Genetic Analysis of Autophagy in the Drosophila Nervous System

描述(申请人提供)：在正常衰老和常见神经退行性疾病如阿尔茨海默氏症和帕金森氏病的进展过程中，含有泛素的细胞内和细胞外蛋白质聚集体都可以在人类的神经元和神经组织中积聚。阻止或抑制这种积聚发生的潜在细胞途径尚不清楚，但越来越多的证据表明，宏观自噬途径(自噬)与年龄相关的细胞损伤和蛋白质聚集体的移除有关。自噬是一种高度保守的溶酶体运输途径，其功能是将受损的细胞成分或聚集的蛋白质隔离到新的囊泡中，这些囊泡被运输到溶酶体进行降解。利用果蝇遗传学，我们已经证明了自噬基因的突变会导致进行性神经退化，并伴随着泛素化蛋白质聚集体的积累。我们还证明，在成熟的中枢神经系统中增加个体自噬基因的表达水平可以显著延长成年寿命。在这项研究提案中，我们将继续我们对新的自噬突变株的表型表征，这些突变株导致寿命缩短和进行性神经缺陷的发展。为了确定自噬途径的哪些成员是神经元维持所必需的，我们将使用遗传和转基因技术(Gal4/UAS)来抑制(UAS-dsRNAi)或增强(UAS-cDNA)成虫大脑中自噬基因的表达水平。成人寿命谱、神经聚集体的形成和积累的不溶性泛素化蛋白(IUP)将被用来检测衰老模式的变化、对环境应激(氧化剂暴露)的反应以及聚集倾向蛋白(PolyQ)对细胞毒表型的抑制。免疫荧光成像和电子显微镜研究将用于确定中枢神经系统泛素化包涵体形成的时间和位置，以及神经元中发生的细胞内运输缺陷的类型和严重程度。这项提议的目标是确定单个自噬基因和一般途径在神经元老化和消除神经元细胞损伤方面的作用。这对人类健康的影响是重大的，因为在许多神经退行性疾病中发现了自噬运输的缺陷，并且通过雷帕霉素治疗上调该途径正在用于亨廷顿病患者的临床试验。相关性：年龄相关性损伤的积累和神经聚集体的形成与影响数百万人的退行性疾病有关。最近对果蝇的遗传学研究表明，一种从神经细胞中移除细胞损伤的细胞途径可以通过保护老化的神经系统显著影响寿命。从这一神经退化和保护的遗传模型中获得的见解将为神经系统提供帮助，这将增强我们对人类复杂过程的理解，并将指导未来旨在促进人类健康和长寿的研究。

DESCRIPTION (provided by applicant): During normal aging and in the progression of common neurodegenerative disorders such as Alzheimer's and Parkinson's disease, both intra-cellular and extra-cellular protein aggregates containing ubiquitin can accumulate in neurons and neural tissues of humans. The underlying cellular pathways that prevent or suppress this accumulation from occurring are not well understood but a growing body of evidence indicates the macroautophagy pathway (autophagy) is involved with the removal of age-related cellular damage and protein aggregates. Autophagy is a highly conserved lysosomal trafficking pathway that functions by sequestering damaged cellular components or aggregated proteins into new vesicles that are transported to the lysosome for degradation. Using Drosophila genetics, we have shown that mutations in autophagy genes result in progressive neural degeneration that is accompanied by the accumulation of ubiquitinated protein aggregates. We also demonstrate that increasing the expression levels of individual autophagy genes in the mature CNS can dramatically extend adult lifespan. In this research proposal we will we continue our phenotypic characterization of new autophagy mutant strains for reduced longevity and the development of progressive neural defects. To determine which members of the autophagic pathway are essential for neuronal maintenance we will use genetic and transgenic techniques (Gal4/UAS) to suppress (UAS- dsRNAi) or enhance (UAS-cDNA) the expression levels of autophagy genes in the adult fly brain. Adult longevity profiles, formation of neural aggregates and accumulation insoluble ubiquitinated proteins (IUP) will be used as assays to detect changes in aging patterns, response to environmental stress (oxidant exposure) and the suppression of cytotoxic phenotypes by aggregate prone proteins (PolyQ). Immunofluorescence imaging and electron microscopy studies will be used to determine the timing and location of ubiquitinated inclusion formation in the CNS and the type and severity of intracellular trafficking defects occurring in neurons. The goal of this proposal is to determine the role that individual autophagy genes and the pathway in general has on neuronal aging and the elimination of cellular damage from neurons. The implications for human health are significant since defects in autophagic trafficking are found in many neural degenerative disorders and that upregulating the pathway by rapamycin treatment is being used in clinical trials on Huntington's disease patients.Relevance: The accumulation of age-dependent damage and the formation of neural aggregates are associated with degenerative disorders affecting millions of people. Recent genetic research in fruit flies has shown a cellular pathway that removes cellular damage from nerve cells can significantly affect longevity by protecting the aging nervous system. Insight from this genetic model of neural degeneration and protection it affords the nervous system will enhance our understanding of complex processes that occur in people and will direct future research that is designed to promote human health and longevity.