Identification of non-essential host genes required for PrP106-126 mediated neurotoxicity

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Stobart, Michael
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Prion diseases are invariably fatal proteinaceous neurodegenerative disorders of the central nervous system. The infectious agent is the host encoded prion protein which has undergone a post-translational refolding from a predominantly alpha-helical to highly beta-sheet containing structure. The mechanism of prion-induced neurotoxicity remains elusive in large part due to the absence of a sufficiently neurotoxic cell culture assay. A modern technique for identifying previously unrecognized mediators of a biological pathway is to screen a commercially available library of gene silencing molecules targeting all known open reading frames. Synthetic gene silencing molecules, such as short hairpin RNA (shRNA), employ the endogenous gene silencing pathway to inhibit protein synthesis. To date, no publication has described the implementation of a large-scale library to screen for genetic mediators of prion neurotoxicity. This project was aimed at developing a cell culture model of acute prion neurotoxicity and screening a library of shRNA molecules in order to identify previously unrecognized gene targets essential to prion-induced neurotoxicity. Using a fragment of the prion protein (PrP106-126 peptide) to mimic prion neurotoxicity, human neuroblastoma cells transduced with a retroviral shRNA library were screened for resistance. Involvement of a subset of library identified gene targets in prion disease was assessed in vivo by quantitative real-time PCR (qPCR) analysis. Validation of the protection conferred by reducing expression of a gene target of interest was accomplished using individual lentiviral vectors expressing shRNA. Of the approximately 54,000 shRNA sequences screened, 80 different shRNA sequences recovered from neurotoxic prion peptide-resistant cells were considered to be of interest. Of these, 49 corresponding gene targets were assessed in vivo by qPCR with the majority demonstrating significant differential expression in brains of prion infected mice. Validation of the protection conferred from knockdown of two identified genes, abcb4 and ube2cbp, was completed. Knockdown of either gene imparted significant protection against prion-induced neurotoxicity, with qPCR analysis confirming significantly reduced mRNA transcript levels. Overall, the validity of the novel assay system developed has been demonstrated, and the first comprehensive list of gene candidates involved in mediating acute prion neurotoxicity has been determined.
prions, RNAi, neurotoxicity, screen