Figure 8A) Cortical neurons were loaded with FM4-64 dye 48 hours after transfection with eGFP-GA50 and transduction with rSV2a-eGFP-pRRL lentivirus. Basal fluorescence was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl. Graphical representation is the change in fluorescence (ΔF) normalized to basal fluorescence (F), ΔF/F. The final 60 seconds of baseline recording and 60 seconds of the depolarization phase are shown, arrow indicates start of depolarization. The lack of synaptic release in the case of GA50 expression (red line) was rescued back to levels comparable to eGFP controls (pink line) in the case of coexpression of rSV2a-eGFP-pRRL (orange line). Statistical significance was determined using the Sidak-Bonferroni method, ***p < 0.001, n=10 puncta regions from each of 4 biological replicates.B) Cortical neurons were co-transfected with mCherry or mCherry-GA50 and GCaMP6f, then transduced with rSV2a-eGFP-pRRL. Green fluorescence intensity was recorded from identified mCherry positive neurons. Basal fluorescence was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl. Quantification of the peak change in fluorescence (ΔF) following ACSF perfusion normalized to basal fluorescence (F), ΔF/F. The significant increase in Ca2+ influx observed following GA50 expression (*p < 0.05) is resolved when rSV2a-eGFP-pRRL is coexpressed. Data presented as mean ± SEM. One-way ANOVA with Sidak's multiple comparisons test, at least 10 cells per condition, pooled from n= 3 independent biological experiments. No significant differences were observed between mCherry or mCherry+SV2 conditions, groups were merged for this analysis.Neurons were co-transfected with mCherry-GAn plasmids or mCherry-alone and transduced with rSV2a-eGFP-pRRL, then imaged at 24-hour intervals. (C) Kaplan-Meier survival analysis of cortical neurons expressing GA50 or GA50+SV2 followed for 13 days. The significant cellular toxicity induced by GA50 is rescued by SV2 replenishment. While the hazard ratio of GA50 was 1.473, this was decreased to 0.7227 for GA50+SV2. At least 190 cells per condition were evaluated from a pooling of n=3 independent experiments, log-rank Mantel-Cox test, ****p < 0.0001. No significant differences were observed between eGFP or eGFP+SV2 conditions, and groups were merged for this analysis as Control. (D) A Kaplan-Meier survival analysis was performed for PR50 and PR50+SV2 containing cells. Under these conditions significant toxicity is observed regardless of increased SV2 levels, with the hazard ratio of PR50 containing cells (1.772) remaining high for those expressing PR50+SV2 (1.784). At least 150 cells per condition were evaluated from a pooling of n=3 independent experiments, log-rank Mantel-Cox test, ****p < 0.0001. No significant differences were observed between eGFP or eGFP+SV2 conditions, and groups were merged for this analysis as Control.(E) Kaplan-Meier survival analysis of motor neurons expressing GA50 or GA50+SV2 followed for 10 days. The significant cellular toxicity induced by GA50 is rescued by SV2 replenishment. While the hazard ratio of GA50 was 1.345, this was decreased to 0.7769 for GA50+SV2. At least 200 cells per condition were evaluated from a pooling of n=3 independent experiments, log-rank Mantel-Cox test, ****p < 0.0001.