Abstracts

Rationale:
RATIONALE: Dravet syndrome (DS) is an intractable epileptic encephalopathy. While mutations in many genes can cause DS, we focus on a mutation in a GABAA receptor (GABR) subunit gene, GABRG2(Q390X).  GABRG2(Q390X) results in a truncated, misfolded protein that has a dominant negative effect on neuronal function. We have demonstrated that the mutant protein is nonfunctional, forms protein aggregates, and causes neurodegeneration in mice. The accumulation of this mutant protein may also trigger a response from cellular protein quality control mechanisms, such as the ubiquitin-proteasome system (UPS). Altered function of the UPS may not be restricted to epilepsies with protein aggregates, as we show in another genetic epilepsy with developmental delay, SLC6A1 epileptic encephalopathy. The mutant GAT1 proteins encoded by the mutated SLC6A1 have not been observed to aggregate, but some of the mutants, such as SLC6A1(S295L), are trafficking-deficient and accumulate within the endoplasmic reticulum (ER). This accumulation suggests that protein quality control processes are affected by this mutation as well.
Method:
METHODS: Protein expression: HEK293T cells were transfected with GABRG2, GABRG2(Q390X), SLC6A1, or SLC6A1(S295L) cDNA. Total protein from cell lysates were subjected to SDS-PAGE and immunoblotted for γ2 and GAT1. Trafficking analysis: Live cells transfected with YFP-tagged plasmids and a fluorescent marker of the ER were imaged, and the colocalization of the mutant GAT1 with the ER was quantified. Glycosylation: HEK cell lysates were digested with Endo-H or PNGase F to remove N-linked glycosylation. UPS function: HEK cells stably expressing the EGFPu construct were transfected with GABRG2, GABRG2(Q390X), SLC6A1, or SLC6A1(S295L) cDNA. Lysates were subjected to SDS-PAGE and immunoblotted for GFP and ubiquitin.
Results:
RESULTS: GAT1(S295L) is primarily colocalized with the ER, in opposition to wildtype GAT1, which is a membrane protein. This retained protein is immature: On immunoblots, the intensity of high molecular weight bands representing mature fully glycosylated protein is barely detectable for GAT1(S295L), while GAT1 presents a strong signal. Both γ2(Q390X) and GAT1(S295L) showed a slightly increased EGFPu signals compared to the respective wildtype, indicating a decreased protein turnover by the UPS.
Conclusion:
CONCLUSIONS: Both GABRG2(Q390X), which causes Dravet syndrome, and SLC6A1(S295), which causes SLC6A1 epileptic encephalopathy, result in nonfunctional proteins that also fail to traffic to the membrane, instead accumulating within the ER. γ2(Q390X) forms aggregates while GAT1(S295L) does not, but both mutants affect the UPS. The decreased turnover by the UPS indicates that the deleterious effects of the mutations extend past their dominant-negative properties on the function of the wildtype protein. More study into the full ramifications of altered UPS activity is required, but decreased UPS activity has been observed in neurodegenerative diseases. Altered UPS activity may be a common phenotype in certain genetic epilepsies.
Funding:
:FUNDING: Dravet Syndrome Foundation, 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