Detailed molecular and structural characterization of the meninges: an approach combining proteomics and Imaging Mass Cytometry

评价脑膜的作用--一种覆盖大脑和脊髓的三层膜 而脑脊液(CSF)通过它循环-已经从被动的物理保护扩大到 炎症性疾病的动态平衡功能及其免疫活性的温床 中枢神经系统(CNS)。然而，尽管他们新获得了作为健康和健康领域积极参与者的认可 疾病，脑膜的成分继续是回避的，因此，充分理解他们的 功能令人失望地没有实现。这种困境在很大程度上是由于一系列技术挑战造成的。 这限制了在分子和结构水平上对脑膜的成分分析。尤其是 问题是缺乏脑膜蛋白标记物，难以保存微妙的排列 用于显微镜分析的脑膜，以及无法同时查看多个分子决定因素。 然而，在解剖脑膜方面的新进展，即在完整脑膜的情况下切割整个脊髓，以及 高参数免疫组织学图像分析将首次允许对 这种组织的分子组织。这项提案利用了这些新的发展，并提出了 一种分叉的方法，允许我们开始测试以下假设：组成和/或 在大脑和脊膜中，蛋白质的排列是不同的，并在疾病期间发生变化。在目标1中， 两个品系的健康小鼠的脑和脊髓脑膜的个别蛋白质谱将是 利用基于蛋白质组学的无偏倚、无标记的定量质谱学揭示。脑膜和脑膜 脊髓将被单独评估，因为它们各自的蛋白质成分的区别可能在于 一些中枢神经系统炎性侮辱为什么会发生在神经轴上的特定部位。和各自的 选择小鼠品系，因为在下一个目标中，每个品系都将被用来模拟特定的中枢神经系统炎症 条件。在目标2中，通过蛋白质组学鉴定的选定蛋白质将通过全面的 免疫组织学成像质量细胞术(IMC)用于描述健康人的脑膜地貌 和疾病，以及它与特定的白细胞群体的关系。病理情况将会模拟 从复发/缓解到继发性进行性多发性硬化症或中枢神经系统狼疮的转变 表现为明显的中枢神经系统炎症和脑膜受累。由于IMC能激活约40种蛋白质 由于同时定位，它提供了特殊的能力，详细说明脑膜和复杂的地形 揭示白细胞和其他免疫细胞的潜在调控部位。这种力量还在进一步增强。 当IMC扩展到3D分析时。因此，通过阐明它们的分子组成以及结构 疾病期间脑膜的变化和免疫相互作用，这些研究有可能揭示 针对目前缺乏有效治疗的多种中枢神经系统炎症性疾病的新治疗靶点。

Appreciation of the role of the meninges – a three-layered membrane that covers the brain and the spinal cord and through which the cerebral spinal fluid (CSF) circulates – has expanded from passive physical protection, to dynamic homeostatic functions and serving as a breeding ground of immune activity in inflammatory diseases of the central nervous system (CNS). Yet, despite their newfound recognition as active players in health and disease, composition of the meninges has continued to be evasive and, as a result, full comprehension of their function left disappointingly unrealized. This dilemma has largely been due to an array of technical challenges that restricted compositional analysis of the meninges at the molecular and structural levels. Particularly problematic has been the lack of meningeal protein markers, difficulty in preserving the delicate arrangement of the meninges for microscopic analysis, and inability to view multiple molecular determinants simultaneously. However, new advances in dissecting the meninges, sectioning entire spinal cord with meninges intact, and high-parameter immunohistological image analysis, will allow – for the first time – exhaustive interrogation of the molecular organization of this tissue. This proposal takes advantage of these new developments and proposes a bifurcated approach that will allow us to begin testing the following hypothesis: The composition and/or arrangement of proteins is distinct in the brain vs spinal meninges, and altered during disease. In Aim 1, the individual protein repertoires of meninges from brain and spinal cord of two strains of healthy mice will be revealed using unbiased, label-free quantitative mass spectrometry-based proteomics. Meninges from brain and spinal cord will be evaluated separately, as distinctions in their respective protein components may lie at the basis of why some CNS inflammatory insults strike at particular sites along the neuroaxis. And the respective mouse strains were selected as each will be used, in the next Aim, to model a specific CNS inflammatory condition. In Aim 2, selected proteins identified by proteomics will be targeted by comprehensive immunohistology using Imaging Mass Cytometry (IMC) to characterize the meningeal landscape during health and disease, and its associations with specific leukocyte populations. Pathological conditions will model either the transition from relapsing/remitting-to-secondary progressive multiple sclerosis, or CNS Lupus, as each present with distinctive patterns of CNS inflammation and meningeal involvement. As IMC enables ~ 40 proteins to be localized simultaneously, it offers exceptional ability to elaborate the intricate terrain of the meninges and unveil potential regulatory sites for leukocytes and other immune cells. Such power is still further heightened when IMC is expanded to 3D analysis. Accordingly, by clarifying their molecular makeup, as well as structural alterations and immune interactions of the meninges during disease, these studies have the potential to uncover novel therapeutic targets for a wide host of CNS inflammatory conditions currently lacking effective treatments.