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.

了解脑膜的作用——覆盖大脑和脊髓的三层膜 脑脊液（CSF）通过它进行彻底改变——已经从被动的物理保护扩展到 动态稳态功能并作为炎症性疾病免疫活动的滋生地 然而，尽管它们被新认识为健康和健康领域的积极参与者。 疾病，脑膜的成分仍然是回避的，因此，对其的充分理解 令人失望的是，这一困境很大程度上是由于一系列技术挑战造成的。 特别是在分子和结构水平上限制了脑膜的成分分析。 问题是缺乏脑膜蛋白标记，难以保存脑膜蛋白的微妙排列 脑膜进行显微分析，并且无法同时查看多个分子决定因素。 然而，解剖脑膜、在脑膜完好无损的情况下切开整个脊髓以及 高参数免疫组织学图像分析，将首次允许对 该提案利用了这些新的发展和提案。 一种分叉的方法，使我们能够开始测试以下假设：组成和/或 蛋白质的排列在大脑和脊髓膜中是不同的，并且在疾病期间发生改变。 两种健康小鼠品系的脑膜和脊髓的各个蛋白质库将是 使用来自大脑和脑膜的无偏见、无标记的基于定量质谱的蛋白质组学揭示了这一点。 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 这就是为什么一些中枢神经系统炎症会袭击神经轴特定部位的基础。 选择小鼠品系，因为在下一个目标中，每种小鼠品系都将用于模拟特定的中枢神经系统炎症 在目标 2 中，通过蛋白质组学鉴定的选定蛋白质将被综合靶向。 使用成像质谱流式细胞术 (IMC) 的免疫组织学来表征健康期间的脑膜景观 和疾病及其与特定白细胞群的关联将模拟其中任何一个。 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 存在中枢神经系统炎症和脑膜受累的独特模式，因为 IMC 能够产生约 40 种蛋白质。 为了同时定位，它提供了特殊的能力来阐述脑膜的复杂地形和 揭示白细胞和其他免疫细胞的潜在调节位点，这种力量仍在进一步发展。 当 IMC 扩展到 3D 分析时，通过阐明它们的分子组成以及结构。 疾病期间脑膜的改变和免疫相互作用，这些研究有可能揭示 针对目前缺乏有效治疗方法的多种中枢神经系统炎症性疾病的新治疗靶点。