18 research outputs found
Role of Glutathione (GSH) in hypoglicaemia-induced cell death
No full textLe diabète est une maladie dont le fardeau économique est important, avec un nombre de personnes diagnostiquées qui devrait atteindre 700 millions en 2045. Les patients diabétiques de type 1 et de type 2 développent des pathologies secondaires, dont la rétinopathie diabétique (RD), qui est l'une des principales causes de cécité acquise dans le monde. La rétinopathie diabétique est une maladie qui a été depuis longtemps associée à l'effet d'une hyperglycémie prolongée, principalement sur les cellules vasculaires et les péricytes. Cependant, il a récemment été proposé que la neurodégénérescence (c'est-à- dire la mort des cellules neuronales) joue un rôle important dans l’établissement de cette maladie. En effet, certaines études indiquent que la mort des cellules rétiniennes pourrait intervenir avant les dommages vasculaires typiques des rétinopathies. Par conséquent, une meilleure compréhension et un contrôle des mécanismes de mort des cellules neuronales pourraient être bénéfiques afin de prévenir l'apparition et/ou la progression de la rétinopathie diabétique.
La production de radicaux libres, produits pendant la respiration mitochondriale, joue un rôle prépondérant dans ces mécanismes. le niveaux de radicaux libres sont généralement bien régulée par les défenses antioxydantes de la cellule. Le glutathion (GSH) est l'un des antioxydants les plus importants de la cellule. Dans la rétine ce sont les photorécepteurs qui consomment la fraction de glucose la plus élevée, car ils ont un métabolisme très actif. Ils sont de ce fait le plus sensible aux radicaux libres. Ne disposant pas de réserves de glycogène l’ensemble du glucose dont ils ont besoin doit être fourni par les vaisseaux rétiniens, ce qui les rend sensible à une diminution du glucose. En 2011, une étude du groupe du Dr Roduit a montré qu’une hypoglycémie de 5 heures induisait la mort des cellules de la rétine qui est corrélée à une diminution des taux de GSH. Une observation similaire a été faite lors de la culture à bas glucose (1mM) des cellules photoréceptrices 661W ; en plus d’une augmentation de la production de radicaux libres. L’administration de GSH éthyl ester était capable de restaurer le pool de GSH et de bloquer l'apoptose ; au contraire, une diminution du pool de GSH était capable d’induire la mort cellulaire.
Dans mon travail de thèse, nous avons analysé les effets de l’hypoglycémie sur le stress du réticulum endoplasmique (RE). Nous avons montré, in vitro et in vivo, que le bas glucose induit une réponse au niveau de RE, et ceci indépendamment des niveaux de GSH de la cellule. Nous avons cherché à déterminer si la modulation stress du RE permettait de protéger les photorécepteurs contre la mort cellulaire. Cette hypothèse fait l’objet d’une étude actuellement en cours dans notre laboratoire. De plus, nous nous sommes intéressé au rôle spécifique du GSH dans notre modèle en utilisant des antioxydants (N-acétylcystéine et N-acétylcystéine amide) afin de restaurer les niveaux de GSH et de prévenir la mort cellulaire induite par le bas glucose. Les deux composés utilisés sont toxique pour les cellules
photoréceptrices à bas glucose, Nous essayons de mieux comprendre les modifications métabolomiques engendrées par le bas glucose afin de déterminer la toxicité surprenante de ces composés.
Nous pensons que la protection des photorécepteurs contre la mort cellulaire induite par l'hypoglycémie peut représenter une fenêtre thérapeutique intéressante pour protéger la vue des patients atteint de RD, en particulier pour ceux qui sont dans les premières phases de cette maladie.
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Diabetes is a disease with a significant economic burden, with an expected number of diagnosed people to rise until 700 million in 2045. Both type 1 and type 2 diabetic patients are at risk of developing secondary side effects, including diabetic retinopathy (DR), which is one of the leading cause of acquired blindness worldwide. Diabetic retinopathy is a disease that has long been associated with the effect of prolonged hyperglycaemia, primarily on vascular cells and pericytes. However, it has recently been proposed that neurodegeneration (i.e. neural cell death) plays an important role in the establishment of the disease. Indeed some studies showed that retinal cell death might happen earlier than vascular damages. Therefore, blocking or delaying neural cell death mechanisms might be beneficial in preventing the onset and/or progression of diabetic retinopathy. It has been recently proposed that the most important molecular mechanism involved in cell death are the increased flux of glucose in the polyol and hexosamine pathways, the increase of advanced glycation end products production and the protein kinase C activation.
All these mechanisms share the unifying reactive oxygen species (ROS) production as a harmful consequence for both vascular and neural cells. ROS are normally produced during mitochondrial respiration: however, usually ROS production is well counteracted by the antioxidant defenses of the cell. Glutathione (GSH) is one of the most important antioxidant defense in the cells. Indeed, the cells which are the most damaged by ROS production are the most metabolically active: in the retina, the highest glucose fraction is consumed by photoreceptors. Despite the huge amount of glucose required for their homeostasis, photoreceptors do not have glycogen reserves. Therefore, all the glucose they need must be supplied by retinal vessels, and this makes them directly damaged in case of glucose decrease. In 2011, a study from Dr.Roduit’s group showed that a 5-hour hypoglycaemia induced retinal cell death which is correlated with a decrease in GSH levels. A similar observation was made when 661W photoreceptor cells were cultured at low glucose (1mM); in addition to an increase in free radical production. Administration of GSH ethyl ester was able to restore the GSH pool and block apoptosis; in contrast, a decrease in the GSH pool was able to induce cell death.
In addition, we addressed the specific role of GSH in our model by using anti-oxidants (N-acetylcysteine and N-acetylcysteine amide) to restore GSH levels and prevent low-glucose induced cell death. Both compounds used are toxic to low photoreceptor cells cultured at low glucose. We are trying to better understand the metabolomic changes caused by low glucose in order to determine the surprising toxicity of these compounds.
In my thesis work, we assessed the effects of hypoglycemia on endoplasmic reticulum (ER) stress. We showed both, in vitro and in vivo, that ER stress was induced by low glucose and this independently of
GSH levels. We sought to investigate whether ER stress modulation is able to protect photoreceptors. This hypothesis is currently under investigation in our laboratory.
We believe that protecting photoreceptors from hypoglycaemia-induced cell death may represent an interesting therapeutic window to protect the eyesight of patients with DR, especially for those in the early phases of this disease
Mutations in the DNA-binding domain of NR2E3 affect in vivo dimerization and interaction with CRX.
No full textBACKGROUND:NR2E3 (PNR) is an orphan nuclear receptor essential for proper photoreceptor determination and differentiation. In humans, mutations in NR2E3 have been associated with the recessively inherited enhanced short wavelength sensitive (S-) cone syndrome (ESCS) and, more recently, with autosomal dominant retinitis pigmentosa (adRP). NR2E3 acts as a suppressor of the cone generation program in late mitotic retinal progenitor cells. In adult rod photoreceptors, NR2E3 represses cone-specific gene expression and acts in concert with the transcription factors CRX and NRL to activate rod-specific genes. NR2E3 and CRX have been shown to physically interact in vitro through their respective DNA-binding domains (DBD). The DBD also contributes to homo- and heterodimerization of nuclear receptors. METHODOLOGY/PRINCIPAL FINDINGS:We analyzed NR2E3 homodimerization and NR2E3/CRX complex formation in an in vivo situation by Bioluminescence Resonance Energy Transfer (BRET(2)). NR2E3 wild-type protein formed homodimers in transiently transfected HEK293T cells. NR2E3 homodimerization was impaired in presence of disease-causing mutations in the DBD, except for the p.R76Q and p.R104W mutant proteins. Strikingly, the adRP-linked p.G56R mutant protein interacted with CRX with a similar efficiency to that of NR2E3 wild-type and p.R311Q proteins. In contrast, all other NR2E3 DBD-mutant proteins did not interact with CRX. The p.G56R mutant protein was also more effective in abolishing the potentiation of rhodospin gene transactivation by the NR2E3 wild-type protein. In addition, the p.G56R mutant enhanced the transrepression of the M- and S-opsin promoter, while all other NR2E3 DBD-mutants did not. CONCLUSIONS/SIGNIFICANCE:These results suggest different disease mechanisms in adRP- and ESCS-patients carrying NR2E3 mutations. Titration of CRX by the p.G56R mutant protein acting as a repressor in trans may account for the severe clinical phenotype in adRP patients
MAP kinase pathways in UV-induced apoptosis of retinal pigment epithelium ARPE19 cells
No full textThe retinal pigment epithelium (RPE) is constantly exposed to external injuries which lead to degeneration, dysfunction or loss of RPE cells. The balance between RPE cells death and proliferation may be responsible for several diseases of the underlying retina, including age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Signaling pathways able to control cells proliferation or death usually involve the MAPK (mitogen-activated protein kinases) pathways, which modulate the activity of transcription factors by phosphorylation. UV exposure induces DNA breakdown and causes cellular damage through the production of reactive oxygen species (ROS) leading to programmed cell death. In this study, human retinal pigment epithelial cells ARPE19 were exposed to 100 J/m(2) stop of UV-C and MAPK pathways were studied. We first showed the expression of the three major MAPK pathways. Then we showed that activator protein-1 (AP-1) was activated through phosphorylation of cJun and cFos, induced by JNK and p38, respectively. Specific inhibitors of both kinases decreased their respective activities and phosphorylation of their nuclear targets (cJun and cFos) and reduced UV-induced cell death. The use of specific kinases inhibitors may provide excellent tools to prevent RPE apoptosis specifically in RPE diseases involving ROS and other stress-related compounds such as in AMD.S
Acute hypoglycemia induces retinal cell death in mouse.
No full textBACKGROUND: Glucose is the most important metabolic substrate of the retina and maintenance of normoglycemia is an essential challenge for diabetic patients. Glycemic excursions could lead to cardiovascular disease, nephropathy, neuropathy and retinopathy. A vast body of literature exists on hyperglycemia namely in the field of diabetic retinopathy, but very little is known about the deleterious effect of hypoglycemia. Therefore, we decided to study the role of acute hypoglycemia in mouse retina. METHODOLOGY/PRINCIPAL FINDINGS: To test effects of hypoglycemia, we performed a 5-hour hyperinsulinemic/hypoglycemic clamp; to exclude an effect of insulin, we made a hyperinsulinemic/euglycemic clamp as control. We then isolated retinas from each group at different time-points after the clamp to analyze cells apoptosis and genes regulation. In parallel, we used 661W photoreceptor cells to confirm in vivo results. We showed herein that hypoglycemia induced retinal cell death in mouse via caspase 3 activation. We then tested the mRNA expression of glutathione transferase omega 1 (Gsto1) and glutathione peroxidase 3 (Gpx3), two genes involved in glutathione (GSH) homeostasis. The expression of both genes was up-regulated by low glucose, leading to a decrease of reduced glutathione (GSH). In vitro experiments confirmed the low-glucose induction of 661W cell death via superoxide production and activation of caspase 3, which was concomitant with a decrease of GSH content. Moreover, decrease of GSH content by inhibition with buthionine sulphoximine (BSO) at high glucose induced apoptosis, while complementation with extracellular glutathione ethyl ester (GSHee) at low glucose restored GSH level and reduced apoptosis. CONCLUSIONS/SIGNIFICANCE: We showed, for the first time, that acute insulin-induced hypoglycemia leads to caspase 3-dependant retinal cell death with a predominant role of GSH content
How do we make indoor environments and healthcare settings healthier?
No full text© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Microbial Biotechnology 10 (2017): 11-13, doi:10.1111/1751-7915.12430.It is now well accepted that our modern lifestyle has certain implications for our health (Schaub et al., 2006), mainly as a result of our willingness to remove ourselves from the biological diversity of our natural environments (Roduit et al., 2016), while still being drawn inextricably to interact with it (Kellert and Wilson, 1995). Much of our interaction with the biological world is shaped by our interaction with the microbiological world. The bacteria, fungi, viruses, archaea and protists that comprise the microbiome of this planet, are also key to the development and normal functioning of our bodies. Our immune system is built to shepherd our microbial exposure, ensuring that microbial organisms that we need are kept close (but not too close), and that less-desirable organisms are expelled or killed before they can do too much damage. By moving from a life interacting with nature on a regular basis, to a life in which we isolate ourselves physically from natural microbial exposure, we may have instigated one of the great plagues of the 21st century; chronic immune disorders.This manuscript was prepared in part with funding from the Alfred P Sloan Foundation
The loss of GLUT2 expression in the pancreatic β-cells of diabetic db/db mice is associated with an impaired DNA-binding activity of islet-specific trans-acting factors
No full textGLUT2 expression is reduced in the pancreatic beta-cells of several diabetic animals. The transcriptional control of the gene in beta-cells involves at least two islet-specific DNA-binding proteins, GTIIa and PDX-1, which also transactivates the insulin, somatostatin and glucokinase genes. In this report, we assessed the DNA-binding activities of GTIIa and PDX-1 to their respective cis-elements of the GLUT2 promoter using nuclear extracts prepared from pancreatic islets of 12 week old db/db diabetic mice. We show that the decreased GLUT2 mRNA expression correlates with a decrease of the GTIIa DNA-binding activity, whereas the PDX-1 binding activity is increased. In these diabetic animals, insulin mRNA expression remains normal. The adjunction of dexamethasone to isolated pancreatic islets, a treatment previously shown to decrease PDX-1 expression in the insulin-secreting HIT-T15 cells, has no effect on the GTIIa and PDX-1 DNA-binding activities. These data suggest that the decreased activity of GTIIa, in contrast to PDX-1, may be a major initial step in the development of the beta-cell dysfunction in this model of diabetes
Bis-Retinoid A2E Induces an Increase of Basic Fibroblast Growth Factor via Inhibition of Extracellular Signal-Regulated Kinases 1/2 Pathway in Retinal Pigment Epithelium Cells and Facilitates Phagocytosis.2
No full textAge-related macular degeneration (ARMD) is the leading cause of vision loss in developed countries. Hallmarks of the disease are well known; indeed, this pathology is characterized by lipofuscin accumulation, is principally composed of lipid-containing residues of lysosomal digestion. The N-retinyl-N-retinylidene ethanolamine (A2E) retinoid which is thought to be a cytotoxic component for RPE is the best- characterized component of lipofuscin so far. Even if no direct correlation between A2E spatial distribution and lipofuscin fluorescence has been established in aged human RPE, modified forms or metabolites of A2E could be involved in ARMD pathology. Mitogen-activated protein kinase (MAPK) pathways have been involved in many pathologies, but not in ARMD. Therefore, we wanted to analyze the effects of A2E on MAPKs in polarized ARPE19 and isolated mouse RPE cells. We showed that long-term exposure of polarized ARPE19 cells to low A2E dose induces a strong decrease of the extracellular signal-regulated kinases' (ERK1/2) activity. In addition, we showed that A2E, via ERK1/2 decrease, induces a significant decrease of the retinal pigment epithelium-specific protein 65 kDa (RPE65) expression in ARPE19 cells and isolated mouse RPE. In the meantime, we showed that the decrease of ERK1/2 activity mediates an increase of basic fibroblast growth factor (bFGF) mRNA expression and secretion that induces an increase in phagocytosis via a paracrine effect. We suggest that the accumulation of deposits coming from outer segments (OS) could be explained by both an increase of bFGF-induced phagocytosis and by the decrease of clearance by A2E. The bFGF angiogenic protein may therefore be an attractive target to treat ARMD
Complement Factor B Polymorphism and the Phenotype of Early Age-related Macular Degeneration
No full textPurpose: Age-related macular degeneration (AMD) has been associated with a number of polymorphisms in genes in the complement pathway. We examined the potential genotype-phenotype correlation of complement factor B (CFB) (R32Q) polymorphisms in Caucasian patients with AMD. Methods: Data from a Central European cohort of 349 patients with early AMD in at least one eye were analyzed for potential associations of the CFB (R32Q/rs641153) polymorphism with phenotypic features of early AMD. Early AMD was classified according to the International Classification and Grading System into predominant drusen size, largest drusen, drusen covered surface, central or ring-like location, peripheral drusen, and pigmentary changes. The potential association with single nucleotide polymorphisms on CFB (R32Q/rs641153) was evaluated for all patients, corrected for age, sex, and the polymorphisms of CFH (Y402H) and ARMS2 (A69S). Results: CFB (R32Q) polymorphisms showed a significant association with smaller drusen size (largest drusen <= 250 mu m, p = 0.021, predominant drusen <= 125 mu m, p = 0.016), with smaller surface covered by drusen (<= 10%; p = 0.02), and with more frequent occurrence of peripheral drusen (p = 0.007). No association was found for pigmentary changes. Conclusions: The CFB (R32Q) polymorphism was associated with AMD characterized by small drusen only, and appeared to be protective of large drusen (OR 0.48/0.45) and of larger drusen covered area (OR 0.34). Furthermore, peripheral drusen were more frequently found (OR 2.27). This result supports the role of complement components and their polymorphisms in drusen formation and may enable a better understanding of AMD pathogenesis.S
Insulin secretion is regulated by the glucose-dependent production of islet β cell macrophage migration inhibitory factor
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