Fig 2: Cx30 regulates hippocampal synaptic levels of laminin proteins. (A) Western blot detection of pre- (synapsin I, SYN1) and post- (PSD95) synaptic proteins showed an enrichment of plasma membrane synaptic proteins in crude synaptosomal membrane fraction (S) compared with total hippocampal fraction (T), whereas actin protein levels were clearly similar in both fractions. (B) Immunoblot detection of laminin expression in synaptosomal membrane fractions from mouse hippocampi. (C) Histogram showing the quantitative analysis of normalized laminin expression levels (+/+, n=3 mice; -/-, n=3 mice). The loss of Cx30 expression increased significantly the synaptic levels of laminin. (D) Immunoblot detection of laminin ß2 protein levels in crude hippocampal membrane fractions. (E) Quantitative analysis of laminin ß2 normalized expressions. Hippocampal synapses lacking Cx30 upregulated laminin ß2 expression. In both experiments, actin was used as a loading control. Asterisks indicate statistical significance (*P<0.05). S+/+, wild-type mice; S-/-, Cx30-/- mice.

Fig 3: PKA-dependent phosphorylation of Syn1 is decreased and insensitive to forskolin in BsnGT A, BRepresentative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted for WT and BsnGT neurons without treatment (black and red) and upon treatment with adenylyl cyclase activator forskolin (grey and pink) or PKA inhibitor H89 (light grey and orange).C, DQuantification of the RRP and TRP fraction from the experiment in (A) and (B).ERepresentative immunoblot with antibody against pSer9Syn1 and total Syn1 and total protein stain of hippocampal P2 fraction from WT and BsnGT mice prepared in the presence of KN93 to isolate PKA-dependent regulation of pSer9Syn1.F, GQuantification of pSer9Syn1/Syn1 ratio (in F) and Syn1 (in G) on blots from (E).HRepresentative images of hippocampal neurons from WT and BsnGT mice labelled with antibodies against pSer9Syn1 and total Syn1. Neurons were pretreated (4 µM, 1 h) with KN93 to isolate PKA-dependent phosphorylation and then treated in addition with forskolin or H89 before fixation.IQuantification of staining in (H).JScheme illustrates the PKA-dependent phosphorylation of Syn1 on Ser9 that promotes recruitment of SVs to the recycling pool. Changes in this signalling confirmed in BsnGT are depicted in red. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets and corresponds to the number of analysed independent imaging experiments performed on three independently prepared culture batches in (C and D), samples prepared from individual animals in (F) and (G), or quantified independent visual fields obtained from two independent culture preparations in (I). The statistical significance was assessed in (C), (D) and (I) using two-way ANOVA with the Tukey's multiple comparison test and in (F) and (G) using the Student's t-test as is depicted in graphs as *P = 0.05, **P < 0.01, ***P < 0.001. The scale bar is 2 µm in (A) and 5 µm in (H).

Fig 4: The effects of LE-derived SynI autoantibodies on excitatory and inhibitory transmission and synapses are abolished in SynI KO hippocampal neurons.a Representative recordings of mEPSCs in SynI KO hippocampal neurons treated with either control CSF (1.5 µg/mL, black trace) or LE-CSF (1.5 µg/mL, green trace) for 72 h. b–d Cumulative distribution of inter-event intervals and mean frequency of mEPSCs (b), cumulative distribution and mean of mEPSC amplitude (c) and rise/decay times of mEPSCs (d). e Representative recordings of mIPSCs in SynI KO hippocampal neurons treated as described in (a). f–h Cumulative distribution of inter-event intervals and mean frequency of mIPSCs (f), cumulative distribution and mean of mIPSC amplitude (g) and rise/decay times of mIPSCs (h). Graphs show means ± SEM, single neuron data points from four independent preparations; not significant, two-tailed Student’s t-test. i Representative images of primary SynI KO hippocampal neurons treated with either control CSF or LE- CSF (1.5 µg/mL) for 72 h and stained for ß-III tubulin (blue), VGLUT1 (green), and VGAT (red). Scale bars, 10 and 1 µm for low and high magnification, respectively. j Density of VGLUT1- positive excitatory synapses (upper panel) and VGAT-positive inhibitory synapses (lower panel) in control and patient CSF-treated SynI KO neurons. Cumulative plots show pooled data from all neurons; graphs show means ± SEM, single neuron data points from three independent preparations; not significant, two-tailed Student’s t-test.

Fig 5: Endocytosed SynI antibodies escape the endocytic compartments and target endogenous SynI.a Hippocampal neurons, incubated daily for three days with either SynI-mAb (1.5 µg/mL) or vehicle, were subjected to subcellular fractionation to obtain the post-nuclear supernatant (S1), a cytosolic fraction (S3) and a membrane-enriched fraction (P3). A representative immunoblot with anti-SynI (SynI), anti-synaptophysin (Syp), anti-ß-tubulin III (ßIII Tub), and anti-mouse secondary antibodies (anti-IgG) to test the presence of the internalized SynI-mAb is shown. b Immunoprecipitation (IP) of the complex between internalized SynI antibodies and endogenous SynI was performed in hippocampal neuronal extracts with Protein G-Sepharose after incubation of neurons with either SynI-mAb or vehicle for 24 h. Equal aliquots of the starting material (input) and of the supernatants (SUP) together with the IP samples were subjected to western blotting with anti-SynI antibodies (SynI) and anti-mouse secondary antibodies (anti-IgG) to test the recovery of the internalized SynI antibodies. Left: A representative immunoblot is shown. Right: quantification of the SynI immunoreactivity signal in the immunoprecipitated samples, normalized to the binding in the control sample (means ± SEM, n = 3 independent experiments). Input, 20 µg cell extract. ***p < 0.001, two-tailed Student’s t-test. c Representative confocal images of in situ proximity ligation assay (PLA) in WT and SynI KO hippocampal neurons incubated with either SynI-mAb (1.5 µg/mL) or vehicle for 72 h. After fixation, cells were stained with Syn I G143 antibody and processed to detect SynI-mAb and synapsin I proximity interactions. Positive interactions are indicated as red dots. DAPI-stained nuclei are indicated in blue. Scale bars, 5 µm. d Quantification of the number of PLA spots. Graphs show means ± SEM, single neuron data points from three independent preparations; *p < 0.05, **p < 0.01, two-way ANOVA/Bonferroni’s tests.