Retinal Pigment Epithelium (retinal + pigment_epithelium)

Distribution by Scientific Domains


Selected Abstracts


Photobleaching of Melanosomes from Retinal Pigment Epithelium: I. Effects on Protein Oxidation

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2007
Janice M. Burke
ABSTRACT Melanin in the long-lived melanosomes of the retinal pigment epithelium (RPE) may undergo photobleaching with aging, which appears to diminish the antioxidant function of melanin and could make photobleached melanosomes less efficient in protecting biomolecules from oxidative modification. Here we analyzed whether photobleaching of melanosomes affects their ability to modify the oxidation state of nearby protein. As conventional methods developed to study soluble antioxidants are not well suited for analysis of granules such as melanosomes, we developed a new analytic method to focus on particle surfaces that involves experimentally coating granules with the cytoskeletal protein ,-actin to serve as a reporter for local protein oxidation. Isolated porcine RPE melanosomes were photobleached with visible light to simulate aging, then photobleached melanosomes, untreated melanosomes and control particles (black latex beads) were actin coated and illuminated in a photosensitized cell free system. Protein was re-stripped from particles and analyzed for carbonylation by Western blotting. Quantitative densitometry showed no reproducible differences for protein associated with untreated melanosomes when compared with control particles. Melanin has both anti- and pro-oxidant functions when light irradiated, but neither of these functions predominated in the protein oxidation assay when untreated melanosomes were used. However, protein extracted from photobleached melanosomes showed markedly increased carbonylation, both of associated actin and of endogenous melanosomal protein(s), and the effect increased with extent of granule photobleaching. Photobleaching of RPE melanosomes therefore changes the oxidation state of protein endogenous to the organelle and reduces the ability of the granule to modify the oxidation of exogenous protein near the particle surface. The results support the growing body of evidence that photobleaching of RPE melanosomes, which is believed to occur with aging, changes the physicochemical properties of the organelle and reduces the likelihood that the granules perform an antioxidant function. [source]


Photobleaching of Melanosomes from Retinal Pigment Epithelium: II.

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2007
Effects on the Response of Living Cells to Photic Stress
Melanosomes of the retinal pigment epithelium (RPE) are long lived organelles that may undergo photobleaching with aging, which can diminish the antioxidant efficiency of melanin. Here, isolated porcine RPE melanosomes were experimentally photobleached with visible light to simulate aging and compared with untreated granules or control particles (black latex beads) for their effects on the survival of photically stressed ARPE-19 cultures. Particles were delivered to cultures for uptake by phagocytosis then cells were exposed to violet light and analyzed by a new live cell imaging method to identify the time of apoptotic blebbing as a dynamic measure of reduced cell survival. Results indicated that untreated melanosomes did not decrease photic injury to ARPE-19 cells when compared with cells lacking particles or with cells containing control particles, as might be expected if melanin performed an antioxidant function. Instead cells with untreated melanosomes showed reduced survival indicated by an earlier onset of blebbing and a lower fraction of surviving cells after photic stress. Cell survival was reduced even further in stressed cells containing melanosomes that were photobleached, and survival decreased with increasing photobleaching time. Photobleaching of RPE melanosomes therefore makes cells containing them more sensitive to light-induced cytotoxicity. This observation raises the possibility that aged melanosomes increase RPE cell photic stress in situ, perhaps contributing to reduced tissue function and to degeneration of the adjacent retina that the RPE supports. How melanosomes (photobleached or not) interact with their local subcellular environment to modify RPE cell survival is poorly understood and is likely determined by the physicochemical state of the granule and its constituent melanin. The live cell imaging method introduced here, which permitted detection of a graded effect of photobleaching, provides a sensitive bioassay for probing the effects of melanosome modifications. [source]


Fine Structure of the Retinal Pigment Epithelium, Bruch's Membrane and Choriocapillaris in the Horse

ANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 3 2000
H. Altunay
Summary The fine structure of the retinal pigment epithelium (RPE), Bruch's membrane and choriocapillaris was investigated by light and transmission electron microscopy in both the tapetal and non-tapetal fundus of the horse eye. In all locations, the RPE consisted of a single layer of low cuboidal cells. The epithelial cells were joined laterally by apically located tight junctions. These cells displayed numerous basal infoldings and abundant thin apical processes which enclosed the rod outer segments. The epithelial cell nuclei were large and located basally. Within the epithelial cells, smooth endoplasmic reticulum was very abundant, while rough endoplasmic reticulum was scarce. polysomes and mitochondria, which often display a ring-shaped struccture, were abundant. Melanosomes were abundant in the non-tapetal area but absent in the tapetal area. Bruch's membrane was pentalaminate throughout the retina. The endothelium of the choriocapillaris was heavily fenestrated. [source]


Dissection and cotransplantation of large pieces of RPE and neural retina; effect of protease K on the development

ACTA OPHTHALMOLOGICA, Issue 1 2000
Rajesh Kumar Sharma
ABSTRACT. Purpose: This study attempts to cotransplant large pieces of the RPE and neural retina in the subretinal space of rabbits by using protease K for dissection of the donor tissue, and to investigate the effect of dissection technique on the development of the grafts. Methods: Eyes from 15-day-old pigmented rabbit embryos were partly digested by protease K to assist dissection of sclera and the choroid from RPE and neural retina. Large pieces of RPE and the neural retina thus obtained were cotransplanted into the eyes of adult albino rabbits who were allowed to survive for up to 63 days. The transplants were examined under light microscope. Results: It was possible to transplant large sheets of RPE and neural retina together. Both the RPE and the neural retina survived after cotransplantation. Retinal pigment epithelium survived in layers, but at places formed clusters. In cotransplants neural retina formed rosettes, developed gliosis, and photoreceptors failed to develop outer segments, possibly due to the action of protease K. Conclusion: Proteases seem to be injurious for the development of the neural retina. [source]


The translocation of signaling molecules in dark adapting mammalian rod photoreceptor cells is dependent on the cytoskeleton

CYTOSKELETON, Issue 10 2008
Boris Reidel
Abstract In vertebrate rod photoreceptor cells, arrestin and the visual G-protein transducin move between the inner segment and outer segment in response to changes in light. This stimulus dependent translocation of signalling molecules is assumed to participate in long term light adaptation of photoreceptors. So far the cellular basis for the transport mechanisms underlying these intracellular movements remains largely elusive. Here we investigated the dependency of these movements on actin filaments and the microtubule cytoskeleton of photoreceptor cells. Co-cultures of mouse retina and retinal pigment epithelium were incubated with drugs stabilizing and destabilizing the cytoskeleton. The actin and microtubule cytoskeleton and the light dependent distribution of signaling molecules were subsequently analyzed by light and electron microscopy. The application of cytoskeletal drugs differentially affected the cytoskeleton in photoreceptor compartments. During dark adaptation the depolymerization of microtubules as well as actin filaments disrupted the translocation of arrestin and transducin in rod photoreceptor cells. During light adaptation only the delivery of arrestin within the outer segment was impaired after destabilization of microtubules. Movements of transducin and arrestin required intact cytoskeletal elements in dark adapting cells. However, diffusion might be sufficient for the fast molecular movements observed as cells adapt to light. These findings indicate that different molecular translocation mechanisms are responsible for the dark and light associated translocations of arrestin and transducin in rod photoreceptor cells. Cell Motil. Cytoskeleton 65: 785,800, 2008. © 2008 Wiley-Liss, Inc. [source]


The unconventional myosin-VIIa associates with lysosomes

CYTOSKELETON, Issue 1 2005
Lily E. Soni
Abstract Mutations in the myosin-VIIa (MYO7a) gene cause human Usher disease, characterized by hearing impairment and progressive retinal degeneration. In the retina, myosin-VIIa is highly expressed in the retinal pigment epithelium, where it plays a role in the positioning of melanosomes and other digestion organelles. Using a human cultured retinal pigmented epithelia cell line, ARPE-19, as a model system, we have found that a population of myosin-VIIa is associated with cathepsin D- and Rab7-positive lysosomes. Association of myosin-VIIa with lysosomes was Rab7 independent, as dominant negative and dominant active versions of Rab7 did not disrupt myosin-VIIa recruitment to lysosomes. Association of myosin-VIIa with lysosomes was also independent of the actin and microtubule cytoskeleton. Myosin-VIIa copurified with lysosomes on density gradients, and fractionation and extraction experiments suggested that it was tightly associated with the lysosome surface. These studies suggest that myosin-VIIa is a lysosome motor. Cell Motil. Cytoskeleton 62:13,26, 2005. © 2005 Wiley-Liss, Inc. [source]


Complete reconstruction of the retinal laminar structure from a cultured retinal pigment epithelium is triggered by altered tissue interaction and promoted by overlaid extracellular matrices

DEVELOPMENTAL NEUROBIOLOGY, Issue 14 2009
Fusako Kuriyama
Abstract The retina regenerates from retinal pigment epithelial (RPE) cells by transdifferentiation in the adult newt and Xenopus laevis when it is surgically removed. This was studied under a novel culture condition, and we succeeded, for the first time, in developing a complete retinal laminar structure from a single epithelial sheet of RPE. We cultured a Xenopus RPE monolayer sheet isolated from the choroid on a filter cup with gels overlaid and found that the retinal tissue structure differentiated with all retinal layers present. In the culture, RPE cells isolated themselves from the culture substratum (filter membrane), migrated, and reattached to the overlaid gel, on which they initiated transdifferentiation. This was exactly the same as observed during in vivo retina regeneration of X. laevis. In contrast, when RPE monolayers were cultured similarly without isolation from the choroid, RPE cells proliferated, but remained pigmented instead of transdifferentiating, indicating that alteration in tissue interaction triggers transdifferentiation. We then examined under the conventional tissue culture condition whether altered RPE-choroid interaction induces Pax6 expression. Pax6 was upregulated in RPE cells soon after they were removed from the choroid, and this expression was not dependent of FGF2. FGF2 administration was needed for RPE cells to maintain Pax6 expression. From the present results, in addition to our previous ones, we propose a two-step mechanism of transdifferentiation: the first step is a reversible process and is initiated by the alteration of the cell-extracellular matrix and/or cell,cell interaction followed by Pax6 upregulation. FGF2 plays a key role in driving RPE cells into the second step, during which they differentiate into retinal stem cells. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009 [source]


Differential effect of dopamine on mitosis in early postnatal albino and pigmented rat retinae

DEVELOPMENTAL NEUROBIOLOGY, Issue 1 2006
Ines Kralj-Hans
Abstract Insufficient levels of L -DOPA, released from the retinal pigment epithelium (RPE), in albino animals are considered responsible for the abnormal development of the underlying neural retina. L -DOPA normalizes retinal neurogenesis by reducing levels of cell proliferation either by acting on the cells directly or by being converted into dopamine. Here we report the effects of dopamine on mitosis in early postnatal neural retinae from albino and pigmented rats, using 4D (x, y, z and time) confocal microscopy. Exogenous dopamine significantly prolongs mitosis in retinae from albino, but not pigmented, animals. As fewer cells move into and divide in the ventricular zone (VZ) in the presence of dopamine, we conclude that the overall cell cycle is affected. The D1 receptor blocker, SCH 23390, inhibits these effects. Thus, the differential effects of dopamine on neural retinae from pigmented and albino rats in vitro must result from the activation of D1 receptors, which are present in the retina from birth. Immunohistochemical labeling of D1 receptors shows that the pattern of their distribution is similar between pigmentation phenotypes, but levels of expression may be elevated in albinos. Labeling is most intense in the inner plexiform layer but is present throughout the neuroblastic layer. These findings are discussed in light of previous reports of reduced catecholamine levels in the albino retina. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2006 [source]


Age-related changes in the dynamics of human albino visual pathways

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2003
Magella M. Neveu
Abstract A deficiency of melanin in the retinal pigment epithelium, which regulates the development of neural retina, leads to chiasmal misrouting such that the uncrossed pathway (to the ipsilateral hemisphere) is reduced relative to the crossed pathway (to the contralateral hemisphere). This study examines age-related changes in the flash and pattern appearance visual evoked potentials (VEP) of human albinos. Scalp recorded cortical VEPs to flash (FVEP) and pattern appearance stimulation were recorded in 58 albino (8 months to 60 years) and 34 normal subjects (4,55 years). VEPs were analysed by amplitude and latency. The contralateral hemisphere FVEP amplitude decreased with age in albino subjects, as in both hemispheres in normals. However, the ipsilateral hemisphere FVEP amplitude was significantly lower in young albino subjects, initially giving a marked interhemispheric asymmetry, but this normalized with age. Significant interhemispheric FVEP latency asymmetries were not observed. The contralateral pattern appearance VEP latency in albino subjects decreased with age, as in both hemispheres in normals; the ipsilateral latency increased significantly with age. Significant interhemispheric pattern appearance VEP amplitude asymmetries were not observed. These novel and unexpected observations indicate significant age-related changes in the retinocortical pathways of the human albino. These changes have implications for our understanding of development and plasticity of the central visual pathways. [source]


Pigment epithelium-derived factor supports normal Müller cell development and glutamine synthetase expression after removal of the retinal pigment epithelium

GLIA, Issue 1 2001
Monica M. Jablonski
Abstract In conditions in which the retinal pigment epithelium (RPE) is dystrophic, carries a genetic mutation, or is removed physically, Müller cells undergo degenerative changes that contribute to the retinal pathology. We previously demonstrated that pigment epithelium-derived factor (PEDF), a glycoprotein secreted by the RPE cells with neuroprotective and differentiation properties, protects against photoreceptor degeneration induced by RPE removal. The purpose of the present study was to analyze the putative gliosupportive activity of PEDF on Müller cells of RPE-deprived retinas and assess whether protection of Müller cells was correlated with improved photoreceptor outer segment assembly. Eyes were dissected from Xenopus laevis tadpoles, and the RPE was removed before culturing in medium containing purified PEDF, PEDF plus anti-PEDF, or medium alone. Control eyes matured with an adherent RPE or in medium containing PEDF plus nonimmune serum. Müller cell ultrastructure was examined. Glial fibrillary acidic protein (GFAP) and glutamine synthetase were localized immunocytochemically, and the corresponding protein levels were quantified. In control retinas, Müller cells were structurally intact and formed adherens junctions with neighboring photoreceptors. In addition, they did not express GFAP, whereas glutamine synthetase expression was high. RPE removal dramatically altered the ultrastructure and biosynthetic activity of Müller cells; Müller cells failed to form adherens junctions with photoreceptors and glutamine synthetase expression was suppressed. PEDF prevented the degenerative glial response; Müller cells were ultrastructurally normal and formed junctional complexes with photoreceptors. PEDF also preserved the expression of glutamine synthetase at near-normal levels. The morphogenetic effects of PEDF were blocked by the anti-PEDF antibody. Our study documents the glioprotective effects of PEDF and suggests that maintenance of the proper Müller cell ultrastructure and expression of glutamine synthetase may be necessary to support the proper assembly of photoreceptor outer segments. GLIA 35:14,25, 2001. © 2001 Wiley-Liss, Inc. [source]


CRB1 mutation spectrum in inherited retinal dystrophies,

HUMAN MUTATION, Issue 5 2004
Anneke I. den Hollander
Abstract Mutations in the Crumbs homologue 1 (CRB1) gene have been reported in patients with a variety of autosomal recessive retinal dystrophies, including retinitis pigmentosa (RP) with preserved paraarteriolar retinal pigment epithelium (PPRPE), RP with Coats-like exudative vasculopathy, early onset RP without PPRPE, and Leber congenital amaurosis (LCA). We extended our investigations of CRB1 in these retinal dystrophies, and identified nine novel CRB1 sequence variants. In addition, we screened patients with "classic" RP and classic Coats disease (without RP), but no pathologic sequence variants were found in the CRB1 gene. In total, 71 different sequence variants have been identified on 184 CRB1 alleles of patients with retinal dystrophies, including amino acid substitutions, frameshift, nonsense, and splice site mutations, in-frame deletions, and large insertions. Recent studies in two animal models, mouse and Drosophila, and in vivo high-resolution microscopy in patients with LCA, have shed light on the role of CRB1 in the pathogenesis of retinal dystrophies and its function in the photoreceptors. In this article, we provide an overview of the currently known CRB1 sequence variants, predict their effect, and propose a genotype,phenotype correlation model for CRB1 mutations. Hum Mutat 24:355,369, 2004. © 2004 Wiley-Liss, Inc. [source]


Subunits of the epithelial sodium channel family are differentially expressed in the retina of mice with ocular hypertension

JOURNAL OF NEUROCHEMISTRY, Issue 1 2005
Frank M. Dyka
Abstract Glaucoma is a prevalent cause of blindness, resulting in the apoptotic death of retinal ganglion cells and optic nerve degeneration. The disease is often associated with elevated intraocular pressure, however, molecular mechanisms involved in ganglion cell death are poorly understood. To identify proteins contributing to this pathological process, we analysed the retinal gene expression of DBA/2J mice that develop an elevated intraocular pressure by the age of 6 months with subsequent ganglion cell loss. In this study, we identified subunits of the epithelial sodium channel (ENaC) family that are specifically expressed under elevated intraocular pressure. Using reverse transcriptase polymerase chain reaction we observed a significant increase of ,-ENaC in the neuronal retina of DBA/2J mice when compared with control animals, while ,-ENaC and ,-ENaC were not detectable in this tissue. Specific immune sera to ENaC subunits showed up-regulation of ,-ENaC in synaptic and nuclear layers of the retina, and in the retinal pigment epithelium. Consistent with our polymerase chain reaction data, ,-ENaC was not detected by specific antibodies in the retina, while ,-ENaC was only present in the retinal pigment epithelium under ocular hypertension. Finally, the increase of ,-ENaC gene expression in the neuronal retina and the retinal pigment epithelium was not observed in other tissues of DBA/2J mice. Since the intraocular pressure is regulated by the transport of aqueous humour across epithelial structures of the eye that in turn is associated with ion flux, the specific up-regulation of ENaC proteins could serve as a protecting mechanism against elevated intraocular pressure. [source]


Targeting of the retinal pigment epithelium (RPE) by means of a rapidly scanned continuous wave (CW) laser beam

LASERS IN SURGERY AND MEDICINE, Issue 4 2003
Ralf Brinkmann
Abstract Background and Objectives Selective treatment of the retinal pigment epithelium (RPE) by repetitively applying green ,s-laser pulses is a new method for retinal diseases associated with a degradation of the RPE, which spares the neural retina. We investigated an alternative approach to realize repetitive ,s-laser exposure by rapidly scanning a continuous wave (CW)-laser beam across the RPE. Study Design/Materials and Methods An Ar+ laser beam (514 nm) with a diameter of 18.75 ,m was repetitively scanned across porcine RPE samples in vitro providing an irradiation time of 1.6 ,s per point on the central scan axis. RPE cell damage was investigated by means of the fluorescence viability assay Calcein-AM. Results The ED50 cell damage is 305 mJ/cm2 when applying 10 scans with a repetition rate of 500 Hz. The threshold decreases with the number of scans, a saturation was found at 135 mJ/cm2 with more than 500 exposures applied. The depth of focus in beam direction is 350 ,m, defined by an increase of the threshold radiant exposure by 20%. Conclusions Targeting of pigmented cells with high local resolution has been proved with a laser-scanning device. Looking ahead selective RPE-treatment, the adaptation of a laser-scanning device on a slit-lamp or into a modified retina angiograph seems to be an attractive alternative to the pulsed ,s laser device. Lasers Surg. Med. 32:252,264, 2003. © 2003 Wiley-Liss, Inc. [source]


Towards metabolic mapping of the human retina

MICROSCOPY RESEARCH AND TECHNIQUE, Issue 5 2007
D. Schweitzer
Abstract Functional alterations are first signs of a starting pathological process. A device that measures parameter for the characterization of the metabolism at the human eye-ground would be a helpful tool for early diagnostics in stages when alterations are yet reversible. Measurements of blood flow and of oxygen saturation are necessary but not sufficient. The new technique of auto-fluorescence lifetime measurement (FLIM) opens in combination with selected excitation and emission ranges the possibility for metabolic mapping. FLIM not only adds an additional discrimination parameter to distinguish different fluorophores but also resolves different quenching states of the same fluorophore. Because of its high sensitivity and high temporal resolution, its capability to resolve multi-exponential decay functions, and its easy combination with laser scanner ophthalmoscopy, multi-dimensional time-correlated single photon counting was used for fundus imaging. An optimized set up for in vivo lifetime measurements at the human eye-ground will be explained. In this, the fundus fluorescence is excited at 446 or 468 nm and the time-resolved autofluorescence is detected in two spectral ranges between 510 and 560 nm as well as between 560 and 700 nm simultaneously. Exciting the fundus at 446 nm, several fluorescence maxima of lifetime t1 were detected between 100 and 220 ps in lifetime histograms of 40° fundus images. In contrast, excitation at 468 nm results in a single maximum of lifetime t1 = 190 ± 16 ps. Several fundus layers contribute to the fluorescence intensity in the short-wave emission range 510,560 nm. In contrast, the fluorescence intensity in the long-wave emission range between 560 and 700 nm is dominated by the fluorescence of lipofuscin in the retinal pigment epithelium. Comparing the lateral distribution of parameters of a tri-exponential model function in lifetime images of the fundus with the layered anatomical fundus structure, the shortest component (t1 = 190 ps) originates from the retinal pigment epithelium and the second lifetime (t2 = 1,000 ps) from the neural retina. The lifetime t3 , 5.5 ns might be influenced by the long decay of the fluorescence in the crystalline lens. In vitro analysis of the spectral properties of expected fluorophores under the condition of the living eye lightens the interpretation of in vivo measurements. Taking into account the transmission of the ocular media, the excitation of NADH is unlikely at the fundus. Microsc. Res. Tech., 2007. © 2007 Wiley-Liss, Inc. [source]


Lipofuscin and Macular Degeneration

NUTRITION REVIEWS, Issue 10 2003
George Wolf DPhil
The accumulation of the autofluorescent pigment lipofuscin in the retina that occurs with aging has been explained as a side effect of the visual cycle. It occurs when two molecules of all- trans -retinal condense with one molecule of phosphatidylethanolamine in the discs of the rod outer segments, and is followed by uptake into retinal pigment epithelium (RPE) and conversion to the stable A2E, a pyridinium bisretinoid that is toxic to RPE cells. The accumulation of A2E, the major component of lipofuscin causes RPE cell apoptosis, thereby explaining age-related macular degeneration and macular degeneration characteristic of Stargardt disease. The drug isotretinoin (13- cis - retinoic acid) prevents accumulation of A2E in mice by slowing down the visual cycle and might therefore be used to prevent macular degeneration. [source]


Functional Expression, Targeting and Ca2+ Signaling of a Mouse Melanopsin-eYFP Fusion Protein in a Retinal Pigment Epithelium Cell Line,

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008
Maikel E. Giesbers
Melanopsin, first discovered in Xenopus melanophores, is now established as a functional sensory photopigment of the intrinsically photosensitive retinal ganglion cells. These ganglion cells drive circadian rhythm and pupillary adjustments through projection to the brain. Melanopsin shares structural similarities with all known opsins. Comprehensive characterization of melanopsin with respect to its spectral properties, photochemical cascade and signaling partners requires a suitable recombinant system and high expression levels. This combination has not yet been described. To address this issue, we have expressed recombinant mouse melanopsin in several cell lines. Using enhanced yellow fluorescent protein (eYFP) as a visualization tag, expression was observed in all cell lines. Confocal microscopy revealed that melanopsin was properly routed to the plasma membrane only in retinal pigment epithelium (RPE)-derived D407 cells and in human embryonic kidney (HEK) cells. Further, we performed intracellular calcium measurements in order to probe the melanopsin signaling activity of this fusion protein. Transfected cells were loaded with the calcium indicator Fura2-AM. Upon illumination, an immediate but transient calcium response was observed in HEK as well as in D407 cells, while mock-transfected cells showed no calcium response under identical conditions. Supplementation with 11- cis retinal or all- trans retinal enhanced the response. After prolonged illumination the cells became desensitized. Thus, RPE-derived cells expressing recombinant melanopsin may constitute a suitable system for the study of the structural and functional characteristics of melanopsin. [source]


Photoprotection by Porcine Eumelanin Against Singlet Oxygen Production,

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 3 2008
Alice Wang
Melanin, a major pigment found in retinal pigment epithelium (RPE) cells, is considered to function in dual roles, one protective and one destructive. By quenching free radical species and reactive oxygen species (ROS) melanin counteracts harmful redox stress. However, melanin is also thought to be capable of creating ROS. In this destructive role, melanin increases redox strain in the cell. This study uses readily available eumelanin extracted from porcine RPE cells as a more authentic model than synthetic melanin to determine specific mechanisms of melanin activity with regard to singlet oxygen in the presence and absence of rose bengal, a singlet-oxygen photosensitizer. Optical detection of singlet-oxygen was determined by monitoring the bleaching of p -nitrosodimethylaniline in the presence of histidine. Production of singlet oxygen in aqueous oxygen-saturated solutions of rose bengal without eumelanin was readily accomplished. In contrast, detection of singlet oxygen in oxygen-saturated solutions of eumelanin without rose bengal failed, consistent with results of others. However, a significant decrease in singlet oxygen production by rose bengal was observed in the presence of eumelanin. After correction for light absorption and chemical bleaching of eumelanin, the results show that eumelanin also provides a photoprotective mode arising from chemistry, that is, not just the physical process of light absorption followed by energy dissipation as heat. [source]


Photobleaching of Melanosomes from Retinal Pigment Epithelium: I. Effects on Protein Oxidation

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2007
Janice M. Burke
ABSTRACT Melanin in the long-lived melanosomes of the retinal pigment epithelium (RPE) may undergo photobleaching with aging, which appears to diminish the antioxidant function of melanin and could make photobleached melanosomes less efficient in protecting biomolecules from oxidative modification. Here we analyzed whether photobleaching of melanosomes affects their ability to modify the oxidation state of nearby protein. As conventional methods developed to study soluble antioxidants are not well suited for analysis of granules such as melanosomes, we developed a new analytic method to focus on particle surfaces that involves experimentally coating granules with the cytoskeletal protein ,-actin to serve as a reporter for local protein oxidation. Isolated porcine RPE melanosomes were photobleached with visible light to simulate aging, then photobleached melanosomes, untreated melanosomes and control particles (black latex beads) were actin coated and illuminated in a photosensitized cell free system. Protein was re-stripped from particles and analyzed for carbonylation by Western blotting. Quantitative densitometry showed no reproducible differences for protein associated with untreated melanosomes when compared with control particles. Melanin has both anti- and pro-oxidant functions when light irradiated, but neither of these functions predominated in the protein oxidation assay when untreated melanosomes were used. However, protein extracted from photobleached melanosomes showed markedly increased carbonylation, both of associated actin and of endogenous melanosomal protein(s), and the effect increased with extent of granule photobleaching. Photobleaching of RPE melanosomes therefore changes the oxidation state of protein endogenous to the organelle and reduces the ability of the granule to modify the oxidation of exogenous protein near the particle surface. The results support the growing body of evidence that photobleaching of RPE melanosomes, which is believed to occur with aging, changes the physicochemical properties of the organelle and reduces the likelihood that the granules perform an antioxidant function. [source]


Photobleaching of Melanosomes from Retinal Pigment Epithelium: II.

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2007
Effects on the Response of Living Cells to Photic Stress
Melanosomes of the retinal pigment epithelium (RPE) are long lived organelles that may undergo photobleaching with aging, which can diminish the antioxidant efficiency of melanin. Here, isolated porcine RPE melanosomes were experimentally photobleached with visible light to simulate aging and compared with untreated granules or control particles (black latex beads) for their effects on the survival of photically stressed ARPE-19 cultures. Particles were delivered to cultures for uptake by phagocytosis then cells were exposed to violet light and analyzed by a new live cell imaging method to identify the time of apoptotic blebbing as a dynamic measure of reduced cell survival. Results indicated that untreated melanosomes did not decrease photic injury to ARPE-19 cells when compared with cells lacking particles or with cells containing control particles, as might be expected if melanin performed an antioxidant function. Instead cells with untreated melanosomes showed reduced survival indicated by an earlier onset of blebbing and a lower fraction of surviving cells after photic stress. Cell survival was reduced even further in stressed cells containing melanosomes that were photobleached, and survival decreased with increasing photobleaching time. Photobleaching of RPE melanosomes therefore makes cells containing them more sensitive to light-induced cytotoxicity. This observation raises the possibility that aged melanosomes increase RPE cell photic stress in situ, perhaps contributing to reduced tissue function and to degeneration of the adjacent retina that the RPE supports. How melanosomes (photobleached or not) interact with their local subcellular environment to modify RPE cell survival is poorly understood and is likely determined by the physicochemical state of the granule and its constituent melanin. The live cell imaging method introduced here, which permitted detection of a graded effect of photobleaching, provides a sensitive bioassay for probing the effects of melanosome modifications. [source]


Origin of the Vertebrate Visual Cycle: III.

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 6 2006
-Monooxygenase Homologues in Ciona intestinalis, -carotene 1, Distinct Distribution of RPE6
We previously identified three genes that encode putative visual cycle proteins that are homologues of retinal G-protein coupled receptor (Ci-opsin3), cellular retinaldehyde-binding protein (Ci-CRALBP) and ,-carotene 15,15,-monooxygenase (Ci-BCO) in the ascidian Ciona intestinalis. Ci-opsin3 and Ci-CRALBP are localized in both ocellus photoreceptor cells and surrounding non-photoreceptor cells in the brain vesicle of the larva. In the present study, we investigated the possible role and evolutionary origin of the BCO/RPE65 family in the visual cycle by analyzing Ci-BCO localization by immunohistochemistry and by identifying a novel gene that encodes a homologue of retinal pigment epithelium,specific 65 kDa protein (Ci-RPE65) in C. intestinalis. In situ hybridization and expressed sequence tag (EST) profiles consistently suggest that Ci-RPE65 is not significantly expressed in the ocellus and brain vesicle of the larva. Ci-RPE65 is expressed in the neural complex, a photoreceptor organ of the adult ascidian, at a level comparable to that of Ci-opsin3 and Ci-CRALBP. Ci-RPE65 is also expressed in various adult tissues, including the gill, body wall and intestine, suggesting that Ci-RPE65 plays a role in addition to that in the visual cycle. In contrast, Ci-BCO is predominantly localized in ocellus photoreceptor cells of the larva. The larval visual cycle seems to use Ci-opsin3 as a photoisomerase. Our results also suggest that the RPE65-dependent visual cycle is used in the adult photoreceptors of a primitive chordate. [source]


Effect of Visible Light on Normal and P23H-3 Transgenic Rat Retinas: Characterization of a Novel Retinoic Acid Derivative Present in the P23H-3 Retina

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 3 2006
Todd Duncan
ABSTRACT Transgenic rats with the P23H mutation in rhodopsin exhibit increased susceptibility to light damage, compared with normal animals. It is known that light-induced retinal damage requires repetitive bleaching of rhodopsin and that photoreceptor cell loss is by apoptosis; however, the underlying molecular mechanism(s) leading to photoreceptor cell death are still unknown. Photoproducts, such as all- trans retinal or other retinoid metabolites, released by the extensive bleaching of rhodopsin could lead to activation of degenerative processes, especially in animals genetically predisposed to retinal degenerations. Using wild-type and transgenic rats carrying the P23H opsin mutation, we evaluated the effects of acute intense visible light on retinoid content, type and distribution in ocular tissues. Rats were exposed to green light (480,590 nm) for 0, 5, 10, 30 and 120 min. Following light treatment, rats were sacrificed and neural retinas were dissected free of the retinal pigment epithelium. Retinoids were extracted from retinal tissues and then subjected to HPLC and mass spectral analysis. We found that the light exposure affected relative levels of retinoids in the neural retina and retinal pigment epithelium of wild-type and P23H rat eyes similarly. In the P23H rat retina but not the wild-type rat retina, we found a retinoic acid-like compound with an absorbance maximum of 357 nm and a mass of 304 daltons. Production of this retinoic acid-like compound in transgenic rats is influenced by the age of the animals and the duration of light exposure. It is possible that this unique retinoid may be involved in the process of light-induced retinal degeneration. [source]


Time-resolved Microspectrofluorimetry and Fluorescence Lifetime Imaging of Hypericin in Human Retinal Pigment Epithelial Cells,

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 3 2005
Paola Taroni
ABSTRACT Hypericin is the active ingredient of the off-the-shelf antidepressant St. John's Wort. It is an effective phototoxic agent and its systemic administration at therapeutic doses could induce particular damage in the eye due to continuous light exposure. Hypercin is strongly fluorescent and its fluorescence properties can be monitored to investigate noninvasively its localization and interactions. To this aim, time-resolved microspectrofluorimetry and fluorescence life-time imaging were used to assess the spectral and temporal properties as well as the spatial distribution of the fluorescence emitted by retinal pigment epithelium (RPE) cells treated with Hyp at concentrations in the micromolar range (0.5,10 ,M). In the presence of hypericin, the emission peaks at 600-605 nm and the fluorescence decay is best fitted with three lifetimes (5.5-7 ns, 1.9-2.5 ns and < 0.8 ns). Spectral and temporal differences were observed between high (,5 ,M) and low hypericin concentrations. In particular, upon increasing concentration, the emission spectrum of the slow component broadens and its lifetime shortens. The latter change is observed also when high concentrations are reached locally, due to more efficient localization within the cell. [source]


Anthocyanins Protect Against A2E Photooxidation and Membrane Permeabilization in Retinal Pigment Epithelial Cells,

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 3 2005
Young P. Jang
ABSTRACT The pyridinium bisretinoid A2E, an autofluorescent pigment that accumulates in retinal pigment epithelial cells with age and in some retinal disorders, can mediate a detergent-like perturbation of cell membranes and light-induced damage to the cell. The photodynamic events initiated by the sensitization of A2E include the generation of singlet oxygen and the oxidation of A2E at carbon-carbon double bonds. To assess the ability of plant-derived anthocyanins to modulate adverse effects of A2E accumulation on retinal pigment epithelium (RPE) cells, these flavylium salts were isolated from extracts of bilberry. Nine anthocyanin fractions reflecting monoglycosides of delphinidin, cyanidin, petunidin and malvidin were obtained and all were shown to suppress the photooxidation of A2E at least in part by quenching singlet oxygen. The anthocyanins tested exhibited antioxidant activity of variable efficiency. The structural characteristics relevant to this variability likely included the ability to form a stable quinonoidal anhydro base at neutral pH, a conjugated diene structure in the C (pyrane) ring, the presence of hydroxyl groups on the B (benzene) ring and the relative hydrophobicity conferred by the arrangement of substituents on the B ring. Cells that had taken up anthocyanins also exhibited a resistance to the membrane permeabilization that occurs as a result of the detergent-like action of A2E. [source]


Environmental Effects on the Photochemistry of A2-E, a Component of Human Retinal Lipofuscin,

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 3 2001
Laura Ragauskaite
ABSTRACT Several retinal dystrophies are associated with the accumulation of lipofuscin, a pigment mixture, in the retinal pigment epithelium (RPE). One of the major fluorophores of this mixture has been identified as the bis-retinoid pyridinium compound, A2-E. Because this compound absorbs incident radiation that is transmitted by the anterior segment of the human eye, photophysical and photochemical studies were performed to determine if A2-E could photosensitize potentially damaging reactions. Steady-state fluorescence measurements indicate that the fluorescence emission maximum and quantum yield are very sensitive to the chemical environment and a correlation between these two parameters and the solvent dielectric constant is observed. Time-resolved absorption experiments of A2-E in pure organic solvents showed no formation of transient species on the timescale of our experiments. However, when these measurements were repeated for A2-E in Triton X-100 micelles, a short-lived (,, 14 ,s), weak absorption was observed. This species is quenched by oxygen (k= 2 × 109M,1 s,1) and by the addition of the antioxidants, cysteine and N,N,N,,N, -tetramethylphenylenediamine. Quenching of this species by 2,3,5-trimethylhydroquinone results in the formation of the 2,3,5-trimethylsemiquinone free radical and an increase in yield of the A2-E,derived species. Sensitization of the A2-E triplet excited state indicates that the species observed in micelles upon direct excitation is not consistent with the triplet excited state. Based on these data we tentatively assign this absorption to a free radical. In the RPE these initial processes can ultimately lead to damage to the tissue through the formation of peroxides and other oxidized species. [source]


Pigmentary function and evolution of tyrp1 gene duplicates in fish

PIGMENT CELL & MELANOMA RESEARCH, Issue 6 2009
Ingo Braasch
Summary The function of the tyrosinase-related protein 1 (Tyrp1) has not yet been investigated in vertebrates basal to tetrapods. Teleost fishes have two duplicates of the tyrp1 gene. Here, we show that the teleost tyrp1 duplicates have distributed the ancestral gene expression in the retinal pigment epithelium (RPE) and melanophores in a species-specific manner. In medaka embryos, tyrp1a expression is found in the RPE and in melanophores while tyrp1b is only expressed in melanophores. In zebrafish embryos, expression of tyrp1 paralogs overlaps in the RPE and in melanophores. Knockdown of each zebrafish tyrp1 duplicate alone does not show pigmentary defects, but simultaneous knockdown of both tyrp1 genes results in the formation of brown instead of black eumelanin accompanied by severe melanosome defects. Our study suggests that the brown melanosome color in Tyrp1-deficient vertebrates is an effect of altered eumelanin synthesis. Black eumelanin formation essentially relies on the presence of Tyrp1 and some of its function is most likely conserved from the common ancestor of bony vertebrates. [source]


MEK mediates in vitro neural transdifferentiation of the adult newt retinal pigment epithelium cells: Is FGF2 an induction factor?

PIGMENT CELL & MELANOMA RESEARCH, Issue 5 2007
Kanako Susaki
Summary Adult newts can regenerate their entire retinas through transdifferentiation of the retinal pigment epithelium (RPE) cells. As yet, however, underlying molecular mechanisms remain virtually unknown. On the other hand, in embryonic/larval vertebrates, an MEK [mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase] pathway activated by fibroblast growth factor-2 (FGF2) is suggested to be involved in the induction of transdifferentiation of the RPE into a neural retina. Therefore, we examined using culture systems whether the FGF2/MEK pathway is also involved in the adult newt RPE transdifferentiation. Here we show that the adult newt RPE cells can switch to neural cells expressing pan-retinal-neuron (PRN) markers such as acetylated tubulin, and that an MEK pathway is essential for the induction of this process, whereas FGF2 seems an unlikely primary induction factor. In addition, we show by immunohistochemistry that the PRN markers are not expressed until the 1,3 cells thick regenerating retina, which contains retinal progenitor cells, appears. Our current results suggest that the activation of an MEK pathway in RPE cells might be involved in the induction process of retinal regeneration in the adult newt, however if this is the case, we must assume complementary mechanisms that repress the MEK-mediated misexpression of PRN markers in the initial process of transdifferentiation. [source]


Expression of Cre Recombinase in Pigment Cells

PIGMENT CELL & MELANOMA RESEARCH, Issue 4 2002
Laurence Guyonneau
Conditional gene targeting using the Cre/loxP system enables specific deletion of a gene in a tissue of interest. For application of Cre-mediated recombination in pigment cells, Cre expression has to be targeted to pigment cells in transgenic mice. So far, no pigment cell-specific Cre transgenic line has been reported and we present and discuss our first results on use of Cre recombinase in pigment cells. A construct was generated where Cre recombinase is controlled by the promoter of the mouse dopachrome tautomerase (Dct) gene. The construct was functionally tested in vitro and introduced into mice. Following breeding to two reporter mouse strains, we detected Cre recombinase activity in telencephalon, melanoblasts, and retinal pigment epithelium (RPE). Our data demonstrate the feasibility of pigment cell-specific Cre/loxP-mediated recombination. [source]


Architecture of cannabinoid signaling in mouse retina

THE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 18 2010
Sherry Shu-Jung Hu
Abstract Cannabinoid receptors and their ligands constitute an endogenous signaling system that is found throughout the body, including the eye. This system can be activated by ,9 -tetrahydrocannabinol, a major drug of abuse. Cannabinoids offer considerable therapeutic potential in modulating ocular immune and inflammatory responses and in regulating intraocular pressure. The location of cannabinoid receptor 1 (CB1) in the retina is known, but recently a constellation of proteins has been identified that produce and break down endocannabinoids (eCBs) and modulate CB1 function. Localization of these proteins is critical to defining specific cannabinoid signaling circuitry in the retina. Here we show the localization of diacylglycerol lipase-, and -, (DGL,/,), implicated in the production of the eCB 2-arachidonoyl glycerol (2-AG); monoacylglycerol lipase (MGL) and ,/,-hydrolase domain 6 (ABHD6), both implicated in the breakdown of 2-AG; cannabinoid receptor-interacting protein 1a (CRIP1a), a protein that may modulate CB1 function; and fatty acid amide hydrolase (FAAH) and N -acylethanolamine-hydrolyzing acid amidase (NAAA), which have been shown to break down the eCB anandamide and related acyl amides. Our most prominent finding was that DGL, is present in postsynaptic type 1 OFF cone bipolar cells juxtaposed to CB1 -containing cone photoreceptor terminals. CRIP1a is reliably presynaptic to DGL,, consistent with a possible role in cannabinoid signaling, and NAAA is restricted to retinal pigment epithelium, whereas DGL, is limited to retinal blood vessels. These results taken together with previous anatomical and functional studies define specific cannabinoid circuitry likely to modulate eCB signaling at the first synapse of the retina as well as in the inner plexiform layer. J. Comp. Neurol. 518:3848,3866, 2010. © 2010 Wiley-Liss, Inc. [source]


Development of photoreceptor-specific promoters and their utility to investigate EIAV lentiviral vector mediated gene transfer to photoreceptors

THE JOURNAL OF GENE MEDICINE, Issue 12 2007
Marjorie Nicoud
Abstract Background We wanted to investigate the ability of recombinant equine infectious anemia virus (EIAV) vectors to transduce photoreceptor cells by developing a series of photoreceptor-specific promoters that drive strong gene expression in photoreceptor cells. Methods Promoter fragments derived from the rhodopsin (RHO), the beta phosphodiesterase (PDE) and the retinitis pigmentosa (RP1) genes were cloned in combination with an enhancer element, derived from the interphotoreceptor retinoid-binding protein gene (IRBP), into luciferase reporter plasmids. An in vitro transient reporter assay was carried out in the human Y-79 retinoblastoma cell line. The optimal promoters from this screen were then cloned into the recombinant EIAV vector for evaluation in vivo following subretinal delivery into mice. Results All promoters maintained a photoreceptor-specific expression profile in vitro and the gene expression was further enhanced in combination with the IRBP enhancer. The use of IRBP-combined RHO or PDE promoters showed modest but exclusive expression in photoreceptors following subretinal delivery to mice. By contrast an EIAV vector containing the cytomegalovirus (CMV) promoter drove reporter gene expression in both photoreceptors and retinal pigment epithelium. Conclusions It may be possible to use recombinant EIAV vectors containing photoreceptor-specific promoters to drive therapeutic gene expression to treat a range of retinal degenerative diseases where the photoreceptor cell is the primary disease target. Copyright © 2007 John Wiley & Sons, Ltd. [source]


Comparison of wild-type and class I integrase mutant-FIV vectors in retina demonstrates sustained expression of integrated transgenes in retinal pigment epithelium

THE JOURNAL OF GENE MEDICINE, Issue 12 2003
Nils Loewen
Abstract Background In neonatal and adult rodent retina, substantial lentiviral vector expression has been detected primarily in retinal pigment epithelium (RPE), except in very young animals (2,5 days post-natal). In non-retinal tissues, studies of lentiviral vectors have utilized various controls. Among the most stringent are class I integrase mutants, which selectively block the integration reaction while leaving all other gag/pol -encoded functions intact. For HIV-1 vectors injected into brain, these have been used to simultaneously control for pseudotransduction and verify that long-term expression requires integration. Such experiments compare particles that differ only in a single amino acid within a single enzyme that forms a very small molar fraction of the virion. Class I integrase mutants have not been described for feline immunodeficiency virus (FIV) integrase, or tested in the eye for any lentiviral vector. Methods We compared subretinally and intravitreally injected FIV vectors and followed animals for up to 7 months, a duration that exceeds prior studies. We also compared the wild-type (WT) vector with one incorporating a single class I amino acid mutation in FIV integrase (D66V). A mock vector (packaging construct absent) was an alternative control. All vectors were vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped and were injected on day 7 of life. One group of animals received either subretinal or intravitreal injections of WT vector in the right eyes. Control left eyes were injected with mock vector. These animals were sacrificed at 2 or 7 days post-injection. A second group received subretinal injections of either WT vector or equivalent D66V vector (reverse transcriptase-normalized to WT), and were analyzed after 2, 3 and 7 months. All eyes were scored for marker gene (,-galactosidase) expression by an observer blinded to vector assignments. Results Subretinal FIV vector injections were much more effective than intravitreal injections. The RPE was the principal retinal layer transduced by the WT vector, and at least 50% of the area of the retina expressed the marker gene at 3 and 7 months. Occasional cells in inner retinal layers also expressed ,-galactosidase at these time points. The sustained retinal expression produced by subretinally injected vector was blocked by the D66V mutation. Conclusions These results show that class I integrase mutant FIV vectors are useful control vectors, and that VSV-G-pseudotyped FIV vectors produce extensive retinal expression for at least 215 days, the longest duration yet reported for lentiviral vectors in retina. Transgene expression is mostly restricted to RPE after post-natal day 7 in rats, suggesting that FIV vectors could be used to target RPE for gene therapy. Copyright © 2003 John Wiley & Sons, Ltd. [source]