The parent R01 is based on our study of medically refractory pediatric epilepsy, where we identified decreased mRNA levels of the transcription factor, Circadian Locomotor Output Cycles Kaput (Clock), compared with non- epileptic brain. Mice with targeted deletion of the Clock gene in excitatory neurons have spontaneous seizures, leading us to hypothesize that loss of Clock leads to circuit dysfunction and epilepsy. In both the parent R01 and the proposed new experiments, we study pediatric epilepsy. In this supplement, we investigate a rare, newly diagnosed form of genetic infantile epilepsy and developmental delay caused by mutations in SLC13A5, a sodium-coupled citrate transporter, using models that we already have in the laboratory, and using the same techniques that we are using in the parent grant i.e. video EEG and whole cell patch-clamp electrophysiology in hippocampal neurons. Slc13a5 mutations result in decreased intracellular citrate levels, indicating metabolic defect. Since we have extended our parent studies of Clock gene into metabolomics, the proposed studies are in keeping with the scope and overall hypothesis that metabolic defects underlie circuit dysfunction in epilepsy. Analysis of Slc13a5 as a less complex, single gene disorder will help us understand important links between metabolic signatures and epilepsy. We generated mouse models containing two of the most commonly found Slc13a5 missense mutations in pediatric patients. Preliminary characterization revealed an unexpected gain-of-function effect of a sodium-binding domain missense mutation i.e. more severe seizures, meeting the definition of status epilepticus, in striking contrast to Slc13a5 gene ablation, which does not produce seizures, indicating altered gene function. We hypothesize that aberrant cortical and hippocampal activity arises from altered neurotransmitter levels and electrophysiological properties at excitatory and inhibitory synapses. We will test our hypothesis in two Aims.
In Aim 1, we will investigate changes in epilepsy associated with Slc13a5 mutations in comparison with Slc13a5 ablation. Using video electroencephalogram (EEG), we plan to measure seizure thresholds in these mice, interictal epileptiform abnormalities, epilepsy severity, and baseline EEG patterns.
In Aim 2, we will determine neurotransmitter changes associated with Slc13a5 mutations, and identify, by patch-clamp electrophysiology, how altered citrate or TCA cycle intermediates lead to depletion of glutamate and GABA levels. We will also investigate action potential generation threshold, firing patterns, and membrane properties to determine changes in excitatory-inhibitory balance. Analysis of hetero- and homozygous mouse null and missense mutants would help determine how the mutant allele gains function or interferes with normal gene activity. These studies constitute a major part of a thesis project for our URM graduate student, whose multi-disciplinary training plans for career growth as a neuroscience investigator are outlined in this proposal. Understanding the genetic and metabolic mechanisms may lead to new treatments for epilepsy and its associated cognitive and behavioral symptoms.
Pediatric epilepsies are common, incompletely understood, debilitating disorders of the developing brain which are accompanied by cognitive and behavioral comorbidities. Our parent proposal investigates metabolic changes underlying seizures due to a circadian regulator deficiency and these new studies will identify metabolic mechanisms of epileptogenesis underlying human mutations of Slc13a5. This supplement outlines research and a training plan to support our URM pre-doctoral trainee, adds depth to our understanding of pediatric epileptic disorders, which may provide therapeutic targets for patients carrying Slc13a5 mutations and circadian-associated epilepsy. .
