Autophagy regulates neuronal excitability by controlling cAMP/Protein Kinase A signaling at the synapse (2022)
Figure 4

Figure 4 | PKA R1α/β subunits of the PKA complex are degraded by autophagy in neurons.

(A) Western Blot analysis of PKA R1α protein levels in NSC34 cells, treated with 250 nM Torin1 or deprived of amino-acids and serum for 16 hours. Lysosomal degradation was blocked in the last 4 hours using 67 nM of Bafilomycin A (BafA).

(B) Starvation significantly reduced PKA R1α protein levels in NSC34 cells (vehicle set to 100%, starvation: 31.19± 5.085%, p<0.0001, one-tailed unpaired t-test). n=5. (C) Application of 67 nM BafA for 4 hours before harvesting was sufficient to stabilize the PKA R1α protein levels in starved NSC34 cells (starvation set to 100%, starvation+BafA: 155.5± 13.93%, p=0.002, one-tailed unpaired t-test). n=5.

(D, E) 16 hours starvation significantly increased intracellular cAMP level measured as mean intensity in MAP2- positive cultured primary neurons (Vehicle: 212.4±5.619, Starvation: 255.1±5.715; p<0.0001, two-tailed paired t-test). nVehicle=314, nStarvation=306 neurons from n=3.

(F, G) Phosphorylation state of proteins containing PKA substrate RRXS/T motif was increased upon 16 hours of amino-acid and serum starvation in primary cortico-hippocampal neurons at DIV14 and was suppressed by 1 µM H89 supplementation in the media (Vehicle set to 100%, Starvation: 326.60±94.53%, H89: 214.80±56.87%, p=0.033, one-way ANOVA with Dunn's multiple comparison test). n=4. The pPKA substrate levels were normalized to the total protein amount stained with Ponceau S.

(H,I) Treatment of NSC34 cells with BafA (67nM) for 6 hours significantly increased PKA R1α protein levels compared to the DMSO-treated group set to 100% (BafA: 198.9± 28.44%, p=0.003, one-tailed unpaired t-test). n=6.

(J) Western blot analysis of purified autophagosomes (AVs) (50μg AVs/lane) with and without Proteinase K (PK) treatment. TX-100 (1% final) was used as a positive control for the activity of PK. Synaptosome lysates (Syn, 30μg/lane) were used as a positive control for the signal of antibodies.

(K, L) Representative confocal images of the hippocampus CA1 area from Atg5flox:CamKIIα-Cre (K) and Atg5flox:Slc32a1-Cre (L) KO mice, immunostained for PKA R1-α/β and co-immunostained for p62 and/or NBR1. White rectangular boxes indicate areas magnified to the right. White arrows indicate p62/NBR1 inclusion bodies positive for PKA R1. Scale bar: large panel 15 µm, small panel: 5 µm. Representative pictures for WT controls are shown in suppl. Fig. 5D-F.

(M, N) Representative fluorescent images and subsequent analysis of PKA R1- α/p62 colocalization (Pearson's correlation coefficient) in primary cortico-hippocampal neurons, which have undergone 16 hours amino-acids and serum starvation and were additionally treated with BafA (67nM) to visualize the lysosomes (control: 0.68± 0.019, starvation: 0.58± 0.015, starvation+BafA: 0.83± 0.014; pcontrol vs starvation<0.0001, pcontrol vs starvation+BafA<0.0001, pstarvation vs starvation+BafA<0.0001 Ordinary one-way ANOVA with Tukey's multiple comparison test). ncontrol=36, nstarvation=38, nstarvation+BafA=36 neurons from n=3. Scale bar: 5 µm.

(O, P) 3D-reconstruction of Atg5flox:CamKIIα-Cre KO soma (O), as well as the immunohistochemical profile and the 3D surface rendering of Atg5flox:CamKIIα-Cre KO neuropil (P) revealing the PKA R1-β/p62 colocalization in the soma (white arrows), but not in processes (yellow arrows). Scale bar: 10 µM.

(Q, R) Co-immunoprecipitation of endogenous PKA R1-β with PKA-Cα from Atg5flox:CamKIIα-Cre WT/KO mouse brain lysates (WT set to 100%, KO: 170.3± 14.88%, p=0.005, one-tailed unpaired t-test, n=3). Input, 1.5% of the total lysate was added to the assay.

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