6-Hydroxydopamine-induced Apoptosis Is Mediated via Extracellular Auto-oxidation and Caspase 3-dependent Activation of Protein Kinase Cδ

Abstract

6-Hydroxydopamine is a neurotoxin commonly used to lesion dopaminergic pathways and generate experimental models for Parkinson disease, however, the cellular mechanism of 6-hydroxydopamine-induced neurodegeneration is not well defined. In this study we have explored how 6-hydroxydopamine neurotoxicity is initiated. We have also investigated downstream signaling pathways activated in response to 6-hydroxydopamine, using a neuronal-like, catecholaminergic cell line (PC12 cells) as an in vitro model system. We have shown that 6-hydroxydopamine neurotoxicity is initiated via extracellular auto-oxidation and the induction of oxidative stress from the oxidative products generated. Neurotoxicity is completely attenuated by preincubation with catalase, suggesting that hydrogen peroxide, at least in part, evokes neuronal cell death in this model. 6-Hydroxydopamine does not initiate toxicity by dopamine transporter-mediated uptake into PC12 cells, because both GBR-12909 and nisoxetine (inhibitors of dopamine and noradrenaline transporters, respectively) failed to reduce toxicity. 6-Hydroxydopamine has previously been shown to induce both apoptotic and necrotic cell-death mechanisms. In this study oxidative stress initiated by 6-hydroxydopamine caused mitochondrial dysfunction, activation of caspases 3/7, nuclear fragmentation, and apoptosis. We have shown that, in this model, proteolytic activation of the proapoptotic protein kinase C␦ (PKC␦) is a key mediator of 6-hydroxydopamine-induced cell death. 6-Hydroxydopamine induces caspase 3-dependent cleavage of full-length PKC␦ (79 kDa) to yield a catalytic fragment (41 kDa). Inhibition of PKC␦ (with rottlerin or via RNA interference-mediated gene suppression) ameliorates the neurotoxicity evoked by 6-hydroxydopamine, implicating this kinase in 6-hydroxydopamine-induced neurotoxicity and Parkinsonian neurodegeneration. Parkinson disease (PD)2 is a neurodegenerative disorder associated with a progressive loss of dopaminergic neurons of the substantia nigra pars compacta and associated depletion of dopamine in the terminal region, the caudate putamen. Although the neuropathological hallmarks of this disease are well described, the etiology is largely undefined. However, a number of biochemical processes and molecular mechanisms have been identified as mediators of neuronal cell death in PD. These include oxidative stress and mitochondrial dysfunction. Dopamine-rich areas of the brain are particularly vulnerable to oxidative stress, because metabolism of dopamine itself (both enzymatic and non-enzymatic) leads to the generation of reactive oxygen species (ROS), including hydrogen peroxide and hydroxyl radicals (1

Rottlerin protected dopaminergic cell line from cytotoxicity of 6-hydroxydopamine by inhibiting PKCδ phosphorylation

“…However, controversy still exists concerning the most important mode of PKC activation [3,4,16,18,19,[28][29][30] . Recent research has suggested that PKCδ phosphorylation at the site of Tyr311 could regulate the proteolytic activation and proapoptotic function of PKCδ in dopaminergic neuronal cells [28] .…”

“…However, some researchers have suggested that phosphorylation at the site of Thr505 in PKCδ might just lead to the allosteric effect, which is one of the prerequisites for PKCδ activation [19] , and the proteolysis of PKCδ by caspase-3 represents the terminal pattern of PKCδ activation. The accumulation of PKCδ catalytic fragment is an important event in cell apoptosis [18] , particularly in H 2 O 2 -or MPP + -induced cytotoxic responses [29,30] . Our studies demonstrate that 6-OHDA could induce PKCδ phosphorylation at the site of Thr505 in PC12 cells (Fig.…”

Neuroprotective effects of dimerumic acid and deferricoprogen fromMonascus purpureusNTU 568-fermented rice against 6-hydroxydopamine-induced oxidative stress and apoptosis in differentiated pheochromocytoma PC-12 cells

“…PC-12 cells exposed to 6-OHDA are widely used as an experimental model of PD in vitro (Blum et al 2001;Hanrott et al 2006;Zhang et al 2012). NGF-induced neurite outgrowth from PC-12 rat pheochromocytoma cells provides a well-characterised model of sympathetic neuron differentiation (Greene & Tischler 1982).…”

“…6-OHDA has been found to initiate cellular oxidative stress, and widely used to create both cellular and animal models of PD (Blum et al 2001). Previous studies suggested that the reactive oxygen species (ROS) involved were derived from auto-oxidation (Hanrott et al 2006). NADPH oxidase (NOX)-derived ROS were also found to play an essential role in 6-OHDA-induced cell death (Choi et al 2012).…”

Oxidative Stress and Neurotoxicity

“…Indeed, injection of 6-OHDA into rat substantia nigra produced the first toxin animal model of PD (236), where neuronal death reflects an apoptotic mechanism (237). 6-OHDA is an active redox-cycling agent (via its derived quinone and semiquinone), and studies on its biochemical mechanism of toxicity in PC12 cells have found catalase attenuation of neurotoxicity, suggesting that the latter is evoked by H 2 O 2 (238). However, factors other than H 2 O 2 also appear to be involved, because catalase did not inhibit the caspase activation contribution to 6-OHDA neurotoxicity (239).…”