Intranuclear localization of apoptosis-inducing factor (AIF) and large scale dna fragmentation after traumatic brain injury in rats and in neuronal cultures exposed to peroxynitrite

Authors

  • Xiaopeng Zhang,

    1. Departments of *Anesthesiology and Critical Care Medicine, †Neurology, §Pediatrics and ¶Cell Biology and Physiology, the Safar Center for Resuscitation Research and the Brain Trauma Research Center, University of Pittsburgh, Pittsburgh, Pennsylvania ‡Geriatric Research Educational and Clinical Center, Veterans Affairs Pittsburgh Health System, Pittsburgh, Pennsylvania **Inotek Corporation, Beverly, Massachusetts
    Search for more papers by this author
  • Jun Chen,

    1. Departments of *Anesthesiology and Critical Care Medicine, †Neurology, §Pediatrics and ¶Cell Biology and Physiology, the Safar Center for Resuscitation Research and the Brain Trauma Research Center, University of Pittsburgh, Pittsburgh, Pennsylvania ‡Geriatric Research Educational and Clinical Center, Veterans Affairs Pittsburgh Health System, Pittsburgh, Pennsylvania **Inotek Corporation, Beverly, Massachusetts
    Search for more papers by this author
  • Steven H. Graham,

    1. Departments of *Anesthesiology and Critical Care Medicine, †Neurology, §Pediatrics and ¶Cell Biology and Physiology, the Safar Center for Resuscitation Research and the Brain Trauma Research Center, University of Pittsburgh, Pittsburgh, Pennsylvania ‡Geriatric Research Educational and Clinical Center, Veterans Affairs Pittsburgh Health System, Pittsburgh, Pennsylvania **Inotek Corporation, Beverly, Massachusetts
    Search for more papers by this author
  • Lina Du,

    1. Departments of *Anesthesiology and Critical Care Medicine, †Neurology, §Pediatrics and ¶Cell Biology and Physiology, the Safar Center for Resuscitation Research and the Brain Trauma Research Center, University of Pittsburgh, Pittsburgh, Pennsylvania ‡Geriatric Research Educational and Clinical Center, Veterans Affairs Pittsburgh Health System, Pittsburgh, Pennsylvania **Inotek Corporation, Beverly, Massachusetts
    Search for more papers by this author
  • Patrick M. Kochanek,

    1. Departments of *Anesthesiology and Critical Care Medicine, †Neurology, §Pediatrics and ¶Cell Biology and Physiology, the Safar Center for Resuscitation Research and the Brain Trauma Research Center, University of Pittsburgh, Pittsburgh, Pennsylvania ‡Geriatric Research Educational and Clinical Center, Veterans Affairs Pittsburgh Health System, Pittsburgh, Pennsylvania **Inotek Corporation, Beverly, Massachusetts
    Search for more papers by this author
  • Romesh Draviam,

    1. Departments of *Anesthesiology and Critical Care Medicine, †Neurology, §Pediatrics and ¶Cell Biology and Physiology, the Safar Center for Resuscitation Research and the Brain Trauma Research Center, University of Pittsburgh, Pittsburgh, Pennsylvania ‡Geriatric Research Educational and Clinical Center, Veterans Affairs Pittsburgh Health System, Pittsburgh, Pennsylvania **Inotek Corporation, Beverly, Massachusetts
    Search for more papers by this author
  • Fengli Guo,

    1. Departments of *Anesthesiology and Critical Care Medicine, †Neurology, §Pediatrics and ¶Cell Biology and Physiology, the Safar Center for Resuscitation Research and the Brain Trauma Research Center, University of Pittsburgh, Pittsburgh, Pennsylvania ‡Geriatric Research Educational and Clinical Center, Veterans Affairs Pittsburgh Health System, Pittsburgh, Pennsylvania **Inotek Corporation, Beverly, Massachusetts
    Search for more papers by this author
  • Paula D. Nathaniel,

    1. Departments of *Anesthesiology and Critical Care Medicine, †Neurology, §Pediatrics and ¶Cell Biology and Physiology, the Safar Center for Resuscitation Research and the Brain Trauma Research Center, University of Pittsburgh, Pittsburgh, Pennsylvania ‡Geriatric Research Educational and Clinical Center, Veterans Affairs Pittsburgh Health System, Pittsburgh, Pennsylvania **Inotek Corporation, Beverly, Massachusetts
    Search for more papers by this author
  • Csaba Szabó,

    1. Departments of *Anesthesiology and Critical Care Medicine, †Neurology, §Pediatrics and ¶Cell Biology and Physiology, the Safar Center for Resuscitation Research and the Brain Trauma Research Center, University of Pittsburgh, Pittsburgh, Pennsylvania ‡Geriatric Research Educational and Clinical Center, Veterans Affairs Pittsburgh Health System, Pittsburgh, Pennsylvania **Inotek Corporation, Beverly, Massachusetts
    Search for more papers by this author
  • Simon C. Watkins,

    1. Departments of *Anesthesiology and Critical Care Medicine, †Neurology, §Pediatrics and ¶Cell Biology and Physiology, the Safar Center for Resuscitation Research and the Brain Trauma Research Center, University of Pittsburgh, Pittsburgh, Pennsylvania ‡Geriatric Research Educational and Clinical Center, Veterans Affairs Pittsburgh Health System, Pittsburgh, Pennsylvania **Inotek Corporation, Beverly, Massachusetts
    Search for more papers by this author
  • Robert S. B. Clark

    1. Departments of *Anesthesiology and Critical Care Medicine, †Neurology, §Pediatrics and ¶Cell Biology and Physiology, the Safar Center for Resuscitation Research and the Brain Trauma Research Center, University of Pittsburgh, Pittsburgh, Pennsylvania ‡Geriatric Research Educational and Clinical Center, Veterans Affairs Pittsburgh Health System, Pittsburgh, Pennsylvania **Inotek Corporation, Beverly, Massachusetts
    Search for more papers by this author

and reprint requests to Robert S. B. Clark, Safar Center for Resuscitation Research, 3434 Fifth Avenue, Pittsburgh, PA 15260, USA. E-mail: clarkrs@ccm.upmc.edu

Abstract

Programmed cell death occurs after ischemic, excitotoxic, and traumatic brain injury (TBI). Recently, a caspase-independent pathway involving intranuclear translocation of mitochondrial apoptosis-inducing factor (AIF) has been reported in vitro; but whether this occurs after acute brain injury was unknown. To address this question adult rats were sacrificed at various times after TBI. Western blot analysis on subcellular protein fractions demonstrated intranuclear localization of AIF in ipsilateral cortex and hippocampus at 2–72 h. Immunocytochemical analysis showed AIF labeling in neuronal nuclei with DNA fragmentation in the ipsilateral cortex and hippocampus. Immunoelectronmicroscopy verified intranuclear localization of AIF in hippocampal neurons after TBI, primarily in regions of euchromatin. Large-scale DNA fragmentation (∼50 kbp), a signature event in AIF-mediated cell death, was detected in ipsilateral cortex and hippocampi by 6 h. Neuron-enriched cultures exposed to peroxynitrite also demonstrated intranuclear AIF and large-scale DNA fragmentation concurrent with impaired mitochondrial respiration and cell death, events that are inhibited by treatment with a peroxynitrite decomposition catalyst. Intranuclear localization of AIF and large-scale DNA fragmentation occurs after TBI and in neurons under conditions of oxidative/nitrosative stress, providing the first evidence of this alternative mechanism by which programmed cell death may proceed in neurons after brain injury.

Ancillary