Abstracts

Rationale:
Mutations of the GABA-ergic system are associated with variable developmental epileptic encephalopathies displaying both epileptic and developmental delay phenotypes. The β subunits of the GABAA receptor are major in the development of the central nervous system and have been linked to Lennox-Gastaut Syndrome (LGS) through patient derived mutations. The impact of the mutation in the brain and how it can cause a developmental and epileptic phenotype are poorly understood, let alone mechanism-based treatment.  Preliminary work in the lab has shown that the overexpression of the ubiquitin-like protein, Plic-1, can rescue mutant subunit containing receptors. We have developed novel mouse models of LGS (Gabrb3+/N328D) and will compare the major defects caused by the mutated GABA-ergic system.  In combination of mouse model with overexpression of the Plic-1, we can determine if molecular and functional phenotypes of the mutant mice can be rescued.
Method:
Subcellular fractionation of whole brain will be used for isolation of synaptosomes from each region in wildtype, mutant, and rescued mice.  Mice are genotyped and sacrificed to extract whole brain and brain tissue from the cortex, cerebellum, thalamus, and hippocampus. Expression of the α1, β3, β2, γ2 and δ subunits of the GABAAreceptor in both total lysates and synaptosomes will be determined in mice without or with overexpression of Plic-1. Video monitoring and synchronized EEG recordings will be conducted. The receptor subunits will be assessed via immunoblot within mice to assess differences in gross protein expression. Brains were also removed from mice and fixed in 4% PFA for immunohistochemistry.  
Results:
To date we have confirmed that the mutant Gabrb3N328D/N328D homozygous are moribund and die within postnatal 7-8 days. There was seen reduced expression of this subunit within heterozygous mice compared to wildtype. The expression of β3 subunits was reduced in both total lysates and synaptosomes in the Gabrb3+/N328D and Gabrb3+/- mice compared to wild-type littermates. There was reduced γ2 subunits in the young but not old Gabrb3+/- mouse brains. There was reduced GABA evoked amplitude in cells harboring the mutant GABRB3(N328D) mutation and reduced GABAergic mIPSCs in the hippocampal CA1 neurons in Gabrb3+/- mice.
Conclusion:
Together we confirmed that a KO and KI of the β3 subunit of the GABAA receptor reduces expression and function of this subunit. Plic-1 rescued the expression and function of mutant β3 subunit containing receptors in vitro. This preliminary data infers that Plic-1 may have a modulating role for GABAergic signaling perturbed by Lennox-Gastaut phenotype by upregulating GABAA receptors despite a present mutation.
Funding:
:This project was supported, in part, by grants from Dravet syndrome foundation (DSF), Vanderbilt Clinical and Translation Science Award, Vanderbilt Brain Institute pilot grant and Vanderbilt Discovery grant, NINDS R01 NS082635 to J.Q.K and NCATS/NIH UL1 TR000445, and NIH RISE Grant #: 5 R25 GM 59994-19