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Characterization of the mechanosensitive cation channel Piezo1 in microglia

University of British Columbia

Microglia are the resident immune cells of the central nervous system. Following injury, infection, or disease, microglia rapidly mobilize to the site of damage and are critically involved in both launching and resolving an immune response. To detect insult and perform their immune functions, microglia interact chemically and mechanically with their surroundings. While the role of chemical signalling in modulating microglial behaviour and immune function has been extensively studied, the impact of mechanical force is less well established. In 2014, a novel mechanosensitive channel was identified in the microglial transcriptome. Piezo1 is a cation channel with significant calcium (Ca²⁺) permeability that activates in response to mechanical force. In addition to its mechanosensitive properties, it is now evident that Piezo1 channels are involved in mediating immune function in astrocytes and peripheral myeloid cells. As highly mechanosensitive and immune competent cells, microglia presented an interesting candidate for investigating Piezo1 expression and function. In this thesis research we obtained evidence that Piezo1 channels are functionally expressed in primary microglia and upregulate in response to immune challenge. Western blot and RT-qPCR data revealed that Piezo1 protein and mRNA increase in response to the toxin lipopolysaccharide (LPS), an effect which is mediated by toll-like receptors 2 and 4. Using immunocytochemistry, we observed that LPS alters the pattern of Piezo1 expression in the peripheral cell membrane and microglial fine processes. Finally, we performed a series of Ca²⁺ imaging experiments to elucidate the functional properties of Piezo1. We observed that the Piezo1 agonist Yoda1 induced an increase in cytosolic Ca²⁺, an effect which was attenuated by blocking Piezo1 with gadolinium and by removing extracellular Ca²⁺. This data suggests that Piezo1 channels directly mediate Ca²⁺ influx from the environment. Interestingly, acute LPS application abolished the Piezo1-mediated Ca²⁺ response, while blocking purinergic receptors potentiated the Ca²⁺ response. Lastly, we induced cell swelling to generate mechanical force on the cell membrane. Our results indicated that mechanical activation of Piezo1 robustly increases cytosolic Ca²⁺. Overall, this thesis provides the first evidence that Piezo1 channels are expressed in primary microglia and are involved in microglial immune function.

Text

2021-01-11

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/77047

Neuroscience

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/