Fig 2: GluA1 function is crucial for behavioral responses to chronic stress(A) Experimental paradigm. BALB mice were subjected to 5-day subchronic and mild social defeat stress (smSDS). An AMPA receptor potentiator was infused into their vCA1 1 hr before smSDS exposure (day 1 to day 5, once per day). SIT, social interaction test; SPT, sucrose preference test.(B and C) Treatment with AMPAR potentiator rescues smSDS-elicited reduction of the time in the interaction zone in the SIT (B) and prevents smSDS-induced anhedonia in the SPT (C). n = 13–14 mice per group.(D) AAV vectors engineered to overexpress a GluA1CT (AAV-GluA1CT-T2A-mCherry) or a control construct (AAV-mCherry). PCamk2a, Camk2a promoter.(E) Experimental paradigm. B6 mice were injected with AAV-GluA1CT-T2A-mCherry or AAV-mCherry and subjected to 5-day exposure to smSDS.(F) AAV microinjection into the vCA1. Region-specific expression of mCherry in the vCA1 is shown. Scale bar, 100 µm.(G) Overexpression of GluA1CT decreases Fos levels in the vCA1 of B6 mice following smSDS exposure. n = 6 mice per group.(H) Representative examples of behavior traces of stressed mice given either AAV-mCherry or AAV-GluA1CT-T2A-mCherry during the SIT (target session).(I and J) GluA1CT overexpression reduces time in the interaction zone in the SIT (I) and induces anhedonia in the SPT (J) after smSDS. n = 14–15 mice per group.two-way ANOVA followed by a Tukey's post hoc test (in B, C, I, and J) and two-tailed Student's t-test (in G) were used for statistical analyses. *p < 0.05. Bar graphs show mean ± SEM. See also Figure S5. Complete statistical summaries are provided in Table S2.

Fig 3: Effects of cannabidiol (CBD) treatment on ketamine (KET)-induced alternations of AMPA receptor subunit protein expression levels in the dorsal hippocampus (top row) and the ventral hippocampus (bottom row). (A,E): GLUA1; (B,F): GLUA2; (C,G): GLUA1/GLUA2 ratio; (D,H): a representative band of the protein of interest and the corresponding loading marker band. Data are presented as percentage of the SAL/VEH group. The optical density (OD) of each band was divided by the corresponding loading marker. Statistical analysis was performed by two-way ANOVA.

Fig 4: Impact of AMPK hyper-activation on synaptic markers expression.Primary neurons were treated at 20 DIV for the indicated times with the AMPK activator, AICAR (1 mM). a Cell lysates were analyzed by WB with antibodies directed against phospho-AMPK-Thr172 (pAMPK), AMPK, phospho-ACC-Ser79 (pACC), ACC, and actin. b, c Quantification of the ratios pAMPK/AMPK (b) and pACC/ACC (c). Results represent mean ± SD, n = 6. d Cytotoxicity was assessed using the lactate dehydrogenase (LDH) assay after 24, 48, 72, and 96 h of AICAR treatment. Treatment with 0.9% Triton X-100 was used as a positive control. Results represent mean ± SD, n = 6. e–m Synaptic markers expression upon AMPK hyper-activation with AICAR treatment. WB analysis (e) and quantification of the pre-synaptic markers SNAP25 (f), Munc (g), synapsin Ia (Syn Ia) (h) and IIb (Syn IIb) (i), and the post-synaptic markers PSD-95 (j), Homer 1bc (k), GluN1 (l), and GluA1 (m). Results represent mean ± SD, n = 6. n–r Post-synaptic densities extractions were performed after 48 h AICAR treatment. Resulting non PSD and PSD fraction were analyzed by WB (n) and expression of the post-synaptic markers PSD-95 (o), Homer 1bc (p), GluN1 (q), and GluA1 (r) was quantified in the PSD fraction. Results represent mean ± SD, n = 4. One-way ANOVA with Bonferroni’s post hoc test was used for b–d and f–m experiments and Student’s t-test was used for o–r experiments; *p < 0.05, **p < 0.01, ***p < 0.001 compared to Ctrl

Fig 5: 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.