Fig 2: 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 3: Increased phosphorylation of pSer551Syn1 in excitatory but not in inhibitory synapses of BsnGT neurons ARepresentative images of 20 DIV cultured hippocampal neurons from WT and BsnGT immunolabelled for pSer551Syn1, total Syn1 and for VGLUT1 or VGAT to identify excitatory and inhibitory synapses.BQuantification of synaptic Syn1 IF intensity in Vglut1-labelled excitatory and VGAT-labelled inhibitory synapses does not differ between BsnGT and WT neurons.CQuantification of IF intensity of pSer551Syn1 relative to synaptic Syn1 IF reveals an increased pSer551Syn1 abundance in excitatory but not in inhibitory synapses of BsnGT neurons, indicating a synapse type-specific effect of Bsn deletion on phosphorylation of synaptic Syn1 at Ser551. 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 quantified independent visual fields obtained from three independent culture preparations (B, C). Statistical significance was assessed using the Student's t-test in (B) and (C) and is given as: ***P < 0.0001. Scale bar is 5 µm.

Fig 4: Decreased phosphorylation of pSer9Syn1 in excitatory but not in inhibitory synapses of BsnGT neurons ARepresentative images of 19 DIV cultured hippocampal neurons from WT and BsnGT immunolabelled for pSer9Syn1 and total Syn1 and for VGLUT1 or VGAT to identify excitatory and inhibitory synapses.BQuantification of IF intensity of Syn1 in excitatory and inhibitory synapses labelled for VGLUT1 and VGAT, respectively, reveals a similar synaptic abundance in BsnGT and WT neurons.CQuantification of IF intensity of pSer9Syn1 relative to the intensity of synaptic Syn1 IF reveals a decreased synaptic abundance of pSer9Syn1 in excitatory but not in inhibitory synapses of BsnGT neurons, indicating synapse type-specific effect of Bsn deletion on Syn1 phosphorylation. 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 quantified independent visual fields obtained from three independent culture preparations (B, C). Statistical significance was assessed using the Student's t-test in (B) and (C) and is given as: ***P < 0.0001. Scale bar is 5 µm.

Fig 5: Increased excitatory transmission and altered GABA currents in G2019S KI cortical neurons. (A-C) Whole-cell patch-clamp recordings of neurons in DIV21 CTX cultures from KI mice. (A) Example traces of mEPSCs. (B) Quantification of mean mEPSC amplitude and frequency shows no significant difference in amplitude, but significantly higher frequency of events in KI neurons (**p = 0.01 by Student's t-test). (C) Cumulative probability analysis found no significant differences in mEPSC amplitudes, but revealed a significant main effect of genotype and interaction between IEI and genotype (2-way RM-ANOVA, **p = 0.01, ****p = 0.0001, values between 40 and 200 ms were also significant by Bonferroni post-test ***p = 0.001), due to shorter IEIs (indicative of higher frequency) in KI neurons. (D) Cultures were stained (as in Figure 2) for MAP2 (green) and VGluT1 (blue) and PSD-95 (red). Left: 60× 2-times zoom of individual neuron staining. Right: expanded ROI from the 63× image showing synaptic markers overlayed with and without MAP2. Co-clusters are highlighted (white arrow heads). (E) KI neuronal densities were similar to those of NT littermates as were total dendritic areas (not shown), there were no differences (or trends) in the density of VGluT1 clusters, PSD-95 clusters or co-clusters (glutamate synapses). Similarly there were no differences in the density, size or intensity of synapsin 1 (Syn1) clusters, present at all glutamatergic and inhibitory synapses. (F) Example traces of miniature inhibitory post-synaptic currents (mIPSCs). (G) Quantification of mean mIPSC amplitude and frequency shows trends, but no significant differences in event amplitude or frequency of events in KI neurons. (H) Cumulative probability analysis revealed a highly significant interaction (and nearly significant genotype effect) due to increased mIPSC amplitudes in KI neurons (2-way RM-ANOVA, ****p = 0.0001, values between 25 and 35pA were significant by Bonferroni post-test **p = 0.01). There was no significant main effect of genotype on mIPSC IEIs or interaction (despite a trend to higher frequency) in KI neurons.