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.

当前对多巴胺能（DA）神经元功能障碍和/或死亡发作的见解（因此，病因 帕金森氏病[PD]）暗示异常的异常 - 呼吸蛋白 - UPS（UPS） 氧化和亚硝化应激，导致蛋白质错误折叠。蛋白质错误折叠似乎是被介导的， 至少部分地是通过parkin或蛋白质 - 二硫化物异构酶（PDI）的S-亚硝基化。因此，这些分子可能 提供基于机制的生物标志物，用于即将发生神经元的灭亡，相反，如果水平下降，则 恢复。功能失调的线粒体会导致活性氧（ROS）和 反应性氮种（RNS）。特别是，有越来越多的证据表明线粒体复合物1 功能障碍导致ROS增加，最终导致A-突触核蛋白的聚集。 A-核蛋白的低聚物/原纤维在神经变性中似乎起着核心作用，尤其是，尤其是 PD病理学 - 可能通过蛋白酶体抑制。 UPS功能障碍可能是家族性PD的基础 以Parkin，Pink1和DJ-1突变为特征。最近，Lipton组（项目3）有 证明帕克蛋白或PDI的S-亚硝基化，这是钥匙应力诱导的内质伴侣 网状（ER）与PD模型中的蛋白质错误折叠和神经变性有关 PD患者。此外，在初步研究中，我们观察到携带突变型核蛋白（ASYN）的小鼠 显示PDI的S-亚硝基化大大增加；即，出现了硝酸/氧化应激增加 在这种突变的背景下存在。 PD发作似乎有一个发展成分。例如，尽管突变体A-syn 存在于具有某些家族形式的PD的患者的最早的中枢神经系统祖细胞中，该疾病不会 通常表现为成年。进行性DA功能障碍似乎也是 衰老过程。未成熟的神经祖细胞似乎以某种方式抵抗氧化应激 当那些相同细胞成熟时观察到。 尽管通常研究人类干细胞的治疗潜力，但它们也提供 （也许更令人信服）人类细胞开发的模型，并为 对人类疾病进行建模（反过来又可以得出新的疗法）。我们已经建立了定义 从未分化的DA神经元发展的迭代步骤建模的培养条件 人类胚胎干细胞（HESC）在体外分化的DA神经元。每个发育阶段的细胞 可以设计以用线粒体复合物在药理学上表达突变体A-syn和/或“病变” 抑制剂。在DA神经元中，此类操作产生模拟PD的特征。因此，我们建议 使用人类干细胞的系统来建模神经前体的发育敏感性 与PD相关的氧化/亚硝化应激，以了解濒临灭绝或危害的机制 功能失调的DA神经元可能最终受到保护。对发展易感性的研究可能有助于 开发可预防内源性和移植神经的氧化/硝化应激的药物 祖先。与尝试重建相比，保留介体电路更容易且更安全 适当的新连接。但是，如果将来需要将其移植到PD患者中，以保护 这些外源干细胞也至关重要。可能需要不同的保护药 取决于使用的神经祖细胞的发育阶段。