Oxidative Modification of Peroxiredoxin Is Associated with Drug-induced Apoptotic Signaling in Experimental Models of Parkinson Disease

Abstract

The aim of this study was to investigate changes in protein profiles during the early phase of dopaminergic neuronal death using two-dimensional gel electrophoresis in conjunction with mass spectrometry. Several protein spots were identified whose expression was significantly altered following treatment of MN9D dopaminergic neuronal cells with 6-hydroxydopamine (6-OHDA). In particular, we detected oxidative modification of thioredoxin-dependent peroxidases (peroxiredoxins; PRX) in treated MN9D cells. Oxidative modification of PRX induced by 6-OHDA was blocked in the presence of N-acetylcysteine, suggesting that reactive oxygen species (ROS) generated by 6-OHDA induce oxidation of PRX. These findings were confirmed in primary cultures of mesencephalic neurons and in rat brain injected stereotaxically. Overexpression of PRX1 in MN9D cells (MN9D/PRX1) exerted neuroprotective effects against death induced by 6-OHDA through scavenging of ROS. Consequently, generation of both superoxide anion and hydrogen peroxide following 6-OHDA treatment was decreased in MN9D/PRX1. Furthermore, overexpression of PRX1 protected cells against 6-OHDA-induced activation of p38 MAPK and subsequent activation of caspase-3. In contrast, 6-OHDA-induced apoptotic death signals were enhanced by RNA interference-targeted reduction of PRX1 in MN9D cells. Taken together, our data suggest that the redox state of PRX may be intimately involved in 6-OHDA-induced dopaminergic neuronal cell death and also provide a molecular mechanism by which PRX1 exerts a protective role in experimental models of Parkinson disease. Parkinson disease (PD)3 is a common neurodegenerative disorder characterized by progressive degeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta (1). Clinical manifestations, such as resting tremor, slowness of movement, stiffness, and postural instability in patients with PD are consequences of the loss of DA neurons and the resulting depletion of dopamine in the striatum. Most PD cases are sporadic, and its etiology is incompletely understood; however, an increasing body of evidence suggests that oxidative stress, mitochondrial dysfunction, and impairment of the ubiquitinproteasome system may be involved in the pathogenesis of PD (2-5). Recent studies indicate that mutations in several genes appear to cause a familial form of PD (6), and mutations in these genes seem to share common pathways underlying the pathogenesis of a sporadic form of PD.DA neurons are vulnerable to neurodegeneration because of their high levels of reactive oxygen species (ROS) (7); enzymatic metabolism of dopamine produces hydrogen peroxide and superoxide radicals, and intracellular autoxidation of dopamine induces the formation of dopamine-quinone, leading to the generation of hydroxyl radical when combined with iron (7, 8). Furthermore, there is a significant alteration in levels or activities of antioxidant enzymes in the substantia nigra (9 -13). Among many potential consequences of oxidative stress, surges in ROS render DA neuron...

Sulfiredoxin Translocation into Mitochondria Plays a Crucial Role in Reducing Hyperoxidized Peroxiredoxin III

“…The role of Prx III in scavenging mitochondrial H 2 O 2 has been emphasized. Previous studies have shown that Prx III is vulnerable to inactivation through hyperoxidation of its active site cysteine residues in cells and rodent tissues under oxidative stress (25)(26)(27)(28)(29)(30)(31)(32)(33). Sulfinic Prx III can be specifically reduced back to the thiol form in mammalian cells (25)(26)(27).…”

“…These three proteins make up a primary line of defense against H 2 O 2 in mitochondria. Like cytosolic Prx I and Prx II, mitochondrial Prx III is hyperoxidized and inactivated in cells under oxidative stress (25)(26)(27)(28)(29)(30)(31)(32)(33). Hyperoxi-dized Prx III is promptly regenerated to protect cells from mitochondrial H 2 O 2 -mediated oxidative damage (25)(26)(27).…”

Nigericin-induced Impairment of Autophagic Flux in Neuronal Cells Is Inhibited by Overexpression of Bak

“…The parent vector was used as a control. Stable cell lines overexpressing Myc-tagged (MN9D/ m-Bak) or FLAG-tagged Bak (MN9D/f-Bak) were also established and characterized, as described previously (31). The stable cell lines were cultured in DMEM supplemented with 10% FBS and 500 g/ml G-418 (A.G. Scientific, San Diego, CA).…”

Cysteine-Based Redox Switches in Enzymes

“…Such highly localized effects-redox signaling is well known to be a highly localized phenomenon-would support a very high gradient of peroxide concentrations around sites of ROS generation, but prevent the flood. Whether or when this floodgate effect would be operational is as yet unclear; while a number of reports link H 2 O 2 treatment of cells with Prx hyperoxidation (21,62,65,92), there are only a few publications that describe physiological stimuli that induce significant amounts of Prx hyperoxidation [e.g., during TNFa treatment of Leydig cells to induce apoptosis, and by 6-hydroxydopamine stimulation of dopaminergic neurons used as a model of Parkinson's disease (44,65)]. If the effect is indeed highly local, however, it may be that only very small amounts of Prx hyperoxidation are needed, amounts that are difficult to detect within the large pool of Prxs.…”