RESUMO
PURPOSE: In the past few years, the essential role of the homeobox gene Pax6 for eye development has been demonstrated unambiguously in a variety of species including humans. In humans, Pax6 mutations lead to a variety of ocular malformations of the anterior and posterior segment. However, little is known about PAX6 expression in the adult human retina. We have therefore investigated PAX6 levels and localization in the human retina at various ages. METHODS: Adult human eyes of various ages (17-79 years) were obtained from the Zurich Eye Bank. PAX6 expression levels and patterns were analysed by Western blot analysis of total retinal protein and by immunohistochemistry on paraffin sections, respectively. RESULTS: PAX6 expression in the retina was detected up to 79 years of donor age and was predominantly localized to the ganglion cell layer and the inner part of the inner nuclear layer. CONCLUSIONS: PAX6 remains distinctly expressed throughout the lifespan of the human retina suggesting a role for PAX6 in the retina after completion of eye morphogenesis.
Assuntos
Envelhecimento/metabolismo , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Repressoras/metabolismo , Retina/metabolismo , Adolescente , Adulto , Idoso , Western Blotting , Proteínas do Olho/genética , Feminino , Expressão Gênica , Genes Homeobox , Proteínas de Homeodomínio/genética , Humanos , Masculino , Pessoa de Meia-Idade , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Proteínas Repressoras/genética , Células Ganglionares da Retina/metabolismoRESUMO
Light accelerates progression of retinal degeneration in many animal models of retinitis pigmentosa (RP). A sequence variant in the Rpe65 gene (Rpe65(450Leu) or Rpe65(450Met)) can act as a modulator of light-damage susceptibility in mice by influencing the kinetics of rhodopsin regeneration and thus by modulating the photon absorption. Depending on exposure duration and light intensity applied, white fluorescent light induces photoreceptor apoptosis and retinal degeneration in wild-type mice by the activation of one of two known molecular pathways. These pathways depend, respectively, on activation of the transcription factor c-Fos/AP-1 and on phototransduction activity. Here we tested Rpe65 as a genetic modifier for inherited retinal degeneration and analysed which degenerative pathway is activated in a transgenic mouse model of autosomal dominant RP. We show that retinal degeneration was reduced in mice expressing the Rpe65(450Met) variant and that these mice retained more visual pigment rhodopsin than did transgenic mice expressing the Rpe65(450Leu) variant. In addition, lack of phototransduction slowed retinal degeneration whereas ablation of c-Fos had no effect. We conclude that sequence variations in the Rpe65 gene can act as genetic modifiers in inherited retinal degeneration, presumably by regulating the daily rate of photon absorption through the modulation of rhodopsin regeneration kinetics. Increased absorption of photons and/or light sensitivity appear to accelerate retinal degeneration via an apoptotic cascade which involves phototransduction but not c-Fos.
Assuntos
Proteínas do Olho/genética , Variação Genética , Retina/patologia , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Fatores Etários , Animais , Proteínas de Transporte , Modelos Animais de Doenças , Proteínas do Olho/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Leucina/genética , Metionina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Rodopsina/genética , Fatores de Tempo , cis-trans-IsomerasesRESUMO
Hereditary retinal degeneration is characterized by apoptotic photoreceptor loss, a process governed by intricate molecular interplay and initiated when proapoptotic signals predominate in the individual cell. Identification of molecules involved and their actions has paved the way for testing the ones with anti-apoptotic functions in models of inherited retinal degeneration. Many of these factors are able to slow the course of the degeneration. However, to date no such treatment has been able to stop or even prevent the devolution of the disorder. Moreover, preservation of morphology does not necessarily correlate with preservation of ERG function. Deepened understanding of the pro- and anti-apoptotic networks is clearly needed for survival factors to be feasible for therapy in humans. In comparison, in a dog model of Leber's congenital amaurosis gene therapy could establish retinal function, thus supplying proof of efficacy of the method.
Assuntos
Proteínas do Olho/metabolismo , Terapia Genética/métodos , Fármacos Neuroprotetores/uso terapêutico , Degeneração Retiniana/genética , Degeneração Retiniana/terapia , Animais , Apoptose , Intervalo Livre de Doença , Cães , Proteínas do Olho/genética , Humanos , Resultado do TratamentoRESUMO
The susceptibility of rats to light-induced retinal degeneration is increased at night. In mice, an important determinant of light damage susceptibility is the efficacy of rhodopsin regeneration after bleaching. The rate of rhodopsin regeneration is at least partly controlled by RPE65, a protein expressed in the retinal pigment epithelium. We therefore tested a potential involvement of RPE65 and rhodopsin regeneration in the increased light damage susceptibility of rats at night. For this purpose, rats were exposed to visible light at noon or at midnight and extent of light damage was determined by retinal morphology and TUNEL staining. Rpe65 gene expression was analyzed by semiquantitative RT-PCR and levels of RPE65 protein were determined by Western blotting. Rhodopsin regeneration kinetics was determined by measuring rhodopsin content immediately after a strong bleach and after different times of recovery in darkness. Rats were more susceptible to light damage at night as described by Organisciak and collegues [Invest. Ophthalmol. Vis. Sci. 41 (2000) 3694]. Rpe65 gene expression followed a day-night rhythm with highest steady-state mRNA levels at the beginning and lowest levels at the end of the day period. However, RPE65 protein levels remained constant. Rhodopsin regeneration kinetics did not differ during day and night. We conclude that levels of RPE65 protein and rhodopsin regeneration kinetics do not correlate with the increased light damage susceptibility observed in rats at night. Additional genetic or physiologic modifiers may exist in rats that regulate the retinal responsiveness to acute light exposure.
Assuntos
Adaptação à Escuridão/fisiologia , Luz/efeitos adversos , Epitélio Pigmentado Ocular/metabolismo , Proteínas/metabolismo , Retina/metabolismo , Rodopsina/fisiologia , Doença Aguda , Animais , Western Blotting/métodos , Proteínas de Transporte , Suscetibilidade a Doenças , Proteínas do Olho , Expressão Gênica , Epitélio Pigmentado Ocular/química , Proteínas/análise , Proteínas/genética , Ratos , Ratos Sprague-Dawley , Regeneração , Reação em Cadeia da Polimerase Via Transcriptase Reversa , cis-trans-IsomerasesRESUMO
Age-related macular degeneration (AMD) is a main causes of severe visual impairment in the elderly in industrialized countries. The pathogenesis of this complex diseases is largely unknown, even though clinical characteristics and histopathology are well described. Because several aging changes are identical to those observed in AMD, there appears to exist an unknown switch mechanism from normal ageing to disease. Recent anatomical studies using elegant innovative techniques reveal that there is a 30% rod loss in normal ageing, which is increased in early AMD. Those and other observations by Curcio and co-workers indicate that early rod loss is an important denominator of AMD (Curcio CA. Eye 2001; 15:376). As in retinitis pigmentosa (RP), rods appear to die by apoptosis. Thus it seems mandatory to study the regulation of rod cell death in animal models to unravel possible mechanisms of rod loss in AMD. Our laboratory investigates signal transduction pathways and gene regulation of rod death in our model of light-induced apoptosis. The transcription factor AP1 is essential, whereas other classical pro- and antiapoptotic genes appear to be less important in our model system. Caspase-1 gene expression is distinctly upregulated after light exposure and there are several factors which completely protect against light-induced cell death, such as the anesthetic halothane, dexamethasone and the absence of bleachable rhodopsin during light exposure. A fast rhodopsin regeneration rate increased damage susceptibility. Our data indicate that rhodopsin is essential for the initiation of light-induced rod loss. Following photon absorption, there may be the generation of photochemically active molecules wich then induce the apoptotic death cascade.
Assuntos
Apoptose , Degeneração Retiniana/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Animais , Apoptose/efeitos da radiação , Caspase 1/genética , Expressão Gênica , Humanos , RNA Mensageiro/biossíntese , Degeneração Retiniana/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/efeitos da radiação , Fator de Transcrição AP-1/fisiologia , Visão OcularRESUMO
A sequence variation in the pigment epithelial protein RPE65 has been shown to correlate with RPE65 protein levels, rhodopsin regeneration kinetics and light damage susceptibility in different mouse strains. Here, we tested whether such a correlation can also be found in rats. We examined four rat strains for RPE65 protein levels and the Rpe65 gene sequence. In two strains, we additionally determined Rpe65 mRNA levels, rhodopsin regeneration and light damage susceptibility (LDS).RPE65 protein levels were higher in Lewis and Brown Norway rats compared to Wistar and Long Evans. The albino strains Wistar and Lewis were investigated further. Lewis had higher Rpe65 mRNA levels than Wistar. Sequence analysis of the coding region of the Rpe65 cDNA revealed no relevant sequence variations in the two strains. Content and regeneration of rhodopsin were comparable in both strains. However, Wistar rats were more susceptible to light damage than Lewis. We conclude that lower RPE65 protein levels in Wistar may have been caused by decreased gene expression and not by a sequence variation as suggested for mice. In rats, RPE65 may not be a limiting factor for rhodopsin regeneration. Since LDS in rats did not directly correlate with RPE65 protein levels and rhodopsin regeneration, other yet unidentified (genetic) factors may account for the susceptibility differences observed in rats.
Assuntos
Proteínas do Olho/análise , Luz/efeitos adversos , Proteínas/análise , Lesões Experimentais por Radiação/etiologia , Sequência de Aminoácidos , Animais , Western Blotting , Proteínas de Transporte , Suscetibilidade a Doenças , Proteínas do Olho/genética , Expressão Gênica , Masculino , Camundongos , Células Fotorreceptoras/metabolismo , Proteínas/genética , RNA Mensageiro/análise , Lesões Experimentais por Radiação/patologia , Ratos , Ratos Endogâmicos , Ratos Wistar , Retina/patologia , Rodopsina/metabolismo , cis-trans-IsomerasesRESUMO
Apoptosis is essential for retinal development but it is also a major mode of cell loss in many human retinal dystrophies. High levels of visible light induce retinal apoptosis in mice and rats. This process is dependent on the induction of the transcription factor AP-1, a dimeric complex composed of c-Fos and c-Jun/JunD phosphoproteins. While c-Fos is essential, JunD is dispensable for light-induced photoreceptor apoptosis. Here we show that N-terminal phosphorylation of c-Jun, the other main partner of c-Fos in induced AP-1 complexes is not required for programmed cell death during retinal development in vivo and is also dispensable for photoreceptor apoptosis induced by the exogenous stimuli "excessive light" and N-nitroso-N-methylurea (MNU). Mice expressing a mutant c-Jun protein (JunAA) that cannot be phosphorylated at its N-terminus are apoptosis competent and their retina is not distinguishable from wild-type mice. Accordingly, Jun kinase, responsible for phosphorylation of wild-type c-Jun protein is at best only marginally induced by the apoptotic stimuli "light" and MNU. Complex composition of light-induced AP-1 complexes is similar in wild-type and JunAA mice. This shows that the mutant c-Jun protein can be part of the DNA binding complex AP-1 and demonstrates that induction of the DNA binding activity of AP-1 after light insult does not depend on N-terminal phosphorylation of c-Jun. Our results suggest that transactivation of target genes by phosphorylated c-jun/AP-1 is not required for MNU- or light-induced apoptosis of photoreceptor cells.
Assuntos
Apoptose/fisiologia , Células Fotorreceptoras/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Transcrição AP-1/metabolismo , Alquilantes/farmacologia , Animais , Apoptose/efeitos dos fármacos , Metilnitrosoureia/farmacologia , Camundongos , Proteína Quinase 8 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Estimulação Luminosa , Células Fotorreceptoras/citologia , Células Fotorreceptoras/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/genética , Proteínas Proto-Oncogênicas c-jun/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-jun/genética , Fator de Transcrição AP-1/efeitos dos fármacos , Fator de Transcrição AP-1/genética , Visão Ocular/efeitos dos fármacos , Visão Ocular/genéticaRESUMO
Leber congenital amaurosis (LCA) is the most serious form of the autosomal recessive childhood-onset retinal dystrophies. Mutations in the gene encoding RPE65, a protein vital for regeneration of the visual pigment rhodopsin in the retinal pigment epithelium, account for 10-15% of LCA cases. Whereas previous studies of RPE65 deficiency in both animal models and patients attributed remaining visual function to cones, we show here that light-evoked retinal responses in fact originate from rods. For this purpose, we selectively impaired either rod or cone function in Rpe65-/- mice by generating double- mutant mice with models of pure cone function (rhodopsin-deficient mice; Rho-/-) and pure rod function (cyclic nucleotide-gated channel alpha3-deficient mice; Cnga3-/-). The electroretinograms (ERGs) of Rpe65-/- and Rpe65-/-Cnga3-/- mice were almost identical, whereas there was no assessable response in Rpe65-/-Rho-/- mice. Thus, we conclude that the rod system is the source of vision in RPE65 deficiency. Furthermore, we found that lack of RPE65 enables rods to mimic cone function by responding under normally cone-isolating lighting conditions. We propose as a mechanism decreased rod sensitivity due to a reduction in rhodopsin content to less than 1%. In general, the dissection of pathophysiological processes in animal models through the introduction of additional, selective mutations is a promising concept in functional genetics.
Assuntos
Atrofias Ópticas Hereditárias/genética , Epitélio Pigmentado Ocular/fisiologia , Proteínas/genética , Células Fotorreceptoras Retinianas Bastonetes/fisiopatologia , Visão Ocular/fisiologia , Animais , Proteínas de Transporte , Modelos Animais de Doenças , Eletrorretinografia , Proteínas do Olho , Camundongos , Camundongos Mutantes , Atrofias Ópticas Hereditárias/fisiopatologia , cis-trans-IsomerasesRESUMO
PURPOSE: Evidence has accumulated that excessive light exposure may promote age-related and inherited retinal degeneration, in which photoreceptor death by apoptosis leads to loss of vision. In the current study, the effect of elevated corticosteroid levels on light-induced apoptosis of photoreceptors was determined. METHODS: Photoreceptor apoptosis was induced in retinas of BALB/c mice by exposure to diffuse white light. High levels of corticosteroids were induced, either endogenously (fasting-mediated stress) or by a single intraperitoneal injection of dexamethasone (DEX). Photoreceptor damage was assessed morphologically and by electroretinography. Glucocorticoid receptor (GR) and activator protein (AP)-1 activities were shown by Western blot analysis and electrophoretic mobility shift assay (EMSA) of retinal nuclear extracts. RESULTS: Fasting and injection of DEX led to an activation of GR in the retina, as judged by its translocation to the nucleus of retinal cells. On induction of GR activity before light exposure, AP-1 activity, normally induced by damaging doses of light, remained at basal levels. Both treatments completely prevented photoreceptor apoptosis and preserved retinal function. CONCLUSIONS: Activity of the transcription factor AP-1 is associated with light-induced apoptosis. In the current study, pharmacologic suppression of AP-1 activity protected against light damage. Inhibition of AP-1 activity may have occurred by the protein-protein interaction of GR and AP-1.
Assuntos
Apoptose , Luz/efeitos adversos , Células Fotorreceptoras de Vertebrados/efeitos da radiação , Lesões Experimentais por Radiação/prevenção & controle , Receptores de Glucocorticoides/metabolismo , Degeneração Retiniana/prevenção & controle , Animais , Apoptose/efeitos da radiação , Western Blotting , Corticosterona/sangue , Dexametasona/farmacologia , Eletrorretinografia , Privação de Alimentos , Glucocorticoides/farmacologia , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/metabolismo , Lesões Experimentais por Radiação/etiologia , Lesões Experimentais por Radiação/metabolismo , Lesões Experimentais por Radiação/patologia , Degeneração Retiniana/etiologia , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Fator de Transcrição AP-1/antagonistas & inibidores , Fator de Transcrição AP-1/metabolismoRESUMO
Excessive light can cause retinal degeneration and may be an environmental cofactor accelerating retinal dystrophies and age-related diseases. In rodent models, the light damage susceptibility (LDS) of the retina is determined genetically. In two mouse strains, with different degrees of LDS, a Leu450Met variation in the pigment epithelial protein RPE65 was shown recently to cosegregate with low LDS. Because light damage is rhodopsin-mediated, and RPE65 is essential for the regeneration of rhodopsin in the visual cycle, we analyzed this variation regarding rhodopsin metabolism and LDS in four mouse strains. We found that, in contrast to previous assertions, LDS does not correlate with the maximal retinal content of rhodopsin present after dark adaptation. Instead, LDS correlated positively with the kinetics of rhodopsin regeneration, which determine rhodopsin availability during light exposure. Light damage occurred after absorption of a threshold dose of photons and thus fast regeneration, as observed in those two strains having Leu at position 450 of RPE65, was correlated with the occurrence of photoreceptor apoptosis after short exposure. In contrast, mice with the Leu450Met variation of Rpe65 regenerated rhodopsin with slow kinetics and showed an increased resistance to light-induced retinal degeneration. In these mice, RPE65 protein levels were reduced by a post-transcriptional mechanism. F(1) hybrid mice, carrying one normal and one variant Rpe65 gene, had intermediate levels of the corresponding protein and showed intermediate rhodopsin regeneration kinetics and an intermediate LDS. Thus, none of the two variants of Rpe65 had a dominant effect.
Assuntos
Luz/efeitos adversos , Proteínas/genética , Retina/metabolismo , Degeneração Retiniana/metabolismo , Rodopsina/metabolismo , Substituição de Aminoácidos , Animais , Apoptose/genética , Apoptose/efeitos da radiação , Proteínas de Transporte , Modelos Animais de Doenças , Relação Dose-Resposta à Radiação , Proteínas do Olho , Predisposição Genética para Doença , Variação Genética , Camundongos , Camundongos Endogâmicos , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Células Fotorreceptoras de Vertebrados/efeitos da radiação , Retina/patologia , Retina/efeitos da radiação , Degeneração Retiniana/patologia , Rodopsina/genética , cis-trans-IsomerasesRESUMO
Structure-function analysis as well as studies with knock-out and transgenic mice have assigned distinct functions to c-Fos and Fra-1, two components of the transcription factor AP-1 (activator protein-1). To test whether Fra-1 could substitute for c-Fos, we generated knock-in mice that express Fra-1 in place of c-Fos. Fra-1 rescues c-Fos-dependent functions such as bone development and light-induced photoreceptor apoptosis. Importantly, rescue of bone cell differentiation, but not photoreceptor apoptosis, is gene-dosage dependent. Moreover, Fra-1 fails to substitute for c-Fos in inducing expression of target genes in fibroblasts. These results show that c-Fos and Fra-1 have maintained functional equivalence during vertebrate evolution.
Assuntos
Proteínas Proto-Oncogênicas c-fos/fisiologia , Fator de Transcrição AP-1/química , Animais , Animais não Endogâmicos , Apoptose/genética , Desenvolvimento Ósseo/genética , Diferenciação Celular/efeitos dos fármacos , Dimerização , Desenvolvimento Embrionário e Fetal/genética , Fibroblastos/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Genes fos , Teste de Complementação Genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Transgênicos , Osteoclastos/patologia , Osteopetrose/genética , Proteínas Proto-Oncogênicas c-fos/deficiência , Células Fotorreceptoras Retinianas Bastonetes/embriologia , Relação Estrutura-Atividade , Fator de Transcrição AP-1/fisiologiaRESUMO
PURPOSE: To determine whether blue light induces photoreversal of rhodopsin bleaching in vivo. METHODS: Eyes of anesthetized albino rats were exposed to either green (550 nm) or deep blue (403 nm) light, and the time course of rhodopsin bleaching was determined. Rhodopsin was isolated from whole retinas by detergent extraction and measured photometrically. To inhibit photoreversal of bleaching, rats were perfused with 70 mM hydroxylamine (NH(2)OH), a known inhibitor of photoreversal. To determine whether blue-absorbing, photoreversible photoproducts were formed, rhodopsin was bleached to near completion with green light and then exposed to blue light. Finally, experimental results were simulated on a computer by means of a simple, three-component model involving a long-lived photoreversible photoproduct. RESULTS: Photoreversal of bleaching in blue light occurs in vivo as evidenced by the following: In the absence of NH(2)OH, bleaching of rhodopsin by blue light was slow and complex. In the presence of NH(2)OH, however, blue light bleached rhodopsin very fast with a simple, pseudo-first-order kinetic. A long-lived bleaching intermediate produced by green light exposure was photoreversed to rhodopsin by exposure to blue light. The three-component computer model, invoking a blue-absorbing, photoreversible, long-lived intermediate accurately described the data. CONCLUSIONS: Because of the instantaneous, nonmetabolic regeneration of rhodopsin by the process of photoreversal of bleaching, blue light exposure permits the absorption of large numbers of photons by rhodopsin and by a photoreversible intermediate of bleaching in vivo. These data may have an important impact on resolving mechanisms of blue light-mediated damage to the retina.
Assuntos
Luz , Células Fotorreceptoras de Vertebrados/fisiologia , Pigmentos da Retina/fisiologia , Rodopsina/fisiologia , Rodopsina/efeitos da radiação , Animais , Simulação por Computador , Hidroxilamina/farmacologia , Masculino , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/efeitos da radiação , Ratos , Ratos Sprague-Dawley , RegeneraçãoRESUMO
Light-induced apoptosis of photoreceptors represents an animal model for retinal degeneration. Major human diseases that affect vision, such as age-related macular degeneration (AMD) and some forms of retinitis pigmentosa (RP), may be promoted by light. The receptor mediating light damage, however, has not yet been conclusively identified; candidate molecules include prostaglandin synthase, cytochrome oxidase, rhodopsin, and opsins of the cones and the retinal pigment epithelium (PE). We exposed to bright light two groups of genetically altered mice that lack the visual pigment rhodopsin (Rpe65-/- and Rho-/-). The gene Rpe65 is specifically expressed in the PE and essential for the re-isomerization of all-trans retinol in the visual cycle and thus for the regeneration of rhodopsin after bleaching. Rho-/- mice do not express the apoprotein opsin in photoreceptors, which, consequently, do not contain rhodopsin. We show that photoreceptors lacking rhodopsin in these mice are completely protected against light-induced apoptosis. The transcription factor AP-1, a central element in the apoptotic response to light, is not activated in the absence of rhodopsin, indicating that rhodopsin is essential for the generation or transduction of the intracellular death signal induced by light.
Assuntos
Luz/efeitos adversos , Epitélio Pigmentado Ocular/efeitos da radiação , Proteínas/genética , Degeneração Retiniana/etiologia , Rodopsina/fisiologia , Animais , Apoptose/efeitos da radiação , Proteínas de Transporte , Proteínas do Olho , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Fotorreceptoras de Vertebrados/citologia , Células Fotorreceptoras de Vertebrados/efeitos da radiação , Degeneração Retiniana/patologia , Rodopsina/deficiência , Rodopsina/genética , Fator de Transcrição AP-1/efeitos da radiação , cis-trans-IsomerasesRESUMO
In 1999, a pioneer in retinal cell biology celebrates his seventieth birthday: Richard W. Young, Professor of Anatomy at the Dept. of Anatomy and Jules Stein Eye Institute, University of Southern California, Los Angeles, California, USA. Against the current dogma of visual cells as static structures he demonstrated that they undergo continual renewal of their light-sensitive outer segments. Entire membranes and/or single molecules are being replaced, and the tips of outer segments are shed (disk-shedding), and phagocytized and degrade by pigment epithelial (PE) cells. About 100 disks are made per rod within 24 hours, and about 30,000 disk membranes from overlying rods are degraded by one PE cell thus rendering the PE one of the most active phagocytic systems of the body. It is not surprising, therefore, that the age pigment lipofuscin accumulates within PE cells, which is mainly composed of undigestible outer segment material. It is generally concluded that lipofuscin can contribute to the pathogenesis of age related macular degeneration (AMD). Early on Young has postulated that light exposure may accelerate AMD and some forms of retinitis pigmentosa (RP). Today we know that indeed in several animal models of RP light exposure can significantly enhance the disease progression. With a similar insight and intuition he described apoptosis of the retina thus preceding the "apoptotic wave" in eye research. Apoptosis now is considered the final common death pathway of many retinal diseases including degenerations and dystrophies. With his work young has created may scientific children, who directly or indirectly were inspired by his pioneering work.
Assuntos
Regeneração Nervosa/fisiologia , Células Fotorreceptoras/fisiologia , Epitélio Pigmentado Ocular/fisiologia , História do Século XX , Humanos , Oftalmologia/história , Estados UnidosRESUMO
Retinitis pigmentosa (RP) is a hereditary retinal dystrophy which leads to severe visual impairment or blindness and affects about 3.5/1000 of individuals in the industrial world. During the past decades, numerous animal models carrying mutations analogous to mutations in human RP have been studied to elucidate the molecular mechanisms leading to apoptotic photoreceptor cell death in this disease. Up to date, there is no effective treatment to influence the fatal outcome of RP. Recent progress in basic research promotes the development of new therapeutic strategies. In order to restore visual function in blind individuals, the development of electronic photoreceptor prosthesis is being investigated by several researchgroups. Other promising approaches are somatic gene therapy, the application of growth factors and/or pharmacological agents and the inhibition of photoreceptor cell death by interfering with the apoptotic pathway. However, a better understanding of the molecular events leading to cell loss due to photoreceptor apoptosis will be essential for the development of effective treatment.
Assuntos
Terapia Genética , Implantes Experimentais/tendências , Retina/transplante , Retinose Pigmentar/genética , Retinose Pigmentar/terapia , Animais , Apoptose/genética , Regulação da Expressão Gênica , Genes fos/genética , Alemanha , Humanos , Microeletrodos , Células Fotorreceptoras de Vertebrados , Retinose Pigmentar/metabolismoRESUMO
PURPOSE: Mice without a functional c-Fos protein (c-fos-/- mice) do not exhibit light-induced apoptotic cell death of rods in contrast to their wild-type littermates (c-fos+/+ mice). To analyze the consequences of the absence of c-fos in the retina, we investigated whether the retinas of c-fos-/- mice have a reduced capacity to absorb and transduce light compared with c-fos+/+ mice. METHODS: Retinal function was evaluated in dark-adapted mice by full-field electroretinograms (ERGs) over more than 6 log units of intensity. Retinal morphology was studied by light- and electron microscopy. Arrestin and the heat shock protein 70 (Hsp70) were detected by Western blot analysis. The rhodopsin content and the kinetics of rhodopsin regeneration were determined in retinal extracts. RESULTS: Although the configuration of the ERGs was comparable in both groups of mice, c-fos-/- mice showed a marked variability in all quantitative ERG-measures with lower mean amplitudes, longer latencies, and a 0.9-log-unit lower b-wave sensitivity on average. Morphometry showed that c-fos-/- mice have 23% fewer rods on average, whereas the number of cones was comparable among c-fos+/+ and c-fos-/- mice. Arrestin levels appeared slightly reduced in c-fos-/- mice when compared with c-fos+/+ mice, whereas Hsp70 levels were comparable in both genotypes. The kinetics of rhodopsin regeneration were similar, but c-fos-/- mice had a 25% lower rhodopsin content on average. CONCLUSIONS: Compared with c-fos+/+ mice, retinal function in c-fos-/- mice is attenuated to a variable but marked degree, which may be, at least in part, related to the reduced number of rods and the reduced rhodopsin content. However, c-fos does not appear to be essential for the ability to absorb photons, nor for phototransduction or the function of second-order neurons. The resistance to light-induced apoptosis of photoreceptor cells in c-fos-/- mice may result from the acute deficit of c-fos in the apoptotic cascade rather than from developmental deficits affecting rod photoreceptor function.
Assuntos
Proteínas Proto-Oncogênicas c-fos/fisiologia , Lesões Experimentais por Radiação/fisiopatologia , Retina , Degeneração Retiniana/fisiopatologia , Rodopsina/metabolismo , Visão Ocular/fisiologia , Animais , Apoptose , Arrestina/metabolismo , Western Blotting , Adaptação à Escuridão/fisiologia , Eletrorretinografia , Deleção de Genes , Proteínas de Choque Térmico HSP70/metabolismo , Luz , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Lesões Experimentais por Radiação/metabolismo , Lesões Experimentais por Radiação/patologia , Retina/metabolismo , Retina/patologia , Retina/fisiologia , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologiaRESUMO
White light (5 klux for 2 hr) induces apoptosis of rod photoreceptors in wild-type mice (c-fos(+/+)) within 24 hr, whereas rods of c-fos knock-out mice (c-fos(-/-)) are protected (). The range of this protection was tested by analyzing retinas of c-fos(+/+) and c-fos(-/-) mice up to 10 d after exposure to threefold increased light intensities (15 klux for 2 hr). In c-fos(-/-) mice, rods were unaffected, whereas they were destroyed in c-fos(+/+) mice. After light exposure, mitochondrial damage in rods was observed exclusively in c-fos(+/+) mice. Electroretinograms recorded 48 hr after exposure revealed a decrease of all components in c-fos(+/+) mice but indicated no light-induced loss of function in c-fos(-/-) mice. Thus, in c-fos(-/-) mice, light-induced apoptosis is blocked or its threshold is elevated more than threefold. Increased activity of the transcription factor activator protein-1 (AP-1) in retinas of light-exposed c-fos(+/+) mice indicated an acute contribution of AP-1 to apoptosis induction. AP-1 activity increased already during exposure and peaked approximately 6 hr thereafter, coinciding with the appearance of major morphological signs of apoptosis. Activated AP-1 mainly consisted of c-Fos/Jun heterodimers. In c-fos(-/-) mice, AP-1 activity remained unchanged, indicating that no other Jun- or Fos-family member could substitute for c-Fos. Like damaging light, N-methyl-N-nitrosourea (MNU) induced AP-1 containing c-Fos in c-fos(+/+) mice and did not induce AP-1 in c-fos(-/-) mice. In contrast to light, however, MNU induced apoptosis in rods of c-fos(-/-) mice. Thus, c-Fos is essential for a specific premitochondrial "private apoptotic pathway" induced by light but not for the execution of apoptosis induced by other stimuli.
Assuntos
Apoptose/fisiologia , Proteínas Proto-Oncogênicas c-fos/genética , Degeneração Retiniana/fisiopatologia , Células Fotorreceptoras Retinianas Bastonetes/citologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Alquilantes/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Adaptação à Escuridão/fisiologia , Modelos Animais de Doenças , Eletrorretinografia , Feminino , Luz/efeitos adversos , Masculino , Metilnitrosoureia/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Mitocôndrias/fisiologia , Mitocôndrias/ultraestrutura , Degeneração Retiniana/genética , Fator de Transcrição AP-1/metabolismoRESUMO
PURPOSE: High levels of visible light induce apoptotic cell death of photoreceptors, a process depending on the activation of the transcription factor AP-1. This suggests that regulation of gene expression might be important for light-induced photoreceptor cell death. We measured expression of AP-1 family members and of several apoptosis-related genes to test their potential involvement in photoreceptor apoptosis. METHODS: Wildtype and c-fos-/- mice were exposed to low (roomlight) or high levels of visible light for up to two hours. Total RNA was prepared from isolated retinas during and after light exposure. Relative mRNA levels were determined semiquantitatively using either competitive or exponential RT-PCR. RESULTS: Expression of c-fos-/- was upregulated by intense light as early as 15 min after lights on. Highest levels (6-fold induction) were detected at 2 h after lights off declining thereafter to basal levels 20 h after the end of exposure. c-jun mRNA was induced at 30 min after lights on and high expression levels (fourfold induction) persisted at least for 8 h. Similarly, expression of caspase-1 was six to 9-fold increased at 6 to 8 h after light exposure in wildtype but not in c-fos knockout mice. The latter mice are protected against light-induced photoreceptor apoptosis. Expression of other apoptosis-related genes (bcl-2, bcl-XL, bax, bad, caspase-3) was not affected by light exposure or the lack of c-Fos in knockout mice. CONCLUSIONS: Expression of c-fos and c-jun mRNA is transiently induced by exposure to damaging light. Induced expression of c-jun persists longer than expression of c-fos. Among the apoptosis-related genes, only caspase-1 expression was upregulated by light exposure and Caspase-1 might therefore be involved in light-induced retinal degeneration.
Assuntos
Caspase 1/genética , Expressão Gênica , Células Fotorreceptoras de Vertebrados/efeitos da radiação , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-jun/genética , Lesões Experimentais por Radiação/genética , Degeneração Retiniana/genética , Fator de Transcrição AP-1/genética , Animais , Apoptose/efeitos da radiação , Caspase 1/biossíntese , Primers do DNA/química , Feminino , Luz/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-fos/biossíntese , Proteínas Proto-Oncogênicas c-jun/biossíntese , RNA Mensageiro/biossíntese , Lesões Experimentais por Radiação/metabolismo , Lesões Experimentais por Radiação/patologia , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição AP-1/biossíntese , Regulação para CimaRESUMO
PURPOSE: To investigate whether regulation of rhodopsin levels as a response to changed lighting environment is performed by autophagic degradation of opsin in rod inner segments (RISs). METHODS: Groups of albino rats were kept in 3 lux or 200 lux. At 10 weeks of age, one group was transferred from 3 lux to 200 lux, another group was switched from 200 lux to 3 lux, and two groups remained in their native lighting (baselines). Rats were killed at days 1, 2, and 3 after switching. Another group was switched from 3 lux to 200 lux, and rats were killed at short intervals after the switch. Numbers of autophagic vacuoles (AVs) in RISs were counted, and immunogold labeling was performed for opsin and ubiquitin in electron microscopic sections. RESULTS: The number of AVs increased significantly after switching from 3 lux to 200 lux at days 1 and 2 and declined at day 3, whereas the reverse intensity change did not cause any increase. Early time points after change from 3 lux to 200 lux showed a significant increase of AVs 2 and 3 hours after switching. Distinct opsin label was observed in AVs of rats switched to 200 lux. Ubiquitin label was present in all investigated specimens and was also seen in AVs especially in 200-lux immigrants. CONCLUSIONS: Earlier studies had shown that an adjustment to new lighting environment is performed by changes in rhodopsin levels in ROSs. Autophagic degradation of opsin or rhodopsin may subserve, at least in part, the adaptation to abruptly increased habitat illuminance by removing surplus visual pigment.