Modeling Childhhood Absence Epilepsy in Mice

描述（由申请人提供）：由于大脑中神经元亚型的多样性，直到最近才能够解决复杂神经系统疾病（如儿童期缺失性癫痫）的细胞基础。噬菌体pi衍生的Cre/loxP重组系统的发展使解决记忆的细胞基础取得了重大进展，也有助于我们解决儿童缺失性癫痫的细胞基础的目标。我们将使用这种方法将人类缺失癫痫基因突变靶向鼠脑中的特定神经元亚型。我们将重点关注最近在t型钙通道Cav3.2基因中发现的儿童期缺失癫痫相关突变。t型钙通道与缺失性癫痫有关。此外，一些疾病突变改变通道门控。基于这些发现，我们假设Cav3.2突变改变了大脑中特定神经元亚型的放电特性，从而导致特征性的3Hz有节奏的峰波复合体放电，以及行为停止。为了验证这一假设，我们将首先使用表位标记的CACNA1H转基因（2424kb，基因组DNA）在小鼠中重建疾病，该基因编码Cav3.2。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，我们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，开发针对特定神经元亚型的疾病基因的技术。我们将通过培养具有神经元亚型特异性Cre重组酶转基因的小鼠来评估基因沉默。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。利用这个工具，我们计划测试皮层锥体或网状丘脑神经元异常的突发放电是否会导致缺失性癫痫。如果再现癫痫的特征体征，结果将确定Cav3.2突变导致缺失性癫痫。一旦沉默元件系统被创建，未来的工作，以确定特定的神经元亚型，其功能障碍导致缺失癫痫将成为可能。确定缺失性癫痫的神经基质将有助于确定其他疾病基因和潜在的药物靶点。

DESCRIPTION (provided by applicant): Due to the multiplicity of neuron subtypes in the brain, solving the cellular basis of a complex neurologic disease such as childhood absence epilepsy could not be achieved until recently. Development of the bacteriophage PI-derived Cre/loxP recombination system enabled significant progress towards solving the cellular basis of memory and should also facilitate our goal of solving the cellular basis of childhood absence epilepsy. We will use this method to target human absence epilepsy gene mutations to specific neuron subtypes in the murine brain. We will focus on the childhood absence epilepsy-associated mutations recently discovered in the T-type calcium channel Cav3.2 gene. T-type calcium channels have been implicated in absence epilepsy. Furthermore, some disease mutations alter channel gating. Based on these findings, we hypothesize Cav3.2 mutations alter the firing properties of specific neuron subtypes in the brain to cause the characteristic 3Hz rhythmic discharge of spike-and-wave complexes, and the behavioral arrests afflicting children with absence epilepsy. To test this hypothesis, we will first, recreate the disease in mice using an epitope-tagged CACNA1H transgene (224 kb, genomic DNA) 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, we will develop technologies for targeting the disease gene to specific neuron subtypes by adding a Cre recombinase delete-able transcriptional and translation silencing element to the transgene. We will assess gene silencing by breeding to mice with neuron subtype specific Cre recombinase transgenes. 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. With this tool we plan to test whether abnormal burst firing in cortical pyramidal or reticular thalamic neurons cause absence epilepsy. If the characteristic signs of epilepsy are reproduced, the results will establish that mutations in Cav3.2 cause absence epilepsy. Once the silencing element system is created, future work to determine the specific neuron subtype whose dysfunction produces absence epilepsy will become possible. Identifying the neural substrate of absence epilepsy will facilitate work to identify other disease genes and potential drug targets.