Fig 2: Loss of C9orf72 reduces levels of synapsin in the hippocampus in vivo. Representative images of hippocampal sections of 12-week-old C9orf72-WT, C9orf72-HET and C9orf72-KO mice stained for a Syn1, b Syn3 or c SV2. Syn1, Syn3, and SV2 levels in the mossy fibre area (outlined) were quantified as the area fraction of positive staining (Area Fraction) and the mean fluorescence intensity level (Intensity) within the outlined area was determined per section. Scale bar 100 µm. Data are presented as box and whisker plots; a Syn1, n (sections analysed) WT = 26, HET = 26, KO = 27 from 5 animals/genotype; b Syn3, n (sections analysed) WT = 30, HET = 28, KO = 26 from 5 animals/genotype; c SV2, n (sections analysed) WT = 26 HET = 24 and KO = 16 from 5 animals/genotype. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test, ns (not significant), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig 3: C9orf72 haploinsufficiency reduces the number of excitatory synapses. Primary rat hippocampal neurons were transduced with EmGFP non-targeting control miRNA (miNTC) or C9orf72 miRNA (miC9) lentivirus at 5DIV. Neurons were immunostained at 12DIV for the dendritic marker MAP2 (white) and pre- (green) and postsynaptic (magenta) pairs: a Syn1 and Homer, b Syn3 and PSD95, c SV2 and Homer. Representative confocal images are shown. Boxes denote zoom area, note: co-localisation of pre- (green) and postsynaptic marker (magenta) appears white. Scale bar: 10 µm. Synapse density and the density of postsynapses was quantified per image for each staining pair and are presented as box and whisker plots. The mean intensity of presynaptic staining within detected synapses was quantified per image and is presented as box and whisker plots. a Syn1/Homer with n (images analysed) miRNA NTC = 41, miRNA C9 = 43 from 4 replicate experiments, b Syn3/PSD95, with n (images analysed) miRNA NTC = 30, miRNA C9 = 30 from three replicate experiments, c SV2/Homer with n (images analysed) miRNA NTC = 29, miRNA C9 n = 30 from three replicate experiments. Statistical significance was determined by unpaired two-tailed t test, ns (not significant), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig 4: Synapsin family proteins are novel binding partners of C9orf72. a Lysates of HEK293 cells co-transfected with Myc-C9orf72L (Myc-C9L) and either empty vector (EV) or V5-tagged Synapsin-3a (V5-Syn3a) were subjected to immunoprecipitation using an anti-Myc antibody. Immune pellets were probed for Myc-C9orf72L and V5-Syn3a on immunoblots. b Lysates of HEK293 cells co-transfected with Myc-C9L and either EV, EGFP or EGFP-Syn1a, YFP-Syn2a, EGFP-Syn3a were subjected to immunoprecipitation using an anti-GFP antibody. Immune pellets were probed for GFP/YFP and Myc-C9orf72L on immunoblots. c HEK293 cells were co-transfected with either Myc-C9L + V5-Syn3a or with Myc-C9L + V5-Syn1a. Transfections were laced with EGFP (green) to identify transfected cells. Cells were fixed and immunostained with both anti-V5 and anti-Myc antibodies and processed for PLA (PLA, magenta), nuclear staining with Hoechst (blue). Images are representative of the individual channels and their overlay. Scale bar 10 µm. Intensity of PLA signals per cell was analysed as corrected total cellular fluorescence (CTCF); data are presented as mean ± SEM; n (cells analysed) EV = 107, C9L = 92, Syn3a = 113, Syn1a = 79, C9L + Syn3a = 129, C9L + Syn1a = 109 from three or 4 replicate experiments. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test, ****P < 0.0001. Images showing the single transfection controls are shown in Supplementary Fig. 2, Online Resource. d Lysates of synaptosomes prepared from 12-week-old mouse brains were subjected to immunoprecipitation using an anti-C9orf72 antibody or an irrelevant antibody (Ctrl) raised in the same species as the C9orf72 antibody. Immune pellets were probed for endogenous SMCR8, C9orf72, Syn3 and Syn1 (* indicates a nonspecific band; enh, enhanced). e Lysates of synaptosomes prepared from 12-week-old C9orf72-WT and C9orf72-KO mouse brains were subjected to immunoprecipitation using an anti-C9orf72 antibody or a control antibody (M2). Immune pellets were probed for endogenous SMCR8, C9orf72, Syn3 and Syn1 (* indicates a nonspecific band; enh, enhanced). f 12DIV primary rat hippocampal neurons were fixed and immunostained with pairs of antibodies against C9orf72 and Syn3 (C9 + Syn3) or C9orf72 and Syn1 (C9 + Syn1) together with an antibody against synaptophysin and processed for PLA. Overlay PLA signals (red) between C9 + Syn3 or C9 + Syn1 and synaptophysin (white). Scale bar 5 µm. For quantification, the number of PLA spots per image was normalised (norm PLA) to the mean grey intensity of the synaptophysin image. Data are presented as mean ± SEM; n (images analysed) C9 = 14, Syn1 = 14, Syn3 = 15, C9 + Syn1 = 59, C9 + Syn3 = 62 from two or three replicate experiments. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test, ****P < 0.0001. Single antibody controls are shown in Supplementary Fig. 2, Online Resource