Biochem. J. (2010) 430, 477–486 (Printed in Great Britain) doi:10.1042/BJ20100615 477

The hydrophobic core region governs mutant prion protein aggregation andintracellular retentionEmiliano BIASINI*†1,2, Laura TAPELLA*†, Elena RESTELLI*†, Manuela POZZOLI*†, Tania MASSIGNAN*‡ and Roberto CHIESA*†2

*Dulbecco Telethon Institute, Milan 20156, Italy, †Department of Neuroscience, Mario Negri Institute for Pharmacological Research, Via G. La Masa 19, Milan, 20156, Italy, and‡Department of Biochemistry and Molecular Pharmacology, Mario Negri Institute for Pharmacological Research, Via G. La Masa 19, Milan, 20156, Italy

Approx. 15% of human prion diseases have a patternof autosomal dominant inheritance, and are linked tomutations in the gene encoding PrP (prion protein), a GPI(glycosylphosphatidylinositol)-anchored protein whose functionis not clear. The cellular mechanisms by which PrP mutationscause disease are also not known. Soon after synthesis in theER (endoplasmic reticulum), several mutant PrPs misfold andbecome resistant to phospholipase cleavage of their GPI anchor.The biosynthetic maturation of the misfolded molecules in the ERis delayed and, during transit in the secretory pathway, they formdetergent-insoluble and protease-resistant aggregates, suggestingthat intracellular PrP aggregation may play a pathogenic role.We have investigated the consequence of deleting residues 114–121 within the hydrophobic core of PrP on the aggregation and

cellular localization of two pathogenic mutants that accumulatein the ER and Golgi apparatus. Compared with their full-lengthcounterparts, the deleted molecules formed smaller protease-sensitive aggregates and were more efficiently transported to thecell surface and released by phospholipase cleavage. These resultsindicate that mutant PrP aggregation and intracellular retentionare closely related and depend critically on the integrity of thehydrophobic core. The discovery that !114–121 counteractsmisfolding and improves the cellular trafficking of mutant PrPprovides an unprecedented model for assessing the role ofintracellular aggregation in the pathogenesis of prion diseases.

Key words: inherited prion disease, prion protein (PrP), proteinaggregation, protein misfolding.

INTRODUCTION

Prion diseases are invariably fatal neurodegenerative disordersof humans and animals that arise because of misfoldingof the PrPC [cellular isoform of PrP (prion protein)], aGPI (glycosylphosphatidylinositol)-anchored glycoprotein whosefunction is not clear [1]. Unique among neurodegenerativedisorders of protein conformation, prion diseases can betransmitted through the infectious route [2]. The transmissibleagent (prion) is composed primarily of PrPSc (scrapie isoformof PrP), a misfolded/aggregated form of PrP that is capableof seeding conformational conversion of PrPC molecules [3,4].Prion infections are quite frequent in animals, but rare inhumans, in whom most prion diseases occur sporadically orare inherited due to mutations in the gene encoding PrP [5].The genetic forms account for approx. 15 % of all human priondiseases, and include fCJD (familial Creutzfeldt–Jakob disease),GSS (Gerstmann–Straussler–Scheinker) syndrome and FFI (fatalfamilial insomnia).

A 72-amino-acid insertion in the N-terminus of PrP isassociated with a mixed CJD-GSS phenotype [6], and areplacement of aspartic acid with asparagine at codon 178 islinked to either fCJD or FFI [7]. Transgenic mice carryingmouse PrP homologues of these mutations (PG14 and D177Nrespectively) develop fatal neurological illnesses that model keyfeatures of the corresponding human disorders [8–10]. Thesemice synthesize misfolded forms of mutant PrP in their brains,with biochemical properties reminiscent of PrPSc, includinginsolubility in non-denaturing detergents, resistance to mild PK

(proteinase K) digestion and resistance to cleavage of their GPIanchor by phospholipase [8,9]. Analysis of PG14 and D177NPrP metabolism in primary neurons shows that the biosyntheticmaturation of these mutants in the ER (endoplasmic reticulum)is delayed [11] and they accumulate abnormally in the ER andGolgi apparatus [9,12].

These observations suggest ways by which mutant PrP mightdamage neurons [13]. Excessive accumulation of misfolded PrPin secretory organelles might saturate the protein folding andtransport machinery leading to defective secretion of proteinsimportant for normal neuronal function [14]. Additionally, build-up of mutant PrP in the ER lumen could stimulate stress-responsemechanisms, such as the unfolded protein response, leading toneuronal death [15]. Alternatively, neuronal dysfunction mightresult from toxicity of mutant PrP molecules that escape quality-control systems in the secretory pathway and reach the cellsurface, where they may interact abnormally with other proteins,such as ion channels, receptors or signalling molecules [16]. Witha view to determining whether the toxic PrP molecules are thoseretained inside the cell or those that escape, we sought ways toinfluence mutant PrP misfolding and intracellular trafficking.

The central HC (hydrophobic core) region between aminoacids 111 and 135 plays a crucial role in PrP misfolding. Afterconformational transition to the PrPSc state, this region becomesinaccessible to antibodies, suggesting that it undergoes a profoundstructural rearrangement [17]. Consistent with this view, syntheticPrP peptides that overlap the HC have an intrinsic tendency tospontaneously adopt a "-sheet-rich structure reminiscent of PrPSc

[18–20]. Conversely, PrP molecules that are deleted in the HC

Abbreviations used: BHK, baby hamster kidney; DAPI, 4!,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; ER, endoplasmicreticulum; FBS, fetal borine serum; fCJD, familial Creutzfeldt–Jakob disease; FFI, fatal familial insomnia; GPI, glycosylphosphatidylinositol; GSS,Gerstmann–Straussler–Scheinker; HC, hydrophobic core; HEK, human embryonic kidney; HRP, horseradish peroxidase; LB, lysis buffer; MEM, minimalessential medium; PDI, protein disulfide isomerase; PIPLC, phosphatidylinositol-specific phospholipase C; PK, proteinase K; PrP, prion protein; PrPC,cellular isoform of PrP; PrP-M6P, PrP with the transmembrane domain of the mannose 6-phosphate receptor; PrPSc, scrapie isoform of PrP; WT, wild-type.

1 Present address: Department of Biochemistry, Boston University School of Medicine, 72 East Concord Street, Boston, MA 02118, U.S.A.2 Correspondence may be addressed to either of these authors (email roberto.chiesa@marionegri.it or biasini@bu.edu).

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have greater conformational stability than WT (wild-type) PrP[21] and are refractory to PrPSc-induced conversion [22,23]. Wetested whether deleting part of the HC of PG14 and D177N PrPsantagonized their intrinsic propensity to misfold and aggregatewithin the cell.

In the present paper, we report that PG14 and D177NPrPs harbouring the 114–121 deletion (hereafter referred to asPG14/!HC and D177N/!HC) have less tendency to aggregateand accumulate in intracellular compartments, and are deliveredto the cell surface more efficiently than their full-lengthcounterparts. This indicates that the HC region is involvedin misfolding, oligomerization and intracellular retention ofmutant PrP. Comparison of the neurotoxicity of PG14/!HC andD177N/!HC and their full-length counterparts may shed lighton how intracellular accumulation and cell-surface expression ofmutant PrP contribute to the pathogenesis, indicating effectivemodalities for therapeutic intervention.

MATERIAL AND METHODS

Plasmids

cDNAs encoding mouse WT, PG14 and D177N PrPscontaining the 3F4 epitope tag have been described previously[12]. Amino acids 114–121 (WT PrP numbering) weredeleted using the GeneTailorTM Site-Directed MutagenesisSystem (Invitrogen) with partially overlapping primers: 5!-ATGAAGCATATGGCAGGGGGGGGCCTTGGTGGC-3! (for-ward) and 5!-CCCTGCCATATGCTTCATGTTGGTTTTTGG-3!

(reverse). The WT and PG14 PrP constructs containing amonomerized version of EGFP (enhanced green fluorescentprotein) inserted after codon 34 of mouse PrP have been describedpreviously [14]. To generate PrP–EGFP fusion molecules carrying!114–121 alone or with the PG14 mutation, KpnI fragments fromthe 5! end of WT PrP–EGFP and PG14 PrP–EGFP constructswere purified and ligated to the 3! end of PrP !114–121 digestedwith the same enzyme. A cDNA construct encoding 3F4-taggedmouse PrP in which the GPI anchor attachment signal hadbeen replaced with the transmembrane domain of the mannose6-phosphate receptor (PrP-M6P) was generously provided byProfessor David A. Harris (Department of Biochemistry, BostonUniversity School of Medicine, Boston, MA, U.S.A.) and willbe described in a forthcoming publication (details available onrequest from Professor David A. Harris). The identity of allconstructs was confirmed by sequencing the entire coding region.All constructs were cloned into pcDNA3.1(+)/Hygro expressionplasmid (Invitrogen).

Cells

BHK (baby hamster kidney)-21 cells (A.T.C.C.; CCL-10) were grown in MEM (minimal essential medium)-#(Gibco) supplemented with 10% (v/v) FBS (fetal bovineserum), 100 µM non-essential amino acids, 1# minimalessential vitamins, 100 units/ml penicillin and 100 µg/mlstreptomycin. HEK (human embryonic kidney)-293 cells(A.T.C.C.; CRL-1573) were grown in a 1:1 mixture ofMEM-# and DMEM (Dulbecco’s modified Eagle’s medium)(Gibco) containing 10% (v/v) FBS, 2 mM glutamine, non-essential amino acids and penicillin/streptomycin. Cells weretransfected with pcDNA3.1(+)/Hygro expression plasmids usingLipofectamineTM 2000 (Invitrogen). Stable transfected cloneswere selected with 200 µg/ml hygromycin for 10–14 days, thenindividual clones were isolated and maintained with 100 µg/ml

hygromycin. The results of the present study were obtained fromat least two independent cloned lines expressing each construct.

Cerebellar granule neurons were prepared from 6-day-oldC57BL/6J mice, as described in [12]. All procedures involvinganimals and their care were conducted according to EuropeanUnion (EEC Council Directive 86/609, OJ L 358, 1; 12 December1987) and Italian (D.L. n.116, G.U. suppl. 40; 18 February1992) laws and policies, and in accordance with the UnitedStates Department of Agriculture Animal Welfare Act and theNational Institute of Health (Bethesda, MA, U.S.A.) policy onHumane Care and Use of Laboratory Animals, and they wereapproved by the Animal Care Committee of the Department ofNeuroscience at Mario Negri Institute (Milan, Italy). Cerebellawere dissected, sliced into $1 mm pieces and incubated in HBSS(Hanks balanced salt solution) (Gibco) containing 0.3 mg/mltrypsin (Sigma–Aldrich) at 37 !C for 15 min. Trypsin inhibitor(Sigma–Aldrich) was added to a final concentration of 0.5 mg/mland the tissue was mechanically dissociated by passing througha flame-polished Pasteur pipette. Cells were transfected usingthe Nucleofector® device and the Mouse Neuron Nucleofector®

Kit (Lonza) following the manufacturer’s instructions. Aftertransfection, cells were pelleted, resuspended in RPMI 1640(Gibco) containing 10% (v/v) dialysed FBS (Sigma–Aldrich) and2 mM glutamine, and plated at 450000 cells/cm2 on poly-L-lysine(0.1 mg/ml)-coated plates. After 2 h, the medium was replacedwith BME (Basal Medium Eagle) (Gibco) supplemented with10% (v/v) FBS, penicillin/streptomycin (as above) and 25 mMKCl, and cells were maintained at 37 !C in an atmosphere of 5 %CO2/95% air. Cells were analysed after 7–10 days in culture.

Antibodies

Monoclonal antibody 3F4 [24] was used at a 1:2000dilution for Western and slot blotting, and at a 1:500dilution for immunofluorescence staining and cell blotting.Monoclonal antibody 12B2 [25] was used at a 1:5000 dilutionfor Western blotting. Anti-giantin (Covance) and anti-PDI(protein disulfide isomerase) (Sigma–Aldrich) rabbit polyclonalantibodies were used at 1:1000 and 1:500 dilutions respectivelyfor immunofluorescence staining.

Western blotting

Samples were diluted in Laemmli sample buffer (4% SDS,20% glycarol, 10% 2-mercaptoethanol, 0.004 % BromophenolBlue and 0.125 M Tris/HCl), heated at 95 !C for 5 minthen resolved by SDS/PAGE (12% gel). Proteins wereelectrophoretically transferred on to PVDF membranes and themembranes were blocked for 10 min in 5% (w/v) non-fatdried skimmed milk powder in Tris-buffered saline containing0.05% Tween 20. After incubation with appropriate primary andHRP (horseradish peroxidase)-conjugated secondary antibodies,signals were revealed using enhanced chemiluminescence(Amersham Biosciences) and visualized using an XRS imagescanner (Bio-Rad Laboratories). Anti-PrP antibodies 3F4 or12B2 were used to develop Western blots, as indicated in theFigure legends. Quantitative densitometry of protein bands wasperformed using Quantity One software (Bio-Rad Laboratories).A one-tailed unpaired Student’s t test was used for statisticalanalysis.

Detergent insolubility and PK resistance

To assay detergent insolubility, cells were lysed in LB (lysisbuffer: 10 mM Tris/HCl, pH 7.5, 100 mM NaCl, 0.5% sodiumdeoxycholate and 0.5% Nonidet P-40), containing a protease

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inhibitor cocktail (SigmaFASTTM S8820; Sigma–Aldrich). Aftera brief centrifugation to remove debris, lysates corresponding to300 µg of protein were centrifuged at 55 000 rev./min for 45 minin a Beckman Optima Max-E ultracentrifuge using a TLA-55rotor. Proteins in the pellet and supernatant were separated bySDS/PAGE (12% gel) and analysed by Western blotting. To assayPK resistance, cell extracts prepared in LB at a concentrationof 1 mg/ml of total proteins were incubated with PK at 4 !C.Compared with the standard incubation at 37 !C, this procedureallows a better detection of weakly protease-resistant forms ofmutant PrP [9,26].

Immunoprecipitation with 15B3

A 100-µl sample of mouse anti-IgM antibody–Dynabeads®

(Dynal) and 50 µg of monoclonal antibody 15B3 (Prionics) werediluted with 1 ml of PBS containing 0.1 % BSA, incubated for2 h, washed three times with PBS and resuspended in 100 µl ofPBS; 10 µl of 15B3-coated Dynabeads® were then added to thecell lysates in LB containing 500 µg of total protein. Sampleswere incubated on a rotating wheel for 24 h at 4 !C, after whichbeads were washed twice with 1 ml of Wash Buffer (Prionics).Immunoprecipitated PrP was eluted by boiling in Laemmli samplebuffer and analysed by Western blotting.

Sedimentation of PrP in sucrose gradients

Cell lysates were diluted to a final concentration of 0.5 mg/ml(total protein) in LB supplemented with protease inhibitors,incubated for 20 min at 4 !C and centrifuged at 13000 g for 5 min.A 0.5-ml portion of the cleared samples was fractionated on a5-ml step sucrose gradient (10, 30, 40, 50 and 60%) in LBby ultracentrifugation at 45000 rev./min at 4 !C in an MLS-50rotor using an Optima MAX-E ultracentrifuge (Beckman). Aftercentrifugation, 0.5-ml fractions of the gradient were collected, andproteins in each fraction were methanol-precipitated and analysedby Western blotting.

Immunofluorescence staining and imaging

Cells were seeded on to glass coverslips and grown for 24 h to50% confluence. For surface staining of PrP, cells were washedwith ice-cold PBS and incubated for 1 h at 4 !C with antibody3F4 diluted 1:500 in Opti-MEM® (Life Technologies), followedby washing with PBS and fixation in 4% paraformaldehyde.Cells were then washed with PBS, incubated with blockingsolution (0.5% BSA and 50 mM NH4Cl in PBS) for 1 h atroom temperature (25 !C) with Alexa Fluor® 488-conjugatedgoat anti-(mouse IgG) antibody (Molecular Probes) diluted1:500 in blocking solution. For co-localization experiments, cellsgrown on glass coverslips were washed with PBS and fixedfor 30 min at room temperature with 4 % paraformaldehydein PBS. Cells were then washed with PBS, incubated withblocking solution containing 0.1% saponin, and incubated withthe primary and Alexa Fluor® 488- or 594-conjugated secondaryantibodies diluted in the same solution. In some experiments, cellswere stained with 1 µg/ml DAPI (4!,6-diamidino-2-phenylindole)(Sigma–Aldrich) for 5 min. Coverslips were mounted with a gelmount (Biomedia), and analysed with an Olympus FV500 laserconfocal scanning system. For imaging PrP–EGFP, cells weregrown on µ-Dishes (Ibidi) and viewed with a CellR imagingstation (Olympus) coupled to an inverted microscope (IX 81;Olympus). The EGFP fluorescent signal was acquired with a high-resolution camera (ORCA) equipped with a 488 nm excitation

Figure 1 PrP molecules carrying !114–121 are efficiently expressed andglycosylated in HEK-293 cells

(A) The total proteins extracted from primary cerebellar granule neurons (CGN)from C57BL/6 mice (lanes 1–3) and stably transfected HEK-293 cells expressingWT mouse PrP (lanes 4–6) were analysed by Western blotting with the anti-PrPantibody 12B2. H, highly glycosylated; M, monoglycosylated; U, unglycosylated. Notethat PrP is glycosylated differently in CGN and HEK-293 cells. (B) Protein extracts(10 µg) of C57BL/6 CGN and stably transfected HEK-293 cells expressing WTmouse PrP were incubated with N-glycosidase F (PNGase-F) to remove the N-linkedoligosaccharides and were analysed by Western blotting using the anti-PrP antibody 12B2.(C) Lysates of stably transfected HEK-293 cells were left untreated (%) or incubated (+) withPNGase-F to remove the N-linked oligosaccharides and were analysed by Western blottingusing the anti-PrP antibody 3F4. Molecular-mass markers are given in kDa.

filter and an emission filter with a range of 510 +% 40 nm. TheDAPI signal was acquired with a 390 nm excitation filter and anemission filter with a range of 430 +% 20 nm.

Assay of PIPLC (phosphatidylinositol-specific phospholipase C)release

For PIPLC-release experiments, cells grown on coverslips wereincubated with 4 µg of recombinant PIPLC (Prozyme) for 2 h at37 !C, followed by surface staining for PrP. PrP released into themedium was quantified by slot blot analysis. After incubation withor without PIPLC, the medium was collected and the adherentcells washed with PBS and lysed in LB. Samples were thentransferred on to a nitrocellulose membrane using a slot blotapparatus (both from Bio-Rad Laboratories), and probed withanti-PrP antibody 3F4 and HRP-conjugated anti-(mouse IgG)secondary antibody. The signals were revealed by enhancedchemiluminescence, visualized by a Bio-Rad Laboratories XRSimage scanner and quantified using Quantity One software.

Cell blotting

The cell blotting technique we used is a modified version of theprocedure described by Bosque and Prusiner [27]. Cells grownto confluence on glass coverslips were washed twice with PBS.For detection of surface PrP, coverslips were placed on ice andincubated for 20 min in Opti-MEM® (Life Technologies) withantibody 3F4 (this step was omitted for detection of total PrP).Coverslips were subsequently removed and placed on Whatman

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Figure 2 PG14 PrP molecules deleted in the 114–121 region have a reduced tendency to aggregate

(A) Lysates of stably transfected HEK-293 cells were ultracentrifuged at 55 000 rev./min for 40 min, and PrP in the supernatant (S) and pellet (P) was analysed by Western blotting with the antibody 3F4.(B) The percentages of soluble and insoluble PrP were determined by densitometric analysis of the Western blots. (C) Aggregated PrP in detergent lysates of HEK-293 cells was immunoprecipitatedwith the antibody 15B3. Equivalent portions of post-immunoprecipitation supernatants (S) and immunoprecipitates (IP) were analysed by Western blotting with the antibody 3F4. (D) The amount ofPrP in post-immunoprecipitation supernatants and immunoprecipitates was quantified by densitometric analysis of the Western blots. Results in (B) and (D) are means +% S.E.M. for three independentexperiments. **P < 0.01 by Student’s t test.

3MM chromatography paper. A nitrocellulose membrane (Bio-Rad Laboratories) was first immersed in distilled water, thensoaked in blotting buffer (0.5% sodium deoxycholate, 0.5%Nonidet-P40 and 50 mM Tris/HCl, pH 7.4) and placed on topof the coverslips. The sandwich was backed with blotting-buffer-soaked chromatography paper and pressed firmly for 1 h. Thecoverslips were carefully removed from the membrane andthe blot was blocked for 10 min in 5% (w/v) non-fat driedskimmed milk powder in Tris-buffered saline containing Tween20. After incubation with the 3F4 antibody (this step was omittedfor cells previously labelled with 3F4) and HRP-conjugatedsecondary antibody, signals were revealed using enhancedchemiluminescence (Amersham Biosciences) and visualizedwith a Bio-Rad Laboratories XRS image scanner. Quantitativedensitometry was performed using Quantity One software.

RESULTS

PG14 PrP molecules carrying !114–121 have a reducedpropensity to misfold and aggregate

In transfected cells, PG14 PrP misfolds spontaneously andforms detergent-insoluble aggregates that are resistant to mildPK digestion [28–31]. On the basis of observations thatdeletion of amino acids 114–121 stabilizes PrP conformation[21], we tested whether !114–121 counteracted the tendencyfor PG14 PrP to misfold. PrP cDNAs carrying !114–121 alone (!HC) or in combination with the PG14mutation (PG14/!HC) were generated by in vitro mutagenesisand expressed in HEK-293 cells. The PrP level intransfected HEK-293 cells was similar to that of endogenousPrP in cerebellar granule neurons from C57BL/6 mice(Figures 1A and B). The glycosylation patterns of !HC andPG14/!HC PrPs were indistinguishable from those of WT

and PG14 PrPs (Figure 1C). After incubation with N-glycosidaseF to remove the N-linked oligosaccharides, !HC and PG14/!HCPrP had an apparent molecular mass approx. 1 kDa lower thantheir full-length counterparts because of the eight-amino-aciddifference between the polypeptide chains (Figure 1C; comparelanes 5 and 6, and 7 and 8).

To test whether deleting residues 114–121 affected PG14 PrPaggregation, detergent cell extracts were ultracentrifuged, andthe amount of PrP in the supernatant and pellet was determinedby Western blotting. Consistent with previous analyses [12,30],approx. 40% of PG14 PrP was aggregated, partitioning in thepellet fraction (Figures 2A, lanes 5 and 6, and 2B). Deletingamino acids 114–121 dramatically improved the solubility,with only $15% of PG14/!HC PrP in the insoluble fraction(Figures 2A, lanes 7 and 8, and 2B). Results were similar whenthe aggregated PrP was measured by 15B3 immunoprecipitation,which selectively detects a variety of aggregated forms of PrP,including PG14 [31,32] (Figures 2C and 2D). Finally, we testedthe effect of !114–121 on the protease resistance of PG14.Detergent cell extracts were incubated with PK at 4 !C to enhancedetection of weakly PK-resistant forms of PrP [9,26]. PG14/!HCwas PK-sensitive, in contrast with the mild protease resistance ofPG14 PrP (Figure 3). There were no differences in solubility,15B3 reactivity or protease sensitivity between WT and !HCPrPs (Figures 2 and 3).

PG14/!HC forms smaller aggregates than PG14 PrP

The differences in solubility, 15B3 reactivity and PK resistancebetween PG14 and PG14/!HC suggested that the two moleculesmight differ in their states of aggregation (quaternary structure).To compare the size distribution of PG14 and PG14/!HC PrPaggregates, we fractionated lysates of stably transfected HEK-293 cells by velocity sedimentation on 0–60% sucrose gradients

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and analysed PrP in the different fractions by Western blotting.WT and !HC PrP sedimented entirely in fractions 1–4 of thegradient (Figures 4A, 4B, 4E and 4F). In contrast, PG14 PrPwas present in all fractions, with a large amount of moleculesin the lower part of the gradient (fractions 7–10) (Figures 4Cand 4G). Deletion of residues 114–121 dramatically changed thesedimentation properties of the mutant protein. Similarly to WTPrP, PG14/!HC distributed in the top half of the gradient, withthe majority of molecules in fractions 1–4 and a small amountin fraction 5 (Figures 4D and 4H). These results indicated thatPG14 and PG14/!HC differed in their quaternary state, the latterforming much smaller aggregates.

PG14/!HC PrP is efficiently expressed on the cell surface andreleased by phospholipase cleavage of its GPI anchor

PG14 PrP is delayed in its export from the ER and at steadystate is present on the cell surface at lower levels than WTPrP [11,12,33]. To investigate the effect of HC deletion on PrPtrafficking, we analysed the protein distribution in transientlytransfected BHK-21 cells in which the subcellular localization ofWT and PG14 PrP had been characterized [33]. To visualize PrPon the plasma membrane, live cells were incubated at 4 !C withanti-PrP antibody 3F4, fixed and reacted with Alexa Fluor® 488(green)-conjugated secondary antibody without permeabilization.To visualize intracellular PrP, cells were fixed and permeabilizedbefore incubation with 3F4. Permeabilized cells were also reactedwith antibodies against PDI or giantin as markers of the ERand Golgi respectively. WT and !HC PrPs were efficientlyexpressed on the cell surface (Figures 5A and 5B) and partly co-localized with PDI and giantin (Figures 5E, 5F, 5I and 5L, yellowcolour). PG14 PrP was weakly detected on the plasma membrane(Figure 5C) and had a perinuclear intracellular distribution that,consistent with previous results [12,33], localized with ER andGolgi markers (Figures 5G and 5M). PG14/!HC PrP, in contrast,showed cell-surface localization similar to WT and !HC PrPs(Figure 5D). However, compared with WT and !HC, a higherfraction of PG14/!HC was found in the ER and Golgi (Figures 5Hand 5N, yellow colour).

Results were similar when PrP distribution was investigated intransfected HEK-293 cells, where we quantified the amount ofsurface PrP using a cell blotting technique. This confirmed thatPG14/!HC PrP was expressed on the cell surface at significantlyhigher levels than PG14, and that there was no difference insurface expression between WT and !HC PrPs (Figure 6).

Next, we analysed PrP distribution in neurons using PrP– EGFPfusion molecules. Primary cultures of cerebellar granule neuronsfrom C57BL/6J mice were transfected with plasmids encodingWT PrP–EGFP, PG14 PrP–EGFP or PG14/!HC PrP–EGFP. Weimaged PrP–EGFP proteins in fixed DAPI-stained cells to analysetheir localization with respect to the nucleus. Consistent withprevious immunolocalization of non-fluorescent versions of WTand PG14 PrP in neurons [12], WT PrP–EGFP distributed on thecell soma and along the neurites (Figure 7A), with PG14 PrP–EGFP mainly in intense perinuclear patches (Figure 7B). Thecellular distribution of PG14/!HC PrP–EGFP was similar to WTPrP–EGFP (Figure 7C), indicating that disrupting the HC domainprevented intracellular aggregation of PG14 PrP, improving itstrafficking to the neuronal surface.

WT PrP can be efficiently released from cell membranes bytreatment with the bacterial enzyme PIPLC, which cleaves theglycerolipid portion of the GPI anchor. In contrast, PG14 PrPis PIPLC-resistant [8,30,34–36]. To test whether the GPI anchorof PG14/!HC PrP was susceptible to cleavage, live HEK-293cells were incubated with or without PIPLC and the surface

Figure 3 PG14/!HC PrP is sensitive to protease digestion

Lysates of stably transfected HEK-293 cells expressing WT, !HC, PG14 or PG14/!HC PrPwere incubated with the amounts indicated of PK for 30 min at 4!C. Proteins were separatedby SDS/PAGE (12% gel) and immunoblotted with the antibody 3F4. A 250-µg portion of initialprotein was digested. The lanes containing undigested samples represent 60 µg of protein.

was immunostained with anti-PrP antibody. As with WT and!HC PrPs, there was a clear fall in surface PG14/!HC PrPimmunofluorescence after PIPLC treatment, indicating that theGPI anchor was efficiently cleaved and the protein released fromthe plasma membrane (Figures 8A and 8F, 8B and 8G, and 8D and8I). Consistent with previous results [34,35], the small amountof PG14 PrP on the cell surface was not released by PIPLC(Figures 8C and 8H). As expected, PrP-M6P, an engineeredform of PrP in which the GPI anchor attachment signal hadbeen replaced with the transmembrane domain of the mannose-6-phosphate receptor protein, was PIPLC-resistant (Figures 8Eand 8L). Quantification of PrP in the medium confirmed thatPIPLC treatment did not release PG14 and PrP-M6P from the cellsurface, whereas it efficiently released WT, !HC and PG14/!HCPrPs (Figure 8M).

Deletion of the HC domain increases solubility and cell-surfaceexpression of D177N PrP

Next, we tested whether deleting the HC region affectedthe biochemical properties and cellular distribution of anotherpathogenic PrP mutant. Like PG14 PrP, mouse PrP carrying theD177N substitution has abnormal biochemical properties, alteredcellular localization [11,12,14,28,33,36] and is pathogenic intransgenic mice [9,10]. When transiently expressed in HEK-293

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Figure 4 PG14 and PG14/!HC PrP aggregates differ in molecular size

(A–D) Lysates of stable transfected HEK-293 cells expressing the PrP molecules were fractionated by centrifugation on a 0–60% sucrose gradient for 1 h at 45 000 rev./min. Fractions of the gradients(lanes 1–10) were collected and analysed by Western blotting with the anti-PrP antibody 3F4. (E–H) The amount of PrP in each fraction was quantified by densitometric analysis of the Western blotsand plotted as percentages of total PrP. Results are means +% S.E.M. for three to four independent experiments.

Figure 5 Cellular localization of PG14 molecules carrying !114–121 is similar to that of WT PrP

To visualize PrP on the cell surface (A–D), transiently transfected BHK-21 cells expressing PrP molecules were stained with the anti-PrP monoclonal antibody 3F4 before fixation and application ofAlexa Fluor® 488-conjugated secondary antibody and staining with DAPI. Cells were viewed with green excitation/emission settings to detect PrP and UV excitation/emission settings to detect thenuclei. Merged images are shown. To investigate the intracellular distribution of PrP, cells were fixed, permeabilized and stained with the monoclonal antibody 3F4, followed by staining with anti-PDI(E–H) or anti-giantin (I–N) rabbit polyclonal antibodies to stain the ER and Golgi respectively. Cells were viewed with green excitation/emission settings to detect PrP, red excitation/emission settingsto detect PDI or giantin and UV excitation/emission settings to detect the nuclei. Merged images are shown in (E–N). Scale bar in (N) (applicable to all panels), 20 µm.

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Figure 6 PG14/!HC molecules are efficiently delivered to the cell surfaceof HEK-293 cells

(A) HEK-293 cells were grown to confluence on glass coverslips. For detection of surface PrP,coverslips were incubated at 0!C with the antibody 3F4 (this step was omitted for detection oftotal PrP). Coverslips were blotted on to a nitrocellulose membrane soaked in lysis buffer andincubated with HRP-conjugated secondary antibody. For detection of total PrP, cell blots wereincubated with the primary and secondary antibodies. The PrP signal was revealed by enhancedchemiluminescence. (B) The PrP signal was quantified by densitometry. The amount of PrP onthe surface was calculated as the ratio of surface to total PrP, and expressed as a percentageof WT. Results are means +% S.E.M. for four to five different samples from three independentexperiments. **P < 0.01 by Student’s t test.

cells, $30% of D177N PrP was detergent-insoluble (Figures 9A,lanes 1–2, and 9B), and selectively immunoprecipitated by the15B3 antibody (Figures 9C, lanes 1–2, and 9D). In contrast,only a small amount of D177N/!HC PrP ($10%) was insoluble(Figures 9A, lanes 3–4, and 9B) and 15B3-reactive (Figures 9C,lanes 3–4, and 9D). Surface immunofluorescence staining oflive (unpermeabilized) cells indicated that D177N/!HC PrP wasexpressed on the plasma membrane at a higher level than D177NPrP (Figures 10A and 10B). Immunofluorescence analysis afterpermeabilization to detect also intracellular PrP showed that,consistent with previous results [12,14], D177N PrP was mostlyconcentrated in perinuclear patches co-localizing mainly withthe Golgi marker giantin (Figures 10C and 10E). In contrast,D177N/!HC was present more on the cell surface and showeda more diffuse intracellular distribution, co-localizing with both

ER and Golgi markers (Figures 10D and 10F). Thus !114–121counteracted aggregation and improved intracellular traffickingof D177N PrP, as with the PG14 mutation.

DISCUSSION

NMR analysis of recombinant and brain-derived PrP showed thatresidues 23–125 form a flexibly disordered tail, and residues 128–230 a globular domain comprising three #-helices and two short"-strands flanking helix 1 [37,38]. The unstructured portion of PrPbetween residues 90 and 120 undergoes profound rearrangementsin PrPSc [17] and spontaneously forms amyloid when synthesizedas a peptide [18–20], indicating that this region is essential toPrP refolding and aggregation. Consistent with this view, PrPmolecules deleted in the HC region between residues 109 and112 or 114 and 121 are refractory to conformational conversioninduced by PrPSc and have a trans-dominant inhibitory effect onconversion of WT PrP [22,23].

In the present study, we found that the biochemical propertiesand cellular trafficking of WT and !114–121 PrPs are similar.This, and the evidence that PrP !114–121 is not toxic in miceand retains the neuroprotective activity of WT PrP [39], supportsthe use of this molecule in gene therapy of prion infections [40].

We also found that !114–121 antagonized the spontaneousconversion of two mutant PrPs carrying either 72 extra aminoacids in the N-terminus or a single residue substitution inthe C-terminal domain. Thus the HC of PrP is vital forprotein misfolding, independently of whether this is triggeredby interaction with PrPSc or by structural information intrinsic tomutant PrP. The fact that region 114–121 governs both mutation-and PrPSc-induced PrP misfolding suggests that similar structuralalterations underlie genetic and infectious prion diseases, aconclusion also emerging from other studies [41]. Since PrPSc-induced conversion is likely to occur in the endosomal recyclingcompartment [42], whereas mutant PrP first adopts an abnormalconformation in the ER [29], our data also suggest that region 114–121 influences PrP misfolding independently of the intracellularmilieu.

In transfected cells, PG14 PrP conversion into the PrPSc-like state proceeds stepwise through a series of identifiablebiochemical intermediates [29]. The initial alteration in themutant molecule occurs in the ER, during or immediatelyafter translation of the polypeptide chain, and is manifested byacquisition of PIPLC resistance. This is likely to reflect PG14 PrPmisfolding and oligomerization, since the GPI anchor becomessensitive to cleavage after protein denaturation [36]. PG14 PrPaggregates further in post-Golgi locations where it first becomes

Figure 7 PG14/!HC PrP–EGFP is delivered to the surface of primary cerebellar neurons

Cerebellar granule neurons from 6-day-old C57BL6/J mice were transiently transfected with the WT (A), PG14 (B) or PG14/!HC (C) PrP–EGFP constructs, fixed, reacted with DAPI to stain thenuclei, and viewed with green excitation/emission settings to detect PrP–EGFP and with UV excitation/emission settings to detect DAPI. Scale bar, 20 µm.

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484 E. Biasini and others

Figure 8 PG14/!HC is released from the plasma membrane by PIPLC cleavage of its GPI anchor

Live HEK-293 cells expressing WT, !HC, PG14, PG14/!HC or PrP-M6P were incubated without (A–E) or with (F–L) PIPLC enzyme, followed by surface staining with antibody 3F4. Scale bar in(F) (applicable to all panels), 25 µm. (M) PrP released into the medium after incubation with (+) or without (%) PIPLC was quantified by slot blot analysis using the antibody 3F4 and is expressedas a percentage of PrP in the corresponding cell lysates (lysate results not shown).

Figure 9 HC deletion interferes with the aggregation of D177N PrP

(A) Lysates of transiently transfected HEK-293 cells expressing D177N or D177N/!HC PrPwere ultracentrifuged at 55 000 rev./min for 40 min, and PrP in the supernatant (S) and pellet(P) was analysed by Western blotting with the antibody 3F4. (B) The percentage of soluble andinsoluble PrP was quantified by densitometric analysis of the Western blots. (C) AggregatedPrP in detergent lysates of HEK-293 cells was immunoprecipitated with the antibody 15B3.Equivalent portions of post-immunoprecipitation supernatants (S) and immunoprecipitates (IP)were analysed by Western blotting with antibody 3F4. (D) The amount of PrP in each lanewas quantified by densitometric analysis of the Western blots. Results in (B) and (D) aremeans +% S.E.M. for three independent experiments. **P < 0.01 by Student’s t test.

detergent-insoluble, then PK-resistant, very probably because ofan incremental change in aggregation [29,43]. We found thatmost PG14/!HC PrP molecules expressed in HEK-293 cellswere susceptible to PIPLC cleavage, suggesting that !114–121inhibited the initial step of conformational conversion in theER, probably by introducing an energy barrier to misfolding[21]. !114–121 also had a dramatic effect on aggregation.Cells expressing PG14/!HC and D177N/!HC PrPs accumulatedonly small amounts of detergent-insoluble and 15B3-reactivemolecules, which sedimented in the lighter fractions of sucrosegradients and were PK-sensitive. These results indicate that theHC deletion changed the aggregation properties of mutant PrP,favouring the formation of small, weakly associated, oligomers.There is evidence that intermolecular PrP interactions involved informing "-pleated sheet are dependent on specific residues in thecentral hydrophobic region [44,45]. These residues are lackingin HC-deleted molecules, suggesting that the intermolecularinteractions necessary for formation and/or stabilization of theaggregates may be disrupted.

Several different pathogenic mutations share a common effecton PrP trafficking, impairing delivery to the cell surface andcausing a portion to accumulate in intracellular compartments[9,11,12,14,33,46–53]. !114–121 greatly improved the secretorytrafficking of PG14 and D177N PrPs. Immunofluorescencemicroscopy in BHK-21 and HEK-293 cells and imaging of PrP–EGFP fusion proteins in live neurons showed that PG14/!HC andD177N/!HC PrPs were efficiently expressed on the cell surface.This observation, in conjunction with the biochemical results,indicates that the effect of the HC deletion on PrP structureand cellular trafficking are closely related, and that impairedtrafficking may be a direct consequence of PrP misfolding and

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The hydrophobic core governs mutant PrP aggregation 485

Figure 10 HC deletion increases the delivery of D177N PrP to the plasmamembrane

To visualize cell-surface PrP (A and B), transiently transfected BHK-21 cells were stained withthe antibody 3F4 before fixation and application of Alexa Fluor® 488-conjugated secondaryantibody and DAPI staining. Cells were viewed with green excitation/emission settings todetect PrP and UV excitation/emission settings to detect the nuclei. Merged images are shown.Intracellular distribution of PrP was studied by immunostaining with anti-PrP and anti-PDI (Cand D) or anti-giantin (E and F) antibodies. Cells were reacted with DAPI to stain the nuclei.Cells were viewed with green excitation/emission settings to detect PrP, red excitation/emissionsettings to detect PDI or giantin and UV excitation/emission settings to detect the nuclei. Mergedimages are shown. Scale bar in (F) (applicable to all panels), 20 µm.

aggregation. However, it is possible that !114–121 preventsintracellular accumulation through a different mechanism, forexample by disrupting a structural motif of PrP essential forrecognition and retention of misfolded molecules in the secretorypathway. In this regard, it could be interesting to study the effectof !114–121 on the trafficking of PrP mutants that accumulate inthe secretory pathway, but are less prone to aggregation and lessPK-resistant, such as E200K and P102L [28,33,49].

The cellular pathways activated by mutant PrP ultimatelyleading to neuronal dysfunction and degeneration in geneticprion diseases are poorly understood. In different non-neuronaland neuronal cells, several mutant PrPs misfold in the earlysteps of the secretory pathway, reside longer in the ER andGolgi apparatus, and are delivered less efficiently to the cellsurface, suggesting that intracellular PrP accumulation may bepathogenic. The discovery that !114–121 counteracts misfoldingand improves the cellular trafficking of mutant PrP providesan unprecedented model for assessing the role of intracellularretention in neurotoxicity. By comparing the effects of HC-deletedand full-length PG14 and D177N molecules on neuronal viability,it should be possible to assess the contributions of intracellularaccumulation and cell-surface localization. Expression of PG14or D177N PrP in cultured cells does not significantly alter cellviability, growth rate or susceptibility to toxic drugs ([54], andT. Massignan, E. Restelli, E. Biasini and R. Chiesa, unpublished

work); thus the only way to test the effect of !114–121 on toxicityis to study the consequences of PG14/!HC or D177N/!HCexpression in transgenic mice. Since !114–121 alone does notinduce neurodegenerative illness in mice [39], any deviation fromthe phenotype of mice expressing the full-length mutants mightbe ascribable to changes in PrP aggregation and/or trafficking.

It is interesting to speculate on the possible outcome of thisexperiment. Should PG14/!HC and D177N/!HC turn out notto be toxic, this would indicate that intracellular aggregationis vital in the disease process, and that therapeutic strategiesaimed at favouring mutant PrP folding and trafficking could bebeneficial [55–57]. Should the deleted molecules be more toxicthan their full-length counterparts, this would imply that mutantPrP expression on the plasma membrane and/or formation of smalloligomers are primarily responsible for toxicity.

In conclusion, the present study indicates that sequence114–121 within the unstructured region of PrP is vital forprotein misfolding and aggregation, and sets the basis for futureinvestigations to ascertain the role of intracellular accumulationof misfolded PrP in the pathogenesis of inherited prion diseases.

AUTHOR CONTRIBUTION

Emiliano Biasini, Laura Tapella, Elena Restelli, Manuela Pozzoli and Tania Massignanperformed the experiments. Emiliano Biasini and Roberto Chiesa conceived and designedthe experiments, analysed the data and wrote the paper.

ACKNOWLEDGEMENTS

We thank Richard Kascsak (New York State Institute for Basic Research, Staten Island,NY, U.S.A.) for the 3F4 antibody, Jan P. Langeveld [Central Veterinary Institute (CVI),Wageningen University and Research Centre, 8200 AB Lelystad, The Netherlands] for the12B2 antibody, Alex Raeber and Bruno Oesch (Prionics, Zurich, Switzerland) for the 15B3antibody, and David A. Harris for the PrP-M6P cDNA construct. We are grateful to PietroVeglianese for advice on live cell imaging, to Gianlugi Forloni for discussion and to JudithBaggott for help in preparing the manuscript.

FUNDING

This work was supported by the Fondazione Telethon [grant number TCR08005 (to R.C.)];and by a fellowship from the Fondazione Monzino to E.B. R.C. is an Associate TelethonScientist (Dulbecco Telethon Institute, Fondazione Telethon).

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Received 20 April 2010/24 June 2010; accepted 14 July 2010Published as BJ Immediate Publication 14 July 2010, doi:10.1042/BJ20100615

c" The Authors Journal compilation c" 2010 Biochemical Society