Engineering the Mouse Nervous System To Decipher Network Mechanisms of Epilepsy

描述（由申请人提供）：候选人和环境：该候选人在哈佛和麻省理工学院完成了广泛的神经科学训练。贝斯以色列神经病学中心提供了一个独立的实验室和启动资金。候选人的K08资助将于11/05结束，他需要继续提供薪金支持以保障他的研究时间。该奖项将使他能够获得获得R01拨款所需的初步数据。他的长期目标是在贝斯以色列/哈佛医学院继续他的全职生物医学研究生涯。在职业发展规划中，候选人提出将自己的训练和经验应用到癫痫的研究中。研究项目总结：儿童期缺失性癫痫（CAE）的峰波发作背后的大脑分子机制长期以来一直存在争议。本研究采用新的噬菌体p1衍生的Cre/loxP重组技术，将缺失癫痫基因突变靶向鼠脑中的特定神经元亚型。最近在t型钙通道Cav3.2基因中发现了儿童期缺失癫痫相关突变。该项目的假设是，Cav3.2突变改变了特定神经元亚型的放电特性，从而导致特征性的3-5赫兹的尖波复合体节律放电，以及使失神性癫痫儿童遭受的行为停滞。为了验证这一假设，候选人将使用表位标记的编码Cav3.2的CACNA1H转基因在小鼠中重建疾病。核苷酸突变将重建癫痫相关的氨基酸变化F161L和V831M，改变Cav3.2通道门控。其次，他将通过在转基因中添加Cre重组酶可删除的转录和翻译沉默元件，将该基因靶向于特定的神经元亚型。由于它们含有细胞类型特异性启动子，Cre转基因在有限的神经元亚型中表达Cre重组酶蛋白。只有在这些神经元中，Cre才会删除沉默元件，引起转基因表达，并产生表位标签染色。在这项研究中，候选人将测试皮质锥体或网状丘脑神经元异常的突发放电是否会导致缺失癫痫。如果再现癫痫的特征性体征，则可以确定Cav3.2基因突变导致癫痫缺失。这种突变基因的靶向表达将识别负责的神经元。识别神经底物将有助于识别其他疾病基因和潜在的药物靶点。

DESCRIPTION (provided by applicant): Candidate and Environment: This candidate has completed extensive neuroscience training at Harvard and MIT. Beth Israel Neurology has provided an independent laboratory and start-up funds. The candidate's K08 funding ends in 11/05 and he needs continued salary support to protect his research time. This Award would enable him to obtain the preliminary data necessary to obtain an R01 grant. His long term goal is to continue his full-time biomedical research career at Beth Israel/ Harvard Medical School. In the career development plan, the candidate proposes to apply his training and experience to the study of epilepsy. Research Project Summary: The molecular mechanisms in the brain that underlie the spike-and-wave seizures of childhood absence epilepsy (CAE) have long been debated. This proposal uses new bacteriophage P1-derived Cre/loxP recombination techniques to target absence epilepsy gene mutations to specific neuron subtypes in the murine brain. Childhood absence epilepsy-associated mutations were recently discovered in the T-type calcium channel Cav3.2 gene. The project's hypothesis is that Cav3.2 mutations alter the firing properties of specific neuron subtypes to cause the characteristic 3-5 Hz rhythmic discharge of spike-and-wave complexes, and the behavioral arrests afflicting children with absence epilepsy. To test this hypothesis, the candidate will recreate the disease in mice using an epitope-tagged CACNA1H transgene that encodes Cav3.2. Nucleotide mutations will be made to recreate the epilepsy-associated amino acid changes F161L and V831M, which alter Cav3.2 channel gating. Second, he will target the gene to specific neuron subtypes by adding a Cre recombinase delete-able trancriptional and translational silencing element to the transgene. Because they contain cell-type specific promoters, the Cre transgenes express Cre recombinase protein in limited neuron subtypes. Only in these neurons will Cre delete the silencing element, cause transgene expression, and generate epitope tag staining. In this study, the candidate will test whether abnormal burst firing in cortical pyramidal or reticular thalamic neurons cause absence epilepsy. If the characteristic signs of epilepsy are reproduced, it will establish that mutations in Cav3.2 cause absence epilepsy. Targeted expression of this mutant gene will identify the responsible neurons. Identifying the neural substrate will facilitate work to identify other disease genes and potential drug targets.