The -Secretase-derivedN-terminalProductofCellularPrion, N1 ... · Cellular prion protein (PrPc)...

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The -Secretase-derived N-terminal Product of Cellular Prion, N1, Displays Neuroprotective Function in Vitro and in Vivo * S Received for publication, July 31, 2009, and in revised form, October 12, 2009 Published, JBC Papers in Press, October 22, 2009, DOI 10.1074/jbc.M109.051086 Marie-Victoire Guillot-Sestier 1,2 , Claire Sunyach 1,3 , Charlotte Druon, Sabine Scarzello, and Fre ´de ´ ric Checler 4 From the Institut de Pharmacologie Mole ´culaire et Cellulaire and Institut de Neurome ´decine Mole ´culaire, UMR6097 CNRS/UNSA, E ´ quipe Labellise ´e Fondation pour la Recherche Me ´dicale, 660 Route des Lucioles, Sophia-Antipolis, 06560 Valbonne, France Cellular prion protein (PrP c ) undergoes a disintegrin-medi- ated physiological cleavage, generating a soluble amino-termi- nal fragment (N1), the function of which remained unknown. Recombinant N1 inhibits staurosporine-induced caspase-3 acti- vation by modulating p53 transcription and activity, whereas the PrP c -derived pathological fragment (N2) remains biologi- cally inert. Furthermore, N1 protects retinal ganglion cells from hypoxia-induced apoptosis, reduces the number of terminal deoxynucleotidyltransferase-mediated biotinylated UTP nick end labeling-positive and p53-immunoreactive neurons in a pressure-induced ischemia model of the rat retina and triggers a partial recovery of b-waves but not a-waves of rat electroretino- grams. Our work is the first demonstration that the -secretase- derived PrP c fragment N1, but not N2, displays in vivo and in vitro neuroprotective function by modulating p53 pathway. It further demonstrates that distinct N-terminal cleavage prod- ucts of PrP c harbor different biological activities underlying the various phenotypes linking PrP c to cell survival. The investigation of the physiological function of PrP c has long been neglected due to the lack of an obvious phenotype in PrP c -deficient mice. Recently, several works have shed light on the putative implication of PrP c in cell adhesion, neurite out- growth, synaptogenesis, and myelinization (for a review see Ref. 1). In addition, albeit debated, a role in the control of cell via- bility has also been proposed. Thus, some reports have sug- gested that PrP c might have a protective function against Bax- induced cell death, oxidative stress, and hypoxic injury (1). Conversely, in several experimental systems, overexpressed or endogenous PrP c both lead to exacerbated cellular responsive- ness to apoptotic insults (2–7). In addition, cell degeneration in the nervous system of old transgenic mice harboring high copy number of the PrP c gene had been observed (8). Overall, these data suggest that PrP c could display both pro- and antiapopto- tic functions, depending on the cell context and/or physiologi- cal situation. A subset of plasma membrane-tethered PrP c molecules undergoes proteolytic processing events. PrP c is mainly endo- proteolyzed at the 110/111 peptidyl bond to produce a 17-kDa C-terminal fragment, C1, which remains membrane-bound (9) and a 9-kDa soluble N-terminal counterpart, referred to as N1, released in the extracellular space (10 –12). In brain from trans- missible spongiform encephalopathy-affected individuals, the PrP c undergoes alternative proteolytic attack. Thus, a 21-kDa C-terminal fragment, C2, and its 7-kDa N2 N-terminal coun- terpart derive from an additional cleavage around the 90/91 residues (9). It is noteworthy that transgenic mice expressing PrP c pro- teins lacking N-terminal residues 33–120 or 33–133 exhibit exacerbated neurodegeneration (13–15). Furthermore, we re- ported that the overexpression of the -secretase-derived PrP c fragment C1 lacking the N-terminal domain was detrimental in vitro (16). Overall, these observations suggest that PrP c N-ter- minal moiety is crucial for its function, but the putative func- tion of secreted N1 has never been delineated. Does proteolytic release of N1 correspond to an inactivating mechanism impair- ing PrP c biological function, or alternatively, does it represent a maturation process allowing N1 to trigger its own physiological function? Here we show that N1 but not N2 displays a protec- tive phenotype by modulating the p53 pathway in vitro as well as in vivo in a pressure-induced ischemia model of rat retina. MATERIALS AND METHODS Animals—Mated wild type black 57 mice were purchased from Charles River (Charles River Breeding Laboratory, St. Aubain les Elbeuf, France). PrP c Zrch-1 knock-out mice were kindly provided by Charles Weissmann (Scripps Florida). Adult male Brown Norway rats weighing 200 g (Charles River Laboratory) were housed in clear plastic cages in a room with controlled temperature (22 1 °C) and fixed 50-1x fluo- rescent (Philips) lighting schedule (lights on from 08:00 to 20:00 h). Food and tap water were provided ad libitum. Rats were acclimated for 1 week before experiments. These studies were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Antibodies and Pharmacological Agents—SAF32 antibody directed against PrP c residues 79 –92 and SAF61, which recog- nizes the C-terminal domain of PrP c (17), was generously pro- vided by J. Grassi (Commissariat a ` l’Energie Atomique/Saclay, Gif sur Yvette, France). Carbachol and staurosporine were pur- chased from Sigma. Atropine was from ICN Biochemicals (Aurora, OH). LY294002 was obtained from Cayman (VWR, Fontenay sous Bois, France). * This work was supported by CNRS, the Fe ´de ´ ration pour la Recherche sur le Cerveau, and the Fondation pour la Recherche Me ´ dicale. S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3. 1 Both authors contributed equally to this work. 2 Supported by the Fondation pour la Recherche Me ´ dicale. 3 Recipient of a France Alzheimer fellowship. Present address: Institut de Neu- robiologie de Mediterranne ´ e, Luminy 13009, France. 4 To whom correspondence should be addressed. Tel.: 33-4-93-95-34-60; Fax: 33-4-93-95-77-08; E-mail: [email protected]. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 51, pp. 35973–35986, December 18, 2009 © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. 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The �-Secretase-derived N-terminal Product of Cellular Prion,N1, Displays Neuroprotective Function in Vitro and in Vivo*□S

Received for publication, July 31, 2009, and in revised form, October 12, 2009 Published, JBC Papers in Press, October 22, 2009, DOI 10.1074/jbc.M109.051086

Marie-Victoire Guillot-Sestier1,2, Claire Sunyach1,3, Charlotte Druon, Sabine Scarzello, and Frederic Checler4

From the Institut de Pharmacologie Moleculaire et Cellulaire and Institut de Neuromedecine Moleculaire, UMR6097 CNRS/UNSA,Equipe Labellisee Fondation pour la Recherche Medicale, 660 Route des Lucioles, Sophia-Antipolis, 06560 Valbonne, France

Cellular prion protein (PrPc) undergoes a disintegrin-medi-ated physiological cleavage, generating a soluble amino-termi-nal fragment (N1), the function of which remained unknown.RecombinantN1 inhibits staurosporine-induced caspase-3 acti-vation by modulating p53 transcription and activity, whereasthe PrPc-derived pathological fragment (N2) remains biologi-cally inert. Furthermore, N1 protects retinal ganglion cells fromhypoxia-induced apoptosis, reduces the number of terminaldeoxynucleotidyltransferase-mediated biotinylated UTP nickend labeling-positive and p53-immunoreactive neurons in apressure-induced ischemiamodel of the rat retina and triggers apartial recovery of b-waves but not a-waves of rat electroretino-grams.Ourwork is the first demonstration that the�-secretase-derived PrPc fragment N1, but not N2, displays in vivo and invitro neuroprotective function by modulating p53 pathway. Itfurther demonstrates that distinct N-terminal cleavage prod-ucts of PrPc harbor different biological activities underlying thevarious phenotypes linking PrPc to cell survival.

The investigation of the physiological function of PrPc haslong been neglected due to the lack of an obvious phenotype inPrPc-deficient mice. Recently, several works have shed light onthe putative implication of PrPc in cell adhesion, neurite out-growth, synaptogenesis, andmyelinization (for a review see Ref.1). In addition, albeit debated, a role in the control of cell via-bility has also been proposed. Thus, some reports have sug-gested that PrPc might have a protective function against Bax-induced cell death, oxidative stress, and hypoxic injury (1).Conversely, in several experimental systems, overexpressed orendogenous PrPc both lead to exacerbated cellular responsive-ness to apoptotic insults (2–7). In addition, cell degeneration inthe nervous system of old transgenic mice harboring high copynumber of the PrPc gene had been observed (8). Overall, thesedata suggest that PrPc could display both pro- and antiapopto-tic functions, depending on the cell context and/or physiologi-cal situation.

A subset of plasma membrane-tethered PrPc moleculesundergoes proteolytic processing events. PrPc is mainly endo-proteolyzed at the 110/111 peptidyl bond to produce a 17-kDaC-terminal fragment, C1, which remains membrane-bound (9)and a 9-kDa soluble N-terminal counterpart, referred to as N1,released in the extracellular space (10–12). In brain from trans-missible spongiform encephalopathy-affected individuals, thePrPc undergoes alternative proteolytic attack. Thus, a 21-kDaC-terminal fragment, C2, and its 7-kDa N2 N-terminal coun-terpart derive from an additional cleavage around the 90/91residues (9).It is noteworthy that transgenic mice expressing PrPc pro-

teins lacking N-terminal residues 33–120 or 33–133 exhibitexacerbated neurodegeneration (13–15). Furthermore, we re-ported that the overexpression of the �-secretase-derived PrPcfragment C1 lacking theN-terminal domainwas detrimental invitro (16). Overall, these observations suggest that PrPc N-ter-minal moiety is crucial for its function, but the putative func-tion of secreted N1 has never been delineated. Does proteolyticrelease of N1 correspond to an inactivatingmechanism impair-ing PrPc biological function, or alternatively, does it represent amaturation process allowingN1 to trigger its own physiologicalfunction? Here we show that N1 but not N2 displays a protec-tive phenotype by modulating the p53 pathway in vitro as wellas in vivo in a pressure-induced ischemia model of rat retina.

MATERIALS AND METHODS

Animals—Mated wild type black 57 mice were purchasedfrom Charles River (Charles River Breeding Laboratory, St.Aubain les Elbeuf, France). PrPc Zrch-1 knock-out mice werekindly provided by Charles Weissmann (Scripps Florida).Adult male Brown Norway rats weighing 200 g (Charles

River Laboratory) were housed in clear plastic cages in a roomwith controlled temperature (22 � 1 °C) and fixed 50-1x fluo-rescent (Philips) lighting schedule (lights on from08:00 to 20:00h). Food and tap water were provided ad libitum. Rats wereacclimated for 1 week before experiments. These studies wereperformed in accordancewith theARVOStatement for theUseof Animals in Ophthalmic and Vision Research.Antibodies and Pharmacological Agents—SAF32 antibody

directed against PrPc residues 79–92 and SAF61, which recog-nizes the C-terminal domain of PrPc (17), was generously pro-vided by J. Grassi (Commissariat a l’Energie Atomique/Saclay,Gif sur Yvette, France). Carbachol and staurosporine were pur-chased from Sigma. Atropine was from ICN Biochemicals(Aurora, OH). LY294002 was obtained from Cayman (VWR,Fontenay sous Bois, France).

* This work was supported by CNRS, the Federation pour la Recherche sur leCerveau, and the Fondation pour la Recherche Medicale.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. S1–S3.

1 Both authors contributed equally to this work.2 Supported by the Fondation pour la Recherche Medicale.3 Recipient of a France Alzheimer fellowship. Present address: Institut de Neu-

robiologie de Mediterrannee, Luminy 13009, France.4 To whom correspondence should be addressed. Tel.: 33-4-93-95-34-60; Fax:

33-4-93-95-77-08; E-mail: [email protected].

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 51, pp. 35973–35986, December 18, 2009© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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Construction of Glutathione S-Transferase (GST)5-N-termi-nal PrPc Fusion-expressing Vector and Purification of PrP N-terminal Recombinant Fragments—cDNA encoding varioussequences of mouse PrPc were generated by PCR: amino acids23–110 (N1), 23–89 (N2), 41–107 (NT), and 23–110 where theKKRPKPG N-terminal domain was replaced by KQHPSPG(NK). Amplicons were cloned into EcoRI and Xho sites ofpGEX-KG (18). To produce and purify PrPc-derived N-termi-nal recombinant fragments, the pGEX-KG GST-N-terminalPrPc-expressing vectors were transformed into BL21 goldstrain of Escherichia coli (Stratagene, Amsterdam Zuidoost,The Netherlands). E. coli were grown in Luria-broth mediumand allowed to reach A600 � 0.6. Fusion proteins were inducedwith 0.5 mM isopropyl 1-thio-�-D-galactopyranoside (Sigma)for 4 h at 37 °C. Cells were pelleted at 5000� g for 20min at 4 °Cand resuspended in phosphate-buffered saline (PBS; 50 �l perml of original culture) supplemented with complete proteaseinhibitor mixture (Sigma), phenylmethylsulfonyl fluoride(Sigma), and lysozyme (150�M/ml; Sigma) and incubated for 30min on ice. Proteins were solubilized by the addition of TritonX-100 (1%), MgCl2 (10 mM), DNase I (5 �g/ml; Promega) andincubation on ice for 30min. Debris were pelleted for 20min at10,000� g. Glutathione-Sepharose beads (GEHealthcare) pre-swollen in PBS (70% slurry) were added to the crude lysate andswirled for 1 h at 4 °C. Beads were pelleted, washed five timeswith 10 volumes of PBS, and resuspended in 1 ml of PBS. Pep-tides were cleaved with thrombin (5 units/ml; GE Healthcare)for 1 h at room temperature. Thrombin was removed usingSepharose benzamidine beads (GEHealthcare). Control exper-iments were carried out with empty pGEX-KG-glutathioneS-transferase, referred to as KG hereafter). The monisotopicmass of each peptide was checked by matrix-assisted laser de-sorption ionization time-of-flight mass spectrometry analysisperformed after reverse phase, solid phase extraction with aC18 ZipTip (Millipore) and was in accordance with the theo-retical mass calculated from the sequence data. Theoreticalmass (TM) N1 10,172.96 Da, measured mass (MM) N110,172.29 Da, N2 TM 7833.36 Da, MM 7832.24 Da, NT TM6844.22 Da, MM 6843.11 Da, NK TM 10153.83 Da, MM10,152.9 Da. GST was produced and purified as describedabove, and then GST bound to Sepharose beads was subjectedto thrombin digestion. Bead supernatant was used as negativecontrol (KG).Cell Systems and Transfections—Human embryonic cells

(HEK293) stably expressing full-length 3F4MoPrP (3F4-taggedmurine PrPc), C-terminal PrPc fragment (C1), and M1 musca-rinic receptors were established and maintained as previouslydetailed (10, 16). p19arf-deficient and p19arf/p53 double knock-out fibroblasts were kindly provided by Dr. M. Roussel (19).ERK1-deficient cells were from Dr. G. Pages (Nice). The PrPc-deficient hippocampus-derived cell line HpL3-4 kindly sup-

plied by Dr. T. Onodera was described previously (20). Primarycultured mouse cortical neurons were transfected usingAmaxa� Nucleofector� kits for primary culture of mouse neu-rons (Lonza, Cologne, Germany) according to the manufactur-er’s specifications. Cells were maintained for 48 h at 37 °Cunder 5% CO2 before being challenged with staurosporine inthe presence or absence of N1 fragment. Treatments andcaspase-3 measurements were performed as described below.Primary Cultured Cortical Neurons—Embryonic cortical

neurons were prepared as previously detailed (21). Briefly, cellsfrom cerebral hemispheres of E14mice fromPrP�/� and PrP0/0embryos were dissociated in Ham’s F-12 (Invitrogen) supple-mented with 0.6% glucose and 10% fetal calf serum. A total of106 cells were seeded in 35-mmdiameter dishes precoated withpolylysine (10 �g/ml; Sigma) and kept for 4 days before beingassayed for apoptosis.Primary Cultured Rat Retinal Cells—Retina cell primary cul-

tures were prepared from Brown Norway newborns. Retinaswere removed and mechanically dissociated at room tempera-ture in neurobasal medium containing 34 units/ml papain, 0.4mg/ml L-cysteine, 0.4 mg/ml bovine serum albumin (all fromSigma), and 0.5 mg/ml DNase I (Promega). Cells washed inneurobasal medium were passed through a 40-mm mesh andseeded at 106 cells/ml on poly-D-lysine-coated dishes (Biocoat;BD Biosciences) or coverslips in neurobasal medium supple-mented with 5% fetal bovine serum, 10 mg/ml basic fibroblastgrowth factor, 1% N2 supplement and kept at 37 °C under 5%CO2 for 7 days before being assayed for survival, caspase-3activity, and terminal deoxynucleotidyltransferase-mediatedbiotinylated UTP nick end labeling (TUNEL) staining.Western Blot Analyses—Recombinant N-terminal fragments

were resolved on 16.5% Tris/Tricine gel and analyzed by stan-dard immunoblotting techniques using SAF32 antibody. Foranalysis of PrPc immunoreactivity, cells were homogenized inlysis buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 0.5% TritonX-100, 0.5% deoxycholate, 5mMEDTA). Equal amounts of pro-teins were resolved on 12% SDS-polyacrylamide gel and ana-lyzed by immunoblotting using and SAF32. Blots were devel-oped using the ECL method according to the manufacturer’sinstructions (Roche Applied Science). Chemiluminescencewas recorded using a luminescence image analyzer LAS-3000 (Raytest, Courbevoie, France), and quantification ofcaptured images was performed using Aida Image Analyzersoftware (Raytest).Immunoprecipitation and Analysis of N1 Fragments—Cells

plated in 35-mm dishes were grown to confluence and incu-bated with required pharmacological agent for 8 h in Dulbec-co’s modified Eagle’s medium. Media were collected, and N1was immunoprecipitated as detailed elsewhere (10). Immuno-captured peptides were subjected to SDS-PAGE analysis on16.5% Tris/Tricine gel as described above. For biochemicaldetection of internalized N1, cells were acid-rinsed, homoge-nized in the above lysis buffer, and then processed for immu-noprecipitation as described previously (10).Cell Viability Assays—Cells were grown in 96-well plates. At

confluence, N1 (1 �M) or an equivalent volume of controlsupernatant produced after thrombin digestion of GST-Sepha-rose beads (KG) was added into fresh medium. After 4 h of

5 The abbreviations used are: GST, glutathione S-transferase; PBS, phosphate-buffered saline; TM, theoretical mass; MM, measured mass; TUNEL, termi-nal deoxynucleotidyltransferase-mediated biotinylated UTP nick endlabeling; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; STS,staurosporine; DAPI, 4�,6-diamino-2-phenylindole; OGD, oxygen glucosedeprivation; RGC, rat retinal ganglion cells; ERK, extracellular signal-regu-lated kinase.

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incubation, N1 was added again to the cells in the presence orabsence of staurosporine (STS; 2 �M), and cells were returnedto 37 °C for 16 h. Fifty �l of 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydrox-ide (XTT) reactive was added to the cells, and absorbance wasmeasured at 452 nm as previously detailed (22).Membrane integrity was evaluated by measuring the lactate

dehydrogenase activity in the culture medium using the cyto-Tox-ONETM kit (Promega). In oxygen glucose deprivation(OGD) experiments (see below), the assay was processedaccording to the manufacturer’s recommendations for 40 �l ofmedium immediately after 2 h of hypoxia and 24 h after returnto complete medium.Caspase-3-like Activity Measurements—Cells were grown in

6-well plates and incubatedwith STS (2�M for 16 h forHEK293cells and primary cortical neurons; 1 �M for 2 h for fibroblasts)after they reached confluence. Samples were processed for acaspase-3-like activity assay as already detailed (22). Fluorime-try was recorded at 360 and 460 nm (excitation and emissionwavelengths) by means of a microtiter plate reader (Lab-systems, Fisher Bioblock Scientific (Illkirch, France)). Caspase-3-like activity was calculated from the linear part of fluorimetryrecorded and expressed in units/h/mg of proteins (establishedby the Bio-Rad procedure). One unit corresponds to 4 nmol of7-amino-4-methylcoumarin released. When blockade of inter-nalization was required, cells were pretreated with 0.45 M

sucrose for 30 min as previously detailed (23) or with 10 �M

Dynasore. When necessary, LY294002 was added simulta-neously to the peptide before staurosporine treatment. Whenstated, cells were treated with carbachol (100 �M) and/or atro-pine (10 �M) together with staurosporine (2 �M) and incubatedfor 16 h. Caspase-3 activity was then monitored as above.TUNEL Analysis—The same number of cells were plated on

glass coverslips until they reached confluence and then werepreincubated with N1 or control supernatant (KG). After 4 h ofincubation, cells were retreated with N1 or KG and with stau-rosporine (1 �M, 2 h). Cells were then fixed for 20 min inparaformaldehyde (4% in PBS), rinsed, and permeabilized over-night in 70% ethanol before being processed for labeling withthe dUTP nick end labeling in situ cell death detection kit, POD(Roche Applied Science) as recommended by the supplier. Forquantification, total cells were counterstained with erythrosineB. Imageswere captured onOlympus BX41microscope (Olym-pus, Rungis, France) using Olympus DP12 software. Cellsundergoing cell death (3,3�-diaminobenzidine-labeled nuclei)were counted on 10 independent optical fields for each experi-mental condition.In situ analyses of apoptotic cell death in rat retina were per-

formed 48 h postischemia, on 10-�m-thick frozen retina sec-tions of ischemia-induced or sham control. TUNEL-positivenuclei were detected with the in situ cell death detection-fluo-rescein kit (Roche Applied Science) essentially as describedpreviously (24). Briefly, sections were fixed in 4% paraformal-dehyde, permeabilized in PBS, 0.1% Tween 20, washed in PBS,and incubated in PBS containing 1.5% H2O2 for 30 min beforeincubation for 1 h at 37 °C with terminal deoxynucleotidyl-transferase to incorporate fluorescein nucleotides into DNAstrand breaks. Sections were rinsed in PBS and mounted in

Vectashield (Vector Laboratories, Burlingame, CA) containing4�,6-diamino-2-phenylindole (DAPI). Images of retinal sec-tions were acquired and photographed using a fluorescentAxioplan2 imaging microscope (Zeiss, Thornwood, NY).p53 Transcriptional Activity and Promoter Transactivation—

Reporter constructs p21waf-1-luciferase and PG13-luciferase(provided by Dr. B. Vogelstein (Baltimore, MD)) used to mea-sure p53 transcriptional activity have been extensively describedelsewhere (4, 16, 25). Briefly, cells grown in 12-well plates wereco-transfected with a 4:1 ratio of p21waf-1 or PG13-luciferasereporter constructs and �-galactosidase expression vector (tonormalize transfection efficiency). Luciferase and�-galactosid-ase activities were assayed 24 h after transfection according toalready described procedures (25) using the luciferase assay sys-tem and �-galactosidase enzyme assay system (Promega). Thep53 promoter-luciferase (pp53) construct (kindly provided byDr.M.Oren (Rehovot, Israel)) described earlier (26)was used todetermine p53 promoter transactivation as above. Whenrequired, cells were treated with carbachol (100 �M), atropine(10 �M), and/or LY294002 (10 �M) for 16 h.Real-time Quantitative PCR—Total RNAs from cells were

isolated using the RNeasy kit (Promega) following the instruc-tions of the manufacturer. After DNase I treatment, 1 �g oftotal RNAwas reverse-transcribed using oligo(dT) priming andavian myeloblastosis virus reverse transcriptase (Promega).Real-time PCRwas performed in an ABI PRISM 5700 sequencedetector system (Applied Biosystems, Courtaboeuf, France)using the SYBR Green detection protocol as outlined by themanufacturer. Human p53-specific primers were designedusing Primer Express software (Applied Biosystems, Courta-boeuf, France): forward, 5�-GAA CCC TTG CTT GCA ATAGG-3�; reverse, 5�-GTG AGG TAG GTG CAA ATG CC-3�.The relative expression level of p53 gene is normalized for RNAconcentrations with housekeeping gene (human glyceralde-hyde-3-phosphate dehydrogenase) using the following primers:forward, 5�-TGG GCT ACA CTG AGC ACC AG-3�; reverse,5�-CAG CGT CAA AGG TGG AGG AG-3�. mRNA values areexpressed in arbitrary units.Immunofluorescence—In situ detection of intracellular N1

fragments was performed by immunohistochemistry onHPL3-4 PrPc-deficient cells cultured on glass coverslips in35-mm dishes. Cells were acid-rinsed and fixed with parafor-maldehyde (1.5%) for 20min and permeabilizedwithTritonX100(0.1%; Sigma). Cells were washed three times in PBS andblocked with milk (1%) in PBS for 30 min, and the primaryantibody (SAF32) was applied for 2 h. After three washes withPBS, cells were incubated for 1 h with goat anti-mouse second-ary antibody conjugated to Alexa Fluor-594 (Interchim,Montlucon, France).Hoechst (Interchim)was added to the PBSduring the first of the last three washes to stain the nuclei. Cov-erslips were then mounted in Vectashield mounting medium(Vector Laboratories, Inc., Burlingame, CA), and staining wasvisualized with an Axioplan 2 imaging microscope (Carl Zeiss,Sartrouville, France) with oil immersion objective �63, cou-pled to a cooled CCD camera (Raper Scientist, Tucson, AZ).Oxygen Glucose Deprivation—Retinal cells were subjected

to oxygen glucose deprivation (OGD) as described previously(27). Cells were treated with an appropriate fragment (1 �M) or

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equivalent volume of KG for 4 h before being washed withdeoxygenated Hanks’ buffered saline solution medium. Cul-tures were retreated with fragment (1 �M) or KG in Hanks’buffered saline solution and placed in a modular incubatorchamber (Billups-Rothenberg, Del Mar, CA) flushed with a4.5% carbon dioxide, 94.5% nitrogen gas mixture for 10 min.The chamber was sealed and placed in a 37 °C incubator for 90min.After 90min of hypoxia,mediumwaswashed and replacedwith neurobasal medium supplemented with 5% fetal bovineserum, 10mg/ml basic fibroblast growth factor, 1%, N2 supple-ment and returned to 37 °C under 5%CO2 atmosphere for 24 h.Control cells were similarly washed and treated with fragmentsin oxygenated Hanks’ buffered saline solution for 6 h beforebeing returned to complete neurobasal medium for 24 h.Intravitreal Injection and Retinal Ischemia—To investigate

the antiapoptotic effect of N1, the rats were treated intravit-really with N1, N2, NT, or KG. Intraocular injection was doneunder dim red light after general anesthesia with sodium pen-tobarbital (60 mg/kg intraperitoneally) and local anesthesia bytopical application of oxybuprocaine (0.4%) hydrochloride inthe right and left eye of adult Brown Norway rats. The esti-mated final vitreal concentration of 1 �M N1 and NT was cal-culated by assuming an average vitreous chamber volume of 30�l. Peptide or control (3�l) was injected in the vitreous at day 0via a 30-gauge needle (Microlance, BD Biosciences) attached toa 10-�l Hamilton syringe under an operating microscope. Theneedlewas inserted through the sclera 1mmbehind the nimbuson the upper pole of the left eye to avoid contact with the lens.Animals with eyes that showed any experimental trauma(opacification of the lens or retinal hemorrhage) when checkedwith an ophthalmoscopewere excluded.Ninetymin after intra-vitreal injection, the rats were maintained under anesthesia byboosts with 50% of the initial dose of anesthetic and placed in astereotaxic frame. After topical instillation of a drop of oxybu-procaine (Laboratoire Chauvin, Montpellier, France), the ante-rior chamber of the right eye was cannulated with a 30-gaugeneedle connected to a reservoir containingHanks’ balanced saltsolution. The left eye served as control. Retinal ischemia wasinduced by increasing the intraocular pressure to 130 mm Hg(28). The increased intraocular pressure was maintained for 45min. At this intraocular pressure, systolic collapse of the centralretinal artery was observed by direct ophthalmoscopy. Thedetailed preconditioning paradigm is diagrammed in Fig. 7a.During the experiment, the animals were kept normothermicwith heated jackets (38 °C). Animals were killed at day 2 or 7. Atleast three animals were used in each group of experimentalconditions. Sham-treated control right eyes underwent a simi-lar procedure but without the elevation of the saline bag, so thatthe normal ocular tension was maintained.Preparation of Retinal Sections—On day 2 or 7, three rats of

each group were dark-adapted for 4 h and were sacrificed withintraperitoneal injection of an overdose of sodium pentobarbi-tal. Eyes were removed, punctured at the limbus, and perfusedwith ice-cold 4% paraformaldehyde in PBS. After 15 min offixation, the cornea, the lens, and the vitreous were removed,and the eye cupswere cryoprotected in sucrose (20%) in PBS for1 h and then embedded into Tissue-Teck� (Sakura Finetek)under frozen isopentane. Frozen sections (10 �m) were next

prepared and frozen at �80 °C until use. Morphological andhistological analyses of the retinas were done by staining withcresyl violet (1%).p53 Immunohistochemistry—The 2-day retinal sections were

preincubated for 45 min in PBS containing 1:500 normal horseserum (Vector laboratories, Inc., Burlingame, CA). After wash-ing three times in PBS, sections were then incubated overnightin PBS containing p53 rabbit polyclonal antibody (1:30; FL-393,Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)) in a darkhumidified chamber maintained at 4 °C. After rinsing, sectionswere incubated with fluorescein isothiocyanate-conjugatedanti-rabbit IgG (1:10; Invitrogen) for 30 min in a dark humidi-fied chamber at room temperature and then with fluorescentDAPI stain for 10 min (1:20,000) and rinsed twice in PBS. Theslides were examined using a fluorescent Axioplan2 Imagingmicroscope (Zeiss).Electroretinography—Full-field electroretinogram (ERG) re-

sponses were obtained after overnight dark adaptation as pre-viously described (the rats were anesthetized with sodium pen-tobarbital (60 mg/kg intraperitoneally)) under dim red light(640 nm) and placed on a heating pad to maintain body tem-perature near 38 °C. Pupils were dilated with 2.5% Neosyneph-rine and 0.5% Mydriaticum, and corneas were kept moist withlocal application of 1% carboxymethylcellulose sodium (Cellu-visc; Allergan (Irvine, CA)). The scotopic ERG responses wererecorded with an ERG test system (UTAS 2000, LKC Technol-ogies (Gaithersburg, MD)). Treated and control animals weresubmitted to 2.98 log candelas/s/m2 flash intensity produced bya Grass PS 22 xenon flash positioned 15 cm from the eye. Theaveraged responses represent the mean of seven flashes deliv-ered 60 min apart. Electroretinograms were recorded beforetreatment and then at different times of recovery (1, 2, or 7days). The amplitude of the a-wave was measured from theprestimulus base line to the apex negative peak of the a-wave.The b-wave amplitude was measured from the a-wave negativepeak to the b-wave positive peak as described previously (29).Statistical Analysis—Statistical analysis was performed with

PRISM software (GraphPad Software, San Diego, CA) by usingthe Newmann-Keuls multiple comparison tests for one-wayanalysis of variance and t test.

RESULTS

Recombinant N1 Protects HEK293 Cells and Primary Cul-tured Neurons against Staurosporine-induced Apoptosis Inde-pendently of Endogenous PrPc—We have produced GST-N1fusion protein and released N1 peptide (residues 23–110 ofPrPc) by thrombin digestion. The molecular mass and integrityof the recovered peptide were confirmed bymass spectrometry(data not shown) and Western blotting using the SAF32 anti-body recognizing residues 79–92 of the PrPc N-terminaldomain (Fig. 1a). As expected, recombinant N1 fragment thatharbors additional amino acid residues adjacent to the throm-bin cleavage site on the pGEX-KG cloning vector (see Fig. 4a)migratesmore slowly than control N1 secreted by PrPc-overex-pressing cells (Fig. 1a). A first set of experiments examined theinfluence of recombinant N1 on STS-induced toxicity inHEK293 cells. Fig. 1b shows that STS triggers a 28.5% reductionof cell viability that was half-rescued by recombinant N1

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(15.25 � 1.90% (p � 0.05, n � 4) increase of N1-treated cellviability). RecombinantN1 triggered similar reductions of stau-rosporine-induced caspase-3 activation (27.22 � 6.46%, p �0.05, n � 6) (Fig. 1c) and TUNEL-positive nuclei (33.13 �7.68%, p � 0.05, n � 10) (Fig. 1d).We and others have reported that cells overexpressing full-

length PrPc (3, 5) or its C-terminal fragment C1 (16) displayenhanced responsiveness to proapoptotic stimuli, including

STS.Wehave therefore investigatedthe potential of recombinant N1 toprotect these cells from STS-in-duced cell death. As expected, PrPcand C1 overexpressions bothincrease STS-induced caspase-3activation (Fig. 1e, compare blackbars). Interestingly, recombinantN1 significantly reduced STS-evoked caspase-3 activation inHEK293 cells (34.8 � 12.8%, p �0.05, n � 4 and 30.5 � 3.5%, p �0.05, n � 4 in 3F4MoPrP- andC1-expressing cells, respectively).We examined the putative anti-

apoptotic function of N1 in primaryculturedmouse cortical neurons. Aswepreviously reported (4), wild typeprimary cultured embryonic corti-cal neurons exhibited an exacer-bated sensitivity to staurosporinewhen compared with PrPc-deficientneurons (see controls in Fig. 1, fand g). Clearly, recombinant N1 sig-nificantly and dose-dependentlylowered STS-induced caspase-3 acti-vation in both wild type and PrPc-deficient neurons (Fig. 1, f andg). In addition, overexpression ofPrPc or C1 fragment in primarycultured mouse cortical neuronsaugments STS-induced caspase-3activity (Fig. 1h, compare controlblack bars). Here again, recombi-nant N1 reduces caspase-3 activ-ity evoked by STS in DNA3,PrP, and C1-expressing neurons(36.27 � 13.40%, p � 0.05, n � 3;23.75 � 6.85%, p � 0.05, n � 5; and35.46 � 10.32%, p � 0.05, n � 6,respectively). Overall, the abovedata suggest that N1 could behaveas a soluble neuroprotective factorin various cell systems indepen-dently of endogenous PrPc.N1 Protective Function Does Not

Require N1 Internalization—TheN-terminal sequence of PrPc directsthe internalization of the molecule(30). We therefore looked at N1

translocation into HEK293 cells and primary cultured neu-rons and investigated whether such putative internalizationcould underlie its neuroprotective function. Kinetic analysis ofthe fate of recombinant N1 added in the cell media indicatesthat N1 is rapidly detected inside the cells. N1 translocationinto intracellular compartments peaked after 30 min and thenreturned to base line (Fig. 2a). This internalization was effi-ciently blocked by 0.45 M sucrose treatment as shown by immu-

FIGURE 1. Recombinant N1 fragment protects cells against staurosporine-induced apoptotic cell deathin HEK293 cells and mouse primary cultured cortical neurons. a, recombinant N1 fragment was producedin E. coli and purified as described under “Materials and Methods.” Secreted N1 was immunocaptured fromHEK293 supernatant as described under “Materials and Methods,” analyzed by 16.5% Tris/Tricine SDS-PAGE,and revealed by immunoblotting with SAF32. b– d, 1 �M recombinant N1 or an equivalent volume of superna-tant produced after thrombin digestion of GST-Sepharose beads (KG) was applied to HEK293 cells. After 4 h ofincubation, cells were treated again with N1 or KG, before being challenged with staurosporine (2 �M, 16 h) andprocessed for XTT measurement (b), caspase-3 activity determination (c), or TUNEL labeling (d). Bars in b and c,means � S.E. of 4 – 6 independent determinations with two technical replicas. Bars in d, means � S.E. of thenumber of labeled nuclei in 10 independent optical fields. e, caspase-3 activity in mock-transfected (Mock)HEK293, 3F4MoPrP, or C1-expressing cells treated with N1 or KG as detailed above. f and g, primary culturedcortical neurons were obtained from the indicated embryonic day 14 mouse embryos as described under“Materials and Methods” and treated as detailed above with increasing concentrations of N1, prior to stauro-sporine challenge (2 �M, 16 h) and measurement of caspase-3 activity. Bars, means � S.E. of 3– 4 independentdeterminations carried out in duplicate. h, caspase-3 activity measured in primary cultured mouse corticalneurons transfected with the cDNA of full-length PrP (PrP), C1 (PrPC1), or parental plasmid (DNA3). Forty-eighth post-transfection, cells were incubated with N1 (1 �M, 4 h) or an equivalent volume of KG. Cells were treatedagain with N1, challenged with staurosporine (2 �M, 16 h), and processed for caspase-3 activity determination.Bars, means of three independent determinations carried out in duplicate. Right, a representative Western blotusing SAF61 antibody of PrPc and C1 expression in cortical neurons after nucleofection. *, p � 0.05; **, p �0.001; ***, p � 0.0001; ns, not significant; IP, immunoprecipitation.

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noprecipitation (Fig. 2b) and in situ labeling (Fig. 2c) of N1 inHEK293 cells and HpL3–4 PrPc-deficient neurons, respec-tively. Blockade of N1 internalization with sucrose does notmodify the N1-induced reduction of STS-stimulated caspase-3activation (Fig. 2d). Furthermore, Dynasore, a pharmacologicalblocker of the dynamin-dependent internalization pathway(31), did not modify N1-dependent inhibition of STS-inducedcaspase-3 activation (Fig. 2e). Overall, the above results indicatethat N1 can translocate into the cytosol, that N1 translocationdoes not require endogenous PrPc, and that N1-associated pro-tective phenotype remains independent of its internalization.N1Down-regulates p53 Transcription—We examined whether

N1 could down-regulate p53 activity, promoter transactivation,andmRNA levels. We first measured p53 transcriptional activ-ity using a luciferase reporter construct (PG13) harboring thecanonical sequence to which p53 binds in its target gene pro-moters. Fig. 3a shows that N1 lowers p53 activity in HEK293cells (45.6 � 9.3% (p � 0.001, n � 6) reduction compared withvehicle-treated cells). This result was confirmed by means ofanother luciferase reporter construct bearing the promoter ofp21waf-1, a natural p53 target (32) (42.7 � 14.0% reduction, p �0.05, n � 4) (Fig. 3b). Importantly, both p53 promoter transac-tivation (Fig. 3c) and p53 mRNA levels (Fig. 3d) were also low-

ered by N1 (45.72 � 8.76%, p �0.001, n � 6 and 59.6 � 3.3% p �0.0001, n � 5 reduction for pro-moter transactivation and mRNAlevels, respectively).In addition to its key role in apo-

ptosis, p53 regulates senescence andcell cycle progression via the tumorsuppressor p19arf. In the absenceof p19arf, p53 apoptotic activityremains functional, whereas p53-mediated growth arrest is impaired.p19arf-deficient cells are thereforeuseful to investigate p53-dependentcell death activity without interfer-ence of cell cycle regulation (19).Interestingly, STS-induced caspase-3activity measurements in p19arf�/�

and p19arf�/�p53�/� fibroblastsindicate that both cell lines remainresponsive to STS although to alesser extent in double knock-out(Fig. 3e). N1 also protects p19arf�/�

cells from STS insult, whereasthe deletion of p53 fully abolishesthe effect of N1 on STS-inducedcaspase-3activation (Fig. 3e).Accord-ingly, the number of TUNEL-positivenuclei was significantly reduced byN1 treatment in p19arf�/� but notin p19arf�/�p53�/� fibroblasts (Fig.3, f and g). Altogether, these resultsindicate that N1-associated protec-tive function is fully dependent onp53.

N2 Does Not Display N1-associated Cytoprotective Func-tion—Recombinant N2 was produced (Fig. 4a) and tested forits capacity to reduce STS-induced caspase-3 activity andimpact on p53 activity and transcription. Interestingly, N2proved unable to protect cells from STS-induced caspase-3activation (Fig. 4b) and did not significantly modulate p53activity (Fig. 4, c and d), promoter transactivation (Fig. 4e), andmRNA levels (Fig. 4f). Other fragments, NT that is deleted ofthe first N-terminal amino acids 23–40 ofN1 and ending at 107and NK, which is as long as N1 but harbors mutations in the Nterminus (supplemental Fig. S1a), also appeared inert in theseparadigms (supplemental Fig. S1, c–f).To rule out a possible impact of C- and N-terminal deletions

on fragment stability, recombinant N1, N2, NT, and NK wereincubated on living cells for up to 24 h. Fig. 4g and supplementalFig. S1b clearly show that the distinct phenotypes exhibited byN2, NT, and NK compared with N1 cannot be ascribed to dis-tinct catabolic fates. The above data demonstrate that NT, NK,and transmissible spongiform encephalopathy-associated N2fragments fail to elicit cytoprotection and therefore that N1 C-and N-terminal integrity is required for its biological function.In order to determine if N2 could block the binding and activityof N1, we carried out experiments where N2 was preincubated

FIGURE 2. N1 translocation in intracellular compartment is not required for protective function. a, recom-binant N1 was added to HEK293 cell medium, and cells were collected at each indicated time point. Internal-ized N1 was immunocaptured from cell lysate using SAF32, as detailed under “Materials and Methods,” appliedon a 16.5% Tris/Tricine SDS-polyacrylamide gel, and probed with SAF32. Bars corresponding to densitometricanalysis of intracellular N1 show the means � S.E. of three independent experiments. b, HEK293 cells weretreated (�) or not (�) for 20 min at 37 °C with 0.45 M sucrose to block internalization. N1 was added for 30 mininto cell medium, and intracellular N1 (I) was immunocaptured from cell lysate. The first lane (Ct) correspondsto the amount of N1 recovered from the cell fluid when cells are maintained at 4 °C (note that upper bands showimmunoglobulin used for immunoprecipitation). c, HpL3– 4 PrP�/� neuronal cell line was seeded on glasscoverslips and cultured for 24 h. Internalization processes were blocked as described above, and N1 wasapplied to the cells for 30 min. Coverslips were then processed for N1 immunolabeling using SAF32 asdescribed under “Materials and Methods.” HEK293 cells were incubated or not with 0.45 M sucrose (Suc, d) orDynasore (Dyn, e) for 20 min at 37 °C, and then recombinant N1 (1 �M) or an equivalent volume of KG wasapplied on cells, and dishes were returned to 37 °C for 30 min before being challenged with staurosporine (2�M, 16 h). Caspase-3 activity was then determined as described under “Materials and Methods.” Bars, means �S.E. of three independent experiments in triplicate (d) and duplicate (e). *, p � 0.05; **, p � 0.01.

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prior to N1 or co-incubated with N1 and therefore used as aputative competitor. Our data show that, in both cases, N2 didnot interfere with the N1-associated protective phenotype(data not shown).Carbachol-stimulated Release of Endogenous N1 Protects

Cells against STS-induced Caspase-3 Activation—Physiologi-cal processing yielding endogenous N1 is increased upon car-bachol activation of endogenous muscarinic receptors M1 andM3 (33). We examined whether endogenous N1 could mimicthe protective p53-dependent phenotype elicited by recombi-nant N1. Fig. 5a confirms that carbachol treatment of HEK293cells expressing M1 muscarinic receptors increases the recov-ery of endogenous N1 that was poorly detectable in basal con-ditions (Fig. 5a, top). Interestingly, carbachol treatment alsotriggers a decrease of caspase-3 activity (29.18 � 4.90% (p �0.0001, n � 6) reduction) (Fig. 5a, bottom) that was fully re-versed by atropine (Fig. 5a). Three lines of data indicate thatcarbachol-associated protection is indeed due to endogenousN1 increase. First, in agreement with the results obtained withrecombinantN1 (Fig. 2d), blockade of endocytosis with sucrosedid not suppress carbachol-induced reduction of caspase-3activity (Fig. 5e). Second, we demonstrated that N1 protectivefunction involved the Akt pathway and was independent ofERK signaling (supplemental Fig. S2). Accordingly, the Akt

inhibitor LY294002 fully preventsthe carbachol effect on caspase-3(Fig. 5b). Third, carbachol reducesp53 activity (Fig. 5c) and promotertransactivation (Fig. 5d) in aLY294002-sensitive manner.It is important to consider that

the activation of M1 muscarinicreceptors also potentiates the lib-eration of neuroprotective solubleamyloid precursor protein � bystimulating the �-secretase cleav-age of the �-amyloid precursorprotein (34). To delineate the gen-uine contribution of endogenousN1 in carbachol-induced reduc-tion of caspase-3 activation, wecompared the effect of carbacholon wild type and PrPc-deficientprimary cultured cortical neurons.Carbachol treatment triggered asignificant reduction of caspase-3activation in wild type neurons(39.71 � 7.86%, p � 0.01, n � 4)(Fig. 5f), whereas the reductionwas statistically smaller (25.8 �4.2%, p � 0.05, n � 4) (Fig. 5f) inPrPc-deficient neurons, indicatingthat about half of the carbachol-induced reduction of caspase-3activation was indeed linked to thepresence of PrPc and therefore prob-ably attributable to M1-stimulatedliberation of endogenous N1.

N1 Protects against OGD in Primary Cultured Retinal Gan-glion Cells and Reduces Pressure-induced Ischemia Effects in theRat Retina—We examined whether N1 could protect primarycultured rat retinal ganglion cells (RGC) against OGD, an invitromodelmimicking ischemia-associated apoptosis. First,N1is recovered in the medium of RGC, and second, 2 h of OGDsignificantly increased the secretion of endogenous N1 (42.1 �11.2%, p� 0.05, n� 3), whereas the total level of PrPc remainedunchanged (Fig. 6a). Twenty-four h post-OGD, recombinantN1 increased cell viability (Fig. 6b) and reduced hypoxia-in-duced caspase-3 activation (Fig. 6c) and the number of TUNEL-positive cells (Fig. 6d) (reductions of 29.92 � 12.22% (p � 0.05,n � 3), 42.82 � 2.82% (p � 0.05, n � 3), and 51.5 � 4.6% (p �0.05, n � 3), respectively).The use of the rat eye as a read out of putative biological

function of N1 was based on five lines of considerations anddata. First, the retina is an open window to the central nervoussystem; second, a previous study indicated that PrPc depletionincreased the photoreceptors susceptibility to light damage;third, the retina’s vulnerability to transient ischemia is associ-ated with increased apoptotic neuronal stigmata; fourth, tran-sient ischemia-induced apoptosis involves up-regulation ofp53 expression; fifth, muscarinic receptors convey protectivesignals against the cytotoxicity triggered by intraocular pres-

FIGURE 3. N1 antiapoptotic activity is strictly p53-dependent. PG13-luciferase (a), p21waf-1-luciferase (b), orp53 promoter-luciferase (pp53) (c) reporter constructs were transiently transfected in HEK293 cells togetherwith �-galactosidase-expressing vector, as detailed under “Materials and Methods.” Twenty-four h after trans-fection, cells were treated for 16 h with recombinant N1 (1 �M) or an equivalent volume of control supernatant(KG). The luciferase and �-galactosidase activities were measured as detailed under “Materials and Methods.”Bars correspond to the ratio of luciferase/�-galactosidase and show the means � S.E. of 3– 6 independentexperiments performed in triplicate. d, cells treated with recombinant N1 (1 �M) or KG for 8 h were analyzed forlevels of p53 mRNA by real time PCR as indicated under “Materials and Methods.” Bars correspond to p53 mRNAlevels expressed as a percentage of control-treated cells and are the means � S.E. of three independentexperiments carried out in duplicate. e, recombinant N1 (1 �M) or equivalent volume of KG were applied top19arf�/� and p19arf�/�p53�/� fibroblasts. After 4 h of incubation, cells were treated with N1 or KG beforebeing challenged with staurosporine (1 �M, 2 h) and processed for caspase-3 activity measurement. Bars,means � S.E. of five independent experiments performed in duplicate. p19arf�/� and p19arf�/�p53�/� cells (f),seeded on glass coverslips, were treated as in e and then processed for TUNEL labeling as described under“Materials and Methods.” f, representative pictures of TUNEL labeling of control (KG) and N1-treated p19arf�/�

cells. g, TUNEL-positive nuclei were counted for 10 independent optical fields. Bars, means � S.E. of labelednuclei/field. *, p � 0.05; **, p � 0.001; ***, p � 0.0001.

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sure. Our demonstration that muscarinic receptor-mediatedproduction of endogenous N1 could lower p53-dependentcaspase-3 activation led us to envision that pressure-inducedischemia could represent a relevant model to establish N1potential, in vivo. The protocol of injection, ischemia, andreperfusion is depicted in Fig. 7a. Fig. 7b clearly shows that N1but not N2 reduced the number of apoptotic cells by about 65%48 h after ischemia. Microphotographs showing fluoresceinandDAPI overlays confirmed thatmost of the TUNEL-positivecells were located in the ganglion cell layer but also to a lesserextent in the inner nuclear layer (Fig. 7c) and that inactive frag-ments N2 (Fig. 7) and NT (supplemental Fig. S3a) did notreduce cell death. ERGs were monitored at 1, 2, and 7 daysfollowing ischemia. ERG traces show that an ischemic insult of45 min decreased the amplitudes of both a- and b-waves oftreated and non-treated rats at all time periods after reperfu-sion (Fig. 7d). Clearly, b-waves but not a-waves were partiallyrescued by N1 but not by N2 (Fig. 7d) and NT (supplementalFig. S3b). Thus, at days 1 and 2 after reperfusion (Fig. 7d), theamplitudes of b-waves in the N1-treated group (32 � 5 and45 � 7%, respectively) were significantly larger (p � 0.05;Mann-Whitney U test) than those of untreated (14.5 � 6 and

19 � 8%, respectively), vehicle-treated (11 � 9 and 15 � 6%,respectively), N2-treated (17 � 12 and 20 � 9%, respectively),and NT-treated (6 � 7 and 15 � 5%, respectively) groups (sup-plemental Fig. S3b). Seven days after recovery when the N1-as-sociated effect on b-waves was maximal (Fig. 7d), retina sliceswere coloredwith cresyl violet to examine their architecture.Asexpected, ischemia induced a pronounced disorganization ofthe retina associated with a cell loss leading to a thinning of allretina layers (Fig. 7e). In N1-injected animals, the retina struc-ture is conserved, and the thickness of the layers is partly res-cued (Fig. 7e), whereas vehicleN2 (Fig. 7e) orNT (supplementalFig. S3c) injections neither prevent the structural disorganiza-tion triggered by ischemia nor rescue retina thickness. Overall,biological data showing important recovery of retina structureand function agree well with the observed protective effect ofN1 on ischemia-induced neuronal cell death. According to ourprevious data obtained in cellulo, one should predict that N1could protect retinal neurons by down-regulating p53, in vivo.First, as expected from previous studies (35), pressure-inducedischemia drastically increased p53-like immunoreactivity incontrol conditions (Fig. 8, a and b). Of greatest interest, 48 hafter ischemia, N1-treated but not vehicle N2- or NT-treated

FIGURE 4. The pathological fragment N2 does not exhibit protective activity. a, schematic representation of the sequences of the various recombinantPrPc-derived N-terminal fragments and their analysis with SAF32 monoclonal antibody (see “Materials and Methods”). Residues shown in boldface type arepresent on endogenous fragments, whereas lightface residues come from the PGEX-KG constructs and are present only on recombinant peptides. Theseadditional residues explain the shift in the migration of fragment N1 recombinant compared with endogenous N1 (see Fig 1a). b, HEK293 cells were treated for4 h with recombinant N1 or N2 fragments (1 �M) or an equivalent volume of control supernatant (KG). Peptides were applied again to the cells before cell deathinduction with staurosporine (2 �M, 16 h). Cells were recovered, lysed, and processed for caspase-3 activity measurement as detailed under “Materials andMethods.” Bars, means � S.E. of four independent experiments carried out in duplicate. PG13-luciferase (c), p21waf-1-luciferase (d), and pp53-luciferase (e)reporter constructs were transiently transfected in HEK293 cells together with �-galactosidase-expressing vector, as detailed under “Materials and Methods.”Twenty-four h after transfection, cells were treated for 16 h with the indicated recombinant fragments (1 �M) or KG. Luciferase and �-galactosidase activitieswere measured as detailed under “Materials and Methods.” The bars correspond to the ratio of luciferase/�-galactosidase and are the means of 3– 6 indepen-dent experiments performed in triplicate. f, confluent HEK293 cells were treated for 8 h with N1, N2 (1 �M), or KG and then monitored for p53 mRNA levels asdetailed under “Materials and Methods.” Bars, means of three independent experiments carried out in duplicate. g, recombinant N1 or N2 was added intoHEK293 cell medium, and then fluids were collected at the indicated time points, and fragments were immunoprecipitated using SAF32 as detailed under“Materials and Methods.” Immunocomplexes were applied on a 16.5% Tris/Tricine SDS-polyacrylamide gel and probed with SAF32. Plots correspond todensitometric analysis of extracellular fragments and show the means � S.E. of three independent experiments. *, p � 0.05; **, p � 0.001.

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eyes exhibited drastically lowered p53 expression (Fig. 8, a andb, and supplemental Fig. S3, d and e). Overall, our data showthat N1 contributed to reduce p53-dependent cell death trig-gered by pressure-induced ischemia and partly rescued retinafunction in vivo.

DISCUSSION

Although several functions have been proposed for PrPc,its genuine physiological roles remained elusive and oftendebated. Several studies suggested an implication in cell sur-vival (for a review, see Ref. 1), whereas, apparently contradict-ing this view, others and we have reported that overexpressedand endogenous PrPc could sensitize cells to proapoptotic stim-uli in several distinct experimental systems involving trans-formed cells and primary cultured neurons (2, 4–7).

One important aspect neglectedwhen considering the PrPc biologyis the fact that PrPc undergoes aset of physiological proteolyticcleavages. Thus, we demonstratedthat PrPc undergoes constitutiveand protein kinase C-regulated hy-drolysis between its residues 110and 111 (10–12). This processingleads to a 9-kDa soluble fragment,referred to as N1, released in theextracellular environment, and itsC-terminal domain counterpart(C1) that remains tethered to theplasma membrane and that wasshown to be generated in vivo (9).The putative influence of this

proteolysis has remained poorlyunderstood. Can it be consideredas a degradationmechanismaimed atclearing full-length PrPc, therebyimpairing its associated function, orcan it be seen as a maturation pro-cess yielding biologically activemetabolites? In the latter case, dothese metabolites account for pro-posed PrPc-associated functions, oralternatively, do they harbor theirown physiological function? If so, isthis function unrelated to the oneassociated with full-length PrPc, ordoes it interfere with it?Our first report showing that the

overexpression of C1 potentiatedstaurosporine-induced caspase-3 ac-tivation through a p53-dependentmechanism (16), as we reported forfull-length PrPc (2), suggested thatsome of the PrPc catabolites couldharbor a functionmimicking that ofthe parent protein. However, nodata were yet available concerningthe secreted N1 product, and evi-

dence that PrPc catabolites could also exhibit their function invivo still awaited demonstration.To our knowledge, our study is the first direct demonstration

of a biological function associatedwith the soluble PrPc-derivedN1 fragment in cells. Thus, we show that recombinant N1attenuates STS-evoked caspase-3 activation and reduces thenumber of TUNEL-positive nuclei in cell cultures by down-regulating the p53 pathway in anAkt-dependent but ERK-inde-pendentmanner. It is interesting to note that the stimulation ofmuscarinic receptors by carbachol, which increases the recov-ery of endogenously secreted N1, also triggers Akt-dependentneuroprotection. Serum-deprived PC12 cells were protectedfrom cell death by M1 stimulation (36), whereas M1 agonistsprotect from DNA damage, oxidative stress, or mitochondrialimpairment in neurons (37). Apparently, M1-mediated neuro-

FIGURE 5. Carbachol-stimulated release of endogenous N1 protects cells against staurosporine-inducedcaspase-3 activation. a, HEK293 cells overexpressing muscarinic receptor M1 (HEK-M1) were treated withcarbachol (100 �M) and/or atropine (10 �M) as indicated, simultaneously treated with staurosporine (2 �M), andincubated for 16 h before being processed for caspase-3 activity measurement. Bars show caspase-3 activityexpressed as a percentage of control and show the means � S.E. of six independent experiments carried out induplicate. Inset, representative Western blot analyses of endogenous N1 immunoprecipitation from the cellmedium and total PrPc expressed in the cells, detected with SAF32 antibody. Tubulin is shown as control of theamount of protein loaded. b, HEK-M1 cells were treated as in a in the absence (�) or in the presence (�) ofLY294002 (10 �M). After 16 h of incubation at 37 °C, caspase-3 activity was measured (see “Materials andMethods”). Bars represent caspase-3 activity expressed as a percentage of staurosporine-treated cells andshow the means � S.E. of six independent experiments carried out in duplicate. HEK-M1 cells were transientlytransfected with PG13-luciferase (c) or pp53-luciferase (d) reporter construct together with �-galactosidase-expressing vector as described under “Materials and Methods.” Twenty-four h post-transfection, cells weretreated for 8 h with carbachol (100 �M) and LY294002 (10 �M) as indicated. Luciferase and �-galactosidaseactivities were measured. Bars correspond to the ratio of luciferase/�-galactosidase expressed as a percentageof untreated cells and show the means � S.E. of 3– 6 independent experiments performed in triplicate. e, cellswere incubated with 0.45 M sucrose and processed as in a. Bars show the means of three independent exper-iments carried out in duplicate. f, wild type or PrP�/� primary cortical neurons were obtained from embryonicday 14 mouse embryos as described under “Materials and Methods.” Neurons were treated as detailed in a andthen processed for caspase-3 activity measurement. Bars represent caspase-3 activity expressed as a percent-age of staurosporine-treated cells and are the means � S.E. of 3– 4 independent experiments carried out inquadruplet. *, p � 0.05; **, p � 0.001; ***, p � 0.0001. ns, not significant.

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protection involves Akt-dependent and extracellular signal-regulated kinase-independent cell survival pathways (38, 39).Altogether, the above studies suggest that carbachol-stimu-lated production of endogenous N1 triggers a neuroprotectivephenotype that strikingly resembles the one obtained withrecombinantN1, suggesting that carbachol-inducedphenotypecould be at least partly mediated by endogenous N1. Thishypothesis is strongly supported by our demonstration thatPrPc deficiency partly rescued carbachol-induced reduction ofcaspase-3 activity in neurons (Fig. 5f).A question arises as to whether N1 could account for the

protective phenotype elicited by PrPc in certain experimental

conditions. Two lines of evidencesuggest that PrPc protective func-tion is transduced by the phospha-tidylinositol 3-kinase/Akt signalingpathway. First, it has been shownthat PrPc can recruit phosphatidyl-inositol 3-kinase/Akt, thereby con-tributing to cell survival (40). Sec-ond, it has recently been suggestedthat PrPc deletion in mice results inaggravation of neuronal injury aftermild focal cerebral ischemia (41).PrPc deletion was later shown toimpair the antiapoptotic phospha-tidylinositol 3-kinase/Akt path-way, resulting in a reduced postis-chemic phospho-Akt expressionand aggravation of neuronal injury(42). These results are in goodagreement with our finding of a sur-vival signal triggered by N1 throughphosphatidylinositol 3-kinase sig-naling pathway and suggest that, inischemic injury, the protective phe-notype of PrPc could be due to therelease of N1 after PrPc cleavage bydisintegrins.N1 also protects primary cultured

retinal ganglion cells from OGD bymodulating cell death. These datafirst show that the N1 protectivephenotype is not restricted to STS-challenged stimulus. It was also veryimportant to correlate cell biologyapproach and in vivo experiments.Thus, we examined the potential ofN1 to protect rat retina from pres-sure-induced ischemia. It is welladmitted that ischemia mainly re-sults from oxygen deprivation andglucose reduction. Interestingly, weclearly established that N1 protectsretina neurons from cell death asso-ciated with pressure-induced ische-mia, as shown by the lowering ofTUNEL-positive neurons, in situ.

This protection was accompanied by a strong reduction in thenumber of p53-positive neurons and by a preservation of theretina architecture. Finally, of utmost importance was ourobservation that N1-induced protection indeed has a func-tional consequence because N1 partly rescued alterationsobserved in ERG traces upon pressure-induced ischemia. Thisis the very first demonstration of a biological function harboredby a PrPc processing product in vivo. Interestingly, OGD aug-ments the recovery of secreted endogenous N1. It is thereforetempting to speculate on a compensatory mechanism aimed atreducing hypoxia-induced stress via the up-regulation of N1production.

FIGURE 6. N1 protects rat RGC against OGD. a, RGC were subjected or not to OGD for 2 h (see “Materials andMethods”) and then returned to complete medium for 24 h. Medium was collected, and N1 was immunopre-cipitated (IP) with the monoclonal antibody SAF32 and analyzed by 16.5% Tris/Tricine SDS-PAGE and Westernblotting using SAF32. RecN1 and RecN2 correspond to the migration pattern of recombinant N1 and N2 (150ng) analyzed under the same conditions. Bars correspond to the densitometric analysis of N1 and are expressedas a percentage of control N1 recovered under normoxic conditions. Values are the means � S.E. of threeindependent experiments. b, 7 days after dissociation, RGC were treated with N1 (2 �M, 4 h) or an equivalentvolume of KG, cells were then retreated and placed under normoxic or hypoxic conditions as in a, viability wasdetermined just before return to complete medium or after 24 h by measurement of release of lactate dehy-drogenase in the culture medium as described under “Materials and Methods.” c, caspase-3 activity was mea-sured 24 h posthypoxia. d, cells plated on coverslips were treated as in a and processed for TUNEL staining. Barsrepresent the means � S.E. of three independent determinations carried out in duplicate. *, p � 0.05.

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Our data could explain the phenotypes observed in severaltransgenic mice. Thus, PrPc deleted of residues 32–121 and32–134 (collectively called �PrP) has been reported to cause

progressive neurodegenerative ill-ness (13). This phenotype is abol-ished when �PrP animals arebackcrossed with wild type mice.On the other hand, the deletion of23–88 N-terminal amino acidsabrogates the ability of PrPc to res-cue mice from �PrP-induced tox-icity (43).Another important aspect of our

study lies with our observation thatthe N1-associated protective func-tion requires the integrity of theN-terminal moiety, both in vitroand in vivo. Thus, NT and NK frag-ments (see supplemental Fig S1a)do not protect cells from staurospo-rine-induced cell death and fail toreduce pressure-induced ischemiain the retina of live animals. Thisagrees well with a recent reportshowing that residues 23–31 areessential for PrPc to protect cerebel-lar granule neurons against �PrPtoxicity and Dpl-induced degenera-tion in mice (44). In the same line ofreasoning, the examination of thesequence domain of PrPc importantfor cytoprotective activity in yeastrevealed that the 23–31 sequencewas crucial for protection againstBax-induced apoptosis (45). It isnoteworthy that the 23–31 motifhas been shown to be important forboth endocytosis (23, 46, 47) andbinding to transmembrane recep-tors (48, 49). However, our datashowing that N1 could indeed beendocytosed but that its protectivephenotype was not prevented byblockade of endocytosis (Figs. 2, dand e, and 5e) or mutations abolish-ing the intracellular translocation(data not shown) strongly suggestthat N1-associated protective func-tion was independent of its endocy-tosis. Alternatively, one could spec-ulate that N1 acts as a soluble factorthat would signal after interactingwith a yet unknownbinding protein.This hypothesis is supported by arecent report showing that the invitro PrP-DA construct inwhich theN terminus is tethered to the mem-brane and consequently not re-

leased in the extracellular space fails to protect cells againstparaquat-induced oxidative injury in neuronal cells (50). Theseresults are in accordance with the need of N-terminal process-

FIGURE 7. N1 protects retina against morphological and functional alterations induced by ischemia. a, sche-matic representation of preconditioning and ischemia procedures. Rats were injected in the vitreous humor withN1, N2 (1 �M final concentration), or KG 90 min before induction of 45-min pressure-induced ischemia. b, quantifi-cation of TUNEL-positive cells in retinas 48 h after ischemia. Bars correspond to percentage of total DAPI-stainednuclei. Counts were performed on six retina sections of the same animal for two independent optical fields. For eachcondition, two animals were analyzed. *, p � 0.01; **, p � 0.001; ***, p � 0.0001. c, representative microphotographsshowing overlay of fluorescein (green) and DAPI (blue) immunostained nuclei in retina slices. In sections, fluoresceinstained the nuclei of apoptotic cells using the TUNEL labeling method. Labeled nuclei appear in ganglion cell andinner nuclear layers. d, ERG recording done as described under “Materials and Methods” after 24 h, 48 h, or 7 days ofrecovery for corresponding representative scotopic ERG traces recorded 24 h (dark line), 48 h (red line), and 7 days(green line) after the end of ischemia and compared with sham control (blue line) obtained for non-ischemic eye.e, histology of the ischemic untreated, N1-, N2-, or KG-treated retinas after 7 days of recovery compared with controlretina, stained by 1% cresyl violet. Specimens were visualized in light microscopy. GCL, ganglion cell layer; IPL, innerplexiform; INL, inner nuclear; OPL, outer plexiform; ONL, outer nuclear; IS, inner segments of rods; OS, outer segmentsof rods and cones. *, p � 0.05; **, p � 0.001; ***, p � 0.0001; ns, not significant.

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ing and subsequent release in the extracellular matrix for PrPcto exert its antiapoptotic activity.We did not yet delineate the molecular intermediate

involved in N1-protective function. However, since N1 func-tion does not involve the ERK1/2 pathway (see above), we couldalready eliminate STSI1 (stress-inducible protein 1) as a candi-date because a recent paper indicated that the PrPc-associatedSTI1-mediated pathway involved ERK1/2 signaling (51).The fact that N2 lacking the 21 last C-terminal amino acids is

inert in all of the tested paradigms suggests that the C-terminalpart of N1 could also be of importance for its neuroprotectiveactivity. However, NK that harbors a strictly identical C termi-nus is inactive. Therefore, one could consider that the deletionof the C-terminal end of N2 may have changed its conforma-tion, thereby leading to the loss of neuroprotective function.Our previous (2, 16) and present works demonstrated that

full-length PrPc and its �-secretase-derived C1 metaboliteexacerbated STS-induced caspase-3 activation and cell deathby up-regulating the p53 pathway. Consequently, one shouldconsider that two PrPc-derived catabolites (i.e.C1 andN1) pro-duced by the same proteolytic attack could display oppositephenotypes and even cross-talk for such a function. Our resultsshowing that recombinant N1 protects cells overexpressingC1 or PrPc full-length from apoptosis suggested that, oncereleased in the extracellular space, N1 protective functionpredominates over the C1-associated proapoptotic pheno-

type. Indeed, when cleavage ofendogenous PrPc yielding equimo-lar amounts of N1 and C1 wasamplified by muscarinic receptorstimulation, the overall phenotypewas that triggered by N1, indicatingthat in these conditions, N1-associ-ated protective function was clearlydominant over the C1-associatedtoxic effect. It is tempting to specu-late that when PrPc cleavage is exac-erbated by various stimuli, includ-ing muscarinic receptor activation,N1 liberation accounts for apparentPrPc-associated antiapoptotic phe-notype, whereas PrPc is biologicallyinert in basal conditions and evencontributes to exacerbate cell deathwhen cells are challenged by variousproapoptotic stimuli.Investigating the role of proteo-

lytic processing and its conse-quences on the function of PrPc is ofgreat importance because the cleav-age is modified in pathological situ-ations. Thus, in infected brains, N1production is conserved, but anadditional cleavage occurs to giverise to the shorter fragment N2 (9,52). The question of transmissiblespongiform encephalopathies beingin part due to a loss of function or a

gain of toxic function is still open. In this regard, our resultsshowing that N2 does not exhibit any protective activity are ofgreat interest and are in accordance with a study by Lee et al.(53) showing that fusion of PrP-(1–124) toDpl (doppel) confersresistance to serum deprivation, whereas fusion construct PrP-(1–95)-Dpl that resembles N2 does not. This was confirmed invivo. Thus, Baumann et al. (54) showed that a chimeric proteinin which the amino-terminal 1–134 sequence of PrPc wasgrafted onto Dpl exerts a neurotrophic function. Importantly,Cronier et al. (55) have recently reported that PrPsc-infectedprimary neurons undergo apoptosis.Whether N1 but not N2 isable to protect infected cells from cell death and slow thedegenerative process will have to be addressed. Moreover, ourresults suggest thatmodulation of desintegrin cleavage could bebeneficial. This strategy has been tested in vivowith success forAlzheimer disease (56, 57).Finally, our data show that even a rather small modulation of

N1 production bymuscarinic stimulation could prove useful totrigger neuroprotection. It is interesting to note that the stim-ulation of soluble amyloid precursor protein � production bygenetic manipulation of the �-secretase ADAM10 lowers anti-body load and rescues cognitive deficits in Alzheimer diseaseanimal models (58). This study and our data open an avenue toexperiments aimed at establishingwhether themanipulation ofN1 could also be beneficial in prion-infected mice. Anotheraspect is correlated to the striking parallel between PrPc/N1

FIGURE 8. Ischemia-induced increase of p53 expression is diminished by N1 pretreatment. a, representa-tive microphotographs showing overlay of p53 immunoreactivity (green) and DAPI-immunostained nuclei(blue) in retina slices, 48 h after pressure-induced ischemia. Ischemic untreated sections; N1-, N2-, or KG-treatedsections; and correspondent control sections are compared. b, quantification of p53-positive cells expressed asa percentage of those obtained in sham control conditions � S.E. of 4 –10 independent sections.

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and�-amyloid precursor protein/A�. Currently, work is in pro-gress in our laboratory to establish a putative protective effectof N1 toward A�-induced toxicity, thereby reinforcing thestrong network, suggesting an intimate cross-talk between PrPcand �-amyloid precursor protein (59).

Acknowledgments—We are grateful to Drs. M. Roussel (Memphis,TN), G. Pages (Nice, France), B. Slack (Boston, MA), and T.Onodera (Tokyo, Japan) for providing cells; Drs. M. Oren and B.Vogelstein for supplying PG13, p21, and p53 promoter reporterconstructs; and Dr. E. Macia (Valbonne) for Dynasore. We sin-cerely thank Dr. M. Ettaiche for advice in in vivo studies.

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Frédéric CheclerMarie-Victoire Guillot-Sestier, Claire Sunyach, Charlotte Druon, Sabine Scarzello and

in Vivo and in VitroNeuroprotective Function -Secretase-derived N-terminal Product of Cellular Prion, N1, DisplaysαThe

doi: 10.1074/jbc.M109.051086 originally published online October 22, 20092009, 284:35973-35986.J. Biol. Chem. 

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