Melatonin in the mammalian retina: Synthesis, mechanisms of action and neuroprotection.

Melatonin is an important player in the regulation of many physiological functions within the body and in the retina. Melatonin synthesis in the retina primarily occurs during the night and its levels are low during the day. Retinal melatonin is primarily synthesized by the photoreceptors, but whether the synthesis occurs in the rods and/or cones is still unclear. Melatonin exerts its influence by binding to G protein-coupled receptors named melatonin receptor type 1 (MT1) and type 2 (MT2). MT1 and MT2 receptors activate a wide variety of signaling pathways and both receptors are present in the vertebrate photoreceptors where they may form MT1/MT2 heteromers (MT1/2h). Studies in rodents have shown that melatonin signaling plays an important role in the regulation of retinal dopamine levels, rod/cone coupling as well as the photopic and scotopic electroretinogram. In addition, melatonin may play an important role in protecting photoreceptors from oxidative stress and can protect photoreceptors from apoptosis. Critically, melatonin signaling is involved in the modulation of photoreceptor viability during aging and other studies have implicated melatonin in the pathogenesis of age-related macular degeneration. Hence melatonin may represent a useful tool in the fight to protect photoreceptors-and other retinal cells-against degeneration due to aging or diseases.

Circadian clocks, retinogenesis and ocular health in vertebrates: new molecular insights.

Circadian clocks are cell-autonomous, molecular pacemakers regulating a wide variety of behavioural and physiological processes in accordance with the 24 â€‹h light/dark cycle. The retina contains a complex network of cell-specific clocks orchestrating many biochemical and cellular parameters to adapt retinal biology and visual function to daily changes in light intensity. The gene regulatory networks controlling proliferation, specification and differentiation of retinal precursors into the diverse retinal cell types are evolutionary conserved among vertebrates. However, how these mechanisms are interconnected with circadian clocks is not well-characterized. Here we explore the existing evidence for the regulation of retinal development by circadian clock-related pathways, throughout vertebrates. We provide evidence for the influence of clock genes, from early to final differentiation steps. In addition, we report that the clock, integrating environmental cues, modulates a number of pathological processes. We highlight its potential role in retinal diseases and its instructive function on eye growth and related disorders.

Core circadian clock genes Per1 and Per2 regulate the rhythm in photoreceptor outer segment phagocytosis.

Retinal photoreceptors undergo daily renewal of their distal outer segments, a process indispensable for maintaining retinal health. Photoreceptor outer segment (POS) phagocytosis occurs as a daily peak, roughly about 1 hour after light onset. However, the underlying cellular and molecular mechanisms which initiate this process are still unknown. Here we show that, under constant darkness, mice deficient for core circadian clock genes (Per1 and Per2) lack a daily peak in POS phagocytosis. By qPCR analysis, we found that core clock genes were rhythmic over 24 hours in both WT and Per1, Per2 double mutant whole retinas. More precise transcriptomics analysis of laser capture microdissected WT photoreceptors revealed no differentially expressed genes between time points preceding and during the peak of POS phagocytosis. In contrast, we found that microdissected WT retinal pigment epithelium (RPE) had a number of genes that were differentially expressed at the peak phagocytic time point compared to adjacent ones. We also found a number of differentially expressed genes in Per1, Per2 double mutant RPE compared to WT ones at the peak phagocytic time point. Finally, based on STRING analysis, we found a group of interacting genes that potentially drive POS phagocytosis in the RPE. This potential pathway consists of genes such as: Pacsin1, Syp, Camk2b, and Camk2d among others. Our findings indicate that Per1 and Per2 are necessary clock components for driving POS phagocytosis and suggest that this process is transcriptionally driven by the RPE.

Rods contribute to the light-induced phase shift of the retinal clock in mammals.

While rods, cones, and intrinsically photosensitive melanopsin-containing ganglion cells (ipRGCs) all drive light entrainment of the master circadian pacemaker of the suprachiasmatic nucleus, recent studies have proposed that entrainment of the mouse retinal clock is exclusively mediated by a UV-sensitive photopigment, neuropsin (OPN5). Here, we report that the retinal circadian clock can be phase shifted by short duration and relatively low-irradiance monochromatic light in the visible part of the spectrum, up to 520 nm. Phase shifts exhibit a classical photon dose-response curve. Comparing the response of mouse models that specifically lack middle-wavelength (MW) cones, melanopsin, and/or rods, we found that only the absence of rods prevented light-induced phase shifts of the retinal clock, whereas light-induced phase shifts of locomotor activity are normal. In a "rod-only" mouse model, phase shifting response of the retinal clock to light is conserved. At shorter UV wavelengths, our results also reveal additional recruitment of short-wavelength (SW) cones and/or OPN5. These findings suggest a primary role of rod photoreceptors in the light response of the retinal clock in mammals.

Dark-adapted light response in mice is regulated by a circadian clock located in rod photoreceptors.

The retinal circadian system consists of a network of clocks located virtually in every retinal cell-type. Although it is established that the circadian clock regulates many rhythmic processes in the retina, the links between retinal cell-specific clocks and visual function remain to be elucidated. Bmal1 is a principal, non-redundant component of the circadian clock in mammals and is required to keep 24 h rhythms in the retinal transcriptome and in visual processing under photopic light condition. In the current study, we investigated the retinal function in mice with a rod-specific knockout of Bmal1. For this purpose, we measured whole retina PER2::Luciferase bioluminescence and the dark-adapted electroretinogram (ERG). We observed circadian day-night differences in ERG a- and b-waves in control mice carrying one allele of Bmal1 in rods, with higher amplitudes during the subjective night. These differences were abolished in rod-specific Bmal1 knockout mice, whose ERG light-responses remained constitutively low (day-like). Overall, PER2::Luciferase rhythmicity in whole retinas was not defective in these mice but was characterized by longer period and higher rhythmic power compared to retinas with wild type Bmal1 gene. Taken together, these data suggest that a circadian clock located in rods regulates visual processing in a cell autonomous manner.

Major role of MT2 receptors in the beneficial effect of melatonin on long-term recognition memory in C57BL/6J male mice.

Melatonin, a major signal of the circadian system, is also involved in brain functions such as learning and memory. Chronic melatonin treatment is known to improve memory performances, but the respective contribution of its central receptors, MT1 and MT2, is still unclear. Here, we used new single receptor deficient MT1-/- and MT2-/- mice to investigate the contribution of each receptor in the positive effect of chronic melatonin treatment on long-term recognition memory. The lack of MT2 receptor precluded memory-enhancing effect of melatonin in the object recognition task and to a lesser extent in the object location task, whereas the lack of MT1 receptor mitigated its effect in the object location task only. Our findings support a key role of MT2 in mediating melatonin's beneficial action on long-term object recognition memory, whereas MT1 may contribute to the effect on object location memory.

The circadian clock regulates RPE-mediated lactate transport via SLC16A1 (MCT1).

Multiple retinal cells harbor a circadian oscillator, including retinal pigment epithelial cells (RPE). However, little is known about the functions that are regulated by the RPE clock. The aim of this study was to investigate whether the circadian clock in the RPE regulates the transport of glucose and its glycolytic metabolic by-product - lactate. To that end, we first characterized the mRNA expression profile of glucose and monocarboxylate transporters in ARPE-19 cells. We found that SLC2A1 and SLC16A1 were, respectively, the most abundantly expressed glucose and lactate (monocarboxylate) transporters. We further observed that the protein products of SLC2A1 (encoding GLUT1) and SLC16A1 (encoding MCT1) localize on the apical membrane of ARPE-19 monolayers. In a subsequent time-course experiment, we found that SLC2A1 and SLC16A1 mRNA oscillated in ARPE-19 monolayers, but not in dispersed cells, suggesting that monolayer cellular organization is necessary for rhythmic regulation of these transporters. In these monolayers, we found that MCT1 proteins varied over time, in contrast to GLUT1 proteins which did not vary over time. Spectrophotometric measurements of supernatants sampled from ARPE-19 monolayer cultures revealed that glucose concentrations did not significantly differ between apical (Api) supernatants and basolateral (BL) ones. In addition, we did not find rhythms in Api or BL glucose concentrations. Conversely, we found higher lactate concentrations in Api supernatants than BL ones. Further, we found that Api lactate concentrations were rhythmic. Pearson's r revealed that the concentration gradients (Api - BL) of glucose and lactate correlated with the gene expression of respective SLC2A1 and SLC16A1 transporters. Incubation with photoreceptor outer segments (POS) affected the mRNA expression of SLC16A1 and SLC2A1 in ARPE-19 monolayers in a time-dependent manner, thus suggesting that the retina might modulate the RPE clock-controlled expression of transporters via interactions with POS. In conclusion, this work provides evidence that the transport of lactate is regulated by the circadian clock in the RPE.

MT1 and MT2 melatonin receptors are expressed in nonoverlapping neuronal populations.

Melatonin (MLT) exerts its physiological effects principally through two high-affinity membrane receptors MT1 and MT2. Understanding the exact mechanism of MLT action necessitates the use of highly selective agonists/antagonists to stimulate/inhibit a given MLT receptor. The respective distribution of MT1 and MT2 within the CNS and elsewhere is controversial, and here we used a "knock-in" strategy replacing MT1 or MT2 coding sequences with a LacZ reporter. The data show striking differences in the distribution of MT1 and MT2 receptors in the mouse brain: whereas the MT1 subtype was expressed in very few structures (notably including the suprachiasmatic nucleus and pars tuberalis), MT2 subtype receptors were identified within numerous brain regions including the olfactory bulb, forebrain, hippocampus, amygdala and superior colliculus. Co-expression of the two subtypes was observed in very few structures, and even within these areas they were rarely present in the same individual cell. In conclusion, the expression and distribution of MT2 receptors are much more widespread than previously thought, and there is virtually no correspondence between MT1 and MT2 cellular expression. The precise phenotyping of cells/neurons containing MT1 or MT2 receptor subtypes opens new perspectives for the characterization of links between MLT brain targets, MLT actions and specific MLT receptor subtypes.

Circadian Analysis of the Mouse Cerebellum Proteome.

The cerebellum contains a circadian clock, generating internal temporal signals. The daily oscillations of cerebellar proteins were investigated in mice using a large-scale two-dimensional difference in gel electrophoresis (2D-DIGE). Analysis of 2D-DIGE gels highlighted the rhythmic variation in the intensity of 27/588 protein spots (5%) over 24 h based on cosinor regression. Notably, the rhythmic expression of most abundant cerebellar proteins was clustered in two main phases (i.e., midday and midnight), leading to bimodal distribution. Only six proteins identified here to be rhythmic in the cerebellum are also known to oscillate in the suprachiasmatic nuclei, including two proteins involved in the synapse activity (Synapsin 2 [SYN2] and vesicle-fusing ATPase [NSF]), two others participating in carbohydrate metabolism (triosephosphate isomerase (TPI1] and alpha-enolase [ENO1]), Glutamine synthetase (GLUL), as well as Tubulin alpha (TUBA4A). Most oscillating cerebellar proteins were not previously identified in circadian proteomic analyses of any tissue. Strikingly, the daily accumulation of mitochondrial proteins was clustered to the mid-resting phase, as previously observed for distinct mitochondrial proteins in the liver. Moreover, a number of rhythmic proteins, such as SYN2, NSF and TPI1, were associated with non-rhythmic mRNAs, indicating widespread post-transcriptional control in cerebellar oscillations. Thus, this study highlights extensive rhythmic aspects of the cerebellar proteome.

Rev-Erbα modulates retinal visual processing and behavioral responses to light.

The circadian clock is thought to adjust retinal sensitivity to ambient light levels, yet the involvement of specific clock genes is poorly understood. We explored the potential role of the nuclear receptor subfamily 1, group D, member 1 (REV-ERBα; or NR1D1) in this respect. In light-evoked behavioral tests, compared with wild-type littermates, Rev-Erbα-/- mice showed enhanced negative masking at low light levels (0.1 lx). Rev-Erbα-/- mouse retinas displayed significantly higher numbers of intrinsically photosensitive retinal ganglion cells (ipRGCs; 62% more compared with wild-type) and more intense melanopsin immunostaining of individual ipRGCs. In agreement with a pivotal role for melanopsin, negative masking at low light intensities was abolished in Rev-Erbα-/- Opn4-/- (melanopsin gene) double-null mice. Rev-Erbα-/- mice showed shortened latencies of both a and b electroretinogram waves, modified scotopic and photopic b-wave and scotopic threshold responses, and increased pupillary constriction, all of which suggested increased light sensitivity. However, wild-type and Rev-Erbα-/- mice displayed no detectable differences by in vivo fundus imaging, retinal histology, or expression of cell type-specific markers for major retinal cell populations. We conclude that REV-ERBα plays a major role in retinal information processing, and we speculate that REV-ERBα and melanopsin set sensitivity levels of the rod-mediated ipRGC pathway to coordinate activity with ambient light.-Ait-Hmyed Hakkari, O., Acar, N., Savier, E., Spinnhirny, P., Bennis, M., Felder-Schmittbuhl, M.-P., Mendoza, J., Hicks, D. Rev-Erbα modulates retinal visual processing and behavioral responses to light.

Circadian organization of the rodent retina involves strongly coupled, layer-specific oscillators.

Rhythmic physiology is central to retinal function and survival and adapts vision to daily light intensity changes. Mammalian retina rhythmically releases melatonin when cultured under constant conditions, and the occurrence of clock gene [e.g., Period (Per)] expression has been shown for most cellular layers. However, contribution of the distinct layers to genesis of circadian rhythms within the retina is still debated. To characterize their endogenous oscillatory capacity and their communication at the whole-tissue level, we used a vibratome-based method to isolate individual or paired retina cellular layers from the mPer2(Luc) mouse and Per1-luciferase (Per1-Luc) rat, and real-time recorded bioluminescence. We report that each layer of the mouse retina harbors a self-sustained oscillator whose period is significantly longer (∼ 26 hours) than in whole-retina explants (∼ 22.9 hours), indicating that the period is correlated with the degree of coupling. Accordingly, the maximal period (∼ 29 hours) is reached upon complete enzymatic dissociation of the retina. By using pharmacological approaches, we demonstrate that connection between retina oscillators involves gap junctions but only minor contribution from the main retina neurochemicals. Taken together with results from Per1-Luc rats, these data show that mammalian retina consists of a network of layer-specific oscillators whose period is determined by their connectivity.

Circadian clocks in rat skin and dermal fibroblasts: differential effects of aging, temperature and melatonin.

As a peripheral tissue localized at the interface between internal and external environments, skin performs functions which are critical for the preservation of body homeostasis, in coordination with environmental changes. Some of these functions undergo daily variations, such as temperature or water loss, suggesting the presence of time-keeping mechanisms. Rhythmic functions are controlled by a network of circadian oscillators present virtually in every cell and coordinated by the central clock located in the suprachiasmatic nuclei. At the molecular level, circadian rhythms are generated by conserved transcriptional-translational feedback loops involving several clock genes, among which Per1 and Per2 play a central role. Here we characterize clock activity in skin of the transgenic Per1-luciferase rat during postnatal development and adulthood, by real-time recording of bioluminescence in explants and primary dermal fibroblasts, and report marked transformation in circadian properties, from early life to aging. Using primary dermal fibroblast cultures we provide evidence that melatonin treatment phase dependently increases the amplitude of circadian oscillations and that ambient temperature impacts on their period, with slight overcompensation. Together, these findings demonstrate that skin contains a self-sustained circadian clock undergoing age-dependent changes. Dermal fibroblasts, one of the major skin cell types, also exhibit robust, yet specific, circadian rhythmicity which can be fine-tuned by both internal (melatonin) and external (temperature) factors.

Rat retina shows robust circadian expression of clock and clock output genes in explant culture.

PURPOSE: Circadian rhythms are central to vision and retinal physiology. A circadian clock located within the retina controls various rhythmic processes including melatonin synthesis in photoreceptors. In the present study, we evaluated the rhythmic expression of clock genes and clock output genes in retinal explants maintained for several days in darkness. METHODS: Retinas were dissected from Wistar rats, either wild-type or from the Per1-luciferase transgenic line housed under a daily 12 h:12 h light-dark cycle (LD12/12), and put in culture at zeitgeber time (ZT) 12 on semipermeable membranes. Explants from wild-type rats were collected every 4 h over 3 days, and total RNA was extracted, quantified, and reverse transcribed. Gene expression was assessed with quantitative PCR, and the periodicity of the relative mRNA amounts was assessed with nonlinear least squares fitting to sine wave functions. Bioluminescence in explants from Per1-luciferase rats was monitored for several days under three different culture protocols. RESULTS: Rhythmic expression was found for all studied clock genes and for clock downstream targets such as c-fos and arylalkylamine N-acetyltransferase (Aanat) genes. Clock and output genes cycled with relatively similar periods and acrophases (peaks of expression during subjective night, except c-fos, which peaked around the end of the subjective day). Data for Per1 were confirmed with bioluminescence monitoring, which also permitted culture conditions to be optimized to study the retina clock. CONCLUSIONS: Our work shows the free-running expression profile of multiple clock genes and potential clock targets in mammalian retinal explants. This research further strengthens the notion that the retina contains a self-sustained oscillator that can be functionally characterized in organotypic culture.

Hyperglycemia and circadian disruption lead to retinal dysfunction in a stabilized colony of the fat sand rat Psammomys obesus.

PURPOSE: The Fat Sand Rat (Psammomys obesus) recapitulates several features of human pre-proliferative diabetic retinopathy, but data are restricted to wild animals, incompatible with stringent biomedical research criteria. To overcome this barrier, we characterized retinal changes in a colony of P. obsesus maintained under strictly controlled housing conditions. METHODS: Animals were maintained on low or high caloric energy diets, and raised under either standard (12 h light/12 h dark) or shortened (5 h light/5 h dark) photoperiods. Visual responses were tested by electroretinography, while structural/molecular changes were assayed by immunochemistry and molecular biology (RNAseq and qPCR). RESULTS: Whereas high calorie diet alone did not induce hyperglycemia, coupled with short photoperiod >80 % animals developed severe hyper-insulinemia by 15 weeks, and 16 % animals further developed hyperglycemia. In these groups, electroretinography showed significant declines in visual responses in both hyper-insulinemic and hyperglycemic animals, especially in photopic (cone) responses. Transcriptomics analysis of hyperglycemic compared to low caloric controls revealed major upregulation in pathways involved in glial activation, extracellular matrix remodeling, inflammation, cytokine production, partial ischemic responses and angiogenesis. Western blotting against rhodopsin and cone opsin also showed decreased levels in both groups, overall decreases being greater for cones than rods in hyperglycemic animals. CONCLUSIONS: P. obesus maintained in rigorously monitored captive conditions, albeit showing attenuated responses to dietary overload compared to wild counterparts, nevertheless do develop some retinal features of diabetic retinopathy-like degeneration. Such a colony with known sanitary status opens their broader use for biomedical research.

Aryl Hydrocarbon Receptor Involvement in the Sodium-Dependent Glutamate/Aspartate Transporter Regulation in Cerebellar Bergmann Glia Cells.

Glutamate, the major excitatory neurotransmitter in the vertebrate brain, exerts its functions through the activation of specific plasma membrane receptors and transporters. Overstimulation of glutamate receptors results in neuronal cell death through a process known as excitotoxicity. A family of sodium-dependent glutamate plasma membrane transporters is responsible for the removal of glutamate from the synaptic cleft, preventing an excitotoxic insult. Glial glutamate transporters carry out more than 90% of the brain glutamate uptake activity and are responsible for glutamate recycling through the GABA/Glutamate/Glutamine shuttle. The aryl hydrocarbon receptor is a ligand-dependent transcription factor that integrates environmental clues through its ability to heterodimerize with different transcription factors. Taking into consideration the fundamental role of glial glutamate transporters in glutamatergic synapses and that these transporters are regulated at the transcriptional, translational, and localization levels in an activity-dependent fashion, in this contribution, we explored the involvement of the aryl hydrocarbon receptor, as a model of environmental integrator, in the regulation of the glial sodium-dependent glutamate/aspartate transporter. Using the model of chick cerebellar Bergmann glia cells, we report herein that the aryl hydrocarbon receptors exert a time-dependent decrease in the transporter mRNA levels and a diminution of its uptake activity. The nuclear factor kappa light chain enhancer of the activated B cell signaling pathway is involved in this regulation. Our results favor the notion of an environmentally dependent regulation of glutamate removal in glial cells and therefore strengthen the notion of the involvement of glial cells in xenobiotic neurotoxic effects.

Mice lacking Period 1 and Period 2 circadian clock genes exhibit blue cone photoreceptor defects.

Many aspects of retinal physiology are modulated by circadian clocks, but it is unclear whether clock malfunction impinges directly on photoreceptor survival, differentiation or function. Eyes from wild-type (WT) and Period1 (Per1) and Period2 (Per2) mutant mice (Per1(Brdm1) Per2(Brdm1) ) were examined for structural (histology, in vivo imaging), phenotypical (RNA expression, immunohistochemistry) and functional characteristics. Transcriptional levels of selected cone genes [red/green opsin (Opn1mw), blue cone opsin (Opn1sw) and cone arrestin (Arr3)] and one circadian clock gene (RORb) were quantified by real-time polymerase chain reaction. Although there were no changes in general retinal histology or visual responses (electroretinograms) between WT and Per1(Brdm1) Per2(Brdm1) mice, compared with age-matched controls, Per1(Brdm1) Per2(Brdm1) mice showed scattered retinal deformations by fundus inspection. Also, mRNA expression levels and immunostaining of blue cone opsin were significantly reduced in mutant mice. Especially, there was an alteration in the dorsal-ventral patterning of blue cones. Decreased blue cone opsin immunoreactivity was present by early postnatal stages, and remained throughout maturation. General photoreceptor differentiation was retarded in young mutant mice. In conclusion, deletion of both Per1 and Per2 clock genes leads to multiple discrete changes in retina, notably patchy tissue disorganization, reductions in cone opsin mRNA and protein levels, and altered distribution. These data represent the first direct link between Per1 and Per2 clock genes, and cone photoreceptor differentiation and function.

Prolonged light exposure induces widespread phase shifting in the circadian clock and visual pigment gene expression of the Arvicanthis ansorgei retina.

PURPOSE: Prolonged periods of constant lighting are known to perturb circadian clock function at the molecular, physiological, and behavioral levels. However, the effects of ambient lighting regimes on clock gene expression and clock outputs in retinal photoreceptors--rods, cones and intrinsically photosensitive retinal ganglion cells--are only poorly understood. METHODS: Cone-rich diurnal rodents (Muridae: Arvicanthis ansorgei) were maintained under and entrained to a 12 h:12 h light-dark cycle (LD; light: ~300 lux). Three groups were then examined: control (continued maintenance on LD); animals exposed to a 36 h dark period before sampling over an additional 24 h period of darkness (DD); and animals exposed to a 36 h light period before sampling over an additional 24 h period of light (~300 lux, LL). Animals were killed every 3 or 4 h over 24 h, their retinas dissected, and RNA extracted. Oligonucleotide primers were designed for the Arvicanthis clock genes Per1, Per2, Cry1, Cry2, and Bmal1, and for transcripts specific for rods (rhodopsin), cones (short- and mid-wavelength sensitive cone opsin, cone arrestin, arylalkylamine N-acetyltransferase) and intrinsically photosensitive retinal ganglion cells (melanopsin). Gene expression was analyzed by real-time PCR. RESULTS: In LD, expression of all genes except cone arrestin was rhythmic and coordinated, with acrophases of most genes at or shortly following the time of lights on (defined as zeitgeber time 0). Arylalkylamine N-acetyltransferase showed maximal expression at zeitgeber time 20. In DD conditions the respective profiles showed similar phase profiles, but were mostly attenuated in amplitude, or in the case of melanopsin, did not retain rhythmic expression. In LL, however, the expression profiles of all clock genes and most putative output genes were greatly altered, with either abolition of daily variation (mid-wavelength cone opsin) or peak expression shifted by 4-10 h. CONCLUSIONS: These data are the first to provide detailed measures of retinal clock gene and putative clock output gene expression in a diurnal mammal, and show the highly disruptive effects of inappropriate (nocturnal) lighting on circadian and photoreceptor gene regulation.

Human skin keratinocytes, melanocytes, and fibroblasts contain distinct circadian clock machineries.

Skin acts as a barrier between the environment and internal organs and performs functions that are critical for the preservation of body homeostasis. In mammals, a complex network of circadian clocks and oscillators adapts physiology and behavior to environmental changes by generating circadian rhythms. These rhythms are induced in the central pacemaker and peripheral tissues by similar transcriptional-translational feedback loops involving clock genes. In this work, we investigated the presence of functional oscillators in the human skin by studying kinetics of clock gene expression in epidermal and dermal cells originating from the same donor and compared their characteristics. Primary cultures of fibroblasts, keratinocytes, and melanocytes were established from an abdominal biopsy and expression of clock genes following dexamethasone synchronization was assessed by qPCR. An original mathematical method was developed to analyze simultaneously up to nine clock genes. By fitting the oscillations to a common period, the phase relationships of the genes could be determined accurately. We thereby show the presence of functional circadian machinery in each cell type. These clockworks display specific periods and phase relationships between clock genes, suggesting regulatory mechanisms that are particular to each cell type. Taken together, our data demonstrate that skin has a complex circadian organization. Oscillators are present not only in fibroblasts but also in epidermal keratinocytes and melanocytes and are likely to act in coordination to drive rhythmic functions within the skin.

Aryl Hydrocarbon Receptor in Glia Cells: A Plausible Glutamatergic Neurotransmission Orchestrator.

Glutamate is the major excitatory amino acid in the vertebrate brain. Glutamatergic signaling is involved in most of the central nervous system functions. Its main components, namely receptors, ion channels, and transporters, are tightly regulated at the transcriptional, translational, and post-translational levels through a diverse array of extracellular signals, such as food, light, and neuroactive molecules. An exquisite and well-coordinated glial/neuronal bidirectional communication is required for proper excitatory amino acid signal transactions. Biochemical shuttles such as the glutamate/glutamine and the astrocyte-neuronal lactate represent the fundamental involvement of glial cells in glutamatergic transmission. In fact, the disruption of any of these coordinated biochemical intercellular cascades leads to an excitotoxic insult that underlies some aspects of most of the neurodegenerative diseases characterized thus far. In this contribution, we provide a comprehensive summary of the involvement of the Aryl hydrocarbon receptor, a ligand-dependent transcription factor in the gene expression regulation of glial glutamate transporters. These receptors might serve as potential targets for the development of novel strategies for the treatment of neurodegenerative diseases.

Combinatorial regulation of photoreceptor differentiation factor, neural retina leucine zipper gene NRL, revealed by in vivo promoter analysis.

Development and homeostasis require stringent spatiotemporal control of gene expression patterns that are established, to a large extent, by combinatorial action of transcription regulatory proteins. The bZIP transcription factor NRL (neural retina leucine zipper) is critical for rod versus cone photoreceptor cell fate choice during retinal development and acts as a molecular switch to produce rods from postmitotic precursors. Loss of Nrl in mouse leads to a cone-only retina, whereas ectopic expression of Nrl in photoreceptor precursors generates rods. To decipher the transcriptional regulatory mechanisms upstream of Nrl, we identified putative cis-control elements in the Nrl promoter/enhancer region by examining cross-species sequence conservation. Using in vivo transfection of promoter-reporter constructs into the mouse retina, we show that a 0.9-kb sequence upstream of the Nrl transcription initiation site is sufficient to drive reporter gene expression in photoreceptors. We further define a 0.3-kb sequence including a proximal promoter (cluster A1) and an enhancer (cluster B) that can direct rod-specific expression in vivo. Electrophoretic mobility shift assays using mouse retinal nuclear extracts, in combination with specific antibodies, demonstrate the binding of retinoid-related orphan nuclear receptor ß (RORß), cone rod homeobox, orthodenticle homolog 2, and cyclic AMP response element-binding protein to predicted consensus elements within clusters A and B. Our studies demonstrate Nrl as a direct transcriptional target of RORß and suggest that combinatorial action of multiple regulatory factors modulates the expression of Nrl in developing and mature retina.