Epilepsy Mechanisms and the Path to Intervention - PROJECT SUMMARY/ABSTRACT Mutations in the SLC13A5 gene, which encodes a plasma membrane citrate transporter, result in a newly diagnosed form of genetic epilepsy termed early infantile epileptic encephalopathy, which is characterized by multi-focal seizures in neonates. These infants subsequently develop cognitive and behavioral deficits. Human genetics has identified both commonly occurring missense and deletion mutations, but it is not known how distinct genetic mutations affect disease presentation and seizure severity. We are addressing this question by characterizing an array of SLC13A5 mutant mouse models carrying: i) ablation of its endogenous murine Slc13a5 gene (knockout), ii) the most common patient mutation, the G222R point mutation (equivalent to the human mutation G219R), and iii) the second most common patient mutation, the T230M (equivalent to the human mutation T227M). Our preliminary data demonstrates that homozygous Slc13a5 knockout mouse demonstrates abnormal epileptiform electroencephalogram (EEG) profiles, while the G222R has seizures and significantly more severe epileptiform activity. Preliminary histopathology reveals differential interneuron reduction between the knockout and G222R, with the G222R additionally showing oligodendroglial loss. These results are consistent with our interpretation that specific missense mutations may acquire dominant gain-of-function effects which exacerbate vulnerability and neuronal hyperexcitability. The knockout has shown reduced excitatory and inhibitory postsynaptic activity suggestive of reduced neurotransmitter levels. We will further characterize these mutant mouse models with the following experiments: experiment 1.1, the characterization of interictal discharges and seizures in the mutant alleles using EEG, in parallel with analysis of brain histopathology. In experiment 1.2, I will apply electrophysiological techniques to investigate whether a deficiency in intracellular citrate transport in Slc13a5 knockout and missense mutations affects neuronal function by altering glutamate and GABA concentrations. A metabolomics screen for neurotransmitters will be performed to corroborate functional data. In experiment 1.3, I will investigate the effect of these SLC13A5 mutant proteins on cellular pathologies. Combined, these experiments will investigate the molecular and cellular mechanisms that contribute to the seizure phenotype in the SLC13A5 disease, which in turn will inform therapeutic approaches. I will acquire training in electrophysiology and molecular biology to follow through with these experiments. Our research training plan and ongoing professional development will provide me with skills to transition me to the next stages of my scientific career. In the postdoctoral stage, I plan to gain experience with gene therapy strategies in experimental models of nervous system disease. I will continue to investigate cellular and molecular mechanisms underlying brain disease, and to apply these findings to optimize gene therapy approaches. Furthermore, I will continue to develop the professional skills required to become an independent primary investigator at an academic research institution.
