RESUMO
Disorders of vascular structure and function play a central role in a wide variety of CNS diseases. Mutations in the Frizzled-4 (Fz4) receptor, Lrp5 coreceptor, or Norrin ligand cause retinal hypovascularization, but the mechanisms by which Norrin/Fz4/Lrp signaling controls vascular development have not been defined. Using mouse genetic and cell culture models, we show that loss of Fz4 signaling in endothelial cells causes defective vascular growth, which leads to chronic but reversible silencing of retinal neurons. Loss of Fz4 in all endothelial cells disrupts the blood brain barrier in the cerebellum, whereas excessive Fz4 signaling disrupts embryonic angiogenesis. Sox17, a transcription factor that is upregulated by Norrin/Fz4/Lrp signaling, plays a central role in inducing the angiogenic program controlled by Norrin/Fz4/Lrp. These experiments establish a cellular basis for retinal hypovascularization diseases due to insufficient Frizzled signaling, and they suggest a broader role for Frizzled signaling in vascular growth, remodeling, maintenance, and disease.
Assuntos
Células Endoteliais/metabolismo , Proteínas do Olho/metabolismo , Receptores Frizzled/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Neovascularização Fisiológica , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Neurônios Retinianos/metabolismo , Transdução de Sinais , Animais , Cerebelo/metabolismo , Receptores Frizzled/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Receptores Acoplados a Proteínas G/genética , Retina/citologia , Retina/metabolismo , Proteínas Wnt/metabolismoRESUMO
Photoreceptors consume glucose supplied by the choriocapillaris to support phototransduction and outer segment (OS) renewal. Reduced glucose supply underlies photoreceptor cell death in inherited retinal degeneration and age-related retinal disease. We have previously shown that restricting glucose transport into the outer retina by conditional deletion of Slc2a1 encoding GLUT1 resulted in photoreceptor loss and impaired OS renewal. However, retinal neurons, glia, and the retinal pigment epithelium play specialized, synergistic roles in metabolite supply and exchange, and the cell-specific map of glucose uptake and utilization in the retina is incomplete. In these studies, we conditionally deleted Slc2a1 in a pan-retinal or rod-specific manner to better understand how glucose is utilized in the retina. Using non-invasive ocular imaging, electroretinography, and histochemical and biochemical analyses we show that genetic deletion of Slc2a1 from retinal neurons and Müller glia results in reduced OS growth and progressive rod but not cone photoreceptor cell death. Rhodopsin levels were severely decreased even at postnatal day 20 when OS length was relatively normal. Arrestin levels were not changed suggesting that glucose uptake is required to synthesize membrane glycoproteins. Rod-specific deletion of Slc2a1 resulted in similar changes in OS length and rod photoreceptor cell death. These studies demonstrate that glucose is an essential carbon source for rod photoreceptor cell OS maintenance and viability.
Assuntos
Transportador de Glucose Tipo 1 , Glucose , Células Fotorreceptoras Retinianas Cones , Degeneração Retiniana , Segmento Externo da Célula Bastonete , Glucose/metabolismo , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Humanos , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Segmento Externo da Célula Bastonete/metabolismo , Segmento Externo da Célula Bastonete/patologiaRESUMO
With recent advancements in artificial intelligence, fundus diseases can be classified automatically for early diagnosis, and this is an interest of many researchers. The study aims to detect the edges of the optic cup and the optic disc of fundus images taken from glaucoma patients, which has further applications in the analysis of the cup-to-disc ratio (CDR). We apply a modified U-Net model architecture on various fundus datasets and use segmentation metrics to evaluate the model. We apply edge detection and dilation to post-process the segmentation and better visualize the optic cup and optic disc. Our model results are based on ORIGA, RIM-ONE v3, REFUGE, and Drishti-GS datasets. Our results show that our methodology obtains promising segmentation efficiency for CDR analysis.
Assuntos
Glaucoma , Disco Óptico , Humanos , Animais , Disco Óptico/diagnóstico por imagem , Inteligência Artificial , Glaucoma/diagnóstico , Fundo de Olho , AbomasoRESUMO
PURPOSE: To find a new approach of pan-retinal photocoagulation (PRP) with less damage to the retina in the treatment of severe non-proliferative diabetic retinopathy (NPDR), this study compared functional changes in the retina after subthreshold and threshold PRP treatment in severe NPDR eyes. METHODS: Post hoc analysis of a randomized clinical trial was conducted in this study. Seventy eyes of 35 patients with bilateral, symmetric, severe NPDR were enrolled. Two eyes from the same patient were randomized into two groups, one eye received subthreshold PRP (S-PRP) and the other eye received threshold PRP (T-PRP). Comprehensive ophthalmological evaluations were performed on the baseline and every 3 months for 1 year. Visual field (VF) and full-field electroretinography (ERG) were performed on the baseline and repeated at month 12. RESULTS: During the 12-month follow-up, 4 eyes (11.4%) in the S-PRP group and 3 eyes (8.6%) in the T-PRP group progressed to proliferative diabetic retinopathy (PDR) stage, and there was no statistical difference in PDR progression rate between the two groups (P = 0.69). In addition, the changes in best-corrected visual acuity (BCVA) from baseline to month 12 between the two groups had no statistical difference (P = 0.30). From baseline to month 12, changes in central VF between the two groups had no statistical difference (P = 0.25), but changes in total score points of peripheral VF in the S-PRP group (- 242.1 ± 210.8 dB) and the T-PRP group (- 308.9 ± 209.7 dB) were statistically significant (P = 0.03). At month 12, ERG records showed that the amplitude of dark-adapted 0.01 ERG, dark-adapted 3.0 ERG, oscillatory potentials, light-adapted 3.0 ERG, and 30 Hz flicker ERG of both groups were significantly decreased from the baseline (P < 0.05). In addition, the amplitude of each ERG record in the S-PRP group decreased significantly less than those in the T-PRP group (P < 0.05). CONCLUSIONS: Subthreshold PRP is as effective as threshold PRP for preventing severe NPDR progress to PDR within 1 year with less damage to periphery VF and retinal function. CLINICALTRIALS: gov Identifier: NCT01759121.
Assuntos
Diabetes Mellitus , Retinopatia Diabética , Humanos , Retinopatia Diabética/cirurgia , Fotocoagulação a Laser , Retina/cirurgia , Eletrorretinografia , Tomografia de Coerência ÓpticaRESUMO
Fluid and solute transporters of the retinal pigment epithelium (RPE) are core components of the outer blood-retinal barrier. Characterizing these transporters and their role in retinal homeostasis may provide insights into ocular function and disease. Here, we describe RPE defects in tvrm77 mice, which exhibit hypopigmented patches in the central retina. Mapping and nucleotide sequencing of tvrm77 mice revealed a disrupted 5' splice donor sequence in Slc4a5, a sodium bicarbonate cotransporter gene. Slc4a5 expression was reduced 19.7-fold in tvrm77 RPE relative to controls, and alternative splice variants were detected. SLC4A5 was localized to the Golgi apparatus of cultured human RPE cells and in apical and basal membranes. Fundus imaging, optical coherence tomography, microscopy, and electroretinography (ERG) of tvrm77 mice revealed retinal detachment, hypopigmented patches corresponding to neovascular lesions, and retinal folds. Detachment worsened and outer nuclear layer thickness decreased with age. ERG a- and b-wave response amplitudes were initially normal but declined in older mice. The direct current ERG fast oscillation and light peak were reduced in amplitude at all ages, whereas other RPE-associated responses were unaffected. These results link a new Slc4a5 mutation to subretinal fluid accumulation and altered light-evoked RPE electrophysiological responses, suggesting that SLC4A5 functions at the outer blood-retinal barrier.
Assuntos
Mutação/genética , Splicing de RNA/genética , Retina/patologia , Descolamento Retiniano/genética , Epitélio Pigmentado da Retina/patologia , Simportadores de Sódio-Bicarbonato/genética , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Descolamento Retiniano/patologia , Tomografia de Coerência Óptica/métodosRESUMO
A single pool of multipotent retinal progenitor cells give rise to the diverse cell types within the mammalian retina. Such cellular diversity is due to precise control of various cellular processes like cell specification, proliferation, differentiation, and maturation. Circadian clock genes can control the expression of key regulators of cell cycle progression and therefore can synchronize the cell cycle state of a heterogeneous population of cells. Here we show that the protein encoded by the circadian clock gene brain and muscle arnt-like protein-1 (Bmal1) is expressed in the embryonic retina and is required to regulate the timing of cell cycle exit. Accordingly, loss of Bmal1 during retinal neurogenesis results in increased S-phase entry and delayed cell cycle exit. Disruption in cell cycle kinetics affects the timely generation of the appropriate neuronal population thus leading to an overall decrease in the number of retinal ganglion cells, amacrine cells, and an increase in the number of the late-born type II cone bipolar cells as well as the Müller glia. Additionally, the mislocalized Müller cells are observed in the photoreceptor layer in the Bmal1 conditional mutants. These changes affect the functional integrity of the visual circuitry as we report a significant delay in visual evoked potential implicit time in the retina-specific Bmal1 null animals. Our results demonstrate that Bmal1 is required to maintain the balance between the neural and glial cells in the embryonic retina by coordinating the timing of cell cycle entry and exit. Thus, Bmal1 plays an essential role during retinal neurogenesis affecting both development and function of the mature retina.-Sawant, O. B., Jidigam, V. K., Fuller, R. D., Zucaro, O. F., Kpegba, C., Yu, M., Peachey, N. S., Rao, S. The circadian clock gene Bmal1 is required to control the timing of retinal neurogenesis and lamination of Müller glia in the mouse retina.
Assuntos
Fatores de Transcrição ARNTL/metabolismo , Células Ependimogliais/metabolismo , Neurogênese , Retina/citologia , Fatores de Transcrição ARNTL/genética , Células Amácrinas/citologia , Células Amácrinas/metabolismo , Animais , Ciclo Celular , Relógios Circadianos , Células Ependimogliais/citologia , Potenciais Evocados Visuais , Camundongos , Retina/embriologia , Retina/metabolismo , Retina/fisiologia , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismoRESUMO
Nucleocytoplasmic transport is dysregulated in sporadic and familial amyotrophic lateral sclerosis (ALS) and retinal ganglion neurons (RGNs) are purportedly involved in ALS. The Ran-binding protein 2 (Ranbp2) controls rate-limiting steps of nucleocytoplasmic transport. Mice with Ranbp2 loss in Thy1+-motoneurons develop cardinal ALS-like motor traits, but the impairments in RGNs and the degree of dysfunctional consonance between RGNs and motoneurons caused by Ranbp2 loss are unknown. This will help to understand the role of nucleocytoplasmic transport in the differential vulnerability of neuronal cell types to ALS and to uncover non-motor endophenotypes with pathognomonic signs of ALS. Here, we ascertain Ranbp2's function and endophenotypes in RGNs of an ALS-like mouse model lacking Ranbp2 in motoneurons and RGNs. Thy1+-RGNs lacking Ranbp2 shared with motoneurons the dysregulation of nucleocytoplasmic transport. RGN abnormalities were comprised morphologically by soma hypertrophy and optic nerve axonopathy and physiologically by a delay of the visual pathway's evoked potentials. Whole-transcriptome analysis showed restricted transcriptional changes in optic nerves that were distinct from those found in sciatic nerves. Specifically, the level and nucleocytoplasmic partition of the anti-apoptotic and novel substrate of Ranbp2, Pttg1/securin, were dysregulated. Further, acetyl-CoA carboxylase 1, which modulates de novo synthesis of fatty acids and T-cell immunity, showed the highest up-regulation (35-fold). This effect was reflected by the activation of ramified CD11b+ and CD45+-microglia, increase of F4\80+-microglia and a shift from pseudopodial/lamellipodial to amoeboidal F4\80+-microglia intermingled between RGNs of naive mice. Further, there was the intracellular sequestration in RGNs of metalloproteinase-28, which regulates macrophage recruitment and polarization in inflammation. Hence, Ranbp2 genetic insults in RGNs and motoneurons trigger distinct paracrine signaling likely by the dysregulation of nucleocytoplasmic transport of neuronal-type selective substrates. Immune-modulators underpinning RGN-to-microglial signaling are regulated by Ranbp2, and this neuronal-glial system manifests endophenotypes that are likely useful in the prognosis and diagnosis of motoneuron diseases, such as ALS.
Assuntos
Microglia/metabolismo , Chaperonas Moleculares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Células Ganglionares da Retina/metabolismo , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Transporte Ativo do Núcleo Celular , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Modelos Animais de Doenças , Potenciais Evocados/efeitos dos fármacos , Regulação da Expressão Gênica , Metabolismo dos Lipídeos , Metaloproteinases da Matriz Secretadas/genética , Metaloproteinases da Matriz Secretadas/metabolismo , Camundongos , Camundongos Knockout , Chaperonas Moleculares/genética , Neurônios Motores/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/deficiência , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Nervo Óptico/anormalidades , Nervo Óptico/patologia , Comunicação Parácrina , Tamoxifeno/farmacologia , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismo , TranscriptomaRESUMO
The monocarboxylate transporter 1 (MCT1) is highly expressed in the outer retina, suggesting that it plays a critical role in photoreceptors. We examined MCT1 +/- heterozygotes, which express half of the normal complement of MCT1. The MCT1 +/- retina developed normally and retained normal function, indicating that MCT1 is expressed at sufficient levels to support outer retinal metabolism.
Assuntos
Transportadores de Ácidos Monocarboxílicos/deficiência , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Simportadores/deficiência , Animais , Eletrorretinografia , Metabolismo Energético , Potenciais Evocados Visuais , Heterozigoto , Lactatos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Neurônios Motores/metabolismo , Oligodendroglia/metabolismo , Células Bipolares da Retina/metabolismo , Simportadores/genética , Simportadores/metabolismoRESUMO
The published online version contains incorrect data in Table 2 caption. Argon should not be mentioned in the caption as this is not used in this paper.
RESUMO
Panretinal photocoagulation (PRP) is a standard method for proliferative diabetic retinopathy (PDR) treatment. However, conventional PRP usually significantly damages the retinal structure and vision. Retinal pattern scanning laser (PASCAL) photocoagulation has emerged as a new technique with fewer complications for the treatment of retinal disorders. This study compares the therapeutic effects of short-pulse PASCAL to conventional single-spot PRP for PDR. Fifty-two PDR patients (104 eyes) were randomly assigned into a short-pulse PASCAL-PRP treatment (SP) group and a conventional PRP treatment (TP) group. The best corrected visual acuity (BCVA) and full-field flash electroretinogram (ERG) data were evaluated before and after the two treatments. The BCVA data between before and after the PRP treatments did not show any significant difference. After the PRP treatment, the b-wave amplitude (b-A) in the dark-adapted 3.0 ERG (p = 0.0005) and the amplitude in the light-adapted 3.0 flicker ERG (p = 0.009) were significantly higher in the SP group compared with that of the TP group. In addition, after the PRP treatment, the a-wave implicit time (a-T) of light-adapted 3.0 ERG prolonged significantly in the TP group compared to the SP group. Compared with the parameters before the treatments, the a-A and b-A under dark-adapted 3.0 ERG and the b-A under the light-adapted 3.0 ERG in both TP and SP groups after the treatments decreased significantly (p < 0.05). Short-pulse PASCAL-PRP significantly attenuated partial vision damage compared to conventional PRP, although it still caused limited retinal injury and mild reduction in retinal function. These findings suggest that short-pulse PASCAL-PRP is a promising technique for PDR treatment.
Assuntos
Retinopatia Diabética/diagnóstico por imagem , Retinopatia Diabética/cirurgia , Fotocoagulação a Laser , Idoso , Eletrorretinografia , Feminino , Humanos , Fotocoagulação a Laser/métodos , Masculino , Pessoa de Meia-Idade , Retina/diagnóstico por imagem , Retina/cirurgia , Resultado do Tratamento , Acuidade VisualRESUMO
Human gene mutations have revealed that a significant number of ADAMTS (a disintegrin-like and metalloproteinase (reprolysin type) with thrombospondin type 1 motifs) proteins are necessary for normal ocular development and eye function. Mutations in human ADAMTSL4, encoding an ADAMTS-like protein which has been implicated in fibrillin microfibril biogenesis, cause ectopia lentis (EL) and EL et pupillae. Here, we report the first ADAMTSL4 mouse model, tvrm267, bearing a nonsense mutation in Adamtsl4. Homozygous Adamtsl4(tvrm267) mice recapitulate the EL phenotype observed in humans, and our analysis strongly suggests that ADAMTSL4 is required for stable anchorage of zonule fibers to the lens capsule. Unexpectedly, homozygous Adamtsl4(tvrm267) mice exhibit focal retinal pigment epithelium (RPE) defects primarily in the inferior eye. RPE dedifferentiation was indicated by reduced pigmentation, altered cellular morphology and a reduction in RPE-specific transcripts. Finally, as with a subset of patients with ADAMTSL4 mutations, increased axial length, relative to age-matched controls, was observed and was associated with the severity of the RPE phenotype. In summary, the Adamtsl4(tvrm267) model provides a valuable tool to further elucidate the molecular basis of zonule formation, the pathophysiology of EL and ADAMTSL4 function in the maintenance of the RPE.
Assuntos
Proteínas ADAM/genética , Ectopia do Cristalino/genética , Pró-Colágeno N-Endopeptidase/genética , Distúrbios Pupilares/genética , Epitélio Pigmentado da Retina/citologia , Proteínas ADAM/fisiologia , Proteína ADAMTS4 , Animais , Comprimento Axial do Olho , Diferenciação Celular , Códon sem Sentido , Colágeno/genética , Modelos Animais de Doenças , Ectopia do Cristalino/patologia , Colágenos Associados a Fibrilas , Regulação da Expressão Gênica , Homozigoto , Humanos , Cristalino/citologia , Cristalino/patologia , Camundongos , Camundongos Mutantes , Pró-Colágeno N-Endopeptidase/fisiologia , Pupila , Distúrbios Pupilares/patologia , Epitélio Pigmentado da Retina/patologiaRESUMO
Mouse models provide important resources for many areas of vision research, pertaining to retinal development, retinal function and retinal disease. The Translational Vision Research Models (TVRM) program uses chemical mutagenesis to generate new mouse models for vision research. In this chapter, we report the identification of mouse models for Grm1, Grk1 and Lrit3. Each of these is characterized by a primary defect in the electroretinogram. All are available without restriction to the research community.
Assuntos
Predisposição Genética para Doença/genética , Mutação , Retina/metabolismo , Doenças Retinianas/genética , Alelos , Animais , Modelos Animais de Doenças , Eletrorretinografia , Oftalmopatias/diagnóstico , Oftalmopatias/genética , Oftalmopatias/fisiopatologia , Receptor Quinase 1 Acoplada a Proteína G/genética , Testes Genéticos/métodos , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Mutagênese , Receptores de Glutamato Metabotrópico/genética , Retina/patologia , Retina/fisiopatologia , Doenças Retinianas/diagnóstico , Pesquisa Translacional Biomédica/métodos , Visão Ocular/genética , Visão Ocular/fisiologiaRESUMO
Non-autonomous cell-death is a cardinal feature of the disintegration of neural networks in neurodegenerative diseases, but the molecular bases of this process are poorly understood. The neural retina comprises a mosaic of rod and cone photoreceptors. Cone and rod photoreceptors degenerate upon rod-specific expression of heterogeneous mutations in functionally distinct genes, whereas cone-specific mutations are thought to cause only cone demise. Here we show that conditional ablation in cone photoreceptors of Ran-binding protein-2 (Ranbp2), a cell context-dependent pleiotropic protein linked to neuroprotection, familial necrotic encephalopathies, acute transverse myelitis and tumor-suppression, promotes early electrophysiological deficits, subcellular erosive destruction and non-apoptotic death of cones, whereas rod photoreceptors undergo cone-dependent non-autonomous apoptosis. Cone-specific Ranbp2 ablation causes the temporal activation of a cone-intrinsic molecular cascade highlighted by the early activation of metalloproteinase 11/stromelysin-3 and up-regulation of Crx and CoREST, followed by the down-modulation of cone-specific phototransduction genes, transient up-regulation of regulatory/survival genes and activation of caspase-7 without apoptosis. Conversely, PARP1+ -apoptotic rods develop upon sequential activation of caspase-9 and caspase-3 and loss of membrane permeability. Rod photoreceptor demise ceases upon cone degeneration. These findings reveal novel roles of Ranbp2 in the modulation of intrinsic and extrinsic cell death mechanisms and pathways. They also unveil a novel spatiotemporal paradigm of progression of neurodegeneration upon cell-specific genetic damage whereby a cone to rod non-autonomous death pathway with intrinsically distinct cell-type death manifestations is triggered by cell-specific loss of Ranbp2. Finally, this study casts new light onto cell-death mechanisms that may be shared by human dystrophies with distinct retinal spatial signatures as well as with other etiologically distinct neurodegenerative disorders.
Assuntos
Morte Celular/genética , Chaperonas Moleculares/genética , Doenças Neurodegenerativas/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Animais , Linhagem da Célula , Humanos , Luz , Camundongos , Camundongos Transgênicos , Rede Nervosa/metabolismo , Doenças Neurodegenerativas/patologia , Retina/patologia , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Células Fotorreceptoras Retinianas Bastonetes/citologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Regulação para CimaRESUMO
Retinal pigment epithelium (RPE) degeneration underpins diseases triggered by disparate genetic lesions, noxious insults, or both. The pleiotropic Ranbp2 controls the expression of intrinsic and extrinsic pathological stressors impinging on cellular viability. However, the physiological targets and mechanisms controlled by Ranbp2 in tissue homeostasis, such as RPE, are ill defined. We show that mice, RPE-cre::Ranbp2(-/-), with selective Ranbp2 ablation in RPE develop pigmentary changes, syncytia, hypoplasia, age-dependent centrifugal and non-apoptotic degeneration of the RPE, and secondary leakage of choriocapillaris. These manifestations are accompanied by the development of F-actin clouds, metalloproteinase-11 activation, deregulation of expression or subcellular localization of critical RPE proteins, atrophic cell extrusions into the subretinal space, and compensatory proliferation of peripheral RPE. To gain mechanistic insights into what Ranbp2 activities are vital to the RPE, we performed genetic complementation analyses of transgenic lines of bacterial artificial chromosomes of Ranbp2 harboring loss of function of selective Ranbp2 domains expressed in a Ranbp2(-/-) background. Among the transgenic lines produced, only Tg(RBD2/3*-HA)::RPE-cre::Ranbp2(-/-)-expressing mutations, which selectively impair binding of RBD2/3 (Ran-binding domains 2 and 3) of Ranbp2 to Ran-GTP, recapitulate RPE degeneration, as observed with RPE-cre::Ranbp2(-/-). By contrast, Tg(RBD2/3*-HA) expression rescues the degeneration of cone photoreceptors lacking Ranbp2. The RPE of RPE-cre::Ranbp2(-/-) and Tg(RBD2/3*-HA)::RPE-cre::Ranbp2(-/-) share proteostatic deregulation of Ran GTPase, serotransferrin, and γ-tubulin and suppression of light-evoked electrophysiological responses. These studies unravel selective roles of Ranbp2 and its RBD2 and RBD3 in RPE survival and functions. We posit that the control of Ran GTPase by Ranbp2 emerges as a novel therapeutic target in diseases promoting RPE degeneration.
Assuntos
Deleção de Genes , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Animais , Capilares/patologia , Proliferação de Células , Sobrevivência Celular , Cromossomos Artificiais Bacterianos/metabolismo , Progressão da Doença , Fenômenos Eletrofisiológicos , Integrases/metabolismo , Camundongos , Camundongos Transgênicos , Mutação/genética , Complexo de Proteínas Formadoras de Poros Nucleares/deficiência , Estrutura Terciária de Proteína , Epitélio Pigmentado da Retina/fisiopatologia , Epitélio Pigmentado da Retina/ultraestrutura , Relação Estrutura-AtividadeRESUMO
The immunophilins, cyclophilins, catalyze peptidyl cis-trans prolyl-isomerization (PPIase), a rate-limiting step in protein folding and a conformational switch in protein function. Cyclophilins are also chaperones. Noncatalytic mutations affecting the only cyclophilins with known but distinct physiological substrates, the Drosophila NinaA and its mammalian homolog, cyclophilin-B, impair opsin biogenesis and cause osteogenesis imperfecta, respectively. However, the physiological roles and substrates of most cyclophilins remain unknown. It is also unclear if PPIase and chaperone activities reflect distinct cyclophilin properties. To elucidate the physiological idiosyncrasy stemming from potential cyclophilin functions, we generated mice lacking endogenous Ran-binding protein-2 (Ranbp2) and expressing bacterial artificial chromosomes of Ranbp2 with impaired C-terminal chaperone and with (Tg-Ranbp2(WT-HA)) or without PPIase activities (Tg-Ranbp2(R2944A-HA)). The transgenic lines exhibit unique effects in proteostasis. Either line presents selective deficits in M-opsin biogenesis with its accumulation and aggregation in cone photoreceptors but without proteostatic impairment of two novel Ranbp2 cyclophilin partners, the cytokine-responsive effectors, STAT3/STAT5. Stress-induced STAT3 activation is also unaffected in Tg-Ranbp2(R2944A-HA)::Ranbp2(-/-). Conversely, proteomic analyses found that the multisystem proteinopathy/amyotrophic lateral sclerosis proteins, heterogeneous nuclear ribonucleoproteins A2/B1, are down-regulated post-transcriptionally only in Tg-Ranbp2(R2944A-HA)::Ranbp2(-/-). This is accompanied by the age- and tissue-dependent reductions of diubiquitin and ubiquitylated proteins, increased deubiquitylation activity, and accumulation of the 26 S proteasome subunits S1 and S5b. These manifestations are absent in another line, Tg-Ranbp2(CLDm-HA)::Ranbp2(-/-), harboring SUMO-1 and S1-binding mutations in the Ranbp2 cyclophilin-like domain. These results unveil distinct mechanistic and biological links between PPIase and chaperone activities of Ranbp2 cyclophilin toward proteostasis of selective substrates and with novel therapeutic potential.
Assuntos
Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/metabolismo , Dobramento de Proteína , Envelhecimento/metabolismo , Animais , Biocatálise , Regulação para Baixo , Potenciais Evocados Visuais , Proteínas Ativadoras de GTPase/metabolismo , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Histona Desacetilases/metabolismo , Humanos , Camundongos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Complexo de Proteínas Formadoras de Poros Nucleares/deficiência , Opsinas/metabolismo , Especificidade de Órgãos , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Fatores de Transcrição STAT/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Relação Estrutura-Atividade , Ubiquitina/metabolismoRESUMO
Activation of hypoxia-inducible factor (HIF) can prevent oxygen-induced retinopathy in rodents. Here we demonstrate that dimethyloxaloylglycine (DMOG)-induced retinovascular protection is dependent on hepatic HIF-1 because mice deficient in liver-specific HIF-1α experience hyperoxia-induced damage even with DMOG treatment, whereas DMOG-treated wild-type mice have 50% less avascular retina (P < 0.0001). Hepatic HIF stabilization protects retinal function because DMOG normalizes the b-wave on electroretinography in wild-type mice. The localization of DMOG action to the liver is further supported by evidence that i) mRNA and protein erythropoietin levels within liver and serum increased in DMOG-treated wild-type animals but are reduced by 60% in liver-specific HIF-1α knockout mice treated with DMOG, ii) triple-positive (Sca1/cKit/VEGFR2), bone-marrow-derived endothelial precursor cells increased twofold in DMOG-treated wild-type mice (P < 0.001) but are unchanged in hepatic HIF-1α knockout mice in response to DMOG, and iii) hepatic luminescence in the luciferase oxygen-dependent degradation domain mouse was induced by subcutaneous and intraperitoneal DMOG. These findings uncover a novel endocrine mechanism for retinovascular protection. Activating HIF in visceral organs such as the liver may be a simple strategy to protect capillary beds in the retina and in other peripheral tissues.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fígado/metabolismo , Oxigênio/toxicidade , Doenças Retinianas/metabolismo , Aminoácidos Dicarboxílicos/farmacologia , Animais , Eritropoetina/genética , Eritropoetina/metabolismo , Hiperóxia/tratamento farmacológico , Hiperóxia/genética , Hiperóxia/metabolismo , Hiperóxia/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Fígado/patologia , Camundongos , Camundongos Knockout , Doenças Retinianas/induzido quimicamente , Doenças Retinianas/tratamento farmacológico , Doenças Retinianas/genética , Doenças Retinianas/patologiaRESUMO
CC chemokine ligand 2 (CCL2) recruits macrophages to reduce inflammatory responses. Decay-accelerating factor (DAF) is a membrane regulator of the classical and alternative pathways of complement activation. In view of the link between complement genes and retinal diseases, we evaluated the retinal phenotype of C57BL/6J mice and mice lacking Ccl2 and/or Daf1 at 12 months of age, using scanning laser ophthalmoscopic imaging, electroretinography (ERG), histology, immunohistochemistry, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis. In comparison to C57BL/6J mice, mutant mice had an increased number of autofluorescent foci, with the greatest number in the Ccl2(-/-)/Daf1(-/-) retina. ERG amplitudes in Ccl2(-/-)/Daf1(-/-), Ccl2(-/-) and Daf1(-/-) mice were reduced, with the greatest reduction in Ccl2(-/-)/Daf1(-/-) mice. TUNEL-positive cells were not seen in C57BL/6J retina, but were prevalent in the outer and inner nuclear layers of Ccl2(-/-)Daf1(-/-) mice and were present at reduced density in Ccl2(-/-) or Daf1(-/-) mice. Cell loss was most pronounced in the outer and inner nuclear layers of Ccl2(-/-)/Daf1(-/-) mice. The levels of the endoplasmic reticulum chaperone GPR78 and transcription factor ATF4 were significantly increased in the Ccl2(-/-)/Daf1(-/-) retina. In comparison to the C57BL/6J retina, the phosphorylation of NF-κB p65, p38, ERK and JNK was significantly upregulated while SIRT1 was significantly downregulated in the Ccl2(-/-)/Daf1(-/-) retina. Our results suggest that loss of Ccl2 and Daf1 causes retinal neuronal death and degeneration which is related to increased endoplasmic reticulum stress, oxidative stress and inflammation.
Assuntos
Antígenos CD55/fisiologia , Quimiocina CCL2/fisiologia , Degeneração Retiniana/etiologia , Degeneração Retiniana/fisiopatologia , Neurônios Retinianos/patologia , Fator 4 Ativador da Transcrição/metabolismo , Animais , Apoptose , Modelos Animais de Doenças , Eletrorretinografia , Chaperona BiP do Retículo Endoplasmático , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Choque Térmico/metabolismo , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Degeneração Retiniana/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
This review covers the utility of electrophysiological studies relevant to inflammatory diseases of the retina in conditions such as acute posterior multifocal placoid pigment epitheliopathy, acute zonal occult outer retinopathy, Adamantiades-Behçet disease, autoimmune retinopathy and neuro-retinopathy, birdshot chorioretinopathy, multiple evanescent white dot syndrome, and Vogt-Koyanagi-Harada disease. Electrophysiological studies can help with the diagnosis, prognostication, evaluation of treatment effects, and follow-up for these conditions.
RESUMO
Macular dystrophies are a heterogeneous group of genetic disorders that often severely threatens the bilateral central vision of the affected patient. While advances in molecular genetics have been instrumental in the understanding and diagnosis of these disorders, there remains significant phenotypical variation among patients within any particular subset of macular dystrophies. Electrophysiological testing remains a vital tool not only to characterize vision loss for differential diagnosis but also to understand the pathophysiology of these disorders and to monitor the treatment effect, potentially leading to therapeutic advances. This review summarizes the application of electrophysiological testing in macular dystrophies, including Stargardt disease, bestrophinopathies, X-linked retinoschisis, Sorsby fundus dystrophy, Doyne honeycomb retina dystrophy, autosomal dominant drusen, occult macular dystrophy, North Carolina macular dystrophy, pattern dystrophy, and central areolar choroidal dystrophy.
RESUMO
Recent research on functional and morphologic features is relevant to the diagnosis of ocular diseases [...].