Authors :
Presenting Author: Kirill Zavalin, PhD – Vanderbilt University Medical Center
Karishma Randhave, BS – Vanderbilt University Medical Center
Alok Karkare, Undergraduate Intern – Vanderbilt University
Wanyi Su, Undergraduate Intern – University of North Carolina Chapel Hill
Luke Wedemeyer, Undergraduate Intern – Vanderbilt University
Rishi Pillai, Undergraduate Intern – Vanderbilt University
Jing-Qiong Kang, MD, PhD – Vanderbilt University Medical Center
Rationale:
A significant need exists for development of new precision therapy for genetic epilepsies (GEs), particularly severe encephalopathies, where co-morbidities and often seizures are resistant to treatment with canonical anti-seizure drugs. In recent years, we found that chemical chaperone 4-phenyl butyrate (4-PBA) treatment mitigated seizures and addressed multiple aspects of underlying pathology in two monogenetic GEs with mutations in vital constituents of GABAergic neurotransmission: 1) developmental and epileptic encephalopathy associated with S295L mutation in SLC6A1, encoding GABA transporter 1, and 2) Dravet syndrome associated with Q390X mutation in GABRG2, encoding ɣ2 subunit of the GABAA receptor.
We hypothesize that 4-PBA acts in a targeted fashion to reverse GABAergic dysfunction in these disorders, making it an excellent candidate for novel precision therapy that is applicable to multiple GEs. To test this hypothesis, we characterized GABAergic dysfunction in the Slc6a1+/S295L and Gabrg2+/Q390X mouse models, and evaluated if 4-PBA treatment can restore this pathology.
Methods:
To evaluate GABAergic dysfunction, we measured GABAergic neurotransmission using patch clamp electrophysiology in ex vivo brain slices from adult 2-4 month old Slc6a1+/S295L and Gabrg2+/Q390X mice and wildtype siblings. PBA treatment included a 7-day intraperitoneal administration or 28-day oral administration with recording on the last day. GABAergic currents were measured in pyramidal neurons in somatosensory cortex in presence of AP5 and NBQX. Results:
We found that evoked and spontaneous inhibitory postsynaptic currents (eIPSCs and sIPSCs) are longer in Slc6a1+/S295L mice. Mice treated with the shorter 7-day treatment still showed these deficits; those treated with the longer 28-day treatment showed a restoration of sIPSC decay closer to wildtype levels but still had prolonged decay of eIPSCs. Direct PBA washon did not affect eIPSC/sIPSC kinetics.
In Gabrg2+/Q390X mice, we found that miniature IPSCs are smaller in amplitude, and that 7-day treatment with PBA leads to a partial restoration of mIPSC amplitude.
Conclusions:
Prolongation of synaptic currents in Slc6a1+/S295L mice is consistent with role of GAT-1 as the primary mechanism of uptake of extrasynaptic GABA after its release. Previously, we reported that 7-day PBA treatment leads to a significant reduction of seizures; we did not observe a matching restoration of IPSCs with PBA washon or 7-day treatment, indicating potentially different mechanisms for these phenomena. The longer 28-day treatment did restore sIPSC kinetics, indicating that long-term treatment can address the synaptic deficits.
In Gabrg2+/Q390X mice, reduction in mIPSC amplitude matches prior reports. Previously, we showed that the 7-day PBA treatment significantly reduced the seizure burden in these mice; concordantly, we see a significant restoration of mIPSC amplitude.
Funding: This work is supported by T32 Vanderbilt Postdoctoral Training Program in Functional Neurogenomics and AES Postdoctoral Research Fellowship.