Oxidative stress, mitochondrial dysfunction and calcium overload in human lamina cribrosa cells from glaucoma donors

Purpose Oxidative stress is implicit in the pathological changes associated with glaucoma. The purpose of this study was to compare levels of oxidative stress in glial fibrillary acid-negative protein (GFAP) lamina cribrosa (LC) cells obtained from the optic nerve head (ONH) region of 5 normal (NLC) and 4 glaucomatous (GLC) human donor eyes and to also examine mitochondrial function and calcium homeostasis in this region of the ONH. Methods Intracellular reactive oxygen species (ROS) production was examined by a thiobarbituric acid reactive substances (TBARS) assay which measures malondialdehyde (MDA), a naturally occurring product of lipid peroxidation and is used as an indicator of oxidative stress. Mitochondrial membrane potential (MMP) and intracellular calcium ([Ca2+]i) levels were evaluated by flow cytometry using the JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetrabenzimidazolecarbocyanine iodide) and fluo-4/AM probes respectively. Anti-oxidant and Ca2+ transport system gene and protein expression were determined by real time polymerase chain reaction (RT-PCR) using gene-specific primer/probe sets and western immunoblotting, respectively. Results Intracellular ROS production was increased in GLC compared to NLC (27.19±7.05 µM MDA versus 14.59±0.82 µM MDA, p<0.05). Expression of the anti-oxidants Aldo-keto reductase family 1 member C1 (AKR1C1) and Glutamate cysteine ligase catalytic subunit (GCLC) were significantly lower in GLC (p=0.02) compared to NLC control. MMP was lower in GLC (57.5±6.8%) compared to NLC (41.8±5.3%). [Ca2+]i levels were found to be higher (p<0.001) in GLC cells compared to NLC. Expression of the plasma membrane Ca2+/ATPase (PMCA) and the sodium-calcium (NCX) exchangers were lower, while intracellular sarco-endoplasmic reticulum Ca2+/ATPase 3 (SERCA) expression was significantly higher in GLC compared to NLC. Subjection of NLC cells to oxidative stress (200 µM H202) reduced expression of Na+/Ca2+ exchanger 1 (NCX 1), plasma membrane Ca2+ ATPase 1 (PMCA 1), and PMCA 4 as determined by RT–PCR. Conclusions Our data finds evidence of oxidative stress, mitochondrial dysfunction and impaired calcium extrusion in GLC cells compared to NLC cells and suggests their importance in the pathological changes occurring at the ONH in glaucoma. Future therapies may target reducing oxidative stress and / or [Ca2+]i.

[1]  C. Burgoyne A biomechanical paradigm for axonal insult within the optic nerve head in aging and glaucoma. , 2011, Experimental eye research.

[2]  M. D. Roberts,et al.  Glaucomatous cupping of the lamina cribrosa: a review of the evidence for active progressive remodeling as a mechanism. , 2011, Experimental eye research.

[3]  H. Uusitalo,et al.  Altered calcium signaling in an experimental model of glaucoma. , 2010, Investigative ophthalmology & visual science.

[4]  Jonathan G. Crowston,et al.  Mechanisms of Retinal Ganglion Cell Injury in Aging and Glaucoma , 2010, Ophthalmic Research.

[5]  N. Osborne Mitochondria: Their role in ganglion cell death and survival in primary open angle glaucoma. , 2010, Experimental eye research.

[6]  Asaad A. Ghanem,et al.  Oxidative Stress Markers in Patients with Primary Open-Angle Glaucoma , 2010, Current eye research.

[7]  Andrew J. Payne,et al.  Control of Intracellular Calcium Signaling as a Neuroprotective Strategy , 2010, Molecules.

[8]  M. Berridge,et al.  Inositol trisphosphate and calcium signalling mechanisms. , 2009, Biochimica et biophysica acta.

[9]  J. Crowston,et al.  Mitochondrial Dysfunction and Glaucoma , 2009, Journal of glaucoma.

[10]  Rebecca M. Sappington,et al.  TRPV1: contribution to retinal ganglion cell apoptosis and increased intracellular Ca2+ with exposure to hydrostatic pressure. , 2009, Investigative ophthalmology & visual science.

[11]  C. O'brien,et al.  Differential global and extra-cellular matrix focused gene expression patterns between normal and glaucomatous human lamina cribrosa cells , 2009, Molecular vision.

[12]  C. O'brien,et al.  Activation of stretch-activated channels and maxi-K+ channels by membrane stress of human lamina cribrosa cells. , 2009, Investigative ophthalmology & visual science.

[13]  J. Ge,et al.  Mitochondrial defects and dysfunction in calcium regulation in glaucomatous trabecular meshwork cells. , 2008, Investigative ophthalmology & visual science.

[14]  N. Osborne Pathogenesis of ganglion "cell death" in glaucoma and neuroprotection: focus on ganglion cell axonal mitochondria. , 2008, Progress in brain research.

[15]  Denis Noble,et al.  The role of the Na+/Ca2+ exchangers in Ca2+ dynamics in ventricular myocytes. , 2008, Progress in biophysics and molecular biology.

[16]  M. Hernandez,et al.  Astrocytes in glaucomatous optic neuropathy. , 2008, Progress in brain research.

[17]  M. Brini Plasma membrane Ca2+-ATPase: from a housekeeping function to a versatile signaling role , 2008, Pflügers Archiv - European Journal of Physiology.

[18]  J. Bruce,et al.  Oxidant-impaired intracellular Ca2+ signaling in pancreatic acinar cells: role of the plasma membrane Ca2+-ATPase. , 2007, American journal of physiology. Cell physiology.

[19]  Tomomi Gotoh,et al.  ER Stress Triggers Apoptosis by Activating BH3-Only Protein Bim , 2007, Cell.

[20]  D. Murchison,et al.  Calcium buffering systems and calcium signaling in aged rat basal forebrain neurons , 2007, Aging cell.

[21]  Anuradha Kalyanasundaram,et al.  SERCA pump isoforms: Their role in calcium transport and disease , 2007, Muscle & nerve.

[22]  E. Strehler,et al.  Rapid Downregulation of NCX and PMCA in Hippocampal Neurons Following H2O2 Oxidative Stress , 2007, Annals of the New York Academy of Sciences.

[23]  A. G. Filoteo,et al.  Plasma Membrane Ca2+ ATPases as Dynamic Regulators of Cellular Calcium Handling , 2007, Annals of the New York Academy of Sciences.

[24]  Deming Sun,et al.  Mechanisms of immune system activation in glaucoma: oxidative stress-stimulated antigen presentation by the retina and optic nerve head glia. , 2007, Investigative ophthalmology & visual science.

[25]  M. Hernandez,et al.  4-Hydroxynonenal, a product of oxidative stress, leads to an antioxidant response in optic nerve head astrocytes. , 2006, Experimental eye research.

[26]  A. Izzotti,et al.  The role of oxidative stress in glaucoma. , 2006, Mutation research.

[27]  D. Kenny,et al.  Trends in blind registration in the adult population of the Republic of Ireland 1996–2003 , 2006, British Journal of Ophthalmology.

[28]  H. Quigley,et al.  The number of people with glaucoma worldwide in 2010 and 2020 , 2006, British Journal of Ophthalmology.

[29]  Catherine Pavoine,et al.  Transcription of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase type 3 gene, ATP2A3, is regulated by the calcineurin/NFAT pathway in endothelial cells. , 2006, The Biochemical journal.

[30]  R. Peters,et al.  Ageing and the brain , 2006, Postgraduate Medical Journal.

[31]  C. O'brien,et al.  Transforming growth factor-beta-regulated gene transcription and protein expression in human GFAP-negative lamina cribrosa cells. , 2005, Glia.

[32]  Ruaidhrí P Kirwan,et al.  Influence of cyclical mechanical strain on extracellular matrix gene expression in human lamina cribrosa cells in vitro. , 2005, Molecular vision.

[33]  L. Coletto,et al.  Exporting calcium from cells. , 2005, Cell calcium.

[34]  Jian Cai,et al.  Proteomic identification of oxidatively modified retinal proteins in a chronic pressure-induced rat model of glaucoma. , 2005, Investigative ophthalmology & visual science.

[35]  R. Ritch,et al.  Dynamic changes in reactive oxygen species and antioxidant levels in retinas in experimental glaucoma. , 2005, Free radical biology & medicine.

[36]  Xiangjun Yang,et al.  Caspase-independent component of retinal ganglion cell death, in vitro. , 2004, Investigative ophthalmology & visual science.

[37]  L. Missiaen,et al.  Thimerosal stimulates Ca2+ flux through inositol 1,4,5-trisphosphate receptor type 1, but not type 3, via modulation of an isoform-specific Ca2+-dependent intramolecular interaction. , 2004, The Biochemical journal.

[38]  R. Rosenstein,et al.  Retinal oxidative stress induced by high intraocular pressure. , 2003, Free radical biology & medicine.

[39]  P. Evelson,et al.  Oxidative stress markers in aqueous humor of glaucoma patients. , 2004, American journal of ophthalmology.

[40]  K. Barnes,et al.  Sarco/endoplasmic reticulum Ca2+-pump isoform SERCA3a is more resistant to superoxide damage than SERCA2b , 2004, Molecular and Cellular Biochemistry.

[41]  M. Berridge,et al.  Calcium signalling: dynamics, homeostasis and remodelling , 2003, Nature reviews. Molecular cell biology.

[42]  S. John,et al.  The Na+/Ca2+ Exchange Molecule , 2002 .

[43]  C. S. Ricard,et al.  Differential gene expression in astrocytes from human normal and glaucomatous optic nerve head analyzed by cDNA microarray , 2002, Glia.

[44]  Ernesto Carafoli,et al.  Calcium signaling: A tale for all seasons , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[45]  S. John,et al.  The Na+/Ca2+ exchange molecule: an overview. , 2002, Annals of the New York Academy of Sciences.

[46]  A. Clark,et al.  Neurotrophin and neurotrophin receptor expression by cells of the human lamina cribrosa. , 2001, Investigative ophthalmology & visual science.

[47]  W. Tatton,et al.  Maintaining mitochondrial membrane impermeability. an opportunity for new therapy in glaucoma? , 2001, Survey of ophthalmology.

[48]  P. Allen,et al.  Transmembrane Redox Sensor of Ryanodine Receptor Complex* , 2000, The Journal of Biological Chemistry.

[49]  K. Gunter,et al.  Mitochondrial calcium transport: mechanisms and functions. , 2000, Cell calcium.

[50]  M. Berridge,et al.  The versatility and universality of calcium signalling , 2000, Nature Reviews Molecular Cell Biology.

[51]  B. Frenguelli,et al.  Mitochondrial Membrane Potential and Glutamate Excitotoxicity in Cultured Cerebellar Granule Cells , 2000, The Journal of Neuroscience.

[52]  W. Tatton,et al.  Retinal damage after 3 to 4 months of elevated intraocular pressure in a rat glaucoma model. , 2000, Investigative ophthalmology & visual science.

[53]  L. Levin,et al.  Differential susceptibility of retinal ganglion cells to reactive oxygen species. , 2000, Investigative ophthalmology & visual science.

[54]  M. Michaelis,et al.  Effects of reactive oxygen species on brain synaptic plasma membrane Ca(2+)-ATPase. , 1999, Free radical biology & medicine.

[55]  M. Michaelis,et al.  Sensitivity of the synaptic membrane Na+/Ca2+ exchanger and the expressed NCX1 isoform to reactive oxygen species. , 1998, Biochimica et biophysica acta.

[56]  J. Mazat,et al.  From calcium signaling to cell death: two conformations for the mitochondrial permeability transition pore. Switching from low- to high-conductance state. , 1998, Biochimica et biophysica acta.

[57]  S. Marklund,et al.  Superoxide dismutase isoenzymes in the human eye. , 1998, Investigative ophthalmology & visual science.

[58]  J. Mazat,et al.  Mitochondria Are Excitable Organelles Capable of Generating and Conveying Electrical and Calcium Signals , 1997, Cell.

[59]  G. E. Marshall,et al.  Antioxidant enzymes in the human iris: an immunogold study , 1997, The British journal of ophthalmology.

[60]  H. Quigley Number of people with glaucoma worldwide. , 1996, The British journal of ophthalmology.

[61]  G. Salama,et al.  Thimerosal Interacts with the Ca2+ Release Channel Ryanodine Receptor from Skeletal Muscle Sarcoplasmic Reticulum (*) , 1995, The Journal of Biological Chemistry.

[62]  M. Berridge,et al.  Luminal Ca2+ promoting spontaneous Ca2+ release from inositol trisphosphate‐sensitive stores in rat hepatocytes. , 1992, The Journal of physiology.

[63]  F. Ursini,et al.  Effect of hydrogen peroxide on calcium homeostasis in smooth muscle cells. , 1992, Archives of biochemistry and biophysics.

[64]  A. Thomas,et al.  Oxidized glutathione causes sensitization of calcium release to inositol 1,4,5-trisphosphate in permeabilized hepatocytes. , 1992, The Biochemical journal.

[65]  A. Thomas,et al.  Beta-adrenergic receptor-mediated phospholipase C activation independent of cAMP formation in turkey erythrocyte membranes. , 1991, The Journal of biological chemistry.

[66]  C. Paterson,et al.  Oxidative inhibition of Ca2+-ATPase in the rabbit lens. , 1989, Investigative ophthalmology & visual science.

[67]  C. Paterson,et al.  Selective inhibition of membrane ATPases by hydrogen peroxide in the lens of the eye. , 1988, Basic life sciences.