**3. Alzheimer´s disease: a brief description and pathological markers (Aβ1-42, 3-42, 11-42)**

Alzheimer's disease (AD) is the most common cause of dementia in the elderly population. It is characterized by a progressive atrophy in several brain areas such as the entorhinal cortex, hippocampus, corpus callosum, and also areas outside the limbic system [1]. This process is irreversible and results in memory loss, inability to learn, performing calculation, unbalanced perception of space, and depression. AD is classified in three stages: mild, moderate, and severe.

AD commences with signs of mild cognitive impairment characterized by memory loss, poor judgment, mood swings, repetitive questions, and difficulty in doing mathematical calcula‐ tions. The symptoms of moderate AD include the inability to learn new things, difficulty to recognized people, hallucinations, delusions, paranoia, and impulsive behavior. Finally, severe AD patients are dependent and bedridden [2].

Pathological hallmarks of AD include the presence of neurofibrillary tangles, senile plaques, neuronal death, synapsis loss, astrogliosis in the enthorinal cortex, hippocampus, amygdala, and frontal, temporal, parietal, and occipital cortex [3].

Neurofibrillary tangles are intracellular deposits of paired helical filaments formed by hyperphosphorylated tau protein. On the other hand, senil plaques are present as diffuse plaques composed of amorphous extracellular deposits of amyloid β (Aβ) that lacks neurites and neuritic plaques composed of extracellular deposits of insoluble Aβ surrounded by dystrophic neurites, reactive astrocytes, and activated microglia [3, 4].

The etiology of AD is not yet fully understood, but genetic and environmental factors are involved in the disease pathogenesis and progression. Early-onset, so-called familial AD occurs in 1% of cases that are linked to autosomal dominant mutation in amyloid β precursor protein (APP), presenillin (PSEN) 1, and PSEN2. The rest of AD cases are sporadic, the lateonset form (so-called "sporadic AD"). There are also genetic risk factors associated with lateonset AD, for example, the presence of the ε4 allele of the gene for apo-lipoprotein E, which has been shown to increase the probability of the development of AD, whereas the presence of an ε2 allele appears to protect against the disease [5, 6]. Nongenetic risk factors include cerebrovascular changes (hemorrhagic or ischemic), hypertension, type 2 diabetes, and metabolic syndrome [4].

Aβ is generated from the amyloid precursor protein, an ~105 kDa single-pass transmembrane glycoprotein found at presynaptic and postsynaptic terminals in the brain. APP gene in human is located on chromosome 21 and alternative splicing of APP transcript generates 8 isoform, of which the 695, 751, and 770 amino acids forms are the most common [7]. APP695 is pre‐ dominantly expressed in neurons, especially during neuronal differentiation whereas APP751 and APP770 are more ubiquitous, although during brain injury their expression increases in astrocytes and microglia [8]. APP plays an important role in neuronal functions such as synapse formation, neuronal migration, neurite outgrowth, synaptic plasticity, synaptic transmission and learning, and memory [9]. APP is synthetized in endoplasmic reticulum and is modified in the Golgi apparatus. The ectodomain contains part of the Aβ sequence, which extends into the transmembrane domain.

Proteolytic processing of APP includes two different pathways (**Figure 1**): (1) nonamyloido‐ genic processing and (2) amyloidogenic processing. The first is the cleavage by α-secretase within the Aβ domain releasing a soluble α-secretase-released N-terminal of APP (sAPPα) and generating a truncated APP CTF (αCTF or C83). The latter is subsequently intramembrane cut by γ-secretase, which liberates a truncated Aβ peptide called p3 and generates the APP intracellular domain (AICD). This process stops the production of β-amyloid peptide and prevents its deposition in plaques. On the other hand, APP can be cleaved by β-secretase at the beginning of Aβ sequence liberating a soluble sAPPβ and generating a membraneassociated *C*-terminal fragment (βCTF or C99) whose subsequent cleavage by γ-secretase activity results in the generation of Aβ peptides ranging in length from 38 to 42 residues, where Aβ1-42 is the most neurotoxic form [10–13]. The resulting peptides are liberated into extrac‐ ellular fluids such as cerebrospinal fluid (CSF), plasma, or interstitial fluid [14].

**Figure 1.** APP schematic structure and processing by secretases.

Mutation in APP molecule has differential effect depending on the location of the mutated residue. Amino acid substitution flanking the Aβ region close to β-secretase cleavage site like Swedish mutation modulates the rate of enzymatic processing of APP maintaining the ratio Aβ-42/Aβ-40. In contrast, mutation occurring in close proximity to γ-secretase cleavage site (such as the so-called Austrian, Iranian, French, German, London, and Florida mutations) is associated with increasing production of Aβ-42 and lower levels of Aβ-40 [15]. Mutations in the mid region of Aβ domain affect the primary sequence of Aβ peptide resulting in enhanced aggregation propensity. Some of these intraAβ mutations can lead to mixed amyloid pathol‐ ogies: marked cerebral angiopathy and marked amyloid plaque formation [11]. The α-secretase activity is mediated by a series of membrane-bound proteases, which are member of the ADAM (a disintegrin and metalloprotease) family. In neuron, the principal constitutive αsecretase activity is exerted by ADAM10. The processing of APP by α-secretase is postulated to be protective in the context of AD because the enzymes cleave within the Aβ sequence, thereby preventing the production of Aβ [16].

cerebrovascular changes (hemorrhagic or ischemic), hypertension, type 2 diabetes, and

Aβ is generated from the amyloid precursor protein, an ~105 kDa single-pass transmembrane glycoprotein found at presynaptic and postsynaptic terminals in the brain. APP gene in human is located on chromosome 21 and alternative splicing of APP transcript generates 8 isoform, of which the 695, 751, and 770 amino acids forms are the most common [7]. APP695 is pre‐ dominantly expressed in neurons, especially during neuronal differentiation whereas APP751 and APP770 are more ubiquitous, although during brain injury their expression increases in astrocytes and microglia [8]. APP plays an important role in neuronal functions such as synapse formation, neuronal migration, neurite outgrowth, synaptic plasticity, synaptic transmission and learning, and memory [9]. APP is synthetized in endoplasmic reticulum and is modified in the Golgi apparatus. The ectodomain contains part of the Aβ sequence, which

Proteolytic processing of APP includes two different pathways (**Figure 1**): (1) nonamyloido‐ genic processing and (2) amyloidogenic processing. The first is the cleavage by α-secretase within the Aβ domain releasing a soluble α-secretase-released N-terminal of APP (sAPPα) and generating a truncated APP CTF (αCTF or C83). The latter is subsequently intramembrane cut by γ-secretase, which liberates a truncated Aβ peptide called p3 and generates the APP intracellular domain (AICD). This process stops the production of β-amyloid peptide and prevents its deposition in plaques. On the other hand, APP can be cleaved by β-secretase at the beginning of Aβ sequence liberating a soluble sAPPβ and generating a membraneassociated *C*-terminal fragment (βCTF or C99) whose subsequent cleavage by γ-secretase activity results in the generation of Aβ peptides ranging in length from 38 to 42 residues, where Aβ1-42 is the most neurotoxic form [10–13]. The resulting peptides are liberated into extrac‐

Mutation in APP molecule has differential effect depending on the location of the mutated residue. Amino acid substitution flanking the Aβ region close to β-secretase cleavage site like

ellular fluids such as cerebrospinal fluid (CSF), plasma, or interstitial fluid [14].

metabolic syndrome [4].

172 Update on Dementia

extends into the transmembrane domain.

**Figure 1.** APP schematic structure and processing by secretases.

**Figure 2.** Amyloid plaques of 1-42, AβN3(pE), and AβN11(pE) present in human brain. (A) Merge (yellow) between βA1-42 plaque (green) and the fibrillar βA1-42 marker TR (red). (B) Merge (yellow) between AβN3(pE) plaque (green) and TR (red). (C) Merge between AβN3(pE) (green), AβN11(pE) (red), and the nuclear marker DAPI (blue). (D) Merge between AβN3(pE) (red) and the glial cell marker GFAP (green) showing a glial cell surrounding AβN3(pE) aggre‐ gates. These amyloid aggregates were observed in 50 mm thick brain tissue sections of temporal cortex from AD pa‐ tients. Scale bar represents 20 μm; A–D.

The major neuronal β-secretase, termed BACE-1, is a transmembrane 501 amino acid aspartyl protease. After synthesis, BACE-1 is transported to the cell surface via the endoplasmic reticulum and Golgi. APP and BACE-1 are both endocytosed where the APP cleavage occurs as the optimum pH of BACE is 3.5–4.4. Mutations of BACE-1 have not been identified in familial AD cases, but the activity of BACE is increased in both familial and sporadic AD [17]. The γ-secretase activity is executed by a high molecular weight, membrane-embedded protein complex consisting of PSEN, nicastrin, anterior pharynx defective (APH1), and presenilin enhancer (PEN2), although PSEN seems to provide the active core γ-secretase complex functioning as an aspartyl protease [18]. In mammals, two homologous, PSEN1 and PSEN2, are found whose mutation alters the biochemical character of the γ-secretase complex and its interaction with APP substrate to skew the transmembrane cleavage toward longer more aggregation-prone forms of Aβ, increasing the ratio Aβ-42/Aβ-40, which is associated to early onset of AD [19]. The proteolytic APP processing preferentially generates Aβ1-40/1-42; however, there is a great diversity of Aβ peptides depending on γ-secretase and shorter peptides resulting from γ-secretase activity upon C99. In addition, a significant proportion of Aβ consist of N-terminal truncated/modified species, which increase the Aβ propensity to form aggregates [14, 20] with the most prominent forms identified starting at position 3 or 11 and possessing N-terminal pyroglutamic acid (pyroE), generated by glutamic acid [21]. The Nterminal truncated Aβ3-42/Aβ3 is generated by the zinc-metalloprotease neutral endopepti‐ dase or neprilysin (NeP)-40 cleaving Aβ between Arg-2 and Glu-3. On the other hand, BACE-1 is also capable of cleaving between Tyr-10 and Glu-11, leading to the release of Aβ11-42/ Aβ11-40 peptides [22]. Then, the GluN-terminal undergoes N-terminal pyroglutamate (pGlu) modification catalyzed by glutaminylcyclase (**Figure 2**) [23].
