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. 2021 May 14;12:2849. doi: 10.1038/s41467-021-23133-9

Fig. 5. PSD-95 Phosphorylation at T19 position by GSK3β targets it to autophogosomes.

Fig. 5

A and B Western blot analysis of total PSD-95 and T19 phosphorylated PSD-95 in purified synaptosomes and in PK-treated autophagic vesicles (AVs) purified before and after induction of the LTD by NMDA (A) or ATP (B) application. PSD-95 and T19PSD-95 levels were normalized to the levels of p62, an autophagic cargo. C Quantification of normalized T19PSD-95 and PSD-95 levels obtained in (A and B) reveals that both phosphorylated and global form of PSD-95 is over-accumulated in autophagic vesicles after NMDA treatment but not after ATP (mean ± SEM, n = 3, one-way ANOVA, p = 0.0005 for pT19P and p = 0.0180 for the Total. Dunnett’s post-test results are realized between each condition and the control condition. For pT19P, control vs ATP-LTD p = 0.85 and control vs NMDA-LTD p = 0.0008; For Total, control vs ATP-LTD p = 0.996 and control vs NMDA-LTD p = 0.022). D Representative images of Dual-color dSTORM experiments with LC3 labelled with alexa-647 (upper panels) and PSD-95 labeled with alexa-532 (bottom panels). PSD-95 intensity inside the AVs shown a threefold increase following LTD induction by NMDA treatment (Right panel, mean ± SEM, unpaired t-test, p = 0.0022, n = 15 and 29). E Evolution in function of time of the mESPC amplitude after NMDAR-dependent LTD in the presence of TDZD8 (10 µM), an inhibitor of GSK3β. After 10 min, a normal LTD is induced but TDZD8 block the maintenance of the LTD (mean ± SEM, one-way ANOVA, p < 0.0001 and Dunnett’s post-test results are realized between each conditions and the control condition, N = 10, 10, 9, 9, 8). F Average of the mESPC amplitude recorded on WT neurons 0 and 30 min after NMDA or ATP treatment, in the absence or the presence of SBI (a specific blocker of autophagy, 0.5 µm). SBI alone does not impact on mEPSC amplitude, while it fully blocks NMDAR-dependent LTD. At the opposite, ATP-induced LTD is preserved in the presence of SBI (mean ± SEM, one-way ANOVA, p < 0.0001 and Dunnett’s post-test results are realized between each conditions and the control condition, N = 19, 7, 7, 17, 11, 11).