The function and mechanisms of voltage-gated proton channel Hv1 in spinal cord injury

项目摘要 尽管在过去的30年里进行了大量的研究，但仍然没有建立有效的治疗方法， 改善脊髓损伤（SCI）后恢复。在某种程度上，这反映了对 涉及复杂的继发性病理生物学机制。我们研究的目的是了解细胞 以及负责损伤后神经炎症的分子机制， 新的疗法。电压门控质子通道Hv 1是一种新发现的离子通道，高表达 大脑中的小胶质细胞。在病理条件下，NADPH氧化酶需要小胶质细胞Hv 1 （NOX）依赖性的ROS（活性氧物质）的产生，通过提供电荷补偿， 输出电子和缓解细胞内酸中毒。因此，Hv 1是用于控制多种NOx的唯一目标 活性和ROS的产生。然而，这一发现背后的精确信号机制， Hv 1在SCI病理生理学中的关键作用已被充分理解。根据我们的初步数据， 测试假设小胶质细胞Hv 1作为神经炎症的关键机制，通过改变 NOX 2/ROS/IFN-γ信号调节小胶质细胞-星形胶质细胞相互作用，从而影响长期 脊髓损伤后的神经功能 我们将使用全身性或小胶质细胞Hv 1 KO、小胶质细胞NOX 2 KO转基因小鼠和在体内和体外 创新技术，以确定SCI触发的Hv 1升高的机制， 神经炎症目的1探讨Hv 1在神经炎症中的作用及机制 SCI之后对小胶质细胞介导的神经炎症的多项定量评估将与 以Hv 1为靶点的遗传或药物干预，以检验SCI诱导的小胶质细胞Hv 1 激活通过改变小胶质细胞介导有害的神经炎症和功能缺陷 NOX 2/ROS信号传导。目的2阐明小胶质细胞NOX 2在损伤后神经炎症中的作用。 我们将利用遗传干预来消除小胶质细胞中Hv 1依赖的NOX 2上调，并评估 小胶质细胞NOX 2与Hv 1偶联对SCI后神经炎症的影响。目标3将确定关键 Hv 1/NOX 2源性ROS/IFN-γ在通过小胶质-星形胶质细胞的SCI慢性神经炎症中的作用 互动互补的细胞，分子和遗传学方法将被用来测试的假设 Hv 1/NOX 2介导的小胶质细胞ROS激活促炎性星形胶质细胞，导致分泌IFN γ， 反过来又加强小胶质细胞炎症，从而导致星形胶质细胞功能障碍和神经元损伤。 我们的研究将是第一个涉及小胶质细胞Hv 1/NOX 2/ROS/IFN-γ信号转导的病理生理学。 导致新的SCI治疗方法。考虑到Hv 1在其他炎症中的作用， 模型中，Hv 1信号转导代表了与其他神经炎症状态相关的一般机制。

Project Summary Despite considerable research over the past 30 years, there is still no established effective treatment to improve recovery following spinal cord injury (SCI). In part, this reflects incomplete understanding of the complex secondary pathobiological mechanisms involved. The aim of our research is to understand the cellular and molecular mechanisms responsible for post-injury neuroinflammation in order to allow future development of novel therapies. The voltage-gated proton channel Hv1 is a newly discovered ion channel, highly expressed in resting microglia of the brain. Under pathological conditions, microglial Hv1 is required for NADPH oxidase (NOX)-dependent generation of ROS (reactive oxygen species) by providing charge compensation for exported electrons and relieving intracellular acidosis. Thus, Hv1 is a unique target for controlling multiple NOX activities and ROS production. However, neither the precise signaling mechanisms underlying this finding nor critical role of Hv1 in the pathophysiology of SCI are fully understood. Based on our preliminary data, we will test the hypothesis that microglial Hv1 functions as a key mechanism in neuroinflammation, through altered NOX2/ROS/IFN- signaling that modulates microglia-astrocyte interaction, thus affecting long-term neurological outcomes after SCI. We will use systemic or microglial Hv1 KO, microglial NOX2 KO transgenic mice and in vivo and in vitro innovatively technologies to determine the mechanisms of SCI-triggered Hv1 elevation on post-injury neuroinflammation. Aim 1 will determine the function and mechanisms of the Hv1 in neuroinflammation after SCI. Multiple quantitative assessments of microglia-mediated neuroinflammation will be combined with genetic or pharmacological intervention targeting Hv1 to test the hypothesis that SCI-induced microglial Hv1 activation mediates detrimental neuroinflammation and functional deficits through altered microglial NOX2/ROS signaling. Aim 2 will elucidate the role of microglial NOX2 in post-injury neuroinflammation. We will utilize genetic intervention to delete Hv1-dependent up-regulation of NOX2 in microglia, and evaluate the effects on microglial NOX2 coupling to Hv1 on neuroinflammation after SCI. Aim 3 will identify critical role of Hv1/NOX2-derived ROS/IFN- in SCI-chronic neuroinflammation through microglia-astrocyte interaction. Complimentary cellular, molecular, and genetic approaches will be used to test the hypothesis that Hv1/NOX2-mediated microglial ROS activates pro-inflammatory astrocytes resulting in secreting IFN that in turn reinforces microglial inflammation, thus contributes to astrocytes dysfunction and neuronal damage. Our study will be the first to implicate microglial Hv1/NOX2/ROS/IFN- signaling in the pathophysiology of SCI, leading to novel treatment approaches for SCI. Given the proposed roles for Hv1 in other inflammatory models, Hv1 signaling represents a generic mechanism relevant to other neuroinflammatory states.