Genetic Analysis of Neurodegeneration

神经退行性疾病的遗传分析

• 批准号: 6849232
• 负责人: SUSAN L ACKERMAN
• 金额: $ 31.62万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6849232
• 关键词: ataxia; cell cycle; free radical oxygen; gene expression; gene mutation; granule cell; immunocytochemistry; laboratory mouse; mitochondria; mitogens; neural degeneration; neurogenetics; oxidative stress; oxidoreductase

Although neurodegenerative disorders are prevalent in the aging human population, the molecular mechanisms underlying these diseases are not well understood. As in humans, genetic lesions have been associated with neurodegeneration in mice. Several mouse mutations exist that result in the abnormal death of embryonic or early postnatal neurons. In contrast, our studies of mice homozygous for the spontaneous mutation harlequin (Hq) have shown that this mutation causes progressive neuron loss in adult mice. Hq mutant mice are characterized initially by a loss of hair in homozygous females and hemizygous males. These mice develop progressive ataxia concomitant with loss of cerebellar neurons. Cell loss in Hq mutants peaks at 5-7 months and is initially confined to granule cells in the caudal cerebellum. Analysis of cell cycle markers demonstrates that granule cell apoptosis is accompanied by abortive cell cycle re-entry. These abnormally cycling cells express sonic hedgehog, a potent mitogen expressed by granule cell precursors, but down-regulated upon terminal differentiation of these cells. The Hq critical region has been refined to a 0.61 cM region of the X Chromosome, and a genomic contig across this region has been assembled. Genetic analysis of transcripts within the Hq region demonstrated that the gene encoding the apoptosis-inducing factor (Aif ), a mitochondrial oxidoreductase, cosegregates with the Hq gene. Further, Aif transcript levels are greatly reduced in pre-ataxic mutant mice, making Aif a likely candidate for the Hq gene. Immunohistochemistry results demonstrate oxidized DNA is present in mutant but not control granule cells, suggesting down-regulation of Aif results in oxidative stress. Experiments outlined in this grant will identify the molecular lesion in the Aif gene in Hq mutant mice. In addition, the effect of the Hq mutation on mitochondrial function and oxidative stress will be analyzed. Lastly, to test whether the ectopic expression of sonic hedgehog is sufficient to cause cell cycle re-entry and subsequent apoptosis, transgenic mice will be generated that abnormally express this molecule in terminally differentiated granule cells. The results of these experiments will allow validation of Hq mutant mice as a model for elucidation of the molecular interplay between oxidative stress, mitogen activation and cell cycle re-entry, and neuronal death in the adult nervous system.

尽管神经退行性疾病在老龄化人群中普遍存在，但这些疾病背后的分子机制尚不清楚。 与人类一样，基因损伤与小鼠的神经变性有关。存在几种小鼠突变，导致胚胎或出生后早期神经元的异常死亡。 相比之下，我们对自发突变 harlequin (Hq) 纯合小鼠的研究表明，这种突变会导致成年小鼠进行性神经元损失。 Hq突变小鼠的最初特征是纯合雌性和半合子雄性的毛发脱落。 这些小鼠出现进行性共济失调并伴有小脑神经元的丧失。 Hq 突变体的细胞损失在 5-7 个月时达到峰值，最初仅限于小脑尾部的颗粒细胞。 细胞周期标记物的分析表明，颗粒细胞凋亡伴随着细胞周期重新进入失败。 这些异常循环的细胞表达声波刺猬，这是一种由颗粒细胞前体表达的有效有丝分裂原，但在这些细胞的终末分化时下调。 Hq 关键区域已细化为 X 染色体的 0.61 cM 区域，并且已组装了跨该区域的基因组重叠群。 对 Hq 区域内转录本的遗传分析表明，编码凋亡诱导因子 (Aif)（一种线粒体氧化还原酶）的基因与 Hq 基因共分离。 此外，Aif 转录水平在共济失调前突变小鼠中大大降低，使得 Aif 可能成为 Hq 基因的候选者。 免疫组织化学结果表明，突变体颗粒细胞中存在氧化 DNA，但对照颗粒细胞中不存在，这表明 Aif 的下调会导致氧化应激。本次资助中概述的实验将鉴定 Hq 突变小鼠中 Aif 基因的分子损伤。 此外，还将分析Hq突变对线粒体功能和氧化应激的影响。 最后，为了测试音刺猬的异位表达是否足以引起细胞周期重入和随后的细胞凋亡，将产生在终末分化的颗粒细胞中异常表达该分子的转基因小鼠。 这些实验的结果将验证 Hq 突变小鼠作为阐明氧化应激、有丝分裂原激活和细胞周期重新进入以及成体神经系统中神经元死亡之间分子相互作用的模型。