Rationale:
The S295L mutant variant of
SLC6A1, encoding ɣ-aminobutyric acid (GABA) transporter 1 (GAT-1), is associated with developmental and epileptic encephalopathy (DEE) in human patients. DEEs encapsulate debilitating disorders marked by interrelated phenotypes of epilepsy and developmental delay, for which pathophysiologies are often poorly defined, and treatment options are lacking, requiring urgent investigation of both. Previously, we demonstrated that GAT-1(S295L) has diminished expression and ability to transport GABA
in vitro. Moreover, we showed reduced GABA uptake in synaptosomes and reduced GAT-1 expression in brain tissue of
Slc6a1S295L knock-in mice, which recapitulate the DEE phenotype observed in human patients. Based on these results, we hypothesize that the core pathologic dysfunction in
SLC6A1(S295L) patients is reduced GABA uptake and associated changes in GABAergic neurotransmission. To test this hypothesis, we investigated pathologic changes in GABA uptake, and tonic and synaptic GABAergic events
ex vivo in
Slc6a1S295L mice.
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. GABA uptake was measured by scintillation counts after incubating slices in H3-GABA for 10 minutes at 37 C. Evoked and spontaneous inhibitory postsynaptic currents (eIPSCs and sIPSCs) were recorded at 32 C and room temperature, respectively, in whole cell patch clamp configuration in presence of glutamatergic blockers 50 µM AP5 and 10 µM NBQX, after which.30 µM bicuculline was added to distinguish tonic current as a baseline shift.
Results:
We found reduced GABA uptake in most brain regions in two to four month old (mo) Slc6a1+/S295L mice compared to wildtype (WT) siblings. Surprisingly, we did not find a change in GABAergic tonic currents in pyramidal neurons of layer 6 somatosensory cortex (L6) in two to four mo and seven+ mo Slc6a1+/S295L and Slc6a1 S295L /S295L mice, or in ventrobasal thalamic neurons in one mo Slc6a1+/S295L mice compared to WT siblings. However, we did find a reduction in sIPSC frequency in L6 pyramidal neurons of two to four mo Slc6a1+/S295L mice. eIPSCs in L2 pyramidal neurons elicited by L4 stimulation showed a progressive increase in area under the curve and lingering current in two to four mo Slc6a1+/S295L and Slc6a1 S295L /S295L mice, respectively, compared to WT siblings.
Conclusions:
We found that brain slices from Slc6a1+/S295L mice exhibit significantly reduced GABA uptake, corroborating our in vitro and GAT-1 expression data. In agreement with this observation, we saw a prolonged GABAergic current in eIPSCs. Additionally, we observed a decrease in sIPSC frequency. Despite our hypothesis, reduced uptake did not result in increased GABAergic tonic current, but this could be due to a limitation of recording at room temperature. Together, these observations indicate a change in GABAergic neurotransmission that could be the core pathologic dysfunction in SLC6A1(S295L) patients, but currently requires further investigation.
Funding:
The work was supported by NIH NINDS (R01 NS121718).