N-Acetyl cysteine blunts proteotoxicity in a heat shock protein-dependent manner

•N-Acetyl cysteine (NAC) protects N2a cells against the proteasome inhibitor MG132.•NAC does not raise glutathione in this model.•NAC facilitates the Hsp70 response in MG132-treated cells.•NAC attenuates the MG132-induced rise in ubiquitin-conjugated proteins.•Hsp70/Hsc70 inhibition abolishes protection and the effect on ubiquitinated proteins.

N-Acetyl cysteine, a glutathione precursor, has been shown to benefit patients with Alzheimer's disease and reduce the symptoms of traumatic brain injury in soldiers. Parkinson's and Alzheimer's disease are both characterized by stress from protein misfolding, or proteotoxicity. We have developed a high-throughput model of proteotoxicity by treating neuroblastoma N2a cells with the proteasome inhibitor MG132 and performing three independent assays for viability. Our previous study showed that N-acetyl cysteine protects N2a cells against two sequential treatments of MG132 and raises glutathione levels in a two-hit model of synergistic neurodegeneration. In the present study, however, N-acetyl cysteine was found to reduce the toxicity of a single hit of MG132 independent of its effect on glutathione. All three viability assays confirmed this protection. We measured heat shock protein 70 (Hsp70) levels because Hsp70 is a protective chaperone that helps refold proteins or guides ubiquitinated proteins toward degradation by the proteasome. Hsp70 levels were higher in MG132-treated cells when N-acetyl cysteine was applied. No parallel change in heat shock cognate 70 (Hsc70) was elicited. Inhibition of Hsp70/Hsc70 activity with VER 155008 attenuated the protection afforded by N-acetyl cysteine in a dose-responsive manner. MG132 induced a large rise in ubiquitinated proteins and N-acetyl cysteine reduced this effect. Consistent with the chaperone functions of Hsp70, VER 155008 also prevented the reduction in ubiquitin-conjugated proteins by N-acetyl cysteine. These data reveal a new role for N-acetyl cysteine: this compound may reduce misfolded protein levels and ameliorate proteotoxicity through heat shock proteins. These findings broaden the potential mechanisms of action for this dietary supplement in neurodegenerative proteinopathies.