cytochrome-c-t and Retinal-Degeneration

Exposure to reactive oxygen species (ROS) leads to the development and progression of retinal degenerative diseases. However, the exact mechanisms are not fully understood. In this article, the role of angiotensin II type 1 receptor (AT1R) signaling in H(2)O(2)-induced retinal damage was examined. Mouse photoreceptor-derived 661 W cells were treated with the AT1R blockers valsartan, losartan and candesartan before exposure to H(2)O(2). Cell viability, intracellular ROS level, mitochondrial membrane potential (MMP), cytochrome-c level, DNA fragmentation, caspase activity and gene expression were detected. Pre-treatment of 661 W cells with AT1R blockers significantly decreased H(2)O(2)-mediated toxicity and reduced the ROS level. In addition, apoptosis-related biochemical indicators showed that pre-incubation of AT1R blockers would elevate the MMP, decrease the release of cytochrome-c and formation of DNA fragmentation, and inhibit activities of caspase-3 and caspase-9 in exogenous H(2)O(2)-treated 661 W cells. Moreover, treatment with AT1R blockers suppressed the expression of Egr1, Fosl1 and Lox12. These results suggest that AT1R signaling mediates H(2)O(2)-induced apoptosis, at least partially through generating the ROS and increasing the levels of proapoptotic molecules in 661 W cells. AT1R blockade may provide a new therapeutic approach for preventing oxidative stress-induced retinal neural damage.

Topics: Angiotensin II Type 1 Receptor Blockers; Animals; Apoptosis; Caspases; Cell Culture Techniques; Cell Line; Cell Survival; Cytochromes c; Gene Expression; Hydrogen Peroxide; Membrane Potential, Mitochondrial; Mice; Oxidative Stress; Photoreceptor Cells, Vertebrate; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Receptor, Angiotensin, Type 1; Retinal Degeneration; Signal Transduction

Optical coherence tomography (OCT) has revolutionises the diagnosis of retinal disease based on the detection of microscopic rather than subcellular changes in retinal anatomy. However, currently the technique is limited to the detection of microscopic rather than subcellular changes in retinal anatomy. However, coherence based imaging is extremely sensitive to both changes in optical contrast and cellular events at the micrometer scale, and can generate subtle changes in the spectral content of the OCT image. Here we test the hypothesis that OCT image speckle (image texture) contains information regarding otherwise unresolvable features such as organelle changes arising in the early stages of neuronal degeneration. Using ultrahigh resolution (UHR) OCT imaging at 800 nm (spectral width 140 nm) we developed a robust method of OCT image analyses, based on spatial wavelet and texture-based parameterisation of the image speckle pattern. For the first time we show that this approach allows the non-invasive detection and quantification of early apoptotic changes in neurons within 30 min of neuronal trauma sufficient to result in apoptosis. We show a positive correlation between immunofluorescent labelling of mitochondria (a potential source of changes in cellular optical contrast) with changes in the texture of the OCT images of cultured neurons. Moreover, similar changes in optical contrast were also seen in the retinal ganglion cell- inner plexiform layer in retinal explants following optic nerve transection. The optical clarity of the explants was maintained throughout in the absence of histologically detectable change. Our data suggest that UHR OCT can be used for the non-invasive quantitative assessment of neuronal health, with a particular application to the assessment of early retinal disease.

Topics: Apoptosis; Caspases; Cell Line; Cytochromes c; Early Diagnosis; Mitochondria; Neurons; Retinal Degeneration; Tomography, Optical Coherence

Sustained elevation in cGMP and a concomitant increase in intracellular Ca(2+) levels in the rd1 photoreceptors are followed by a rapid loss of photoreceptors. In a murine-derived photoreceptor cell line, 661W, treated with the phosphodiesterase inhibitor IBMX or the cyclic GMP-gated channel agonist 8-bromo-cGMP, it was previously found that the induced cell death was mediated by calpain and caspase-3. Because oxidative stress is a common product of ionic imbalance or elevated Ca(2+), we tested the role of oxidative stress in cGMP-induced photoreceptor cell death. In the rd1 mouse retina, oxidative stress was found to precede calpain and caspase-3 activation. In 661W cells, the increase in intracellular cGMP and Ca(2+) resulted in the generation of reactive oxygen species (ROS), the activation of oxidative stress enzymes, and the activation of calpain, followed by apoptosis mediated by the effector caspase-3. All these events, including calpain activation, were ameliorated by docosahexanoic acid (DHA). The cell-permeable inhibitor of calpain, SJA6017, while inhibiting cell death, had no effect on the generation of oxidative stress. These results establish a central role for oxidative stress in cGMP-induced cell death and suggest a ROS-mediated sequential activation of signal transduction events, which provide targets for future treatment strategies.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Apoptosis; Calcium; Calpain; Caspase 3; Catalase; Cell Line; Cell Survival; Cyclic GMP; Cytochromes c; Docosahexaenoic Acids; Glutathione Peroxidase; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Oxidative Stress; Phosphodiesterase Inhibitors; Photoreceptor Cells, Vertebrate; Reactive Oxygen Species; Retinal Degeneration

Photoreceptor cell death is an irreversible, pathologic event in many blinding retinal diseases including retinitis pigmentosa, age-related macular disease, and retinal detachment. Light exposure can exacerbate a variety of human retinal diseases by increasing the rate of photoreceptor cell death. In the present study, we characterize the kinetics of photoreceptor cell death in Tubby (homozygous tub/tub, which have inherited, progressive retinal degeneration) mice born and raised in a bright cyclic light environment. Our data show that raising tub/tub mice in a bright cyclic light environment induces rapid loss of photoreceptors. This effect can be slowed, but not prevented, by raising animals in constant darkness, which suggests the involvement of phototransduction in the accelerated death of photoreceptors in this animal. We further demonstrated that the activities of cytosolic cytochrome c and caspases-3 and -9 were significantly increased in the retinas of tub/tub mice. Raising animals in darkness significantly reduced the increased activities of caspases-3 and -9, as well as cytosolic cytochrome c. We also observed that rhodopsin, a phototransduction protein, is not restricted to the rod outer segment, but is distributed throughout the rod cell, including the inner segments, cell bodies, and synapses. In addition, the light-dependent translocation and compartmentalization of arrestin and transducin are affected by the tubby mutation. Our results support the interpretation that problems in protein trafficking in the photoreceptors of the tub/tub mouse may contribute to retinal degeneration.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Arrestin; Caspases; Cytochromes c; Electroretinography; Fluorescent Antibody Technique; Light; Mice; Photoreceptor Cells, Vertebrate; Protein Transport; Proteins; Retinal Degeneration; Transducin; Vision, Ocular

Apoptosis plays a crucial role in the sculpture of the mammalian retina during development. However, once the retina is fully differentiated, the emphasis must shift towards survival and mechanisms have to be put in place to prevent inappropriate cell death. In this study, we identify a potential control point at the level of mitochondrial permeability. We show that pro-apoptotic Bcl-2 family members known to be involved in the regulation of permeability transition and physiological cell death in the retina are down regulated during postnatal retinal development. In addition, we demonstrate an age-dependent susceptibility to retinal cell death induced by various stimuli known to target mitochondrion. These results potentially explain why retinal cells employ different death pathways depending on their stage of development. In contrast to developmental apoptosis, pathological retinal cell death in several animal models has been reported to occur independently of caspase activation. Here, we show that not only is cytochrome c release precluded from degenerating retinas but other pro-death molecules such as Omi/HtrA2 and AIF also remain in the mitochondrion. Our results indicate that transcriptional regulation of 'death genes' such as pro-apoptotic Bcl-2 family members during retinal development affords protection in adult post-mitotic neurons by preventing execution of the archetypal mitochondrial death pathway.

Topics: Age Factors; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Apoptosis Inducing Factor; Caspase 9; Caspase Inhibitors; Caspases; Cytochromes c; Down-Regulation; Enzyme Activation; High-Temperature Requirement A Serine Peptidase 2; Light; Mice; Mice, Inbred C57BL; Mitochondria; Mitochondrial Proteins; Organ Culture Techniques; Photoreceptor Cells, Vertebrate; Proto-Oncogene Proteins c-bcl-2; Retina; Retinal Degeneration; Serine Endopeptidases

To characterize the time course of apoptosis and degeneration in a transgenic mouse model of retinal degeneration based on truncated mutant HRG4; to investigate the nature of binding of the mutant HRG4 to its target, ADP-ribosylation factor-like (ARL)2; to study its effects on the downstream molecules Binder-of-ARL2 (BART) and adenine nucleotide transporter (ANT)-1 and on the induction of apoptosis.. Saturation binding, microscopic morphometric, Western blot, immunofluorescence, and TUNEL analyses were used.. Increased apoptosis did not occur until 20 months in the transgenic retina, consistent with the delayed-onset degeneration in this model. The truncated HRG4 protein exhibited approximately threefold greater affinity for ARL2 than the wild-type HRG4, likely resulting in nonfunctional sequestration of ARL2. A significant decrease in ARL2 was present by 20 months, accompanied by a 50% decrease in ANT-1 in the photoreceptor synaptic mitochondria, with evidence of mitochondrial dysfunction. Preapoptotic degeneration in the photoreceptor synapse was demonstrated with cytochrome c release and caspase 3 activation within the synapse-without evidence of TUNEL-positive apoptosis in the photoreceptor cell body-indicating an initial event in the synapse leading to apoptosis. Caspase 3 was activated in the accompanying secondary neuron, consistent with transsynaptic degeneration.. The results support a novel mechanism of retinal degeneration in which preapoptotic degeneration starts in the photoreceptor synapse because of a deficiency in ANT-1 and spreads to the secondary neuron transsynaptically, followed by apoptosis and degeneration in the cell body of the photoreceptor.

Topics: Adaptor Proteins, Signal Transducing; Adenine Nucleotide Translocator 1; Animals; Apoptosis; Blotting, Western; Caspase 3; Caspases; Codon, Nonsense; Cytochromes c; Enzyme Activation; In Situ Nick-End Labeling; Intracellular Signaling Peptides and Proteins; Mice; Mice, Transgenic; Microscopy, Fluorescence; Microtubule Proteins; Mitochondria; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Synapses