Fig. 4

Expression of VAPB or PTPIP51 rescues mutant TDP43 induced disruption to dendritic spine numbers. (a) Characterisation of mutant TDP43-IRES-EGFP lentivirus. HEK293 cells were transfected with control vector (CTRL), EGFP, Myc-TDP43-Q331K or Myc-TDP43-A382T as negative and positive controls, and samples run on SDS-PAGE alongside samples of neurons infected with lentivirus expressing Myc-tagged TDP43-Q331K-IRES-EGFP or TDP43-A382T-IRES-EGFP as indicated. Samples were probed on immunoblots for TDP43 via the Myc tags and EGFP; molecular masses are shown on the right. (b and c) Representative super resolution SIM images of dendritic spines in neurons treated with lentivirus expressing (b) EGFP + CTRL, TDP43-Q331K-IRES-EGFP + CTRL, TDP43-Q331K-IRES-EGFP + HA tagged VAPB or TDP43-Q331K-IRES-EGFP + HA tagged PTPIP51 or (c) EGFP + CTRL, TDP43-A383T-IRES-EGFP + CTRL, TDP43-A382T-IRES-EGFP + HA tagged VAPB or TDP43-A382T-IRES EGFP + HA tagged PTPIP51. Neurons were infected with lentivirus at DIV 12 and analysed at DIV 15. Exogenous VAPB and PTPIP51 were identified by immunostaining for the HA tags. Scale bars = 5 μm. Bar charts show spine densities calculated as described (spines/µm) [17]. Data were analysed by one-way ANOVA with Tukey’s post hoc test. N = 20–26 neurons from 3 independent experiments; error bars are SEM. * p≤0.05, ** p≤0.01, *** p≤0.001