Pathologic Prion Protein Infects Cells by Lipid-Raft Dependent Macropinocytosis

Jehangir S Wadia,Steven F Dowdy,Monica Schaller,R Anthony Williamson,Mikhail V Blagosklonny

doi:10.1371/journal.pone.0003314

Abstract

Transmissible spongiform encephalopathies, including variant-Creutzfeldt-Jakob disease (vCJD) in humans and bovine spongiform encephalopathies in cattle, are fatal neurodegenerative disorders characterized by protein misfolding of the host cellular prion protein (PrPC) to the infectious scrapie form (PrPSc). However, the mechanism that exogenous PrPSc infects cells and where pathologic conversion of PrPC to the PrPSc form occurs remains uncertain. Here we report that similar to the mechanism of HIV-1 TAT-mediated peptide transduction, processed mature, full length PrP contains a conserved N-terminal cationic domain that stimulates cellular uptake by lipid raft-dependent, macropinocytosis. Inhibition of macropinocytosis by three independent means prevented cellular uptake of recombinant PrP; however, it did not affect recombinant PrP cell surface association. In addition, fusion of the cationic N-terminal PrP domain to a Cre recombinase reporter protein was sufficient to promote both cellular uptake and escape from the macropinosomes into the cytoplasm. Inhibition of macropinocytosis was sufficient to prevent conversion of PrPC to the pathologic PrPSc form in N2a cells exposed to strain RML PrPSc infected brain homogenates, suggesting that a critical determinant of PrPC conversion occurs following macropinocytotic internalization and not through mere membrane association. Taken together, these observations provide a cellular mechanism that exogenous pathological PrPSc infects cells by lipid raft dependent, macropinocytosis.

Highlights

The mammalian prion protein (PrP) is a GPI-anchored cell surface [1] protein expressed predominantly on neurons, neuroendocrine cells and within the lymphoreticular system [2]
Confocal microscopy on live cells showed that both rPrP-546 and TAT-Cre-546 proteins rapidly accumulated within the cell and co-localized within the same intracellular vesicles (Figure 1B), suggesting that exogenous rPrP protein and TAT Peptide transduction domains (PTDs) are internalized within the same intracellular compartments in these cells
Transmission of disease is thought to involve PrPC located within lipid rafts since cholesterol depletion at the cell surface has been reported to attenuate the conversion of PrPC to the scrapie form [19,39]

Summary

Introduction

The mammalian prion protein (PrP) is a GPI-anchored cell surface [1] protein expressed predominantly on neurons, neuroendocrine cells and within the lymphoreticular system [2]. PrP knock-out mice, that appear phenotypically normal, remain disease-free and do not produce PrPSc following inoculation with scrapie brain homogenates [9,10,11,12,13]. The mechanism and cellular requirements that govern PrPSc-mediated conversion of PrPC remain unclear. PrPC is predominantly found on the cell surface within detergent-insoluble lipid rafts [14]. Lipid raft microdomains within the plasma membrane are composed of cholesterol and sphingolipid-rich complexes that associate with GPI-anchored proteins making them important sites for transmembrane signaling [14–

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Conclusion