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

我们对获得的神经损伤的病理检查发现了大脑活化免疫细胞的杆小胶质细胞变体。一个多世纪以前，手工绘制了类似的杆小胶质细胞，以表明一般性减少，伤寒和化学暴露的神经病理学，然后在很大程度上被忽略。在我们的2012年出版之后，除癫痫，精神健康疾病等外，还对阿尔茨海默氏病和相关痴呆症（ADRD）的临床和神经病理学确认，在验尸后进行了杆状小胶质细胞。然而，尚无分子工具来精确的杆小胶质细胞分子投资。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，因此在ADRD/脑损伤神经炎症的背景下，我们无法检测和分析Rod小胶质细胞。关于杆小胶质细胞的知识仅依赖于组织学染色和非特异性小胶质细胞抗体（例如Iba1，CD68，CX3CR1）用于验尸后显微镜识别。我们最近来自阿尔茨海默氏病（AD）和控制受试者的最新单细胞RNASEQ数据确定了由基因定义的独特的小胶质细胞簇，这些小胶质细胞由运动，细胞骨架肌动蛋白动力学以及小胶质细胞过程的运动。如果没有新的分子工具来表现出这种难以捉摸的小胶质细胞变体，则不能发生该簇，因为杆小胶质细胞不可能发生。 对于这项生物工程研究赠款（PAR 19-158），将开发，验证和验证Rod小胶质细胞变体所特有的分子工具和标志物的集合，以实现ADRD和大脑损伤病例中神经毒素进展的未来诊断，预后和机械研究。为了开发这些工具，我们提出了一个弥漫性脑损伤模型，该模型生成与神经元树突相邻的杆小胶质细胞场。从该组织中，我们将捕获（LCM）CX3CR1-EGFP杆小胶质细胞和非ROD小胶质细胞，用于从人体衍生的可变重链结合结构抗体（DAB）的库中显示出生物固定。合成环化的HCDR3 Pepperides将被开发为杆小胶质细胞独有的DAB MIMETICS（目标1）。使用纳米弦数字数字空间分析师的杆小胶质细胞变体的空间转录组学将确定杆和非rod小胶质细胞之间不同表达的基因（目标2）。途径分析将确定与杆小胶质细胞的结构，功能，运动和起源相关的细胞过程。对于每个目标，新的分子工具在伤害后，性别之间以及在混合病理学的实验模型中进行了验证和验证，TBI，中风和AD（家族性和晚期淀粉样变性）以及历史后人类AD/ADRD组织。本应用程序中提出的新工具将能够识别，隔离和询问杆小胶质细胞的变体，同时扩大衰老，损伤和疾病中神经炎症的概念，包括呈现出与神经树突相邻的被忽视的杆小胶质细胞变体。使用精制的分子工具，可以在许多神经系统条件下研究杆小胶质细胞，并纳入解释小胶质细胞激活和神经炎症的统一原理。 33