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

描述(申请人提供)：氧化应激在神经退行性疾病和中风的发病机制中起着重要作用。然而，低水平的ROS在许多神经元信号转导通路中作为第二信使发挥作用，因此可能受到疾病过程本身或内源性抗氧化反应激活的影响。这项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。该应用的具体目的是阐明Nrf2突触功能的分子机制，Nrf2是可诱导的抗氧化反应的转录调节因子，是神经元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加通过ROS敏感的谷氨酸能NMDA受体(NMDARs，突触可塑性和谷氨酸兴奋毒性的关键介质)的电流来响应神经元的活动，同时激活Nrf2通路来保护神经元免受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大初步数据的基础上提出的，并将通过追求四个具体目标来检验。首先，我们将阐明在神经元-星形胶质细胞共培养中，活性介导的Nrf2通路诱导的分子机制。其次，我们将确定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得到加强。第三，我们将确定神经胶质细胞如何在神经元-神经胶质细胞混合环境中增加神经元NMDAR电流密度。第四，我们将剖析Nrf2介导的NMDAR信号调节下的神经元-神经胶质细胞信号转导级联，并确定Nrf2通路激活对电路可塑性的影响。这些目标将通过分子、生化、电生理、细胞生物学和毒理学方法的组合来实现，这些方法的可行性已经通过初步数据在我们手中建立；为了剖析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区培养，以及确定细胞组成的神经元-神经胶质细胞共培养。整个方法将该领域带入了一个新的方向，专注于神经元-胶质细胞相互作用在大脑中Nrf2信号的调节和功能中的作用，这是Nrf2生物学尚未被研究的一个方面。这项拟议研究的完成有望促进我们对ROS信号和Nrf2在大脑中的生理学的理解；最终，这些知识将使药物治疗的发展能够利用内源性抗氧化剂的神经保护力量，而不会对神经元活动和突触信号产生负面影响。 公共卫生相关性：通过促进我们对大脑中Nrf2途径生物学的理解，拟议中的研究将发现预防和/或治疗阿尔茨海默病和其他突触可塑性受损的神经疾病的最佳分子和细胞靶点。因此，它与美国国立卫生研究院的使命相一致，即促进基础科学发现和创新研究战略，形成保护和改善公共健康的基础。

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. PUBLIC HEALTH RELEVANCE: By advancing our understanding of Nrf2 pathway biology in the brain, the proposed research will uncover optimal molecular and cellular targets for prevention and/or treatment of Alzheimer disease and other neurologic disorders with impaired synaptic plasticity. Thus, it is aligned with the NIH mission to foster fundamental scientific discoveries and innovative research strategies that form a basis for protection and improvement of public health.