Glial cell response to constant low light exposure in rat retina.

To study the macroglia and microglia and the immune role in long-time light exposure in rat eyes, we performed glial cell characterization along the time-course of retinal degeneration induced by chronic exposure to low-intensity light. Animals were exposed to light for periods of 2, 4, 6, or 8 days, and the retinal glial response was evaluated by immunohistochemistry, western blot and real-time reverse transcription polymerase chain reaction. Retinal cells presented an increased expression of the macroglia marker GFAP, as well as increased mRNA levels of microglia markers Iba1 and CD68 after 6 days. Also, at this time-point, we found a higher number of Iba1-positive cells in the outer nuclear layer area; moreover, these cells showed the characteristic activated-microglia morphology. The expression levels of immune mediators TNF, IL-6, and chemokines CX3CR1 and CCL2 were also significantly increased after 6 days. All the events of glial activation occurred after 5-6 days of constant light exposure, when the number of photoreceptor cells has already decreased significantly. Herein, we demonstrated that glial and immune activation are secondary to neurodegeneration; in this scenario, our results suggest that photoreceptor death is an early event that occurs independently of glial-derived immune responses.

Oxidative Stress in Retinal Degeneration Promoted by Constant LED Light.

Light pollution by artificial light, might accelerate retinal diseases and circadian asynchrony. The excess of light exposure is a growing problem in societies, so studies on the consequences of long-term exposure to low levels of light are needed to determine the effects on vision. The possibility to understand the molecular mechanisms of light damage will contribute to the knowledge about visual disorders related to defects in the phototransduction. Several animal models have been used to study retinal degeneration (RD) by light; however, some important aspects remain to be established. Previously, we demonstrated that cool white treatment of 200 lux light-emitting diode (LED) induces retinal transformation with rods and cones cell death and significant changes in opsin expression in the inner nuclear layer (INL) and ganglion cell layer (GCL). Therefore, to further develop describing the molecular pathways of RD, we have examined here the oxidative stress and the fatty acid composition in rat retinas maintained at constant light. We demonstrated the existence of oxidative reactions after 5 days in outer nuclear layer (ONL), corresponding to classical photoreceptors; catalase (CAT) enzyme activity did not show significant differences in all times studied and the fatty acid study showed that docosahexaenoic acid decreased after 4 days. Remarkably, the docosahexaenoic acid diminution showed a correlation with the rise in stearic acid indicating a possible association between them. We assumed that the reduction in docosahexaenoic acid may be affected by the oxidative stress in photoreceptors outer segment which in turn affects the stearic acid composition with consequences in the membrane properties. All these miss-regulation affects the photoreceptor survival through unknown mechanisms involved. We consider that oxidative stress might be one of the pathways implicated in RD promoted by light.

Non-Visual Photopigments Effects of Constant Light-Emitting Diode Light Exposure on the Inner Retina of Wistar Rats.

The retina is part of the central nervous system specially adapted to capture light photons and transmit this information to the brain through photosensitive retinal cells involved in visual and non-visual activities. However, excessive light exposure may accelerate genetic retinal diseases or induce photoreceptor cell (PRC) death, finally leading to retinal degeneration (RD). Light pollution (LP) caused by the characteristic use of artificial light in modern day life may accelerate degenerative diseases or promote RD and circadian desynchrony. We have developed a working model to study RD mechanisms in a low light environment using light-emitting diode (LED) sources, at constant or long exposure times under LP conditions. The mechanism of PRC death is still not fully understood. Our main goal is to study the biochemical mechanisms of RD. We have previously demonstrated that constant light (LL) exposure to white LED produces a significant reduction in the outer nuclear layer (ONL) by classical PRC death after 7 days of LL exposure. The PRCs showed TUNEL-positive labeling and a caspase-3-independent mechanism of cell death. Here, we investigate whether constant LED exposure affects the inner-retinal organization and structure, cell survival and the expression of photopigments; in particular we look into whether constant LED exposure causes the death of retinal ganglion cells (RGCs), of intrinsically photosensitive RGCs (ipRGCs), or of other inner-retinal cells. Wistar rats exposed to 200 lx of LED for 2 to 8 days (LL 2 and LL 8) were processed for histological and protein. The results show no differences in the number of nucleus or TUNEL positive RGCs nor inner structural damage in any of LL groups studied, indicating that LL exposure affects ONL but does not produce RGC death. However, the photopigments melanopsin (OPN4) and neuropsin (OPN5) expressed in the inner retina were seen to modify their localization and expression during LL exposure. Our findings suggest that constant light during several days produces retinal remodeling and ONL cell death as well as significant changes in opsin expression in the inner nuclear layer.

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

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

Rhythms of glycerophospholipid synthesis in retinal inner nuclear layer cells.

The present study demonstrates that the biosynthesis of phospholipids in the inner nuclear layer cells of the chicken retina displays daily rhythms under constant illumination conditions. The vertebrate retina contains circadian oscillators and photoreceptors (PRCs) that temporally regulate its own physiology and synchronize the whole organism to the daily environmental changes. We have previously reported that chicken photoreceptors and retinal ganglion cells (RGCs) present significant daily variations in their phospholipid biosynthesis under constant illumination conditions. Herein, we demonstrate that cell preparations highly enriched in inner nuclear layer cells also exhibit a circadian-regulated phospholipid labeling after the in vivo administration of [(32)P]phosphate or [(3)H]glycerol both in animals maintained under constant darkness or light for at least 48h. In constant darkness, there was a significant incorporation of both precursors into phospholipids with the highest levels of labeling around midday and dusk. In constant light, the labeling of (32)P-phospholipids was also significantly higher during the day and early night whereas the incorporation of [(3)H]glycerol into phospholipids, that indicates de novo biosynthesis, was greater during the day but probably reflecting a higher precursor availability at those phases. We also measured the in vitro activity of phosphatidate phosphohydrolase and diacylglycerol lipase in preparations obtained from the dark condition. The two enzymes exhibited the highest activity levels late in the day. When we assessed the in vitro incorporation of [(14)C]oleate into different lysophospholipids from samples collected at different phases in constant darkness, reaction catalyzed by lysophospholipid acyltransferases II, labeling showed a complex pattern of daily activity. Taken together, these results demonstrate that the biosynthesis of phospholipids in cells of the chicken retinal inner nuclear layer exhibits a daily rhythmicity under constant illumination conditions, which is controlled by a circadian clock.

Retinal ganglion cells are autonomous circadian oscillators synthesizing N-acetylserotonin during the day.

Retinal ganglion cells send visual and circadian information to the brain regarding the environmental light-dark cycles. We investigated the capability of retinal ganglion cells of synthesizing melatonin, a highly reliable circadian marker that regulates retinal physiology, as well as the capacity of these cells to function as autonomous circadian oscillators. Chick retinal ganglion cells presented higher levels of melatonin assessed by radioimmunoassay during both the subjective day in constant darkness and the light phase of a light-dark cycle. Similar changes were observed in mRNA levels and activity of arylalkylamine N-acetyltransferase, a key enzyme in melatonin biosynthesis, with the highest levels of both parameters during the subjective day. These daily variations were preceded by the elevation of cyclic-AMP content, the second messenger involved in the regulation of melatonin biosynthesis. Moreover, cultures of immunopurified retinal ganglion cells at embryonic day 8 synchronized by medium exchange synthesized a [3H]melatonin-like indole from [3H]tryptophan. This [3H]indole was rapidly released to the culture medium and exhibited a daily variation, with levels peaking 8 h after synchronization, which declined a few hours later. Cultures of embryonic retinal ganglion cells also showed self-sustained daily rhythms in arylalkylamine N-acetyltransferase mRNA expression during at least three cycles with a period near 24 h. These rhythms were also observed after the application of glutamate. The results demonstrate that chick retinal ganglion cells may function as autonomous circadian oscillators synthesizing a melatonin-like indole during the day.

Inhibitors of protein phosphatase 1 and 2A decrease the level of tubulin carboxypeptidase activity associated with microtubules.

The association of tubulin carboxypeptidase with microtubules may be involved in the determination of the tyrosination state of the microtubules, i.e. their proportion of tyrosinated vs. nontyrosinated tubulin. We investigated the role of protein phosphatases in the association of carboxypeptidase with microtubules in COS cells. Okadaic acid and other PP1/PP2A inhibitors, when added to culture medium before isolation of the cytoskeletal fraction, produced near depletion of the carboxypeptidase activity associated with microtubules. Isolation of the native assembled and nonassembled tubulin fractions from cells treated and not treated with okadaic acid, and subsequent in vitro assay of the carboxypeptidase activity, revealed that the enzyme was dissociated from microtubules by okadaic acid treatment and recovered in the soluble fraction. There was no effect by nor-okadaone (an inactive okadaic acid analogue) or inhibitors of PP2B and of tyrosine phosphatases which do not affect PP1/PP2A activity. When tested in an in vitro system, okadaic acid neither dissociated the enzyme from microtubules nor inactivated it. In living cells, prior stabilization of microtubules with taxol prevented the dissociation of carboxypeptidase by okadaic acid indicating that dynamic microtubules are needed for okadaic acid to exert its effect. On the other hand, stabilization of microtubules subsequent to okadaic acid treatment did not reverse the dissociating effect of okadaic acid. These results suggest that dephosphorylation (and presumably also phosphorylation) of the carboxypeptidase or an intermediate compound occurs while it is not associated with microtubules, and that the phosphate content determines whether or not the carboxypeptidase is able to associate with microtubules.

Post-translational incorporation of the antiproliferative agent azatyrosine into the C-terminus of alpha-tubulin.

Detyrosination/tyrosination of tubulin is a post-translational modification that occurs at the C-terminus of the alpha-subunit, giving rise to microtubules rich in either tyrosinated or detyrosinated tubulin which coexist in the cell. We hereby report that the tyrosine analogue, azatyrosine, can be incorporated into the C-terminus of alpha-tubulin instead of tyrosine. Azatyrosine is structurally identical to tyrosine except that a nitrogen atom replaces carbon-2 of the phenolic group. Azatyrosine competitively excluded incorporation of [14C]tyrosine into tubulin of soluble brain extract. A newly developed rabbit antibody specific to C-terminal azatyrosine was used to study incorporation of azatyrosine in cultured cells. When added to the culture medium (Ham's F12K), azatyrosine was incorporated into tubulin of glioma-derived C6 cells. This incorporation was reversible, i.e. after withdrawal of azatyrosine, tubulin lost azatyrosine and reincorporated tyrosine. Azatyrosinated tubulin self-assembled into microtubules to a similar degree as total tubulin both in vitro and in vivo. Studies by other groups have shown that treatment of certain types of cultured cancer cells with azatyrosine leads to reversion of phenotype to normal, and that administration of azatyrosine into animals harbouring human proto-oncogenic c-Ha- ras prevents tumour formation. These interesting observations led us to study this phenomenon in relation to tubulin status. Under conditions in which tubulin was mostly azatyrosinated, C6 cells remained viable but did not proliferate. After 7-10 days under these conditions, morphology changed from a fused, elongated shape to a rounded soma with thin processes. Incorporation of azatyrosine into the C-terminus of alpha-tubulin is proposed as one possible cause of reversion of the malignant phenotype.

Incorporation of 3-nitrotyrosine into the C-terminus of alpha-tubulin is reversible and not detrimental to dividing cells.

The C-terminus of the alpha-chain of tubulin is subject to reversible incorporation of tyrosine by tubulin tyrosine ligase and removal by tubulin carboxypeptidase. Thus, microtubules rich in either tyrosinated or detyrosinated tubulin can coexist in the cell. Substitution of the terminal tyrosine by 3-nitrotyrosine has been claimed to cause microtubule dysfunction and consequent injury of epithelial lung carcinoma A549 cells. Nitrotyrosine is formed in cells by nitration of tyrosine by nitric oxide-derived species. We studied properties of tubulin modified by in vitro nitrotyrosination at the C-terminus of the alpha-subunit, and the consequences for cell functioning. Nitrotyrosinated tubulin was a good substrate of tubulin carboxypeptidase, and showed a similar capability to assemble into microtubules in vitro to that of tyrosinated tubulin. Tubulin of C6 cells cultured in F12K medium in the presence of 500 micro m nitrotyrosine became fully nitrotyrosinated. This nitrotyrosination was shown to be reversible. No changes in morphology, proliferation, or viability were observed during cycles of nitrotyrosination, denitrotyrosination, and re-nitrotyrosination. Similar results were obtained with CHO, COS-7, HeLa, NIH-3T3, NIH-3T3(TTL-), and A549 cells. C6 and A549 cells were subjected to several passages during 45 days or more in the continuous presence of 500 micro m nitrotyrosine without noticeable alteration of morphology, viability, or proliferation. The microtubular networks visualized by immunofluorescence with antibodies to nitrotyrosinated and total tubulin were identical. Furthermore, nitrotyrosination of tubulin in COS cells did not alter the association of tubulin carboxypeptidase with microtubules. Our results demonstrate that substitution of C-terminal tyrosine by 3-nitrotyrosine has no detrimental effect on dividing cells.