One of the major focus areas of the Tuberous Sclerosis Complex Research Program is the neurocognitive, sleep, and behavioral issues associated with tuberous sclerosis complex (TSC). A white paper prepared by Dr. Vicky Whittemore in 2012 states that one of the critical priorities in TSC research is "to understand the role of gene dysfunction that leads to specific manifestations of TSC, specifically cognitive deficits and epilepsy, such that prevention strategies can be identified." To achieve this goal, it would be ideal to have a specific cell population that could be used to identify neurodevelopmental disorder-related phenotypes in a cell culture dish. However, it has been difficult until recently to identify what cells in the brain contribute to autistic behavior in patients with TSC. Until recently, research in TSC was focused on the cerebral gray matter pathology (i.e., cortical tubers). However, accumulating evidence suggests that another part of the brain, cerebellum, plays a crucial role in the neurocognitive deficits associated with TSC. One in three TSC patients displays cerebellar pathology, and the presence of cerebellar tubers has been correlated with autism spectrum disorder (ASD) symptomatology in patients with TSC. The proposed research will use the tools of mouse genetics to identify developmental pathways that are affected in TSC and human stem cell biology to study human cerebellar neurons from patients with TSC.

These studies build on studies by the Sahin lab, which demonstrated that deletion of the Tsc1 gene in cerebellar Purkinje cells recapitulates key features of autism: lack of interest in socializing, repetitive behaviors, restricted interests, and abnormal communication. Our discovery that Purkinje cells play a critical role in autistic behavior in a mouse model of TSC is an important advance in unraveling the contribution of the cerebellum to autistic behaviors in affected TSC patients. Importantly, this advance is supported by imaging studies, which provide evidence of structural changes in the cerebellum of TSC patients with autistic symptoms. Here, we propose to collaborate with the Hatten lab, which has carried out pioneering studies on cerebellar development, to generate Purkinje neurons from induced pluripotent stem cells from TSC patients with autism. We will use these patient Purkinje cells to analyze changes in gene expression in Purkinje cells with TSC mutations and to identify defects in the development of these neurons after implanting them into mouse cerebellum. Importantly, we will use novel genetic engineering methods to correct the TSC mutations and test whether this reverses defects in gene expression and in the development of the cells. Successful completion of this project will generate human TSC cerebellar neurons that can be utilized to identify novel molecular targets for the development of new drugs or other therapies for TSC and autism.