Can altering cholesterol homeostasis alleviate prion disease? Authors: Jessica Cashion, Tahir Ali, Anja Itum, Samia Hannaoui, Sabine Gilch

Like many other neurodegenerative diseases, prion disease boasts aberrantly high levels of neuronal cholesterol. We found reduced levels of cholesterol 24-hydroxylase (CYP46A1), the major brain cholesterol elimination enzyme acting by metabolizing cholesterol to 24-hydroxycholesterol (24-HC), in both in vivo models and human Creutzfeldt-Jakob disease (CJD) patient samples. We have shown that utilizing the FDA-approved pharmaceutical efavirenz (EFV), an allosteric activator of CYP46A1, reduces PrP^Sc accumulation in in vitro and in vivo models while prolonging disease progression, even in CJD-infected humanized mice. Our current objective is to confirm the mechanism of CYP46A1-based PrP^Sc reduction and to determine the regulatory effects of increased 24-HC on cholesterol metabolism in astrocytes. N2a and CAD5 cells infected with RML or 22L prions, were transiently transfected with plasmids encoding CYP46A1 to allow for its overexpression. Western blotting analysis demonstrated decreased PrP^Sc content along with increased CYP46A1 expression when compared to control cells. ELISA analysis of media samples exhibited drastically increased 24-HC, confirming the successful overexpression of CYP46A1. To verify these findings in vivo, we utilized AAV-CYP46A1-mediated overexpression along with control AAV-GFP in 22L-infected FVB mice, using PrP^Sc levels and survival times as readouts. To better understand how altering CYP46A1 impacts the environment of the brain, we applied 24S-HC to 22L-infected astrocyte cultures. We utilized RT-qPCR to determine changes to the expression of cholesterogenic genes including HMG-CoA Reductase, ABCA1, and ApoE within prion infection.  Our findings point to CYP46A1 and cholesterol modulation as a logical target for therapeutic development in prion disease.