Gandhi S, Muqit M M K, Stanyer L, Healy D G, Abou-Sleiman P M, Hargreaves I, Heales S, Ganguly M, Parsons L, Lees A J, Latchman D S, Holton J L, Wood N W, Revesz T
Department of Molecular Neuroscience, Institute of Neurology University College London, London, UK.
Brain. 2006 Jul;129(Pt 7):1720-31. doi: 10.1093/brain/awl114. Epub 2006 May 15.
Parkinson's disease is a common incurable neurodegenerative disease whose molecular aetiology remains unclear. The identification of Mendelian genes causing rare familial forms of Parkinson's disease has revealed novel proteins and pathways that are likely to be relevant in the pathogenesis of sporadic Parkinson's disease. Recently, mutations in a novel gene, PINK1, encoding a 581 amino acid protein with both mitochondrial targeting and serine/threonine kinase domains, were identified as a cause of autosomal recessive parkinsonism. This provided important evidence for the role of the mitochondrial dysfunction and kinase pathways in neurodegeneration. In this study, we report the first characterization of the PINK1 protein in normal human and sporadic Parkinson's brains, in addition to Parkinson's cases with heterozygous PINK1 mutations. The possible role of the PINK1 protein was also assessed in a number of neurodegenerative diseases characterized by proteinaceous inclusions. For these studies, rabbit polyclonal antibodies were raised against two peptide sequences within the N-terminal hydrophilic loops of PINK1 protein. Using immunohistochemistry and western blotting we were able to demonstrate that PINK1 is a ubiquitous protein expressed throughout the human brain and it is found in all cell types showing a punctate cytoplasmic staining pattern consistent with mitochondrial localization. Fractionation studies of human and rat brain confirm that PINK1 is localized to the mitochondrial membranes. In addition, we show that PINK1 is detected in a proportion of Lewy bodies in cases of sporadic Parkinson's disease and Parkinson's disease associated with heterozygous mutations in the PINK1 gene, which are clinically and pathologically indistinguishable from the sporadic cases. PINK1 was absent in cortical Lewy bodies, in neurofibrillary tangles in Alzheimer's disease, progressive supranuclear palsy and corticobasal degeneration, and in the glial and neuronal alpha-synuclein positive inclusions in multiple system atrophy. These studies provide for the first time in vivo morphological and biochemical evidence to support a mitochondrial localization of PINK1 and underpin the significance of mitochondrial dysfunction in the pathogenesis of nigral cell degeneration in Parkinson's disease.
帕金森病是一种常见的无法治愈的神经退行性疾病，其分子病因尚不清楚。导致罕见家族性帕金森病的孟德尔基因的鉴定揭示了可能与散发性帕金森病发病机制相关的新蛋白质和信号通路。最近，一个新基因PINK1发生突变，该基因编码一种含有线粒体靶向结构域和丝氨酸/苏氨酸激酶结构域的581个氨基酸的蛋白质，被确定为常染色体隐性帕金森综合征的病因。这为线粒体功能障碍和激酶信号通路在神经退行性变中的作用提供了重要证据。在本研究中，我们报告了PINK1蛋白在正常人和散发性帕金森病患者大脑中的首次特征描述，以及伴有PINK1杂合突变的帕金森病患者大脑中的情况。我们还评估了PINK1蛋白在一些以蛋白质包涵体为特征的神经退行性疾病中的可能作用。为进行这些研究，我们针对PINK1蛋白N端亲水环内的两个肽序列制备了兔多克隆抗体。通过免疫组织化学和蛋白质印迹法，我们能够证明PINK1是一种在全脑广泛表达的普遍存在的蛋白质，在所有细胞类型中均有发现，呈现出与线粒体定位一致的点状胞质染色模式。对人和大鼠大脑的分级分离研究证实PINK1定位于线粒体膜。此外，我们发现，在散发性帕金森病患者以及与PINK1基因杂合突变相关的帕金森病患者的一部分路易小体中可检测到PINK1，这些患者在临床和病理上与散发性病例无法区分。在皮质路易小体、阿尔茨海默病的神经原纤维缠结、进行性核上性麻痹和皮质基底节变性以及多系统萎缩的胶质细胞和神经元α-突触核蛋白阳性包涵体中未检测到PINK1。这些研究首次提供了体内形态学和生物化学证据，支持PINK1定位于线粒体，并强调了线粒体功能障碍在帕金森病黑质细胞变性发病机制中的重要性。
