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，但仍然没有确定有效的治疗方法 促进脊髓损伤(SCI)后的恢复。在一定程度上，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 以及导致损伤后神经炎症的分子机制，以便未来发展 新的治疗方法。电压门控质子通道Hv1是新近发现的一种高表达的离子通道 在静息的大脑小胶质细胞中。在病理条件下，小胶质细胞Hv1需要NADPH氧化酶 (NOx)依赖的ROS(活性氧物种)的产生，通过提供电荷补偿 输出电子，缓解细胞内酸中毒。因此，Hv1是控制多个NOX的唯一目标 活动和ROS生产。然而，无论是这一发现背后的确切信号机制，还是 Hv1在脊髓损伤的病理生理学中的重要作用已被充分了解。根据我们的初步数据，我们将 验证小胶质细胞Hv1作为神经炎症的关键机制的假说，通过改变 NOX2/ROS/干扰素-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 脊髓损伤后的神经学结果。 我们将使用全身性或小胶质细胞Hv1 KO、小胶质细胞NOX2 KO转基因小鼠以及体内和体外 确定脊髓损伤后Hv1升高机制的创新技术 神经炎。目标1将确定Hv1在神经炎症中的功能和机制 在SCI之后。小胶质细胞介导的神经炎症的多项定量评估将与 针对Hv1的基因或药物干预以检验脊髓损伤诱导的小胶质细胞Hv1的假设 激活通过改变的小胶质细胞介导有害的神经炎症和功能缺陷 NOX2/ROS信令。目的2阐明小胶质细胞NOX2在损伤后神经炎症中的作用。 我们将利用基因干预来删除Hv1依赖的小胶质细胞中NOX2的上调，并评估 小胶质细胞NOX2与Hv1偶联在脊髓损伤后神经炎症中的作用目标3将确定关键 Hv1/NOX2来源的ROS/干扰素-通过小胶质细胞-星形胶质细胞在脊髓慢性神经炎中的作用 互动。互补的细胞、分子和遗传学方法将被用来检验这一假说。 Hv1/NOX2介导的小胶质细胞ROS激活促炎星形胶质细胞，导致分泌干扰素，从而 反过来又会加剧小胶质细胞的炎症，从而导致星形胶质细胞功能障碍和神经元损伤。 我们的研究将首次将小胶质细胞Hv1/NOX2/ROS/干扰素-信号与病理生理学联系起来 导致了脊髓损伤的新的治疗方法。鉴于Hv1在其他炎症性疾病中的拟议作用 模型中，Hv1信号代表了与其他神经炎性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.