Authors :
Presenting Author: Michael Kavanaugh, PhD – University of Montana
Gregory Leary, PhD – Postdoctoral Fellow, Neuroscience, University of Montana; Alessandra Jester, Undergraduate – Student Research Assistant, Neuroscience, University of Montana; Leonid Kalachev, PhD – Professor, Mathematics, University of Montana
Rationale:
An increasing number of spontaneous mutations in the major GABA transporter gene SLC6A1 (GAT1) have been associated with pediatric epilepsy and neurodevelopmental disorders. The molecular mechanisms and impact of these presumed loss-of-function mutations on GABA homeostasis and neuronal circuit function is poorly understood. Furthermore, there are currently no therapeutic approaches to treat patients. Studies of SLC6A1 mutations in heterologous expression systems and preclinical transgenic mouse models afford an opportunity to better understand pathogenic mechanisms and develop novel small-molecule therapies.
Methods:
A de novo mutation (S295L) in the GABA transporter gene SLC6A1 that was identified in a pediatric patient was introduced into a human cDNA construct and mutant and wild-type transporters were expressed and studied in Xenopus oocytes by voltage clamp and radiolabeled GABA flux measurement. In addition, the effect of the mutation on behavior and on GABA homeostasis and neuronal signaling was studied in acute cortical slices from transgenic SLC6A1 S295L knock-in mice using patch clamp recording of neurons and by measuring radiolabeled GABA flux in brain tissue from wild-type, heterozygous, and homozygous mutant mice.
Results:
The S295L mutation caused a profound loss of function in the mutant GABA transporter. Heterologous co-expression of mutant and wild-type transporter showed no dominant negative effect of the mutation. Consistent with this, there was a 50% loss of SLC6A1 activity in heterozygous S295L mutant mice. This loss was partially compensated by an increase in SLCA11 activity. No change was seen in miniature inhibitory synaptic currents in heterozygous mutants, but an increase in ambient extrasynaptic GABA-A receptor signaling was observed indicating that extracellular GABA levels were increased approximately two fold. The dysregulation of GABA homeostasis in the mutant mice resulted in neurophysiological deficits including abnormal burst spiking and impaired hippocampal synaptic plasticity. We tested the ability of 4-phenylbutyrate, an FDA-approved drug that acts as a chemical chaperone, to rescue impaired GABA uptake and homeostasis by increasing surface trafficking. Instead, the drug unexpectedly acted as a direct allosteric activator of SLC6A1 and SLC6A11. Some analogs and fatty acids were more efficacious, increasing transport rates up to 100%. Addition of the naturally occurring fatty acid butyrate to acute brain slices from heterozygous mutant animals was capable of restoring GABA uptake to normal wild-type levels, suggesting a novel therapeutic strategy.
Conclusions:
We demonstrated that a series of fatty acids and analogs act as positive allosteric activators of GABA uptake. These molecules are capable of restoring normal uptake levels in a preclinical genetic model of epilepsy and neurological impairment caused by a representative de novo SLC6A1 mutation. The naturally occurring fatty acid butyrate represents a potentially promising and novel treatment for patients with SLC6A1 loss-of-function mutations.
Funding: This study was funded by SLC1A6connect.org