**2. Experimental therapeutic strategies to prevent Alzheimer progression to Alzheimer Disease (AD) states**

Several reviews have focused on various aspects related to habits and dietary elements which may act as protective factors against AD, including physical and mental exercise, low caloric intake, various diets with low fat content, and vitamin complements [2, 3]. It is worth noting that neuropathological studies in old-aged individuals usually present combined pathologies, and combination of Alzheimer changes and vascular lesions are very common [4]. It is well documented that vascular pathology potentiates primary neurodegenerative pathology and that vascular factors may be causative of cognitive impairment and dementia [5]. Therefore, therapies geared to reduce vascular risk factors are also protective factors against AD clinical manifestations.

### **2.1. Targeting Aβ**

production and deposition of hyper-phosphorylated tau. Therefore, Aβ and hyper-phos‐ phorylated tau promote the progression of the process and this may occur in an exponential

In addition to these pathological hallmarks, multiple alterations play roles in the degen‐ erative process. Several genetic factors, such as apolipoprotein ε4 (APOE4), and external factors, such as vascular and circulatory alterations and repeated cerebral traumatisms, among others, facilitate disease progression in sporadic forms. Furthermore, metabolic components mainly, but not merely, associated with aging have a cardinal influence, in‐ cluding mitochondrial defects and energy production deficiencies, production of free rad‐ icals (oxidative and nitrosative reactive species: ROS and NOS) and oxidative and nitrosative damage, increased reticulum stress damage, altered composition of mem‐ branes, inflammatory responses and impaired function of degradation pathways such as

It has been proven that the degenerative process, at least the presence of neurofibrillary tangles, starts in middle age in selected nuclei of the brain stem and entorhinal cortex, and then progresses to other parts of the brain. Instrumental stages of Braak cover stages I and II with involvement of the entorhinal and transentorhinal cortices; stages II and IV also affect the hippocampus and limbic system together with the basal nucleus of Meynert; and stages V and VI involve the whole brain although neurofibrillary tangles are not found in selected regions such as the cerebellar cortex and the dentate gyrus. The distribution of senile plaques is a bit different as they first appear in the orbitofrontal cortex and temporal cortex and then progress

A concomitant decline in neuronal organization occurs most often in parallel with senile plaques and neurofibrillary tangles manifested as synaptic dysfunction and synaptic loss, and

An important observation is that about 80% of individuals aged 65 years have Alzheimerrelated changes, at least at stages I-III, whereas only 5% have cognitive impairment and dementia. About 25% of individuals aged 85 years suffer from cognitive impairment and dementia of Alzheimer type. Stages I-IV are often silent with no clinical symptoms. Cognitive impairment and dementia usually occur at stages V and VI when the neurodegenerative process is very advanced. Importantly, the progression from stage I to stage IV may last decades, whereas the progression to stages V and VI is much more rapid. Therefore, Alzheimer is a well-tolerated degenerative process during a relatively long period of time, but it may have devastating effects once thresholds are crossed. Moreover, clinical symptoms may be compli‐

Several attempts have been made to predict the evolution to disease states. Neuroimaging, including high resolution and functional magnetic resonance imaging, positron emission tomography and the use of relative selective markers of β-amyloid and tau deposition in the brain, together with reduced levels of Aβ and increased index of phospho-tau/total tau in the cerebrospinal fluid, are common complementary probes (biomarkers) in addition to the data

way once these abnormal proteins are accumulated in the brain.

neuronal death and progressive isolation of remaining neurons.

autophagy and ubiquitin-proteasome system.

cated by concomitant vascular pathology.

to the whole convexity.

252 Understanding Alzheimer's Disease

Most of the current drug development for the prevention or treatment of AD is based on the β-amyloid cascade hypothesis and aims at reducing the levels of Aβ in the brain. Overpro‐ duction, aggregation and deposition of the Aβ peptide begin before the onset of symptoms and they are considered an essential early event in AD pathogenesis. Thus, targeting these early Aβ alterations is assumed to reduce the progression to disease states. The different strategies developed to achieve this objective include decreasing Aβ production through modulating secretase activity, interfering with Aβ aggregation, and promoting Aβ clearance.

#### *2.1.1. Secretase-targeting therapies*

APP is processed in the brain exclusively by three membrane-bound proteases, α-, β- and γsecretase. Therefore, specifically modifying such enzyme activity should result in a reduction of Aβ production [6].

and deposition in APP mouse models and in humans [15-17]. However, target-based toxicity of GSIs has been a major obstacle to the clinical development of these compounds. In fact, two large Phase III clinical trials of *Semagacestat*, the only GSI extensively studied in AD, were prematurely interrupted because of the observation of detrimental cognitive and functional effects of the drug [18]. Several dozen γ-secretase substrates have been identified, including Notch1 trans-membrane receptor, which plays an important role in a variety of developmental and physiological processes by controlling cell fate decisions. To overcome these toxicity issues, pharmaceutical companies have been trying to develop a second generation of 'Notch-sparing' GSIs, which revealed beneficial effects in *in vitro* and in animal models of AD [19-21]. They are currently under clinical studies. Such 'Notch-sparing' GSIs have higher pharmacological selectivity than the first GSIs probably due to the distinct binding to the substrate docking site on γ-secretase of Notch and APP. Identification of

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Almost 20 enzymes are currently known to contribute to Aβ degradation in the brain, although the most studied are two zinc metalloproteases, neprilysin (NEP) and insulin-degrading enzyme (IDE). NEP is one of the major Aβ-degrading enzymes in the brain [23] and NEP levels are decreased in the brain of AD and animal models [24, 25]. Lentiviral delivery of the NEP gene to the brain of AD transgenic mice reduced Aβ pathology [26]. A number of subsequent studies with NEP and other related peptidases such as endothelin-converting enzymes 1 and 2 (ECE-1 and ECE-2) further supported this observation [27]. Similarly, over-expression of IDE in neurons significantly reduces brain Aβ levels, prevents Aβ plaque formation and its associated cytopathology, and rescues the premature lethality present in these particular APP transgenic mice [28]. A growing body of evidence has been accumulated supporting the

Other specific Aβ-cleaving proteases such as angiotensin-converting enzyme (ACE), matrix metalloproteinase-9 (MMP-9) and the serine protease plasmin, which have distinct sub-cellular localizations and differential responses to aging, oxidative stress and pharmacological agents, are also potential candidates to become novel therapeutic strategies for AD prevention and

Targeting the delivery of these compounds to the brain remains a major challenge. The most promising current approaches include peripheral administration of agents that enhance the activity of Aβ-degrading enzymes and direct intra-cerebral release of enzymes by convectionenhanced delivery. Genetic procedures geared at increasing cerebral expression of Aβ-

Compounds that suppress the aggregation or reduce the stability of Aβ oligomers may bind monomers in order to attenuate formation of both the oligomeric and senile plaque fibrillar Aβ constituents. One of the amyloid-binding drugs more extensively studied in animal models

several γ-secretase inhibitors has been reviewed elsewhere [22].

potential therapeutic properties of IDE in AD [29].

degrading enzymes may offer additional advantages [30].

*2.1.2. Aβ degrading enzymes*

treatment [27].

*2.1.3. Decreasing Aβ aggregation*


and deposition in APP mouse models and in humans [15-17]. However, target-based toxicity of GSIs has been a major obstacle to the clinical development of these compounds. In fact, two large Phase III clinical trials of *Semagacestat*, the only GSI extensively studied in AD, were prematurely interrupted because of the observation of detrimental cognitive and functional effects of the drug [18]. Several dozen γ-secretase substrates have been identified, including Notch1 trans-membrane receptor, which plays an important role in a variety of developmental and physiological processes by controlling cell fate decisions. To overcome these toxicity issues, pharmaceutical companies have been trying to develop a second generation of 'Notch-sparing' GSIs, which revealed beneficial effects in *in vitro* and in animal models of AD [19-21]. They are currently under clinical studies. Such 'Notch-sparing' GSIs have higher pharmacological selectivity than the first GSIs probably due to the distinct binding to the substrate docking site on γ-secretase of Notch and APP. Identification of several γ-secretase inhibitors has been reviewed elsewhere [22].

#### *2.1.2. Aβ degrading enzymes*

*2.1.1. Secretase-targeting therapies*

of Aβ production [6].

254 Understanding Alzheimer's Disease

APP is processed in the brain exclusively by three membrane-bound proteases, α-, β- and γsecretase. Therefore, specifically modifying such enzyme activity should result in a reduction

**•** *α-secretase activators*: α-secretase initiates the non-amyloidogenic pathway by cleaving APP within the Aβ sequence, thereby preventing the production of Aβ and producing a nontoxic form of APP derivative which is neuroprotective and growth- promoting [7]. There‐ fore, compounds that stimulate α-secretase activity could become an attractive strategy to reduce Aβ production. In fact, some indirect methods of promoting α-secretase activity, such as the stimulation of the protein kinase C (PKC) or Mitogen-activated protein kinases (MAPK) pathways, the use of α-7-nicotinic acetylcholine (ACh) receptor and 5-hydroxi‐ tryptamine (5-HT) receptor 4 agonists, and γ-aminobutyric acid A receptor modulators, result in α-secretase-mediated cleavage of APP and reduced Aβ levels *in vivo* [8]. However, the development of a direct activator of α-secretase as a drug treatment for AD seems premature because of the lack of knowledge about the consequences of chronic up-regula‐

**•** *β-secretase inhibitors*: the β-secretase enzyme initiates the amyloidogenic pathway, cleaving APP at the amino terminus of the Aβ peptide. Further cleavage of the resulting carboxyterminal fragment by γ-secretase results in the release of Aβ. β-secretase activity is specifi‐ cally mediated by the β-site APP cleaving enzyme 1 (BACE1), which is also involved in the processing of numerous substrates in addition to APP. The research of drugs inhibiting BACE1 activity was encouraged by studies revealing that the expression of mutated BACE1 reduces amyloidogenesis and cognitive impairment in APP transgenic mice [9, 10]. The first generation of BACE1 inhibitors was peptide-based mimetics of the APP β-cleavage site. Unfortunately, these compounds exhibited some difficulties because of the large substrate binding site of BACE1 and because of the difficulty in crossing the blood–brain barrier (BBB) and penetrating the plasma and endosomal membranes to gain access to the intracellular compartments where endogenous BACE1 plays its function. Recently, non-peptide smallmolecule BACE1 inhibitors have been reported to improve bioavailability and to lower cerebral Aβ levels in animal models of AD [11, 12]. However, the involvement of BACE1 in other important physiological processes raises concerns about minimizing the potential

**•** *γ-secretase inhibitors (GSIs)*: γ-secretase is a complex composed of presenilin 1 and presenilin 2 (PS1 and PS2) forming the catalytic core and three accessory proteins, anterior pharynxdefective 1 (APH-1), nicastrin and presenilin enhancer protein 2 (PEN2). The γ-secretase complex displays a high degree of subunit heterogeneity and little is known about the physiological roles of the diverse complexes and how they process different trans-mem‐ brane substrates in addition to APP. This heterogeneity suggests that selective targeting of one particular subunit might be a more effective treatment strategy than non-selective γsecretase inhibition [13]. Thus, removal of APH-1B and APH-1C isoforms in a mouse model of AD decreased Aβ plaque formation and improved behavioral deficits [14]. A number of orally bioavailable and brain-penetrating GSIs have been shown to decrease Aβ production

tion of α-secretase-mediated cleavage on other substrates [6].

adverse effects derived from generalized BACE1 inhibition.

Almost 20 enzymes are currently known to contribute to Aβ degradation in the brain, although the most studied are two zinc metalloproteases, neprilysin (NEP) and insulin-degrading enzyme (IDE). NEP is one of the major Aβ-degrading enzymes in the brain [23] and NEP levels are decreased in the brain of AD and animal models [24, 25]. Lentiviral delivery of the NEP gene to the brain of AD transgenic mice reduced Aβ pathology [26]. A number of subsequent studies with NEP and other related peptidases such as endothelin-converting enzymes 1 and 2 (ECE-1 and ECE-2) further supported this observation [27]. Similarly, over-expression of IDE in neurons significantly reduces brain Aβ levels, prevents Aβ plaque formation and its associated cytopathology, and rescues the premature lethality present in these particular APP transgenic mice [28]. A growing body of evidence has been accumulated supporting the potential therapeutic properties of IDE in AD [29].

Other specific Aβ-cleaving proteases such as angiotensin-converting enzyme (ACE), matrix metalloproteinase-9 (MMP-9) and the serine protease plasmin, which have distinct sub-cellular localizations and differential responses to aging, oxidative stress and pharmacological agents, are also potential candidates to become novel therapeutic strategies for AD prevention and treatment [27].

Targeting the delivery of these compounds to the brain remains a major challenge. The most promising current approaches include peripheral administration of agents that enhance the activity of Aβ-degrading enzymes and direct intra-cerebral release of enzymes by convectionenhanced delivery. Genetic procedures geared at increasing cerebral expression of Aβdegrading enzymes may offer additional advantages [30].

#### *2.1.3. Decreasing Aβ aggregation*

Compounds that suppress the aggregation or reduce the stability of Aβ oligomers may bind monomers in order to attenuate formation of both the oligomeric and senile plaque fibrillar Aβ constituents. One of the amyloid-binding drugs more extensively studied in animal models and AD patients is tramiprosate (3-amino-1-propanesulfonic acid; Alzhemed). Tramiprosate was effective in reducing Aβ polymerisation *in vitro*, inhibiting the formation of neurotoxic aggregates, and decreasing Aβ plaque formation in animal models [31]. However, recent phase III clinical trials did not produce any significant improvement in cognition in AD patients chronically treated with tramiprosate in spite of the significant reduction in hippocampus volume loss [32]. Similarly, some other compounds known to inhibit Aβ aggregation and fibril formation showed positive effects in animal and *in vitro* models of AD but failed to produce conclusive results in human clinical trials. This is the case with scyllo-inositol and PBT2. Scylloinositol inhibited cognitive deficits in TgCRND8 mice and significantly ameliorated disease pathology, even in animals at advanced stages of AD-like pathology, without interfering with endogenous phosphatidylinositol lipid production [33, 34]. Yet a phase II clinical trial failed in supporting or refuting a benefit of scyllo-inositol in mild to moderate AD patients [35]. PBT2 is a copper/zinc ionophore which targets metal-induced aggregation of Aβ. When given orally to two models of Aβ-bearing transgenic mice, PTB2 was able to markedly decrease soluble brain Aβ levels within hours and to improve cognitive performance within days [36]. These results correlated with a rapid cognitive improvement in AD patients in a recent phase IIa clinical trial [37], an observation that argues for large-scale testing of PBT2 for AD.

vascular Aβ deposits [47]. Yet important conclusions were drawn from the studies in humans: immunization reduced the number of Aβ plaques and the number of dystrophic neurites, including tau phosphorylation around plaques, but not Aβ burden in blood vessels; however,

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New vaccines containing immunodominant B-cell epitopes of Aβ [51] and recognizing other Aβ residues [52, 53], and the use of passive immunization with deglycosylated an‐ tibodies [54] have demonstrated positive effects in the clearance of Aβ without causing inflammatory response or hemorrhages in animal models of AD [55]. These findings have prompted new clinical trials which are currently evaluating the toxicity and effec‐ tiveness of at least ten vaccines in mild-to-moderate AD patients worldwide [56]. While vaccines hold great hope as AD therapies, it is important to stress that immunization at pre-symptomatic stages is essential in order to avoid the irreversible brain damage occur‐

The interest in tau-related therapies is still emerging and very few clinical studies are under‐ way, in part because of the difficulties encountered with anti-Aβ strategies that captured most efforts in the two last decades, but also because of the challenging identification of tractable therapeutic targets related to tau. Current research in the prevention of tau pathology devel‐ oped in animal models of AD has resulted in some promising results [58]. Main rationales in tau pathology are based on: 1: inhibition of tau aggregation, 2: reduction of tau phosphoryla‐ tion by inhibition of tau kinases or activation of phosphatases (including PP2a activity), 3: reduction of tau levels by increasing tau degradation or by using active immunization, and 4:

Some compounds that are known to inhibit tau-tau interactions have been tested as agents aimed at slowing Alzheimer progression to disease states. Among them, phenothiaziazine methylene blue inhibits tau-tau interactions, is neuroprotective and is able to facilitate soluble tau clearance in a mouse model of human tauopathy [60, 61]. Moreover, phenothiaziazine methylene blue has shown beneficial effects in a phase II clinical trial conducted for one year [62]. Another promising inhibitor of tau aggregation is the immunosuppressant FK506, which exerts its beneficial effects in transgenic mice by directly binding tau to the FK506 binding

However, some concerns araise from the use of tau aggregation inhibitors in that at least some tau aggregation inhibitors enhance the formation of potentially toxic tau oligomers [65].

Kinases which participate in the phosphorylation of tau and phosphatases which dephos‐ phorylate tau are clear putative therapeutic targets for AD [66]. The most widely studied tau kinases in AD pathogenesis are Glycogen synthase kinase 3 beta (GSK-3β) and Cyclin-

immunization increased intracerebral levels of soluble Aβ [48-50].

ring even at the early symptomatic stages [57].

**2.2. Targeting tau**

stabilization of microtubule [59].

*2.2.1. Inhibition of tau aggregation*

protein 52 and by modulating microglial activation [63, 64].

*2.2.2. Reduction of tau hyperphosphorylation*

Another promising recent experimental approach is the use of dendrimers as agents interfering with Aβ fibrilization. Dendrimers are globular branched polymers, typically symmetric around the core with a spherical three-dimensional morphology. Their chemical structure allows dendrimers to couple to active amyloid species through hundreds of possible sites. Dendrimers have been shown to be able to modulate Aβ peptide aggregation by interfering in different ways with the polymerization process, including fibril breaking, inhibition of fibril formation and acceleration of fibril formation [38, 39]. However, some dendrimers assayed in amyloidogenic systems are toxic to cells. The development of non-toxic glycodendrimers, which reduce toxicity by clumping fibrils together [40], opens the possibility of using den‐ drimers with low intrinsic toxicity in AD. Additional difficulties in dendrimer administration involve the crossing of the BBB so as to reach their targets in the brain.

#### *2.1.4. Facilitating Aβ clearance: Immunotherapy against Aβ*

Active and passive immunotherapy against Aβ peptide has been explored as a therapeutic approach to stimulate the clearance of Aβ in the brain at the preclinical and clinical stages of the disease in animal models. Pioneering studies proved that vaccination of young APP transgenic mice using a synthetic aggregated form of Aβ42 (AN-1792) effectively prevented Aβ plaque formation, neuritic dystrophy and astrogliosis in adult brains [41]. Subsequent studies further demonstrated improvement of memory loss in those APP transgenic mice vaccinated against Aβ [42, 43]. Different models, methods and ways of administration showed the beneficial effects of active and passive immunization in animal models of AD. Neverthe‐ less, the phase II trial in humans was discontinued because of the occurrence of aseptic meningoencephalitis in a number of cases [44-46]. The cause of the meningoencephalitis was a concomitant T-cell-mediated autoimmune response [45, 46]. Moreover, several studies in APP transgenic mice have reported an increased risk of microhemorrhages at sites of cerebro‐ vascular Aβ deposits [47]. Yet important conclusions were drawn from the studies in humans: immunization reduced the number of Aβ plaques and the number of dystrophic neurites, including tau phosphorylation around plaques, but not Aβ burden in blood vessels; however, immunization increased intracerebral levels of soluble Aβ [48-50].

New vaccines containing immunodominant B-cell epitopes of Aβ [51] and recognizing other Aβ residues [52, 53], and the use of passive immunization with deglycosylated an‐ tibodies [54] have demonstrated positive effects in the clearance of Aβ without causing inflammatory response or hemorrhages in animal models of AD [55]. These findings have prompted new clinical trials which are currently evaluating the toxicity and effec‐ tiveness of at least ten vaccines in mild-to-moderate AD patients worldwide [56]. While vaccines hold great hope as AD therapies, it is important to stress that immunization at pre-symptomatic stages is essential in order to avoid the irreversible brain damage occur‐ ring even at the early symptomatic stages [57].

#### **2.2. Targeting tau**

and AD patients is tramiprosate (3-amino-1-propanesulfonic acid; Alzhemed). Tramiprosate was effective in reducing Aβ polymerisation *in vitro*, inhibiting the formation of neurotoxic aggregates, and decreasing Aβ plaque formation in animal models [31]. However, recent phase III clinical trials did not produce any significant improvement in cognition in AD patients chronically treated with tramiprosate in spite of the significant reduction in hippocampus volume loss [32]. Similarly, some other compounds known to inhibit Aβ aggregation and fibril formation showed positive effects in animal and *in vitro* models of AD but failed to produce conclusive results in human clinical trials. This is the case with scyllo-inositol and PBT2. Scylloinositol inhibited cognitive deficits in TgCRND8 mice and significantly ameliorated disease pathology, even in animals at advanced stages of AD-like pathology, without interfering with endogenous phosphatidylinositol lipid production [33, 34]. Yet a phase II clinical trial failed in supporting or refuting a benefit of scyllo-inositol in mild to moderate AD patients [35]. PBT2 is a copper/zinc ionophore which targets metal-induced aggregation of Aβ. When given orally to two models of Aβ-bearing transgenic mice, PTB2 was able to markedly decrease soluble brain Aβ levels within hours and to improve cognitive performance within days [36]. These results correlated with a rapid cognitive improvement in AD patients in a recent phase IIa

clinical trial [37], an observation that argues for large-scale testing of PBT2 for AD.

involve the crossing of the BBB so as to reach their targets in the brain.

*2.1.4. Facilitating Aβ clearance: Immunotherapy against Aβ*

256 Understanding Alzheimer's Disease

Another promising recent experimental approach is the use of dendrimers as agents interfering with Aβ fibrilization. Dendrimers are globular branched polymers, typically symmetric around the core with a spherical three-dimensional morphology. Their chemical structure allows dendrimers to couple to active amyloid species through hundreds of possible sites. Dendrimers have been shown to be able to modulate Aβ peptide aggregation by interfering in different ways with the polymerization process, including fibril breaking, inhibition of fibril formation and acceleration of fibril formation [38, 39]. However, some dendrimers assayed in amyloidogenic systems are toxic to cells. The development of non-toxic glycodendrimers, which reduce toxicity by clumping fibrils together [40], opens the possibility of using den‐ drimers with low intrinsic toxicity in AD. Additional difficulties in dendrimer administration

Active and passive immunotherapy against Aβ peptide has been explored as a therapeutic approach to stimulate the clearance of Aβ in the brain at the preclinical and clinical stages of the disease in animal models. Pioneering studies proved that vaccination of young APP transgenic mice using a synthetic aggregated form of Aβ42 (AN-1792) effectively prevented Aβ plaque formation, neuritic dystrophy and astrogliosis in adult brains [41]. Subsequent studies further demonstrated improvement of memory loss in those APP transgenic mice vaccinated against Aβ [42, 43]. Different models, methods and ways of administration showed the beneficial effects of active and passive immunization in animal models of AD. Neverthe‐ less, the phase II trial in humans was discontinued because of the occurrence of aseptic meningoencephalitis in a number of cases [44-46]. The cause of the meningoencephalitis was a concomitant T-cell-mediated autoimmune response [45, 46]. Moreover, several studies in APP transgenic mice have reported an increased risk of microhemorrhages at sites of cerebro‐

The interest in tau-related therapies is still emerging and very few clinical studies are under‐ way, in part because of the difficulties encountered with anti-Aβ strategies that captured most efforts in the two last decades, but also because of the challenging identification of tractable therapeutic targets related to tau. Current research in the prevention of tau pathology devel‐ oped in animal models of AD has resulted in some promising results [58]. Main rationales in tau pathology are based on: 1: inhibition of tau aggregation, 2: reduction of tau phosphoryla‐ tion by inhibition of tau kinases or activation of phosphatases (including PP2a activity), 3: reduction of tau levels by increasing tau degradation or by using active immunization, and 4: stabilization of microtubule [59].

#### *2.2.1. Inhibition of tau aggregation*

Some compounds that are known to inhibit tau-tau interactions have been tested as agents aimed at slowing Alzheimer progression to disease states. Among them, phenothiaziazine methylene blue inhibits tau-tau interactions, is neuroprotective and is able to facilitate soluble tau clearance in a mouse model of human tauopathy [60, 61]. Moreover, phenothiaziazine methylene blue has shown beneficial effects in a phase II clinical trial conducted for one year [62]. Another promising inhibitor of tau aggregation is the immunosuppressant FK506, which exerts its beneficial effects in transgenic mice by directly binding tau to the FK506 binding protein 52 and by modulating microglial activation [63, 64].

However, some concerns araise from the use of tau aggregation inhibitors in that at least some tau aggregation inhibitors enhance the formation of potentially toxic tau oligomers [65].

#### *2.2.2. Reduction of tau hyperphosphorylation*

Kinases which participate in the phosphorylation of tau and phosphatases which dephos‐ phorylate tau are clear putative therapeutic targets for AD [66]. The most widely studied tau kinases in AD pathogenesis are Glycogen synthase kinase 3 beta (GSK-3β) and Cyclindependent kinase (CDK5) [67, 68]. Several GSK-3β inhibitors, including lithium, aloisines, flavopiridol, hymenialdisine, paullones, and staurosporine, are under active investigation and development [69]. Lithium revealed some promising results when administered in transgenic mice expressing the P301L human 4R0N tau at pre-symptomatic stages; it improved behavior and reduced the levels of phosphorylation, aggregation and insoluble tau in transgenic mice [70]. However, several concerns have arisen in relation of the use of GSK-3β in the treatment of AD; these are based on the fact that lithium lacks specificity over GSK-3β activity and it has a narrow safety margin [71]. Moreover, GSK-3β acts on multiple metabolic pathways that are also impaired with unknown consequences after chronic treatment.

Tau degradation can also be enhanced by immunization. Active immunization targeting phosphorylated tau reduces filamentous tau inclusions and neuronal dysfunction in JNPL3 transgenic mice [85, 86]. Moreover, recent studies have raised the possibility of modulating tau pathology by passive immunization revealing reduced behavioral impairment and tau

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Since microtubule disruption occurs in several models of AD and is associated with tau dysfunction, microtubule stabilizers have been assayed in preclinical and clinical trials for AD [88]. The anti-mitotic drug paclitaxel prevents Aβ-induced toxicity in cell culture [89], as well as axonal transport deficits and behavioral impairments in tau transgenic mice [90]. Unfortu‐ nately, paclitaxel is a P-glycoprotein substrate and it has very low capacity to cross the BBB, making it unsuitable for the treatment of human tauopathies. Epothilone D, which has better BBB permeability, improves microtubule density and cognition in tau transgenic mice [91]. Finally, the peptide NAP stabilizes microtubules and reduces tau hyper-phosphorylation [92]. NAP can be administered intra-nasally and has shown promising results in a phase II clinical

Several pieces of evidence demonstrate that oxidative stress precedes other hallmarks of the neurodegenerative process in human brains and animal models of AD, including Aβ deposi‐ tion, NFT formation, and metabolic dysfunction and cognitive decline. It plays a functional role in the pathogenesis of the disease [94-100]. These findings sustain the possibility of using anti-oxidants in the prevention and treatment of Alzheimer [101, 102]. Several studies in AD transgenic mouse models support the potential beneficial effect of antioxidant compounds as

Several nutritional antioxidants such as resveratrol, curcumin, epigallocatechin gallate, Lacetyl-carnitine, RRR-α-tocopherol (vitamin E) and ascorbic acid (vitamin C) have been tested

**•** *Resveratrol* is a polyphenolic compound found in grapes, berries and peanuts with well known anti-oxidant, anti-cancer, anti-inflammatory and estrogenic activities. *In vitro* and animal experiments reveal that resveratrol protects against Aβ toxicity by promoting the non-amyloidogenic cleavage of APP, thus enhancing the clearance of Aβ peptides by promoting their degradation through the ubiquitin-proteasome system, as well as reducing neuronal damage by decreasing the expression of inducible nitric oxide synthase (iNOS) and cyclooxigenase 2 (COX-2), and the pro-apoptotic factors Bax and c-Jun N-terminal kinase (JNK). Moreover, the capacity of resveratrol to induce the overexpression of sirtuins, proteins having a role in cell survival, probably contributes to its

pathology in two transgenic models of taupathies [87].

*2.2.4. Microtubule stabilizers*

trial [93].

**2.3. Oxidative stress**

preventive drugs.

*2.3.1. Naturally-occurring anti-oxidants*

neuroprotective effect [103, 104].

to counteract oxidative stress-induced brain damage in AD.

CDK5 inhibitors prevent Aβ-induced tau hyper-phosphorylation and cell death *in vitro* [72, 73]. A recent *in vivo* study further demonstrates that inhibition of CDK5 activates GSK-3β, which plays a more dominant role in overall tau phosphorylation than does CDK5 [74]. Thus, considering that CDK5 inhibitors might be unable to reverse abnormal hyper-phosphorylation of tau and treat neurofibrillary degeneration because of the interplay between CDK5 and GSK-3β, as well as the essential role played by CDK5 in multiple cell signaling pathways [75], the interest of such compounds as a tau-targeting therapy for AD is limited.

Another approach to reverse tau hyper-phosphorylation is up-regulation of tau phosphatases [66]. The major tau phosphatase, PP2A, is down-regulated in AD brain. In consequence, correct‐ ing PP2A levels is the primary target to be considered. Among the compounds known to re‐ verse PP2A inhibition, memantine is the most outstanding because of the demonstrated clinical benefit in AD. In an animal model, memantine was able to reverse okadaic acid–induced PP2A inhibition and to prevent tau hyper-phosphorylation, restoring MAP2 expression [76]. Similar‐ ly, melatonin has also been shown to restore PP2A activity and reverse tau hyper-phosphoryla‐ tion, both *in vitro* and in experimental animals [77]. One important concern in considering PP2A as a potential therapeutic target is that all protein phosphatases have much broader substrate specificities than protein kinases. Thus, more undesirable effects might be expected than when using kinase inhibitors [66]. A further intriguing point is that PP2A function and activity de‐ pend on multiple subunits and cofactors which are dysregulated in AD [78]. It is not clear how all these elements can be resolved to result in maintained balanced activity.

#### *2.2.3. Reduction of tau levels*

A potential alternative to modulate tau phosphorylation is reducing overall tau levels [58]. Experiments carried out in genetically-modified mice expressing reduced tau levels revealed diminished cognitive impairment and Aβ-induced neuronal damage [79-81]. An alternative method to reduce tau levels could is by targeting molecules that regulate the expression or clearance of tau. Tau can be degraded via the ubiquitin-proteasome system and the lysosomal pathways. Reduction of the levels of the tau ubiquitin-ligase CHIP increases the accumulation of tau aggregates in JNPL3 mice, suggesting that increasing the expression of CHIP could result in reduced tau levels [82]. Acetylation of tau inhibits its degradation [83], alters its microtubule binding, and enhances aggregation [84]. Thus, the combination of tau acetylation inhibition and ubiquitination-proteasome enhancement might produce a synergy that lowers the levels of pathogenic tau species.

Tau degradation can also be enhanced by immunization. Active immunization targeting phosphorylated tau reduces filamentous tau inclusions and neuronal dysfunction in JNPL3 transgenic mice [85, 86]. Moreover, recent studies have raised the possibility of modulating tau pathology by passive immunization revealing reduced behavioral impairment and tau pathology in two transgenic models of taupathies [87].

#### *2.2.4. Microtubule stabilizers*

dependent kinase (CDK5) [67, 68]. Several GSK-3β inhibitors, including lithium, aloisines, flavopiridol, hymenialdisine, paullones, and staurosporine, are under active investigation and development [69]. Lithium revealed some promising results when administered in transgenic mice expressing the P301L human 4R0N tau at pre-symptomatic stages; it improved behavior and reduced the levels of phosphorylation, aggregation and insoluble tau in transgenic mice [70]. However, several concerns have arisen in relation of the use of GSK-3β in the treatment of AD; these are based on the fact that lithium lacks specificity over GSK-3β activity and it has a narrow safety margin [71]. Moreover, GSK-3β acts on multiple metabolic pathways that are

CDK5 inhibitors prevent Aβ-induced tau hyper-phosphorylation and cell death *in vitro* [72, 73]. A recent *in vivo* study further demonstrates that inhibition of CDK5 activates GSK-3β, which plays a more dominant role in overall tau phosphorylation than does CDK5 [74]. Thus, considering that CDK5 inhibitors might be unable to reverse abnormal hyper-phosphorylation of tau and treat neurofibrillary degeneration because of the interplay between CDK5 and GSK-3β, as well as the essential role played by CDK5 in multiple cell signaling pathways [75],

Another approach to reverse tau hyper-phosphorylation is up-regulation of tau phosphatases [66]. The major tau phosphatase, PP2A, is down-regulated in AD brain. In consequence, correct‐ ing PP2A levels is the primary target to be considered. Among the compounds known to re‐ verse PP2A inhibition, memantine is the most outstanding because of the demonstrated clinical benefit in AD. In an animal model, memantine was able to reverse okadaic acid–induced PP2A inhibition and to prevent tau hyper-phosphorylation, restoring MAP2 expression [76]. Similar‐ ly, melatonin has also been shown to restore PP2A activity and reverse tau hyper-phosphoryla‐ tion, both *in vitro* and in experimental animals [77]. One important concern in considering PP2A as a potential therapeutic target is that all protein phosphatases have much broader substrate specificities than protein kinases. Thus, more undesirable effects might be expected than when using kinase inhibitors [66]. A further intriguing point is that PP2A function and activity de‐ pend on multiple subunits and cofactors which are dysregulated in AD [78]. It is not clear how

A potential alternative to modulate tau phosphorylation is reducing overall tau levels [58]. Experiments carried out in genetically-modified mice expressing reduced tau levels revealed diminished cognitive impairment and Aβ-induced neuronal damage [79-81]. An alternative method to reduce tau levels could is by targeting molecules that regulate the expression or clearance of tau. Tau can be degraded via the ubiquitin-proteasome system and the lysosomal pathways. Reduction of the levels of the tau ubiquitin-ligase CHIP increases the accumulation of tau aggregates in JNPL3 mice, suggesting that increasing the expression of CHIP could result in reduced tau levels [82]. Acetylation of tau inhibits its degradation [83], alters its microtubule binding, and enhances aggregation [84]. Thus, the combination of tau acetylation inhibition and ubiquitination-proteasome enhancement might produce a synergy that lowers the levels

also impaired with unknown consequences after chronic treatment.

the interest of such compounds as a tau-targeting therapy for AD is limited.

all these elements can be resolved to result in maintained balanced activity.

*2.2.3. Reduction of tau levels*

258 Understanding Alzheimer's Disease

of pathogenic tau species.

Since microtubule disruption occurs in several models of AD and is associated with tau dysfunction, microtubule stabilizers have been assayed in preclinical and clinical trials for AD [88]. The anti-mitotic drug paclitaxel prevents Aβ-induced toxicity in cell culture [89], as well as axonal transport deficits and behavioral impairments in tau transgenic mice [90]. Unfortu‐ nately, paclitaxel is a P-glycoprotein substrate and it has very low capacity to cross the BBB, making it unsuitable for the treatment of human tauopathies. Epothilone D, which has better BBB permeability, improves microtubule density and cognition in tau transgenic mice [91]. Finally, the peptide NAP stabilizes microtubules and reduces tau hyper-phosphorylation [92]. NAP can be administered intra-nasally and has shown promising results in a phase II clinical trial [93].

#### **2.3. Oxidative stress**

Several pieces of evidence demonstrate that oxidative stress precedes other hallmarks of the neurodegenerative process in human brains and animal models of AD, including Aβ deposi‐ tion, NFT formation, and metabolic dysfunction and cognitive decline. It plays a functional role in the pathogenesis of the disease [94-100]. These findings sustain the possibility of using anti-oxidants in the prevention and treatment of Alzheimer [101, 102]. Several studies in AD transgenic mouse models support the potential beneficial effect of antioxidant compounds as preventive drugs.

### *2.3.1. Naturally-occurring anti-oxidants*

Several nutritional antioxidants such as resveratrol, curcumin, epigallocatechin gallate, Lacetyl-carnitine, RRR-α-tocopherol (vitamin E) and ascorbic acid (vitamin C) have been tested to counteract oxidative stress-induced brain damage in AD.

**•** *Resveratrol* is a polyphenolic compound found in grapes, berries and peanuts with well known anti-oxidant, anti-cancer, anti-inflammatory and estrogenic activities. *In vitro* and animal experiments reveal that resveratrol protects against Aβ toxicity by promoting the non-amyloidogenic cleavage of APP, thus enhancing the clearance of Aβ peptides by promoting their degradation through the ubiquitin-proteasome system, as well as reducing neuronal damage by decreasing the expression of inducible nitric oxide synthase (iNOS) and cyclooxigenase 2 (COX-2), and the pro-apoptotic factors Bax and c-Jun N-terminal kinase (JNK). Moreover, the capacity of resveratrol to induce the overexpression of sirtuins, proteins having a role in cell survival, probably contributes to its neuroprotective effect [103, 104].

**•** *Curcumin* is a polyphenolic compound present in the rhizome of *Curcuma longa*, commonly used as a spice to color and flavor food, which has anti-inflammatory, anti-carcinogenic and anti-infectious properties. The first evidence of a protective role of curcumin in AD was derived from epidemiological studies based on populations subjected to a curcuminenriched diet. Additionally, *in vitro* studies have shown that curcumin protects neurons from Aβ toxicity whereas the use of AD transgenic mouse models show that curcumin suppresses inflammation and oxidative damage as well as accelerating the Aβ rate of clearance and inhibiting Aβ aggregation. Curcumin is considered a bi-functional antioxidant because it is a direct scavenger of oxidants as well as a long-lasting protector promoting the expression of cytoprotective proteins through the induction of Nrf2 dependent genes [105, 106]. Regrettably, no significant improvement in cognitive function between placebo and curcumin-treated groups has been observed in the only two clinical trials carried out until now [107].

results: whereas vitamin E supplementation partially prevents the memory loss associated with the progression of the disease in some cases, the same treatment was detrimental in

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**•** *Ascorbic acid (Vitamin C)* is an essential nutrient since it acts as a cofactor in elemental enzymatic reactions, but in contrast to most of organisms, humans are not able to synthesize ascorbic acid. The main dietary source of vitamin C is fresh fruit and vegetables. The main interest in vitamin C for the treatment of neurodegenerative processes is related to its potent anti-oxidant properties. Some studies have revealed that vitamin C supplementation reduces oxidative stress, and mitigates Aβ oligomer formation and behavioral decline, but it did not decrease plaque deposition in AD mouse models [113, 114]. Despite epidemio‐ logical studies reporting reduced prevalence and incidence of AD in consumers of vitamin supplements [115], meta-analyses revealed the risks of chronic consumption of high doses

**•** *Egb76* is a standardized *Ginkgo biloba* extract already approved in some countries as symptomatic treatment for dementia although the evidence for its effectiveness remains inconclusive [117]. However, Egb761 has anti-oxidant properties, inhibits Aβ oligomeriza‐ tion *in vitro*, reduces impaired memory and learning capacities and enhances hippocampal neurogenesis in AD transgenic mice [118]. For these reasons, *Ginkgo biloba* extract is currently

In spite of the experimental evidence of beneficial effects of natural anti-oxidants in cultured cells and transgenic models, clinical studies have demonstrated only minimal effect in humans probably due to the bioavailability and pharmacokinetics of these substances [102, 105]. What's more, a slight acceleration in cognitive decline has been observed in patients treated for 16

In contrast to other antioxidants, those designed to target the free radical damage to mito‐

**•** *Lipoic acid (LA)* is a naturally-occurring precursor of an essential cofactor of many mitochondrial enzymes, including pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase, which is found in almost all foods. LA has been shown to present a variety of properties that can interfere with pathogenic processes of AD. LA increases ACh production, stimulates glucose uptake, protects against Aβ toxicity, chelates redoxactive transition metals, scavenges reactive oxygen species (ROS) and induces antioxidant protective enzymes probably through the activation of the transcription factor Nrf2. Via the same mechanisms, down-regulation of redox-sensitive inflammatory processes is also achieved [120]. Data from cell culture and animal models suggest that LA can be combined with other dietary anti-oxidants to synergistically decrease oxidative stress, inflammation, Aβ levels, and thus provide a combined benefit in the treatment of AD. However, clinical benefits after LA administration were quite small in

of vitamin C thus discouraging its routine use in AD. [116]

under evaluation as a preventive drug in AD.

weeks with a cocktail of natural antioxidants [119].

chondria provide greater therapeutic potential.

patients with mild or moderate dementia [121].

*2.3.2. Mitochondrial antioxidants*

others [112].


results: whereas vitamin E supplementation partially prevents the memory loss associated with the progression of the disease in some cases, the same treatment was detrimental in others [112].


In spite of the experimental evidence of beneficial effects of natural anti-oxidants in cultured cells and transgenic models, clinical studies have demonstrated only minimal effect in humans probably due to the bioavailability and pharmacokinetics of these substances [102, 105]. What's more, a slight acceleration in cognitive decline has been observed in patients treated for 16 weeks with a cocktail of natural antioxidants [119].

#### *2.3.2. Mitochondrial antioxidants*

**•** *Curcumin* is a polyphenolic compound present in the rhizome of *Curcuma longa*, commonly used as a spice to color and flavor food, which has anti-inflammatory, anti-carcinogenic and anti-infectious properties. The first evidence of a protective role of curcumin in AD was derived from epidemiological studies based on populations subjected to a curcuminenriched diet. Additionally, *in vitro* studies have shown that curcumin protects neurons from Aβ toxicity whereas the use of AD transgenic mouse models show that curcumin suppresses inflammation and oxidative damage as well as accelerating the Aβ rate of clearance and inhibiting Aβ aggregation. Curcumin is considered a bi-functional antioxidant because it is a direct scavenger of oxidants as well as a long-lasting protector promoting the expression of cytoprotective proteins through the induction of Nrf2 dependent genes [105, 106]. Regrettably, no significant improvement in cognitive function between placebo and curcumin-treated groups has been observed in the only two clinical

**•** *Epigallocatechin gallate (EGCG)* is a polyphenolic flavonoid encountered in green tea. Human epidemiological and animal data suggest that tea may decrease the incidence of dementia and AD. EGCG has been demonstrated to exert its neuroprotective activity by reducing Aβ production and inflammation, and increasing mitochondrial stabilization, iron chelation and ROS scavenging [108]. However, to date no clinical trials have been performed to verify whether EGCG neuroprotective/neurorestorative actions can be successfully translated into

**•** *Acetyl-L-Carnitine (ALC)* is a natural compound found in red meat whose biological role is to facilitate the transport of fatty acids to the mitochondria. Thus, the main mechanism of action of ALC is the improvement of mitochondrial respiration, which allows the neurons to produce the necessary ATP to maintain normal membrane potential. Yet ALC is neuro‐ protective through a variety of additional effects, including an increase in protein kinase C activity and modulation of synaptic plasticity by counteracting the loss of NMDA receptors in the neuronal membrane and by increasing the production of neurotrophins [105]. Moreover, ALC reduces Aβ toxicity in primary cortical neuronal cultures by increasing both heme-oxygenase 1 (HO-1) and heat-shock protein 70 (Hsp70) expression, probably through transcription factor Nrf2. In two clinical studies, ALC administered for one year significantly reduced cognitive decline in early-onset AD patients [109, 110] thus sustaining the potential

**•** *RRR-a-tocopherol (Vitamin E)* is probably the most important lipid-soluble natural antioxi‐ dant in mammalian cells. Most vegetable oils, nuts and some fruits are important dietary sources of vitamin E. The interest in evaluating its potential beneficial properties in AD is also sustained by its known ability to cross the BBB and to accumulate in the central nervous system. Deficiency in the α-tocopherol transfer protein mediating vitamin E activity induces an increase in brain lipid peroxidation, earlier and more severe cognitive dysfunction, and increased Aβ deposits in the brain of Tg2576 mice; this phenotype was ameliorated with vitamin E supplementation [111]. However, although epidemiological studies have demonstrated that increasing the intake of fruit and vegetables rich in vitamins prevents or retards the onset of AD, clinical trials for vitamin E treatment have revealed paradoxical

use of ALC in AD prevention and treatment at early stages.

trials carried out until now [107].

human beings.

260 Understanding Alzheimer's Disease

In contrast to other antioxidants, those designed to target the free radical damage to mito‐ chondria provide greater therapeutic potential.

**•** *Lipoic acid (LA)* is a naturally-occurring precursor of an essential cofactor of many mitochondrial enzymes, including pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase, which is found in almost all foods. LA has been shown to present a variety of properties that can interfere with pathogenic processes of AD. LA increases ACh production, stimulates glucose uptake, protects against Aβ toxicity, chelates redoxactive transition metals, scavenges reactive oxygen species (ROS) and induces antioxidant protective enzymes probably through the activation of the transcription factor Nrf2. Via the same mechanisms, down-regulation of redox-sensitive inflammatory processes is also achieved [120]. Data from cell culture and animal models suggest that LA can be combined with other dietary anti-oxidants to synergistically decrease oxidative stress, inflammation, Aβ levels, and thus provide a combined benefit in the treatment of AD. However, clinical benefits after LA administration were quite small in patients with mild or moderate dementia [121].

**•** *N-acetyl-cysteine (NAC)* is a precursor of glutathione (GSH), the most abundant endogenous anti-oxidant. NAC acts itself as an anti-oxidant by directly interacting with free radicals, as well as by increasing GSH levels. NAC protects against Aβ-induced cognitive deficits by decreasing the associated oxidative stress and related neuroinflammation, but also by activating anti-apoptotic signaling pathways in neuronal cultures [122]. Late-stage AD patients supplemented with NAC over a period of six months showed significantly improved performance in some cognitive tasks, although levels of oxidative stress in peripheral blood did not differ significantly from untreated patients [123].

in the Tg2576 mice [136, 137]. In contrast, the selective COX-2 inhibitor celecoxib failed to reduce the inflammatory burden and, even worse, increased the Aβ<sup>42</sup> levels when administered

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In spite of the promising results in animal models and the data from retrospective human epidemiological studies identifying long-term use of NSAIDs as being protective against AD, prospective clinical trials have not confirmed the efficiency of this group of drugs in the

Other anti-inflammatory agents such as trifusal have been shown to be beneficial in certain

Several findings indicate that brain glucose hypometabolism, deficient bioenergetics and mitochondrial dysfunction precede clinical symptoms in AD [1, 141-143]. The energetic failure observed even in the prodromal phase of the Alzheimer process is thought to be produced by the combination of mitochondria dysfunction, alteration of energy metabolism at poremitochondrial level, and increase in energetic demands of altered nerve cells. Thus, strategies to improve brain energy supply and to preserve mitochondrial functions becomes relevant in

The primary fuel for the brain under normal conditions is glucose, whereas the energetic contribution made by fatty acids is minor. Therefore, facilitation of energy metabolism and energy availability has been assayed in animal models and AD by facilitating glucose metab‐

**•** *Targeting reduced glucose metabolism*: Reduction in the utilization of glucose in AD [147] can be due to several causes including deficient insulin signaling, impairment in glucose transport mechanisms and dysfunction in glucolysis. Preclinical studies in animal models of AD have revealed some beneficial effects of anti-diabetic treatments. Thus, the use of the insulin sensitizer rosiglitazone, an activator of peroxisome-proliferator-activated receptor gamma (PPARγ) receptor, resulted in the rescue of behavioral deficits and insulin respon‐ siveness in Tg2576 mice [148, 149]. Similarly, exendin-4, an antidiabetic agent that stimulates the insulin signaling pathway through activation of glucagon-like peptide -1 (GLP1) receptors, shows beneficial effects in AD, and reduces brain soluble Aβ levels, amyloid plaque burden, and cognitive impairment in treated APP/PS1 transgenic mice [150, 151]. Therefore, it seems that the positive effects of targeting insulin signaling in AD are related to the role played by insulin receptor in memory formation, inflammation and Aβ neuro‐ protective effects rather than to the facilitation of glucose transport into the brain [149, 150]. This hypothesis seems also to be supported by a recent study revealing that insulin did not ameliorate the disruption of energetic homeostasis induced by Aβ oligomers in cultured neurons [152]. In the end, clinical trials designed to test whether PPARγ agonists could be

**2.5. Energetic failure: Metabolic deficiency and mitochondrial impairment**

amelioration of symptoms and in the progression of AD [139].

the prevention of progression to disease states [1, 144-146].

olism and shifting towards the use of alternative fuels.

beneficial in AD patients provided negative results [153].

to young Tg2576 mice [138].

AD transgenic mice models [140].

*2.5.1. Metabolic deficiency*

**•** *Coenzyme Q10(CoQ10)* is a small electron-carrier of the respiratory chain with anti-oxidant properties due to its role in carrying high-energy electrons from complex I to complex II during oxidative phosphorylation. CoQ10 and its analogues, idebenone and mitoquinone (or MitoQ), have been widely used for the treatment of mitochondrial disorders, as well as for the treatment of Friedreich's ataxia, and they are also being tested in other neurodege‐ nerative disorders such as amyotrophic lateral sclerosis, and Huntington's, Parkinson's and Alzheimer's diseases [124]. CoQ10 reduces oxidative stress damage and Aβ plaque burden, and ameliorates behavioral performance in mouse models of AD [125, 126]. However, CoQ10 presents two major weaknesses. First, the function of the enzyme is entirely depend‐ ent on the electron transport chain (ETC) which is usually damaged in AD mitochondria. Second, CoQ10 does not efficiently cross the BBB when administered systemically, being unable to directly protect neurons from damage. Consequently, CoQ10 derivatives such as MitoQ, which is a more soluble compound able to penetrate the BBB and that does not depend on ETC, are seen to offer more promising results [127].

#### **2.4. Inflammation**

There is a general consensus that neuroinflammation is a prominent feature in AD with activated microglia being one of the main manifestations. Neuroinflammation is a complex process that has both beneficial effects, in terms of maintaining brain homeostasis after various kinds of insults, and detrimental effects when sustained chronically [128]. This latter situation is what occurs in AD, in which neuroinflammation is driven by different mechanisms includ‐ ing Aβ production and plaque formation, tau pathology, oxidative stress, and autocrine and paracrine release of cytokines and other inflammatory molecules which contribute to a feedforward spiral favoring the self-propagation of neuroinflammation.

Early epidemiological studies suggesting that long-term use of antiinflammatories might reduce the risk for developing AD [129] prompted several studies designed to evaluate the preventive properties of non-steroid anti-inflammatory drugs (NSAIDs). The main NSAID mechanism of action is to inhibit the activity of cyclooxigenase-1 and -2 (COX-1 and COX-2) which are the enzymes responsible of the production of prostaglandins and other inflamma‐ tory agents [130]. The administration of the NSAID ibuprofen at early stages of the pathological process resulted in the reduction of the Aβ burden, dystrophic neurites and activated microglia in at least three different AD transgenic models [131-134]. Another study indicated that ibuprofen was effective even in older mice once lesions are well established [135]. Other NSAIDs such as indomethacin and nimuselide exhibit milder effects compared to ibuprofen in the Tg2576 mice [136, 137]. In contrast, the selective COX-2 inhibitor celecoxib failed to reduce the inflammatory burden and, even worse, increased the Aβ<sup>42</sup> levels when administered to young Tg2576 mice [138].

In spite of the promising results in animal models and the data from retrospective human epidemiological studies identifying long-term use of NSAIDs as being protective against AD, prospective clinical trials have not confirmed the efficiency of this group of drugs in the amelioration of symptoms and in the progression of AD [139].

Other anti-inflammatory agents such as trifusal have been shown to be beneficial in certain AD transgenic mice models [140].

#### **2.5. Energetic failure: Metabolic deficiency and mitochondrial impairment**

Several findings indicate that brain glucose hypometabolism, deficient bioenergetics and mitochondrial dysfunction precede clinical symptoms in AD [1, 141-143]. The energetic failure observed even in the prodromal phase of the Alzheimer process is thought to be produced by the combination of mitochondria dysfunction, alteration of energy metabolism at poremitochondrial level, and increase in energetic demands of altered nerve cells. Thus, strategies to improve brain energy supply and to preserve mitochondrial functions becomes relevant in the prevention of progression to disease states [1, 144-146].

#### *2.5.1. Metabolic deficiency*

**•** *N-acetyl-cysteine (NAC)* is a precursor of glutathione (GSH), the most abundant endogenous anti-oxidant. NAC acts itself as an anti-oxidant by directly interacting with free radicals, as well as by increasing GSH levels. NAC protects against Aβ-induced cognitive deficits by decreasing the associated oxidative stress and related neuroinflammation, but also by activating anti-apoptotic signaling pathways in neuronal cultures [122]. Late-stage AD patients supplemented with NAC over a period of six months showed significantly improved performance in some cognitive tasks, although levels of oxidative stress in

**•** *Coenzyme Q10(CoQ10)* is a small electron-carrier of the respiratory chain with anti-oxidant properties due to its role in carrying high-energy electrons from complex I to complex II during oxidative phosphorylation. CoQ10 and its analogues, idebenone and mitoquinone (or MitoQ), have been widely used for the treatment of mitochondrial disorders, as well as for the treatment of Friedreich's ataxia, and they are also being tested in other neurodege‐ nerative disorders such as amyotrophic lateral sclerosis, and Huntington's, Parkinson's and Alzheimer's diseases [124]. CoQ10 reduces oxidative stress damage and Aβ plaque burden, and ameliorates behavioral performance in mouse models of AD [125, 126]. However, CoQ10 presents two major weaknesses. First, the function of the enzyme is entirely depend‐ ent on the electron transport chain (ETC) which is usually damaged in AD mitochondria. Second, CoQ10 does not efficiently cross the BBB when administered systemically, being unable to directly protect neurons from damage. Consequently, CoQ10 derivatives such as MitoQ, which is a more soluble compound able to penetrate the BBB and that does not

There is a general consensus that neuroinflammation is a prominent feature in AD with activated microglia being one of the main manifestations. Neuroinflammation is a complex process that has both beneficial effects, in terms of maintaining brain homeostasis after various kinds of insults, and detrimental effects when sustained chronically [128]. This latter situation is what occurs in AD, in which neuroinflammation is driven by different mechanisms includ‐ ing Aβ production and plaque formation, tau pathology, oxidative stress, and autocrine and paracrine release of cytokines and other inflammatory molecules which contribute to a feed-

Early epidemiological studies suggesting that long-term use of antiinflammatories might reduce the risk for developing AD [129] prompted several studies designed to evaluate the preventive properties of non-steroid anti-inflammatory drugs (NSAIDs). The main NSAID mechanism of action is to inhibit the activity of cyclooxigenase-1 and -2 (COX-1 and COX-2) which are the enzymes responsible of the production of prostaglandins and other inflamma‐ tory agents [130]. The administration of the NSAID ibuprofen at early stages of the pathological process resulted in the reduction of the Aβ burden, dystrophic neurites and activated microglia in at least three different AD transgenic models [131-134]. Another study indicated that ibuprofen was effective even in older mice once lesions are well established [135]. Other NSAIDs such as indomethacin and nimuselide exhibit milder effects compared to ibuprofen

peripheral blood did not differ significantly from untreated patients [123].

depend on ETC, are seen to offer more promising results [127].

forward spiral favoring the self-propagation of neuroinflammation.

**2.4. Inflammation**

262 Understanding Alzheimer's Disease

The primary fuel for the brain under normal conditions is glucose, whereas the energetic contribution made by fatty acids is minor. Therefore, facilitation of energy metabolism and energy availability has been assayed in animal models and AD by facilitating glucose metab‐ olism and shifting towards the use of alternative fuels.

**•** *Targeting reduced glucose metabolism*: Reduction in the utilization of glucose in AD [147] can be due to several causes including deficient insulin signaling, impairment in glucose transport mechanisms and dysfunction in glucolysis. Preclinical studies in animal models of AD have revealed some beneficial effects of anti-diabetic treatments. Thus, the use of the insulin sensitizer rosiglitazone, an activator of peroxisome-proliferator-activated receptor gamma (PPARγ) receptor, resulted in the rescue of behavioral deficits and insulin respon‐ siveness in Tg2576 mice [148, 149]. Similarly, exendin-4, an antidiabetic agent that stimulates the insulin signaling pathway through activation of glucagon-like peptide -1 (GLP1) receptors, shows beneficial effects in AD, and reduces brain soluble Aβ levels, amyloid plaque burden, and cognitive impairment in treated APP/PS1 transgenic mice [150, 151]. Therefore, it seems that the positive effects of targeting insulin signaling in AD are related to the role played by insulin receptor in memory formation, inflammation and Aβ neuro‐ protective effects rather than to the facilitation of glucose transport into the brain [149, 150]. This hypothesis seems also to be supported by a recent study revealing that insulin did not ameliorate the disruption of energetic homeostasis induced by Aβ oligomers in cultured neurons [152]. In the end, clinical trials designed to test whether PPARγ agonists could be beneficial in AD patients provided negative results [153].

**•** *Shift to alternative energy source*: Under metabolically challenging conditions neurons can utilize acetyl-CoA generated from ketone body metabolism, produced distally in the liver or locally in the brain by glial cells. In this way, ketone bodies can bypass defects in glucose metabolism and enter the tricarboxylic acid cycle in the mitochondria of neurons as a source of ATP. The use of ketogenic diets reduces Aβ40 and Aβ42 levels in young AD transgenic mice [154] and enhances mitochondrial bioenergetic capacity, reducing Aβ generation and increasing mechanisms of Aβ clearance in a mouse model of AD [155]. The ketogenic compound AC-1202 administered in patients with AD has shown a significant improvement in some cognitive parameters more notable in individuals APOE4(-) [156]. Another possible alternative source of ATP is creatine. Preliminary studies have shown that creatine has protective effects against Aβ *in vitro* [157] and against injury *in vivo* by maintaining ATP levels and mitochondrial function [158], suggesting a potential therapeutic effect of creatine supplementation in AD.

Finally, another potential drug in the treatment of AD that acts on mitochondrial path‐ ways is latrepirdine, also known as Dimebon™ [165]. Latrepirdine reduces Aβ-induced mitochondrial impairment and increases the threshold of inductors to mitochondrial pore transition, making mitochondria more resistant to lipid peroxidation and increasing neu‐ ronal survival *in vitro* [166-168]. The interest in developing latrepirdine as a drug against AD is also supported by its multiple potential mechanism of action apart from mitochon‐ drial effects, including anti-excitotoxic agent, inhibitor of AChE, channel-regulatior and neurotrophic stimulator [165]. A preliminary clinical trial revealed that latrepirdine was safe and well tolerated, and significantly improved the clinical course of the disease in patients with mild-to-moderate AD [169]. Current phase III clinical trials are already be‐

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The alteration of several transmitter systems is assumed to trigger both cognitive and neuro‐ psychiatric symptoms in AD. A number of *post-mortem* studies indicate that neurotransmitter systems are not uniformly affected in AD. Thus, while cholinergic, serotonergic and glutama‐ tergic deficits are present at relatively early stages of AD, dopaminergic and GABAergic

A large body of evidence has shown that basal forebrain cholinergic neurons are vulnerable to AD leading to a progressive cholinergic denervation of the cerebral neocortex [171, 172]. Taking into account the involvement of this system in the cognitive processing of memory and attention, the current attempts in cholinergic therapy in AD are justified [172, 173]. The various cholinergic strategies include the use of ACh precursors, inhibitors of cholinesterases, mus‐ carinic and nicotinic agonists, and ACh releasers, in addition to the rescue of cholinergic

**•** *ACh precursor.* Animal studies report that choline and lecithin increased the production of brain ACh which argues for their use in the treatment of cholinergic deficits in AD. However,

**•** *Cholinesterase inhibitors (ChEIs).* Physostigmine, tacrine and derivatives donepezil, galanta‐ mine and rivastigmine have been tested in AD patients during the last three decades. Their therapeutic properties have been profusely reviewed [172, 175-177] and for this reason a detailed revision of ChEIs is beyond the scope of this chapter. Nevertheless, it is worth briefly indicating additional mechanisms of action of these compounds beyond inhibition of cholinesterases, including increase of nicotinc ACh receptor expression, facilitation of APP processing and attenuation of Aβ-induced toxicity [173, 178]. In spite of the fact that their efficacy has been proved in several clinical trials, only approximately 50% of patients respond positively. This limited effect of ChEIs on cognitive decline, together with the occurrence of undesirable side-effects such as diarrhea, nausea, insomnia, fatigue and loss

function by nerve growth factor (NGF) which is reviewed in section 2.8.

evidence from randomized trials did not sustain this hypothesis [174].

of appetite, reduces the therapeutic capacities of ChEIs.

ing conducted [165].

*2.6.1. Cholinergic system*

**2.6. Neurotransmitter dysfunction**

systems appear to be affected later [170].

#### *2.5.2. Mitochondrial dysfunction*

In addition to the already discussed antioxidant compounds, other potential drugs targeting mitochondrial dysfunction in AD are available. Several findings point towards a role for Aβ toxicity in the mitochondrial dysfunction found in AD.

The progressive Aβ accumulation in mitochondria is associated with diminished enzy‐ matic activity of respiratory chain complexes (III and IV) and reduction in the rate of oxygen consumption, contributing to cellular dysfunction in AD [159]. Aβ in mitochon‐ dria binds to Aβ-binding alcohol dehydrogenase (ABAD) to block ABAD activity, in‐ creasing the production of ROS, reducing the mitochondrial membrane potential and the activity of the respiratory chain complex IV, and ultimately leading to a decrease in ATP levels [160]. In fact, double transgenic mice over-expressing mutated APP and ABAD ex‐ hibit exaggerated oxidative stress and memory impairment [160]. Therefore, compounds designed to block Aβ-ABAD interactions are considered putative therapeutic agents in AD. In line with this hypothesis, a recent study has shown that AG18051, a novel small ABAD-specific compound inhibitor, partially blocked the Aβ-ABAD interaction, prevent‐ ed the Aβ42-induced down-regulation of ABAD activity and protected cultured neurons against Aβ42 toxicity by reducing Aβ42-induced impairment of mitochondrial function and oxidative stress [161]. Furthermore, the introduction of an ABAD-decoy peptide into transgenic APP mice reduces Aβ-ABAD interaction and protects against Aβ-mediated mitochondrial toxicity [162].

Another line of research suggests that drugs that activate ATP-sensitive potassium (K*ATP*) channels present in the mitochondrial inner membrane exhibit therapeutic potential in the treatment of AD, as K*ATP* channels are activated when cellular ATP levels fall below a critical value thereby reducing excitability so as to maintain ion homeostasis and pre‐ serve ATP levels [163]. Long-term administration of diazoxide improves neuronal bioen‐ ergetics, suppresses Aβ and tau pathologies, and ameliorates memory deficits in the 3xTgAD mouse model of AD [164].

Finally, another potential drug in the treatment of AD that acts on mitochondrial path‐ ways is latrepirdine, also known as Dimebon™ [165]. Latrepirdine reduces Aβ-induced mitochondrial impairment and increases the threshold of inductors to mitochondrial pore transition, making mitochondria more resistant to lipid peroxidation and increasing neu‐ ronal survival *in vitro* [166-168]. The interest in developing latrepirdine as a drug against AD is also supported by its multiple potential mechanism of action apart from mitochon‐ drial effects, including anti-excitotoxic agent, inhibitor of AChE, channel-regulatior and neurotrophic stimulator [165]. A preliminary clinical trial revealed that latrepirdine was safe and well tolerated, and significantly improved the clinical course of the disease in patients with mild-to-moderate AD [169]. Current phase III clinical trials are already be‐ ing conducted [165].

#### **2.6. Neurotransmitter dysfunction**

The alteration of several transmitter systems is assumed to trigger both cognitive and neuro‐ psychiatric symptoms in AD. A number of *post-mortem* studies indicate that neurotransmitter systems are not uniformly affected in AD. Thus, while cholinergic, serotonergic and glutama‐ tergic deficits are present at relatively early stages of AD, dopaminergic and GABAergic systems appear to be affected later [170].

#### *2.6.1. Cholinergic system*

**•** *Shift to alternative energy source*: Under metabolically challenging conditions neurons can utilize acetyl-CoA generated from ketone body metabolism, produced distally in the liver or locally in the brain by glial cells. In this way, ketone bodies can bypass defects in glucose metabolism and enter the tricarboxylic acid cycle in the mitochondria of neurons as a source of ATP. The use of ketogenic diets reduces Aβ40 and Aβ42 levels in young AD transgenic mice [154] and enhances mitochondrial bioenergetic capacity, reducing Aβ generation and increasing mechanisms of Aβ clearance in a mouse model of AD [155]. The ketogenic compound AC-1202 administered in patients with AD has shown a significant improvement in some cognitive parameters more notable in individuals APOE4(-) [156]. Another possible alternative source of ATP is creatine. Preliminary studies have shown that creatine has protective effects against Aβ *in vitro* [157] and against injury *in vivo* by maintaining ATP levels and mitochondrial function [158], suggesting a potential therapeutic effect of creatine

In addition to the already discussed antioxidant compounds, other potential drugs targeting mitochondrial dysfunction in AD are available. Several findings point towards a role for Aβ

The progressive Aβ accumulation in mitochondria is associated with diminished enzy‐ matic activity of respiratory chain complexes (III and IV) and reduction in the rate of oxygen consumption, contributing to cellular dysfunction in AD [159]. Aβ in mitochon‐ dria binds to Aβ-binding alcohol dehydrogenase (ABAD) to block ABAD activity, in‐ creasing the production of ROS, reducing the mitochondrial membrane potential and the activity of the respiratory chain complex IV, and ultimately leading to a decrease in ATP levels [160]. In fact, double transgenic mice over-expressing mutated APP and ABAD ex‐ hibit exaggerated oxidative stress and memory impairment [160]. Therefore, compounds designed to block Aβ-ABAD interactions are considered putative therapeutic agents in AD. In line with this hypothesis, a recent study has shown that AG18051, a novel small ABAD-specific compound inhibitor, partially blocked the Aβ-ABAD interaction, prevent‐ ed the Aβ42-induced down-regulation of ABAD activity and protected cultured neurons against Aβ42 toxicity by reducing Aβ42-induced impairment of mitochondrial function and oxidative stress [161]. Furthermore, the introduction of an ABAD-decoy peptide into transgenic APP mice reduces Aβ-ABAD interaction and protects against Aβ-mediated

Another line of research suggests that drugs that activate ATP-sensitive potassium (K*ATP*) channels present in the mitochondrial inner membrane exhibit therapeutic potential in the treatment of AD, as K*ATP* channels are activated when cellular ATP levels fall below a critical value thereby reducing excitability so as to maintain ion homeostasis and pre‐ serve ATP levels [163]. Long-term administration of diazoxide improves neuronal bioen‐ ergetics, suppresses Aβ and tau pathologies, and ameliorates memory deficits in the

supplementation in AD.

264 Understanding Alzheimer's Disease

*2.5.2. Mitochondrial dysfunction*

mitochondrial toxicity [162].

3xTgAD mouse model of AD [164].

toxicity in the mitochondrial dysfunction found in AD.

A large body of evidence has shown that basal forebrain cholinergic neurons are vulnerable to AD leading to a progressive cholinergic denervation of the cerebral neocortex [171, 172]. Taking into account the involvement of this system in the cognitive processing of memory and attention, the current attempts in cholinergic therapy in AD are justified [172, 173]. The various cholinergic strategies include the use of ACh precursors, inhibitors of cholinesterases, mus‐ carinic and nicotinic agonists, and ACh releasers, in addition to the rescue of cholinergic function by nerve growth factor (NGF) which is reviewed in section 2.8.


**•** *Muscarinic receptor 1 agonist.* The cholinergic deficiency in AD appears to be mainly presynaptic. Thus, the pharmacological stimulation of the post-synaptic M1 muscarinic receptors, which are preserved until late stages of AD, may balance the degeneration of presynaptic cholinergic terminals unable to properly synthesize and release ACh [173]. In fact, the selective M1 agonist AF267B reduces memory impairment, Aβ42 levels, and tau hyperphosphorylation in AD triple transgenic mice [179], corroborating some early studies *in vitro* [180, 181]. This selective agonist is currently under clinical evaluation for safety and tolerability and a number of other M1 agonists are being investigated [173].

functional synaptic AMPA receptors reduces fast excitatory transmission and eventually triggers spine retraction and synaptic loss [198]. Moreover, glutamate receptors are not only involved in the process of Aβ-mediated synaptic dysfunction but also play important roles in

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Based on these observations, several studies have been designed in an attempt to correct glutamatergic dysfunction in AD, including the modulation of both AMPA and NMDA receptors [201]. First attempts were carried out with AMPAKines [202], which are drugs that prolong the action of glutamate on AMPA receptors by increasing their sensitivity. Interestingly, AMPAKines proved effective in restoring cognitive deficits in aging rats [203, 204]. These compounds were tested in AD patients [205]. The modulation of the NMDA receptor was assessed via the glycine co-agonist site in rats with disrupted gluta‐ matergic temporal systems resulting in improved learning and memory [206]. Prelimina‐ ry clinical studies suggested some promising effects in AD [207] but full-scale trials have

The most relevant glutamatergic strategy against AD is the non-competitive NMDA antagonist memantine [201, 208], which has succeeded in clinical trials in moderate and severe AD as reviewed in detail elsewhere [209, 210]. Several studies performed in animal models of AD corroborate the beneficial properties of memantine as a symptomatological and neuroprotec‐ tive treatment in AD [211-215]. Nevertheless, memantine has no benefits in cases with mild AD [216] suggesting that this drug is not a good choice for preventing the progression to

Loss of serotonergic nerve terminals in AD was described several years ago [217, 218]. Although the suggested serotonergic dysfunction was initially related almost exclusively with the neuropsychiatric symptoms of AD, including anxiety, irritability, fear and depression, recent studies have demonstrated that serotonin signaling also plays an important role in

Antidepressant compounds, acting through serotonin signaling, result in cognitive im‐ provements and reduce the levels of Aβ and tau pathology in animal models of AD [220, 221]. Similar compounds reduce amyloid burden in humans [221]. Additional sero‐ tonergic compounds that are currently being investigated in AD are 5-hydroxytrypta‐ mine (5-HT or serotonin) receptors: 5-HT1 and 5-HT6 antagonists, and 5-HT4 agonists. The 5-HT1A antagonist lecozotan (SRA-333) enhances cognition in primates and is now being tested in AD [222-224]. The pro-cognitive effects of 5-HT1A antagonists are proba‐ bly due to the facilitation of glutamategic and cholinergic transmission after reduction of the inhibitory effects of serotonin. Similarly, 5-HT6 antagonists improve cognitive per‐ formance in animal models and human beings by modulating multiple neurotransmitter systems [225]. These properties mark 5-HT6 antagonists as potential symptomatic drugs in AD. In addition, 5-HT4 receptor agonists are neuroprotective, modulating the produc‐

tion of Aβ, and have the property of ameliorating cognitive deficits [226, 227].

cognition and in the development of Aβ and tau pathologies [219].

Aβ production [199, 200].

not yet been initiated.

disease states.

*2.6.3. Serotonergic system*


#### *2.6.2. Glutamatergic system*

Low concentrations of Aβ oligomers are able to activate certain glutamate receptors including NMDA receptors. The activation of NMDA receptors may increase glutamate activity, raise intracellular Ca2+ concentration and promote excitotoxicity and neuronal damage [194, 195]. Another process contributing to the excessive glutamate activity in AD is the impairment of glial cells to remove glutamate form the synaptic cleft possibly due to the action of free radicals on the glutamate transporter 1 (GLT-1) [196]. Glutamatergic activation, in turn, may disrupt synaptic plasticity promoting long term depression (LTD) and inhibiting long term potentia‐ tion (LTP) of 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid (AMPA) receptormediated synaptic transmission [197]. The associated persistent reduction in the number of functional synaptic AMPA receptors reduces fast excitatory transmission and eventually triggers spine retraction and synaptic loss [198]. Moreover, glutamate receptors are not only involved in the process of Aβ-mediated synaptic dysfunction but also play important roles in Aβ production [199, 200].

Based on these observations, several studies have been designed in an attempt to correct glutamatergic dysfunction in AD, including the modulation of both AMPA and NMDA receptors [201]. First attempts were carried out with AMPAKines [202], which are drugs that prolong the action of glutamate on AMPA receptors by increasing their sensitivity. Interestingly, AMPAKines proved effective in restoring cognitive deficits in aging rats [203, 204]. These compounds were tested in AD patients [205]. The modulation of the NMDA receptor was assessed via the glycine co-agonist site in rats with disrupted gluta‐ matergic temporal systems resulting in improved learning and memory [206]. Prelimina‐ ry clinical studies suggested some promising effects in AD [207] but full-scale trials have not yet been initiated.

The most relevant glutamatergic strategy against AD is the non-competitive NMDA antagonist memantine [201, 208], which has succeeded in clinical trials in moderate and severe AD as reviewed in detail elsewhere [209, 210]. Several studies performed in animal models of AD corroborate the beneficial properties of memantine as a symptomatological and neuroprotec‐ tive treatment in AD [211-215]. Nevertheless, memantine has no benefits in cases with mild AD [216] suggesting that this drug is not a good choice for preventing the progression to disease states.

#### *2.6.3. Serotonergic system*

**•** *Muscarinic receptor 1 agonist.* The cholinergic deficiency in AD appears to be mainly presynaptic. Thus, the pharmacological stimulation of the post-synaptic M1 muscarinic receptors, which are preserved until late stages of AD, may balance the degeneration of presynaptic cholinergic terminals unable to properly synthesize and release ACh [173]. In fact, the selective M1 agonist AF267B reduces memory impairment, Aβ42 levels, and tau hyperphosphorylation in AD triple transgenic mice [179], corroborating some early studies *in vitro* [180, 181]. This selective agonist is currently under clinical evaluation for safety and

**•** *Nicotinic agonists.* Preclinical studies in animal models and some pilot studies in AD have shown that the activation of pre-synaptic nicotinic ACh receptors may reduce cognitive impairment by increasing ACh release and may have beneficial effects on Aβ metabolism [182, 183]. Thus, chronic nicotine treatment results in a significant reduction in plaque burden and in cortical Aβ concentrations in Tg2575/PS1-A246E mice [184]. However, nicotine exacerbates tau pathology in 3xTg-AD mice [185]. These apparently contradictory results may be due to the presence of several subtypes of nicotinic receptors, the activation of which may have disparate effects in AD. Therefore, more specific nicotine agonists are needed to act exclusively on determinate subtypes of nicotinic receptor [186]. In this line, α7 nAChR gene delivery into mouse hippocampal neurons leads to functional receptor expression and improves spatial memory-related performance and hyperphosphorylation of tau [187]. Regarding α4β2 nicotinic receptor, the selective agonist cytisine inhibits Aβ

**•** *ACh releasers.* Facilitation of ACh release can be achieved with depolarizing agents of the cholinergic neurons acting via potassium-channel blockade as happens with linopirdine and analogues [189] or by the blockade of the pre-synaptic inhibitory M2 muscarinic receptor via specific antagonists [190, 191]. However, clinical trials using linopirdine did not demonstrate effectiveness in improving cognitive function [192]. On the other hand, certain selective M2 antagonists, such as SCH-57790 and SC-72788, restore memory impairments in animal models that mimic to some extent the cholinergic failure in AD [193]. It must be kept in mind that the potential benefit of M2 antagonists is limited because of the progressive pre-synaptic cholinergic degeneration in AD and because of the possible side-effects derived

from the blockade of peripheral M2 receptors including cardiac M2 receptors.

Low concentrations of Aβ oligomers are able to activate certain glutamate receptors including NMDA receptors. The activation of NMDA receptors may increase glutamate activity, raise intracellular Ca2+ concentration and promote excitotoxicity and neuronal damage [194, 195]. Another process contributing to the excessive glutamate activity in AD is the impairment of glial cells to remove glutamate form the synaptic cleft possibly due to the action of free radicals on the glutamate transporter 1 (GLT-1) [196]. Glutamatergic activation, in turn, may disrupt synaptic plasticity promoting long term depression (LTD) and inhibiting long term potentia‐ tion (LTP) of 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid (AMPA) receptormediated synaptic transmission [197]. The associated persistent reduction in the number of

tolerability and a number of other M1 agonists are being investigated [173].

cytotoxicity in cortical neurons [188].

*2.6.2. Glutamatergic system*

266 Understanding Alzheimer's Disease

Loss of serotonergic nerve terminals in AD was described several years ago [217, 218]. Although the suggested serotonergic dysfunction was initially related almost exclusively with the neuropsychiatric symptoms of AD, including anxiety, irritability, fear and depression, recent studies have demonstrated that serotonin signaling also plays an important role in cognition and in the development of Aβ and tau pathologies [219].

Antidepressant compounds, acting through serotonin signaling, result in cognitive im‐ provements and reduce the levels of Aβ and tau pathology in animal models of AD [220, 221]. Similar compounds reduce amyloid burden in humans [221]. Additional sero‐ tonergic compounds that are currently being investigated in AD are 5-hydroxytrypta‐ mine (5-HT or serotonin) receptors: 5-HT1 and 5-HT6 antagonists, and 5-HT4 agonists. The 5-HT1A antagonist lecozotan (SRA-333) enhances cognition in primates and is now being tested in AD [222-224]. The pro-cognitive effects of 5-HT1A antagonists are proba‐ bly due to the facilitation of glutamategic and cholinergic transmission after reduction of the inhibitory effects of serotonin. Similarly, 5-HT6 antagonists improve cognitive per‐ formance in animal models and human beings by modulating multiple neurotransmitter systems [225]. These properties mark 5-HT6 antagonists as potential symptomatic drugs in AD. In addition, 5-HT4 receptor agonists are neuroprotective, modulating the produc‐ tion of Aβ, and have the property of ameliorating cognitive deficits [226, 227].

#### **2.7. Synaptic dysfunction**

Synaptic dysfunction and failure are processes that occur early in the Alzheimer process and progress during the course of the disease from an initially reversible functionally-re‐ sponsive stage of down-regulated synaptic function to stages irreversibly associated with degeneration.

proteins, phosphatases and kinases, thereby facilitating signal-transduction cascades. Evi‐ dence from *in vitro* cell and animal models of AD indicates that reductions in the post-synaptic density membrane-associated guanylate kinase (PSD-MAGUK) proteins are linked to synaptic dysfunction that might trigger plastic changes at early stages of the Alzheimer process [240]. However, specific molecules that affect interactions between scaffolding proteins and neuro‐ transmitter receptors are still in development and further research is necessary to evaluate

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269

Neurotrophins represent a family of proteins that play a pivotal role in the mechanisms underlying neuronal survival, differentiation, modulation of dendritic branching and den‐ dritic spine morphology as well as synaptic plasticity and apoptosis [241]. All the members of the neurotrophin family, including NGF, brain-derived neurotrophic factor (BDNF) and neurotrophins 3 to 7, transduce their biological effects by interacting with two types of cell surface receptors, the tyrosine kinase receptor (Trk) and the p75 pan-neurotrophin receptor (p75NTR) [241]. Other growth factor families also related to synaptic plasticity include the cytokine family of growth factors, the transforming growth factor-β (TGFβ) family, the fibroblast growth factor family and the insulin-like growth factor family. Evidence accumu‐ lated during recent years suggests that targeting neurotrophic factor signaling can retard nerve cell degeneration and to some extent preserve synaptic function. The most studied neurotro‐

**•** *NGF*: Mature basal forebrain cholinergic neurons are highly dependent on the availability of NGF for the maintenance of their biochemical and morphological phenotype, and for survival after lesions or variegated insults [242, 243]. For this reason, exploitation of NGF activity on cholinergic neurons may provide an attractive therapeutic option for preventing cholinergic cell degeneration in AD. Levels of proNGF, the precursor form of NGF, are highly elevated in AD brains and animal models, a feature that may be associated with a reduced conversion to NGF and augmented degradation of mature NGF. These combined effects have been interpreted as causative of cholinergic atrophy in AD [244]. A role for Aβ peptide in the induction of such NGF altered metabolism has been described [245]. Minocycline, a second-generation tetracycline antibiotic known to potentiate NGF activity, is able to normalize proNGF levels and to reverse the increased activity of the NGFdegrading enzyme matrix metalloproteinase 9, as well as to increase the expression of iNOS and microglial activation, leading to improved cognitive behavior in a transgenic mouse model of AD [245]. Yet a disturbing finding is the demonstration of AD proNGF when compared to proNGF of control individuals [246-248]. Whether this abnormal form of ADrelated proNGF has any impact on the pathogenesis of AD needs further investigation. Another putative therapy is the use NGF, but NGF does not readily cross the BBB and requires intra-cerebroventricular infusion to reach targeted brain areas. Pilot clinical trials were discontinued because of the side-effects of NGF infusions [249]. Therefore, the development of NGF therapy is constrained by the need to achieve adequate concentrations in the relevant brain areas with susceptible target neurons while preventing unwanted

their potential benefit in AD.

phic factors in AD are NGF, BDNF and TGFβ1.

**2.8. Neurotrophic factors**

These alterations are manifested early as impaired metabotropic glutamate receptor/phos‐ pholipase C signaling pathway [230] and up-regulation of adenosine receptors in the frontal cortex in AD [231].

The initial reversible stages are important targets for protective treatments to slow progression and preserve cognitive and functional abilities [232, 233]. *In vivo* and *in vitro* studies have demonstrated that high levels of Aβ impair structural and functional plasticity of synapses by affecting the balance between excitation and inhibition and contributing to the destabilization of neuronal networks, eventually causing synaptic loss [234]. Two main designs have been proposed to antagonize synaptic plasticity-disrupting actions of Aβ oligomers in preclinical AD: maintenance of the structure and fluidity of the lipid membranes forming the synaptic buttons, and stimulation of synaptic plasticity by neurotrophic factors.

Minor changes in the fluidity of phospholipidic membranes might have an important impact on the function of synapses by influencing neurotransmitter receptor activity. In fact, AD brains exhibit altered lipid composition of lipid rafts, key membrane microdomains that facilitate the transfer of substrates and protein-protein and lipid-protein interactions, as a result of the abnormally low levels of n-3 long-chain polyunsaturated fatty acids, mainly docosa‐ hexaenoic acid (DHA), increasing viscosity and energy consumption and contributing to synaptic dysfunction [142, 235]. Abnormal lipid raft composition may also modify the activity of key enzymes that modulate the cleavage of APP to form toxic Aβ. Thus, the preservation of adequate membrane composition has become an alternative way to prevent the deleterious effect of Aβ at the synapses. DHA is a major lipid constituent of synaptic end-sites and its delivery is a prerequisite for the conversion of nerve growth cones to mature synapses [236]. Numerous epidemiological studies have highlighted the beneficial influence of DHA on the preservation of synaptic function and memory capacity in aged individuals or after Aβ exposure, whereas DHA deficiency is presented as a risk factor for AD [237]. Moreover, a number of studies have reported the beneficial effects of dietary DHA supplementation on cognition and synaptic integrity in various AD models [238]. According to thes evidence, DHA, which can be synthesized or obtained directly from fish oil, appear to be one of the most valuable diet ingredients whose neuroprotective properties contribute to preventing AD.

Cytidine 5'-diphosphocholine, CDP-choline, or citicoline is an essential intermediate in the biosynthetic pathway of structural phospholipids in cell membranes, particularly phosphati‐ dylcholine. Chronic administration has been beneficial in patients with mild cognitive impairment [239].

Another emerging potential line to preserve synaptic function is the targeting of scaffolding proteins that modulate neurotransmitter receptor activity at the synapses. Scaffolding proteins stabilize post-synaptic receptors at the spines in close proximity to their intracellular signaling proteins, phosphatases and kinases, thereby facilitating signal-transduction cascades. Evi‐ dence from *in vitro* cell and animal models of AD indicates that reductions in the post-synaptic density membrane-associated guanylate kinase (PSD-MAGUK) proteins are linked to synaptic dysfunction that might trigger plastic changes at early stages of the Alzheimer process [240]. However, specific molecules that affect interactions between scaffolding proteins and neuro‐ transmitter receptors are still in development and further research is necessary to evaluate their potential benefit in AD.

#### **2.8. Neurotrophic factors**

**2.7. Synaptic dysfunction**

268 Understanding Alzheimer's Disease

degeneration.

cortex in AD [231].

impairment [239].

Synaptic dysfunction and failure are processes that occur early in the Alzheimer process and progress during the course of the disease from an initially reversible functionally-re‐ sponsive stage of down-regulated synaptic function to stages irreversibly associated with

These alterations are manifested early as impaired metabotropic glutamate receptor/phos‐ pholipase C signaling pathway [230] and up-regulation of adenosine receptors in the frontal

The initial reversible stages are important targets for protective treatments to slow progression and preserve cognitive and functional abilities [232, 233]. *In vivo* and *in vitro* studies have demonstrated that high levels of Aβ impair structural and functional plasticity of synapses by affecting the balance between excitation and inhibition and contributing to the destabilization of neuronal networks, eventually causing synaptic loss [234]. Two main designs have been proposed to antagonize synaptic plasticity-disrupting actions of Aβ oligomers in preclinical AD: maintenance of the structure and fluidity of the lipid membranes forming the synaptic

Minor changes in the fluidity of phospholipidic membranes might have an important impact on the function of synapses by influencing neurotransmitter receptor activity. In fact, AD brains exhibit altered lipid composition of lipid rafts, key membrane microdomains that facilitate the transfer of substrates and protein-protein and lipid-protein interactions, as a result of the abnormally low levels of n-3 long-chain polyunsaturated fatty acids, mainly docosa‐ hexaenoic acid (DHA), increasing viscosity and energy consumption and contributing to synaptic dysfunction [142, 235]. Abnormal lipid raft composition may also modify the activity of key enzymes that modulate the cleavage of APP to form toxic Aβ. Thus, the preservation of adequate membrane composition has become an alternative way to prevent the deleterious effect of Aβ at the synapses. DHA is a major lipid constituent of synaptic end-sites and its delivery is a prerequisite for the conversion of nerve growth cones to mature synapses [236]. Numerous epidemiological studies have highlighted the beneficial influence of DHA on the preservation of synaptic function and memory capacity in aged individuals or after Aβ exposure, whereas DHA deficiency is presented as a risk factor for AD [237]. Moreover, a number of studies have reported the beneficial effects of dietary DHA supplementation on cognition and synaptic integrity in various AD models [238]. According to thes evidence, DHA, which can be synthesized or obtained directly from fish oil, appear to be one of the most valuable diet ingredients whose neuroprotective properties contribute to preventing AD.

Cytidine 5'-diphosphocholine, CDP-choline, or citicoline is an essential intermediate in the biosynthetic pathway of structural phospholipids in cell membranes, particularly phosphati‐ dylcholine. Chronic administration has been beneficial in patients with mild cognitive

Another emerging potential line to preserve synaptic function is the targeting of scaffolding proteins that modulate neurotransmitter receptor activity at the synapses. Scaffolding proteins stabilize post-synaptic receptors at the spines in close proximity to their intracellular signaling

buttons, and stimulation of synaptic plasticity by neurotrophic factors.

Neurotrophins represent a family of proteins that play a pivotal role in the mechanisms underlying neuronal survival, differentiation, modulation of dendritic branching and den‐ dritic spine morphology as well as synaptic plasticity and apoptosis [241]. All the members of the neurotrophin family, including NGF, brain-derived neurotrophic factor (BDNF) and neurotrophins 3 to 7, transduce their biological effects by interacting with two types of cell surface receptors, the tyrosine kinase receptor (Trk) and the p75 pan-neurotrophin receptor (p75NTR) [241]. Other growth factor families also related to synaptic plasticity include the cytokine family of growth factors, the transforming growth factor-β (TGFβ) family, the fibroblast growth factor family and the insulin-like growth factor family. Evidence accumu‐ lated during recent years suggests that targeting neurotrophic factor signaling can retard nerve cell degeneration and to some extent preserve synaptic function. The most studied neurotro‐ phic factors in AD are NGF, BDNF and TGFβ1.

**•** *NGF*: Mature basal forebrain cholinergic neurons are highly dependent on the availability of NGF for the maintenance of their biochemical and morphological phenotype, and for survival after lesions or variegated insults [242, 243]. For this reason, exploitation of NGF activity on cholinergic neurons may provide an attractive therapeutic option for preventing cholinergic cell degeneration in AD. Levels of proNGF, the precursor form of NGF, are highly elevated in AD brains and animal models, a feature that may be associated with a reduced conversion to NGF and augmented degradation of mature NGF. These combined effects have been interpreted as causative of cholinergic atrophy in AD [244]. A role for Aβ peptide in the induction of such NGF altered metabolism has been described [245]. Minocycline, a second-generation tetracycline antibiotic known to potentiate NGF activity, is able to normalize proNGF levels and to reverse the increased activity of the NGFdegrading enzyme matrix metalloproteinase 9, as well as to increase the expression of iNOS and microglial activation, leading to improved cognitive behavior in a transgenic mouse model of AD [245]. Yet a disturbing finding is the demonstration of AD proNGF when compared to proNGF of control individuals [246-248]. Whether this abnormal form of ADrelated proNGF has any impact on the pathogenesis of AD needs further investigation. Another putative therapy is the use NGF, but NGF does not readily cross the BBB and requires intra-cerebroventricular infusion to reach targeted brain areas. Pilot clinical trials were discontinued because of the side-effects of NGF infusions [249]. Therefore, the development of NGF therapy is constrained by the need to achieve adequate concentrations in the relevant brain areas with susceptible target neurons while preventing unwanted adverse effects in non-target regions or cells. Alternative strategies that are currently under development include gene therapy and nasal delivery of recombinant forms of NGF, the use of small molecules with NGF agonist activity, NGF synthesis inducers, NGF processing modulators, and proNGF antagonists [250].

**2.9. Autophagy**

**2.10. Multi-target treatments**

Autophagy is a catabolic process occurring in all cell types in which the machinery of the lysosome degrades cellular components such as long-lived or damaged proteins and organ‐ elles. Thus, a failure of autophagy in neurons results in the accumulation of aggregate-prone proteins that might exacerbate neurodegenerative process [271, 272]. Autophagy is also implicated in the accumulation of altered mitochondria and polymorphous inclusions in the

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Indeed, autophagic dysfunction is implicated in the progression of Alzheimer from the earliest stage, when a defective lysosomal clearance of autophagic substrates and impaired autophagy initiation occurs and leads to massive buildup of incompletely digested substrates within dystrophic axons and dendrites [279]. The pharmacological induction of 'preserved' autoph‐ agy might enhance the clearance of intracytoplasmic aggregate-prone proteins and therefore ameliorate pathology [272]. Attempts to restore more normal lysosomal proteolysis and autophagy efficiency in mouse models of AD pathology have revealed promising therapeutic effects on neuronal function and cognitive performance, demonstrating the relevance of the failure of autophagy in the pathogenesis of AD, and the potential of autophagy modulation as a therapeutic strategy. Autophagy induction with the mTOR-inhibiting drug rapamycin in young mice resulted in a reduction in Aβ plaques, NFT and cognitive deficits in the adulthood in two different models of AD [280-283]. Interestingly, rapamycin did not alter any of those parameters when administered in old animals once the pathology was established, highlight‐ ing the importance of early treatmenting in the disease progression [282]. However, the kinase mTOR plays an important role in multiple signaling pathways apart from negatively regulat‐ ing autophagy [284]. Therefore, rapamycin treatment is also a putative inducer of undesirable side-effects. Other drugs including lithium, sodium valproate and carbamazepine acting have ben proved to induce autophagy through the inhibition of of inositol monophosphatase in an mTOR-independent pathway [285]. These compunds reveal positive effects by reducing the accumulation and toxic effects of aggregation-prone proteins in cell models as well as by protecting against neurodegeneration in *in vivo* models of Huntington's disease [286]. Further research is needed to learn whether they can also be useful tools in the treatment of AD.

Considering the multifactorial etiology of AD, and the numerous and complex pathologi‐ cal mechanisms involved in the progression of the disease, it is quite reasonable that treatments targeting a single causal or modifying factor may have limited benefits. Therefore, growing interest is focused on therapeutic agents with pleiotropic activity, which will be able to target, in parallel, several processes affected in AD [287, 288]. Sev‐ eral compounds already mentioned in the previous sections fulfill these properties, such as DHA which presents anti-inflammatory, anti-oxidant, neuroprotective and anti-tau phosphorylation properties apart from the modulation of synaptic membrane composi‐ tion [289], and curcumin, which in addition to anti-oxidant properties also exhibits antiinflammatory and Aβ- and tau-binding properties [106]. Similarly, rosiglitazone and dimebon are known to produce beneficial effects through insulin receptor signaling mod‐

dystrophic neurites around amyloid plaques [273-278].


A final point must be considered. A generalized sprouting is produced around β-amyloid deposits in senile plaques in both humans and in animal models [268-270]. The reasons for such sprouting are not well defined but amyloid species may play a trigger role. In any case, trophic factors might increase aberrant sprouting at the senile plaques through receptors expressed at these localizations.

#### **2.9. Autophagy**

adverse effects in non-target regions or cells. Alternative strategies that are currently under development include gene therapy and nasal delivery of recombinant forms of NGF, the use of small molecules with NGF agonist activity, NGF synthesis inducers, NGF processing

**•** *BDNF*: This neurotrophin is normally produced in the cerebral cortex with high levels in the entorhinal cortex and hippocampus in adulthood [241]. BDNF levels are reduced in the cerebral cortex and hippocampus in AD [251-254]. Several studies have shown beneficial effects of BDNF in animal models of AD [255]. For instance, sustained BDNF gene delivery using viral vectors after disease onset resulted in elevated BDNF levels in the entorhinal cortex and hippocampus which were associated with improvement in learning and memory, and with restoration of most genes altered as a result of mutant APP expression in that specific transgenic mice model [256]. Similar results were obtained in a different mouse model of AD, and in aged rats and primates by using distinct BDNF delivery systems [256, 257]. It is worth pointing out that BDNF did not change β-amyloid plaque density in any case suggesting that the therapeutic effects of BDNF occur independently of direct action on APP processing. However, the multiple variegated effects of BDNF on neuronal function also raise the hypothetical possibility that unintended adverse effects of BDNF may limit its clinical efficacy in AD [256]. An additional point must be considered; BDNF signaling pathway is also altered in AD as TrkB expression is reduced and truncated TrkB is highly expressed in astrocytes at least in advanced stages of the disease [251]. Therefore, regarding BDNF function in AD, there is not only an alteration in the expression of BDNF but also an impaired downstream pathway that may corrupt the signal of the trophic factor acting on inappropriate receptors. Preliminary clinical trials are currently in progress to

**•** *TGFβ1*: Astrocytes and microglia are the major sources of TGF-β1 in the injured brain [258, 259]. Impaired TGF-β1 signaling has been demonstrated in AD brain, particularly at the early phase of the disease; this is associated with Aβ pathology and neurofibril‐ lary tangle formation in animal models [260]. Reduced TGF-β1 seems to induce microglial activation [259] and ectopic cell-cycle re-activation in neurons [261]. Several drugs may induce TGF-β1 release by glial cells, including estrogens [262], mGlu2/3 agonists [263], lithium [264], the antidepressant venlafaxine [265] and glatiramer, which is a synthetic amino acid co-polymer currently approved for the treatment of multiple sclerosis [266]. All of them have neuroprotective effects in different *in vitro* and *in vivo* models of AD pathology [260]. Additionally, small molecules with specific TGF-β1-like

A final point must be considered. A generalized sprouting is produced around β-amyloid deposits in senile plaques in both humans and in animal models [268-270]. The reasons for such sprouting are not well defined but amyloid species may play a trigger role. In any case, trophic factors might increase aberrant sprouting at the senile plaques through receptors

modulators, and proNGF antagonists [250].

270 Understanding Alzheimer's Disease

evaluate the safety and efficacy of BDNF.

activity are being developed as neuroprotectors [267].

expressed at these localizations.

Autophagy is a catabolic process occurring in all cell types in which the machinery of the lysosome degrades cellular components such as long-lived or damaged proteins and organ‐ elles. Thus, a failure of autophagy in neurons results in the accumulation of aggregate-prone proteins that might exacerbate neurodegenerative process [271, 272]. Autophagy is also implicated in the accumulation of altered mitochondria and polymorphous inclusions in the dystrophic neurites around amyloid plaques [273-278].

Indeed, autophagic dysfunction is implicated in the progression of Alzheimer from the earliest stage, when a defective lysosomal clearance of autophagic substrates and impaired autophagy initiation occurs and leads to massive buildup of incompletely digested substrates within dystrophic axons and dendrites [279]. The pharmacological induction of 'preserved' autoph‐ agy might enhance the clearance of intracytoplasmic aggregate-prone proteins and therefore ameliorate pathology [272]. Attempts to restore more normal lysosomal proteolysis and autophagy efficiency in mouse models of AD pathology have revealed promising therapeutic effects on neuronal function and cognitive performance, demonstrating the relevance of the failure of autophagy in the pathogenesis of AD, and the potential of autophagy modulation as a therapeutic strategy. Autophagy induction with the mTOR-inhibiting drug rapamycin in young mice resulted in a reduction in Aβ plaques, NFT and cognitive deficits in the adulthood in two different models of AD [280-283]. Interestingly, rapamycin did not alter any of those parameters when administered in old animals once the pathology was established, highlight‐ ing the importance of early treatmenting in the disease progression [282]. However, the kinase mTOR plays an important role in multiple signaling pathways apart from negatively regulat‐ ing autophagy [284]. Therefore, rapamycin treatment is also a putative inducer of undesirable side-effects. Other drugs including lithium, sodium valproate and carbamazepine acting have ben proved to induce autophagy through the inhibition of of inositol monophosphatase in an mTOR-independent pathway [285]. These compunds reveal positive effects by reducing the accumulation and toxic effects of aggregation-prone proteins in cell models as well as by protecting against neurodegeneration in *in vivo* models of Huntington's disease [286]. Further research is needed to learn whether they can also be useful tools in the treatment of AD.

#### **2.10. Multi-target treatments**

Considering the multifactorial etiology of AD, and the numerous and complex pathologi‐ cal mechanisms involved in the progression of the disease, it is quite reasonable that treatments targeting a single causal or modifying factor may have limited benefits. Therefore, growing interest is focused on therapeutic agents with pleiotropic activity, which will be able to target, in parallel, several processes affected in AD [287, 288]. Sev‐ eral compounds already mentioned in the previous sections fulfill these properties, such as DHA which presents anti-inflammatory, anti-oxidant, neuroprotective and anti-tau phosphorylation properties apart from the modulation of synaptic membrane composi‐ tion [289], and curcumin, which in addition to anti-oxidant properties also exhibits antiinflammatory and Aβ- and tau-binding properties [106]. Similarly, rosiglitazone and dimebon are known to produce beneficial effects through insulin receptor signaling mod‐ ulation and mitochondrial protection [153, 165]. Other multi-target potential treatments currently under development for AD are based on the use of the following compounds:

receptor (carbamylated EPO) or that have such a brief half-life in the circulation that they do not stimulate erythropoiesis (asialo EPO and neuro EPO) have demonstrated neuropro‐ tective activities without the potential adverse effects on circulation associated with EPO [318]. Therefore, these new compounds are considered as potential treatments in AD. **•** *Statins*: Evidence has accumulated that a high cholesterol level may increase the risk of developing AD and that the use of statins to treat hyper-cholesterolemia is useful in treating and preventing AD [319]. Statins reduce the production of cholesterol and isoprenoid intermediates. These isoprenoids modulate the turnover of small GTPase molecules that are essential in numerous cell-signaling pathways, including vesicular trafficking and inflam‐ mation [320]. Thus, statins reduce the production of Aβ by disrupting secretase enzyme function and by curbing neuroinflammation in experimental models of AD [321, 322]. **•** *Ladostigil* is a dual acetylcholine-butyrylcholineesterase and brain selective monoamine oxidase (MAO)-A and -B inhibitor *in vivo*. Interest in this compound in AD treatment research is sustained by the potential increase in brain cholinergic activity properties but also by the capacity of ladostigil to prevent gliosis and oxidative-nitrosative stress damage. Moreover, ladostigil has been demonstrated to possess potent anti-apoptotic and neuro‐ protective properties *in vitro* and in various neurodegenerative animal models including AD transgenic mice [323]. These neuroprotective activities involve regulation of APP processing, activation of protein kinase C and mitogen-activated protein kinase signaling pathways, inhibition of neuronal death markers, prevention of the fall in mitochondrial membrane potential, up-regulation of neurotrophic factors, and anti-oxidative activity. **•** *Huperzine A* is an extract of the Chinese plant *Huperzia serrata*. Huperzine A is a selective potent inhibitor of AChE [324]. In addition, some studies have shown that huperzine A may shift APP metabolism towards the non-amyloidogenic α-secretase pathway [325]. In addition, huperzine A reduces glutamate-induced cytotoxicity by antagonizing cerebral NMDA receptors [326]. Finally, huperzine A reverses or attenuates cognitive deficits in some animal models of AD [325]. Large-scale, randomized, placebo-controlled trials are

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necessary to establish the role of huperzine A in the treatment of AD [327].

matory, anti-oxidant and reductor of amyloouid via BACE 1 [331, 332].

NFkappaB [328-330].

**3. Concluding remarks**

**•** *Phytochemicals* as curcumin, catechins and resveratrol beyond their antioxidant activity are also involved in antiamyloidogenic, anti-inflammatory mechanisms and inhibitors of

**•** *Celastrol* is another compound whicha appears to have multiple functions as anti-inflam‐

Main targets of therapeutic intervention at early stages of Alzheimer are summarized in Figure 1. Based on the presently available data several conclusions can be drawn. Combination therapies with drugs targeting different pathological factors or the use of multi-target com‐ pounds appear to be the most effective strategy in the treatment of the neurodegenerative


receptor (carbamylated EPO) or that have such a brief half-life in the circulation that they do not stimulate erythropoiesis (asialo EPO and neuro EPO) have demonstrated neuropro‐ tective activities without the potential adverse effects on circulation associated with EPO [318]. Therefore, these new compounds are considered as potential treatments in AD.

