Abstract
Purpose :
Glaucoma is characterized by the progressive degeneration of retinal ganglion cells (RGC) leading to irreversible blindness. Elevated intraocular pressure (IOP) in glaucoma can lead to optic nerve head (ONH) strain. In response, local astrocytes, rearrange their actin cytoskeleton and upregulate reactivity markers such as intermediate filament glial fibrillary acidic protein (GFAP), potentially exacerbating RGC loss. One important mechanism by which astrocytes can sense biomechanical strains is via mechanosensitive channels such as Piezo1. Here, we investigated whether stimulation of Piezo1 increases astrocyte cytoskeletal remodeling, alters morphology, and increases levels of reactivity markers.
Methods :
Primary mouse ONH astrocytes (MONHAs) were isolated from 6–8-week-old C57BL/6J mice and purity confirmed by GFAP-positive immunostaining. MONHA-hydrogels were generated by mixing 2.5 x 106 cells with photoactive ECM polymers (i.e., collagen type I, hyaluronic acid) and crosslinked using 0.025% Riboflavin and UV light (405-500 nm, 10.3mW/cm2) for 5 minutes. MONHA-hydrogels were cultured for 2 weeks before treating with vehicle control, 5uM or 10uM Yoda1 (a Piezo1 specific agonist), 500nM GsMTx4 (a nonspecific mechanosensitive channel inhibitor), or GsMTx4+Yoda1 co-treatment for 24 and 72 hrs. Cell viability was determined using Live/Dead and MTS assays. MONHA morphology was analyzed from Phalloidin-stained z-stacks via confocal microscopy and Sholl analysis. MONHA reactivity was measured by GFAP stained z-stacks using confocal microscopy and intensity analysis.
Results :
Live/Dead and MTS assay confirmed stable astrocyte viability in all groups (~87%). Treatment with 10uM Yoda1 for 72h altered F-actin cytoskeletal morphology resulting in retracted process length (p<0.0001) and decreased process complexity as quantified by Sholl analysis (p<0.0001). Additionally, levels of reactivity marker GFAP were increased in cells treated with 10uM Yoda1 (p <0.005). Cotreatment of GsMTx4 with Yoda1 reversed these responses.
Conclusions :
Our data supports that Piezo1 mechanosensitive channel activation alters MONHA cytoskeletal and cellular morphology and increases cell reactivity. Future studies will determine the role of this important mechanosensitive channel in regulating astrocyte response to mechanical strain.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.