Piezo1 plays a role in optic nerve head astrocyte mechanotransduction

Abstract

Purpose : Elevated intraocular pressure (IOP) is a primary risk factor for glaucoma; however, the mechanisms by which elevated IOP causes glaucomatous neurodegeneration are poorly understood. Optic nerve head (ONH) astrocytes are the mediators of glaucomatous axonal damage. In this study, we investigated the role of the mechanosensing channel piezo1 in ONH astrocyte mechanotransduction, the potential mechanism linking IOP elevation and astrocyte reactivity.

Methods : Primary mouse ONH astrocytes were cultured from C57BL/6J mice. Real time quantitative PCR was performed to assess the expression levels of mechanosensing channels in cultured astrocytes. Cells were further labeled with fura-2-AM and treated with various concentrations of Yoda1, a selective agonist for piezo1. The intracellular calcium concentration was determined by the 340/380 nm excitation ratio for fura-2. To determine mechanical stretch induced astrocyte reactivity, astrocytes were cultured on oxygen plasma treated polydimethylsiloxane (PDMS) membranes. Mechanical stretch was applied at a speed of 100 µm/s and distance of 100 µm to induce 10% strain. After 24 hours of stretch, cells were immunostained for piezo1, fibronectin, and phalloidin.

Results : Nine mechanically activated ion channels from piezo, transient receptor potential and potassium mechanosensing channel families were selected and gene expression levels were compared. Piezo1 showed the highest expression level in ONH astrocytes among all genes examined. Yoda1 induced robust Ca²⁺ responses in a dose dependent manner. Following Yoda1 (100, 300, and 500 nM) stimulation, the intracellular Ca²⁺ concentration (198.1 ± 145.5 nM) in astrocytes increased to 1552.1 ± 620.3, 4696.4 ± 623.6 and 5996.4 ± 213.0 nM, respectively (Mean ± S.D, n=5-7, p<0.001). After 24 hours of mechanical stretch, the morphology of astrocytes did not show significant changes; however, there was a dramatically increased expression of piezo1 and fibronectin in stretch stimulated cells. In addition, phalliodin staining showed a reorganization of F-actin cytoskeleton in stretched cells.

Conclusions : Piezo1 is a major mechanosensing ion channel in ONH astrocytes and plays a role in astrocyte reactivity in response to mechanical stretch. This study provides a new insight in ONH astrocyte mechanotransduction.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.