BioMed Research International

Volume 2014 (2014), Article ID 837157, 22 pages

http://dx.doi.org/10.1155/2014/837157

Review Article

Potential Therapeutic Strategies for Alzheimer’s Disease Targeting or Beyond β-Amyloid: Insights from Clinical Trials

Qiutian Jia, Yulin Deng, and Hong Qing

School of Life Science, Beijing Institute of Technology, 5 South Zhongguancun Street, Haidian District, Beijing 100081, China

Received 4 May 2014; Revised 23 June 2014; Accepted 25 June 2014; Published 17 July 2014

Academic Editor: Tan E. King

Copyright © 2014 Qiutian Jia et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder with two hallmarks: β-amyloid plagues and neurofibrillary tangles. It is one of the most alarming illnesses to elderly people. No effective drugs and therapies have been developed, while mechanism-based explorations of therapeutic approaches have been intensively investigated. Outcomes of clinical trials suggested several pitfalls in the choice of biomarkers, development of drug candidates, and interaction of drug-targeted molecules; however, they also aroused concerns on the potential deficiency in our understanding of pathogenesis of AD, and ultimately stimulated the advent of novel drug targets tests. The anticipated increase of AD patients in next few decades makes development of better therapy an urgent issue. Here we attempt to summarize and compare putative therapeutic strategies that have completed clinical trials or are currently being tested from various perspectives to provide insights for treatments of Alzheimer’s disease.

1. Introduction

Alzheimer’s disease (AD) is named after the German physiologist who first presented neuropathological characteristics of the dementia at the 37th meeting of Society of Southwest German Psychiatrists in 1906 [1]. Alzheimer’s studied a patient with progressive memory loss for five years and analyzed the brain post mortem using silver staining [2]. This contributed to the identification of neuritic plagues and neurofibrillary tangles (NFT) [1], two characteristics employed to identify the dementia to date. It took another 20 years to determine β-amyloid (Aβ) and tau which are major components of neuritic plagues and NFTs, respectively [3], marking the modern era of study of AD research.

AD, as a progressive neurodegenerative disorder, deprives patients of their memory and even lives. Memory loss is the most notable symptom [4, 5] at the early stage but as the disorder advances, difficulties with language, perception, and execution of movement become prominent [6], followed by neuropsychiatric and behavioral abnormality, muscle mass loss, and mobility deterioration [6]. Loss of normal daily living in those with dementia is inevitable.

In addition to the affliction and sufferings to patients, Alzheimer’s disease can cost society substantially, especially in developed countries. The expenditure of AD was around $100 billion per year [7]; the bill was about 177 billion in Europe solely in 2008 [8]. Due to deteriorating abilities to live on their own, caregivers are necessary for progressed AD patients. Burdens on these caregivers’ life including physical, psychological, and economic aspects [9–11] can be a major concern.

The global prevalence of dementia for people over the age of 60 is estimated as high as 40 million in 2001, and the figure is forecasted to double every 20 years [12, 13], indicating that Alzheimer’s disease has become a modern epidemic. In the near future, surging number of AD patients will become an overbearing social issue. Therefore, the need for therapeutic strategies for this devastating disease is urgent.

Currently, Food and Drug Administration (FDA) approved AD drugs are still limited within two categories: cholinesterase inhibitors and memantine [14–16] (a NMDA receptor antagonist). Unfortunately, the effects and benefits of these drugs are marginal and work only to alleviate the symptoms [17–19]. However, in recent years, fundamental researches focusing on the pathogenesis of AD paved the way for development of new treatments targeting the radical source of Alzheimer’s disease [20]. Numerous trials have been or are currently being conducted to determine effects of various compounds on AD in different stages.

Alzheimer’s disease causes major impairment of individual health and social economy due to the limited effective therapeutic approaches. With the explosive explorations based on two hallmarks of AD, numerous clinical trials targeting on or off Aβ have been or are being conducted. In this paper, we will briefly summarize successes and failures in clinical trials in Alzheimer’s disease and try to give a systematic review in an attempt to derive insights from previous experience.

2. Therapeutic Targets Focusing on Aβ Cascade Hypothesis (Table 1)

Table 1: RCTs based on -amyloid in recent years.

Mechanism
RCT
Status
Estimated end
Dementia stage
Enrollment
Duration
Reported outcomes
Details of drugs/RCTs

↓A production

    BACE1 inhibitor
Pioglitazone
Phase 2; completed
2005.1
Mild-to-moderate
25
18 months

Insulin sensitizer, class of PPAR agonists

CTS-21166
Phase 1; completed
2008.2
Healthy
56

MK8931
Phase 3; ongoing
2018.3
Mild-to-moderate
1960
≈6.5 years

With enhanced BBB permeability

E2609
Phase 1; completed
2013.9
MCI/mild AD
65

    GSI/GSM
NIC5-15
Phase 2; ongoing
2013.12

40

Notch-sparing, insulin-sensitizer

Begacestat
Phase 1; completed
2009.10
Elder healthy
49

Dose-dependent changes in plasma A levels
Selectively inhibits cleavage of APP over Notch [208]

CHF 5074
Phase 2; completed
2012.4
MCI
96
12 weeks

NSAID

EVP-0962
Phase 2; completed
2013.10
MCI/early stage
52
14 days

    -secretase activator
Atorvastatin
Phase 3; completed*
2007.7
Mild-to-moderate
600
80 weeks

Tested with AchEI

Simvastatin
Phase 3; completed
2007.10
Mild-to-moderate
400
18 months

Etazolate
Phase 2a; completed
2009.8
Mild-to-moderate
159

Safe and well tolerated
↑-secretase activity, acting as a GABA-A receptor modulator and a PDE-4 inhibitor [209]

Epigallocatechin-3-gallate (EGCg)
Phase 2/3; ongoing
2015.6
Early stage
50
18 months

Prevents the A aggregation via binding to the unfolded peptide

    ↓A aggregation/oligomers
Scyllo-inositol (ELND005/AZD103)
Phase 2; completed
2010.5
Mild-to-moderate
350
18 months
Insufficient to support/refute benefits [210]

Tramiprosate (3APS)
Phase 3
unknown
Mild-to-moderate
950

Suggesting disease-modifying effects [211]

PBT2
Phase 2; completed
2007.12
Mild AD
80
12 weeks
Well-tolerated, ↓CSF A42, and improved executive function [212]

↑Aβ clearance

    Active immunotherapy
Affitope AD02
Phase 2; completed
2013.12
Early stage
335
>1 year

N-terminal A1-6, a synthetic peptide

Affitope AD03
Phase 1; completed
2011.11
Mild-to-moderate
28

i.h. with or without adjuvant aluminum

UB 311
Phase 1; completed
2011.4
Mild-to-moderate
19

N-terminal A1-14

V 950
Phase 1; completed
2012.1

86

formulated on Aluminum-containing adjuvant

CAD 106
Phase 2; completed
2012.12
Mild AD
177

A favourable safety profile [213]
N-terminal A1-6; i.m. of adjuvanted CAD106;

    Passive     immunotherapy
BAN2401
Phase 2; ongoing
2016.12
MCI/mild AD
800
18 months

mAb against A oligomers

BIIB037
Phase 1; ongoing
2014.11
Prodromal to mild
160

Administered via intravenous (IV) infusions in subjects

Ponezumab
Phase 2; completed
2011.8
Mild-to-moderate
198
24 months

Crenezumab
Phase 2/3; ongoing
2016.5
Mild-to-moderate
361
24 months

Gammagard (IVIg)
Phase 2, completed
2010.4
Mild-to-moderate
24
6 months
Improved cognition

Phase 3; completed
2012.12
Mild-to-moderate
390
70 weeks
Showed no significant effect

Phase 2; ongoing
2014.10
MCI
50
24 months

AMBAR
Phase 2/3; ongoing
2016.12
Mild-to-moderate
350

Gantenerumab
Phase 3; ongoing
2019.3
Mild
1000
>5 months
Phase 1 RCT↓brain A; high doses, AE
Mainly targets A plagues

Solanezumab
Phase 3; ongoing
2016.12
Mild
2100

No benefits in primary outcomes
Mainly targets soluble oligomeric A

AAB-003
Phase 1; ongoing
2014.8
Mild-to-moderate
104
52 weeks

Previously treated with AAB-003

GSK933776
Phase 1; completed
2011.5

50

SAR228810
Phase 1; ongoing
2015.1
Mild-to-moderate
48
14.5–22 months

Anti-tau

    ↓tau production
Valproate
Phase 3; complete
2009.12
Mild-to-moderate
313
2 years
Did not show cognitive benefits and prevention of behavioral defects; associated with reduced brain volumes

Lithium
Phase 2; ongoing
2019.4

80

A pilot study was insufficient to support or refute the efficacy [214]

    ↓tau     fibrillization/deposition
Nicotinamide
Phase 1/2; ongoing
2014.7
Mild-to-moderate
50
24 weeks

Vitamin B3

TRx0237
Phase 3; ongoing
2015.12
Mild/mild-to-moderate
700/833
18 months/15 months

Methylene blue (Rember)
Phase 2; completed

Mild-to-moderate
321
6 months
Showed uncertain results

Davunetide (AL108)
Phase 2; completed
2008.1
MCI
144
12 weeks
Showed benefits on memory

BMS-241027
Phase 1; completed
2013.10
Mild
40
9 weeks

RCT: randomized controlled trial; PPAR: peroxisome proliferators activated receptor; BBB: blood brain barrier; MCI: mild cognitive impairment; GSI: secretase inhibitor; GSM: secretase modulator; NSAID: nonsteroidal anti-inflammatory drugs; AChEI: acetylcholinesterase inhibitor; GABA: -aminobutyric acid; PDE: phosphodiesterase; CSF: cerebrospinal fluid; i.h.: subcutaneous injection; i.m.: intramuscular injection; mAb: monoclonal antibody; AE: adverse event.

*RCTs with a combination of another drug.

Data sources: http://www.clinicaltrials.gov/.

2.1. Inhibition of Aβ Production

Studies of familial Alzheimer’s disease (FAD) motivate the discovery of responsible genetic factors, establishing Aβ-centered theory for AD. Amyloid precursor protein (APP) experiences sequential cleavages by β-secretase and γ-secretase and gives rise to the dementia culprit β amyloid (Aβ) that is thought to initiate soluble oligomers, insoluble fibrils, and accumulated plagues (Figure 1). APP can be alternatively processed by α-secretase within the Aβ region and generate a longer C-terminal fragment under the first cleavage. In terms of curbing production of Aβ, the three crucial enzymes processing APP have been therapeutic targets in drug development. The rationale is to inhibit β-/γ-secretase while promoting the α-secretase activity to become the priority strategy.

Figure 1: β-amyloid hypothesis based therapeutic targets. APP, after sequentially being cleaved by BACE1 and γ-secretase, gives rise to a neuron toxic molecule Aβ42. This peptide can exist as monomers or aggregates into oligomers and plagues. The assembly of Aβ42 triggers downstream effects and induces tau phosphorylation. BACE1 inhibitors and GSI/GSM aim to prohibit the production of pathological Aβ, and vaccines or Aβ antibodies promote clearance mechanism. As for tau, GSK-3β inhibitors and other antiaggregates are potential therapeutics targeting on blocking tau hyperphosphorylation or aggregation.

2.1.1. β-Secretase (BACE1) Inhibitor

Beta-site APP-cleaving enzyme 1 (BACE1) is the protease responsible for the initial cleavage of APP, giving rise to the production of neurotoxic suspect Aβ [21, 22]. Mounting evidence corroborate the availability of BACE1 inhibition. BACE1 knock-out mice indicated a close correlation between the BACE1 inhibition and the Aβ decline [23, 24]. It is reported that BACE1 inhibition improved memory deficits [25] and rescued Aβ-driven cholinergic dysfunction [26] in APP transgenic mice. Although the BACE1-deficient animal model presented a relatively benign phenotype with high viability, suggesting that the possibility of targeting β-secretase would be a safe therapeutic approach, further testing indicated that the drastic inhibition would result in hypomyelination and behavioral abnormalities such as seizures [27–30]. This is because, except from APP, BACE1 has a series of substrates, like neuregulin-1, related to myelination [29, 31]. AD pathology onset was postponed in the APP × BACE1+/− mice; however, it hinted at a partial inhibition that might mitigate the potential safety problems [32, 33]. It has been noted that the discrepancy between potency-required molecular weight and CNS penetration-required size [34, 35] poses another challenge.

Many BACE1 inhibitors are derived from approved drugs for type 2 diabetes with properties regulating insulin metabolism. Nuclear peroxisome proliferator activated receptor gamma (PPARγ) functions as a transcription factor regulating gene expression [36], modulating inflammation response, promoting microglia-mediated Aβ endocytosis, and declining cytokine secretion [37]. Thiazolidinediones can activate PPARγ to inhibit β-secretase and promote ubiquitination to degrade amyloid load [38]. PPARγ agonists like thiazolidinediones derivatives rosiglitazone and pioglitazone soften the peripheral insulin resistance [39], which aggravates AD neuropathology, and this decline of insulin sensitivity helps in Aβ proteolysis. The study of rosiglitazone has been developed to a large phase 3 trial; however, it has been discontinued due to cardiac risk concerns [40]. Pioglitazone has recently progressed into a phase 3 clinical trial after precluding a previously reported bladder risk. But due to the involvement of substrate complexity and some adverse effects, other phase 3 clinical trials for BACE1 inhibitors are still lacking.

However, several novel drugs are currently under investigation. Based on conjugation to a penetrant carrier peptide [41, 42], the potent CNS impermeable compound, CTS-21166, has completed the phase 1 trial. It showed a good tolerance and a reduction of plasma Aβ level in healthy volunteers [43]. A phase 1b dose-escalating study for MK-8931 demonstrated a positive effect in reducing the level of toxic proteins in addition to safety and good tolerance. A phase 2 trial recruiting 200 mild-to-moderate patients was expanded to a larger 1960-participant phase 3 trial, including conventional cognitive and functional primary outcomes, and it recently passed an interim safety evaluation.

Another BACE1 inhibitor, LY2886721, though it appeared to be safe and lowered Aβ42 in cerebrospinal fluid by more than two-thirds in phase 1 trial [44], was terminated due to the fact that 4 out of 45 patients showed liver abnormalities during the phase 2 trial. Besides, RG-7129 was also terminated in its phase 3 trials in 2013. These terminations again signaled that significant challenges are remaining: whether BACE1 inhibitors will be safe in the long run and if lowering BACE1 activity will slow cognitive decline.

2.1.2. γ-Secretase Inhibitors (GSI) and Modulators (GSM)

γ-secretase is a transmembrane protease responsible for the eventual cleavage of amyloid precursor protein (APP) to generate Aβ (Figure 1), thus it is considered a principal therapeutic target in Alzheimer’s disease [45, 46]. This enzyme complex consists of four components: Aph1, Pen2, glycosylated nicastrin, and endoproteolyzed presenilin as the catalytic core [47], and it is involved in myriads of physiological process. The versatility places hurdles in the way of γ-secretase targeted drug development. In the human body, aside from APP, there are more than 50 different substrates that γ-secretase is capable of reacting with, many of which are neuronal substrates [48]. Importantly, γ-secretase is also responsible for cleavage of Notch 1, which leads to the release of the Notch intracellular domain (NICD), subsequently translocated to the nucleus to regulate genes involved in cell development, cell survival, and cell fate determination [49]. Thus, inhibition of γ-secretase needs to be cautiously designed to particularly circumvent the drawbacks caused by Notch signaling abnormality. Haematological [50] and gastrointestinal [51] toxicity, skin reactions [52, 53], and changes to hair color [54] are the most commonly reported adverse effects of γ-secretase inhibitor.

Several γ-secretase inhibitors (GSIs) have been launched in clinical trials. Many reduced the Aβ production in plasma or CSF (cerebrospinal fluid), but few successfully avoided the Notch-induced side-effects. Semagacestat decreases Aβ level in plasma and downregulates its generation in the central nervous system (CNS) [55]. Semagacestat is the first γ-secretase inhibitor that have been taken into Phase 3 clinical trials. While phase 1 trial suggested a dose-dependent decline of Aβ synthesis in CSF [55], phase 2 trial began exhibiting skin-related side effects. Although Aβ level in plasma has significantly decreased, it was not duplicated in CSF and no effects on cognition and function were found. Two pivotal phase 3 trials were reluctantly started; however they were discontinued due to increased risk of skin cancer and infection and lack of efficacy [56]. Fall of semagacestat, a potentially promising drug candidate, repeated disappointing results of other GSIs, which deemed that a deeper understanding of interaction between 4 subunits and their substrates is necessary.

Different GSIs present favor to interact with subunits of γ-secretase, exhibiting target specificity. DAPT and L685458 indicated the smallest selectivity, while MRK-560 and sulfonamide based GSIs strongly prefer to inhibit PS1 instead of PS2 [57, 58]. Aph1 heterogeneity is critical for individual survival, suggesting that targeting of Aph1b γ-secretase specifically would be more tolerated [59], although the feasibility of drug design still remains difficult to determine.

Accordingly, the second generation Notch-sparing γ-secretase inhibitors aimed at selective inhibition of specific sites took the spotlight. Avagacestat (BMS-708163), begacestat, and NIC5-15 are such Notch-sparing GSIs under clinical trials. It was reported that avagacestat (BMS-708163) has 137-fold selectivity for APP over Notch in cell culture and robustly reduces CSF Aβ levels without causing Notch-related toxicity in rats and dogs, although this is still being researched [60]. Phase 2 trials have to be terminated due to the adverse effects of gastrointestinal and dermatological system in addition to the lack of cognitive improvement compared to placebo counterparts. Begacestat decreased the Aβ concentration in the plasma but not in CSF [49, 61], and a phase 1 clinical trial in combination with cholinesterase inhibitor donepezil has been completed, further data was unavailable. Another Notch sparing GSI candidate, NIC5-15, a natural monosaccharide [62], is currently under a phase 2 trial and demonstrated good tolerance and safety [63].

Given that the unresolved adverse effects brought on by GSIs are tricky to address, the concept of γ-secretase modulators (GSMs) was established with the expectation of nonsteroidal anti-inflammatory drugs (NSAIDs). A subset of NSAIDs, like ibuprofen, indomethacin, and sulindac sulfide, disconnected from their cyclooxygenase (COX) properties were discovered to be able to selectively reduce the production of Aβ42 at the cost of elevated shorter peptide Aβ38 [64, 65]. Surprisingly, this downregulation of toxic Aβ level lacks the inhibitory effect on Notch or other substrates [64]. This finding promoted the GSMs as promising therapeutic candidates for Alzheimer’s disease, because the Notch-induced drawbacks may be avoided and on the other hand, the overproduction of shorter and more soluble A38 seems less likely to aggregate and is less pathogenic.

Among the compounds described above, tarenflurbil (R-flurbiprofen) relates to ibuprofen structurally and pharmacologically. Phase 1 trials with a broad dose range (400 to 1600 mg/day) revealed a low drug exposure in the brain [66], while phase 2 trials narrowed this spectrum (400–800 mg, twice daily) and showed trivial benefits on function with the lowest dosage (400 mg). Although undesirable side effects like nausea, dizziness, and diarrhea were observed, this compound is still considered tolerable [67]. After modification, phase 3 trial suggested neither functional improvement nor clinical efficacy in the mild AD patients [68] and thus the results were disappointing. The weak potency of tarenflurbil can be attributable to low CNS penetration as shown in phase 1 trial, and on the other hand, NSAID residue activity curbed Aβ clearance mechanism mediated by microglia [69].

Another GSM CHF-5074 based on R-flurbiprofen ameliorated brain Aβ load and improved the animals’ performance in behavior tests. The drug’s safety and tolerability have been evaluated and are undergoing a phase 2 trial. Published data indicated that this compound may have an additional function of acting independently of Aβ42 [70, 71]. Nevertheless, a balance between lipophilicity and potency of these compounds must be considered. The remarkably increased potency in 2nd and 3rd generations of GSMs relies heavily on the increase of lipophilicity, which has been proved to result in off-targets, like hepatotoxicity [72].

2.1.3. α-Secretase Activator

APP can be cleaved by an alternative α-secretase rather than β-secretase in the first step to circumvent the generation of pathological Aβ peptide. Hence, increasing the chance of α-cleavage could be an effective approach to decrease the Aβ formation and promote soluble APP production to protect neurons [73]. Agonists of muscarinic, glutamate, and serotonin receptors (and the agonists or antagonists of transmitters receptors would be discussed in following section), statins, oestrogens, testosterone, and protein kinase C activators belong to this drug classification that can motivate α-secretase activity, and they have been launched in clinical trials, but data indicating their use in AD is limited [74].

Etazolate (EHT-0202), a selective [75] receptor modulator, has completed a phase 2 trial in patients with mild to moderate AD. It presented a good oral bioavailability and an elevation of sAPPα [76]. Bryostatin-1, a macrocyclic lactone, caused a decline of brain Aβ40/42, improved behavior test in AD mouse model [77], and was under a phase 2 trial, but the specific information is inaccessible.

Statin drugs such as atorvastatin and simvastatin lower peripheral cholesterol production to prevent heart attacks and other expressions of cardiovascular disease. Atorvastatin, in combination with cholinesterase inhibitor, has completed a phase 2 clinical trial and achieved a beneficial cognition and function [78], but failed to repeat the outcome in a 641-patient phase 3 clinical trial [79, 80]. Simvastatin can penetrate BBB and long-term statin treatment can decline Aβ level. In a 35-normal participant phase 4 trial of Simvastatin, it was reported to reduce phospho-tau-181 in CSF, while not total tau or Aβ level [81]. A follow-up study evaluating one year simvastatin treatment in 120 cognitively normal and middle-aged adults, effect on CSF levels of Aβ42, t-tau, and p-tau181, is ongoing.

2.2. Anti-β-Amyloid Aggregation

The pathological Aβ peptides, prone to assembly into aggregate as neuro-/synaptic toxic products spur the idea of inhibition of Aβ aggregation or destabilization of the Aβ oligomers species. However, Aβ aggregations are characterized with a high stability resistance to disaggregation [82] and remain insoluble even with heat or SDS [83]. The fact that amyloid fibrils have an extremely low energy state [82] and the lack of thorough understanding of Aβ aggregation process have complicated the issue. Besides, another challenge would be to access the compounds with high CNS bioavailability and low immunogenicity and toxicity. It is generally believed there are three strategies that block Aβ aggregation: antiaggregate compounds, metal complexing agents and immunization. They can disturb the formation of either soluble oligomers or insoluble plagues.

2.2.1. Nonpeptidic Antiaggregates

The first class of mentioned inhibiting aggregation compounds is nonpeptidic antiaggregates, tramiprosate, derived from proprionic acid that is a primitive representative. The promising outcomes of this agent from the safety and tolerance [84] were neutralized by two following phase 3 trials: the European trial precluded methodological problems that might lead to the negative in the North American trial and demonstrated the poor CNS penetration and the weak potency [85] of this drug.

The second generation of nonpeptidic antiaggregates was expected to meet those challenges. Scyllo-inositol is thought to effectively impede Aβ aggregation, promote misfolding modulation, and accelerate aggregates disassociation [86]. Because this compound can cross blood brain barrier (BBB), with the assistance of inositol transporters, it can achieve a high concentration in CNS via peripheral administration. This drug is being tested in the phase 2 trial with mild-to-moderate Alzheimer’s disease patients on the basis of good tolerance and safety profile [87]. Although high doses (1000 mg and 2000 mg) resulted in serious adverse effects, the studies continued to test the low dose (250 mg) cohorts [88]. Epigallocatechin-3-gallate (EGCg), a polyphenol from green tea, via disrupting unfolded peptide, stimulated α-secretase activity and inhibited Aβ aggregation in animal models [89]. This agent was also involved in modulation of cell transduction, regulation of cell survival and death [89], and protection of mitochondrial function. The multiple effects of this natural compound make it a promising candidate, and a phase 3 trial with early AD patients with EGCg is being conducted.

2.2.2. Metal Complexing Agents

After Aβ peptides were produced and released into extracellular fluids, metals like Zn and Cu can motivate oligomerization into fibrils. So metal chelators or metal complexing agents that can interfere with reaction of metal ions with Aβ are likely to be a therapeutic strategy. Clioquinol (PBT2), metal-induced Aβ inhibitors, also has a potent CNS permeability. PBT2 can redistribute metal ions to neurons promoting metalloproteinase expression and thus an increment of Aβ degradation. A phase 2 trial was completed and it proved a decrease of Aβ42 concentration in CSF and an improvement of cognitive and behavioral performance [90].

2.2.3. Active Immunization

It is conventionally thought that clearance of CNS Aβ requires a BBB permeability property, confining the therapeutic targets in a very narrow realm: medicinal chemistry-driven and small molecules. Nonetheless, incredible work done by Schenk et al. revealed that immunization of PDAPP transgenic mice markedly mitigated amyloid plaque burden, improved neuritic dystrophy, and even reduced existed Aβ plagues [91]. This striking breakthrough suggested that Aβ immunotherapy would be a potential strategy to remove both soluble and aggregated β amyloid [92].

AN-1792, the first anti-Aβ vaccine (with full length Aβ 1–42) tested in active immunization clinical trial, was terminated in the phase 2 trial in patients with early AD due to the fact that some participants developed aseptic meningoencephalitis and cerebral microhemorrhage [93]. The complication is attributed to cytotoxic T cell or autoimmune response [94–96]. Therefore, employing only fragments instead of full length of Aβ or other cell epitopes to circumvent toxicity and inflammation is highlighted. Additionally, the security of adjuvant and delivery approaches must be cautiously considered.

The next generation of vaccine is devoid of any T-cell epitopes. CAD-106, consisting Aβ 1–6 peptides coupled to a Qβ virus-like particle, has recently completed the phase 2 trial in patients with mild AD and did not lead to meningoencephalitis [97]. Two other vaccines, UB 311 (Aβ 1–14) and V950 (Aβ N-terminal conjugated to ISCO-MATRIX), both containing B-cell epitopes, have also recently finished phase 1 trial. However, another vaccine AC-001(Aβ 1–7 conjugated to inactivated diphtheria toxin) discontinued its phase 2 trial in August 2013, because the studied drug elicited a strong antibody response. Another active immunization approach is developed on the foundation of Affitope using short six-amino acid peptides that imitate the native Aβ sequence. AD-01 and AD-02, targeting N-terminal fragments of Aβ, were proved to rescue AD-like symptoms in animal models [98]. Recently, AD-02 has progressed into a phase 2 clinical trial.

2.2.4. Passive Immunization

Another strategy to avoid immune response is direct administration of antibodies. This passive immunization has an approximate potency to remove amyloid plaques and rescue neuritic and glial pathology [99], reduce early tau hyperphosphorylation [100] and cytopathology [101], and reverse abnormal hippocampus synaptic plasticity [102].

Bapineuzumab (AAB-001) is a humanized monoclonal antibody, derived from 3D6, published to promote removal of Aβ plagues and rescue synapse loss in APP transgenic mice brain [99]. However, in a 234-patient phase 2A safety and tolerability trial, this agent indicated no significant alteration on primary measures of cognition and daily activity. For apolipoprotein E (ApoE) ε4 carriers, there is a temporary vasogenic oedema, an adverse effect correlated with dose administration [87]. Given that 4,000 mild AD patients across North America and Europe showed no treatment effect on either cognitive or functional outcomes, the phase 3 trial was terminated. Solanezumab (LY2062430) is a humanized monoclonal IgG1 antibody directed against the mid-domain of the Aβ peptide (Aβ 16–24) and designed to specifically bind soluble species of Aβ. Phase 2 study showed dose-dependent increases of various Aβ species in plasma and CSF, an indication that insoluble Aβ is released from plagues and leaches into fluid [103]. Two trials in phase 3 suggested a limited benefit for cognitive performance as compared to cholinesterase-inhibitor drugs. A third trial started from July 2013 to test demonstrated brain amyloid burden, and data is expected to be read out in December 2016. Gantenerumab, a human IgG1 antibody binding to Aβ fibrils, can elicit phagocytosis to remove Aβ plagues in brain and rescue Aβ oligomers that induced impaired long-term potentiation (LTP) in rats model. An expanded phase 2/3 trial of 770 participants is being conducted and is estimated to be completed in 2016. Crenezumab, a novel humanized antibody with IgG4 backbone, is believed to limit microglia mediated inflammatory cytokines release to avoid vasogenic oedema. It can recognize β amyloid oligomers, fibrils, and plagues with a high binding affinity. Phase 1 study confirmed safety and tolerance, followed by an ongoing phase 2 trial in patients with mild to moderate AD using elevated dose as well as a test for prevention of this progressive dementia. There are several antibodies which have completed or have undergone the early clinical trials, as shown in Table 1.

Many monoclonal antibodies are delivered intravenously, whereas passive immunization can be also accomplished via infusion of intravenous of immunoglobulins (IVIg) from healthy donor. A small study in 8 patients showed increase of Aβ antibodies in serum, decrease of Aβ in CSF, and stabilization of MMSE (mini-mental-state exam) scores over 18 months. A phase 2 trial with 24 patients suggested beneficial cytokine concentrations alteration in plasma. However, two critical phase 3 trials showed no difference between study drug and placebo, though a trend toward benefit for the higher dose, and thus were halted. Another published phase 2/3 clinical trial evaluating infusion of albumin in combination of IVIg is currently conducted in patients with mild to moderate AD.

Active immunization maintains the body with a constant high concentration of immunoglobulin, so this strategy calls for fewer follow-up injections with a reasonable expense. But to tackle with the T-cell induced inflammation would be a tricky issue. Passive immunization is a more effective method especially for elderly people considering their weakened responsiveness to vaccines [104]. Selection of safe epitopes can be readily met, as well as a better control of antibody titer; however, antibody delivery could be inconvenient and costly, and the risk of vasogenic oedema and cerebral amyloid angiopathy might increase.

2.3. Tau

According to Aβ hypothesis, intracellular neurofibrillar tangles (NFTs) induced by altered phosphatase/kinase activity is a downstream event of aggregation of β-amyloid (Figure 1), and NFTs as a catalyst will aggravate the oxidation and further result in neuronal dysfunction, cell death, and transmitter deficits. Tau is normally a highly soluble protein in cytoplasm binding to microtubules as a stabilizer. Formation of NFTs as a result of hyperphosphorylated and misfolded tau protein aggregation is toxic to neurons. The pathological tau proteins lose the capability to aid microtubules in transporting neuronal substance, leading to neuronal dysfunction and apoptosis [105, 106].

2.3.1. Kinase Inhibitors

Protein kinase, a group of critical enzymes responsible for tau overphosphorylation, is a prerequisite for the tau-induced toxicity. However, myriads of kinases mutually play a central role in regulating cell function and guaranteeing a normal physiological condition. The development of tau-targeted therapy is therefore challenging due to redundancy of kinase interactions and uncertainty of which enzyme specifically catalyzes the phosphorylation that we are focusing on [107, 108].

The first class of tau inhibitors aims to modulate tau phosphorylation via decreasing the activity of related kinase since imbalanced interaction between glycogen synthase kinase 3 beta (GSK3β) and protein phosphate 2 (PP2A) enhances tau hyperphosphorylation and NFT formation [109]. GSK3β appears to engage in AD pathogenesis given its impact on cellular signaling and gene description [109]. Recently, it has been reported that GSK3β is responsible for 31% of the pathological phosphorylation sites of tau protein [110] and is found colocalized with NFTs in postmortem brain [111, 112]. Toxic Aβ that promotes GSK3β activity bridges a link between the two hallmarks of Alzheimer’s disease [110], implicating that GSK3β inhibitor is a potential drug target.

Lithium and valproate reduced tau phosphorylation and prevented reversed aspects of tauopathy in animal models [113] but did not show cognitive improvement in clinical trials with AD patients [114]. NP-031112 (NP-12), a non-ATP competitive inhibitor of GSK3β, counteracts tau phosphorylation, reverses amyloid burden in brain, prevents cell loss, and rescues spatial memory deficits using animal models [115]. But the phase 2b trial was terminated due to the negative results. Development of some paullone, indirubin, and maleimide family-derived GSK3β inhibitors is in the pipeline, yet stuck in the preclinical trials concerning the cytotoxic effects.

Cyclin dependent kinase 5 (cdk5) is another kinase tightly associated with tau pathology. Cdk5 regulating protein was found in AD brain and thus is probably causing a pathophysiological tau phosphorylation [116]. Cdk5-selective inhibitors were demonstrated to penetrate BBB and reduce elevated Aβ level by regulating cdk5 [117] and are at preclinical status. The test of several compounds targeting other protein kinases, like cdk1/2/9, p38, Erk1/2, JNK, casein kinase, and DYRKIA brought disappointing outcomes, and trials were discontinued due to the poor efficacy or severe adverse effects.

2.3.2. Inhibition of Tau Aggregation

Another scenario to interfere with tau-induced NFT is to inhibit tau aggregation or promote tau assembly disassociation. Rember (methylene blue) is such a tau antiaggregant [118]. Preclinical data revealed a learning deficit reversing property and a completed phase 2 trial proved that this agent can slow down AD progression with a good bioavailability [119, 120]. TRx0237, another methylene blue, has an improved drug absorption, bioavailability, and tolerability. Since 2008, intensive investigation of this agent began, and growing evidence indicated that TRx0237 benefits neuroprotection [121] and Aβ clearance in transgenic mice and improves spatial learning in rats [119, 122]. The antiaggregation properties were reported by some papers, and three phase 3 studies are ongoing.

Epothilone D (BMS-241027) is a microtubule stabilizer, via inhibition of tau release from microtubule to maintain the transportation function of axon, and on the other hand, precludes formation of tau aggregation. This agent restored behavioral and cognitive deficits, inhibited neuron loss, and curbed the tauopathy in animal models [123, 124]. Epothilone can penetrate BBB and exert a better efficacy at low concentration and now undergoes a phase 1 clinical trial. Nicotinamide, the precursor of coenzyme NAD+, reduces phosphorylated tau and protects microtubules stabilization in mouse model [125]. Nicotinamide has been launched into clinical studies suggesting that it is safe and well tolerated and a phase 2 clinical trial is ongoing in patients with mild-to-moderate Alzheimer’s disease.

3. Putative Therapies Still Derived from Neurotransmitter System

Neurotransmitters depletion (basically referring to acetylcholine, ACh) and synaptic dysfunction are two classical features of AD [126]. Thus, two hypotheses have been established—cholinergic hypothesis [127] and glutamatergic hypothesis [128], based on which FDA approved therapies—AchE inhibitors and NMDA receptor antagonists—to mitigate AD symptoms were developed. Although drugs regulating transmitters’ production, release, and recycling cannot prevent the progression of AD, pursuit of searching novel receptor agonists and antagonists has never stopped (Table 2).

Table 2: RCTs targeting neurotransmitter systems in recent years.

Mechanism
RCT
Status
Estimated end
Dementia stage
Enrollment
Duration
Reported outcomes
Details of drugs/RCTS

Cholinergic agents

    AchE inhibitor
Huperzine A
Phase 2/3; completed
2012.6
Mild-to-moderate
390
6 months
↑cognitive function, daily living activity, global clinical assessment
A natural AChEI; antioxidant and neuroprotective properties [215]

Ladostigil hemitartrate
Phase 2; ongoing
2016.9
MCI
200
36 months

Antioxidant properties; modulates APP processing

    Nicotinic receptor     agonist
EVP-6124
Phase 3; ongoing
2017.7
Mild-to-moderate
790
26 weeks
Positive outcomes in a 24-week phase 2b RCT

RO5313534
Phase 2; completed
2010.11
Mild-to-moderate
389
6 months

α7 nicotinic receptor agonist; as add-on therapy to donepezil

Ispronicline (AZD3480)
Phase 2; ongoing
2014.7
Mild-to-moderate
300
1 year

α4β2 and α2β2 nicotinic receptor agonist

MT-4666
Phase 2; ongoing
2015.5
Mild-to-moderate
450
24 weeks

ABT-089
Phase 2; terminated
2013.10
Mild-to-moderate
434
24 weeks

MK-7622
Phase 2b; ongoing
2017.8
Mild-to-moderate
830
12–24 weeks
As adjunctive therapy to donepezil
α7 receptor modulator

Glutamatergic agents
AVP-923
Phase 2; ongoing
2014.9
Mild-to-moderate
200
10 weeks
Behavioral problems
NMDA receptor antagonist

Serotoninergic agents
Lu AE58054
Phase 3; ongoing*
2016.1
Mild-to-moderate
≈2500

Positive results in a phase 2 RCT, 278 participants, 6 months;
Several phase3 RCTS with donepezil (AchEI);

SB-742457
Phase 2; completed
2011.8
Mild-to-moderate
684
6 months
showed positive results

RCT: randomized controlled trial; AChEI: acetylcholinesterase inhibitor; MCI: mild cognitive impairment; NMDA: N-methyl-D-aspartic acid.

Data sources: http://www.clinicaltrials.gov/.

*RCTs with a combination of another drug.

Cholinergic neurons impairment accompanies the early progression of dementia. From animal and human studies, cholinesterase inhibitors administration stimulated memory and learning process [129]. Besides, a marked correlation between loss of cholinergic neurons and deterioration of defected memory was proved in animal models later [130, 131]. Therefore, improvement of cholinergic system, including potentiating effects of acetylcholine (Ach) and inhibiting activity of cholinesterase, is a potential therapeutic goal.

Ach is a ligand for nicotine receptors and exerts an excitatory effect on the postsynaptic neuron, an essential event for long-term potentiation (LTP) and memory formation. Several nicotinic receptor agonists to reinforce this event are being tested in clinical trials. EVP-6124, a selective agonist of the α-7 nicotinic acetylcholine receptor, has finished a phase 1/2 trial showing safe and well tolerated results and recently (Oct 2013) entered two phase 3 trials to test the cognitive benefits. Quite a few other clinical trials testing nicotinic agonists are ongoing (ladostigil hemitartrate, phase 2; ispronicline, phase 1), completed (RO5313534), or terminated (ABT-089).

A transmitter that indirectly modulates neuron degeneration and memory deficits is serotonin (5-HT). Growing evidence indicated that inhibition of 5-HT₆ could facilitate Ach release and via elevated cholinergic transmission, memory and learning defects were likely to be ameliorated. 5-HT₆ antagonists were widely reported in many studies to rescue anticholinergic drugs-induced amnesia [132]. Recently, two agents, PRX-03140(5-HT₄ antagonist) and SB-742457(5-HT₆ antagonist), completed the phase 2 trials. Lu AE58054, an antagonist of the serotonin 6 (5-HT₆) receptor was recently progressed into a phase 3 trial with 930 mild to moderate AD patients in combination with AchE inhibitor donepezil.

4. Potential Findings of Therapeutics for Alzheimer’s Disease from Other Perspectives

In addition to the two hallmarks and neurotransmitter system impairment, there are several other features found in Alzheimer’s disease, including inflammation, oxidative stress, mitochondrial dysfunction, neurotrophin deficiency, and so forth. These aspects are not systematically and thoroughly summarized and are likely to be neglected though; they do provide new perspectives in developing AD treatments. Many drugs of great therapeutic potential are under clinical trials (Table 3).

Table 3: Other novel approaches in AD clinical trials.

Mechanism
RCT
Status
Estimated end
Dementia stage
Enrollment
Duration
Reported outcomes
Details of drugs/RCTS

Anti-inflammation and antioxidation
Curcumin
Phase 2; completed
2007.12
Mild-to-moderate
33

NSAID, cholesterol-lowering properties

Etanercept
Phase 1; ongoing
2015.6
Mild-to-moderate
12
12 months
↑cognitive function with other nutrients.
Approved drug for arthritis; may modulate immune system; benefit AD patients

dl-alpha-tocopherol (vitamin E)
Phase 3; completed
2012.10
Mild-to-moderate
613

PUFA
Phase 1/2; ongoing*
2015.1

100
18 months

Tested alone or together with lipoic acid

RO4602522
Phase 1; completed
2013.5

17

PDE inhibitors
PF-04447943
Phase 2, completed
2010.9
Mild-to-moderate
198

Selective PDE 9A inhibitor

MK0952
Phase 2; completed
2007.11
Mild-to-moderate

Selective PDE 4 inhibitor

Cilostazol
Phase 4; completed
2013.7
Mild-to-moderate
46

PDE3 inhibitor, Antiplatelet agent in WMHI; ↑pCREB

Tyrosine kinase inhibitor
Masitinib
Phase 3; ongoing*
2015.12
Mild-to-moderate
396

In combination with AChEI and/or memantine

Insulin and GLP1-R agonists
Intranasal insulin (glulisine)
Phase 2/3; ongoing
2015.2
MCI/mild AD
240
12 months

Exendin-4 (exenatide)
Phase 2; ongoing
2016.7
MCI/early stage
100
3 years
Showed neuroprotection
Diabetes agent

Liraglutide
Phase 2; ongoing
2017.1
early stage
206
12 months

Modulating mitochondrial function
AC-1204
Phase 2/3; ongoing
2015.1
Mild-to-moderate
480
26 weeks

Latrepirdine (Dimebon)
Phase 3; completed
2009.12
Mild-to-moderate
598
6 months

RXR agonist
Bexarotene
Phase 2; ongoing
2014.3
Mild-to-moderate
20

Approved anticancer drug; linked to key pathways relevant to AD and Aβ

NGF delivery
CERE-110
Phase 2; ongoing
2014.12
Mild-to-moderate
50
24 months
24 months
Designed to help neurons function better; uses a virus to transfer NGF gene

Encapsulated Cell biodelivery of NGF
Phase 1b
2011.12

6
12 months

RCT: randomized controlled trial; NSAID: nonsteroidal anti-inflammatory drugs; PDE: phosphodiesterase; WMHI: subcortical vascular disease; pCREB: phosphorylated cAMP-response element binding protein; AChEI: acetylcholinesterase inhibitor; GLP1-R: glucagon-likepeptide1 receptor; MCI: mild cognitive impairment; RXR: retinoid X receptors; NGF: nerve growth factor.

*RCTs with a combination of another drug.

Data sources: http://www.clinicaltrials.gov/.

4.1. Anti-Inflammation and Antioxidants

Chronic inflammation is an essential feature of AD and contributes to its pathogenesis in numerous ways. Microglia are brain’s resident macrophages that monitor brain activity and play a contributing role in removal of redundant and apoptotic neurons [133, 134], remodeling of normal synapse [135], and protection of CNS from pathogens and detritus [136]. However, they can shift to another phenotype to secrete series of inflammatory factors, exerting detrimental effects on bystander neurons and processes they are involved in. Aggregated Aβ appears to be a robust agent driving this alteration, since markers of activated microglia were densely colocalized within the deposits [137, 138]. Microglia seem incapable of degrading Aβ that they intake [139, 140], leading to a frustrated phagocytosis instead. As clinical trials have been a major disappointment, agents that drive microglia to a phenotype that favors attack on pathogens rather than bystander neurons may hold therapeutic potential.

Based on compelling evidence of the involvement of inflammation in AD pathogenesis, anti-inflammatory drugs have been investigated. COX inhibitors, aiming to reverse the elevated Aβ burden and cognitive deficits caused by overexpression of COX2 [141, 142], showed limited efficacy [143]. Glucocorticoid steroids, considered as potent drugs by declining overexpression of proinflammatory mediators [144], showed poor benefits [145] or adverse effects [146]. Flavonoid administration prevented cognitive impairment associated with inflammation in animal studies [147, 148]; however, the beneficial effects cannot be repeated in human [149].

Another anti-inflammatory agent etanercept, an approved arthritis drug, is a TNF-α antagonist to neutralize the activated microglia secreted cytokines. Modulation of immune system may have benefits for Alzheimer’s disease patients and a phase1 clinical trial in combination with supplementation of some specific nutrients is ongoing in mild to moderate AD patients. Curcumin, a natural polyphenol, has anti-inflammatory and antioxidant properties and exhibits other neuroprotective functions like promoting metal chelation, curbing tau aggregation, and facilitating neurogenesis. It undergoes a phase 2 study, but details are not available.

Oxidative injury is the following causal event of inflammation and the study of antioxidants in treatment of AD achieved little success. Alpha-tocopherol, a synthetic vitamin E, is thought to prevent brain cell damage by destroying toxic free radicals and slowing down the cognitive decline in the finished phase 3 trial. In addition, a phase 3 trial of DHA (docosahexaenoic acid), an omega-3 fatty acid, was terminated because cognitive decline was not changed compared to placebo group.

4.2. Mitochondrial Dysfunction

Mitochondrial dysfunction taking place in early AD enhances synaptic damages and neuron apoptosis, so it is considered a causal factor of neurodegeneration [150]. APP and Aβ are transported into mitochondrion reacting with mitochondrial components, leading to an impaired ATP processing and increased oxidative stress level [150, 151]. ApoE4, a risk factor for sporadic AD, harms mitochondrial trafficking and function and promotes mitochondrial apoptosis [152, 153]. Replacing mitochondrial DNA (mtDNA) form one cell line with mtDNA from AD patients supported a mitochondrion cascade hypothesis [154], offering new therapeutic targets. Latrepirdine (dimebon), an antihistamine that preserves mitochondrial structure and function and protects against Aβ induced pore apoptosis, has been tested in a clinical trial in Russia and phase 2 data showed improvement of all outcomes [155] while phase 3 trial did not confirm it [156]. However, a combination of therapy with donepezil was demonstrated as well tolerated from preliminary results in phase 1 trial and further information awaits analysis [157]. AC-1204 is designed to improve mitochondrial metabolism [158] by induction of chronic ketosis, thus rescuing regional cerebral hypometabolism presented in early Alzheimer’s disease, and this agent is undergoing a phase 3 clinical.

4.3. Diabetes

Diabetes is another risk factor for Alzheimer’s disease [159] in which the insulin resistance and disrupted glucose metabolism [160] can be attributed to a tumor necrosis factor (TNF) induced inflammation pathway [161, 162]. Insulin can mediate Aβ degradation by activating insulin-degrading enzyme (IDE) [163]. A CSF insulin decline in prodromal female AD patients [164], the presence of insulin resistance, and the dysfunctional insulin signaling pathway in dementia brain [165] are documented. Incretin and liraglutide, two drugs for hyperglycemia, implicating beneficial effects on AD mice [166, 167], reinforced the relationship between diabetes and AD, and a phase 2 study of liraglutide, a glycogen like peptide 1 agonist is still ongoing. These evidences brought the advent of concept “type 3 diabetes,” [168] and an intranasal insulin delivery with an ameliorating cognitive function effect [169] has completed its phase 2 study.

4.4. ApoE (Apolipoprotein) and Aβ Export

ApoE (apolipoprotein) is a powerful genetic factor [170, 171] for sporadic AD beyond APP, PS1, and PS2 genes. The isoform ApoE4 substantially promotes the risk of AD and decreases the age of onset [172]. ApoE is generally thought to regulate Aβ clearance and thus influence fibrillogenesis. In CNS, ApoE, responsible for transportation of cholesterol to neurons, is primarily produced in astrocytes [173]. Aβ aggregation and clearance are differently affected in an isoform (ε2, ε3, and ε4) dependent manner; frequency of AD and mean age at clinical onset are 91% and 68 years of age in ε4 homozygote, 47% and 76 years of age in ε4 heterozygote, and 20% and 84 years in ε4 noncarriers [172, 174]. ApoE was found colocalized with amyloid plagues [175] and this coexistence is more abundant in ApoE4 carriers [176]. Additionally, ApoE4 is associated with cognition decline before clinically apparent syndromes [177, 178]. ApoE4, as previously described, can work synergically with other risk factors, like insulin resistance and peripheral vascular diseases [179, 180], thus exerts a confounding effect on AD and triggers inflammatory cascade. After being synthesized, ApoE is lipidated by the ABCA1, a process regulated by nuclear receptor liver X receptor (LXR) or retinoid X receptor (RXR), and transported to form lipoprotein particles. The complex particle binds soluble Aβ, promoting transfer via neuron surface receptors such as low-density lipoprotein receptor (LDLR), low-density lipoprotein receptor-related protein 1 (LRP1), and heparin sulphate proteoglycan (HSPG) [181, 182] into neurons where degradation can be finished with proteolysis in lysosome. ApoE ε4 isoform has less affinity of binding Aβ compared to ε3, showing a less efficient clearance phenotype [183, 184]. Stimulation of LXR/RXR enhances removal of Aβ [185, 186] while inhibition of ABCA1 impairs Aβ clearance in ApoE4 rather than ApoE3 mice [187]. Therefore, the molecules and receptors involved in ApoE metabolism can be potential therapeutic targets for drug development.

Recent studies demonstrated that oral administration of bexarotene, a RXR agonist and a FDA approved anticancer drug, reduces Aβ plaques and improves cognitive function in an ApoE-dependent manner in amyloid mouse model [186], and a phase 2 clinical trial is currently ongoing to determine its safety and effect on abnormal proteins in the brain with 300 mg for one month compared to placebo. Other drugs that aim to regulate ApoE expression (LXR agonist TO901317) [185, 188], block ApoE-Aβ interaction, disrupt ApoE4 domain (CB9032258, phthalazinone analogue) [189], mimic the receptor binding region [190] (COG112), and so forth, have shown benefits of reversing Aβ burden in vivo or in vitro, but did not reach the clinical trials yet. ApoE-targeted therapies are still at the early stage of development and relevant approaches and strategies are required to carefully evaluate them though, showing a huge promising battle with Alzheimer’s disease.

4.5. Neurotrophin

Nerve growth factor (NGF) as a neurotrophin plays a critical role promoting survival and maintaining the function of cholinergic neurons [191, 192]. In AD patients, transcription and translation levels of NGF were changed [193, 194], suggesting that NGF supplementation probably is a treatment approach for Alzheimer’s disease. NGF with unfavorable size and polarity is a peptide that cannot cross BBB [193, 195], so to safely and efficiently deliver it to the brain will be a great challenge [196, 197]. However, efforts have been made to overcome this obstacle. An example of strategy is as follows: CERE-110 uses adeno-associated virus to transfer a gene that makes NGF and is injected into AD patients’ brain. This approach undergoes a phase 2 study.

5. Concluding Remarks

Aβ cascade hypothesis was firstly proposed in 1992 [198] assuming that β-amyloid would be the suspect initiating pathogenesis of Alzheimer’s disease. So a series of explorations focusing on physiological and pathological processes that participate in the production, aggregation, and clearance of Aβ have been widely studied. The identification of two crucial enzymes (γ-secretase and BACE1), responsible for the cleavage of the presumably pathogenic Aβ from its precursor, suggests that the cure of AD may be around the corner.

However, failures in many large clinical trials using Aβ-targeted drugs (Table 4) and FDA approved compounds with marginal efficacy questioned the validity of Aβ cascade hypothesis. Indeed, Aβ hypothesis, having dominated the AD realm for two decades, has always been controversial. One of the most unfavorable evidences was the finding that amyloid plagues were diffused in AD patients’ brain postmortem (and neuroimaging outcomes confirmed the autopsy findings), which is abundant in healthy people [199, 200]. Nevertheless, plenty subsequent investigations put forward the oligomeric form of Aβ, rather than plagues, as the actual culprit for synapse dysfunction [201, 202] and the following amplifying events. This significant finding, at least partially, defended the validity of Aβ cascade hypothesis. But, still, why do therapeutic strategies targeting the secretases only have marginal efficacy? First, the two versatile secretases (BACE1 and -secretase) are at the same time responsible for processing other substrates, which unfortunately are either vital to metabolism normality or tricky to avoid targeting. The undesirable side effects are so overwhelming that they prohibit drug’s efficacy and approval. Second, the drug permeability through blood brain barrier (BBB) is another considerable problem. Most drugs described above have a poor capability to cross BBB, so it is reasonable to see numerous clinical trials, including those having progressed to phase 3, fail. Instead of questioning the plausible hypothesis, it is more imperative to cautiously design clinical studies and interpret the outcomes.

Table 4: Terminated trials targeting Aβ hypothesis.

Mechanism
RCT
End
Enrollment
Duration
Main reasons

BACE1 inhibitor
Rosiglitazone
2009.2
693
24 weeks
Unimproved cognitive status

LY2886721
2013.8
128

AE: 4 cases of liver damage in a phase 2 study in June 2013

GSI/GSM
Semagacestat
2011.5
164
>7 months
Unimproved cognitive status, but worsening functional ability; AE: skin cancers and infections

Avagacestat
2010.6
209
24 weeks
AE: gastrointestinal and dermatological abnormalities like diarrhea, nausea, vomiting, rash, and itching skin; nonmelanoma skin cancers; and worsened cognition

tarenflurbil
2008.5
1684
18 months
Insufficient pharmacodynamics: poor capability to penetrate the BBB

Active immunology
AN1792
2003.9
375

AE: 6 patients developed aseptic meningoencephalitis due cytotoxic T cell response in phase 2a trial

ACC-001
2014.2
126
24 months
Showed a serious side effect in phase 2 trial

Passive immunology
Bapineuzumab (AAB-001)
2012.6
1331
18 months
Showed no treatment effect on either cognitive or functional outcomes in two phase 3 trials

GSK3 inhibitor
Tideglusib (NP12)
2012.6
306
45 weeks
Missed its primary endpoint and some secondary endpoints

RCT: randomized controlled trial; AE: adverse event; BBB: blood brain barrier.

Data sources: http://www.clinicaltrials.gov/; http://www.alzforum.org/.

Given limited benefits from inhibition of Aβ production, more focus should be converted to the clearance strategy. Delivery of antibodies may be a good choice due to the safety leverage compared to vaccine. Besides, there are quite a few ongoing clinical trials using passive immunization. From Table 1, antibodies are capable of binding and clearing multiple forms of Aβ. It is important because there is equilibrium between oligomers and plagues of Aβ [203]. For a single-target antiaggregate disrupting formation or enhancing disassembly of Aβ oligomers, plagues as a reservoir will replenish and maintain the balance [204, 205]. So the property of simultaneously interfering different processes during Aβ aggregation suggested that passive immunization might be of a promising value.

In recent years other AD risk factors have been widely studied. Though no groundbreaking outcomes have been shown, it provided quite a few unprecedented opportunities. First, the validated AD specific biomarkers need to be carefully developed and examined. Biomarkers should be able to at least precisely indicate the response to therapeutic intervention to avoid misinterpretation of clinical trial data. Besides, current animal models have serious limitations. Most transgenic mouse models published in AD studies overproduce Aβ solely mimicking familial Alzheimer’s disease, might not suffice phenotypes of sporadic AD accounting for the dominant populations.

In addition, AD is a disorder that is too intricate and too factor-driven to be entirely understood from its pathogenesis. As we discussed previously, various factors (A, tau, inflammation, and apoE) complicatedly interact with each other. So the conventional “one protein, one drug, one disease” hypothesis would not work for Alzheimer’s disease. From the successful experience in therapeutic development in multifactorial diseases like AIDS, atherosclerosis, cancer, and depression, multitarget drugs or combination therapy can possibly generate more benefits. Since drugs with more than one target could possibly mitigate a redundancy effect in such a complex nerve network, this combination therapy or similar approach multitarget-directed ligands (MTDLs) might bring new hope in search of therapeutics for Alzheimer’s diseases [206, 207]. In this novel fashion, some combinations with approved drug are under clinical trials (Tables 1, 2, and 3, RCTs marked with ).

Notwithstanding these challenges, with more scientific insights from basic researches and cooperation between laboratories and pharmaceutical companies, it is very likely to find the optimum treatment for Alzheimer’s disease in the near future.

Abbreviation

AD: | Alzheimer’s disease
Aβ: | Amyloid β
NFT: | Intracellular neurofibrillar tangles
FDA: | Food and Drug Administration
APP: | Amyloid precursor protein
NMDA: | N-Methyl-D-aspartic acid
PS: | Presenilin
NICD: | Notch intracellular domain
GSI: | γ-Secretase inhibitors
CSF: | Cerebrospinal fluid
CNS: | Central nervous system
CSF: | Cerebrospinal fluid
GSM: | γ-Secretase modulators
NSAID: | Nonsteroidal anti-inflammatory drugs
BACE1: | Beta-site APP-cleaving enzyme 1
FAD: | Familial Alzheimer’s disease
BBB: | Blood brain barrier
COX: | Cyclooxygenase
GABA: | γ-Aminobutyric acid
ApoE: | Apolipoprotein E
MMSE: | Mini-mental-state exam
GSK3β: | Glycogen synthase kinase 3 beta
PP2A: | Protein phosphatase 2
cdk5: | Cyclin dependent kinase 5
TNF: | Tumor necrosis factor
IDE: | Insulin-degrading enzyme
LXR: | Liver X receptor
RXR: | Retinoid X Receptor
LDLR: | Low-density lipoprotein receptor
LRP1: | Low-density lipoprotein receptor-related protein 1
HSPG: | Low-density lipoprotein receptor-related protein 1
ABCA1: | ATP-binding cassette transporter 1
Ach: | Acetylcholine
LTP: | Long-term potentiation
5-HT: | Serotonin
NGF: | Nerve growth factor.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

References

M. Goedert and M. G. Spillantini, “A century of Alzheimer's disease,” Science, vol. 314, no. 5800, pp. 777–781, 2006.

G. E. Berrios, “Alzheimer's disease: a conceptual history,” International Journal of Geriatric Psychiatry, vol. 5, no. 6, pp. 355–365, 1990.

M. Hashimoto, E. Rockenstein, L. Crews, and E. Masliah, “Role of protein aggregation in mitochondrial dysfunction and neurodegeneration in Alzheimer's and Parkinson's diseases,” NeuroMolecular Medicine, vol. 4, no. 1-2, pp. 21–36, 2003.

L. Bäckman, S. Jones, A. Berger, E. J. Laukka, and B. J. Small, “Multiple cognitive deficits during the transition to Alzheimer's disease,” Journal of Internal Medicine, vol. 256, no. 3, pp. 195–204, 2004.

E. Arnáiz and O. Almkvist, “Neuropsychological features of mild cognitive impairment and preclinical Alzheimer's disease,” Acta Neurologica Scandinavica, vol. 107, no. 179, pp. 34–41, 2003.

H. Förstl and A. Kurz, “Clinical features of Alzheimer's disease,” European Archives of Psychiatry and Clinical Neuroscience, vol. 249, no. 6, pp. 288–290, 1999.

P. D. Meek, E. K. McKeithan, and G. T. Schumock, “Economic considerations in Alzheimer's disease,” Pharmacotherapy, vol. 18, no. 2, pp. 68–82, 1998.

A. Wimo, L. Jönsson, A. Gustavsson et al., “The economic impact of dementia in Europe in 2008-cost estimates from the Eurocode project,” International Journal of Geriatric Psychiatry, vol. 26, no. 8, pp. 825–832, 2011.

C. A. Thompson, K. Spilsbury, J. Hall, Y. Birks, C. Barnes, and J. Adamson, “Systematic review of information and support interventions for caregivers of people with dementia,” BMC Geriatrics, vol. 7, article 18, 2007.

J. Schneider, J. Murray, S. Banerjee, and A. Mann, “EUROCARE: a cross-national study of co-resident spouse carers for people with Alzheimer's disease: I—Factors associated with carer burden,” International Journal of Geriatric Psychiatry, vol. 14, pp. 651–661, 1999.

J. Murray, J. Schneider, S. Banerjee, and A. Mann, “EUROCARE, a cross-national study of co-resident spouse carers for people with Alzheimer's disease: II—a qualitative analysis of the experience of caregiving,” International Journal of Geriatric Psychiatry, vol. 14, pp. 662–667, 1999.

C. P. Ferri, M. Prince, C. Brayne et al., “Global prevalence of dementia: a Delphi consensus study,” The Lancet, vol. 366, no. 9503, pp. 2112–2117, 2005.

C. Reitz, C. Brayne, and R. Mayeux, “Epidemiology of Alzheimer disease,” Nature Reviews Neurology, vol. 7, no. 3, pp. 137–152, 2011.

M. Pohanka, “Cholinesterases, a target of pharmacology and toxicology,” Biomedical Papers of the Medical Faculty of the University Palacký, Olomouc, Czechoslovakia, vol. 155, no. 3, pp. 219–230, 2011.

P. M. Doraiswamy and G. L. Xiong, “Pharmacological strategies for the prevention of Alzheimer's disease,” Expert Opinion on Pharmacotherapy, vol. 7, no. 1, pp. 1–10, 2006.

A. Lleó, S. M. Greenberg, and J. H. Growdon, “Current pharmacotherapy for Alzheimer's disease,” Annual Review of Medicine, vol. 57, pp. 513–533, 2006.

C. W. Ritchie, D. Ames, T. Clayton, and R. Lai, “Metaanalysis of randomized trials of the efficacy and safety of donepezil, galantamine, and rivastigmine for the treatment of Alzheimer disease,” The American Journal of Geriatric Psychiatry, vol. 12, no. 4, pp. 358–369, 2004.

A. Whitehead, C. Perdomo, R. D. Pratt, J. Birks, G. K. Wilcock, and J. G. Evans, “Donepezil for the symptomatic treatment of patients with mild to moderate Alzheimer's disease: a meta-analysis of individual patient data from randomised controlled trials,” International Journal of Geriatric Psychiatry, vol. 19, no. 7, pp. 624–633, 2004.

R. Bullock, “Efficacy and safety of memantine in moderate-to-severe Alzheimer disease: the evidence to date,” Alzheimer Disease and Associated Disorders, vol. 20, no. 1, pp. 23–29, 2006.

M. L. Michaelis, “Drugs targeting Alzheimer's disease: some things old and some things new,” Journal of Pharmacology and Experimental Therapeutics, vol. 304, no. 3, pp. 897–904, 2003.

R. Vassar, B. D. Bennett, S. Babu-Khan et al., “β-Secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE,” Science, vol. 286, no. 5440, pp. 735–741, 1999.

R. Yan, M. J. Blenkowski, M. E. Shuck et al., “Membrane-anchored aspartyl protease with Alzheimer's disease -secretase activity,” Nature, vol. 402, no. 6761, pp. 533–537, 1999.

S. L. Roberds, J. Anderson, G. Basi et al., “BACE knockout mice are healthy despite lacking the primary β-secretase activity in brain: implications for Alzheimer's disease therapeutics,” Human Molecular Genetics, vol. 10, no. 12, pp. 1317–1324, 2001.

Y. Luo, B. Bolon, S. Kahn et al., “Mice deficient in BACE1, the Alzheimer's β-secretase, have normal phenotype and abolished β-amyloid generation,” Nature Neuroscience, vol. 4, no. 3, pp. 231–232, 2001.

M. Ohno, L. Chang, W. Tseng et al., “Temporal memory deficits in Alzheimer's mouse models: rescue by genetic deletion of BACE1,” European Journal of Neuroscience, vol. 23, no. 1, pp. 251–260, 2006.

M. Ohno, E. A. Sametsky, L. H. Younkin et al., “BACE1 deficiency rescues memory deficits and cholinergic dysfunction in a mouse model of Alzheimer's disease,” Neuron, vol. 41, no. 1, pp. 27–33, 2004.

S. M. Harrison, A. J. Harper, J. Hawkins et al., “BACE1 (β-secretase) transgenic and knockout mice: identification of neurochemical deficits and behavioral changes,” Molecular and Cellular Neuroscience, vol. 24, no. 3, pp. 646–655, 2003.

X. Hu, C. W. Hicks, W. He et al., “Bace1 modulates myelination in the central and peripheral nervous system,” Nature Neuroscience, vol. 9, no. 12, pp. 1520–1525, 2006.

M. Willem, A. N. Garratt, B. Novak et al., “Control of peripheral nerve myelination by the β-secretase BACE1,” Science, vol. 314, no. 5799, pp. 664–666, 2006.

D. Kobayashi, M. Zeller, T. Cole et al., “BACE1 gene deletion: impact on behavioral function in a model of Alzheimer's disease,” Neurobiology of Aging, vol. 29, no. 6, pp. 861–873, 2008.

X. Hu, W. He, C. Diaconu et al., “Genetic deletion of BACE1 in mice affects remyelination of sciatic nerves,” The FASEB Journal, vol. 22, no. 8, pp. 2970–2980, 2008.

F. M. Laird, H. Cai, A. V. Savonenko et al., “BACE1, a major determinant of selective vulnerability of the brain to amyloid-β amyloidogenesis, is essential for cognitive, emotional, and synaptic functions,” Journal of Neuroscience, vol. 25, no. 50, pp. 11693–11709, 2005.

L. McConlogue, M. Buttini, J. P. Anderson et al., “Partial reduction of BACE1 has dramatic effects on Alzheimer plaque and synaptic pathology in APP transgenic mice,” Journal of Biological Chemistry, vol. 282, no. 36, pp. 26326–26334, 2007.

I. Hussain, “The potential for BACE1 inhibitors in the treatment of Alzheimer's disease,” IDrugs, vol. 7, no. 7, pp. 653–658, 2004.

Y. Hamada and Y. Kiso, “Recent progress in the drug discovery of non-peptidic BACE1 inhibitors,” Expert Opinion on Drug Discovery, vol. 4, no. 4, pp. 390–416, 2009.

L. Michalik, J. Auwerx, J. P. Berger et al., “International union of pharmacology. LXI. Peroxisome proliferator-activated receptors,” Pharmacological Reviews, vol. 58, no. 4, pp. 726–741, 2006.

S. Mandrekar-Colucci, J. Colleen Karlo, and G. E. Landreth, “Mechanisms underlying the rapid peroxisome proliferator-activated receptor-γ-mediated amyloid clearance and reversal of cognitive deficits in a murine model of Alzheimer's disease,” The Journal of Neuroscience, vol. 32, no. 30, pp. 10117–10128, 2012.

G. Landreth, Q. Jiang, S. Mandrekar, and M. Heneka, “PPARγ agonists as therapeutics for the treatment of Alzheimer's disease,” Neurotherapeutics, vol. 5, no. 3, pp. 481–489, 2008.

S. Craft, “The role of metabolic disorders in Alzheimer disease and vascular dementia: two roads converged,” Archives of Neurology, vol. 66, no. 3, pp. 300–305, 2009.

Senate CoFUS, Staff Report on GlaxoSmithKline and the Diabetes Drug Avandia, US Government Printing Office, Washington, DC, USA, 2010.

W. Chang, G. Koelsch, S. Wong et al., “In vivo inhibition of Abeta production by memapsin 2 (beta-secretase) inhibitors,” Journal of Neurochemistry, vol. 89, no. 6, pp. 1409–1416, 2004.

A. K. Ghosh, N. Kumaragurubaran, L. Hong, G. Koelsh, and J. Tang, “Memapsin 2 (beta-secretase) inhibitors: drug development,” Current Alzheimer Research, vol. 5, no. 2, pp. 121–131, 2008.

J. J. N. Tang, “Beta-secretase as target for amyloid-reduction therapy,” Alzheimer's & Dementia, vol. 5, no. 4, p. 74, 2009.

P. C. May, “Preclinical to clinical translation of APP biomarker responses to LY2886721, a potent, oral BACE1 inhibitor in phase II development for Alzheimer's disease,” in Proceedings of the Neuroscience Meeting, Nanosymposium, Society for Neuroscience, vol. 542, p. 334.

M. Shoji, T. E. Golde, J. Ghiso et al., “Production of the Alzheimer amyloid β protein by normal proteolytic processing,” Science, vol. 258, no. 5079, pp. 126–129, 1992.

C. Haass, M. G. Schlossmacher, A. Y. Hung et al., “Amyloid beta-peptide is produced by cultured cells during normal metabolism,” Nature, vol. 359, no. 6393, pp. 322–325, 1992.

B. De Strooper, W. Annaert, P. Cupers et al., “A presenilin-1-dependent γ-secretase-like protease mediates release of notch intracellular domain,” Nature, vol. 398, no. 6727, pp. 518–522, 1999.

D. B. Henley, P. C. May, R. A. Dean, and E. R. Siemers, “Development of semagacestat (LY450139), a functional γ-secretase inhibitor, for the treatment of Alzheimer's disease,” Expert Opinion on Pharmacotherapy, vol. 10, no. 10, pp. 1657–1664, 2009.

B. P. Imbimbo, “Therapeutic potential of γ-secretase inhibitors and modulators,” Current Topics in Medicinal Chemistry, vol. 8, no. 1, pp. 54–61, 2008.

I. Maillard, S. H. Adler, and W. S. Pear, “Notch and the immune system,” Immunity, vol. 19, no. 6, pp. 781–791, 2003.

B. Z. Stanger, R. Datar, L. C. Murtaugh, and D. A. Melton, “Direct regulation of intestinal fate by Notch,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 35, pp. 12443–12448, 2005.

X. Xia, S. Qian, S. Soriano et al., “Loss of presenilin 1 is associated with enhanced β-catenin signaling and skin tumorigenesis,” Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 19, pp. 10863–10868, 2001.

M. Nicolas, A. Wolfer, K. Raj et al., “Notch1 functions as a tumor suppressor in mouse skin,” Nature Genetics, vol. 33, no. 3, pp. 416–421, 2003.

A. S. Fleisher, R. Raman, E. R. Siemers et al., “Phase 2 safety trial targeting amyloid β production with a γ-secretase inhibitor in Alzheimer disease,” Archives of Neurology, vol. 65, no. 8, pp. 1031–1038, 2008.

R. J. Bateman, E. R. Siemers, K. G. Mawuenyega et al., “A γ-secretase inhibitor decreases amyloid-β production in the central nervous system,” Annals of Neurology, vol. 66, no. 1, pp. 48–54, 2009.

R. S. Doody, R. Raman, M. Farlow et al., “A phase 3 trial of semagacestat for treatment of Alzheimer's disease,” The New England Journal of Medicine, vol. 369, no. 4, pp. 341–350, 2013.

T. Borgegård, S. Gustavsson, C. Nilsson et al., “Alzheimer's disease: presenilin 2-sparing γ-secretase inhibition is a tolerable Aβ peptide-lowering strategy,” The Journal of Neuroscience, vol. 32, no. 48, pp. 17297–17305, 2012.

B. Zhao, M. Yu, M. Neitzel et al., “Identification of γ-secretase inhibitor potency determinants on presenilin,” The Journal of Biological Chemistry, vol. 283, no. 5, pp. 2927–2938, 2008.

L. Serneels, J. van Biervliet, K. Craessaerts et al., “γ-Secretase heterogeneity in the aph1 subunit: relevance for alzheimer's disease,” Science, vol. 324, no. 5927, pp. 639–642, 2009.

C. J. Crump, S. V. Castro, F. Wang et al., “BMS-708,163 targets presenilin and lacks notch-sparing activity,” Biochemistry, vol. 51, no. 37, pp. 7209–7211, 2012.

L. Ereshefsky, S. Jhee, M. Yen, S. Moran, S. Pretorius, and J. Adams, “Cerebrospinal fluid -amyloid and dynabridging in Alzheimer's disease drug development,” Biomarkers in Medicine, vol. 3, no. 6, pp. 711–721, 2009.

J. Wang, L. Ho, and G. M. Passinetti, “The development of NIC5-15.A natural anti-diabetic agent, in the treatment of Alzheimer’s disease,” Alzheimer’s & Dementia: The Journal of the Alzheimer's Association, vol. 1, article 62, 2005.

H. Grossman, G. Marzloff, X. Luo, D. LeRoith, M. Sano, and G. Pasinetti, “NIC5-15 as a treatment for Alzheimer's: safety, pharmacokinetics and clinical variables,” Alzheimer's & Dementia, vol. 5, no. 4, supplement, p. P259, 2009.

S. Weggen, J. L. Eriksen, P. Das et al., “A subset of NSAIDs lower amyloidogenic Aβ42 independently of cyclooxygenase activity,” Nature, vol. 414, no. 6860, pp. 212–216, 2001.

T. Tomita, “Secretase inhibitors and modulators for Alzheimer's disease treatment,” Expert Review of Neurotherapeutics, vol. 9, no. 5, pp. 661–679, 2009.

D. R. Galasko, N. Graff-Radford, S. May et al., “Safety, tolerability, pharmacokinetics, and Aβ levels after short-term administration of R-flurbiprofen in healthy elderly individuals,” Alzheimer Disease and Associated Disorders, vol. 21, no. 4, pp. 292–299, 2007.

G. K. Wilcock, S. E. Black, S. B. Hendrix, K. H. Zavitz, E. A. Swabb, and M. A. Laughlin, “Efficacy and safety of tarenflurbil in mild to moderate Alzheimer's disease: a randomised phase II trial,” The Lancet Neurology, vol. 7, no. 6, pp. 483–493, 2008.

R. C. Green, L. S. Schneider, D. A. Amato et al., “Effect of tarenflurbil on cognitive decline and activities of daily living in patients with mild Alzheimer disease: a randomized controlled trial,” Journal of the American Medical Association, vol. 302, no. 23, pp. 2557–2564, 2009.

B. P. Imbimbo, “Why did tarenflurbil fail in alzheimer's disease?” Journal of Alzheimer's Disease, vol. 17, no. 4, pp. 757–760, 2009.

S. Sivilia, L. Lorenzini, A. Giuliani et al., “Multi-target action of the novel anti-Alzheimer compound CHF5074: in vivo study of long term treatment in Tg2576 mice,” BMC Neuroscience, vol. 14, article 44, 2013.

G. Poli, E. Corda, B. Lucchini et al., “Therapeutic effect of CHF5074, a new -secretase modulator, in a mouse model of scrapie,” Prion, vol. 6, no. 1, pp. 62–72, 2012.

A. Hall, R. L. Elliott, G. M. P. Giblin et al., “Piperidine-derived γ-secretase modulators,” Bioorganic & Medicinal Chemistry Letters, vol. 20, no. 3, pp. 1306–1311, 2010.

R. J. van Marum, “Current and future therapy in Alzheimer's disease,” Fundamental & Clinical Pharmacology, vol. 22, no. 3, pp. 265–274, 2008.

H. H. Griffiths, I. J. Morten, and N. M. Hooper, “Emerging and potential therapies for Alzheimer's disease,” Expert Opinion on Therapeutic Targets, vol. 12, no. 6, pp. 693–704, 2008.

M. Marcade, J. Bourdin, N. Loiseau et al., “Etazolate, a neuroprotective drug linking GABAA receptor pharmacology to amyloid precursor protein processing,” Journal of Neurochemistry, vol. 106, no. 1, pp. 392–404, 2008.

L. Desire, M. Marcade, H. Peillon, D. Drouin, and O. Sol, “Clinical trials of EHT 0202, a neuroprotective and procognitive alpha-secretase,” Alzheimer's & Dementia, vol. 5, no. 4, pp. P255–P256, 2009.

R. Etcheberrigaray, M. Tan, I. Dewachtert et al., “Therapeutic effects of PKC activators in Alzheimer's disease transgenic mice,” Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 30, pp. 11141–11146, 2004.

D. L. Sparks, M. N. Sabbagh, D. J. Connor et al., “Atorvastatin for the treatment of mild to moderate Alzheimer disease: preliminary results,” Archives of Neurology, vol. 62, no. 5, pp. 753–757, 2005.

R. W. Jones, M. Kivipelto, H. Feldman et al., “The Atorvastatin/Donepezil in Alzheimer's Disease Study (LEADe): design and baseline characteristics,” Alzheimer's and Dementia, vol. 4, no. 2, pp. 145–153, 2008.

H. H. Feldman, R. S. Doody, M. Kivipelto et al., “Randomized controlled trial of atorvastatin in mild to moderate Alzheimer disease: LEADe,” Neurology, vol. 74, no. 12, pp. 956–964, 2010.

R. G. Riekse, G. Li, E. C. Petrie et al., “Effect of statins on Alzheimer's disease biomarkers in cerebrospinal fluid,” Journal of Alzheimer's Disease, vol. 10, no. 4, pp. 399–406, 2006.

M. Calamai, F. Chiti, and C. M. Dobson, “Amyloid fibril formation can proceed from different conformations of a partially unfolded protein,” Biophysical Journal, vol. 89, no. 6, pp. 4201–4210, 2005.

A. E. Roher, K. C. Palmer, E. C. Yurewicz, M. J. Ball, and B. D. Greenberg, “Morphological and biochemical analyses of amyloid plaque core proteins purified from Alzheimer disease brain tissue,” Journal of Neurochemistry, vol. 61, no. 5, pp. 1916–1926, 1993.

P. S. Aisen, D. Saumier, R. Briand et al., “A Phase II study targeting amyloid-β with 3APS in mild-to-moderate Alzheimer disease,” Neurology, vol. 67, no. 10, pp. 1757–1763, 2006.

H. Amijee and D. I. C. Scopes, “The quest for small molecules as amyloid inhibiting therapies for Alzheimer's disease,” Journal of Alzheimer's Disease, vol. 17, no. 1, pp. 33–47, 2009.

J. McLaurin, M. E. Kierstead, M. E. Brown et al., “Cyclohexanehexol inhibitors of A aggregation prevent and reverse Alzheimer phenotype in a mouse model,” Nature Medicine, vol. 12, no. 7, pp. 801–808, 2006.

S. Salloway, R. Sperling, S. Gilman et al., “A phase 2 multiple ascending dose trial of bapineuzumab in mild to moderate Alzheimer disease,” Neurology, vol. 73, no. 24, pp. 2061–2070, 2009.

Elan, “Elan and Transition Therapeutics announce modifications to ELND005 phase II clinical trials in Alzheimer’s disease,” 2009

S. A. Mandel, T. Amit, L. Kalfon, L. Reznichenko, O. Weinreb, and M. B. H. Youdim, “Cell signaling pathways and iron chelation in the neurorestorative activity of green tea polyphenols: Special reference to epigallocatechin gallate (EGCG),” Journal of Alzheimer's Disease, vol. 15, no. 2, pp. 211–222, 2008.

P. A. Adlard, R. A. Cherny, D. I. Finkelstein et al., “Rapid restoration of cognition in Alzheimer's transgenic mice with 8-hydroxy quinoline analogs is associated with decreased interstitial Abeta,” Neuron, vol. 59, no. 1, pp. 43–55, 2008.

D. Schenk, R. Barbour, W. Dunn et al., “Immunization with amyloid-β attenuates Alzheimer disease-like pathology in the PDAPP mouse,” Nature, vol. 400, no. 6740, pp. 173–177, 1999.

M. N. Pangalos, S. J. Jacobsen, and P. H. Reinhart, “Disease modifying strategies for the treatment of Alzheimer's disease targeted at modulating levels of the β-amyloid peptide,” Biochemical Society Transactions, vol. 33, no. 4, pp. 553–558, 2005.

J.-M. Orgogozo, S. Gilman, J.-F. Dartigues et al., “Subacute meningoencephalitis in a subset of patients with AD after A42 immunization,” Neurology, vol. 61, no. 1, pp. 46–54, 2003.

J. A. R. Nicolll, D. Wilkinson, C. Holmes, P. Steart, H. Markham, and R. O. Weller, “Neuropathology of human Alzheimer disease after immunization with amyloid- peptide: a case report,” Nature Medicine, vol. 9, no. 4, pp. 448–452, 2003.

I. Ferrer, M. B. Rovira, M. L. S. Guerra, M. J. Rey, and F. Costa-Jussá, “Neuropathology and pathogenesis of encephalitis following amyloid-β immunization in Alzheimer's disease,” Brain Pathology, vol. 14, no. 1, pp. 11–20, 2004.

E. Masliah, L. Hansen, A. Adame et al., “Aβ vaccination effects on plaque pathology in the absence of encephalitis in Alzheimer disease,” Neurology, vol. 64, no. 1, pp. 129–131, 2005.

B. Winblad, L. Minthon, and A. Floesser, “Results of the first-in-man study with the active Aβ immunotherapy CAD106 in Alzheimer patients,” Alzheimer's & Dementia, vol. 5, no. 4, supplement, pp. P113–P114, 2009.

A. Schneeberger, M. Mandler, O. Otava, W. Zauner, F. Mattner, and W. Schmidt, “Development of AFFITOPE vaccines for Alzheimer's Disease (AD)—from concept to clinical testing,” Journal of Nutrition, Health and Aging, vol. 13, no. 3, pp. 264–267, 2009.

F. Bard, C. Cannon, R. Barbour et al., “Peripherally administered antibodies against amyloid β-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease,” Nature Medicine, vol. 6, no. 8, pp. 916–919, 2000.

S. Oddo, L. Billings, J. P. Kesslak, D. H. Cribbs, and F. M. LaFerla, “Aβ immunotherapy leads to clearance of early, but not late, hyperphosphorylated tau aggregates via the proteasome,” Neuron, vol. 43, no. 3, pp. 321–332, 2004.

D. M. Wilcock, N. Gharkholonarehe, W. E. Van Nostrand, J. Davis, M. P. Vitek, and C. A. Colton, “Amyloid reduction by amyloid-β vaccination also reduces mouse tau pathology and protects from neuron loss in two mouse models of Alzheimer's disease,” The Journal of Neuroscience, vol. 29, no. 25, pp. 7957–7965, 2009.

I. Klyubin, D. M. Walsh, C. A. Lemere et al., “Amyloid β protein immunotherapy neutralizes Aβ oligomers that disrupt synaptic plasticity in vivo,” Nature Medicine, vol. 11, no. 5, pp. 556–561, 2005.

E. R. Siemers, S. Friedrich, R. A. Dean, et al., “P4-346: Safety, tolerability and biomarker effects of an Abeta monoclonal antibody administered to patients with Alzheimer's disease,” Alzheimer's & Dementia, vol. 4, no. 4, supplement, p. T774, 2008.

J. E. McElhaney and R. B. Effros, “Immunosenescence: what does it mean to health outcomes in older adults?” Current Opinion in Immunology, vol. 21, no. 4, pp. 418–424, 2009.

K. Iqbal and I. Grundke-Iqbal, “Alzheimer neurofibrillary degeneration: significance, etiopathogenesis, therapeutics and prevention,” Journal of Cellular and Molecular Medicine, vol. 12, no. 1, pp. 38–55, 2008.

M. A. Meraz-Ríos, K. I. Lira-de León, V. Campos-Peña, M. A. de Anda-Hernández, and R. Mena-López, “Tau oligomers and aggregation in Alzheimer's disease,” Journal of Neurochemistry, vol. 112, no. 6, pp. 1353–1367, 2010.

A. J. Myers, M. Kaleem, L. Marlowe et al., “The H1c haplotype at the MAPT locus is associated with Alzheimer's disease,” Human Molecular Genetics, vol. 14, no. 16, pp. 2399–2404, 2005.

N. Brouwers, K. Sleegers, S. Engelborghs et al., “Genetic risk and transcriptional variability of amyloid precursor protein in Alzheimer's disease,” Brain, vol. 129, no. 11, pp. 2984–2991, 2006.

J. J. Pei, M. Sjögren, and B. Winblad, “Neurofibrillary degeneration in Alzheimer’s disease: from molecular mechanisms to identification of drug targets,” Current Opinion in Psychiatry, vol. 21, no. 6, pp. 555–561, 2008.

L. Martin, X. Latypova, C. M. Wilson et al., “Tau protein kinases: involvement in Alzheimer's disease,” Ageing Research Reviews, vol. 12, no. 1, pp. 289–309, 2013.

J. Pei, T. Tanaka, Y. Tung, E. Braak, K. Iqbal, and I. Grundke-Iqbal, “Distribution, levels, and activity of glycogen synthase kinase-3 in the Alzheimer disease brain,” Journal of Neuropathology and Experimental Neurology, vol. 56, no. 1, pp. 70–78, 1997.

K. Leroy, Z. Yilmaz, and J. P. Brion, “Increased level of active GSK-3β in Alzheimer's disease and accumulation in argyrophilic grains and in neurones at different stages of neurofibrillary degeneration,” Neuropathology and Applied Neurobiology, vol. 33, no. 1, pp. 43–55, 2007.

P. N. Tariot and P. S. Aisen, “Can lithium or valproate untie tangles in Alzheimer's disease?” The Journal of Clinical Psychiatry, vol. 70, no. 6, pp. 919–921, 2009.

H. Hampel, M. Ewers, K. Bürger et al., “Lithium trial in Alzheimer's disease: a randomized, single-blind, placebo-controlled, multicenter 10-week study,” The Journal of Clinical Psychiatry, vol. 70, no. 6, pp. 922–931, 2009.

L. Serenó, M. Coma, M. Rodríguez et al., “A novel GSK-3 inhibitor reduces Alzheimer's pathology and rescues neuronal loss in vivo,” Neurobiology of Disease, vol. 35, no. 3, pp. 359–367, 2009.

W. Noble, V. Olm, K. Takata et al., “Cdk5 is a key factor in tau aggregation and tangle formation in vivo,” Neuron, vol. 38, no. 4, pp. 555–565, 2003.

Y. Wen, E. Planel, M. Herman et al., “Interplay between cyclin-dependent kinase 5 and glycogen synthase kinase 3β mediated by neuregulin signaling leads to differential effects on tau phosphorylation and amyloid precursor protein processing,” Journal of Neuroscience, vol. 28, no. 10, pp. 2624–2632, 2008.

C. M. Wischik, P. C. Edwards, R. Y. K. Lai, M. Roth, and C. R. Harrington, “Selective inhibition of Alzheimer disease-like tau aggregation by phenothiazines,” Proceedings of the National Academy of Sciences of the United States of America, vol. 93, no. 20, pp. 11213–11218, 1996.

S. Deiana, C. R. Harrington, C. M. Wischik, and G. Riedel, “Methylthioninium chloride reverses cognitive deficits induced by scopolamine: comparison with rivastigmine,” Psychopharmacology, vol. 202, no. 1–3, pp. 53–65, 2009.

C. Wischik, “Rember: issues in design of a phase 3 disease modifying clinical trial of tau aggregation inhibitor therapy in Alzheimer’s disease,” Alzheimer's & Dementia: The Journal of the Alzheimer's Association, vol. 5, no. 4, p. P74, 2009.

Y. Wen, W. Li, E. C. Poteet et al., “Alternative mitochondrial electron transfer as a novel strategy for neuroprotection,” The Journal of Biological Chemistry, vol. 286, no. 18, pp. 16504–16515, 2011.

P. D. Riha, J. C. Rojas, and F. Gonzalez-Lima, “Beneficial network effects of methylene blue in an amnestic model,” NeuroImage, vol. 54, no. 4, pp. 2623–2634, 2011.

D. E. Hurtado, L. Molina-Porcel, M. Iba et al., “A accelerates the spatiotemporal progression of tau pathology and augments tau amyloidosis in an Alzheimer mouse model,” The American Journal of Pathology, vol. 177, no. 4, pp. 1977–1988, 2010.

B. Zhang, J. Carroll, J. Q. Trojanowski et al., “The microtubule-stabilizing agent, epothilone D, reduces axonal dysfunction, neurotoxicity, cognitive deficits, and alzheimer-like pathology in an interventional study with aged tau transgenic mice,” Journal of Neuroscience, vol. 32, no. 11, pp. 3601–3611, 2012.

K. N. Green, J. S. Steffan, H. Martinez-Coria et al., “Nicotinamide restores cognition in Alzheimer's disease transgenic mice via a mechanism involving sirtuin inhibition and selective reduction of Thr231-phosphotau,” The Journal of Neuroscience, vol. 28, no. 45, pp. 11500–11510, 2008.

P. H. St George-Hyslop, “Piecing together Alzheimer's,” Scientific American, vol. 283, no. 6, pp. 76–83, 2000.

L. A. Craig, N. S. Hong, and R. J. McDonald, “Revisiting the cholinergic hypothesis in the development of Alzheimer's disease,” Neuroscience and Biobehavioral Reviews, vol. 35, no. 6, pp. 1397–1409, 2011.

I. Bezprozvanny and M. P. Mattson, “Neuronal calcium mishandling and the pathogenesis of Alzheimer's disease,” Trends in Neurosciences, vol. 31, no. 9, pp. 454–463, 2008.

A. C. Santucci, P. D. Kanof, and V. Haroutunian, “Effect of physostigmine on memory consolidation and retrieval processes in intact and nucleus basalis-lesioned rats,” Psychopharmacology, vol. 99, no. 1, pp. 70–74, 1989.

C. L. Hunter, H. A. Bimonte-Nelson, M. Nelson, C. B. Eckman, and A. Granholm, “Behavioral and neurobiological markers of Alzheimer's disease in Ts65Dn mice: effects of estrogen,” Neurobiology of Aging, vol. 25, no. 7, pp. 873–884, 2004.

L. M. Veng, A.-C. Granholm, and G. M. Rose, “Age-related sex differences in spatial learning and basal forebrain cholinergic neurons in F344 rats,” Physiology & Behavior, vol. 80, no. 1, pp. 27–36, 2003.

W. D. Hirst, B. Abrahamsen, F. E. Blaney et al., “Differences in the central nervous system distribution and pharmacology of the mouse 5-hydroxytryptamine-6 receptor compared with rat and human receptors investigated by radioligand binding, site-directed mutagenesis, and molecular modeling,” Molecular Pharmacology, vol. 64, no. 6, pp. 1295–1308, 2003.

A. Bessis, C. Béchade, D. Bernard, and A. Roumier, “Microglial control of neuronal death and synaptic properties,” Glia, vol. 55, no. 3, pp. 233–238, 2007.

J. Calderó, N. Brunet, D. Ciutat, M. Hereu, and J. E. Esquerda, “Development of microglia in the chick embryo spinal cord: implications in the regulation of motoneuronal survival and death,” Journal of Neuroscience Research, vol. 87, no. 11, pp. 2447–2466, 2009.

H. Wake, A. J. Moorhouse, S. Jinno, S. Kohsaka, and J. Nabekura, “Resting microglia directly monitor the functional state of synapses in vivo and determine the fate of ischemic terminals,” Journal of Neuroscience, vol. 29, no. 13, pp. 3974–3980, 2009.

T. Wyss-Coray, “Inflammation in Alzheimer disease: driving force, bystander or beneficial response?” Nature Medicine, vol. 12, no. 9, pp. 1005–1015, 2006.

J. Luber-Narod and J. Rogers, “Immune system associated antigens expressed by cells of the human central nervous system,” Neuroscience Letters, vol. 94, no. 1-2, pp. 17–22, 1988.

J. Rogers, J. Luber-Narod, S. D. Styren, and W. H. Civin, “Expression of immune system-associated antigens by cells of the human central nervous system: relationship to the pathology of Alzheimer's disease,” Neurobiology of Aging, vol. 9, pp. 339–349, 1988.

D. G. Walker and L. F. Lue, “Investigations with cultured human microglia on pathogenic mechanisms of Alzheimer's disease and other neurodegenerative diseases,” Journal of Neuroscience Research, vol. 81, no. 3, pp. 412–425, 2005.

A. Majumdar, H. Chung, G. Dolios et al., “Degradation of fibrillar forms of Alzheimer's amyloid β-peptide by macrophages,” Neurobiology of Aging, vol. 29, no. 5, pp. 707–715, 2008.

K. I. Andreasson, A. Savonenko, S. Vidensky et al., “Age-dependent cognitive deficits and neuronal apoptosis in cyclooxygenase-2 transgenic mice,” Journal of Neuroscience, vol. 21, no. 20, pp. 8198–8209, 2001.

X. Liang, L. Wu, Q. Wang et al., “Function of COX-2 and prostaglandins in neurological disease,” Journal of Molecular Neuroscience, vol. 33, no. 1, pp. 94–99, 2007.

P. S. Aisen, “The potential of anti-inflammatory drugs for the treatment of Alzheimer's disease,” Lancet Neurology, vol. 1, no. 5, pp. 279–284, 2002.

I. R. A. Mackenzie, “Postmortem studies of the effect of anti-inflammatory drugs on Alzheimer-type pathology and associated inflammation,” Neurobiology of Aging, vol. 22, no. 6, pp. 819–822, 2001.

M. Hüll, K. Lieb, and B. L. Fiebich, “Pathways of inflammatory activation in Alzheimer's disease: potential targets for disease modifying drugs,” Current Medicinal Chemistry, vol. 9, no. 1, pp. 83–88, 2002.

M. E. Harris-White, T. Chu, S. A. Miller et al., “Estrogen (E2) and glucocorticoid (Gc) effects on microglia and Aβ clearance in vitro and in vivo,” Neurochemistry International, vol. 39, no. 5-6, pp. 435–448, 2001.

P. Goyarzu, D. H. Malin, F. C. Lau et al., “Blueberry supplemented diet: effects on object recognition memory and nuclear factor-kappa B levels in aged rats,” Nutritional Neuroscience, vol. 7, no. 2, pp. 75–83, 2004.

D. F. Obregon, K. Rezai-Zadeh, Y. Bai et al., “ADAM10 activation is required for green tea (-)-epigallocatechin-3-gallate- induced α-secretase cleavage of amyloid precursor protein,” The Journal of Biological Chemistry, vol. 281, no. 24, pp. 16419–16427, 2006.

D. Laurin, K. H. Masaki, D. J. Foley, L. R. White, and L. J. Launer, “Midlife dietary intake of antioxidants and risk of late-life incident dementia: the Honolulu-Asia Aging Study,” American Journal of Epidemiology, vol. 159, no. 10, pp. 959–967, 2004.

P. H. Reddy and M. F. Beal, “Amyloid beta, mitochondrial dysfunction and synaptic damage: implications for cognitive decline in aging and Alzheimer's disease,” Trends in Molecular Medicine, vol. 14, no. 2, pp. 45–53, 2008.

C. A. Hansson Petersen, N. Alikhani, H. Behbahani et al., “The amyloid β-peptide is imported into mitochondria via the TOM import machinery and localized to mitochondrial cristae,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 35, pp. 13145–13150, 2008.

R. E. Davis and M. Williams, “Mitochondrial function and dysfunction: an update,” The Journal of Pharmacology and Experimental Therapeutics, vol. 342, no. 3, pp. 598–607, 2012.

D. J. Triggle, “Nous sommes tous des bacteries: implications for medicine, pharmacology and public health,” Biochemical Pharmacology, vol. 84, no. 12, pp. 1543–1550, 2012.

R. H. Swerdlow and S. M. Khan, “A “mitochondrial cascade hypothesis” for sporadic Alzheimer's disease,” Medical Hypotheses, vol. 63, no. 1, pp. 8–20, 2004.

R. S. Doody, S. I. Gavrilova, M. Sano et al., “Effect of dimebon on cognition, activities of daily living, behaviour, and global function in patients with mild-to-moderate Alzheimer's disease: a randomised, double-blind, placebo-controlled study,” The Lancet, vol. 372, no. 9634, pp. 207–215, 2008.

R. A. Sperling, C. R. Jack Jr., and P. S. Aisen, “Testing the right target and right drug at the right stage,” Science Translational Medicine, vol. 3, no. 111, Article ID 111cm33, 2011.

P. Tariot, M. Sabbagh, S. Flitman, P. Reyes, L. Taber, and L. Seely, “A safety, tolerability and pharmacokinetic study of dimebon in patients with Alzheimer’s disease already receiving donepezil,” Alzheimer's & Dementia, vol. 5, no. 4, supplement, p. P251, 2009.

E. M. Reiman, K. Chen, G. E. Alexander et al., “Functional brain abnormalities in young adults at genetic risk for late-onset Alzheimer's dementia,” Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 1, pp. 284–289, 2004.

P. A. Maher and D. R. Schubert, “Metabolic links between diabetes and Alzheimer’s disease,” Expert Review of Neurotherapeutics, vol. 9, no. 5, pp. 617–630, 2009.

S. C. Correia, R. X. Santos, G. Perry, X. Zhu, P. IMoreira, and M. A. Smith, “Insulin-resistant brain state: the culprit in sporadic Alzheimer's disease?” Ageing Research Reviews, vol. 10, no. 2, pp. 264–273, 2011.

I. Clark, C. Atwood, R. Bowen, G. Paz-Filho, and B. Vissel, “Tumor necrosis factor-induced cerebral insulin resistance in Alzheimer's disease links numerous treatment rationales,” Pharmacological Reviews, vol. 64, no. 4, pp. 1004–1026, 2012.

J. Butchart and C. Holmes, “Systemic and central immunity in Alzheimer's disease: therapeutic implications,” CNS Neuroscience and Therapeutics, vol. 18, no. 1, pp. 64–76, 2012.

W. Q. Qiu and M. F. Folstein, “Insulin, insulin-degrading enzyme and amyloid-β peptide in Alzheimer's disease: review and hypothesis,” Neurobiology of Aging, vol. 27, no. 2, pp. 190–198, 2006.

F. J. Gil-Bea, M. Solas, A. Solomon et al., “Insulin levels are decreased in the cerebrospinal fluid of women with prodomal alzheimer's disease,” Journal of Alzheimer's Disease, vol. 22, no. 2, pp. 405–413, 2010.

P. Dandona, I. Mohamed, H. Ghanim et al., “Insulin suppresses the expression of amyloid precursor protein, presenilins, and glycogen synthase kinase-3β in peripheral blood mononuclear cells,” The Journal of Clinical Endocrinology & Metabolism, vol. 96, no. 6, pp. 1783–1788, 2011.

P. L. Mcclean, V. Parthsarathy, E. Faivre, and C. Holscher, “The diabetes drug liraglutide prevents degenerative processes in a mouse model of Alzheimer's disease,” Journal of Neuroscience, vol. 31, no. 17, pp. 6587–6594, 2011.

K. N. Manolopoulos, L.-O. Klotz, P. Korsten, S. R. Bornstein, and A. Barthel, “Linking Alzheimer's disease to insulin resistance: the FoxO response to oxidative stress,” Molecular Psychiatry, vol. 15, no. 11, pp. 1046–1052, 2010.

S. M. De La Monte, M. Tong, N. Lester-Coll, M. Plater Jr., and J. R. Wands, “Therapeutic rescue of neurodegeneration in experimental type 3 diabetes: relevance to Alzheimer's disease,” Journal of Alzheimer's Disease, vol. 10, no. 1, pp. 89–109, 2006.

S. Craft, L. D. Baker, T. J. Montine et al., “Intranasal insulin therapy for Alzheimer disease and amnestic mild cognitive impairment: A pilot clinical trial,” Archives of Neurology, vol. 69, no. 1, pp. 29–38, 2012.

M. Chartier-Harlin, M. Parfitt, S. Legrain et al., “Apolipoprotein E, 4 allele as a major risk factor for sporadic early and late-onset forms of Alzheimer's disease: analysis of the 19q13.2 chromosomal region,” Human Molecular Genetics, vol. 3, no. 4, pp. 569–574, 1994.

H. Houlden, R. Crook, H. Backhovens, et al., “ApoE genotype is a risk factor in nonpresenilin early-onset Alzheimer's disease families,” The American Journal of Medical Genetics, vol. 81, pp. 117–121, 1998.

E. H. Corder, A. M. Saunders, W. J. Strittmatter et al., “Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families,” Science, vol. 261, no. 5123, pp. 921–923, 1993.

G. Bu, “Apolipoprotein e and its receptors in Alzheimer's disease: pathways, pathogenesis and therapy,” Nature Reviews Neuroscience, vol. 10, no. 5, pp. 333–344, 2009.

G. W. Rebeck, J. S. Reiter, D. K. Strickland, and B. T. Hyman, “Apolipoprotein E in sporadic Alzheimer's disease: allelic variation and receptor interactions,” Neuron, vol. 11, no. 4, pp. 575–580, 1993.

Y. Namba, M. Tomonaga, H. Kawasaki, E. Otomo, and K. Ikeda, “Apolipoprotein E immunoreactivity in cerebral amyloid deposits and neurofibrillary tangles in Alzheimer's disease and kuru plaque amyloid in Creutzfeldt-Jakob disease,” Brain Research, vol. 541, no. 1, pp. 163–166, 1991.

E. Kok, S. Haikonen, T. Luoto et al., “Apolipoprotein E-dependent accumulation of alzheimer disease-related lesions begins in middle age,” Annals of Neurology, vol. 65, no. 6, pp. 650–657, 2009.

R. J. Caselli, A. C. Dueck, D. Osborne et al., “Longitudinal modeling of age-related memory decline and the APOE ε4 effect,” The New England Journal of Medicine, vol. 361, no. 3, pp. 255–263, 2009.

G. J. Izaks, R. T. Gansevoort, A. M. van der Knaap, G. Navis, R. P. F. Dullaart, and J. P. J. Slaets, “The association of APOE genotype with cognitive function in persons aged 35 years or older,” PLoS ONE, vol. 6, no. 11, Article ID e27415, 2011.

M. N. Haan, L. Shemanski, W. J. Jagust, T. A. Manolio, and L. Kuller, “The role of APOE 4 in modulating effects of other risk factors for cognitive decline in elderly persons,” Journal of the American Medical Association, vol. 282, no. 1, pp. 40–46, 1999.

R. Peila, B. L. Rodriguez, and L. J. Launer, “Type 2 diabetes, APOE gene, and the risk for dementia and related pathologies: the Honolulu-Asia Aging Study,” Diabetes, vol. 51, no. 4, pp. 1256–1262, 2002.

T. Kanekiyo, J. Zhang, Q. Liu, C. Liu, L. Zhang, and G. Bu, “Heparan sulphate proteoglycan and the low-density lipoprotein receptor-related protein 1 constitute major pathways for neuronal amyloid-β uptake,” Journal of Neuroscience, vol. 31, no. 5, pp. 1644–1651, 2011.

J. Kim, J. M. Castellano, H. Jiang et al., “Overexpression of low-density lipoprotein receptor in the brain markedly inhibits amyloid deposition and increases extracellular Aβ clearance,” Neuron, vol. 64, no. 5, pp. 632–644, 2009.

J. M. Castellano, J. Kim, F. R. Stewart et al., “Human apoE isoforms differentially regulate brain amyloid-β peptide clearance,” Science Translational Medicine, vol. 3, no. 89, Article ID 89ra57, 2011.

A. Rapp, B. Gmeiner, and M. Hüttinger, “Implication of apoE isoforms in cholesterol metabolism by primary rat hippocampal neurons and astrocytes,” Biochimie, vol. 88, no. 5, pp. 473–483, 2006.

T. Vanmierlo, K. Rutten, J. Dederen et al., “Liver X receptor activation restores memory in aged AD mice without reducing amyloid,” Neurobiology of Aging, vol. 32, no. 7, pp. 1262–1272, 2011.

P. E. Cramer, J. R. Cirrito, D. W. Wesson et al., “ApoE-directed therapeutics rapidly clear β-amyloid and reverse deficits in AD mouse models,” Science, vol. 335, no. 6075, pp. 1503–1506, 2012.

N. F. Fitz, A. A. Cronican, M. Saleem et al., “Abca1 deficiency affects Alzheimer's disease-like phenotype in human ApoE4 but not in ApoE3-targeted replacement mice,” Journal of Neuroscience, vol. 32, no. 38, pp. 13125–13136, 2012.

D. R. Riddell, H. Zhou, T. A. Comery et al., “The LXR agonist TO901317 selectively lowers hippocampal Aβ42 and improves memory in the Tg2576 mouse model of Alzheimer's disease,” Molecular and Cellular Neuroscience, vol. 34, no. 4, pp. 621–628, 2007.

H. Chen, Z. Liu, A. Meyer-Franke et al., “Small molecule structure correctors abolish detrimental effects of apolipoprotein E4 in cultured neurons,” The Journal of Biological Chemistry, vol. 287, no. 8, pp. 5253–5266, 2012.

M. Aono, E. R. Bennett, K. S. Kim et al., “Protective effect of apolipoprotein E-mimetic peptides on N-methyl-D-aspartate excitotoxicity in primary rat neuronal-glial cell cultures,” Neuroscience, vol. 116, no. 2, pp. 437–445, 2003.

P. E. Batchelor, D. M. Armstrong, S. N. Blaker, and F. H. Gage, “Nerve growth factor receptor and choline acetyltransferase colocalization in neurons within the rat forebrain: response to fimbria-fornix transection,” The Journal of Comparative Neurology, vol. 284, no. 2, pp. 187–204, 1989.

M. V. Sofroniew, C. L. Howe, and W. C. Mobley, “Nerve growth factor signaling, neuroprotection, and neural repair,” Annual Review of Neuroscience, vol. 24, pp. 1217–1281, 2001.

A. Granholm, D. Albeck, C. Bäckman et al., “A non-invasive system for delivering neural growth factors across the blood-brain barrier: a review,” Reviews in the Neurosciences, vol. 9, no. 1, pp. 31–55, 1998.

T. Nonomura, C. Nishio, R. M. Lindsay, and H. Hatanaka, “Cultured basal forebrain cholinergic neurons from postnatal rats show both overlapping and non-overlapping responses to the neurotrophins,” Brain Research, vol. 683, no. 1, pp. 129–139, 1995.

P. M. Friden, L. R. Walus, P. Watson et al., “Blood-brain barrier penetration and in vivo activity of an NGF conjugate,” Science, vol. 259, no. 5093, pp. 373–377, 1993.

F. H. Gage, M. B. Rosenberg, M. H. Tuszynski et al., “Gene therapy in the CNS: intracerebral grafting of genetically modified cells,” Progress in Brain Research, vol. 86, pp. 205–217, 1990.

F. Hefti, “Development of effective therapy for Alzheimer's disease based on neurotrophic factors,” Neurobiology of Aging, vol. 15, supplement 2, pp. S193–S194, 1994.

J. A. Hardy and G. A. Higgins, “Alzheimer's disease: the amyloid cascade hypothesis,” Science, vol. 256, no. 5054, pp. 184–185, 1992.

P. Edison, H. A. Archer, R. Hinz et al., “Amyloid, hypometabolism, and cognition in Alzheimer disease: an [11C]PIB and [18F]FDG PET study,” Neurology, vol. 68, no. 7, pp. 501–508, 2007.

Y. Li, J. O. Rinne, L. Mosconi et al., “Regional analysis of FDG and PIB-PET images in normal aging, mild cognitive impairment, and Alzheimer's disease,” European Journal of Nuclear Medicine and Molecular Imaging, vol. 35, no. 12, pp. 2169–2181, 2008.

W. L. Klein, “Aβ toxicity in Alzheimer's disease: globular oligomers (ADDLs) as new vaccine and drug targets,” Neurochemistry International, vol. 41, no. 5, pp. 345–352, 2002.

D. J. Selkoe, “Alzheimer's disease is a synaptic failure,” Science, vol. 298, no. 5594, pp. 789–791, 2002.

J. Hardy and D. J. Selkoe, “The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics,” Science, vol. 297, no. 5580, pp. 353–356, 2002.

R. M. Koffie, M. Meyer-Luehmann, T. Hashimoto et al., “Oligomeric amyloid β associates with postsynaptic densities and correlates with excitatory synapse loss near senile plaques,” Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 10, pp. 4012–4017, 2009.

T. Oide, T. Kinoshita, and K. Arima, “Regression stage senile plaques in the natural course of Alzheimer's disease,” Neuropathology and Applied Neurobiology, vol. 32, no. 5, pp. 539–556, 2006.

G. R. Zimmermann, J. Lehár, and C. T. Keith, “Multi-target therapeutics: when the whole is greater than the sum of the parts,” Drug Discovery Today, vol. 12, no. 1-2, pp. 34–42, 2007.

M. J. Millan, “Multi-target strategies for the improved treatment of depressive states: Conceptual foundations and neuronal substrates, drug discovery and therapeutic application,” Pharmacology and Therapeutics, vol. 110, no. 2, pp. 135–370, 2006.

R. L. Martone, H. Zhou, K. Atchison et al., “Begacestat (GSI-953): a novel, selective thiophene sulfonamide inhibitor of amyloid precursor protein γ-secretase for the treatment of Alzheimer's disease,” The Journal of Pharmacology and Experimental Therapeutics, vol. 331, no. 2, pp. 598–608, 2009.

B. Vellas, O. Sol, P. J. Snyder et al., “EHT0202 in Alzheimer's disease: a 3-Month, randomized, placebo-controlled, double-blind study,” Current Alzheimer Research, vol. 8, no. 2, pp. 203–212, 2011.

S. Salloway, R. Sperling, R. Keren et al., “A phase 2 randomized trial of ELND005, scyllo-inositol, in mild to moderate Alzheimer disease,” Neurology, vol. 77, no. 13, pp. 1253–1262, 2011.

C. Caltagirone, L. Ferrannini, N. Marchionni, G. Nappi, G. Scapagnini, and M. Trabucchi, “The potential protective effect of tramiprosate (homotaurine) against Alzheimer's disease: a review,” Aging Clinical and Experimental Research, vol. 24, no. 6, pp. 580–587, 2012.

N. G. Faux, C. W. Ritchie, A. Gunn et al., “PBT2 rapidly improves cognition in alzheimer's disease: additional phase II analyses,” Journal of Alzheimer's Disease, vol. 20, no. 2, pp. 509–516, 2010.

B. Winblad, N. Andreasen, L. Minthon et al., “Safety, tolerability, and antibody response of active A immunotherapy with CAD106 in patients with Alzheimer's disease: randomised, double-blind, placebo-controlled, first-in-human study,” The Lancet Neurology, vol. 11, no. 7, pp. 597–604, 2012.

T. Del Ser, K. C. Steinwachs, H. J. Gertz et al., “Treatment of Alzheimer's disease with the GSK-3 inhibitor tideglusib: a pilot study,” Journal of Alzheimer's Disease, vol. 33, no. 1, pp. 205–215, 2013.

G. Yang, Y. Wang, J. Tian, and J. P. Liu, “Huperzine a for Alzheimer's disease: a systematic review and meta-analysis of randomized clinical trials,” PloS ONE, vol. 8, Article ID e74916, 2013.