Mechanisms of Resistance to Excitotoxic Cell Death

抵抗兴奋性毒性细胞死亡的机制

• 批准号: 6898463
• 负责人: PAULA E SCHAUWECKER
• 金额: $ 30.06万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6898463
• 关键词: cell death; cytotoxicity; epilepsy; genetic mapping; genetic markers; genetic susceptibility; genotype; inbreeding; kainate; laboratory mouse; molecular cloning; neurons; neuroprotectants; neurotoxicology; quantitative trait loci; single nucleotide polymorphism

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.

描述（由申请人提供）：兴奋性毒性对于癫痫、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至关重要。在癫痫症中，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不受控制，并随后导致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且高度重复的癫痫发作引起的。然而，我们对癫痫发作后调节细胞死亡的分子途径的理解仍处于起步阶段，并且很大程度上落后于脑损伤其他领域的研究。我们已经证明，近交系小鼠在对兴奋性氨基酸诱导的细胞死亡的易感性方面表现出显着的遗传差异。在目前 RO1 资助的最后四年中，我们在源自 C57BL/6 和 FVB/N 近交小鼠品系的回交群体中使用基于 DNA 的标记进行基因组排除图谱，确定了小鼠基因组中 18、15 和 4 号染色体上三个重要数量性状基因座 (QTL) 的位置，这些基因座负责癫痫诱导的细胞死亡易感性。这一更新应用的目标是利用精细作图、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易感性遗传变异的基因。为了识别每个 QTL 背后的基因，我们提出了三个具体目标。 Specific Aim 1 的同源作图研究将用于确认这些小鼠细胞死亡易感性位点的存在并对其进行精细绘制。我们将严格评估临时遗传图谱分配的强度，确认图谱位置，并重新评估这些基因座的表型效应。具体目标 2 中概述的精细作图研究将使用区间特异性同源菌株将每个 QTL 区间缩小到小于 1 cM 区间。这种具有已识别供体染色体的个体的扩增将使我们能够统计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渗入区域中的候选基因。对于基因鉴定，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鉴定候选基因。将根据与癫痫发作诱导的兴奋性毒性细胞死亡有关的已知或推断的功能来寻找候选基因。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鉴定。然后，我们将测试近交系小鼠品系中的单核苷酸多态性 (SNP) 单倍型与癫痫诱导的细胞死亡的易感性之间的关系，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。