Abstract
Tuberous sclerosis complex 1 (TSC1) is a tumour suppressor gene that inhibits the mechanistic target of rapamycin (mTOR) pathway. Mutations in TSC1 lead to a rare complex disorder of the same name, in which up to 50% of patients present with autism spectrum disorder (ASD). ASD is a highly prevalent, early-onset neurodevelopmental disorder, characterized by social deficits and repetitive behaviours, although the type and severity of symptoms show wide variability across individuals. Amongst different brain areas proposed to play a role in the development of ASD, the cerebellum is commonly reported to be altered, and cerebellar-specific deletion of Tsc1 in mice is sufficient to induce an ASD-like phenotype. Given that the mTOR pathway is crucial for proper cell replication and migration, this suggests that dysregulation of this pathway, particularly during critical phases of cerebellar development, could contribute to the establishment of ASD.
Here, we used a mouse model of TSC to investigate gene and protein expression during embryonic and early postnatal periods of cerebellar development. We found that, at E18 and P7, mRNA levels of the cerebellar inhibitory interneuron marker Pax2 were dysregulated. This was accompanied by changes in the expression of mTOR pathway-related genes and downstream phosphorylation of S6. Differential gene correlation analysis revealed dynamic changes in correlated gene pairs across development, with an overall loss of correlation between mTOR- and cerebellar-related genes in Tsc1 mutants compared to controls. We corroborated the genetic findings by characterizing the mTOR pathway and cerebellar development on protein and cellular levels with Western blot and immunohistochemistry. We found that Pax2-expressing cells were hypertrophic at E18 while, at P7, their number was increased and maturation into parvalbumin-expressing cells delayed. Our findings indicate that E18 and P7 are crucial time points in cerebellar development in mice that are particularly susceptible to mTOR pathway dysregulation.
Manuscript contribution to the field ASD is one of the most prevalent neurodevelopmental disorders, however little is known about the shared mechanisms underlying its aetiology. At the anatomical level, the cerebellum has been identified as one of the key structures involved in the development of ASD, whereas at the molecular level, mutations in the mTOR signalling pathway, essential for cell growth and proliferation, carry a high genetic risk for this disorder. We used a haploinsufficient tuberous sclerosis complex 1 (Tsc1) mouse model to investigate the effects of mTOR overactivation in the developing cerebellum. Tsc1 inhibits the mTOR pathway, and mice with cerebellar-specific deletion of Tsc1 have been shown to harbour an ASD-like phenotype. We found that Pax2 expression in the cerebellum is dysregulated at prenatal and early postnatal time points, leading to a delayed maturation of inhibitory interneurons. Our findings indicate that mTOR overactivity in the cerebellum selectively affects the development of cerebellar interneurons. This finding is in line with other studies, which found decreased numbers of inhibitory interneurons in other models of ASD. Therefore, deficits in the maturation of the inhibitory signalling could be one of the mechanisms integrating high-risk mutations that underlie ASD aetiology.
Competing Interest Statement
The authors have declared no competing interest.