Minimal Peroxide Exposure of Neuronal Cells Induces Multifaceted Adaptive Responses Wayne Chadwick1, Yu Zhou1, Sung-Soo Park1, Liyun Wang1, Nicholas Mitchell2, Matthew D. Stone1, Kevin G. Becker3, Bronwen Martin4, Stuart Maudsley1* 1 Receptor Pharmacology Unit, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, United States of America, 2 Department of Biology, Saint Bonaventure University, Saint Bonaventure, New York, United States of America, 3 Gene Expression and Genomics Unit, Research Resources Branch, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, United States of America, 4 Metabolism Unit, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, United States of America Abstract Oxidative exposure of cells occurs naturally and may be associated with cellular damage and dysfunction. Protracted low level oxidative exposure can induce accumulated cell disruption, affecting multiple cellular functions. Accumulated oxidative exposure has also been proposed as one of the potential hallmarks of the physiological/pathophysiological aging process. We investigated the multifactorial effects of long-term minimal peroxide exposure upon SH-SY5Y neural cells to understand how they respond to the continued presence of oxidative stressors. We show that minimal protracted oxidative stresses induce complex molecular and physiological alterations in cell functionality. Upon chronic exposure to minimal doses of hydrogen peroxide, SH-SY5Y cells displayed a multifactorial response to the stressor. To fully appreciate the peroxide-mediated cellular effects, we assessed these adaptive effects at the genomic, proteomic and cellular signal processing level. Combined analyses of these multiple levels of investigation revealed a complex cellular adaptive response to the protracted peroxide exposure. This adaptive response involved changes in cytoskeletal structure, energy metabolic shifts towards glycolysis and selective alterations in transmembrane receptor activity. Our analyses of the global responses to chronic stressor exposure, at multiple biological levels, revealed a viable neural phenotype in-part reminiscent of aged or damaged neural tissue. Our paradigm indicates how cellular physiology can subtly change in different contexts and potentially aid the appreciation of stress response adaptations. Citation: Chadwick W, Zhou Y, Park S-S, Wang L, Mitchell N, et al. (2010) Minimal Peroxide Exposure of Neuronal Cells Induces Multifaceted Adaptive Responses. PLoS ONE 5(12): e14352. doi:10.1371/journal.pone.0014352 Editor: Adrian Harwood, Cardiff University, United Kingdom Received June 14, 2010; Accepted November 24, 2010; Published December 17, 2010 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: This research was supported by the Intramural Research Program of the National Institute on Aging. The funding authority 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. * E-mail: [email protected] Introduction system, including both small molecules (tocopherols, vitamin C, glutathione, etc.) and antioxidant enzymes (the superoxide Cellular adaptations to environmental changes are likely to be dismutases (SOD), the glutathione peroxidases, catalase). The highly complex and involve many of the basic cellular functions. It balance between these pathways determines the absolute level of is crucial for cellular/organismal homeostasis during lifespan that oxidative stress. In aging, complex accumulated systemic imbal- molecular systems can adapt and retain functionality despite long- ances may result in the generation of excess free radicals that term variation of environment. Aging is a complex multifactorial overwhelm cellular antioxidant defenses, thereby causing oxidative process, unique in its exact etiology to each individual. There are stress [2]. Aging has also been associated with both a disruption of however several key factors common amongst current hypotheses mitochondrial function [3] along with the steady increase in ROS of aging, one of them being accumulated oxidative stresses. The species, a state that may seem paradoxical as the mitochondria Harman free radical/oxidative stress theory of aging underpins may be the prime source of the ROS. Therefore, it is likely that one of the most popular concepts regarding the biochemical/ there are complex interactions between the ROS generating and molecular factors in aging [1]. Harman proposed that physiolog- buffering systems in the aging process. Studies have shown an age- ical iron and other metals would cause reactive oxygen species related increase in oxidative damage to a variety of molecules, (ROS) to form in cells as a by-product of normal redox reactions. lipid, protein or DNA, in multiple organisms [4–6]. Age- ROS are a by-product of a variety of pathways in aerobic dependent oxidative damage has been implicated in the pathology metabolism. The mitochondrial electron transport chain accounts of age-related disorders in multiple organ systems, e.g. sporadic and for the majority of the total oxygen metabolized by the cell, and familial Alzheimer’s disease, Huntington’s and Parkinson’s disease, the by-products produced by the electron transport chain (e.g., amyotrophic lateral sclerosis, cardiovascular disease, Type II superoxide anion radicals, hydrogen peroxide, and hydroxyl diabetes and cancer [7–12]. Experimental excessive ROS stress radicals) are potential sources of oxidative damage to the can trigger cellular senescence in multiple human cell lines mitochondrion itself and other cellular compartments. Endoge- [13,14]. After exposure to high concentrations of hydrogen nous ROS-scavenging pathways represent an antioxidant defense peroxide (0.2–1 M) human cells undergo premature senescence, PLoS ONE | www.plosone.org 1 December 2010 | Volume 5 | Issue 12 | e14352 Neural Oxidative Adaptation demonstrate lack of response to mitogenic stimuli and show the formation of classical actin stress fibres (Figure 1B, 1–2) significant changes in gene expression [15,16]. Metabolic polymerized microtubule structures (Figure 1B, 3–4). Along with inhibitors, e.g. oligomycin or antimycin A, also induce ROS cytoskeletal alterations we also assessed the effects of the CMP production and induce cellular senescence, demonstrating that paradigm upon another primary cellular compartment often defective mitochondria are involved in oxidative cellular senes- linked with oxidative stress, i.e. mitochondria. Using Mitotracker cence [17]. High concentration (0.25 M), acute (90 minute) (10 nM) to identify mitochondrial expression we noted a peroxide exposure has also been shown to switch energy significant reduction of Mitotracker-fluorescence (Figure 1B,C) in generation in human cells from aerobic metabolism to glycolysis. cells exposed to the CMP paradigm. The extant mitochondria in This functional energetic shift appears to be an important CMP-treated cells appeared functional when assessed using hallmark of aged tissues in numerous species, as proposed by the tetramethylrhodamine ethyl ester-associated fluorescence (data epigenetic oxidative redox shift theory of aging [18–21]. The not shown). In addition to this alteration of energy-controlling disruption of energy regulation therefore may be a hallmark of organelles we also investigated the ability of CMP cells to uptake aging and neurodegeneration [22–24] however, the specific and utilize glucose. CMP-treated cells, compared to control, molecular connections between these two events still remain to demonstrated a significant reduction of glucose uptake (Figure 1D) be comprehensively identified. From a therapeutic point of view, as well as reductions of membrane GLUT 1 and 2 transporter interventions ameliorating aging/neurodegeneration-related pa- expression (Figure 1E). In addition, we determined the metabolic thologies have therefore been targeted to modulating anti-oxidant fate of glucose in CMP cells and found that CMP treatment mechanisms as well as inflammatory processes, DNA repair induced a proportional increase in lactate production for a given mechanisms and modulation of neurotrophic receptor systems level of glucose uptake after introduction of a 5 mM bolus dose [25–28]. Disruption of the neurotrophin brain-derived neuro- (4 hours) (Figure 1F). trophic factor (BDNF) activity has been associated with aging and multiple neurodegenerative diseases that demonstrate oxidative CMP-induced alterations of calcium homeostasis pathological aspects [29–35]. It has also been shown that many Using the membrane-permeant calcium sensitive dye, Fluo- other profound deficits in other receptor systems, e.g. cholinergic, 4AM, a significantly elevated resting calcium level was identified in serotoninergic, dopaminergic, histaminic, are also implicated in CMP-treated SH-SY5Y cells (Figure 2A). CMP cells were still able aging and neurodegeneration processes [36–39]. to respond to excitatory glutamate however. In both CMP and Cell death and atrophy have been strongly associated with the control cells glutamate stimulation resulted in long-lasting aging process and neurological