Authors :
Presenting Author: Paige Hall, BS – Baylor College of Medicine
Kristy Jay, PhD – Massachusetts General Hospital, Harvard Medical School
Jonathan Andrews, PhD – Baylor College of Medicine
Sharayu Jangam, MS – Baylor College of Medicine
Vanessa Gomez, MS – Baylor College of Medicine
Ryan German, MS, CGC – Baylor College of Medicine
Hongling Pan, BS – Baylor College of Medicine
Shinya Yamamoto, PhD, DVM – Baylor College of Medicine
Michael Wangler, MD – Baylor College of Medicine
Rationale:
Variants in SLC6A1 cause a rare neurodevelopmental disorder (SLC6A1-NDD) with nearly 200 affected individuals identified. Patients often present with myoclonic-atonic epilepsy, autism spectrum disorder, developmental delay, sleep disturbances, and other symptoms. Epilepsy is one of the most severe co-morbidities and greatly reduces quality of life. In fact, many patients undergo years of trial-and-error treatment due to our limited understanding of disease mechanism and pathogenesis. In humans, SLC6A1 encodes the GABA transporter 1 (GAT1) protein that is responsible for GABA reuptake at the synapse. GABA is the main inhibitory neurotransmitter in humans, thus impaired GABA reuptake is thought to result in altered inhibitory signaling which contributes to seizures.Methods:
In this study, we are leveraging Drosophila melanogaster to better understand the disease mechanism of four patients’ variants that were chosen for their high frequency or lack of functional study: p.S295L, p.G297R, p.A334S, and p.F339L. The Drosophila ortholog of SLC6A1 is Gat, which encodes the single GABA transporter in the fly, Gat. Drosophila make an excellent tool for modeling SLC6A1-NDD due to the similarity of GABA regulation between humans and the fly and ability to capture neurobehaviors arising from GABA dysregulation.
Results:
To study Gat, we generated a Trojan-Gal4 (GatTG4) allele, which creates a truncated Gat protein while simultaneously producing a Gal4 transcriptional activator protein in the same spatiotemporal expression pattern as the Gat gene. We molecularly confirmed that this allele is loss-of-function (LOF) through RT-qPCR and confirmed that Gat is expressed exclusively in repo-positive glial cells. Using the GatTG4 allele to drive expression of our SLC6A1 constructs, we identified significant sleep defects. Compared to the SLC6A1Ref, GatTG4/+ flies have a significantly decreased sleep latency and experience fragmented sleep patterns. The SLC6A1F339L flies are the only variant-expressing line that rescued these sleep phenotypes to reference levels, indicating that all other variants are likely strong LOF. We observe more subtle differences in other sleep phenotypes between all variants, including changes in total sleep and night vs. day sleep, suggesting that each variant line induces unique physiological changes. These changes in sleep phenotypes are hypothesized to be attributed to GABA dysregulation within the central nervous system and these readouts provide rare, in vivo insights into how these variants impact the brain.
Conclusions:
We have previously screened these flies for seizure phenotypes with no significant results. Thus, next steps for this project include unbiased heat-shock seizure screening and testing for spontaneous seizures in our fly models. We plan to use these readouts, along with sleep, to evaluate SLC6A1 targeted therapeutics. The significance of this work is not limited to only SLC6A1-NDD, as other forms of epilepsy can also result from GABA dysregulation; thus, findings can inform molecular outcomes and potential therapeutic options for a vast range of epilepsies and related disorders.
Funding:
5T32GM139534-04