The ketogenic diet and hypoxia promote mitophagy in the context of glaucoma.

Mitochondrial homeostasis includes balancing organelle biogenesis with recycling (mitophagy). The ketogenic diet protects retinal ganglion cells (RGCs) from glaucoma-associated neurodegeneration, with a concomitant increase in mitochondrial biogenesis. This study aimed to determine if the ketogenic diet also promoted mitophagy. MitoQC mice that carry a pH-sensitive mCherry-GFP tag on the outer mitochondrial membrane were placed on a ketogenic diet or standard rodent chow for 5 weeks; ocular hypertension (OHT) was induced via magnetic microbead injection in a subset of control or ketogenic diet animals 1 week after the diet began. As a measure of mitophagy, mitolysosomes were quantified in sectioned retina immunolabeled with RBPMS for RGCs or vimentin for Müller glia. Mitolysosomes were significantly increased as a result of OHT and the ketogenic diet (KD) in RGCs. Interestingly, the ketogenic diet increased mitolysosome number significantly higher than OHT alone. In contrast, OHT and the ketogenic diet both increased mitolysosome number in Müller glia to a similar degree. To understand if hypoxia could be a stimulus for mitophagy, we quantified mitolysosomes after acute OHT, finding significantly greater mitolysosome number in cells positive for pimonidazole, an adduct formed in cells exposed to hypoxia. Retinal protein analysis for BNIP3 and NIX showed no differences across groups, suggesting that these receptors were equivocal for mitophagy in this model of OHT. Our data indicate that OHT and hypoxia stimulate mitophagy and that the ketogenic diet is an additive for mitophagy in RGCs. The different response across RGCs and Müller glia to the ketogenic diet may reflect the different metabolic needs of these cell types.

Long-term HIF-1α stabilization reduces respiration, promotes mitophagy, and results in retinal cell death.

Ocular hypertension during glaucoma can lead to hypoxia, activation of the HIF transcription factors, and a metabolic shift toward glycolysis. This study aims to test whether chronic HIF activation and the attendant metabolic reprogramming can initiate glaucoma-associated pathology independently of ocular hypertension. HIF-1α stabilization was induced in mice for 2 and 4 weeks by inhibiting prolyl hydroxylases using the small molecule Roxadustat. HIF-1α stabilization and the expression of its downstream bioenergetic targets were investigated in the retina by immunofluorescence, capillary electrophoresis, and biochemical enzyme activity assays. Roxadustat dosing resulted in significant stabilization of HIF-1α in the retina by 4 weeks, and upregulation in glycolysis-associated proteins (GLUT3, PDK-1) and enzyme activity in both neurons and glia. Accordingly, succinate dehydrogenase, mitochondrial marker MTCO1, and citrate synthase activity were significantly decreased at 4 weeks, while mitophagy was significantly increased. TUNEL assay showed significant apoptosis of cells in the retina, and PERG amplitude was significantly decreased with 4 weeks of HIF-1α stabilization. A significant increase in AMPK activation and glial hypertrophy, concomitant with decreases in retinal ganglion cell function and inner retina cell death suggests that chronic HIF-1α stabilization alone is detrimental to retina metabolic homeostasis and cellular survival.

Stretch stress propels glutamine dependency and glycolysis in optic nerve head astrocytes.

Glaucoma is an optic neuropathy that leads to irreversible blindness, the most common subtype of which is typified by a chronic increase in intraocular pressure that promotes a stretch injury to the optic nerve head. In rodents, the predominant glial cell in this region is the optic nerve head astrocyte that provides axons with metabolic support, likely by releasing lactate produced through astrocytic glycolysis. Our primary hypothesis is that stretching of the optic nerve head astrocytes alters their metabolic activity, thereby advancing glaucoma-associated degeneration by compromising the metabolic support that the astrocytes provide to the axons in the optic nerve head. Metabolic changes in optic nerve head astrocytes were investigated by subjecting them to 24 h of 12% biaxial stretch at 1 Hz then measuring the cells' bioenergetics using a Seahorse XFe24 Analyzer. We observed significant glycolytic and respiratory activity differences between control and stretched cells, including greater extracellular acidification and lower ATP-linked respiration, yet higher maximal respiration and spare capacity in stretched optic nerve head astrocytes. We also determined that both control and stretched optic nerve head astrocytes displayed a dependency for glutamine over pyruvate or long-chain fatty acids for fuel. The increased use of glycolysis as indicated by the extracellular acidification rate, concomitant with a dependency on glutamine, suggests the need to replenish NAD + for continued glycolysis and provision of carbon for TCA cycle intermediates. Stretch alters optic nerve astrocyte bioenergetics to support an increased demand for internal and external energy.