Kalirin-7 prevents dendritic spine dysgenesis induced by amyloid beta-derived oligomers.

Synapse degeneration and dendritic spine dysgenesis are believed to be crucial early steps in Alzheimer's disease (AD), and correlate with cognitive deficits in AD patients. Soluble amyloid beta (Aß)-derived oligomers, also termed Aß-derived diffusible ligands (ADDLs), accumulate in the brain of AD patients and play a crucial role in AD pathogenesis. ADDLs bind to mature hippocampal neurons, induce structural changes in dendritic spines and contribute to neuronal death. However, mechanisms underlying structural and toxic effects are not fully understood. Here, we report that ADDLs bind to cultured mature cortical pyramidal neurons and induce spine dysgenesis. ADDL treatment induced the rapid depletion of kalirin-7, a brain-specific guanine-nucleotide exchange factor for the small GTPase Rac1, from spines. Kalirin-7 is a key regulator of dendritic spine morphogenesis and maintenance in forebrain pyramidal neurons and here we show that overexpression of kalirin-7 prevents ADDL-induced spine degeneration. Taken together, our results suggest that kalirin-7 may play a role in the early events leading to synapse degeneration, and its pharmacological activation may prevent or delay synapse pathology in AD.

A human scFv antibody that targets and neutralizes high molecular weight pathogenic amyloid-ß oligomers.

Brain accumulation of soluble oligomers of the amyloid-ß peptide (AßOs) is increasingly considered a key early event in the pathogenesis of Alzheimer's disease (AD). A variety of AßO species have been identified, both in vitro and in vivo, ranging from dimers to 24mers and higher order oligomers. However, there is no consensus in the literature regarding which AßO species are most germane to AD pathogenesis. Antibodies capable of specifically recognizing defined subpopulations of AßOs would be a valuable asset in the identification, isolation, and characterization of AD-relevant AßO species. Here, we report the characterization of a human single chain antibody fragment (scFv) denoted NUsc1, one of a number of scFvs we have identified that stringently distinguish AßOs from both monomeric and fibrillar Aß. NUsc1 readily detected AßOs previously bound to dendrites in cultured hippocampal neurons. In addition, NUsc1 blocked AßO binding and reduced AßO-induced neuronal oxidative stress and tau hyperphosphorylation in cultured neurons. NUsc1 further distinguished brain extracts from AD-transgenic mice from wild type (WT) mice, and detected endogenous AßOs in fixed AD brain tissue and AD brain extracts. Biochemical analyses indicated that NUsc1 targets a subpopulation of AßOs with apparent molecular mass greater than 50 kDa. Results indicate that NUsc1 targets a particular AßO species relevant to AD pathogenesis, and suggest that NUsc1 may constitute an effective tool for AD diagnostics and therapeutics.

Intraneuronal aggregation of the ß-CTF fragment of APP (C99) induces Aß-independent lysosomal-autophagic pathology.

Endosomal-autophagic-lysosomal (EAL) dysfunction is an early and prominent neuropathological feature of Alzheimers's disease, yet the exact molecular mechanisms contributing to this pathology remain undefined. By combined biochemical, immunohistochemical and ultrastructural approaches, we demonstrate a link between EAL pathology and the intraneuronal accumulation of the ß-secretase-derived ßAPP fragment (C99) in two in vivo models, 3xTgAD mice and adeno-associated viral-mediated C99-infected mice. We present a pathological loop in which the accumulation of C99 is both the effect and causality of impaired lysosomal-autophagic function. The deleterious effect of C99 was found to be linked to its aggregation within EAL-vesicle membranes leading to disrupted lysosomal proteolysis and autophagic impairment. This effect was Aß independent and was even exacerbated when γ-secretase was pharmacologically inhibited. No effect was observed in inhibitor-treated wild-type animals suggesting that lysosomal dysfunction was indeed directly linked to C99 accumulation. In some brain areas, strong C99 expression also led to inflammatory responses and synaptic dysfunction. Taken together, this work demonstrates a toxic effect of C99 which could underlie some of the early-stage anatomical hallmarks of Alzheimer's disease pathology. Our work also proposes molecular mechanisms likely explaining some of the unfavorable side-effects associated with γ-secretase inhibitor-directed therapies.

Clathrin adaptor CALM/PICALM is associated with neurofibrillary tangles and is cleaved in Alzheimer's brains.

PICALM, a clathrin adaptor protein, plays important roles in clathrin-mediated endocytosis in all cell types. Recently, genome-wide association studies identified single nucleotide polymorphisms in PICALM gene as genetic risk factors for late-onset Alzheimer disease (LOAD). We analysed by western blotting with several anti-PICALM antibodies the pattern of expression of PICALM in human brain extracts. We found that PICALM was abnormally cleaved in AD samples and that the level of the uncleaved 65-75 kDa full-length PICALM species was significantly decreased in AD brains. Cleavage of human PICALM after activation of endogenous calpain or caspase was demonstrated in vitro. Immunohistochemistry revealed that PICALM was associated in situ with neurofibrillary tangles, co-localising with conformationally abnormal and hyperphosphorylated tau in LOAD, familial AD and Down syndrome cases. PHF-tau proteins co-immunoprecipitated with PICALM. PICALM was highly expressed in microglia in LOAD. These observations suggest that PICALM is associated with the development of AD tau pathology. PICALM cleavage could contribute to endocytic dysfunction in AD.

Visualization of co-localization in Aß42-administered neuroblastoma cells reveals lysosome damage and autophagosome accumulation related to cell death.

Aß42 [amyloid-ß peptide-(1-42)] plays a central role in Alzheimer's disease and is known to have a detrimental effect on neuronal cell function and survival when assembled into an oligomeric form. In the present study we show that administration of freshly prepared Aß42 oligomers to a neuroblastoma (SH-SY5Y) cell line results in a reduction in survival, and that Aß42 enters the cells prior to cell death. Immunoconfocal and immunogold electron microscopy reveal the path of the Aß42 with time through the endosomal system and shows that it accumulates in lysosomes. A 24 h incubation with Aß results in cells that have damaged lysosomes showing signs of enzyme leakage, accumulate autophagic vacuoles and exhibit severely disrupted nuclei. Endogenous Aß is evident in the cells and the results of the present study suggest that the addition of Aß oligomers disrupts a crucial balance in Aß conformation and concentration inside neuronal cells, resulting in catastrophic effects on cellular function and, ultimately, in cell death.

Aß oligomer-induced synapse degeneration in Alzheimer's disease.

Aß oligomers cause a collection of molecular events associated with memory loss in Alzheimer's disease, centering on disrupting the maintenance of synapse structure and function. In this brief review of the synaptotoxic effects of Aß oligomers, we focus on the neuronal properties governing oligomer targeting and toxicity-especially with respect to binding sites and mechanisms of binding. We also discuss ways in which mechanistic insights from other diseases offer clues in the pursuit of the molecular basis of Alzheimer's disease.

Alzheimer's-associated Abeta oligomers show altered structure, immunoreactivity and synaptotoxicity with low doses of oleocanthal.

It now appears likely that soluble oligomers of amyloid-beta1-42 peptide, rather than insoluble fibrils, act as the primary neurotoxin in Alzheimer's disease (AD). Consequently, compounds capable of altering the assembly state of these oligomers (referred to as ADDLs) may have potential for AD therapeutics. Phenolic compounds are of particular interest for their ability to disrupt Abeta oligomerization and reduce pathogenicity. This study has focused on oleocanthal (OC), a naturally-occurring phenolic compound found in extra-virgin olive oil. OC increased the immunoreactivity of soluble Abeta species, when assayed with both sequence- and conformation-specific Abeta antibodies, indicating changes in oligomer structure. Analysis of oligomers in the presence of OC showed an upward shift in MW and a ladder-like distribution of SDS-stable ADDL subspecies. In comparison with control ADDLs, oligomers formed in the presence of OC (Abeta-OC) showed equivalent colocalization at synapses but exhibited greater immunofluorescence as a result of increased antibody recognition. The enhanced signal at synapses was not due to increased synaptic binding, as direct detection of fluorescently-labeled ADDLs showed an overall reduction in ADDL signal in the presence of OC. Decreased binding to synapses was accompanied by significantly less synaptic deterioration assayed by drebrin loss. Additionally, treatment with OC improved antibody clearance of ADDLs. These results indicate oleocanthal is capable of altering the oligomerization state of ADDLs while protecting neurons from the synaptopathological effects of ADDLs and suggest OC as a lead compound for development in AD therapeutics.

Abeta oligomer-induced aberrations in synapse composition, shape, and density provide a molecular basis for loss of connectivity in Alzheimer's disease.

The basis for memory loss in early Alzheimer's disease (AD) seems likely to involve synaptic damage caused by soluble Abeta-derived oligomers (ADDLs). ADDLs have been shown to build up in the brain and CSF of AD patients and are known to interfere with mechanisms of synaptic plasticity, acting as gain-of-function ligands that attach to synapses. Because of the correlation between AD dementia and synaptic degeneration, we investigated here the ability of ADDLs to affect synapse composition, structure, and abundance. Using highly differentiated cultures of hippocampal neurons, a preferred model for studies of synapse cell biology, we found that ADDLs bound to neurons with specificity, attaching to presumed excitatory pyramidal neurons but not GABAergic neurons. Fractionation of ADDLs bound to forebrain synaptosomes showed association with postsynaptic density complexes containing NMDA receptors, consistent with observed attachment of ADDLs to dendritic spines. During binding to hippocampal neurons, ADDLs promoted a rapid decrease in membrane expression of memory-related receptors (NMDA and EphB2). Continued exposure resulted in abnormal spine morphology, with induction of long thin spines reminiscent of the morphology found in mental retardation, deafferentation, and prionoses. Ultimately, ADDLs caused a significant decrease in spine density. Synaptic deterioration, which was accompanied by decreased levels of the spine cytoskeletal protein drebrin, was blocked by the Alzheimer's therapeutic drug Namenda. The observed disruption of dendritic spines links ADDLs to a major facet of AD pathology, providing strong evidence that ADDLs in AD brain cause neuropil damage believed to underlie dementia.

Advances on the understanding of the origins of synaptic pathology in AD.

Although Alzheimer's disease (AD) was first discovered a century ago, we are still facing a lack of definitive diagnosis during the patient's lifetime and are unable to prescribe a curative treatment. However, the past 10 years have seen a "revamping" of the main hypothesis about AD pathogenesis and the hope to foresee possible treatment. AD is no longer considered an irreversible disease. A major refinement of the classic beta-amyloid cascade describing amyloid fibrils as neurotoxins has been made to integrate the key scientific evidences demonstrating that the first pathological event occurring in AD early stages affects synaptic function and maintenance. A concept fully compatible with synapse loss being the best pathological correlate of AD rather than other described neuropathological hallmarks (amyloid plaques, neurofibrillary tangles or neuronal death). The notion that synaptic alterations might be reverted, thus offering a potential curability, was confirmed by immunotherapy experiments targeting beta-amyloid protein in transgenic AD mice in which cognitive functions were improved despite no reduction in the amyloid plaques burden. The updated amyloid cascade now integrates the synapse failure triggered by soluble Abeta-oligomers. Still no consensus has been reached on the most toxic Abeta conformations, neither on their site of production nor on their extra- versus intra-cellular actions. Evidence shows that soluble Abeta oligomers or ADDLs bind selectively to neurons at their synaptic loci, and trigger major changes in synapse composition and morphology, which ultimately leads to dendritic spine loss. However, the exact mechanism is not yet fully understood but is suspected to involve some membrane receptor(s).

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.

Acute amnestic encephalopathy in amyloid-ß oligomer-injected mice is due to their widespread diffusion in vivo.

Amyloid-ß (Aß) oligomers are the suspected culprit as initiators of Alzheimer's disease (AD). However, their diffusion in the brain remains unknown. Here, we studied Aß oligomers' dissemination and evaluated their in vivo toxicity. Wild-type mice were injected with 50 pmol of synthetic Aß oligomers (of different size) in the hippocampus. Oligomers diffused largely in the brain as soon as 1 hour and up to 7 days after injection. A transient encephalopathy with memory impairment was induced by this unique injection. The immunoreactivity of the postsynaptic marker PSD95 was diffusely decreased. Similar results (both on memory and PSD95 immunoreactivity) were obtained with delipidated and high molecular weight oligomers (>50 kDa) but not with smaller assemblies. Tau hyperphosphorylation was observed in the oligomer-injected brains. Finally, fos immunostaining was increased in Aß-derived diffusible ligands-injected mice, suggesting neuronal hyperactivity. Rapid and widespread diffusion of Aß oligomers was demonstrated in vivo and associated with decreased synaptic markers and memory deficits which gives new insight to the pathogenicity of Aß.

Towards non-invasive diagnostic imaging of early-stage Alzheimer's disease.

One way to image the molecular pathology in Alzheimer's disease is by positron emission tomography using probes that target amyloid fibrils. However, these fibrils are not closely linked to the development of the disease. It is now thought that early-stage biomarkers that instigate memory loss are composed of Aß oligomers. Here, we report a sensitive molecular magnetic resonance imaging contrast probe that is specific for Aß oligomers. We attach oligomer-specific antibodies onto magnetic nanostructures and show that the complex is stable and binds to Aß oligomers on cells and brain tissues to give a magnetic resonance imaging signal. When intranasally administered to an Alzheimer's disease mouse model, the probe readily reached hippocampal Aß oligomers. In isolated samples of human brain tissue, we observed a magnetic resonance imaging signal that distinguished Alzheimer's disease from controls. Such nanostructures that target neurotoxic Aß oligomers are potentially useful for evaluating the efficacy of new drugs and ultimately for early-stage Alzheimer's disease diagnosis and disease management.

Brain transit and ameliorative effects of intranasally delivered anti-amyloid-ß oligomer antibody in 5XFAD mice.

Alzheimer's disease (AD) is a global health crisis with limited treatment options. Despite major advances in neurotherapeutics, poor brain penetration due to the blood-brain barrier continues to pose a big challenge in overcoming the access of therapeutics to the central nervous system. In that regard, the non-invasive intranasal route of brain targeting is gaining considerable attention. The nasal mucosa offers a large surface area, rapid absorption, and avoidance of first-pass metabolism increasing drug bioavailability with less systemic side effects. Intranasal delivery is known to utilize olfactory, rostral migratory stream, and trigeminal routes to reach the brain. This investigation confirmed that intranasal delivery of oligomeric amyloid-ß antibody (NU4) utilized all three routes to enter the brain with a resident time of 96 hours post single bolus intranasal administration, and showed evidence of perikaryal and parenchymal uptake of NU4 in 5XFAD mouse brain, confirming the intranasal route as a non-invasive and efficient way of delivering therapeutics to the brain. In addition, this study demonstrated that intranasal delivery of NU4 antibody lowered cerebral amyloid-ß and improved spatial learning in 5XFAD mice.

Disruption of neocortical histone H3 homeostasis by soluble Aß: implications for Alzheimer's disease.

Amyloid-ß peptide (Aß) fragment misfolding may play a crucial role in the progression of Alzheimer's disease (AD) pathophysiology as well as epigenetic mechanisms at the DNA and histone level. We hypothesized that histone H3 homeostasis is disrupted in association with the appearance of soluble Aß at an early stage in AD progression. We identified, localized, and compared histone H3 modifications in multiple model systems (neural-like SH-SY5Y, primary neurons, Tg2576 mice, and AD neocortex), and narrowed our focus to investigate 3 key motifs associated with regulating transcriptional activation and inhibition: acetylated lysine 14, phosphorylated serine 10 and dimethylated lysine 9. Our results in vitro and in vivo indicate that multimeric soluble Aß may be a potent signaling molecule indirectly modulating the transcriptional activity of DNA by modulating histone H3 homeostasis. These findings reveal potential loci of transcriptional disruption relevant to AD. Identifying genes that undergo significant epigenetic alterations in response to Aß could aid in the understanding of the pathogenesis of AD, as well as suggesting possible new treatment strategies.

Alzheimer's disease pathology in the neocortex and hippocampus of the western lowland gorilla (Gorilla gorilla gorilla).

The two major histopathologic hallmarks of Alzheimer's disease (AD) are amyloid beta protein (Aß) plaques and neurofibrillary tangles (NFT). Aß pathology is a common feature in the aged nonhuman primate brain, whereas NFT are found almost exclusively in humans. Few studies have examined AD-related pathology in great apes, which are the closest phylogenetic relatives of humans. In the present study, we examined Aß and tau-like lesions in the neocortex and hippocampus of aged male and female western lowland gorillas using immunohistochemistry and histochemistry. Analysis revealed an age-related increase in Aß-immunoreactive plaques and vasculature in the gorilla brain. Aß plaques were more abundant in the neocortex and hippocampus of females, whereas Aß-positive blood vessels were more widespread in male gorillas. Plaques were also Aß40-, Aß42-, and Aß oligomer-immunoreactive, but only weakly thioflavine S- or 6-CN-PiB-positive in both sexes, indicative of the less fibrillar (diffuse) nature of Aß plaques in gorillas. Although phosphorylated neurofilament immunostaining revealed a few dystrophic neurites and neurons, choline acetyltransferase-immunoreactive fibers were not dystrophic. Neurons stained for the tau marker Alz50 were found in the neocortex and hippocampus of gorillas at all ages. Occasional Alz50-, MC1-, and AT8-immunoreactive astrocyte and oligodendrocyte coiled bodies and neuritic clusters were seen in the neocortex and hippocampus of the oldest gorillas. This study demonstrates the spontaneous presence of both Aß plaques and tau-like lesions in the neocortex and hippocampus in old male and female western lowland gorillas, placing this species at relevance in the context of AD research.

Different ß-amyloid oligomer assemblies in Alzheimer brains correlate with age of disease onset and impaired cholinergic activity.

In this study, we examined the relationship between various ß-amyloid (Aß) oligomer assemblies in autopsy brain with the levels of fibrillar Aß and cholinergic synaptic function. Brain tissues obtained from the frontal cortex of 14 Alzheimer's disease (AD) patients grouped into early-onset AD (EOAD) and late-onset AD (LOAD) and 12 age-matched control subjects were used to extract and quantify Aß oligomers in soluble (TBS), detergent soluble (TBST), and insoluble (GuHCl) fractions. The predominant oligomeric Aß assemblies detected were dodecamers, decamers, and pentamers, and different patterns of expression were observed between EOAD and LOAD patients. There was no association between any of the detected Aß oligomer assemblies and fibrillar Aß levels measured by N-methyl[(3)H] 2-(40-methylaminophenyl)-6-hydroxy-benzothiazole ([(3)H]PIB) binding. Levels of pentamers in the soluble fraction significantly correlated with a reduction in choline acetyltransferase activity in AD patients. The number of nicotinic acetylcholine receptors negatively correlated with the total amount of Aß oligomers in the insoluble fraction in EOAD patients, and with decamers in the soluble fraction in LOAD patients. These novel findings suggest that distinct Aß oligomers induce impairment of cholinergic neurotransmission in AD pathogenesis.

Endoplasmic reticulum stress occurs downstream of GluN2B subunit of N-methyl-d-aspartate receptor in mature hippocampal cultures treated with amyloid-ß oligomers.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder affecting both the hippocampus and the cerebral cortex. Reduced synaptic density that occurs early in the disease process seems to be partially due to the overactivation of N-methyl-d-aspartate receptors (NMDARs) leading to excitotoxicity. Recently, we demonstrated that amyloid-beta oligomers (AßO), the species implicated in synaptic loss during the initial disease stages, induce endoplasmic reticulum (ER) stress in cultured neurons. Here, we investigated whether AßO trigger ER stress by an NMDAR-dependent mechanism leading to neuronal dysfunction and analyzed the contribution of GluN2A and GluN2B subunits of this glutamate receptor. Our data revealed that AßO induce ER stress in mature hippocampal cultures, activating ER stress-associated sensors and increasing the levels of the ER chaperone GRP78. We also showed that AßO induce NADPH oxidase (NOX)-mediated superoxide production downstream of GluN2B and impairs ER and cytosolic Ca2+ homeostasis. These events precede changes in cell viability and activation of the ER stress-mediated apoptotic pathway, which was associated with translocation of the transcription factor GADD153 / CHOP to the nucleus and occurred by a caspase-12-independent mechanism. Significantly, ER stress took place after AßO interaction with GluN2B subunits. In addition, AßO-induced ER stress and hippocampal dysfunction were prevented by ifenprodil, an antagonist of GluN2B subunits, while the GluN2A antagonist NVP-AAM077 only slightly attenuated AßO-induced neurotoxicity. Taken together, our results highlight the role of GluN2B subunit of NMDARs on ER stress-mediated hippocampal dysfunction caused by AßO suggesting that it might be a potential therapeutic target during the early stages of AD.

Synapse-binding subpopulations of Aß oligomers sensitive to peptide assembly blockers and scFv antibodies.

Amyloid ß42 self-assembly is complex, with multiple pathways leading to large insoluble fibrils or soluble oligomers. Oligomers are now regarded as most germane to Alzheimer's pathogenesis. We have investigated the hypothesis that oligomer formation itself occurs through alternative pathways, with some leading to synapse-binding toxins. Immediately after adding synthetic peptide to buffer, solutions of Aß42 were separated by a 50 kDa filter and fractions assessed by SDS-PAGE silver stain, Western blot, immunoprecipitation, and capacity for synaptic binding. Aß42 rapidly assembled into aqueous-stable oligomers, with similar protein abundance in small (<50 kDa) and large (>50 kDa) oligomer fractions. Initially, both fractions were SDS-labile and resolved into tetramers, trimers, and monomers by SDS-PAGE. Upon continued incubation, the larger oligomers developed a small population of SDS-stable 10-16mers, and the smaller oligomers generated gel-impermeant complexes. The two fractions associated differently with neurons, with prominent synaptic binding limited to larger oligomers. Even within the family of larger oligomers, synaptic binding was associated with only a subset of these species, as a new scFv antibody (NUsc1) immunoprecipitated only a small portion of the oligomers while eliminating synaptic binding. Interestingly, low doses of the peptide KLVFFA blocked assembly of the 10-16mers, and this result was associated with loss of the smaller clusters of oligomers observed at synaptic sites. What distinguishes these smaller clusters from the unaffected larger clusters is not yet known. Results indicate that distinct species of Aß oligomers are generated by alternative assembly pathways and that synapse-binding subpopulations of Aß oligomers could be specifically targeted for Alzheimer's therapeutics.

A glycine zipper motif mediates the formation of toxic ß-amyloid oligomers in vitro and in vivo.

BACKGROUND: The ß-amyloid peptide (Aß) contains a Gly-XXX-Gly-XXX-Gly motif in its C-terminal region that has been proposed to form a "glycine zipper" that drives the formation of toxic Aß oligomers. We have tested this hypothesis by examining the toxicity of Aß variants containing substitutions in this motif using a neuronal cell line, primary neurons, and a transgenic C. elegans model. RESULTS: We found that a Gly37Leu substitution dramatically reduced Aß toxicity in all models tested, as measured by cell dysfunction, cell death, synaptic alteration, or tau phosphorylation. We also demonstrated in multiple models that Aß Gly37Leu is actually anti-toxic, thereby supporting the hypothesis that interference with glycine zipper formation blocks assembly of toxic Aß oligomers. To test this model rigorously, we engineered second site substitutions in Aß predicted by the glycine zipper model to compensate for the Gly37Leu substitution and expressed these in C. elegans. We show that these second site substitutions restore in vivo Aßtoxicity, further supporting the glycine zipper model. CONCLUSIONS: Our structure/function studies support the view that the glycine zipper motif present in the C-terminal portion of Aß plays an important role in the formation of toxic Aß oligomers. Compounds designed to interfere specifically with formation of the glycine zipper could have therapeutic potential.