Poly(ADP-Ribose)synthetase activation and NAD depletion after traumatic brain injury in rats

Introduction: Poly(ADP-Ribose) synthetase (PARS; also known as PARP) deficient mice are dramatically protected from functional deficits after traumatic brain injury (TBI)¹. PARS activation generates poly(ADP-ribose)polymers (PADPR) on damaged DNA and nuclear proteins consuming NAD in the process; accordingly, we hypothesized that PARS activation contributes to energy failure after TBI. Methods: Anesthetized adult rats were subjected to controlled cortical impact with imposed secondary insult using our standard model. Rats were killed at 6, 8, 24, and 72 h after TBI; naïve rats were used as controls (n = 3-5/group). To determine whether the potent trigger of PARS activation, peroxynitrite, is generated after TBI, Western Wots were analyzed for nitrotyrosine residues on proteins. In addition, brain tissue was examined for evidence of oxidative DNA damage using DNA polymerase mediated dATP nick translation (PANT). Activation of PARS was measured indirectly using Western blot and immunohistochemistry to detect PADPR. NAD levels in cortex and hippocampus were measured using an enzymatic cycling method. Results: Nitrotyrosine residues were detected on ∼30 and ∼80kD protein bands from ipsilateral cortex at 8, 24 and 72 h after TBI but not in controls. PANT showed single-strand DNA nicks in ipsilateral cortex and hippocampus at 6 and 24 h after TBI. An increase in PADPR on multiple protein bands was seen at 8 and 24 h but not in controls. By 72 h, PADPR on proteins were reduced (vs. 24 h). PADPR were also detected immunohistochemically in injured brain. NAD levels in ipsilateral cortex and hippocampus were depleted by 86% at 24 h after TBI vs. control. Conclusions: These data support the hypothesis that NAD depletion after TBI is a consequence of PARS activation triggered by peroxynitrite-mediated DNA damage. Thus, PARS activation after TBI may exacerbate energy failure in injured brain, which might in turn explain the protective effects observed in PARS deficient mice. Further study using clinically relevant PARS inhibitors after TBI is warranted. ¹Whalen et al, J Cereb Blood Flow Metab 1999;19:825-42.