Amyloid ß Proteoforms Elucidated by Quantitative LC/MS in the 5xFAD Mouse Model of Alzheimer's Disease.

Numerous Aß proteoforms, identified in the human brain, possess differential neurotoxic and aggregation propensities. These proteoforms contribute in unknown ways to the conformations and resultant pathogenicity of oligomers, protofibrils, and fibrils in Alzheimer's disease (AD) manifestation owing to the lack of molecular-level specificity to the exact chemical composition of underlying protein products with widespread interrogating techniques, like immunoassays. We evaluated Aß proteoform flux using quantitative top-down mass spectrometry (TDMS) in a well-studied 5xFAD mouse model of age-dependent Aß-amyloidosis. Though the brain-derived Aß proteoform landscape is largely occupied by Aß1-42, 25 different forms of Aß with differential solubility were identified. These proteoforms fall into three natural groups defined by hierarchical clustering of expression levels in the context of mouse age and proteoform solubility, with each group sharing physiochemical properties associated with either N/C-terminal truncations or both. Overall, the TDMS workflow outlined may hold tremendous potential for investigating proteoform-level relationships between insoluble fibrils and soluble Aß, including low-molecular-weight oligomers hypothesized to serve as the key drivers of neurotoxicity. Similarly, the workflow may also help to validate the utility of AD-relevant animal models to recapitulate amyloidosis mechanisms or possibly explain disconnects observed in therapeutic efficacy in animal models vs humans.

AAV-mediated neuronal expression of an scFv antibody selective for Aß oligomers protects synapses and rescues memory in Alzheimer models.

The accumulation of soluble oligomers of the amyloid-ß peptide (AßOs) in the brain has been implicated in synapse failure and memory impairment in Alzheimer's disease. Here, we initially show that treatment with NUsc1, a single-chain variable-fragment antibody (scFv) that selectively targets a subpopulation of AßOs and shows minimal reactivity to Aß monomers and fibrils, prevents the inhibition of long-term potentiation in hippocampal slices and memory impairment induced by AßOs in mice. As a therapeutic approach for intracerebral antibody delivery, we developed an adeno-associated virus vector to drive neuronal expression of NUsc1 (AAV-NUsc1) within the brain. Transduction by AAV-NUsc1 induced NUsc1 expression and secretion in adult human brain slices and inhibited AßO binding to neurons and AßO-induced loss of dendritic spines in primary rat hippocampal cultures. Treatment of mice with AAV-NUsc1 prevented memory impairment induced by AßOs and, remarkably, reversed memory deficits in aged APPswe/PS1ΔE9 Alzheimer's disease model mice. These results support the feasibility of immunotherapy using viral vector-mediated gene delivery of NUsc1 or other AßO-specific single-chain antibodies as a potential therapeutic approach in Alzheimer's disease.

An Essential Role for Alzheimer's-Linked Amyloid Beta Oligomers in Neurodevelopment: Transient Expression of Multiple Proteoforms during Retina Histogenesis.

Human amyloid beta peptide (Aß) is a brain catabolite that at nanomolar concentrations can form neurotoxic oligomers (AßOs), which are known to accumulate in Alzheimer's disease. Because a predisposition to form neurotoxins seems surprising, we have investigated whether circumstances might exist where AßO accumulation may in fact be beneficial. Our investigation focused on the embryonic chick retina, which expresses the same Aß as humans. Using conformation-selective antibodies, immunoblots, mass spectrometry, and fluorescence microscopy, we discovered that AßOs are indeed present in the developing retina, where multiple proteoforms are expressed in a highly regulated cell-specific manner. The expression of the AßO proteoforms was selectively associated with transiently expressed phosphorylated Tau (pTau) proteoforms that, like AßOs, are linked to Alzheimer's disease (AD). To test whether the AßOs were functional in development, embryos were cultured ex ovo and then injected intravitreally with either a beta-site APP-cleaving enzyme 1 (BACE-1) inhibitor or an AßO-selective antibody to prematurely lower the levels of AßOs. The consequence was disrupted histogenesis resulting in dysplasia resembling that seen in various retina pathologies. We suggest the hypothesis that embryonic AßOs are a new type of short-lived peptidergic hormone with a role in neural development. Such a role could help explain why a peptide that manifests deleterious gain-of-function activity when it oligomerizes in the aging brain has been evolutionarily conserved.

Antibody Fragments as Tools for Elucidating Structure-Toxicity Relationships and for Diagnostic/Therapeutic Targeting of Neurotoxic Amyloid Oligomers.

The accumulation of amyloid protein aggregates in tissues is the basis for the onset of diseases known as amyloidoses. Intriguingly, many amyloidoses impact the central nervous system (CNS) and usually are devastating diseases. It is increasingly apparent that neurotoxic soluble oligomers formed by amyloidogenic proteins are the primary molecular drivers of these diseases, making them lucrative diagnostic and therapeutic targets. One promising diagnostic/therapeutic strategy has been the development of antibody fragments against amyloid oligomers. Antibody fragments, such as fragment antigen-binding (Fab), scFv (single chain variable fragments), and VHH (heavy chain variable domain or single-domain antibodies) are an alternative to full-length IgGs as diagnostics and therapeutics for a variety of diseases, mainly because of their increased tissue penetration (lower MW compared to IgG), decreased inflammatory potential (lack of Fc domain), and facile production (low structural complexity). Furthermore, through the use of in vitro-based ligand selection, it has been possible to identify antibody fragments presenting marked conformational selectivity. In this review, we summarize significant reports on antibody fragments selective for oligomers associated with prevalent CNS amyloidoses. We discuss promising results obtained using antibody fragments as both diagnostic and therapeutic agents against these diseases. In addition, the use of antibody fragments, particularly scFv and VHH, in the isolation of unique oligomeric assemblies is discussed as a strategy to unravel conformational moieties responsible for neurotoxicity. We envision that advances in this field may lead to the development of novel oligomer-selective antibody fragments with superior selectivity and, hopefully, good clinical outcomes.

Early intraneuronal amyloid triggers neuron-derived inflammatory signaling in APP transgenic rats and human brain.

Chronic inflammation during Alzheimer's disease (AD) is most often attributed to sustained microglial activation in response to amyloid-ß (Aß) plaque deposits and cell death. However, cytokine release and microgliosis are consistently observed in AD transgenic animal models devoid of such pathologies, bringing into question the underlying processes that may be at play during the earliest AD-related immune response. We propose that this plaque-independent inflammatory reaction originates from neurons burdened with increasing levels of soluble and oligomeric Aß, which are known to be the most toxic amyloid species within the brain. Laser microdissected neurons extracted from preplaque amyloid precursor protein (APP) transgenic rats were found to produce a variety of potent immune factors, both at the transcript and protein levels. Neuron-derived cytokines correlated with the extent of microglial activation and mobilization, even in the absence of extracellular plaques and cell death. Importantly, we identified an inflammatory profile unique to Aß-burdened neurons, since neighboring glial cells did not express similar molecules. Moreover, we demonstrate within disease-vulnerable regions of the human brain that a neuron-specific inflammatory response may precede insoluble Aß plaque and tau tangle formation. Thus, we reveal the Aß-burdened neuron as a primary proinflammatory agent, implicating the intraneuronal accumulation of Aß as a significant immunological component in the AD pathogenesis.

Deficiency of TYROBP, an adapter protein for TREM2 and CR3 receptors, is neuroprotective in a mouse model of early Alzheimer's pathology.

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-ß 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.

Alzheimer's Toxic Amyloid Beta Oligomers: Unwelcome Visitors to the Na/K ATPase alpha3 Docking Station.

Toxic amyloid beta oligomers (AßOs) are known to accumulate in Alzheimer's disease (AD) and in animal models of AD. Their structure is heterogeneous, and they are found in both intracellular and extracellular milieu. When given to CNS cultures or injected ICV into non-human primates and other non-transgenic animals, AßOs have been found to cause impaired synaptic plasticity, loss of memory function, tau hyperphosphorylation and tangle formation, synapse elimination, oxidative and ER stress, inflammatory microglial activation, and selective nerve cell death. Memory loss and pathology in transgenic models are prevented by AßO antibodies, while Aducanumab, an antibody that targets AßOs as well as fibrillar Aß, has provided cognitive benefit to humans in early clinical trials. AßOs have now been investigated in more than 3000 studies and are widely thought to be the major toxic form of Aß. Although much has been learned about the downstream mechanisms of AßO action, a major gap concerns the earliest steps: How do AßOs initially interact with surface membranes to generate neuron-damaging transmembrane events? Findings from Ohnishi et al (PNAS 2005) combined with new results presented here are consistent with the hypothesis that AßOs act as neurotoxins because they attach to particular membrane protein docks containing Na/K ATPase-α3, where they inhibit ATPase activity and pathologically restructure dock composition and topology in a manner leading to excessive Ca++ build-up. Better understanding of the mechanism that makes attachment of AßOs to vulnerable neurons a neurotoxic phenomenon should open the door to therapeutics and diagnostics targeting the first step of a complex pathway that leads to neural damage and dementia.

Amyloid-ß oligomers transiently inhibit AMP-activated kinase and cause metabolic defects in hippocampal neurons.

AMP-activated kinase (AMPK) is a key player in energy sensing and metabolic reprogramming under cellular energy restriction. Several studies have linked impaired AMPK function to peripheral metabolic diseases such as diabetes. However, the impact of neurological disorders, such as Alzheimer disease (AD), on AMPK function and downstream effects of altered AMPK activity on neuronal metabolism have been investigated only recently. Here, we report the impact of Aß oligomers (AßOs), synaptotoxins that accumulate in AD brains, on neuronal AMPK activity. Short-term exposure of cultured rat hippocampal neurons or ex vivo human cortical slices to AßOs transiently decreased intracellular ATP levels and AMPK activity, as evaluated by its phosphorylation at threonine residue 172 (AMPK-Thr(P)172). The AßO-dependent reduction in AMPK-Thr(P)172 levels was mediated by glutamate receptors of the N-methyl-d-aspartate (NMDA) subtype and resulted in removal of glucose transporters (GLUTs) from the surfaces of dendritic processes in hippocampal neurons. Importantly, insulin prevented the AßO-induced inhibition of AMPK. Our results establish a novel toxic impact of AßOs on neuronal metabolism and suggest that AßO-induced, NMDA receptor-mediated AMPK inhibition may play a key role in early brain metabolic defects in AD.

Unexpected partial correction of metabolic and behavioral phenotypes of Alzheimer's APP/PSEN1 mice by gene targeting of diabetes/Alzheimer's-related Sorcs1.

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 (Aß) 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.

Alzheimer-associated Aß oligomers impact the central nervous system to induce peripheral metabolic deregulation.

Alzheimer's disease (AD) is associated with peripheral metabolic disorders. Clinical/epidemiological data indicate increased risk of diabetes in AD patients. Here, we show that intracerebroventricular infusion of AD-associated Aß oligomers (AßOs) in mice triggered peripheral glucose intolerance, a phenomenon further verified in two transgenic mouse models of AD. Systemically injected AßOs failed to induce glucose intolerance, suggesting AßOs target brain regions involved in peripheral metabolic control. Accordingly, we show that AßOs affected hypothalamic neurons in culture, inducing eukaryotic translation initiation factor 2α phosphorylation (eIF2α-P). AßOs further induced eIF2α-P and activated pro-inflammatory IKKß/NF-κB signaling in the hypothalamus of mice and macaques. AßOs failed to trigger peripheral glucose intolerance in tumor necrosis factor-α (TNF-α) receptor 1 knockout mice. Pharmacological inhibition of brain inflammation and endoplasmic reticulum stress prevented glucose intolerance in mice, indicating that AßOs act via a central route to affect peripheral glucose homeostasis. While the hypothalamus has been largely ignored in the AD field, our findings indicate that AßOs affect this brain region and reveal novel shared molecular mechanisms between hypothalamic dysfunction in metabolic disorders and AD.

TNF-α mediates PKR-dependent memory impairment and brain IRS-1 inhibition induced by Alzheimer's ß-amyloid oligomers in mice and monkeys.

Alzheimer's disease (AD) and type 2 diabetes appear to share similar pathogenic mechanisms. dsRNA-dependent protein kinase (PKR) underlies peripheral insulin resistance in metabolic disorders. PKR phosphorylates eukaryotic translation initiation factor 2α (eIF2α-P), and AD brains exhibit elevated phospho-PKR and eIF2α-P levels. Whether and how PKR and eIF2α-P participate in defective brain insulin signaling and cognitive impairment in AD are unknown. We report that ß-amyloid oligomers, AD-associated toxins, activate PKR in a tumor necrosis factor α (TNF-α)-dependent manner, resulting in eIF2α-P, neuronal insulin receptor substrate (IRS-1) inhibition, synapse loss, and memory impairment. Brain phospho-PKR and eIF2α-P were elevated in AD animal models, including monkeys given intracerebroventricular oligomer infusions. Oligomers failed to trigger eIF2α-P and cognitive impairment in PKR(-/-) and TNFR1(-/-) mice. Bolstering insulin signaling rescued phospho-PKR and eIF2α-P. Results reveal pathogenic mechanisms shared by AD and diabetes and establish that proinflammatory signaling mediates oligomer-induced IRS-1 inhibition and PKR-dependent synapse and memory loss.

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.

An anti-diabetes agent protects the mouse brain from defective insulin signaling caused by Alzheimer's disease- associated Aß oligomers.

Defective brain insulin signaling has been suggested to contribute to the cognitive deficits in patients with Alzheimer's disease (AD). Although a connection between AD and diabetes has been suggested, a major unknown is the mechanism(s) by which insulin resistance in the brain arises in individuals with AD. Here, we show that serine phosphorylation of IRS-1 (IRS-1pSer) is common to both diseases. Brain tissue from humans with AD had elevated levels of IRS-1pSer and activated JNK, analogous to what occurs in peripheral tissue in patients with diabetes. We found that amyloid-ß peptide (Aß) oligomers, synaptotoxins that accumulate in the brains of AD patients, activated the JNK/TNF-α pathway, induced IRS-1 phosphorylation at multiple serine residues, and inhibited physiological IRS-1pTyr in mature cultured hippocampal neurons. Impaired IRS-1 signaling was also present in the hippocampi of Tg mice with a brain condition that models AD. Importantly, intracerebroventricular injection of Aß oligomers triggered hippocampal IRS-1pSer and JNK activation in cynomolgus monkeys. The oligomer-induced neuronal pathologies observed in vitro, including impaired axonal transport, were prevented by exposure to exendin-4 (exenatide), an anti-diabetes agent. In Tg mice, exendin-4 decreased levels of hippocampal IRS-1pSer and activated JNK and improved behavioral measures of cognition. By establishing molecular links between the dysregulated insulin signaling in AD and diabetes, our results open avenues for the investigation of new therapeutics in AD.

Aß oligomers-induced toxicity is attenuated in cells cultured with NbActiv4™ medium.

Pathogenic Aß-derived diffusible ligands (ADDLs) bind to post-synaptic targets, induce excessive reactive oxygen species (ROS) and stimulate tau hyperphosphorylation in cultured neurons. Recently, NbActiv4™ medium was reported to increase neuron synapse densities in cultured hippocampal neurons. We aimed to investigate the effect of this novel medium on ADDL-induced toxicity. We found that ADDL-induced ROS was attenuated in cells cultured with NbActiv4™. ADDL binding assay was performed in neurons cultured by different feeding conditions with NbActiv4™. Feeding cells with 30 % medium once a week, ADDL binding sites were abundant at days in vitro (DIV) 18. However, changing 50 % medium once a week decreased ADDL binding about 80 %. NbActiv4™ produced about 40 % more glial fibrillary acidic protein (GFAP) positive astrocytes than the widely used hippocampal culture medium, neurobasal supplemented with B27 (neurobasal/B27). Astrocytes are reported to produce kinds of trophic factors including insulin-like growth factor 1 (IGF-1). Consistently, when cultured with NbActiv4™, neurons were sensitive to inhibitors of insulin/IGF-1 signaling in response to ADDL attack. Overall, this study supports the important role of astrocytes in neuroprotection and indicates that targeting astrocytes dysfunction may lead to new therapeutic strategies for Alzheimer's disease.

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.

Novel demonstration of amyloid-ß oligomers in sporadic inclusion-body myositis muscle fibers.

Accumulation of amyloid-ß (Aß) within muscle fibers has been considered an upstream step in the development of the s-IBM pathologic phenotype. Aß42, which is considered more cytotoxic than Aß40 and has a higher propensity to oligomerize, is preferentially increased in s-IBM muscle fibers. In Alzheimer disease (AD), low-molecular weight Aß oligomers and toxic oligomers, also referred to as "Aß-Derived Diffusible Ligands" (ADDLs), are considered strongly cytotoxic and proposed to play an important pathogenic role. ADDLs have been shown to be increased in AD brain. We now report for the first time that in s-IBM muscle biopsies Aß-dimer, -trimer, and -tetramer are identifiable by immunoblots. While all the s-IBM samples we studied had Aß-oligomers, their molecular weights and intensity varied between the patient samples. None of the control muscle biopsies had Aß oligomers. Dot-immunoblots using highly specific anti-ADDL monoclonal antibodies also showed highly increased ADDLs in all s-IBM biopsies studied, while controls were negative. By immunofluorescence, in some of the abnormal s-IBM muscle fibers ADDLs were accumulated in the form of plaque-like inclusions, and were often increased diffusely in very small fibers. Normal and disease-controls were negative. By gold-immuno-electron microscopy, ADDL-immunoreactivities were in close proximity to 6-10 nm amyloid-like fibrils, and also were immunodecorating amorphous and floccular material. In cultured human muscle fibers, we found that inhibition of autophagy led to the accumulation of Aß oligomers. This novel demonstration of Aß42 oligomers in s-IBM muscle biopsy provides additional evidence that intra-muscle fiber accumulation of Aß42 oligomers in s-IBM may contribute importantly to s-IBM pathogenic cascade.

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.

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.

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.

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.