| Budget Amount *help |
¥10,900,000 (Direct Cost: ¥10,900,000)
Fiscal Year 2002: ¥4,900,000 (Direct Cost: ¥4,900,000)
Fiscal Year 2001: ¥6,000,000 (Direct Cost: ¥6,000,000)
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| Research Abstract |
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
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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 PCR
In 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 study
Then 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
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