**1. Introduction**

Alzheimer's disease (AD) is the main neurological cause of dementia, and it affects about 46 million people worldwide, mostly elderly adults. The incidence of AD increases exponen‐ tially every 5 years from 65 years of age, and it is estimated that 74.7 and 131.5 million people will be living with AD by 2030 and 2050, respectively (World Alzheimer Report, 2015). Patients with AD undergo progressive memory loss, reduced cognitive capacity and eventually, dementia. The debilitating effects of AD, especially at advanced disease stages, impose a substantial financial burden on AD patient's families, primarily due to the cost associated with medical care. However, the etiology of AD still remains largely unclear and although there has been much effort to elucidate the pathophysiological mechanisms underlying this devastat‐ ing condition over the last 20 years, the principal cause remains unknown, representing an important unmet clinical need. Therefore, AD is undoubtedly one of today's most challeng‐ ing global public health problems, and there is a pressing need to develop novel therapeutic agents to prevent and treat this disease.

The neuropathological hallmarks of AD include the formation of extracellular senile plaques due to the aggregation of amyloid-β (Aβ; normally associated with local inflammation and dystrophy/swelling of neurites), the formation of intracellular neurofibrillary tangles of hyperphosphorylated tau protein, as well as a loss of synaptic connections and neuronal degenera‐ tion [1]. Clinically, AD can be classified into two categories: familial AD (FAD, also known as early-onset AD) and sporadic AD (SAD, also known as late-onset AD). FAD generally accounts for <1% of the total AD cases, and they correspond to a disease variant with onset prior to 65 years of age [2]. This familial form of AD is inherited in an autosomal dominant pattern, and it is caused by mutations in three genes involved in Aβ generation: the amyloid precursor protein (APP), presenilin 1 (PS1) and presenilin 2 (PS2) [3]. In contrast to FAD, no single gene mutation has been found to be directly responsible for the onset and pathogenesis of SAD [4]. For the late-onset cases, the principal risk factors are ageing and the apolipoprotein E (ApoE) allele ε4 (see Section 3.1.).

The identification of clinical mutations in APP and presenilins in association with FAD has contributed to our understanding of AD pathogenesis. APP is a transmembrane protein that undergoes primary enzymatic cleavage by an α- or β-secretase in its extracellular (or intraluminal) domain, as well as secondary cleavage by a γ-secretase within the transmem‐ brane region (**Figure 1**). The metalloproteases ADAM10 and/or ADAM17 appear to be responsible for this α-secretase activity and the aspartyl protease BACE-1 (beta-site APP cleaving enzyme 1) corresponds to the β-secretase activity, whereas γ-secretase is an aspartyl proteolytic complex containing four subunits (PS1 or 2, nicastrin, APH1, and PEN-2) [5]. APP cleavage may be produced by β- and γ-secretases in a pathway known as the amyloidogen‐ ic route of APP. First, APP β-cleavage produces soluble APP-β (sAPPβ) and a transmem‐ brane C-terminal fragment known as β-CTF or C99. The latter then undergoes γ-secretase cleavage to generate the APP intracellular domain (AICD) and the Aβ peptide, preferential‐ ly the Aβ40 and 42 isoforms. Alternatively, APP may be cleaved by α- and γ-secretases in a pathway known as the non-amyloidogenic route of APP where α-secretase cleaves APP right

in the middle of the Aβ sequence (**Figure 1**) to generate soluble APPα (sAPPα) and a transmembrane C-terminal fragment known as α-CTF or C83. The latter undergoes further γ-cleavage to produce AICD and p3 (also known as Aβ17–40/42). In this context, it has been widely reported that FAD mutations induce alterations in APP processing that increased the cellular production of Aβ and augment the Aβ 42/40 ratio. Since mutations in both APP and presenilins are the major causal factors in FAD etiology, altered APP metabolism was assumed to be the principal cause triggering AD, leading to the formulation of the amyloid cascade hypothesis more than 20 years ago. Finally, it is notable that all these participants in APP metabolism, APP and secretases, are membrane-associated proteins influenced by the composition and structure of cell membrane lipids that in turn modulate APP metabolism [6].

**1. Introduction**

128 Update on Dementia

agents to prevent and treat this disease.

allele ε4 (see Section 3.1.).

Alzheimer's disease (AD) is the main neurological cause of dementia, and it affects about 46 million people worldwide, mostly elderly adults. The incidence of AD increases exponen‐ tially every 5 years from 65 years of age, and it is estimated that 74.7 and 131.5 million people will be living with AD by 2030 and 2050, respectively (World Alzheimer Report, 2015). Patients with AD undergo progressive memory loss, reduced cognitive capacity and eventually, dementia. The debilitating effects of AD, especially at advanced disease stages, impose a substantial financial burden on AD patient's families, primarily due to the cost associated with medical care. However, the etiology of AD still remains largely unclear and although there has been much effort to elucidate the pathophysiological mechanisms underlying this devastat‐ ing condition over the last 20 years, the principal cause remains unknown, representing an important unmet clinical need. Therefore, AD is undoubtedly one of today's most challeng‐ ing global public health problems, and there is a pressing need to develop novel therapeutic

The neuropathological hallmarks of AD include the formation of extracellular senile plaques due to the aggregation of amyloid-β (Aβ; normally associated with local inflammation and dystrophy/swelling of neurites), the formation of intracellular neurofibrillary tangles of hyperphosphorylated tau protein, as well as a loss of synaptic connections and neuronal degenera‐ tion [1]. Clinically, AD can be classified into two categories: familial AD (FAD, also known as early-onset AD) and sporadic AD (SAD, also known as late-onset AD). FAD generally accounts for <1% of the total AD cases, and they correspond to a disease variant with onset prior to 65 years of age [2]. This familial form of AD is inherited in an autosomal dominant pattern, and it is caused by mutations in three genes involved in Aβ generation: the amyloid precursor protein (APP), presenilin 1 (PS1) and presenilin 2 (PS2) [3]. In contrast to FAD, no single gene mutation has been found to be directly responsible for the onset and pathogenesis of SAD [4]. For the late-onset cases, the principal risk factors are ageing and the apolipoprotein E (ApoE)

The identification of clinical mutations in APP and presenilins in association with FAD has contributed to our understanding of AD pathogenesis. APP is a transmembrane protein that undergoes primary enzymatic cleavage by an α- or β-secretase in its extracellular (or intraluminal) domain, as well as secondary cleavage by a γ-secretase within the transmem‐ brane region (**Figure 1**). The metalloproteases ADAM10 and/or ADAM17 appear to be responsible for this α-secretase activity and the aspartyl protease BACE-1 (beta-site APP cleaving enzyme 1) corresponds to the β-secretase activity, whereas γ-secretase is an aspartyl proteolytic complex containing four subunits (PS1 or 2, nicastrin, APH1, and PEN-2) [5]. APP cleavage may be produced by β- and γ-secretases in a pathway known as the amyloidogen‐ ic route of APP. First, APP β-cleavage produces soluble APP-β (sAPPβ) and a transmem‐ brane C-terminal fragment known as β-CTF or C99. The latter then undergoes γ-secretase cleavage to generate the APP intracellular domain (AICD) and the Aβ peptide, preferential‐ ly the Aβ40 and 42 isoforms. Alternatively, APP may be cleaved by α- and γ-secretases in a pathway known as the non-amyloidogenic route of APP where α-secretase cleaves APP right

**Figure 1.** APP processing by secretases. In the non-amyloidogenic pathway, APP is first cleaved by α-secretase at a sequence of amino acids within the Aβ peptide, releasing the sAPPα ectodomain. Further processing of the resulting membrane-associated C-terminal C83 fragment (α-CTF) by γ-secretase leads to the release of the p3 fragment and the APP intracellular domain (AICD). This processing takes place preferentially at the plasma membrane. Conversely, the amyloidogenic pathway is initiated when β-secretase cleaves APP at the amino terminus of the Aβ peptide to release the sAPPβ ectodomain. Further processing of the resulting membrane-associated C-terminal C99 fragment (β-CTF) by γ-secretase releases the Aβ peptide and AICD. This processing normally takes place in acidic cellular compartments like late endosomes. The Aβ peptide produced is normally 40 or 42 amino acids long (Aβ40 or 42) and the Aβ42/40 ratio increases in AD.

#### **2. Historical perspective on the pathophysiology of Alzheimer's disease**

For more than 20 years, the accumulation of the Aβ peptide has been considered to be the main cellular/molecular event that triggers AD-related neurodegeneration. Amyloid plaques were first thought to cause AD pathogenesis, and more recently, Aβ-soluble oligomers have gained more attention as key players in AD etiology [7]. Regardless of the form of amyloid, the amyloid cascade hypothesis postulates that Aβ accumulation in the brain is the major upstream event in AD pathophysiology, whereas other neuropathological features are a result of this primary amyloid pathology, including the formation of neurofibrillary tangles, neuroinflam‐ mation, synaptic failure, and eventually neural death [8, 9].

According to the amyloid cascade hypothesis, enhanced amyloidogenic activity of secretases and/or reduced clearance of the Aβ peptide may trigger Aβ accumulation. As a result, the secretases involved in Aβ generation have been extensively targeted by the pharmaceutical industry to develop new compounds to treat AD [10]. In particular, the Aβ42/40 ratio may increase due to FAD mutations and this increase enhances oligomer formation, which may in turn impair synaptic function and provoke neuronal degeneration [7]. At the same time, secreted Aβ42 forms primary extracellular Aβ deposits in the brain parenchyma, first as diffuse plaques and later as insoluble fibrillary plaques. A concomitant local inflammatory response develops around these amyloid deposits (involving microglial and astroglial activation), coupled to synaptic spine loss and neurite dystrophy (neuritic pathology) [11, 12]. Over time, these events result in oxidative stress and altered ion homeostasis. Neurofibrillary tangles appear as a consequence of the altered kinase and phosphatase activities that cause tau protein hyperphosphorylation, and likely its subsequent dysfunction in axonal transport, as well as neurite dystrophy [13, 14]. Finally, the cascade ends with extensive synaptic and neuronal dysfunction, which precedes the well-characterized neuronal death associated with the Aβ and tau pathologies [7]. It is this neuronal degeneration that is responsible for memory loss and dementia in patients with AD.

Amyloid burden in the brain parenchyma is closely associated with tau hyperphosphorylation, axonal dystrophy and inflammatory reaction around amyloid plaques (**Figure 2**). Both, inflammation and axonal dystrophy can promote neuronal degeneration [15, 16]. However, it is still largely unknown which of these events (amyloid, inflammation, or neurite dystrophy) appear first during disease development and how these three events are connected. The amyloid cascade hypothesis postulates that amyloid accumulation, first intracellular and then extracellular, leads to the generation of amyloid plaques. Given the close relationship between Aβ plaque number and size with the surrounding dystrophies and gliosis, these two latter events were proposed to progress in conjunction with Aβ plaque formation. However, evidence is now accumulating against the amyloid cascade hypothesis. On the one hand, therapeutic approaches focused on combating amyloid pathology have generally failed to prevent AD progression in clinical trials (see Section 4, [17, 18]), while on the other hand, transgenic AD animal models, mostly created by incorporating human mutated APP and/or PS1 into the animal genome, do not recapitulate all the neuropathological features of AD, and not even the large scale neuronal death that occurs during this pathology [19]. Moreover, the alterations to membrane lipids in neurons of patients with AD suggest that the changes to lipid bilayer could be the first event in the amyloid cascade and related pathways [6]. Indeed, the normalization of membrane lipids is associated with cognitive restoration (see Section 5.2). Accordingly, the amyloid pathology may not actually be the first initial event driving the events that provoke neuronal degeneration.

more attention as key players in AD etiology [7]. Regardless of the form of amyloid, the amyloid cascade hypothesis postulates that Aβ accumulation in the brain is the major upstream event in AD pathophysiology, whereas other neuropathological features are a result of this primary amyloid pathology, including the formation of neurofibrillary tangles, neuroinflam‐

According to the amyloid cascade hypothesis, enhanced amyloidogenic activity of secretases and/or reduced clearance of the Aβ peptide may trigger Aβ accumulation. As a result, the secretases involved in Aβ generation have been extensively targeted by the pharmaceutical industry to develop new compounds to treat AD [10]. In particular, the Aβ42/40 ratio may increase due to FAD mutations and this increase enhances oligomer formation, which may in turn impair synaptic function and provoke neuronal degeneration [7]. At the same time, secreted Aβ42 forms primary extracellular Aβ deposits in the brain parenchyma, first as diffuse plaques and later as insoluble fibrillary plaques. A concomitant local inflammatory response develops around these amyloid deposits (involving microglial and astroglial activation), coupled to synaptic spine loss and neurite dystrophy (neuritic pathology) [11, 12]. Over time, these events result in oxidative stress and altered ion homeostasis. Neurofibrillary tangles appear as a consequence of the altered kinase and phosphatase activities that cause tau protein hyperphosphorylation, and likely its subsequent dysfunction in axonal transport, as well as neurite dystrophy [13, 14]. Finally, the cascade ends with extensive synaptic and neuronal dysfunction, which precedes the well-characterized neuronal death associated with the Aβ and tau pathologies [7]. It is this neuronal degeneration that is responsible for memory loss

Amyloid burden in the brain parenchyma is closely associated with tau hyperphosphorylation, axonal dystrophy and inflammatory reaction around amyloid plaques (**Figure 2**). Both, inflammation and axonal dystrophy can promote neuronal degeneration [15, 16]. However, it is still largely unknown which of these events (amyloid, inflammation, or neurite dystrophy) appear first during disease development and how these three events are connected. The amyloid cascade hypothesis postulates that amyloid accumulation, first intracellular and then extracellular, leads to the generation of amyloid plaques. Given the close relationship between Aβ plaque number and size with the surrounding dystrophies and gliosis, these two latter events were proposed to progress in conjunction with Aβ plaque formation. However, evidence is now accumulating against the amyloid cascade hypothesis. On the one hand, therapeutic approaches focused on combating amyloid pathology have generally failed to prevent AD progression in clinical trials (see Section 4, [17, 18]), while on the other hand, transgenic AD animal models, mostly created by incorporating human mutated APP and/or PS1 into the animal genome, do not recapitulate all the neuropathological features of AD, and not even the large scale neuronal death that occurs during this pathology [19]. Moreover, the alterations to membrane lipids in neurons of patients with AD suggest that the changes to lipid bilayer could be the first event in the amyloid cascade and related pathways [6]. Indeed, the normalization of membrane lipids is associated with cognitive restoration (see Section 5.2). Accordingly, the amyloid pathology may not actually be the first initial event driving the

mation, synaptic failure, and eventually neural death [8, 9].

and dementia in patients with AD.

130 Update on Dementia

events that provoke neuronal degeneration.

**Figure 2.** Dystrophic neurites surrounding β-amyloid plaques in AD patient's brain. (a–c) Double-labeling immuno‐ fuorescence and confocal microscopy to mitochondrial porin (a; green) and β-amyloid plaques (b; red). Porin immu‐ nostaining revealed mitochondrial enrichment in dystrophic neurites surrounding amyloid plaques (c). (d–f) Doublelabeling immunofluorescence and confocal microscopy to mitochondrial porin (d; red), and phosphorylated tau (pThr181) (b; green) show co-segregation of porin and hyperphosphorylated tau in dystrophic neurites (long arrow in f). (g–i) Double-labeling immunofuorescence and confocal microscopy to lysosomal associated protein 1 (LAMP-1) (a; green) and mitochondrial porin (b; red). LAMP-1 and porin co-localize in a subset of cellular processes (c; arrowheads) suggesting engulfment of mitochondria into matured autophagic vesicles and participation of lysosomes in its degra‐ dation in distrophic neurites. β-Amyloid is stained in blue. Bar 10 μm (a–c and d–f), and 20 μm (g–i). Adapted from [12] with permission of Springer.

It also appears that axon swelling or dystrophy can precede extracellular amyloid deposition in certain animal models, in which autophagic vesicles with all the necessary enzymatic machinery to produce the Aβ peptide are evident [20–23]. In this sense, dystrophic axons have been proposed to be an intracellular source of secreted Aβ that would seed extracellular amyloid plaques. Protein deposits containing APP fragments can be seen in the brain

parenchyma of aged wild-type mice, originating from axonal varicosities, further supporting this hypothesis. These data suggest that axonal dystrophy occurs first, leading thereafter to extracellular amyloid deposition in the early stages of the disease. In fact, it has been proposed that neurite dystrophy could reflect a conserved neuroprotective strategy to overcome the agerelated accumulation of misfolded proteins, which in turn may represent a molecular mechanism of Aβ plaque deposition that potentially underlies the shift from normal to pathological aging [24, 25]. Nevertheless, Aβ alone may promote axonal atrophy through its interactions with the p75 neurotrophin receptor (p75NTR) in axon membranes [26]. Together, the evidence suggests that dystrophy and extracellular Aβ deposition are involved in a positive feedback loop whereby axon dystrophy is a source of extracellular Aβ, and the latter promotes axonal atrophy.

In terms of neuroinflammation, it is widely accepted that Aβ deposition alone might be sufficient to induce an inflammatory reaction that subsequently contributes to neuronal death and cognitive decline in AD [15]. However, this fact does not necessarily imply that Aβ plaque formation precedes microglial activation in AD. During normal aging, microglial activation aims to clear the misfolded proteins contained in fragmented neurites and aggregated into senile plaques. Interestingly, during AD-related pathological ageing, microglia cells recruited around plaques phagocytose Aβ and this could constitute part of the microglial mechanism to clear misfolded proteins, also during normal ageing [25]. Thus, in a scenario characterized by age-related chronic inflammation, microglia would be highly responsive to further activation which would drive their differentiation toward a classic phenotype characterized by pro-inflammatory cytokine secretion, in turn impairing axon trafficking, promoting Aβ accumulation and cell death [25, 27]. However, this putative role for AD-associated neuroin‐ flammation is not supported by evidence showing that the inflammatory response is not neurotoxic and, indeed, it is even neuroprotective in a transgenic mouse model of AD [28]. In fact, from early in the amyloid pathology, alternative neuroprotective microglia are activated around amyloid plaques supporting neuronal survival, and this alternative phenotype is also present during animal ageing. By contrast, the classic microglial phenotype that is character‐ ized by cytotoxic cytokine secretion only appears at advanced ages, associated with the presence of soluble Aβ oligomers and neuronal loss [27, 28]. Thus, these evidences show that alternative neuroprotective microglia may be present at advanced ages and coexist with classic microglial activation. In summary, although it is widely accepted that neuroinflammation promotes neuronal degeneration, it remains unclear how brain inflammation participates in the shift from normal to pathological ageing.

Hence, determining whether amyloid pathology is the first event in the pathway to ADassociated neuronal degeneration and dementia appears to be a particularly relevant issue, especially after the repeated fiascos in clinical trials of drugs targeting Aβ and related molec‐ ular entities. There is a close relationship among Aβ, inflammatory and neurite pathologies in AD because they all appear at early stages of the disease and all three are involved in neuronal death. In the present chapter, we will review how these neuropathological hallmarks are related to AD-associated membrane lipid alterations, as there can now be no shadow of doubt that brain lipids and the pathways they are involved in influence the pathophysiology of AD.
