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

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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