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Cited by 1,146Genetics and Neurodevelopmental DisordersAutism Spectrum Disorder ResearchAutophagy in Disease and Therapy

Loss of mTOR-Dependent Macroautophagy Causes Autistic-like Synaptic Pruning Deficits

Open access
Tang, Guomei, Gudsnuk, Kathryn, Kuo, Sheng-Han, Cotrina, Marisa L., Rosoklija, Gorazd, Sosunov, Alexander, Sonders, Mark S., Kanter, Ellen +9 more·2014·Neuron
DOI 10.1016/j.neuron.2014.07.040PMC PMC4159743
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Abstract

Summary 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 Atg7CKO neuronal autophagy deficient mice or Tsc2+/-:Atg7CKO 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.

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Crossref · 10.1016/j.neuron.2014.07.040

Cites

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Claims the paper makes

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  • Findingown data

    The decrease in PSD95 level with age was absent in ASD patients.

    “This decrease in PSD95 level with age was absent in ASD patients (Fig 2D), consistent with the lower spine pruning in ASD patients.”
    Quote in the paperSubstantial claim
  • Findingown data

    Linear regression indicated substantially greater net spine pruning in controls than in ASD patients.

    “Linear regression of spine density with age indicated a substantially greater level of net spine pruning in controls (slope = -0.40 spines / 10 μm /year, R2 = 0.93) than in ASD patients (slope = -0.19 / 10 μm / year, R2 = 0.55; difference from linear regression of controls, F = 9.4, p = 0.007) (Fig 1C).”
    Quote in the paperSubstantial claim
  • Findingown data

    The cellular area occupied by LC3 puncta and the integrated intensity of LC3 puncta were lower in pyramidal neurons from ASD patients than age-matched controls during both early childhood and adolescence.

    “The impairment in autophagy in ASD patients was confirmed by immunolabel of LC3-positive puncta in BA21 layer V: the cellular area occupied by LC3 puncta and the integrated intensity of LC3 puncta were lower in pyramidal neurons from ASD patients than age-matched controls during both early childhood and adolescence (Figs S1E, S1F, S1G).”
    Quote in the paperSubstantial claim
  • Findingown data

    Higher spine density is found in basal dendrites of layer V pyramidal neurons in ASD patients than in controls.

    “We found a higher spine density in basal dendrites of layer V pyramidal neurons in ASD patients than in controls.”
    • Also stated in: § Dendritic spine pruning deficits in ASD human brain · plain
      “The mean net spine density per individual was significantly higher in ASD patients than in controls (Figs 1A and 1B: mean ± SD: 8.81 ± spines/10 μm vs. 11.32 ± spines/10 μm, p=0.017, two-tailed t-test).”
    Quote in the paperSubstantial claim
  • Findingown data

    From childhood through adolescence, dendritic spines decreased by ∼45% in control subjects but only by ∼16% in ASD patients.

    “From childhood through adolescence, dendritic spines decreased by ∼45% in control subjects but only by ∼16% in ASD patients (Fig 1E), demonstrating a developmental defect in net spine pruning in ASD.”
    Quote in the paperSubstantial claim
  • May combine two claims. 55
  • Classified as probably not a substantial claim. 20

  • May combine two claims.

    Findingown data

    Spine density was slightly higher in childhood ASD patients than controls but markedly higher in adolescent ASD patients than controls.

    “The spine density was slightly higher in childhood ASD patients than controls (mean ± SD: 12.32 ± 0.60 spines /10 μm in ASD cases vs. 11.37 ± 0.68 spines /10 μm in controls) but markedly higher in adolescent ASD patients than controls (10.33 ± 0.74 spines/10 μm in ASD cases vs. 6.24 ± 0.59 spines/10 μm in controls).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    LC3-II level was significantly lower in ASD patients than controls throughout childhood and adolescence, while p62 level was higher in both childhood and adolescent ASD patients than controls.

    “The level of LC3-II, a biomarker that indicates the abundance of autophagosomes, was significantly lower in ASD patients than controls throughout childhood and adolescence (Figs 2F and 2G), while the level of p62, a protein substrate for autophagy, was higher in both childhood and adolescent ASD patients that controls (Figs 2F and 2H).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Postmortem ASD temporal lobe shows increased dendritic spine density and reduced developmental spine pruning in layer V pyramidal neurons.

    “Here, we report increased dendritic spine density with reduced developmental spine pruning in layer V pyramidal neurons in postmortem ASD temporal lobe.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The increased spine density in ASD is associated with a defect in net postnatal spine pruning and correlated with hyperactivated mTOR and impaired autophagy.

    “The increased spine density was associated with a defect in net postnatal spine pruning that was correlated with hyperactivated mTOR and impaired autophagy.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The spine deficits in ASD temporal lobe correlate with hyperactivated mTOR and impaired autophagy.

    “These spine deficits correlate with hyperactivated mTOR and impaired autophagy.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Tsc2+/- ASD mice with constitutively overactive mTOR show postnatal spine pruning defects, autophagy blockade, and ASD-like social behaviors.

    “In Tsc2+/- ASD mice where mTOR is constitutively overactive, we observed postnatal spine pruning defects, blockade of autophagy, and ASD-like social behaviors.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Neuronal autophagy enables spine elimination without affecting spine formation.

    “Neuronal autophagy furthermore enabled spine elimination with no effects on spine formation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Rapamycin corrects ASD-like behaviors and spine pruning defects in Tsc2+/- mice but not in Atg7CKO neuronal autophagy deficient mice or Tsc2+/-:Atg7CKO double mutants.

    “The mTOR inhibitor rapamycin corrected ASD-like behaviors and spine pruning defects in Tsc2+/ mice, but not in Atg7CKO neuronal autophagy deficient mice or Tsc2+/-:Atg7CKO double mutants.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Two-way ANOVA revealed profound effects of disease and age on spine density, with a significant disease × age interaction.

    “Analysis revealed profound effects of both disease and age on spine density (p<0.001, 2-way ANOVA, effect of disease: F (1, 16) =73.11, p<0.001; effect of age: F (1, 16) =145.7, p<0.001; disease X age interaction: F (1, 16) =28.35, p<0.001).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    p-mTOR level decreases with age in controls and is higher in ASD patients than controls at 13-20 years.

    “We observed a decrease in p-mTOR level with age in controls, and higher p-mTOR level in ASD patients than in controls at 13-20 y (Fig 2B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    PSD95 protein level was higher in controls aged 2-10 y than controls aged 13-20 y, consistent with normal developmental spine pruning.

    “PSD95 protein level was higher in controls aged 2-10 y than controls aged 13-20 y, consistent with normal developmental spine pruning.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the 3-chamber test, Tsc2+/- mice showed preference for social target over non-social target, but the preference index was decreased.

    “While Tsc2+/- mice showed a preference for interacting with a social target compared with non-social target (Fig 3D, left), the preference index (the ratio of time sniffing mouse vs. empty cage) was decreased (Fig 3D, right).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    LC3-II protein levels decreased in Tsc2+/- cortex and were normalized by rapamycin.

    “We confirmed suppression of basal autophagy due to mTOR disinhibition in the Tsc mutant mouse brain by 1) a decrease in protein levels of LC3-II in Tsc2+/- cortex, which was normalized by rapamycin (Fig 4A);”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At P19-20, Atg7CKO basal dendrites from layer V A1/S2 pyramidal neurons exhibited a similar number of spines as controls, but by P29-30 they exhibited more spines than controls.

    “At P19-20, Atg7CKO basal dendrites from layer V A1/S2 pyramidal neurons exhibited a similar number of spines as those in the Atg7flox/flox control mice (Figs 5G and 5H).”
    • Also stated in: § Autophagy deficiency results in ASD like social behaviors and spine pruning defects · plain
      “In contrast, by P29-30 the Atg7CKO basal dendrites exhibited more spines than controls.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Rapamycin normalized pS6 levels and numbers of GFP-LC3 puncta.

    “Rapamycin normalized pS6 levels and numbers of GFP-LC3 puncta;”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In Atg7siRNA infected neurons, ∼12% of spines were formed but only ∼5% of pre-existing spines were pruned.

    “In contrast, ∼12% of spines were formed, but only ∼5% of pre-existing spines were pruned in Atg7siRNA infected neurons (p<0.05, t-test).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    By P30, Atg7CKO mice exhibited less conjugated ATG5-12 and LC3-II protein and more p62 protein than controls.

    “By P30, however, Atg7CKO mice exhibited less conjugated ATG5-12 and LC3-II protein and more p62 protein than the Atg7flox/flox controls.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At P19-20, spine density in basal dendrites of A1/S2 layer V pyramidal neurons was equivalent for Tsc1flox/flox control and Tsc1CKO mice, but at P29-30 spines in control mice were significantly less dense than in Tsc1CKO mice.

    “At P19-20, the density of spines in basal dendrites of A1/S2 layer V pyramidal neurons was equivalent for both Tsc1flox/flox control and Tsc1CKO mice (p>0.05), but spines in control mice were significantly less dense than in Tsc1CKO mice at P29-30 (p<0.01, Figs 3I and 3J).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Atg7CKO mice displayed occasional p62/Ub positive inclusions in pyramidal neurons at P20 and prominent p62/Ub positive aggregates in layer II-III and layer V-VI pyramidal neurons at P30.

    “Atg7CKO mice displayed occasional p62/Ub positive inclusions in pyramidal neurons at P20 and prominent p62/Ub positive aggregates in layer II-III and layer V-VI pyramidal neurons at P30 (Fig 5B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    pS6 level increased and GFP-LC3 puncta were reduced in cortical pyramidal neurons from Tsc2+/-:GFP-LC3 mice, indicating mTOR hyperactivation and impaired autophagy.

    “2) an increase in the level of phospho-S6 (pS6), indicating mTOR hyperactivation, and a reduction in GFP-LC3 puncta, a fluorescent marker for autophagosomes, in cortical pyramidal neurons from Tsc2+/-: GFP-LC3 mice (Fig S5A), indicating impaired autophagy.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In novel object recognition, Tsc2+/- mice spent less time exploring the novel object than wild-type littermates, with no difference in time exploring the familiar object.

    “In the novel object recognition test, Tsc2+/- mice spent less time exploring the novel object than their wild type (wt) littermates, with no difference in time spent exploring the familiar object (Fig 3A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In the 3-chamber test, Atg7CKO mice displayed impaired preference for sniffing the social target and for social novelty.

    “In the 3-chamber test, Atg7CKO mice displayed impaired preference for sniffing the social target (Fig 5E) and for social novelty (Fig 5F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    High levels of p62 and ubiquitin were observed in Tsc2+/-, Atg7CKO, and Tsc2+/-:Atg7CKO double mutant cortices at P30, consistent with a deficit of autophagy between P20 and P30.

    “We observed high levels of p62 and ubiquitin in Tsc2+/-, Atg7CKO and Tsc2+/-:Atg7CKO double mutant cortices at P30, consistent with a deficit of autophagy between P20 and P30 (Fig 6A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    DiOlistic labeling revealed similar spine numbers at P19-20 in Tsc2+/- and wild-type mice, but far more spines in P29-30 Tsc2+/- mice than in wild-type mice.

    “DiOlistic labeling revealed similar numbers of spines at P19-20 in Tsc2+/- and wt mice, but far more spines in P29-30 Tsc2+/- mice than in wt (Figs 3G and 3H).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Atg7CKO mice exhibited ASD-like social recognition and social interaction deficits similar to Tsc2+/- mutants.

    “Atg7CKO mice exhibited ASD-like social recognition (Fig 5C) and social interaction deficits similar to those seen in Tsc2+/- mutants.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Rapamycin reversed spine pruning defects in Tsc2+/- mice to control level but did not rescue spine pruning in Atg7CKO mice or Tsc2+/-:Atg7CKO double mutants.

    “Rapamycin reversed spine pruning defects in Tsc2+/- mice to the level of control mice, but did not rescue spine pruning in Atg7CKO mice or in Tsc2+/-:Atg7CKO double mutants, demonstrating that neuronal autophagy is required for spine elimination in Tsc2+/- mice.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Rapamycin showed no effects in wild-type controls but corrected the pruning defect in Tsc2+/- mutants to control level.

    “Inhibition of mTOR by i.p. injection of rapamycin showed no effects in wt controls (Fig S3B) but corrected the pruning defect in Tsc2+/- mutants to the control level.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At P20, there were no differences in LC3-II and conjugated ATG5-12 levels between genotypes, although p62 levels were higher in Atg7CKO mice.

    “At P20 there were no differences in LC3-II and conjugated ATG5-12 levels between genotypes, although p62 levels were higher in Atg7flox/flox:CamKII-Cre (Atg7CKO) autophagy-deficient mice (Fig 5A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In the social novelty test, Tsc2+/- mice spent similar time sniffing novel and familiar social targets, with decreased preference index.

    “In the social novelty test, Tsc2+/- mice spent a similar amount of time sniffing both novel and familiar social targets (Fig 3E, left), with decreased preference index (the ratio of time sniffing a stranger mouse vs. a familiar mouse, Fig 3E, right), indicating a reduced preference for social novelty.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Rapamycin did not affect spine density in Tsc1flox/flox control mice but corrected spine pruning in Tsc1CKO mice to control levels, with no effect on basal dendritic branching.

    “Rapamycin treatment did not affect spine density in Tsc1flox/flox control mice (Fig S5B), but corrected spine pruning in Tsc1CKO mice to control levels (Figs 3I and 3J), with no effect on basal dendritic branching.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Many ASD brains exhibit both disrupted mTOR signaling and synaptic defects during childhood and adolescence.

    “We find that ASD brains exhibit both disrupted mTOR signaling and synaptic defects.”
    • Also stated in: § Autophagy and spine pruning · plain
      “In summary, we find that many ASD brains exhibit both disrupted mTOR signaling and synaptic defects during childhood and adolescence”
    Quote in the paperSubstantial claim
    • Rests on an assumption: ASD brain samples are representative of ASD patients generally.

  • May combine two claims.

    Findingown data

    A deficit in autophagy in Atg7CKO mice was confirmed by western blot of conjugated ATG5-12, p62, and LC3-II and by immunolabel of p62 and ubiquitin.

    “A deficit in autophagy was confirmed by western blot analysis of conjugated ATG5-12, p62 and LC3-II proteins (Fig 5A) and by immunolabel of p62 and ubiquitin (Ub) (Figs 5B and 6A), proteins that form aggregates after autophagy inhibition (Komatsu et al., 2006; Komatsu et al., 2007).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Komatsu et al., 2006 source

      Not checked in the free run.

    • cites Komatsu et al., 2007

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Atg7flox/flox control mice treated with vehicle preferred to sniff the novel mouse more than the non-social object in the sociability test, and preferred to sniff the stranger mouse more than the familiar mouse in the social novelty test.

    “Atg7flox/flox control mice treated with vehicle preferred to sniff the novel mouse more than the non-social object in the sociability test (Figs 6E and 6F), and preferred to sniff the stranger mouse more than the familiar mouse in the social novelty test (Figs 6G and 6H).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In control neurons, ∼12% of spines were formed and ∼13% eliminated, indicating equivalent rates of spine formation and elimination that reflect stabilized spine densities.

    “In control neurons infected with viral vector controls, ∼12% of spines were formed and ∼13% eliminated, indicating equivalent rates of spine formation and elimination that reflect stabilized spine densities (Fig 5K).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Inhibition of neuronal autophagy in mouse models produced ASD-like inhibition of normal developmental spine pruning and ASD-like behaviors.

    “We have confirmed in mouse models that inhibition of neuronal autophagy produced ASD-like inhibition of normal developmental spine pruning and ASD-like behaviors.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Mouse models of ASD-like behaviors and spine pruning deficits recapitulate human ASD.

  • May combine two claims.

    Findingown data

    Autophagy regulates spine elimination but not spine formation during developmental pruning of dendritic spines in vitro.

    “Using an in vitro primary neuronal culture system, we find that autophagy regulates spine elimination but not spine formation during developmental pruning of dendritic spines.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Increased spine density is observed in frontal, temporal, and parietal lobes in ASD brains, and synaptic structure changes are detected in multiple ASD model mice.

    “Consistently, increased spine density is observed in frontal, temporal, and parietal lobes in ASD brains (Hutsler and Zhang, 2010) and changes in synaptic structure are detected in multiple ASD model mice (Zoghbi and Bear, 2012).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Hutsler and Zhang, 2010 source

      Not checked in the free run.

    • cites Zoghbi and Bear, 2012 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Synaptic mTOR integrates signaling from various ASD synaptic and regulatory proteins, including SHANK3, FMRP, and glutamate receptors mGluR1/5.

    “Synaptic mTOR integrates signaling from various ASD synaptic and regulatory proteins, including SHANK3, FMRP and the glutamate receptors mGluR1/5 (Peca et al., 2012; Bourgeron, 2009).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Peca et al., 2012

      Not checked in the free run.

    • cites Bourgeron, 2009 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    mTOR-regulated autophagy is required for developmental spine pruning, and activating neuronal autophagy corrects synaptic pathology and social behavior deficits in ASD models with hyperactivated mTOR.

    “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.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Dendritic spine density peaks in early childhood and declines steeply during late childhood and adolescence to adult levels, providing selection and maturation of synapses and neural circuits.

    “Consistently, the density of dendritic spines peaks in early childhood and is followed by a steep decline during late childhood and adolescence to adult levels (Penzes et al., 2011), a process that provides selection and maturation of synapses and neural circuits.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Penzes et al., 2011 source

      Not checked in the free run.

  • May combine two claims.

    Study design and conductown data

    No effects of postmortem interval, cause of death, brain pH, or length of storage on protein levels were detected.

    “No effects of PMI, cause of death, brain pH or length of storage on protein levels were detected (Table S2).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    BA21 is implicated in ASD due to its participation in brain networks for social and communicative processes, including language, social and speech perception, auditory and visual processing, and comprehension of intentions.

    “We measured dendritic spines of basal dendrites of layer V pyramidal neurons in the superior middle temporal lobe, Brodmann Area 21 (BA21), a region implicated in ASD due to its participation in brain networks involved in social and communicative processes, including language, social and speech perception, auditory and visual processing, and comprehension of intentions (Redcay, 2008; Zahn et al.,”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Redcay, 2008 source

      Not checked in the free run.

    • cites Zahn et al., 2007

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Multiple ASD susceptibility genes converge on cellular pathways that intersect at the postsynaptic site of glutamatergic synapses, implicating dendritic spine abnormalities in ASD pathogenesis.

    “Multiple ASD susceptibility genes converge on cellular pathways that intersect at the postsynaptic site of glutamatergic synapses (Bourgeron, 2009; Peca et al., 2012), implicating abnormalities in dendritic spines in ASD pathogenesis.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Bourgeron, 2009 source

      Not checked in the free run.

    • cites Peca et al., 2012

      Not checked in the free run.

  • May combine two claims.

    Study design and conductown data

    LC3-II and p62 protein levels were not correlated with any confounding factor for tissue preservation but were correlated with seizure activity.

    “LC3-II and p62 protein levels were not correlated with any confounding factor for tissue preservation but were correlated with seizure activity (Table S2), a common feature of ASD.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Postnatal synaptic development in mammalian cerebral cortex involves concurrent formation and elimination/pruning.

    “Postnatal synaptic development in mammalian cerebral cortex is a dynamic process involving concurrent formation and elimination/pruning (Purves and Lichtman, 1980; Rakic et al., 1986).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Purves and Lichtman, 1980 source

      Not checked in the free run.

    • cites Rakic et al., 1986 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Autophagy has been implicated in synaptic remodeling in C. elegans and Drosophila.

    “Autophagy has been implicated in synaptic remodeling in C. elegans (Rowland et al., 2006) and Drosophila (Shen and Ganetzky, 2009), but a role in mammalian synaptic development is unexplored.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Rowland et al., 2006

      Not checked in the free run.

    • cites Shen and Ganetzky, 2009 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Abnormalities in ASD temporal lobe have been confirmed by functional imaging and pathological studies, including disturbed gene transcription profiles, increased dendritic spine densities in pyramidal neurons, and reduced functional specialization.

    “Abnormalities in ASD temporal lobe have been confirmed by functional imaging and pathological studies, including disturbed gene transcription profiles (Garbett et al., 2008; Voineagu et al., 2011), increased dendritic spine densities in pyramidal neurons (Hutsler and Zhang, 2010), and reduced functional specialization (Shih et al., 2011).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Garbett et al., 2008 source

      Not checked in the free run.

    • cites Voineagu et al., 2011 source

      Not checked in the free run.

    • cites Hutsler and Zhang, 2010 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Aberrant autophagy and mTOR hyperactivation underlie ASD-like synaptic pathology, and correcting autophagy signaling could normalize developmental dendritic spine pruning defects and social behaviors.

    “Using Tsc1/2 mutant ASD mice and Atg7CKO neuronal autophagy deficient mice, we find that aberrant autophagy and mTOR hyperactivation underlies ASD-like synaptic pathology and correcting autophagy signaling could normalize developmental dendritic spine pruning defects and social behaviors.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    An ongoing deficiency in autophagy and impaired spine elimination at younger ages would increase net spine density and interfere with dynamic synapse turnover.

    “An ongoing deficiency in autophagy and impaired spine elimination at younger ages would be expected to increase net spine density and interfere with the dynamic turnover of synapses that organizes neural circuits.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Prolonged use of mTOR inhibitors may cause adverse effects.

    “prolonged use of mTOR inhibitors may cause adverse effects (Rodrik-Outmezguine et al., 2011).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Rodrik-Outmezguine et al., 2011 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Atg7 knockdown produces excessive dendritic spines by inhibiting elimination, with no effect on formation.

    “Therefore, Atg7 knockdown produces excessive dendritic spines by inhibiting elimination, but exerted no effect on formation.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    mTOR signaling contributes to protein synthesis required for neuronal survival, development, synaptic plasticity, learning, and memory.

    “mTOR signaling, however, contributes to protein synthesis required for neuronal survival, development, synaptic plasticity, learning, and memory (Hoeffer and Klann, 2010)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Hoeffer and Klann, 2010 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    There is a period of massive spine pruning between P19-20 and P29-30 in wild-type mice, but a lack of normal pruning in Tsc2+/- mice.

    “These results suggest that there is a period of massive spine pruning between the ages of P19-20 and P29-30 in wt, but a lack of normal pruning in Tsc2+/- mice.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Mutations in both Tsc1 and Tsc2 cause mTOR hyperactivation and ASD-like behaviors in mice.

    “Mutations in both Tsc1 and Tsc2 cause mTOR hyperactivation and ASD-like behaviors in mice (Chevere-Torres et al., 2012; Tsai et al., 2012; Goorden et al., 2007; Sato et al., 2012).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Chevere-Torres et al., 2012

      Not checked in the free run.

    • cites Tsai et al., 2012

      Not checked in the free run.

    • cites Goorden et al., 2007

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Both Tsc1CKO and Tsc2+/- mutants showed a lack of efficient postnatal spine pruning, indicating that TSC inhibition of mTOR is required for postnatal spine pruning.

    “Thus, both Tsc1CKO and Tsc2+/- mutants showed a lack of efficient postnatal spine pruning, indicating that TSC inhibition of mTOR is required for postnatal spine pruning.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Spine pruning during early postnatal development occurs in cerebral cortex, cerebellum, olfactory bulb, and hippocampus.

    “spine pruning during early postnatal development occurs in cerebral cortex, cerebellum, olfactory bulb, and hippocampus (Purves and Lichtman, 1980; Shinoda et al., 2010).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Purves and Lichtman, 1980 source

      Not checked in the free run.

    • cites Shinoda et al., 2010

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationreasoning

    The extended duration for normal spine pruning in human brain may provide an opportunity for therapeutic intervention for ASD after diagnosis.

    “The extended duration for normal spine pruning in human brain may provide an opportunity for therapeutic intervention of multiple functional domains associated with ASD after the disease is diagnosed.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    Additional ASD brain regions may feature spine pruning defects during different periods of synaptic development.

    “it is likely that additional ASD brain regions may feature spine pruning defects during different periods of synaptic development.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Each of these conditions features altered dendritic spine densities.

    “each of which features altered dendritic spine densities.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Peca et al., 2012

      Not checked in the free run.

    • cites Bourgeron, 2009 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Basal neuronal autophagy is required for normal spine pruning during postnatal development and for the development of normal social behaviors.

    “Thus, basal neuronal autophagy is required for normal spine pruning during postnatal development and for the development of normal social behaviors.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Atg7 is an E1-like activating enzyme required for autophagosome formation.

    “Atg7 is an E1-like activating enzyme required for autophagosome formation (Komatsu et al., 2006).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Komatsu et al., 2006 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Primary cultures of hippocampal neurons have been used as an in vitro system to investigate formation, maturation, and pruning of dendritic spines, with spine density increasing between DIV6-10, peaking at DIV14-21, and decreasing after weeks 3 or 4.

    “Primary cultures of hippocampal neurons have been used as an in vitro system to investigate the formation, maturation and pruning of dendritic spines, in which dendritic spines are formed and pruned during a developmental period similar to that in vivo in mouse brain (Orefice et al., 2013; Ko et al., 2011; Papa et al., 1995), with spine density increases between 6-10 days in vitro (DIV6-10), peaks”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Orefice et al., 2013 source

      Not checked in the free run.

    • cites Ko et al., 2011

      Not checked in the free run.

    • cites Papa et al., 1995

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Tsc2+/- mice exhibit normal social preference in a 3-chamber test but deficient social interaction in a dyadic reciprocal social test.

    “Tsc2+/- mice however exhibit normal social preference in a 3-chamber test (Ehninger et al., 2008; Ehninger et al., 2012) but deficient social interaction in a dyadic reciprocal social test (Sato et al., 2012).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Ehninger et al., 2008 source

      Not checked in the free run.

    • cites Ehninger et al., 2012

      Not checked in the free run.

    • cites Sato et al., 2012

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationnone given

    It is highly unlikely that these ASD patients possessed TSC mutations.

    “It is highly unlikely that these patients possessed TSC mutations”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationreasoning

    Enhancing autophagy downstream of mTOR during development may provide the basis for novel ASD therapeutics.

    “developing targeted means to enhance autophagy downstream of mTOR during development may provide the basis for novel ASD therapeutics.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    Glial non-cell autonomous autophagic regulation of spine morphogenesis is possible.

    “pointing to the possibility of glial non-cell autonomous autophagic regulation of spine morphogenesis.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationcitation

    Autophagy may degrade proteins that suppress spine elimination, and loss of autophagy could accumulate proteins that block spine pruning.

    “autophagy may degrade proteins that suppress spine elimination, and the loss of autophagy could accumulate proteins that block spine pruning”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Banerjee et al., 2009

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A link has been established between eIF4E dependent translational control downstream of mTOR and ASD-like phenotypes in mouse models.

    “a link has been established between eIF4E dependent translational control downstream of mTOR and ASD-like phenotypes in mouse models (Santini et al., 2012).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Santini et al., 2012

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationreasoning

    Targeting neuronal autophagy could provide therapeutic benefit.

    “suggest that targeting neuronal autophagy could provide therapeutic benefit.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    Changes in mTOR-autophagy signaling and spine pruning defects may represent a secondary mechanism in response to an imbalance between excitatory and inhibitory neurotransmission.

    “changes in mTOR-autophagy signaling and spine pruning defects may represent a secondary mechanism in response to an imbalance between excitatory and inhibitory neurotransmission”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Chao et al., 2010

      Not checked in the free run.

    • cites Fu et al., 2011

      Not checked in the free run.

    • cites Yizhar et al., 2011 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    Altered synaptic function is consistent with the recent finding that chronic lack of neuronal autophagy enhances evoked neurotransmitter release and rate of synaptic recovery.

    “Altered synaptic function is consistent with our recent finding that chronic lack of neuronal autophagy enhances evoked neurotransmitter release and rate of synaptic recovery (Hernandez et al., 2012).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Hernandez et al., 2012

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Defective neuronal autophagy can be induced by infected microglia.

    “Defective neuronal autophagy can be induced by infected microglia (Alirezaei et al., 2008)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Alirezaei et al., 2008

      Not checked in the free run.

72 of 72

  • The paper relies on this without support.

    The reasons for spine pathology and its link to the onset and progression of ASD symptoms are unknown.

    “It remains however unclear why spine pathology occurs and how it is associated with the onset and progression of ASD related symptoms.”

  • The paper relies on this without support.

    The role of autophagy in mammalian synaptic development has not been explored.

    “Autophagy has been implicated in synaptic remodeling in C. elegans (Rowland et al., 2006) and Drosophila (Shen and Ganetzky, 2009), but a role in mammalian synaptic development is unexplored.”

  • The paper relies on this without support.

    mTOR controls multiple downstream processes besides autophagy, including protein synthesis, ribosome biogenesis, transcription for lysosome biogenesis, and mitochondrial metabolism.

    “mTOR regulates a number of downstream biological processes including protein synthesis, autophagy, ribosome biogenesis, and activation of transcription leading to lysosome biogenesis or mitochondrial metabolism.”

  • The paper relies on this without support.

    Autophagy deficiency is a consequence of mTOR overactivation.

    “autophagy deficiency, which is a consequence of mTOR overactivation”

  • The paper relies on this without support.

    Mouse models of ASD-like behaviors and spine pruning deficits recapitulate human ASD.

    “We have confirmed in mouse models that inhibition of neuronal autophagy produced ASD-like inhibition of normal developmental spine pruning and ASD-like behaviors.”

  • The paper relies on this without support.

    Cross-sectional age-related spine density data reflect developmental spine pruning trajectories.

    “We therefore relied on correlations among an age range of available pathological specimens, synaptic density, and biochemical markers for analysis.”

  • The paper relies on this without support.

    Postmortem brain tissue measurements of spine density reflect in vivo synaptic density.

    “Neuronal morphology in postmortem human brain was analyzed by Golgi-Kopsch technique.”

  • The paper relies on this without support.

    ASD brain samples are representative of ASD patients generally.

    “We assessed spine density across development and confirm an increase in basal dendrite spine density in layer V pyramidal neurons in ASD temporal lobe.”
8 of 8

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Autophagy helps eliminate synapses during development.

    “autophagy helps eliminate synapses during development [377,378]”

    From Autophagy and ALS: mechanistic insights and therapeutic implications (2022) · cites this paper as [378]

  • This finding is consistent with other evidence from genetic and environmental models of ASD as well as evidence showing that mTOR protein levels in post-mortem cortical tissue of ASD patients can be increased or decreased.

    “Although this finding is in the opposite direction of known hyperactivation of mTOR by some highly ASD-penetrant mutations, it is consistent with other evidence from genetic103 and environmental models104 of ASD as well as evidence showing that mTOR protein levels in post-mortem cortical tissue of ASD patients can be increased105 or decreased.104”

    From Maternal immune activation dysregulation of the fetal brain transcriptome and relevance to the pathophysiology of autism spectrum disorder (2018) · cites this paper as [105]

  • Tsc2+/− mice show increased spine density and decreased pruning with age but comparable cell size and dendritic branching to wild-type.

    “Layer V neurons in the temporal cortex of Tsc2+/− mice show increased spine density and decreased pruning with age but comparable cell size and dendritic branching to the WT [349].”

    From Autism spectrum disorder: neuropathology and animal models (2017) · cites this paper as [349]

  • Conditional KO of Tsc1 in forebrain pyramidal neurons increases spine density in basal dendrites of temporal cortex neurons due to reduced synaptic pruning during development.

    “Conditional KO of Tsc1 in forebrain pyramidal neurons (Tsc1CKO mice) does not result in change in soma size or dendritic arborization but does increase spine density in basal dendrites of temporal cortex neurons. This defect is due to reduced synaptic pruning during development [349].”

    From Autism spectrum disorder: neuropathology and animal models (2017) · cites this paper as [349]

Statistics recomputed

Every reported test result, recomputed from its test statistic and degrees of freedom. A result shows as not adding up only once an independent check has confirmed it.

  • ConsistentF(1, 16) = 73.11reported p < .001 · recomputed p < .001

    “F (1, 16) =73.11, p < .001”

  • ConsistentF(1, 16) = 145.7reported p < .001 · recomputed p < .001

    “F (1, 16) =145.7, p < .001”

  • ConsistentF(1, 16) = 28.35reported p < .001 · recomputed p < .001

    “F (1, 16) =28.35, p < .001”

  • ConsistentF(3, 16) = 15.38reported p < .001 · recomputed p < .001

    “F(3,16)=15.38, p < .001”

  • ConsistentF(1, 16) = 32.16reported p < .001 · recomputed p < .001

    “F(1,16)=32.16, p < .001”

  • ConsistentF(3, 16) = 56.17reported p < .001 · recomputed p < .001

    “F(3,16)=56.17, p < .001”

  • ConsistentF(1, 85) = 4.156reported p < .05 · recomputed p = .045

    “F(1,85)=4.156, p < .05”

  • ConsistentF(3, 85) = 6.775reported p < .001 · recomputed p < .001

    “F(3,85)=6.775, p < .001”

  • ConsistentF(3, 85) = 3.085reported p < .05 · recomputed p = .032

    “F(3,85)=3.085, p < .05”

  • ConsistentF(1, 85) = 13.02reported p < .001 · recomputed p < .001

    “F(1,85)=13.02, p < .001”

  • ConsistentF(3, 85) = 25.64reported p < .001 · recomputed p < .001

    “F(3,85)=25.64, p < .001”

11 of 11

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • The stated direction of the mTOR-spine association reverses

    Minor

    The paper says lower mTOR activity is associated with higher spine density, but it also reports higher p-mTOR in adolescent ASD patients, the group with markedly higher spine density than controls. The stated direction of the association is therefore opposite to the reported group pattern.

1 of 1

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Authors
Tang, G., Gudsnuk, K., Kuo, S.-H., Cotrina, M. L., Rosoklija, G., Sosunov, A., Sonders, M. S., Kanter, E., Castagna, C., Yamamoto, A., Yue, Z., Arancio, O., Peterson, B. S., Champagne, F., Dwork, A. J., Goldman, J., & Sulzer, D.
Affiliations
  • Columbia University Irving Medical Center (US)
  • Icahn School of Medicine at Mount Sinai (US)
Corresponding author
Sulzer, David
ORCID
Venue
Neuron
Publisher
Elsevier BV
Published
2014-09
Volume
83
Issue
5
Pages
1131-1143
ISSN
0896-6273 (ISSN-L)
DOI
10.1016/j.neuron.2014.07.040
PMC
PMC4159743
OpenAlex
W2133470905
Semantic Scholar
f57d2433b68dbe26936008e588e03509c7f18c4e
Cited by
1,146
References
66
Citations by year
2026: 74 · 2025: 105 · 2024: 100 · 2023: 116 · 2022: 102 · 2021: 115 · 2020: 100 · 2019: 103 · 2018: 92 · 2017: 90 · 2016: 79 · 2015: 60 · 2014: 8 · 2012: 1
FWCI
51.89
Type
journal article
Language
en
Update notices
erratum (2014-09-17)
Open access
Licence
Locations
  • Neuron (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • U.S. Department of Defense
  • Simons Foundation
  • National Institutes of Health (1dp2od001674-01)
  • National Institute of Mental Health (R01MH064168, MH64168, K01MH096956)
  • National Institute on Drug Abuse (P01DA010154, R01DA007418)
  • National Institute of Neurological Disorders and Stroke (R01NS077111, R01NS049442, NS049442, K08NS083738, R01NS060123)
  • National Center for Research Resources (S10RR026895)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetics and Neurodevelopmental Disorders
  • Life Sciences › Neuroscience › Cognitive Neuroscience › Autism Spectrum Disorder Research
  • Health Sciences › Medicine › Epidemiology › Autophagy in Disease and Therapy
Keywords
autism spectrum disorder, mTOR, autophagy, macroautophagy, synaptic pruning, dendritic spines, TSC2, ATG7, rapamycin, layer 5 pyramidal neurons, social behavior deficits, excitatory synapses
MeSH terms
Tuberous Sclerosis Complex 2 Protein, Adolescent, Age Factors, Animals, Autistic Disorder, Autophagy, Child, Child, Preschool, Disease Models, Animal, Exploratory Behavior, Female, Humans, Immunosuppressive Agents, Male, Mice, Transgenic, Neurons, Synapses, Temporal Lobe, Sirolimus, Tumor Suppressor Proteins, Dendritic Spines, Mice, Young Adult, TOR Serine-Threonine Kinases

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-06.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-06.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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