Ординатура / Офтальмология / Английские материалы / The Retina and its Disorders_Besharse, Bok_2011

RPE layer

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RPE layer

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Figure 7 (a) Time domain optical coherence tomogram from patient with normal macula showing good definition of macular layers and normal foveal contour. (b) Optical coherence tomogram from patient with neurosensory retinal choroidal neovascularization, showing disruption of ordered architecture of the retinal layers, increased hyperreflectance (red) in layer near RPE/Bruch’s membrane and intraretinal cysts, indicating intraretinal fluid (arrowhead). (c) Optical coherence tomogram showing subretinal fluid (dotted arrow – beneath the photoreceptors and above the RPE) and sub-RPE fluid (solid arrow – beneath the RPE and above Bruch’s membrane).

endothelial growth factor (VEGF)). All these stimuli can lead to increased secretion of VEGF, which also increases permeability of blood vessels and RPE and choroidal endothelial migration, proliferation, and chemotaxis, all processes believed important in the development of neurosensory retinal choroidal neovascularization. Furthermore, RPE can release other cytokines that recruit leukocytes, including macrophages that can then release VEGF, or that interact with other processes important in the pathogenesis of AMD.

Diabetes Mellitus

In diabetic retinopathy, the inner BRB is impaired and is most easily appreciated clinically on fluorescein angiography, as leakage from microaneurysms, dilated capillaries, intraretinal microvascular abnormalities, and

neovascularization that grows above the inner limiting membrane into the vitreous (Figure 9). However, fluorescein staining at the level of the RPE is seen on fluorescein angiograms of patients with diabetes. Also, hyperglycemia has been shown to impair the function of tight junctions of the RPE in vitro. Diabetic retinopathy also impairs vision through the development of macular edema, which occurs when there is fluid and solutes that leak from the vasculature into the neurosensory retina (Figure 10). It can occur through a breakdown of the inner and potentially outer BRBs. Besides the finding that hyperglycemia can reduce TER in cultured RPE, animal models of diabetic retinopathy have found that there is reduced Na,K-ATPase activity in the RPE. So, once retinal blood vessels leak fluid, lipids, and protein into the neurosensory retina, mechanisms to transport fluid and compounds out of the retina also appear to be impaired in the diabetic state.

Inflammation has been shown to play a role in the pathophysiology of diabetic retinopathy. In animal models, leukostasis or adherence of white blood cells to retinal capillaries has been found and postulated to be a mechanism of later capillary nonperfusion and endothelial damage, which precede the development of proliferative diabetic retinopathy and macular edema. Furthermore, diabetes can also cause a choroidal vasculopathy associated with leukostasis, which may cause choroidal ischemia and later angiogenesis both of which can interfere with intrinsic ocular flow from the vitreous toward the choroid.

Proliferative Vitreoretinopathy

Proliferative vitreoretinopathy (PVR) occurs when cellular contractile membranes develop on the surface of the retina and contract it and pull open breaks in the retina, which can lead to complex retinal detachments. It is the most common cause of failed retinal detachment repair. Vitreous fluorophotometry readings in animal models of PVR show a breakdown in the BRB associated with released cells into the vitreous cavity. It is believed that RPE cells, serum, and other factors have access to the vitreous cavity and are responsible for further breakdown of the BRB and growth of preretinal membranes.

The treatment for PVR, currently, is surgical, requiring vitrectomy and stripping of the membranes from the retina, and then methods to reattach the retina and create a permanent chorioretinal adhesion. However, ongoing research may provide medical means to prevent the formation of preretinal membranes.

Drug Toxicity

Several drugs, including thioridazine (Mellaril), thorazine, hydroxychloroquine, and chloroquine, have been shown to cause vision loss and toxicity with pigmentary changes. The exact effects on the RPE and BRB are unclear.

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Figure 9 (a) Color images of right eye of patient with diabetic retinopathy previously treated with laser (examples of pigmented laser spots shown by arrows). (b) Fluorescein angiogram of same eye showing intraretinal microvascular abnormalities (arrow) and areas of avascular retina with dilated capillaries, an irregular foveal avascular zone, and microaneurysms (arrowhead). Dotted arrow shows area of hyperfluorescence from leaking neovascularization likely growing above the inner limiting membrane.

Central Serous Retinopathy

Central serous retinopathy (CSR) is a clinical disease often occurring in young to middle-aged individuals, although it can also manifest or recur and become chronic in later life. Symptoms include reduced vision or inability to focus, and clinical examination shows the presence of a neurosensory retinal detachment within the macula. Fluorescein angiography shows focal areas of RPE leaks (Figure 11) or diffuse RPE disturbances. In chronic CSR, RPE decompensation occurs (Figure 12) and can lead to chronic subretinal leakage and accumulation of subretinal

Figure 10 Example of intraretinal edema in a time domain optical coherence tomogram from a patient with diabetic macular edema. Note that the RPE reflective layer is intact in contrast to Figure 7(b) in which invasion of cells into the neurosensory retina has occurred in neovascular AMD.

and intraretinal fluid. Although cases of CSR usually resolve without permanent vision loss, recurrent or chronic CSR can lead to permanent loss of visual acuity. Even when a sole leak appears to be present, CSR is believed to be associated with broad RPE dysfunction, because if only one area of dysfunction were present, the Na,K-ATPases of surrounding healthy RPE would pump out fluid from the subretinal space.

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Figure 12 RPE decompensation showing broad area of hyperfluorescence in fluorescein angiogram from chronic longstanding central serous retinopathy in 60 year old male.

Angiograms using indocyanine green dye, which permits visualization of the choroidal vasculature, show that areas of choroidal hypoperfusion and later choroidal hyperpermeability are present in CSR. Although one might suspect inflammation to be a cause of the hyperpermeability, treatment with steroids can severely worsen CSR and should be avoided. Corticosteroids can affect the expression of adrenergic receptor genes and it is thought that this contributes to the overall effect of catecholamines on CSR. Some have postulated that the pathology may involve the adrenocorticotrophic hormone. Another unusual aspect of CSR is that treatment of a sole RPE leak on fluorescein angiography can hasten resolution of the serous detachment, even though it is believed that broad RPE dysfunction is present. Besides corticosteroids, hypertension also increases the risk.

CSR has long been believed to be associated with breakdown in the BRB particularly of the RPE. The cause remains unknown but it is associated with increased stress and

a type A personality and is believed to be related to elevated cortisol and epinephrine, which affect the autoregulation of the choroidal circulation. In early studies, adult Japanese monkeys that received multiple daily (>30) injections of intravenous adrenalin developed serous retinal detachments and leaking RPE spots by fluorescein angiography similar in appearance to that seen in CSR. An intramuscular injection of prednisolone led to the same findings on fluorescein angiography but required fewer doses of adrenalin.

Retinitis Pigmentosa

Evidence of abnormalities in the localization of ZO-1, beta-catenin, and other associated adherens proteins in the rho / mouse, a model of autosomal dominant retinitis pigmentosa, provides support for tight junction and adherens junction-associated protein modifications in retinitis pigmentosa. Furthermore, in retinitis pigmentosa, there is cystoid macular edema often associated with hyperfluorescence of the RPE cells on fluorescein angiography, suggesting a breakdown of the RPE barrier. Toxins such as sodium iodate selectively poison the RPE and have been used to test the role of the RPE BRB in animal models in which fluid had been injected into the subretinal space or for studies in PVR. Although the toxin is selective for RPE, it poisons the RPE and therefore affects all functions of the RPE, not just the tight junctions.

Urethrane was used to test the BRB in earlier studies and was reported to lead to inhibition of intervesicular transport across endothelia and loss of RPE.

Growth Factors

Besides the role of VEGF in reducing RPE barrier properties, other growth factors play a role. Insulin-like growth factor-1 (IGF-1) can induce VEGF-related RPE barrier breakdown. Also, hepatocyte growth factor (HGF) can lead to disassembly of tight and adherens junctions in

association with reduced barrier properties. HGF has also been shown to be increased in human PVR.

Studies to Increase the Function of the BRB

Carbonic anhydrase inhibitors lead to acidification of the subretinal space, which, in turn, leads to an increase in chloride ion transport into the choroid, thus eliminating water from the subretinal space and retina and increasing the adhesiveness of the RPE. Carbonic anhydrase inhibitors have been associated with improved BRB function, based on reduced fluorescein leakage into the retina, in small clinical studies. However, larger clinical trials have shown a smaller benefit.

See also: Breakdown of the Blood–Retinal Barrier; Photopic, Mesopic and Scotopic Vision and Changes in Visual Performance; Phototransduction in Limulus Photoreceptors.

Further Reading

Alberts, B., Johnson, A., Lewis, J., et al. (2002). Cell junctions, cell adhesion, and the extracellular matrix. In: Alberts, B., Johnson, A., Lewis, J., et al. (eds.) Molecular Biology of the Cell, pp. 949–1009. New York: Garland Science.

Bian, Z. M., Elner, S. G., Yoshida, A., and Elner, V. M. (2003). Human RPE-monocyte co-culture induces chemokine gene expression through activation of MAPK and NIK cascade. Experimental Eye Research 76: 573–583.

Campbell, M., Humphries, M., Kennan, A., et al. (2006). Aberrant retinal tight junction and adherens junction protein expression in an animal model of autosomal dominant retinitis pigmentosa: The Rho(–/–) mouse. Experimental Eye Research 83: 484–492.

Crane, I. J., Wallace, C. A., McKillop-Smith, S., and Forrester, J. V. (2000). CXCR4 receptor expression on human retinal pigment epithelial cells from the blood–retina barrier leads to chemokine

secretion and migration in response to stromal cell-derived factor 1 alpha. Journal of Immunology 165: 4372–4378.

Dibas, A. and Yorio, T. (2008). Regulation of transport in the RPE. In: Tombran-Tink, J. and Barnstable, C. (eds.) Ocular Transporters in Ophthalmic Diseases and Drug Delivery, 1st edn., pp. 157–184.

Berlin: Springer/Humana Press.

Fubuoka, Y., Strainic, M., and Medof, M. E. (2003). Differential cytokine expression of human retinal pigment epithelial cells in response to stimulation by C5a. Clinical and Experimental Immunology

131: 248–253.

Hartnett, M. E., Lappas, A., Darland, D., et al. (2003). Retinal pigment epithelium and endothelial cell interaction causes retinal pigment epithelial barrier dysfunction via a soluble VEGF-dependent mechanism. Experimental Eye Research 77: 593–599.

Hu, J. and Bok, D. (2001). A cell culture medium that supports the differentiation of human retinal pigment epipthelium into functionally polarized monolayers. Molecular Vision 7: 14–19.

Jin, M., Barron, E., He, S., Ryan, S. J., and Hinton, D. R. (2002). Regulation of RPE intercellular junction integrity and function by hepatocyte growth factor. Investigative Ophthalmology and Visual Science 43: 2782–2790.

Lodish, H., Berk, A., Zipursky, S. L., et al. (2000). Transport across cell membranes. In: Molecular Cell Biology. Basingstokes:

WH Freeman.

Marmor, M. F. and Wolfensberger, T. J. (eds.) (1998). The Retinal Pigment Epithelium. New York: Oxford University Press.

Penn, J. S., Madan, A., Caldwell, R. B., et al. (2008). Vascular endothelial growth factor in eye disease. Progress in Retina and Eye Research 27(4): 331–371.

Rajasekaran, S. A., Hu, J., Gopal, J., et al. (2003). Na,K-ATPase inhibition alters tight junction structure and permeability in human retinal pigment epithelial cells. American Journal of Physiology – Cell Physiology 284: C1497–C1507.

Rizzolo, L. J. (2007). Development and role of tight junctions in the retinal pigment epithelium. In: Jeon, K. W. (ed.) International Review of Cytology, a Survey of Cell Biology vol. 258, pp. 195–234. San Diego, CA: Elsevier.

Sen, H. A., Robertson, T. J., Conway, B. P., and Campochiaro, P. A. (1988). The role of breakdown of the blood–retinal barrier in cellinjection models of proliferative vitreoretinopathy. Archives of Ophthalmology 106: 1291–1294.

Xu, H., Dawson, R., Crane, I. J., and Liversidge, J. (2005). Leukocyte diapedesis in vivo induces transient loss of tight junction protein at the blood–retina barrier. Investigative Ophthalmology and Visual Science 46: 2487–2494.

Yoshioka, H., Katsume, Y., and Akune, H. (1982). Experimental central serous chorioretinopathy in monkey eyes: Fluorescein angiographic findings. Ophthalmologica 185(3): 168–178.

Glossary

Basic helix–loop–helix-Per-ARNT-Sim (bHLHPAS) domain transcription factors – A family of transcription factors that heterodimerize and bind to E box enhancer elements in gene promoters. The family includes the clock gene products CLOCK, neuronal PAS domain protein 2 (NPAS2), and brain and muscle aryl hydrocarbon receptor nuclear translocator 1 (BMAL1), as well as the arylhydrocarbon nuclear receptor and the hypoxiainducible factors.

Circadian rhythms – Changes in biological processes that occur on a daily basis; they are driven by autonomous circadian clocks; these rhythms provide selective advantage to organisms by allowing them to anticipate temporal changes in their environment.

Clock-controlled genes – Genes that are regulated by circadian oscillators via clock gene transcription factors; the proteins encoded by clock-controlled genes are rhythmically expressed and generate rhythms of physiology.

Clock genes – Genes that encode proteins that form the molecular basis of circadian oscillators; most clock gene proteins are transcription factors. Scotopic vision – Vision in dim light that is mediated by rod photoreceptors and rod bipolar cell pathways. Zeitgeber time – Time of day relative to the light–dark cycle; light onset corresponds to ZT0.

Introduction

The chick retina has been used extensively to study ocular circadian rhythms. It displays particularly robust rhythms of clock gene expression, clock-controlled gene expression, and several biochemical and physiological clock outputs. In addition, embryonic chick retinal cells can be cultured, and they maintain light responsiveness and circadian rhythm generation in vitro. These cell cultures facilitate pharmacological and molecular studies of clock signaling pathways.

Clock Gene Expression

The current model for the molecular basis of circadian clocks involves transcriptional–translational feedback loops

that are comprised of highly conserved clock genes and the proteins that they encode (Figure 1). The clock gene proteins are characterized as positive and negative elements, based on their transcriptional activity. The positive elements include basic helix–loop–helix–PAS-domain (bHLH-PAS) transcription factors that heterodimerize and bind to circadian E box enhancer elements in promoters of other clock genes and clock-controlled genes. These positive elements include brain and muscle aryl hydrocarbon receptor nuclear translocator 1 (BMAL1; also called ARNTL, MOP3) and CLOCK. The BMAL1 heterodimerizes with CLOCK and the dimerized transcription factors stimulate the transcription of the genes encoding the negative elements, the cryptochromes (CRY1 and CRY2) and periods (PER1, PER2, and PER3). The CRY and PER proteins are imported into the nucleus and inhibit transactivation of their own promoters by BMAL1:CLOCK. In some clocks, neuronal PAS domain protein 2 (NPAS2, also called MOP4) can substitute for CLOCK and form active heterodimers with BMAL1. A second feedback loop involves E-box-mediated transcriptional activation of the orphan retinoic-acid-related receptor family genes, Rev-erba and Rora; the protein products of these genes contribute to the rhythmic regulation of Bmal1 gene transcription. These feedback loops, coupled with a variety of post translational modifications, generate daily rhythms of gene expression that ultimately generate physiological circadian rhythms.

Most of the clock gene transcripts identified in mammalian circadian clocks have been identified in the chick retina and in cultured chick photoreceptors, and the regulation of their expression has been analyzed (Figures 2 and 3). Of the genes encoding the positive elements of the oscillator, Bmal1 and Npas2 transcripts show robust daily rhythms under light–dark cycles or in constant (24 h day 1) darkness (Figure 2). These circadian rhythms peak near the time of subjective dusk ( ZT12) in vivo and in vitro. In contrast, Clock mRNA appears to be constitutively expressed in dark–dark (DD). Transcripts encoding the negative elements, the cryptochrome and period proteins, also show rhythmic expression (Figure 3(a) and 3(b)). The Cry1 mRNA peaks in the middle of the day ( ZT8) in vivo and in photoreceptor cultures. In vivo, the most robust rhythms of Cry1 mRNA expression are observed in the ganglion cell and photoreceptor cell layers. The Cry2 mRNA is also expressed in retina, but analyses of whole retina mRNA suggest that it is not rhythmically expressed. The Per2 mRNA expression is maximal in the early morning, while Per3 transcript level appears maximal late in the night.

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Thus far, Per1 has not been identified in the chick retina, and the gene may be missing from the chicken genome. In addition to oscillating in a clock-dependent manner, Per2 and Cry1 are rapidly induced by light exposure (Figure 3(c) and 3(d)). It is generally thought that this induction is a mechanism for circadian clock entrainment by light.

The rhythmic and light-regulated expression of circadian clock genes in the chick retina, particularly in cultured retinal cells, provides conclusive evidence that the retina contains autonomous circadian oscillators that can function independently of oscillators in the brain and pineal gland. Nevertheless, in the intact organism, retinal, pineal, and brain clocks are thought to interact to regulate physiology.

Circadian Regulation of Cyclic AMP

in Retina

Cyclic AMP is a ubiquitous second messenger molecule that regulates multiple aspects of cellular metabolism and

function. Effects of cyclic AMP are mediated by activation of cyclic AMP-dependent protein kinase (PKA), which phosphorylates proteins to regulate their function or activity. In so doing, cyclic AMP regulates intermediary metabolism, neurotransmission, and gene expression. Cyclic AMP can also affect cellular function by regulating cyclic nucleotidegated channels or activating Epac, a cyclic AMP-dependent Rap GTP exchange factor. The Rap is a guanine nucleotidebinding protein of the Ras family.

In chick photoreceptor cell cultures, cyclic AMP levels are regulated by light and circadian clocks. Photoreceptor cyclic AMP levels are high in darkness and reduced by light exposure. Thus, when cells are exposed to a daily light–dark cycle, cyclic AMP fluctuates as a daily rhythm with high levels at night in darkness and low levels during the daytime in light (Figure 4). Exposure to light at night rapidly reduces cyclic AMP. The daily rhythm of cyclic AMP persists, albeit with reduced amplitude, when cells are transferred from a light–dark cycle to constant darkness. Thus, the combined effects of illumination and

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circadian influences interact to generate the daily rhythm of cyclic AMP.

The regulation of cyclic AMP formation in chick photoreceptor cells is Ca2+-dependent, at least in part. Depolarization of the plasma membrane with high concentrations of extracellular K+ stimulates cyclic AMP formation. This effect requires Ca2+ influx through L-type voltage-gated Ca2+ channels. The plasma membrane of photoreceptors is partially depolarized in darkness and is hyperpolarized by light. Thus, the dark–light difference in cyclic AMP formation in photoreceptors is likely due to high Ca2+ conductance in darkness and decreased Ca2+conductance

following light exposure, due to closure of the voltage-gated channels. Accordingly, the circadian fluctuation in cyclic AMP levels is eliminated by nitrendipine, an L-type Ca2+ channel blocker.

Cyclic AMP is synthesized from ATP by adenylyl cyclase. There are 10 isoforms of adenylyl cyclase that are regulated by multiple mechanisms. The findings described above indicate that photoreceptor cyclic AMP levels may be regulated by a Ca2+/calmodulin-stimulated cyclase. The type 1 and type 8 adenylyl cyclases are both stimulated by Ca2+/calmodulin and are both expressed in the chick retina. There is a circadian rhythm in the expression of

Adcy1, the transcript that encodes the type 1 adenylyl cyclase, in photoreceptor cell cultures and in the chick retina in vivo. In addition, there is a circadian rhythm in Ca2+/calmodulin-stimulated adenylyl cyclase activity in membranes prepared from photoreceptor cell cultures, with high activity at night. Thus, the circadian regulation of cyclic AMP is due to clock-controlled expression of Ca2+/calmodulin-stimulated adenylyl cyclase. The mammalian Adcy1 gene contains an E-box in its promoter that can be activated by BMAL1:CLOCK heterodimers, and a similar mechanism may contribute to the circadian regulation of Adcy1 expression in chick photoreceptor cells. However, this hypothesis has not yet been tested directly in the chick. The circadian rhythm of cyclic AMP formation may also be influenced by the clock-controlled expression and activity of the L-type Ca2+ channels in photoreceptors.

Circadian Regulation of Melatonin

Biosynthesis

Melatonin is a neurohormone that is synthesized in the retinal photoreceptor cells and in the pineal gland. Melatonin synthesis in retinas of most vertebrate species, including chicken, is regulated in a circadian fashion, with high levels at night in darkness. Melatonin functions in the retina to optimize nighttime visual function. Like cyclic AMP formation, melatonin levels are regulated by both illumination and circadian clocks.

The key regulatory enzyme in melatonin biosynthesis is arylalkylamine N-acetyltransferase (AANAT). In chicken retina, AANAT activity undergoes a robust circadian rhythm with peak activity at night (Figure 5). Exposure to light at night causes a rapid decline in AANAT activity

Figure 4 Circadian fluctuation of intracellular cAMP level. Cells were prepared from embryonic neural retinas and incubated for 8 days under LD. Illumination was switched from LD to DD before expected onset of light at the beginning of day 9 in vitro (DIV 9). White symbols represent cAMP level at Zeitgeber time (ZT) 10 in light; black symbols represent ZT 20 in darkness; gray symbols represent subjective day, ZT 10, in darkness. The horizontal white and black bars above the x-axis represent times of light and darkness, respectively. Level of cAMP was significantly higher at night than during the day in LD on DIV 8 and this fluctuation persisted in DD on DIV 9 and DIV 10. Reproduced from Ivanova, T. N. and Iuvone, P. M. (2003). Circadian rhythm and photic control of cAMP level in chick retinal cell cultures: A mechanism for coupling the circadian oscillator to the melatonin-synthesizing enzyme, arylalkylamine N-acetyltransferase, in photoreceptor cells. Brain Research 991: 96–103, with permission from Elsevier.

(t½ 20 min), to insure that significant melatonin synthesis occurs in darkness only. Chicken AANAT activity is regulated by transcriptional and post-translational mechanisms (Figure 6). The retina displays daily rhythms of Aanat mRNA and AANAT protein in chickens exposed to a light–dark cycle or constant darkness. Levels of transcript, protein, enzyme activity, and melatonin all peak at night. The proximal promoter of the chicken Aanat gene contains a circadian E-box that can be activated by either BMAL1: CLOCK or BMAL1:NPAS2 heterodimers, and it is generally thought that this directly couples the circadian clock to the rhythmic expression of Aanat. In addition, the chicken Aanat 50-flanking region contains cyclic AMP response elements. Thus, the circadian rhythm of cyclic AMP may also contribute to the rhythm of Aanat mRNA.

The AANAT protein is regulated by PKA-dependent phosphorylation and proteasomal degradation (Figure 6). The AANATcontains two consensus PKA phosphorylation sites. When cyclic AMP levels are high at night, AANAT is phosphorylated. Phospho-AANAT binds to 14-3-3 proteins, which are ubiquitous signaling proteins involved in

Light pulse Control

Figure 5 Daily rhythm of retinal AANAT activity: effects of light.

(a) AANAT activity fluctuates during the 12 h light–12 h dark cycle (filled circles). Unexpected light exposure at night (open circles) rapidly inhibits activity. (b) AANAT activity in constant

(24 h day 1) darkness. The activity rhythm persists on the second day of constant darkness (filled circles). The rhythm is phase advanced by a 6 h light pulse from 18 to 24 h 2 days prior to sampling in constant darkness (open circles). Filled bars on the x- axis represent darkness; open bars represent light. Activity was measured in retinal homogenates of 2-week old chickens. Reproduced from Iuvone, P. M. and Alonso-Go´mez, A. L. (1998). Melatonin in the vertebrate retina. In: Christen Y., Doly, M., and Droy-Lefaix, M. -T. (eds.) Retine, Luminiere, et Radiations, vol. 9, pp. 49–62. Paris: Irvinn; and Iuvone, P. M., Tosini, G., Pozdeyev, N., et al. (2005). Circadian clocks, clock networks, arylalkylamine N-acetyltransferase, and melatonin in the retina. Progress in Retinal and Eye Research 24: 433–456, with permission from Elsevier.

multiple cellular functions. The interaction of 14-3-3 with AANAT increases the affinity of the enzyme for its substrate, increasing catalytic activity. Exposure to light at night causes a very rapid decrease in AANAT activity and protein level (t½ 20 min) without any initial change of Aanat mRNA (Figure 6). The decrease of AANAT is accompanied by a similarly rapid decline in melatonin levels, insuring that melatonin only functions in darkness. The decrease of protein and activity results from the lightinduced decrease of cyclic AMP levels, resulting in dephosphorylation of AANAT, unbinding of 14-3-3, and rapid proteolytic degradation of the enzyme. The degradation can be blocked by proteasome inhibitors, indicating that this is a proteasome-dependent event.

During the daytime, multiple mechanisms cooperate to keep melatonin biosynthesis at a minimum (Figure 6). Low levels of cyclic AMP appear to play key roles in these mechanisms. The Aanat transcript levels are low, presumably due to reduced cyclic AMP-directed transcriptional activation and suppression by Cry1 of E-box- mediated transactivation (Figure 6). In addition, the low levels of cyclic AMP during the daytime, especially in light, favor the dephosphorylated state of the AANAT protein, resulting in its rapid degradation by proteasomes.

The majority of AANAT in the chick retina is expressed at night in photoreceptor cells. However, there is some

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evidence that small amounts of AANAT may be expressed in ganglion cells during the daytime. The role of ganglion cell-derived AANAT is not known.

Other Rhythms of Gene Expression and Metabolism in the Chick Retina

Circadian clocks regulate phospholipid metabolism in the chick retina. Daily rhythms of phospholipid labeling with either 32P or [3H]glycerol have been observed in chicks kept on a light–dark cycle or in constant darkness. The 32P labeling of total phospholipids in photoreceptors and ganglion cells peaks in the late night. The major phospholipid labeled under these conditions is phosphotidylinositol. In contrast, labeling with [3H]glycerol peaks midday, with phosphatidylcholine as the major labeled species.

Melanopsin (Opn4) is a photopigment first discovered in Xenopus melanocytes, where it mediates photic regulation of melanin pigment aggregation. Subsequent cloning of orthologous melanopsin genes revealed that it is expressed in mammalian retina exclusively in a small subset of ganglion cells that are intrinsically photosensitive. These ganglion cells project to the suprachiasmatic nucleus (SCN) of the hypothalamus, the inferior olivary nucleus, and other brain regions involved in nonimage forming vision. The intrinsically photosensitive ganglion

cells mediate the pupillary light reflex and photic entrainment of the circadian oscillator in the SCN.

In contrast to mammals, chickens express two melanopsin genes, designated Opn4x and Opn4m because of their sequence homology to the Xenopus and mammalian melanopsin genes, respectively. The Opn4x and Opn4m transcripts are more widely distributed than their mammalian counterpart. Chicken melanopsin transcripts are found in a subpopulation of cells in the ganglion cell layer, and also in photoreceptors, retinal pigment epithelium (RPE) cells, inner nuclear layer (INL) cells, the pineal gland, and areas of the brain known to contain deep brain photoreceptors. The Opn4x is rhythmically expressed in the chicken retina in light–dark cycles and in constant darkness. Patterns of rhythmicity differ among retinal layers; Opn4x peaks in the morning in RPE and INL cells, but at night in photoreceptors. Similar to the retinal photoreceptors, Opn4x also peaks at night in chicken pinealocytes, which are directly photosensitive and contain a circadian clock that is entrained by light. The regulation and localization of Opn4 is consistent with the hypothesis that this novel photopigment plays a role in circadian regulation in the retina and pineal gland.

Iodopsin is the photopigment of red-sensitive cone photoreceptors of avian species. Iodopsin mRNA levels fluctuate as a circadian rhythm in retinal photoreceptors in vivo and in vitro, in cultured photoreceptors, and in