Eye / Vision
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/30810
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Browsing Eye / Vision by Author "Inman, Denise"
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Item Glaucoma-associated stretch of optic nerve head astrocytes drives changes in glycolysis bioenergetics and glutamine dependency(2022) Yin, Eric; Pappenhagen, Nate; Inman, DenisePurpose: 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.Item The Role of Oxidative Phosphorylation in Müller Glia Functions and Survival(2022) Nsiah, Nana Yaa; Inman, DenisePurpose The importance of mitochondria to the energy production of Müller glia (MG), the main glial cells of the retina, is controversial. Previous studies showed MG are mainly glycolytic. Others challenge this view because MG are deficient in key glycolytic enzymes. Our goal is to potentially settle this debate by destabilizing the electron transport chain in MG mitochondria and assessing how retinal metabolism may be impacted. Methods MG that lack oxidative phosphorylation in vivo through destabilization of Complex IV were generated using GLASTCreERT2::Cox10fl/fl transgenic mice. Mice received daily tamoxifen injections for 5 consecutive days beginning at P30. Confirmation of recombination of the floxed Cox10 locus and enzyme activity was performed using PCR analysis of genomic DNA isolated from the retina and sequential cytochrome c oxidase (COX)/succinate dehydrogenase (SDH) histochemistry, respectively. Cell lysates from primary Müller cells were used for western blotting and total protein analysis. Full-field electroretinography (ERG) was performed to assess MG function from transgenic and wild-type mice in vivo. Scotopic ERGs were recorded (OcuScience® HMsERG, Xenotec Inc., Henderson, NV) in response to six light flash intensities ranging from −3 to 1 log cd x s/m2 on a dark background. Each stimulus was presented in a series of three. Data were analyzed with GraphPad Prism and ERG b-wave amplitudes were compared using a paired two-tailed Student’s t-test. The b-wave amplitude was measured from the trough of the a-wave to the peak of the b-wave. Results A 465bp DNA fragment amplified from genomic DNA of mutant mice, with no corresponding fragment from control, confirmed Cox10 locus recombination. Total protein analysis, with normalization to the mitochondrial protein VDAC1, showed lower levels of cytochrome c oxidase protein from mutant mice compared to controls. Scotopic ERG b-wave was not significantly different between mutant and wild-type age-controlled mice at all light intensities. No overt retinal abnormalities were observed in GLASTCreERT2::Cox10fl/fl transgenic mice. Conclusion Our results show that cre recombinase induction in GLASTCreERT2::Cox10fl/fl successfully inhibits cytochrome c oxidase activity in MG from adult mice. Our in vivo experiments suggest that oxidative phosphorylation is not necessary for Müller glia energy metabolism under physiological conditions.