INIBIZIONE DELL' ALDOSO REDUTTASI DA EMIACETALI

L'aldoso reduttasi (alditol:NADP oxidoreductase, EC 1.1.1.21) (AR) è un enzima citosolico monomerico, appartenente alla famiglia delle aldo-cheto reduttasi, in grado di agire su aldeidi sia aromatiche che alifatiche. Esso catalizza la prima tappa (che è anche quella limitante) della via dei polioli, che consiste nella riduzione NADPH-dipendente del glucosio in sorbitolo. La seconda tappa della suddetta via, catalizzata dalla sorbitolo deidrogenasi, converte il sorbitolo in fruttosio, in una reazione NAD+-dipendente. In condizioni fisiologiche, la via dei polioli coinvolge meno del 3% del glucosio totale presente nella cellula, poiché la quasi totalità dello zucchero viene fosforilato dalla esochinasi, che lo convoglia nella via glicolitica. In condizioni di iperglicemia, come nel caso dei soggetti diabetici, una quota di di glucosio diventa substrato dell'aldoso reduttasi e il flusso di glucosio attraverso la via dei polioli può interessare percentuali fino al 30%. del glucosio totale L’aumento di flusso attraverso la via dei polioli è considerato dannoso per una serie di ragioni: la prima riguarda l'accumulo di sorbitolo all'interno della cellula, che causa uno squilibrio osmotico tale da richiamare acqua dall'esterno, con gravi alterazioni del normale equilibrio elettrolitico. La seconda riguarda l’aumento della produzione di fruttosio che , essendo un agente glicante addirittura migliore del glucosio stesso, può indurre fenomeni di glicazione proteica. Infine, l’aumento del consumo di NADPH ad opera dell’AR determina una riduzione delle difese antiossidanti della cellula. Queste alterazioni metaboliche correlate con l’aumentato flusso attraverso la via dei polioli sono considerate responsabili dell’insorgenza delle cosiddette complicanze diabetiche, che includono cataratta, neuropatie, nefropatie e retinopatie. Per questo l’AR è da tempo considerata un bersaglio per l’identificazione di inibitori che possano portare allo sviluppo di farmaci contro le complicanze del diabete. Sebbene il glucosio rappresenti uno dei substrati di AR più rilevanti dal punto di vista fisiopatologico, la reazione di riduzione catalizzata dall’enzima mostra ancora alcuni aspetti non completamente chiariti. Tra questi, l’apparente cooperatività negativa che è stata osservata con questo substrato e che è stata ascritta all’esistenza di più forme enzimatiche. Il lavoro sperimentale riportato in questa tesi ha fatto uso sia di AR ricombinante umana. I risultati ottenuti indicano che le strutture emiacetaliche degli aldosi esercitano un effetto modulatorio sulla riduzione della forma aldeidica libera catalizzata dall’AR e che questo effetto è in grado di dare spiegazione dell’apparente negatività cooperativa osservata per l’enzima

Role of Glutathione (GSH) in hypoglicaemia-induced cell death

Le 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. -- 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

GSH-Independent Induction of ER Stress during Hypoglycaemia in the Retinal Cells of Mice

Glucose is one of the most important metabolic substrates of the retina, and glycaemic imbalances can lead to serious side effects, including retinopathy. We previously showed that hypoglycaemia induces retinal cell death in mice, as well as the implication of glutathione (GSH) in this process. This study aimed to analyse the role of low glucose-induced decrease in GSH levels in endoplasmic reticulum (ER) stress. We cultured 661W photoreceptor-like cells under various glucose conditions and analysed ER stress markers at the mRNA and protein levels. We used the ERAI ("ER stress-activated indicator") mouse model to test ER stress in both ex vivo, on retinal explants, or in vivo, in mice subjected to hypoglycaemia. Moreover, we used buthionine sulfoximine (BSO) and glutamate cysteine ligase (Gclm)-KO mice as models of low GSH to test its effects on ER stress. We show that the unfolded protein response (UPR) is triggered in 661W cells and in ERAI mice under hypoglycaemic conditions. Low GSH levels promote cell death, but have no impact on ER stress. We concluded that low glucose levels induce ER stress independently of GSH levels. Inhibition of ER stress could prevent neurodegeneration, which seems to be an early event in the pathogenesis of diabetic retinopathy