Title: Crosstalk between innate-immunity human microglia and adaptive-immunity Tregs in Alzheimer’s disease (RFA-AG-22-017) Project Summary This application is in response to RFA-AG-22-017, looking at crosstalk between innate and acquired immunity in Alzheimer’s disease (AD). AD affects ~5% of people over age 65, and its prevalence is increasing. Neuroinflammation has been described in neurodegenerative disorders, and particularly in AD. Microglial cells, the resident macrophages of the central nervous system (CNS), respond to different pro- inflammatory stimuli by releasing glutamate, as well as pro-inflammatory cytokines and chemokines. Glutamate release from astrocytes and microglia has also been observed after exposure to Aβ and to αSyn, proteins that are misfolded in AD. T lymphocytes, express several NMDA-type glutamate receptors (NMDARs), and we have also detected their expression in Tregs, whose activity can reportedly ameliorate neurodegenerative disorders. Using patch-clamp recording, our Preliminary Studies show for the first time the presence of functional NMDAR-operated ion channels on human Tregs. Glutamate is known to affect T cell migration and activation; however, the effect of glutamate on Treg migration/activation has not been reported, and, specifically, it is not known how this might impact neurons in AD brain. We hypothesize that glutamate exposure of Tregs will limit their release of anti-inflammatory and neuroprotective factors, thus contributing to neuronal synaptic damage and cell loss. This crosstalk between the innate and acquired immune system may represent a significant contributor to the pathogenesis of AD. We have recently developed a protocol for generating human iPSC-derived microglia (hiMG) following a yolk sac differentiation method (brain microglia originate from the yolk sac and not from the bone marrow). We will test our hypothesis using a platform of hiPSC-derived microglia (hiMG), human Tregs (isolated from normal blood donors), and hiPSC-derived cerebrocortical neurons (hiN) bearing AD mutations (vs. their isogenic/gene- corrected controls). Using this platform, we show in our Preliminary Studies that hiMG release glutamate following activation with oligomeric Aβ and αSyn. We will systematically investigate the mechanism of the activation of Tregs by glutamate released by hiMG, and the effect of this cellular crosstalk on neuronal function. We will evaluate neuronal synaptic integrity and function as well as neuronal survival. The knowledge gained from this study will shed light on the effect of glutamate on neuroinflammation in a human context, and will provide insight into the effect of crosstalk between human microglial and Tregs in stimulating Treg activation. Thus, the work may aid in the development of future novel therapeutic interventions based on human microglial and Treg function and interaction.