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Previously we showed that anti-Aβ peptide immunotherapy significantly attenuated Alzheimer's-like amyloid deposition in the central nervous system of aged canines. In this report we have characterized the changes that occurred in the humoral immune response over 2.4 years in canines immunized repeatedly with aggregated Aβ1–42 (AN1792) formulated in alum adjuvant. We observed a rapid and robust induction of anti-Aβ antibody titers, which were associated with an anti-inflammatory T helper type 2 (Th2) response. The initial antibody response was against dominant linear epitope at the N-terminus region of the Aβ1–42 peptide, which is identical to the one in humans and vervet monkeys. After multiple immunizations the antibody response drifted toward the elevation of antibodies that recognized conformational epitopes of assembled forms of Aβ and other types of amyloid. Our findings indicate that prolonged immunization results in distinctive temporal changes in antibody profiles, which may be important for other experimental and clinical settings.

Strong evidence indicates that amyloid beta (Aβ) inflicts its toxicity in Alzheimer's disease (AD) by promoting uncontrolled elevation of cytosolic Ca 2+ in neurons. We have previously shown that synthetic Aβ42 oligomers stimulate abnormal intracellular Ca 2+ release from the endoplasmic reticulum stores, suggesting that a similar mechanism of Ca 2+ toxicity may be common to the endogenous Aβs oligomers. Here, we use human postmortem brain extracts from AD-affected patients and test their ability to trigger Ca 2+ fluxes when injected intracellularly into Xenopus oocytes. Immunological characterization of the samples revealed the elevated content of soluble Aβ oligomers only in samples from AD patients. Intracellular injection of brain extracts from control patients failed to trigger detectable changes in intracellular Ca 2+ . Conversely, brain extracts from AD patients triggered Ca 2+ events consisting of local and global Ca 2+ fluorescent transients. Pre-incubation with either the conformation-specific OC antiserum or caffeine completely suppressed the brain extract's ability to trigger cytosolic Ca 2+ events. Computational modeling suggests that these Ca 2+ fluxes may impair cells bioenergetic by affecting ATP and ROS production. These results support the hypothesis that Aβ oligomers contained in neurons of AD-affected brains may represent the toxic agents responsible for neuronal malfunctioning and death associated with the disruption of Ca 2+ homeostasis.

Amyloid precursor protein (APP) metabolism is central to Alzheimer's disease (AD) pathogenesis, but the key etiological driver remains elusive. Recent failures of clinical trials targeting amyloid-β (Aβ) peptides, the proteolytic fragments of amyloid precursor protein (APP) that are the main component of amyloid plaques, suggest that the proteostasis-disrupting, key pathogenic species remain to be identified. Previous studies suggest that APP C-terminal fragment (APP.C99) can cause disease in an Aβ-independent manner. The mechanism of APP.C99 pathogenesis is incompletely understood. We used Drosophila models expressing APP.C99 with the native ER-targeting signal of human APP, expressing full-length human APP only, or co-expressing full-length human APP and β-secretase (BACE), to investigate mechanisms of APP.C99 pathogenesis. Key findings are validated in mammalian cell culture models, mouse 5xFAD model, and postmortem AD patient brain materials. We find that ribosomes stall at the ER membrane during co-translational translocation of APP.C99, activating ribosome-associated quality control (RQC) to resolve ribosome collision and stalled translation. Stalled APP.C99 species with C-terminal extensions (CAT-tails) resulting from inadequate RQC are prone to aggregation, causing endolysosomal and autophagy defects and seeding the aggregation of amyloid β peptides, the main component of amyloid plaques. Genetically removing stalled and CAT-tailed APP.C99 rescued proteostasis failure, endolysosomal/autophagy dysfunction, neuromuscular degeneration, and cognitive deficits in AD models. Our finding of RQC factor deposition at the core of amyloid plaques from AD brains further supports the central role of defective RQC of ribosome collision and stalled translation in AD pathogenesis. These findings demonstrate that amyloid plaque formation is the consequence and manifestation of a deeper level proteostasis failure caused by inadequate RQC of translational stalling and the resultant aberrantly modified APP.C99 species, previously unrecognized etiological drivers of AD and newly discovered therapeutic targets., (© 2021. The Author(s).)

Antibodies against Aß amyloid are indispensable research tools and potential therapeutics for Alzheimer's disease. They display several unusual properties, such as specificity for aggregated forms of the peptide, the ability to distinguish polymorphic aggregate structures, and the ability to recognize generic aggregation-related epitopes formed by unrelated amyloid sequences. Understanding the mechanisms underlying these unusual properties and the structures of their corresponding epitopes is crucial for the understanding why antibodies display different therapeutic activities and for the development of more effective therapeutic agents. Here we employed a novel "epitomic" approach to map the fine structure of the epitopes of 28 monoclonal antibodies against amyloid-beta using immunoselection of random sequences from a phage display library, deep sequencing, and pattern analysis to define the critical sequence elements recognized by the antibodies. Although most of the antibodies map to major linear epitopes in the amino terminal 1 to 14 residues of Aß, the antibodies display differences in the target sequence residues that are critical for binding and in their individual preferences for nontarget residues, indicating that the antibodies bind to alternative conformations of the sequence by different mechanisms. Epitomic analysis also identifies discontinuous, nonoverlapping sequence Aß segments that may constitute the conformational epitopes that underlie the aggregation specificity of antibodies. Aggregation-specific antibodies recognize sequences that display a significantly higher predicted propensity for forming amyloid than antibodies that recognize the monomer, indicating that the ability of random sequences to aggregate into amyloid is a critical element of their binding mechanism., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Apolipoprotein E (apoE) colocalizes with amyloid-β (Aβ) in Alzheimer disease (AD) plaques and in synapses, and evidence suggests that direct interactions between apoE and Aβ are important for apoE's effects in AD. The present work examines the hypothesis that apoE receptors mediate uptake of apoE/Aβ complex into synaptic terminals. Western blot analysis shows multiple SDS-stable assemblies in synaptosomes from human AD cortex; apoE/Aβ complex was markedly increased in AD compared with aged control samples. Complex formation between apoE and Aβ was confirmed by coimmunoprecipitation experiments. The apoE receptors low-density lipoprotein receptor (LDLR) and LDLR-related protein 1 (LRP1) were quantified in synaptosomes using flow cytometry, revealing up-regulation of LRP1 in early- and late-stage AD. Dual-labeling flow cytometry analysis of LRP1- and LDLR positives indicate most (approximately 65%) of LDLR and LRP1 is associated with postsynaptic density-95 (PSD-95)-positive synaptosomes, indicating that remaining LRP1 and LDLR receptors are exclusively presynaptic. Flow cytometry analysis of Nile red labeling revealed a reduction in cholesterol esters in AD synaptosomes. Dual-labeling experiments showed apoE and Aβ concentration into LDLR and LRP1-positive synaptosomes, along with free and esterified cholesterol. Synaptic Aβ was increased by apoE4 in control and AD samples. These results are consistent with uptake of apoE/Aβ complex and associated lipids into synaptic terminals, with subsequent Aβ clearance in control synapses and accumulation in AD synapses., (Copyright © 2019 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The monoclonal antibodies 6E10 and 4G8 are among the first anti-amyloid monoclonal antibodies against Aβ and the most widely used antibodies in Alzheimer's disease research. Although the epitopes for 6E10 and 4G8 have been reported to correspond to residues 1-16 and 17-24, a more recent high-resolution mapping approach indicates that 6E10 maps to residues 4-10 while 4G8 maps to residues 18-23. To characterize the binding specificity of both antibodies in greater detail, we used immunoselection of random sequences from phage display library followed by deep sequencing and analysis of resulting patterns from thousands of immunoselected sequences. We found that the minimum sequence required for 6E10 binding is R-x-D with over half (53%) of the immunoselected sequences conforming to this pattern. The vast majority of these sequences contain an H at position x (R-H-D), corresponding to residues 5-7 of the Aβ target sequences, but Y is also permitted at this position in a minority of sequences. For 4G8 we found that the most frequent pattern is F-x-A contained in approximately 30% of the sequences, followed by F-A, L-x(3)-A, L-x-F, and F-F each accounting for approximately 18% of the sequences. The F-x-A motif also occurs in islet amyloid poly peptide which may explain why 4G8 also recognizes amyloid fibrils of this peptide. Immunoselection of random sequences and deep sequencing may also be a facile and efficient means of determining residues critical for antibody binding and validating the specificity of monoclonal antibodies and polyclonal antisera.

Conventional genetic approaches and computational strategies have converged on immune-inflammatory pathways as key events in the pathogenesis of late onset sporadic Alzheimer's disease (LOAD). Mutations and/or differential expression of microglial specific receptors such as TREM2, CD33, and CR3 have been associated with strong increased risk for developing Alzheimer's disease (AD). DAP12 (DNAX-activating protein 12)/TYROBP, a molecule localized to microglia, is a direct partner/adapter for TREM2, CD33, and CR3. We and others have previously shown that TYROBP expression is increased in AD patients and in mouse models. Moreover, missense mutations in the coding region of TYROBP have recently been identified in some AD patients. These lines of evidence, along with computational analysis of LOAD brain gene expression, point to DAP12/TYROBP as a potential hub or driver protein in the pathogenesis of AD. Using a comprehensive panel of biochemical, physiological, behavioral, and transcriptomic assays, we evaluated in a mouse model the role of TYROBP in early stage AD. We crossed an Alzheimer's model mutant APP KM670/671NL /PSEN1 Δexon9 (APP/PSEN1) mouse model with Tyrobp -/- mice to generate AD model mice deficient or null for TYROBP (APP/PSEN1; Tyrobp +/- or APP/PSEN1; Tyrobp -/- ). While we observed relatively minor effects of TYROBP deficiency on steady-state levels of amyloid-β peptides, there was an effect of Tyrobp deficiency on the morphology of amyloid deposits resembling that reported by others for Trem2 -/- mice. We identified modulatory effects of TYROBP deficiency on the level of phosphorylation of TAU that was accompanied by a reduction in the severity of neuritic dystrophy. TYROBP deficiency also altered the expression of several AD related genes, including Cd33. Electrophysiological abnormalities and learning behavior deficits associated with APP/PSEN1 transgenes were greatly attenuated on a Tyrobp-null background. Some modulatory effects of TYROBP on Alzheimer's-related genes were only apparent on a background of mice with cerebral amyloidosis due to overexpression of mutant APP/PSEN1. These results suggest that reduction of TYROBP gene expression and/or protein levels could represent an immune-inflammatory therapeutic opportunity for modulating early stage LOAD, potentially leading to slowing or arresting the progression to full-blown clinical and pathological LOAD.

Introduction: Insulin resistance and type 2 diabetes mellitus (T2D) are associated with increased risk for cognitive impairment, Alzheimer's disease (AD) and vascular dementia. SORCS1 encodes a protein-sorting molecule genetically linked to both T2D and AD. The association of SORCS1 with both AD and T2D is sexually dimorphic in humans, with both disease associations showing more robust effects in females. Based on published evidence that manipulation of the mouse genome combining multiple genes related to cerebral amyloidosis, to T2D, or both, might provide novel mouse models with exacerbated amyloid and/or diabetes phenotypes, we assessed memory, glucose homeostasis, and brain biochemistry and pathology in male and female wild-type, Sorcs1 -/-, APP/PSEN1, and Sorcs1 -/- X APP/PSEN1 mice., Results: Male mice with either the APP/PSEN1 or Sorcs1 -/- genotype displayed earlier onset and persistent impairment in both learning behavior and glucose homeostasis. Unlike prior examples in the literature, the behavioral and metabolic abnormalities in male mice were not significantly exacerbated when the two disease model mice (Sorcs1 -/- models T2D; APP/PSEN1 models AD) were crossed. However, female Sorcs1 -/- X APP/PSEN1 mice exhibited worse metabolic dysfunction than Sorcs1 -/- knockout mice and worse memory than wild-type mice. The deletion of Sorcs1 from APP/PSEN1 mutant mice led to no obvious changes in brain levels of total or oligomeric amyloid-beta (Aβ) peptide., Conclusions: In general, unexpectedly, there was a trend for gene targeting of Sorcs1-/- to partially mitigate, not exacerbate, the metabolic and amyloid pathologies. These results indicate that crossing AD model mice and T2D model mice may not always cause exacerbation of both the amyloidosis phenotype and the metabolic phenotype and highlight the unexpected pitfalls of creating mixed models of disease.

Recently we reported that several monoclonal antibodies that recognize linear segments of amyloid-β (Aβ) also recognize amyloid fibrils, but not monomers of unrelated sequences, indicating that recognition of a linear sequence segment is not a reliable indicator of sequence specificity. We asked whether any of the commonly used commercially available Aβ antibodies also recognize fibrils of unrelated sequence. Here we report that 4G8, which recognizes residues 18-23 of the Aβ sequence and is widely believed to be sequence-specific, also recognizes fibrils formed from α-synuclein and islet amyloid polypeptide (IAPP). The recognition of amyloid fibrils is aggregation-dependent because 4G8 does not recognize α-synuclein or IAPP monomer. 4G8 also stains fibrillar α-synuclein aggregates in human multiple system atrophy brain where it colocalizes with anti-α-synuclein monoclonal antibody LB509 immunoreactivity. We also found that LB509 recognizes Aβ fibrils, but not monomer, indicating that generic epitope-reactive antibodies are also produced in response to α-synuclein immunization. Taken together, our results indicate that generic fibril conformational epitope specificity may be a pervasive property among monoclonal antibodies raised against amyloid-forming antigens and that the specificity of their immunoreactivity should be rigorously established and otherwise interpreted with caution.

Amyloid-β (Aβ) and hyperphosphorylated tau (p-tau) aggregates form the two discrete pathologies of Alzheimer disease (AD), and oligomeric assemblies of each protein are localized to synapses. To determine the sequence by which pathology appears in synapses, Aβ and p-tau were quantified across AD disease stages in parietal cortex. Nondemented cases with high levels of AD-related pathology were included to determine factors that confer protection from clinical symptoms. Flow cytometric analysis of synaptosome preparations was used to quantify Aβ and p-tau in large populations of individual synaptic terminals. Soluble Aβ oligomers were assayed by a single antibody sandwich enzyme-linked immunosorbent assay. Total in situ Aβ was elevated in patients with early- and late-stage AD dementia, but not in high pathology nondemented controls compared with age-matched normal controls. However, soluble Aβ oligomers were highest in early AD synapses, and this assay distinguished early AD cases from high pathology controls. Overall, synapse-associated p-tau did not increase until late-stage disease in human and transgenic rat cortex, and p-tau was elevated in individual Aβ-positive synaptosomes in early AD. These results suggest that soluble oligomers in surviving neocortical synaptic terminals are associated with dementia onset and suggest an amyloid cascade hypothesis in which oligomeric Aβ drives phosphorylated tau accumulation and synaptic spread. These results indicate that antiamyloid therapies will be less effective once p-tau pathology is developed., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Amyloidogenic proteins generally form intermolecularly hydrogen-bonded β-sheet aggregates, including parallel, in-register β-sheets (recognized by antiserum OC) or antiparallel β-sheets, β-solenoids, β-barrels, and β-cylindrins (recognized by antiserum A11). Although these groups share many common properties, some amyloid sequences have been reported to form polymorphic structural variants or strains. We investigated the humoral immune response to Aβ42 fibrils and produced 23 OC-type monoclonal antibodies recognizing distinct epitopes differentially associated with polymorphic structural variants. These mOC antibodies define at least 18 different immunological profiles represented in aggregates of amyloid-β (Aβ). All of the antibodies strongly prefer amyloid aggregates over monomer, indicating that they recognize conformational epitopes. Most of the antibodies react with N-terminal linear segments of Aβ, although many recognize a discontinuous epitope consisting of an N-terminal domain and a central domain. Several of the antibodies that recognize linear Aβ segments also react with fibrils formed from unrelated amyloid sequences, indicating that reactivity with linear segments of Aβ does not mean the antibody is sequence-specific. The antibodies display strikingly different patterns of immunoreactivity in Alzheimer disease and transgenic mouse brain and identify spatially and temporally unique amyloid deposits. Our results indicate that the immune response to Aβ42 fibrils is diverse and reflects the structural polymorphisms in fibrillar amyloid structures. These polymorphisms may contribute to differences in toxicity and consequent effects on pathological processes. Thus, a single therapeutic monoclonal antibody may not be able to target all of the pathological aggregates necessary to make an impact on the overall disease process., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Few quantitative diagnostic and monitoring, tools are available to clinicians treating patients with Alzheimer's disease. Further, many of the promising quantitative imaging tools under development lack clear specificity toward different types of Amyloid-β (Aβ) pathology such as vascular or oligomeric species. Antibodies offer an opportunity to image specific types of Aβ pathology because of their excellent specificity. In this study, we developed a method to translate a panel of anti-Aβ antibodies, which show excellent histological performance, into live animal imaging contrast agents. In the TgCRND8 mouse model of Alzheimer's disease, we tested two antibodies, M64 and M116, that target parenchyma aggregated Aβ plaques and one antibody, M31, that targets vascular Aβ. All three antibodies were administered intravenously after labeling with both poly(ethylene glycol) to enhance circulation and (64)Cu to allow detection via positron emission tomography (PET) imaging. We were clearly able to differentiate TgCRND8 mice from wild type controls by PET imaging using either M116, the anti-Aβ antibody targeting parenchymal Aβ or M31, the antivascular Aβ antibody. To confirm the validity of the noninvasive imaging of specific Aβ pathology, brains were examined after imaging and showed clear evidence of binding to Aβ plaques.

Background: Although rabbit antibodies are widely used in research, no structures of rabbit antigen-binding fragments (Fab) have been reported. M204 is a rabbit monoclonal antibody that recognizes a generic epitope that is common to prefibrillar amyloid oligomers formed from many different amyloidogenic sequences. Amyloid oligomers are widely suspected to be a primary causative agent of pathogenesis in several age-related neurodegenerative diseases, such as Alzheimer's disease. The detailed structure of these amyloid oligomers is not known nor is the mechanism for the recognition of the generic epitope by conformation-dependent monoclonal antibodies., Method: As a first approach to understanding the mechanism of conformation-dependent antibody recognition, we have crystallized the Fab of M204., Results: We have determined the structure of the Fab of M204 at 1.54Å resolution. The crystal structure reveals details of the M204 antigen combining site and features unique to rabbit Fabs such as an interdomain disulfide bond on its light chain., General Significance: Based on the structural features of the antigen-combining site of the M204, we rule out a "steric zipper" formation, as found in numerous amyloid fibril structures, as a mechanism of antibody-antigen recognition. The details of the first rabbit immunoglobulin Fab structure might also be useful for exploiting the potential of rabbit monoclonal antibodies for the development of humanized rabbit antibodies as therapeutic agents., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Huntington's disease (HD) is a devastating neurodegenerative disorder with no disease-modifying treatments available. The disease is caused by expansion of a CAG trinucleotide repeat and manifests with progressive motor abnormalities, psychiatric symptoms, and cognitive decline. Expression of an expanded polyglutamine repeat within the Huntingtin (Htt) protein impacts numerous cellular processes, including protein folding and clearance. A hallmark of the disease is the progressive formation of inclusions that represent the culmination of a complex aggregation process. Methylene blue (MB), has been shown to modulate aggregation of amyloidogenic disease proteins. We investigated whether MB could impact mutant Htt-mediated aggregation and neurotoxicity. MB inhibited recombinant protein aggregation in vitro, even when added to preformed oligomers and fibrils. MB also decreased oligomer number and size and decreased accumulation of insoluble mutant Htt in cells. In functional assays, MB increased survival of primary cortical neurons transduced with mutant Htt, reduced neurodegeneration and aggregation in a Drosophila melanogaster model of HD, and reduced disease phenotypes in R6/2 HD modeled mice. Furthermore, MB treatment also promoted an increase in levels of BDNF RNA and protein in vivo. Thus, MB, which is well tolerated and used in humans, has therapeutic potential for HD.

Amyloid diseases, including Alzheimer's, Parkinson's, and the prion conditions, are each associated with a particular protein in fibrillar form. These amyloid fibrils were long suspected to be the disease agents, but evidence suggests that smaller, often transient and polymorphic oligomers are the toxic entities. Here, we identify a segment of the amyloid-forming protein αB crystallin, which forms an oligomeric complex exhibiting properties of other amyloid oligomers: β-sheet-rich structure, cytotoxicity, and recognition by an oligomer-specific antibody. The x-ray-derived atomic structure of the oligomer reveals a cylindrical barrel, formed from six antiparallel protein strands, that we term a cylindrin. The cylindrin structure is compatible with a sequence segment from the β-amyloid protein of Alzheimer's disease. Cylindrins offer models for the hitherto elusive structures of amyloid oligomers.

In Parkinson's disease (PD) and other α-synucleinopathies, prefibrillar α-synuclein (αS) oligomer is implicated in the pathogenesis. However, toxic αS oligomers observed using in vitro systems are not generally seen to be associated with α-synucleinopathy in vivo. Thus, the pathologic significance of αS oligomers to αS neurotoxicity is unknown. Herein, we show that, αS that accumulate within endoplasmic reticulum (ER)/microsome forms toxic oligomers in mouse and human brain with the α-synucleinopathy. In the mouse model of α-synucleinopathy, αS oligomers initially form before the onset of disease and continue to accumulate with the disease progression. Significantly, treatment of αS transgenic mice with Salubrinal, an anti-ER stress compound that delays the onset of disease, reduces ER accumulation of αS oligomers. These results indicate that αS oligomers with toxic conformation accumulate in ER, and αS oligomer-dependent ER stress is pathologically relevant for PD.

The paradoxical appearance of aggregated α-synuclein (αsyn) in naive transplanted embryonic stem cells in Parkinson's disease (PD) brains has recently been reported, highlighting the possibility of neuron to neuron transmission of αsyn in PD. Here, we demonstrate in a cellular model the presence of αsyn oligomers in the extracellular space, their uptake by neurons, retrograde axonal transport to cell soma, and detrimental effects on neighboring cells. Moreover, we demonstrate that Hsp70 chaperones αsyn in the extracellular space and reduces extracellular αsyn oligomer formation and related toxicity. These novel findings provide evidence that extracellular αsyn oligomers may represent a crucial player in the propagation of pathology in PD, with their modulation by Hsp70 representing a potential new target for therapeutic interventions.

Background: Age-related neurodegenerative diseases share a number of important pathological features, such as accumulation of misfolded proteins as amyloid oligomers and fibrils. Recent evidence suggests that soluble amyloid oligomers and not the insoluble amyloid fibrils may represent the primary pathological species of protein aggregates., Results: We have produced several monoclonal antibodies that specifically recognize prefibrillar oligomers and do not recognize amyloid fibrils, monomer or natively folded proteins. Like the polyclonal antisera, the individual monoclonals recognize generic epitopes that do not depend on a specific linear amino acid sequence, but they display distinct preferences for different subsets of prefibrillar oligomers. Immunological analysis of a number of different prefibrillar Aβ oligomer preparations show that structural polymorphisms exist in Aβ prefibrillar oligomers that can be distinguished on the basis of their reactivity with monoclonal antibodies. Western blot analysis demonstrates that the conformers defined by the monoclonal antibodies have distinct size distributions, indicating that oligomer structure varies with size. The different conformational types of Aβ prefibrillar oligomers can serve as they serve as templates for monomer addition, indicating that they seed the conversion of Aβ monomer into more prefibrillar oligomers of the same type., Conclusions: These results indicate that distinct structural variants or conformers of prefibrillar Aβ oligomers exist that are capable of seeding their own replication. These conformers may be analogous to different strains of prions.

Inclusion bodies of aggregated mutant huntingtin (htt) fragments are a neuropathological hallmark of Huntington disease (HD). The molecular chaperones Hsp70 and Hsp40 colocalize to inclusion bodies and are neuroprotective in HD animal models. How these chaperones suppress mutant htt toxicity is unclear but might involve direct effects on mutant htt misfolding and aggregation. Using size exclusion chromatography and atomic force microscopy, we found that mutant htt fragments assemble into soluble oligomeric species with a broad size distribution, some of which reacted with the conformation-specific antibody A11. Hsp70 associated with A11-reactive oligomers in an Hsp40- and ATP-dependent manner and inhibited their formation coincident with suppression of caspase 3 activity in PC12 cells. Thus, Hsp70 and Hsp40 (DNAJB1) dynamically target specific subsets of soluble oligomers in a classic ATP-dependent reaction cycle, supporting a pathogenic role for these structures in HD.

Objective: Recent evidence suggests that high molecular weight soluble oligomeric Abeta (oAbeta) assemblies (also known as Abeta-derived diffusible ligands, or ADDLs) may represent a primary neurotoxic basis for cognitive failure in Alzheimer disease (AD). To date, most in vivo studies of oAbeta/ADDLs have involved injection of assemblies purified from the cerebrospinal fluid of human subjects with AD or from the conditioned media of Abeta-secreting cells into experimental animals. We sought to study the bioactivities of endogenously formed oAbeta/ADDLs generated in situ from the physiological processing of human amyloid precursor protein (APP) and presenitin1 (PS1) transgenes., Methods: We produced and histologically characterized single transgenic mice overexpressing APP(E693Q) or APP(E693Q) X PS1DeltaE9 bigenic mice. APP(E693Q) mice were studied in the Morris water maze (MWM) task at 6 and 12 months of age. Following the second MWM evaluation, mice were sacrificed, and brains were assayed for Abetatotal, Abeta40, Abeta42, and oAbeta/ADDLs by enzyme-linked immunosorbent assay (ELISA) and were also histologically examined. Based on results from the oAbeta/ADDL ELISA, we assigned individual APP(E693Q) mice to either an undetectable oAbeta/ADDLs group or a readily detectable oAbeta/ADDLs group. A days to criterion (DTC) analysis was used to determine delays in acquisition of the MWM task., Results: Both single transgenic and bigenic mice developed intraneuronal accumulation of APP/Abeta, although only APP(E693Q) X PS1Delta9 bigenic mice developed amyloid plaques. The APP(E693Q) mice did not develop amyloid plaques at any age studied, up to 30 months. APP(E693Q) mice were tested for spatial learning and memory, and only 12-month-old APP(E693Q) mice with readily detectable oAbeta/ADDLs displayed a significant delay in acquisition of the MWM task when compared to nontransgenic littermates., Interpretation: These data suggest that cerebral oAbeta/ADDL assemblies generated in brain in situ from human APP transgenes may be associated with cognitive impairment. We propose that a DTC analysis may be a sensitive method for assessing the cognitive impact in mice of endogenously generated oligomeric human Abeta assemblies. ANN NEUROL 2010.

The cognitive impairment in patients with Alzheimer's disease is closely associated with synaptic loss in the neocortex and limbic system. Although the neurotoxic effects of aggregated amyloid-beta oligomers in Alzheimer's disease have been studied extensively in experimental models, less is known about the characteristics of these aggregates across the spectrum of Alzheimer's disease. In this study, postmortem frontal cortex samples from controls and patients with Alzheimer's disease were fractionated and analyzed for levels of oligomers and synaptic proteins. We found that the levels of oligomers correlated with the severity of cognitive impairment (blessed information-memory-concentration score and mini-mental state examination) and with the loss of synaptic markers. Reduced levels of the synaptic vesicle protein, vesicle-associated membrane protein-2, and the postsynaptic protein, postsynaptic density-95, correlated with the levels of oligomers in the various fractions analyzed. The strongest associations were found with amyloid-beta dimers and pentamers. Co-immunoprecipitation and double-labeling experiments supported the possibility that amyloid-beta and postsynaptic density-95 interact at synaptic sites. Similarly, in transgenic mice expressing high levels of neuronal amyloid precursor protein, amyloid-beta co-immunoprecipitated with postsynaptic density-95. This was accompanied by a decrease in the levels of the postsynaptic proteins Shank1 and Shank3 in patients with Alzheimer's disease and in the brains of amyloid precursor protein transgenic mice. In conclusion, this study suggests that the presence of a subpopulation of amyloid-beta oligomers in the brains of patients with Alzheimer's disease might be related to alterations in selected synaptic proteins and cognitive impairment.

Soluble amyloid oligomers are potent neurotoxins that are involved in a wide range of human degenerative diseases, including Alzheimer disease. In Alzheimer disease, amyloid beta (Abeta) oligomers bind to neuronal synapses, inhibit long term potentiation, and induce cell death. Recent evidence indicates that several immunologically distinct structural variants exist as follows: prefibrillar oligomers (PFOs), fibrillar oligomers (FOs), and annular protofibrils. Despite widespread interest, amyloid oligomers are poorly characterized in terms of structural differences and pathological significance. FOs are immunologically related to fibrils because they react with OC, a conformation-dependent, fibril-specific antibody and do not react with antibodies specific for other types of oligomers. However, fibrillar oligomers are much smaller than fibrils. FOs are soluble at 100,000 x g, rich in beta-sheet structures, but yet bind weakly to thioflavin T. EPR spectroscopy indicates that FOs display significantly more spin-spin interaction at multiple labeled sites than PFOs and are more structurally similar to fibrils. Atomic force microscopy indicates that FOs are approximately one-half to one-third the height of mature fibrils. We found that Abeta FOs do not seed the formation of thioflavin T-positive fibrils from Abeta monomers but instead seed the formation of FOs from Abeta monomers that are positive for the OC anti-fibril antibody. These results indicate that the lattice of FOs is distinct from the fibril lattice even though the polypeptide chains are organized in an immunologically identical conformation. The FOs resulting from seeded reactions have the same dimensions and morphology as the initial seeds, suggesting that the seeds replicate by growing to a limiting size and then splitting, indicating that their lattice is less stable than fibrils. We suggest that FOs may represent small pieces of single fibril protofilament and that the addition of monomers to the ends of FOs is kinetically more favorable than the assembly of the oligomers into fibrils via sheet stacking interaction. These studies provide novel structural insight into the relationship between fibrils and FOs and suggest that the increased toxicity of FOs may be due to their ability to replicate and the exposure of hydrophobic sheet surfaces that are otherwise obscured by sheet-sheet interactions between protofilaments in a fibril.

Beta-amyloid (Abeta) is thought to be a key contributor to the pathogenesis of Alzheimer disease (AD) in the general population and in adults with Down syndrome (DS). Different assembly states of Abeta have been identified that may be neurotoxic. Abeta oligomers can assemble into soluble prefibrillar oligomers, soluble fibrillar oligomers and insoluble fibrils. Using a novel antibody, OC, recognizing fibrils and soluble fibrillar oligomers, we characterized fibrillar Abeta deposits in AD and DS cases. We further compared human specimens to those obtained from the Tg2576 mouse model of AD. Our results show that accumulation of fibrillar immunoreactivity is significantly increased in AD relative to nondemented aged subjects and those with select cognitive impairments (p < 0.0001). Further, there was a significant correlation between the extent of frontal cortex fibrillar deposit accumulation and dementia severity (MMSE r = -0.72). In DS, we observe an early age of onset and age-dependent accumulation of fibrillar OC immunoreactivity with little pathology in similarly aged non-DS individuals. Tg2576 mice show fibrillar accumulation that can be detected as young as 6 months. Interestingly, fibril-specific immunoreactivity was observed in diffuse, thioflavine S-negative Abeta deposits in addition to more mature neuritic plaques. These results suggest that fibrillar deposits are associated with disease in both AD and in adults with DS and their distribution within early Abeta pathology associated with diffuse plaques and correlation with MMSE suggest that these deposits may not be as benign as previously thought.

Microtubule-based neuronal transport pathways are impaired during the progression of Alzheimer's disease and other neurodegenerative conditions. However, mechanisms leading to defects in transport remain to be determined. We quantified morphological changes in neuronal cells following treatment with fibrils and unaggregated peptides of beta-amyloid (Abeta). Abeta fibrils induce axonal and dendritic swellings indicative of impaired transport. In contrast, Abeta peptides induce a necrotic phenotype in both neurons and non-neuronal cells. We tested several popular hypotheses by which aggregated Abeta could disrupt transport. Using fluorescent polystyrene beads, we developed experimental models of physical blockage and localized release of reactive oxygen species (ROS) that reliably induce swellings. Like the beads, Abeta fibrils localize in close proximity to swellings; however, fibril internalization is not required for disrupting transport. ROS and membrane permeability are also unlikely to be responsible for fibril-mediated toxicity. Collectively, our results indicate that multiple initiating factors converge upon pathways of defective transport.

Amyloid oligomers are believed to play causal roles in several types of amyloid-related neurodegenerative diseases. Several different types of amyloid oligomers have been reported that differ in morphology, size, or toxicity, raising the question of the pathological significance and structural relationships between different amyloid oligomers. Annular protofibrils (APFs) have been described in oligomer preparations of many different amyloidogenic proteins and peptides as ring-shaped or pore-like structures. They are interesting because their pore-like morphology is consistent with numerous reports of membrane-permeabilizing activity of amyloid oligomers. Here we report the preparation of relatively homogeneous preparations of APFs and an antiserum selective for APFs (alphaAPF) compared with prefibrillar oligomers (PFOs) and fibrils. PFOs appear to be precursors for APF formation, which form in high yield after exposure to a hydrophobic-hydrophilic interface. Surprisingly, preformed APFs do not permeabilize lipid bilayers, unlike the precursor PFOs. APFs display a conformation-dependent, generic epitope that is distinct from that of PFOs and amyloid fibrils. Incubation of PFOs with phospholipids vesicles results in a loss of PFO immunoreactivity with a corresponding increase in alphaAPF immunoreactivity, suggesting that lipid vesicles catalyze the conversion of PFOs into APFs. The annular anti-protofibril antibody also recognizes heptameric alpha-hemolysin pores, but not monomers, suggesting that the antibody recognizes an epitope that is specific for a beta barrel structural motif.

Background: An arg120gly (R120G) missense mutation in HSPB5 (alpha-beta-crystallin ), which belongs to the small heat shock protein (HSP) family, causes desmin-related cardiomyopathy (DRM), a muscle disease that is characterized by the formation of inclusion bodies, which can contain pre-amyloid oligomer intermediates (amyloid oligomer). While we have shown that small HSPs can directly interrupt amyloid oligomer formation, the in vivo protective effects of the small HSPs on the development of DRM is still uncertain., Methodology/principal Findings: In order to extend the previous in vitro findings to in vivo, we used geranylgeranylacetone (GGA), a potent HSP inducer. Oral administration of GGA resulted not only in up-regulation of the expression level of HSPB8 and HSPB1 in the heart of HSPB5 R120G transgenic (R120G TG) mice, but also reduced amyloid oligomer levels and aggregates. Furthermore, R120G TG mice treated with GGA exhibited decreased heart size and less interstitial fibrosis, as well as improved cardiac function and survival compared to untreated R120G TG mice. To address possible mechanism(s) for these beneficial effects, cardiac-specific transgenic mice expressing HSPB8 were generated. Overexpression of HSPB8 led to a reduction in amyloid oligomer and aggregate formation, resulting in improved cardiac function and survival. Treatment with GGA as well as the overexpression of HSPB8 also inhibited cytochrome c release from mitochondria, activation of caspase-3 and TUNEL-positive cardiomyocyte death in the R120G TG mice., Conclusions/significance: Expression of small HSPs such as HSPB8 and HSPB1 by GGA may be a new therapeutic strategy for patients with DRM.

Alzheimer's disease (AD) is characterized by neuronal atrophy caused by soluble amyloid beta protein (Abeta) peptide "oligomers" and a microglial-mediated inflammatory response elicited by extensive amyloid deposition in the brain. We show that CNI-1493, a tetravalent guanylhydrazone with established antiinflammatory properties, interferes with Abeta assembly and protects neuronal cells from the toxic effect of soluble Abeta oligomers. Administration of CNI-1493 to TgCRND8 mice overexpressing human amyloid precursor protein (APP) for a treatment period of 8 wk significantly reduced Abeta deposition. CNI-1493 treatment resulted in 70% reduction of amyloid plaque area in the cortex and 87% reduction in the hippocampus of these animals. Administration of CNI-1493 significantly improved memory performance in a cognition task compared with vehicle-treated mice. In vitro analysis of CNI-1493 on APP processing in an APP-overexpressing cell line revealed a significant dose-dependent decrease of total Abeta accumulation. This study indicates that the antiinflammatory agent CNI-1493 can ameliorate the pathophysiology and cognitive defects in a murine model of AD.

Aged canines (dogs) accumulate human-type beta-amyloid (Abeta) in diffuse plaques in the brain with parallel declines in cognitive function. We hypothesized that reducing Abeta in a therapeutic treatment study of aged dogs with preexisting Abeta pathology and cognitive deficits would lead to cognitive improvements. To test this hypothesis, we immunized aged beagles (8.4-12.4 years) with fibrillar Abeta(1-42) formulated with aluminum salt (Alum) for 2.4 years (25 vaccinations). Cognitive testing during this time revealed no improvement in measures of learning, spatial attention, or spatial memory. After extended treatment (22 vaccinations), we observed maintenance of prefrontal-dependent reversal learning ability. In the brain, levels of soluble and insoluble Abeta(1-40) and Abeta(1-42) and the extent of diffuse plaque accumulation was significantly decreased in several cortical regions, with preferential reductions in the prefrontal cortex, which is associated with a maintenance of cognition. However, the amount of soluble oligomers remained unchanged. The extent of prefrontal Abeta was correlated with frontal function and serum anti-Abeta antibody titers. Thus, reducing total Abeta may be of limited therapeutic benefit to recovery of cognitive decline in a higher mammalian model of human brain aging and disease. Immunizing animals before extensive Abeta deposition and cognitive decline to prevent oligomeric or fibrillar Abeta formation may have a greater impact on cognition and also more directly evaluate the role of Abeta on cognition in canines. Alternatively, clearing preexisting Abeta from the brain in a treatment study may be more efficacious for cognition if combined with a second intervention that restores neuron health.

The amyloid hypothesis of Alzheimer's toxicity has undergone a resurgence with increasing evidence that it is not amyloid fibrils but a smaller oligomeric species that produces the deleterious results. In this paper we address the mechanism of this toxicity. Only oligomers increase the conductance of lipid bilayers and patch-clamped mammalian cells, producing almost identical current-voltage curves in both preparations. Oligomers increase the conductance of the bare bilayer, the cation conductance induced by nonactin, and the anion conductance induced by tetraphenyl borate. Negative charge reduces the sensitivity of the membrane to amyloid, but cholesterol has little effect. In contrast, the area compressibility of the lipid has a very large effect. Membranes with a large area compressibility modulus are almost insensitive to amyloid oligomers, but membranes formed from soft, highly compressible lipids are highly susceptible to amyloid oligomer-induced conductance changes. Furthermore, membranes formed using the solvent decane (instead of squalane) are completely insensitive to the presence of oligomers. One simple explanation for these effects on bilayer conductance is that amyloid oligomers increase the area per molecule of the membrane-forming lipids, thus thinning the membrane, lowering the dielectric barrier, and increasing the conductance of any mechanism sensitive to the dielectric barrier.

Characterization of soluble oligomeric amyloid beta (Abeta) species in the brains of Alzheimer's disease (AD) patients and transgenic models has raised the possibility that different conformations of Abeta may contribute to AD pathology via different mechanisms. To characterize the toxic effect of different Abeta conformations, we tested side by side the effect of well characterized Abeta oligomers (AbetaOs), Abeta-derived diffusible ligands (ADDLs), and fibrillar Abeta (Abetaf) preparations in human cortical neurons (HCNs). Both AbetaOs and ADDLs bind rapidly and with high affinity to synaptic contacts and cellular membranes. AbetaOs (5 microm) induced rapid and massive neuronal death. Calcium influx accelerated, but was not required for, AbetaO toxicity. AbetaOs elicited a stereotyped succession of cellular changes consistent with the activation of a mitochondrial death apoptotic pathway. At low concentrations AbetaOs caused chronic and subtler mitochondrial alterations but minimal cell death. ADDLs induced similar toxic changes as AbetaOs but on a fivefold longer time scale. Higher concentrations of Abetaf and longer incubation times were required to produce widespread neuritic dystrophy but modest HCN cell death. Thus various Abeta species may play relevant roles in AD, causing neurotoxicity by distinct non-overlapping mechanisms affecting neuronal function and viability over multiple time courses.