Figure 6: SALM4 inhibits SALM2-dependent excitatory synapse facilitation and SALM3/5-dependent presynaptic differentiation.

(a) SALM4 inhibits SALM2-dependent promotion of excitatory synapse number. (Top panels) Representative images of cultured hippocampal neurons transfected with control (EGFP alone), SALM2 and EGFP (SALM2), SALM4 and EGFP (SALM4) or co-transfected with SALM2 and SALM4 (SALM2+SALM4) at DIV10. Neurons were analysed at DIV14 by triple immunofluorescence for EGFP (green), Shank (an excitatory postsynaptic marker protein; red) and SALM4 (blue). Scale bar, 10 μm, applies to all images. (Bottom panels) Bar graphs summarizing the effects of SALM4 overexpression on SALM2-induced postsynaptic development, quantified using Shank immunoreactivity (pan-Shank). n=15 for control (EGFP alone), SALM2, SALM4 and SALM2+SALM4, **P<0.01, ***P<0.001, ns, not significant, ANOVA with Tukey’s test. (b) SALM4 suppresses SALM3/5-dependent presynaptic differentiation. (Top panels) Representative images of cocultures. Hippocampal neurons were cocultured for 2 days (DIV 10–12) with HEK293T cells expressing EGFP alone (control), SALM3/5, SALM3/5+SALM4, LRRTM2 or LRRTM2+SALM4, and stained for EGFP/HA (blue), SALM4 (green) and synapsin I (red). Scale bar, 10 μm. (Bottom panels) Quantification of heterologous synapse-forming activities of SALM3/5 and LRRTM2, by measuring the ratio of synapsin I to surface HA immunofluorescence (absolute red/synapsin and blue/HA fluorescence values are also indicated). Note that SALM4 coexpression does not affect the surface expression of SALM3, SALM5 or LRRTM2, as indicated by fluorescence intensity of HA signals. n=12 for control, 11 for SALM3, 10 for SALM3+SALM4, 10 for SALM5, 10 for SALM5+SALM4, 12 for LRRTM2 and 11 for LRRTM2+SALM4, **P<0.01, ***P<0.001, ns, not significant, ANOVA with Tukey’s test. (c) SALM4 does not affect surface levels of SALM3 or SALM5, as determined by surface biotinylation assays. HEK293T cells transfected with SALM3/5 alone, or SALM3/5+SALM4, were biotin-labelled, followed by avidin precipitation and immunoblotting. Input and surface, 1% and 8%, respectively. n=3 for SALM3 and SALM3+SALM4, ns, not significant, Student’s t-test.