Disease-related Prion Protein Forms Aggresomes in Neuronal Cells

Disease-related Prion Protein Forms Aggresomes in NeuronalCells Leading to Caspase Activation and Apoptosis*□S

Received for publication, June 17, 2005, and in revised form, September 8, 2005 Published, JBC Papers in Press, September 12, 2005, DOI 10.1074/jbc.M506600200

Mark Kristiansen, Marcus J. Messenger, Peter-Christian Klohn, Sebastian Brandner, Jonathan D. F. Wadsworth,John Collinge, and Sarah J. Tabrizi1

From the Medical Research Council Prion Unit and Department of Neurodegenerative Disease, Institute of Neurology, UniversityCollege London, Queen Square, London WC1N 3BG, United Kingdom

The molecular basis for neuronal death in prion disease is notestablished, but putative pathogenic roles for both disease-relatedprion protein (PrPSc) and accumulated cytosolic PrPC have beenproposed. Here we report that only prion-infected neuronal cellsbecome apoptotic after mild inhibition of the proteasome, and thisis strictly dependent upon sustained propagation of PrPSc.Whereascells overexpressing PrPC developed cytosolic PrPC aggregates, thisdid not cause cell death. In contrast, only in prion-infected cells,mild proteasome impairment resulted in the formation of largecytosolic perinuclear aggresomes that contained PrPSc, heat shockchaperone 70, ubiquitin, proteasome subunits, and vimentin. Sim-ilar structures were found in the brains of prion-infected mice.PrPSc aggresome formation was directly associated with activationof caspase 3 and 8, resulting in apoptosis. These data suggest thatneuronal propagation of prions invokes a neurotoxic mechanisminvolving intracellular formationof PrPSc aggresomes.This, in turn,triggers caspase-dependent apoptosis and further implicates pro-teasome dysfunction in the pathogenesis of prion diseases.

Prion diseases are rare fatal neurodegenerative disorders, whichinclude Creutzfeldt-Jakob disease in humans, bovine spongiformencephalopathy (BSE)2 in cattle and scrapie in sheep. The molecularhallmark of these disorders is the accumulation of abnormal prion pro-tein conformers (PrPSc) derived from normal cellular host prion protein(PrPC) (1). The cause of neurodegeneration in these disorders is not wellunderstood, and a major gap exists in the understanding of how theconversion of PrPC to PrPSc ultimately kills neurons. Whereas PrPC isabsolutely required for prion conversion and neurotoxicity (2), knock-out of PrPC in adult brain (3) and in embryonicmodels (4, 5) has no overtphenotypic effect, effectively excluding loss of PrPC function in neuronsas a significantmechanism in prion neurodegeneration. However, thereis much evidence that also argues against the direct neurotoxicity ofPrPSc or prions (whether or not they are identical). PrPC-null tissueremains healthy and free of pathology when exposed to PrPSc (6, 7), andthere is no direct correlation between neuronal loss and PrPSc plaques in

Creutzfeldt-Jakob disease brains (8). Similarly, prion diseases in whichPrPSc is barely detectable have been described (9–11), and subclinicalinfectionwhere high levels of PrPSc accumulate in the absence of clinicalsymptoms are also recognized (12). In such “subclinical disease states,”the majority of the accumulated PrPSc may be inert; alternatively, PrPSc

may not be the toxic entity, but instead a toxic oligomeric PrP interme-diate species (PrPL for lethal) may be produced during prion conversion(12). Either this intermediate species or PrPSc itself may then only elicitneurotoxic effects when present at sufficient concentrations in partic-ular subcellular compartments.Various mechanisms have been proposed to explain neuronal death

in prion disease (reviewed in Ref. 13), which is thought to occur via anapoptoticmechanism (14–16). In vitro studies have suggested that bothfull-length PrPSc (16, 17) and shorter peptide fragments (18) are toxicwhen applied to primary cultured neurons. Other mechanisms sug-gested relate to altered PrPC trafficking. It has been described that PrPC

may assume a transmembrane topology (CtmPrP), the concentration ofwhich has been suggested to correlate with neurotoxicity (19). Morerecently, it was proposed that prion-associated toxicity involves alteredtrafficking of PrPC, where inhibition of the ubiquitin-proteasome sys-tem (UPS) results in extensive PrPC accumulation in the cytoplasm andassociated neuronal cell death (20). However, the data is conflicting,with evidence both for (21, 22) and against (23–25) this cytoplasmicaccumulation of PrPC having neurotoxic sequelae. One of the majordrawbacks ofmany of these studies on cytosolic PrPC is the high levels ofproteasome inhibition used, which may limit any physiological rele-vance to the situation in vivo (26).The concept that UPS inhibition may contribute to neurodegenera-

tion is not new. Degradation of intracellular proteins via the UPS is ahighly complex and tightly regulated process that plays a major role in avariety of cellular processes (27). Aberrations in this system have beenimplicated, either as a primary or secondary event, in the pathogenesisof a range of neurodegenerative diseases including Parkinson disease,Alzheimer disease, and Huntington disease (28). The degradativecapacity of the UPS in the nervous system is known to become impairedin these neurodegenerative diseases as well as during the aging processitself (29) (reviewed in Ref. 48).We therefore set out to examine further the nature of the PrP species

responsible for neurotoxicity and to what extent low level or “physio-logical” UPS inhibition may be involved in prion disease pathogenesis.We chose to study these effects both in cell models of prion infection aswell as examining overexpression of native PrPC. We found thatwhereas neuronal cells overexpressing PrPC developed cytosolic PrPC

aggregates under conditions of mild proteasome inhibition, this did notcause cell death. However, under similar conditions, we found that neu-ronal propagation of prions invokes a neurotoxic mechanism involvingintracellular formation of compartmentalized cytosolic PrPSc aggre-somes that triggers caspase-dependent apoptosis and implicates protea-

* This work was supported by the United Kingdom Department of Health and MedicalResearch Council. The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked “advertise-ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) contains supple-mental Table S1 and Figs. S1–S4.

1 A United Kingdom Department of Health National Clinician Scientist. To whom corre-spondence should be addressed. Tel.: 44-207-837-3611; Fax: 44-207-676-2180;E-mail: [email protected].

2 The abbreviations used are: BSE, bovine spongiform encephalopathy; DAPI, 4�,6-dia-midino-2-phenylindole; Hsc70, heat shock chaperone 70; LAC, lactacystin; prnp,mouse prion protein gene; PrP, prion protein; PrPL, PrP lethal; SCA, scrapie cell assay;fmk, fluoromethylketone; UPS, ubiquitin-proteasome system; PBS, phosphate-buff-ered saline; RML, Rocky Mountain Laboratory.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 46, pp. 38851–38861, November 18, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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some dysfunction in the pathogenesis of prion diseases. The aggresomehas emerged as a key organelle in the clearance of toxic cytoplasmicmisfolded protein aggregates (30). Interestingly, we found evidence forsimilar structures in vivo in brains of prion-infected mice.

MATERIALS AND METHODS

Cell Culture and Scrapie Infection—GT-1 and N2aPD88mouse neu-ronal cells were infected with mouse-adapted RML scrapie prions orwild-type CD-1 mouse brain homogenate as described (31, 32) andpassaged to remove the initial brain inoculum. Cells were cultured inOpti-MEM (Invitrogen) with 10% fetal calf serum supplemented with1% penicillin/streptomycin and maintained at 37 °C in 5% CO2. Cul-tures were tested for the presence of newly generated PrPSc by thescrapie cell assay (SCA) (32). The SCA was used to determine the per-centage of infected cells (32). Cells were diluted in duplicate so that nomore than 1000 cells were seeded into one well of a scrapie cell assayplate. To determine the number of infected cells, the assay was devel-oped using the standard color reaction as described (32). To determinethe total number of cells, the assaywas developed using a 1:10 dilution oftrypan blue as an indicator of cell viability. The percentage of infectedcells was calculated as the number of infected cells expressed as a per-centage of the total number of cells. Both ScN2aPD88 and ScGT-1 cellswere cured of PrPSc with 0.5 �g/ml anti-PrP antibody ICSM18 (D-GenLtd., London, UK) for 14 days. Confirmation of clearance of PrPSc wasdetermined using the scrapie cell assay as described (32).

Generation of N2amoPrPC Cells—Exon 3 (bp 7–1316) encoding the fullopen reading frame ofmouse prnpwas cloned into theNotI-ClaI sites ofthe retroviral vector LNCX2 (Clontech). This vector was then packagedinto the GP-E86 line using Fugene 6 (Roche Applied Science) andselected using G418. Viral supernatant was used to infect N2aPD88 andGT-1 cells with 4 �g/ml polybrene (Sigma). At 24 h after retroviraltransduction, stable exon 3 moprnp- expressing clones were selectedusing G418. Expression levels were quantified by immunoblotting asdescribed below.

Cell Death and Apoptosis Assays—For cell death studies, dose-re-sponse curves were established using proteasome inhibitors, and con-centrations causing �20% cell death in wild-type cells were chosen.Analysis of cell death using the lactate dehydrogenase kit was as recom-mended by themanufacturer (Alexis, Nottingham, UK). Quantificationof apoptosis using annexinV and propidium iodide staining and caspase3 and 8 assays was performed according to the manufacturer’s instruc-tions (Oncogene). For caspase inhibition experiments, cells were prein-cubated for 2 h with benzyloxycarbonyl-DEVD-fmk or benzyloxycar-bonyl-IETD-fmk before the addition of proteasome inhibitors. AnnexinV-FITC binding was quantified using flow cytometry (fluorescence-activated cell sorting). Cells were treated with 1 �M lactacystin for 24 hand then harvested by trypsinization. A total of 10,000 cells/samplewere analyzed for cell death by a FACSCalibur (BD Biosciences) withthe Cell Quest software.

SDS-PAGE and Immunoblot Analysis—Cells were harvested, andbrain tissue was homogenized on ice in PBS, freeze-thawed three times,and treated with benzonase (50 units/ml) to digest DNA. Protein con-centration was determined by BCA assay (Pierce). The equivalent of 25�g of total protein was loaded onto 16% SDS-PAGE minigels (Novex,Paisley, UK) and analyzed by electrophoresis and immunoblotting asdescribed (33). For proteinase K (Roche Applied Science) digestion,lysates were incubated with proteinase K at 1 �g/mg protein (cells) or 5�g/mg protein (brain) at 37 °C for 90 min. Equivalent protein loadingwas confirmed in non-proteinase K-treated lanes by stripping mem-branes and reprobing with anti-�-actin antibody (Sigma).

Immunofluorescence and Confocal Analysis—Cells were fixed ontopoly-L-lysine-coated glass coverslips using 4% paraformaldehyde for 20min at room temperature, washed three times with PBS, and then per-meabilized inmethanol at�20 °C for 15min. Cells were then incubatedin 10%normal goat serum for 30min. Incubationwith primary antibodywas at 37 °C for 1 h. After washing, cells were incubated for 45min withthe appropriate secondary antibody at 37 °C, washed several times inPBS, and mounted in Antifade (Sigma) containing 1 �g/ml 4�,6-dia-midino-2-phenylindole (DAPI; Sigma). To remove PrPC and revealPrPSc, cells were exposed to 98% formic acid for 5 min after fixing andbefore permeabilization. For confocal analysis, images were obtainedusing an LSM510 confocal microscopy system (Zeiss). A �63 oilimmersion objective was used for all imaging. For some experiments,ICSM18 was conjugated to a fluorescent Alexa-488 FITC secondaryantibody (Molecular Probes, Inc., Eugene, OR). Details of all antibodiesare shown in supplemental Table S1.

Subcellular Fractionation and PrP Analysis—Analysis of PrP solubil-ity and aggregationwas performedwithmodifications to published pro-cedures (20, 34). Briefly, after 24 h of lactacystin treatment, cells wereharvested, washed in ice-cold PBS, freeze-thawed three times, and thencentrifuged at 1000� g for 10min at 4 °C to remove cellular debris. Thesupernatant was collected, adjusted with an equal volume of �2 lysisbuffer (100 mM Tris, pH 7.4, 300 mM NaCl, 4 mM EDTA, 1% TritonX-100, 1% deoxycholate), and incubated with benzonase (50 units/ml)for 20 min at 4 °C before centrifugation at 100,000 � g for 45 min.Proteins in the supernatant were precipitated at �20 °C with methanol,air-dried, and then boiled in�2 SDS-sample buffer, whereas proteins inthe pellet fraction were boiled in �2 SDS-sample buffer. Each fractionwas analyzed by immunoblotting as described above.

Affinity Purification of PrPSc Aggresomes—Magnetic tosyl-activatedbeads were coated with either mouse monoclonal antibody to vimentinor Brc126, an IgG isotype control antibody, according to the manufac-turer’s instructions (Dynal, Bromborough, UK). ScGT-1 cells (with orwithout proteasome inhibition) were harvested in ice-cold PBS, freeze-thawed three times before treatment with benzonase (50 units/ml) onice for 15 min. Equivalent aliquots of sample (10 �l containing 25 �g oftotal cell protein) were incubated with vimentin antibody-coated beads,Brc126-coated beads, or magnetic beads alone (25-�l bead bed volume)for 2 h at 37 °C on an orbital shaker. Beads were concentrated in amagnetic particle concentrator (Dynal) and washed three times withPBS (3 � 5 min, 500 �l of PBS). Washed beads were resuspended in 20�l of PBS and analyzed after proteinase K digestion (final protease con-centration of 1 �g/mg protein, 90 min, 37 °C) or in the absence of pro-tease digestion. Beads were treated with 2� sample buffer at 100 °C for10 min, and the supernatant was analyzed by immunoblotting withbiotinylated anti-PrP monoclonal antibody ICSM35 (D-Gen). For anal-ysis of brain, 10% brain homogenate was prepared in PBS and freeze-thawed three times before digestionwith benzonase (50 units/ml on ice,15 min) and centrifugation at 1000 � g for 10 min to remove cellulardebris. Aliquots of supernatant (25 �l) were adjusted with an equalvolume of 2� lysis buffer (100 mM Tris, pH 7.4, 300 mM NaCl, 4 mM

EDTA, 1% Triton-X-100, 1% deoxycholate) and incubated with eithervimentin antibody-coated beads, Brc126 antibody-coated beads, ormagnetic beads alone (25-�l bead bed volume) for 2 h at 37 °C on anorbital shaker. Beads were concentrated in a magnetic particle concen-trator (Dynal) and washed three times with PBS (3 � 5 min, 500 �l ofPBS). Washed beads were resuspended in 20 �l of PBS and analyzedafter proteinase K digestion (final protease concentration 5 �g/mg pro-tein, 90 min, 37 °C) or in the absence of protease digestion. Beads weretreated with 2� sample buffer at 100 °C for 10min, and the supernatant

PrPSc Forms Apoptosis-inducing Aggresomes

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was analyzed by immunoblotting with biotinylated anti-PrP mono-clonal antibody ICSM35.

Statistical Analysis—Data were analyzed by two-tailed nonparamet-ricMann-WhitneyU test, and significancewas expressed as p� 0.01 (*),p � 0.001 (**), and p � 0.0001 (***) unless otherwise specified. For allgraphs (see Figs. 1–3, 5, and 6), bars representmeans� S.D.; *, p� 0.01;**, p � 0.001; ***, p � 0.0001 (two-tailed Mann-Whitney U test).

Image Acquisition—Fluorescence images were obtained using a con-focal microscope (Zeiss microscope LSM510 META) equipped with a“plan-Apochromat” �63/1.40 oil differential interference contrastobjective at room temperature and is controlled by Zeiss LSM software.Fluorescence was recorded at 488 nm using a 30-milliwatt argon laserfor excitation or at 543 nmusing a 1-milliwattHeNE laser for excitation.Zeiss ImmersolTM 518 F was used as imaging medium. Images notrequiring confocal analysis were obtained using an Axioplan 2 MOTmicroscope (Zeiss) with filters for fluorescein isothiocyanate, rhoda-mine, and DAPI and Plan Neofluar �10/0.30 Ph1 objective at room

temperature. An AxioCamMRm (Zeiss) camera was used and was con-trolled using the Axiovision Control software (Zeiss).

RESULTS

PrPSc Infection Sensitizes both GT-1 and N2aPD88 Cells to Mild Pro-teasome Inhibition—Mouse hypothalamic neuronal GT-1 (31) andhighly prion-susceptible N2aPD88 cells (32) were infected with mouse-adapted RML scrapie prions or wild-type CD-1 brain homogenate andpassaged to remove the brain inoculum (31, 32) (Fig. 1A). Cells mock-infected with wild-type CD-1 brain homogenate were negative on theSCA, indicating that they were not prion-infected.3 Cells were thentreated with a range of doses of the irreversible proteasome inhibitorlactacystin (Fig. 1B, graph i). Significant differences in cell death wereobserved. At 1 �M lactacystin, a highly significant difference (p �

3 M. Kristiansen and S. J. Tabrizi, unpublished data.

FIGURE 1. Mild proteasome inhibition sensitizes prion-infected GT-1 and N2aPD88 cells to apoptosis, and curing the cells of prions abrogates this effect. A, cell lysates fromwild type mouse hypothalamic neuronal GT-1 cells (lanes 1 and 2), RML scrapie-infected GT-1 cells (lanes 3 and 4), wild type mouse neuroblastoma N2aPD88 cells (lanes 5 and 6), andRML scrapie-infected N2aPD88 cells (lanes 7 and 8) were incubated in the absence (�) or presence (�) of proteinase K and immunoblotted using anti-PrP antibody ICSM35 todemonstrate PrPSc infection in these cells. B, i, dose-response curves for proteasome inhibition by lactacystin (LAC) in GT-1 cells, ScGT-1 cells, or ScGT-1-18 cells. Cell death wasdetermined after 24 h by measuring lactate dehydrogenase release (n � 12). B, ii, the percentage of cell death after mild LAC treatment (1 �M) is significantly different (p � 0.0001;n � 12) in ScGT-1 cells compared with uninfected GT-1 cells or antibody-cured ScGT-1-18 cells. C, i, dose-response curves for proteasome inhibition by lactacystin in N2aPD88 cells,ScN2aPD88 cells, or antibody-cured ScN2aPD88-18 cells. Cell death was determined after 24 h by measuring lactate dehydrogenase release (n � 12). C, ii, the percentage of cell deathafter mild LAC treatment (1 �M) is significantly different (p � 0.0001; n � 12) in ScN2aPD88 cells compared with uninfected N2aPD88 cells or antibody-cured ScN2aPD88-18 cells.Analysis by the SCA showed that �52 and �42% of the ScGT-1 and ScN2aPD88 cell populations were scrapie-infected, which is consistent with the percentages of cell death seen inB (ii) and C (ii). The prefix Sc denotes scrapie infection; the suffix -18 indicates the clonal line that has been cured of prion infection with anti-PrP antibody ICSM18.




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0.0001) was observed in cell death in the ScGT-1 cells (52%) comparedwith uninfected GT-1 cells (17%) (Fig. 1B, graph ii). Mock-infectedGT-1 cells resulted in levels of cell death similar to those of wild-typeGT-1 cells, confirming the specificity of prion infection in sensitizing

cells tomild proteasome inhibition (supplemental Fig. S1). This concen-tration of lactacystin was selected to mimic the degree of proteasomeimpairment that may occur in vivo (26, 35). These results were repro-duced with another specific proteasome inhibitor, epoxomicin (supple-

FIGURE 2. Cytosolic accumulation of detergent-insoluble PrPC aggregates after mild protea-some inhibition is not neurotoxic. A, PrP expres-sion in N2aPD88 cells before (lane 1) or aftertransfection with wild-type mouse PrPC to pro-duce N2amoPrPC cells (lane 2). B, intracellular local-ization of PrPC in untreated N2amoPrPC cells. Anti-PrP monoclonal antibody ICSM18 (red) andmarkers for the lysosome (lysosome-associatedmembrane protein-1 (LAMP-1), green), ER (anti-�-protein disulfide isomerase (PDI), (green), andnucleus (DAPI, blue). PrP is located in lysosomes(top panel), in the ER and Golgi (middle panel), andon the cell surface (bottom panel). C, PrPC aggre-gates in N2amoPrPC cells co-localize with cytosolicHsc70 and are predominantly located in the cyto-plasm after LAC treatment (1 �M; 24 h). Anti-PrPmonoclonal antibody ICSM18 (red), DAPI nuclearstaining (blue), cytosolic chaperone Hsc70 (green).D, i, cell death in both N2aPD88 and N2amoPrPC

cells after LAC treatment (1 �M; 24 h) is not signif-icantly different, but levels of death in both celltypes are significantly different from LAC-treatedScN2aPD88 cells (p � 0.0001). D, ii, after treatmentwith 10 �M LAC for 24 h, N2amoPrPC cells showed asignificantly lower percentage of cell death com-pared with wild type N2aPD88 cells (p � 0.001; n �12), suggesting that overexpressed cytosolic PrPC

may have a neuroprotective effect. E, subcellularfractionation of PrP (S, soluble supernatant; P,insoluble pellet). In the absence of LAC treatment,PrP in N2amoPrPC cells is detergent-soluble (lane 1);after low dose (1 �M) LAC treatment, a proportionof PrP becomes insoluble and is isolated in the pel-let fraction (lane 4); after high dose (10 �M) LACtreatment, all PrP becomes detergent-insolubleand aggregated and is found only in the pelletfraction (lane 6). F, infecting these N2amoPrPC cellswith RML scrapie prions produces a significant(p � 0.0001; n � 6) increase in cell death after 1 �M

LAC treatment, indicating that the presence ofPrPSc sensitizes these cells to apoptosis. D and F,open boxes, vehicle only; closed boxes, LAC treat-ment. Scale bars, 20 �m.




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mental Fig. S2). To ensure that the effects we observed were not con-fined to a subgroup of ScGT-1 cells known to have an apoptoticphenotype after scrapie infection (31), experiments were repeated usingscrapie-infected clonal N2aPD88 cells (32). Again, RML-infectedN2aPD88 cells were significantly more susceptible to induction of apo-ptosis after mild proteasome inhibition compared with uninfectedN2aPD88 cells (p � 0.0001) (Fig. 1C, graphs i and ii). Analysis by theSCA (32) showed that �52 and �42% of the ScGT-1 and ScN2aPD88cell populations were scrapie-infected,3 which is consistent with thepercentages of cell death seen in Fig. 1B (graph ii) and Fig. 1C (graph ii).

Curing Prion-infected Cells with Anti-PrP Monoclonal AntibodiesAbrogates the Neurotoxic Effect of Proteasome Inhibition—To investi-gate whether the sensitivity of scrapie-infected cells was due to PrPSc,cells were treated for 14 days with 0.5 �g/ml anti-PrP monoclonal anti-body ICSM18 (36) and confirmed to have undetectable levels of infec-tivity using the SCA.3 Curing cells of prion infection abrogated the

sensitivity to proteasome inhibition and resulted in the same degree ofcell death as uninfected GT-1, mock-infected GT-1, and N2aPD88 cells(Fig. 1, B and C, and supplemental Fig. S1). Thus, prion propagationappears to sensitize these neuronal cells to mild proteasome inhibition.

Cytosolic Accumulation of Detergent-insoluble PrPC Aggregates Is NotNeurotoxic after Mild Proteasome Inhibition—To further investigatewhether our findings were due to PrPSc, we generated N2aPD88 cellsoverexpressing �3-fold full-length wild-type mouse PrPC (N2amoPrPC)(Fig. 2A). In non-lactacystin-treatedN2amoPrPC cells, the PrPCwas local-ized on the cell surface, in the lysosomal system, and in an ER-Golgipattern with partial ER co-localization (63) (Fig. 2B). After low doselactacystin treatment, the pattern of PrPC immunostaining changed inthe N2amoPrPC cells, with the majority of PrPC deposition now in thecytoplasm co-localizing with the cytosolic protein Hsc70 (Fig. 2C).However, despite the presence of cytosolic PrPC, there was no signif-

icant difference in cell death when compared with wild-type N2aPD88

FIGURE 3. Prion infection induces caspase 3- and 8-dependent apoptosis, which is abrogated by specific caspase inhibitors. A, ScGT-1 cells undergo apoptosis after 1 �M LACtreatment. Nuclear fragments (DAPI, blue) are shown by arrows. The percentage of apoptotic cells (annexin V-positive, green) and necrotic cells (propidium iodide-positive, red;annexin V-positive, green) were determined at 0, 12, and 24 h (scale bar (top panel), 20 �m; scale bar (bottom panel), 126 �m). B, the percentage of apoptotic cells increases by �30%after LAC treatment as quantified by fluorescence-activated cell sorting analysis (p � 0.03; n � 4). C, during the course of apoptosis, the activity of caspase 3 and 8 activation increasedin a time-dependent manner. From 1 h, there was a highly significant increase in caspase 3 activity (p � 0.001) in the ScGT-1 cells as compared with uninfected GT-1 cells. D, there wasalso a highly significant increase in caspase 8 activation over time (p � 0.001). E, cell death was completely abrogated in ScGT-1 cells (p � 0.0001; n � 12) after treatment with highlyspecific caspase 3 or 8 inhibitors.




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cells (Fig. 2D, graph i), suggesting that at low level proteasome inhibi-tion, cytoplasmic accumulation of PrPC is not neurotoxic. Notably, inour N2amoPrPC cells, there appeared to be a neuroprotective effect ofPrPC overexpression (p � 0.001) after treatment with 10 �M lactacystincomparedwith wild-typeN2aPD88 cells (Fig. 2D, graph ii) as previouslyreported (24, 25). At high doses of lactacystin (10 �M), there were highlevels of cell death in both the scrapie-infected and uninfectedN2aPD88(�90% in ScN2aPD88 and �75% in N2aPD88) (Fig. 1C, graph i) inagreement with previous studies (20). To assess whether the cytosolicPrPC observed in our lactacystin-treated cells had formed detergent-insoluble aggregates, we performed subcellular fractionation with anal-ysis of detergent solubility and aggregation status of PrPC in cytosolicandmembrane fractions (Fig. 2E).With no proteasome treatment, all ofthe PrPC expressed in our N2amoPrPC was fully detergent-soluble (Fig.2E, lane 1); after low dose lactacystin treatment (1 �M), there was anincrease in detergent-insoluble aggregated PrPC isolated in the pelletfraction (Fig. 2E, lane 4). After high dose lactacystin treatment (10 �M),all of the PrPC was aggregated and detergent-resistant (Fig. 2E, lane 6).

We then infected our N2amoPrPC cells with RML scrapie prions andobserved correlation between the presence of PrPSc and neurotoxiceffect after 1 �M lactacystin treatment (Fig. 2F). Thus, the presence ofPrPSc, rather than cytoplasmic aggregates of wild-type PrPC, appears tobe associated with apoptosis after mild proteasome inhibition.

Prion Infection InducesCaspase 3- and 8-dependentApoptosis,WhichIs Abrogated by Specific Caspase Inhibitors—To evaluate cell death aftermild proteasome inhibition, nuclear DNA fragmentation analysis andannexin V and propidium iodide staining were performed in lactacys-tin-treated ScGT-1 cells, and apoptosis was quantified using fluores-cence-activated cell sorting analysis. These results demonstrated thatthe ScGT-1 cells were dying by apoptosis (Figs. 3, A and B). Apoptosismay be initiated through a number of different pathways, and in vivostudies in prion disease have suggested that caspase 3- and 8-dependentpathways are activated (37, 38). To study this process further, a timecourse study of caspase 3 and 8 activation was undertaken. From 1 h,there was a highly significant rise in caspase 3 and 8 activities in theScGT-1 cells versus uninfected cells in a time-dependentmanner (Fig. 3,C and D). At 24 h, there was a 120% increase in caspase 8 activationcompared with uninfected GT-1 cells (Fig. 3D); this finding supports invivo data suggesting that caspase 8-mediated apoptotic cell death playsa significant role in prion-mediated neuronal cell death (39). Apoptoticcell death was completely abrogated in the scrapie-infected cells usingcell-permeable specific caspase 3 and 8 inhibitors (DEVD-fmk andIETD-fmk, respectively), supporting their pivotal role in scrapie-medi-ated neuronal cell death (Fig. 3E).

PrPSc, but Not Aggregated PrPC, Forms Large Cytoplasmic PerinuclearAggresomes, Which Appear Directly Neurotoxic—Aggresomes arelocated near the microtubule-organizing center at the centrosome (40),reflecting the fact that aggresome formation needs an intact microtu-bule network (40, 41). They are also distinguished by a cage of theintermediate filament protein vimentin, which is an invariant feature ofthese structures (40, 42).We used stringent formic acid pretreatment ofcells to remove PrPC immunoreactivity and to reveal PrPSc deposits inour scrapie-infected cells (Fig. 4A). Double-labeling immunostainingdemonstrated that after mild proteasome inhibition, PrPSc accumulatesin ScGT-1 cells as large cytoplasmic perinuclear aggresomes and co-localizes with vimentin, Hsc70, 20 S proteasome, and ubiquitin (Fig. 4,B–E). Similar structures were found in lactacystin-treated ScN2aPD88cells (supplemental Fig. S3). Using confocal microscopy, cytoplasmiclocalization was confirmed by colocalization with the cytosolic chaper-

one Hsc70 (Fig. 4D) and the absence of immunostaining with markersfor the ER and nucleus.3

To confirm that PrPSc itself was a major constituent of these aggre-somes, we performed an affinity purification of the ScGT-1 aggresomesusing vimentin antibody-coated magnetic beads. Vimentin is a type-IIIintermediate filament protein that normally displays an extended cyto-plasmic distribution. In aggresome-containing cells, vimentin is redis-tributed to form a cagelike structure wrapped around the exterior of theinclusion (41); it has been suggested that this contributes to the stabilityof the aggresome (42). Vimentin antibody-coated beads purified PrPSc

from lactacystin-treated ScGT-1 cells (Fig. 5A, lane 2), the specificity ofthis interaction was confirmed using isotype control antibody-coatedbeads that did not purify PrPSc (Fig. 5A, lanes 3 and 4) and beads alone(Fig. 5A, lanes 5 and 6). Previous reports have suggested that cytosolicPrPC forms aggresomes after cyclosporinA treatment (43); we thereforeperformed immunostaining for aggresomes in our N2amoPrPC cells.Importantly, we found that cytoplasmic PrPC aggregates did not formaggresomes.3 Mock-infected cells also did not form aggresomes andwere indistinguishable from wild-type uninfected cells.3

The role of aggresomes in cellular neurotoxicity is controversial; theirformation in cells has been reported to be a neuroprotectivemechanismto sequester toxic misfolded proteins (44), whereas others suggest thatthey are a toxic species (45, 46). To investigate this further, we usedagents that inhibit the formation of aggresomes by disrupting retro-grade microtubule-mediated transport (47). Prior to treatment withcolchicine, nocodazole, and cytochalasin D, we undertook dose-re-sponse curves inN2aPD88 andGT-1 cells to optimize treatment of cellswith these agents (supplemental Fig. S4). Colchicine is an antimitoticagent that disrupts microtubule function. Treatment with colchicine (5�g/ml) prevented cell death in prion-infected cells after lowproteasomeinhibition (p � 0.0001) (Fig. 5B, graph i). To ensure that colchicinetreatment had also prevented aggresome formation, we performedimmunofluorescence, which confirmed that the prevention of PrPSc

aggresome formation had abrogated cell death (Fig. 5C, panels 1 and 2).We also examined the effect of nocodazole treatment on the formationof PrPSc aggresomes; nocodazole is an agent that also disrupts microtu-bule dynamics (47). This supported the argument that the effect ofcolchicine treatment was via this mechanism, since cell death was alsoabrogated by 0.5 �M nocodazole treatment (p � 0.0001) (Fig. 5B, graphii) with prevention of aggresome formation (Fig. 5C, panel 3). To ensurethat the abrogation of cell death induced by PrPSc aggresomes by themicrotubule-disrupting agents was specific, we treated our cells with 50ng/ml cytochalasin D, which disrupts actin microfilaments that are notinvolved in aggresome formation (47). Treatment with cytochalasin Ddid not affect cell death (Fig. 5B, graph iii) or remove aggresomes (Fig.5C, panel 4), confirming our data indicating that clearance of PrPSc

aggresomes selectively abrogates cell death. To ensure that colchicineand nocodazole were not exerting their antiapoptotic effect throughclearance of PrPSc, we treated ScGT-1 cells for 5 days with these drugs,which had no effect on PrPSc levels in these cells assessed using theSCA.3

Formation of PrPSc Aggresomes Is Temporally Associated withCaspase 3 and 8 Activation—To examine whether aggresome forma-tion is directly related to caspase activation, we measured caspase 3 and8 activities after treatment with colchicine and nocodazole to preventaggresome formation. This treatment also abrogated caspase activationin prion-infected cells (Fig. 6A), suggesting a direct relationshipbetween the formation of PrPSc aggresomes and caspase activation lead-ing to apoptosis. We then performed a time course analysis of PrPScaggresome formation and caspase 3 and 8 activation in ScGT-1 cells




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after mild proteasome inhibition (Fig. 6, B–F, panels 1–3). At time 0,there are no PrPSc aggresomes (Fig. 6B, panel 3) and no evidence ofcaspase 8 (Fig. 6B, panel 1) or caspase 3 activation (Fig. 6B, panel 2).

PrPSc aggresome formation initiates as small perinuclear structures at6 h (Fig. 6C, panel 3), which directly correlates with the presence of denovo caspase 8 and 3 immunostaining in the cells at the same time point

FIGURE 4. PrPSc forms large cytoplasmic perinuclear aggresomes. A, to analyze the subcellular localization of PrPSc, cells were treated with 98% formic acid for 2 and 5 min and thenimmunostained with anti-PrP antibody (ICSM18) to reveal PrPSc. In GT-1 cells, formic acid treatment removed all detectable PrPC after 2 min (top panel); in ScGT-1 cells, PrPSc is present on thecell surface and intracellularly (bottom panel). Nuclear staining was done with DAPI (blue). B–E, after treatment with 1 �M LAC, PrPSc (green) accumulates in ScGT-1 cells in large perinuclearaggresomes; co-localizes with ubiquitin (C), Hsc70 (D), 20 S proteasome (E); and is surrounded by a vimentin (B) cage. Scale bars, 20 �m.




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FIGURE 5. Aggresomes are composed of PrPSc and are neurotoxic to the cell. A, for co-immunoprecipitation experiments, ScGT-1 cells were harvested 24 h after treatment with 1 �M

lactacystin. PrPSc was co-precipitated from lactacystin-treated ScGT-1 cells by anti-vimentin antibody-coated beads (lanes 1 and 2) but not by Brc126 isotype control antibody-coated beads(lanes 3 and 4) or beads alone (lanes 5 and 6), demonstrating that aggresomes contain PrPSc. PK, protein kinase. B, cell death in lactacystin-treated (1 �M; 24 h) ScGT-1 cells was abrogated inthe presence of 5�g/ml colchicine (B, i) (p�0.0001; n�12) and reduced by�20% in the presence of 0.5�M nocodazole (B, ii) (p�0.0001; n�12) and was unaffected by 50 ng/ml cytochalasinD (B, iii) (p not significant), indicating that clearance of aggresomes by colchicine and nocodazole ameliorates cell death. C, ScGT-1 cells were incubated for 24 h in the presence of 1 �M

lactacystin (LAC) alone (panel 1) or in combination with colchicine (5 �g/ml) (panel 2) or nocodazole (0.5 �M) (panel 3). Treatment with colchicine or nocodazole prevented aggresomeformation (panels 2 and 3). Treatment with cytochalasin D did not clear aggresomes (panel 4), confirming our data indicating that clearance of PrPSc aggresomes selectively abrogates celldeath. PrPSc-containing aggresomes were detected using anti-PrP antibody ICSM18 (green) and anti-vimentin antibodies (red). Nuclear staining was done with DAPI (blue). Scale bar, 20 �m.




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(Fig. 6C, panels 1 and 2). After 12 h, the size of the PrPSc aggresomeincreases (Fig. 6D, panel 3) with marked caspase 8 and 3 immuno-staining in the cells containing PrPSc aggresomes (Fig. 6D, panels 1 and2). By 18 and 24 h, there is widespread intracellular caspase 8 and 3immunostaining, and large perinuclear PrPSc aggresomes are seen (Fig.6, E and F, panels 1–3). These data are supported by the time coursestudy of caspase 3 and 8 activation in ScGT-1 cells (Figs. 3, C and D),where from 6 h onward there was a significant increase in caspase 3 and8 activity levels (p� 0.001; Figs. 3,C andD). These data support a directrelationship between the formation of PrPSc aggresomes and caspase 8-and 3-dependent apoptosis in neuronal cells after mild proteasomeinhibition.

PrPSc Is Also Associated with Aggresome-like Structures in Vivo—Toassess whether PrPSc aggresome structures occurred in vivo, weattempted to affinity-purify PrPSc from terminal scrapie-infected CD-1mice brains using vimentin antibody-coated magnetic beads (Fig. 7, Aand B). We demonstrate that there is a specific association betweenPrPSc and the intracellular protein vimentin in vivo, since we were ableto affinity-purify PrPSc from scrapie-infected CD-1 mouse brain usingvimentin antibody-coated beads (Fig. 7A, lane 3). Using densitometryand quantitative immunoblotting, the estimated proportion of totalbrain PrPSc recovered by the vimentin beads is �4%, which representsthe intracellular PrPSc associated with aggresomes in these terminalscrapie-infectedmouse brains (Fig. 7B, lane 7). Isotype control antibodyor beads alone did not isolate PrPSc (Fig. 7A, lanes 1 and 4). Therewas noassociation between PrPC and vimentin in uninfected CD-1 brain, again

FIGURE 6. PrPSc aggresomes are temporally linked to caspase 3 and 8 activation. A,treatment with nocodazole (NOC; 0.5 �M) and colchicine (COL; 5 �g/ml), which preventaggresome formation, abrogated caspase 3 and 8 activation in lactacystin (LAC; 1 �M)-treated prion-infected cells. B–F, after formic acid treatment, cells were immunostainedwith antibodies directed against PrP (ICSM18, red in panels 1 and 2; and green in panel 3),vimentin (red), activated caspase 8 (green), and activated caspase 3 (green) to demon-strate the time course relationship between aggresome formation and caspase 8 and 3activation. Nuclear DAPI staining is blue. B, at time 0, there are no PrPSc aggresomes(panel 3) and no evidence of caspase 8 (panel 1) or caspase 3 activation (panel 2). C, earlyPrPSc containing aggresomes are formed as small perinuclear structures after 6 h (panel3) with early caspase 8 and 3 activation (panels 1 and 2). D, after 12 h, the size of the PrPSc

aggresome increases (panel 3) with marked caspase 8 and 3 immunostaining in the cells

containing PrPSc aggresomes (panels 1 and 2). E and F, by 18 and 24 h, there is widespreadintracellular caspase 8 and 3 immunostaining, and large perinuclear PrPSc aggresomesare seen (panels 1–3). Scale bars, 40 �m.

FIGURE 7. Association of PrPSc and vimentin in scrapie-infected mouse brain. A andB, immunoblots developed with anti-PrP monoclonal antibody ICSM35. A, co-immuno-precipitation experiments were performed using uninfected CD-1 mouse brain homo-genate and RML-infected CD-1 mouse brain homogenate. Lanes 1– 4 show PrP immuno-reactivity from RML-infected CD-1 mouse brain homogenate after co-precipitation withvimentin antibody-coated magnetic beads (lanes 2 and 3 show total PrP (lane 2) andPrPSc (lane 3) affinity-purified using vimentin-coated beads) or isotype-control antibody-coated magnetic beads (lane 1, negative) or magnetic beads alone (lane 4, negative).Lanes 5 and 6 show that no PrP immunoreactivity is recovered from uninfected CD-1mouse brain homogenate by co-precipitation with vimentin antibody-coated magneticbeads. B, quantification of PrPSc isolated from RML-infected CD-1 mouse brain. Lanes 1–5show the amount of PrPSc present in 12.5, 6.25, 3.13, 1.56, and 0.78 �l of 10% RML-infected brain homogenate, respectively. Lane 7 shows the amount of PrPSc recoveredby vimentin antibody-coated beads from 25 �l of 10% RML-infected brain homogenate.Densitometry indicates recovery of �4% of total PrPSc, representing intracellular PrPSc

bound to intracellular vimentin.




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confirming the specificity of the interaction with PrPSc and vimentin(Fig. 7A, lanes 5 and 6).

DISCUSSION

This study aimed to define further the cellular basis of neurotoxicityin prion-mediated neuronal death and the subcellular compartments inwhich toxicity may be generated. To investigate the role of the UPS inprion-mediated toxicity, we studied much milder levels of proteasomeinhibition than reported in previous studies (20–22, 34). Levels of pro-teasome impairment that we investigated are more compatible with theloss of proteasome activity associated with either senescence (45, 48) orthat may be seen in prion-infected brain (26, 35).We chose to study two separate mouse prion-propagating neuronal

cell lines (N2aPD88 and GT-1) (31, 32) to allow validation of the data indifferent prion-infected cell systems. There are very few cell lines able tostably propagate prions in vitro, and these two neuronal cell systems arewell characterized and represent a valuable tool for analysis (49). Weconfirmed that both the N2aPD88 and GT-1 cells are able to propagatelow levels of scrapie infectivity and suffer no obvious cytotoxic effects(31, 32, 50, 51). This may be due to possible cell-specific properties ofenhanced degradative capacity, where accumulation of PrPSc does notoccur to a level where it may become neurotoxic (52), which mayaccount for the reason these neuronal cells are uniquely able to stablypropagate low levels of mouse prions.Here we show that prion-infected N2aPD88 and GT-1 neuronal cells

were significantly more susceptible to cell death when treated with lowdose proteasome inhibitors than uninfected or mock-infected cells.These cells underwent caspase 3- and 8-dependent apoptotic cell deaththat was abrogated by specific caspase 3 and 8 inhibitors. Curing thesecells of prion infection with an anti-PrP antibody abrogated neurotox-icity; however, when the same cells were reinfectedwith prions, apopto-sis occurred under conditions ofmild proteasome inhibition. Therefore,neurotoxicity was dependent on continued PrPSc propagation. Toinvestigate whether apoptosis was due to a nonspecific cellular pro-teinopathy, we studied N2aPD88 cells overexpressing PrPC. Under thesame low level of proteasome inhibition, these cells developed largecytoplasmic PrPC aggregates but did not undergo apoptosis. Neurotox-icity occurred only when these PrPC-overexpressing cells were infectedwith prions, arguing that prion infectionwas a prerequisite for apoptosisunder these conditions.Under conditions of mild proteasome impairment, both prion-in-

fected cell lines accumulated large cytoplasmic perinuclear aggresomescontaining PrPSc, heat shock protein 70, ubiquitin, proteasome sub-units, and vimentin, characteristic of these structures. PrPSc aggresomeformationwas temporally associatedwith caspase 3 and 8 activation andsubsequent apoptosis. Inhibition of aggresome formation with differentmicrotubule inhibitors abrogated both caspase activation and cell death,indicating that aggresome formation triggers apoptosis. Importantly,PrPSc was associatedwith vimentin in RMLprion-infectedmouse brain,suggesting that similar PrPSc aggresome structures may have relevancein vivo. Recently, granular deposits of disease-related prion protein havealso been reported in the cell body of neurons, suggesting intraneuronalprion aggregates may play a role in Creutzfeldt-Jakob disease pathogen-esis (53).Our data suggest a neurotoxic mechanism in prion disease where

formation of intraneuronal cytosolic PrPSc-containing aggresomes isassociated with caspase 3- and 8-dependent apoptosis. They support arole for UPS dysfunction in the neuropathogenesis of prion disease butnot a role for cytosolic aggregation of wild-type PrPC.Whereas we con-firm in this study that high levels of proteasome inhibition (20) can

result in accumulation of misfolded cytosolic PrPC and resultant neuro-toxicity, such a degree of proteasome inhibition is unlikely to occur invivo during prion pathogenesis (26, 45, 48).It has been proposed that aggresome formation is a specific and active

cellular response to cope with excessive levels of misfolded and aggre-gated proteins (40–42). In support of the role for aggresomes in proc-essing intracellular misfolded protein aggregates, proteasome compo-nents and molecular chaperones are actively recruited to aggresomes.Here we have identified for the first time the formation of cytosolicPrPSc aggresomes and shown that their presence is deleterious to neu-ronal cells. Aggresomes contain ubiquitin, chaperones, and proteasomecomponents, thereby lowering the degradative ubiquitin and protea-some-dependent proteolysis in the cell in a negative feedback loop,resulting in an autocatalytic chain leading to the induction of apoptosis(41, 46) Accumulation of misfolded proteins at the centrosomes mayalso severely impair their function and therefore interfere with cell divi-sion (54). Recently, it has also been shown that UPS impairment byprotein aggregates is global and that the capacity of the entire cellularUPS is compromised by the presence of aggregates that are restricted toeither the cytoplasmic or nuclear compartments (55). In our cell system,prion aggresome formation appears directly related to caspase 8 activa-tion, which then proteolytically activates downstream caspase 3 andinduces neuronal apoptotic cell death. Similar intraneuronal caspase8-mediated apoptosis in response to aggregated proteins has beendescribed in Huntington disease (56) and Alzheimer disease (57).Whether PrPSc aggresome structures occur in vivo is not known, but ourdata showing a specific association between PrPSc and the aggresome-associated intracellular protein vimentin in RML-infected CD-1 mousebrain suggest that this may be the case.We also propose that itmay specifically be the cytosolic accumulation

of PrPSc aggresomes that is particularly proapopotic. In support of this,inhibition of specific lysosomal cysteine proteases in GT-1 cells inhib-ited the degradation of PrPSc and resulted in an accumulation of com-partmentalized lysosomal PrPSc (58, 59); however, thiswas not cytotoxic(58). Whereas pathogenic prion protein mutants have been reported toform intracellular aggresomes in response to proteasome inhibition (43,60) and misfolded cytosolic PrP has been reported to form aggresome-like structures after cyclosporin A treatment (43), there have been noreports to date of PrPSc aggresome formation or of the effects of aggre-somes on cell viability in prion disease. How PrPSc may enter the cyto-plasm to form aggresomes has not been established, but little is knownabout the exact details of cellular PrPSc trafficking (recently reviewed inRef. 61). Possible sites of entry include retrotranslocation from the ER(62), as has been described for PrPC and some pathogenic prionmutants(34, 63–65), during its intracellular trafficking pathway or by intracel-lular trafficking from outside the cell. PrPSc may then accumulate in thecytoplasm when the proteasome is inhibited as may occur in aging orduring prion infection in vivo (35, 48) and generate toxic aggresomestructures as demonstrated in this study. A fundamental question raisedby the present study is whether PrPSc accumulation in aggresomes isaccompanied by concomitant accumulation of a distinct neurotoxicPrPL species. Detailed physico-chemical characterization of PrPSc

aggresome formation will be required to pinpoint the neurotoxic PrPentity. The neurotoxic intracellular mechanism suggested by the pres-ent study is likely to be part of a multifactorial prion disease pathogen-esis, which may also involve synaptic dysfunction and alterations tocellular membrane permeability (66). Given the critical role that hasemerged for the UPS in protein misfolding disorders (28), combinedwith the age-dependent decrease in UPS activity, the design of drugsthat improve UPS function in neurons may help provide effective inter-




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vention to slow or prevent these diseases in which toxic proteins aremisfolded.

Acknowledgments—We thank Charles Weissmann for advice and criticalreview of the manuscript, Nico Dantuma and Giovanna Mallucci for helpfulcomments on the manuscript, Liza Sutton and Jennifer Podesta for technicalhelp, Andy Hill for GT-1 cells, and Ray Young for graphics.

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Jonathan D. F. Wadsworth, John Collinge and Sarah J. TabriziMark Kristiansen, Marcus J. Messenger, Peter-Christian Klöhn, Sebastian Brandner,

Caspase Activation and ApoptosisDisease-related Prion Protein Forms Aggresomes in Neuronal Cells Leading to

doi: 10.1074/jbc.M506600200 originally published online September 12, 20052005, 280:38851-38861.J. Biol. Chem.

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Disease-related Prion Protein Forms Aggresomes in Neuronal Cells