Browsing by Subject "age-related macular degeneration"
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Item Mechanisms of Photoreceptor Cell Apoptosis(2000-05-01) Crawford, Matthew John; Neeraj Agarwal; Victoria Rudick; Raghu KrishnamoorthyCrawford, Matthew John, Mechanisms of Photoreceptor Cell Apoptosis. Doctor of Philosophy (Biomedical Sciences), May 2000; 168 pp; 3 tables; 23 figures; bibliography, 282 titles. Photoreceptor cell death mediated by programmed cell death pathways is responsible for many disease states of the retina, which result in vision loss. Examples of this include retinal dystrophies and age-related macular degeneration. Correspondingly, the understanding of programmed cell death, or apoptosis, in these cells is important in the formulation of preventative and treatment options. The goals of this dissertation are to characterize a suitable in vitro photoreceptor cell model and explore the molecular mechanisms resulting in apoptotic cell death secondary to oxidative cell death paradigm. Means of interrupting the cell death process were also investigated. An immortalized clonal mouse retinal cell line was shown to express photoreceptor-specific genes and proteins by RT-PCR amplification, Western blot analysis, and immunocytochemical localization. Exposing these cultured cells to visible light resulted in oxidative stress, as exhibited by elevated malonyldialdehyde and reduced gluthathione levels, as well light exposure-dependent apoptosis was shown using multiple techniques which identified fragmentation of chromosomal DNA, a key finding in the apoptotic cell death process. Molecular regulators of apoptotic cell death, including bcl-2 family proto-oncogenes and the nuclear transcription factor NF-kB, were found to be important in oxidative stress-induced pathogenesis of 661 W photoreceptor cells. mRNA and protein levels of the anti-apoptotic proto-oncogene bcl-2 declined following oxidative stress disturbing the balance proto-oncogene regulators and initiating the apoptotic pathway. The nuclear transcription factor NF-kB was found to be constitutionally expressed in the photoreceptor cells with its down-regulation during apoptosis. Permanent transfection of the photoreceptor cells with bcl-2 gene imparted protection from apoptosis and sustained NF-kB levels. The results presented in this dissertation help define the molecular mechanisms which occur during apoptosis of photoreceptor cells. Photo-oxidative stress results in programmed cell death mediated through changes in NF-kB binding activity and bcl-2 family genes. The involvement of caspase-1 in the degradation of NF-kB and the execution of apoptosis is also demonstrated. Over-expression of the proto-oncogene bcl-2 interrupts the apoptotic events, protecting against down-modulation of NF-kb binding activity and cell death. Our proposed mechanism for apoptosis in photoreceptor cells provides several points at which targeted gene expression (bcl-2 or NF-kB), or pharmaceuticals (anti-oxidants, caspase inhibitors, or calcium channel blockers) may prevent apoptotic cell death.Item Molecular Mechanisms of and Potential Therapies for Oxidative Damage to the Retinal Pigment Epithelium(2007-09-01) Wang, Zhaohui; Roque, Rouel S.; Wordinger, Robert J.; Das, HridayWang, Zhaohui, Molecular Mechanisms of and Potential Therapies for Oxidative Damage to the Retinal Pigment Epithelium. Doctor of Philosophy (Biomedical Sciences), September 2007, 161 pages, 34 illustrations, bibliography, 119 titles. Age-related macular degeneration (AMD), the most common cause of irreversible vision loss in the elderly, results mainly from degeneration of the retinal pigment epithelium (RPE) and loss of photoreceptor cells. Oxidative stress has been acknowledged as a leading cause of RPE degeneration and concomitant photoreceptor cell loss, but the exact role of reactive oxygen species (ROS) in RPE cell death remains to be established. Moreover, while mitogen-activated protein kinases (MAPKs) are suggested to be involved in RPE degeneration induced by oxidative stress, the precise functions and molecular mechanisms of MAPKs in RPE degeneration remain elusive. In spite of the numerous therapeutic modalities proposed for AMD, the treatment of AMD remains unsatisfactory. Recent studies suggesting stem cells as a potential source for trophic factors in damaged murine hearts led us to investigate a possible role for stem/progenitor cell-derived factors in protecting RPE cells from oxidative damage. Furthermore, human retinal progenitor cells promote RPE cell survival by regulating p42/p44 MAPK activity. When exposed to oxidative stress produced by glucose oxidase/glucose, human RPE cells exhibited membrane blebbing and cytoskeleton remodeling in the early phase of oxidative stress. Prolonged exposure to oxidative stress induced mitochondrial membrane potential depolarization, cell death and DNA condensation, but not DNA fragmentation. Furthermore, both p38 MAPK and p42/p44 MAPK were activated by oxidative injury. P38 MAPK inhibitor, but not p38 MAPK siRNA, inhibited RPE cell death induced by oxidative stress. Overexpression of constitutively active MEK1 inhibited RPE cell death exposed to oxidative damage. In contrast, interfering p42/p44 MAPK expression accelerated oxidative-stress induced RPE cell death. To investigate the effects of human retinal progenitor cells (hRPC) on RPE cells, we isolated and expanded hRPC in vitro. The hRPCs expressed markers of neuronal and retinal progenitor cells, and were capable of differentiating into neuronal phenotype in defined medium. In the presence of 10% fetal bovine serum, hPRC suppressed RPE cell death induced by oxidative damage. Furthermore, conditioned medium of hRPC induced activation of p42/p44 MAPK, and the protective effect of hRPC and conditioned medium was suppressed by p42/p44 MAPK inhibitor. Our studies increase our understanding of the molecular mechanisms that could be employed to rescue RPE cells from degeneration and support the therapeutic potential of retinal progenitor cells. It will provide further insight into molecular mechanisms of AMD and establish a foundation for the long-term prevention and treatment of AMDItem Role of mitophagy in ocular neurodegeneration(Frontiers Media S.A., 2023-11-15) Brooks, Calvin D.; Kodati, Bindu; Stankowska, Dorota L.; Krishnamoorthy, Raghu R.Neurons in the central nervous system are among the most metabolically active cells in the body, characterized by high oxygen consumption utilizing glucose both aerobically and anaerobically. Neurons have an abundance of mitochondria which generate adequate ATP to keep up with the high metabolic demand. One consequence of the oxidative phosphorylation mechanism of ATP synthesis, is the generation of reactive oxygen species which produces cellular injury as well as damage to mitochondria. Mitochondria respond to injury by fusion which serves to ameliorate the damage through genetic complementation. Mitochondria also undergo fission to meet an increased energy demand. Loss of mitochondria is also compensated by increased biogenesis to generate new mitochondria. Damaged mitochondria are removed by mitophagy, an autophagic process, in which damaged mitochondria are surrounded by a membrane to form an autophagosome which ultimately fuses with the lysosome resulting in degradation of faulty mitochondria. Dysregulation of mitophagy has been reported in several central nervous system disorders, including, Alzheimer's disease and Parkinson's disease. Recent studies point to aberrant mitophagy in ocular neurodegenerative disorders which could be an important contributor to the disease etiology/pathology. This review article highlights some of the recent findings that point to dysregulation of mitophagy and it's underlying mechanisms in ocular neurodegenerative diseases, including, glaucoma, age-related macular degeneration and diabetic retinopathy.