PrP Knockout Cells Expressing Transmembrane PrP Resist Prion Infection

Abstract

Glycosylphosphatidylinositol (GPI) anchoring of the prion protein (PrP C ) influences PrP C misfolding into the disease-associated isoform, PrP res , as well as prion propagation and infectivity. GPI proteins are found in cholesterol-and sphingolipid-rich membrane regions called rafts. Exchanging the GPI anchor for a nonraft transmembrane sequence redirects PrP C away from rafts. Previous studies showed that nonraft transmembrane PrP C variants resist conversion to PrP res when transfected into scrapie-infected N2a neuroblastoma cells, likely due to segregation of transmembrane PrP C and GPI-anchored PrP res in distinct membrane environments. Thus, it remained unclear whether transmembrane PrP C might convert to PrP res if seeded by an exogenous source of PrP res not associated with host cell rafts and without the potential influence of endogenous expression of GPI-anchored PrP C . To further explore these questions, constructs containing either a C-terminal wild-type GPI anchor signal sequence or a nonraft transmembrane sequence containing a flexible linker were expressed in a cell line derived from PrP knockout hippocampal neurons, NpL2. NpL2 cells have physiological similarities to primary neurons, representing a novel and advantageous model for studying transmissible spongiform encephalopathy (TSE) infection. Cells were infected with inocula from multiple prion strains and in different biochemical states (i.e., membrane bound as in brain microsomes from wild-type mice or purified GPI-anchorless amyloid fibrils). Only GPI-anchored PrP C supported persistent PrP res propagation. Our data provide strong evidence that in cell culture GPI anchor-directed membrane association of PrP C is required for persistent PrP res propagation, implicating raft microdomains as a location for conversion.IMPORTANCE Mechanisms of prion propagation, and what makes them transmissible, are poorly understood. Glycosylphosphatidylinositol (GPI) membrane anchoring of the prion protein (PrP C ) directs it to specific regions of cell membranes called rafts. In order to test the importance of the raft environment on prion propagation, we developed a novel model for prion infection where cells expressing either GPI-anchored PrP C or transmembrane-anchored PrP C , which partitions it to a different location, were treated with infectious, misfolded forms of the prion protein, PrP res . We show that only GPI-anchored PrP C was able to convert to PrP res and able to serially propagate. The results strongly suggest that GPI anchoring and the localization of PrP C to rafts are crucial to the ability of PrP C to propagate as a prion.

Volatile Anesthetic Sevoflurane Precursor 1,1,1,3,3,3-Hexafluoro-2-Propanol (HFIP) Exerts an Anti-Prion Activity in Prion-Infected Culture Cells

“…Biosynthesis of cholesterol has been reported to be enhanced by prion infection [ 48 ], suggesting a link between the metabolism of cholesterol and the transmission of prion. Cholesterol is also required for PrP cell surface expression and stabilization [ 49 ], and PrP Sc with GPI anchors is present in lipid rafts [ 50 ]; thus, cholesterol depletion inhibits prion replication by inhibiting the transport of PrP to lipid rafts [ 51 , 52 ].…”

GPI-anchor signal sequence influences PrPC sorting, shedding and signalling, and impacts on different pathomechanistic aspects of prion disease in mice

“…It is known that not only the presence of PrP C but also its type of membrane anchorage is fundamental for the pathophysiology of prion disease. Briefly, (i) presence of extracellular PrP Sc does not lead to prion-induced neurodegeneration in the absence of PrP C on neurons [51, 52], (ii) cells expressing either anchorless PrP or PrP with a transmembrane domain instead of the GPI-anchor are resistant to prion infection [17, 18, 53], and (iii) prion-infected transgenic mice expressing low amounts of anchorless PrP do not show clinical symptoms of prion disease despite high titers of infectivity and high levels of PrP Sc [21, 54]. When the expression of anchorless PrP is increased, this leads to delayed onset of disease and generation of a new prion strain [22, 55].…”

Endogenous Brain Lipids Inhibit Prion Amyloid Formation In Vitro

“…Cell culture and transgenic-mouse studies have established that the C-terminal GPI anchor is not required for PrP RES formation but is necessary for PrP RES propagation between cells and for cytopathology (28). Altering the location of PrP C from the cell surface to the transmembrane domain prevents PrP C conversion (5,29,30). In in vivo studies, GPI anchorless PrP RES formed large amyloid accumulations in mice without the formation of smaller oligomer and fibril forms (31).…”