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. Posttranslational 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 γ-H2AX) occurs rapidly following DNA doublestrand breaks. We investigated γ-H2AX formation in rat cortical neurons (day in vitro 14) following activation of NMDA or AMPA/Kainate glutamate receptors using fluorescent immunohistochemical techniques. Moreover, we evaluated the co-localization of γ-H2AX "foci" with Mre11, a doublestrand 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 is sufficient to evoke γ-H2AX in neurons and that NMDA-induced γ-H2AX foci formation was attenuated by pretreatment with the antioxidant, Vitamin E, and the intracellular calcium chelator, BAPTA-AM. Moreover, a subset of γ-H2AX foci co-localized with Mre11, indicating that Corresponding Author: Alexei Kondratyev, Ph.D., Assistant Professor, Department of Pediatrics, 3970 Reservoir Rd, NW, The Research Building, W217, Washington, D.C. 20057, Phone: 202-687-0204; Fax: 801-469-5580, e-mail: kondrata@georgetown.edu. We would like to thank Dr. Karen Gale for her valuable feedback and helpful comments in the preparation of this manuscript. We would also like to thank Irina Kats for technical assistance. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscriptat least a portion of γ-H2AX foci is damage-dependent. The extent of γ-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 μM)). These data suggest that insults not necessarily resulting in neuronal death induce the DNA damage-evoked chromatin modification, γ-H2AX, and implicate a role for histone alterations in determining neuronal vulnerability following neurological insults.Keywords histone modification; rat; cortical neurons; gamma-H2AX; glutamate receptorsIn addition to DNA repair protein regulation, post-translational histone modifications can mediate the cellular response to DNA damage (Hassa & Hottiger, 2005). Although the importance of functional DNA-repair pathways is underscored by the abnormal neuronal development and vulnerability to injury observed in DNA repair-deficient mice (Vemuri et al., 2001;Culmsee et al., 2001;Meira et al., 2001;Laposa & Cleaver, 2001) and humans (Rolig & McKinnon, 2000;Brooks, 2002), DNA damage-related histone alterations remain relatively unexamined in neurons.It is well documented in non-neuronal cells ...

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“…Note that presence of γH2A.X could also be involved in mitosis and meiosis, neural development, and neurogenesis in the adult brain [50–52]. The formation of γH2A.X is associated with cell cycle suspension in proliferating cells [29], as well as NMDA/AMPA receptor stimulation in differentiated neuronal cells [53]. Finally, the presence of γH2A.X predicts neuronal endangerment and death following a pathological state in the adult brain [54].…”

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“…Moreover, phosphorylation of histone H2A.X (γ-H2A.X) occurs 'generically' after DNA damage (Rogakou et al, 1998), but the same is also observed under physiologically unique circumstances, e.g. stimulation of the inotropic glutamate receptors in cortical neurons (Crowe et al, 2006).…”

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