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(Abst. 3.032), 2019

Dampened Cellular Stress Response in GABRG2 Mutations Associated with Variable Epilepsy Syndromes and Therapeutic Implications
Authors: Jing-Qiong K. Kang, Vanderbilt; Robert L. Macdonald, Vanderbilt University Medical Center; Melissa Lee, Vanderbilt University Medical Center; Lan Xiao, Vanderbilt University Medical Center; Wangzhen Shen, Vanderbilt University Medical Center
Content: Rationale: We have extensively studied the trafficking of GABAA receptor subunits associated with various genetic epilepsy syndromes.  GABRG2 (Q390X) is a mutation associated with generalized epilepsy with febrile seizure plus (GEFS+) and Dravet syndrome while other mutations in the same gene are associated with relatively mild epilepsy syndromes or with simple febrile seizures.  We previously found that impaired trafficking is a major molecular defect for GABRG2 epilepsy mutations  and the steady state amount of nonfunctional mutant subunits modifies disease severity. Heat shock proteins (Hsps), such as Hsp90, Hsp70, Hsc70  and Hsp40 are a group of proteins that assist in protein folding and disposal and prevent protein aggregation. We thus investigate the role of Hsps in GABRG2 associated epilepsies. Methods: We used both cell and mouse models harboring GABRG2 mutations and determined the expression level of Hsps. We used transit transfection, knockin mouse generation, confocal microscopy and subcellular fractionation to determine the subcellular location and expression of Hsps in cells and mouse brain tissues. Patch-clamp whole cell recordings were performed to determine the mutant receptor function. Results: In this study, we compared the γ2 (Q390X) subunit with other trafficking deficient GABAA receptor subunits (γ2 (W429X) and γ2 (W461X)) subunits. The three mutant subunits are assoociated with epilepsy with different severity. The γ2 (Q390X) subunit is associated with Dravet syndrome, γ2 (W429X) is associated with generalized tonic clonic seizures with febrile seizure (FS) plus and FS while γ2 (W461X) subunit is associated with simple FS. We found that the γ2 (Q390X) and γ2(W429X), but not the mutant γ2(W461X), subunits disturbed heat shock stress chaperone expression. Specifically, Hsp90, Hsc70 was reduced at total lystae level in both cells expressing the mutant subunits (N=6 different transfections, P<0.001, wildtype=1 vs 0.63± 0.05 for Hsp90 and 0.57± 0.08 for Hsc70 ) and the mutation carrying mice (n=4 pairs of mice) while Hsc70 was reduced at the cytosolic and light membrane fraction with subcellular fractionation (S2) (wildtype=1 vs 0.63± 0.05 for Hsp90 and 0.32±0.04 ).  Importantly, the γ2(Q390X) subunit reduced stress chaperones more than the γ2(W429X) subunits and had more impaired channel function. The reduced chaperone expression was correlated with the reduced surface channel expression and function. Finally, we identified the Hsp activator HSF-1 was reduced in cells expressing the mutant γ2(Q390X) subunit associated with Dravet syndrome but not in the subunits associated with FS (N=7 transfection, wildtype=1 vs 0.47± 0.06 for HSF-1, P<0.001). Conclusions: We conclude that the cellular stress response is impaired in epilepsy associated with the GABRG2 mutations. The reduction of chaperones was inversely correlated with the amount of the mutant protein in both cell and mouse models carrying different GABAG2 mutations (Gabrg2+/Q390X knockin and Gabrg2+/- knockout mice.  The cells harboring the mutant γ2 subunits had reduced heat shock response to external cellular challenges.  This study thus provides novel insights into pathophysiology of genetic epilepsy associated with GABRG2 mutations and suggest modulation of chaperones could be beneficial.  Funding: The work is supported by research grants from CURE, Dravet Syndrome Foundation and NINDS R01 NS0082635 to KJQ, and NINDS R01 NS0051590 to RLM.