Regulation of microglial cell function by the Piezo1 channel

Microglial cells are the major immune cells in the central nervous system (CNS) and regulation of their pro-inflammatory function is crucial to maintaining CNS homeostasis. Increasing evidence suggests that alterations in the mechanical properties of CNS microenvironments influence glial cell phenotypes. This thesis therefore aims to examine expression of the mechanosensitive Piezo1 channel in microglial cells and its role in regulating pro-inflammatory functions. Firstly, Piezo1 mRNA and protein expression was detected in BV2 cells and primary mouse microglial cells using RT-PCR, Western blotting and immunocytochemistry. Exposure to Yoda1, a Piezo1 channel agonist, induced an increase in intracellular Ca2+ concentration, measured using Fura-2 ratiometry, but almost no Ca2+ response in extracellular Ca2+-free solution. Yoda1-induced Ca2+ responses were reduced by treatment with ruthenium red, a Piezo1 channel inhibitor, and by transfection with Piezo1-specific siRNA, indicating that the Piezo1 channel in microglial cells functions as a Ca2+-permeable channel on the cell surface. Next, ELISA was used to measure the release of pro-inflammatory cytokines from microglial cells after priming with lipopolysaccharide (LPS). Priming with LPS, while having a minimal effect on IL-1β production, induced production of TNF-α and IL-6. In primary microglial cells, LPS priming also induced morphological changes. Such responses were reduced by treatment with Yoda1, suggesting that Piezo1 channel activation inhibits the pro-inflammatory function of microglial cells. Finally, shown using Western blotting or immunocytochemistry, LPS priming increased the phosphorylation and nuclear translocation of NF-κB p65, which was inhibited by treatment with Yoda1. Additionally, Yoda1-induced inhibition of NF-κB activation in LPS-primed cells was blunted by treatment with BAPTA-AM, an intracellular Ca2+ chelator, indicating that Piezo1-mediated Ca2+ signalling inhibits the NF-κB signalling pathway. In summary, this thesis provides evidence that activation of the Piezo1 channel downregulates the pro-inflammatory function of microglial cells, raising an interesting perspective for alleviating neuroinflammation by targeting such a mechanism.