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理后发生的神经元死亡中至关重要。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，并随后导致兴奋性毒性细胞死亡。脑损伤是由持续和高度重复的癫痫发作引起的，这些癫痫发作与兴奋性毒性细胞死亡机制有关。然而，我们对癫痫发作后调节细胞死亡的分子通路的了解仍处于起步阶段，在很大程度上落后于脑损伤的其他领域的工作。我们发现近交系小鼠对兴奋性氨基酸诱导的细胞死亡的敏感性存在显著的遗传差异。在最近四年的RO1资助中，我们在C57BL/6和FVB/N近交系小鼠的回交群体中，利用基于DNA的标记，利用基因组排斥作图，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基因座(QTL)的位置，这些QTL与癫痫诱导的细胞死亡易感性有关。这一更新应用的目标是利用精细定位、位置候选和位置克隆技术来识别导致癫痫诱发的兴奋性毒性细胞死亡的遗传变异的基因。为了确定每个QTL背后的基因，我们提出了三个特定的目标。特定目的1的同源定位研究将被用来证实这些小鼠细胞死亡易感基因的存在和精细定位。我们将严格评估临时遗传作图任务的强度，确定作图位置，并重新评估这些基因座的表型效应。在《特定目标2》中概述的精细作图研究将使用特定于区间的同源品系将每个QTL区间缩小到1 cM以下。这种具有确定的供体染色体的个体的扩大将使我们能够在统计上测试每个染色体片段与红藻氨酸诱导的细胞死亡的联系。在具体目标3中，我们将使用两种方法评估和识别导入区域中的候选基因。在基因识别方面，我们将首先利用迅速出现的与基因和基因图谱相关的小鼠和人类基因组资源来识别候选基因。候选基因将基于已知或推断的与癫痫诱导的兴奋性毒性细胞死亡相关的功能来寻找。新的和已知的基因将通过使用单链构象多态性(SSCP)分析的菌株序列比较来识别。然后，我们将测试近交系小鼠的单核苷酸多态(SNP)单倍型与癫痫诱导的细胞死亡的易感性之间的关联，并使用RT-PCR和原位杂交方法评估候选基因的差异表达。这些研究的结果将有助于我们理解神经元敏感性的生物学机制，并为人类癫痫的发病机制提供信息。

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.