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

Abstract

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.

A functionalized hydroxydopamine quinone links thiol modification to neuronal cell death

“…Another early study attributed the protective effect of cysteine to very quickly replenishing the GSH/GSSG balance upon challenge with 6-OHDA but did not consider a nucleophilic attack of cysteine's thiol on the 6-OHDA quinone [43]. In addition, a large number of studies investigated the role of different antioxidants and found that compounds containing thiols protect against ROS [43,[88], [89], [90], [91]]. Our finding that thiol modification is a major factor contributing to cell death provides a simple and clear explanation for all these observations.…”

N-acetylcysteine protects against motor, optomotor and morphological deficits induced by 6-OHDA in zebrafish larvae

“…Because of its mechanism of action, NAC is likely to have protected these pathways, preventing the morphological changes caused by 6-OHDA. NAC (100 mg/kg) was able to increase the levels of the dopaminergic marker TH in the striatum of mice exposed to 6-OHDA, reinforcing the important effect of NAC in the preservation of the dopaminergic system ( Nouraei et al, 2016 ). We observed main effects of NAC in the direction of increased forebrain width and (consequently) eye distance ( Table 2 )—this is very different from the damaging effects of 6-OHDA and other toxins, which are expected to decrease such values.…”

“…NAC also reduces oxidative damage markers ( Alboni et al, 2013 ), increases the number of brain synapses ( Samuni et al, 2013 ) and activates the mitochondrial complex I ( Samuni et al, 2013 ). Although there is limited research about NAC in Parkinson’s disease, some studies have demonstrated that NAC has potential as a therapeutic strategy for PD prevention and treatment in humans and PD animal models ( Muñoz et al, 2004 ; Martínez-Banaclocha, 2012 ; Katz et al, 2015 ; Nouraei et al, 2016 ; Coles et al, 2017 ).…”

Mechanistic Research for the Student or Educator (Part II of II)

“…There are many more complicated examples of real-life data than depicted here, including data from non-mechanistic experiments. For an example of controls for full-factorial ANOVAs in purely descriptive (non-mechanistic) studies, the student is guided to a report showing an unexpected lack of lasting therapeutic effects of N-acetyl cysteine (and evidence of some toxicity) in an animal model of acute dopaminergic neuron loss (5). Although there are many caveats in this simple study, the inclusion of all control groups in full-factorial ANOVAs mitigated the risk of Type I and II errors.…”