Endosome and exosome enrichment

Endosomes were enriched to detect internalized α-syn-biotin PFF as previously described (5). Primary cultured neurons from WT or PARP-1 KO were incubated with α-syn-biotin PFF for 1.5 h, followed by adding trypsin to remove the membrane-bound α-syn-biotin PFF. After washing with PBS, neurons were harvested and lysed by aspirating a syringe 20 times in lysis buffer [250 mM sucrose, 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1mM EDTA, 1mM EGTA] with a complete protease inhibitor mixture (Roche). The last pellet containing the endosomes were used for immunoblot analysis after sequential centrifugation at 1000 x g for 10 min, 16,000 x g for 20 min, and 100,000 x g for 60 min at 4°C). Exosomes were enriched to detect secreted α-syn as previously described (18). Culture supernatants of primary cortical neurons transduced with AAV-α-syn followed by incubation with 500 μM NMDA for 5 min were collected and spun at 300 x g for 10 min to remove cells. The supernatants were then sequentially centrifuged at 2000 × g for 10 min, 10,000 × g for 30 min, and 100,000 × g for 90 min at 4°C). The last pellet containing exosomes was washed once with PBS and centrifuged again at 100,000 × g for 90 min. The remaining pellet was resuspended with lysis buffer.