Project 4: A Developmental Perspective to Nitrosative/Oxidative Susceptibility

项目 4：亚硝化/氧化敏感性的发展视角

• 批准号: 8292290
• 负责人: EVAN Y SNYDER
• 金额: $ 27.94万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8292290
• 关键词: Adult; Aging-Related Process; Biological Markers; Brain; Cells; Cessation of life; Complex; Development; Disease; Disease model; Endoplasmic Reticulum; Engineering; Etiology; Functional disorder; Future; Generations; Grant; Human; In Vitro; Lead; Lesion; Link; Mediating; Mitochondria; Modeling; Molecular Chaperones; Mus; Mutation; Nerve Degeneration; Neuronal Dysfunction; Neurons; Onset of illness; Oxidative Stress; PINK1 gene; Parkinson Disease; Parkinsonian Disorders; Pathology; Patients; Pharmaceutical Preparations; Play; Predisposition; Proteasome Inhibition; Protective Agents; Protein Disulfide Isomerase; Proteins; Reactive Nitrogen Species; Reactive Oxygen Species; Recovery; Resistance; Role; SKIL gene; Staging; Stem cells; Stress; System; Therapeutic; Transplantation; Undifferentiated; adduct; alpha synuclein; base; cellular development; dopaminergic neuron; human disease; human embryonic stem cell; human stem cells; inhibitor/antagonist; insight; multicatalytic endopeptidase complex; mutant; nerve stem cell; neuron development; nitrosative stress; novel; parkin gene/protein; prevent; progenitor; protein misfolding; response

Current insights into the onset of dopaminergic (DA) neuronal dysfunction and/or death (hence, the etiology of Parkinson's Disease [PD]) implicate abnormalities in the unbiquitin-proteasome system (UPS) in response to oxidative and nitrosative stress, leading to protein misfolding. Protein misfolding appears to be mediated, at least in part, by S-nitrosylation of parkin or protein-disulfide isomerase (PDI). Hence, these molecules may provide mechanism-based biomarkers for impending neuronal demise or, conversely, if levels go down, their recovery. Dysfunctional mitochondria can lead to the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). In particular, there is growing evidence that mitochondrial complex 1 dysfunction results in an increase in ROS, eventually leading to the aggregation of a-synuclein. Oligomers/protofibrils of a-synuclein appear to play a central role in neurodegeneration - and, particularly, PD pathology -- likely through proteasome inhibition. Dysfunction of the UPS is likely the basis for familial PD characterized by mutations in Parkin, PINK1 and DJ-1. Recently, the Lipton group (Project 3) has demonstrated that S-nitrosylation of parkin or PDI, a key stress-induced chaperone in the endoplasmic reticulum (ER), has been linked to protein misfolding and neurodegeneration in PD models and in brains of PD patients. In addition, in preliminary studies, we have observed that mice carrying mutant a-synuclein (asyn) show dramatically increased S-nitrosylation of PDI; i.e., increased nitrosative/oxidative stress appears to be present in the context of such a mutation. There appears to be a developmental component to PD onset. For example, although, mutant a-syn is present in the earliest CNS progenitors of patients with some familial forms of PD, the disease does not typically manifest itself until adulthood. Progressive DA dysfunction also appears to be a component of the aging process. Immature neural progenitor cells appear to be resistant to oxidative stress in a manner not observed when those same cells become mature. Although human stem cells are typically studied for their therapeutic potential, they also provide (perhaps even more compellingly) models of human cellular development and offer the prospect for modeling human disease (from which novel therapies may, in turn, be derived). We have established defined culture conditions for modeling the iterative steps of DA neuronal development from an undifferentiated human embryonic stem cell (hESC) to a differentiated DA neuron in vitro. Cells at each developmental stage can be engineered to express mutant a-syn and/or "lesioned" pharmacologically with mitochondrial complex inhibitors. In DA neurons, such manipulations produce features emulating PD. Therefore, we propose to use a human stem cell-based system to model the developmental susceptibility of neural precursors to oxidative/nitrosative stress relevant to PD in order to understand mechanisms by which endangered or dysfunctional DA neurons may ultimately be protected. A study of developmental susceptibility may help to develop drugs that will prevent oxidative/nitrosative stress in both endogenous and transplanted neural progenitors. Preserving mesostriatal circuitry is more tractable and safer than attempting to reconstruct proper new connections. However, if, in the future, transplantation into PD patients is required, protecting these exogenous stem cells will also be crucial. It is possible that different protective drugs will be necessary depending on the developmental stage of the neural progenitors used.

目前对多巴胺(DA)能神经元功能障碍和/或死亡(因此，病因)的研究进展 帕金森氏病[PD])与Unbiquitin-Proteasome系统(UPS)的异常有关 氧化和亚硝化应激，导致蛋白质错误折叠。蛋白质的错误折叠似乎是被中介的， 至少部分是通过S-帕金亚硝化或蛋白质-二硫键异构酶。因此，这些分子可能 为即将到来的神经元死亡提供基于机制的生物标记物，或者相反，如果水平下降，其 恢复。线粒体功能障碍可导致活性氧物种(ROS)的产生和 活性氮物种(RNS)。特别是，越来越多的证据表明线粒体复合体1 功能障碍导致ROS增加，最终导致α-突触核蛋白聚集。 α-突触核蛋白的寡聚体/原纤维似乎在神经退行性变中起着核心作用--尤其是， 帕金森病的病理--可能是通过蛋白酶体抑制。UPS功能障碍可能是家族性帕金森病的基础 以Parkin、PINK1和DJ-1突变为特征。最近，立顿集团(Project 3)已经 内质中应激诱导的伴侣蛋白Pdi或Pdi的S亚硝化 内质网(ER)与帕金森病模型中的蛋白质错误折叠和神经退行性变有关，在帕金森病患者的大脑中。 帕金森病患者。此外，在初步研究中，我们观察到携带突变的a-突触核蛋白(Asyn)的小鼠 显示PDI的S亚硝化显著增加，即亚硝化/氧化应激增加 出现在这样一种突变的背景下。 帕金森病的发病似乎有发育因素。例如，尽管突变的a-syn 是存在于早期中枢神经系统祖细胞的一些家族性帕金森病患者，本病不存在 通常会一直表现到成年。进行性DA功能障碍似乎也是 老化过程。未成熟的神经前体细胞似乎以一种不同的方式抵抗氧化应激 观察到这些相同的细胞何时成熟。 尽管人类干细胞通常被研究为其治疗潜力，但它们也提供了 (也许更令人信服的)人类细胞发育的模型，并提供了 对人类疾病进行建模(由此可能衍生出新的治疗方法)。我们已经确定了 模拟未分化DA神经元发育迭代步骤的培养条件 人胚胎干细胞(HESC)体外分化为DA神经元。各个发育阶段的细胞 可以被改造来表达突变的a-syn和/或与线粒体复合体药理上的“受损” 抑制剂。在DA神经元中，这样的操作产生了模拟PD的特征。因此，我们建议 使用基于人类干细胞的系统来模拟神经前体的发育易感性 与帕金森病相关的氧化/亚硝化应激，以了解危害或 功能失调的DA神经元最终可能会受到保护。对发育易感性的研究可能有助于 开发药物，防止内源性和移植神经的氧化/亚硝化应激 祖先。保留中纹状体环路比尝试重建更容易和更安全 合适的新关系。然而，如果将来需要对帕金森病患者进行移植，保护 这些外源干细胞也将是至关重要的。有可能需要不同的保护性药物 取决于所使用的神经前体细胞的发育阶段。