**4. Proteins in the pathophysiology of AD**

#### **4.1. The amyloid precursor protein (APP)**

The APP is a type 1 transmembrane glycoprotein of 110–130 kDa, one of the most abundant proteins in the central nervous system (CNS), and is cut by α‐secretase within the sequence of amino acids that comprise the Aβ peptide, precluding formation of amyloid peptides [49]. In the amyloidogenic pathway, APP is cleaved instead by β‐secretase, releasing a smaller N‐ terminal fragment (sAPPβ) and a longer C‐terminal fragment (C99) that contains the full amyloidogenic sequence of amino acids. A further cleavage of APP by γ‐secretase yields the Aβ peptide. In brain, there is an equilibrium between Aβ peptide production and its clearance [50]. How Aβ is removed from the brain is not entirely clear, but is mediated by two proteins: apolipoprotein E (APOE) and the insulin‐degrading enzyme (IDE) that may inhibit its aggregation [51]. Disadvantageous genetic polymorphisms (such as the ε4 allele of APOE) and pathological conditions related to abnormal IDE homeostasis (e.g., diabetes mellitus) that may favor the amyloidogenic cleavage of APP and/or decrease Aβ clearance from the brain will therefore facilitate Aβ accumulation in neural tissues and the downstream effects of the amyloid cascade [52].

#### **4.2. Deposition of Aβ**

**3.3. Synaptopathy**

196 Update on Dementia

**4. Proteins in the pathophysiology of AD**

**4.1. The amyloid precursor protein (APP)**

Activated Arc/Arg3.1 is targeted to the post‐synaptic density of synaptically active dendritic spines where it associates with polysomes. Arc interacts with endophilin 2/3 and dynamin, contributing to α‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazole‐propionate (AMPA) type gluta‐ mate receptor (AMPAR) modulation by enhancing receptor endocytosis. The Arc‐endosome also traffics APP and physically associates with PS 1, thereby increasing the amount of activity‐ dependent Aβ [45]. This may be a positive feedback mechanism in which removal of the AMPAR from the synapse will produce a significant loss of dendritic spines and synaptic activity, resulting in synaptic failure similar to that observed in AD. Activity of the *N*‐methyl‐ D‐aspartate receptor (NMDAR) in the hippocampus is also known to be crucial for long‐term spatial memory formation and to play a role in AD pathogenesis. The NMDAR is localized at synaptic and extra‐synaptic sites where it has diverse functions, from modulating memory strength to neurotoxicity and neuroprotection, and one of the components of the NMDAR‐ associated signaling complex is Arc/Arg3.1. Other postsynaptic elements are the lipid rafts (subdomains of the plasma membrane that contain high concentrations of cholesterol and glycosphingolipids), which are involved in cell signaling and with the NMDAR complex. Thus, physiological and pathological events such as ischemia and spatial learning can induce movements of NMDAR signaling complexes between the postsynaptic density and lipid raft subdomains. Synaptopathy and lipid raft disruption may be related to the onset of episodic memory deficits during the early stages of AD [46–48]. In order to analyze this possibility, studies have been initiated to determine the content of NMDA and AMPA receptors as well as Arc/Arg3.1 levels in the lipid raft microdomains of the 3xTg‐AD murine model of AD at the pre‐plaque stage and to understand perturbations in neurons, which may help to explain the synaptic plasticity deficits and long‐term memory impairments observed in AD models.

The APP is a type 1 transmembrane glycoprotein of 110–130 kDa, one of the most abundant proteins in the central nervous system (CNS), and is cut by α‐secretase within the sequence of amino acids that comprise the Aβ peptide, precluding formation of amyloid peptides [49]. In the amyloidogenic pathway, APP is cleaved instead by β‐secretase, releasing a smaller N‐ terminal fragment (sAPPβ) and a longer C‐terminal fragment (C99) that contains the full amyloidogenic sequence of amino acids. A further cleavage of APP by γ‐secretase yields the Aβ peptide. In brain, there is an equilibrium between Aβ peptide production and its clearance [50]. How Aβ is removed from the brain is not entirely clear, but is mediated by two proteins: apolipoprotein E (APOE) and the insulin‐degrading enzyme (IDE) that may inhibit its aggregation [51]. Disadvantageous genetic polymorphisms (such as the ε4 allele of APOE) and pathological conditions related to abnormal IDE homeostasis (e.g., diabetes mellitus) that may favor the amyloidogenic cleavage of APP and/or decrease Aβ clearance from the brain will Aβ is produced by endoproteolysis, post‐translational processing of the amyloid precursor protein (APP), which is achieved by the sequential cleavage of APP by groups of enzymes or enzyme complexes termed α‐, β‐ and γ‐secretases [53]. The first transgenic mouse (PDAPP) model that developed amyloid plaque pathology was generated by Games and colleagues to express human APP containing mutations associated with early‐onset AD; results obtained in these mice support a primary role for APP/Aβ in the genesis of AD and show they could provide a preclinical model for testing therapeutic drugs [13]. Since then, other mouse models have been created that recapitulate all aspects of AD including processing of the APP. However, not all APP transgenic mice have cognitive impairment, cellular loss and other AD characteristics, and they fail to replicate the full human disease. Some models actually confirm that the reduction of Aβ is insufficient to rescue memory function once downstream processes are underway. Conversely, other studies in mice predict that immunization against Abfix might prevent cognitive decline if administered early enough [54]. Also, Schenk et al. [55] studied transgenic mouse models and reported that their active immunization alleviated the burden of amyloid plaque, suggesting a potential therapeutic strategy [56].

Brain injury is reported to accelerate Aβ deposition and exacerbate Alzheimer's disease associated with impairment of cognition prior to the emergence of Aβ plaques. However, the relationships between Aβ levels (Aβ 40, A β42, or the ratio of Aβ 42 to Aβ 40), gender, age and cognitive function were measured in five mouse models (Tg2576, APP, PS 1, APP(OSK)‐Tg, 3xTg‐AD), see reference [57]. They used behavior tests such as escape latency times in the Morris water maze or exploratory preference percentage in the novel object recognition test. Tg2576 mice, overexpressing human APP695 concentration six times greater than that of normal mouse APP levels, show higher levels of Aβ40 and Aβ42 and Aβ deposits that begin at 9 months of age [58]. The APP models express hAPPSw and APP751 isoforms under the control of the murine Thy1 promoter. As a result, this mouse exhibits levels of human APP seven times greater than that of wild‐type mice, and its Aβ plaques begin at 6 months of age. The APP(OSK)‐Tg mouse expresses APP harboring the Osaka (E693) mutation, and it exhibits intraneuronal Aβ oligomers and memory impairment from 8 months of age. The PS 1 model expresses human PS with the mutation M146L or M146V via the PDGF‐β promoter and higher levels of endogenous mouse Aβ1‐42/43 [59]. The 3xTg‐AD, triple‐transgenic model exhibits both Aβ and tau pathologies and mimics human AD [60]. Thus, the possible role of Aβ in AD cognitive decline needs to be further investigated, fueled by other possible hypotheses and explanations [57].

#### **4.3. Apolipoprotein E**

Genetic association studies reveal that several genes such as ApoE are associated with multiple age‐related disorders, indicating that these genes could play a crucial role in their causation. The e4 allele of the apolipoprotein E (ApoE) gene is the best‐known genetic risk factor for AD, because it has been suggested to affect both Aβ and NFT pathology in AD. ApoE is a 34‐kDa lipid‐binding protein that functions in the transport of triglycerides and cholesterol in multiple tissues by interacting with lipoprotein receptors on target cells; these functions are particularly critical for the central nervous system where ApoE transport of cholesterol is important for the maintenance of myelin and neuronal membranes [60]. Polymorphism of the ApoE gene has been implicated in many chronic cardiovascular (myocardial infarction, hypertension, coronary heart) and neuronal diseases. The ApoE ε4 genotype not only is a risk factor for cardiovascular disease but also it combines synergistically with age, atherosclerosis, peripheral vascular disease or type‐2 diabetes to increase the risk of AD [62–66].

The ApoE gene is expressed most highly in the liver and brain; genome‐wide association studies have confirmed the ε4 allele of ApoE as the strongest genetic risk factor for AD [67, 68], because over 60% of persons with AD harbor at least one ApoE‐ε4 allele, and recent data indicate complex interactions between age, ApoE genotype and gender [61]. In reference [69], Dowell et al. used NMR to study two age groups: a young group (average age, 21 years) and a mid‐age group (average age, 50 years); they reported that there are regional white matter brain volume and cortical thickness differences between genotype groups at each age. They raised the possibility that an over‐engagement with these regions by e4+ individuals in youth may have a neurogenic effect that is observable later in life. According to a genome‐wide association study of cerebrospinal fluid (CSF) from AD subjects, several single nucleotide polymorphisms (SNPs) in the ApoE gene region of the brain were also associated with phosphorylated tau (p tau) elevated levels in the CSF. When cerebrospinal fluid levels of Aβ 1–42 were analyzed together with tau/ p tau, a significant correlation was found with SNPs of the ApoE gene. ApoE is also a crucial regulator of the innate immune system, which promotes pro‐inflammatory responses that could exacerbate AD pathogenesis [70].

In 2002, Colton et al. demonstrated that ApoE regulates the production of nitric oxide (NO), a critical cytoactive factor released by active macrophages. Thus, due to greater NO production, ApoE4 carriers characteristically have high levels of oxidative/nitrosative stress and a higher incidence of AD, a mechanism that explains the genetic association between ApoE4 and human diseases [71].

#### **4.4. Tau accumulation**

Besides the accumulation of soluble and toxic Aβ‐aggregates, tau accumulation causes oxidative stress and mitochondrial dysfunction, and it is linked to the initiation of the tau cascade. The tauopathies are a group of degenerative diseases with histopathology character‐ ized by filamentary inclusions composed of tau protein in neurons (NFT pathology). These are abundant in many neurodegenerative diseases, including AD, Pick's disease, argyrophilic grain disease and frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP‐17) [72]. In AD, the presence of neurofibrillary tangles (NFT) composed of tau is prominent, and their density correlates with neuronal loss and clinical severity [72, 74]. Dystrophic neurites are all sites of accumulation of pathological paired helical filaments (PHFs) that appear to be central to neurofibrillary degeneration of neuropathology and that contain (the microtubule‐associated protein) tau as an integral structural component [75–78]. Also, tau processing in AD, leading to the formation of paired helical filaments, is driven by aggregation and polymerization, and appears to be associated with abnormal phosphorylation and truncation processes [79]. Mouse models expressing the P301L mutation causing neurofibril‐ lary degeneration have been generated to study neurofibrillary pathologies [80]; and this mutation facilitates the development in transgenic mice of tauopathies [81–83] that recapitulate human tauopathies [83]; these mice provided the opportunity to test experimentally whether the distribution or timing of neurofibrillary pathology is influenced by the pathogenic mutations that cause AD. However, the physiology of tau protein is different in adult mice and humans; because mouse brain contains only isoforms like 4R, while in normal adult human there is a balance between 3R and 4R isoforms [85].

There is a clear link between type 2 diabetes mellitus and AD, and the use of antidiabetic drugs such as metformin has been proposed as a potential therapy for AD. There is also experimental evidence that metformin may have beneficial effects on cognition [86]. However, it remains unknown whether, in the absence of insulin resistance or diabetes, chronic treatment with metformin ameliorates tau pathology and behavioral performance in a transgenic model of neurodegenerative tauopathy *in vivo*. A recent study by Barini et al. shows how metformin modulates tau pathology in vivo. In P301S mice, they found similar levels of tau and ptau in the cortex and hippocampus with or without metformin, but metformin enhanced hyperactive behavior in the open field test. Due to dual actions on tau phosphorylation and aggregation, metformin may unpredictably impact the development of tauopathy in elderly diabetic patients at risk for AD [87].

In order to elucidate the molecular mechanisms underlying the post‐translational modifica‐ tions of Aβ and tau, several transgenic mouse models have been developed. One of these models is the 3xTg‐AD transgenic mouse, carrying three transgenes encoding the APPSWE, S1M146V and TauP301L proteins. Ontiveros‐Torres et al. reported the hippocampal accumu‐ lation of fibrillar Aβ as a function of age and hyperphosphorylation patterns of TauP301L at both its N‐ and C‐termini: the expression of activated protein kinases and mediators of inflammation was monitored from 3 to 28 months as well. These authors reported that the accumulation of Aβ oligomers results in an inflammatory environment that upregulates kinases involved in hyperphosphorylation of the TauP301L polypeptide. The 3xTg‐AD mouse is an excellent model for further studying pathological modifications of key factors in AD [88].
