Protein fingerprints of brain-derived extracellular vesicles predict types of tau pathology

Tauopathies are a heterogeneous group of neurodegenerative disorders characterized by the brain-regional aggregation of three-repeat (3R) or four-repeat (4R) tau isoforms. Current fluid and imaging biomarkers rarely discriminate these isoforms, hampering early, pathology‑specific diagnosis. To determine whether proteomic fingerprints of brain‑derived extracellular vesicles (BD‑EVs) isolated from the prefrontal cortex can (i) distinguish 3R from 4R tauopathies and (ii) mirror the histopathological burden of phosphorylated tau. BD‑EVs were purified from post‑mortem prefrontal cortex interstitial fluid of Pick’s disease (PiD; 3R), progressive supranuclear palsy (PSP; 4R), and control cases (CTRL). Nanoparticle tracking analysis quantified the concentration and size of vesicles. Label‑free LC–MS/MS profiled BD‑EV proteomes, followed by differential expression, gene set enrichment (GSEA), weighted gene co‑expression network analysis (WGCNA), and machine‑learning classification. AT8 immunohistochemistry quantified cortical tau pathology, enabling protein–pathology correlations. Tau pathology did not alter overall BD‑EV yield but shifted vesicle size distribution in PiD (higher small/large EV ratio). Proteomic analysis identified two discriminant modules: an astrocyte-derived mitochondrial cluster enriched in PiD and a neuron-derived microtubule cluster depleted in PiD relative to PSP and control groups. Combined glial protein abundance (e.g., GFAP, AQP4, S100β, GLAST, ANXA1) classified PiD, PSP, and controls with perfect accuracy (F1 = 1.0). Several BD‑EV proteins—including CAMKV, TMEM30A, NMT1, AK1 (PiD‑specific), and CALB2 (PSP‑specific)—correlated strongly with regional AT8 burden (|ρ| ≥ 0.70, FDR < 0.05). BD‑EV proteomic fingerprints robustly differentiate 3R and 4R tauopathies and track disease severity, unveiling astrocytic mitochondrial proteins as candidate biomarkers. Overall, our results indicate that BD-EV profiling may complement existing approaches for distinguishing tau isoforms and, pending further validation, could ultimately be adapted for use in more accessible biofluids.