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

Jiang, Yihui; Rumble, Jennifer L.; Gleixner, Amanda M.; Unnithan, Ajay S.; Pulugulla, Sree H.; Posimo, Jessica M.; Choi, Hailey J.; Crum, Tyler S.; Pant, Deepti B.; Leak, Rehana K.

doi:10.1016/j.neuroscience.2013.09.049

Cited by 14 publications

(26 citation statements)

References 81 publications

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“…MG132 was applied in the presence or absence of the glutathione precursor N-acetyl cysteine. These data can also be found in our recent publication on the protective effects of N-acetyl cysteine 30 . Cells were assayed 48 hr following MG132 treatments.…”

Section: Representative Resultssupporting

confidence: 72%

Viability Assays for Cells in Culture

Posimo

Unnithan

Gleixner

et al. 2014

JoVE

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Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16 bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (α-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function. Video LinkThe video component of this article can be found at

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Section: Representative Resultssupporting

confidence: 72%

Viability Assays for Cells in Culture

Posimo

Unnithan

Gleixner

et al. 2014

JoVE

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“…Our recent in vitro studies demonstrate that NAC can robustly protect cortical neurons and neuroblastoma cells against proteasome inhibitors and oxidative stress (Jiang et al, 2013; Posimo et al, 2013; Unnithan et al, 2012; Unnithan et al, 2014). Indeed, NAC was so protective that it prevented cell death in response to severe, dual hits of the proteasome inhibitor MG132 and hydrogen peroxide (Unnithan et al, 2012; Unnithan et al, 2014).…”

Section: Discussionmentioning

confidence: 99%

“…Indeed, NAC was so protective that it prevented cell death in response to severe, dual hits of the proteasome inhibitor MG132 and hydrogen peroxide (Unnithan et al, 2012; Unnithan et al, 2014). We also discovered that NAC can prevent loss of heat shock protein defenses in neuroblastoma cells treated with high concentrations of MG132 and that inhibitors of heat shock protein 70 (Hsp70) abolished NAC-mediated protection (Jiang et al, 2013). In the present study we established that intraperitoneal NAC only transiently protects nigrostriatal neurons from oxidative stress in the 6-OHDA animal model and that it exerts some degree of mild toxicity over longer periods.…”

Section: Discussionmentioning

confidence: 99%

“…NAC can induce AP-1 activity by the sequential activation of ERK and Elk-1, which, in turn, binds to the c- Fos promoter and induces c-Fos expression (Meyer et al, 1993; Muller et al, 1997). We have also shown that NAC can protect cells in a heat shock protein 70-dependent manner (Jiang et al, 2013), perhaps through thiol exchanges with transcription factors that regulate synthesis of this protein, such as heat shock factor 1. Thus, the goal of the present study was to develop an animal model in which the molecular mechanisms underlying the protective effects of NAC could be dissected.…”

Section: Introductionmentioning

confidence: 96%

“…For example, we recently showed that NAC prevents neurodegeneration in multiple cellular models of neurodegenerative disorders (Jiang et al, 2013; Posimo et al, 2013; Unnithan et al, 2012; Unnithan et al, 2014). Consistent with these observations, Clark and colleagues have shown that NAC can prevent dopaminergic terminal loss and α-synucleinopathy in a transgenic mouse model of Parkinson's disease (Clark et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

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Investigation of the therapeutic potential of N-acetyl cysteine and the tools used to define nigrostriatal degeneration in vivo

Nouraei

Zarger

Weilnau

et al. 2016

Toxicology and Applied Pharmacology

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The glutathione precursor N-acetyl-L-cysteine (NAC) is currently being tested on Parkinson's patients for its neuroprotective properties. Our studies have shown that NAC can elicit protection in glutathione-independent manners in vitro. Thus, the goal of the present study was to establish an animal model of NAC-mediated protection in which to dissect the underlying mechanism. Mice were infused intrastriatally with the oxidative neurotoxicant 6-hydroxydopamine (6-OHDA; 4 μg) and administered NAC intraperitoneally (100 mg/kg). NAC-treated animals exhibited higher levels of the dopaminergic terminal marker tyrosine hydroxylase (TH) in the striatum 10d after 6-OHDA. As TH expression is subject to stress-induced modulation, we infused the tracer FluoroGold into the striatum to retrogradely label nigrostriatal projection neurons. As expected, nigral FluoroGold staining and cell counts of FluoroGold+ profiles were both more sensitive measures of nigrostriatal degeneration than measurements relying on TH alone. However, NAC failed to protect dopaminergic neurons 3 weeks following 6-OHDA, an effect verified by four measures: striatal TH levels, nigral TH levels, nigral TH+ cell counts, and nigral FluoroGold levels. Some degree of mild toxicity of FluoroGold and NAC was evident, suggesting that caution must be exercised when relying on FluoroGold as a neuron-counting tool and when designing experiments with long-term delivery of NAC—such as clinical trials on patients with chronic disorders. Finally, the strengths and limitations of the tools used to define nigrostriatal degeneration are discussed.

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N‐Acetyl Cysteine Is Associated With Dopaminergic Improvement in Parkinson's Disease

Monti

Zabrecky

Kremens

et al. 2019

Clin Pharma and Therapeutics

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This study assessed the biological and clinical effects in patients with Parkinson's disease (PD) of N‐acetyl‐cysteine (NAC), the prodrug to l‐cysteine, a precursor to the natural biological antioxidant glutathione. Forty‐two patients with PD were randomized to either weekly intravenous infusions of NAC (50 mg/kg) plus oral doses (500 mg twice per day) for 3 months or standard of care only. Participants received prebrain and postbrain imaging with ioflupane (DaTscan) to measure dopamine transporter (DAT) binding. In the NAC group, significantly increased DAT binding was found in the caudate and putamen (mean increase from 3.4% to 8.3%) compared with controls (P < 0.05), along with significantly improved PD symptoms (P < 0.0001). The results suggest NAC may positively affect the dopaminergic system in patients with PD, with corresponding positive clinical effects. Larger scale studies are warranted.

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

Cited by 14 publications

References 81 publications

Viability Assays for Cells in Culture

Viability Assays for Cells in Culture

Investigation of the therapeutic potential of N-acetyl cysteine and the tools used to define nigrostriatal degeneration in vivo

N‐Acetyl Cysteine Is Associated With Dopaminergic Improvement in Parkinson's Disease

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