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Mutation and functional analysis of parkin responsible for AR-JP

负责AR-JP的parkin突变和功能分析

基本信息

批准号：
13470136
负责人：
HATTORI Nobutaka
金额：
$ 6.98万
依托单位：
Juntendo University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

We conducted mutational analysis on more than 700 families with Parkinson's disease. We also established a method to detect compound heterozygotes of parkin mutations using gene dosage technique. Mutinous of the parkin gene were found in approximately 50% of autosomal recessive families. Many kinds of exonic deletions and point mutations were found. This type of familial Parkinson's disease had been considered to be unique among Japanese, but since we started mutational analysis of the parkin gene, we confirmed the world wide distribution of parkin gene mutations. In addition, even though autosomal dominant mode of inheritance, the parkin mutations could be detected in such families. Furthermore, parkin gene mutations were observed in so many solitary cases like sporadic cases. Thus, parkin gene mutations are the most popular form in familial Parkinson's disease. In families with parkin gene mutations, only heterozygous mutations are present in one allele, indicating such patients have … More phenotypes owing to haploinsufficiency or dominant negative effects. Moreover, this finding indicates that parkin gene is a risk factor for developing Parkinson's disease, more common sporadic Parkinson's disease. We now search the exact site of break points or insertion points to screen the parkin cariiers using conventional PCRIn process of screening the parkin gene mutations in young-onset Parkinson's disease, we could collect the families without parkin geme mutations. Very recently, a novel causative gene, DJ-1 for Park7 have been identified. We also screened its mutation in the families that linked to Park7 based on the haplotype analysis. However, we could not find out the DJ-1 mutations in such families. Thus, DJ-1 mutations are rare frequent in young-onset Parkinson's disease compared to parkin mutations. In addition, several families may be link to Park6 or other loci We try to identify causative genes for familial Parkinson' s disease using linkage studyThen we analyzed functions of parkin protein with the collaboration with Dr. Keiji Tanaka of Tokyo Metropolitan Institute of Medical Sciences. We found that parkin protein was a ubiquitin-protein ligase of the ubiquitin system. Now we are working on the candidate substrates of parkin protein as a ubiquitin ligase. We found that CDCrel 1, a synaptic vesicle protein, was a candidate substrate of parkin protein. In addition, we found two additional candidate proteins, i.e., alpha-synuclein 22 and PAEL receptor, with the collaboration of Professor Denis Selcoe of Harvard Medical School and Dr. Rhosuke Takahashi of RIKEN, respectively. Accumulation of PAEL receptor in the endoplasmic reticulum causes endoplasmic reticulum stress and apoptotic cell death. We found evidence to indicate accumulation of PAEL receptor and the presence of endoplasmic reticulum stress in one patient with AR-JP (Park2). In addition, we made immunohistochemical studies in five autopsied brains with Park2. However, no accumulation was not observed in the remaing brains. It is unclear that only one brain with Park2 had accumulation of Pael receptor. It would be possible that different mutations might be related to its accumulation. Different from candidate substrates as mentioned above, we identified 14 clones using yeast two hybrid screening. Among them, a few proteins were ubiquitinated by parkin in vivo ubiqutination system. We now prepared the antibodies for such substrates Less

我们对700多个帕金森病家庭进行了突变分析。我们还建立了一种利用基因剂量技术检测parkin突变复合杂合子的方法。大约50%的常染色体隐性遗传家族中发现了parkin基因的突变。发现了多种外显子缺失和点突变。这种家族性帕金森病曾被认为是日本人特有的疾病，但自从我们开始对parkin基因进行突变分析后，我们证实了parkin基因突变在全球范围内的分布。此外，即使是常染色体显性遗传方式，在此类家族中也可以检测到parkin突变。此外，在许多孤立病例（如散发病例）中观察到parkin基因突变。因此，parkin 基因突变是家族性帕金森病中最常见的形式。在具有parkin基因突变的家族中，一个等位基因中仅存在杂合突变，表明此类患者由于单倍体不足或显性负效应而具有表型。此外，这一发现表明parkin基因是患帕金森病（更常见的是散发性帕金森病）的危险因素。我们现在通过常规PCR寻找断裂点或插入点的确切位点来筛选parkin载体。在筛选年轻发病帕金森病的parkin基因突变的过程中，我们可以收集没有parkin基因突变的家系。最近，Park7 的一种新致病基因 DJ-1 被鉴定出来。我们还根据单倍型分析筛选了与 Park7 相关的家族中的突变。然而，我们无法找出这些家族中的DJ-1突变。因此，与 Parkin 突变相比，DJ-1 突变在年轻发病的帕金森病中很少见。此外，几个家族可能与Park6或其他基因座有联系。我们尝试利用连锁研究来鉴定家族性帕金森病的致病基因。然后，我们与东京都医学科学研究所的Keiji Tanaka博士合作分析了parkin蛋白的功能。我们发现parkin蛋白是泛素系统的泛素蛋白连接酶。现在我们正在研究作为泛素连接酶的parkin蛋白的候选底物。我们发现CDCrel 1（一种突触小泡蛋白）是parkin 蛋白的候选底物。此外，我们分别与哈佛医学院的 Denis Selcoe 教授和 RIKEN 的 Rhosuke Takahashi 博士合作，发现了另外两种候选蛋白，即 α-synuclein 22 和 PAEL 受体。 PAEL 受体在内质网中的积累导致内质网应激和细胞凋亡。我们发现证据表明一名 AR-JP (Park2) 患者体内存在 PAEL 受体积累和内质网应激。此外，我们还使用 Park2 对 5 个尸检大脑进行了免疫组织化学研究。然而，在剩余的脑中没有观察到积累。目前还不清楚只有一个带有 Park2 的大脑积累了 Pael 受体。不同的突变可能与其积累有关。与上述候选底物不同，我们利用酵母二杂交筛选鉴定了14个克隆。其中，少数蛋白质被parkin体内泛素化系统泛素化。我们现在准备了针对此类底物的抗体 Less