Abstracts

Rationale: GABA transporters reuptake γ-aminobutyric acid (GABA) from the synaptic cleft. GABA transporter 1 (GAT-1), encoded by SLC6A1, is highly expressed in GABAergic neurons and astrocytes. Mutations in SLC6A1 are linked to various neurological disorders, including epilepsy, neurodevelopmental delay, autism spectrum disorder, anxiety disorders, and motor and language delays. Many cases are treatment-resistant, underscoring the need for viable therapeutic options for SLC6A1 variant-related disorders. This research aims to uncover the mechanisms of optimal rescue-of-function treatment for SLC6A1 variants, evaluating potential pharmacological interventions.


Methods:
The study employed ³H-labeled GABA radiobinding assays, Western blotting, and microsome isolation to measure GAT-1 uptake function and protein expression in HEK293T cells expressing recombinant GAT-1. Various drug treatments were tested, including 4-phenylbutyrate (PBA), butyrate, BiX, and SAHA. PBA was tested to determine if optimal rescue comes from alleviating ER stress, increasing GAT-1 wild-type protein expression, diminishing mutant variant expression in heterozygous models, and enhancing GABA reuptake functionality. Butyrate, a short-chain fatty acid, was evaluated for its potential as a positive allosteric modulator or histone deacetylase inhibitor. BiX was tested for its role in protein assembly and folding. SAHA, an HDAC inhibitor, was assessed for its ability to increase histone protein acetylation, leading to relaxed chromatin structure and altered gene expression.




Results:
Data indicate that PBA [2mM], 24 hrs significantly improves GABA uptake, alleviates ER stress, and increases GAT-1 functionality and surface expression. This aligns with our previous findings that PBA acts as a chronic treatment by promoting protein trafficking and relieving ER stress. Patient data showed improvements in seizure activity and neurodevelopmental delay following PBA treatment. Butyrate demonstrated potential as a PAM or HDAC inhibitor, reversing the hyperexcited state seen in epilepsy models and showing modest improvements in GABA uptake in vitro. BiX facilitated protein folding and processing, restoring normal trafficking and GAT-1 expression, and increasing GABA uptake. SAHA treatment reduced seizure activity and improved cognitive function, supporting its role as an HDAC inhibitor and increasing GABA uptake in vitro.




Conclusions: The reduction in GABA reuptake likely contributes to the observed phenotypes in patients with SLC6A1 variant-related disorders, including epilepsy, neurodevelopmental delay, and autism spectrum disorder. These findings suggest that the pharmacological agents tested may offer viable therapeutic options. Ravicti (glycerol-phenylbutyrate) is currently in clinical trials for SLC6A1-related disorders, but this study aims to tease out mechanisms to identify alternative therapeutic treatments. This study provides evidence that pharmacological interventions can rescue impaired function and reduced expression of GAT-1 in disease conditions, addressing the urgent need for effective treatments.


Funding: T32AI007281, T32GM14492, T32HL00773, and NS82635; Cure GABAA, Taysha Gene Therapies, and NIH R01 NS121718.