Abstract
In many neurodegenerative disorders, aggregates of ubiquitinated proteins are detected in neuronal inclusions, but their role in neurodegeneration remains to be defined. To identify intracellular mechanisms associated with the appearance of ubiquitin-protein aggregates, mouse neuronal HT4 cells were treated with cadmium. This heavy metal is a potent cell poison that mediates oxidative stress and disrupts the ubiquitin/proteasome pathway. In the current studies, the following intracellular events were found to be also induced by cadmium: (i) a specific rise in cyclooxygenase-2 (COX-2) gene expression but not COX-1; (ii) an increase in the extracellular levels of the proinflammatory prostaglandin E2, a product of COX-2; and (iii) production of 4-hydroxy-2-nonenal-protein adducts, which result from lipid peroxidation. In addition, cadmium treatment led to the accumulation of high molecular weight ubiquitin-COX-2 conjugates and perturbed COX-2 glycosylation. The thiol-reducing antioxidant N-acetylcysteine, and, to a lesser extent, the COX-2 inhibitor celecoxib, attenuated the loss of cell viability induced by cadmium demonstrating that oxidative stress and COX-2 activation contribute to cadmium cytotoxicity. These findings establish that disruption of the ubiquitin/proteasome pathway is not the only event triggered by cadmium. This oxidative stressor also activates COX-2 function. Both events could be triggered by formation of 4-hydroxy-2-nonenal as a result of cadmium-induced lipid peroxidation. Proinflammatory responses stimulated by oxidative stressors that mimic the cadmium effects may, therefore, be important initiators of the neurodegenerative process and exacerbate its progress.
Highlights
The ubiquitin/proteasome pathway plays a major role in the intracellular quality control process by degrading mutated or abnormally folded proteins to prevent their accumulation as aggregates
Based on the observations that Cd2ϩ-induced oxidative stress is closely associated with COX-2 upregulation and neuronal cell death, we evaluated the protective effect of two antioxidants (N-acetylcysteine (NAC) and ascorbic acid) and a COX-2-specific inhibitor on Cd2ϩ cytotoxicity
In cells treated with higher Cd2ϩ concentrations, such as 3, 15, 30 and, in some cases, 45 M, two additional bands were identified as corresponding to COX-2 forms that are N-glycosylated at four sites (74 kDa) and nonglycosylated (65 kDa), respectively [27]
Summary
The ubiquitin/proteasome pathway plays a major role in the intracellular quality control process by degrading mutated or abnormally folded proteins to prevent their accumulation as aggregates (for review, see Ref. 1). In this model, transfected rat striatal neurons coexpressing a nonfunctional ubiquitin-conjugating enzyme and a mutant huntingtin containing polyglutamine expansions showed a greater loss of cell viability than transfectants expressing the huntingtin mutant alone [9] Together, these results strongly support the view that disruption of the ubiquitin/proteasome pathway plays an important role in neurodegeneration. The production of free radicals by oxidative stress promotes partial unfolding of cellular proteins, resulting in exposure of previously buried hydrophobic domains to proteolytic enzymes [12,13,14] and to ubiquitin-conjugating enzymes [15] This sudden increase in protein substrates may compromise the capacity of the ubiquitin/proteasome pathway to clear the abnormal proteins and cause their aggregation and accumulation within the cell. The link between oxidative stress and the accumulation of ubiquitinated proteins in neurodegeneration remains to be established
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