Epilepsy affects more than 0.5 % of the population in the world. Genetic factors play an important role in many of the idiopathic generalized epilepsies (IGEs) and in some partial epilepsies. GABAA receptors (GABARs) are the major inhibitory receptors in the CNS, and mutations in ?2, d and al GABAR genes are associated with IGEs. To understand the bases for IGEs associated with GABAR mutations, it is necessary to determine the functional, assembly and trafficking errors produced by the mutations. Many of these are single nucleotide missense mutations, but recently mutations that introduce a premature translation- termination codon (PTC) and mutations in splice donor sites have been reported. PTCs might produce nonsense mediated decay (NMD) of mRNA if the mutation is not in the last exon or produce a truncated subunit if it is in the last exon. Splice donor site mutations produce mutant protein by: (1) exon skipping, (2) use of cryptic splice site within the down stream intron, or (3) intron inclusion if the intron is small. It is possible all three mechanisms would generate a PTC, thus triggering NMD. The goals of this proposal are to characterize the altered expression, function and trafficking produced by ?2 epilepsy PTC and splice donor site mutations. Hypotheses are that the: a) ?2S(Q351X) PTC mutation reduces het al?2?2S(Q351X) current by producing a C-terminal truncated subunit that assembles to form mutant receptors that reduce trafficking of wt receptors, are co trafficked with wt receptors to the cell surface and have altered function, b) ?2S(Q1X) PTC mutation reduces het al?2?2L(Q1X) receptor current by triggering NMD of mutant mRNA, thus producing haploinsufficiency. c) ?2S (IVS6+2T-G) splice donor site mutation reduces het al?2?2L (IVS6+2T-G) receptor current via haploinsufficiency by triggering NMD through either exon 6 skipping, resulting in a PTC at the joining site of exon 5 and exon 7, or by using intron 6 downstream cryptic splice sites, also resulting in a PTC, or by generating a truncated protein due to incomplete NMD (NMD inefficiency).
Specific aims are to determine the pathophysiological alterations in translation, trafficking, surface expression and pharmacological and biophysical properties of het and horn a) al?2?2 (Q351X), b) al?2?2 (Q1X), and c) al?2?2 (IVS6+2T-G) receptors expressed in fibroblasts and ?2S siRNA treated cultured hippocampal neurons.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS051590-04
Application #
7737352
Study Section
Clinical Neuroplasticity and Neurotransmitters Study Section (CNNT)
Program Officer
Stewart, Randall R
Project Start
2007-02-01
Project End
2011-02-28
Budget Start
2009-12-01
Budget End
2011-02-28
Support Year
4
Fiscal Year
2010
Total Cost
$332,424
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Neurology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
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Golden, Lisa C; Voskuhl, Rhonda (2017) The importance of studying sex differences in disease: The example of multiple sclerosis. J Neurosci Res 95:633-643
Huang, Xuan; Zhou, Chengwen; Tian, Mengnan et al. (2017) Overexpressing wild-type ?2 subunits rescued the seizure phenotype in Gabrg2+/Q390X Dravet syndrome mice. Epilepsia 58:1451-1461
Xia, Geqing; P Pourali, Sarah; Warner, Timothy A et al. (2016) Altered GABAA receptor expression in brainstem nuclei and SUDEP in Gabrg2(+/Q390X) mice associated with epileptic encephalopathy. Epilepsy Res 123:50-4
Warner, Timothy A; Shen, Wangzhen; Huang, Xuan et al. (2016) Differential molecular and behavioural alterations in mouse models of GABRG2 haploinsufficiency versus dominant negative mutations associated with human epilepsy. Hum Mol Genet 25:3192-3207
Wang, Juexin; Shen, Dingding; Xia, Geqing et al. (2016) Differential protein structural disturbances and suppression of assembly partners produced by nonsense GABRG2 epilepsy mutations: implications for disease phenotypic heterogeneity. Sci Rep 6:35294
Kang, Jing-Qiong; Macdonald, Robert L (2016) Molecular Pathogenic Basis for GABRG2 Mutations Associated With a Spectrum of Epilepsy Syndromes, From Generalized Absence Epilepsy to Dravet Syndrome. JAMA Neurol 73:1009-16
Kang, Jing-Qiong; Shen, Wangzhen; Zhou, Chengwen et al. (2015) The human epilepsy mutation GABRG2(Q390X) causes chronic subunit accumulation and neurodegeneration. Nat Neurosci 18:988-96
Lo, Wen-Yi; Lagrange, Andre H; Hernandez, Ciria C et al. (2014) Co-expression of ?2 subunits hinders processing of N-linked glycans attached to the N104 glycosylation sites of GABAA receptor ?2 subunits. Neurochem Res 39:1088-103
Tian, Mengnan; Mei, Davide; Freri, Elena et al. (2013) Impaired surface ??? GABA(A) receptor expression in familial epilepsy due to a GABRG2 frameshift mutation. Neurobiol Dis 50:135-41

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