Destabilization of the delicate balance between inhibition and excitation in the nervous system may underlie many neurological disorders, including temporal lobe epilepsy (TLE). Gamma-aminobutyric acid (GABA) is the major transmitter at inhibitory chemical synapses in the central nervous system. Alteration in type A GABA receptor (GABA/AR) function due to change in subunit composition has been hypothesized to be a critical component of epileptogenesis. Little is known, however, about the genetic mechanisms that regulate granule cells of adult rats following pilocarpine-induced status epilepticus (SE), it has yet to be demonstrated that these changes are either necessary or sufficient for the development of epilepsy. The presence of an alpha4 subunit (GABRA4) and the lack of an alpha1 subunit (GABRA) in the GABAalphaR complex has been associated with a decrease in benzodiazepine sensitivity and a heightened sensitivity to blockade by zinc. Both of these features are also seen in adult rats with TLE following pilocarpine-induced SE. The broad objective of this project is to test the hypothesis that alterations in GABAA4 subunit gene expression play a critical role in the process of epileptogenesis by re- establishing normal levels of GABRA4 and GABRA1 following pilocarpine-induced SE and determining whether development of spontaneous seizures is subsequently prevented. To accomplish this objective we will further characterize the 5'flanking region of the GABRA4 gene to identify the boundaries of the promoter and its regulatory sequences that are critical for transcriptional activity in primary cultures of dentate granule cells. Adeno-associated parvovirus (AAV) vectors will then be designed to contain the GABRA4 promoter driving the transcription of GABRA1 transgene to up-regulate alpha1 subunit levels, or a GABRA4 antisense RNA, to down-regulate alpha4 levels. GABA/AR subunit levels will be examined following viral delivery of these vectors to dentate granule cells in culture and in vivo. An alternative strategy of decoy oligonucleotides containing regulatory sequences found in the GABRA4 promoter will also be tested in vitro and in vivo for its ability to down-regulate endogenous GABRA4 promoter will also be tested in vitro and in vivo for its ability to down- regulate sequences found in the GABRA4 promoter will also be tested in vitro and in vivo for its ability to down-regulate endogenous GABRA4 gene expression. These vectors will then be introduced into dentate granule cells of pilocarpine-treated rats to determine whether GABA/AR alpha-subunit expression can be normalized, and if so whether subsequent development of epilepsy can be prevented. Results of these studies should enhance our understanding of GABA/AR subunit gene regulation, establish if subunit changes are a necessary component of epileptogenesis and provide a basis for novel therapeutic strategies for the prevention or cure of epilepsy.
