SV2A/B KO neurons are resistant to TeNT.

<p>(A) Mouse spinal cord neurons with the following genotypes: WT, SV2B KO [SV2A (+/+) SV2B (−/−)], and SV2A/B KO [SV2A (−/−) SV2B (−/−)], were exposed to HCR/T (50 nM). HCR/T fluorescence in SV2A/B KO neurons was dramatically reduced as compared to WT and SV2B KO. (B) Quantification of HCR/T binding: fluorescence was reduced by 30% and 50% for SV2B KO and SV2A/B KO neurons, respectively. Error bars represent SD, WT n = 9, SV2B KO n = 11, SV2A/B KO n = 12, ***p≤0.001. (C) SV2B KO and SV2A/B KO cultures were exposed to TeNT (20 nM) and BoNT/A (10 nM). Cell lysates were subjected to immunoblot analysis and probed for syb II, SV2, SNAP-25, and actin. Syb II in SV2A/B KO neurons was largely protected from TeNT action until resensitized through lentiviral expression of SV2A or SV2B; arrow indicates the BoNT/A cleaved form of SNAP-25. (D) SV2B KO and SV2A/B KO spinal cord neurons from were assayed for susceptibility to TeNT. Syb II was cleaved by 5 nM TeNT in SV2B KO neurons, while syb II was protected from TeNT in SV2A/B KO neurons. SV2A/B KO neurons could be resensitized to TeNT, upon lentiviral expression of SV2A. (E) Three putative glycosylation sites in SV2A were removed by creating N to Q mutants (residues 498, 548 and 573) and were expressed in SV2A/B KO neurons along with WT SV2A. Syb II was cleaved by TeNT in neurons reinfected with WT SV2A as well as the three mutants. (F) WT and SV2B KO mice were injected with the indicated amounts of TeNT and their time-to-death was recorded. SV2B KO mice were more than five-times more resistant to TeNT as compared to their WT counterparts. (G) WT and SV2A/B KO neurons were cultured and treated with BoNT/F at the indicated concentrations. Cell lysates were probed for syb II and syp by immunoblot analysis. WT and SV2A/B KO neurons exhibited similar sensitivities to BoNT/F.