The role of meningeal immune cells in the efficacy of CGRP-based migraine therapies

项目摘要/摘要 偏头痛是一种痛苦的慢性神经疾病，是全球第二大致残性疾病。 导致偏头痛的两种机制是免疫细胞激活和降钙素基因相关 多肽(CGRP)信号转导。这项建议调查了这两种机制之间的联系。大脑皮层 传播性抑郁症(CSD)，被认为是偏头痛发作前的先兆的基础 偏头痛，与脑膜巨噬细胞激活增加有关。巨噬细胞可以激活 脑膜初级传入神经元通过释放促炎细胞因子促进偏头痛， 在偏头痛期间，患者脑脊液(CSF)中的这些物质会增加。CSD还能增加CGRP的合成 和释放，单细胞RNA测序数据支持降钙素基因相关肽受体亚单位mRNA的表达 小鼠脑膜免疫细胞。患者血浆和脑脊液中CGRP的升高及CGRP受体的作用 作为偏头痛治疗药物的拮抗剂支持CGRP在偏头痛发生发展中的中心作用。 然而，这些药物的作用机制还不完全清楚。鉴于惩教署对 免疫细胞激活和CGRP释放，免疫细胞上CGRP受体的存在，以及 CGRP受体拮抗剂治疗偏头痛，我假设CGRP受体拮抗剂的疗效 是通过它们抑制脑膜中促炎症巨噬细胞的激活而起作用的。 在三个具体目标下描述的一系列实验中，我将使用解剖学(目标1)、功能(目标2)、 和行为学(目标3)方法，在偏头痛微创模型的背景下测试我的假设 有AURA(光遗传扩散抑制，OSD)。我将描述降钙素基因相关肽的OSD依赖性变化 用荧光原位杂交和RT-qPCR检测脑膜免疫细胞受体的表达(目标1)。我 将评估CGRP受体拮抗剂预防OSD诱导的巨噬细胞变化的能力 形态和表型与活体实时双光子显微镜的激活一致(AIM 2A) 和流式细胞术(Aim 2B)。最后，我将评估巨噬细胞CGRP受体在OSD中的作用。 使用选择性耗竭策略诱导疼痛行为(目标3)。这些实验的结果可能不会 仅揭示了CGRP拮抗剂治疗效果的潜在机制，但可能是新的靶点 以及改善现有偏头痛疗法的机制。在完成这项提议的过程中，我将发展出有价值的 技术技能和接受成为独立调查员所必需的严格的智力培训，以及 我已经组建了一支由专家科学家和临床医生导师组成的团队，以帮助确保我的成功。因此，这一点 奖学金将使我实现我的长期目标，成为一名学术神经学家-在 疼痛研究和临床神经学的交叉点。

Project Summary/Abstract Migraine is a painful, chronic neurological disorder that represents the second most disabling illness worldwide. Two of the mechanisms that to contribute to migraine pain are immune cell activation and calcitonin gene-related peptide (CGRP) signaling. This proposal investigates the connection between these two mechanisms. Cortical spreading depression (CSD), thought to underlie the aura that precedes migraine attack in a subpopulation of migraineurs, is associated with increased meningeal macrophage activation. Macrophages can activate meningeal primary afferent neurons and promote migraine pain through release of pro-inflammatory cytokines, which are increased in patient cerebrospinal fluid (CSF) during migraine. CSD also increases CGRP synthesis and release, and single-cell RNA sequencing data support the expression of CGRP receptor subunit mRNA in mouse meningeal immune cells. Elevated CGRP in patient plasma and CSF and the efficacy of CGRP receptor antagonists as migraine therapies support the central role of CGRP in the development of migraine pain. However, the mechanism of action of these drugs is incompletely understood. Given the effect of CSD on immune cell activation and CGRP release, the presence of CGRP receptor on immune cells, and the use of CGRP receptor antagonists to treat migraine, I hypothesize that the efficacy of CGRP receptor antagonists is mediated by their ability to inhibit the activation of pro-inflammatory macrophages in the meninges. In a set of experiments described under three specific aims, I will use anatomical (Aim 1), functional (Aim 2), and behavioral (Aim 3) approaches to test my hypothesis in the context of a minimally invasive model of migraine with aura (optogenetic spreading depression, OSD). I will characterize OSD-dependent changes in CGRP receptor expression in meningeal immune cells using fluorescent in situ hybridization and RT-qPCR (Aim 1). I will assess the ability of CGRP receptor antagonists to prevent OSD-induced changes in macrophage morphology and phenotype consistent with activation using real-time in vivo two-photon microscopy (Aim 2A) and flow cytometry (Aim 2B). Finally, I will assess the contribution of macrophage CGRP receptors to OSD- induced pain behavior using a selective depletion strategy (Aim 3). The outcomes of these experiments may not only reveal a mechanism underlying the therapeutic efficacy of CGRP antagonists but potentially novel targets and mechanisms to improve existing migraine therapies. In completing this proposal, I will develop valuable technical skills and receive rigorous intellectual training necessary to becoming an independent investigator, and I have assembled a team of expert scientist and clinician mentors to help ensure my success. Thus, this fellowship will allow me to achieve my long-term goal of becoming an academic neurologist-scientist working at the intersection of pain research and clinical neurology.