Abstract
Purpose :
Glaucoma is the leading cause of irreversible blindness worldwide, strongly associated with elevated intraocular pressure (IOP). Astrocytes of the ONH play a significant role in the response to mechanical strain in glaucoma. The linker of nucleoskeleton and cytoskeleton (LINC) complex regulates how the nucleus senses and responds to strain. The overall goal of this project is to investigate the effect of acute tensile strain on nuclear morphology and expression of nuclear envelope proteins to characterize nuclear mechanosensation in ONH astrocytes.
Methods :
Primary mouse ONH astrocytes were isolated and characterized by immunostaining on glass coverslips for GFAP, OSP, F4/80, and aquaporin-4. Cells were plated at passages 2-3 on BioFlex plates with 0.2% gelatin coating at a seeding density of 50,000 cells per well and grown to semi-confluence for 5 days. Serum deprivation was performed for 24 hours before tension application. Using the Flex Cell-4000T tension system, cyclic 5% multiaxial tensile strain was applied for 0, 1, or 6 hours at 1Hz frequency. Expression levels of LINC complex proteins and F-actin were quantified via immunostaining and confocal microscopy. Nuclear morphology was assessed using Imaris. Chromatin condensation was assessed using the ChromCon code in Matlab.
Results :
Acute tensile strain increased nuclear volume ~2-fold (p<0.05) after 6 hours and decreased sphericity (p<0.0001). Chromatin condensation decreased ~2-fold (p<0.05) after 6 hours of strain. Emerin expression levels decreased (p<0.05) after 1 hour of strain, while nesprin-2 expression levels decreased (p<0.05) after 6 hours of strain. Total F-actin (cytoplasmic and nuclear) intensity increased after 6 hours of strain (p<0.05).
Conclusions :
In response to tensile strain, astrocytes acutely alter nuclear volume and sphericity which correlates with changes in F-actin and LINC complex protein expression and decreased chromatin condensation. These data suggest interplay between actin and nuclear envelope proteins may mediate changes in nuclear morphology, resulting in changes in chromatin condensation and transcriptional activation.
Future studies will further investigate transcriptomic and chromatin accessibility changes in response to acute tensile strain, and analyze LINC complex and actin interactions using live cell imaging.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.