Promoting Protein Trafficking with 4-phenylbutyrate to Treat Genetic Epilepsy

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Despite the discovery of hundreds of genetic mutations associated with epilepsy and neurodevelopmental comorbidities – including autism and intellectual disability (ID) – no effective treatments are currently available. Our recent work has identified the primary common mechanisms across mutations at a large scale in astrocytes and inhibitory neurons that express the GABA transporter 1 (GAT-1) encoding SLC6A1. Preliminary findings suggest 4-phenylbutyrate (PBA), a previously FDA-approved chaperone inducer for pediatric use, displays a mechanism-based rescue in those mutations. Specifically, PBA appears to repair mildly misfolded proteins and increase membrane expression of the wildtype allele across all tested mutations. Moreover, our success has prompted a pilot trial with very promising results. This study aims to fully characterize the effect and detailed mechanisms of PBA rescue. Our central hypothesis states that protein misfolding and impaired trafficking are standard mechanisms for SLC6A1 mutations, which display rescue potential through pharmacological intervention by restoring effective protein activity. As a part of our research, our lab has developed highly relevant preclinical model systems, including a plasmid library of SLC6A1 mutations, mutation- bearing patient cell lines, and knockin mouse models. Future studies focused on these model systems will provide critical insights into disease mechanisms with high potential of translating the findings to treatment. Moving forward, our lab aims to (1) evaluate the effect of PBA on restoring GAT-1 function in vitro for 20 patient mutations, (2) gauge the effect of PBA on restoring GAT-1 function in vivo in Slc6a1 mutation knockin mice, and (3) elucidate the underlying mechanism of PBA rescue on GAT-1 functioning to establish a foundation for novel treatment approaches. Research design and methods. We will employ a plasmid library containing >50 SLC6A1 mutations identified from patients across a wide spectrum of disease phenotypes, two knockin mice, and two lines of patient induced pluripotent stem cells (iPSCs) derived neurons and astrocytes to determine the impact of PBA on the mutant GAT-1 trafficking and function. We will utilize a multidisciplinary approach, including in vivo microdialysis, to determine the dynamic interplay of GAT-1 inhibition, GABA levels, and seizures in knockin mice. All patients carrying SLC6A1 mutations are heterozygous, which suggests there is benefit in either boosting the remaining wildtype allele or rescuing the mutant copy or both. In either case, the overall GABA uptake activity should be improved in patients. The large-scale study will provide us a broad view of the impact of PBA. In contrast, the in-depth investigation of mice and patient-derived cells will provide critical insights into PBA's rescue mechanism in partial and complete loss-of-function mutations. We propose to test this hypothesis in vitro and in vivo with high-throughput assays and cutting-edge new techniques in iPSCs and mutation knockin mice. Our goal is to identify a novel treatment target for genetic epilepsy using SLC6A1 as example. We believe the impact of this study is broad as it can be scaled up for many genetic epilepsy syndromes and others.