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

神经元和非神经元细胞由一个外部边界组成，该外部边界由称为质膜的蛋白质和脂类的复杂混合物定义。这包围了一个充满流体的三维空间，称为胞浆，它包含额外的被膜包围的隔室，每个隔间由蛋白质和脂类的独特组合组成。为了让细胞正常运作，蛋白质和脂类必须被有效地输送到这个迷宫般的膜中正确的富含膜的隔室。毫不奇怪，如果这种转运受到干扰，以至于错误的蛋白质和脂类被输送到错误的膜富集室，细胞功能可能会受到不利影响，进而导致各种人类疾病的发生。因此，建立细胞实现调节蛋白质和脂肪运输的机制是细胞生物学中的一个重大挑战，对我们理解人类疾病具有直接意义。在过去的10年里，我们的实验室一直专注于描述控制蛋白质和脂类在细胞膜迷宫中特定方面的调节运输的机制细节，称为内吞网络。特别是，我们研究了一种称为逆转录增殖体的蛋白质家族。我们和其他人的研究开始定义逆转录功能，并在这样做的过程中揭示了它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，逆转聚体的缺陷是包括神经退行性疾病在内的各种人类疾病的基础。逆转聚体的一个核心成分是一种名为VPS35的蛋白质。VPS35基因编码天冬氨酸天冬氨酸620位氨基酸的转换，是家族性常染色体显性遗传性帕金森氏病(VPS35(D620N)突变)的罕见原因(估计在0.1-1.0%之间)。有了这样的发现，现在迫切需要进行功能研究，以确定VPS35(D620N)在帕金森病发病机制中的作用。在目前的提案中，我们试图利用我们研究逆转聚体的丰富经验来确定帕金森氏病相关VPS35突变对逆转聚体功能的功能影响，最终目的是揭示这如何影响神经细胞功能和死亡。我们拟议的研究将解决以下问题：1)VPS35(D620N)突变对多蛋白逆转录复合体的组装有何影响？(S)VPS35(D620N)突变对逆转录增殖体调节特定蛋白质在内吞网络中运输的能力有什么影响？正常的VPS35和VPS35(D620N)突变如何影响通常用于研究帕金森氏病的人类多巴胺能神经元培养物中的蛋白质运输和细胞活性？总体而言，来自拟议研究的数据将通过同行评审出版物和在国际会议上的口头陈述来传播，将定义与帕金森氏病相关的VPS35突变对神经细胞活性的功能影响，可能导致识别新的生物标记物的知识，并为携带VPS35(D620N)突变的患者提供治疗干预的理论基础。

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.