Regulation of susceptibility and severity of inflammatory diseases of the central nervous system by novel innate immune signaling pathways in human myeloid cells

炎症反应的个体差异调节多发性硬化症（MS）和其他疾病的发病和严重程度。 脑损伤的类型。这些炎症反应的启动、放大和消退部分发生在 通过巨噬细胞和相关免疫细胞介导的先天免疫信号传导。本实验室曾 在人类巨噬细胞中发现了新的先天免疫信号通路， 电压门控钠通道的剪接变体。这些通道调节dsRNA的模式识别， 细胞内信号传导、囊泡运输和抗病毒基因的转录。在MS的小鼠模型中， 在小鼠巨噬细胞中表达这些通道之一，人巨噬细胞SCN 5A， 严重性和增强的组织修复。最近发表的研究表明，一个新发现的通道， 一种变异体，人巨噬细胞SCN 10A，以协同方式与SCN 5A作用，调节RNA加工， 一种编码DNA修复蛋白PPP 1 R10的转录物。新的初步数据显示， PPP 1 R10表达调节的变化。新的数据还表明，SCN 10A定位于线粒体 在细胞损伤过程中并调节ATP的产生。本订正提案的目的是， 这些先天性免疫信号机制在人类细胞和动物模型中。核心假设是 人巨噬细胞SCN 10A和SCN 5A通过增强DNA保护细胞和组织损伤 细胞生物能量学的修复和维护。这个假设将在三个目标进行评估：1）分析 人巨噬细胞通道变体如何调节PPP 1 R10蛋白表达，2）确定人巨噬细胞通道变体如何调节PPP 1 R10蛋白表达。 巨噬细胞通道变体调节线粒体功能，和3）表征巨噬细胞SN 10A如何 和SCN 5A防止组织损伤。对于目标1，所提出的模型是细胞损伤的内源性信号 激活人巨噬细胞SCN 10 A和SCN 5A以启动钙依赖性核信号传导途径 调节DNA修复蛋白PPP 1 R10的表达。据推测，在这方面的个体差异 在炎症性疾病如MS中，该途径增加了组织损伤的风险。对于目的2，提出 人巨噬细胞SCN 10A在细胞损伤期间定位于线粒体， 线粒体ATP生成。还假设SCN 10A和SCN 5A调节线粒体自噬，细胞自噬是一种细胞免疫调节。 保护机制对于目的3，假设表达SCN 5A的人变体的巨噬细胞 和SCN 10A通过增强DNA修复防止炎性损伤中的组织损伤， 维持生物能量学。这些假设将使用多学科方法在小学进行测试。 人巨噬细胞的培养物;巨噬细胞、小胶质细胞和来源于人诱导的- 多能干细胞;在多发性硬化症的小鼠模型中，实验性自身免疫 脑脊髓炎我们的期望是，我们将确定新的调节机制的生物能学和 与MS和相关疾病相关的组织修复。长期目标是开发新的生物标志物 疾病的易感性和严重性，并确定新的治疗策略，预防和减少长期- 退伍军人的长期残疾与MS。

Individual variation in inflammatory responses regulates onset and severity of multiple sclerosis (MS) and other types of brain injury. Initiation, amplification, and resolution of these inflammatory responses occur in part through innate immune signaling mediated by macrophages and related immune cells. This laboratory has discovered novel innate immune signaling pathways in human macrophages that are regulated by intracellular splice variants of voltage-gated sodium channels. These channels regulate pattern recognition of dsRNA, intracellular signaling, vesicular trafficking, and transcription of anti-viral genes. In a mouse model of MS, expression of one of these channels, human macrophage SCN5A, in mouse macrophages reduced disease severity and enhanced tissue repair. Recently published work demonstrates that a newly discovered channel variant, human macrophage SCN10A, acts in a synergistic manner with SCN5A to regulate RNA processing of a transcript that encodes a DNA repair protein, PPP1R10. New preliminary data demonstrate individual variation in regulation of PPP1R10 expression. New data also reveal that SCN10A localizes to mitochondria during cellular injury and regulates ATP production. The objective of this revised proposal is to characterize these innate immune signaling mechanisms in human cells and an animal model. The central hypothesis is that human macrophage SCN10A and SCN5A prevent cell and tissue injury through enhancement of DNA repair and maintenance of cellular bioenergetics. This hypothesis will be assessed in three aims: 1) Analyze how human macrophage channel variants regulate PPP1R10 protein expression, 2) Determine how the human macrophage channel variants regulate mitochondrial function, and 3) Characterize how macrophage SN10A and SCN5A prevent tissue injury. For Aim 1, the proposed model is that endogenous signals of cellular injury activate human macrophage SCN10A and SCN5A to initiate a calcium-dependent nuclear signaling pathway that regulates expression of the DNA repair protein PPP1R10. It is hypothesized that individual variation in this pathway increases the risk of tissue injury in inflammatory diseases such as MS. For Aim 2, it is proposed that human macrophage SCN10A localizes to mitochondria during cellular injury to transiently increase mitochondrial ATP production. It is also hypothesized that SCN10A and SCN5A regulate mitophagy, a cellular protective mechanism. For Aim 3, it is postulated that macrophages that express human variants of SCN5A and SCN10A prevent tissue injury in inflammatory lesions through enhancement of DNA repair and maintenance of bioenergetics. These hypotheses will be tested using multidisciplinary approaches in primary cultures of human macrophages; macrophages, microglia, and neurons derived from human induced- pluripotent stem cells; and in the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis. The expectations are that we will identify novel regulatory mechanisms of bioenergetics and tissue repair that are relevant to MS and related diseases. The long-term goals are to develop new biomarkers of disease susceptibility and severity and identify novel therapeutic strategies that prevent and reduce long- term disability of Veterans with MS.