Mutation and functional analysis of parkin responsible for AR-JP

我们对700多个帕金森病家族进行了突变分析。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。在约50%的常染色体隐性遗传家系中发现parkin基因突变。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，但自从我们开始parkin基因的突变分析以来，我们证实了parkin基因突变的世界性分布。此外，即使是常染色体显性遗传，也可以在这些家系中检测到parkin突变。此外，parkin基因突变在许多孤立病例中观察到，如散发病例。因此，parkin基因突变是家族性帕金森病中最常见的形式。在parkin基因突变的家族中，只有一个等位基因存在杂合突变，这表明此类患者有 关于我们 表型由于单倍不足或显性负效应。此外，这一发现表明parkin基因是帕金森病的危险因素，更常见的是散发性帕金森病。本研究采用常规PCR技术，通过寻找突变位点或插入位点的准确位置，对帕金森病的parkin基因突变进行筛查，收集到无parkin基因突变的家系。最近，一个新的致病基因，DJ-1的公园7已被确定。我们还根据单倍型分析在与Park7连锁的家系中筛选了其突变。然而，我们无法在这些家庭中发现DJ-1突变。因此，与parkin突变相比，DJ-1突变在帕金森病中是罕见的。此外，有几个家族可能与Park6或其他基因座相关。我们试图通过连锁研究来确定家族性帕金森病的致病基因，然后与东京都医学科学研究所的Keiji Tanaka博士合作分析parkin蛋白的功能。我们发现parkin蛋白是泛素系统中的一种泛素-蛋白质连接酶。目前我们正在研究帕金蛋白作为泛素连接酶的候选底物。我们发现，突触囊泡蛋白CDCrel 1是parkin蛋白的候选底物.此外，我们发现了另外两种候选蛋白，即，α-突触核蛋白22和PAEL受体，分别与哈佛医学院的Denis Selcoe教授和RIKEN的Rhosuke Takahashi博士合作。PAEL受体在内质网中的积累引起内质网应激和凋亡性细胞死亡。我们发现的证据表明，PAEL受体的积累和内质网应激的存在与AR-JP（帕克2）的患者。此外，我们还对5例尸检脑进行了Park2免疫组化研究。然而，在其余脑中未观察到蓄积。目前尚不清楚只有一个具有Park2的大脑具有Pael受体的积累。不同的突变可能与其积累有关。与上述候选底物不同，我们利用酵母双杂交筛选法鉴定了14个克隆。其中，有几种蛋白质在体内被parkin泛素化系统泛素化。我们现在制备了这些底物的抗体减

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