Genetic Dissection of Age-dependent Neuroprotection Mechanisms in Drosophila

果蝇年龄依赖性神经保护机制的遗传解析

• 批准号: 7799697
• 负责人: BARRY S GANETZKY
• 金额: $ 38.04万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-15 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7799697
• 关键词: Adolescence; Advanced Glycosylation End Products; Affect; Autophagocytosis; Biochemical; Biological; Biological Assay; Brain; Cells; Cessation of life; Chimeric Proteins; Collection; Coma; Communication; Defect; Dihydroxyacetone Phosphate; Dissection; Drosophila genus; Enzymes; Event; Exhibits; Experimental Models; Functional disorder; Gene Proteins; Generations; Genes; Genetic; Genetic screening method; Glyceraldehyde 3-Phosphate; Glycolysis; Goals; Health; Histological Techniques; Human; Isomerase; Link; Longevity; Lysosomes; Maintenance; Maleimides; Maps; Mediating; Medical; Membrane Fusion; Molecular; Molecular Analysis; Molecular Genetics; Motor; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Oxidative Stress; Paralysed; Pathway interactions; Phenotype; Process; Proteins; Pyruvaldehyde; Recycling; Role; Signal Transduction; Surveys; Synaptic Transmission; Synaptic Vesicles; System; Testing; Toxic effect; Triose-Phosphate Isomerase; age related; base; biological adaptation to stress; crosslink; dopaminergic neuron; dosage; fly; in vivo; mutant; nervous system disorder; neuromechanism; neuron loss; neuronal survival; neuroprotection; novel; overexpression; prevent; progressive neurodegeneration; protein complex; relating to nervous system; research study; synuclein; wasting

DESCRIPTION (provided by applicant): Our long-term aim is to elucidate molecular mechanisms of neural signaling in Drosophila. Here we focus on analysis of mutants with defects in maintenance of neuronal viability. We have discovered several mutations among our collection, including wstd and comt, that exhibit shortened lifespan and age-dependent, progressive neurodegeneration providing us with novel starting points to dissect neuroprotective mechanisms. Our goals are to determine the in vivo roles of the affected proteins using genetic, molecular, biochemical, and histological techniques to analyze how defects in these proteins result in the observed phenotypes. wstd encodes the glycolytic enzyme, triose phosphate isomerase (Tpi) responsible for the interconversion of DHAP (dihydroxyacetone phosphate) and GAP (glyceraldehyde 3-phosphate), only the latter of which is able to continue through glycolysis. Mutations of Tpi in humans result in Triosephosphate isomerase deficiency, characterized by early death and neurodegeneration but the underlying mechanism has remained unclear. We hypothesize that the enzymatic block in Tpi-deficient flies and humans leads to excess accumulation of methylglyoxal (MG), which reacts with target proteins to generate advanced glycation end products (AGEs) causing loss of protein activity, cross-linking, aggregation, and ultimately neuronal death. We propose genetic and biochemical experiments to test and refine this hypothesis. comt, which encodes NSF-1 (N-ethyl- maleimide sensitive fusion protein), exhibits a deficit in lysosomes and accumulation of ubiquitinated protein complexes in parallel with neurodegeneration. We hypothesize that comt is deficient in autophagy. Experiments are proposed to test this hypothesis and to dissect the step(s) in the process that are impaired. Additional mechanisms of neuroprotection will be investigated by phenotypic and molecular analysis of other mutants in our collection that exhibit neurodegeneration. Neuroprotective mechanisms are essential for proper neural communication and their disruption leads to some of the most devastating human neurological disorders. Detailed understanding of these mechanisms is thus of fundamental biological as well as medical importance. Drosophila has already proven to be a potent experimental system for elucidating these mechanisms. Moreover, both wstd and comt have direct links with human neurodegenerative disorders. Consequently, our proposed analyses should have broad biological and medical significance by contributing important new information that will advance our understanding of the underlying molecules and mechanisms that maintain neuronal viability and integrity.

描述（申请人提供）：我们的长期目标是阐明果蝇神经信号的分子机制。在这里，我们重点分析在维持神经元活力方面有缺陷的突变体。我们已经在我们的收集中发现了一些突变，包括wstd和comt，它们表现出寿命缩短和年龄依赖的进行性神经变性，为我们剖析神经保护机制提供了新的起点。我们的目标是利用遗传、分子、生化和组织学技术来确定受影响蛋白质的体内作用，分析这些蛋白质的缺陷如何导致观察到的表型。wstd编码糖酵解酶，三糖磷酸异构酶（Tpi），负责DHAP（二羟丙酮磷酸）和GAP（3-磷酸甘油醛）的相互转化，只有后者能够通过糖酵解继续进行。人类Tpi突变导致三磷酸异构体酶缺乏，以早期死亡和神经变性为特征，但其潜在机制尚不清楚。我们假设，tpi缺陷果蝇和人类的酶阻滞导致甲基乙二醛（MG）的过量积累，甲基乙二醛与靶蛋白反应产生晚期糖基化终产物（AGEs），导致蛋白质活性丧失、交联、聚集，最终导致神经元死亡。我们提出基因和生化实验来检验和完善这一假设。comt编码NSF-1 （n -乙基-马来酰亚胺敏感融合蛋白），在神经变性过程中表现出溶酶体的缺陷和泛素化蛋白复合物的积累。我们假设comt缺乏自噬。提出了实验来验证这一假设，并剖析过程中受损的步骤。神经保护的其他机制将通过我们收集的其他表现出神经变性的突变体的表型和分子分析来研究。神经保护机制对于适当的神经交流至关重要，它们的破坏导致一些最具破坏性的人类神经系统疾病。因此，详细了解这些机制具有基本的生物学和医学重要性。果蝇已经被证明是阐明这些机制的一个强有力的实验系统。此外，wstd和comt都与人类神经退行性疾病有直接联系。因此，我们提出的分析应该具有广泛的生物学和医学意义，通过提供重要的新信息，将推进我们对维持神经元活力和完整性的潜在分子和机制的理解。