Double knock-in reporter mice: a novel tool to study age-associated NF-κB dynamics in primary microglia

Abstract

Abstract Microglia are immune sentinels in the brain that are capable of orchestrating potent inflammatory responses in aging and neurodegenerative diseases like Alzheimer’s and Parkinson’s diseases. NF-κB signaling pathway is commonly recognized as a significant regulator of inflammation and aging. Mapping the spatiotemporal complexity of NF-κB signaling is crucial to understand its impact and function in vivo, but the lack of tools to directly monitor NF-κB protein components has hindered such efforts. Our lab has generated reporter mice with the endogenous RelA (p65) and c-Rel labeled with distinct fluorescent proteins and a double knock-in line with both labeled subunits. To understand how aging affects microglial function, we isolated primary microglia from young and old animals. We cultured them in vitro and validated by P2RY12 +, CD11b +and CD45 int. Interestingly, we observed two different microglial populations, one motile, and the other forming clusters. Microglia from aged animals showed a higher prevalence of motile, free-roaming microglia. We also observed these distinct microglial populations in brain slices from the mice ex-vivo. Live-cell imaging of NF-kB dynamics in primary microglia from young and old mice revealed a shift towards c-Rel in old brains. Further, we stained for Ki67 as a proliferation marker to functionally characterize these microglia populations. We observed only the free-roaming microglia to be proliferative in nature. Furthermore, we are investigating the senescence pattern between the two populations to better understand the molecular mechanisms associated with age-dependent and inflammation-derived changes in young and old brains.