RESUMO
Dark-rearing has been found to slow the rate of retinal degeneration in albino P23H but not S334ter mutant rhodopsin transgenic (Tg) rats. Since eye pigmentation has the same protective slowing effect as dark-rearing in RCS rats, we examined whether eye pigmentation has a comparable slowing effect in the different mutant rhodopsin Tg rats. Different lines of albino P23H and S334ter Tg rats on the Sprague-Dawley (SD) background were bred to Long-Evans (LE) rats to produce pigmented Tg rats. These were compared to albino Tg rats at postnatal days of different ages using the outer nuclear layer (ONL) as a morphological measure of photoreceptor number and electroretinogram (ERG) a- and b-wave amplitudes as a measure of retinal function. When compared to albino P23H rats, pigmented P23H rats had a slower rate of degeneration as measured by greater ONL thicknesses and greater ERG a- and b-wave amplitudes. By contrast, pigmented S334ter rats showed no difference in ONL thicknesses or ERG a- and b-wave amplitudes when compared to their albino equivalents. Thus, degeneration of photoreceptors in P23H Tg rats is slowed by eye pigmentation as measured by ONL thickness, while it is not in the S334ter Tg rats. Eye pigmentation also protects functional changes in ERG a- and b-waves for the P23H lines, but not for the S334ter lines.
Assuntos
Cor de Olho/genética , Retina/fisiopatologia , Degeneração Retiniana/genética , Degeneração Retiniana/fisiopatologia , Rodopsina/genética , Animais , Eletrorretinografia , Mutação , Fenótipo , Células Fotorreceptoras de Vertebrados/patologia , Ratos , Ratos Long-Evans , Ratos Sprague-Dawley , Ratos TransgênicosRESUMO
We produced 8 lines of transgenic (Tg) rats expressing one of two different rhodopsin mutations in albino Sprague-Dawley (SD) rats. Three lines were generated with a proline to histidine substitution at codon 23 (P23H), the most common autosomal dominant form of retinitis pigmentosa in the United States. Five lines were generated with a termination codon at position 334 (S334ter), resulting in a C-terminal truncated opsin protein lacking the last 15 amino acid residues and containing all of the phosphorylation sites involved in rhodopsin deactivation, as well as the terminal QVAPA residues important for rhodopsin deactivation and trafficking. The rates of photoreceptor (PR) degeneration in these models vary in proportion to the ratio of mutant to wild-type rhodopsin. The models have been widely studied, but many aspects of their phenotypes have not been described. Here we present a comprehensive study of the 8 Tg lines, including the time course of PR degeneration from the onset to one year of age, retinal structure by light and electron microscopy (EM), hemispheric asymmetry and gradients of rod and cone degeneration, rhodopsin content, gene dosage effect, rapid activation and invasion of the outer retina by presumptive microglia, rod outer segment disc shedding and phagocytosis by the retinal pigmented epithelium (RPE), and retinal function by the electroretinogram (ERG). The biphasic nature of PR cell death was noted, as was the lack of an injury-induced protective response in the rat models. EM analysis revealed the accumulation of submicron vesicular structures in the interphotoreceptor space during the peak period of PR outer segment degeneration in the S334ter lines. This is likely due to the elimination of the trafficking consensus domain as seen before as with other rhodopsin mutants lacking the C-terminal QVAPA. The 8 rhodopsin Tg lines have been, and will continue to be, extremely useful models for the experimental study of inherited retinal degenerations.
Assuntos
Modelos Animais de Doenças , Células Fotorreceptoras de Vertebrados/patologia , Mutação Puntual , Retina/fisiologia , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Rodopsina/genética , Animais , Eletrorretinografia , Microscopia , Microscopia Eletrônica , Fenótipo , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Degeneração Retiniana/fisiopatologiaRESUMO
Inherited photoreceptor degenerations (IPDs) are the most genetically heterogeneous of Mendelian diseases. Many IPDs exhibit substantial phenotypic variability, but the basis is usually unknown. Mutations in MERTK cause recessive IPD phenotypes associated with the RP38 locus. We have identified a murine genetic modifier of Mertk-associated photoreceptor degeneration, the C57BL/6 (B6) allele of which acts as a suppressor. Photoreceptors degenerate rapidly in Mertk-deficient animals homozygous for the 129P2/Ola (129) modifier allele, whereas animals heterozygous for B6 and 129 modifier alleles exhibit an unusual intermixing of degenerating and preserved retinal regions, with females more severely affected than males. Mertk-deficient mice homozygous for the B6 modifier allele display degeneration only in the far periphery, even at 8 months of age, and have improved retinal function compared to animals homozygous for the 129 allele. We genetically mapped the modifier to an approximately 2-megabase critical interval that includes Tyro3, a paralog of Mertk. Tyro3 expression in the outer retina varies with modifier genotype in a manner characteristic of a cis-acting expression quantitative trait locus (eQTL), with the B6 allele conferring an approximately three-fold higher expression level. Loss of Tyro3 function accelerates the pace of photoreceptor degeneration in Mertk knockout mice, and TYRO3 protein is more abundant in the retinal pigment epithelium (RPE) adjacent to preserved central retinal regions of Mertk knockout mice homozygous for the B6 modifier allele. Endogenous human TYRO3 protein co-localizes with nascent photoreceptor outer segment (POS) phagosomes in a primary RPE cell culture assay, and expression of murine Tyro3 in cultured cells stimulates phagocytic ingestion of POS. Our findings demonstrate that Tyro3 gene dosage modulates Mertk-associated retinal degeneration, provide strong evidence for a direct role for TYRO3 in RPE phagocytosis, and suggest that an eQTL can modify a recessive IPD.
Assuntos
Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Degeneração Retiniana/genética , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Knockout , Fagocitose , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patologia , Proteínas Proto-Oncogênicas/biossíntese , Receptores Proteína Tirosina Quinases/biossíntese , Retina/metabolismo , Retina/patologia , Degeneração Retiniana/patologia , Epitélio Pigmentado da Retina/patologia , c-Mer Tirosina QuinaseRESUMO
Retinal degenerations, including age-related macular degeneration and the retinitis pigmentosa family of diseases, are among the leading causes of legal blindness in the United States. We previously found that Stanniocalcin-1 (STC-1) reduced photoreceptor loss in the S334ter-3 and Royal College of Surgeons rat models of retinal degeneration. The results were attributed in part to a reduction in oxidative stress. Herein, we tested the hypothesis that long-term delivery of STC-1 would provide therapeutic rescue in more chronic models of retinal degeneration. To achieve sustained delivery, we produced an adeno-associated virus (AAV) construct to express STC-1 (AAV-STC-1) under the control of a retinal ganglion cell targeting promoter human synapsin 1 (hSYN1). AAV-STC-1 was injected intravitreally into the P23H-1 and S334ter-4 rhodopsin transgenic rats at postnatal day 10. Tissues were collected at postnatal day 120 for confirmation of STC-1 overexpression and histologic and molecular analysis. Electroretinography (ERG) was performed in a cohort of animals at that time. Overexpression of STC-1 resulted in a significant preservation of photoreceptors as assessed by outer nuclear thickness in the P23H-1 (P < 0.05) and the S334ter-4 (P < 0.005) models compared to controls. Additionally, retinal function was significantly improved in the P23H-1 model with overexpressed STC-1 as assessed by ERG analysis (scotopic b-wave P < 0.005 and photopic b-wave P < 0.05). Microarray analysis identified common downstream gene expression changes that occurred in both models. Genes of interest based on their function were selected for validation by quantitative real-time PCR and were significantly increased in the S334ter-4 model.
Assuntos
Dependovirus , Glicoproteínas/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Retinose Pigmentar/tratamento farmacológico , Animais , Modelos Animais de Doenças , Eletrorretinografia , Glicoproteínas/administração & dosagem , Fármacos Neuroprotetores/administração & dosagem , Células Fotorreceptoras de Vertebrados/patologia , Ratos , Ratos Transgênicos , Retinose Pigmentar/patologiaRESUMO
Retinal degeneration (RD) affects millions of people and is a major cause of ocular impairment and blindness. With a wide range of mutations and conditions leading to degeneration, targeting downstream processes is necessary for developing effective treatments. Ceramide and sphingosine-1-phosphate, a pair of bioactive sphingolipids, are involved in apoptosis and its prevention, respectively. Apoptotic cell death is a potential driver of RD, and in order to understand the mechanism of degeneration and potential treatments, we studied rhodopsin mutant RD model, P23H-1 rats. Investigating this genetic model of human RD allows us to investigate the association of sphingolipid metabolites with the degeneration of the retina in P23H-1 rats and the effects of a specific modulator of sphingolipid metabolism, FTY720. We found that P23H-1 rat retinas had altered sphingolipid profiles that, when treated with FTY720, were rebalanced closer to normal levels. FTY720-treated rats also showed protection from RD compared with their vehicle-treated littermates. Based on these data, we conclude that sphingolipid dysregulation plays a secondary role in retinal cell death, which may be common to many forms of RDs, and that the U.S. Food and Drug Administration-approved drug FTY720 or related compounds that modulate sphingolipid metabolism could potentially delay the cell death.
Assuntos
Cloridrato de Fingolimode/farmacologia , Distrofias Retinianas/metabolismo , Esfingolipídeos/metabolismo , Animais , Vias Biossintéticas , Progressão da Doença , Avaliação Pré-Clínica de Medicamentos , Cloridrato de Fingolimode/uso terapêutico , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Ratos Sprague-Dawley , Distrofias Retinianas/tratamento farmacológico , Esfingomielina Fosfodiesterase/metabolismoRESUMO
MERTK-associated retinal degenerations are thought to have defects in phagocytosis of shed outer segment membranes by the retinal pigment epithelium (RPE), as do the rodent models of these diseases. We have subretinally injected an RPE-specific AAV2 vector, AAV2-VMD2-hMERTK, to determine whether this would provide long-term photoreceptor rescue in the RCS rat, which it did for up to 6.5 months, the longest time point examined. Moreover, we found phagosomes in the RPE in the rescued regions of RCS retinas soon after the onset of light. The same vector also had a major protective effect in Mertk-null mice, with a concomitant increase in ERG response amplitudes in the vector-injected eyes. These findings suggest that planned clinical trials with this vector will have a favorable outcome.
Assuntos
Terapia Genética/métodos , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Degeneração Retiniana/genética , Degeneração Retiniana/terapia , Animais , Bestrofinas , Canais de Cloreto/genética , Dependovirus/genética , Modelos Animais de Doenças , Eletrorretinografia , Proteínas do Olho/genética , Vetores Genéticos/genética , Humanos , Camundongos Knockout , Fagocitose/genética , Fagocitose/fisiologia , Fagossomos/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/metabolismo , Ratos Mutantes , Ratos Sprague-Dawley , Receptores Proteína Tirosina Quinases/deficiência , Receptores Proteína Tirosina Quinases/metabolismo , Degeneração Retiniana/fisiopatologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/fisiopatologia , Resultado do Tratamento , c-Mer Tirosina QuinaseRESUMO
RHO (Rod opsin) encodes a G-protein coupled receptor that is expressed exclusively by rod photoreceptors of the retina and forms the essential photopigment, rhodopsin, when coupled with 11-cis-retinal. Many rod opsin disease -mutations cause rod opsin protein misfolding and trigger endoplasmic reticulum (ER) stress, leading to activation of the Unfolded Protein Response (UPR) signal transduction network. Chop is a transcriptional activator that is induced by ER stress and promotes cell death in response to chronic ER stress. Here, we examined the role of Chop in transgenic mice expressing human P23H rhodopsin (hP23H Rho Tg) that undergo retinal degeneration. With the exception of one time point, we found no significant induction of Chop in these animals and no significant change in retinal degeneration by histology and electrophysiology when hP23H Rho Tg animals were bred into a Chop (-/-) background. Our results indicate that Chop does not play a significant causal role during retinal degeneration in these animals. We suggest that other modules of the ER stress-induced UPR signaling network may be involved photoreceptor disease induced by P23H rhodopsin.
Assuntos
Degeneração Retiniana/genética , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Rodopsina/genética , Fator de Transcrição CHOP/genética , Animais , Sobrevivência Celular/genética , Eletrorretinografia , Expressão Gênica , Humanos , Camundongos Knockout , Camundongos Transgênicos , Degeneração Retiniana/metabolismo , Degeneração Retiniana/fisiopatologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rodopsina/metabolismo , Fator de Transcrição CHOP/deficiência , Transgenes/genéticaRESUMO
Oxidative stress and photoreceptor apoptosis are prominent features of many forms of retinal degeneration (RD) for which there are currently no effective therapies. We previously observed that mesenchymal stem/stromal cells reduce apoptosis by being activated to secrete stanniocalcin-1 (STC-1), a multifunctional protein that reduces oxidative stress by upregulating mitochondrial uncoupling protein-2 (UCP-2). Therefore, we tested the hypothesis that intravitreal injection of STC-1 can rescue photoreceptors. We first tested STC-1 in the rhodopsin transgenic rat characterized by rapid photoreceptor loss. Intravitreal STC-1 decreased the loss of photoreceptor nuclei and transcripts and resulted in measurable retinal function when none is otherwise present in this rapid degeneration. We then tested STC-1 in the Royal College of Surgeons (RCS) rat characterized by a slower photoreceptor degeneration. Intravitreal STC-1 reduced the number of pyknotic nuclei in photoreceptors, delayed the loss of photoreceptor transcripts, and improved function of rod photoreceptors. Additionally, STC-1 upregulated UCP-2 and decreased levels of two protein adducts generated by reactive oxygen species (ROS). Microarrays from the two models demonstrated that STC-1 upregulated expression of a similar profile of genes for retinal development and function. The results suggested that intravitreal STC-1 is a promising therapy for various forms of RD including retinitis pigmentosa and atrophic age-related macular degeneration (AMD).
Assuntos
Glicoproteínas/farmacologia , Degeneração Retiniana/tratamento farmacológico , Animais , Eletrorretinografia , Ensaio de Imunoadsorção Enzimática , Humanos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Degeneração Macular/tratamento farmacológico , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Degeneração Retiniana/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Retinose Pigmentar/tratamento farmacológico , Proteína Desacopladora 2RESUMO
The early loss of photoreceptors in some retinal degenerations in mice has been shown to have a profound effect on vascular development of the retina. To better characterize this relationship, we have examined the formation of retinal blood vessels during the first month of life in 8 lines of transgenic rats with different ages of onset and rates of photoreceptor cell loss mediated by the expression of mutant rhodopsin (P23H and S334ter). The number of capillary profiles in the superficial plexus (SP) and deep capillary plexus (DCP) of the retina were quantified in retinal sections taken at postnatal day (P) 8, 10, 12, 15 and 30. In normal wild-type rats, the SP and DCP had mostly established mature, adult patterns by P15, as previously shown. In the transgenic rats, the loss of photoreceptors had relatively little effect on the SP. By contrast, the loss of photoreceptors during vascular development had a major impact on the DCP. In the two lines with early and most rapid photoreceptor loss, S334ter-7 and S334ter-3, where about 90% and 65%, respectively, of the photoreceptors were already lost by P15, the DCP either failed to form (S334ter-7) or the number of capillary profiles was less than 7% of controls (S334ter-3). In lines where almost all photoreceptors were still present at P15 (S334ter-4, S334ter-9, P23H-2 and P23H-3), the number of profiles in the DCP were the same as in wild-type controls at P30. In two lines with an intermediate rate of degeneration (S334ter-5 and P23H-1), where only about 25% of the photoreceptors were lost by P15, there was an intermediate number of vascular profiles in the DCP at P30. Thus, a very close relationship between the number of photoreceptors and vessel profiles in the DCP during its development exists in the transgenic rats, and the loss of photoreceptors results in the failure or inhibition of the DCP to develop. Several mechanisms may explain this relationship including changes in the level of physiological oxygen tension or alteration in the release of angiogenic factors that normally drive vessel development. Analysis of older transgenic retinas up to 1 year of age revealed that (1) vascular profiles are lost from the DCP in essentially all lines once fewer than about 30-33% of photoreceptors remain; (2) in those lines where the DCP essentially did not develop (S334ter-7 and S334ter-3), the effect of photoreceptor absence was permanent, and there was no late vascularization of the DCP; (3) the number of capillary profiles in the SP remained no different from controls in any of the lines, despite long-standing loss of photoreceptors; and (4) neovascularization of the RPE by retinal capillaries occurred with a latency of 60-180 days after the loss of photoreceptors, except in S334ter-7 rats, where neovascularization essentially did not occur. Analysis of RCS rats was carried out for comparison.
Assuntos
Mutação , Células Fotorreceptoras de Vertebrados/patologia , Degeneração Retiniana/patologia , Vasos Retinianos/crescimento & desenvolvimento , Rodopsina/genética , Envelhecimento/patologia , Animais , Neovascularização Patológica/patologia , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Degeneração Retiniana/fisiopatologia , Epitélio Pigmentado da Retina/irrigação sanguínea , Epitélio Pigmentado da Retina/patologia , Segmento Externo da Célula Bastonete/patologiaRESUMO
PURPOSE: A prior study showed that brain-derived neurotrophic factor (BDNF) rescues photoreceptors from collateral retinal damage caused by photodynamic therapy (PDT). This study was conducted to determine whether ciliary neurotrophic factor (CNTF), a combination of BDNF and CNTF, or pigment epithelial cell-derived growth factor (PEDF) might protect photoreceptors and retinal function more effectively than BDNF. Also investigated was whether protection would be observed after a second round of PDT with adjunctive BDNF treatment. METHODS: Normal rats received intravitreal injections of BDNF, CNTF, a combination of BDNF and CNTF, or PEDF in one eye and PBS in the other 2 days before PDT. Retinal function and photoreceptor survival were assessed with multifocal ERG (mfERG) and histology 1 week after PDT. Another group of rats received two courses of PDT 3 months apart, with injection of BDNF 2 days before each treatment. RESULTS: All factors significantly increased photoreceptor survival. The combination of BDNF and CNTF rescued more photoreceptors than either factor alone. Only BDNF improved retinal function 1 week after PDT, with CNTF and the combination of BDNF and CNTF reducing mfERG responses. BDNF injection before a second round of PDT improved mfERG responses and retinal structure. CONCLUSIONS: BDNF is the most effective single factor among those tested for neuroprotection and improvement of retinal function after PDT, although a combination of BDNF and CNTF rescues more photoreceptors. Adjunctive treatment with BDNF also protects retinal structure and function through two rounds of PDT, suggesting its potential value for patients who require multiple treatments.
Assuntos
Fatores de Crescimento Neural/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Fotoquimioterapia , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Fármacos Fotossensibilizantes/toxicidade , Porfirinas/toxicidade , Doenças Retinianas/prevenção & controle , Animais , Fator Neurotrófico Derivado do Encéfalo/uso terapêutico , Sobrevivência Celular/efeitos dos fármacos , Fator Neurotrófico Ciliar/uso terapêutico , Quimioterapia Combinada , Eletrorretinografia/efeitos dos fármacos , Proteínas do Olho/uso terapêutico , Injeções , Células Fotorreceptoras de Vertebrados/patologia , Ratos , Ratos Endogâmicos BN , Retina/efeitos dos fármacos , Retina/fisiologia , Doenças Retinianas/induzido quimicamente , Doenças Retinianas/fisiopatologia , Serpinas/uso terapêutico , Verteporfina , Corpo VítreoRESUMO
PURPOSE: CNTF is a neuroprotective agent for retinal degenerations that can cause reduced electroretinogram (ERG) amplitudes. The goal of the present study was to determine the effects of intraocular delivery of CNTF on normal rat visual function. METHODS: Full-field scotopic and photopic ERG amplitudes and spatial frequency thresholds of the optokinetic response (OKR) of adult Long-Evans rats were measured before and after intravitreous injection of CNTF or subretinal delivery of adenoassociated virus-vectored CNTF (AAV-CNTF) into one eye. Visual acuity was also measured by using the Visual Water Task in AAV-CNTF-injected animals. Multiunit luminance thresholds were recorded in the superior colliculus after CNTF injection, and the eyes were examined histologically. RESULTS: In eyes injected with a high dose of CNTF, ERG amplitudes and OKR thresholds measured through CNTF-injected eyes were decreased by 45% to 70% within 6 days after injection. ERG amplitudes had begun to recover by 21 days, whereas OKR thresholds only began to recover after 56 days. Neither OKR thresholds nor ERG amplitudes fully recovered until 90 to 100 days. When measured in the superior colliculus at 2 weeks after CNTF injection, luminance thresholds were elevated by 0.35 log units. In AAV-CNTF-injected eyes, OKR thresholds, and visual acuity were reduced by approximately 50% for at least 6 months, and scotopic and photopic ERG b-waves were reduced by 30% to 50%. Photoreceptor loss occurred in the injected regions in some of the eyes. By contrast, comparison of dose-response analysis with a dose-response study of light damage strongly suggests that therapeutic doses of CNTF exist that do not suppress ERG responses. CONCLUSIONS: Intraocular delivery of CNTF, which preserves photoreceptors in animal models of retinal degeneration, impairs visual function in normal rats at very high doses, but not at lower doses that still provide protection from constant light damage.
Assuntos
Fator Neurotrófico Ciliar/toxicidade , Eletrorretinografia/efeitos dos fármacos , Nistagmo Optocinético/efeitos dos fármacos , Transtornos da Visão/induzido quimicamente , Acuidade Visual/efeitos dos fármacos , Animais , Fator Neurotrófico Ciliar/genética , Dependovirus/genética , Relação Dose-Resposta a Droga , Vetores Genéticos , Injeções , Luz/efeitos adversos , Células Fotorreceptoras de Vertebrados/efeitos da radiação , Lesões Experimentais por Radiação/etiologia , Lesões Experimentais por Radiação/prevenção & controle , Ratos , Ratos Long-Evans , Limiar Sensorial/efeitos dos fármacos , Colículos Superiores/efeitos dos fármacos , Corpo VítreoAssuntos
Dependovirus/genética , Terapia Genética/métodos , Proteínas de Filamentos Intermediários/genética , Glicoproteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , RNA Interferente Pequeno/genética , Retinose Pigmentar/genética , Retinose Pigmentar/terapia , Animais , Modelos Animais de Doenças , Eletrorretinografia , Genes Dominantes/genética , Células HEK293 , Humanos , Camundongos , Periferinas , Células Fotorreceptoras de Vertebrados/fisiologia , RNA Mensageiro/genética , RNA Interferente Pequeno/farmacologiaRESUMO
PURPOSE: Endoplasmic reticulum (ER) stress activates inositol requiring enzyme 1 (IRE1), a key regulator of the unfolded protein response. The ER stress activated indicator (ERAI) transgenic mouse expresses a yellow fluorescent GFP variant (Venus) when IRE1 is activated by ER stress. We tested whether ERAI mice would allow for real-time longitudinal studies of ER stress in living mouse eyes. METHODS: We chemically and genetically induced ER stress, and qualitatively and quantitatively studied the Venus signal by fluorescence ophthalmoscopy. We determined retinal cell types that contribute to the signal by immunohistology, and we performed molecular and biochemical assays using whole retinal lysates to assess activity of the IRE1 pathway. RESULTS: We found qualitative increase in vivo in fluorescence signal at sites of intravitreal tunicamycin injection in ERAI eyes, and quantitative increase in ERAI mice mated to RhoP23H mice expressing ER stress-inducing misfolded rhodopsin protein. As expected, we found that increased Venus signal arose primarily from photoreceptors in RhoP23H/+;ERAI mice. We found increased Xbp1S and XBP1s transcriptional target mRNA levels in RhoP23H/+;ERAI retinas compared to Rho+/+;ERAI retinas, and that Venus signal increased in ERAI retinas as a function of age. CONCLUSIONS: Fluorescence ophthalmoscopy of ERAI mice enables in vivo visualization of retinas undergoing ER stress. ER stress activated indicator mice enable identification of individual retinal cells undergoing ER stress by immunohistochemistry. ER stress activated indicator mice show higher Venus signal at older ages, likely arising from amplification of basal retinal ER stress levels by GFP's inherent stability.
Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Retina/diagnóstico por imagem , Animais , Retículo Endoplasmático/diagnóstico por imagem , Retículo Endoplasmático/patologia , Retículo Endoplasmático/fisiologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estudos Longitudinais , Proteínas de Membrana/análise , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Transgênicos , Oftalmoscopia , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/fisiologia , Retina/química , Retina/efeitos dos fármacos , Retina/patologia , Retina/fisiologia , Transdução de Sinais/fisiologia , Tomografia de Coerência Óptica , Tunicamicina/farmacologia , UltrassonografiaRESUMO
Rhodopsin is a G protein-coupled receptor essential for vision and rod photoreceptor viability. Disease-associated rhodopsin mutations, such as P23H rhodopsin, cause rhodopsin protein misfolding and trigger endoplasmic reticulum (ER) stress, activating the unfolded protein response (UPR). The pathophysiologic effects of ER stress and UPR activation on photoreceptors are unclear. Here, by examining P23H rhodopsin knock-in mice, we found that the UPR inositol-requiring enzyme 1 (IRE1) signaling pathway is strongly activated in misfolded rhodopsin-expressing photoreceptors. IRE1 significantly upregulated ER-associated protein degradation (ERAD), triggering pronounced P23H rhodopsin degradation. Rhodopsin protein loss occurred as soon as photoreceptors developed, preceding photoreceptor cell death. By contrast, IRE1 activation did not affect JNK signaling or rhodopsin mRNA levels. Interestingly, pro-apoptotic signaling from the PERK UPR pathway was also not induced. Our findings reveal that an early and significant pathophysiologic effect of ER stress in photoreceptors is the highly efficient elimination of misfolded rhodopsin protein. We propose that early disruption of rhodopsin protein homeostasis in photoreceptors could contribute to retinal degeneration.
Assuntos
Degradação Associada com o Retículo Endoplasmático , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Rodopsina/metabolismo , Animais , Animais Recém-Nascidos , Apoptose , Estresse do Retículo Endoplasmático , Técnicas de Introdução de Genes , Imunoprecipitação , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Retina/metabolismo , Retina/patologia , Retina/ultraestrutura , Segmento Interno das Células Fotorreceptoras da Retina/metabolismo , Segmento Interno das Células Fotorreceptoras da Retina/patologia , Segmento Interno das Células Fotorreceptoras da Retina/ultraestrutura , Rodopsina/genética , Transdução de Sinais , Fator de Transcrição CHOP/metabolismo , UbiquitinaçãoRESUMO
PURPOSE: To preserve photoreceptor cell structure and function in a rodent model of retinitis pigmentosa with P23H rhodopsin by selective inhibition of the mutant rhodopsin allele using a second generation antisense oligonucleotide (ASO). METHODS: Wild-type mice and rats were treated with ASO by intravitreal (IVT) injection and rhodopsin mRNA and protein expression were measured. Transgenic rats expressing the murine P23H rhodopsin gene (P23H transgenic rat Line 1) were administered either a mouse-specific P23H ASO or a control ASO. The contralateral eye was injected with PBS and used as a comparator control. Electroretinography (ERG) measurements and analyses of the retinal outer nuclear layer were conducted and correlated with rhodopsin mRNA levels. RESULTS: Rhodopsin mRNA and protein expression was reduced after a single ASO injection in wild-type mice with a rhodopsin-specific ASO. Transgenic rat eyes that express a murine P23H rhodopsin gene injected with a murine P23H ASO had a 181 ± 39% better maximum amplitude response (scotopic a-wave) as compared with contralateral PBS-injected eyes; the response in control ASO eyes was not significantly different from comparator contralateral eyes. Morphometric analysis of the outer nuclear layer showed a significantly thicker nuclear layer in eyes injected with murine P23H ASO (18%) versus contralateral PBS-injected eyes. CONCLUSIONS: Allele-specific ASO-mediated knockdown of mutant P23H rhodopsin expression slowed the rate of photoreceptor degeneration and preserved the function of photoreceptor cells in eyes of the P23H rhodopsin transgenic rat. Our data indicate that ASO treatment is a potentially effective therapy for the treatment of retinitis pigmentosa.
Assuntos
Regulação da Expressão Gênica , Degeneração Macular/prevenção & controle , Oligonucleotídeos Antissenso/genética , RNA Mensageiro/genética , Rodopsina/genética , Alelos , Animais , Western Blotting , Modelos Animais de Doenças , Eletrorretinografia , Degeneração Macular/genética , Degeneração Macular/metabolismo , Masculino , Camundongos , Ratos , Ratos Transgênicos , Reação em Cadeia da Polimerase em Tempo Real , Rodopsina/biossínteseRESUMO
PURPOSE: Verteporfin photodynamic therapy (PDT) is the most effective treatment for age-related macular degeneration, using laser activation of a photosensitizing dye to achieve closure of choroidal neovascularization. Although PDT preferentially affects pathologic vessels, it can also cause collateral damage to the overlying retina. In the current study, it was found that the neuroprotective agent brain-derived neurotrophic factor (BDNF) reduces this retinal damage. METHODS: Normal adult rats received intravitreal BDNF in one eye and PBS or no injection in the other eye 2 days before PDT. RESULTS: Control eyes exhibited choroidal hypofluorescence, moderate to severe photoreceptor loss, and depression of local retinal function measured using multifocal ERG in the laser-treated area. BDNF-injected eyes had more surviving photoreceptors and improved multifocal ERG responses 1 week after PDT. BDNF did not diminish the effect of PDT on the choroidal circulation as assessed by fluorescein angiography, and there was no evidence of retinal toxicity due to BDNF treatment. CONCLUSIONS: These results suggest that adjunctive neuroprotective therapy may reduce collateral damage to photoreceptors and improve visual outcome after PDT.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , Fármacos Neuroprotetores/farmacologia , Fotoquimioterapia , Fármacos Fotossensibilizantes/toxicidade , Porfirinas/toxicidade , Retina/efeitos dos fármacos , Animais , Corioide/irrigação sanguínea , Corioide/efeitos dos fármacos , Corioide/patologia , Eletrorretinografia/efeitos dos fármacos , Angiofluoresceinografia , Fluorescência , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/patologia , Ratos , Ratos Endogâmicos BN , Retina/patologia , VerteporfinaRESUMO
PURPOSE: To determine whether mice that are homozygous for a targeted disruption of the Mer receptor tyrosine kinase gene (mer(kd)) manifest a retinal dystrophy phenotype similar to RCS rats, which carry a mutation in the orthologous gene MERTK: METHODS: Eyes of mer(kd) and C57BL/6 wild-type (WT) mice were examined by light and electron microscopy, whole-eye rhodopsin measurement, and Ganzfeld electroretinography (ERG). RESULTS: The mer(kd) mice showed rapid, progressive degeneration of the photoreceptors (PRs). Features of the phenotype common to mer(kd) mice and RCS rats included the absence or near absence of phagosomes in the retinal pigment epithelium (RPE) at the peak of outer segment (OS) disc shedding, accumulation of debris and whorls of membranes at the RPE-OS interface, transient supernormal rhodopsin content and OS lengths, the presence of OS vacuoles beginning at early ages, and a relatively slow removal of pyknotic PR nuclei. Most PRs were missing, and OS debris was removed by approximately postnatal day (P)45. Scotopic ERG responses were lower than age-matched WT responses and declined with PR loss. Photopic responses were preserved better than scotopic responses, corresponding with preferential cone preservation as judged histologically. ERG amplitudes were usually unmeasurable beyond P40, although a small-amplitude scotopic threshold response (STR) could still be elicited at P253 in some mice when only scattered PR nuclei remained. CONCLUSIONS: Ablation of Mer function in mer(kd) mice results in a retinal phenotype almost identical with that of RCS rats. The similarity in phenotypes between the two rodent models suggests that an RPE phagocytic defect is a feature of all types of retinal degeneration caused by loss of function of Mer tyrosine kinase, perhaps including mutations in human MERTK.
Assuntos
Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases , Retina/ultraestrutura , Degeneração Retiniana/enzimologia , Degeneração Retiniana/patologia , Animais , Eletrorretinografia , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose , Fagossomos/patologia , Fenótipo , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/metabolismo , Retina/enzimologia , Rodopsina/metabolismo , c-Mer Tirosina QuinaseRESUMO
PURPOSE: Previous studies have shown that the level of docosahexaenoic acid (22:6n-3, DHA) is lower in the rod outer segment (ROS) membranes of dogs and mice with inherited retinal degeneration than in ROS from appropriate controls. In the present study, we analyzed the ROS fatty composition of several lines of transgenic rats with P23H and S334ter rhodopsin mutations. Lines were chosen that have different rates of retinal degeneration. METHODS: At 21-22 days of age, animals were perfused and eyes fixed and sectioned for morphologic examination. Others were killed and retinas isolated for preparation of ROS by sucrose step-gradient centrifugation. Fatty acid composition of ROS phospholipids was determined by gas-liquid chromatography. Membrane purity was assessed by polyacrylamide gel electrophoresis. RESULTS: Retinas of the slow degenerating lines were indistinguishable from controls, whereas there was a 15-20% and 50-60% loss of photoreceptor cell nuclei in intermediate and fast degenerating lines, respectively. Except for the slow P23H line, all mutant lines had lower levels of 22:6n-3 and total n-3 fatty acids in ROS phospholipids, compared to wild-type controls, and the level of 22:6n-3 was lowest in those lines with the fastest rate of degeneration. The relative levels of the other fatty acid families (saturated, monoenoic, and n-6) increased proportionately. The n-6/n-3 ratio increased in the more rapidly degenerating lines, but the phospholipid/protein ratios did not change. The low levels of 22:6n-3 in the ROS membranes were not compensated for by an increase in 22:5n-6, which always occurs in the retina of animals where 22:6n-3 levels are reduced by dietary manipulation. CONCLUSIONS: Rats that express mutant rhodopsins have lower levels of 22:6n-3 in their ROS phospholipids than wild-type animals. We propose that photoreceptor-specific mutations provoke a metabolic stress in rod photoreceptor cells that generates an oxidant stress in these cells. The retina responds to this stress by reducing the level of substrate for lipid peroxidation (22:6n-3).
Assuntos
Ácidos Docosa-Hexaenoicos/metabolismo , Mutação , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Rodopsina/genética , Segmento Externo da Célula Bastonete/metabolismo , Animais , Animais Geneticamente Modificados , Membrana Celular/metabolismo , Cromatografia Gasosa , Eletroforese em Gel de Poliacrilamida , Ratos , Ratos Sprague-Dawley , Degeneração Retiniana/patologiaRESUMO
Huntington disease (HD) is an inherited neurodegenerative disease that affects multiple brain regions. It is caused by an expanded polyglutamine tract in huntingtin (Htt). The development of therapies for HD and other neurodegenerative diseases has been hampered by multiple factors, including the lack of clear therapeutic targets, and the cost and complexity of testing lead compounds in vivo. The R6/2 HD mouse model is widely used for pre-clinical trials because of its progressive and robust neural dysfunction, which includes retinal degeneration. Profilin-1 is a Htt binding protein that inhibits Htt aggregation. Its binding to Htt is regulated by the rho-associated kinase (ROCK), which phosphorylates profilin at Ser-137. ROCK is thus a therapeutic target in HD. The ROCK inhibitor Y-27632 reduces Htt toxicity in fly and mouse models. Here we characterized the progressive retinopathy of R6/2 mice between 6-19 weeks of age to determine an optimal treatment window. We then tested a clinically approved ROCK inhibitor, HA-1077, administered intravitreally via liposome-mediated drug delivery. HA-1077 increased photopic and flicker ERG response amplitudes in R6/2 mice, but not in wild-type littermate controls. By targeting ROCK with a new inhibitor, and testing its effects in a novel in vivo model, these results validate the in vivo efficacy of a therapeutic candidate, and establish the feasibility of using the retina as a readout for CNS function in models of neurodegenerative disease.