Analysing the cell biology of the Parkinson's Disease-linked missense mutation in the retromer VPS35 subunit.

神经元细胞和非神经元细胞由一种叫做质膜的蛋白质和脂质的复杂混合物组成的外边界组成。它围绕着一个充满液体的三维空间，称为细胞质溶胶，其中包含额外的膜包围的隔间，每个隔间由蛋白质和脂质的独特组合组成。为了使细胞正常工作，蛋白质和脂质必须有效地运输到这个迷宫般的膜中正确的富含膜的隔室。不足为奇的是，如果这种运输受到干扰，导致错误的蛋白质和脂质被输送到不正确的富含膜的隔室，细胞功能就会受到不利影响，从而导致各种人类疾病的发展。因此，建立细胞实现受调节的蛋白质和脂质转运的机制是细胞生物学的一个主要挑战，对我们对人类疾病的理解有直接的影响。在过去的10年里，我们的实验室一直专注于描述在被称为内吞网络的细胞膜迷宫的特定方面中控制蛋白质和脂质运输的机制细节。特别地，我们研究了一个叫做逆转录酶的蛋白质家族。我们的研究，以及其他人的研究，正在开始定义逆转录酶的功能，并在此过程中揭示其在各种细胞过程中的重要性，这些过程对正常细胞功能至关重要。此外，很明显，逆转录酶的缺陷是包括神经退行性疾病在内的多种人类疾病的基础。逆转录酶的一个核心成分是一种叫做VPS35的蛋白质。VPS35基因中有一种特殊的突变，它编码了620位天冬氨酸氨基酸的开关，产生了天冬酰胺，这种突变被认为是家族性常染色体显性帕金森病的一种不常见的原因（估计在0.1%到1.0%之间）（这被称为VPS35（D620N）突变）。有了这样的发现，现在进行功能研究以确定VPS35（D620N）在帕金森病发病机制中的作用变得势在必行。在目前的建议中，我们试图利用我们研究逆转录酶的丰富经验来定义与帕金森病相关的VPS35突变对逆转录酶功能的功能含义，最终目的是揭示其如何影响神经细胞功能和死亡。我们提出的研究将解决以下问题：1)。VPS35（D620N）突变如何影响多蛋白反转录复合物的组装？VPS35（D620N）突变对逆转录物调节内吞网络中特定蛋白质运输的能力有什么影响？正常VPS35和VPS35（D620N）突变如何影响通常用于研究帕金森病的人类多巴胺样神经元培养物中的蛋白质转运和细胞活力？总的来说，来自拟议研究的数据将通过同行评审出版物和国际会议上的口头报告传播，将定义帕金森病相关VPS35突变对神经细胞活力的功能含义，这些知识可能导致鉴定新的生物标志物，并为携带VPS35（D620N）突变的患者的治疗干预提供理论依据。

Neuronal and non-neuronal cells are composed of an outer boundary that is defined by a complex mixture of protein and lipids called the plasma membrane. This encircles a fluid filled 3-dimensional space, termed the cytosol, which contains additional membrane-encircled compartments each composed of a unique combination of proteins and lipids. For cells to function normally, proteins and lipids must be efficiently transported to the correct membrane-enriched compartment within this maze of membranes. Not surprisingly, if such transport is perturbed, so that the wrong proteins and lipids are delivered to the incorrect membrane-enriched compartment, cell function can be adversely affected which in turn leads to the development of various human diseases. Establishing the mechanisms through which cells achieve regulated protein and lipid transport is therefore a major challenge in cell biology with direct implication for our understanding of human disease. For the last 10 years our laboratory has focused on describing the mechanistic details that govern regulated transport of proteins and lipids within a specific aspect of the cell's membraneous maze termed the endocytic network. In particular, we have studied a family of proteins called the retromers. Our research, and that of others, is beginning to define retromer function and in so doing revealing its importance in a variety of cellular processes that are vital for normal cell function. Furthermore, it has become apparent that defects in retromer underlie a variety of human diseases including neurodegenerative diseases.One central component of retromer is a protein called VPS35. A specific mutation in the VPS35 gene, which encodes for a switch of the aspartic acid amino acid at position 620 for the amino acid asparagine, has been observed as an uncommon (estimated between 0.1 and 1.0%) cause of familial autosomal dominant Parkinson's Disease (this is termed the VPS35(D620N) mutation). With such a discovery, it becomes imperative that functional studies are now performed to determine the role of VPS35(D620N) in the pathogenesis of Parkinson's Disease. In the current proposal we seek to utilize our extensive experience of studying retromer to define the functional implication of the Parkinson's Disease-linked VPS35 mutation for retromer function, with the ultimate aim of revealing how this affects nerve cell function and death.Our proposed research will address the following questions:1). How does the VPS35(D620N) mutation affect the assemble of the multi-protein retromer complex?2). What effect(s) does the VPS35(D620N) mutation have on the ability of retromer to regulate the transport of specific proteins within the endocytic network?3). How does normal VPS35 and the VPS35(D620N) mutation affect protein transport and cellular viability within human dopaminergic-like neuronal cultures that are commonly used to study Parkinson's Disease? Overall, data derived from the proposed research, which will be disseminated through peer-review publications and oral presentation at international meetings, will define the functional implication of the Parkinson's Disease-linked VPS35 mutation for neuronal cell viability, knowledge which may lead to the identification of novel biomarkers and provide a rationale for therapeutic intervention in patients carrying the VPS35(D620N) mutation.