Glycosylphosphatidylinositol Anchor Analogues Sequester Cholesterol and Reduce Prion Formation

Clive Bate,Mourad Tayebi,Alun Williams

doi:10.1074/jbc.m110.108548

Abstract

A hallmark of prion diseases is the conversion of the host-encoded prion protein (PrP(C) where C is cellular) into an alternatively folded, disease-related isoform (PrP(Sc), where Sc is scrapie), the accumulation of which is associated with synapse degeneration and ultimately neuronal death. The formation of PrP(Sc) is dependent upon the presence of PrP(C) in specific, cholesterol-sensitive membrane microdomains, commonly called lipid rafts. PrP(C) is targeted to these lipid rafts because it is attached to membranes via a glycosylphosphatidylinositol anchor. Here, we show that treatment of prion-infected neuronal cell lines (ScN2a, ScGT1, or SMB cells) with synthetic glycosylphosphatidylinositol analogues, glucosamine-phosphatidylinositol (glucosamine-PI) or glucosamine 2-O-methyl inositol octadecyl phosphate, reduced the PrP(Sc) content of these cells in a dose-dependent manner. In addition, ScGT1 cells treated with glucosamine-PI did not transmit infection following intracerebral injection to mice. Treatment with glucosamine-PI increased the cholesterol content of ScGT1 cell membranes and reduced activation of cytoplasmic phospholipase A(2) (PLA(2)), consistent with the hypothesis that the composition of cell membranes affects key PLA(2)-dependent signaling pathways involved in PrP(Sc) formation. The effect of glucosamine-PI on PrP(Sc) formation was also reversed by the addition of platelet-activating factor. Glucosamine-PI caused the displacement of PrP(C) from lipid rafts and reduced expression of PrP(C) at the cell surface, putative sites for PrP(Sc) formation. We propose that treatment with glucosamine-PI modifies local micro-environments that control PrP(C) expression and activation of PLA(2) and subsequently inhibits PrP(Sc) formation.

Highlights

Network of Excellence. 1 To whom correspondence should be addressed
Because lipid rafts exist as heterogeneous subsets [10], we examined the potential of compounds to alter specific lipid raft subsets involved in PrPSc formation
Treatment with 5 ␮M glucosamine-PI or glucosamine 2-O-methyl inositol octadecyl phosphate did not affect the survival of ScGT1 cells as measured by thiazyl blue tetrazolium

Summary

GPI and Prion Formation

Some GPI anchors are more than a simple mechanism of attaching proteins to the cell membranes and have cell signaling functions. GPIs from the protozoan Plasmodium falciparum stimulate macrophages [20], whereas other GPIs stimulate lipogenesis in adipocytes [21], and GPIs isolated from PrPC stimulate phospholipase A2 (PLA2) [22]. Because PLA2 affected the formation of prions [23], we used synthetic GPI analogues to examine the relationship between GPI anchors, lipid rafts, cell activation, and prion formation. We report that two synthetic GPI anchor analogues altered the composition of cell membranes in three prion-infected neuronal cell lines, reduced cell signaling and the formation of PrPSc, and greatly diminished infectivity of ScGT1 cells

EXPERIMENTAL PROCEDURES

RESULTS

GPI analogues reduced extracellular PrPSc

Cholesterol Cholesterol esters PrPSc

DISCUSSION