**Alzheimer Disease: The Role of Aβ in the Glutamatergic System**

Victoria Campos-Peña and Marco Antonio Meraz-Ríos

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/57367

## **1. Introduction**

The beta amyloid hypothesis is the most accepted theory explaining the pathophysiology of Alzheimer's disease (AD). In general terms, it is known that AD is characterized by a chronic and progressive neurodegenerative process involving the intracellular and extracellular accumulation of fibrillary proteins. The presence of these aggregates leads to synaptic and neuronal loss observed in Alzheimer's patients. Although the precise etiology of AD is unknown, the main risk factor is advanced age. It is also known that a small proportion of AD patients have an autosomal dominant inheritance pattern in three genes – amyloid precursor protein (AβPP), presenilin 1 (PS1) and presenilin 2 (PS2) [1-6]. The presence of specific mutations in these genes leads to the premature development of the disease, known as Early Onset Alzheimer's Disease (EOAD) or Familial Alzheimer's Disease (FAD). The most common mutations are located in the presenilin genes [1, 7-9], mainly in PS1. Currently more than 185 mutations in PS1 have been reported, with only 13 mutations in PS2. While these mutations are located along the length of the protein sequence, the majority is found in the transmem‐ brane area, and affects protein function. To date, approximately 36 different missense muta‐ tions in the APP gene have been identified in 85 families and are located near sites that are recognized by alpha, beta and gamma secretases, thus affecting protein processing and increasing the production of amyloid peptides [10]. The presence of these mutations is a causal factor in the development of AD, and, although they are all related to the disruption of the normal functioning of proteins and an increased formation of beta amyloid, together they are present in less than 10% of all Alzheimer's cases, suggesting that there are many other nongenetic factors involved in the development of the pathology. The remaining 90% of AD cases are known as Sporadic Alzheimer's Disease or Late Onset Alzheimer's disease (LOAD). These

© 2014 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.

patients also exhibit genetic risk factors, such as the presence of allele 4 of the Apolipoprotein E (Apoε4), where individuals with one copy of ε4 allele are three times more at risk of developing the disease, while those with two copies (ε4/ε4) are 10–15 times more likely to develop AD [11-14]. While other non-genetic factors are head trauma, hypertension, athero‐ sclerosis, metabolic disorders such as hypercholesterolemia, obesity and diabetes [15-17], the main risk factor is age. It has been reported that the incidence of the disease increases by 5% in people over 65 and 20% in those over 80. Other factors have also been associated with the development of the disease, such as female gender, smoking, educational level, and a low level of physical and mental activity during the early stages of life.

of cognitive impairment. Also has been observed to inhibit the normal critical neuron func‐ tions, such as long-term potentiation (LTP)[30]. The amyloid βpeptide, also increases Tau phosphorylation [33-35], oxidative stress and altered homeostasis of Ca2+ [36-37] and excito‐ toxicity [38]. It has been documented that these oligomeric forms of Aβ interact with receptors from the glutamatergic system such as the NMDA-receptors, which are responsible for

Alzheimer Disease: The Role of Aβ in the Glutamatergic System

http://dx.doi.org/10.5772/57367

287

Aβ comes from the normal proteolytic processing of AβPP, a type 1 transmembrane glyco‐ protein [25] whose gene is located on chromosome 21 [41-42]. AβPP processing and the "efficiency" of Aβ formation could be affected by the subcellular localization of the protein. AβPP is synthesized in the endoplasmic reticulum (ER) and transported through the constit‐ utive secretory pathway, and only a small fraction of this protein (10%) goes to the plasma membrane. All AβPP isoforms undergo posttranslational modifications involving N and Oglycosylation, phosphorylation and sulfation. Aβpeptide formation is carried out by the action of β- and γ-secretase, in which the peptides formed vary from 39 to 43 amino-acid residues (Aβ39, 40, 42, 43). Although Aβ40 is the most abundant, Aβ42 is the most hydrophobic and is found in a greater proportion of the NPs observed in AD patients [43]. These peptides are continuously released into the extracellular space at possibly low concentrations, and, in soluble form, could carry out normal physiological functions in the cell including those related

According to the amyloid hypothesis, the Aβ accumulation in the patient's brain is the key event that leads to the development of the disease, while other pathological findings (NFT formation and neuronal death) are secondary events occurring after the amyloid aggregation. Most of the studies reported in the literature have focused on the toxicity and neuronal death induced by the presence of amyloid aggregates. However, in recent years a great importance has been attached to the role of these peptides as responsible in the etiology of synaptic dysfunction[40]. In this sense, it has been widely documented that the presence of soluble oligomeric forms of Aβ responsible for synaptic damage and neurodegeneration [29, 45-46]. The results reported in the literature indicate that Aβoligomers ranging in size from 2 to 12 subunits may be responsible for the synaptic damage and memory loss observed in patients with Alzheimer's disease [47]. These oligomeric forms may be produced through several routes, either in the extracellular space or inside of the cell organelles such as the endoplasmic reticulum and mitochondria, which complicates the analysis and understanding of the pathophysiology [48-50]. Several types of soluble Aβ oligomers have been described in the brains of AD patients and in transgenic mouse models of AD, however it has been reported that the putative dodecamer Aβ\*56 correlated with markers of neuronal dysfunction or injury in cognitively normal subjects [51]. In addition, the role of Aβ oligomers (in the absence of amyloid fibers) in neurodegenerative processes was demonstrated in a transgenic model expressing mutant hAPPE693Δ. This mouse has the ability to form high levels of Aβ oligomers without fibrillization, indicating that the intracellular deposits of Aβ oligomers from 8 months of age onwards correlate with the alterations in synaptic plasticity and memory impairment

maintaining glutamate homeostasis [39-40].

to plasticity and memory processes [44].

**1.1. β—Amyloid**

The pathological markers for AD are the presence of neurofibrillary tangles (NFT) and neuritic plaques (NP). NFT are intracellular and insoluble fibril deposits of paired helical filaments (PHF). As these filaments occupy the cytoplasm of the neuron, the nucleus is displaced and the dendrites disappear, in the absence of which, the filaments takes on the pyramidal shape of the soma and then go on to destroy the neuron itself. Each filament is formed from the association of 6-7 Tau protein fragments, and each fragment consists of 93-95 amino acid residues and has a molecular weight of 12.5Kd [18-21]. In normal conditions, Tau stabilizes microtubules in the cytoskeleton of neurons through a cell process that involves the phos‐ phorylation and dephosphorylation of the protein. In pathological conditions, Tau is abnor‐ mally hyperphosphorylated and loses its ability to bind to microtubules, generating insoluble aggregates within the neuron, altering the axonal transport and eventually leading to neuronal death. Generally NFT formation begins in the allocortex of the medial temporal lobe (entorhi‐ nal cortex and hippocampus) and spreads to the associative isocortex. In this way, the amount and distribution of NFTs correlate with the severity and duration of dementia.

NPs are extracellular deposits of 10-100μm formed by an insoluble fibrillary core surrounded by activated microglia, reactive astrocytes and dystrophic neurites [22]. Unlike NFTs, amyloid plaques accumulate mainly in the isocortex. The main component in the NP is the amyloidbeta peptide (Aβ); a fragment of 39-42 amino acids with a molecular weight of 4KD [23-24], which arises as a result of the normal secretion derived from amyloid-β precursor protein (AβPP) [25]. Aβ formation occurs as a result of the proteolytic processing of AβPP by the sequential action of β- and γ-secretase. Three AβPP isoforms consisting of 695, 770 and 751 amino acids (APP695, APP751 and APP770) are mainly expressed in the Central Nervous System (CNS). The shortest of these isoforms, APP695, is mostly expressed in neurons, whereas isoforms APP770 and APP751 are expressed in glial cells.

It has been proposed that the progressive accumulation of NP and NFT in the brains of AD patients are responsible for the neurodegeneration observed in the hippocampal, cortical and subcortical neurons. This neurodegenerative damage involves the loss of neuropil networks, selective neuron death, decreased synaptic density and alterations in neurotransmitters and the homeostasis of calcium. An important feature of the NFTs is that the density of such lesions directly correlates with the degree of dementia observed in AD patients [26]. Conversely, it is observed that the number of NP present in a particular region does not correlate with neuronal death, synaptic loss or with cognitive impairment [27-29]. However, the presence of Aβ oligomer deposits has a very important role in synaptic loss [30-32] determining the severity of cognitive impairment. Also has been observed to inhibit the normal critical neuron func‐ tions, such as long-term potentiation (LTP)[30]. The amyloid βpeptide, also increases Tau phosphorylation [33-35], oxidative stress and altered homeostasis of Ca2+ [36-37] and excito‐ toxicity [38]. It has been documented that these oligomeric forms of Aβ interact with receptors from the glutamatergic system such as the NMDA-receptors, which are responsible for maintaining glutamate homeostasis [39-40].

#### **1.1. β—Amyloid**

patients also exhibit genetic risk factors, such as the presence of allele 4 of the Apolipoprotein E (Apoε4), where individuals with one copy of ε4 allele are three times more at risk of developing the disease, while those with two copies (ε4/ε4) are 10–15 times more likely to develop AD [11-14]. While other non-genetic factors are head trauma, hypertension, athero‐ sclerosis, metabolic disorders such as hypercholesterolemia, obesity and diabetes [15-17], the main risk factor is age. It has been reported that the incidence of the disease increases by 5% in people over 65 and 20% in those over 80. Other factors have also been associated with the development of the disease, such as female gender, smoking, educational level, and a low level

The pathological markers for AD are the presence of neurofibrillary tangles (NFT) and neuritic plaques (NP). NFT are intracellular and insoluble fibril deposits of paired helical filaments (PHF). As these filaments occupy the cytoplasm of the neuron, the nucleus is displaced and the dendrites disappear, in the absence of which, the filaments takes on the pyramidal shape of the soma and then go on to destroy the neuron itself. Each filament is formed from the association of 6-7 Tau protein fragments, and each fragment consists of 93-95 amino acid residues and has a molecular weight of 12.5Kd [18-21]. In normal conditions, Tau stabilizes microtubules in the cytoskeleton of neurons through a cell process that involves the phos‐ phorylation and dephosphorylation of the protein. In pathological conditions, Tau is abnor‐ mally hyperphosphorylated and loses its ability to bind to microtubules, generating insoluble aggregates within the neuron, altering the axonal transport and eventually leading to neuronal death. Generally NFT formation begins in the allocortex of the medial temporal lobe (entorhi‐ nal cortex and hippocampus) and spreads to the associative isocortex. In this way, the amount

and distribution of NFTs correlate with the severity and duration of dementia.

isoforms APP770 and APP751 are expressed in glial cells.

NPs are extracellular deposits of 10-100μm formed by an insoluble fibrillary core surrounded by activated microglia, reactive astrocytes and dystrophic neurites [22]. Unlike NFTs, amyloid plaques accumulate mainly in the isocortex. The main component in the NP is the amyloidbeta peptide (Aβ); a fragment of 39-42 amino acids with a molecular weight of 4KD [23-24], which arises as a result of the normal secretion derived from amyloid-β precursor protein (AβPP) [25]. Aβ formation occurs as a result of the proteolytic processing of AβPP by the sequential action of β- and γ-secretase. Three AβPP isoforms consisting of 695, 770 and 751 amino acids (APP695, APP751 and APP770) are mainly expressed in the Central Nervous System (CNS). The shortest of these isoforms, APP695, is mostly expressed in neurons, whereas

It has been proposed that the progressive accumulation of NP and NFT in the brains of AD patients are responsible for the neurodegeneration observed in the hippocampal, cortical and subcortical neurons. This neurodegenerative damage involves the loss of neuropil networks, selective neuron death, decreased synaptic density and alterations in neurotransmitters and the homeostasis of calcium. An important feature of the NFTs is that the density of such lesions directly correlates with the degree of dementia observed in AD patients [26]. Conversely, it is observed that the number of NP present in a particular region does not correlate with neuronal death, synaptic loss or with cognitive impairment [27-29]. However, the presence of Aβ oligomer deposits has a very important role in synaptic loss [30-32] determining the severity

of physical and mental activity during the early stages of life.

286 Neurochemistry

Aβ comes from the normal proteolytic processing of AβPP, a type 1 transmembrane glyco‐ protein [25] whose gene is located on chromosome 21 [41-42]. AβPP processing and the "efficiency" of Aβ formation could be affected by the subcellular localization of the protein. AβPP is synthesized in the endoplasmic reticulum (ER) and transported through the constit‐ utive secretory pathway, and only a small fraction of this protein (10%) goes to the plasma membrane. All AβPP isoforms undergo posttranslational modifications involving N and Oglycosylation, phosphorylation and sulfation. Aβpeptide formation is carried out by the action of β- and γ-secretase, in which the peptides formed vary from 39 to 43 amino-acid residues (Aβ39, 40, 42, 43). Although Aβ40 is the most abundant, Aβ42 is the most hydrophobic and is found in a greater proportion of the NPs observed in AD patients [43]. These peptides are continuously released into the extracellular space at possibly low concentrations, and, in soluble form, could carry out normal physiological functions in the cell including those related to plasticity and memory processes [44].

According to the amyloid hypothesis, the Aβ accumulation in the patient's brain is the key event that leads to the development of the disease, while other pathological findings (NFT formation and neuronal death) are secondary events occurring after the amyloid aggregation. Most of the studies reported in the literature have focused on the toxicity and neuronal death induced by the presence of amyloid aggregates. However, in recent years a great importance has been attached to the role of these peptides as responsible in the etiology of synaptic dysfunction[40]. In this sense, it has been widely documented that the presence of soluble oligomeric forms of Aβ responsible for synaptic damage and neurodegeneration [29, 45-46]. The results reported in the literature indicate that Aβoligomers ranging in size from 2 to 12 subunits may be responsible for the synaptic damage and memory loss observed in patients with Alzheimer's disease [47]. These oligomeric forms may be produced through several routes, either in the extracellular space or inside of the cell organelles such as the endoplasmic reticulum and mitochondria, which complicates the analysis and understanding of the pathophysiology [48-50]. Several types of soluble Aβ oligomers have been described in the brains of AD patients and in transgenic mouse models of AD, however it has been reported that the putative dodecamer Aβ\*56 correlated with markers of neuronal dysfunction or injury in cognitively normal subjects [51]. In addition, the role of Aβ oligomers (in the absence of amyloid fibers) in neurodegenerative processes was demonstrated in a transgenic model expressing mutant hAPPE693Δ. This mouse has the ability to form high levels of Aβ oligomers without fibrillization, indicating that the intracellular deposits of Aβ oligomers from 8 months of age onwards correlate with the alterations in synaptic plasticity and memory impairment observed in the mouse model. Other results observed were abnormal Tau phosphorylation, present at 8 months, microglial activation at 12 months, astrocytes activation at 18 months and neuronal loss at 24 months. The results suggest that the presence of oligomeric forms of β amyloid are able to induce many of the changes observed in the brains of patients with AD, even in the absence of NP [52].

nerative process [65]. In 1993, Wertkin and *et. al.* demonstrated that most significantly, the NT2N neurons constitutively generated intracellular Aβ peptide and released it into the culture medium, which demonstrated the intracellular production of Aβ peptide [66]. The presence of mutations in AβPP (AβPPswe), as well as by the duplication of the AβPP gene on chromosome 21 (which has been observed in patients with Down syndrome [67-68]) could be favorable to the accumulation of intracellular amyloid. Although there is evidence to support the assertion that amyloid accumulation precedes the formation of extracellular Aβ deposits and the microtubule-related pathology, the link between Aβ and Tau remains unclear [67-69]. It has also been demonstrated that the pathological accumulation of Aβ and hyperphosphor‐ ylation of Tau within synaptic terminals [70] is associated with early changes in MAP2 in neurites and synapses [71]. Finally, the position of soluble oligomers in cellular processes could help to explain their role in the synaptic dysfunction observed in patients with AD [72]. Several reports in the literature have indicated that amyloid can be formed intracellularly [73-75]. Aside from the plasma membrane, it is known that AβPP as well as β- and γ-secretase activity are located in the trans-Golgi network, the endoplasmic reticulum, and the endosomal, lysosomal and mitochondrial membranes. Aβ is generated mostly in the sub-cellular region and then secreted through exocytosis. It has been proposed that production of Aβ42 occurs in the endoplasmic reticulum, while the Aβ40 is formed in the trans-Golgi network. It has also been observed that non-neuronal cells produce both Aβ isoforms on the cell surface [73].

Alzheimer Disease: The Role of Aβ in the Glutamatergic System

http://dx.doi.org/10.5772/57367

289

Secreted amyloid forms extracellular deposits and may also be able to enter the cell through transporters and membrane receptors such as the acetylcholine receptor, the low-density lipoprotein receptor (LPR), the N-methyl-D–aspartate receptor (NMDAR), and the scavenger receptor for advanced glycation end products (RAGE) [57, 69]. The interaction between

The neuronal toxicity mediated by Aβ has been documented *in vitro* and in vivo. *In vitro* studies have demonstrated that the direct administration of Aβ to cell cultures has a neurotoxic effect because it increases oxidative stress levels and apoptosis [76-78]. The accumulation of amyloid also leads to proteasomal dysfunction and the consequent accumulation of damaged, mis‐ sfolded, and aggregated proteins, including Aβ and Tau [79-81]. The reactive oxygen species (ROS) affect membrane proteins, mitochondrial DNA, lipids, and cytoplasmic proteins, and also contribute to the vascular damage observed in AD patients [57, 61, 82-85]. Oxidative stress has been observed in the early stages of AD and has been directly associated with Aβ accu‐ mulation. Moreover, Aβ1-42, enhanced glutamate toxicity in human cerebral cortical cell

Importantly, the alterations in these patients were observed in specific brain areas such as the hippocampus, the entorhinal cortex, the amygdala, the neocortex and some sub-cortical areas, such as the cholinergic neurons in the basal forebrain, the serotonergic neurons of the dorsal raphe nucleus and the noradrenergic neurons of the locus coeruleus. The glutamatergic neurons located in the hippocampus and in the frontal, temporal and parietal cortex are severely affected. As we know, the hippocampus and cortex regions are important for the

amyloid and these receptors can trigger neurotoxicity and neuronal dysfunction.

cultures and was associated with changes in intracellular Ca2+ levels [86].

**1.4. Aβ Toxicity**

#### **1.2. Extracellular Aβ**

The extracellular deposits of amyloid are a specific marker for AD and are involved in synaptic dysfunction and neurotoxicity; however, the complete signaling mechanism involved remains unclear. Importantly, the amyloid oligomers interact with a variety of receptors on the surface, activating or inhibiting several neuronal signaling pathways and possibly contributing to neuronal death [35]. Furthermore, it is also known that the damage caused by amyloid is mainly determined by the level of peptide aggregation. In this way, several studies reported in the literature suggest that extracellular Aβ oligomers could be formed by several biocom‐ ponents, such as proteins and ganglioside. For example, the distribution of ganglioside GM1 has the ability to affect the spatial arrangement of the oligosaccharide chains in a molecule. In 2007 Yamamoto *et al.* showed that GM1 provides a microenvironment that favors the formation of amyloid oligomers [53]. These oligomers are spherical structures with a 10-20nm diameter and 200-300kDa that form complexes with the GM1, similar to those identified in the tissue of AD patients. Previous studies have shown that, initially, the peptides adopt a random structure, which then changes when interacting with GM1, and enables the transition from α-helix to β-pleated sheets [54]. Similarly, nonfibrillar Aβ can be produced in presence of αBcrystalline and ApoJ [55-56]. These oligomeric forms interact with the nerve growth factor receptor (NGF), triggering a toxic mechanism that causes cell death. Moreover, the oligomeric forms bind to Frizzled (*Fz*) receptors, inhibiting the *Wnt* signaling pathway, and affecting cell proliferation and neuronal differentiation during development of the brain. Furthermore, the inhibition of Wnt signaling by Aβ oligomers causes Tau phosphorylation and the formation of neuro fibrillary tangles, which suggests a Wnt/β-catenin toxicity pathway [35, 57].

On the other hand, it has also been observed that Aβ oligomers are able to destabilize the plasma membrane, forming pores which alter the normal flow of ions and permitting the entry of extracellular Ca2+ and leading to neuronal death [58-60]. Another mechanism of neuronal receptor-mediated damage is the binding of Aβ oligomers to N-methyl-D-aspartate (NMDA) type glutamate receptor (NMDAR), which generates altered calcium homeostasis, increased oxidative stress and loss of synapses [61-63].

#### **1.3. Intracellular Aβ**

The presence of intracellular Aβ deposits was first observed by Iqbal *et al* in 1989 [64]. They identified the presence of intraneuronal Aβ, by using an antibody against residues 17–24 of Aβ peptide in tissue from AD patients. Importantly, they also observed the presence of these immuno-positive deposits in neurons that preferentially contained NFT [64]. The discovery of the coexistence of amyloid and NFT in the same neuron allowed the development of several lines of research that attempt to show how a protein can induce or accelerate the neurodege‐ nerative process [65]. In 1993, Wertkin and *et. al.* demonstrated that most significantly, the NT2N neurons constitutively generated intracellular Aβ peptide and released it into the culture medium, which demonstrated the intracellular production of Aβ peptide [66]. The presence of mutations in AβPP (AβPPswe), as well as by the duplication of the AβPP gene on chromosome 21 (which has been observed in patients with Down syndrome [67-68]) could be favorable to the accumulation of intracellular amyloid. Although there is evidence to support the assertion that amyloid accumulation precedes the formation of extracellular Aβ deposits and the microtubule-related pathology, the link between Aβ and Tau remains unclear [67-69]. It has also been demonstrated that the pathological accumulation of Aβ and hyperphosphor‐ ylation of Tau within synaptic terminals [70] is associated with early changes in MAP2 in neurites and synapses [71]. Finally, the position of soluble oligomers in cellular processes could help to explain their role in the synaptic dysfunction observed in patients with AD [72]. Several reports in the literature have indicated that amyloid can be formed intracellularly [73-75]. Aside from the plasma membrane, it is known that AβPP as well as β- and γ-secretase activity are located in the trans-Golgi network, the endoplasmic reticulum, and the endosomal, lysosomal and mitochondrial membranes. Aβ is generated mostly in the sub-cellular region and then secreted through exocytosis. It has been proposed that production of Aβ42 occurs in the endoplasmic reticulum, while the Aβ40 is formed in the trans-Golgi network. It has also been observed that non-neuronal cells produce both Aβ isoforms on the cell surface [73].

Secreted amyloid forms extracellular deposits and may also be able to enter the cell through transporters and membrane receptors such as the acetylcholine receptor, the low-density lipoprotein receptor (LPR), the N-methyl-D–aspartate receptor (NMDAR), and the scavenger receptor for advanced glycation end products (RAGE) [57, 69]. The interaction between amyloid and these receptors can trigger neurotoxicity and neuronal dysfunction.

#### **1.4. Aβ Toxicity**

observed in the mouse model. Other results observed were abnormal Tau phosphorylation, present at 8 months, microglial activation at 12 months, astrocytes activation at 18 months and neuronal loss at 24 months. The results suggest that the presence of oligomeric forms of β amyloid are able to induce many of the changes observed in the brains of patients with AD,

The extracellular deposits of amyloid are a specific marker for AD and are involved in synaptic dysfunction and neurotoxicity; however, the complete signaling mechanism involved remains unclear. Importantly, the amyloid oligomers interact with a variety of receptors on the surface, activating or inhibiting several neuronal signaling pathways and possibly contributing to neuronal death [35]. Furthermore, it is also known that the damage caused by amyloid is mainly determined by the level of peptide aggregation. In this way, several studies reported in the literature suggest that extracellular Aβ oligomers could be formed by several biocom‐ ponents, such as proteins and ganglioside. For example, the distribution of ganglioside GM1 has the ability to affect the spatial arrangement of the oligosaccharide chains in a molecule. In 2007 Yamamoto *et al.* showed that GM1 provides a microenvironment that favors the formation of amyloid oligomers [53]. These oligomers are spherical structures with a 10-20nm diameter and 200-300kDa that form complexes with the GM1, similar to those identified in the tissue of AD patients. Previous studies have shown that, initially, the peptides adopt a random structure, which then changes when interacting with GM1, and enables the transition from α-helix to β-pleated sheets [54]. Similarly, nonfibrillar Aβ can be produced in presence of αBcrystalline and ApoJ [55-56]. These oligomeric forms interact with the nerve growth factor receptor (NGF), triggering a toxic mechanism that causes cell death. Moreover, the oligomeric forms bind to Frizzled (*Fz*) receptors, inhibiting the *Wnt* signaling pathway, and affecting cell proliferation and neuronal differentiation during development of the brain. Furthermore, the inhibition of Wnt signaling by Aβ oligomers causes Tau phosphorylation and the formation

of neuro fibrillary tangles, which suggests a Wnt/β-catenin toxicity pathway [35, 57].

oxidative stress and loss of synapses [61-63].

**1.3. Intracellular Aβ**

On the other hand, it has also been observed that Aβ oligomers are able to destabilize the plasma membrane, forming pores which alter the normal flow of ions and permitting the entry of extracellular Ca2+ and leading to neuronal death [58-60]. Another mechanism of neuronal receptor-mediated damage is the binding of Aβ oligomers to N-methyl-D-aspartate (NMDA) type glutamate receptor (NMDAR), which generates altered calcium homeostasis, increased

The presence of intracellular Aβ deposits was first observed by Iqbal *et al* in 1989 [64]. They identified the presence of intraneuronal Aβ, by using an antibody against residues 17–24 of Aβ peptide in tissue from AD patients. Importantly, they also observed the presence of these immuno-positive deposits in neurons that preferentially contained NFT [64]. The discovery of the coexistence of amyloid and NFT in the same neuron allowed the development of several lines of research that attempt to show how a protein can induce or accelerate the neurodege‐

even in the absence of NP [52].

**1.2. Extracellular Aβ**

288 Neurochemistry

The neuronal toxicity mediated by Aβ has been documented *in vitro* and in vivo. *In vitro* studies have demonstrated that the direct administration of Aβ to cell cultures has a neurotoxic effect because it increases oxidative stress levels and apoptosis [76-78]. The accumulation of amyloid also leads to proteasomal dysfunction and the consequent accumulation of damaged, mis‐ sfolded, and aggregated proteins, including Aβ and Tau [79-81]. The reactive oxygen species (ROS) affect membrane proteins, mitochondrial DNA, lipids, and cytoplasmic proteins, and also contribute to the vascular damage observed in AD patients [57, 61, 82-85]. Oxidative stress has been observed in the early stages of AD and has been directly associated with Aβ accu‐ mulation. Moreover, Aβ1-42, enhanced glutamate toxicity in human cerebral cortical cell cultures and was associated with changes in intracellular Ca2+ levels [86].

Importantly, the alterations in these patients were observed in specific brain areas such as the hippocampus, the entorhinal cortex, the amygdala, the neocortex and some sub-cortical areas, such as the cholinergic neurons in the basal forebrain, the serotonergic neurons of the dorsal raphe nucleus and the noradrenergic neurons of the locus coeruleus. The glutamatergic neurons located in the hippocampus and in the frontal, temporal and parietal cortex are severely affected. As we know, the hippocampus and cortex regions are important for the establishment of memory and learning, and so, therefore, the specific loss of glutamatergic neurons could play an important role in the progression of the pathology. Since the 1980's, it has been proposed that Alzheimer's disease may be caused by the over-activity of glutama‐ tergic neurons causing excitotoxic damage in cortical afferent neurons [87-88]. Several studies have shown that Aβ accumulates in certain synapses in micro molar concentrations of Aβ, and has the ability to bind to NMDA receptors, thus inducing the internalization and deregulation of the NMDA signaling pathway [63, 89-91].

development of the central nervous system, as well as synapse induction and elimination, cell migration, differentiation and death [101-102]. Most of the glutamate in the brain is located intracellularly inside nerve terminals and only a tiny fraction of this glutamate is normally present outside or between the cells [103-105]. The extracellular elevation of glutamate causes alterations in the glutamate-mediated neurotransmission, activating receptors and inducing the depolarization of neurons which in turn triggers a sequence of intracellular events that conclude in Ca2+ and Na2+ influx. This leads to the exocytosis of glutamate and ultimately cell death, which correlates with the loss of memory function and learning ability in AD patients [106-107]. Recently it has been shown that there is a close correlation between reduced glutamatergic presynaptic button density and cognitive deficits. A study of brain tissue from subjects with no cognitive impairment, mild cognitive impairment, or mild/severe-stage Alzheimer's disease; demonstrated that glutamatergic synaptic remodeling, presents a pattern- dependent pathology, according to disease progression by comparing the mini mental status examination scores of healthy individuals to those of individuals with mild or severe

Alzheimer Disease: The Role of Aβ in the Glutamatergic System

http://dx.doi.org/10.5772/57367

291

Glutamate excitotoxicity has also been implicated in other neurodegenerative diseases such as Huntington's disease, epilepsy, ischemia, and trauma [109-111]. In this sense, it is crucial to maintain adequate extracellular levels of glutamate, as it is continuously released from the cells and must therefore be continually removed from the extracellular fluid [93-94, 105]. It has been documented that glutamatergic neurotransmission in neocortical regions and the hippocampus is severely disrupted in Alzheimer's disease. So far, it is unknown whether molecular abnormalities observed in patients are a cause or a consequence of other changes that allow the development of neurodegeneration. Another proposed hypothesis is that alterations in the expression of neurotransmitter transporters could contribute to neurotrans‐

Under normal conditions, the low concentration of glutamate into the extracellular space is regulated by specific transporters, localized in both nerve endings and surrounding glial cells. This transport system prevents cell damage generated by excessive activation of glutamate receptors [105, 112-113]. There are two glutamate transport systems: the Vesicular GluTs (VGLUT) and the Excitatory Amino Acid Transporters (EAAT) located in the plasma mem‐ brane. The VGLUTs are crucial for the storage of glutamate in synaptic vesicles. When a neuron is depolarized, glutamate is released into the synaptic cleft where it binds glutamate receptors to pre and post-synaptic neurons. There are three isoforms; VGLUT1, VGLUT2, VGLUT3. The transport of glutamate into secretory vesicles is highly dependent on Cl- [114]. This anion stimulates glutamate transport, but is inhibitory at higher concentrations. This process is

the VGLUT activity, affect vesicular glutamate content and subsequently the glutamatergic

Studies have also identified five different 'high-affinity' glutamate excitatory amino acid (EAATs) transporters (EAAT1, EAAT2, EAAT3, EAAT4 and EAAT5). Residing on postsy‐

established by V-ATPase, which, together with

Alzheimer's disease [99, 108] (figure 1).

mission imbalances in the AD brain [112].

driven by an electrochemical gradient of H+

**2.2. Glutamate transporters**

signaling [115].

#### **2. Glutamatergic system**

#### **2.1. Glutamate**

Glutamate is a nonessential amino acid that does not cross the blood-brain barrier (BBB), and is produced primarily by neurons and glial cells from local precursors derived from glucose and α-ketoglutarate. Glutamate participates in balance with GABA to modulate the activity of GABAergic and glutamatergic neurons [92]. The majority of excitatory neurons in the CNS are glutamatergic; moreover, it is estimated that over half of nerve-endings release glutamate. Presynaptic depolarization promotes vesicles to release their contents of glutamate into the synapses through exocytosis, where upon the released glutamate binds to post-synaptic ionotropic receptors, stimulating an influx of cations which depolarizes the post-synaptic cell [93]. To prevent over-stimulation, glutamate is removed by astrocytes and converted to Lglutamine through the action of glutamine synthetase, which is released to the extracellular fluid taken up by neurons. Glutamine, normally found in the extracellular space, is, unlike glutamate, a non-toxic molecule and lacks the ability to activate glutamate receptors. The glutamine transferred back to the neuron is recycled by phosphate-activated glutaminase and, once again, forms L-glutamate, which is taken by vesicular transporters into synaptic vesicles to be available for use in the excitatory neurotransmission [93-96]. This trafficking of glutamate and glutamine between astrocytes and neurons is the primary route by which glutamate may be recycled (glutamine–glutamate cycle). The removal of this neurotransmitter from the synaptic cleft is carried out through high-affinity transporters. These transport proteins are the only existing mechanism for extracellular glutamate removal, and are of vital importance in maintaining low and non-toxic concentrations of this neurotransmitter [94]. Both neurons and glial cells express glutamate transporters. Glutamate taken up by cells may be used for metabolic purposes (protein synthesis, energy metabolism, ammonia fixation) or be reused as a neurotransmitter [94]. It is important to clarify that glutamate is not necessarily derived from glutamine nor it is necessarily converted to glutamine by astrocytes, nor does glutamine necessarily acts as a precursor to glutamate. While the mechanisms involved and the resulting metabolites are more complex, they are not mentioned in this chapter.

Glutamate is the major excitatory neurotransmitter in the CNS (approximately 8–10 mM/kg), and is found in more than 80% of all neurons [92, 97-99]. It is involved in most normal brain function, especially in the cortical and hippocampal regions, which deal with cognition, memory and learning [100] among other functions. Glutamate also plays a major role in the development of the central nervous system, as well as synapse induction and elimination, cell migration, differentiation and death [101-102]. Most of the glutamate in the brain is located intracellularly inside nerve terminals and only a tiny fraction of this glutamate is normally present outside or between the cells [103-105]. The extracellular elevation of glutamate causes alterations in the glutamate-mediated neurotransmission, activating receptors and inducing the depolarization of neurons which in turn triggers a sequence of intracellular events that conclude in Ca2+ and Na2+ influx. This leads to the exocytosis of glutamate and ultimately cell death, which correlates with the loss of memory function and learning ability in AD patients [106-107]. Recently it has been shown that there is a close correlation between reduced glutamatergic presynaptic button density and cognitive deficits. A study of brain tissue from subjects with no cognitive impairment, mild cognitive impairment, or mild/severe-stage Alzheimer's disease; demonstrated that glutamatergic synaptic remodeling, presents a pattern- dependent pathology, according to disease progression by comparing the mini mental status examination scores of healthy individuals to those of individuals with mild or severe Alzheimer's disease [99, 108] (figure 1).

Glutamate excitotoxicity has also been implicated in other neurodegenerative diseases such as Huntington's disease, epilepsy, ischemia, and trauma [109-111]. In this sense, it is crucial to maintain adequate extracellular levels of glutamate, as it is continuously released from the cells and must therefore be continually removed from the extracellular fluid [93-94, 105]. It has been documented that glutamatergic neurotransmission in neocortical regions and the hippocampus is severely disrupted in Alzheimer's disease. So far, it is unknown whether molecular abnormalities observed in patients are a cause or a consequence of other changes that allow the development of neurodegeneration. Another proposed hypothesis is that alterations in the expression of neurotransmitter transporters could contribute to neurotrans‐ mission imbalances in the AD brain [112].

#### **2.2. Glutamate transporters**

establishment of memory and learning, and so, therefore, the specific loss of glutamatergic neurons could play an important role in the progression of the pathology. Since the 1980's, it has been proposed that Alzheimer's disease may be caused by the over-activity of glutama‐ tergic neurons causing excitotoxic damage in cortical afferent neurons [87-88]. Several studies have shown that Aβ accumulates in certain synapses in micro molar concentrations of Aβ, and has the ability to bind to NMDA receptors, thus inducing the internalization and deregulation

Glutamate is a nonessential amino acid that does not cross the blood-brain barrier (BBB), and is produced primarily by neurons and glial cells from local precursors derived from glucose and α-ketoglutarate. Glutamate participates in balance with GABA to modulate the activity of GABAergic and glutamatergic neurons [92]. The majority of excitatory neurons in the CNS are glutamatergic; moreover, it is estimated that over half of nerve-endings release glutamate. Presynaptic depolarization promotes vesicles to release their contents of glutamate into the synapses through exocytosis, where upon the released glutamate binds to post-synaptic ionotropic receptors, stimulating an influx of cations which depolarizes the post-synaptic cell [93]. To prevent over-stimulation, glutamate is removed by astrocytes and converted to Lglutamine through the action of glutamine synthetase, which is released to the extracellular fluid taken up by neurons. Glutamine, normally found in the extracellular space, is, unlike glutamate, a non-toxic molecule and lacks the ability to activate glutamate receptors. The glutamine transferred back to the neuron is recycled by phosphate-activated glutaminase and, once again, forms L-glutamate, which is taken by vesicular transporters into synaptic vesicles to be available for use in the excitatory neurotransmission [93-96]. This trafficking of glutamate and glutamine between astrocytes and neurons is the primary route by which glutamate may be recycled (glutamine–glutamate cycle). The removal of this neurotransmitter from the synaptic cleft is carried out through high-affinity transporters. These transport proteins are the only existing mechanism for extracellular glutamate removal, and are of vital importance in maintaining low and non-toxic concentrations of this neurotransmitter [94]. Both neurons and glial cells express glutamate transporters. Glutamate taken up by cells may be used for metabolic purposes (protein synthesis, energy metabolism, ammonia fixation) or be reused as a neurotransmitter [94]. It is important to clarify that glutamate is not necessarily derived from glutamine nor it is necessarily converted to glutamine by astrocytes, nor does glutamine necessarily acts as a precursor to glutamate. While the mechanisms involved and the resulting

metabolites are more complex, they are not mentioned in this chapter.

Glutamate is the major excitatory neurotransmitter in the CNS (approximately 8–10 mM/kg), and is found in more than 80% of all neurons [92, 97-99]. It is involved in most normal brain function, especially in the cortical and hippocampal regions, which deal with cognition, memory and learning [100] among other functions. Glutamate also plays a major role in the

of the NMDA signaling pathway [63, 89-91].

**2. Glutamatergic system**

**2.1. Glutamate**

290 Neurochemistry

Under normal conditions, the low concentration of glutamate into the extracellular space is regulated by specific transporters, localized in both nerve endings and surrounding glial cells. This transport system prevents cell damage generated by excessive activation of glutamate receptors [105, 112-113]. There are two glutamate transport systems: the Vesicular GluTs (VGLUT) and the Excitatory Amino Acid Transporters (EAAT) located in the plasma mem‐ brane. The VGLUTs are crucial for the storage of glutamate in synaptic vesicles. When a neuron is depolarized, glutamate is released into the synaptic cleft where it binds glutamate receptors to pre and post-synaptic neurons. There are three isoforms; VGLUT1, VGLUT2, VGLUT3. The transport of glutamate into secretory vesicles is highly dependent on Cl- [114]. This anion stimulates glutamate transport, but is inhibitory at higher concentrations. This process is driven by an electrochemical gradient of H+ established by V-ATPase, which, together with the VGLUT activity, affect vesicular glutamate content and subsequently the glutamatergic signaling [115].

Studies have also identified five different 'high-affinity' glutamate excitatory amino acid (EAATs) transporters (EAAT1, EAAT2, EAAT3, EAAT4 and EAAT5). Residing on postsy‐

system as well as genetic mutation in the transporters and enzymes involved in the glutamate metabolism can lead to excitotoxic damage due to an excessive release of glutamate, which in

Alzheimer Disease: The Role of Aβ in the Glutamatergic System

http://dx.doi.org/10.5772/57367

293

Glutamate-mediated neurotransmission occurs through specific receptors. There are 2 families of glutamate receptors located on the plasma membrane of the neurons: ionotropic (iGluR) glutamate receptors, which act as ion channels, and metabotropic (mGluR) glutamate receptors

The iGluR family is divided into three kinds of receptors, depending on their permeabili‐ ty to different cations. NMDA receptors (NR1, NR2A–D and NR3A–B) are predominantly Ca2+ ion permeable, whereas α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; GluR1–4) and Kainate (KA; GluR5–7, KA1–2) receptors are predominantly permeable to Na+ and K<sup>+</sup> ions [99, 120]. Each of these receptors is composed of four subunits, and variations in the expression of each subunit have different types of response in the receptor function [92, 119]. AMPA receptors have a lower affinity for Glutamate than NMDA receptors, and are responsible for an initial excitatory potential when Glutamate is present in the synapse [92]. Kainate receptors play a role in synaptic neurotransmission,

NMDA and AMPA receptors are present in most of the synapses in mammalian brains (approximately 70%). This type of receptor is preferentially localized in the cerebral cortex, hippocampus, amygdala, striatum, and septum. The specific location of these receptors is of great importance, since glutamatergic signaling has a very important role in both the plasticity and excitotoxicity processes, and, therefore, changes in their function lead to the development

The NMDA receptor is the most important and studied ionotropic receptor to date, and participates in several functions such as synaptogenesis, synaptic plasticity, learning and memory, as well as in the pathogenesis of several central nervous system disorders [121-123]. Calcium influx through the NMDA receptor is capable of modulating physiological and pathological conditions in the neuron. The increase in intracellular Ca2+ concentration triggers a cascade of events that dramatically modifies synaptic efficacy and neuronal morphology. Functional NMDA receptors are heterotetrameric complexes composed of different subunits (GluN1, GluN2A-D, GluN3A-B). Typically, each NMDAR comprises two obligatory GluN1 subunits and two GluN2 subunits, which can a form a dimer themselves, or alternatively one GluN2 can combine with one GluN3 subunit to do the same [121, 123-124]. GluN1 occurs as eight distinct isoforms encoded by a single gene [125]. The functional significance of the differential expression of GluN1 isoforms is not clear. GluN2 and GluN3 also exist in several alternatively spliced forms, although the functional differences between them are complex. There are four genes encoding GluN2 subunits and each has a unique spatiotemporal profile. In addition, GluN2A and GluN2B are expressed primarily in the cortex and hippocampus and differ in their kinetic properties, developmental expression pattern, subcellular localization and trafficking regulation. GluN3 subunits also display differential expression patterns, with

which are linked to the intracellular second messenger systems [92, 99, 118-119].

turn can lead to neuronal death.

but the exact nature of this role is unclear [119].

of neurodegenerative processes.

**2.3. Glutamate receptors**

**Figure 1.** Glutamatergic Transmission in Normal Brain. Glutamate released from presynaptic terminals acts through the activation of glutamate receptors located at the postsynaptic terminal. The interaction between glutamate and NMDA receptor favors the activation of several metabolic pathways such as CaMK, ERK, and CREB, which are responsi‐ ble for anabolic activation with subsequent activation of long-term potentiation (LTP) mechanisms. Glutamate excess is transported via the EAAT into astrocytes, where is transformed to glutamine by the glutamine synthase. Subse‐ quently, glutamine it is converted into glutamate by glutaminase and packaged into vesicles through specific trans‐ porters (VGlut). VGlut (vesicular glutamate transporter); EAAT (excitatory amino acid transporter); NMDANR2A (Nmethyl-D-aspartate NR2A subunit); NMDANR2B (N-methyl- D-aspartate NR2B subunit); ERK (extracellular signalrelated kinase); CaMKII (calcium calmodulin-dependent kinase II); pCREB (phosphorylated cyclic AMP response element binding protein); GSK3b (glycogen synthase kinase 3b); p38-MAPK (p38 mitogen-activated protein kinase).

naptic GABAergic neurons, EAATs transport glutamate and serve as a precursor for the synthesis of GABA. These two transporter families differ in many of their functional properties, including substrate specificity and ion requirements [113]. EAATs-mediated glutamate transport is Na+ dependent, where, for each transport cycle, one glutamate molecule is transported together with two or three Na+ ions and one H+ in exchange for one K+ ion. These transporters also interact with other proteins at the plasma membrane and are regulated by protein kinases, growth factors and second messengers [116-117]. Alterations in this regulatory system as well as genetic mutation in the transporters and enzymes involved in the glutamate metabolism can lead to excitotoxic damage due to an excessive release of glutamate, which in turn can lead to neuronal death.

#### **2.3. Glutamate receptors**

naptic GABAergic neurons, EAATs transport glutamate and serve as a precursor for the synthesis of GABA. These two transporter families differ in many of their functional properties, including substrate specificity and ion requirements [113]. EAATs-mediated glutamate

**Figure 1.** Glutamatergic Transmission in Normal Brain. Glutamate released from presynaptic terminals acts through the activation of glutamate receptors located at the postsynaptic terminal. The interaction between glutamate and NMDA receptor favors the activation of several metabolic pathways such as CaMK, ERK, and CREB, which are responsi‐ ble for anabolic activation with subsequent activation of long-term potentiation (LTP) mechanisms. Glutamate excess is transported via the EAAT into astrocytes, where is transformed to glutamine by the glutamine synthase. Subse‐ quently, glutamine it is converted into glutamate by glutaminase and packaged into vesicles through specific trans‐ porters (VGlut). VGlut (vesicular glutamate transporter); EAAT (excitatory amino acid transporter); NMDANR2A (Nmethyl-D-aspartate NR2A subunit); NMDANR2B (N-methyl- D-aspartate NR2B subunit); ERK (extracellular signalrelated kinase); CaMKII (calcium calmodulin-dependent kinase II); pCREB (phosphorylated cyclic AMP response element binding protein); GSK3b (glycogen synthase kinase 3b); p38-MAPK (p38 mitogen-activated protein kinase).

transporters also interact with other proteins at the plasma membrane and are regulated by protein kinases, growth factors and second messengers [116-117]. Alterations in this regulatory

transported together with two or three Na+ ions and one H+ in exchange for one K+

dependent, where, for each transport cycle, one glutamate molecule is

ion. These

transport is Na+

292 Neurochemistry

Glutamate-mediated neurotransmission occurs through specific receptors. There are 2 families of glutamate receptors located on the plasma membrane of the neurons: ionotropic (iGluR) glutamate receptors, which act as ion channels, and metabotropic (mGluR) glutamate receptors which are linked to the intracellular second messenger systems [92, 99, 118-119].

The iGluR family is divided into three kinds of receptors, depending on their permeabili‐ ty to different cations. NMDA receptors (NR1, NR2A–D and NR3A–B) are predominantly Ca2+ ion permeable, whereas α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; GluR1–4) and Kainate (KA; GluR5–7, KA1–2) receptors are predominantly permeable to Na+ and K<sup>+</sup> ions [99, 120]. Each of these receptors is composed of four subunits, and variations in the expression of each subunit have different types of response in the receptor function [92, 119]. AMPA receptors have a lower affinity for Glutamate than NMDA receptors, and are responsible for an initial excitatory potential when Glutamate is present in the synapse [92]. Kainate receptors play a role in synaptic neurotransmission, but the exact nature of this role is unclear [119].

NMDA and AMPA receptors are present in most of the synapses in mammalian brains (approximately 70%). This type of receptor is preferentially localized in the cerebral cortex, hippocampus, amygdala, striatum, and septum. The specific location of these receptors is of great importance, since glutamatergic signaling has a very important role in both the plasticity and excitotoxicity processes, and, therefore, changes in their function lead to the development of neurodegenerative processes.

The NMDA receptor is the most important and studied ionotropic receptor to date, and participates in several functions such as synaptogenesis, synaptic plasticity, learning and memory, as well as in the pathogenesis of several central nervous system disorders [121-123]. Calcium influx through the NMDA receptor is capable of modulating physiological and pathological conditions in the neuron. The increase in intracellular Ca2+ concentration triggers a cascade of events that dramatically modifies synaptic efficacy and neuronal morphology. Functional NMDA receptors are heterotetrameric complexes composed of different subunits (GluN1, GluN2A-D, GluN3A-B). Typically, each NMDAR comprises two obligatory GluN1 subunits and two GluN2 subunits, which can a form a dimer themselves, or alternatively one GluN2 can combine with one GluN3 subunit to do the same [121, 123-124]. GluN1 occurs as eight distinct isoforms encoded by a single gene [125]. The functional significance of the differential expression of GluN1 isoforms is not clear. GluN2 and GluN3 also exist in several alternatively spliced forms, although the functional differences between them are complex. There are four genes encoding GluN2 subunits and each has a unique spatiotemporal profile. In addition, GluN2A and GluN2B are expressed primarily in the cortex and hippocampus and differ in their kinetic properties, developmental expression pattern, subcellular localization and trafficking regulation. GluN3 subunits also display differential expression patterns, with GluN3A peaking in early postnatal life and GluN3B increasing throughout development [123]. Finally, although receptor subunits have structural similarities, the composition of the different receptor subtypes confer distinct functional and biophysical properties that are reflected in the ion permeability, protein interactions and membrane localization (synaptic or extrasynaptic). They may also have different roles in modulating synaptic plasticity and development of pathologies [122-123]. In fact, the expression of individual subunits is highly dependent on brain area and developmental stage, thus, alterations in the expression of each subunit can lead to a pathological condition which may be reflected in the development of neurodegenerative diseases.

**2.5. Neurotoxicity of Aβ: Synaptic dysfunction in AD**

neurons in the cortex and hippocampus (Figure 2).

ERK, pCREB).

Aside from the above toxic effects, it is known that amyloid has the ability to inhibit normal function of the glutamatergic system. It can also interact with glutamine synthetase (GS) to induce the inactivation of the enzyme [137], chronically depolarize neurons through its action on the metabotropic glutamate receptor 1 [138], and partially relieve the voltage-dependent Mg2+ block of NMDA receptors, which allows the continuous entry of calcium into neurons by altering the homeostasis and thus causing cell death. This also causes that neurons to express NMDA receptors selectively and become vulnerable to glutamatergic stimulation. In AD patients, it has been observed that glutamatergic transmission is severely affected by

Alzheimer Disease: The Role of Aβ in the Glutamatergic System

http://dx.doi.org/10.5772/57367

295

**Figure 2.** Glutamatergic transmission in Alzheimer's disease. Aβ oligomers enhance the pre-synaptic release of gluta‐ mate together with the simultaneous blockade of glutamate uptake by astrocytes through glutamate transporters (EAAT), due to this, glutamate concentration in synaptic cleft increases. In addition, Aβ form complexes with alpha7 nicotinic receptors, increasing levels of glutamate release. Activation of NMDA receptors increases the influx of calci‐ um and activates signaling pathways responsible for neuronal shrinkage and synaptic loss (p38-MbAPK, GSK-3b, JNK), leading to Tau phosphorylation and neuronal death. Finally, there is an inhibition of the survival pathways (CAMK II,

#### **2.4. Glutamate/NMDAR: Role in learning and memory**

In the CNS, it is known that the hippocampus is closely related to learning and memory, and has a very high density of glutamate receptors, particularly the NMDA-type, which are significantly involved in this type of neuronal plasticity. Glutamate is essential for the establishment of new neural networks, forming memory and learning through a process known as long-term potentiation (LTP) or long-term depression (LTD) of synaptic strength, which occurs upon activation of NMDA receptors.

NMDA receptors are characterized by their high Ca2+ ions' permeability, their voltage dependent blockade by Mg2+ ions, and their slower gating kinetics. At rest, the NMDA receptor is blocked by Mg2+, while prolonged activation by the presence of glutamate allows the release of the Mg2+, opening the NMDA receptor and allowing the Ca2+ ions to freely enter into postsynaptic neuron. Calcium entering through the NMDA receptors activates CaMKII, PKA, PKC and mitogen-activated protein kinase (MAPK), and protein phosphatases. Acti‐ vated CaMKII phosphorylates the AMPA-type glutamate receptor 1 (GluR1) subunit, which, in turn, promotes synaptic incorporation of GluR1-containing AMPARs, thereby increasing AMPAR number and channel conductance [107, 121, 126]. The fundamental role of the NMDAreceptor system in the establishment of learning and memory has been demonstrated in various animal models [127-131]. However, pharmacological studies and the manipulation of experimental models have shown that, although this system is important in memory induction, it does not participate in the maintenance of memory [132-135] (figure 1).

These features make NMDA receptors quite suitable for mediating plastic changes in the brain, such as learning. However, they may also contribute to the excitotoxicity processes produced by a massive influx of Ca2+. Under these conditions, the continuous presence of glutamate induces constant activation of the NMDA receptor, and the ensuing massive influx of Ca2+ may trigger a cascade of events leading to neuronal injury and death[136]. Chronic depolarization of the membrane on vulnerable neurons, as observed in AD patients, is accompanied by other disorders such as neuronal oxidative stress, mitochondrial damage, and inflammation, and the presence of amyloid beta and possibly hyperphosphorylated-tau, which may eventually increase the sensitivity of the glutamatergic system and result in neuronal dysfunction and cell death [97, 106-107].

#### **2.5. Neurotoxicity of Aβ: Synaptic dysfunction in AD**

GluN3A peaking in early postnatal life and GluN3B increasing throughout development [123]. Finally, although receptor subunits have structural similarities, the composition of the different receptor subtypes confer distinct functional and biophysical properties that are reflected in the ion permeability, protein interactions and membrane localization (synaptic or extrasynaptic). They may also have different roles in modulating synaptic plasticity and development of pathologies [122-123]. In fact, the expression of individual subunits is highly dependent on brain area and developmental stage, thus, alterations in the expression of each subunit can lead to a pathological condition which may be reflected in the development of

In the CNS, it is known that the hippocampus is closely related to learning and memory, and has a very high density of glutamate receptors, particularly the NMDA-type, which are significantly involved in this type of neuronal plasticity. Glutamate is essential for the establishment of new neural networks, forming memory and learning through a process known as long-term potentiation (LTP) or long-term depression (LTD) of synaptic strength,

NMDA receptors are characterized by their high Ca2+ ions' permeability, their voltage dependent blockade by Mg2+ ions, and their slower gating kinetics. At rest, the NMDA receptor is blocked by Mg2+, while prolonged activation by the presence of glutamate allows the release of the Mg2+, opening the NMDA receptor and allowing the Ca2+ ions to freely enter into postsynaptic neuron. Calcium entering through the NMDA receptors activates CaMKII, PKA, PKC and mitogen-activated protein kinase (MAPK), and protein phosphatases. Acti‐ vated CaMKII phosphorylates the AMPA-type glutamate receptor 1 (GluR1) subunit, which, in turn, promotes synaptic incorporation of GluR1-containing AMPARs, thereby increasing AMPAR number and channel conductance [107, 121, 126]. The fundamental role of the NMDAreceptor system in the establishment of learning and memory has been demonstrated in various animal models [127-131]. However, pharmacological studies and the manipulation of experimental models have shown that, although this system is important in memory induction,

These features make NMDA receptors quite suitable for mediating plastic changes in the brain, such as learning. However, they may also contribute to the excitotoxicity processes produced by a massive influx of Ca2+. Under these conditions, the continuous presence of glutamate induces constant activation of the NMDA receptor, and the ensuing massive influx of Ca2+ may trigger a cascade of events leading to neuronal injury and death[136]. Chronic depolarization of the membrane on vulnerable neurons, as observed in AD patients, is accompanied by other disorders such as neuronal oxidative stress, mitochondrial damage, and inflammation, and the presence of amyloid beta and possibly hyperphosphorylated-tau, which may eventually increase the sensitivity of the glutamatergic system and result in neuronal dysfunction and

it does not participate in the maintenance of memory [132-135] (figure 1).

neurodegenerative diseases.

294 Neurochemistry

cell death [97, 106-107].

**2.4. Glutamate/NMDAR: Role in learning and memory**

which occurs upon activation of NMDA receptors.

Aside from the above toxic effects, it is known that amyloid has the ability to inhibit normal function of the glutamatergic system. It can also interact with glutamine synthetase (GS) to induce the inactivation of the enzyme [137], chronically depolarize neurons through its action on the metabotropic glutamate receptor 1 [138], and partially relieve the voltage-dependent Mg2+ block of NMDA receptors, which allows the continuous entry of calcium into neurons by altering the homeostasis and thus causing cell death. This also causes that neurons to express NMDA receptors selectively and become vulnerable to glutamatergic stimulation. In AD patients, it has been observed that glutamatergic transmission is severely affected by neurons in the cortex and hippocampus (Figure 2).

**Figure 2.** Glutamatergic transmission in Alzheimer's disease. Aβ oligomers enhance the pre-synaptic release of gluta‐ mate together with the simultaneous blockade of glutamate uptake by astrocytes through glutamate transporters (EAAT), due to this, glutamate concentration in synaptic cleft increases. In addition, Aβ form complexes with alpha7 nicotinic receptors, increasing levels of glutamate release. Activation of NMDA receptors increases the influx of calci‐ um and activates signaling pathways responsible for neuronal shrinkage and synaptic loss (p38-MbAPK, GSK-3b, JNK), leading to Tau phosphorylation and neuronal death. Finally, there is an inhibition of the survival pathways (CAMK II, ERK, pCREB).

Neurochemical analysis performed on tissue from patients with AD revealed deficits in numerous neurotransmitters. Early symptoms appear to correlate with dysfunction of cholinergic and glutamatergic synapses. Furthermore, morphometric studies of temporal and frontal cortical biopsies from AD have revealed that there is a 25-30% decrease in the density of synapses. In the same way, it has been observed that the degree of cognitive impairment observed correlates with changes in the protein synaptophysin in the hippocampus. In fact, it has also been shown that the presence of soluble amyloid correlates with cognitive deficits and synapse loss [139-140].

**2.6. Animal models in AD and synaptic dysfunction**

dendritic spine density in a normal rodent hippocampus [154].

cognitive alteration manifests in an age-related manner [157].

Several studies have shown that amyloid oligomers – both synthetic [150] and those isolated from the brains of patients [63] – have the ability to induce synaptic alterations in neuronal cultures and organotypic hippocampal slice cultures. The transgenic mice models that express different APP mutated forms show extensive neuritic dystrophy and loss of synapses, important features that suggest a neurodegenerative process. In this way, it has been suggested that Aβ oligomers could modulate both pre and post-synaptic structures and functions in a dose and assembly-dependent manner [151-152]. The results indicate that protofibrils and oligomeric forms of Aβ most likely generate neuronal death through a nucleation-dependent process rather than acting as direct neurotoxic ligands [153]. In 2008, Shankar and colleagues showed that the presence of Aβ oligomers in slice cultures blocked the LTP, while NP-derived aggregates had no effect unless they were treated with formic acid. The oligomers potently inhibited long-term potentiation (LTP), enhanced long-term depression (LTD), and reduced

Alzheimer Disease: The Role of Aβ in the Glutamatergic System

http://dx.doi.org/10.5772/57367

297

Animal models overexpressing hAβPP protein also show a decrease in synaptophysin-positive presynaptic terminals, approximately 30% less than that observed in non-transgenic mice. Is important to note that these decreases in presynaptic terminals are dependent on soluble amyloid levels rather than on plaques *per se* [63, 152, 154-156], which would also explain the cognitive deficits observed. In a triple transgenic mice model which presented PS1(M146V), APP(Swe), and tau(P301L) transgenes (3xTg-AD), it was possible to show that the intraneuro‐ nal amyloid deposition correlates with the cognitive deficits observed in these mice. At six months, the 3xTg-AD mice showed a profound LTP deficit and intraneuronal Aβ accumulation occurring within pyramidal neurons. This cognitive deficit occurs before the accumulation of extracellular amyloid aggregates, suggesting that cognitive impairment occurs before the formation of neuritic plaques [157-160]. The synaptic dysfunction, including LTP deficits and

Moreover, it has also been observed that Aβ oligomers bind to high-affinity cell-surface receptors (cellular prion protein or PrP(C) and block hippocampal long-term potentiation and dendritic spine retraction from pyramidal cells at nanomolar concentrations of oligomers. Anti-PrP antibodies prevent the Aβ-oligomer from binding to PrPC and rescue synaptic plasticity in hippocampal slices from oligomeric Aβ [161]. Other studies also have shown that Aβ/PrPC interaction leads to activation of Fyn kinase. PrPC /Fyn signaling yields phosphor‐ ylation of the NR2B subunit of NMDA receptors, which is coupled with an initial increase and then a loss of surface NMDA-receptors. Thus, Aβ generates changes in GluR function and dendritic spine anatomy. Additionally, Fyn activation might suggest correlation with Tau pathology and the epileptiform phenotype observed in some patients with AD [162]. In this sense, it has been reported that oligomers of Aβ lead the activation of AMPK. The increased intracellular Ca2+ induced by membrane depolarization or NMDA receptor activation triggers AMP-activated kinase (AMPK) activation in a CAMKK2-dependent manner. CAMKK2 or AMPK overactivation is sufficient to induce dendritic spine loss [163]. The roles of AMPK in the pathogenesis of AD include β-amyloid protein (Aβ) generation and tau phosphorylation [164]. AMPK phosphorylates Tau in S262, while expression of Tau S262A inhibits the synap‐

In 2005, Kokubo *et al* investigated the ultrastructural localization of soluble Aβ oligomers in human brain tissue. They used post-embedding immunoelectron microscopy (IEM) [72] and an antibody that specifically recognizes soluble oligomers [141]. The results showed that approximately 80% of oligomers are found in active cellular processes. In addition, oligomers were found in both the presynaptic active zone and in postsynaptic densities, and their presence may be related to synaptic dysfunction [72]. This might suggest that the Aβ oligomers are released from the presynaptic site into extracellular space or are synaptically transported from neuron to neuron. These results agree with the hypothesis that the oligomerization of Aβ begins intracellularly [50]. The amyloid that is released from presynaptic terminals and not degraded efficiently accumulates in extracellular deposits and could serve as a seed to induce further accumulation of Aβ aggregates that culminates in the formation of neuritic plaques [142-143]. Neprilysin is an enzyme which is located in the presynaptic sites and participates in the Aβ clearance. In AD, Neprilysin is decreased and may contribute to AD pathogenesis increasing the amyloid levels in the presynaptic sites [144-145]. This was demonstrated in a transgenic mouse model that expressed low levels of APP and had one or both NEP genes silenced. The analysis of the brains and plasma in young and old mice showed elevated levels of human Aβ1-40 and Aβ1-42, an increase in Aβ dimer concentration, and a markedly increased hippocampal amyloid plaque burden, and led to development of severe amyloid angiopathy, supporting the hypothesis that primary defects in Aβ clearance can cause or contribute to AD pathogenesis [146].

In 2012, Koffie *et al* analyzed more than 50,000 synapses in 11 AD brains and 5 control subjects, and found that the synapse loss directly correlated with the presence of oligomeric amyloid. This was confirmed by the use of specific antibodies, such as NAB61, which recognizes oligomeric Aβ, and the R1282 antibody which recognizes all conformations of amyloid and the 82E1 antibody [147]. Extensive neuronal loss is another important feature in the Alzheim‐ er's pathology and, is observed as being restricted to the cell bodies and dendrites of gluta‐ matergic neurons located in layers III and IV of the neocortex and the glutamatergically innervated cortical and hippocampal neurons [38, 148].

The mechanism by which Aβ oligomers induce synaptic dysfunction remains unknown; however, it has been proposed that this alteration in synaptic transmission may be performed through non-excitotoxic glutamatergic mechanisms [149]. In this way, the accumulation of Aβ oligomers in synaptic components, especially in the axon terminal, results in synaptic and cognitive dysfunction seen in AD [72].

#### **2.6. Animal models in AD and synaptic dysfunction**

Neurochemical analysis performed on tissue from patients with AD revealed deficits in numerous neurotransmitters. Early symptoms appear to correlate with dysfunction of cholinergic and glutamatergic synapses. Furthermore, morphometric studies of temporal and frontal cortical biopsies from AD have revealed that there is a 25-30% decrease in the density of synapses. In the same way, it has been observed that the degree of cognitive impairment observed correlates with changes in the protein synaptophysin in the hippocampus. In fact, it has also been shown that the presence of soluble amyloid correlates with cognitive deficits and

In 2005, Kokubo *et al* investigated the ultrastructural localization of soluble Aβ oligomers in human brain tissue. They used post-embedding immunoelectron microscopy (IEM) [72] and an antibody that specifically recognizes soluble oligomers [141]. The results showed that approximately 80% of oligomers are found in active cellular processes. In addition, oligomers were found in both the presynaptic active zone and in postsynaptic densities, and their presence may be related to synaptic dysfunction [72]. This might suggest that the Aβ oligomers are released from the presynaptic site into extracellular space or are synaptically transported from neuron to neuron. These results agree with the hypothesis that the oligomerization of Aβ begins intracellularly [50]. The amyloid that is released from presynaptic terminals and not degraded efficiently accumulates in extracellular deposits and could serve as a seed to induce further accumulation of Aβ aggregates that culminates in the formation of neuritic plaques [142-143]. Neprilysin is an enzyme which is located in the presynaptic sites and participates in the Aβ clearance. In AD, Neprilysin is decreased and may contribute to AD pathogenesis increasing the amyloid levels in the presynaptic sites [144-145]. This was demonstrated in a transgenic mouse model that expressed low levels of APP and had one or both NEP genes silenced. The analysis of the brains and plasma in young and old mice showed elevated levels of human Aβ1-40 and Aβ1-42, an increase in Aβ dimer concentration, and a markedly increased hippocampal amyloid plaque burden, and led to development of severe amyloid angiopathy, supporting the hypothesis that primary defects in Aβ clearance can cause or

In 2012, Koffie *et al* analyzed more than 50,000 synapses in 11 AD brains and 5 control subjects, and found that the synapse loss directly correlated with the presence of oligomeric amyloid. This was confirmed by the use of specific antibodies, such as NAB61, which recognizes oligomeric Aβ, and the R1282 antibody which recognizes all conformations of amyloid and the 82E1 antibody [147]. Extensive neuronal loss is another important feature in the Alzheim‐ er's pathology and, is observed as being restricted to the cell bodies and dendrites of gluta‐ matergic neurons located in layers III and IV of the neocortex and the glutamatergically

The mechanism by which Aβ oligomers induce synaptic dysfunction remains unknown; however, it has been proposed that this alteration in synaptic transmission may be performed through non-excitotoxic glutamatergic mechanisms [149]. In this way, the accumulation of Aβ oligomers in synaptic components, especially in the axon terminal, results in synaptic and

synapse loss [139-140].

296 Neurochemistry

contribute to AD pathogenesis [146].

cognitive dysfunction seen in AD [72].

innervated cortical and hippocampal neurons [38, 148].

Several studies have shown that amyloid oligomers – both synthetic [150] and those isolated from the brains of patients [63] – have the ability to induce synaptic alterations in neuronal cultures and organotypic hippocampal slice cultures. The transgenic mice models that express different APP mutated forms show extensive neuritic dystrophy and loss of synapses, important features that suggest a neurodegenerative process. In this way, it has been suggested that Aβ oligomers could modulate both pre and post-synaptic structures and functions in a dose and assembly-dependent manner [151-152]. The results indicate that protofibrils and oligomeric forms of Aβ most likely generate neuronal death through a nucleation-dependent process rather than acting as direct neurotoxic ligands [153]. In 2008, Shankar and colleagues showed that the presence of Aβ oligomers in slice cultures blocked the LTP, while NP-derived aggregates had no effect unless they were treated with formic acid. The oligomers potently inhibited long-term potentiation (LTP), enhanced long-term depression (LTD), and reduced dendritic spine density in a normal rodent hippocampus [154].

Animal models overexpressing hAβPP protein also show a decrease in synaptophysin-positive presynaptic terminals, approximately 30% less than that observed in non-transgenic mice. Is important to note that these decreases in presynaptic terminals are dependent on soluble amyloid levels rather than on plaques *per se* [63, 152, 154-156], which would also explain the cognitive deficits observed. In a triple transgenic mice model which presented PS1(M146V), APP(Swe), and tau(P301L) transgenes (3xTg-AD), it was possible to show that the intraneuro‐ nal amyloid deposition correlates with the cognitive deficits observed in these mice. At six months, the 3xTg-AD mice showed a profound LTP deficit and intraneuronal Aβ accumulation occurring within pyramidal neurons. This cognitive deficit occurs before the accumulation of extracellular amyloid aggregates, suggesting that cognitive impairment occurs before the formation of neuritic plaques [157-160]. The synaptic dysfunction, including LTP deficits and cognitive alteration manifests in an age-related manner [157].

Moreover, it has also been observed that Aβ oligomers bind to high-affinity cell-surface receptors (cellular prion protein or PrP(C) and block hippocampal long-term potentiation and dendritic spine retraction from pyramidal cells at nanomolar concentrations of oligomers. Anti-PrP antibodies prevent the Aβ-oligomer from binding to PrPC and rescue synaptic plasticity in hippocampal slices from oligomeric Aβ [161]. Other studies also have shown that Aβ/PrPC interaction leads to activation of Fyn kinase. PrPC /Fyn signaling yields phosphor‐ ylation of the NR2B subunit of NMDA receptors, which is coupled with an initial increase and then a loss of surface NMDA-receptors. Thus, Aβ generates changes in GluR function and dendritic spine anatomy. Additionally, Fyn activation might suggest correlation with Tau pathology and the epileptiform phenotype observed in some patients with AD [162]. In this sense, it has been reported that oligomers of Aβ lead the activation of AMPK. The increased intracellular Ca2+ induced by membrane depolarization or NMDA receptor activation triggers AMP-activated kinase (AMPK) activation in a CAMKK2-dependent manner. CAMKK2 or AMPK overactivation is sufficient to induce dendritic spine loss [163]. The roles of AMPK in the pathogenesis of AD include β-amyloid protein (Aβ) generation and tau phosphorylation [164]. AMPK phosphorylates Tau in S262, while expression of Tau S262A inhibits the synap‐ totoxic effects of Aβ42 oligomers, which suggests that the CAMKK2-AMPK-Tau pathway could be a critical mediator of the synaptotoxic effects of Aβ42 oligomers [163]

**2.7. Aβ and synaptic plasticity**

of several neurodegenerative diseases.

**on memantine**

Although for many years the theory has been maintained that beta amyloid deposits are the main factor in AD pathology, recent years have seen an increase in the evidence pointing to the fact that its accumulation in certain brain regions may participate importantly in memory and cognition [176]. This dual concept of amyloid, where, at low doses it can positively stimulate the normal physiological processes of the cells, and at high doses it can cause toxic effects has also been observed in a very large number of molecules. This idea has been strongly supported by the observation that APP knock-out mice show long-term potentiation (LTP) and memory impairment [44, 177]. Glutamate, the main excitatory neurotransmitter is undoubtedly another example, as it is known that at low doses, it has the ability to stimulate synaptic plasticity and memory [178], while at high doses it is toxic and favors the development

Alzheimer Disease: The Role of Aβ in the Glutamatergic System

http://dx.doi.org/10.5772/57367

299

We have already mentioned that the amyloid is generated the proteolytic processing of APP throughthe actionofβ-andγ-secretases.Inrecentyears,ithasbeenreportedthatthese enzymes are involved in memory and synaptic plasticity. In 2004, Saura and colleagues developed conditional double knockout mice lacking the expression of both presenilins in the postnatal forebrain. The results showed impairments in hippocampal memory and synaptic plasticity. These alterations are associated with decreases in NMDAreceptor-mediated responses and the synaptic levels of NMDA receptors and αCaMKII. Also, a decrease was observed in the expression of CBP and CREB/CBP target genes, such as *c-fos* and BDNF, while, increased levels oftheCdk5 activatorp25 andhyperphosphorylatedTauwere also observed.Finally,thesemice develop a process of neurodegeneration, which increases with age. These results indicate that the inhibition of presenilin could accelerate memory loss and neurodegeneration [179]. Other trials have suggested that synaptic plasticity and memory depend on BACE1-mediated APP processing, which may facilitate memory andsynapticplasticity [180].In the same way,BACE1 null mice exhibit alterations in hippocampal synaptic plasticity as well as in their perform‐ ance in tests of cognition and emotion [181]. Recently it has been suggested that concentra‐ tions of picomolar amyloid are capable of inducing synaptic plasticity and memory in the hippocampus, and that the exposure of amyloid to Aß did not affect the NMDA receptor. The action mechanism of picomolar Aβ42 on synaptic plasticity and memory involves α7-nicotin‐ ic acetylcholine receptors [44], suggestingthatAβ42maybe animportantmodulatorof synaptic plasticity and memory in the normal brain. Furthermore it has been observed that many of the

effects on amyloid NMDA receptors can be blocked by antagonists of this receptor.

**3. Glutamatergic system-targeted treatment in Alzheimer's disease: Focus**

Aβ peptide is able to interact with a whole variety of proteins [97], and this interaction may cause dysfunction of the protein to which Aβ is binding. One group of proteins with which Aβ is able to interact is the glutamatergic NMDA receptors. Texidó *et al.* [182] showed that the Aβ peptide directly binds and activates NMDA receptors expressed in *Xenopus laevis* oocytes,

Dendritic spines are certainly the sites with more excitatory synapses, and their loss correlates with the cognitive impairment observed in Alzheimer's patients [165]. A large body of evidence suggests that amyloid oligomers may cause loss of dendritic spines [47, 166-172]. The exposure of cultured pyramidal neurons to Aβ oligomers showed decreased synaptic activity and a decrease in the density of dendritic spines [154]. Multiphoton imaging of GFP-labeled neurons in living Tg2576 APP mice showed disrupted neurite trajectories and reductions in dendritic spine density compared with age-matched control mice. Spine loss is most pro‐ nounced near plaques, indicating focal toxicity and also that the decrease in the density of dendritic spines may contribute to the altered neuronal function observed in these mice [166]. It has also been found that Aβ trimmers fully inhibit LTP, whereas dimers and tetramers have an intermediate potency and support the hypothesis that diffusible oligomers of Aβ initiate a synaptic dysfunction that may be an early event in AD [173]. It is known that the presence of oligomers of Aβ induces the loss of synapses, although little is known whether synapse loss precedes or follows plaque formation. In 2012, Bittner *et al* conducted an *in vivo* study using two-photon microscopy through a cranial window in double transgenic APPPS mice. Using this technique, they observed the manner in which the amyloid is deposited to form neuritic plaques and determined the loss of dendritic spines in the vicinity of these deposits. They found that the rate of dendritic spine loss in proximity to plaques is the same in both young and older animals. The plaque size only increases in young animals, while spine loss persists even many months after the initial appearance of the plaque. Finally, they found that spine loss occurs, with a significant time delay, after the birth of new plaques, and persists in the vicinity of amyloid plaques over many months [168].

A key aspect that determines the functionality of dendritic spines is their morphology. It is known that Calcineurin (CaN) activation is critically involved in regulating both the mor‐ phology of the spines in response to oligomeric Aβ, and the synaptic plasticity in normal memory. When adding oligomers derived from Tg2576 murine transgenic neurons or human AD brains to wild-type murine primary cortical neurons, CaN activation in spines was observed and led to rapid but reversible morphological changes in spines and postsynaptic proteins, suggesting that Calcineurin might have an important role in regulating the synaptic alterations associated with Alzheimer's disease [174]. Finally, it has been shown that APP has an important role in regulating synaptic and structure function. Analysis of dendritic spines in the primary cultures of hippocampal neurons and the CA1 neurons of hippocampi of APP −/− mice showed a significant decrease in spine density (35%), compared to control cultures. This spine loss was partially restored with sAPPα-conditioned medium. These abnormalities in neuronal morphology were also accompanied by a reduction in long-term potentiation. These results suggest that sAPPα is necessary for the maintenance of dendritic integrity in the hippocampus [172].

Thechanges indendriticspinesobservedinvariousdiseases impactheavilyonsynapsefunction and circuit-level connectivity in the form of altered connectivity or changes in connection strength [175]. Changes in the number and morphology of the spines can start a cascade of symptoms and effects that lead to the pathological changes observed in Alzheimer's disease.

#### **2.7. Aβ and synaptic plasticity**

totoxic effects of Aβ42 oligomers, which suggests that the CAMKK2-AMPK-Tau pathway

Dendritic spines are certainly the sites with more excitatory synapses, and their loss correlates with the cognitive impairment observed in Alzheimer's patients [165]. A large body of evidence suggests that amyloid oligomers may cause loss of dendritic spines [47, 166-172]. The exposure of cultured pyramidal neurons to Aβ oligomers showed decreased synaptic activity and a decrease in the density of dendritic spines [154]. Multiphoton imaging of GFP-labeled neurons in living Tg2576 APP mice showed disrupted neurite trajectories and reductions in dendritic spine density compared with age-matched control mice. Spine loss is most pro‐ nounced near plaques, indicating focal toxicity and also that the decrease in the density of dendritic spines may contribute to the altered neuronal function observed in these mice [166]. It has also been found that Aβ trimmers fully inhibit LTP, whereas dimers and tetramers have an intermediate potency and support the hypothesis that diffusible oligomers of Aβ initiate a synaptic dysfunction that may be an early event in AD [173]. It is known that the presence of oligomers of Aβ induces the loss of synapses, although little is known whether synapse loss precedes or follows plaque formation. In 2012, Bittner *et al* conducted an *in vivo* study using two-photon microscopy through a cranial window in double transgenic APPPS mice. Using this technique, they observed the manner in which the amyloid is deposited to form neuritic plaques and determined the loss of dendritic spines in the vicinity of these deposits. They found that the rate of dendritic spine loss in proximity to plaques is the same in both young and older animals. The plaque size only increases in young animals, while spine loss persists even many months after the initial appearance of the plaque. Finally, they found that spine loss occurs, with a significant time delay, after the birth of new plaques, and persists in the

A key aspect that determines the functionality of dendritic spines is their morphology. It is known that Calcineurin (CaN) activation is critically involved in regulating both the mor‐ phology of the spines in response to oligomeric Aβ, and the synaptic plasticity in normal memory. When adding oligomers derived from Tg2576 murine transgenic neurons or human AD brains to wild-type murine primary cortical neurons, CaN activation in spines was observed and led to rapid but reversible morphological changes in spines and postsynaptic proteins, suggesting that Calcineurin might have an important role in regulating the synaptic alterations associated with Alzheimer's disease [174]. Finally, it has been shown that APP has an important role in regulating synaptic and structure function. Analysis of dendritic spines in the primary cultures of hippocampal neurons and the CA1 neurons of hippocampi of APP −/− mice showed a significant decrease in spine density (35%), compared to control cultures. This spine loss was partially restored with sAPPα-conditioned medium. These abnormalities in neuronal morphology were also accompanied by a reduction in long-term potentiation. These results suggest that sAPPα is necessary for the maintenance of dendritic integrity in the

Thechanges indendriticspinesobservedinvariousdiseases impactheavilyonsynapsefunction and circuit-level connectivity in the form of altered connectivity or changes in connection strength [175]. Changes in the number and morphology of the spines can start a cascade of symptoms and effects that lead to the pathological changes observed in Alzheimer's disease.

could be a critical mediator of the synaptotoxic effects of Aβ42 oligomers [163]

vicinity of amyloid plaques over many months [168].

hippocampus [172].

298 Neurochemistry

Although for many years the theory has been maintained that beta amyloid deposits are the main factor in AD pathology, recent years have seen an increase in the evidence pointing to the fact that its accumulation in certain brain regions may participate importantly in memory and cognition [176]. This dual concept of amyloid, where, at low doses it can positively stimulate the normal physiological processes of the cells, and at high doses it can cause toxic effects has also been observed in a very large number of molecules. This idea has been strongly supported by the observation that APP knock-out mice show long-term potentiation (LTP) and memory impairment [44, 177]. Glutamate, the main excitatory neurotransmitter is undoubtedly another example, as it is known that at low doses, it has the ability to stimulate synaptic plasticity and memory [178], while at high doses it is toxic and favors the development of several neurodegenerative diseases.

We have already mentioned that the amyloid is generated the proteolytic processing of APP throughthe actionofβ-andγ-secretases.Inrecentyears,ithasbeenreportedthatthese enzymes are involved in memory and synaptic plasticity. In 2004, Saura and colleagues developed conditional double knockout mice lacking the expression of both presenilins in the postnatal forebrain. The results showed impairments in hippocampal memory and synaptic plasticity. These alterations are associated with decreases in NMDAreceptor-mediated responses and the synaptic levels of NMDA receptors and αCaMKII. Also, a decrease was observed in the expression of CBP and CREB/CBP target genes, such as *c-fos* and BDNF, while, increased levels oftheCdk5 activatorp25 andhyperphosphorylatedTauwere also observed.Finally,thesemice develop a process of neurodegeneration, which increases with age. These results indicate that the inhibition of presenilin could accelerate memory loss and neurodegeneration [179]. Other trials have suggested that synaptic plasticity and memory depend on BACE1-mediated APP processing, which may facilitate memory andsynapticplasticity [180].In the same way,BACE1 null mice exhibit alterations in hippocampal synaptic plasticity as well as in their perform‐ ance in tests of cognition and emotion [181]. Recently it has been suggested that concentra‐ tions of picomolar amyloid are capable of inducing synaptic plasticity and memory in the hippocampus, and that the exposure of amyloid to Aß did not affect the NMDA receptor. The action mechanism of picomolar Aβ42 on synaptic plasticity and memory involves α7-nicotin‐ ic acetylcholine receptors [44], suggestingthatAβ42maybe animportantmodulatorof synaptic plasticity and memory in the normal brain. Furthermore it has been observed that many of the effects on amyloid NMDA receptors can be blocked by antagonists of this receptor.

## **3. Glutamatergic system-targeted treatment in Alzheimer's disease: Focus on memantine**

Aβ peptide is able to interact with a whole variety of proteins [97], and this interaction may cause dysfunction of the protein to which Aβ is binding. One group of proteins with which Aβ is able to interact is the glutamatergic NMDA receptors. Texidó *et al.* [182] showed that the Aβ peptide directly binds and activates NMDA receptors expressed in *Xenopus laevis* oocytes, thereby causing an increase in cytosolic calcium concentration. These results are coincident with previous results by McDermott *et al.* [183], who reported increased calcium intracellular concentration in spinal cord mouse neuron cultures, after adding NMDA. Cytosolic calcium overload causes mitochondrial dysfunction and leads to an increase in ROS production, which in turn generates oxidative stress and leads to cell death [184]. For these reasons, the idea was born that NMDAR antagonists could be a promising therapeutic target in AD treatment.

Morris water maze, in an object recognition task and in a passive avoidance task, showing that

Alzheimer Disease: The Role of Aβ in the Glutamatergic System

http://dx.doi.org/10.5772/57367

301

In contrast, few studies have failed in obtaining a significant cognitive function improvement after memantine treatment. Dong et al. [193], using Tg2576 mice (which also express the Swedish mutation), did not find any treatment effect in a conditioned fear experiment after six months of daily memantine administration. Interestingly, histological analysis revealed that the memantine-treated group exhibited less Aβ plaque deposition, less axonal degeneration

Because memantine appeared to be effective and safe in animal model assays, clinical trials soon began to be developed. In general, results from these trials showed a modest effect increasing the preservation of cognitive function. Rive et al. [194] classified a group of 252 AD patients in "autonomous" or "dependant" according to their punctuation by the ADCS-ADL (Alzheimer's Disease Cooperative Study-Activities of Daily Living) scale and found that, after a 28-week treatment with memantine or placebo, memantine-treated patients had 3 times more probability of remaining autonomous than placebo-treated patients. Peskind et al. [195] measured the outcomes of 403 AD patients for the ADAS-cog (Alzheimer's Disease Assess‐ ment Scale-cognitive subscale), the CIBIC-Plus (Clinician's Interview-Based Impression of Change-Plus caregiver input) scale and the NPI (Neuro-Psychiatric Inventory) scale. Meas‐ urements were taken at the beginning of the study and after 24 weeks of memantine or placebo treatment. Results showed that memantine-treated patients exhibited better performance in all of these scales, when compared to placebo treated patients. Another study [196] showed that after 24 weeks of treatment with memantine or placebo, memantine-treated AD patients exhibited significantly slower cognitive decline compared with those treated with placebo, as measured by the SIB (Severe Impairment Battery). Memantine also showed to be moderately effective in the improvement of semantic memory. The study by Ferris et al. [197] found a significant amelioration of language impairment (assessed by the language subscale of the SIB) in AD patients after 28 weeks of memantine treatment. Another study [18] followed 295 AD patients receiving memantine or placebo during 52 weeks. Their results show that memantinetreated patients scored 1.2 points higher in the MMSE (Mini-Mental State Examination) than

All the aforementioned results point out that memantine is a safe disease-modifying drug to use in AD treatment, and its effectiveness has turned out to be slight, but significant, and comparable to that of other AD treatment drugs, such as cholinesterase inhibitors. Clinical trials in order to assess the effectiveness of combined treatment of memantine with other drugs

Finally, studies reported in the literature suggest that Aβ, the glutamatergic system, and in particular NMDA receptors have a major role in the processes of learning and memory. Synaptic plasticity can be regulated positively or negatively, depending on the levels and

and increased synaptic density, when compared with the placebo-treated group.

memantine has an effect on different types of memory.

placebo treated patients.

are currently being implemented.

**4. Conclusion**

Among all the known NMDAR antagonists, the most widely studied and used in the treatment of AD is the molecule known as memantine. Memantine (1-amine-3, 5, dimethyladamantane) was first synthesized in 1963 [185]. The drug is a derivative of amantadine, an antiviral used in influenza treatment. Like amantadine, memantine has a three ring structure, with an amine group and two methyl groups [186]. Memantine NMDAR antagonist properties remained unknown until Kornhuber et al. [187] reported that memantine had the same properties and same binding site of the well known NMDAR antagonist MK-801. Chen and Lipton [186] observed that memantine affinity towards NMDA receptors was sensitive to NMDA concen‐ tration, leading to the conclusion that memantine NMDA receptor antagonism is uncompeti‐ tive. It is this uncompetitivity and the fact that his binding is voltage-dependent which makes memantine an effective and safe therapeutic agent. For memantine to be able to exhibit its inhibitory activity, the receptor channel must be in an open state. Memantine blocks NMDAR activity by entering and binding to the cation pore, thus preventing cation flux and inhibiting functional NMDAR activity. Memantine binding to the receptor is voltage-dependant, in such a way that it leaves the channel pore in depolarization conditions, i.e. during excitatory postsynaptic potential, this way allowing synaptic activity to be maintained [188].

Memantine disease-modifying efficacy and safety has been proven in many studies. Most assays using a variety of AD animal models have lead to promising results. Minkeviciene et al. [189] showed that a 4 week oral treatment with memantine (via drinking water) improved the performance in the Morris water maze of mice carrying both a human APP transgene with the Swedish mutation and a human PS1 transgene with the A246E mutation, when compared with placebo-treated mice. In fact, this study showed that memantine-treated transgenic mice performed well in the water maze as well as WT mice, thus revealing a complete rescue of cognitive function due to memantine. Surprisingly, a later study [190] using this same mouse model did not find an effect of memantine treatment on performance in the Morris water maze, but memantine-treated mice performed better in a left-right discrimination task when compared with placebo-treated mice. Another study [191], which used heterozygous APP23 mice (mice carrying one copy of a human APP transgene with the Swedish mutation), reported an increase in spatial accuracy of memantine-treated mice in the Morris water maze, as measured by the time mice spent in the target quadrant of the maze. However, in this study, memantine failed to decrease escape latency (time that takes to mice to reach the target platform of the maze). Martínez-Coria et al. [192], using 3x-TgAD mice (mice that express simultaneously a human APP transgene carrying the Swedish mutation, a PS1 gene carrying the M146V mutation and a human tau transgene carrying a P301L mutation), showed that treatment with memantine caused a significant improvement in mice performance in the Morris water maze, in an object recognition task and in a passive avoidance task, showing that memantine has an effect on different types of memory.

In contrast, few studies have failed in obtaining a significant cognitive function improvement after memantine treatment. Dong et al. [193], using Tg2576 mice (which also express the Swedish mutation), did not find any treatment effect in a conditioned fear experiment after six months of daily memantine administration. Interestingly, histological analysis revealed that the memantine-treated group exhibited less Aβ plaque deposition, less axonal degeneration and increased synaptic density, when compared with the placebo-treated group.

Because memantine appeared to be effective and safe in animal model assays, clinical trials soon began to be developed. In general, results from these trials showed a modest effect increasing the preservation of cognitive function. Rive et al. [194] classified a group of 252 AD patients in "autonomous" or "dependant" according to their punctuation by the ADCS-ADL (Alzheimer's Disease Cooperative Study-Activities of Daily Living) scale and found that, after a 28-week treatment with memantine or placebo, memantine-treated patients had 3 times more probability of remaining autonomous than placebo-treated patients. Peskind et al. [195] measured the outcomes of 403 AD patients for the ADAS-cog (Alzheimer's Disease Assess‐ ment Scale-cognitive subscale), the CIBIC-Plus (Clinician's Interview-Based Impression of Change-Plus caregiver input) scale and the NPI (Neuro-Psychiatric Inventory) scale. Meas‐ urements were taken at the beginning of the study and after 24 weeks of memantine or placebo treatment. Results showed that memantine-treated patients exhibited better performance in all of these scales, when compared to placebo treated patients. Another study [196] showed that after 24 weeks of treatment with memantine or placebo, memantine-treated AD patients exhibited significantly slower cognitive decline compared with those treated with placebo, as measured by the SIB (Severe Impairment Battery). Memantine also showed to be moderately effective in the improvement of semantic memory. The study by Ferris et al. [197] found a significant amelioration of language impairment (assessed by the language subscale of the SIB) in AD patients after 28 weeks of memantine treatment. Another study [18] followed 295 AD patients receiving memantine or placebo during 52 weeks. Their results show that memantinetreated patients scored 1.2 points higher in the MMSE (Mini-Mental State Examination) than placebo treated patients.

## **4. Conclusion**

thereby causing an increase in cytosolic calcium concentration. These results are coincident with previous results by McDermott *et al.* [183], who reported increased calcium intracellular concentration in spinal cord mouse neuron cultures, after adding NMDA. Cytosolic calcium overload causes mitochondrial dysfunction and leads to an increase in ROS production, which in turn generates oxidative stress and leads to cell death [184]. For these reasons, the idea was born that NMDAR antagonists could be a promising therapeutic target in AD treatment.

300 Neurochemistry

Among all the known NMDAR antagonists, the most widely studied and used in the treatment of AD is the molecule known as memantine. Memantine (1-amine-3, 5, dimethyladamantane) was first synthesized in 1963 [185]. The drug is a derivative of amantadine, an antiviral used in influenza treatment. Like amantadine, memantine has a three ring structure, with an amine group and two methyl groups [186]. Memantine NMDAR antagonist properties remained unknown until Kornhuber et al. [187] reported that memantine had the same properties and same binding site of the well known NMDAR antagonist MK-801. Chen and Lipton [186] observed that memantine affinity towards NMDA receptors was sensitive to NMDA concen‐ tration, leading to the conclusion that memantine NMDA receptor antagonism is uncompeti‐ tive. It is this uncompetitivity and the fact that his binding is voltage-dependent which makes memantine an effective and safe therapeutic agent. For memantine to be able to exhibit its inhibitory activity, the receptor channel must be in an open state. Memantine blocks NMDAR activity by entering and binding to the cation pore, thus preventing cation flux and inhibiting functional NMDAR activity. Memantine binding to the receptor is voltage-dependant, in such a way that it leaves the channel pore in depolarization conditions, i.e. during excitatory post-

synaptic potential, this way allowing synaptic activity to be maintained [188].

Memantine disease-modifying efficacy and safety has been proven in many studies. Most assays using a variety of AD animal models have lead to promising results. Minkeviciene et al. [189] showed that a 4 week oral treatment with memantine (via drinking water) improved the performance in the Morris water maze of mice carrying both a human APP transgene with the Swedish mutation and a human PS1 transgene with the A246E mutation, when compared with placebo-treated mice. In fact, this study showed that memantine-treated transgenic mice performed well in the water maze as well as WT mice, thus revealing a complete rescue of cognitive function due to memantine. Surprisingly, a later study [190] using this same mouse model did not find an effect of memantine treatment on performance in the Morris water maze, but memantine-treated mice performed better in a left-right discrimination task when compared with placebo-treated mice. Another study [191], which used heterozygous APP23 mice (mice carrying one copy of a human APP transgene with the Swedish mutation), reported an increase in spatial accuracy of memantine-treated mice in the Morris water maze, as measured by the time mice spent in the target quadrant of the maze. However, in this study, memantine failed to decrease escape latency (time that takes to mice to reach the target platform of the maze). Martínez-Coria et al. [192], using 3x-TgAD mice (mice that express simultaneously a human APP transgene carrying the Swedish mutation, a PS1 gene carrying the M146V mutation and a human tau transgene carrying a P301L mutation), showed that treatment with memantine caused a significant improvement in mice performance in the

All the aforementioned results point out that memantine is a safe disease-modifying drug to use in AD treatment, and its effectiveness has turned out to be slight, but significant, and comparable to that of other AD treatment drugs, such as cholinesterase inhibitors. Clinical trials in order to assess the effectiveness of combined treatment of memantine with other drugs are currently being implemented.

Finally, studies reported in the literature suggest that Aβ, the glutamatergic system, and in particular NMDA receptors have a major role in the processes of learning and memory. Synaptic plasticity can be regulated positively or negatively, depending on the levels and degrees of amyloid oligomerization. The negative effect of these oligomeric forms may be reversed by the presence of NMDA receptor antagonists. In this regard, it has been reported that the noncompetitive antagonist memantine is able to block the "pathological" receptor activation exerted by these oligomers. In this view, an early pharmacological treatment with memantine, or even a memantine associated treatment combined with AChE inhibitors, might represent a very good option for the treatment of patients with AD.

[8] Sherrington, R., et al., Alzheimer's disease associated with mutations in presenilin 2

Alzheimer Disease: The Role of Aβ in the Glutamatergic System

http://dx.doi.org/10.5772/57367

303

[9] Sherrington, R., et al., Cloning of a gene bearing missense mutations in early-onset

[10] Kowalska, A., Amyloid precursor protein gene mutations responsible for early-onset autosomal dominant Alzheimer's disease. Folia Neuropathol, 2003. 41(1): p. 35-40.

[11] Corder, E.H., et al., Gene dose of apolipoprotein E type 4 allele and the risk of Alz‐

[12] Schupf, N. and G.H. Sergievsky, Genetic and host factors for dementia in Down's

[13] Tang, M.X., et al., Relative risk of Alzheimer disease and age-at-onset distributions, based on APOE genotypes among elderly African Americans, Caucasians, and His‐

[14] Zahs, K.R. and K.H. Ashe, beta-Amyloid oligomers in aging and Alzheimer's disease.

[15] Chen, J.H., K.P. Lin, and Y.C. Chen, Risk factors for dementia. J Formos Med Assoc,

[16] de la Torre, J.C., How do heart disease and stroke become risk factors for Alzheimer's

[17] Holscher, C., Diabetes as a risk factor for Alzheimer's disease: insulin signalling im‐ pairment in the brain as an alternative model of Alzheimer's disease. Biochem Soc

[18] Goedert, M., et al., Cloning and sequencing of the cDNA encoding a core protein of the paired helical filament of Alzheimer disease: identification as the microtubule-as‐

[19] Jakes, R., et al., Identification of 3- and 4-repeat tau isoforms within the PHF in Alz‐

[20] Wischik, C.M., et al., Structural characterization of the core of the paired helical fila‐ ment of Alzheimer disease. Proc Natl Acad Sci U S A, 1988. 85(13): p. 4884-8.

[21] Wischik, C.M., et al., Isolation of a fragment of tau derived from the core of the paired helical filament of Alzheimer disease. Proc Natl Acad Sci U S A, 1988. 85(12):

[22] Iversen, L.L., et al., The toxicity in vitro of beta-amyloid protein. Biochem J, 1995. 311

[23] Glenner, G.G., et al., The amyloid deposits in Alzheimer's disease: their nature and

sociated protein tau. Proc Natl Acad Sci U S A, 1988. 85(11): p. 4051-5.

heimer's disease in late onset families. Science, 1993. 261(5123): p. 921-3.

panics in New York City. Am J Hum Genet, 1996. 58(3): p. 574-84.

is rare and variably penetrant. Hum Mol Genet, 1996. 5(7): p. 985-8.

familial Alzheimer's disease. Nature, 1995. 375(6534): p. 754-60.

syndrome. Br J Psychiatry, 2002. 180: p. 405-10.

Front Aging Neurosci, 2013. 5: p. 28.

disease? Neurol Res, 2006. 28(6): p. 637-44.

heimer's disease. EMBO J, 1991. 10(10): p. 2725-9.

pathogenesis. Appl Pathol, 1984. 2(6): p. 357-69.

2009. 108(10): p. 754-64.

Trans, 2011. 39(4): p. 891-7.

p. 4506-10.

( Pt 1): p. 1-16.

## **Author details**

Victoria Campos-Peña1 and Marco Antonio Meraz-Ríos1\*

\*Address all correspondence to: mmeraz@cinvestav.mx

1 Laboratorio Experimental de Enfermedades Neurodegenerativas, Instituto Nacional de Neurología y Neurocirugía, Manuel Velasco Suárez, México City, México

Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados del IPN, (CINVESTAV-IPN), México City, México

#### **References**


[8] Sherrington, R., et al., Alzheimer's disease associated with mutations in presenilin 2 is rare and variably penetrant. Hum Mol Genet, 1996. 5(7): p. 985-8.

degrees of amyloid oligomerization. The negative effect of these oligomeric forms may be reversed by the presence of NMDA receptor antagonists. In this regard, it has been reported that the noncompetitive antagonist memantine is able to block the "pathological" receptor activation exerted by these oligomers. In this view, an early pharmacological treatment with memantine, or even a memantine associated treatment combined with AChE inhibitors, might

represent a very good option for the treatment of patients with AD.

\*Address all correspondence to: mmeraz@cinvestav.mx

del IPN, (CINVESTAV-IPN), México City, México

Neurosci Lett, 1991. 129(1): p. 134-5.

ence, 1995. 269(5226): p. 970-3.

presenilin 1. Nature, 1996. 383(6602): p. 710-3.

lins. Am J Hum Genet, 1999. 65(1): p. 7-12.

transport. J Neurosci, 2003. 23(11): p. 4499-508.

and Marco Antonio Meraz-Ríos1\*

Neurología y Neurocirugía, Manuel Velasco Suárez, México City, México

1 Laboratorio Experimental de Enfermedades Neurodegenerativas, Instituto Nacional de

Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados

[1] Borchelt, D.R., et al., Familial Alzheimer's disease-linked presenilin 1 variants elevate

[2] Chartier-Harlin, M.C., et al., Screening for the beta-amyloid precursor protein muta‐ tion (APP717: Val----Ile) in extended pedigrees with early onset Alzheimer's disease.

[3] Duff, K., et al., Increased amyloid-beta42(43) in brains of mice expressing mutant

[4] Levy-Lahad, E., et al., A familial Alzheimer's disease locus on chromosome 1. Sci‐

[5] Murrell, J., et al., A mutation in the amyloid precursor protein associated with he‐

[6] Sisodia, S.S., S.H. Kim, and G. Thinakaran, Function and dysfunction of the preseni‐

[7] Pigino, G., et al., Alzheimer's presenilin 1 mutations impair kinesin-based axonal

reditary Alzheimer's disease. Science, 1991. 254(5028): p. 97-9.

Abeta1-42/1-40 ratio in vitro and in vivo. Neuron, 1996. 17(5): p. 1005-13.

**Author details**

302 Neurochemistry

**References**

Victoria Campos-Peña1


[24] Selkoe, D.J., Alzheimer's disease: a central role for amyloid. J Neuropathol Exp Neu‐ rol, 1994. 53(5): p. 438-47.

[38] Hynd, M.R., H.L. Scott, and P.R. Dodd, Glutamate-mediated excitotoxicity and neu‐ rodegeneration in Alzheimer's disease. Neurochem Int, 2004. 45(5): p. 583-95.

Alzheimer Disease: The Role of Aβ in the Glutamatergic System

http://dx.doi.org/10.5772/57367

305

[39] Crimins, J.L., et al., The intersection of amyloid beta and tau in glutamatergic synap‐ tic dysfunction and collapse in Alzheimer's disease. Ageing Res Rev, 2013.

[40] Boehm, J., A 'danse macabre': tau and Fyn in STEP with amyloid beta to facilitate in‐ duction of synaptic depression and excitotoxicity. Eur J Neurosci, 2013. 37(12): p.

[41] Robakis, N.K., et al., Chromosome 21q21 sublocalisation of gene encoding beta-amy‐ loid peptide in cerebral vessels and neuritic (senile) plaques of people with Alzheim‐

[42] Yoshikai, S., et al., Genomic organization of the human-amyloid beta-protein precur‐

[43] Jarrett, J.T., E.P. Berger, and P.T. Lansbury, Jr., The carboxy terminus of the beta amyloid protein is critical for the seeding of amyloid formation: implications for the

[44] Puzzo, D., et al., Picomolar amyloid-beta positively modulates synaptic plasticity and

[45] Lambert, M.P., et al., Diffusible, nonfibrillar ligands derived from Abeta1-42 are po‐ tent central nervous system neurotoxins. Proc Natl Acad Sci U S A, 1998. 95(11): p.

[46] Walsh, D.M. and D.J. Selkoe, Oligomers on the brain: the emerging role of soluble protein aggregates in neurodegeneration. Protein Pept Lett, 2004. 11(3): p. 213-28.

[47] Lacor, P.N., et al., Abeta oligomer-induced aberrations in synapse composition, shape, and density provide a molecular basis for loss of connectivity in Alzheimer's

[48] Cuello, A.C., Intracellular and extracellular Abeta, a tale of two neuropathologies.

[49] Selkoe, D.J., et al., The role of APP processing and trafficking pathways in the forma‐

[50] Walsh, D.M., et al., The oligomerization of amyloid beta-protein begins intracellular‐ ly in cells derived from human brain. Biochemistry, 2000. 39(35): p. 10831-9.

[51] Lesne, S.E., et al., Brain amyloid-beta oligomers in ageing and Alzheimer's disease.

[52] Tomiyama, T., et al., A mouse model of amyloid beta oligomers: their contribution to synaptic alteration, abnormal tau phosphorylation, glial activation, and neuronal loss

tion of amyloid beta-protein. Ann N Y Acad Sci, 1996. 777: p. 57-64.

pathogenesis of Alzheimer's disease. Biochemistry, 1993. 32(18): p. 4693-7.

er disease and Down syndrome. Lancet, 1987. 1(8529): p. 384-5.

memory in hippocampus. J Neurosci, 2008. 28(53): p. 14537-45.

sor gene. Gene, 1991. 102(2): p. 291-2.

disease. J Neurosci, 2007. 27(4): p. 796-807.

Brain Pathol, 2005. 15(1): p. 66-71.

Brain, 2013. 136(Pt 5): p. 1383-98.

in vivo. J Neurosci, 2010. 30(14): p. 4845-56.

1925-30.

6448-53.


[38] Hynd, M.R., H.L. Scott, and P.R. Dodd, Glutamate-mediated excitotoxicity and neu‐ rodegeneration in Alzheimer's disease. Neurochem Int, 2004. 45(5): p. 583-95.

[24] Selkoe, D.J., Alzheimer's disease: a central role for amyloid. J Neuropathol Exp Neu‐

[25] Kang, J., et al., The precursor of Alzheimer's disease amyloid A4 protein resembles a

[26] Samuel, W., et al., Hippocampal connectivity and Alzheimer's dementia: effects of synapse loss and tangle frequency in a two-component model. Neurology, 1994.

[27] Arriagada, P.V., et al., Neurofibrillary tangles but not senile plaques parallel dura‐ tion and severity of Alzheimer's disease. Neurology, 1992. 42(3 Pt 1): p. 631-9.

[28] Giannakopoulos, P., et al., Tangle and neuron numbers, but not amyloid load, pre‐ dict cognitive status in Alzheimer's disease. Neurology, 2003. 60(9): p. 1495-500. [29] Serrano-Pozo, A., et al., Neuropathological alterations in Alzheimer disease. Cold

[30] Puzzo, D. and O. Arancio, Fibrillar beta-amyloid impairs the late phase of long term

[31] Puzzo, D., A. Palmeri, and O. Arancio, Involvement of the nitric oxide pathway in synaptic dysfunction following amyloid elevation in Alzheimer's disease. Rev Neu‐

[32] Prasansuklab, A. and T. Tencomnao, Amyloidosis in Alzheimer's Disease: The Toxic‐ ity of Amyloid Beta (A beta ), Mechanisms of Its Accumulation and Implications of Medicinal Plants for Therapy. Evid Based Complement Alternat Med, 2013. 2013: p.

[33] Manczak, M. and P.H. Reddy, Abnormal Interaction of Oligomeric Amyloid-beta with Phosphorylated Tau: Implications to Synaptic Dysfunction and Neuronal Dam‐

[34] Reddy, P.H., Amyloid beta-induced glycogen synthase kinase 3beta phosphorylated VDAC1 in Alzheimer's disease: Implications for synaptic dysfunction and neuronal

[35] Magdesian, M.H., et al., Amyloid-beta binds to the extracellular cysteine-rich domain of Frizzled and inhibits Wnt/beta-catenin signaling. J Biol Chem, 2008. 283(14): p.

[36] Supnet, C. and I. Bezprozvanny, The dysregulation of intracellular calcium in Alz‐

[37] Supnet, C. and I. Bezprozvanny, Neuronal calcium signaling, mitochondrial dysfunc‐ tion, and Alzheimer's disease. J Alzheimers Dis, 2010. 20 Suppl 2: p. S487-98.

cell-surface receptor. Nature, 1987. 325(6106): p. 733-6.

Spring Harb Perspect Med, 2011. 1(1): p. a006189.

age. J Alzheimers Dis, 2013. 36(2): p. 285-95.

heimer disease. Cell Calcium, 2010. 47(2): p. 183-9.

damage. Biochim Biophys Acta, 2013.

potentiation. Curr Alzheimer Res, 2006. 3(3): p. 179-83.

rol, 1994. 53(5): p. 438-47.

rosci, 2006. 17(5): p. 497-523.

413808.

9359-68.

44(11): p. 2081-8.

304 Neurochemistry


[53] Yamamoto, N., et al., GM1-ganglioside-induced Abeta assembly on synaptic mem‐ branes of cultured neurons. Biochim Biophys Acta, 2007. 1768(5): p. 1128-37.

[67] Gouras, G.K., et al., Intraneuronal beta-amyloid accumulation and synapse patholo‐

Alzheimer Disease: The Role of Aβ in the Glutamatergic System

http://dx.doi.org/10.5772/57367

307

[68] Gouras, G.K., et al., Intraneuronal Abeta42 accumulation in human brain. Am J Path‐

[69] LaFerla, F.M., K.N. Green, and S. Oddo, Intracellular amyloid-beta in Alzheimer's

[70] Takahashi, R.H., et al., Co-occurrence of Alzheimer's disease ss-amyloid and tau

[71] Takahashi, R.H., et al., Accumulation of intraneuronal beta-amyloid 42 peptides is associated with early changes in microtubule-associated protein 2 in neurites and

[72] Kokubo, H., et al., Soluble Abeta oligomers ultrastructurally localize to cell processes and might be related to synaptic dysfunction in Alzheimer's disease brain. Brain Res,

[73] Hartmann, T., et al., Distinct sites of intracellular production for Alzheimer's disease

[74] Skovronsky, D.M., R.W. Doms, and V.M. Lee, Detection of a novel intraneuronal pool of insoluble amyloid beta protein that accumulates with time in culture. J Cell

[75] Wild-Bode, C., et al., Intracellular generation and accumulation of amyloid beta-pep‐

[77] Forloni, G., et al., Apoptosis mediated neurotoxicity induced by chronic application

[78] Loo, D.T., et al., Apoptosis is induced by beta-amyloid in cultured central nervous

[79] Almeida, C.G., R.H. Takahashi, and G.K. Gouras, Beta-amyloid accumulation im‐ pairs multivesicular body sorting by inhibiting the ubiquitin-proteasome system. J

[80] Oh, S., et al., Amyloid peptide attenuates the proteasome activity in neuronal cells.

[81] Tseng, B.P., et al., Abeta inhibits the proteasome and enhances amyloid and tau accu‐

[82] Butterfield, D.A. and R. Sultana, Methionine-35 of abeta(1-42): importance for oxida‐

tive stress in Alzheimer disease. J Amino Acids, 2011. 2011: p. 198430.

tide terminating at amino acid 42. J Biol Chem, 1997. 272(26): p. 16085-8.

[76] Forloni, G., beta-Amyloid neurotoxicity. Funct Neurol, 1993. 8(3): p. 211-25.

of beta amyloid fragment 25-35. Neuroreport, 1993. 4(5): p. 523-6.

system neurons. Proc Natl Acad Sci U S A, 1993. 90(17): p. 7951-5.

gy in Alzheimer's disease. Acta Neuropathol, 2010. 119(5): p. 523-41.

pathologies at synapses. Neurobiol Aging, 2010. 31(7): p. 1145-52.

A beta40/42 amyloid peptides. Nat Med, 1997. 3(9): p. 1016-20.

disease. Nat Rev Neurosci, 2007. 8(7): p. 499-509.

synapses. PLoS One, 2013. 8(1): p. e51965.

ol, 2000. 156(1): p. 15-20.

2005. 1031(2): p. 222-8.

Biol, 1998. 141(4): p. 1031-9.

Neurosci, 2006. 26(16): p. 4277-88.

Mech Ageing Dev, 2005. 126(12): p. 1292-9.

mulation. Neurobiol Aging, 2008. 29(11): p. 1607-18.


[67] Gouras, G.K., et al., Intraneuronal beta-amyloid accumulation and synapse patholo‐ gy in Alzheimer's disease. Acta Neuropathol, 2010. 119(5): p. 523-41.

[53] Yamamoto, N., et al., GM1-ganglioside-induced Abeta assembly on synaptic mem‐ branes of cultured neurons. Biochim Biophys Acta, 2007. 1768(5): p. 1128-37.

[54] McLaurin, J., et al., Structural transitions associated with the interaction of Alzheimer beta-amyloid peptides with gangliosides. J Biol Chem, 1998. 273(8): p. 4506-15. [55] Oda, T., et al., Clusterin (apoJ) alters the aggregation of amyloid beta-peptide (A beta 1-42) and forms slowly sedimenting A beta complexes that cause oxidative stress.

[56] Stege, G.J., et al., The molecular chaperone alphaB-crystallin enhances amyloid beta

[57] Sakono, M. and T. Zako, Amyloid oligomers: formation and toxicity of Abeta oligom‐

[58] Butterfield, S.M. and H.A. Lashuel, Amyloidogenic protein-membrane interactions: mechanistic insight from model systems. Angew Chem Int Ed Engl, 2010. 49(33): p.

[59] Kawahara, M., Neurotoxicity of beta-amyloid protein: oligomerization, channel for‐ mation, and calcium dyshomeostasis. Curr Pharm Des, 2010. 16(25): p. 2779-89. [60] Valincius, G., et al., Soluble amyloid beta-oligomers affect dielectric membrane prop‐ erties by bilayer insertion and domain formation: implications for cell toxicity. Bio‐

[61] De Felice, F.G., et al., Abeta oligomers induce neuronal oxidative stress through an N-methyl-D-aspartate receptor-dependent mechanism that is blocked by the Alz‐

[62] Reddy, P.H. and M.F. Beal, Amyloid beta, mitochondrial dysfunction and synaptic damage: implications for cognitive decline in aging and Alzheimer's disease. Trends

[63] Shankar, G.M., et al., Natural oligomers of the Alzheimer amyloid-beta protein in‐ duce reversible synapse loss by modulating an NMDA-type glutamate receptor-de‐

[64] Grundke-Iqbal, I., et al., Amyloid protein and neurofibrillary tangles coexist in the same neuron in Alzheimer disease. Proc Natl Acad Sci U S A, 1989. 86(8): p. 2853-7.

[65] Blurton-Jones, M. and F.M. Laferla, Pathways by which Abeta facilitates tau patholo‐

[66] Wertkin, A.M., et al., Human neurons derived from a teratocarcinoma cell line ex‐ press solely the 695-amino acid amyloid precursor protein and produce intracellular beta-amyloid or A4 peptides. Proc Natl Acad Sci U S A, 1993. 90(20): p. 9513-7.

heimer drug memantine. J Biol Chem, 2007. 282(15): p. 11590-601.

pendent signaling pathway. J Neurosci, 2007. 27(11): p. 2866-75.

gy. Curr Alzheimer Res, 2006. 3(5): p. 437-48.

neurotoxicity. Biochem Biophys Res Commun, 1999. 262(1): p. 152-6.

Exp Neurol, 1995. 136(1): p. 22-31.

ers. FEBS J, 2010. 277(6): p. 1348-58.

phys J, 2008. 95(10): p. 4845-61.

Mol Med, 2008. 14(2): p. 45-53.

5628-54.

306 Neurochemistry


[83] Christen, Y., Oxidative stress and Alzheimer disease. Am J Clin Nutr, 2000. 71(2): p. 621S-629S.

[97] Danysz, W. and C.G. Parsons, Alzheimer's disease, beta-amyloid, glutamate, NMDA receptors and memantine--searching for the connections. Br J Pharmacol, 2012.

Alzheimer Disease: The Role of Aβ in the Glutamatergic System

http://dx.doi.org/10.5772/57367

309

[98] Gao, S.F. and A.M. Bao, Corticotropin-releasing hormone, glutamate, and gamma-

[99] Revett, T.J., et al., Glutamate system, amyloid ss peptides and tau protein: functional interrelationships and relevance to Alzheimer disease pathology. J Psychiatry Neuro‐

[100] Collingridge, G.L. and R.A. Lester, Excitatory amino acid receptors in the vertebrate

[101] Komuro, H. and P. Rakic, Modulation of neuronal migration by NMDA receptors.

[102] Rossi, D.J. and N.T. Slater, The developmental onset of NMDA receptor-channel ac‐ tivity during neuronal migration. Neuropharmacology, 1993. 32(11): p. 1239-48.

[103] Ottersen, O.P., et al., Ischemic disruption of glutamate homeostasis in brain: quanti‐ tative immunocytochemical analyses. J Chem Neuroanat, 1996. 12(1): p. 1-14.

[104] Ottersen, O.P., et al., Molecular organization of a type of peripheral glutamate syn‐ apse: the afferent synapses of hair cells in the inner ear. Prog Neurobiol, 1998. 54(2):

[105] Gasparini, C.F. and L.R. Griffiths, The biology of the glutamatergic system and po‐

[106] Wenk, G.L., Neuropathologic changes in Alzheimer's disease: potential targets for

[107] Wenk, G.L., C.G. Parsons, and W. Danysz, Potential role of N-methyl-D-aspartate re‐ ceptors as executors of neurodegeneration resulting from diverse insults: focus on

[108] Bell, K.F., D.A. Bennett, and A.C. Cuello, Paradoxical upregulation of glutamatergic presynaptic boutons during mild cognitive impairment. J Neurosci, 2007. 27(40): p.

[109] Fujikawa, D.G., Prolonged seizures and cellular injury: understanding the connec‐

[110] Won, S.J., D.Y. Kim, and B.J. Gwag, Cellular and molecular pathways of ischemic

[111] Yi, J.H. and A.S. Hazell, Excitotoxic mechanisms and the role of astrocytic glutamate transporters in traumatic brain injury. Neurochem Int, 2006. 48(5): p. 394-403.

aminobutyric acid in depression. Neuroscientist, 2011. 17(1): p. 124-44.

central nervous system. Pharmacol Rev, 1989. 41(2): p. 143-210.

tential role in migraine. Int J Biomed Sci, 2013. 9(1): p. 1-8.

treatment. J Clin Psychiatry, 2006. 67 Suppl 3: p. 3-7; quiz 23.

memantine. Behav Pharmacol, 2006. 17(5-6): p. 411-24.

neuronal death. J Biochem Mol Biol, 2002. 35(1): p. 67-86.

tion. Epilepsy Behav, 2005. 7 Suppl 3: p. S3-11.

167(2): p. 324-52.

sci, 2013. 38(1): p. 6-23.

p. 127-48.

10810-7.

Science, 1993. 260(5104): p. 95-7.


[97] Danysz, W. and C.G. Parsons, Alzheimer's disease, beta-amyloid, glutamate, NMDA receptors and memantine--searching for the connections. Br J Pharmacol, 2012. 167(2): p. 324-52.

[83] Christen, Y., Oxidative stress and Alzheimer disease. Am J Clin Nutr, 2000. 71(2): p.

[84] Ramassamy, C., et al., Oxidative insults are associated with apolipoprotein E geno‐

[85] Sultana, R., et al., Increased protein and lipid oxidative damage in mitochondria iso‐ lated from lymphocytes from patients with Alzheimer's disease: insights into the role of oxidative stress in Alzheimer's disease and initial investigations into a potential bi‐

[86] Mattson, M.P., et al., beta-Amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity. J Neurosci, 1992. 12(2): p.

[87] Hardy, J., et al., Region-specific loss of glutamate innervation in Alzheimer's disease.

[88] Maragos, W.F., et al., Loss of hippocampal [3H]TCP binding in Alzheimer's disease.

[89] Deshpande, A., et al., A role for synaptic zinc in activity-dependent Abeta oligomer formation and accumulation at excitatory synapses. J Neurosci, 2009. 29(13): p.

[90] Snyder, E.M., et al., Regulation of NMDA receptor trafficking by amyloid-beta. Nat

[91] Roselli, F., et al., Soluble beta-amyloid1-40 induces NMDA-dependent degradation of postsynaptic density-95 at glutamatergic synapses. J Neurosci, 2005. 25(48): p.

[92] Frisardi, V., F. Panza, and A.A. Farooqui, Late-life depression and Alzheimer's dis‐ ease: the glutamatergic system inside of this mirror relationship. Brain Res Rev, 2011.

[93] Walton, H.S. and P.R. Dodd, Glutamate-glutamine cycling in Alzheimer's disease.

[95] Hazell, A.S., et al., Selective down-regulation of the astrocyte glutamate transporters GLT-1 and GLAST within the medial thalamus in experimental Wernicke's encephal‐

[96] Moriyama, Y. and H. Omote, Vesicular glutamate transporter acts as a metabolic reg‐

[94] Danbolt, N.C., Glutamate uptake. Prog Neurobiol, 2001. 65(1): p. 1-105.

omarker for this dementing disorder. J Alzheimers Dis, 2011. 24(1): p. 77-84.

type in Alzheimer's disease brain. Neurobiol Dis, 2000. 7(1): p. 23-37.

621S-629S.

308 Neurochemistry

376-89.

4004-15.

11061-70.

67(1-2): p. 344-55.

Neurosci Lett, 1987. 73(1): p. 77-80.

Neurosci Lett, 1987. 74(3): p. 371-6.

Neurosci, 2005. 8(8): p. 1051-8.

Neurochem Int, 2007. 50(7-8): p. 1052-66.

opathy. J Neurochem, 2001. 78(3): p. 560-8.

ulator. Biol Pharm Bull, 2008. 31(10): p. 1844-6.


[112] Chen, K.H., et al., Disturbed neurotransmitter transporter expression in Alzheimer's disease brain. J Alzheimers Dis, 2011. 26(4): p. 755-66.

[128] Maleszka, R., P. Helliwell, and R. Kucharski, Pharmacological interference with glu‐ tamate re-uptake impairs long-term memory in the honeybee, apis mellifera. Behav

Alzheimer Disease: The Role of Aβ in the Glutamatergic System

http://dx.doi.org/10.5772/57367

311

[129] May-Simera, H. and E.D. Levin, NMDA systems in the amygdala and piriform cortex and nicotinic effects on memory function. Brain Res Cogn Brain Res, 2003. 17(2): p.

[130] Si, A., P. Helliwell, and R. Maleszka, Effects of NMDA receptor antagonists on olfac‐ tory learning and memory in the honeybee (Apis mellifera). Pharmacol Biochem Be‐

[131] Wong, R.W., et al., Overexpression of motor protein KIF17 enhances spatial and working memory in transgenic mice. Proc Natl Acad Sci U S A, 2002. 99(22): p.

[132] Izquierdo, I. and J.H. Medina, Role of the amygdala, hippocampus and entorhinal cortex in memory consolidation and expression. Braz J Med Biol Res, 1993. 26(6): p.

[133] Izquierdo, I., et al., Memory processing by the limbic system: role of specific neuro‐

[134] Rickard, N.S., K.T. Ng, and M.E. Gibbs, A nitric oxide agonist stimulates consolida‐ tion of long-term memory in the 1-day-old chick. Behav Neurosci, 1994. 108(3): p.

[135] Rickard, N.S., et al., Both non-NMDA and NMDA glutamate receptors are necessary for memory consolidation in the day-old chick. Behav Neural Biol, 1994. 62(1): p.

[136] Bezprozvanny, I. and P.R. Hiesinger, The synaptic maintenance problem: membrane recycling, Ca2+ homeostasis and late onset degeneration. Mol Neurodegener, 2013. 8:

[137] Aksenov, M.Y., et al., Oxidative modification of glutamine synthetase by amyloid be‐

[138] Blanchard, B.J., V.L. Thomas, and V.M. Ingram, Mechanism of membrane depolari‐ zation caused by the Alzheimer Abeta1-42 peptide. Biochem Biophys Res Commun,

[139] Lue, L.F., et al., Soluble amyloid beta peptide concentration as a predictor of synaptic

[140] McLean, C.A., et al., Soluble pool of Abeta amyloid as a determinant of severity of neurodegeneration in Alzheimer's disease. Ann Neurol, 1999. 46(6): p. 860-6.

[141] Kayed, R., et al., Common structure of soluble amyloid oligomers implies common

change in Alzheimer's disease. Am J Pathol, 1999. 155(3): p. 853-62.

mechanism of pathogenesis. Science, 2003. 300(5618): p. 486-9.

transmitter systems. Behav Brain Res, 1993. 58(1-2): p. 91-8.

ta peptide. Free Radic Res, 1997. 27(3): p. 267-81.

Brain Res, 2000. 115(1): p. 49-53.

hav, 2004. 77(2): p. 191-7.

475-83.

14500-5.

573-89.

640-4.

33-40.

p. 23.

2002. 293(4): p. 1197-203.


[128] Maleszka, R., P. Helliwell, and R. Kucharski, Pharmacological interference with glu‐ tamate re-uptake impairs long-term memory in the honeybee, apis mellifera. Behav Brain Res, 2000. 115(1): p. 49-53.

[112] Chen, K.H., et al., Disturbed neurotransmitter transporter expression in Alzheimer's

[113] Shigeri, Y., R.P. Seal, and K. Shimamoto, Molecular pharmacology of glutamate transporters, EAATs and VGLUTs. Brain Res Brain Res Rev, 2004. 45(3): p. 250-65.

[114] Kalariti, N., N. Pissimissis, and M. Koutsilieris, The glutamatergic system outside the CNS and in cancer biology. Expert Opin Investig Drugs, 2005. 14(12): p. 1487-96. [115] Omote, H., et al., Vesicular neurotransmitter transporter: bioenergetics and regula‐

[116] Kalandadze, A., Y. Wu, and M.B. Robinson, Protein kinase C activation decreases cell surface expression of the GLT-1 subtype of glutamate transporter. Requirement of a carboxyl-terminal domain and partial dependence on serine 486. J Biol Chem, 2002.

[117] Robinson, M.B., Regulated trafficking of neurotransmitter transporters: common

[118] Schoepfer, R., et al., Molecular biology of glutamate receptors. Prog Neurobiol, 1994.

[119] Mitchell, N.D. and G.B. Baker, An update on the role of glutamate in the pathophysi‐

[120] Kew, J.N. and J.A. Kemp, Ionotropic and metabotropic glutamate receptor structure

[121] Lau, C.G. and R.S. Zukin, NMDA receptor trafficking in synaptic plasticity and neu‐

[122] Mellone, M. and F. Gardoni, Modulation of NMDA receptor at the synapse: Promis‐ ing therapeutic interventions in disorders of the nervous system. Eur J Pharmacol,

[123] Sanz-Clemente, A., R.A. Nicoll, and K.W. Roche, Diversity in NMDA receptor com‐ position: many regulators, many consequences. Neuroscientist, 2013. 19(1): p. 62-75.

[124] Traynelis, S.F., et al., Glutamate receptor ion channels: structure, regulation, and

[125] Horak, M. and R.J. Wenthold, Different roles of C-terminal cassettes in the trafficking of full-length NR1 subunits to the cell surface. J Biol Chem, 2009. 284(15): p. 9683-91.

[126] Luscher, C., et al., Synaptic plasticity and dynamic modulation of the postsynaptic

[127] Lozano, V.C., C. Armengaud, and M. Gauthier, Memory impairment induced by cholinergic antagonists injected into the mushroom bodies of the honeybee. J Comp

tion of glutamate transport. Biochemistry, 2011. 50(25): p. 5558-65.

notes but different melodies. J Neurochem, 2002. 80(1): p. 1-11.

ology of depression. Acta Psychiatr Scand, 2010. 122(3): p. 192-210.

and pharmacology. Psychopharmacology (Berl), 2005. 179(1): p. 4-29.

ropsychiatric disorders. Nat Rev Neurosci, 2007. 8(6): p. 413-26.

function. Pharmacol Rev, 2010. 62(3): p. 405-96.

membrane. Nat Neurosci, 2000. 3(6): p. 545-50.

Physiol A, 2001. 187(4): p. 249-54.

disease brain. J Alzheimers Dis, 2011. 26(4): p. 755-66.

277(48): p. 45741-50.

310 Neurochemistry

42(2): p. 353-7.

2013.


[142] Kakio, A., et al., Formation of a membrane-active form of amyloid beta-protein in raft-like model membranes. Biochem Biophys Res Commun, 2003. 303(2): p. 514-8.

[158] Hsia, A.Y., et al., Plaque-independent disruption of neural circuits in Alzheimer's disease mouse models. Proc Natl Acad Sci U S A, 1999. 96(6): p. 3228-33.

Alzheimer Disease: The Role of Aβ in the Glutamatergic System

http://dx.doi.org/10.5772/57367

313

[159] Tseng, B.P., M. Kitazawa, and F.M. LaFerla, Amyloid beta-peptide: the inside story.

[160] LaFerla, F.M. and S. Oddo, Alzheimer's disease: Abeta, tau and synaptic dysfunction.

[161] Lauren, J., et al., Cellular prion protein mediates impairment of synaptic plasticity by

[162] Um, J.W., et al., Alzheimer amyloid-beta oligomer bound to postsynaptic prion pro‐ tein activates Fyn to impair neurons. Nat Neurosci, 2012. 15(9): p. 1227-35.

[163] Mairet-Coello, G., et al., The CAMKK2-AMPK kinase pathway mediates the synapto‐ toxic effects of Abeta oligomers through Tau phosphorylation. Neuron, 2013. 78(1): p.

[164] Cai, Z., et al., Roles of AMP-activated protein kinase in Alzheimer's disease. Neuro‐

[165] Scheff, S.W., et al., Synaptic alterations in CA1 in mild Alzheimer disease and mild

[166] Spires, T.L., et al., Dendritic spine abnormalities in amyloid precursor protein trans‐ genic mice demonstrated by gene transfer and intravital multiphoton microscopy. J

[167] Lacor, P.N., Advances on the understanding of the origins of synaptic pathology in

[168] Bittner, T., et al., Amyloid plaque formation precedes dendritic spine loss. Acta Neu‐

[169] Selkoe, D.J., Soluble oligomers of the amyloid beta-protein impair synaptic plasticity

[170] Kirkwood, C.M., et al., Dendritic Spine Density, Morphology, and Fibrillar Actin Content Surrounding Amyloid-beta Plaques in a Mouse Model of Amyloid-beta

[171] Sivanesan, S., A. Tan, and J. Rajadas, Pathogenesis of Abeta oligomers in synaptic

[172] Tyan, S.H., et al., Amyloid precursor protein (APP) regulates synaptic structure and

[173] Townsend, M., et al., Effects of secreted oligomers of amyloid beta-protein on hippo‐ campal synaptic plasticity: a potent role for trimers. J Physiol, 2006. 572(Pt 2): p.

amyloid-beta oligomers. Nature, 2009. 457(7233): p. 1128-32.

cognitive impairment. Neurology, 2007. 68(18): p. 1501-8.

and behavior. Behav Brain Res, 2008. 192(1): p. 106-13.

failure. Curr Alzheimer Res, 2013. 10(3): p. 316-23.

function. Mol Cell Neurosci, 2012. 51(1-2): p. 43-52.

Deposition. J Neuropathol Exp Neurol, 2013. 72(8): p. 791-800.

Curr Alzheimer Res, 2004. 1(4): p. 231-9.

Trends Mol Med, 2005. 11(4): p. 170-6.

molecular Med, 2012. 14(1): p. 1-14.

Neurosci, 2005. 25(31): p. 7278-87.

ropathol, 2012. 124(6): p. 797-807.

AD. Curr Genomics, 2007. 8(8): p. 486-508.

94-108.

477-92.


[158] Hsia, A.Y., et al., Plaque-independent disruption of neural circuits in Alzheimer's disease mouse models. Proc Natl Acad Sci U S A, 1999. 96(6): p. 3228-33.

[142] Kakio, A., et al., Formation of a membrane-active form of amyloid beta-protein in raft-like model membranes. Biochem Biophys Res Commun, 2003. 303(2): p. 514-8.

[143] Lazarov, O., et al., Evidence that synaptically released beta-amyloid accumulates as extracellular deposits in the hippocampus of transgenic mice. J Neurosci, 2002.

[144] Wang, D.S., et al., Oxidized neprilysin in aging and Alzheimer's disease brains. Bio‐

[145] Yasojima, K., et al., Reduced neprilysin in high plaque areas of Alzheimer brain: a possible relationship to deficient degradation of beta-amyloid peptide. Neurosci Lett,

[146] Farris, W., et al., Loss of neprilysin function promotes amyloid plaque formation and causes cerebral amyloid angiopathy. Am J Pathol, 2007. 171(1): p. 241-51.

[147] Koffie, R.M., et al., Apolipoprotein E4 effects in Alzheimer's disease are mediated by

[148] Albin, R.L. and J.T. Greenamyre, Alternative excitotoxic hypotheses. Neurology,

[149] Hu, N.W., T. Ondrejcak, and M.J. Rowan, Glutamate receptors in preclinical research on Alzheimer's disease: update on recent advances. Pharmacol Biochem Behav, 2012.

[150] Lacor, P.N., et al., Synaptic targeting by Alzheimer's-related amyloid beta oligomers.

[151] Palop, J.J. and L. Mucke, Amyloid-beta-induced neuronal dysfunction in Alzheimer's disease: from synapses toward neural networks. Nat Neurosci, 2010. 13(7): p. 812-8.

[152] Selkoe, D.J., Alzheimer's disease is a synaptic failure. Science, 2002. 298(5594): p.

[153] Wogulis, M., et al., Nucleation-dependent polymerization is an essential component of amyloid-mediated neuronal cell death. J Neurosci, 2005. 25(5): p. 1071-80.

[154] Shankar, G.M., et al., Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory. Nat Med, 2008. 14(8): p. 837-42.

[155] Mucke, L. and D.J. Selkoe, Neurotoxicity of amyloid beta-protein: synaptic and net‐ work dysfunction. Cold Spring Harb Perspect Med, 2012. 2(7): p. a006338.

[156] Cullen, W.K., et al., Block of LTP in rat hippocampus in vivo by beta-amyloid precur‐

[157] Oddo, S., et al., Triple-transgenic model of Alzheimer's disease with plaques and tan‐ gles: intracellular Abeta and synaptic dysfunction. Neuron, 2003. 39(3): p. 409-21.

sor protein fragments. Neuroreport, 1997. 8(15): p. 3213-7.

synaptotoxic oligomeric amyloid-beta. Brain, 2012. 135(Pt 7): p. 2155-68.

chem Biophys Res Commun, 2003. 310(1): p. 236-41.

22(22): p. 9785-93.

312 Neurochemistry

2001. 297(2): p. 97-100.

1992. 42(4): p. 733-8.

100(4): p. 855-62.

789-91.

J Neurosci, 2004. 24(45): p. 10191-200.


[174] Wu, H.Y., et al., Distinct dendritic spine and nuclear phases of calcineurin activation after exposure to amyloid-beta revealed by a novel fluorescence resonance energy transfer assay. J Neurosci, 2012. 32(15): p. 5298-309.

[189] Minkeviciene, R., P. Banerjee, and H. Tanila, Memantine improves spatial learning in a transgenic mouse model of Alzheimer's disease. J Pharmacol Exp Ther, 2004.

Alzheimer Disease: The Role of Aβ in the Glutamatergic System

http://dx.doi.org/10.5772/57367

315

[190] Filali, M., R. Lalonde, and S. Rivest, Subchronic memantine administration on spatial learning, exploratory activity, and nest-building in an APP/PS1 mouse model of Alz‐

[191] Van Dam, D. and P.P. De Deyn, Cognitive evaluation of disease-modifying efficacy of galantamine and memantine in the APP23 model. Eur Neuropsychopharmacol,

[192] Martinez-Coria, H., et al., Memantine improves cognition and reduces Alzheimer'slike neuropathology in transgenic mice. Am J Pathol, 2010. 176(2): p. 870-80.

[193] Dong, H., et al., Effects of memantine on neuronal structure and conditioned fear in the Tg2576 mouse model of Alzheimer's disease. Neuropsychopharmacology, 2008.

[194] Rive, B., et al., Memantine enhances autonomy in moderate to severe Alzheimer's

[195] Peskind, E.R., et al., Memantine treatment in mild to moderate Alzheimer disease: a 24-week randomized, controlled trial. Am J Geriatr Psychiatry, 2006. 14(8): p. 704-15.

[196] Reisberg, B., et al., A 24-week open-label extension study of memantine in moderate

[197] Ferris, S., et al., Treatment effects of Memantine on language in moderate to severe

Alzheimer's disease patients. Alzheimers Dement, 2009. 5(5): p. 369-74.

heimer's disease. Neuropharmacology, 2011. 60(6): p. 930-6.

disease. Int J Geriatr Psychiatry, 2004. 19(5): p. 458-64.

to severe Alzheimer disease. Arch Neurol, 2006. 63(1): p. 49-54.

311(2): p. 677-82.

2006. 16(1): p. 59-69.

33(13): p. 3226-36.


[189] Minkeviciene, R., P. Banerjee, and H. Tanila, Memantine improves spatial learning in a transgenic mouse model of Alzheimer's disease. J Pharmacol Exp Ther, 2004. 311(2): p. 677-82.

[174] Wu, H.Y., et al., Distinct dendritic spine and nuclear phases of calcineurin activation after exposure to amyloid-beta revealed by a novel fluorescence resonance energy

[175] Penzes, P., et al., Dendritic spine pathology in neuropsychiatric disorders. Nat Neu‐

[176] Puzzo, D., L. Privitera, and A. Palmeri, Hormetic effect of amyloid-beta peptide in synaptic plasticity and memory. Neurobiol Aging, 2012. 33(7): p. 1484 e15-24.

[177] Seabrook, G.R., et al., Mechanisms contributing to the deficits in hippocampal synap‐ tic plasticity in mice lacking amyloid precursor protein. Neuropharmacology, 1999.

[178] Rezvani, A.H., Involvement of the NMDA System in Learning and Memory. 2006.

[179] Saura, C.A., et al., Loss of presenilin function causes impairments of memory and synaptic plasticity followed by age-dependent neurodegeneration. Neuron, 2004.

[180] Ma, H., et al., Involvement of beta-site APP cleaving enzyme 1 (BACE1) in amyloid precursor protein-mediated enhancement of memory and activity-dependent synap‐

[181] Laird, F.M., et al., BACE1, a major determinant of selective vulnerability of the brain to amyloid-beta amyloidogenesis, is essential for cognitive, emotional, and synaptic

[182] Texido, L., et al., Amyloid beta peptide oligomers directly activate NMDA receptors.

[183] MacDermott, A.B., et al., NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurones. Nature, 1986. 321(6069): p. 519-22.

[184] Peng, T.I. and M.J. Jou, Oxidative stress caused by mitochondrial calcium overload.

[185] Gerzon, K., et al., The Adamantyl Group in Medicinal Agents. I. Hypoglycemic N-

[186] Chen, H.S. and S.A. Lipton, Mechanism of memantine block of NMDA-activated channels in rat retinal ganglion cells: uncompetitive antagonism. J Physiol, 1997. 499

[187] Kornhuber, J., et al., Memantine displaces [3H]MK-801 at therapeutic concentrations in postmortem human frontal cortex. Eur J Pharmacol, 1989. 166(3): p. 589-90.

[188] Rogawski, M.A. and G.L. Wenk, The neuropharmacological basis for the use of mem‐ antine in the treatment of Alzheimer's disease. CNS Drug Rev, 2003. 9(3): p. 275-308.

Arylsulfonyl-N' -Adamantylureas. J Med Chem, 1963. 6: p. 760-3.

tic plasticity. Proc Natl Acad Sci U S A, 2007. 104(19): p. 8167-72.

functions. J Neurosci, 2005. 25(50): p. 11693-709.

Cell Calcium, 2011. 49(3): p. 184-90.

Ann N Y Acad Sci, 2010. 1201: p. 183-8.

transfer assay. J Neurosci, 2012. 32(15): p. 5298-309.

rosci, 2011. 14(3): p. 285-93.

38(3): p. 349-59.

314 Neurochemistry

42(1): p. 23-36.

( Pt 1): p. 27-46.


**Chapter 11**

**Genetics of Alzheimer´S Disease**

Victoria Campos-Peña, Rocío Gómez and

Additional information is available at the end of the chapter

anatomical damage and the clinical phases of the disease.

Alzheimer's disease, which was first described in 1907 by Alois Alzheimer, is a progressive neurodegenerative disorder characterized by memory loss and other cognitive functions, and is the most common cause of dementia in old age. Histopathologically, AD is defined by the presence of two specific features: neuritic plaques (NP), containing beta amyloid (Aβ) deposits and neurofibrillary tangles (NTF), containing hyperphosphorylated tau protein [1-3] (Figure 1). The pathological changes observed in the brains of AD patients are not distributed uniformly over the cerebral cortex. Instead, these changes are located in specific cortical areas, indicating a relationship between disease progression and the connectivity of affected areas [2, 4-5]. These changes follow a pattern that correspond to the information transmission routes between cortical and subcortical areas of the brain, suggesting a direct correlation between

There are two subtypes of AD: 1) familial Alzheimer´s disease which is associated with mutations in three different genes and 2) sporadic Alzheimer´s disease, which is much more common and the causes for it, are not yet completely understood. In recent decades, numerous genome-wide association studies (GWAS) have been performed in an attempt to identify new risk loci related with the development of sporadic cases. In this regard, genetic association studies of cases and controls, have proven the existence of polymorphic variants in genes which could be interpreted as genetic susceptibility factors contributing to the development of LOAD. However, these results are not replicated in all populations, suggesting the importance of geographical and environmental factors in the phenotypic expression of the disease. For this purpose and in order to validate the data obtained, it is necessary to take in account con‐ founding factors as genetic admixture in population-based genetic association studies. This review, describe the genetics of Alzheimer´s disease and some of the most relevant GWAS

> © 2014 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.

Marco Antonio Meraz Ríos

http://dx.doi.org/10.5772/58286

**1. Introduction**

conducted to date.

## **Chapter 11**

## **Genetics of Alzheimer´S Disease**

Victoria Campos-Peña, Rocío Gómez and Marco Antonio Meraz Ríos

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/58286

#### **1. Introduction**

Alzheimer's disease, which was first described in 1907 by Alois Alzheimer, is a progressive neurodegenerative disorder characterized by memory loss and other cognitive functions, and is the most common cause of dementia in old age. Histopathologically, AD is defined by the presence of two specific features: neuritic plaques (NP), containing beta amyloid (Aβ) deposits and neurofibrillary tangles (NTF), containing hyperphosphorylated tau protein [1-3] (Figure 1). The pathological changes observed in the brains of AD patients are not distributed uniformly over the cerebral cortex. Instead, these changes are located in specific cortical areas, indicating a relationship between disease progression and the connectivity of affected areas [2, 4-5]. These changes follow a pattern that correspond to the information transmission routes between cortical and subcortical areas of the brain, suggesting a direct correlation between anatomical damage and the clinical phases of the disease.

There are two subtypes of AD: 1) familial Alzheimer´s disease which is associated with mutations in three different genes and 2) sporadic Alzheimer´s disease, which is much more common and the causes for it, are not yet completely understood. In recent decades, numerous genome-wide association studies (GWAS) have been performed in an attempt to identify new risk loci related with the development of sporadic cases. In this regard, genetic association studies of cases and controls, have proven the existence of polymorphic variants in genes which could be interpreted as genetic susceptibility factors contributing to the development of LOAD. However, these results are not replicated in all populations, suggesting the importance of geographical and environmental factors in the phenotypic expression of the disease. For this purpose and in order to validate the data obtained, it is necessary to take in account con‐ founding factors as genetic admixture in population-based genetic association studies. This review, describe the genetics of Alzheimer´s disease and some of the most relevant GWAS conducted to date.

© 2014 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. Neuritic plaques and the β-amyloid precursor protein**

Neuritic plaques are extracellular deposits of 10-100 μm in diameter, contain an insoluble core consisting of a peptide known as amyloid-β (Aβ), surrounded by microglia, reactive astrocytes and dystrophic neurites [7]. Aβ is a peptide of 39-42 amino acids [8-9] that originates as a normal secretory product derived from amyloid-β precursor protein (AβPP) [10]. AβPP is an integral membrane protein that is widely expressed in epithelial cells of various organs, such as the thyroid gland, skin and the central nervous system. AβPP is a type I integral membrane glycoprotein that resembles a signal-transduction receptor [10]. This protein is conformed by a large extracellular domain, a hydrophobic transmembrane domain and a short cytoplasmic carboxyl terminus (Figure 2). The gene is located on chromosome 21q21 and consists of 18 exons. Alternative splicing generates several isoforms with lengths varying between 365 and 770 amino acid residues. In the central nervous system, four isoforms are expressed: APP695, APP714, APP751 and APP770. Amyloid-β is present only in APP695, APP751 and APP770 (Figure 3A, 3B). The APP695 isoform is mainly expressed in neuronal cells [11], while the APP751 and APP770 isoforms are expressed in glial cells [12-13]. To date, the primary function of the protein has not been defined yet, but it has been proposed that it could participate as a growth factor in cultured fibroblasts [14] and play role in cell adhesion [15], intraneuronal calcium regulation [16], neural plasticity [17] and act as a regulator of synapse formation [18]. AβPP, is posttranslationally modified by N-and O-glycosylation, phosphorylation and tyrosine sulphation and undergoes two types of proteolytic processing [19] through three

Genetics of Alzheimer´S Disease http://dx.doi.org/10.5772/58286 319

**Figure 2. Schematic representation of AβPP**. AβPP is a member of a family of conserved type I membrane proteins, consists of a large extracellular domain, a hydrophobic transmembrane domain, and a short cytoplasmic carboxyl ter‐ minus. Amyloid sequence contains 40-and 43-amino acid residues that extend from the ectodomain into the trans‐ membrane domain of the protein. The Aβ sequence lies partially outside the cell membrane (amino acids 1–17 of Aβ)

and the some identified mutations in the protein are indicated in bold.

**Figure 1. Pathological changes observed in AD patients brains. (A)** Cross-section on the left represents a normal brain and the one on the right represents a brain with Alzheimer's disease. The picture shows the generalized brain atrophy in AD, characterized by widening in sulcus, ventricles dilatation and extensive cell loss. **(B)** Silver stain showing the presence of neurofibrillary tangles (NFT) the Tau protein aggregates are indicated by white arrows. We observe the formation of these deposits at different stages of neurodegeneration. **(C)** Double staining showing a neuritic pla‐ que (NP), amyloid deposits are seen in red and marked with an asterisk; neurofibrillary aggregates surrounding the amyloid are marked with the arrow.

#### **2. Diagnosis**

There are currently several clinical tools for the diagnosis of AD, including the minimum mental state examination (MMSE) [6] and the Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition [DSM-IV]). In general terms, these tools consist of a semi-structured interview with an appropriate reporter and the patient with damage being described as loss of two or more of the following cognitive areas: memory, orientation, calculation and language. Other aspects are similarly evaluated, such as problem solving, social relationships, work, hobbies and personal care. Another commonly used criterion for diagnosis is that of the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer´s Disease and Related Disorders Association (NINCDSADRDA). Under this criterion, the state of dementia is clinically determined based on the loss of two cognitive areas and the absence of other systemic disorders, accompanied by a progressive loss of memory. These criteria are sufficient to determine *probable Alzheimer´s disease.* However, diagnosis of AD requires exclusion of other neurodegenerative diseases, such as frontotemporal dementia, Parkinson's disease and Lewy Body disease. Discrimination between AD and other types of dementia are usually achieved based on clinical history and through neurological examinations that require imaging studies. Nevertheless, definitive diagnosis of AD requires postmortem confirmation by histopathological examination to demonstrate the presence of NP and NFT (Figure 1).

## **2. Neuritic plaques and the β-amyloid precursor protein**

Neuritic plaques are extracellular deposits of 10-100 μm in diameter, contain an insoluble core consisting of a peptide known as amyloid-β (Aβ), surrounded by microglia, reactive astrocytes and dystrophic neurites [7]. Aβ is a peptide of 39-42 amino acids [8-9] that originates as a normal secretory product derived from amyloid-β precursor protein (AβPP) [10]. AβPP is an integral membrane protein that is widely expressed in epithelial cells of various organs, such as the thyroid gland, skin and the central nervous system. AβPP is a type I integral membrane glycoprotein that resembles a signal-transduction receptor [10]. This protein is conformed by a large extracellular domain, a hydrophobic transmembrane domain and a short cytoplasmic carboxyl terminus (Figure 2). The gene is located on chromosome 21q21 and consists of 18 exons. Alternative splicing generates several isoforms with lengths varying between 365 and 770 amino acid residues. In the central nervous system, four isoforms are expressed: APP695, APP714, APP751 and APP770. Amyloid-β is present only in APP695, APP751 and APP770 (Figure 3A, 3B). The APP695 isoform is mainly expressed in neuronal cells [11], while the APP751 and APP770 isoforms are expressed in glial cells [12-13]. To date, the primary function of the protein has not been defined yet, but it has been proposed that it could participate as a growth factor in cultured fibroblasts [14] and play role in cell adhesion [15], intraneuronal calcium regulation [16], neural plasticity [17] and act as a regulator of synapse formation [18]. AβPP, is posttranslationally modified by N-and O-glycosylation, phosphorylation and tyrosine sulphation and undergoes two types of proteolytic processing [19] through three

**2. Diagnosis**

318 Neurochemistry

amyloid are marked with the arrow.

There are currently several clinical tools for the diagnosis of AD, including the minimum mental state examination (MMSE) [6] and the Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition [DSM-IV]). In general terms, these tools consist of a semi-structured interview with an appropriate reporter and the patient with damage being described as loss of two or more of the following cognitive areas: memory, orientation, calculation and language. Other aspects are similarly evaluated, such as problem solving, social relationships, work, hobbies and personal care. Another commonly used criterion for diagnosis is that of the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer´s Disease and Related Disorders Association (NINCDSADRDA). Under this criterion, the state of dementia is clinically determined based on the loss of two cognitive areas and the absence of other systemic disorders, accompanied by a progressive loss of memory. These criteria are sufficient to determine *probable Alzheimer´s disease.* However, diagnosis of AD requires exclusion of other neurodegenerative diseases, such as frontotemporal dementia, Parkinson's disease and Lewy Body disease. Discrimination between AD and other types of dementia are usually achieved based on clinical history and through neurological examinations that require imaging studies. Nevertheless, definitive diagnosis of AD requires postmortem confirmation by histopathological examination to demonstrate the presence of NP and NFT (Figure 1).

**Figure 1. Pathological changes observed in AD patients brains. (A)** Cross-section on the left represents a normal brain and the one on the right represents a brain with Alzheimer's disease. The picture shows the generalized brain atrophy in AD, characterized by widening in sulcus, ventricles dilatation and extensive cell loss. **(B)** Silver stain showing the presence of neurofibrillary tangles (NFT) the Tau protein aggregates are indicated by white arrows. We observe the formation of these deposits at different stages of neurodegeneration. **(C)** Double staining showing a neuritic pla‐ que (NP), amyloid deposits are seen in red and marked with an asterisk; neurofibrillary aggregates surrounding the

**Figure 2. Schematic representation of AβPP**. AβPP is a member of a family of conserved type I membrane proteins, consists of a large extracellular domain, a hydrophobic transmembrane domain, and a short cytoplasmic carboxyl ter‐ minus. Amyloid sequence contains 40-and 43-amino acid residues that extend from the ectodomain into the trans‐ membrane domain of the protein. The Aβ sequence lies partially outside the cell membrane (amino acids 1–17 of Aβ) and the some identified mutations in the protein are indicated in bold.

enzyme activities α-secretase, β-secretase which cleave AβPP within the luminal domain, and a third activity, termed γ-secretase which cleaves APP within the transmembrane domain. All three AβPP secretases are transmembrane proteases.

The first step of amyloidogenic processing is carried out by the action of β-secretase, (BACE1), which generates the formation of two products: 1) a soluble product (sAβPP) that is released into the extracellular space and 2) a carboxyl terminal membrane-anchored called C99. In the same way C99 is cut by the γ-secretase, generating the AICD fragment into the cytoplasm and

Genetics of Alzheimer´S Disease http://dx.doi.org/10.5772/58286 321

Although AβPP metabolism and amyloid peptide accumulation represent central events in the pathogenesis of AD, in animal models, it has not been possible to demonstrate that their

**Figure 4. AβPP Processing.** The AβPP is an integral membrane protein, is sequentially processed by the three proteas‐ es α-, β-, and γ-secretase. **(A)** The non amyloidogenic pathway involves the α-secretase, which made the cut at the middle portion of the fragment corresponding to the amyloid sequence; preventing the amyloid peptides generation. **(B)** The amyloidogenic pathway involves β-secretase, leading to the formation of C-terminal fragments (CTFs) that are subsequently cleaved by the "γ-secretase-complex" which is responsible for the formation of Aβ (40 or 42 amino acids

in length) and the AβPP intracellular domain peptide (AICD) of 58 or 56 amino acids.

occurrence *per se* is capable of generating the complete pathology of the disease.

the neurotoxic fragment amyloid beta (Aß) (Figure 4B) [22].

**Figure 3. Human APP gene structure. (A)** The APP gene consist of 18 exons, is located on chromosome 21 (21q21.2-3). The region encoding the amyloid sequence comprises part of exons 16 and 17 (yellow box). **(B)** APP is alternatively spliced into several products, named according to their length in amino acids (ie, APP695, APP714, APP751, APP770, and APP563) that are expressed differentially by tissue type. The major APP derivatives in the CNS are APP695, APP751 and APP770. Some isoforms contain a 57 amino acid KPI domain and a 19 aminoacid MRC OX-2 antigen in the extracellular sequences (pink box).

It is believed that the principal proteolytic cleavage of AβPP is non-amyloidogenic pathway, which is performed by the action of a protease known as alpha-secretase. This protease cleaves, at residues 612-613 corresponding to the middle portion Aβ (Lys16 and Leu17 in Aβ peptide), thereby preventing amyloid formation [20-21]. α-secretase (ADAM10) generates two products: a soluble fragment (sAPPα) that is released into the extracellular space and a carboxyl terminal membrane-anchored product, called C83. Finally, the C83 fragment is cut by γ-secretase generating a 6 KDa fragment, called AICD (APP intracellular domain-) and a ~3kDa peptide (p3) that is released into the extracellular space (Figure 4A).

The first step of amyloidogenic processing is carried out by the action of β-secretase, (BACE1), which generates the formation of two products: 1) a soluble product (sAβPP) that is released into the extracellular space and 2) a carboxyl terminal membrane-anchored called C99. In the same way C99 is cut by the γ-secretase, generating the AICD fragment into the cytoplasm and the neurotoxic fragment amyloid beta (Aß) (Figure 4B) [22].

enzyme activities α-secretase, β-secretase which cleave AβPP within the luminal domain, and a third activity, termed γ-secretase which cleaves APP within the transmembrane domain. All

**Figure 3. Human APP gene structure. (A)** The APP gene consist of 18 exons, is located on chromosome 21 (21q21.2-3). The region encoding the amyloid sequence comprises part of exons 16 and 17 (yellow box). **(B)** APP is alternatively spliced into several products, named according to their length in amino acids (ie, APP695, APP714, APP751, APP770, and APP563) that are expressed differentially by tissue type. The major APP derivatives in the CNS are APP695, APP751 and APP770. Some isoforms contain a 57 amino acid KPI domain and a 19 aminoacid MRC OX-2

It is believed that the principal proteolytic cleavage of AβPP is non-amyloidogenic pathway, which is performed by the action of a protease known as alpha-secretase. This protease cleaves, at residues 612-613 corresponding to the middle portion Aβ (Lys16 and Leu17 in Aβ peptide), thereby preventing amyloid formation [20-21]. α-secretase (ADAM10) generates two products: a soluble fragment (sAPPα) that is released into the extracellular space and a carboxyl terminal membrane-anchored product, called C83. Finally, the C83 fragment is cut by γ-secretase generating a 6 KDa fragment, called AICD (APP intracellular domain-) and a ~3kDa peptide

three AβPP secretases are transmembrane proteases.

320 Neurochemistry

antigen in the extracellular sequences (pink box).

(p3) that is released into the extracellular space (Figure 4A).

Although AβPP metabolism and amyloid peptide accumulation represent central events in the pathogenesis of AD, in animal models, it has not been possible to demonstrate that their occurrence *per se* is capable of generating the complete pathology of the disease.

**Figure 4. AβPP Processing.** The AβPP is an integral membrane protein, is sequentially processed by the three proteas‐ es α-, β-, and γ-secretase. **(A)** The non amyloidogenic pathway involves the α-secretase, which made the cut at the middle portion of the fragment corresponding to the amyloid sequence; preventing the amyloid peptides generation. **(B)** The amyloidogenic pathway involves β-secretase, leading to the formation of C-terminal fragments (CTFs) that are subsequently cleaved by the "γ-secretase-complex" which is responsible for the formation of Aβ (40 or 42 amino acids in length) and the AβPP intracellular domain peptide (AICD) of 58 or 56 amino acids.

## **3. Neurofibrillary tangles and tau**

Neurofibrillary tangles are simpler and yet more enigmatic than neuritic plaques. Unlike NP, the density of NFT in the brains of AD patients is closely related to the severity of dementia [23-24]. In particular, neurofibrillary degeneration is a prerequisite for the clinical expression of AD pathology, i.e., *dementia*, whereas amyloid accumulation in the absence of neurofibrillary tangles does not produce the AD pathology. The structural units of NFT are paired helical filaments (FHA), which are formed by the association of five to six6 fragments of the micro‐ tubule binding protein Tau. The gene that encodes this protein is located on chromosome 17 and comprises 16 exons of which-1 and 14 exons, can be transcribed but not translated [25-27]. Alternative RNAm splicing of exons 2, 3 and 10, of the *MAPT* gene generates the formation of six isoforms which are expressed in adult brain [28]. Each isoform differs from each other by the presence or absence of a 29-aminoacid or 58-aminoacid inserts in the amino-terminal half and by the inclusion or not in the carboxy-terminal half of the protein of a 31-aminoacid repeat encoded by exon 10 of *MAPT* [25, 29-30]. When exon 10 is excluded, the result is a protein with three repeats of the microtubule-binding domain (3RMBD). When exon 10 is included, a fourth microtubule binding domain is added to generate four-repeat tau (4RMBD) [28, 31-32] (Figure 5). Under normal conditions, Tau is a highly soluble protein, since it contains no apparent secondary structure [33-34]. However, in pathological conditions, Tau tends to self-assemble into the insoluble filament structures [32]. To date have been identified and MAPT gene mutations, however, none of these mutations have been associated with the development of AD. This type of mutations in the Tau gene cause frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) [35-39]

that among patients who develop LOAD, approximately 40-65% present an indirect genetic agent in the form of the 4 allele of apolipoprotein E (ApoE/4) [44-47]. However, the effect of APOEe4 as a genetic risk factor is not sufficient or necessary for developing the disease [48-49].

Genetics of Alzheimer´S Disease http://dx.doi.org/10.5772/58286 323

**Figure 5. Human Tau-protein. (A)** Schematic representation human tau gene (MAPT). The human gene comprises 16 exons of which exons-1 and 14, can be transcribed but not translated. In central nervous system are expressed 6 iso‐ forms, which are obtained by alternative splicing of exons 2, 3 and 10; the exons 1, 4, 5, 7, 9, 11, 12 and 13 are ex‐ pressed in all isoforms. In boxes, are indicated the mutations found in the gene which have been associated with FTDP-17. **(B)** Representation of the 6 Tau isoforms. The different isoforms differ from each other by the presence of one or two inserts located in the region N (yellow and orange boxes) and the presence of 3 or 4 repeated domains, located in C-terminus of the molecule (blue box) and termed microtubule binding domain (MTBD). The expression of the different isoforms is regulated during development; in fetal stages are expressed only isoforms containing 3 re‐

peated whereas adult stages, all isoforms are expressed.

## **4. Genetics of AD**

Conventionally, AD is divided into two forms depending on the age of onset: early onset Alzheimer´s disease (EOAD) and late onset Alzheimer´s disease (LOAD). EOAD or familial cases, which account for only 5-10% of all cases, exhibit an autosomal dominant mode of inheritance, high penetrance of clinical symptoms and onset before 55 years of age. LOAD or sporadic cases account for 90-95% of all AD cases, usually present a later onset age (≥ 65 years) and apparently do not show familial aggregation associated with the development of the disease. Twin studies provide insight into the relative contributions of genetic and environ‐ mental influences for Alzheimer´s disease and other types of dementia [40-42]. It has long been argued that a twin study design is advantageous for identifying risk and protective factors because this type of study has suggested the existence of a genetic component associated with the development of LOAD cases [43]. The results of these studies have shown that the pairwise concordance rate for Alzheimer´s disease is 78% (7/9) among monozygotic and 39% (9/23) among dizygotic twin pairs [40]. In 2006, Gatz adjusted their findings for age and also included like-and unlike-sex pairs, and the results showed that the age–adjusted heritability for AD was estimated to be 58-79%, and there were no significant differences between men and women regarding prevalence or heritability after controlling for age [41]. Nevertheless, it was observed that among patients who develop LOAD, approximately 40-65% present an indirect genetic agent in the form of the 4 allele of apolipoprotein E (ApoE/4) [44-47]. However, the effect of APOEe4 as a genetic risk factor is not sufficient or necessary for developing the disease [48-49].

**3. Neurofibrillary tangles and tau**

322 Neurochemistry

linked to chromosome 17 (FTDP-17) [35-39]

**4. Genetics of AD**

Neurofibrillary tangles are simpler and yet more enigmatic than neuritic plaques. Unlike NP, the density of NFT in the brains of AD patients is closely related to the severity of dementia [23-24]. In particular, neurofibrillary degeneration is a prerequisite for the clinical expression of AD pathology, i.e., *dementia*, whereas amyloid accumulation in the absence of neurofibrillary tangles does not produce the AD pathology. The structural units of NFT are paired helical filaments (FHA), which are formed by the association of five to six6 fragments of the micro‐ tubule binding protein Tau. The gene that encodes this protein is located on chromosome 17 and comprises 16 exons of which-1 and 14 exons, can be transcribed but not translated [25-27]. Alternative RNAm splicing of exons 2, 3 and 10, of the *MAPT* gene generates the formation of six isoforms which are expressed in adult brain [28]. Each isoform differs from each other by the presence or absence of a 29-aminoacid or 58-aminoacid inserts in the amino-terminal half and by the inclusion or not in the carboxy-terminal half of the protein of a 31-aminoacid repeat encoded by exon 10 of *MAPT* [25, 29-30]. When exon 10 is excluded, the result is a protein with three repeats of the microtubule-binding domain (3RMBD). When exon 10 is included, a fourth microtubule binding domain is added to generate four-repeat tau (4RMBD) [28, 31-32] (Figure 5). Under normal conditions, Tau is a highly soluble protein, since it contains no apparent secondary structure [33-34]. However, in pathological conditions, Tau tends to self-assemble into the insoluble filament structures [32]. To date have been identified and MAPT gene mutations, however, none of these mutations have been associated with the development of AD. This type of mutations in the Tau gene cause frontotemporal dementia with Parkinsonism

Conventionally, AD is divided into two forms depending on the age of onset: early onset Alzheimer´s disease (EOAD) and late onset Alzheimer´s disease (LOAD). EOAD or familial cases, which account for only 5-10% of all cases, exhibit an autosomal dominant mode of inheritance, high penetrance of clinical symptoms and onset before 55 years of age. LOAD or sporadic cases account for 90-95% of all AD cases, usually present a later onset age (≥ 65 years) and apparently do not show familial aggregation associated with the development of the disease. Twin studies provide insight into the relative contributions of genetic and environ‐ mental influences for Alzheimer´s disease and other types of dementia [40-42]. It has long been argued that a twin study design is advantageous for identifying risk and protective factors because this type of study has suggested the existence of a genetic component associated with the development of LOAD cases [43]. The results of these studies have shown that the pairwise concordance rate for Alzheimer´s disease is 78% (7/9) among monozygotic and 39% (9/23) among dizygotic twin pairs [40]. In 2006, Gatz adjusted their findings for age and also included like-and unlike-sex pairs, and the results showed that the age–adjusted heritability for AD was estimated to be 58-79%, and there were no significant differences between men and women regarding prevalence or heritability after controlling for age [41]. Nevertheless, it was observed

**Figure 5. Human Tau-protein. (A)** Schematic representation human tau gene (MAPT). The human gene comprises 16 exons of which exons-1 and 14, can be transcribed but not translated. In central nervous system are expressed 6 iso‐ forms, which are obtained by alternative splicing of exons 2, 3 and 10; the exons 1, 4, 5, 7, 9, 11, 12 and 13 are ex‐ pressed in all isoforms. In boxes, are indicated the mutations found in the gene which have been associated with FTDP-17. **(B)** Representation of the 6 Tau isoforms. The different isoforms differ from each other by the presence of one or two inserts located in the region N (yellow and orange boxes) and the presence of 3 or 4 repeated domains, located in C-terminus of the molecule (blue box) and termed microtubule binding domain (MTBD). The expression of the different isoforms is regulated during development; in fetal stages are expressed only isoforms containing 3 re‐ peated whereas adult stages, all isoforms are expressed.

## **5. Early onset alzheimer´s disease**

While the vast majority of cases of AD occur late in life and are sporadic, approximately 5– 10% of cases exhibit an early onset. EOAD or Familial Alzheimer´s disease is associated with mutations in proteins such as presenilin 1 and 2 (PS1 and PS2) and amyloid precursor protein (APP) [50-58]. Currently, more than 200 distinct disease-causing mutations have been identi‐ fied across these genes, which exhibit an autosomal dominant transmission pattern.

#### **5.1. APP mutations**

To date, approximately 36 different missense mutations in the APP gene have been identified among 85 families (Table 1). AβPP mutations account for 0.1% of AD patients, all missense mutations influence APP processing since they are positioned in or near the Aβ coding exons 16-17, in the transmembranal domain, where the sites recognized by the α, β and γ-secretases are found (Figure 2). This alters the APPβ processing and causes the accumulation of Aβ42 fragments [54-55]. The major mutations in APPβ include the Swedish double mutation (APPSW: APPK670N, APPM671L) [59] and the London mutation (V717I) [55]. The Swedish mutation is located just outside the N-terminus of the Aβ domain of APP, favors β-secretase cleavage and it is associated with increased levels and deposition of Aβ1-42 in the brains of AD patients [60-61]. London mutation is located in exon 17 and leads to a valine to isoleucine change at amino acid 717 (V717I) [55], corresponding to the transmembrane domain near the γ-secretase cleavage site. Other mutations in APP linked to EOAD include the Dutch (E693Q) [62], Indiana (V717F) [58], Florida (I716V) [63], Iowa (D694N) [64] and Arctic (E693G) [65] mutations. Besides the mutations identified in the APP gene is known that duplication of APP regions containing several genes [66-68] or APP [69] were clinically linked to EOAD.

The transgenic animal models developed to date that overexpress these mutations have the potential to develop extracellular deposits of amyloid beta and exhibit different types of neurological abnormalities [55, 70-73]. For example, transgenic mouse line APP/V717I displays deficits in the maintenance of long-term potentiation, premature death and cognitive impair‐ ment, which is directly correlated with amyloid plaque formation [74]. Another transgenic mouse line used to investigate the pathology of AD is Tg2576, which carries the Swedish mutation. These mice exhibit memory loss starting at 6 months of age, which coincides with the appearance of detergent-insoluble amyloid aggregates [73]. Overexpression of mutated AβPP in cell cultures induces DNA synthesis and apoptosis [75], suggesting that APPβ could induce the apoptotic events observed in Alzheimer´s disease patients via activation of specific pathways of neuronal signaling.

#### **5.2. Presenilin mutation**

Presenilin represent the catalytic component of the gamma-secretase complex, which also includes nicastrin, anterior pharynx-defective 1 (Aph-1) and presenilin enhancer 2 (Pen-2) [76]. Presenilins are expressed in several tissues and in the brain, where are mainly expressed in neurons [52]. Presenilins localize into the endoplasmic reticulum (ER), Golgi apparatus, endosomes, lysosomes, phagosome plasma membranes and mitochondria [77-79]. During assembly and maturation of the complex, presenilin undergoes endoproteolitic processes generating stable N-and C-terminal fragments (NTF and CTF, respectively). Both fragments (NTF and CTF) contribute with one of the two catalytic aspartates that are, the active site which is responsible for the intramembranal proteolysis of AβPP and some other proteins as well [60, 80-85]. Both presenilins (PS1 and PS2) possess these conserved aspartate residues required for γ-secretase activity [85]. In addition, presenilins directly or indirectly regulate the trafficking and metabolism of select membrane proteins in neurons [86], as well as having a role in

**Mutation Phenotype Age of**

**T714I (Austrian)** Affects γ-secretase cleavage directly,

vitro.

**Table 1.** Amyloid Precursor Protein Mutations

**KM670/671NL (Swedish)**

**E665D** AD, but may not be pathogenic 86? Peacock, et al., 1994

**H677R** AD 55 (55-56) Janssen, et al. 2003 **D678N (Tottori)** FAD 60 Wakutani, et al. 2004 **E693Δ** AD Tomiyama et al., 2008 **D694N (Iowa)** AD or Cerebral Hemorrhage 69 Grabowski, et al. 2001

**A713T** AD, but may not be pathogenic 59 Carter, et al., 1992 **T714A (Iranian)** AD 52 (40-60) Pasaler, et al., 2002

**V715M (French)** AD 52 (40-60) Ancolio, et al., 1999 **I716T** AD 55 Terrini, et al., 2002 **I716V (Florida)** AD 55 Eckman, et al., 1997 **V717F (Indiana)** AD 47 (42-52) Murrell, et al. 1991

**V717G** AD 55 (45-62) Chartier-Harlin, et al. 1991

**V717I (London)** AD 55 (50-60) Goate, et al. 1991 **T719P** AD 46 Ghidoni et al., 2009

**L723P (Australian)** AD 56 (45-60) Kwok JB, 2000

**V715A (German)** AD 47 De Jonghe, et al., 2001; Cruts, et al.,

11X increase in Aβ(42)/Aβ(40) ratio in

**Onset**

AD 52 (44-59) Mullan, et al. 1992

**References**

Genetics of Alzheimer´S Disease http://dx.doi.org/10.5772/58286 325

Kumar-Singh, et al.

2003


**Table 1.** Amyloid Precursor Protein Mutations

**5. Early onset alzheimer´s disease**

**5.1. APP mutations**

324 Neurochemistry

pathways of neuronal signaling.

**5.2. Presenilin mutation**

While the vast majority of cases of AD occur late in life and are sporadic, approximately 5– 10% of cases exhibit an early onset. EOAD or Familial Alzheimer´s disease is associated with mutations in proteins such as presenilin 1 and 2 (PS1 and PS2) and amyloid precursor protein (APP) [50-58]. Currently, more than 200 distinct disease-causing mutations have been identi‐

To date, approximately 36 different missense mutations in the APP gene have been identified among 85 families (Table 1). AβPP mutations account for 0.1% of AD patients, all missense mutations influence APP processing since they are positioned in or near the Aβ coding exons 16-17, in the transmembranal domain, where the sites recognized by the α, β and γ-secretases are found (Figure 2). This alters the APPβ processing and causes the accumulation of Aβ42 fragments [54-55]. The major mutations in APPβ include the Swedish double mutation (APPSW: APPK670N, APPM671L) [59] and the London mutation (V717I) [55]. The Swedish mutation is located just outside the N-terminus of the Aβ domain of APP, favors β-secretase cleavage and it is associated with increased levels and deposition of Aβ1-42 in the brains of AD patients [60-61]. London mutation is located in exon 17 and leads to a valine to isoleucine change at amino acid 717 (V717I) [55], corresponding to the transmembrane domain near the γ-secretase cleavage site. Other mutations in APP linked to EOAD include the Dutch (E693Q) [62], Indiana (V717F) [58], Florida (I716V) [63], Iowa (D694N) [64] and Arctic (E693G) [65] mutations. Besides the mutations identified in the APP gene is known that duplication of APP

fied across these genes, which exhibit an autosomal dominant transmission pattern.

regions containing several genes [66-68] or APP [69] were clinically linked to EOAD.

The transgenic animal models developed to date that overexpress these mutations have the potential to develop extracellular deposits of amyloid beta and exhibit different types of neurological abnormalities [55, 70-73]. For example, transgenic mouse line APP/V717I displays deficits in the maintenance of long-term potentiation, premature death and cognitive impair‐ ment, which is directly correlated with amyloid plaque formation [74]. Another transgenic mouse line used to investigate the pathology of AD is Tg2576, which carries the Swedish mutation. These mice exhibit memory loss starting at 6 months of age, which coincides with the appearance of detergent-insoluble amyloid aggregates [73]. Overexpression of mutated AβPP in cell cultures induces DNA synthesis and apoptosis [75], suggesting that APPβ could induce the apoptotic events observed in Alzheimer´s disease patients via activation of specific

Presenilin represent the catalytic component of the gamma-secretase complex, which also includes nicastrin, anterior pharynx-defective 1 (Aph-1) and presenilin enhancer 2 (Pen-2) [76]. Presenilins are expressed in several tissues and in the brain, where are mainly expressed in neurons [52]. Presenilins localize into the endoplasmic reticulum (ER), Golgi apparatus, endosomes, lysosomes, phagosome plasma membranes and mitochondria [77-79]. During

assembly and maturation of the complex, presenilin undergoes endoproteolitic processes generating stable N-and C-terminal fragments (NTF and CTF, respectively). Both fragments (NTF and CTF) contribute with one of the two catalytic aspartates that are, the active site which is responsible for the intramembranal proteolysis of AβPP and some other proteins as well [60, 80-85]. Both presenilins (PS1 and PS2) possess these conserved aspartate residues required for γ-secretase activity [85]. In addition, presenilins directly or indirectly regulate the trafficking and metabolism of select membrane proteins in neurons [86], as well as having a role in synaptic function [87-88], learning and memory [89], neuronal survival in the adult brain, regulation of calcium homeostasis [90-91] and presynaptic neurotransmitter release [92].

deletion of exon 9. However, the biochemical consequences of these mutations for γ-secretase assembly seem to be limited [103-104]. PS1 also appears to modulate GSK3β activity and the release of kinesin-I from membrane-bound organelles at sites of vesicle delivery and mem‐ brane insertion. These findings suggest that mutations in PS1 may compromise neuronal function, affecting GSK-3 activity and kinesin-I-based motility and, thus, leading to neurode‐ generation [105]. Although PS2 shows close homology to PS1, PS2 is less efficient with respect to amyloid peptide production [106]. *In vitro* expression of PSEN2 V393M cDNA did not result in a detectable increase in the secreted Aβ42/40 peptide ratio. However, patients heterozygous for this missense mutation are characterized by profound language impairment [107]. Although mutations are found throughout the protein, most are located in the transmembrane

Genetics of Alzheimer´S Disease http://dx.doi.org/10.5772/58286 327

Allele 4 of apolipoprotein E (ApoE4) has been reported to represent the main identified risk factor for sporadic AD [44, 49, 108]. This gene has been associated with EOAD and LOAD in multiple ethnic groups, and carriers of APOE4 exhibit an earlier age of onset for AD [44, 109-110]. The frequency of the APOE4 allele varies among ethnic groups and it has been shown that ApoE4 is determinant for AD risk in white's individuals; however, in Hispanic and African populations, there is no correlation between the presence of the pathology and this allele. These results suggest that other genes or risk factors may contribute to the increased risk of AD in

The ApoE4 gene is located at chromosome 19q13.2 [115] and consists of 4 exons encoding a protein of 299 amino acid residues with a molecular weight of 34 kDa. APOE is a glycoprotein exhibiting variable levels of posttranslational sialylation due to O-linked glycosylation at threonine 194 [116]. The gene contains several single-nucleotide polymorphisms (SNPs) [117] leading to changes in the amino acid sequence of the protein, resulting in the production of three isoforms: ApoE2, ApoE3 and ApoE4, which are associated with different alleles (ε2, ε3, ε4). The three isoforms differ only by one or two amino acids, with the changes occurring at amino acid residues 112 and 158: ApoE2 (cys112, cys158), ApoE3 (cys112, arg158) and ApoE4 (arg112, arg158) [118-120]. The allelic distribution varies among ethnic groups, although it has generally been observed that allele 3 is the most frequent (77%), followed by allele 4 (15%),

ApoE is a plasma protein that plays an important role in lipid metabolism and cholesterol transport in various tissues [108, 121-122]. The amino acid changes observed in the different isoforms of ApoE alter the 3-dimensional structure of the protein, affecting its lipid-binding properties, indicating that each isoform is metabolically different and varies in its affinity to bind to lipoprotein particles [123-124]. Apolipoproteins are synthesized primarily in the liver but can be processed and secreted in the brain by astrocytes and microglia. They are involved in neuronal regeneration [125], an increase in the synthesis of these proteins has been observed

region.

**6.1. Apoe risk gene**

**6. Late onset alzheimer´s disease**

African and Hispanics [111-114].

while allele 2 is less frequently observed (8%).

PS1 is an integral membrane protein with eight transmembranal domains and a hydrophilic domain between the 6 and 7 domains. The PSEN1 gene is located on chromosome 14q24.2, comprises 12 exons and generates a protein with a length of 467 amino acids. To date, more than 180 mutations in PSEN1 have been described in 405 families (http:// www.molgen.ua.ac.be/ADmutations), all of which are related to the appearance of the disease at younger ages (Figure 6A) [93-94]. The PSEN2 gene is located on chromosome 1q42.13 and comprises 12 exons, of which only 10 are translated to generate a protein with a length of 448 amino acid residues. This protein exhibits 9 transmembrane domains and displays tissuespecific alternative splicing [95]. Only 13 mutations in PS2 have been described among 22 families (Figure 6B). (http://www.molgen.ua.ac.be/ADmutations)

**Figure 6. Schematic representation of Presenilis. (A)** Presenilins are membrane proteins that form the catalytic core of the γ-secretase complex. The PSEN1 gene is located on chromosome 14q24.2 and comprises 12 exons. PS1 is an integral membrane protein with eight transmembrane domains and a hydrophilic domain between domains 6 and 7. Two aspartate residues in transmembrane domains (TMs) 6 and 7 constituting the catalytic site. To date, more than 185 mutations in PSEN1 have been described in 405 families all of which are related to the appearance of the disease at younger ages. Although mutations are found throughout the protein, most are located in the transmembrane re‐ gion. **(B)** The PSEN2 gene is located on chromosome 1q42.13 and comprises 12 exons, of which only 10 are translated to generate a protein with a length of 448 amino acid residues. This protein exhibits 9 transmembrane domains and displays tissue-specific alternative splicing, major mutations found in the protein are identified.

Most familial cases of Alzheimer´s disease are associated with mutations in presenilins [50, 53, 96], the majority of PSEN mutations are single-nucleotide substitutions, but small deletions and insertions have been described as well. It has been suggested that these mutations induce overproduction of β-amyloid, apparently by increasing γ-secretase activity [51, 53, 97-102]. Although transgenic mice expressing presenilin mutations do not develop the formation of neuritic plaques, these animals showed changes similar to those observed in AD patients, such as neuronal damage, synaptic loss and vascular disease. The most severe mutation in *PSEN1* is a donor-acceptor splice mutation that causes a two-aminoacid substitution and an in-frame deletion of exon 9. However, the biochemical consequences of these mutations for γ-secretase assembly seem to be limited [103-104]. PS1 also appears to modulate GSK3β activity and the release of kinesin-I from membrane-bound organelles at sites of vesicle delivery and mem‐ brane insertion. These findings suggest that mutations in PS1 may compromise neuronal function, affecting GSK-3 activity and kinesin-I-based motility and, thus, leading to neurode‐ generation [105]. Although PS2 shows close homology to PS1, PS2 is less efficient with respect to amyloid peptide production [106]. *In vitro* expression of PSEN2 V393M cDNA did not result in a detectable increase in the secreted Aβ42/40 peptide ratio. However, patients heterozygous for this missense mutation are characterized by profound language impairment [107]. Although mutations are found throughout the protein, most are located in the transmembrane region.

## **6. Late onset alzheimer´s disease**

#### **6.1. Apoe risk gene**

synaptic function [87-88], learning and memory [89], neuronal survival in the adult brain, regulation of calcium homeostasis [90-91] and presynaptic neurotransmitter release [92].

PS1 is an integral membrane protein with eight transmembranal domains and a hydrophilic domain between the 6 and 7 domains. The PSEN1 gene is located on chromosome 14q24.2, comprises 12 exons and generates a protein with a length of 467 amino acids. To date, more than 180 mutations in PSEN1 have been described in 405 families (http:// www.molgen.ua.ac.be/ADmutations), all of which are related to the appearance of the disease at younger ages (Figure 6A) [93-94]. The PSEN2 gene is located on chromosome 1q42.13 and comprises 12 exons, of which only 10 are translated to generate a protein with a length of 448 amino acid residues. This protein exhibits 9 transmembrane domains and displays tissuespecific alternative splicing [95]. Only 13 mutations in PS2 have been described among 22

**Figure 6. Schematic representation of Presenilis. (A)** Presenilins are membrane proteins that form the catalytic core of the γ-secretase complex. The PSEN1 gene is located on chromosome 14q24.2 and comprises 12 exons. PS1 is an integral membrane protein with eight transmembrane domains and a hydrophilic domain between domains 6 and 7. Two aspartate residues in transmembrane domains (TMs) 6 and 7 constituting the catalytic site. To date, more than 185 mutations in PSEN1 have been described in 405 families all of which are related to the appearance of the disease at younger ages. Although mutations are found throughout the protein, most are located in the transmembrane re‐ gion. **(B)** The PSEN2 gene is located on chromosome 1q42.13 and comprises 12 exons, of which only 10 are translated to generate a protein with a length of 448 amino acid residues. This protein exhibits 9 transmembrane domains and

Most familial cases of Alzheimer´s disease are associated with mutations in presenilins [50, 53, 96], the majority of PSEN mutations are single-nucleotide substitutions, but small deletions and insertions have been described as well. It has been suggested that these mutations induce overproduction of β-amyloid, apparently by increasing γ-secretase activity [51, 53, 97-102]. Although transgenic mice expressing presenilin mutations do not develop the formation of neuritic plaques, these animals showed changes similar to those observed in AD patients, such as neuronal damage, synaptic loss and vascular disease. The most severe mutation in *PSEN1* is a donor-acceptor splice mutation that causes a two-aminoacid substitution and an in-frame

displays tissue-specific alternative splicing, major mutations found in the protein are identified.

families (Figure 6B). (http://www.molgen.ua.ac.be/ADmutations)

326 Neurochemistry

Allele 4 of apolipoprotein E (ApoE4) has been reported to represent the main identified risk factor for sporadic AD [44, 49, 108]. This gene has been associated with EOAD and LOAD in multiple ethnic groups, and carriers of APOE4 exhibit an earlier age of onset for AD [44, 109-110]. The frequency of the APOE4 allele varies among ethnic groups and it has been shown that ApoE4 is determinant for AD risk in white's individuals; however, in Hispanic and African populations, there is no correlation between the presence of the pathology and this allele. These results suggest that other genes or risk factors may contribute to the increased risk of AD in African and Hispanics [111-114].

The ApoE4 gene is located at chromosome 19q13.2 [115] and consists of 4 exons encoding a protein of 299 amino acid residues with a molecular weight of 34 kDa. APOE is a glycoprotein exhibiting variable levels of posttranslational sialylation due to O-linked glycosylation at threonine 194 [116]. The gene contains several single-nucleotide polymorphisms (SNPs) [117] leading to changes in the amino acid sequence of the protein, resulting in the production of three isoforms: ApoE2, ApoE3 and ApoE4, which are associated with different alleles (ε2, ε3, ε4). The three isoforms differ only by one or two amino acids, with the changes occurring at amino acid residues 112 and 158: ApoE2 (cys112, cys158), ApoE3 (cys112, arg158) and ApoE4 (arg112, arg158) [118-120]. The allelic distribution varies among ethnic groups, although it has generally been observed that allele 3 is the most frequent (77%), followed by allele 4 (15%), while allele 2 is less frequently observed (8%).

ApoE is a plasma protein that plays an important role in lipid metabolism and cholesterol transport in various tissues [108, 121-122]. The amino acid changes observed in the different isoforms of ApoE alter the 3-dimensional structure of the protein, affecting its lipid-binding properties, indicating that each isoform is metabolically different and varies in its affinity to bind to lipoprotein particles [123-124]. Apolipoproteins are synthesized primarily in the liver but can be processed and secreted in the brain by astrocytes and microglia. They are involved in neuronal regeneration [125], an increase in the synthesis of these proteins has been observed in the central and peripheral nervous system during neuronal damage. The distinct human ApoE isoforms differ significantly in their long-term effects on neuronal integrity as well as in their ability to protect against exocitotoxicity [126-128]. When ApoE isoforms are expressed in brain cells of ApoE-knockout (APOE-/-) mice, it can be observed that ApoE3 has a protective effect against age-related Aβ toxicity and neurodegeneration [129-130]. These differences in the neuroprotective capacities of apoE3 and apoE4 could contribute to the increased suscept‐ ibility of human ApoE4 carriers to AD [131]. Cholesterol homeostasis in hippocampal neurons is affected by the presence of apoE4, while the presence of ApoE2 and ApoE3 is not associated with any alterations in homeostasis [132]. Other roles of APOE isoforms include proliferation, synaptogenesis, myelination and amyloid elimination and tau phosphorylation.

and behavioral deficits *in vivo* [145]. Transgenic mice expressing apoE4Δ272–299 displayed AD-like neurodegenerative alterations in the cortex and hippocampus, including abnormally phosphorylated tau (p-tau) and Gallyas silver-positive neurons that contained cytosolic straight filaments with diameters of 15–20 nm, resembling pre-neurofibrillary tangles [145]. Similarly, overexpression of human ApoE4 in neurons results in hyperphosphorylation of the

Genetics of Alzheimer´S Disease http://dx.doi.org/10.5772/58286 329

Finally, although the presence of allele 4 of ApoE is not a deterministic factor for AD, it has been observed that this allele may favor the development of the disease at younger ages [148].

**Figure 7. Interaction of Amyloid and ApoE.** The ApoE4 gene is located on chromosome 19q13.2. It has been sug‐ gested that ApoE, could be involved in the Aβ aggregation and clearance. This process can be regulated of ApoE iso‐ form and thereby promote the onset of Aβ aggregation. In this way other pathologic mechanisms could be favored

The genetic causes of AD can be highly variable, even for familial forms. While EOAD is characterized by the presence of mutations with high penetrance in specific genes, the genetics of sporadic cases (LOAD) are more complex. LOAD susceptibility is determined by an

**7. Genome-wide association studies (gwas)**

tau protein, which increases with age [146-147].

#### **6.2. Apoe and amyloid**

Overexpression of a mutated form of human APP has shown that the levels of amyloid and ApoE increase in the brain with age, which is associated with decreasing Aβ levels in plasma [133]. It is possible that ApoE increases Aβ sequestration, deregulating the clearance of amyloid and leading to cognitive impairment in transgenic mice expressing a mutant form of human APP [134-135]. Recent studies have shown that apolipoprotein E (ApoE) receptor 2 and other members of the low-density lipoprotein receptor family (LRP, LRP1B, SorLA/LR11) interact with AβPP and regulates its endocytic trafficking [136-137]. Stable expression of human APP in B103 rat neuroblastoma cells (B103-APP); demonstrated that the isoform-specific effects of ApoE on Aβ production result from an alteration of AβPP recycling due to more pronounced stimulation of AβPP recycling by apoE4 than ApoE3 [138]. However, other authors have noted that there is no clear evidence upon which to base conclusions regarding the isoform-specific effects on AβPP processing [127, 139].

Although clearance of Aβ by ApoE has not been extensively studied, ApoE may modulate the removal of Aβ from the brain (Figure 7). Nevertheless, it has been suggested that clearance of Aβ is regulated by low-density lipoprotein receptor related protein-1 (LRP) and the receptor for advanced glycation end products (RAGE); this function is compromised in AD, which may contribute to elevation of the levels of amyloid in the brain [135, 140-141].

#### **6.3. Apoe and tau**

Neither the mechanisms by which the tau and ApoE4 proteins confer pathogenicity nor the nature of the interaction between these proteins has yet been established. Some authors have suggested that there is a relationship between the dosage of the ApoE4 allele and the density of NTFs [142-143]. It is known that ApoE3 has the ability to form a stable complex with Tau protein, and this association is believed to decrease Tau phosphorylation, preventing abnormal phosphorylation of Tau protein and their aggregation into paired helical filaments (PHF) [144]. When tau is phosphorylated, it loses its ability to interact with ApoE3. In contrast, ApoE4 does not interact with Tau.

It has recently been shown that the expression of a carboxy-terminal truncated fragment of the ApoE4 protein (Δ272-299 carboxyl terminal) is sufficient to elicit AD-like neurodegeneration and behavioral deficits *in vivo* [145]. Transgenic mice expressing apoE4Δ272–299 displayed AD-like neurodegenerative alterations in the cortex and hippocampus, including abnormally phosphorylated tau (p-tau) and Gallyas silver-positive neurons that contained cytosolic straight filaments with diameters of 15–20 nm, resembling pre-neurofibrillary tangles [145]. Similarly, overexpression of human ApoE4 in neurons results in hyperphosphorylation of the tau protein, which increases with age [146-147].

in the central and peripheral nervous system during neuronal damage. The distinct human ApoE isoforms differ significantly in their long-term effects on neuronal integrity as well as in their ability to protect against exocitotoxicity [126-128]. When ApoE isoforms are expressed in brain cells of ApoE-knockout (APOE-/-) mice, it can be observed that ApoE3 has a protective effect against age-related Aβ toxicity and neurodegeneration [129-130]. These differences in the neuroprotective capacities of apoE3 and apoE4 could contribute to the increased suscept‐ ibility of human ApoE4 carriers to AD [131]. Cholesterol homeostasis in hippocampal neurons is affected by the presence of apoE4, while the presence of ApoE2 and ApoE3 is not associated with any alterations in homeostasis [132]. Other roles of APOE isoforms include proliferation,

Overexpression of a mutated form of human APP has shown that the levels of amyloid and ApoE increase in the brain with age, which is associated with decreasing Aβ levels in plasma [133]. It is possible that ApoE increases Aβ sequestration, deregulating the clearance of amyloid and leading to cognitive impairment in transgenic mice expressing a mutant form of human APP [134-135]. Recent studies have shown that apolipoprotein E (ApoE) receptor 2 and other members of the low-density lipoprotein receptor family (LRP, LRP1B, SorLA/LR11) interact with AβPP and regulates its endocytic trafficking [136-137]. Stable expression of human APP in B103 rat neuroblastoma cells (B103-APP); demonstrated that the isoform-specific effects of ApoE on Aβ production result from an alteration of AβPP recycling due to more pronounced stimulation of AβPP recycling by apoE4 than ApoE3 [138]. However, other authors have noted that there is no clear evidence upon which to base conclusions regarding the isoform-specific

Although clearance of Aβ by ApoE has not been extensively studied, ApoE may modulate the removal of Aβ from the brain (Figure 7). Nevertheless, it has been suggested that clearance of Aβ is regulated by low-density lipoprotein receptor related protein-1 (LRP) and the receptor for advanced glycation end products (RAGE); this function is compromised in AD, which may

Neither the mechanisms by which the tau and ApoE4 proteins confer pathogenicity nor the nature of the interaction between these proteins has yet been established. Some authors have suggested that there is a relationship between the dosage of the ApoE4 allele and the density of NTFs [142-143]. It is known that ApoE3 has the ability to form a stable complex with Tau protein, and this association is believed to decrease Tau phosphorylation, preventing abnormal phosphorylation of Tau protein and their aggregation into paired helical filaments (PHF) [144]. When tau is phosphorylated, it loses its ability to interact with ApoE3. In contrast, ApoE4 does

It has recently been shown that the expression of a carboxy-terminal truncated fragment of the ApoE4 protein (Δ272-299 carboxyl terminal) is sufficient to elicit AD-like neurodegeneration

contribute to elevation of the levels of amyloid in the brain [135, 140-141].

synaptogenesis, myelination and amyloid elimination and tau phosphorylation.

**6.2. Apoe and amyloid**

328 Neurochemistry

effects on AβPP processing [127, 139].

**6.3. Apoe and tau**

not interact with Tau.

Finally, although the presence of allele 4 of ApoE is not a deterministic factor for AD, it has been observed that this allele may favor the development of the disease at younger ages [148].

**Figure 7. Interaction of Amyloid and ApoE.** The ApoE4 gene is located on chromosome 19q13.2. It has been sug‐ gested that ApoE, could be involved in the Aβ aggregation and clearance. This process can be regulated of ApoE iso‐ form and thereby promote the onset of Aβ aggregation. In this way other pathologic mechanisms could be favored

## **7. Genome-wide association studies (gwas)**

The genetic causes of AD can be highly variable, even for familial forms. While EOAD is characterized by the presence of mutations with high penetrance in specific genes, the genetics of sporadic cases (LOAD) are more complex. LOAD susceptibility is determined by an uncertain number of genetic risk factors exhibiting low penetrance that are present at a high frequency. This is particularly important because although patients who develop this subtype of the disease have been considered to represent *sporadic cases*, the genetic component of these cases is a feature that has not been established. A possible explanation for the difficulty involved in the identification of genetic risk factors is that LOAD is a multifactorial complex disorder that involves both genetic and environmental components.

**Genome-Wide Association Studies (GWAS)**

Harold, 2009 CC GWAS Europe & USA

Heinzen, 2009 CC GWAS

<sup>2011</sup> CC GWAS

Hu, 2011 CC GWAS

Poduslo, 2009 CC,

Seshadri, 2010 CC

Wijsman, 2011 CC,

Hollingworth,

**Study Design Type Population # of SNPs**

(GERAD1)

Europe & USA (GERAD1+2, EADI1+2, ADNI, TGEN1, MAYO2, CHARGE)

USA (Pfizer, ADNI), Canada (GenADA, Genizon)

Naj, 2011 CC GWAS USA (ADGC) 2,324,889

Reiman, 2007 CC GWAS USA, Netherlands

GWAS + metaanalysis

Sherva, 2011 CC GWAS Israel (Wadi Ara) 2,540,000

(TGEN1)

Europe & USA (CHARGE, EADI1, GERAD1)

**Table 2.** Genome-Wide Association Studies in Alzheimer's Disease.

**AD Cases Normal Controls**

**# Subjects GWAS**

529205 M 3941 2023 7848 2340 APOE, CLU, PICALM

**# Subjects (followup)**

**Featured Genes**

Genetics of Alzheimer´S Disease http://dx.doi.org/10.5772/58286 331

GWA\_7p15.2, LMNA, LOC651924, MY13, PCK1, PGBD1, TNK1, TRAK2, UBD

ABCA7, BIN1, CD2AP, CD33, CR1, EPHA1, MS4A4E, MS4A6A

APOE, GOLM1, GWA\_15q21.2, GWA\_9p24.3

APOE, BIN1, CD2AP, CD33, CLU, CR1, EPHA1, MS4A4A, PICALM

APOE, ARSB, CAND1, EFNA5, MAG12, PRUNE2 TOMM40

EXOC3L2, PICALM

AGPAT1, ATPVOA4, GLOD4, RGS6, TMEM132C

**# Subjects (followup)**

496763 M 6688 13182 13685 26261

(imputed) <sup>M</sup> <sup>8309</sup> <sup>3531</sup> <sup>7366</sup> <sup>3565</sup>

312316 M 446 415 290 260 GAB2

(imputed) <sup>C</sup> <sup>124</sup> - <sup>142</sup> -

FBAT GWAS USA (NIA, NCRAD) <sup>565336</sup> <sup>M</sup> <sup>1848</sup> <sup>617</sup> <sup>1991</sup> <sup>573</sup> APOE, CELF2

(imputed) <sup>M</sup> <sup>3006</sup> <sup>6505</sup> <sup>22604</sup> <sup>13532</sup> APOE, BIN1, CLU,

509376 C 1831 751 1764 751 APOE, BIN1

**DX # Subjects GWAS**

+CNV USA (CAP, DUKE) n.g. <sup>U</sup> <sup>331</sup> - <sup>368</sup> -

Lambert, 2009 CC GWAS Europe (EADI1) 537029 C 2032 3978 5328 3297 APOE, CLU, CR1

FBAT GWAS USA <sup>489218</sup> <sup>C</sup> <sup>9</sup> <sup>199</sup> <sup>10</sup> <sup>225</sup> TRCP4AP

Li, 2008 CC GWAS Canada (GenADA) 469438 C 753 418 736 249

Potkin, 2009 CC, QT GWAS USA (ADNI) 516645 C 172 - 209 -

2,540,000

In the last thirty years, a considerable number of studies have been developed aimed at identifying risk factors that confer susceptibility for developing AD. In this regard, genomewide association studies (GWAS) represent a powerful approach for identifying putative candidate genes for common complex diseases, such as LOAD. These studies simultaneously analyze a large number of genetic markers, typically consisting of single-nucleotide polymor‐ phisms (SNPs). Although they have also involved arrays for assessing copy-number variants (deletions or multiplications of chromosomal segments), other GWAS arrays only contain SNPs located in predicted or known coding regions (cSNPs). The Affymetrix GeneChip 500K platform exhibits 60% coverage of the phase II HapMAp (Affymetrix, Santa Clara, CA, USA), whereas the Illumina Hap300 platform presents 77% coverage (Illumina, Inc., San Diego, CA, USA). At least 12 GWAS addressing Alzheimer´s disease have been published to date, which have identified more than 40 genetic variants that might confer risk for developing this pathology. However, much remains to be learned regarding the pathology and the genetic risk factors associated with late onset Alzheimer´s disease. The main studies investigating the associations between cases and controls with LOAD using such platforms are described below (Table 2).



uncertain number of genetic risk factors exhibiting low penetrance that are present at a high frequency. This is particularly important because although patients who develop this subtype of the disease have been considered to represent *sporadic cases*, the genetic component of these cases is a feature that has not been established. A possible explanation for the difficulty involved in the identification of genetic risk factors is that LOAD is a multifactorial complex

In the last thirty years, a considerable number of studies have been developed aimed at identifying risk factors that confer susceptibility for developing AD. In this regard, genomewide association studies (GWAS) represent a powerful approach for identifying putative candidate genes for common complex diseases, such as LOAD. These studies simultaneously analyze a large number of genetic markers, typically consisting of single-nucleotide polymor‐ phisms (SNPs). Although they have also involved arrays for assessing copy-number variants (deletions or multiplications of chromosomal segments), other GWAS arrays only contain SNPs located in predicted or known coding regions (cSNPs). The Affymetrix GeneChip 500K platform exhibits 60% coverage of the phase II HapMAp (Affymetrix, Santa Clara, CA, USA), whereas the Illumina Hap300 platform presents 77% coverage (Illumina, Inc., San Diego, CA, USA). At least 12 GWAS addressing Alzheimer´s disease have been published to date, which have identified more than 40 genetic variants that might confer risk for developing this pathology. However, much remains to be learned regarding the pathology and the genetic risk factors associated with late onset Alzheimer´s disease. The main studies investigating the associations between cases and controls with LOAD using such platforms are described below

**AD Cases Normal Controls**

561494 C 1082 - 1239 1400 APOE, LRAT

502627 N 664 - 422 - APOE

**# Subjects GWAS**

**# Subjects (followup)**

**Featured Genes**

APOE ATXN1 CD33 GWA 14q31.2

ACAN, APOE, BCR, CTSS, EBF3, FAM63A,

GALP, GWA\_14q32.13,

**# Subjects (followup)**

**DX # Subjects GWAS**

<sup>2009</sup> CC GWAS USA (CAP) <sup>532000</sup> <sup>M</sup> <sup>492</sup> <sup>238</sup> <sup>496</sup> <sup>220</sup> APOE, FAM113B

<sup>2009</sup> CC GWAS USA (Mayo) <sup>313504</sup> <sup>M</sup> <sup>844</sup> <sup>1547</sup> <sup>1255</sup> <sup>1209</sup> APOE PCDH11X

Bertram, 2008 FBAT GWAS USA (NIMH) 484522 M 941 1767 404 838

Grupe, 2007 CC GWAS USA & UK 17343 M 380 1428 396 1666

disorder that involves both genetic and environmental components.

(Table 2).

330 Neurochemistry

CAUSASIAN Abraham,

Beecham,

Carrasquillo,

<sup>2008</sup> CC GWAS

**Genome-Wide Association Studies (GWAS)**

**Study Design Type Population # of SNPs**

Overlaps with Harold, 2009

(TGEN1)

pooled

Coon, 2007 CC GWAS USA, Netherlands

**Table 2.** Genome-Wide Association Studies in Alzheimer's Disease.

#### **7.1. Grupe 2007**

The first GWAS addressing Alzheimer´s disease was reported in 2007 by Grupe *et al*. A total of 17, 343 SNPs, located in 11 221 unique genes were tested for an association with LOAD in a case–control discovery sample from the UK (1808 LOAD cases and 2062 controls) [149]. These researchers reported the identification of several candidate SNPs showing a significant association with LOAD. Three of these SNPs (**rs157581, rs405509** and **rs1132899**) are located on chromosome 19, close to the APOE gene, and exhibit genome-wide significance (P val‐ ue=6.94E-81 to 0.0001) and linkage disequilibrium (LD) with the APOE4 and 2/3 variants (0.09 < D0 < 1). Furthermore, sixteen additional SNPs showed evidence of an association with LOAD [P=0.0010-0.00006; odds ratio (OR)=1.07–1.45].

In a subsequent study, the same group of researchers divided each cohort of LOAD cases and controls into two subgroups: allelic APOE ε4 carriers and APOE ε4 noncarriers. The results showed an association with six SNPs of the GRB-associated binding protein 2 (GAB2) gene and a common haplotype encompassing the entire **GAB2** gene [158]. SNP **rs2373115** was associated with an odds ratio of 4.06 (confidence interval 2.81–14.69) and interacts with APOE

Genetics of Alzheimer´S Disease http://dx.doi.org/10.5772/58286 333

The GAB2 protein is involved in a number of different pathways, and thus, it is possible that GAB2 could affect mechanisms involved in cell survival, Tau phosphorylation and NFT formation. Additionally, GAB2 may be involved in the production of Aβ [158], contributing to the development of AD pathology. Finally, GAB2 has been found to be coexpressed with

The GWAS conducted by Abraham in 2008 differs from all other currently published GWAS addressing AD in that, in the initial screening in this study, DNA pools were utilized for genotyping rather than individual DNA samples [160]. DNA samples were collected from 1,082 individuals with LOAD and 1,239 control subjects. The age at onset ranged from 60 to 95 years, and controls were matched for age (mean=76.53 years, SD=33), gender and ethnicity. The construction of the pools was validated using the SNaPshot method. The pools were genotyped using Illumina HumanHap300 and Illumina Sentrix HumanHap240S arrays, testing 561,494 SNPs. The results showed an association of several SNPs close to the *APOE* locus with LOAD, including 7 SNPs within 71 kb, with allele frequency differences of between 6% – 14%. Five of the seven SNPs were individually genotyped and were confirmed to present highly significant associations with LOAD. Although these studies using pooled DNA samples considerably reduce costs, their results may not accurately represent real allele frequency

Another GWAS addressing AD was performed by Bertram *et al.* in 2008. This study repre‐ sented the first to employ family-based methods for the initial screening. This case, a genomewide association (GWA) analysis was performed using 484,522 single-nucleotide polymorphisms (SNPs) on a large (1,376 samples from 410 families) sample of AD families of self-reported European descent. All 10,388 X chromosome markers were eliminated, as also were 5,758 SNPs that did not pass genotype quality assessment or showed a minor allele frequency (MAF). A total of 404,604 (80.8%) SNPs were finally used for screening [161].

In this study, five SNPs were identified as showing either a significant or marginally significant genome-wide association with a multivariate phenotype combining affection status and onset age. Four of these markers were not related to APOE4. The first marker, **rs4420638**, is located 340 bp 3' of APOC1 on chromosome 19q13 and very likely reflects the effects of the APOE4 allele (**rs429358**). The other markers are **rs11159647** (located in predicted gene NT\_026437.1360 on chromosome 14q31.2), **rs179943** (located in **ATXN1** [MIM 601556] on chromosome 6p22.3,

ε4 to further modify risk.

**7.3. Abraham 2008**

distributions.

**7.4. Bertram 2008.**

other putative AD-related genes [159].

Of these SNPs, one was a missense mutation (**rs3745833**) located in the galanin-like peptide precursor (**GALP**) gene. The associated SNP encodes a non-synonymous substitution (Ili72Met) in exon 4. In the Caucasian population, the common minor C-allele increases the risk for AD in 10% of individuals. The galanin gene has been implicated in neuronal survival, regeneration and neuroprotection as well as the inhibition of cholinergic neurotransmission and suppression of long-term potentiation [150-151]. In limbic brain regions of AD patients, galanin expression is upregulated and could conceivably worsen the symptoms of the disease. Transgenic mice overexpressing galanin display cognitive and neurochemical deficits similar to those observed in AD patients [152].

Another important SNP was found to be located in PGBD1 (piggyBack transposable element derived 1). The associated SNP (**rs3800324**) encodes a non-synonymous substitution (Gly244Glu) in exon 5, and the presence of the minor A (Glu) allele significantly increases the risk of AD by 20%. The function of this protein is not known, but it is specifically expressed in the brain. Finally, in this study, the authors showed that four additional SNPs showed evidence of association with LOAD. These variants include SNPs located in TNK1 and PCK1 as well as an intergenic SNP near SERPINA13.

TNK1 is a non-receptor tyrosine kinase that mediates phospholipid signal transduction. In addition, together with TRAK2, TNK1 may be involved in protein trafficking and signal transduction [153] and participate in the processing of amyloid precursor protein and amyloid β-production [154-155]. Aberrant TNK1 activity may increase the risk of LOAD [156].

#### **7.2. Coon 2007, Reiman 2007**

In the same year, Coon *et al.* employed an ultra-high-density whole-genome association analysis, demonstrating the ability to identify the APOE locus as a major susceptibility gene for late onset AD [157]. This study used the Affymetrix 500K platform, including 502,627 SNPs, and was performed in a population of 1086 histopathologically verified AD cases and controls. The results obtained showed that the APOE locus is the major susceptibility gene for late onset AD in the human genome, with an OR significantly greater than any other locus in the human genome (Bonferroni corrected OR=4.01). The polymorphism identified in this study (**rs4420638**) is located on chromosome 19 and is 14 kilobase pairs distal to the APOE epsilon variant.

In a subsequent study, the same group of researchers divided each cohort of LOAD cases and controls into two subgroups: allelic APOE ε4 carriers and APOE ε4 noncarriers. The results showed an association with six SNPs of the GRB-associated binding protein 2 (GAB2) gene and a common haplotype encompassing the entire **GAB2** gene [158]. SNP **rs2373115** was associated with an odds ratio of 4.06 (confidence interval 2.81–14.69) and interacts with APOE ε4 to further modify risk.

The GAB2 protein is involved in a number of different pathways, and thus, it is possible that GAB2 could affect mechanisms involved in cell survival, Tau phosphorylation and NFT formation. Additionally, GAB2 may be involved in the production of Aβ [158], contributing to the development of AD pathology. Finally, GAB2 has been found to be coexpressed with other putative AD-related genes [159].

#### **7.3. Abraham 2008**

**7.1. Grupe 2007**

332 Neurochemistry

[P=0.0010-0.00006; odds ratio (OR)=1.07–1.45].

to those observed in AD patients [152].

an intergenic SNP near SERPINA13.

**7.2. Coon 2007, Reiman 2007**

variant.

The first GWAS addressing Alzheimer´s disease was reported in 2007 by Grupe *et al*. A total of 17, 343 SNPs, located in 11 221 unique genes were tested for an association with LOAD in a case–control discovery sample from the UK (1808 LOAD cases and 2062 controls) [149]. These researchers reported the identification of several candidate SNPs showing a significant association with LOAD. Three of these SNPs (**rs157581, rs405509** and **rs1132899**) are located on chromosome 19, close to the APOE gene, and exhibit genome-wide significance (P val‐ ue=6.94E-81 to 0.0001) and linkage disequilibrium (LD) with the APOE4 and 2/3 variants (0.09 < D0 < 1). Furthermore, sixteen additional SNPs showed evidence of an association with LOAD

Of these SNPs, one was a missense mutation (**rs3745833**) located in the galanin-like peptide precursor (**GALP**) gene. The associated SNP encodes a non-synonymous substitution (Ili72Met) in exon 4. In the Caucasian population, the common minor C-allele increases the risk for AD in 10% of individuals. The galanin gene has been implicated in neuronal survival, regeneration and neuroprotection as well as the inhibition of cholinergic neurotransmission and suppression of long-term potentiation [150-151]. In limbic brain regions of AD patients, galanin expression is upregulated and could conceivably worsen the symptoms of the disease. Transgenic mice overexpressing galanin display cognitive and neurochemical deficits similar

Another important SNP was found to be located in PGBD1 (piggyBack transposable element derived 1). The associated SNP (**rs3800324**) encodes a non-synonymous substitution (Gly244Glu) in exon 5, and the presence of the minor A (Glu) allele significantly increases the risk of AD by 20%. The function of this protein is not known, but it is specifically expressed in the brain. Finally, in this study, the authors showed that four additional SNPs showed evidence of association with LOAD. These variants include SNPs located in TNK1 and PCK1 as well as

TNK1 is a non-receptor tyrosine kinase that mediates phospholipid signal transduction. In addition, together with TRAK2, TNK1 may be involved in protein trafficking and signal transduction [153] and participate in the processing of amyloid precursor protein and amyloid

In the same year, Coon *et al.* employed an ultra-high-density whole-genome association analysis, demonstrating the ability to identify the APOE locus as a major susceptibility gene for late onset AD [157]. This study used the Affymetrix 500K platform, including 502,627 SNPs, and was performed in a population of 1086 histopathologically verified AD cases and controls. The results obtained showed that the APOE locus is the major susceptibility gene for late onset AD in the human genome, with an OR significantly greater than any other locus in the human genome (Bonferroni corrected OR=4.01). The polymorphism identified in this study (**rs4420638**) is located on chromosome 19 and is 14 kilobase pairs distal to the APOE epsilon

β-production [154-155]. Aberrant TNK1 activity may increase the risk of LOAD [156].

The GWAS conducted by Abraham in 2008 differs from all other currently published GWAS addressing AD in that, in the initial screening in this study, DNA pools were utilized for genotyping rather than individual DNA samples [160]. DNA samples were collected from 1,082 individuals with LOAD and 1,239 control subjects. The age at onset ranged from 60 to 95 years, and controls were matched for age (mean=76.53 years, SD=33), gender and ethnicity. The construction of the pools was validated using the SNaPshot method. The pools were genotyped using Illumina HumanHap300 and Illumina Sentrix HumanHap240S arrays, testing 561,494 SNPs. The results showed an association of several SNPs close to the *APOE* locus with LOAD, including 7 SNPs within 71 kb, with allele frequency differences of between 6% – 14%. Five of the seven SNPs were individually genotyped and were confirmed to present highly significant associations with LOAD. Although these studies using pooled DNA samples considerably reduce costs, their results may not accurately represent real allele frequency distributions.

#### **7.4. Bertram 2008.**

Another GWAS addressing AD was performed by Bertram *et al.* in 2008. This study repre‐ sented the first to employ family-based methods for the initial screening. This case, a genomewide association (GWA) analysis was performed using 484,522 single-nucleotide polymorphisms (SNPs) on a large (1,376 samples from 410 families) sample of AD families of self-reported European descent. All 10,388 X chromosome markers were eliminated, as also were 5,758 SNPs that did not pass genotype quality assessment or showed a minor allele frequency (MAF). A total of 404,604 (80.8%) SNPs were finally used for screening [161].

In this study, five SNPs were identified as showing either a significant or marginally significant genome-wide association with a multivariate phenotype combining affection status and onset age. Four of these markers were not related to APOE4. The first marker, **rs4420638**, is located 340 bp 3' of APOC1 on chromosome 19q13 and very likely reflects the effects of the APOE4 allele (**rs429358**). The other markers are **rs11159647** (located in predicted gene NT\_026437.1360 on chromosome 14q31.2), **rs179943** (located in **ATXN1** [MIM 601556] on chromosome 6p22.3, **rs3826656** (located in predicted gene NT\_011109.848 on 19q13.33), and **rs2049161** (located in cDNA BC040718 on 18p11.31). These four SNPs were tested in three additional independent AD family samples composed of nearly 2700 individuals from almost 900 families. SNP **rs11159647** on chromosome 14q31 was primarily associated with age of onset (p=0.006), with a median reduction in onset age of 1.1 years being observed. Evidence of an association with this allele was also found in GWA data generated in an independent sample of ~1,400 AD cases and controls (p=0.04). None of these markers were previously described as modifiers of AD risk or onset age (Bertram 2008). The SNP **rs179943** on chromosome 6p22.3 is located within an intron of the ataxin 1 (ATXN1) gene. Although the function of ataxin1 is not known, it has been proposed to be associated with spinocerebellar ataxia type 1 (**SCA1**), a progressive neurodegenerative disease. The SNP **rs3826656** on 19q33 is located less than 2 kb proximal of the transcription initiation site of **CD33**. This protein is a cell-surface receptor on cells of monocytic or myeloid lineages. Additionally, it is a member of the SIGLEC family of lectins that bind sialic acid and regulate the innate immune system via the activation of caspasedependent and caspase-independent cell death pathways.

**KCNMA1, NOS2A, SORCS2, SORCS3, SORL1,** and **WWC1)** exhibited p values ranging from

Genetics of Alzheimer´S Disease http://dx.doi.org/10.5772/58286 335

Of these genes, the main candidate associated with the development of LOAD in several populations is the *sortlin-related receptor* (**SORL1**) gene. The mechanism by which SORL1 affects the development of Alzheimer´s disease is unknown, but it has been established to have the ability to interact with APP and APOE, possibly affecting the formation and accumulation

This genome-wide association study was performed in two stages using the Illumina Human‐ Hap300 array. In stage I, 313,504 SNPs were analyzed in 844 cases and 1,255 controls (2100 subjects from the Mayo clinic), and only six *APOE*-linked SNPs showed genome-wide significance in this stage of study. Of these polymorphisms, only rs2075650 (located on chromosome 19) showed genome-wide significance, and this SNP shows strong linkage disequilibrium (LD) with APOE (P value 4.8x10-46). In stage II, the 25 SNPs showing the most significant associations in stage I were genotyped in an additional 845 cases and 1,000 controls. These 25 SNPs included 10 SNPs in the APOE region on chromosome 19, all of which presented *P* values ranging from 9.5X10-79 to 0.05. The other 15 SNPs are located on other chromosomes. One of two SNPs on the X chromosome, rs5984894 (P value 0.0006), is located within the gene encoding protocadherin 11, X-linked (**PCDH11X**) in the Xq21.3/Yp11.2 region. To extend the analysis of *PCDH11X*, three *PCDH11X* SNPs (rs5941047, rs4568761 and rs2573905) residing in the same haplotype block as rs5984894 were genotyped in all stages. Highly significant associations were observed for all three SNPs, with *P* values of 1.6×10-7 (rs2573905), 8.0×10-5 (rs5941047) and 0.001 (rs4568761) being obtained. rs2573905 is located 8,483 bp 3′ of rs5984894 and is in strong linkage disequilibrium with rs5984894 (r2=0.98, D'=0.99). Analysis of rs5984894 by multivariable logistic regression adjusted by sex showed that the association was stronger in female homozygotes (OR=1.75, P=2.0x10-7) and heterozygotes (OR=1.26, P=0.01). For hemi‐ zygous males, a similar trend was observed (OR=1.18), although this did not reach statistical

The *PCDH11X* gene contains at least 17 exons spanning over 700 kb. Alternative splicing of *PCDH11X* produces several isoforms that are mainly expressed in the brain, with particularly strong expression being detected in the cortex and hippocampus and weaker expression being observed in the cerebellum. The PCDH11X protein plays a fundamental role in cell-cell recognition and it is essential for the segmental development and function of the central nervous system. However, among all published and reported AD GWASs, this is the only one that reports involvement of an X chromosome locus, which, if confirmed, could at least partially explain the well-established increased disease prevalence in women versus men.

In the first stage of this study, an association with the APOE locus (rs2075650, p=1.8×10−157) was established in 3,941 patients and 7,848 controls. Additionally, this GWA analysis identified

0.003 to 0.05 in the individual GWAS and from 0.0001 to 0.01 in the joint analysis.

of amyloid beta peptides.

significance (P-value 0.07) [163].

**7.7. Harold 2009**

**7.6. Carrasquillo 2009**

#### **7.5. Beecham 2009**

Another GWAS was carried out by Beecham in 2009. This GWAS included 998 individuals of European descent, including 492 LOAD cases and 496 cognitive controls, using Illumina's HumanHap550 BeadChip. An additional 238 cases and 220 controls were also used in this study as a validation dataset for single-nucleotide polymorphisms (SNPs) that met the genome-wide significance criteria. The results showed associations of 38 SNPs with LOAD with uncorrected p values < 0.00005, six of which were in or near the APOE gene [162].

The most significant non-APOE SNP was rs11610206 on chromosome 12q13 (45.92 Mb), which presented an uncorrected p=1.93X10-6. This SNP was genotyped in an independent replication dataset of 238 cases and 220 controls, resulting in a p value of 3.452X10-7, which was more significant than in the initial dataset. This SNP is not located in a known gene but is less than 10 kb from the FAM113B gene. Additionally, there are a number of nearby candidate genes, such as the *vitamin D* (1,25-dihydroxyvitamin D3) *receptor* (*VDR* [MIM 601769]) and *adhesion molecule with Ig-like domain 2* (*AMIGO2*) genes.

These authors also compared their results with those obtained by Reiman, and four polymor‐ phisms were found that showed an association in both studies. Two of these SNPs, 1q42 and 19q13, are located within genes; the two other signals replicated in both datasets are not in known genes. The 1q42 SNP (rs12044355) resides in the DISC1 gene, which has been associated with schizophrenia and is linked to bipolar disorder, depression, and cognitive function. The 19q13 signal is located in and near exon 6 of *zinc finger protein 224* (*ZNF224* [MIM 194555]); two of the associated markers (rs4508518 and rs3746319) are within the exon. The first SNP (rs4508518) is a coding but synonymous polymorphism, whereas the second (rs3746319) leads to a missense mutation.

Finally, nine candidate genes from the over 500 genes in the AlzGene candidate gene list present SNPs with a nominal association in both GWASs. These genes (**ADAM12, CSF1, GBP2,** **KCNMA1, NOS2A, SORCS2, SORCS3, SORL1,** and **WWC1)** exhibited p values ranging from 0.003 to 0.05 in the individual GWAS and from 0.0001 to 0.01 in the joint analysis.

Of these genes, the main candidate associated with the development of LOAD in several populations is the *sortlin-related receptor* (**SORL1**) gene. The mechanism by which SORL1 affects the development of Alzheimer´s disease is unknown, but it has been established to have the ability to interact with APP and APOE, possibly affecting the formation and accumulation of amyloid beta peptides.

#### **7.6. Carrasquillo 2009**

**rs3826656** (located in predicted gene NT\_011109.848 on 19q13.33), and **rs2049161** (located in cDNA BC040718 on 18p11.31). These four SNPs were tested in three additional independent AD family samples composed of nearly 2700 individuals from almost 900 families. SNP **rs11159647** on chromosome 14q31 was primarily associated with age of onset (p=0.006), with a median reduction in onset age of 1.1 years being observed. Evidence of an association with this allele was also found in GWA data generated in an independent sample of ~1,400 AD cases and controls (p=0.04). None of these markers were previously described as modifiers of AD risk or onset age (Bertram 2008). The SNP **rs179943** on chromosome 6p22.3 is located within an intron of the ataxin 1 (ATXN1) gene. Although the function of ataxin1 is not known, it has been proposed to be associated with spinocerebellar ataxia type 1 (**SCA1**), a progressive neurodegenerative disease. The SNP **rs3826656** on 19q33 is located less than 2 kb proximal of the transcription initiation site of **CD33**. This protein is a cell-surface receptor on cells of monocytic or myeloid lineages. Additionally, it is a member of the SIGLEC family of lectins that bind sialic acid and regulate the innate immune system via the activation of caspase-

Another GWAS was carried out by Beecham in 2009. This GWAS included 998 individuals of European descent, including 492 LOAD cases and 496 cognitive controls, using Illumina's HumanHap550 BeadChip. An additional 238 cases and 220 controls were also used in this study as a validation dataset for single-nucleotide polymorphisms (SNPs) that met the genome-wide significance criteria. The results showed associations of 38 SNPs with LOAD with uncorrected p values < 0.00005, six of which were in or near the APOE gene [162].

The most significant non-APOE SNP was rs11610206 on chromosome 12q13 (45.92 Mb), which presented an uncorrected p=1.93X10-6. This SNP was genotyped in an independent replication dataset of 238 cases and 220 controls, resulting in a p value of 3.452X10-7, which was more significant than in the initial dataset. This SNP is not located in a known gene but is less than 10 kb from the FAM113B gene. Additionally, there are a number of nearby candidate genes, such as the *vitamin D* (1,25-dihydroxyvitamin D3) *receptor* (*VDR* [MIM 601769]) and *adhesion*

These authors also compared their results with those obtained by Reiman, and four polymor‐ phisms were found that showed an association in both studies. Two of these SNPs, 1q42 and 19q13, are located within genes; the two other signals replicated in both datasets are not in known genes. The 1q42 SNP (rs12044355) resides in the DISC1 gene, which has been associated with schizophrenia and is linked to bipolar disorder, depression, and cognitive function. The 19q13 signal is located in and near exon 6 of *zinc finger protein 224* (*ZNF224* [MIM 194555]); two of the associated markers (rs4508518 and rs3746319) are within the exon. The first SNP (rs4508518) is a coding but synonymous polymorphism, whereas the second (rs3746319) leads

Finally, nine candidate genes from the over 500 genes in the AlzGene candidate gene list present SNPs with a nominal association in both GWASs. These genes (**ADAM12, CSF1, GBP2,**

dependent and caspase-independent cell death pathways.

*molecule with Ig-like domain 2* (*AMIGO2*) genes.

to a missense mutation.

**7.5. Beecham 2009**

334 Neurochemistry

This genome-wide association study was performed in two stages using the Illumina Human‐ Hap300 array. In stage I, 313,504 SNPs were analyzed in 844 cases and 1,255 controls (2100 subjects from the Mayo clinic), and only six *APOE*-linked SNPs showed genome-wide significance in this stage of study. Of these polymorphisms, only rs2075650 (located on chromosome 19) showed genome-wide significance, and this SNP shows strong linkage disequilibrium (LD) with APOE (P value 4.8x10-46). In stage II, the 25 SNPs showing the most significant associations in stage I were genotyped in an additional 845 cases and 1,000 controls. These 25 SNPs included 10 SNPs in the APOE region on chromosome 19, all of which presented *P* values ranging from 9.5X10-79 to 0.05. The other 15 SNPs are located on other chromosomes. One of two SNPs on the X chromosome, rs5984894 (P value 0.0006), is located within the gene encoding protocadherin 11, X-linked (**PCDH11X**) in the Xq21.3/Yp11.2 region. To extend the analysis of *PCDH11X*, three *PCDH11X* SNPs (rs5941047, rs4568761 and rs2573905) residing in the same haplotype block as rs5984894 were genotyped in all stages. Highly significant associations were observed for all three SNPs, with *P* values of 1.6×10-7 (rs2573905), 8.0×10-5 (rs5941047) and 0.001 (rs4568761) being obtained. rs2573905 is located 8,483 bp 3′ of rs5984894 and is in strong linkage disequilibrium with rs5984894 (r2=0.98, D'=0.99). Analysis of rs5984894 by multivariable logistic regression adjusted by sex showed that the association was stronger in female homozygotes (OR=1.75, P=2.0x10-7) and heterozygotes (OR=1.26, P=0.01). For hemi‐ zygous males, a similar trend was observed (OR=1.18), although this did not reach statistical significance (P-value 0.07) [163].

The *PCDH11X* gene contains at least 17 exons spanning over 700 kb. Alternative splicing of *PCDH11X* produces several isoforms that are mainly expressed in the brain, with particularly strong expression being detected in the cortex and hippocampus and weaker expression being observed in the cerebellum. The PCDH11X protein plays a fundamental role in cell-cell recognition and it is essential for the segmental development and function of the central nervous system. However, among all published and reported AD GWASs, this is the only one that reports involvement of an X chromosome locus, which, if confirmed, could at least partially explain the well-established increased disease prevalence in women versus men.

#### **7.7. Harold 2009**

In the first stage of this study, an association with the APOE locus (rs2075650, p=1.8×10−157) was established in 3,941 patients and 7,848 controls. Additionally, this GWA analysis identified strong associations of SNPs in two new loci: rs11136000, which is located in the *CLU,* or *APOJ*, gene (p=1.4×10−9), and rs3851179, a SNP 5′ to the *PICALM* gene (p=1.9×10−8). rs11136000 is located within an intron of the clusterin (*CLU*, also known as *APOJ*) gene on chromosome 8, and rs3851179 is found 88.5 kb 5′ of *PICALM* on chromosome 11. In stage 2, these new SNPs were genotyped in 2,023 AD cases and 2,340 age-matched controls from an independent sample. Associations were found for both polymorphisms, with p=0.017 and OR=0.905 for rs11136000 and p=0.014 and OR=0.897 for rs3851179. A meta-analysis of stages 1 and 2 was also conducted in this study, and the results showed highly significant evidence of associations for the *CLU* and *PICALM* loci (rs11136000 p=8.5×10−10 and rs3851179 p=1.3×10−9, respectively). Finally, no significant interactions of novel SNPs associated with *APOE* status were observed to influence AD risk (rs11136000x*APOE*4 interaction p=0.674; rs3851179x*APOE*4 interaction p=0.735) [164].

reported in previous studies: CYP19A1 (rs2899472, p=1.90 × 10-7) and NCAM2 (rs1022442,

**8. Population genetics and genetic association studies: crucial issues to**

Although efforts to obtain genetic biomarkers that help in anticipate diagnostic of Alzheimer disease, present-day the clinical research not have the results that expected. The publications that relate genes with Alzheimer disease has increased exponentially however, numerous lines of evidence have demostrated discrepant results among populations. These findings suggest that it is neccesary diminish the confounder factors and focus on identify the cause [169]. Once the causes are established, could integrated in practice of medicine helping with anticipate

In order to avoid spurious associations Little J. et al published an initiative that pretends increase the quality of reporting genetic association studies dissemining this information in different journals (epidemiology, clinical investigation, internal medicine and basic research) [170-176]. The publication refered as STREGA report (STrenghthening the REporting of Geenetic Association studies) provides additional comments to 22 items reported previously by STROBE (STrengthening the Reporting of OBservational Studies in Epidemiology) [177]. These comments include different items, however population genetics topics are crucial issues

One of the most important topics in genetic association studies (GAS) is Hardy-Weinberg equilibrium. Hardy-Weinberg equilibrium (HWE) is represented by the equation (p+q)2

represented homozygous state, whereas 2pq represented heterozygous state. Under random maiting and non-overlapping, homozygous and heterozygous states are in equal proportions (0.5 each one) maintaining the HWE. This equilibrium are also maintained when evolutionary forces are absents (mutation, random drift, genetic flow, natural selection), the population size it is nearby to the infinitum, and when frequencies of alleles in both sexes are equal [178]. However, some conditions could modify these proportions provoking a Hardy-Weinberg departure (HWD). HWD is related with an excess of homozygous individuals (with subse‐ quent heterozygous deficit) or heterozygous individuals (with subsequent homozygous deficit). Therefore, Hardy-Weinberg model is an essential element used to analyze genetic data, and is the initial step for check the quality of genotyping, because genotyping errors due to poor quality provoke HWD as a consequence of distort in genotype distribution [179]. Nevertheless, HWD are not only related with genotyping mistakes because some factors as demographic events, young population, founder effect, inbreeding, and population stratifi‐

Population stratification is the consequence of populations with a recent miscegenation. Admixture populations show different allele frequencies among different subpopulations that conform the whole population, which consequently is not a homogeneous population [180].

=1,

and q2

+2pq+q2

=1, where p2

Genetics of Alzheimer´S Disease http://dx.doi.org/10.5772/58286 337

whose depreciation, increase statistical mistakes type I and II [173].

whose perfect square binomial equation it is represented by p2

p=2.75 × 10-7).

diagnostic.

cation may provoke HWD.

**enhance the transparency of results.**

CLU is a secreted chaperone that can also be found in the cytosol under some stress conditions. It has been suggested that CLU is involved in several basic biological events, including cell death, tumor progression, and neurodegenerative disorders. The genetic risk allele (C) of CLU gene variant rs11136000 is carried by ∼88% of Caucasians; the C allele confers 1.16 greater odds of developing late onset AD than the T allele [165].

PICALM is a phosphatidylinositol-binding clathrin assembly protein. This protein plays a role in altering synaptic vesicle cycling or APP endocytosis. Although the presence of polymor‐ phism rs3851179 was associated with high significance related to the development of AD in the Caucasian population, these results could not be replicated in Chinese or Italian popula‐ tions [166-167]. The results obtained in recent studies by Piaceri showed that the segregation of the PICALM rs3851179 variant did not show a statistically significant difference between LOAD cases and controls, suggesting a reduced risk of developing late onset Alzheimer´s disease (LOAD).

#### **7.8. Han 2010**

Unlike the studies described above, this study additionally established a relationship between the allelic variants found by GWAS and cerebrospinal fluid (CSF) levels of amyloid Ab1-42, T-tau, and P-tau181P [168]. The data used in this study was obtained from the Alzheimer´s Disease Neuroimaging Initiative (ADNI). This database consists of approximately 800 adults with ages between 55-90 years, 243 of whom were normal subjects, while 235 presented mild cognitive impairment, and 340 had been diagnosed with Alzheimer´s disease. These partici‐ pants were genotyped using Illumina Human Genome 610 Quad BeadChips, and the CSF levels of amyloid Ab1-42, T-tau, and P-tau181P were determined in 410 subjects (119 normal, 115 MCI and 247 AD), of which 247 were men and 163 were women. An association analysis using age and APOE4 genotype as covariates was also performed, but did not incorporate principal component analysis.

The results showed that T-Tau levels are higher in AD patients than in control subjects. When the results were adjusted using APOE and the age of individuals as covariates, it was not possible to observe an association between SNPs and CSF levels among patients. This study also identified polymorphisms associated with the development of AD that had been already reported in previous studies: CYP19A1 (rs2899472, p=1.90 × 10-7) and NCAM2 (rs1022442, p=2.75 × 10-7).

strong associations of SNPs in two new loci: rs11136000, which is located in the *CLU,* or *APOJ*, gene (p=1.4×10−9), and rs3851179, a SNP 5′ to the *PICALM* gene (p=1.9×10−8). rs11136000 is located within an intron of the clusterin (*CLU*, also known as *APOJ*) gene on chromosome 8, and rs3851179 is found 88.5 kb 5′ of *PICALM* on chromosome 11. In stage 2, these new SNPs were genotyped in 2,023 AD cases and 2,340 age-matched controls from an independent sample. Associations were found for both polymorphisms, with p=0.017 and OR=0.905 for rs11136000 and p=0.014 and OR=0.897 for rs3851179. A meta-analysis of stages 1 and 2 was also conducted in this study, and the results showed highly significant evidence of associations for the *CLU* and *PICALM* loci (rs11136000 p=8.5×10−10 and rs3851179 p=1.3×10−9, respectively). Finally, no significant interactions of novel SNPs associated with *APOE* status were observed to influence AD risk (rs11136000x*APOE*4 interaction p=0.674; rs3851179x*APOE*4 interaction

CLU is a secreted chaperone that can also be found in the cytosol under some stress conditions. It has been suggested that CLU is involved in several basic biological events, including cell death, tumor progression, and neurodegenerative disorders. The genetic risk allele (C) of CLU gene variant rs11136000 is carried by ∼88% of Caucasians; the C allele confers 1.16 greater

PICALM is a phosphatidylinositol-binding clathrin assembly protein. This protein plays a role in altering synaptic vesicle cycling or APP endocytosis. Although the presence of polymor‐ phism rs3851179 was associated with high significance related to the development of AD in the Caucasian population, these results could not be replicated in Chinese or Italian popula‐ tions [166-167]. The results obtained in recent studies by Piaceri showed that the segregation of the PICALM rs3851179 variant did not show a statistically significant difference between LOAD cases and controls, suggesting a reduced risk of developing late onset Alzheimer´s

Unlike the studies described above, this study additionally established a relationship between the allelic variants found by GWAS and cerebrospinal fluid (CSF) levels of amyloid Ab1-42, T-tau, and P-tau181P [168]. The data used in this study was obtained from the Alzheimer´s Disease Neuroimaging Initiative (ADNI). This database consists of approximately 800 adults with ages between 55-90 years, 243 of whom were normal subjects, while 235 presented mild cognitive impairment, and 340 had been diagnosed with Alzheimer´s disease. These partici‐ pants were genotyped using Illumina Human Genome 610 Quad BeadChips, and the CSF levels of amyloid Ab1-42, T-tau, and P-tau181P were determined in 410 subjects (119 normal, 115 MCI and 247 AD), of which 247 were men and 163 were women. An association analysis using age and APOE4 genotype as covariates was also performed, but did not incorporate

The results showed that T-Tau levels are higher in AD patients than in control subjects. When the results were adjusted using APOE and the age of individuals as covariates, it was not possible to observe an association between SNPs and CSF levels among patients. This study also identified polymorphisms associated with the development of AD that had been already

odds of developing late onset AD than the T allele [165].

p=0.735) [164].

336 Neurochemistry

disease (LOAD).

principal component analysis.

**7.8. Han 2010**

## **8. Population genetics and genetic association studies: crucial issues to enhance the transparency of results.**

Although efforts to obtain genetic biomarkers that help in anticipate diagnostic of Alzheimer disease, present-day the clinical research not have the results that expected. The publications that relate genes with Alzheimer disease has increased exponentially however, numerous lines of evidence have demostrated discrepant results among populations. These findings suggest that it is neccesary diminish the confounder factors and focus on identify the cause [169]. Once the causes are established, could integrated in practice of medicine helping with anticipate diagnostic.

In order to avoid spurious associations Little J. et al published an initiative that pretends increase the quality of reporting genetic association studies dissemining this information in different journals (epidemiology, clinical investigation, internal medicine and basic research) [170-176]. The publication refered as STREGA report (STrenghthening the REporting of Geenetic Association studies) provides additional comments to 22 items reported previously by STROBE (STrengthening the Reporting of OBservational Studies in Epidemiology) [177]. These comments include different items, however population genetics topics are crucial issues whose depreciation, increase statistical mistakes type I and II [173].

One of the most important topics in genetic association studies (GAS) is Hardy-Weinberg equilibrium. Hardy-Weinberg equilibrium (HWE) is represented by the equation (p+q)2 =1, whose perfect square binomial equation it is represented by p2 +2pq+q2 =1, where p2 and q2 represented homozygous state, whereas 2pq represented heterozygous state. Under random maiting and non-overlapping, homozygous and heterozygous states are in equal proportions (0.5 each one) maintaining the HWE. This equilibrium are also maintained when evolutionary forces are absents (mutation, random drift, genetic flow, natural selection), the population size it is nearby to the infinitum, and when frequencies of alleles in both sexes are equal [178]. However, some conditions could modify these proportions provoking a Hardy-Weinberg departure (HWD). HWD is related with an excess of homozygous individuals (with subse‐ quent heterozygous deficit) or heterozygous individuals (with subsequent homozygous deficit). Therefore, Hardy-Weinberg model is an essential element used to analyze genetic data, and is the initial step for check the quality of genotyping, because genotyping errors due to poor quality provoke HWD as a consequence of distort in genotype distribution [179]. Nevertheless, HWD are not only related with genotyping mistakes because some factors as demographic events, young population, founder effect, inbreeding, and population stratifi‐ cation may provoke HWD.

Population stratification is the consequence of populations with a recent miscegenation. Admixture populations show different allele frequencies among different subpopulations that conform the whole population, which consequently is not a homogeneous population [180]. Indeed, admixture population is an heterogeneous population with dissimilar ancestry proportions. Consequently, the population stratification may lead to spurious associations because each subpopulation are not equally represented [169, 179]. Applying these premises to GAS, the differences of frequencies between cases and controls populations could be related with dissimilar frequencies among different population strata rather than association of genes with disease. Therefore, population stratification is the most common problem and one of the most important confounder factors in GAS [180-181].

association rates (40%) [193]. However, these methods help to identify rare variants that could have a role in common disease etiology [194]. Hence, all of these variants have an implications in desing, analysis and interpretation of GAS, and are a good strategy for developing markers to elucidate the origins of many human genetic diseases. This alternative approach of antici‐ pated diagnosis can significantly reduce treatment costs by providing preventive medicine

Genetics of Alzheimer´S Disease http://dx.doi.org/10.5772/58286 339

Alzheimer´s disease is one of the main causes of dementia. This disease is clinically charac‐ terized by the irreversible and progressive loss of memory and it is histopathologically characterized by the presence of neuritic plaques (NP) and neurofibrillary tangles (NFT). Both types of lesions are formed due to the accumulation of insoluble protein aggregates, consisting of beta amyloid peptide and the microtubule binding protein Tau, respectively. Studies performed in the last thirty years have provided important advances in understanding the molecular mechanisms involved in the pathology of AD. Through genetic studies, it has been possible to identify the presence of mutations in the APP, PS1 and PS2 genes as causal factors for early onset Alzheimer´s disease (EOAD). These mutations are associated with beta amyloid peptide accumulation, which generates a series of molecular events that lead to a neurodege‐ nerative process. With respect to late onset Alzheimer ́s disease (LOAD), the results obtained to date do not support amyloid overproduction as a cause; in this case, it has been proposed that alterations in the mechanisms responsible for peptide clearance indirectly favor the amyloid accumulation. Amyloids have the ability to interact with several different receptor types, including the Frizzled, insulin, NMDA and NGF receptors, triggering events leading to neuronal death. Additionally, it is known that molecules such as APOE play an important role in the clearance and aggregation of amyloid beta and other risk factors that may eventually determine the conformational changes that allow amyloids to aggregate and form neuritic plaques. For LOAD, APOE is the single most important risk factor. However, a recent GWAS identified several susceptibility loci associated with disease development in different popu‐ lations, although these studies provide a better understanding of the pathophysiology of the disease, these new genetic markers seem to have a weak genetic effect. Therefore, it is necessary to consider using other tools to detect genotyping errors that can be caused among other

**9. Conclusions**

reasons, by population stratification.

, Rocío Gómez2

Neurología y Neurocirugía Manuel Velasco Suárez. Mexico City, Mexico

\*Address all correspondence to: mmeraz@cinvestav.mx

and Marco Antonio Meraz Ríos3\*

1 Laboratorio Experimental de Enfermedades Neurodegenerativas, Instituto Nacional de

**Author details**

Victoria Campos-Peña1

At first glance could appear that HWD are only related with false associations nevertheless, HWD could also be a singnature of disease association, principally in case-control studies, because if some allele is associated with a disease this association break the random maiting provoking HWD [182-183]. This HWD is the result of differences between allele frequencies, where one allele are overrepresented in cases group (excess of homozygous), whereas the same allele are underrepresented in control cases (excess of heterozygous). In order to suport these findings it is neccesary to know the frequency of distribution of this allele in the general population. If the allele show a high frequency in the general population, this finding could be not related with the disease [182, 184-185]. Conversely, if the prevalence of the allele is low in the general population, these data may support a relationship between the allele and the disease suggesting the allele could be a risk allele. As a consequence, the HWD is particularly relevant in GAS.

In light of these evidence, several methods have been developed to detect HWD. The most used method is chi square, however this statistic only must be used in homogeneous popula‐ tion [186]. Other approach is detect the intrapoblational variance (*Fis*), where *Fis*> 0 means a homozygous excess, whereas *Fis*< 0 means heterozygous deficit [187]. Recently, Li M and Li C have developed a likelihood test that allows assessment of HWD taking into account potential association with the disease [182]. This method can differentitate HWD caused by disease association, diminishing the over estimation of type I error and avoiding the false exclusion of associated markers. Hence, is necessary diminish the effect of genetic structure in order to detect susceptibility loci for complex disease. Studies to date suggest different methods, among which are:


All of these bioinformatic models have been an excelent help to clarify the genetic associations in population-based genetic associations increasing the statistical power. These methods have detected limitations or errors in assessments genotypes (20-70%) [192], as well as spurious association rates (40%) [193]. However, these methods help to identify rare variants that could have a role in common disease etiology [194]. Hence, all of these variants have an implications in desing, analysis and interpretation of GAS, and are a good strategy for developing markers to elucidate the origins of many human genetic diseases. This alternative approach of antici‐ pated diagnosis can significantly reduce treatment costs by providing preventive medicine

## **9. Conclusions**

Indeed, admixture population is an heterogeneous population with dissimilar ancestry proportions. Consequently, the population stratification may lead to spurious associations because each subpopulation are not equally represented [169, 179]. Applying these premises to GAS, the differences of frequencies between cases and controls populations could be related with dissimilar frequencies among different population strata rather than association of genes with disease. Therefore, population stratification is the most common problem and one of the

At first glance could appear that HWD are only related with false associations nevertheless, HWD could also be a singnature of disease association, principally in case-control studies, because if some allele is associated with a disease this association break the random maiting provoking HWD [182-183]. This HWD is the result of differences between allele frequencies, where one allele are overrepresented in cases group (excess of homozygous), whereas the same allele are underrepresented in control cases (excess of heterozygous). In order to suport these findings it is neccesary to know the frequency of distribution of this allele in the general population. If the allele show a high frequency in the general population, this finding could be not related with the disease [182, 184-185]. Conversely, if the prevalence of the allele is low in the general population, these data may support a relationship between the allele and the disease suggesting the allele could be a risk allele. As a consequence, the HWD is particularly

In light of these evidence, several methods have been developed to detect HWD. The most used method is chi square, however this statistic only must be used in homogeneous popula‐ tion [186]. Other approach is detect the intrapoblational variance (*Fis*), where *Fis*> 0 means a homozygous excess, whereas *Fis*< 0 means heterozygous deficit [187]. Recently, Li M and Li C have developed a likelihood test that allows assessment of HWD taking into account potential association with the disease [182]. This method can differentitate HWD caused by disease association, diminishing the over estimation of type I error and avoiding the false exclusion of associated markers. Hence, is necessary diminish the effect of genetic structure in order to detect susceptibility loci for complex disease. Studies to date suggest different methods, among

**•** Genomic control. This method diminish the population heterogeneity due to cryptic relatedmess or correlation across individuals, correcting the variance inflation, which is

**•** Infer the number of populations. This Bayesian analysis inferrers the number of subpopu‐

**•** Summarize the genetic background using hierarchical clustering through principal com‐ ponent analysis (PCA) and its variants enable the detecction of differences between samples, detect clinal distributions and suggest other demographic events as isolation-by-distance

All of these bioinformatic models have been an excelent help to clarify the genetic associations in population-based genetic associations increasing the statistical power. These methods have detected limitations or errors in assessments genotypes (20-70%) [192], as well as spurious

lations (*k*) and correct them, decreasing the effect of admixture over GAS [189].

previously detected with unlinked null markers [188].

most important confounder factors in GAS [180-181].

relevant in GAS.

338 Neurochemistry

which are:

[190-191].

Alzheimer´s disease is one of the main causes of dementia. This disease is clinically charac‐ terized by the irreversible and progressive loss of memory and it is histopathologically characterized by the presence of neuritic plaques (NP) and neurofibrillary tangles (NFT). Both types of lesions are formed due to the accumulation of insoluble protein aggregates, consisting of beta amyloid peptide and the microtubule binding protein Tau, respectively. Studies performed in the last thirty years have provided important advances in understanding the molecular mechanisms involved in the pathology of AD. Through genetic studies, it has been possible to identify the presence of mutations in the APP, PS1 and PS2 genes as causal factors for early onset Alzheimer´s disease (EOAD). These mutations are associated with beta amyloid peptide accumulation, which generates a series of molecular events that lead to a neurodege‐ nerative process. With respect to late onset Alzheimer ́s disease (LOAD), the results obtained to date do not support amyloid overproduction as a cause; in this case, it has been proposed that alterations in the mechanisms responsible for peptide clearance indirectly favor the amyloid accumulation. Amyloids have the ability to interact with several different receptor types, including the Frizzled, insulin, NMDA and NGF receptors, triggering events leading to neuronal death. Additionally, it is known that molecules such as APOE play an important role in the clearance and aggregation of amyloid beta and other risk factors that may eventually determine the conformational changes that allow amyloids to aggregate and form neuritic plaques. For LOAD, APOE is the single most important risk factor. However, a recent GWAS identified several susceptibility loci associated with disease development in different popu‐ lations, although these studies provide a better understanding of the pathophysiology of the disease, these new genetic markers seem to have a weak genetic effect. Therefore, it is necessary to consider using other tools to detect genotyping errors that can be caused among other reasons, by population stratification.

## **Author details**

Victoria Campos-Peña1 , Rocío Gómez2 and Marco Antonio Meraz Ríos3\*

\*Address all correspondence to: mmeraz@cinvestav.mx

1 Laboratorio Experimental de Enfermedades Neurodegenerativas, Instituto Nacional de Neurología y Neurocirugía Manuel Velasco Suárez. Mexico City, Mexico

2 Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, De‐ partamennto de Toxicología, Mexico

[12] Graebert, K.S., et al., *Localization and regulated release of Alzheimer amyloid precursor-like protein in thyrocytes.* Laboratory investigation; a journal of technical methods and

Genetics of Alzheimer´S Disease http://dx.doi.org/10.5772/58286 341

[13] Hoffmann, J., et al., *A possible role for the Alzheimer amyloid precursor protein in the regu‐ lation of epidermal basal cell proliferation.* European journal of cell biology, 2000. 79(12):

[14] Schmitz, A., et al., *The biological role of the Alzheimer amyloid precursor protein in epithe‐*

[15] Schubert, D., et al., *The regulation of amyloid beta protein precursor secretion and its modu‐*

[16] Crowther, R.A. and C.M. Wischik, *Image reconstruction of the Alzheimer paired helical*

[17] Turner, P.R., et al., *Roles of amyloid precursor protein and its fragments in regulating neu‐ ral activity, plasticity and memory.* Progress in neurobiology, 2003. 70(1): p. 1-32.

[18] Priller, C., et al., *Synapse formation and function is modulated by the amyloid precursor protein.* The Journal of neuroscience : the official journal of the Society for Neuro‐

[19] De Strooper, B. and W. Annaert, *Proteolytic processing and cell biological functions of the*

[20] Esch, F.S., et al., *Cleavage of amyloid beta peptide during constitutive processing of its pre‐*

[21] Sisodia, S.S., et al., *Evidence that beta-amyloid protein in Alzheimer's disease is not derived*

[22] Dries, D.R. and G. Yu, *Assembly, maturation, and trafficking of the gamma-secretase com‐*

[23] Blessed, G., B.E. Tomlinson, and M. Roth, *The association between quantitative measures of dementia and of senile change in the cerebral grey matter of elderly subjects.* The British

journal of psychiatry : the journal of mental science, 1968. 114(512): p. 797-811.

[24] Braak, H. and E. Braak, *Staging of Alzheimer-related cortical destruction.* International

[25] Andreadis, A., W.M. Brown, and K.S. Kosik, *Structure and novel exons of the human tau*

[26] Neve, R.L., et al., *Identification of cDNA clones for the human microtubule-associated pro‐ tein tau and chromosomal localization of the genes for tau and microtubule-associated protein*

*lial cells.* Histochemistry and cell biology, 2002. 117(2): p. 171-80.

*amyloid precursor protein.* J Cell Sci, 2000. 113 (Pt 11): p. 1857-70.

*plex in Alzheimer's disease.* Curr Alzheimer Res, 2008. 5(2): p. 132-46.

psychogeriatrics / IPA, 1997. 9 Suppl 1: p. 257-61; discussion 269-72.

*by normal processing.* Science, 1990. 248(4954): p. 492-5.

*latory role in cell adhesion.* Neuron, 1989. 3(6): p. 689-94.

*filament.* The EMBO journal, 1985. 4(13B): p. 3661-5.

pathology, 1995. 72(5): p. 513-23.

science, 2006. 26(27): p. 7212-21.

*cursor.* Science, 1990. 248(4959): p. 1122-4.

*gene.* Biochemistry, 1992. 31(43): p. 10626-33.

*2.* Brain Res, 1986. 387(3): p. 271-80.

p. 905-14.

3 Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, De‐ partamento de Biomedicina Molecular. México City, Mexico

#### **References**


[12] Graebert, K.S., et al., *Localization and regulated release of Alzheimer amyloid precursor-like protein in thyrocytes.* Laboratory investigation; a journal of technical methods and pathology, 1995. 72(5): p. 513-23.

2 Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, De‐

3 Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, De‐

[1] Tomlinson, B.E.C., JAN, *Ageing and the dementias*. Greenfield's Neuropathology, ed.

[2] Roberts, G.W., et al., *On the origin of Alzheimer's disease: a hypothesis.* Neuroreport,

[3] Terry, R.D., et al., *Physical basis of cognitive alterations in Alzheimer's disease: synapse loss is the major correlate of cognitive impairment.* Annals of neurology, 1991. 30(4): p.

[4] Heyman, A., et al., *Early-onset Alzheimer's disease: clinical predictors of institutionaliza‐*

[5] McKhann, G., et al., *Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADR‐ DA Work Group under the auspices of Department of Health and Human Services Task*

[6] Folstein, M.F., S.E. Folstein, and P.R. McHugh, *"Mini-mental state". A practical method for grading the cognitive state of patients for the clinician.* Journal of psychiatric research,

[7] Iversen, L.L., et al., *The toxicity in vitro of beta-amyloid protein.* The Biochemical journal,

[8] Glenner, G.G., et al., *The amyloid deposits in Alzheimer's disease: their nature and patho‐*

[9] Selkoe, D.J., *Alzheimer's disease: a central role for amyloid.* Journal of neuropathology

[10] Kang, J., et al., *The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-*

[11] Sandbrink, R., C.L. Masters, and K. Beyreuther, *Beta A4-amyloid protein precursor mRNA isoforms without exon 15 are ubiquitously expressed in rat tissues including brain,*

*but not in neurons.* The Journal of biological chemistry, 1994. 269(2): p. 1510-7.

J.C. Hume Adams, JAN; Duchen, L.W.. 1984, London Edward Arnold.

partamennto de Toxicología, Mexico

1993. 4(1): p. 7-9.

1975. 12(3): p. 189-98.

1995. 311 (Pt 1): p. 1-16.

572-80.

**References**

340 Neurochemistry

partamento de Biomedicina Molecular. México City, Mexico

*tion and death.* Neurology, 1987. 37(6): p. 980-4.

*genesis.* Applied pathology, 1984. 2(6): p. 357-69.

and experimental neurology, 1994. 53(5): p. 438-47.

*surface receptor.* Nature, 1987. 325(6106): p. 733-6.

*Force on Alzheimer's Disease.* Neurology, 1984. 34(7): p. 939-44.


[27] Neve, R.L., et al., *A cDNA for a human microtubule associated protein 2 epitope in the Alz‐ heimer neurofibrillary tangle.* Brain Res, 1986. 387(2): p. 193-6.

[41] Gatz, M., et al., *Role of genes and environments for explaining Alzheimer disease.* Archives

Genetics of Alzheimer´S Disease http://dx.doi.org/10.5772/58286 343

[42] Raiha, I., et al., *Alzheimer's disease in Finnish twins.* Lancet, 1996. 347(9001): p. 573-8.

[43] Gatz, M., et al., *Dementia in Swedish twins: predicting incident cases.* Behavior genetics,

[44] Corder, E.H., et al., *Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's*

[45] Saunders, A.M., et al., *Association of apolipoprotein E allele epsilon 4 with late-onset fami‐*

[46] Aleshkov, S., C.R. Abraham, and V.I. Zannis, *Interaction of nascent ApoE2, ApoE3, and ApoE4 isoforms expressed in mammalian cells with amyloid peptide beta (1-40). Relevance to*

[47] Strittmatter, W.J. and A.D. Roses, *Apolipoprotein E and Alzheimer disease.* Proceedings of the National Academy of Sciences of the United States of America, 1995. 92(11): p.

[48] Ertekin-Taner, N., *Genetics of Alzheimer's disease: a centennial review.* Neurologic clin‐

[49] Kim, J., J.M. Basak, and D.M. Holtzman, *The role of apolipoprotein E in Alzheimer's dis‐*

[50] Levy-Lahad, E., et al., *A familial Alzheimer's disease locus on chromosome 1.* Science,

[51] Duff, K., et al., *Increased amyloid-beta42(43) in brains of mice expressing mutant presenilin*

[52] Kovacs, D.M., et al., *Alzheimer-associated presenilins 1 and 2: neuronal expression in brain and localization to intracellular membranes in mammalian cells.* Nature medicine, 1996.

[53] Borchelt, D.R., et al., *Familial Alzheimer's disease-linked presenilin 1 variants elevate Abe‐*

[54] Scheuner, D., et al., *Secreted amyloid beta-protein similar to that in the senile plaques of Alzheimer's disease is increased in vivo by the presenilin 1 and 2 and APP mutations linked*

[55] Goate, A., et al., *Segregation of a missense mutation in the amyloid precursor protein gene*

[56] Sisodia, S.S., S.H. Kim, and G. Thinakaran, *Function and dysfunction of the presenilins.*

*ta1-42/1-40 ratio in vitro and in vivo.* Neuron, 1996. 17(5): p. 1005-13.

*to familial Alzheimer's disease.* Nature medicine, 1996. 2(8): p. 864-70.

*with familial Alzheimer's disease.* Nature, 1991. 349(6311): p. 704-6.

American journal of human genetics, 1999. 65(1): p. 7-12.

*disease in late onset families.* Science, 1993. 261(5123): p. 921-3.

*Alzheimer's disease.* Biochemistry, 1997. 36(34): p. 10571-80.

*lial and sporadic Alzheimer's disease.* Neurology, 1993. 43(8): p. 1467-72.

of general psychiatry, 2006. 63(2): p. 168-74.

2010. 40(6): p. 768-75.

4725-7.

ics, 2007. 25(3): p. 611-67, v.

1995. 269(5226): p. 970-3.

2(2): p. 224-9.

*ease.* Neuron, 2009. 63(3): p. 287-303.

*1.* Nature, 1996. 383(6602): p. 710-3.


[27] Neve, R.L., et al., *A cDNA for a human microtubule associated protein 2 epitope in the Alz‐*

[28] Goedert, M., et al., *Multiple isoforms of human microtubule-associated protein tau: sequen‐ ces and localization in neurofibrillary tangles of Alzheimer's disease.* Neuron, 1989. 3(4): p.

[29] Goedert, M. and R. Jakes, *Expression of separate isoforms of human tau protein: correlation with the tau pattern in brain and effects on tubulin polymerization.* EMBO J, 1990. 9(13): p.

[30] Spillantini, M.G. and M. Goedert, *Tau pathology and neurodegeneration.* Lancet Neurol,

[31] Ennulat, D.J., et al., *Two separate 18-amino acid domains of tau promote the polymerization*

[32] Goedert, M. and A. Klug, *Tau protein and the paired helical filament of Alzheimer's dis‐*

[33] Cleveland, D.W., S.Y. Hwo, and M.W. Kirschner, *Physical and chemical properties of pu‐ rified tau factor and the role of tau in microtubule assembly.* Journal of molecular biology,

[34] Schweers, O., et al., *Structural studies of tau protein and Alzheimer paired helical filaments show no evidence for beta-structure.* The Journal of biological chemistry, 1994. 269(39):

[35] Buee, L. and A. Delacourte, *Comparative biochemistry of tau in progressive supranuclear palsy, corticobasal degeneration, FTDP-17 and Pick's disease.* Brain pathology, 1999. 9(4):

[36] Poorkaj, P., et al., *Tau is a candidate gene for chromosome 17 frontotemporal dementia.* An‐

[37] Spillantini, M.G., T.D. Bird, and B. Ghetti, *Frontotemporal dementia and Parkinsonism linked to chromosome 17: a new group of tauopathies.* Brain pathology, 1998. 8(2): p.

[38] Hutton, M., *Molecular genetics of chromosome 17 tauopathies.* Annals of the New York

[39] Hutton, M., et al., *Association of missense and 5'-splice-site mutations in tau with the in‐*

[40] Bergem, A.L., K. Engedal, and E. Kringlen, *The role of heredity in late-onset Alzheimer disease and vascular dementia. A twin study.* Archives of general psychiatry, 1997. 54(3):

*of tubulin.* The Journal of biological chemistry, 1989. 264(10): p. 5327-30.

*ease.* Brain research bulletin, 1999. 50(5-6): p. 469-70.

nals of neurology, 1998. 43(6): p. 815-25.

Academy of Sciences, 2000. 920: p. 63-73.

*herited dementia FTDP-17.* Nature, 1998. 393(6686): p. 702-5.

*heimer neurofibrillary tangle.* Brain Res, 1986. 387(2): p. 193-6.

519-26.

342 Neurochemistry

4225-30.

2013. 12(6): p. 609-22.

1977. 116(2): p. 227-47.

p. 24290-7.

p. 681-93.

387-402.

p. 264-70.


[57] Chartier-Harlin, M.C., et al., *Screening for the beta-amyloid precursor protein mutation (APP717: Val----Ile) in extended pedigrees with early onset Alzheimer's disease.* Neuro‐ science letters, 1991. 129(1): p. 134-5.

[72] Irizarry, M.C., et al., *APPSw transgenic mice develop age-related A beta deposits and neu‐ ropil abnormalities, but no neuronal loss in CA1.* Journal of neuropathology and experi‐

Genetics of Alzheimer´S Disease http://dx.doi.org/10.5772/58286 345

[73] Westerman, M.A., et al., *The relationship between Abeta and memory in the Tg2576 mouse model of Alzheimer's disease.* The Journal of neuroscience : the official journal of the So‐

[74] Moechars, D., et al., *Transgenic mice expressing an alpha-secretion mutant of the amyloid precursor protein in the brain develop a progressive CNS disorder.* Behavioural brain re‐

[75] McPhie, D.L., et al., *DNA synthesis and neuronal apoptosis caused by familial Alzheimer disease mutants of the amyloid precursor protein are mediated by the p21 activated kinase PAK3.* The Journal of neuroscience : the official journal of the Society for Neuro‐

[76] Wolfe, M.S., *The gamma-secretase complex: membrane-embedded proteolytic ensemble.* Bio‐

[77] Rechards, M., et al., *Presenilin-1 exists in both pre-and post-Golgi compartments and recy‐*

[78] Chyung, J.H., D.M. Raper, and D.J. Selkoe, *Gamma-secretase exists on the plasma mem‐ brane as an intact complex that accepts substrates and effects intramembrane cleavage.* The

[79] Vetrivel, K.S., et al., *Association of gamma-secretase with lipid rafts in post-Golgi and endo‐ some membranes.* The Journal of biological chemistry, 2004. 279(43): p. 44945-54.

[80] Kulic, L., et al., *Separation of presenilin function in amyloid beta-peptide generation and en‐ doproteolysis of Notch.* Proceedings of the National Academy of Sciences of the United

[81] Vassar, R. and M. Citron, *Abeta-generating enzymes: recent advances in beta-and gamma-*

[82] Ebinu, J.O. and B.A. Yankner, *A RIP tide in neuronal signal transduction.* Neuron, 2002.

[83] Marambaud, P., et al., *A presenilin-1/gamma-secretase cleavage releases the E-cadherin in‐ tracellular domain and regulates disassembly of adherens junctions.* The EMBO journal,

[84] Li, T., et al., *Nicastrin is required for assembly of presenilin/gamma-secretase complexes to mediate Notch signaling and for processing and trafficking of beta-amyloid precursor protein in mammals.* The Journal of neuroscience : the official journal of the Society for Neuro‐

*cles via COPI-coated membranes.* Traffic, 2003. 4(8): p. 553-65.

Journal of biological chemistry, 2005. 280(6): p. 4383-92.

States of America, 2000. 97(11): p. 5913-8.

34(4): p. 499-502.

2002. 21(8): p. 1948-56.

science, 2003. 23(8): p. 3272-7.

*secretase research.* Neuron, 2000. 27(3): p. 419-22.

mental neurology, 1997. 56(9): p. 965-73.

search, 1998. 95(1): p. 55-64.

science, 2003. 23(17): p. 6914-27.

chemistry, 2006. 45(26): p. 7931-9.

ciety for Neuroscience, 2002. 22(5): p. 1858-67.


[72] Irizarry, M.C., et al., *APPSw transgenic mice develop age-related A beta deposits and neu‐ ropil abnormalities, but no neuronal loss in CA1.* Journal of neuropathology and experi‐ mental neurology, 1997. 56(9): p. 965-73.

[57] Chartier-Harlin, M.C., et al., *Screening for the beta-amyloid precursor protein mutation (APP717: Val----Ile) in extended pedigrees with early onset Alzheimer's disease.* Neuro‐

[58] Murrell, J., et al., *A mutation in the amyloid precursor protein associated with hereditary*

[59] Mullan, M., et al., *A pathogenic mutation for probable Alzheimer's disease in the APP gene*

[60] Nunan, J. and D.H. Small, *Regulation of APP cleavage by alpha-, beta-and gamma-secre‐*

[61] Perez, R.G., S.L. Squazzo, and E.H. Koo, *Enhanced release of amyloid beta-protein from codon 670/671 "Swedish" mutant beta-amyloid precursor protein occurs in both secretory and endocytic pathways.* The Journal of biological chemistry, 1996. 271(15): p. 9100-7.

[62] Levy, E., et al., *Mutation of the Alzheimer's disease amyloid gene in hereditary cerebral*

[63] Eckman, C.B., et al., *A new pathogenic mutation in the APP gene (I716V) increases the rel‐ ative proportion of A beta 42(43).* Human molecular genetics, 1997. 6(12): p. 2087-9. [64] Grabowski, T.J., et al., *Novel amyloid precursor protein mutation in an Iowa family with dementia and severe cerebral amyloid angiopathy.* Annals of neurology, 2001. 49(6): p.

[65] Nilsberth, C., et al., *The 'Arctic' APP mutation (E693G) causes Alzheimer's disease by en‐ hanced Abeta protofibril formation.* Nature neuroscience, 2001. 4(9): p. 887-93.

[66] Kasuga, K., et al., *Identification of independent APP locus duplication in Japanese patients with early-onset Alzheimer disease.* J Neurol Neurosurg Psychiatry, 2009. 80(9): p.

[67] Rovelet-Lecrux, A., et al., *APP locus duplication in a Finnish family with dementia and intracerebral haemorrhage.* J Neurol Neurosurg Psychiatry, 2007. 78(10): p. 1158-9. [68] Rovelet-Lecrux, A., et al., *APP locus duplication causes autosomal dominant early-onset Alzheimer disease with cerebral amyloid angiopathy.* Nat Genet, 2006. 38(1): p. 24-6. [69] Sleegers, K., et al., *APP duplication is sufficient to cause early onset Alzheimer's dementia*

[70] Higgins, G.A. and H. Jacobsen, *Transgenic mouse models of Alzheimer's disease: pheno‐*

[71] Mineur, Y.S., et al., *Genetic mouse models of Alzheimer's disease.* Neural plasticity, 2005.

*with cerebral amyloid angiopathy.* Brain, 2006. 129(Pt 11): p. 2977-83.

*type and application.* Behavioural pharmacology, 2003. 14(5-6): p. 419-38.

*at the N-terminus of beta-amyloid.* Nature genetics, 1992. 1(5): p. 345-7.

science letters, 1991. 129(1): p. 134-5.

*tases.* FEBS letters, 2000. 483(1): p. 6-10.

697-705.

344 Neurochemistry

1050-2.

12(4): p. 299-310.

*Alzheimer's disease.* Science, 1991. 254(5028): p. 97-9.

*hemorrhage, Dutch type.* Science, 1990. 248(4959): p. 1124-6.


[85] Yu, G., et al., *Nicastrin modulates presenilin-mediated notch/glp-1 signal transduction and betaAPP processing.* Nature, 2000. 407(6800): p. 48-54.

[100] Storey, E. and R. Cappai, *The amyloid precursor protein of Alzheimer's disease and the Abeta peptide.* Neuropathology and applied neurobiology, 1999. 25(2): p. 81-97.

Genetics of Alzheimer´S Disease http://dx.doi.org/10.5772/58286 347

[101] Wolfe, M.S., et al., *Are presenilins intramembrane-cleaving proteases? Implications for the molecular mechanism of Alzheimer's disease.* Biochemistry, 1999. 38(35): p. 11223-30.

[102] Wolfe, M.S., et al., *Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity.* Nature, 1999. 398(6727): p. 513-7.

[103] Steiner, H., et al., *A loss of function mutation of presenilin-2 interferes with amyloid betapeptide production and notch signaling.* J Biol Chem, 1999. 274(40): p. 28669-73.

[104] Bentahir, M., et al., *Presenilin clinical mutations can affect gamma-secretase activity by dif‐*

[105] Pigino, G., et al., *Alzheimer's presenilin 1 mutations impair kinesin-based axonal transport.* The Journal of neuroscience : the official journal of the Society for Neuroscience,

[106] Bentahir, M., et al., *Presenilin clinical mutations can affect gamma-secretase activity by dif‐*

[107] Lindquist, S.G., et al., *A novel presenilin 2 mutation (V393M) in early-onset dementia with profound language impairment.* European journal of neurology : the official journal of

[108] Holtzman, D.M., J. Herz, and G. Bu, *Apolipoprotein E and apolipoprotein E receptors: normal biology and roles in Alzheimer disease.* Cold Spring Harb Perspect Med, 2012.

[109] Schupf, N. and G.H. Sergievsky, *Genetic and host factors for dementia in Down's syn‐ drome.* The British journal of psychiatry : the journal of mental science, 2002. 180: p.

[110] Tang, M.X., et al., *Relative risk of Alzheimer disease and age-at-onset distributions, based on APOE genotypes among elderly African Americans, Caucasians, and Hispanics in New York*

[111] Farrer, L.A., et al., *Association between bleomycin hydrolase and Alzheimer's disease in*

[112] Tang, M.X., et al., *The APOE-epsilon4 allele and the risk of Alzheimer disease among Afri‐ can Americans, whites, and Hispanics.*JAMA : the journal of the American Medical As‐

[113] Graff-Radford, N.R., et al., *Association between apolipoprotein E genotype and Alzheimer disease in African American subjects.* Archives of neurology, 2002. 59(4): p. 594-600.

[114] Murrell, J.R., et al., *Association of apolipoprotein E genotype and Alzheimer disease in Afri‐*

*City.* American journal of human genetics, 1996. 58(3): p. 574-84.

*caucasians.* Annals of neurology, 1998. 44(5): p. 808-11.

*can Americans.* Archives of neurology, 2006. 63(3): p. 431-4.

sociation, 1998. 279(10): p. 751-5.

the European Federation of Neurological Societies, 2008. 15(10): p. 1135-9.

*ferent mechanisms.* Journal of neurochemistry, 2006. 96(3): p. 732-42.

*ferent mechanisms.* J Neurochem, 2006. 96(3): p. 732-42.

2003. 23(11): p. 4499-508.

2(3): p. a006312.

405-10.


[100] Storey, E. and R. Cappai, *The amyloid precursor protein of Alzheimer's disease and the Abeta peptide.* Neuropathology and applied neurobiology, 1999. 25(2): p. 81-97.

[85] Yu, G., et al., *Nicastrin modulates presenilin-mediated notch/glp-1 signal transduction and*

[86] Naruse, S., et al., *Effects of PS1 deficiency on membrane protein trafficking in neurons.*

[87] Saura, C.A., E. Servian-Morilla, and F.G. Scholl, *Presenilin/gamma-secretase regulates*

[88] Pratt, K.G., et al., *Presenilin 1 regulates homeostatic synaptic scaling through Akt signal‐*

[89] Toda, T., et al., *Presenilin-2 mutation causes early amyloid accumulation and memory im‐ pairment in a transgenic mouse model of Alzheimer's disease.* Journal of biomedicine & bi‐

[90] Supnet, C. and I. Bezprozvanny, *Presenilins function in ER calcium leak and Alzheimer's*

[91] Zhang, H., et al., *Role of presenilins in neuronal calcium homeostasis.* The Journal of neu‐ roscience : the official journal of the Society for Neuroscience, 2010. 30(25): p. 8566-80.

[92] Zhang, C., et al., *Presenilins are essential for regulating neurotransmitter release.* Nature,

[93] Campion, D., et al., *A novel presenilin 1 mutation resulting in familial Alzheimer's disease*

[94] Wisniewski, T., et al., *A novel Polish presenilin-1 mutation (P117L) is associated with fam‐ ilial Alzheimer's disease and leads to death as early as the age of 28 years.* Neuroreport,

[95] Prihar, G., et al., *Structure and alternative splicing of the presenilin-2 gene.* Neuroreport,

[96] Sherrington, R., et al., *Cloning of a gene bearing missense mutations in early-onset familial*

[97] Citron, M., et al., *Mutant presenilins of Alzheimer's disease increase production of 42-resi‐ due amyloid beta-protein in both transfected cells and transgenic mice.* Nature medicine,

[98] Busciglio, J., et al., *Neuronal localization of presenilin-1 and association with amyloid pla‐ ques and neurofibrillary tangles in Alzheimer's disease.* The Journal of neuroscience : the

[99] Jacobsen, H., et al., *The influence of endoproteolytic processing of familial Alzheimer's dis‐ ease presenilin 2 on abeta42 amyloid peptide formation.* The Journal of biological chemis‐

official journal of the Society for Neuroscience, 1997. 17(13): p. 5101-7.

*betaAPP processing.* Nature, 2000. 407(6800): p. 48-54.

*ing.* Nature neuroscience, 2011. 14(9): p. 1112-4.

*disease pathogenesis.* Cell calcium, 2011. 50(3): p. 303-9.

*with an onset age of 29 years.* Neuroreport, 1996. 7(10): p. 1582-4.

*Alzheimer's disease.* Nature, 1995. 375(6534): p. 754-60.

*neurexin processing at synapses.* PloS one, 2011. 6(4): p. e19430.

Neuron, 1998. 21(5): p. 1213-21.

346 Neurochemistry

otechnology, 2011. 2011: p. 617974.

2009. 460(7255): p. 632-6.

1998. 9(2): p. 217-21.

1996. 7(10): p. 1680-4.

1997. 3(1): p. 67-72.

try, 1999. 274(49): p. 35233-9.


[115] Weisgraber, K.H., *Apolipoprotein E: structure-function relationships.* Advances in pro‐ tein chemistry, 1994. 45: p. 249-302.

[129] Buttini, M., et al., *Expression of human apolipoprotein E3 or E4 in the brains of Apoe-/ mice: isoform-specific effects on neurodegeneration.* The Journal of neuroscience : the offi‐

Genetics of Alzheimer´S Disease http://dx.doi.org/10.5772/58286 349

[130] Keller, J.N., et al., *Amyloid beta-peptide effects on synaptosomes from apolipoprotein E-defi‐*

[131] Deane, R., et al., *apoE isoform-specific disruption of amyloid beta peptide clearance from mouse brain.* The Journal of clinical investigation, 2008. 118(12): p. 4002-13.

[132] Rapp, A., B. Gmeiner, and M. Huttinger, *Implication of apoE isoforms in cholesterol me‐ tabolism by primary rat hippocampal neurons and astrocytes.* Biochimie, 2006. 88(5): p.

[133] Kuo, Y.M., et al., *Elevated A beta and apolipoprotein E in A betaPP transgenic mice and its relationship to amyloid accumulation in Alzheimer's disease.* Molecular medicine, 2000.

[134] Nilsson, L.N., et al., *Cognitive impairment in PDAPP mice depends on ApoE and ACTcatalyzed amyloid formation.* Neurobiology of aging, 2004. 25(9): p. 1153-67.

[135] Castellano, J.M., et al., *Human apoE isoforms differentially regulate brain amyloid-beta pep‐*

[136] Cam, J.A. and G. Bu, *Modulation of beta-amyloid precursor protein trafficking and process‐ ing by the low density lipoprotein receptor family.* Molecular neurodegeneration, 2006. 1:

[137] Marzolo, M.P. and G. Bu, *Lipoprotein receptors and cholesterol in APP trafficking and pro‐ teolytic processing, implications for Alzheimer's disease.* Seminars in cell & developmen‐

[138] Ye, S., et al., *Apolipoprotein (apo) E4 enhances amyloid beta peptide production in cultured neuronal cells: apoE structure as a potential therapeutic target.* Proceedings of the Nation‐ al Academy of Sciences of the United States of America, 2005. 102(51): p. 18700-5.

[139] Irizarry, M.C., et al., *Apolipoprotein E affects the amount, form, and anatomical distribution of amyloid beta-peptide deposition in homozygous APP(V717F) transgenic mice.* Acta neu‐

[140] Deane, R., et al., *Clearance of amyloid-beta peptide across the blood-brain barrier: implica‐ tion for therapies in Alzheimer's disease.* CNS & neurological disorders drug targets,

[141] Sagare, A., et al., *Clearance of amyloid-beta by circulating lipoprotein receptors.* Nature

[142] Ohm, T.G., et al., *Apolipoprotein E polymorphism influences not only cerebral senile plaque load but also Alzheimer-type neurofibrillary tangle formation.* Neuroscience, 1995. 66(3): p.

cial journal of the Society for Neuroscience, 1999. 19(12): p. 4867-80.

*cient mice.* Journal of neurochemistry, 2000. 74(4): p. 1579-86.

*tide clearance.* Sci Transl Med, 2011. 3(89): p. 89ra57.

tal biology, 2009. 20(2): p. 191-200.

ropathologica, 2000. 100(5): p. 451-8.

medicine, 2007. 13(9): p. 1029-31.

2009. 8(1): p. 16-30.

583-7.

473-83.

p. 8.

6(5): p. 430-9.


[129] Buttini, M., et al., *Expression of human apolipoprotein E3 or E4 in the brains of Apoe-/ mice: isoform-specific effects on neurodegeneration.* The Journal of neuroscience : the offi‐ cial journal of the Society for Neuroscience, 1999. 19(12): p. 4867-80.

[115] Weisgraber, K.H., *Apolipoprotein E: structure-function relationships.* Advances in pro‐

[116] Wernette-Hammond, M.E., et al., *Glycosylation of human apolipoprotein E. The carbohy‐ drate attachment site is threonine 194.* The Journal of biological chemistry, 1989. 264(15):

[117] Nickerson, D.A., et al., *Sequence diversity and large-scale typing of SNPs in the human*

[118] Seripa, D., et al., *The genetics of the human APOE polymorphism.* Rejuvenation Res,

[119] Persico, A.M., et al., *Enhanced APOE2 transmission rates in families with autistic pro‐*

[120] Avramopoulos, D., *Genetics of Alzheimer's disease: recent advances.* Genome Med, 2009.

[121] Davignon, J., R.E. Gregg, and C.F. Sing, *Apolipoprotein E polymorphism and atherosclero‐*

[122] Mahley, R.W. and Z.S. Ji, *Remnant lipoprotein metabolism: key pathways involving cellsurface heparan sulfate proteoglycans and apolipoprotein E.* Journal of lipid research, 1999.

[123] Fullerton, S.M., et al., *Apolipoprotein E variation at the sequence haplotype level: implica‐ tions for the origin and maintenance of a major human polymorphism.* American journal of

[124] Hui, D.Y., T.L. Innerarity, and R.W. Mahley, *Defective hepatic lipoprotein receptor bind‐ ing of beta-very low density lipoproteins from type III hyperlipoproteinemic patients. Impor‐ tance of apolipoprotein E.* The Journal of biological chemistry, 1984. 259(2): p. 860-9.

[125] Strittmatter, W.J., et al., *Binding of human apolipoprotein E to synthetic amyloid beta pep‐ tide: isoform-specific effects and implications for late-onset Alzheimer disease.* Proceedings of the National Academy of Sciences of the United States of America, 1993. 90(17): p.

[126] Buttini, M., et al., *Cellular source of apolipoprotein E4 determines neuronal susceptibility to excitotoxic injury in transgenic mice.* The American journal of pathology, 2010. 177(2):

[127] Cedazo-Minguez, A., M. Huttinger, and R.F. Cowburn, *Beta-VLDL protects against A beta(1-42) and apoE toxicity in human SH-SY5Y neuroblastoma cells.* Neuroreport, 2001.

[128] Wilhelmus, M.M., et al., *Apolipoprotein E genotype regulates amyloid-beta cytotoxicity.* J

*apolipoprotein E gene.* Genome research, 2000. 10(10): p. 1532-45.

tein chemistry, 1994. 45: p. 249-302.

*bands.* Psychiatr Genet, 2004. 14(2): p. 73-82.

*sis.* Arteriosclerosis, 1988. 8(1): p. 1-21.

human genetics, 2000. 67(4): p. 881-900.

p. 9094-101.

348 Neurochemistry

1(3): p. 34.

40(1): p. 1-16.

8098-102.

p. 563-9.

12(2): p. 201-6.

Neurosci, 2005. 25(14): p. 3621-7.

2011. 14(5): p. 491-500.


[143] Polvikoski, T., et al., *Apolipoprotein E, dementia, and cortical deposition of beta-amyloid protein.* The New England journal of medicine, 1995. 333(19): p. 1242-7.

[155] Wolf, B.A., et al., *Muscarinic regulation of Alzheimer's disease amyloid precursor protein secretion and amyloid beta-protein production in human neuronal NT2N cells.* The Journal

Genetics of Alzheimer´S Disease http://dx.doi.org/10.5772/58286 351

[156] Arvanitakis, Z., et al., *Diabetes mellitus and risk of Alzheimer disease and decline in cogni‐*

[157] Coon, K.D., et al., *A high-density whole-genome association study reveals that APOE is the major susceptibility gene for sporadic late-onset Alzheimer's disease.* The Journal of clinical

[158] Reiman, E.M., et al., *GAB2 alleles modify Alzheimer's risk in APOE epsilon4 carriers.*

[159] Li, K.C., et al., *A system for enhancing genome-wide coexpression dynamics study.* Pro‐ ceedings of the National Academy of Sciences of the United States of America, 2004.

[160] Abraham, R., et al., *A genome-wide association study for late-onset Alzheimer's disease us‐*

[161] Bertram, L., et al., *Genome-wide association analysis reveals putative Alzheimer's disease susceptibility loci in addition to APOE.* American journal of human genetics, 2008. 83(5):

[162] Beecham, G.W., et al., *Genome-wide association study implicates a chromosome 12 risk lo‐ cus for late-onset Alzheimer disease.* American journal of human genetics, 2009. 84(1): p.

[163] Carrasquillo, M.M., et al., *Genetic variation in PCDH11X is associated with susceptibility*

[164] Harold, D., et al., *Genome-wide association study identifies variants at CLU and PICALM associated with Alzheimer's disease.* Nature genetics, 2009. 41(10): p. 1088-93.

[165] Braskie, M.N., et al., *Common Alzheimer's disease risk variant within the CLU gene affects white matter microstructure in young adults.* The Journal of neuroscience : the official

[166] Piaceri, I., et al., *Implication of a genetic variant at PICALM in Alzheimer's disease patients and centenarians.* Journal of Alzheimer's disease : JAD, 2011. 24(3): p. 409-13.

[167] Yu, J.T., et al., *Genetic association of PICALM polymorphisms with Alzheimer's disease in Han Chinese.* Journal of the neurological sciences, 2011. 300(1-2): p. 78-80.

[168] Han, M.R., G.D. Schellenberg, and L.S. Wang, *Genome-wide association reveals genetic effects on human Abeta42 and tau protein levels in cerebrospinal fluids: a case control study.*

*to late-onset Alzheimer's disease.* Nature genetics, 2009. 41(2): p. 192-8.

journal of the Society for Neuroscience, 2011. 31(18): p. 6764-70.

of biological chemistry, 1995. 270(9): p. 4916-22.

psychiatry, 2007. 68(4): p. 613-8.

Neuron, 2007. 54(5): p. 713-20.

BMC neurology, 2010. 10: p. 90.

101(44): p. 15561-6.

p. 623-32.

35-43.

*tive function.* Archives of neurology, 2004. 61(5): p. 661-6.

*ing DNA pooling.* BMC medical genomics, 2008. 1: p. 44.


[155] Wolf, B.A., et al., *Muscarinic regulation of Alzheimer's disease amyloid precursor protein secretion and amyloid beta-protein production in human neuronal NT2N cells.* The Journal of biological chemistry, 1995. 270(9): p. 4916-22.

[143] Polvikoski, T., et al., *Apolipoprotein E, dementia, and cortical deposition of beta-amyloid*

[144] Strittmatter, W.J., et al., *Hypothesis: microtubule instability and paired helical filament for‐ mation in the Alzheimer disease brain are related to apolipoprotein E genotype.* Experimen‐

[145] Harris, F.M., et al., *Carboxyl-terminal-truncated apolipoprotein E4 causes Alzheimer's dis‐ ease-like neurodegeneration and behavioral deficits in transgenic mice.* Proceedings of the National Academy of Sciences of the United States of America, 2003. 100(19): p.

[146] Brecht, W.J., et al., *Neuron-specific apolipoprotein e4 proteolysis is associated with increased tau phosphorylation in brains of transgenic mice.* The Journal of neuroscience : the official

[147] Tesseur, I., et al., *Expression of human apolipoprotein E4 in neurons causes hyperphosphor‐ ylation of protein tau in the brains of transgenic mice.* The American journal of pathology,

[148] Leoni, V., *The effect of apolipoprotein E (ApoE) genotype on biomarkers of amyloidogenesis, tau pathology and neurodegeneration in Alzheimer's disease.* Clinical chemistry and labo‐

[149] Grupe, A., et al., *Evidence for novel susceptibility genes for late-onset Alzheimer's disease from a genome-wide association study of putative functional variants.* Human molecular

[150] Counts, S.E., et al., *Galanin hyperinnervation upregulates choline acetyltransferase expres‐ sion in cholinergic basal forebrain neurons in Alzheimer's disease.* Neuro-degenerative dis‐

[151] Lang, R., A.L. Gundlach, and B. Kofler, *The galanin peptide family: receptor pharmacolo‐ gy, pleiotropic biological actions, and implications in health and disease.* Pharmacology &

[152] Steiner, R.A., et al., *Galanin transgenic mice display cognitive and neurochemical deficits characteristic of Alzheimer's disease.* Proceedings of the National Academy of Sciences

[153] Felschow, D.M., C.I. Civin, and G.T. Hoehn, *Characterization of the tyrosine kinase Tnk1 and its binding with phospholipase C-gamma1.* Biochemical and biophysical research

[154] Emmerling, M.R., et al., *Phospholipase A2 activation influences the processing and secre‐ tion of the amyloid precursor protein.* Biochemical and biophysical research communica‐

journal of the Society for Neuroscience, 2004. 24(10): p. 2527-34.

ratory medicine : CCLM / FESCC, 2011. 49(3): p. 375-83.

of the United States of America, 2001. 98(7): p. 4184-9.

*protein.* The New England journal of medicine, 1995. 333(19): p. 1242-7.

tal neurology, 1994. 125(2): p. 163-71; discussion 172-4.

10966-71.

350 Neurochemistry

2000. 156(3): p. 951-64.

genetics, 2007. 16(8): p. 865-73.

eases, 2008. 5(3-4): p. 228-31.

therapeutics, 2007. 115(2): p. 177-207.

communications, 2000. 273(1): p. 294-301.

tions, 1993. 197(1): p. 292-7.


[169] Jiang, Y., M.P. Epstein, and K.N. Conneely, *Assessing the Impact of Population Stratifi‐ cation on Association Studies of Rare Variation.* Hum Hered, 2013. 76(1): p. 28-35.

[185] Ryckman, K. and S.M. Williams, *Calculation and use of the Hardy-Weinberg model in as‐*

Genetics of Alzheimer´S Disease http://dx.doi.org/10.5772/58286 353

[186] Chen, W., et al., *A rapid association test procedure robust under different genetic models ac‐*

[187] Rousset, F. and M. Raymond, *Testing heterozygote excess and deficiency.* Genetics, 1995.

[188] Devlin, B. and K. Roeder, *Genomic control for association studies.* Biometrics, 1999.

[189] Falush, D., M. Stephens, and J.K. Pritchard, *Inference of population structure using mul‐ tilocus genotype data: linked loci and correlated allele frequencies.* Genetics, 2003. 164(4): p.

[190] Athanasiadis, G. and P. Moral, *Spatial principal component analysis points at global ge‐*

[191] Matsen, F.A.t. and S.N. Evans, *Edge principal components and squash clustering: using the special structure of phylogenetic placement data for sample comparison.* PLoS One, 2013.

[192] Salanti, G., et al., *Hardy-Weinberg equilibrium in genetic association studies: an empirical evaluation of reporting, deviations, and power.* Eur J Hum Genet, 2005. 13(7): p. 840-8.

[193] O'Connor, T.D., et al., *Fine-scale patterns of population stratification confound rare variant*

[194] He, H., et al., *Effect of population stratification analysis on false-positive rates for common*

*netic structure in the Western Mediterranean.* J Hum Genet, 2013.

*association tests.* PLoS One, 2013. 8(7): p. e65834.

*and rare variants.* BMC Proc, 2011. 5 Suppl 9: p. S116.

*sociation studies.* Curr Protoc Hum Genet, 2008. Chapter 1: p. Unit 1 18.

*counting for population stratification.* Hum Hered, 2013. 75(1): p. 23-33.

140(4): p. 1413-9.

55(4): p. 997-1004.

1567-87.

8(3): p. e56859.


[185] Ryckman, K. and S.M. Williams, *Calculation and use of the Hardy-Weinberg model in as‐ sociation studies.* Curr Protoc Hum Genet, 2008. Chapter 1: p. Unit 1 18.

[169] Jiang, Y., M.P. Epstein, and K.N. Conneely, *Assessing the Impact of Population Stratifi‐ cation on Association Studies of Rare Variation.* Hum Hered, 2013. 76(1): p. 28-35.

[170] Little, J., et al., *STrengthening the REporting of Genetic Association studies (STREGA)--an extension of the STROBE statement.* Eur J Clin Invest, 2009. 39(4): p. 247-66.

[171] Little, J., et al., *STrengthening the REporting of Genetic Association Studies (STREGA)--an extension of the STROBE statement.* Genet Epidemiol, 2009. 33(7): p. 581-98.

[172] Little, J., et al., *Strengthening the reporting of genetic association studies (STREGA): an ex‐ tension of the strengthening the reporting of observational studies in epidemiology*

[173] Little, J., et al., *STrengthening the REporting of Genetic Association Studies (STREGA): an*

[174] Little, J., et al., *STrengthening the REporting of Genetic Association studies (STREGA): an extension of the STROBE Statement.* Ann Intern Med, 2009. 150(3): p. 206-15.

[175] Little, J., et al., *Strengthening the reporting of genetic association studies (STREGA): an ex‐*

[176] Little, J., et al., *Strengthening the reporting of genetic association studies (STREGA): an ex‐*

[177] Noah, N., *The STROBE initiative: STrengthening the Reporting of OBservational studies in*

[178] Schaap, T., *The applicability of the Hardy-Weinberg principle in the study of populations.*

[179] Zang, Y. and Y. Yuan, *A Shrinkage Method for Testing the Hardy-Weinberg Equilibrium*

[180] Pritchard, J.K. and P. Donnelly, *Case-control studies of association in structured or ad‐*

[181] Voight, B.F. and J.K. Pritchard, *Confounding from cryptic relatedness in case-control asso‐*

[182] Li, M. and C. Li, *Assessing departure from Hardy-Weinberg equilibrium in the presence of*

[183] Deng, H.W., W.M. Chen, and R.R. Recker, *Population admixture: detection by Hardy-Weinberg test and its quantitative effects on linkage-disequilibrium methods for localizing*

[184] Sha, Q. and S. Zhang, *A test of Hardy-Weinberg equilibrium in structured populations.*

*tension of the STROBE statement.* Eur J Epidemiol, 2009. 24(1): p. 37-55.

*tension of the STROBE Statement.* Hum Genet, 2009. 125(2): p. 131-51.

*Epidemiology (STROBE).* Epidemiol Infect, 2008. 136(7): p. 865.

*mixed populations.* Theor Popul Biol, 2001. 60(3): p. 227-37.

*disease association.* Genet Epidemiol, 2008. 32(7): p. 589-99.

*genes underlying complex traits.* Genetics, 2001. 157(2): p. 885-97.

Ann Hum Genet, 1980. 44(Pt 2): p. 211-5.

352 Neurochemistry

*in Case-Control Studies.* Genet Epidemiol, 2013.

*ciation studies.* PLoS Genet, 2005. 1(3): p. e32.

Genet Epidemiol, 2011. 35(7): p. 671-8.

*(STROBE) statement.* J Clin Epidemiol, 2009. 62(6): p. 597-608 e4.

*extension of the STROBE statement.* PLoS Med, 2009. 6(2): p. e22.


**Chapter 12**

**Accumulation of Abnormally Processed Tau Protein in**

Among neurodegenerative diseases, dementias are an heterogeneous group in terms of their symptoms and pathological findings. One of the main risk factors for developing neurodege‐ nerative disease is aging. Currently there is no cure for these diseases, mainly due to the lack of knowledge of the causes and mechanisms of the accumulation of abnormal protein aggre‐ gates within the cellular or extracellular body. This is a common characteristic pathological feature in several neurodegenerative diseases. Pathological protein accumulations not only define the characteristics of a particular neurodegenerative disease, but also are associated

Research in this field had been focused on finding potential highly specific biomarkers that correlates with the disease and can be detected at early stages of the pathology. In medicine, a biomarker is defined as a featured specific somatic or measurable biological change related to a health condition or disease [2]. A biomarker can be measured and objectively evaluated as an indicator of normal biological processes or disease, as well as the pharmacological response to treatment. In general, we can say that a biomarker can be used to diagnose the disease, or to establish its severity and allow monitoring its progression and response [1, 3, 4]. A biomarker must adhere to the following statements: 1) detect a fundamental feature of the neuropathology of the disease, 2) must be validated in cases confirmed by neuropathological examination, 3) have a high sensitivity and specificity, above 80% for discriminating the

> © 2014 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.

with clinical progression, including cognitive impairment or motor disorders [1].

**Neuronal Cells as a Biomarker for Dementia**

J. Luna-Muñoz, A. Martínez-Maldonado,

M. del C. Cárdenas-Aguayo, R. Mena and

Additional information is available at the end of the chapter

I. Ferrer, B. Floran-Garduño,

http://dx.doi.org/10.5772/58305

M.A. Meraz Ríos

**1. Introduction**

V. Ibarra-Bracamontes, M. A. Ontiveros-Torres,
