4-Phenylbutyrate Rescue in GABRA1 Variants Associated with Developmental Epileptic Encephalopathies: From Cell and...

Disease variants in GABR genes encoding γ-aminobutyric acid type A receptor (GABA_AR) subunits are major causes of developmental and epileptic encephalopathies (DEEs). There is no effective treatment for these DEEs, although the GABA_AR is a major target for antiseizure drugs. We previously identified the therapeutic effect of 4-phenylbutyrate (PBA) in Gabrg2^+/Q390X knockin DEE mice and in this study tested the effect of the drug in GABRA1 variants that encode the α1 subunit of GABA_AR. We used a multidisciplinary approach including in silico structural modeling, flow cytometry, patch-clamp recordings and biochemistry in conjunction with differential tagging of the wildtype (WT) and the mutant alleles to evaluate the effect of PBA on rescue of GABA_AR subunit expression, surface trafficking, and function in vitro in a heterologous HEK293T cell model and in vivo in Gabra1^+/A322D mice. We found that the α1 subunit expression at both the total level and the cell surface was reduced when the variant α1 protein was present, suggesting reduced functional receptor availability on the cell membrane and synapse. Patch-clamp recordings identified that α1 variants reduced GABA-evoked current amplitude. In silico prediction indicated reduced protein stability for GABRA1 variants by negative ∆∆G values. PBA increased both total and surface expression of WT α1 and α1 variants and improved expression of both WT and variant α1 alleles when these were co-expressed. Importantly, PBA also increased the GABA_AR expression in the cortex and thalamus of the Gabra1^+/A322D mice. This study indicates that PBA is a promising treatment option for DEEs associated with GABRA1 mutations. Our previous work has demonstrated that PBA improves proteostasis by enhancing expression of the WT allele, repairing the mutant allele, and reducing endoplasmic reticulum stress in other DEEs associated with GABRG2 and SLC6A1 mutations. Importantly, it can mitigate seizures and improve neurobehavioral phenotypes at behavioral levels. Based on this and our previous work on GABRG2 and SLC6A1 mutations, we propose that PBA holds promise as a common medicine for multiple genetic neurologic disorders that share the proteostasis pathology with a broad clinical application in DEEs.