Role of mTORC1 dependent translation in neurological deficits of TSC

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ABSTRACT Tuberous sclerosis complex (TSC) is a developmental disorder caused by a mutation in the TSC1 or TSC2 gene. Mutations in TSC genes result in hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1), a signaling pathway vital for development. Individuals with TSC present with a high incidence of neurological conditions, among the most prevalent of which are epilepsy and autism spectrum disorder (ASD). While mTORC1 inhibitors can effectively attenuate seizures, they do not improve TSC’s autistic comorbidity. Thus, there is a critical need to better understand the etiology of autism traits to identify novel treatments. Our laboratoryhasdevelopedamousemodelofTSCin which constitutively active Rheb is expressed in developing cortical neurons of the medial prefrontal cortex (mPFC), a region that is dysregulated in TSC and implicated in ASD. This model allows for a more precise activation of mTORC1 than transgenic models and is thus ideally suited to parse out complex etiology. Using this model, our lab found alterations in local mPFC connectivity that resemble the pathological changes in TSC’s autistic comorbidity. Moreover, when aberrant cap dependent translation was normalized via the activation of a translational repressor, the observed alterations were prevented. While connectivity has been examined within the mPFC in a hyperactive mTORC1 state, the accompanying behavioral phenotype has not been assessed nor have mTORC1’s effects on specific subcortical projections from the mPFC. To this end, the striatum is a compelling target, as it is heavily interconnected with the mPFC and is integral to socio-communicative behaviors (core deficits in ASD). However, axonal connectivity in TSC is understudied and mPFC-striatal projections have not been assessed. Furthermore, while normalizing translation can mitigate cellular alterations in hyperactive mTORC1 conditions, the specific downstream molecular alterations that account for these changes are unknown, as are their efficacy to attenuate behavioral deficits. Therefore, the specific aims are to: 1). Determine whether hyperactive mTORC1 in mPFC-striatal projections contributes to socio-communicative deficits. 2). Determine whether increasing mTORC1 activity during early mPFC development leads to alterations in cortico-striatal projections resulting in an excitation-inhibition imbalance in the striatum. 3). Determine whether altered cap-dependent translation and downstream molecules contribute to alterations in axonal connectivity and socio-communicative deficits. To address these aims, in utero electroporation (IUE) will be used to increase mTORC1 in pyramidal neurons of the mPFC. Vocalization and social interaction tests will assess the phenotype, whereas lightsheet microscopy, axonal tracing, and electrophysiology will assess mPFC-striatal connectivity. Potential candidate molecules downstream of translation that control axonal growth will be identified via Trap, and shRNA will be designed to assess the effects of normalizing the identified genes on socio-communicative deficits and alterations in mPFC-striatal connectivity.