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Poly(ADP-ribose) polymerase-1 (PARP-1) is a homeostatic enzyme that paradoxically contributes to disturbances in spatial memory acquisition after traumatic brain injury (TBI) in transgenic mice, thought to be related to depletion of its substrate nicotinamide adenine dinucleotide (NAD + ). In this study, systemic administration of the PARP-1 inhibitor 5-iodo-6-amino-1,2-benzopyrone (INH2BP) after TBI preserved brain NAD + levels and dose-dependently reduced poly-ADPribosylation 24 h after injury. While moderate-dose INH2BP improved spatial memory acquisition after TBI; strikingly, both injured-and sham-mice receiving high-dose INH2BP were unable to learn in the Morris-water maze. Poly-ADP-ribosylated peptides identified using a proteomics approach yielded several proteins potentially associated with memory, including structural proteins (tubulin a and b, c-actin, and a-internexin neuronal intermediate filament protein) and 14-3-3c. Nuclear poly-ADP-ribosylation of 14-3-3c was completely inhibited by the dose of INH2BP that produced profound memory disturbances. Thus, partial inhibition of poly-ADP-ribosylation preserves NAD + and improves functional outcome after TBI, whereas more complete inhibition impairs spatial memory acquisition independent of injury, and is associated with ribosylation of 14-3-3c. Keywords: 14-3-3, DNA damage, head injury, 5-iodo-6-amino-1,2-benzopyrone, nicotinamide adenine dinucleotide, poly(ADP-ribose) synthetase. Poly(ADP-ribose) polymerase-1 (PARP-1; E.C.2.4.2.30) is an abundant nuclear protein that is activated by single-strand DNA breaks, produced by a variety of insults including reactive oxygen and nitrogen species, chemical agents, and UV irradiation (Dawson and Dawson 1995;Szabo 1996). Activated PARP-1 utilizes nicotinamide adenine dinucleotide (NAD + ) as a substrate for generating large branching adenosine diphosphate (ADP)-ribose polymers on various proteins including histones, topoisomerases, and PARP-1 itself (Szabo 1998). PARP-1 activation facilitates DNA repair by enabling other enzymes to locate and replace missing base pairs (Szabo et al. 1997;Bauer et al. 2000). Neuronal death and neurologic dysfunction after CNS injury appears to be mediated in part by activation of PARP-1, particularly CNS insults associated with DNA damage and energy failure such as cerebral ischemia (Eliasson et al. 1997;Endres et al. 1998a) and traumatic brain injury (TBI;Whalen et al. 1999;LaPlaca et al. 2001). While disruption of the PARP-1 gene Received December 17, 2002; revised manuscript received January 14, 2003; accepted January 15, 2003. Address correspondence 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 Abbreviations used: 2-AB, 3-aminobenzamide; ADP, adenosine diphosphate; CCI, controlled cortical impact; DMSO, dimethyl sulphoxide; DTT, dithiothreitol; Hsc54, heat shock cognate protein 54; IEF, isoelectric focusing; INH2BP, 5-iodo-6-amino-1,2-benzopyrone; IPG, i...

Previous studies suggest that delayed neuronal death occurs in patients with inflicted traumatic brain injury (TBI) from child abuse. It is unknown whether the mode of this delayed neuronal death represents apoptosis or necrosis, a distinction that carries therapeutic ramifications. Cytochrome c, an electron transport chain component, can be released from mitochondria under conditions of cellular stress, whereupon it can initiate and serve as a biomarker of apoptosis. To resolve this issue, cytochrome c concentration was determined in 167 ventricular cerebrospinal fluid (CSF) samples from 67 infants and children with TBI (including 15 patients diagnosed with child abuse) by ELISA. Controls included lumbar CSF from 19 infants and children without trauma or meningitis. A multivariate model adjusted for multiple within-subject observations was used to identify clinical variables associated with CSF cytochrome c. Other apoptosis-related proteins were also examined in a subset of TBI patients. Increased CSF cytochrome c was independently associated with inflicted TBI (P=0.0001) and female gender (P=0.04), but not age, Glasgow coma scale score, or survival. Other apoptosis-related proteins including Fas and caspase-1 were increased in CSF after TBI, but did not independently discriminate between accidental and inflicted TBI. These data suggest that apoptosis, as detected by the presence of cytochrome c in CSF, is uniquely prominent among the subset of TBI patients diagnosed with child abuse. The degree of apoptosis after TBI also appears to be gender-dependent. Development of strategies targeting apoptosis after TBI, particularly in victims of child abuse and in girls, appears justified.

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