Neuroscience Letters 560 (2014) 71–76
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N-Acetyl cysteine prevents synergistic, severe toxicity from two hits of oxidative stress Ajay S. Unnithan, Yiran Jiang, Jennifer L. Rumble, Sree H. Pulugulla, Jessica M. Posimo, Amanda M. Gleixner, Rehana K. Leak ∗ Division of Pharmaceutical Sciences, Duquesne University, Pittsburgh, PA 15282, USA
h i g h l i g h t s • • • • •
A severe stress model of neurodegeneration was established with dual H2 O2 hits. Three independent viability assays confirmed severe, synergistic cell loss. Catalase defenses were raised by the first hit. Glutathione defenses were reduced by the first and second hit. N-acetyl cysteine prevented glutathione loss and robustly protected against severe H2 O2 toxicity.
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Article history: Received 17 June 2013 Received in revised form 12 November 2013 Accepted 10 December 2013 Keywords: Dual hit Parkinson’s Alzheimer’s Glutathione Antioxidant
a b s t r a c t The two hit hypothesis of neurodegeneration states that cells that have been severely stressed once are more vulnerable to the negative impact of a second hit. In other words, the toxicity of two hits of severe stress may be synergistic in neurons. We previously developed a two hit model of proteotoxic neurodegeneration using the proteasome inhibitor MG132. In that study, we found that the potent antioxidant N-acetyl cysteine was able to protect against the toxicity associated with dual MG132 hits. N-acetyl cysteine has been shown to ameliorate cognitive deficits in Alzheimer’s patients and to reduce the symptoms of blast injury in soldiers. These studies and many others in experimental models of neurodegeneration suggest that N-acetyl cysteine can protect neurons even when they are severely injured. In the present study, we tested the hypotheses that dual hits of hydrogen peroxide and paraquat would elicit synergistic neurodegeneration and that this extreme toxicity would be prevented by N-acetyl cysteine. The findings reveal for the first time that neuronal N2a cells are much more sensitive to oxidative stress from hydrogen peroxide treatment when they have been exposed previously to the same toxin. Two hits of hydrogen peroxide also caused severe loss of glutathione. N-acetyl cysteine attenuated the loss of glutathione and reduced the near-complete loss of cells after exposure to dual hydrogen peroxide hits. The present study supports the notion that N-acetyl cysteine can robustly protect against severe, unremitting oxidative stress in a glutathione-dependent manner. © 2013 Elsevier Ireland Ltd. All rights reserved.
Introduction The cellular stress response is dependent upon the nature of the insult, stressor dose and duration, cell type, previous exposures to stress, and many other factors. For example, exposure to mild stress can protect against subsequent challenges of the same or different nature, a phenomenon known as preconditioning or tolerance [1–4]. In contrast, when the stress is severe, adaptive
Abbreviations: DMSO, dimethyl sulfoxide; GSH, glutathione; NAC, N-acetyl cysteine. ∗ Corresponding author. Tel.: +1 412 396 4734; fax: +1 412 396 4660. E-mail address: [email protected] (R.K. Leak). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.12.023
defenses may be irreparably weakened, leading to stress exacerbation. This latter phenomenon is the subject of the two hit hypothesis of neurodegeneration, which states that two insults may be synergistic in their toxic effects [5–10]. For example, early life infections and inflammation [11–13] as well as traumatic brain injuries [14] can predispose cells to future injury. We recently developed a two hit model of proteotoxic neurodegeneration in the neuronal cell line N2a [15]. Proteotoxicity is a hallmark of neurodegenerative disorders and is defined as stress from protein misfolding and aggregation [16,17]. We elicited proteotoxicity in N2a cells with the proteasome inhibitor MG132 and found that exposure to high concentrations of MG132 exacerbated cell loss in response to a second hit of the same toxin. In addition to proteotoxicity, many neurodegenerative diseases are also characterized by severe oxidative
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stress [18,19]. In the present study, we tested the hypothesis that a dual hit model of oxidative neurodegeneration could be generated by treating N2a cells with sequential hits of high concentrations of paraquat or hydrogen peroxide. In our previous two hit model, we observed that the glutathione precursor N-acetyl cysteine protected N2a cells from two MG132 hits [15]. These findings are relevant to Alzheimer’s and Parkinson’s diseases because both conditions elicit loss of glutathione [19–26]. N-acetyl cysteine has been shown to improve cognition in Alzheimer’s patients [27] and is also used in psychiatric conditions with disruptions in glutathione metabolism. For example, N-acetyl cysteine-treated bipolar patients showed improvements in depressive symptoms [28], whereas schizophrenic patients taking N-acetyl cysteine experienced fewer negative symptoms and less akathisia [29]. Recently, a study of soldiers in combat found that N-acetyl cysteine doubled the chances of symptom resolution after blast injury [30]. N-acetyl cysteine can also increase brain glutathione levels in Parkinson’s patients [31]. N-acetyl cysteine is so effective against oxidative damage that it is commonly used as a positive control for antioxidant defense in experimental models of disease. In the present study, we challenged N2a cells with two hits of severe oxidative stress to test the hypothesis that N-acetyl cysteine would still be able to raise glutathione levels and prevent cell death even under conditions where almost complete cell loss was elicited.
(160280250, Acros Organics, Pittsburgh, PA) or vehicle (water) was coadministered with H2 O2 . Immunoblots and viability assays Cell lysates were prepared for infrared immunoblotting as described previously [15]. Briefly, proteins were run on polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were incubated in primary anti-catalase and anti-actin IgGs overnight at 4 ◦ C. This was followed by secondary antibody incubation for 1 h at room temperature. Infrared signal was quantified on an Odyssey Imager (LI-COR). Cells were also prepared for infrared In-Cell Westerns as described previously [15]. Briefly, cells were fixed and incubated in ␣-tubulin antibodies followed by anti-mouse IgGs (800 nm). Nuclei were then stained with DRAQ5 (700 nm, 0.25 M; Biostatus Limited, Leicestershire, UK). In-Cell Western staining was also visualized on the Odyssey Imager. In addition, cells were stained with Hoechst 33258 (10 g/mL; Sigma-Aldrich) and photographed on an EVOS microscope (Advanced Microscopy Group, Bothell, WA). For ATP measurements, the Cell Titer Glo manufacturer’s directions (Promega Inc., Madison, WI, USA) were modified, such that 25 L of reagent were added to 50 L of media, as described previously [15]. Statistics
Materials and methods Antibodies Primary and secondary antibodies were purchased and used as follows: anti-␣-tubulin (1:10,000 for immunocytochemistry; T5168, Sigma-Aldrich, St. Louis, MO), anti-catalase (1:1000 for immunoblotting; C0979, Sigma-Aldrich), anti-glutathione (1:300 for immunocytochemistry; AB5010, EMD Millipore, Billerica, MA), anti--actin (1:50,000 for Western blotting; A5441, SigmaAldrich), donkey or goat anti-mouse IgG 800 nm (1:2000 for immunocytochemistry, 1:10,000 for Western blotting; 92632212, 926-32210, LI-COR Biosciences, Lincoln, NE), donkey or goat anti-rabbit IgG 800 nm (1:500 for immunocytochemistry, 1:10,000 for Western blotting; 926-32213, 926-32211, LI-COR), and donkey or goat anti-mouse 700 nm (1:2000 for immunocytochemistry, 1:10,000 for Western blotting; 926-32222, 926-32220, LI-COR). Cell maintenance and toxin treatments Naïve N2a cells were maintained at 37 ◦ C with 5% CO2 in Dulbecco’s modified Eagle’s medium (12100-061, Life Technologies, Grand Island, NY), with 0.37% sodium bicarbonate (S5761, Sigma-Aldrich), 10% fetal clone-III (SH30109.03, ThermoScientific Hyclone, Logan, UT), 50 U/mL penicillin and 50 g/mL streptomycin (15070, Life Technologies). Cells were plated at a density of 35,000 cells per cm2 on Costar plates (3595, Corning Incorporated, Corning, NY). Cells were treated with hydrogen peroxide (H324, ThermoFisher Scientific, Pittsburgh, PA) or paraquat (36541, SigmaAldrich) 24 h after plating in a 10× stock added to existing media (1st hit) and/or 48 h after plating in a fresh media exchange at a 1× concentration (2nd hit). All cells were assayed 72 h after plating. The group that received the 1st hit by itself (0 M H2 O2 or 0 M paraquat for the 2nd hit) received only water (vehicle for H2 O2 ) or phosphate-buffered saline (PBS; vehicle for paraquat) for the 2nd hit. H2 O2 and paraquat were removed at the time of the 2nd hit in this control group because we needed to avoid contrasting two short hits with one long hit of twice the duration. N-acetyl cysteine
Data are presented as the mean ± SEM. Each experiment was run in triplicate wells and then repeated 3–7 times. Data were analyzed by two or three-way ANOVA (SPSS Ver 20.0, IBM, Armonk, NY), always employing the Bonferroni post hoc correction. Differences were deemed significant when p ≤ 0.05. Results Three independent viability assays were used to survey cellular fitness. We have verified that all three assays are linearly correlated with cell number [15]. Combining two hits of paraquat did not elicit extensive synergistic exacerbation of toxicity (Fig. 1). As in our previous report [15], we have illustrated the data in two ways. First, all raw values were expressed as a percentage of the group that received no toxin (Fig. 1A–C). Second, raw values were expressed as a percentage of the group that did not receive a 2nd hit (the 0 M 2nd hit group; Fig. 1D–F). Data were expressed in this way so that the synergy of two hits could be statistically evaluated. Fig. 1D–F reveal that the two paraquat hits were only synergistic after pretreatment with 400 M paraquat because the 2nd hits of 400 and 800 M paraquat became significantly more toxic in this group (see ␣-tubulin assay, Fig. 1E and H). These findings suggest that paraquat toxicity is not an ideal model of the two hit hypothesis. In contrast to paraquat, two hits of H2 O2 elicited synergistic cell loss according to all three assays (Fig. 2). Pretreatment with 100–150 M H2 O2 enhanced the toxicity of 300–400 M H2 O2 in the 2nd hit according to the DRAQ5 (Fig. 2A, D and G) and ␣-tubulin assays (Fig. 2B, E and H). The ATP assay also revealed that cells treated with 200–300 M H2 O2 as a 2nd hit were more functionally compromised when previously stressed. Photomicrographs of N2a cells hit once or twice with H2 O2 are shown in Fig. 2I to illustrate the synergistic toxicity of two hits at the cellular level. These findings suggest that oxidative stress from H2 O2 can be used to model the two hit hypothesis of neurodegeneration. Catalase and glutathione are known to break down H2 O2 into less toxic by-products. If pretreatment with H2 O2 led to a loss in either, this might explain why stressed cells were predisposed to a 2nd hit. However, catalase levels exhibited a rise in response to
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Fig. 1. N2a cells were sequentially treated with one paraquat hit 24 h after plating and a 2nd paraquat hit 48 h after plating. Cells were assayed for DRAQ5 ((A), (D) and (G)), ␣-tubulin ((B), (E) and (H)), and ATP ((C) and (F)) 72 h after plating or 24 h after the last hit. All infrared images were pseudocolored red or green. Data are expressed in (D), (E) and (F) as a percentage of the group that did not receive a 2nd hit (0 M 2nd hit) to contrast the drop in viability following two hits with the viability drop in cells that did not receive the 1st hit (0 M 1st hit). Shown are the mean + S.E.M. from 3 to 4 experiments. * p ≤ 0.05 versus 0 M 2nd hit, + p ≤ 0.05 versus 0 M 1st hit, two-way ANOVA followed by Bonferroni post hoc test.
pretreatment with H2 O2 , not a loss (Fig. 3A). Therefore, the toxicity of this concentration of H2 O2 could not be explained by a loss of catalase defenses. In contrast to catalase, glutathione levels fell in response to the 1st and 2nd hits of H2 O2 and the combination of two hits (Fig. 3B–D). As expected, N-acetyl cysteine reduced the drop in glutathione in response to two hits. Therefore, we tested the hypothesis that N-acetyl cysteine would mitigate the severe toxicity of two H2 O2 hits (Fig. 3E–I). Although treatment with 125 M H2 O2 and 300 M H2 O2 elicited almost complete loss of signal in all three assays, N-acetyl cysteine was still able to protect against these two H2 O2 hits by all three viability measures. Discussion We have developed a two hit model of synergistic neurodegeneration with the oxidative stressor H2 O2 and found that N-acetyl cysteine prevented the severe toxicity of two oxidative hits. Stressed N2a cells did not lose catalase defenses but were nevertheless compromised, perhaps because of excessive glutathione loss. N-acetyl cysteine attenuated the drop in glutathione and robustly
protected against almost complete loss of cells. These results support the hypothesis that N-acetyl cysteine may be effective in neurological conditions by rescuing severely compromised cells from an unremitting burden of oxidative stress. Multiple double blind, placebo-controlled trials support this notion. For example, Alzheimer’s patients taking N-acetyl cysteine performed better in the Letter Fluency Task and Wechsler Memory Scale Immediate Number Recall [27]. Patients on a supplement containing, among other substances, N-acetyl cysteine, also performed better on cognitive endpoints [32]. N-acetyl cysteine reduced mitochondrial oxidative stress in fibroblasts from Alzheimer’s patients [33], perhaps by raising glutathione. Oxidized glutathione levels are higher in mild cognitive impairment, increase with Alzheimer’s progression, and correlate with declining cognition [20,26]. Loss of glutathione is also evident in Parkinson’s disease [22,23]. A recent study showed that N-acetyl cysteine can raise glutathione in the brain and serum of Parkinson’s patients [31]. In addition, traumatic brain injury is associated with loss of glutathione [34], providing rationale for the use of N-acetyl cysteine in combat. Soldiers exposed to blast injury had an 86% chance of symptom resolution
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Fig. 2. N2a cells were treated with the 1st H2 O2 hit 24 h after plating and a 2nd hit 48 h after plating. Cells were assayed for DRAQ5 ((A), (D) and (G)), ␣-tubulin ((B), (E) and (H)), and ATP ((C) and (F)) 72 h after plating or 24 h after the last hit. Data are expressed in (D), (E) and (F) as a percentage of the group that did not receive a 2nd hit (0 M 2nd hit) to contrast the drop in viability following two hits with the viability drop in cells that did not receive the 1st hit (0 M 1st hit). Shown are the mean + S.E.M. from 3 to 4 experiments. * p ≤ 0.05 versus 0 M 2nd hit, + p ≤ 0.05 versus 0 M 1st hit, two-way ANOVA followed by Bonferroni post hoc test. (I) N2a cells were treated with one or two hits of H2 O2 delivered 24 h (1st hit, 125 M) or 48 h (2nd hit, 300 M) after plating and stained with Hoechst 72 h after plating.
when administered N-acetyl cysteine, compared to a 42% chance of symptom resolution on placebo [30]. In addition to raising glutathione levels, N-acetyl cysteine can react directly with hydrogen peroxide [35]. Furthermore, N-acetyl cysteine may also protect cells in glutathione-independent manners. For example, we have shown that N-acetyl cysteine can protect cells in a heat shock proteindependent manner [36]. Most studies of the two hit hypothesis use two different insults to elicit synergistic toxicity [5–10]. However, our previous studies show that two hits of the same insult can also be synergistic when the stressor dose is high enough [15]. Similarly, the present study supports the notion that the two hit hypothesis can be extended to sequential insults from the same H2 O2 toxin. However, we did not gather evidence that paraquat elicits extensive synergistic toxicity when administered as two sequential hits. Paraquat generates reactive oxygen species by induction of redox cycling with a cellular diaphorase, such as nitric oxide synthase [37]. Paraquat elicits toxicity by the formation of both superoxide and nitric oxide free radicals [38,39]. Unlike paraquat, hydrogen peroxide is a normal product of mitochondrial respiration and plays a role in
cell signaling [40]. However, hydrogen peroxide can also directly oxidize targets, especially at high concentrations [41]. The discrepancies between hydrogen peroxide and paraquat in the present study suggest that the two hit phenomenon is not universal but stress-specific. In addition, it should be noted that not all cell types respond to severe stress with synergistic toxicity. For example, the impact of a second toxic hit is blunted in highly stressed astrocyte survivors [42]. Thus, the response to two hits may depend upon multiple factors, such as cell type, type of toxin, and dose and duration of stress. In the present study, H2 O2 elicited a rise in catalase. This is inconsistent with the hypothesis that a loss in catalase defenses was responsible for toxicity. However, the data are still instructive in that they reveal that stressed cells protect themselves by upregulating defenses. This finding is consistent with previous studies showing that astrocytes and dopaminergic cells raise several pro-survival proteins to adapt to proteotoxic stress [2,42]. The catalase data are also consistent with studies demonstrating adaptive responses to oxidative stress for example, see [43]. Without this antioxidative response, we would expect H2 O2 -pretreated cells to
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Fig. 3. (A) N2a cells were treated with vehicle or one hit of H2 O2 (125 M) and assayed 24 h later for catalase and -actin by immunoblotting. Shown are the mean + S.E.M. from six experiments. + p ≤ 0.05 versus 0 M H2 O2 treatment, two-tailed t-test. N2a cells were treated with one or two hits of H2 O2 at the indicated concentrations 24 h after plating (1st hit) or 48 h after plating (2nd hit), in the absence or presence of N-acetyl cysteine (NAC, 3 mM) and assayed 72 h after plating for glutathione (GSH) levels ((B)–(D)) or for DRAQ5 ((E) and (G)), ␣-tubulin ((F) and (H)) and ATP (I). GSH was expressed as a fraction of DRAQ5+ cells to control for differences in cell numbers across groups. Shown are the mean + S.E.M. from 5 to 7 experiments. * p ≤ 0.05 versus 0 M 2nd hit, + p ≤ 0.05 versus 0 M 1st hit, ˆ p ≤ 0.05 versus 0 M NAC (H2 O vehicle), three-way ANOVA followed by Bonferroni post hoc test.
be all the more vulnerable to a 2nd hit and all three dose response curves to be shifted to the left. We performed multiple viability assays because cells can change their metabolic functions and cytoskeletal protein expression before actual cell death occurs. Cell counts, such as of DRAQ5stained nuclei, are also not sufficient by themselves because cells may be present without being metabolically or structurally healthy [44]. In other words, cytoskeletal disruption, metabolic compromise, and cell death can be uncoupled from each other. The use of three independent assays therefore gives us greater confidence that
H2 O2 elicits synergistic cell loss. Furthermore, all three assays are in agreement that N-acetyl cysteine was highly protective. Therefore, we can conclude that both structure and function are protected by this compound, even under conditions of extreme oxidative stress. Acknowledgments RKL designed the experiments and wrote the manuscript. ASU performed the majority of experiments. Additional experiments and technical support were provided by YJ, JR, SH, AG, and JP.
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
N-Acetyl cysteine prevents synergistic, severe toxicity from two hits of oxidative stress Ajay S. Unnithan, Yiran Jiang, Jennifer L. Rumble, Sree H. Pulugulla, Jessica M. Posimo, Amanda M. Gleixner, Rehana K. Leak ∗ Division of Pharmaceutical Sciences, Duquesne University, Pittsburgh, PA 15282, USA
h i g h l i g h t s • • • • •
A severe stress model of neurodegeneration was established with dual H2 O2 hits. Three independent viability assays confirmed severe, synergistic cell loss. Catalase defenses were raised by the first hit. Glutathione defenses were reduced by the first and second hit. N-acetyl cysteine prevented glutathione loss and robustly protected against severe H2 O2 toxicity.
a r t i c l e
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Article history: Received 17 June 2013 Received in revised form 12 November 2013 Accepted 10 December 2013 Keywords: Dual hit Parkinson’s Alzheimer’s Glutathione Antioxidant
a b s t r a c t The two hit hypothesis of neurodegeneration states that cells that have been severely stressed once are more vulnerable to the negative impact of a second hit. In other words, the toxicity of two hits of severe stress may be synergistic in neurons. We previously developed a two hit model of proteotoxic neurodegeneration using the proteasome inhibitor MG132. In that study, we found that the potent antioxidant N-acetyl cysteine was able to protect against the toxicity associated with dual MG132 hits. N-acetyl cysteine has been shown to ameliorate cognitive deficits in Alzheimer’s patients and to reduce the symptoms of blast injury in soldiers. These studies and many others in experimental models of neurodegeneration suggest that N-acetyl cysteine can protect neurons even when they are severely injured. In the present study, we tested the hypotheses that dual hits of hydrogen peroxide and paraquat would elicit synergistic neurodegeneration and that this extreme toxicity would be prevented by N-acetyl cysteine. The findings reveal for the first time that neuronal N2a cells are much more sensitive to oxidative stress from hydrogen peroxide treatment when they have been exposed previously to the same toxin. Two hits of hydrogen peroxide also caused severe loss of glutathione. N-acetyl cysteine attenuated the loss of glutathione and reduced the near-complete loss of cells after exposure to dual hydrogen peroxide hits. The present study supports the notion that N-acetyl cysteine can robustly protect against severe, unremitting oxidative stress in a glutathione-dependent manner. © 2013 Elsevier Ireland Ltd. All rights reserved.
Introduction The cellular stress response is dependent upon the nature of the insult, stressor dose and duration, cell type, previous exposures to stress, and many other factors. For example, exposure to mild stress can protect against subsequent challenges of the same or different nature, a phenomenon known as preconditioning or tolerance [1–4]. In contrast, when the stress is severe, adaptive
Abbreviations: DMSO, dimethyl sulfoxide; GSH, glutathione; NAC, N-acetyl cysteine. ∗ Corresponding author. Tel.: +1 412 396 4734; fax: +1 412 396 4660. E-mail address: [email protected] (R.K. Leak). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.12.023
defenses may be irreparably weakened, leading to stress exacerbation. This latter phenomenon is the subject of the two hit hypothesis of neurodegeneration, which states that two insults may be synergistic in their toxic effects [5–10]. For example, early life infections and inflammation [11–13] as well as traumatic brain injuries [14] can predispose cells to future injury. We recently developed a two hit model of proteotoxic neurodegeneration in the neuronal cell line N2a [15]. Proteotoxicity is a hallmark of neurodegenerative disorders and is defined as stress from protein misfolding and aggregation [16,17]. We elicited proteotoxicity in N2a cells with the proteasome inhibitor MG132 and found that exposure to high concentrations of MG132 exacerbated cell loss in response to a second hit of the same toxin. In addition to proteotoxicity, many neurodegenerative diseases are also characterized by severe oxidative
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stress [18,19]. In the present study, we tested the hypothesis that a dual hit model of oxidative neurodegeneration could be generated by treating N2a cells with sequential hits of high concentrations of paraquat or hydrogen peroxide. In our previous two hit model, we observed that the glutathione precursor N-acetyl cysteine protected N2a cells from two MG132 hits [15]. These findings are relevant to Alzheimer’s and Parkinson’s diseases because both conditions elicit loss of glutathione [19–26]. N-acetyl cysteine has been shown to improve cognition in Alzheimer’s patients [27] and is also used in psychiatric conditions with disruptions in glutathione metabolism. For example, N-acetyl cysteine-treated bipolar patients showed improvements in depressive symptoms [28], whereas schizophrenic patients taking N-acetyl cysteine experienced fewer negative symptoms and less akathisia [29]. Recently, a study of soldiers in combat found that N-acetyl cysteine doubled the chances of symptom resolution after blast injury [30]. N-acetyl cysteine can also increase brain glutathione levels in Parkinson’s patients [31]. N-acetyl cysteine is so effective against oxidative damage that it is commonly used as a positive control for antioxidant defense in experimental models of disease. In the present study, we challenged N2a cells with two hits of severe oxidative stress to test the hypothesis that N-acetyl cysteine would still be able to raise glutathione levels and prevent cell death even under conditions where almost complete cell loss was elicited.
(160280250, Acros Organics, Pittsburgh, PA) or vehicle (water) was coadministered with H2 O2 . Immunoblots and viability assays Cell lysates were prepared for infrared immunoblotting as described previously [15]. Briefly, proteins were run on polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were incubated in primary anti-catalase and anti-actin IgGs overnight at 4 ◦ C. This was followed by secondary antibody incubation for 1 h at room temperature. Infrared signal was quantified on an Odyssey Imager (LI-COR). Cells were also prepared for infrared In-Cell Westerns as described previously [15]. Briefly, cells were fixed and incubated in ␣-tubulin antibodies followed by anti-mouse IgGs (800 nm). Nuclei were then stained with DRAQ5 (700 nm, 0.25 M; Biostatus Limited, Leicestershire, UK). In-Cell Western staining was also visualized on the Odyssey Imager. In addition, cells were stained with Hoechst 33258 (10 g/mL; Sigma-Aldrich) and photographed on an EVOS microscope (Advanced Microscopy Group, Bothell, WA). For ATP measurements, the Cell Titer Glo manufacturer’s directions (Promega Inc., Madison, WI, USA) were modified, such that 25 L of reagent were added to 50 L of media, as described previously [15]. Statistics
Materials and methods Antibodies Primary and secondary antibodies were purchased and used as follows: anti-␣-tubulin (1:10,000 for immunocytochemistry; T5168, Sigma-Aldrich, St. Louis, MO), anti-catalase (1:1000 for immunoblotting; C0979, Sigma-Aldrich), anti-glutathione (1:300 for immunocytochemistry; AB5010, EMD Millipore, Billerica, MA), anti--actin (1:50,000 for Western blotting; A5441, SigmaAldrich), donkey or goat anti-mouse IgG 800 nm (1:2000 for immunocytochemistry, 1:10,000 for Western blotting; 92632212, 926-32210, LI-COR Biosciences, Lincoln, NE), donkey or goat anti-rabbit IgG 800 nm (1:500 for immunocytochemistry, 1:10,000 for Western blotting; 926-32213, 926-32211, LI-COR), and donkey or goat anti-mouse 700 nm (1:2000 for immunocytochemistry, 1:10,000 for Western blotting; 926-32222, 926-32220, LI-COR). Cell maintenance and toxin treatments Naïve N2a cells were maintained at 37 ◦ C with 5% CO2 in Dulbecco’s modified Eagle’s medium (12100-061, Life Technologies, Grand Island, NY), with 0.37% sodium bicarbonate (S5761, Sigma-Aldrich), 10% fetal clone-III (SH30109.03, ThermoScientific Hyclone, Logan, UT), 50 U/mL penicillin and 50 g/mL streptomycin (15070, Life Technologies). Cells were plated at a density of 35,000 cells per cm2 on Costar plates (3595, Corning Incorporated, Corning, NY). Cells were treated with hydrogen peroxide (H324, ThermoFisher Scientific, Pittsburgh, PA) or paraquat (36541, SigmaAldrich) 24 h after plating in a 10× stock added to existing media (1st hit) and/or 48 h after plating in a fresh media exchange at a 1× concentration (2nd hit). All cells were assayed 72 h after plating. The group that received the 1st hit by itself (0 M H2 O2 or 0 M paraquat for the 2nd hit) received only water (vehicle for H2 O2 ) or phosphate-buffered saline (PBS; vehicle for paraquat) for the 2nd hit. H2 O2 and paraquat were removed at the time of the 2nd hit in this control group because we needed to avoid contrasting two short hits with one long hit of twice the duration. N-acetyl cysteine
Data are presented as the mean ± SEM. Each experiment was run in triplicate wells and then repeated 3–7 times. Data were analyzed by two or three-way ANOVA (SPSS Ver 20.0, IBM, Armonk, NY), always employing the Bonferroni post hoc correction. Differences were deemed significant when p ≤ 0.05. Results Three independent viability assays were used to survey cellular fitness. We have verified that all three assays are linearly correlated with cell number [15]. Combining two hits of paraquat did not elicit extensive synergistic exacerbation of toxicity (Fig. 1). As in our previous report [15], we have illustrated the data in two ways. First, all raw values were expressed as a percentage of the group that received no toxin (Fig. 1A–C). Second, raw values were expressed as a percentage of the group that did not receive a 2nd hit (the 0 M 2nd hit group; Fig. 1D–F). Data were expressed in this way so that the synergy of two hits could be statistically evaluated. Fig. 1D–F reveal that the two paraquat hits were only synergistic after pretreatment with 400 M paraquat because the 2nd hits of 400 and 800 M paraquat became significantly more toxic in this group (see ␣-tubulin assay, Fig. 1E and H). These findings suggest that paraquat toxicity is not an ideal model of the two hit hypothesis. In contrast to paraquat, two hits of H2 O2 elicited synergistic cell loss according to all three assays (Fig. 2). Pretreatment with 100–150 M H2 O2 enhanced the toxicity of 300–400 M H2 O2 in the 2nd hit according to the DRAQ5 (Fig. 2A, D and G) and ␣-tubulin assays (Fig. 2B, E and H). The ATP assay also revealed that cells treated with 200–300 M H2 O2 as a 2nd hit were more functionally compromised when previously stressed. Photomicrographs of N2a cells hit once or twice with H2 O2 are shown in Fig. 2I to illustrate the synergistic toxicity of two hits at the cellular level. These findings suggest that oxidative stress from H2 O2 can be used to model the two hit hypothesis of neurodegeneration. Catalase and glutathione are known to break down H2 O2 into less toxic by-products. If pretreatment with H2 O2 led to a loss in either, this might explain why stressed cells were predisposed to a 2nd hit. However, catalase levels exhibited a rise in response to
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Fig. 1. N2a cells were sequentially treated with one paraquat hit 24 h after plating and a 2nd paraquat hit 48 h after plating. Cells were assayed for DRAQ5 ((A), (D) and (G)), ␣-tubulin ((B), (E) and (H)), and ATP ((C) and (F)) 72 h after plating or 24 h after the last hit. All infrared images were pseudocolored red or green. Data are expressed in (D), (E) and (F) as a percentage of the group that did not receive a 2nd hit (0 M 2nd hit) to contrast the drop in viability following two hits with the viability drop in cells that did not receive the 1st hit (0 M 1st hit). Shown are the mean + S.E.M. from 3 to 4 experiments. * p ≤ 0.05 versus 0 M 2nd hit, + p ≤ 0.05 versus 0 M 1st hit, two-way ANOVA followed by Bonferroni post hoc test.
pretreatment with H2 O2 , not a loss (Fig. 3A). Therefore, the toxicity of this concentration of H2 O2 could not be explained by a loss of catalase defenses. In contrast to catalase, glutathione levels fell in response to the 1st and 2nd hits of H2 O2 and the combination of two hits (Fig. 3B–D). As expected, N-acetyl cysteine reduced the drop in glutathione in response to two hits. Therefore, we tested the hypothesis that N-acetyl cysteine would mitigate the severe toxicity of two H2 O2 hits (Fig. 3E–I). Although treatment with 125 M H2 O2 and 300 M H2 O2 elicited almost complete loss of signal in all three assays, N-acetyl cysteine was still able to protect against these two H2 O2 hits by all three viability measures. Discussion We have developed a two hit model of synergistic neurodegeneration with the oxidative stressor H2 O2 and found that N-acetyl cysteine prevented the severe toxicity of two oxidative hits. Stressed N2a cells did not lose catalase defenses but were nevertheless compromised, perhaps because of excessive glutathione loss. N-acetyl cysteine attenuated the drop in glutathione and robustly
protected against almost complete loss of cells. These results support the hypothesis that N-acetyl cysteine may be effective in neurological conditions by rescuing severely compromised cells from an unremitting burden of oxidative stress. Multiple double blind, placebo-controlled trials support this notion. For example, Alzheimer’s patients taking N-acetyl cysteine performed better in the Letter Fluency Task and Wechsler Memory Scale Immediate Number Recall [27]. Patients on a supplement containing, among other substances, N-acetyl cysteine, also performed better on cognitive endpoints [32]. N-acetyl cysteine reduced mitochondrial oxidative stress in fibroblasts from Alzheimer’s patients [33], perhaps by raising glutathione. Oxidized glutathione levels are higher in mild cognitive impairment, increase with Alzheimer’s progression, and correlate with declining cognition [20,26]. Loss of glutathione is also evident in Parkinson’s disease [22,23]. A recent study showed that N-acetyl cysteine can raise glutathione in the brain and serum of Parkinson’s patients [31]. In addition, traumatic brain injury is associated with loss of glutathione [34], providing rationale for the use of N-acetyl cysteine in combat. Soldiers exposed to blast injury had an 86% chance of symptom resolution
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Fig. 2. N2a cells were treated with the 1st H2 O2 hit 24 h after plating and a 2nd hit 48 h after plating. Cells were assayed for DRAQ5 ((A), (D) and (G)), ␣-tubulin ((B), (E) and (H)), and ATP ((C) and (F)) 72 h after plating or 24 h after the last hit. Data are expressed in (D), (E) and (F) as a percentage of the group that did not receive a 2nd hit (0 M 2nd hit) to contrast the drop in viability following two hits with the viability drop in cells that did not receive the 1st hit (0 M 1st hit). Shown are the mean + S.E.M. from 3 to 4 experiments. * p ≤ 0.05 versus 0 M 2nd hit, + p ≤ 0.05 versus 0 M 1st hit, two-way ANOVA followed by Bonferroni post hoc test. (I) N2a cells were treated with one or two hits of H2 O2 delivered 24 h (1st hit, 125 M) or 48 h (2nd hit, 300 M) after plating and stained with Hoechst 72 h after plating.
when administered N-acetyl cysteine, compared to a 42% chance of symptom resolution on placebo [30]. In addition to raising glutathione levels, N-acetyl cysteine can react directly with hydrogen peroxide [35]. Furthermore, N-acetyl cysteine may also protect cells in glutathione-independent manners. For example, we have shown that N-acetyl cysteine can protect cells in a heat shock proteindependent manner [36]. Most studies of the two hit hypothesis use two different insults to elicit synergistic toxicity [5–10]. However, our previous studies show that two hits of the same insult can also be synergistic when the stressor dose is high enough [15]. Similarly, the present study supports the notion that the two hit hypothesis can be extended to sequential insults from the same H2 O2 toxin. However, we did not gather evidence that paraquat elicits extensive synergistic toxicity when administered as two sequential hits. Paraquat generates reactive oxygen species by induction of redox cycling with a cellular diaphorase, such as nitric oxide synthase [37]. Paraquat elicits toxicity by the formation of both superoxide and nitric oxide free radicals [38,39]. Unlike paraquat, hydrogen peroxide is a normal product of mitochondrial respiration and plays a role in
cell signaling [40]. However, hydrogen peroxide can also directly oxidize targets, especially at high concentrations [41]. The discrepancies between hydrogen peroxide and paraquat in the present study suggest that the two hit phenomenon is not universal but stress-specific. In addition, it should be noted that not all cell types respond to severe stress with synergistic toxicity. For example, the impact of a second toxic hit is blunted in highly stressed astrocyte survivors [42]. Thus, the response to two hits may depend upon multiple factors, such as cell type, type of toxin, and dose and duration of stress. In the present study, H2 O2 elicited a rise in catalase. This is inconsistent with the hypothesis that a loss in catalase defenses was responsible for toxicity. However, the data are still instructive in that they reveal that stressed cells protect themselves by upregulating defenses. This finding is consistent with previous studies showing that astrocytes and dopaminergic cells raise several pro-survival proteins to adapt to proteotoxic stress [2,42]. The catalase data are also consistent with studies demonstrating adaptive responses to oxidative stress for example, see [43]. Without this antioxidative response, we would expect H2 O2 -pretreated cells to
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Fig. 3. (A) N2a cells were treated with vehicle or one hit of H2 O2 (125 M) and assayed 24 h later for catalase and -actin by immunoblotting. Shown are the mean + S.E.M. from six experiments. + p ≤ 0.05 versus 0 M H2 O2 treatment, two-tailed t-test. N2a cells were treated with one or two hits of H2 O2 at the indicated concentrations 24 h after plating (1st hit) or 48 h after plating (2nd hit), in the absence or presence of N-acetyl cysteine (NAC, 3 mM) and assayed 72 h after plating for glutathione (GSH) levels ((B)–(D)) or for DRAQ5 ((E) and (G)), ␣-tubulin ((F) and (H)) and ATP (I). GSH was expressed as a fraction of DRAQ5+ cells to control for differences in cell numbers across groups. Shown are the mean + S.E.M. from 5 to 7 experiments. * p ≤ 0.05 versus 0 M 2nd hit, + p ≤ 0.05 versus 0 M 1st hit, ˆ p ≤ 0.05 versus 0 M NAC (H2 O vehicle), three-way ANOVA followed by Bonferroni post hoc test.
be all the more vulnerable to a 2nd hit and all three dose response curves to be shifted to the left. We performed multiple viability assays because cells can change their metabolic functions and cytoskeletal protein expression before actual cell death occurs. Cell counts, such as of DRAQ5stained nuclei, are also not sufficient by themselves because cells may be present without being metabolically or structurally healthy [44]. In other words, cytoskeletal disruption, metabolic compromise, and cell death can be uncoupled from each other. The use of three independent assays therefore gives us greater confidence that
H2 O2 elicits synergistic cell loss. Furthermore, all three assays are in agreement that N-acetyl cysteine was highly protective. Therefore, we can conclude that both structure and function are protected by this compound, even under conditions of extreme oxidative stress. Acknowledgments RKL designed the experiments and wrote the manuscript. ASU performed the majority of experiments. Additional experiments and technical support were provided by YJ, JR, SH, AG, and JP.
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