N21 max

The hippocampus was dissected from E18 rat embryos (WT) or E16.5 mouse embryos (WT, PrP^C KO or p47 KO), digested with 0.06% trypsin (Sigma-Aldrich, T4799) in Hanks’ balanced salt solution (HBSS, Sigma, H9394) for 15 min at 37 °C. Following digestion, neurons were dissociated by gentle trituration and resuspended in neurobasal medium (Invitrogen, 21103049) supplemented with 2% N21-MAX (R&D Systems, AR008), 1% P/S and 2.5 mM L-Glutamine (Lonza, 17-605E). Cells were then counted and plated at a density of 15,000 cells/coverslip in a 24-well plate for immunostaining analysis, or at a density of 200,000 cells/well in a 6-well plate for western blot analysis and qPCR. Coverslips and plates were precoated with 20 µg/mL poly-D-lysine (Sigma, P0899). For neuronal transduction, DIV3 hippocampal neurons were treated with 1 µM of (+)-MK-801 hydrogen maleate (Sigma, M107) for 30 min at 37 °C to reduce spontaneous rod formation. At DIV4, neurons were infected either with WT αSyn-IRES-GFP or IRES-GFP lentiviruses (1 TU/cell). DIV7 or DIV14 neurons were fixed for imaging or lysed for cell extracts.

ReN VM neural progenitor (catalog number SCC008) and Neuro-2a mouse neuroblast (catalog number CCL-131) cells were obtained from Millipore and ATCC (VA, USA), respectively. The undifferentiated ReN VM cells were grown on Matrigel basement membrane matrix (catalog number 354230, Corning, NY, USA) and maintained in DMEM/F12 (catalog number 11320033, Thermo Fisher Scientific) supplemented with 20ng/ml basic fibroblast growth factor (catalog number 8910, Cell Signaling, MA, USA), 20ng/ml epidermal growth factor (catalog number RKP01133, Reprokine, FL, USA), 10 Units/ml heparin sodium salt (catalog number H3149, Sigma-Aldrich) and 1X N21-MAX (catalog number AR008, R&D Systems, MN, USA) or 1X B27 media supplement (catalog number 17504044, Thermo Fisher Scientific). The cells were differentiated into neuronal and glial populations with the removal of heparin and growth factors from media for at least 7 days. Passage-matched N2a and RML-infected N2a cells were created as described previously [64 (link)] and maintained in DMEM (catalog number 11995065, Thermo Fisher Scientific), supplemented with 10% fetal bovine serum (catalog number 12483020, Thermo Fisher Scientific) and 1% GlutaMAX (catalog number 35050061, Thermo Fisher Scientific). CRISPR-Cas9-generated N2a knockout cells were described previously [65 (link)].