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

脑膜的详细分子和结构表征：蛋白质组学和成像质量细胞术相结合的方法

• 批准号: 10462994
• 负责人: Joel S Pachter
• 金额: $ 20.5万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10462994
• 关键词: Adult; Alzheimer' s Disease; Anatomy; Architecture; Autoimmune; Awareness; Blood; Blood - brain barrier anatomy; Brain; Breeding; Cells; Central Nervous System Diseases; Cerebral Amyloid Angiopathy; Cerebrospinal Fluid; Comprehension; Cytometry; Development; Developmental Process; Disease; Elements; Emerging Technologies; Experimental Autoimmune Encephalomyelitis; Exploratory/Developmental Grant; Foundations; Generations; Health; Higher Order Chromatin Structure; Image; Image Analysis; Immune; Immune system; Immunologics; Immunophenotyping; In Situ; Individual; Infiltration; Inflammation; Inflammatory; Knowledge; Label; Laboratories; Lead; Left; Leukocytes; Locales; Lupus; Lymphoid Follicle; Mass Spectrum Analysis; Membrane; Meningeal; Meninges; Meningothelial Cell; Microscopic; Migraine; Modeling; Molecular; Molecular Analysis; Mouse Strains; Multiple Sclerosis; Mus; Nature; Nervous system structure; Neuraxis; Neuroglia; Neurologic; Neuropsychiatric Systemic Lupus Erythematosus; Pathogenicity; Pathologic; Pattern; Play; Population; Population Heterogeneity; Preparation; Proteins; Proteomics; Regulation; Relapse; Research; Role; Scanning Electron Microscopy; Secondary Progressive Multiple Sclerosis; Secondary to; Site; Spinal; Spinal Cord; Spinal meninges; Stroke; Structure; Testing; Therapeutic; Three-dimensional analysis; Time; Tissue membrane; Tissues; Trauma; United States National Institutes of Health; Vascular System; Vasculitis; base; cell motility; cell type; cohort; design; effective therapy; exhaustion; experience; extracellular; link protein; nerve stem cell; nervous system disorder; new therapeutic target; novel; novel strategies; novel therapeutics; prenatal; preservation; protein biomarkers; single-cell RNA sequencing

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.

欣赏脑膜的作用-一种覆盖大脑和脊髓的三层膜 并且通过其脑脊髓液（CSF）循环-已经从被动的物理保护扩展到 动态稳态功能，并在炎症性疾病中作为免疫活性的滋生地 中枢神经系统（CNS）。然而，尽管他们新发现的认识，作为积极的球员在健康和 由于疾病的存在，脑膜的组成仍然是难以捉摸的，因此，人们无法完全理解它们的组成。 功能令人不安地没有实现。这种困境在很大程度上是由于一系列技术挑战 这限制了脑膜在分子和结构水平上的成分分析。特别 问题是缺乏脑膜蛋白标记物，难以保存脑膜的精细排列， 用于显微镜分析的脑膜，以及无法同时观察多个分子决定因素。 然而，在脑膜解剖、完整的脊髓切片和 高参数免疫组织学图像分析，将允许-第一次-详尽的询问， 组织的分子结构该提案利用这些新的发展， 一个分叉的方法，使我们能够开始测试以下假设： 蛋白质的排列在脑与脊膜中是不同的，并且在疾病期间改变。在目标1中， 两种品系健康小鼠的脑和脊髓脑膜的单个蛋白质库将被 使用无偏的，无标记的定量质谱法为基础的蛋白质组学揭示。脑脊膜和 脊髓将分别进行评估，因为它们各自蛋白质组分的区别可能在于 这是为什么某些CNS炎性损伤会袭击沿着神经轴的特定部位的基础。和相应的 选择小鼠品系，因为每一种都将在下一个目标中用于模拟特定的CNS炎性 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 免疫组织学使用成像质量细胞术（IMC）来表征健康期间的脑膜景观 以及它与特定白细胞群的关系。病理条件将模拟 从复发/缓解到继发进行性多发性硬化症或CNS狼疮的过渡， 表现出中枢神经系统炎症和脑膜受累的独特模式。由于IMC使约40种蛋白质 同时定位，它提供了特殊的能力，阐述脑膜的复杂地形， 揭示了白细胞和其他免疫细胞的潜在调节位点。这种力量还在进一步增强 当IMC扩展到3D分析时。因此，通过澄清它们的分子组成以及结构， 这些研究有可能揭示疾病期间脑膜的变化和免疫相互作用 目前缺乏有效治疗的多种CNS炎性病症的新治疗靶点。