Hypercholesterolemia accelerates intraneuronal accumulation of Aß oligomers resulting in memory impairment in Alzheimer's disease model mice.

AIMS: Hypercholesterolemia is known to be a risk factor for Alzheimer's disease (AD), and diet-induced hypercholesterolemia has been shown to accelerate amyloid pathology in animals. While growing evidence has shown that synaptic and cognitive dysfunction in AD is associated with intraneuronal accumulation of Aß, the relationships between hypercholesterolemia, memory impairment, and intraneuronal Aß remains unclear. The present study aims to clarify this association. MAIN METHODS: Transgenic mice expressing amyloid precursor protein (APP) harboring the Osaka (E693∆) mutation (APP(OSK)-Tg mice) were used. These mice exhibit intraneuronal Aß oligomers and memory impairment from 8months of age. Five-month-old male APP(OSK)-Tg mice and non-Tg littermates were fed a high-cholesterol diet for 1 month to induce hypercholesterolemia. At 6 months of age, their cognitive function was evaluated by the Morris water maze. Intraneuronal Aß, synaptic density, and tau phosphorylation were examined by immunohistochemistry. KEY FINDINGS: Serum and brain cholesterol levels were significantly higher in APP(OSK)-Tg mice and non-Tg littermates that were fed a high-cholesterol diet than in control mice that were fed normal chow, indicating that hypercholesterolemia was successfully induced. Hypercholesterolemic APP(OSK)-Tg mice, but not control APP(OSK)-Tg mice or hypercholesterolemic non-Tg littermates, exhibited impaired spatial reference memory, which was accompanied with intraneuronal accumulation of Aß oligomers, reduced synaptophysin immunoreactivity, and abnormal tau phosphorylation in the hippocampus. Hypercholesterolemia-accelerated accumulation of intraneuronal Aß oligomers was also observed in another model mouse, Tg2576. SIGNIFICANCE: Our findings suggest that hypercholesterolemia accelerates intraneuronal accumulation of Aß oligomers and subsequent synapse loss, resulting in memory impairment.

Intraneuronal amyloid ß oligomers cause cell death via endoplasmic reticulum stress, endosomal/lysosomal leakage, and mitochondrial dysfunction in vivo.

Intraneuronal accumulation of amyloid ß (Aß) is an early pathological change in Alzheimer's disease. Previously, we showed that the E693Δ mutation (referred to as the "Osaka" mutation) of amyloid precursor protein (APP) caused intracellular accumulation of Aß oligomers and apoptosis in transfected COS-7 cells. We also showed that transgenic mice expressing APP(E693Δ) (APP(OSK) ) displayed both an age-dependent accumulation of intraneuronal Aß oligomers from 8 months of age and apparent neuronal loss in the hippocampus at 24 months of age. These findings indicate that intraneuronal Aß oligomers cause cell death, but the mechanism of this process remains unclear. Accordingly, here we investigated the subcellular localization and toxicity of intraneuronal Aß oligomers in APP(OSK) -transgenic mice. We found Aß oligomer accumulation in the endoplasmic reticulum (ER), endosomes/lysosomes, and mitochondria in hippocampal neurons of 22-month-old mice. We also detected up-regulation of Grp78 and HRD1 (an E3 ubiquitin ligase), leakage of cathepsin D from endosomes/lysosomes into cytoplasm, cytochrome c release from mitochondria, and activation of caspase-3 in the hippocampi of 18-month-old mice. Collectively, our findings suggest that intraneuronal Aß oligomers cause cell death by inducing ER stress, endosomal/lysosomal leakage, and mitochondrial dysfunction in vivo. © 2011 Wiley-Liss, Inc.

A mouse model of amyloid beta oligomers: their contribution to synaptic alteration, abnormal tau phosphorylation, glial activation, and neuronal loss in vivo.

Although amyloid beta (Abeta) oligomers are presumed to cause synaptic and cognitive dysfunction in Alzheimer's disease (AD), their contribution to other pathological features of AD remains unclear. To address the latter, we generated APP transgenic mice expressing the E693Delta mutation, which causes AD by enhanced Abeta oligomerization without fibrillization. The mice displayed age-dependent accumulation of intraneuronal Abeta oligomers from 8 months but no extracellular amyloid deposits even at 24 months. Hippocampal synaptic plasticity and memory were impaired at 8 months, at which time the presynaptic marker synaptophysin began to decrease. Furthermore, we detected abnormal tau phosphorylation from 8 months, microglial activation from 12 months, astrocyte activation from 18 months, and neuronal loss at 24 months. These findings suggest that Abeta oligomers cause not only synaptic alteration but also other features of AD pathology and that these mice are a useful model of Abeta oligomer-induced pathology in the absence of amyloid plaques.

Soluble state high resolution atomic force microscopy study of Alzheimer's beta-amyloid oligomers.

We report here the direct observation of high resolution structures of assemblies of Alzheimer beta-amyloid oligomers and monomers using liquid atomic force microscopy (AFM). Visualization of nanoscale features of Abeta oligomers (also known as ADDLs) was carried out in tapping mode AFM in F12 solution. Our results indicate that ADDL preparations exist in solution primarily as a mixture of monomeric peptides and higher molecular mass oligomers. Our study clearly reveals that the size and shape of these oligomer aggregates exhibit a pronounced dependence on concentration. These studies show that wet AFM enables direct assessment of oligomers in physiological fluids and suggests that this method may be developed to visualize Abeta oligomers from human fluids.

CCL2 accelerates microglia-mediated Abeta oligomer formation and progression of neurocognitive dysfunction.

BACKGROUND: The linkages between neuroinflammation and Alzheimer's disease (AD) pathogenesis are well established. What is not, however, is how specific immune pathways and proteins affect the disease. To this end, we previously demonstrated that transgenic over-expression of CCL2 enhanced microgliosis and induced diffuse amyloid plaque deposition in Tg2576 mice. This rodent model of AD expresses a Swedish beta-amyloid (Abeta) precursor protein mutant. METHODOLOGY/PRINCIPAL FINDINGS: We now report that CCL2 transgene expression accelerates deficits in spatial and working memory and hippocampal synaptic transmission in beta-amyloid precursor protein (APP) mice as early as 2-3 months of age. This is followed by increased numbers of microglia that are seen surrounding Abeta oligomers. CCL2 does not suppress Abeta degradation. Rather, CCL2 and tumor necrosis factor-alpha directly facilitated Abeta uptake, intracellular Abeta oligomerization, and protein secretion. CONCLUSIONS/SIGNIFICANCE: We posit that CCL2 facilitates Abeta oligomer formation in microglia and propose that such events accelerate memory dysfunction by affecting Abeta seeding in the brain.

Insulin receptor dysfunction impairs cellular clearance of neurotoxic oligomeric a{beta}.

Accumulation of amyloid beta (Abeta) oligomers in the brain is toxic to synapses and may play an important role in memory loss in Alzheimer disease. However, how these toxins are built up in the brain is not understood. In this study we investigate whether impairments of insulin and insulin-like growth factor-1 (IGF-1) receptors play a role in aggregation of Abeta. Using primary neuronal culture and immortal cell line models, we show that expression of normal insulin or IGF-1 receptors confers cells with abilities to reduce exogenously applied Abeta oligomers (also known as ADDLs) to monomers. In contrast, transfection of malfunctioning human insulin receptor mutants, identified originally from patient with insulin resistance syndrome, or inhibition of insulin and IGF-1 receptors via pharmacological reagents increases ADDL levels by exacerbating their aggregation. In healthy cells, activation of insulin and IGF-1 receptor reduces the extracellular ADDLs applied to cells via seemingly the insulin-degrading enzyme activity. Although insulin triggers ADDL internalization, IGF-1 appears to keep ADDLs on the cell surface. Nevertheless, both insulin and IGF-1 reduce ADDL binding, protect synapses from ADDL synaptotoxic effects, and prevent the ADDL-induced surface insulin receptor loss. Our results suggest that dysfunctions of brain insulin and IGF-1 receptors contribute to Abeta aggregation and subsequent synaptic loss.

AAV1/2-mediated CNS gene delivery of dominant-negative CCL2 mutant suppresses gliosis, beta-amyloidosis, and learning impairment of APP/PS1 mice.

Accumulation of aggregated amyloid-beta (Abeta) peptide was studied as an initial step for Alzheimer's disease (AD) pathogenesis. Following amyloid plaque formation, reactive microglia and astrocytes accumulate around plaques and cause neuroinflammation. Here brain chemokines play a major role for the glial accumulation. We have previously shown that transgenic overexpression of chemokine CCL2 in the brain results in increased microglial accumulation and diffuse amyloid plaque deposition in a transgenic mouse model of AD expressing Swedish amyloid precursor protein (APP) mutant. Here, we report that adeno-associated virus (AAV) serotype 1 and 2 hybrid efficiently deliver 7ND gene, a dominant-negative CCL2 mutant, in a dose-response manner and express >1,000-fold higher recombinant CCL2 than basal levels after a single administration. AAV1/2 hybrid virus principally infected neurons without neuroinflammation with sustained expression for 6-months. 7ND expressed in APP/presenilin-1 (APP/PS1) bigenic mice reduced astro/microgliosis, beta-amyloidosis, including suppression of both fibrillar and oligomer Abeta accumulation, and improved spatial learning. Our data support the idea that the AAV1/2 system is a useful tool for CNS gene delivery, and suppression of CCL2 may be a therapeutic target for the amelioration of AD-related neuroinflammation.

Targeting generation of antibodies specific to conformational epitopes of amyloid beta-derived neurotoxins.

Individuals with early Alzheimer's disease (AD) suffer from a selective and profound failure to form new memories. A novel molecular mechanism with implications for therapeutics and diagnostics is now emerging in which the specificity of AD for memory derives from disruption of plasticity at synapses targeted by toxic Abeta oligomers (also known as ADDLs). ADDLs accumulate in AD brain and constitute long-lived alternatives to the disease-defining Abeta fibrils deposited in amyloid plaques. The AD-like cellular pathologies induced by ADDLs suggest their impact could provide a unifying mechanism for AD pathogenesis, explaining why early stage disease is specific for memory and accounting for major facets of AD neuropathology. Discovery of these new toxins has provided an appealing target for disease-modifying immunotherapy. For optimal protection against these toxins, antibodies should bind to the pathological oligomers without being depleted by their monomeric subunits, which are rapidly generated by membrane protein turnover. A solution to this problem is likely to come from the continued development of conformation-specific antibodies, as described here. Prototype conformation-specific antibodies, not yet in the clinic, have been introduced and utilized in multiple applications for their ability to bind with high specificity and affinity to ADDLs. It can be anticipated that further development of such antibodies for use in clinical trials will come in the near future.

Protection of synapses against Alzheimer's-linked toxins: insulin signaling prevents the pathogenic binding of Abeta oligomers.

Synapse deterioration underlying severe memory loss in early Alzheimer's disease (AD) is thought to be caused by soluble amyloid beta (Abeta) oligomers. Mechanistically, soluble Abeta oligomers, also referred to as Abeta-derived diffusible ligands (ADDLs), act as highly specific pathogenic ligands, binding to sites localized at particular synapses. This binding triggers oxidative stress, loss of synaptic spines, and ectopic redistribution of receptors critical to plasticity and memory. We report here the existence of a protective mechanism that naturally shields synapses against ADDL-induced deterioration. Synapse pathology was investigated in mature cultures of hippocampal neurons. Before spine loss, ADDLs caused major downregulation of plasma membrane insulin receptors (IRs), via a mechanism sensitive to calcium calmodulin-dependent kinase II (CaMKII) and casein kinase II (CK2) inhibition. Most significantly, this loss of surface IRs, and ADDL-induced oxidative stress and synaptic spine deterioration, could be completely prevented by insulin. At submaximal insulin doses, protection was potentiated by rosiglitazone, an insulin-sensitizing drug used to treat type 2 diabetes. The mechanism of insulin protection entailed a marked reduction in pathogenic ADDL binding. Surprisingly, insulin failed to block ADDL binding when IR tyrosine kinase activity was inhibited; in fact, a significant increase in binding was caused by IR inhibition. The protective role of insulin thus derives from IR signaling-dependent downregulation of ADDL binding sites rather than ligand competition. The finding that synapse vulnerability to ADDLs can be mitigated by insulin suggests that bolstering brain insulin signaling, which can decline with aging and diabetes, could have significant potential to slow or deter AD pathogenesis.

Conformation-dependent single-chain variable fragment antibodies specifically recognize beta-amyloid oligomers.

Increasing evidence indicates that beta-amyloid (Abeta) oligomers rather than monomers or fibrils are the major toxic agents that specifically inhibit synaptic plasticity and long-term potentiation (LTP) in Alzheimer's disease (AD). Neutralization of Abeta oligomeric toxicity was found to reverse memory deficits. Here, we report four single-chain variable fragment (scFv) antibodies isolated from the naive human scFv library by phage display that specifically recognized Abeta oligomers but not monomers and fibrils. These conformation-dependent scFv antibodies inhibit both Abeta fibrillation and cytotoxicity and bind to the same type of eptitope displayed on the Abeta oligomers. Such scFv antibodies specifically targeting toxic Abeta oligomers may have potential therapeutic and diagnostic applications for AD.

The E693Delta mutation in amyloid precursor protein increases intracellular accumulation of amyloid beta oligomers and causes endoplasmic reticulum stress-induced apoptosis in cultured cells.

The E693Delta mutation within the amyloid precursor protein (APP) has been suggested to cause dementia via the enhanced formation of synaptotoxic amyloid beta (Abeta) oligomers. However, this mutation markedly decreases Abeta secretion, implying the existence of an additional mechanism of neuronal dysfunction that is independent of extracellular Abeta. We therefore examined the effects of this mutation on both APP processing to produce Abeta as well as subcellular localization and accumulation of Abeta in transfected HEK293 and COS-7 cells. Both beta- and gamma-cleavage of mutant APP increased, indicating a lack of inhibition in Abeta production. Instead, this mutation promoted Abeta accumulation within cells, including the endoplasmic reticulum (ER), Golgi apparatus, early and late endosomes, lysosomes, and autophagosomes, all of which have been proposed as intracellular sites of Abeta generation and/or degradation, suggesting impairment of APP/Abeta trafficking. Notably, the intracellular mutant Abeta was found to predominantly form oligomers. Concomitant with this accumulation, the ER stress markers Grp78 and phosphorylated eIF2alpha were both strongly induced. Furthermore, the activation of caspase-4 and -3 as well as DNA fragmentation were detected in these cells. These results suggest that mutant Abeta induces alteration of Abeta trafficking and subsequent ER stress-induced apoptosis via enhancement of its intracellular oligomerization. Our findings suggest that Abeta oligomers exhibit toxicity in the extracellular space and within the cells themselves.

Detection and Identification of Bioanalytes with High Resolution LSPR Spectroscopy and MALDI Mass Spectrometry.

High resolution localized surface plasmon resonance (HR-LSPR) sensors were combined with matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) for the first time. LSPR sensors provide real-time label-free detection of molecular adsorption. Subsequent MALDI-MS analysis enables identification of the adsorbed molecules. This synergistic LSPR-MS approach was applied to the detection and identification of amyloid beta oligomers which play an important role in the molecular pathogenesis of Alzheimer's Disease.

Alzheimer's disease-type neuronal tau hyperphosphorylation induced by A beta oligomers.

Alzheimer's disease (AD) is characterized by presence of extracellular fibrillar A beta in amyloid plaques, intraneuronal neurofibrillary tangles consisting of aggregated hyperphosphorylated tau and elevated brain levels of soluble A beta oligomers (ADDLs). A major question is how these disparate facets of AD pathology are mechanistically related. Here we show that, independent of the presence of fibrils, ADDLs stimulate tau phosphorylation in mature cultures of hippocampal neurons and in neuroblastoma cells at epitopes characteristically hyperphosphorylated in AD. A monoclonal antibody that targets ADDLs blocked their attachment to synaptic binding sites and prevented tau hyperphosphorylation. Tau phosphorylation was blocked by the Src family tyrosine kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7(t-butyl)pyrazol(3,4-D)pyramide (PP1), and by the phosphatidylinositol-3-kinase inhibitor LY294002. Significantly, tau hyperphosphorylation was also induced by a soluble aqueous extract containing A beta oligomers from AD brains, but not by an extract from non-AD brains. A beta oligomers have been increasingly implicated as the main neurotoxins in AD, and the current results provide a unifying mechanism in which oligomer activity is directly linked to tau hyperphosphorylation in AD pathology.

Amyloid beta oligomers induce impairment of neuronal insulin receptors.

Recent studies have indicated an association between Alzheimer's disease (AD) and central nervous system (CNS) insulin resistance. However, the cellular mechanisms underlying the link between these two pathologies have not been elucidated. Here we show that signal transduction by neuronal insulin receptors (IR) is strikingly sensitive to disruption by soluble Abeta oligomers (also known as ADDLs). ADDLs are known to accumulate in AD brain and have recently been implicated as primary candidates for initiating deterioration of synapse function, composition, and structure. Using mature cultures of hippocampal neurons, a preferred model for studies of synaptic cell biology, we found that ADDLs caused a rapid and substantial loss of neuronal surface IRs specifically on dendrites bound by ADDLs. Removal of dendritic IRs was associated with increased receptor immunoreactivity in the cell body, indicating redistribution of the receptors. The neuronal response to insulin, measured by evoked IR tyrosine autophosphorylation, was greatly inhibited by ADDLs. Inhibition also was seen with added glutamate or potassium-induced depolarization. The effects on IR function were completely blocked by NMDA receptor antagonists, tetrodotoxin, and calcium chelator BAPTA-AM. Downstream from the IR, ADDLs induced a phosphorylation of Akt at serine473, a modification associated with neurodegenerative and insulin resistance diseases. These results identify novel factors that affect neuronal IR signaling and suggest that insulin resistance in AD brain is a response to ADDLs, which disrupt insulin signaling and may cause a brain-specific form of diabetes as part of an overall pathogenic impact on CNS synapses.

Abeta oligomers induce neuronal oxidative stress through an N-methyl-D-aspartate receptor-dependent mechanism that is blocked by the Alzheimer drug memantine.

Oxidative stress is a major aspect of Alzheimer disease (AD) pathology. We have investigated the relationship between oxidative stress and neuronal binding of Abeta oligomers (also known as ADDLs). ADDLs are known to accumulate in brain tissue of AD patients and are considered centrally related to pathogenesis. Using hippocampal neuronal cultures, we found that ADDLs stimulated excessive formation of reactive oxygen species (ROS) through a mechanism requiring N-methyl-d-aspartate receptor (NMDA-R) activation. ADDL binding to neurons was reduced and ROS formation was completely blocked by an antibody to the extracellular domain of the NR1 subunit of NMDA-Rs. In harmony with a steric inhibition of ADDL binding by NR1 antibodies, ADDLs that were bound to detergent-extracted synaptosomal membranes co-immunoprecipitated with NMDA-R subunits. The NR1 antibody did not affect ROS formation induced by NMDA, showing that NMDA-Rs themselves remained functional. Memantine, an open channel NMDA-R antagonist prescribed as a memory-preserving drug for AD patients, completely protected against ADDL-induced ROS formation, as did other NMDA-R antagonists. Memantine and the anti-NR1 antibody also attenuated a rapid ADDL-induced increase in intraneuronal calcium, which was essential for stimulated ROS formation. These results show that ADDLs bind to or in close proximity to NMDA-Rs, triggering neuronal damage through NMDA-R-dependent calcium flux. This response provides a pathologically specific mechanism for the therapeutic action of memantine, indicates a role for ROS dysregulation in ADDL-induced cognitive impairment, and supports the unifying hypothesis that ADDLs play a central role in AD pathogenesis.

Monoclonal antibodies that target pathological assemblies of Abeta.

Amyloid beta (Abeta) immunotherapy for Alzheimer's disease has shown initial success in mouse models of Alzheimer's disease and in human patients. However, because of meningoencephalitis in clinical trials of active vaccination, approaches using therapeutic antibodies may be preferred. As a novel antigen to generate monoclonal antibodies, the current study has used Abeta oligomers (amyloid beta-derived diffusible ligands, ADDLs), pathological assemblies known to accumulate in Alzheimer's disease brain. Clones were selected for the ability to discriminate Alzheimer's disease from control brains in extracts and tissue sections. These antibodies recognized Abeta oligomers and fibrils but not the physiologically prevalent Abeta monomer. Discrimination derived from an epitope found in assemblies of Abeta1-28 and ADDLs but not in other sequences, including Abeta1-40. Immunoneutralization experiments showed that toxicity and attachment of ADDLs to synapses in culture could be prevented. ADDL-induced reactive oxygen species (ROS) generation was also inhibited, establishing this response to be oligomer-dependent. Inhibition occurred whether ADDLs were prepared in vitro or obtained from Alzheimer's disease brain. As conformationally sensitive monoclonal antibodies that selectively immunoneutralize binding and function of pathological Abeta assemblies, these antibodies provide tools by which pathological Abeta assemblies from Alzheimer's disease brain might be isolated and evaluated, as well as offering a valuable prototype for new antibodies useful for Alzheimer's disease therapeutics.

Amyloid-beta-induced pathological behaviors are suppressed by Ginkgo biloba extract EGb 761 and ginkgolides in transgenic Caenorhabditis elegans.

Amyloid-beta (Abeta) toxicity has been postulated to initiate synaptic loss and subsequent neuronal degeneration seen in Alzheimer's disease (AD). We previously demonstrated that the standardized Ginkgo biloba extract EGb 761, commonly used to enhance memory and by AD patients for dementia, inhibits Abeta-induced apoptosis in neuroblastoma cells. In this study, we use EGb 761 and its single constituents to associate Abeta species with Abeta-induced pathological behaviors in a model organism, Caenorhabditis elegans. We report that EGb 761 and one of its components, ginkgolide A, alleviates Abeta-induced pathological behaviors, including paralysis, and reduces chemotaxis behavior and 5-HT hypersensitivity in a transgenic C. elegans. We also show that EGb 761 inhibits Abeta oligomerization and Abeta deposits in the worms. Moreover, reducing oxidative stress is not the mechanism by which EGb 761 and ginkgolide A suppress Abeta-induced paralysis because the antioxidant L-ascorbic acid reduced intracellular levels of hydrogen peroxide to the same extent as EGb 761, but was not nearly as effective in suppressing paralysis in the transgenic C. elegans. These findings suggest that (1) EGb 761 suppresses Abeta-related pathological behaviors, (2) the protection against Abeta toxicity by EGb 761 is mediated primarily by modulating Abeta oligomeric species, and (3) ginkgolide A has therapeutic potential for prevention and treatment of AD.

Temporal profile of amyloid-beta (Abeta) oligomerization in an in vivo model of Alzheimer disease. A link between Abeta and tau pathology.

Accumulation of amyloid-beta (Abeta) is one of the earliest molecular events in Alzheimer disease (AD), whereas tau pathology is thought to be a later downstream event. It is now well established that Abeta exists as monomers, oligomers, and fibrils. To study the temporal profile of Abeta oligomer formation in vivo and to determine their interaction with tau pathology, we used the 3xTg-AD mice, which develop a progressive accumulation of plaques and tangles and cognitive impairments. We show that SDS-resistant Abeta oligomers accumulate in an age-dependent fashion, and we present evidence to show that oligomerization of Abeta appears to first occur intraneuronally. Finally, we show that a single intrahippocampal injection of a specific oligomeric antibody is sufficient to clear Abeta pathology, and more importantly, tau pathology. Therefore, Abeta oligomers may play a role in the induction of tau pathology, making the interference of Abeta oligomerization a valid therapeutic target.

Synaptic targeting by Alzheimer's-related amyloid beta oligomers.

The cognitive hallmark of early Alzheimer's disease (AD) is an extraordinary inability to form new memories. For many years, this dementia was attributed to nerve-cell death induced by deposits of fibrillar amyloid beta (Abeta). A newer hypothesis has emerged, however, in which early memory loss is considered a synapse failure caused by soluble Abeta oligomers. Such oligomers rapidly block long-term potentiation, a classic experimental paradigm for synaptic plasticity, and they are strikingly elevated in AD brain tissue and transgenic-mouse AD models. The current work characterizes the manner in which Abeta oligomers attack neurons. Antibodies raised against synthetic oligomers applied to AD brain sections were found to give diffuse stain around neuronal cell bodies, suggestive of a dendritic pattern, whereas soluble brain extracts showed robust AD-dependent reactivity in dot immunoblots. Antigens in unfractionated AD extracts attached with specificity to cultured rat hippocampal neurons, binding within dendritic arbors at discrete puncta. Crude fractionation showed ligand size to be between 10 and 100 kDa. Synthetic Abeta oligomers of the same size gave identical punctate binding, which was highly selective for particular neurons. Image analysis by confocal double-label immunofluorescence established that >90% of the punctate oligomer binding sites colocalized with the synaptic marker PSD-95 (postsynaptic density protein 95). Synaptic binding was accompanied by ectopic induction of Arc, a synaptic immediate-early gene, the overexpression of which has been linked to dysfunctional learning. Results suggest the hypothesis that targeting and functional disruption of particular synapses by Abeta oligomers may provide a molecular basis for the specific loss of memory function in early AD.

Self-assembly of Abeta(1-42) into globular neurotoxins.

Amyloid beta 1-42 (Abeta(1-42)) is a self-associating peptide that becomes neurotoxic upon aggregation. Toxicity originally was attributed to the presence of large, readily formed Abeta fibrils, but a variety of other toxic species are now known. The current study shows that Abeta(1-42) can self-assemble into small, stable globular assemblies free of fibrils and protofibrils. Absence of large molecules was verified by atomic force microscopy (AFM) and nondenaturing gel electrophoresis. Denaturing electrophoresis revealed that the globular assemblies comprised oligomers ranging from trimers to 24mers. Oligomers prepared at 4 degrees C stayed fibril-free for days and remained so when shifted to 37 degrees C, although the spectrum of sizes shifted toward larger oligomers at the higher temperature. The soluble, globular Abeta(1-42) oligomers were toxic to PC12 cells, impairing reduction of MTT and interfering with ERK and Rac signal transduction. Occasionally, oligomers were neither toxic nor recognized by toxicity-neutralizing antibodies, suggesting that oligomers could assume alternative conformations. Tests for oligomerization-blocking activity were carried out by dot-blot immunoassays and showed that neuroprotective extracts of Ginkgo biloba could inhibit oligomer formation at very low doses. The observed neurotoxicity, structure, and stability of synthetic Abeta(1-42) globular assemblies support the hypothesis that Abeta(1-42) oligomers play a role in triggering nerve cell dysfunction and death in Alzheimer's disease.