Original Contribution
6-Hydroxydopamine induces mitochondrial ERK activation

https://doi.org/10.1016/j.freeradbiomed.2007.04.028Get rights and content

Abstract

Reactive oxygen species (ROS) are implicated in 6-hydroxydopamine (6-OHDA) injury to catecholaminergic neurons; however, the mechanism(s) are unclear. In addition to ROS generated during autoxidation, 6-OHDA may initiate secondary cellular sources of ROS that contribute to toxicity. Using a neuronal cell line, we found that catalytic metalloporphyrin antioxidants conferred protection if added 1 h after exposure to 6-OHDA, whereas the hydrogen peroxide scavenger catalase failed to protect if added more than 15 min after 6-OHDA. There was a temporal correspondence between loss of protection and loss of the ability of the antioxidant to inhibit 6-OHDA-induced ERK phosphorylation. Time course studies of aconitase inactivation, an indicator of intracellular superoxide, and MitoSOX red, a mitochondria targeted ROS indicator, demonstrate early intracellular ROS followed by a delayed phase of mitochondrial ROS production, associated with phosphorylation of a mitochondrial pool of ERK. Furthermore, on initiation of mitochondrial ROS and ERK activation, 6-OHDA-injured cells became refractory to rescue by metalloporphyrin antioxidants. Together with previous studies showing that inhibition of the ERK pathway confers protection from 6-OHDA toxicity, and that phosphorylated ERK accumulates in mitochondria of degenerating human Parkinson's disease neurons, these studies implicate mitochondrial ERK activation in Parkinsonian oxidative neuronal injury.

Introduction

Parkinson's disease (PD) is a common age-related neurodegenerative disease characterized by selective neuronal cell death within several regions of the brain (reviewed in [1]). Degeneration of the dopaminergic neurons of the nigral-striatal projection accounts for many of the major symptoms. Although the molecular mechanisms leading to neuronal cell death in PD remain unclear, studies of postmortem human tissues, genetic studies of familial PD, and toxin/pesticide-based models of PD suggest common pathways involving oxidative stress, mitochondrial dysfunction, disrupted protein turnover, and altered kinase signaling in dopaminergic neuron degeneration [2], [3], [4], [5], [6], [7], [8], [9].

6-Hydroxydopamine (6-OHDA), a redox cycling dopamine analog [10], is an oxidative neurotoxin that causes a parkinsonian pattern of neuronal loss in rodents following intrastriatal injection [11], [12]. Although used as an exogenous neurotoxin in this model, 6-OHDA can be formed from dopamine in vivo, and elevated levels have been detected in body fluids of patients with PD [13]. Notably, 6-OHDA injury recapitulates several features of degenerating neurons observed in human PD tissues. These include proteasome inhibition, α-synuclein aggregation, oxidation and nitration of proteins, increased protein ubiquitination, cleaved caspase 3 expression, glutathione depletion, and cytoplasmic accumulation of activated signaling proteins [14], [15], [16], [17], [18], [19], [20], [21]. A better understanding of 6-OHDA-mediated neurotoxicity could lend important insights into injury and degeneration pathways shared among different causes of dopaminergic neuron degeneration.

The mechanisms by which 6-OHDA elicits its neurotoxic effects have yet to be fully elucidated, although studies implicate a role for oxidative mediators [22], [23]. 6-OHDA metabolism generates a series of ROS at physiologic pH including hydrogen peroxide, para-quinone, and superoxide (reviewed in [2]). The role of these oxidative species in 6-OHDA toxicity and their intracellular sites of action remain ill defined.

We have previously shown that catalase and metalloporphyrin antioxidants that are capable of affecting intracellular compartments conferred protection against cytotoxicity in 6-OHDA-treated B65 cells [24]. Furthermore, activation of the ERK signaling pathway contributes to 6-OHDA toxicity [25], [26]. Moreover, mitochondrial, but not extracellular, superoxide dismutase protects against delayed retrograde substantia nigra cell death following intrastriatal injection of 6-OHDA [12]. Taking into account the temporal and spatial considerations in this model, we hypothesize that 6-OHDA elicits a secondary wave of mitochondrial ROS associated with neurotoxic ERK activation. We found that catalase and metalloporphyrins exhibit different kinetics of protection against 6-OHDA-mediated cytotoxicity that correlate with the ability of the antioxidant to inhibit sustained ERK phosphorylation. In addition, 6-OHDA elicits two phases of intracellular superoxide production associated with increased mitochondrial ROS production and phosphorylation of ERK in mitochondrial fractions. The cells become refractory to metalloporphyrin antioxidant protection on initiation of mitochondrial ROS and ERK phosphorylation, implicating these intracellular events in 6-OHDA neurotoxicity.

Section snippets

Materials and methods

Chemical reagents (except where specified) were purchased from Sigma (St. Louis, MO).

Kinetics of 6-OHDA autoxidation in culture media

Under physiologic conditions, 6-OHDA is capable of undergoing oxidation to produce several reactive oxygen species (ROS) as well as quinones [2], [10]. Stock solutions of 6-OHDA prepared in water showed a low rate of quinone formation at 37°C, but 6-OHDA prepared in 0.05% (w/v) ascorbate was stable for 2 h (Fig. 1A). However, in the presence of culture media, 6-OHDA rapidly oxidized to its quinone form by 15 min, and there were no differences between 6-OHDA prepared in water and 6-OHDA prepared

Discussion

While oxidative mediators have been implicated in 6-OHDA toxicity, 6-OHDA metabolism is capable of generating a series of ROS at physiologic pH [10], and the role of these oxidative species in 6-OHDA toxicity remains ill defined. Previously we have demonstrated that 6-OHDA is cytotoxic to B65 cells and that cytotoxicity is associated with sustained ERK phosphorylation [25]. In addition, extracellular application of either catalase or metalloporphyrins, but not of superoxide dismutase, was able

Acknowledgments

We thank Amy Sartori, Charlotte Diges, Prajakta Sonalker, and Jianhui Zhu for technical assistance. We thank Incara Pharmaceuticals (Research Triangle Park, NC) for providing AEOL 11013. This work was supported by a Veterans Administration Advanced Research Career Development Award (S.M.K.), the National Institutes of Health NS40817, NS053777, AG026389 (C.T.C.), NS045748 (M.P.), and the University of Pittsburgh Pathology Post-doctoral Research Training Program (S.M.K.).

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