Synaptic Mechanisms in Drosophila Neurodegeneration

描述（由申请人提供）：最近的进展表明，胆固醇和鞘脂，膜脂筏结构域的组成部分，在突触前功能和神经退行性疾病中都起着关键作用。我们建议研究鞘脂代谢酶/转运体在神经传递和神经元活力的同时维持中的作用。在上一个资助周期中，我们确定了果蝇slug-a-bed （slab）基因，该基因编码神经酰胺酶，是鞘脂代谢的核心。SLAB神经酰胺酶促进囊泡运输和融合介导神经递质释放。我们还建立了Niemann Pick C （NPC）病的果蝇模型，这是一种以鞘脂和胆固醇的错误传递和积累为特征的脂质储存性神经退行性疾病。95%的人鼻咽癌病例是由NPC1基因突变引起的，该基因编码内体细胞样细胞器中的鞘脂转运蛋白。两种果蝇NPC1蛋白dNPC1a/b同样存在于突触前细胞器中，并且是独立必需的。dNPC1突变体损害突触前末端的囊泡和蛋白质运输并导致年龄进行性神经变性。因此，推动这一提议的假设是鞘脂结构域的维持对于突触前功能下的蛋白质和囊泡运输是必不可少的，而这一途径的破坏会引发突触功能障碍，导致神经退行性变。我们提出了四个具体目标来检验这一假设。首先，利用共聚焦成像和亚细胞分离技术研究SLAB神经酰胺酶和dNPC1a/b在神经元脂质/蛋白质转运调节中的作用。其次，利用电生理学、染料成像和电子显微镜分析鞘脂转换在调节突触前功能中的作用。第三，利用克隆技术生成SLAB神经酰胺酶和dNPC1a/b缺失的神经元群体，以检测其对细胞活力和神经退行性疾病进展的影响。最后，利用遗传和分子相互作用研究来探索这些蛋白质介导突触前功能的分子机制，或者在它们缺失的情况下导致神经元死亡。我们的方法独特地结合了鞘脂通路突变体，复杂的细胞生物学方法和果蝇遗传学来探索鞘脂依赖通路在神经元功能中的新作用。这项工作有望大大增加我们对蛋白质和囊泡运输机制的理解，这些机制对神经传递至关重要，并揭示了许多不同神经退行性疾病的共同因果关系缺陷。

DESCRIPTION (provided by applicant): Recent advances suggest that cholesterol and sphingolipids, components of membrane lipid raft domains, play critical roles in both presynaptic function and neurodegenerative disorders. We propose to investigate the roles of sphingolipid metabolizing enzymes/transporters in the coincident maintenance of neurotransmission and neuronal viability. In the last funding cycle, we identified the Drosophila slug-a-bed (slab) gene, which encodes the ceramidase enzyme at the heart of sphingolipid metabolism. SLAB ceramidase facilitates vesicular trafficking and fusion mediating neurotransmitter release. We also established a Drosophila model of Niemann Pick C (NPC) disease, a lipid-storage neurodegenerative disorder characterized by mistrafficking and accumulation of sphingolipids and cholesterol. 95% of human NPC cases are caused by mutation of the NPC1 gene, which encodes a putative sphingolipid transporter in endosomal-ike organelles. Two Drosophila NPC1 proteins, dNPC1a/b, similarly reside in presynaptic organelles and are independently essential. dNPC1 mutants impair vesicular and protein trafficking in the presynaptic terminal and cause age-progressive neurodegeneration. Thus, the hypothesis driving this proposal is that maintenance of sphingolipid domains is essential for protein and vesicle trafficking underlying presynaptic function, and that disruption of this pathway triggers synaptic dysfunction causative to neurodegeneration. We propose four Specific Aims to test this hypothesis. First, to use confocal imaging and subcellular fractionation to investigate the role of SLAB ceramidase and dNPC1a/b in lipid/protein trafficking regulation in neurons. Second, to use electrophysiology, dye imaging and electron microscopy to assay roles of sphingolipid turnover in regulating presynaptic function. Third, to use clonal techniques to generate SLAB ceramidase and dNPC1a/b deficient populations of neurons to assay the consequence on cell viability and the progression of neurodegeneration. Finally, to employ genetic and molecular interaction studies to probe the molecular mechanisms by which these proteins mediate presynaptic function or, in their absence, cause neuronal death. Our approach uniquely combines sphingolipid-pathway mutants, sophisticated cell biological approaches and Drosophila genetics to probe the emerging role of sphingolipid-dependent pathways in neuronal function. The proposed work promises to substantially increase our understanding of protein and vesicle trafficking mechanisms critical to neurotransmission, and to reveal defects of common causality to a number of disparate neurodegenerative diseases.