TRANSCRIPTIONAL DYSREGULATION OF T-TYPE CALCIUM CHANNELS IN CHILDHOOD ABSENCE EPILEPSY

项目摘要/摘要 本研究旨在明确儿童失神癫痫(CAE)的病理生理机制。 使用转基因小鼠模型。CAE是最常见的儿童癫痫，超过三分之一的病例是 耐药。虽然临床消失发作是非惊厥性的，但广泛性的3-5赫兹棘波 癫痫发作(SWS)伴随着部分意识丧失和行为停止，发生数百次 每天，并与显著的注意力和认知缺陷有关。这不是良性疾病。 在人类和小鼠模型中的电生理学证据表明，SWS是在 大脑丘脑皮质回路，其中升高的T型钙电流在 反弹爆发式发射活动被认为是推动CAE发作活动的原因。来自几个单基因小鼠的数据 CAE的模型，从P/Q型钙通道突变开始，支持这样的假设 在SWS发生前，突触对丘脑神经元的异常输入使低阈值T-电流升高。然而， 调节丘脑神经元T通道活性下游重塑的分子机制是 还没有明确的定义。本申请中提出的实验将剖析特定T通道的作用 丘脑皮质环路中的异构体，导致过度兴奋和棘波发作。我要测试三个 P/Q型钙通道突变导致突触释放缺陷导致T电流升高的假说 突触后丘脑细胞通过改变1)T型亚单位基因的转录，2)它们的剪接形式比率， 和/或3)T-型通道的辅助基因转录，该辅助基因编码功能上相互作用的蛋白质 调节T通道的表达。为了实现这些目标，我提出了以下两个研究目标：1)确定 Cacna 1a突变模型丘脑Cacna 1g和Cacna 1h通道异构体的表达水平和模式 2)研究已建立的T通道修饰物基因Stac1在T电流升高中的作用 和SWS。第一个目标将研究已确定的T通道异构体表达模式 丘脑皮质环路神经元，并确定它们是否有表达比例异常 在摇摇欲坠的小鼠癫痫发作后。第二个目标将调查Stac1是否有助于SWS活动， Stac1缺失是否会改变体内丘脑T通道的表达，以及Stac1缺失是否会 改变或防止摇摇欲坠的小鼠的癫痫发作。在初步研究中，我获得了定量证据，使用 灵敏、单细胞分辨的RNAScope原位杂交法检测Cacna 1g、 丘脑中继核表达的主要T型通道α亚基在摇摇欲坠的丘脑中升高 与电生理证据一致。我还确认了Stac1的删除，它是 T型通道亚型的表面表达在丘脑神经元中表达，产生CAE表型。 这些研究可能有助于理解T型钙通道重构的分子机制。 扩大丘脑皮质节律紊乱的基因组。

PROJECT SUMMARY/ABSTRACT The goal of my research is to identify pathophysiological mechanisms of Childhood Absence Epilepsy (CAE) using transgenic mouse models. CAE is the most common pediatric epilepsy, and over one-third of cases are pharmaco-resistant. While clinical absence episodes are non-convulsive, the generalized 3-5 Hz spike-wave seizures (SWs) are accompanied by partial loss of consciousness and behavioral arrest, occur hundreds of times per day, and are linked to significant attention and cognitive deficits. This is not a benign disorder. Electrophysiological evidence in human and mouse models show SWs are generated within the thalamocortical circuit of the brain, where elevated T-type calcium currents have a well-characterized role in rebound burst firing activity that is thought to drive CAE seizure activity. Data from several monogenic mouse models of CAE, beginning with the P/Q-type calcium channel mutant tottering, support the hypothesis that abnormal synaptic input onto thalamic neurons elevates low threshold T-currents prior to SWs onset. However, the molecular mechanisms mediating the downstream remodeling of T-channel activity in thalamic neurons are not yet clearly defined. The experiments proposed in this application will dissect the roles of specific T-channel isoforms within the thalamocortical circuit that lead to hyperexcitability and spike-wave seizures. I will test three hypotheses that defective synaptic release due to P/Q-type calcium channel mutations elevate T-currents in postsynaptic thalamic cells by altering 1) transcription of T-type subunit genes, 2) their splice form ratios, and/or 3) transcription of auxiliary genes for T-type channels that encode functionally interacting proteins that regulate T-channel expression. To achieve these goals, I propose the following 2 research aims: 1) Determine the level and pattern of thalamic Cacna1g and Cacna1h channel isoform expression in Cacna1a mutant model tottering, and 2) Investigate the role of an established T-channel modifier gene, Stac1, in T-current elevation and SWs. The first aim will investigate the T-channel isoform expression patterns within identified thalamocortical circuit neurons and determine whether they have expression ratio abnormalities before and after seizure onset in tottering mice. The second aim will investigate whether Stac1 contributes to SWs activity, whether Stac1 deletion modifies thalamic T-channel expression in vivo, and whether deletion of Stac1 will modify or prevent seizures in tottering mice. In preliminary studies, I have obtained quantitative evidence using the sensitive, single cell resolution RNAscope in situ hybridization method that mRNA for Cacna1g, the predominant T-type channel alpha-subunit expressed in thalamic relay nuclei, is elevated in tottering thalamus in concordance with electrophysiological evidence. I have also identified that deletion of Stac1, a modifier of surface expression of T-type channel subtype expressed in thalamic neurons, generates a CAE phenotype. These studies may help understand the molecular mechanisms of T-type calcium channel remodeling and expand the genome of thalamocortical rhythm disorders.