Neuroglobin protects astroglial cells from hydrogen peroxide-induced oxidative stress and apoptotic cell death

International audienceOxidative stress, resulting from accumulation of reactive oxygen species, plays a critical role in astroglial cell death occurring in diverse neuropathological conditions. Numerous studies indicate that neuroglobin (Ngb) promotes neuron survival, but nothing is known regarding the action of Ngb in astroglial cell survival. Thus, the purpose of this study was to investigate the potential glioprotective effect of Ngb on hydrogen peroxide (H 2 O 2)-induced oxidative stress and apop-tosis in cultured mouse astrocytes. Incubation of cells with subnanomolar concentrations of Ngb (10 À14 –10 À10 M) was found to prevent both H 2 O 2-evoked reduction in surviving cells number and accumulation of reactive oxygen species in a concentration-dependent manner. Furthermore, Ngb treatment abolishes H 2 O 2-induced increase in mitochondrial oxygen consumption rates. Concomitantly, Ngb treatment rescues H 2 O 2-associated reduced expression of endogenous antiox-idant enzymes (superoxide dismutases and catalase) and prevents the stimulation of the expression of pro-inflammatory genes (inducible nitric oxide synthase, cyclooxygenase-2, and interleukin (IL) IL-6 and IL-33). Moreover, Ngb blocks the stimulation of Bax (pro-apoptotic) and the inhibition of Bcl-2 (anti-apoptotic) gene expression induced by H 2 O 2 , which in turn abolishes caspase 3 activation. The protective effect of Ngb upon H 2 O 2 induced activation of caspase 3 activity and cell death can be accounted for by activation of protein kinase A and mitogen-activated protein kinase transduction cascade. Finally, we demonstrate that Ngb increases Akt phosphoryla-tion and prevents H 2 O 2-provoked inhibition of ERK and Akt phosphorylation. Taken together, these data demonstrate for the first time that Ngb is a glioprotective agent that prevents H 2 O 2-induced oxidative stress and apoptotic astroglial cell death. Protection of astrocytes from oxidative insult may thus contribute to the neuroprotective effect of Ngb

Hemoglobin-Improved Protection in Cultured Cerebral Cortical Astroglial Cells: Inhibition of Oxidative Stress and Caspase Activation

International audienceOxidative stress plays a major role in triggering astroglial cell death in diverse neuropathological conditions such as ischemia and neurodegenerative diseases. Numerous studies indicate that hemoglobin (Hb) is expressed in both resting and reactive glia cells, but nothing is known regarding a possible role of Hb on astroglial cell survival. Thus, the purpose of the present study was to investigate the potential glioprotective effect of Hb on hydrogen peroxide (H2O2)-induced oxidative stress and apoptosis in cultured rat astrocytes. Our study demonstrates that administration of graded concentrations of Hb (10−12 to 10−6 M) to H2O2-treated astrocytes reduces cell death in a concentration-dependent manner. H2O2 treatment induces the accumulation of reactive oxygen species (ROS) and nitric oxide (NO), a drop of the mitochondrial membrane potential, and a stimulation of caspase-3/7 activity. Exposure of H2O2-treated cells to Hb was accompanied by marked attenuations of ROS and NO surproductions, mitochondrial membrane potential reduction, and caspase-3/7 activity increase. The protective action of Hb was blocked by the protein kinase A (PKA) inhibitor H89, the protein kinase C (PKC) inhibitor chelerythrine, and the mitogen-activated protein (MAP)-kinase kinase (MEK) inhibitor U0126. Taken together, these data demonstrate for the first time that Hb is a glioprotective factor that protects astrocytes from apoptosis induced by oxidative stress and suggest that Hb may confer neuroprotection in neurodegenerative diseases. The anti-apoptotic activity of Hb on astrocytes is mediated through the PKA, PKC, and MAPK transduction pathways and can be accounted for by inhibition of oxidative stress-induced mitochondrial dysfunctions and caspase activation

Role of PACAP and VIP in astroglial functions

International audienceAstrocytes represent at least 50% of the volume of the human brain. Besides their roles in various supportive functions, astrocytes are involved in the regulation of stem cell proliferation, synaptic plasticity and neuroprotection. Astrocytes also influence neuronal physiology by responding to neurotransmitters and neuropeptides and by releasing regulatory factors termed gliotransmitters. In particular, astrocytes express the PACAP-specific receptor PAC1-R and the PACAP/VIP mutual receptors VPAC1-R and VPAC2-R during development and/or in the adult. There is now clear evidence that PACAP and VIP modulate a number of astrocyte activities such as proliferation, plasticity, glycogen production, and biosynthesis of neurotrophic factors and gliotransmitters

PACAP Stimulates Biosynthesis and Release of Endozepines from Rat Astrocytes

International audienceAstrocytes synthesize and release endozepines, a family of neuropeptides related to diazepam-binding inhibitor (DBI). Astroglial cells also express the receptors of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP). In the present article, we show that PACAP dose dependently increases DBI gene expression and stimulates endozepine release through activation of PAC1-R. PACAP increases cAMP formation, enhances polyphosphoinositide turnover, and evokes calcium mobilization from intracellular Ca2+ pools. The effect of PACAP on endozepine release is mediated through the adenylyl cyclase/PKA pathway while the downregulation of astrocyte response to PACAP can be ascribed to activation of the PLC/PKC pathway

Neuroprotective effects of PACAP against paraquat-induced oxidative stress in the Drosophila central nervous system

International audienceParkinson's disease (PD) is a progressive neurodegenerative movement disorder that can arise after long-term exposure to environmental oxidative stressors, such as the herbicide paraquat (PQ). Here we investigated the potential neuroprotective action of vertebrate pituitary adenylate cyclase-activating polypeptide (PACAP) against PQ in Drosophila. We found that pre-treatment with this neuropeptide applied to the ventral nerve cord (VNC) at low doses markedly extended the survival of wild-type decapitated flies exposed to neurotoxic levels of PQ or dopamine (DA). In contrast and interestingly, application of a PACAP receptor antagonist, PACAP-6-38, had opposite effects, significantly decreasing the resistance of flies to PQ. PACAP also reduced PQ-induced caspase activation and reactive oxygen species (ROS) accumulation in the VNC. We then searched for the endogenous neuropeptide receptor potentially involved in PACAP-mediated neuroprotection in Drosophila. Knocking down the gene encoding the receptor Han/PDFR of the neuropeptide pigment-dispersing factor (PDF) in all neurons conferred to flies higher resistance to PQ, whereas PDFR downregulation restricted to PDF or DA neurons did not increase PQ resistance, but remarkably suppressed the neuroprotective action of PACAP. Further experiments performed with Pdf and Pdfr-deficient mutant strains confirmed that PDF and its receptor are required for PACAP-mediated neuroprotection in flies. We also provide evidence using split-green fluorescent protein (split-GFP) reconstitution that PDF neurons make synaptic contacts onto DA neurons in the abdominal VNC. Our results therefore suggest that the protective action of PACAP against PQ-induced defects in the Drosophila nervous system involves the modulation of PDFR signaling in a small number of interconnected neurons

Functional role of ODN, an endogenous peptide released by astrocyte, under oxydatif stress

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Induction of Neuronal Differentiation of Murine N2a Cells by Two Polyphenols Present in the Mediterranean Diet Mimicking Neurotrophins Activities: Resveratrol and Apigenin

In the prevention of neurodegeneration associated with aging and neurodegenerative diseases (Alzheimer’s disease, Parkinson’s disease), neuronal differentiation is of interest. In this context, neurotrophic factors are a family of peptides capable of promoting the growth, survival, and/or differentiation of both developing and immature neurons. In contrast to these peptidyl compounds, polyphenols are not degraded in the intestinal tract and are able to cross the blood–brain barrier. Consequently, they could potentially be used as therapeutic agents in neurodegenerative pathologies associated with neuronal loss, thus requiring the stimulation of neurogenesis. We therefore studied the ability to induce neuronal differentiation of two major polyphenols present in the Mediterranean diet: resveratrol (RSV), a major compound found in grapes and red wine, and apigenin (API), present in parsley, rosemary, olive oil, and honey. The effects of these compounds (RSV and API: 6.25–50 µM) were studied on murine neuro-2a (N2a) cells after 48 h of treatment without or with 10% fetal bovine serum (FBS). Retinoic acid (RA: 6.25–50 µM) was used as positive control. Neuronal differentiation was morphologically evaluated through the presence of dendrites and axons. Cell growth was determined by cell counting and cell viability by staining with fluorescein diacetate (FDA). Neuronal differentiation was more efficient in the absence of serum than with 10% FBS or 10% delipidized FBS. At concentrations inducing neuronal differentiation, no or slight cytotoxicity was observed with RSV and API, whereas RA was cytotoxic. Without FBS, RSV and API, as well as RA, trigger the neuronal differentiation of N2a cells via signaling pathways simultaneously involving protein kinase A (PKA)/phospholipase C (PLC)/protein kinase C (PKC) and MEK/ERK. With 10% FBS, RSV and RA induce neuronal differentiation via PLC/PKC and PKA/PLC/PKC, respectively. With 10% FBS, PKA and PLC/PKC as well as MEK/ERK signaling pathways were not activated in API-induced neuronal differentiation. In addition, the differentiating effects of RSV and API were not inhibited by cyclo[DLeu5] OP, an antagonist of octadecaneuropeptide (ODN) which is a neurotrophic factor. Moreover, RSV and API do not stimulate the expression of the diazepam-binding inhibitor (DBI), the precursor of ODN. Thus, RSV and API are able to induce neuronal differentiation, ODN and its receptor are not involved in this process, and the activation of the (PLC/PKC) signaling pathway is required, except with apigenin in the presence of 10% FBS. These data show that RSV and API are able to induce neuronal differentiation and therefore mimic neurotrophin activity. Thus, RSV and API could be of interest in regenerative medicine to favor neurogenesis

Functional role of ODN, an endogenous peptide released by astrocyte, under oxydatif stress

National audienc

Characterization of Cell Death Induced by Imine Analogs of Trans-Resveratrol: Induction of Mitochondrial Dysfunction and Overproduction of Reactive Oxygen Species Leading to, or Not, Apoptosis without the Increase in the S-Phase of the Cell Cycle

Trans-resveratrol (RSV) is a non-flavonoid polyphenol (stilbene) with numerous biological activities, such as anti-tumor activities. However, RSV is rapidly metabolized, which limits its therapeutic use. The availability of RSV analogues with similar activities for use in vivo is therefore a major challenge. For this purpose, several isomeric analogues of RSV, aza-stilbenes (AZA-ST 1a–g), were synthesized, and their toxicities were characterized and compared to those of RSV on murine N2a neuronal cells using especially flow cytometric methods. All AZA-ST 1a–g have an inhibitory concentration 50 (IC50) between 11.3 and 25 µM when determined by the crystal violet assay, while that of RSV is 14.5 µM. This led to the characterization of AZA-ST 1a–g—induced cell death, compared to RSV, using three concentrations encompassing the IC50s (6.25, 12.5 and 25 µM). For AZA-ST 1a–g and RSV, an increase in plasma membrane permeability to propidium iodide was observed, and the proportion of cells with depolarized mitochondria measured with DiOC6(3) was increased. An overproduction of reactive oxygen species (ROS) was also observed on whole cells and at the mitochondrial level using dihydroethidium and MitoSox Red, respectively. However, only RSV induced a mode of cell death by apoptosis associated with a marked increase in the proportion of cells with condensed and/or fragmented nuclei (12.5 µM: 22 ± 9%; 25 µM: 80 ± 10%) identified after staining with Hoechst 33342 and which are characteristic of apoptotic cells. With AZA-ST, a slight but significant increase in the percentage of apoptotic cells was only detected with AZA-ST 1b (25 µM: 17 ± 1%) and AZA-ST 1d (25 µM: 26 ± 4%). Furthermore, only RSV induced significant cell cycle modifications associated with an increase in the percentage of cells in the S phase. Thus, AZA-ST 1a–g—induced cell death is characterized by an alteration of the plasma membrane, an induction of mitochondrial depolarization (loss of ΔΨm), and an overproduction of ROS, which may or may not result in a weak induction of apoptosis without modification of the distribution of the cells in the different phases of the cell cycle

Octadecaneuropeptide ODN prevents hydrogen peroxide-induced oxidative damage of biomolecules in cultured rat astrocytes.

International audienceOxidative stress, associated with a variety of disorders including neurodegenerative diseases, is a major cause of cellular dysfunction and biomolecule damages which play a crucial role in neuronal apoptosis. Astrocytes specifically synthesize and release endozepines, a family of regulatory peptides, including the octadecaneuropeptide ODN. We have recently shown that ODN is a potent glioprotective agent that prevents hydrogen peroxide (H2O2)-induced oxidative stress and apoptosis. The purpose of the present study was to investigate the potential protective effect of ODN on oxidative-generated damage of biomolecules in cultured rat astrocytes. Incubation of cells with subnanomolar concentrations of ODN (0.1fM-0.1nM) inhibited H2O2-evoked reactive oxygen species accumulation and cell death in a concentration-dependent manner. Exposure of H2O2-treated cells to 0.1nM ODN inhibited superoxide anion generation and blocked oxidative damage of cell molecules caused by H2O2i.e. formation and accumulation of lipid oxidation products, malondialdehydes and conjugated dienes, and protein carbonyl compounds. Taken together, these data demonstrate for the first time that ODN prevents oxidative stress-induced alteration of cellular constituents. ODN is thus a potential candidate to reduce neuronal damage in various pathological conditions involving oxidative neurodegeneration