Neuronal cholesterol upregulation affects prion propagation through altering PrPC metabolism

Prion conversion occurs through a direct interaction between PrP^C and PrP^Sc in cholesterol-rich lipid raft domains of membranes. We have previously demonstrated the prion-induced loss of active Rab7a, which results in delayed LDL trafficking and increased cholesterol synthesis in neurons. We hypothesize that the elevated cholesterol levels affect PrP^C metabolism and trafficking, thus contributing to prion propagation.

To model the effect of increased cholesterol on PrP^C, we treated N2a and CAD5 cells with water-soluble cholesterol. Cholesterol levels were measured with the Amplex™ Red Cholesterol Assay Kit and its localization was determined using the D4H cholesterol probe. Changes in PrP^C levels were determined by immunoblotting.

The cholesterol treatment overcame negative feedback regulation, increasing cellular cholesterol, with most being localized at the plasma membrane. Interestingly, the treatment resulted in decreased PrP^C levels in N2a cells, but increased levels in CAD5 cells. These changes were not due to transcriptional regulation. Therefore, we will investigate if high membrane cholesterol treatment affects proteolytic cleavage and turnover of PrP^C. Despite this, cholesterol loading prior to de novo infection with 22L-infected brain homogenate dramatically reduced PrP^Sc levels in both cell lines.

For further mechanistic insights, we will determine alterations in the Rab7a interactome in prion infection using the BioID approach, with the aim of elucidating the cause and further downstream effects of reduced Rab7a activation. Since cholesterol plays a key role in prion disease, its role warrants more research. As there are no effective prophylaxes or therapeutics, improving our mechanistic understanding of prion disease pathogenesis is crucial.