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.

Neuroprotective effects of the gliopeptide ODN in an in vivo model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by a progressive loss of dopamine (DA) neurons through apoptotic, inflammatory and oxidative stress mechanisms. The octadecaneuropeptide (ODN) is a diazepam-binding inhibitor (DBI)-derived peptide, expressed by astrocytes, which protects neurons against oxidative cell damages and apoptosis in an in vitro model of PD. The present study reveals that a single intracerebroventricular injection of 10 ng ODN 1 h after the last administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) prevented the degeneration of DA neurons induced by the toxin in the substantia nigra pars compacta of mice, 7 days after treatment. ODN-mediated neuroprotection was associated with a reduction of the number of glial fibrillary acidic protein-positive reactive astrocytes and a strong inhibition of the expression of pro-inflammatory genes such as interleukins 1ß and 6, and tumor necrosis factor-α. Moreover, ODN blocked the inhibition of the anti-apoptotic gene Bcl-2, and the stimulation of the pro-apoptotic genes Bax and caspase-3, induced by MPTP in the substantia nigra pars compacta. ODN also decreased or even in some cases abolished MPTP-induced oxidative damages, overproduction of reactive oxygen species and accumulation of lipid oxidation products in DA neurons. Furthermore, DBI knockout mice appeared to be more vulnerable than wild-type animals to MPTP neurotoxicity. Taken together, these results show that the gliopeptide ODN exerts a potent neuroprotective effect against MPTP-induced degeneration of nigrostriatal DA neurons in mice, through mechanisms involving downregulation of neuroinflammatory, oxidative and apoptotic processes. ODN may, thus, reduce neuronal damages in PD and other cerebral injuries involving oxidative 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.

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.