Role of Parkin in Regulating Manganese Toxicity

Parkin 在调节锰毒性中的作用

• 批准号: 7814314
• 负责人: JEROME Allan ROTH
• 金额: $ 21.01万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-26 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7814314
• 关键词: Address; Alleles; Apoptotic; Applications Grants; Area; Basal Ganglia; Biochemical; Biological Assay; Brain; Cell Culture Techniques; Cell Death; Cell Line; Cell Survival; Cells; Cerebellum; Characteristics; Chronic; Complex; Corpus striatum structure; Coupled; DNA Fragmentation; Development; Disease; Dose; Elements; Environmental Exposure; Equilibrium; Exons; Exposure to; Functional disorder; Gene Mutation; Generations; Genes; Genetic; Globus Pallidus; Grant; Health; Hippocampus (Brain); Human; Individual; Influentials; Injury; JNK-activating protein kinase; JUN gene; Knockout Mice; Lesion; Ligands; Link; Literature; Lymphocyte; MAPK11 gene; MAPK14 gene; MAPK8 gene; Manganese; Measures; Membrane Potentials; Metals; Mitochondria; Modeling; Monitor; Moods; Morphology; Mutate; Mutation; Neuroblastoma; Neurologic; Neurons; Nuclear Structure; Obsessive compulsive behavior; Occupational Diseases; Outcome; Oxidative Stress; Parkin gene; Parkinson Disease; Parkinsonian Disorders; Pathway interactions; Play; Population; Predisposition; Process; Protein Family; Protein Isoforms; Proteins; Recessive Genes; Reporting; Risk; Role; SLC11A2 gene; Signal Pathway; Signal Transduction; Speed; Staging; Substantia nigra structure; Symptoms; Syndrome; System; Time; Toxic effect; Transfection; Translational Research; Ubiquitin-Proteasomal Pathway; Ubiquitination; base; caspase-3; central nervous system injury; computerized data processing; cytotoxic; design; dopaminergic neuron; early onset; knock-down; mind control; mutant; nervous system disorder; neuron loss; neurotoxicity; numb protein; overexpression; parkin gene/protein; parkin protein; pars compacta; prevent; protein transport; public health relevance; research study; response; ubiquitin-protein ligase; vector; vector control

DESCRIPTION (provided by applicant): The application addresses the broad Challenge Area (15) Translational Science and Specific Challenge Topic, 15-ES-101; Effects of environmental exposures on phenotypic outcomes using non-human models. This application will apply non-human modeling to examine a genetic mutation which may be responsible for increasing susceptibility to develop manganese (Mn) toxicity. Chronic exposure to the Mn has been linked to development of a severe, irreversible neurological disorder known as manganism consisting of reduced response speed, intellectual deficits and mood changes in the initial stages of the disorder to more prominent and irreversible extrapyramidal dysfunction resembling Parkinson's disease upon protracted exposure. Manganism is primarily an occupational disorder associated with environmental conditions in which workers are exposed to chronic high levels of the metal and include Mn miners, welders and those involved in ferroalloy processing. The CNS injury caused by Mn manifests in a diverse set of symptoms indicative of the fact that the basal ganglia is one of the most complex areas of the brain. When in excess, Mn disrupt the delicate balance between the well organized and interrelated components within the basal ganglia as a disturbance in any one of the elements, whether it occurs in the globus pallidus as in manganism or the nigro-striatal neurons as in Parkinson's disease, manifests in insidious neurological deficits that, in some respect, can appear remarkably similar. Differences in response to Mn overexposure in the human population, most likely, are due to underlying genetic variability which ultimately presents in deviations in both susceptibility as well as the characteristics of the neurological lesions and symptoms expressed. Although the underlying genetic defect responsible for increased susceptibility to develop manganism is not known, our preliminary findings revealed that the gene for early onset of Parkinson's disease, parkin, has the potential to regulate Mn toxicity and, thus for the first time, identify a potential genetic link between manganism and Parkinsonism. Parkin is an E3 ligase responsible for ubiquitinating a number of proteins destined for degradation via the proteasomal pathway. Of the known Parkinson's disease -linked genetic mutations correlating with early onset, those involving parkin are the most prevalent encompassing approximately 50% of all recessive Parkinson's disease cases. Our preliminary studies have now established (unpublished findings) that parkin is the E3 ligase responsible for the ubiquitination of the 1B isoforms of divalent metal transporter 1 (DMT1), the major protein for transport of Mn. These studies reveal that neuroblastoma cells overexpressing parkin display both decreased expression of DMT1 as well as increased toxicity towards Mn. Non-transformed human lymphocytes that are homozygous for the mutation in exon 4 of parkin express increased levels of the transporter as do brains from parkin knock-out mice. Although considered to be an autosomal recessive gene there is evidence in the literature suggesting that mutations in a single allele of the parkin gene may exert sufficient imbalance in dopaminergic activity to cause subclinical features of Parkinsonism and play a significant role in idiopathic Parkinson's disease. Thus, individuals that are heterozygous for the parkin mutation may similarly be at greater risk to develop Mn toxicity. In addition to the ability of parkin to alter DMT1 expression, mutations in the gene have also been reported to regulate activation of a number of signaling processes associated with Mn toxicity. The contribution of these parkin-induced changes in response to oxidative and toxic insults and their subsequent role in stimulating Mn toxicity is not known but we hypothesize they may likely accelerate the biochemical systems initiating cell death. The major mechanism contributing to Mn toxicity is via disruption of mitochondrial function and the subsequent activation of oxidative stress-related processes which ultimate manifests in changes in classical markers and signaling pathways associated with apoptotic cell death. Accordingly, the experiments proposed in this grant application will address this issue as we propose to determine the influence that mutations in the parkin gene have on cell signaling as it relates to Mn toxicity in non-human models. Changes in known Mn-inducing apoptotic and mitochondrial signaling processes will be monitored in the SH-SY5Y human neuroblastoma cells transfected with vector only, wild-type and mutant constructs of the parkin gene. For the knock-down model, we will use three human lymphocyte cell lines (wt and heterozygous and homozygous for the parkin mutation) as application of these latter cells eliminates any problems that may be associated with transfection effects in the SH-SY5Y cells and assures that we have totally abolished parkin activity. In addition, we will compare signaling processes in brains of control and parkin knockout mice treated with Mn. The litany of assays described in this application using non- human models is designed to obtain a comprehensive overview of the changes in the Mn-stimulated pathways that may be altered by mutations in the parkin gene. PUBLIC HEALTH RELEVANCE: Overexposure to high atmospheric levels of manganese leads to a syndrome known as manganism which is characterized by an irreversible extrapyramidal dysfunction resembling that of Parkinson's disease. Our preliminary studies, for the first time, reveal a link between manganism and Parkinson's disease in that the gene evoking early onset of Parkinsonism, parkin, can prevent Mn toxicity. Thus, the studies proposed in this grant are relevant to human health in that they will investigate the mechanism by which mutations in the parkin gene regulate and increase vulnerability to develop manganism.

描述（由申请人提供）：该申请涉及广泛的挑战领域（15）转化科学和特定挑战主题，15- es -101；环境暴露对非人类模型表型结果的影响。本应用程序将应用非人类建模来检查可能负责增加对锰（Mn）毒性的易感性的基因突变。长期接触锰与一种严重的、不可逆的神经系统疾病的发展有关，这种疾病被称为锰中毒，包括在疾病的初始阶段反应速度降低、智力缺陷和情绪变化，在长期接触锰后，会出现更突出的、不可逆的锥体外系功能障碍，类似于帕金森病。锰主要是一种职业病，与工人长期暴露于高水平金属的环境条件有关，包括矿工、焊工和参与铁合金加工的人员。Mn引起的中枢神经系统损伤表现为多种症状，表明基底神经节是大脑最复杂的区域之一。当锰过量时，基底神经节内组织良好和相互关联的成分之间的微妙平衡就会被破坏，任何一个元素都会受到干扰，无论是在白球（如锰）还是黑纹状体神经元（如帕金森病）中，锰都表现为潜伏的神经功能缺陷，在某些方面，这些缺陷可能表现得非常相似。人群中对锰过度暴露的反应差异，很可能是由于潜在的遗传变异，最终表现为易感性、神经病变特征和症状表现的偏差。尽管导致锰中毒易感性增加的潜在遗传缺陷尚不清楚，但我们的初步研究结果显示，帕金森病早期发病的基因parkin具有调节锰毒性的潜力，从而首次确定了锰中毒和帕金森病之间的潜在遗传联系。Parkin是一种E3连接酶，负责泛素化一些蛋白质，这些蛋白质通过蛋白酶体途径降解。在已知的与早期发病相关的帕金森病相关基因突变中，涉及帕金森氏病的基因突变最为普遍，约占所有隐性帕金森病病例的50%。我们的初步研究已经确定（未发表的发现），帕金是E3连接酶，负责二价金属转运蛋白1 (DMT1) 1B亚型的泛素化，DMT1是锰运输的主要蛋白。这些研究表明，过表达parkin的神经母细胞瘤细胞既表现出DMT1表达的降低，也表现出对Mn的毒性增加。与帕金基因外显子4突变纯合的非转化人类淋巴细胞表达的转运蛋白水平增加，与帕金基因敲除小鼠的大脑一样。虽然被认为是一种常染色体隐性基因，但文献证据表明，parkin基因的单个等位基因突变可能使多巴胺能活性充分失衡，导致帕金森病的亚临床特征，并在特发性帕金森病中发挥重要作用。因此，帕金突变的杂合个体可能同样具有更大的锰毒性风险。除了帕金改变DMT1表达的能力外，该基因的突变也被报道调节与锰毒性相关的一些信号传导过程的激活。这些帕金森诱导的变化在氧化和毒性损伤反应中的作用及其随后在刺激锰毒性中的作用尚不清楚，但我们假设它们可能加速生化系统启动细胞死亡。锰毒性的主要机制是通过线粒体功能的破坏和随后氧化应激相关过程的激活，最终表现为与凋亡细胞死亡相关的经典标志物和信号通路的改变。因此，本拨款申请中提出的实验将解决这一问题，因为我们建议确定帕金基因突变对细胞信号传导的影响，因为它与非人类模型中的锰毒性有关。已知mn诱导凋亡和线粒体信号传导过程的变化将在转染载体、野生型和突变型parkin基因构建的SH-SY5Y人神经母细胞瘤细胞中进行监测。对于敲除模型，我们将使用三种人类淋巴细胞系（wt、杂合子和纯合子用于帕金突变），因为后一种细胞的应用消除了SH-SY5Y细胞中可能与转染效应相关的任何问题，并确保我们完全消除了帕金活性。此外，我们将比较Mn处理的对照组和帕金基因敲除小鼠大脑中的信号过程。本应用程序中描述的使用非人类模型的一系列测定旨在全面概述mn刺激通路的变化，这些变化可能被帕金基因突变所改变。