Glaucoma-associated stretch of optic nerve head astrocytes drives changes in glycolysis bioenergetics and glutamine dependency

Abstract

Purpose: Glaucoma is an optic neuropathy that leads to irreversible blindness, often through a chronic increase in intraocular pressure which promotes a stretch injury to the optic nerve head. In rodents and humans, the predominant glial cell in this region is the optic nerve head astrocyte (ONHA). Since this region of the optic nerve is unmyelinated, the ONHAs provide neighboring axons with metabolic support, likely in the form of lactate produced through astrocytic glycolysis. Previously, we found that exposing astrocytes to glaucoma-associated deformation altered their metabolism in ways that indicated stronger commitment to and upregulation of glycolysis. Here, we explore the predominant source supplying the requisite carbon for TCA cycle intermediates that this stretch-induced glycolysis upregulation demands; our hypothesis is that glutamine metabolism plays a major role in this mechanism. Methods: Primary ONHAs were cultured from P5 rat pup optic nerve head explants. Metabolic changes in ONHAs were investigated by subjecting them to 24h of 12% biaxial stretch at 1Hz. The cells' bioenergetics were measured using a Seahorse XFe24 Analyzer. Protein markers for glycolysis and other cellular metabolism pathways were measured using a ProteinSimple Jess Automated Western Blot Analyzer. Results: We observed significant glycolytic and respiratory activity differences between control and stretched ONHAs, including greater extracellular acidification and lower ATP-linked respiration, yet higher maximal respiration and spare capacity in stretched ONHAs. We determined that both control and stretched ONHAs displayed a dependency upon glutamine over pyruvate or long-chain fatty acids for fuel. We also found increased proteome markers of glutamine metabolism such as glutamine synthetase, and glycolytic lactate production through increased lactate dehydrogenase-a, in stretched ONHAs when compared against that of control. Conclusions: Our results of extracellular acidification rate, fuel flexibility studies, and various metabolic proteome markers suggest that ONHAs, after being subjected to glaucoma-associated stretch deformation, show a preference for the increased use of glycolysis over oxidative phosphorylation, and glutamine over other sources of TCA cycle carbon intermediates. Therefore, stretch alters ONHA bioenergetics to support an increased demand for internal and external energy. This is significant as these altered bioenergetics could potentially inhibit ONHAs from providing metabolic support to neighboring retinal ganglion cell axons, further advancing the axonal degeneration commonly associated with glaucoma.