Activity-Dependent Gene Expression in Human Epilepsy

描述（由申请人提供）：癫痫是一种常见但通常具有破坏性的神经系统疾病，其中异常的大脑兴奋导致无法控制和不可预测的正常功能丧失，通常以强直阵挛发作结束。在大多数情况下，癫痫发作始于大脑的一个小区域，没有明确的潜在遗传或结构缺陷。这种“癫痫病灶”可以定位并通过手术切除，以潜在地治愈癫痫发作。究竟是什么导致并维持了人类新皮层的某些区域的癫痫尚不清楚，也不知道发生了什么结构和功能变化导致并维持了这种状态。一种越来越受到关注的可能性是，这些变化是通过生长和分化因子启动的途径中的活动依赖性变化来实现的。这些因子通过信号中间体来调节转录因子，这些转录因子开启了可以使皮层过度兴奋的基因。利用硬脑膜下记录电极绘制难治性癫痫患儿的人类新皮层癫痫灶，采用高通量筛选方法，结合聚焦高密度cDNA阵列和广泛搜索寡核苷酸阵列，鉴定癫痫灶与附近无独立癫痫样活动区域的基因差异表达。使用这些方法确定了两个重要的信号通路；一种由神经营养因子（BDNF）启动，另一种由神经调节蛋白启动。这些因子分别激活NFkB和ETS-2转录因子，进而调节已知的调节神经元兴奋性的蛋白质。本文提出的研究将利用实时RT-PCR、原位杂交和免疫组织化学来测量人类癫痫组织中神经营养因子和神经调节因子信号通路基因的差异表达，并利用cDNA和寡核苷酸微阵列将这些重要发现扩展到更大的患者系列。假设这些基因表达的变化与脑电活动直接相关，并将在癫痫扩散区域和癫痫样活动的间隔梯度中发现。这些变化会诱发并维持组织的高兴奋状态。了解调控基因的活性依赖性表达，并确定表达这些基因的细胞类型，将有助于了解人类癫痫发作的发展过程，这将有助于临床管理，并为癫痫患者揭示新的治疗途径。

DESCRIPTION (provided by applicant): Epilepsy is a common but often devastating neurological condition where abnormal brain excitation leads to uncontrolled and unpredictable loss of normal function often ending in a tonic-clonic seizure. In a majority of cases the seizures are initiated in a small region of the brain with no clear underlying genetic or structural defect. This "epileptic focus" can be localized and surgically removed to potentially cure the seizures. Exactly what makes and maintains certain areas of human neocortex epileptic is not known, nor is it known what structural and functional changes occur that lead to and maintain this state. One possibility that has gained increasing attention is that these changes come about through activity-dependent changes in pathways that are initiated through growth and differentiation factors. These factors act through signaling intermediates to regulate transcription factors that turn on genes that can make the cortex hyperexcitable. Using subdural recording electrodes to map human neocortical epileptic foci in children with intractable seizures, a high-throughput screening method that combines focused, high-density cDNA arrays with wide searching oligonucleotide arrays was used to identify genes differentially expressed at epileptic foci compared to nearby areas with no independent epileptiform activity. Using these methods two important signaling pathways were identified; one initiated by neurotrophins (BDNF) and the other by neuregulin. These factors activate the NFkB and ETS-2 transcription factors, respectively, which in turn regulate proteins known to modulate neuronal excitability. The studies proposed here will measure the differential expression of neurotrophin and neuregulin signaling pathway genes in human epileptic tissues using real-time RT-PCR, in situ hybridization, and immunohistochemistry and will extend these important findings to a larger series of patients using cDNA and oligonucleotide microarrays. The hypothesis is that these gene expression changes correlate directly with electrical activity and will be found in regions of seizure spread and along interictal gradients of epileptiform activity. These changes would then induce and maintain the hyperexcitable state of the tissue. Understanding activity-dependent expression of regulatory genes together with defining the cell types expressing these genes will provide insights into how seizures develop in humans that will aid in clinical management and reveal new therapeutic avenues for patients with epilepsy.