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
SLC6A1 are associated with a broad spectrum of neurodevelopmental disorders. While the association of
SLC6A11 with disease is uncertain, both
SLC6A1 and
SLC6A11 are located at the same 3p25.3 chromosome region. A proximal haploinsufficient microdeletion of
SLC6A1 and
SLC6A11 resulting has been reported; however, the functional consequence is unknown. This research aims to characterize the functional consequence of the microdeletion of
SLC6A1 and
SLC6A11 related to 3p- syndrome and a possible pharmacochaperone rescue using 4-phenylbutyrate acid (PBA).
Methods:
The impact of the microdeletion on GAT-1 and GAT-3 function is evaluated by
3H radiolabeling to measure GABA uptake using HEK293T cells expressing recombinant GAT-1 and GAT-3. The effect of the half-gene dose was determined by transfecting a mixture of GAT-1 or GAT-3 cDNAs with pcDNA. The microdeletion condition was mimicked through co-expression conditions by co-transfecting GAT-1/GAT-3 to compare this data with previously identified missense GAT-1 loss-of-function mutations. The specific GABA uptake activity of GAT-1 vs. GAT-3 is determined by applying Cl-966 (50μM, GAT-1 inhibitor), SNAP5114 (30μM, GAT-3 inhibitor). We treated cells with PBA (2mM, 24hrs) to elucidate the effect of this chaperone on GABA uptake as a potential rescue-of-function. The functional consequence at the protein level is evaluated by microsome isolation followed to quantify the amount of protein retained in the endoplasmic reticulum (ER).
Results:
Our preliminary data suggests that the loss of a half-gene dose due to the microdeletion of both
SLC6A1 and
SLC6A11 resulted in reduced GABA uptake from both GAT-1 and GAT-3. PBA treatment increases the GABA uptake in both the wildtype and the condition of the microdeletion related to 3p- syndrome. Our previous work in GAT-1 mutations has shown excessive mutant protein is retained in the ER, thus reducing the amount of GABA transporters recruited to the cell surface. Microsome experiments are ongoing and preliminary data shows promising effects of PBA treatment.
Conclusions:
This reduction in GABA reuptake would account for the observed phenotype in patients. 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. Continued experiments are necessary to characterize 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 examining if PBA can provide rescue-of-function properties for both transporters.Funding:
National Institute of Health (NINDS) NS82635,
SLC6A1 Connect, Taysha Gene Therapies, and NIH R01NS 121718.