Degree Type

Dissertation

Date of Award

2003

Degree Name

Doctor of Philosophy

Department

Biomedical Sciences

Major

Toxicology

First Advisor

Anumantha G. Kanthasamy

Abstract

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, alpha-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 muM) or MMT (30--200 muM) exposure. This ROS generation was partially blocked by alpha-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 Cdelta (PKCdelta) following dieldrin, MMT, and manganese exposure. PKCdelta (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 PKCalpha, PKCbetaII, and PKCzeta, were not cleaved during dieldrin or MMT exposure, indicating that the proteolytic cleavage of PKCdelta was isozyme specific. Both pharmacological and genetic modulation of PKCdelta resulted in attenuation of toxicant-induced DNA fragmentation and apoptosis, suggesting that PKCdelta plays an important role in the execution of apoptosis. Additional experimental results indicate that PKCdelta amplifies the caspase cascade by positive feedback activation during the neurotoxic insult. Another regulatory role of PKCdelta was observed during dieldrin or MMT treatment; translocation of PKCdelta 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 PKCdelta by TPA or pretreatment with rottlerin significantly blocked dieldrin-induced cytochrome c release, yet rottlerin did not inhibit translocation of PKCdelta into mitochondria. These results strongly support that PKCdelta modulates mitochondrial function and triggers the initiation process of apoptosis. Delivery of recombinant active PKCdelta into dopaminergic cells mimicked the effect of dieldrin-induced PKCdelta translocation into mitochondria, indicating that PKCdelta 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 PKCdelta, and (iii) amplification of the caspase cascade by positive feedback regulation of upstream molecules associated with mitochondrial mediated apoptotic cell death.

DOI

https://doi.org/10.31274/rtd-180813-12391

Publisher

Digital Repository @ Iowa State University, http://lib.dr.iastate.edu

Copyright Owner

Masashi Kitazawa

Language

en

Proquest ID

AAI3085924

File Format

application/pdf

File Size

341 pages

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