Jan Simak and Larisa CervenakovaCastro, Irina Vasilyeva, Silvia H. De Paoli, Paula Saá, Oksana Yakovleva, Jorge de 

Amplificationin VitroDisease by Mouse-adapted Variant Creutzfeldt-JakobVesicles from Plasma of Mice Infected with

) in ExtracellularTSEPrion Protein (PrPSpongiform Encephalopathy-associated First Demonstration of TransmissibleMolecular Bases of Disease:

doi: 10.1074/jbc.M114.589564 originally published online August 25, 20142014, 289:29247-29260.J. Biol. Chem. 

  10.1074/jbc.M114.589564Access the most updated version of this article at doi:

  .JBC Affinity SitesFind articles, minireviews, Reflections and Classics on similar topics on the

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/289/42/29247.full.html#ref-list-1

This article cites 93 references, 27 of which can be accessed free at

at NO

RT

H D

AK

OT

A ST

AT

E U

NIV

on October 28, 2014

http://ww

w.jbc.org/

Dow

nloaded from

at NO

RT

H D

AK

OT

A ST

AT

E U

NIV

on October 28, 2014

http://ww

w.jbc.org/

Dow

nloaded from

First Demonstration of Transmissible SpongiformEncephalopathy-associated Prion Protein (PrPTSE) inExtracellular Vesicles from Plasma of Mice Infected withMouse-adapted Variant Creutzfeldt-Jakob Disease by in VitroAmplification*

Received for publication, June 16, 2014, and in revised form, August 12, 2014 Published, JBC Papers in Press, August 25, 2014, DOI 10.1074/jbc.M114.589564

Paula Saá‡1, Oksana Yakovleva‡, Jorge de Castro‡, Irina Vasilyeva‡, Silvia H. De Paoli§, Jan Simak§,and Larisa Cervenakova‡

From the ‡Transmissible Diseases Department, Biomedical Services Holland Laboratory, American National Red Cross, Rockville,Maryland 20855 and the §Laboratory of Cellular Hematology, Center for Biologics Evaluation and Research, United States Foodand Drug Administration, Silver Spring, Maryland 20993

Background: Prions can be transmitted by blood transfusion, but their origin and distribution in blood are unknown.Results: Prions were detected in plasma extracellular vesicles from preclinical and clinically sick mice.Conclusion: Prions associate with blood-circulating extracellular vesicles.Significance: These findings provide information about prion distribution in blood and set the groundwork for novel prionremoval and disease diagnosis technologies.

The development of variant Creutzfeldt-Jakob disease (vCJD)in three recipients of non-leukoreduced red blood cells fromasymptomatic donors who subsequently developed the diseasehas confirmed existing concerns about the possible spread oftransmissible spongiform encephalopathies (TSEs) via bloodproducts. In addition, the presence of disease-associated mis-folded prion protein (PrPTSE), generally associated with infec-tivity, has been demonstrated in the blood of vCJD patients.However, its origin and distribution in this biological fluid arestill unknown. Various studies have identified cellular prionprotein (PrPC) among the protein cargo in human blood-circu-lating extracellular vesicles released from endothelial cells andplatelets, and exosomes isolated from the conditioned media ofTSE-infected cells have caused the disease when injected intoexperimental mice. In this study, we demonstrate the detectionof PrPTSE in extracellular vesicles isolated from plasma samplescollected during the preclinical and clinical phases of the diseasefrom mice infected with mouse-adapted vCJD and confirm thepresence of the exosomal marker Hsp70 in these preparations.

Human transmissible spongiform encephalopathies (TSEs)2

are fatal neurodegenerative disorders that can develop as spo-

radic, genetic, or infectious diseases. In the United Kingdomand France, which have reported the highest number of variantCreutzfeldt-Jakob disease (vCJD) cases due to exposure tobovine spongiform encephalopathy (BSE)-contaminated prod-ucts (1), uncertainty exists in regard to the number of infectedpeople (2, 3), raising concerns about the safety of blood- andplasma-derived products.

Transmission of vCJD through transfusion of non-leukore-duced red blood cells (RBCs) has been shown in four individu-als, three of whom developed clinical disease and one who diedfrom non-TSE-related causes, but in which the presence of dis-ease-associated misfolded prion protein (PrPTSE) in lymphore-ticular tissues was indicative of preclinical or subclinical disease(4). In addition, one hemophilic patient might have beeninfected through treatment with plasma-derived products (5).Importantly, PrPTSE has been detected in whole blood of vCJDpatients (6 – 8). Moreover, a number of healthy people havebeen found to harbor this agent in lymphoreticular tissues(3, 9, 10); however, it is not known whether PrPTSE circulatesin the blood of these individuals and whether they willdevelop the disease later in life. Recently, infectivity wasreported in the plasma of two out of four patients affected bysporadic CJD (sCJD) (11). Although similar levels of PrPTSE

have been detected in spleens, tonsils, and lymph nodes ofvCJD and sCJD patients by Western blotting (12), the iden-tification of this protein in the blood of individuals afflictedwith the latter by various methods, including PrPTSE capturecoupled to direct immunodetection of surface-bound mate-rial (6), capillary electrophoresis (13), and protein misfold-ing cyclic amplification (PMCA) coupled to surroundingoptical fiber immunoassay (12), has been highly elusive for

* This study was supported by research funding from the American NationalRed Cross (to L. C.) and the Fondation Alliance BioSecure (to P. S.).

1 To whom correspondence should be addressed: Transmissible DiseasesDept., Biomedical Services, Holland Laboratory, American National RedCross, 15601 Crabbs Branch Way, Rockville, MD 20855. Tel.: 240-314-3529;Fax: 301-610-4120; E-mail: Paula.Saa@redcross.org.

2 The abbreviations used are: TSE, transmissible spongiform encephalopathy;vCJD, variant Creutzfeldt-Jakob disease; PrPTSE, TSE-associated prion pro-tein; PrPC, cellular prion protein; EV, extracellular vesicle; BSE, bovine spon-giform encephalopathy; MoBH, mouse brain homogenate; sCJD, sporadicCJD; PMCA, protein misfolding cyclic amplification; CWD, chronic wastingdisease; saPMCA, serial automated PMCA; wpi, weeks post-inoculation;

EVP, EV protein; NTA, nanoparticle tracking analysis; FVB, FVB/NCr; C57BL,C57BL/6; WT, wild-type.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 42, pp. 29247–29260, October 17, 2014Published in the U.S.A.

OCTOBER 17, 2014 • VOLUME 289 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 29247

at NO

RT

H D

AK

OT

A ST

AT

E U

NIV

on October 28, 2014

http://ww

w.jbc.org/

Dow

nloaded from

decades, with only two cases recently reported (8). Neverthe-less, extensive epidemiological data continue to provide noevidence of human-to-human transmission of sCJD throughblood transfusion (14).

Multiple studies showed that TSE infectivity is present in theblood of experimental rodents, sheep with natural and experi-mental scrapie and experimental BSE, and deer affected withchronic wasting disease (CWD) (15–20). PrPTSE has been suc-cessfully detected in the following: in whole blood of experi-mentally infected mice and hamsters, in sheep with naturalscrapie and experimental BSE, and in white-tailed deer afflictedwith CWD (21–23); in buffy coats of hamsters and sheep exper-imentally infected with scrapie (24 –26), as well as in plasma ofmice and hamsters with scrapie; and in sheep and white-taileddeer naturally and experimentally infected with scrapie andCWD, respectively (27–30). However, it is still not fully under-stood in what blood component TSE infectivity and/or PrPTSE

reside and whether and how blood contributes to the spread ofthe disease from the periphery to the brain.

Exosomes are extracellular membrane vesicles of 40 to 200nm in diameter, secreted by most cell types upon fusion ofmultivesicular bodies with the plasma membrane. Originally,they were considered to be a cellular mechanism to eliminateunwanted proteins during reticulocyte maturation (31–33).Further studies have shown the presence of nucleic acid andprotein cargo inside exosomes (34 –36). These extracellularvesicles (EVs) are believed to participate in intercellular com-munication processes and to execute functions regulating theimmune response, angiogenesis, coagulation, inflammation,and programmed cell death (37). Importantly, it has beenshown that viruses like HIV-1 can take over the cellularmachinery for exosome secretion and use it as another mecha-nism for viral release (38, 39). This “Trojan exosome” hypoth-esis, posited by Gould et al. (39), is currently being evaluated inprotein-misfolding neurodegenerative diseases as a possiblemechanism for the spread of misfolded proteins (40 – 45). Cel-lular prion protein (PrPC) and misfolded PrPTSE have beenidentified in exosomes from various TSE models (46 –55) andintracerebral inoculation of exosomes obtained from TSE-in-fected cell cultures has caused clinical disease in mice (47, 49).Little is known about the distribution of PrPTSE in blood. PrPC

has been detected in exosomes isolated from platelets andannexin V-positive EVs released from apoptotic endothelialcells (48, 56). These findings raise the possibility that endothe-lial and blood cells other than platelets may be capable of releas-ing PrPC and possibly PrPTSE in association with exosomes andother types of EVs, contributing to the transmission of TSEsthrough blood-derived products (48, 56 –58). Because of theiravailability in biological fluids, their stability, and their ability tocarry specific cargo, exosomes are ideal targets for detection ofbiomarkers for the diagnosis of various diseases (34, 59 – 61).Detection of PrPTSE in EVs obtained from blood or other bodyfluids (urine, saliva, nasal secretions, and tears) will enable thedesign of minimally invasive or noninvasive diagnostic tests forTSEs. In this study, EVs, containing exosomes, were isolatedfrom the conditioned media of cell cultures infected withmouse-adapted vCJD (Mo-vCJD) (16) or Fukuoka-1, a mouse-adapted isolate from a Gerstmann-Sträussler-Scheinker dis-

ease patient (62, 63), and from plasma collected periodicallyduring the preclinical and clinical phases from Mo-vCJD-in-fected mice. They were used to seed serial automated proteinmisfolding cyclic amplification (saPMCA) reactions followedby PrPTSE detection by Western blotting.

Our findings represent the first evidence of the presence ofPrPTSE in EVs obtained from plasma samples from preclinicaland clinically sick mice, and they provide proof-of-concept forthe design of a novel microvesicle-based diagnostic test forprion diseases.

EXPERIMENTAL PROCEDURES

Ethics Statement—The Institutional Animal Care and UseCommittee of the American National Red Cross reviewed andapproved the animal protocols numbered 0807-023 and 1006-045. The American National Red Cross maintains a centralizedanimal care and use program registered by the United StatesFood and Drug Administration, ensured with the Office of Lab-oratory Animal Welfare, and accredited by the Association forAssessment and Accreditation of Laboratory Animal Care,International. Housing and care of animals are consistent withthe Public Health Service Policy on Humane Care and Use ofLaboratory Animals, Guide for the Care and Use of LaboratoryAnimals, the Animal Welfare Act, and other applicable stateand local regulations.

Human blood was collected into citrate/phosphate dextroseupon informed written consent under protocol number 1998-18, approved by the Institutional Review Board of the AmericanNational Red Cross.

Animals

Mouse Inoculations—Wild type (WT), C57BL/6 (C57BL),and FVB/NCr (FVB) mice (Charles River Laboratories, Ando-ver, MA) were intracerebrally injected with 30 �l of a 10�2

diluted or a 10-fold serially diluted (10�1 to 10�7) Mo-vCJD-infected brain homogenate, respectively. Control mice receiveda similar injection of physiological saline. The inoculum titerwas determined by the method of Reed and Muench (64) basedon the survival rate of FVB mice inoculated with 10�1 to 10�8

dilutions of Mo-vCJD. FVB mice in the experimental groupswere euthanized when they developed clinical signs of TSE.C57BL mice were euthanized periodically during the incuba-tion period (6, 9, 12, 15, and 20 weeks post inoculation (wpi))and at clinical onset (�23 wpi). Negative controls were eutha-nized at the end of the experiment, together with the last mousefrom the experimental group. TSE in mice was confirmed bydetecting PrPTSE in brain extracts by Western blotting and/orimmunohistochemistry as described elsewhere (65).

Blood Collection and Processing—Mouse blood was collectedinto citrate/phosphate dextrose by cardiac puncture under iso-flurane (Patterson Veterinary, Devens, MA) anesthesia. Bloodsamples were usually processed separately, but on some occa-sions samples from a few mice were pooled together. Sampleswere spun at 2,300 � g for 10 min, at room temperature in aFA45-24-11 rotor (Eppendorf, Hauppauge, NY). Plasma wascollected, aliquoted, and stored at �80 °C.

Substrate Preparation for saPMCA—Mice were euthanizedby CO2 inhalation and perfused with 40 ml of 5 mmol/liter

Detection of PrPTSE in Exosomes from TSE-infected Mice

29248 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 42 • OCTOBER 17, 2014

at NO

RT

H D

AK

OT

A ST

AT

E U

NIV

on October 28, 2014

http://ww

w.jbc.org/

Dow

nloaded from

EDTA in PBS by intra-cardiac injection. Brains were collectedand frozen at �80 °C until further processing. Brains werehomogenized in appropriate volumes of conversion buffer (1%Triton X-100 in PBS with 1� Complete protease inhibitor mix-ture (Roche Applied Science)) to prepare a 10% (w/v)WT-mouse brain homogenate (MoBH) that was then split into1-ml aliquots and stored at �80 °C.

Human Samples

Human blood was separated into RBCs, buffy coat, and plate-let-rich plasma by centrifugation at 800 � g for 30 min. Platelet-rich plasma was centrifuged at 3,900 � g for 16 min. Platelet-poor plasma was collected, aliquoted, and stored at �80 °C. Allcentrifugations were performed at room temperature in anA-4-44 rotor (Eppendorf).

Cell Culture

Murine spleen-derived stromal cell culture persistentlyinfected with Mo-vCJD (Mo-vCJD/SP-SC), murine bone mar-row-derived stromal cell culture persistently infected withFukuoka-1 (OF1-BMS), and appropriate uninfected control cellcultures (NB/SP-SC and BMS, respectively) were developed inour laboratory and have been described previously (66, 67).Cells were plated in 25-cm2 flasks with 5 ml of Opti-MEMmedia (Invitrogen) not supplemented with fetal bovine serumand allowed to grow to confluence at 37 °C and 5% CO2 for 2–3days for EV preparation.

EV Isolation

EV Isolation from Conditioned Media by ExoQuickTM-TC,Optimized for Culture Media and Urine Samples (System Bio-sciences, Mountain View, CA)—The medium from each cellculture was collected when cells reached confluence and wascentrifuged at 3,000 � g for 15 min to remove cells and celldebris. Supernatants were collected, split in 5-ml aliquots, fro-zen on dry ice, and stored at �80 °C until further processing.Supernatant aliquots of 5 ml were thawed at room temperature,mixed with 1 ml of ExoQuickTM-TC, and incubated at 4 °Covernight. The mixture was centrifuged at 1,500 � g for 30 min,and the EV pellets were collected and stored at �80 °C untilanalysis. All centrifugations were done at 4 °C in an A-4-44rotor.

EV Isolation from Plasma by ExoQuickTM, Optimized forSerum or Ascites (System Biosciences)—Plasma samples werethawed at 37 °C and spun for 15 min at 3,000 � g. The resultingsupernatant was mixed with ExoQuickTM (63 �l of ExoQuickper 250 �l of plasma) and incubated overnight at 4 °C. Sampleswere centrifuged at 1,500 � g for 30 min and EV pellets werecollected and stored at �80 °C until analysis. All centrifuga-tions were performed at 4 °C in a FA45-24-11 rotor.

Exosome Purification Using a 30% Sucrose Cushion

EV pellets obtained after ExoQuickTM were processedthrough a 30% sucrose cushion as indicated in the text, follow-ing a protocol adapted from Thery et al. (68). The ExoQuickTM

pellet was thawed at room temperature, resuspended in 4 ml ofPBS, and layered on top of 0.7 ml of 30% sucrose prepared in aTris/D2O solution (30 g of sucrose, 2.4 g of Tris base, D2O up to

100 ml). After an initial ultracentrifugation at 110,000 � g for 75min, three fractions were collected as follows: a top (“top”) layerof �0.2 ml, the “middle” layer containing the sucrose layer andthe PBS/sucrose interface, and the pellet (“bottom”). The pelletwas resuspended in PBS, and all three fractions were broughtup to 5 ml with PBS. Samples were subjected to a second ultra-centrifugation at 110,000 � g for 70 min. The three resultingpellets were collected and stored at �80 °C. All centrifugationswere performed at 4 °C in an SW55 Ti rotor.

saPMCA Amplification

To determine the presence of PrPTSE, EV preparations fromdifferent sources (source of PrPTSE) were resuspended into 10%WT-MoBHs (source of PrPC) that were prepared as indicatedabove. The volume of 10% WT-MoBH used to resuspend theEV pellets is specified for each individual experiment under“Results.” Furthermore, samples were aliquoted into 0.2-mlPCR tubes containing zirconia/silica beads of 1 mm in diameter(Biospec Products Inc., Bartlesville, OK). Samples were ampli-fied by 48 cycles (one round) of incubation at 37 °C, followed bya 20-s pulse of sonication at power 4 in a Q700MPX microplatehorn sonicator (QSonica, Newtown, CT) (69, 70). After eachround, sample aliquots were mixed 1:1 or 1:3, as indicatedbelow for each experiment, with 10% WT-MoBH to performthe next round of saPMCA. The number of rounds varied foreach experiment and is indicated in the appropriate sections.

Proteinase K (PK) Digestion

Aliquots of 9 �l of samples subjected to saPMCA weretreated with 20 �g/ml PK (Novagen�, Darmstadt, Germany) for1 h at 37 °C with agitation at 450 rpm. PK treatment wasstopped by sample denaturation in NuPAGE LDS sample buffer(Invitrogen) at 100 °C for 10 min.

Western Blotting

Proteins were diluted in NuPAGE LDS sample buffer (Invit-rogen) and separated by SDS-PAGE, electroblotted onto anitrocellulose membrane (Invitrogen), and probed with variousprimary and secondary antibodies as indicated in the text.Immunoreactive bands were visualized using West Pico(Pierce). The primary antibodies used were as follows: mousemonoclonal anti-PrP antibody 6D11 (Covance�, Berkeley, CA),and rabbit monoclonal antibodies anti-Hsp70 (Epitomics, Bur-lingame, CA) and anti-GM130 (Abcam, Cambridge, MA). Sec-ondary antibodies used were as follows: HRP-conjugated goatanti-mouse IgG (Kirkegaard & Perry, Gaithersburg, MD); HRP-conjugated rat anti-mouse IgG Mouse Trueblot� (Rockland,Gilbertsville, PA); and HRP-conjugated mouse anti-rabbit IgGrabbit Trueblot� (Rockland).

Methanol Precipitation

Where applicable, samples were mixed with 5 volumes ofprechilled 100% methanol and incubated overnight at �20 °C.Samples were centrifuged at 25,000 � g for 30 min in a FA45-24-11 rotor and resuspended in 1% Sarkosyl.

EV Protein (EVP) Solubilization

EV proteins were solubilized by resuspending extracellularvesicle pellets in EVP lysis buffer consisting of 100 mmol/liter

Detection of PrPTSE in Exosomes from TSE-infected Mice

OCTOBER 17, 2014 • VOLUME 289 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 29249

at NO

RT

H D

AK

OT

A ST

AT

E U

NIV

on October 28, 2014

http://ww

w.jbc.org/

Dow

nloaded from

NaCl, 10 mmol/liter EDTA, 0.5% Triton X-100, 0.5% sodiumdeoxycholate, and 10 mmol/liter Tris, pH 7.4.

Nanoparticle Tracking Analysis (NTA)

The EV pellet obtained from the plasma of a healthy FVBmouse after purification with ExoQuickTM was resuspended in200 �l of PBS, and the suspension was diluted 1,000-fold priorto being analyzed. Alternatively, the EV pellet obtained asabove, was further purified through a 30% sucrose cushion asdescribed under “Exosome Purification Using a 30% SucroseCushion.” The three fractions collected (top, middle, and bot-tom) were resuspended in 700 �l of PBS prior to being analyzed.The particle hydrodynamic diameter of the EVs was measuredfor 90 s using a NanoSight LM10 system (Malvern, Worcester-shire, UK). The Nanoparticle Tracking Analysis 2.3 analyticalsoftware version was used for capturing and analyzing the data.Particle number per ml and standard deviation were calculatedfrom three measurements from the same sample.

RESULTS

saPMCA Detection of PrPTSE in EVs Isolated from Condi-tioned Media Collected from TSE-infected Cells—We firstinvestigated the presence of PrPTSE in EVs isolated from con-ditioned media collected from Mo-vCJD/SP-SC and OF1-BMSand corresponding uninfected cells (NB/SP-SC and BMS,respectively) used as control. EVs were isolated from 5 ml ofconditioned media with ExoQuickTM-TC as described under“Experimental Procedures” (EV Isolation from ConditionedMedia by ExoQuickTM-TC, Optimized for Culture Media andUrine Samples (System Biosciences, Mountain View, CA)). EVpellets from spleen-derived and bone marrow-derived cellswere resuspended in 250 and 200 �l, respectively, of 10% WT-MoBH. Samples were amplified by saPMCA through 48 cycles(one round). After each round, aliquots of 30 �l were mixed 1:1with 10% WT-MoBH to perform the next round of saPMCA.Additionally, aliquots of 9 �l of amplified material were takenfrom each sample and processed for the detection of PrPTSE byPK digestion followed by Western blotting as under “Experi-mental Procedures.”

EVs from 5 ml of conditioned media contained sufficientamounts of PrPTSE to trigger PrPC conversion in saPMCA reac-tions, validating the applicability of this approach to detectPrPTSE in these preparations (Fig. 1). One round of saPMCA ledto PrPTSE detection in samples generated from Mo-vCJD/SP-SC cells (Fig. 1A), whereas two rounds were required todetect this protein in samples purified from OF1-BMS cells(Fig. 1C). The difference in number of rounds required for eachcell model, which differ in their tissue of origin, might be due tovariations in PrPTSE levels in the EV preparations and/or in thenumber of EVs secreted by the cells. Alternatively, it may reflectdifferences in the conversion efficiency of the two strains, Mo-vCJD and Fukuoka-1.

Detection of PrPTSE in Uninfected Human and Mouse PlasmaSamples Supplemented with EVs from Conditioned Media ofTSE-infected Cells—Earlier studies have shown that some bloodcomponents impair PMCA amplification (24, 25). This inhibi-tion can be overcome by performing several rounds of saP-MCA. Under these conditions, inhibitors are diluted after each

round, at the same time that new PrPTSE is generated. Thisapproach enabled PrPTSE detection as follows: in buffy coatsamples from 263K-infected hamsters during the preclinicaland clinical phases of prion disease (24, 25); in plasma prepara-tions from preclinical and clinically sick scrapie-affected sheep,CWD-infected white-tailed deer (28), and from clinically illmice infected with mouse-adapted BSE (71); in blood leuko-cytes from clinically sick VRQ/VRQ scrapie-affected sheep(26); and in whole blood from clinical mice infected withscrapie (21). To evaluate the effect of plasma on the detection ofEV-associated PrPTSE by saPMCA, EVs isolated from condi-tioned media of infected cells as described above were added to250 �l of human plasma obtained from a healthy donor. Humanplasma was used because of large volumes available to ensureexperimental consistency during the study. EVs were re-iso-lated from EV-supplemented plasma samples with Exo-QuickTM as described under “Experimental Procedures” (“EVIsolation from Plasma by ExoQuickTM, Optimized for Serum orAscites (System Biosciences)”). Re-isolated EVs were thawedand mixed with 250 �l of 10% WT-MoBH. In parallel, EV sam-ples that were not added to plasma were mixed with 250 �l of10% WT-MoBH and used as positive controls for saPMCA.Four aliquots of 58 �l were prepared from control and experi-mental samples and amplified by saPMCA as described under“Experimental Procedures.” Samples were diluted 1:1 betweensaPMCA rounds.

Although PrPTSE was readily demonstrated in EVs from theconditioned media of Mo-vCJD/SP-SC cells after one round ofsaPMCA (Fig. 2A), a high unspecific signal background pre-vented detection of PrPTSE in plasma samples supplemented

FIGURE 1. Detection by saPMCA of PrPTSE in EV preparations isolatedfrom the conditioned media of chronically infected cells. EVs were puri-fied with ExoQuickTM-TC (System Biosciences) from 5 ml of conditionedmedia collected from Mo-vCJD-infected (A) and -uninfected (B) spleen-de-rived stromal cells (Mo-vCJD/SP-SC and NB/SP-SC, respectively), and fromFukuoka-1 (FU)-infected (C) and -uninfected (D) bone marrow-derived stro-mal cells (OF1-BMS and BMS, respectively). EV pellets were mixed with 250and 200 �l, respectively, of 10% wild-type mouse brain homogenate (used assource of PrPC). Aliquots of 9 �l were taken from each sample and used ascontrols not subjected to saPMCA (�). The remaining BMS and SP-SC sampleswere split into 3 and 4 aliquots, respectively, and amplified by saPMCA (�).PrPTSE was specifically detected in EV samples from Mo-vCJD/SP-SC cells (A)and from OF1-BMS cells (C) after one and two rounds of saPMCA, respectively,but not in samples from uninfected NB/SP-SC (B) and BMS (D) cells. All sam-ples were treated with 20 �g/ml of proteinase K, resolved by SDS-PAGE, andsubjected to Western blotting using the PrP-specific antibody 6D11 (Cova-nce). Molecular mass is shown on the right.

Detection of PrPTSE in Exosomes from TSE-infected Mice

29250 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 42 • OCTOBER 17, 2014

at NO

RT

H D

AK

OT

A ST

AT

E U

NIV

on October 28, 2014

http://ww

w.jbc.org/

Dow

nloaded from

with EVs isolated from the conditioned media of infected cells(Fig. 2B). Nevertheless, dilution and amplification of these sam-ples for three additional rounds of saPMCA resulted in the reli-able detection of PrPTSE (Fig. 2A). Similar findings wereobtained when EVs, prepared from the conditioned media ofFukuoka-1-infected OF1-BMS cells, were added to plasma col-lected from uninfected wild-type FVB mice, re-isolated, andused in saPMCA (Fig. 3).

Presence of IgG in Plasma EV Preparations Disables Detectionof PrPTSE after the Initial Rounds of saPMCA—IgG is one of themost abundant proteins in plasma, and its presence in samplesis a recognized problem in Western blotting applications due toreaction with secondary antibodies. We observed high back-ground signal when performing Western blotting analysis ofsamples after initial rounds of saPMCA that were seeded withplasma EV preparations. Therefore, we attempted to overcomethis problem by using the mouse Trueblot� HRP-conjugatedanti-mouse IgG (Rockland) secondary antibody, which doesnot detect the reduced and SDS-denatured forms of IgG,instead of the commonly used secondary HRP-conjugated anti-mouse IgG (Kirkegaard & Perry) antibody. EVs were isolated

with ExoQuickTM from 250 �l of plasma samples from twoterminally ill FVB mice infected with Mo-vCJD. EV pellets wereresuspended in 250 �l of 10% WT-MoBH and amplified bysaPMCA. Selected saPMCA-amplified plasma EV samples cor-responding to rounds one and two were treated with PK, sub-jected to electrophoresis, and immunoblotted with either anti-body (Fig. 4A). The antibody from Kirkegaard & Perry revealedthe presence of IgG and other reactive bands, in all samplesfrom the two mice after one round of saPMCA. The intensity ofthe signal significantly decreased after two rounds demonstrat-ing that cycles of dilution/conversion efficiently eliminatedunspecific proteins, including IgG, from the samples. Bandscorresponding to IgG were not detected with the antibody fromRockland after rounds one and two. To demonstrate the appro-priate binding to the primary antibody 6D11, which is specificto PrP, antibodies from both Kirkegaard & Perry and Rocklandwere used to reveal PrPTSE in Western blotting of samples sub-jected to five rounds of saPMCA. Three bands corresponding todi-, mono-, and unglycosylated PrPTSE were observed witheither antibody in samples treated with PK; however, the inten-sity of the signal obtained with the antibody from Rockland wassignificantly lower, and longer exposure times were necessaryto obtain results comparable with those generated with theantibody from Kirkegaard & Perry (Fig. 4B).

Isolation and Characterization of Plasma EVs, PrPTSE Asso-ciates with Plasma EVs—To investigate association of PrPTSE

with plasma EVs, we took advantage of the flotation propertiesof nanovesicles on a 30% sucrose cushion. Ultracentrifugationof EVs in the presence of 30% sucrose eliminates nonspecificallyassociated proteins, or protein aggregates, which are sedi-mented by centrifugation but do not float on a sucrose gradientand allow isolation of highly purified exosomes (68). Two500-�l aliquots of pooled plasma from clinically sick FVB miceinfected with 10�1 and 10�2 dilutions of Mo-vCJD were pro-cessed with ExoQuickTM to isolate EVs. The pellet from 1 ali-quot was stored at �80 °C until analysis and was labeled as“infected non-sucrose.” The pellet from the second aliquot wasprocessed through a 30% sucrose cushion as described under“Experimental Procedures.” Three fractions were collectedafter ultracentrifugation as follows: a thin infected top layer of�0.2 ml of white material of lipidic appearance; the infectedmiddle layer containing the PBS/sucrose interface and thesucrose layer; and the infected bottom pellet, which was notvisible. All three fractions were brought up to 5 ml of PBS andsubjected to a second ultracentrifugation. Plasma samples fromuninfected FVB mice were processed similarly and used as neg-ative controls (“uninfected non-sucrose” and “uninfected top,middle, and bottom”). Infected and uninfected “non-sucrose”fractions were resuspended in 250 �l of 10% WT-MoBH;infected and uninfected “sucrose” fractions were resuspendedin 125 �l of 10% WT-MoBH. All samples were subjected tovarious rounds of saPMCA.

Samples were evaluated for the presence of PrPTSE by West-ern blotting with the anti-PrP primary antibody 6D11 andmouse Trueblot� secondary antibody (Fig. 5). This allowedPrPTSE detection in plasma EVs at early rounds of saPMCAwithout IgG interference. Under these conditions, PrPTSE wasspecifically detected in “non-sucrose” plasma exosome frac-

FIGURE 2. Detection by saPMCA of PrPTSE in EV preparations isolatedfrom cell-conditioned media of chronically infected cells and added tonormal human plasma. A, EV samples were obtained from 5 ml of condi-tioned media from Mo-vCJD-infected and -uninfected spleen-derived stro-mal cells (Mo-vCJD/SP-SC and NB/SP-SC, respectively) using ExoQuickTM-TC(System Biosciences). EV pellets were either mixed with 250 �l of 10% wild-type mouse brain homogenate (WT-MoBH) (A, top and bottom left panels, andB) or added to 250 �l of human plasma. Samples added to plasma were sub-jected to repeated EV extraction with ExoQuickTM, and the pellets were mixedwith 1,250 �l of WT-MoBH (A, top and bottom right panels, and B). Each samplewas split into 4 aliquots and subjected to cycles of saPMCA (�). The presenceof heavy background signal in EV preparations from plasma prevented PrPTSE

detection after one round of saPMCA (B). Dilution of samples in three addi-tional rounds allowed reliable detection of PrPTSE (A, top right panel). Samplealiquots of 9 �l were treated with 20 �g/ml proteinase K, resolved by SDS-PAGE, and subjected to Western blotting using the PrP-specific antibody6D11 (Covance). Molecular mass is shown on the right.

Detection of PrPTSE in Exosomes from TSE-infected Mice

OCTOBER 17, 2014 • VOLUME 289 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 29251

at NO

RT

H D

AK

OT

A ST

AT

E U

NIV

on October 28, 2014

http://ww

w.jbc.org/

Dow

nloaded from

FIGURE 3. Detection by saPMCA of PrPTSE in extracellular vesicles EVs isolated from cell-conditioned media of OF1-BMS cells that were added tonormal FVB mouse plasma. EV samples were obtained from 5 ml of conditioned media from Fukuoka-infected bone marrow-derived stromal cells (OF1-BMS)using ExoQuickTM-TC (System Biosciences). Samples were either mixed with 250 �l of 10% wild-type mouse brain homogenate (WT-MoBH) (A, lanes 2 and 3,and B, lanes 3– 6) or added to 250 �l of FVB mouse plasma. Samples added to plasma were subjected to repeated EV extraction with ExoQuickTM, and the pelletswere mixed with 150 �l (A, lanes 4 and 5, and B, lanes 7 and 8), 250 �l (A, lanes 6 –9, and B, lanes 9 –12), and 1,250 �l of WT-MoBH (A, lanes 10 and 11, and B, lanes13 and 14). Each sample was split into aliquots prior to saPMCA (�). The presence of heavy background signal in EVs re-isolated from plasma prevented PrPTSE

by Western blotting detection after one round of saPMCA. Dilution of samples in three and four additional rounds allowed reliable detection of PrPTSE (A andB, respectively). Samples were treated with 20 �g/ml of proteinase K (PK), resolved by SDS-PAGE, and subjected to Western blotting using the PrP-specificantibody 6D11 (Covance). �PK, WT-MoBH not treated with PK. Molecular mass (M) is shown on the left. Samples were diluted 1:5 between saPMCA rounds.

FIGURE 4. Presence of IgG in plasma-derived EV preparations. Western blotting analysis of the initial rounds of saPMCA showed high background thatprevented the detection of PrPTSE. A, presence of IgG was evaluated by assessing sample reactivity with two secondary antibodies: HRP-conjugated goatanti-mouse IgG (Kirkegaard & Perry antibody (Ab)) and mouse Trueblot� HRP-conjugated rat anti-mouse IgG (Rockland antibody), which does not detect thereduced SDS-denatured forms of IgG. saPMCA-amplified plasma EVs from terminally ill FVB/NCr mice infected with Mo-vCJD after rounds one and two weredigested with proteinase K and separated on SDS-PAGE prior to immunoblotting with either antibody (A, left and right panels, respectively). Immunoblottingwith the antibody from Kirkegaard & Perry revealed the presence of IgG in all aliquots from both mice after one round of saPMCA. The intensity of the signalsignificantly decreased after two rounds of saPMCA, demonstrating that cycles of dilution/conversion effectively eliminate IgG in plasma EV-seeded samples.No signal was detected with mouse Trueblot� antibody. B, same plasma EV preparations were probed with the PrP-specific antibody 6D11 (Covance) as theprimary antibody was followed by secondary antibodies from either Kirkegaard & Perry or Rockland. Molecular mass is shown on the right. Note the differencein the intensity signals between the antibodies used from Kirkegaard & Perry and Rockland, with the antibody from Kirkegaard & Perry giving significantlybetter signal readout. Lanes 1–3 represent sample triplicates.

Detection of PrPTSE in Exosomes from TSE-infected Mice

29252 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 42 • OCTOBER 17, 2014

at NO

RT

H D

AK

OT

A ST

AT

E U

NIV

on October 28, 2014

http://ww

w.jbc.org/

Dow

nloaded from

tions from infected mice after just one round of saPMCA (Fig.5A, infected non-sucrose). A second round of saPMCA showedthe presence of PrPTSE in the pellet fraction obtained after cen-trifugation through a sucrose cushion (Fig. 5B, lanes 3 and 4). Inaddition, PrPTSE was detected in the “top” layer (Fig. 5B, lanes 1and 2), which represents a fraction of co-purified PrPTSE that islikely to be associated with lipids, very low density and lowdensity lipoproteins (72), and/or because the ExoQuickTM pro-tocol does not include a filtration step, probably with mem-brane fragments. Most importantly, PrPTSE was detected in the“middle” fraction (Fig. 5B, lanes 5 and 6), containing plasmaexosomes. No PrPTSE was detected in corresponding fractionsfrom plasma EV preparations from uninfected mice (Fig. 5, A,lanes 5– 8, and B, lanes 7–12). Samples were diluted 1:1between rounds.

Exosomal Marker Hsp70 Associates with Plasma EVs—Tocharacterize EV preparations, we analyzed the presence of theexosomal marker Hsp70 and the absence of the Golgi markerGM130. EVs were isolated from 500 �l of plasma from a healthyFVB mouse using ExoQuickTM. The EV pellet was subjected toultracentrifugation through a 30% sucrose cushion andrepeated ultracentrifugation as described above. The three pel-lets, representing sucrose top, middle, and bottom fractions,were resuspended in EVP lysis buffer to solubilize membraneproteins. Proteins were concentrated by methanol precipita-tion and resuspended in 1% Sarkosyl in PBS. For comparison,

EVs were also isolated from the conditioned media ofNB/SP-SC cells with ExoQuickTM-TC, resuspended in EVPlysis buffer, methanol-precipitated, and resuspended in 1%Sarkosyl in PBS (“cell EVs”). This preparation was used as posi-tive control for Hsp70. Additionally, NB/SP-SC cells were lysed inEVP lysis buffer (“NB/SP-SC cell lysate”) and used as control forGM130. The same amount of total protein (12 �g) was loaded persample. Western blotting analysis of samples obtained fromplasma, conditioned media, and cell lysates revealed the presenceof Hsp70 in the conditioned media sample (cell EVs) and sucrosemiddle plasma fraction (Fig. 6A, lanes 1 and 4, respectively) but notin sucrose top and bottom plasma fractions obtained after ultra-centrifugation (Fig. 6A, lanes 3 and 5), thus confirming that sam-ples prepared from plasma using ExoQuickTM contained exo-somes. As expected, the Golgi marker GM130 was exclusivelydetected in cell lysates (Fig. 6B, lane 1) but was absent in samplesprepared from conditioned media and from sucrose top, middle,and bottom fractions (Fig. 6B, lanes 2–5). The specificity of theimmunoreactive bands was confirmed by the absence of signalwhile probing the samples with the secondary rabbit Trueblot�antibody only (data not shown).

FIGURE 5. Detection by saPMCA of PrPTSE in EV preparations from plasmasamples of Mo-vCJD-infected mice. EV samples were prepared using Exo-QuickTM (System Biosciences) from 500 �l of plasma from clinically sick oruninfected control mice as described in the text. A, EV pellets were mixed into 250�l of 10% wild-type mouse brain homogenate (WT-MoBH), split into 4 aliquots,and subjected to one round of saPMCA (non-sucrose). B, EV pellets were used topurify exosomes by ultracentrifugation on a 30% sucrose cushion. Three fractionswere collected (top, middle (containing exosomes), and bottom) and subjected toa second round of ultracentrifugation. Pellets were mixed into 125 �l of 10%WT-MoBH, split into 2 aliquots, and subjected to two rounds of saPMCA. Aliquotsof 9 �l were collected from each sample, treated with 20 �g/ml proteinase K,resolved by SDS-PAGE, and subjected to Western blotting using the PrP-specificantibody 6D11 (Covance) and mouse Trueblot� (Rockland) secondary antibody.�PK, WT-MoBH not treated with PK. Molecular mass is shown on the right. Sam-ples were diluted 1:1 between rounds.

FIGURE 6. Demonstration of the presence of the exosomal marker Hsp70in EV preparations from normal FVB mouse plasma. EVs were isolatedfrom 500 �l of plasma from uninfected FVB mice with ExoQuickTM (SystemBiosciences) and purified as described in the text. Proteins from the top,middle, and bottom fractions were concentrated by methanol precipita-tion and resuspended in 1% Sarkosyl in PBS. The exosomal marker Hsp70(A) and the Golgi marker GM130 (B) were detected with correspondingrabbit primary antibodies and the rabbit Trueblot� (Rockland) secondaryantibodies. A, lane 1, cell EVs: non-sucrose EVs from uninfected NB/SP-SCconditioned media (positive control); lane 2, molecular mass marker; lanes3–5, sucrose top, middle (containing EVs), and bottom fractions, respec-tively. B, lane 1, NB/SP-SC cell lysate (positive control); lane 2, cell EVs:non-sucrose-cleaned EVs from NB/SP-SC-conditioned media; lanes 3–5,sucrose top, middle (containing EVs), and bottom fractions, respectively.Twelve micrograms of total protein were loaded per well in A and B.Molecular mass is shown on the left.

Detection of PrPTSE in Exosomes from TSE-infected Mice

OCTOBER 17, 2014 • VOLUME 289 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 29253

at NO

RT

H D

AK

OT

A ST

AT

E U

NIV

on October 28, 2014

http://ww

w.jbc.org/

Dow

nloaded from

Detection of Particle Hydrodynamic Diameter in EV Prepa-rations Purified by ExoQuickTM—To identify the particlehydrodynamic diameter of EVs, samples prepared from theplasma of healthy FVB mice were subjected to NTA. The vesiclehydrodynamic diameter ranged from 50 to 300 nm, with anaverage diameter of 114 nm, in a sample with a vesicle count of5.46 � 108 particles/ml (Fig. 7A). Established EV hydrodynamicdiameter was within the range reported for human plasma EVs(73). Next, we evaluated the presence of EVs in the three frac-tions collected following centrifugation through a 30% sucrosecushion. To this end, EVs were isolated from the plasma ofhealthy FVB mice with ExoQuickTM and further purifiedthrough a 30% sucrose cushion as described above. The top,middle, and bottom fractions were resuspended in PBS andsubjected to NTA. We identified the presence of EVs in allfractions analyzed, albeit with differences in particle concentra-tion and hydrodynamic diameter. The top fraction was charac-terized by the presence of a single population of EVs rangingfrom 38 to 350 nm and an average hydrodynamic diameter of103 nm (Fig. 7B). The middle fraction contained EVs with ahydrodynamic diameter range of 25 to 450 nm and a majorparticle population of 102 nm. Two other minor peaks weredetected in this preparation showing average hydrodynamicdiameters of 124 and 145 nm (Fig. 7C). The bottom fractioncontained EVs of 40 to 500 nm in diameter. Various peaks couldbe identified in this fraction with average hydrodynamic diam-eters of 87, 114, 139, and 170 nm (Fig. 7D). The particle con-centration also varied between fractions, with the bottom frac-tion containing the lowest number of particles, 7.2 � 0.21 � 108

particles/ml, followed by the top fraction with 11.6 � 0.21 �108 particles/ml, and the middle fraction with 17.3 � 1.33 � 108

particles/ml.Detection of PrPTSE in Plasma EVs Isolated from Sympto-

matic Mo-vCJD-infected FVB Mice—After demonstrating theefficiency of ExoQuickTM to isolate PrPTSE-containing EVs andthe potential of saPMCA to detect PrPTSE in plasma EV prepa-rations (Fig. 5), we aimed to confirm the presence of PrPTSE inplasma preparations from a larger number of samples collectedfrom terminally sick mice infected with Mo-vCJD.

EVs were isolated from 200 and 250 �l of plasma from Mo-vCJD-infected FVB mice and appropriate uninfected controls,using ExoQuickTM as described under “Experimental Proce-dures.” Samples were resuspended in an equal volume of 10%WT-MoBH, split into 4 aliquots, and subjected to multiplerounds of saPMCA. Samples were diluted 3-fold betweensaPMCA rounds. Results of PrPTSE detection, starting fromround five, in which the first positive samples were clearly iden-tified, are summarized in Table 1 and Fig. 8. PrPTSE wasdetected in all plasma EV pellets from infected mice in whichTSE had been biochemically confirmed. Samples from mouse 4,which received a 10�7 dilution of Mo-vCJD, were negativethrough all seven rounds. In agreement with this result, mouse4 showed no clinical signs of TSE and tested negative for PrPTSE

in the brain by Western blotting. The calculated LD50 value forthe Mo-vCJD brain homogenate, used for inoculation, was esti-mated to be 10�6.3. The other three mice in the group infectedwith a 10�7 dilution exhibited clinical signs and tested positive forPrPTSE in the brain by Western blotting. Samples from uninfected

FIGURE 7. Analysis of plasma-derived EVs. A, EVs were purified from 250 �l of uninfected mouse plasma, resuspended in 200 �l of PBS, and further diluted1,000 times in PBS. B–D, EVs were isolated from 500 �l of uninfected mouse plasma. EVs were further purified through a 30% sucrose cushion. Top (B), middle(C), and bottom (D) fractions were collected and resuspended in 700 �l of PBS. All samples were analyzed in a NanoSight LM-10 instrument. The particlehydrodynamic diameter distribution is represented relative to the particle concentration. A, NTA analysis of the total EVs isolated from plasma shows a mainparticle population of 114 nm, which is consistent with the exosome hydrodynamic diameter range of 40 to 200 nm. B, top fraction was characterized by thepresence of a single population of EVs with an average hydrodynamic diameter of 103 nm. C, middle fraction contained EVs with a major particle populationof 102 nm in diameter and two minor peaks with average hydrodynamic diameters of 124 and 145 nm. D, bottom fraction was characterized by the presenceof various peaks of 87, 114, 139, and 170 nm in diameter.

Detection of PrPTSE in Exosomes from TSE-infected Mice

29254 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 42 • OCTOBER 17, 2014

at NO

RT

H D

AK

OT

A ST

AT

E U

NIV

on October 28, 2014

http://ww

w.jbc.org/

Dow

nloaded from

control mice remained negative for all rounds of saPMCA (Fig. 8,FVB-I and FVB-II). Likewise, PrPTSE was specifically detected inplasma EVs isolated from FVB mice inoculated with Fukuoka-1;samples collected from noninfected mice remained negative forfive rounds of saPMCA (data not shown).

Detection of PrPTSE in EVs Isolated from Presymptomatic andSymptomatic Mo-vCJD-infected C57BL Mice—Plasma EV pelletswere obtained from groups of Mo-vCJD-infected C57BL mice that

were euthanized at 6, 9, 12, 15, and 20 wpi and at clinical stage ofTSE (�23 wpi) and from uninfected control mice. Samples wereevaluated for the presence of PrPTSE after several rounds of saP-MCA as described above. The results of this experiment are sum-marized in Table 2 and Fig. 9. All mice had tested positive forPrPTSE in the brain by Western blotting (Fig. 10). Four rounds ofsaPMCA reliably revealed the presence of PrPTSE in all aliquots ofplasma EVs obtained from mice euthanized at 20 wpi and during

TABLE 1Detection by saPMCA of PrPTSE in plasma-derived EV preparations isolated from clinically sick Mo-vCJD-infected FVB miceThe presence of PrPTSE was tested in 4 aliquots of EV preparations from individual or pooled plasma samples obtained from terminally ill Mo-vCJD-infected mice. Allinfected mice had tested positive (�) for PrPTSE in the brain by Western blotting, except mouse 4 (�). EVs were isolated from 200 �l of plasma by ExoQuickTM (SystemBiosciences). EV pellets were mixed with 200 �l of 10% wild-type mouse brain homogenate and split into 4 aliquots that were subjected to up to seven rounds of saPMCA.A summary of the data from rounds 5 to 7 is presented. Background interference prevented identification of PrPTSE-positive samples prior to round five. Presence of PrPTSE

was evaluated by Western blotting using the PrP-specific antibody 6D11 (Covance). FVB-I and FVB-II indicate plasma samples from FVB control mice.Mouse ID/inoculum

dilutionRound 5, aliquots

positive/totalRound 6, aliquots

positive/totalRound 7, aliquots

positive/totalPrPTSE

in brain

Mouse 1/10�7 4/4 4/4 4/4 �Mouse 2/10�7 4/4 4/4 3/4 �Mouse 3/10�7 UDa 4/4 4/4 �Mouse 4/10�7 0/4 0/4 0/4 �Mouse 5/10�6 3/4 3/4 3/4 �Plasma pool/10�5 4/4 4/4 4/4 �Mouse 6/10�5 4/4 4/4 4/4 �Mouse 7/10�4 3/4 3/4 3/4 �Mouse 8/10�4 4/4 4/4 4/4 �Mouse 9/10�3 4/4 4/4 4/4 �Mouse 10/10�3 4/4 4/4 4/4 �Plasma pool/10�1 and 10�2 16/16 16/16 16/16 �FVB-I/control 0/4 0/4 0/4 �FVB-II/control 0/4 0/4 0/4 �

a UD, PrPTSE presence undetermined due to unspecific background; � or �, the presence or absence of PrPTSE in the brain. Boldface indicates plasma EV samples thattested positive for PrPTSE in all 4 aliquots.

FIGURE 8. Detection by saPMCA of PrPTSE in plasma EV preparations isolated from clinically sick Mo-vCJD-infected FVB mice. EVs were isolated withExoQuickTM (System Biosciences) from 200 �l of plasma collected from terminally sick FVB mice intracerebrally injected with 10-fold diluted samples of aMo-vCJD-infected brain homogenate (the inoculum dose is indicated below each mouse number). EV pellets were mixed with 200 �l of 10% wild-type mousebrain homogenate (10% WT-MoBH), split into 4 aliquots, and amplified by saPMCA. After each round, aliquots of 20 �l were taken from each sample, mixed with40 �l of 10% WT-MoBH, and amplified in a new round of saPMCA; additionally, aliquots of 9 �l were collected from each sample, treated with 20 �g/ml ofproteinase K (PK), resolved by SDS-PAGE, and subjected to Western blotting using the PrP-specific antibody 6D11 (Covance). The figure illustrates thepresence/absence of PrPTSE signal in each of 4 sample aliquots from each mouse after six rounds of saPMCA. FVB-I and FVB-II, samples from uninfected wild-typemice used as negative control; �PK, WT-MoBH not treated with PK. Molecular mass is shown on the right.

Detection of PrPTSE in Exosomes from TSE-infected Mice

OCTOBER 17, 2014 • VOLUME 289 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 29255

at NO

RT

H D

AK

OT

A ST

AT

E U

NIV

on October 28, 2014

http://ww

w.jbc.org/

Dow

nloaded from

the clinical phase. Moreover, PrPTSE was demonstrated in at least 1of 4 aliquots from mice from earlier disease stages starting from 6wpi, except for mouse 1. One additional round of conversionincreased the number of positive aliquots from animals 3, 5, and12, euthanized at 6, 9, and 15 wpi. Mouse 1 again tested negativefor PrPTSE. Aliquots from samples generated from uninfectedmice remained negative for all five rounds of saPMCA. Conclu-sively, the presence of PrPTSE was demonstrated in plasma EVsfrom Mo-vCJD-infected mice during the preclinical phase, as earlyas 6 wpi. This finding is in agreement with the work of Tattum et al.(21) showing detection of PrPTSE, after four rounds of saPMCA, inwhole blood collected at 60 days post-inoculation from scrapie-infected CD1 mice.

DISCUSSION

The finding of four human vCJD infections related to bloodtransfusion (4) highlights the need to develop a method that

FIGURE 9. Detection by saPMCA of PrPTSE in plasma EV preparations isolated from presymptomatic and symptomatic Mo-vCJD-infected C57BL mice.EVs were isolated with ExoQuickTM (System Biosciences) from 200 and 250 �l of plasma collected from presymptomatic and symptomatic C57BL miceintracerebrally injected with a 1% Mo-vCJD-infected brain homogenate. EV pellets were mixed with 200 and 250 �l, respectively, of 10% wild-type mouse brainhomogenate (WT-MoBH), split into 4 aliquots, and amplified by saPMCA. After each round, aliquots of 20 �l were taken from each sample, mixed with 40 �l of10% WT-MoBH, and amplified in a new round of saPMCA; additionally, aliquots of 9 �l were collected from each sample, treated with 20 �g/ml of proteinaseK (PK), resolved by SDS-PAGE, and subjected to Western blotting using the PrP-specific antibody 6D11 (Covance). The figure illustrates the presence/absenceof PrPTSE signal in each of 4 sample aliquots from each mouse after five rounds of saPMCA. C57BL-I and C57BL-II, samples from uninfected wild-type mice usedas negative control; �PK, WT-MoBH not treated with PK. Molecular mass is shown on the right.

FIGURE 10. Detection of PrPTSE in brain tissue from presymptomatic andsymptomatic Mo-vCJD-infected C57BL mice. 10% mouse brain homoge-nates were treated with 20 �g/ml of proteinase K, resolved by SDS-PAGE, andsubjected to Western blotting using the PrP-specific antibody 6D11 (Cova-nce). Molecular mass is shown on the left.

TABLE 2Detection by saPMCA of PrPTSE in plasma EV preparations isolatedfrom Mo-vCJD-infected C57BL/6 (C57CL) mice, pre-symptomatic andclinical phaseThe presence of PrPTSE was tested after four to five saPMCA rounds in 4 aliquots of EVpreparations from individual plasma samples obtained at various post-inoculation timesfrom Mo-vCJD-infected C57BL mice. All infected mice had tested positive for PrPTSE inthe brain. Plasma samples from uninfected mice were used as negative controls(C57BL-I and C57BL-II). EVs were isolated from 200 or 250 �l of plasma by Exo-QuickTM (System Biosciences). EV pellets were mixed with 200 or 250 �l of 10% wild-type mouse brain homogenate, respectively, and split into 4 aliquots that were subjectedto saPMCA. Repeated rounds of saPMCA were performed. The presence of PrPTSE wasevaluated by Western blotting using the PrP-specific antibody 6D11 (Covance). Bold-face indicates plasma extracellular vesicle samples that tested positive for PrPTSE in all 4aliquots.

Bloodcollection time

post-inoculationMouse

ID

Round 4,aliquots

positive/total

Round 5,aliquots

positive/total

6 weeks 1 0/4 0/42 3/4 2/43 2/4 3/4

9 weeks 4 4/4 4/45 1/4 4/46 4/4 4/4

12 weeks 7 2/4 2/48 3/4 3/49 4/4 4/4

15 weeks 10 4/4 4/411 4/4 4/412 1/4 4/4

20 weeks 13 4/4 4/414 4/4 4/415 4/4 4/4

Clinical phase (�23 weeks) 16 4/4 4/417 4/4 4/4

Uninfected controls C57BL-I 0/4 0/4C57BL-II 0/4 0/4

Detection of PrPTSE in Exosomes from TSE-infected Mice

29256 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 42 • OCTOBER 17, 2014

at NO

RT

H D

AK

OT

A ST

AT

E U

NIV

on October 28, 2014

http://ww

w.jbc.org/

Dow

nloaded from

can reliably identify preclinical cases to minimize the risk ofiatrogenic disease transmission via blood-derived products.Experimental detection of PrPTSE in animal cerebrospinalfluid (CSF) (29), whole blood (21, 22), plasma (27–30, 71),buffy coat (24 –26), and urine (74) and in human CSF (12,75), whole blood (6 – 8), and urine (76, 77) has been achievedby different methods that rely on the concentration oramplification techniques to bring PrPTSE levels to the detec-tion threshold of biochemical assays. Although the presenceof PrPTSE in blood exosomes has been suggested previously(48), biochemical detection has been complicated by the lowlevels of PrPTSE in blood and the concomitantly large vol-umes required for exosome isolation by standard methods.Traditionally, exosome isolation has been achieved by aseries of differential centrifugation and filtration steps fromlarge sample volumes (68). This approach has been com-bined with saPMCA to demonstrate the presence of PrPTSE

in exosomes isolated from the conditioned media of cell cul-tures infected with M1000, a mouse-adapted sCJD strain(52). In the context of disease diagnosis using body fluids,limitations exist as to the sample volume available for eval-uation. In recent years, new reagents have become availablefor isolating exosomes from small volumes expected insuch clinical applications. Two of these reagents, Exo-QuickTM-TC and ExoQuickTM, have been repeatedly shownto successfully isolate exosomes from conditioned mediaand biological fluids for various downstream applications(78 – 82). By combining extracellular vesicle isolation usingExoQuickTM with PrPTSE amplification by saPMCA, we wereable to demonstrate the presence of PrPTSE in plasma sam-ples collected from preclinical and sick mice infected withMo-vCJD, and we showed for the first time its associationwith blood-circulating EVs. Biochemical characterization ofEV preparations obtained from plasma and conditionedmedia showed the presence of the exosomal marker Hsp70and the absence of the Golgi marker GM130, thus demon-strating the efficiency of ExoQuickTM reagents to purify EV-containing exosomes. Moreover, processing of EVs by ultra-centrifugation through a 30% sucrose cushion, coupled withsaPMCA amplification, allowed the detection of PrPTSE inEVs that were confirmed to carry the exosomal markerHsp70, further indicating the possible association of PrPTSE

with these vesicles. There are three types of EVs released bythe cell that differ in their biogenesis and that can be classi-fied as follows: (i) apoptotic vesicles (50 –5000 nm in diam-eter) released by apoptotic cells; (ii) exosomes (40 –200 nm)released upon fusion of multivesicular bodies with theplasma 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 toseparate them by the current isolation methods. In particularExoQuick isolates EVs ranging in size from 17 to 200 nm.Nevertheless, the demonstration of the exosomal markerHsp70 exclusively in the middle fraction, where exosomesfractionate following centrifugation in a 30% sucrose cush-ion, confirms the presence of these vesicles in our prepara-tions. The fact that PrPTSE was also identified after saPMCAin the top and bottom fraction where other EVs have been

detected by NTA suggests that it can associate with otherclasses of EVs, although this conclusion requires furtherinvestigation because, as mentioned above, the top and bot-tom fraction may contain lipoprotein-associated PrPTSE andPrPTSE aggregates, respectively, not associated with EVs.Interestingly, on the bases of the accumulated evidence thatPrPC is present at the exosomal membrane (46 –50, 53, 58,84 – 87), this protein is being used to characterize exosomalpreparations (88). Additionally, we demonstrated that EVisolation can be scaled down to volumes compatible withclinical applications. Two hundred microliters of plasmaprovided enough material to allow PrPTSE detection by saP-MCA in murine samples. The challenge will be to detectPrPTSE in similar volumes of human plasma.

Western blotting analysis of plasma EVs, isolated withExoQuickTM, revealed the presence of heavy and light chainsof IgG in our samples. These findings are in line with variousstudies that showed association of immunoglobulin lightchains (89) and various types and subtypes of heavy chainswith exosomes isolated from tumors and body fluids (89 –92). The presence of IgG in EVs isolated using ExoQuickTM

hindered the detection of PrPTSE in the initial rounds of saP-MCA by Western blotting due to a high nonspecific back-ground signal. Serial dilution/amplification significantlyreduced IgG, effectively eliminating the signal interferenceand revealing the presence of misfolded PrPTSE. Detection ofPrPTSE in plasma EVs after just one round of saPMCA wasachieved with a secondary antibody that does not recognizeheavy and light chains of IgG under reduced conditions.However, this antibody was only used in our confirmatoryassays due to practical reasons, i.e. high cost and low readingsignal in Western blotting. Additional methods to removeIgG from plasma sample preparations are currently underevaluation; we expect that these approaches will reduce thenumber of rounds required to detect PrPTSE. The number ofamplification rounds needed to identify PrPTSE variedbetween animals infected with the same TSE agent andbetween cell cultures infected with Mo-vCJD or Fukuoka-1.These findings suggest that variability exists between indi-vidual mice as far as the quantities of PrPTSE present inplasma at the terminal stages of disease as well as differencesin conversion efficiencies between TSE strains. However, wecannot exclude differences in the PrPTSE replication capacityof our cell models.

In summary, our experiments provide a valuable foundationfor the development of new diagnostics and potential targetsfor TSE treatment. Moreover, our data provide evidence thata fraction of blood-circulating PrPTSE co-localizes with exo-somes. This finding opens new avenues for further TSEresearch. Because exosomes are known to not only participatein cell to cell communication processes (34, 58, 59, 93), but toalso cross the blood-brain barrier (94, 95), association of PrPTSE

with exosomes may serve as a platform for TSE spread from theperiphery to the CNS (47, 56, 58). Additionally, we are furthercharacterizing PrPTSE-bearing microvesicles with the goal ofidentifying the cellular origin of this protein in blood. Thisinformation may advance the development of new strategies toensure the safety of blood- and plasma-derived products.

Detection of PrPTSE in Exosomes from TSE-infected Mice

OCTOBER 17, 2014 • VOLUME 289 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 29257

at NO

RT

H D

AK

OT

A ST

AT

E U

NIV

on October 28, 2014

http://ww

w.jbc.org/

Dow

nloaded from

Acknowledgments—We thank Dr. Paul Brown for providing aFukuoka-infected mouse brain and Dr. Moira Bruce for providing aMo-vCJD-infected mouse brain for the infectivity studies. We alsothank Donna Sobieski for editorial assistance, Anton Cervenak fortechnical support, and the American National Red Cross vivariumstaff for providing excellent animal care. We are grateful to Dr.Andrew Hill for critical reading of the manuscript and for helpfuldiscussions.

REFERENCES1. Brandel, J. P., Heath, C. A., Head, M. W., Levavasseur, E., Knight, R.,

Laplanche, J. L., Langeveld, J. P., Ironside, J. W., Hauw, J. J., Mackenzie, J.,Alpérovitch, A., Will, R. G., and Haïk, S. (2009) Variant Creutzfeldt-Jakobdisease in France and the United Kingdom: evidence for the same agentstrain. Ann. Neurol. 65, 249 –256

2. Hilton, D. A., Ghani, A. C., Conyers, L., Edwards, P., McCardle, L., Ritchie,D., Penney, M., Hegazy, D., and Ironside, J. W. (2004) Prevalence of lym-phoreticular prion protein accumulation in UK tissue samples. J. Pathol.203, 733–739

3. Gill, O. N., Spencer, Y., Richard-Loendt, A., Kelly, C., Dabaghian, R.,Boyes, L., Linehan, J., Simmons, M., Webb, P., Bellerby, P., Andrews, N.,Hilton, D. A., Ironside, J. W., Beck, J., Poulter, M., Mead, S., and Brandner,S. (2013) Prevalent abnormal prion protein in human appendixes afterbovine spongiform encephalopathy epizootic: large scale survey. BMJ 347,f5675

4. Knight, R. (2010) The risk of transmitting prion disease by blood or plasmaproducts. Transfus. Apher. Sci. 43, 387–391

5. Peden, A., McCardle, L., Head, M. W., Love, S., Ward, H. J., Cousens, S. N.,Keeling, D. M., Millar, C. M., Hill, F. G., and Ironside, J. W. (2010) VariantCJD infection in the spleen of a neurologically asymptomatic UK adultpatient with haemophilia. Haemophilia 16, 296 –304

6. Edgeworth, J. A., Farmer, M., Sicilia, A., Tavares, P., Beck, J., Campbell, T.,Lowe, J., Mead, S., Rudge, P., Collinge, J., and Jackson, G. S. (2011) Detec-tion of prion infection in variant Creutzfeldt-Jakob disease: a blood-basedassay. Lancet 377, 487– 493

7. Jackson, G. S., Burk-Rafel, J., Edgeworth, J. A., Sicilia, A., Abdilahi, S.,Korteweg, J., Mackey, J., Thomas, C., Wang, G., Mead, S., and Collinge, J.(2014) A highly specific blood test for vCJD. Blood 123, 452– 453

8. Jackson, G. S., Burk-Rafel, J., Edgeworth, J. A., Sicilia, A., Abdilahi, S.,Korteweg, J., Mackey, J., Thomas, C., Wang, G., Schott, J. M., Mummery,C., Chinnery, P. F., Mead, S., and Collinge, J. (2014) Population screeningfor variant Creutzfeldt-Jakob disease using a novel blood test: diagnosticaccuracy and feasibility study. JAMA Neurology 71, 421– 428

9. Wadsworth, J. D., Dalmau-Mena, I., Joiner, S., Linehan, J. M., O’Malley, C.,Powell, C., Brandner, S., Asante, E. A., Ironside, J. W., Hilton, D. A., andCollinge, J. (2011) Effect of fixation on brain and lymphoreticular vCJDprions and bioassay of key positive specimens from a retrospective vCJDprevalence study. J. Pathol. 223, 511–518

10. de Marco, M. F., Linehan, J., Gill, O. N., Clewley, J. P., and Brandner, S.(2010) Large-scale immunohistochemical examination for lymphoreticu-lar prion protein in tonsil specimens collected in Britain. J. Pathol. 222,380 –387

11. Douet, J. Y., Zafar, S., Perret-Liaudet, A., Lacroux, C., Lugan, S., Aron, N.,Cassard, H., Ponto, C., Corbière, F., Torres, J. M., Zerr, I., and Andreoletti,O. (2014) Detection of infectivity in blood of persons with variant andsporadic Creutzfeldt-Jakob disease. Emerg. Infect. Dis. 20, 114 –117

12. Rubenstein, R., and Chang, B. (2013) Re-assessment of PrP(Sc) distribu-tion in sporadic and variant CJD. PLoS One 8, e66352

13. Cervenakova, L., Brown, P., Soukharev, S., Yakovleva, O., Diringer, H.,Saenko, E. L., and Drohan, W. N. (2003) Failure of immunocompetitivecapillary electrophoresis assay to detect disease-specific prion protein inbuffy coat from humans and chimpanzees with Creutzfeldt-Jakob disease.Electrophoresis 24, 853– 859

14. Dorsey, K., Zou, S., Schonberger, L. B., Sullivan, M., Kessler, D., Notari, E.,4th, Fang, C. T., and Dodd, R. Y. (2009) Lack of evidence of transfusion

transmission of Creutzfeldt-Jakob disease in a US surveillance study.Transfusion 49, 977–984

15. Brown, P., Cervenáková, L., and Diringer, H. (2001) Blood infectivity andthe prospects for a diagnostic screening test in Creutzfeldt-Jakob disease.J. Lab. Clin. Med. 137, 5–13

16. Cervenakova, L., Yakovleva, O., McKenzie, C., Kolchinsky, S., McShane,L., Drohan, W. N., and Brown, P. (2003) Similar levels of infectivity in theblood of mice infected with human-derived vCJD and GSS strains oftransmissible spongiform encephalopathy. Transfusion 43, 1687–1694

17. Andréoletti, O., Litaise, C., Simmons, H., Corbière, F., Lugan, S., Costes, P.,Schelcher, F., Vilette, D., Grassi, J., and Lacroux, C. (2012) Highly efficientprion transmission by blood transfusion. PLoS Pathog. 8, e1002782

18. McCutcheon, S., Alejo Blanco, A. R., Houston, E. F., de Wolf, C., Tan, B. C.,Smith, A., Groschup, M. H., Hunter, N., Hornsey, V. S., MacGregor, I. R.,Prowse, C. V., Turner, M., and Manson, J. C. (2011) All clinically-relevantblood components transmit prion disease following a single blood trans-fusion: a sheep model of vCJD. PLoS One 6, e23169

19. Mathiason, C. K., Powers, J. G., Dahmes, S. J., Osborn, D. A., Miller, K. V.,Warren, R. J., Mason, G. L., Hays, S. A., Hayes-Klug, J., Seelig, D. M., Wild,M. A., Wolfe, L. L., Spraker, T. R., Miller, M. W., Sigurdson, C. J., Telling,G. C., and Hoover, E. A. (2006) Infectious prions in the saliva and blood ofdeer with chronic wasting disease. Science 314, 133–136

20. Hunter, N., Foster, J., Chong, A., McCutcheon, S., Parnham, D., Eaton, S.,MacKenzie, C., and Houston, F. (2002) Transmission of prion diseases byblood transfusion. J. Gen. Virol. 83, 2897–2905

21. Tattum, M. H., Jones, S., Pal, S., Collinge, J., and Jackson, G. S. (2010)Discrimination between prion-infected and normal blood samples by pro-tein misfolding cyclic amplification. Transfusion 50, 996 –1002

22. Terry, L. A., Howells, L., Hawthorn, J., Edwards, J. C., Moore, S. J., Bell-worthy, S. J., Simmons, H., Lizano, S., Estey, L., Leathers, V., and Everest,S. J. (2009) Detection of PrPsc in blood from sheep infected with thescrapie and bovine spongiform encephalopathy agents. J. Virol. 83,12552–12558

23. Elder, A. M., Henderson, D. M., Nalls, A. V., Wilham, J. M., Caughey,B. W., Hoover, E. A., Kincaid, A. E., Bartz, J. C., and Mathiason, C. K.(2013)In vitro detection of prionemia in TSE-infected cervids and ham-sters. PLoS One 8, e80203

24. Castilla, J., Saá, P., and Soto, C. (2005) Detection of prions in blood. Nat.Med. 11, 982–985

25. Saá, P., Castilla, J., and Soto, C. (2006) Presymptomatic detection of prionsin blood. Science 313, 92–94

26. Thorne, L., and Terry, L. A. (2008) In vitro amplification of PrPSc derivedfrom the brain and blood of sheep infected with scrapie. J. Gen. Virol. 89,3177–3184

27. Vascellari, S., Orrù, C. D., Hughson, A. G., King, D., Barron, R., Wilham,J. M., Baron, G. S., Race, B., Pani, A., and Caughey, B. (2012) Prion seedingactivities of mouse scrapie strains with divergent PrPSc protease sensitiv-ities and amyloid plaque content using RT-QuIC and eQuIC. PLoS One 7,e48969

28. Rubenstein, R., Chang, B., Gray, P., Piltch, M., Bulgin, M. S., Sorensen-Melson, S., and Miller, M. W. (2010) A novel method for preclinical de-tection of PrPSc in blood. J. Gen. Virol. 91, 1883–1892

29. Orrú, C. D., Wilham, J. M., Raymond, L. D., Kuhn, F., Schroeder, B., Rae-ber, A. J., and Caughey, B. (2011) Prion disease blood test using immuno-precipitation and improved quaking-induced conversion. mBio 2,e00078 – e00011

30. Bannach, O., Birkmann, E., Reinartz, E., Jaeger, K. E., Langeveld, J. P.,Rohwer, R. G., Gregori, L., Terry, L. A., Willbold, D., and Riesner, D. (2012)Detection of prion protein particles in blood plasma of scrapie infectedsheep. PLoS One 7, e36620

31. Pan, B.-T., and Johnstone, R. M. (1983) Fate of the transferrin receptorduring maturation of sheep reticulocytes in vitro: Selective externalizationof the receptor. Cell 33, 967–978

32. Pan, B. T., and Johnstone, R. (1984) Selective externalization of the trans-ferrin receptor by sheep reticulocytes in vitro. Response to ligands andinhibitors of endocytosis. J. Biol. Chem. 259, 9776 –9782

33. Johnstone, R. M., Adam, M., Hammond, J. R., Orr, L., and Turbide, C.(1987) Vesicle formation during reticulocyte maturation. Association of

Detection of PrPTSE in Exosomes from TSE-infected Mice

29258 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 42 • OCTOBER 17, 2014

at NO

RT

H D

AK

OT

A ST

AT

E U

NIV

on October 28, 2014

http://ww

w.jbc.org/

Dow

nloaded from

plasma membrane activities with released vesicles (exosomes). J. Biol.Chem. 262, 9412–9420

34. Valadi, H., Ekström, K., Bossios, A., Sjöstrand, M., Lee, J. J., and Lötvall,J. O. (2007) Exosome-mediated transfer of mRNAs and microRNAs is anovel mechanism of genetic exchange between cells. Nat. Cell Biol. 9,654 – 659

35. Zitvogel, L., Regnault, A., Lozier, A., Wolfers, J., Flament, C., Tenza, D.,Ricciardi-Castagnoli, P., Raposo, G., Amigorena, S. (1998) Eradication ofestablished murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes. Nat. Med. 4, 594 – 600

36. Raposo, G., Nijman, H. W., Stoorvogel, W., Liejendekker, R., Harding,C. V., Melief, C. J., and Geuze, H. J. (1996) B lymphocytes secrete antigen-presenting vesicles. J. Exp. Med. 183, 1161–1172

37. Vlassov, A. V., Magdaleno, S., Setterquist, R., and Conrad, R. (2012) Exo-somes: current knowledge of their composition, biological functions, anddiagnostic and therapeutic potentials. Biochim. Biophys. Acta 1820,940 –948

38. Nguyen, D. G., Booth, A., Gould, S. J., and Hildreth, J. E. (2003) Evidencethat HIV budding in primary macrophages occurs through the exosomerelease pathway. J. Biol. Chem. 278, 52347–52354

39. Gould, S. J., Booth, A. M., and Hildreth, J. E. (2003) The Trojan exosomehypothesis. Proc. Natl. Acad. Sci. U.S.A. 100, 10592–10597

40. Schneider, A., and Simons, M. (2013) Exosomes: vesicular carriers forintercellular communication in neurodegenerative disorders. Cell TissueRes. 352, 33– 47

41. Tasaki, M., Ueda, M., Ochiai, S., Tanabe, Y., Murata, S., Misumi, Y., Su, Y.,Sun, X., Shinriki, S., Jono, H., Shono, M., Obayashi, K., and Ando, Y. (2010)Transmission of circulating cell-free AA amyloid oligomers in exosomesvectors via a prion-like mechanism. Biochem. Biophys. Res. Commun. 400,559 –562

42. Vingtdeux, V., Sergeant, N., and Buee, L. (2012) Potential contribution ofexosomes to the prion-like propagation of lesions in Alzheimer’s disease.Front. Physiol. 3, 1–16

43. Rajendran, L., Honsho, M., Zahn, T. R., Keller, P., Geiger, K. D., Verkade,P., and Simons, K. (2006) Alzheimer’s disease �-amyloid peptides are re-leased in association with exosomes. Proc. Natl. Acad. Sci. U.S.A. 103,11172–11177

44. Surgucheva, I., Sharov, V. S., and Surguchov, A. (2012) �-Synuclein: seed-ing of �-synuclein aggregation and transmission between cells. Biochem-istry 51, 4743– 4754

45. Saman, S., Kim, W., Raya, M., Visnick, Y., Miro, S., Saman, S., Jackson, B.,McKee, A. C., Alvarez, V. E., Lee, N. C., and Hall, G. F. (2012) Exosome-associated Tau is secreted in tauopathy models and is selectively phosphor-ylated in cerebrospinal fluid in early Alzheimer disease. J. Biol. Chem. 287,3842–3849

46. Ecroyd, H., Sarradin, P., Dacheux, J.-L., and Gatti, J.-L. (2004) Compart-mentalization of prion isoforms within the reproductive tract of the ram.Biol. Reprod. 71, 993–1001

47. Fevrier, B., Vilette, D., Archer, F., Loew, D., Faigle, W., Vidal, M., Laude,H., and Raposo, G. (2004) Cells release prions in association with exo-somes. Proc. Natl. Acad. Sci. U.S.A. 101, 9683–9688

48. Robertson, C., Booth, S. A., Beniac, D. R., Coulthart, M. B., Booth, T. F.,and McNicol, A. (2006) Cellular prion protein is released on exosomesfrom activated platelets. Blood 107, 3907–3911

49. Vella, L. J., Sharples, R. A., Lawson, V. A., Masters, C. L., Cappai, R., andHill, A. F. (2007) Packaging of prions into exosomes is associated with anovel pathway of PrP processing. J. Pathol. 211, 582–590

50. Alais, S., Simoes, S., Baas, D., Lehmann, S., Raposo, G., Darlix, J. L., andLeblanc, P. (2008) Mouse neuroblastoma cells release prion infectivityassociated with exosomal vesicles. Biol. Cell 100, 603– 615

51. Castro-Seoane, R., Hummerich, H., Sweeting, T., Tattum, M. H., Linehan,J. M., Fernandez de Marco, M., Brandner, S., Collinge, J., and Klöhn, P.-C.(2012) Plasmacytoid dendritic cells sequester high prion titres at earlystages of prion infection. PLoS Pathog. 8, e1002538

52. Coleman, B. M., Hanssen, E., Lawson, V. A., and Hill, A. F. (2012) Prion-infected cells regulate the release of exosomes with distinct ultrastructuralfeatures. FASEB J. 26, 4160 – 4173

53. Wik, L., Klingeborn, M., Willander, H., and Linne, T. (2012) Separate

mechanisms act concurrently to shed and release the prion protein fromthe cell. Prion 6, 498 –509

54. Veith, N. M., Plattner, H., Stuermer, C. A., Schulz-Schaeffer, W. J., andBürkle, A. (2009) Immunolocalisation of PrPSc in scrapie-infected N2amouse neuroblastoma cells by light and electron microscopy. Eur. J. CellBiol. 88, 45– 63

55. Mattei, V., Barenco, M. G., Tasciotti, V., Garofalo, T., Longo, A., Boller, K.,Löwer, J., Misasi, R., Montrasio, F., and Sorice, M. (2009) Paracrine diffu-sion of PrPC and propagation of prion infectivity by plasma membrane-derived microvesicles. PLoS One 4, e5057

56. Simák, J., Holada, K., D’Agnillo, F., Janota, J., and Vostal, J. G. (2002)Cellular prion protein is expressed on endothelial cells and is releasedduring apoptosis on membrane microparticles found in human plasma.Transfusion 42, 334 –342

57. Brouckova, A., Holada, K. (2009) Cellular prion protein in blood plateletsassociates with both lipid rafts and the cytoskeleton. Thromb. Haemost.102, 966 –974

58. Février, B., Vilette, D., Laude, H., and Raposo, G. (2005) Exosomes: abubble ride for prions? Traffic 6, 10 –17

59. Skog, J., Würdinger, T., van Rijn, S., Meijer, D. H., Gainche, L., Sena-Esteves, M., Curry, W. T., Jr., Carter, B. S., Krichevsky, A. M., and Breake-field, X. O. (2008) Glioblastoma microvesicles transport RNA and pro-teins that promote tumour growth and provide diagnostic biomarkers.Nat. Cell Biol. 10, 1470 –1476

60. Kalra, H., Adda, C. G., Liem, M., Ang, C.-S., Mechler, A., Simpson, R. J.,Hulett, M. D., and Mathivanan, S. (2013) Comparative proteomics evalu-ation of plasma exosome isolation techniques and assessment of the sta-bility of exosomes in normal human blood plasma. Proteomics 13,3354 –3364

61. Cheng, L., Sharples, R. A., Scicluna, B. J., and Hill, A. F. (2014) Exosomesprovide a protective and enriched source of miRNA for biomarker profil-ing compared to intracellular and cell-free blood. J. Extracell. Vesicles 3,23743

62. Brown, P., Cervenáková, L., McShane, L. M., Barber, P., Rubenstein, R.,and Drohan, W. N. (1999) Further studies of blood infectivity in an exper-imental model of transmissible spongiform encephalopathy, with an ex-planation of why blood components do not transmit Creutzfeldt-Jakobdisease in humans. Transfusion 39, 1169 –1178

63. Brown, P., Rohwer, R. G., Dunstan, B. C., MacAuley, C., Gajdusek, D. C.,and Drohan, W. N. (1998) The distribution of infectivity in blood compo-nents and plasma derivatives in experimental models of transmissiblespongiform encephalopathy. Transfusion 38, 810 – 816

64. Reed, L. J., and Muench, H. (1938) A simple method of estimating fifty percent endpoints. Am. J. Epidemiol. 27, 493– 497

65. Cervenakova, L., Akimov, S., Vasilyeva, I., Yakovleva, O., McKenzie, C.,Cervenak, J., Piccardo, P., and Asher, D. M. (2011) Fukuoka-1 strain oftransmissible spongiform encephalopathy agent infects murine bone mar-row-derived cells with features of mesenchymal stem cells. Transfusion51, 1755–1768

66. Akimov, S., Yakovleva, O., Vasilyeva, I., McKenzie, C., and Cervenakova,L. (2008) Persistent propagation of variant Creutzfeldt-Jakob diseaseagent in murine spleen stromal cell culture with features of mesenchymalstem cells. J. Virol. 82, 10959 –10962

67. Akimov, S., Vasilyeva, I., Yakovleva, O., McKenzie, C., and Cervenakova,L. (2009) Murine bone marrow stromal cell culture with features of mes-enchymal stem cells susceptible to mouse-adapted human TSE agent,Fukuoka-1. Folia Neuropathol. 47, 205–214

68. Thery, C., Clayton, A., Amogorena, S., and Raposo, G. (2006) CurrentProtocols in Cell Biology, pp. 3.22.1–3.22.29, John Wiley & Sons, Inc., NewYork

69. Castilla, J., Saá, P., Hetz, C., and Soto, C. (2005) In vitro generation ofinfectious scrapie prions. Cell 121, 195–206

70. Saá, P., Castilla, J., and Soto, C. (2006) Ultra-efficient replication of infec-tious prions by automated protein misfolding cyclic amplification. J. Biol.Chem. 281, 35245–35252

71. Fujihara, A., Atarashi, R., Fuse, T., Ubagai, K., Nakagaki, T., Yamaguchi,N., Ishibashi, D., Katamine, S., and Nishida, N. (2009) Hyperefficient PrPSc amplification of mouse-adapted BSE and scrapie strain by protein mis-

Detection of PrPTSE in Exosomes from TSE-infected Mice

OCTOBER 17, 2014 • VOLUME 289 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 29259

at NO

RT

H D

AK

OT

A ST

AT

E U

NIV

on October 28, 2014

http://ww

w.jbc.org/

Dow

nloaded from

folding cyclic amplification technique. FEBS J. 276, 2841–284872. Safar, J. G., Wille, H., Geschwind, M. D., Deering, C., Latawiec, D., Serban,

A., King, D. J., Legname, G., Weisgraber, K. H., Mahley, R. W., Miller, B. L.,Dearmond, S. J., and Prusiner, S. B. (2006) Human prions and plasmalipoproteins. Proc. Natl. Acad. Sci. U.S.A. 103, 11312–11317

73. Dragovic, R. A., Gardiner, C., Brooks, A. S., Tannetta, D. S., Ferguson, D. J.,Hole, P., Carr, B., Redman, C. W., Harris, A. L., Dobson, P. J., Harrison, P.,and Sargent, I. L. (2011) Sizing and phenotyping of cellular vesicles usingnanoparticle tracking analysis. Nanomedicine 7, 780 –788

74. Rubenstein, R., Chang, B., Gray, P., Piltch, M., Bulgin, M. S., Sorensen-Melson, S., and Miller, M. W. (2011) Prion disease detection, PMCA ki-netics, and IgG in urine from sheep naturally/experimentally infected withscrapie and deer with preclinical/clinical chronic wasting disease. J. Virol.85, 9031–9038

75. Atarashi, R., Satoh, K., Sano, K., Fuse, T., Yamaguchi, N., Ishibashi, D.,Matsubara, T., Nakagaki, T., Yamanaka, H., Shirabe, S., Yamada, M., Miz-usawa, H., Kitamoto, T., Klug, G., McGlade, A., Collins, S. J., and Nishida,N. (2011) Ultrasensitive human prion detection in cerebrospinal fluid byreal-time quaking-induced conversion. Nat. Med. 17, 175–178

76. Dabaghian, R., Zerr, I., Heinemann, U., and Zanusso, G. (2008) Detection ofproteinase K-resistant proteins in the urine of patients with Creutzfeldt-Jakoband other neurodegenerative diseases. Prion 2, 170–178

77. Gonzalez-Romero, D., Barria, M. A., Leon, P., Morales, R., and Soto, C.(2008) Detection of infectious prions in urine. FEBS Lett. 582, 3161–3166

78. Tadokoro, H., Umezu, T., Ohyashiki, K., Hirano, T., and Ohyashiki, J. H.(2013) Exosomes derived from hypoxic leukemia cells enhance tube for-mation in endothelial cells. J Biol. Chem. 288, 34343–34351

79. Huang, X., Yuan, T., Tschannen, M., Sun, Z., Jacob, H., Du, M., Liang, M.,Dittmar, R. L., Liu, Y., Liang, M., Kohli, M., Thibodeau, S. N., Boardman,L., and Wang, L. (2013) Characterization of human plasma-derived exo-somal RNAs by deep sequencing. BMC Genomics 14, 319

80. Taylor, D., Zacharias, W., and Gercel-Taylor, C. (2011) in Serum/PlasmaProteomics (Simpson, R. J., and Greening, D. W., eds) pp. 235–246, Hu-mana Press Inc., Totowa, NJ

81. Epple, L. M., Griffiths, S. G., Dechkovskaia, A. M., Dusto, N. L., White, J.,Ouellette, R. J., Anchordoquy, T. J., Bemis, L. T., and Graner, M. W. (2012)Medulloblastoma exosome proteomics yield functional roles for extracel-lular vesicles. PLoS ONE 7, e42064

82. Haney, M. J., Suresh, P., Zhao, Y., Kanmogne, G. D., Kadiu, I., Sokolsky-Papkov, M., Klyachko, N. L., Mosley, R. L., Kabanov, A. V., Gendelman,H. E., and Batrakova, E. V. (2012) Blood-borne macrophage–neural cellinteractions hitchhike on endosome networks for cell-based nanozymebrain delivery. Nanomedicine 7, 815– 833

83. Cocucci, E., Racchetti, G., and Meldolesi, J. (2009) Shedding microvesicles:

artefacts no more. Trends Cell Biol. 19, 43–5184. Porto-Carreiro, I., Février, B., Paquet, S., Vilette, D., and Raposo, G. (2005)

Prions and exosomes: from PrPc trafficking to PrPsc propagation. BloodCells Mol. Dis. 35, 143–148

85. Vella, L. J., Sharples, R. A., Nisbet, R. M., Cappai, R., and Hill, A. F. (2008)The role of exosomes in the processing of proteins associated with neu-rodegenerative diseases. Eur. Biophys. J. 37, 323–332

86. Vella, L. J., Greenwood, D. L., Cappai, R., Scheerlinck, J. P., and Hill, A. F.(2008) Enrichment of prion protein in exosomes derived from ovine cer-ebral spinal fluid. Vet. Immunol. Immunopathol. 124, 385–393

87. Ritchie, A. J., Crawford, D. M., Ferguson, D. J., Burthem, J., and Roberts,D. J. (2013) Normal prion protein is expressed on exosomes isolated fromhuman plasma. Br. J. Haematol. 163, 678 – 680

88. Silva, C. J., Dynin, I., Erickson, M. L., Requena, J. R., Balachandran, A., Hui,C., Onisko, B. C., and Carter, J. M. (2013) Oxidation of methionine 216 insheep and elk prion protein is highly dependent upon the amino acid atposition 218 but is not important for prion propagation. Biochemistry 52,2139 –2147

89. Gonzalez-Begne, M., Lu, B., Han, X., Hagen, F. K., Hand, A. R., Melvin,J. E., and Yates, J. R. (2009) Proteomic analysis of human parotid glandexosomes by multidimensional protein identification technology (MudPIT).J. Proteome Res. 8, 1304–1314

90. Admyre, C., Johansson, S. M., Qazi, K. R., Filén, J.-J., Lahesmaa, R., Nor-man, M., Neve, E. P., Scheynius, A., and Gabrielsson, S. (2007) Exosomeswith immune modulatory features are present in human breast milk. J. Im-munol. 179, 1969 –1978

91. Mathivanan, S., Lim, J. W., Tauro, B. J., Ji, H., Moritz, R. L., and Simpson,R. J. (2010) Proteomics analysis of A33 immunoaffinity-purified exosomesreleased from the human colon tumor cell line LIM1215 reveals a tissue-specific protein signature. Mol. Cell. Proteomics 9, 197–208

92. Bard, M. P., Hegmans, J. P., Hemmes, A., Luider, T. M., Willemsen, R.,Severijnen, L.-A., van Meerbeeck, J. P., Burgers, S. A., Hoogsteden, H. C.,and Lambrecht, B. N. (2004) Proteomic analysis of exosomes isolated fromhuman malignant pleural effusions. Am. J. Respir. Cell Mol. Biol. 31,114 –121

93. Stoorvogel, W., Kleijmeer, M. J., Geuze, H. J., and Raposo, G. (2002) Thebiogenesis and functions of exosomes. Traffic 3, 321–330

94. Kalani, A., Tyagi, A., and Tyagi, N. (2014) Exosomes: mediators of neuro-degeneration, neuroprotection and therapeutics. Mol. Neurobiol. 49,590 – 600

95. Sun, D., Zhuang, X., Xiang, X., Liu, Y., Zhang, S., Liu, C., Barnes, S., Griz-zle, W., Miller, D., and Zhang, H.-G. (2010) A novel nanoparticle drugdelivery system: the anti-inflammatory activity of curcumin is enhancedwhen encapsulated in exosomes. Mol. Ther. 18, 1606 –1614

Detection of PrPTSE in Exosomes from TSE-infected Mice

29260 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 42 • OCTOBER 17, 2014

at NO

RT

H D

AK

OT

A ST

AT

E U

NIV

on October 28, 2014

http://ww

w.jbc.org/

Dow

nloaded from