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

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels that can modulate various neuronal processes by altering intracellular Ca 2+ levels. Following nAChR stimulation Ca 2+ can enter cells either directly, through the intrinsic ion channel, or indirectly following voltage-operated Ca 2+ channel (VOCC) activation; Ca 2+ levels can subsequently be amplified via Ca 2+ -induced Ca 2+ release from intracellular stores. We have used subtype-selective nAChR agonists to investigate the Ca 2+ sources contributing to a7 and non-a7 nAChR-mediated increases in intracellular Ca 2+ in PC12 cells. Application of the a7 nAChR positive allosteric modulator PNU 120596 (10 lM), in conjunction with the a7 nAChR agonist, compound A [(R)-N-(1-azabicyclo [2.2.2]oct-3-yl)(5-(2-pyridyl)thiophene-2-carboxamide), 10 nM], produces a rapid increase in fluo-3 fluorescence that is prevented by the selective a7 nAChR antagonist a-bungarotoxin. The non-a7 nAChR agonist 5-Iodo-A-85380 produces a-bungarotoxin-insensitive increases in intracellular Ca 2+ (EC 50 ¼ 11.2 lM). Using these selective agonists or KCl in conjunction with general and selective VOCC inhibitors, we demonstrate that the primary route of Ca 2+ entry following either non-a7 nAChR activation or KCl stimulation is via L-type VOCCs. In contrast, the a7 nAChR-mediated response is unaffected by VOCC blockers but is inhibited by modulators of intracellular Ca 2+ stores. These results indicate that a7 and non-a7 nAChRs are differentially coupled to Ca 2+ -induced Ca 2+ release and VOCCs, respectively.

Parkinsonian neurodegeneration is associated with heightened levels of oxidative stress and the activation of apoptotic pathways. In an in vitro cellular model, we reported that 6-hydroxydopamine (6-OHDA) induces apoptotic cell death via the induction of mitochondrial dysfunction, the activation of caspase 3 and the consequent proteolytic activation of the redox-sensitive kinase, protein kinase C (PKC)delta, in PC12 cells. Here we have investigated the involvement of PKCdelta in 6-OHDA-induced cell death in vivo. The nigrostriatal pathway of rats was lesioned by unilateral infusion of 6-OHDA into either the striatum or substantia nigra pars compacta (SNpc). Infusion into the SNpc resulted in rapid loss of tyrosine hydroxylase (TH)-positive cells (87% decrease after 4 days), consistent with a necrotic-like mode of cell death. In contrast, striatal infusion initiated a slower, progressive decline in TH immunoreactivity (25% decrease in the SNpc after 4 days); cell appearance was characteristic of apoptosis. This is consistent with a transient increase in active caspase 3 immunoreactivity at 4 days post-infusion, and a concomitant proteolytic activation of PKCdelta in the SNpc of striatal-lesioned rats. Cleavage of PKCdelta did not occur in the striatum or cerebellum of lesioned animals, or in the SNpc of sham-operated controls. No increase in caspase 3 immunoreactivity or proteolytic activation of PKCdelta was detected in nigral-lesioned rats. These results suggest that after 6-OHDA infusion into the striatum, retrograde neurotoxicity induces caspase 3-dependent PKCdelta proteolytic activation in the cell bodies of the SNpc, implicating this kinase in the neurodegenerative process.