Authors :
Presenting Author: Melissa DeLeeuw, MS – Vanderbilt University Medical Center
Karishma Randhave, BS – Vanderbilt University Medical Center
Ekta Anand, BS – Vanderbilt Unviersity
Ziang Song, BS – Vanderbilt University Medical Center
Wangzhen Shen, MD – Vanderbilt University Medical Center
Jing-Qiong Kang, MD, PhD – Vanderbilt University Medical Center
Rationale:
GABA transporter 1 (GAT-1), encoded by SLC6A1, is responsible for reuptake of γ-aminobutyric acid (GABA) from the synaptic cleft and is critical for maintaining inhibitory tone in the brain. Pathogenic variants in SLC6A1 result in a spectrum of neurodevelopmental disorders, including epilepsy, autism, intellectual disability, anxiety, and motor and language delays. Many of these conditions are treatment-resistant, emphasizing the urgent need for targeted therapeutics. We previously demonstrated that 4-phenylbutyrate (PBA) improves GAT-1 function and reduces seizures in preclinical models. However, it remains unclear whether its benefit is driven by pharmacochaperoning, which facilitates protein folding and trafficking, or histone deacetylase (HDAC) inhibition. This study evaluates the mechanistic basis of PBA's therapeutic activity by comparing it to known pharmacochaperones and HDAC inhibitors across multiple SLC6A1 variants.
Methods:
We evaluated 32 SLC6A1 variants in HEK293T cells using 3H-labeled GABA uptake assays and Western blot. PBA and tauroursodeoxycholic acid (TUDCA) were tested as pharmacochaperones, while butyrate and suberoylanilide hydroxamic acid (SAHA) were tested as HDAC inhibitors. In vivo seizure activity was recorded using EEG in heterozygous Slc6a1+/S295L and Slc6a1+/A288V knock-in mice.
Results:
Pharmacochaperones, PBA and TUDCA, significantly increased GABA uptake across all heterozygous variants tested, with PBA demonstrating the most robust effect. Both drugs significantly increased mature GAT-1 expression and improved trafficking by reducing endoplasmic reticulum (ER) retention of WT protein in heterozygous conditions. In contrast, HDAC inhibitors, butyrate and SAHA, showed limited or no increase in GABA uptake and failed to rescue trafficking defects. Western blot quantification revealed that PBA selectively increased the mature form of GAT-1, whereas butyrate caused non-specific increases in both mature and immature forms. EEG analysis demonstrated that PBA significantly reduced seizure frequency in both Slc6a1+/S295L and Slc6a1+/A288V mice. Butyrate significantly increased seizure events in Slc6a1+/S295L mice and had no effect in Slc6a1+/A288V mice. Synaptosome assays confirmed that PBA, but not butyrate, restored GABA uptake in forebrain preparations. Together, these findings provide in vitro and in vivo evidence that PBA enhances both function and trafficking of WT GAT-1 under heterozygous variant burden.
Conclusions:
PBA acts as a pharmacochaperone that restores GAT-1 function and trafficking by stabilizing the mature protein and reducing ER stress. Its therapeutic effect is not attributable to HDAC inhibition, as HDAC inhibitors alone failed to rescue GABA uptake and, in some cases, exacerbated seizure burden. This work provides the first integrated mechanistic and in vivo evidence that pharmacochaperoning can overcome variant-induced trafficking defects and restore GABAergic inhibition. These findings support the continued development of PBA and other pharmacochaperones as a disease-modifying strategy for SLC6A1-related disorders.
Funding:
NIH R01 NS121718, T32AI007281, T32GM14492, and T32HL00773, and NS82635; SLC6A1 Connect; Taysha Gene Therapies.