Mutation and functional analysis of parkin responsible for AR-JP

我们对700多个帕金森氏症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帕金突变复合杂合子的方法。约50%的常染色体隐性家族存在帕金基因突变。发现了多种外显子缺失和点突变。这种家族性帕金森病一直被认为是日本独有的，但自从我们开始对帕金基因进行突变分析以来，我们证实了帕金基因突变在世界范围内的分布。此外，即使是常染色体显性遗传模式，帕金突变也可以在这些家庭中检测到。此外，帕金基因突变在许多孤立病例中观察到，如散发性病例。因此，帕金森氏基因突变是家族性帕金森氏病中最常见的形式。在帕金基因突变的家庭中，只有一个等位基因存在杂合突变，表明这类患者由于单倍不全或显性负作用而具有更多的表型。此外，这一发现表明帕金森氏基因是发生帕金森病的危险因素，更常见的是散发性帕金森病。我们现在利用传统的PCRIn筛选年轻发病帕金森病患者帕金基因突变的方法，搜索断点或插入点的确切位置来筛选帕金基因携带者，我们可以收集没有帕金基因突变的家庭。最近，一种新的致病基因，Park7的DJ-1被发现。我们还根据单倍型分析在与Park7相关的家族中筛选了它的突变。然而，我们无法在这些家族中发现DJ-1突变。因此，与帕金突变相比，DJ-1突变在年轻发病的帕金森病中是罕见的。此外，一些家族可能与Park6或其他位点有关。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。然后我们与东京都医学研究所的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，我们还与哈佛医学院的Denis Selcoe教授和RIKEN的Rhosuke Takahashi博士合作，发现了另外两个候选蛋白，即α -synuclein 22和PAEL受体。内质网中PAEL受体的积累引起内质网应激和凋亡细胞死亡。我们发现有证据表明在一例AR-JP患者中存在PAEL受体积累和内质网应激（Park2）。此外，我们用Park2对5个解剖脑进行了免疫组化研究。然而，在剩余的大脑中没有观察到积累。目前尚不清楚是否只有一个具有Park2的大脑有Pael受体的积累。不同的突变可能与它的积累有关。与上述候选底物不同，我们利用酵母双杂交筛选鉴定了14个克隆。其中，少数蛋白在体内泛素化系统中被parkin泛素化。我们现在为这种底物制备抗体

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