Rationale: Pathogenic variants of
SLC6A1, encoding GABA transporter 1 (GAT-1), are a common etiology of developmental and epileptic encephalopathies (DEE), debilitating childhood epilepsies with drug-resistant seizures and severe cognitive co-morbidities for which few treatments exist. Severe nature of these neurologic pathologies stems from the essential brain function of GAT-1 as the main transporter of ɣ-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in brain. Previously, we showed significant pathologies in GAT-1 trafficking, expression, and ability to uptake GABA in cell and animal models of various
SLC6A1 variant disorders, including the severe loss of function S295L variant. In
Slc6a1+/S295L knock-in mice, these pathologies underlie absence seizures and other behavioral co-morbidities similar to the patient phenotype. We showed that treatment with chemical chaperone 4-phenylbutyrate is able to rescue seizures and molecular pathologies, and it is currently showing promise in an ongoing clinical trial in patients with
SLC6A1-variant disorders (
NCT04937062). Here, we test our hypothesis that
Slc6a1+/S295L exhibit significant pathologic changes in GABAergic neurotransmission, which are rescued when 4—PBA treatment is able to correct the molecular pathologies.
Methods:
Male and female
Slc6a1+/S295L mice (Shanghai Model Organisms, NM-KI-190014) in C57BL/6J background were used. Acute
ex vivo brain slices were prepared per Ting et al, 2018. Evoked and spontaneous inhibitory postsynaptic currents (eIPSCs and sIPSCs) were recorded at 32 C or room temperature in whole cell patch clamp configuration in presence of glutamatergic blockers 50 µM AP5 and 10 µM NBQX. Tonic current was distinguished with 30-100 µM bicuculline washon. 4-PBA treatment was administered as daily i.p. (7-day ) or oral in peanut butter (28-day) at 100 mg/kg body weight.
Results: We found a profound pathology in GABAergic neurotransmission in
Slc6a1+/S295L and
Slc6a1S295L/S295L mice associated with timely clearance of synaptic GABA. Both evoked and spontaneous IPSC responses were greatly prolonged in
Slc6a1+/S295L and
Slc6a1S295L/S295L mice compared to wildtype siblings. Moreover, duration of responses in wildtype mice significantly depended on GAT-1 activity, since application of GAT-1 blocker tiagabine greatly prolonged events. In comparison, GAT-1 function was greatly reduced in
Slc6a1+/S295L mice, where tiagabine affected eIPSCs significantly less and did not affect sIPSCs. Tonic currents appeared normal in cortex and thalamus. These changes persisted in mice treated with 4-PBA for 7 days.
Conclusions: Our results show profound pathologic changes in GABAergic neurotransmission in
Slc6a1+/S295L mice that are consistent with the role of GAT-1 in clearing extracellular GABA and several previous studies of GAT-1 loss of function. While a 7-day treatment with 4-PBA did not restore these changes, a longer 28-day regiment holds promise at a rescue, which we are currently evaluating.
Funding: The work was supported by NIH NINDS (R01 NS121718), Vanderbilt Postdoctoral Training Program in Functional Neurogenomics (NIMH T32 2022-2024), and the AES Postdoctoral Fellowship Award (2024-2025).