Research

Mutant Prion Protein Expression Is Associated with an Alteration of the Rab GDP Dissociation Inhibitor ␣ (GDI)/Rab11 Pathway*□ S

Tania Massignan‡§, Emiliano Biasini‡¶, Eliana Lauranzano‡§, Pietro Veglianese¶, Mauro Pignataro‡§, Luana Fioriti‡¶, David A. Harris储**, Mario Salmona§, Roberto Chiesa‡¶‡‡, and Valentina Bonetto‡§‡‡§§ The prion protein (PrP) is a glycosylphosphatidylinositolanchored membrane glycoprotein that plays a vital role in prion diseases, a class of fatal neurodegenerative disorders of humans and animals. Approximately 20% of human prion diseases display autosomal dominant inheritance and are linked to mutations in the PrP gene on chromosome 20. PrP mutations are thought to favor the conformational conversion of PrP into a misfolded isoform that causes disease by an unknown mechanism. The PrP mutation D178N/Met-129 is linked to fatal familial insomnia, which causes severe sleep abnormalities and autonomic dysfunction. We showed by immunoelectron microscopy that this mutant PrP accumulates abnormally in the endoplasmic reticulum and Golgi of transfected neuroblastoma N2a cells. To investigate the impact of intracellular PrP accumulation on cellular homeostasis, we did a two-dimensional gel-based differential proteomics analysis. We used wide range immobilized pH gradient strips, pH 4 –7 and 6 –11, to analyze a large number of proteins. We found changes in proteins involved in energy metabolism, redox regulation, and vesicular transport. Rab GDP dissociation inhibitor ␣ (GDI) was one of the proteins that changed most. GDI regulates vesicular protein trafficking by acting on the activity of several Rab proteins. We found a specific reduction in the level of functional Rab11 in mutant PrP-expressing cells associated with impaired post-Golgi trafficking. Our data are consistent with a model by which mutant PrP induces overexpression of GDI, activating a cytotoxic feedback loop that leads to protein accumulation in the secretory pathway. Molecular & Cellular Proteomics 9:611– 622, 2010.

Familial Creutzfeldt-Jakob disease (fCJD),1 GerstmannStra¨ussler-Scheinker syndrome, and fatal familial insomnia (FFI) are dominantly inherited degenerative disorders of the central nervous system (CNS) linked to mutations in the prion protein (PrP) gene on chromosome 20 (1). The pathogenic mutations favor conversion of PrP into a misfolded pathogenic isoform that accumulates in the CNS, ultimately leading to neuronal dysfunction and degeneration by a mechanism still unknown (2). A mutation at PrP codon 178, resulting in the substitution of aspartic acid for asparagine is linked to two different inherited prion diseases, depending on the amino acid specified at the polymorphic site 129 of the mutant allele where either methionine or valine can be present. The D178N/ Val-129 haplotype is linked to fCJD, whereas D178N/Met-129 is associated with FFI (3). PrP is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein of uncertain function (4). Like other membrane proteins, PrP is synthesized in the rough endoplasmic reticulum (ER), transits the Golgi, and is delivered to the cell surface where it resides in lipid rafts (5, 6). Several mutant PrP molecules, in contrast, misfold soon after synthesis in the ER (7), accumulate in the secretory pathway, and are less efficiently delivered to the cell surface (8 –15). Mutant PrPs expressed in transfected cells and primary neurons from transgenic mice acquire biochemical properties of pathogenic PrP, including insolubility in non-denaturing detergents and protease resistance (14, 16 –19). These observations suggest that mutant PrP misfolding and abnormal intracellular localization may trigger pathogenic processes (2, 20). In line with this view, ER accumulation of mutant PrP and alteration of ER morphology have been found

From the ‡Dulbecco Telethon Institute (DTI) c/o Istituto di Ricerche Farmacologiche “Mario Negri,” Via G. La Masa 19, 20156 Milan, Italy, §Department of Biochemistry and Molecular Pharmacology, Istituto di Ricerche Farmacologiche “Mario Negri,” Via G. La Masa 19, 20156 Milan, Italy, ¶Department of Neuroscience, Istituto di Ricerche Farmacologiche “Mario Negri,” Via La Masa 19, 20156 Milan, Italy, and 储Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110 Received, June 15, 2009, and in revised form, November 20, 2009 Published, MCP Papers in Press, December 7, 2009, DOI 10.1074/ mcp.M900271-MCP200

1 The abbreviations used are: fCJD, familial Creutzfeldt-Jakob disease; BCA, bicinchoninic acid; CNS, central nervous system; 2D, two-dimensional; 2DE, two-dimensional gel electrophoresis; EGFP, enhanced green fluorescent protein; ER, endoplasmic reticulum; FFI, fatal familial insomnia; GDI, Rab GDP dissociation inhibitor ␣; GFP, green fluorescent protein; GPI, glycosylphosphatidylinositol; N2a, Neuro-2a; PrP, prion protein; WB, Western blot; WT, wild-type; Z, benzyloxycarbonyl; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; EM, electron microscopy; TIRF, total internal reflection fluorescence; siRNA, small interfering RNA; RNAi, RNA interference; TRAP, transposon-associated protein.

© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at http://www.mcponline.org

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in the CNS of a transgenic mouse model of fCJD (15). However, further studies are needed to decipher the cellular and molecular pathways activated by mutant PrPs that ultimately result in neuronal dysfunction and degeneration. To investigate the impact of mutant PrP on neuronal homeostasis, we carried out a proteomics analysis of mouse neuroblastoma N2a cells expressing either wild-type (WT) PrP or the mouse homologue of the human D178N/Met-129 mutation linked to FFI (referred to as D177N/Met-128 PrP). N2a cells have been extensively used as a model system to study the cellular biology of prion disease (21, 22). Proteomics data indicated changes in proteins involved in energy metabolism, redox regulation, and vesicular transport, including significant up-regulation of the Rab GDP dissociation inhibitor ␣ (GDI). GDI regulates the function of several Rab proteins, which are key regulators of intracellular vesicular trafficking (23, 24). Rabs are small GTPases found solely in specific membrane compartments that function as molecular switches, cycling from an inactive, cytosolic GDP-bound state to an active membrane-associated, GTP-bound state. Excess GDI induces dissociation of GDP-bound Rabs from membranes, inhibiting vesicular transport and recycling (25–27). We therefore investigated how GDI overexpression induced by mutant PrP affected intracellular trafficking. EXPERIMENTAL PROCEDURES

Cells—Generation of N2a cells expressing WT or D177N/Met-128 mouse PrP carrying the epitope tag for the monoclonal antibody 3F4 has been described (14). N2a cells were grown in Dulbecco’s modified Eagle’s medium and modified Eagle’s medium ␣ 1:1 supplemented with 10% fetal bovine serum (Invitrogen), non-essential amino acids, and penicillin/streptomycin (Sigma) and maintained in an atmosphere of 5% CO2, 95% air. Plasmids—pcDNA 3.1 plasmids encoding WT and D177N/Met-128 PrPs containing the 3F4 epitope tag have been previously described (14). The WT and D177N PrP-EGFP constructs were generated by inserting a monomerized version of enhanced green fluorescent protein (EGFP), containing a GS linker (GGGGS, repeated four times) at its 3⬘-end, after codon 34 of mouse PrP. The plasmid carrying the GPI-anchored green fluorescent protein (GFP-GPI) cDNA (28) was kindly provided by Chiara Zurzolo (Pasteur Institute, Paris, France). Protein Extraction—Confluent cells were scraped into ice-cold buffer containing 250 mM sucrose and 10 mM Tris-Cl, pH 7.4 and lysed in ice-cold lysis buffer containing 0.5% sodium deoxycholate, 0.5% Zwittergent, 10 mM Tris, pH 7.5, and protease inhibitors (1 ␮g/ml pepstatin and leupeptin, 0.5 mM PMSF, 2 mM EDTA, and 80 ␮M Z-Leu-Leu-Leu-al also called MG132 (Sigma-Aldrich)). Lysates were further disrupted by 10 passages through a 26-gauge needle, and protein concentration was determined by bicinchoninic acid (BCA) assay (Pierce). Two-dimensional Gel Electrophoresis (2DE)—For analysis of acidic proteins, protein extracts corresponding to 0.8 mg were precipitated with a clean-up kit (GE Healthcare) and then dissolved in DeStreakTM rehydration solution (GE Healthcare) with 2% DTT and 2% carrier ampholytes, pH 4 –7. For analysis of basic proteins, 80 ␮g of protein extracts were precipitated by adding chloroform/methanol/water (1: 4:3). The protein fraction was redissolved in 8 M urea, 2 M thiourea, and 2% CHAPS; dialyzed against a solution containing 8 M urea using a minidialysis kit (GE Healthcare); and then diluted in DeStreak rehy-

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dration solution containing 4% DTT and 4% carrier ampholytes, pH 6 –11. IEF was done using linear IPG strips (GE Healthcare) at different pH ranges: pH 4 –7, 13 cm long for separation of acidic proteins, and pH 6 –11, 7 cm long for basic proteins. IPG strips were run in an IPGphor system (GE Healthcare). Two different IEF protocols were used for acidic and basic proteins: a total of 65 kV-h (1 h at 500 V, 1 h at 1500 V, and 3 h of ramping scale to 8000 V followed by 8000 V for 7 h) for the separation of acidic proteins and a total of 47 kV-h (0.5 h at 200 V, 0.5 h at 500 V, 0.5 h at 1000 V, 1.5 h of a linear gradient to 3500 V, 2 h at 3500 V, and 2.5 h of a linear gradient to 8000 V followed by 8000 V for 4 h) for the separation of basic proteins. For the second dimension, pH 4 –7 IPG strips were run on 7.5–17.5% polyacrylamide-SDS gels. Proteins were stained using Gel-Code Blue Coomassie stain (Pierce). The pH 6 –11 IPG strips were run on a precast 4 –12% Bis-Tris-acrylamide gradient gel (Invitrogen). Proteins were visualized by SYPRO Ruby staining (Invitrogen). Image Analysis and Statistics—Triplicates of 2D maps were obtained for the acidic and the basic proteins. Gel images were digitized with a 1680 Pro scanner (Epson) at 16-bit and 300-dpi resolution. Quantitative densitometry was carried out using Progenesis PG240 v2006 software (Nonlinear Dynamics). Detection, warping, and matching of the protein spots were done using the Combined warp and match algorithm, which uses a non-parametric pattern recognition clustering technique to align different gel images. The Total spot volumes normalization algorithm was used to calculate each protein spot volume as the sum of the intensities of the pixels within the spot’s boundary minus the background level within that same boundary normalized to the total spot volumes in the gel (29). Observed pI and Mr were calculated by the software on the basis of protein spots of known characteristics. To detect differentially expressed proteins, two proteomics analyses were done, each based on a different cell clone. Only protein spots with a statistically significant change (p ⬍ 0.05 by Student’s t test) of at least 1.4-fold in both analyses were further identified by mass spectrometry. In-gel Digestion of Protein Spots—Protein spots were excised using an EX-Quest spot cutter (Bio-Rad); destained for 2 h in a solution containing 25 mM ammonium bicarbonate, 40% ethanol; and then washed with sequentially increasing percentages of acetonitrile. Proteins were in-gel digested overnight at 37 °C with trypsin (Promega) at a concentration of 10 ng/␮l in a solution of 25 mM ammonium bicarbonate and 10% acetonitrile. MALDI-TOF MS—Peptide mass fingerprinting was done on a Bruker Reflex III MALDI-TOF mass spectrometer equipped with a SCOUT 384 multiprobe inlet and a 337 nm nitrogen laser using ␣-cyano-4-hydroxycinnamic acid (Bruker Daltonics) as the matrix, prepared as described (30). Tryptic digests were concentrated and desalted using ZipTip pipette tips with C18 resin and a 0.2-␮l bed volume (Millipore). Aliquots of tryptic digests were mixed with 2:1 matrix solution and directly deposited on the target. All mass spectra were obtained in positive reflector mode with a delayed extraction of 200 ns. The reflector voltage was set to 23 kV, and the detector voltage was set to 1.7 kV. All the other parameters were set for optimized mass resolution. To avoid detector saturation, low mass material (500 Da) was deflected. The mass spectra were internally calibrated with trypsin autolysis fragments (842.51, 1045.56, 2211.10, and 2283.18 m/z). The mass spectra were obtained by averaging 150 –350 individual laser shots and then automatically processed by the FlexAnalysis software, version 2.0, using the following parameters: the Savitzky-Golay smoothing algorithm and the SNAP peak detection algorithm. Spectra originating from parallel protein digestions were compared pairwise to discard common peaks derived from trypsin autodigestion or from contamination with keratins. Data searches (Swiss-Prot) were done using the MASCOT software package available on the internet (Matrix Science), allowing up to one

Proteomics of Mutant PrP N2a Cells

FIG. 1. Ultrastructural localization of PrP and quantification of organelle volumes. Electron microscopic images of N2a cells expressing WT (A) or D177N/Met-128 PrP (B–D) are shown. White arrowheads point to the plasma membrane (PM), black filled arrowheads point to cisternae of the ER, and white arrows point to the Golgi complex. In WT cells, gold particles are mainly localized on plasma membrane (A), whereas in D177N/Met-128 cells, they are preferentially associated with the ER and Golgi (B–D). Scale bar, 250 nm. E, quantification of gold particles in different cellular compartments. Data are the mean of at least 10 cells per specimen. Error bars represent S.D. F, quantification of ER and Golgi volumes showed a selective enlargement of the Golgi compartment in cells expressing D177N/Met-128 PrP. Data are the mean of at least 10 cells per specimen. Error bars represent S.D. *, p ⬍ 0.05 by Student’s t test.

missed trypsin cleavage, carbamidomethylation of Cys and oxidation of Met as variable modifications, and a mass tolerance of ⫾0.1 Da. In the MASCOT program, probability-based molecular weight search (MOWSE) scores (31) greater than 55 were considered significant

(p ⬍ 0.05) searching Mus musculus sequences deposited in the Swiss-Prot database. Western Blotting (WB)—Proteins were transferred onto PVDF membranes (Millipore). For the reaction with the primary antibodies,

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FIG. 2. Experimental design of proteomics analysis. Two separate experiments (1 and 2) were done using two pairs of N2a clones (WT1, WT2, D177N1, and D177N2). Image analysis of 2D map triplicates was done separately for each experiment. Acidic (pI 4 –7) and basic (pI 6 –11) proteins from WT cells and D177N cells were compared. 117 and 202 spots, respectively, were significantly different in experiments 1 and 2. 42 spots were different in both experiments and were identified (see Table I).

FIG. 3. 2D maps showing spots with significantly different volumes. Representative 2D gels of acidic (pI 4 –7) and basic (pI 6 –11) proteins from N2a cells expressing WT and D177N PrP are shown. Proteins in the gels were stained with Coomassie Blue (A) or SYPRO Ruby (B), digitized, and analyzed by quantitative computer-assisted densitometry. Proteins whose levels were significantly different are numbered from 1 to 20 (acidic proteins) (A) and from 21 to 42 (basic proteins) (B) (Table I).

membranes were incubated for 1 h at room temperature with a blocking buffer (5% milk in Tris-buffered saline containing 0.1% Tween 20) and probed with the primary antibody diluted in the same solution overnight at 4 °C. Primary antibodies were as follows: monoclonal anti-PrP 3F4 (1:5000) (32), polyclonal anti-transposon-associated protein (TRAP) (1:5000) from Upstate, polyclonal anti-GDI (1: 2500) from Zymed Laboratories Inc., monoclonal anti-Rab11 (1:2000) from Upstate, anti-monoclonal ATPase ␣ (1:1000), polyclonal antiAHSA1 (1:1000), monoclonal anti-annexin A2 (1:1000) from Santa Cruz Biotechnology, polyclonal anti-Rab5 (1:2000) kindly provided by Marino Zerial (Max Planck Institute, Dresden, Germany), and poly-

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clonal anti-ERp19 (1:1000) (33) kindly provided by Marek Michalak (University of Alberta, Edmonton, Alberta, Canada). The blots were probed with goat anti-rabbit or anti-mouse peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) and developed by the ECL protein detection system (GE Healthcare). Densitometry was done with Progenesis PG240 software. Immunoreactivity was normalized to the actual amount of proteins loaded onto the membrane as detected after Coomassie Blue or red Ponceau staining (Fluka). Subcellular Fractionation—Cell pellets were resuspended in homogenization buffer (10 mM Tris, pH 7.4, 0.1 M NaCl, and 0.01 M EDTA) and homogenized using a Teflon/glass apparatus. Lysates

Proteomics of Mutant PrP N2a Cells

TABLE I Differential protein spots in common to the two proteomics experiments Spot no. 1 2 3 4 5 6 7 8a 8b 8c 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Gene name

Protein name

Hspa4 Vcp

Heat shock 70-kDa protein 4 Transitional endoplasmic reticulum ATPase P4ha1 Prolyl 4-hydroxylase ␣-1 subunit P4ha2 Prolyl 4-hydroxylase ␣-2 subunit Pdia3 Protein-disulfide isomerase A3 (ERp57) Gdi1 GDI Prph Peripherin Nudcd3 NudC domain-containing protein 3 Dctn2 Dynactin subunit 2 Atp5b ATP synthase subunit ␤ Ruvbl2 RuvB-like 2 (p47 protein) Ahsa1 Activator of 90-kDa heat shock protein ATPase (AHSA1) Ddah1 Dimethylarginine dimethylaminohydrolase 1 Rplp0 60 S acidic ribosomal P0 Ppa1 Pyrophosphatase Tpm4 Tropomyosin ␣-4 chain Prdx4 Peroxiredoxin 4 Prdx4 Peroxiredoxin 4 Ndufv2 NADH-ubiquinone oxidoreductase, 24 kDa Psmb6 Proteasome subunit ␤ type 6 Atp5h ATP synthase subunit d Txndc12 Thioredoxin domain-containing protein 12 (ERp19) Acly ATP-citrate synthase Aco2 Aconitate hydratase Tkt Transketolase Hnrnpl Heterogeneous nuclear ribonucleoprotein L Pkm2 Pyruvate kinase isozyme M2 Dld Dihydrolipoyl dehydrogenase Atp5a1 ATP synthase ␣ chain (ATPase ␣) Atp5a1 ATPase ␣ Atp5a1 ATPase ␣ Eno1 ␣-Enolase Acot9 Acyl-coenzyme A thioesterase 9 Fh Fumarate hydratase Metap1 Methionine aminopeptidase 1 Ppid 40-kDa peptidyl-prolyl cis-trans isomerase Adh5 Alcohol dehydrogenase class 3 Aldoa Fructose-bisphosphate aldolase A Anxa2 Annexin A2 Mthfd2 Bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase Ldha L-Lactate dehydrogenase A chain Memo1 Protein MEMO1 Vdac1 Voltage-dependent anion-selective channel protein 1 Ran GTP-binding nuclear protein Ran

Acc.a

Molecular Molecular pI weight calc. weight c calc.b obs.d

pI obs.e

Cov.f

Matc./ unMatc.g

Scoreh (Exp. 1)

-Foldi (Exp. 2)

-Foldi

Q61316 Q01853

94 89.1

5.1 5.1

95 90

5.3 5.3

27 39

22/37 36/63

148 232

0.4 0.6

0.6 0.6

Q60715 Q60716 P27773 P50396 P15331 Q8R1N4 Q99KJ8 P56480 Q9WTM5 Q8BK64

59 58.4 56.6 50.5 52.7 40.9 44 56 51.1 38.1

5.6 5.5 6.6 5 5.4 5.1 5.1 5.2 5.5 5.4

60 57 52 54 51 50 50 50 50 38

6.1 6 6 5 5.6 5.2 5.2 5.2 5.8 5.7

40 33 40 48 53 30 38 33 50 39

20/62 20/54 23/41 25/59 32/73 10/59 16/59 18/59 25/59 15/55

151 137 211 158 254 243 243 243 182 101

2 1.9 1.8 3.8 0.6 0.6 0.6 0.6 1.6 1.5

2 1.7 1.8 2.3 0.5 0.5 0.5 0.5 1.6 1.5

Q9CWS0

31.2

5.6

34

5.9

57

18/49

143

1.9

1.6

P14869 Q9D819 Q6IRU2 O08807 O08807 Q9D6J6

34.2 32.6 28.4 31 31 27.3

5.9 5.4 4.6 6.7 6.7 22

33 31 28 25 27 23

5.9 5.5 4.5 6 6.4 5.6

47 59 29 39 34 52

17/50 17/57 9/39 11/19 9/48 14/60

139 115 69 149 72 93

2 1.5 2 2.9 2 0.6

1.6 1.5 1.6 2.5 1.7 0.7

Q60692 Q9DCX2 Q9CQU0

22 18.6 16.5

5 5.5 5.1

22 21 15

5.1 5.8 5.4

34 67 47

9/37 13/51 9/45

69 118 67

0.6 0.7 1.5

0.7 0.7 1.4

Q91V92 Q99KI0 P40142 Q8R081

119.6 85.4 67.6 60

7.1 8.1 7.2 6.6

119 85 67 60

7.2 7.5 7.3 6.9

29 29 24 19

23/53 19/41 12/22 11/25

235 141 123 88

0.6 0.7 0.6 1.7

0.5 0.4 0.6 1.9

P52480 O08749 Q03265 Q03265 Q03265 P17182 Q9R0X4 P97807 Q8BP48 Q9CR16

57.7 54.2 59.7 59.7 59.7 46.9 50.5 54.3 43.2 40.6

7.4 8 9.2 9.2 9.2 6.4 8.7 9.1 6.7 7

58 55 54 54 54 50 50 51 48 47

7.6 6.6 7.5 7.6 8 6.4 7.5 7.8 6.8 7.2

34 30 37 29 26 36 38 21 29 23

16/47 13/44 19/36 14/35 9/32 12/25 19/50 10/37 8/25 10/33

116 99 187 123 71 120 126 66 71 83

1.6 0.4 0.6 0.5 0.4 1.5 0.4 0.6 1.8 1.7

2.2 0.6 0.2 0.3 0.3 2 0.3 0.5 2 2.4

P28474 P05064 P07356 P18155

39.5 39.2 38.5 37.8

7.6 8.4 7.5 9

40 40 37 36

7.1 8.5 7.5 7.8

23 32 49 24

9/50 14/50 20/56 8/30

58 99 176 71

1.5 1.5 0.4 0.6

2.2 2.6 0.4 0.3

P06151 Q91VH6 Q60932

36.3 33.7 32.3

7.8 6.7 8.5

35 32 31

7.1 6.8 8.3

32 31 52

10/58 7/36 12/40

65 58 124

1.7 1.7 0.5

2.1 1.9 0.6

P62827

24.3

7.2

26

7

46

11/36

124

1.6

1.7

a

Accession number from UniProtKB database. b Calculated molecular weight. c Calculated pI. d Observed molecular weight. e Observed pI. f Percentage of sequence coverage. g Peptide matched. h Mascot score. i Increases and decreases in experiments 1 and 2.

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were further disrupted through a 26-gauge needle. Cell debris and nuclei were removed by centrifugation, and the protein concentration was determined with BCA. The resulting postnuclear supernatant was ultracentrifuged at 4 °C for 1 h in a Beckman TLA 55 rotor at 100,000 ⫻ g to separate the cytosol from the membrane fraction. The membrane pellet was then resuspended in homogenization buffer, and each fraction was methanol-precipitated. Pellets were finally resuspended in Laemmli sample buffer. Equivalent volumes of the cytosolic and membrane fractions were analyzed by WB. Immunogold Microscopy—Cells grown on glass coverslips were washed with PBS and fixed in a solution of 4% paraformaldehyde and 0.1% glutaraldehyde in 0.2 M Hepes buffer, pH 7.4, for 15 min at room temperature. After washing with PBS, cells were incubated for 30 min in blocking solution (50 mM NH4Cl, 0.1% saponin, and 1% BSA in Hepes buffer) and overnight at 4 °C with anti-PrP monoclonal antibody SA65 (34) diluted 1:250 in blocking solution. Cells were washed and incubated for 1 h at room temperature with Nanogold-conjugated anti-mouse IgG Fab⬘ fragment diluted 1:100 in blocking solution and processed according to the Nanogold enhancement protocol (Nanoprobes). Stained cells were embedded in Epon 812 and cut as described (35). Electron microscopy (EM) images were acquired from thin sections using a Philips Tecnai 12 electron microscope equipped with an Ultra View CCD digital camera (Philips). Gold particles were quantified in the different compartments of the secretory pathway, and total cell, ER, and Golgi volumes were analyzed using analySIS software (Soft Imaging Systems GmbH). Total Internal Reflection Fluorescence (TIRF) Microscopy—N2a cells stably expressing WT or D177N/Met-128 PrP were seeded on ␮-Dishes (Ibidi, Martinsried, Germany) and transiently transfected with a plasmid carrying the GFP-GPI cDNA (28) using LipofectamineTM 2000 (Invitrogen) according to the manufacturer’s instructions. The day after transfection, cells expressing similar levels of GFP-GPI were analyzed by TIRF microscopy. In some experiments, N2a cells were seeded on ␮-Dishes and were transiently co-transfected with 250 ng of plasmid carrying the WT or D177N/Met-128 PrP-EGFP cDNA and/or with StealthTM small interfering RNA (siRNA) duplexes using Lipofectamine RNAiMAX (Invitrogen). Images were acquired with an objective-based Olympus (Tokyo, Japan) TIRF illumination arm attached to an IX81 inverted microscope using a 1.45 numerical aperture 60⫻ oil immersion lens. For GFP imaging, excitation was from the 488 nm line of a 200-milliwatt argon ion laser at ⬃5% power. Specialized U-MF2 filters were from Olympus. Images were acquired using Cell M software (Olympus) and an Orca camera (Hamamatsu, Hamamatsu City, Japan). For wide field images, the laser was tuned to be focused straight through the sample. Live cells were maintained at 37 °C with 5% CO2 using a Solent Scientific Ltd. incubator (Segensworth, UK). Trafficking of GFP-GPI or PrP-EGFP fusion molecules close to the plasma membrane was measured using the track particles module of Imaris software (Bitplane, Zurich, Switzerland). Fluorescent spots were visualized and automatically detected by segmentation, and the motion path of a single spot was evaluated at consecutive time points (60 s). The total number of spots detected in 60 s was normalized to the surface area analyzed. siRNA—siRNA duplex oligonucleotides targeting PrP and a scrambled duplex were kindly provided by Giovanna Mallucci (Medical Research Council Toxicology Unit, Leicester, UK) and used as described (36). To knock down GDI expression, a set of three Stealth RNAi siRNAs was purchased from Invitrogen. For siRNA experiments, N2a cells were treated at ⬃30% confluency with 25 nM siRNA against GDI or with Stealth RNAi Negative Control Duplexes (Invitrogen) with matching GC content using Lipofectamine RNAiMAX (Invitrogen) as the transfection reagent according to the manufacturer’s instructions. Forty-eight hours after transfec-

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FIG. 4. Functional blocks. The pie chart indicates the impact of a single functional alteration on the total functions classified. Percentages on slices are calculated as the number of proteins associated with a particular functional block normalized to all the proteins associated with functional blocks, set as 100. tion, protein extracts were analyzed for protein knockdown efficiency by slot blot analysis as described (37). RESULTS

D177N/Met-128 PrP Is Retained in ER and Golgi—In primary neurons and non-neuronal cell lines, PrP molecules carrying pathogenic mutations start aggregating soon after biosynthesis in the ER and accumulate in intracellular organelles (7, 14, 15). Consistent with previous observations (14), D177N/Met-128 PrP expressed in N2a cells acquired distinctive biochemical characteristics of pathogenic PrP, such as insolubility in non-denaturing detergents (supplemental Fig. 1A). We used immuno-EM to characterize the intracellular localization of mutant PrP in N2a cells in detail. Although ⬃85% of WT PrP localized on the cell surface, only ⬃25% of D177N/Met-128 PrP resided on the plasma membrane with the rest distributed between endosomes (⬃5%), the ER (⬃30%), and the Golgi complex (⬃40%) (Fig. 1). Because WT and mutant PrP were expressed at similar levels as indicated by WB (supplemental Fig. 1B), the alterations in protein localization documented by EM are very likely the result of abnormal cellular trafficking and/or metabolism of D177N/Met-128 PrP. Proteomics Analysis of N2a Cells Expressing WT and Mutant PrP—To check for molecular changes associated with expression of D177N/Met-128 PrP, we used a differential proteomics analysis to compare N2a cells expressing mutant and WT PrP. We selected two different clones for each genotype, named WT1, WT2, D177N1, and D177N2, which expressed PrP at similar levels (supplemental Fig. 1B). Two independent proteomics experiments were done (Fig. 2). 2D map triplicates were obtained for each different N2a cell clone. We used two wide range immobilized pH gradient strips (pH 4 –7 and 6 –11) to resolve a large number of proteins accurately (⬃1400 spots). Two optimized protocols were used to prepare and separate acidic and basic proteins. Optimal IEF in the alkaline region remains challenging in 2DE. Focusing basic proteins was successful only after the introduction of a dialysis step in the sample preparation and limited sample loading in the 7-cm strip (80 ␮g). The 2DE map

Proteomics of Mutant PrP N2a Cells

FIG. 5. GDI is up-regulated in cells expressing D177N/Met-128. A, quantification of GDI expression in both pairs of N2a clones (1 and 2) from 2D gel data. Each bar indicates the mean (n ⫽ 3). Error bars represent S.D. Representative GDI spot images from 2D gels of WT and D177N clones 1 and 2 are shown. B, quantification of GDI expression by WB in both pair of N2a clones. 30 ␮g of cell lysate was probed with anti-GDI antibody. Each bar indicates the mean of three independent experiments. Error bars represent S.D. Chemiluminescence signals were normalized to the amount of protein loaded as revealed by Coomassie staining of the membrane (*, p ⬍ 0.05 by Student’s t test). C, N2a cells expressing WT or D177N/Met-128 PrP were treated with siRNA against PrP or a scrambled sequence (scr), and the GDI expression was quantified by WB. A representative blot

image analysis detected 117 and 202 spots, respectively, with significantly different volumes in the first and second experiment (Fig. 2). We decided to further analyze only the protein spots that were significantly over- or under-represented in both experiments, changing more than 1.4-fold (Fig. 3 and Table I). Of these, 23 were over- and 19 were under-represented in D177N/Met-128 cells compared with WT controls. The corresponding proteins were identified by MALDI-TOF mass spectrometry (Table I). The alteration of ATPase ␣, AHSA1, annexin A2, ERp19, and GDI was also confirmed by WB (see Fig. 5B and supplemental Fig. 2). To reconstruct the pathways altered by mutant PrP, identified proteins were classified on the basis of gene ontology annotations provided by Protein Knowledgebase (UniProtKB). Supplemental Table 1 reports the gene ontology terms for the general biological processes associated with the identified proteins, including their up- or down-regulation. In view of the multifunctionality of the proteins, some of them were associated with multiple biological processes. The functional annotations most enriched were linked to energy metabolism, redox regulation, and protein transport (Fig. 4). The altered proteins associated with energy metabolism were mainly in the mitochondrion, whereas the ones associated with stress response, protein folding, and redox regulation were mainly ER-associated (supplemental Table 1). GDI, which belongs to the protein transport functional block and is involved in vesicular protein trafficking, was one of the proteins that changed most in both experiments (3.8-fold in experiment 1 and 2.3-fold in experiment 2) (Fig. 5A). The level of GDI reverted to normal after siRNA-mediated D177N/Val-128 PrP silencing (Fig. 5C and supplemental Fig. 3), confirming that mutant PrP was directly responsible for the GDI increase. We then decided to investigate any functional changes associated with GDI overexpression. GDI Overexpression in Mutant Cells Is Associated with Reduced Levels of Membrane-associated Rab11—GDI controls protein trafficking by regulating the function of Rab proteins. Rabs are functionally active when located on membranes in the GTP-bound state but are inactive when associated with GDI in the cytoplasm in the GDP-bound state. Thus, the distribution of a Rab protein between membranes and the cytoplasm indicates its functional state. GDI overexpression causes preferential release of Rab11 from membranes, possibly because it remains longer than other Rabs in the GDPbound state (27); therefore, we investigated the functional state of Rab11. We also looked at Rab5 because it is involved in post-Golgi trafficking and possibly in PrP recycling through

is shown where 15 ␮g of cell lysate was probed with anti-GDI antibody. Each bar indicates the mean ⫾ S.E. relative immunoreactivity (IR) of at least five independent experiments. The chemiluminescence signal was normalized to the total amount of protein loaded as revealed by red Ponceau staining of the membrane. *, p ⬍ 0.05 by one-way analysis of variance followed by a Tukey multiple comparison test.

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FIG. 6. Rab5 and Rab11 distribution. Cytosolic and total membrane fractions of WT and D177N cells were separated by ultracentrifugation at 100,000 ⫻ g, and the amount of Rab11 and Rab5 in the two fractions was evaluated by WB (A–C). A, equivalent volumes of membrane and cytosolic fractions were loaded and probed with anti-Rab11 (upper panel) and anti-Rab5 (middle panel) antibodies. The efficiency of the separation of the membrane fraction was verified with anti-TRAP, a protein associated with ER membrane (lower panel). B–D, the chemiluminescence signal was normalized to the total amount of protein loaded as revealed by Coomassie staining of the membrane. In C and B, values are expressed as percentages of the total immunoreactivity (cytosol ⫹ membrane). In D, the amounts of Rab5 and Rab11 associated with membranes in WT and D177N/Met-128 cells, corresponding to the active protein fraction, are compared. In all experiments, each bar indicates the mean of three independent experiments. Error bars represent S.D. *, p ⬍ 0.05 by Student’s t test.

clathrin-coated vesicles (38 – 41). Rab5 was equally distributed between the cytosolic and membrane fractions (Fig. 6, A and B) with no difference in the amount of active, membranebound Rab5 between WT and mutant cells (Fig. 6D). In contrast, Rab11 was mostly in the cytosol of cells expressing D177N/Met-128 PrP (Fig. 6, A and C). In these cells, the active, membrane-bound fraction of Rab11 was significantly lower than in WT cells (Fig. 6D), suggesting that GDI overexpression was associated with reduced Rab11 function. Expression of D177N/Met-128 PrP Is Associated with Swelling of Golgi Compartment—Reduction in the function of Rab11 inhibits protein transport to the cell membrane and causes proteins to accumulate in the Golgi (27). We measured the volume of intracellular organelles in N2a cells expressing WT and D177N/Met-128 PrP. The Golgi volume was significantly increased up to 3.3 times higher in D177N/ Met-128 cells than in WT cells, but no significant differences were found in the ER volume (Fig. 1F). These data suggested impaired protein trafficking beyond the Golgi network. Trafficking of GPI-anchored Proteins Is Altered in N2a Cells Expressing Mutant PrP—To directly test whether post-Golgi trafficking was altered in cells expressing mutant PrP, we investigated the transport of a GFP-GPI reporter. The GFP-

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GPI lacks identifiable sorting elements other than the C-terminal signal for addition of the GPI anchor, allowing the protein to rapidly cycle between the Golgi and the plasma membrane (28). A plasmid carrying the GFP-GPI cDNA was transiently transfected in WT and D177N/Met-128 PrP-expressing N2a cells, and the trafficking dynamics of GFP-GPI was measured by counting the fluorescent spots on the cell surface during 1 min by TIRF microscopy. Despite similar GFP-GPI expression as judged by total cellular green fluorescence (data not shown), the amount of GFP-GPI on the plasma membrane was strikingly lower in D177N/Met-128 than in WT cells (Fig. 7, A and B). This indicated that D177N/ Met-128 PrP expression was associated with impaired postGolgi trafficking of GPI-anchored proteins. Because PrP is GPI-linked, we also measured the trafficking dynamics of PrP at the cell surface. For this experiment, we transiently transfected N2a cells with WT and D177N/Met128 PrP-EGFP fusion molecules. TIRF analysis showed a significant impairment of D177N/Met-128 PrP-EGFP trafficking compared with WT PrP-EGFP that was partially rescued when GDI overexpression was knocked down by siRNA (Fig. 7C and supplemental Fig. 3). This result suggested that GDI overexpression in mutant N2a cells stimulates a feedback loop affecting the trafficking of PrP itself.

Proteomics of Mutant PrP N2a Cells

FIG. 7. Trafficking dynamics of GPI-anchored proteins by TIRF. N2a cells expressing WT or D177N/Met-128 PrP were transiently transfected with GFP-GPI. Cells expressing similar levels of the fluorescent protein were used in the TIRF experiment. A, representative TIRF images of live N2a cells expressing WT or D177N/Met-128 PrP. The fluorescent signal represents protein that has reached the plasma membrane. B, quantification of the GFP-GPI molecules on the cell surface. Each bar of the histogram represents the total number of spots detected in 60 s normalized to the surface area analyzed. Data are the mean of 10 cells (*, p ⬍ 0.05 by Mann-Whitney U test). Error bars represent S.E. C, quantification of the EGFP-PrP molecules on the cell surface. Each bar of the histogram represents the total number of spots detected in 60 s normalized to the surface area analyzed. Data are the mean of at least eight cells per condition. Error bars represent S.E. *, p ⬍ 0.05 by one-way analysis of variance followed by a Tukey multiple comparison test. scr, scrambled sequence. DISCUSSION

The cellular pathways activated by mutant PrP ultimately leading to neuronal dysfunction and degeneration in genetic prion diseases are poorly understood. In different non-neuronal and neuronal cells, several mutant PrPs aggregate in the early steps of the secretory pathway, reside longer in the ER and Golgi, and are delivered less efficiently to the cell surface (10, 13–15). This held true for D177N/Met-128 PrP expressed

in N2a cells that formed insoluble aggregates and was mainly localized in the ER and Golgi. We used a proteomics approach to investigate the molecular alterations induced by expression of mutant PrP in N2a cells. We optimized the analysis within two pH ranges to explore proteomic alterations by 2DE with high resolution and sensitivity. The alkaline 2D maps allowed us to analyze a portion of the proteome that is hard to detect in conventional 2DE. For example, several mitochondrial proteins have pI values greater than 6 (42). The proteomics analysis detected several protein changes indicative of alterations in different biological processes. Because N2a cells expressing PrP D177N/Met-128 survive normally in culture, these protein changes are not due to degenerative events but can be directly ascribed to expression of the mutant PrP. The most affected proteins were associated with energy metabolism, redox regulation, and protein transport (Fig. 4). Low expression of mitochondrial proteins involved in ATP synthesis is consistent with reduced ATP levels in N2a cells expressing D177N/Met-128 PrP (data not shown) and may indicate a reduction in energy metabolism. This alteration can be expected because cell lines are able to adapt their metabolism in response to cytotoxic insults (43). Low ATP levels, in fact, may allow mutant N2a to survive longer because high ATP is necessary for apoptosis (44). Proteins involved in redox regulation, including peroxiredoxin 4, which is implicated in cellular protection against oxidative stress (45), were up-regulated in the mutant cells. Peroxiredoxins also increased in a transgenic mouse model of inherited prion disease (30) and in prion-infected mice (46) and may be regarded as a cellular response to oxidative stress induced by various forms of pathogenic PrP (47). Many ER-resident proteins involved in redox regulation, stress response, and protein folding were also up-regulated in mutant N2a cells, consistent with evidence of altered ER homeostasis in prion diseases (15, 48, 49). We found upregulation of ERp57, an ER-associated chaperone with protein-disulfide isomerase-like activity. In N2a cells, inhibition of ERp57 enhanced PrP-induced toxicity, whereas overexpression was protective and correlated with a lower rate of caspase-12 activation (49). This suggests that N2a cells expressing D177N/Met-128 PrP can activate cellular responses that counteract PrP toxicity. One of the proteins that changed most was GDI, an important regulator of Rab proteins, which govern intracellular vesicular trafficking. GDI is believed to interact differently with several Rabs depending on their binding constants and on the time they spend in the GDP-bound state (50, 51). We found that the amount of cytosolic Rab11, but not Rab5, increased in cells expressing D177N/Met-128 PrP, indicating a selective effect of GDI overexpression on Rab11 localization and function, consistent with previous findings (27). Reduced Rab11 function impairs delivery of proteins, such as rhodopsin, protease-activated receptor 2, and E-cadherin, to the cell surface

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FIG. 8. Model for alterations in GDI/Rab11 pathway produced by mutant PrP. D177N/Met-128 PrP is less efficiently trafficked to the cell surface and accumulates in the Golgi complex, which becomes bigger. N2a cells expressing D177N/Met-128 PrP have a higher level of GDI and show a dramatic change in the intracellular localization of Rab11. GDI sequesters Rab11 in the cytosol, keeping it in the inactive state and affecting the post-Golgi trafficking of secreted proteins and possibly of PrP itself.

with deleterious consequence for cells (52–54). Mutant PrP expression was also associated with enlargement of the Golgi, leading us to investigate the functionality of post-Golgi transport. We analyzed by TIRF microscopy the trafficking of a GFP-GPI reporter, which recycles between the plasma membrane and the Golgi complex (28). We found defective cell membrane delivery of the reporter in N2a cells expressing D177N/Met-128 PrP, confirming a possible link between mutant PrP and dysregulation of post-Golgi transport. Furthermore, we observed that the alteration of mutant PrP trafficking at the cell surface was partially rescued by partially silencing GDI, indicating that alteration of the GDI/Rab11 pathway stimulated a feedback loop leading to further intracellular accumulation of mutant PrP. Intriguingly, it has been shown recently that PrP knockdown in zebrafish embryos causes loss of cell adhesion due to accumulation of E-cadherin in a Rab11-positive post-Golgi compartment (55). This was associated with activation of Src-related kinases, suggesting that zebrafish PrP may serve a signaling function that regulates post-Golgi trafficking (56). PrP-dependent activation of the tyrosine kinase Fyn has been described in mammalian cells (57). It is therefore tempting to speculate that PrP may fulfill a conserved signaling function involved in regulating intracellular protein trafficking and that this function may be altered by the pathogenic mutations. Studies are now necessary to determine whether an altered level of GDI can affect the trafficking of other proteins besides the GFP-GPI reporter and D177N/Met-128 PrP and to dissect the underlying mechanism. In conclusion, our proteomics screening revealed a general up-regulation of the proteins involved in response to cellular insults, such as oxidative and ER stresses. This and the

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effects on energy metabolism indicated by changes in proteins involved in ATP synthesis may contribute to the survival of mutant PrP cells in culture. Initial studies aimed at defining the cellular processes affected by mutant PrP highlighted a potential toxic feedback involving overexpression of GDI and sequestration of Rab11 in the cytosol, which could lead to alterations of protein trafficking between the Golgi and the plasma membrane and induce further accumulation of mutant PrP (Fig. 8). Impairment of protein transport could have adverse cellular effects by preventing correct delivery to the plasma membrane of adhesion molecules, ion channels, or receptors important for normal neuronal function. Further studies are now needed to evaluate the role of defective protein trafficking in the pathogenesis of inherited prion diseases. Acknowledgments—We thank Gianluigi Zanusso, Richard Kascsak, Marek Michalak, Marino Zerial, and Patrizia D’Adamo for providing antibodies; Giovanna Mallucci for the siRNA duplex oligonucleotides targeting PrP; Chiara Zurzolo for the GFP-GPI construct and scientific advice; and Manuela Basso for scientific advice. We thank Marco Gobbi for comments on the manuscript and Judith Baggott for help in preparing the manuscript. We are grateful to Roman S. Polishchuk of the Telethon Microscopy and Bio-Imaging Facility (Consorzio Mario Negri Sud) for immunogold staining of N2a cells. * This work was supported, in whole or in part, by National Institutes of Health Grants NS040975 and NS052526 (to D. A. H.). This work was also supported by Telethon Foundation Grants TCR08002 (to V. B.) and TCR08005 (to R. C.), Cariplo Foundation Grant 20050632, the Compagnia di San Paolo Foundation (to V. B. and R. C.) and the E.C. Network of Excellence NeuroPrion (to R. C.). □ S This article contains supplemental Table 1 and Figs. 1–3. ** Present address: Dept. of Biochemistry, Boston University School of Medicine, Boston, MA. ‡‡ Associate Telethon Scientist. §§ To whom correspondence should be addressed: Dulbecco Telethon Inst. and “Mario Negri” Inst. for Pharmacological Research, Via G. La Masa 19, 20156 Milan, Italy. Tel.: 39-02-39014548; Fax: 39-02-39014744; E-mail: [email protected]. REFERENCES 1. Young, K., Piccardo, P., Dlouhy, S., Bugiani, O., Tagliavini, F., and Ghetti, B. (1999) The human genetic prion diseases, in Prions: Molecular and Cellular Biology (Harris, D. A., ed) pp. 139 –175, Horizon Scientific Press, Wymondham, UK 2. Chiesa, R., and Harris, D. A. (2001) Prion diseases: what is the neurotoxic molecule? Neurobiol. Dis. 8, 743–763 3. Goldfarb, L. G., Petersen, R. B., Tabaton, M., Brown, P., LeBlanc, A. C., Montagna, P., Cortelli, P., Julien, J., Vital, C., Pendelbury, W. W., Haltia, M., Willis, P. R., Hauw, J. J., McKeever, P. E., Monari, L., Schrank, B., Swergold, G. D., Autilio-Gambetti, L., Gajdusek, D. C., Lugaresi,E., and Gambetti, P. (1992) Fatal familial insomnia and familial Creutzfeldt-Jakob disease: disease phenotype determined by a DNA polymorphism. Science 258, 806 – 808 4. Westergard, L., Christensen, H. M., and Harris, D. A. (2007) The cellular prion protein (PrP(C)): its physiological function and role in disease. Biochim. Biophys. Acta 1772, 629 – 644 5. Harris, D. A. (2003) Trafficking, turnover and membrane topology of PrP. Br. Med. Bull. 66, 71– 85 6. Campana, V., Sarnataro, D., and Zurzolo, C. (2005) The highways and byways of prion protein trafficking. Trends Cell Biol. 15, 102–111

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