Research ArticleNEUROSCIENCE

GABARAPs dysfunction by autophagy deficiency in adolescent brain impairs GABAA receptor trafficking and social behavior

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Science Advances  10 Apr 2019:
Vol. 5, no. 4, eaau8237
DOI: 10.1126/sciadv.aau8237
  • Fig. 1 Autophagy deficiency in forebrain GABAergic inhibitory or excitatory neurons resulted in an overlapping set of ASD-like behavioral deficits.

    (A) Atg7 conditional deletion by Dlx5-creERT2 and CaMKII-cre led to increased p62 levels in affected brain regions. (B) p62+ aggregate formation was observed in all major subclasses of cortical GABAergic interneurons of Dlx5-creERT2 Atg7 cKO mice. (C and D) Resident-intruder social interaction test showed deficits in both Dlx5-creERT2 and CaMKII-cre Atg7 cKO mice. n = 10 (Dlx5-control), n = 8 (Dlx5-cKO), n = 16 (CaMKII-control), n = 16 (CaMKII-cKO). (E and F) Three-chamber social interaction test revealed a difference in Dlx5-creERT2 and CaMKII-cre Atg7 cKO mice for social novelty (middle) but not social approach (left). A social interaction preference index was also calculated, which showed a statistical significant difference for social novelty across genotypes for CaMKII-cre but not Dlx5-creERT2 Atg7 cKO animals (right). n = 12 (Dlx5-control), n = 10 (Dlx5-cKO), n = 16 (CaMKII-control), n = 16 (CaMKII-cKO). (G and H) Deficits in nesting behavior were observed for both Dlx5-creERT2 and CaMKII-cre Atg7 cKO mice. n = 12 (Dlx5-control), n = 10 (Dlx5-cKO), n = 14 (CaMKII-control), n = 10 (CaMKII-cKO). (I and J) Elevated plus maze showed that both Dlx5-creERT2 and CaMKII-cre Atg7 cKO mice have increased anxiety. n = 5 (Dlx5-control), n = 4 (Dlx5-cKO), n = 14 (CaMKII-control), n = 16 (CaMKII-cKO). (K) Localized Atg7 deletion in mPFC excitatory neurons in 12-week-old Atg7flox/flox mice led to deficits in social behavior as measured by the resident-intruder social interaction test. n = 7 animals per genotype. (L and M) mPFC-specific autophagy deficiency in excitatory neurons did not affect nesting or anxiety-related behaviors. n = 7 animals per genotype. Data are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired two-tailed Student’s t test; §P < 0.05, between genotypes, two-way repeated-measures analysis of variance (ANOVA). n.s., not statistically significant.

  • Fig. 2 Mislocalization and altered conformational status of GABARAPs in Atg7 cKO brains.

    (A) Schematic diagram of SDD-AGE/SILAM experiment. (B) Abundance of proteins identified in high molecular weight fraction (>250 kDa) only in CaMKII-cre Atg7 cKO but not control brains by SDD-AGE/SILAM experiments (6-month-old mice). (C) Candidate proteins meeting the selection criteria are listed with SILAM scores representing relative abundance to SILAM-labeled sample. Selection criteria are depicted schematically in fig. S2B. Candidate proteins identified by these criteria at all three ages examined are specifically marked by asterisks. (D) GABARAPL2 and p62 were consistently identified by SDD-AGE/SILAM in CaMKII-cre Atg7 cKO brain homogenates at 3 months (left) and 12 months (right) of age. (E) p62-like accumulation of GABARAPs was observed in Dlx5-creERT2 and CaMKII-cre Atg7 cKO mice. (F) Native PAGE analysis revealed shifts of GABARAPL2 and GABARAP into high–molecular weight (MW) complexes in the brains of CaMKII-cre Atg7 cKO mice (cortex). (G to I) GABARAPs were observed to mislocalize to p62+ aggregates in affected neurons of Dlx5-creERT2 Atg7 cKO mice. Scale bars, 20 μm (G to I).

  • Fig. 3 Autophagy deficiency led to a reduction of surface GABAA receptors and increased GABA release by Atg7 cKO GABAergic interneurons.

    (A) Significant reductions in surface expression of GABAA receptors (GABAAR) were observed in striata of Dlx5-creERT2 Atg7 cKO mice (see fig. S3A). n = 4 animals per genotype. (B) mIPSCs recorded from CA1 pyramidal cells in Dlx5-creERT2 Atg7 cKO hippocampi showed a significant increase in frequency (left) but no differences in amplitude (right). Representative traces are shown at the bottom. n = 35 cells recorded from five different animals. Data are presented as means ± SEM. * P < 0.05, paired (A) or unpaired (B) two-tailed Student’s t test.

  • Fig. 4 GABAA receptor trafficking was disrupted by alterations of GABARAPL2 due to autophagy deficiency.

    (A) Atg7 deletion in cultured cortical GABAergic interneurons (DIV15) derived from the MGEs of Atg7flox/flox embryos followed by infection with lentivirus expressing Venus reporter with or without Cre recombinase. Disrupted LC3 processing and accumulations of p62 and GABARAPs were observed as a result of autophagy deficiency induced by Atg7 deletion. (B) A significant reduction of surface GABAA receptors was observed in Atg7 cKO cultured cortical GABAergic interneurons, whereas NMDA and AMPA receptors were unchanged in both total and surface fractions. n = 4 independent experiments using distinct preparations of primary cultured neurons. (C) Immunofluorescence under nonpermeable conditions showed a significant reduction of surface GABAA receptors in Atg7 cKO cultured cortical GABAergic interneurons. Venus expression (green fluorescence) was used as an indicator of infected neurons. n = 30 cells analyzed per genotype. (D) A significant increase in the frequency of spontaneous action potentials fired by Atg7 cKO cultured cortical GABAergic interneurons was observed compared to controls (top). n > 25 cells per genotype; data shown for n = 10 and 9 cells for control and Atg7 cKO, respectively, from three distinct preparations of primary cultured neurons, which fired spontaneous action potentials. Resting membrane potential was not altered by Atg7 deletion in cultured cortical GABAergic interneurons (bottom). n > 30 cells per genotype from three distinct preparations of primary cultured neurons. (E) Gabarapl2 knockdown in WT cultured cortical GABAergic interneurons resulted in a significant reduction of surface GABAA receptors. n = 3 independent experiments using distinct preparations of primary cultured neurons. (F) GABARAPL2-gephyrin interaction was significantly reduced in the brains of Dlx5-creERT2 (striatum) Atg7 cKO mice (see fig. S4D). Quantifications shown represent the amount of gephyrin coimmunoprecipitated normalized by total gephyrin and amount of GABARAPL2 immunoprecipitated from individual samples. n = 3 independent experiments using tissue homogenates from distinct animals. (G) GABARAPL2 was mislocalized to p62+ aggregates formed in Atg7 cKO cultured cortical GABAergic interneurons in a similar manner as in affected neurons of Atg7 cKO brains. (H) GABARAPL2 was observed by immunofluorescence to localize to the TGN marked by TGN38 in controls, but this localization was reduced in Atg7 cKO cultured cortical GABAergic interneurons as the protein became mislocalized into foci. Venus signal was reduced in merge images to allow better visualization of signals from other channels. (I) GABARAPL2 ΔN overexpression (O/E) reversed the reduction of surface GABAA receptors observed in Atg7 cKO cultured cortical GABAergic interneurons. n = 5 independent experiments using distinct preparations of primary cultured neurons. (J) Intracellular accumulations of GABAA receptors were observed in BIG-2+ structures of Atg7 cKO cultured cortical GABAergic interneurons but not in controls. Data are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, paired (B, E, F, and I) or unpaired (C) two-tailed Student’s t test or Kolmogorov-Smirnov test (D). Scale bars, 2 μm (C), 5 μm (G), 5 μm (H and J), and 2 μm (insets).

  • Fig. 5 Surface GABAA receptor expression depended on p62 levels.

    (A) p62 overexpression in WT cultured cortical GABAergic interneurons resulted in aggregates that recruited GABARAPL2 (see fig. S7, B and C). EGFP, enhanced green fluorescent protein. (B) p62 overexpression in WT cultured cortical GABAergic interneurons led to a significant reduction of surface GABAA receptors. n = 3 independent experiments using distinct preparations of primary cultured neurons. (C) Sqstm1 knockdown in Atg7 cKO cultured cortical GABAergic interneurons rescued the reduction in surface GABAA receptors. n = 5 independent experiments using distinct preparations of primary cultured neurons (D) mTOR hyperactivation via Tsc2 knockdown in WT cultured cortical excitatory neurons resulted in p62+ aggregates that sequestered GABARAPL2 (see fig. S7, E and F). (E) Tsc2 knockdown resulted in a significant reduction of surface GABAA receptors in WT cultured cortical excitatory neurons. n = 4 independent experiments using distinct preparations of primary cultured neurons. Data are presented as means ± SEM. *P < 0.05, **P < 0.01, paired two-tailed Student’s t test. Scale bars, 15 μm (A and D).

  • Fig. 6 Increased aggregation of GABARAPs in human ASD brain samples.

    (A to C) No significant difference was observed in total expression of GABARAPs between ASD and matched (age, sex, and ethnicity) control brains (BA40); however, significant increases in the detergent-insoluble fraction were observed for all three proteins. Insoluble p62 also showed a trend for increase in ASD brain samples. n = 22 pairs of ASD and matched controls. (D and E) A significant reduction in TSC2 expression was detected in ASD brain samples. n = 22 pairs of ASD and matched controls. (F to I) Negative correlations were observed between insoluble GABARAPs, insoluble p62, and TSC2 expression. No correlation with age was observed. Data are presented as means ± SEM. *P < 0.05, **P < 0.01, paired two-tailed Student’s t test.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/4/eaau8237/DC1

    Fig. S1. Atg7 deletion in forebrain GABAergic neurons by Dlx5-creERT2.

    Fig. S2. GABARAPs mislocalized to p62+ aggregates in affected neurons of Dlx5-creERT2 and CaMKII-cre Atg7 cKO mice.

    Fig. S3. Autophagy deficiency led to a reduction of surface GABAA receptors but no change in excitatory signaling.

    Fig. S4. Reduction of GABAA receptors in cortical GABAergic interneurons due to compromised functions of GABARAPL2 by autophagy deficiency.

    Fig. S5. Levels and localizations of BIG-2 and NSF were not affected by autophagy deficiency.

    Fig. S6. GABAA receptors are not substantially localized to endosomal and lysosomal compartments in cortical GABAergic interneurons and are not altered by autophagy deficiency.

    Fig. S7. Reduced surface GABAA receptor expression by manipulation of p62 levels.

    Table S1. shRNA sequences used in this study.

    Table S2. Antibodies used in this study.

    Table S3. Patient information for frozen postmortem human brain samples.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Atg7 deletion in forebrain GABAergic neurons by Dlx5-creERT2.
    • Fig. S2. GABARAPs mislocalized to p62+ aggregates in affected neurons of Dlx5-creERT2 and CaMKII-cre Atg7 cKO mice.
    • Fig. S3. Autophagy deficiency led to a reduction of surface GABAA receptors but no change in excitatory signaling.
    • Fig. S4. Reduction of GABAA receptors in cortical GABAergic interneurons due to compromised functions of GABARAPL2 by autophagy deficiency.
    • Fig. S5. Levels and localizations of BIG-2 and NSF were not affected by autophagy deficiency.
    • Fig. S6. GABAA receptors are not substantially localized to endosomal and lysosomal compartments in cortical GABAergic interneurons and are not altered by autophagy deficiency.
    • Fig. S7. Reduced surface GABAA receptor expression by manipulation of p62 levels.
    • Table S1. shRNA sequences used in this study.
    • Table S2. Antibodies used in this study.
    • Table S3. Patient information for frozen postmortem human brain samples.

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