Extracellular Vesicles Derived from Postmortem Human Brain Tissue Contain Seed‐competent C‐terminal Tau Fragments, and Provide Proteomic Clues to the Identity of Selectively Vulnerable Cell Populations in Human Tauopathies

AbstractBackgroundPathological tau is spread along anatomically connected networks, originating in areas containing particularly vulnerable cell populations. It is hypothesized that tau is released and spread as free tau, or via extracellular vesicles (EVs) that include exosomes (30‐150 nm), microvesicles (100‐1000 nm), and apoptotic bodies (1000‐5000 nM). While a variety of different EV tau species have been identified in tissue, cells, and bio‐fluids, EV tau has not yet been described from human post‐mortem tauopathy brain tissue.MethodUsing a differential centrifugation and sucrose flotation method described by Perez‐Gonzalez et al., EVs were isolated from post‐mortem human frontal cortex tissue blocks from Alzheimer’s Disease (AD), Corticobasal Degeneration (CBD), and Control cases (BA9, n=4/group).ResultElectron microscopy (EM) confirmed the presence of vesicles with characteristic shape and size of exosomes and microvesicles. As demonstrated by immunoblotting, EV tau from tauopathy cases contains very little full‐length tau, but is specifically enriched in tau fragments of 10‐15 and 30‐35 kDa. EVs from control cases contain low levels of full‐length tau, and no tau fragments. EV tau fragments are sarkosyl‐insoluble, protected by the lipid membrane of the vesicle, and are seed‐competent (RD‐tau biosensor FRET seeding assay, seeding of primary neurons, and significant spread of AT8+ tau in EV‐injected PS19 mouse hippocampi). Epitope mapping and mass spectrometry sequencing confirm the presence of C‐terminal tau fragments, and further proteomic analyses confirm the identity of EVs as secreted vesicles. Together, these data provide the first clues to tauopathy‐specific proteomic signatures of EVs, and identify exosome components derived from the most vulnerable cell populations. This work complements the biomarker literature showing that N‐terminal regions of tau are secreted into the cerebral spinal fluid, while C‐terminal regions are retained in the brain.ConclusionThe identification of a seed‐competent species encased in a vesicle that readily fuses with cell membranes confirms that EVs are likely a significant mediator of pathological tau spread in the human brain. Our continued investigation of EV tau cryo‐EM structure, and of EV tau‐containing vesicles will focus on identifying novel immunological targets for pathological tau, and the vesicles that facilitate its spread.