Exacerbation of Copper Toxicity in Primary Neuronal Cultures Depleted of Cellular Glutathione

Abstract

Perturbations to glutathione (GSH) metabolism may play an important role in neurodegenerative disorders such as Alzheimer's, Parkinson's, and prion diseases. A primary function of GSH is to prevent the toxic interaction between free radicals and reactive transition metals such as copper (Cu). Due to the potential role of Cu in neurodegeneration, we examined the effect of GSH depletion on Cu toxicity in murine primary neuronal cultures. Depletion of cellular GSH with L-buthionine-[S,R]-sulfoximine resulted in a dramatic potentiation of Cu toxicity in neurons without effect on iron (Fe) toxicity. Similarly, inhibition of glutathione reductase (GR) activity with 1,3-bis(2-chloroethyl)-l -nitrosurea also increased Cu toxicity in neurons. To determine if the Alzheimer's amyloid-p (Ap) peptide can affect neuronal resistance to transition metal toxicity, we exposed cultures to nontoxic concentrations of Ap25-35 in the presence or absence of Cu or Fe. Ap25-35 pretreatment was found to deplete neuronal GSH and increase GR activity, confirming the ability of Ap to perturb neuronal GSH homeostasis.Ap25-35 pretreatment potently increased Cu toxicity but had no effect on Fe toxicity. These studies demonstrate an important role for neuronal GSH homeostasis in selective protection against Cu toxicity, a finding with widespread implications for neurodegenerative disorders.

The mitochondrial permeability transition, and oxidative and nitrosative stress in the mechanism of copper toxicity in cultured neurons and astrocytes

“…The latter possibility is consistent with reports documenting copper-induced lipid peroxidation and associated cell death in neurons and glioma cells. 47,48 Our observation that vitamin E blocked copper-induced cell death in astrocytes is consistent with the above reports. Copper has also been shown to cause DNA damage and cell death in brain in an animal model of Wilson's disease.…”

“…21,22,47,48 Our observation that PBN, which is known to trap several free radical species, including superoxide, 49 significantly blocked astrocyte death suggests that multiple species of free radicals produced by copper play a role in astrocyte death. In addition, the fact that vitamin E blocked cell death by copper suggests that lipid peroxidation represents one mechanism by which OS results in cell death.…”

“…As noted above, copper is well known to cause oxidative and nitrosative stress (ONS). 21,22,38,47,48 Copper has also been shown to deplete glutathione levels in cultured neurons, 47 an event likely to aggravate OS and neuronal death.…”

Clioquinol Promotes Cancer Cell Toxicity through Tumor Necrosis Factor α Release from Macrophages

“…Although we did not investigate the cell toxicity pathway in this study, it is known that TNF␣ can induce apoptosis in cancer cells. However, activation of the cell death pathway in HeLa cells normally requires inhibition of protein synthesis in addition to TNF␣ treatment (White et al, 1999). Protein synthesis inhibition was not required to induce the toxic effects observed in our cultures by CQ macrophageconditioned media or recombinant TNF␣.…”

“…For coculture experiments, HeLa cells on coverslips were added to wells containing macrophages. CQ (10 mM stock in dimethyl sulfoxide) and/or Cu(II) (10 mM in dH2O) was added to macrophageHeLa cell cocultures at indicated concentrations for 24 h. Coverslips of HeLa cells were then removed, and cell viability of macrophages and HeLa cells was determined separately using the MTT assay as described previously (White et al, 1999) or Trypan blue staining with 0.4% Trypan blue (Sigma-Aldrich, St. Louis, MO). For experiments involving transfer of conditioned medium, macrophages were treated with CQ or metals at indicated concentrations for 2 or 24 h, and medium was transferred to separate cultures of HeLa cells for a further 24 h. This was followed by MTT or Trypan blue assay of cell viability.…”