Investigation of the GATOR1 complex genes in focal cortical dysplasia and focal epilepsy

Abstract

Epilepsy is a complex disease characterised by seizures due to abnormal neuronal activity. Both hereditable and non-hereditable epilepsy forms of epilepsy exist, and understanding their underlying mechanisms leads to more effective and targeted treatments. Following advances in DNA sequencing technologies, more cases of epilepsy caused by genetic mutations have been identified. Therefore, the investigation into how these mutations result in seizures is a worthwhile avenue of research, and will improve diagnosis and treatment of affected individuals. DEPDC5, NPRL2 and NPRL3 encode the GATOR1 complex and mutations in each of these genes have been found in patients with familial focal epilepsy and focal cortical dysplasia (FCD) type II. Heterozygous patients are affected with variable severity, variable foci and incomplete penetrance. The function of these genes and the mechanisms of how their mutation cause the disease are not well understood. In vitro studies have recently found that GATOR1 functions to downregulate the mTORC1 signalling pathway in response to depleted amino acid levels. The mTORC1 pathway is involved in regulating many cellular processes including protein synthesis, cell growth and autophagy. Many diseases, including some epilepsies, arise from mTORC1 dysregulation and are termed ‘mTORopothies’. mTOR dysregulation is therefore hypothesised to be the major factor in the pathology of GATOR1-related epilepsy. To better understand the roles these genes play in causing epilepsy and FCD, this thesis uses mutant mouse models and cell lines engineered with CRISPR/CAS9 genome editing technology. Firstly, a knockout mouse model for Depdc5 highlights the gene’s in vivo role in regulating the mTORC1 signalling pathway and in embryonic development. Depdc5 homozygous null mice do not survive past the mid-late stages of gestation, showing a range of gross malformations in the head and brain. Brain lysates show hyperactivity of the mTORC1 pathway, establishing a role for the deregulation of this pathway in disease. Heterozygous mice, however, develop normally and do not show any susceptibility to seizures despite patients of this genotype being affected. Interestingly, heterozygous GATOR1 patients present with focal neurological pathologies. It is hypothesised that this may result from a second somatic mutation occurring during the brain development of germline heterozygotes, causing a region of tissue lacking GATOR1 function. We investigate this mechanism and accurately model the features of the disease with a Depdc5 conditional mouse. Using CRISPR/CAS9, we generated a floxed allele which, following the unilateral electroporation of Cre into developing embryo brains, recombines to result in discrete regions of null neuron ‘clones’. This successfully modelled FCD and epilepsy phenotypes including increased soma growth, increased dendritic complexity, abnormal morphology, migration defects and lower seizure thresholds. mTORC1 hyperactivity is evident in regions of brain pathology, where the gene function has been abolished, which further supports deregulation of mTORC1 as the major pathogenic mechanism. To investigate the functional outcomes of specific patient variants of all three GATOR1 genes, we develop a functional assay using GATOR1 null cell lines, measured by the ability to rescue of null phenotypes in vitro and in vivo contexts. We confirm that some variants for each of the GATOR1 genes have lost the ability to inhibit mTORC1 activity in amino acid starvation conditions. Additionally, using the Depdc5 conditional mouse model, we confirm the functional outcome of two DEPDC5 variants in vivo, and the downstream effects their loss-of-function has on disease phenotypes. Importantly, we also find the majority of missense variants were not functionally compromised. This highlights the importance of functional screening of GATOR1 gene variants in regard to pathogenic classification and accurate diagnosis, which in turn leads to improved therapies and genetic counselling . Collectively, these investigations using mutant cell lines and mutant mice support the involvement of mTORC1 dysregulation in GATOR1-related epilepsy and FCD through a second-hit disease mechanism.