A loss-of-function approach to investigate the role of cholesterol in prion replication

Prions, the infectious agents of prion diseases, are abnormal conformational variants of the host-encoded prion protein (PrPC) and are thought to replicate by template-directed conversion of native PrPC. There are multiple prion strains thought to arise from different conformations of PrP; of particular interest to this thesis different prion strains are differently able to propagate in cultured cell lines. This PhD focused on two main projects. Firstly, as PrPC segregates into cholesterol-rich rafts at the plasma membrane, the question arises whether cholesterol affects the conversion of PrPC to prions. Using transcriptional gene silencing we investigated whether downregulation of genes with a role in biosynthesis and trafficking of cholesterol affect the rate of prion replication. Silencing of Hmgcr and Dhcr24, two genes that encode for enzymes of cholesterol biosynthesis, reduced the total cholesterol levels by about 50% whilst increasing PrP conversion rates by two-fold, suggesting an inhibitory role of cholesterol. Interestingly, perturbation of cholesterol trafficking by knockdown of Npc1 and Npc2, which causes accumulation of cholesterol in late endosomes/lysosomes, led to diametrically opposing effects on prion conversion. Silencing of Npc1 in N2a cells decreased, while Npc2 loss of function increased, conversion rates. Notably, Npc1 knockdown led to a dramatic loss of surface PrPC expression, a phenotype that was absent upon Npc2 silencing, suggesting that the Npc1-associated decrease in conversion rates is cholesterol-independent and accounted for by decreased PrPC, the substrate for conversion. A secondary project focused on identifying cellular factors underlying selective propagation of prion strains. We subcloned the CAD5 cell line, susceptible to both RML and 22L prion strains, and showed selective prion strain propagation in a small number of isolated clones. Selective propagation could not be stably obtained and this project was halted in favour of further investigation into the role of cholesterol in prion replication