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Purpose: Inflammation generates changes in the protein constituents of the aqueous humor. Proteins that change in multiple models of uveitis may be good biomarkers of disease or targets for therapeutic intervention. The present study was conducted to identify differentially-expressed proteins in the inflamed aqueous humor., Methods: Two models of uveitis were induced in Lewis rats: experimental autoimmune uveitis (EAU) and primed mycobacterial uveitis (PMU). Differential gel electrophoresis was used to compare naïve and inflamed aqueous humor. Differentially-expressed proteins were separated by using 2-D gel electrophoresis and excised for identification with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Expression of select proteins was verified by Western blot analysis in both the aqueous and vitreous., Results: The inflamed aqueous from both models demonstrated an increase in total protein concentration when compared to naïve aqueous. Calprotectin, a heterodimer of S100A8 and S100A9, was increased in the aqueous in both PMU and EAU. In the vitreous, S100A8 and S100A9 were preferentially elevated in PMU. Apolipoprotein E was elevated in the aqueous of both uveitis models but was preferentially elevated in EAU. Beta-B2-crystallin levels decreased in the aqueous and vitreous of EAU but not PMU., Conclusions: The proinflammatory molecules S100A8 and S100A9 were elevated in both models of uveitis but may play a more significant role in PMU than EAU. The neuroprotective protein β-B2-crystallin was found to decline in EAU. Therapies to modulate these proteins in vivo may be good targets in the treatment of ocular inflammation.

Cryptochrome (CRY) is the primary circadian photoreceptor in Drosophila. It resets the circadian clock by promoting light-induced degradation of the clock proteins Timeless and Period, as well as its own proteolysis. The E3 ligases that ubiquitylate Timeless and Period before degradation are known and it is known that Drosophila (d) CRY is degraded by the ubiquitin-proteasome system as well. To identify the E3 ligase for dCRY we screened candidates in S2 cells by RNAi. Knockdown of each of the 25 putative F-box proteins identified by bioinformatics did not attenuate the light-induced degradation of dCRY. However, knockdown of a WD40 protein, Bromodomain and WD repeat domain containing 3 (Brwd3) (CG31132/Ramshackle) caused strong attenuation of dCRY degradation following light exposure. We found that BRWD3 functions as a Damage-specific DNA binding protein 1 (DDB1)- and CULLIN (CUL)4-associated factor in a Cullin4-RING Finger E3 Ligase (CRL4) that mediates light-dependent binding of dCRY to CUL4-ROC1-DDB1-BRWD3, inducing ubiquitylation of dCRY and its light-induced degradation. Thus, this study identifies a light-activated E3 ligase complex essential for light-mediated CRY degradation in Drosophila cells.

A subset of intrinsically photosensitive retinal ganglion cells transduce information about ambient lighting conditions to areas of the brain involved in tasks including entrainment of the circadian clock, pupillary light reflexes and melatonin synthesis. The phototransduction system(s) utilized by these cells are unknown. Melanopsin and cryptochromes have been proposed as candidate photopigments for this system. Recent analyses of retinal degenerate mice lacking melanopsin or cryptochromes indicates that outer and inner photoreceptors can both contribute to non-visual photoresponses, and that both melanopsin and cryptochromes play important roles in this process.

The basic molecular mechanisms underlying circadian oscillators follow the same general plan across the phylogenetic spectrum: oscillating feedback loops in which clock gene products negatively regulate their own expression. The circadian clocks of animals involve at least two interacting feedback loops. This Viewpoint compares and contrasts the circadian clocks of mammals and of Drosophila, emphasizing how different players are used to create the same basic script. Both the general script and the specific details of the murine and Drosophila circadian pathways are available at Science's Signal Transduction Knowledge Environment Connections Maps.

Nearly all circadian clocks have free-running periods that differ significantly from 24 hours. To maintain synchrony with the 24 h day, the mammalian circadian clock is reset by light. Unlike other animals, mammalian circadian entrainment occurs exclusively via the eyes and optic nerves. Remarkably, the classical photoreceptors--the rods and cones--are not necessary for photic entrainment. Instead, a subset of inner retinal ganglion cells are directly photoresponsive and transmit photic information specifically to brain centres involved in irradiance detection, including the master circadian pacemaker in the suprachiasmatic nucleus of the hypothalamus. The photopigment(s) responsible for inner retinal phototransduction are unknown. Several lines of evidence constrain candidate photopigments. First, near-total vitamin A depletion does not diminish retinohypothalamic signalling. Second, loss of cryptochrome function in retinal-degenerate mice substantially decreases photic signalling to the suprachiasmatic nucleus, and markedly decreases pupillary light responses. Third, vitamin A depletion of cryptochrome mutant mice leads to loss of photic signalling to the suprachiasmatic nucleus. These findings suggest a model where either classical photopigments or inner retinal photopigments are sufficient for non-visual irradiance detection.

Cryptochromes function in both light entrainment of circadian rhythms and in a peripheral circadian clock mechanism in Drosophila. Mice have two closely related cryptochrome genes (mCry1 and mCry2). To further understand the roles of mammalian cryptochromes, we bred mice to carry all possible combinations of wild-type and cryptochrome knockout alleles, and tested these mice for free-running and entrained circadian rhythmicity. We find that a single wild-type copy of mCry1, but not mCry2, is sufficient for free running circadian rhythmicity; however, these mice show markedly variable free-running periods. Two wild-type copies of either mCry1 or mCry2 are sufficient to establish a stable free-running clock. A subset of mCry1-/mCry-; mCry2-/mCry2- mice have a diurnal activity preference, suggesting that cryptochromes function in light-dependent behavioral masking. We also analyzed mice lacking both cryptochromes and carrying the homozygous rd retinal degeneration mutation. These mice have markedly depressed behavioral photoresponses in light-dark conditions, despite having an anatomically intact retinohypothalamic tract and normal expression of melanopsin. These results suggest that, similar to insect cryptochromes, mammalian cryptochromes function pleiotropically in both circadian rhythm generation and in photic entrainment and behavioral responses.

Cryptochromes are a family of flavoproteins found in organisms ranging from Arabidopsis to man. Across phylogeny, these proteins have been used for pleiotropic functions ranging from blue-light-dependent development in plants and blue-light-mediated phase shifting of the circadian clock in insects to a core circadian clock component in mammals. Review of the roles of cryptochromes in model organisms reveals several common themes: Multiple cryptochrome family members within individual organisms have redundant functions; cryptochromes used in photic entrainment pathways of the circadian clock are partially redundant with other photopigments; and cryptochromes may function in circadian phototransduction and core clock mechanisms in the same organism, with different functions in different tissues. The present review summarizes recent research on the functions of cryptochrome in the circadian timekeeping and photic entrainment pathways.

At least six light-regulated phenomena are preserved in the eyes of retinally degenerate mice, including the entrainment of circadian rhythms, the gating of ocular immune response, and pupillary reactivity. Some of these phenomena have also been observed in blind human patients. These findings have prompted the search for a non-visual ocular phototransduction mechanism. Molecular genetic studies have identified several candidate genes for these effects. These include genes encoding novel ocular opsins, such as melanopsin, as well as potential flavin-based photopigments. Data linking these potential photoreceptors to these phenomena are discussed, and the clinical implications of these findings are explored.

The daily light-dark (LD) cycle exerts a powerful influence on the temporal organization of behavior and physiology. Much of this influence is preserved in behaviorally blind retinally degenerate mice; the photoreceptors underlying this nonvisual phototransduction are unknown. The mammalian eye contains at least two classes of photoactive pigments, the vitamin A-based opsins and the vitamin B(2)-based cryptochromes. To genetically define the roles of these pigments in light modulation of behavior, we generated rd/rd;mCry1(-)/mCry1(-);mCry2(-)/mCry2(-) mutant mice lacking rods and most cones as well as both cryptochrome proteins. The response of the mutant mouse to photic input was analyzed at both behavioral and molecular levels. Behaviorally, mice lacking either classical photoreceptors or cryptochromes exhibited strongly rhythmic locomotor responses to 10 and 100 lux daily LD 12 h/12-h cycles; however, triple mutant mice carrying both cryptochrome and retinal degenerate mutations were nearly arrhythmic under both LD cycles and in constant darkness. At the molecular level, the light induction of c-fos transcription in the suprachiasmatic nucleus was markedly reduced in the triple mutant mouse compared with either rd/rd or cryptochrome mutant mice. These data indicate that classical opsins and cryptochromes serve functionally redundant roles in the transduction of light information to behavioral modulation and suggest a pleomorphic role for cryptochromes in both photoreception and central clock mechanism.