Prion Propagation and Toxicity Occur In Vitro with Two-PhaseKinetics Specific to Strain and Neuronal Type

Samia Hannaoui,a,b,c,d Layal Maatouk,a,b,c,d Nicolas Privat,a,b,c,d Etienne Levavasseur,a,b,c,d Baptiste A. Faucheux,a,b,c,d,e

Stéphane Haïka,b,c,d,e

Université Pierre et Marie Curie-Paris 6, Centre de Recherche de l’Institut du Cerveau et de la Moelle Épinière (CRICM), UMRS 975, Equipe Alzheimer’s and Prion Diseases,Paris, Francea; INSERM, U 975, Paris, Franceb; CNRS, UMR 7225, Paris, Francec; Centre National de Référence des Agents Transmissibles Non Conventionnels, Hôpital de laSalpêtrière, Paris, Franced; AP-HP, Laboratoire de Neuropathologie, Hôpital de la Salpêtrière, Paris, Francee

Prion diseases, or transmissible spongiform encephalopathies (TSEs), are fatal neurodegenerative disorders that occur in hu-mans and animals. The neuropathological hallmarks of TSEs are spongiosis, glial proliferation, and neuronal loss. The onlyknown specific molecular marker of TSEs is the abnormal isoform (PrPSc) of the host-encoded prion protein (PrPC), which accu-mulates in the brain of infected subjects and forms infectious prion particles. Although this transmissible agent lacks a specificnucleic acid component, several prion strains have been isolated. Prion strains are characterized by differences in disease out-come, PrPSc distribution patterns, and brain lesion profiles at the terminal stage of the disease. The molecular factors and cellu-lar mechanisms involved in strain-specific neuronal tropism and toxicity remain largely unknown. Currently, no cellular modelexists to facilitate in vitro studies of these processes. A few cultured cell lines that maintain persistent scrapie infections havebeen developed, but only two of them have shown the cytotoxic effects associated with prion propagation. In this study, we havedeveloped primary neuronal cultures to assess in vitro neuronal tropism and toxicity of different prion strains (scrapie strains139A, ME7, and 22L). We have tested primary neuronal cultures enriched in cerebellar granular, striatal, or cortical neurons.Our results showed that (i) a strain-specific neuronal tropism operated in vitro; (ii) the cytotoxic effect varied among strains andneuronal cell types; (iii) prion propagation and toxicity occurred in two kinetic phases, a replicative phase followed by a toxicphase; and (iv) neurotoxicity peaked when abnormal PrP accumulation reached a plateau.

Prion diseases, or transmissible spongiform encephalopathies(TSEs), are fatal neurodegenerative disorders that include

Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiformencephalopathy in cattle, chronic wasting disease in deer, andscrapie in sheep. They cause central nervous system damage char-acterized by neuronal vacuolation, reactive gliosis, and neuronalcell death. Prion diseases are marked by the accumulation of amisfolded isoform of the host-encoded prion protein. The cellularprion protein (PrPC) is a membrane-bound, sialoglycoprotein ex-pressed primarily in neurons but also found in other cell types,including astrocytes (1). The misfolded prion protein (PrPSc) re-sults from the conformational transition of PrPC into a beta-sheet-enriched form. Compared to PrPC, PrPSc is prone to aggre-gation and partially resistant to proteases, like proteinase K (PK)(1). PK digestion of PrPSc generates an amino-terminally trun-cated fragment called PrPres. According to the “protein only” hy-pothesis, the transmissible agent is thought to be composed solelyof PrPSc and propagates through a PrP conversion process (2, 3).

Despite the absence of a specific nucleic acid genome, variousprion strains with specific biological properties have been identi-fied from different, experimentally transmitted animal isolates(4). These strains, after serial passages in the same host, are phe-notypically stable and retain their properties when transmitted toanother species. Prion strains are differentiated by the incubationtime, the clinical signs of the disease, the localization of lesions(lesion profile), and PrPSc accumulation in specific brain struc-tures (5, 6). Lesion profiling has been extensively used to charac-terize prion strains and contributed to the identification of thebovine agent as the strain responsible for variant Creutzfeldt-Ja-kob epidemics in humans (6–9).

Neuronal vacuolation and neuronal death in a given brain

structure imply two distinct phenomena: the propagation of pri-ons, which typically leads to PrPSc accumulation, and the involve-ment of a neurotoxic process (10). It remains largely unknownwhether these processes are dissociated and how their kineticsvary among different strains (5). Interestingly, very recent in vivodata have suggested that they may be uncoupled; i.e., PrPSc accu-mulation alone is not neurotoxic, but it can lead to the formationof toxic PrP species by a separate but linked pathway (11). In thatstudy, the production of neurotoxic species may be triggeredwhen prion replication reached its maximum level. However, sev-eral transmission studies have shown that significant pathologyand/or clinical dysfunction developed with little or undetectablePrPSc accumulation (12–14). This suggests that PrPSc is not theprimary effector of prion-induced neurodegeneration. Otherstudies that related PrP deposits and neuronal loss in the brains ofpatients with sporadic CJD are consistent with the view that vari-ous molecular PrP species with distinct toxic properties may beproduced in the terminal phase of the disease (15, 16).

The present study aimed to mimic strain-specific neuronal tro-pism and to explore the relationship between prion propagationand toxicity in a cellular system. We took advantage of the abilityof primary neuronal cultures to propagate different prion strains.

Received 5 November 2012 Accepted 7 December 2012

Published ahead of print 19 December 2012

Address correspondence to Stéphane Haïk, stephane.haik@upmc.fr.

L.M. and N.P. have equal contributions.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.03082-12

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In this model, prion propagation can be monitored by PrPSc ac-cumulation and leads to neuronal loss (17–19). We compared invitro prion propagation and neurotoxicity of three differentscrapie strains (139A, ME7, and 22L) that had been stabilized inC57BL/6 mice and had demonstrated distinct lesion profiles (20).Moreover, stereotaxic inoculations of C57BL/6 mice had clearlydemonstrated that the cerebellum was selectively vulnerable to the22L strain compared to the 139A and ME7 strains (20). We stud-ied the behavior of these strains in three different primary neuro-nal cultures (cerebellar, cortical, and striatal cell cultures) pre-pared from C57BL/6 mice. Our results demonstrated that (i) theselective cerebellar vulnerability to 22L strain propagation couldbe reproduced in vitro, (ii) the toxicity associated with in vitroprion propagation varied in a strain- and neuronal type-specificmanner, (iii) prion-induced cell damage occurred in two kineticphases (a replicative phase followed by a toxic phase), and (iv)maximal neuronal loss occurred when PrPres accumulationreached a plateau (toxic phase). Interestingly, for a given strain,the toxic phase occurred differently in the three neuronal models.

MATERIALS AND METHODSPrimary cell cultures. Cerebellar granule neurons (CGN) were mechan-ically extracted from the cerebella of 6- to 7-day-old C57BL/6 mice (RenéJanvier, Le Genest-St-Isle, France) and enzymatically dissociated, as pre-viously described (19). Cells were plated at a density of 1.9 � 103 cells/mm2 on plastic culture wells precoated with 10 �g/ml poly-D-lysine(PDL) (Sigma-Aldrich, St. Quentin Fallavier, France). Cells were culturedin Dulbecco’s modified Eagle’s medium-Glutamax I high glucose(DMEM) (Life Technologies-Gibco, Villebon sur Yvette, France) supple-mented with penicillin and streptomycin (Life Technologies), 10% fetalcalf serum (Life Technologies), 20 mM KCl (Sigma-Aldrich), and N2 andB27 supplements (Life Technologies). Cells were incubated at 37°C in ahumidified 5% CO2 atmosphere. Every week, the medium was supple-mented with glucose (1 mg/ml); in addition, the antimitotics uridine andfluorodeoxyuridine (10 �M) (Sigma-Aldrich) were added to reduce as-trocyte proliferation.

Striatal and cortical neurons were obtained by dissecting 15-day-oldembryos extracted from pregnant C57BL/6 mice (21, 22). Briefly, embry-onic brains were removed and transferred into phosphate-buffered saline(PBS) supplemented with glucose. Striata or cortices were dissected andthen dissociated mechanically and enzymatically. After decanting for 5min, cells were collected by centrifugation at 115 � g (5417R rotor; Ep-pendorf) for 7 min at 4°C. Cell pellets were resuspended in DMEM sup-plemented with penicillin and streptomycin, 3% fetal calf serum, and N2and B27 supplements and then plated onto plastic culture wells precoatedwith PDL. Cells were cultured at 37°C in a humidified atmosphere with5% CO2. After 2 days, the culture medium was replaced with serum-freeDMEM supplemented with N2 and B27.

Prior to the infection, cerebellar granule cultures comprised 95% neu-rons and 5% astrocytes, striatal cultures comprised 97% neurons and 3%astrocytes, and cortical cultures comprised 94% neurons and 6% astro-cytes. None of these cultures showed detectable microglial cells.

Prion infection. Prion infection sources were prepared from thebrains of terminally ill C57BL/6 mice that had been inoculated with the139A, ME7, or 22L scrapie strain and showed comparable levels of PrPres

(Fig. 1). Prion-infected or normal brain homogenates were added to pri-mary neuronal cultures, as described previously (19). Briefly, brain ho-mogenates were sonicated and added at a final concentration of 0.01% toprimary cultures 48 h after plating. Four days later, the medium was re-moved from the cultures, and cells were washed twice in fresh culturemedium. Fresh medium was then added, and no medium changes wereperformed for the remaining experiments.

PrPres detection. Brain tissue homogenates were prepared from thebrains of terminally ill C57BL/6 mice that had been inoculated with the

139A, ME7, or 22L scrapie strain. Brain tissue was homogenized at 20%(wt/vol) in 5% glucose with a ribolyser (FastPrep-24; MP Biomedicals,Illkirch, France) by one run for 45 s at 6.5 m/s. Samples were treated byusing solutions A and B from TeSeE kits, according to the procedures ofthe manufacturer (Bio-Rad, Marnes-la-Coquette, France), and submittedto proteinase K treatment (200 �g/ml for 10 min at 37°C). After centrif-ugation, pellets were resuspended in loading buffer and heated at 100°Cfor 5 min.

On different days postexposure (dpe), cells were washed twice withPBS and then incubated in lysis buffer (50 mM Tris-HCl [pH 7.4], 0.5%Triton X-100, 0.5% sodium deoxycholate) (Sigma-Aldrich) for 10 min at4°C. The protein concentration of each cell lysate was measured with thebicinchoninic acid (BCA) protein assay (ThermoFisher Scientific-Pierce,Brebières, France). Next, as previously described (19), 50 �g of proteinwas digested with 5 �g/ml of PK (Promega, Charbonnières, France) for 30min at 37°C, and the reaction was stopped by adding 10 mM phenylmeth-ylsulfonyl fluoride (PMSF) to the mixture. Proteins were precipitated bythe addition of methanol to the samples for 1 h at �20°C. The sampleswere then centrifuged at 16,000 � g (5417R rotor; Eppendorf) for 20 min.Pellets were resuspended in loading buffer and heated at 100°C for 5 min.

Protein extracts (from the brain tissues and the cell lysates) wereloaded onto precast 4 to 12% Bis-Tris gels (Invitrogen). The separatedproteins were transferred onto nitrocellulose membranes (Whatman,Fontenay-sous-Bois, France). Prion proteins were first immunostainedwith anti-PrP monoclonal antibody (MAb) 6D11 (1/5,000; EurogentecFrance, Angers, France) and anti-mouse Ig secondary antibody (GEHealthcare Europe GmbH, Saclay, France).

Labeled proteins from the cell lysate were detected with the ECLchemiluminescence kit (GE Healthcare Europe GmbH). PrPres levels weresemiquantified with a GS-800 calibrated densitometer and dedicatedQuantity One software (Bio-Rad Laboratories, Marnes-la-Coquette,France), as described previously (23, 24). Values for the three PrPSc gly-coforms were measured on blots with unsaturated signals and summed.For each experiment, we studied the kinetics of PrPres accumulation dur-ing infection by sampling at different time points. Four independent ex-periments performed in duplicate were analyzed.

Immunofluorescence experiments. Cells grown on PDL-coated glasscoverslips and exposed to brain homogenates were washed in PBS andfixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hat-field, PA) for 10 min at room temperature (RT). Fixed cells were perme-abilized with 0.1% Triton X-100 for 5 min at RT and treated with 3 Mguanidine thiocyanate (Sigma-Aldrich) for 5 min at RT to expose aggre-gated forms of PrP (25). PrP was detected with MAb Sha31 (1/1,000;SpiBio, Massy, France). Neuronal cells were labeled with anti-microtu-bule-associated protein (MAP2) MAb (1/500; Sigma-Aldrich), astrocytes

FIG 1 Detection of PrPres in brain homogenates used as inocula in cell infec-tion experiments. Brain homogenates from C57BL/6 mice infected with the139A, ME7, and 22L scrapie strains at the terminal stage of the disease weredigested with proteinase K, and PrPres was detected by using monoclonal an-tibody 6D11.

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were labeled with anti-glial fibrillary acidic protein (GFAP) polyclonalantibody (pAb) (1/500; Dako, Trappes, France) and microglial cells werelabeled by using two different microglial-specific markers: an anti-ionizedcalcium binding adaptor molecule 1 (Iba1) pAb (1/800; Wako Chemicals,Neuss, Germany) and an anti-macrophage antigen 1 (MAC-1) MAb (Se-rotec, Colmar, France). The sensitivity and specificity of these markerswere confirmed by using a pure microglia primary culture as a positivecontrol. Tetramethyl rhodamine-isothiocyanate (TRITC) or Alexa-con-jugated secondary antibodies were added to the appropriate primary an-tibodies. Cells were mounted in Prolong Gold antifade reagent with 4=,6-diamidino-2-phenylindole (DAPI) (Life Technologies-Invitrogen).Immunolabeling was detected with a Zeiss Axiovert 200 epifluorescencemicroscope and a 40� lens; images were acquired with a cooled digitalcamera (CoolSnap HQ; Roper Scientific Princeton Instrument) andMetaview software using the same gain values for all culture conditions.Three independent experiments were performed, and a total of 30 imageswere captured under each condition, with approximately 50 cells per im-age.

Double immunolabeling was performed for subcellular localization ofPrP aggregates with various cell compartment markers (Abcam, Paris,France), including anti-LAMP2 pAb for lysosomal membranes, anti-ca-thepsin D pAb for lysosome lumens, anti-GRP78/BiP pAb for endoplas-mic reticulum, anti-giantin pAb for the Golgi apparatus, and anti-EEA1pAb for early endosomes. TRITC- or Alexa-conjugated secondary anti-bodies were added to the appropriate primary antibodies. Cells weremounted in Prolong Gold antifade reagent with DAPI (Life Technolo-gies). Immunolabeling was detected with a Leica confocal microscope(Leica TCS SP2 AOBS) equipped with an argon laser (excitation wave-lengths at 488, 568, and 647 nm). Confocal z stacks were captured in0.6-�m increments. Images were processed with Image J software. Colo-calization patterns were determined with the colocalization threshold al-gorithm in WCIF Image J. Three-dimensional reconstructions and mea-surements were performed with Volocity software. To avoid changes incolocalization coefficients due to artifacts, threshold levels were main-tained constant across all images analyzed. Values were expressed as themeans of the threshold Mander coefficient for each image. A total of 240confocal acquisitions (12 z stacks of 21 images for each fluorochrome withan increment of 0.6 �m) were performed, corresponding to 5,000 images.

Quantification of PrP aggregates. Immunostaining of PrP was per-formed as described above, using monoclonal antibody Sha31 (1/1,000)after guanidine thiocyanate (3 M) treatment. Immunolabeling was ob-served by using a Zeiss Axiovert 200 epifluorescence microscope. Images,from three independent experiments, were acquired under the same con-ditions, and threshold levels were maintained constant across all images toavoid artifacts. As described previously (26), we quantified the numberand the size of neuronal and astrocytic PrP aggregates in 30 images undereach condition using cell profiler software.

Total cell viability assay. Cell viability was measured with CellTiter 96Aqueous One solution (Promega). Cells were grown in 96-well platesprecoated with PDL and then exposed to different brain homogenates. Atdifferent times postexposure, cell viability was quantified with 3-(4,5-dimethylthiazol-2-yl)-5-3-carboxymethoxy-phenyl)-2-(4-sulfophe-nyl)-2H tetrazolium (MTS), according to the manufacturer’s recommen-dations (Promega). The absorbance was measured at 570 nm on amicroplate reader (Sunrise; Tecan, Männedorf, Switzerland).

Quantification of neurodegeneration by immunocytochemistry.Cells were fixed at various intervals postexposure, labeled with anti-MAP2and anti-GFAP antibodies, and then mounted with Prolong Gold antifadereagent with DAPI. The neuronal population was determined by countingMAP2-positive cells with neuronal morphology, the astrocyte populationwas determined by counting GFAP-positive cells with astrocytic mor-phology, and the presence of abnormal nuclei suggesting apoptosis wasassessed by counting pyknotic or fragmented nuclei.

Statistical analysis. Statview software (version 5; SAS Institute Inc.,Cary, NC) was used. We used a Mann-Whitney test to compare two

groups and one-way analysis of variance (ANOVA) (Dunnett and Scheffétests) to compare strains to controls and one strain to another one.

RESULTSPropagation of 139A, ME7, and 22L scrapie strains inCGNC57BL/6 cultures. Cronier et al. previously developed a cellu-lar model of prion propagation that led to neuronal death in pri-mary cultured cerebellar granule neurons (CGN) (18, 19). Wetested the permissiveness of CGNC57BL/6 cultures to three experi-mental scrapie strains: 139A, ME7, and 22L. These strains causedthe death of C57BL/6 mice after intracerebral inoculation withinapproximately 125, 145, and 175 days, respectively (20). More-over, it was clearly established that these strains showed differenttropisms in vivo. The 22L strain, for instance, was associated withmarked cerebellar lesions.

Two days after plating, CGNC57BL/6 cultures were exposed todifferent brain homogenates from terminally ill mice that hadbeen inoculated with strain 139A, ME7, or 22L, and to uninfectedbrain homogenate (control), at a concentration of 0.01%. Fourdays later, the cells were infected, and the culture medium wasreplaced with fresh medium to ascertain that further increases inthe PrPres signal corresponded to newly synthesized PrPres.

After PK digestion, Western blots showed a progressive accu-mulation of PrPres in infected CGNC57BL/6 cultures (Fig. 2). In139A-infected CGNC57BL/6 cultures, PrPres started accumulatingwithin 14 dpe and progressively increased until the endpoint of thestudy (Fig. 2A). In ME7-infected CGNC57BL/6 cultures (Fig. 2B),

FIG 2 Accumulation of PrPres in CGNC57BL/6 cultures exposed to differentscrapie strains, assessed by Western blotting. Shown are kinetics of PrPres ac-cumulation after exposure to brain homogenates from terminally ill mice in-fected with scrapie strains at a final concentration of 0.01% (wt/vol). Shownare Western blot results from duplicate culture wells of a representative exper-iment. Fifty micrograms of proteins from cell lysates was digested with PK, andPrPres was detected with monoclonal antibody 6D11. PrPres accumulation wasobserved from 14 dpe to 28 dpe for the 139A scrapie strain (A), from 21 dpe to28 dpe for the ME7 scrapie strain (B), and from 14 dpe to 28 dpe for the 22Lscrapie strain (C).

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PrPres started accumulating within 21 dpe; this suggested thatME7 propagation was less effective than 139A propagation inCGNC57BL/6 cultures. This was consistent with results previouslydescribed for CGN cultures derived from tga20 transgenic micethat overexpressed murine PrP (18) and other cell models (27–29). In 22L-infected CGNC57BL/6 cultures, PrPres started accumu-lating at 14 dpe and reached maximum levels more rapidly thanwith the two other strains (Fig. 2C). Control cultures exposed touninfected brain homogenates did not show any PrPres signal.

Neuronal loss in CGNC57BL/6 cultures after infection with139A, ME7, and 22L scrapie strains. We next explored the impactof prion propagation on primary CGNC57BL/6 culture viability.

Two different approaches were used to assess neuronal viability:an MTS assay, which measured the activity of mitochondrial de-hydrogenases of viable cells, and counting of MAP2-immunopo-sitive cells.

Results of MTS assays indicated that the kinetics of cell survivalafter prion exposure showed a roughly similar pattern among thedifferent strains. Compared to the control cultures, infected-cellsurvival significantly decreased for the first 21 dpe and then in-creased at later times (Fig. 3A).

The most significant decrease in cell viability was observed forCGNC57BL/6 cells infected with the 22L strain. This decrease wasdetectable as early as 14 dpe. Interestingly, exposure to the 139A

FIG 3 Neurotoxicity and kinetics of prion propagation in infected CGNC57BL/6 cells. (A) Cellular viability assessed with the MTS assay. CGNC57BL/6 cultures wereplated into 96-well microtiter plates at 105 cells/well, exposed to normal or scrapie brain homogenates at a final concentration of 0.01% (wt/vol) for 4 days, andthen washed twice and incubated for various times. The percentage of cellular viability was calculated relative to that of the control (CTR). Each value representsthe mean percentage of cell survival from five independent experiments performed in triplicate � the standard error of the mean. Differences from control values(�) and between strains (‡) were significant at a P value of �0.05 (� and ‡) and at a P value of �0.001 (‡‡), determined by a Mann-Whitney test and one-wayANOVA. (B and C) Neurons and astrocytes (B) and abnormal nuclei (C) were quantified in CGNC57BL/6 cultures infected with the 139A, ME7, and 22L strains.CGNC57BL/6 cultures were grown on glass coverslips. Immunofluorescence was performed at different days postexposure (dpe). The proportion of neurons in thecultures was determined as the number of MAP2-positive nuclei (MAP2�), expressed as a percentage of the total number of nuclei. The proportion of astrocytesin the cultures was determined as the number of GFAP-positive nuclei (GFAP�), expressed as a percentage of the total number of nuclei, and the proportion ofabnormal nuclei was determined as the number of pyknotic or fragmented nuclei, expressed as a percentage of the total number of nuclei. Each value representsthe mean of three independent experiments performed in triplicate � the standard error of the mean. Differences from control values (�) and between strains(‡) were significant at a P value of �0.05, determined by a Mann-Whitney test and one-way ANOVA. In CGNC57BL/6 cells infected with the 22L strain, from 21dpe, the proportion of neurons was significantly decreased and the proportions of astrocytes and abnormal nuclei were significantly increased compared toCGNC57BL/6 cells infected with the ME7 and the 139A strains (B). (D) Variations in neurons and astrocytes (left) and in abnormal nuclei (right) in infectedCGNC57BL/6 cultures quantified at the end point (28 dpe), as described above for panels B and C, are recapitulated. Significant differences (P � 0.05, determinedby a Mann-Whitney test and one-way ANOVA) compared to the control (�) and between strains (‡) were observed. (E) PrPres variations observed at various dayspostexposure relative to the level of PrPres observed at 4 dpe. Each value represents the mean of four independent experiments performed in duplicate � thestandard error of the mean. The 22L strain, the most toxic one for CGNC57BL/6 cultures, reached a plateau of PrPres accumulation.

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and ME7 strains had less of an impact on viability at 14 and 21 dpe.Later, compared to control cultures, cell survival in infected cul-tures increased at 28 dpe. However, the MTS assay does not spe-cifically quantify neuronal viability. Thus, given the fact that CGNare postmitotic, the observed increase in MTS values could be dueto the proliferation of astrocytes present in the primary neuronalcultures. The antimitotics added to the culture medium couldcontrol, but not totally inhibit, astrocyte proliferation.

To evaluate neuronal loss and glial proliferation after infectionof CGNC57BL/6 cultures with the three scrapie strains, we sepa-rately counted the neurons, astrocytes, and microglial cells in in-fected cultures. No microglial cells were detected by using specificmarkers. In contrast, the proportion of astrocytes and neuronalcells changed significantly in infected CGNC57BL/6 cultures.

Neurons were identified with the dendritic marker MAP2, andastrocytes were identified with GFAP labeling. In addition, wequantified the presence of abnormal nuclei (pyknotic or frag-mented nuclei) indicative of apoptotic cells. Histograms showedthat the proportions of neurons and astrocytes (Fig. 3B) and ab-normal nuclei (Fig. 3C) varied over time, with significant strain-dependent differences. Compared to control cells, CGNC57BL/6

cultures infected with the 22L strain showed an early, significantdecline in the neuronal population, associated with a significantincrease in the proportion of abnormal nuclei within 14 dpe. TheME7 and 139A strains did not cause a significant decrease in theneuronal population and a significant increase in abnormal nucleiuntil 21 dpe. After 21 dpe, the 22L strain caused significantly moreneuronal loss than that induced by the ME7 and the 139A strains(Fig. 3B to D). Interestingly, an increase in the glial populationalso occurred significantly earlier in cultures exposed to the 22Lstrain than in cultures exposed to the other strains.

In the CGNC57BL/6 model, the 22L strain was the most neuro-toxic, the ME7 strain caused intermediate neurotoxicity, and the139A strain was the least neurotoxic (Fig. 3D).

Next, we measured the kinetics of PrPres accumulation relativeto the PrPres level observed at 4 dpe (Fig. 3E). Interestingly, afterinfection with the 22L strain, PrPres accumulation reached a pla-teau at 14 dpe. After infection with 139A, PrPres accumulationincreased significantly at 14 dpe and continued to increase signif-icantly each week. After infection with the ME7 strain, PrPres ac-cumulation increased only after 21 dpe.

Thus, in this CGNC57BL/6 model, among the three studiedstrains, the first to reach a plateau of PrPres accumulation (the 22Lstrain) showed the highest level of neurotoxicity.

Monitoring of PrP aggregates in neurons and astrocytes dur-ing infection with 139A, ME7, and 22L scrapie strains inCGNC57BL/6 cultures. Neuronal permissiveness to the threestrains was confirmed by PrP immunostaining. CGNC57BL/6 cellsfixed at different times postexposure were treated with 3 M gua-nidine thiocyanate to expose epitopes of aggregated PrP (25, 30).Control cells showed uniform PrP labeling (Fig. 4A). As expected,PrP immunostaining was strikingly different in infected cells (Fig.4A). In 139A-, ME7-, and 22L-infected CGNC57BL/6 cultures, pre-dominantly punctate labeling was observed, while the homoge-neous staining of PrP, as observed in control cultures, markedlydecreased. This was assumed to reflect the accumulation of abnor-mal PrP aggregates (19, 25, 31). We quantified the formation ofPrP aggregates in neurons and astrocytes during the time course ofcell infection by the three experimental scrapie strains (139A,ME7, and 22L) (Fig. 4B to D). First, we observed that as soon as 4

dpe, all the cells had at least one PrP aggregate, probably corre-sponding to the trapping of PrPres from the inoculum. We thenmonitored the variation of the number and the size of PrP aggre-gates in neurons and astrocytes at each point of the kinetics. Weobserved that the variations of the number of aggregates per neu-ron are consistent with the results of PrPres accumulation for eachstrain (Fig. 4B): an early increase followed by a plateau-like phasewith 22L, a slight and late increase with ME7, and a continuousand marked increase in the last 2 weeks of cultures with 139A.When the number of aggregates in neurons increased, their sizedecreased (Fig. 4C). In contrast, neither the number (Fig. 4D) northe size (data not shown) of aggregates per astrocyte significantlyvaried during infection. These results strongly suggest that theneuronal loss that is associated with prion replication in thesecultures is related to the formation of PrP aggregates within neu-rons and did not correlate with PrP accumulation in astrocytes.

Subcellular localization of PrP aggregates in scrapie-infectedCGNC57BL/6 cells. To determine the subcellular localization of in-tracellular PrP aggregates, we used double immunostaining andconfocal microscopy. Cells were coimmunostained for PrP (afterguanidine thiocyanate treatment) and several subcellular com-partment markers at 21 dpe. Based on previous results for chron-ically infected cell lines, we investigated four subcellular compart-ments: lysosomes (lysosomal lumen and lysosomal membrane),endoplasmic reticulum, Golgi apparatus, and early endosomes. Aspreviously described (25, 32), uninfected CGNC57BL/6 cultures(controls) showed PrPC in all the studied subcellular compart-ments. PrPC was localized mainly in the endoplasmic reticulum,the lysosomal lumen, and the early endosomes. It was rarely foundin the Golgi apparatus (Table 1). In infected CGNC57BL/6 cultures,the punctate PrP signals revealed different distributions (Table 1).

For all three scrapie strains, aggregated PrP was associated pri-marily with lysosomal markers (Table 1 and Fig. 5 for the 139Astrain). In ME7- and 22L-infected CGNC57BL/6 cultures (Fig. 6 and7), the punctate PrP signal was also associated with the Golgi ap-paratus. This localization was consistent with data from previousreports (25, 33) and most likely involved retrograde transport viathe Golgi apparatus. Aggregated PrP was rarely associated with theendoplasmic reticulum or early endosome markers.

Propagation and neurotoxicity of scrapie strains in neuronalcells derived from the striatum and cortex. To determinewhether the propagation and neurotoxic effects of the differentscrapie strains were neuron specific, we investigated their effects intwo additional neuronal cell models.

First, we tested the permissiveness of primary striatal cultures(StNC57BL/6) to the 139A, ME7, and 22L scrapie strains using thesame inocula and under conditions of exposure similar to thoseused previously. StNC57BL/6 cultures can be maintained for only 21days. After PK digestion, Western blots showed PrPres accumula-tion in StNC57BL/6 cultures infected with the 139A and 22L strains(Fig. 8A and D). In contrast, in StNC57BL/6 cultures exposed to theME7 strain, we did not detect PrPres formation (data notshown). Thus, we investigated StNC57BL/6 cultures infectedwith the 139A and 22L strains to assess the neurotoxicity asso-ciated with prion propagation and PrPres formation (Fig. 8).Compared to control cultures, we observed a significant de-crease in the proportion of neurons, associated with a signifi-cant increase in the proportion of abnormal nuclei and a slightincrease that was not significant in the astroglial population in139A-infected cultures. The proportion of neurons and the

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presence of abnormal nuclei varied significantly in the culturesinfected with 139A compared to those infected with 22L, whereno significant neuronal loss was observed. In cultures infectedwith the 139A strain, the kinetics of PrPres accumulation nearly

reached the maximum at 11 dpe. In cultures exposed to the 22Lstrain, PrPres accumulated slowly, increased later, and did notreach a plateau phase (Fig. 8H).

Of the two strains tested, in StNC57BL/6 cultures, the 139A strain

FIG 4 Monitoring of PrP aggregates in neurons and astrocytes in CGNC57BL/6 cultures infected with the 139A, ME7, or 22L scrapie strain. (A) Cultures wereexposed to 139A-, ME7-, or 22L-infected brain homogenates at a final concentration of 0.01% (wt/vol). At different times postexposure, cells were fixed,permeabilized, and treated with 3 M guanidine thiocyanate. Immunostaining of PrP (in green) was performed by using monoclonal antibody Sha31. DAPIstaining appears in blue. CGNC57BL/6 cultures infected with the 139A, ME7, and 22L strains are compared to control cultures (CT). Control and infectedCGNC57BL/6 cultures shown in the figure were labeled at 21 dpe. In infected cultures, cells showed punctate fluorescence signals, suggesting intracellular PrPaggregates. In contrast, control cultures showed diffuse, homogenous PrP labeling. (B) Data presented in the box plots show the distribution of the quantifiednumber of intraneuronal PrP aggregates at different days postexposure for each scrapie strain. The number of PrP aggregates per neuron significantly increasedduring the time course of infection in CGNC57BL/6 cells infected with the 139A, ME7, and 22L strains. In CGNC57BL/6 cells infected with the 22L strain, thevariation of the number of PrP aggregates per neuron occurred earlier, as soon as 7 dpe, than with the other strains. The variations observed in ME7-infectedCGNC57BL/6 cells were slighter than those observed with the two other strains. (C) Data represented in the box plot show the distribution of the size ofintraneuronal PrP aggregates at different days after exposure to each scrapie strain. Regardless of the strain, infected CGNC57BL/6 cells showed a significantdecrease in the size of neuronal PrP aggregates. (D) Data presented in the box plots show the distribution of the number of intra-astrocyte PrP aggregates atdifferent days postexposure for each scrapie strain. Regardless of the strain, the number of PrP aggregates per astrocyte did not vary during the time course ofinfection. Box plots represent data from three independent experiments performed in triplicate � the standard error of the mean. Differences compared betweeneach point (�) were significant at a P value of �0.05.

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showed a higher level of toxicity and reached a plateau-like phase,unlike the 22L strain.

Next, we investigated the permissiveness of primary corticalcultures (CxNC57BL/6) to the 139A, ME7, and 22L scrapie strains.Because the survival of these cell cultures was limited to 2 1/2weeks after plating, corresponding to 11 dpe, long-term accumu-lation could not be measured. After PK digestion, Western blotsshowed that newly synthesized PrPres accumulated in CxNC57BL/6

cultures after exposure to the 139A and the 22L strains (Fig. 9Aand D). As observed for StNC57BL/6 cultures, no PrPres accumula-tion was detected in CxNC57BL/6 cultures infected with the ME7strain (data not shown). 139A and 22L propagation caused signif-icant neuronal loss compared to control cultures at 11 dpe (Fig.9B, C, and E to G). As observed for StNC57BL/6 cultures, no signif-icant change in the astrocyte population was detected in infectedCxNC57BL/6 cultures. This was probably due to the early use ofserum-free culture medium, which strongly limited astrocyticproliferation. Interestingly, in CxNC57BL/6 cultures, the 139A and22L strains showed similar PrPres accumulation kinetics (none ofthem reached a plateau) (Fig. 9F) and showed similar neurotoxiceffects.

DISCUSSION

We investigated the effects of prion propagation by infecting pri-mary neuronal cultures with three different strains of experimen-tal scrapie (139A, ME7, and 22L). We also studied the potentialneurotoxicity of these strains toward different neuronal cell types(CGNC57BL/6, StNC57BL/6, and CxNC57BL/6). We found that boththe kinetics of PrPres accumulation and the neurotoxicity associ-ated with prion propagation were specific to the strain and theneuronal cell type. Furthermore, the severity of neurotoxicitycaused by prion infection was related to the occurrence of a pla-teau phase in PrPres accumulation.

First, we assessed the propagation of the three scrapie strains inCGNC57BL/6 cultures. For each strain, Western blotting after PKdigestion demonstrated the accumulation of newly synthesizedPrPres. Immunostaining of PrP after guanidine thiocyanate treat-ment revealed a punctate signal, indicative of abnormal PrP ag-gregates (19, 25). In subcellular colocalization studies, we foundthat these aggregates were typically associated with the lysosomalcompartment of infected cells but also with the Golgi apparatus ofcells infected with the ME7 and 22L strains. These results wereconsistent with those previously described for cell line models ofprion infection (25, 33).

Interestingly, the behavior of the 22L and 139A strains in neu-ronal cell cultures which we observed was consistent with resultsfrom in vivo infections. Kim et al. (20) reported previously thatthese strains showed well-defined tropism in different brain re-

gions. A stereotaxic injection of the 22L strain in the cerebellum ofC57BL/6 mice caused a significantly different disease than thosecaused by other strains and injections of 22L at different inocula-tion sites. Indeed, cerebellar injection of 22L in mice showedshorter incubation periods and vacuolization scores only in thecerebellum. Stereotaxic inoculation of the 139A strain in the stria-tum showed a shorter incubation period than the other strains.These results suggest that prion strains show a distinct tropism fordifferent types of neurons, which contributes to a strain-specificlesion profile. In this study, we found that maximum PrPres accu-mulation was achieved more rapidly with the 22L strain inCGNC57BL/6 neurons and with the 139A strain in StNC57BL/6 neu-rons than in other neuronal cell cultures. In addition, both the 22Lstrain in CGNC57BL/6 cells and the 139A strain in StNC57BL/6 cellsshowed higher-level cytopathogenic effects than the other strains.It is remarkable that our in vitro results with strains stabilized inC57BL/6 mice and C57BL/6-derived neurons matched those de-scribed in vivo in the same genetic background. However, themechanisms underlying these phenomena remain unclear, partlybecause no in vitro model that recapitulates these strain-specificfeatures has been available. This study strongly suggests that pri-mary neuronal cultures isolated from different brain structurescould serve as novel models for studying strain-specific cell tro-pism in vitro. A recent study by Ayers et al. compared the anatom-ical route of spread of two mink prion strains from the inoculationsite in the sciatic nerve to the brain of the Syrian hamster (34).Their results suggested that the time required for PrPSc to spreadwithin the brain and to reach various neuronal populations mayinfluence prion distribution. In contrast, prion propagation inneuronal cultures does not involve anatomical pathways. Thus,our results provide support for the notion that, in addition toneuroanatomical spread (34–36), strain-specific neuronal tro-pism may influence prion brain targeting.

Our results are also consistent with previous studies that sug-gested that local cofactors could influence the prion conversionprocess in the brains of infected individuals (37). These conver-sion cofactors could be strain and neuron specific. In in vivo stud-ies, it was hypothesized that a molecular factor, protein X, which ispresumably a chaperone protein (38, 39), may interact with PrPC

to form a complex that binds PrPSc during the PrP conversionprocess. In some in vitro systems, the conversion of PrPC intoPrPSc required the addition of total cell extracts (40). Several PrPligands have been proposed to influence the conversion process(41), including heparan sulfate proteoglycans (42–44) or the high-affinity laminin receptor (LRP/LR) (45, 46). The addition of poly-anionic molecules to a mixture of PrPC/PrPSc was shown to im-prove the efficiency of the transconformation process in a cell-freeconversion model (47) and protein-misfolding cyclic amplifica-

TABLE 1 Colocalization study of organelle markers and PrP immunostaining detected by confocal microscopy and Image J software analysis(CGNC57BL/6 cultures at 21 dpe)

Organelle marker antibody Cellular compartment

% PrP colocalized (�SEM)

Control 139A ME7 22L

Giantin (MG120) Golgi apparatus 4.0 (�0.02) 8.3 (�0.01) 16.5 (�0.01) 15.9 (�0.01)EEA1 Early endosomes 13.1 (�0.03) 9.0 (�0.03) 8.7 (�0.03) 8.9 (�0.02)LAMP2 Lysosome membrane 5.1 (�0.05) 18.8 (�0.04) 7.5 (�0.03) 16.5 (�0.03)Cathepsin D Enriched in lysosome lumen 12.9 (�0.04) 24.0 (�0.03) 13.1 (�0.03) 5.3 (�0.01)GRP78 BiP Endoplasmic reticulum 14.0 (�0.02) 5.0 (�0.01) 5.8 (�0.02) 2.4 (�0.02)

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tion (48, 49). Our in vitro approach offers the alternative of usingcomplementary neuronal cell models that are permissive to dif-ferent prions for studying strain-specific neuronal tropism. Thisapproach may also provide a valuable tool for further identifyingcellular cofactors involved in this process.

Neuronal loss occurs during central nervous system degenera-tion in prion diseases. Although several cell models chronicallyinfected with prions have been reported, very few have demon-strated neurodegeneration related to prion propagation (50, 51),and the mechanisms involved remain controversial (10, 13, 16,

52). In vitro data that compare the neurotoxicity of differentstrains are lacking. In this study, we showed for the first time invitro that different prion strains may induce different degrees ofneurodegeneration, and these effects may vary for each strainamong different neuronal cell types.

The analysis of scrapie strain behaviors in our three neuronalcell models suggested the existence of a first replication phase(phase 1), during which PrPres progressively accumulated but didnot induce severe neurotoxicity, followed by a plateau phase(phase 2), where PrPres production reached a steady state and the

FIG 5 Double immunofluorescence of PrP and organelle markers in CGNC57BL/6 cultures infected with the 139A strain at 21 dpe. Immunolabeled PrP (A, D, G,J, and M) and organelle markers (B, E, H, K, and N) were visualized by confocal microscopy (green and red, respectively). In merged images (C, F, I, L, and O),yellow areas indicate colocalization, and DAPI staining appears in blue. Immunostaining was performed with the following antibodies: Sha31 monoclonalantibody (PrP), giantin monoclonal antibody (Golgi apparatus) (B and C), early endosome autoantigen 1 monoclonal antibody (EEA1) (E and F), lysosome-associated membrane protein 2 monoclonal antibody (LAMP2) (H and I), cathepsin D monoclonal antibody (lysosomal lumen) (K and L), and GRP78monoclonal antibody (endoplasmic reticulum) (N and O).

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cytopathogenic effect intensified. Indeed, the strain that reached aplateau phase of PrPres accumulation most rapidly was the mostdeleterious to neurons. This behavior was suggested for the 22Lstrain propagated in CGNC57BL/6 cultures and the 139A strainpropagated in StNC57BL/6 cultures. In CxNC57BL/6 cultures, neitherthe 22L nor the 139A strain reached a plateau phase, and bothstrains showed similar degrees of neurotoxicity. These results sup-port the idea that the occurrence of a plateau phase is critical forsevere neuronal damage to occur in response to prion infection.

Recent in vivo and in vitro studies also suggested the occurrence

of a plateau phase during prion replication. Indeed, one study of achronically infected cell line (N2a) proposed a limited conversionmodel for prion formation kinetics that led to a plateau phase,possibly due to various factors, including the saturation of subcel-lular sites of PrP conversion and the depletion of key cellular co-factors (53). In our neuron-enriched cell cultures, the steady-statelevel of PrPres during phase 2 may also result from the balancebetween PrPres formation in neurons, neuronal death, PrPres

clearance, and PrPres production by proliferating astrocytes. In thecell culture model where we observed a significant variation of the

FIG 6 Double immunofluorescence of PrP and organelle markers in CGNC57BL/6 cultures infected with the ME7 strain at 21 dpe. Immunolabeled PrP (A, D, G,J, and M) and organelle markers (B, E, H, K, and N) were visualized by confocal microscopy (green and red, respectively). In merged images (C, F, I, L, and O),yellow areas indicate colocalization, and DAPI staining appears in blue. Immunostaining was performed with the following antibodies: Sha31 monoclonalantibody (PrP), giantin monoclonal antibody (Golgi apparatus) (B and C), early endosome autoantigen 1 monoclonal antibody (EEA1) (E and F), lysosome-associated membrane protein 2 monoclonal antibody (LAMP2) (H and I), cathepsin D monoclonal antibody (lysosomal lumen) (K and L), and GRP78monoclonal antibody (endoplasmic reticulum) (N and O).

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astrocytic population during prion infection, (i.e., infectedCGNC57BL/6 cultures), we observed that the increase followed by aplateauing of PrPres accumulation was associated with similarvariations in the number of PrP aggregates in infected neurons,although the number and the size of PrP aggregates in astrocytesremained unchanged during the course of infection. This suggeststhat in our cell model, the stabilization of PrPres was probablyrelated to various and opposite factors: a plateau of PrPres accu-mulation within neurons and neuronal loss that compensated forthe production of PrPres by an increase in the astrocytic popula-tion. In infected CGNC57BL/6 cultures, intense neuronal loss was

associated with a plateauing of PrP aggregates in neurons(Fig. 4B). This suggests that neurodegeneration may be related tointraneuronal PrPres formation and that astrocytic proliferationand PrP accumulation within astrocytes are not key factors ofprion-induced neurotoxicity. This hypothesis is supported by ourresults for infected striatal and cortical cultures, where significantneuronal loss was observed despite the absence of a significantincrease in the astrocytic population, and by the results of a recentstudy which proposed that the efficient conversion of neuronalPrPC is the main culprit in prion-induced neurodegeneration(31). However, a contribution of infected astrocytes to the neuro-

FIG 7 Double immunofluorescence of PrP and organelle markers in CGNC57BL/6 cultures infected with the 22L strain at 21 dpe. Immunolabeled PrP (A, D, G,J, and M) and organelle markers (B, E, H, K, and N) were visualized by confocal microscopy (green and red, respectively). In merged images (C, F, I, L, and O),yellow areas indicate colocalization, and DAPI staining appears in blue. Immunostaining was performed with the following antibodies: Sha31 monoclonalantibody (PrP), giantin monoclonal antibody (Golgi apparatus) (B and C), early endosome autoantigen 1 monoclonal antibody (EEA1) (E and F), lysosome-associated membrane protein 2 monoclonal antibody (LAMP2) (H and I), cathepsin D monoclonal antibody (lysosomal lumen) (K and L), and GRP78monoclonal antibody (endoplasmic reticulum) (N and O).

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degenerative process cannot be excluded and may operatethrough various mechanisms, such as the production of proin-flammatory cytokines (54) or an impairment of their ability tomaintain neuronal homeostasis and to protect neurons from ox-idative stress.

Interestingly, a study of mice infected with the RML straindemonstrated that prion propagation in the brain also proceededvia two distinct phases (an exponential phase followed by a pla-teau phase). This strikingly resembled the phases that we observedfor primary infected neurons (11). Furthermore, in that study,clinical onset occurred only during phase 2, after an incubationperiod inversely proportional to the level of PrPC expression.Based on that result, those authors suggested an uncoupling ofprion replication and toxicity. Here, we have provided the first invitro evidence that supports that hypothesis. In addition, we ob-served that the kinetics of the two mechanistic phases were strainand neuronal type dependent. Thus, we can speculate that the

lesion profile of a given prion strain may partly be due to thedistinct prion kinetics that occur in different parts of the brain.

It remains unclear how the propagation of PrP misfolding, whichleads to the formation of protein aggregates, triggers a neurodegen-erative process. The putative toxic entity remains unknown. ForTSEs, and in other brain proteinopathies, it was suggested previouslythat the most toxic entities are not the large protein aggregates butdiffusible oligomeric species (55–58). Several in vivo studies suggestedthat PrP species other than PrPSc may be key players in prion-inducedneurotoxicity, and the relationship between abnormal PrP accumu-lation and lesion intensity remains unclear (13, 56, 59, 60). Theseobservations also led to the proposal that PrPSc production may beuncoupled from neurotoxicity. According to that hypothesis, whenprion propagation reaches a plateau phase (phase 2 in our neuronalcell model), a pathway switch may induce the production of a highlytoxic molecular species, PrPL (61, 62).

The results of our study showed two-phase kinetics in prion

FIG 8 Accumulation of PrPres and neuronal loss in striatal neuronal cultures (StNC57BL/6) infected with the 139A and 22L strains. Cultures were exposed to 139A(A to C, G, and H) or 22L-infected brain homogenates (D to H) at a final concentration of 0.01% (wt/vol). (A and D) Western blot results from duplicate culturewells of a representative experiment are shown. The accumulation of PrPres occurred earlier after infection with the 139A strain than with the 22L strain.StNC57BL/6 cultures exposed to normal or infected brain homogenates were immunostained at different days post exposure (dpe). The proportion of neurons inthe cultures was determined as the number of MAP2-positive nuclei (MAP2�), expressed as a percentage of the total number of nuclei; the proportion ofastrocytes in the cultures was determined as the number of GFAP-positive nuclei (GFAP�), expressed as a percentage of the total number of nuclei; and theproportion of abnormal nuclei was determined as the number of pyknotic condensed or fragmented nuclei, expressed as a percentage of the total number ofnuclei. Each value represents the mean of three independent experiments performed in triplicate � the standard error of the mean. Differences compared tocontrol values (�) and compared to the other strains (‡) were significant at a P value of �0.05, determined by a Mann-Whitney test and one-way ANOVA. (B,C, E, and F) After 11 dpe, the proportion of neurons was significantly lower in StNC57BL/6 cells infected with the 139A strain (B) than in those infected with the22L strain (E), and after 14 dpe, the proportion of abnormal nuclei was significantly higher in StNC57BL/6 cells infected with the 139A strain (C) than in thoseinfected with the 22L strain (F). (G) The variations in neurons and astrocytes and abnormal nuclei in infected StNC57BL/6 cells quantified at the end point (14 dpe),as described for panels B, C, E, and F, are recapitulated. Significant differences (P � 0.05, determined by a Mann-Whitney test and one-way ANOVA) wereobserved compared to controls (�) and between strains (‡). (H) PrPres accumulation observed at various days postexposure is expressed relative to the PrPres levelobserved at 4 dpe. Each value represents the mean of at least four independent experiments performed in duplicate � the standard error of the mean.

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propagation specific to the prion strain and the neuronal type.Considering that infected-cell cultures are useful in the search ofantiprion molecules (63) and that CGN cultures can be infectedwith prion strains of human origin (18), the model of prion infec-tion described in this study may facilitate investigations of thera-peutic candidates for treating prion propagation (phase 1) andprion-induced neuronal death (phase 2) in a neuron-specificmanner.

ACKNOWLEDGMENTS

This work was supported by the Institut de Veille Sanitaire and the Pôle deCompétitivité Medicen-Paris Région.

We thank M. Rosset and P. Aucouturier for kindly providing us the139A and ME7 scrapie strains, E. Comoy for the 22L scrapie strain, thePlate-Forme d’Imagerie Cellulaire-Pitié Salpêtrière for confocal image ac-quisition, D. Langui for critical discussion and assistance, VanessaDiouron and Emilie Morain for excellent technical help, M. Valente for

his help in image analysis with CellProfiler software, and all the membersof the R. Escourolle Laboratory.

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FIG 9 PrPres accumulation and neurotoxicity in cortical neuronal cultures infected with the 139A and 22L strains. Cultures were exposed to 139A-infected (Ato C, G, and H) or 22L-infected (D to H) brain homogenates at a final concentration of 0.01% (wt/vol). (A and D) Western blot results from duplicate culturewells of a representative experiment are shown. The accumulation of PrPres occurred up to 11 dpe for both the 139A and 22L strains. CxNC57BL/6 cultures exposedto normal or infected brain homogenates were immunostained at different days postexposure. The proportion of neurons in the cultures was determined as thenumber of MAP2-positive nuclei (MAP2�), expressed as a percentage of the total number of nuclei. The proportion of astrocytes in the cultures was determinedas the number of GFAP-positive nuclei (GFAP�), expressed as a percentage of the total number of nuclei, and the proportion of abnormal nuclei was determinedas the number of pyknotic condensed or fragmented nuclei, expressed as a percentage of the total number of nuclei. Each value represents the mean of threeindependent experiments performed in triplicate � the standard error of the mean. Differences compared to control values (�) were significant at a P value of�0.05, determined by a Mann-Whitney test and one-way ANOVA. (B, C, E, and F) The proportions of neurons and astrocytes and of abnormal nuclei weresimilar in CxNC57BL/6 cultures infected with the 139A (B and C) and 22L (E and F) strains. (G) The variations in neurons and astrocytes and abnormal nuclei ininfected CxNC57BL/6 cells quantified at the end point (11 dpe), as described for panels B, C, E, and F, are recapitulated. Significant differences (�, P � 0.05 by aMann-Whitney test and one-way ANOVA) were observed compared to controls. (H) PrPres accumulation observed at various days postexposure is expressedrelative to the PrPres level observed at 4 dpe. Each value represents the mean of four independent experiments performed in duplicate � the standard error of themean.

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