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Abstract

L-Lysine (Lysine) is an essential, cationic amino acid and therefore must be acquired through a protein-rich diet to allow for normal growth. Intracellularly, lysine is either used in protein biosynthesis or is metabolized through one of two pathways, the mitochondrial saccharopine pathway or the cytosolic pipecolic acid pathway. Pyridoxine-dependent epilepsy (PDE), a rare autosomal recessively inherited lysine metabolic disorder, is caused by loss-of-function mutations to the gene ALDH7A1, which encodes the mitochondrial enzyme α-aminoadipic semialdehyde dehydrogenase (ALDHA1). The reduction of enzymatic activity due to these mutations causes the accumulation of the lysine metabolites α-aminoadipic semialdehyde (α-AASA) and piperideine-6-carboxylate (P6C), which in turn cause various forms of oxidative damage within brain tissues and metabolic dysregulation from the inactivation of pyridoxal-5-phosphate (PLP) by accumulating levels of P6C. Children that present with PDE are prescribed a treatment known as triple therapy which consists of lifelong dietary lysine restriction, arginine supplementation, and pyridoxine supplementation. Unfortunately, large concentrations of arginine must be taken orally to sufficiently lower levels of plasma lysine. I aim to better understand lysine metabolism and transport in hopes of improving therapeutic strategies targeted towards lysine catabolic disorders. Using cells and tissues isolated from mice, I showed that the brain, kidney and liver highly express Aldh7a1, thereby supporting the notion that these organs are major hubs for lysine metabolism. I developed an in vitro PDE model in HEK293 cells and through stable isotope tracing revealed that both my model and primary mouse astrocytes from Aldh7a1⁻/⁻ mice primarily catabolized lysine through the saccharopine pathway. I developed a fluorescent sensor which could measure the effectiveness of various amino acid derivatives as competitive inhibitors for the lysine transporters. Through liquid chromatography-mass spectrometry (LC-MS) I further characterized the capability of successful compounds in limiting the accumulation of lysine and lysine-derived metabolites in cells and in Aldh7a1-/- mice. Through my results we obtained a better understanding of lysine metabolism and transport. In addition, I laid the groundwork for future studies which aim to identify more potent competitive inhibitors of lysine transport.