Genetic Analysis of Neurodegeneration

尽管神经退行性疾病在老龄化人口中普遍存在，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退化有关。存在几种导致胚胎或出生后早期神经元异常死亡的小鼠突变。相反，我们对自发突变小丑(HQ)纯合子小鼠的研究表明，这种突变会导致成年小鼠进行性神经元丢失。HQ突变小鼠最初的特征是纯合子雌性和半合子雄性脱毛。这些小鼠发展为进行性共济失调，并伴有小脑神经元的丧失。HQ突变体的细胞丢失在5-7个月时达到高峰，最初仅限于小脑尾侧的颗粒细胞。对细胞周期标志物的分析表明，颗粒细胞的凋亡伴随着流产的细胞周期重新进入。这些异常循环的细胞表达Sonic Hedgehog，一种由颗粒细胞前体表达的强大的有丝分裂原，但在这些细胞的终末分化时下调。HQ临界区已被细化到X染色体的0.61 cM区域，并组装了跨越该区域的基因组重叠群。对HQ区域内转录产物的遗传分析表明，编码凋亡诱导因子(AIF)的基因是一种线粒体氧化还原酶，与HQ基因共分离。此外，在共济失调前期突变小鼠中，AIF转录水平大大降低，使AIF可能成为HQ基因的候选基因。免疫组织化学结果显示，在突变的颗粒细胞中存在氧化的DNA，而不是对照颗粒细胞，这表明AIF的下调导致了氧化应激。这项资助中概述的实验将确定HQ突变小鼠的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sonic hedgehog的异位表达是否足以导致细胞周期重新进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。这些实验的结果将使HQ突变小鼠成为阐明氧化应激、有丝分裂原激活和细胞周期重新进入以及成年神经系统神经元死亡之间的分子相互作用的模型。

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.