Fig. 5
M1-Pep restored cell surface expression of GABAB receptors after OGD by inhibiting the MARCH1/GABAB receptor interaction. (a) Neurons/glia co-cultures were subjected to 1 h of OGD, then treated with M1-Pep or Ctrl-Pep for 16 h and analyzed for cell surface expression of GABAB receptors and MARCH1 expression. Left: representative images (scale bar: 10 μm). Right: quantification of fluorescence intensities (mean ± SD of 36 neurons per condition, 3 independent experiments). One-way ANOVA followed by Tukey’s multiple comparison test (ns, p > 0.05; ***, p < 0.001; ****, p < 0.0001). (b) Western blot analysis of total GABAB receptor expression using GABAB2 antibodies. Left: representative Western blot. Cultures were subjected for 1 h to OGD, then immediately treated or not with M1-Pep or Ctrl-Pep and harvested after 16–24 h for analysis. Right: quantification of Western blot signals (mean ± SD of 3 independent cultures per condition and one technical replicate). Signals were normalized to untreated control cultures (Ctrl). One-way ANOVA followed by Tukey’s multiple comparison test (ns, p > 0.05; ***, p < 0.001; ****, p < 0.0001). (c) M1-Pep inhibited the increased interaction between GABAB receptors and MARCH1 after OGD as analyzed by in situ PLA using antibodies directed against GABAB2 and MARCH1. Signals were normalized to untreated cultures (Ctrl). Left: representative images; top images: in situ PLA signals (white dots), bottom images: MARCH1 staining (scale bar: 10 μm). Right: quantification of in situ PLA and MARCH1 signals (mean ± SD of 25 neurons per condition, 3 independent experiments). One-way ANOVA followed by Tukey’s multiple comparison test (ns, p > 0.05; ***, ****, p < 0.0001). (d) M1-Pep induced lysosomal degradation MARCH1. Cultures were subjected to OGD for 1 h and immediately thereafter remained untreated or were treated with M1-Pep in the presence of proteasomal inhibitors (50 µM lactacystin or 50 µM MG132) or the lysosomal inhibitor leupeptin (50 µM). Neurons were tested for MARCH1 expression. Left: representative images (scale bar: 10 μm). Right: quantification of fluorescence intensities (mean ± SD of 30 neurons per condition, 2 independent experiments). The fluorescence intensity of untreated neurons served as control. Brown-Forsythe and Welch’s one-way ANOVA followed by Dunnett’s T3 multiple comparisons test (ns, p > 0.05; ****, p < 0.0001).
