Using gene targeting to address the role of LRRK2 in Parkinson's disease

描述(申请人提供)：仅在美国，每年就有大约50,000例新的帕金森氏症病例。帕金森氏病(PD)是由负责产生神经递质多巴胺的脑细胞发生故障或死亡引起的。多巴胺缺乏会导致震颤和运动控制能力下降。帕金森氏症的病因很复杂，在大多数情况下，可能涉及环境和遗传因素的组合。强大的基因技术的出现使得快速识别可能与帕金森病发展相关的基因成为可能。然而，这些基因编码的蛋白质随后的生化特征一直是了解它们在疾病中作用的瓶颈。这项建议的主要目标是开发一种新的方法，以促进PD相关蛋白的表征。基因表达中断是确定基因产物生理作用的一种非常有效的方法。最近，一种名为RNA干扰的技术已经在各种实验系统中抑制了基因表达。然而，使用这种方法通常不可能完全消除蛋白质的表达。或者，基因序列可以通过一种称为同源重组的过程直接打乱。这种方法提供了完全和永久的靶向基因破坏，但只在有限数量的细胞系中起到了良好的作用。我们已经修改了方法，潜在地允许将其用于许多额外的细胞系，包括对帕金森病研究有用的细胞系。作为原则的证明，我们将扰乱神经细胞系中编码Dardarin的基因。尽管Dardarin的功能目前尚不清楚，但编码Dardarin蛋白的基因(LRRK2)的突变与大约5%的帕金森病家族病例有关。利用Dardarin缺失的细胞系，我们将开始研究这种新的蛋白质在神经细胞生理学中的作用。一种适用于神经元细胞系的同源重组方法的发展将有助于其他PD相关蛋白的表征。对已知的PD相关蛋白的生理作用的表征将有助于识别产生多巴胺的神经元中易受损伤的生化途径。这一知识将有助于识别参与帕金森氏病发展的环境触发因素和其他遗传因素。

DESCRIPTION (provided by applicant): Each year in the United States alone there are approximately 50,000 new.cases of Parkinson's disease. Parkinson's disease (PD) results from the malfunction or death of brain cells responsible for the production of the neurotransmitter dopamine. Dopamine deficiency results in tremor and degraded motor control. The etiology of Parkinson's is complex, and in most cases probably involves a combination of environmental and genetic factors. The advent of powerful genetic techniques has allowed the rapid identification of genes potentially associated with the development of PD. However, the subsequent biochemical characterization of the proteins encoded by these genes has been a bottleneck in understanding their roles in disease. The major goal of this proposal is to develop a new approach that will facilitate the characterization of PD associated proteins. The disruption of gene expression is an extremely powerful approach for defining the physiological roles of gene products. Recently, a technique known as RNA interference has allowed suppression of gene expression in a variety of experimental systems. However, it is often not possible to entirely eliminate the expression of a protein using this approach. Alternatively, gene sequences can be disrupted directly using a process known as homologous recombination. This approach offers complete and permanent disruption of targeted genes but has worked well only in a limited number of cell lines. We have modified the approach to potentially allow its use in many additional cell lines, including cell lines useful in PD research. As proof of principle, we will disrupt the gene encoding dardarin in a neuronal cell line. Although the function of dardarin is currently unknown, mutations in the gene (LRRK2) encoding the protein are associated with approximately 5% of all familial cases of Parkinson's disease. Using the dardarin deficient cell line, we will begin to examine the role of this novel protein in the physiology of neuronal cells. The development of a homologous recombination approach amenable to neuronal cell lines will facilitate the characterization of other PD-associated proteins. Characterization of the physiological roles of known PD associated proteins will lead to the identification of the biochemical pathways susceptible to damage in dopamine-producing neurons. This knowledge will aid in the identification of the environmental triggers and additional genetic factors that participate in the development of Parkinson's disease.