Regulation of Retinal Melanopsin on Lens-induced Myopia in Guinea Pigs.

SIGNIFICANCE: Investigation of the mechanism and the role of melanopsin in lens-induced myopia is necessary to find out potential targets in the prevention of myopia development. PURPOSE: We aimed to study the effect and mechanism of retinal melanopsin on lens-induced myopia in guinea pigs, as well as the interactions between melanopsin and other myopic regulation neurotransmitters such as dopamine and melatonin, and to explore the possible role of melanopsin in the prevention of myopia development. METHODS: Twenty-day-old tricolor guinea pigs were randomly divided into four groups: control group, defocus group, defocus + AA92593 group, and defocus + dimethyl sulfoxide (DMSO) group. The defocus eyes wore -6.00 D lens. In the defocus + AA92593 group, the vitreous cavities were injected with melanopsin antagonist AA92593. In the defocus + DMSO group, the vitreous cavities were injected with 5% DMSO as the administration control. The expression of retinal melanopsin protein was measured with immunofluorescence staining and Western blot. The content of dopamine and melatonin in the retina was determined by the high-performance liquid chromatography electrochemical method. RESULTS: Compared with the defocus group, intravitreal injection of AA92593 resulted in increased axial length of the defocus eyes (defocus, 8.05 ± 0.09 mm; defocus + AA92593, 8.15 ± 0.11 mm; P = .008), lower refractive degree (defocus, -1.98 ± 0.82 D; defocus + AA92593, -2.59 ± 0.97 D; P = .05), decreased relative expression of retinal melanopsin protein (defocus, 0.67 ± 0.11; defocus + AA92593, 0.20 ± 0.06; P < .0001), and increased melatonin content in the defocus eyes (defocus, 0.38 ± 0.09 ng/mg; defocus + AA92593, 0.55 ± 0.13 ng/mg; P = .01), but it had no obvious effect on dopamine content (defocus, 0.64 ± 0.18 ng/mg; defocus + AA9259, 0.61 ± 0.17 ng/mg; P > .99). The melatonin content of retina in the defocus + AA92593 group was correlated with refractive error (Pearson correlation coefficient = -0.68, P = .006) and eye axis length (Pearson correlation coefficient = 0.74, P = .02). CONCLUSIONS: Retinal melanopsin has inhibitory effect on lens-induced myopia development in guinea pigs, and such effect may be related to retinal melatonin.

Yellow Filter Effect on Melatonin Secretion in the Eye: Role in IOP Regulation.

Purpose: Melatonin is a neurohormone mainly synthesized in the pineal gland; however, it is also present in the aqueous humor. One of melatonins' functions in the eye is the regulation of intraocular pressure (IOP). Melatonin is known to be sensitive to light. Recently, the photopigment which controls melatonin synthesis, melanopsin, was found in the crystalline lens. Therefore, light conditions are an interesting possible way of regulating melatonin levels in the aqueous humor. The current study used yellow filters, since melanopsin is activated by short wavelength (blue light). Methods: New Zealand white rabbits were used, divided in two groups, one under controlled 12 h light/dark cycles, while the rest had their cages encased with a yellow filter (λ 465-480). IOP measurements were taken every week at the same time before they were anesthetized for aqueous humor extraction. Results: Keeping the rabbits under the yellow filter resulted in a decrease in IOP up to 43.8 ± 7.8% after 3 weeks. This effect was reversed after the topical application of selective and nonselective melatonin receptors antagonists, 4PPDOT and luzindole. Also, blocking melanopsin by its antagonist AA92593 under white light condition decreased IOP. Finally, melatonin levels were found significantly higher in the aqueous humor of rabbits developed under yellow filter compared to controls (37.4 ± 4.2 and 15.3 ± 3.1 ng/ml, respectively). Conclusion: Yellow filters modulate melatonin levels in the aqueous humor due to deactivating melanopsin activity. This effect leads to a decrease in IOP mediated by melatonin receptors.

Light-induced ATP release from the lens.

The recent discovery of the photoreceptor melanopsin in lens epithelial cells has opened the possibility of modulating this protein by light stimulation. Experiments carried out on New Zealand white rabbits have demonstrated that the release of ATP from the lens to the aqueous humor can be reduced either when a yellow filter or a melanopsin antagonist is used. Compared to control (1.10 ± 0.15 µM ATP), the application of a yellow filter (λ465-480) reduced ATP in the aqueous humor 70%, while the melanopsin antagonist AA92593 reduced the presence of ATP 63% (n = 5), an effect which was also obtained with the PLC inhibitor U73122. These results indicate that when melanopsin is blocked either by the lack of light, a filter, or an antagonist, the extracellular presence of ATP is significantly reduced. This discovery may be relevant, on the one hand, because many ocular physiological processes are controlled by ATP and, on the other hand, because it is possible to stimulate ATP release with just light and without using any added substance.

Melanopsin, a Canonical Light Receptor, Mediates Thermal Activation of Clock Genes.

Melanopsin (OPN4) is a photo-pigment found in a small subset of intrinsically photosensitive ganglion cells (ipRGCs) of the mammalian retina. These cells play a role in synchronizing the central circadian pacemaker to the astronomical day by conveying information about ambient light to the hypothalamic suprachiasmatic nucleus, the site of the master clock. We evaluated the effect of a heat stimulus (39.5 °C) on clock gene (Per1 and Bmal1) expression in cultured murine Melan-a melanocytes synchronized by medium changes, and in B16-F10 melanoma cells, in the presence of the selective OPN4 antagonist AA92593, or after OPN4 knockdown by small interfering RNA (siRNA). In addition, we evaluated the effects of heat shock on the localization of melanopsin by immunocytochemistry. In both cell lines melanopsin was found in a region capping the nucleus and heat shock did not affect its location. The heat-induced increase of Per1 expression was inhibited when melanopsin was pharmacologically blocked by AA92593 as well as when its protein expression was suppressed by siRNA in both Melan-a and B16-F10 cells. These data strongly suggest that melanopsin is required for thermo-reception, acting as a thermo-opsin that ultimately feeds the local circadian clock in mouse melanocytes and melanoma cells.

Pharmacological induction of skin pigmentation unveils the neuroendocrine circuit regulated by light.

Light-regulated skin colour change is an important physiological process in invertebrates and lower vertebrates, and includes daily circadian variation and camouflage (i.e. background adaptation). The photoactivation of melanopsin-expressing retinal ganglion cells (mRGCs) in the eye initiates an uncharacterized neuroendocrine circuit that regulates melanin dispersion/aggregation through the secretion of alpha-melanocyte-stimulating hormone (α-MSH). We developed experimental models of normal or enucleated Xenopus embryos, as well as in situ cultures of skin of isolated dorsal head and tails, to analyse pharmacological induction of skin pigmentation and α-MSH synthesis. Both processes are triggered by a melanopsin inhibitor, AA92593, as well as chloride channel modulators. The AA9253 effect is eye-dependent, while functional data in vivo point to GABAA receptors expressed on pituitary melanotrope cells as the chloride channel blocker target. Based on the pharmacological data, we suggest a neuroendocrine circuit linking mRGCs with α-MSH secretion, which is used normally during background adaptation.

Using siRNA to define functional interactions between melanopsin and multiple G Protein partners.

Melanopsin expressing photosensitive retinal ganglion cells (pRGCs) represent a third class of ocular photoreceptors and mediate a range of non-image forming responses to light. Melanopsin is a G protein coupled receptor (GPCR) and existing data suggest that it employs a membrane bound signalling cascade involving Gnaq/11 type G proteins. However, to date the precise identity of the Gα subunits involved in melanopsin phototransduction remains poorly defined. Here we show that Gnaq, Gna11 and Gna14 are highly co-expressed in pRGCs of the mouse retina. Furthermore, using RNAi based gene silencing we show that melanopsin can signal via Gnaq, Gna11 or Gna14 in vitro, and demonstrate that multiple members of the Gnaq/11 subfamily, including Gna14 and at least Gnaq or Gna11, can participate in melanopsin phototransduction in vivo and contribute to the pupillary light responses of mice lacking rod and cone photoreceptors. This diversity of G protein interactions suggests additional complexity in the melanopsin phototransduction cascade and may provide a basis for generating the diversity of light responses observed from pRGC subtypes.

ß-Arrestin-dependent deactivation of mouse melanopsin.

In mammals, the expression of the unusual visual pigment, melanopsin, is restricted to a small subset of intrinsically photosensitive retinal ganglion cells (ipRGCs), whose signaling regulate numerous non-visual functions including sleep, circadian photoentrainment and pupillary constriction. IpRGCs exhibit attenuated electrical responses following sequential and prolonged light exposures indicative of an adaptational response. The molecular mechanisms underlying deactivation and adaptation in ipRGCs however, have yet to be fully elucidated. The role of melanopsin phosphorylation and ß-arrestin binding in this adaptive process is suggested by the phosphorylation-dependent reduction of melanopsin signaling in vitro and the ubiquitous expression of ß-arrestin in the retina. These observations, along with the conspicuous absence of visual arrestin in ipRGCs, suggest that a ß-arrestin terminates melanopsin signaling. Here, we describe a light- and phosphorylation- dependent reduction in melanopsin signaling mediated by both ß-arrestin 1 and ß-arrestin 2. Using an in vitro calcium imaging assay, we demonstrate that increasing the cellular concentration of ß-arrestin 1 and ß-arrestin 2 significantly increases the rate of deactivation of light-activated melanopsin in HEK293 cells. Furthermore, we show that this response is dependent on melanopsin carboxyl-tail phosphorylation. Crosslinking and co-immunoprecipitation experiments confirm ß-arrestin 1 and ß-arrestin 2 bind to melanopsin in a light- and phosphorylation- dependent manner. These data are further supported by proximity ligation assays (PLA), which demonstrate a melanopsin/ß-arrestin interaction in HEK293 cells and ipRGCs. Together, these results suggest that melanopsin signaling is terminated in a light- and phosphorylation-dependent manner through the binding of a ß-arrestin within the retina.

Small-molecule antagonists of melanopsin-mediated phototransduction.

Melanopsin, expressed in a subset of retinal ganglion cells, mediates behavioral adaptation to ambient light and other non-image-forming photic responses. This has raised the possibility that pharmacological manipulation of melanopsin can modulate several central nervous system responses, including photophobia, sleep, circadian rhythms and neuroendocrine function. Here we describe the identification of a potent synthetic melanopsin antagonist with in vivo activity. New sulfonamide compounds inhibiting melanopsin (opsinamides) compete with retinal binding to melanopsin and inhibit its function without affecting rod- and cone-mediated responses. In vivo administration of opsinamides to mice specifically and reversibly modified melanopsin-dependent light responses, including the pupillary light reflex and light aversion. The discovery of opsinamides raises the prospect of therapeutic control of the melanopsin phototransduction system to regulate light-dependent behavior and remediate pathological conditions.

Mislocalized opsin and cAMP signaling: a mechanism for sprouting by rod cells in retinal degeneration.

PURPOSE: In human retinal degeneration, rod photoreceptors reactively sprout neurites. The mechanism is unknown in part because of the paucity of animal models displaying this feature of human pathology. We tested the role of cAMP and opsin in sprouting by tiger salamander rod cells, photoreceptors that can produce reactive growth. METHODS: In vitro systems of isolated photoreceptor cells and intact neural retina were used. cAMP signaling was manipulated with nucleotide analogues, enzyme stimulators, agonists for adenosine and dopamine receptors, and the opsin agonist, ß-ionone. Levels of cAMP were determined by radioimmunoassay, and protein levels by Western blot and quantitative immunocytochemistry. Neuritic growth was assayed by image analysis and conventional and confocal microscopy. RESULTS: cAMP analogues and stimulation of adenylyl cyclase (AC) directly or through G-protein-coupled receptors resulted in significant increases in neuritic growth of isolated rod, but not cone, cells. The signaling pathway included protein kinase A (PKA) and phosphorylation of the transcription factor cAMP response element-binding protein (pCREB). Opsin, a G-linked receptor, is present throughout the plasmalemma of isolated cells; its activation also induced sprouting. In neural retina, rod sprouting was significantly increased by ß-ionone with concomitant increases in cAMP, pCREB, and synaptic proteins. Notably, opsin stimulated sprouting only when mislocalized to the plasmalemma of the rod cell body. CONCLUSIONS: cAMP causes neuritic sprouting in rod, but not cone, cells through the AC-PKA-CREB pathway known to be associated with synaptic plasticity. We propose that in retinal disease, mislocalized rod opsin gains access to cAMP signaling, which leads to neuritic sprouting.

Senseless functions as a molecular switch for color photoreceptor differentiation in Drosophila.

A major question in development is how different specialized cell types arise from a common progenitor. In the adult Drosophila compound eye, color discrimination is achieved by UV-, blue- and green-sensitive photoreceptors (PRs). These different PR subsets arise from neuronal precursors called R7 and R8 cells. Recent studies have demonstrated that R7-based UV-sensitive PRs require the repression of R8-based blue/green-sensitive PR characteristics to properly develop. This repression is mediated by the transcription factor Prospero (Pros). Here, we report that Senseless (Sens), a Drosophila ortholog of the vertebrate Gfi1 transcription factor, plays an opposing role to Pros by both negatively regulating R7-based features and positively enforcing R8-based features during terminal differentiation. In addition, we demonstrate that Pros and Sens function together with the transcription factor Orthodenticle (Otd) to oppositely regulate R7 and R8 PR Rhodopsin gene expression in vitro. These data show that sens, previously shown to be essential for neuronal specification, also controls differentiation of specific neuronal subtypes in the retina. Interestingly, Pros has recently been shown to function as a tumor suppressor, whereas Gfi1 is a well-characterized oncogene. Thus, we propose that sens/pros antagonism is important for regulating many biological processes.

Making the gradient: thyroid hormone regulates cone opsin expression in the developing mouse retina.

Most mammals have two types of cone photoreceptors, which contain either medium wavelength (M) or short wavelength (S) opsin. The number and spatial organization of cone types varies dramatically among species, presumably to fine-tune the retina for different visual environments. In the mouse, S- and M-opsin are expressed in an opposing dorsal-ventral gradient. We previously reported that cone opsin patterning requires thyroid hormone beta2, a nuclear hormone receptor that regulates transcription in conjunction with its ligand, thyroid hormone (TH). Here we show that exogenous TH inhibits S-opsin expression, but activates M-opsin expression. Binding of endogenous TH to TRbeta2 is required to inhibit S-opsin and to activate M-opsin. TH is symmetrically distributed in the retina at birth as S-opsin expression begins, but becomes elevated in the dorsal retina at the time of M-opsin onset (postnatal day 10). Our results show that TH is a critical regulator of both S-opsin and M-opsin, and suggest that a TH gradient may play a role in establishing the gradient of M-opsin. These results also suggest that the ratio and patterning of cone types may be determined by TH availability during retinal development.