Alicia Torriglia

We present here an improvement in the classical molybdate method for inorganic phosphate determination. This method has high sensitivity, with 1 nmol of Pi giving an absorbance change of 0.060 A850 unit. It is highly reproducible and the color remains stable for at least 3.5 h. In addition the use of sodium dodecyl sulfate makes it possible to stop enzymatic reactions without organic phosphate hydrolysis. It also shows an extremely low interference by highly concentrated solutions of different origins. Of special interest is its high tolerance to protein, permitting as much as 50 mg/ml of human serum protein in the sample without precipitation or color interference. For these reasons, it proves to be very useful in the determination of ATPases in tissues such as the ocular lens with low specific activity.

Spectra of “white LEDs” are characterized by an intense emission in the blue region of the visible spectrum, absent in daylight spectra. This blue component and the high intensity of emission are the main sources of concern about the health risks of LEDs with respect to their toxicity to the eye and the retina. The aim of our study was to elucidate the role of blue light from LEDs in retinal damage. Commercially available white LEDs and four different blue LEDs (507, 473, 467, and 449 nm) were used for exposure experiments on Wistar rats. Immunohistochemical stain, transmission electron microscopy, and Western blot were used to exam the retinas. We evaluated LED-induced retinal cell damage by studying oxidative stress, stress response pathways, and the identification of cell death pathways. LED light caused a state of suffering of the retina with oxidative damage and retinal injury. We observed a loss of photoreceptors and the activation of caspase-independent apoptosis, necroptosis, and necrosis. A wavelength dependence of the effects was observed. Phototoxicity of LEDs on the retina is characterized by a strong damage of photoreceptors and by the induction of necrosis.

The present review draws together wide-ranging studies performed over the last decades that catalogue the effects of artificial-light-at-night (ALAN) upon living species and their environment. We provide an overview of the tremendous variety of light-detection strategies which have evolved in living organisms - unicellular, plants and animals, covering chloroplasts (plants), and the plethora of ocular and extra-ocular organs (animals). We describe the visual pigments which permit photo-detection, paying attention to their spectral characteristics, which extend from the ultraviolet into infrared. We discuss how organisms use light information in a way crucial for their development, growth and survival: phototropism, phototaxis, photoperiodism, and synchronization of circadian clocks. These aspects are treated in depth, as their perturbation underlies much of the disruptive effects of ALAN. The review goes into detail on circadian networks in living organisms, since these fundamental features are of critical importance in regulating the interface between environment and body. Especially, hormonal synthesis and secretion are often under circadian and circannual control, hence perturbation of the clock will lead to hormonal imbalance. The review addresses how the ubiquitous introduction of light-emitting diode technology may exacerbate, or in some cases reduce, the generalized ever-increasing light pollution. Numerous examples are given of how widespread exposure to ALAN is perturbing many aspects of plant and animal behaviour and survival: foraging, orientation, migration, seasonal reproduction, colonization and more. We examine the potential problems at the level of individual species and populations and extend the debate to the consequences for ecosystems. We stress, through a few examples, the synergistic harmful effects resulting from the impacts of ALAN combined with other anthropogenic pressures, which often impact the neuroendocrine loops in vertebrates. The article concludes by debating how these anthropogenic changes could be mitigated by more reasonable use of available technology - for example by restricting illumination to more essential areas and hours, directing lighting to avoid wasteful radiation and selecting spectral emissions, to reduce impact on circadian clocks. We end by discussing how society should take into account the potentially major consequences that ALAN has on the natural world and the repercussions for ongoing human health and welfare.

Cornea transplantation is one of the most performed graft procedures worldwide with an impressive success rate of 90%. However, for "high-risk" patients with particular ocular diseases in addition to the required surgery, the success rate is drastically reduced to 50%. In these cases, cyclosporin A (CsA) is frequently used to prevent the cornea rejection by a systemic treatment with possible systemic side effects for the patients. To overcome these problems, it is a challenge to prepare well-tolerated topical CsA formulations. Normally high amounts of oils or surfactants are needed for the solubilization of the very hydrophobic CsA. Furthermore, it is in general difficult to obtain ocular therapeutic drug levels with topical instillations due to the corneal barriers that efficiently protect the intraocular structures from foreign substances thus also from drugs. The aim of this study was to investigate in vivo the effects of a novel CsA topical aqueous formulation. This formulation was based on nanosized polymeric micelles as drug carriers. An established rat model for the prevention of cornea graft rejection after a keratoplasty procedure was used. After instillation of the novel formulation with fluorescent labeled micelles, confocal analysis of flat-mounted corneas clearly showed that the nanosized carriers were able to penetrate into all corneal layers. The efficacy of a 0.5% CsA micelle formulation was tested and compared to a physiological saline solution and to a systemic administration of CsA. In our studies, the topical CsA treatment was carried out for 14 days, and the three parameters (a) cornea transparency, (b) edema, and (c) neovascularization were evaluated by clinical observation and scoring. Compared to the control group, the treated group showed a significant higher cornea transparency and significant lower edema after 7 and 13 days of the surgery. At the end point of the study, the neovascularization was reduced by 50% in the CsA-micelle treated animals. The success rate of cornea graft transplantation was 73% in treated animals against 25% for the control group. This result was as good as observed for a systemic CsA treatment in the same animal model. This new formulation has the same efficacy like a systemic treatment but without the serious CsA systemic side effects. Ocular drug levels of transplanted and healthy rat eyes were dosed by UPLC/MS and showed a high CsA value in the cornea (11710 ± 7530 ng(CsA)/g(tissue) and 6470 ± 1730 ng(CsA)/g(tissue), respectively). In conclusion, the applied formulation has the capacity to overcome the ocular surface barriers, the micelles formed a drug reservoir in the cornea from, where a sustained release of CsA can take place. This novel formulation for topical application of CsA is clearly an effective and well-tolerated alternative to the systemic treatment for the prevention of corneal graft rejection.

To save energy, the European directives from the Eco-design of Energy Using Products (2005/32/CE) have recommended the replacement of incandescent lamps by more economic devices such as Light Emitting Diodes (LEDs). However, the emission spectrum of these devices is enriched in blue radiations, known to be potentially dangerous to the retina. Recent studies showed that light exposure contributes to the onset of early stages of age-related macular degeneration (AMD). Here, we investigate, in albinos and pigmented rats, the effects of different exposure protocols. Twenty-four hours exposure at high luminance was compared to a cyclic (dark/light) exposure at domestic levels for 1week and 1month, using different LEDs (Cold-white, blue and green), as well as fluorocompact bulbs and fluorescent tubes. The data suggest that the blue component of the white-LED may cause retinal toxicity at occupational domestic illuminance and not only in extreme experimental conditions, as previously reported. It is important to note that the current regulations and standards have been established on the basis of acute light exposure and do not take into account the effects of repeated exposure.

ABSTRACTThe most widely recognized biochemical change associated with the majority of apoptotic systems is the degradation of genomic DNA. Among the enzymes that may participate in this cleavage, the acidic cation-independent DNase II is a likely candidate since it is activated in many apoptotic cells. To better understand its role, we purified and sequenced a DNase II extracted from porcine spleen. Protein sequencing of random peptides demonstrated that this enzyme is derived from a ubiquitous serpin, the leukocyte elastase inhibitor (LEI), by an acidic-dependent posttranslational modification or by digestion with elastase. We call this novel enzyme L-DNase II. In vitro experiments with purified recombinant LEI show that the native form has no effect on purified nuclei whereas its posttranslationally activated form induces pycnosis and DNA degradation. Antibodies directed against L-DNase II showed, in different cell lines, an increased expression and a nuclear translocation of this enzyme during apoptosis. Since the appearance of the endonuclease activity results in a loss of the anti-protease properties of LEI, the transformation from LEI to L-DNase II may act as a switch of protease and nuclease pathways, each of which is activated during apoptosis.View correction statement:L-DNase II, a Molecule That Links Proteases and Endonucleases in Apoptosis, Derives from the Ubiquitous Serpin Leukocyte Elastase Inhibitor ACKNOWLEDGMENTSWe acknowledge Pascal Egger and Delphine Goux for their help with DNA sequencing and David McDevitt for correcting the English in the manuscript.A.T. was supported by Retina France-AFRP and Rhône-Poulenc Rorer, and P.P. was supported by Association Claude Bernard and Association pour Recherche sur le Cancer-ARC.

We have isolated, from a human tumor cDNA library, a gene encoding a putative receptor-like protein-tyrosine kinase that we call TK14. The amino acid sequence of the TK14 protein is closely related to the available partial sequence of the mouse protein bek, and more distantly related to the sequences of a chicken basic fibroblast growth factor receptor (73% sequence homology) and the apparent human equivalent of this receptor, the FLG protein (encoded by the fms-like tyrosine kinase gene). Overexpression of the TK14 protein by transfection of COS-1 cells with the corresponding cDNA in a simian virus 40-based expression vector leads to the appearance of new cell-surface binding sites for both acidic and basic fibroblast growth factors. This has been demonstrated by specific binding assays and chemical cross-linking experiments using 125I-labeled growth factors. It appears, therefore, that the human genome contains at least two distinct genes, for TK14 and FLG, that code for related fibroblast growth factor receptors.

The characterization of DNase II and DNase I activity was undertaken to discriminate their different roles in physiological nuclear degradation during lens fiber cell differentiation.The activity of both nucleases determined in a new assay allows to discriminate DNase II from DNase I in the same extract.In fibers, both types of nuclease activities are found and appear higher than in epithelial cells.Specific polyclonal antibodies directed against these two nucleases reveal by Western blot analysis the presence of various DNase isoforms.DNase II like-nuclease, present in fibers, is represented by three major bands (60, 23, and 18 kDa), which are not detected, at least for two of them (60 and 23 kDa), in epithelial cells.DNase I like-nuclease pattern in fiber cells shows a single 32-kDa band, while several bands can be detected in epithelial cells.Immunocytochemistry studies show both nucleases present in lens cell sections.DNase II is, as usual, in cytoplasm of epithelial cells, but it appears strikingly concentrated in the nuclei of fibers.DNase I is always concentrated in nuclei of epithelial and fiber cells.DNA degradation observed in agarose gels shows that DNase II-activating medium cleaves the DNA from fiber cells more efficiently than DNase I-activating buffer.In addition, DNase II antibody is able to prevent this degradation.These results suggest a specific involvement of DNase II in nuclear degradation during lens cell differentiation.Apoptosis or programmed cell death occurs in many physiological and pathological situations where selection of cells is required (1-4).In 1980, a landmark study (5) revealed that glucocorticoids induced extensive DNA degradation in rat thymocytes in vitro at the onset of cell death.DNA cleavage occurred in a very specific pattern producing fragments of DNA that were multiples of 180 -200 base pairs.This indicated that the chromatin was cleaved at the linker DNA between nucleosomic cores.The characteristic ladder was first shown by Hewish and Burgoyne (6) when they demonstrated the Ca 2ϩ -and Mg 2ϩ -dependent autodigestion of DNA in isolated liver cell nuclei.To explain this process, a Ca 2ϩ -and Mg ϩ2 -specific endonuclease was postulated by Wyllie et al. (5,7).To date, three different endonucleases have been involved in DNA fragmentation leading to nucleosomal appearance.Some authors, such as Peitsch et al. (8), claimed that the well characterized pancreatic deoxyribonuclease (DNase I) was consti-

In light induced retinal degeneration (LIRD) photoreceptor cell death is mediated by caspase independent mechanisms. The activation of LEI/L-DNase II pathway in this model, is due to cathepsin D release from lysosomes, although the underlying mechanism remains poorly understood. In this paper we studied the involvement of calpains in lysosomal permeabilization. We investigated, for the first time, the calpain targets at lysosomal membrane level. We found that calpain 1 is responsible for lysosomal permeabilization by cleavage of the lysosomal associated membrane protein 2 (LAMP 2). Moreover, LAMP 2 degradation and lysosomal permeabilization were rescued by calpain inhibition and the use of MEF(-/-)lamp 2 cells indicates that the cleavage of LAMP 2A is essential for this permeabilization. Finally, we found that LAMP 2 is cleaved in LIRD, suggesting that the mechanism of calpain induced lysosomal permeabilization is not exclusive of a single cell death model. Overall, these data shed new light on understanding the mechanisms of lysosomal and caspase-independent cell death and point to the original targets for development of the new therapeutic protocols.

Topical ocular drug delivery has always been a challenge for pharmaceutical technology scientists. In the last two decades, many nano-systems have been studied to find ways to overcome the typical problems of topical ocular therapy, such as difficult corneal penetration and poor drug availability. In this study, methoxy poly(ethylene glycol)-hexylsubstituted poly(lactides) (MPEG-hexPLA) micelle formulations, which are promising nanocarriers for poorly water soluble drugs, were investigated for the delivery of Cyclosporin A (CsA) to the eye. As a new possible pharmaceutical excipient, the ocular compatibility of MPEG-hexPLA micelle formulations was evaluated. An in vitro biocompatibility assessment on human corneal epithelial cells was carried out using different tests. Cytotoxicity was studied by using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT), and clonogenic tests and revealed that the CsA formulations and copolymer solutions were not toxic. After incubation with MPEG-hexPLA micelle formulations, the activation of caspase-dependent and -independent apoptosis as well as autophagy was evaluated using immunohistochemistry by analyzing the localization of four antibodies: (1) anti-caspase 3; (2) anti-apoptotic inducing factor (AIF); (3) anti-IL-Dnase II and (4) anti-microtubule-associated protein 1 light chain 3 (LC3). No apoptosis was induced when the cells were treated with the micelle solutions that were either unloaded or loaded with CsA. The ocular tolerance was assessed in vivo on rabbit eyes by Confocal Laser Scanning Ophthalmoscopy (CLSO), and very good tolerability was seen. The observed corneal surface was comparable to a control surface that was treated with a 0.9% NaCl solution. In conclusion, these results demonstrate that MPEG-hexPLA micelles are promising drug carriers for ocular diseases involving the activation of cytokines, such as dry eye syndrome and autoimmune uveitis, or for the prevention of corneal graft rejection.

Ageing and alteration of the functions of the retinal pigment epithelium (RPE) are at the origin of lost of vision seen in age-related macular degeneration (AMD). The RPE is known to be vulnerable to high-energy blue light. The white light-emitting diodes (LED) commercially available have relatively high content of blue light, a feature that suggest that they could be deleterious for this retinal cell layer. The aim of our study was to investigate the effects of "white LED" exposure on RPE. For this, commercially available white LEDs were used for exposure experiments on Wistar rats. Immunohistochemical stain on RPE flat mount, transmission electron microscopy and Western blot were used to exam the RPE. LED-induced RPE damage was evaluated by studying oxidative stress, stress response pathways and cell death pathways as well as the integrity of the outer blood-retinal barrier (BRB). We show that white LED light caused structural alterations leading to the disruption of the outer blood-retinal barrier. We observed an increase in oxidized molecules, disturbance of basal autophagy and cell death by necrosis. We conclude that white LEDs induced strong damages in rat RPE characterized by the breakdown of the BRB and the induction of necrotic cell death.

Here we review the different apoptotic DNases. From a functional point of view, DNases implicated in apoptosis may be classified into three groups: the Ca 2+ /Mg 2+ endonucleases, the Mg 2+ -endonucleases, and the cation-independent endonucleases. The first group includes DNase I which has no specificity for the linker region, DNase gamma which has some homology with DNase I, and other DNases which cleave DNA in the linker region. Both DNase I and DNase gamma have been cloned. The other nucleases of this category have dispersed molecular weights. Their sequences are unknown and it is difficult to determine their role(s) in apoptosis. It seems that different pathways are present and that these nucleases may be activated either by caspases or serine proteases. The caspase 3 activated DNase (CAD, CPAN, or DFF40) belongs to the Mg 2+ -dependent endonucleases. DNase II belongs to the third group of acid endonucleases or cation-independent DNases. We have shown the involvement of DNase II in lens cell differentiation. Recently, the molecular structure of two different enzymes has been elucidated, one of which has a signal peptide and appears to be secreted. The other, called L-DNase II, is an intracellular protein having two enzymatic activities; in its native form, it is an anti-protease, and after posttranslational modification, it becomes a nuclease.Key words: endonucleases, apoptosis, caspases, serine proteases.

SERPINB1, also called Leukocyte Elastase Inhibitor (LEI) is a member of the clade B of SERPINS. It is an intracellular protein and acts primarily to protect the cell from proteases released into the cytoplasm during stress. Its role in inflammation is clear due to its involvement in the resolution of chronic inflammatory lung and bowel diseases. LEI/SERPINB1 intrinsically possesses two enzymatic activities: an antiprotease activity dependent on its reactive site loop, which is analogous to the other proteins of the family and an endonuclease activity which is unveiled by the cleavage of the reactive site loop. The conformational change induced by this cleavage also unveils a bipartite nuclear localization signal allowing the protein to translocate to the nucleus. Recent data indicate that it has also a role in cell migration suggesting that it could be involved in diverse processes like wound healing and malignant metastases.

The cornified layer, the stratum corneum, of the epidermis is an efficient barrier to the passage of genetic material, i.e. nucleic acids. It contains enzymes that degrade RNA and DNA which originate from either the living part of the epidermis or from infectious agents of the environment. However, the molecular identities of these nucleases are only incompletely known at present. Here we performed biochemical and genetic experiments to determine the main DNase activity of the stratum corneum. DNA degradation assays and zymographic analyses identified the acid endonucleases L-DNase II, which is derived from serpinB1, and DNase 2 as candidate DNases of the cornified layer of the epidermis. siRNA-mediated knockdown of serpinB1 in human in vitro skin models and the investigation of mice deficient in serpinB1a demonstrated that serpinB1-derived L-DNase II is dispensable for epidermal DNase activity. By contrast, knockdown of DNase 2, also known as DNase 2a, reduced DNase activity in human in vitro skin models. Moreover, the genetic ablation of DNase 2a in the mouse was associated with the lack of acid DNase activity in the stratum corneum in vivo. The degradation of endogenous DNA in the course of cornification of keratinocytes was not impaired by the absence of DNase 2. Taken together, these data identify DNase 2 as the predominant DNase on the mammalian skin surface and indicate that its activity is primarily targeted to exogenous DNA.

Glucocorticoids (GCs) are routinely administered systemically or injected into the eye when treating numerous ocular diseases; however, their toxicity on the retinal microvasculature has not been previously investigated. In this article, the effects of hydrocortisone (Hydro), dexamethasone, dexamethasone-phosphate and triamcinolone acetonide (TA) were evaluated in vitro on human skin microcirculation cells and, bovine endothelial retinal cells, ex-vivo, on flat mounted rat retinas. The degree of GCs induced endothelial cell death varied according to the endothelial cell type and GCs chemical properties. GCs toxicity was higher in skin microvascular endothelial cells and for hydrophobic GC formulations. The mechanism of cell death differed between GCs, Hydro and TA activated the leukocyte elastase inhibitor/L-DNase II pathways but did not activate caspases. The mechanisms of cell death observed in cell cultures were similar to those observed in rat retinal explants. Taken together these results indicate that particular attention should be paid to the potential vascular side effects when administrating GCs clinically and in particular when developing sustained-release intraocular devices.

Abstract Exposure Limit Values (ELV) for artificial lighting were defined in order to prevent light-induced damage to the retina. The evaluation of the lighting devices include the correction of their spectra by the B(λ) function or blue light hazard function, representing the relative spectral sensitivity of the human eye to the blue light. This weighting function peaks between 435 and 440 nm. In this study we evaluate a new generation of light emitting diode (LED), the GaN-on-GaN (gallium nitride on gallium nitride) LED, that present an emission peak in the purple part of the spectrum. Wistar rats were exposed to GaN-on-GaN and conventional diodes at different retinal doses (from 2.2 to 0.5 J/cm 2 ). We show that GaN-on-GaN diodes are more toxic than conventional LED for the rat neural retina and the rat retinal pigment epithelium, indicating that the BLH (blue light hazard) weighting is not adapted to this type of diodes. One of the reasons of this increased toxicity is the effects of shorter wavelengths on mitochondria polarization. We also show that the threshold of phototoxic retinal dose in the rat (fixed at 11 J/cm 2 , BLH weighted) is overestimated, suggesting that the values used for regulations, calculated in primates using the same methods than in rats, should be revised.

Lens cells demonstrate a terminal differentiation process with loss of their organelles including nuclei. Chromatin disappearance is characterised by the same changes as most apoptotic cells, i.e. condensation of chromatin and cleavage into high molecular weight fragments and oligonucleosomes. The endo-deoxyribonucleases (bicationic (Ca2+, Mg2+), mono-cationic (Ca2+ or Mg2+) and acidic non-cationic dependent nucleases) are present in lens fibre cells. Our results suggest that the acidic non-cationic nuclease (DNase II) plays a major role in chromatin cleavage. This nuclease, known to be lysosomal, is found in lens fibre nuclei and only an antibody directed against DNase II inhibits the acidic DNA cleavage of lens fibre nuclei. In addition, there must be another DNase implicated in the process which is not DNase I but appears to be a Ca2+, Mg2+ dependent molecule. Regulation of these DNase activities may be accomplished by the effect of post-translational modifications, acidic pH, mitochondrial release molecules, growth factors or oncogenes. Finally, fibre cells lose organelles without cytoplasmic elimination. The survival of these differentiated cells might be due to the action of survival factors such as FGF 1.

Using L1210 murine leukemia cells, we have previously shown that in response to treatment with drugs having different targets, apoptotic cell death occurs through at least two different signaling pathways. Here, we present evidence that nuclear extracts from staurosporine-treated cells elicit DNase II activity that is not detected in nuclear extracts from cisplatin-treated cells. This activity correlates with the accumulation of two nuclear proteins (70 and 30 kDa) which are detected by an anti-L-DNase II antibody. Partial purification of this DNase II activity suggests that the 30-kDa protein could be the nuclease responsible for staurosporine-induced DNA fragmentation. The 70-kDa protein is also recognized by an anti-elastase antibody, suggesting that it carries residues belonging to both L-DNase II and elastase. Since previous findings showed that L-DNase II was generated from the leukocyte inhibitor of elastase, we propose that the 70-kDa protein results from an SDS-stable association between these two proteins and is translocated from the cytoplasm to the nucleus during staurosporine-induced apoptosis.

Apoptosis is characterized by cell shrinkage, nuclear condensation and internucleosomal DNA cleavage. Besides the central role of caspases and other proteases, cell death triggers DNA degradation so that DNases have an active role in apoptotic cell death. The best-characterized apoptotic DNase is CAD, a neutral Mg-dependent endonuclease. Its activity is regulated by its inhibitor, ICAD, which is cleaved by caspases. Other neutral DNases have been shown to cleave nuclear DNA in apoptotic conditions: endonuclease G, GADD. In cells, the cytosolic pH is maintained to 7.2, mostly due to the activity of the Na+/H+ exchanger. In many apoptotic conditions, a decrease of the intracellular pH has been shown. This decrease may activate different acid DNases, mostly when pH decreases below 6.5. Three acidic DNases II are so far known: DNase II α, DNase II β and L-DNase II, a DNase II, derived from the serpin LEI (Leukocyte Elastase Inhibitor). Their activation during cell death is discussed in this review.

Corticosteroids are hormones involved in many physiological responses such as stress, immune modulation, protein catabolism and water homeostasis. The subfamily of glucocorticoids is used systemically in the treatment of inflammatory diseases or allergic reactions. In the eye, glucocorticoides are used to treat macular edema, inflammation and neovascularization. The most commonly used glucocorticoid is triamcinolone acetonide (TA). The pharmaceutical formulation of TA is not adapted for intravitreal administration but has been selected by ophthalmologists because its very low intraocular solubility provides sustained effect. Visual benefits of intraocular TA do not clearly correlate with morpho-anatomical improvements, suggesting potential toxicity. We therefore studied, non-common, but deleterious effects of glucocorticoids on the retina. We found that the intravitreal administration of TA is beneficial in the treatment of neovascularization because it triggers cell death of endothelial cells of neovessels by a caspase-independent mechanism. However, this treatment is toxic for the retina because it induces a non-apoptotic, caspase-independent cell death related to paraptosis, mostly in the retinal pigmented epithelium cells and the Müller cells.

The pathogenesis of age-related macular degeneration (AMD) involves demise of the retinal pigment epithelium and death of photoreceptors. In this article, we investigated the response of human adult retinal pigmented epithelial (ARPE-19) cells to 5-(N,N-hexamethylene)amiloride (HMA), an inhibitor of Na(+) /H(+) exchangers. We observed that ARPE-19 cells treated with HMA are unable to activate 'classical' apoptosis but they succeed to activate autophagy. In the first 2 hrs of HMA exposure, autophagy is efficient in protecting cells from death. Thereafter, autophagy is impaired, as indicated by p62 accumulation, and this protective mechanism becomes the executioner of cell death. This switch in autophagy property as a function of time for a single stimulus is here shown for the first time. The activation of autophagy was observed, at a lesser extent, with etoposide, suggesting that this event might be a general response of ARPE cells to stress and the most important pathway involved in cell resistance to adverse conditions and toxic stimuli.

Abstract Polycyclic aromatic hydrocarbons (PAH), such as benzo[a]pyrene (B[a]P), are ubiquitous genotoxic environmental pollutants. Their DNA‐damaging effects lead to apoptosis induction, through similar pathways to those identified after exposure to other DNA‐damaging stimuli with activation of p53‐related genes and the involvement of the intrinsic apoptotic pathway. However, at a low concentration of B[a]P (50 nM), our previous results pointed to the involvement of intracellular pH (pH i ) variations during B[a]P‐induced apoptosis in a rat liver epithelial cell line (F258). In the present work, we identified the mitochondrial F0F1‐ATPase activity reversal as possibly responsible for pH i decrease. This acidification not only promoted executive caspase activation, but also activated leucocyte elastase inhibitor/leucocyte‐derived DNase II (LEI/L‐DNase II) pathway. p53 appeared to regulate mitochondria homeostasis, by initiating F0F1‐ATPase reversal and endonuclease G (Endo G) release. In conclusion, a low dose of B[a]P induced apoptosis by recruiting a large panel of executioners apparently depending on p53 phosphorylation and, for some of them, on acidification. J. Cell. Physiol. 208: 527–537, 2006. © 2006 Wiley‐Liss, Inc.

L-DNase II is derived from its precursor leucocyte elastase inhibitor (LEI) by post-translational modification. In vitro, the conversion of LEI into L-DNase II can be induced by incubation of LEI at an acidic pH. In this study, we proposed to analyze the effects of intracellular acidification on this transformation. Amiloride derivatives, like hexamethylene amiloride (HMA), are known to provoke a decrease of cytosolic pH by inhibiting the Na(+)/H(+) antiport. In BHK cells, treatment with HMA-induced apoptosis accompanied by an increase in L-DNase II immunoreactivity and L-DNase II enzymatic activity. Overexpression of L-DNase II precursor led to a significant increase of apoptosis in these cells supporting the involvement of L-DNase II in HMA induced apoptosis. As previously shown in other cells, etoposide-induced apoptosis did not activate L-DNase. On the contrary, LEI overexpression significantly increased cell survival in etoposide-induced apoptosis. Together these results suggest differential roles of LEI and L-DNase II in response to different types of apoptotic inducers.

Corticosteroids have recorded beneficial clinical effects and are widely used in medicine. In ophthalmology, besides their treatment benefits, side effects, including ocular toxicity have been observed especially when intraocular delivery is used. The mechanism of these toxic events remains, however, poorly understood. In our present study, we investigated the mechanisms and potential pathways of corticosteroid-induced retinal cell death.Rats were sacrificed 24 h and 8 days after an intravitreous injection of 1 microl (40 microg) of Kenacort Retard. The eyes were processed for ultra structure analysis and detection of activated caspase-3, cytochrome-C, apoptosis-inducing factor (AIF), LEI-L-Dnase II, terminal transferase dUTP nick end labeling (TUNEL), and microtubule-associated protein 1-light chain 3 (MAP-LC3). In vitro, rat retinal pigment epithelial cells (RPE), retinal Müller glial cells (RMG) and human ARPE-19 cells were treated with triamcinolone acetonide (TA) or other glucocorticoids. Cell viability was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5 phenyltetrazolium bromide test (MTT) assay and cell counts. Nuclei staining, TUNEL assay, annexin-V binding, activated caspase-3 and lactate dehydrogenase (LDH) production characterized cell death. Localization of cytochrome-C, AIF, LEI-and L-Dnase II, and staining with MAP-LC3 or monodansylcadaverine were also carried out. Finally, ARPE-19 cells transfected with AIP-1/Alix were exposed to TA.In vitro incubation of retinal cell in the presence of corticosteroids induced a specific and dose-dependent reduction of cell viability. These toxic events were not associated with the anti-inflammatory activity of these compounds but depended on the hydro solubility of their formulation. Before cell death, extensive cytoplasmic vacuolization was observed in the retinal pigment epithelial (RPE) cells in vivo and in vitro. The cells however, did not show known caspase-dependent or caspase-independent apoptotic reactions. These intracellular vacuoles were negative for MAP-LC3 but some stained positive for monodansylcadaverine. Furthermore, over expression of AIP-1/Alix inhibited RPE cell death.These observations suggest that corticosteroid-induced retinal cell death may be carried out mainly through a paraptosis pathway.

©2003, European Journal of HistochemistryCell death by apoptosis requires a precise plan of destruction of DNA and proteins.In this paper, we review the current knowledge on the different DNA-degrading enzymes which are activated in apoptotic cells.The activation of DNases by upstream proteases is also discussed.

Alzheimer's disease (AD) develops undiagnosed for 10–15 years due to the lack of early diagnostic biomarkers. Visual deficits are common and crippling in AD patients and histopathological alterations found in the retina and brain are similar. We hypothesize that subtle morphological and functional changes in microglial and neuronal activities, such as those recently reported in the hippocampus, may also occur in retina during the preclinical stages of AD. These alterations are likely much more accessible to modern imaging and electrophysiological exploration than those occurring in the hippocampus and therefore, may serve as the earliest diagnostic biomarkers for AD.

We have applied to human HeLa cells two different stimuli of apoptosis: the antitumoral drug etoposide, and a more 'physiological' death condition, obtained by growing cells in the same medium for long time periods, for up to 10 days. Analysis of different parameters demonstrated that in both experimental systems the same apoptotic features are visible. However, the DNA degradation pattern appeared to be different, suggesting the involvement of different DNases. In this view, we have analyzed the activity and expression of Ca2+-Mg2+-dependent and acid DNases. We have observed that DNase I is not modulated during apoptosis. In contrast, the acid L-DNase II (derived from Leukocyte Elastase Inhibitor by post-translational modification), recently identified in our laboratory, is mainly active in the apoptotic pathway induced by long term-culture. Furthermore, we have provided evidence that while caspase 3 is activated by both inducers, caspase 1 is essential only for the etoposide-induced apoptosis.

Tightly controlled proteolysis is a defining feature of apoptosis and caspases are critical in this regard. Significant roles for non-caspase proteases in cell death have been highlighted. Staurosporine causes a rapid induction of apoptosis in virtually all mammalian cell types. Numerous studies demonstrate that staurosporine can activate cell death under caspase-inhibiting circumstances. The aim of this study was to investigate the proteolytic mechanisms responsible for cell death under these conditions. To that end, we show that inhibitors of serine proteases can delay cell death in one such system. Furthermore, through profiling of proteolytic activation, we demonstrate, for the first time, that staurosporine activates a chymotrypsin-like serine protease-dependent cell death in HL-60 cells independently, but in parallel with the caspase controlled systems. Features of the serine protease-mediated system include cell shrinkage and apoptotic morphology, regulation of caspase-3, altered nuclear morphology, generation of an endonuclease and DNA degradation. We also demonstrate a staurosporine-induced activation of a putative 16 kDa chymotrypsin-like protein during apoptosis.

The best characterized biochemical feature of apoptosis is degradation of genomic DNA into oligonucleosomes.The endonuclease responsible for DNA degradation in caspasedependent apoptosis is Caspase Activated DNase (CAD).In caspase-independent apoptosis, different endonucleases may be activated according to the cell line and the original insult.Among the known effectors of caspase-independent cell death, L-DNase II (LEI derived DNase II) has been previously characterized by our laboratory.We have thus shown that this endonuclease derives from a serpin super-family member LEI (Leukocyte Elastase Inhibitor) by post-translational modification [Torriglia et al, (1998) Mol.Cell.Biol 18, 3612-3619].In this work we assessed the molecular mechanism involved in the change of the enzymatic activity of this molecule from an anti-protease to an endonuclease.We report that the cleavage of LEI by elastase at its reactive center loop abolishes its anti-protease activity and leads to a conformational modification that exposes an endonuclease active site and a Nuclear Localization Signal (NLS).This represents a novel molecular mechanism for a complete functional conversion induced by the conformational change of a serpin.We also show that this molecular transformation affects cellular fate and that both endonuclease activity and nuclear translocation of L-DNase II, are needed to induce cell death.

Neuroprotection strategies in the retina aim at interference with regulatory mechanisms of cell death. To successfully target these mechanisms it is necessary to understand the molecular pathways activated in the degenerating retina. Induced retinal degeneration models, like the light damage model, give a synchronized response allowing their detailed investigation. In this study we exposed Fisher rats to a continuous white light. This induced a caspase-independent cell death in which the activation of cathepsin D has an important role via the activation of L-DNase II. Inhibition of this enzyme by intravitreal administration of pepstatin A protects photoreceptors indicating that this enzyme might be an interesting target for neuroprotection.

Recent evidence has suggested that Alzheimer's disease (AD)-associated neuronal loss may occur via the caspase-independent route of programmed cell death (PCD) in addition to caspase-dependent mechanisms. However, the brain region specificity of caspase-independent PCD in AD-associated neurodegeneration is unknown. We therefore used the transgenic CRND8 (TgCRND8) AD mouse model to explore whether the apoptosis inducing factor (AIF), a key mediator of caspase-independent PCD, contributes to cell loss in selected brain regions in the course of aging.Increased expression of truncated AIF (tAIF), which is directly responsible for cell death induction, was observed at both 4- and 6-months of age in the cortex. Concomitant with the up-regulation of tAIF was an increase in the nuclear translocation of this protein. Heightened tAIF expression or translocation was not observed in the hippocampus or cerebellum, which were used as AD-vulnerable and relatively AD-spared regions, respectively. The cortical alterations in tAIF levels were accompanied by increased Bax expression and mitochondrial translocation. This effect was preceded by a significant reduction in ATP content and an increase in reactive oxygen species (ROS) production, detectable at 2 months of age despite negligible amounts of amyloid-beta peptides (Aβ).Taken together, these data suggest that AIF is likely to play a region-specific role in AD-related caspase-independent PCD, which is consistent with aging-associated mitochondrial impairment and oxidative stress.

One approach to discriminate among specific DNases in apoptosis is to use inhibitors specific for each endonuclease. Zn2+ is known to inhibit Ca(2+)- and Mg(2+)-dependent endonuclease enzymatic activities during apoptosis. Acidic DNases were thought to be insensitive to Zn2+. In this paper, we analyse the effects of Zn2+ on activity of DNase II, either purified or in nuclei from lens fiber cells. These cells follow a physiological nuclear degeneration with DNase II accumulation in their nuclei. We show that Zn2+ is able to inhibit also this acidic endonuclease at a concentration of 1-6 mM. At a higher concentration of Zn2+, DNA is extensively degraded during the assay, masking the inhibition of the enzyme. This DNA degradation in the presence of Zn2+ has led to an overestimation of the activity of DNase II in studies of apoptosis. Hence, Zn2+ cannot be used to specifically identify one endonuclease among the different DNases involved in nuclear degradation during programmed cell death.

The influence of in vitro near-UV radiation exposure on the physical state of the rat lens and on its membrane-bound Na-K-ATPase activity was investigated. Lens swelling was correlated to the appearance of opacities and the inactivation of the enzyme. The results show a significant decrease in the Na-K-ATPase activity which may be an early change leading to osmotic type cataracts. The dose-effect curves obtained for cortical and epithelial enzymes were different. Since the data do not follow a monoexponential function, the existence of two forms of Na-K-ATPase in the lens is discussed.

The discovery of caspase activation counts as one of the most important finds in the biochemistry of apoptosis. However, targeted disruption of caspases does not impair every type of apoptosis. Other proteases can replace caspases and several so called “caspase independent” pathways are now described. Here we review our current knowledge on one of these pathways, the LEI/L-DNase II. It is a serine protease-dependent pathway and its key event is the transformation of LEI (leukocyte elastase inhibitor, a serine protease inhibitor) into L-DNase II (an endonuclease). The molecular events leading to this change of enzymatic function as well as the cross-talk and interactions of this molecule with other apoptotic pathway, including caspases, are discussed.

The review intends to draw the attention of researchers working in retinal degenerations on the fact that classical apoptosis, for example, apoptosis triggering caspase activation, may not be the main pathway of cellular demise in this tissue.Former work already showed the difficulty of proving the activation of apoptosis effectors in different models of retinal degeneration. However, these results were not really considered because of the lack of an alternative explanation for cell death. Nowadays, the description of many pathways of cellular demise is filling the gap and other forms of cell death are now described in the retina.The knowledge on the molecular mechanisms of cell death is very important for the development of new therapeutic strategies, as well as for the evaluation of cell death onset in retinal degeneration.

Retinal phototoxicity is the main mechanism by which light induces damage to the retina, the sensitive part of the eye. The impact of light on the different retinal layers varies depending on several factors including the wavelength of the incident light, its energy, and the exposure time. We propose an overview of the factors modulating the amount of light that reaches the retina, the type of damage, and the different cell death mechanisms triggered by phototoxicity to mediate cell demise in the retina. We also provide an analysis of the phototoxicity mechanisms induced by light depending on the lighting settings. Special interest is given to the influence of the emission spectrum on the induction of specific cell death pathways. Moreover, the existing literature on phototoxicity is reviewed by taking into consideration the used doses of light.

Nowadays artificial light highly increases human exposure to light leading to circadian rhythm and sleep perturbations. Moreover, excessive exposure of ocular structures to photons can induce irreversible retinal damage. Meta-analyses showed that sunlight exposure influences the age of onset and the progression of Age-related macular degeneration (AMD), the leading cause of blindness in people over fifty-year old. Currently, the blue-light hazard (BLH) curve is used in the evaluation of the phototoxicity of a light source for domestic lighting regulations.Here, we analyze the phototoxicity threshold in rats and investigate the role played by the light spectrum, assessing the relevance of the use of the BLH-weighting to define phototoxicity.We exposed albino rats to increasing doses of blue and white light, or to lights of different colors to evaluate the impact of each component of the white light spectrum on phototoxicity. Cellular mechanisms of cell death and cellular stress induced by light were analyzed.Our results show that the phototoxicity threshold currently accepted for rats is overestimated by a factor of 50 when considering blue light and by a factor of 550 concerning white light. This is the result of the toxicity induced by green light that increases white light toxicity by promoting an inflammatory response. The content of green in white light induces 8 fold more invasion of macrophages in the retina than the content of blue light. Moreover, the use of BLH-weighting does not evaluate the amount of red radiations contained in white light that mitigates damage by inhibiting the nuclear translocation of L-DNase II and reducing by 33% the number of TUNEL-positive cells.These findings question the current methods to determine the phototoxicity of a light source and show the necessity to take into account the entire emission spectrum. As current human phototoxicity thresholds were estimated with the same methods used for rats, our results suggest that they might need to be reconsidered.

The analysis of the current safety standards for electric toys showed that the normative intensity limits for visible light emitted by LEDs integrated in toys are overestimated. These limits were originally set in a scientific article published in 2012 that was adapted into the international safety standard published in 2017, and into the subsequent European and national standards, all published in 2020. The overestimation of the intensity limits results from several errors made in the original article. Accordingly, the current normative intensity limits for visible light used in toys do not protect the children’s eyes against adverse effects of exposure to high intensity LEDs which may compromise their visual health. Updating the safety standards for electric toys using a method based on robust scientific data is recommended to protect children's visual health and ensure their long-term well-being.

The analysis of the current safety standards for electric toys showed that the normative intensity limits for visible light emitted by LEDs integrated in toys are overestimated. These limits were originally set in a scientific article published in 2012 that was adapted into the international safety standard published in 2017, and into the subsequent European and national standards, all published in 2020. The overestimation of the intensity limits results from several errors made in the original article. Accordingly, the current normative intensity limits for visible light used in toys do not protect the children’s eyes against adverse effects of exposure to high intensity LEDs which may compromise their visual health. Updating the safety standards for electric toys using a method based on robust scientific data is recommended to protect children's visual health and ensure their long-term well-being.

Light is known to induce retinal damage affecting photoreceptors and retinal pigment epithelium. For polychromatic light, the blue part of the spectrum is thought to be the only responsible for photochemical damage, leading to the establishment of a phototoxicity threshold for blue light (445 nm). For humans it corresponds to a retinal dose of 22 J/cm2. Recent studies on rodents and non-human primates suggested that this value is overestimated. In this study, we aim at investigating the relevance of the current phototoxicity threshold and at providing new hints on the role of the different components of the white light spectrum on phototoxicity. We use an in vitro model of human induced pluripotent stem cells (hiPSC)-derived retinal pigment epithelial (iRPE) cells and exposed them to white, blue and red lights from LED devices at doses below 22 J/cm2. We show that exposure to white light at a dose of 3.6 J/cm2 induces an alteration of the global cellular structure, DNA damage and an activation of cellular stress pathways. The exposure to blue light triggers DNA damage and the activation of autophagy, while exposure to red light modulates the inflammatory response and inhibits autophagy.

The spectral distribution is a fundamental property of non-monochromatic optical radiation. It is commonly used in research and practical applications when studying how light interacts with matter and living organisms, including humans. In the field of lighting, misconceptions about the spectral distribution of light are responsible for unfounded claims, which pervade the scientific and technical communities. Starting from the definition of the spectral distribution, this paper describes the ambiguities and errors associated with a purely graphical analysis of the spectral distribution. It also emphasizes the importance of considering the particle nature of light in research involving both visual and non-visual effects, which implies using the spectral distribution expressed in the photon system of units, a system that has been seldom used in lighting research for historical reasons. The authors encourage lighting engineers and researchers to determine which system is best suited to their work and then proceed with the correct use of spectral distributions and of spectral weighting functions for applications involving optical radiation.

The discovery of caspase-mitochondrial pathway counts as one of the most important discovery in apoptosis biochemistry. Today, however, we begin to recognize its limits. Inhibition of caspase does not prevent cell death in many mammalian models. Targeted disruption of caspases does not impair every type of apoptosis. Other pathways, caspase independent, are now described. Here we present one of these pathways. It is a serine-protease dependent pathway and its key event is the transformation of LEI (a serine protease inhibitor) into L-DNase II (an endonuclease). When using this apoptotic pathway the cell activates, at the same time, its endonuclease activity (L-DNase II appears) and its protease activity (there is a release of inhibition of proteases).

Retinal death induced by light seems to be a caspase-independent process. In this work we investigate the LEI/L-DNase II pathway, a caspase-independent pathway, in light-induced retinal degeneration in Fischer rats. Measurement of DNase activity in total retinal extracts of light exposed Fischer rats was performed by analysing a plasmid degradation on an agarose gel. The same method was used to measure the in vitro activity of recombinant LEI (reticulocyte lysate) after incubation with calpains. L-DNase II activity is observed in retinal extracts of light exposed Fischer rats and increases with time illumination. In this apoptotic death, the activation of calpains has been shown. Here we show that L-DNase II activation is not catalized by calpains. The present study indicates that the LEI/L-DNase II may be a possible pathway activated during photoreceptor apoptosis in light-induced retinal degeneration but that this pathway is not directly activated by calpains.

purpose. To evaluate the antimitotic and toxic effects of 5-chlorouracil (5-CU) and 5-fluorouracil (5-FU) and study their potential to delay filtering bleb closure in the rabbit eye when released by poly(ortho esters) (POE). methods. Rabbit Tenon fibroblasts and human conjunctival cells were incubated with various 5-CU and 5-FU concentrations. Antiproliferative effects and toxicity were evaluated at 24 and 72 hours by monotetrazolium, neutral red, and Hoechst tests and cell counting. Mechanisms of cell death were evaluated using TUNEL assay, annexin V binding, immunohistochemistry for anti–apoptosis-inducing factor (AIF) and LEI/L-DNase II. Trabeculectomy was performed in pigmented rabbits. Two hundred microliters of POE loaded with 1% wt/wt 5-FU or 5-CU was injected into the subconjunctival space after surgery. Intraocular pressure (IOP) and bleb persistence were monitored for 150 days. results. In vitro, 5-FU showed a higher antiproliferative effect and a more toxic effect than 5-CU. 5-FU induced cell necrosis, whereas 5-CU induced mostly apoptosis. The apoptosis induced by 5-CU was driven through a non-caspase–dependent pathway involving AIF and LEI/L-DNase II. In vivo, at 34 days after surgery, the mean IOP in the POE/5-CU–treated group was 83% of the baseline level and only 40% in the POE/5-FU–treated group. At 100 days after surgery, IOP was still decreased in the POE/5-CU group when compared with the controls and still inferior to the preoperative value. The mean long-term IOP, with all time points considered, was significantly (P < 0.0001) decreased in the POE/5-CU–treated group (6.0 ± 2.4 mm Hg) when compared with both control groups, the trabeculectomy alone group (7.6 ± 2.9 mm Hg), and the POE alone group (7.5 ± 2.6 mm Hg). Histologic analysis showed evidence of functioning blebs in the POE-5-CU–treated eyes along with a preserved structure of the conjunctiva epithelium. conclusions. The slow release of 5-CU from POE has a longstanding effect on the decrease of IOP after glaucoma-filtering surgery in the rabbit eye. Thus, the slow release of POE/5-CU may be beneficial for the prevention of bleb closure in patients who undergo complicated trabeculectomy.

Apoptosis is an essential cellular mechanism involved in many processes such as embryogenesis, metamorphosis, and tissue homeostasis. DNA fragmentation is one of the key markers of this form of cell death. DNA fragmentation is executed by endogenous endonucleases such as caspase-activated DNase (CAD) in caspase-dependent apoptosis. The TUNEL (TdT-mediated dUTP–biotin nick end labeling) technique is the most widely used method to identify apoptotic cells in a tissue or culture and to assess drug toxicity. It is based on the detection of 3′-OH termini that are labeled with dUTP by the terminal deoxynucleotidyl transferase. Although the test is very reliable and sensitive in caspase-dependent apoptosis, it is completely useless when cell death is mediated by pathways involving DNA degradation that generates 3′-P ends as in the LEI/L-DNase II pathway. Here, we propose a modification in the TUNEL protocol consisting of a dephosphorylation step prior to the TUNEL labeling. This allows the detection of both types of DNA breaks induced during apoptosis caspase-dependent and independent pathways, avoiding underestimating the cell death induced by the treatment of interest.

LEI (Leukocyte Elastase Inhibitor), the precursor of the pro-apoptotic molecule L-DNase II, belongs to the ovalbumin subgroup of serpins. Several serpins can inhibit apoptosis: the viral serpin Crm A inhibits Fas or TNFα-induced apoptosis, and overexpression of PAI-2 or PI-9 protects cells from TNFα or granzyme B induced apoptosis. We have previously shown that LEI overexpression protects cells from etoposide-induced apoptosis. The molecular reason of this anti-apoptotic activity is now investigated. We show that, in BHK-21 and HeLa cells, LEI anti-protease activity is essential for its anti-apoptotic effect. The protease inhibited is cathepsin D, released from the lysosome during etoposide treatment. Cathepsin D enhances caspase activity in the cell by cleaving procaspase-8 and LEI overexpression slows down this cleavage, protecting cells from apoptosis. This let us presume that high expression of LEI in tumor cells may reduce the efficiency of etoposide as a chemotherapeutic agent.

This study investigates the effects of triamcinolone acetonide (TA) on retinal endothelial cells in vitro and explores the potential vascular toxic effect of TA injected into the vitreous cavity of rats in vivo.Subconfluent endothelial cells were treated with either 0.1 mg/ml or 1 mg/ml TA in 1% ethanol. Control cells were either untreated or exposed to 1% ethanol. Cell viability was evaluated at 24 h, 72 h, and five days using the tetrazolium 3-(4,5-dimethylthiazol-2-yl)-2,5 phenyltetrazolium bromide test (MTT) and lactate dehydrogenase (LDH) assays. Cell proliferation was evaluated by 5-bromo-2-deoxyuridine (BrdU) test. Apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL assay), annexin-binding, and caspase 3 activation. Caspase-independent cell deaths were investigated by immunohistochemistry using antibodies against apoptosis inducing factor (AIF), cytochrome C, microtubule-associated protein (MAP)-light chain 3 (MAP-LC3), and Leukocyte Elastase Inhibitor/Leukocyte Elastase Inhibitor-derived DNase II (LEI/L-DNase II). In vivo, semithin and ultrathin structure analysis and vascular casts were performed to examine TA-induced changes of the choroidal vasculature. In addition, outer segments phagocytosis assay on primary retinal pigment epithelium (RPE) cells was performed to assess cyclooxygenase (COX-2) and vascular endothelial growth factor (VEGF) mRNAs upregulation with or without TA.The inhibitory effect of TA on cell proliferation could not explain the significant reduction in cell viability. Indeed, TA induced a time-dependent reduction of bovine retinal endothelial cells viability. Annexin-binding positive cells were observed. Cytochrome C was not released from mitochondria. L-DNase II was found translocated to the nucleus, meaning that LEI was changed into L-DNase II. AIF was found nuclearized in some cells. LC3 labeling showed the absence of autophagic vesicles. No autophagy or caspase dependent apoptosis was identified. At 1 mg/ml TA induced necrosis while exposure to lower concentrations for 3 to 5 days induced caspase independent apoptosis involving AIF and LEI/L-DNase II. In vivo, semithin and ultrathin structure analysis and vascular casts revealed that TA mostly affected the choroidal vasculature with a reduction of choroidal thickness and increased the avascular areas of the choriocapillaries. Experiments performed on primary RPE cells showed that TA downregulates the basal expression of COX-2 and VEGF and inhibits the outer segments (OS)-dependent COX-2 induction but not the OS-dependent VEGF induction.This study demonstrates for the first time that glucocorticoids exert direct toxic effect on endothelial cells through caspase-independent cell death mechanisms. The choroidal changes observed after TA intravitreous injection may have important implications regarding the safety profile of TA use in human eyes.

Caspase activation has been seen, for several years, as the biochemical marker of apoptosis. However, in 2005 the Nomenclature Committee on Cell Death (NCCD) established that the 'official' classification of cell death had to rely on morphological criteria owing to the absence of a clear-cut equivalence between structural alterations and biochemical pathways. Actually, the controlled destruction of the cell is coordinated by a proteolytic system involving caspases but also other proteases like cathepsins, calpains and serine proteases. These enzymes participate in an activation cascade that culminates in cleavage of a set of proteins resulting in disassembly of the cell. This disassembling also includes the activation of endonucleases that will destroy a potentially harmful DNA. A caspase-activated DNase performs DNA degradation in caspase-dependent apoptosis, but other endonucleases like L-DNase II or GAAD are activated in caspase-independent apoptosis, allowing the complete dismantling of the cell.

During the development of the neural retina, 50% of the neurons die physiologically by apoptosis. In the chick embryo, the apoptotic wave starts at E8 and ends at E18, with a peak at E11. The onset of apoptosis is accompanied by the activation of several degradative enzymes. Among these, the activation of the endonucleases leads to the degradation of the genomic DNA of the cell which is thought to be the final event in apoptosis. Here, we have investigated the endonucleases activated during apoptosis associated with retinal development. We have found that Ca2+-Mg2+-dependent endonucleases, as well as acid endonucleases are activated. The results obtained in vitro using purified nuclei from chicken retina indicate that the endonuclease activity resulting from the activation of L-DNase II, an acid DNase is responsible for most of the DNA degradation observed in these cells.

Purpose. To look for apoptosis pathways involved in corneal endothelial cell death during acute graft rejection and to evaluate the potential role of nitric oxide in this process. Materials and Methods. Corneal buttons from Brown-Norway rats were transplanted into Lewis rat corneas. At different time intervals after transplantation, apoptosis was assessed by diamino-2-phenylindol staining and annexin-V binding on flat-mount corneas, and by terminal transferase dUTP nick end labeling (TUNEL), caspase-3 dependent and leukocyte elastase inhibitor (LEI)/LDNase II caspase-independent pathways on sections. Inducible nitric oxide synthase (NOS-II) expression and the presence of nitrotyrosine were assayed by immunohistochemistry. Results. Graft endothelial cells demonstrated nuclear fragmentation and LEI nuclear translocation, annexin-V binding, and membranes bleb formation. Apoptosis associated with caspase-3 activity or TUNEL-positive reaction was not observed at any time either in the graft or in the recipient corneal endothelial cells. During 14 days posttransplantation, the recipient corneal endothelial cells remained unaltered and their number unchanged in all studied corneas. NOS-II was expressed in infiltrating cells present within the graft. This expression was closely associated with the presence of nitrotyrosine in endothelial and infiltrating cells. Conclusion. During the time course of corneal graft rejection, graft endothelial cells undergo apoptosis. Apoptosis is caspase 3 independent and TUNEL negative and is, probably, carried out by an alternative pathway driven by an LEI/L-Dnase II. Peroxynitrite formation may be an additional mechanism for cell toxicity and programmed cell death of the graft endothelial cells during the rejection process in this model.

HMA (5-(N,N-hexamethylene)amiloride), which belongs to a family of novel amiloride derivatives, is one of the most effective inhibitors of Na+/H+ exchangers, while uneffective against Na+ channels and Na+/Ca2+ exchangers. In this study, we provided evidence that HMA can act as a fluorescent probe. In fact, human retinal ARPE19 cells incubated with HMA show an intense bluish fluorescence in the cytoplasm when observed at microscope under conventional UV-excitation conditions. Interestingly, a prolonged observation under continuous exposure to excitation lightdoes not induce great changes in cells incubated with HMA for times up to about 5 min, while an unexpected rapid increase in fluorescence signal is observed in cells incubated for longer times. The latter phenomenon is particularly evident in the perinuclear region and in discrete spots in the cytoplasm. Since HMA modulates intracellular acidity, the dependence of its fluorescence properties on medium pH and response upon irradiation have been investigated in solution, at pH 5.0 and pH 7.2. The changes in both spectral shape and amplitude emission indicate a marked pH influence on HMA fluorescence properties, making HMA exploitable as a self biomarker of pH alterations in cell studies, in the absence of perturbations induced by the administration of other exogenous dyes.

Light-induced retinal degeneration is characterized by photoreceptor cell death. Many studies showed that photoreceptor demise is caspase-independent. In our laboratory we showed that leucocyte elastase inhibitor/LEI-derived DNase II (LEI/L-DNase II), a caspase-independent apoptotic pathway, is responsible for photoreceptor death. In this work, we investigated the activation of a pro-survival kinase, the protein kinase C (PKC) zeta. We show that light exposure induced PKC zeta activation. PKC zeta interacts with LEI/L-DNase II and controls its DNase activity by impairing its nuclear translocation. These results highlight the role of PKC zeta in retinal physiology and show that this kinase can control caspase-independent pathways.

Nitrosative stress takes place in endothelial cells (EC) during corneal acute graft rejection. The purpose of this study was to evaluate the potential role of peroxynitrite on corneal EC death.The effect of peroxynitrite was evaluated in vivo. Fifty, 250, and 500 microM in 1.5 microL of the natural or denatured peroxynitrite in 50 microM NaOH, 50 microM NaOH alone, or balanced salt solution were injected into the anterior chamber of rat eyes (n=3/group). Corneal toxic signs after injection were assessed by slit-lamp, in vivo confocal imaging, pachymetry, and EC count. The effect of peroxynitrite was also evaluated on nitrotyrosine and leucocyte elastase inhibitor/LDNase II immunohistochemistry. Human corneas were incubated with peroxynitrite and the effect on EC viability was evaluated. A specific inducible nitric oxide synthase inhibitor (iNOS) was administered systemically in rats undergoing allogeneic corneal graft rejection and the effect on EC was evaluated by EC count.Rat eyes receiving as little as 50 microM peroxynitrite showed a specific dose-dependent toxicity on EC. We observed an intense nitrotyrosine staining of human and rat EC exposed to peroxynitrite associated with leucocyte elastase inhibitor nuclear translocation, a noncaspase dependent apoptosis reaction. Specific inhibition of iNOS generation prevented EC death and enhanced EC survival of the grafted corneas. However, inhibition of iNOS did not have a significant influence on the incidence of graft rejection.Nitrosative stress during acute corneal graft rejection in rat eyes induces a noncaspase dependent apoptotic death in EC. Inhibition of nitric oxide production during the corneal graft rejection has protective effects on the corneal EC survival.

Poly(ADP-ribose) polymerase-1 (PARP-1) is an important regulator of apoptosis. Its over-activation at the onset of apoptosis can inhibit the action of apoptotic endonucleases like caspase-activated DNase and DNAS1L3. Therefore, controlled PARP-1 proteolysis during caspase-dependent apoptosis is considered essential to promote DNA degradation. Yet, little is known about the interplay of PARP-1 and endonucleases that operate during caspase-independent cell death. Here we show that in the long-term cultured HeLa cells which undergo caspase-independent death, PARP-1 co-immunoprecipitates with leukocyte elastase inhibitor-derived DNase II (L-DNase II), an acid DNase implicated in this death pathway and activated by serine proteases. Our results indicate that, despite having putative poly(ADP-ribose)-acceptor sites, LEI/L-DNase II is neither significantly poly(ADP-ribosyl)ated nor inhibited by PARP-1 during caspase-independent apoptosis. Unexpectedly, caspase-independent apoptosis induced by hexa-methylene amiloride, LEI/L-DNase II can activate PARP-1 and promote its auto-poly(ADP-ribosyl)ation, thus inhibiting PARP-1 activity. Moreover, overexpression of LEI blocks the pro-survival effect of PARP-1 in this model of cell death. Our results provide the original evidence for a new mechanism of PARP-1 activity regulation in the caspase-independent death pathway involving LEI/L-DNase II.

Leukocyte Elastase Inhibitor (LEI, also called serpin B1) is a protein involved in apoptosis among other physiological processes. We have previously shown that upon cleavage by its cognate protease, LEI is transformed into L-DNase II, a protein with a pro-apoptotic activity. The caspase independent apoptotic pathway, in which L-DNase II is the final effector, interacts with other pro-apoptotic molecules like Poly-ADP-Ribose polymerase (PARP) or Apoptosis Inducing Factor (AIF). The screening of LEI/L-DNase II interactions showed a possible interaction with several members of the BCL-2 family of proteins which are known to have a central role in the regulation of caspase dependent cell death. In this study, we investigated the regulation of LEI/L-DNase II pathway by two members of this family of proteins: BAX and BCL-2, which have opposite effects on cell survival. We show that, in both BHK and HeLa cells, LEI/L-DNase II can interact with BCL-2 and BAX in apoptotic and non-apoptotic conditions. These proteins which are usually thought to be anti-apoptotic and pro-apoptotic respectively, both inhibit the L-DNase II pro-apoptotic activity. These results give further insight in the regulation of caspase independent pathways and highlight the involvement of the intracellular environment of a given protein in the determinism of its function. They also add a link between caspase-dependent and independent pathways of apoptosis.

The distribution of fibroblast growth factor (FGF) receptor in bovine retina was established using a polyclonal antibody against the extracellular domain of this receptor. Different conditions of tissue fixation and development of the secondary antibody were tested. The ability of the antiserum to map precisely the receptor was obtained on fresh-frozen sections which had been treated with paraformaldehyde prior to incubation with this antiserum. Positive staining was confined mainly to the synaptic and ganglion axon layers. These results suggest that the FGF receptor might act in the transmitter stability, and the plasticity of synapses and ganglion cell axons in adult retina.

During retinal development, the neuronal death is carried out by the mechanism of apoptosis. Among the different endonucleases activated, L-DNase II seems to be responsible for most of DNA degradation in this tissue. L-DNase II derives from LEI (Leukocyte Elastase Inhibitor) by a post-translational modification carried out by elastase in apoptosis induced in vitro. In this study, we investigated whether elastase could be implicated in apoptosis occurring during retinal development. Although elastase and LEI/elastase complex are colocalized in retinal sections, the LEI/elastase complex, detected by Western blot, does not change at all stages of development. However, at pH 4 retinal extracts show an enhanced activation of the L-DNase II. These results suggest that an acid protease, such as a cathepsin, may be implicated in neuronal retinal apoptosis.

We have recently demonstrated that a high c-myc endogenous amplification level confers an apoptosis-prone phenotype to serum-deprived colon carcinoma SW613-S cells. The aim of this study was to gain new insights into the features of c-myc-dependent apoptosis, by extending our analysis to different apoptogenic stimuli. The study was carried out on clones, derived from the human colon carcinoma SW613-S cell line, which harbor different levels of endogenous c-myc amplification, and on isogenic cell lines with an enforced c-myc overexpression. Our results indicate that cells with endogenous or transfected exogenous c-myc overexpression (SW613-12A1 and -2G1mycP2Tu1 cell lines, respectively), activate the apoptotic machinery in response to the treatment with etoposide, doxorubicin and vitamin D3, which induce apoptosis through the death receptor Fas. The low levels of c-myc expression present in SW613-B3 and -B3mycC5, seem to be unable to activate Fas-mediated apoptosis, thus suggesting that only a high c-myc expression can bypass the lack of Fas receptor. Apoptosis induction mediated by DNA damage and long-term culture was independent of c-myc expression. A pathway of apoptosis characterized by the activation of the enzyme L-DNase II, was observed in both 12A1 and B3 cell lines.

To analyse the mechanism of ethanol-induced cell death, and particularly, the activation of the leucocyte elastase inhibitor (LEI) pathway.Cultured ARPE-19 cells were exposed to 0-13% ethanol for 24 h. Cytotoxicity was estimated by morphologic changes within the nucleus and breakdown of DNA, assessed by agarose gel electrophoresis or flow cytometry cell sorter. Poly(ADP-ribose)polymerase cleavage (PARP) was determined by western blot analysis. Changes in transcription and translation of LEI were assessed by analysis of mRNA levels and expression of protein product (immunohistochemistry), respectively.We established the ability of ethanol to induce cell death in ARPE-19 cells. After a 24 h incubation with 4% ethanol, 50% of the cells died; all the cells died in the presence of 10% ethanol. After ethanol incubation, we observed nuclear condensation and DNA fragmentation; the amount of fragmentation was proportional to the ethanol level. By flow cytometry analysis and agarose gel electrophoresis, the pattern of DNA cleavage exhibited a sub-G1 peak, suggesting necrotic cell death. However, other observations, i.e. nuclei shrinkage, PARP cleavage and inhibition of cell death by cycloheximide, and activation of a caspase independent LEI/DNase II pathway were observed and are features associated with apoptotic cell death. During ethanol stress, an LEI/L-DNase II intermediate was lost, leading to complete activation L-DNase II (24 kDa). RT-PCR analysis showed an early and specific increase of the LEI mRNA. Cycloheximide inhibited LEI synthesis and protected cells against apoptosis.Our data indicate that ethanol stress on ARPE-19 cells can induce a pathway which is a form of programmed cell death with characteristics of both apoptosis and necrosis, possibly by triggering conversion of LEI to L-DNase II.

Poly(ADP‐ribose) polymerase‐1 (PARP‐1) uses NAD + as a substrate to form ADP‐ribose. During apoptosis, caspases cleave PARP‐1 to avoid excessive NAD consumption. Because PARP‐1 is a key regulator of the activity of DNases involved in caspase‐dependent apoptosis, its cleavage is required to promote DNA degradation. To explore the situation in caspase‐independent cell death, we investigated the effect of PARP‐1 on the acid endonuclease leukocyte elastase inhibitor (LEI)–derived DNase II (L‐DNase II). We found for the first time an association between PARP‐1 and LEI/L‐DNase II. Unexpectedly, we observed that LEI influenced the automodification of PARP‐1.

Spino-cerebellar ataxia type 7 (SCA7) is a polyglutamine (polyQ) disorder characterized by neurodegeneration of the brain, cerebellum, and retina caused by a polyglutamine expansion in ataxin7. The presence of an expanded polyQ tract in a mutant protein is known to induce protein aggregation, cellular stress, toxicity, and finally cell death. However, the consequences of the presence of mutant ataxin7 in the retina and the mechanisms underlying photoreceptor degeneration remain poorly understood. In this study, we show that in a retinal SCA7 mouse model, polyQ ataxin7 induces stress within the retina and activates Muller cells. Moreover, unfolded protein response and autophagy are activated in SCA7 photoreceptors. We have also shown that the photoreceptor death does not involve a caspase-dependent apoptosis but instead involves apoptosis inducing factor (AIF) and Leukocyte Elastase Inhibitor (LEI/L-DNase II). When these two cell death effectors are downregulated by their siRNA, a significant reduction in photoreceptor death is observed. These results highlight the consequences of polyQ protein expression in the retina and the role of caspase-independent pathways involved in photoreceptor cell death.

LEI/L-DNase II is the key protein of a caspase-independent pathway activated by serine proteases. LEI (Leukocyte elastase inhibitor), L-DNase II precursor, is a member of the clade B serpins (also called serpin b1). In its native conformation it inhibits several intracellular proteases and has an anti-apoptotic activity. Following a metabolic stress and the increase of protease activity in the cell, LEI is cleaved and transformed into L-DNase II (LEI-derived DNase II). This transformation is due to a conformational modification that exposes a nuclear localization signal and an endonuclease active site. In this paper we show that LEI can bind the exportin Crm1, and we identify on LEI a nuclear export signal involved in the control of LEI/L-DNase II nuclearization in healthy cells. Point mutation of this site increases the accumulation of the molecule in the nucleus and triggers cell death.

Ocular opacity, associated with reluctance to move and inability to feed properly, was observed in approximately 1% of all newly hatched females from several related flocks of Mulard ducks. A 5-week follow-up study of 10 1-day-old affected females was performed, and they were compared with 10 control animals. Clinical, ocular and ultrasonographic examinations, and a complete necropsy of two animals per group with histological examination of the eye, were performed weekly. A bilateral immature cortical anterior cataract was diagnosed at ocular examination and confirmed by ultrasonography in affected ducks. Dyscoria was occasionally observed in affected animals. Severe cataract, with Morgagnian globules, severe anterior fibre liquefaction and disorganization were observed by photonic microscopy. No retinal or choroidal lesions were observed. No progression or repair of ultrasonographic and microscopic lesions could be detected during the 5 weeks of examination. The female predisposition for the ocular lesions suggests a congenital sex-linked recessive cataract.

Leukocyte elastase inhibitor (LEI) is a cytosolic component of lung macrophages and blood leukocytes that inhibits neutrophil elastase. LEI is a member of the serpin superfamily, these proteins, mostly protease inhibitors, are thought to undergo a conformational change upon complex formation with proteinase that involves partial insertion of the hinge region of the reactive centre loop into a beta-sheet of the inhibitor. In this work three mutations were produced in the hinge region of elastase inhibitor that abolish the inhibition activity of LEI and transform the protein in a substrate of the elastase. This result demonstrates that the inhibitory mechanism of serpin is common to LEI.

Evidence for fibroblast growth factor receptors in the central nervous system has only been obtained using autoradiographic localization of fibroblast growth factor binding sites and messenger RNA. To clarify those neuronal functions that are regulated by fibroblast growth factor receptors, we have localized immunocytochemically the fibroblast growth factor receptor protein in bovine retina, a neural tissue of well-defined structure and function. The extracellular domain of the gene product referred to as fibroblast growth factor receptor 2 was expressed genetically in bacteria to obtain a polyclonal antibody. Positive staining was confined almost exclusively to the synaptic and optic fiber layers. Such a specific association suggests a role for this receptor in modulation of synaptic terminals and ganglion cell axons of the optic nerve, especially with respect to glutamate release.

Dans la rétine, couche neurosensorielle de l’œil, les photorécepteurs transforment le signal lumineux en influx nerveux interprétable par le cerveau. Malgré sa spécialisation dans le traitement des signaux lumineux, la rétine peut subir des dommages, à la suite d’une exposition excessive à la lumière ; on parle alors de phototoxicité rétinienne. Ces dernières années, l’apparition de dispositifs d’éclairage riches en longueurs d’onde de forte énergie (ce que l’on nomme lumière bleue), remet le problème de la phototoxicité rétinienne à l’ordre du jour. Nous discutons des pathologies oculaires induites par la lumière et de la possible influence des nouvelles technologies d’éclairage sur notre santé visuelle.

We developed a mathematical model to describe healing processes in bovine corneal endothelial (BCE) cells in culture, triggered by mechanical wounds with parallel edges. Previous findings from our laboratory show that, in these cases, BCE monolayers exhibit an approximately constant healing velocity. Also, that caspase-dependent apoptosis occurs, with the fraction of apoptotic cells increasing with the distance traveled by the healing edge. In addition, in this study we report the novel findings that, for wound scratch assays performed preserving the basal extracellular matrix: i) the healing cells increase their en face surface area in a characteristic fashion, and ii) the average length of the segments of the cell columns actively participating in the healing process increases linearly with time. These latter observations preclude the utilization of standard traveling wave formalisms to model wound healing in BCE cells. Instead, we developed and studied a simple phenomenological model based on a plausible formula for the spreading dynamics of the individual healing cells, that incorporates original evidence about the process in BCE cells. The model can be simulated to: i) obtain an approximately constant healing velocity; ii) reproduce the profile of the healing cell areas, and iii) obtain approximately linear time dependences of the mean cell area and average length of the front active segments per column. In view of its accuracy to account for the experimental observations, the model can also be acceptably employed to quantify the appearance of apoptotic cells during BCE wound healing. The strategy utilized here could offer a novel formal framework to represent modifications undergone by some epithelial cell lines during wound healing.

Acidic and basic fibroblast growth factors (FGFs) influence cell division and differentiation in retina cells. Their effects are thought to be mainly mediated through stimulation of a specific membrane receptor and subsequent generation of an intracellular signal pathway. In this study, we purified a FGF receptor of 130 kDa from bovine neural retina using wheat germ agglutinin affinity chromatography followed by FGF-affinity chromatography. The isolated receptor showed ligand binding activity with dissociation constants of 0.8 nM and 2 nM for aFGF and bFGF, respectively. Furthermore, binding of aFGF and bFGF to purified receptor resulted in self-phosphorylation, demonstrating that the isolated receptor had an unaltered intrinsic kinase activity.

La LEI <i>(leukoçyte elastase inhibitor)<i/> est une protéine appartenant à la superfamille des serpines, qui regroupe des inhibiteurs de protéases d’un type particulier impliqués dans de nombreux processus physiologiques et pathologiques. L’originalité de leur mode d’action réside dans la modification conformationnelle qu’ils subissent après avoir été reconnus comme substrats puis coupés par la protéase qu’ils inhibent, celle-ci étant alors piégée dans un complexe particulièrement stable. Au cours de l’évolution, plusieurs serpines ont mis à profit cette modification conformationnelle pour acquérir une nouvelle fonction, sans rapport avec la première. Ainsi nous avons montré que la LEI acquiert une activité DNase après coupure par l’élastase, activité mise évidence au cours de processus apoptotiques. Nous montrons ici comment l’analyse structurale de la molécule est en accord avec cette deuxième fonction, en révélant des structures compatibles avec un site actif DNase et une translocation nucléaire.

Macular edema is an increase in volume of the central area of the retina, responsible for visual acuity. Visual symptoms handicap the lives of millions of patients with macular edema secondary to chronic and sometimes acute retinal disease. Proteins that neutralize the vascular endothelial growth factor (VEGF) pathway or glucocorticoids, at the cost of repeated intraocular injections over years, limit visual symptoms. A better understanding of why and how edema forms and how therapeutic molecules exert an anti-edematous effect will help prevent this disabling and blinding retinal complication from occurring.

L’œdème maculaire est une augmentation de volume de la macula, zone centrale de la rétine, responsable de l’acuité visuelle. Des symptômes visuels handicapent la vie de millions de patients atteints d’œdème maculaire secondaire à une maladie chronique et parfois aiguë de la rétine. Les protéines qui neutralisent la voie du facteur de croissance de l’endothélium vasculaire (VEGF) ou les glucocorticoïdes, au prix d’injections intraoculaires répétées pendant des années, limitent les symptômes visuels. Mieux comprendre pourquoi et comment l’œdème se forme et comment les molécules thérapeutiques exercent un effet anti-œdémateux permettra de mieux prévenir la survenue de cette complication rétinienne handicapante et cécitante.

Il est actuellement admis que l'induction de l'apoptose est suivie par la liberation d'elements mitochondriaux, l'activation de proteases et d'endonucleases. Dans ce processus, l'activation des proteases, et en particulier des caspases, est consideree comme l'etape decisive. L'activation des endonucleases est concue comme une etape finale de degradation de l'ADN devenu accessible. Or, dans l'apoptose des neurones retiniens, induite pendant le developpement embryonnaire, et dans la differenciation terminale des cellules fibres du cristallin, nous avons decrit l'augmentation de l'activite d'une DNase de type II comme etant une etape critique. L'etude moleculaire de cette enzyme, presente dans de nombreux tissus, a montre qu'elle est synthetisee sous la forme d'une proteine de la famille des serpines, la leukocyte elastase inhibitor (LEI), qui a une activite anti-protease. Lors de l'activation de l'apoptose, la LEI se transforme, par modification post-traductionnelle en L-DNase II, une proteine sans activite antiprotease mais avec une activite endonuclease. Il est donc possible que, lors du declenchement de l'apoptose, cette voie LEI-L-DNase II agisse comme un switch qui entraine, a la fois l'activation des endonucleases par apparition dans la cellule de la L-DNase II et l'activation des proteases par liberation de leur inhibition, Dans ce cas, l'evenement decisif serait situe a ce niveau et non au niveau des caspases.

De nombreuses etudes ont mis en evidence le role de la lumiere comme un facteur potentiellement deletere pour la retine humaine et la vision. Ainsi, l’exposition intense au soleil, comme c’est le cas pour les marins et les guides de haute montagne, augmente le risque de degenerescence maculaire liee a l’âge (DMLA). Cette maladie, qui apparait a partir de 65 ans, affecte la vision centrale, alterant la vision des details et la lecture.La directive europeenne pour l’ecologie [...]

L’injection intra-vitréenne de triamcinolone acétonide est réalisée en pratique courante pour traiter des affections oculaires d’origine diverses. Des travaux pré cliniques ont montré que la triamcinolone acétonide exerçait in vitro et in vivo une toxicité rétinienne, alors que d’autres études n’ont pas mis en évidence de toxicité fonctionnelle. Les mécanismes d’une éventuelle toxicité restent imparfaitement compris. Le but de ce travail a été d’analyser de façon systématique les effets de différents glucocorticoïdes in vitro et in vivo sur différents paramètres de la survie cellulaire et les mécanismes de toxicité. Nous avons injecté 1 microl (40 microg) de Kenacort retard dans le vitré de rat Lewis et avons analysé les yeux 24 heures et 8 jours plus tard en ultra-structure et par détection de l’apoptose par l’activation des caspases, du cytochrome C, du facteur AIF, et par la méthode TUNEL. Nous avons également recherché la présence d’autophagie. In vitro, sur des cultures de lignées cellules d’épithélium pigmentaire de la rétine humaine et sur des cultures primaires d’épithélium pigmentaire de la rétine et de cellules gliales de Müller de rat (RMG), nous avons testé les effets à différentes doses de triamcinolone acétonide et d’autres glucocorticoïdes et avons également recherché les différents marqueurs d’apoptose, de nécrose et d’autres formes atypiques de mort cellulaire. Nous avons évalué les effets d’excipient du Kenacort et des cristaux de triamcinolone acétonide sur la toxicité. L’analyse ultra structurale de la rétine après injection intra-vitréenne met en évidence une souffrance de l’épithélium pigmentaire de la rétine et des cellules gliales mais pas d’altération des photorécepteurs. Aucun des marqueurs d’apoptose n’est positif. In vitro, la triamcinolone acétonide entraîne une réduction de la survie des cellules gliales plus importante que celle observée sur les cellules d’épithélium pigmentaire de la rétine. Les formes de glucocorticoïdes les plus hydrophobes sont les plus toxiques et l’excipient par lui-même n’entraîne pas de mort cellulaire. La toxicité observée met en jeu des mécanismes de mort atypique par paraptose, comme cela a été observé dans certains neurones. Le type de mort que nous décrivons ici n’est pas détecté par les méthodes de toxicologie classique, expliquant que d’autres études ont observé des signes de toxicité sans pouvoir les caractériser. Une toxicité directe de la triamcinolone acétonide est observée sur la rétine par un mécanisme non classique de mort cellulaire. D’autres glucocorticoïdes sont mieux tolérés et pourraient efficacement être utilisés en limitant les risques qui se cumulent avec la répétition des injections.

La directive européenne pour l’écologie incite à l’amélioration des performances énergétiques des produits d’éclairage domestique. De ce fait elle a adopté la suppression des ampoules incandescentes et leur remplacement par des ampoules fluo-compactes ou des diodes électroluminescentes, « Light Emitting Diodes » (LED). Du point de vue énergétique cette décision est indiscutable. Cependant, les risques potentiels représentés par ces nouvelles sources lumineuses ont fait l’objet d’un avis réservé émis par l’ANSES indiquant le besoin d’études approfondies sur la question de la photo toxicité des LED sur la rétine. En effet, ces dispositifs présentent une forte luminance et un spectre d’émission avec un déséquilibre spectral vers les faibles longueurs d’onde (lumière bleue) ce qui expose la rétine à des rayonnements dangereux. Ainsi, « The Beaver Dam Eye Study », par exemple, a montré que l’exposition à la lumière du soleil pouvait être un facteur de risque pour les stades précoces de la dégénérescence maculaire liée à l’âge (DMLA). Nous avons montré, chez le rat, que les LED sont plus toxiques pour la rétine que d’autres sources de lumière et que ceci est lié à la composante bleue des LED. Nous avons montré une mort des photorécepteurs mais aussi des altérations au niveau de l’épithélium pigmentaire, des altérations qui risquent de devenir dramatiques lors d’expositions répétées. The European directive for ecology encourages the improvement of the energy performance of home lighting products. As a result, it has adopted the suppression of incandescent bulbs and their replacement with compact fluorescent bulbs or light emitting diodes (LED). From an energetic point of view this decision is indisputable. However, the potential risks represented by these new light sources were the subject of a reserved opinion issued by ANSES indicating the need for in-depth studies on the retinal phototoxicity of LED. Indeed, these devices have a high luminance and an emission spectrum with a spectral imbalance towards low wavelengths (blue light) which exposes the retina to the so called “ blue light hazard ”. Moreover, studies such as “ The Beaver Dam Eye Study ” have shown that exposure to sunlight can be a risk factor for the early stages of age-related macular degeneration (AMD). We have shown, using a rat model, that LED is more toxic to the retina than other light sources and this depends on their blue component. We have shown not only death of photoreceptors but also damages at the retinal pigmented epithelium, damages that risk having dramatics effects after repeated exposures.

The authors regret there were several errors in Figs. 4, 6, 8, 9 and 10. The correct versions, and the errors, are given below. The correct version of Fig. 4 appears below: The incorrect version of Fig. 4 appeared as: The correct version of Fig. 6 includes labels in panel A. Fig. 6 appears as follows: The incorrect version of Fig. 6 appeared as: The correct version of Fig. 8 includes amendments to the x-axis labels of panels A and C: The incorrect version of Fig. 8 appeared as: The correct version of Fig. 9 includes amendments to the x-axis labels of panels A, B and C: The incorrect version appeared as follows: The correct version of Fig. 10 includes an amendment to the x-axis of Panel B: The incorrect version of Fig. 10 appeared as: The authors would like to apologise for any inconvenience caused. Cell Death Mechanisms in a Mouse Model of Retinal Degeneration in Spinocerebellar Ataxia 7NeuroscienceVol. 400PreviewSpinocerebellar ataxia type 7 (SCA7) belongs to the heterogeneous family of neurodegenerative disorders. Clinically, SCA7 is characterized by a progressive ataxia and a gradual vision loss leading to blindness. It is a rare disease caused by an unstable CAG expansion in the ATXN7 gene leading to the expression of ataxin7 with an abnormal polyglutamine tract (polyQ) (David et al., 1997). Nine polyQ neurodegenerative disorders have been described so far, including SCA1, SCA2, SCA6, SCA7, SCA 17, Huntington's disease, spinal and bulbar muscular atrophy (SBMA) and dentatorubralpallidoluysian atrophy (DRPLA). Full-Text PDF

RESUME Contrairement aux autres sources de pollution, l’agression lumineuse par les nouvelles sources de lumiere artificielle sont moins souvent denoncees. Et cependant, la retine y est constamment exposee. A tout le moins une photo-protection par des verres filtrant les rayonnements nocifs pour la retine pourrait etre a recommander, tout particulierement chez les enfants et apres intervention de la cataracte.