Deregulation of calcium homeostasis mediates secreted aesynuclein - induced neurotoxicity

α-Synuclein (AS) plays a crucial role in Parkinson's disease pathogenesis. AS is normally secreted from neuronal cells and can thus exert paracrine effects. We have previously demonstrated that naturally secreted AS species, derived from SH-SY5Y cells inducibly overexpressing human wild type AS, can be toxic to recipient neuronal cells. In the current study, we show that application of secreted AS alters membrane fluidity and increases calcium (Ca2+) entry. This influx is reduced on pharmacological inhibition of voltage-operated Ca2+ channels. Although no change in free cytosolic Ca2+ levels is observed, a significantly increased mitochondrial Ca2+ sequestration is found in recipient cells. Application of voltage-operated Ca2+ channel blockers or Ca2+ chelators abolishes AS-mediated toxicity. AS-treated cells exhibit increased calpain activation, and calpain inhibition greatly alleviates the observed toxicity. Collectively, our data suggest that secreted AS exerts toxicity through engagement, at least in part, of the Ca2+ homeostatic machinery. Therefore, manipulating Ca2+ signaling pathways might represent a potential therapeutic strategy for Parkinson's disease

Rhinovirus-induced basic fibroblast growth factor release mediates airway remodeling features

BACKGROUND: Human rhinoviruses, major precipitants of asthma exacerbations, induce lower airway inflammation and mediate angiogenesis. The purpose of this study was to assess the possibility that rhinoviruses may also contribute to the fibrotic component of airway remodeling. METHODS: Levels of basic fibroblast growth factor (bFGF) mRNA and protein were measured following rhinovirus infection of bronchial epithelial cells. The profibrotic effect of epithelial products was assessed by DNA synthesis and matrix metalloproteinase activity assays. Moreover, epithelial cells were exposed to supernatants from cultured peripheral blood mononuclear cells, obtained from healthy donors or atopic asthmatic subjects and subsequently infected by rhinovirus and bFGF release was estimated. bFGF was also measured in respiratory secretions from atopic asthmatic patients before and during rhinovirus-induced asthma exacerbations. RESULTS: Rhinovirus epithelial infection stimulated mRNA expression and release of bFGF, the latter being positively correlated with cell death under conditions promoting rhinovirus-induced cytotoxicity. Supernatants from infected cultures induced lung fibroblast proliferation, which was inhibited by anti-bFGF antibody, and demonstrated increased matrix metalloproteinase activity. Rhinovirus-mediated bFGF release was significantly higher in an in vitro simulation of atopic asthmatic environment and, importantly, during rhinovirus-associated asthma exacerbations. CONCLUSIONS: Rhinovirus infection induces bFGF release by airway epithelium, and stimulates stroma cell proliferation contributing to airway remodeling in asthma. Repeated rhinovirus infections may promote asthma persistence, particularly in the context of atopy; prevention of such infections may influence the natural history of asthma

Towards an alternative testing strategy for nanomaterials used in nanomedicine: lessons from NanoTEST.

In spite of recent advances in describing the health outcomes of exposure to nanoparticles (NPs), it still remains unclear how exactly NPs interact with their cellular targets. Size, surface, mass, geometry, and composition may all play a beneficial role as well as causing toxicity. Concerns of scientists, politicians and the public about potential health hazards associated with NPs need to be answered. With the variety of exposure routes available, there is potential for NPs to reach every organ in the body but we know little about the impact this might have. The main objective of the FP7 NanoTEST project ( www.nanotest-fp7.eu ) was a better understanding of mechanisms of interactions of NPs employed in nanomedicine with cells, tissues and organs and to address critical issues relating to toxicity testing especially with respect to alternatives to tests on animals. Here we describe an approach towards alternative testing strategies for hazard and risk assessment of nanomaterials, highlighting the adaptation of standard methods demanded by the special physicochemical features of nanomaterials and bioavailability studies. The work has assessed a broad range of toxicity tests, cell models and NP types and concentrations taking into account the inherent impact of NP properties and the effects of changes in experimental conditions using well-characterized NPs. The results of the studies have been used to generate recommendations for a suitable and robust testing strategy which can be applied to new medical NPs as they are developed

Etude de la differenciation des cellules erytroleucemiques de souris : methylation du DNA, regulation de l'expression de l'ornithine decarboxylase

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Anti-apoptotic effects of allopregnanolone on P19 neurons

Progesterone and its metabolites are potent allosteric modulators of GABAA receptor function, through a direct, non-genomic interaction with specific receptor subtypes. In addition, fluctuations in the concentration of progesterone, and allopregnanolone in particular, have been shown to modulate GABAA receptor gene expression and activity. In this study, mouse P19 cells were induced to differentiate into post-mitotic neurons which express specific neuronal markers, including GABAA and N-methyl-d-aspartate (NMDA) receptors. Apoptotic cell death, induced in the presence of NMDA, was efficiently prevented by allopregnanolone and dehydroepiandrosterone (DHEA) but not DHEA sulfate. Apoptosis was accompanied by cytochrome c release to the cytoplasm and Bax translocation to the mitochondria, while the levels of the anti-apoptotic proteins Bcl-2 and Bcl-xL remained unchanged. In the presence of the most potent neurosteroid, allopregnanolone, DNA fragmentation as well as cytochrome c and Bax translocation were prevented. On the other hand, short-term exposure (1-20 μm, 24 h) of P19-derived neurons to allopregnanolone and DHEA significantly increased the levels of α1 and β2 mRNAs of GABA A receptor, while the levels of NR1 mRNA of NMDA receptor were not altered. These results suggest that neurosteroids, interfering with the mitochondrial apoptotic pathway, are able to act as survival factors in neuronal cells, while they contribute to GABAA receptor plasticity modulating the expression of its subunits. © The Authors (2006)

Hypoxia-inducible factor-1α increase is an early and sensitive marker of lung cells responding to benzo[a] pyrene

Hypoxia inducible factor-1α (HIF-1α) is a central regulator of tumor survival and metastasis, responsible for metabolic adaptation to hypoxic conditions and promotion of angiogenesis. It has been also shown to respond to non-hypoxic stimuli, such as growth factors and moderate oxidative stress. We examined the protein levels of HIF-1α in A549 human lung cells exposed to the typical carcinogen benzo[a]pyrene (B[a]P). Our results revealed that B[a]P, at low, non-cytotoxic concentrations, induced a transient increase of nuclear HIF-1α and its target, GLUT1. HIF-1α upregulation was partly mediated by Akt kinase and coincided with increased nuclear levels of the redox-sensitive marker, nuclear factor erythroid 2-related factor-2 (NrF-2). B[a]P-induced HIF-1α was also detected during serum depletion or treatment with the hypoxia-mimicking agent, CoCl2. In addition, exposure of A549 cells to B[a]P containing diesel exhaust particles enhanced HIF-1α accumulation, probably due to the presence of additional carcinogenic compounds. B[a]P-induced increase of HIF-1α was further confirmed in normal rat and human lung fibroblasts. Our findings indicate that HIF-1α stimulation may act as an early and sensitive marker of exposure to low, non-cytotoxic concentrations of B[a]P and/or other carcinogens. © 2012 Begell House, Inc

Developmental and age-related alterations of calcium homeostasis in human fibroblasts

Calcium homeostasis, in terms of both cytosolic calcium concentration ([Ca2+](i)) and capacitative calcium entry, has been investigated in human skin and lung fibroblasts at different developmental and ageing stages by employing the Fura-2 based measurements. [Ca2+](i) in foetal lung or skin cells were nearly similar. However, significant changes were observed between foetal and adult fibroblasts and interestingly in opposite directions depending on the tissue of origin. In particular, in adult lung cells [Ca2+](i) was more than three-fold higher compared to adult skin fibroblasts, a difference which may contribute to tissue-specific functions. Capacitative Ca2+ entry, i.e. the transient [Ca2+](i) increase induced by re-addition of extracellular calcium after depletion of thapsigargin-sensitive calcium stores, was found to exhibit the same pattern of differences during foetal-to-adult transition (it is two-fold higher in adult lung cells than in adult skin cells). At variance, we found capacitative Ca2+ entry to be significantly attenuated during in vivo or in vitro ageing of fibroblasts; while minor alterations of [Ca2+](i) were observed. These findings indicate that capacitative calcium entry rather than [Ca2+](i), is mainly affected during the ageing process. (C) 2002 Elsevier Science Inc. All rights reserved

Induction of Akt by endogenous neurosteroids and calcium sequestration in P19 derived neurons

Neuronal cell death caused by pathophysiological over-activation of glutamate receptors and the subsequent Ca2+ overloading, has been implicated in neurodegeneration after stroke, cerebral trauma and epileptic seizures. Recent findings suggest that certain progesterone metabolites (neurosteroids) such as allopregnanolone and dehydroepiandrosterone can protect neuronal cells from such insults. In the present study, murine P19 cells were induced to differentiate into postmitotic neurons expressing specific neuronal markers, including GABAA and NMDA receptors. Activation of NMDA receptors in P19-N neurons resulted in excitotoxic cell death, which involved suppression of the phosphorylation of the survival kinase PKB/ Akt. Allopregnanolone and DHEA induced a rapid and prolonged phosphorylatio0n of the Akt kinase and they were able to reverse the NMDAinduced suppression of the PI3-K/Akt pathway. The specificity of the neuroprotective effects of these neurosteroids was confirmed by the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin, as well as by the GABAA receptor antagonist, bicuculline. The neurotoxic effect of NMDA on P19-N neurons was directly correlated with increased Ca2+ entry, since the addition of EGTA or BAPTA-AM, significantly suppressed the NMDA-induced decrease of phospho-Akt and subsequent neuronal death. These results suggest that neurosteroids are able to act as survival factors on P19-N neurons, promoting the activation of the PI3-K/Akt pathway through a calcium-entry dependent mechanism. © Springer 2008