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Decreased mTOR signaling pathway in human idiopathic autism and in rats exposed to valproic acid.

Nicolini C, Ahn Y, Michalski B, Rho JM, Fahnestock M - Acta Neuropathol Commun (2015)

Bottom Line: The molecular mechanisms underlying autistic behaviors remain to be elucidated.Full-length TrkB, PI3K, Akt, phosphorylated and total mTOR, p70S6 kinase, eIF4B and PSD-95 were reduced in autistic versus control fusiform gyrus.However, no changes in 4E-BP1 or eIF4E were found in autistic brains.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry & Behavioural Neurosciences, McMaster University, 1280 Main Street West, Hamilton L8S 4K1, ON, Canada. fahnest@mcmaster.ca.

ABSTRACT

Background: The molecular mechanisms underlying autistic behaviors remain to be elucidated. Mutations in genes linked to autism adversely affect molecules regulating dendritic spine formation, function and plasticity, and some increase the mammalian target of rapamycin, mTOR, a regulator of protein synthesis at spines. Here, we investigated whether the Akt/mTOR pathway is disrupted in idiopathic autism and in rats exposed to valproic acid, an animal model exhibiting autistic-like behavior.

Methods: Components of the mTOR pathway were assayed by Western blotting in postmortem fusiform gyrus samples from 11 subjects with idiopathic autism and 13 controls and in valproic acid versus saline-exposed rat neocortex. Additionally, protein levels of brain-derived neurotrophic factor receptor (TrkB) isoforms and the postsynaptic organizing molecule PSD-95 were measured in autistic versus control subjects.

Results: Full-length TrkB, PI3K, Akt, phosphorylated and total mTOR, p70S6 kinase, eIF4B and PSD-95 were reduced in autistic versus control fusiform gyrus. Similarly, phosphorylated and total Akt, mTOR and 4E-BP1 and phosphorylated S6 protein were decreased in valproic acid- versus saline-exposed rats. However, no changes in 4E-BP1 or eIF4E were found in autistic brains.

Conclusions: In contrast to some monogenic disorders with high rates of autism, our data demonstrate down-regulation of the Akt/mTOR pathway, specifically via p70S6K/eIF4B, in idiopathic autism. These findings suggest that disruption of this pathway in either direction is widespread in autism and can have adverse consequences for synaptic function. The use of valproic acid, a histone deacetylase inhibitor, in rats successfully modeled these changes, implicating an epigenetic mechanism in these pathway disruptions.

No MeSH data available.


Related in: MedlinePlus

Quantification of (A) full-length (FL)/truncated and (B) truncated/full-length (FL) TrkB isoform protein ratios in fusiform gyrus of autism and control samples. **p = 0.002 and **p = 0.009, respectively, 2-tailed t tests. Bars indicate mean ± SE. Autism, n = 11; control, n = 13. (C) Quantification of full-length (FL) TrkB and (E) truncated TrkB isoform protein expression in fusiform gyrus of autism and control samples by Western blotting. Each sample was normalized to its β-actin. **p = 0.003 for TrkB-FL and p = 0.09 for truncated TrkB isoforms, 2-tailed t tests. Bars indicate mean ± SE. Autism, n = 11; control, n = 13. The mean from two independent Western blots per sample was used for statistical analysis. (D) and (F) Representative Western blots of fusiform gyrus showing autism (A) and control (C) cases. Lanes 2 to 5: standard curve consisting of different amounts of total protein from a single normal human cortex sample. Lanes 6 to 13: 35 μg of total protein from each autism and control sample.
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Fig5: Quantification of (A) full-length (FL)/truncated and (B) truncated/full-length (FL) TrkB isoform protein ratios in fusiform gyrus of autism and control samples. **p = 0.002 and **p = 0.009, respectively, 2-tailed t tests. Bars indicate mean ± SE. Autism, n = 11; control, n = 13. (C) Quantification of full-length (FL) TrkB and (E) truncated TrkB isoform protein expression in fusiform gyrus of autism and control samples by Western blotting. Each sample was normalized to its β-actin. **p = 0.003 for TrkB-FL and p = 0.09 for truncated TrkB isoforms, 2-tailed t tests. Bars indicate mean ± SE. Autism, n = 11; control, n = 13. The mean from two independent Western blots per sample was used for statistical analysis. (D) and (F) Representative Western blots of fusiform gyrus showing autism (A) and control (C) cases. Lanes 2 to 5: standard curve consisting of different amounts of total protein from a single normal human cortex sample. Lanes 6 to 13: 35 μg of total protein from each autism and control sample.

Mentions: The TrkB/Akt pathway is known to influence PSD-95 trafficking to spines [37]. Thus, differences in relative TrkB protein isoform levels in fusiform gyrus between autism and control groups were examined by Western blotting. A significant reduction in full-length TrkB (TrkB-FL)/truncated TrkB (TrkB-T1 + TrkB-Shc) isoform ratio and increase in the ratio of truncated TrkB isoforms/TrkB-FL (**p = 0.002, Figure 5A; **p = 0.009, Figure 5B; 2-tailed t tests) were observed in the fusiform gyrus of individuals with idiopathic autism versus controls. This may be due to significantly decreased TrkB-FL (**p = 0.003, Figure 5C,D; 2-tailed t test) as well as a trend towards increased truncated TrkB (p = 0.09, Figure 5E,F; 2-tailed t test). TrkB-FL and truncated TrkB are differentially expressed in different cell types: TrkB-FL is neuronal whereas truncated TrkB isoforms are widely expressed in glia. Therefore, we measured protein levels of GFAP (glial) and βIII-Tubulin (neuronal) markers. There were no significant differences in GFAP or βIII-Tubulin protein expression in the fusiform gyrus (p = 0.4, Figure 6A,B; p = 0.5, Figure 6C,D; 2-tailed t tests) between control and autism subjects, which precludes differential cell loss as a mechanism for TrkB isoform alterations. There were also no differences between groups in β-actin levels (p = 0.7, 2-tailed t test, data not shown), which were used to normalize samples on the Western blots.Figure 5


Decreased mTOR signaling pathway in human idiopathic autism and in rats exposed to valproic acid.

Nicolini C, Ahn Y, Michalski B, Rho JM, Fahnestock M - Acta Neuropathol Commun (2015)

Quantification of (A) full-length (FL)/truncated and (B) truncated/full-length (FL) TrkB isoform protein ratios in fusiform gyrus of autism and control samples. **p = 0.002 and **p = 0.009, respectively, 2-tailed t tests. Bars indicate mean ± SE. Autism, n = 11; control, n = 13. (C) Quantification of full-length (FL) TrkB and (E) truncated TrkB isoform protein expression in fusiform gyrus of autism and control samples by Western blotting. Each sample was normalized to its β-actin. **p = 0.003 for TrkB-FL and p = 0.09 for truncated TrkB isoforms, 2-tailed t tests. Bars indicate mean ± SE. Autism, n = 11; control, n = 13. The mean from two independent Western blots per sample was used for statistical analysis. (D) and (F) Representative Western blots of fusiform gyrus showing autism (A) and control (C) cases. Lanes 2 to 5: standard curve consisting of different amounts of total protein from a single normal human cortex sample. Lanes 6 to 13: 35 μg of total protein from each autism and control sample.
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Fig5: Quantification of (A) full-length (FL)/truncated and (B) truncated/full-length (FL) TrkB isoform protein ratios in fusiform gyrus of autism and control samples. **p = 0.002 and **p = 0.009, respectively, 2-tailed t tests. Bars indicate mean ± SE. Autism, n = 11; control, n = 13. (C) Quantification of full-length (FL) TrkB and (E) truncated TrkB isoform protein expression in fusiform gyrus of autism and control samples by Western blotting. Each sample was normalized to its β-actin. **p = 0.003 for TrkB-FL and p = 0.09 for truncated TrkB isoforms, 2-tailed t tests. Bars indicate mean ± SE. Autism, n = 11; control, n = 13. The mean from two independent Western blots per sample was used for statistical analysis. (D) and (F) Representative Western blots of fusiform gyrus showing autism (A) and control (C) cases. Lanes 2 to 5: standard curve consisting of different amounts of total protein from a single normal human cortex sample. Lanes 6 to 13: 35 μg of total protein from each autism and control sample.
Mentions: The TrkB/Akt pathway is known to influence PSD-95 trafficking to spines [37]. Thus, differences in relative TrkB protein isoform levels in fusiform gyrus between autism and control groups were examined by Western blotting. A significant reduction in full-length TrkB (TrkB-FL)/truncated TrkB (TrkB-T1 + TrkB-Shc) isoform ratio and increase in the ratio of truncated TrkB isoforms/TrkB-FL (**p = 0.002, Figure 5A; **p = 0.009, Figure 5B; 2-tailed t tests) were observed in the fusiform gyrus of individuals with idiopathic autism versus controls. This may be due to significantly decreased TrkB-FL (**p = 0.003, Figure 5C,D; 2-tailed t test) as well as a trend towards increased truncated TrkB (p = 0.09, Figure 5E,F; 2-tailed t test). TrkB-FL and truncated TrkB are differentially expressed in different cell types: TrkB-FL is neuronal whereas truncated TrkB isoforms are widely expressed in glia. Therefore, we measured protein levels of GFAP (glial) and βIII-Tubulin (neuronal) markers. There were no significant differences in GFAP or βIII-Tubulin protein expression in the fusiform gyrus (p = 0.4, Figure 6A,B; p = 0.5, Figure 6C,D; 2-tailed t tests) between control and autism subjects, which precludes differential cell loss as a mechanism for TrkB isoform alterations. There were also no differences between groups in β-actin levels (p = 0.7, 2-tailed t test, data not shown), which were used to normalize samples on the Western blots.Figure 5

Bottom Line: The molecular mechanisms underlying autistic behaviors remain to be elucidated.Full-length TrkB, PI3K, Akt, phosphorylated and total mTOR, p70S6 kinase, eIF4B and PSD-95 were reduced in autistic versus control fusiform gyrus.However, no changes in 4E-BP1 or eIF4E were found in autistic brains.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry & Behavioural Neurosciences, McMaster University, 1280 Main Street West, Hamilton L8S 4K1, ON, Canada. fahnest@mcmaster.ca.

ABSTRACT

Background: The molecular mechanisms underlying autistic behaviors remain to be elucidated. Mutations in genes linked to autism adversely affect molecules regulating dendritic spine formation, function and plasticity, and some increase the mammalian target of rapamycin, mTOR, a regulator of protein synthesis at spines. Here, we investigated whether the Akt/mTOR pathway is disrupted in idiopathic autism and in rats exposed to valproic acid, an animal model exhibiting autistic-like behavior.

Methods: Components of the mTOR pathway were assayed by Western blotting in postmortem fusiform gyrus samples from 11 subjects with idiopathic autism and 13 controls and in valproic acid versus saline-exposed rat neocortex. Additionally, protein levels of brain-derived neurotrophic factor receptor (TrkB) isoforms and the postsynaptic organizing molecule PSD-95 were measured in autistic versus control subjects.

Results: Full-length TrkB, PI3K, Akt, phosphorylated and total mTOR, p70S6 kinase, eIF4B and PSD-95 were reduced in autistic versus control fusiform gyrus. Similarly, phosphorylated and total Akt, mTOR and 4E-BP1 and phosphorylated S6 protein were decreased in valproic acid- versus saline-exposed rats. However, no changes in 4E-BP1 or eIF4E were found in autistic brains.

Conclusions: In contrast to some monogenic disorders with high rates of autism, our data demonstrate down-regulation of the Akt/mTOR pathway, specifically via p70S6K/eIF4B, in idiopathic autism. These findings suggest that disruption of this pathway in either direction is widespread in autism and can have adverse consequences for synaptic function. The use of valproic acid, a histone deacetylase inhibitor, in rats successfully modeled these changes, implicating an epigenetic mechanism in these pathway disruptions.

No MeSH data available.


Related in: MedlinePlus