Mechanisms of Resistance to Excitotoxic Cell Death

描述（由申请方提供）：兴奋性毒性在癫痫、中风、创伤性脑损伤和一系列其他CNS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，导致神经元组的不受控制的兴奋性和随后的兴奋性毒性细胞死亡。脑损伤是由与兴奋性毒性细胞死亡机制相关的持续性和高度重复性癫痫发作引起的。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脑损伤其他领域的工作。我们已经表明，近交系小鼠表现出显着的遗传差异，兴奋性氨基酸诱导的细胞死亡的易感性。在过去的四年中，目前的RO 1资金，我们已经确定了三个显着的数量性状位点（QTL）的位置在染色体18，15和4的小鼠基因组中，负责烟草诱导的细胞死亡的易感性，使用基因组排阻作图与DNA为基础的标记在回交群体来自C57 BL/6和FVB/N近交系小鼠品系。该更新申请的目标是使用精细定位、位置候选和位置克隆技术来鉴定导致对尿素诱导的兴奋性毒性细胞死亡的易感性的遗传变异的基因。为了确定每个QTL的基因，我们提出了三个具体的目标。特异性目的1的同源基因定位研究将用于确认这些小鼠细胞死亡易感性基因座的存在并进行精细定位。我们将严格评估临时遗传定位的强度，确认图位，并重新评估这些位点的表型效应。具体目标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。这种具有已鉴定供体染色体的个体的扩增将使我们能够在统计学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鉴定渗入区域中的候选基因。对于基因鉴定，我们将首先通过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的或推断的功能寻找，关于尿素诱导的兴奋性毒性细胞死亡。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，我们将测试单核苷酸多态性（SNP）单倍型与易感性的近交系小鼠品系之间的关联，以诱导细胞死亡，并评估候选基因的差异表达，使用RT-PCR和原位杂交方法。从这些研究中收集的结果将有助于我们了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.