Cellular prion protein is released on exosomes from activated platelets

Abstract

Cellular prion protein (PrP C ) is a glycophosphatidylinositol (GPI)-anchored protein, of unknown function, found in a number of tissues throughout the body, including several blood components of which platelets constitute the largest reservoir in humans. It is widely believed that a misfolded, protease-resistant form of PrP C , PrP Sc , is responsible for the transmissible spongiform encephalopathy (TSE) group of fatal neurodegenerative diseases. Although the pathogenesis of TSEs is poorly understood, it is known that PrP C must be present in order for the disease to progress; thus, it is important to determine the physiologic function of PrP C . Resolving the location of PrP C in blood will provide valuable clues as to its function. PrP C was previously shown to be on the alpha granule membrane of resting platelets. In the current study platelet activation led to the transient expression of PrP C on the platelet surface and its subsequent release on both microvesicles and exosomes. The presence of PrP C on platelet-derived exosomes suggests a possible mechanism for PrP C transport in blood and for cellto-cell transmission. IntroductionCellular prion protein (PrP C ) is a membrane-bound, glycophosphatidylinositol (GPI)-anchored protein 1 found primarily in lipid rafts on the cell membrane of neuronal and non-neuronal cells, including tonsils, spleen, and of the secretory granules of epithelial cells in the stomach, as well as in cultured cell lines. 2,3 Although PrP C has been shown to be present on the surface of a number of peripheral blood cells, 4 the relative levels on individual cell types have been contentious. Individual studies have reported that the majority of PrP C is associated with both platelets 5,6 and red blood cells. 7 In the former case the surface expression of PrP C is increased following stimulation, suggesting an additional internal membrane source of the protein, 8 recently shown to be alpha granule membranes. 9 Furthermore, platelet activation is associated with the accumulation of PrP C in releasates, 10 and in platelet concentrates, stored for up to 10 days, there is an increase in initially the microsomal, then plasma levels of PrP C . 11 Transmissible spongiform encephalopathies (TSEs) are a family of neurodegenerative disorders, including CreutzfeldtJakob disease (CJD), Gerstmann-Straussler-Schienker syndrome, and fatal familial insomnia in humans; scrapie in sheep; and bovine spongiform encephalopathy (BSE) in cattle. 12,13 They are all characterized by the accumulation of a proteaseresistant isomer (PrP Sc ) of PrP C in the brain of affected individuals. It is generally considered that PrP Sc acts as a template inducing the same structural changes within other normally folded PrP C molecules on contact, thus propagating the misfolded state of the protein. 14 The CNS is the site at which TSE pathology is apparent in prion infections; however, the agent must first replicate and be transported to the CNS after peripheral infection. The spread of PrP Sc has been tracked from the gas...

Membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication

“…In fact, recent studies using the dissociation-enhanced lanthanide fluoroimmunoassay showed that platelets and plasma are the main reservoir of cellassociated prion protein in human blood. 76 Similar studies also indicate that this protein is released from platelets obtained by apheresis during storage. 77 Evidence that these infectious prion particles are associated with platelet-derived MP suggests that the latter play a role in their spread.…”

“…Furthermore, as MV membranes engulf some cytoplasm during membrane blebbing, they may also contain proteins derived from it and mRNA. 17,18 Moreover, they may 'hijack' infectious particles (e.g., human immuno deficiency virus (HIV) or prions) from the cytoplasm [19][20][21][22] or possibly even whole intact organelles such as the mitochondria. 23 MV are released by various cell types and differ in composition depending on cell origin and status.…”

First Demonstration of Transmissible Spongiform Encephalopathy-associated Prion Protein (PrPTSE) in Extracellular Vesicles from Plasma of Mice Infected with Mouse-adapted Variant Creutzfeldt-Jakob Disease by in Vitro Amplification

“…Little is known about the distribution of PrP TSE in blood. PrP C has been detected in exosomes isolated from platelets and annexin V-positive EVs released from apoptotic endothelial cells (48,56). These findings raise the possibility that endothelial and blood cells other than platelets may be capable of releasing PrP C and possibly PrP TSE in association with exosomes and other types of EVs, contributing to the transmission of TSEs through blood-derived products (48, 56 -58).…”

“…Experimental detection of PrP TSE in animal cerebrospinal fluid (CSF) (29), whole blood (21,22), plasma (27)(28)(29)(30)71), buffy coat (24 -26), and urine (74) and in human CSF (12,75), whole blood (6 -8), and urine (76,77) has been achieved by different methods that rely on the concentration or amplification techniques to bring PrP TSE levels to the detection threshold of biochemical assays. Although the presence of PrP TSE in blood exosomes has been suggested previously (48), biochemical detection has been complicated by the low levels of PrP TSE in blood and the concomitantly large volumes required for exosome isolation by standard methods. Traditionally, exosome isolation has been achieved by a series of differential centrifugation and filtration steps from large sample volumes (68).…”

“…Moreover, processing of EVs by ultracentrifugation through a 30% sucrose cushion, coupled with saPMCA amplification, allowed the detection of PrP TSE in EVs that were confirmed to carry the exosomal marker Hsp70, further indicating the possible association of PrP TSE with these vesicles. There are three types of EVs released by the cell that differ in their biogenesis and that can be classified as follows: (i) apoptotic vesicles (50 -5000 nm in diameter) released by apoptotic cells; (ii) exosomes (40 -200 nm) released upon fusion of multivesicular bodies with the plasma membrane, and (iii) microvesicles (50 -1000 nm) that directly bud from the plasma membrane (37,48,83). Because some of these vesicles overlap in size, it is difficult to separate them by the current isolation methods.…”