Epileptic phenotypes inslc13a5loss-of-function zebrafish are rescued by blocking NMDA receptor signaling

Abstract

AbstractSLC13A5encodes a citrate transporter highly expressed in the brain important for regulating intra- and extracellular citrate levels. Mutations in this gene cause a rare infantile epilepsy characterized by lifelong seizures, developmental delays, behavioral deficits, poor motor progression, and language impairments. SLC13A5 individuals respond poorly to treatment options; yet drug discovery programs are limited due to a paucity of animal models that phenocopy human symptoms. Here, we used CRISPR/Cas9 to create loss-of-function mutations inslc13a5aandslc13a5b, the zebrafish paralogs to humanSLC13A5.slc13a5mutant larvae showed cognitive dysfunction and sleep disturbances, consistent with SLC13A5 individuals. These mutants also exhibited fewer neurons and a concomitant increase in apoptosis across the optic tectum, a region important for sensory processing.slc13a5mutants displayed hallmark features of epilepsy, including an imbalance in glutamatergic and GABAergic excitatory-inhibitory gene expression, disrupted neurometabolism, and neuronal hyperexcitation as measuredin vivoby extracellular field recordings and live calcium imaging. Mechanistically, we tested the involvement of NMDA signaling inslc13a5mutant epilepsy-like phenotypes. Slc13a5 protein co-localizes with excitatory NMDA receptors in wild-type zebrafish and blocking NMDA receptors inslc13a5mutant larvae rescued bioenergetics, hyperexcitable calcium events, and behavioral defects. These data provide empirical evidence in support of the hypothesis that excess extracellular citrate over-chelates the ions needed to regulate NMDA receptor function, leading to sustained channel opening and an exaggerated excitatory response that manifests as seizures. These data show the utility ofslc13a5mutant zebrafish for studying SLC13A5 epilepsy and open new avenues for drug discovery.