Project 4: A Developmental Perspective to Nitrosative/Oxidative Susceptibility

目前对多巴胺能（DA）神经元功能障碍和/或死亡（因此，病因学）发病的认识 帕金森氏病[PD]）的发病机制涉及到泛素-蛋白酶体系统（UPS）的异常， 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由， 至少部分通过帕金或蛋白质二硫键异构酶（PDI）的S-亚硝基化。因此，这些分子可以 为即将到来的神经元死亡提供基于机制的生物标志物，或者相反，如果水平下降， 复苏线粒体功能障碍可导致活性氧（ROS）的产生， 活性氮类（RNS）。特别是，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α-突触核蛋白的聚集。 α-突触核蛋白的寡聚体/原纤维似乎在神经变性中起核心作用，特别是， 帕金森病理学可能是通过蛋白酶体抑制UPS功能障碍可能是家族性PD的基础 其特征在于Parkin、PINK 1和DJ-1的突变。最近，立顿集团（项目3） 证明了parkin或PDI的S-亚硝基化，这是内质网中关键的应激诱导的伴侣蛋白。 网状细胞（ER）与PD模型和帕金森病患者大脑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α-突触核蛋白（asyn）的小鼠， 显示PDI的S-亚硝基化显著增加;即，亚硝化/氧化应激增加 出现在这种突变的背景下。 PD发作似乎有发育成分。例如，虽然突变的a-syn 存在于某些家族性PD患者的最早CNS祖细胞中，但该疾病不 通常会在成年后才显现出来进行性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对氧化应激具有抵抗力， 当这些相同的细胞成熟时观察到。 虽然人类干细胞通常被研究其治疗潜力，但它们也提供了 （也许更有说服力）人类细胞发育的模型，并提供了前景， 模拟人类疾病（由此可以衍生出新的疗法）。我们已经建立了定义 用于模拟DA神经元从未分化的神经元发育的迭代步骤的培养条件 体外诱导人胚胎干细胞（hESC）分化为DA神经元。每个发育阶段的细胞 可以被工程化以表达突变的α-syn和/或具有线粒体复合物的“损伤的”β-syn 抑制剂的在DA神经元中，这样的操作产生模拟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PD相关的氧化/亚硝化应激，以了解危害或 功能失调的DA神经元可能最终得到保护。对发育易感性的研究可能有助于 开发药物，防止内源性和移植的神经细胞中的氧化/亚硝化应激， 祖先保留中纹状体回路比试图重建 正确的新连接。然而，如果将来需要移植到PD患者中， 这些外源性干细胞也将是至关重要的。可能需要不同的保护性药物 这取决于所用神经祖细胞的发育阶段。

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.