Molecular characterization of signalling complexes involved in G-protein coupled receptor-induced cardiac hypertrophy

In response to pathological stresses, the heart undergoes a remodelling process associated with cardiac hypertrophy. Since sustained hypertrophy can progress to heart failure, there is an intense investigation about the intracellular signalling pathways that control cardiomyocyte growth. Accumulating evidence has demonstrated that most stimuli known to initiate pathological changes associated with the development of cardiac hypertrophy activate G protein-coupled receptors (GPCRs) including the αl-adrenergic- (αl-AR), Angiotensin II- (AT-R) and endothelin-1- (ET-R) receptors. In this context, we have previously identified a cardiac scaffolding protein, called AKAP-Lbc (Α-kinase anchoring protein), with an intrinsic Rho specific guanine nucleotide exchange factor activity, that plays a key role in integrating and transducing hypertrophic signals initiated by these GPCRs (Appert-Collin, Cotecchia et al. 2007). Activated RhoA controls the transcriptional activation of genes involved in cardiomyocyte hypertrophy through signalling pathways that remain to be characterized. Here, we identified the nuclear factor-Kappa Β (NF-κΒ) activating kinase ΙΚΚβ as a novel AKAP-Lbc interacting protein. This raises the hypothesis that AKAP-Lbc might promote cardiomyocyte growth by maintaining a signalling complex that promotes the activation of the pro-hypertrophic transcription factor NF-κΒ. In fact, the activation of NF- κΒ-dependent transcription has been detected in numerous disease contexts, including hypertrophy, ischemia/reperfusion injury, myocardial infarction, allograft rejection, myocarditis, apoptosis, and more (Hall, Hasday et al. 2006). While it is known by more than a decade that NF-κΒ is a critical mediator of cardiac hypertrophy, it is currently poorly understood how pro-hypertrophic signals controlling NF-κΒ transcriptional activity are integrated and coordinated within cardiomyocytes. In this study, we show that AKAP-Lbc and ΙΚΚβ form a transduction complex in cardiomyocytes that couples activation of αl-ARs to NF-κB-mediated transcriptional reprogramming events associated with cardiomyocyte hypertrophy. In particular, we can show that activation of ΙΚΚβ within the AKAP-Lbc complex promotes NF-κB-dependent production of interleukine-6 (IL-6), which, in turn, enhances foetal gene expression. These findings indicate that the AKAP-Lbc/ΙΚΚβ complex is critical for selectively directing catecholamine signals to the induction of cardiomyocyte hypertrophy.

Effects of triamcinolone acetonide on vessels of the posterior segment of the eye.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

The Effect Of Induced Intraocular Straylight (cataract Simulation) On Sensitivity Measures Of Standard Automated Perimetry, Pulsar Perimetry And Moorfields Motion Displacement Test

Purpose: To investigate the effect of incremental increases in intraocular straylight on threshold measurements made by three modern forms of perimetry: Standard Automated Perimetry (SAP) using Octopus (Dynamic, G-Pattern), Pulsar Perimetry (PP) (TOP, 66 points) and the Moorfields Motion Displacement Test (MDT) (WEBS, 32 points).Methods: Four healthy young observers were recruited (mean age 26yrs [25yrs, 28yrs]), refractive correction [+2 D, -4.25D]). Five white opacity filters (WOF), each scattering light by different amounts were used to create incremental increases in intraocular straylight (IS). Resultant IS values were measured with each WOF and at baseline (no WOF) for each subject using a C-Quant Straylight Meter (Oculus, Wetzlar, Germany). A 25 yr old has an IS value of ~0.85 log(s). An increase of 40% in IS to 1.2log(s) corresponds to the physiological value of a 70yr old. Each WOFs created an increase in IS between 10-150% from baseline, ranging from effects similar to normal aging to those found with considerable cataract. Each subject underwent 6 test sessions over a 2-week period; each session consisted of the 3 perimetric tests using one of the five WOFs and baseline (both instrument and filter were randomised).Results: The reduction in sensitivity from baseline was calculated. A two-way ANOVA on mean change in threshold (where subjects were treated as rows in the block and each increment in fog filters was treated as column) was used to examine the effect of incremental increases in straylight. Both SAP (p<0.001) and Pulsar (p<0.001) were significantly affected by increases in straylight. The MDT (p=0.35) remained comparatively robust to increases in straylight.Conclusions: The Moorfields MDT measurement of threshold is robust to effects of additional straylight as compared to SAP and PP.

Bitumens in the late Variscan hydrothermal vein-type uranium deposit of Pribram, Czech Republic: Sources, radiation-induced alteration, and relation to mineralization

The late Variscan (275-278 Ma) Pribram uranium deposit is one of the largest known accumulations of uraniferous bitumens in hydrothermal veins. The deposit extends along the northwestern boundary of the Central Bohemian pluton (345-335 Ma) with low-grade metamorphosed Late Proterozoic and unmetamorphosed Cambrian rocks. From a net uranium production of 41,742 metric tons (t), more than 6,000 t were extracted from bitumen-uraninite ores during 43 years of exploration and mining. Three morphological varieties of solid bitumen are recognized: globular, asphaltlike, and cokelike. While the globular bitumen is uranium free, the other two types are uraniferous. The amount of bitumen in ore veins gradually decreases toward the contact with the plutonic body and increases with depth. Two types of bitumen microtextures are recognized using high-resolution transmission electron microscopy: amorphous and microporous, the former being less common in uraniferous samples. A lower Raman peak area ratio (1,360/1,575 cm(-1)) in mineralized bitumens (0.9) compared with uranium-free samples (2.0) indicates a lower degree of microtextural organization in the latter The H/C and O/C atomic ratios in uranium-free bitumens (0.9-1.1 and 0.09, respectively) are higher than those in mineralized samples (H/C = 0.3-0.8, O/C = 0.03-0.09). The chloroform extractable matter yield is Very low in uranium-free bitumens (0.30-0.35% of the total organic carbon,TOC) and decreases with uranium content increase. The extracted solid uraniferous bitumen infrared spectra show depletion in aliphatic CH2 and CH3 groups compared to uranium-free samples. The concentration of oxygen-bearing functional groups relative to aromatic bonds in the IR spectra of uranium-free and mineralized bitumen, however, do not differ significantly. C-13 NMR confirmed than the aromaticity of a uraniferous sample is higher (F-ar = 0.61) than in the uranium-free bitumen (F-ar = 0.51). Pyrolysates from uraniferous and nonuraniferous bitumens do not differ significantly, being predominantly cresol, alkylphenols, alkylbenzenes, and alkylnaphthalenes. The liquid pyrolysate yield decreases significantly with increasing uranium content. The delta(13)C Values of bulk uranium-free bitumens and low-grade uraniferous, asphaltlike bitumens range from -43.6 to 52.3 per mil. High-grade, cokelike, uraniferous bitumens are more C-13 depleted (54.5 to -58.4 parts per thousand). In contrast to the very light isotopic ratios of the high-grade uraniferous cokelike bitumen bulk carbon, the individual n-alkanes and isoprenoids (pristane and phytane) extracted from the same sample are significantly C-13 enriched. The isotopic composition of the C13-24 n-alkanes extracted from the high-grade uraniferous sample (delta(13)C = -28.0 to 32.6 parts per thousand) are heavier compared with the same compounds in a uranium-free sample (delta(13)C = 31.9 to 33.8 parts per thousand). It is proposed that the bitumen source was the isotopically light (delta(13)C = 35.8 to 30.2 parts per thousand) organic matter of the Upper Proterozoic host rocks that were pyrolyzed during intrusion of the Central Bohemian pluton. The C-13- depleted pyrolysates were mobilized from the innermost part of the contact-metamorphic aureole, accumulated in structural traps in less thermally influenced parts of the sedimentary complex and were later extracted by hydrothermal fluids. Bitumens at the Pribram deposit are younger than the main part of the uranium mineralization and were formed through water-washing and radiation-induced polymerization of both the gaseous and liquid pyrolysates. Direct evidence for pyrolysate reduction of uranium in the hydrothermal system is difficult to obtain as the chemical composition of the original organic fluid phase was modified during water-washing and radiolytic alteration. However, indirect evidence-e.g., higher O/C atomic ratios in uranium-free bitumens (0.1) relative to the Upper Proterozoic source rocks (0.02-0.05), isotopically very light carbon in associated whewellite (delta(13)C = 31.7 to -28.4 parts per thousand), and the striking absence of bitumens in the pre-uranium, hematite stage of the mineralization-indicates that oxidation of organic fluids may have contributed to lowering of aO(2) and uraninite precipitation.

Fas-associated death domain protein (FADD) and caspase-8 mediate up-regulation of c-Fos by Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via a FLICE inhibitory protein (FLIP)-regulated pathway.

Fas, a death domain-containing member of the tumor necrosis factor receptor family and its ligand FasL have been predominantly studied with respect to their capability to induce cell death. However, a few studies indicate a proliferation-inducing signaling activity of these molecules too. We describe here a novel signaling pathway of FasL and the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) that triggers transcriptional activation of the proto-oncogene c-fos, a typical target gene of mitogenic pathways. FasL- and TRAIL-mediated up-regulation of c-Fos was completely dependent on the presence of Fas-associated death domain protein (FADD) and caspase-8, but caspase activity seemed to be dispensable as a pan inhibitor of caspases had no inhibitory effect. Upon overexpression of the long splice form of cellular FADD-like interleukin-1-converting enzyme (FLICE) inhibitory protein (cFLIP) in Jurkat cells, FasL- and TRAIL-induced up-regulation of c-Fos was almost completely blocked. The short splice form of FLIP, however, showed a rather stimulatory effect on c-Fos induction. Together these data demonstrate the existence of a death receptor-induced, FADD- and caspase-8-dependent pathway leading to c-Fos induction that is inhibited by the long splice form FLIP-L.

Neutrophils and TNF: two key players in the immune response induced by "Leishmania major" infection

Summary Resolution of the inflammation is as important as its induction. In this thesis, we investigated the contributions of two prominent factors involved in inflammation, Tumour Necrosis Factor (TNF) and neutrophils. We studied their role in the resolution óf the inflammatory lesion induced by the infection with the protozoan parasite Leishmania major. In mice susceptible to infection with L. major, unhealing lesions are characterized by an elevated number and sustained presence of inflammatory neutrophils in the infected tissue, illustrating an acute inflammatory process. In contrast, mice from resistant strains, which resolve their lesions, can control the presence of neutrophils at the site of infection. Neutrophil persistence in the infected tissue may result from several events including an increased survival of neutrophils mediated by factors produced by the pathogen or the microenvironment. Following infection with L. major, the cellular composition of the inflammatory lesion differs significantly between susceptible and resistant mice and a higher proportion of macrophages is present in the lesions of resistant strains. In an attempt to clarify the factors involved in neutrophil persistence, we investigated the mechanisms modulating neutrophil cell death. We demonstrated that macrophages could induce neutrophil apoptosis in a process involving TNF. TNF is an essential cytokine with pro- and anti-inflammatory properties, which is expressed as a transmembrane protein that can be cleaved releasing the secreted form. Our data show the essential role of the transmembrane form of TNF (mTNF) in the induction of neutrophil apoptosis by macrophages, revealing macrophages and mTNF as important regulators of neutrophil apoptosis. TNF is critical in the resolution of the inflammatory lesion induced by L. major infection, and in L. major resistant strains its absence results in increased swelling of the lesions. We investigated the contribution of mTNF in the outcome of L. major infection. Our data demonstrate that following infection with L. major, mTNF is sufficient to support the resolution of the inflammatory lesion and optimal parasite killing. In addition, we show that the presence of mTNF is essential to induce neutrophil clearance in the infected tissue. While the persistence of neutrophils is deleterious for the host, we could demonstrate an early anti-inflammatory role of neutrophils. Altogether, this study demonstrates the importance of mTNF in the induction of neutrophil apoptosis, a process involved in the resolution of the inflammatory lesion induced by L. major infection. Résumé La résolution de l'inflammation est toute aussi importante que son initiation. Durant ce travail de thèse, nous avons étudié les contributions de deux facteurs importants impliqués dans l'inflammation, le TNF (Facteur Nécrosant des Tumeurs) et les neutrophiles, dans la résolution de la lésion inflammatoire induite par l'infection avec le parasite protozoaire Leishmania major. Chez les souris sensibles à l'infection avec L. major, des lésions importantes qui ne guérissent pas se développent ; celles-ci sont caractérisées par un nombre élevé et une présence soutenue de neutrophiles dans les tissus infectés, ce qui illustre un processus inflammatoire aigu. Au contraire, les souris résistantes à l'infection qui guérissent leurs lésions, sont capables de contrôler la présence des neutrophiles au site d'infection. La persistance des neutrophiles dans la lésion inflammatoire peut être la conséquence de plusieurs événements, dont une augmentation de la survie des neutrophiles induite par des facteurs produits par le pathogène ou le micro-environnement. Suite à l'infection avec L. major, la composition cellulaire de la lésion inflammatoire est significativement différente entre les souris sensibles et résistantes à l'infection, et une plus grande proportion de macrophages est présente dans les lésions des souris résistantes. Dans l'objectif de clarifier les facteurs impliqués dans la persistance des neutrophiles dans les tissus infectés par L. major, nous avons étudié les mécanismes de régulation de la mort des neutrophiles. Nous avons démontré que les macrophages pouvaient induire l'apoptose des neutrophiles dans un procédé impliquant le TNF. Le TNF est une cytokine aux propriétés pro- et anti-inflammatoires, exprimée sous une forme transmembranaire qui peut être clivée pour relâcher la forme sécrétée. Nos expériences illustrent le rôle essentiel de la forme transmembranaire du TNF (mTNF) dans l'induction de l'apoptose des neutrophiles par les macrophages. Lé TNF est une cytokine importante dans la résolution de la réaction inflammatoire induite par L. major, et chez les souris résistantes l'absence de TNF provoque des lésions inflammatoires plus importantes. Nous avons étudié la contribution du mTNF dans la résolution de l'infection avec L. major. Nos résultats démontrent que suite à une infection avec le parasite, la présence du mTNF est suffisante pour guérir la lésion inflammatoire et contrôler efficacement la réplication du parasite. De plus, le mTNF joue un rôle essentiel dans l'élimination des neutrophiles du tissu infecté. Alors que la persistance des neutrophiles est nocive pour l'hôte, nous avons montré que les neutrophiles avaient un rôle précoce anti-inflammatoire. En résumé, cette étude révèle l'importance du mTNF dans l'induction de l'apoptose des neutrophiles par les macrophages, un procédé impliqué dans la résolution de la lésion inflammatoire induite par l'infection avec L. major.

Connexin36 contributes to INS-1E cells survival through modulation of cytokine-induced oxidative stress, ER stress and AMPK activity.

Cell-to-cell communication mediated by gap junctions made of Connexin36 (Cx36) contributes to pancreatic β-cell function. We have recently demonstrated that Cx36 also supports β-cell survival by a still unclear mechanism. Using specific Cx36 siRNAs or adenoviral vectors, we now show that Cx36 downregulation promotes apoptosis in INS-1E cells exposed to the pro-inflammatory cytokines (IL-1β, TNF-α and IFN-γ) involved at the onset of type 1 diabetes, whereas Cx36 overexpression protects against this effect. Cx36 overexpression also protects INS-1E cells against endoplasmic reticulum (ER) stress-mediated apoptosis, and alleviates the cytokine-induced production of reactive oxygen species, the depletion of the ER Ca(2+) stores, the CHOP overexpression and the degradation of the anti-apoptotic protein Bcl-2 and Mcl-1. We further show that cytokines activate the AMP-dependent protein kinase (AMPK) in a NO-dependent and ER-stress-dependent manner and that AMPK inhibits Cx36 expression. Altogether, the data suggest that Cx36 is involved in Ca(2+) homeostasis within the ER and that Cx36 expression is downregulated following ER stress and subsequent AMPK activation. As a result, cytokine-induced Cx36 downregulation elicits a positive feedback loop that amplifies ER stress and AMPK activation, leading to further Cx36 downregulation. The data reveal that Cx36 plays a central role in the oxidative stress and ER stress induced by cytokines and the subsequent regulation of AMPK activity, which in turn controls Cx36 expression and mitochondria-dependent apoptosis of insulin-producing cells.

The role of Bmi1 in CNS development and in retinal degeneration

SUMMARY : The present work addresses several aspects of cell cycle regulation, cell fate specification and cell death in the central nervous system (CNS), specifically the cortex and the retina. More precisely, we investigated the role of Bmi1, a polycomb family gene required for stem cell proliferation and self-renewal, in the development of the cerebral cortex, as well as in the genesis of the retina. These data, together with studies published during the last two decades concerning cell cycle re-activation in apoptotic neurons in the CNS, raised the question of a possible link between regulation of the cell cycle during development and during retinal degeneration. 1. The effects of Bmi1 loss in the cerebral cortex : Consistently with our and others' observations on failure of Bmi9-/- stem cells to proliferate and self-renew in vitro, the Bmi9-/- cerebral cortex presented slight defects in proliferation in stem/progenitor cells compartments in vivo. This was in accordance with the pattern of Bmi1 expression in the developing forebrain. The modest proliferation defects, compared to the drastic consequences of Bmi9 loss in vitro, suggest that cell-extrinsic mechanisms may partially compensate for Bmi1 deletion in vivo during cortical histogenesis. Nevertheless, we observed a decreased proliferating activity in neurogenic regions of the adult telencephalon, more precisely in the subventricular zone, showing that Bmi1 controls neural stem/progenitor proliferation during adulthood in vivo. Our data also highlight an increased production of astrocytes at birth, and a generalized gliosis in the adult Bmi9-/- brain. Importantly, glial progenitors and astrocytes retained the ability to proliferate in the absence of Bmi1. 2. The effects of Bmi1 loss in the retina : The pattern of expression of Bmi1 during development and in the adult retina suggests a role for Bmi1 in cell fate specification and differentiation rather than in proliferation. While the layering and the global structure of the retina appear normal in Bmi1 /adult mice, immunohistochemìcal analysis revealed defects in the three major classes of retinal interneurons, namely: horizontal, bipolar and amacrine cells. Electroretinogram recordings in Bmi9-/- mice are coherent with the defects observed at the histological level, with a reduced b-wave and low-profile oscillatory potentials. These results show that Bmi1 controls not only proliferation, but also cell type generation, as previously observed in the cerebellum. 3. Cell cycle events and related neuroprotective strategies in retinal degeneration : In several neurodegenerative disorders, neurons re-express cell cycle proteins such as cyclin dependent kinases (Cdks) prior to apoptosis. Here, we show for the first time that this is also the case during retinal degeneration. Rd1 mice carry a recessive defect (Pdeóbrd/rd) that causes retinal degeneration and serves as a model of retinitis pigmentosa. We found that photoreceptors express Cdk4 and Cdk2, and undergo DNA synthesis prior to cell death. To interfere with the reactivation of Cdk-related pathways, we deleted E2fs or Brni1, which normally allow cell cycle progression. While deleting E2f1 (downstream of Cdk4/6) in Rd1 mice provides only temporary protection, knocking out Bmi1 (upstream of Cdks) leads to an extensive neuroprotective effect, independent of p16ink4a or p19arf, two tumor suppressors regulated by Bmi1. Analysis of Cdks and the DNA repair-related protein Ligase IV showed that Bmi1 acts downstream of DNA repair events and upstream of Cdks in this neurodegenerative mechanism. Expression of Cdks during an acute model of retinal degeneration, light damage-induced photoreceptor death, points to a role for Bmi1 and cell cycle proteins in retinal degeneration. Considering the similarity with the cell cycle-related apoptotic pathway observed in other neurodegenerative diseases, Bmi1 is a possible general target to prevent or delay neuronal death. RESUME : Ce travail aborde plusieurs aspects de la régulation du cycle cellulaire, de la spécification du devenir des cellules et de la mort cellulaire dans le système nerveux centrale (SNC), plus particulièrement dans le cortex cérébral et dans la rétine. Nous nous sommes intéressés au gène Bmi1, appartenant à la famille polycomb et nécessaire à la prolifération et au renouvellement des cellules souches. Nous avons visé à disséquer son rôle dans le développement du cortex et de la rétine. Ces données, ainsi qu'une série de travaux publiés au cours des deux dernières décennies concernant la réactivation du cycle cellulaire dans les neurones en voie d'apoptose dans le SNC, nous ont ensuite poussé à chercher un lien entre la régulation du cycle cellulaire pendant le développement et au cours de la dégénérescence rétinienne. 1. Les effets de l'inactivation de Bmi1 dans le cortex cérébral : En accord avec l'incapacité des cellules souches neurales in vitro à proliférer et à se renouveler en absence de Bmi1, le cortex cérébral des souris Bmi1-/- présente de légers défauts de prolifération dans les compartiments contenant les cellules souches neurales. Ceci est en accord avec le profil d'expression de Bmi1 dans le télencéphale. Les conséquences de la délétion de Bmi1 sont toutefois nettement moins prononcées in vivo qu'in vitro ; cette différence suggère l'existence de mécanismes pouvant partiellement compenser l'absence de Bmi1 pendant la corticogenèse. Néanmoins, l'observation d'une réduction de la prolifération dans la zone sous-ventriculaire, la zone majeure de neurogenèse dans le télencéphale adulte, montre que Bmi1 contrôle la prolifération des cellules souche/progénitrices neurales chez la souris adulte. Nos résultats démontrent par ailleurs une augmentation de la production d'astrocytes à la naissance ainsi qu'une gliose généralisée à l'état adulte chez les souris Bmi1-/-. Les progéniteurs gliaux et les astrocytes conservent donc leur capacité à proliférer en absence de Bmi1. 2. Les effets de l'inactivation de Bmi1 dans la rétine : Le profil d'expression de Bmi1 pendant fe développement ainsi que dans la rétine adulte suggère un rôle de Bmi1 dans la spécification de certains types cellulaires et dans la différentiation plutôt que dans la prolifération. Alors que la structure et la lamination de la rétine semblent normales chez les souris Bmi1-/-, l'analyse par immunohistochimie amis en évidence des défauts au niveau des trois classes d'interneurones rétiniens (les cellules horizontales, bipolaires et amacrines). Les électrorétinogrammes des souris Bmi1-/- sont cohérents avec les défauts observés au niveau histologique et montrent une réduction de l'onde « b » et des potentiels oscillatoires. Ces résultats montrent que Bmi1 contrôle la génération de certaines sous-populations de neurones, comme démontré auparavant au niveau de cervelet. 3. Réactivation du cycle cellulaire et stratégies théraoeutiaues dans les dégénérescences rétiniennes : Dans plusieurs maladies neurodégénératives, les neurones ré-expriment des protéines du cycle cellulaire telles que les kinases cycline-dépendantes (Cdk) avant d'entrer en apoptose. Nous avons démontré que c'est aussi le cas dans les dégénérescences rétiniennes. Les souris Rd1 portent une mutation récessive (Pde6brd/rd) qui induit une dégénérescence de la rétine et sont utilisées comme modèle animal de rétinite pigmentaire. Nous avons observé que les photorécepteurs expriment Cdk4 et Cdk2, et entament une synthèse d'ADN avant de mourir par apoptose. Pour interférer avec la réactivation les mécanismes Cdk-dépendants, nous avons inactivé les gènes E2f et Bmi1, qui permettent normalement la progression du cycle cellulaire. Nous avons mis en évidence que la délétion de E2f1 (en aval de Cdk4/6) dans les souris Rd1 permet une protection transitoire des photorécepteurs. Toutefois, l'inactivation de Bmi1 (en amont des Cdk) est corrélée à une neuroprotection bien plus durable et ceci indépendamment de p16ink4a et p19arf, deux suppresseurs de tumeurs normalement régulés par Bmi1. L'analyse des Cdk et de la ligase IV (une protéine impliquée dans les mécanismes de réparation de l'ADN) a montré que Bmi1 agit en aval des événements de réparation de l'ADN et en amont des Cdk dans la cascade apoptotique dans les photorécepteurs des souris Rd1. Nous avons également observé la présence de Cdk dans un modèle aigu de dégénérescence rétinienne induit par une exposition des animaux à des niveaux toxiques de lumière. Nos résultats suggèrent donc un rôle général de Bmi1 et des protéines du cycle cellulaire dans les dégénérescences de la rétine. Si l'on considère la similarité avec les événements de réactivation du cycle cellulaire observés dans d'autres maladies neurodégénératives, Bmi1 pourrait être une cible thérapeutique générale pour prévenir la mort neuronale.

Survival response in the skin

Apoptosis is defined as a programmed cell death process operating in multicellular organisms in order to maintain proper homeostasis of tissues. Caspases are among the best characterized proteases to execute apoptosis although lately many studies have associated them with non-apoptotic functions. In the laboratory an antiapoptotic pathway relying on caspase-3 activation and RasGAP has been described in vitro. RasGAP bears two conserved caspase-3 cleavage sites. Under low stress conditions, RasGAP is first cleaved by low caspase-3 activity generating an N terminal fragment (fragment N) that induces a potent anti-apoptotic response mediated by the Ras/PI3K/Akt pathway. High levels of active caspase-3, associated with increased stress conditions, induce further cleavage of fragment N abrogating this anti-apoptotic response. In the present work I studied the functionality of fragment N-mediated protection in physiological conditions as well as the mechanism by which fragment N induces an anti-apoptotic response, with a focus on survivin, an inhibitor of apoptosis. During my work in the laboratory I found that mice lacking caspase-3 or unable to cleave RasGAP (KI mice) are deficient in Akt activation and more sensitive to apoptosis than wild-type mice in response to stress. This higher sensitivity to stress led to augmented tissue damage, highlighting the importance of this pathway in protection against low stress. In parallel I focused on the study of survivin expression in the skin in response to UV-B light and I found that survivin is induced in the cytoplasm of keratinocytes in response to stress where it may fulfill a cyto-protective role. However fragment N had no effect on survivin expression. In addition, cytoplasmic survivin was increased in keratinocytes exposed to UV-B light, whether RasGAP is cleaved (WT mice) or not (KI mice), indicating that survivin is not involved in fragment N mediated protection. Altogether these data indicate that fragment N is pivotal for cell protection against pathophysiologic damage and can encourage the development of therapies aimed to strengthen the resistance of cells against aggressive treatments. Importantly, this finding contributes to the characterization of how caspase-3 can be activated without inducing cell death, although further studies need to be conducted in order to completely characterize this pro-survival molecular mechanism.

Comparative resilience of clinical perimetric tests to induced levels of intraocular straylight.

PURPOSE: To investigate the effect of intraocular straylight (IOS) induced by white opacity filters (WOF) on threshold measurements for stimuli employed in three perimeters: standard automated perimetry (SAP), pulsar perimetry (PP) and the Moorfields motion displacement test (MDT).¦METHODS: Four healthy young (24-28 years old) observers were tested six times with each perimeter, each time with one of five different WOFs and once without, inducing various levels of IOS (from 10% to 200%). An increase in IOS was measured with a straylight meter. The change in sensitivity from baseline was normalized, allowing comparison of standardized (z) scores (change divided by the SD of normative values) for each instrument.¦RESULTS: SAP and PP thresholds were significantly affected (P < 0.001) by moderate to large increases in IOS (50%-200%). The drop in motion displacement (MD) from baseline with WOF 5, was approximately 5 dB, in both SAP and PP which represents a clinically significant loss; in contrast the change in MD with MDT was on average 1 minute of arc, which is not likely to indicate a clinically significant loss.¦CONCLUSIONS: The Moorfields MDT is more robust to the effects of additional straylight in comparison with SAP or PP.

Improvement of the specificity of cancer detection by autofluorescence imaging in the tracheo-bronchial tree using backscattered violet light.

BACKGROUND: Autofluorescence bronchoscopy (AFB) is a highly sensitive tool for the detection of early bronchial cancers. However, its specificity remains limited due to primarily false positive results induced by hyperplasia, metaplasia and inflammation. We have investigated the potential of blue-violet backscattered light to eliminate false positive results during AFB in a clinical pilot study. METHODS: The diagnostic autofluorescence endoscopy (DAFE) system was equipped with a variable band pass filter in the imaging detection path. The backscattering properties of normal and abnormal bronchial mucosae were assessed by computing the contrast between the two tissue types for blue-violet wavelengths ranging between 410 and 490 nm in 12 patients undergoing routine DAFE examination. In a second study including 6 patients we used a variable long pass (LP) filter to determine the spectral design of the emission filter dedicated to the detection of this blue-violet light with the DAFE system. RESULTS: (Pre-)neoplastic mucosa showed a clear wavelength dependence of the backscattering properties of blue-violet light while the reflectivity of normal, metaplastic and hyperplastic autofluorescence positive mucosa was wavelength independent. CONCLUSIONS: Our results showed that the detection of blue-violet light has the potential to reduce the number of false positive results in AFB. In addition we determined the spectral design of the emission filter dedicated to the detection of this blue-violet light with the DAFE system.

Association of nuclear matrix proteins with granular and threaded nuclear bodies in cell lines undergoing apoptosis.

The granules which appear in the nucleolar area in apoptotic HL-60 cells after camptothecin administration (Zweyer et al., Exp. Cell Res. 221,27-40, 1995) were detected also in several other cell lines induced to undergo apoptosis by different stimuli, such as MOLT-4 treated with staurosporine, K-562 incubated with actinomycin D, P-815 exposed to temperature causing heat shock, Jurkat cells treated with EGTA, U-937 growing in the presence of cycloheximide and tumor necrosis factor-alpha, and HeLa cells treated with etoposide. Using immunoelectron microscopy techniques, we demonstrate that, besides the already described nuclear matrix proteins p125 and p160, these granules contain other nucleoskeletal polypeptides such as proliferating cell nuclear antigen, a component of ribonucleoprotein particles, a 105-kDa constituent of nuclear spliceosomes, and the 240-kDa nuclear mitotic apparatus-associated protein referred to as NuMA. Moreover, we also found in the granules SAF-A/hn-RNP-U and SATB1 proteins, two polypeptides that have been reported to bind scaffold-associated regions DNA sequences in vitro, thus mediating the formation of looped DNA structures in vivo. Fibrillarin and coilin are not present in these granules or the PML protein. Thus, the granules seen during the apoptotic process apparently are different from coiled bodies or other types of nuclear bodies. Furthermore, these granules do not contain chromatin components such as histones and DNA. Last, Western blotting analysis revealed that nuclear matrix proteins present in the granules are not proteolytically degraded except for the NuMA polypeptide. We propose that these granules might represent aggregates of nuclear matrix proteins forming during the apoptotic process. Moreover, since the granules are present in several cell lines undergoing apoptosis, they could be considered a previously unrecognized morphological hallmark of the apoptotic process.

Zoledronate sensitizes endothelial cells to tumor necrosis factor-induced programmed cell death: evidence for the suppression of sustained activation of focal adhesion kinase and protein kinase B/Akt.

Bisphosphonates are potent inhibitors of osteoclast function widely used to treat conditions of excessive bone resorption, including tumor bone metastases. Recent evidence indicates that bisphosphonates have direct cytotoxic activity on tumor cells and suppress angiogenesis, but the associated molecular events have not been fully characterized. In this study we investigated the effects of zoledronate, a nitrogen-containing bisphosphonate, and clodronate, a non-nitrogen-containing bisphosphonate, on human umbilical vein endothelial cell (HUVEC) adhesion, migration, and survival, three events essential for angiogenesis. Zoledronate inhibited HUVEC adhesion mediated by integrin alphaVbeta3, but not alpha5beta1, blocked migration and disrupted established focal adhesions and actin stress fibers without modifying cell surface integrin expression level or affinity. Zoledronate treatment slightly decreased HUVEC viability and strongly enhanced tumor necrosis factor (TNF)-induced cell death. HUVEC treated with zoledronate and TNF died without evidence of enhanced annexin-V binding, chromatin condensation, or nuclear fragmentation and caspase dependence. Zoledronate inhibited sustained phosphorylation of focal adhesion kinase (FAK) and in combination with TNF, with and without interferon (IFN) gamma, of protein kinase B (PKB/Akt). Constitutive active PKB/Akt protected HUVEC from death induced by zoledronate and TNF/IFNgamma. Phosphorylation of c-Src and activation of NF-kappaB were not affected by zoledronate. Clodronate had no effect on HUVEC adhesion, migration, and survival nor did it enhanced TNF cytotoxicity. Taken together these data demonstrate that zoledronate sensitizes endothelial cells to TNF-induced, caspase-independent programmed cell death and point to the FAK-PKB/Akt pathway as a novel zoledronate target. These results have potential implications to the clinical use of zoledronate as an anti-angiogenic or anti-cancer agent.

A-Kinase Anchoring Protein (AKAP)-Lbc Anchors a PKN-based Signaling Complex Involved in α1-Adrenergic Receptor-induced p38 Activation.

The mitogen-activated protein kinases (MAPKs) pathways are highly organized signaling systems that transduce extracellular signals into a variety of intracellular responses. In this context, it is currently poorly understood how kinases constituting these signaling cascades are assembled and activated in response to receptor stimulation to generate specific cellular responses. Here, we show that AKAP-Lbc, an A-kinase anchoring protein (AKAP) with an intrinsic Rho-specific guanine nucleotide exchange factor activity, is critically involved in the activation of the p38α MAPK downstream of α(1b)-adrenergic receptors (α(1b)-ARs). Our results indicate that AKAP-Lbc can assemble a novel transduction complex containing the RhoA effector PKNα, MLTK, MKK3, and p38α, which integrates signals from α(1b)-ARs to promote RhoA-dependent activation of p38α. In particular, silencing of AKAP-Lbc expression or disrupting the formation of the AKAP-Lbc·p38α signaling complex specifically reduces α(1)-AR-mediated p38α activation without affecting receptor-mediated activation of other MAPK pathways. These findings provide a novel mechanistic hypothesis explaining how assembly of macromolecular complexes can specify MAPK signaling downstream of α(1)-ARs.

The anti-lymphoma activity of APO866, an inhibitor of nicotinamide adenine dinucleotide biosynthesis, is potentialized when used in combination with anti-CD20 antibody.

Abstract APO866 is an inhibitor of nicotinamide adenine dinucleotide (NAD) biosynthesis that exhibits potent anti-lymphoma activity. Rituximab (RTX), an anti-CD20 antibody, kills lymphoma cells by direct apoptosis and antibody- and complement-dependent cell-mediated cytotoxicities, and has clinical efficacy in non-Hodgkin cell lymphomas. In the present study, we evaluated whether RTX could potentiate APO866-induced human B-lymphoma cell death and shed light on death-mediated mechanisms associated with this drug combination. We found that RTX significantly increases APO866-induced death in lymphoma cells from patients and lines. Mechanisms include enhancement of autophagy-mediated cell death, activation of caspase 3 and exacerbation of mitochondrial depolarization, but not increase of reactive oxygen species (ROS) production, when compared with those induced by each drug alone. In vivo, combined administration of APO866 with RTX in a laboratory model of human aggressive lymphoma significantly decreased tumor burden and prolonged survival over single-agent treatment. Our study demonstrates that the combination of RTX and APO866 optimizes B-cell lymphoma apoptosis and therapeutic efficacy over both compounds administered separately.