(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.

(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.

(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).

(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).

(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.

(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).