Primary motor neuron culture

Motor neurons were cultured as previously described⁵⁹. Briefly, spinal cord explants from SOD1^G93A, WT, or Sig1R^−/− E12.5 mouse embryos were collected in 1X HBSS buffer (Gibco), then trypsinized and consequently triturated in L-15 medium (Life Technologies) containing 0.4% BSA (Sigma) and 2–10% DNAse (Sigma). Cells were centrifuged through a 4% BSA cushion and then resuspended in Complete Neurobasal Medium (CNB) containing 2% Horse serum (Biological Industries, Ltd.), 2% B27 Supplement, 1% Glutamax, 1% Penicillin-Streptomycin, 0.5% 2-mercaptoethanol, 1 ng/mL BDNF (Alamone labs, B-250), 1 ng/mL GDNF (Alamone labs; G-240), and 0.25 ng/mL CNTF (Alamone labs; C-240). Motor neurons were isolated by centrifugation through Optiprep gradient (10.4% Optiprep (Sigma-Aldrich), 10 mM Tricine, 4% glucose) for 20 min at 760 × g with the brake turned off. Culture dishes were pre-coated with 1.5 g/mL poly D-L- ornithine (PLO; Sigma-Aldrich) overnight at 37 °C and with 3 g/mL Laminin (Sigma-Aldrich) overnight at 37 °C.

For western blotting, 400,000 cells were plated in two 24-well plate wells per condition. MN cultures at 2DIV were starved for 16 h in PNB medium prior to applying pridopidine or BDNF. At 3DIV, BDNF 50 ng/mL or pridopidine (0.1 µM; 1 µM) was diluted in PNB medium and added to the cultures for 30 min. PNB medium was used as a control.