Identification of candidate markers for the isolation of neuron-specific extracellular vesicles

Extracellular vesicles (EVs) are present in biofluids and may be useful biomarkers to non-invasively monitor brain cells, but isolating cell type-specific EVs remains challenging. Here we introduce an approach to immuno-isolate EVs from neurons and glia based on protein surface markers. We first selected transmembrane proteins suitable as cell type-specific EV markers based on gene expression and EV proteomic data. We developed a high-purity EV isolation method and cataloged the proteins present on EVs in human cerebrospinal fluid and plasma. We then computationally identified cell type-specific candidate markers for isolating EVs from neurons, astrocytes, oligodendrocytes and microglia. Using ultrasensitive immunoassays against several predicted neuron-specific proteins, we confirmed one marker, NRXN3, as present on EVs in cerebrospinal fluid and plasma. Finally, we developed efficient EV immuno-isolation methods and applied them to isolate NRXN3 + EVs. Our work provides a general methodology for the isolation of cell type-specific EVs, advancing the goal of using EVs to study brain diseases.