Simultaneous isolation of intact brain cells and cell-specific extracellular vesicles from cryopreserved Alzheimer's disease cortex.

BACKGROUND: The neuronal and gliaI populations within the brain are tightly interwoven, making isolation and study of large populations of a single cell type from brain tissue a major technical challenge. Concurrently, cell-type specific extracellular vesicles (EVs) hold enormous diagnostic and therapeutic potential in neurodegenerative disorders including Alzheimer's disease (AD). NEW METHOD: Postmortem AD cortical samples were thawed and gently dissociated. Following filtration, myelin and red blood cell removal, cell pellets were immunolabeled with fluorescent antibodies and analyzed by flow cytometry. The cell pellet supernatant was applied to a triple sucrose cushion for brain EV isolation. RESULTS: Neuronal, astrocyte and microglial cell populations were identified. Cell integrity was demonstrated using calcein AM, which is retained by cells with esterase activity and an intact membrane. For some experiments cell pellets were fixed, permeabilized, and immunolabeled for cell-specific markers. Characterization of brain small EV fractions showed the expected size, depletion of EV negative markers, and enrichment in positive and cell-type specific markers. COMPARISON WITH EXISTING METHODS AND CONCLUSIONS: We optimized and integrated established protocols, aiming to maximize information obtained from each human autopsy brain sample. The uniqueness of our method lies in its capability to isolate cells and EVs from a single cryopreserved brain sample. Our results not only demonstrate the feasibility of isolating specific brain cell subpopulations for RNA-seq but also validate these subpopulations at the protein level. The accelerated study of EVs from human samples is crucial for a better understanding of their contribution to neuron/glial crosstalk and disease progression.

Integrated Multiomics Analysis of Salivary Exosomes to Identify Biomarkers Associated with Changes in Mood States and Fatigue.

Fatigue and other deleterious mood alterations resulting from prolonged efforts such as a long work shift can lead to a decrease in vigilance and cognitive performance, increasing the likelihood of errors during the execution of attention-demanding activities such as piloting an aircraft or performing medical procedures. Thus, a method to rapidly and objectively assess the risk for such cognitive fatigue would be of value. The objective of the study was the identification in saliva-borne exosomes of molecular signals associated with changes in mood and fatigue that may increase the risk of reduced cognitive performance. Using integrated multiomics analysis of exosomes from the saliva of medical residents before and after a 12 h work shift, we observed changes in the abundances of several proteins and miRNAs that were associated with various mood states, and specifically fatigue, as determined by a Profile of Mood States questionnaire. The findings herein point to a promising protein biomarker, phosphoglycerate kinase 1 (PGK1), that was associated with fatigue and displayed changes in abundance in saliva, and we suggest a possible biological mechanism whereby the expression of the PGK1 gene is regulated by miR3185 in response to fatigue. Overall, these data suggest that multiomics analysis of salivary exosomes has merit for identifying novel biomarkers associated with changes in mood states and fatigue. The promising biomarker protein presents an opportunity for the development of a rapid saliva-based test for the assessment of these changes.

Multi-Omics Analysis of Microglial Extracellular Vesicles From Human Alzheimer's Disease Brain Tissue Reveals Disease-Associated Signatures.

Alzheimer's disease (AD) is the most common cause of dementia, yet there is no cure or diagnostics available prior to the onset of clinical symptoms. Extracellular vesicles (EVs) are lipid bilayer-delimited particles that are released from almost all types of cell. Genome-wide association studies have linked multiple AD genetic risk factors to microglia-specific pathways. It is plausible that microglia-derived EVs may play a role in the progression of AD by contributing to the dissemination of insoluble pathogenic proteins, such as tau and Aß. Despite the potential utility of EVs as a diagnostic tool, our knowledge of human brain EV subpopulations is limited. Here we present a method for isolating microglial CD11b-positive small EVs from cryopreserved human brain tissue, as well as an integrated multiomics analysis of microglial EVs enriched from the parietal cortex of four late-stage AD (Braak V-VI) and three age-matched normal/low pathology (NL) cases. This integrated analysis revealed 1,000 proteins, 594 lipids, and 105 miRNAs using shotgun proteomics, targeted lipidomics, and NanoString nCounter technology, respectively. The results showed a significant reduction in the abundance of homeostatic microglia markers P2RY12 and TMEM119, and increased levels of disease-associated microglia markers FTH1 and TREM2, in CD11b-positive EVs from AD brain compared to NL cases. Tau abundance was significantly higher in AD brain-derived microglial EVs. These changes were accompanied by the upregulation of synaptic and neuron-specific proteins in the AD group. Levels of free cholesterol were elevated in microglial EVs from the AD brain. Lipidomic analysis also revealed a proinflammatory lipid profile, endolysosomal dysfunction, and a significant AD-associated decrease in levels of docosahexaenoic acid (DHA)-containing polyunsaturated lipids, suggesting a potential defect in acyl-chain remodeling. Additionally, four miRNAs associated with immune and cellular senescence signaling pathways were significantly upregulated in the AD group. Our data suggest that loss of the homeostatic microglia signature in late AD stages may be accompanied by endolysosomal impairment and the release of undigested neuronal and myelin debris, including tau, through extracellular vesicles. We suggest that the analysis of microglia-derived EVs has merit for identifying novel EV-associated biomarkers and providing a framework for future larger-scale multiomics studies on patient-derived cell-type-specific EVs.

Exosomal tau with seeding activity is released from Alzheimer's disease synapses, and seeding potential is associated with amyloid beta.

Synaptic transfer of tau has long been hypothesized from the human pathology pattern and has been demonstrated in vitro and in vivo, but the precise mechanisms remain unclear. Extracellular vesicles such as exosomes have been suggested as a mechanism, but not all tau is exosomal. The present experiments use a novel flow cytometry assay to quantify depolarization of synaptosomes by KCl after loading with FM2-10, which induces a fluorescence reduction associated with synaptic vesicle release; the degree of reduction in cryopreserved human samples equaled that seen in fresh mouse synaptosomes. Depolarization induced the release of vesicles in the size range of exosomes, along with tetraspanin markers of extracellular vesicles. A number of tau peptides were released, including tau oligomers; released tau was primarily unphosphorylated and C-terminal truncated, with Aß release just above background. When exosomes were immunopurified from release supernatants, a prominent tau band showed a dark smeared appearance of SDS-stable oligomers along with the exosomal marker syntenin-1, and these exosomes induced aggregation in the HEK tau biosensor assay. However, the flow-through did not seed aggregation. Size exclusion chromatography of purified released exosomes shows faint signals from tau in the same fractions that show a CD63 band, an exosomal size signal, and seeding activity. Crude synaptosomes from control, tauopathy, and AD cases demonstrated lower seeding in tauopathy compared to AD that is correlated with the measured Aß42 level. These results show that AD synapses release exosomal tau that is C-terminal-truncated, oligomeric, and with seeding activity that is enhanced by Aß. Taken together with previous findings, these results are consistent with a direct prion-like heterotypic seeding of tau by Aß within synaptic terminals, with subsequent loading of aggregated tau onto exosomes that are released and competent for tau seeding activity.

Pharmacological inhibition of nSMase2 reduces brain exosome release and α-synuclein pathology in a Parkinson's disease model.

AIM: We have previously reported that cambinol (DDL-112), a known inhibitor of neutral sphingomyelinase-2 (nSMase2), suppressed extracellular vesicle (EV)/exosome production in vitro in a cell model and reduced tau seed propagation. The enzyme nSMase2 is involved in the production of exosomes carrying proteopathic seeds and could contribute to cell-to-cell transmission of pathological protein aggregates implicated in neurodegenerative diseases such as Parkinson's disease (PD). Here, we performed in vivo studies to determine if DDL-112 can reduce brain EV/exosome production and proteopathic alpha synuclein (αSyn) spread in a PD mouse model. METHODS: The acute effects of single-dose treatment with DDL-112 on interleukin-1ß-induced extracellular vesicle (EV) release in brain tissue of Thy1-αSyn PD model mice and chronic effects of 5 week DDL-112 treatment on behavioral/motor function and proteinase K-resistant αSyn aggregates in the PD model were determined. RESULTS/DISCUSSION: In the acute study, pre-treatment with DDL-112 reduced EV/exosome biogenesis and in the chronic study, treatment with DDL-112 was associated with a reduction in αSyn aggregates in the substantia nigra and improvement in motor function. Inhibition of nSMase2 thus offers a new approach to therapeutic development for neurodegenerative diseases with the potential to reduce the spread of disease-specific proteopathic proteins.

Dual Neutral Sphingomyelinase-2/Acetylcholinesterase Inhibitors for the Treatment of Alzheimer's Disease.

We report the discovery of a novel class of compounds that function as dual inhibitors of the enzymes neutral sphingomyelinase-2 (nSMase2) and acetylcholinesterase (AChE). Inhibition of these enzymes provides a unique strategy to suppress the propagation of tau pathology in the treatment of Alzheimer's disease (AD). We describe the key SAR elements that affect relative nSMase2 and/or AChE inhibitor effects and potency, in addition to the identification of two analogs that suppress the release of tau-bearing exosomes in vitro and in vivo. Identification of these novel dual nSMase2/AChE inhibitors represents a new therapeutic approach to AD and has the potential to lead to the development of truly disease-modifying therapeutics.

Apolipoprotein E/Amyloid-ß Complex Accumulates in Alzheimer Disease Cortical Synapses via Apolipoprotein E Receptors and Is Enhanced by APOE4.

Apolipoprotein E (apoE) colocalizes with amyloid-ß (Aß) in Alzheimer disease (AD) plaques and in synapses, and evidence suggests that direct interactions between apoE and Aß are important for apoE's effects in AD. The present work examines the hypothesis that apoE receptors mediate uptake of apoE/Aß complex into synaptic terminals. Western blot analysis shows multiple SDS-stable assemblies in synaptosomes from human AD cortex; apoE/Aß complex was markedly increased in AD compared with aged control samples. Complex formation between apoE and Aß 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 Aß concentration into LDLR and LRP1-positive synaptosomes, along with free and esterified cholesterol. Synaptic Aß was increased by apoE4 in control and AD samples. These results are consistent with uptake of apoE/Aß complex and associated lipids into synaptic terminals, with subsequent Aß clearance in control synapses and accumulation in AD synapses.

A small molecule ApoE4-targeted therapeutic candidate that normalizes sirtuin 1 levels and improves cognition in an Alzheimer's disease mouse model.

We describe here the results from the testing of a small molecule first-in-class apolipoprotein E4 (ApoE4)-targeted sirtuin1 (SirT1) enhancer, A03, that increases the levels of the neuroprotective enzyme SirT1 while not affecting levels of neurotoxic sirtuin 2 (SirT2) in vitro in ApoE4-transfected cells. A03 was identified by high-throughput screening (HTS) and found to be orally bioavailable and brain penetrant. In vivo, A03 treatment increased SirT1 levels in the hippocampus of 5XFAD-ApoE4 (E4FAD) Alzheimer's disease (AD) model mice and elicited cognitive improvement while inducing no observed toxicity. We were able to resolve the enantiomers of A03 and show using in vitro models that the L-enantiomer was more potent than the corresponding D-enantiomer in increasing SirT1 levels. ApoE4 expression has been shown to decrease the level of the NAD-dependent deacetylase and major longevity determinant SirT1 in brain tissue and serum of AD patients as compared to normal controls. A deficiency in SirT1 level has been recently implicated in increased tau acetylation, a dominant post-translational modification and key pathological event in AD and tauopathies. Therefore, as a novel approach to therapeutic development for AD, we targeted identification of compounds that enhance and normalize brain SirT1 levels.

Suppression of tau propagation using an inhibitor that targets the DK-switch of nSMase2.

Targeting of molecular pathways involved in the cell-to-cell propagation of pathological tau species is a novel approach for development of disease-modifying therapies that could block tau pathology and attenuate cognitive decline in patients with Alzheimer's disease and other tauopathies. We discovered cambinol through a screening effort and show that it is an inhibitor of cell-to-cell tau propagation. Our in vitro data demonstrate that cambinol inhibits neutral sphingomyelinase 2 (nSMase2) enzyme activity in dose response fashion, and suppresses extracellular vesicle (EV) production while reducing tau seed propagation. Our in vivo testing with cambinol shows that it can reduce the nSMase2 activity in the brain after oral administration. Our molecular docking and simulation analysis reveals that cambinol can target the DK-switch in the nSMase2 active site.

Evaluation of an Allosteric BACE Inhibitor Peptide to Identify Mimetics that Can Interact with the Loop F Region of the Enzyme and Prevent APP Cleavage.

The aspartyl protease BACE1 (BACE) has emerged as an appealing target for reduction of amyloid-ß in Alzheimer's disease. The clinical fate of active-site BACE inhibitors may depend on potential side effects related to enzyme and substrate selectivity. One strategy to reduce this risk is through development of allosteric inhibitors that interact with and modulate the Loop F region unique to BACE1. Previously, a BACE-inhibiting antibody (Ab) was shown by co-crystallization to bind and induce conformational changes of Loop F, resulting in backbone perturbations at the distal S6 and S7 subsites, preventing proper binding of a long APP-like substrate to BACE and inhibiting its cleavage. In an effort to discover small Loop F-interacting molecules that mimic the Ab inhibition, we evaluated a peptide series with a YPYF(I/L)P(L/Y) motif that was reported to bind a BACE exosite. Our studies show that the most potent inhibitor from this series, peptide 65007, has a similar substrate cleavage profile to the Ab and reduces sAPPß levels in cell models and primary neurons. As our modeling indicates, it interacts with the Loop F region causing a conformational shift of the BACE protein backbone near the distal subsites. The peptide-bound enzyme adopts a conformation that closely overlays with the crystal structure (PDB: 3R1G) from Ab binding. Importantly, peptide 65007 appears to be BACE substrate and enzyme selective, showing little inhibition of NRG1, PSGL1, CHL1, or Cat D. Thus, peptide 65007 is a promising lead for discovery of Loop F-interacting small-molecule mimetics as allosteric inhibitors of BACE.

Neuronal pentraxin 1: A synaptic-derived plasma biomarker in Alzheimer's disease.

Synaptic neurodegeneration is thought to be an early event initiated by soluble ß-amyloid (Aß) aggregates that closely correlates with cognitive decline in Alzheimer disease (AD). Apolipoprotein ε4 (APOE4) is the most common genetic risk factor for both familial AD (FAD) and sporadic AD; it accelerates Aß aggregation and selectively impairs glutamate receptor function and synaptic plasticity. However, its molecular mechanisms remain elusive and these synaptic deficits are difficult to monitor. AD- and APOE4-dependent plasma biomarkers have been proposed, but synapse-related plasma biomarkers are lacking. We evaluated neuronal pentraxin 1 (NP1), a potential CNS-derived plasma biomarker of excitatory synaptic pathology. NP1 is preferentially expressed in brain and involved in glutamate receptor internalization. NP1 is secreted presynaptically induced by Aß oligomers, and implicated in excitatory synaptic and mitochondrial deficits. Levels of NP1 and its fragments were increased in a correlated fashion in both brain and plasma of 7-8 month-old E4FAD mice relative to E3FAD mice. NP1 was also found in exosome preparations and reduced by dietary DHA supplementation. Plasma NP1 was higher in E4FAD+ (APOE4+/+/FAD+/-) relative to E4FAD- (non-carrier; APOE4+/+/FAD-/-) mice, suggesting NP1 is modulated by Aß expression. Finally, relative to normal elderly, plasma NP1 was also elevated in patients with mild cognitive impairment (MCI) and elevated further in the subset who progressed to early-stage AD. In those patients, there was a trend towards increased NP1 levels in APOE4 carriers relative to non-carriers. These findings indicate that NP1 may represent a potential synapse-derived plasma biomarker relevant to early alterations in excitatory synapses in MCI and early-stage AD.

A Small Molecule Mimetic of the Humanin Peptide as a Candidate for Modulating NMDA-Induced Neurotoxicity.

Humanin (HN), a 24-amino acid bioactive peptide, has been shown to increase cell survival of neurons after exposure to Aß and NMDA-induced toxicity and thus could be beneficial in the treatment of Alzheimer's disease (AD). The neuroprotection by HN is reported to be primarily through its agonist binding properties to the gp130 receptor. However, the peptidic nature of HN presents challenges in its development as a therapeutic for AD. We report here for the first time the elucidation of the binding site of Humanin (HN) peptide to the gp130 receptor extracellular domain through modeling and the synthesis of small molecule mimetics that interact with the HN binding site on the gp130 receptor and provide protection against NMDA-induced neurotoxicity in primary hippocampal neurons. A brain permeable small molecule mimetic was identified through exploratory medicinal chemistry using microfluidic flow chemistry to facilitate the synthesis of new analogues for screening and SAR optimization.

Pharmacological Rescue of Long-Term Potentiation in Alzheimer Diseased Synapses.

Long-term potentiation (LTP) is an activity-dependent and persistent increase in synaptic transmission. Currently available techniques to measure LTP are time-intensive and require highly specialized expertise and equipment, and thus are not well suited for screening of multiple candidate treatments, even in animal models. To expand and facilitate the analysis of LTP, here we use a flow cytometry-based method to track chemically induced LTP by detecting surface AMPA receptors in isolated synaptosomes: fluorescence analysis of single-synapse long-term potentiation (FASS-LTP). First, we demonstrate that FASS-LTP is simple, sensitive, and models electrically induced LTP recorded in intact circuitries. Second, we conducted FASS-LTP analysis in two well-characterized Alzheimer's disease (AD) mouse models (3xTg and Tg2576) and, importantly, in cryopreserved human AD brain samples. By profiling hundreds of synaptosomes, our data provide the first direct evidence to support the idea that synapses from AD brain are intrinsically defective in LTP. Third, we used FASS-LTP for drug evaluation in human synaptosomes. Testing a panel of modulators of cAMP and cGMP signaling pathways, FASS-LTP identified vardenafil and Bay-73-6691 (phosphodiesterase-5 and -9 inhibitors, respectively) as potent enhancers of LTP in synaptosomes from AD cases. These results indicate that our approach could provide the basis for protocols to study LTP in both healthy and diseased human brains, a previously unattainable goal. SIGNIFICANCE STATEMENT: Learning and memory depend on the ability of synapses to strengthen in response to activity. Long-term potentiation (LTP) is a rapid and persistent increase in synaptic transmission that is thought to be affected in Alzheimer's disease (AD). However, direct evidence of LTP deficits in human AD brain has been elusive, primarily due to methodological limitations. Here, we analyze LTP in isolated synapses from AD brain using a novel approach that allows testing LTP in cryopreserved brain. Our analysis of hundreds of synapses supports the idea that AD-diseased synapses are intrinsically defective in LTP. Further, we identified pharmacological agents that rescue LTP in AD, thus opening up a new avenue for drug screening and evaluation of strategies for alleviating memory impairments.

Flow Cytometry Analysis and Quantitative Characterization of Tau in Synaptosomes from Alzheimer's Disease Brains.

Synaptosomes, resealed nerve terminals that form when tissue is homogenized in isotonic medium, are a model system that has been a key source of knowledge about neurotransmission. Synaptosomes contain mitochondria, cytoskeletal proteins, and release neurotransmitters; many have postsynaptic elements. Cryopreservation at the time of autopsy makes it possible to prepare synaptosomes from human samples. Flow cytometry is a powerful analytic technique that precisely measures fluorescence on a cell-by-cell basis, and also indicates particle size and complexity with a routine parameter that measures light scattering. We describe here a procedure for flow cytometry analysis of tau in synaptosomes, a procedure that enables (1) "purification" of synaptosomes from the P-2 fraction (crude synaptosomes) by gating on particle size, and (2) quantitative measure of tau immunofluorescence in individual terminals. Application of flow cytometry to study of synaptosomes has yielded important information, not possible with routine biochemistry, about synaptic pathology in Alzheimer's disease.

Nanoscale Extracellular Vesicle Analysis in Alzheimer's Disease Diagnosis and Therapy.

Diagnostic assays that leverage bloodborne neuron-derived (neuronal) nanoscale extracellular vesicles (nsEVs) as "windows into the brain" can predict incidence of Alzheimer's Disease (AD) many years prior to onset. Beyond diagnostics, bloodborne neuronal nsEVs analysis may have substantial translational impact by revealing mechanisms of AD pathology; such knowledge could enlighten new drug targets and lead to new therapeutic approaches. The potential to establish three-dimensional nsEV analysis methods that characterize highly purified bloodborne nsEV populations in method of enrichment, cell type origin, and protein or RNA abundance dimensions could bring this promise to bear by yielding nsEV "omics" datasets that uncover new AD biomarkers and enable AD therapeutic development. In this review we provide a survey of both the current status of and new developments on the horizon in the field of neuronal nsEV analysis. This survey is supplemented by a discussion of the potential to translate such neuronal nsEV analyses to AD clinical diagnostic applications and drug development.

Synaptic Amyloid-ß Oligomers Precede p-Tau and Differentiate High Pathology Control Cases.

Amyloid-ß (Aß) 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, Aß 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 Aß and p-tau in large populations of individual synaptic terminals. Soluble Aß oligomers were assayed by a single antibody sandwich enzyme-linked immunosorbent assay. Total in situ Aß 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 Aß 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 Aß-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 Aß drives phosphorylated tau accumulation and synaptic spread. These results indicate that antiamyloid therapies will be less effective once p-tau pathology is developed.

Dietary DHA supplementation in an APP/PS1 transgenic rat model of AD reduces behavioral and Aß pathology and modulates Aß oligomerization.

Increased dietary consumption of docosahexaenoic acid (DHA) is associated with decreased risk for Alzheimer's disease (AD). These effects have been postulated to arise from DHA's pleiotropic effects on AD pathophysiology, including its effects on ß-amyloid (Aß) production, aggregation, and toxicity. While in vitro studies suggest that DHA may inhibit and reverse the formation of toxic Aß oligomers, it remains uncertain whether these mechanisms operate in vivo at the physiological concentrations of DHA attainable through dietary supplementation. We sought to clarify the effects of dietary DHA supplementation on Aß indices in a transgenic APP/PS1 rat model of AD. Animals maintained on a DHA-supplemented diet exhibited reductions in hippocampal Aß plaque density and modest improvements on behavioral testing relative to those maintained on a DHA-depleted diet. However, DHA supplementation also increased overall soluble Aß oligomer levels in the hippocampus. Further quantification of specific conformational populations of Aß oligomers indicated that DHA supplementation increased fibrillar (i.e. putatively less toxic) Aß oligomers and decreased prefibrillar (i.e. putatively more toxic) Aß oligomers. These results provide in vivo evidence suggesting that DHA can modulate Aß aggregation by stabilizing soluble fibrillar Aß oligomers and thus reduce the formation of both Aß plaques and prefibrillar Aß oligomers. However, since fibrillar Aß oligomers still retain inherent neurotoxicity, DHA may need to be combined with other interventions that can additionally reduce fibrillar Aß oligomer levels for more effective prevention of AD in clinical settings.

Loss of endophilin-B1 exacerbates Alzheimer's disease pathology.

Endophilin-B1, also known as Bax-interacting factor 1 (Bif-1, and encoded by SH3GLB1), is a multifunctional protein involved in apoptosis, autophagy and mitochondrial function. We recently described a unique neuroprotective role for neuron-specific alternatively spliced isoforms of endophilin-B1. To examine whether endophilin-B1-mediated neuroprotection could be a novel therapeutic target for Alzheimer's disease we used a double mutant amyloid precursor protein and presenilin 1 (APPswe/PSEN1dE9) mouse model of Alzheimer's disease and observed that expression of neuron-specific endophilin-B1 isoforms declined with disease progression. To determine if this reduction in endophilin-B1 has a functional role in Alzheimer's disease pathogenesis, we crossed endophilin-B1(-/-) mice with APPswe/PSEN1dE9 mice. Deletion of endophilin-B1 accelerated disease onset and progression in 6-month-old APPswe/PSEN1dE9/endophilin-B1(-/-) mice, which showed more plaques, astrogliosis, synaptic degeneration, cognitive impairment and mortality than APPswe/PSEN1dE9 mice. In mouse primary cortical neuron cultures, overexpression of neuron-specific endophilin-B1 isoforms protected against amyloid-ß-induced apoptosis and mitochondrial dysfunction. Additionally, protein and mRNA levels of neuron-specific endophilin-B1 isoforms were also selectively decreased in the cerebral cortex and in the synaptic compartment of patients with Alzheimer's disease. Flow sorting of synaptosomes from patients with Alzheimer's disease demonstrated a negative correlation between amyloid-ß and endophilin-B1 levels. The importance of endophilin-B1 in neuronal function was further underscored by the development of synaptic degeneration and cognitive and motor impairment in endophilin-B1(-/-) mice by 12 months. Our findings suggest that endophilin-B1 is a key mediator of a feed-forward mechanism of Alzheimer's disease pathogenesis where amyloid-ß reduces neuron-specific endophilin-B1, which in turn enhances amyloid-ß accumulation and neuronal vulnerability to stress.

Pre-synaptic C-terminal truncated tau is released from cortical synapses in Alzheimer's disease.

The microtubule-associated protein tau has primarily been associated with axonal location and function; however, recent work shows tau release from neurons and suggests an important role for tau in synaptic plasticity. In our study, we measured synaptic levels of total tau using synaptosomes prepared from cryopreserved human postmortem Alzheimer's disease (AD) and control samples. Flow cytometry data show that a majority of synaptic terminals are highly immunolabeled with the total tau antibody (HT7) in both AD and control samples. Immunoblots of synaptosomal fractions reveal increases in a 20 kDa tau fragment and in tau dimers in AD synapses, and terminal-specific antibodies show that in many synaptosome samples tau lacks a C-terminus. Flow cytometry experiments to quantify the extent of C-terminal truncation reveal that only 15-25% of synaptosomes are positive for intact C-terminal tau. Potassium-induced depolarization demonstrates release of tau and tau fragments from pre-synaptic terminals, with increased release from AD compared to control samples. This study indicates that tau is normally highly localized to synaptic terminals in cortex where it is well-positioned to affect synaptic plasticity. Tau cleavage may facilitate tau aggregation as well as tau secretion and propagation of tau pathology from the pre-synaptic compartment in AD. Results demonstrate the abundance of tau, mainly C-terminal truncated tau, in synaptic terminals in aged control and in Alzheimer's disease (AD) samples. Tau fragments and dimers/oligomers are prominent in AD synapses. Following depolarization, tau release is potentiated in AD nerve terminals compared to aged controls. We hypothesize (i) endosomal release of the different tau peptides from AD synapses, and (ii) together with phosphorylation, fragmentation of synaptic tau exacerbates tau aggregation, synaptic dysfunction, and the spread of tau pathology in AD. Aß = amyloid-beta.