Browsing by Author "Morgan, Autumn B."
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Item Identification of proteins affected by increased intraocular pressure in the glaucomatous female mouse retina by label-free proteomics(2023) Zaman, Khadiza; Morgan, Autumn B.; Nguyen, Vien; Prokai-Tatrai, Katalin; Inman, Denise; Prokai, LaszloPurpose: Mass spectrometry-based retina proteomics using animal models of human diseases has enabled novel insights into ocular neuropathology’s such as in glaucoma, as it holds promise for disease biomarker discovery. However, publicly accessible data on retina proteins affected by ocular hypertension (OHT) in animal models utilized males, or sex was not disclosed. Recently, female animals were chosen to advance therapeutic antibody development against glaucomatous neurodegeneration with retina proteomics support. Therefore, our retinal proteomics-based investigation intended to fill a knowledge gap by focusing on OHT-induced changes of protein expressions in the glaucomatous female retinae compared to normotensive controls. Methods: Proteins were extracted from the retinae of normotensive female mice (control, n=5) and OHT mice (n=5) in which increase of intraocular pressure was induced by the magnetic microbead method. After reduction, alkylation and digestion by trypsin, bottom-up shotgun proteomics analyses of the samples were done using data-dependent nanoflow liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) on a hybrid Orbitrap instrument (Thermo Fisher Scientific). MS/MS spectra were searched against the UniProt mouse protein sequence database using the SEQUEST search engine in Proteome Discoverer (version 2.4; Thermo Fisher Scientific). Validation of proteins identifications using stringent criteria and label-free quantifications (LFQ) employing spectral counting to detect regulated proteins between groups using t-tests were performed using Scaffold (version 5.1.2; Proteome Software). Targeted proteomics on selected biomarkers was designed and analyzed using SkylineTM (MacCoss Lab software). Mapping to protein interaction networks and biological processes was done through Ingenuity Pathway Analysis® (IPA®, Qiagen). Results: Our discovery driven data-dependent nanoflow LC–ESI-MS/MS analyses covered nearly 1200 retinal proteins with <1% false discovery rate. Among these proteins, 168 were significantly affected by OHT based on LFQ. Bioinformatics analyses by IPA® revealed important diseases and functions triggered by OHT pertaining to neurological and ophthalmic pathologies. The topmost protein interaction network represented neurological disease, organismal injury and abnormalities. The molecule activity predictor of IPA® revealed important canonical pathways, including inhibition of synaptogenesis signaling and mitochondrial dysfunction leading to degeneration of central nervous system tissue. Another prominent protein interaction network represented nervous system development and function, as well as organ development. In addition, this network also displayed downregulation of neuroprotective crystallins owing to OHT. Neuronal crystallins have been identified not only as biomarkers to monitor the progression of OHT-induced retinal neuropathy and evaluate neuroprotective interventions, but also as potential druggable targets or possible protein therapeutics to prevent glaucomatous neurodegeneration. Parallel reaction monitoring-based targeted proteomics validation of significant OHT-regulated retina proteins are currently underway to establish them as potential preclinical biomarkers and/or therapeutic targets. In addition, our studies will be expanded to investigate sex as a biological variable affecting ocular neurodegeneration associated with glaucoma. Conclusion: We anticipate that biological information one can derive from our dataset at the protein expression level will provide inspiration for future hypothesis-driven experimental studies focusing on knowledge gaps involving the biology of glaucomatous neurodegeneration.Item Long-term HIF-1alpha stabilization reduces respiration, promotes mitophagy, and results in retinal cell death(Springer Nature, 2023-11-24) Nsiah, Nana Yaa; Morgan, Autumn B.; Donkor, Nina; Inman, Denise M.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-1alpha stabilization was induced in mice for 2 and 4 weeks by inhibiting prolyl hydroxylases using the small molecule Roxadustat. HIF-1alpha 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-1alpha 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-1alpha 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-1alpha stabilization alone is detrimental to retina metabolic homeostasis and cellular survival.Item Stretch stress propels glutamine dependency and glycolysis in optic nerve head astrocytes(Frontiers Media S.A., 2022-08-05) Pappenhagen, Nathaniel; Yin, Eric; Morgan, Autumn B.; Kiehlbauch, Charles C.; Inman, Denise M.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.