Pathway Analysis Reveals Common Pro-Survival Mechanisms of Metyrapone and Carbenoxolone after Traumatic Brain Injury

et al. (2013) Pathway Analysis Reveals Common Pro-Survival Mechanisms of Metyrapone and Carbenoxolone after Traumatic Brain Injury. PLoS ONE 8(1): e53230. doi:10.1371/journal.pone.0053230 Pathway Analysis Reveals Common Pro-Survival Mechanisms of Metyrapone and Carbenoxolone after Traumatic Brain Injury Helen L. Hellmich 0 Daniel R. Rojo 0 Maria-Adelaide Micci 0 Stacy L. Sell 0 Deborah R. Boone 0 Jeanna M. Crookshanks 0 Douglas S. DeWitt 0 Brent E. Masel 0 Donald S. Prough 0 Ioannis P. Androulakis, Rutgers University, United States of America 0 Department of Anesthesiology, University of Texas Medical Branch , Galveston, Texas , United States of America Developing new pharmacotherapies for traumatic brain injury (TBI) requires elucidation of the neuroprotective mechanisms of many structurally and functionally diverse compounds. To test our hypothesis that diverse neuroprotective drugs similarly affect common gene targets after TBI, we compared the effects of two drugs, metyrapone (MT) and carbenoxolone (CB), which, though used clinically for noncognitive conditions, improved learning and memory in rats and humans. Although structurally different, both MT and CB inhibit a common molecular target, 11b hydroxysteroid dehydrogenase type 1, which converts inactive cortisone to cortisol, thereby effectively reducing glucocorticoid levels. We examined injuryinduced signaling pathways to determine how the effects of these two compounds correlate with pro-survival effects in surviving neurons of the injured rat hippocampus. We found that treatment of TBI rats with MT or CB acutely induced in hippocampal neurons transcriptional profiles that were remarkably similar (i.e., a coordinated attenuation of gene expression across multiple injury-induced cell signaling networks). We also found, to a lesser extent, a coordinated increase in cell survival signals. Analysis of injury-induced gene expression altered by MT and CB provided additional insight into the protective effects of each. Both drugs attenuated expression of genes in the apoptosis, death receptor and stress signaling pathways, as well as multiple genes in the oxidative phosphorylation pathway such as subunits of NADH dehydrogenase (Complex1), cytochrome c oxidase (Complex IV) and ATP synthase (Complex V). This suggests an overall inhibition of mitochondrial function. Complex 1 is the primary source of reactive oxygen species in the mitochondrial oxidative phosphorylation pathway, thus linking the protective effects of these drugs to a reduction in oxidative stress. The net effect of the drug-induced transcriptional changes observed here indicates that suppressing expression of potentially harmful genes, and also, surprisingly, reduced expression of pro-survival genes may be a hallmark of neuroprotective therapeutic effects. - Funding: This study was supported by the Department of Anesthesiology, the Moody Foundation and the Moody Center for Traumatic Brain Injury and Spinal Cord Injury Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. To date, all pharmacotherapeutic agents in clinical trials for treatment of traumatic brain injury (TBI) have failed to show efficacy, suggesting a need for more effective pre-clinical screening of novel therapeutic compounds [1]. Significant factors that contribute to lifelong disability after TBI are learning and memory deficits associated with damage to the hippocampus. Thus, our screening efforts focused on compounds that provide neuroprotection in the hippocampus [2]. Although the large group of compounds that have been shown to reduce neuronal damage in animal models of hippocampal injury are structurally and functionally diverse and target multiple cell signaling pathways [3], their common neuroprotective effects suggest a common mechanism of action. Elucidating the common pro-survival mechanisms shared by these drugs could provide selective criteria to choose compounds as potential treatments for TBI. Rather than a reductionist gene-by-gene strategy, recent advances in systems biology allow us to interrogate disease-relevant networks on a genome-wide scale [4,5]. Systematically comparing common changes in cell signaling pathways generated by otherwise diverse neuroprotective compounds could provide useful mechanistic insights into the essential elements of neuroprotection. Ideally, in order to counteract hippocampal dysfunction, an effective therapeutic agent would have both neuroprotective and nootropic (memory and cognitive enhancing) properties. In order to focus on the drug-induced alterations in known cell signaling pathways, as opposed to the functional consequences of drug treatment, we investigated two compounds, metyrapone (MT) and carbenoxolone (CB), that possess neuroprotective and nootropic properties but have been used clinically for nonneurologic indications. Metyrapone is used to test for adrenal insufficiency [6] and CBa derivative of 18-glycyrrhetinic acid and a mineralocorticoid agonisthas been used for a variety of purposes, including treatment of peptic ulcers [7]. Both exert their neuroprotective and memory enhancing effects, in part, by inhibition of a common molecular target. Each compound inhibits the gene coding for 11b hydroxysteroid dehydrogenase type 1 (11bHSD1), which converts inactive cortisone to active cortisol in the brain [8,9]. Both also act through additional unidentified cellular signals [10,11]. Chronically elevated cortisol levels are traditionally associated with both hippocampal atrophy and hippocampal-dependent learning and memory deficits in aging humans [12]. Thus, both the effects of MT on improving memory consolidation and retrieval in rats [13,14], and CB on improving verbal fluency and memory in normal and diabetic elderly men [9] imply that these effects are mediated through inhibition of 11bHSD1 [12]. Additionally, glucocorticoid-mediated oxidative damage in the rat hippocampus has been associated with cognitive deficits [15]. Other studies have also shown that injury- and stress-induced release of glucocorticoids increases glutamate release in the prefrontal cortex and hippocampus. Through stimulation of endocannabinoids, glucocorticoids also influence GABAergic, noradrenergic, cholinergic and serotonergic neurotransmission in the brain [16]. However, other reported pro-survival effects of CB and MT appear to be mediated by divergent mechanisms. For example, CB is often associated with induction of heat shock proteins and non-gap junction mediated effects on synaptic transmission [1719]. Similarly, MT is found to influence sleep, depression, cytochrome P450 genes, and cyclic AMP response element binding protein [8,2023]. These effects provide evidence that these two drugs have multiple mechanisms of action associated with their neuroprotective and nootropic e (...truncated)