Genetic and Functional Mechanisms in Citrate Transporter Disorder associated with SLC13A5 - ABSTRACT SLC13A5 epilepsy is a newly recognized form of Developmental Epileptic Encephalopathy 25 (DEE25) with seizures beginning within the first days of life along with subsequent intellectual and motor symptoms. In these patients, mutations in the SLC13A5 gene, which encodes a plasma membrane citrate transporter, result in a severe, early onset multi-focal epilepsy and cognitive and behavioral symptoms. How disruption of SLC13A5 function results in dysfunction of neural circuitry is unknown. However, SLC13A5 loss of function may not account for the full severity of the disorder, and truncations of SLC13A5 are rarely observed in patients. Instead, human genetics show that certain mutations are over-represented as known causative mutations; SLC13A5 G219R (DNA G655A) and T227M (DNA C680T) are the most common recurrent mutations found in approximately two-thirds of all known patients. While the epilepsy is associated with bi-allelic mutations, the presence of recurrent missense mutations suggests mechanisms more complex than simple autosomal recessive genetics. However, these have not been fully investigated. In order to better understand the genetics of SLC13A5 epilepsy, we have developed novel experimental systems containing mouse alleles with mutations in Slc13a5. In this supplement, we describe plans to examine the cellular and molecular properties of human SLC13A5 and the human SLC13A5 G219R disease variant in a novel mouse model. Based on known species differences between mouse and human SLC13A5 proteins, and our observations of pathogenicity of the human G219R mutation in Drosophila and in cultured mouse neurons, we hypothesize that the expression of the G219R disease variant of human SLC13A5 in mice impairs neural cell development, and is associated with aberrant protein properties, such as localization and stability. We will determine pathogenicity of a novel knock- in SLC13A5-G219R mouse allele expressing the human SLC13A5-G219R cDNA from the mouse Slc13a5 locus (MMRRC-hSLC13A5-G219R). In Aim 1 we will analyze neural cell specific expression of hSLC13A5- G219R protein in the mouse brain, and determine whether gray and white matter regions of the MMRRC- hSLC13A5-G219R show significant neuropathological changes at different postnatal ages. In Aim 2, we will analyze SLC13A5 protein expression to determine whether the hSLC13A5-G219R variant is expressed at lower levels than non-transgenic and/or humanized knockin SLC13A5-WT controls (MMRRC-hSLC13A5-WT), or is associated with cellular stress. These studies will enhance our investigation into pathogenic mechanisms of SLC13A5 mutations, and form the research component in the training of our post-baccalaureate research assistant, Jayden Johnson. He will be integrated into our research team on the SLC13A5 project while he gains experience in histological techniques and advanced microscopy, as well as biochemical and molecular analyses. Together with conference activities and training in rigor, ethics and scientific communication, Jayden will build research skills in preparation for entry into a graduate program in Neuroscience.
