Autophagy regulates neuronal excitability by controlling cAMP/Protein Kinase A signaling at the synapse (2022)
Figure 6

Figure 6 | ATG5 regulates PKA-mediated phosphorylation of proteins enriched at the PSD of excitatory synapses.

(A-B) Western Blot analysis of PKA substrates carrying RRXS/T motif in primary cortico-hippocampal Atg5flox:CAG-CreTmx WT/KO neurons at DIV14, treated with Forskolin (WTDMSO set to 100%, WTForskolin: 291.85± 17.02%, KODMSO: 152.97± 25.26%, KOForskolin: 182.56± 31.89%, pWTDMSO/WTForskolin: 0.001, pWTForskolin/KOForskolin: 0.033, two-way ANOVA with Tukey's multiple comparisons). n=3.

(C) The top downregulated phosphopeptides (cut-off p<0.05, Fold change<-1.5) in either cortical lysates of Atg5flox:CamKIIα-Cre KO (n=3) or striatal lysates of Atg5flox:Slc32a1-Cre KO mice (n=5), identified by global phosphoproteome analysis. The levels of phosphopeptides were normalized to their total protein levels. See also Dataset EV3.

(D) WebGestalt- based GO analysis of "cellular component"-enriched terms among significantly downregulated phosphopeptides (cut-off p<0.05, Fold change <-1.2) in Atg5flox:CamKIIα-Cre KO (left) and Atg5flox:Slc32a1-Cre KO (right) brains.

(E) Volcano plot of phosphopeptides identified using label-free phosphoproteome analysis of acute cortico-hippocampal slices from Atg5flox:CamKIIα-Cre WT mice treated either with DMSO or 50 µM Forskolin for 15 min (n=5). Phosphopeptides significantly upregulated (p<0.05) upon Forskolin treatment are highlighted in dark gray.

(F) Heat map representation of LFQ intensities of common phosphopeptides, identified in (E) and containing putative PKA phosphorylation motif (predicted by Perseus software) in Atg5flox:CamKIIα-Cre WT and KO acute slices, treated either with DMSO or 50 µM Forskolin for 15 min (n=5).

(G) Venn diagram showing the number of phosphopeptides significantly downregulated in Atg5flox:CamKIIα-Cre KO and Atg5flox:Slc32a1-Cre KO brains (cut-off p<0.05, Fold-change<-1.2) and the phosphophopeptides, which were "irresponsive" to Forskolin treatment in Atg5flox:CamKIIα-Cre KO acute slices (p>0.05).

(H) Top three enriched "cellular component" terms obtained by GO analysis of common phosphopeptides identified in (G).

(I) Fluorescence lifetime images of AKARet-PSD at spines of ATG5 WT and KO cortico-hippocampal neurons before and 4 min after Forskolin and IBMX administration. Scale bar: 1µm. Drawn contours represent single spines of WT or KO neurons.

(J) Averaged timecourses of fluorescence lifetime change of AKARet-PSD in WT and ATG5 KO spines (WT6 min: -0.053±0.018, KO6 min: 0.009±0.015; p=0.026, two-tailed unpaired t-test. nWT: 50/5, nKO: 58/6 (spines/neurons) from n=3 independent experiments.

(K-M) Representative STED images and Pearson's colocalization coefficient analysis of SHANK3 (K) and CASKIN1 (L) and PSD95 in Atg5flox:CamKIIα-Cre:Ai9 WT/KO neurons. Scale bar: 5µm, 1µm (small panels). WTSHANK3: 0.324±0.022, KOSHANK3: 0.415±0.024; p=0.0067, two-tailed unpaired t-test. nWT=46, nKO=46. WTCASKIN1: 0.255±0.024, KO CASKIN1: 0.326±0.025; p=0.0432, two-tailed unpaired t-test. nWT=39, nKO=43 from n=2 independent experiments.

Data information: *P < 0.05; **P < 0.01; ***P < 0.001. All data represent mean ± SEM. All n represent biological replicates.