Rescue from a two hit, high-throughput model of neurodegeneration with N-acetyl cysteine

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Abstract

Postmortem tissue from patients with neurodegeneration exhibits protein-misfolding stress and reduced proteasome activity. This hallmark burden of proteotoxic stress has led to the term “proteinopathies” for neurodegenerative diseases. Proteinopathies may also be exacerbated by previous insults, according to the two hit hypothesis of accelerated neurodegeneration. In order to model the response to two successive insults in a high-throughput fashion, we exposed the neuronal cell line N2a to two hits of the proteasome inhibitor MG132 and performed three unbiased viability assays. MG132 toxicity was synergistically exacerbated following sequential hits provided the first hit was high enough to be toxic. This accelerated viability loss was apparent by (1) a nuclear and cytoplasmic stain (DRAQ5 + Sapphire), (2) immunocytochemistry for a cytoskeletal marker (α-tubulin), and (3) ATP levels (Cell Titer Glo). Ubiquitin-conjugated proteins were raised by toxic, but not subtoxic MG132, and were thus correlated with toxicity exacerbation at higher doses. We hypothesized that levels of autophagic and antioxidant defenses would be reduced with toxic, but not subtoxic MG132, explaining their differential impact on a second hit. However, proteins involved in chaperone-mediated autophagy were raised by toxic MG132, not reduced. Furthermore, inhibiting autophagy enhanced the toxicity of both subtoxic and toxic MG132 as well as of dual hits, suggesting that autophagic removal of cellular debris protected against proteasome inhibition. Two toxic hits of MG132 synergistically decreased the antioxidant glutathione. The glutathione precursor N-acetyl cysteine reversed this glutathione loss and prevented the toxic response to dual hits by all three assays. Dietary supplementation with N-acetyl cysteine benefits Alzheimer’s patients and is currently undergoing clinical trials in Parkinson’s disease. The present report is the first demonstration that this versatile compound is protective against synergistic loss of viability as well as of glutathione following unrelenting, sequential hits of proteotoxic stress as may occur in the diseased brain.

Highlights

► The first dual hit model of synergistic proteotoxic stress was developed in N2a cells. ► Toxic, but not subtoxic MG132 exacerbated the response to a second MG132 hit. ► Toxic MG132 raised adaptive autophagic defenses. ► Two hits of toxic MG132 caused a synergistic loss of glutathione. ► N-acetyl cysteine protected against synergistic proteotoxic stress by three viability assays.

Introduction

The response to cellular stress is not linear or monophasic; stress can elicit robust adaptations or severe toxicity depending on the dose and duration of treatment, commonly leading to U- or J-shaped dose response curves (Giordano et al., 2008, Mattson, 2008, Calabrese, 2010). Whether adaptations or toxic responses are elicited by a particular stress can be determined by challenging the previously stressed cells with a subsequent toxic hit and then measuring viability. In such protocols, subtoxic stress below the threshold required to elicit cell death often protects against subsequent challenges. This phenomenon is called preconditioning or tolerance and is well studied in ischemia, where short duration ischemic episodes protect against subsequent infarcts and can save animal lives (Dirnagl et al., 2003, Stenzel-Poore et al., 2007, Yang et al., 2010, Candilio et al., 2011). Although best studied in ischemia, preconditioning is also relevant to neurodegenerative conditions (Texel and Mattson, 2011). For example, preconditioning protocols using sublethal toxins in place of short duration ischemia can also elicit adaptations in models of neurodegeneration (Cannon et al., 2005, Xiao-Qing et al., 2005, Leak et al., 2006, Leak et al., 2008, Ding and Li, 2008, Collins et al., 2010). Whereas subtoxic or sublethal stress often elicits protection, toxic stress (defined as lethal stress above a threshold to elicit cell death) may weaken the remaining cells irreparably so that they are sensitized to subsequent hits, not protected. This phenomenon is theorized to occur in the brain of patients with neurodegenerative disorders and is varyingly referred to as the two hit, dual hit, or multiple hit model of neurodegeneration.

Many have theorized that early hits sensitize the brain to subsequent injury and pave the way for future neurodegeneration (Cory-Slechta et al., 2005, Carvey et al., 2006, Manning-Bog and Langston, 2007, Sulzer, 2007, Zhu et al., 2007a, Weidong et al., 2009, Boger et al., 2010, Gao and Hong, 2011). For example, aging may result in progressive cell loss in cerebral cortex (Pakkenberg and Gundersen, 1997) or phenotypic neurotransmitter loss in midbrain (McCormack et al., 2004), and toxic insults to the brain may shift this kinetic profile towards earlier and more aggressive loss. This is important to study because the threshold for displaying a frank clinical syndrome is reached at an earlier age than otherwise. Some examples of sensitizing stressors which predispose towards neurodegeneration include early life infections and inflammatory sequelae (Gao et al., 2003, Gao et al., 2011, Ling et al., 2004), traumatic brain injuries (Kiraly and Kiraly, 2007), and dual exposures to environmental poisons (Thiruchelvam et al., 2000, Cory-Slechta et al., 2005). While such dual insults can be spaced over decades, they may also occur in rapid succession or chronically. Specific examples of daily exposures include the frequent use of pesticides and herbicides by workers in agriculture, or exposure to toxicants in careers in industry. Similarly, protein-misfolding or proteotoxic stress is an unremitting burden in neurodegenerative diseases, as evidenced by their hallmark protein aggregations. Indeed, neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease are often called proteinopathies because proteotoxic stress is so intimately linked to their etiology (Walker and LeVine, 2000, Menzies et al., 2006, Walker et al., 2006, Dickson, 2009, Jellinger, 2009, Uversky, 2009, Angot et al., 2010, Xilouri and Stefanis, 2010). Furthermore, proteotoxic stress in proteinopathies and in aging is associated with chronic inhibition of the ubiquitin proteasome system (Conconi and Friguet, 1997, McNaught and Jenner, 2001, Stolzing and Grune, 2001, Carrard et al., 2002, Ding and Keller, 2003, Zeng et al., 2005).

The primary goal of the present study was to test the hypothesis that lethal proteotoxic stress weakens adaptive defenses against a second challenge in the remaining cells and to assess whether this phenomenon was dose-dependent. To accomplish this goal, we elicited protein-misfolding stress in the neuroblastoma cell line N2a with a reversible proteasome inhibitor, the substrate analog MG132. This is the first examination of the dual hit phenomenon with proteasome inhibition. In our model, a toxic hit of MG132 exacerbated the response to a second MG132 challenge by three independent measures. In contrast, low concentrations of MG132 failed to synergize with a second hit. In order to identify potential mechanisms underlying this synergistic toxicity, we assessed whether there was loss of protective autophagic and antioxidant defenses with lethal MG132. We chose these two cellular defenses because antioxidants can protect the cell from proteotoxicity and autophagy can aid in the removal of damaged proteins when the proteasome is inhibited (Iwata et al., 2005, Ding et al., 2007, Rubinsztein et al., 2007, Breusing and Grune, 2008, Janen et al., 2010, Wong and Cuervo, 2010). In contrast to a loss in these adaptations with high, toxic MG132 concentrations, one might expect low MG132 concentrations to raise such adaptations, leading to U-shaped dose response curves (Giordano et al., 2008, Mattson, 2008, Calabrese, 2010). We therefore measured two markers of autophagy following subtoxic as well as toxic MG132 and also inhibited autophagy. If autophagy is an adaptive compensatory response to proteasome inhibition, autophagy inhibitors ought to further exacerbate MG132 toxicity. However, autophagic self-digestion can also lead to cell death in times of severe stress, although this is far less common (Menzies et al., 2006, Xilouri and Stefanis, 2010). In the latter scenario, one would expect toxic MG132 to raise markers of autophagy instead and autophagy inhibitors to protect against MG132 toxicity in the two hit protocol.

Similar to proteotoxic stress, signs of oxidative stress are omnipresent in postmortem tissue from patients with Parkinson’s and Alzheimer’s disease (Alam et al., 1997, Gabbita et al., 1998, Jenner, 2003, Zhu et al., 2005, Lovell and Markesbery, 2007, Butterfield et al., 2010). Antioxidant defenses are expected to protect against proteotoxicity because proteasome inhibition is closely associated with oxidative damage (Breusing and Grune, 2008). We expected a loss of antioxidant defenses with lethal MG132 and focused on the ferroxidase and copper chaperone ceruloplasmin as well as the ubiquitous and abundant tripeptide glutathione (γ-l-Glutamyl-l-cysteinylglycine). We chose ceruloplasmin because Alzheimer’s and Parkinson’s patients suffer from deficits in serum ceruloplasmin (Brewer et al., 2010, Torsdottir et al., 2010). We were particularly interested in this protein because the effects of proteasome inhibitors on ceruloplasmin have not been investigated. We also chose glutathione because both Alzheimer’s and Parkinson’s disease elicit an early loss of glutathione defenses (Sofic et al., 1992, Sian et al., 1994, Calabrese et al., 2006, Baldeiras et al., 2008, Zeevalk et al., 2008, Lloret et al., 2009, Martin and Teismann, 2009). Loss of glutathione is expected to be catastrophic because it so effectively detoxifies reactive oxygen and nitrogen species and is present in the millimolar range (Dringen, 2000, Pompella et al., 2003, Pocernich and Butterfield, 2011).

The second major goal of the present study was to find an effective means of neuroprotection in sequentially challenged cells. A high-throughput two hit model such as ours, where weakened cells are rendered highly prone towards cellular demise, is an ideal platform to screen potential neuroprotective compounds in a rapid and cost-effective manner. Because we observed that two hits of toxic MG132 caused a synergistic loss in glutathione levels, we tested whether the glutathione precursor N-acetyl cysteine could rescue cells in the two hit model. N-acetyl cysteine crosses the blood brain barrier and provides the rate-limiting cysteine precursor for glutathione synthesis (Pocernich et al., 2000, Neuwelt et al., 2001, Farr et al., 2003). N-acetyl cysteine antioxidant supplementation has been tested in clinical trials against Alzheimer’s disease, with measurable success (Adair et al., 2001). N-acetyl cysteine is also in clinical use for non-neurodegenerative conditions (Dodd et al., 2008, Dean et al., 2011). Notably, this compound is currently being tested in Parkinson’s patients (Clinicaltrials.gov. ID: NCT01470027). Although N-acetyl cysteine is known to protect against singular insults, it is not known if this compound will protect against the sensitization of cells to a second hit in more realistic, multiple hit models of proteotoxic neurodegeneration. We found that both the loss of glutathione as well as the loss of viability with dual hits of MG132 were abrogated by N-acetyl cysteine. As expected, N-acetyl cysteine also completely protected against single hits of MG132. These data support the use of N-acetyl cysteine in neurodegenerative conditions where protein-misfolding stress is rampant, and demonstrate that this supplement can even prevent the loss of cells following unrelenting sequential hits of high-dose proteotoxic stress.

Section snippets

Chemicals and antibodies

Chemicals were purchased from Sigma–Aldrich (St. Louis, MO), unless specified otherwise. Peptide aldehyde MG132 (carbobenzoxy-Leu-Leu-leucinal), a reversible substrate analog and inhibitor of the chymotrypsin-like activity of the proteasome particle, was purchased from EMD Millipore (Billerica, MA). Primary and secondary antibodies were purchased and used as follows: mouse anti-α-tubulin (1:10,000 for immunocytochemistry, Cat. No. T5168, Lot No. 078K4781, Sigma–Aldrich), mouse anti-heat shock

Validation of multiple assays

In order to validate our high-throughput assays, we plated a range of cellular densities and checked for a linear rise in signal strength with increasing number of cells plated (Fig. 1). All three assays were linear under these normal, untreated conditions. The first assay was the infrared nuclear (DRAQ5) and cytoplasmic (Sapphire) stain for approximate assessments of cell number (Fig. 1A; R2 = 0.9998, p = 0.0001). Second, cells were immunostained for cytoskeletal α-tubulin with infrared

Discussion

The present study reports a novel high-throughput two hit model of neurodegenerative toxicity that is exquisitely concentration dependent. The findings demonstrate for the first time that cells can be placed in a severely compromised state when exposed to successive, high levels of proteotoxic stress and provide potential insights into similar stress sensitization in proteinopathies. The toxic response to high-dose hits was correlated with a considerable rise in ubiquitin-conjugated proteins,

Conclusions

The present report reveals that two hits of proteotoxic stress combine to elicit synergistic toxicity, but only if a threshold concentration is reached. This was accomplished using a novel, high-throughput two hit model of neurodegeneration and multiple unbiased viability assays. We found that a toxic concentration of MG132 elicited a rise in ubiquitin-conjugated proteins, ceruloplasmin, LAMP2a, and Hsc70, with the latter three responses presumably being adaptive in nature. Inhibition of

Acknowledgments and Disclosures

This work was supported by a startup award from Duquesne University to Rehana Leak. Planning of the experiments and writing of the manuscript was performed by Rehana Leak. The majority of experiments were performed jointly by Ajay Unnithan and Hailey Choi. Some additional experiments were performed by Amanda Titler and Jessica Posimo. We are deeply grateful to Deb Wilson, Mary Caruso, and Jackie Farrer for superb administrative support. The authors have approved the final version of the article

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