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 to protect from cytotoxicity in this cell culture model [24
]. The current studies indicate two phases of ROS generation, occurring concurrent with 6-OHDA autoxidation and followed by delayed onset of mitochondrial ROS production. Interestingly, this delayed mitochondrial component is temporally correlated with the onset of ERK phosphorylation by 2 h [25
], and metalloporphyrin antioxidants added at this time can no longer prevent ERK phosphorylation and cell death. Furthermore, there is mitochondrial ERK activation in response to 6-OHDA.
As demonstrated in , 6-OHDA undergoes rapid oxidation in cell culture medium. Moreover, catalase data indicates that a component of cytotoxicity results from the generation of hydrogen peroxide, findings that are consistent with previous reports from other laboratories using 6-OHDA with PC12 cells [40
]. In these model systems extracellular catalase exhibits similar kinetics of protection [41
] and, as in our previously reported observations in B65 cells [24
], extracellular SOD fails to protect against cytotoxicity [40
]. The potential role of intracellular superoxide in mediating cytotoxicity in these model systems was not addressed, and data from other model systems suggest that dopaminergic neurons may be particularly vulnerable to extracellular ROS compared to other cells [42
]. Our current data indicates that there are both extracellular and intracellular sources of ROS that are responsible for 6-OHDA toxicity. Moreover, delayed protection assays suggest that catalase is not able to protect the cells upon completion of 6-OHDA autoxidation whereas metalloporphyrin antioxidants conferred protection when added after the completion of 6-OHDA autoxidation, but prior to the onset of mitochondrial superoxide production.
Several classes of small molecular antioxidant mimetics have been shown to protect against CNS injuries, including pesticide-induced DA neuron degeneration [34
]. A comparison of the time dependence of protection from 6-OHDA toxicity of catalase and metalloporphyrins demonstrated that metalloporphyrins can be added 45 minutes later than catalase and still afford protection. Metalloporphyrins differ from catalase in several ways including possessing SOD activity and having the ability to permeate cells [34
]. The observed protective effect of metalloporphyrins and the absence of protection of extracellularly applied SOD [24
] raise the possibility that compartmentalization of ROS may be important in mediating both cytotoxicity and sustained ERK activation. The results from this study confirm the role of a delayed phase of superoxide production in mediating 6-OHDA toxicity, findings consistent with those observed with 6-OHDA in primary neuronal cultures [36
]. Interestingly, in this study, the peak of intracellular superoxide production, as detected by dihydroethidium staining, occurred at 1 hour after the addition of 6-OHDA, the latest time at which metalloporphyrin addition was protective.
While these results suggest a role for intracellular superoxide, alternative interpretations must be considered based on the reported properties of metalloporphyrins, which include their ability to scavenge peroxynitrite [46
]. Also, metalloporphyrins possess low levels of catalase activity and have been shown to protect against H2
]. Since extracellular and intracellular hydrogen peroxide are in dynamic equilibrium, extracellular catalase can lower intracellular levels of hydrogen peroxide [48
]. Thus it is possible that metalloporphyrins protect by providing a source of intracellular catalase activity. However, when assessed using in vitro
activity assays, the catalase activity of the metalloporphyrins is similar to that of heat-inactivated catalase, findings that support a role for another mediator, such as intracellular superoxide, in mediating cytotoxicity and sustained ERK activation.
Based upon numerous lines of experimental evidence, mitochondrial dysfunction has been proposed to play an important role in Parkinson’s disease (Reviewed in [49
]). MPTP and rotenone are two toxins used to recapitulate Parkinsonian injury in experimental model systems [50
]. Both neurotoxins are capable of inhibiting mitochondrial electron transport chain complex I, which exhibits decreased activity in PD patient material [51
]. While 6-OHDA is capable of inhibiting complex I in isolated mitochondria [23
], the ability of 6-OHDA to induce mitochondrial dysfunction in intact cells is less clear [36
]. Our results suggest that mitochondria serve as an important source of intracellular superoxide production in 6-OHDA toxicity, which is supported by studies showing that MnSOD transgenic mice are protected from delayed 6-OHDA-induced retrograde neuron cell death [12
]. While the molecular mechanisms leading to delayed mitochondrial superoxide production is currently unclear, a variety of potential mechanisms may be involved.
6-OHDA-induced toxicity in B65 cells bears some similarities to glutamate-induced non-apoptotic programmed cell death. Schubert and coworkers have demonstrated that glutamate-induced cytotoxicity in HT22 cells exhibits a secondary phase of ROS generation, and prevention of this by inhibitors of transcription and translation as well as inhibitors of mitochondrial complex III confers protection from cytotoxicity [58
]. Similarly, Luetjens et al have reported a delayed ROS production in response to glutamate toxicity in primary hippocampal neurons which can be prevented by delayed addition of MnTBAP [59
]. In this system, this delayed phase of ROS production is preceded by mitochondrial cytochrome c release and can be prevented by mitochondrial complex III inhibition.
Recently, it has become established that ERK activation can contribute neurotoxicity, particularly in the context of oxidative insults (Reviewed in [26
]). Depending on the injury, different sources of ROS generation lead to redox-activation of the ERK signaling pathway [60
]. The present study demonstrates that the sustained ERK activation and cytotoxicity induced by the redox active molecule 6-OHDA is mediated by catalase- and metalloporphyrin-sensitive events. Given the similar redox cycling of dopamine and other catecholamines [65
], abnormal ERK activation via a redox sensitive mechanism may contribute to neuronal cell death in PD. In support of this hypothesis, we observed a correlation between the ability of catalase and metalloporphyrins to protect B65 cells from 6-OHDA toxicity and the ability to inhibit sustained ERK activation. Furthermore, increased levels of phosphorylated ERK and increased ERK activity are observed in substantia nigra tissue from patients with PD and/or dementia with Lewy bodies as compared to age-matched controls [21
]. Finally, inhibitors of the MEK 1/2 kinases that activate ERK, protects neuronal cells from 6-OHDA toxicity [25
] and from MPP+ toxicity [66
]. These observations suggest that understanding the oxidative mechanisms involved in and the identifying members of the signal transduction cascade that are affected by 6-OHDA to yield sustained ERK activation in B65 cells will be important for understanding the molecular pathogenesis of neuronal loss in PD and related neurodegenerative diseases.
The temporal and/or spatial pattern of signaling molecules in the cell is known to influence cellular responses to stimuli. Such a paradigm is known to exist with regards to ERK signaling in PC12 cells in response to different growth factors (Reviewed in [68
]). Treatment of PC12 cells with epidermal growth factor (EGF) results in a transient activation of ERK whereas nerve growth factor (NGF) treatment results in a sustained ERK activation [69
]. In addition, this sustained ERK activation is associated with nuclear translocation and cellular differentiation, while transient EGF-mediated ERK activation remains predominantly cytosolic and is associated with a proliferative response [69
]. In pathologic conditions, different cell fates can also be mediated by different temporal and spatial patterns of kinase activation, with nuclear localization promoting neuroprotective signaling responses not observed during cytoplasmic accumulation of activated phosphoproteins [20
]. In recent years, there has been intensifying interest in the intersection between kinase signaling pathways and mitochondria [38
], particularly with the recognition that kinases and other proteins implicated genetically in parkinsonian neurodegeneration localize to mitochondria [73
]. In addition to altered subcellular distributions of signaling proteins observed in PD neurons [20
] normal mitochondrial functions for phosphoproteins traditionally thought of as nuclear regulators have begun to be elucidated [75
]. Poderosos and colleagues have demonstrated the presence of mitochondrial ERK in the matrix, intermembrane space, and outer membrane of the rat CNS [39
]. Interestingly, the levels of mitochondrial ERK peaked in the late in utero
and early post natal periods, suggesting mitochondrial ERK may play a particularly important role in the developing nervous system.
The function of mitochondrial ERK is currently unclear. The results of our study suggest that mitochondrial ERK activation is not required to elicit 6-OHDA-induced delayed mitochondrial ROS generation, as concentrations of MEK 1/2 inhibitors capable of complexly inhibiting mitochondrial ERK activation did not attenuate the increased MitoSOX fluorescence elicited by 6-OHDA (). These results are consistent with reports from other laboratories where ERK activation lies downstream of mitochondrially-induced ROS [76
], although , these studies did not examine ERK activation within mitochondria. Nowak et al have suggested that mitochondrially activated ERK suppresses mitochondrial respiration in oxidatively damaged tert
-butylhydroperoxide treated renal epithelial cells [80
]. Interestingly, while decreased mitochondrial respiration and ATP production were reversed by inhibition of the MEK/ERK pathway, these inhibitors did not restore mitochondrial aconitase activity [80
].As mitochondrial aconitase inactivation is a sensitive indicator for mitochondrial superoxide, activation of the MEK/ERK pathway in this study was downstream of mitochondrial oxidative stress, but upstream of other aspects of mitochondrial dysfunction.
Catecholamine metabolism also generates reactive species which may differ from those generated by tert
]. Thus, it is possible that the delayed component of 6-OHDA-mediated mitochondrial dysfunction may involve additional metabolites such as dopamine quinone, a compound which has been previously shown to be capable of eliciting mitochondrial dysfunction in purified brain mitochondria [81
]. Coupled with the observation that phosphorylated ERK is present in mitochondria of dopaminergic neurons of patients dying from Parkinson and Lewy Body Diseases [29
], the identification of the cellular targets of mitochondrial ERK activated during this delayed phase of 6-OHDA-induced mitochondrial ROS may yield important insight as to the role played by activated mitochondrial ERK in mediating mitochondrial dysfunction during oxidative neuronal injury.
In summary, the catalase and metalloporphyrin antioxidants exhibit different kinetics of protection from 6-OHDA in B65 cells, exhibiting a temporal correspondence between loss of protection and loss of the ability of the antioxidant to inhibit sustained ERK phosphorylation. Based upon time course studies of aconitase inactivation and MitoSOX red fluorescence, this difference suggests that catalase protects only during early phases of toxin injury, while metalloporphyrin antioxidants, which penetrate mitochondrial fractions [34
], may act to prevent a delayed phase of mitochondrial ROS production important for 6-OHDA-mediated ERK activation. Subcellular fractionation studies further revealed that 6-OHDA induced phosphorylation of mitochondrial ERK. These results implicate a role for localized redox-activation of ERK in 6-OHDA cytotoxicity.