Mitochondrial dysregulation is strongly implicated in

induce mitophagy to a degree comparable with that elicited by 6-OHDA, while constitutively active ERK2 (ERK2-CA) had a greater effect. We developed green fluorescent protein (GFP) fusion constructs of WT, CA, and kinase-deficient (KD) ERK2 to study the role of ERK2 localization in regulating mitophagy and cell death. Under basal conditions, cells transfected with GFP-ERK2-WT or GFP-ERK2-CA, but not GFP-ERK2-KD, displayed discrete cytoplasmic ERK2 granules of which a significant fraction colocalized with mitochondria and markers of autophagolysosomal maturation. The colocalizing GFP-ERK2/mitochondria granules are further increased by 6-OHDA and undergo autophagic degradation, as bafilomycin-A, an inhibitor of autolysosomal degradation, robustly increased their detection. Interestingly, increasing ERK2-WT or ERK2-CA expression was sufficient to promote comparable levels of macroautophagy as assessed by analysis of the autophagy marker microtubule-associated protein 1 light chain 3 (LC3). In contrast, the level of mitophagy was more tightly correlated with ERK activity levels, potentially explained by the greater localization of ERK2-CA to mitochondria compared to ERK2-WT. These data indicate that mitochondrial localization of ERK2 activity is sufficient to recapitulate the effects of 6-OHDA on mitophagy and autophagic cell death.

PKA puts the brakes on autophagy by inhibiting LC3 recruitment to autophagosomes.

Although the toxin 6-hydroxydopamine (6-OHDA) is utilized extensively in animal models of Parkinson's disease, the underlying mechanism of its toxic effects on dopaminergic neurons is not completely understood. We examined the effects of 6-OHDA on the CNS-derived tyrosine hydroxylase expressing B65 cell line, with particular attention to the regulation of the extracellular signal-regulated protein kinases (ERK). 6-OHDA elicited a dose-dependent cytotoxicity in B65 cells. Toxic doses of 6-OHDA also elicited a biphasic pattern of ERK phosphorylation with a prominent sustained phase, a pattern that differed from that observed with hydrogen peroxide (H 2 O 2 ) treatment. 6-OHDA-elicited ERK phosphorylation was blocked by PD98059, an inhibitor of the upstream mitogen activated protein kinase kinase (MEK) that phosphorylates and activates ERK. PD98059 also conferred protection against 6-OHDA cytotoxicity, but did not affect H 2 O 2 toxicity in B65 cells. These results suggest that ERK activation plays a direct mechanistic role in 6-OHDA toxicity, rather than representing a protective compensatory response, and raise the possibility that abnormal patterns of ERK activation may contribute to dopaminergic neuronal cell death.

A better understanding of cellular mechanisms that occur in Parkinson's disease and related Lewy body diseases is essential for development of new therapies. We previously found that 6-hydroxydopamine (6-OHDA) elicits sustained extracellular signal-regulated kinase (ERK) activation that contributes to neuronal cell death in vitro. As subcellular localization of activated kinases affect accessibility to downstream targets, we examined spatial patterns of ERK phosphorylation in 6-OHDA-treated cells and in human postmortem tissues representing the full spectrum of Lewy body diseases. All diseased human cases exhibited striking granular cytoplasmic aggregates of phospho-ERK (P-ERK) in the substantia nigra (involving 28 ؎ 2% of neurons), which were largely absent in control cases (0.3 ؎ 0.3%). Double-labeling studies and examination of preclinical cases suggested that these P-ERK alterations could occur relatively early in the disease process. Development of granular cytoplasmic P-ERK staining in 6-OHDA-treated cells was blocked by neuroprotective doses of catalase, supporting a role for oxidants in eliciting neurotoxic patterns of ERK activation. Evidence of nuclear translocation was not observed in degenerating neurons. Moreover, granular cytoplasmic P-ERK was associated with alterations in the distribution of downstream targets such as P-RSK1, but not of P-Elk-1, suggesting functional diversion of ERK-signaling pathways in Lewy body diseases. Parkinson's disease (PD) is a debilitating movement disorder characterized by degenerating neurons containing cytoplasmic inclusions called Lewy bodies.

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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 hour 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, as 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, upon 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.