Cellular Prion Protein Sensitizes Neurons to Apoptotic Stimuli through Mdm2-regulated and p53-dependent Caspase 3-like Activation
2003; Elsevier BV; Volume: 278; Issue: 12 Linguagem: Inglês
10.1074/jbc.m211580200
ISSN1083-351X
AutoresErwan Paitel, Robin Fåhræus, Frédéric Checler,
Tópico(s)Trace Elements in Health
ResumoWe examined the influence of cellular prion protein (PrPc) in the control of cell death in stably transfected TSM1 cells. PrPc expression enhanced staurosporine-stimulated neuronal toxicity and DNA fragmentation, caspase 3-like activity and immunoreactivity, and p53 immunoreactivity and transcriptional activities. Caspase activation was reduced by the chemical inhibitor of p53, pifithrin-α, as well as by PrPc- or p53-antisense approaches but remained insensitive to the Fyn kinase inhibitor PP2 (4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine). We establish that PrPc controls p53 at a post-transcriptional level and is reversed by Mdm2 transfection and p38 MAPK inhibitor. We propose that endogenous cellular prion protein sensitizes neurons to apoptotic stimuli through a p53-dependent caspase 3-mediated activation controlled by Mdm2 and p38 MAPK. We examined the influence of cellular prion protein (PrPc) in the control of cell death in stably transfected TSM1 cells. PrPc expression enhanced staurosporine-stimulated neuronal toxicity and DNA fragmentation, caspase 3-like activity and immunoreactivity, and p53 immunoreactivity and transcriptional activities. Caspase activation was reduced by the chemical inhibitor of p53, pifithrin-α, as well as by PrPc- or p53-antisense approaches but remained insensitive to the Fyn kinase inhibitor PP2 (4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine). We establish that PrPc controls p53 at a post-transcriptional level and is reversed by Mdm2 transfection and p38 MAPK inhibitor. We propose that endogenous cellular prion protein sensitizes neurons to apoptotic stimuli through a p53-dependent caspase 3-mediated activation controlled by Mdm2 and p38 MAPK. cellular prion protein mitogen-activated protein kinase antisense propidium iodide (4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) staurosporine sodium 3′-[1-(phenylamino)carbonyl-3,4-tetrazolium]-bis-(4-methoxy-6-nitro)benzene sulphonic acid hydrate Prion-associated pathologies are transmissible diseases that all have fatal issues (1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5168) Google Scholar, 2Aguzzi A. Montrasio F. Kaeser P.S. Nat. Rev. Mol. Cell. Biol. 2001; 2: 118-126Crossref PubMed Scopus (133) Google Scholar). The elucidation of their etiology has revealed a fully original transmission mechanism. It is now generally admitted that the pathological infectious mechanism is due to a protein present ubiquitously in the brain (referred to as PrPc)1 that becomes deleterious after its biophysical conversion into a protease-resistant protein referred to as PrPres or PrP-scrapie (3Ghetti B. Piccardo P. Frangione B. Bugiani O. Giaccone G. Young K. Prelli F. Farlow M.R. Dlouhy S.R. Tagliavni F. Brain Pathol. 1996; 6: 127-145Crossref PubMed Scopus (172) Google Scholar). This "bio-transformation" and subsequent pathologies absolutely require the presence of PrPc because the absence of endogenous PrPc totally precludes the PrPsc-mediated infectivity and neurotoxicity (4Büeler H. Aguzzi A. Sailer A. Greiner R. Autenried P. Aguet M. Weissmann C. Cell. 1993; 73: 1339-1347Abstract Full Text PDF PubMed Scopus (1814) Google Scholar, 5Brandner S. Isenmann S. Raeber A. Fischer M. Sailer A. Kobayashi Y. Marino S. Weissmann C. Aguzzi A. Nature. 1996; 379: 339-343Crossref PubMed Scopus (721) Google Scholar). Most of the works on prions have focused on these intriguing disease-related features, particularly their conformation propertiesin vitro (6Telling G.C. Parchi P. DeArmond S.J. Cortelli P. Montagna P. Gabizon R. Mastrianni J. Lugaresi E. Gambetti P. Prusiner S.B. Science. 1996; 274: 2079-2082Crossref PubMed Scopus (757) Google Scholar, 7Safar J. Wille H. Itri V. Groth D. Serban H. Torchia M. Cohen F. Prusiner S. Nat. Med. 1998; 4: 1157-1165Crossref PubMed Scopus (1081) Google Scholar, 8Lawson V.A. Priola S.A. Wehrly K. Chesebro B. J. Biol. Chem. 2001; 276: 35265-35271Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) and the species barrier allowing or limiting the propagation of the various prion strains (for reviews see Refs. 1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5168) Google Scholarand 2Aguzzi A. Montrasio F. Kaeser P.S. Nat. Rev. Mol. Cell. Biol. 2001; 2: 118-126Crossref PubMed Scopus (133) Google Scholar). The invalidation of the PrP gene (Prnp) led to the conclusion that the Prnp −/− mice exhibit normal development and unaltered behavioral phenotype (9Bueler H. Fischer M. Lang Y. Bluethmann H. Lipp H. DeArmond S. Prusiner S.B. Aguet M. Weissmann C. Nature. 1992; 356: 577-582Crossref PubMed Scopus (1441) Google Scholar,10Mallucci G.R. Ratté S. Asante E.A. Linehan J. Gowland I. Jefferys J.G.R. Collinge J. EMBO J. 2002; 21: 202-210Crossref PubMed Scopus (327) Google Scholar). Several clues of a possible participation of PrPc in programmed cell death came from the identification of a significant but restricted sequence homology of PrPc octapeptide repeats with the anti-apoptotic oncogene Bcl-2 protein and its ability to bind to Bcl-2 in double hybrid approach (11Yin X.-M. Oltvai Z.N. Korsmeyer S.J. Nature. 1994; 369: 321-323Crossref PubMed Scopus (1221) Google Scholar, 12Kurschner C. Morgan J.I. Mol. Brain Res. 1995; 30: 165-168Crossref PubMed Scopus (150) Google Scholar). A recent study (13Bounhar Y. Zhang Y. Goodyer C.G. LeBlanc A. J. Biol. Chem. 2001; 276: 39145-39149Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar) also indicated that prion protein could rescue human neurons from Bax-induced apoptosis. Alternatively, the possibility that a restricted sequence corresponding to the 106–126 part of the molecule could elicit a cellular toxic response has also been extensively documented (14Forloni G. Angeretti N. Chiesa R. Monzani E. Salmona M. Bugiani O. Tagliavini F. Nature. 1993; 362: 543-546Crossref PubMed Scopus (896) Google Scholar, 15Brown D.R. J. Neurochem. 1999; 73: 1105-1113Crossref PubMed Scopus (93) Google Scholar, 16Jobling M.F. Stewart L.R. White A.R. McLean C. Friedhuber A. Maher F. Beyreuther K. Masters C.L. Barrow C.J. Collins S.J. Cappai R. J. Neurochem. 1999; 73: 1557-1565Crossref PubMed Scopus (152) Google Scholar). It is noteworthy that the 106–126 domain of the prion protein has no genuine physiological or pathological existence as it has never been described as a catabolite of PrPc that would have been proteolytically generated upon normal or pathological conditions. Therefore, the "toxic" potential of the 106–126 peptide could either be not relevant to any normal or altered situation or, alternatively, could reflect a phenotype that would be also triggered by the whole parent prion protein. The latter hypothesis would be in contradiction with the above studies, suggesting an anti-apoptotic PrPc-related phenotype, but would be in line with the work of Westaway et al. (17Westaway D. DeArmond S.J. Cayetano-Canlas J. Groth D. Foster D. Yang S.-L. Torchia M. Carlson G.A. Prusiner S.B. Cell. 1994; 76: 117-129Abstract Full Text PDF PubMed Scopus (293) Google Scholar), showing that transgenic mice over-expressing "normal" wild type PrPcexhibit degeneration in central and peripheral nervous system as well as in skeletal muscle. Interestingly, the 106–126 domain appears to be targeted by a set of proteolytic activities called disintegrins, ADAM10 and ADAM17, that cleave PrPc at the 110/111 peptide bond and thereby "inactivate" the putative toxic core of the PrPc protein (18Vincent B. Paitel E. Frobert Y. Lehmann S. Grassi J. Checler F. J. Biol. Chem. 2000; 275: 35612-35616Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). To establish a pro- or anti-apoptotic PrPc-mediated phenotype, we have examined the possible toxicity of PrPc and its putative involvement in the control of cell death. We show that in TSM1 cells, over-expressed and endogenous PrPc all trigger p53-dependent caspase 3 activation leading to a pro-apoptotic phenotype. Murine TSM1 neuronal cells (19Chun J. Jaenisch R. Mol. Cell. Neurosci. 1996; 7: 304-321Crossref PubMed Scopus (61) Google Scholar) stably expressing mouse 3F4-tagged MoPrPc (3F4MoPrPc) were obtained and cultured as previously described (18Vincent B. Paitel E. Frobert Y. Lehmann S. Grassi J. Checler F. J. Biol. Chem. 2000; 275: 35612-35616Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). PrPc- or p53-antisense cDNAs (referred to as ASPrP or ASp53, respectively) were obtained after transfection of 1 μg of antisense pcDNA3 vector bearing 3F4MoPrPc or p53 with DAC30 (Eurogentec). Doubly transfected 3F4MoPrPc/ASp53 TSM1 cells were obtained after transfection of 3F4MoPrPc-expressing cells with ASp53 cDNA. p53-inactive cells were obtained from ATCC (NCI-H1299 clone) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cells were scraped and homogenized in lysis buffer (50 mm Tris-HCl, pH7.5, 150 mmNaCl, 5 mm EDTA containing 0.5% Triton X-100 and 0.5% sodium deoxycholate). Equal amounts of protein (50 μg) determined by the Bradford method (20Bradford M.M. Anal. Biochem. 1976; 72: 248-259Crossref PubMed Scopus (217544) Google Scholar) were separated on 12% SDS-PAGE gels and analyzed for their p53, active caspase 3, Mdm2, phospho-p38, and PrPc immunoreactivities by Western blot and hybridization with anti-p53 (mouse monoclonal, Pab24, Santa Cruz Biotechnology), anti-active caspase 3 (rabbit polyclonal R&D System), mouse monoclonal anti-Mdm2, anti-phospho-p38 (Promega, Charbonnières-les Bains, France) and anti-PrPc (SAF32, Ref. 21Demart S. Fournier J.G. Cremignon C. Frobert Y. Lamoury F. Marce D. Lasmézas C. Dormont D. Grassi J. Deslys J.-P. Biochem. Biophys. Res. Commun. 1999; 265: 652-657Crossref PubMed Scopus (128) Google Scholar), respectively. The secreted N-terminal fragment (N1) of PrPcwas recovered after incubation for 8 h at 37 °C/5% CO2 in serum-free medium. Medium was then incubated overnight with 2 μg/ml SAF32 monoclonal and protein A-Sepharose (Zymed Laboratory Inc.) and analyzed using sheep anti-mouse peroxydase-conjugated secondary antibody (dilution 1:10,000, AmershamBiosciences) as described (18Vincent B. Paitel E. Frobert Y. Lehmann S. Grassi J. Checler F. J. Biol. Chem. 2000; 275: 35612-35616Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Cells were grown in 6-well plates and incubated for 15 h at 37 °C in the presence or absence of staurosporine (0.5 μm). Cells were rinsed with phosphate-buffered saline, resuspended with 750 μl of buffer (20 mm Tris, 0.1% tri-natrium citrate, 0.05% Triton X-100) containing 50 μg/ml propidium iodide (PI) and incubated overnight at 4 °C. The PI fluorescence of individual nuclei was measured using a FACSCalibur flow cytometer (CellQuest software; BD Biosciences). Nuclei were characterized for their side angle scatter and forward-angle scatter parameters. Red fluorescence due to PI staining of DNA was then counted on a logarithmic scale. All measurements were performed under identical conditions. This technique allows discrimination of populations of fragmented nuclei from debris and non-viable cells and also from intact diploid nuclei those show higher fluorescence staining. The number of apoptotic nuclei is expressed as a percentage of the hundred thousand events gated. Cells were grown in a 5% CO2 atmosphere in 96-well plates in a final volume of a 100 μl per well and incubated for 24 h at 37 °C in the presence or absence of 2 μm of staurosporine. Briefly, XTT is metabolized by mitochondrial dehydrogenase to a water soluble formazan salt only by metabolically active viable cells. XTT metabolizing activity was determined as previously described (22Alves da Costa C. Ancolio K. Checler F. J. Biol. Chem. 2000; 275: 24065-24069Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Cells were cultured in 6-well plates for 15 h at 37 °C in the absence or presence of etoposide, ceramide C2, or staurosporine (0.5 μm) or 15 μm of the Fyn kinase inhibitor PP2. In some cases, cells were pre-incubated for 24 h with Ac-DEVD-al (100 μm) or for 2 h with the p38 MAPK inhibitor SB203580 (1 μm, Calbiochem). Cells were then rinsed, gently scraped, pelleted by centrifugation, then resuspended in 40 μl of lysis buffer and analyzed as previously detailed (22Alves da Costa C. Ancolio K. Checler F. J. Biol. Chem. 2000; 275: 24065-24069Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). The PG13-luciferase p53 gene reporter construct (provided by Dr. B. Vogelstein) has been previously described (23El-Deiry W. Kern S. Pietenpol J. Kinzler K. Vogelstein B. Nat. Gen. 1992; 1: 45-49Crossref PubMed Scopus (1752) Google Scholar). One μg of PG13-luciferase was co-transfected with 1 μg of a β-galactosidase transfection vector (to normalize transfections efficiencies) in TSM1 neurons and in PrP+/+and PrP−/− primary cultured neurons. Forty-eight hours after transfection, luciferase and β-galactosidase activities were measured according to previously described procedures (23El-Deiry W. Kern S. Pietenpol J. Kinzler K. Vogelstein B. Nat. Gen. 1992; 1: 45-49Crossref PubMed Scopus (1752) Google Scholar). Statistical analyses were performed with Prism software (Graphpad Software, San Diego, CA) using Newman-Keuls multiple comparison test for one-way analysis of variance and the unpaired Student's t test for pair-wise comparisons. We have obtained stable TSM1 transfectants over-expressing 3F4MoPrPc (Fig.1 A, upper panel). As expected, these cells secrete higher amounts of a 11–12-kDa fragment (Fig. 1 A, lower panel), the immunological characterization of which previously led to its identification as the N-terminal product (called N1) derived from proteolysis of PrPc at the 110/111–112 peptide bond (18Vincent B. Paitel E. Frobert Y. Lehmann S. Grassi J. Checler F. J. Biol. Chem. 2000; 275: 35612-35616Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The over-expression of PrPc in TSM1 neurons increases both basal and staurosporine-induced toxicity (Fig. 1 B) and DNA fragmentation (Fig. 1 C) as measured by XTT and PI incorporation, respectively. This phenotype has been observed for all clones examined (not shown). Over-expression of PrPc increases staurosporine-stimulated but not basal Ac-DEVD-al-sensitive Ac-DEVD-7AMC-hydrolyzing caspase 3-like activity in TSM1 neurons (Fig.1 D). This was accompanied by an augmentation of staurosporine-induced active caspase 3-like immunoreactivity (Fig.1 D). It should be emphasized that PrPcexpression potentiated the increase of caspase 3 activity triggered by several other apoptotic stimuli such as ceramide C2 and etoposide in TSM1 (Fig. 1 E). PP2, a selective inhibitor of kinases belonging to the Src family, such as Lck and Fyn, (24Hanke J.H. Gardner J.P. Dow R.L. Changelian P.S. Brissette W.H. Weringer E.J. Pollock B.A. Connelly P.A. J. Biol. Chem. 1996; 271: 695-701Abstract Full Text Full Text PDF PubMed Scopus (1790) Google Scholar) did not affect the extent of PrPc-induced staurosporine-stimulated caspase 3 activation in TSM1 neurons (Fig. 1 F). To examine the endogenous contribution of PrPc to TSM1 susceptibility to staurosporine, we set up stable antisense-PrPc (ASPrPc) TSM1 transfectants. This antisense approach led to a drastic reduction of endogenous PrPc expression and to a barely detectable N1 secretion (Fig. 2 A). Interestingly, the staurosporine-stimulated, but not the basal caspase 3 activity observed in mock-transfected TSM1 neurons, was drastically reduced in ASPrPc-TSM1 cells (Fig. 2 B). Concomitantly, PrPc-antisense abolished the staurosporine-induced augmentation of active caspase 3-like immuno-reactivity (Fig. 2 B). In line with these observations, the staurosporine-induced DNA fragmentation was reduced by about 50% in ASPrPc-expressing TSM1 neurons (Fig.2 C), a percentage that matched well the extent of the reduction of caspase 3 activity (see Fig. 2 B). We examined the putative contribution of the tumor suppressor oncogene p53 to the PrPc-induced caspase 3 activation and toxicity by antisense and pharmacological approaches. We set up TSM1 stable transfectants expressing p53 antisense cDNA either alone (ASp53) or in combination with 3F4MoPrPc(3F4MoPrPc/ASp53)(see Fig. 4 B). Our data show that all 3F4MoPrPc-related phenotypes can be prevented by antisense down-regulation of p53. Thus, Fig.3 A clearly shows that ASp53 drastically protects TSM1 cells from both basal and staurosporine-stimulated PrPc-mediated toxicity measured by the XTT assay (compare 3F4MoPrPc with 3F4MoPrPc/ASp53). Furthermore, ASp53 fully prevents the 3F4MoPrPc-induced DNA fragmentation (Fig. 3 D, compare 3F4MoPrPc-sts with3F4MoPrPc/ASp53-sts). Finally, ASp53 totally reverses the staurosporine-induced caspase activation triggered by 3F4MoPrPc (Fig. 3 B, compare3F4MoPrPc and3F4MoPrPc/ASp53 transfectants).Figure 3PrPc-induced caspase 3 is p53-dependent. A, mock-, 3F4MoPrPc, or 3F4MoPrPc-TSM1 cells stably transfected with p53 antisense cDNA (3F4MoPrP/ASp53), respectively, were checked for their cell viability in the absence (−) or presence (+) of staurosporine (Sts) as described in the legend to Fig. 1. B–D, TSM1 cells stably transfected with p53 antisense cDNA (ASp53 and 3F4MoPrP/ASp53, respectively) were measured for their caspase activity (B and C) and DNA fragmentation (D) in the absence (−) or presence (+) of staurosporine (B and D) or pifithrin-α (Pft, 10 μm, B). Barsrepresent the means ± S.E. of 3–6 independent determinations (carried out in duplicates).View Large Image Figure ViewerDownload (PPT) We took advantage of the recent design of a specific p53 inhibitor, pifithrin-α (25Komarov P.G. Komarova E.A. Kondratov R.V. Christov-Tselkov K. Coon J.S. Chernov M.V. Gudkov A.V. Science. 1999; 285: 1733-1737Crossref PubMed Scopus (1122) Google Scholar), to further confirm the role of endogenous p53 in PrPc-mediated caspase activation. This pharmacological approach allowed us to show that pifithrin-α abolishes the 3F4MoPrPc-mediated staurosporine-stimulated increase of caspase 3 activity (Fig. 3 C, compare3F4MoPrPc and Mock−/+pft). Interestingly, pifithrin-α appeared inactive in ASp53-TSM1 cells (Fig. 3 C, compare ASp53−/+pft), indicating that the antisense and pharmacological approaches indeed inhibited the same expected molecular target,i.e. p53. This conclusion was reinforced by the fact that pifithrin-α did not significantly modify caspase 3 activity displayed by doubly transfected 3F4MoPrPc/ASp53-TSM1 neurons that remain at the level of pifithrin-α-treated mock- and 3F4MoPrP-transfected cells (Fig. 3 C). Because PrPc-proapoptoptic phenotype appeared p53-dependent, we examined whether PrPc could also affect p53 expression and transcriptional activity. First, our data show that TSM1 over-expressing 3F4MoPrPc displays higher endogenous p53-like immunoreactivity than mock-transfected cells (Fig.4 A). This was also observed in transfected HEK293 cells (Fig. 4 E). Conversely, ASPrPc-TSM1 cells exhibit lower p53-like immunoreactivity (Fig. 4 A). To correlate the modulation of p53 immunoreactivity with its biological activity, we examined the p53 transcriptional activity by means of the PG13-luciferase construct classically used as the p53 gene reporter (23El-Deiry W. Kern S. Pietenpol J. Kinzler K. Vogelstein B. Nat. Gen. 1992; 1: 45-49Crossref PubMed Scopus (1752) Google Scholar). Interestingly, p53 transcriptional activity was drastically enhanced in 3F4MoPrPc-TSM1 cells (Fig. 4 C) and lowered in ASPrPc-expressing neurons (Fig. 4 C). As expected, ASp53-expressing cells exhibit lower p53 immunoreactivity (Fig. 4 B, compare ASp53 and Mock) and totally prevents the p53 increase observed with singly transfected 3F4MoPrPc cells (Fig. 4 B, compare3F4MoPrP and 3F4MoPrP/ASp53 withASp53). We took advantage of a cell system in which p53 is functionally deficient (26Mitsudomi T. Steinberg S.M. Nau M.M. Carbone D. D'Amico D. Bodner S. Oie H.K. Linnoila R.I. Mulshine J.L. Minna J.D. Oncogene. 1992; 7: 171-180PubMed Google Scholar) to examine the ability of PrPcto increase p53 expression in co-transfection experiments. Any PrPc-dependent increase in p53 could not be explained by a transcriptional activation as the p53 cDNA is driven by a cytomegalovirus promoter and therefore would imply post-transcriptional PrPc-dependent p53 modulation. As expected, mock-transfected cells do not display any p53 transcriptional activity (Fig.5 A). PrPc cDNA transfection does not modify this null phenotype. However, we establish that transient co-transfection of PrPc and p53 cDNAs drastically potentiates the p53 transcriptional activity associated with p53 cDNA transfection alone (compare p53 and3F4/p53 in Fig. 5 A). These data led us to search for post-transcriptional modifications triggered by PrPc that would have altered p53 transcriptional activity. Mdm2 was recently shown to decrease p53 metabolic stability thereby lowering its activity (27Yin Y. Luciani M.G. Fahraeus R. Nat. Cell Biol. 2002; PubMed Google Scholar). We demonstrate here that Mdm2 fully reverses the PrPc-induced increase of p53 transcriptional activity (Fig. 5 E). As p38 MAPK was reported to up-regulate p53 activity via upstream decrease of Mdm2 expression (28Zhu Y. Mao X.O. Sun Y. Xia Z. Greenberg D.A. J. Biol. Chem. 2002; 277: 22909-22914Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), we examined the effect of a selective p38 MAPK inhibitor (29Young P.R. McLaughlin M.M. Kumar S. Kassis S. Doyle M.L. McNulty D. Gallagher T.F. Fisher S. McDonnell P.C. Carr S.A. Huddleston M.J. Seibel G. Porter T.G. Livi G.P. Adams J.L. Lee J.C. J. Biol. Chem. 1997; 272: 12116-12121Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar) and the endogenous levels of the active phosphorylated counterpart of p38 MAPK. SB203580 significantly reduced but did not abolish the staurosporine-induced caspase activation in mock- and 3F4MoPrPc-expressing neurons (Fig.5 D). Accordingly, over-expression of PrPcincreases phospho-p38 MAPK immunoreactivity in both TSM1 neurons (Fig.5 B) and p53-inactive fibroblasts (Fig. 5 C). Altogether, our data suggest that PrPc increases p53 activity by p38 MAPK activation and concomitant reduction of Mdm2. Although a series of studies (1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5168) Google Scholar, 2Aguzzi A. Montrasio F. Kaeser P.S. Nat. Rev. Mol. Cell. Biol. 2001; 2: 118-126Crossref PubMed Scopus (133) Google Scholar, 3Ghetti B. Piccardo P. Frangione B. Bugiani O. Giaccone G. Young K. Prelli F. Farlow M.R. Dlouhy S.R. Tagliavni F. Brain Pathol. 1996; 6: 127-145Crossref PubMed Scopus (172) Google Scholar, 4Büeler H. Aguzzi A. Sailer A. Greiner R. Autenried P. Aguet M. Weissmann C. Cell. 1993; 73: 1339-1347Abstract Full Text PDF PubMed Scopus (1814) Google Scholar, 5Brandner S. Isenmann S. Raeber A. Fischer M. Sailer A. Kobayashi Y. Marino S. Weissmann C. Aguzzi A. Nature. 1996; 379: 339-343Crossref PubMed Scopus (721) Google Scholar, 6Telling G.C. Parchi P. DeArmond S.J. Cortelli P. Montagna P. Gabizon R. Mastrianni J. Lugaresi E. Gambetti P. Prusiner S.B. Science. 1996; 274: 2079-2082Crossref PubMed Scopus (757) Google Scholar, 7Safar J. Wille H. Itri V. Groth D. Serban H. Torchia M. Cohen F. Prusiner S. Nat. Med. 1998; 4: 1157-1165Crossref PubMed Scopus (1081) Google Scholar, 8Lawson V.A. Priola S.A. Wehrly K. Chesebro B. J. Biol. Chem. 2001; 276: 35265-35271Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 9Bueler H. Fischer M. Lang Y. Bluethmann H. Lipp H. DeArmond S. Prusiner S.B. Aguet M. Weissmann C. Nature. 1992; 356: 577-582Crossref PubMed Scopus (1441) Google Scholar) attempted to delineate the prions-related pathogenic mechanisms, very few data concern the putative physiological function of PrPc. This lack of attention perhaps came in part from the fact that the absence of the protein does not seem absolutely crucial because mice in which the prnp gene had been invalidated are safe and healthy, with apparently poorly detectable alterations in their development and behavior (9Bueler H. Fischer M. Lang Y. Bluethmann H. Lipp H. DeArmond S. Prusiner S.B. Aguet M. Weissmann C. Nature. 1992; 356: 577-582Crossref PubMed Scopus (1441) Google Scholar). However, Kuwahara et al. (30Kuwahara C. Takeuchi A.M. Nishimura T. Haraguchi K. Kubosaki A. Matsumoto Y. Saeki K. Matsumoto Y. Yokoyama T. Itohara S. Onodera T. Nature. 1999; 400: 225-226Crossref PubMed Scopus (374) Google Scholar) reported that hippocampal neurons prepared fromPrnp −/− mice undergo exacerbated cell death triggered by serum deprivation, thereby suggesting that PrPc could display an anti-apoptotic tonus. More recently, an interesting study documented the fact that PrPcprotected human neurons from Bax-induced cell death (13Bounhar Y. Zhang Y. Goodyer C.G. LeBlanc A. J. Biol. Chem. 2001; 276: 39145-39149Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). These data are apparently in opposition with a previous work demonstrating that transgenic mice over-expressing wild type PrPc could exhibit severe neurodegeneration (17Westaway D. DeArmond S.J. Cayetano-Canlas J. Groth D. Foster D. Yang S.-L. Torchia M. Carlson G.A. Prusiner S.B. Cell. 1994; 76: 117-129Abstract Full Text PDF PubMed Scopus (293) Google Scholar). On the other hand, a recent article indicated that the post-natal deletion of the PrPcalters CA1 hippocampal neuron excitability but does not induce neurodegeneration (10Mallucci G.R. Ratté S. Asante E.A. Linehan J. Gowland I. Jefferys J.G.R. Collinge J. EMBO J. 2002; 21: 202-210Crossref PubMed Scopus (327) Google Scholar) as would have been expected if the protein displayed an important anti-apoptotic function at adulthood. Furthermore, an infected hypothalamic neuronal cell line exhibits increased apoptosis (31Schätzl H.M. Laszlo L. Holtzman D.M. Tatzelt J. DeArmond S.J. Weiner R.I. Mobley W.C. Prusiner S.B. J. Virol. 1997; 71: 8821-8831Crossref PubMed Google Scholar). Whether this results from a dysfunction in the normal control of cell death by PrPc remains a possibility. The 106–126 domain of PrPc was shown to be toxic (14Forloni G. Angeretti N. Chiesa R. Monzani E. Salmona M. Bugiani O. Tagliavini F. Nature. 1993; 362: 543-546Crossref PubMed Scopus (896) Google Scholar, 15Brown D.R. J. Neurochem. 1999; 73: 1105-1113Crossref PubMed Scopus (93) Google Scholar) and appears to activate several pro-apoptotic markers (16Jobling M.F. Stewart L.R. White A.R. McLean C. Friedhuber A. Maher F. Beyreuther K. Masters C.L. Barrow C.J. Collins S.J. Cappai R. J. Neurochem. 1999; 73: 1557-1565Crossref PubMed Scopus (152) Google Scholar, 32White A.R. Guirguis R. Brazier M.W. Jobling M.F. Hill A.F. Beyreuther K. Barrow C.J. Masters C.L. Colins S.J. Cappai R. Neurobiol. Dis. 2001; 8: 299-316Crossref PubMed Scopus (66) Google Scholar). This fragment is never proteolytically released as such from PrPc. In this context, either the bulk of studies examining the effect of 106–126 just describe not relevant and artifactual data, or, more likely, the studies carried out with this fragment can be seen as a revelator of a PrPc function that would be pro-apoptotic. Our study clearly establishes by means of neuronal cell systems, stable transfections, antisense, and pharmacological approaches that PrPc drastically exacerbates cell responsiveness to pro-apoptotic stimuli. Thus, over-expression or specific induction of PrPc enhances staurosporine-induced cell toxicity, DNA fragmentation, and increases both caspase 3-like activity and immuno-reactivity that are all reversed by PrPc antisense approach. The fact that PrPc-mediated caspase activation also occurs in HEK293 cells (33Paitel E. Alves da Costa C. Vilette D. Grassi J. Checler F. J. Neurochem. 2002; 83: 1208-1214Crossref PubMed Scopus (62) Google Scholar) importantly indicates that the pro-apoptotic PrPc-related phenotype is not cell-specific. A recent study (34Ma J. Wollmann R. Lindquist S. Science. 2002; 298: 1781-1785Crossref PubMed Scopus (432) Google Scholar) suggested that PrPc could behave as a signaling molecule, transducing the signal after its coupling to the Src kinase Fyn. In our experiments, the selective Src inhibitor PP2 does not affect the PrPc-induced caspase activation, indicating that Fyn kinase phosphorylating activity was clearly not essential for PrPc-induced apoptosis in our experimental conditions. It should be noted that distinct cell types were used in the two studies, and more particularly the Fyn-dependent PrPc-mediated signaling appeared drastically dependent on the differentiation state of the 1C11 cells used in Mouillet-Richardet al. (35Mouillet-Richard S. Ermonval M. Chebassier C. Laplanche J.L. Lehmann S. Launay J.M. Kellermann O. Science. 2000; 289: 1925-1928Crossref PubMed Scopus (678) Google Scholar). Clearly the lack of Fyn dependence observed for PrPc-mediated phenotype in TSM1 cells indicates that Fyn requirement is likely restricted to few specialized PrPc phenotypes much more than for more general PrP function in cell death control. We demonstrate that the PrPc-induced caspase 3 activation is mediated by p53. Indeed, PrPc expression increases p53 immuno-reactivity and transcriptional activity, whereas the PrPc antisense approach led to the opposite phenotype. Furthermore, p53 antisense blocked the PrPc-induced toxicity, DNA fragmentation, and caspase 3 activation. The opposite phenotype triggered by down-regulation of endogenous PrPcimportantly indicates that the pro-apoptotic phenotype observed in PrPc-transfected cells was not artifactually related to the procedure involving over-expression of the protein. This is in agreement with our other data showing that Rov9 cells display a pro-apoptotic phenotype only after selective PrPc induction (33Paitel E. Alves da Costa C. Vilette D. Grassi J. Checler F. J. Neurochem. 2002; 83: 1208-1214Crossref PubMed Scopus (62) Google Scholar). PrPc controls p53 expression and activity at a post-transcriptional level. Thus we were able to demonstrate that in a cell line deficient in active p53, PrPc drastically increases p53 activity after co-transfection of p53 and PrPc cDNAs. As p53 and PrPc cDNA are driven by a constitutively active cytomegalovirus promoter, p53 enhanced activity cannot be derived from increased transcription of p53, the genuine promoter of which leads to an inactive protein in our cell system. Interestingly, Mdm2 fully prevents the PrPc-induced p53 increase observed in the above cell system. Mdm2 was recently shown to control p53 expression at post-transcriptional levels (27Yin Y. Luciani M.G. Fahraeus R. Nat. Cell Biol. 2002; PubMed Google Scholar) and particularly modulates p53 ubiquitination and degradation. Therefore, one could conclude that PrPc increases p53 expression and transcriptional activity via a decrease of the p53 down-regulator Mdm2. To support this hypothesis we examined the putative contribution of the p38 mitogen-activated protein kinase that was recently shown to down-regulate Mdm2 and concomitantly increase p53 expression in neurons in a model of hypoxia. Indeed, we clearly showed that phospho-38 MAPK immuno-reactivity, the activated form of this kinase, was higher in PrPc-expressing TSM1 cells, in PrP+/+ than in PrP−/− neurons, and could be increased by PrPc in fibroblasts (see Fig. 5). Furthermore, p38 inhibitor could partly block the PrPc-associated caspase 3 activation in transfected TSM1 neurons. Altogether, our data show that PrPc exerts a pro-apoptotic phenotype through the modulation of caspase 3 activity via a p53-dependent pathway that is positively and negatively controlled by p38 MAPK and Mdm2, respectively. It is interesting to note that a recent study indicated that misfolded PrPc could be neurotoxic and could induce neurodegenerescence when accumulating in the cytosol, even at small amounts (35Mouillet-Richard S. Ermonval M. Chebassier C. Laplanche J.L. Lehmann S. Launay J.M. Kellermann O. Science. 2000; 289: 1925-1928Crossref PubMed Scopus (678) Google Scholar). Whether such a small amount of PrPc could account for the phenotype observed in our over-expression system is difficult to examine. However, the fact that our antisense approach led to an anti-apoptotic phenotype would suggest that even in normal conditions, a small fraction of endogenous PrPc could be responsible for our observed p53-dependent caspase 3 activation. We recently documented the fact that PrPcundergoes basal and protein kinase C-regulated cleavage in the middle of its 106–126 domain by proteases of the disintegrins family, thereby releasing a fragment that we called N1 (for review see Ref. 36Checler F. Vincent B. Trends Neurosci. 2002; 25: 616-620Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). This PrPc cleavage also observed by Chen et al.(37Chen S.G. Teplow D.B. Parchi P. Teller J.K. Gambetti P. Autilio-Gambetti L. J. Biol. Chem. 1995; 270: 19173-19180Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar) is reminiscent of the one taking place in the middle of the Aβ domain borne by the β-amyloid precursor protein (38Checler F. J. Neurochem. 1995; 65: 1431-1444Crossref PubMed Scopus (423) Google Scholar). Interestingly, this intra-Aβ cleavage not only lowers Aβ production but also gives rise to a cytotrophic and neuroprotective secreted fragment called sAPPα. Whether the cleavage generating N1 not only abolishes a pro-apoptotic control of PrP but also produces a fragment with opposite neuroprotective influence is currently being examined in our laboratory. We thank Prof. Weissmann (Imperial College, London, UK) for providing Prnp −/− mice. PG13-luciferase- and ASp53-cDNAs were generously provided by Dr. B. Vogelstein (Howard Hughes Medical Institute, Baltimore, MD). The p53-inactive NCI-H1299 cells were kindly provided by Drs. L. Mercken and L. Pradier (Aventis Pharma, Vitry sur Seine, France). We thank Drs. Y. Frobert and J. Grassi (Commissariatà l'Energie Atomique, Saclay, France) for the kind gift SAF32 monoclonals and Dr. P. Auberger (Nice, France) and M. P. Mattson (National Institute of Aging, Baltimore, MD) for the gift of PP2 and pifithrin-α inhibitors, respectively.
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