Loss of mTOR-dependent macroautophagy causes autistic-like synaptic pruning deficits

Abstract

Developmental alterations of excitatory synapses are implicated in autism spectrum disorders (ASDs). Here, we report increased dendritic spine density with reduced developmental spine pruning in layer V pyramidal neurons in postmortem ASD temporal lobe. These spine deficits correlate with hyperactivated mTOR and impaired autophagy. In Tsc2 ± ASD mice where mTOR is constitutively overactive, we observed postnatal spine pruning defects, blockade of autophagy, and ASD-like social behaviors. The mTOR inhibitor rapamycin corrected ASD-like behaviors and spine pruning defects in Tsc2 ± mice, but not in Atg7(CKO) neuronal autophagy-deficient mice or Tsc2 ± :Atg7(CKO) double mutants. Neuronal autophagy furthermore enabled spine elimination with no effects on spine formation. Our findings suggest that mTOR-regulated autophagy is required for developmental spine pruning, and activation of neuronal autophagy corrects synaptic pathology and social behavior deficits in ASD models with hyperactivated mTOR.

Fig 1. Dendritic spine pruning in temporal lobe of ASD patients and controls

(A)Representative Golgi images for postmortem human temporal lobe (left, 10X, stitched from 9 separate image stacks), Layer V pyramidal neurons with basal dendritic tree (upper middle, 20X, pseudocolored in red; lower middle, 40X, pseudocolored in green; scale bar: 50 μm). The right four panels (100X, scale bar: 5 μm) are representative images of proximal basal dendritic segments from 2 control subjects (C, aged 8 yrs and 18 yrs) and 2 ASD cases (A, aged 7 yrs and 15 yrs); (B) Distribution of spine density (mean ± SD) in basal dendrites after the 1^st bifurcation. Age and diagnosis are indicated for each sample. Controls aged 2-8yrs [C(2-8yrs)]: n=5; controls aged 13-18yrs [C(13-18yrs)]: n=5; ASD cases aged 2-8yrs [A(2-8yrs)]: n=5; ASD cases aged 13-18yrs [A(13-18yrs)]: n=5. Each point represents the average spine density for each individual neuron measured from each individual; (C) A linear regression of spine density with age in the control subjects (n = 10) and ASD patients (n = 10). The number of spines per 10 μm was plotted against the age of each individual. Broken lines indicate 95% confidence intervals; (D) Spine density (Mean ± SD) for the controls and ASD patients in childhood and adolescence. Each point represents the mean spine density for an individual. 2-way ANOVA, Bonferroni post hoc test. ***, p<0.001, *, p<0.05; (E) The decrease of spine density with age was greater in the controls than the ASD patients (Mean ±SD). ***, p<0.001.

Fig 2. Dysregulated mTOR-autophagy signaling and spine pruning in ASD temporal lobe

(A) Representative western blots of p-mTOR, t-mTOR, p-S6, t-S6, PSD95 and synapsin I in temporal lobe of ASD patients and control subjects aged 2-9y (ASD, n = 8; Controls, n=7) and 13-19y (ASD, n = 5; Controls, n=9). A, ASD patients; C, controls; (B-E) The relative density (Mean±SD) for p-mTOR (B) and p-S6 (C) were normalized to t-mTOR and t-S6, respectively. PSD95 (D) and synapsin I (E) levels were normalized to actin and are presented as scatter plots for ASD patients and controls in two age groups. Each point represents each individual subject. ** p<0.01; *** p<0.001 (2-way ANOVA, Bonferroni's post hoc test); (F) Western blot of autophagy markers, LC3-II and p62, in temporal lobe of ASD patients and control subjects aged 2-9 yrs and 13-20 yrs; (G) LC3-II levels normalized to actin in controls and patients. ** p<0.01; *** p<0.001 (2-way ANOVA, Bonferroni post hoc test); (H) p62 levels normalized to actin in controls and patients. ** p<0.01; *** p<0.001 (2-way ANOVA, Bonferroni post hoc test); (I) Correlation between p-mTOR and PSD95 (r² =0.598, P<0.001); (J) Correlation between p-mTOR and LC3-II in individuals younger than 10 yrs (R² =0.347, p<0.0001), indicating that LC3-II is regulated by mTOR in both ASD patients and controls; (K) Correlation between LC3-II and PSD95 in individuals younger than 10 years (R² =0.422, p<0.0001), suggesting a relationship between synaptic structure protein levels and autophagy.

Fig 3. Spine pruning defects in Tsc1/2 deficient cortical projection neurons

(A-D) Social behaviors in P30-33 male adolescent TSC2+/- mice. Tsc2+/+wt: n=15, Tsc2+/-: n=14. (A) Novel object recognition test showing time spent investigating a familiar vs. novel object; (B) ASD-like repetitive behavior; (C) Dyadic reciprocal social interaction test showing time spent sniffing a stimulus mouse; (D) Sociability in the 3-chamber test showing time spent (left) and preference (right) for sniffing a stimulus mouse or an object; (E) Social novelty in the 3-chamber test showing time spent (left) and preference (right) for sniffing a stranger mouse vs. a familiar mouse. Compared to wt, ** p<0.01; * p<0.05 (unpaired t test); (F) A confocal image of a DiI-labeled layer V cortical pyramidal neuron. Scale: 20 μm; (G) Typical confocal images of DiI labeled dendrites in wt, Tsc2+/- and rapamycin treated Tsc2+/- mice at P19-20 and P29-30. Rapamycin was administered at 3mg/kg/day intraperitoneally from P21 to P28, and the mice were labeled for spine analysis on P29-30. Scale bar: 2 μm. (H) Spine pruning in Tsc2+/- mice. ** compared with wt at P29-30, p<0.01 (2-way ANOVA, Bonferroni post hoc test). n=7-10 mice per group; (I) Representative images of DiI labeled dendrites from Tsc1^CKO mutants and Tsc1^flox/flox controls. Scale bar: 2 μm; (J) Spine density in Tsc1^flox/flox and Tsc1^CKO mice at P19-20 and P29-30. ** compared to P29-30 Tsc1^flox/flox controls, p<0.01 (2-way ANOVA, Bonferroni post hoc test).

Fig 4. TSC ablation downregulates autophagic activity and rapamycin reconstitutes normal autophagy

(A)Western blot analysis of p-mTOR, t-mTOR and LC3-II in P29 Tsc2+/- mouse brain. Tsc2 wt and Tsc2+/-mice were i.p. injected with DMSO vehicle or rapamycin from p20 to p28. Right: quantification of p-mTOR and LC3-II levels. Mean ± SD. * p<0.05; ** p<0.01; *** p<0.001 (2-way ANOVA, Bonferroni post hoc test). n=5-6 animals per group. Tsc2+/- mutant cortex on P29-30 showed increased p-mTOR levels and decreased levels of LC3-II. Inhibiting mTOR with rapamycin decreased p-mTOR and increased LC3II in both wild type and mutant lines; (B) Impaired autophagic flux in Tsc2+/-; GFP-LC3 cortical neurons. Right: mean number of GFP-LC3 puncta per soma of cortical neurons. 8-10 neurons per group in triplicates were analyzed. Scale bar: 10 μm. * p<0.05; ** p<0.01; *** p<0.001 (2-way ANOVA, Bonferroni post hoc test).

Fig 5. Dendritic spine pruning defects and ASD like behaviors in Atg7^CKO mice

(A)Western blot analysis of autophagy markers in the Atg7^CKO cortex. Brain homogenates from P19-20 and P29-30 mice were immunoblotted with antibodies against Atg12-Atg5, LC3 and autophagy substrate p62. Data shown are representative of three separate experiments. Loss of autophagy was indicated by a decrease in levels of Atg5-12 conjugation and LC3-II protein, and an increase in p62 protein; (B) Immunofluorescent labeling of p62 and ubiquitin (Ub) in P30 Atg7^CKO mouse cortex. Scale bar: 10 μm. (C-F) ASD like social behaviors in Atg7^CKO mice. Atg7^flox/flox males, n=15; Atg7^CKO males, n=13; (C) Novel object recognition test showing time spent sniffing a familiar object vs. a novel object; (D) Dyadic social interaction test showing the time testing mice spent sniffing a stimulus mouse. ** Compared to Atg7^flox/flox; p<0.01; unpaired t-test; (E) Sociability in the 3-chamber test showing time spent (left) and preference (right) for a stimulus mouse or an object; (F) Social novelty in the 3-chamber test showing time spent (left) and preference (right) for sniffing a stranger mouse vs. a familiar mouse. Compared to wt, ** p<0.01 (unpaired t test); (G) Dendritic segments from Atg7^flox/flox and Atg7^CKO pyramidal neurons at P19-20 and P29-30. n=7-10 animals per group. Scale bar: 2 μm; (H) Fewer spines were pruned in Atg7^CKO mice. ** Compared to P29-30 Atg7^flox/flox, p<0.01 (2-way ANOVA, Bonferroni post hoc test). (I) Timeline of infection and spine analysis; (J) Cultured control and Atg7 SiRNA lentiviral infected mouse hippocampal neurons at DIV20, visualized by GFP and PSD95 fluorescence. Atg7SiRNA expressing neurons exhibited more PSD95 puncta than controls; (K) Spine formation and elimination in control and Atg7SiRNA infected neurons during a 12-hour time window at DIV19-20.

Fig 6. Autophagy deficiency underlies spine pruning defects and ASD-like social deficits in Tsc2+/- mice

(A) p62 and Ub positive immunolabeled inclusions in autophagy deficient neurons. Note that P62⁺ and Ub⁺ inclusions were occasionally present in Tsc2+/-, Atg7C^KO and Tsc2+/-:Atg7^CKO cortex at p20, but appear in a majority of cortical neurons in these three lines at P30. No p62⁺ and Ub⁺ inclusions were seen in P30 cortical neurons from Atg7^flox/flox control mice; (B-D) Rapamycin normalized dendritic spine pruning in an autophagy-dependent manner; (B) Representative images of dendrites from different mouse lines treated with DMSO vehicle or rapamycin. Scale: 2 μm; (C) Graphic representation of dendritic spine densities in each condition; (D) Percentage of spines pruned in control and mutant mouse lines. Percentage change in mean spine density (MSD) between P20 and P29 was calculated as: (MSD (P20) - MSD (P29)) / MSD (P20) *100%. Rapamycin rescued spine pruning deficits in Tsc2+/- mice, but not in Atg7^CKO and had relatively little effect in Tsc2+/-:Atg7^CKO double mutants. n=7-10 animals per group. *** Compared to DMSO treated Atg7^flox/flox controls, p<0.001; ^### Compared to rapamycin treated Atg7^flox/flox controls, p<0.001; (IMG) compared to DMSO vehicle controls, p<0.001 (2-way ANOVA, Bonferroni post hoc test); (E-H) Autophagy deficiency blocks the rescue by rapamycin on ASD like social behaviors in Tsc2+/- mice. n=10-14 animals per group; (E, F) Sociability: DMSO treated Tsc2+/-, Atg7C^KO and Tsc2+/-:Atg7^CKO mice each spent less time sniffing social target versus non-social target (E) and exhibited decreased preference (F) for the social target vs. non-social target. Rapamycin treatment ameliorated impaired sociability in Tsc2+/- mice, but not in Atg7^CKO and Tsc2+/-:Atg7^CKO mice; (G, H) Social novelty: DMSO treated Tsc2+/-, Atg7C^KO and Tsc2+/-:Atg7^CKO mice all spent less time sniffing novel mice during social novelty test (G) and displayed a decrease in preference for social novelty (H). Rapamyin treatment prevented the loss of preference for social novelty in Tsc2+/- mice, but not in Atg7C^KO and Tsc2+/-:Atg7C^KO mice. *, p<0.05; **, p<0.01; ***, p<0.001 (2-way ANOVA, Bonferroni post hoc test).