Inhibition of cholesterol recycling impairs cellular PrPSc propagation

The infectious agent in prion diseases consists of an aberrantly folded isoform of the cellular prion protein (PrPc), termed PrPSc, which accumulates in brains of affected individuals. Studies on prion-infected cultured cells indicate that cellular cholesterol homeostasis influences PrPSc propagation. Here, we demonstrate that the cellular PrPSc content decreases upon accumulation of cholesterol in late endosomes, as induced by NPC-1 knock-down or treatment with U18666A. PrPc trafficking, lipid raft association, and membrane turnover are not significantly altered by such treatments. Cellular PrPSc formation is not impaired, suggesting that PrPSc degradation is increased by intracellular cholesterol accumulation. Interestingly, PrPSc propagation in U18666A-treated cells was partially restored by overexpression of rab 9, which causes redistribution of cholesterol and possibly of PrPSc to the trans-Golgi network. Surprisingly, rab 9 overexpression itself reduced cellular PrPSc content, indicating that PrPSc production is highly sensitive to alterations in dynamics of vesicle trafficking

Activation Mobilizes the Cholesterol in the Late Endosomes-Lysosomes of Niemann Pick Type C Cells

A variety of intercalating amphipaths increase the chemical activity of plasma membrane cholesterol. To test whether intracellular cholesterol can be similarly activated, we examined NPC1 and NPC2 fibroblasts, since they accumulate large amounts of cholesterol in their late endosomes and lysosomes (LE/L). We gauged the mobility of intracellular sterol from its appearance at the surface of the intact cells, as determined by its susceptibility to cholesterol oxidase and its isotope exchange with extracellular 2-(hydroxypropyl)-β-cyclodextrin-cholesterol. The entire cytoplasmic cholesterol pool in these cells was mobile, exchanging with the plasma membrane with an apparent half-time of ∼3–4 hours, ∼4–5 times slower than that for wild type human fibroblasts (half-time ∼0.75 hours). The mobility of the intracellular cholesterol was increased by the membrane-intercalating amphipaths chlorpromazine and 1-octanol. Chlorpromazine also promoted the net transfer of LE/L cholesterol to serum and cyclodextrin. Surprisingly, the mobility of LE/L cholesterol was greatly stimulated by treating intact NPC cells with glutaraldehyde or formaldehyde. Similar effects were seen with wild type fibroblasts in which the LE/L cholesterol pool had been expanded using U18666A. We also showed that the cholesterol in the intracellular membranes of fixed wild-type fibroblasts was mobile; it was rapidly oxidized by cholesterol oxidase and was rapidly replenished by exogenous sterol. We conclude that a) the cholesterol in NPC cells can exit the LE/L (and the extensive membranous inclusions therein) over a few hours; b) this mobility is stimulated by the activation of the cholesterol with intercalating amphipaths; c) intracellular cholesterol is even more mobile in fixed cells; and d) amphipaths that activate cholesterol might be useful in treating NPC disease