Brain development in experimental epilepsy

描述（由申请人提供）：该项目的长期目标是了解癫痫发作对发育中的大脑的影响。在上一个资助期，我们发现严重的癫痫发作（癫痫持续状态，SE）可导致未成熟大脑中的神经元死亡，这种死亡通常是凋亡，但也可能是坏死。坏死性死亡的比例随着年龄的增长而增加。我们的研究结果表明，缺氧神经元坏死始于线粒体的细胞色素释放，并需要一系列半胱天冬酶的激活。即将到来的拨款的目标是验证癫痫引起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，但确实涉及半胱天冬酶级联的线粒体激活的假设。这一过程是由线粒体故障触发的，线粒体故障将细胞色素C释放到细胞质中，从而激活caspase-9，然后（通过特定的、节省能量的蛋白质水解），从它们的结构表达的前酶中激活caspase-3。这一假设将在P15大鼠和成年小鼠中进行测试，这些小鼠受到锂和匹罗卡品或kainic酸诱导的癫痫持续状态。我们将使用电镜、免疫细胞化学、生物化学等方法对这一序列的程序激活进行检测。我们还预测caspase-3或caspase-9活性抑制剂将减少癫痫引起的神经元坏死，我们预计caspase-3零突变的转基因小鼠的神经元坏死将少于匹配的对照组，而p53的KOs不会受到影响。我们还预测，表达杆状病毒p35 caspase抑制剂的转基因小鼠将显示出海马中硒诱导的神经元坏死（和凋亡）的减少。这将与癫痫引起的脑损伤问题密切相关，因为更好地了解癫痫引起的神经元坏死的机制有助于预防或减少这种损伤。

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.