A Disease-Causing Mutation in the Human GABA Transporter SLC6A1
Presentation Type
Oral Presentation
Category
STEM (science, technology, engineering, mathematics)
Abstract/Artist Statement
GABA (gamma-aminobutyrate) is the primary inhibitory neurotransmitter in mammalian brain. Mutations in the major GABA transporter gene SLC6A1 are associated with pediatric neurological disorders including autism and epilepsy. Understanding the molecular and electrophysiological changes associated with these mutations is crucial for developing patient therapies. In this study we characterized the effects of a spontaneous mutation (S295L) discovered in a heterozygous child experiencing severe neurological symptoms. Expression of the mutant transporter in Xenopus oocytes indicated a complete loss-of-function mutation. Using CRISPR-transgenic SLC6A1 mice, we characterized GABA homeostasis, seizure activity, and synaptic transmission in wild-type, heterozygous, and homozygous animals by measuring radioactive GABA uptake in brain and by patch-clamping cortical neurons. Neither the amplitude nor frequency of miniature GABA-mediated postsynaptic currents (mIPSCs) were changed in the S295L mutants. However, tonic GABAA and GABAB receptor signaling were modestly increased, reflecting an S295L allele-dependent increase in ambient extracellular GABA. Because SLC6A1 is believed to be the primary GABA transporter in brain, the relatively modest effect of its loss on ambient [GABA] was surprising. The potential compensating effect of SLC6A11, another brain GABA transporter, was examined in the mutant mice. The SLC6A11 blocker SNAP5114 did not affect [3H]GABA uptake in wild-type brain, while a significant SNAP5114-sensitive component of GABA uptake was revealed in brains from mutant mice, suggesting that an increase in SLC6A11 transport partially compensates for the loss of SLC6A1. Surprisingly, a similar effect of SNAP5114 was also revealed in wild-type mouse brain when it was applied in combination with SKF89976, an SLC6A1-selective blocker. Using MATLAB, a model incorporating transporter kinetics and GABA diffusion was constructed that can account for this non-linear interaction. We are currently pursuing pre-clinical studies with the SLC6A1-S295L transgenic mice to test the therapeutic efficacy of increasing GABA transport using novel pharmacological activators or gene replacement with viral vectors.
Mentor Name
Michael Kavanaugh
Personal Statement
In December of 2022, I had the opportunity to attend the SLC6A1 annual symposium to present our lab’s research to scientists, doctors, and to the families of children who have been diagnosed with SLC6A1. SLC6A1 is a rare disease, and as with all rare diseases, limited knowledge limits treatment options. A handful of parents who had a child with SLC6A1 spoke about the struggles of living with the disease: both the devastating symptoms in their children and the limited knowledge and attention given from healthcare providers. The emotions were palpable, and many doctors and scientists, including myself, were moved to tears. The people in that room are the only people researching this disease, and current research directly helps the children affected. Because this is a rare disease, clinical trials are easily reachable once data from our lab alone demonstrates efficacy on transgenic mice. Our lab will be researching the efficacy of the drug, phenylbutyrate, which preliminary data shows could help reduce the seizures of children with SLC6A1. We also will be exploring the use of viral vectors to test the therapeutic efficacy of gene replacement on transgenic mice, which might lead to a cure of this devastating disease. After listening to the parent’s heartbreaking testimonies, it was obvious to all who heard them that the importance of the work we are doing cannot be overstated.
A Disease-Causing Mutation in the Human GABA Transporter SLC6A1
UC 331
GABA (gamma-aminobutyrate) is the primary inhibitory neurotransmitter in mammalian brain. Mutations in the major GABA transporter gene SLC6A1 are associated with pediatric neurological disorders including autism and epilepsy. Understanding the molecular and electrophysiological changes associated with these mutations is crucial for developing patient therapies. In this study we characterized the effects of a spontaneous mutation (S295L) discovered in a heterozygous child experiencing severe neurological symptoms. Expression of the mutant transporter in Xenopus oocytes indicated a complete loss-of-function mutation. Using CRISPR-transgenic SLC6A1 mice, we characterized GABA homeostasis, seizure activity, and synaptic transmission in wild-type, heterozygous, and homozygous animals by measuring radioactive GABA uptake in brain and by patch-clamping cortical neurons. Neither the amplitude nor frequency of miniature GABA-mediated postsynaptic currents (mIPSCs) were changed in the S295L mutants. However, tonic GABAA and GABAB receptor signaling were modestly increased, reflecting an S295L allele-dependent increase in ambient extracellular GABA. Because SLC6A1 is believed to be the primary GABA transporter in brain, the relatively modest effect of its loss on ambient [GABA] was surprising. The potential compensating effect of SLC6A11, another brain GABA transporter, was examined in the mutant mice. The SLC6A11 blocker SNAP5114 did not affect [3H]GABA uptake in wild-type brain, while a significant SNAP5114-sensitive component of GABA uptake was revealed in brains from mutant mice, suggesting that an increase in SLC6A11 transport partially compensates for the loss of SLC6A1. Surprisingly, a similar effect of SNAP5114 was also revealed in wild-type mouse brain when it was applied in combination with SKF89976, an SLC6A1-selective blocker. Using MATLAB, a model incorporating transporter kinetics and GABA diffusion was constructed that can account for this non-linear interaction. We are currently pursuing pre-clinical studies with the SLC6A1-S295L transgenic mice to test the therapeutic efficacy of increasing GABA transport using novel pharmacological activators or gene replacement with viral vectors.