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The purpose of this study is threefold, first to determine whether abnormal prion protein treatment activates protein kinase by capase-3-dependent proteolytic cleavage, and to investigate whether the proteolytic activation of protein kinase plays a role in abnormal prion protein-induced neuronal apoptosis. Second we will further investigate whether the proteolytic activation of protein kinase amplifies abnormal prion protein-induced apoptotic signaling through positive feedback activation of the caspase cascade. Finally, we seek to determine whether protein kinase knockout mice are resistant to abnormal prion protein-induced neurological and neurodegenerative changes.
Prion diseases are fatal neurodegenerative conditions caused by transmissible protein-resistant prions. Animal prion diseases have become epidemic and caused worldwide economic impacts. Unlike other neurodegenerative diseases, prion diseases are transmissible and thus threatening to humans and animals. The purpose of this study is to better understand the cellular/molecular mechanisms underlying the neurodegenerative process of prion diseases in order to devise effective treatment strategies.
For the proposed in vitro studies, we will be using SH-SY5Y cells derived from human neuroblastoma. SH-SY5Y cells are an excellent in vitro model for studying PrPsc-induced neurodegeneration because i) these cell express key proapoptotic and antiapoptotic molecules, ii) these cells are currently used in many laboratories, including our own, to study the cell death mechanisms of oxidative stress-induced neuronal damage, and iii) PrPsc has recently been shown to induce caspase-3 activity and DNA fragmentation in SH-SY5Y cells. We will be using the synthetic peptide fragment corresponding to residues of human PrP, which has been used extensively in neurotoxic prion disease research, and induces apoptosis in a number of different neuronal cell types including SH-SY5Y. The results obtained from the SH-SY5Y cells will be confirmed via in vivo experiments with protein kinaseKO and naive C-57 black mice.