Epilepsy affects more than 0.5% of the population worldwide. Genetic factors play an important role in many of the epilepsy syndromes. GABAA receptors (GABAARs) are the major inhibitory receptors in the brain and mutations in GABAAR subunit genes (GABRs) coding for the 32, 11 and 4 GABAAR subunits are associated with idiopathic generalized epilepsy syndromes (IGEs). To understand the bases for IGEs associated with GABR mutations, it is necessary to determine the errors in receptor biogenesis and function that are produced by the mutations. Many of them are single nucleotide missense mutations, but recently nonsense mutations that introduce a premature translation-termination codon (PTC) and mutations in splice donor sites have been reported. PTCs might produce nonsense mediated mRNA decay (NMD) if the mutation is not in the last exon or produce a truncated subunit if it is in the last exon that could have dominant negative effects on wt subunits. Splice donor site mutations produce mutant subunits by: (1) exon skipping, (2) use of a cryptic splice site within the downstream intron, or (3) intron inclusion if the intron is small. It is possible that all three mechanisms would generate a PTC, thus triggering NMD. There are several approaches that can be taken to treat patients with nonsense epilepsy mutations. If the mutation produces no functional protein and therefore is haplo-insufficient, the subunit could be over expressed in neurons to overcome the loss of subunit or if the PTC is in frame, a read through strategy could be applied to read through the PTC and express a full length functional subunit. If the mutation produces a truncated, nonfunctional dominant negative protein, the subunit could be over expressed in neurons to overcome the loss of subunit or a read through strategy could be applied to read through the PTC and express a full length functional subunit. The goals of this proposal are to characterize the altered receptor biogenesis and function produced by GABRG2 epilepsy PTC and splice donor site mutations and to develop strategies to overcome the resultant deficits as potential therapies for the associated epilepsies. The hypotheses of the proposal are: 1) The GABRG2(IVS6+2T`G) mutation leads to aberrant mRNA splicing that creates a PTC and activates NMD and produces a stable, nonfunctional, truncated dominant negative protein whose effects can be reversed by siRNA knock down and/or GABRG2 over expression;2) The GABRG2(Q1X) mutation activates NMD to degrade the mutant mRNA, producing loss of function that can be reversed by PTC read through and/or GABRG2 over expression;and 3) The GABRG2(Q351X) mutation produces a stable, nonfunctional, truncated dominant negative protein that can be reversed by PTC read through and/or GABRG2 over expression.
The specific aims of the proposal are: 1) To determine the aberrant mRNA splicing pattern produced by the GABRG2(IVS6+2T`G) mutation and how the mutant translation products affect g2 subunit expression and GABAAR function and to evaluate potential treatment strategies;2) To determine the impairment of g2 subunit biogenesis produced by the GABRG2(Q1X) mutation and to evaluate potential treatment strategies, and 3) To characterize GABRG2(Q351X) knock in mice and evaluate PTC read through and/or GABRG2 over expression as potential treatment strategies.

Public Health Relevance

Patients with epilepsy have recurring, unprovoked brain seizures that impair bodily functions and often cause unconsciousness and/or injury. Many forms of epilepsy appear to be inherited, thus having a genetic basis, and have similar behaviors, associated impairments and electrical brain discharges and have been called idiopathic generalized epilepsy syndromes (IGEs). We propose studying the basis for IGEs caused by nonsense gene mutations that interrupt the formation of inhibitory GABAA receptor subunits, thereby causing seizures, and to develop new treatments for these IGEs.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS051590-07
Application #
8415950
Study Section
Clinical Neuroplasticity and Neurotransmitters Study Section (CNNT)
Program Officer
Stewart, Randall R
Project Start
2005-04-01
Project End
2015-02-28
Budget Start
2013-03-01
Budget End
2014-02-28
Support Year
7
Fiscal Year
2013
Total Cost
$329,307
Indirect Cost
$118,213
Name
Vanderbilt University Medical Center
Department
Neurology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
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
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
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 γ2 subunits hinders processing of N-linked glycans attached to the N104 glycosylation sites of GABAA receptor β2 subunits. Neurochem Res 39:1088-103
Tian, Mengnan; Mei, Davide; Freri, Elena et al. (2013) Impaired surface αβγ GABA(A) receptor expression in familial epilepsy due to a GABRG2 frameshift mutation. Neurobiol Dis 50:135-41
Kang, Jing-Qiong; Shen, Wangzhen; Macdonald, Robert L (2013) Trafficking-deficient mutant GABRG2 subunit amount may modify epilepsy phenotype. Ann Neurol 74:547-59
Tian, Mengnan; Macdonald, Robert L (2012) The intronic GABRG2 mutation, IVS6+2T->G, associated with childhood absence epilepsy altered subunit mRNA intron splicing, activated nonsense-mediated decay, and produced a stable truncated γ2 subunit. J Neurosci 32:5937-52
Macdonald, Robert L; Kang, Jing-Qiong (2012) mRNA surveillance and endoplasmic reticulum quality control processes alter biogenesis of mutant GABAA receptor subunits associated with genetic epilepsies. Epilepsia 53 Suppl 9:59-70
Huang, Xuan; Tian, Mengnan; Hernandez, Ciria C et al. (2012) The GABRG2 nonsense mutation, Q40X, associated with Dravet syndrome activated NMD and generated a truncated subunit that was partially rescued by aminoglycoside-induced stop codon read-through. Neurobiol Dis 48:115-23

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