Prion Peptide 106–126 Modulates the Aggregation of CellularPrion Protein and Induces the Synthesis of Potentially NeurotoxicTransmembrane PrP*

Received for publication, May 14, 2001, and in revised form, October 5, 2001Published, JBC Papers in Press, October 26, 2001, DOI 10.1074/jbc.M104345200

Yaping Gu, Hisashi Fujioka, Ravi Shankar Mishra, Ruliang Li, and Neena Singh‡

From the Institute of Pathology, Case Western Reserve University, Cleveland, Ohio

In infectious and familial prion disorders, neurode-generation is often seen without obvious deposits of thescrapie prion protein (PrPSc), the principal cause ofneuronal death in prion disorders. In such cases, neuro-toxicity must be mediated by alternative pathways ofcell death. One such pathway is through a transmem-brane form of PrP. We have investigated the relation-ship between intracellular accumulation of prion pro-tein aggregates and the consequent up-regulation oftransmembrane prion protein in a cell model. Here, wereport that exposure of neuroblastoma cells to the prionpeptide 106–126 catalyzes the aggregation of cellularprion protein to a weakly proteinase K-resistant formand induces the synthesis of transmembrane prion pro-tein, the proposed mediator of neurotoxicity in certainprion disorders. The N terminus of newly synthesizedtransmembrane prion protein is cleaved spontaneouslyon the cytosolic face of the endoplasmic reticulum, andthe truncated C-terminal fragment accumulates on thecell surface. Our results suggest that neurotoxicity inprion disorders is mediated by a complex pathway in-volving transmembrane prion protein and not by depos-its of aggregated and proteinase K-resistant PrP alone.

Prion disorders manifest when the prion protein (PrPC),1 anormal cell surface glycoprotein, undergoes a conformationalchange from a predominantly �-helical to a �-sheet-rich struc-ture that is pathogenic (PrPSc). This transformation is initiatedby an exogenous source of PrPSc in cases acquired by infection,triggered by mutation(s) in the prion protein gene in inheritedforms, and is a random, spontaneous event in sporadic cases.Following the initial conversion, subsequent transformation ofadditional PrPC molecules progresses autocatalytically, result-ing in deposits of PrPSc in the brain parenchyma. Unlike PrPC,PrPSc aggregates easily, is insoluble in nonionic detergents,

and is partially resistant to limited digestion by proteinase K.Deposits of PrPSc in the brain parenchyma are believed to bethe principal cause of neuronal toxicity in prion disorders (1–3).

Although PrPSc is believed to be responsible for both trans-mission and pathogenicity in all prion disorders, the molecularevents leading to PrPSc-induced transformation of additionalPrPC molecules and the consequent neuronal toxicity arepoorly understood. Because neurodegenerative changes typicalof prion disorders are often seen without detectable PrPSc,alternative mechanisms of neuronal death besides PrPSc dep-osition have been suggested (4–6). One such mechanism isthrough the preferred synthesis of CtmPrP, a transmembraneform of PrP that spans the endoplasmic reticulum (ER) mem-brane at residues 113–135 with its N terminus in the cytosol,rather than the normal glycolipid-linked PrPC that is translo-cated co-translationally into the ER lumen. Mice carrying themutant PrP transgene A117V that has an increased predilec-tion for CtmPrP synthesis show spontaneous neurodegenerationwithout detectable PrPSc and, when challenged with infectiousprions, show neurodegeneration earlier and with smalleramounts of accumulated PrPSc than the corresponding animalswith a deleted transmembrane domain. In fact, in these casesthe extent of neurodegeneration correlates directly with theamount of CtmPrP rather than PrPSc load, indicating that Ctm

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PrP, and not accumulated PrPSc, is responsible for the observedneurodegeneration (7, 8).

We have examined the initial events of PrPC aggregation ina cell model, and the correlation between intracellular accumu-lation of aggregated PrPC and CtmPrP generation. To initiatethe aggregation of endogenous PrPC, we have used an internalpeptide of PrP comprising residues 106–126 (PrP106–126) in-stead of the proteinase K (PK)-resistant core of PrPSc thatconstitutes the infectious prion particle. PrP106–126 offers theadvantage of being similar to PrPSc in several respects and atthe same time is more soluble and easy to manipulate for cellculture studies. For example, like PrPSc, PrP106–126 is rich in�-sheet structure, forms aggregates that are detergent-insolu-ble and PK-resistant, and is toxic to primary cultures of neu-rons (9–11). Furthermore, residues 90–141 of PrP have beenshown to be sufficient for initiating or blocking the transfor-mation of PrPC, probably because these constitute the principalsite of binding of PrPSc to PrPC during the conversion process(12–15). This region can be further narrowed down to residues112–120, which are considered particularly important for thebinding and inhibitory effect (11, 14).

In this study, we demonstrate that when PrPC-expressingneuroblastoma cells are exposed to nontoxic concentrations ofPrP106–126, micro-aggregates of PrP106–126 “seed” the aggrega-tion of endogenous, cellular PrPC into thioflavin-binding depos-its that accumulate in the lysosomes. Subsequently, there isincreased synthesis of the potentially neurotoxic transmem-

* This work was supported by National Institutes of Health GrantsNS35962 and NS39089 (to N. S.). The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

‡ To whom correspondence should be addressed: Institute of Pathol-ogy, Case Western Reserve University, 2085 Adelbert Rd., Cleveland,OH 44106. Tel.: 216-368-2617; E-mail: nxs2@po.cwru.edu.

1 The abbreviations used are: PrPC, normal cell-associated prion pro-tein; PrP106–126, PrP peptide including residues 106–126 biotin-taggedat the N terminus; PrPScr106–126, PrP peptide with a scrambled 106–126sequence biotin-tagged at the N terminus; PrPSc, conformationallytransformed scrapie form of PrP; A�, �-amyloid peptide of amyloidprecursor protein; PK, proteinase K; PI-PLC, phosphatidylinositol-spe-cific phospholipase C; ER, endoplasmic reticulum; DAB, 3,3�-diamino-benzidine; GPI, glycosylphosphatidylinositol; PNGase-F, N-glycosidaseF; CtmPrP, transmembrane PrP; L, lysosomes; N, nucleus; E,endosomes.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 3, Issue of January 18, pp. 2275–2286, 2002© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org 2275

brane CtmPrP in these cells. Our data provide the first directevidence for nucleation-dependent transformation of PrPC intoaggregated and weakly PK-resistant forms in a cell model.More importantly, our findings show a direct correlation be-tween intracellular PrPC aggregation and CtmPrP up-regula-tion, indicating that prion-related neuropathology is mediatedby complex cellular pathway(s) involving CtmPrP and not sim-ply by deposits of PrPSc.

EXPERIMENTAL PROCEDURES

Materials and Cell Culture Conditions—All cell culture supplieswere obtained from Invitrogen. Hygromycin B was from Calbiochem;Hoechst and Lysotracker dye were from Molecular Probes (Eugene,Oregon). Biotin-tagged PrP106 –126 and biotin-tagged scrambledPrP106–126 were custom synthesized (Genemed Corp., San Francisco).Glutaraldehyde, osmium tetroxide, uranyl acetate, lead citrate, andepoxy resin were from Polysciences Inc. (Warrington, PA). Anti-PrPmonoclonal antibody 3F4 (specific to PrP residues 109–112) was fromRichard Kascsak (New York State Institute for Basic Research in De-velopmental Disabilities). Anti-PrP monoclonal antibody 8H4, whichbinds to a site between residues 145–180 of PrP, was raised in ourfacility (20). All other chemicals, including antibodies to the glial fibril-lary acidic protein (anti-GFAP) and neurofilament-specific (NF68) an-tibodies were purchased from Sigma. PrPC-expressing human neuro-blastoma cells were generated as described previously (17) and culturedin the presence of 500 �g/ml hygromycin B. Fetal brain cells wereobtained from the Birth Defects Research Laboratory (University ofWashington). Discarded brain tissue from human therapeutic abortionswas washed with cold Hanks’ buffer containing gentamycin and disso-ciated by trituration through a fire-polished glass pipette. Cells wereseparated from large aggregates of connective tissue by a brief centrif-ugation at 200 � g for 5 min. The supernatant containing suspendedcells was recentrifuged, and the cells were cultured on Matrigel-coateddishes in minimum essential medium containing 10% fetal bovine se-rum, 1% penicillin/streptomycin, and 0.01% gentamycin for 24 h. Theculture medium was then replaced with neurobasal medium containingB27 supplements. To enrich for neuronal cells, the mitotic inhibitorcytosine arabinoside (10 �M) was added to the medium after 2 days ofculture. For immunofluorescence analysis, the cells were plated onMatrigel-coated coverslips rather than in Petri dishes and subjected tothe required experimental conditions.

Treatment of Neuroblastoma and Mixed Brain Cells with PrP106–126—The following peptides were custom synthesized for the study: biotin-tagged PrP106–126 (PrP106–126), biotin-106KTNMKHMAGAAAAGAVVG-GLG126; and biotin-tagged scrambled PrP106–126 (PrPScr106–126), biotin-NGAMALMGGHGATKVKVGAAA. Biotinylation of PrP106–126 does notalter its biochemical properties (11, 16).

Peptide stocks were dissolved in Me2SO and kept frozen until use. Atthe time of subculture, PrP106–126 or PrPScr106–126 (5 �M) was dissolvedin culture medium and centrifuged at 1000 � g for 10 min to eliminatelarge aggregates. The supernatant was used to culture PrPC-over-ex-pressing neuroblastoma cells or primary brain cultures. Every 3 days,the neuroblastoma cells were trypsinized and subcultured with freshpeptide solution. Brain cells were cultured in the presence of peptide for1–4 weeks.

Immunofluorescence Staining and Confocal Microscopy—For all im-munostaining experiments, anti-PrP antibody 8H4 was used to detectcellular PrP. This antibody is specific to residues 145–180 of PrP andthus does not react with PrP106–126. The specificity of this antibody hasbeen confirmed in several studies by independent laboratories (18–20).For detecting biotin-tagged PrP106–126 or biotin-tagged PrPScr106–126,streptavidin-conjugated-Texas red was used because of the specificbinding of streptavidin with biotin. For immunostaining cell surfaceproteins, permeabilization of the membrane with Triton X-100 wasomitted. Neuroblastoma cells cultured in normal medium or in 5 �M

biotin-PrP106–126 or biotin-PrPScr106–126 were fixed and stained with8H4 (1:25)-anti-mouse-fluorescein isothiocyanate (1:25) (Southern Bio-technology Associates) followed by streptavidin-conjugated-Texas red(1:40) (Pierce). For intracellular staining, fixed cells were permeabilizedwith 0.1% Triton X-100 prior to immunostaining as described above.Stained cells were incubated with Hoechst 33342 (1 �g/ml) (MolecularProbes) for 5 min to detect apoptotic nuclei and mounted in Gel-mount(Biomeda Corp., Foster City, CA). All samples were observed with alaser scanning confocal microscope (Bio-Rad MRC 600). A single 5 �moptical section and a composite of several sections were evaluated ineach case.

Electron Microscopy—For immunoelectron microscopy, cells treatedwith biotin-PrPScr106–126 (“control”) or biotin-PrP106–126 (“treated”) werefixed with 4% paraformaldehyde and 0.01% glutaraldehyde, and endog-enous peroxidase activity was quenched by exposing fixed cells to 0.3%H2O2. The cells were then immunostained with 8H4 (1:20) followed byperoxidase-conjugated anti-mouse (1:50) and mouse peroxidase-anti-peroxidase (1:250), exposed to 3,3�-diaminobenzidine (DAB) containing0.1 M imidazole to obtain an electron-dense reaction product and fixedagain with 2.5% glutaraldehyde. Subsequently, the cells were post-fixedin 1% osmium tetroxide, dehydrated, embedded in Epon 812, and pro-cessed for electron microscopy. Post-staining with uranyl acetate andlead citrate was omitted to enhance the contrast for the PrP-specificDAB stain. For intracellular immunostaining, fixed cells were perme-abilized with 0.1% Triton X-100 before processing as described above.For ultrastructural analysis, cells were processed by conventionalmethods and examined with a Zeiss electron microscope (Zeiss CEM902; Carl Zeiss Inc., Thornwood, NY).

SDS-PAGE and Western Blotting—Cells treated with biotin-PrPScr106–126 (control) or biotin-PrP106–126 (treated) were processed forWestern blotting as described (17, 37). Membranes containing trans-ferred proteins were probed with either anti-PrP antibody 8H4 (1:1000)or 3F4 (1:40,000) followed by anti-mouse-horseradish peroxidase (1:4000) or streptavidin-conjugated horseradish peroxidase (1:40,000). Re-active bands were visualized on an autoradiographic film by ECL (Am-ersham Biosciences Inc.).

Metabolic Labeling and Immunoprecipitation—Cells treated withbiotin-PrPScr106–126 (control) or biotin-PrP106–126 (treated) were radiola-beled with 50 �Ci/ml of Tran35S-label overnight in Dulbecco’s modifiedEagle’s medium containing 5% dialyzed serum or with 75 �Ci/ml of[3H]ethanolamine in complete medium. Labeled cells were treated withPI-PLC (Oxford Glycosystems) in Opti-MEM for 1 h at 37 °C. Celllysates and PI-PLC-released proteins in the medium were subjected toimmunoprecipitation with anti-PrP antibody 3F4 or 8H4 as described(17, 37). Metabolic labeling at 15 °C was performed in a refrigeratedCO2 incubator for 2 h, and lysates were immunoprecipitated withanti-PrP antibody 2301 specific for C-terminal residues 220–230 of PrP(17, 37, 38). Anti-PrP antibody 3F4 binds to residues 109 and 112 of PrPand therefore detects truncated CtmPrP and the conventional PK-resist-ant 20-kDa fragment of PrP. Anti-PrP antibodies 8H4 and 2301 bind toboth the 18- and 20-kDa fragments of PrP (Fig. 8).

Assay of Detergent Insolubility and Proteinase K Resistance—Cellstreated with biotin-PrPScr106–126 (control) or biotin-PrP106�126 (treated)were lysed in a buffer containing nonionic detergents Nonidet P-40 (1%)and deoxycholate (0.5%). Large aggregates and nuclei were pelleted bylow speed centrifugation at 500 � g, and the low speed supernatant wasfurther ultracentrifuged at 100,000 � g in a Beckman SW50 rotor for1 h at 4 °C. The low and high speed pellet and supernatant sampleswere analyzed by Western blotting. Duplicate sets of samples wereelectroblotted and probed with 8H4, 3F4, or streptavidin-horseradishperoxidase as described above. Only the low speed supernatant fractionis shown here because all of the aggregated PrPC and PrP106–126 werefound in the low speed pellet and supernatant fractions rather than inthe high speed pellet as observed for conventional aggregated PrP.

For evaluation of proteinase K resistance, lysates of control (biotin-PrPScr106–126) or treated (PrP106–126) cells were exposed to 3 �g/ml PKfor 0, 2, or 4 min at 37 °C. The reaction was stopped with 1 mM

phenylmethylsulfonyl fluoride, and proteins were electroblotted andprobed with 3F4 as described (17, 37).

Enzymatic Deglycosylation—Deglycosylation with N-glycosidase F(PNGase-F) was performed essentially as described (17, 37).

Cell Homogenization and PK Treatment—Cells treated with biotin-PrPScr106–126 (control) or PrP106–126 (treated) were washed and homog-enized on ice in a buffer containing 10 mM HEPES (pH 7.9), 1.5 mM

MgCl2, 10 mM KCl, and 0.5 mM dithiothreitol by 20 strokes of a Kontesall-glass Dounce homogenizer. The homogenate was checked microscop-ically for cell breakage and centrifuged to pellet nuclei. The resultingsupernatant was centrifuged at 20,000 � g to pellet membrane vesicles.The pelleted vesicles were resuspended in 0.5 ml of transport buffer (25mM HEPES, pH 7.4, 115 mM KOAc, 2.5 mM MgCl2, 10 mM KCl, 2.5 mM

CaCl2, and 1 mM dithiothreitol) and treated with 20 �g/ml proteinase Kon ice for 30 min. After the addition of 5 mM phenylmethylsulfonylfluoride to stop the reaction, membrane vesicles were pelleted again,solubilized in lysis buffer, and immunoblotted with 3F4.

Staining with Thioflavin S—Cells treated with biotin-PrPScr106–126

(control) or biotin-PrP106–126 (treated) for 1 year were fixed and perme-abilized as described above and immunostained with 8H4 (1:25 dilu-tion)-anti-mouse-rhodamine isothiocyanate (1:40 dilution). Subse-quently, the cells were incubated with a 1% aqueous solution of

Induction of CtmPrP by PrPSc-like Forms in a Cell Model2276

thioflavin S for 20 min and washed three times with phosphate-bufferedsaline followed by one quick wash with 70% ethanol. Cells stained onlywith thioflavin S were prestained with the basic dye hematoxylin toreduce the background binding to nucleic acids, and processed as above.

RESULTS

PrP106–126 Induces the Aggregation and Intracellular Accu-mulation of PrPC—In a previous report, we showed that expo-sure of neuroblastoma cells to nontoxic concentrations ofPrP106–126 leads to intracellular accumulation of aggregatedpeptide, whereas scrambled PrP106–126 is degraded rapidly(16). To study the effect of long-term exposure of cells toPrP106–126, neuroblastoma cells expressing high levels of trans-fected PrPC were cultured for 4–12 months in complete me-dium containing 5 �M PrP106–126 or PrPScr106–126 biotinylatedat the N terminus. The cells were subcultured every 3 days,and the peptide was replenished. Cell viability was checkedevery day for the first month by staining with the nuclear dyeHoechst, and the rate of cell division was monitored by count-ing the number of cells every 3 days before subculturing. Atthis low concentration, the peptide dissolves easily in culturemedium at pH 7.4 and is not toxic to neuroblastoma cells evenafter prolonged periods of constant exposure.

Interaction of PrP106–126 and the corresponding scrambledpeptide with PrPC-expressing cells was studied by immunoflu-orescence analysis. Monoclonal antibody 8H4 was used to de-tect cellular PrPC and Texas red-conjugated streptavidin tovisualize biotin-tagged PrP106–126 or the corresponding scram-bled peptide. Because the epitope for 8H4 lies between residues145–180 of PrP, it does not bind to PrP106–126, and it providesa convenient method of differentiation between PrPC andPrP106–126. After 4 months of exposure to biotin-PrP106–126,cells were fixed with paraformaldehyde and immunostained forPrPC (green) or PrP106–126 (red; Fig. 1). To visualize immuno-stained surface proteins, permeabilization with detergent wasomitted. A composite confocal image was taken to detect stain-ing at different depths, including invaginations of the plasmamembrane. Small to large aggregates of PrP106–126 are evidentat the cell surface (red), most of which co-localize with PrPC

(yellow; Fig. 1A, panels 1 and 2). Some aggregates appear to beundergoing endocytosis (*, Fig. 1A, panels 1 and 2), whereasothers are larger and more diffuse (Fig. 1A, panel 2). Controlcells cultured with biotin-PrPScr106–126 show no staining for thepeptide but a uniform surface staining for PrPC with 8H4 and3F4 antibodies (Fig. 1A, panels 3 and 4). Nontransfected M17cells show no reactivity with 8H4 (Fig. 1A, panel 5).

These results were confirmed by immunoelectron micros-copy. Cells treated with biotin-PrPScr106 –126 or biotin-PrP106–126 were fixed and stained with 8H4 without prior per-meabilization as described above. The bound antibody wasreacted with the appropriate secondary antibody and DAB toobtain an electron-dense deposit as described under “Experi-mental Procedures.” Subsequent staining with uranyl acetateand lead citrate was omitted to obtain a better contrast withDAB staining. Cells cultured in the presence of scrambledpeptide show a uniformly distributed, punctate staining ofPrPC on the cell surface (PM: Fig. 1A, panel 6), whereasPrP106–126-treated cells show localized aggregation of PrPC atthe plasma membrane Fig. 1A, panels 7 and 8). The aggregatesincrease in size gradually and are internalized in large, mem-brane-bound vesicles (Fig. 1A, panels 9–11). In some cases theaggregates are so large as to distort the nuclear membrane(Fig. 1A, panel 10).

The intracellular localization of aggregated PrP106–126 andPrPC was assessed by immunofluorescence analysis of TritonX-100-permeabilized cells. Cells treated with scrambled pep-tide or PrP106–126 for 4 months were immunostained with 8H4

and Texas red-conjugated streptavidin as described above.Composite confocal images were taken to visualize the aggre-gates at different depths in the cell. Control cells treated withscrambled peptide show the expected intracellular immunore-activity of PrPC in the Golgi area (Fig. 1B, panel 1), whereasPrP106–126-treated cells show aggregates of PrP106–126 (red)and PrPC (green) or complex aggregates containing PrPC andPrP106–126 (yellow) at the cell surface and in intracellular ves-icles (Fig. 1B, panels 2 and 3). Immunostaining with an alter-nate anti-PrP antibody 3F4 shows similar results. Scrambledpeptide-treated cells show normal PrP immunoreactivity (Fig.1B, panel 4), whereas PrP106–126-treated cells show large in-tracellular aggregates with immunoreactivity for PrPC (green)(Fig. 1B, panels 5 and 6). (Because 3F4 also binds to PrP106–126

(at residues 109 and 112), the aggregates with a yellow stainingpattern could be combined aggregates of PrP106–126-PrPC or theaggregated peptide alone.)

Immunoelectron microscopy shows a uniformly distributedpunctate staining of PrPC on the plasma membrane of cellstreated with scrambled peptide (Fig. 1B, panel 7). In contrast,PrP106–126-treated cells show large intracellular aggregates ofPrPC enclosed by a membrane (Fig. 1B, panel 8), in some caseslarge enough to distort the nucleus. Nontransfected M17 cellstreated with PrP106–126 and processed in parallel show noreactivity for PrPC (Fig. 1B, panel 9).

The above described results show that exogenously addedPrP106–126 induces the aggregation and internalization of cellsurface PrPC in large membrane-bound vesicles. The aggre-gates degrade slowly and cause a reactive proliferation of lyso-somes in cells treated with PrP106–126 (Fig. 1C, panel 2) ascompared with controls exposed to PrPScr106–126 (Fig. 1C,panel 1).

Aggregates of PrPC Are Insoluble in Nonionic Detergents—Insolubility in non-ionic detergents is one of the earliestchanges observed during the transition of PrPC to PrPSc. Con-ventionally, detergent insolubility is determined by preparingcell lysates in a buffer containing Nonidet P-40 (1%) and de-oxycholate (0.5%), and subjecting the lysates clarified at lowspeed (500 � g) to ultracentrifugation at 100,000 � g. Aggre-gated PrP is usually detected in the pellet fraction of ultracen-trifuged samples. To check whether aggregated PrPC is insol-uble in non-ionic detergents, control cells cultured withscrambled peptide and cells exposed to PrP106–126 for 4–12months were subjected to the above treatment, and the pelletand supernatant fractions from low and high speed centrifuga-tion were immunoblotted with streptavidin or 8H4 to detectPrP106–126 or PrPC, respectively. Because most of the aggre-gates partitioned in the low speed pellet and supernatant frac-tion, the supernatant obtained after centrifugation at 500 � gis shown in Fig. 2A. Immunoreaction with 8H4 and 3F4 anti-bodies shows normal PrP glycoforms for both control cells cul-tured with biotin-tagged PrPScr106–126 and treated cells cul-tured in the presence of biotin-tagged PrP106–126 (Fig. 2A, lanes1–4). In addition, protein bands that react with both 8H4 and3F4 antibodies are detected in the stacking gel of PrP106–126-treated cells (Fig. 2A, lanes 2 and 4, overexposed lanes 9 and11). Strikingly, the PrP-reactive bands cross-react withstreptavidin (Fig. 2A, lane 7), indicating that the protein ag-gregates in PrP106–126-treated cells are too large to be resolvedby the stacking or the separating gel and that these are com-plex aggregates of PrPC and PrP106–126 (Fig. 2A, lanes 2, 4, 7, 9,and 11, top two arrows). A significant amount of monomeric,detergent-soluble PrP106–126 is detected at �3 kDa in treatedlysates (Fig. 2A, lanes 7 and 11) that co-migrates with purifiedPrP106–126 (Fig. 2A, lane 5), confirming that PrP106–126 addedto the culture medium is not pre-assembled into large aggre-

Induction of CtmPrP by PrPSc-like Forms in a Cell Model 2277

FIG. 1. PrP106–126 induces the aggregation and internalization of cell surface PrPC. A, surface immunofluorescence analysis of cells treated withbiotin-PrP106–126 for 4 months with 8H4-fluorescein isothiocyanate to identify PrPC (green) followed by Texas red-streptavidin to detect biotin-PrP106–126

(red) shows co-localization of PrPC and PrP106–126 aggregates on the plasma membrane (panels 1 and 2). Large aggregates of irregular shape that co-stainfor PrPC and PrP106–126 (panel 2) and smaller aggregates that appear to be undergoing endocytosis are evident (panels 1 and 2, insets). Immunostaining ofcontrol cells treated with biotin-PrPScr106–126 for a similar period of time with 3F4 or 8H4 shows a normal pattern of PrP immunoreactivity on the cell surfaceand in the Golgi area (panels 3 and 4). Nontransfected M17 cells treated with biotin-PrP106–126 and immunostained with 8H4 show no PrP immunoreactivity(panel 5). Bar, panels 1 and 2, 25 �m; insets, 10 �m. Immunoelectron microscopy of the above cells using 8H4 shows a uniform, punctate staining of PrPC

on the surface of control cells treated with biotin-PrPScr106–126 (panel 6). In contrast, cells treated with biotin-PrP106–126 show aggregates of PrPC on the cellsurface (panels 7 and 8) that are gradually internalized in membrane-enclosed vesicles (panels 9–11). In some cells, the aggregates assume large proportions(panels 9–11), enough to distort the nucleus (panel 10). The aggregate in panel 10 is in a plasma membrane invagination, although it appears intracellular.PM, plasma membrane; N, nucleus. Bar, 1 �m. B, immunofluorescence analysis of Triton-permeabilized cells treated with biotin-PrPScr106–126 (control) orbiotin-PrP106–126 with 8H4 shows normal staining pattern of PrP in control cells (green, panel 1). Cells treated with PrP106–126 show engorgement with PrPC

(green), PrP106–126 (red), and combined (yellow) aggregates (panels 2 and 3). A similar analysis with 3F4 shows normal PrP staining pattern in control cells(green, panel 4), whereas PrP106–126-treated cells show intracellular aggregates of PrPC (green) or PrP106–126 (red). (Because 3F4 cross-reacts with PrP106–126,the yellow aggregates may represent only PrP106–126 mixed aggregates as in panels 2 and 3; immunoelectron microscopy of Triton-permeabilized cells showssimilar results. A uniform, punctate reaction of PrPC can be detected on the plasma membrane of cells treated with biotin-PrPScr106–126 (panel 7). In contrast,biotin-PrP106–126-treated cells show large intracellular aggregates of PrPC (panel 8). Nontransfected M17 cells treated with PrP106–126 show no PrP reactivity

Induction of CtmPrP by PrPSc-like Forms in a Cell Model2278

gates. Thus, the PrPC aggregates in treated cells are large andinsoluble in non-ionic detergents and SDS. The aggregated PrPconstitutes less than 10% of the total cellular PrP, which, asshown above, is detected in the detergent-soluble supernatantfraction. (A longer exposure of lanes 1–4 is shown in lanes 8–11

to show PrP106–126/PrPC aggregates in the stacking gel.)To corroborate the above results, cells treated with biotin-

PrP106–126 or biotin-PrPScr106–126 for 4–12 months were platedon glass coverslips and treated with Triton X-100 (0.1%) for 5min on ice prior to fixation with paraformaldehyde. The resid-

FIG. 1—continued

(panel 9). Bar, panels 1–6, 25 �m; panels 7–9, 1 �m. C, electron microscopy of cells treated with biotin-PrPScr106–126 (control) or biotin-PrP106–126

for 1 year shows extensive proliferation of electron-dense lysosomes in PrP106–126-treated cells (panel 2) compared with the control sample (panel1). There are no obvious signs of cellular toxicity, such as swollen mitochondria or condensation of nuclear chromatin in treated cells (panel 2). PM,plasma membrane; N, nucleus; L, lysosomes; M, mitochondria; G, Golgi apparatus. Bar, 0.7 �m.

Induction of CtmPrP by PrPSc-like Forms in a Cell Model 2279

ual insoluble material was immunostained with 8H4 andstreptavidin. Detergent-insoluble aggregates of PrPC (green)and PrP106–126 (red) are detected in residual cell structures,some of which appear to be membrane ghosts (Fig. 2B, panel 1),whereas others bear the round shape of cellular organelles thathave been solubilized by the detergent (Fig. 2B, panel 2). Cellstreated with scrambled peptide show only occasional mem-brane ghosts, but conspicuously they lack large aggregates(Fig. 2B, panel 3).

Aggregated PrPC Binds the Amyloid-specific Dye ThioflavinS—To evaluate whether intracellular aggregates of biotin-PrP106–126 and PrPC bind the amyloid-specific dye thioflavin S,cells treated with biotin-PrP106–126 for �1 year were fixed inparaformaldehyde, permeabilized with detergent, and stainedwith the basic dye hematoxylin followed by the amyloid-bind-ing dye thioflavin S. Green fluorescence of thioflavin-stainedaggregates can be seen in intracellular structures (Fig. 3, pan-els 1–4), similar to the large endocytic vesicles observed in Fig.1, A and B. Co-immunostaining with 8H4 followed by rhoda-

mine isothiocyanate-conjugated secondary antibody shows thatmost of the thioflavin-positive aggregates co-immunostain forPrPC (Fig. 3, panels 3 and 4, yellow). Some of the intenselythioflavin-positive aggregates do not show PrPC staining andare either adjacent to or surrounded by aggregated PrPC (Fig.3, panels 2 and 3). These aggregates do not co-stain with thePrP106–126 marker streptavidin either (data not shown), sug-gesting that the epitopes for these indicators are not accessiblebecause of aggregation or change in conformation of PrPC andPrP106–126. No thioflavin-positive staining was detected in un-treated cells or in cells treated with scrambled peptide (datanot shown).

Immunoelectron microscopy of cells treated with PrP106–126

for �1 year shows a large intracellular aggregate that immu-noreacts with 8H4, showing amyloid-like fibrils in the center ofthe aggregate (Fig. 3, panel 5, arrow) . Thus, after prolongedexposure of cells to PrP106–126, the internalized aggregates ofboth PrP106–126 and PrPC acquire some of the properties ofamyloid.

FIG. 2. Aggregates of PrPC are insoluble in non-ionic detergents. A, lysates prepared from cells treated with biotin-PrPScr106–126 (Control)or biotin-PrP106–126 (Treated) for 4–12 months were centrifuged at low speed (500 � g), and the supernatant was immunoblotted with 8H4 (lanes1 and 2), 3F4 (lanes 3 and 4), or streptavidin (lanes 6 and 7). Purified biotin-PrP106–126 was added as a control in lane 5. Normal PrP glycoformsconsisting of unglycosylated (27 kDa), intermediate, and highly glycosylated forms are detected in 3F4 and 8H4 immunoblots (lanes 1–4). Inaddition, as expected, the 18- and 20-kDa fragments of PrP are evident in the 8H4 blot (lanes 1 and 2), whereas only the 20-kDa fragment is seenin the 3F4 blot (lanes 3 and 4). More importantly, treated samples show protein aggregates in the stacking gel and at the top of separating gel thatimmunoreact with 8H4, 3F4, and streptavidin (lanes 2, 4, and 7 or over-exposed lanes 9, 11, and 7). These aggregates are notably absent in controllysates (lanes 1, 3, and 6 or over-exposed lanes 8 and 10). A 3-kDa band of monomeric PrP106–126 can be detected by streptavidin in treated cells(lane 7) which, as expected, immunoreacts with 3F4 (lane 11) and co-migrates with purified PrP106–126 (lane 5). B, cells treated with biotin-PrP106–126

for 4 months were plated on glass coverslips and treated with Triton X-100 on ice prior to fixation with paraformaldehyde. The residual membranesand insoluble cell debris were immunostained with 8H4-anti-mouse-fluorescein isothiocyanate to visualize PrPC (green) or with Texas red-streptavidin to detect PrP106–126 (red). Detergent-insoluble aggregates of PrPC that co-localize with PrP106–126 can be seen on residual membranesand as large aggregates (panels 1 and 2). Cells treated with biotinPrPScr106–126 and processed similarly show immunostaining of residualmembranes, conspicuously lacking any aggregates of PrPC or PrP106–126 (panel 3). Bar, 10 �m.

Induction of CtmPrP by PrPSc-like Forms in a Cell Model2280

Primary Cultures of Human Neurons Show Similar Aggre-gation of PrPC—Although the neuroblastoma cells used abovetolerate relatively large intracellular aggregates of PrPC andPrP106–126 without significant toxicity (assessed by Hoechststaining), these are tumor cells that divide in culture and donot exactly replicate the metabolism of primary neuronal cells.To confirm whether similar aggregation of PrPC can be inducedin primary neurons, mixed cultures of human brain cells pre-pared from discarded fetal tissue and enriched for neuronswere treated with biotin-PrP106–126 for 2 weeks as describedabove. The cells were washed thoroughly to remove precipi-tated peptide, fixed, permeabilized, and immunostained forPrPC and PrP106–126 as described above. Parallel cultures wereimmunostained with anti-glial fibrillary acidic protein (anti-GFAP) and anti-neurofilament (anti-NF68)-specific antibodiesto identify specific cell types in the mixed brain cultures. Ac-cumulation of PrPC and PrP106–126 can be seen in neuronal cellbodies, neuronal processes, and astrocytes (Fig. 4, panels 1, 2,and 3, respectively). Most of the intracellular deposits co-stain

for PrPC (green) and PrP106–126 (red) (Fig. 4, panels 1–3,yellow), indicating the presence of mixed aggregates as seenabove for neuroblastoma cells. Most of the cells in primarybrain cultures show nuclear fragmentation after 4 weeks ofPrP106–126 treatment, as opposed to cells cultured withPrPScr106–126, which are healthy for up to 8 weeks (data notshown).

A C-terminal 20-kDa Fragment of PrPC Accumulates on theSurface of PrP106–126-treated Cells—To investigate the metab-olism of aggregated PrPC in cells treated with PrP106–126, equalnumber of cells treated with PrPScr106–126 (control) or PrP106–126

(treated) for 4–12 months were radiolabeled with [35S]methi-onine overnight and treated with PI-PLC to cleave cell surfaceGPI-linked proteins. Both the lysate and PI-PLC-released sam-ples were subjected to immunoprecipitation with anti-PrP an-tibody 3F4 (specific to residues 109–112). Analysis by SDS-PAGE fluorography shows significantly more PrP in the lysatesample of PrP106–126-treated cells compared with controls, in-dicating that some of the intracellular accumulated PrPC issoluble and immunoprecipitable before it finally aggregates(Fig. 5A, lanes 1 and 2). The amount of PrPC released from thecell surface is similar in control and treated cells, except for asignificant increase in the 20-kDa fragment in treated cells(Fig. 5A, lanes 3 and 4). This fragment becomes more promi-nent after deglycosylation with PNGase-F (Fig. 5A, lanes 5 and6), indicating the presence of glycosylated forms of 20 kDa onthe cell surface, which accumulate in treated cells. Radiolabel-ing with the GPI anchor component [3H]ethanolamine and

FIG. 3. Intracellular PrP106–126 and PrPC aggregates bind thio-flavin S. Cells treated with biotin-PrP106–126 for 1 year were fixed,permeabilized with Triton X-100, and stained with thioflavin S to detectamyloid (green, panel 1) or immunostained with 8H4-anti-mouse-rho-damine isothiocyanate to detect PrPC (red) followed by staining withthioflavin S (panels 2–4). Green fluorescence of thioflavin S-positiveaggregates of different sizes is seen in vesicular structures, some moreintense than others (panel 1). Co-immunostaining for PrPC shows co-localization in some cells (panels 3 and 4, yellow), whereas in others thegreen fluorescence of amyloid is surrounded by PrPC (panel 2). Immu-noelectron micrograph of PrP106–126-treated cells reacted with 8H4-peroxidase-DAB shows an intracellular vesicle containing aggregatedPrP with a fibrillar, amyloid-like appearance in the center (panel 5,arrow). Inset, 0.5 �m. Bar, panels 1–4, 25 �m; panel 5, 1 �m.

FIG. 4. Primary cultures of human neurons show similar ag-gregation of PrPC. Mixed cultures of human brain cells treated withbiotin-PrP106–126 for 2 weeks were immunostained for PrPC andPrP106–126. Aggregates of PrPC (green) and PrP106–126 (red) can be seenin neuronal cell bodies (panel 1), in axonal processes and nodal swell-ings (panel 2), and in astrocytes (panel 3). Most of the intracellulardeposits co-stain for PrPC and PrP106–126 (yellow). Bar, 25 �m.

Induction of CtmPrP by PrPSc-like Forms in a Cell Model 2281

immunoprecipitation with 3F4 confirms that the 20-kDa frag-ment is GPI-anchored and therefore C-terminal (Fig. 5, lanes 7and 8). Quantitative estimation of the relative percentage of20-kDa fragment in comparison with full-length PrP formsshows that in control cells, 20-kDa constitutes 2% of the totalPrP, more than half of which is at the cell surface (Fig. 5B,lanes 1 and 3). After treatment with PrP106–126, the totalamount of 20 kDa increases to 10%, more than half of which isat the cell surface (Fig. 5B, lanes 2 and 4). Treatment of PI-PLC-cleaved surface proteins with PNGase-F shows that theamount of 20-kDa fragment on the surface of treated cells is atleast four-fold more compared to control cells (Fig. 5B, lanes 5and 6). A similar ratio of 20-kDa versus full-length PrP isobserved in ethanolamine-labeled samples (Fig. 5B, lanes 7 and8).

The C-terminal 20-kDa Fragment Is Truncated CtmPrP—Previous reports have documented that PrPC exists in differenttopological forms at the ER. Increased synthesis of one of thetransmembrane forms, CtmPrP, is believed to mediate the neu-rodegeneration observed in certain inherited and infectiousprion disorders (7, 8). As opposed to PrPC, CtmPrP is inserted inthe ER membrane through its hydrophobic domain, includingresidues 113–135, with its N terminus in the cytosol and Cterminus in the ER lumen. Thus, PK treatment of microsomesspares an �19-kDa C-terminal fragment, which is significantlyincreased in brain tissue obtained from diseased animals (7).Because this fragment retains reactivity to 3F4, the proteolyticclip must spare residue 109. To determine whether the 20-kDafragment observed in PrP106–126-treated cells is the proteolyti-cally cleaved CtmPrP, microsomes prepared from cells culturedin the presence of PrPScr106–126 (control) or PrP106–126 (treated)for 4–12 months were treated with 20 �g/ml PK on ice for 30min as described in previous reports (7, 8). The reaction wasstopped with phenylmethylsulfonyl fluoride, and the micro-somes were solubilized in detergent and subjected to immuno-blotting with 3F4 or anti-calnexin antibody. As expected, full-length GPI-linked forms of PrPC are protected from theprotease in both control and treated cells (Fig. 6A, lanes 2 and4). In contrast, there is a small but significant increase in the20-kDa fragment after protease digestion of treated cells (Fig.6A, lane 3 versus 4), indicating that the 20-kDa fragment isgenerated by limited proteolysis of CtmPrP and, more im-portantly, that the number of PrP molecules inserted in theCtmPrP orientation is higher in treated cells than in controls.Cleavage of calnexin to produce a faster migrating specieslacking the cytosolic C-terminal domain confirms the efficiencyof PK treatment and the intactness of the microsomes (Fig. 6A,lanes 2 and 4). Because the number of PrPC molecules in theCtm orientation constitute less than 2% of the total PrPC inChinese hamster ovary and baby hamster kidney cells (34), theincrease observed in peptide treated cells is noteworthy. Aquantitative estimation of the percentage of 20-kDa fragmentcompared with full-length PrP forms shows a 4-fold increase inthe 20-kDa fragment in treated cells as compared with thecontrols (Fig. 6C, lane 1 versus 3). More significantly, proteasetreatment of the control sample results in an increase of �2%in the amount of 20 kDa, as compared with a 33% increase inthe treated sample (Fig. 6C, lanes 1 versus 2 and 3 versus 4). (Itis difficult to visualize the 20-kDa fragment in control cells byimmunoblotting, but is detected by densitometry.)

To confirm that the 20-kDa fragment indeed originates in theER, control and treated cells were radiolabeled for 2 h at 15 °Cto block transport of secretory proteins from the ER. Lysateswere subjected to immunoprecipitation with anti-PrP antibody2301 and analyzed by SDS-PAGE-fluorography. Because trans-port of PrP beyond the ER-cis-Golgi is blocked under theseconditions, as expected, only PrP glycoforms with high man-nose core glycans are detected. The highly glycosylated formand the 18-kDa C-terminal fragment that is a product of nor-mal recycling of PrP from the plasma membrane are also ab-sent (Fig. 6B, lanes 1 and 2). However, there is a clear accu-mulation of the 20-kDa fragment, which is significantly more intreated cells as compared with the control sample (Fig. 6B, lane1 versus 2). Because 2301 is specific for the C-terminal residues220–230 of PrP, these results confirm that the 20 kDa is indeeda C-terminal fragment of PrP, and it originates in the ER. Uponquantitative analysis, the 20-kDa fragment constitutes 0.8% oftotal full-length PrP in control cells, as compared with 10.5% intreated cells, an increase of �10-fold due to treatment of thecells with PrP106–126 (Fig. 6D, lanes 1 and 2).

To exclude the possibility that 20 kDa is a proteolytic product

FIG. 5. A C-terminal 20-kDa fragment of PrPC accumulates onthe surface of PrP106–126-treated cells. A, cells treated with biotin-PrPScr106–126 (Control) or biotin-PrP106–126 (Treated) for 4–12 monthswere radiolabeled overnight with [35S]methionine and treated withPI-PLC to cleave surface-expressed PrP. Lysate and PI-PLC-releasedsamples were immunoprecipitated with 3F4 and deglycosylated withPNGase-F. The amount of PrPC immunoprecipitated from treated cellsis significantly more than with control cells (lane 1 versus 2). The20-kDa fragment of PrP is detected only in treated cells (lane 2) but issignificantly more in the PI-PLC-released sample compared with cell-associated proteins (lane 2 versus 4). The intensity of the 20-kDa frag-ment increases following PNGase-F treatment, indicating the presenceof glycosylated forms that migrate at 20-kDa when glycans are removed(lane 6). Radiolabeling with the GPI anchor component [3H]ethano-lamine and immunoprecipitation with 3F4 show that the 20-kDa frag-ment is GPI-linked (lane 8). B, quantitative measurement of the 20-kDafragment versus full-length PrP forms in control and treated sampleswas done by densitometric scanning of autoradiograms as described inA above. The data represent the percentage of 20-kDa fragment incomparison with total full-length PrP in each lane. The differencesamong mean values were tested by analysis-of-variance for repeatedmeasurements. Statistical analysis within groups was carried out usingStudent’s t test. Each bar represents the mean � S.D. of three experi-ments. *, p � 0.01.

Induction of CtmPrP by PrPSc-like Forms in a Cell Model2282

of full-length PrP in the ER, PrPC cells were pulsed for 30 minat 37 °C, and chased for 4 h at the same temperature in thepresence of brefeldin-A to block transport of proteins from the

ER. The 20-kDa fragment is detected soon after the pulse, butdoes not accumulate with chase, indicating that it does notarise from full-length PrPC (data not shown). If the brefeldinblock is removed and radiolabeled proteins are allowed to exitthe ER, the 20-kDa fragment, along with the full-length PrPC

forms, can be recovered from the cell surface by PI-PLC treat-ment after 1 h of chase, and follows similar kinetics of turnoveras PrPC (data not shown).

Together, the above results show that the 20-kDa fragmentis not a proteolytic product of full-length PrPC in the ER.Instead, it originates from an alternate form of PrP, probablyCtmPrP, which is up-regulated in PrP106–126-treated cells. The20-kDa fragment is probably generated because of spontaneouscleavage of CtmPrP at the cytosolic face of the ER, and istransported along the secretory path to the plasma membrane.

Aggregated PrPC Is Resistant to Digestion by ProteinaseK—Resistance to limited digestion by PK is considered thehallmark of PrPSc, and is one of the primary diagnostic testsavailable at this time for identifying prion-infected tissue. Al-though the amount of PK used for evaluating PK-resistant PrPin cell models is orders of magnitude lower, and the conditionsless harsh than the ones used to detect PrPSc in the brain, thistest allows an evaluation of a change in conformation of PrPC,an important step toward the final transformation to PrPSc. Toevaluate if aggregated PrPC generated in our cell model isPK-resistant, lysates of control and treated cells were exposedto 3 �g/ml of PK for 0, 2, or 4 min. The protease-resistantproteins remaining in the lysate were precipitated with coldmethanol and analyzed by immunoblotting with 3F4. In thecontrol sample treated with scrambled peptide, PrPC is di-gested completely after 4 min of PK treatment, with only tracesof the unglycosylated form remaining (Fig. 7, lane 1 versus 3).In contrast, a significant amount of PrP from cells treated withPrP106–126 is resistant to PK-digestion after 4 min at 37 °C(Fig. 7, lane 5 versus 7). Deglycosylation of 2 min PK-treatedsamples with PNGase-F shows that the resistant species intreated cells comprises of full-length PrPC and a 20-kDa C-terminal fragment of PrP, both of which are at least 10-foldmore in treated cells as compared with the control sample (Fig.7, lane 4 versus 8). Because the PK-resistant fragment of PrPSc

has a ragged N terminus near residue 90 (21) and CtmPrP isalso cleaved at around residue 104 in intact microsomes (7),both fragments would have a similar molecular mass of �20kDa on SDS-PAGE, making it difficult to distinguish betweenthe two. However, judging from the sensitivity of truncatedCtmPrP to PI-PLC cleavage and its solubility in non-ionic de-tergents (data not shown), it does not appear to be aggregatedor PK-resistant. Thus, the PK-resistant 20-kDa fragment de-tected above represents the digested product of aggregatedPrPC rather than truncated CtmPrP. In addition, a considerableamount of full-length PrP is also resistant to mild proteasedigestion in the treated sample, probably because of its state ofaggregation.

DISCUSSION

This report extends the current understanding of possiblemechanisms of PrPSc-induced aggregation of PrPC and conse-quent neurodegeneration in three distinct ways. First, we showthat a �-sheet-rich peptide of PrP catalyzes the aggregation ofendogenous full-length PrPC in a cell model, confirming thenucleation hypothesis of PrP aggregation. Second, we demon-strate that intracellular accumulation of PrP aggregates leadsto up-regulation of the synthesis of CtmPrP, and finally, weshow that majority of CtmPrP accumulates as a C-terminalfragment on the cell surface. These results suggest that theactive mediator of neurotoxicity in prion disorders is perhapstruncated CtmPrP, which probably accentuates the neurotoxic

FIG. 6. The C-terminal 20-kDa fragment represents truncatedCtmPrP. Cells treated with biotin-PrPScr106–126 (Control) or biotin-PrP106–126 (Treated) for 4–12 months were homogenized, and the re-sulting microsomes were treated with 20 �g/ml PK for 30 min on ice.After inactivating PK, microsomes were solubilized with detergent andimmunoblotted with 3F4 or anti-calnexin antibody. As expected, full-length PrPC is protected from PK digestion in all samples (lanes 1–4).The amount of 20-kDa fragment is too low to be detected in control cellsby immunoblotting either before or after PK treatment (lanes 1 and 2).In peptide-treated cells, on the other hand, the 20-kDa fragment in-creases in amount after PK treatment of microsomes (lanes 3 and 4). Asexpected, the C terminus of calnexin is cleaved by PK, increasing itsmigration by �10 kDa (lanes 2 and 4). B, control and treated cells asabove were radiolabeled with [35S]methionine for 2 h at 15 °C, lysed,and subjected to immunoprecipitation with anti-C-terminal PrP anti-body 2301 to detect both the 18- and 20-kDa fragments. As expected,full-length PrP forms do not exit the ER, indicated by the absence ofhighly glycosylated forms of PrP in both cell lines (lanes 1 and 2).Strikingly, in comparison with control cells, there is a marked increasein the 20-kDa fragment in peptide-treated cells (lane 1 versus 2). A faintband of 18 kDa is detected in peptide-treated cells (arrowhead) probablybecause of the exit of a small amount of PrP to the plasma membrane(*, lanes 1 and 2). C and D, quantitative analysis of the amount of20-kDa versus full-length PrP forms in control and treated samples wascarried out by densitometric scanning of autoradiograms as describedin A and B above. The data represent the percentage of 20-kDa frag-ment in comparison with total full-length PrP. The differences amongmean values were tested by analysis-of-variance. Statistical analysiswithin groups was carried out by Student’s t test. Each bar representsthe mean � S.D. of three experiments. *, p � 0.01.

Induction of CtmPrP by PrPSc-like Forms in a Cell Model 2283

effect of intracellular aggregated PrP by initiating the aggre-gation of additional PrPC molecules at the cell surface, orfunctions as a novel receptor for as yet unidentified factors thataccelerate the neurodegenerative process.

The Conformational Change of PrPC to PrPSc—This report isthe first direct demonstration of a change in conformation ofendogenous PrPC into an aggregated, partially PK-resistantform by an exogenously added �-sheet rich peptide of PrP.Although the cellular and biochemical processes of PrPSc prop-agation and neuronal toxicity remain contentious, substantialevidence indicates that transmission of PrPSc occurs by induc-ing the conversion of host PrPC into the PrPSc conformation.Two largely unsubstantiated hypotheses have been proposed toexplain the mechanism of this conformational change: 1) tem-plate-assisted conversion or re-folding of endogenous PrPC toPrPSc, and 2) the nucleation or seeding hypothesis (22, 23). Weshow that a �-sheet-rich internal fragment of PrP, PrP106–126

seeds the aggregation of PrPC on the plasma membrane of cells,and catalyzes its accumulation in an exponential manner. Webelieve that PrP106–126 intercalates within the lipid bilayer,where the charged phospholipid environment induces its ag-gregation. Here, it acts as a seed for the deposition of additionalPrPC molecules. Because �-sheet rich peptides have an affinityfor cholesterol (9, 24), aggregated PrP106–126 probably concen-trates in cholesterol-rich lipid domains of the plasma mem-brane. Incidentally, this is also the preferred location for en-dogenous PrPC and the site for PrPC to PrPSc conversion (25–27), thus providing an ideal environment for the interaction ofPrP106–126 and PrPC, and subsequent aggregation of the latter.

Following the initial aggregation of PrPC at the plasma mem-brane, the complex aggregates of PrPC and PrP106–126 aretransported to the endosomal/lysosomal compartment, wherelow pH and the negatively charged membrane microenviron-ment probably plays a major role in promoting their transfor-mation to thioflavin-S-binding, �-sheet rich structures. Suchan accumulation of virtually nondegradable PrPC andPrP106–126 aggregates explains the extensive proliferation oflysosomes observed in our cell model, and the presence ofabundant PrP immunoreactive lysosomes in the neurons of

scrapie-infected animals and new variant Creutzfeldt-Jakobdisease patients (25, 28). A similar change in conformationfrom an �-helical to a �-sheet structure has been reported forrecombinant PrP91–231 and for certain other peptides whenexposed to an acidic pH (24, 29) and is consistent with thenotion that lysosomotropic agents inhibit the accumulation andbranched polyamines stimulate the degradation of PrPSc inscrapie-infected cells (30, 31).

The intracellular aggregates of PrPC thus generated displayseveral of the biochemical characteristics typical of PrPSc-de-tergent insolubility, partial resistance to digestion by protein-ase K (PK), and amyloid-like characteristics as demonstratedby thioflavin-S binding. Some of the aggregated PrPC is insol-uble even in SDS, and cannot be resolved by conventionalSDS-PAGE. However, as opposed to conventional PrPSc thatcan transmit disease, the apparently similar biochemical char-acteristics of PrPC aggregates observed in our cell model depicta particular conformational state of PrPC rather than infectiv-ity per se. Whether this conformational state will eventuallyevolve into other PrP conformations leading to PrPSc is unclear.It is interesting to note that clinical signs of prion disease havebeen produced in transgenic 196 mice injected with MoPrPpeptide (89–143, P101L) without significant correlation withPK-resistant PrPSc deposition (32), in part supporting the re-sults obtained in our study. A similar aggregation of PrPC inprimary human fetal brain cells exposed to PrP106–126 confirmsour results in neuroblastoma cells, and suggests the possibilityof a similar sequence of events during infection of animals andhuman beings exposed to exogenous PrPSc infection. The ag-gregates of PrP106–126 and PrPC in brain cells are not onlypresent in neuronal and glial cell bodies, but also in neuronalprocesses and axonal swellings. It is plausible that these ag-gregates travel along axons to neighboring cells by vesiculartraffic, or, alternately, are extruded into the extracellular mi-lieu and are subsequently internalized by other cells. Thus,intracellular PrP aggregates may be directly toxic to neurons,or alternately, the surrounding glial cells may release cyto-kines and other factors that participate in the observed neuro-toxicity. Microglia cultured in the presence of A� show similar

FIG. 7. Aggregated PrPC is resistantto digestion by proteinase K. Lysatesof cells treated with biotin-tagged scram-bled PrP106–126 (Control) or biotin-taggedPrP106–126 (Treated) for 4–12 monthswere exposed to 3 �g/ml PK for 0, 2, and 4min at 37 °C, and the 2-min sample wasfurther subjected to deglycosylation withPNGase-F. Immunoblotting with anti-PrP antibody 3F4 shows almost completedigestion of PrPC in control lysates after 2and 4 min of PK treatment (lane 1 versuslanes 2 and 3), whereas a significantamount of undigested PrP is present inpeptide-treated lysates (lane 5 versuslanes 6 and 7). Deglycosylation of 2-minPK-digested samples shows an increasedamount of 20-kDa fragment in peptide-treated cells (lane 4 versus 8). (3F4 is spe-cific to PrP residues 109–112 included inthe PK-resistant fragment of PrPSc.)

Induction of CtmPrP by PrPSc-like Forms in a Cell Model2284

results. Aggregated A� accumulates in lysosomes and is ulti-mately released from the cell without degradation (33). Be-cause most PrPSc in diseased tissue is likely sequestered inplaques and therefore unable to interact with neighboring cells,the propagation of PrPSc may occur through fragments likePrP106–126 that are relatively soluble, resistant to proteases,and have the potential to fold into �-sheet upon contact withthe membrane micro-environment. The occurrence of such aphenomenon would explain the spread of PrPSc infection in thebrain in an exponential manner.

CtmPrP As a Mediator of Neurotoxicity by PrPSc—This studyshows, for the first time, that intracellular accumulation ofaggregated PrPC up-regulates the synthesis of CtmPrP, theproposed mediator of neurodegeneration in certain inheritedand infectious prion disorders (7, 8). Several lines of evidencesupport our assertion that the 20-kDa C-terminal fragment ofPrP that accumulates on the surface of PrP106–126 treated cellsarises from CtmPrP. 1) PK treatment of intact microsomesprepared from peptide-treated cells shows a small but signifi-cant increase in the expected 20-kDa C-terminal fragment, anda further increase in peptide treated cells, confirming its trans-membrane orientation with N terminus in the cytosol. In PrP-expressing Chinese hamster ovary and baby hamster kidneycells, �2% PrP has been reported to exist in the transmem-brane orientation (34), consistent with the small increase in the20-kDa fragment observed in our cell model. 2) There is at leasta 4-fold increase in the surface expression of 20-kDa fragmentin PrP106–126-treated cells that is similar in molecular massand immunoreactivity to the fragment of CtmPrP obtained afterPK treatment. 3) The amount of 20-kDa and not full-length PrPincreases in the presence of a proteasomal inhibitor,2 consist-ent with its origin from CtmPrP, which is normally degraded bythe proteasomes (35). 4) The 20-kDa fragment is GPI-linked (asreported for CtmPrP) and is transported to the plasma mem-brane within 1 h of synthesis.2 5) This fragment does notaccumulate with chase under conditions in which transport ofPrP from the ER is blocked, arguing against its origin fromfull-length PrP.2 6) Finally, in a separate cell model expressingmutant PrP(S231T), which lacks the GPI anchor and aggre-gates in the ER, a similar 20-kDa fragment is generated that,remarkably, contains an intact GPI anchor.3 BecausePrPS231T is anchorless, the GPI-linked 20 kDa probably arisesfrom CtmPrP, which, because of its transmembrane orientation,is probably in a suitable conformation to receive the GPI anchordespite the mutation at codon 231. Overall, the evidence pre-sented above strongly favors the possibility that the 20-kDafragment is derived from CtmPrP and not from fully translo-cated full-length PrP in the ER.

Although the involvement of CtmPrP in mediating neurotox-icity has already been demonstrated by an indirect method inmice infected with PrPSc (8), this study is the first direct dem-onstration of a correlation between intracellular accumulationof PrP aggregates and up-regulation of CtmPrP synthesis. Thefact that majority of CtmPrP is cleaved spontaneously on thecytosolic face of the ER and the truncated C-terminal fragmentis expressed on the cell surface provides novel and importantinformation about the metabolism of CtmPrP and possiblemechanisms of neuronal toxicity. A recent study suggests thatCtmPrP perhaps exerts its neurotoxic effect through cell mem-brane perturbation (36). However, truncated CtmPrP in our cellmodel is GPI-linked and does not appear to be inserted throughthe transmembrane domain because it is released into themedium by PI-PLC treatment. Perhaps cleavage of the N-

terminal domain of CtmPrP at the ER membrane destabilizesits association with the membrane at nearby residues 113–135of PrP, leaving GPI anchor as the sole membrane linkage of the20-kDa truncated CtmPrP. Moreover, because CtmPrP has beenreported to partition into the aqueous phase of Triton X-114partitioning after cleavage of the GPI anchor (34), the trans-membrane domain does not appear to be hydrophobic enoughto maintain membrane association by itself. Thus, truncatedCtmPrP must use an alternative mechanism for neurotoxicity inour cell model. Because this fragment includes the amyloido-genic region of PrP, comprising residues 106–126, that is nor-mally disrupted during recycling of PrP from the cell surface(38), the truncated CtmPrP may initiate aggregation of addi-tional PrPC molecules at the cell surface by acting as a nidus,or it may function as a novel surface receptor. Although theprecise mechanism by which CtmPrP mediates neurotoxicity isnot clear from our data, this study demonstrates a direct cor-relation between aggregation of PrPC and induction of CtmPrPand clarifies the downstream pathway of CtmPrP transport andmetabolism, thus laying the groundwork for future investiga-tions on the cellular pathways of CtmPrP-mediatedneurodegeneration.

Although truncated CtmPrP appears similar in molecularmass to the mildly PK-resistant fragment generated from de-tergent-solubilized lysates of peptide-treated cells, the twofragments differ in important aspects. First, the limited PKresistance of PrP observed in the presence of non-ionic deter-gents reflects a change in the protease susceptibility of PrPC

because of its conformational transition to PrPSc. TruncatedCtmPrP, on the other hand, results from a proteolytic clip oftransmembrane PrP in the ER and is unlikely to resist PKtreatment in the presence of detergent. Second, protease-resis-tant PrPSc accumulates in intracellular compartments,whereas truncated CtmPrP is present on the cell surface and isreleasable by PI-PLC treatment. Thus, the PK-resistant 20-

2 Y. Gu and N. Singh, unpublished observations.3 Y. Gu, et al., manuscript in preparation.

FIG. 8. Proposed model of PrPC aggregation and induction ofCtmPrP. Step 1, micro-aggregates of PrP106–126 bind to the plasmamembrane and initiate the aggregation of PrPC. Step 2, the aggregatedproteins are endocytosed in large vesicular structures and transportedto lysosomes. Step 3, the intracellular PrP aggregates induce the syn-thesis of CtmPrP through trans-activating factors. Step 4, through an asyet unknown mechanism, the N-terminal region of CtmPrP is cleaved bycytosolic enzymes. Step 5, the C-terminal 20-kDa fragment of CtmPrP istransported to the cell surface through the secretory path. Step 6, thetruncated CtmPrP is inserted in the outer leaflet of the plasma mem-brane through the GPI anchor. Step 7, during the normal re-cycling ofPrP, full-length PrPC is cleaved at residue 111 or 112, resulting in atruncated 18-kDa C-terminal fragment that accumulates on the cellsurface (38). PM, plasma membrane; E, endosomes; L, lysosomes; N,nucleus.

Induction of CtmPrP by PrPSc-like Forms in a Cell Model 2285

kDa fragment in our model likely arises from aggregated andconformationally transformed full-length PrPC and does notrepresent truncated CtmPrP. It is unclear, however, whetherintracellular aggregated PrPC, full-length CtmPrP, or truncatedCtmPrP is the key player in the pathogenesis of prion disorders.

In conclusion, our data suggest the following sequence ofevents when PrPC-expressing cells are exposed to an amyloi-dogenic peptide of PrP, simulating, in some regards, PrPSc

infection in a cell model (Fig. 8). Micro-aggregates of PrP106–126

bind to the plasma membrane and initiate the aggregation ofPrPC, which is endocytosed and transported to lysosomes,where it polymerizes into thioflavin-binding, �-sheet-richfibrils. The intracellular PrP aggregates induce the synthesis ofCtmPrP, possibly through trans-activating factors. By an as yetunknown mechanism, the N-terminal region of CtmPrP iscleaved by a cytosolic enzyme(s), and the C-terminal 20-kDafragment of CtmPrP accumulates on the cell surface, linked tothe plasma membrane by the GPI anchor. Together, theseobservations emphasize that aggregated and PK-resistantPrPSc is not the sole causative agent of neurotoxicity and thatseveral factors, acting through complex and multiple path-ways, contribute to the final outcome of neuronal death inscrapie-infected animals. Thus, the cell model reported herenot only enhances our understanding of the mechanism of PrPC

aggregation but also clarifies the possible cellular pathways ofPrPSc-mediated neurodegeneration.

Acknowledgments—We thank Dr. P. Gambetti for his support,Dr. M. S. Sy for providing the anti-PrP 8H4 antibody, Dr. J. Andersonfor the use of confocal microscope, Anil Kumar for help in scanning andpreparing some of the figures, and Kiet Luc for technical assistancewith electronmicroscopy.

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