Rationale:
We previously identified that loss of GABA uptake is a major pathophysiology for neurodevelopmental disorders mediated by A288V and S295L mutations in
SLC6A1, coding for GABA transporter 1 (GAT-1). Patients with these mutations suffer from epilepsy plus complex neurodevelopmental comorbidities, including autism and learning disabilities. Our previous
in vitro work has shown that
SLC6A1(A288V) and
SLC6A1(S295L) result in respective partial or complete loss of GABA uptake function of GAT1. However, the
in vivo effect of these mutations on neurophenotype of patients and animal models is unclear. In this study, we characterize the neurophenotypes of two knockin mouse models,
Slc6a1+/A288V and
Slc6a1+/S295L, through rigorous longitudinal monitoring to investigate the developmental impact of
SLC6A1(A288V) and
SLC6A1(S295L) mutations and the long-term outcome of the associated disorders.
Methods:
We monitored weight, survival, righting reflex, and ultrasonic vocalization of
Slc6a1+/A288V and
Slc6a1+/S295L pups during postnatal development. For mice at 1, 4, and 10 months old (mo) of both sexes, we designed a battery of neurobehavioral assays, including grip strength, marble burying, nest building, elevated zero maze, three-chamber test, open field test, Barnes maze, light-dark cycle, and home cage scan (HCS). Additionally, we evaluated GABA uptake by measuring GABA level in prefrontal cortex of live mice via microdialysis before, during, or after 50mM KCl stimulation and determined the cumulative GABA level.
Results:
We observed reduced weight specific to early development (postnatal day 7) and prolonged righting reflex for the knockin pups for both mouse lines. A battery of neurobehavioral tests indicated that both mouse models exhibit subtle neurobehavioral phenotypes.
Slc6a1+/S295L mice showed reduced travelled distance on Open Field Test at one mo. However, both
Slc6a1+/A288V and
Slc6a1+/S295L exhibited reduced travelled distance on HCS at 7 mo, and reduced hanging at seven and ten mo, most prominently at night.
Slc6a1+/S295L mice had increased sleeping during daytime at seven mo, but no change in grooming time. By contrast,
Slc6a1+/A288V mice exhibited reduced sleep and increased grooming at seven mo suggesting repetitive behaviors. Microdialysis showed increased GABA in
Slc6a1+/S295L mice at 3 mo during KCl stimulation, but not at 1, 7, or 10 mo and increased cumulative GABA in the
Slc6a1+/S295L mice at seven mo but no changes in
Slc6a1+/A288V mice.
Conclusions:
Our results indicate an early developmental effect in
Slc6a1+/A288V and
Slc6a1+/S295L mice and significant behavioral abnormalities at adulthood associated with locomotion, exploration, repetitive behaviors, and sleep. Increased GABA levels in
Slc6a1+/S295L mice are suggestive of a deficient GABA uptake that underlies the behavioral phenotypes. Further studies are required to evaluate a link between extracellular GABA concentrations and phenotype.
Funding:
The work was supported by research grants from SLC6A1 Connect, UCB and NIH R01 NS121718 to KJQ.