Fig. 3

Loss of VAPB and PTPIP51 disrupts ER-mitochondria contacts and reduces dendritic spine and active spine numbers in hippocampal neurons. a Immunoblots showing VAPB and PTPIP51 levels in hippocampal neurons either untreated (UT) or treated with control (Ctrl), 4 different VAPB/PTPIP51 siRNAs or a pool of these siRNAs. PTPIP51 migrates at approximately 61 kD in agreement with previous studies [9, 45]. The weakly staining upper minor species (*) on the PTPIP51 immunoblot does not display any consistent changes in response to the PTPIP51 siRNAs and so we believe it to be non-specific protein. We did not detect any other PTPIP51 species in the neurons. b Super resolution SIM images of neurons either untreated or treated with control, VAPB or PTPIP51 pooled siRNAs, and then immunostained for ER and mitochondria with PDI and TOM20 antibodies. Zooms are of boxed regions with merge and co-localisation of signals. Scale bars = 5 μm. Bar chart shows ER–mitochondria co-localisation normalized to control siRNA in the different samples. Data were analysed by one-way ANOVA with Tukey’s post hoc test. N = 19 neurons UT, N = 42 neurons Ctrl siRNA, N = 42 neurons VAPB siRNA, N = 40 neurons PTPIP51 siRNA from 3 independent experiments; error bars are SEM, **p ≤ 0.01. c Representative images of dendritic spines in EGFP transfected neurons either untreated or treated with control, VAPB or PTPIP51 siRNAs. Bar chart shows spine densities (spines/μm). Data were analysed by one-way ANOVA with Tukey’s post hoc test. N = 14 neurons UT, N = 21 neurons Ctrl siRNA, N = 17 neurons VAPB siRNA, N = 21 neurons PTPIP51 siRNA from 3 independent experiments; error bars are SEM, *p ≤ 0.05. Scale bar = 5 μm. d Representative images of active dendritic spines in EGFP transfected neurons either untreated or treated with control, VAPB or PTPIP51 siRNAs. Active spines were identified by immunostaining for the presynaptic marker synaptophysin (red) and monitoring spine and synaptophysin apposition. Bar chart shows % of active spines normalised to control siRNA treatment. Data were analysed by one-way ANOVA with Tukey’s post hoc test. N = 19 neurons UT, N = 20 neurons Ctrl siRNA, N = 22 neurons VAPB siRNA, N = 27 neurons PTPIP51 siRNA from 3 independent experiments; error bars are SEM, *p ≤ 0.05, **p ≤ 0.01. Scale bar = 3 μm