Abstracts

Rationale: GABA transporters are responsible for the reuptake of γ-aminobutyric acid (GABA) from the synaptic cleft. GABA transporter 1 (GAT-1), encoded by SLC6A1, is abundantly expressed in GABAergic neurons and astrocytes, and GABA transporter 3 (GAT-3), encoded by SLC6A11, is primarily expressed in astrocytes. Mutations in GAT-1 encoding SLC6A1 are associated with a wide spectrum of neurodevelopmental disorders, such as epilepsy syndromes, intellectual disabilities, and autism. While the association of SLC6A11 with disease is uncertain, both SLC6A1 and SLC6A11 are located at the same 3p25.3 chromosome region. A proximal microdeletion of SLC6A1 and SLC6A11 has previously been reported and is reoccurring; however, the functional consequence of the loss of GAT-1 and GAT-3 is unknown, nor the differential contribution to GABA uptake. We thus characterized the functional consequence of the microdeletion of SLC6A1 and SLC6A11 related to 3p syndrome and possible rescue.

Methods: The impact of the microdeletion on GAT-1 and GAT-3 function is evaluated by ³H radiolabeling to measure GABA uptake and transporter expression using mouse cortical astrocytes and HEK293T cell lines expressing the recombinant GAT-1 and GAT-3 transporters. The specific GABA uptake activity of GAT-1 versus GAT-3 is determined by applying Cl-966 (50µM, GAT-1 inhibitor), SNAP5114 (30µM, GAT-3 inhibitor). We also tested the effect of chaperones such as 4-Phenylbutyrateacid (PBA) and Menthol to elucidate the effects of these treatments on GABA uptake. We will determine the cell surface and total expression of GAT-1 and GAT-3 of microdeletion in HEK293T cells and mouse cortical astrocytes.

Results: It is anticipated that this mutation will result in a loss of function of both transporters, thus resulting in reduced GABA reuptake from the synaptic cleft. Our preliminary data indicate that the loss of a half gene dose due to the microdeletion of SLC6A1 and SLC6A11 resulted in reduced GABA uptake. With specific GAT-1 or GAT-3 inhibitors, we identified that GABA uptake from both GAT-1 and GAT-3 is reduced. Pharmaco-chaperoning approach with PBA (2mM, 24 hrs) or menthol (500nM, 24 hrs) increases the GABA uptake in both the wildtype and the condition of the microdeletion related to 3p syndrome.

Conclusions: This reduction in GABA reuptake would account for the observed phenotype in patients as all reported cases exhibit neurodevelopmental delay and seizure activity. Given seizures and neurodevelopmental delay are prominent features for patients with point mutations in SLC6A1 alone, this study suggests that the deletion of both SLC6A1 and SLC6A11 is likely to contribute to a great extent, if not causative for the observed phenotype. Further experiments are necessary to continue studying this novel mutation and its functional deficit. This study provides the first and concrete evidence that reduced GABA uptake contributes to disease phenotype associated with 3p syndrome containing SLC6A1 and SLC6A11 and suggests a mechanism-based treatment option.

Funding: Please list any funding that was received in support of this abstract.: This work was supported by research grants from National Institute of Health (NINDS) NS82635, SLC6A1 Connect, Taysha Gene Therapies, and NIH RISE Grant #: 5 R25 GM 59994-19.