Long-term protective effect of PACAP in a fetal alcohol syndrome (FAS) model.

Prenatal ethanol exposure provokes teratogenic effects, due to oxidative stress and massive neuronal apoptosis in the developing brain that result in lifelong behavioral abnormalities. PACAP exerts anti-oxidative and neuroprotective activities on neuronal cells, and prevents ethanol neurotoxicity. The present study focused on the ability of PACAP to protect the brain of 30-day-old mice (P30) from prenatal alcohol exposure induced oxidative damage and toxicity. Pregnant mice were divided randomly into 4 groups, i.e. control group, ethanol group (1.5 g/kg ip daily injection), PACAP group (5 µg intrauterine daily injection) and an ethanol plus PACAP group. Offspring prenatally exposed to ethanol had decreased body weight and reduced cell survival. Moreover, production of ROS was sharply enhanced in the brain of prenatal ethanol-exposed animals, associated with an elevation in the activity of the antioxidant enzymes, and an increase of oxidative damages as shown by the accumulation of the lipid oxidation marker malondialdehyde and of protein carbonyl compounds. Intrauterine administration of PACAP during the gestational period restored the endogenous antioxidant system, prevented ROS overproduction and promoted the survival of dissociated cells from animals prenatally exposed to ethanol. Behavioral tests revealed that P30 animals exposed to ethanol during the prenatal period exhibited reduced motor activity, altered exploratory interest and increased anxiety. However, PACAP treatment significantly attenuated these behavioral impairments. This study demonstrates that PACAP exerts a potent neuroprotective effect against alcohol toxicity during brain development, and indicates that PACAP and/or PACAP analogs might be a useful tool for treatment of alcohol intoxication during pregnancy.

Cytoprotective and Neurotrophic Effects of Octadecaneuropeptide (ODN) in in vitro and in vivo Models of Neurodegenerative Diseases.

Octadecaneuropeptide (ODN) and its precursor diazepam-binding inhibitor (DBI) are peptides belonging to the family of endozepines. Endozepines are exclusively produced by astroglial cells in the central nervous system of mammals, and their release is regulated by stress signals and neuroactive compounds. There is now compelling evidence that the gliopeptide ODN protects cultured neurons and astrocytes from apoptotic cell death induced by various neurotoxic agents. In vivo, ODN causes a very strong neuroprotective action against neuronal degeneration in a mouse model of Parkinson's disease. The neuroprotective activity of ODN is based on its capacity to reduce inflammation, apoptosis, and oxidative stress. The protective effects of ODN are mediated through its metabotropic receptor. This receptor activates a transduction cascade of second messengers to stimulate protein kinase A (PKA), protein kinase C (PKC), and mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) signaling pathways, which in turn inhibits the expression of proapoptotic factor Bax and the mitochondrial apoptotic pathway. In N2a cells, ODN also promotes survival and stimulates neurite outgrowth. During the ODN-induced neuronal differentiation process, numerous mitochondria and peroxisomes are identified in the neurites and an increase in the amount of cholesterol and fatty acids is observed. The antiapoptotic and neurotrophic properties of ODN, including its antioxidant, antiapoptotic, and pro-differentiating effects, suggest that this gliopeptide and some of its selective and stable derivatives may have therapeutic value for the treatment of some neurodegenerative diseases.

Antioxidant and Anti-Apoptotic Activity of Octadecaneuropeptide Against 6-OHDA Toxicity in Cultured Rat Astrocytes.

Oxidative stress, associated with various neurodegenerative diseases, promotes ROS generation, impairs cellular antioxidant defenses, and finally, triggers both neurons and astroglial cell death by apoptosis. Astrocytes specifically synthesize and release endozepines, a family of regulatory peptides, including the octadecaneuropeptide (ODN). We have previously reported that ODN acts as a potent neuroprotective agent that prevents 6-OHDA-induced apoptotic neuronal death. The purpose of the present study was to investigate the potential glioprotective effect of ODN on 6-OHDA-induced oxidative stress and cell death in cultured rat astrocytes. Incubation of astrocytes with graded concentrations of ODN (10-14 to 10-8 M) inhibited 6-OHDA-evoked cell death in a concentration- and time-dependent manner. In addition, ODN prevented the decrease of mitochondrial activity and caspase-3 activation induced by 6-OHDA. 6-OHDA-treated cells also exhibited enhanced levels of ROS associated with a generation of H2O2 and O2°-, and a reduction of both superoxide dismutase (SOD) and catalase (CAT) activities. Co-treatment of astrocytes with low concentrations of ODN dose-dependently blocked 6-OHDA-evoked production of ROS and inhibition of antioxidant enzyme activities. Concomitantly, ODN stimulated Mn-SOD, CAT, glutathione peroxidase-1, and sulfiredoxin-1 gene transcription and rescued 6-OHDA-associated reduced expression of endogenous antioxidant enzymes. Taken together, these data indicate that, in rat astrocytes, ODN exerts anti-apoptotic and anti-oxidative activities, and hence prevents 6-OHDA-induced oxidative assault and cell death. ODN is thus a potential candidate to delay neuronal damages in various pathological conditions involving oxidative neurodegeneration.

Neuroprotection with the Endozepine Octadecaneuropeptide, ODN.

The term endozepines designates a family of astroglia-secreted proteins including the diazepambinding inhibitor (DBI) and its processing products, which have been originally isolated and characterized as endogenous ligands of benzodiazepine receptors. It is now clearly established that the octadecaneuropeptide ODN (DBI33-50), acting through the central-type benzodiazepine receptor or a metabotropic receptor, exerts important functions such as proconflict behavior, induction of anxiety, inhibition of pentobarbital-provoked sleep, decrease of water consumption and reduction of food intake. To mediate its effects, ODN regulates both glial cell and neuronal activities by acting on neurosteroid biosynthesis and/or neuropeptide expression. In addition, ODN stimulates astrocyte proliferation and protects both neurons and astrocytes from oxidative stress-induced cell death. The antiapoptotic effect of ODN on neural cells is mediated through activation of the ODN metabotropic receptor positively coupled to PKA, PKC and MAPK/ERK transduction pathways, which ultimately reduces the pro-apoptotic gene Bax and stimulates Bcl-2 expressions, and inhibits intracellular reactive oxygen species accumulation. The imbalance in favor of Bcl2 promotes mitochondria functions and blocks in turn caspases activation while at the same time, ODN also activates the endogenous antioxidant system i.e. glutathione biosynthesis, and expression and activities of antioxidant enzymes. In cultured astrocytes, DBI expression is up-regulated during moderate oxidative stress, and authentic ODN production is increased, suggesting that ODN may act as a paracrine factor protecting neighboring neurons. Taken together, the remarkable effect of ODN on the apoptotic cascade suggests that innovative ODN derivatives could potentially be useful for treatment of cerebral injuries involving oxidative stress and neurodegeneration.

Endogenous Expression of ODN-Related Peptides in Astrocytes Contributes to Cell Protection Against Oxidative Stress: Astrocyte-Neuron Crosstalk Relevance for Neuronal Survival.

Astroglial cells are important actors in the defense of brain against oxidative stress injuries. Glial cells synthesize and release the octadecaneuropeptide ODN, a diazepam-binding inhibitor (DBI)-related peptide, which acts through its metabotropic receptor to protect neurons and astrocytes from oxidative stress-induced apoptosis. The purpose of the present study is to examine the contribution of the endogenous ODN in the protection of astrocytes and neurons from moderate oxidative stress. The administration of H2O2 (50 µM, 6 h) induced a moderate oxidative stress in cultured astrocytes, i.e., an increase in reactive oxygen species, malondialdehyde, and carbonyl group levels, but it had no effect on astrocyte death. Mass spectrometry and QPCR analysis revealed that 50 µM H2O2 increased ODN release and DBI mRNA levels. The inhibition of ODN release or pharmacological blockage of the effects of ODN revealed that in these conditions, 50 µM H2O2 induced the death of astrocytes. The transfection of astrocytes with DBI siRNA increased the vulnerability of cells to moderate stress. Finally, the addition of 1 nM ODN to culture media reversed cell death observed in DBI-deficient astrocytes. The treatment of neurons with media from 50 µM H2O2-stressed astrocytes significantly reduced the neuronal death induced by H2O2; this effect is greatly attenuated by the administration of an ODN metabotropic receptor antagonist. Overall, these results indicate that astrocytes produce authentic ODN, notably in a moderate oxidative stress situation, and this glio- and neuro-protective agent may form part of the brain defense mechanisms against oxidative stress injury.

Involvement of endogenous antioxidant systems in the protective activity of pituitary adenylate cyclase-activating polypeptide against hydrogen peroxide-induced oxidative damages in cultured rat astrocytes.

Astroglial cells possess an array of cellular defense mechanisms, including superoxide dismutase (SOD) and catalase antioxidant enzymes, to prevent damages caused by oxidative stress. Nevertheless, astroglial cell viability and functionality can be affected by significant oxidative stress. We have previously shown that pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent glioprotective agent that prevents hydrogen peroxide (H2 O2 )-induced apoptosis in cultured astrocytes. The purpose of this study was to investigate the potential protective effect of PACAP against oxidative-generated alteration of astrocytic antioxidant systems. Incubation of cells with subnanomolar concentrations of PACAP inhibited H2 O2 -evoked reactive oxygen species accumulation, mitochondrial respiratory burst, and caspase-3 mRNA level increase. PACAP also stimulated SOD and catalase activities in a concentration-dependent manner, and counteracted the inhibitory effect of H2 O2 on the activity of these two antioxidant enzymes. The protective action of PACAP against H2 O2 -evoked inhibition of antioxidant systems in astrocytes was protein kinase A, PKC, and MAP-kinase dependent. In the presence of H2 O2 , the SOD blocker NaCN and the catalase inhibitor 3-aminotriazole, both suppressed the protective effects of PACAP on SOD and catalase activities, mitochondrial function, and cell survival. Taken together, these results indicate that the anti-apoptotic effect of PACAP on astroglial cells can account for the activation of endogenous antioxidant enzymes and reduction in respiration rate, thus preserving mitochondrial integrity and preventing caspase-3 expression provoked by oxidative stress. Considering its powerful anti-apoptotic and anti-oxidative properties, the PACAPergic signaling system should thus be considered for the development of new therapeutical approaches to cure various pathologies involving oxidative neurodegeneration. We propose the following cascade for the glioprotective action of Pituitary adenylate cyclase-activating polypeptide (PACAP) against H2 O2 -induced astrocyte damages and cell apoptosis in cultured rat astrocytes. PACAP, through activation of its receptor, PAC1-R, and the protein kinase A (PKA), protein kinase C (PKC), and MAP-kinases signaling pathways, prevents accumulation of ROS and inhibition of SOD and catalase activities. This allows the preservation of mitochondrial membrane integrity and the reduction in caspase-3 activation induced by H2 O2 . These data may lead to the development of new strategies for cerebral injury treatment. Cat, catalase; Cyt. C, cytochrome C; SOD, superoxide dismutase.

Prenatal exposure to cigarette smoke enhances oxidative stress in astrocytes of neonatal rat.

Oxidative stress is involved in the pathogenesis of smoking-related disease. Protection of astrocytes from oxidative insult appears essential to maintain brain function. In this study, we have investigated the effect of gestational cigarette exposure on astrocyte survival. Pregnant female were randomly allocated to the control group or to the cigarette smoke group in which they were placed in an exposure chamber and inhale three cigarettes smoke twice a day for a period of 20 days. The control group was kept in the exposure chamber for the same duration, but without exposure to cigarette smoke. Newborn rats from both groups were weighed 24 h after birth and then cerebral hemispheres were collected for astrocyte culture. Incubation of astrocytes isolated from animals exposed to cigarette smoke with 300 µM H2O2 for 1 h induced a significant decrease of the proportion of surviving cells compared to cells isolated form control animals. We have observed that H2O2-treated astroglial cells derived from cigarette smoke exposure showed more reduced superoxide dismutase and catalase activities than H2O2-treated astroglial cells from control animals. In conclusion, this study indicates that astroglial cells derived from newborn rats exposed in utero to cigarette smoke are more vulnerable to oxidative assault than cultured astrocytes obtained from control animals. These results point out the existence of excitotoxic lesions in newborn exposed in utero to cigarette smoke and suggest that despite their high antioxidative activities, astrocytes cannot survive and protect neurons under massive oxidative stress.

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

Oxidative 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.

The octadecaneuropeptide ODN prevents 6-hydroxydopamine-induced apoptosis of cerebellar granule neurons through a PKC-MAPK-dependent pathway.

Oxidative stress, induced by various neurodegenerative diseases, initiates a cascade of events leading to apoptosis, and thus plays a critical role in neuronal injury. In this study, we have investigated the potential neuroprotective effect of the octadecaneuropeptide (ODN) on 6-hydroxydopamine (6-OHDA)-induced oxidative stress and apoptosis in cerebellar granule neurons (CGN). ODN, which is produced by astrocytes, is an endogenous ligand for both central-type benzodiazepine receptors (CBR) and a metabotropic receptor. Incubation of neurons with subnanomolar concentrations of ODN (10⁻¹8 to 10⁻¹² M) inhibited 6-OHDA-evoked cell death in a concentration-dependent manner. The effect of ODN on neuronal survival was abrogated by the metabotropic receptor antagonist, cyclo1₋8 [DLeu5]OP, but not by a CBR antagonist. ODN stimulated polyphosphoinositide turnover and ERK phosphorylation in CGN. The protective effect of ODN against 6-OHDA toxicity involved the phospholipase C/ERK MAPK transduction cascade. 6-OHDA treatment induced an accumulation of reactive oxygen species, an increase of the expression of the pro-apoptotic gene Bax, a drop of the mitochondrial membrane potential and a stimulation of caspase-3 activity. Exposure of 6-OHDA-treated cells to ODN blocked all the deleterious effects of the toxin. Taken together, these data demonstrate for the first time that ODN is a neuroprotective agent that prevents 6-OHDA-induced oxidative stress and apoptotic cell death.

The stimulatory effect of the octadecaneuropeptide ODN on astroglial antioxidant enzyme systems is mediated through a GPCR.

Astroglial cells possess an array of cellular defense systems, including superoxide dismutase (SOD) and catalase antioxidant enzymes, to prevent damage caused by oxidative stress on the central nervous system. Astrocytes specifically synthesize and release endozepines, a family of regulatory peptides including the octadecaneuropeptide (ODN). ODN is the ligand of both central-type benzodiazepine receptors (CBR), and an adenylyl cyclase- and phospholipase C-coupled receptor. We have recently shown that ODN is a potent protective agent that prevents hydrogen peroxide (H(2)O(2))-induced inhibition of SOD and catalase activities and stimulation of cell apoptosis in astrocytes. The purpose of the present study was to investigate the type of receptor involved in ODN-induced inhibition of SOD and catalase in cultured rat astrocytes. We found that ODN induced a rapid stimulation of SOD and catalase gene transcription in a concentration-dependent manner. In addition, 0.1 nM ODN blocked H(2)O(2)-evoked reduction of both mRNA levels and activities of SOD and catalase. Furthermore, the inhibitory actions of ODN on the deleterious effects of H(2)O(2) on SOD and catalase were abrogated by the metabotropic ODN receptor antagonist cyclo(1-8)[Dleu(5)]OP, but not by the CBR antagonist flumazenil. Finally, the protective action of ODN against H(2)O(2)-evoked inhibition of endogenous antioxidant systems in astrocytes was protein kinase A (PKA)-dependent, but protein kinase C-independent. Taken together, these data demonstrate for the first time that ODN, acting through its metabotropic receptor coupled to the PKA pathway, prevents oxidative stress-induced alteration of antioxidant enzyme expression and activities. The peptide ODN is thus a potential candidate for the development of specific agonists that would selectively mimic its protective activity.

The octadecaneuropeptide ODN protects astrocytes against hydrogen peroxide-induced apoptosis via a PKA/MAPK-dependent mechanism.

Astrocytes synthesize and release endozepines, a family of regulatory peptides, including the octadecaneuropeptide (ODN) an endogenous ligand of both central-type benzodiazepine (CBR) and metabotropic receptors. We have recently shown that ODN exerts a protective effect against hydrogen peroxide (H(2)O(2))-induced oxidative stress in astrocytes. The purpose of the present study was to determine the type of receptor and the transduction pathways involved in the protective effect of ODN in cultured rat astrocytes. We have first observed a protective activity of ODN at very low concentrations that was abrogated by the metabotropic ODN receptor antagonist cyclo(1-8)[DLeu(5)]OP, but not by the CBR antagonist flumazenil. We have also found that the metabotropic ODN receptor is positively coupled to adenylyl cyclase in astrocytes and that the glioprotective action of ODN upon H(2)O(2)-induced astrocyte death is PKA- and MEK-dependent, but PLC/PKC-independent. Downstream of PKA, ODN induced ERK phosphorylation, which in turn activated the expression of the anti-apoptotic gene Bcl-2 and blocked the stimulation by H(2)O(2) of the pro-apoptotic gene Bax. The effect of ODN on the Bax/Bcl-2 balance contributed to abolish the deleterious action of H(2)O(2) on mitochondrial membrane integrity and caspase-3 activation. Finally, the inhibitory effect of ODN on caspase-3 activity was shown to be PKA and MEK-dependent. In conclusion, the present results demonstrate that the potent glioprotective action of ODN against oxidative stress involves the metabotropic ODN receptor coupled to the PKA/ERK-kinase pathway to inhibit caspase-3 activation.

Protective effect of the octadecaneuropeptide on hydrogen peroxide-induced oxidative stress and cell death in cultured rat astrocytes.

Oxidative stress, resulting from accumulation of reactive oxygen species (ROS), plays a critical role on astrocyte death associated with neurodegenerative diseases. Astroglial cells produce endozepines, a family of biologically active peptides that have been implicated in cell protection. Thus, the purpose of the present study was to investigate the potential protective effect of one of the endozepines, the octadecaneuropeptide ODN, on hydrogen peroxide (H(2) O(2) )-induced oxidative stress and cell death in rat astrocytes. Incubation of cultured astrocytes with graded concentrations of H(2) O(2) for 1 h provoked a dose-dependent reduction of the number of living cells as evaluated by lactate dehydrogenase assay. The cytotoxic effect of H(2) O(2) was associated with morphological modifications that were characteristic of apoptotic cell death. H(2) O(2) -treated cells exhibited high level of ROS associated with a reduction of both superoxide dismutases (SOD) and catalase activities. Pre-treatment of astrocytes with low concentrations of ODN dose-dependently prevented cell death induced by H(2) O(2) . This effect was accompanied by a marked attenuation of ROS accumulation, reduction of mitochondrial membrane potential and activation of caspase 3 activity. ODN stimulated SOD and catalase activities in a concentration-dependent manner, and blocked H(2) O(2) -evoked inhibition of SOD and catalase activities. Blockers of SOD and catalase suppressed the effect of ODN on cell survival. Taken together, these data demonstrate for the first time that ODN is a potent protective agent that prevents oxidative stress-induced apoptotic cell death.

Pituitary adenylate cyclase-activating polypeptide protects astroglial cells against oxidative stress-induced apoptosis.

Oxidative stress, associated with a variety of disorders including neurodegenerative diseases, results from accumulation of reactive oxygen species (ROS). Oxidative stress is not only responsible for neuron apoptosis, but can also provoke astroglial cell death. Numerous studies indicate that pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neuron survival, but nothing is known regarding the action of PACAP on astroglial cell survival. Thus, the purpose of the present study was to investigate the potential glioprotective effect of PACAP on H(2)O(2)-induced astrocyte death. Pre-treatment of cultured rat astrocytes with nanomolar concentrations of PACAP prevented cell death provoked by H(2)O(2) (300 µM), whereas vasoactive intestinal polypeptide was devoid of protective activity. The effect of PACAP on astroglial cell survival was abolished by the type 1 PACAP receptor antagonist, PACAP6-38. The protective action of PACAP was blocked by the protein kinase A inhibitor H89, the protein kinase C inhibitor chelerythrine and the mitogen-activated protein (MAP)-kinase kinase (MEK) inhibitor U0126. PACAP stimulated glutathione formation, and blocked H(2)O(2)-evoked ROS accumulation and glutathione content reduction. In addition, PACAP prevented the decrease of mitochondrial activity and caspase 3 activation induced by H(2)O(2). Taken together, these data indicate for the first time that PACAP, acting through type 1 PACAP receptor, exerts a potent protective effect against oxidative stress-induced astrocyte death. The anti-apoptotic activity of PACAP on astrocytes is mediated through the protein kinase A, protein kinase C and MAPK transduction pathways, and can be accounted for by inhibition of ROS-induced mitochondrial dysfunctions and caspase 3 activation.