Mechanisms of environmental chemical-induced apoptosis in dopaminergic cells: Critical roles of protein kinase C-delta and relevance to Parkinson's disease.

摘要

We have investigated the dopaminergic toxicity and cell death signaling mechanisms of the potential environmental risk factors, dieldrin, methylcyclopentadienyl manganese tricarbonyl (MMT), and manganese, for Parkinson's disease (PD) in the dopaminergic rat pheochromocytoma (PC12) and rat mesencephalic (1RB₃AN₂₇ or N27) cell lines. Dopaminergic cells were more susceptible to both dieldrin and MMT toxicity as compared to non-dopaminergic cells, such as M213–20 (rat striatal GABAergic) cells, α-TC (rat clonal pancreatic) cells, and HCN-2 (human cortical neuronal) cells. Acute exposure to dieldrin or MMT altered dopamine catabolism, as observed by an increase in DOPAC formation and dopamine release, and subsequent decrease in dopamine content. Also, a rapid generation of reactive oxygen species (ROS) was observed within 5 min of dieldrin (30–300 μM) or MMT (30–200 μM) exposure. This ROS generation was partially blocked by α-methyl-p-tyrosine or selegiline, inhibitors of tyrosine hydroxylase or monoamine oxidase-B, respectively, indicating that the presence of dopamine and disruption of dopamine catabolism and degradation may serve as an additional source of ROS. Dieldrin, MMT or manganese treatment in dopaminergic cells triggered apoptotic cell death process, as measured by mitochondrial depolarization, release of cytochrome c, and caspase-9 and caspase-3 activation. These initial pro-apoptotic processes were almost completely blocked by the over-expression of the anti-apoptotic protein, Bcl-2. Thus, one of the primary cellular targets of dieldrin, MMT, and manganese could be the mitochondria; specifically, mitochondrial function was inhibited to initiate the apoptotic cascade. Interestingly, we observed proteolytic cleavage of the novel protein kinase Cδ (PKCδ) following dieldrin, MMT, and manganese exposure. PKCδ (72–74 kDa) was cleaved into the regulatory (42 kDa) and catalytic (38 kDa) subunits by caspase-3, resulting in increased kinase activity. Other PKC family proteins, including PKCα, PKCβII, and PKCζ, were not cleaved during dieldrin or MMT exposure, indicating that the proteolytic cleavage of PKCδ was isozyme specific. Both pharmacological and genetic modulation of PKCδ resulted in attenuation of toxicant-induced DNA fragmentation and apoptosis, suggesting that PKCδ plays an important role in the execution of apoptosis. Additional experimental results indicate that PKCδ amplifies the caspase cascade by positive feedback activation during the neurotoxic insult. Another regulatory role of PKCδ was observed during dieldrin or MMT treatment; translocation of PKCδ into mitochondrial membranes was increased, and it was followed by release of pro-apoptotic molecules such as cytochrome c and Smac, and time-dependent activation of caspase-9 and caspase-3. Down-regulation of PKCδ by TPA or pretreatment with rottlerin significantly blocked dieldrin-induced cytochrome c release, yet rottlerin did not inhibit translocation of PKCδ into mitochondria. These results strongly support that PKCδ modulates mitochondrial function and triggers the initiation process of apoptosis. Delivery of recombinant active PKCδ into dopaminergic cells mimicked the effect of dieldrin-induced PKCδ translocation into mitochondria, indicating that PKCδ plays a critical role not only in the execution process, but also in the initiation of apoptosis. Taken together, these experimental results suggest that environmental neurotoxic agents (dieldrin, MMT, and manganese) promote dopaminergic degeneration by sequentially activating the following cellular events: (i) generation of oxidative stress to initiate the apoptotic cascade, (ii) induction of apoptotic cell death by caspase-3 dependent proteolytic activation of PKCδ, and (iii) amplification of the caspase cascade by posi