Oral Sodium Hyaluronate Reshapes Gut Microbiota Composition and Suppresses the LPS-TLR4/NF-κB Pathway to Exert Neuroprotection in MPTP-Induced Parkinson's Disease.

Pathogenic variants in genes involved in inhibitory neurotransmission comprise a substantial fraction of known monogenetic causes of neurologic disorders, including epilepsies and more severe developmental and epileptic encephalopathies (DEEs) [,]. Among these, variants in genes that encode subunits of γ-aminobutyric acid (GABA) type A receptors (GABARs) are particularly prominent. GABARs mediate most synaptic inhibition and a significant portion of tonic inhibition in the brain and are essential for controlling neuronal excitability. GABARs are ionotropic ligand-gated chloride channels comprising five subunits with nineteen known isoforms. Most synaptic GABA Rs are comprised of two α1-3, two β2/3, and one γ2S subunit. The α1β2/3γ2S combination is by far the most prevalent in the brain, and the , , , and genes encoding these subunits are the most frequently occurring pathogenic variants []. Most known cases are dominant mutations, with a smaller proportion of familial cases []. In recent years, our lab has begun developing a promising new therapy for monogenetic epilepsies, including variant-mediated disorders, using 4-phenylbutyrate (PBA), a small molecule with chemical chaperone and histone deacetylase inhibitor properties that is approved by the FDA for treatment of urea cycle disorders [,]. mutations destabilize the subunit protein, impacting intracellular trafficking and/or interaction with partnering subunits, which affects GABAR expression and functional properties [,,,]. PBA remedies this pathomechanism two-fold by stabilizing mutant protein in mild cases where this is possible or by assisting the clearance of severely misfolded proteins and improving expression of the functional wildtype (WT) allele and partnering subunits in a fashion similar to its effects in other monogenetic disorders [,,,,,,,,,,] and acquired injuries [,]. Previously, we demonstrated this for the (Q390X) variant associated with Dravet syndrome, which exhibits a severe trafficking pathology where mutant protein is retained in the endoplasmic reticulum and has a dominant negative effect on partnering subunits, significantly reducing GABAR expression and synaptic inhibition and causing seizures []. Treatment with PBA promoted clearance of the mutant protein and improved expression of the functional WT allele and partnering subunits in cell and mouse models, improving synaptic inhibition and significantly reducing the seizure burden []. Additionally, we have been successful in using PBA to treat variant-mediated disorders in both preclinical models [,] and humans [,], which have a similar pathomechanism. For these disorders, mutant variants in GABA transporter 1 are frequently misfolded, resulting in reduced expression and transporter-mediated GABA uptake. Treatment with PBA improved intracellular trafficking and expression of the transporter, increased GABA uptake in cells and the (S295L) mouse model, and reduced the mouse seizure burden and neurobehavioral phenotypes [,,,,]. These studies formed the basis for a clinical trial for PBA treatment, which reported seizure reduction or resolution in 70% of the patients tested []. Clinical trials for variant-mediated disorders are also under way, and a significant need exists for translational studies evaluating the efficacy of PBA for mutations, including subunits that have not yet been evaluated, including . In this study, we evaluate the effect that mutations have on GABAR subunit stability and expression and function. We also determine if PBA can restore these deficits. We focus on the variants I148F, R214C, R214H, M253T, T292S, and F325L (A; ) obtained from the CURE GABA-A patient organization. Additionally, we include the (A322D) variant associated with autosomal dominant juvenile myoclonic epilepsy. A322D was the first variant to be identified, and it has been extensively characterized, displaying a misfolding/trafficking pathology with a modest dominant negative effect on partnering subunits [,,,,]. Functional studies have also been done on R214C, R214H, T292S; R214C and R214H, which were identified in patients with Dravet syndrome and exhibit gating defects, showing diminished GABAR currents, with differing reports regarding expression [,,,]. The T292S variant is associated with severe developmental delay without epilepsy and shows increased GABA sensitivity and normal expression, indicating a gain in GABAR function []. No functional characterization has yet been done for I148F, F325L identified in a patient with epilepsy [], and M253T identified in a West syndrome patient []. To predict the destabilizing effects these variants have on the subunit protein, we used computational tools and measured the expression of these variants along with the WT allele and partnering subunits in the HEK293 heterologous expression system and mice. Then, we tested if PBA treatment can improve GABAR subunit expression.