Dataset Information

Optimizing Small and Large Brain-Derived Extracellular Vesicle Purification Reveals Critical Pathways in Alzheimer's Disease Patients

ABSTRACT: At this day, Alzheimer’s disease is the most prominent form of dementia worldwide. It is characterized by tau lesions that spread throughout the brain in a spatio-temporal manner. This has led to the prion-like propagation hypothesis implicating a transfer of pathological tau seeds from cell-to-cell. Extracellular vesicles have been described as one of the mechanisms contributing to this intercellular transfer of tau seeds. Thus far, we and others demonstrated that human EVs from the brain-derived fluid (BD-EVs) of AD patients contain the capacity to induce tau seeding in vitro and in vivo. Based on these findings and the rich diversity of BD-EVs, we studied BD-EVs sub-populations in AD patients. Here, enriched-small and enriched-large EVs were isolated from two distinct enzymatic brain dissociation protocols. Proteomic analysis then aimed to define the optimal brain dissociation enzyme resulting in well-preserved EVs. We demonstrated that collagenase enzymatic brain dissociation not only results in a high EVs protein yield but also allowed us to detect more transmembrane proteins for both enriched-large and enriched-small EVs in comparison to papain dissociation. Proteomic content analysis of collagenase-derived AD-EVs revealed the increased presence of integrin-mediated synaptic signalling, brain-immunity and GWAS-associated proteins. Although, enriched-large EVs contain more GWAS-associated proteins then enriched-small EVs. This enriched small and large EV sub-population characterization drives comprehension of AD associated pathophysiologic mechanisms potentially mediated through EVs.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Brain, Extracellular Vesicle

DISEASE(S): Alzheimer's Disease

SUBMITTER: Soulaimane Aboulouard

LAB HEAD: Michel Salzet

PROVIDER: PXD055734 | Pride | 2025-12-15

REPOSITORIES: Pride

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Stratification of brain-derived extracellular vesicles of Alzheimer's disease patients indicates a unique proteomic content and a higher seeding capacity of small extracellular vesicles.

Oosterlynck Marie M Leroux Elodie E Namasivayam Balasubramaniam B Bouillet Thomas T Caillierez Raphaelle R Loyens Anne A Mazur Daniele D Perbet Romain R Lefebvre Christophe C Aboulouard Soulaimane S Maurage Claude-Alain CA Accart Bertrand B Buée Luc L Colin Morvane M

Translational neurodegeneration 20251205 1

<h4>Background</h4>Alzheimer's disease (AD) is the most prominent form of dementia worldwide. It is characterized by tau lesions that spread throughout the brain in a spatio-temporal manner. This has led to the prion-like propagation hypothesis implicating a transfer of pathological tau seeds from cell to cell. Human brain-derived extracellular vesicles (BD-EVs) isolated from the brain-derived fluid of AD patients contain seeds that contribute to this tau pathology spreading. Knowing the rich di ...[more]

PMID: 41345709

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Project description:The abnormal higher-order assembly of tau protein in neurons and glia underlies a large group of neurodegenerative diseases, including Alzheimer's disease (AD). Tau assemblies are first detected in localised brain regions and subsequently accumulate in connected networks. Prion-like propagation is thought to underlie the temporospatial accumulation of assembled tau, encompassing intracellular trafficking and trans-synaptic transfer of assembled tau species that seed the assembly of normally folded tau. Evidence suggests that extracellular vesicles (EVs), a diverse group of lipid vesicles that facilitate the intercellular transfer of bioactive molecules, mediate transfer of assembled tau seeds. However, the molecular species of assembled tau seeds in human brain are unknown. Furthermore, the identities of the EVs that mediate intercellular transfer of assembled tau and how this is achieved have not been determined. Here, we isolated sub-populations of EVs from human brain enriched in plasma membrane; endo-lysosomal; or mitochondrial proteins. We used electron cryo-tomography (cryo-ET) and single-particle electron cryo-microscopy (cryo-EM) to directly analyse the structure and molecular organisation of assembled tau seeds associated with intact EVs isolated from the brains of individuals who had AD. We found that short tau paired helical filaments (PHFs) were packaged within the lumen of large EVs enriched in endo-lysosomal proteins. The PHFs incorporated anionic molecules not observed in cell-derived PHFs, leading to a more compact filament fold. Multiple PHFs associated with one another and were linked to the luminal membrane at their ends. The PHF-containing EVs initiated propagation in directly-converted neurons, as well as in transgenic mice and a biosensor cell line. This work suggests that short PHFs are responsible for EV-mediated propagation of assembled tau in human brain. These results have broad implications for diagnostic and therapeutic strategies targeting extracellular assembled tau seeds in AD and other neurodegenerative diseases.

Project description:Alzheimer’s Disease (AD) is the most common cause of dementia in the elderly population without a cure or early diagnostics currently available. Despite the demonstrated ability of extracellular vesicles (EVs) to spread tau and Aβ pathology in AD models and their potential utility as a diagnostic and treatment-monitoring tool, our knowledge of human brain EV subpopulations, their molecular composition and roles in disease progression is very limited. Genome-wide association studies linked multiple AD genetic risk factors to microglia-specific pathways suggesting a potential role of microglia-derived EVs in AD progression. Here we are presenting a method for isolation of microglial CD11b-positive small EVs from cryopreserved human brain tissue and multi-omics analysis of the EVs from the parietal cortex of 4 late-stage AD (Braak V-VI) and 3 age-matched normal/low pathology (NL) cases. We identified a total of 1000 proteins by quantitative proteomics, analyzed 594 individual lipid species by targeted lipidomics, and 105 miRNAs using a NanoString miRNA expression panel. We found significant reduction in abundance of homeostatic microglia markers, P2RY12 and TMEM119, and increased levels of disease associated microglia markers, FTH1 and TREM2, in microglial EVs from AD brain compared to NL cases. Protein tau abundance was also significantly higher in AD brain-derived microglial EVs. These changes were accompanied by upregulation of synaptic and neuron-specific proteins in the AD group. Levels of free-cholesterol were elevated in microglial EVs from AD brain. Lipidomic analysis also revealed a pro-inflammatory lipid profile, endolysosomal dysfunction and significant AD-associated decrease in levels of docosahexaenoic acid (DHA)-containing polyunsaturated lipids of different classes, suggesting a potential defect in acyl-chain remodeling. Several immune pathways and senescence were among the top pathways controlled by 4 miRNAs significantly upregulated in the AD group. Our data suggest that loss of the homeostatic microglia signature in late AD stages may accompany endolysosomal impairment and release of undigested neuronal and myelin debris, including tau, through extracellular vesicles. These data validate a method of isolation of small cell-type specific EVs from human brain tissue, suggests new potential EV-associated markers and disease-related pathways, and provides a framework for future large-scale multi-omics study.

Project description:Alzheimer’s disease (AD) and vascular dementia (VaD) are the two most common forms of dementia, neither of which can be effectively treated. There is growing evidence on vascular contributions to cognitive impairment and dementia such as AD, but their pathogenic molecular links are not defined yet. Notably, neurofibrillary tangles made of hyperphosphorylated tau (P-tau) are a hallmark lesion of AD, but are not found in VaD. Although brain ischemia induces some tau changes and tau knockout reduces stroke-induced acute brain damage, little is known about the role of tau in mediating progression from vascular insufficiency to later development of VaD. Cis P-tau is a pre-tangle pathology in AD and an early driver of neurodegeneration resulting from brain injury, but its role in AD treatment or in VaD is unknown. Here we identify cis P-tau as an antibody-neutralizable major common early driver of AD and VaD. We show that cis P-tau elimination using cis antibody not only prevents, but also treats AD-like neurodegeneration and memory loss in a hTau mouse model of AD. Purified cis P-tau causes and spreads neurodegeneration with behavioral changes when injected into wild-type mouse brains, but is prevented by cis antibody. Surprisingly, we also find robust cis P-tau with no evidence of tau tangles in human VaD brains and a mouse model of chronic cerebral hypoperfusion that mimics key aspects of clinical VaD. Cis mAb treatment of hypoperfusive mice for 1 or 6 months blocks VaD-like neuropathological and functional outcomes. Single-cell transcriptomic profiling reveals that cerebral hypoperfusion induces numerous global changes in diverse brain cells including those of human AD brains. Remarkably, ~90% of these global changes are fully recovered with cis antibody, correlating with tau expression in cells. Thus, cis P-tau is a major common early driver of AD and VaD, and cis antibody has a potential role in the early detection, prevention and treatment of these devastating diseases.

Project description:Alzheimer’s disease (AD) is an irreversible neurodegenerative disease defined by its molecular hallmarks - amyloid beta peptide plaques and neurofibrillary Tau tangles. Despite significant progress that has been made in uncovering a large number of genetic risk factors through extensive genomic sequencing and genetic studies, the molecular mechanisms driving AD-associated pathology and cognitive decline remain poorly understood. Therefore, alongside the identification of more risk genes, it is also paramount to study how these genes function and influence each other within the cellular pathways and overall molecular networks in AD-relevant brain cell types. However, current human protein-protein interactome datasets were all generated in either yeast or generic human cell lines. Consequently, many important neuronal interactions, especially neuron-specific ones, have yet been discovered. To address this critical gap, we developed a highly scalable, high-quality interactome mapping pipeline in human excitatory neurons derived from induced pluripotent stem cells (iPSC), and generated a comprehensive, neuron-specific interactome map, named ADNeuronNet, for key AD risk genes. ADNeuronNet consists of 1,767 high-confidence interactions among 1,189 proteins and is the only dataset enriched with neuron-specific genes when compared to known protein interactions, including previous large-scale interactome maps, for the same baits in the literature. Within ADNeuronNet, we identified 1,375 novel interactions, many of which are likely neuron specific. For example, we identified a neuron-specific interactor, RIN2, for major AD risk factor BIN1 and confirmed RIN2’s function in recruiting BIN1 to early endosomes, a process that has been well-associated with AD etiology. Additionally, we performed quantitative interaction perturbation analyses on AD risk genes with AD-associated mutations or isoforms and identified significant changes in 91 protein interactions among 11 different protein variants. Finally, we found that subunits from the anaphase-promoting complex/cyclosome (APC/C), other novel BIN1 interactors identified by ADNeuronNet, mediated modulation of Tau-aggregation in neurons via regulation of APOE expression, uncovering a previously unrecognized BIN1-APC/C-APOE regulatory axis in AD pathobiology. Overall, these findings illustrate how our neuron-specific ADNeuronNet can be leveraged to uncover new risk gene candidates and cellular pathways that help advance our understanding of molecular mechanisms underlying AD risk.

			Action	DRS
	AD-eLEVs-collagenase-1.raw	Raw
	AD-eLEVs-collagenase-2.raw	Raw
	AD-eLEVs-collagenase-3.raw	Raw
	AD-eLEVs-papain-1.raw	Raw
	AD-eLEVs-papain-2.raw	Raw