RESUMO
The presence of melanopsin (OPN4) has been shown in cultured murine melanocytes and was associated with ultraviolet A radiation (UVA) reception. Here we demonstrated the protective role of OPN4 in skin physiology and the increased UVA-induced damage in its absence. Histological analysis showed a thicker dermis and thinner hypodermal white adipose tissue layer in Opn4-/- (KO) mice than in wild-type (WT) animals. Proteomics analyses revealed molecular signatures associated with proteolysis, remodeling chromatin, DNA damage response (DDR), immune response, and oxidative stress coupled with antioxidant responses in the skin of Opn4 KO mice compared to WT. Skin protein variants were found in Opn4 KO mice and Opn2, Opn3, and Opn5 gene expressions were increased in the genotype. We investigated each genotype response to UVA stimulus (100 kJ/m2). We found an increase of Opn4 gene expression following stimulus on the skin of WT mice suggesting melanopsin as a UVA sensor. Proteomics findings suggest that UVA decreases DDR pathways associated with ROS accumulation and lipid peroxidation in the skin of Opn4 KO mice. Relative changes in methylation (H3-K79) and acetylation sites of histone between genotypes and differentially modulated by UVA stimulus were also observed. We also identified alterations of molecular traits of the central hypothalamus-pituitary- adrenal (HPA) and the skin HPA-like axes in the absence of OPN4. Higher skin corticosterone levels were detected in UVA-stimulated Opn4 KO compared to irradiated WT mice. Taken altogether, functional proteomics associated with gene expression experiments allowed a high-throughput evaluation that suggests an important protective role of OPN4 in regulating skin physiology in the presence and absence of UVA radiation.
Assuntos
Opsinas de Bastonetes , Pele , Animais , Camundongos , Homeostase , Melanócitos/metabolismo , Proteínas de Membrana/metabolismo , Opsinas de Bastonetes/genética , Opsinas de Bastonetes/metabolismo , Pele/metabolismo , Raios Ultravioleta/efeitos adversosRESUMO
Skin melanocytes harbor a complex photosensitive system comprised of opsins, which were shown, in recent years, to display light- and thermo-independent functions. Based on this premise, we investigated whether melanopsin, OPN4, displays such a role in normal melanocytes. In this study, we found that murine Opn4KO melanocytes displayed a faster proliferation rate compared to Opn4WT melanocytes. Cell cycle population analysis demonstrated that OPN4KO melanocytes exhibited a faster cell cycle progression with reduced G0-G1, and highly increased S and slightly increased G2/M cell populations compared to the Opn4WT counterparts. Expression of specific cell cycle-related genes in Opn4KO melanocytes exhibited alterations that corroborate a faster cell cycle progression. We also found significant modification in gene and protein expression levels of important regulators of melanocyte physiology. PER1 protein level was higher while BMAL1 and REV-ERBα decreased in Opn4KO melanocytes compared to Opn4WT cells. Interestingly, the gene expression of microphthalmia-associated transcription factor (MITF) was upregulated in Opn4KO melanocytes, which is in line with a higher proliferative capability. Taken altogether, we demonstrated that OPN4 regulates cell proliferation, cell cycle, and affects the expression of several important factors of the melanocyte physiology; thus, arguing for a putative tumor suppression role in melanocytes.
Assuntos
Ciclo Celular/genética , Melanócitos/metabolismo , Opsinas de Bastonetes/deficiência , Animais , Biomarcadores , Proteínas CLOCK/genética , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Proliferação de Células , Células Cultivadas , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Melanócitos/efeitos dos fármacos , Camundongos , Pele/citologia , Pele/metabolismoRESUMO
Glaucoma is a blindness-causing disease that involves selective damage to retinal ganglion cells (RGCs) and their axons. A subset of RGCs expressing the photopigment melanopsin regulates non-image-forming visual system functions, such as pupillary light reflex and circadian rhythms. We analyzed the effect of melatonin on the non-image-forming visual system alterations induced by experimental glaucoma. For this purpose, male Wistar rats were weekly injected with vehicle or chondroitin sulfate into the eye anterior chamber. The non-image-forming visual system was analyzed in terms of (1) melanopsin-expressing RGC number, (2) anterograde transport from the retina to the olivary pretectal nucleus and the suprachiasmatic nuclei, (3) blue- and white light-induced pupillary light reflex, (4) light-induced c-Fos expression in the suprachiasmatic nuclei, (5) daily rhythm of locomotor activity, and (6) mitochondria in melanopsin-expressing RGC cells. Melatonin prevented the effect of experimental glaucoma on melanopsin-expressing RGC number, blue- and white light-induced pupil constriction, retina-olivary pretectal nucleus, and retina- suprachiasmatic nuclei communication, light-induced c-Fos expression in the suprachiasmatic nuclei, and alterations in the locomotor activity daily rhythm. In addition, melatonin prevented the effect of glaucoma on melanopsin-expressing RGC mitochondrial alterations. These results support that melatonin protected the non-image-forming visual system against glaucoma, probably through a mitochondrial protective mechanism.
Assuntos
Antioxidantes/administração & dosagem , Glaucoma/prevenção & controle , Melatonina/administração & dosagem , Células Ganglionares da Retina/efeitos dos fármacos , Visão Ocular/efeitos dos fármacos , Animais , Glaucoma/induzido quimicamente , Glaucoma/metabolismo , Luz/efeitos adversos , Masculino , Ratos , Ratos Wistar , Células Ganglionares da Retina/metabolismo , Opsinas de Bastonetes/metabolismo , Núcleo Supraquiasmático/efeitos dos fármacos , Núcleo Supraquiasmático/metabolismo , Visão Ocular/fisiologiaRESUMO
The aim of this pilot study was to determine the association of the P10L (rs2675703) polymorphism of the OPN4 gene with chronic insomnia in uncertain etiology in a Mexican population. A case control study was performed including 98 healthy subjects and 29 individuals with chronic insomnia not related to mental disorders, medical condition, medication or substance abuse. Samples were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Genetic analyses showed that the T allele of P10L increased risk to chronic insomnia in a dominant model (p = 1 ×10-4; odds ratio (OR) = 9.37, CI = 8.18-335.66, Kelsey statistical power (KSP) = 99.9%), and in a recessive model (p = 7.5 × 10-5, OR = 9.37, KSP = 99.3%, CI = 2.7-34.29). In the insomnia group, we did not find a correlation between genotypes and chronotype (p = 0.219 Fisher's exact test), severity of chronic insomnia using ISI score (p = 0.082 Fisher's exact test) and ESS score (p Ë 0.999 Fisher's exact test). However, evening chronotype was correlated to daytime sleepiness severity, individuals with an eveningness chronotype had more severe drowsiness according to their insomnia severity index (ISI) score (p = 0.021 Fisher's exact test) and Epworth sleepiness scale (ESS) score (p = 0.015 Fisher's exact test) than the morningness and intermediate chronotype. We demonstrated that the T allele of the P10L polymorphism in the OPN4 gene is associated with chronic insomnia in Mexicans. We suggest the need to conduct larger studies in different ethnic populations to test the probable association and function of P10L and other SNPs in the OPN4 gene and in the onset of chronic insomnia.
Assuntos
Distúrbios do Início e da Manutenção do Sono , Estudos de Casos e Controles , Humanos , Projetos Piloto , Opsinas de Bastonetes , Distúrbios do Início e da Manutenção do Sono/genéticaRESUMO
Optic neuritis (ON) is an inflammatory condition of the optic nerve, which leads to retinal ganglion cell (RGC) loss. A subset of RGCs expressing the photopigment melanopsin regulates non-image-forming visual system (NIFVS) functions such as pupillary light reflex (PLR) and circadian rhythms. Melatonin is a chronobiotic agent able to regulate the circadian system. We analyzed the effect of ON on the NIFVS, and the effect of melatonin on the NIFVS alterations induced by ON. For this purpose, optic nerves from male Wistar rats received vehicle or bacterial lipopolysaccharide (LPS), and one group of animals received a subcutaneous pellet of melatonin or a sham procedure. The NIFVS was analyzed in terms of: i) blue light-evoked PLR, ii) the communication between the retina and the suprachiasmatic nuclei (by anterograde transport, and ex vivo magnetic resonance images), iii) locomotor activity rhythm, and iv) Brn3a(+) and melanopsin(+) RGC number (by immunohistochemistry). Experimental ON significantly decreased the blue light-evoked PLR, induced a misconnection between the retina and the suprachiasmatic nuclei, decreased Brn3a(+) RGCs, but not melanopsin(+) RGC number. A bilateral injection of LPS significantly increased the light (but not dark) phase locomotor activity, rhythm periodicity, and time of offset activity. Melatonin prevented the decrease in blue light-evoked PLR, and locomotor activity rhythm alterations induced by ON. These results support that ON provoked alterations of the circadian physiology, and that melatonin could restore the circadian system misalignment.
Assuntos
Antioxidantes/administração & dosagem , Fenômenos Cronobiológicos/efeitos dos fármacos , Ritmo Circadiano/efeitos dos fármacos , Melatonina/administração & dosagem , Neurite Óptica/tratamento farmacológico , Neurite Óptica/metabolismo , Animais , Antioxidantes/metabolismo , Fenômenos Cronobiológicos/fisiologia , Ritmo Circadiano/fisiologia , Implantes de Medicamento , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Masculino , Melatonina/metabolismo , Neurite Óptica/induzido quimicamente , Ratos , Ratos Wistar , Opsinas de Bastonetes/metabolismoRESUMO
BACKGROUND: A number of non-visual responses to light in vertebrates, such as circadian rhythm control and pupillary light reflex, are mediated by melanopsins, G-protein coupled membrane receptors, conjugated to a retinal chromophore. In non-mammalian vertebrates, melanopsin expression is variable within the retina and extra-ocular tissues. Two paralog melanopsin genes were classified in vertebrates, Opn4x and Opn4m. Snakes are highly diversified vertebrates with a wide range of daily activity patterns, which raises questions about differences in structure, function and expression pattern of their melanopsin genes. In this study, we analyzed the melanopsin genes expressed in the retinas of 18 snake species from three families (Viperidae, Elapidae, and Colubridae), and also investigated extra-retinal tissue expression. RESULTS: Phylogenetic analysis revealed that the amplified gene belongs to the Opn4x group, and no expression of the Opn4m was found. The same paralog is expressed in the iris, but no extra-ocular expression was detected. Molecular evolutionary analysis indicated that melanopsins are evolving primarily under strong purifying selection, although lower evolutionary constraint was detected in snake lineages (ω = 0.2), compared to non-snake Opn4x and Opn4m (ω = 0.1). Statistical analysis of selective constraint suggests that snake phylogenetic relationships have driven stronger effects on melanopsin evolution, than the species activity pattern. In situ hybridization revealed the presence of melanopsin within cells in the outer and inner nuclear layers, in the ganglion cell layer, and intense labeling in the optic nerve. CONCLUSIONS: The loss of the Opn4m gene and extra-ocular photosensitive tissues in snakes may be associated with a prolonged nocturnal/mesopic bottleneck in the early history of snake evolution. The presence of melanopsin-containing cells in all retinal nuclear layers indicates a globally photosensitive retina, and the expression in classic photoreceptor cells suggest a regionalized co-expression of melanopsin and visual opsins.
Assuntos
Proteínas de Répteis/genética , Retina/metabolismo , Opsinas de Bastonetes/genética , Serpentes/genética , Animais , Relógios Circadianos , Evolução Molecular , Regulação da Expressão Gênica , Filogenia , Opsinas de Bastonetes/fisiologia , Serpentes/classificação , Serpentes/fisiologia , Visão OcularRESUMO
The vertebrate retina contains typical photoreceptor (PR) cones and rods responsible for day/night vision, respectively, and intrinsically photosensitive retinal ganglion cells (ipRGCs) involved in the regulation of non-image-forming tasks. Rhodopsin/cone opsin photopigments in visual PRs or melanopsin (Opn4) in ipRGCs utilizes retinaldehyde as a chromophore. The retinoid regeneration process denominated as "visual cycle" involves the retinal pigment epithelium (RPE) or Müller glial cells. Opn4, on the contrary, has been characterized as a bi/tristable photopigment, in which a photon of one wavelength isomerizes 11-cis to all-trans retinal (Ral), with a second photon re-isomerizing it back. However, it is unknown how the chromophore is further metabolized in the inner retina. Nor is it yet clear whether an alternative secondary cycle occurs involving players such as the retinal G-protein-coupled receptor (RGR), a putative photoisomerase of unidentified inner retinal activity. Here, we investigated the role of RGR in retinoid photoisomerization in Opn4x (Xenopus ortholog) (+) RGC primary cultures free of RPE and other cells from chicken embryonic retinas. Opn4x (+) RGCs display significant photic responses by calcium fluorescent imaging and photoisomerize exogenous all-trans to 11-cis Ral and other retinoids. RGR was found to be expressed in developing retina and in primary cultures; when its expression was knocked down, the levels of 11-cis, all-trans Ral, and all-trans retinol in cultures exposed to light were significantly higher and those in all-trans retinyl esters lower than in dark controls. The results support a novel role for RGR in ipRGCs to modulate retinaldehyde levels in light, keeping the balance of inner retinal retinoid pools.
Assuntos
Proteínas do Olho/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Retina/metabolismo , Vias Visuais/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Embrião de Galinha , Galinhas , Isomerismo , Modelos Biológicos , Células Ganglionares da Retina/metabolismo , Retinaldeído/metabolismo , Retinoides/metabolismoRESUMO
In the vertebrate retina, three types of photoreceptors-visual photoreceptor cones and rods and the intrinsically photosensitive retinal ganglion cells (ipRGCs)-converged through evolution to detect light and regulate image- and nonimage-forming activities such as photic entrainment of circadian rhythms, pupillary light reflexes, etc. ipRGCs express the nonvisual photopigment melanopsin (OPN4), encoded by two genes: the Xenopus (Opn4x) and mammalian (Opn4m) orthologs. In the chicken retina, both OPN4 proteins are found in ipRGCs, and Opn4x is also present in retinal horizontal cells (HCs), which connect with visual photoreceptors. Here we investigate the intrinsic photosensitivity and functioning of HCs from primary cultures of embryonic retinas at day 15 by using calcium fluorescent fluo4 imaging, pharmacological inhibitory treatments, and Opn4x knockdown. Results show that HCs are avian photoreceptors with a retinal-based OPN4X photopigment conferring intrinsic photosensitivity. Light responses in HCs appear to be driven through an ancient type of phototransduction cascade similar to that in rhabdomeric photoreceptors involving a G-protein q, the activation of phospholipase C, calcium mobilization, and the release of the inhibitory neurotransmitter GABA. Based on their intrinsic photosensitivity, HCs may have a key dual function in the retina of vertebrates, potentially regulating nonvisual tasks together with their sister cells, ipRGCs, and with visual photoreceptors, modulating lateral interactions and retinal processing.
Assuntos
Células Fotorreceptoras de Vertebrados/fisiologia , Células Horizontais da Retina/fisiologia , Opsinas de Bastonetes/fisiologia , Animais , Cálcio/fisiologia , Células Cultivadas , Galinhas , Embrião não Mamífero , Luz , Retinaldeído/fisiologia , Opsinas de Bastonetes/genética , Ácido gama-Aminobutírico/fisiologiaRESUMO
PURPOSE: The vertebrate inner retina has a subset of intrinsically photosensitive retinal ganglion cells (ipRGCs) that express the nonvisual photopigment melanopsin. The intrinsically photosensitive retinal ganglion cells send light information from the environment to the brain to control, among other parameters, the amount of energy entering the eyes through the pupillary light reflex (PLR). A daily variation in the PLR in both mice and humans has recently been shown, indicating circadian control of this response. In a previous work involving the sensitivity spectra for the PLR, we showed that blind chickens (GUCY1*) display the highest sensitivity to light of 480 nm. The aim of the present study was to evaluate the potential circadian control of PLRs in blind birds under scotopic conditions. METHODS: Circadian PLR was performed on GUCY1* chickens with lights of different wavelengths (white or blue light of 475 nm) under scotopic conditions. RESULTS: We found a significant daily variation in the PLRs of chickens exposed to white or blue light of 475 nm, with increased sensitivity at circadian time 6 during the subjective day. CONCLUSIONS: Our observations clearly point to circadian control of PLRs even in blindness, strongly indicating that both the entry of light into the eyes and its quality are differentially regulated during the day in diurnal animals.
Assuntos
Cegueira/fisiopatologia , Ritmo Circadiano , Pupila/fisiologia , Reflexo Pupilar/fisiologia , Células Ganglionares da Retina/fisiologia , Animais , Galinhas , Modelos Animais de Doenças , Transdução de Sinal Luminoso/fisiologia , Estimulação LuminosaRESUMO
Retinal ischemic injury is an important cause of visual impairment. The loss of retinal ganglion cells (RGCs) is a key sign of retinal ischemic damage. A subset of RGCs expressing the photopigment melanopsin (mRGCs) regulates non-image-forming visual functions such as the pupillary light reflex (PLR), and circadian rhythms. We studied the effect of retinal ischemia on mRGCs and the non-image-forming visual system function. For this purpose, transient ischemia was induced by raising intraocular pressure to 120 mm Hg for 40 min followed by retinal reperfusion by restoring normal pressure. At 4 weeks post-treatment, animals were subjected to electroretinography and histological analysis. Ischemia induced a significant retinal dysfunction and histological alterations. At this time point, a significant decrease in the number of Brn3a(+) RGCs and in the anterograde transport from the retina to the superior colliculus and lateral geniculate nucleus was observed, whereas no differences in the number of mRGCs, melanopsin levels, and retinal projections to the suprachiasmatic nuclei and the olivary pretectal nucleus were detected. At low light intensity, a decrease in pupil constriction was observed in intact eyes contralateral to ischemic eyes, whereas at high light intensity, retinal ischemia did not affect the consensual PLR. Animals with ischemia in both eyes showed a conserved locomotor activity rhythm and a photoentrainment rate which did not differ from control animals. These results suggest that the non-image forming visual system was protected against retinal ischemic damage.
Assuntos
Isquemia/patologia , Retina/fisiopatologia , Animais , Toxina da Cólera/química , Ritmo Circadiano/fisiologia , Eletrorretinografia , Corpos Geniculados/patologia , Luz , Masculino , Movimento , Ratos , Ratos Wistar , Retina/patologia , Células Ganglionares da Retina/metabolismo , Opsinas de Bastonetes/metabolismo , Núcleo Supraquiasmático/metabolismo , Fatores de Tempo , Visão OcularRESUMO
PURPOSE: To investigate the pupillary light reflex (PLR) of patients with severe loss of vision due to Leber's Hereditary Optic Neuropathy (LHON) in the context of a proposed preservation of melanopsin-expressing retinal ganglion cells (mRGCs). METHODS: Ten LHON patients (7 males; 51.6 ± 14.1 years), with visual acuities ranging from 20/400 to hand motion perception and severe visual field losses, were tested and compared with 16 healthy subjects (7 males; 42.15 ± 15.4 years) tested as controls. PLR was measured with an eye tracker and the stimuli were controlled with a Ganzfeld system. Pupil responses were measured monocularly, to 1 second of blue (470 nm) and red (640 nm) flashes with 1, 10, 100, and 250 cd/m² luminances. The normalized amplitude of peak of the transient PLR and the amplitude of the sustained PLR at 6 seconds after the flash offset were measured. In addition, optical coherence topography (OCT) scans of the peripapillary retinal nerve fiber layer were obtained. RESULTS: The patient's peak PLR responses were on average 15% smaller than controls (P < 0.05), but 5 out of 10 patients had amplitudes within the range of controls. The patients' sustained PLRs were comparable with controls at lower flash intensities, but on average, 27% smaller to the 250 cd/m² blue light, although there was considerable overlap with the PLR amplitudes of control. All patients had severe visual field losses and the retinal nerve fiber layer thickness was reduced to a minimum around the optic disc in 8 of the 10 patients. CONCLUSIONS: The PLR is maintained overall in LHON patients despite the severity of optic atrophy. These results are consistent with previous evidence of selective preservation of mRGCs.
Assuntos
Atrofia Óptica Hereditária de Leber/fisiopatologia , Reflexo Pupilar/fisiologia , Células Ganglionares da Retina/metabolismo , Opsinas de Bastonetes/metabolismo , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estimulação Luminosa/métodos , Células Ganglionares da Retina/patologia , Tomografia de Coerência ÓpticaRESUMO
Vertebrates have a central clock and also several peripheral clocks. Light responses might result from the integration of light signals by these clocks. The dermal melanophores of Xenopus laevis have a photoreceptor molecule denominated melanopsin (OPN4x). The mechanisms of the circadian clock involve positive and negative feedback. We hypothesize that these dermal melanophores also present peripheral clock characteristics. Using quantitative PCR, we analyzed the pattern of temporal expression of Opn4x and the clock genes Per1, Per2, Bmal1, and Clock in these cells, subjected to a 14-h light:10-h dark (14L:10D) regime or constant darkness (DD). Also, in view of the physiological role of melatonin in the dermal melanophores of X. laevis, we determined whether melatonin modulates the expression of these clock genes. These genes show a time-dependent expression pattern when these cells are exposed to 14L:10D, which differs from the pattern observed under DD. Cells kept in DD for 5 days exhibited overall increased mRNA expression for Opn4x and Clock, and a lower expression for Per1, Per2, and Bmal1. When the cells were kept in DD for 5 days and treated with melatonin for 1 h, 24 h before extraction, the mRNA levels tended to decrease for Opn4x and Clock, did not change for Bmal1, and increased for Per1 and Per2 at different Zeitgeber times (ZT). Although these data are limited to one-day data collection, and therefore preliminary, we suggest that the dermal melanophores of X. laevis might have some characteristics of a peripheral clock, and that melatonin modulates, to a certain extent, melanopsin and clock gene expression.