Altered mTORC1 signaling in multipotent stem cells from nearly 25% of patients with nonsyndromic autism spectrum disorders

Recent autism spectrum disorders (ASD) research supports a model wherein disinhibited mTORC1 signaling and dysregulated protein synthesis in neurons contribute to the clinical features of ASD, mainly through disturbances in synaptic connectivity and plasticity.^{1, 2} Most insights into this pathomechanism came from ASD-related monogenic syndromes caused by loss of function mutations in negative regulators of PI3K-mTOR pathway (such as TSC1/TSC2, tuberous sclerosis syndrome; FMR1, fragile-X syndrome).^{3, 4} In addition, it has been recently reported aberrant mTORC1-dependent translation in a mouse model for nonsyndromic ASD (eIF4E).⁵ However, functional studies addressing mTORC1-signaling activity in accessible sources of cells from patients with nonsyndromic ASD are lacking. In this study we have made use of the cultured stem cells from human exfoliated deciduous teeth (SHEDs) derived from nonsyndromic ASD patients, a model system that, albeit non-neural, we have recently shown to be suitable to explore dysregulated pathways and biological processes in ASD,⁶ to investigate this important and yet poorly explored question.

Because mTORC1 signaling functions as an environmental sensor of nutrient availability, we took advantage of the use of cultured SHEDs derived from 13 patients (ASD1–13) and 11 age- and sex-matched controls and examined the status of mTORC1 activity in serum-deprived and serum-stimulated (after starvation) cells (see Supplementary Information). Phosphorylation analysis of key mTORC1 cascade components (p-mTOR^S2448 and its downstream effector p-rpS6^{S235/236 and S240/244}) suggested hyperfunction of mTORC1 signaling in SHEDs derived from three patients (ASD1–3), 23% of our patient sample (Figure 1a, Supplementary Figure 1a). Notably, no differences in the phosphorylation levels of mTOR and rpS6 between ASD1–3 and control cells were observed when the cells were maintained in standard growth medium without a prior step of serum starvation followed by serum stimulation (Supplementary Figure 1a), suggesting that the extracellular environment has an important role in determining ASD cell phenotype.

Figure 1

(a) mTORC1-signaling pathway analysis in SHEDs derived from patients (ASD1–13) and controls (C1–11). Western blot analysis of p-mTOR^S2448, p-rpS6^S235/236 and p-rpS6^S240/244 was conducted in serum-deprived and serum-stimulated cells and the bar graphs represent the fold changes in the phosphorylation levels of these proteins in the fetal bovine serum (FBS)-stimulated condition as compared with the FBS-deprived condition (the activation ratio, see Supplementary Information). (b) Proliferation of SHEDs derived from patients and controls. SHEDs were cultured in growth medium with 20% FBS and were collected and counted at the indicated time points. Results are expressed as the fold increase in cell number relative to 0 h. Similar results were obtained using 0.5, 5 and 10% FBS (Supplementary Figure 1b). (c) Effect of mTOR and PI3K-mTOR inhibition on the proliferation of SHEDs derived from patients and controls. SHEDs were cultured in growth medium (20% FBS) supplemented with either the vehicle or 100 nM rapamycin, and with either the vehicle or 100 nM rapamycin plus 1 uM wortmannin, collected and counted at the indicated time points. Bar graphs represent the fold change of the number of drug-treated versus untreated cells (vehicle only). Patient samples were divided into two groups according to the phosphorylation state of mTORC1-signaling components (a), and response to serum (b) or drug treatment (c). It is noteworthy that, as we have not analyzed stem cells from two different teeth of the same individual, the extent of intra-individual variation is unknown. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001; see Supplementary Information.

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In accordance with the hyperresponsive mTORC1 signaling, growth curves of ASD1–3 SHEDs in response to different fetal bovine serum concentrations showed that these cells could be clearly distinguished from the other samples analyzed, and displayed a 1.5–2-fold greater increase in cell number, mainly at the later time points of the curve (Figure 1b, Supplementary Figure 1b), and reduced response to the antiproliferative effect of rapamycin, a specific mTOR inhibitor (Figure 1c, Supplementary Figure 1c). Curiously, whereas dual PI3K-mTOR inhibition (wortmannin+rapamycin treatment) continued to be less effective in reducing to a similar extent the proliferation of ASD1–2 and control cells, it was able to restore the enhanced proliferation of ASD3 cells (Figure 1c, Supplementary Figure 1c), suggesting a possible different regulatory mechanism in ASD3 cells. Knockout mouse models of both syndromic and nonsyndromic ASD with disinhibited mTOR signaling and dysregulated protein synthesis present dysplastic and enlarged neurons with increased spine density in several brain regions.^{5, 7, 8} Although we did not notice increased ASD1–3 SHED volumes, it will now be important to determine and explore further whether the altered proliferative phenotype observed in these cells may also be observed in neuronal cell types, which could be a potential additional mechanism whereby disrupted mTORC1 signaling contributes to ASD neuropathology.

Conventional karyotyping and analysis of copy number variations at 15q11-q13, 16p11 and 22q13, found to occur more often in ASD, did not reveal any genomic aberrations in all patients except ASD3, who presents an inverted duplication of 15q11-q13. It is possible that genes located at this region may contribute to the aberrant molecular and cellular phenotypes observed in ASD3 cells. In addition, TSC1/2, FMR1, PTEN, NF1 and MeCP2 genes (ASD-associated genes known to be negative regulators of PI3K-mTOR signaling pathway) were screened for coding and splice-site mutations in ASD1–13 patients and no potentially deleterious variants were identified. Therefore, the causative genetic architecture underlying mTORC1 overactivity in ASD1–3 cells remains to be determined. It is also noteworthy that patients ASD1–3, who seem to some extent share overlapping pathophysiological mechanisms, show different degree of cognitive and social impairments: ASD1 was diagnosed with Asperger syndrome and ASD2–3 with low-functioning autism (Supplementary Table S1), suggesting that other genetic and environmental modifier factors may also have a role in cognitive development in these patients.

In conclusion, our results suggest that dysregulation of mTORC1 signaling has an important role in the pathogenesis of a subgroup of nonsyndromic ASD, and that mTOR pathway components might be promising therapeutic targets for these patients. Importantly, during the revision process of this manuscript, it was reported hyperactivated mTOR signaling in postmortem brain tissue from both adolescent patients with idiopathic ASD⁹ and 15q11-q13 duplication patients with ASD,¹⁰ which provide further strong support for this hypothesis. Finally, our results suggest that SHEDs are an alternative and more readily accessible source of patient material to study disease pathophysiology and to refine treatment approaches for individual patients.

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