The Effects of Soluble Aβ Oligomers on ...

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The Effects of Soluble A
Oligomers on Neurodegeneration in Alzheimer's Disease Jonathan Brouillette* Yale University, School of Medicine, 06519 New Haven, Connecticut, USA Abstract: The neurodegenerative process that defines Alzheimer’s disease (AD) is initially characterized by synaptic alterations followed by synapse loss and ultimately cell death. Decreased synaptic density that precedes neuronal death is the strongest pathological correlate of cognitive deficits observed in AD. Substantial synapse and neuron loss occur early in disease progression in the entorhinal cortex (EC) and the CA1 region of the hippocampus, when memory deficits become clinically detectable. Mounting evidence suggests that soluble amyloid- (A
) oligomers trigger synapse dysfunction both in vitro and in vivo. However, the neurodegenerative effect of A
species observed on neuronal culture or organotypic brain slice culture has been more challenging to mimic in animal models. While most of the transgenic mice that overexpress A show abundant amyloid plaque pathology and early synaptic alterations, these models have been less successful in recapitulating the spatiotemporal pattern of cell loss observed in AD. Recently we developed a novel animal model that revealed the neurodegenerative effect of soluble low-molecular-weight A
oligomers in vivo. This new approach may now serve to determine the molecular and cellular mechanisms linking soluble A species to neurodegeneration in animals. In light of the low efficiency of AD therapies based on the amyloid cascade hypothesis, a novel framework, the aging factor cascade hypothesis, is proposed in an attempt to integrate the new data and concepts that emerged from recent research to develop disease modifying therapies.

Keywords: A oligomers, neurodegeneration, Alzheimer’s disease, animal models, aging, hippocampus, aging factor cascade hypothesis, memory deficits, synaptic dysfunction, tau. 1. INTRODUCTION 1.1. Phenotypes and Hallmarks of AD AD is an age-related neurodegenerative disorder that affects nearly 2% of the population and one in three individuals over 85 years old in industrialized countries [1, 2]. AD represents the most common cause of dementia among people over 65 years old and is the eighth leading cause of death in the United States [3]. Prevalence studies have estimated that 35 million people worldwide are currently affected by AD [4, 5]. The National Institute on Aging indicates that 4.5 million people in the United States alone are suffering from this devastating illness with annual direct and indirect care costs estimated to at least $100 billion. As the population continues to age, baby boomers reach retirement, and the proportion of aged individuals in countries such as Japan is expected to nearly double in the next 50 years, the number of AD patients worldwide is intended to increase to 114 million by 2050 if new therapies do not emerge [4, 5]. AD is a dementia that primarily impairs episodic memory [6, 7], and other cognitive deficits manifest as the disease progresses, particularly in attention and executive functions, semantic memory, language and spatial orientation [8, 9]. Synapse loss that precedes neuronal death is considered the strongest correlate for memory decline in AD [10-14]. Individuals with mild cognitive impairment (MCI), a prodromal state of AD, and early AD have substantial neuron loss in layer II of the EC [15-17]. Cell death was also observed in the CA1 region of the hippocampus in very mild AD [17]. The disproportionate decreases in synapse density compared to the loss of neuronal cell bodies in biopsied tissue [18, 19] could be attributed to synapse loss on neurons that are still living before undergoing neurodegeneration in AD [13]. Two other neuropathological hallmarks of the disease are the extracellular senile plaque deposition and neurofibrillary tangles (NFTs) [20]. At their core, plaques are composed of A species *Address correspondence to this author at the Yale University, School of Medicine, 230 South Frontage Road, SHM I room 269, New Haven, CT 06519 USA; Tel: (203)737-4292; Fax: (203)737-3318; E-mail: [email protected]

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produced by the proteolytic cleavage of the transmembrane amyloid precursor protein (APP) by - and -secretases [21]. A is a natural soluble component of serum and CSF that was shown to be up-regulated during neuronal and synaptic activity [22]. In AD brains, A 42 species, and to a lesser extent A40, tend to accumulate and form insoluble senile plaques. However, because significant amyloid deposits are also observed in some cognitively intact aged individuals, plaques are known to correlate poorly with cognitive status [10, 23-26]. NFTs mostly consist of a highly polymerized, hyperphosphorylated form of microtubule-associated tau proteins [27-30]. Some reports have shown that hyperphosphorylation induces detachment of tau from microtubules and intracellular aggregation that perturbs axonal transport of synaptic vesicles and mitochondria, resulting in neuronal degeneration [26, 31]. The presence of NFTs correlates better than A deposits with cognitive declines in AD but to a lesser degree than synapse loss [32]. In most instances, the definitive diagnosis of AD can only be established by examining those neuropathological hallmarks at autopsy. The most robust risk factors for AD are age and a family history of the disease [33]. Genetic screens of families in which the disease occurs at an elevated frequency, familial AD (FAD), have revealed autosomal dominant mutations in three distinct genes: APP [34], presenilin 1 (PS1) [35, 36], and presenilin 2 (PS2) [37, 38]. Mutations in the APP and PS1 genes are the most frequent and penetrating (i.e. most carriers of the mutations develop AD before the age of 60), whereas PS2 mutations are rare and not fully penetrating. However, 99.5% of AD patients do not have mutations in any one of these three genes and FAD accounts for only about 5% of the total number of AD cases [39]. For the more common sporadic lateonset AD, the
4 allele of the apolipoprotein E (APOE) has been identified as the major genetic risk factor for AD [40, 41]. People who are homozygous for the APOE
4 allele increases risk for AD by 15 times, whereas those that are heterozygous carriers have 3 times the risk for AD compared to APOE
2 and APO E
3 carriers [42-44].

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1.2. Soluble High- and Low-Molecular-Weight A
Oligomers in Alzheimer’ Disease The amyloid cascade hypothesis suggests that accumulation of A
in the brain is central to the pathogenesis of AD [45, 46]. Initially, this theory proposed that insoluble A
was causing the disease. However, A
deposition is also observed in some cognitively normal elderly individuals and senile plaques are observed long after marked neuronal loss in AD [10, 23, 25]. The poor correlation between senile plaque deposition and the cognitive status of the patients has been confirmed with modern neuroimaging techniques [47, 48]. To overcome this discrepancy, more recent studies have emphasized the neurotoxic role of soluble A
oligomers that naturally arise at the very early stage of AD, leading to what has been called the "A
oligomer cascade hypothesis" (Fig. 1) [23, 49-52]. Nonetheless, the nature of the soluble A
species that trigger the pathophysiological alterations that characterize the disease remains uncertain. Many A
intermediates such as dimers, trimers, tetramers, nonamers, dodecamers, higher molecular weight oligomers named A
-derived diffusible ligands (ADDLs), protofibrils, fibrils and lipid-induced oligomers have all been individually identified as main contributors to neurotoxicity [53-62]. Most of the higher A
conformations were shown to be toxic, although the mechanism of their deleterious effect on neurons remains elusive. The extracellular accumulation of a 56 kDa soluble A
assembly (termed A
*56) was shown to correlate with the development of memory deficits but without producing any neuronal loss in an

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animal model of AD (Tg2576 mice) that expresses human APP containing the Indiana mutation [53]. As observed with A
*56, soluble low-molecular-weight A
oligomers were also found to disrupt synaptic plasticity, exert neurotoxic effects and induce memory impairment. These effects have been found using synthetic A
oligomers as well as small soluble A
species extracted directly from AD brains or purified from cell culture expressing a mutant form of human APP (hAPP). Naturally secreted A
dimers and trimers were described to induce synapse dysfunction and cognitive deficits [57, 58, 63], whereas another study has observed that tetramers may have more potent neurotoxicity than dimers in cell culture [55]. It should be noted that most of these reports have investigated the synaptotoxic and neurodegenerative role of soluble A
oligomers mainly on neuronal cultures or organotypic brain slices and that the toxic effect of these various A
preparations as yet to be established in vivo [53-58]. Nonetheless, two of these studies have done single brain infusion of dimers or A
*56 species and both have found synapse alterations and memory impairment [53, 57]. Most of these studies have characterized the toxic role of a precise stable toxic A
species with a defined size and structure. Because of the rapid and dynamic oligomerization of A
, others have suggested that a series of soluble A
intermediates present in the brain at the same time may explain more accurately the global neurotoxic effect of A
[55, 64, 65]. Soluble A
oligomers ranging

Fig. (1). The A
oligomer cascade hypothesis. Extracellular soluble A
oligomers accumulate and bind to neuronal surface inducing synaptic dysfunction. Intracellular soluble A
oligomers are also suspected to contribute to neurotoxicity in AD. Soluble A
species tend to dynamically oligomerize to form insoluble fibrils that aggregate into senile plaques surrounded by glia. Neurofibrillary tangles (NFTs) are composed of highly polymerized, hyperphosphorylated forms of tau and accumulate in neurons. The A
oligomer cascade hypothesis suggests that intra- and/or extra-accumulation of soluble A
oligomers may cause early cognitive deficits that define AD.

The Effects of A
Oligomers on Alzheimer's Disease Neurodegeneration

from 10 to 100 kDa in weight have been observed at the very early stage of AD and strongly correlate with the clinical state of AD [52, 66, 67]. 2. NEURODEGENERATION IN AD Many studies have convincingly shown that soluble A oligomers are able to induce alteration and physical degeneration of the synapse in cell cultures, brain slices as well as in animal models [53-58]. However, the relationship between A species and the neuronal loss that accompanied synapse loss in AD remains elusive. Indeed, the precise spatial and temporal pattern of progressive neurodegeneration observed in AD could not be recapitulated in the animal models overexpressing A that have been developed so far. One critical feature of AD pathogenesis is the massive, widespread cell death in the advanced form of the disease and the significant synapse and neuronal loss observed in localized brain regions when memory deficits become clinically detectable. This suggests that pruning of synaptic endings may precede the demise of the neuron but both synapse and neuronal loss are needed to induce cognitive deficits that are clinically sufficient to diagnosis AD. Decreased synaptic density is observed in the mesiotemporal regions of early AD patients as well as in MCI [10, 15, 16, 18, 19, 68-70], and is the pathological feature that most closely correlates with the severity of memory impairment and ensuing dementia [1013]. Quantification using electron microscopy or immunohistochemical staining for synaptic markers has reported significant decrease in synaptic density in the hippocampus and association cortices of AD brain [10, 18, 19]. Changes in synaptic markers in the brains of AD patients and transgenic mouse models of AD suggest that synapse function is compromised prior to its physical degeneration [68, 71-73]. An early event in AD is the neuronal death that is tightly associated with synapse loss that begins in the entorhinal cortex to eventually extend to the hippocampus and later on to the temporal cortex [18, 74]. The most vulnerable circuit is the medial perforant pathway that originates in layer II of the EC and terminates in the outer molecular layer of the dentate gyrus (DG) [75, 76]. Unbiased stereological counts indicate that individuals with MCI and very early AD have substantial atrophy and losses of layer II EC neurons and show synaptic loss in the DG, which correlates with cognitive deficits on clinical tests [15-17, 70]. In severe AD cases up to 90% of neurons are lost in the EC while in healthy elderly individuals no neuronal loss is observed in this brain region [15]. The substantial cell death observed in the EC in early AD is detected in the absence of significant amounts of extracellular plaque deposition and NFTs

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[20]. Neurodegeneration and atrophy of the CA1 region of the hippocampus has also been observed in very mild AD [77-79]. Neuronal and synaptic losses along with pathology of cholinergic basal forebrain neurons are considered to correlate strongly with memory decline in AD [10, 11]. Some studies have shown that the memory deficits in AD and aging are closely associated with decreased choline acetyltransferase (ChAT) activity and reduced number of cholinergic neurons [80-84]. The patterns of hippocampal subfield atrophy in the CA1 region and in the EC observed in early AD and MCI patients using the novel high field magnetic resonance imaging (MRI) technology are consistent with decreased synaptic density and cell loss described by histopathology [85-91]. During the progression of the illness, synapse and cell losses gradually become more widespread to ultimately encompass the whole cortex [92-94]. At the end-stage of the disease, a drastic atrophy of the entire brain is accompanied by substantial ventricular enlargement (Fig. 2) [92-94]. 2.1. Neurodegenerative Patterns in Transgenic AD Mouse Models Many different transgenic AD mouse models have been developed over the last two decades (Tg2576, TgCRND8, PDAPP, APP/Ld, APP23, TgAPPArcSwe, APP-Au, APP E693
, APP751SL/ PS1M146L, APP/PS1, APP/PS1KI, PS2APP, TauPS2APP, APP/ tau, APP/tau, NF-L-A, TBA2, 3xTg-AD, 5XFAD, etc.) (for review see [95, 96]). Most of the transgenic AD mouse models overexpressing A present extensive amounts of senile plaques, tau hyperphosphorylation, early synaptic deficiency and memory impairment. However, these models remain less successful in modeling the significant cell death in the CA1 and EC at the onset of memory decline and the drastic brain atrophy seen at the endstage of AD. Despite their high level expression of mutated or full-length form of APP many transgenic models lack complete neuronal loss or exhibit only modest, late-onset cell death long after the occurrence of memory deficits. Very modest or no neuronal loss was reported in the Tg2576, TgCRND8, PDAPP, TauPS2APP mice models [97-100]. In the APP23, APP751SL/PS1M146L and APPE693
transgenic animals, hippocampal cell death has been observed in relatively old animals (14 to 24 months of age) after plaque depositions and occurrence of cognitive impairment [101103]. The APP/PS1KI, APP/tau and 5XFAD animal models were shown to have neuronal loss at younger ages with the onset of plaque formation preceding or appearing concomitantly with cell death [104-106]. Recently, degeneration of the cholinergic septohippocampal pathway was found in 4 months old tripletransgenic AD (3xTg-AD) model [107], but no widespread neuro-

Fig. (2). Neuronal and synaptic degenerations in the pathogenesis of AD. Synapse loss that precedes neuronal death is the strongest pathological correlate of cognitive deficits observed in AD. The most vulnerable circuit is the perforant fiber pathway that originates in layer II of the entorhinal cortex (EC) and terminates in the outer molecular layer (OML) of the dentate gyrus (DG). Individuals with MCI and very early AD have substantial cell death in layer II of the EC as well as synaptic loss in the DG. Neurodegeneration in the CA1 region of the hippocampus is also observed in very mild AD. In severe AD cases massive cell loss is observed throughout the brain, the cortex shrivels up, the hippocampus is severely shrunk and ventricles grow larger.

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AD (3xTg-AD) model [107], but no widespread neuro-degeneration as observed in AD patients was reported in these mice although they are harboring three mutant genes: beta-amyloid precursor protein (betaAPPSwe), presenilin-1 (PS1M146V), and tauP301L [108, 109]. The first evidence of the neurodegenerative effect of A
came from the NF-L-A
mouse model that developed neuronal loss in the CA3 and DG regions of the hippocampus as well as in the neocortex, thalamus and to a lesser extent the hindbrain of 6 months old animals [110]. The TBA2 mice were shown to have abundant neuronal loss in the cerebellar Purkinje cells, but not in the hippocampus, and die prematurely [111]. It should be noted that in many publications, intraneuronal A
has been proposed to induce some of the phenotypes observed in these mice [101, 102, 104, 105, 110112]. However, if intracellular A
arises at the same time as cognitive deficits, its levels have been shown to decline with the progression of AD in both human and animal models and it remains to be determined if toxic intraneuronal A
observed in mouse models can account for the pathogenesis in humans [113, 114]. Although these transgenic models could not precisely recapitulate the spatiotemporal pattern of neurodegeneration occurring in AD, some of them have proven to be useful for understanding early synaptic alterations and revealing the neurodegenerative properties of A
in vivo. The lack of cell death in many AD models could be attributed to compensatory mechanisms present in rodents. While this last hypothesis still needs to be validated, an alternative possibility is that despite the unquestionable neurotoxic effect of A
oligomers, the accumulation of A
species may not represent the only neurodegenerative triggering event in most late-onset sporadic cases. Because plaque deposition is observed before neuronal death in most of these animal models, it is difficult to discriminate the neuropathological features that can be attributed to either the soluble A
oligomers or the insoluble A
fibrils. Thus, to determine the nature of toxic soluble A
oligomers other studies have opted to administer well-defined A
preparations directly into the brain of animals and analysed their molecular mechanisms of action in cell culture or organotypic brain slice. 3. NEUROTOXICITY OF SOLUBLE A OLIGOMERS 3.1. Neurotoxicity of Soluble A Preparations in Cell Culture and Brain Slice Although the primary pathogenic event that leads to synaptic loss and ultimately to cell death in AD remains a subject of debate, recent studies have emphasized the toxic role of soluble high- and low-molecular-weight A
oligomers [53, 55-57, 115]. To prove this "A
oligomer cascade hypothesis", many research groups have elaborate different protocols to generate in vitro A
preparations or oligomeric species derived from AD brains. Numerous studies on cell culture or organotypic brain slice have revealed synaptic dysfunction and cell death following application of different A
oligomer solutions [54-56, 65, 115-117]. The cellular and molecular mechanisms underpinning the neurotoxic effect of A
oligomers remain elusive but different molecules have been shown to be implicated in this cascade of events leading to synapse and neuronal loss. The potentiation or depression of synapse activity following cell stimulation depends in part on the level of calcium (Ca2+) influx through the N-methyl-Daspartate (NMDA) receptors and/or the activation of the metabotropic glutamate receptors (mGluRs) [118-121]. High entry of Ca2+ through the NMDA receptors has been shown to induce long-term potentiation (LTP), an electrophysiological correlate of memory formation [56-58, 63, 122]. Inversely, long-term depression (LTD) occurs when low levels of Ca2+ influx occur through NMDA receptors [123]. These cellular phenomena impact synaptic morphology since LTP was shown to favor spine enlargement,

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whereas facilitation of LTD induces shrinkage of dendritic spines [124-127]. The application of A
dimers and trimers on organotypic hippocampal slices was found to potently inhibit LTP, enhance LTD and limit dendritic spine density [57, 58, 63, 128]. Previous studies have shown that synaptic loss following treatment with different A
preparations on primary cell culture or brain slices was associated with rapid decrease in membrane expression of NMDA receptors [58, 115, 129, 130]. The activation of the calcineurin phosphatase was reported to induce LTD by promoting the internalization of NMDA and AMPA receptors [129, 131-134]. Some studies have found that calcineurin inhibitors were able to reverse the endocytosis of NMDA and AMPA receptors and rescued dendritic spines loss induced by A
[135-137]. The internalization of these receptors by calcineurin in the disease process is driven at least in part by STEP (striatal-enriched tyrosine phosphatase), an indirect substrate of calcineurin that dephosphorylates both receptors prior to their endocytosis [129, 138-142]. Interestingly, A
administration failed to induce NMDA receptor internalization in STEP knock-out cultures [140]. Moreover, the level of STEP was found to be increased in three mouse models of AD (Tg2576, J20, 3xTg) and in the prefrontal cortex of AD patients [140, 143, 144]. Although the association between these signaling events and the integrity of the synapse functioning is quite well defined, their implication in the neurodegenerative process observed in AD still needs to be determined. Several reports indicate that the activation of the caspase-3 enzyme participates in the molecular mechanism leading to synaptic dysfunction and cell death in AD [115, 117, 145-147]. Recently, we observed that the level of caspase-3 activity was proportional to the level of synapse loss and cell death following the application of soluble low-molecular-weight A
oligomers to primary hippocampal cultures [115]. These results are in line with another study showing that enhanced caspase-3 activity correlates with spine degeneration in the CA1 neurons at the onset of memory deficits in Tg2576 mouse model, via a calcineurindependent pathway [146]. A number of other proteins were shown to be involved in synapse loss and neurodegeneration in AD, including glycogen synthase kinase-3
(GSK-3
) [148, 149], fyn kinase [143, 150, 151], cyclin-dependent kinase-5 (CDK-5) [152-154], extracellular signal-regulated kinase (ERK) and c-jun Nterminal kinase (JNK) [155-157]. There are some indications that tau is required for A
toxicity in cell cultures. Indeed, hippocampal neurons depleted in tau were shown to be more resistant to the toxicity of fibrillar A
than normal cultures [158, 159]. Another report has shown that tau mutant mimicking AD-like hyperphosphorylation had larger neurotoxic effect on cultured neurons, and that phosphorylation of tau is the limiting factor in A
-induced cell death [160]. More recently, synthetic soluble low-molecular-weight A
oligomers and dimers isolated from AD cortex were both found to trigger neurodegenerative process in primary hippocampal culture in association with tau hyperphosphorylation at specific AD-relevant epitopes [115, 116]. These neuronal effects were prevented by knocking down endogenous tau, whereas the expression of wild-type human tau precipitates these events in culture [116]. Thus, many molecules seem to be involved in the pathogenic events leading to AD but their effects still need to be extensively validated in animal models. It also remains to be established how these events relate to synaptic dysfunctions, neurodegeneration and cognitive impairment seen in AD. 3.2. Neurotoxicity of Soluble A Oligomers In vivo In transgenic animal models, the nature of toxic A
intermediates can hardly be studied because low-molecular-weight A
oligomers, protofibrils and fibrillar A
species as well as senile plaques are often all simultaneously present in the brain when synapse and neuronal loss become apparent in some of these models. Alternatively, several groups have used amyloid-infused animal

The Effects of A
Oligomers on Alzheimer's Disease Neurodegeneration

models. Using a single injection of synthetized or natural A
oligomers isolated from human brain, some studies have reported synaptic alterations and memory impairment but no A
accumulation or neuronal death were detected [53, 57]. Single injection of A
1-42 fibrils in humanP301L tau mice was found to induce NFTs and neuropil threads but not neuronal loss [161]. To increase toxicity of the solution, others have used a mixture of A
species and ibotenic acid to induce neuronal loss, which complicates the interpretation of the respective neurodegenerative effect attributed to each toxin [162]. Osmotic pump system has also been used for prolonged administration of A
species. Two weeks of continual intraventricular infusion of A
1-42 was able to induce age-dependent learning deficits but no A
accumulation or neuronal loss was reported [163]. As determined in this study A
dynamically continues to oligomerize during the infusion period, making this approach not suitable to control the nature of A
species injected over time, unless the A
oligomers are structurally modified to preserve a stable conformation. Moreover, during the oligomerization process A
peptides are constantly evolving from a soluble to an insoluble state and may perturb the release of higher molecular weight aggregates which stick into the pump, as observed in the former study. We observed the same phenomenon on the side of low-adhesion tubes during the incubation of the A
1-42 peptide [115]. Others have reported a decrease in hippocampal LTP [122], accompanied by caspase-3 activation following chronic intracerebroventricular injection of A
1-40 species over 28 days [164]. It should also be noted that most of these reports have studied the impact of fibrils or more mature A
species [163-166], incubated during one or several days before injection, instead of soluble lowmolecular-weight A
oligomers which were recently shown to be the more neurotoxic species. In some studies a lack of characterization of the A
species infused makes it hard to interpret the nature of the toxic intermediates. Recently, we developed a novel approach to more accurately control the nature of A
intermediates injected in mice and study the neurodegenerative effect of A
preparation in vivo [115]. We found that repeated hippocampal injections of soluble lowmolecular-weight A
1-42 oligomers in awake, freely-moving mice induced gradual and robust neuronal loss in the vicinity of oligomer deposition, tau hyperphosphorylation and memory impairment. These key neuropathological hallmarks of AD were accompanied by decreased levels of the NMDA receptor subunit NR2B and increased levels of active caspase-3 enzyme. Many advantages can be attributed to this novel and flexible in vivo approach: The preparation of soluble A
oligomers can be characterized before and after chronic injection under denaturing and non-denaturing conditions using western blot, transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance spectroscopy (NMR), electrospray-ionization mass spectrometry (ESI-MS) and atomic force microscopy (AFM). Since the injections are perform in awake, freely-moving mice there are no confounding interference effects between anesthetic agents and the A
solution on signaling pathways. Numerous studies have shown that anesthesia can increase tau phosphorylation, promote fibril formation and the neurotoxicity of A
, augment senile plaque accumulation in AD mouse model, induce memory deficits and alter many other molecules that were shown to be involved in AD pathogenesis, such as caspase-3, GSK-3
, Akt, Erk1/2, JNK and PP2A [167-171]. Injections in freely-moving mice also allow testing them in behavioural task any time before and following the infusions. The neurodegenerative effect of A
1–42 species were determined in 12 months old mice to take into account that aging is the most robust risk factor associated with AD.

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Because neurodegeneration occurs in the vicinity of the A
injection site, synapse and neuronal loss can be induced in different and localized brain regions. The collateral infusion of A
1–42 oligomers and vehicles allows controlling for any alteration within the same mouse. Because A
oligomers are gradually cleared out following infusion, the long-term effects of A
intermediates after removal can be determined both at the molecular and behavioural levels. The effect of A
species on molecular pathways can be determined before and after neuronal loss within a reasonably short time frame. Since A
deposition occurs in a time-dependent manner, the dose and number of A
injections can be adjusted to obtain more or less severe readouts of A
pathogenicity. This novel in vivo approach can be used for preclinical validation of agents designed to prevent A
neurodegeneration. In summary, our new animal model recapitulates many neuropathological hallmarks of AD and proves useful in the determination of the neurotoxicity of A
preparations as a function of their temporal profile. Since currently available techniques to characterize A
species are very limited and provide only semi-quantitative information, it is difficult to compare different oligomeric preparations in terms of concentration, conformation, and their potential relevance to the disease (for review see [172]). The preparation that was injected in this model derived from synthetic A
1-42 and is almost exclusively composed of soluble low-molecular-weight A
species, including monomers, dimers, trimers and tetramers [115, 117, 173]. In future studies, it would be interesting to determine if A
solutions isolated directly from AD brains can induce similar neurodegenerative effects using this approach [57, 58]. Since the mixture of A
1-42 and A
1-40 in a 3:7 molar ratio has been shown to exert synaptotoxic and neurotoxic effect in primary hippocampal neurons [117], it would also be worth it to analyze the impact of this preparation in our model. Since the neurotoxicity of A
oligomers was mainly studied so far in cell cultures or brain slices, our approach allows a new field of investigation to evaluate the neurotoxicity of different A
preparations in vivo. 4. SUBSTRATES OF SOLUBLE A
OLIGOMERS In the past, the accumulation of A
into plaques has been proposed to be central in the pathogenesis of AD. However, a poor correlation was observed between the levels of senile plaques and the degree of neurodegeneration in the patients [10, 25]. More recently a lot of attention has gone to the neurotoxic effect of soluble A
species. Different groups have identified the dimers, trimers, tetramers, nonamers, dodecamers, ADDLs, protofibrils, fibrils and lipid-induced oligomers as the main culprit in inducing neurotoxicity [53-62]. Many of the publications have identified one particular toxic A
oligomer that was presented as a stable and defined structure able to trigger the pathophysiological alterations observed in AD. This would argue in favor of a ligand-receptor mechanism where a specific A
species can act on intracellular signaling pathway to induce synaptotoxicity, neuronal loss and memory impairment. Alternatively, some authors have suggested that given the rapid and dynamic oligomerization of A
, the toxicity of A
is attributable to a series of soluble species present in the brain at the same time, rather than to just one precise type of oligomer [52, 55, 64, 65, 115, 117, 174]. Indeed, naturally occurring A
1-42 oligomers ranging from 10 to 100 kDa in weight have been found in AD brains [66, 67, 174]. These studies suggest that the assembly and disassembly of A
is a dynamic and continuous process, unless structural changes are made to stabilize its conformation, and that the toxicity of soluble A
oligomers should be regarded as a

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whole since they occur simultaneously in the brain. Consistent with this hypothesis, various synthetic or naturally secreted A preparations containing different types of soluble oligomers or protofibrillar intermediates were able to induce synapse dysfunction, LTP and LTD alterations, synapse and neuronal loss as well as cognitive deficits [57, 65, 115, 117, 122, 130, 175-177]. This dynamic interpretation of A neurotoxicity does not exclude the possibility that some A species may be more toxic than others. Another possibility is that each individual species has more or less different effects and once combine they are able to recapitulate more closely the large spectrum of molecular alterations induced by soluble A oligomers that lead to the multiple pathogenic events observed in AD. Compelling evidence indicates that soluble A oligomers are highly hydrophobic and could induce neurotoxicity by unspecific interaction with lipid membranes [54, 178]. Similarly, the hydrophobic properties of soluble A species were also observed in tube assays where they adhere to the walls of low-adhesion tubes during the oligomerization process [115]. Thus, the accumulation of several soluble forms of A was proposed to disturb the normal functioning of neurons by binding non-specifically to lipid membrane at the levels of the synapse and consequently altering membrane and receptor trafficking as well as synaptic transmission and plasticity [54, 178]. Another possibility is that the hydrophobic interaction of A intermediates with the cell membrane induces the formation of pore-like structures with channel activity that could alter intracellular signaling pathways [62, 179-181]. Because of their high propensity to stick at the membrane, we observed that soluble A species infused in the hippocampus do not diffuse broadly and tend to accumulate in the vicinity of the injection site, mostly in the granule cell layer of the dentate gyrus where synaptic connections are formed [115]. Different groups have shown that various A species were able to exert toxic effects by binding different membrane proteins such as the NMDA receptors [182], the 7
-nicotinic acetylcholine receptors [183, 184], the cellular prion proteins (PrPc) [185], the metabotropic glutamate receptors (mGLuR5) [182], the angiotensin II receptors [186], the voltage-gated calcium channels [185], the fibronectin domain of EphB2 [187] and the p75 neurotrophin receptor (p75NTR) [188]. The retrieval of low-molecular-weight A1-42 oligomers following six days of injection in mice suggests that the interaction with receptors and/or lipid membranes stabilized their conformation, as observed when soluble A1-42 species bind to the sequestering agent transthyretin (TTR) [115]. Unifying all these findings, one could argue that different types of soluble A oligomers are deleterious and may affect the normal functioning of neuronal circuitry by interacting with the membrane and by acting on a plethora of receptors and intracellular signaling pathways. It is not clear at the moment how extracellular soluble A oligomers may affect intracellular signaling following their binding to the cell membrane and receptors. Some investigators have found that A can induce mitochondrial failure [189, 190], lysosomal malfunction [191], and alter molecular pathways involved in cell death, synaptic plasticity and neurogenesis [95]. Interestingly, another group has found that low or high levels of A are able to either facilitate or inhibit the release of various neurotransmitters (dopamine, glycine, glutamate, aspartate and GABA) elicited by the activation of cholinergic muscarinic and nicotinic receptors in different brain areas [192-195]. Thus, it is conceivable that the accumulation of A observed in AD may perturb the fine regulation of multiple neurotransmitter release, which in turn may lead to cognitive deficits and ultimately to neurodegeneration. 5. AD TREATMENT 5.1. Current Medication At the moment there is no cure for AD and treatments only provide modest symptomatic benefits to some individuals with AD

Jonathan Brouillette

[196, 197]. Two classes of drugs have been approved thus far for the treatment of AD. The findings of deficits in cholinergic neurotransmission in AD led to the development of cholinesterase inhibitors as the first approved treatment for AD’s dementia symptoms [198-201]. The general mechanism of action for this type of agents (including donepezil, rivastigmine and galantamine) is to increase the availability of acetylcholine (ACh) by preventing its degradation through the inhibition of the catabolic enzyme acetylcholinesterase [202]. Another approach to the treatment of AD is to inhibit excessive glutamatergic neurotransmission. In 2004, the Food and Drug Administration (FDA) of the USA approved memantine, a noncompetitive NMDA receptor antagonist, for treating dementia symptoms in moderate to severe AD cases [203]. Pharmacological studies suggested that memantine prevents excitotoxicity due to the high entry of extracellular calcium via NMDA receptors causing neuronal dysfunctions and cell death [204]. Since memantine has low- to moderate-affinity for NMDA receptors, this drug leaves the calcium channel relatively open for neurotransmission at a low stimulation rate necessary for LTP and memory processes [205]. Among other treatments that appear promising but yet remain to prove their effectiveness are other cholinergic agents such as M1 muscarinic receptor agonists, antioxidants, nonsteroidal antiinflammatory compounds, estrogen replacement therapy, cholesterol-lowering agents, neurotrophic agents, tau pathology approaches and A-related treatments [197, 206, 207]. Since synapse dysfunction is an early event in AD and the best correlate of cognitive declines associated with the illness, one may predict that the identification of the primary phenomenon responsible for the neurodegeneration seen in AD will lead to the development of more effective therapies. 5.2. Treatment Based on the A
Oligomer Cascade Hypothesis Over the past few years, pharmacological companies have predominantly focused their efforts in AD therapies based on the A oligomer cascade hypothesis to develop a disease modifying strategy. New therapeutic agents are currently being evaluated in clinical trials to prevent the formation and accumulation of toxic A oligomers in the brain [208]. However, none of the antiamyloid treatments tested so far has proven more beneficial than current symptomatic therapies and many were halted in Phase II and Phase III clinical studies. Inhibitors of the -secretase have been investigated to decrease the levels of A after it was observed that this enzyme is part of the process leading to the constitutive production of A peptide [209]. Recently, semagacestat failed in Phase III trial because of lack of efficacy and increased risk of skin cancer [210]. This agent is a first-generation, low-specificity inhibitor that has side effects probably because of its interaction with Notch1 protein. FlurizanTM (also named R-flurbiprofen or tarenflurbil), a -secretase modulator developed by Myriad, exhibited low potency and poor brain penetrance and failed also in Phase III [211]. The clinical trials for Elan’s ELND006 were also halted in October 2010 because of liver side effects that may not be related to the mechanism of action of the drug. After announcing the halt of semagacestat trials, Eli Lilly and Company (Lilly) decided to test a -secretase inhibitor (LY2886721) but recently they voluntarily stopped its Phase II study due to abnormal liver biochemical tests. To inhibit A aggregation Neurochem developed AlzhemedTM (also named tramiprosate or homotaurine), a small molecule that binds A monomers and prevents their oligomerization [212]. However, this agent did not exhibit efficacy and was halted in Phase III trials. To enhance A clearance Pfizer has tested an inhibitor (PF-04494700) of the receptor for advanced glycation end products (RAGE) that was shown to bind and move A from the brain to the periphery for degradation [213]. However, the trials were discontinued at Phase

The Effects of A
Oligomers on Alzheimer's Disease Neurodegeneration

II because at the highest dose it worsened cognitive performance. Elan is about to begin Phase III trials with a cyclohexanehexol isomer (ELND005), a class of molecule that inhibits A
fibril formation [214]. A main area of new therapeutic development is active and passive A
immunotherapy. The first A
immunotherapy to progress in Phase II trials was the AN1792 from Elan and Wyeth’s [215, 216]. The clinical treatment was halted in 2002 after some patients developed meningoencephalitis. Recently Lilly announced that solanezumab (humanized 266 antibody) did not meet primary endpoints, both cognitive and functional, in the two independent Phase III, double-blind, placebo-controlled trials in patients with mild to moderate AD [217]. Elan’s and Wyeth’s bapineuzumab (humanized 3D6 antibody) failed in Phase III because it did not show any benefit [218, 219]. Baxter’s intravenous immunoglobulin G (IVIG) preparation containing polyclonal A
antibodies was tested in Phase III trials but showed no cognitive improvement in mild-to-moderate AD patients [220]. Many reasons can explain the failure of these AD therapies based on the A
oligomer cascade hypothesis. First, many of the anti-amyloid agents that failed in Phase III showed only modest disease-modifying activity in preclinical and clinical studies. For instance, AlzhemedTM was moved to Phase III without showing strong evidence of efficacy in transgenic AD mouse models and also displayed weak action as an inhibitor of A
aggregation [212]. FlurizanTM did not seem to penetrate the brain in sufficient amounts to have any therapeutic effect [221]. Second, the population of patients used in these clinical trials may not be adequate to test the efficiency of the agents that are currently developed. Most clinical trials recruited patients with mild to moderate AD, instead of patients at earlier disease stages. Since many in vitro and in vivo studies have shown that A
induces synapse dysfunction, it is conceivable that A
-related treatment may be most effective at the very early stage of AD. Novel biomarkers are urgently needed to reliably detect the prodromal state of the disease and target more effective patient populations for these clinical trials [222]. The unsuccessful AD therapies that were developed so far served as references to improve the next generation of treatment and to adequately test the amyloid hypothesis. That being said, as long as the toxic A
oligomer cascade hypothesis is not fully validated in clinical trials other therapeutic avenues should not be disregarded to slow down, delay or halt the progression of the disease. 6. THE AGING FACTOR CASCADE HYPOTHESIS The ability of A
oligomers to induce neuropathological hallmarks of AD is without doubt critical in disease process. However, many pieces of evidence that are required to firmly establish the causative role of A
oligomers in AD are missing. A fundamental question that remains unanswered relates to the missing link between A
accumulation and aging, the most robust risk factor for AD. Changes in the steady-state levels of A
are due to alteration in its production and/or clearance and degradation during aging, but the factor responsible for this alteration remains to be determined. Recently it was observed that the clearance, but not the production rates, of A
42 and A
40 were decreased in lateonset form of AD [223]. Increased A
production was only reported in some rare FAD cases and in Down syndrome with trisomy of chromosome 21, which contain the APP gene [33, 224]. Although this aging factor has not been isolated, results from epidemiological and biochemical studies may be helpful to reveal the accurate candidate. Because of the high prevalence of the illness (approximately one person out of three over 85 years old [1, 2]), it is more likely that the accumulation of A
would be the result of a signal that is naturally altered during aging but who gets even

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7

more perturbed in the individual that will slowly and progressively develop AD during aging. This does not imply that an individual will necessarily develop AD with age, since this signal may continue to function adequately in elderly who will never show any symptoms of AD. In this aging factor cascade hypothesis, the series of events that are produced by A
is responsible for only a fraction of the total phenomena occurring in AD (Fig. 3). Indeed, the aging factor would induce the accumulation of A
but at the same time would initiate another cascade of events that interact in close association with the one cause by A
. To trigger the neurodegenerative process, accumulation of soluble A
oligomers would be necessary to induce synapse dysfunction and eventually lead to synapse loss. However, to create a massive and widespread cell death the action of A
would need to be associated with the deterioration of the signaling pathway triggered more directly by the aging factor. Because A
is only one component of the events causing neuron death, this could account for the difficulty of developing a transgenic mouse model overexpressing A
that shows neuronal losses and gross brain atrophy in the same spatiotemporal pattern as observed in AD. It would also explain why repetitive injections of relatively high dose of A
oligomers are needed to induce neuron loss in vivo [115], and why long exposure with A
species is necessary to observe significant cell death in neuronal cultures and brain slices [54-56, 65, 115-117]. These results suggest that A
is able to induce cell death on its own when time of exposure and doses are sufficiently high in cell culture, transgenic and infused animal models as well as in Down syndrome and rare FAD cases, but is unable to do so in the pathological conditions that define the more common sporadic late-onset AD form. Senile plaques are also involved in the degenerative process by inducing cell death in their vicinity and may serve as a reservoir of toxic A
intermediates as proposed by others [65, 225, 226]. Since the primary role of soluble A
oligomers is to alter synapse function, this hypothesis predicts that A
-related therapies may slowdown pathogenesis only in the early stage of the disease and not with mild-to-moderate AD as observed so far in the clinical trials. By targeting the right aging factor, this hypothesis suggests that it will be possible to halt the progression of the disease in mildto-moderate AD patients. The critical and essential impact of the aging factor on the neurodegenerative process would explain why A
immuno-therapies have been found so far to clear the senile plaques and A
effectively but without significantly altering the neurodegenerative process and the cognitive status of AD patients [227]. These last results are in contrast with the cognitive improvement observed in animal models following A
immunotherapy most probably because the pathology induced in transgenic mice is directly caused by an overexpression of A
, a situation only observed in rare FAD cases as well as in Down syndrome, but not in the more common sporadic late-onset AD form. Another area of intense investigation is the association between A
and tau pathology in AD. Although tau hyperphosphorylation at specific AD-relevant epitopes has been found in some animals overexpressing A
[95, 101, 228-231], none of them showed NFTs except when APP, tau and presenilin mutations are combined in 3xTg-AD mouse model [108, 109]. Tau hyperphosphorylation was also observed following administration of soluble A
oligomers in the hippocampus of mice, in cell cultures and brain slices but no NFT formation was reported except when infused in a mouse model with tau mutation [115, 116, 161]. Thus it appears that A
can lead to tau hyperphosphorylation but requires the intervention of other molecular events to fully induce tau pathology as observed in AD. The cascade of events initiated by the aging factor could fill this gap by concomitantly contributing to the phosphorylation of tau on the same epitopes as A
and/or another set of sites that are phosphorylated in physiological or pathological conditions.

8 Current Pharmaceutical Design, 2014, Vol. 20, No. 00

Jonathan Brouillette

Fig. (3). The aging factor cascade hypothesis for the more common late-onset form of AD. The signaling pathway induced by the aging factor is naturally altered during lifespan but is even more perturbed in an individual that will progressively develop sporadic late-onset AD. This factor is sensitive to environmental, physical and mental factors and acts in close association with APOE
4. This factor can lead to synapse loss, cell death and cognitive deficits by two complementary pathways: 1) a signaling pathway directly under its control and 2) the accumulation of A oligomers. In the first pathway synapse dysfunction may results from intracellular modifications such as increased or decreased activity of kinases and phosphatases, receptor trafficking perturbation, oxidative injury, mitochondrial malfunction, lysosomal failure as well as tau hyperphosphorylation or pathological phosphorylation. These events are concomitantly triggered by the accumulation of A oligomers in the second pathway to initiate a progressive and robust neurodegeneration that can be either dependent or independent of NFTs. The senile plaques that appear later on in the pathogenesis of AD also contribute to cell loss by releasing toxic A species which induce synapse dysfunction. Reactive astrocytes surrounding some of these plaques release cytokines and other toxic agents during the neuroinflammatory process that induce cell death in the vicinity of the plaque. Altogether these phenomena lead to the cognitive impairment that defines late-onset AD.

The overall pathological alterations triggered by the aging factor would lead to progressive synaptotoxicity, cell loss, gross brain atrophy and cognitive impairment that worsen as AD patients age. This hypothesis does not exclude the possibility that a group of aging factors may contribute to the series of phenomena described above, but because of the high prevalence of the disease it is more likely that only a very limited subset of factors may directly initiate the pathogenic cascade. Any relevant aging factor candidate will at least need to show: 1) a natural tendency to be altered during lifespan, which in turn will have a critical impact on the multiple aspects of neuron health, 2) a close association with APOE
4, the most robust genetic factor in AD [40], 3) a direct or indirect sensitivity to environmental, physical and mental factors that were found to increase or decrease the incidence of the disease (such as gender, diabetes, physical activity, intellectual stimulation, midlife hypertension, depression, etc.) (for review see [232]). The aging factor cascade hypothesis is presented here as a new framework to open the avenue on other areas of research in the field of AD where the disease modifying strategies tested so far in clinical trials did not produce the beneficial effects expected.

Because the novel therapeutics that are currently in the pipelines of the pharmacological companies seem more relevant to adequately test the amyloid hypothesis, it will be interesting to see the output of all these new approaches on the pathogenesis of AD. The theory proposed here is not in opposition with the A oligomer cascade hypothesis but instead incorporates it in attempt to integrate the new data and concepts that emerged from recent research in the AD field. 7. CONCLUSION Many essential and exciting pieces of data were obtained over the past few years to more accurately understand the fundamental mechanisms that are altered in the age-related neurodegenerative process that characterizes AD. Although the spatiotemporal pattern of neuronal loss is not mimicked in transgenic AD mouse model overexpressing A, these animals are necessary tools to understand the synapse dysfunction that is initiated by A species. The new in vivo approach that we developed recently can now be used to investigate various A preparations in animal models and can be a valuable tool in preclinical trials to test potential drug therapies for AD. Much evidence supports the important role of soluble A oligomers in AD and the upcoming Phase II and III clinical trials will be critical to validate the A oligomer cascade hypothesis. The aging factor cascade hypothesis is proposed here as a new

The Effects of A
Oligomers on Alzheimer's Disease Neurodegeneration

framework to develop disease modifying therapies by taking into consideration the new data and emerging concepts from recent research in the field of AD. CONFLICT OF INTEREST The authors confirm that this article content has no conflicts of interest. ACKNOWLEDGEMENTS The author is grateful to Mitchell Powell and Kristina Sakers for their technical support and for correcting this manuscript. ABBREVIATIONS A
= Amyloid-
AD = Alzheimer’s disease ADDLs = A
-derived diffusible ligands AFM = Atomic force microscopy APOE = Apolipoprotein E APP = Amyloid precursor protein CDK-5 = Cyclin-dependent kinase-5 ChAT = Choline acetyltransferase CSF = Cerebrospinal fluid DG = Dentate gyrus EC = Entorhinal cortex ERK = Extracellular signal-regulated kinase ESI-MS = Electrospray-ionization mass spectrometry FAD = Familial Alzheimer’s disease FT-IR = Fourier transform infrared spectroscopy GSK-3
= Glycogen synthase kinase-3
JNK = c-Jun N-terminal kinases LTD = Long-term depression LTP = Long-term potentiation MCI = Mild cognitive impairment mGluRs = Metabotropic glutamate receptors MRI = Magnetic resonance imaging NFTs = Neurofibrillary tangles NMDA = N-methyl-D-aspartate NMR = Nuclear magnetic resonance spectroscopy = Cellular prion proteins PrPc PS1 = Presenilin 1 PS2 = Presenilin 2 p75NTR = p75 neurotrophin receptor STEP = Striatal-enriched tyrosine phosphatase TEM = Transmission electron microscopy TTR = Transthyretin 3xTg-AD = Triple-transgenic Alzheimer’s disease model REFERENCES [1]

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Received: May 10, 2013

Accepted: July 8, 2013

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