Mechanisms of Resistance to Excitotoxic Cell Death

描述（由申请人提供）：兴奋性毒性在癫痫、中风、创伤性脑损伤和一系列其他中枢神经系统病变后发生的神经元死亡中起着至关重要的作用。在癫痫中，大脑兴奋和抑制之间的平衡被破坏，导致神经元群的兴奋性失控和随后的兴奋毒性细胞死亡。脑损伤是由与兴奋性毒性细胞死亡机制相关的持续和高度重复的癫痫发作引起的。然而，我们对癫痫活动后调节细胞死亡的分子途径的理解仍处于起步阶段，并且在很大程度上落后于其他脑损伤领域的工作。我们已经证明，近交系小鼠对兴奋性氨基酸诱导的细胞死亡的易感性表现出显著的遗传差异。在目前RO1资助的过去四年中，我们已经确定了小鼠基因组中18、15和4号染色体上三个重要的数量性状位点（QTL）的位置，这些位点负责癫痫诱导的细胞死亡易感性，使用基于dna的标记在C57BL/6和FVB/N近交系小鼠的回交群体中进行基因组排除定位。这项更新申请的目的是利用精细定位、位置候选和位置克隆技术鉴定引起癫痫诱发的兴奋性毒性细胞死亡易感性遗传变异的基因。为了确定每个QTL背后的基因，我们提出了三个具体目标。特异靶蛋白1的基因定位研究将用于证实这些小鼠细胞死亡易感性位点的存在并精细定位。我们将严格评估临时遗传作图分配的强度，确认作图位置，并重新评估这些位点的表型效应。在Specific Aim 2中概述的精细定位研究将使用间隔特异性同源菌株将每个QTL间隔缩小到小于1 cM的间隔。这种扩增了供体染色体的个体将使我们能够在统计学上测试每个染色体片段与盐酸盐诱导的细胞死亡的联系。在Specific Aim 3中，我们将使用两种方法评估和鉴定渗入区域中的候选基因。对于基因鉴定，我们将首先利用与基因和基因图谱相关的快速出现的小鼠和人类基因组资源来鉴定候选基因。候选基因将根据已知或推断的与癫痫诱发的兴奋性毒性细胞死亡有关的功能来寻找。新的和已知的基因将通过菌株序列比较使用单链构象多态性（SSCP）分析。然后，我们将在自交系小鼠株中检测单核苷酸多态性（SNP）单倍型是否与癫痫诱导的细胞死亡易感性相关，并使用RT-PCR和原位杂交方法评估候选基因的差异表达。从这些研究中收集到的结果将有助于我们理解神经元敏感性的生物学机制，并提供有关人类癫痫发病机制的信息。

DESCRIPTION (provided by applicant): Excitotoxicity is of critical importance in the neuronal death that occurs after epilepsy, stroke, traumatic brain injury, and a range of other CNS pathologies. In epilepsy, the balance between cerebral excitation and inhibition is disrupted, leading to uncontrolled excitability of groups of neurons and subsequent excitotoxic cell death. Brain damage is caused by persistent and highly repetitive seizures that are associated with excitotoxic cell death mechanisms. However, our understanding of the molecular pathways that regulate cell death after seizure activity remain in their infancy and largely lag behind work in other areas of brain injury. We have shown that inbred strains of mice show remarkable genetic differences in susceptibility to excitatory amino acid-induced cell death. During the last four years of present RO1 funding, we have identified the location of three significant quantitative trait loci (QTL) on chromosomes 18, 15, and 4 in the mouse genome, responsible for seizure-induced cell death susceptibility, using genome exclusion mapping with DNA-based markers in a backcross population derived from the C57BL/6 and FVB/N inbred mouse strains. The goal of this renewal application is to identify the genes causing genetic variation in susceptibility to seizure-induced excitotoxic cell death using fine-mapping, positional candidate and positional cloning techniques. To identify the genes that underlie each QTL, we have proposed three specific aims. The congenic mapping studies of Specific Aim 1 will be used to confirm the existence of and fine map these murine cell death susceptibility loci. We will rigorously assess the strength of the provisional genetic mapping assignments, confirm the map positions, and reassess the phenotypic effects of these loci. The fine mapping studies outlined in Specific Aim 2 will narrow each QTL interval down to less than a 1 cM interval using interval-specific congenic strains. This expansion of individuals with an identified donor chromosome will allow us to test each chromosomal segment statistically for linkage to kainate-induced cell death. In Specific Aim 3, we will assess and identify candidate genes in the introgressed regions using two approaches. For gene identification, we will first identify candidate genes by utilizing the rapidly emerging mouse and human genome resources relating to genes and gene maps. Candidate genes will be sought based on known or deduced function with regard to seizure-induced excitotoxic cell death. New and known genes will be identified through strain sequence comparison using single-stranded conformation polymorphism (SSCP) analysis. We will then test for single nucleotide polymorphism (SNP) haplotype among inbred mouse strains for association with susceptibility to seizure-induced cell death and also assess differential expression of candidate genes using RT-PCR and in situ hybridization approaches. Results gleaned from these studies will facilitate our understanding of the biological mechanisms that underlie neuronal sensitivity and provide information regarding the pathogenesis of human epilepsy.