Autism spectrum disorder (ASD) is a complex neurodevelopmental monogenic disorder with a strong genetic influence. Idiopathic autism could be defined as a type of autism that does not have a specific causative agent. Among signalling cascades, mTOR signalling pathway plays a pivotal role not only in cell cycle, but also in protein synthesis and regulation of brain homeostasis in ASD patients. The present review highlights, underlying mechanism of mTOR and its role in altered signalling cascades as a triggering factor in the onset of idiopathic autism. Further, this review discusses how distorted mTOR signalling pathway stimulates truncated translation in neuronal cells and leads to downregulation of protein synthesis at dendritic spines of the brain. This review concludes by suggesting downstream regulators such as p70S6K, eIF4B, eIF4E of mTOR signalling pathway as promising therapeutic targets for idiopathic autistic individuals.
Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder categorized by concomitant manifestation of diminished social communication, restricted/perseverative/stereotypical behaviour and repetitive pattern of activities. The incidence rate of ASD was 58.7 per 10,000 individuals in 2013 (1). However, the number of reported cases with mild to severe autism has gradually increased over the years in various countries. In the United States alone, 1 in 68 children has ASD (2). ASD can be differentiated into four different disorders based on the Diagnostic and Statistical Manual of Mental Disorders IV (DSM – IV): Autistic disorder, Asperger’s disorder, Childhood disintegrative disorder and Pervasive developmental disorder (PDD) (3–6). According to DSM – V manual, autism is broadly classified into syndromic and non–syndromic types (7). Syndromic autism comprises of disorders with known causative agents, including monogenic disorders (8) such as Phelan-McDermid syndrome, Noonan syndrome, Rett-syndrome, Timothy syndrome, Tuberous sclerosis complex (TSC), TSC associated Poly hydramnios megalencephaly and symptomatic epilepsy syndrome (PMSE), Phosphatase and tensin homolog (PTEN), Fragile X Syndrome (FXS), Neurofibromatosis, and a number of developmental disorders covered by ASD (3, 9). Idiopathic autism is the non–syndromic type with no known genetic or epigenetic cause for manifestation of the disease (9). Probable causative factors for idiopathic autism include environmental factors (10) such as toxins, pesticides, infection, and in utero exposure to drugs like valproic acid (9–14). There has been various hypotheses, mutational targets, and pathways about idiopathic autism (9, 12). However, none of them could explain the exact reason behind the onset and expression of this disorder.
Signalling pathways are important for the regulation of specific molecular mechanism after activation through a target molecule. Alteration in some signalling pathways can lead to expression of certain features in the human brain, including megalocephaly, axonal misregulation, alteration in neuron size, connectivity of neuronal circuits, proliferation of cerebral cells, protein synthesis and dendritic spine density variation at different regions of the brain (15, 16). Among various pathways, mechanistic target of rapamycin (mTOR) plays a centralized role as a signalling hub that can lead to regulation of certain physiological features of autism. Specifically, in the brain, mTOR pathway is involved in the regulation of synaptogenesis, corticogenesis, and associated functions of neurons (2). Several studies (9, 13, 16) have indicated that the Akt/mTOR pathway which regulates translation at dendritic spines is a potential molecular substrate of autism. Indeed, mutations in genes encoding Akt-mTOR cascade components cause disorders with higher rates of autistic characteristics. Nicolini
Neurodevelopment comprises of certain crosslinked molecular mechanisms including neurogenesis, corticogenesis, and synaptogenesis. Adult neurogenesis is the development of new neurons in specific regions of the brain such as the hippocampus and olfactory bulb. This process is particularly important for the conversion of Neural Stem Cells (NSCs) and neural precursors into functional neurons (17). Corticogenesis that leads to mammalian neurocortex development is a part of embryonic neurogenesis. It is required for the origin of six layers of the cortex where neuronal migration begins at the ventricular zone and proceeds towards their final position in the brain (18, 19). Synaptogenesis is the concluding step in neural development which comprises of initiation and linkage of pre- and post-synaptic domains in target neurons as well as regulation of synaptic development through mTOR and Wnt signalling pathways. Following synaptogenesis, ratio of excitatory to inhibitory synapses (E/I ratio) is also important for neural circuit assembly and it analysis the synaptic output (20, 21). Alterations in these key regulatory processes has been reported in neurodegenerative and developmental disorders such as ASD, Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, Amyotrophic Lateral Sclerosis and Schizophrenia (17). During corticogenesis, neuronal migration and laminar formation are critical for brain function. Their disruption due to overexpression of certain glycoproteins such as reelin secreted by cajal-retzius cells and GABAergic interneurons can lead to ASD, bipolar disorder, and schizophrenia (22). Oblak
Autism is linked to certain signalling pathways such as Wnt pathway, calcium and calmodulin pathway and PI3K/mTOR signalling pathway. Wnt pathway gives a better understanding of cellular differentiation, polarity, and proliferation in different tissues. Its deregulation leads to various types of cancers and certain cases of ASD as reported by Oron and Elliot (34). Maturation and development of brain are associated with the canonical Wnt pathway. In individuals with ASD, the Wnt signalling pathway is linked to cortical patterning, upregulation of dendritic spine density, and alteration of spine morphology (35). Other than Wnt signalling, calcium concentrations and calmodulin binding capacity can also affect various components of the Central Nervous System (CNS), specifically targeting the pre- and post-synaptic responses of neurons (36). Wen
mTOR plays a centralised role in inducing and activating various interlinked pathways. Thus, it acts as a signalling hub and regulates certain physiological features of the cell. Various intracellular and extracellular processes including protein synthesis, RNA biogenesis, autophagy, cell growth, and survival (39) are pictorially presented in Fig. 1. mTOR is also associated with various diseases including various cancers (40, 41) such as prostate cancer (42), kidney cancer (43), and breast cancer (44) due to mutations and misregulations in the mTOR pathway. It is also associated with other diseases including cardiovascular disease (45), renal diseases (46), pulmonary fibrosis (47), and diabetes mellitus (48). Associations of mTOR signalling pathway with normal and abnormal brain, neurite development, synapse formation and associated functions have been discussed in detail. mTOR signalling pathway is also associated with abnormal developmental features of the brain including Megalencephaly (49, 50), hemimegalencephaly (51), pigmentary mosaicism (50), glioblastoma multiforme (52), astrocytoma (52), and focal cortical dysplasia (53). These defects are mainly associated with malfunction of mTOR associated cells either upstream or downstream of the signalling cascade (50, 54). Specifically, in the brain, mTOR is involved in the regulation of synaptogenesis, corticogenesis, and associated functions of neurons. mTOR is also related to a number of neuropsychatric and neurodevelopmental disorders such as Alzheimer’s disease, ASD, and idiopathic autism (55). Altered mTORC1 activity due to mutations in TSC has been linked to Alzheimer’s disease characterised by accumulation of cellular amyloid-β proteins, decreased soma size, and decreased activities of mTORC1 and mTORC2 (56). Activation and phosphorylation of Akt, a protein kinase, play a major role in the onset of mTOR pathway because Akt targets TSC and regulates mTORC1 activity (57). Mutations in TSC1 and TSC2 can lead to alteration in the activity and size of brain, leading to megalencephaly and dysmorphisms of developing neurons, glia, and progenitor cells (58). In contrary to Alzheimer’s where altered soma size is the main characteristic feature, hyperactivity of mTOR signalling in ASD leads to megalencephaly, overconnectivity of neurons and increases the spine growth (15).
Overlaps among cellular phenotypes, genes, and molecular and biochemical pathways have led to the classification of a number of monogenetic syndromes as part of the broader spectrum of ASD as depicted in Fig. 2 (7). Mutations in mTOR signalling including TSC1, TSC2, PTEN, and Phosphoinositide 3-kinases (PI3K) components have also led to expression of these disorders (59) as explained in detail in Table 1. One of the major upstream signalling components that activate mTORC1 is Ras homolog enriched in brain (RHEB) in brain cells. It could be blocked by mutated TSC, leading to tumorous outgrowth in the brain. TSC complex inhibits RHEB and mTORC. When this upstream process is altered, there is hyperactivation of mTORC1. Another regulating molecule is PTEN which is involved in Akt activation and lipid signalling by regulating PI3K levels. Misregulation of PI3K leads to hyperactivity of not just mTORC1, but also Akt pathway (60). Another not well explored aspect of mTOR pathway is the fragile X mental retardation protein (FMRP) that plays a role in activating fragile X syndrome (FXS). It is known that 50% of FXS individuals have ASD and mutation in the
Despite there are increased knowledge about pathways that create havoc in syndromic autism, there are far fewer researches related to the physiology and manifestation of idiopathic autism. VPA (66) induced knockout mouse models and induced Pluripotent Stem Cells (iPSC) (67) are the widely used models to study idiopathic autism. Similar to knockout mice models, BTBR mice can serve a model organism for studying ASD and idiopathic autism. Meyza and Blanchard (68) have highlighted the importance of BTBR T+Itpr3tf/J mouse for studying idiopathic autism as convergence of multiple signalling pathways and exhibition of distinct neuroanatomical features.
mTOR is involved in various neurodevelopmental processes, including neuronal differentiation, axon guidance, cell migration, and neural region patterning. These processes are altered in idiopathic autism. Roles of dendritic spine formation, function, and neural plasticity at the fusiform gyrus in idiopathic autism have been studied by Nicolini
Novel therapeutic target identification and implementation against diseases have become mandatory for various neurodevelopmental conditions, especially ASD and idiopathic autism since the number of individuals affected by them is increasing globally (3). mTOR is involved in the regulation of various intracellular processes as explained in Fig. 1. However, its key role in ASD and idiopathic autism is the maintenance of amino acid pool by regulating protein translation at dendritic spines in the brain (71).
mTOR-S6K is also involved in the regulation of translation by eIF4F, a protein composed of eukaryotic initiation factors eIF4E and eIF4A (71, 72). Inhibition of cap binding to eIF4F by 4EBPs as a major rate altering process could be prevented when mTORC1 phosphorylates 4EBPs which hinders the inhibitory role of this molecule, thereby activating the translation initiation at specific regions of the brain such as dendritic spines (73). Phosphorylation of specific subunits of mTOR involved in translational initiation is necessary to regulate various cellular processes as shown in Fig. 1. These molecules include eIF4B which activates eIF4A, S6 ribosomal subunit (74), PDCD4 that inhibits eIF4A translation by phosphorylation, and various mRNA splicing factors (75). Presence of certain postsynaptic proteins including postsynaptic density protein 95 kDa (PSD-95), NMDA, metabotropic glutamate receptor subtype 5 (mGlu R5) that take part in translational initiation is important for structural organisation of prefrontal cortex of the brain (76). Phosphorylation of these downstream regulators has been studied by Jernigan
mTOR plays a critical role in not only the regulation of various cellular functions such as cell growth, cell proliferation, lipid synthesis, and protein synthesis, but also plays a critical role in neurodevelopmental processes such as regulation of neurogenesis, corticogenesis, synaptogenesis, axon guidance, and cellular migration (2). Truncation of signalling molecules of mTOR that govern both upstream and downstream processes in the cascade can lead to developmental disorders such as ASD and idiopathic autism. Subjects with idiopathic autism exhibit drastic decreases in synaptic activity, dendritic spine growth, cellular proliferation, protein synthesis, and spiking activity due to significant downregulation of mTOR signalling molecules in the brain. The major process that could alter the downregulation of mTOR signalling cascade is truncated mRNA translation through binding to protein 4E-BP1 and altering 5′ capped mRNA translation initiation by initiation factor eIF4E. eIF4E either up-regulates or decreases p70S6K and eIF4B expression which in turn can modulate mTOR dependent translation (9). As no alteration was reported in 4E-BP1 and eIF4E but a demur in p70S6K signalling was observed in the mTOR pathway, p70S6K could be targeted as a regulator of idiopathic autism. Collectively, we conclude that proper regulation of p70S6K and associated down regulators of mTOR signalling pathway is a critical in the maintenance of brain homeostasis.
The authors would like to thank Bharathiar University and the Science and Engineering Research Board (SERB) (ECR/2016/001688), Government of India, New Delhi for providing necessary help in carrying out this Autism review process.
The authors have no conflicting interests.
Effect of various mTOR associated signalling molecules autism spectrum disorders
|Affected region in brain||Effect of mTOR associated molecule/Results||Model system||Process involved||Disease induced||Analytical methods||Reference|
|PTEN||Hippocampus, Cerebral cortex||Macrocephaly, Neurohypertrophy||Mouse||AKT/mTOR pathway activation and Gsk3β inactivation||-||Cre mediated recombination in mice||86|
|Increased length and thickness in dendritic arbors||Behavioural testing of mutant mice|
|Variation in response to sensory stimuli, learning and anxiety.||Immunohistochemical staining|
|PTEN||Hippocampus, Cerebral cortex||Macrocephaly, Neurohypertrophy, Increased seizures, Decreased adaptability to environmental stimuli, Pten’s effect on PI3K cascade, inturn affects the circadian rhythm.||Mouse||Circadian rhythm||-||EEG/EMG recording||87|
|Wheel running test|
|Tsc1||Neural progenitor cells in Sub Ventricular Zone||Heterotropia in RMS and OB Enlarged microglia||Mouse||Increased mTOR signaling||Tuberous Sclerosis||Electrophoration of plasmid||88|
|Rheb||Cell migration disrupted by Rheb with Mash1 cells at the RMS||Migration, morphometric, micronodule assays|
|Neuronal migration speed decreased||Immunostaining|
|Increased mTOR signalling does not affect the action potential||Confocal microscopy|
|Tsc1||Hippocampal pyramidal neurons||Increased phosphorylation of S6||Mouse||Tsc up/down regulation||Tuberous Sclerosis||Immunostaining||89|
|Tsc2||Absence of Tsc1 leads to increase in soma size but decrease, decrease in dendritic spines, increased synaptic current||Rat||Two-photon laser scanning microscopy|
|Rictor||Central Nervous System||mTORC2 affects cell size, Neuronal morphology and function||Mouse||Rictor deficiency||-||Immunohistochemical analysis||90|
|Purkinje cells||Only RiPuKO mice had synaptic functional changes||Electrophysiological analysis|
|mTORC2 plays key role in synaptic homeostasis||Biochemical analysis|
|Mouse behaviour analysis|
|Tsc1||Hippocampal neurons||Increase in action potential, dendrite length and soma size||Mouse||Excitatory and inhibitory synaptic transmission||-||Electrophysiological analysis||91|
|Pten||Tsc1 loss does not affect the excitatory neurons, unlike pten which increases it||Immunocytochemistry|
|Excitation to inhibition ration in the neural network is altered|
|Tsc1||Axon||Tsc1/2 plays an important role in axon formation, neural polarity||Mouse||Polarised activation and inactivation of Tsc pathway||Tuberous Sclerosis||Transfection||92|
|Tsc2||Tsc2 inhibition leads to mTOR activation||Immunocytochemistry|
|Tsc/mTOR pathway leads to axonal regeneration||Immunohistochemistry|