Molecular mechanisms of PKC[delta] in neurotoxin-induced apoptotic death of nigral dopaminergic neurons : relevance to the pathogenesis of Parkinson's disease /
Abstract (Summary)We investigated the molecular role of PKC[Delta] in neurotoxic models of Parkinson's disease (PD). Both the protein and mRNA levels of PKC[Delta] were selectively higher in the dopaminergic neurons in the substantia nigra (SN) region of rodent brains. siRNA designed against PCK[Delta] effectively silenced the expression of PKC[Delta] and reduced dieldrin (a neurotoxic pesticide) or MPP+ (Parkinsonian toxin)-induced apoptotic death in rat mesencephalic dopaminergic neuronal (N27) cells and degeneration of primary mesencephalic dopaminergic neurons. Following dieldrin exposure in N27 cells, we determined that the full-length PKC[Delta] is first cleaved in the cytoplasm, and the activated PKC[Delta] catalytic fragment subsequently translocates into the nucleus. Blockade of proteolytic activation of PKC[Delta] by PKC[Delta]-CRM also protected dopaminergic neurons from MPP+-induced degeneration. Moreover, using PKC[Delta]-CF, PKC[Delta]-CRM, PKC[Delta]-[Delta]NLS constructs, we demonstrated that activated cleaved PKC[Delta] mediates Ser14 phosphorylation of histone H2B followed by its nuclear translocation in apoptotic dopaminergic neuronal cells. Additionally, we also demonstrated that the N- and the C-terminals of PKC[Delta] are in close proximity in the tertiary structure, as demonstrated by fluorescence resonance energy transfer (FRET). By employing the C1 or C2-like domain deletion mutant of PKC[Delta]), we showed that deletion of the C2-like domain, but not the C1 domain, induces strong nuclear localization of PKC[Delta]. Moreover, the C2-like domain alone was fused to the N-terminal of a NES mutated form of Rev protein to generate the Rev chimera, which is still primarily localized in the nucleus. Thus, the N-terminal C2-like domain apparently regulates the subcellular localization of PKC[Delta] by masking the C-terminal NLS through the position obstacle in the tertiary structure, but not through NES or interaction with anchoring proteins. Taken together, our studies show that i) the novel PKC isoform family member PKC[Delta] is highly expressed in nigral dopaminergic neurons, ii) PKC[Delta] mainly localizes in the cytoplasm by the position obstacle effect of the C2-like domain to the NLS, iii) neurotoxin exposure induces proteolytic activation of full-length PKC[Delta] and subsequent nuclear translocation of the PKC[Delta] cleaved fragment to cause histone H2B phosphorylation and apoptotic cell death, and iv) RNAi mediated suppression of PKC[Delta] protects dopaminergic neurons against neurotoxic insults.
School:Iowa State University
School Location:USA - Iowa
Source Type:Master's Thesis
Date of Publication: