Disruption of mTOR and MAPK pathways correlates with severity in idiopathic autism

The molecular signature underlying autism spectrum disorder remains largely unknown. This study identifies differential expression of mTOR and MAPK pathways in patients affected by mild and severe idiopathic autism. A total of 55 subjects were enrolled, of which 22 were typically developing individuals and 33 were patients aged between 3 and 11 years, with autism spectrum disorder. A detailed history, including physical examination, developmental evaluation, mental health history and autism diagnostic observation schedule were performed for each patient. Components of the mTOR and MAPK signalling pathways were analysed from peripheral blood at the protein level. Patients were then stratified according to their clinical phenotypes, and the molecular profiling was analysed in relation to the degree of autism severity. In this cohort of patients, we identified increased activity of mTOR and the MAPK pathways, key regulators of synaptogenesis and protein synthesis. Specifically, rpS6, p-eIF4E, TSC1 and p-MNK1 expression discriminated patients according to their clinical diagnosis, suggesting that components of protein synthesis signalling pathways might constitute a molecular signature of clinical severity in autism spectrum disorder.


Introduction
Autism spectrum disorder (ASD) is characterized by high phenotypic heterogeneity, including deficits in social interaction and communication, as well as repetitive and unusual behaviours 1,2 . The aetiology of ASD is still largely unknown, although a complex genetic basis is present 3 . Some insights have been gained through the study of specific genetic disorders, such as fragile X syndrome (FXS) and tuberous sclerosis (TSC), two monogenic disorders characterized by a high incidence of ASD ranging from 25 to 60% [4][5][6] . FXS, the most common form of inherited intellectual disability, is caused by the loss or mutation of the fragile X mental retardation protein (FMRP) 7,8 . FMRP regulates several aspects of mRNA metabolism [9][10][11] . At synapses, FMRP regulates local protein synthesis at different levels 9 , with one of the characterized mechanisms based on the binding to CYFIP1 (cytoplasmic FMRP interacting protein 1) and eIF4E (eukaryotic translation initiation factor 4E) [12][13][14][15] . TSC is a dominantly inherited multisystem disorder characterized by the formation of hamartomas in different organs and the brain, caused by mutations in the TSC1 or TSC2 genes encoding hamartin and tuberin, respectively. TSC1 and TSC2 form a biochemical complex that inhibits the mammalian (also named mechanistic) target of rapamycin mTOR signalling pathway 16 . A large majority (85%) of children and adolescents with TSC display epilepsy, 50% cognitive disorders and~30-40% ASD 16,17 .
Several forms of syndromic autism and intellectual disabilities (IDs), including FXS and TSC, are associated with mutations in genes that regulate protein synthesis and affect structure, transmission and plasticity of synapses 18 . Thus, it has been hypothesized that aberrant synaptic protein synthesis might contribute to ASD and other IDs that share ASD-like clinical features 19,20 . In the brain, mTOR and MAPK signalling pathways regulate synaptogenesis and local protein synthesis 21 . The MAPK pathway regulates neural progenitor biogenesis, learning and memory 22,23 and mRNA translation, by phosphorylation of TSC2 and eIF4E via MAPK-interacting kinase 1 and 2 (MNK1 and MNK2) 14,24 . The MAPK pathway is upregulated in patients with syndromic ASD 25 . Furthermore, the deletion of the 16p11.2 locus, which includes the MAPK3 (mitogen-activated protein kinase 3) gene, is associated with ASD 26,27 .
The mTOR kinase is part of two functionally distinct biochemical complexes: mechanistic target of rapamycin complex 1 (mTORC1) and mechanistic target of rapamycin complex 2 (mTORC2) 24,28 . mTORC1 affects protein synthesis by phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (eIF4E-BP1) and p70 ribosomal protein S6 kinase (S6K1) 21,28 . The mTOR pathway is dysregulated in several human diseases, including cancer and diabetes 29,30 as well as intellectual disabilities, such as FXS, Rett syndrome and ASD 30 . Thus, there is a wealth of evidence implicating mTOR and MAPK pathways, and ultimately protein synthesis, in syndromic ASD. Surprisingly, however, less is known about the function of these pathways in idiopathic ASD.
Here, we sought to characterize mTOR and MAPK pathways in children with idiopathic autism who had no other identifiable clinical syndromes. We investigated the expression of the above-described two pathways in peripheral blood mononuclear cells (PBMCs) of children with idiopathic autism and in typically developing individuals (TDI). In addition to performing a global analysis of patients versus TDI, we also analysed the molecular profile according to the clinical severity of ASD to identify a potential molecular signature of the disease severity.

Subjects
This research was approved by the ethical committee of the University Hospital of Tor Vergata (Rome, Italy) and all participants provided written informed consent. Clinical data were gathered from 33 patients (27 males and 6 females) with idiopathic autism, recruited among those attending the Division of Child Neurology and Psychiatry, University Hospital of Tor Vergata (Rome, Italy). Patients ranged in age from 3 to 11 years, and met clinical criteria for a diagnosis of ASD, based on the diagnostic and statistical manual of mental disorders, fifth edition (DSM5) 31 . The diagnosis of ASD was also confirmed by using the autism diagnostic observation schedule (ADOS) test. Exclusion criteria included (1) neurodevelopmental disorders of known aetiology (as FXS, TSC, chromosomal abnormalities), (2) significant sensory or motor impairment, (3) significant medical conditions known to affect brain development, (4) low birth weight (< 2200g) or prematurity (<36 week of gestation).
The neuropsychological evaluation of children with ASD, involving development and cognitive assessment, was conducted by means of a diagnostic protocol, which included the administration of (1) the psychoeducational profile third edition (PEP-3) 32 to determine the developmental level of young children with autism, (2) Leiter International Performance Scale-Revised (Leiter-R) 33 to evaluate non-verbal-cognitive levels and (3) the autism diagnostic observation schedule (ADOS) 34 for measuring the severity of autistic symptoms. The patient group was compared with a control group of 22 TDI, children and adolescents, ranging from 3 to 17 years of age (10 males and 12 females), who attended the outpatient clinic of the "Tor Vergata" University Hospital (Rome) for routine visits. The medical history of these children did not report evidence of any neurological or psychiatric disease. All children underwent a medical workup, including neurological examination, awake/sleep EEG, standard genetic analysis and auxological measurements. In addition, medical record examinations and parental interviews about the child history were performed. Consistency was required between parental information and medical records.
PEP-3: a standardised norm-referenced scale to assess the development of communication and motor skills and the presence of maladaptive behaviours of children suspected of ASD, with a developmental age between 2 and 7 years, who may be non-verbal, have limited attention skills and poor concentration, and who are not used to a formal testing situation. The PEP-3 does not provide a development quotient (DQ) like PEP-R, obtained by dividing the child's age-equivalent score by his/her chronological age. We generated the scale by estimating a development level (DL) by the ratio of developmental age/ chronological age 35 for each developmental subtest.
Leiter-R: expressively non-verbal measure of global intelligence with fair cross-cultural applicability. Its engaging, non-verbal format makes it ideal for use with individuals with ASD. It provides an IQ score, as well as percentile and age-equivalent scores for each subtest. For cases where the IQ score was not possible to evaluate, DL was assessed.
In accordance with a recent study, DLs of each subtest of the PEP-3, can be compared with Leiter-R IQ and can be considered as indicators of cognitive functioning in subjects with ASD 36 . The decision to administer the PEP-3 or the Leiter-R scale or both, was based on the child's developmental skills, attention skills and age.
ADOS: a semi-structured assessment of communication, social interaction and relatedness, play, imagination and stereotyped or repetitive behaviours. This measure yields scores in the social domain, communication domain, as well as a combined score. A child can be classified as having autistic disorder, pervasive development disorder not otherwise specified (PDD-NOS), or as non-autistic.
The modules provide social-communicative sequences that combine a series of unstructured and structured situations. The examiner selects the module that is most appropriate for a child on the basis of his/her expressive language level. Module 1 is intended for individuals who do not have verbal language or use just simple words; Module 2 for individuals with some phrase speech who are not verbally fluent; Module 3 for verbally fluent children.
CSS (Calibrated Score System): a standardized severity score based on codes within the domains, that can be calculated to compare autism symptoms across modules 37 . Based on the CSS, each child was assigned to AUT, for severe symptoms, ASD, for mild symptoms and NS for non-significant symptoms. In our clinical severity analysis, we separated patients into "Mild" and "Severe" groups based on their CSS designation as "ASD" or "AUT", respectively. The division into groups based on cognitive function was performed using the scores obtained by the cognitive evaluation Leiter-VR or development assessment PEP-3, in accordance with recent data 36 .

Peripheral blood mononuclear cell (PBMCs) isolation
Blood samples were collected by venepuncture into EDTA-coated tubes. PBMCs were isolated from whole blood (9 ml) using Histopaque-1077 (Sigma-Aldrich) according to manufacturer's instructions. All samples in this study represent patient peripheral blood mononuclear cells (PBMCs) that were freshly purified after blood collection and not cultured.

Statistical analysis
Sample sizes were based on our preliminary results, where we determined the need for a sample size of at least n = 21/group for a power of 85%, alpha = 0.05, and effect size: Hedges' g = 0.67. Randomization of subjects was not possible in this study, as the goal was to compare patients versus controls. Comparisons between patients with ASD and controls were performed using nonparametric Mann-Whitney U test. When more than two groups were compared, analyses were performed using generalized linear models with maximum likelihood estimation followed by post hoc Tukey's multiple comparison test.
InStat statistical software (GraphPad Softwares Inc., La Jolla, CA) was used for the statistical analysis. Canonical discriminant analysis (CDA) was used to investigate whether a set of differentially expressed proteins could discriminate among three groups of patients according to their clinical disease status (controls, mild ASD and severe ASD), using SPSS (IBM Corp. Armonk, NY, US) software, version 24.0. The analysis employed a stepwise technique, based on the Wilks' ʎ test with the standard probabilities of F (3.84 to enter and 2.71 to be removed) for variable selection, keeping variables with a statistically significant classification performance (p < 0.05) in the model. Crossvalidation was performed using a leave-one-out method to assess the model performance.
Significance was denoted as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars represent the standard error of the mean (SEM). Variances between groups were similar. Outliers were calculated using the Grubbs' outlier test and were excluded from the figures and the statistical analysis.

Clinical summary
Comprehensive clinical data were gathered from 33 patients with idiopathic autism (Table 1).
At the time of the evaluation, 21 children had acquired verbal language. Of these, 9 children were able to use phrase speech, while the remaining 12 were able to use only simple words. Five children showed comorbid hyperactivity and attention problems, and two children were suffering from celiac disease. All patients had an established diagnosis of ASD at the time of enrolment. For this study, they were re-evaluated for core clinical features of ASD using the ADOS; for developmental skills using standardised scale psychoeducational profile third edition (PEP-3); and for nonverbal-cognitive abilities using the Leiter-R scale. Three patients were unable to complete the neuropsychiatric evaluations (Table 1). Their data were included in the global analyses, but excluded in analyses based on disease severity.
Thirty subjects were evaluated for the presence of ASD symptoms using the ADOS. According to the expressive language level of the child, the examiner selected the most appropriate ADOS module. ADOS-module 1 was used for 22 patients (10 non-verbal children; 12 verbal children whose language was constituted by simple words); ADOSmodule 2 (for individuals with some phrase speech not verbally fluent) for two children; ADOS-module 3 (for verbally fluent children) for six patients.
All subjects, except one (ID 15), exceeded cut-off criteria scores for ASD on the ADOS. For each child, a score was calculated according to the Calibrated Score System (CSS), a standardized severity score based on codes within the domains, calculated to compare autism symptoms across ADOS-modules 37 . Each child was then assessed for cognitive and development. The PEP-3 or the Leiter-R scales or both were applied based on the child's developmental skills, attention skills and age. Children were grouped according to the scores obtained in the cognitive evaluation Leiter-VR or functional assessment PEP-3, in accordance with recent data 36 (Table 1).

Expression of the components of mTOR and MAPK signalling pathways in patients with idiopathic autism
We first performed a global analysis of the expression levels of 17 proteins involved in the mTOR and MAPK pathways in the blood of patients with ASD compared to typically developing controls ( Fig. 1 and Supplementary  Fig. 1). We targeted proteins with a strong link to local protein synthesis modulation.
The key effector mTOR did not show any significant differences either in the total level or in the phosphorylation status (Ser2448 site) between ASD patients and controls ( Supplementary Fig. 1a). The two downstream mediators of the mTORC1 complex activity, 4E-BP1 and the p70S6K1 kinase, were also similar between groups ( Supplementary Fig. 1a,b). Moreover, no differences were observed in the translational repressor FMRP (Supplementary Fig. 1b). Interestingly, the 40S ribosomal subunit component, rpS6, was increased in ASD patients compared to controls (p = 0.03) (Fig. 1a). Moreover, while the translation initiation factor eIF4E did not exhibit differential expression between ASD patients and TDI, its activated phosphorylated form (Ser209) was significantly higher in ASD patients compared to TDI (p < 0.0001) (Fig. 1a).
Since mTORC1 activity and protein synthesis initiation are inhibited by the TSC complexes 38 , we next investigated if TSC1 and TSC2 levels were dysregulated in ASD patients. However, no differences were observed in TSC1 and TSC2 protein levels ( Supplementary Fig. 1c). We also investigated the expression level of RHEB, a protein known to interact and activate mTORC1 38 . RHEB levels also did not differ between ASD patients and TDI (Supplementary Fig. 1c).
ERK1-2 (extracellular signal-regulated kinase 1-2) signalling is also involved in the translational control downstream of the activation of cell surface receptors 23,39 . The expression of p44/42 MAPK (ERK1-2) was significantly increased in ASD patients compared to controls (p = 0.04), however there were no differences in its phosphorylated status on the Thr202/Tyr204 residues (p-ERK1-2) (Fig. 1b). Interestingly, while the downstream effector of ERK1-2, MNK1, did not show differences between the groups, we observed a significant increase in MNK1 phosphorylation levels (Thr197/202) in ASD patients compared with controls (p = 0.004) (Fig. 1b), suggesting increased activity at the end of this pathway. Taken together, our findings point to increased activity in mTOR and MAPK pathways in children with idiopathic ASD.

Clinical-molecular correlations
Given the heterogeneity of phenotypes found in autism spectrum disorder, and the variability of protein expression observed within our cohort, we hypothesized that protein expression may change in relation to the severity of the disease. To address this question, we analysed the molecular profile according to the clinical phenotype for each patient. Based on the CSS classification, we identified several of the previously analysed 17 proteins that correlated with the severity of the disease. Among those, p-eIF4E, rpS6 and p-MNK1 proteins, which were increased in the global analysis, exhibited significantly increased levels in the severe group compared with controls (p-eIF4E: p = 0.0003; rpS6: p = 0.03; p-MNK1: p = 0.01; Fig. 2a-c). While ERK1-2 levels were changed in the global analysis, we did not observe any correlation with the severity of ASD. Interestingly, we observed an increase of TSC1 levels in the mild ASD subgroup but not in the severe subgroup (p = 0.048; Fig. 2d).
Together, these findings suggest that mild and severe autism may be differentiated according to different protein expression profiles.
Finally, as we identified four proteins (p-eIF4E, rpS6, TSC1 and p-MNK1) that demonstrated an association with the clinical severity, we wanted to investigate if, together, those proteins could constitute a molecular signature for autism severity. Therefore, we performed a canonical discriminant analysis between the disease groups and healthy controls. Remarkably, the analysis generated two canonical functions that significantly separated the patient groups with 71.9% accuracy (p = 0.01; Fig. 2e). A cross-validation of the analysis revealed a robust model with 68.8% of cross-validated cases correctly classified. Thus, these four proteins are able to distinguish, with high accuracy, patients with ASD from typically developing controls as well as patients with mild versus severe autistic clinical features.

Discussion
The complexity and heterogeneity of ASD clinical phenotypes makes it difficult to determine the severity of ASD symptomology, predict prognoses and stratify individuals for research purposes 40 . In this study, we investigated mTOR and MAPK signalling pathways in the peripheral tissue of autism spectrum disorder patients in order to identify a molecular signature both for the disease and for ASD severity. Globally, we found a significant increase of rpS6, p-eIF4E, ERK1-2 and p-MNK1 levels in  Table 1) patients with ASD compared with controls ( Fig. 1). Interestingly, we observed a large variability in the protein expression levels of ASD patients compared with typically developing controls that was related to their clinical phenotype. When patients were stratified according to their CSS classification, we found several proteins (p-eIF4E, rpS6, TSC1 and p-MNK1) that appear to constitute a molecular signature of severity. Indeed, a canonical discriminant analysis revealed that p-eIF4E, rpS6, p-MNK1 and TSC1 proteins could discriminate between the three subgroups (controls, mild ASD and severe ASD) (Fig. 2e). We suggest that this set of proteins is a molecular readout, which correlates with ASD severity. The identified proteins are positive translational regulators, suggesting a peripheral hyperactivation of protein synthesis in autism. Compelling evidence in mice and humans indicates that aberrant synaptic protein synthesis is associated with several forms of neurodevelopmental disorders 19,[41][42][43] . At the molecular level, a dysregulation of MAPK and mTOR pathways has been suggested to disrupt protein synthesis [19][20][21][44][45][46] . Additionally, a link between two direct regulators of protein synthesis, eIF4E-BPS and S6K1 kinase, in autistic-like phenotypes in mice 20 and human cells 46,47 was proposed. Moreover, our findings are supported by studies in post-mortem brains and T cells from idiopathic ASD patients, where a hyperactivation of the mTOR pathway was observed 47,48 .
Functionally, in the brain, mTOR and MAPK are downstream effectors of group I mGluRs, NMDA and TrkB receptors 24 . MAPKs modulate the activation of MNK1, which phosphorylates eIF4E on serine 209 49 and enhances protein synthesis 50 . Notably, our cohort of ASD patients displayed a significant increase of p-MNK1 and p-eIF4E, both in the global expression and in the clinical correlations (Figs. 1a, b and 2a, c). Surprisingly, the increase in p-MNK1 in ASD patients was not accompanied by an increase in ERK1-2 activation, suggesting that the activation of MNK1 is ERK-independent. MNK1 can also be regulated by the upstream MAP kinase p38 51 , thus, future studies should examine the possibility for increased activation of this MAP kinase. Furthermore, we analysed the phosphorylation of 4E-BP1 and did not see any significant differences between groups (Supplementary Fig. 1a), suggesting that the upregulation of eIF4E phosphorylation is linked to the MNK axis rather than the mTOR signalling. Ultimately, our findings indicate a hyperactivation of the MAPK pathway leading to hyperphosphorylation of p-eIF4E, which arguably results in aberrant protein synthesis.
Studies in humans and mice indicate that an increased activity of eIF4E leads to ASD and autistic-like phenotypes respectively 52-55 eIF4E activity is modulated by the mitogen-activated protein kinase (MAPK)-interacting kinases (MNKs) 49 . Specifically, MNK1 is required for translational activation in cell migration, cancer metastasis 56 and in response to BDNF in the mouse cortical neurons and in the dentate gyrus 14,57 . Our work here identifies a new role for p-MNK1 in idiopathic ASD, providing further evidence for the contribution of the MAPK pathway to idiopathic ASD 58 .
Patients affected by FXS and TSC disorders meet the diagnostic criteria of ASD, suggesting that mutations in these pathways may cause ASD 21,59 . The TSC complex inhibits mTOR activity, leading to translational repression 38,60 . Tsc1 deletion in neurons has been linked to macrocephaly and embryonic lethality, and Tsc1 models present defects in synaptic plasticity [61][62][63] . Despite the fact that the small sample size limits interpretations, the observed increase of TSC1 levels may indicate an increased inhibition of protein synthesis in the mild subgroup only. Moreover, these results suggest a possible bidirectional disruption of protein synthesis in ASD. A bidirectional alteration in protein synthesis has been previously proposed in two mouse models for ASD. Fmr1 -/y and TSC2 +/mice displayed opposing effects in synaptic protein synthesis that compensate each other in the double mutant Fmr1 -/y TSC2 +/mice 19 . While we observed increased levels of some positive regulators of protein synthesis in the severe subgroup, our data also found an increased level in the negative protein synthesis regulator, TSC1, in the mild ASD group (Fig. 2d). Our study strengthens the bidirectional disruption hypothesis, and indicates that similar mechanisms may occur in humans.
Our findings suggest an overlap in the molecular mechanisms between syndromic and idiopathic ASD. Recently, we demonstrated an increase in protein synthesis levels in non-neuronal cells of individuals with FXS 64 . As similar findings were obtained in neuronal and nonneuronal cells within the same mouse 64 , we suggest that peripheral levels of protein synthesis may function as a representative neuronal molecular signature for future clinical trials aiming to restore translational control in FXS and other neurodevelopmental disorders. Interestingly, several studies have explored the use of human peripheral blood cells as a surrogate for the neuronal molecular signature in neurological diseases, suggesting that lymphocytes may constitute an easily accessible "neural probe" to study neuropsychiatric disorders such as autism 65,66 . Overall, our results suggest that defective protein synthesis may constitute the potential molecular mechanism underlying ASD pathology across different etiologies 67 .
Our data propose that an increase in both MAPK and mTOR pathways possibly leads to aberrant protein synthesis (Fig. 3) in subjects with ASD, indicating that alteration of mTOR and the MAPK pathways may contribute to intellectual disabilities and to both syndromic and non-syndromic ASD. Although further studies in different cohorts are required, we suggest that measurements of key protein synthesis regulators may constitute a molecular signature in PBMCs that may be predictive for early diagnosis of autism, particularly across severity groups. This may facilitate the identification of suitable targets for future personalised therapies.