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

• Published in: Neurochemistry international Vol. 61; no. 3; pp. 356 - 368
• Main Authors: Unnithan, Ajay S., Choi, Hailey J.H., Titler, Amanda M., Posimo, Jessica M., Leak, Rehana K.
• Format: Journal Article
• Language: English
• Published: Kidlington Elsevier Ltd 01.08.2012; Elsevier
• Subjects: Acetylcysteine - pharmacology; Alzheimer's disease; Alzheimer’s; Antioxidants; ATP; Autophagy; Biological and medical sciences; Blotting, Western; Brain; Cell Line, Tumor; Ceruloplasmin; Clinical trials; Cysteine; Cytoskeleton; Degenerative and inherited degenerative diseases of the nervous system. Leukodystrophies. Prion diseases; Dietary supplements; Dual hit; Fundamental and applied biological sciences. Psychology; Glutathione; Humans; Immunocytochemistry; Immunohistochemistry; In cell western; Medical sciences; Movement disorders; Neurodegenerative diseases; Neurology; Neurons - cytology; Neurons - drug effects; Neurotoxicity; Parkinson's disease; Parkinson’s; Phagocytosis; proteasome inhibitors; proteasomes; Stains; Stress; Tubulin; Vertebrates: nervous system and sense organs; Ceruloplasmin; DMSO; Autophagy; Alzheimer’s; LAMP2a; Parkinson’s; NAC; Hsp70; In cell western; GSH; Hsc70; Dual hit; Glutathione; Parkinson's; Nervous system diseases; Cysteine; Alzheimer disease; Parkinson disease; Cerebral disorder; Central nervous system disease; Degenerative disease; Degeneration; Models; Extrapyramidal syndrome; Alzheimer's
• ISSN: 0197-0186; 1872-9754
• DOI: 10.1016/j.neuint.2012.06.001

► 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. 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.