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

项目总结/摘要 本研究的目的是探讨儿童失神癫痫（CAE）的病理生理机制 使用转基因小鼠模型。CAE是最常见的小儿癫痫，超过三分之一的病例是 抗药性虽然临床失神发作是非惊厥性的，但一般3-5 Hz棘波 癫痫发作（SW）伴随着部分意识丧失和行为停止，发生数百起 这些都与显著的注意力和认知缺陷有关。这不是良性疾病。 人类和小鼠模型中的电生理学证据表明，SW是在 大脑的丘脑皮层回路，其中升高的T型钙电流在以下方面具有良好的特征性作用： 被认为是驱动CAE发作活动的反弹爆发放电活动。来自几个单基因小鼠的数据 从P/Q型钙通道突变体开始的CAE模型支持这一假设， 在SW发作之前，对丘脑神经元的异常突触输入升高了低阈值T电流。然而，在这方面， 调节丘脑神经元中T通道活性的下游重塑的分子机制是 尚未明确定义。本申请中提出的实验将剖析特定T通道的作用， 在丘脑皮层回路内的亚型，导致过度兴奋和棘波癫痫发作。我将测试三个 假设由于P/Q型钙通道突变引起的有缺陷的突触释放升高了T电流， 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型通道的辅助基因的转录，所述T型通道编码功能性相互作用蛋白， 调节T通道表达。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突变模型中丘脑Cacna 1g和Cacna 1h通道亚型表达水平和模式 2）研究T通道修饰基因Stac 1在T电流升高中的作用 和SW。第一个目的是研究T通道亚型的表达模式， 丘脑皮层回路神经元，并确定它们在之前和之后是否有表达比率异常。 在蹒跚小鼠癫痫发作后。第二个目标将调查Stac 1是否有助于SW活动， Stac 1缺失是否会改变体内丘脑T通道的表达，以及Stac 1缺失是否会 改变或防止蹒跚小鼠的癫痫发作。在初步研究中，我已经获得了定量证据， 敏感、单细胞分辨率RNA显微镜原位杂交方法检测Cacna 1g的mRNA， 在丘脑中继核中表达的主要T型通道α亚单位，在蹒跚的丘脑中升高 与电生理学证据一致。我还发现，删除Stac 1，一个修饰符， 在丘脑神经元中表达的T型通道亚型的表面表达产生CAE表型。 这些研究有助于理解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.