Isolation and Characterization of Neuronal-Derived Extracellular Vesicles

Total EVs were isolated from frozen serum aliquots and enriched for neuronal-derived EVs as previously described^{22 (link)}. Briefly, EVs were isolated from 0.5 mL of frozen human serum following ExoQuick manufacturer instructions (System Biosciences, Cat # EXOQ5TM-1, Palo Alto, CA, USA) and MISEV2018 reporting guidelines⁴⁰ as previously described^{41 (link)}. For EV characterization data, see supplementary material and supplemental fig. S1. Neuronal-derived EVs were enriched by using 4 μg of biotinylated antibodies against neuronal surface markers CD171 (L1CAM) (clone 5G3; eBioscience, San Diego, CA, USA) in 50 μL of 3% Bovine Serum Albumin (BSA) (Thermo-Fisher Scientific Inc., Rockford, IL, USA), and followed by adding 15 μL of Streptavidin-agarose Ultralink resin (Thermo-Fisher Scientific Inc., Rockford, IL, USA) in 25 μL of 3% BSA per tube. The resin pellet was resuspended in 200 μL of 0.1 M glycine–HCl and centrifuged at 4 °C (for 10 min at 4500 × g). Supernatant fluid was then harvested to new collecting tubes containing 25 μL of 10% BSA and 15 μL of 1 M Tris–HCl and mixed. Neuronal-derived EVs were lysed with equal volume of mammalian protein extraction reagent (M-PER) (Thermo-Fisher Scientific Inc., Rockford, IL, USA), and then the lysis was ready for downstream analysis.