Etude du rôle des récepteurs des anaphylatoxines C3a et C5a dans le remodelage et le développement tissulaires

Les anaphylatoxines du complément et leurs récepteurs, RC3a et RC5a, jouent un rôle lors de la régénération et l ontogénèse tissulaires. Les hépatocytes n expriment le RC5a qu après un stress inflammatoire ; l administration de C5a augmente la prolifération hépatocytaire in vitro et in vivo, au cours de la régénération hépatique. Les RC3a et RC5a sont exprimés dans les neurones en grain du cervelet de rat en cours de développement. In vitro, le RC5a protège les neurones en grain de l apoptose induite par appauvrissement du milieu ; in vivo, l injection sous durale de C5a induit une prolifération accrue des neuroblastes par une action directe sur le RC5a neuronal. L injection de C3a provoque une diminution de la couche externe du cervelet et une augmentation concomitante de celle de la couche interne pouvant s expliquer par une accélération de la migration des neurones en grain comme mesurée in vitro par une augmentation de la motilité des neuroblastes.Complement anaphylatoxines and their receptors, C3Ar and C5aR, play a role in regeneration and ontogenesis. Hepatocytes express C5aR only after inflammatory stress. C5a administration increase the hepatocyte proliferation in vitro and in vivo, during liver generation. C3aR and C5aR are expressed in granular neurons of rat cerebellum during development. In vitro, C5aR protects granular neurons against apoptosis induced by lack of substract in medium ; in vivo, sub-dural injection of C5a induces an increase of neuroblast proliferation by a direct action of this peptide on C5aR. C3a injection provokes a decrease of cerebellar external layer and a concomitant increase of internal layer that could be explained by an acceleration of granular neuron migration as measured in vitro by an increase of neuroblast motility.ROUEN-BU Sciences (764512102) / SudocROUEN-BU Sciences Madrillet (765752101) / SudocSudocFranceF

Isolation of human epidermal layers by laser capture microdissection: application to the analysis of gene expression by quantitative real-time PCR

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Role of root cap-derived cells and secretions in root protection

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Role of complement anaphylatoxin receptors (C3aR, C5aR) in the development of the rat cerebellum

International audienceThere is now strong evidence for non-immune or inflammatory functions of complement, notably in the central nervous system. In particular, it has been recently reported that the anaphylatoxin receptors C3aR and C5aR are transiently expressed in the cerebellar cortex of newborn rat, suggesting that anaphylatoxins are involved in the histogenesis of the cerebellum. In the present study, we have investigated the effects of C3aR and C5aR agonists and antagonists on the development of the cerebellum of 11-12-day-old rats in vivo and in vitro. Sub-dural injection of C3aR and C5aR agonists at the surface of the cerebellum transiently modified the thickness of the cortical layers. The C5aR agonist provoked an enlargement of the external granule cell layer (EGL) that was due to increased proliferation of immature granule neurons. Conversely, the C3aR agonist decreased the thickness of the EGL and increased the thickness of the internal granule cell layer (IGL), suggesting that C3a accelerates the migration process of granule cells from the EGL to the IGL. Video-microscopy examination of cultured granule neurons confirmed the role of C3aR in cell motility. These results provide clear evidence for the involvement of anaphylatoxin receptors in the histogenesis of the cerebellar cortex

Probe, Sample, and Instrument (PSI): The Hat-Trick for Fluorescence Live Cell Imaging

International audienceCell Imaging Platforms (CIPs) are research infrastructures offering support to a number of scientific projects including the choice of adapted fluorescent probes for live cell imaging. What to detect in what type of sample and for how long is a major issue with fluorescent probes and, for this, the “hat-trick” “Probe–Sample–Instrument” (PSI) has to be considered. We propose here to deal with key points usually discussed in CIPs including the properties of fluorescent organic probes, the modality of cell labeling, and the best equipment to obtain appropriate spectral, spatial, and temporal resolution. New strategies in organic synthesis and click chemistry for accessing probes with enhanced photophysical characteristics and targeting abilities will also be addressed. Finally, methods for image processing will be described to optimize exploitation of fluorescence signals

Interleukin-1beta and anaphylatoxins exert a synergistic effect on NGF expression by astrocytes.

C3a and C5a anaphylatoxins are proinflammatory polypeptides released during complement activation. They exert their biological activities through interaction with two G protein-coupled receptors named C3aR and C5aR, respectively. In the brain, these receptors are expressed on glial cells, and some recent data have suggested that anaphylatoxins could mediate neuroprotection. In this study, we used RT-PCR and ribonuclease protection assays (RPA) to investigate the role of anaphylatoxins on neurotrophin expression by the human glioblastoma cell line T98G and by rat astrocytes. Our data show that for both cell types, anaphylatoxins upregulate expression of NGF mRNA. This response depended on a G protein-coupled pathway since pre-treatment of cells with pertussis toxin (PTX) completely blocked NGF mRNA increases. This effect was anaphylatoxin-specific since pre-incubation with anti-C3a or anti-C5aR antibodies abolished the effects of C3a and C5a, respectively. The regulation of NGF mRNA by anaphylatoxins was not accompanied by translation into protein expression, but there was a significant synergic effect of anaphylatoxins/IL-1b costimulation. Our demonstration of involvement of anaphylatoxins in the NGF release process by astrocytes suggests that C3a and C5a could modulate neuronal survival in the CNS

Peroxiredoxin 2 is Involved in the Neuroprotective Effects of PACAP in Cultured Cerebellar Granule Neurons

International audienceThe neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is known to counteract in vitro the deleterious effects of toxic agents on cerebellar granule cell survival and differentiation. The potent antiapoptotic action of PACAP is mediated through inhibition of caspase-3 activity; however, additional proteins are likely involved and remain to be identified. Two-dimensional gel electrophoresis analysis coupled with mass spectrometry characterization led to the identification of a protein, peroxiredoxin 2, which was induced after a 6-h treatment with PACAP. Western blot analysis confirmed the regulation of peroxiredoxin 2 by PACAP and revealed that this protein is induced by both cyclic AMP and protein kinase C stimulators. Inhibition of peroxiredoxin 2 expression, using two distinct small-interfering RNAs (siRNAs), reduced the effect of PACAP on caspase-3 activity and cerebellar granule cell survival. Peroxiredoxin 2 expression was also induced in vivo and in vitro by ethanol. Although ethanol and PACAP exert opposite effects on caspase-3 activity, inhibition of peroxiredoxin 2 expression, using siRNAs, only reduced the ability of PACAP to prevent ethanol-induced caspase-3 activity. Taken together, these data indicate that peroxiredoxin 2 is probably involved in the neurotrophic effect of PACAP and suggest that this protein may have a therapeutic potential for the treatment of some neurodegenerative disease

Sustained activation of P2X7 induces MMP-2-evoked cleavage and functional purinoceptor inhibition.

From the 1School of Allied Health Sciences, Faculty of Health and Life Sciences, De Montfort University, Leicester, LE1 5RR, UK, the 2Molecular Medicine Laboratory, Institute of Biomedical and Biomolecular Sciences, School of Pharmacy and Biomedical Sciences, University of Portsmouth, PO1 2DT, UK, 3Laboratory of Cellular Metabolism, Department of Biochemistry, Nencki Institute of Experimental Biology of the Polish Academy of Sciences, Pasteur Str., 02-093 Warsaw, Poland, the 4PRIMACEN, Cell Imaging Platform of Normandy, Inserm, IBiSA and PISSARO Proteomic Platform, Institute for Research and Innovation in Biomedicine, University of Rouen; 76821 Mont-Saint-Aignan, France.P2X7 purinoceptor promotes survival or cytotoxicity depending on extracellular ATP (eATP) stimulus intensity controlling its ion channel or P2X7-dependent large pore (LP) functions. Mechanisms governing this operational divergence and functional idiosyncrasy are ill-understood. We have discovered a feedback loop where sustained activation of P2X7 triggers release of active MMP-2, which halts ion channel and LP responses via the MMP-2-dependent receptor cleavage. This mechanism operates in cells as diverse as macrophages, dystrophic myoblasts, P2X7-transfected HEK293 and human tumor cells. Given that serum-born MMP-2 activity also blocked receptor functions, P2X7 responses in vivo may decrease in organs with permeable capillaries. Therefore, this mechanism represents an important fine-tuning of P2X7 functions, reliant on both cell-autonomous and extraneous factors. Indeed, it allowed evasion from the ATP-induced cytotoxicity in macrophages and human cancer cells with high P2X7 expression levels. Finally, we demonstrate that P2X7 ablation eliminated gelatinase activity in inflamed dystrophic muscles in vivo. Thus, P2X7 antagonists could be used as an alternative to highly toxic MMP inhibitors in treatments of inflammatory diseases and cancers