Regulation of gene expression by melatonin: a microarray survey of the rat retina

The pineal secretory product melatonin is synthesized by pinealocytes and retinal photoreceptors on a cyclic rhythm, with highest levels occurring at night. Our previous work has demonstrated that melatonin treatment increases the sensitivity of the rat retina to light-induced photoreceptor cell death. This raises the possibility that inappropriate exposure of photoreceptors to melatonin may result in visual impairment, caused by a loss of retinal photoreceptors. We hypothesize that retinal genes whose expression levels are altered in response to melatonin may be involved in processes that contribute to light-induced photoreceptor cell death. To identify retinal genes that are up- or down-regulated in response to melatonin receptor binding, rats were treated with or without melatonin, and the RNA from the neural retinas and retinal pigment epithelium (RPE) were analyzed for differential gene expression by hybridization of labeled cRNA probes to an Affymetrix rat genome microarray set. GeneChip algorithms were applied to measured hybridization intensities of compared samples and showed that in the neural retina, six genes were up-regulated, and eight were down-regulated. In the RPE, 15 genes were up-regulated, and two genes were down-regulated. The protein products of these specific genes are potentially involved in the molecular mechanism of melatonin action in the retina, and may play a role in the effect of melatonin on light-induced photoreceptor cell death. Identification of these candidate genes and their response to melatonin administration may provide a foundation for further studies on gene regulation by melatonin, the function of melatonin in the retina, and the role of circadian signaling in inherited and environmentally induced photoreceptor degenerations.     

Receptor interacting protein kinases mediate retinal detachment-induced photoreceptor necrosis and compensate for inhibition of apoptosis

Apoptosis has been shown to be a significant form of cell loss in many diseases. Detachment of photoreceptors from the retinal pigment epithelium, as seen in various retinal disorders, causes photoreceptor loss and subsequent vision decline. Although caspase-dependent apoptotic pathways are activated after retinal detachment, caspase inhibition by the pan-caspase inhibitor Z-VAD fails to prevent photoreceptor death; thus, we investigated other pathways leading to cell loss. Here, we show that receptor interacting protein (RIP) kinase-mediated necrosis is a significant mode of photoreceptor cell loss in an experimental model of retinal detachment and when caspases are inhibited, RIP-mediated necrosis becomes the predominant form of death. RIP3 expression, a key activator of RIP1 kinase, increased more than 10-fold after retinal detachment. Morphological assessment of detached retinas treated with Z-VAD showed decreased apoptosis but significantly increased necrotic photoreceptor death. RIP1 kinase inhibitor necrostatin-1 or Rip3 deficiency substantially prevented those necrotic changes and reduced oxidative stress and mitochondrial release of apoptosis-inducing factor. Thus, RIP kinase-mediated programmed necrosis is a redundant mechanism of photoreceptor death in addition to apoptosis, and simultaneous inhibition of RIP kinases and caspases is essential for effective neuroprotection and may be a novel therapeutic strategy for treatment of retinal disorders.     

Restoration of visual function in retinal degeneration mice by ectopic expression of melanopsin

The rod and cone cells of the mammalian retina are the principal photoreceptors for image-forming vision. They transmit information by means of a chain of intermediate cells to the retinal ganglion cells, which in turn send signals from the retina to the brain. Loss of photoreceptor cells, as happens in a number of human diseases, leads to irreversible blindness. In a mouse model (rd/rd) of photoreceptor degeneration, we used a viral vector to express in a large number of retinal ganglion cells the light sensitive protein melanopsin, normally present in only a specialized subset of the cells. Whole-cell patch–clamp recording showed photoresponses in these cells even after degeneration of the photoreceptors and additional pharmacological or Cd²⁺ block of synaptic function. Interestingly, similar responses were observed across a wide variety of diverse types of ganglion cell of the retina. The newly melanopsin-expressing ganglion cells provided an enhancement of visual function in rd/rd mice: the pupillary light reflex (PLR) returned almost to normal; the mice showed behavioral avoidance of light in an open-field test, and they could discriminate a light stimulus from a dark one in a two-choice visual discrimination alley. Recovery of the PLR was stable for at least 11 months. It has recently been shown that ectopic retinal expression of a light sensitive bacterial protein, channelrhodopsin-2, can restore neuronal responsiveness and simple visual abilities in rd/rd mice. For therapy in human photodegenerations, channelrhodopsin-2 and melanopsin have different advantages and disadvantages; both proteins (or modifications of them) should be candidates.     

Noncell-autonomous photoreceptor degeneration in a zebrafish model of choroideremia

Choroideremia is an X-linked hereditary retinal degeneration resulting from mutations in the Rab escort protein-1 (REP1). The Rep1 protein facilitates posttranslational modification of Rab proteins, which regulate intracellular trafficking in the retinal pigment epithelium (RPE) and photoreceptors and are likely involved in the removal of outer segment disk membranes by the RPE. A critical question for potential treatment of choroideremia is whether photoreceptor degeneration results from autonomous defects in opsin transport within the photoreceptor or as a nonautonomous and secondary consequence of RPE degeneration. To address this question, we have characterized the retinal pathology in zebrafish rep1 mutants, which carry a recessive nonsense mutation in the REP1 gene. Zebrafish rep1 mutants exhibit degeneration of the RPE and photoreceptors and complete loss of visual function as measured by electroretinograms. In the mutant RPE, photoreceptor outer segment material was not effectively eliminated, and large vacuoles were observed. However, opsin trafficking in photoreceptors occurred normally. Mosaic analysis revealed that photoreceptor degeneration was nonautonomous and required contact with the mutant RPE as mutant photoreceptors were rescued in wild-type hosts and wild-type photoreceptors degenerated in mutant hosts. We conclude that mutations in REP1 disrupt cellular processes in the RPE, which causes photoreceptor death as a secondary consequence. These results suggest that therapies that correct the RPE may successfully rescue photoreceptor loss in choroideremia.     

Ultrastructure and opsin immunocytochemistry of the pineal complex of the larval Arctic charr Salvelinus alpinus: A comparison with the retina

The fine structure and opsin immunocytochemistry of the pineal and parapineal organs of the salmonid fish Salvelinus alpinus, the landlocked Arctic charr, were studied and compared with the retina in various developmental stages, from prehatching to two-month-old. For opsin immunocytochemistry two polyclonal antibovine rhodopsin and the monoclonal antichicken opsin antibodies OS-2 (detecting blue and green pigments) and OS-1 (detecting green and red pigments) were used. Histologically, the pineal organ consists of nervous tissue like that of the retina. It is composed of photoreceptor pinealocytes, which formed axon terminals containing synaptic ribbons, on the dendrites and perikarya of secondary pineal neurons. Already in prehatching embryos, both the pineal and retinal photoreceptors display well-developed outer segments and form synaptic terminals. The distal part of the pineal organ differentiates earlier than its proximal stalk. The differentiation of the retina starts centrally, but the caudal and dorsal retinae are differentiated earlier than the rostral and ventral ones. At the end of the larval period, the lateral retina is still undifferentiated. In all stages studied, (rhod)opsin immunoreactivity was found in the outer segments of the pineal organ and rod-type retinal photoreceptors, a finding speaking in favour of the presence of the opsin of a rhodopsin/porphyropsin. Cone-type retinal photoreceptors identified morphologically in the pre- and posthatching stages were opsin-immunonegative with the four primary antisera used. This result suggests that in the charr the opsins of cone visual pigments differ in their chemical nature from those of rhodopsin/porphyropsin. The parapineal organ was opsin immunonegative. Using the monoclonal antibody OS-2 opsin immunoreactivity was also detected in inner segments, perikarya, and pedicles of rod-type photoreceptors of both retina and pineal organ of embryos and 1- to 4-day-old larvae. This may indicate a high level of opsin gene expression during photoreceptor growth around hatching. The well-developed pineal organ and its opsin content are discussed in connection with the photonegative behaviour of the larval charr.     

bcl-2 overexpression reduces apoptotic photoreceptor cell death in three different retinal degenerations.

Apoptosis of photoreceptors occurs infrequently in adult retina but can be triggered in inherited and environmentally induced retinal degenerations. The protooncogene bcl-2 is known to be a potent regulator of cell survival in neurons. We created lines of transgenic mice overexpressing bcl-2 to test for its ability to increase photoreceptor survival. Bcl-2 increased photoreceptor survival in mice with retinal degeneration caused by a defective opsin or cGMP phosphodiesterase. Overexpression of Bcl-2 in normal photoreceptors also decreased the damaging effects of constant light exposure. Apoptosis was induced in normal photoreceptors by very high levels of bcl-2. We conclude that bcl-2 is an important regulator of photoreceptor cell death in retinal degenerations.     

Increased macrophage chemoattractant protein-1 in cerebrospinal fluid precedes and predicts simian immunodeficiency virus encephalitis

Macrophage chemoattractant protein-1 (MCP-1) may be a key trigger for the influx of macrophages into the brain in human immunodeficiency virus (HIV) encephalitis. In this study, simian immunodeficiency virus-infected macaques that developed moderate-to-severe encephalitis had significantly higher MCP-1 levels in cerebrospinal fluid (CSF) than in plasma as early as 28 days after inoculation, which was before the development of brain lesions. In contrast, CSF:plasma MCP-1 ratios remained constant at preinoculation levels in macaques that developed minimal or no encephalitis. Abundant MCP-1 protein and mRNA were detected in both macrophages and astrocytes in the brain. Macaques with increased MCP-1 in CSF had significantly greater expression of markers of macrophage and microglia activation and infiltration (CD68; P= .003) and astrocyte activation (glial fibrillary acidic protein; P= .019 and P= .031 in white and gray matter, respectively). The results suggest that the CSF:plasma MCP-1 ratio may be a valuable prognostic marker for the development of HIV-induced central nervous system disease.     

Stable transgene expression in rod photoreceptors after recombinant adeno-associated virus-mediated gene transfer to monkey retina

Recombinant adeno-associated virus (rAAV) is a promising vector for therapy of retinal degenerative diseases. We evaluated the efficiency, cellular specificity, and safety of retinal cell transduction in nonhuman primates after subretinal delivery of an rAAV carrying a cDNA encoding green fluorescent protein (EGFP), rAAV. CMV.EGFP. The treatment results in efficient and stable EGFP expression lasting >1 year. Transgene expression in the neural retina is limited exclusively to rod photoreceptors. There is neither electroretinographic nor histologic evidence of photoreceptor toxicity. Despite significant serum antibody responses to the vector, subretinal readministration results in additional transduction events. The findings further characterize the retinal cell tropism of rAAV. They also support the development of studies aimed ultimately at treating inherited retinal degeneration by using rAAV-mediated gene therapy.     

A typology of photoreceptor gene expression patterns in the mouse

Mutations in photoreceptor-enriched genes have been implicated in dozens of human retinal diseases, yet no systematic analysis of rod and cone gene expression patterns has been carried out. In addition, although cone photoreceptor loss accounts for much of the morbidity of retinal disease, relatively few cone-specific genes are known. In this study, we carried out microarray and in situ hybridization analyses of the mouse Neural retina leucine zipper gene (Nrl) mutant, which shows an en masse conversion of rods into cones, to establish a typology of photoreceptor gene expression and to identify novel cone-specific genes. We found a total of 18 new cone-enriched genes, some of which map near uncloned retinal disease loci. Several of these genes have a dorsal-ventral (D-V) pattern of expression similar to that of short- or medium-wavelength opsins. We carried out microarray analysis of dorsal and ventral microdissected WT retina and found additional photoreceptor genes with an asymmetric distribution. Overall, we found that photoreceptor genes fall on an expression spectrum from rod-specific to cone-specific, with many showing varying degrees of rod and cone coexpression. These expression patterns can be reliably predicted from microarray data alone. Our results demonstrate definitive molecular differences between rods and cones that may underlie the physiological differences between these two classes of photoreceptors.     

CD74 indicates microglial activation in experimental diabetic retinopathy and exogenous methylglyoxal mimics the response in normoglycemic retina

Diabetes induces vasoregression, neurodegeneration and glial activation in the retina. Formation of advanced glycation endoproducts (AGEs) is increased in diabetes and contributes to the pathogenesis of diabetic retinopathy. CD74 is increased in activated microglia in a rat model developing both neurodegeneration and vasoregression. In this study, we aimed at investigating whether glucose and major AGE precursor methylglyoxal induce increased CD74 expression in the retina. Expression of CD74 in retinal microglia was analyzed in streptozotocin-diabetic rats by wholemount immunofluorescence. Nondiabetic mice were intravitreally injected with methylglyoxal. Expression of CD74 was studied by retinal wholemount immunofluorescence and quantitative real-time PCR, 48 h after the injection. CD74-positive cells were increased in diabetic 4-month retinas. These cells represented a subpopulation of CD11b-labeled activated microglia and were mainly located in the superficial vascular layer (13.7-fold increase compared to nondiabetic group). Methylglyoxal induced an 9.4-fold increase of CD74-positive cells in the superficial vascular layer and elevated gene expression of CD74 in the mouse retina 2.8-fold. In summary, we identified CD74 as a microglial activation marker in the diabetic retina. Exogenous methylglyoxal mimics the response in normoglycemic retina. This suggests that methylglyoxal is important in mediating microglial activation in the diabetic retina.     

Inhibition of ceramide biosynthesis preserves photoreceptor structure and function in a mouse model of retinitis pigmentosa

Retinitis pigmentosa (RP) is a genetic disease causing progressive apoptotic death of photoreceptors and, ultimately, incurable blindness. Using the retinal degeneration 10 (rd10) mouse model of RP, we investigated the role of ceramide, a proapoptotic sphingolipid, in retinal degeneration. We also tested the possibility that photoreceptor loss can be slowed or blocked by interfering with the ceramide signaling pathway of apoptosis in vivo. Retinal ceramide levels increased in rd10 mice during the period of maximum photoreceptor death. Single intraocular injections of myriocin, a powerful inhibitor of serine palmitoyl-CoA transferase, the rate-limiting enzyme of ceramide biosynthesis, lowered retinal ceramide levels to normal values and rescued photoreceptors from apoptotic death. Noninvasive treatment was achieved using eye drops consisting of a suspension of solid lipid nanoparticles loaded with myriocin. Short-term noninvasive treatment lowered retinal ceramide in a manner similar to intraocular injections, indicating that nanoparticles functioned as a vector permitting transcorneal drug administration. Prolonged treatment (10-20 d) with solid lipid nanoparticles increased photoreceptor survival, preserved photoreceptor morphology, and extended the ability of the retina to respond to light as assessed by electroretinography. In conclusion, pharmacological targeting of ceramide biosynthesis slowed the progression of RP in a mouse model, and therefore may represent a therapeutic approach to treating this disease in humans. Transcorneal administration of drugs carried in solid lipid nanoparticles, as experimented in this study, may facilitate continuous, noninvasive treatment of patients with RP and other retinal pathologies.     

Apoptotic photoreceptor cell death in mouse models of retinitis pigmentosa.

Retinitis pigmentosa (RP) is a group of inherited human diseases in which photoreceptor degeneration leads to visual loss and eventually to blindness. Although mutations in the rhodopsin, peripherin, and cGMP phosphodiesterase genes have been identified in some forms of RP, it remains to be determined whether these mutations lead to photoreceptor cell death through necrotic or apoptotic mechanisms. In this paper, we report a test of the hypothesis that photoreceptor cell death occurs by an apoptotic mechanism in three mouse models of RP: retinal degeneration slow (rds) caused by a peripherin mutation, retinal degeneration (rd) caused by a defect in cGMP phosphodiesterase, and transgenic mice carrying a rhodopsin Q344ter mutation responsible for autosomal dominant RP. Two complementary techniques were used to detect apoptosis-specific internucleosomal DNA fragmentation: agarose gel electrophoresis and in situ labeling of apoptotic cells by terminal dUTP nick end labeling. Both methods showed extensive apoptosis of photoreceptors in all three mouse models of retinal degeneration. We also show that apoptotic death occurs in the retina during normal development, suggesting that different mechanisms can cause photoreceptor death by activating an intrinsic death program in these cells. These findings raise the possibility that retinal degenerations may be slowed by interfering with the apoptotic mechanism itself.     

A unique pattern of photoreceptor degeneration in cyclin D1 mutant mice

Cyclin D1-deficient mice have small eyes with thin retinas. We observed that there was a lower level of retinal cell proliferation and a unique pattern of photoreceptor cell death. Death was first observed in scattered clusters of cells in the retina. It then appeared to spread from these few cells to nearby photoreceptors, eventually producing extensive holes in the photoreceptor layer. These holes appeared to be filled with interneurons from the inner nuclear layer. The death mainly occurred during the second to fourth postnatal weeks. Other models of photoreceptor degeneration in rodents differ in that they occur more uniformly across the retina, with death proceeding over a longer period of time until all, or nearly all, of the photoreceptors degenerate. We also tested whether expression of a bcl-2 transgene could prevent the death and found that it could not.     

Effects of intraarticular glucocorticoids on macrophage infiltration and mediators of joint damage in osteoarthritis synovial membranes: findings in a double-blind, placebo-controlled study

OBJECTIVE: To investigate the effects of intraarticular glucocorticoid treatment on macrophage infiltration, the expression of the chemokines monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 1alpha (MIP-1alpha), and the expression of matrix metalloproteinases 1 and 3 (MMPs 1 and 3) and their inhibitors, the tissue inhibitors of metalloproteinases 1 and 2 (TIMPs 1 and 2), in osteoarthritis (OA) synovial membranes. METHODS: Forty patients underwent arthroscopic biopsy before and 1 month after intraarticular injection of glucocorticoids. Twenty-one patients received 120 mg of methylprednisolone acetate (Depo-Medrol; Upjohn, Kalamazoo, MI), and 20 patients received placebo (1 patient received placebo in 1 knee and methylprednisolone acetate in the other). Immunoperoxidase staining for the expression of CD68, MCP-1, MIP-1alpha, MMP-1, MMP-3, TIMP-1, and TIMP-2 was performed, and the immunostaining was quantified by color video image analysis. RESULTS: CD68, MCP-1, MIP-1alpha, MMP-1, MMP-3, TIMP-1, and TIMP-2 immunostaining was observed in all synovial membranes. Intraarticular glucocorticoid treatment was associated with a small (30%) but statistically significant (P = 0.048) reduction in CD68+ macrophage staining in the synovial lining layer, but there was no change in the CD68 expression in the synovial sublining layer. No significant differences were observed for MCP-1, MIP-1alpha, MMP-1, MMP-3, TIMP-1, and TIMP-2 immunostaining in the synovial lining or sublining layers. CONCLUSION: Intraarticular glucocorticoids may reduce CD68+ macrophage infiltration into the synovial lining layer, but not the expression of MCP-1, MIP-1alpha, MMP-1, MMP-3, TIMP-1, and TIMP-2 in the synovial membrane, in patients with OA.     

Identifying photoreceptors in blind eyes caused by RPE65 mutations: Prerequisite for human gene therapy success

Mutations in RPE65, a gene essential to normal operation of the visual (retinoid) cycle, cause the childhood blindness known as Leber congenital amaurosis (LCA). Retinal gene therapy restores vision to blind canine and murine models of LCA. Gene therapy in blind humans with LCA from RPE65 mutations may also have potential for success but only if the retinal photoreceptor layer is intact, as in the early-disease stage-treated animals. Here, we use high-resolution in vivo microscopy to quantify photoreceptor layer thickness in the human disease to define the relationship of retinal structure to vision and determine the potential for gene therapy success. The normally cone photoreceptor-rich central retina and rod-rich regions were studied. Despite severely reduced cone vision, many RPE65-mutant retinas had near-normal central microstructure. Absent rod vision was associated with a detectable but thinned photoreceptor layer. We asked whether abnormally thinned RPE65-mutant retina with photoreceptor loss would respond to treatment. Gene therapy in Rpe65(-/-) mice at advanced-disease stages, a more faithful mimic of the humans we studied, showed success but only in animals with better-preserved photoreceptor structure. The results indicate that identifying and then targeting retinal locations with retained photoreceptors will be a prerequisite for successful gene therapy in humans with RPE65 mutations and in other retinal degenerative disorders now moving from proof-of-concept studies toward clinical trials.     

Role of macrophage infiltration in the orbital fat of patients with Graves' ophthalmopathy

Objective  Infiltration of the retro-ocular space by inflammatory cells, accumulation of glycosaminoglycans, and the overabundance of orbital adipose tissue are characteristic findings in Graves' ophthalmopathy (GO). The cause of macrophage infiltration in the orbital adipose tissue of patients with GO remains to be elucidated.
Design  Immunohistochemistry of orbital adipose tissues with anti-CD68 was used for determining macrophage infiltration pattern and cell counts. Quantitative real-time PCR was used for analysing mRNA expression. Correlation of macrophage infiltration with the duration of GO and mRNA expression were also determined.
Patients  Fifteen subjects with GO who underwent orbital decompression were recruited. Six patients without thyroid history who underwent elective orbital surgery were enrolled as controls.
Measurements  Histological distribution of macrophages, macrophage cell counts, CD68 and monocyte chemoattractant protein-1 (MCP-1) mRNA levels, and duration of GO.
Results  We demonstrated that macrophage infiltration in orbital fat from patients with GO was higher than controls ( P  = 0·005). The infiltration of macrophages was located primarily around blood vessels and between mature adipocytes. Macrophage infiltration did not attenuate in GO of long duration. We also found that the expression of MCP-1 was higher in GO orbital fat than that in the orbital fat of controls ( P =  0·047) and the infiltration of macrophages in adipose tissue from patients with GO was positively correlated with expression of MCP-1 mRNA ( r  = 0·546, P  = 0·035).
Conclusion  Macrophage infiltration may play an important role in the pathogenesis of GO via over-expression of MCP-1.     

Expression of chemokines and matrix metalloproteinases in early rheumatoid arthritis

OBJECTIVE: To compare macrophage infiltration and expression of chemokines and matrix metalloproteinases (MMPs) in synovial tissue between patients with early and long-standing rheumatoid arthritis (RA). METHODS: Knee synovial biopsies were taken from 22 patients with early (<1 yr) and 22 patients with long-standing (>5 yr) RA and immunostained with antibodies specific for CD68; macrophage inflammatory protein (MIP)-1alpha and monocyte chemoattractant protein (MCP)-1; MMP-1 and -3 and the tissue inhibitors of metalloproteinases (TIMP)-l and -2. Immunostaining was quantified using a colour video image analysis system. RESULTS: CD68+ macrophage infiltration and the expression of MIP-1alpha, MCP-1, MMP-1, MMP-3, TIMP-1, and TIMP-2 were observed in synovial tissue of patients with early RA. In long-standing RA, there was a further increase in CD68+ macrophage infiltration and MIP-1alpha expression in the synovial lining layer. CD68 expression correlated with MIP-1alpha (R=0.39, P=0.01), but not with MCP-1 expression. CONCLUSION: Macrophage accumulation, and the expression of chemokines and MMPs in synovial tissue occur in early RA. Targeting chemokines which play a role in the migration of macrophages into the joints may be of therapeutic benefit in RA patients.