Autophagy regulates neuronal excitability by controlling cAMP/Protein Kinase A signaling at the synapse (2022)
Figure 2

Figure 2 | PKA R1α/β subunits of the PKA complex are upregulated in autophagy-deficient excitatory and inhibitory neurons in-vivo.

(A, B) Volcano plot of differentially expressed proteins in Atg5flox:CamKIIα-Cre KO cortical brain lysates (A) and Atg5flox:Slc32a1-Cre KO striatal brain lysates (B), analyzed using a SILAC-based proteomic approach (nAtg5flox:CamKIIα-Cre=3, nAtg5flox:Slc32a1-Cre=5 mice per genotype). Orange- and blue-color-coded circles indicate all protein deregulated at p<0.05 and log2 fold change of >1.

(C, D) Volcano plot of differentially expressed proteins in FACS-sorted Atg5flox:CamKIIα-Cre:Ai9 (C) and Atg5flox:Slc32a1-Cre:Ai9 (D) KO neurons, analyzed using label-free proteomic analysis (nAtg5flox:CamKIIα-Cre:Ai9=4, nAtg5flox:Slc32a1-Cre:Ai9= 5 mice per genotype). Orange- and blue-color-coded circles highlight highly deregulated proteins at p<0.05 and log2 fold change of >1. See also Dataset EV1.

(E) ShinyGO v0.741-based GO analysis of "cellular component" -enriched terms in the proteome (cut-off p<0.05, log2FC>0.5) of FACS sorted Atg5flox:CamKIIα-Cre:Ai9 KO and Atg5flox:Slc32a1-Cre:Ai9 KO neurons.

(F, G) PKA R1α (F) and PKA R1β (G) protein levels are significantly increased in cortical brain lysates from 13-week-old Atg5flox:CamKIIα-Cre KO mice compared to the WT set to 100% (KOPKA R1α: 286.1± 50.53%, p=0.005, KOPKA R1β: 194.7± 27.82%, p=0.007, one-tailed unpaired t-test). n=4 for each genotype.

(H, I) PKA R1α (H) and PKA R1β (I) protein levels are significantly increased in striatal lysates of Atg5flox:Slc32a1-Cre KO mice compared to the WT set to 100% (KOPKA R1α: 524.1± 8.35%, p<0.0001, KOPKA R1β: 247.9± 26.26%, p=0.0007, one-tailed unpaired t-test). n=4 for each genotype.

(J) Immunohistochemistry for PKA R1β on horizontal brain sections from Atg5flox:CamKIIα-Cre WT/KO and Atg5flox:Slc32a1-Cre WT/KO mice. White rectangular boxes indicate areas magnified to the right. Scale bars: 200 µM in large panels, 50 µM in small panels.

(K, L) Immunohistochemistry for PKA R1β and PKA R1α on horizontal brain sections from Atg5flox:CamKIIα-Cre:Ai9 (K) and Atg5flox:Slc32a1-Cre:Ai9 WT/KO (L) mice. White rectangular boxes indicate areas magnified to the right. Scale bars: 50µm, 25µm (small panels in K), 20µm (small panels in L). Drawn contours indicate representative WT or KO neuronal cell body.

(M, N) Immunohistochemistry analysis of PKA R1α levels in NeuN-posititve neurons in the hippocampus of Atg5flox:CamKIIα-Cre WT/KO mice. Scale bar: 50µm, 20µm (small panels). WT: 54.70±0.693, KO: 89.79±1.831; p<0.0001, two-tailed unpaired t-test. nWT= 64; nKO= 71 neurons from n=3 mice per genotype.

(O, P) Immunohistochemistry analysis of PKA R1α levels in GFAP-positive cells in the hippocampus of Atg5flox:CamKIIα-Cre WT/KO mice. Scale bar: 50µm, 30µm (small panels). WT: 63.86±2.477, KO: 61.71±2.653; p=0.5549, two-tailed unpaired t-test. nWT= 45; nKO= 40 cells from n=3 mice per genotype.

(Q, R) Immunohistochemistry analysis of PKA R1α in GFAP-positive cells in the hippocampus of Atg5flox:Slc32a1-Cre WT and KO mice. Scale bar: 50µm, 30µm (small panels). WT: 45.28±1.018, KO: 48.50±1.440; p=0.0636, two-tailed unpaired t-test. nWT= 61; nKO= 48 cells from n=3 mice per genotype.

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