Genetic Analysis of Neurodegeneration

虽然神经退行性疾病在老龄化人群中普遍存在，但这些疾病的分子机制尚不清楚。与人类一样，遗传损伤也与小鼠的神经退行性变有关。几种小鼠突变可导致胚胎或出生后早期神经元的异常死亡。相反，我们对自发突变harlequin （Hq）的小鼠纯合子的研究表明，这种突变会导致成年小鼠的进行性神经元丢失。Hq突变小鼠最初的特征是纯合子雌性和半合子雄性的毛发脱落。这些小鼠发生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个月时达到顶峰，最初仅限于尾侧小脑的颗粒细胞。细胞周期标志物分析表明，颗粒细胞凋亡伴随着流产细胞周期的重新进入。这些异常循环的细胞表达音hedgehog基因，这是一种由颗粒细胞前体表达的强效有丝分裂原，但在这些细胞的终末分化中下调。Hq关键区域已被细化到X染色体的0.61 cM区域，并在该区域组装了一个基因组contig。Hq区转录本的遗传分析表明，编码凋亡诱导因子（一种线粒体氧化还原酶）的基因与Hq基因共分离。此外，在共济失调前突变小鼠中，Aif转录水平大大降低，这使得Aif可能是Hq基因的候选基因。免疫组织化学结果显示，突变体颗粒细胞中存在氧化DNA，而控制颗粒细胞中不存在氧化DNA，这表明Aif的下调导致氧化应激。本拨款中概述的实验将确定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，为了验证超音hedgehog基因的异位表达是否足以引起细胞周期再进入和随后的细胞凋亡，我们将在终末分化的颗粒细胞中产生异常表达该分子的转基因小鼠。这些实验的结果将允许验证Hq突变小鼠作为一个模型来阐明氧化应激、有丝分裂原激活和细胞周期再进入以及成人神经系统中神经元死亡之间的分子相互作用。

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.