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

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。在世纪以前，类似的杆状小胶质细胞被手绘出来，用来指示一般性轻瘫、伤寒和化学暴露的神经病理学，然后在很大程度上被忽视了。在我们2012年的出版物之后，除了癫痫、精神健康障碍等之外，在具有阿尔茨海默病和相关痴呆（ADRD）的临床和神经病理学确认的死后组织中已经观察到杆状小胶质细胞。然而，没有分子工具存在精确的分子研究杆小胶质细胞。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活化，因此出现了一个关键问题，即我们无法在ADRD/脑损伤神经炎症的背景下检测和分析杆小胶质细胞。关于视杆小胶质细胞的知识体系仅依赖于组织学染色和非特异性小胶质细胞抗体（例如，Iba 1、CD 68、CX 3CR 1）用于死后显微镜鉴定。我们最近从100名阿尔茨海默病（AD）和对照受试者中获得的单细胞RNAseq数据确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运动的基因定义;如果没有新的分子工具来表型这种难以捉摸的小胶质细胞变体，则无法确认该簇为杆状小胶质细胞。 对于这项生物工程研究资助（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的集合，以实现ADRD和脑损伤病例中神经炎症进展的未来诊断，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，该模型产生了与神经元树突相邻的杆状小胶质细胞。从该组织中，我们将捕获（LCM）CX 3CR 1-eGFP杆状小胶质细胞和非杆状小胶质细胞，用于从人源性可变重链结合结构域抗体（dAB）文库中进行噬菌体展示生物淘选。将开发合成的环化HCDR 3肽作为杆状小胶质细胞特有的dAb模拟物（目的1）。使用nanoString GeoMX Digital Spatial Profiler对杆状小胶质细胞变体进行空间转录组学分析，将确定杆状和非杆状小胶质细胞之间的差异表达基因（目标2）。通路分析将识别与视杆细胞小胶质细胞的结构、功能、运动性和起源相关的细胞过程。对于每一个目标，新的分子工具在损伤后的时间内，在性别之间，在混合病理TBI、中风和AD（家族性和迟发性淀粉样变性）的实验模型中，以及在死后的人AD/ADRD组织中进行验证和验证。本申请中提出的新工具将能够识别、分离和询问视杆细胞小胶质细胞变体，同时扩展衰老、损伤和疾病中神经炎症的概念，以包括被忽视的视杆细胞小胶质细胞变体，该变体存在于神经元树突附近。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，并将其纳入解释小胶质细胞活化和神经炎症的统一原则。 33

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