Glycogen Synthase Kinase-3β Regulates Cellular Prion Protein Levels

The normal cellular prion protein (PrP ^C ) is an essential substrate in all forms of prion diseases and a receptor for Aβ oligomers in Alzheimer’s disease. However, it is not fully understood how cells regulate PrP ^C levels. Recently, we identified glycogen synthase kinase-3β (GSK-3β) as a potential regulator of PrP ^C levels in a whole genome knockout screen. Here, we show that both cell surface and total PrP ^C levels can be reduced either by siRNA-mediated Gsk3b (but not Gsk3a ) knockdown or by CRISPR-mediated Gs3b knockout. Whole cell mass spectrometric analysis showed that PrP ^C was the 60 ^th most significantly reduced protein (out of 7227 total proteins detected) in Gsk3b knockout cells, compared to wild-type cells. Two different GSK-3 inhibitors, laduviglusib (CHIR-99021) and AZD-1080, reduced PrP ^C levels in mouse CAD5 and human BE(2)-C cells, both in undifferentiated and differentiated states. PrP ^C levels were similarly reduced by cycloheximide treatment in both Gsk3b knockout and WT cells, indicating that GSK-3β regulates PrP ^C levels through a post-translational mechanism. Finally, treatment with either laduviglusib or AZD-1080 reduced PrP ^Sc levels in CAD5 cells infected with three different rodent prion strains. Overall, the results reveal that GSK-3β activity controls PrP ^C levels in living cells, revealing a novel regulatory mechanism and promising therapeutic target.