Previous studies have shown that

Previous studies have shown that Cu(I) neurotoxicity includes induction of ROS with involvement of the NF-kB pathway and apoptosis (Butterworth, 2010) and that, in its Cu(I) state, Cu(I) alters microglia phenotype and inhibits nitric oxide release (Rossi-George et al., 2012). Cu(I) has a great affinity for thiols (SH groups) and is an effective catalyst for the formation and degradation of SNOs (Burg et al., 2000). Thus, high levels of Cu(I) can be toxic for the CNS. Cu(I) is an essential transition metal whose ability to accept and donate electrons makes it an intrinsic component of numerous essential enzymatic reactions (Burg et al., 2000, Field et al., 2002). Its importance for general metabolic processes, antioxidant properties, iron metabolism, neurotransmitter and neuropeptide synthesis, and neuromodulatory effects is well established (reviewed in (Scheiber et al., 2014). However, its high redox activity can also lead to the generation of toxic reactive oxygen species (Rubino and Franz, 2012). Peripheral Cu(I) is transported into the z vad fmk as free Cu(I) ions (Choi and Zheng, 2009); under physiological conditions, binding of Cu(I) to metallothioneins and to other low molecular mass ligands including glutathione (GSH) keeps intracellular free levels low (Rae et al., 1999). Impairment of homeostatic Cu(I) concentration leads to brain degeneration – with Wilson’s and Menkes’ diseases at the extremes of Cu(I) excess and deficiency. Disruption of homeostatic Cu(I) concentrations and/or distribution in the brain has also been observed in several neurodegenerative diseases, including Alzheimer’s disease (AD; (Ayton et al., 2013), familial amyotrophic lateral sclerosis (Lovejoy and Guillemin, 2014), Parkinson’s disease (Dusek et al., 2015), and prion diseases (Haigh et al., 2010). In AD, for example, while the total brain concentration of soluble extractable Cu(I) is reduced (Rembach et al., 2013), high levels are found in senile plaques (Noda et al., 2013). In addition, because Aβ binds to Cu(I) with great affinity, Aβ can reduce Cu(II) to Cu(I) thereby promoting Aβ oligomer toxicity (Eskici and Axelsen, 2012). While various mechanisms have been proposed, the exact role Cu(I) plays in neurodegeneration is still elusive (Scheiber et al., 2014). Elevated levels of Cu(I) in the neuronal microenvironment will determine the fate of NO, possibly by dysregulating the balance kept by GSNO and GSNOR. Because of the unique expression of GSNOR in the brain, we sought to examine whether Cu(I) affects GSNOR and SNO content thus regulating NO metabolism in microglia. Our results show that a high dose of Cu(I) inhibits GSNOR activity and reduces SNO accumulation in activated microglia.
Materials and methods
Results
Discussion Neuroinflammation plays an important role in the pathogenesis of many neurodegenerative diseases. Activated microglia have been implicated both in the initiation and progression of neurodegeneration by acquiring different phenotypes that range from induction of neuronal injury and death through the release of cytotoxic products to promotion of injury resolution through the release of anti-inflammatory products. The landmark of inflammation is a massive induction of iNOS with the subsequent release of NO by activated microglia. S-nitrosylation of protein targets has been associated with neurodegenerative disorders including Alzheimer’s and Parkinson’s (Brown, 2010). Cu(I) dysfunction has also been observed in neurodegeneration (Squitti et al., 2013). The ‘metal-based neurodegeneration hypothesis’ is based on the observation that the generation of oxidative stress results from the production of reactive oxygen (ROS) and nitrogen species (RNS) by redox-active metal ions including Cu(I). Proteins are a major target of ROS and RNS. This study sought to extend our previous results on the effects of Cu(I) on microglia. Our results confirmed that exposure to a supra-physiologic dose of Cu(I) significantly reduces the production of nitrite in LPS-stimulated BV2 microglia, a result consistent with our previous finding showing that Cu(I) alters microglia phenotype through release of NO. Interestingly, a study by Colasanti and colleagues (Colasanti et al., 2000) found that exposure of LPS-stimulated C6 glioma cells to CuSO4 for 1 h, and measured 48 h later, had no effect on nitrite release. The different outcome could be due to the dose of LPS being one order of magnitude higher than in the present study and administered in combination with interferon (IFN) γ, the different species of Cu(I) used, or the length of exposure to Cu(I). In light of our finding that treatment with 1 μM Cu(I) inhibits nitrite release elicited by both 100 and 1000 ng/mL LPS, we attribute the lack of effect reported by the Colasanti and colleagues to a dose of Cu(I) that is insufficient to inhibit NO formation brought about by 10,000 ng/mL LPS plus IFN γ. The addition of IFN-γ to LPS is an important difference as it was shown that while stimulation with LPS alone does not induce cell death, the combination of LPS (100 ng/mL) and IFN γ does, and it involves two distinct pathways: one mediated by NO and the other by Caspase-11 (Mayo and Stein, 2007). Our results showing that 100 μM Cu(II) reduces nitrite release in microglia stimulated with a high dose of LPS (1000 ng/mL) is, most likely, due to Cu(II) having been reduced by copper-reducing molecules within the cell, including ascorbate, reduced glutathione and other endogenous thiols (Aliaga et al., 2016).