Researches of calcium-activated chloride channel ANO1 intervening amyotrophic lateral sclerosis progression by activating EGFR and CaMKII signaling

Abstract

Background: ANO1 is closely correlated with the activation of EGFR and CaMKII, while EGFR and CaMKII show low activation in amyotrophic lateral sclerosis (ALS) models. Therefore, we designed experiments to verify that ANO1 may play a protective role on motor neurons in ALS by activating EGFR and CaMKII.

Methods: The expression changes of ANO1, EGFR, CaMKII, pEGFR, and pCaMKII, cell survival status, and apoptosis were studied by western blot, real-time quantitative PCR, immunofluorescence, immunohistochemistry, CCK-8, and flow cytometry. The role of ANO1 in the ALS model by activating EGFR and CaMKII was studied by applying corresponding activators, inhibitors, gene silencing, and overexpression.

Results: In hSOD1^G93A transgenic animals and cell lines, low expression of ANO1 and low activation of EGFR and CaMKII were identified. ANO1 expression decreased gradually with the progression of ALS. Overexpression of ANO1 in the hSOD1^G93A cell line and primary neurons of hSOD1^G93A transgenic mice increased cell viability and decreased cell apoptosis. After the application of ANO1 inhibitor CaCC-inhA01 in hSOD1^G93A cell line and primary neurons of hSOD1^G93A transgenic mice, EGFR activator EGF and CaMKII activator Carbachol, increased cell viability and reduced cell apoptosis. After ANO1 was overexpressed in the hSOD1^G93A cell line and primary neurons of hSOD1^G93A transgenic mice, EGFR inhibitor AEE788 and CaMKII inhibitor KN93 decreased cell viability and increased cell apoptosis.

Conclusions: Our results suggest that ANO1 plays an important role in the survival of ALS motor neurons. ANO1 can increase cell activity and reduce apoptosis by activating EGFR and CaMKII signals.

Keywords: Amyotrophic lateral sclerosis; Anoctamin1; Calmodulin-dependent protein kinase II; Epidermal growth factor receptor.