Light-dependent phosphorylation of the carboxy tail of mouse melanopsin

Melanopsin-based phototransduction is involved in non-image forming light responses including circadian entrainment, pupil constriction, suppression of pineal melatonin synthesis, and direct photic regulation of sleep in vertebrates. Given that the functions of melanopsin involve the measurement and summation of total environmental luminance, there would appear to be no need for the rapid deactivation typical of other G-protein coupled receptors. In this study, however, we demonstrate that heterologously expressed mouse melanopsin is phosphorylated in a light-dependent manner, and that this phosphorylation is involved in regulating the rate of G-protein activation and the lifetime of melanopsin’s active state. Furthermore, we provide evidence for light-dependent phosphorylation of melanopsin in the mouse retina using an in situ proximity ligation assay. Finally, we demonstrate that melanopsin preferentially interacts with the GRK2/3 family of G-protein coupled receptor kinases through co-immunoprecipitation assays. Based on the complement of G-protein receptor kinases present in the melanopsin-expressing retinal ganglion cells, GRK2 emerges as the best candidate for melanopsin’s cognate GRK.     

Functional diversity of melanopsins and their global expression in the teleost retina

Melanopsin (OPN4) is an opsin photopigment that, in mammals, confers photosensitivity to retinal ganglion cells and regulates circadian entrainment and pupil constriction. In non-mammalian species, two forms of opn4 exist, and are classified into mammalian-like (m) and non-mammalian-like (x) clades. However, far less is understood of the function of this photopigment family. Here we identify in zebrafish five melanopsins (opn4m-1, opn4m-2, opn4m-3, opn4x-1 and opn4x-2), each encoding a full-length opsin G protein. All five genes are expressed in the adult retina in a largely non-overlapping pattern, as revealed by RNA in situ hybridisation and immunocytochemistry, with at least one melanopsin form present in all neuronal cell types, including cone photoreceptors. This raises the possibility that the teleost retina is globally light sensitive. Electrophysiological and spectrophotometric studies demonstrate that all five zebrafish melanopsins encode a functional photopigment with peak spectral sensitivities that range from 470 to 484 nm, with opn4m-1 and opn4m-3 displaying invertebrate-like bistability, where the retinal chromophore interchanges between cis- and trans-isomers in a light-dependent manner and remains within the opsin binding pocket. In contrast, opn4m-2, opn4x-1 and opn4x-2 are monostable and function more like classical vertebrate-like photopigments, where the chromophore is converted from 11-cis to all-trans retinal upon absorption of a photon, hydrolysed and exits from the binding pocket of the opsin. It is thought that all melanopsins exhibit an invertebrate-like bistability biochemistry. Our novel findings, however, reveal the presence of both invertebrate-like and vertebrate-like forms of melanopsin in the teleost retina, and indicate that photopigment bistability is not a universal property of the melanopsin family. The functional diversity of these teleost melanopsins, together with their widespread expression pattern within the retina, suggests that melanopsins confer global photosensitivity to the teleost retina and might allow for direct “fine-tuning” of retinal circuitry and physiology in the dynamic light environments found in aquatic habitats.     

Role of a novel photopigment,melanopsin, in behavioral adaptation to light

Adaptation to changes in the ambient light is of critical importance to life. In mammals, three principal photoadaptation mechanisms depend on ocular photoreception and exhibit spectral sensitivity suggestive of the opsin class of photopigment(s). These include rapid adaptation of the visual system to the ambient light by pupil constriction, direct modulation of neuroendocrine function and entrainment of the circadian clock to the day:night cycle. Surprisingly, these processes can largely function independent of classical rod/cone photoreceptors, suggesting a novel opsin-based signaling mechanism. They appear to involve a recently discovered network of intrinsically photosensitive retinal ganglion cells that make direct or indirect axonal connections to brain centers regulating photoadaptive behaviors. The discovery of a novel opsin, melanopsin, in these cells has offered an exciting entry point to explore, at the molecular level, how mammals adapt to their light environment. There is now genetic proof of a principal role for melanopsin in all three major photoadaptation processes.     

Functional characterisation of naturally occurring mutations in human melanopsin

Melanopsin is a blue light-sensitive opsin photopigment involved in a range of non-image forming behaviours, including circadian photoentrainment and the pupil light response. Many naturally occurring genetic variants exist within the human melanopsin gene (OPN4), yet it remains unclear how these variants affect melanopsin protein function and downstream physiological responses to light. Here, we have used bioinformatic analysis and in vitro expression systems to determine the functional phenotypes of missense human OPN4 variants. From 1242 human OPN4 variants collated in the NCBI Short Genetic Variation database (dbSNP), we identified 96 that lead to non-synonymous amino acid substitutions. These 96 missense mutations were screened using sequence alignment and comparative approaches to select 16 potentially deleterious variants for functional characterisation using calcium imaging of melanopsin-driven light responses in HEK293T cells. We identify several previously uncharacterised OPN4 mutations with altered functional properties, including attenuated or abolished light responses, as well as variants demonstrating abnormal response kinetics. These data provide valuable insight into the structure–function relationships of human melanopsin, including several key functional residues of the melanopsin protein. The identification of melanopsin variants with significantly altered function may serve to detect individuals with disrupted melanopsin-based light perception, and potentially highlight those at increased risk of sleep disturbance, circadian dysfunction, and visual abnormalities.     

Nucleolar localization and circadian regulation of Per2S,a novel splicing variant of the Period 2 gene

In this work, we show for the first time that a second splicing variant of the core clock gene Period 2 (Per2), Per2S, is expressed at both the mRNA and protein levels in human keratinocytes and that it localizes in the nucleoli. Moreover, we show that a reversible perturbation of the nucleolar structure acts as a resetting stimulus for the cellular clock. Per2S expression and periodic oscillation upon dexamethasone treatment were assessed by qRT-PCR using specific primers. Western blot (WB) analysis using an antibody against the recombinant human PER2 (abRc) displayed an intense band at a molecular weight of ~55 kDa, close to the predicted size of Per2S, and a weaker band at the expected size of Per2 (~140 kDa). The antibody raised against PER2 pS662 (abS662), an epitope absent in PER2S, detected only the higher band. Immunolocalization studies with abRc revealed a peculiar nucleolar signal colocalizing with the nucleolar marker nucleophosmin, whereas with abS662 the signal was predominantly diffuse all over the nucleus and partially colocalized with abRc in the nucleolus. The analysis of cell fractions by WB confirmed the enrichment of PER2S and the presence of PER2 in the nucleolar compartment. Finally, a pulse (1 h) of actinomycin D (0.01 μg/ml) induced reversible nucleolar disruption, PER2S de-localization and circadian synchronization of clock and Per2S genes. Our work represents the first evidence that the Per2S splicing isoform is a clock component expressed in human cells localizing in the nucleolus. These results suggest a critical role for the nucleolus in the process of circadian synchronization in human keratinocytes.     

Vertebrate circadian rhythms: Retinal and extraretinal photoreception

Summary ERRs Both the pineal and the SCN are elements of the vertebrate multioscillator system although the relative importance of these 2 areas probably varies between, and possibly within, the different vertebrate classes. Extraretinal photoreception is a universal feature of submammalian vertebrates, and possibly of neonatal mammals, but is absent in adult mammals. Although the pineal systems of sumammalian vertebrates are photosensitive, the pineal system has been directly implicated as an extraocular site for the perception of entraining light cycles only in amphibians. In all other submammalian vertebrates extraretinal entrainment can occur in the absence of the pineal system although it is certainly conceivable that the pineal system may act as an alternate route of photoreception. These extraretinal-extrapineal receptors are located within the brain but the exact location(s) of these receptors within the brain is unknown. The hypothalamus would be likely area for this extraretinal photoreception, however, for several reasons: 1. Neurophysiological studies have identified light sensitive neurons in the frog's hypothalamus⁴³. 2. The avian hypothalamus is a site of photoperiodic photoreception^100–103. 3. The only other light sensitive structures known in vertebrates—the pineal system and the lateral eyes—are all derived embryologically from the hypothalamus. 4. The hypothalamus appears to be the site of a circadian clock and there may be advantages in having the photoreceptors and the clock anatomically close to one another. These considerations, of course, do not exclude the possibility that other brain areas may be involved as well. The reason behind the loss of extraretinal photoreception in mammals is uncertain. The shift to exclusive retinal photoreception in mammals may have been dictated by the extensive reorganization that occurred during the evolution of the mammalian brain. Or, perhaps, the increased size of the mammalian skull and overlying tissue made direct photoreception difficult and necessitated a shift to retinal photoreception. The persistence of extraretinal photoreceptors in submammalian vertebrates, however, underscores their importance in the sensory repertoire of vertebrates.     

Bilaterale Projektion der Netzhaut auf die Sehrinde des Kaninchens

Summary The bilateral representation of the retina on the visual cortex was investigated by analysing the amplitude, latency and topic distribution of the potentials evoked in the visual cortex by monocular photic stimulation of the retina in the conscious rabbit. The amplitudes of the potentials evoked on both ipsi- and contralateral hemispheres, show a relation 1:4. A small field for bilateral retinal projection of the retina was mapped in the anterolateral part of the visual cortex. These differences in amplitude, latency and topic distribution may be significant for stereoscopic vision.     

Knockout of the Bcmo1 gene results in an inflammatory response in female lung, which is suppressed by dietary beta-carotene

Beta-carotene 15,15′-monooxygenase 1 knockout (Bcmo1 ^?/?) mice accumulate beta-carotene (BC) similarly to humans, whereas wild-type (Bcmo1 ^+/+) mice efficiently cleave BC. Bcmo1 ^?/? mice are therefore suitable to investigate BC-induced alterations in gene expression in lung, assessed by microarray analysis. Bcmo1 ^?/? mice receiving control diet had increased expression of inflammatory genes as compared to BC-supplemented Bcmo1 ^?/? mice and Bcmo1 ^+/+ mice that received either control or BC-supplemented diets. Differential gene expression in Bcmo1 ^?/? mice was confirmed by real-time quantitative PCR. Histochemical analysis indeed showed an increase in inflammatory cells in lungs of control Bcmo1 ^?/? mice. Supported by metabolite and gene-expression data, we hypothesize that the increased inflammatory response is due to an altered BC metabolism, resulting in an increased vitamin A requirement in Bcmo1 ^?/? mice. This suggests that effects of BC may depend on inter-individual variations in BC-metabolizing enzymes, such as the frequently occurring human polymorphisms in BCMO1.     

Photoreceptors in the pineal of lower vertebrates: Functional aspects

Summary The pineal of lower vertebrates characteristically contains true and modified photoreceptors with functional und structural homologies to retinal photoreceptors. Afferent nerves convey photic information from the pineal to sensory areas of the brain stem. Light also influences synthetic activity within the organ, controlling the rhythm in melatonin production which is generated endogenously. The molecular mechanisms underlying this rhythmic event are described and the hypothesis advanced that the pineal transduces several forms of environmental stimulus involved in the regulation of rhythmic function.     

Endocrine-dependent expression of circadian clock genes in insects

Current models state that insect peripheral oscillators are directly responsive to light, while mammalian peripheral clock genes are coordinated by a master clock in the brain via intermediate factors, possibly hormonal. We show that the expression levels of two circadian clock genes, period (per) and Par Domain Protein 1 (Pdp1) in the peripheral tissue of an insect model species, the linden bug Pyrrhocoris apterus, are inversely affected by contrasting photoperiods. The effect of photoperiod on per and Pdp1 mRNA levels was found to be mediated by the corpus allatum, an endocrine gland producing juvenile hormone. Our results provide the first experimental evidence for the effect of an endocrine gland on circadian clock gene expression in insects. Received 31 October 2007; received after revision 7 January 2008; accepted 9 January 2008 D. Dolezel, L. Zdechovanova: These authors contributed equally to this work.     

Vitamin B12 amplifies circadian phase shifts induced by a light pulse in rats

Vitamin B₁₂ (VB₁₂) is a putative modulator of the human circadian clock, improving entrainability to the 24 h light-dark cycle. The present study was intended to elucidate the mechanism of VB₁₂ action in an animal model. In male rats free-running under constant dim illumination, a single light pulse of 50–1000 lux for 20 min given at circadian time (CT) 20 induced a 0.28 to 1.08 h phase advance and at CT 14 induced a 0.54 to 2.10 h phase delay. A 3 h intracerebroventricular (icv) infusion of 30 nmol VB₁₂ starting 2 h prior to a 20 min 200 lux light pulse significantly amplified phase shifts in comparison with saline-treated or untreated controls. The mean phase advance (1.13 h) was 1.8-fold greater than that of saline-infused controls, whereas the mean phase delay (2.28 h) was 2.9-fold greater. These values were comparable to the maximal phase shifts caused by 1000 lux light pulses in untreated rats. Since the same VB₁₂ treatment alone had failed to induce a phase shift in a previous experiment, these results indicate that VB₁₂ strongly enhanced light pulse-induced phase shifts and thus augmented the entrainability of the circadian clock to light.     

Soluble factors and the development of rod photoreceptors

Photoreceptors are the most abundant cell type in the vertebrate neural retina. Like the other retinal neurons and the Müller glia, they arise from a population of precursor cells that are multipotent and intrinsic to the retina. Approximately 10 years ago, several studies demonstrated that retinal precursor cells (RPCs) are competent to respond to environmental factors that promote cell type determination and differentiation. Since those studies, significant effort has been directed at identifying the molecular nature of these environmental signals and understanding the precise mechanisms they employ to drive RPCs towards the different retinal fates. In this review, we describe the recent progress toward understanding how environmental factors influence the development of vertebrate rod photoreceptors.     

The circadian clock and metabolic homeostasis: entangled networks

The circadian clock exerts an important role in systemic homeostasis as it acts a keeper of time for the organism. The synchrony between the daily challenges imposed by the environment needs to be aligned with biological processes and with the internal circadian clock. In this review, it is provided an in-depth view of the molecular functioning of the circadian molecular clock, how this system is organized, and how central and peripheral clocks communicate with each other. In this sense, we provide an overview of the neuro-hormonal factors controlled by the central clock and how they affect peripheral tissues. We also evaluate signals released by peripheral organs and their effects in the central clock and other brain areas. Additionally, we evaluate a possible communication between peripheral tissues as a novel layer of circadian organization by reviewing recent studies in the literature. In the last section, we analyze how the circadian clock can modulate intracellular and tissue-dependent processes of metabolic organs. Taken altogether, the goal of this review is to provide a systemic and integrative view of the molecular clock function and organization with an emphasis in metabolic tissues.

     

Diffusion of d-glucose measured in the cytosol of a single astrocyte

Astrocytes interact with neurons and endothelial cells and may mediate exchange of metabolites between capillaries and nerve terminals. In the present study, we investigated intracellular glucose diffusion in purified astrocytes after local glucose uptake. We used a fluorescence resonance energy transfer (FRET)-based nano sensor to monitor the time dependence of the intracellular glucose concentration at specific positions within the cell. We observed a delay in onset and kinetics in regions away from the glucose uptake compared with the region where we locally super-fused astrocytes with the d-glucose-rich solution. We propose a mathematical model of glucose diffusion in astrocytes. The analysis showed that after gradual uptake of glucose, the locally increased intracellular glucose concentration is rapidly spread throughout the cytosol with an apparent diffusion coefficient (D _app) of (2.38 ± 0.41) × 10^?10 m² s^?1 (at 22–24 °C). Considering that the diffusion coefficient of d-glucose in water is D = 6.7 × 10^?10 m² s^?1 (at 24 °C), D _app determined in astrocytes indicates that the cytosolic tortuosity, which hinders glucose molecules, is approximately three times higher than in aqueous solution. We conclude that the value of D _app for glucose measured in purified rat astrocytes is consistent with the view that cytosolic diffusion may allow glucose and glucose metabolites to traverse from the endothelial cells at the blood–brain barrier to neurons and neighboring astrocytes.     

Gonyauline: A novel endogenous substance shortening the period of the circadian clock of a unicellular alga

Summary The circadian clock in the unicellular algaGonyaulax polyedra is accelerated by a substance in extracts from the cells themselves. The extracts have been fractionated using the circadian rhythm of bioluminescence as bioassay. The active substance, termed gonyauline, has been isolated and characterized as a novel low molecular weight cyclopropanecarboxylic acid (S-methyl-cis-2-(methylthio) cyclopropanecarboxylic acid). Synthetic gonyauline has a similar shortening effect on the period of the circadian clock.     

The opsins of the vertebrate retina: insights from structural,biochemical, and evolutionary studies

The vertebrate retina contains several classes of visual pigments responsible for such diverse functions as image- and nonimage-forming vision, the entrainment of circadian cycles, and the pupilary light response. With vision being vital to the survival of many species, the elucidation of the structural and biochemical properties of visual pigments has been the focus of a large body of research that has led to rapid advances in the field of photoreception. In this review, the current understanding of the structure, function, biochemistry, and evolution of the opsins that make up the photopigments in the vertebrate retina will be reviewed. These include the rod and cone opsins, melanopsin, RGR, peropsin, and VA-opsin. The goal is to highlight important questions that have been answered and to define some of the remaining questions in the field that will provide future directions for research.