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Contribution of Piezo1 mechanosensitive channels to astrocyte calcium signaling

University of British Columbia

Astrocyte calcium (Ca²⁺) signaling is involved in the regulation of physiological processes such as synaptic activity and vascular tone in the brain. Recent developments in tools to monitor Ca²⁺ signals have revealed a novel type of spontaneous Ca²⁺ transient that is localized to microdomains in the fine astrocytic processes. However, the molecular mechanisms underlying these signals have not been fully characterized. Based on data from rodent brain transcriptomic and proteomic studies, we identified Piezo1, a mechanosensitive cation channel, as a potential candidate for mediating these Ca²⁺ transients. In Chapter 2, we performed two-photon imaging of the membrane-tethered, genetically encoded Ca²⁺ indicator Lck-GCaMP5 in cultured astrocytes and developed an algorithm for extracting and analyzing the microdomain Ca²⁺ signals. Using a combination of pharmacological and siRNA approaches, we showed that Piezo1 channels contribute to these spontaneous Ca²⁺ transients. We also conducted preliminary imaging experiments in brain slice astrocytes and found that spontaneous Ca²⁺ signals in the endfoot compartments were sensitive to pharmacological modulators of Piezo1. In Chapter 3, we performed immunostaining using strategies that were optimized to target subcellular locations where Piezo1 expression had previously been reported in other cell types. Our results indicated that Piezo1 is localized to subcellular compartments relevant to mechanosensation; Piezo1 immunoreactivity was localized to discrete clusters on the plasma membrane and associated with focal adhesion and actin stress fibers in cultured astrocytes, and Piezo1 expression was observed within the endfoot processes of brain slice astrocytes. Lastly, in Chapter 4, we showed that an osmotic stress model of astrocyte swelling could activate Piezo1-mediated Ca²⁺ microdomain signals in cultured astrocytes. Taken together, the data provide evidence that Piezo1 contributes to spontaneous Ca²⁺ microdomain signals in astrocytes in both cell culture and acute brain slices, and suggest that astrocyte Ca²⁺ signaling may play a role in integrating mechanical stimuli to regulate brain function in physiological and pathological processes involving changes in mechanical force.

Text

2018-07-03

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/66389

Neuroscience

University of British Columbia

UBCV

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