Brain development in experimental epilepsy

描述(由申请人提供)：本项目的长期目标是了解癫痫发作对发育中的大脑的影响。在上一次授予期间，我们证明了严重癫痫发作(癫痫持续状态，SE)可以导致未成熟大脑中的神经元死亡，这种死亡通常是细胞凋亡，但也可以是坏死性的。坏死性死亡的比例随着年龄的增长而增加。我们的结果表明，缺氧性神经元坏死始于线粒体中的细胞色素释放，需要caspase级联的激活。即将到来的拨款的目标是检验这样一个假设，即癫痫诱导的神经元坏死是一个有序的过程，不涉及基因表达、RNA或蛋白质合成，但确实涉及caspase级联的线粒体激活。这一过程是由线粒体失效触发的，线粒体失效将细胞色素C释放到细胞质中，从而激活caspase-9，然后(通过特定的、节省能量的蛋白分解)从其固有表达的前酶中激活caspase-3。这一假设将在P15大鼠和成年小鼠身上进行测试，这些小鼠由锂、匹罗卡品或红藻氨酸诱导的癫痫持续状态。我们将使用电子显微镜、免疫细胞化学、生物化学等方法来检测这一程序激活序列。我们还预测，caspase-3或caspase-9活性的抑制剂将减少癫痫诱导的神经元坏死，我们预计caspase-3零突变转基因小鼠的神经元坏死将比对照小鼠少，而p53的KO不受影响。我们还预测，表达杆状病毒p35半胱氨酸酶抑制物的转基因小鼠将显示Se诱导的海马神经元坏死(和凋亡)数量减少。这将与癫痫诱发的脑损伤问题密切相关，因为更好地了解癫痫诱发神经元坏死的机制有助于防止或最大限度地减少这种损害。

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the effects of epileptic seizures on the developing brain. During the last grant period, we showed that severe seizures (status epilepticus, SE) can cause neuronal death in the immature brain, and that this death is often apoptotic, but can also be necrotic. The proportion of necrotic death increases with advancing age. Our results suggest that hypoxic neuronal necrosis starts with cytochrome liberation from mitochondria, and requires the activation of a cascade of caspases. The goal of the upcoming grant is to test the hypothesis that seizure-induced neuronal necrosis is an orderly process that does not involve gene expression, RNA or protein synthesis, but does involve the mitochondrial activation of a caspase cascade. This process is triggered by mitochondrial failure that liberates cytochrome C into the cytoplasm, thereby activating caspase-9, then (by specific, energy-sparing proteolysis), caspase-3 from their constitutionally expressed proenzymes. This hypothesis will be tested in P15 rats and in adult mice subjected to status epilepticus induced by lithium and pilocarpine or by kainic acid. We will use electron microscopy immunocytochemistry, biochemistry and other methods to test this sequence of program activation. We also predict that inhibitors of caspase-3 or caspase-9 activity will reduce seizure-induced neuronal necrosis, We expect that transgenic mice with a null mutation for caspase-3 will have less neuronal necrosis than matched controls, while KOs for p53 will not be affected. We also predict that transgenic mice expressing the baculovirus p35 caspase inhibitor will show a reduced amount of Se-induced neuronal necrosis (and apoptosis) in the hippocampus. This will be of great relevance to the problem of seizure-induced brain damage, since a better understanding of the mechanisms involved in seizure induced neuronal necrosis could help to prevent or minimize that damage.