Fig 2: Aberrant Glu-GluR2-Parkin pathway triggered mitophagy-mediated hippocampal neurons apoptosis in DD rats. (A)Mitophagy and autophagosome detected by transmission electron microscope in each group. The ‘blue arrow’ represents for the normal mitochondria and the ‘yellow arrow’ shows the mitophagy. (B-F)Expression of Glu-GluR2-Parkin pathway protein and endogenous apoptotic protein for Cyt-c detected by Western blot assay in each group (n = 3; **P < .001vs Con; # P < .05, ## P < .01 vs DD)

Fig 3: RNF220 directly ubiquitinates AMPARs to regulate the receptor protein stability.(A) Polyubiquitination analyses of endogenous GluA1 and GluA2 protein in hippocampus of Emx1-Cre;RNF220fl/fl mice and littermate controls. (B and C) Coimmunoprecipitation analyses of Flag-tagged RNF220 or HA-tagged glutamate receptor interaction HEK293 cells with transfection as indicated. The whole-cell lysates (WCLs) were immunoprecipitated by Flag or HA antibody, and the WCLs and immunoprecipitations (IPs) were immunoblotted by the indicated antibodies. (D) Coimmunoprecipitation analyses of the interaction between RNF220 and glutamate receptors from RNF220-BAP and littermate mice hippocampus, and the input and IP samples were immunoblotted with the indicated antibodies. (E and F) Polyubiquitination analyses of GluA1 and GluA2 by coexpressed RNF220 or E3 ligase-dead mutants in HEK293 cells.

Fig 4: RNF220-mediated ubiquitination is involved in AMPAR neuronal surface trafficking.(A and B) BS3 cross-linking assay of the surface expression of GluA1 and GluA2 from Emx1-Cre;RNF220fl/fl mice (n = 3) and littermate control (n = 3) hippocampal (A) and cortical (B) acute slices. The surface receptor levels (top band, red) are normalized against the cytoplasmic protein levels (bottom band, blue) as indicated. Bar graphs (mean ± SEM) show relative surface GluA1 and GluA2 (sGluA1 and sGluA2) protein levels normalized against each control with BS3 treatment. Statistical analyses are compared to controls with unpaired t test. (C and D) Impermeable immunofluorescence staining shows the cell surface GluA1 expression levels in primary cultured rat cortical neurons with indicated transfection. EGFP construct was coexpressed as marker for transfection. CRISPR-Cas9 was used for RNF220 KO, and gRNA-resistant RNF220 constructs were used for the rescue experiments. Bar graphs (mean ± SEM) show relative surface GluA1 intensity normalized against only EGFP transfection control [(C) EGFP, n = 42; RNF220 gRNA, n = 39; RNF220 gRNA + RNF220, n = 26; (D) EGFP, n = 32; RNF220, n = 34; RNF220(W539R), n = 36; RNF220(?RING), n = 29]. Statistical analyses are compared between indicated treatments with unpaired t test. Scale bars, 2.35 µm. ***P < 0.001 and *P < 0.05.

Fig 5: Aberrant Glu-GluR2-Parkin pathway leads to monoamine neurotransmitter deficiency and depressive-like behaviour in DD rats. (A-B) 5-HT and DA levels detected by ELISA in each group (n = 4; *P < .05,**P < .001vs Con; # P < .05, ## P < .01 vs DD). (C-D)Number of activities in open-field test and the immobility time in forced swimming test among each group (n = 5; *P < .05, **P < .001 vs Con; # P < .05 vs DD)