Autophagy regulates neuronal excitability by controlling cAMP/Protein Kinase A signaling at the synapse (2022)
Figure 7

Figure 7 | Autophagy-dependent PKA signaling regulates synaptic GLUR1 localization and AMPAR function.

(A) g:Profiler-based analysis of GO molecular function and human phenotype enriched terms of "irresponsive" PKA target proteins in autophagy-deficient neurons identified in Fig. 6G (cut-off p<0.05, FC<-1.2). See also Dataset EV4.

(B) Representation of proteins from GO molecular function term "cytoskeletal protein binding" shown in (A). Highlighted in blue are proteins with a published function in GLUR1 function and trafficking.

(C,D) Analysis of GLUR1 fluorescent levels at the spines of 10-11 week-old Atg5wt/wt:Ai9 and Atg5flox/flox:Ai9 mice injected with CamKIIα-Cre AAV into the hippocampus. Scale bar: 5µm. GLUR1 level at PSD95-positve spines were assessed measuring mean gray values after background subtraction (WT: 64.55±7.988%, KO: 99.14±6.863%; p=0.0029, two-tailed unpaired t-test). nWT=13, nKO=14 images from n=3 mice (>31 dendritic spines analyzed per each mouse). sp, spine.

(E) AMIRA-based color coded 3D-reconstrution of GLUR1 localization in dendrites of excitatory neurons of Atg5wt/wt:Ai9 and Atg5flox/flox:Ai9 mice injected with CamKIIα-Cre AAV into the hippocampus. sp, spine. Scale bar: 5µm.

(F) Analysis of GLUR1 concentration in the dendritic spines versus shaft. The mean gray value of GLUR1 at spines and shafts was measured separately and the spine value was divided by the shaft. (WT: 0.778±0.030, KO: 1.012±0.046; p=0.0003, nWT: 13, nKO: 12 images from n=3 mice (>31 dendritic spines/ shaft analyzed per mice).

(G) Representative fluorescence images of Atg5flox:CAG-CreTmx WT/KO neurons transfected with eGFP or eGFP-ATG5 and immunostained for GLUR1, PSD95 and GFP. Scale bar: 5µm.

(H) The overexpression of eGFP-ATG5 in Atg5flox:CAG-CreTmx KO neurons diminished the of GLUR1/PSD95 colocalization compared to the eGFP-overexpressing KO neurons (KOeGFP: 70.07±1.473%, KOeGFP-ATG5: 65.42±1.721%,; p=0.048, two-tailed unpaired t-test). nKO+GFP=42, nKO+GFP-ATG5=50 images from n=5.

(I, J) The coexpression of eGFP-PKA R1α and eGFP-PKA R1β in cultured Atg5flox:CAG-CreTmx WT neurons results in significantly increased colocalization between GLUR1 and PSD95 compared to the eGFP-expressing neurons (WT: 42.30±2.453%, KO: 51.32±2.624%, p=0.014, two-tailed unpaired t-test). nWT+GFP=46, nWTGFP+PKA R1 OE=46 images from n=3. Analysis in H, J is performed using a unbiased custom-written Plugin for ImageJ.

(K-M) GCAMP7f responses to tetanic stimulation (four tetani, 100 APs at 100Hz, 3s interval) in cell bodies of CamKIIα-Cre-transduced primary cortico-hippocampal Atg5wt:wt/Ai9 (WT) and Atg5flox:flox/Ai9 (KO) neurons expressing mCamKIIα-jGCaMP7f and treated either with DMSO (K) or with 10 µM CNQX for 5 min (L). Autophagy deficient cells showed significantly increased facilitation of GCAMP7f signal to electrical stimulation (ΔF/Fpeak1) compared to the WT, a phenotype which was blocked by the application of CNQX (WTDMSO: 0.28± 0.12, WTCNQX: -0.07± 0.03, KODMSO: 3.74± 0.86, KOCNQX: -0.014± 0.13, p WT DMSO/ KO DMSO<0.0001, pKO DMSO/KO CNQX<0.0001, two-way ANOVA with Tukey's multiple comparisons test). nWT DMSO= 25, nWT CNQX= 27, nKO DMSO= 33, nKO CNQX= 30 neurons from n=3. See also Appendix Figure S9L for changes in calcium signals in dendrites.

(N, O) GCAMP7f responses to tetanic stimulation (four tetani, 100 APs at 100Hz, 3s interval) in primary cortico-hippocampal neurons expressing mCamKIIα-jGCaMP7f and treated either with DMSO or with 10 µM of H89 for 20 min (DMSO: 0.069± 0.042, H89: 1.668± 0.28, p <0.0001, unpaired two-tailed t-test). nDMSO=48, nH89=47 neurons from n=3 independent experiments.

(P) Example traces of the network activity of primary cortico-hippocampal CamKIIα-eGFP AAV (WT)/ CamKIIα-Cre-eGFP AAV (KO)-transduced Atg5flox:flox neurons analyzed using the MEA system.

(Q, R) Increased spike rate in ATG5 KO excitatory neurons is rescued by long-term application of Forskolin (10µM for 72 hours) (WTDMSO: 0.59± 0.29, WTForskolin: 1.35± 0.88, KODMSO: 1.27± 0.62, KOForskolin: 0.02± 0.001, pKO DMSO/KO Forskolin=0.027, unpaired two-tailed t-test). nWT DMSO=9, nWT Forskolin=8, nKO DMSO: 7, nKO Forskolin=10 MEA chambers from 3 mice.

(S, T) AMPAR-mediated sEPSCs in cortical pyramidal neurons of 12-week-old mice Atg5flox:flox mice, which were stereotactically injected either with CamKIIα-eGFP AAV (WT) or with CamKIIα-Cre-eGFP AAV (KO) into the perirhinal cortex layer II/III. (S) Amplitude of AMPAR sEPSC is increased in pyramidal KO neurons compared to the WT (WT: 7.75± 0.72 pA, KO: 9.79± 2.75 pA; nWT=11, nKO=9; unpaired mean difference between WT and KO (ΔWT-KO): 2.04 [95%CI: 0.629, 4.22]; pWT/KO=0.0152 (two-sided permutation t-test)). Inset: representative traces of a WT sEPSC (petrol) and a KO sEPSC (ochre). (T) Frequency of AMPAR sEPSC is increased in pyramidal KO neurons compared to the WT (WT: 5.27± 2.66 Hz, KO: 9.47± 5.16 Hz; nWT=11, nKO=9; ΔWT-KO: 4.19 [95%CI: 0.57, 7.56]; pWT/KO=0.0326 (two-sided permutation t-test)). Each data point represents the mean of a 1 min recording interval of an individual neuron. The filled curve indicates the resampled Δ distribution (5000 bootstrap samples), given the observed data. Horizontally aligned with the mean of the test group, the Δ is indicated by the black circle. The 95% confidence interval of the mean difference is illustrated by the black vertical line.

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