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parkin突变复合杂合子的方法。在大约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。这种类型的家族性帕金森病在日本人中被认为是独一无二的，但自从我们开始对parkin基因进行突变分析以来，我们证实了parkin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，也可以在这些家系中检测到parkin突变。此外，在许多孤立病例中也观察到parkin基因突变，如散发性病例。因此，parkin基因突变是家族性帕金森病最常见的形式。在Parkin基因突变的家系中，只有一个等位基因存在杂合突变，这表明这些患者患有…由于单倍体不足或显性负效应导致的表型较多。此外，这一发现表明parkin基因是患帕金森氏病的危险因素，帕金森氏症是更常见的散发性帕金森氏病。我们现在用传统的PCR法搜索断裂点或插入点的确切位置来筛查帕金森病患者的Parkin基因突变。在筛选年轻发病的帕金森病患者Parkin基因突变的过程中，我们可以收集没有Parkin Geme突变的家系。最近，一个新的Park7致病基因DJ-1被发现。我们还在单倍型分析的基础上，在与Park7连锁的家系中筛选了它的突变。然而，我们无法在这些家系中发现DJ-1突变。因此，与帕金森突变相比，DJ-1突变在年轻发病的帕金森病中很少见。此外，几个家系可能与Park6或其他基因座连锁。我们试图通过连锁研究来确定家族性帕金森病S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蛋白是泛素系统的一种泛素-蛋白质连接酶。现在我们正在研究Parkin蛋白作为泛素连接酶的候选底物。我们发现CDCrel 1是一种突触囊泡蛋白，是Parkin蛋白的候选底物。此外，在哈佛医学院的Denis Selcoe教授和RIKEN的Rhosuke Takahashi博士的合作下，我们还发现了另外两个候选蛋白质，即α-突触核蛋白22和Pael受体。PAEL受体在内质网中的积聚导致内质网应激和细胞凋亡。我们发现有证据表明1例AR-JP(Park2)患者PAEL受体积聚和内质网压力的存在。此外，我们还对5例Park2脑组织进行了免疫组织化学研究。然而，在剩余的脑中没有观察到蓄积。目前还不清楚是否只有一个Park2的大脑积累了Pael受体。不同的突变可能与它的积累有关。与上面提到的候选底物不同，我们用酵母双杂交筛选的方法鉴定了14个克隆。其中，少数蛋白质被Parkin体内泛素化系统泛素化。我们现在为这些底物准备了更少的抗体

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