ABSTRACT
Group I metabotropic glutamate (mGlu) receptors (i.e. mGlu1 and mGlu5) coupled to phospholipase C have been widely investigated for their possible role in excitotoxic and post-ischemic neuronal death. Recently, phospholipase C has been shown to directly stimulate the activity of poly(ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair that has been proposed to play a key role in necrotic cell death. In this study, we investigated whether the stimulation of group I mGlu receptors leads to an increase in PARP activity, as detected by flow cytometry, immunodot blot and immunocytochemistry, both in baby hamster kidney cells transfected with mGlu1a or mGlu5a receptors and in cultured cortical cells. Our results show that the group I mGlu receptor agonist DHPG elicited a significant increase in PARP activity that was completely abolished by the administration of the mGlu1 antagonist 3-MATIDA and partially prevented, in cortical neurons, by the mGlu5 antagonist MPEP. To evaluate whether this pathway is involved in post-ischemic neuronal death, we used a sublethal model of oxygen-glucose deprivation in mixed cortical cell cultures. DHPG exacerbated neuronal death, and this effect was significantly prevented by the application of the PARP inhibitor DPQ. This novel pathway may contribute to the effects of mGlu1 receptors in the mechanisms leading to post-ischemic neuronal death.
Subject(s)
Cerebral Cortex/cytology , Neuroglia/enzymology , Neurons/enzymology , Poly(ADP-ribose) Polymerases/metabolism , Receptors, Metabotropic Glutamate/physiology , Animals , Animals, Newborn , Blotting, Western/methods , Cells, Cultured , Cricetinae , Drug Interactions , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Fluorescent Antibody Technique/methods , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Enzymologic/physiology , Glial Fibrillary Acidic Protein/metabolism , Glucose/deficiency , Hydrogen Peroxide/pharmacology , Hypoxia/enzymology , Isoquinolines/pharmacology , Methoxyhydroxyphenylglycol/analogs & derivatives , Methoxyhydroxyphenylglycol/pharmacology , Mice , Neuroglia/drug effects , Neurons/drug effects , Piperidines/pharmacology , Pyridines/pharmacology , Thiophenes/pharmacology , Time Factors , Transfection/methodsABSTRACT
Overactivation of the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) plays a key role in the mechanisms responsible for neuronal death. In the present study, we examined the effects of the PARP-1 inhibitor 3,4-dihydro-5-[4-1(1-piperidinyl)buthoxy]-1(2H)-isoquinolinone (DPQ) in two models of N-methyl-d-aspartate (NMDA)-induced neurotoxicity. The exposure of mixed cultured cortical cells to 300 microM NMDA for 10 min induced a caspase-dependent type of apoptotic neuronal death. Conversely, exposure to 2 mM NMDA for 10 min led to the appearance of morphological features of necrosis, with no increase in caspase-3 activity and depletion in adenosine triphosphate (ATP) levels. DPQ (10 microM) reduced the NMDA-induced PARP activation, restored ATP to near control levels and significantly attenuated neuronal injury only in the severe NMDA exposure model. Similar results were obtained when pure neuronal cortical cultures were used. PARP-1 activation thus appears to play a preferential role in necrotic than in caspase-dependent apoptotic neuronal death.
Subject(s)
Apoptosis/physiology , Cerebral Cortex/metabolism , Neurons/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Adenosine Triphosphate/metabolism , Animals , Apoptosis/drug effects , Caspase 3 , Caspases/drug effects , Caspases/metabolism , Cells, Cultured , Cerebral Cortex/drug effects , Cerebral Cortex/physiopathology , Coculture Techniques , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Isoquinolines/pharmacology , Mice , Models, Biological , N-Methylaspartate/toxicity , Necrosis , Neurons/drug effects , Neurons/pathology , Neurotoxins/toxicity , Piperidines/pharmacology , Poly(ADP-ribose) Polymerase InhibitorsABSTRACT
Poly(ADP-ribose) polymerases (PARPs) are a group of protein-modifying and nucleotide-polymerizing enzymes able to catalyze the transfer of multiple ADP-ribose units from NAD to substrate proteins. In the human genome, 16 different genes encoding for members of this emerging family of enzymes have been identified. Known family members are PARP-1, PARP-2, PARP-3, vPARP, tankyrase 1 and tankyrase 2, each of them with a possible specific role in cell biology. The most studied member of the family is PARP-1, which is abundantly present in the nucleus and is involved in the maintenance of genomic stability. In pathological conditions, highly reactive radical species may cause DNA damage and PARP-1 hyperactivation. This may lead to necrotic cell death through massive NAD consumption. We show that following middle cerebral artery occlusion, rats treated with PARP inhibitors displayed reduced brain infarct volumes. Similarly, PARP inhibitors reduced neuronal death induced by oxygen-glucose deprivation (OGD) or excitotoxins in primary cultures of murine cortical cells. On the contrary, PARP inhibitors did not attenuate the OGD-induced selective loss of CA1 pyramidal cells in rat organotypic hippocampal slices. In addition, they were not neuroprotective against transient bilateral carotid occlusion in gerbils. We observed that post-ischemic brain damage was predominally necrotic in cultured cortical cells, whereas a caspase-dependent apoptotic process was responsible for the CA1 pyramidal cell loss in hippocampal slices. Hence, it appears reasonable to propose PARP inhibitors as useful therapeutic agents in pathological brain conditions were necrosis predominates.
