Mechanisms of Photoreceptor Cell Apoptosis




Crawford, Matthew John


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Crawford, 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.