Modeling Childhhood Absence Epilepsy in Mice

描述(申请人提供)：由于大脑中神经元亚型的多样性，直到最近才能解决儿童失神癫痫等复杂神经疾病的细胞基础。噬菌体PI衍生的Cre/loxP重组系统的发展使我们在解决记忆的细胞基础方面取得了重大进展，也应该有助于我们解决儿童失神癫痫的细胞基础的目标。我们将使用这种方法将人类失神癫痫基因突变定位到小鼠大脑中特定的神经元亚型。我们将关注最近在T型钙通道Cav3.2基因中发现的儿童失神癫痫相关突变。失神癫痫与T型钙通道有关。此外，一些疾病突变会改变通道门控。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，导致特征的3赫兹棘波复合波的节律性放电，并导致困扰失神癫痫儿童的行为抑制。为了验证这一假设，我们首先将使用编码Cav3.2的表位标记的CACNA1H转基因(224kb，基因组DNA)在小鼠身上重建疾病。核苷酸突变将重新创造癫痫相关的氨基酸变化F161L和V831M，这将改变Cav3.2通道门控。其次，我们将开发针对特定神经元亚型的疾病基因的技术，方法是在转基因中添加Cre重组酶可删除的转录和翻译沉默元件。我们将通过培育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。由于含有细胞型特异性启动子，Cre转基因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，导致转基因表达，并产生表位标记染色。利用这一工具，我们计划测试皮质锥体或网状丘脑神经元的异常放电是否会导致失神癫痫。如果癫痫的特征体征被复制，结果将确定Cav3.2的突变导致失神癫痫。一旦沉默元件系统被创建，未来确定其功能障碍导致失神癫痫的特定神经元亚型的工作将成为可能。识别失神癫痫的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): 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.