Original Contribution6-Hydroxydopamine induces mitochondrial ERK activation
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.).
References (81)
- et al.
Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology
Neuron
(2004) - et al.
Enhanced substantia nigra mitochondrial pathology in human alpha-synuclein transgenic mice after treatment with MPTP
Exp. Neurol.
(2004) - et al.
The generation of hydrogen peroxide, superoxide radical, and hydroxyl radical by 6-hydroxydopamine, dialuric acid, and related cytotoxic agents
J. Biol. Chem.
(1974) 6-Hydroxy-dopamine induced degeneration of central monoamine neurons
Eur. J. Pharmacol.
(1968)- et al.
Manganese superoxide dismutase protects against 6-hydroxydopamine injury in mouse brains
J. Biol. Chem.
(2005) - et al.
Reactive oxidative and nitrogen species in the nigrostriatal system following striatal 6-hydroxydopamine lesion in rats
Brain Res.
(2005) - et al.
Effects of 6-hydroxydopamine on mitochondrial function and glutathione status in SH-SY5Y human neuroblastoma cells
Toxicol. In Vitro
(2005) - et al.
Functional repression of cAMP response element in 6-hydroxydopamine-treated neuronal cells
J. Biol. Chem.
(2006) - et al.
Cytoplasmic aggregates of phosphorylated extracellular signal-regulated protein kinases in Lewy body diseases
Am. J. Pathol.
(2002) - et al.
Attenuation of 6-hydroxydopamine-induced dopaminergic nigrostriatal lesions in superoxide dismutase transgenic mice
Neuroscience
(1998)