N-acetyl-L-cysteine (NAC) has been shown to improve aspects of cognitive function in Alzheimer's disease (AD), increase brain glutathione (GSH) levels in Parkinson's disease (PD), and double the chances of symptom resolution in soldiers suffering traumatic brain injury. Our recent studies reveal that NAC can have both GSH-dependent and GSH-independent actions. We have discovered that NAC can protect neuronal cell lines, primary astrocytes, and primary neurons through heat shock proteins. In all 3 cellular models, the pan-Hsp70 inhibitor VER155008 abolished the protective effects of NAC against the proteasome inhibitor MG132. NAC upregulated HspA1/2, the major member of the Hsp70 family, in MG132-treated N2a cells. NAC did not interfere with direct or acute MG132 inhibition of chymotrypsin-like proteasome activity in N2a cells, but did eventually reduce the MG132-induced increase in ubiquitinated proteins in an Hsp70- dependent manner. Thus, we hypothesize that NAC protects neurons against protein-misfolding stress through Hsp70.
In Aim 1, we will generate a comprehensive NAC concentration-response curve in primary cortical and hippocampal neurons treated with two proteasome inhibitors: MG132 and lactacystin. We will perform three independent, unbiased viability assays that we have shown to be linearly correlated with cell density. NAC-induced changes in HspA1/2, Hsc70, mitochondrial Hsp70, and GRP78 will be assessed in both models by two techniques: immunoblotting and RT-PCR. Toxin action will be verified by measuring both proteasome activity and K48-linked ubiquitinated proteins. We will test if three pan-Hsp70 inhibitors, VER155008, MAL3-101, and 2-phenylethyne-sulfonamide attenuate NAC-mediated protection. We will then perform subcellular fractionation followed by Westerns to determine if NAC elicits nuclear translocation of Nrf2 or HSF1, both of which are known to control Hsp70 gene transcription. Nrf2 is normally inhibited by Keap1. Keap1 and HSF1 have reactive cysteine residues that may react with nucleophiles such as NAC in thiol exchange reactions. We will also assess HSF1 activation via phosphorylation at Ser230. If NAC elicits Nrf2 or HSF1 activation, we will knock them down to see if NAC-mediated protection and Hsp70 induction is abolished.
In Aim 2, we will generate a dose-response curve with hippocampal and cortical infusions of lactacystin in mice and perform stereological counts of NeuN+ cells to determine the ~LD50 for neuron loss. We will then inject vehicle or various doses of NAC into lactacystin or vehicle-treated mice to see if NAC reduces lactacystin toxicity in vivo. We will also measure Hsp70 proteins, Nrf2/HSF1 activation, proteasome activity, and ubiquitinated proteins in vivo. These studies are expected to shed light on why NAC may be efficacious in the clinic and seed future grants to a) inhibit Hsp70 proteins in vivo, 2) administe -amyloid oligomers and ?-synuclein fibrils to NAC-treated cells, 3) examine the impact of NAC in fibroblasts from PD and AD patients, and 4) mutate cysteine residues in HSF1 and Keap1 to alanine to see if NAC action is thereby abolished.

Public Health Relevance

N-acetyl-L-cysteine (NAC) has been used to ameliorate the symptoms of multiple neurological and psychiatric conditions. NAC is tolerable and inexpensive (9/capsule), helping to improve patient compliance and reduce health disparities. We recently discovered that NAC may elicit protection in a heat shock protein- dependent manner. Our goal is to examine this possibility in hippocampal and cortical neurons in vitro and in vivo and to identify whether it increases heat shock proteins through Nrf2 or HSF1 activation. Our studies may identify potential mechanisms underlying NAC-mediated effects in the clinic.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Small Research Grants (R03)
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Neural Oxidative Metabolism and Death Study Section (NOMD)
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Corriveau, Roderick A
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Duquesne University
Schools of Pharmacy
United States
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Gleixner, Amanda M; Hutchison, Daniel F; Sannino, Sara et al. (2017) N-Acetyl-l-Cysteine Protects Astrocytes against Proteotoxicity without Recourse to Glutathione. Mol Pharmacol 92:564-575
Heinemann, Scott D; Posimo, Jessica M; Mason, Daniel M et al. (2016) Synergistic stress exacerbation in hippocampal neurons: Evidence favoring the dual-hit hypothesis of neurodegeneration. Hippocampus 26:980-94
Nouraei, Negin; Zarger, Lauren; Weilnau, Justin N et al. (2016) Investigation of the therapeutic potential of N-acetyl cysteine and the tools used to define nigrostriatal degeneration in vivo. Toxicol Appl Pharmacol 296:19-30
Mason, Daniel M; Nouraei, Negin; Pant, Deepti B et al. (2016) Transmission of ?-synucleinopathy from olfactory structures deep into the temporal lobe. Mol Neurodegener 11:49