Fig 2: The level of NLGN1 and NLGN3 proteins is elevated in synaptoneurosomes and hippocampal cultures from Fmr1 KO mice. a–c Western blot analysis of a NLGN1, b NLGN2, and c NLGN3 protein levels in synaptoneurosomes isolated from WT and Fmr1 KO mice. Below, the graphs representing mean values ± SEM from densitometric analysis of immunoblots, n = 6 mice/genotype, NLGN1 p = 0.0016; NLGN2 p = 0.4293 (not significant); NLGN3 p = 0.0112; *p < 0.05, **p < 0.01 by unpaired Student’s t test. d–f Western blot analysis of d NLGN1, e NLGN2, and f NLGN3 protein levels in DIV21 WT and Fmr1 KO hippocampal cultures. Below, the graphs representing mean values ± SEM from densitometric analysis of immunoblots, n = 5–9, NLGN1 p = 0.0086; NLGN2 p = 0.577 (not significant); NLGN3 p = 0.0189; *p < 0.05, **p < 0.01, by unpaired Student’s t test. For protein loading control, see Fig. S3. g Comparative western blots presenting excitatory and inhibitory synapses’ markers in brain homogenates (H) and synaptoneurosomes (SN) from WT mice

Fig 3: Enhanced postsynaptic membrane targeting of NLGN1 and its activity-dependent cleavage in synaptoneurosomes from WT and Fmr1 KO mice. a Schematic representation of NLGN1 local protein synthesis, membrane trafficking and proteolytic cleavage in response to the stimulation. NLGN1 species detected on the western blot after cross-linking experiment are depicted. b Western blot with anti-NLGN1 antibody recognizing NLGN1 extracellular domain. Cross-linked NLGN1 surface dimers (sNLGN1) are detected at about 300 kDa (corresponding to homo- or heterodimers containing NLGN1), intracellular NLGN1 monomers (iNLGN1) at about 120 kDa and cleaved N-terminal fragment of NLGN1 (cleaved NLGN1 NTF) at about 95 kDa. c TGX acrylamide gel visualization by Bio-Rad Gel Doc XR+ to confirm equal protein loading on the gel. d Western blot showing deglycosylation of cross-linked NLGN1, NLGN2, and NLGN3 proteins with PNGase F and Endo H. e–g Densitometric quantification of the western blot bands from independent experiments, n = 6. The graphs represent the mean values ± SEM of e surface, f intracellular, and g cleaved NLGN1 protein levels relative to WT untreated sample (WT Bsl), *p < 0.05, **p < 0.01 by unpaired Student’s t test for comparisons between genotypes and paired Student’s t test for comparisons within the genotype. h Western blot showing surface (sNLGN1) and intracellular (iNLGN1) NLGN1 protein levels in cross-linked DIV19 hippocampal cultures. Below, the protein loading. The graphs represent mean values ± SEM from densitometric analysis of immunoblots, n = 12; *p < 0.05, ***p < 0.001 by unpaired Student’s t test. i Scheme of biotinylation experiment. j Immunoblot probed with anti-NLGN1 antibody showing biotinylated surface NLGN1 (sNLGN1) level in WT and Fmr1 KO synaptoneurosomes. The graph shows densitometric quantification of the bands from six independent experiments ± SEM, n = 6; **p < 0.01, by unpaired Student’s t test

Fig 4: Baseline measurements of resting colon diameter and numbers of colonic migrating motor complexes (CMMCs) between WT and Nlgn3-/- mice. (A, B) Spatiotemporal maps showing baseline CMMCs in WT (A) and Nlgn3-/- (B) mice. (C) Resting gut diameter was higher in Nlgn3-/- mice (n = 16) compared to WT mice (n = 15, P = 0.03). (D) No difference in the number of CMMCs/15 min was observed between WT mice (n = 16) and Nlgn3-/- (n = 19, P = 0.38). (E) Nlgn3-/-mice (n = 17) had increased speed of CMMCs compared to WT mice (n = 15, P = 0.04). Statistics: Student's t test, Bonferroni post hoc test, Number of CMMCs assessed via Mann–Whitney test, *P < 0.05. Horizontal scale bar = Time (1 min). Vertical scale bar = 0.5 cm (length of colon). The color scale on the right of each map indicates the width of the gut for each captured frame during the 15-min recording. Arrows indicate individual CMMCs. The broken lines within the enlarged regions of the spatiotemporal maps in A and B highlight the gradient (speed) of the CMMCs.

Fig 5: Confirmation of Neuroligin-3 expression in colon tissue. Neuroligin-3 protein expression is present in colon and brain of WT but absent in Nlgn3-/- (KO) mice. Neuroligin-3 was immunoprecipitated from the brain, proximal colon, and distal colon of a WT mouse and a Nlgn3-/- mouse, and subjected to SDS-PAGE and Western immunoblotting with an antibody to Neuroligin-3. n.s., non-specific band; IgG HC, IgG heavy chain.