Molecular Tool Development to Identify, Isolate, and Interrogate the Rod Microglia Phenotype in Neurological Disease and Injury

开发分子工具来识别、分离和询问神经系统疾病和损伤中的杆状小胶质细胞表型

• 批准号: 10599762
• 负责人: JONATHAN LIFSHITZ
• 金额: $ 74.62万
• 依托单位: ARIZONA VETERANS RESEARCH AND EDUCATION FOUNDATION
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10599762
• 关键词: Actins; Address; Aging; Alzheimer' s Disease; Alzheimer' s disease related dementia; Amyloidosis; Antibodies; Antigens; Autopsy; Binding; Biological; Biomedical Engineering; Brain; Brain Injuries; Cd68; Cell physiology; Cell surface; Cells; Chemical Exposure; Clinical; Collection; Cyclization; Cytoskeleton; Data; Data Set; Dendrites; Diagnosis; Diagnostic; Diffuse Brain Injury; Disease; Disease Progression; Disease model; Epilepsy; Evolution; Experimental Models; Female; Fluorescent in Situ Hybridization; Functional disorder; Future; Gene Expression; Genes; Goals; Hand; Harvest; Human; Immune; Immunohistochemistry; Impairment; Inflammation; Inflammatory; Injury; Intervention; Investigation; Knowledge; Laboratories; Libraries; Mental disorders; Microglia; Microscopic; Modeling; Molecular; Morphology; Movement; Nervous System Trauma; Neurodegenerative Disorders; Neurologic; Neurons; Paresis; Pathogenicity; Pathologic; Pathology; Pathway Analysis; Peptides; Phage Display; Pharmacologic Substance; Phenotype; Process; Prognosis; Proteins; Publications; RNA; Regional Disease; Regulation; Reporting; Research Project Grants; Resolution; Rod; Role; Signal Induction; Site; Stroke; Structure; Surface; Swelling; Techniques; Thick; Time; Tissues; Traumatic Brain Injury; Typhoid Fever; Variant; Visualization; Western Blotting; antibody mimetics; antigen binding; biomarker panel; cell motility; design; differential expression; digital; fluid percussion injury; glial activation; histological stains; laser capture microdissection; male; mimetics; mouse model; nano-string; nerve injury; nervous system disorder; neural repair; neuroinflammation; neuromechanism; neuronal cell body; neuropathology; pharmacologic; prevent; prognostic; repaired; sex; single-cell RNA sequencing; theories; tool; tool development; transcriptome sequencing; transcriptomics; verification and validation

Our pathological examination of acquired neurological injury uncovered the rod microglia variant of activated immune cells in the brain. Over a century ago, similar rod microglia were hand drawn to indicate neuropathology in general paresis, typhoid, and chemical exposure, and then largely ignored. Following our 2012 publication, rod microglia have been visualized in post-mortem tissue with clinical and neuropathological confirmation of Alzheimer’s Disease and Related Dementias (ADRD), in addition to epilepsy, mental health disorders, and others. Yet no molecular tools exist for precise molecular investigation of rod microglia. As cellular mechanisms of ADRD and injury include neuroinflammation and concomitant microglia activation, a critical problem emerges from our inability to detect and analyze rod microglia within the context of ADRD/brain injury neuroinflammation. The body of knowledge on rod microglia relies solely on histological stains and non-specific microglia antibodies (e.g., Iba1, CD68, CX3CR1) for post-mortem microscopic identification. Our most recent single-cell RNAseq data from 100 Alzheimer’s disease (AD) and control subjects identified a unique cluster of microglia defined by genes for motility, cytoskeletal actin dynamics, and movement of microglial processes; confirmation of this cluster as rod microglia cannot occur without new molecular tools to phenotype this elusive microglia variant. For this Bioengineering Research Grant (PAR 19-158), collections of molecular tools and markers unique to the rod microglia variant will be developed, validated, and verified to enable future diagnostic, prognostic, and mechanistic studies of neuroinflammation progression in cases of ADRD and brain injury. To develop these tools, we propose a diffuse brain-injury model that generates fields of rod microglia adjacent to neuronal dendrites. From this tissue, we will capture (LCM) CX3CR1-eGFP rod microglia and non-rod microglia for phage display biopanning from a library of human-derived variable heavy-chain binding domain antibodies (dAB). Synthetic cyclized HCDR3 peptides will be developed as dAb mimetics unique to rod microglia (Objective 1). Spatial transcriptomics of the rod microglia variant using the nanoString GeoMX Digital Spatial Profiler will determine differentially expressed genes between rod and non-rod microglia (Objective 2). Pathway analysis will identify cellular processes associated with the structure, function, motility, and origin of rod microglia. For each objective, new molecular tools are validated and verified across time post-injury, between sexes, and in experimental models of mixed pathology TBI, stroke, and AD (familial and late-onset amyloidosis), as well as post-mortem human AD/ADRD tissue. The new tools proposed in this application will be able to identify, isolate, and interrogate the rod microglia variant, while expanding the concept of neuroinflammation in aging, injury, and disease to include the overlooked rod microglia variant that presents adjacent to neuronal dendrites. With refined molecular tools, rod microglia can be investigated across many neurological conditions and incorporated into unifying principles that explain microglial activation and neuroinflammation. 33

我们对获得性神经损伤的病理检查发现了大脑中激活的免疫细胞的杆状小胶质细胞变体，一个多世纪前，类似的杆状小胶质细胞被手工绘制以指示一般性麻痹、伤寒和化学暴露的神经病理学，然后在很大程度上被忽视。我们 2012 年的出版物中，在死后组织中观察到了杆状小胶质细胞，并通过临床和神经病理学证实了阿尔茨海默病和相关痴呆症 (ADRD)，以及癫痫、精神健康障碍和然而，由于 ADRD 和损伤的细胞机制包括神经炎症和伴随的小胶质细胞激活，尚不存在用于精确分子研究的分子工具，因此我们无法在 ADRD/脑损伤的背景下检测和分析杆状小胶质细胞，从而出现了一个关键问题。关于杆状小胶质细胞的知识体系仅依赖于尸检的组织学染色和非特异性小胶质细胞抗体（例如 Iba1、CD68、CX3CR1）。我们最新的来自 100 名阿尔茨海默病 (AD) 和对照受试者的单细胞 RNAseq 数据鉴定了一个独特的小胶质细胞簇，该簇由运动性、细胞骨架肌动蛋白动力学和小胶质细胞过程的运动基因定义，并确认该簇为​​杆状小胶质细胞；如果没有新的分子工具来对这种难以捉摸的小胶质细胞变异进行表型分析，这种情况就不可能发生。 对于这项生物工程研究补助金（PAR 19-158），将开发、验证和验证杆状小胶质细胞变体特有的分子工具和标记物集合，以实现未来 ADRD 和 ADRD 病例中神经炎症进展的诊断、预后和机制研究。为了开发这些工具，我们提出了一种弥漫性脑损伤模型，该模型可以生成与神经树突相邻的杆状小胶质细胞区域，我们将从该组织中捕获（LCM）。 CX3CR1-eGFP 杆状小胶质细胞和非杆状小胶质细胞，用于从人源可变重链结合域抗体 (dAB) 库中进行噬菌体展示生物淘选 合成环化 HCDR3 肽将被开发为杆状小胶质细胞特有的 dAb 模拟物（目标 1）。使用 nanoString GeoMX 数字空间分析仪对杆状小胶质细胞变体进行空间转录组学，将确定杆状细胞和非杆状细胞之间的差异表达基因。小胶质细胞（目标 2）。通路分析将识别与杆状小胶质细胞的结构、功能、运动和起源相关的细胞过程。对于每个目标，新的分子工具都会在损伤后、性别之间和实验中进行验证和验证。该应用中提出的新工具将能够识别、分离和诊断混合病理学 TBI、中风和 AD（家族性和迟发性淀粉样变性）以及死后人类 AD/ADRD 组织。研究杆状小胶质细胞变异，同时扩展衰老、损伤和疾病中神经炎症的概念，以包括与神经元树突相邻的被忽视的杆状小胶质细胞变异，利用精细的分子工具，可以在许多神经系统疾病中研究杆状小胶质细胞并将其纳入其中。解释小胶质细胞激活和神经炎症的统一原理。 33