Lysosomes in Drosophila melanogaster: Characterization of key components and analysis of their impact for neuronal development.

该项目的总体目标是利用果蝇作为研究神经元发育中溶酶体转运的功能和影响的遗传模型系统。已知约40种人类溶酶体遗传性胆积症是由单一或多种溶酶体水解酶缺乏引起的。除了溶酶体功能紊乱外，最突出的表型特征是神经退行性缺陷、进行性认知衰退和智力迟钝。然而，在神经元细胞中几乎没有研究溶酶体。在过去的几年中，在阿尔茨海默病患者的神经元细胞的内体/溶酶体系统中发现了病理变化。神经元的发育在黑腹果蝇（Drosophila melanogaster）中得到了很好的表征，因此黑腹果蝇被用作研究神经退行性疾病（阿尔茨海默病、亨廷顿病和帕金森病）的模式生物，然而在果蝇中还没有表征溶酶体组分。 我们已经克隆并表达了哺乳动物和果蝇细胞中哺乳动物溶酶体关键组分的果蝇直向同源物：溶酶体酶受体蛋白，一种GGA接头蛋白，溶酶体蛋白酶组织蛋白酶D，B和L以及溶酶体膜蛋白（sun和lamp）。将通过RNAi技术分析分选LERP和单个组分的作用。LERP结合结构域的表征将通过表达在其管腔结构域中含有缺失的突变体来完成。将表征组织蛋白酶的底物特异性和活性。这一基本的生物化学工作将被用来描述果蝇中的溶酶体区室。这些溶酶体成分在果蝇发育过程中的表达将被研究，重点是神经元的发育。我们还将分析单个组织蛋白酶在其特异性抑制剂CER调控的长时程记忆形成中的作用。

The overall goal of the project is to make use of Drosophila as a genetic model system for the investigation of the function and the influence of lysosomal transport in neuronal development. About 40 lysosomal inherited storage disorders in humans are known resulting from the deficiency of single or multiple lysosomal hydrolases. Besides the disturbed lysosomal function most prominent phenotypic features are neurodegenerative defects, progressive cognitive decline and mental retardation. However, lysosomes are hardly studied in neuronal cells. During the last years pathological changes were found in the endosomal/lysosomal system of neuronal cells from Alzheimer¿s disease patients. The development of neurons is well characterized in Drosophila melanogaster which is used therefore as a model organism for the investigation of neurodegenerative disorders (Alzheimer¿s, Huntington¿s and Parkinson¿s-diseases), however lysosomal components have not been characterized in the fly. We have cloned and expressed the Drosophila orthologs of the mammalian lysosomal key components in mammalian and Drosophila cells: the lysosomal enzyme receptor protein ¿LERP¿, a GGA-adaptor protein, the lysosomal proteinases cathepsin D, B und L and lysosomal membrane proteins (sun and lamp). The roles of the sorting LERP and individual components will be analyzed by RNAi techniques. The characterization of LERP binding domain will be done by expression of mutants that contain deletions in its luminal domain. Substrate specificity and activity of the cathepsins will be characterized. This basic biochemical work will be used to characterize the lysosomal compartment in Drosophila. The expression of these lysosomal components during the development of the fly will be studied with a focus on the neuronal development. We will also analyze the role of individual cathepsins in the long term memory formation regulated by their specific inhibitor CER.