Markers and Enrichment Strategies for Brain-Derived Extracellular Vesicles: Current Evidence, Limitations and Future Directions.

Brain-derived extracellular vesicles (bdEVs) have gained attention as key mediators of central nervous system communication and as minimally invasive biomarkers for neurological and neuropsychiatric disorders due to their presence in cerebrospinal fluid and peripheral biofluids. Despite this promise, their translational application is constrained by difficulties in selective enrichment and reliable assignment of cellular origin. This review summarizes the progression of bdEV isolation methodologies, from bulk physicochemical approaches to immunoaffinity-based, combinatorial, and single-vesicle-resolved strategies, with an emphasis on marker specificity. Using L1CAM as a representative example, we highlight how peripheral expression, extensive proteolytic cleavage, and dominance of soluble forms undermine its utility as a definitive neuronal EV marker. We further evaluate alternative surface and cargo markers proposed for neuron, astrocyte, microglial, and oligodendrocyte-derived bdEVs, identifying candidates such as ATP1A3, AMPA receptor subunits, GLAST, and AQP4 as more biologically grounded, while noting their limitations. Overall, current evidence indicates that no single marker can reliably define bdEV populations and that marker performance is strongly context dependent. Advancing the field will therefore require brain-restricted marker discovery, multiplexed and orthogonal enrichment strategies, single-vesicle validation, and systematic benchmarking against neural reference datasets to establish bdEVs as robust biomarkers and mechanistic reporters in neurobiology and neurological disease research.