Environmental exposure to high concentrations of manganese (Mn) results in adverse neurological deficits commonly referred to as Manganism. Manganese neurotoxicity is a significant toxicological problem resulting from the use of Mn as a gasoline additive, and its use in welding rods, pesticides, pharmaceutical preparations, alloy manufacturing, and in dry cell battery production, for instance. Mn predominantly accumulates in the basal ganglia structures including the globus pallidus and striatum area, but the cellular mechanisms underlying the neurotoxic effect of Mn in these target neurons are yet to be established. Recently, we have identified that protein kinase C-delta (PKCd), a member of the novel PKC isoform family, is a key pro-apoptotic kinase that is highly expressed in the basal ganglia, including striatal neurons, and serves as a key substrate for caspase-3. We also found that PKCd is proteolytically cleaved into regulatory and catalytic subunits by caspase-3 following Mn treatment to persistently increase its kinase activity, which further contributes to Mn-induced apoptosis in cell lines. Therefore, we propose to extend these preliminary findings to primary neuronal cultures obtained from striatum and mesencephalon and animal models by studying the following specific aims: i) To determine whether proteolytically cleaved PKCd by caspase-3 translocates to the nucleus in order to initiate key nuclear apoptotic events such as histone 2B, STAT1 and lamin B phosphorylation and DNA- PK inactivation, ii) To examine whether Mn exposure in an animal model induces caspase-3 dependent PKCd proteolytic activation, PKCd nuclear translocation, DNA-PK inactivation, and histone 2B, STAT1 and lamin B phosphorylation and finally, apoptotic cell death, and iii) To further confirm the involvement of PKCd in Mn-induced neurotoxicity by comparing the behavioral deficits, neurotransmitter depletion and neuronal degeneration in naive PKCd (+/+) and PKCd (-/-) animals.
These specific aims will be delineated using cellular, molecular, and neurochemical approaches in relevant cell culture and animal models of Mn neurotoxicity. Collectively, experimental results of the proposed systematic studies will define the biochemical mechanisms underlying the apoptotic cell death process in Mn toxicity, and this knowledge will advance the development of interventional strategies for Mn neurotoxicity in humans.
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