Many different and useful experimental animal models of epilepsy have led to the characterization of the nature of epileptiform discharges. However, the defects or alterations in the human disease remain far from being established. To begin to address this problem, the proposal adopts a strategy of first defining physiologically relevant phenomena in humans and then studying the underlying molecular mechanisms in a well- controlled model system. Initial experiments show that transcript levels of the genes for human brain voltage-gated sodium channel (HBSC) subtypes I and Ii are significantly altered in patients undergoing surgery for relief of longstanding epileptic seizures. The potential physiological relevance is highlighted by the facts that i) these ion channels are intimately involved in neuronal excitability, a property that is pathologically altered in epilepsy, and ii) these same channels are an important target of the majority of front-line anticonvulsants used to treat the disorder. To study this further, the proposal has three parts. First, RNA from additional specimens of resected human brain tissues obtained at surgery will be analyzed by the ligase detection reaction and in situ hybridization to determine absolute changes of HBSC I, II and III mRNA in the epileptic focus and the surrounding perimeter, establishing any cytoarchitectural subregion or HBSC subtype specificity. The relationship of the mRNA changes to variables of gliosis, cell death, seizure and medication history will also be determined by analysis of variance. Secondly, the relationship between seizures and the observed changes will be further tested in a controlled manner using the rat kainate-induced seizure model. Additionally, rat primary neocortical cultures and human cell cultures known to express HBSC's will be subjected to electric field stimulation (EFS) protocols to test the hypothesis that electrical activity feeds back to affect transcription of sodium channel genes. The time course, frequency dependence and selectivity vis a vis the different subtypes of any such changes will be established. Pharmacologic manipulations will be used to test the potential involvement of cAMP and Ca++ as downstream mediators of stimulation-induced transcriptional changes. Thirdly, a basic molecular biological approach will be used to provide an initial comparative description of the transcriptional promotion and regulation sites encoded within (primarily) 5'-untranslated DNA sequences for human brain sodium channels I and II. The methods include functional transcription assays using reporter gene constructs and structural assays such as DNA footprinting. The results of these studies may provide additional insight into the possible mechanisms leading to altered sodium channel mRNA levels in epilepsy.
| Lu, C M; Brown, G B (1998) Isolation of a human-brain sodium-channel gene encoding two isoforms of the subtype III alpha-subunit. J Mol Neurosci 10:67-70 |
| Lu, C M; Eichelberger, J S; Beckman, M L et al. (1998) Isolation of the 5'-flanking region for human brain sodium channel subtype II alpha-subunit. J Mol Neurosci 11:179-82 |
