title: Rapid phosphorylation of histone H2A.X following ionotropic glutamate receptor activation.
abstract: Excessive activation of ionotropic glutamate receptors increases oxidative stress, contributing to the neuronal death observed following neurological insults such as ischemia and seizures. Post-translational histone modifications may be key mediators in the detection and repair of damage resulting from oxidative stress, including DNA damage, and may thus affect neuronal survival in the aftermath of insults characterized by excessive glutamate release. In non-neuronal cells, phosphorylation of histone variant H2A.X (termed gamma-H2AX) occurs rapidly following DNA double-strand breaks. We investigated gamma-H2AX formation in rat cortical neurons (days in vitro 14) following activation of N-methyl-D-aspartate (NMDA) or alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate glutamate receptors using fluorescent immunohistochemical techniques. Moreover, we evaluated the co-localization of gamma-H2AX 'foci' with Mre11, a double-strand break repair protein, to provide further evidence for the activation of this DNA damage response pathway. Here we show that minimally cytotoxic stimulation of ionotropic glutamate receptors was sufficient to evoke gamma-H2AX in neurons, and that NMDA-induced gamma-H2AX foci formation was attenuated by pretreatment with the antioxidant, Vitamin E, and the intracellular calcium chelator, BAPTA-AM. Moreover, a subset of gamma-H2AX foci co-localized with Mre11, indicating that at least a portion of gamma-H2AX foci is damage dependent. The extent of gamma-H2AX induction following glutamate receptor activation corresponded to the increases we observed following conventional DNA damaging agents [i.e. non-lethal doses of gamma-radiation (1 Gy) and hydrogen peroxide (10 microm)]. These data suggest that insults not necessarily resulting in neuronal death induce the DNA damage-evoked chromatin modification, gamma-H2AX, and implicate a role for histone alterations in determining neuronal vulnerability following neurological insults.
authors.affiliation: Interdisciplinary Program in Neuroscience, Georgetown University, Washington, DC, USA.
authors.forename: Samantha L
authors.initials: SL
authors.lastname: Crowe
authors.forename: Vilen A
authors.initials: VA
authors.lastname: Movsesyan
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Jorgensen
authors.forename: Alexei
authors.initials: A
authors.lastname: Kondratyev
mesh_terms: D015763:2-Amino-5-phosphonovalerate / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D002470:Cell Survival / Q000187:drug effects; D002540:Cerebral Cortex / Q000166:cytology; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins / Q000378:metabolism; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D004347:Drug Interactions; D005455:Fluorescent Antibody Technique / Q000379:methods; D005720:Gamma Rays; D015870:Gene Expression / Q000187:drug effects / Q000528:radiation effects; D006657:Histones / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D000076228:MRE11 Homologue Protein; D016202:N-Methylaspartate / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D010766:Phosphorylation / Q000187:drug effects / Q000528:radiation effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D018091:Receptors, AMPA / Q000819:agonists / Q000037:antagonists & inhibitors / Q000502:physiology*; D016194:Receptors, N-Methyl-D-Aspartate / Q000819:agonists / Q000037:antagonists & inhibitors / Q000502:physiology*; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000494:pharmacology
chemical_list: D004268:DNA-Binding Proteins; D006657:Histones; C512690:Mre11 protein, rat; D018091:Receptors, AMPA; D016194:Receptors, N-Methyl-D-Aspartate; D016202:N-Methylaspartate; D016291:Dizocilpine Maleate; D015763:2-Amino-5-phosphonovalerate; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D006861:Hydrogen Peroxide; D000076228:MRE11 Homologue Protein; D045643:DNA Repair Enzymes