Cell-type-specific regulation of neuronal intrinsic excitability by macroautophagy

Abstract

The basal ganglia are a group of subcortical nuclei that contribute to action selection and reinforcement learning. The principal neurons of the striatum, spiny projection neurons of the direct (dSPN) and indirect (iSPN) pathways, maintain low intrinsic excitability, requiring convergent excitatory inputs to fire. Here, we examined the role of autophagy in mouse SPN physiology and animal behavior by generating conditional knockouts of Atg7 in either dSPNs or iSPNs. Loss of autophagy in either SPN population led to changes in motor learning but distinct effects on cellular physiology. dSPNs, but not iSPNs, required autophagy for normal dendritic structure and synaptic input. In contrast, iSPNs, but not dSPNs, were intrinsically hyperexcitable due to reduced function of the inwardly rectifying potassium channel, Kir2. These findings define a novel mechanism by which autophagy regulates neuronal activity: control of intrinsic excitability via the regulation of potassium channel function.

Keywords: autophagy; basal ganglia; cell biology; intrinsic excitability; mouse; neuronal activity; neuroscience; potassium channel; striatum.

Figure 1.. Specific loss of Atg7-mediated autophagy in dSPNs or iSPNs in the absence of neurodegeneration in dSPN^Atg7cKO mice and iSPN^Atg7cKO, respectively.

(A) Schematic representation of the role of Atg3, 5, 7, 8, 10, and Atg12 in a cascade leading to autophagosome formation. (B) Schematic of Atg7 locus in Atg7^Fl/Fl mice and following Cre-mediated recombination. (Adapted from Komatsu et al., 2006). (C) Immunofluorescent images of striatal sections from Atg7^Fl/Fl (Control), D1-cre Atg7^Fl/Fl (dSPN^Atg7cKO) or A2Acre-Atg7^Fl/Fl (iSPN^Atg7cKO) mice. (D) p62⁺ cells per field in control, dSPN^Atg7cKO or iSPN^Atg7cKO mice. Control: N=6 mice, dSPN^Atg7cKO: N = 6 mice, iSPN^Atg7cKO: N=4 mice. Data analyzed by one-way ANOVA, F_(2,13)=65.73, p = 0.001. (E-F) Number of (E) D1-tomato+ or (F) D1-Tomato-, p62⁺ cells in dSPNAtg7cKO and iSPN^Atg7cKO mice. N = 6 dSPN^Atg7cKO mice and N=4 iSPN^Atg7cKO mice. Data in (E-F) were analyzed by two-tailed unpaired t test. (E) t₈=8.816, p = 0.0001. (F) t₈=24.94, p = 0.0001. (G-H) There were no differences in NeuN⁺ [Control: N = 3 mice, dSPN^Atg7cKO: N = 3 mice, iSPN^Atg7cKO: N = 3 mice; analyzed by one-way ANOVA F_(2,6) = 1.019, p = 0.4160] or D1-tomato+ cells per field [Control: N=6 mice, dSPNAtg7cKO: N = 6 mice, iSPNAtg7cKO: N=4 mice; analyzed by one-way ANOVA F(2,13) = 0.3144, p = 0.7356]. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, † p<0.0001.

Figure 2.. Atg7 in SPNs is required for motor performance and learning.

(A) Nine-week-old male and female dSPN^Atg7cKO mice, but not iSPN^Atg7cKO mice, weigh less than controls. Data were analyzed by two-way ANOVA followed by Bonferroni post-hoc test. Sex x Genotype: F_(2,111) = 0.1073, p = 0.8983; Genotype: F_(2,111) = 17.58, p<0.0001; Sex: F_(1,111) = 87.65, p<0.0001. (B) Both dSPN^Atg7cKO and iSPN^Atg7cKO mice have a lower latency to fall off the accelerating rotarod. Control n = 50, dSPN^Atg7cKOn = 34, iSPN^Atg7cKOn = 23. Data were analyzed by two-way ANOVA followed by Bonferroni post-hoc test. Trial x Genotype: F_(4,208) = 6.198, p<0.0001; Trial: F_(2,208) = 26.34, p<0.0001; Genotype: F_(2,104) = 20.73, p<0.0001. (C) iSPN^Atg7cKO mice, but not dSPN^Atg7cKO, demonstrate locomotor hyperactivity in the open field arena. One-way ANOVA followed by Bonferroni post-hoc test. (D) Time course of locomotor activity in the open field. Control n = 32, dSPN^Atg7cKOn = 15, iSPN^Atg7cKOn = 14. Data were analyzed by two-way ANOVA followed by Bonferroni post-hoc test. Time x Genotype: F_(8,240) = 2.547, p = 0.0111; Time: F_(4,240) = 21.99, p<0.0001; Genotype: F_(2,60) = 6.270, p = 0.0034. (E) Automated scoring of stereotypies in the open field. Control: n = 15; iSPN^Atg7cKO: n = 16. Data analyzed by two-tailed, unpaired t test. t₂₉ = 2.994, p = 0.0056. (F) Manual scoring of grooming bouts over thirty minutes following habituation during a separate session. Control: n = 23; iSPN^Atg7cKO: n = 26. Data analyzed by two tailed, unpaired t test. t₄₇ = 3.623, p = 0.0007. See Table 1 for detailed statistics split by sex. ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, † p<0.0001.

Figure 3.. Atg7 contributes to synaptic function and dendritic complexity in dSPNs but not iSPNs.

(A) Sample dendritic trees of dSPNs from control of dSPN^Atg7cKO mice. Reconstructions of neurobiotin filled neurons (left) and dendritic segment (right). (B) Sample dendritic trees from reconstructed iSPNs in control or iSPN^Atg7cKO mice. Left: reconstructed dendritic tree, scale bar 100 μm. Right: dendritic segment, scale bar 1 μm. (C) Cumulative dendritic length is significantly reduced in dSPNs from dSPN^Atg7cKO mice compared to control (left) but not in iSPNs from iSPN^Atg7cKO mice compared to control (right). dSPN^Ctrl: n = 22 cells, five mice, dSPN^cKO: n = 12,4. iSPN^Ctrl: n = 12,3; iSPN^cKO: n = 16,3. (D) Sholl analysis reveals a significant reduction in dendritic complexity in dSPNs from dSPN^Atg7cKO mice compared to control (left) but not in iSPNs from iSPN^Atg7cKO mice compared to control (right). dSPN^Ctrl: n = 22, 5, dSPN^cKO: n = 12,4. iSPN^Ctrl: n = 13,3; iSPN^cKO: n = 16,3. (E) Dendritic spine density on dendritic segments 50–100 µm from the soma. dSPN^Ctrl: n = 15, 5, dSPN^cKO: n = 13,4. iSPN^Ctrl: n = 8,3; iSPN^cKO: n = 8,3. (F) Representative traces of mEPSCs in dSPNs (top) and iSPNs (bottom). (G-H) A significant reduction in (G) mEPSC frequency and (H) mEPSC amplitude in dSPNs from dSPN^Atg7cKO mice compared to control but no difference in iSPN mEPSC frequency or amplitude between genotypes. Frequency: dSPN^Ctrl: n = 21, 5, dSPN^cKO: n = 25,4. iSPN^Ctrl: n = 22,3; iSPN^cKO: n = 26,4. Amplitude: dSPN^Ctrl: n = 19, 5, dSPN^cKO: n = 26,4. iSPN^Ctrl: n = 23,5; iSPN^cKO: n = 26,4. (I) Representative traces of mIPSCs in dSPNs (top) and iSPNs (bottom) (J-K) No difference in mIPSC frequency or amplitude after loss of autophagy in either dSPNs or iSPNs. Frequency: dSPN^Ctrl: n = 17, 5, dSPN^cKO: n = 23,4. iSPN^Ctrl: n = 20,5; iSPN^cKO: n = 25,4. Amplitude: dSPN^Ctrl: n = 17,5, dSPN^cKO: n = 22,4. iSPN^Ctrl: n = 21,5; iSPN^cKO: n = 24,4. See Table 2 for detailed statistics. ns p>0.05, *p<0.05, **p<0.01, † p<0.0001.

Figure 4.. Loss of Atg7 leads to intrinsic hyperexcitability due to reduced Kir2 currents in iSPNs.

(A) Representative current clamp traces in iSPNs from control or iSPN^Atg7cKO mice. (B-F) iSPNs lacking Atg7 display (B) depolarized resting membrane potential (t₇₀=3.617, p = 0.0006; iSPN^Ctrl: n=34 (8), iSPN^Atg7cKO: n=38 (9)), (C) elevated input resistance (t₆₈=3.630, p = 0.0005; iSPN^Ctrl: n=32 (8), iSPN^Atg7cKO: n=38 (9)), (D) decreased rheobase (t₆₁=4.456, p<0.0001; iSPN^Ctrl: n=26 (7), iSPN^Atg7cKO: n=37(9)), (E) no change in capacitance (t₆₇ = 0.8096, p = 0.4210; iSPN^Ctrl: n=33 (8), iSPN^Atg7cKO: n=39 (9)) and (F) a left-shifted current-response curve [APs/500 msec (Current x Genotype: F_(13,772) = 1.538, p = 0.0983; Current F_(13,772)=421.5, p<0.0001; Genotype: F_(1,772) = 6.586, p = 0.0141); iSPN^Ctrl: n=25(7), iSPN^Atg7cKO: n=36(8)]. Data in (B-F) analyzed with a two-tailed, unpaired t test. Data in (F) analyzed with two-way repeated measures ANOVA. (G) Representative voltage clamp recordings of Kir2 currents. (H) iSPNs have lower Kir2 current density in iSPN^Atg7cKO mice compared to iSPNs in iSPN^Ctrl mice. iSPN^Ctrl: n = 17(5), iSPN^Atg7cKO: n=25(6). Data analyzed with a two-way repeated measures ANOVA, followed by Bonferooni post-hoc test. Voltage x Genotype: F_(10,400) = 13.39, p<0.0001. ns p>0.05, * p<0.05, *** p<0.001, † p<0.0001.

Figure 4—figure supplement 1.. Atg7 is required for maximal Kir2 current but does not affect Kir2 voltage dependence.

(A–C) Representative traces of voltage clamp recordings of iSPNs in (A) ACSF, (B) ACSF + BaCl₂, and (C) BaCl₂-sensitive (Kir2) currents. (D-F) Reduction in Ba²⁺-sensitive current Kir2 current, but not the Ba²⁺-resistant leak current, in iSPNs lacking autophagy. iSPN^Ctrl: N = 19,5; iSPN^Atg7cKO: N = 25,6. (D) Data analyzed with repeated measure two-way ANOVA. Voltage x Genotype: F_(10,400) = 13.31, p<0.0001. (E) Data analyzed with repeated measure two-way ANOVA. Voltage x Genotype: F_(10,400) = 1.852, p = 0.0503; Voltage: F_{(10, 400)}=250.1, p<0.0001; Genotype: F_(1,40)=2.942, p = 0.0941. (F) Data analyzed with repeated measure two-way ANOVA. Voltage x Genotype: F_(10,400) = 17.33, p<0.0001. (G) No difference in the voltage dependence or (H) voltage at half-maximal activation for Kir2 currents between genotypes. (G) Data analyzed with repeated measure, two-way ANOVA. Voltage x Genotype: F_(10,400) = 0.8522, p = 0.5785; Voltage: F_(10,400)=2599, p<0.0001; Genotype: F_(1,40) = 0.01361, p = 0.9077. (I) Slope of Kir2 current is greater in iSPN^Control than iSPN^Atg7cKO cells. Data in (H-I) analyzed with a two-tailed, unpaired t test. Data in D-G were analyzed with a repeated measures two-way ANOVA followed by Bonferroni post-hoc tests. Data in H and I were analyzed with a two-tailed unpaired t test. ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, † p<0.0001.

Figure 5.. Autophagy is required for Kir2 degradation.

(A–C) There was no difference in Kir2.1 or Kir2.3 mRNA expression in SPNs of iSPN^Atg7cKO mice in an RNAscope assay (N = 77–173 cells from three mice per group). Inset shows just RNAscope and DAPI. Scale bar 30 µm. Data analyzed by the Kolmogorov-Smirnov test, Genotype effect: p>0.05 for Kir2.1 and Kir2.3. (D) Representative western blots of specified proteins from total striatal lysates from iSPN^Ctrl or iSPN^Atg7cKO mice. Quantifications of (E) p62 (t₂₀ = 3.551, p = 0.0020), (F) DARPP32 (t₂₃ = 1.984, p = 0.0593), (G) Kir2.1 (t₁₄ = 3.435, p = 0.0040), and (H) Kir2.3 (t₁₄ = 4.492, p = 0.0005) relative to actin. p62: iSPN^ctrl: n = 11, iSPN^Atg7cKO: n = 11. DARPP32: iSPN^ctrl: n = 14, iSPN^Atg7cKO: n = 11. Kir2.1: iSPN^ctrl: n = 7, iSPN^Atg7cKO: n = 9. Kir2.3: iSPN^ctrl: n = 7, iSPN^Atg7cKO: n = 9. Data analyzed by two-tailed, unpaired t test. (I-J) Kir2.1 is localized in LC3-GFP+ puncta in Atg5^WT but not Atg5^KO MEFs. BafilomycinA1 (BafA1; 100 nM 2 hr) treatment increases the number of LC3/Kir2.1-double labeled puncta in Atg5^WT MEFs. Scale bar 20 µm. Inset scale bar 1 µm. Analyzed by one-way ANOVA followed by Bonferroni post-hoc test. F_(2,26)=25.64, p<0.0001. (K-L) A reduction of Lamp1⁺Kir2.1⁺ puncta in Atg5^KO MEFs. Scale bar 20 µm, inset scale bar 1 µm. Data analyzed by two-tailed, unpaired t test. t₂₃ = 3.083, p = 0.0053. (M) Reduced degradation of SNAP-tag labeled Kir2.1 in Atg5^KO MEFs. N: (WT,KO): T = 0 min (35,30), T = 30 min (30,27), T = 60 min (25,38), T = 180 min (33,67), T = 360 min (40,20). Data analyzed by two-way ANOVA. Genotype x time: F_4,335 = 7.880, p<0.0001. (N) No significant difference in the internalization of antibody-labeled surface Kir2.1 channels in Atg5^KO MEFs. N: (WT,KO): T = 0 min (76,56), T = 30 min (52,37), T = 60 min (41,42), T = 120 min (28,38). Data analyzed by two-way ANOVA. Genotype x time: F_3,362 = 0.8038, ns; Time: F_(3,362) = 25.88, p = 0.0001; Genotype: F_(1,362) = 1.877, ns. ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, † p<0.0001.

Figure 5—figure supplement 1.. Loss of autophagy does not affect Kir2.1 expression in dSPNAtg7cKO mice.

(A) Representative western blots from control or dSPN^Atg7cKO mice. (B-G) Quantification of relative protein levels in control and dSPN^Atg7cKO mice shows no significant difference between (B) Kir2.1 [dSPN^Ctrl: n = 10, dSPN^Atg7cKO: n = 11; t₁₉ = 0.5816, p = 0.5677], (C) Kir2.3 [dSPN^Ctrl: n = 12, dSPN^Atg7cKO: n = 10; t₂₀ = 0.08775, p = 0.9309], (D) Kv1.2 [dSPN^Ctrl: n = 10, dSPN^Atg7cKO: n = 11; t₁₉ = 0.04627, p = 0.9636], (E) PSD95 [dSPN^Ctrl: n = 11, dSPN^Atg7cKO: n = 12; t₂₁ = 0.3730, p = 0.7129], or (G) DARPP32 [dSPN^Ctrl: n = 6, dSPN^Atg7cKO: n=5; t₉ = 0.6178, p = 0.5520], but a significant increase in (F) p62 [dSPN^Ctrl: n = 11, dSPN^Atg7cKO: n = 11; t₂₀=6.936, p<0.0001]. (H) Representative western blots for Kv1.2 and PSD95 in control or iSPN^Atg7cKO mice. (I-J) Quantification of Kv1.2 [iSPN^Ctrl: n = 11, iSPN^Atg7cKO: n = 11; t₂₀ = 0.3213, p = 0.7513] and PSD95 [iSPN^Ctrl: n = 12, iSPN^Atg7cKO: n = 11; t₂₁ = 1.264, p = 0.2202] shows no difference between control and iSPNAtg7cKO mice. Data analyzed by two-tailed unpaired t test. ns p>0.05, *** p<0.0001.

Figure 5—figure supplement 2.. Higher steady-state levels of Kir2.1 and reduced degradation of Kir2.1 in Atg5^KO MEFs.

(A) Schematic of Kir2.1-externalHA-Flag-SNAP (abb. Kir2.1 below) construct. (B) Representative western blots and (C) quantification for Flag, Kir2.1, LC3B and actin show elevated steady state levels of Kir2.1 in transiently transfected in Atg5^KO MEFs compared to Atg5^WT MEFs. N = 3 per genotype. Data analyzed with two-tailed unpaired t test, t₄ = 3.390, p = 0.0275. (D and E) Representative blots and aggregate data from six independent replicates per timepoint treated with cycloheximide at t = 0 min. Data were analyzed with a two-way ANOVA followed by Bonferroni post-hoc test. Time x genotype: F_(4,44)=2.764, p = 0.0391. ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, † p<0.0001.

Figure 5—figure supplement 3.. Kir2.1 degradation is disrupted in Atg7^KO MEFs.

(A) Representative western blot showing the absence of Atg7 in Atg7^KO primary MEFs. (B) Representative images of primary MEFs transfected with Kir2.1-Flag-SNAP and labeled with SNAPcell ligand. (C) Snap-labeled Kir2.1 is significantly reduced in Atg7^WT cells but not Atg7^KO cells. N: (0,120 min) WT: (7,6), KO: (7,6). Data analyzed by two-way ANOVA. Genotype x time: F_(1,22)=2.323, p = 0.1417; Genotype: F_(1,22)=2.323, p = 0.1417. Time: F_(1,22)=7.675, p = 0.0112. (D) The distribution of Kir2.1 is not different between Atg7^WT and Atg7^KO cells. Data are combined from two independent replicates. *p<0.05.

Figure 6.. Atg7 reexpression but not Kir2.1 overexpression rescues changes in iSPN physiology.

(A) Immunofluorescent images of striatal hemisections from mice injected with AAV1-DIO-mCherry or AAVDJ-DIO-Kir2.1-t2A-zsGreen stained against mCherry (left) or GFP (right). Scale bar 500 µm. (B) Significant effect of Kir2.1 overexpression on Kir2 current density in dSPNs from control and dSPNs from dSPN^Atg7cKO mice. Data analyzed with two-way ANOVA. Genotype x virus: F_(1,47)=0.01278, p = 0.9105; Genotype: F_(1,47)=0.01840, p = 0.8937; Virus: F_(1,47) = 13.23, p=0.0007. N = dSPN^Ctrl (D1cre Atg7^wt/wt, mCh: n = 13(4), Kir2.1: n = 14(4)) or dSPN^Atg7cKO (D1cre Atg7^Fl/Fl, mCh: n = 13(4), Kir2.1: n = 11 (3)). (C) Significant effect of Kir2.1 overexpression on Kir2 current density in iSPNs from control but not from iSPN^Atg7cKO mice. Data analyzed with two-way ANOVA. Genotype x virus: F_(1,54) = 17.62, p = 0.0001. N = iSPN^Ctrl (A2Acre Atg7^wt/wt, mCh: n = 9 (3), Kir2.1: n = 14(3)) or iSPN^Atg7cKO (A2Acre Atg7^Fl/Fl, mCh: n = 18(5), Kir2.1: n = 17(4)). (D) Representative hemisections from iSPN^Atg7cKO mice injected with AAV1-DIO-mCherry (left) or AAVDJ-Flex-Atg7-2A-ZsGreen (right) showing reduction in p62 puncta in cells expressing Atg7 (right) compared to mCherry (left). Scale Bar 500 µm. Inset, scale bar 20 µm. (E) Representative current clamp traces from mCherry-expressing iSPN in a control (A2Acre) mouse, mCherry expressing iSPN in an iSPN^Atg7cKO mouse or an Atg7-expressing iSPN from an iSPN^Atg7cKO mouse. (F-J) Reexpression of Atg7 normalizes RMP [N = iSPN^Ctrl: mCh: n = 25(5), Kir2.1: n = 26(5)) or iSPN^Atg7cKO: mCh: n = 13(3), Kir2.1: n = 15(3). Genotype x virus: F_(1,76)=9.491, p = 0.0029], rheobase [N = iSPN^Ctrl: mCh: n = 25(5), Kir2.1: n = 26(5)) or iSPN^Atg7cKO: mCh: n = 14(3), Kir2.1: n = 15(3). Genotype x virus: F_(1,75)=36.54, p<0.0001], R_in [N = iSPN^Ctrl: mCh: n = 25(5), Kir2.1: n = 26(5)) or iSPN^Atg7cKO: mCh: n = 13(3), Kir2.1: n = 16(3). Genotype x virus: F_(1,76)=55.55, p<0.0001], and Kir2 current in iSPNs from iSPN^Atg7cKO mice compared to mCherry control but only reduces R_in in controls. All data analyzed by two-way ANOVA. In (I), N = iSPN^Ctrl: mCh: n = 9 (5), Kir2.1: n = 9 (5). Voltage x virus: F_(10,160)=0.1170, p = 0.9996; Voltage: F_(10,160) = 178.8, p<0.0001; Virus: F_(1,16)=0, p = 0.9929. In (J), N = iSPN^Atg7cKO: mCh: n = 10(3), Kir2.1: n = 12(3). Genotype x virus: F_(10,200) = 18.56, p<0.0001. ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, † p<0.0001.

Figure 7.. Autophagy is not required for Kir2 trafficking.

(A) Sample traces from MEFs transfected with Kir2.1 demonstrate an inwardly rectifying, barium sensitive current that is absent in untransfected cells. (B) Atg5^KO MEFs have reduced Kir2.1 current compared to Atg5^WT MEFs. WT: N = 8, KO: N = 12. Voltage step x genotype: F_{(14, 252)}=8.985, p<0.0001. (C) Representative micrographs of Atg5^WT and Atg5^KO MEFs. On right, the Kir2.1 channel in the WT image has been contrasted to compare staining pattern. Scale bar 20 µm. (D-F) Elevated levels of both surface and total levels of Kir2.1 in Atg5^KO MEFs. Scale bar 20 µm, inset scale bar 1 µm. (E) t₁₄₇=4.511, p<0.0001. (F) t₁₄₇=4.511, p<0.0001. (G-J) Subcellular fractionation reveals elevated levels of Kir2.1 and Kir2.3 in all fractions of iSPN^Atg7cKO mice compared to controls but no change in the relative distribution of Kir2.1 or Kir2.3 between genotypes. (H) p62 is elevated in the total homogenate of iSPN^Atg7KO mice. (I) No change in distribution or level of Kv1.2 between genotypes. iSPN^Ctrl: N = 5. iSPN^Atg7cKO: N = 3. TH, total homogenate; PNS, post-nuclear supernatant; S2, 20,000xg supernatant; P2, 20,000xg pellet; P3, resuspended P2 spun at 100,000xg; SPM, synaptic plasma membranes isolated from 1.0M and 1.2M sucrose interface. Data were analyzed by two-way ANOVA for each analyzed protein with fraction and genotype as factors. No significant interaction between fraction and genotype was found for any protein. Fraction was significant for each protein. Genotype was only significant for Kir2.1 and Kir2.3. Kir2.1: Genotype: F_(1,6)=9.373, p = 0.0222. Kir2.3: Genotype: F_(1,6)=6.615, p = 0.0422. See Table 1 for detailed statistics. ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, † p<0.0001. Experiments in B, E, and F were combined from at least three independent experiments.

Figure 7—figure supplement 1.. Plasma membrane floatation and MBCD treatment.

(A) Schematic of iodixanol gradient. (B,D) representative blots of gradient fractions. (C) Aggregate data of Kir2.1 in each fraction normalized to the total amount of Kir2.1 across the gradient. No difference in Kv1.2 levels or distribution and no difference in Kir2.1 distribution was seen. N = 3 mice per genotype. Data analyzed by repeated measure, two-way ANOVA. Genotype x fraction: F_(8,48) = 0.3456, p = 0.9433; Genotype: F_(1,6)=2.455, p = 0.1682; Fraction: F_(8,48) = 16.28, p<0.0001. (E) Kir2 currents from iSPNs from slices pretreated with methyl-β-cyclodextrin (MBCD; 10 mM) were recorded. No effect of MBCD was seen at either genotype. iSPN^Ctrl: Veh N = 8, MBCD N = 6. iSPN^Atg7cKO: Veh N = 5, MBCD N = 7 (Cells from two mice per condition).

Figure 8.. Hyperacetylation of Kir2.1 at K334 in the absence of autophagy inhibits channel activity.

(A) Immunoprecipitation of Kir2.1 reveals elevated levels of acetylated lysines on Kir2.1 in Atg5^KOMEFs without a change in ubiquitination. Representative blots shown from at least three independent replicates. (B) A conserved motif in the C-terminal tail of Kir2.1 and Kir2.3 contains three modifiable lysines and has previously been implicated in Kir2 channel degradation. (C) A degradation screen in which K334, K338 and K346 were mutated to the unmodifiable residue, arginine, reveals that K334 is required for Kir2.1 degradation in Atg5^WT MEFs. N: (T=0min, T = 120 min) WT (52,79), K334R (27,51), K338R (40,41), K346R (23,11). Data analyzed by two-way ANOVA followed by Bonferroni post-hoc test. Kir genotype x time: F_(3,390) = 3.211, p = 0.0230. (D-E) Kir2.1 K334R current is normalized in Atg5^KO MEFs but does not affect Kir2.1 current in Atg5^WT MEFs. Data analyzed by two-way ANOVA followed by Bonferroni post-hoc test. Cell genotype x Kir genotype: F_(1,42) = 9.603, p = 0.0035. (F-G) Kir2.1 K334Q, with an acetylation-mimic at K334, has reduced current in Atg5^WT MEFs. Voltage step protocol is the same as in (D). Data analyzed by two-tailed unpaired t test. t₂₄=2.707, p=0.0123. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, † p<0.0001. Experiments in C, E and G were combined from at least three independent experiments.

Figure 8—figure supplement 1.. Reduced acetylation of Kir2.1 K334R in Atg5^KO MEFs but acetylation status at K334 does not affect surface residence of Kir2.1.

(A) Representative western blots of immunoprecipitated WT Kir2.1^{extHA-Flag-SNAP} or Kir2.1^{extHA-Flag-SNAP} K334R. Immunoblots for acetylated lysines (AceK) or Flag. (B) Quantification of acetylated lysine signal normalized to WT or K334R Kir2.1^{extHA-Flag-SNAP} in immunoprecipiates from Atg5^KO MEFs shows a reduction in total Kir2.1 acetylation when K334 is mutated to arginine. Data analyzed with two-tailed, unpaired t test. t₁₀ = 2.452; p = 0.0341. (C) Surface staining for WT Kir2.1, Kir2.1 K334R, and Kir2.1 K334Q in Atg5^WT or Atg5^KO MEFs reveals that mutations at K334 does not affect surface localization of Kir2.1 in either MEF cell line. Data analyzed with two-way ANOVA. Cell genotype x Channel genotype: F_(2,142)=0.04505, p = 0.9560; Cell genotype: F_(1,142)=75.66, p<0.0001; Channel Genotype: F_(2,142)=0.6539, p = 0.5216. *p<0.05, † p<0.0001.

Author response image 1.. Similar numbers of p62+ cells in dSPN^Atg7cKO and iSPN^Atg7cKO striatum at P28.

Data were analyzed with a one-way ANOVA followed by a Bonferroni post-hoc test.

Author response image 2.. No change in mEPSC frequency or amplitude in iSPNs from dSPN^Atg7cKO mice.

mEPSCs were recorded from iSPNs in dSPN^Atg7cKO mice and compared to control iSPNs. We show here that iSPNs from dSPN^Atg7cKO mice have equivalent (A) mEPSC frequency and (B) mEPSC amplitude as iSPNs from control mice. Data from dSPNs arising from control or dSPN^Atg7cKO mice are reproduced here for reference from Figure 3. Control iSPN data, also reproduced from Figure 3. Data are analyzed by two-way ANOVA followed by Bonferroni posthoc test. (A) Subtype x Genotype: F_(1,75)=11.57, p=0.0011. (B) Subtype x Genotype: F_(1,77)=4.444, p=0.0383. iSPNs from dSPN^Atg7cKO mice: n=11,3. N for other group are the same as in Figure 3.

Author response image 3.. No change in paired-pulse ratio (PPR) of excitatory inputs to dSPNs from dSPN^Atg7cKO mice.

(A) Representative traces of evoked EPSCs in dSPNs from control or dSPN^Atg7cKO mice. EPSCs were evoked in the presence of 25 μM picrotoxin following electrical stimulation within the striatum >100μm from the cell body. (B) No effect of loss of dSPN autophagy on the paired pulse ratio of evoked EPSCs. Control: n=7,2; dSPN^Atg7cKO: n=11,3. Latency of EPSC is between 5-10 ms in all cells examined.

Author response image 4.. Increasing cellular PIP₂ levels does not rescue Kir2 currents in Atg7-deficient iSPNs.

(A) diC8-PIP2 (50 μM) was included in the intracellular solution. Percent change in Kir2 currents after 12 minutes of dialysis with the internal solution ((Lieberman et al., 2018). No effect of diC8-PIP2 was found in either genotype. Data analyzed with two-way ANOVA followed by Bonferroni test. Genotype x PIP2: F_(1,29)=1.124, p=0.2978; Genotype: F_(1,29)=0.8626, p=0.3607; PIP2: F_(1,29)=0.03962, p=0.8436. Each condition arose from >3 mice each.

Author response image 5.. Motor learning differences between control, dSPN^Atg7cKO and iSPN^Atg7cKO mice.

The difference in latency to fall between trial 3 and trial 1 were plotted for each mouseincluded in Figure 2B. Data was analysed with a one-way ANOVA followed by a Bonferroni post-hoc test.

Author response image 6.. Specificity of Kir2.1 antibody.

Lysates were generated from Atg5^WT and Atg5^KO MEFs, with or without transfection with Kir2.1^FlagSNAP and blotted for Flag, Kir2.1, Kir2.3, p62 and actin.