root ganglia sensory neurons from diabetic patients [134]. An increase of NF phosphorylation, correlated with activation of JNK, was also detected in lumbar dorsal root ganglia from rat models [135]. Finally, there were a substantial decline in the mRNA levels of all three NF subunits as well as reduced NF numbers and densities within large myelinated sensory of long-term diabetic models [136]. All these results suggest that NF abnormalities may contribute to the development of diabetic neuropathy, or may be affected by this disease. However, slowing of conduction velocity in diabetic models occurs much earlier than loss of NF investment or axonal atrophy [136]. To further elucidate the contribution of NFs to diabetic neuropathy pathogenesis, the effect of streptozotocin-induced diabetes was analyzed in NFH-LacZ transgenic mice characterized by axons completely lacking NFs [137]. Interestingly, di‐ abetic mice lacking NFs developed progressive slowing of conduction velocity in their motor and sensory fibres and displayed decreased nerve action potential amplitudes earlier than diabetic mice with normal IF cytoskeleton. Moreover, superimposing diabetes on axons with‐ out NFs also accentuated axonal atrophy. Administration of insulin that restored normogly‐ cemia reversed conduction slowing and restored sensory axon caliber. These findings indicate that changes in NF expression, transport or post-translational modifications cannot account alone for neurological features of diabetic neuropathy, but these IFs may help axons to better resist the negative effects of diabetes [137].

145], arguing against the implication of this *NEFM* mutation in pathogenesis of PD. Interest‐ ingly, research has shown that changes in the levels of NFL in the cerebrospinal fluid may be used as a biomarker for the identification of PD [146] and that the serum levels of anti-NF protein antibodies increase significantly in patients with PD [147]. Finally, it seemed that the serum level of NFs in patients with PD was significantly correlated with duration of the disease and age [148]. These findings support the idea that axonal injury causes the release of cytos‐ keleton proteins, and changes in the concentrations of serum NFs are probably related to the

Intermediate Filaments in Neurodegenerative Diseases

http://dx.doi.org/10.5772/54676

471

Neuronal IFs are not the only class of IF to be responsible for the development of neurological disorders. Glial IF can also be the primary cause of a CNS disorder. Indeed, GFAP, the major constituent of astrocytic IFs, is directly involved in the development of the AXD. This disease is a fatal, progressive white matter disorder that has been classified into three types based on the age of onset: infantile, juvenile and adult. The infantile type, with onset between birth and about two years of age, is the most frequent form of the disease and is fatal either within that period or by around the age of 10 years. Clinical symptoms comprise progressive megalence‐ phaly, seizures and impaired cognitive function, which may be associated with ataxia and hydrocephalus. Such phenotypes become progressively less common for the juvenile and adult forms (for recent reviews, see [149, 150]). Both the infantile and juvenile forms usually appear

AXD is a primary astrocytic disease and its manifestations are the result of astrocyte dysfunc‐ tions leading to both myelin damage and neuron dysfunction. Neuronal loss is often reported but axons are relatively well preserved in demyelinated areas. The pathological hallmark of AXD is the presence of protein aggregates known as Rosenthal fibers within the cytoplasm of astrocytes throughout the CNS, but especially those located in the subpial, periventricular and subependymal zones. Different constituents were identified in Rosenthal fibers: GFAP, αBcrystallin, HSP27 and ubiquitin [151-153]. Although GFAP is also expressed in glial cells of the PNS and in several other organs, Rosenthal fibers were not reported outside the CNS of AXD

To examine the function of GFAP *in vivo*, GFAP knock-out mice were generated [154-157]. These studies showed that mice lacking GFAP displayed astrocytes devoid of the IF, but still developed and reproduced normally. Only subtle phenotypes emerged with age, arguing for a role of GFAP in the white matter architecture, blood-brain barrier integrity, astrocyte-neu‐ ronal interactions and in modulating synaptic efficacy in the CNS [156, 157]. This is consistent with the known roles of astrocytes that help to form blood brain barrier, promote synaptic plasticity and coordinate neuronal activity. To determine the influence of increased GFAP expression on astrocyte function, mice overexpressing the human *GFAP* gene were produced [158]. Mice in the highest expressing lines developed a phenotype close to that observed in AXD. Indeed, their brains contain many inclusion bodies indistinguishable from human

**3. Glial intermediate filament GFAP and Alexander disease**

to be sporadic while the adult form is often familial.

severity of axonal injuries.

patients.

#### **2.6. Parkinson disease**

Parkinson's disease (PD) is the second most common neurodegenerative disorder after AD, with a prevalence of about 2% among people over the age of 65 years. This disease is marked by the depletion of dopaminergic melanin-containing neurons in the substantia nigra pars compacta and a consequent loss of dopamine in the striatum. Another important pathological feature is the presence, especially in substantia nigra pars compacta neurons, of eosinophilic cytoplasmic inclusion bodies named Lewy bodies, composed of α-synuclein, NF proteins, ubiquitin and proteasome subunits. Various features distinguish NFs in PD, including inap‐ propriate phosphorylation and proteolysis in Lewy bodies [138, 139], decreased NFL and NFH mRNA levels [140], and reduced protein level of NFL and NFM [141]. A point mutation in the *NEFM* gene was reported in a case of PD with early onset [142]. This mutation consisted in a substitution of Ser for Gly at residue 336, a highly conserved region in the rod domain 2B of NFM, and was argued to disrupt NF assembly. Although three other unaffected family mem‐ bers also carried this mutation, the authors had then proposed that aberrations in neuronal IFs could lead to the development of the pathology seen in PD. However, the G336S mutation does not disrupt the assembly and the distribution of NFs *in vitro* [143] and the screenings of PD patients of similar or different ethnic background failed to identify this mutations [144, 145], arguing against the implication of this *NEFM* mutation in pathogenesis of PD. Interest‐ ingly, research has shown that changes in the levels of NFL in the cerebrospinal fluid may be used as a biomarker for the identification of PD [146] and that the serum levels of anti-NF protein antibodies increase significantly in patients with PD [147]. Finally, it seemed that the serum level of NFs in patients with PD was significantly correlated with duration of the disease and age [148]. These findings support the idea that axonal injury causes the release of cytos‐ keleton proteins, and changes in the concentrations of serum NFs are probably related to the severity of axonal injuries.

### **3. Glial intermediate filament GFAP and Alexander disease**

features of nerve function depend on the integrity of the axonal cytoskeleton and particularly on NFs. In agreement with this, multiple abnormalities of NF biology were identified in models of diabetes. An impairment of the axonal transport of NFs, actin and tubulin concomitant with a proximal increase and a distal decrease of axonal calibers were observed in rats with strep‐ tozotocin-induced diabetes and in BioBreeding rats (a model of spontaneous type I diabetes) [131, 132]. The distal axonal atrophy is accompanied by a concomitant NF loss in this region [133], and accumulations of highly phosphorylated NF epitopes are present in proximal axonal segments of dorsal root ganglia sensory neurons from diabetic patients [134]. An increase of NF phosphorylation, correlated with activation of JNK, was also detected in lumbar dorsal root ganglia from rat models [135]. Finally, there were a substantial decline in the mRNA levels of all three NF subunits as well as reduced NF numbers and densities within large myelinated sensory of long-term diabetic models [136]. All these results suggest that NF abnormalities may contribute to the development of diabetic neuropathy, or may be affected by this disease. However, slowing of conduction velocity in diabetic models occurs much earlier than loss of NF investment or axonal atrophy [136]. To further elucidate the contribution of NFs to diabetic neuropathy pathogenesis, the effect of streptozotocin-induced diabetes was analyzed in NFH-LacZ transgenic mice characterized by axons completely lacking NFs [137]. Interestingly, di‐ abetic mice lacking NFs developed progressive slowing of conduction velocity in their motor and sensory fibres and displayed decreased nerve action potential amplitudes earlier than diabetic mice with normal IF cytoskeleton. Moreover, superimposing diabetes on axons with‐ out NFs also accentuated axonal atrophy. Administration of insulin that restored normogly‐ cemia reversed conduction slowing and restored sensory axon caliber. These findings indicate that changes in NF expression, transport or post-translational modifications cannot account alone for neurological features of diabetic neuropathy, but these IFs may help axons to better

Parkinson's disease (PD) is the second most common neurodegenerative disorder after AD, with a prevalence of about 2% among people over the age of 65 years. This disease is marked by the depletion of dopaminergic melanin-containing neurons in the substantia nigra pars compacta and a consequent loss of dopamine in the striatum. Another important pathological feature is the presence, especially in substantia nigra pars compacta neurons, of eosinophilic cytoplasmic inclusion bodies named Lewy bodies, composed of α-synuclein, NF proteins, ubiquitin and proteasome subunits. Various features distinguish NFs in PD, including inap‐ propriate phosphorylation and proteolysis in Lewy bodies [138, 139], decreased NFL and NFH mRNA levels [140], and reduced protein level of NFL and NFM [141]. A point mutation in the *NEFM* gene was reported in a case of PD with early onset [142]. This mutation consisted in a substitution of Ser for Gly at residue 336, a highly conserved region in the rod domain 2B of NFM, and was argued to disrupt NF assembly. Although three other unaffected family mem‐ bers also carried this mutation, the authors had then proposed that aberrations in neuronal IFs could lead to the development of the pathology seen in PD. However, the G336S mutation does not disrupt the assembly and the distribution of NFs *in vitro* [143] and the screenings of PD patients of similar or different ethnic background failed to identify this mutations [144,

resist the negative effects of diabetes [137].

**2.6. Parkinson disease**

470 Neurodegenerative Diseases

Neuronal IFs are not the only class of IF to be responsible for the development of neurological disorders. Glial IF can also be the primary cause of a CNS disorder. Indeed, GFAP, the major constituent of astrocytic IFs, is directly involved in the development of the AXD. This disease is a fatal, progressive white matter disorder that has been classified into three types based on the age of onset: infantile, juvenile and adult. The infantile type, with onset between birth and about two years of age, is the most frequent form of the disease and is fatal either within that period or by around the age of 10 years. Clinical symptoms comprise progressive megalence‐ phaly, seizures and impaired cognitive function, which may be associated with ataxia and hydrocephalus. Such phenotypes become progressively less common for the juvenile and adult forms (for recent reviews, see [149, 150]). Both the infantile and juvenile forms usually appear to be sporadic while the adult form is often familial.

AXD is a primary astrocytic disease and its manifestations are the result of astrocyte dysfunc‐ tions leading to both myelin damage and neuron dysfunction. Neuronal loss is often reported but axons are relatively well preserved in demyelinated areas. The pathological hallmark of AXD is the presence of protein aggregates known as Rosenthal fibers within the cytoplasm of astrocytes throughout the CNS, but especially those located in the subpial, periventricular and subependymal zones. Different constituents were identified in Rosenthal fibers: GFAP, αBcrystallin, HSP27 and ubiquitin [151-153]. Although GFAP is also expressed in glial cells of the PNS and in several other organs, Rosenthal fibers were not reported outside the CNS of AXD patients.

To examine the function of GFAP *in vivo*, GFAP knock-out mice were generated [154-157]. These studies showed that mice lacking GFAP displayed astrocytes devoid of the IF, but still developed and reproduced normally. Only subtle phenotypes emerged with age, arguing for a role of GFAP in the white matter architecture, blood-brain barrier integrity, astrocyte-neu‐ ronal interactions and in modulating synaptic efficacy in the CNS [156, 157]. This is consistent with the known roles of astrocytes that help to form blood brain barrier, promote synaptic plasticity and coordinate neuronal activity. To determine the influence of increased GFAP expression on astrocyte function, mice overexpressing the human *GFAP* gene were produced [158]. Mice in the highest expressing lines developed a phenotype close to that observed in AXD. Indeed, their brains contain many inclusion bodies indistinguishable from human Rosenthal fibers, astrocytes are hypertrophic and these animals died from an encephalopathy at an age that is inversely correlated with the level of expression of the transgene. However, no myelin abnormalities were observed. Microarray analysis performed on olfactory bulbs of these animals recently highlighted the appearance of an initial stress response by astrocytes which results in the activation of microglia and compromised neuronal function [159]. All these results suggested that a primary alteration in GFAP may be responsible for AXD.

While the genetic basis for AXD is now firmly recognized, there is little information con‐ cerning the mechanisms by which GFAP mutations lead to disease. Several study showed that mutations of GFAP alters the normal solubility and organization of GFAP networks [163, 167]. When expressed alone, these mutant proteins lost their ability to form filament *in vitro*. But in presence of assembly partners, such as wild-type GFAP or vimentin, they were still capable of incorporation into filament networks in transfected cells. If wild-type GFAP is prone to aggregate, mutations of GFAP exacerbates this accu‐ mulation [168]. Insufficient amounts of plectin, due to R239C GFAP expression, were al‐ so proposed to promote GFAP aggregation and Rosenthal fibers formation in AXD [169]. Both inhibited proteasome activity and activated stress pathways seemed to be important consequences of GFAP accumulation [168]. As a positive feedback response, both the proteasome hypofunction and JNK activation exacerbated GFAP accumulation, increasing susceptibility of the cell to stressful stimuli. It thus appeared that accumulations of GFAP protein would be more deleterious to the astrocytes than the mutant protein itself. How‐ ever, as a positive consequence, up-regulation of αB-crystallin and HSP27 were also asso‐ ciated to the aggregation of GFAP in AXD patients [153, 170] as well as in cell and animal models [159, 165, 168]. Increased αB-crystallin levels would contribute to the dis‐ aggregation of GFAP aggregates and could protect cells from apoptotic events [171]. Moreover, a recent study demonstrated that AXD mutant GFAP accumulation stimulates autophagy which in turn contributes to decrease GFAP levels [172]. The balance between the positive and negative effects of GFAP accumulation might define the survival or death of the cell. Compounds known to reduce GFAP expression *in vitro*, such as querce‐ tin, might be useful as therapeutics. For instance, treatment with the antibiotic ceftriax‐ one alleviates intracytoplasmic aggregates of mutant GFAP by inducing the upregulation of HSP27 and αB-crystallin, poly-ubiquitination and autophagy, and by reducing the *GFAP* promoter transcriptional regulation [173]. Curcumin was also report‐ ed to have beneficial effects in an *in vitro* model of AXD. Indeed, curcumin is able to in‐ duce both HSP27 and αB-crystallin, to reduce expression of both RNA and protein of endogenous GFAP, to induce autophagy and, finally, to rescue the filamentous organiza‐ tion of the GFAP mutant protein, thus suggesting a role of this spice in counteracting the

Intermediate Filaments in Neurodegenerative Diseases

http://dx.doi.org/10.5772/54676

473

The *GFAP* gene is known to generate different splice variants, including the most abundant isoform GFAP-α, and seven other differentially expressed transcripts including GFAP-δ (hu‐ man homologous GFAP-ε). GFAP-δ is incapable of self-assembly into IF *per se*, but can incor‐ porate a filament network composed of GFAP-α if the proportion of GFAP-δ to GFAP-α remains <10% [175]. However, elevating the proportion of GFAP-δ perturbs association of αBcrystallin with the IF fraction and induced IF bundling and aggregation in transiently trans‐ fected cells. Interestingly, GFAP-δ isoform is preferentially expressed in the same populations of astrocytes that contain the most Rosenthal fibers in AXD. This raises the possibility that GFAP-δ may play a key role in aggregate formation in combination with mutated GFAP. It

remains to determine whether GFAP-α:GFAP-δ ratio is perturbed in AXD tissues.

pathogenic effects of *GFAP* mutations [174].

Sequence analysis of DNA samples from AXD patients was thus performed and revealed that most cases are associated with mutations in the *GFAP* gene [160]. Since then, numerous mu‐ tations of this gene were identified; many of them being located in highly conserved domains of the encoded protein that play specific roles in the assembly of IF network [8, 150]. It was estimated that more than 95% of AXD cases are due to *GFAP* mutation. To date, all the iden‐ tified mutations are heterozygous and nearly all of them involve amino acid substitutions, but several insertion or deletion/insertion alterations have also been reported (a continually up‐ dated list of all published mutations is maintained at the Waisman Center of the University of Wisconsin-Madison; www.waisman.wisc.edu/alexander). Numerous mutations cluster in the coils 1A and 2B of GFAP and two sites (R79 and R239) account for approximately half of all patients affected. The comparison of mutations occurring in the various IF proteins revealed that frequent mutations lying in the 2A segment seem to be unique to GFAP. It is possible that molecular partners specifically interact with this region of GFAP but not with the equivalent region of other IFs. The calcium-binding protein S100B binds to the N-terminal part of GFAPcoil 2A [161]. As S100B prevents GFAP assembly [162], mutations in this domain could impair GFAP-S100B interactions, resulting in the accumulation of GFAP polymers and possibly ag‐ gregates. It seems that a correlation exists between the different mutations and the severity of the disease. However, there also exists significant phenotypic variability and age of onset for the same mutation [163], suggesting that epigenetic and environmental factors influence the appearance and timing of disease symptoms. It should also be noted that in rare cases of AXD, no mutations in the *GFAP* gene has been found [164], indicating that there may be additional causes of the disease.

The discovery of GFAP mutations led to the generation of knock-in mice with missense mu‐ tations homologous to those found in humans (R76H and R236H, which correspond to the R79H and R239H mutations in human) [165, 166]. If the presence of mutant GFAP *perse* seemed insufficient for aggregate formation, a 30% increase in GFAP content over that in wild-type induced the formation of Rosenthal fibers in multiples sites throughout the CNS [166]. These animals were also more susceptible to kainate-induced seizures. Nevertheless, they had a nor‐ mal lifespan, showed no overt behavioral defects and general white matter architecture and myelination appeared normal. These features resemble those found in the adult form of AXD rather than in the infantile form. This indicates that the presence of GFAP aggregates contain‐ ing mutant GFAP is not sufficient to induce a major phenotype of AXD, even though it causes some abnormalities in the mouse. Interestingly, further elevation of GFAP via crosses to GFAP transgenic animals led to a shift in GFAP solubility, an increased stress response, and ulti‐ mately death [165]. This correlates GFAP protein levels to the severity of the disease.

While the genetic basis for AXD is now firmly recognized, there is little information con‐ cerning the mechanisms by which GFAP mutations lead to disease. Several study showed that mutations of GFAP alters the normal solubility and organization of GFAP networks [163, 167]. When expressed alone, these mutant proteins lost their ability to form filament *in vitro*. But in presence of assembly partners, such as wild-type GFAP or vimentin, they were still capable of incorporation into filament networks in transfected cells. If wild-type GFAP is prone to aggregate, mutations of GFAP exacerbates this accu‐ mulation [168]. Insufficient amounts of plectin, due to R239C GFAP expression, were al‐ so proposed to promote GFAP aggregation and Rosenthal fibers formation in AXD [169]. Both inhibited proteasome activity and activated stress pathways seemed to be important consequences of GFAP accumulation [168]. As a positive feedback response, both the proteasome hypofunction and JNK activation exacerbated GFAP accumulation, increasing susceptibility of the cell to stressful stimuli. It thus appeared that accumulations of GFAP protein would be more deleterious to the astrocytes than the mutant protein itself. How‐ ever, as a positive consequence, up-regulation of αB-crystallin and HSP27 were also asso‐ ciated to the aggregation of GFAP in AXD patients [153, 170] as well as in cell and animal models [159, 165, 168]. Increased αB-crystallin levels would contribute to the dis‐ aggregation of GFAP aggregates and could protect cells from apoptotic events [171]. Moreover, a recent study demonstrated that AXD mutant GFAP accumulation stimulates autophagy which in turn contributes to decrease GFAP levels [172]. The balance between the positive and negative effects of GFAP accumulation might define the survival or death of the cell. Compounds known to reduce GFAP expression *in vitro*, such as querce‐ tin, might be useful as therapeutics. For instance, treatment with the antibiotic ceftriax‐ one alleviates intracytoplasmic aggregates of mutant GFAP by inducing the upregulation of HSP27 and αB-crystallin, poly-ubiquitination and autophagy, and by reducing the *GFAP* promoter transcriptional regulation [173]. Curcumin was also report‐ ed to have beneficial effects in an *in vitro* model of AXD. Indeed, curcumin is able to in‐ duce both HSP27 and αB-crystallin, to reduce expression of both RNA and protein of endogenous GFAP, to induce autophagy and, finally, to rescue the filamentous organiza‐ tion of the GFAP mutant protein, thus suggesting a role of this spice in counteracting the pathogenic effects of *GFAP* mutations [174].

Rosenthal fibers, astrocytes are hypertrophic and these animals died from an encephalopathy at an age that is inversely correlated with the level of expression of the transgene. However, no myelin abnormalities were observed. Microarray analysis performed on olfactory bulbs of these animals recently highlighted the appearance of an initial stress response by astrocytes which results in the activation of microglia and compromised neuronal function [159]. All these

Sequence analysis of DNA samples from AXD patients was thus performed and revealed that most cases are associated with mutations in the *GFAP* gene [160]. Since then, numerous mu‐ tations of this gene were identified; many of them being located in highly conserved domains of the encoded protein that play specific roles in the assembly of IF network [8, 150]. It was estimated that more than 95% of AXD cases are due to *GFAP* mutation. To date, all the iden‐ tified mutations are heterozygous and nearly all of them involve amino acid substitutions, but several insertion or deletion/insertion alterations have also been reported (a continually up‐ dated list of all published mutations is maintained at the Waisman Center of the University of Wisconsin-Madison; www.waisman.wisc.edu/alexander). Numerous mutations cluster in the coils 1A and 2B of GFAP and two sites (R79 and R239) account for approximately half of all patients affected. The comparison of mutations occurring in the various IF proteins revealed that frequent mutations lying in the 2A segment seem to be unique to GFAP. It is possible that molecular partners specifically interact with this region of GFAP but not with the equivalent region of other IFs. The calcium-binding protein S100B binds to the N-terminal part of GFAPcoil 2A [161]. As S100B prevents GFAP assembly [162], mutations in this domain could impair GFAP-S100B interactions, resulting in the accumulation of GFAP polymers and possibly ag‐ gregates. It seems that a correlation exists between the different mutations and the severity of the disease. However, there also exists significant phenotypic variability and age of onset for the same mutation [163], suggesting that epigenetic and environmental factors influence the appearance and timing of disease symptoms. It should also be noted that in rare cases of AXD, no mutations in the *GFAP* gene has been found [164], indicating that there may be additional

The discovery of GFAP mutations led to the generation of knock-in mice with missense mu‐ tations homologous to those found in humans (R76H and R236H, which correspond to the R79H and R239H mutations in human) [165, 166]. If the presence of mutant GFAP *perse* seemed insufficient for aggregate formation, a 30% increase in GFAP content over that in wild-type induced the formation of Rosenthal fibers in multiples sites throughout the CNS [166]. These animals were also more susceptible to kainate-induced seizures. Nevertheless, they had a nor‐ mal lifespan, showed no overt behavioral defects and general white matter architecture and myelination appeared normal. These features resemble those found in the adult form of AXD rather than in the infantile form. This indicates that the presence of GFAP aggregates contain‐ ing mutant GFAP is not sufficient to induce a major phenotype of AXD, even though it causes some abnormalities in the mouse. Interestingly, further elevation of GFAP via crosses to GFAP transgenic animals led to a shift in GFAP solubility, an increased stress response, and ulti‐

mately death [165]. This correlates GFAP protein levels to the severity of the disease.

results suggested that a primary alteration in GFAP may be responsible for AXD.

causes of the disease.

472 Neurodegenerative Diseases

The *GFAP* gene is known to generate different splice variants, including the most abundant isoform GFAP-α, and seven other differentially expressed transcripts including GFAP-δ (hu‐ man homologous GFAP-ε). GFAP-δ is incapable of self-assembly into IF *per se*, but can incor‐ porate a filament network composed of GFAP-α if the proportion of GFAP-δ to GFAP-α remains <10% [175]. However, elevating the proportion of GFAP-δ perturbs association of αBcrystallin with the IF fraction and induced IF bundling and aggregation in transiently trans‐ fected cells. Interestingly, GFAP-δ isoform is preferentially expressed in the same populations of astrocytes that contain the most Rosenthal fibers in AXD. This raises the possibility that GFAP-δ may play a key role in aggregate formation in combination with mutated GFAP. It remains to determine whether GFAP-α:GFAP-δ ratio is perturbed in AXD tissues.

#### **4. Conclusion**

IFs abnormalities are reminiscent in multiple human neurodegenerative disorders. Despite extensive efforts over the past 40 years, processes leading to these abnormalities as well as their precise contribution to disease pathogenesis often remain poorly understood. For in‐ stance, if it is clearly established that mutation in IF genes can be a primary cause of neurode‐ generative disorders, the question as to how they induce neurodegeneration frequently remain unsolved. Although transgenic mouse models have been somewhat helpful in understanding some mechanisms, most of these animals displayed a much less severe phenotype than patients and results have not always been completely clear-cut. A growing body of evidence suggests that perturbation of IF axonal transport and/or stoichiometry are directly involved in the for‐ mation of intracellular IF aggregates. Destabilization of IF mRNA could be responsible for alteration in IF stoichiometry whereas aberrant post-translational modifications could affect their transport. More investigations are also necessary to identify IF partners. The importance of IF-associated proteins in the development of neurodegenerative disorders was also high‐ lighted by the identification of mutations in genes encoding IF partners that mimic IF-related disease. This is particularly the case of gigaxonin in GAN. A particular attention should also be paid to elucidate the role that IF proteins may play in signaling. Finally, it will be important to elucidate why certain types of IF accumulations appear more toxic than others. While per‐ ikaryal accumulations are generally well tolerated, axonal inclusions are often noxious. The more deleterious effect of axonal aggregates on axonal transport could be a promising avenue to explore in the future and the identification of compounds able to remove these IF aggregates is crucial to the development of new therapeutic approaches.

ated with the neurofilament triplet proteins in the mature CNS. J Neurosci 2006;26

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475

[3] Yan Y, Jensen K and Brown A. The polypeptide composition of moving and station‐ ary neurofilaments in cultured sympathetic neurons. Cell Motil Cytoskeleton 2007;64

[4] Perrot R, Berges R, Bocquet A and Eyer J. Review of the multiple aspects of neurofila‐ ment functions, and their possible contribution to neurodegeneration. Mol Neurobiol

[5] Nixon RA and Shea TB. Dynamics of neuronal intermediate filaments: a develop‐

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### **Author details**

Rodolphe Perrot1 and Joel Eyer2

1 Service Commun d'Imageries et d'Analyses Microscopiques, Université d'Angers, Institut de Biologie en Santé – IRIS, CHU, France

2 Laboratoire de Neurobiologie & Transgenese, UPRES-EA3143, Institut de Biologie en Santé – IRIS, CHU, France

#### **References**


ated with the neurofilament triplet proteins in the mature CNS. J Neurosci 2006;26 (39) 10006-19

[3] Yan Y, Jensen K and Brown A. The polypeptide composition of moving and station‐ ary neurofilaments in cultured sympathetic neurons. Cell Motil Cytoskeleton 2007;64 (4) 299-309

**4. Conclusion**

474 Neurodegenerative Diseases

**Author details**

Rodolphe Perrot1

**References**

Santé – IRIS, CHU, France

IFs abnormalities are reminiscent in multiple human neurodegenerative disorders. Despite extensive efforts over the past 40 years, processes leading to these abnormalities as well as their precise contribution to disease pathogenesis often remain poorly understood. For in‐ stance, if it is clearly established that mutation in IF genes can be a primary cause of neurode‐ generative disorders, the question as to how they induce neurodegeneration frequently remain unsolved. Although transgenic mouse models have been somewhat helpful in understanding some mechanisms, most of these animals displayed a much less severe phenotype than patients and results have not always been completely clear-cut. A growing body of evidence suggests that perturbation of IF axonal transport and/or stoichiometry are directly involved in the for‐ mation of intracellular IF aggregates. Destabilization of IF mRNA could be responsible for alteration in IF stoichiometry whereas aberrant post-translational modifications could affect their transport. More investigations are also necessary to identify IF partners. The importance of IF-associated proteins in the development of neurodegenerative disorders was also high‐ lighted by the identification of mutations in genes encoding IF partners that mimic IF-related disease. This is particularly the case of gigaxonin in GAN. A particular attention should also be paid to elucidate the role that IF proteins may play in signaling. Finally, it will be important to elucidate why certain types of IF accumulations appear more toxic than others. While per‐ ikaryal accumulations are generally well tolerated, axonal inclusions are often noxious. The more deleterious effect of axonal aggregates on axonal transport could be a promising avenue to explore in the future and the identification of compounds able to remove these IF aggregates

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2 Laboratoire de Neurobiologie & Transgenese, UPRES-EA3143, Institut de Biologie en

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**Chapter 20**

**Astrocytes Role in Parkinson: A Double-Edged Sword**

Parkinson Disease (PD) is the second most chronic neurodegenerative disorder in the world, after Alzheimer´s Disease (AD), and is estimated to affect about 1% of the population over 60 years of age. PD is caused by the disruption of dopaminergic neurotransmission in the basal ganglia, which causes a reduction in the numbers of dopaminergic neurons in the sub‐

Both in normal and pathological circumstances, astrocytes are critical supporters of neuro‐ nal function in processes such as antioxidant protection, glutamate clearance, the develop‐ ment and/or maintenance of blood brain barrier characteristics, the release of gliotransmitters and cytokines [2-4]. In recent years, much research on PD has focused on the astrocytic-neuronal crosstalk, suggesting that this interaction is important for future therapies against neurodegenerative processes. During brain damage events, astrocytes be‐ come transiently or permanently impaired, and the subsequent impact on neuronal cells

In the present chapter, we provide a brief overview of the astrocytic functions and the path‐ ophysiological events elicited during PD. Additionally, we explore the beneficial and dam‐ aging consequences of reactive astrogliosis in dopaminergic neurons during PD, particularly on oxidative damage, which is a main component of numerous neuropathological condi‐ tions, and that may have a damaging effect in astrocytic functions. We also highlight some of the cellular and animal models currently used in Parkinson research, such rotenone, 1 methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and paraquat as inducers, which have many similar features with this disease. Finally, a brief overview of the future perspectives

and reproduction in any medium, provided the original work is properly cited.

© 2013 Cabezas et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

stantia nigra and formation of cytoplasmic inclusions called Lewy bodies [1].

Ricardo Cabezas, Marco Fidel Avila, Daniel Torrente,

Ramon Santos El-Bachá, Ludis Morales, Janneth Gonzalez and George E. Barreto

http://dx.doi.org/10.5772/54305

**1. Introduction**

Additional information is available at the end of the chapter

may lead to pathological conditions such as PD [5-7].

in astrocytic protection during Parkinson development is discussed.

### **Astrocytes Role in Parkinson: A Double-Edged Sword**

Ricardo Cabezas, Marco Fidel Avila, Daniel Torrente, Ramon Santos El-Bachá, Ludis Morales, Janneth Gonzalez and George E. Barreto

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54305

### **1. Introduction**

Parkinson Disease (PD) is the second most chronic neurodegenerative disorder in the world, after Alzheimer´s Disease (AD), and is estimated to affect about 1% of the population over 60 years of age. PD is caused by the disruption of dopaminergic neurotransmission in the basal ganglia, which causes a reduction in the numbers of dopaminergic neurons in the sub‐ stantia nigra and formation of cytoplasmic inclusions called Lewy bodies [1].

Both in normal and pathological circumstances, astrocytes are critical supporters of neuro‐ nal function in processes such as antioxidant protection, glutamate clearance, the develop‐ ment and/or maintenance of blood brain barrier characteristics, the release of gliotransmitters and cytokines [2-4]. In recent years, much research on PD has focused on the astrocytic-neuronal crosstalk, suggesting that this interaction is important for future therapies against neurodegenerative processes. During brain damage events, astrocytes be‐ come transiently or permanently impaired, and the subsequent impact on neuronal cells may lead to pathological conditions such as PD [5-7].

In the present chapter, we provide a brief overview of the astrocytic functions and the path‐ ophysiological events elicited during PD. Additionally, we explore the beneficial and dam‐ aging consequences of reactive astrogliosis in dopaminergic neurons during PD, particularly on oxidative damage, which is a main component of numerous neuropathological condi‐ tions, and that may have a damaging effect in astrocytic functions. We also highlight some of the cellular and animal models currently used in Parkinson research, such rotenone, 1 methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and paraquat as inducers, which have many similar features with this disease. Finally, a brief overview of the future perspectives in astrocytic protection during Parkinson development is discussed.

© 2013 Cabezas et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **2. Parkinson´s disease**

PD is a progressive neurodegenerative disorder caused by the neuronal death in the sub‐ stantia nigra (SN), degeneration of dopaminergic neurotransmission, and the presence of αsynuclein and protein inclusions in neuronal cell bodies (Lewy bodies) [4-5,7]. Main symptoms of Parkinson are asymmetrical bradikinesia, rigidity, resting tremor and postural instability. Other non-motor symptoms that generate serious disability problems have also been noted, including fatigue, pain, Lewy Body dementia, psychosis, depression, and apa‐ thy [1]. Although there is not a cure for the disease, the most used and cheaper treatment for PD continues to be Levodopa [1,8]. However, about 40% of patients developed motor fluctu‐ ations and dyskinesias after 4 to 6 years of treatment [1], demonstrating that further phar‐ macological research is needed in order to counterbalance side effects. In this aspect, treatments using long-acting dopaminergic agents or a continuous dopaminergic effect in the striatum have been associated with less severe motor complications, given alone or in combination with L-dopa [9]. Some pharmacological agents that have shown promising ap‐ plications, include dopamine agonist like apomorphine and ropinirole, and catechol-Omethyltransferase (COMT; EC 2.1.1.6) inhibitors [9].

also have beneficial roles during PD progression [21-22]. For example, astrocytes express different antioxidant molecules such as glutathione peroxidase (EC 1.11.1.9), which have been inversely correlated with the severity of dopaminergic cell loss in the respective cell

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Astrocytes are the most common cell type in the mammalian brain, conforming the glia with oligodendrocytes and microglia [23]. They are characterized by the expression of the inter‐ mediate filaments glial fibrillary acidic protein (GFAP) and vimentin (Vim). Astrocytes are essential for the metabolism of the brain, transporting multiple nutrients and metabolic pre‐ cursors to the neurons by the malate-asparte shuttle and other transporters [24]. There are two main types of astrocytes in the SNC: Protoplasmic astrocytes, which envelope neuronal bodies and synapses and fibrous astrocytes which interact with the nodes of Ranvier and oligodendroglia [7]. Current research has shown that only protoplasmic astrocytes have an

increase in the accumulation of α-synuclein, whereas fibrous astrocytes do not [7,19].

elevation in intracellular Ca2+ levels in the endfeets [24,29].

creased neuronal death [30].

Current knowledge indicates that astrocytes are critical for some cellular processes, such as the development and/or maintenance of blood–brain barrier characteristics, the promotion of neurovascular coupling, the attraction of cells through the release of chemokines, K+ buf‐ fering, release of gliotransmitters, release of glutamate by calcium signaling, maintenance of general metabolism, control of the brain pH, metabolization of dopamine and other sub‐ strates by monoamine oxidases (MAOs; EC 1.4.3.4), uptake of glutamate and γ-aminobuty‐ ric acid (GABA) by specific transporters and production of antioxidants [2-3,25-27] (Figure 1). Recent evidence has shown that astrocytes are arranged in non-overlapping domains forming a syncytial network that may contact approximately 160.000 synapses, thus inte‐ grating neural activity with the vascular network [4,28]. In this aspect, astrocytic terminal processes, known as endfeet, contact the brain vasculature and enwrap the neuronal synap‐ ses, enabling the modulation of both neuronal activity and cerebral blood flow, following an

During brain damage (including diseases, brain injury and oxidative stress), these astrocytic functions become transiently or permanently impaired, and the subsequent impact on neu‐ ronal cells may lead to pathological conditions and neurodegenerative diseases [3,26]. Neu‐ rons are more susceptible to injury than astrocytes, as they have limited antioxidant capacity, and rely heavily on their metabolic coupling with astrocytes to combat oxidative stress [3]. However, severe brain damage also results in astrocyte dysfunction, leading to in‐

As previously stated, astrocytes exert both neuroprotective and neurodegenerative roles, de‐ pending on the molecules released by them, and the pathological or normal circumstances of their microenvironment [6]. For example, astrocytes release antioxidant molecules like

groups in patients with PD [4].

**3. Astrocytes in PD**

**3.1. Astrocytic functions**

Numerous reviews and articles agree that the exact cause of PD remains unknown [1,9-10]. Mutations in various proteins such as leucine-rich repeat kinase 2 (LRRK2; EC 2.7.11.1), Par‐ kinson protein 2 (PARK2), probable cation-transporting ATPase type 13A2 (ATP13A2; EC 3.6.3-), phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1; EC 2.7.11.1), and Parkinson disease (autosomal recessive, early onset) 7 protein (DJ-1) have been observed in familiar cases of Parkinson, which only accounts for 10-15% of diagnosed cases [6,11-12]. Interestingly, LRRK2, PINK1, and DJ-1, which are present in mitochondrial mem‐ branes, have been suggested to play a role in reactive oxygen species (ROS) production by a defective maintenance of the mitochondrial membrane potential [12-13].

A number of environmental factors have been found to induce PD-like symptoms, and are currently used in animal and cellular models of the disease. Environmental factors include vascular insults to the brain, oxidative stress, neuroleptic drugs and repeated head trauma. [6,14]. Additionally, the exposure to pesticides like rotenone or 1-methyl-4-phenylpyridini‐ um (MPP+ ) and heavy metals (manganese) increases the risk of PD development [6, 10, 14-15]. In this aspect, numerous epidemiologic and toxicologic studies have examined pesti‐ cides as a risk factor for PD and parkinsonism and the possible mechanisms by which pesti‐ cides may act [14-17].

Initiation and progression of PD is dependent upon cellular events, including failures in the protein degradation machinery, oxidative stress, mitochondrial dysfunction, defects in mito‐ chondrial autophagy (mitophagy) and the continuous accumulation of α-synuclein, driven through cell to cell interactions between glial cells and neurons that ultimately lead to apop‐ tosis [7,10,18]. Previous studies pointed that astrocytic α-synuclein deposition initiates the recruitment of phagocyte microglia that attacks and kills neurons in restricted brain regions [7,19], correlating this α-synuclein accumulation with nigral neuronal cell death [20], and suggest the importance of astrocytes in the initiation of the disease. Conversely, astrocytes also have beneficial roles during PD progression [21-22]. For example, astrocytes express different antioxidant molecules such as glutathione peroxidase (EC 1.11.1.9), which have been inversely correlated with the severity of dopaminergic cell loss in the respective cell groups in patients with PD [4].

### **3. Astrocytes in PD**

**2. Parkinson´s disease**

492 Neurodegenerative Diseases

um (MPP+

cides may act [14-17].

methyltransferase (COMT; EC 2.1.1.6) inhibitors [9].

PD is a progressive neurodegenerative disorder caused by the neuronal death in the sub‐ stantia nigra (SN), degeneration of dopaminergic neurotransmission, and the presence of αsynuclein and protein inclusions in neuronal cell bodies (Lewy bodies) [4-5,7]. Main symptoms of Parkinson are asymmetrical bradikinesia, rigidity, resting tremor and postural instability. Other non-motor symptoms that generate serious disability problems have also been noted, including fatigue, pain, Lewy Body dementia, psychosis, depression, and apa‐ thy [1]. Although there is not a cure for the disease, the most used and cheaper treatment for PD continues to be Levodopa [1,8]. However, about 40% of patients developed motor fluctu‐ ations and dyskinesias after 4 to 6 years of treatment [1], demonstrating that further phar‐ macological research is needed in order to counterbalance side effects. In this aspect, treatments using long-acting dopaminergic agents or a continuous dopaminergic effect in the striatum have been associated with less severe motor complications, given alone or in combination with L-dopa [9]. Some pharmacological agents that have shown promising ap‐ plications, include dopamine agonist like apomorphine and ropinirole, and catechol-O-

Numerous reviews and articles agree that the exact cause of PD remains unknown [1,9-10]. Mutations in various proteins such as leucine-rich repeat kinase 2 (LRRK2; EC 2.7.11.1), Par‐ kinson protein 2 (PARK2), probable cation-transporting ATPase type 13A2 (ATP13A2; EC 3.6.3-), phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1; EC 2.7.11.1), and Parkinson disease (autosomal recessive, early onset) 7 protein (DJ-1) have been observed in familiar cases of Parkinson, which only accounts for 10-15% of diagnosed cases [6,11-12]. Interestingly, LRRK2, PINK1, and DJ-1, which are present in mitochondrial mem‐ branes, have been suggested to play a role in reactive oxygen species (ROS) production by a

A number of environmental factors have been found to induce PD-like symptoms, and are currently used in animal and cellular models of the disease. Environmental factors include vascular insults to the brain, oxidative stress, neuroleptic drugs and repeated head trauma. [6,14]. Additionally, the exposure to pesticides like rotenone or 1-methyl-4-phenylpyridini‐

14-15]. In this aspect, numerous epidemiologic and toxicologic studies have examined pesti‐ cides as a risk factor for PD and parkinsonism and the possible mechanisms by which pesti‐

Initiation and progression of PD is dependent upon cellular events, including failures in the protein degradation machinery, oxidative stress, mitochondrial dysfunction, defects in mito‐ chondrial autophagy (mitophagy) and the continuous accumulation of α-synuclein, driven through cell to cell interactions between glial cells and neurons that ultimately lead to apop‐ tosis [7,10,18]. Previous studies pointed that astrocytic α-synuclein deposition initiates the recruitment of phagocyte microglia that attacks and kills neurons in restricted brain regions [7,19], correlating this α-synuclein accumulation with nigral neuronal cell death [20], and suggest the importance of astrocytes in the initiation of the disease. Conversely, astrocytes

) and heavy metals (manganese) increases the risk of PD development [6, 10,

defective maintenance of the mitochondrial membrane potential [12-13].

#### **3.1. Astrocytic functions**

Astrocytes are the most common cell type in the mammalian brain, conforming the glia with oligodendrocytes and microglia [23]. They are characterized by the expression of the inter‐ mediate filaments glial fibrillary acidic protein (GFAP) and vimentin (Vim). Astrocytes are essential for the metabolism of the brain, transporting multiple nutrients and metabolic pre‐ cursors to the neurons by the malate-asparte shuttle and other transporters [24]. There are two main types of astrocytes in the SNC: Protoplasmic astrocytes, which envelope neuronal bodies and synapses and fibrous astrocytes which interact with the nodes of Ranvier and oligodendroglia [7]. Current research has shown that only protoplasmic astrocytes have an increase in the accumulation of α-synuclein, whereas fibrous astrocytes do not [7,19].

Current knowledge indicates that astrocytes are critical for some cellular processes, such as the development and/or maintenance of blood–brain barrier characteristics, the promotion of neurovascular coupling, the attraction of cells through the release of chemokines, K+ buf‐ fering, release of gliotransmitters, release of glutamate by calcium signaling, maintenance of general metabolism, control of the brain pH, metabolization of dopamine and other sub‐ strates by monoamine oxidases (MAOs; EC 1.4.3.4), uptake of glutamate and γ-aminobuty‐ ric acid (GABA) by specific transporters and production of antioxidants [2-3,25-27] (Figure 1). Recent evidence has shown that astrocytes are arranged in non-overlapping domains forming a syncytial network that may contact approximately 160.000 synapses, thus inte‐ grating neural activity with the vascular network [4,28]. In this aspect, astrocytic terminal processes, known as endfeet, contact the brain vasculature and enwrap the neuronal synap‐ ses, enabling the modulation of both neuronal activity and cerebral blood flow, following an elevation in intracellular Ca2+ levels in the endfeets [24,29].

During brain damage (including diseases, brain injury and oxidative stress), these astrocytic functions become transiently or permanently impaired, and the subsequent impact on neu‐ ronal cells may lead to pathological conditions and neurodegenerative diseases [3,26]. Neu‐ rons are more susceptible to injury than astrocytes, as they have limited antioxidant capacity, and rely heavily on their metabolic coupling with astrocytes to combat oxidative stress [3]. However, severe brain damage also results in astrocyte dysfunction, leading to in‐ creased neuronal death [30].

As previously stated, astrocytes exert both neuroprotective and neurodegenerative roles, de‐ pending on the molecules released by them, and the pathological or normal circumstances of their microenvironment [6]. For example, astrocytes release antioxidant molecules like glutathione (GSH) and superoxide dismutases (SODs; EC 1.15.1.1), and supply neurons with neurotrophic factors, such as nerve growth factor (NGF), basic fibroblast growth factor (bFG), that constitute an important attempt to protect neurons during brain damaging proc‐ esses, including PD [6, 31-32]. On the other hand, during the process of reactive astrogliosis, astrocytes release inflammatory cytokines that may affect the surrounding neurons, both by the induced production of ROS and lipid peroxidation, and by the activation of apoptotic mechanisms that induce neuronal dopaminergic death [6,10]. These unusual, and sometimes contradictory, features of astrocytes in PD will be further explored in this chapter.

Astrogliosis and microgliosis in the SN of Parkinson patients are key features of the disease, which is a nonspecific consequence of neuronal degeneration [10]. Cellular and animal models using environmental and biological toxins, especially lipopolysacchar‐ ides (LPS), herbicides and pesticides like rotenone or MPTP (1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine), can induce both astrogliosis and microgliosis, which is accompanied by neuronal death, mitochondrial dysfunction and nuclear fragmentation [41-45]. Additionally, it was previously shown that the injection of LPS in rat brains was followed by an increase in the inducible nitric oxide synthase (iNOS; EC 1.14.13.39), suggesting that chronic glial activation can cause oxidative stress in the brain, similarly to that seen in neurodegenerative processes like AD and Parkinson [10, 39, 45]. A previous report showed that activated glial cells can participate in the death of dopaminergic neurons, probably by the activation of apoptosis by cytokines like TNF-α, IL-1B, IL-6 and interferon-γ and the subsequent production of nitric oxide (NO) by the iNOS that may diffuse toward the neurons and induce lipid peroxidation, DNA strands breaks and inhibition of mitochondrial metabolism [6,10]. Furthermore, cytokines released by astrocytes may bind to their specific receptors in the dopaminer‐ gic neurons, such as TNFR1 and 2, and activate proapoptotic mechanisms through the activation of caspase 3, caspase 8, and cytochrome c [10]. Interestingly, the excessive uptake of neuronal α-synuclein by astrocytes can lead to accumulation of aggregates of this protein in astrocytes, and cause an upregulation of IL-1 α, IL-1β and IL-6, fol‐ lowed by the release of TNF-α and IL-6 [6]. These results suggest that the inhibition of glial reaction to damage and further inflammatory processes could be considered as

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495

a promising therapy to reduce neuronal damage during PD [10].

In the brain, oxidative stress and other toxic insults can trigger the overexpression and acti‐ vation of neuronal nitric oxide synthase that increases NO production and may cause apop‐ totic cell death by inducing the release of cytochrome c from mitochondrial impairment, loss of membrane potential, the opening of permeability transition pores, and the release of proapoptotic molecules [46,47]. After brain damaging processes, neurons experience greater metabolic deterioration than glial cells. For instance, astrocytes contain glycogen stores that allow them to maintain ATP production through glycolysis and mitochondrial membrane potential by reversal of the F0-F1-ATPase (EC 3.6.3.14) [48]. For example, cultured astrocytes subjected to oxygen and glucose deprivation showed a decrease in mitochondrial membrane potential, possibly caused by the mitochondrial permeability transition pore (mtPTP) open‐ ing, which leads to a loss of intramitochondrial contents, mitochondrial respiration and ATP

Nowadays there is much evidence of the role of oxidative stress in the development of neurodegenerative diseases, such as AD, PD, Amyotrophic Lateral Sclerosis (ALS) and Huntington's disease (HD). Much of these oxidative damaging processes are associated with an imbalance on the production of ROS that leads to mitochondrial stress and im‐

), can be produced

pairment in energy production [47,49]. ROS, such as superoxide (O•2-

**3.3. Oxidative stress and Parkinson: Role of astrocytes**

production [48].

**Figure 1.** Astrocytes support neuronal function by multiple ways, including the development and maintenance of blood–brain barrier and promoting the neurovascular coupling. Astrocytes regulate the levels of ions, neurotransmit‐ ters and fueling molecules such as K+, glutamate, GABA, dopamine, lactate and piruvate. Furthermore, astrocytes pro‐ mote the attraction of cells through the release of chemokines, and produce beneficial antioxidants, including glutathione, superoxide dismutases (SODs 1, 2 and 3), and ascorbate.

#### **3.2. Astrogliosis and parkinson**

Reactive astrogliosis is the main reaction of astrocytes following brain insults such as infec‐ tion, trauma [33-34], α-synuclein accumulation [35], ischemia [36-37] and neurodegenerative diseases [3]. This process involves both molecular and morphological changes in the astro‐ cytes, including increased expression of GFAP, vimentin and nestin, uptake of excitotoxic glutamate, protection from oxidative stress by the production of GSH, neuroprotection by release of adenosine, degradation of amyloid-beta peptides, facilitation of blood-brain barri‐ er, increased formation of gap junctions between astrocytes, formation of scars and, in some cases release of inflammatory cytokines, including tumor necrosis factor-α (TNF-α), and production of ROS [3,35,38-40].

Astrogliosis and microgliosis in the SN of Parkinson patients are key features of the disease, which is a nonspecific consequence of neuronal degeneration [10]. Cellular and animal models using environmental and biological toxins, especially lipopolysacchar‐ ides (LPS), herbicides and pesticides like rotenone or MPTP (1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine), can induce both astrogliosis and microgliosis, which is accompanied by neuronal death, mitochondrial dysfunction and nuclear fragmentation [41-45]. Additionally, it was previously shown that the injection of LPS in rat brains was followed by an increase in the inducible nitric oxide synthase (iNOS; EC 1.14.13.39), suggesting that chronic glial activation can cause oxidative stress in the brain, similarly to that seen in neurodegenerative processes like AD and Parkinson [10, 39, 45]. A previous report showed that activated glial cells can participate in the death of dopaminergic neurons, probably by the activation of apoptosis by cytokines like TNF-α, IL-1B, IL-6 and interferon-γ and the subsequent production of nitric oxide (NO) by the iNOS that may diffuse toward the neurons and induce lipid peroxidation, DNA strands breaks and inhibition of mitochondrial metabolism [6,10]. Furthermore, cytokines released by astrocytes may bind to their specific receptors in the dopaminer‐ gic neurons, such as TNFR1 and 2, and activate proapoptotic mechanisms through the activation of caspase 3, caspase 8, and cytochrome c [10]. Interestingly, the excessive uptake of neuronal α-synuclein by astrocytes can lead to accumulation of aggregates of this protein in astrocytes, and cause an upregulation of IL-1 α, IL-1β and IL-6, fol‐ lowed by the release of TNF-α and IL-6 [6]. These results suggest that the inhibition of glial reaction to damage and further inflammatory processes could be considered as a promising therapy to reduce neuronal damage during PD [10].

#### **3.3. Oxidative stress and Parkinson: Role of astrocytes**

glutathione (GSH) and superoxide dismutases (SODs; EC 1.15.1.1), and supply neurons with neurotrophic factors, such as nerve growth factor (NGF), basic fibroblast growth factor (bFG), that constitute an important attempt to protect neurons during brain damaging proc‐ esses, including PD [6, 31-32]. On the other hand, during the process of reactive astrogliosis, astrocytes release inflammatory cytokines that may affect the surrounding neurons, both by the induced production of ROS and lipid peroxidation, and by the activation of apoptotic mechanisms that induce neuronal dopaminergic death [6,10]. These unusual, and sometimes

**Figure 1.** Astrocytes support neuronal function by multiple ways, including the development and maintenance of blood–brain barrier and promoting the neurovascular coupling. Astrocytes regulate the levels of ions, neurotransmit‐ ters and fueling molecules such as K+, glutamate, GABA, dopamine, lactate and piruvate. Furthermore, astrocytes pro‐ mote the attraction of cells through the release of chemokines, and produce beneficial antioxidants, including

Reactive astrogliosis is the main reaction of astrocytes following brain insults such as infec‐ tion, trauma [33-34], α-synuclein accumulation [35], ischemia [36-37] and neurodegenerative diseases [3]. This process involves both molecular and morphological changes in the astro‐ cytes, including increased expression of GFAP, vimentin and nestin, uptake of excitotoxic glutamate, protection from oxidative stress by the production of GSH, neuroprotection by release of adenosine, degradation of amyloid-beta peptides, facilitation of blood-brain barri‐ er, increased formation of gap junctions between astrocytes, formation of scars and, in some cases release of inflammatory cytokines, including tumor necrosis factor-α (TNF-α), and

glutathione, superoxide dismutases (SODs 1, 2 and 3), and ascorbate.

**3.2. Astrogliosis and parkinson**

494 Neurodegenerative Diseases

production of ROS [3,35,38-40].

contradictory, features of astrocytes in PD will be further explored in this chapter.

In the brain, oxidative stress and other toxic insults can trigger the overexpression and acti‐ vation of neuronal nitric oxide synthase that increases NO production and may cause apop‐ totic cell death by inducing the release of cytochrome c from mitochondrial impairment, loss of membrane potential, the opening of permeability transition pores, and the release of proapoptotic molecules [46,47]. After brain damaging processes, neurons experience greater metabolic deterioration than glial cells. For instance, astrocytes contain glycogen stores that allow them to maintain ATP production through glycolysis and mitochondrial membrane potential by reversal of the F0-F1-ATPase (EC 3.6.3.14) [48]. For example, cultured astrocytes subjected to oxygen and glucose deprivation showed a decrease in mitochondrial membrane potential, possibly caused by the mitochondrial permeability transition pore (mtPTP) open‐ ing, which leads to a loss of intramitochondrial contents, mitochondrial respiration and ATP production [48].

Nowadays there is much evidence of the role of oxidative stress in the development of neurodegenerative diseases, such as AD, PD, Amyotrophic Lateral Sclerosis (ALS) and Huntington's disease (HD). Much of these oxidative damaging processes are associated with an imbalance on the production of ROS that leads to mitochondrial stress and im‐ pairment in energy production [47,49]. ROS, such as superoxide (O•2- ), can be produced in mitochondrial complexes I and III in components of the tricarboxylic-acid cycle, in‐ cluding α-ketoglutarate dehydrogenase (EC 1.2.4.2), and in the outter mitochondrial membrane, damaging cell components such as lipids, proteins and DNA [25, 47]. In PD, oxidative damage is a common feature, as demonstrated by increased levels of ROS in post-mortem PD brain samples [25]. Oxidative stress seems to affect various brain re‐ gions, including the SN and caudate nucleus, and it is accompanied by an increase in GFAP and astrocytic proliferation [50]. Additionally, PD patients present deficiencies in mitochondrial complex I in the SN, suggesting that a defect in this complex could con‐ tribute to neuronal degeneration in PD [25]. However, it is not clear whether the damage induced by ROS is a cause or a consequence of other cellular dysfunctions [25]. For ex‐ ample, a previous study on PD brains showed an increase in lipid peroxidation prod‐ ucts, such as 4-hydroxinonenal, and in protein crosslinking and fragmentation [51], suggesting that oxidative stress may affect other brain regions apart from the SN.

Astrocytes produce numerous antioxidant molecules, such as GSH, catalase (EC 1.11.1.6) and SODs, providing further antioxidant protection to neurons. Unfortunately, it is known that the astrocytic protection afforded to neurons is limited, possibly due to a decline in GSH trafficking by chronic iNOS induction [52]. This depletion of GSH may facilitate the production of ROS and reactive nitrogen species (RNS) by astrocytes, causing alterations in neuronal proteins such as α-synuclein [25]. Furthermore, the nitration of α-synuclein by RNS can significantly enhance the synuclein fibril formation *in vitro,* similarly to what hap‐ pens in PD brains [25]. In sum, the antioxidant properties of astrocytes have a fundamental role in the development of neurodegenerative diseases, and are considered as promising therapeutically targets.

> **Figure 2.** Experimental models in PD. Many molecules are currently used in cellular and animal models of PD, includ‐ ing pesticides as paraquat or rotenone and neurotoxins such as 6-OHDA and MPP+. Paraquat, 6-hydroxydopamine (6- OHDA) and MPP+ easily cross cell membrane through the dopamine transporter (DA) thus inducing the formation of α-synuclein aggregates and mitochondrial impairment with the subsequent production of ROS and quinones. Com‐ pounds, as rotenone, are extremely hydrophobic and penetrate easily the cellular membrane of neurons and astro‐ cytes. Rotenone may promote processes such as the formation of α-synuclein aggregates, and the genetic activation through the nuclear translocation of NF-κB. Additionally, as an inhibitor of mitochondrial complex I, rotenone causes the impairment of ATP, the generation of ROS and the release of proapoptotic molecules, such as cytochrome c that

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497

Rotenone is one of the most studied neurotoxic substances used as a model for PD features and oxidative stress events in cellular and animal models [14,57]. Rotenone is a naturally occurring isoflavonoid produced in the leaves, roots and rhizomes of the tropical legumes from the gen‐ res *Derris*, *Lonchocarpus,* and T*ephrosia*. It is extremely hydrophobic and crosses biological membranes and serves as a high-affinity noncompetitive inhibitor of complex I, thus affecting ATP generation [58]. Rotenone is commonly used in solution as a pesticide, insecticide, or in

activate caspase 9, which trigger caspases 3, 6 and 7, and induce apoptosis.

**4.1. Rotenone as a Parkinson model**

emulsified liquid form as a piscicide [59,60].

#### **4. Experimental models in Parkinson**

Various pesticides, herbicides and drugs have been used in animal and *in vitro* models of Parkinson, as their effects mimic similar features of that seen in PD. Different epidemiologi‐ cal studies have shown a correlation between the exposure of these substances (especially in the case of pesticides) and appearance of PD [14-15, 17, 53]. A common feature of many of these neurotoxic compounds, such as rotenone, paraquat, or MPTP, is the inhibition of mito‐ chondrial complex I, followed by the overproduction of ROS, ATP exhaustion, and induc‐ tion of a wide range of abnormalities that can elicit neuronal and astrocytic cell death [54]. Additionally, neurotoxins induce nuclear fragmentation, endoplasmic reticulum (ER) stress and unfolded protein response in catecholaminergic cells, which are associated with changes in proteasomal and chaperone activities, similar to those observed in PD [45,55]. Other molecules used in PD models include the fungicide maneb, cyclodienes, organophos‐ phates such as deltamethrin, DDT (dichlorodiphenyltrichloroethane), 2,4-dichlorophenoxy‐ acetic acid, dieldrin, deguelin, diethyldithiocarbamate, paraquat, maneb, trifluralin and parathion (Figure 2) [15,56].

**Figure 2.** Experimental models in PD. Many molecules are currently used in cellular and animal models of PD, includ‐ ing pesticides as paraquat or rotenone and neurotoxins such as 6-OHDA and MPP+. Paraquat, 6-hydroxydopamine (6- OHDA) and MPP+ easily cross cell membrane through the dopamine transporter (DA) thus inducing the formation of α-synuclein aggregates and mitochondrial impairment with the subsequent production of ROS and quinones. Com‐ pounds, as rotenone, are extremely hydrophobic and penetrate easily the cellular membrane of neurons and astro‐ cytes. Rotenone may promote processes such as the formation of α-synuclein aggregates, and the genetic activation through the nuclear translocation of NF-κB. Additionally, as an inhibitor of mitochondrial complex I, rotenone causes the impairment of ATP, the generation of ROS and the release of proapoptotic molecules, such as cytochrome c that activate caspase 9, which trigger caspases 3, 6 and 7, and induce apoptosis.

#### **4.1. Rotenone as a Parkinson model**

in mitochondrial complexes I and III in components of the tricarboxylic-acid cycle, in‐ cluding α-ketoglutarate dehydrogenase (EC 1.2.4.2), and in the outter mitochondrial membrane, damaging cell components such as lipids, proteins and DNA [25, 47]. In PD, oxidative damage is a common feature, as demonstrated by increased levels of ROS in post-mortem PD brain samples [25]. Oxidative stress seems to affect various brain re‐ gions, including the SN and caudate nucleus, and it is accompanied by an increase in GFAP and astrocytic proliferation [50]. Additionally, PD patients present deficiencies in mitochondrial complex I in the SN, suggesting that a defect in this complex could con‐ tribute to neuronal degeneration in PD [25]. However, it is not clear whether the damage induced by ROS is a cause or a consequence of other cellular dysfunctions [25]. For ex‐ ample, a previous study on PD brains showed an increase in lipid peroxidation prod‐ ucts, such as 4-hydroxinonenal, and in protein crosslinking and fragmentation [51],

suggesting that oxidative stress may affect other brain regions apart from the SN.

therapeutically targets.

496 Neurodegenerative Diseases

parathion (Figure 2) [15,56].

**4. Experimental models in Parkinson**

Astrocytes produce numerous antioxidant molecules, such as GSH, catalase (EC 1.11.1.6) and SODs, providing further antioxidant protection to neurons. Unfortunately, it is known that the astrocytic protection afforded to neurons is limited, possibly due to a decline in GSH trafficking by chronic iNOS induction [52]. This depletion of GSH may facilitate the production of ROS and reactive nitrogen species (RNS) by astrocytes, causing alterations in neuronal proteins such as α-synuclein [25]. Furthermore, the nitration of α-synuclein by RNS can significantly enhance the synuclein fibril formation *in vitro,* similarly to what hap‐ pens in PD brains [25]. In sum, the antioxidant properties of astrocytes have a fundamental role in the development of neurodegenerative diseases, and are considered as promising

Various pesticides, herbicides and drugs have been used in animal and *in vitro* models of Parkinson, as their effects mimic similar features of that seen in PD. Different epidemiologi‐ cal studies have shown a correlation between the exposure of these substances (especially in the case of pesticides) and appearance of PD [14-15, 17, 53]. A common feature of many of these neurotoxic compounds, such as rotenone, paraquat, or MPTP, is the inhibition of mito‐ chondrial complex I, followed by the overproduction of ROS, ATP exhaustion, and induc‐ tion of a wide range of abnormalities that can elicit neuronal and astrocytic cell death [54]. Additionally, neurotoxins induce nuclear fragmentation, endoplasmic reticulum (ER) stress and unfolded protein response in catecholaminergic cells, which are associated with changes in proteasomal and chaperone activities, similar to those observed in PD [45,55]. Other molecules used in PD models include the fungicide maneb, cyclodienes, organophos‐ phates such as deltamethrin, DDT (dichlorodiphenyltrichloroethane), 2,4-dichlorophenoxy‐ acetic acid, dieldrin, deguelin, diethyldithiocarbamate, paraquat, maneb, trifluralin and

Rotenone is one of the most studied neurotoxic substances used as a model for PD features and oxidative stress events in cellular and animal models [14,57]. Rotenone is a naturally occurring isoflavonoid produced in the leaves, roots and rhizomes of the tropical legumes from the gen‐ res *Derris*, *Lonchocarpus,* and T*ephrosia*. It is extremely hydrophobic and crosses biological membranes and serves as a high-affinity noncompetitive inhibitor of complex I, thus affecting ATP generation [58]. Rotenone is commonly used in solution as a pesticide, insecticide, or in emulsified liquid form as a piscicide [59,60].

Rotenone, and other complex I inhibitors, such as MPTP, paraquat and maneb, are used as models for assessing the environmental causes of PD [12]. Previous epidemiological studies have supported the hypothesis that a prolonged exposure to pesticides is a risk factor for PD [17, 57,61]. Furthermore, a recent case-control study from the NIH, which reviewed 110 PD cas‐ es and 358 controls, and observed that PD incidence was increased 2.5 times in individuals who reported use of rotenone compared with nonusers [17]. Another study in agricultural workers from East Texas identified a significant increased risk (OR = 10.9) of PD with the continuous use of rotenone [53]. Although these reports raised important concerns on the use of rotenone, fur‐ ther studies are needed to assess the detailed global epidemiology of PD by this pesticide.

Alternatively, it has been postulated that rotenone-induced dopaminergic neuronal death could be dependent on the inflammatory process associated with microglial activation [64] thus indicating that rotenone differentially affects various types of CNS cells. Other previ‐ ous experiments have shown that subcutaneous administration of rotenone resulted in a highly selective dopaminergic damage in neurons and α-synuclein aggregation, similar to the Lewy bodies of PD [63,65]. The mechanisms by which rotenone upregulates α-synuclein and causes its aggregation, are not well understood. A possible hypothesis is that aggrega‐ tion is probably a consequence of oxidative modifications of α-synuclein [69]. For instance, neurons and astrocytes treated with rotenone (25 to 50 nM) showed an altered expression of g-tubulin and a disorganization of the centrosome with aggregates of α-synuclein [70]. Simi‐ larly, other studies suggest that inhibition of mitochondrial complex I activity and facilita‐ tion of α-synuclein aggregation may be closely associated with rotenone's selective dopaminergic toxicity in neurons [14,65]. Furthermore, a different approach using intragas‐ trically administered rotenone (5 mg/Kg) in mice showed that the accumulation and aggre‐ gation of α-synuclein in neurons of the dorsal motor nucleus of the vagus (DMV) and the intermediolateral nucleus (IML) in the spinal cord was accompanied by the selective loss of dopaminergic neurons and astrogliosis, suggesting that the gastric administration of rote‐ none through the connection of the enteric nervous system (ENS) with anatomical structures of the CNS also induces PD-like features [11,19]. Rotenone has also been shown to cause in‐ creased expression of connexin43 (Cx43), which forms gap junctions, and P2X7 receptors that modulate cytokine secretion and gamma tubulin; these are important for the adequate function of the cytoskeleton and organelles such as the Golgi apparatus [70-73]. Moreover, rotenone induces astrogliosis and alterations in the expression of g-tubulin, signal transduc‐

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499

er and activator of transcription 3 (STAT3), and connexin 43 in astrocytes [70, 72, 74].

creased neuronal death [2-4,74].

**4.2. MPTP and Parkinson**

In sum, the *in vitro* and *in vivo* evidences presented here show that dopaminergic neurons are more sensitive to rotenone toxicity than non-dopaminergic neurons, amacrine cells of retina and astrocytes [55, 75-77], possibly due to their lesser effective oxidative mechanisms and reduced supply of antioxidants [30,78]. However, astrocytes are more resilient to rote‐ none treatment than neurons, being its mitochondrial dysfunction tightly associated with in‐

MPTP is a widely used neurotoxicant, known for the induction of Parkinson-like symptoms such as bradikinesia, movement disorders, α-synuclein bodies, mitochondrial abnormalities, sustained inflammation in the substantia nigra and activation of the microglia [6,10,15, 79-80]. It was initially shown that in drug addicts, who were accidentally exposed to MPTP, there was a depletion of pigmented nerve cells in the substantia nigra, accompanied by astrogliosis and clustering of microgliosis around nerve cells [41], thus presenting some PD-like features.

MPTP is an alipophilic prototoxin that rapidly crosses the blood-brain barrier and damage

amine transporter [80]. Similarly to rotenone, its neurotoxicity is induced by the inhibition of mitochondrial complex I, and subsequent energy depletion [80-81]. Additionally, MPP+ has

via the dop‐

dopaminergic neurons due to the selective uptake of the active metabolite MPP+

Much of the research on rotenone has used animal models and different routes of adminis‐ tration for evaluating its effects in the Central Nervous System (CNS), especially in neurons [14,57]. Several groups have demonstrated that continuous systemic administration of rote‐ none to rats and mice reproduces key features of PD, including selective degeneration of the nigrostriatal dopaminergic system, activation of astroglia and microglia, formation of cyto‐ plasmic inclusions in neurons, movement disorders, and defects in mitochondrial complex I [11, 14, 57, 62-64]. Previous studies have shown that intracerebral administration of rotenone damages the nigrostriatal dopaminergic pathway in rats, including the striatum fibers and neurons [14,57]. However, the doses employed in those experiments were much higher than the standard IC50 for rotenone. For example, doses of 2-3 mg/kg/day, similar to that reported in platelets from PD patients, produced complex I inhibition with selective nigrostriatal de‐ generation and astrocyte activation [14,65]. In this matter, neuronal death is thought to be a consequence of the inhibition of mitochondrial complex I, which leads to a reduction in the energy supply and subsequent collapse of the mitochondrial membrane potential [66]. A re‐ cent study suggests that rotenone administration activates caspase-2 in mice neurons induc‐ ing the activation of downstream apoptotic effectors such as Bid, Bax, caspase 3 and 9, thus initiating apoptosis [67]. Similarly, the exposure of human dopaminergic SH- SY5Y cells to rotenone caused the nuclear translocation of nuclear factor κB (NF-κB) and the activation of caspase-3, suggesting that complex I deficiency induced by rotenone can induce NF-κBmediated apoptosis (Figure 3) [68].

**Figure 3.** Rotenone-induced cell death. Astrocytic cell line ESP12 cells were treated with 30 nM of rotenone (right) or control (right), and stained for Hoetsch 33258 to assess nuclear fragmentation. Rotenone-treated cells showed in‐ creased nuclear damage compared to controls. Scale bar, 50 μm.

Alternatively, it has been postulated that rotenone-induced dopaminergic neuronal death could be dependent on the inflammatory process associated with microglial activation [64] thus indicating that rotenone differentially affects various types of CNS cells. Other previ‐ ous experiments have shown that subcutaneous administration of rotenone resulted in a highly selective dopaminergic damage in neurons and α-synuclein aggregation, similar to the Lewy bodies of PD [63,65]. The mechanisms by which rotenone upregulates α-synuclein and causes its aggregation, are not well understood. A possible hypothesis is that aggrega‐ tion is probably a consequence of oxidative modifications of α-synuclein [69]. For instance, neurons and astrocytes treated with rotenone (25 to 50 nM) showed an altered expression of g-tubulin and a disorganization of the centrosome with aggregates of α-synuclein [70]. Simi‐ larly, other studies suggest that inhibition of mitochondrial complex I activity and facilita‐ tion of α-synuclein aggregation may be closely associated with rotenone's selective dopaminergic toxicity in neurons [14,65]. Furthermore, a different approach using intragas‐ trically administered rotenone (5 mg/Kg) in mice showed that the accumulation and aggre‐ gation of α-synuclein in neurons of the dorsal motor nucleus of the vagus (DMV) and the intermediolateral nucleus (IML) in the spinal cord was accompanied by the selective loss of dopaminergic neurons and astrogliosis, suggesting that the gastric administration of rote‐ none through the connection of the enteric nervous system (ENS) with anatomical structures of the CNS also induces PD-like features [11,19]. Rotenone has also been shown to cause in‐ creased expression of connexin43 (Cx43), which forms gap junctions, and P2X7 receptors that modulate cytokine secretion and gamma tubulin; these are important for the adequate function of the cytoskeleton and organelles such as the Golgi apparatus [70-73]. Moreover, rotenone induces astrogliosis and alterations in the expression of g-tubulin, signal transduc‐ er and activator of transcription 3 (STAT3), and connexin 43 in astrocytes [70, 72, 74].

In sum, the *in vitro* and *in vivo* evidences presented here show that dopaminergic neurons are more sensitive to rotenone toxicity than non-dopaminergic neurons, amacrine cells of retina and astrocytes [55, 75-77], possibly due to their lesser effective oxidative mechanisms and reduced supply of antioxidants [30,78]. However, astrocytes are more resilient to rote‐ none treatment than neurons, being its mitochondrial dysfunction tightly associated with in‐ creased neuronal death [2-4,74].

#### **4.2. MPTP and Parkinson**

Rotenone, and other complex I inhibitors, such as MPTP, paraquat and maneb, are used as models for assessing the environmental causes of PD [12]. Previous epidemiological studies have supported the hypothesis that a prolonged exposure to pesticides is a risk factor for PD [17, 57,61]. Furthermore, a recent case-control study from the NIH, which reviewed 110 PD cas‐ es and 358 controls, and observed that PD incidence was increased 2.5 times in individuals who reported use of rotenone compared with nonusers [17]. Another study in agricultural workers from East Texas identified a significant increased risk (OR = 10.9) of PD with the continuous use of rotenone [53]. Although these reports raised important concerns on the use of rotenone, fur‐ ther studies are needed to assess the detailed global epidemiology of PD by this pesticide.

Much of the research on rotenone has used animal models and different routes of adminis‐ tration for evaluating its effects in the Central Nervous System (CNS), especially in neurons [14,57]. Several groups have demonstrated that continuous systemic administration of rote‐ none to rats and mice reproduces key features of PD, including selective degeneration of the nigrostriatal dopaminergic system, activation of astroglia and microglia, formation of cyto‐ plasmic inclusions in neurons, movement disorders, and defects in mitochondrial complex I [11, 14, 57, 62-64]. Previous studies have shown that intracerebral administration of rotenone damages the nigrostriatal dopaminergic pathway in rats, including the striatum fibers and neurons [14,57]. However, the doses employed in those experiments were much higher than the standard IC50 for rotenone. For example, doses of 2-3 mg/kg/day, similar to that reported in platelets from PD patients, produced complex I inhibition with selective nigrostriatal de‐ generation and astrocyte activation [14,65]. In this matter, neuronal death is thought to be a consequence of the inhibition of mitochondrial complex I, which leads to a reduction in the energy supply and subsequent collapse of the mitochondrial membrane potential [66]. A re‐ cent study suggests that rotenone administration activates caspase-2 in mice neurons induc‐ ing the activation of downstream apoptotic effectors such as Bid, Bax, caspase 3 and 9, thus initiating apoptosis [67]. Similarly, the exposure of human dopaminergic SH- SY5Y cells to rotenone caused the nuclear translocation of nuclear factor κB (NF-κB) and the activation of caspase-3, suggesting that complex I deficiency induced by rotenone can induce NF-κB-

**Figure 3.** Rotenone-induced cell death. Astrocytic cell line ESP12 cells were treated with 30 nM of rotenone (right) or control (right), and stained for Hoetsch 33258 to assess nuclear fragmentation. Rotenone-treated cells showed in‐

mediated apoptosis (Figure 3) [68].

498 Neurodegenerative Diseases

creased nuclear damage compared to controls. Scale bar, 50 μm.

MPTP is a widely used neurotoxicant, known for the induction of Parkinson-like symptoms such as bradikinesia, movement disorders, α-synuclein bodies, mitochondrial abnormalities, sustained inflammation in the substantia nigra and activation of the microglia [6,10,15, 79-80]. It was initially shown that in drug addicts, who were accidentally exposed to MPTP, there was a depletion of pigmented nerve cells in the substantia nigra, accompanied by astrogliosis and clustering of microgliosis around nerve cells [41], thus presenting some PD-like features.

MPTP is an alipophilic prototoxin that rapidly crosses the blood-brain barrier and damage dopaminergic neurons due to the selective uptake of the active metabolite MPP+ via the dop‐ amine transporter [80]. Similarly to rotenone, its neurotoxicity is induced by the inhibition of mitochondrial complex I, and subsequent energy depletion [80-81]. Additionally, MPP+ has high affinity for noradrenergic and serotonergic uptake transporters [6,82], and its precur‐ sor, MPTP, has been mainly used in neuronal models with dopaminergic characteristics, such as the dopaminergic neuroblastoma cell line SH-SY5Y [83]. In astrocytes, MPTP has shown different (and sometimes contradictory) effects according to the experimental evi‐ dence collected in cellular and animal models. For instance, Rappold and Tieu (2010) showed that MPTP is metabolized by the astrocytic monoamineoxidase-B (MAO-B) and converted to the toxic cation MPP+ , which is extruded to the extracellular space through the organic cation transporter 3 (oct3) [6, 84]. Afterwards, MPP+ is taken by neighboring dopa‐ minergic neurons, thus inducing neuronal death [84]. Interestingly, silencing of oct 3 trans‐ porter in mice attenuates both the MPP+ release from astrocytes and the subsequent impairment of dopaminergic neurons, in which makes oct3 as an important molecular target for dopaminergic related pathologies [6,84]. On the other hand, other authors have shown that MPP+ induces negative effects in astrocytes, such as loss of viability, impairment of en‐ ergetic metabolism of mitochondria, ROS generation and decrease in the glutamate clear‐ ance by astrocytes [81,85,86]. Taking into account the importance of MPTP, as a model for PD, it seems that further epidemiological research is needed to address more thoroughly the role of MPTP in astrocytic damage and PD development.

cules targeting neuronal survival, it is possible that genetic manipulation of these functions in astrocytes may represent a promising strategy to improve dopaminergic neurons or neu‐ ral progenitor cells survival [4,23]. These neuroprotective features of astrocyte in Parkinson

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501

Over the last years, much research has focused on specific molecules produced by astrocytes that exert neuroprotection during brain injuries and diseases including PD, both through the reuptake of glutamate, or by producing gliotransmitters, antioxidant enzimes such as SODs, growth factors, peptide hormones and heat shock proteins [4,94-98]. Many of them have shown protective effects both in dopaminergic neurons and glial cells, and have been used

Astrocytes produce beneficial antioxidants, including glutathione, superoxide dismutases (SODs 1, 2 and 3), and ascorbate, which are important for neuronal survival during neuro‐

The tripeptid glutathione, as the main antioxidant in the brain, is needed for the conversion of methylglyoxal, a toxic by-product of metabolism, into d-lactate by glyoxalase 1 (EC 4.4.1.5) [94,95]. GSH is also important in limiting and repairing the deleterious actions of NO, but unfortunately GSH levels can be depleted by extremely high concentrations of NO [23]. For example, glutathione becomes rapidly oxidized to glutathione disulfide either by glutathione peroxidase (GPx) or by enzyme-independent chemical reactions [102]. This is an important effect against ROS formation in PD, as it helps reducing the inhibition of complex I by NO [103]. Astrocytes possess a greater concentration of glutathione (3,8 mmol/L) than neurons (2.5 mmol), probably due to a higher content of the astrocytic enzyme y-glutamyl‐ cysteine synthethase (EC 6.3.2.2) [6]. For example, neurons co-cultured with astrocytes ex‐ hibit higher levels of glutathione compared to neurons cultured alone, demonstrating that astrocytes provide additional antioxidant defenses to neurons [104-106]. Additionally, an in‐ crease in glutathione peroxidase-containing cells shows to be inversely correlated with the severity of dopaminergic cell loss in cell populations from patients with PD, suggesting that the quantity of glutathione peroxidase in cells might be critical for a protective effect against

The greater production of GSH by astrocytes seems to be dependent on the preferential acti‐ vation of transcription factor Nrf2 in astrocytes, which leads to a more efficient GSH synthe‐ sis and higher GSH content in astrocytes than in neurons [108]. Interestingly, Nrf2 is known to regulate the expression of cytoprotective genes, and factors essential to neuronal survival [6,108]. Additionally, Nrf2 knockout mice are more sensitive to mitochondrial complex in‐ hibitors such as MPTP and 3-nitropropionic acid [108], suggesting an important role of this transcription factor in scavenging free radicals. On the other hand, decrease in glutathione is

in animal models and clinical trials with remarkable results (Figure 4) [31,32].

are further explored in the following topic.

**5.1. Glutathione and Parkinson**

degenerative processes [95,99-101].

oxidative stress during PD [107].

**5. Astrocytic neuroprotection in Parkinson**

#### **4.3. Other toxic compounds involved in Parkinson development: Paraquat and 6-OHDA**

The pesticide *N*,*N*′-dimethyl-4,4′-bipyridinium dichloride (paraquat), which shares similar structure with MPP+ , impairs mitochondrial functions by inducing an augmented produc‐ tion of oxidative stress and 4-hydroxynenal *in vivo* [87]. Although paraquat may not be an efficient inhibitor of mitochondrial complex I, and so does not affect dopamine uptake [87,88], it does cause α-synuclein aggregation in C57Bl/6 mice, and alters Parkin solubility, decreasing proteasome activity and causing cellular damage [87].

Paraquat has been previously shown to induce PD-like neuronal dopaminergic lesions in ani‐ mal models and neuronal cell lines (Brown et al., 2006; Berry et al., 2010). Additionally, epide‐ miological studies suggest that long-term exposure to paraquat is associated with PD development [15,89]. To counteract this oxidative damage induced by paraquat, and MPTP, as‐ trocytes seem to protect dopaminergic neurons by increasing expression of antioxidant mole‐ cules, such as heme oxigenase1 (EC 1.14.99.3), glutathione S-transferase P (EC 2.5.1.18) and glutathione [90,91]. Although this protective role of astrocytes on neuronal death by paraquat is quite promising, only few studies address this interaction and further research is needed in order to establish the precise effect of paraquat in astrocytes metabolism and neuroprotection.

Similarly to paraquat, 6-Hydroxydopamine (6-OHDA) is another widely used for *in vivo* and *in vitro* animal models of PD [92]. This compound has a structure similar to dopamine and norepinephrine and exhibits a high affinity for catecholaminergic transporters such as dopamine DAT (Dopamine transporter). 6-OHDA induces dopaminergic neuronal death by the increased generation of H2O2 and quinones [92]. Additionally, it causes both microgliosis and astrogliosis, which is characterized by increased astrocytic proliferation in rat cortex and striatum accompanied by a marked expression of GFAP [92,93]. Taking into account that reactive astrocytes may produce various neurotrophic factors and antioxidant mole‐ cules targeting neuronal survival, it is possible that genetic manipulation of these functions in astrocytes may represent a promising strategy to improve dopaminergic neurons or neu‐ ral progenitor cells survival [4,23]. These neuroprotective features of astrocyte in Parkinson are further explored in the following topic.

### **5. Astrocytic neuroprotection in Parkinson**

Over the last years, much research has focused on specific molecules produced by astrocytes that exert neuroprotection during brain injuries and diseases including PD, both through the reuptake of glutamate, or by producing gliotransmitters, antioxidant enzimes such as SODs, growth factors, peptide hormones and heat shock proteins [4,94-98]. Many of them have shown protective effects both in dopaminergic neurons and glial cells, and have been used in animal models and clinical trials with remarkable results (Figure 4) [31,32].

#### **5.1. Glutathione and Parkinson**

high affinity for noradrenergic and serotonergic uptake transporters [6,82], and its precur‐ sor, MPTP, has been mainly used in neuronal models with dopaminergic characteristics, such as the dopaminergic neuroblastoma cell line SH-SY5Y [83]. In astrocytes, MPTP has shown different (and sometimes contradictory) effects according to the experimental evi‐ dence collected in cellular and animal models. For instance, Rappold and Tieu (2010) showed that MPTP is metabolized by the astrocytic monoamineoxidase-B (MAO-B) and

minergic neurons, thus inducing neuronal death [84]. Interestingly, silencing of oct 3 trans‐

impairment of dopaminergic neurons, in which makes oct3 as an important molecular target for dopaminergic related pathologies [6,84]. On the other hand, other authors have shown

ergetic metabolism of mitochondria, ROS generation and decrease in the glutamate clear‐ ance by astrocytes [81,85,86]. Taking into account the importance of MPTP, as a model for PD, it seems that further epidemiological research is needed to address more thoroughly the

**4.3. Other toxic compounds involved in Parkinson development: Paraquat and 6-OHDA**

The pesticide *N*,*N*′-dimethyl-4,4′-bipyridinium dichloride (paraquat), which shares similar

tion of oxidative stress and 4-hydroxynenal *in vivo* [87]. Although paraquat may not be an efficient inhibitor of mitochondrial complex I, and so does not affect dopamine uptake [87,88], it does cause α-synuclein aggregation in C57Bl/6 mice, and alters Parkin solubility,

Paraquat has been previously shown to induce PD-like neuronal dopaminergic lesions in ani‐ mal models and neuronal cell lines (Brown et al., 2006; Berry et al., 2010). Additionally, epide‐ miological studies suggest that long-term exposure to paraquat is associated with PD development [15,89]. To counteract this oxidative damage induced by paraquat, and MPTP, as‐ trocytes seem to protect dopaminergic neurons by increasing expression of antioxidant mole‐ cules, such as heme oxigenase1 (EC 1.14.99.3), glutathione S-transferase P (EC 2.5.1.18) and glutathione [90,91]. Although this protective role of astrocytes on neuronal death by paraquat is quite promising, only few studies address this interaction and further research is needed in order to establish the precise effect of paraquat in astrocytes metabolism and neuroprotection.

Similarly to paraquat, 6-Hydroxydopamine (6-OHDA) is another widely used for *in vivo* and *in vitro* animal models of PD [92]. This compound has a structure similar to dopamine and norepinephrine and exhibits a high affinity for catecholaminergic transporters such as dopamine DAT (Dopamine transporter). 6-OHDA induces dopaminergic neuronal death by the increased generation of H2O2 and quinones [92]. Additionally, it causes both microgliosis and astrogliosis, which is characterized by increased astrocytic proliferation in rat cortex and striatum accompanied by a marked expression of GFAP [92,93]. Taking into account that reactive astrocytes may produce various neurotrophic factors and antioxidant mole‐

induces negative effects in astrocytes, such as loss of viability, impairment of en‐

, impairs mitochondrial functions by inducing an augmented produc‐

, which is extruded to the extracellular space through the

release from astrocytes and the subsequent

is taken by neighboring dopa‐

converted to the toxic cation MPP+

that MPP+

500 Neurodegenerative Diseases

structure with MPP+

porter in mice attenuates both the MPP+

organic cation transporter 3 (oct3) [6, 84]. Afterwards, MPP+

role of MPTP in astrocytic damage and PD development.

decreasing proteasome activity and causing cellular damage [87].

Astrocytes produce beneficial antioxidants, including glutathione, superoxide dismutases (SODs 1, 2 and 3), and ascorbate, which are important for neuronal survival during neuro‐ degenerative processes [95,99-101].

The tripeptid glutathione, as the main antioxidant in the brain, is needed for the conversion of methylglyoxal, a toxic by-product of metabolism, into d-lactate by glyoxalase 1 (EC 4.4.1.5) [94,95]. GSH is also important in limiting and repairing the deleterious actions of NO, but unfortunately GSH levels can be depleted by extremely high concentrations of NO [23]. For example, glutathione becomes rapidly oxidized to glutathione disulfide either by glutathione peroxidase (GPx) or by enzyme-independent chemical reactions [102]. This is an important effect against ROS formation in PD, as it helps reducing the inhibition of complex I by NO [103]. Astrocytes possess a greater concentration of glutathione (3,8 mmol/L) than neurons (2.5 mmol), probably due to a higher content of the astrocytic enzyme y-glutamyl‐ cysteine synthethase (EC 6.3.2.2) [6]. For example, neurons co-cultured with astrocytes ex‐ hibit higher levels of glutathione compared to neurons cultured alone, demonstrating that astrocytes provide additional antioxidant defenses to neurons [104-106]. Additionally, an in‐ crease in glutathione peroxidase-containing cells shows to be inversely correlated with the severity of dopaminergic cell loss in cell populations from patients with PD, suggesting that the quantity of glutathione peroxidase in cells might be critical for a protective effect against oxidative stress during PD [107].

The greater production of GSH by astrocytes seems to be dependent on the preferential acti‐ vation of transcription factor Nrf2 in astrocytes, which leads to a more efficient GSH synthe‐ sis and higher GSH content in astrocytes than in neurons [108]. Interestingly, Nrf2 is known to regulate the expression of cytoprotective genes, and factors essential to neuronal survival [6,108]. Additionally, Nrf2 knockout mice are more sensitive to mitochondrial complex in‐ hibitors such as MPTP and 3-nitropropionic acid [108], suggesting an important role of this transcription factor in scavenging free radicals. On the other hand, decrease in glutathione is one of the earliest biochemical changes in PD and incidental Lewy body disease [109]. Addi‐ tionally, the GSH content was significantly reduced in the substantia nigra of PD patients, suggesting that GSH depletion enhances neuronal death under certain pathological condi‐ tions [6]. Interestingly, this evidence is consistent with the data in PD patients, in which glu‐ tathione-containing cells are in regions with preserved dopaminergic neurons [52].

It is possible that the recovery of glutathione levels may enhance the survival of affected neurons, either by increasing synthesis of GSH or by slowing its degradation [25]. However, the GSH blood-brain barrier permeability is low, and clinical trials using injections of GSH have shown little benefits [6,25,110]. Alternatively, it has been demonstrated that the use of GSH precursors, such as glutamyl cysteine ethyl ester (GCEE) and gluthathione ethyl ester (GEE), increases significantly the intracellular glutathione levels in neuronal cells, protecting dopaminergic neurons against oxidative an nitrosative stress, both in animal and cellular models [25,109]. Finally, the modulation of Nrf2 downstream signaling may be considered as a promising strategy for enhancing the astrocytic production of GSH [108], which may counteract the oxidative imbalance that likely affects neurons in neurodegenerative process‐

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Superoxide dismutases catalyze the dismutation of superoxide ions into oxygen and hy‐ drogen peroxide [23]. As such, they are an important antioxidant defense in nearly all cells exposed to oxygen. In most mammalian cells, SOD is present in three isoforms: a cytosolic copper, zinc superoxide dismutase (SOD1); a mitochondrial manganese super‐ oxide dismutase (SOD2); and an extracellular copper, zinc superoxide dismutase (SOD3) [23, 112]. Given its importance in neuroprotection, SODs and other antioxidant molecules released by astrocytes are highly studied in neurodegenerative diseases like PD and in other oxidative-related events. Evidence that SODs defend against oxidative stress *in situ* has been obtained using transgenic mutants that either overexpress or lack these antioxi‐ dant enzymes [111]. For example, the overexpression of Cu/Zn SOD was able to rescue dopaminergic neurons and diminishes locomotor disabilities in a *Drosophila* mutant mod‐ el for α-synuclein overexpression [112]. Interestingly in PD patients, it has been shown, an specific increase in SOD levels in the substantia nigra, with no changes in activities of glutathione peroxidase, catalase and glutathione reductase (EC 1.8.1.7) [25]. A similar in‐ crease was observed in the mitochondrial isoform of SOD in the motor cortex from PD patients [113], suggesting that SODs have a greater importance than other antioxidant enzymes during PD development. Further research is needed in order to address the

Chaperones belonging to the conserved family of Heat shock proteins (Hsps) are proteins involved in the regulation of protein folding, translocation of proteins across membranes, regulation of cell death and assembling of protein [114]. Interestingly, protein aggregates, and misfolded proteins have been found in AD, Huntington, PD, prion disease, ALS and other neurological injuries [115-117]. Furthermore, previous evidence suggests that forma‐ tion of unfolded proteins in astrocytes could induce the inflammatory responses previously

Many Hsps are currently being considered for the potential treatment of diseases involving protein aggregation and misfolding such as the case of PD [116,118]. These include the chap‐

es such as PD.

**5.2. Superoxide dismutases and Parkinson**

therapeutic application of SOD in PD and other diseases.

**5.3. Astrocytic chaperones and Parkinson**

mentioned [117].

**Figure 4.** Astrocytes-mediate neuroprotection through multiple signaling pathways Astrocytes release glutathione, which serves as precursors for neuronal GSH synthesis, and trophic growth factors such as bFGF, GDNF, and MANF. Activa‐ tion of the transcription factor Nrf2 leads to the expression of antioxidant genes, including γ-glutamylcysteine synthetase (GS), which is involved in GSH synthesis and removal or degradation of cytotoxic molecules, such as α-synuclein.

It is possible that the recovery of glutathione levels may enhance the survival of affected neurons, either by increasing synthesis of GSH or by slowing its degradation [25]. However, the GSH blood-brain barrier permeability is low, and clinical trials using injections of GSH have shown little benefits [6,25,110]. Alternatively, it has been demonstrated that the use of GSH precursors, such as glutamyl cysteine ethyl ester (GCEE) and gluthathione ethyl ester (GEE), increases significantly the intracellular glutathione levels in neuronal cells, protecting dopaminergic neurons against oxidative an nitrosative stress, both in animal and cellular models [25,109]. Finally, the modulation of Nrf2 downstream signaling may be considered as a promising strategy for enhancing the astrocytic production of GSH [108], which may counteract the oxidative imbalance that likely affects neurons in neurodegenerative process‐ es such as PD.

#### **5.2. Superoxide dismutases and Parkinson**

one of the earliest biochemical changes in PD and incidental Lewy body disease [109]. Addi‐ tionally, the GSH content was significantly reduced in the substantia nigra of PD patients, suggesting that GSH depletion enhances neuronal death under certain pathological condi‐ tions [6]. Interestingly, this evidence is consistent with the data in PD patients, in which glu‐

**Figure 4.** Astrocytes-mediate neuroprotection through multiple signaling pathways Astrocytes release glutathione, which serves as precursors for neuronal GSH synthesis, and trophic growth factors such as bFGF, GDNF, and MANF. Activa‐ tion of the transcription factor Nrf2 leads to the expression of antioxidant genes, including γ-glutamylcysteine synthetase

(GS), which is involved in GSH synthesis and removal or degradation of cytotoxic molecules, such as α-synuclein.

tathione-containing cells are in regions with preserved dopaminergic neurons [52].

502 Neurodegenerative Diseases

Superoxide dismutases catalyze the dismutation of superoxide ions into oxygen and hy‐ drogen peroxide [23]. As such, they are an important antioxidant defense in nearly all cells exposed to oxygen. In most mammalian cells, SOD is present in three isoforms: a cytosolic copper, zinc superoxide dismutase (SOD1); a mitochondrial manganese super‐ oxide dismutase (SOD2); and an extracellular copper, zinc superoxide dismutase (SOD3) [23, 112]. Given its importance in neuroprotection, SODs and other antioxidant molecules released by astrocytes are highly studied in neurodegenerative diseases like PD and in other oxidative-related events. Evidence that SODs defend against oxidative stress *in situ* has been obtained using transgenic mutants that either overexpress or lack these antioxi‐ dant enzymes [111]. For example, the overexpression of Cu/Zn SOD was able to rescue dopaminergic neurons and diminishes locomotor disabilities in a *Drosophila* mutant mod‐ el for α-synuclein overexpression [112]. Interestingly in PD patients, it has been shown, an specific increase in SOD levels in the substantia nigra, with no changes in activities of glutathione peroxidase, catalase and glutathione reductase (EC 1.8.1.7) [25]. A similar in‐ crease was observed in the mitochondrial isoform of SOD in the motor cortex from PD patients [113], suggesting that SODs have a greater importance than other antioxidant enzymes during PD development. Further research is needed in order to address the therapeutic application of SOD in PD and other diseases.

#### **5.3. Astrocytic chaperones and Parkinson**

Chaperones belonging to the conserved family of Heat shock proteins (Hsps) are proteins involved in the regulation of protein folding, translocation of proteins across membranes, regulation of cell death and assembling of protein [114]. Interestingly, protein aggregates, and misfolded proteins have been found in AD, Huntington, PD, prion disease, ALS and other neurological injuries [115-117]. Furthermore, previous evidence suggests that forma‐ tion of unfolded proteins in astrocytes could induce the inflammatory responses previously mentioned [117].

Many Hsps are currently being considered for the potential treatment of diseases involving protein aggregation and misfolding such as the case of PD [116,118]. These include the chap‐

erones, DJ-1, Hsp70, Hsp9- and the co-chaperone Hsp40, and members from the Bag family, such as Bag 5, CHIP and suppression of tumorigenicity 13 (ST13) [118]. Several of these chaperones are colocalized or associated with the PD related proteins, E3-ubiquitin ligase (E 6.3.2.19), parkin, α-synuclein and the dopamine transporter (DAT) [119].

activation of the transcription factor NF-kB, which induces the expression of antioxidant en‐ zymes such as Mn-SOD and the anti-apoptotic proteins, Bcl-2 and inhibitor of apoptosis pro‐ teins IAPs [123,124]. Additionally, the endogenous administration of BDNF was shown to protect neurons within the substantia nigra from 6-OHDA and MPTP toxicity, both in rat

Astrocytes Role in Parkinson: A Double-Edged Sword

http://dx.doi.org/10.5772/54305

505

The family of glial cell line-derived neurotrophic factor (GDNF) comprises ligands such as GDNF, neurturin (NRTN), artemin (ARTN) and persephin. GDNF, secreted by astrocytes, is essential for the survival of dopaminergic neurons [32]. It has been shown that GDNF ad‐ ministration by catheter increases dopaminergic neuronal resistance against 6-OHDA toxici‐ ty, but with preservation of motor functions in rat and rhesus monkey models [96]. However, clinical trials in patients that were administered GDNF in different regions of the

Insulin-like growth factors (IGFs) signaling through the phosphatidylinositol 3-kinase (PI3K/Akt) downstream pathway can protect neurons against LPS excitotoxicity in cell cul‐ ture and *in vivo* [124, 128,129]. Furthermore, the activation of this signaling pathway by IGF-I can suppress α-synuclein aggregation and toxicity, suggesting a possible therapeutically strategy in PD [130]. Similarly to IGF-I, vascular endothelial growth factor (VEGF) affects the survival and proliferation of endothelial cells, neurons and astrocytes in the brain, sug‐ gesting a potential therapeutic application in PD [32]. Additionally, VEGF-B (isorform B) was found activated in a rat brain model following treatment with 40 nM rotenone, and showed neuroprotective actions by improving neuronal survival (Falk et al., 2009). Some studies suggest that VEGF promotes neuroprotection by signaling through the neuropilin receptor expressed in DA neurons, and indirectly by activating the proliferation of astroglia and by promoting angiogenesis [32,131,132]. Furthermore, the striatal injection of an adenoassociated virus (AAV)-mediated VEGF expression provided neuroprotection and behavio‐

Basic fibroblast growth factor (bFGF) protects hippocampal and cortical neurons against glutamate toxicity by changing the expression of *N*-methyl-D-aspartic acid (NMDA) recep‐ tors and antioxidant enzymes like superoxide dismutases and glutathione reductase [124]. Furthermore, a coculture of transgenic overexpressing FGF-2 Schwann cells with dopami‐ nergic neurons improved the survival of dopaminergic neurons and the behavioral outcome in a parkinsonian rat model lesioned with 6-OHDA [134]. Finally, there are other neurotro‐ phic factors that have shown dopaminergical neuronal protection in Parkinson-like models, including hepatocyte growth factor (HGF), mesencephalic astrocyte-derived neurotrophic factor (MANF), cerebral dopaminergic neurotrophic factor (CDNF), granulocyte colonystimulating factor (G-CSF), and platelet derived growth factor (PDGF-CC) [31-32, 135-136].

In recent years a growing body of evidence has demonstrated that the malfunctioning of as‐ trocytes may contribute to various neurodegenerative diseases, including Alzheimer, ALS,

brain have shown mixed results and further research is needed [31, 125-127].

ral improvement in rats treated with 6-OHDA [133].

**6. Conclusions and future perspectives**

and primate Parkinson models [31].

DJ-1, also known as PARK7, is upregulated in reactive astrocytes and serves as a redox-sen‐ sitive chaperone with antiapoptotic properties [119]. DJ-1, both in normal and mutant forms, colocalizes with Hsp70 and CHIP in the cytosol. Following oxidative stress, this molecule is translocated to the mitochondria, where it becames associated with the chaperone GRP75 [119,120]. It has been previously shown that DJ-1 can suppress the aggregation and oligome‐ rization of α-synuclein, thus promoting its degradation, which is dependent on the redox state of the cell environment [119,121]. Additionally, DJ-1 regulates signaling pathways such P38 mitogen-activated protein kinases (MAPK; EC 2.7.11.24), apoptosis signal-regulating kinase 1 (ASK1; EC 2.7.11.25) and protein kinase B (AKT) following cellular production of ROS, suggesting that this chaperone is an important redox-reactive molecule during oxida‐ tive stress in PD and other age-related disorders [120].

Hsp70 family of chaperones are thought to be critical in the regulation of protein oligomeriza‐ tion and aggregation, which are believed to be central in the molecular pathogenesis of PD and other neurodegenerative diseases [118]. For example, the overexpression of Hsp70 has been found to protect PC12 cells, and dopaminergic neurons against MPTP toxicity [118,119]. Addi‐ tionally, the overexpression of Hsp70 in mice has been shown to reduce the amount of misfold‐ ed and aggregated α-synuclein species, suggesting a protection of this chaperone against αsynuclein-dependent toxicity [122]. It seems that α-synuclein degradation mediated by Hsp70 occurs in the proteasome or in the lisosomes by a selective process called chaperone-mediated autophagy (CMA) [114]. The wild type, but not a mutant form of α-synuclein is degradated by CMA, suggesting that this mechanism is important in the formation of α-synuclein aggregates during PD [114]. Importantly, the astrocytic clearance of α-synuclein by chaperones, like Hsp70, may confer additional neuroprotection to dopaminergic neurons [6,114].

Chaperones located in other organelles, such as the ER, have also been studied in the devel‐ opment of neurodegenerative processes. For example, homocysteine-induced endoplasmic reticulum protein, which is located in the ER membrane of neurons and astrocytes in the Central Nervous System (SNC), is found accumulated in Lewy bodies, suggesting a role in their formation and further development of PD [117]. In sum, given the central importance of chaperones in protein homeostasis, or proteostasis, they may serve as rational targets for the design of therapeutic strategies in neurodegenerative diseases associated with aberrant protein folding including PD.

#### **5.4. Growth factors and Parkinson**

Several neurotrophic and growth factors have been shown to protect dopaminergic neurons and glial cells against induced excitotoxicity by the activation of specific signaling pathways that are responsible for cell survival and axonal sprouting [31,32]. Some of them have also been tested in PD clinical trials with some promising results [31,32]. For example, brain de‐ rived neurotrophic factor (BDNF) and TNF protect neurons against excitotoxicity through activation of the transcription factor NF-kB, which induces the expression of antioxidant en‐ zymes such as Mn-SOD and the anti-apoptotic proteins, Bcl-2 and inhibitor of apoptosis pro‐ teins IAPs [123,124]. Additionally, the endogenous administration of BDNF was shown to protect neurons within the substantia nigra from 6-OHDA and MPTP toxicity, both in rat and primate Parkinson models [31].

erones, DJ-1, Hsp70, Hsp9- and the co-chaperone Hsp40, and members from the Bag family, such as Bag 5, CHIP and suppression of tumorigenicity 13 (ST13) [118]. Several of these chaperones are colocalized or associated with the PD related proteins, E3-ubiquitin ligase (E

DJ-1, also known as PARK7, is upregulated in reactive astrocytes and serves as a redox-sen‐ sitive chaperone with antiapoptotic properties [119]. DJ-1, both in normal and mutant forms, colocalizes with Hsp70 and CHIP in the cytosol. Following oxidative stress, this molecule is translocated to the mitochondria, where it becames associated with the chaperone GRP75 [119,120]. It has been previously shown that DJ-1 can suppress the aggregation and oligome‐ rization of α-synuclein, thus promoting its degradation, which is dependent on the redox state of the cell environment [119,121]. Additionally, DJ-1 regulates signaling pathways such P38 mitogen-activated protein kinases (MAPK; EC 2.7.11.24), apoptosis signal-regulating kinase 1 (ASK1; EC 2.7.11.25) and protein kinase B (AKT) following cellular production of ROS, suggesting that this chaperone is an important redox-reactive molecule during oxida‐

Hsp70 family of chaperones are thought to be critical in the regulation of protein oligomeriza‐ tion and aggregation, which are believed to be central in the molecular pathogenesis of PD and other neurodegenerative diseases [118]. For example, the overexpression of Hsp70 has been found to protect PC12 cells, and dopaminergic neurons against MPTP toxicity [118,119]. Addi‐ tionally, the overexpression of Hsp70 in mice has been shown to reduce the amount of misfold‐ ed and aggregated α-synuclein species, suggesting a protection of this chaperone against αsynuclein-dependent toxicity [122]. It seems that α-synuclein degradation mediated by Hsp70 occurs in the proteasome or in the lisosomes by a selective process called chaperone-mediated autophagy (CMA) [114]. The wild type, but not a mutant form of α-synuclein is degradated by CMA, suggesting that this mechanism is important in the formation of α-synuclein aggregates during PD [114]. Importantly, the astrocytic clearance of α-synuclein by chaperones, like

Chaperones located in other organelles, such as the ER, have also been studied in the devel‐ opment of neurodegenerative processes. For example, homocysteine-induced endoplasmic reticulum protein, which is located in the ER membrane of neurons and astrocytes in the Central Nervous System (SNC), is found accumulated in Lewy bodies, suggesting a role in their formation and further development of PD [117]. In sum, given the central importance of chaperones in protein homeostasis, or proteostasis, they may serve as rational targets for the design of therapeutic strategies in neurodegenerative diseases associated with aberrant

Several neurotrophic and growth factors have been shown to protect dopaminergic neurons and glial cells against induced excitotoxicity by the activation of specific signaling pathways that are responsible for cell survival and axonal sprouting [31,32]. Some of them have also been tested in PD clinical trials with some promising results [31,32]. For example, brain de‐ rived neurotrophic factor (BDNF) and TNF protect neurons against excitotoxicity through

Hsp70, may confer additional neuroprotection to dopaminergic neurons [6,114].

6.3.2.19), parkin, α-synuclein and the dopamine transporter (DAT) [119].

tive stress in PD and other age-related disorders [120].

504 Neurodegenerative Diseases

protein folding including PD.

**5.4. Growth factors and Parkinson**

The family of glial cell line-derived neurotrophic factor (GDNF) comprises ligands such as GDNF, neurturin (NRTN), artemin (ARTN) and persephin. GDNF, secreted by astrocytes, is essential for the survival of dopaminergic neurons [32]. It has been shown that GDNF ad‐ ministration by catheter increases dopaminergic neuronal resistance against 6-OHDA toxici‐ ty, but with preservation of motor functions in rat and rhesus monkey models [96]. However, clinical trials in patients that were administered GDNF in different regions of the brain have shown mixed results and further research is needed [31, 125-127].

Insulin-like growth factors (IGFs) signaling through the phosphatidylinositol 3-kinase (PI3K/Akt) downstream pathway can protect neurons against LPS excitotoxicity in cell cul‐ ture and *in vivo* [124, 128,129]. Furthermore, the activation of this signaling pathway by IGF-I can suppress α-synuclein aggregation and toxicity, suggesting a possible therapeutically strategy in PD [130]. Similarly to IGF-I, vascular endothelial growth factor (VEGF) affects the survival and proliferation of endothelial cells, neurons and astrocytes in the brain, sug‐ gesting a potential therapeutic application in PD [32]. Additionally, VEGF-B (isorform B) was found activated in a rat brain model following treatment with 40 nM rotenone, and showed neuroprotective actions by improving neuronal survival (Falk et al., 2009). Some studies suggest that VEGF promotes neuroprotection by signaling through the neuropilin receptor expressed in DA neurons, and indirectly by activating the proliferation of astroglia and by promoting angiogenesis [32,131,132]. Furthermore, the striatal injection of an adenoassociated virus (AAV)-mediated VEGF expression provided neuroprotection and behavio‐ ral improvement in rats treated with 6-OHDA [133].

Basic fibroblast growth factor (bFGF) protects hippocampal and cortical neurons against glutamate toxicity by changing the expression of *N*-methyl-D-aspartic acid (NMDA) recep‐ tors and antioxidant enzymes like superoxide dismutases and glutathione reductase [124]. Furthermore, a coculture of transgenic overexpressing FGF-2 Schwann cells with dopami‐ nergic neurons improved the survival of dopaminergic neurons and the behavioral outcome in a parkinsonian rat model lesioned with 6-OHDA [134]. Finally, there are other neurotro‐ phic factors that have shown dopaminergical neuronal protection in Parkinson-like models, including hepatocyte growth factor (HGF), mesencephalic astrocyte-derived neurotrophic factor (MANF), cerebral dopaminergic neurotrophic factor (CDNF), granulocyte colonystimulating factor (G-CSF), and platelet derived growth factor (PDGF-CC) [31-32, 135-136].

#### **6. Conclusions and future perspectives**

In recent years a growing body of evidence has demonstrated that the malfunctioning of as‐ trocytes may contribute to various neurodegenerative diseases, including Alzheimer, ALS, multiple sclerosis, and Parkinson. Importantly, astrocytes are involved in both exacerbation of damage and in neuroprotective mechanisms that are crucial for neuronal survival. In this matter, astrocytes are essential for the regulation of oxidative stress and ROS production, both in normal and in pathological circumstances.

[2] Volterra A, Meldolesi J. Astrocytes, from brain glue to communication elements: the

Astrocytes Role in Parkinson: A Double-Edged Sword

http://dx.doi.org/10.5772/54305

507

[3] Hamby ME, Sofroniew MV. Reactive astrocytes as therapeutic targets for CNS disor‐

[4] Barreto GE, Gonzalez J, Capani F, Morales L. Role of Astrocytes in Neurodegenera‐ tive Diseases. In: Raymond Chuen-Chung Chang. (ed.) Neurodegenerative Diseases -

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[7] Halliday GM, Stevens CH. Glia: initiators and progressors of pathology in Parkin‐

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[13] Knott AB, Bossy-Wetzel E. Impairing the mitochondrial fission and fusion balance: a new mechanism of neurodegeneration. Annals of the New York Academy of Scien‐

[14] Betarbet R, Sherer TB, MacKenzie G, Garcia-Osuna M, Panov, AV, Greenamyre, JT. Chronic systemic pesticide exposure reproduces features of Parkinson's disease. Na‐

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175-179

The overexpression of antioxidant molecules such as GSH and SOD2, or chaperones such as Hsp70 has proved to be a successful experimental approach in brain diseases, including PD. The use of growth factors, both in animal models and in clinical trials, has shown promising effects in protecting dopaminergic neurons and astrocytes in damaged regions by the activa‐ tion of different signaling pathways important in neuronal survival and regeneration. It is important to mention that mitochondrial protection in astrocytes is an important asset to maintain the energetic balance of the brain and the antioxidant production that contribute to neuronal protection. Therefore future efforts in neuroprotective strategies should emphasize the mitochondrial protection in astrocytes. Finally, the combination of novel drug therapies, a better understanding of the α-synuclein clearance by astrocytes, the use of neurotoxic models, growth factors use and other therapies that increase astrocyte survival and its anti‐ oxidant function may shed light on a prospective cure of PD in the near future.

#### **Acknowledgements**

This work was supported in part by grants PUJ IDs 4509 and 4327 to GEB.

### **Author details**

Ricardo Cabezas1 , Marco Fidel Avila1 , Daniel Torrente1 , Ramon Santos El-Bachá2 , Ludis Morales1 , Janneth Gonzalez1 and George E. Barreto1

\*Address all correspondence to: gsampaio@javeriana.edu.co

1 Departamento de Nutrición y Bioquímica, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá D.C., Colombia

2 Laboratorio de Neuroquímica e Biologia Celular, Universidade Federal da Bahía, Salvador, Bahia, Brazil

### **References**

[1] Fernandez HH. Updates in the medical management of Parkinson disease. Cleveland Clinic Journal of Medicine 2012; 79(1) 28-35.

[2] Volterra A, Meldolesi J. Astrocytes, from brain glue to communication elements: the revolution continues. Nature Reviews Neuroscience 2005; 6(8) 626-640.

multiple sclerosis, and Parkinson. Importantly, astrocytes are involved in both exacerbation of damage and in neuroprotective mechanisms that are crucial for neuronal survival. In this matter, astrocytes are essential for the regulation of oxidative stress and ROS production,

The overexpression of antioxidant molecules such as GSH and SOD2, or chaperones such as Hsp70 has proved to be a successful experimental approach in brain diseases, including PD. The use of growth factors, both in animal models and in clinical trials, has shown promising effects in protecting dopaminergic neurons and astrocytes in damaged regions by the activa‐ tion of different signaling pathways important in neuronal survival and regeneration. It is important to mention that mitochondrial protection in astrocytes is an important asset to maintain the energetic balance of the brain and the antioxidant production that contribute to neuronal protection. Therefore future efforts in neuroprotective strategies should emphasize the mitochondrial protection in astrocytes. Finally, the combination of novel drug therapies, a better understanding of the α-synuclein clearance by astrocytes, the use of neurotoxic models, growth factors use and other therapies that increase astrocyte survival and its anti‐

oxidant function may shed light on a prospective cure of PD in the near future.

This work was supported in part by grants PUJ IDs 4509 and 4327 to GEB.

, Daniel Torrente1

and George E. Barreto1

1 Departamento de Nutrición y Bioquímica, Facultad de Ciencias, Pontificia Universidad

2 Laboratorio de Neuroquímica e Biologia Celular, Universidade Federal da Bahía, Salvador,

[1] Fernandez HH. Updates in the medical management of Parkinson disease. Cleveland

, Ramon Santos El-Bachá2

,

, Marco Fidel Avila1

\*Address all correspondence to: gsampaio@javeriana.edu.co

Clinic Journal of Medicine 2012; 79(1) 28-35.

, Janneth Gonzalez1

Javeriana, Bogotá D.C., Colombia

both in normal and in pathological circumstances.

**Acknowledgements**

506 Neurodegenerative Diseases

**Author details**

Ricardo Cabezas1

Ludis Morales1

Bahia, Brazil

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**Chapter 21**

**Zinc and Neurodegenerative Diseases**

Masahiro Kawahara, Keiko Konoha,

Yutaka Sadakane

**1. Introduction**

http://dx.doi.org/10.5772/54489

Hironari Koyama, Susumu Ohkawara and

Additional information is available at the end of the chapter

Zinc (Zn) is an essential trace element for most organisms. It plays important roles in vari‐ ous physiological functions such as the mitotic cell division, the immune system, the synthe‐ sis of proteins and DNA as a co-factor of more than 300 enzymes or metalloproteins [1]. Recent studies revealed that Zn signaling plays crucial roles in various biological systems of humans [2]. Zn deficiency in human childhood is known to cause the dwarfism, the retarda‐ tion of mental and physical development, the immune dysfunction, and the learning disabil‐

The human body contains approximately 2 g of Zn, mostly in the testes, muscle, liver, and brain tissues. In the brain, Zn is found at the highest concentrations in the hippocampus, amygdala, cerebral cortex, thalamus, and olfactory cortex [4]. The total Zn content of the hippocampus is estimated to be 70–90 ppm (dry weight). Although some Zn in the brain binds firmly to metalloproteins or enzymes, a substantial fraction (approximately 10% or more) either forms free Zn ions (Zn2+) or is loosely bound, and is histochemically detectable by staining using chelating reagents. This chelatable Zn is stored in presynaptic vesicles of specific excitatory glutamatergic neurons and is secreted from these vesicles into synaptic clefts along with glutamate during neuronal excitation. Recent studies have suggested that this secreted Zn2+ plays crucial roles in information processing, synaptic plasticity, learning, and memory (Fig. 1A). Indeed, Zn2+ in the hippocampus is essential for the induction of long-term potentiation (LTP), a form of synaptic information storage that has become a well-

However, despite its importance, excess Zn is neurotoxic and implicated in neurodegenera‐ tive diseases. In this chapter, we review the current understanding about the link between

and reproduction in any medium, provided the original work is properly cited.

© 2013 Kawahara et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

ities [3]. In adults, Zn deficiency causes the taste and odor disorders.

known paradigm for the mechanisms underlying memory formation [5].

### **Chapter 21**

### **Zinc and Neurodegenerative Diseases**

Masahiro Kawahara, Keiko Konoha, Hironari Koyama, Susumu Ohkawara and Yutaka Sadakane

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54489

#### **1. Introduction**

Zinc (Zn) is an essential trace element for most organisms. It plays important roles in vari‐ ous physiological functions such as the mitotic cell division, the immune system, the synthe‐ sis of proteins and DNA as a co-factor of more than 300 enzymes or metalloproteins [1]. Recent studies revealed that Zn signaling plays crucial roles in various biological systems of humans [2]. Zn deficiency in human childhood is known to cause the dwarfism, the retarda‐ tion of mental and physical development, the immune dysfunction, and the learning disabil‐ ities [3]. In adults, Zn deficiency causes the taste and odor disorders.

The human body contains approximately 2 g of Zn, mostly in the testes, muscle, liver, and brain tissues. In the brain, Zn is found at the highest concentrations in the hippocampus, amygdala, cerebral cortex, thalamus, and olfactory cortex [4]. The total Zn content of the hippocampus is estimated to be 70–90 ppm (dry weight). Although some Zn in the brain binds firmly to metalloproteins or enzymes, a substantial fraction (approximately 10% or more) either forms free Zn ions (Zn2+) or is loosely bound, and is histochemically detectable by staining using chelating reagents. This chelatable Zn is stored in presynaptic vesicles of specific excitatory glutamatergic neurons and is secreted from these vesicles into synaptic clefts along with glutamate during neuronal excitation. Recent studies have suggested that this secreted Zn2+ plays crucial roles in information processing, synaptic plasticity, learning, and memory (Fig. 1A). Indeed, Zn2+ in the hippocampus is essential for the induction of long-term potentiation (LTP), a form of synaptic information storage that has become a wellknown paradigm for the mechanisms underlying memory formation [5].

However, despite its importance, excess Zn is neurotoxic and implicated in neurodegenera‐ tive diseases. In this chapter, we review the current understanding about the link between

© 2013 Kawahara et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

the disruption of Zn homeostasis and the pathogenesis of various neurodegenerative diseas‐ es including senile dementia.

rons, the permeablity of Zn2+ and Ca2+ through AMPA/kainate channels is greater than

In a normal condition, most hippocampal neurons express AMPA receptors with subunit GluR2, which are poorly permeable to divalent cations including Ca2+ and Zn2+(A/K-R). However, after ischemia, the acute reduction in the expression of GluR2 subunit occurs, and neurons possess specific type of AMPA receptors which channels are directly Ca2+ permeable (Ca-AMPA/kainate channels; Ca-A/K-R)) [12]. The appearance of Ca-AMPA/ kainate channels causes the increased permeability of Ca2+ and enhances the toxicity. Therefore, the expression of Zn2+-permeable Ca-AMPA/kainite channels and the entry of Ca2+ and/or Zn2+ through the channels are mediators of the delayed neuronal death after ischemia. Considering that Ca EDTA, a zinc chelator, attenuates the ischemia-induced down-regulation of GluR2 gene [10], Zn is also implicated in the transcriptional regula‐

> : Glutamate : Zinc

Synapse

B: After ischemia

ZnT-1

**Figure 1.** Zinc in normal or pathological conditions in the brain. Under normal conditions (A), neuronal excitation causes the release of glutamate and Zn. Zn regulates the postsynaptic excitability by binding to NMDA-type glutamate receptors (NMDA-R). However, under pathological conditions such as ischemia (B), oxygen-glucose deprivation indu‐ ces the release of excess glutamate as well as Zn into the synaptic clefts. Excess Zn enhances the expression of Ca-AMPA/kainite channels (Ca-A/K-R), and is translocated through the Ca-A/K-R or through other pathways such as voltage-gated L-type Ca2+ channels (VGLC) into the target neuron, where Zn acts to inhibit various enzymes, inhibit mitochondrial respiration, cause energy depletion, and produce reactive reactive oxygen species (ROS). Excess gluta‐ mate induces elevation of intracellular Ca2+ levels in the target neuron. Elevated levels of intracellular Ca2+ then trigger various apoptotic pathways such as the activation of calpain, caspases or other enzymatic pathways related to apop‐

Ca-A/K-R

Ca2+

[Zn2+]i [Ca2+]i

Neuronal death

Vascular type of dementia

VGLC

ZnT-3

Zinc and Neurodegenerative Diseases http://dx.doi.org/10.5772/54489 521

: Glutamate : Zinc

Ca2+

NMDA-R

NMDA-R

Ca2+

NMDA-receptor channels [11].

tion in Ca-AMPA/kainite channels.

A/K-R

Memory formation

tosis; ultimately this leads to neuronal death.

Information processing

VGLC

ZnT-3

Synapse

A: Normal condition

ZnT-1

### **2. Zinc and vascular type of dementia**

#### **2.1. Zn-induced neurodegeneration after ischemia**

Senile dementia is a serious problem in a rapidly aging world. Its prevalence increases with age. Approximately 25% of elderly individuals are affected by the diseases. In Japan, 3 mil‐ lion people have been estimated to be affected by senile dementia by 2025, and the number continues to grow annually. Senile dementia is mainly divided to Alzheimer's disease (AD) and vascular-type dementia (VD). VD is a degenerative cerebrovascular disease, and its risk factors include aging, sex difference (male), diabetes, and high blood pressure*.* The most common type of VD is caused by a series of small strokes or ischemia [6]. Following transi‐ ent global ischemia or stroke, the interruption of blood flow and the resulting oxygen-glu‐ cose deprivation induce long-lasting membrane depolarization and cause an excessive release of glutamate into synaptic clefts. Thereafter, the excess glutamate causes over-stimu‐ lation of its receptors, namely, *N*-methyl-D-aspartate (NMDA)-type receptors, amino-3-hy‐ droxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type receptors, and kainite-type receptors. Finally, Ca2+ dyshomeostasis, *i.e.*, the entry of large quantities of Ca2+ occurring in glutamate-responsive neurons, triggers the delayed death of vulnerable populations of neu‐ rons such as pyramidal neurons in the hippocampus —an area associated with learning and memory. Thereafter, the development of an infarct and the subsequent cognitive dysfunc‐ tion mark the pathogenesis of VD in elderly people. Approximately 30% of stroke patients show symptoms of dementia within 3 months of the initial stroke [7].

Increasing evidence suggests that Zn is central to ischemia-induced neuronal death and fi‐ nally the pathogenesis of VD [8]. In ischemic conditions, a considerable amount of Zn (up to 300µM) is co-released with glutamate into synaptic clefts by membrane depolarization. Zn caused the apoptotic death of primary cultured cortical neurons. Furthermore, the chelata‐ ble Zn reportedly moved from presynaptic terminals into postsynaptic neuronal cell bodies. The increase in intracellular Zn2+ levels ([Zn2+]i ), namely, "Zn translocation," occurs in vul‐ nerable neurons in the CA1 or CA3 regions of the hippocampus prior to the onset of the de‐ layed neuronal death after transient global ischemia [9]. This Zn translocation is reported to enhance the appearance of the infarct. Administration of calcium EDTA (Ca EDTA), a mem‐ brane-impermeable chelator that chelates cations except for calcium, blocked the transloca‐ tion of Zn, protected the hippocampal neurons after transient global ischemia, and reduced the infarct volume [10]. Thus, Zn translocation is recognized to be the primary event in the pathway of Zn-induced neuronal death. Sensi *et al.* observed a temporal change of [Zn2+]i in cultured cortical neurons using a zinc-sensitive fluorescent dye; those results revealed that at least three major routes of Zn2+ entry have been identified; voltage-gated Ca2+ channels (VGLC), NMDA-type glutamate receptors (NMDA-R), and AMPA/kainite-type glutamate receptors (A/K-R). Although the NMDA-type glutamate receptors are present in most neu‐ rons, the permeablity of Zn2+ and Ca2+ through AMPA/kainate channels is greater than NMDA-receptor channels [11].

the disruption of Zn homeostasis and the pathogenesis of various neurodegenerative diseas‐

Senile dementia is a serious problem in a rapidly aging world. Its prevalence increases with age. Approximately 25% of elderly individuals are affected by the diseases. In Japan, 3 mil‐ lion people have been estimated to be affected by senile dementia by 2025, and the number continues to grow annually. Senile dementia is mainly divided to Alzheimer's disease (AD) and vascular-type dementia (VD). VD is a degenerative cerebrovascular disease, and its risk factors include aging, sex difference (male), diabetes, and high blood pressure*.* The most common type of VD is caused by a series of small strokes or ischemia [6]. Following transi‐ ent global ischemia or stroke, the interruption of blood flow and the resulting oxygen-glu‐ cose deprivation induce long-lasting membrane depolarization and cause an excessive release of glutamate into synaptic clefts. Thereafter, the excess glutamate causes over-stimu‐ lation of its receptors, namely, *N*-methyl-D-aspartate (NMDA)-type receptors, amino-3-hy‐ droxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type receptors, and kainite-type receptors. Finally, Ca2+ dyshomeostasis, *i.e.*, the entry of large quantities of Ca2+ occurring in glutamate-responsive neurons, triggers the delayed death of vulnerable populations of neu‐ rons such as pyramidal neurons in the hippocampus —an area associated with learning and memory. Thereafter, the development of an infarct and the subsequent cognitive dysfunc‐ tion mark the pathogenesis of VD in elderly people. Approximately 30% of stroke patients

Increasing evidence suggests that Zn is central to ischemia-induced neuronal death and fi‐ nally the pathogenesis of VD [8]. In ischemic conditions, a considerable amount of Zn (up to 300µM) is co-released with glutamate into synaptic clefts by membrane depolarization. Zn caused the apoptotic death of primary cultured cortical neurons. Furthermore, the chelata‐ ble Zn reportedly moved from presynaptic terminals into postsynaptic neuronal cell bodies.

nerable neurons in the CA1 or CA3 regions of the hippocampus prior to the onset of the de‐ layed neuronal death after transient global ischemia [9]. This Zn translocation is reported to enhance the appearance of the infarct. Administration of calcium EDTA (Ca EDTA), a mem‐ brane-impermeable chelator that chelates cations except for calcium, blocked the transloca‐ tion of Zn, protected the hippocampal neurons after transient global ischemia, and reduced the infarct volume [10]. Thus, Zn translocation is recognized to be the primary event in the pathway of Zn-induced neuronal death. Sensi *et al.* observed a temporal change of [Zn2+]i in cultured cortical neurons using a zinc-sensitive fluorescent dye; those results revealed that at least three major routes of Zn2+ entry have been identified; voltage-gated Ca2+ channels (VGLC), NMDA-type glutamate receptors (NMDA-R), and AMPA/kainite-type glutamate receptors (A/K-R). Although the NMDA-type glutamate receptors are present in most neu‐

), namely, "Zn translocation," occurs in vul‐

es including senile dementia.

520 Neurodegenerative Diseases

**2. Zinc and vascular type of dementia**

**2.1. Zn-induced neurodegeneration after ischemia**

show symptoms of dementia within 3 months of the initial stroke [7].

The increase in intracellular Zn2+ levels ([Zn2+]i

In a normal condition, most hippocampal neurons express AMPA receptors with subunit GluR2, which are poorly permeable to divalent cations including Ca2+ and Zn2+(A/K-R). However, after ischemia, the acute reduction in the expression of GluR2 subunit occurs, and neurons possess specific type of AMPA receptors which channels are directly Ca2+ permeable (Ca-AMPA/kainate channels; Ca-A/K-R)) [12]. The appearance of Ca-AMPA/ kainate channels causes the increased permeability of Ca2+ and enhances the toxicity. Therefore, the expression of Zn2+-permeable Ca-AMPA/kainite channels and the entry of Ca2+ and/or Zn2+ through the channels are mediators of the delayed neuronal death after ischemia. Considering that Ca EDTA, a zinc chelator, attenuates the ischemia-induced down-regulation of GluR2 gene [10], Zn is also implicated in the transcriptional regula‐ tion in Ca-AMPA/kainite channels.

**Figure 1.** Zinc in normal or pathological conditions in the brain. Under normal conditions (A), neuronal excitation causes the release of glutamate and Zn. Zn regulates the postsynaptic excitability by binding to NMDA-type glutamate receptors (NMDA-R). However, under pathological conditions such as ischemia (B), oxygen-glucose deprivation indu‐ ces the release of excess glutamate as well as Zn into the synaptic clefts. Excess Zn enhances the expression of Ca-AMPA/kainite channels (Ca-A/K-R), and is translocated through the Ca-A/K-R or through other pathways such as voltage-gated L-type Ca2+ channels (VGLC) into the target neuron, where Zn acts to inhibit various enzymes, inhibit mitochondrial respiration, cause energy depletion, and produce reactive reactive oxygen species (ROS). Excess gluta‐ mate induces elevation of intracellular Ca2+ levels in the target neuron. Elevated levels of intracellular Ca2+ then trigger various apoptotic pathways such as the activation of calpain, caspases or other enzymatic pathways related to apop‐ tosis; ultimately this leads to neuronal death.

Zn-specific membrane transporter proteins (Zn transporters) also control Zn homeostasis; they facilitate zinc influx in deficiency and efflux during zinc excess. Recent genetic and molecular approaches revealed the implications of abnormalities in Zn transporters in various human diseases [13]. Zn transporter 1 (ZnT-1), a membrane protein with six transmembrane domains, is widely distributed in mammalian cells, and is co-localized with chelatable Zn in the brain. ZnT-1 is activated by excess Zn and the expression of ZnT-1 is induced after transient global ischemia. On the contrary, dietary Zn deficiency decreases expression of ZnT-1. Consequently, it is provable that ZnT-1 plays a pivotal role in efflux of Zn and in protection from Zn toxicity. Another important Zn transporter in the brain is ZnT-3, which localizes in the membranes of presynaptic vesicles, trans‐ ports Zn into synaptic vesicles, and maintains high Zn concentrations in the vesicles. Al‐ though the physiological role of ZnT-3 and vesicular zinc remain elusive, recent studies have suggested the implication of ZnT-3 or other Zn transporters in the pathogenesis of AD and other neurodegenerative diseases [14].

of GT1-7 cells. Neither antagonists nor agonists of excitatory neurotransmitters (D-APV, glu‐ tamate, and CNQX), or those of inhibitory neurotransmitters (bicuculline, muscimol, baclo‐ fen, and GABA) attenuated the viability of GT1-7 cells after Zn exposure. Our findings in GT1-7 cells, which lack such glutamate receptors, are inconsistent with previous studies that agonists of glutamate receptors, such as NMDA or AMPA, enhance Zn-induced neurotoxici‐

ty in cultured cortical neurons [21].

0

B-50 Cerebral

exposed to 50 µM Zn, and were observed with TUNEL staining after 24h.

cortex PC12 GT1-7 Hippo-

**Figure 2.** Apoptotic death of GT1-7 cells after exposure to Zn. A: Viability of various neuronal cells after exposure to Zn. Cultured neuronal cells (GT1-7 cells, PC-12 cells, B-50 cells (a neuroblastoma cell line), primary cultured neurons of the rat cerebral cortex, and primary cultured neurons of the rat hippocampus) were administered to 50 µM of Zn. Af‐ ter 24h, cell viability was analyzed by WST-1 method. B: TUNEL staining of Zn intoxicated GT1-7 cells GT1-7 cells were

To evaluate the involvement of other metal ions in Zn neurotoxicity, we investigated the vi‐ ability of GT1-7 cells with or without various metal ions after exposure to Zn [22]. The equi‐ molar addition of Al3+ and Gd3+ significantly inhibited Zn-induced neurotoxicity. Moreover, overloading of Ca2+ and Mg2+ inhibited the Zn-induced death of GT1-7 cells; Zn protected GT1-7 cells from neurotoxicity induced by Ca2+ overload, and *vice versa* (Fig. 3B). Further‐ more, Kim *et al.* reported that Zn neurotoxicity in PC-12 cells was attenuated by an L-type Ca2+ channel blocker, nimodipine, and enhanced by the L-type Ca2+ channel activator, S(-)- Bay K 8644 [16]. Additionally, Zn neurotoxicity was attenuated by aspirin, which prevents Zn2+ entry through voltage-gated Ca2+ channels. These pharamacological evidence suggests

that Ca dyshomeostasis is involved in the mechanism of Zn-induced neurotoxicity.

campus

control

B

Zinc and Neurodegenerative Diseases http://dx.doi.org/10.5772/54489 523

Zn

50 µm

20

40

60

Viability (%)

80

100

120

A

#### **2.2. Molecular mechanism of Zn-induced neurotoxicity: GT1-7 cells as an** *in vitro* **model system**

Understanding the molecular mechanism of Zn-induced neuronal death is of great impor‐ tance for the treatment of VD. Numerous studies have been undertaken to elucidate the mechanism of Zn-induced neuronal death. To this end, many researchers have investigated Zn neurotoxicity *in vitro* mainly using primary cultured neurons from rat cerebral cortex or hippocampus [15] or PC-12 cells, a pheochromocytoma cell line [16]. However, the roles of Zn are highly complex. For example, Zn reportedly inhibits NMDA-type glutamate recep‐ tors and regulates the excitability of glutamatergic neurons, which are toxic to neurons. Therefore, distinguishing of the effects of Zn and glutamate by using neuronal cells which possess glutamate receptors has proved difficult.

We found that GT1-7 cells, immortalized hypothalamic neurons, are much more sensitive to Zn than other neuronal cells are [17,18] (Fig. 2A). Zn caused the apoptotic death of GT1-7 cells in a dose-dependent and time-dependent manner. The degenerated GT1-7 cells were terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling (TU‐ NEL) positive and exhibited the DNA fragmentation.

The GT1-7 cells were originally developed by Mellon *et al.* by genetically targeting tu‐ morigenesis of mouse hypothalamic neurons [19]. The cells possess neuronal characteris‐ tics such as the extension of neurites, secretion of gonadotropin-releasing hormone (GnRH), and expression of neuron-specific proteins or receptors including microtubuleassociated protein 2 (MAP2), tau protein, neurofilament, synaptophysin, GABAA recep‐ tors, dopamine receptors, and L-type Ca2+ channels. Additionally, the GT1-7 cells either lack or possess low levels of ionotropic glutamate receptors and do not exhibit glutamate toxicity [20]. These properties make the GT1-7 cell line an excellent model system for the investigation of Zn-induced neurotoxicity.

We investigated the detailed characteristics of Zn-induced death in GT1-7 cells and its mech‐ anisms. First, we tested the effects of various pharmacological agents prior to Zn treatment of GT1-7 cells. Neither antagonists nor agonists of excitatory neurotransmitters (D-APV, glu‐ tamate, and CNQX), or those of inhibitory neurotransmitters (bicuculline, muscimol, baclo‐ fen, and GABA) attenuated the viability of GT1-7 cells after Zn exposure. Our findings in GT1-7 cells, which lack such glutamate receptors, are inconsistent with previous studies that agonists of glutamate receptors, such as NMDA or AMPA, enhance Zn-induced neurotoxici‐ ty in cultured cortical neurons [21].

Zn-specific membrane transporter proteins (Zn transporters) also control Zn homeostasis; they facilitate zinc influx in deficiency and efflux during zinc excess. Recent genetic and molecular approaches revealed the implications of abnormalities in Zn transporters in various human diseases [13]. Zn transporter 1 (ZnT-1), a membrane protein with six transmembrane domains, is widely distributed in mammalian cells, and is co-localized with chelatable Zn in the brain. ZnT-1 is activated by excess Zn and the expression of ZnT-1 is induced after transient global ischemia. On the contrary, dietary Zn deficiency decreases expression of ZnT-1. Consequently, it is provable that ZnT-1 plays a pivotal role in efflux of Zn and in protection from Zn toxicity. Another important Zn transporter in the brain is ZnT-3, which localizes in the membranes of presynaptic vesicles, trans‐ ports Zn into synaptic vesicles, and maintains high Zn concentrations in the vesicles. Al‐ though the physiological role of ZnT-3 and vesicular zinc remain elusive, recent studies have suggested the implication of ZnT-3 or other Zn transporters in the pathogenesis of

**2.2. Molecular mechanism of Zn-induced neurotoxicity: GT1-7 cells as an** *in vitro* **model**

Understanding the molecular mechanism of Zn-induced neuronal death is of great impor‐ tance for the treatment of VD. Numerous studies have been undertaken to elucidate the mechanism of Zn-induced neuronal death. To this end, many researchers have investigated Zn neurotoxicity *in vitro* mainly using primary cultured neurons from rat cerebral cortex or hippocampus [15] or PC-12 cells, a pheochromocytoma cell line [16]. However, the roles of Zn are highly complex. For example, Zn reportedly inhibits NMDA-type glutamate recep‐ tors and regulates the excitability of glutamatergic neurons, which are toxic to neurons. Therefore, distinguishing of the effects of Zn and glutamate by using neuronal cells which

We found that GT1-7 cells, immortalized hypothalamic neurons, are much more sensitive to Zn than other neuronal cells are [17,18] (Fig. 2A). Zn caused the apoptotic death of GT1-7 cells in a dose-dependent and time-dependent manner. The degenerated GT1-7 cells were terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling (TU‐

The GT1-7 cells were originally developed by Mellon *et al.* by genetically targeting tu‐ morigenesis of mouse hypothalamic neurons [19]. The cells possess neuronal characteris‐ tics such as the extension of neurites, secretion of gonadotropin-releasing hormone (GnRH), and expression of neuron-specific proteins or receptors including microtubuleassociated protein 2 (MAP2), tau protein, neurofilament, synaptophysin, GABAA recep‐ tors, dopamine receptors, and L-type Ca2+ channels. Additionally, the GT1-7 cells either lack or possess low levels of ionotropic glutamate receptors and do not exhibit glutamate toxicity [20]. These properties make the GT1-7 cell line an excellent model system for the

We investigated the detailed characteristics of Zn-induced death in GT1-7 cells and its mech‐ anisms. First, we tested the effects of various pharmacological agents prior to Zn treatment

AD and other neurodegenerative diseases [14].

possess glutamate receptors has proved difficult.

NEL) positive and exhibited the DNA fragmentation.

investigation of Zn-induced neurotoxicity.

**system**

522 Neurodegenerative Diseases

**Figure 2.** Apoptotic death of GT1-7 cells after exposure to Zn. A: Viability of various neuronal cells after exposure to Zn. Cultured neuronal cells (GT1-7 cells, PC-12 cells, B-50 cells (a neuroblastoma cell line), primary cultured neurons of the rat cerebral cortex, and primary cultured neurons of the rat hippocampus) were administered to 50 µM of Zn. Af‐ ter 24h, cell viability was analyzed by WST-1 method. B: TUNEL staining of Zn intoxicated GT1-7 cells GT1-7 cells were exposed to 50 µM Zn, and were observed with TUNEL staining after 24h.

To evaluate the involvement of other metal ions in Zn neurotoxicity, we investigated the vi‐ ability of GT1-7 cells with or without various metal ions after exposure to Zn [22]. The equi‐ molar addition of Al3+ and Gd3+ significantly inhibited Zn-induced neurotoxicity. Moreover, overloading of Ca2+ and Mg2+ inhibited the Zn-induced death of GT1-7 cells; Zn protected GT1-7 cells from neurotoxicity induced by Ca2+ overload, and *vice versa* (Fig. 3B). Further‐ more, Kim *et al.* reported that Zn neurotoxicity in PC-12 cells was attenuated by an L-type Ca2+ channel blocker, nimodipine, and enhanced by the L-type Ca2+ channel activator, S(-)- Bay K 8644 [16]. Additionally, Zn neurotoxicity was attenuated by aspirin, which prevents Zn2+ entry through voltage-gated Ca2+ channels. These pharamacological evidence suggests that Ca dyshomeostasis is involved in the mechanism of Zn-induced neurotoxicity.

decreased the levels of NAD+ and ATP in cultured cortical neurons, and that treatment with

dye and a mitochondrial marker revealed that Zn is localized within mitochondria. Zn is re‐ ported to inhibit various mitochondrial enzymes and the intracellular trafficking of mito‐ chondria. It has also been reported that Zn produced ROS and caused oxidative damage resulting from mitochondrial impairments. Therefore, energy failure and the inhibition of

Considering the implication of Zn in transient global ischemia, substances that protect against Zn-induced neuronal death could be potential candidates for the prevention or treat‐ ment of neurodegeneration following ischemia, and ultimately provide a lead to treatments for VD. With the aim of exploring this idea, we developed a rapid, sensitive, and convenient assay system for the mass-screening of such substances by using GT1-7 cells. We examined the potential inhibitory effects of various agricultural products such as vegetable extracts, fruits extracts, and fish extracts, and found that extracts from eel muscles significantly pro‐ tected against Zn-induced neurotoxicity [26]. Finally, we demonstrated that carnosine (ßalanyl histidine), a small hydrophilic peptide abundant in eel muscles, protected GT1-7 cells from Zn-induced neurotoxicity in a dose-dependent manner. Therefore, we applied for the patent on carnosine as a drug for the treatment of VD or for slowing the progress of cogni‐ tive decline after ischemia (the application No. 2006-145857; the publication No. 2007-314467 in Japan) [27]. Carnosine is a naturally occurring dipeptide and is commonly present in ver‐ tebrate tissues, particularly within the skeletal muscles and nervous tissues [28]. It is found at high concentrations in the muscles of animals or fish which exhibit high levels of exercise, such as horses, chickens, and whales. The concentration of carnosine in the muscles of such animals is estimated to be 50–200 mM, and carnosine is believed to play important roles in the buffering capacities of muscle tissue. During high-intensity anaerobic exercise, proton accumulation causes a decrease in intracellular pH, which influences various metabolic

glycolysis in mitochondria may be involved in Zn neurotoxicity [25].

functions. The p*K*a value of carnosine is 7.01, close to intracellular pH.

ment was attenuated by Zn and carnosine [30].

Therefore, carnosine contributes to physicochemical non-bicarbonate buffering in skeletal muscles, and the administration of carnosine has been reported to induce hyperactivity in

Carnosine reportedly has various functions including anti oxidant, anti glycation, anti crosslink, and considered to be an endogenous neuroprotective, anti-aging substances. Considering the advantegeous properties of carnosine Considering the advantegeous properties of carnosine (relatively non-toxic, heat-stable, and water-soluble), the dietary supplementation of carnosine might be an effective strategy for the prevention or treat‐ ment of neurodegenerative diseases such as ischemia, VD, AD, and prion diseases. Coro‐ na et al. reported that supplementation of carnosine improved learning abilities of Alzheimer's model mice [29]. We demonstrated that neurotoxicity of prion protein frag‐

**2.4. Carnosine as an endogenous protective substance against Zn neurotoxicity**

level [24]. An imaging study using a Zn-sensitive fluorescent

Zinc and Neurodegenerative Diseases http://dx.doi.org/10.5772/54489 525

pyruvate restored the NAD+

animals.

**Figure 3.** Effects of various pharmacological substances on Zn-induced death of GT1-7 cells. A: GT1-7 cells were ex‐ posed to 50µM of Zn2+ with agonists or antagonisits of neurotransmitters (D-APV (D-2-amino-5-phosphonovalerate), glutamate, CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), bicuculline, muscimol, baclofen, GABA (gamma-aminobuty‐ ric acid)), channel blockers (TTX(tetrodotoxin), nimodipine), etc. B: GT1-7 cells were exposed to Zn2+ with or without Ca2+.

#### **2.3. Implication of Ca dyshomeostasis in Zn-induced neuronal death**

To address this issue, we employed a high-resolution multi-site video imaging system with fura-2 as the cytosolic free calcium reporter fluorescent probe for the observation of tempo‐ ral changes in [Ca2+]i after exposure to Zn (Fig. 4). This multisite fluorometry system enables the simultaneous long-term observation of temporal changes in [Ca2+]i of more than 50 neu‐ rons. The elevations in [Ca2+]i were observed among GT1-7 cells after 3-30 min of the expo‐ sure to Zn [18]. Detailed analysis of Zn-induced [Ca2+]i revealed that pretreatment of Al3+ significantly blocked the Zn-induced [Ca2+]i elevations. Thus, it is possible that Al3+, a known blocker of various types of Ca2+ channels, attenuate Zn-induced neurotoxicity by blocking Zn-induced elevations in [Ca2+]i .

We also showed that the administration of sodium pyruvate, an energy substrate, signifi‐ cantly inhibited the Zn-induced death of GT1-7 cells [17]. The results are consistent with findings of other studies using primary cultured cortical neurons, oligodendrocyte progeni‐ tor cells, or retinal cells. Furthermore, the administration of pyruvate attenuated the neuro‐ nal death after ischemia *in vivo* [23]. Shelline and his colleagues reported that Zn exposure decreased the levels of NAD+ and ATP in cultured cortical neurons, and that treatment with pyruvate restored the NAD+ level [24]. An imaging study using a Zn-sensitive fluorescent dye and a mitochondrial marker revealed that Zn is localized within mitochondria. Zn is re‐ ported to inhibit various mitochondrial enzymes and the intracellular trafficking of mito‐ chondria. It has also been reported that Zn produced ROS and caused oxidative damage resulting from mitochondrial impairments. Therefore, energy failure and the inhibition of glycolysis in mitochondria may be involved in Zn neurotoxicity [25].

#### **2.4. Carnosine as an endogenous protective substance against Zn neurotoxicity**

Zn (50 µM) + Glutamate + D-APV + CNQX + Muscimol + Baclofen + Bicuculline + TTX + Nimodipine + KCl + pyruvate + Vitamine E + Deferoxamine + *o-*phenanthroline

524 Neurodegenerative Diseases

Ca2+.

rons. The elevations in [Ca2+]i

Zn-induced elevations in [Ca2+]i

Cell viability (%) 0 5 10 15 20 25 30 35 40

**2.3. Implication of Ca dyshomeostasis in Zn-induced neuronal death**

sure to Zn [18]. Detailed analysis of Zn-induced [Ca2+]i

.

\*

\*

Relative viability (%)

0

were observed among GT1-7 cells after 3-30 min of the expo‐

20

40

Zn2+ Ca2+ cont Zn2+

revealed that pretreatment of Al3+

+ Ca2+

40

60

80

100

\* \*

**Figure 3.** Effects of various pharmacological substances on Zn-induced death of GT1-7 cells. A: GT1-7 cells were ex‐ posed to 50µM of Zn2+ with agonists or antagonisits of neurotransmitters (D-APV (D-2-amino-5-phosphonovalerate), glutamate, CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), bicuculline, muscimol, baclofen, GABA (gamma-aminobuty‐ ric acid)), channel blockers (TTX(tetrodotoxin), nimodipine), etc. B: GT1-7 cells were exposed to Zn2+ with or without

To address this issue, we employed a high-resolution multi-site video imaging system with fura-2 as the cytosolic free calcium reporter fluorescent probe for the observation of tempo‐ ral changes in [Ca2+]i after exposure to Zn (Fig. 4). This multisite fluorometry system enables the simultaneous long-term observation of temporal changes in [Ca2+]i of more than 50 neu‐

significantly blocked the Zn-induced [Ca2+]i elevations. Thus, it is possible that Al3+, a known blocker of various types of Ca2+ channels, attenuate Zn-induced neurotoxicity by blocking

We also showed that the administration of sodium pyruvate, an energy substrate, signifi‐ cantly inhibited the Zn-induced death of GT1-7 cells [17]. The results are consistent with findings of other studies using primary cultured cortical neurons, oligodendrocyte progeni‐ tor cells, or retinal cells. Furthermore, the administration of pyruvate attenuated the neuro‐ nal death after ischemia *in vivo* [23]. Shelline and his colleagues reported that Zn exposure

\*

BA

Considering the implication of Zn in transient global ischemia, substances that protect against Zn-induced neuronal death could be potential candidates for the prevention or treat‐ ment of neurodegeneration following ischemia, and ultimately provide a lead to treatments for VD. With the aim of exploring this idea, we developed a rapid, sensitive, and convenient assay system for the mass-screening of such substances by using GT1-7 cells. We examined the potential inhibitory effects of various agricultural products such as vegetable extracts, fruits extracts, and fish extracts, and found that extracts from eel muscles significantly pro‐ tected against Zn-induced neurotoxicity [26]. Finally, we demonstrated that carnosine (ßalanyl histidine), a small hydrophilic peptide abundant in eel muscles, protected GT1-7 cells from Zn-induced neurotoxicity in a dose-dependent manner. Therefore, we applied for the patent on carnosine as a drug for the treatment of VD or for slowing the progress of cogni‐ tive decline after ischemia (the application No. 2006-145857; the publication No. 2007-314467 in Japan) [27]. Carnosine is a naturally occurring dipeptide and is commonly present in ver‐ tebrate tissues, particularly within the skeletal muscles and nervous tissues [28]. It is found at high concentrations in the muscles of animals or fish which exhibit high levels of exercise, such as horses, chickens, and whales. The concentration of carnosine in the muscles of such animals is estimated to be 50–200 mM, and carnosine is believed to play important roles in the buffering capacities of muscle tissue. During high-intensity anaerobic exercise, proton accumulation causes a decrease in intracellular pH, which influences various metabolic functions. The p*K*a value of carnosine is 7.01, close to intracellular pH.

Therefore, carnosine contributes to physicochemical non-bicarbonate buffering in skeletal muscles, and the administration of carnosine has been reported to induce hyperactivity in animals.

Carnosine reportedly has various functions including anti oxidant, anti glycation, anti crosslink, and considered to be an endogenous neuroprotective, anti-aging substances. Considering the advantegeous properties of carnosine Considering the advantegeous properties of carnosine (relatively non-toxic, heat-stable, and water-soluble), the dietary supplementation of carnosine might be an effective strategy for the prevention or treat‐ ment of neurodegenerative diseases such as ischemia, VD, AD, and prion diseases. Coro‐ na et al. reported that supplementation of carnosine improved learning abilities of Alzheimer's model mice [29]. We demonstrated that neurotoxicity of prion protein frag‐ ment was attenuated by Zn and carnosine [30].

### **3. Zn and Alzheimer's disease diseases**

#### **3.1. Amyloid cascade hypothesis and Zn**

AD is a severe senile type of dementia first reported in 1906. The pathological hallmarks of AD are the deposition of extracellular senile plaques, intracellular neurofibrillary tangles (NFTs), and the selective loss of synapses and neurons in the hippocampal and cerebral cort‐ ical regions. The major component of NFTs is the phosphorylated tau protein. Senile pla‐ ques are largely comprised of ß-amyloid protein (AßP) [31]. Numerous biochemical, toxicological, cell biological, and genetic studies have supported the idea termed "amyloid cascade hypothesis" which suggests that the neurotoxicity caused by AßP play a central role in AD [32,33]. AβP is a small peptide with 39–43 amino acid residues. It is derived from the proteolytic cleavage of a large precursor protein (amyloid precursor protein; APP). AβP has an intrinsic tendency to self-assemble to form sodium dodecyl sulfate (SDS)-stable oligom‐ ers. Moreover, oligomerization and conformational changes in AßP are important for its neurodegeneration process. In an aqueous solution, freshly prepared and dissolved AβP ex‐ ists as a monomeric protein with a random coil structure. However, following incubation at 37°C for several days (*aging*), AβPs form aggregates (oligomers) with β-pleated sheet struc‐ tures, and finally form insoluble aggregates, termed amyloid fibrils (Fig. 5). The *aged* AβP peptides were considerably more toxic to cultured neurons than *fresh* (freshly prepared just before the experiment) AβP. AßP is secreted in the cerebrospinal fluid (CSF) of young indi‐ viduals as well as in aged or dementia patients [34]. Therefore, factors that accelerate or in‐ hibit the oligomerization may play essential roles in the pathogenesis of AD. Several factors such as the concentration of peptides, pH, composition of solvents, temperature, oxidations, mutations, and racemization of AßP can influence the oligomerization processes [35].

Interestingly, rodent AßP exhibits less tendency to oligomerization than human AßP *in vitro* and the accumulation of AßP is rarely observed in the brains of rodents (rats or mice) as compared to primates (humans or monkeys). As shown in Fig. 5, the amino acid sequences of human and rodent AßP are similar, but rodent AßP differs from primate only 3 amino acids (Arg5 , Tyr10, and His13) from primate AßP. All three amino acids have the ability to bind metals. Therefore, trace elements including Al, Zn, Cu, Fe as the accelerating factor of AßP are of particularly interest.

cent approaches using size-exclusion chromatography, gel electrophoresis, and atomic force microscopy have demonstrated that identified soluble oligomers are neurotoxic, further

**Figure 5.** Amyloid cascade hypothesis and the implications of Zn and other metals. AßP is secreted from its precursor APP, a Zn- or Cu- binding protein. AßP monomers exhibit random-coil structures. However, during aging or in the presence of some acceleratory factors, AßP self-aggregates and forms several types of oligomers (SDS-soluble oligom‐ ers, ADDLS, globulomers, protofibrils, etc.) and finally forms insoluble aggregates, which are termed amyloid fibrils.

• ß-sheet • soluble • toxic

Toxicity

*protofibril ADDLs*

*globulomer etc.*

• ß-sheet • insoluble • less or non-toxic

Zinc and Neurodegenerative Diseases http://dx.doi.org/10.5772/54489 527

Primate AßP: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKK-

*monomer Soluble oligomer Amyloid fibril*

Rodent AßP: DAEF*G*HDSG*F*EV*R*HQKLVFFAEDVGSNKGAIIGLMVGGVVIAT

APP membrane

• aging (incubation) • metals (Al, Cu, Zn, *etc.*)

Fe2+ Fe3+

Zn2+ Cu2+

Acceleratory factors

• oxidation • Mutation • racemization *etc.*

AßP

• rifampicin • curcumine • polyphenol • aspirin

Inhibitory factors

• ß-sheet breaker peptide *etc.*

APP also possesses copper/zinc binding sites in its amino-terminal domain and in the AßP domain and may be involved in homeostasis of these metals [42]. Duce *et al.* demonstrated that APP has ferroxidase activity, which converts Fe2+ to Fe3+ and regulates free pro-oxidant Fe2+ concentrations. They also found that Zn2+ inhibits the ferroxidase activity of APP [43]. Thus, the interaction with Zn and other metals in the functions of APP are of great interst.

Zn is involved in the mechanism of AßP-induced neurotoxicity. There is considerable inter‐ est regarding the mechanism by which AßPs cause neuronal death. In 1993, Arispe *et al.* first

studies about metal-induced oligomerization are necessary.

Oligomeric soluble AßP s are toxic, although monomers and fibrils are rather nontoxic.

**3.2. AßP -induced neuronal death and Zn**

We have investigated the metal-induced oligomerization of AßP and found that the metals including Al, Zn, Fe, Cu, and Cd enhanced the oligomerization. However, the oligomeriza‐ tion induced by Al is more marked than that induced by other metals [36,37]. Furthermore, while Zn-aggregated AßPs are rarely observed on the surface of cultured neurons several days after its exposure, Al-aggregated AßPs bind tightly to the surface of cultured neurons and form fibrillar deposits. Bush et al. reported the Zn- or Cu- induced oligomerization of AßP [38,39], and have developed the chelation therapy for AD treatment [40]. Clioquinol (quinoform), a chelator of Cu2+ or Zn2+, inhibits oligomerization of AßP and attenuates the accumulation of amyloid in the brains of experimental animals. Clinical trials using its ana‐ logue PBT2 are under investigation. However, considering that the morphology of AßP oligomers treated with metals including Al, Cu, Fe, Zn are quite different [41] and that re‐

**Figure 5.** Amyloid cascade hypothesis and the implications of Zn and other metals. AßP is secreted from its precursor APP, a Zn- or Cu- binding protein. AßP monomers exhibit random-coil structures. However, during aging or in the presence of some acceleratory factors, AßP self-aggregates and forms several types of oligomers (SDS-soluble oligom‐ ers, ADDLS, globulomers, protofibrils, etc.) and finally forms insoluble aggregates, which are termed amyloid fibrils. Oligomeric soluble AßP s are toxic, although monomers and fibrils are rather nontoxic.

cent approaches using size-exclusion chromatography, gel electrophoresis, and atomic force microscopy have demonstrated that identified soluble oligomers are neurotoxic, further studies about metal-induced oligomerization are necessary.

APP also possesses copper/zinc binding sites in its amino-terminal domain and in the AßP domain and may be involved in homeostasis of these metals [42]. Duce *et al.* demonstrated that APP has ferroxidase activity, which converts Fe2+ to Fe3+ and regulates free pro-oxidant Fe2+ concentrations. They also found that Zn2+ inhibits the ferroxidase activity of APP [43]. Thus, the interaction with Zn and other metals in the functions of APP are of great interst.

#### **3.2. AßP -induced neuronal death and Zn**

**3. Zn and Alzheimer's disease diseases**

AD is a severe senile type of dementia first reported in 1906. The pathological hallmarks of AD are the deposition of extracellular senile plaques, intracellular neurofibrillary tangles (NFTs), and the selective loss of synapses and neurons in the hippocampal and cerebral cort‐ ical regions. The major component of NFTs is the phosphorylated tau protein. Senile pla‐ ques are largely comprised of ß-amyloid protein (AßP) [31]. Numerous biochemical, toxicological, cell biological, and genetic studies have supported the idea termed "amyloid cascade hypothesis" which suggests that the neurotoxicity caused by AßP play a central role in AD [32,33]. AβP is a small peptide with 39–43 amino acid residues. It is derived from the proteolytic cleavage of a large precursor protein (amyloid precursor protein; APP). AβP has an intrinsic tendency to self-assemble to form sodium dodecyl sulfate (SDS)-stable oligom‐ ers. Moreover, oligomerization and conformational changes in AßP are important for its neurodegeneration process. In an aqueous solution, freshly prepared and dissolved AβP ex‐ ists as a monomeric protein with a random coil structure. However, following incubation at 37°C for several days (*aging*), AβPs form aggregates (oligomers) with β-pleated sheet struc‐ tures, and finally form insoluble aggregates, termed amyloid fibrils (Fig. 5). The *aged* AβP peptides were considerably more toxic to cultured neurons than *fresh* (freshly prepared just before the experiment) AβP. AßP is secreted in the cerebrospinal fluid (CSF) of young indi‐ viduals as well as in aged or dementia patients [34]. Therefore, factors that accelerate or in‐ hibit the oligomerization may play essential roles in the pathogenesis of AD. Several factors such as the concentration of peptides, pH, composition of solvents, temperature, oxidations,

mutations, and racemization of AßP can influence the oligomerization processes [35].

Interestingly, rodent AßP exhibits less tendency to oligomerization than human AßP *in vitro* and the accumulation of AßP is rarely observed in the brains of rodents (rats or mice) as compared to primates (humans or monkeys). As shown in Fig. 5, the amino acid sequences of human and rodent AßP are similar, but rodent AßP differs from primate only 3 amino

bind metals. Therefore, trace elements including Al, Zn, Cu, Fe as the accelerating factor of

We have investigated the metal-induced oligomerization of AßP and found that the metals including Al, Zn, Fe, Cu, and Cd enhanced the oligomerization. However, the oligomeriza‐ tion induced by Al is more marked than that induced by other metals [36,37]. Furthermore, while Zn-aggregated AßPs are rarely observed on the surface of cultured neurons several days after its exposure, Al-aggregated AßPs bind tightly to the surface of cultured neurons and form fibrillar deposits. Bush et al. reported the Zn- or Cu- induced oligomerization of AßP [38,39], and have developed the chelation therapy for AD treatment [40]. Clioquinol (quinoform), a chelator of Cu2+ or Zn2+, inhibits oligomerization of AßP and attenuates the accumulation of amyloid in the brains of experimental animals. Clinical trials using its ana‐ logue PBT2 are under investigation. However, considering that the morphology of AßP oligomers treated with metals including Al, Cu, Fe, Zn are quite different [41] and that re‐

, Tyr10, and His13) from primate AßP. All three amino acids have the ability to

**3.1. Amyloid cascade hypothesis and Zn**

526 Neurodegenerative Diseases

acids (Arg5

AßP are of particularly interest.

Zn is involved in the mechanism of AßP-induced neurotoxicity. There is considerable inter‐ est regarding the mechanism by which AßPs cause neuronal death. In 1993, Arispe *et al.* first demonstrated that AßP directly incorporates into artificial lipid bilayer membranes and forms cation-selective (including Ca2+) ion channels [44,45]. We revealed that AßP formed amyloid channels on the GT1-7 cell membranes and their characteristics were considerably similar to those observed on artificial lipid bilayers; cation-selective, multilevel [46]., and that AßP causes the increase of intracellular Ca2+ in GT1-7 cells and degeneration [47]. These results strongly support the hypothetical idea termed 'amyloid channel hypothesis', namely, that the direct incorporation of AßPs and the subsequent imbalances of Ca2+ and other ions through amyloid channels may be the primary event in AßP neurotoxicity [48].

Inorganic cations such as Al3+ or Zn2+ inhibit current induced by amyloid channels [44,45]. Zn reportedly inhibited AßP-induced Ca2+ increase. We have revealed that the amyloid channel activity formed on membranes of GT1-7 cells was inhibited by addition of Zn2+, and recovered by Zn chelator, *o*-phenanthroline [47]. Considering that Zn binds to His residues of AßP, Arispe *et al.* found that histidine-related peptide derivatives such as His-His are effective in the inhibition of amyloid channels, the attenuation of AßP-induced [Ca2+]i changes, and the protection of neurons from AßP toxicity. Among various com‐ pounds tested, small amphiphilic pyridinium salts were revealed to block the amyloid

Zinc and Neurodegenerative Diseases http://dx.doi.org/10.5772/54489 529

Based on our and other findings about the link between Zn and the pathogenesis of AD, we made a hypothetical scheme about the link between AD pathogenesis and Zn (Fig. 6). AßPs are normally secreted from APP, which exists in the synapse. Secreted AßPs are usually de‐ graded proteolytically by proteases within a short period. However, Zn or other metals en‐ hance the oligomerization and accumulation of AßP. After incorporation into the membrane, the conformation of AßPs change and the accumulated AßPs aggregate on the membranes.. Finally, aggregated AßP oligomers form ion channels leading to the various neurodegenerative processes.Unlike endogenous Ca2+ channels, these AßP channels are not regulated by usual blockers. Thus, once formed on membranes, a continuous flow of [Ca2+]i is initiated. Disruption of calcium homeostasis triggers several apoptotic pathways and pro‐ motes numerous degenerative processes, including free radical formation and tau phos‐ phorylation, thereby accelerating neuronal death. Meanwhile, Zn2+, which are secreted into synaptic clefts in a neuronal activity-dependent manner, inhibit AßP-induced Ca2+ entry,

Based on results of our own and other numerous studies, the disruption of Zn homeostasis, namely both zinc depletion and excess zinc, cause severe damage to neurons and linked with various neurodegenerative diseases including VD and AD. Increasing evidence sug‐ gests the implications of Zn in the pathogenesis of other neurodegenerative disease includ‐ ing prion diseases, Parkinson disease, ALS etc. Zn acts as a contributor of the disease in one part, and as a protector in another part. Thus, Zn might play a role like that of Janus, an an‐

Our new approach to ischemia-induced neurodegeneration from the perspective of the Zn hypothesis will lead to new therapeutic tools for the treatment and/or prevention of VD. Further research about the role of Zn in neuronal injury and the significance of Zn homeo‐ stasis might give rise to the development of new treatments for neurodegenerative diseases. In this context, the advantegeous properties of carnosine (relatively non-toxic, heat-stable, and water-soluble) as a possible candidate for the prevention or treatment of neurodegener‐

cient Roman god of doorways with two different faces, in the brain (Fig. 7).

ative diseases such as ischemia, VD, AD, and prion diseases are important.

channel and protect neurons [49].

and thus have a protective function in AD.

**4. Conclusion**

**Figure 6.** Zn and other metals in the pathogenesis of Alzheimer's disease. Details are shown in the text.

Inorganic cations such as Al3+ or Zn2+ inhibit current induced by amyloid channels [44,45]. Zn reportedly inhibited AßP-induced Ca2+ increase. We have revealed that the amyloid channel activity formed on membranes of GT1-7 cells was inhibited by addition of Zn2+, and recovered by Zn chelator, *o*-phenanthroline [47]. Considering that Zn binds to His residues of AßP, Arispe *et al.* found that histidine-related peptide derivatives such as His-His are effective in the inhibition of amyloid channels, the attenuation of AßP-induced [Ca2+]i changes, and the protection of neurons from AßP toxicity. Among various com‐ pounds tested, small amphiphilic pyridinium salts were revealed to block the amyloid channel and protect neurons [49].

Based on our and other findings about the link between Zn and the pathogenesis of AD, we made a hypothetical scheme about the link between AD pathogenesis and Zn (Fig. 6). AßPs are normally secreted from APP, which exists in the synapse. Secreted AßPs are usually de‐ graded proteolytically by proteases within a short period. However, Zn or other metals en‐ hance the oligomerization and accumulation of AßP. After incorporation into the membrane, the conformation of AßPs change and the accumulated AßPs aggregate on the membranes.. Finally, aggregated AßP oligomers form ion channels leading to the various neurodegenerative processes.Unlike endogenous Ca2+ channels, these AßP channels are not regulated by usual blockers. Thus, once formed on membranes, a continuous flow of [Ca2+]i is initiated. Disruption of calcium homeostasis triggers several apoptotic pathways and pro‐ motes numerous degenerative processes, including free radical formation and tau phos‐ phorylation, thereby accelerating neuronal death. Meanwhile, Zn2+, which are secreted into synaptic clefts in a neuronal activity-dependent manner, inhibit AßP-induced Ca2+ entry, and thus have a protective function in AD.

#### **4. Conclusion**

demonstrated that AßP directly incorporates into artificial lipid bilayer membranes and forms cation-selective (including Ca2+) ion channels [44,45]. We revealed that AßP formed amyloid channels on the GT1-7 cell membranes and their characteristics were considerably similar to those observed on artificial lipid bilayers; cation-selective, multilevel [46]., and that AßP causes the increase of intracellular Ca2+ in GT1-7 cells and degeneration [47]. These results strongly support the hypothetical idea termed 'amyloid channel hypothesis', namely, that the direct incorporation of AßPs and the subsequent imbalances of Ca2+ and other ions

monomer oligomer

Al Cu Zn Fe

Ca2+

Disruption of Ca2+ homeostasis

Synaptotoxicity

Alzheimer's disease

**Figure 6.** Zn and other metals in the pathogenesis of Alzheimer's disease. Details are shown in the text.

Neurotoxicity

Senile plaque

Fe Al

through amyloid channels may be the primary event in AßP neurotoxicity [48].

APP

528 Neurodegenerative Diseases

Zn

AßP

Fe

Cu

Zn

Al

synapse

Zn

Based on results of our own and other numerous studies, the disruption of Zn homeostasis, namely both zinc depletion and excess zinc, cause severe damage to neurons and linked with various neurodegenerative diseases including VD and AD. Increasing evidence sug‐ gests the implications of Zn in the pathogenesis of other neurodegenerative disease includ‐ ing prion diseases, Parkinson disease, ALS etc. Zn acts as a contributor of the disease in one part, and as a protector in another part. Thus, Zn might play a role like that of Janus, an an‐ cient Roman god of doorways with two different faces, in the brain (Fig. 7).

Our new approach to ischemia-induced neurodegeneration from the perspective of the Zn hypothesis will lead to new therapeutic tools for the treatment and/or prevention of VD. Further research about the role of Zn in neuronal injury and the significance of Zn homeo‐ stasis might give rise to the development of new treatments for neurodegenerative diseases. In this context, the advantegeous properties of carnosine (relatively non-toxic, heat-stable, and water-soluble) as a possible candidate for the prevention or treatment of neurodegener‐ ative diseases such as ischemia, VD, AD, and prion diseases are important.

[4] Frederickson CJ et al. Importance of zinc in the central nervous system: the zinc-con‐

Zinc and Neurodegenerative Diseases http://dx.doi.org/10.5772/54489 531

[5] Tamano H and Takeda A. Dynamic action of neurometals at the synapse. Metallo‐

[7] de Haan EH et al. Cognitive function following stroke and vascular cognitive impair‐

[8] Weiss JH, Sensi SL, Koh JY. Zn2+: a novel ionic mediator of neural injury in brain dis‐

[9] Koh JY et al. The role of zinc in selective neuronal death after transient global cere‐

[10] Calderone A et al. Late calcium EDTA rescues hippocampal CA1 neurons from glob‐

[11] Sensi SL et al. Measurement of intracellular free zinc in living cortical neurons: routes

[12] Pellegrini-Giampietro DE et al. The GluR2 (GluR-B) hypothesis: Ca2+-permeable AM‐ PA receptors in neurological disorders. Trends Neurosci 1997; 20: 464-70.

[13] Fukada T and Kambe T. Molecular and genetic features of zinc transporters in physi‐

[14] Lovell MA. A potential role for alterations of zinc and zinc transport proteins in the

[15] Koh JY and Choi DW. Zinc toxicity of cultured cortical neurons: involvement of N-

[16] Kim AH. L-type Ca2+ channel-mediated Zn2+ toxicity and modulation by ZnT-1 in

[17] Kawahara M et al. Pyruvate blocks zinc-induced neurotoxicity in immortalized hy‐ pothalamic neurons. Cellular and Molecular Neurobiology 2002; 22: 87-93.

[18] Koyama H et al. Zinc neurotoxicity and the pathogenesis of vascular-type dementia: Involvement of calcium dyshomeostasis and carnosine. J Clin Toxicol 2012 S3: 002.

[19] Mellon PL et al. Immortalization of hypothalamic GnRH neurons by genetically tar‐

[20] Mahesh VB et al. Characterization of ionotropic glutamate receptors in rat hypothala‐ mus, pituitary and immortalized gonadotropin-releasing hormone (GnRH) neurons

progression of Alzheimer's disease. J Alzheimers Dis. 2009; 16(3): 471-83.

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[6] Lee JM et al. Brain tissue responses to ischemia. J Clin Invest 2000; 106: 723-31.

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mics 2011; 3(7):656-61..

**Figure 7.** Zn as Janus in brain

As described here, Zn plays important roles in memory formation, and protects neurons from various neurodegenerative diseases. Meanwhile, excess Zn is neurotoxic and may en‐ hance the pathogenesis of the diseases.

### **Author details**

Masahiro Kawahara1 , Keiko Konoha2 , Hironari Koyama2 , Susumu Ohkawara2 and Yutaka Sadakane3

1 Department of Bio-Analytical Chemistry, Musashino University, Research Institute of Pharmaceutical Sciences, Musashino University, Nishitokyo-shi, Tokyo, Japan

2 Department of Analytical Chemistry, School of Pharmaceutical Sciences, Kyushu Universi‐ ty of Health and Welfare, Miyazaki, Japan

3 Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Mie, Japan

### **References**


Zn

Prion disease

As described here, Zn plays important roles in memory formation, and protects neurons from various neurodegenerative diseases. Meanwhile, excess Zn is neurotoxic and may en‐

, Hironari Koyama2

1 Department of Bio-Analytical Chemistry, Musashino University, Research Institute of

2 Department of Analytical Chemistry, School of Pharmaceutical Sciences, Kyushu Universi‐

[2] Hirano T, Murakami M, Fukada T, et al. Roles of zinc and zinc signaling in immuni‐ ty: zinc as an intracellular signaling molecule. Adv Immunol 2008; 97: 149-76.

[3] Prasad AS. Impact of the discovery of human zinc deficiency on health. J Am Coll

3 Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Mie, Japan

Pharmaceutical Sciences, Musashino University, Nishitokyo-shi, Tokyo, Japan

[1] Hambidge M. Human zinc deficiency. J Nutr 2000; 130: 1344S-9S.

Vascular-type dementia

, Susumu Ohkawara2

and

Alzheimer's disease

**Figure 7.** Zn as Janus in brain

530 Neurodegenerative Diseases

**Author details**

Masahiro Kawahara1

Yutaka Sadakane3

**References**

hance the pathogenesis of the diseases.

ty of Health and Welfare, Miyazaki, Japan

Nutr 2009; 28: 257-65.

, Keiko Konoha2


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[47] Kawahara M et al. Alzheimer's ß-amyloid, human islet amylin and prion protein fragment evoke intracellular free-calcium elevations by a common mechanism in a

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[22] Konoha K, Sadakane Y, Kawahara M. Effects of gadolinium and other metal on the neurotoxicity of immortalized hypothalamic neurons induced by zinc. Biomed Res

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[25] Sensi SL et al. Modulation of mitochondrial function by endogenous Zn2+ pools. Proc

[26] Konoha K, Sadakane Y, Kawahara M. Carnosine protects GT1-7 cells against zinc-in‐ duced neurotoxicity: a possible candidate for treatment for vascular type of demen‐

[27] Kawahara M et al. Protective substances against zinc-induced neuronal death after ischemia: carnosine a target for drug of vascular type of dementia. Recent Patents on

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532 Neurodegenerative Diseases


**Chapter 22**

**Oligodendrocyte Metabolic Stress in**

**Neurodegeneration**

http://dx.doi.org/10.5772/54461

**1. Introduction**

a single class of drugs.

Daniel Radecki and Alexander Gow

Additional information is available at the end of the chapter

Neurodegeneration can be viewed in general terms as a common endpoint for a large and diverse group of nervous system diseases that arise in patients with disparate clinical symp‐ toms. As such, neurodegeneration is a convergent pathology wherein clinical signs are largely dependent on the location and identity of the degenerating cells. For example, in pa‐ tients for whom substantia nigra neurons are degenerating, the accompanying symptoms re‐ flect Parkinson disease (PD). Many of the symptoms are unique to PD, thereby enabling diagnosis, and would rarely be confused with those of patients suffering from amyotrophic lateral sclerosis (ALS), for whom ventral horn motor neurons in the spinal cord are lost. In the same vein, disease in patients with Alzheimer disease (AD) or multiple sclerosis (MS)

Despite these disease specific phenotypes, recent evidence indicates that their underlying pathophysiology, and that of many others, involves activation of a signaling pathway known as the unfolded protein response (UPR). This suggests the exciting possibility of a shared disease mechanism and, potentially, a common treatment strategy such as the use of

Research efforts from many laboratories have begun to elucidate the importance of the UPR to disease etiology. For example, causative mutations in familial forms of PD and AD are found in genes that encode components of protein aggregation and degradation pathways such as the ubiquitin-proteasome pathway, which strongly suggests that sporadic forms of these diseases also arise from perturbed protein folding or degradation [1-3]. In addition, the etiology of MS, which was once understood to be entirely caused by autoimmune at‐ tacks on the central nervous system (CNS), is becoming increasingly unclear because of new evidence pointing to an underlying degenerative pathology in oligodendrocytes that in‐

and reproduction in any medium, provided the original work is properly cited.

© 2013 Radecki and Gow; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

stem from the loss of distinct cell populations which confer unique phenotypes.

**Chapter 22**

### **Oligodendrocyte Metabolic Stress in Neurodegeneration**

Daniel Radecki and Alexander Gow

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54461

### **1. Introduction**

Neurodegeneration can be viewed in general terms as a common endpoint for a large and diverse group of nervous system diseases that arise in patients with disparate clinical symp‐ toms. As such, neurodegeneration is a convergent pathology wherein clinical signs are largely dependent on the location and identity of the degenerating cells. For example, in pa‐ tients for whom substantia nigra neurons are degenerating, the accompanying symptoms re‐ flect Parkinson disease (PD). Many of the symptoms are unique to PD, thereby enabling diagnosis, and would rarely be confused with those of patients suffering from amyotrophic lateral sclerosis (ALS), for whom ventral horn motor neurons in the spinal cord are lost. In the same vein, disease in patients with Alzheimer disease (AD) or multiple sclerosis (MS) stem from the loss of distinct cell populations which confer unique phenotypes.

Despite these disease specific phenotypes, recent evidence indicates that their underlying pathophysiology, and that of many others, involves activation of a signaling pathway known as the unfolded protein response (UPR). This suggests the exciting possibility of a shared disease mechanism and, potentially, a common treatment strategy such as the use of a single class of drugs.

Research efforts from many laboratories have begun to elucidate the importance of the UPR to disease etiology. For example, causative mutations in familial forms of PD and AD are found in genes that encode components of protein aggregation and degradation pathways such as the ubiquitin-proteasome pathway, which strongly suggests that sporadic forms of these diseases also arise from perturbed protein folding or degradation [1-3]. In addition, the etiology of MS, which was once understood to be entirely caused by autoimmune at‐ tacks on the central nervous system (CNS), is becoming increasingly unclear because of new evidence pointing to an underlying degenerative pathology in oligodendrocytes that in‐

© 2013 Radecki and Gow; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

volves UPR induction and secondary activation of the immune system. Finally, the etiology of oligodendrocyte metabolic diseases including at least two of the leukodystrophies, van‐ ishing white matter disease (VWM) and Pelizaeus-Merzbacher disease (PMD), is known to involve UPR activation.

ever, if the synthesis of these polypeptides surpasses the rate of degradation, they accumu‐

Oligodendrocyte Metabolic Stress in Neurodegeneration

http://dx.doi.org/10.5772/54461

537

The importance of UPR signaling to cell homeostasis and survival is highlighted by the de‐ gree of conservation of this pathway in prokaryotes and in eukaryotes from yeast to mam‐ mals. Significant increases in signaling complexity in higher eukaryotes also indicates the growing importance of this pathway in multicellular organisms through evolution [13]. In most cases for eukaryotes, activation of the UPR by misfolded proteins causes rapid shut‐ down of global protein translation, expansion of intracellular membrane-bound compart‐ ments, induction of molecular chaperone expression and increased degradation of

In the event that such comprehensive changes in cell structure, reprogramming and metabo‐ lism are ineffective at curbing UPR signaling and reestablishing homeostasis, cells will in‐ evitably undergo apoptosis in a manner that limits damage to neighboring cells and the survival of the organism [14]. Surprisingly, the nature of the trigger that induces apoptosis appears to be divergent in different cell types, and several hypotheses have been developed

In general terms, the UPR signaling cascade maintains cell homeostasis, metabolism and cell survival [13, 15, 16]. In higher eukaryotes, the UPR can be divided into three path‐ ways named for the proteins that initiate signaling: IRE1α (inositol requiring protein 1α), ATF6 (activating transcription factor 6) and PERK (pancreatic endoplasmic reticulum kin‐ ase). Together, these pathways increase expression of molecular chaperone proteins, pro‐ tein degradation and decrease global translation to alleviate misfolding and restore

The IRE1α receptor is a transmembrane protein that is localized to the ER and detects the accumulation of misfolded proteins or, in more general terms, serves as a sensor of changes in secretory pathway protein flux. IRE1α was the first component of the UPR cascade to be identified in any eukaryote and is the only UPR sensor present in yeast [14, 18]. The ER lu‐ minal domain is topographically similar to the Major Histocompatibility Class (MHC) pro‐ teins of the immune system and appears to bind to the molecular chaperone protein, BiP, which maintains IRE1α as a monomer and prevents its activation. However, misfolded pro‐ tein accumulation in the ER lumen sequesters BiP from IRE1α and allows this receptor to homodimerize leading to transautophosphorylation and activation of its cytoplasmic endor‐

A major downstream target of the IRE1α nuclease domain is an mRNA that encodes the bzip transcription factor, X-box Binding Protein 1 (XBP1) [20]. Processing of this mRNA re‐ moves a short internal 26 base intron and completes the major open reading frame that enc‐ odes functional XBP1. The major target genes of XBP1 include molecular chaperones which

late in the ER, causing metabolic stress and induction of the UPR [12].

to explain the bulk of published studies as detailed below.

misfolded proteins.

**3.1. UPR signaling in mammals**

homeostasis. [12, 17].

*3.1.1. The IRE1 pathway*

ibonuclease P domain [19].

In this review, we begin with a general definition of normal versus disease states in terms of cell homeostasis and its relation to UPR signaling, metabolic stress and neurodegeneration. Next, we examine essential aspects of the UPR signaling cascade, as well as emerging con‐ cepts about UPR activation and function, and conclude with an examination of MS as a pri‐ mary UPR disease rather than its typical consideration as a primary autoimmune disease.

### **2. Metabolic stress and the concept of homeostasis**

An increasing awareness of the pathophysiology of neurodegeneration has led to the reali‐ zation that metabolic stress is a major contributor to disease etiology. This novel view can be conceptualized as follows. Cells under normal metabolic conditions are described as main‐ taining homeostasis. Metabolic stress is viewed as a loss of homeostasis, defined as any pathological process that impedes cell function.

Intracellular signaling pathways have evolved to detect and counteract many forms of meta‐ bolic stress. These pathways modify cell activity and impart significant protection under pathological conditions, thereby maintaining homeostasis. However, when metabolic stress disrupts homeostasis, cells become vulnerable to apoptosis leading to brain atrophy and dis‐ ease. These concepts have been principally developed to account for the pathophysiology and disease severity that we observe in animal models of PMD [4-7]. However, they are also relevant to other oligodendrocyte diseases as well as major neurological diseases like AD, PD and ALS.

### **3. Misfolded proteins trigger UPR signaling**

Two of the most important homeostatic features of normal cell function are the consistent and efficient translation of proteins and the post-translational folding and processing of those proteins into their stable higher-ordered conformations. However, not all protein mol‐ ecules achieve native conformations after translation even in normal cells, and particularly in genetic diseases when missense or nonsense mutations in coding exons of genes confer distinct misfolded conformations on the translated products [8, 9].

In cases of transmembrane or secreted proteins that are synthesized on the ER (endoplasmic reticulum) in eukaryotes, misfolded or abnormal folding intermediates are prevented from being transported beyond this compartment by the quality control machinery of the cell. These nascent polypeptide chains are either removed from the ER and degraded via the ubiquitin-proteasome system or are shunted into the lysosome by autophagy [10, 11]. How‐ ever, if the synthesis of these polypeptides surpasses the rate of degradation, they accumu‐ late in the ER, causing metabolic stress and induction of the UPR [12].

The importance of UPR signaling to cell homeostasis and survival is highlighted by the de‐ gree of conservation of this pathway in prokaryotes and in eukaryotes from yeast to mam‐ mals. Significant increases in signaling complexity in higher eukaryotes also indicates the growing importance of this pathway in multicellular organisms through evolution [13]. In most cases for eukaryotes, activation of the UPR by misfolded proteins causes rapid shut‐ down of global protein translation, expansion of intracellular membrane-bound compart‐ ments, induction of molecular chaperone expression and increased degradation of misfolded proteins.

In the event that such comprehensive changes in cell structure, reprogramming and metabo‐ lism are ineffective at curbing UPR signaling and reestablishing homeostasis, cells will in‐ evitably undergo apoptosis in a manner that limits damage to neighboring cells and the survival of the organism [14]. Surprisingly, the nature of the trigger that induces apoptosis appears to be divergent in different cell types, and several hypotheses have been developed to explain the bulk of published studies as detailed below.

#### **3.1. UPR signaling in mammals**

volves UPR induction and secondary activation of the immune system. Finally, the etiology of oligodendrocyte metabolic diseases including at least two of the leukodystrophies, van‐ ishing white matter disease (VWM) and Pelizaeus-Merzbacher disease (PMD), is known to

In this review, we begin with a general definition of normal versus disease states in terms of cell homeostasis and its relation to UPR signaling, metabolic stress and neurodegeneration. Next, we examine essential aspects of the UPR signaling cascade, as well as emerging con‐ cepts about UPR activation and function, and conclude with an examination of MS as a pri‐ mary UPR disease rather than its typical consideration as a primary autoimmune disease.

An increasing awareness of the pathophysiology of neurodegeneration has led to the reali‐ zation that metabolic stress is a major contributor to disease etiology. This novel view can be conceptualized as follows. Cells under normal metabolic conditions are described as main‐ taining homeostasis. Metabolic stress is viewed as a loss of homeostasis, defined as any

Intracellular signaling pathways have evolved to detect and counteract many forms of meta‐ bolic stress. These pathways modify cell activity and impart significant protection under pathological conditions, thereby maintaining homeostasis. However, when metabolic stress disrupts homeostasis, cells become vulnerable to apoptosis leading to brain atrophy and dis‐ ease. These concepts have been principally developed to account for the pathophysiology and disease severity that we observe in animal models of PMD [4-7]. However, they are also relevant to other oligodendrocyte diseases as well as major neurological diseases like AD,

Two of the most important homeostatic features of normal cell function are the consistent and efficient translation of proteins and the post-translational folding and processing of those proteins into their stable higher-ordered conformations. However, not all protein mol‐ ecules achieve native conformations after translation even in normal cells, and particularly in genetic diseases when missense or nonsense mutations in coding exons of genes confer

In cases of transmembrane or secreted proteins that are synthesized on the ER (endoplasmic reticulum) in eukaryotes, misfolded or abnormal folding intermediates are prevented from being transported beyond this compartment by the quality control machinery of the cell. These nascent polypeptide chains are either removed from the ER and degraded via the ubiquitin-proteasome system or are shunted into the lysosome by autophagy [10, 11]. How‐

**2. Metabolic stress and the concept of homeostasis**

pathological process that impedes cell function.

**3. Misfolded proteins trigger UPR signaling**

distinct misfolded conformations on the translated products [8, 9].

involve UPR activation.

536 Neurodegenerative Diseases

PD and ALS.

In general terms, the UPR signaling cascade maintains cell homeostasis, metabolism and cell survival [13, 15, 16]. In higher eukaryotes, the UPR can be divided into three path‐ ways named for the proteins that initiate signaling: IRE1α (inositol requiring protein 1α), ATF6 (activating transcription factor 6) and PERK (pancreatic endoplasmic reticulum kin‐ ase). Together, these pathways increase expression of molecular chaperone proteins, pro‐ tein degradation and decrease global translation to alleviate misfolding and restore homeostasis. [12, 17].

#### *3.1.1. The IRE1 pathway*

The IRE1α receptor is a transmembrane protein that is localized to the ER and detects the accumulation of misfolded proteins or, in more general terms, serves as a sensor of changes in secretory pathway protein flux. IRE1α was the first component of the UPR cascade to be identified in any eukaryote and is the only UPR sensor present in yeast [14, 18]. The ER lu‐ minal domain is topographically similar to the Major Histocompatibility Class (MHC) pro‐ teins of the immune system and appears to bind to the molecular chaperone protein, BiP, which maintains IRE1α as a monomer and prevents its activation. However, misfolded pro‐ tein accumulation in the ER lumen sequesters BiP from IRE1α and allows this receptor to homodimerize leading to transautophosphorylation and activation of its cytoplasmic endor‐ ibonuclease P domain [19].

A major downstream target of the IRE1α nuclease domain is an mRNA that encodes the bzip transcription factor, X-box Binding Protein 1 (XBP1) [20]. Processing of this mRNA re‐ moves a short internal 26 base intron and completes the major open reading frame that enc‐ odes functional XBP1. The major target genes of XBP1 include molecular chaperones which are ER-resident proteins that bind to unfolded or misfolded polypeptides [14]. Accordingly, the IRE1α pathway detects changes in protein flux and acts to increase the folding capacity of the ER, ultimately completing a negative feedback loop on the UPR.

lational changes that reduce the accumulation of misfolded polypeptides and ultimately negatively feedback to switch off the UPR. Collectively, these activities comprise the adaptive arm of the UPR cascade, which adjusts cell metabolism to maintain homeostasis and promote cell survival. However, the UPR cascade also appears to include a maladap‐ tive arm, the major function of which is to trigger apoptosis in the event that cells fail to

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539

Although the maladaptive arm of the UPR is widely known and discussed in published studies, the molecular mechanisms underlying its activation are poorly understood. Many studies identify CHOP or a decoy kinase known as Tribbles3 (Trib3) as major components of the maladaptive trigger for apoptosis [23, 24]; however, this view reveals a significant co‐ nundrum. Thus, if PERK signaling requires CHOP expression to complete the negative feed‐ back loop that dephosphorylates eIF2α as part of the adaptive response, why would CHOP

There are three principal hypotheses that address this issue. The first proposes that CHOP is a molecular rheostat that drives distinct downstream pathways as a function of expression level [25, 26]. The second suggests that the IRE1 and PERK pathways act in concert to effect cell survival but drive apoptosis when the activities of these pathways are unbalanced [20]. The third hypothesizes that apoptosis is triggered stochastically at a restriction point in the PERK pathway, which is more-or-less coincident with the reinitiation of protein translation

Studies in human embryonic kidney 293 (HEK293) cells utilizing genetic and chemical in‐ duction of the UPR have led to the hypothesis of graded activation, mediated by CHOP, with apoptosis resulting from the highest levels of expression [25, 26]. Transient ER stress requires a UPR; however, the response itself would be modulated so that mild stress generates tapered transient CHOP induction, and severe prolonged stress causes sustained CHOP expression. Indeed, a modulated CHOP response has been observed during molecular and mechanical stress in vitro that activates PERK in the ER, with sus‐ tained PERK activation causing sustained CHOP expression and increased apoptosis. In contrast, oligodendrocytes undergoing severe metabolic stress and widespread apoptosis do not express CHOP, suggesting that its induction is transient even during severe stress

From their in vitro manipulation of the IRE1 and PERK pathways in HEK293 cells, Walter and colleagues [29] identified disparate roles for each pathway that could account for diver‐ gent UPR phenotypes in animal models of disease. The results showed that activating the PERK pathway alone decreased cell proliferation in vitro and triggered a morphological de‐ differentiation characterized by a loss of cell processes. In contrast, unilateral IRE1 activation increased cell numbers. Because activation of the PERK and IRE1 pathways stem from the

expression trigger apoptosis as part of the maladaptive response?

upon eIF2α dephosphorylation [17, 27].

*3.2.2. Balanced IRE1 and PERK signaling*

*3.2.1. CHOP as a rheostat*

[4, 6, 28].

maintain homeostasis.

#### *3.1.2. The ATF6 pathway*

A second UPR pathway is initiated through activation of the membrane-tethered ATF6 pro‐ tein and converges with IRE1α signaling. ATF6 interacts with BiP, similar to IRE1α. Misfold‐ ed proteins displace BiP from the ATF6 luminal domain and enable the protein to traffic from the ER to the Golgi apparatus where it is cleaved by the site 1 and site 2 proteases (S1P and S2P, respectively). The resulting cytosolic N-terminal fragment of ATF6 is the functional bzip transcription factor that heterodimerizes with XBP1 and induces expression of molecu‐ lar chaperone genes including BiP, glucose-regulated protein 94 (GRP94) and other genes encoding protein folding pathway proteins [15, 19]. ATF6 also upregulates proteins associat‐ ed with the ER Associated Degradation (ERAD) pathway, which is a checkpoint in the ER that ubiquitinates proteins and shuttles them into the cytoplasm for proteasome-mediated degradation [21]. Thus, ATF6 helps to upregulate chaperones to relieve mild protein mis‐ folding, but can also activate degradation of proteins that are severely misfolded and cannot be rescued by chaperones.

#### *3.1.3. The PERK pathway*

A third UPR pathway is regulated by an ER-resident receptor known as PERK. The luminal domain of PERK functions analogously to that of IRE1α in binding BiP, and is also activated by dimerization and transautophosphorylation. The cytoplasmic domain of PERK is a pro‐ tein kinase, a major target of which is the alpha subunit of eukaryotic initiation factor 2 (eIF2α). eIF2α is a critical component in ribosome assembly and can be inactivated by phos‐ phorylation, which leads to the shut down of global protein synthesis [17].

Despite global translation arrest, a small number of proteins that are critical to the UPR signal‐ ing are actively translated, including the bzip transcription factors, activating transcription fac‐ tor 4 (ATF4), ATF3 and the CCAAT-enhancer-binding protein homologous protein (CHOP) as well as the regulatory subunit of protein phosphatase 1 (PP1), known as growth-arrest and DNA damage protein 34 (GADD34) [17, 22]. The GADD34-PP1 complex is targeted to the ER membrane to dephosphorylate p-eIF2α and reinitiate protein translation. Thus, the PERK path‐ way temporarily halts protein synthesis to suppress additional accumulation of misfolded pro‐ teins in the ER. The pathway subsequently reactivates protein synthesis by opposing the phosphorylation activity of PERK. Thus, because of the time that is necessary to complete each of the steps downstream of eIF2α phosphorylation, the PERK pathway can be considered to be a time-delay circuit that forms a negative feedback loop to regulate UPR signaling.

#### **3.2. Adaptive and maladaptive facets of UPR signaling**

A common theme among the three branches of the UPR cascade is the similar activation of the ER-resident receptors by changes in protein flux leading to transcriptional or trans‐ lational changes that reduce the accumulation of misfolded polypeptides and ultimately negatively feedback to switch off the UPR. Collectively, these activities comprise the adaptive arm of the UPR cascade, which adjusts cell metabolism to maintain homeostasis and promote cell survival. However, the UPR cascade also appears to include a maladap‐ tive arm, the major function of which is to trigger apoptosis in the event that cells fail to maintain homeostasis.

Although the maladaptive arm of the UPR is widely known and discussed in published studies, the molecular mechanisms underlying its activation are poorly understood. Many studies identify CHOP or a decoy kinase known as Tribbles3 (Trib3) as major components of the maladaptive trigger for apoptosis [23, 24]; however, this view reveals a significant co‐ nundrum. Thus, if PERK signaling requires CHOP expression to complete the negative feed‐ back loop that dephosphorylates eIF2α as part of the adaptive response, why would CHOP expression trigger apoptosis as part of the maladaptive response?

There are three principal hypotheses that address this issue. The first proposes that CHOP is a molecular rheostat that drives distinct downstream pathways as a function of expression level [25, 26]. The second suggests that the IRE1 and PERK pathways act in concert to effect cell survival but drive apoptosis when the activities of these pathways are unbalanced [20]. The third hypothesizes that apoptosis is triggered stochastically at a restriction point in the PERK pathway, which is more-or-less coincident with the reinitiation of protein translation upon eIF2α dephosphorylation [17, 27].

#### *3.2.1. CHOP as a rheostat*

are ER-resident proteins that bind to unfolded or misfolded polypeptides [14]. Accordingly, the IRE1α pathway detects changes in protein flux and acts to increase the folding capacity

A second UPR pathway is initiated through activation of the membrane-tethered ATF6 pro‐ tein and converges with IRE1α signaling. ATF6 interacts with BiP, similar to IRE1α. Misfold‐ ed proteins displace BiP from the ATF6 luminal domain and enable the protein to traffic from the ER to the Golgi apparatus where it is cleaved by the site 1 and site 2 proteases (S1P and S2P, respectively). The resulting cytosolic N-terminal fragment of ATF6 is the functional bzip transcription factor that heterodimerizes with XBP1 and induces expression of molecu‐ lar chaperone genes including BiP, glucose-regulated protein 94 (GRP94) and other genes encoding protein folding pathway proteins [15, 19]. ATF6 also upregulates proteins associat‐ ed with the ER Associated Degradation (ERAD) pathway, which is a checkpoint in the ER that ubiquitinates proteins and shuttles them into the cytoplasm for proteasome-mediated degradation [21]. Thus, ATF6 helps to upregulate chaperones to relieve mild protein mis‐ folding, but can also activate degradation of proteins that are severely misfolded and cannot

A third UPR pathway is regulated by an ER-resident receptor known as PERK. The luminal domain of PERK functions analogously to that of IRE1α in binding BiP, and is also activated by dimerization and transautophosphorylation. The cytoplasmic domain of PERK is a pro‐ tein kinase, a major target of which is the alpha subunit of eukaryotic initiation factor 2 (eIF2α). eIF2α is a critical component in ribosome assembly and can be inactivated by phos‐

Despite global translation arrest, a small number of proteins that are critical to the UPR signal‐ ing are actively translated, including the bzip transcription factors, activating transcription fac‐ tor 4 (ATF4), ATF3 and the CCAAT-enhancer-binding protein homologous protein (CHOP) as well as the regulatory subunit of protein phosphatase 1 (PP1), known as growth-arrest and DNA damage protein 34 (GADD34) [17, 22]. The GADD34-PP1 complex is targeted to the ER membrane to dephosphorylate p-eIF2α and reinitiate protein translation. Thus, the PERK path‐ way temporarily halts protein synthesis to suppress additional accumulation of misfolded pro‐ teins in the ER. The pathway subsequently reactivates protein synthesis by opposing the phosphorylation activity of PERK. Thus, because of the time that is necessary to complete each of the steps downstream of eIF2α phosphorylation, the PERK pathway can be considered to be a

A common theme among the three branches of the UPR cascade is the similar activation of the ER-resident receptors by changes in protein flux leading to transcriptional or trans‐

phorylation, which leads to the shut down of global protein synthesis [17].

time-delay circuit that forms a negative feedback loop to regulate UPR signaling.

**3.2. Adaptive and maladaptive facets of UPR signaling**

of the ER, ultimately completing a negative feedback loop on the UPR.

*3.1.2. The ATF6 pathway*

538 Neurodegenerative Diseases

be rescued by chaperones.

*3.1.3. The PERK pathway*

Studies in human embryonic kidney 293 (HEK293) cells utilizing genetic and chemical in‐ duction of the UPR have led to the hypothesis of graded activation, mediated by CHOP, with apoptosis resulting from the highest levels of expression [25, 26]. Transient ER stress requires a UPR; however, the response itself would be modulated so that mild stress generates tapered transient CHOP induction, and severe prolonged stress causes sustained CHOP expression. Indeed, a modulated CHOP response has been observed during molecular and mechanical stress in vitro that activates PERK in the ER, with sus‐ tained PERK activation causing sustained CHOP expression and increased apoptosis. In contrast, oligodendrocytes undergoing severe metabolic stress and widespread apoptosis do not express CHOP, suggesting that its induction is transient even during severe stress [4, 6, 28].

#### *3.2.2. Balanced IRE1 and PERK signaling*

From their in vitro manipulation of the IRE1 and PERK pathways in HEK293 cells, Walter and colleagues [29] identified disparate roles for each pathway that could account for diver‐ gent UPR phenotypes in animal models of disease. The results showed that activating the PERK pathway alone decreased cell proliferation in vitro and triggered a morphological de‐ differentiation characterized by a loss of cell processes. In contrast, unilateral IRE1 activation increased cell numbers. Because activation of the PERK and IRE1 pathways stem from the accumulation of misfolded proteins, it is likely that the relative activation levels of these pathways generates a balance between proliferation and differentiation that determines the fate of the cells.

**4.1. Leukodystrophy and metabolic stress as a model of neurodegenerative disease**

consequences of this disease mechanism beyond the primary cell type involved.

Arguably, PMD is one of the most extensively characterized neurodegenerative UPR disease in terms of molecular and cellular etiology. In virtually all patients, disease stems from ge‐ netic lesions in the *X*-linked *Plp1* gene [44]. The gene products are polytopic membrane pro‐ teins that constitute approximately 50% of the total protein in the CNS myelin sheath and the developmental expression levels of this gene are among the most abundantly expressed

Mutations in the *Plp1* gene arises from three types of genetic lesions: duplications, deletions and missense/nonsense mutations. These lesions confer disease symptoms with a wide range of clinical severity that are mild in the case of deletions, severe in the case of duplica‐ tions and mild or severe for coding region mutations. In general, mild phenotypes are asso‐ ciated with reduced oligodendrocyte function but relatively little cell death while severe

Mild forms of disease caused by deletion of the entire *Plp1* gene or nonsense mutations in exon 1 are characterized by clinical presentation in middle age patients, often in the form of cognitive decline [43, 44] and a length-dependent dying back neuropathology. Although the absence of *Plp1* expression in patients does not significantly reduce oligodendrocyte func‐ tion and the amount of myelin formed during development, the absence of this protein re‐ duces the long-term stability of long myelinated tracts such as the corticospinal tract, which degenerate in later life. Importantly, the stability of the CNS specifically requires the PLP1 protein, and cannot be conferred by the alternatively-spliced PLP1 isoform, called DM-20,

forms cause widespread apoptosis and a virtual absence of white matter [45, 46].

which lacks a 35 amino acid segment in the cytoplasmic domain of the protein [47].

The leukodystrophies are a group of diseases characterized by a systemic absence of white matter in the CNS resulting in sensorimotor deficits, ataxia, hypotonia and eventual decline in cognitive function [36]. Although leukodystrophies affect all white matter tracts to vary‐ ing extents, they differ in their primary causes. For example, in the case of PMD the absence of white matter stems from mutations in the gene encoding the most abundant myelin pro‐ tein, proteolipid protein-1 (PLP1) [37-39], while VWM disease is caused by mutations in genes that encode subunits of the eIF2 complex [40]. In many cases, metabolic stress is se‐ vere enough that the disease develops in childhood and dramatically affects the life span of the patient [6, 38, 39, 41, 42]. The common mechanism between these leukodystrophies is the failure to manage and remove misfolded proteins, some of which rapidly activate the UPR leading to metabolic stress and apoptosis [38, 39]. Importantly, metabolic stress in oligoden‐ drocytes also leads to secondary neuron loss [43], which demonstrates the potentially severe

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**etiology**

*4.1.1. Pelizaeus-Merzbacher disease*

*4.1.1.1. Gene duplications and deletions in PMD*

in mammals.

#### *3.2.3. Stochastic apoptosis*

The third hypothesis stems from the results of several in vivo studies involving CNS and PNS myelin mutant mice [17]. In contrast to in vitro studies in many cell types where CHOP expression drives apoptosis and CHOP loss-of-function promotes cell survival, ablation of the *Chop* gene in oligodendrocytes renders them much more susceptible to apoptosis under UPR conditions [6, 30]. Thus, CHOP promotes cell survival. In addition, induction of CHOP in Schwann cells does not induce cell death but, rather, causes dedifferentiation of these cells to promote their survival [27]. A similar mechanism is also observed in osteoblasts [31]. Together, these and other studies [29, 32] indicate that PERK signaling protects myelinating cells from apoptosis. If so, how do these cells undergo apoptosis?

One possibility is that myelinating cells become vulnerable to apoptosis at a restriction point in the PERK pathway as protein translation is restarted. At this restriction, the PP1-GADD34 complex dephosphorylates eIF2α and demand for ATP, GTP, NADH and other high-energy intermediates would dramatically increase. Sub-threshold levels of these critical molecules, perhaps also exacerbated by dissipation of the mitochondrial membrane potential, would occur stochastically in individual cells during translation-suppression and cause a loss of homeostasis leading to cell death. Under mild metabolic stress conditions, most cells would maintain supra-threshold levels of critical molecules and survive beyond the restriction point. Some of these cells would undergo apoptosis during subsequent UPR-induction cy‐ cles. Ultimately, the stronger the stress, the greater the number of UPR cycles, and the high‐ er the likelihood that cells would undergo apoptosis.

### **4. Oligodendrocyte metabolic stress and neurodegenerative disease**

Oligodendrocytes play a critical role in the CNS by myelinating axons to ensure efficient saltatory conduction and reliable communication between neurons over long distances as well as to promote neuronal survival [33]. The surface area of myelin membrane that is synthesized by each oligodendrocyte within a few days during development exceeds that of the cell body by several hundred fold, which makes oligodendrocytes one of the most metabolically active cell types [34]. Thus, it is not surprising that these cells are vulnera‐ ble to metabolic stress and undergo apoptosis associated with protein misfolding [4, 33, 35]. Genetic diseases that disrupt oligodendrocyte metabolism are associated with UPR signaling and are well characterized at the molecular level. It is also becoming increas‐ ingly clear that other diseases of oligodendrocytes, such as MS, involve this signaling pathway.

#### **4.1. Leukodystrophy and metabolic stress as a model of neurodegenerative disease etiology**

The leukodystrophies are a group of diseases characterized by a systemic absence of white matter in the CNS resulting in sensorimotor deficits, ataxia, hypotonia and eventual decline in cognitive function [36]. Although leukodystrophies affect all white matter tracts to vary‐ ing extents, they differ in their primary causes. For example, in the case of PMD the absence of white matter stems from mutations in the gene encoding the most abundant myelin pro‐ tein, proteolipid protein-1 (PLP1) [37-39], while VWM disease is caused by mutations in genes that encode subunits of the eIF2 complex [40]. In many cases, metabolic stress is se‐ vere enough that the disease develops in childhood and dramatically affects the life span of the patient [6, 38, 39, 41, 42]. The common mechanism between these leukodystrophies is the failure to manage and remove misfolded proteins, some of which rapidly activate the UPR leading to metabolic stress and apoptosis [38, 39]. Importantly, metabolic stress in oligoden‐ drocytes also leads to secondary neuron loss [43], which demonstrates the potentially severe consequences of this disease mechanism beyond the primary cell type involved.

#### *4.1.1. Pelizaeus-Merzbacher disease*

accumulation of misfolded proteins, it is likely that the relative activation levels of these pathways generates a balance between proliferation and differentiation that determines the

The third hypothesis stems from the results of several in vivo studies involving CNS and PNS myelin mutant mice [17]. In contrast to in vitro studies in many cell types where CHOP expression drives apoptosis and CHOP loss-of-function promotes cell survival, ablation of the *Chop* gene in oligodendrocytes renders them much more susceptible to apoptosis under UPR conditions [6, 30]. Thus, CHOP promotes cell survival. In addition, induction of CHOP in Schwann cells does not induce cell death but, rather, causes dedifferentiation of these cells to promote their survival [27]. A similar mechanism is also observed in osteoblasts [31]. Together, these and other studies [29, 32] indicate that PERK signaling protects myelinating

One possibility is that myelinating cells become vulnerable to apoptosis at a restriction point in the PERK pathway as protein translation is restarted. At this restriction, the PP1-GADD34 complex dephosphorylates eIF2α and demand for ATP, GTP, NADH and other high-energy intermediates would dramatically increase. Sub-threshold levels of these critical molecules, perhaps also exacerbated by dissipation of the mitochondrial membrane potential, would occur stochastically in individual cells during translation-suppression and cause a loss of homeostasis leading to cell death. Under mild metabolic stress conditions, most cells would maintain supra-threshold levels of critical molecules and survive beyond the restriction point. Some of these cells would undergo apoptosis during subsequent UPR-induction cy‐ cles. Ultimately, the stronger the stress, the greater the number of UPR cycles, and the high‐

**4. Oligodendrocyte metabolic stress and neurodegenerative disease**

Oligodendrocytes play a critical role in the CNS by myelinating axons to ensure efficient saltatory conduction and reliable communication between neurons over long distances as well as to promote neuronal survival [33]. The surface area of myelin membrane that is synthesized by each oligodendrocyte within a few days during development exceeds that of the cell body by several hundred fold, which makes oligodendrocytes one of the most metabolically active cell types [34]. Thus, it is not surprising that these cells are vulnera‐ ble to metabolic stress and undergo apoptosis associated with protein misfolding [4, 33, 35]. Genetic diseases that disrupt oligodendrocyte metabolism are associated with UPR signaling and are well characterized at the molecular level. It is also becoming increas‐ ingly clear that other diseases of oligodendrocytes, such as MS, involve this signaling

cells from apoptosis. If so, how do these cells undergo apoptosis?

er the likelihood that cells would undergo apoptosis.

fate of the cells.

540 Neurodegenerative Diseases

pathway.

*3.2.3. Stochastic apoptosis*

Arguably, PMD is one of the most extensively characterized neurodegenerative UPR disease in terms of molecular and cellular etiology. In virtually all patients, disease stems from ge‐ netic lesions in the *X*-linked *Plp1* gene [44]. The gene products are polytopic membrane pro‐ teins that constitute approximately 50% of the total protein in the CNS myelin sheath and the developmental expression levels of this gene are among the most abundantly expressed in mammals.

Mutations in the *Plp1* gene arises from three types of genetic lesions: duplications, deletions and missense/nonsense mutations. These lesions confer disease symptoms with a wide range of clinical severity that are mild in the case of deletions, severe in the case of duplica‐ tions and mild or severe for coding region mutations. In general, mild phenotypes are asso‐ ciated with reduced oligodendrocyte function but relatively little cell death while severe forms cause widespread apoptosis and a virtual absence of white matter [45, 46].

#### *4.1.1.1. Gene duplications and deletions in PMD*

Mild forms of disease caused by deletion of the entire *Plp1* gene or nonsense mutations in exon 1 are characterized by clinical presentation in middle age patients, often in the form of cognitive decline [43, 44] and a length-dependent dying back neuropathology. Although the absence of *Plp1* expression in patients does not significantly reduce oligodendrocyte func‐ tion and the amount of myelin formed during development, the absence of this protein re‐ duces the long-term stability of long myelinated tracts such as the corticospinal tract, which degenerate in later life. Importantly, the stability of the CNS specifically requires the PLP1 protein, and cannot be conferred by the alternatively-spliced PLP1 isoform, called DM-20, which lacks a 35 amino acid segment in the cytoplasmic domain of the protein [47].

In contrast, *Plp1* duplications cause severe phenotypes perinatally or within the first year of life. Children and adolescents with duplications exhibit severe cognitive decline in conjunc‐ tion with physical disabilities including loss of motor function and coordination [6, 48]. Be‐ cause of extremely high PLP1 expression levels during normal development, duplications may effectively overwhelm the secretory pathway in oligodendrocytes and disrupt cell func‐ tion or survival. Whether this disruption involves defective cholesterol trafficking [49] or immune activation [50] is currently unclear.

from *rsh* myelin sheaths, but DM-20 is present at normal levels[54]. This selective trafficking defect is consistent with the protein misfolding hypothesis [4, 5, 7, 51]. Oligodendrocyte metabolic stress leading to apoptosis is observed to occur at a moderate level in this strain

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http://dx.doi.org/10.5772/54461

543

The *msd* mutation was originally described by Baumann and colleagues [65] and is charac‐ terized by an alanine to valine substitution at codon 243 (A243V) in PLP1 [66]. Mice harbor‐ ing this mutation exhibit severe symptoms, with behavioral changes evident by P13 and a short life span of 3 - 4 weeks. The amount of myelin in the *msd* CNS is severely reduced to approximately 5% of normal and the phenotype is very similar to that of *jimpy* mice on the same genetic background [62, 67]. Oligodendrocyte apoptosis is widespread in these mice and, similar to *rsh* mice, involves metabolic stress and activation of the UPR [4, 6, 12]. PMD patients with the corresponding mutation have a severe form of the disease with systemic

Importantly, some patients with severe forms of PMD such as those corresponding to the *msd* mutation also show signs of neuron loss as a consequence of profound hypomyelina‐ tion. These observations establish the principle that the survival of each of the major neural cell types is interdependent; thus, a primary insult in oligodendrocytes in the form of meta‐ bolic stress has secondary consequences for neurons [4, 6, 37]. Furthermore, symptoms in PMD can include autoimmune disease [68], which has major significance for the classifica‐ tion of MS as a neurodegenerative disease and suggests that the etiology may arise, at least in some instances, from primary metabolic stress in oligodendrocytes leading to secondary

Multiple sclerosis is the most common neurological disease in young adults worldwide and is typically described as an autoimmune attack on CNS white matter tracts resulting in focal lesions and degeneration of myelin throughout the CNS [69]. There are three major forms of this disease, relapse remitting MS (RRMS), secondary progressive MS (SPMS) and primary progressive MS (PPMS). RRMS is the most prevalent form and is characterized by patients for which lesions develop spontaneously and cause transient loss of neurological function (also known as a relapse) followed by essentially full recovery (known as remission). Dis‐ ease in RRMS patients eventually transitions from these transient symptoms to SPMS, when patients do not fully recover neurological function after relapses and sensorimotor deficits become more continuous and progressive. PPMS defines the third category, which is clini‐ cally similar to SPMS but without a preceding RRMS phase. Thus, patients experience rapid

Results from recent long-term clinical trials in RRMS patients that were medicated with any of several new immune suppressant therapies demonstrate that dramatic reductions in the number of new demyelinating lesions is accompanied by only modest amelioration of clini‐

**4.2. Is MS a neurodegenerative metabolic stress disease of oligodendrocytes?**

[6, 28] despite early claims to the contrary [64].

demyelination and widespread oligodendrocyte death.

*4.1.2.2. Severe disease in msd mice*

immune activation.

severe degeneration [69-71].

#### *4.1.1.2. Plp1 mutations in PMD*

Approximately 30% of PMD patients harbor mutations in the *Plp1* coding region that cause missense or nonsense changes in the protein primary structure. These changes arise throughout the coding region and cause a spectrum of disease severities in patients [43, 44]. Although there does not appear to be a correlation between the location of a mutation and disease severity, most mutations in the transmembrane domains cause severe disease. This is a general feature of membrane domain mutations in many secretory pathway proteins. Accordingly, the underlying cell biology of coding region mutations is proposed to stem from a failure of protein folding and trafficking through the secretory pathway, leading to metabolic stress and activation of the UPR [6, 12, 43, 44, 51]. Two missense mutations in PMD patients have also been identified in mice. Although similarities of disease symptoms and pathology conferred by each mutation might be anticipated because the PLP1 primary structure is identical in rodents and humans, the robustness of these findings provides a strong basis for using the animal models to model PMD [4, 52-55].

#### *4.1.2. Animal models of PMD*

A common goal in the analysis and development of therapeutic strategies to treat many neu‐ rodegenerative diseases is the generation of animal models, particularly in rodents which are amenable to genetic manipulation. Naturally-occurring animal models of PMD have been described in multiple species including dog, rabbit, rat and mouse [56-59], and engi‐ neered mutations have been generated in rats and mice [47, 52, 58, 60, 61].

The *jimpy* mouse, which exhibits a severe behavioral phenotype, is the original *Plp1* allele identified and has been characterized in greatest detail [57]. More recently, the *rumpshaker* (*rsh*) and *myelin synthesis-deficient* (*msd*) alleles have become popular not only because they exhibit mild and severe phenotypes, respectively, but also because the specific single amino acid changes harbored by these strains are also found in humans [54, 62].

#### *4.1.2.1. Mild disease in rsh mice*

This allele was originally described by Griffiths and colleagues and harbors an isoleucine to threonine mutation at codon 187 (I187T) in the second extracellular domain of PLP1 [54, 63]. These mice are fertile and exhibit a normal life span with behavioral changes becoming evi‐ dent between 13 – 19 days after birth (P13 – 19), depending on the background strain of the colony. The total myelin content of the brain is reduced to 40–50%. PLP1 is virtually absent from *rsh* myelin sheaths, but DM-20 is present at normal levels[54]. This selective trafficking defect is consistent with the protein misfolding hypothesis [4, 5, 7, 51]. Oligodendrocyte metabolic stress leading to apoptosis is observed to occur at a moderate level in this strain [6, 28] despite early claims to the contrary [64].

#### *4.1.2.2. Severe disease in msd mice*

In contrast, *Plp1* duplications cause severe phenotypes perinatally or within the first year of life. Children and adolescents with duplications exhibit severe cognitive decline in conjunc‐ tion with physical disabilities including loss of motor function and coordination [6, 48]. Be‐ cause of extremely high PLP1 expression levels during normal development, duplications may effectively overwhelm the secretory pathway in oligodendrocytes and disrupt cell func‐ tion or survival. Whether this disruption involves defective cholesterol trafficking [49] or

Approximately 30% of PMD patients harbor mutations in the *Plp1* coding region that cause missense or nonsense changes in the protein primary structure. These changes arise throughout the coding region and cause a spectrum of disease severities in patients [43, 44]. Although there does not appear to be a correlation between the location of a mutation and disease severity, most mutations in the transmembrane domains cause severe disease. This is a general feature of membrane domain mutations in many secretory pathway proteins. Accordingly, the underlying cell biology of coding region mutations is proposed to stem from a failure of protein folding and trafficking through the secretory pathway, leading to metabolic stress and activation of the UPR [6, 12, 43, 44, 51]. Two missense mutations in PMD patients have also been identified in mice. Although similarities of disease symptoms and pathology conferred by each mutation might be anticipated because the PLP1 primary structure is identical in rodents and humans, the robustness of these findings provides a

A common goal in the analysis and development of therapeutic strategies to treat many neu‐ rodegenerative diseases is the generation of animal models, particularly in rodents which are amenable to genetic manipulation. Naturally-occurring animal models of PMD have been described in multiple species including dog, rabbit, rat and mouse [56-59], and engi‐

The *jimpy* mouse, which exhibits a severe behavioral phenotype, is the original *Plp1* allele identified and has been characterized in greatest detail [57]. More recently, the *rumpshaker* (*rsh*) and *myelin synthesis-deficient* (*msd*) alleles have become popular not only because they exhibit mild and severe phenotypes, respectively, but also because the specific single amino

This allele was originally described by Griffiths and colleagues and harbors an isoleucine to threonine mutation at codon 187 (I187T) in the second extracellular domain of PLP1 [54, 63]. These mice are fertile and exhibit a normal life span with behavioral changes becoming evi‐ dent between 13 – 19 days after birth (P13 – 19), depending on the background strain of the colony. The total myelin content of the brain is reduced to 40–50%. PLP1 is virtually absent

strong basis for using the animal models to model PMD [4, 52-55].

neered mutations have been generated in rats and mice [47, 52, 58, 60, 61].

acid changes harbored by these strains are also found in humans [54, 62].

immune activation [50] is currently unclear.

*4.1.1.2. Plp1 mutations in PMD*

542 Neurodegenerative Diseases

*4.1.2. Animal models of PMD*

*4.1.2.1. Mild disease in rsh mice*

The *msd* mutation was originally described by Baumann and colleagues [65] and is charac‐ terized by an alanine to valine substitution at codon 243 (A243V) in PLP1 [66]. Mice harbor‐ ing this mutation exhibit severe symptoms, with behavioral changes evident by P13 and a short life span of 3 - 4 weeks. The amount of myelin in the *msd* CNS is severely reduced to approximately 5% of normal and the phenotype is very similar to that of *jimpy* mice on the same genetic background [62, 67]. Oligodendrocyte apoptosis is widespread in these mice and, similar to *rsh* mice, involves metabolic stress and activation of the UPR [4, 6, 12]. PMD patients with the corresponding mutation have a severe form of the disease with systemic demyelination and widespread oligodendrocyte death.

Importantly, some patients with severe forms of PMD such as those corresponding to the *msd* mutation also show signs of neuron loss as a consequence of profound hypomyelina‐ tion. These observations establish the principle that the survival of each of the major neural cell types is interdependent; thus, a primary insult in oligodendrocytes in the form of meta‐ bolic stress has secondary consequences for neurons [4, 6, 37]. Furthermore, symptoms in PMD can include autoimmune disease [68], which has major significance for the classifica‐ tion of MS as a neurodegenerative disease and suggests that the etiology may arise, at least in some instances, from primary metabolic stress in oligodendrocytes leading to secondary immune activation.

#### **4.2. Is MS a neurodegenerative metabolic stress disease of oligodendrocytes?**

Multiple sclerosis is the most common neurological disease in young adults worldwide and is typically described as an autoimmune attack on CNS white matter tracts resulting in focal lesions and degeneration of myelin throughout the CNS [69]. There are three major forms of this disease, relapse remitting MS (RRMS), secondary progressive MS (SPMS) and primary progressive MS (PPMS). RRMS is the most prevalent form and is characterized by patients for which lesions develop spontaneously and cause transient loss of neurological function (also known as a relapse) followed by essentially full recovery (known as remission). Dis‐ ease in RRMS patients eventually transitions from these transient symptoms to SPMS, when patients do not fully recover neurological function after relapses and sensorimotor deficits become more continuous and progressive. PPMS defines the third category, which is clini‐ cally similar to SPMS but without a preceding RRMS phase. Thus, patients experience rapid severe degeneration [69-71].

Results from recent long-term clinical trials in RRMS patients that were medicated with any of several new immune suppressant therapies demonstrate that dramatic reductions in the number of new demyelinating lesions is accompanied by only modest amelioration of clini‐ cal symptoms [72-75]. Moreover, patients continue to experience disease progression. These data indicate that the number of autoimmune attacks on the CNS is not strongly correlated with increasing disease severity and that there may be additional unknown mechanisms in‐ volved in the pathogenesis. If so, then immune attacks may actually be secondary to an un‐ derlying primary etiology.

proinflammatory cytokine release [79, 91-93]. However, these models have significant shortcomings in modeling MS pathology. For example, the neurological phenotypes in affected mice largely stems from tissue edema rather than demyelination. Although some models generate immune mediated demyelination, symptoms are monophasic rather than multiphasic and relapsing-remitting, in contrast to the most common form of MS [94]. Finally, in the absence of gray matter lesions and subsequent neuronal degenera‐ tion, these models fail to recapitulate the most debilitating features of MS that contribute

Oligodendrocyte Metabolic Stress in Neurodegeneration

http://dx.doi.org/10.5772/54461

545

To overcome such shortcomings, we have developed a novel genetic mouse model of MS pathology that is based on primary metabolic stress in oligodendrocytes [95]. The etiology of disease in these mice has been characterized in mechanistic detail [4-7, 51] and we are cur‐ rently determining if we can recapitulate the degenerative white and gray matter lesions that arise in MS patients without specifically provoking the immune system to attack the CNS. Furthermore, we are determining if our primary insult in oligodendrocytes can secon‐ darily induce a relapsing-remitting or progressive autoimmune phenotype in the mice that would account for the pathophysiology observed in MS patients in terms of metabolic stress

**5. Identifying metabolic stress for the diagnosis of neurodegenerative**

For many neurodegenerative diseases, progress toward finding treatments and cures is painstakingly slow. This is in part limited by current capabilities for real-time imaging of the CNS as well as by ethical constraints that protect the health of patients and often exclude invasive procedures such as biopsies. These limitations largely confine research studies to post-mortem tissue, or generating in vitro and in vivo animal models, to develop treatments for disease. In many cases, these approaches have proved only partially effective for the

Recently, several imaging technologies have advanced significantly and become sufficiently widespread in hospitals for routine application to neurodegenerative diseases like AD and MS, including magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS),

Magnetic resonance techniques are widely used in clinical diagnostics of many diseases since their development approximately 40 years ago [86, 101]. Nevertheless, there are signif‐ icant drawbacks for their use in neurodegenerative diseases, particularly with respect to ear‐ ly disease detection [69, 102]. MRI is the most common technique used, and is particularly important for identifying white matter pathology such as hypomyelination or demyelinat‐ ing lesions, as well as gray matter degeneration, because it can easily detect differences in

to the declining quality of life for patients [77].

rather than primary autoimmune activation.

study of neurodegenerative diseases [79, 94, 96, 97].

and positron emission tomography (PET) [98-100].

**5.1. Magnetic resonance imaging**

**diseases**

Clues about the nature of such an unknown etiology in MS are scarce, but may be found in the clinical literature. For example, a few case reports detailing the misdiagnosis of PMD as childhood MS indicate that the symptoms of these two diseases overlap significantly. In‐ deed, the responsiveness of one of these patients to steroids suggests that PMD symptoms can be exacerbated by immune system activation at some level and perhaps similar to MS. Together, these reports provide tantalizing, if anecdotal, evidence that metabolic stress in oligodendrocytes could be one form of a primary etiology that secondarily activates the im‐ mune system [76-78].

#### *4.2.1. Neurodegeneration in MS*

The immune demyelinating lesion in white matter is an important component of MS pathol‐ ogy that has been studied extensively [76, 79-81]. However, a plethora of the clinical symp‐ toms, particularly those affecting the daily activities of patients and significantly reducing their quality of life, stem from axonal transection and loss of neurons in gray matter regions [82]. The significance of this degenerative feature is that emergent immune suppressive therapies might not be expected to have a major impact in halting symptom progression [81, 83, 84]. Cognitive decline, memory loss, partial paralysis, and optic neuritis are caused by the loss of neurons in different brain regions that are spared from direct immune attacks but still contribute to disease, especially for the more severe SPMS and PPMS forms [78, 85].

Gray matter cortical atrophy may constitute the majority of the total tissue atrophy observed in MS patients, especially those with SPMS and PPMS [86-88]. Although this pathological feature has been known for decades, one of the most important advances contributing to our understanding and acceptance of neuron loss as a major, if not the principal, symptom of MS is the increasing sensitivity for detecting gray matter lesions using clinical diagnostic MRIs. Thus, with renewed interest and appreciation for this issue, there is an urgent need to understand the underlying pathogenesis. In this regard, the development of novel animal models will lead to new hypotheses and the development of novel therapeutic strategies.

#### *4.2.2. Current and future MS models*

Because of the characterization of MS as a primary autoimmune disease, a large propor‐ tion of animal model studies, particularly in mice, have focused on developing and char‐ acterizing immune models such as experimental autoimmune encephalomyelitis (EAE) [79, 81, 89, 90]. These models rely on priming the peripheral immune system with inject‐ ed peptides from various myelin proteins to stimulate the immune system to attack and demyelinate white matter tracts. Damage is largely confined to spinal cord and is charac‐ terized by immune cell profiles of CD4+ , CD8+ T-cells and CD68+ macrophages as well as proinflammatory cytokine release [79, 91-93]. However, these models have significant shortcomings in modeling MS pathology. For example, the neurological phenotypes in affected mice largely stems from tissue edema rather than demyelination. Although some models generate immune mediated demyelination, symptoms are monophasic rather than multiphasic and relapsing-remitting, in contrast to the most common form of MS [94]. Finally, in the absence of gray matter lesions and subsequent neuronal degenera‐ tion, these models fail to recapitulate the most debilitating features of MS that contribute to the declining quality of life for patients [77].

To overcome such shortcomings, we have developed a novel genetic mouse model of MS pathology that is based on primary metabolic stress in oligodendrocytes [95]. The etiology of disease in these mice has been characterized in mechanistic detail [4-7, 51] and we are cur‐ rently determining if we can recapitulate the degenerative white and gray matter lesions that arise in MS patients without specifically provoking the immune system to attack the CNS. Furthermore, we are determining if our primary insult in oligodendrocytes can secon‐ darily induce a relapsing-remitting or progressive autoimmune phenotype in the mice that would account for the pathophysiology observed in MS patients in terms of metabolic stress rather than primary autoimmune activation.

### **5. Identifying metabolic stress for the diagnosis of neurodegenerative diseases**

For many neurodegenerative diseases, progress toward finding treatments and cures is painstakingly slow. This is in part limited by current capabilities for real-time imaging of the CNS as well as by ethical constraints that protect the health of patients and often exclude invasive procedures such as biopsies. These limitations largely confine research studies to post-mortem tissue, or generating in vitro and in vivo animal models, to develop treatments for disease. In many cases, these approaches have proved only partially effective for the study of neurodegenerative diseases [79, 94, 96, 97].

Recently, several imaging technologies have advanced significantly and become sufficiently widespread in hospitals for routine application to neurodegenerative diseases like AD and MS, including magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS), and positron emission tomography (PET) [98-100].

#### **5.1. Magnetic resonance imaging**

cal symptoms [72-75]. Moreover, patients continue to experience disease progression. These data indicate that the number of autoimmune attacks on the CNS is not strongly correlated with increasing disease severity and that there may be additional unknown mechanisms in‐ volved in the pathogenesis. If so, then immune attacks may actually be secondary to an un‐

Clues about the nature of such an unknown etiology in MS are scarce, but may be found in the clinical literature. For example, a few case reports detailing the misdiagnosis of PMD as childhood MS indicate that the symptoms of these two diseases overlap significantly. In‐ deed, the responsiveness of one of these patients to steroids suggests that PMD symptoms can be exacerbated by immune system activation at some level and perhaps similar to MS. Together, these reports provide tantalizing, if anecdotal, evidence that metabolic stress in oligodendrocytes could be one form of a primary etiology that secondarily activates the im‐

The immune demyelinating lesion in white matter is an important component of MS pathol‐ ogy that has been studied extensively [76, 79-81]. However, a plethora of the clinical symp‐ toms, particularly those affecting the daily activities of patients and significantly reducing their quality of life, stem from axonal transection and loss of neurons in gray matter regions [82]. The significance of this degenerative feature is that emergent immune suppressive therapies might not be expected to have a major impact in halting symptom progression [81, 83, 84]. Cognitive decline, memory loss, partial paralysis, and optic neuritis are caused by the loss of neurons in different brain regions that are spared from direct immune attacks but still contribute to disease, especially for the more severe SPMS and PPMS forms [78, 85].

Gray matter cortical atrophy may constitute the majority of the total tissue atrophy observed in MS patients, especially those with SPMS and PPMS [86-88]. Although this pathological feature has been known for decades, one of the most important advances contributing to our understanding and acceptance of neuron loss as a major, if not the principal, symptom of MS is the increasing sensitivity for detecting gray matter lesions using clinical diagnostic MRIs. Thus, with renewed interest and appreciation for this issue, there is an urgent need to understand the underlying pathogenesis. In this regard, the development of novel animal models will lead to new hypotheses and the development of novel therapeutic strategies.

Because of the characterization of MS as a primary autoimmune disease, a large propor‐ tion of animal model studies, particularly in mice, have focused on developing and char‐ acterizing immune models such as experimental autoimmune encephalomyelitis (EAE) [79, 81, 89, 90]. These models rely on priming the peripheral immune system with inject‐ ed peptides from various myelin proteins to stimulate the immune system to attack and demyelinate white matter tracts. Damage is largely confined to spinal cord and is charac‐

, CD8+ T-cells and CD68+ macrophages as well as

derlying primary etiology.

544 Neurodegenerative Diseases

mune system [76-78].

*4.2.1. Neurodegeneration in MS*

*4.2.2. Current and future MS models*

terized by immune cell profiles of CD4+

Magnetic resonance techniques are widely used in clinical diagnostics of many diseases since their development approximately 40 years ago [86, 101]. Nevertheless, there are signif‐ icant drawbacks for their use in neurodegenerative diseases, particularly with respect to ear‐ ly disease detection [69, 102]. MRI is the most common technique used, and is particularly important for identifying white matter pathology such as hypomyelination or demyelinat‐ ing lesions, as well as gray matter degeneration, because it can easily detect differences in tissue structure or composition between normal and diseased regions. Applications to ex‐ pand the utility of this technique beyond the structural realm include injectable biomarkers to detect subclinical disease or to follow the evolution of lesions in real time, but these are currently nonexistent except for animal studies [103].

The lessons learned from PMD and other neurodegenerative diseases could be relevant to MS and may help to explain why this disease is recalcitrant to treatments that only target the immunological aspects of the pathophysiology. Thus, considering MS as a gain-of-func‐ tion disease with an underlying condition of unknown etiology that is exacerbated by auto‐ immune activation may shed new light on the pathophysiology and lead to novel

Oligodendrocyte Metabolic Stress in Neurodegeneration

http://dx.doi.org/10.5772/54461

547

Although neurodegenerative diseases have typically been defined as a disparate group of diseases involving neurons, there is clear evidence in the clinical and basic science literature against such a narrow viewpoint. For example, diseases of the white matter such as PMD and MS arise from primary insults to oligodendrocytes and cause neuron loss in gray matter and lead to behavioral changes and memory loss. This reflects a broader consideration that all major cell types of the CNS are interdependent and degenerative changes in one of these

In similar vein, the fundamental belief by immunologists that MS is a primary autoimmune disease is no longer tenable. Clear evidence from large clinical trials demonstrates that the elimination of adaptive immune cells from the CNS by various forms of immune suppres‐ sion does not halt the progression of disease at early or late stages. The simplest interpreta‐ tion of these data is that there is an underlying etiology that is poorly understood and must be recognized. In light of overlapping symptoms between PMD and MS, it is plausible that metabolic stress could play a primary role in oligodendrocyte degeneration with secondary activation of immune cells. Indeed, several studies have demonstrated induction of the UPR

UPR (unfolded protein response), IRE (inositol requiring protein), PERK (pancreatic endo‐ plasmic reticulum kinase), ATF (activating transcription factor), XBP (X-box binding pro‐ tein), CHOP (CCAAT/-enhancer binding protein homologous protein), PLP1 (proteolipid protein-1), GADD34 (growth arrest and DNA damage protein 34), BiP (chaperone protein), eIF2α (eukaryotic initiation factor 2 α), PP1 (protein phosphatase 1), GRP94 (glucose-regu‐ lated protein 94), S1P (site 1 protease), S2P (site 2 protease), ERAD (endoplasmic reticulum associated degradation), ataxia (lack of voluntary muscle coordination), hypotonia (low muscle tone), RRMS (relapse-remitting Multiple Sclerosis), SPMS (secondary progressive Multiple Sclerosis), PPMS (primary progressive Multiple Sclerosis), CD4,8,68 (cluster of dif‐ ferentiation, immune cell specific glycoproteins), *rumpshaker* mutation (*rsh*), *myelin-synthesis deficient* mutation (*msd*), MRI (magnetic resonance imaging), MRS (magnetic resonance spec‐

therapeutic strategies to ameliorate the symptoms [70, 108, 109].

will lead to loss of at least some of the other cell types.

troscopy), PET (positron emission tomography).

**7. Conclusion**

in MS tissue.

**Nomenclature**

The imaging of metabolic changes in structurally normal regions of the CNS can be achieved using MRS [100, 104], but this technology is currently limited to a few major neurochemicals at low resolution. MRS can be used to detect neuron cell loss by monitoring levels of the neurochemical, N-acetyl aspartate (NAA), which is specific to this cell type [99]. However, the time, expense and difficulty of scanning more than one region of the CNS at a time se‐ verely limits the use of MRS for early disease detection when clinicians are uncertain about the specific location of lesions.

#### **5.2. Positron emission tomography**

Positron emission tomography involves the incorporation of radioactive molecules into me‐ tabolites that are selectively taken up by defined cell populations so that their location and metabolic activity can be analyzed [105]. This technique has the potential to generate de‐ tailed information about the molecular basis of neurodegeneration because the metabolism of affected cells changes dramatically as they lose homeostasis. PET has been used success‐ fully in diseases such as AD and PD where degeneration of specific neuronal populations can be monitored *in vivo* even before patients experience significant symptoms [106]. How‐ ever, a significant drawback with this technique is its low resolution, which renders the technique very limited for small animal model studies.

### **6. Treatments for metabolic stress in neurodegenerative diseases**

Increasing awareness and more sophisticated technologies have enabled earlier detection of neurodegenerative processes. However, the development of treatment strategies often has been hampered, in large measure because of the enormous plasticity of the CNS which enables neuronal circuits to compensate for ongoing damage and cell loss. Thus, these diseases only become clinically apparent at advanced stages when damage is wide‐ spread and irreparable.

The treatment of neurodegenerative diseases is also hampered by the fact that a number of these diseases stem from toxic gain-of-function, rather than loss-of-function, phenotypes. For example, deletion of the *Plp1* gene is the most mild form of PMD; thus, the loss of the protein in myelin does not confer a strong phenotype. However, mutations that cause PLP1 to misfold are toxic to oligodendrocytes because of the extremely rapid accumulation of the intermediates in the ER, which overwhelm the capacity of the UPR to eliminate them through the ubiquitin-proteasome system [107]. Therapeutic strategies to insert a wild type *Plp1* allele into these patients would fail unless the toxic protein from the mutant allele were also eliminated.

The lessons learned from PMD and other neurodegenerative diseases could be relevant to MS and may help to explain why this disease is recalcitrant to treatments that only target the immunological aspects of the pathophysiology. Thus, considering MS as a gain-of-func‐ tion disease with an underlying condition of unknown etiology that is exacerbated by auto‐ immune activation may shed new light on the pathophysiology and lead to novel therapeutic strategies to ameliorate the symptoms [70, 108, 109].

### **7. Conclusion**

tissue structure or composition between normal and diseased regions. Applications to ex‐ pand the utility of this technique beyond the structural realm include injectable biomarkers to detect subclinical disease or to follow the evolution of lesions in real time, but these are

The imaging of metabolic changes in structurally normal regions of the CNS can be achieved using MRS [100, 104], but this technology is currently limited to a few major neurochemicals at low resolution. MRS can be used to detect neuron cell loss by monitoring levels of the neurochemical, N-acetyl aspartate (NAA), which is specific to this cell type [99]. However, the time, expense and difficulty of scanning more than one region of the CNS at a time se‐ verely limits the use of MRS for early disease detection when clinicians are uncertain about

Positron emission tomography involves the incorporation of radioactive molecules into me‐ tabolites that are selectively taken up by defined cell populations so that their location and metabolic activity can be analyzed [105]. This technique has the potential to generate de‐ tailed information about the molecular basis of neurodegeneration because the metabolism of affected cells changes dramatically as they lose homeostasis. PET has been used success‐ fully in diseases such as AD and PD where degeneration of specific neuronal populations can be monitored *in vivo* even before patients experience significant symptoms [106]. How‐ ever, a significant drawback with this technique is its low resolution, which renders the

**6. Treatments for metabolic stress in neurodegenerative diseases**

Increasing awareness and more sophisticated technologies have enabled earlier detection of neurodegenerative processes. However, the development of treatment strategies often has been hampered, in large measure because of the enormous plasticity of the CNS which enables neuronal circuits to compensate for ongoing damage and cell loss. Thus, these diseases only become clinically apparent at advanced stages when damage is wide‐

The treatment of neurodegenerative diseases is also hampered by the fact that a number of these diseases stem from toxic gain-of-function, rather than loss-of-function, phenotypes. For example, deletion of the *Plp1* gene is the most mild form of PMD; thus, the loss of the protein in myelin does not confer a strong phenotype. However, mutations that cause PLP1 to misfold are toxic to oligodendrocytes because of the extremely rapid accumulation of the intermediates in the ER, which overwhelm the capacity of the UPR to eliminate them through the ubiquitin-proteasome system [107]. Therapeutic strategies to insert a wild type *Plp1* allele into these patients would fail unless the toxic protein from the mutant allele were

currently nonexistent except for animal studies [103].

technique very limited for small animal model studies.

the specific location of lesions.

546 Neurodegenerative Diseases

spread and irreparable.

also eliminated.

**5.2. Positron emission tomography**

Although neurodegenerative diseases have typically been defined as a disparate group of diseases involving neurons, there is clear evidence in the clinical and basic science literature against such a narrow viewpoint. For example, diseases of the white matter such as PMD and MS arise from primary insults to oligodendrocytes and cause neuron loss in gray matter and lead to behavioral changes and memory loss. This reflects a broader consideration that all major cell types of the CNS are interdependent and degenerative changes in one of these will lead to loss of at least some of the other cell types.

In similar vein, the fundamental belief by immunologists that MS is a primary autoimmune disease is no longer tenable. Clear evidence from large clinical trials demonstrates that the elimination of adaptive immune cells from the CNS by various forms of immune suppres‐ sion does not halt the progression of disease at early or late stages. The simplest interpreta‐ tion of these data is that there is an underlying etiology that is poorly understood and must be recognized. In light of overlapping symptoms between PMD and MS, it is plausible that metabolic stress could play a primary role in oligodendrocyte degeneration with secondary activation of immune cells. Indeed, several studies have demonstrated induction of the UPR in MS tissue.

### **Nomenclature**

UPR (unfolded protein response), IRE (inositol requiring protein), PERK (pancreatic endo‐ plasmic reticulum kinase), ATF (activating transcription factor), XBP (X-box binding pro‐ tein), CHOP (CCAAT/-enhancer binding protein homologous protein), PLP1 (proteolipid protein-1), GADD34 (growth arrest and DNA damage protein 34), BiP (chaperone protein), eIF2α (eukaryotic initiation factor 2 α), PP1 (protein phosphatase 1), GRP94 (glucose-regu‐ lated protein 94), S1P (site 1 protease), S2P (site 2 protease), ERAD (endoplasmic reticulum associated degradation), ataxia (lack of voluntary muscle coordination), hypotonia (low muscle tone), RRMS (relapse-remitting Multiple Sclerosis), SPMS (secondary progressive Multiple Sclerosis), PPMS (primary progressive Multiple Sclerosis), CD4,8,68 (cluster of dif‐ ferentiation, immune cell specific glycoproteins), *rumpshaker* mutation (*rsh*), *myelin-synthesis deficient* mutation (*msd*), MRI (magnetic resonance imaging), MRS (magnetic resonance spec‐ troscopy), PET (positron emission tomography).

### **Acknowledgements**

This work was supported by grants to A.G. from the National Institutes of Health, NINDS, NS067157 and the National Multiple Sclerosis Society, RG4078 and RG4639.

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### **Author details**

Daniel Radecki1 and Alexander Gow1,2,3\*

\*Address all correspondence to: agow@med.wayne.edu

1 Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI, USA

2 Carman and Ann Adams Department of Pediatrics, Wayne State University School of Medicine, Detroit, MI, USA

3 Department of Neurology, Wayne State University School of Medicine, Detroit, MI, USA

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**Acknowledgements**

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**Author details**

Daniel Radecki1

Detroit, MI, USA

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2 Carman and Ann Adams Department of Pediatrics, Wayne State University School of

3 Department of Neurology, Wayne State University School of Medicine, Detroit, MI, USA

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**Section 4**

**Miscellaneous**

**Section 4**

### **Miscellaneous**

**Chapter 23**

**Spinal Muscular Atrophy: Classification,**

**and Development of Therapeutics**

Faraz Tariq Farooq, Martin Holcik and

Additional information is available at the end of the chapter

Alex MacKenzie

**1. Introduction**

of SMA.

**2. Epidemiology**

of infant death globally [1-5].

http://dx.doi.org/10.5772/53800

**Diagnosis, Background, Molecular Mechanism**

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease and one of the most common genetic causes of infant death. The loss or mutation of the SMN1 gene results in reduced SMN protein level leading to motor neuron death and progressive muscle atrophy. Although recent progress has been made in our understanding of the molecular mechanisms underlying the pathogenesis of the disease, there is currently no cure for SMA. In this review, we summarize the clinical manifestations, molecular pathogenesis, diagnostic strategy and development of therapeutic regimes for the better understanding and treatment

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder character‐ ized by the loss of motor neurons from the anterior horn of the spinal cord which leads to muscle weakness, hypotonia and ultimately muscle atrophy [1]. With a pan ethnic incidence of 1:11,000 live births and a carrier frequency of 1:50, SMA is one of the leading genetic causes

> © 2013 Farooq et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.
