Microglia are long-lived, self-replicating, yolk sac-derived immune cells of the central nervous system (CNS) parenchyma that are critical modulators of immunity and neuropathology. Microglia dysfunction contributes to several neurologic diseases relevant to the VA, including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). In our preliminary studies, we have unexpectedly identified microglia circulating with the cerebrospinal fluid (CSF) of patients with MS and ALS. We hypothesize that CSF microglia are endogenous CNS cells that exhibit signatures unique to neurologic disease states. We aim to identify any disease- associated characteristics of CSF microglia. We will explore the number and immunologic features of CSF microglia in MS and ALS by flow cytometric analysis. To explore the molecular signature of CSF microglia in various disease states, we will perform single cell RNA sequencing on sorted cells. Comparisons will be made between gene expression patterns in CSF microglia between MS and ALS and in relation to healthy control subjects to determine the health- and disease-specific profile of these cells. Finally, we will pursue flow cytometric and RNA sequencing studies in rats, with a particular focus on models of MS and ALS, to uncover the origins and function of CSF microglia. This proposal will lay the groundwork for the characterization of a novel cellular population capable of regulating CNS immunity.