Cilia disassembly and dysfunction in malformations of cortical development

About This Grant

PROJECT SUMMARY Malformations of cortical development (MCDs) are neurological disorders characterized by altered organization and function of cortical neurons, often leading to intractable epileptic seizures. Somatic mutations in more than 60 genes have been found in MCDs, with pathogenic variants causing focal impairment of cortical function. This genetic heterogeneity is reflected in the delineation of multiple types of MCDs, including focal cortical dysplasia type I (FCDI) and type II (FCDII) and Tuberous Sclerosis Complex (TSC). These MCD classes have overlapping and distinct genetic bases and cortical neuron phenotypes. For example, FCDII and TSC exhibit hyperactive mTORC1 signaling due to activating mutations in pathway components (or inactivating mutations in negative regulators such as TSC1 and TSC2), while FCDI does not exhibit mTORC1 alteration. Critically, the mechanisms by which MCD-associated mutations drive disease pathogenesis remain incompletely understood, and the degree to which these mechanisms are shared across MCD types is unclear. Better understanding the basis of MCDs promises to not only shed light on the fundamental biology of cortical development, but also to offer improved diagnostic tools and therapeutic strategies for MCD patients. Here, we propose a new model for MCD pathogenesis based on our recent finding that dysregulation of primary cilia is a shared feature of gene variants responsible for FCDI, FCDII, and TSC. This model is based upon the convergence of two unbiased genetic studies: a consortium-led sequencing study that identified 69 genes mutated in MCD patients and a genome-wide CRISPR activation screen we conducted that identified a novel pathway for cilia disassembly. Based on the significant overlap of these gene sets and the established importance of cilia in cortical development, we hypothesize that aberrant disassembly of primary cilia is a shared pathological basis for MCDs. In support of this hypothesis, our preliminary data show that a FCDI- associated variant in SARM1 induces cilia loss, while Sarm1 inhibition restores cilia in cells harboring mutations in SARM1 or in FCDII gene TSC2. From these findings, several questions emerge, including: how does FCDI-associated mutation of SARM1 impact cilia and cortical development in vivo; what is the functional relationship between cilia loss induced by Sarm1 versus by mTORC1; can inhibition of cilia disassembly mitigate FCD-associated pathology; and do other ciliary regulators contribute to MCDs? We will address these questions in three aims: 1) test the effect of an FCDI-associated SARM1 variant on neuronal ciliation, cortical development, and seizure activity, 2) test the hypothesis that mutation of FCDII gene TSC2 alters neuronal development and function through Sarm1-mediated cilia disassembly, and 3) use CRISPR screening to define additional genes that promote cilia loss in MCD. These studies will be performed through a collaborative effort of the Breslow and Bordey labs that combines their unique and complementary expertise in cilia, ciliopathies, functional genomic screening, MCD pathogenesis, and murine cortical development and function.