Ana Maria de Lauro Castrucci

Morphology and mosaics of melanopsin-expressing retinal ganglion cell types in mice

Melanopsin is the photopigment of intrinsically photosensitive retinal ganglion cells (ipRGCs). Melanopsin immunoreactivity reveals two dendritic plexuses within the inner plexiform layer (IPL) and morphologically heterogeneous retinal ganglion cells. Using enhanced immunohistochemistry, we provide a fuller description of murine cell types expressing melanopsin, their contribution to the plexuses of melanopsin dendrites, and mosaics formed by each type. M1 cells, corresponding to the originally described ganglion‐cell photoreceptors, occupy the ganglion cell or inner nuclear layers. Their large, sparsely branched arbors (mean diameter 275 μm) monostratify at the outer limit of the OFF sublayer. M2 cells also have large, monostratified dendritic arbors (mean diameter 310 μm), but ramify in the inner third of the IPL, within the ON sublayer. There are ≈900 M1 cells and 800 M2 cells per retina; each type comprises roughly 1–2% of all ganglion cells. The cell bodies of M1 cells are slightly smaller than those of M2 cells (mean diameters: 13 μm for M1, 15 μm for M2). Dendritic field overlap is extensive within each type (coverage factors ≈3.8 for M1 and 4.6 for M2 cells). Rare bistratified cells deploy terminal dendrites within both melanopsin‐immunoreactive plexuses. Because these are too sparsely distributed to permit complete retinal tiling, they lack a key feature of true ganglion cell types and may be anomalous hybrids of the M1 and M2 types. Finally, we observed weak melanopsin immunoreactivity in other ganglion cells, mostly with large somata, that may constitute one or more additional types of melanopsin‐expressing cells. J. Comp. Neurol. 518:2405–2422, 2010.

MT‐1 melatonin receptor expression increases the antiproliferative effect of melatonin on S‐91 murine melanoma cells

Abstract:  Melatonin, a derivative of tryptophan that is present in all vertebrates, was first described in bovine pineal gland. It is known that melatonin is a highly conserved molecule, present also in unicellular organisms and plants. Several effects of melatonin have been described, including receptor‐ and non‐receptor‐mediated actions. Herein, we studied the effects of melatonin on in vitro and in vivo cell proliferation of Cloudman S‐91 murine melanoma cells. We demonstrated that melatonin treatment significantly inhibits S‐91 melanoma cell proliferation in vitro (EC50 = 10−7 m) as well as reduces tumor growth in vivo. We also demonstrated that melatonin directly increases the activity of the antioxidant enzymes catalase and glutathione peroxidase. These effects are most likely triggered through the direct intracellular action of melatonin, since the presence of receptors could not be demonstrated in this cell line. Expression of MT‐1 melatonin receptor by stable transfection, mediated a dramatic antiproliferative melatonin effect (EC50 = 10−10 m) in S‐91 cells. The expressed receptor is negatively coupled to the adenylyl cyclase/cyclic AMP signaling pathway via Gi protein. These results suggest that expression of the MT‐1 melatonin receptor in melanoma cells is a potential alternative approach to specifically target cells in cancer therapeutic treatment.

Nonvisual light responses in the Rpe65 knockout mouse: Rod loss restores sensitivity to the melanopsin system

Intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment melanopsin (OPN4), together with rods and cones, provide light information driving nonvisual light responses. We examined nonvisual photoreception in mice lacking RPE65, a protein that is required for regeneration of visual chromophore in rods and cones. Although Rpe65 knockouts retain a small degree of rod function, we show here that circadian phase shifting responses in Rpe65−/− mice are attenuated far beyond what has been reported for rodless/coneless mice. Furthermore, the number of melanopsin-immunoreactive perikarya and the extent of dendritic arborizations were decreased in Rpe65 knockout mice compared with controls. To assess the nature of the photoreceptive defect in Rpe65 null mice, we eliminated either rods or melanopsin from Rpe65−/− retinas by generating (i) Rpe65−/− mice carrying a transgene (rdta) that results in selective elimination of rods and (ii) double knockout Rpe65−/−;Opn4−/− mice. Surprisingly, rod loss in Rpe65 knockout mice resulted in restoration of circadian photosensitivity. Normal photoentrainment was lost in Rpe65−/−;Opn4−/− mice, and, instead, a diurnal phenotype was observed. Our findings demonstrate that RPE65 is not required for ipRGC function but reveal the existence of a mechanism whereby rods may influence the function of ipRGCs.

MELANOPSIN AND CLOCK GENES: REGULATION BY LIGHT AND ENDOTHELIN IN THE ZEBRAFISH ZEM-2S CELL LINE

It is well known that clocks are present in brain regions other than the suprachiasmatic nucleus and in many peripheral tissues. In the teleost, Danio rerio, peripheral oscillators can be directly synchronized by light. Danio rerio ZEM-2S embryonic cells respond to light with differential growth: cells kept in constant light exhibited a strong inhibition of proliferation, whereas in cells kept in light:dark (LD) cycles (14L:10D and 10L:14D) or in constant darkness (DD), the doubling times were not statistically different. We demonstrated by RT-PCR followed by PCR that ZEM-2S cells express two melanopsins, Opn4x and Opn4m, and the six Cry genes. The presence of the protein OPN4x was demonstrated by immunocytochemistry. The pattern of temporal expression of the genes Opn4x, Per1, Cry1b, and Clock was studied in ZEM-2S cells kept for five days in 12L:12D or DD. In 12L:12D, the clock genes Per 1 and Cry1b exhibited robust circadian expression, while Opn4x and Clock expression seemed to vary in an ultradian pattern. Both Per1 and Cry1b genes had higher expression during the L phase; Clock gene had an increase in expression coincident with the D phase, and during the subjective night. In DD, the temporal variation of Per1 and Cry1b genes was greatly attenuated but not extinguished, and the higher expressions were shifted to the transition times between subjective day and night, demonstrating that Per and Cry1b were synchronized by the LD cycle. Clock and Opn4x kept the ultradian oscillation, but the rhythm was not statistically significant. As endothelins (ET) have been reported to be a potent stimulator of Per genes in rodents, we investigated the effect of endothelin on ZEM-2S cells, which express ETA receptors. Cells were kept in 12D:12L for five days, and then treated with 10 − 11 to 10 − 8M ET-1 for 24 h. ET-1 exhibited a biphasic effect on Opn4x expression. At 10 − 11M, the hormone exerted a highly significant stimulation of Opn4x expression during the L phase and introduced a circadian oscillatory pattern. At 10 − 10M, a significant increase was seen at ZT21 and ZT0 (i.e., at the end of the D phase and beginning of the L phase), whereas 10 − 9 and 10 − 8M ET-1 inhibited the expression of Opn4x at most ZTs. Clock expression was unaffected by 10 − 8M ET-1; however, in the presence of lower concentrations, the expression was enhanced at some ZTs, strengthening the ultradian oscillation. ET-1 at 10 − 11 and 10 − 10M had no effect on Per1 circadian expression; however, 10 − 9 and 10 − 8M ET-1 reduced the amplitude of Per1 expression in the beginning of the L phase. ET-1 effects were less evident on Cry 1b. For both genes, the reduction in expression was not sufficient to abolish the circadian oscillatory pattern. Based on these results and data in the literature, a link between ET-1 stimulation of ETA receptors may be established by E4BP4 binding to the promoters and consequent inhibition of gene expression. (Author correspondence: amdlcast@ib.usp.br)

Melanopsin in chicken melanocytes and retina

Abstract The vertebrate pigment cell, with the exception of mammals and birds, is able to provide the animal with rapid colour changes, which involve dispersion and aggregation of pigment granules in response to hormonal and neuronal agents, and in some cases as a direct response to light. The search for the mechanisms through which Xenopus leavis melanophores respond to light led to the discovery of a new photopigment, melanopsin, with a different spectral sensitivity to that of rhodopsin. This photopigment was also found in mammalian retinal ganglion cells that project to the suprachiasmatic nucleus and other non-visual retinorecipient areas. Herein we demonstrate (by RT-PCR, cloning and sequencing) for the first time that chick melanocytes express melanopsin, and confirmed the presence of the protein by immunocytochemistry. In the chicken retina, we revealed by immunocytochemistry that ganglion cells express melanopsin, but the highest density of immunopositive cells was found in the inner nuclear layer. Quantitative PCR showed that the retina of animals kept in 6 h light: 18 h dark possessed three-fold higher melanopsin mRNA content than animals kept in longer photoperiod, thus demonstrating that light modulates melanopsin expression in chickens.

Seasonal pelage color change: news based on a South American Rodent

Mammalian seasonal molting and color change are known to be influenced by photoperiod changes. Calomys laucha, a South American rodent, exhibits seasonal pelage color change; however, unlike Northern hemisphere rodents, which present a gray or brown color during summer and a whitish color during winter, C. laucha pelage changes from an orange color during summer to a dark gray color during winter. Animals maintained for over a year in stationary photoperiod (LD 12:12h, 22 degrees C) presented orange pelage color during the summer corresponding month (January), and gray color during the winter corresponding month (July). Same age animals were evaluated during summer or winter months, and also showed different colors. Animals exposed for 12 weeks to summer or winter artificial conditions displayed color change, not according to the environmental conditions, as expected, but similar to that of animals maintained in stationary photoperiod. These results suggest that pelage color change in C. laucha is controlled by an endogenous circannual rhythm. The adaptive function of C. laucha color change is discussed.

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.

Cold-sensing TRPM8 channel participates in circadian control of the brown adipose tissue

Transient receptor potential (TRP) channels are known to regulate energy metabolism, and TRPM8 has become an interesting player in this context. Here we demonstrate the role of the cold sensor TRPM8 in the regulation of clock gene and clock controlled genes in brown adipose tissue (BAT). We investigated TrpM8 temporal profile in the eyes, suprachiasmatic nucleus and BAT; only BAT showed temporal variation of TrpM8 transcripts. Eyes from mice lacking TRPM8 lost the temporal profile of Per1 in LD cycle. This alteration in the ocular circadian physiology may explain the delay in the onset of locomotor activity in response to light pulse, as compared to wild type animals (WT). Brown adipocytes from TrpM8 KO mice exhibited a larger multilocularity in comparison to WT or TrpV1 KO mice. In addition, Ucp1 and UCP1 expression was significantly reduced in TrpM8 KO mice in comparison to WT mice. Regarding circadian components, the expression of Per1, Per2, Bmal1, Pparα, and Pparβ oscillated in WT mice kept in LD, whereas in the absence of TRPM8 the expression of clock genes was reduced in amplitude and lack temporal oscillation. Thus, our results reveal new roles for TRPM8 channel: it participates in the regulation of clock and clock-controlled genes in the eyes and BAT, and in BAT thermogenesis. Since disruption of the clock machinery has been associated with many metabolic disorders, the pharmacological modulation of TRPM8 channel may become a promising therapeutic target to counterbalance weight gain, through increased thermogenesis, energy expenditure, and clock gene activation.

Lack of Melanopsin Is Associated with Extreme Weight Loss in Mice upon Dietary Challenge

Metabolic disorders have been established as major risk factors for ocular complications and poor vision. However, little is known about the inverse possibility that ocular disease may cause metabolic dysfunction. To test this hypothesis, we assessed the metabolic consequences of a robust dietary challenge in several mouse models suffering from retinal mutations. To this end, mice null for melanopsin (Opn4-/-), the photopigment of intrinsically photosensitive retinal ganglion cells (ipRGCs), were subjected to five weeks of a ketogenic diet. These mice lost significantly more weight than wild-type controls or mice lacking rod and cone photoreceptors (Pde6brd1/rd1). Although ipRGCs are critical for proper circadian entrainment, and circadian misalignment has been implicated in metabolic pathology, we observed no differences in entrainment between Opn4-/- and control mice. Additionally, we observed no differences in any tested metabolic parameter between these mouse strains. Further studies are required to establish the mechanism giving rise to this dramatic phenotype observed in melanopsin-null mice. We conclude that the causality between ocular disease and metabolic disorders merits further investigation due to the popularity of diets that rely on the induction of a ketogenic state. Our study is a first step toward understanding retinal pathology as a potential cause of metabolic dysfunction.

Melanopsin and rhodopsin mediate UVA-induced immediate pigment darkening: Unravelling the photosensitive system of the skin

The mammalian skin has a photosensitive system comprised by several opsins, including rhodopsin (OPN2) and melanopsin (OPN4). Recently, our group showed that UVA (4.4 kJ/m2) leads to immediate pigment darkening (IPD) in murine normal and malignant melanocytes. We show the role of OPN2 and OPN4 as UVA sensors: UVA-induced IPD was fully abolished when OPN4 was pharmacologically inhibited by AA9253 or when OPN2 and OPN4 were knocked down by siRNA in both cell lines. Our data, however, demonstrate that phospholipase C/protein kinase C pathway, a classical OPN4 pathway, is not involved in UVA-induced IPD in either cell line. Nonetheless, in both cell types we have shown that: a) intracellular calcium signal is necessary for UVA-induced IPD; b) the involvement of CaMK II, whose inhibition, abolished the UVA-induced IPD; c) the role of CAMK II/NOS/sGC/cGMP pathway in the process since inhibition of either NOS or sGC abolished the UVA-induced IPD. Taken altogether, we show that OPN2 and OPN4 participate in IPD induced by UVA in murine normal and malignant melanocytes through a conserved common pathway. Interestingly, upon knockdown of OPN2 or OPN4, the UVA-driven IPD is completely lost, which suggests that both opsins are required and cooperatively signal in murine both cell lines. The participation of OPN2 and OPN4 system in UVA radiation-induced response, if proven to take place in human skin, may represent an interesting pharmacological target for the treatment of depigmentary disorders and skin-related cancer.