Neuron-glia interactions in regulation of activity-dependent signaling pathways

描述（由申请人提供）：氧化应激在神经退行性疾病和卒中的发病机制中起重要作用。然而，低水平的活性氧（ROS）作为第二信使在许多神经元信号转导途径，这可能因此受到疾病过程本身或激活内源性抗氧化反应。本研究的长期目标是确定内源性抗氧化剂信号如何调节突触传递，从而形成突触网络对氧化应激的脆弱性。本申请的具体目的是阐明Nrf 2突触功能的分子机制，Nrf 2是诱导型抗氧化反应的转录调节因子，是神经元对损伤易感性的关键决定因素。我们的中心假设是，星形胶质细胞通过增强通过ROS敏感的谷氨酸能NMDA受体（NMDARs，突触可塑性和谷氨酸兴奋性毒性的关键介质）的电流来响应神经元活动，同时激活Nrf 2通路以保护神经元免受ROS诱导的损伤和神经毒性。这一假设是在我们实验室获得的强有力的初步数据的基础上提出的，并将通过追求四个具体目标进行检验。首先，我们将阐明神经元-星形胶质细胞共培养物中活性介导的Nrf 2通路诱导的分子机制。其次，我们将确定当神经元与星形胶质细胞共培养时，突触活动诱导的神经保护是否增强。第三，我们将确定神经胶质细胞如何增加神经元NMDAR电流密度在混合神经元-神经胶质细胞环境。第四，我们将剖析神经元-神经胶质细胞信号转导级联反应的基础Nrf 2介导的调节NMDAR信号传导，并确定Nrf 2通路激活电路可塑性的影响。这些目标将通过分子，生物化学，电生理，细胞生物学和毒理学的方法，其可行性在我们手中已经建立了通过初步数据的组合来实现;解剖单个细胞类型的作用，我们将使用原代神经元，神经胶质细胞和混合海马培养物，以及神经元-神经胶质细胞共培养物的定义的细胞组成。整体方法通过关注神经元-胶质细胞相互作用在脑中Nrf 2信号传导的调节和功能中的作用，将该领域带入了一个新的方向，这是Nrf 2生物学尚未研究的一个方面。完成拟议的研究预计将促进我们对大脑中ROS信号传导和Nrf 2生理学的理解;最终，这些知识将能够开发能够利用内源性抗氧化剂的神经保护能力而不会对神经元活性和突触信号传导产生负面影响的药理学治疗。

DESCRIPTION (provided by applicant): Oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases and stroke. However, low levels of reactive oxygen species (ROS) function as second messengers in many neuronal signal transduction pathways, which could thus be affected either by the disease process itself or by activation of endogenous antioxidant responses. The long term goal of this research is to define how endogenous antioxidant signaling regulates synaptic transmission and thus shapes vulnerability of synaptic networks to oxidative stress. The specific objective of this application is to elucidate molecular mechanisms underlying synaptic function of Nrf2, the transcriptional regulator of inducible antioxidant response that is a key determinant of neuronal susceptibility to injury. Our central hypothesis is that astrocytes respond to neuronal activity by enhancing current flow through ROS-sensitive glutamatergic NMDA receptors (NMDARs, the key mediators of both synaptic plasticity and glutamate excitotoxicity), while simultaneously activating Nrf2 pathway to protect neurons from ROS-induced damage and neurotoxicity. This hypothesis was formulated on the basis of the strong preliminary data obtained in our laboratory and will be tested by pursuing four specific aims. First, we will elucidate the molecular mechanism that underlies activity-mediated induction of Nrf2 pathway in neuron-astrocyte co-cultures. Second, we will establish whether neuroprotection induced by synaptic activity is enhanced when neurons are co-cultured with astrocytes. Third, we will determine how glial cells increase neuronal NMDAR current density in the mixed neuron-glia environment. Fourth, we will dissect the neuron-glia signal transduction cascade that underlies Nrf2-mediated regulation of NMDAR signaling and determine the effect of Nrf2 pathway activation on circuit plasticity. These aims will be accomplished through a combination of molecular, biochemical, electrophysiological, cell biological, and toxicological approaches whose feasibility in our hands has been established through the preliminary data; to dissect the roles of individual cell types, we will use primary neuronal, glial, and mixed hippocampal cultures, as well as neuron-glia co-cultures of defined cellular composition. The overall approach takes the field in a new direction by focusing on the role of neuron- glia interactions in the regulation and function of Nrf2 signaling in the brain, an aspect of Nrf2 biology that has not yet been investigated. Completion of the proposed research is expected to advance our understanding of ROS signaling and Nrf2 physiology in the brain; ultimately, such knowledge will enable development of pharmacologic treatments capable of harnessing neuroprotective power of endogenous antioxidants without negatively affecting neuronal activity and synaptic signaling.