Research Description
The regeneration of the 11-cis-retinyl imine chromophore of rhodopsin during the visual cycle and mechanisms that control this process are central questions in the field of vision research. The retinal pigment epithelium (RPE)-specific protein RPE65 is centrally involved in the isomerization and hydrolysis of all-trans-retinyl esters. We investigate potential regulatory mechanisms of the visual cycle under oxidative stress conditions.
PART I: The Visual Cycle
Light induces isomerization of the 11-cis-retinal Schiff base of the visual pigment chromophore to a sterically less hindered all-trans isomer to initiate vertebrate vision. This light-induced photoisomerization activates the G-protein-coupled receptor pathway, converting light energy into an electrical signal that, in turn, hyperpolarizes photoreceptor cells. For continued vision, 11-cis-retinoid must be regenerated in retinal pigment epithelial (RPE) and photoreceptor cells by a series of enzymes that includes retinol dehydrogenases (RDHs), lecithin retinol acyltransferase (LRAT) and RPE65. Steady-state retinoid concentrations and photosensitivity are controlled by the biochemical reactions that lead to 11-cis-retinal regeneration, known as the visual cycle.
Figure 1. The mammalian visual cycle
Active functioning of the visual cycle seems to be involved in light-induced retinal degeneration in a mouse model. Light damage only occurs when the retina is supplied with 11-cis-retinal, whereas inhibition of retinoid regeneration causes resistance to light damage. RPE65 L450M mutant mice, which exhibit slow rhodopsin regeneration, are more resistant to light damage than are wild-type mice, indicating that induction of light damage requires continuous isomerization/hydrolysis of all-trans retinyl ester and consecutive bleaching of rhodopsin. The retinal pigment epithelium (RPE) is susceptible to oxidative stress due to its high oxygen consumption, the generation of reactive oxygen species (ROS), the presence of a high percentage of unsaturated fatty acids, and exposure to light. Bright light may generate free radicals and increase ROS production, which further induces oxidative stress in RPE cells.
Although the overall pathway of retinoid resynthesis in the eye is generally well characterized, the mechanisms that regulate it under oxidative stress conditions remain elusive. For example, it is unknown how light and oxygen imbalances influence proteins related to retinoid metabolism in the RPE. Recently, we found that levels of RPE65 and its truncated form, RPE45, were increased after short-term exposure to intense light in the human RPE cultures. We found that the level of RPE65 is increased in light compared with dark conditions, and RPE45, a 45-kDa fragment of RPE65, is present only in cells exposed to light or high oxidative stress. We further suggest that the RPE45 fragment may be generated via an ubiquitination mechanism involving interaction of specific proteases with RPE65.
Prolonged exposure to light has been implicated as an important risk factor for the development of retinal neurodegeneration through apoptosis. Reactions in the visual cycle include major chemical conversions of retinoids, such as isomerizationof cis-trans retinoid by rhodopsin and RPE65, oxidation/reduction by retinol dehydrogenases, and ester formation/hydrolysis by LRAT and RPE65. However, it is not known how light regulates retinoid flux in the RPE.
RPE65 is reported to be an all-trans-retinyl ester isomerohydrolase necessary for the synthesis of 11-cis-retinol, and is clearly important for 11-cis-retinal biosynthesis in vivo.Ablation of RPE65 expression in a mouse knock-out model severely reduces 11-cis-retinal biosynthesis and results in the accumulation of all-trans-retinyl esters as oil droplets in the RPE, indicating that RPE65 may be involved in retinyl ester processing. Several mutations in the RPE65 gene have been shown to be associated with eye diseases, including autosomal recessive childhood-onset severe retinal dystrophy, Leber’s congenital amaurosis (LCA) and retinitis pigmentosa.
Figure 2. Light induced up-regulation of RPE65
We study the effects of oxidative stress and light on RPE65 regulation, showing that treatment with H2O2 or exposure to high-intensity light promotes RPE65 up-regulation and cleavage in vitro. Changes in RPE65 expression were also seen in vivo during cycles of light and dark conditions, indicating a possible relationship between light cues and RPE65 regulation. The dose-dependent induction of RPE45, a fragment of RPE65 generated by caspase-mediated cleavage in response to H2O2 exposure, indicates that RPE45 is a potential biomarker of oxidative stress- and light-induced apoptosis. Stress-induced proteolysis is an important signal transduction pathway mediated by caspases, presenilin and amyloid precursor protein that can lead to neurodegenerative disorders and inflammation. Caspase-3, a common mediator of different apoptotic signaling pathways, also generates the same fragmentation pattern as H2O2, suggesting that both caspase-3 and H2O2 promote proteolysis of RPE65 at similar sites. Both mitochondria-mediated and cell death receptor-mediated apoptotic pathways converge on caspase-3. RPE65 is a highly uveitogenic and antigenic protein that may be among those involved in certain autoimmune diseases of the eye. Thus, adequate turnover of this specific and abundant protein in the RPE would have a significant role in various pathologic contexts, including oxidative stress or intense light exposure. Although each retinoid reaction in the visual cycle has been studied in detail, the mechanisms that control this cycle have not been clearly defined. 11-cis-retinol is known to inhibit all-trans-retinyl ester isomerohydrolase, whereas addition of retinol binding proteins stimulates this process. Changes in retinoid concentration as a result of light exposure have been shown to correlate with RPE65 expression levels in a mouse model.
Toxic organic molecules including lipofuscin (A2E) are generated as byproducts of the visual cycle. It is still not known why macular deteriorates and why macular degeneration progresses. Lipofuscin accumulation might be one of the major risk factors for macular degeneration. In our study, biochemical reactions of A2E and drusen will be addressed focusing on metarhodopsin decay vs. retinal release.
Figure 3. Biochemical reactions of A2E formation.
PART II: Retinal Degeneration
Apoptosis is the primary mechanism of abnormal cell death of photoreceptors, retinal ganglion cells (RGC), or retinal pigment epithelium (RPE) cells in degenerative retinal diseases such as age-related macular degeneration (AMD), retinitis pigmentosa (RP), or glaucoma, and can be triggered by even moderate oxidation. Biochemical reactions of light–electrical energy conversion in the retina and the RPE require increased metabolism to satisfy the energy demands. As such, the eye needs increased levels of oxygen, especially when the eye is continuously exposed to light. Light-induced degeneration of photoreceptors depends on rhodopsin bleaching and phototransduction. Light insults result in increased production of H2O2 in the outer retina, and the production of reactive oxygen species including free radicals has been shown to be involved as an early event in light-induced retinal degeneration.
The RPE is exposed to an oxidative environment due to its high oxygen tensions of 70–90 mmHg, a high metabolic rate, and accumulation of lipofuscin. Moreover, continuous exposure to light causes RPE cells to consume a large amount of oxygen in order to complete the complex processes in the visual cycle, nutrient transport, and phagocytosis. These processes put the RPE into a relatively hypoxic condition with high oxygen demand. In response to light triggers, neuroprotective proteins provide an anti-apoptotic effect within the eye. The mechanisms to regulate the expression of these retinal proteins that protect against light-induced oxidative stress remain elusive.
Erythropoietin (EPO) is an oxygen-regulated glycoprotein and a hematopioetic cytokine that stimulates the proliferation, survival, and differentiation of erythroid progenitor cells. It is known to prevent apoptosis and protect neuronal cells from neuronal damage, including experimental CNS models of hypoxic and ischemic insults. EPO in the brain and the kidney is regulated by oxygen-dependent mechanisms. These data are derived from models such as traumatic brain injury, spinal cord injury, Parkinson's disease, excitotoxicity, oxidative stress, and chemical neurotoxicity. EPO can protect RGC from degeneration induced by acute ischemia-reperfusion injury and axotomy injury, and promote survival of RGCs in glaucoma mouse models. EPO is also neuroregenerative and stimulates neurogenesis and post-stroke recovery. In vitro studies have reported that EPO stimulates neuritic outgrowth by postnatal and adult RGCs. EPO is known to be produced in the retina in response to acute hypoxia via hypoxia inducible factor-1α (HIF-1α) stabilization, which confers protection from light-induced retinal degeneration. The specific role of EPO was highlighted because only EPO gene expression was significantly affected among the various angiogenic factors in a HIF-1alpha-like factor (HLF) knockdown model.
Figure 4. Exogenous EPO treatment reduces retinal cell death under oxidative stress.
We hypothesize that light, reactive oxygen species, and hypoxic and hyperoxic conditions may regulate the protection mechanisms in retinal and RPE cells through EPO by anti-apoptosis. The neuroprotective effect of EPO has been documented in cell cultures as well as in vivo; however, its effect on primary retinal cell cultures was not reported, including concurrent detailed molecular events. Moreover, the effect of various oxidative environment conditions such as hypoxia, hyperoxia, bright light, or hydrogen peroxide has never been studied or compared in retinal cell cultures or human RPE cell culture systems. We investigate whether EPO and EPOR levels might be regulated by time of day and light conditions in vivo. The antiapoptotic effect of EPO further compared in the primary rat retinal culture and low passage human RPE culture under oxidative stress conditions.
Figure 5. Up-regulation of EPO/EPOR expression in the retina exposed to hypoxia (MAP2 or GFAP: green vs. EPO/EPOR: red).
Our data demonstrate that the circadian organization of retinal EPO and EPOR elaboration, anticipating daily light onset, is a potentially effective endogenous physiologic strategy for retinal protection from light-induced oxygen-mediated damage. These findings are consistent with documented circadian organization of the full range of oxidative damage protecting enzyme systems in a number of tissues.
PART III: Functional Proteomics
Retinal pigment epithelial cells (RPE), located between photoreceptors and choroid, are in a unique position to mediate transport of nutrients, oxygen, and retinoid from blood to photoreceptors. For continued vision, the RPE is responsible for retinoid recycling and removal of molecular components that shed from photoreceptor outer segment. Dysfunction of the RPE leads to depletion of nutrients and oxygen from photoreceptor cells to initiate apoptosis or accumulation of retinoid to block visual signaling. Continuous exposure to light causes RPE to consume a large amount of oxygen in order to complete the complex processes of the visual cycle, nutrient transport, and phagocytosis. This oxidative environment in the RPE may contribute to the pathogenesis and progress of retinal diseases. It is still not known why the initial retinal degeneration begins and how degenerative signals progress as a result of continued oxidative stress. Adaptation to changes in oxidative environment is critical for the survival of the RPE. Clinical trials demonstrated a significant reduction in the rate of progression toward retinal degeneration upon intake of antioxidants such as carotenoid (lutein, zeaxanthin), vitamin C, E, and zinc containing supplements. Oxidation of polyunsaturated fatty acids (PFA) and abundant photosensitizers in the RPE induce generation of ROS upon exposure to visible light. Hydrogen peroxide (H2O2) is generated in the RPE during phagocytosis of photoreceptor outer segment, and it has been used as a direct oxidative inducing reagent to initiate cellular oxidative stress.10 Understanding of molecular mechanisms that mediate oxidative stress-induced proteome changes in the RPE may provide insight into pathogenesis of retinal degeneration. In the current study, comparative and differential proteomics has been applied to investigate global changes of RPE proteome under oxidative stress induced by H2O2. Two dimensional fluorescent differential gel electrophoresis (2D-DIGE) is an advanced 2D technology that protein samples were prelabeled with different fluorescent dyes and two different samples (control vs. treated) run simultaneously on the same gel. By using 2D-electrophoresis and 2D-DIGE coupled with mass spectrometry, unbiased system-wide analysis of proteome changes in oxidative stress is investigated in our lab. Identification of target proteins in the RPE under oxidative stress implies new insights of signaling mechanisms at the molecular level.
Figure 6. Very sensitive fluorescent labeling (A) compared to traditional Coomassie blue staining (B, C) allowed us to use 5 microg of total protein extracts/2D gel for reliable analysis.
Functional proteomic data obtained from our studies are essential to understand early molecular signaling events of oxidative stress in the RPE. Targeting early signaling proteins would be a useful therapeutic strategy for the treatment of diverse diseases of the retina and the RPE.
PART IV: Melatonin and Circadian Clock
Melatonin (N-acetyl-5-methoxytryptamine) is a paracoid/autocoid small molecule, nocturnally synthesized in the pineal gland and the retina of mammals. When produced in retinal photoreceptor cells, melatonin acts as a paracrine signaling molecule and participates in many physiological processes within in the eye. Several effects of melatonin result from its interaction with its membrane receptors (MT1/ MT2). These G protein coupled receptors are found in several cell types in the retina, including amacrine cells, horizontal cells, ganglion cells and photoreceptor cells. Melatonin also interacts with nuclear receptors and intracellular proteins such as calmodulin, tubulin and calreticulin. Daily production and secretion of melatonin shows striking daily organization with peaks during each night and very low levels during each day. This pattern is regulated by both the endogenous circadian clock and environmentally tuned by the light. As a circadian pacemaker, melatonin modulates many rhythmic processes in the eye such as retinomotor movements, neurotransmitter release, rod outer segment disc shedding and RPE phagocytosis. Furthermore, ocular melatonin is an effective anti-oxidant crossing cell membranes and the blood-brain-barrier to react with many reactive oxygen/nitrogen species including hydroxyl radicals, superoxide anions, and nitric oxide. Since many eye diseases are caused or aggravated by oxidative stress, the retinal melatonin cycle is a prime candidate for normal maintenance of retinal health and a candidate therapy for these oxidative retinal diseases. We show that profound and interesting protein changes occur with constant light (at night), many of which are modulated or reversed by melatonin. Our study unambiguously demonstrates that in vivo supplementation of melatonin to mice with constant light-exposed retinas partially reverses potentially important light-induced protein changes. Since constant light eventually results in retinal dysfunction and degeneration and since human beings are more and more often exposed to light at night, this study provides a stimulus to better understand constant light-induced retinal pathologies as well as whether and how melatonin at night might prevent or alleviate them.
Publications
Jahng, W. J., Cheung, E., Rando, R. R. (2002) Lecithin Retinol Acyltransferase Forms Functional Homodimers, Biochemistry 41, 6311-6319.
Jahng, W. J., David, C., Nesnas, N., Nakanishi, K., Rando, R. R. (2003) A Cleavable Affinity Biotinylating Agent Reveals A Retinoid Binding Role For RPE65, Biochemistry 42, 6159-6168.
Bok, D., Ruiz, A., Yaron, O., Jahng, W. J., Ray, A., Xue, L., Rando, R. R. (2003) Purification and Characterization of a Transmembrane Domain-deleted Form of Lecithin Retinol Acyltransferase, Biochemistry 42, 6090-6098.
Krosky, P. M., Baek, M. C., Jahng, W. J., Barrera, I., Harvey, R. J., Biron, K. K., Coen, D. M., Sethna, P. B. (2003) The Human Cytomegalovirus UL44 Protein is a Substrate for the UL97 Protein Kinase, Journal of Virology 77, 7720-7727.
Jahng, W. J., Xue, L., Rando, R. R. (2003) Lecithin Retinol Acyltransferase Is a Founder Member of a Novel Family of Enzymes, Biochemistry 42, 12805-12812.
Berkowitz, D. B., de la Salud-Bea, R., Jahng, W. J. (2004) Synthesis of Quaternary Amino Acids Bearing a (2’Z)-Fluorovinyl alpha-Branch: Potential PLP Enzyme Inactivators. Organic Letters 6, 1821-1824.
Xue, L., Gollapalli, D., Maiti, P., Jahng, W. J., and Rando, R. R. (2004) A Palmitoylation Switch Mechanism in the Regulation of the Visual Cycle, Cell 117, 761-771.
Furukawa, N., Ongusaha, P., Jahng, W. J., Choi, C. –S., Kim, H.-J., Araki, K., Lee, Y. H. Kaibuchi, K., Kahn, B. B., Masuzaki, H., Kim, J. K., Lee, S. W., Kim, Y.-B. (2005) Role of Rho-kinase in Regulation of Insulin Action and Glucose Homeostasis, Cell Metabolism, 2, 119-129.
Xue, L., Jahng, W. J., Gollapalli, D., Rando, R. R. (2006) The Palmitoyl Transferase Activity of Lecithin Retinol Acyltransferase, Biochemistry 45, 10716-10718.
Jahng, W. J. (2006) Functional Proteomics of the Eye: From Understanding the Visual Cycle to Drug Development, Mol. Cell. Bio. News (Korean Society for Molecular and Cellular Biology), 26, 115-121.
Karukurichi, K. R., de la Salud-Bea, R., Jahng, W. J., Berkowitz, D. B. (2007) Examination of the New -(2’ Z-Fluoro)vinyl Trigger with Lysine Decarboxylase : The Absolute Stereochemistry Dictates the Reaction Course, Journal of the American Chemical Society 129, 258-259.
Kim, J., Jahng, W. J., Vizio, D. D., Lee, J., Rubin, M. A., Shisheva, A., and Freeman, M. R. (2007) The Phosphoinositide Kinase PIKfyve Mediates EGF Receptor Trafficking to the Nucleus, Cancer Research 67, 9229-9237.
Hamirally, S., Kamil, J.P., Ndassa-Colday, Y.M., Lin, A.J., Jahng, W.J., Baek, M.C., Noton, S., Silva, L.A., Simpson-Holley, M., Knipe, D.M., Golan, D.E., Marto, J.A., Coen, D.M. (2009) Viral mimicry of Cdc2/cyclin-dependent kinase 1 mediates disruption of nuclear lamina during human cytomegalovirus nuclear egress. PLoS Pathog. 2009 Jan;5(1):e1000275. Epub 2009 Jan 23.
Chung, H., Lee, H., Lamoke, F., Hrushesky, W.J., Wood, P.A., Jahng, W.J. (2009) Neuroprotective role of erythropoietin by antiapoptosis in the retina. J. Neurosci. Res. 87, 2365-2374. |
![[Michigan Tech - Department of Biological Sciences]](../img/bio3d.jpg)
