Linking microtubules to the activation of CDC42 in fission yeast

Cell polarity is an essential property of most cell types and relies on a dynamic cytoskeleton of actin filaments and microtubules. In rod-shaped S. pombe cells microtubules are organized along the length of the cell and transport polarity factors to cell tips to regulate cell polarity. An important cell polarity factor is the protein Tea4, which is responsible for correct cell morphogenesis and bipolar growth. During my research I confirmed the known transport mechanism of Tea4 and I also showed alternative localization and anchoring mechanisms at the cell ends. Tea4 contains a conserved SH3 domain, the function of which was unknown and my results show that the SH3 domain of Tea4 is essential for Tea4 function in vivo. First, cells with tea4SH3 mutations show aberrant cell shapes and monopolar growth patterns similar to tea4A and in addition SH3 domain is important for proper localization of multiple cell polarity proteins. Second, I showed that Tea4 associates with Type 1 Phosphatase Dis2 through both its SH3 domain and an RVxF motif. Tea4 also binds the DYRK kinase Pomi through its SH3 domain. In addition Tea4 is proposed to promote the local dephosphorylation of Pomi by Dis2 to induce the formation of a cortical gradient from cell ends essential for cell size homeostasis. Polarized growth is also controlled by cell tip-localized Cdc42. This Rho- family GTPase is activated by the Guanine Exchange Factors Gef1 and Scd1 and inactivated by the Rho GTPase Activating Protein Rga4. In this study, I investigated the mechanisms of how Tea4 promotes Cdc42 activation. My work suggests that Tea4 promotes the local exclusion of Rga4, which in turn allows the accumulation of active Cdc42, which may result in growth. Exclusion of Rga4 by Tea4 is likely to be mediated by Dis2-dependent dephosphorylation. These results suggest a molecular pathway that links the microtubule- associated factor Tea4 with Cdc42 to promote cell polarization and morphogenesis. - La polarité cellulaire est une propriété essentielle de la plupart des types cellulaires et s'appuie sur une dynamique des cytosquelettes d'actine et de microtubules. Dans les cellules en forme de bâtonnet de S. pombe les microtubules sont alignés selon l'axe longitudinal de la cellule et les facteurs de polarité transportés aux extrémité cellulaires afin de réguler la polarité cellulaire. Un facteur important de polarité cellulaire est la protéine Tea4, qui est responsable de la morphogenèse des cellules et leur croissance bipolaire. Au cours de mes recherches, j'ai confirmé les mécanismes connus de transport de Tea4 et j'ai aussi mis en évidence d'autres mechanismes de localisation et d'ancrage de Tea4 aux extrémités cellulaires. Tea4 contient un domaine SH3 conservé, dont la fonction était inconnue et mes résultats montrent que le domaine SH3 est essentiel pour la fonction de Tea4 in vivo. Tout d'abord, les cellules avec des mutations tea4sm ont des formes aberrantes et leur croissance est monopolaire de manière similaire au mutant tea4A. De plus ce domaine SH3 est important pour la localisation correcte de plusieurs protéines de polarité cellulaire. Deuxièmement, j'ai montré que Tea4 s'associe avec la Phosphatase de Type-1 Dis2 par son domaine SH3 et un motif RVxF. Tea4 se lie également la kinase DYRK Pomi par son domaine SH3. De plus, Tea4 pourrait favoriser la déphosphorylation locale de Pomi par Dis2 afin d'induire la formation d'un gradient cortical de Pomi essentiel pour l'homéostasie de la longueur des cellules. La croissance polarisée est également contrôlée par la protéine Cdc42 localisée aux extrémités cellulaires. Cette GTPase de la famille de Rho GTPase est activée par les facteurs échange de guanine Gef1 et Scd1 et inactivée par la protéine "Rho GTPase activating" Rga4. Dans cette étude, j'ai étudié les mécanismes d' activation de Cdc42 par Tea4. Mes résultats suggèrent que Tea4 favorise l'exclusion locale de Rga4, ce qui permet l'accumulation de Cdc42 active, nécessaire à la croissance. L' exclusion de Rga4 par Tea4 est vraisemblablement médiée par une déphosphorylation Dis2- dépendente. Ces résultats suggèrent une voie moléculaire qui lie le facteur associé aux microtubules Tea4 à Cdc42 pour promouvoir la polarisation cellulaire et la morphogenèse. - Cell polarity is important for several essential biological functions such as generation of distinct cell fates during development and function of differentiated cells. Defective cell polarity has been related to uncontrolled cell division and subsequently to cancer initiation. Cell polarity depends on a functional cytoskeleton that consists of actin filaments and microtubules, which maintains cell shape, helps cellular motion, enables intracellular protein transport and plays a vital role in cell division. A component of cytoskeleton is microtubules that regulate cell polarization in diverse cell types. During my research, I worked with Schizosaccharomyces pombe, also named fission yeast, a powerful unicellular model organism that allows combination of genetic, biochemical and microscopic analysis for the proper study of cell polarity. Microtubule-associated protein Tea4 is transported to cell tips where it is thought to organize polarized growth. I showed that Tea4 and its evolutionarily conserved SH3 domain play an important role for maintenance of fission yeast cells shape and growth. Furthermore, Tea4 is responsible for the proper localization of multiple polarity proteins and acts as a mediator to control the local activity of an essential polarity regulator called Cdc42. Thus, my results provide a better understanding of the molecular mechanisms that regulate cell polarity. - La polarité cellulaire est importante pour plusieurs fonctions biologiques essentielles telles que la différenciation cellulaires au cours du développement et de la fonction de cellules différenciées. Les défauts de la polarité cellulaire ont été liés à des divisions cellulaires incontrôlées et à l'initiation de tumeur. La polarité cellulaire dépend d'un cytosquelette fonctionnel, qui maintient la forme des cellules, aide à la migration cellulaire, permet le transport intracellulaire des protéines et joue un rôle essentiel dans la division cellulaire. Un composant du cytosquelette est constitué de microtubules qui régissent la polarisation cellulaire dans divers types cellulaires. Au cours de mes recherches, j'ai travaillé avec Schizosaccharomyces pombe, appelé également levure fissipare, un modèle unicellulare puissant qui permet la combinaison de différentes d'approches expérimentales: génétiques, biochimiques et microscopiques pour l'étude de la polarité cellulaire. La protéine Tea4 associée aux microtubules est transportée aux extrémités cellulaires où elle organise la croissance polarisée. J'ai montré que Tea4 et son domaine conservé SH3 jouent un rôle important pour le maintien de la forme des cellules de levure et leur croissance. De plus, Tea4 est responsable de la localisation correcte de multiples facteurs de polarité et agit comme un médiateur pour contrôler l'activité locale d'un régulateur de polarité essentiel appelé Cdc42. Ainsi, mes résultats permettent de mieux comprendre les mécanismes moléculaires qui régulent la polarité cellulaire.

A coherent neurobiological framework for functional neuroimaging provided by a model integrating compartmentalized energy metabolism.

Functional neuroimaging has undergone spectacular developments in recent years. Paradoxically, its neurobiological bases have remained elusive, resulting in an intense debate around the cellular mechanisms taking place upon activation that could contribute to the signals measured. Taking advantage of a modeling approach, we propose here a coherent neurobiological framework that not only explains several in vitro and in vivo observations but also provides a physiological basis to interpret imaging signals. First, based on a model of compartmentalized energy metabolism, we show that complex kinetics of NADH changes observed in vitro can be accounted for by distinct metabolic responses in two cell populations reminiscent of neurons and astrocytes. Second, extended application of the model to an in vivo situation allowed us to reproduce the evolution of intraparenchymal oxygen levels upon activation as measured experimentally without substantially altering the initial parameter values. Finally, applying the same model to functional neuroimaging in humans, we were able to determine that the early negative component of the blood oxygenation level-dependent response recorded with functional MRI, known as the initial dip, critically depends on the oxidative response of neurons, whereas the late aspects of the signal correspond to a combination of responses from cell types with two distinct metabolic profiles that could be neurons and astrocytes. In summary, our results, obtained with such a modeling approach, support the concept that both neuronal and glial metabolic responses form essential components of neuroimaging signals.

Reggies/flotillins regulate E-cadherin-mediated cell contact formation by affecting EGFR trafficking.

The reggie/flotillin proteins are implicated in membrane trafficking and, together with the cellular prion protein (PrP), in the recruitment of E-cadherin to cell contact sites. Here, we demonstrate that reggies, as well as PrP down-regulation, in epithelial A431 cells cause overlapping processes and abnormal formation of adherens junctions (AJs). This defect in cell adhesion results from reggie effects on Src tyrosine kinases and epidermal growth factor receptor (EGFR): loss of reggies reduces Src activation and EGFR phosphorylation at residues targeted by Src and c-cbl and leads to increased surface exposure of EGFR by blocking its internalization. The prolonged EGFR signaling at the plasma membrane enhances cell motility and macropinocytosis, by which junction-associated E-cadherin is internalized and recycled back to AJs. Accordingly, blockage of EGFR signaling or macropinocytosis in reggie-deficient cells restores normal AJ formation. Thus, by promoting EGFR internalization, reggies restrict the EGFR signaling involved in E-cadherin macropinocytosis and recycling and regulate AJ formation and dynamics and thereby cell adhesion.

Role of peroxynitrite in the redox regulation of cell signal transduction pathways.

Peroxynitrite is a potent oxidant and nitrating species formed from the reaction between the free radicals nitric oxide and superoxide. An excessive formation of peroxynitrite represents an important mechanism contributing to cell death and dysfunction in multiple cardiovascular pathologies, such as myocardial infarction, heart failure and atherosclerosis. Whereas initial works focused on direct oxidative biomolecular damage as the main route of peroxynitrite toxicity, more recent evidence, mainly obtained in vitro, indicates that peroxynitrite also behaves as a potent modulator of various cell signal transduction pathways. Due to its ability to nitrate tyrosine residues, peroxynitrite affects cellular processes dependent on tyrosine phosphorylation. Peroxynitrite also exerts complex effects on the activity of various kinases and phosphatases, resulting in the up- or downregulation of signalling cascades, in a concentration- and cell-dependent manner. Such roles of peroxynitrite in the redox regulation of key signalling pathways for cardiovascular homeostasis, including protein kinase B and C, the MAP kinases, Nuclear Factor Kappa B, as well as signalling dependent on insulin and the sympatho-adrenergic system are presented in detail in this review.

Progenitor cell therapy for heart disease.

Many cell types are currently being studied as potential sources of cardiomyocytes for cell transplantation therapy to repair and regenerate damaged myocardium. The question remains as to which progenitor cell represents the best candidate. Bone marrow-derived cells and endothelial progenitor cells have been tested in clinical studies. These cells are safe, but their cardiogenic potential is controversial. The functional benefits observed are probably due to enhanced angiogenesis, reduced ventricular remodeling, or to cytokine-mediated effects that promote the survival of endogenous cells. Human embryonic stem cells represent an unlimited source of cardiomyocytes due to their great differentiation potential, but each step of differentiation must be tightly controlled due to the high risk of teratoma formation. These cells, however, confront ethical barriers and there is a risk of graft rejection. These last two problems can be avoided by using induced pluripotent stem cells (iPS), which can be autologously derived, but the high risk of teratoma formation remains. Cardiac progenitor cells have the advantage of being cardiac committed, but important questions remain unanswered, such as what is the best marker to identify and isolate these cells? To date the different markers used to identify adult cardiac progenitor cells also recognize progenitor cells that are outside the heart. Thus, it cannot be determined whether the cardiac progenitor cells identified in the adult heart represent resident cells present since fetal life or extracardiac cells that colonized the heart after cardiac injury. Developmental studies have identified markers of multipotent progenitors, but it is unknown whether these markers are specific for adult progenitors when expressed in the adult myocardium. Cardiac regeneration is dependent on the stability of the cells transplanted into the host myocardium and on the electromechanical coupling with the endogenous cells. Finally, the promotion of endogenous regenerative processes by mobilizing endogenous progenitors represents a complementary approach to cell transplantation therapy.

Regulated exocytosis from astrocytes physiological and pathological related aspects.

Astrocytes have traditionally been considered ancillary, satellite cells of the nervous system. However, it is a very recent acquisition that glial cells generate signaling loops which are integral to the brain circuitry and participate, interactively with neuronal networks, in the processing of information. Such a conceptual breakthrough makes this field of investigation one of the hottest in neuroscience, as it calls for a revision of past theories of brain function as well as for new strategies of experimental exploration of brain function. Glial cells are electrically not excitable, and it was only the use of optical recording techniques together with calcium sensitive dyes, that allowed the chemical excitability of glial cells to become apparent. Studies using these new techniques have shown for the first time that glial cells are activated by surrounding synaptic activity and translate neuronal signals into their own calcium code. Intracellular calcium concentration([Ca2+]i) elevations in glial cells have then shown to underlie spatial transfer of information in the glial network, accompanied by release of chemical transmitters (gliotransmitters) such as glutamate and back-signaling to neurons. As a consequence, optical imaging techniques applied to cell cultures or intact tissue have become a state-of-the-art technology for studying glial cell signaling. The molecular mechanisms leading to release of "gliotransmitters," especially glutamate, from glia are under debate. Accumulating evidence clearly indicates that astrocytes secrete numerous transmitters by Ca(2+)-dependent exocytosis. This review will discuss the mechanisms underlying the release of chemical transmitters from astrocytes with a particular emphasis to the regulated exocytosis processes.

Involvement of the RasGAP derived fragment N in the resistance of pancreatic beta cells towards apoptosis

RESUME DESTINE AUX NON SCIENTIFIQUESLe diabète est une maladie associée à un excès de glucose (sucre) dans le sang. Le taux de glucose sanguin augmente lorsque l'action d'une hormone, l'insuline, responsable du transport du glucose du sang vers les tissus de l'organisme diminue, ou lorsque les quantités d'insuline à disposition sont inadéquates.L'une des causes communes entre les deux grands types de diabète connus, le type 1 et le type 2, est la disparition des cellules beta du pancréas, spécialisées dans la sécrétion d'insuline, par mort cellulaire programmée aussi appelée apoptose. Alors que dans le diabète de type 1, la destruction des cellules beta est causée par notre propre système immunitaire, dans le diabète de type 2, la mort de ces cellules, est principalement causée par des concentrations élevées de graisses saturés ou de molécules impliquées dans l'inflammation que l'on rencontre en quantités augmentées chez les personnes obèses. Etant donné l'augmentation épidémique du nombre de personnes obèses de par le monde, on estime que le nombre de personnes diabétiques (dont une majorité sont des diabétiques de type 2), va passer de 171 million en l'an 2000, à 366 million en l'an 2030, expliquant la nécessité absolue de mettre au point de nouvelles stratégies thérapeutique pour combattre cette maladie.L'apoptose est un processus complexe dont la dérégulation induit de nombreuses affections allant du cancer jusqu'au diabète. L'activation de caspase 3, une protéine clé contrôlant la mort cellulaire, était connue pour systématiquement mener à la mort cellulaire programmée. Ces dernières années, notre laboratoire a décrit des mécanismes de survie qui sont activés par caspase 3 et qui expliquent sans doute pourquoi son activation ne mène pas systématiquement à la mort cellulaire. Lorsqu'elle est faiblement activée, caspase 3 clive une autre protéine appelée RasGAP en deux protéines plus courtes dont l'une, appelée le fragment Ν a la particularité de protéger les cellules contre l'apoptose.Durant ma thèse, j'ai été impliqué dans divers projets destinés à mieux comprendre comment le fragment Ν protégeait les cellules contre l'apoptose et à savoir s'il pouvait être utilisé comme outil thérapeutique dans les conditions de survenue d'un diabète expérimental. C'est dans ce but que nous avons créé une souris transgénique, appelée RIP-N, exprimant le fragment Ν spécifiquement dans les cellules beta. Comme attendu, les cellules beta de ces souris étaient plus résistantes à la mort induite par des composés connus pour induire le diabète, comme certaines molécules induisant l'inflammation ou les graisses saturées. Nous avons ensuite pu montrer que les souris RIP-N étaient plus résistantes à la survenue d'un diabète expérimental que ce soit par l'injection d'une drogue induisant l'apoptose des cellules beta, que ce soit dans un fond génétique caractérisé par une attaque spontanée des cellules beta par le système immunitaire ou dans le contexte d'un diabète de type 2 induit par l'obésité. Dans plusieurs des modèles animaux étudiés, nous avons pu montrer que le fragment Ν protégeait les cellules en activant une voie protectrice bien connue impliquant successivement les protéines Ras, PI3K et Akt ainsi qu'en bloquant la capacité d'Akt d'activer le facteur NFKB, connu pour être délétère pour la survie de la cellule beta. La capacité qu'a le fragment Ν d'activer Akt tout en prévenant l'activation de NFKB par Akt est par conséquent particulièrement intéressante dans l'intégration des signaux régulant la mort cellulaire dans le contexte de la survenue d'un diabète.La perspective d'utiliser le fragment Ν comme outil thérapeutique dépendra de notre capacité à activer les signaux protecteurs induits par le fragment Ν depuis l'extérieur de la cellule ou de dériver des peptides perméables aux cellules possédant les propriétés du fragment N.2 SUMMARYDiabetes mellitus is an illness associated with excess blood glucose. Blood glucose levels raise when the action of insulin decreases or when insulin is provided in inappropriate amounts. In type 1 diabetes (T1D) as well as in type 2 diabetes (T2D), the insulin secreting beta cells in the pancreas undergo controlled cell death also called apoptosis. Whereas in T1D, beta cells are killed by the immune system, in T2D, they are killed by several factors, among which are increased blood glucose levels, increased levels of harmful lipids or pro-inflammatory cytokines that are released by the dysfunctional fat tissue of obese people. Given the epidemic increase in the number of obese people throughout the world, the number of diabetic people (a majority of which are type 2 diabetes) is estimated to rise from 171 million affected people in the year 2000 to 366 million in 2030 explaining the absolute requirement for new therapies to fight the disease.Apoptosis is a very complex process whose deregulation leads to a wide range of diseases going from cancer to diabetes. Caspase 3 although known as a key molecule controlling apoptosis, has been shown to have various other functions. In the past few years, our laboratory has described a survival mechanism, that takes place at low caspase activity and that might explain how cells that activate their caspases for reasons other than apoptosis survive. In such conditions, caspase 3 cleaves another protein called RasGAP into two shorter proteins, one of which, called fragment N, protects cells from apoptosis.We decided to check whether fragment Ν could be used as a therapeutical tool in the context of diabetes inducing conditions. We thus derived a transgenic mouse line, called RIP-N, in which the expression of fragment Ν is restricted to beta cells. As expected, the beta cells of these mice were more resistant ex-vivo to cell death induced by diabetes inducing factors. We then showed that the RIP-N transgenic mice were resistant to streptozotocin induced diabetes, a mouse model mimicking type 1 diabetes, which correlated to fewer number of apoptotic beta cells in the pancreas of the transgenic mice compared to their controls. The RIP-N transgene also delayed overt diabetes development in the NOD background, a mouse model of autoimmune type 1 diabetes, and delayed the occurrence of obesity induced hyperglycemia in a mouse model of type 2-like diabetes. Interestingly, fragment Ν was mediating its protection by activating the protective Akt kinase, and by blocking the detrimental NFKB factor. Our future ability to activate the protective signals elicited by fragment Ν from the outside of cells or to derive cell permeable peptides bearing the protective properties of fragment Ν might condition our ability to use this protein as a therapeutic tool.3 RESUMELe diabète est une maladie associée à un excès de glucose plasmatique. La glycémie augmente lorsque l'action de l'insuline diminue ou lorsque les quantités d'insuline à disposition sont inadéquates. Dans le diabète de type 1 (D1) comme dans le diabète de type 2 (D2), les cellules beta du pancréas subissent la mort cellulaire programmée aussi appelée apoptose. Alors que dans le D1 les cellules beta sont tuées par le système immunitaire, dans le D2 elles sont tuées par divers facteurs parmi lesquels on trouve des concentrations élevées de glucose, d'acides gras saturés ou de cytokines pro-inflammatoires qui sont sécrétées en concentrations augmentées par le tissu adipeux dysfonctionnel des personnes obèses. Etant donné l'augmentation épidémique du nombre de personnes obèses de par le monde, on estime que le nombre de personnes diabétiques (dont une majorité sont des diabétiques de type 2), va passer de 171 million en l'an 2000, à 366 million en l'an 2030, justifiant la nécessité absolue de mettre au point de nouvelles stratégies thérapeutique pour combattre cette maladie.L'apoptose est un processus complexe dont la dérégulation induit de nombreuses affections allant du cancer jusqu'au diabète. Caspase 3, bien que connue comme étant une protéine clé contrôlant l'apoptose a bien d'autres fonctions démontrées. Ces dernières années, notre laboratoire a décrit un mécanisme de survie qui est activé lorsque caspase 3 est faiblement activée et qui explique probablement comment des cellules qui ont activé leurs caspases pour une autre raison que l'apoptose peuvent survivre. Dans ces conditions, caspase 3 clive une autre protéine appelée RasGAP en deux protéines plus courtes dont l'une, appelée le fragment Ν a la particularité de protéger les cellules contre l'apoptose.Nous avons donc décidé de vérifier si le fragment Ν pouvait être utilisé comme outil thérapeutique dans les conditions de survenue d'un diabète expérimental. Pour se faire, nous avons créé une souris transgénique, appelée RIP-N, exprimant le fragment Ν spécifiquement dans les cellules beta. Comme attendu, les cellules beta de ces souris étaient plus résistantes ex-vivo à la mort induite par des facteurs pro-diabétogènes. Nous avons ensuite pu montrer que les souris RIP-N étaient plus résistantes à la survenue d'un diabète induit par la streptozotocine, un drogue mimant la survenue d'un D1 et que ceci était corrélée à une diminution du nombre de cellules en apoptose dans le pancréas des souris transgéniques comparé à leurs contrôles. L'expression du transgène a aussi eu pour effet de retarder la survenue d'un diabète franc dans le fond génétique NOD, un modèle génétique de diabète de type 1 auto-immun, ainsi que de retarder la survenue d'une hyperglycémie dans un modèle murin de diabète de type 2 induit par l'obésité. Dans plusieurs des modèles animaux étudiés, nous avons pu montrer que le fragment Ν protégeait les cellules en activant la kinase protectrice Akt ainsi qu'en bloquant le facteur délétère NFKB. La perspective d'utiliser le fragment Ν comme outil thérapeutique dépendra de notre capacité à activer les signaux protecteurs induits par le fragment Ν depuis l'extérieur de la cellule ou de dériver des peptides perméables aux cellules possédant les propriétés du fragment

Mechanism of reduction of virus release and cell-cell fusion in persistent canine distemper virus infection.

Canine distemper virus (CDV), a mobillivirus related to measles virus causes a chronic progressive demyelinating disease, associated with persistence of the virus in the central nervous system (CNS). CNS persistence of morbilliviruses has been associated with cell-to-cell spread, thereby limiting immune detection. The mechanism of cell-to-cell spread remains uncertain. In the present study we studied viral spread comparing a cytolytic (non-persistent) and a persistent CDV strain in cell cultures. Cytolytic CDV spread in a compact concentric manner with extensive cell fusion and destruction of the monolayer. Persistent CDV exhibited a heterogeneous cell-to-cell pattern of spread without cell fusion and 100-fold reduction of infectious viral titers in supernatants as compared to the cytolytic strain. Ultrastructurally, low infectious titers correlated with limited budding of persistent CDV as compared to the cytolytic strain, which shed large numbers of viral particles. The pattern of heterogeneous cell-to-cell viral spread can be explained by low production of infectious viral particles in only few areas of the cell membrane. In this way persistent CDV only spreads to a small proportion of the cells surrounding an infected one. Our studies suggest that both cell-to-cell spread and limited production of infectious virus are related to reduced expression of fusogenic complexes in the cell membrane. Such complexes consist of a synergistic configuration of the attachment (H) and fusion (F) proteins on the cell surface. F und H proteins exhibited a marked degree of colocalization in cytolytic CDV infection but not in persistent CDV as seen by confocal laser microscopy. In addition, analysis of CDV F protein expression using vaccinia constructs of both strains revealed an additional large fraction of uncleaved fusion protein in the persistent strain. This suggests that the paucity of active fusion complexes is due to restricted intracellular processing of the viral fusion protein.

Kinase signaling cascades that modulate peroxisome proliferator-activated receptors.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors involved in lipid and glucose homeostasis, inflammation and wound healing. In addition to ligand binding, phosphorylation can also regulate PPARs; the biological effects of phosphorylation depend on the stimulus, the kinase, the PPAR isotype, the residue modified, the cell type and the promoter investigated. The study of this dual regulation mode, which allows PPARs to integrate signals conveyed by lipophilic ligands with those coming from the plasma membrane, may ultimately offer new therapeutic strategies.

Autophagic cell death exists.

The term autophagic cell death (ACD) initially referred to cell death with greatly enhanced autophagy, but is increasingly used to imply a death-mediating role of autophagy, as shown by a protective effect of autophagy inhibition. In addition, many authors require that autophagic cell death must not involve apoptosis or necrosis. Adopting these new and restrictive criteria, and emphasizing their own failure to protect human osteosarcoma cells by autophagy inhibition, the authors of a recent Editor's Corner article in this journal argued for the extreme rarity or nonexistence of autophagic cell death. We here maintain that, even with the more stringent recent criteria, autophagic cell death exists in several situations, some of which were ignored by the Editor's Corner authors. We reject their additional criterion that the autophagy in ACD must be the agent of ultimate cell dismantlement. And we argue that rapidly dividing mammalian cells such as cancer cells are not the most likely situation for finding pure ACD.

Reduction of ARNT in myeloid cells causes immune suppression and delayed wound healing.

Aryl hydrocarbon receptor nuclear translocator (ARNT) is a transcription factor that binds to partners to mediate responses to environmental signals. To investigate its role in the innate immune system, floxed ARNT mice were bred with lysozyme M-Cre recombinase animals to generate lysozyme M-ARNT (LAR) mice with reduced ARNT expression. Myeloid cells of LAR mice had altered mRNA expression and delayed wound healing. Interestingly, when the animals were rendered diabetic, the difference in wound healing between the LAR mice and their littermate controls was no longer present, suggesting that decreased myeloid cell ARNT function may be an important factor in impaired wound healing in diabetes. Deferoxamine (DFO) improves wound healing by increasing hypoxia-inducible factors, which require ARNT for function. DFO was not effective in wounds of LAR mice, again suggesting that myeloid cells are important for normal wound healing and for the full benefit of DFO. These findings suggest that myeloid ARNT is important for immune function and wound healing. Increasing ARNT and, more specifically, myeloid ARNT may be a therapeutic strategy to improve wound healing.

Sustained NKG2D engagement induces cross-tolerance of multiple distinct NK cell activation pathways

NKG2D is a multisubunit activation receptor that allows natural killer (NK) cells to detect and eliminate stressed, infected, and transformed host cells. However, the chronic exposure of NK cells to cell-bound NKG2D ligands has been shown to impair NKG2D function both in vitro and in vivo. Here we have tested whether continuous NKG2D engagement selectively impacted NKG2D function or whether heterologous NK cell activation pathways were also affected. We found that sustained NKG2D engagement induced cross-tolerization of several unrelated NK cell activation receptors. We show that receptors that activate NK cells via the DAP12/KARAP and DAP10 signaling adaptors, such as murine NKG2D and Ly49D, cross-tolerize preferentially NK cell activation pathways that function independent of DAP10/12, such as antibody-dependent cell-mediated cytotoxicity and missing-self recognition. Conversely, DAP10/12-independent pathways are unable to cross-tolerize unrelated NK cell activation receptors such as NKG2D or Ly49D. These data define a class of NK cell activation receptors that can tolerize mature NK cells. The reversible suppression of the NK cells' cytolytic function probably reduces the NK cells' efficacy to control endogenous and exogenous stress yet may be needed to limit tissue damage

Altering α-dystroglycan receptor affinity of LCMV pseudotyped lentivirus yields unique cell and tissue tropism.

BACKGROUND: The envelope glycoprotein of lymphocytic choriomeningitis virus (LCMV) can efficiently pseudotype lentiviral vectors. Some strains of LCMV exploit high affinity interactions with α-dystroglycan (α-DG) to bind to cell surfaces and subsequently fuse in low pH endosomes. LCMV strains with low α-DG affinity utilize an unknown receptor and display unique tissue tropisms. We pseudotyped non-primate feline immunodeficiency virus (FIV) vectors using LCMV derived glycoproteins with high or low affinity to α-DG and evaluated their properties in vitro and in vivo. METHODS: We pseudotyped FIV with the LCMV WE54 strain envelope glycoprotein and also engineered a point mutation in the WE54 envelope glycoprotein (L260F) to diminish α-DG affinity and direct binding to alternate receptors. We hypothesized that this change would alter in vivo tissue tropism and enhance gene transfer to neonatal animals. RESULTS: In mice, hepatic α- and β-DG expression was greatest at the late gestational and neonatal time points. When displayed on the surface of the FIV lentivirus the WE54 L260F mutant glycoprotein bound weakly to immobilized α-DG. Additionally, LCMV WE54 pseudotyped FIV vector transduction was neutralized by pre-incubation with soluble α-DG, while the mutant glycoprotein pseudotyped vector was not. In vivo gene transfer in adult mice with either envelope yielded low transduction efficiencies in hepatocytes following intravenous delivery. In marked contrast, neonatal gene transfer with the LCMV envelopes, and notably with the FIV-L260F vector, conferred abundant liver and lower level cardiomyocyte transduction as detected by luciferase assays, bioluminescent imaging, and β-galactosidase staining. CONCLUSIONS: These results suggest that a developmentally regulated receptor for LCMV is expressed abundantly in neonatal mice. LCMV pseudotyped vectors may have applications for neonatal gene transfer. ABBREVIATIONS: Armstrong 53b (Arm53b); baculovirus Autographa californica GP64 (GP64); charge-coupled device (CCD); dystroglycan (DG); feline immunodeficiency virus (FIV); glycoprotein precursor (GP-C); firefly luciferase (Luc); lymphocytic choriomeningitis virus (LCMV); nuclear targeted β-galactosidase (ntLacZ); optical density (OD); PBS/0.1% (w/v) Tween-20 (PBST); relative light units (RLU); Rous sarcoma virus (RSV); transducing units per milliliter (TU/ml); vesicular stomatitis virus (VSV-G); wheat germ agglutinin (WGA); 50% reduction in binding (C50).

Entamoeba lysyl-tRNA synthetase contains a cytokine-like fomain with chemokine activity towards human endothelial cells

Immunological pressure encountered by protozoan parasites drives the selection of strategies to modulate or avoid the immune responses of their hosts. Here we show that the parasite Entamoeba histolytica has evolved a chemokine that mimics the sequence, structure, and function of the human cytokine HsEMAPII (Homo sapiens endothelial monocyte activating polypeptide II). This Entamoeba EMAPII-like polypeptide (EELP) is translated as a domain attached to two different aminoacyl-tRNA synthetases (aaRS) that are overexpressed when parasites are exposed to inflammatory signals. EELP is dispensable for the tRNA aminoacylation activity of the enzymes that harbor it, and it is cleaved from them by Entamoeba proteases to generate a standalone cytokine. Isolated EELP acts as a chemoattractant for human cells, but its cell specificity is different from that of HsEMAPII. We show that cell specificity differences between HsEMAPII and EELP can be swapped by site directed mutagenesis of only two residues in the cytokines' signal sequence. Thus, Entamoeba has evolved a functional mimic of an aaRS-associated human cytokine with modified cell specificity.

Cell death and autophagy after severe hypoxic-ischemic encephalopathy in term newborns

Introduction: Various studies from hypoxic-ischemic animals haveinvestigated neuroprotection by targeting necrosis and apoptosis with inconclusive results. Three types of cell death have been described: apoptosis, necrosis and more recently, autophagic cell death. While autophagy is a physiological process of degradation of cellular components, excessive autophagy may be involved in cell death. Recent studies showed that inhibition of autophagy is neuroprotective in rodent neonatal models of cerebral ischemia. Furthermore, neonatal hypoxia-ischemia strongly increased neuronal autophagic flux which is linked to cell death in a rat model of perinatal asphyxia. Following our observations in animals, the aim of the present study was to characterize the different neuronal death phenotypes and to clarify whether autophagic cell death could be also involved in neuronal death in the human newborns after perinatal asphyxia. Methods: we selected retrospectively and anonymously all newborns who died in our unit of neonatology between 2004 and 2009, with the following criteria: gestational age >36 weeks, diagnosis of perinatal asphyxia (Apgar <5 at 5 minutes, arterial pH <7.0 at 1 hour of life and encephalopathy Sarnat III) and performed autopsy. The brain of 6 cases in asphyxia group and 6 control cases matching gestational age who died of pulmonary or other malformations were selected. On histological sections of thalamus, frontal cortex and hippocampus, different markers of apoptosis (caspase 3, TUNEL), autophagosomes (LC3-II) and lysosomes (LAMP1, Cathepsin D) were tested by immunohistochemistry. Results: Preliminary studies on markers of apoptosis (TUNEL, caspase 3) and of autophagy (Cathepsin D, LC3II, LAMP1) showed an expected increase of apoptosis, but also an increase of neuronal autophagic flux in the selected areas. The distribution seems to be region specific. Conclusion: This is the first time that autophagic flux linked with cell death is shown in brain of human babies, in association with hypoxicischemic encephalopathy. This work leads to a better understanding of the mechanisms associated with neuronal death following perinatal asphyxia and determines whether autophagy could be a promising therapeutic target.