Circadian Oscillations in the Murine Preoptic Area Are Reset by Temperature, but Not Light.

Mammals maintain their internal body temperature within a physiologically optimal range. This involves the regulation of core body temperature in response to changing environmental temperatures and a natural circadian oscillation of internal temperatures. The preoptic area (POA) of the hypothalamus coordinates body temperature by responding to both external temperature cues and internal brain temperature. Here we describe an autonomous circadian clock system in the murine ventromedial POA (VMPO) in close proximity to cells which express the atypical violet-light sensitive opsin, Opn5. We analyzed the light-sensitivity and thermal-sensitivity of the VMPO circadian clocks ex vivo. The phase of the VMPO circadian oscillations was not influenced by light. However, the VMPO clocks were reset by temperature changes within the physiological internal temperature range. This thermal-sensitivity of the VMPO circadian clock did not require functional Opn5 expression or a functional circadian clock within the Opn5-expressing cells. The presence of temperature-sensitive circadian clocks in the VMPO provides an advancement in the understanding of mechanisms involved in the dynamic regulation of core body temperature.

Non-visual Opsins and Novel Photo-Detectors in the Vertebrate Inner Retina Mediate Light Responses Within the Blue Spectrum Region.

In recent decades, a number of novel non-visual opsin photopigments belonging to the family of G protein- coupled receptors, likely involved in a number of non-image-forming processes, have been identified and characterized in cells of the inner retina of vertebrates. It is now known that the vertebrate retina is composed of visual photoreceptor cones and rods responsible for diurnal/color and nocturnal/black and white vision, and cells like the intrinsically photosensitive retinal ganglion cells (ipRGCs) and photosensitive horizontal cells in the inner retina, both detecting blue light and expressing the photopigment melanopsin (Opn4). Remarkably, these non-visual photopigments can continue to operate even in the absence of vision under retinal degeneration. Moreover, inner retinal neurons and Müller glial cells have been shown to express other photopigments such as the photoisomerase retinal G protein-coupled receptor (RGR), encephalopsin (Opn3), and neuropsin (Opn5), all able to detect blue/violet light and implicated in chromophore recycling, retinal clock synchronization, neuron-to-glia communication, and other activities. The discovery of these new photopigments in the inner retina of vertebrates is strong evidence of novel light-regulated activities. This review focuses on the features, localization, photocascade, and putative functions of these novel non-visual opsins in an attempt to shed light on their role in the inner retina of vertebrates and in the physiology of the whole organism.

The molecular clockwork of mammalian cells.

Most organisms contain self-sustained circadian clocks. These clocks can be synchronized by environmental stimuli, but can also oscillate indefinitely in isolation. In mammals this is true at the molecular level for the majority of cell types that have been examined. A core set of "clock genes" form a transcriptional/translational feedback loop (TTFL) which repeats with a period of approximately 24 h. The exact mechanism of the TTFL differs slightly in various cell types, but all involve similar family members of the core cohort of clock genes. The clock has many outputs which are unique for different tissues. Cells in diverse tissues will convert the timing signals provided by the TTFL into uniquely orchestrated transcriptional oscillations of many clock-controlled genes and cellular processes.

Evolutionary Constraint on Visual and Nonvisual Mammalian Opsins.

Animals have evolved light-sensitive G protein-coupled receptors, known as opsins, to detect coherent and ambient light for visual and nonvisual functions. These opsins have evolved to satisfy the particular lighting niches of the organisms that express them. While many unique patterns of evolution have been identified in mammals for rod and cone opsins, far less is known about the atypical mammalian opsins. Using genomic data from over 400 mammalian species from 22 orders, unique patterns of evolution for each mammalian opsins were identified, including photoisomerases, RGR-opsin (RGR) and peropsin (RRH), as well as atypical opsins, encephalopsin (OPN3), melanopsin (OPN4), and neuropsin (OPN5). The results demonstrate that OPN5 and rhodopsin show extreme conservation across all mammalian lineages. The cone opsins, SWS1 and LWS, and the nonvisual opsins, OPN3 and RRH, demonstrate a moderate degree of sequence conservation relative to other opsins, with some instances of lineage-specific gene loss. Finally, the photoisomerase, RGR, and the best-studied atypical opsin, OPN4, have high sequence diversity within mammals. These conservation patterns are maintained in human populations. Importantly, all mammalian opsins retain key amino acid residues important for conjugation to retinal-based chromophores, permitting light sensitivity. These patterns of evolution are discussed along with known functions of each atypical opsin, such as in circadian or metabolic physiology, to provide insight into the observed patterns of evolutionary constraint.

Wounding Induces Facultative Opn5-Dependent Circadian Photoreception in the Murine Cornea.

Purpose: Autonomous molecular circadian clocks are present in the majority of mammalian tissues. These clocks are synchronized to phases appropriate for their physiologic role by internal systemic cues, external environmental cues, or both. The circadian clocks of the in vivo mouse cornea synchronize to the phase of the brain's master clock primarily through systemic cues, but ex vivo corneal clocks entrain to environmental light cycles. We evaluated the underlying mechanisms of this difference. Methods: Molecular circadian clocks of mouse corneas were evaluated in vivo and ex vivo for response to environmental light. The presence of opsins and effect of genetic deletion of opsins were evaluated for influence on circadian photoresponses. Opn5-expressing cells were identified using Opn5Cre;Ai14 mice and RT-PCR, and they were characterized using immunocytochemistry. Results: Molecular circadian clocks of the cornea remain in phase with behavioral circadian locomotor rhythms in vivo but are photoentrainable in tissue culture. After full-thickness incision or epithelial debridement, expression of the opsin photopigment Opn5 is induced in the cornea in a subset of preexisting epithelial cells adjacent to the wound site. This induction coincides with conferral of direct, short-wavelength light sensitivity to the circadian clocks throughout the cornea. Conclusions: Corneal circadian rhythms become photosensitive after wounding. Opn5 gene function (but not Opn3 or Opn4 function) is necessary for induced photosensitivity. These results demonstrate that opsin-dependent direct light sensitivity can be facultatively induced in the murine cornea.

Activated α2-Macroglobulin Regulates LRP1 Levels at the Plasma Membrane through the Activation of a Rab10-dependent Exocytic Pathway in Retinal Müller Glial Cells.

Activated α2-macroglobulin (α2M*) and its receptor, low-density lipoprotein receptor-related protein 1 (LRP1), have been linked to proliferative retinal diseases. In Müller glial cells (MGCs), the α2M*/LRP1 interaction induces cell signaling, cell migration, and extracellular matrix remodeling, processes closely associated with proliferative disorders. However, the mechanism whereby α2M* and LRP1 participate in the aforementioned pathologies remains incompletely elucidated. Here, we investigate whether α2M* regulates both the intracellular distribution and sorting of LRP1 to the plasma membrane (PM) and how this regulation is involved in the cell migration of MGCs. Using a human Müller glial-derived cell line, MIO-M1, we demonstrate that the α2M*/LRP1 complex is internalized and rapidly reaches early endosomes. Afterward, α2M* is routed to degradative compartments, while LRP1 is accumulated at the PM through a Rab10-dependent exocytic pathway regulated by PI3K/Akt. Interestingly, Rab10 knockdown reduces both LRP1 accumulation at the PM and cell migration of MIO-M1 cells induced by α2M*. Given the importance of MGCs in the maintenance of retinal homeostasis, unravelling this molecular mechanism can potentially provide new therapeutic targets for the treatment of proliferative retinopathies.

The Visual Cycle in the Inner Retina of Chicken and the Involvement of Retinal G-Protein-Coupled Receptor (RGR).

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.

Horizontal cells expressing melanopsin x are novel photoreceptors in the avian inner retina.

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.

Melanopsin and the Non-visual Photochemistry in the Inner Retina of Vertebrates.

Melanopsin (Opn4), a member of the G-protein-coupled receptor family, is a vitamin A-based opsin in the vertebrate retina that has been shown to be involved in the synchronization of circadian rhythms, pupillary light reflexes, melatonin suppression and other light-regulated tasks. In nonmammalian vertebrates there are two Opn4 genes, Opn4m and Opn4x, the mammalian and Xenopus orthologs respectively. Opn4x is only expressed in nonmammalian vertebrates including reptiles, fish and birds, while Opn4m is found in a subset of retinal ganglion cells (RGCs), the intrinsically photosensitive (ip) RGCs of the inner retina of both mammals and nonmammalian vertebrates. All opsins described utilize retinaldehyde as chromophore, photoisomerized from 11-cis- to all-trans-retinal upon light exposure. Visual retinal photoreceptor cones and rods, responsible for day and night vision respectively, recycle retinoids through a process called the visual cycle that involves the retinal pigment epithelium or glial Müller cells. Although Opn4 has been characterized as a bistable photopigment, little is known about the mechanism/s involved in its chromophore regeneration. In this review, we will attempt to shed light on the visual cycle taking place in the inner retina and discuss the state of the art in the nonvisual photochemistry of vertebrates.

Early appearance of nonvisual and circadian markers in the developing inner retinal cells of chicken.

The retina is a key component of the vertebrate circadian system; it is responsible for detecting and transmitting the environmental illumination conditions (day/night cycles) to the brain that synchronize the circadian clock located in the suprachiasmatic nucleus (SCN). For this, retinal ganglion cells (RGCs) project to the SCN and other nonvisual areas. In the chicken, intrinsically photosensitive RGCs (ipRGCs) expressing the photopigment melanopsin (Opn4) transmit photic information and regulate diverse nonvisual tasks. In nonmammalian vertebrates, two genes encode Opn4: the Xenopus (Opn4x) and the mammalian (Opn4m) orthologs. RGCs express both Opn4 genes but are not the only inner retinal cells expressing Opn4x: horizontal cells (HCs) also do so. Here, we further characterize primary cultures of both populations of inner retinal cells (RGCs and HCs) expressing Opn4x. The expression of this nonvisual photopigment, as well as that for different circadian markers such as the clock genes Bmal1, Clock, Per2, and Cry1, and the key melatonin synthesizing enzyme, arylalkylamine N-acetyltransferase (AA-NAT), appears very early in development in both cell populations. The results clearly suggest that nonvisual Opn4 photoreceptors and endogenous clocks converge all together in these inner retinal cells at early developmental stages.

Differential regulation of feeding rhythms through a multiple-photoreceptor system in an avian model of blindness.

All organisms have evolved photodetection systems to synchronize their physiology and behavior with the external light-dark (LD) cycles. In nonmammalian vertebrates, the retina, the pineal organ, and the deep brain can be photoreceptive. Inner retinal photoreceptors transmit photic information to the brain and regulate diverse nonvisual tasks. We previously reported that even after preventing extraretinal photoreception, blind GUCY1* chickens lacking functional visual photoreceptors could perceive light that modulates physiology and behavior. Here we investigated the contribution of different photoreceptive system components (retinal/pineal and deep brain photoreceptors) to the photic entrainment of feeding rhythms. Wild-type (WT) and GUCY1* birds with head occlusion to avoid extraocular light detection synchronized their feeding rhythms to a LD cycle with light >12 lux, whereas at lower intensities blind birds free-ran with a period of >24 h. When released to constant light, both WT and blind chickens became arrhythmic; however, after head occlusion, GUCY1* birds free-ran with a 24.5-h period. In enucleated birds, brain illumination synchronized feeding rhythms, but in pinealectomized birds only responses to high-intensity light (≥800 lux) were observed, revealing functional deep brain photoreceptors. In chickens, a multiple photoreceptive system, including retinal and extraretinal photoreceptors, differentially contributes to the synchronization of circadian feeding behavior.

Differential regulation of arylalkylamine N-acetyltransferase activity in chicken retinal ganglion cells by light and circadian clock.

Retinal ganglion cells (RGCs) contain circadian clocks driving melatonin synthesis during the day, a subset of these cells acting as nonvisual photoreceptors sending photic information to the brain. In this work, the authors investigated the temporal and light regulation of arylalkylamine N-acetyltransferase (AA-NAT) activity, a key enzyme in melatonin synthesis. The authors first examined this activity in RGCs of wild-type chickens and compared it to that in photoreceptor cells (PRs) from animals maintained for 48 h in constant dark (DD), light (LL), or regular 12-h:12-h light-dark (LD) cycle. AA-NAT activity in RGCs displayed circadian rhythmicity, with highest levels during the subjective day in both DD and LL as well as in the light phase of the LD cycle. In contrast, AA-NAT activity in PRs exhibited the typical nocturnal peak in DD and LD, but no detectable oscillation was observed under LL, under which conditions the levels were basal at all times examined. A light pulse of 30-60 min significantly decreased AA-NAT activity in PRs during the subjective night, but had no effect on RGCs during the day or night. Intraocular injection of dopamine (50 nmol/eye) during the night to mimic the effect of light presented significant inhibition of AA-NAT activity in PRs compared to controls but had no effect on RGCs. The results clearly demonstrate that the regulation of the diurnal increase in AA-NAT activity in RGCs of chickens undergoes a different control mechanism from that observed in PRs, in which the endogenous clock, light, and dopamine exhibited differential effects.

Acetylated tubulin associates with the fifth cytoplasmic domain of Na(+)/K(+)-ATPase: possible anchorage site of microtubules to the plasma membrane.

We showed previously that NKA (Na(+)/K(+)-ATPase) interacts with acetylated tubulin resulting in inhibition of its catalytic activity. In the present work we determined that membrane-acetylated tubulin, in the presence of detergent, behaves as an entity of discrete molecular mass (320-400 kDa) during molecular exclusion chromatography. We also found that microtubules assembled in vitro are able to bind to NKA when incubated with a detergent-solubilized membrane preparation, and that isolated native microtubules have associated NKA. Furthermore, we determined that CD5 (cytoplasmic domain 5 of NKA) is capable of interacting with acetylated tubulin. Taken together, our results are consistent with the idea that NKA may act as a microtubule-plasma membrane anchorage site through an interaction between acetylated tubulin and CD5.