Interplay between 20S proteasomes and prion proteins in scrapie disease

Interplay Between 20S Proteasomes andPrion Proteins in Scrapie Disease

Manila Amici,1 Valentina Cecarini,1 Massimiliano Cuccioloni,1 Mauro Angeletti,1

Simone Barocci,2 Giacomo Rossi,3 Evandro Fioretti,1 Jeffrey N. Keller,4 andAnna Maria Eleuteri1*1Department of Molecular, Cellular and Animal Biology, University of Camerino, Camerino, Italy2Istituto Zooprofilattico Sperimentale Umbria-Marche, Perugia, Italy3Department of Veterinary Science, Faculty of Veterinary Medicine, University of Camerino,Matelica, Italy4Pennington Biomedical Research Center, Baton Rouge, Louisiana

Scrapie is a transmissible spongiform encephalopathyaffecting the central nervous system in sheep. The keyevent in such neurodegeneration is the conversion ofthe normal prion protein (PrPC) into the pathologicalisoform (PrPSc). Misfolded prion proteins are normallydegraded by the proteasome. This work, analyzingmodels of scrapie disease, describes the in vivo rela-tionship between the proteasome and prions. We reportthat the disease is associated with an increase of pro-teasome functionality, most likely as a means of coun-teracting the increased levels of oxidative stress. Here,we show that prions coprecipitate with the 20S protea-some and that they colocalize within the same neuron,thus raising the possibility that PrP interacts with theproteasome in both normal and diseased brain, affect-ing substrate trafficking and proteasome functionality.This interaction, inducing proteasome activation, leadsto different neuronal alterations and triggers apoptosis.Furthermore, testing the effects of isolated PrPC onpurified 20S proteasomes, we obtain a concentration-and proteasome composition-dependent decrease inthe complex activity. VVC 2009 Wiley-Liss, Inc.

Key words: 20S proteasome; prion protein; proteinoxidation; colocalization; apoptosis

Prion diseases are infectious, inherited, or sporadicneurodegenerative central nervous system (CNS) disor-ders affecting both animals and humans (Prusiner, 1998;Aguzzi and Polymenidou, 2004). Animal prion diseasesinclude most notably scrapie in sheep and bovine spon-giform encephalopathy or ‘‘mad cow’’ disease in cattle(Prusiner, 2004). All forms of prion diseases are charac-terized by various neurological signs and common histo-pathological features such as spongiform degeneration ofCNS, reactive gliosis, neuronal loss, and in some casesformation of amyloid plaques (Prusiner et al., 1983; Huret al., 2002). The central event in prion propagation isthe conversion of the normal PrP isoform (PrPC), whichis rich in a-helices, into the pathogenic form (PrPSc),which consists mainly of b-sheet conformation (Pan

et al., 1993; Prusiner, 2004; Thackray et al., 2007).PrPSc accumulation in the cytosol is favored by itshigher resistance to detergents and proteolytic digestion(Caughey et al., 1991; Prusiner, 1998).

PrPC is processed in the secretory pathway, andincorrectly assembled PrPC molecules are diverted to thecytosol, deglycosylated, ubiquinated, and degraded bythe proteasome, which is the major structure responsiblefor the degradation of oxidized, aggregated, andmisfolded proteins (Davies, 2001; Jung et al., 2007). The20S proteasome is a four-ring structure with sevendifferent subunits in each ring, arranged as a1–7b1–7b1–7a1–7 (Baumeister et al., 1998; DeMartino andSlaughter, 1999). Binding of the 20S proteasome to aregulatory particle, the 19S cap, results in the formationof a larger complex, known as the 26S proteasome, re-sponsible for the ATP-mediated degradation of polyubi-quitinated proteins (DeMartino and Slaughter, 1999).The best characterized proteasomal activities include the‘‘chymotrypsin-like’’ (ChT-L), the ‘‘peptidylglutamyl-peptide hydrolizing’’ (PGPH), and the ‘‘trypsin-like’’(T-L) (Orlowski and Wilk, 2000). Additionally, it alsoshows two other catalytic components, the BrAAP(branched-chain amino acids preferring) and the SNAAPactivities (small neutral amino acids preferring) (Orlowskiet al., 1993). In cells exposed to interferon-g, the consti-tutive b subunits (b1, b2, and b5) are replaced by othercatalytic subunits (b1i, b2i, and b5i, respectively)incorporated cooperatively into a de novo synthesizedproteasome form, the immunoproteasome, with an

Contract grant sponsor: Istituto Zooprofilattico Sperimentale Umbria-

Marche (Perugia, Italy).

*Correspondence to: Dr. Anna Maria Eleuteri, Department of Molecular,

Cellular and Animal Biology, University of Camerino, Via Gentile III da Var-

ano, 62032 Camerino (MC), Italy. E-mail: [email protected]

Received 16 September 2008; Revised 6 May 2009; Accepted 4 June

2009

Published online 5 August 2009 in Wiley InterScience (www.

interscience.wiley.com). DOI: 10.1002/jnr.22186

Journal of Neuroscience Research 88:191–201 (2010)

' 2009 Wiley-Liss, Inc.

enhanced capacity to generate antigenic peptides(Kloetzel et al., 1999).

Prion-associated diseases are characterized by analtered trafficking of PrPC, and the ubiquitin-proteasomepathway inhibition is responsible for the cytoplasmicaccumulation of PrPC (Ma et al., 2002). Yedidia et al.(2001) showed that the treatment of cultured cells withproteasome inhibitors resulted in a cytoplasmic increaseof misfolded PrP, with consequent aggresome formationand neurotoxicity. Furthermore, the prion inhibitoryeffect was demonstrated in pure proteasomes and in neu-ronal cell lines (Kristiansen et al., 2007). Although thesefindings contribute to clarifying the relationship betweenprion proteins and proteasomes system, they do not elu-cidate the eventual in vivo interaction in models of PrPtoxicity such as scrapie disease. To gain insight into suchmechanisms, we have investigated the composition andfunctionality of 20S proteasomes in brains of scrapie-pos-itive sheep in comparison with young and aged healthyanimals. We have verified protein oxidation levels, thepresence of ubiquitinated proteins, and proteasome-prionprotein coprecipitation. Moreover, we have examinedthe regional and subcellular distributions of the 20S pro-teasome and prion proteins in healthy and scrapie sheepbrainstem and evaluated the correlation with neuronallesions and apoptosis. Finally, the effects of isolated PrPC

on purified constitutive and immunoproteasomes havebeen measured.

MATERIALS AND METHODS

Materials

Tissue samples from brainstem of sheep were obtainedfrom the Istituto Zooprofilattico Sperimentale Umbria-Marche.Substrates Suc-LLVY-AMC, Z-LLE-2NA, and Z-GGR-2NAfor assaying the ChT-L, PGPH, and T-L activities were pur-chased from Sigma (St. Louis, MO). The Z-GPALA-pAB sub-strate and the Z-GPFL-CHO inhibitor were the kind gift ofProf. M. Orlowski (Department of Pharmacology, Mount SinaiSchool of Medicine, New York, NY). The anti-20S and theantisubunit b5, b1, b2, b5i, b1i, and b2i antibodies were pur-chased from Biomol International LP (Matford Court, Exeter,United Kingdom). The Prionics-Check Western and the mousemonoclonal anti-PrP antibody (mAB) 6H4 were obtained fromPrionics (Switzerland). The polyclonal anti-GAPDH (FL-335;glyceraldehyde-3-phosphate dehydrogenase) antibody and themouse monoclonal anti-ubiquitin antibody were purchasedfrom Santa Cruz Biotechnology (Santa Cruz, CA). The Oxi-dized Protein Detection Kit (OxyBlot) was purchased fromAppligene-Oncor (Strasbourg, France). The polyclonal anti-body raised against prion protein PrP-ab3531 was obtainedfrom Abcam (Cambridge, United Kingdom) and used forimmunohistochemical staining of PrP in formalin-fixed sheeptissues. The peroxidase/DAB kit (En Vision; DAKO, Glostrup,Denmark) was employed in the immunostaining assays;Vectamount mounting medium (Vector Laboratories,Burlingame, CA) was used for sections counterstaining.

Apoptosis was highlighted through a TUNEL colori-metric staining (DeadEnd; Promega Italy) according to the

manufacturer’s instructions. The Silver Staining Kit Proteinwas obtained from Amersham Biosciences. Other reagentswere obtained from Sigma.

Sampling of Brainstem and Scrapie Test

All the analyzed ovine samples were obtained from theZooprofilactic Institute of Marche region, which applies, forsheep killing, rules from the Italian active surveillance plan.All efforts were made to minimize both the suffering (deepsurgical anesthesia) and the numbers of animals. The practicefor acquiring the samples follows a European proceduredescribed by the European commission in ‘‘The Evaluation oftests for the diagnosis of transmissible spongiform encephalop-athy in bovines,’’ 8 July 1999. After the beheading of eachsheep, the whole brainstem was removed using a specialspoonlike instrument through the foramen magnum, and 0.5g from the brainstem, specifically cut in the obex region(Hejazi and Danyluk, 2005), was cut out and then homoge-nized in a specific buffer and subsequently treated withproteinase K, according to the Prionics-Check Western SRprotocol (Prionics, CH) used to establish the scrapie positivity.This allows the degradation of the normal prion proteinisoform, and only the PrPSc protein remains in the test sample.After digestion, the PrPSc was revealed by Western blot analy-sis using the antibody 6H4. The monoclonal antibody 6H4recognizes the sequence DYEDRYYRE in the prion protein(human PrP; amino acids 144–152): this sequence is conservedin most known mammalian PrP sequences (human, cattle,sheep, rabbit, mink, and a variety of primates).

PrPC Purification

PrPC purification was performed on brainstem tissuelysates from different sheep using the Pan protocol (Pan et al.,1992). After removal of the brainstems through the spinalcord canal, tissues were rapidly frozen in liquid nitrogen andmaintained at 2708C; all purification steps were performed at48C, and ice-cold buffers were used.

Purity of the obtained protein was assessed by perform-ing an SDS-PAGE (12% gel), visualizing proteins by silverstaining, and by standard Western Blot analysis performedwith anti-PrP mAB 6H4 (Fig. 1). No copurifying proteinbands were detectable. Final protein concentration was meas-ured by the optical density at 280 nm, using as extinctioncoefficient a value of 58718.0 M21cm21. The obtained PrPC

was divided into aliquots and stored at –808C.

Measurement of Proteasome Activities

Fractions of the brainstem were homogenized in 50mM Tris, 0.1 M KCl, 2 mM EDTA, pH 7.5, and ice-coldbuffer and subsequently passed 15 times through a 20-gaugeneedle, maintaining the samples at 48C. The insoluble materialwas removed by refrigerated centrifugation for 60 min at13,000g, and the supernatant was further analyzed. Proteinconcentration was determined by the Lowry protocol (Lowryet al., 1951). Isolation and purification of 20S proteasomesfrom bovine brain and thymus were carried out as previouslyreported (Eleuteri et al., 2000; Amici et al., 2003). Proteasomeactivities were measured upon incubation of an aliquot of

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each sample homogenate (250 lg of total proteins) with syn-thetic substrates specific for the T-L and PGPH components(Z-GGR-2NA and Z-LLE-2NA, respectively) in the activitybuffer (50 mM Tris-EDTA, pH 7.8) for 1 hr at 378C in atotal volume of 250 ll, as reported previously (Orlowskiet al., 1993; Eleuteri et al., 2000). Absorbance values weredetected on a spectrophotometer Cary 1.

The ChT-L and BrAAP activities were determined withthe fluorogenic substrates Suc-LLVY-AMC and Z-GPALG-pAB, respectively, in a final volume of 500 ll. After 1 hr ofincubation at 378C, the fluorescence was detected on a spec-trofluorimeter Shimadzu RF5301 (AMC: kexc 5 365 nm, kem5 449 nm; PABA: kexc 5 304 nm, kem 5 664 nm). TheChT-L and the BrAAP activities were measured also in thepresence of specific inhibitors Z-GPFL-CHO and lactacystin(5 lM in the assay; Dick et al., 1996; Eleuteri et al., 1997).Aminopeptidase N (EC 3.4.11.2), used in the coupled assayfor the detection of the BrAAP activity, was purified from pigkidney, as reported elsewhere (Pfleiderer, 1970; Almenoff andOrlowski, 1983). Specific activities are expressed as the num-ber of micromoles of substrate degraded per milligram of pro-tein per hour.

In the same manner, catalytic activities of constitutiveand IFNg-inducible isolated 20S proteasomes (10 lg ofenzyme) were then measured in the presence of increasingconcentrations of PrPC (0–300 nM) previously isolated. Toeliminate the possible interference of PrPC dissolving buffer,control assays were carried out in the presence of the sameamounts of dissolving buffer (0.01 M MES, 0.12% ZW 3-12,pH 6.2).

Proteasome Immunoprecipitation From BrainstemHomogenates

Proteasome immunoprecipitation was conducted onbrainstem tissue lysates from different sheep using anti-20S pro-teasome antibody coupled with protein A-Sepharose CL-4B

(Sigma). The immunoprecipitation was carried out accordinglyto previously published protocols (Zwilling et al., 1995; Harlowand Lane, 1999; Keck et al., 2003). After immunoprecipitation,each pellet was resuspended in 50 ll Laemmli sample buffer,heated at 958C for 5 min, centrifuged for 30 sec at 12,000g, andpooled supernatants were used for Western blot analyses withanti-PrP antibody (mAB) 6H4. An immunoblot detection withan anti-20S proteasome antibody was performed to verify theefficiency of the immunoprecipitation procedure and to controlthe equal protein loading.

Polyacrylamide Gel Electrophoresis and Western Blot

Sodium dodecyl sulfate-polyacrylamide gel electrophore-sis (SDS-PAGE) was performed in 12% gels. Immunoblotexperiments with anti-20S proteasome, anti-b5, b1, and b2subunit or anti-b5i, b1i, and b2i subunit antibodies were per-formed electroblotting tissue lysates, previously separated on a12% SDS gel, onto a PVDF membrane (Millipore Corpora-tion, Bedford, MA) according to Towbin and Burnette(Towbin et al., 1979; Burnette, 1991). Mouse monoclonalanti-PrP antibody (mAB) 6H4 was utilized for immunoblotdetections of PrP protein in 20S proteasome-immunoprecipi-tated samples to verify their coimmunoprecipitation.

Every gel was loaded with molecular weight markersincluding proteins with MW from 6.5 to 205 kDa (SigmaMarker-Wide Molecular Weight Range; Sigma-Aldrich S.r.l.,Milano, Italy). GAPDH was utilized as a control for equalprotein loading: membranes were stripped and reprobed forGAPDH with a monoclonal antibody diluted 1:500. A densi-tometric algorithm has been developed to quantitate theWestern blot results. Each Western blot film has been scanned(16-bit gray-scale), and the obtained digital data were proc-essed to calculate the background mean value and its standarddeviation. The background-free image was then obtained bysubtracting the background intensity mean value from theoriginal digital data. The integrated densitometric value asso-ciated with each band was then calculated as the sum of thedensity values over all the pixels belonging to the consideredband having a density value higher than the backgroundstandard deviation. The band densitometric value was thennormalized to the relative GAPDH signal intensity. The ratiosof band intensities were calculated within the same Westernblot. All the calculations were carried out in the Matlab envi-ronment (The MathWorks Inc., Natick, MA) (Marchiniet al., 2005).

Immunohistochemistry

Formalin-fixed, paraffin-embedded tissue blocks frombrainstem portions, sampled adjacent to portions used for West-ern blot evaluations, were selected for immunohistochemistryto estimate the expression of the 20S proteasome core particlein infected and control sheep. Serial sections were used to eval-uate, respectively, proteasome expression, prion expression(using the prion protein antibody PrP-ab3531, 1:20 dilution),and apoptosis by using the TUNEL colorimetric staining(DeadEnd; Promega Italy) according to the manufacturer’sinstructions. Finally, adjacent sections were also employed forthe evaluation of immunoreactive ubiquitin-protein conjugates,

Fig. 1. PrPC purification. PrPC was purified following Pan protocol(Pan et al., 1992). Purity of the obtained protein was assessed byperforming 12% SDS-PAGE, visualizing proteins by silver staining(gel lane) and a Western Blot analysis with an anti-PrP mAB, 6H4(wb lane).

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by using an anti-ubiquitin antibody diluted 1:50 in buffer, incu-bated overnight, and routinely developed as described below.

Briefly, after blocking the endogenous peroxidase, sec-tions were treated by an epitope retrieval method usingmicrowaves in 0.01 M citrate buffer (pH 6.0), then incubatedin PBS containing 1% bovine serum albumin (BSA) and 1%polyvinyl-pyrrolidone (PVP; Sigma) for 30 min at room tem-perature, followed by incubation with primary antibodies. Toenhance PrP immunostaining, sections were pretreated withpicric acid for 15 min, autoclaved in distilled water for 10min, treated with 96% formic acid for 10 min, and thentreated with 4 M guanidine thiocyanate at 48C for 120 min(Kovacs et al., 2002; Adori et al., 2005). This step was omit-ted for sections employed for triple-stain reaction, tested toevaluate the colocalization of PrP, the 20S proteasome, andapoptotic activity in the same neuron. All single reactions ofimmunostaining were developed with a peroxidase/DAB kit(En Vision; DAKO). No-first-antibody control was carriedout with the omission of primary antibody. In sections testedfor multiple antigens, binding of previously mentioned anti-bodies was revealed with ABC-peroxidase (Vector Laborato-ries, Burlingame, CA) or ABC-alkaline phosphatase (VectorLaboratories) techniques with 1:200-diluted biotin-conjugatedgoat anti-rabbit IgG (AO433; DAKO), applied for 45 min atroom temperature as secondary antibodies. The enzymaticreaction was developed with 3-1-diaminobenzydine (DAB)with nickel (Sigma) added to develop the TUNEL colorimet-ric staining, and VIP (Vector Laboratories) or Vector blue(Vector Laboratories) as substrates, respectively, for ABC-per-oxidase and ABC-alkaline phosphatase techniques. All sectionswere counterstained with Meyer’s haematoxylin and coveredwith Vectamount mounting medium (Vector Laboratories).Intensity of immunostaining was assessed semiquantitativelyby a four-grade score (no, weak, moderate, and stronglabeling).

Immunoblot Detection of Carbonyl Groups

Immunoblot detection of carbonyl groups was performedwith the OxyBlot oxidized protein detection kit, according tothe manufacturer. Briefly, 25 lg brainstem lysates were incubatedfor 15 min at room temperature with 2,4-dinitrophenylhydrazine(DNPH) to form the dinitrophenylhydrazone carbonyl deriva-tive and separated on a 12% SDS/PAGE. The modified proteins,blotted on a PVDF membrane, were revealed by anti-DNPantibodies. The immunoblot detection was carried out with anenhanced chemiluminescence (ECL) Western blot analysissystem (Amersham-Pharmacia-Biotech) using peroxidase-conjugated secondary antibody.

Statistical Analysis

Values are expressed as mean and SD of results obtainedfrom separate experiments. Student’s t-test was used to com-pare differences of means among control, aged, and scrapiegroups in the 20S-specific activities measures and in the densi-tometric analyses of Western blots.

RESULTS

Immunoblot Detection of PrPSc Isoform

Tissue samples of brainstems from post-mortemsamplings on slaughtered and fallen stock sheep werefirst subjected to the Prionics-Check Western test toevaluate the presence of the PrPSc protein. These testswere performed at the Istituto Zooprofilattico Sperimen-tale Umbria-Marche (data not shown). Table I summa-rizes data on the samples utilized in the present study.All tested brainstem tissues came from female sheep ofdifferent age. In particular, samples were chosen in orderto consider both various scrapie stages (lethal or not) andmodifications resulting from the normal aging processes.Indeed, besides considering healthy and scrapie-positivesubjects, we analyzed three healthy sheep (samples 6–8),which were much older than the other animals. Thischoice was made with the aim of estimating thecontribution of oxidative stress in the pathologicalcondition.

Proteasome Activities in Brainstem Tissues

Proteasome activities were measured in brainstemtissue lysates comparing controls (n 5 5 different sub-jects), aged samples (n 5 3 different subjects), and sam-ples positive to the Prionics-Check Kit (n 5 5 differentsubjects). Assays were conducted as reported in Materialsand Methods. The BrAAP and ChT-L activities weredetermined in the presence and absence of specificinhibitors, Z-GPLF-CHO and lactacystin, to assess theeffective contribution of these two components to thepeptide degradation.

In comparing the specific activity mean valuesobtained by assaying the proteasome components incontrols and scrapie-positive animals, there was a signifi-cant change in the T-L and ChT-L activities. In particu-lar, from the data reported in Table II, it is evidentthat the ChT-L is the most affected component, with a3.6-fold increase compared with control mean value(P < 0.05), followed by the T-L activity, which shows a

TABLE I. Data related to the animals utilized in the

present study

Sample Prionics1-Check test Type Sex Age (month)

1 Negative Fallen Stock F 52

2 Negative Slaughtered F 54

3 Negative Slaughtered F 34

4 Negative Slaughtered F 35

5 Negative Slaughtered F 32

6 Negative Slaughtered F 170

7 Negative Slaughtered F 165

8 Negative Slaughtered F 183

9 Positive Slaughtered F 34

10 Positive Slaughtered F 33

11 Positive Slaughtered F 35

12 Positive Slaughtered F 39

13 Positive Fallen Stock F 59

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1.4-fold activation (P < 0.05). No significant changes inscrapie-affected animals were detected for the PGPHand BrAAP components.

As mentioned above, samples 6–8 represent con-trol, non-infected animals, but they were much olderthan the other healthy subjects. It is interesting to notethat for these samples the tendency observed for protea-some proteolytic activities was similar to that of positivesheep: the ChT-L and the T-L components showed,respectively, a 1.97-fold and a 1.4-fold increasecompared with control group mean values (P < 0.05).Furthermore, aged subjects presented an altered BrAAPactivity, which was 1.8-fold increased with respect tocontrol group mean value (P < 0.05), and a slight inhi-bition of the PGPH component (20% decrease comparedwith controls, P < 0.05). These results suggest that anincrease of oxidative stress, known to be associated withaging, is involved in the progression of scrapie.However, the scrapie sheep ChT-L showed a more evi-dent activation (3.6-fold increase) than in older samples(1.97-fold activation) compared with the healthy sheepChT-L mean value (P < 0.05).

Variations in proteasome functionality are corre-lated with changes in the cytoplasmic amounts of ubiq-uitin-protein conjugates. To detect such complexes, weperformed immunohistochemical studies on obex sec-tions comparing control and scrapie tissues. Structurescontaining immunoreactive ubiquitin-protein conjugateswere evident in scrapie-affected obex, particularly ininclusion bodies within vacuolated neurons and in areassurrounding anti-PrP-positive plaques. The ubiquinatedintraneuronal inclusion bodies were observed only inhighly vacuolated parts of the scrapie-affected brains,

strictly related to the diffuse deposits of PrP protein(Fig. 2).

Subsequently, with the aim of explaining the dif-ferences observed in proteasome activities between con-trols and pathological samples, we analyzed proteasomecontent in the brainstem samples by immunoblottingassays with an anti-20S ‘‘core’’ proteasome antibody.These results indicate that the variations in proteasomeactivity values are the result of neither modified protea-some expression nor proteasome decomposition duringpost-mortem autolytic tissue degradation (Fig. 3).

Immunoblot Detection of Proteasomalb-Catalytic Subunits

To verify b-subunit expression levels in scrapiesamples in comparison with controls, immunoblots wereperformed with antibodies against constitutive (b5, b1,and b2) and IFNg-inducible (b5i, b1i, and b2i) subu-nits. Each subunit is responsible for the different protea-some activities: b5 for the ChT-L and BrAAP, b2 forthe T-L, and b1 for the PGPH in the constitutiveproteasome; b5i for the ChT-L and BrAAP; b2i for theT-L, and b1i for the PGPH in the immunoproteasome(Nelson et al., 2000; Krause et al., 2006).

20S proteasomes isolated from bovine brain andthymus were used as internal references for a constitutiveand an immunoproteasome, respectively. Immunoblotanalyses revealed that both constitutive and IFNg-induc-ible subunits were expressed in all samples; moreover,no differences were detectable in proteasome subunitexpression between scrapie-positive and control samples(Fig. 3D–I).

TABLE II. 20S proteasome specific activity values. The assays were performed as described in the Materials and Methods section

Specific activity (lmoles of substrate/mg h)

PGPH (Z-LLE-2NA) BrAAP (GPALA-pAB) T-L (Z-GGR-2NA) ChT-L (Suc-LLVY-AMC)

CONTROLS 1 28.05 3 1023 0.847 3 1022 22.95 3 1023 6.57 3 1026

2 26.20 3 1023 1.762 3 1022 18.11 3 1023 9.11 3 1026

3 21.83 3 1023 0.918 3 1022 21.37 3 1023 6.59 3 1026

4 24.20 3 1023 1.412 3 1022 21.80 3 1023 7.25 3 1026

5 22.29 3 1023 1.516 3 1022 24.45 3 1023 7.40 3 1026

Mean value (24.51 6 2.63) 3 1023 (1.291 6 0.39) 3 1022 (21.73 6 2.35) 3 1023 (7.38 6 1.04) 3 1026

AGED SUBJECTS 6 21.14 3 1023 2.384 3 1022 30.09 3 1023 15.42 3 1026

7 17.99 3 1023 2.293 3 1022 29.89 3 1023 13.64 3 1026

8 19.72 3 1023 2.570 3 1022 31.67 3 1023 14.66 3 1026

Mean value * * * *

(19.61 6 1.58) 3 1023 (2.415 6 0.14) 3 1022 (30.55 6 0.98) 3 1023 (14.57 6 0.89) 3 1026

SCRAPIE 9 28.39 3 1023 1.830 3 1022 29.49 3 1023 23.26 3 1026

10 29.85 3 1023 2.638 3 1022 33.86 3 1023 22.22 3 1026

11 24.50 3 1023 1.544 3 1022 29.72 3 1023 24.74 3 1026

12 22.31 3 1023 0.518 3 1022 28.52 3 1023 31.65 3 1026

13 22.67 3 1023 0.645 3 1022 34.75 3 1023 32.22 3 1026

Mean value * *

(25.54 6 3.41) 3 1023 (1.435 6 0.88) 3 1022 (31.27 6 2.83) 3 1023 (26.82 6 4.76) 3 1026

(*) statistical significance (p < 0.05) of scrapie and aged subjects mean values compared to controls.

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Immunoblot Detection of PrP in20S-Immunoprecipitated Samples

Trying to explain the altered proteolytic activitiesobserved in scrapie samples, we evaluated the possibilityof a direct interaction between PrP and 20S protea-somes. To evaluate the presence of the prion protein in20S-immunoprecipitated samples, immunoblot experi-ments using the anti-PrP antibody 6H4 were performed.This monoclonal antibody recognizes the amino acidsequence from 144 to 152, a well-conserved region inmammalian prions. The 20S proteasome was immuno-precipitated following previously published protocols(Zwilling et al., 1995; Keck et al., 2003). Resultsreported in Figure 4 demonstrate that the immunostain-ing of the immunoprecipitated 20S proteasomes withthe anti-PrP antibody produces a protein band in all theloaded samples, young (lanes 1–5), aged (lanes 6–8), andpositive ones (lanes 9–13), indicating that prions copreci-pitate with the 20S proteasome. The control sample(Std) contains the normal isoform of the prion protein:the corresponding diffuse band is spread from 25 to 35

kDa because of the glycosylation of PrPC, which causesa heterogeneous distribution.

Immunohistochemical Distribution of the 20SProteasome Complex and PrP

To further elucidate the proteasome-PrP interac-tion suggested by the Western blot analysis, weexamined the regional and subcellular distribution of 20Sproteasome and prion proteins in scrapie sheep com-pared with healthy, age-matched controls. Topographi-cally, no differences were observed in 20S proteasome

Fig. 3. A–I: Representative Western blot performed with anti-20Sproteasome and anti-b subunits antibodies. Representative autora-diographies of 20S proteasome (B) and proteasomal b-subunits (D–I)expression in brainstem homogenates of control sheep (lanes 1–5),aged sheep (lanes 6–8), and scrapie-positive animals (lanes 9–13).Lanes C were loaded with 5 lg constitutive or immuno-20S protea-some purified from bovine brain or thymus. The densitometric anal-ysis from six separate blots provided for quantitative analysis of theamount of 20S ‘‘core’’ is presented (A) and a representative Westernblot is shown in B. Equal protein loading was verified by using ananti-GAPDH antibody (C). The immunostaining was performedusing anti-20S proteasome (B), anti-b1 (D), anti-b1i (E), anti-b2 (F),anti-b2i (G), anti-b5 (H), and anti-b5i (I) antibodies, and the detec-tion was executed by an ECL Western blot analysis system.

Fig. 2. Representative immunohistochemical analyses showing sec-tions from control sheep 4(A) and scrapie sheep 11(B) stained with theanti-Ub antibody. Scale bars 5 50 lm. [Color figure can be viewed inthe online issue, which is available at www.interscience.wiley.com.]

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signal localization between unaffected and affected brain-stems (Fig. 5A-A0). Variations between groups wereobserved in the neuronal localization of the signal.Controls present a 20S proteasome signal mainly in thecytoplasm, with both perikaryons and neuritis immuno-

reactive (Fig. 5A). In absence of BrdU labeling of nuclei,there was an immunohistochemical negativity of the samebrainstem neurons for PrPSc protein. In scrapie samples,neurons with signs of vacuolation or other degenerativemodifications showed a prominent nuclear 20S immuno-positivity (Fig. 5A0). In brainstems of scrapie-positivesheep, tests performed with an anti-prion protein asprimary antibody revealed a constant cytoplasmic colocali-zation of PrPSc in neurons strongly positive also for 20Sproteasome (Fig. 5B0). To verify that neurons were dyingby apoptosis, cells were labeled with a DeadEnd Kit,evidencing DNA breaks, a hallmark of apoptosis. As seenin Figure 5C, C0, brainstem neurons showing nuclearexpression of 20S proteasome and an evident positivity forPrP exhibited intense 5-bromo-2-deoxy-uridine (BrdU)labeling, indicating that neurons underwent apoptosis.Furthermore, the levels of BrdU labeling also provided adirect comparison of 20S proteasome nuclear expressionvs. cytoplasmic expression, confirming that damagedneurons showed strong nuclear positivity.

To corroborate such data, we conducted triple-staining analyses, evidencing on the same section apopto-tic processes and the localization of the 20S proteasomeand prion proteins (Fig. 5D,D0). In confirmation of thedata discussed above, control sheep show the cytoplasmiccoexpression in the same neurons of 20S proteasomeand PrP in perikarions and neuritis. The absence ofBrdU labeling of nuclei demonstrates the lack of apopto-tic activity. In vacuolated neurons showing PrP cytoplas-mic localization, a prevalent nuclear immunopositivityfor the 20S proteasome can be observed. Furthermore,suffering neurons present the nuclear coexpression of20S proteasome with an intense BrdU labeling,indicating that neurons underwent apoptosis and that thecomplex is involved in such pathway.

Fig. 5. Representative immunohistochemical analyses. A–C,A0–C0: Sections from control sheep 4 andscrapie sheep 11 stained with the anti-20S and the anti-prion protein antibodies and TUNEL assay.D,D0: Triple-staining images: PrP protein, brownish stain; 20S proteasome, purple-blue stain; BrdUlabeling, black stain. Scale bars 5 50 lm in A–C and A0–C0; 25 lm in D. [Color figure can be viewedin the online issue, which is available at www.interscience.wiley.com.]

Fig. 4. A–C: Detection of the PrP isoform in immunoprecipitated20S proteasomes. Representative Western blot showing 20S protea-some-immunoprecipitated samples immunostained with the anti-PrPantibody 6H4. Lanes 1–8 were loaded with immunoprecipitatedobtained from the corresponding young and aged healthy animals;lanes 9–13 were loaded with immunoprecipitated obtained from thecorresponding positive subjects. Lane Std was loaded with the stand-ard mixture of glycosylated PrPC according to the Prionics-CheckWestern Test. Immunostaining with the anti-20S proteasome wasperformed as a control of the immunoprecipitation procedure andequal sample loading (C). Lane C was loaded with a purified 20Sproteasome used as internal control.

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Immunoblot Detection of Carbonyl Groups

Numerous papers have reported that an increase inoxidative conditions and a decrease of antioxidant defen-ces are responsible for damages in the brain of infectedanimals and may therefore contribute to the develop-ment of prion diseases (Guentchev et al., 2000, 2002).In the present study, oxidized proteins levels were ana-lyzed by the immunoblot detection of carbonyl groupsin lysates of brainstem tissues. Infected animals (meanO.D. 12.95 6 0.32) showed a significant increase (P <0.001) in carbonyl groups compared with controls (meanO.D. 8.42 6 0.33), whereas no remarkable differenceswere measurable between positive samples from slaugh-tered and fallen stock sheep (Fig. 6). In line with datafrom the proteolytic assays, aged animals (O.D. 12.8 60.36) showed a trend similar to that of scrapie-positiveanimals, confirming that scrapie is correlated with anincrease in the levels of cellular oxidative stress (Fig. 6).

Moreover, the same experiment was performedwith 20S-immunoprecipitated samples to detect a differ-ent oxidation level of 20S proteasomes from scrapie tis-sues. No differences were found between positive tissuesand controls (data not shown).

Effects of Purified PrPC on Isolated 20SProteasome Activities

The effects of the isolated PrPC isoform on consti-tutive and IFNg-inducible 20S proteasome functionality

were evaluated through enzymatic assays. Given that thesolubility of the pathological prion protein isoform isstrictly dependent on high detergent concentrations,yielding to nonoptimal experimental conditions for pro-teasomal functionality, the effects of PrPSc have not beentested in the present study. Furthermore, instead of usingunglycosylated recombinant PrPC, in our assay we addeda tissue purified PrPC to assess the effect of a naturaloccurring protein on proteasomal systems (Cuccioloniet al., 2005).

The ChT-L, PGPH, T-L, and BrAAP proteasomecomponents were tested in the presence of increasingconcentrations of PrPC (0.0–300.0 nM), using specificsubstrates. As shown in Figure 7, PrPC induced a global,concentration-dependent, and proteasome-composition-dependent inhibition of the tested components in boththe proteasomes. However, in the immunoproteasome,the BrAAP component is particularly sensitive to theexposure to PrPC, with a 50% inhibition detectable at aconcentration of prion protein of 30 nM. In both pro-teasomes, the most affected catalytic activity is the ChT-L which, at low prion protein concentration such as 75nM, showed a 60% inhibition compared with protea-some activity alone. The exposure to higher PrPC con-centrations (300 nM) dramatically decreased the ChT-Lcomponent functionality, since just a 10% and 20% ofthe total ChT-L activity in the immunoproteasome andin the constitutive proteasome, respectively, remaineddetectable.

DISCUSSION

Possible correlations between 20S proteasome andprion proteins were previously proposed, suggesting thatinhibition of the proteasomal system may be a cause forthe cytosolic accumulation of PrP and that PrPSc specifi-cally inhibits proteasome subunit activity (Yedidia et al.,2001; Ma et al., 2002; Kristiansen et al., 2007). Never-theless, these findings do not explain the relationshipbetween proteasome and prion proteins in vivo and inmodels of PrP toxicity, such as scrapie disease. From thisperspective, in the present study the interplay between20S proteasomes and prion proteins in healthy and scra-pie-infected animals has been elucidated.

Brainstem homogenates from both young and agedhealthy sheep and from infected animals were tested forproteasome activities. Scrapie-positive subjects comparedwith young controls presented not only no decrease inproteasome activity but rather an increase, with an evi-dent activation of two of the tested components, theChT-L and T-L. Interestingly, values obtained fromanalyzing aged controls were comparable to those meas-ured in the scrapie-affected subjects, indicating that oxi-dative conditions, which characterize the aging process,are able to modify proteasome activity and are widelyimplicated in the progression of the disease.

The immunohistochemical analysis performed oncontrol and scrapie tissues revealed an accumulation ofubiquitinated proteins in positive sections, most likely

Fig. 6. Carbonyl group levels in brainstem homogenates. Measure-ment of protein carbonyl groups levels in brainstem homogenates fromcontrol sheep (lanes 1–5), aged sheep (lanes 6–8), and scrapie-positiveanimals (lanes 9–13). The densitometric analysis from six separate blotsprovided for quantitative analysis of the amount of protein carbonyls ispresented (A), and a representative Western blot of protein carbonyls isshown (B). Equal protein loading was verified by using an anti-GAPDH antibody (C). Samples were treated according to the OxyBlotprocedure (Oxidized Protein Detection Kit; Oncor). The detectionwas executed with an ECL Western blot analysis system.

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indicating that the increased proteasome functionality isnot able to compensate the ROS-mediated cellular dam-age. No variations in 20S proteasome content or in itssubunit composition were detected, suggesting that,although the infection affects proteasome activity, it doesnot influence proteasome content and subunitsexpression. These observations are in accordance withimmunohistochemical results that reveal no changes inthe 20S proteasome complex expression in affected andhealthy tissues, with the exception of a different signallocalization.

The immunohistochemical analysis shows for neu-ronal nuclei of scrapie samples a strong 20S proteasomeimmunostaining compared with healthy subjects, inparticular in brainstem areas where the PrP cytoplasmicsignal is evident and absent in controls. This signal distri-bution positively correlates also with TUNEL reactivity.Therefore, neurons positive to both 20S proteasome andPrP proteins can be considered a subset of susceptibleneurons in scrapie. Through the TUNEL reaction, amarker of DNA damage, we identified neurons in whichpathways of cell death are triggered. In addition, as pre-viously reported by Adori et al. (2005), the nuclearredistribution and accumulation of 20S proteasome inscrapie neurons suggest that the proteasome takes part inDNA repair and/or cell death mechanisms.

High levels of protein oxidation, consideredresponsible for alterations in cellular homeostasis andneuronal cell death, often occur in neurodegenerative

pathologies (Sayre et al., 2001; Rubinsztein, 2006;Cecarini et al., 2007; Ding et al., 2007). Earlier studieshave described an increased neuronal susceptibility tooxidative stress and an altered free radical metabolism inprion-associated pathologies (Wong et al., 2001; Milha-vet and Lehmann, 2002). Investigating the levels of pro-tein carbonyl groups, we observed a higher incidence ofprotein oxidation in both aged and infected subjectscompared with young individuals, confirming thatprotein oxidation is a common hallmark for these twoconditions, aging and scrapie. Insofar as previous publi-cations have reported that oxidized proteins are prefer-entially degraded by the 20S proteasome (Grune et al.,2003; Jung et al., 2007) and that mild oxidative condi-tions may regulate and enhance proteasome-mediateddegradation (Grune et al., 1998; Davies, 2001; Buchczyket al., 2003; Ding et al., 2003), the activation of theproteasome complex reported here may be considered asa means of counteracting the effects of oxidative stress.

The PrP and 20S proteasome coprecipitation andthe immunohistochemical coexpression in the same neu-rons demonstrate that PrP interacts with the proteasomein healthy and diseased brains and describe a possiblefunctional proteasome–PrP interaction. Even thoughsuch data do not provide sufficient information to clarifythe real nature of the interaction (between prionproteins and the complex) or its specificity, given theexperimental conditions utilized in the immunoprecipi-tation procedure, it is reasonable to think that most of

Fig. 7. Effects of isolated PrPC on 20S proteasomes. Effect of increasing concentrations (from 0.0 to300.0 nM) of PrPC on the ChT-L (A), BrAAP (B), PGPH (C), and T-L (D) components of constitu-tive (solid squares) and IFNg-inducible (open squares) 20S proteasomes. Values are reported as per-centage compared with proteasome activity alone. The data shown are mean values 6 SD from sixdistinct determinations.

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the nonspecific interactions are eliminated throughoutthe assay.

Data obtained by testing the in vitro effects ofPrPC on purified constitutive and immuno-20S protea-somes showed a concentration-dependent and protea-some composition-dependent decrease (particularlyevident for the ChT-L component) in proteasome activ-ity. Quantitative and qualitative distribution of PrPC inseveral tissues from sheep has been reported by Moudjouet al. (2001), and PrPC concentrations employed in ourassays were comparable to those detected in brainregions, ranging from 15 to 300 nM (Moudjou et al.,2001). Interestingly, Ma et al. (2002) have reported thatproteasome inhibition is able to induce PrPC neurotox-icity through the accumulation of prion protein in thecytosol. The observed inhibition may therefore occurunder physiological conditions in the brain.

The different behavior ‘‘in vitro’’ and in brainstemsamples described for the proteasome functionality, that is,respectively, inhibition upon treatment with isolated PrPC

and activation in the homogenates, could be explained bytaking into account that in vivo several pathways contrib-ute to the final evidence. For example, the proteasomeactivation may reflect the presence of lower levels of PrPC

in affected tissues, because of its conversion in the abnor-mal isoform and the incidence of oxidative stress able topromote proteasome functionality. It is likely, taking intoaccount the above-mentioned coprecipitation of the twoproteins in both healthy and pathological samples, thatprion proteins interact with the proteasome interferingwith substrates trafficking and degradation.

Unlike the other activities, the BrAAP componentof the constitutive proteasome was activated by the pres-ence of the prion protein. Previous data indicate that theBrAAP component in the constitutive proteasome canbe assigned to the b5 subunit and that also the b1 subu-nit presents a limited activity related to this component(Groll et al., 2005). It has been earlier reported on thelatent form of the BrAAP activity, whose full expressionrequires activation (Eleuteri et al., 1997). It seems rea-sonable to hypothesize that the observed BrAAP activa-tion in the constitutive proteasome could be due to arearrangement of the proteasome structure induced bythe prion protein that ultimately facilitates substrate entryinto the proteasome active sites (Eleuteri et al., 1997;Kisselev et al., 2002; Amici et al., 2008). This can beascribed to a previously reported selective effect of pro-teasome stability and conformation on its subunits thatinduces different behaviors of its catalytic sites(Ferrington and Kapphahn, 2004).

In summary, our data are consistent with scrapiepromoting an increase in proteasome activity in thebrain, presumably as a means of counteracting the effectsof secondary pathologies of scrapie such as oxidativestress. This is based on previous studies demonstratingthat low-level oxidative stress stimulates proteasomeactivity in neural cells (Ding et al., 2003). These dataraise the possibility that PrP interacts with the protea-some in both normal and diseased brain and potentially

plays a role in substrate trafficking and/or regulation ofproteasome activity, with the proteasome–PrP interac-tions able to induce proteasome activity in a proteasomecomposition-dependent manner. A thoroughly under-standing of this newly reported event will significantlyincrease our knowledge of the relationship betweenPrP and the proteasome in both normal and diseasedconditions.

ACKNOWLEDGMENT

The authors thank Alessandra Di Donato for hertechnical assistance.

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