Fig 2: SNAP29 KD in the CA1 hippocampal region increased indifference to danger and caused social dysfunction. A. The diagrammatic sketch and representative results from the open field test. B. The schematic process diagram and representative results from the three-chamber social test were presented. The total distance traveled, the number of entries into the center and the spent time in the central area of the open field test were shown in C, D and E (***p < 0.001 vs. WT group, ###p < 0.001 vs. NC group, n = 8 per group, one-way ANOVA; Bonferroni post-test). The time in each chamber (Trial1), sniffing time for strange#1 (Trial 2) and sniffing time for strange#2 (Trial 3) of the three-chamber social test were present in F, G and H. (*p < 0.001 vs. control group, #p < 0.001 vs. NC group, n = 8 per group, one-way ANOVA; Bonferroni post-test). Data are shown as mean and SEM.

Fig 3: The SNAP29 protein levels significantly decreased after exposed to OGD. A. SNAP29 in neurons was detected following OGD exposure and during subsequent reperfusion (1, 2, 4, 6, and 12 h) with western blot assay, and quantitative analysis was shown in B (*p < 0.05, **p < 0.01, and ***p < 0.001 vs. normoxia group, n = 6 per group, one-way ANOVA; Bonferroni post-test). C. Immunofluorometric assay of SNAP29 levels following OGD/R exposure (**p < 0.01 and ***p < 0.001 vs. normoxia group, n = 6 per group, one-way ANOVA; Bonferroni post-test). D. RT-qPCR analysis of Snap29 mRNA in neurons following OGD/R exposure (**p < 0.01, n = 3 per group, one-way ANOVA; Bonferroni post-test). E. SNAP29 in neurons was detected after pretreatment of MG132 (10 µM for 6 h) and then OGD/R (0, 1 and 12 h) exposure using western blot, and quantitative analysis was shown in F (*p < 0.01, n = 6 per group, Student's t-test; one-tailed). G. After over-expressed with SNAP29 plasmid, the survival of neurons upon OGD or during subsequent reperfusion were detected with CCK8 assay (n = 9 per group, one-way ANOVA; Bonferroni post-test). H. The survival of SNAP29 overexpressed neurons following OGD/R exposure were detected with LDH assay (n = 3 per group, one-way ANOVA; Bonferroni post-test). Data are shown as mean and SEM.

Fig 4: CUR5g blocks incorporation of STX17 on autophagosomes.A Western blot analysis of STX17, SNAP29, and VAMP8 levels in A549 cells treated with CUR5g (10 µM) for 0–24 h. GAPDH was used as a loading control. B, C Representative fluorescence images of the colocalization of LC3B (green) and STX17 (red) (B) or LC3B (green) and SNAP29 (red) (C). Nuclei were stained with DAPI. The line-scanned profiles show the distribution of fluorescence for each channel in the white line in the corresponding confocal images. Scale bar = 10 µm.

Fig 5: O-GlcNAc modification of SNAP29 inhibits SNAP29-STX17-VAMP8 complex formation and inhibits autophagy-mediated degradation. NRCMs were exposed to high glucose (25 mM) and were treated with TG (5 µM) or Don (40 µM) for 24 h. The combination of O-GlcNAc-modified SNAP29 into the SNAP29-STX17-VAMP8 complex was observed by co-IP. (A) SNAP29 antibody was used as pull down the VAMP8 and STX17, SNAP29 and Tubulin were used as the loading control; (B) VAMP8 antibody was applied to pull down the complex SNAP29 and STX17, VAMP8 and Tubulin were used as the loading control. (C) STX17 antibody was used to pull down the complex SANP29 and VAMP8, STX17 and Tubulin acted as the loading control. (**P<0.01, ***P<0.001 vs. Vehicle+Glu group; ##P<0.01, ###P<0.001 vs. TG+Glu group); NRCMs were transfected with sh-OGT or ad-OGA for 48 h and to high glucose for another 24 h, following which the formation of the SNAP29-STX17-VAMP8 complex was observed by co-IP. (D) SNAP29 antibody was used as pull down the VAMP8 and STX17, SNAP29 and Tubulin were used as the loading control; (E) VAMP8 antibody was applied to pull down the complex SNAP29 and STX17, VAMP8 and Tubulin were used as the loading control. (F) STX17 antibody was used to pull down the complex SANP29 and VAMP8, STX17 and Tubulin acted as the loading control. (*P<0.05, **P<0.01, ***P<0.001 vs. sh-Ctrl+Glu group; ##P<0.01, ###P<0.001 vs. sh-OGT+Glu group); NRCMs, neonatal rat cardiomyocytes; O-GlcNAc, O-linked ß-N-acetylglucosamine; SNAP29, synaptosomal-associated protein 29; vesicle-associated membrane protein 8; STX17, syntaxin-17; Don, 6-diazo-5-oxo-L-norleucine; TG, thiamet G; OGT, O-GlcNAc transferase; sh-OGT, OGT-knockdown adenovirus; ad-OGA, OGA-overexpression adeno-virus; Ctrl, control; co-IP, coimmunoprecipitation.