RESUMO
The diurnal peak of phagocytosis by the retinal pigment epithelium (RPE) of photoreceptor outer segments (POS) is under circadian control and believed that this process involves interactions from the retina and RPE. Previous studies have demonstrated that a functional circadian clock exists within multiple retinal cell types and RPE. Thereby, the aim of this study was to determine whether the clock in the retina or RPE controls the diurnal phagocytic peak and whether disruption of the circadian clock in the RPE would affect cellular function and the viability during aging. To that, we generated and validated an RPE tissue-specific KO of the essential clock gene, Bmal1, and then determined the daily rhythm in phagocytic activity by the RPE in mice lacking a functional circadian clock in the retina or RPE. Then, using electroretinography, spectral domain-optical coherence tomography, and optomotor response of visual function we determined the effect of Bmal1 removal in young (6 months) and old (18 months) mice. RPE morphology and lipofuscin accumulation was determined in young and old mice. Our data shows that the clock in the RPE, rather than the retina clock, controls the diurnal phagocytic peak. Surprisingly, absence of a functional RPE clock and phagocytic peak does not result in any detectable age-related degenerative phenotype in the retina or RPE. Thus, our results demonstrate that the circadian clock in the RPE controls the daily peak of phagocytic activity. However, the absence of the clock in the RPE does not result in deterioration of photoreceptors or the RPE during aging.
Assuntos
Relógios Circadianos , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Animais , Ritmo Circadiano/fisiologia , Camundongos , Fagócitos , Epitélio Pigmentado da Retina/metabolismoRESUMO
Purpose: The present study tested the hypothesis that connexin-36 (Cx36) and gap junctions between photoreceptor cells contribute to the circadian rhythm of the photopic electroretinogram (ERG) b-wave amplitude. Methods: Cone-specific disruption of Cx36 was obtained in mice with a floxed Gjd2 gene and human red/green pigment promoter (HRGP)-driven Cre recombinase. Standard ERG, spectral-domain optical coherence tomography (SD-OCT) and histochemical methods were used. Results: HRGPcreGjd2fl/fl mice had a selective reduction in Cx36 protein in the outer plexiform layer; no reduction in Cx36 was observed in the inner plexiform layer. Cx36 disruption had no effect on the number of cones, the thickness of the photoreceptor layer, or the scotopic ERG responses. However, there was a reduction of the photopic ERG circadian rhythm, with b-wave amplitudes in the day and the night locked in the daytime, light-adapted state. In HRGPcreGjd2+/+and Gjd2fl/fl controls, the circadian rhythm of light-adapted ERG persisted, similar to that in wild type mice. Conclusions: Cx36 regulation contributes to the circadian rhythm of light-adapted ERG; in the absence of photoreceptor gap junctions, mice appear to be in a fully light-adapted state regardless of the time of day. The higher amplitudes and reduced circadian regulation of the b-wave of HRGPcreGjd2fl/fl mice may be due to increased synaptic strength at the cone to ON bipolar cell synapse due to electrotonic isolation of the terminals lacking gap junctions.
Assuntos
Adaptação Ocular/fisiologia , Ritmo Circadiano/fisiologia , Conexinas/metabolismo , Adaptação à Escuridão/fisiologia , Eletrorretinografia/métodos , Células Fotorreceptoras Retinianas Cones/metabolismo , Animais , Junções Comunicantes , Camundongos , Camundongos Transgênicos , Modelos Animais , Proteína delta-2 de Junções ComunicantesRESUMO
Purpose: To explore the impact of ocular surface insults on the immunomodulatory capacity and phenotype of corneal epithelial cells (CECs) with a focus on epithelial-mesenchymal transition (EMT). Methods: Corneas were harvested from mice 6 days following scratch injury, ragweed pollen-induced allergy, or herpes simplex virus type 1 (HSV-1) infection and compared to healthy tissue controls. Corneas were enzymatically digested and CECs phenotypically characterized using flow cytometry. CECs were defined as epithelial cell adhesion molecule (EpCAM)-positive CD45-negative cells. CECs were assessed by PCR to evaluate EMT-associated transcripts. Recombinant HSV-1 and transgenic mice were utilized to investigate the role of vascular endothelial growth factor A (VEGFA) on the phenotype observed. The immunomodulatory potential of CECs was assessed in coculture assays with ovalbumin-specific CD4 T cells. Results: Ectopic expression of classic "myeloid" antigens Ly6G, CCR2, and CX3CR1 was identified in CEC subsets from all groups with evidence supporting an underlying partial EMT event resulting from loss of cell-cell contacts. Corneal HSV-1 infection induced Ly6C expression and major histocompatibility complex (MHC)-II upregulation in CECs through a VEGFA-linked mechanism. These Ly6C+ MHC-II+ CECs were found to function as amateur antigen-presenting cells and induced CD4 T cell proliferation in vitro. Conclusions: This study characterizes a novel immunomodulatory CEC phenotype with possible implications for immune privilege, chronic inflammation, and tissue fibrosis. Moreover, the identification of CECs masquerading with multiple "myeloid" antigens warrants careful evaluation of flow cytometry data involving corneal digests.
Assuntos
Doenças da Córnea/imunologia , Células Epiteliais/imunologia , Transição Epitelial-Mesenquimal/imunologia , Epitélio Corneano/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Células Mieloides/imunologia , Animais , Modelos Animais de Doenças , CamundongosRESUMO
Recent work suggested that the activity of extracellular signal-regulated kinase 1/2 (ERK1/2) is increased in the retinal pigment epithelium (RPE) of age-related macular degeneration (ARMD) patients and therefore could be an attractive therapeutic target. Notably, ERK1/2 pathway inhibitors are used in cancer therapy, with severe and noncharacterized ocular side effects. To decipher the role of ERK1/2 in RPE cells, we conditionally disrupted the Erk1 and Erk2 genes in mouse RPE. The loss of ERK1/2 activity resulted in a significant decrease in the level of RPE65 expression, a decrease in ocular retinoid levels concomitant with low visual function, and a rapid disorganization of RPE cells, ultimately leading to retinal degeneration. Our results identify the ERK1/2 pathway as a direct regulator of the visual cycle and a critical component of the viability of RPE and photoreceptor cells. Moreover, our results caution about the need for a very fine adjustment of kinase inhibition in cancer or ARMD treatment in order to avoid ocular side effects.
Assuntos
Sistema de Sinalização das MAP Quinases , Degeneração Macular/metabolismo , Epitélio Pigmentado da Retina/metabolismo , cis-trans-Isomerases/metabolismo , Animais , Degeneração Macular/terapia , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Modelos Animais , Retina/metabolismo , Retinoides/genética , Retinoides/metabolismo , cis-trans-Isomerases/genéticaRESUMO
This overview introduces contributions to a special issue on causes of vision loss from diabetes mellitus, focusing on the retina and also the cornea. Diabetic retinopathy is the most common and leading cause of vision loss among people with diabetes. Research to detect early symptoms, understand mechanisms leading to diabetic eye disease, and the development of treatments is a highly active research area, with currently about 2000 scientific publication per year. We provide a series of 27 comprehensive reviews and research articles from leading experts in the field.
Assuntos
Retinopatia Diabética , Diabetes Mellitus/fisiopatologia , Humanos , Transtornos da Visão/fisiopatologiaRESUMO
An appropriate animal model is essential to screening drugs or designing a treatment strategy for geographic atrophy. Since oxidative stress contributes to the pathological changes of the retinal pigment epithelium (RPE), we are reporting a new mouse AMD model of retinal degeneration by inducing mitochondrial oxidative stress in RPE. Sod2 the gene for manganese superoxide dismutase (MnSOD) was deleted in RPE layer using conditional knockout strategy. Fundus microscopy, SD-OCT and electroretinography were used to monitor retinal structure and function in living animals and microscopy was used to assess pathology post mortem. Tissue specific deletion of Sod2 caused elevated signs of oxidative stress, RPE dysfunction and showed some key features of AMD. Due to induction of oxidative stress, the conditional knockout mice show progressive reduction in ERG responses and thinning of outer nuclear layer (ONL) compared to non-induced littermates.
Assuntos
Modelos Animais de Doenças , Degeneração Macular/genética , Estresse Oxidativo , Degeneração Retiniana/genética , Epitélio Pigmentado da Retina/metabolismo , Superóxido Dismutase/genética , Animais , Bestrofinas , Eletrorretinografia , Proteínas do Olho/genética , Feminino , Humanos , Imuno-Histoquímica , Canais Iônicos/genética , Degeneração Macular/metabolismo , Degeneração Macular/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Oftalmoscópios , Oftalmoscopia/métodos , Degeneração Retiniana/metabolismo , Degeneração Retiniana/fisiopatologia , Epitélio Pigmentado da Retina/patologia , Epitélio Pigmentado da Retina/fisiopatologia , Superóxido Dismutase/deficiência , Superóxido Dismutase/metabolismo , Tomografia de Coerência ÓpticaRESUMO
Cones are the primary photoreceptor (PR) cells responsible for vision in humans. They are metabolically highly active requiring phosphoinositide 3-kinase (PI3K) activity for long-term survival. One of the downstream targets of PI3K is the kinase mammalian target of rapamycin (mTOR), which is a key regulator of cell metabolism and growth, integrating nutrient availability and growth factor signals. Both PI3K and mTOR are part of the insulin/mTOR signaling pathway, however if mTOR is required for long-term PR survival remains unknown. This is of particular interest since deregulation of this pathway in diabetes results in reduced PR function before the onset of any clinical signs of diabetic retinopathy. mTOR is found in two distinct complexes (mTORC1 & mTORC2) that are characterized by their unique accessory proteins RAPTOR and RICTOR respectively. mTORC1 regulates mainly cell metabolism in response to nutrient availability and growth factor signals, while mTORC2 regulates pro-survival mechanisms in response to growth factors. Here we analyze the effect on cones of loss of mTORC1, mTORC2 and simultaneous loss of mTORC1 & mTORC2. Interestingly, neither loss of mTORC1 nor mTORC2 affects cone function or survival at one year of age. However, outer and inner segment morphology is affected upon loss of either complex. In contrast, concurrent loss of mTORC1 and mTORC2 leads to a reduction in cone function without affecting cone viability. The data indicates that PI3K mediated pro-survival signals diverge upstream of both mTOR complexes in cones, suggesting that they are independent of mTOR activity. Furthermore, the data may help explain why PR function is reduced in diabetes, which can lead to deregulation of both mTOR complexes simultaneously. Finally, although mTOR is a key regulator of cell metabolism, and PRs are metabolically highly active, the data suggests that the role of mTOR in regulating the metabolic transcriptome in healthy cones is minimal.
Assuntos
Complexos Multiproteicos/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Animais , Sobrevivência Celular , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/fisiopatologia , Modelos Animais de Doenças , Eletrorretinografia , Proteínas do Olho/metabolismo , Imunossupressores/uso terapêutico , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/fisiologia , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Sirolimo/uso terapêuticoRESUMO
Retinitis pigmentosa (RP) is an inherited photoreceptor degenerative disorder that results in blindness. The disease is often caused by mutations in genes that are specific to rod photoreceptors; however, blindness results from the secondary loss of cones by a still unknown mechanism. Here, we demonstrated that the mammalian target of rapamycin complex 1 (mTORC1) is required to slow the progression of cone death during disease and that constitutive activation of mTORC1 in cones is sufficient to maintain cone function and promote long-term cone survival. Activation of mTORC1 in cones enhanced glucose uptake, retention, and utilization, leading to increased levels of the key metabolite NADPH. Moreover, cone death was delayed in the absence of the NADPH-sensitive cell death protease caspase 2, supporting the contribution of reduced NADPH in promoting cone death. Constitutive activation of mTORC1 preserved cones in 2 mouse models of RP, suggesting that the secondary loss of cones is caused mainly by metabolic deficits and is independent of a specific rod-associated mutation. Together, the results of this study address a longstanding question in the field and suggest that activating mTORC1 in cones has therapeutic potential to prolong vision in RP.
Assuntos
Complexos Multiproteicos/fisiologia , Células Fotorreceptoras Retinianas Cones/patologia , Retinose Pigmentar/patologia , Serina-Treonina Quinases TOR/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Animais , Apoptose , Caspase 2/deficiência , Caspase 2/fisiologia , Sobrevivência Celular , Glucose/metabolismo , Insulina/farmacologia , Insulina/uso terapêutico , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Modelos Neurológicos , NADP/fisiologia , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/fisiologia , Proteína Regulatória Associada a mTOR , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologia , Retinose Pigmentar/genética , Retinose Pigmentar/terapia , Transdução de Sinais/fisiologiaRESUMO
Dysfunction or death of photoreceptors is the primary cause of vision loss in retinal and macular degenerative diseases. As photoreceptors have an intimate relationship with the retinal pigment epithelium (RPE) for exchange of macromolecules, removal of shed membrane discs and retinoid recycling, an improved understanding of the development of the photoreceptor-RPE complex will allow better design of gene- and cell-based therapies. To explore the epigenetic contribution to retinal development we generated conditional knockout alleles of DNA methyltransferase 1 (Dnmt1) in mice. Conditional Dnmt1 knockdown in early eye development mediated by Rx-Cre did not produce lamination or cell fate defects, except in cones; however, the photoreceptors completely lacked outer segments despite near normal expression of phototransduction and cilia genes. We also identified disruption of RPE morphology and polarization as early as E15.5. Defects in outer segment biogenesis were evident with Dnmt1 exon excision only in RPE, but not when excision was directed exclusively to photoreceptors. We detected a reduction in DNA methylation of LINE1 elements (a measure of global DNA methylation) in developing mutant RPE as compared with neural retina, and of Tuba3a, which exhibited dramatically increased expression in mutant retina. These results demonstrate a unique function of DNMT1-mediated DNA methylation in controlling RPE apicobasal polarity and neural retina differentiation. We also establish a model to study the epigenetic mechanisms and signaling pathways that guide the modulation of photoreceptor outer segment morphogenesis by RPE during retinal development and disease.
Assuntos
Permeabilidade da Membrana Celular/fisiologia , DNA (Citosina-5-)-Metiltransferases/genética , Morfogênese/genética , Segmento Externo das Células Fotorreceptoras da Retina/fisiologia , Epitélio Pigmentado da Retina/fisiologia , Animais , Permeabilidade da Membrana Celular/genética , Polaridade Celular/genética , DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA/genética , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Transgênicos , Análise em Microsséries , Morfogênese/fisiologia , Especificidade de Órgãos/genética , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Epitélio Pigmentado da Retina/embriologia , Epitélio Pigmentado da Retina/crescimento & desenvolvimento , Epitélio Pigmentado da Retina/metabolismo , TranscriptomaRESUMO
PURPOSE: Phosphoinositide 3-kinase (PI3K) consists of a p110 catalytic protein and a p85α regulatory protein, required for the stabilization and localization of p110-PI3K activity. The biological significance of PI3K was investigated in vertebrate rod photoreceptors by deleting its regulatory p85α protein and examining its role in photoreceptor structure, function, and protein trafficking. METHODS: Mice that expressed Cre recombinase in rods were bred to mice with a floxed p85α (pik3r1) regulatory subunit of PI3K to generate a conditional deletion of pik3r1 in rods. Functional and structural changes were determined by ERG and morphometric analysis, respectively. PI3K activity was measured in retinal homogenates immunoprecipitated with an anti-PY antibody. Akt activation was determined by Western blot analysis with a pAkt antibody. RESULTS: Light-induced stress increased PI3K activity in retinal immunoprecipitates and phosphorylation of Akt. There was no effect of pik3r1 deletion on retinal structure. However, twin flash electroretinography revealed a slight delay in recovery kinetics in pik3r1 knockout (KO) mice compared with wild-type controls. The movement of arrestin in the pik3r1 KO mice was slower than that in the wild-type mouse retinas at 5 minutes of exposure to light. At 10 minutes of exposure, the ROS localization of arrestin was almost identical between the wild-type and pik3r1 KO mice. CONCLUSIONS: The results provide the first direct evidence that rods use PI3K-generated phosphoinositides for photoreceptor function. The lack of phenotype in pik3r1 KO rod photoreceptors suggests a redundant role in controlling PIP(3) synthesis.
Assuntos
Fosfatidilinositol 3-Quinase/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/enzimologia , Transdução de Sinais/fisiologia , Animais , Arrestina/metabolismo , Western Blotting , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Eletrorretinografia , Deleção de Genes , Integrases/genética , Luz/efeitos adversos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Fosforilação , Estimulação Luminosa , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Lesões Experimentais por Radiação/enzimologia , Ratos , Degeneração Retiniana/enzimologia , Células Fotorreceptoras Retinianas Bastonetes/efeitos da radiação , Rodopsina/metabolismo , Transducina/metabolismoRESUMO
PURPOSE: Downregulation of the retinal insulin/mTOR pathway in mouse models of retinitis pigmentosa is linked to cone cell death, which can be delayed by systemic administration of insulin. A classic survival kinase linking extracellular trophic/growth factors with intracellular antiapoptotic pathways is phosphoinositide 3-kinase (PI3K), which the authors have shown to protect rod photoreceptors from stress-induced cell death. The role of PI3K in cones was studied by conditional deletion of its p85α regulatory subunit. METHODS: Mice expressing Cre recombinase in cones were bred to mice with a floxed pi3k gene encoding the p85α regulatory subunit of the PI3K and were back-crossed to ultimately generate offspring with cone-specific p85α knockout (cKO). Cre expression and cone-specific localization were confirmed by Western blot analysis and immunohistochemistry (IHC), respectively. Cone structural integrity was determined by IHC using peanut agglutinin and an M-opsin-specific antibody. Electroretinography (ERG) was used to assess rod and cone photoreceptor function. Retinal structure was examined by light and electron microscopy. RESULTS: An age-related cone degeneration was found in cKO mice, evidenced by a reduction in photopic ERG amplitudes and loss of cone cells. By 12 months of age, approximately 78% of cones had died, and progressive disorganization of synaptic ultrastructure was noted in surviving cone terminals in cKO retinas. Rod viability was unaffected in p85α cKO mice. CONCLUSIONS: The present study suggests that PI3K signaling pathway is essential for cone survival in the mouse retina.
Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/fisiologia , Células Fotorreceptoras Retinianas Cones/enzimologia , Degeneração Retiniana/enzimologia , Animais , Arrestina/metabolismo , Western Blotting , Linhagem Celular , Sobrevivência Celular , Eletrorretinografia , Feminino , Deleção de Genes , Imuno-Histoquímica , Integrases , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Opsinas/metabolismo , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Degeneração Retiniana/patologiaRESUMO
Heterotrimeric kinesin-II is a molecular motor localized to the inner segment, connecting cilium and axoneme of mammalian photoreceptors. Our purpose was to identify the role of kinesin-II in anterograde intraflagellar transport by photoreceptor-specific deletions of kinesin family member 3A (KIF3A), its obligatory motor subunit. In cones lacking KIF3A, membrane proteins involved in phototransduction did not traffic to the outer segments resulting in complete absence of a photopic electroretinogram and progressive cone degeneration. Rod photoreceptors lacking KIF3A degenerated rapidly between 2 and 4 weeks postnatally, but the phototransduction components including rhodopsin trafficked to the outer segments during the course of degeneration. Furthermore, KIF3A deletion did not affect synaptic anterograde trafficking. The results indicate that trafficking of membrane proteins to the outer segment is dependent on kinesin-II in cone, but not rod photoreceptors, even though rods and cones share similar structures, and closely related phototransduction polypeptides.
Assuntos
Cinesinas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Animais , Cinesinas/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Transporte Proteico/fisiologia , Retina/crescimento & desenvolvimento , Retina/fisiopatologia , Retina/ultraestrutura , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Degeneração Retiniana/fisiopatologia , Pigmentos da Retina/metabolismo , Rodopsina/metabolismo , Sinapses/fisiologia , Fatores de TempoRESUMO
Insulin receptor (IR) signaling provides a trophic signal for transformed retinal neurons in culture, but the role of IR activity in vivo is unknown. We previously reported that light causes increased tyrosine phosphorylation of the IR in vivo, which leads to the downstream activation of the phosphoinositide 3-kinase and Akt pathway in rod photoreceptor cells. The functional role of IR in rod photoreceptor cells is not known. We observed that light stress induced tyrosine phosphorylation of the IR in rod photoreceptor cells, and we hypothesized that IR activation is neuroprotective. To determine whether IR has a neuroprotective role on rod photoreceptor cells, we used the Cre/lox system to specifically inactivate the IR gene in rod photoreceptors. Rod-specific IR knock-out mice have reduced the phosphoinositide 3-kinase and Akt survival signal in rod photoreceptors. The resultant mice exhibited no detectable phenotype when they were raised in dim cyclic light. However, reduced IR expression in rod photoreceptors significantly decreased retinal function and caused the loss of photoreceptors in mice exposed to bright light stress. These results indicate that reduced expression of IR in rod photoreceptor cells increases their susceptibility to light-induced photoreceptor degeneration. These data suggest that the IR pathway is important for photoreceptor survival and that activation of the IR may be an essential element of photoreceptor neuroprotection.