Title: Prion Protein Protects Human Neurons against Bax-mediated Apoptosis
Abstract: The function of the cellular prion protein (PrP) is still poorly understood. We present here an unprecedented role for PrP against Bax-mediated neuronal apoptosis and show that PrP potently inhibits Bax-induced cell death in human primary neurons. Deletion of four octapeptide repeats of PrP (PrPΔOR) and familial D178N and T183A PrP mutations completely or partially eliminate the neuroprotective effect of PrP. PrP remains anti-apoptotic despite truncation of the glycosylphosphatidylinositol (GPI) anchor signal peptide, indicating that the neuroprotective form of PrP does not require the abundant cell surface GPI-anchored PrP. Our results implicate PrP as a potent and novel anti-apoptotic protein against Bax-mediated cell death. The function of the cellular prion protein (PrP) is still poorly understood. We present here an unprecedented role for PrP against Bax-mediated neuronal apoptosis and show that PrP potently inhibits Bax-induced cell death in human primary neurons. Deletion of four octapeptide repeats of PrP (PrPΔOR) and familial D178N and T183A PrP mutations completely or partially eliminate the neuroprotective effect of PrP. PrP remains anti-apoptotic despite truncation of the glycosylphosphatidylinositol (GPI) anchor signal peptide, indicating that the neuroprotective form of PrP does not require the abundant cell surface GPI-anchored PrP. Our results implicate PrP as a potent and novel anti-apoptotic protein against Bax-mediated cell death. prion protein glycosylphosphatidylinositol octapeptide repeats terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling translocation accessory factors fluorescein isothiocyanate N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine brefeldin A analysis of variance dextran Texas Red polymerase chain reaction amyloid precursor protein Bcl-2-associated protein x Prion protein (PrP)1 is a sialoglycoprotein that is highly expressed in brain, heart, lungs, and lymphoid system and at lower levels in several other tissues such as muscle (1Oesch B. Westaway D. Walchi M. McKinley M. Kent S. Aebersold R. Barry R. Tempst P. Teplow D. Hood L. Prusiner S. Weissmann C. Cell. 1985; 40: 735-746Abstract Full Text PDF PubMed Scopus (1238) Google Scholar, 2Kretzschmar H.A. Stowring L.E. Westaway D. Stubblebine W.H. Prusiner S.B. Dearmond S.J. DNA. 1986; 5: 315-324Crossref PubMed Scopus (294) Google Scholar). Mature PrP contains two N-linked glycans and a disulfide bond (reviewed in Ref. 3Prusiner S. Scott M. DeArmond S. Cohen F. Cell. 1998; 93: 337-348Abstract Full Text Full Text PDF PubMed Scopus (813) Google Scholar). PrP possesses a C-terminal GPI-anchoring signal and a transmembrane domain that can generate type I (CtmPrP) or type II (NtmPrP) transmembrane-spanning isoforms in isolated endoplasmic reticulum microsomes or phospholiposomes (4Stahl N. Borchelt D.R. Hsiao K. Prusiner S.B. Cell. 1987; 51: 229-240Abstract Full Text PDF PubMed Scopus (889) Google Scholar, 5Hay B. Barry R.A. Lieberburg I. Prusiner S.B. Lingappa V.R. Mol. Cell. Biol. 1987; 7: 914-920Crossref PubMed Scopus (100) Google Scholar, 6Hegde R. Mastrianni J. Scott M. Defea K. Tremblay P. Torchia M. DeArmond S. Prusiner S. Lingappa V. Science. 1998; 279: 827-834Crossref PubMed Scopus (613) Google Scholar, 7Hegde R.S. Voigt S. Lingappa V.R. Mol. Cell. 1998; 2: 85-91Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In most cells, the majority of the PrP localizes to the cell surface as a GPI-anchored protein (8Borchelt D.R. Scott M. Taraboulos A. Stahl N. Prusiner S.B. J. Cell Biol. 1990; 110: 743-752Crossref PubMed Scopus (435) Google Scholar). The complete translocation of PrP is dependent on translocation accessory factors (TrAF). In the absence of TrAF, PrP is exclusively synthesized in a transmembrane topology (7Hegde R.S. Voigt S. Lingappa V.R. Mol. Cell. 1998; 2: 85-91Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Whereas the role of the infectious form of PrP in a number of human and animal neurodegenerative diseases has been extensively studied, the normal function of PrP is still poorly understood. PrP-null mice display no dramatic phenotype (9Bueler H. Fischer M. Lang Y. Bluethmann H. Lipp H. DeArmond S. Prusiner S. Aguet M. Weissmann C. Nature. 1992; 356: 577-582Crossref PubMed Scopus (1429) Google Scholar). However, evidence indicates that PrP may promote sleep continuity (10Tobler I. Gaus S. Deboer T. Achermann P. Fischer M. Rulicke T. Moser M. Oesch B. McBride P. Manson J. Nature. 1996; 380: 639-642Crossref PubMed Scopus (563) Google Scholar). PrP is involved in the regulation of presynaptic copper concentration, intracellular calcium concentration, activation of lymphocytes, astrocyte proliferation, and signal transduction and has antioxidant properties (11Whatley S.A. Powell J.F. Politopoulos G. Campbell I. Brammer M. Percy N. Neurorep. 1995; 6: 2333-2337Crossref PubMed Scopus (53) Google Scholar, 12Cashman N. Loertscher R. Nalbantoglu J. Shaw I. Kascsak R. Bolton D. Bendheim P. Cell. 1990; 61: 185-192Abstract Full Text PDF PubMed Scopus (278) Google Scholar, 13Forloni G. Del Bo R. Angeretti N. Chiesa R. Smiroldo S. Doni R. Ghibaudi E. Salmona M. Porro M. Verga L. Giaccone G. Bugiani O. Tagliavini F. Eur. J. Neurosci. 1994; 6: 1415-1422Crossref PubMed Scopus (107) Google Scholar, 14Mouillet-Richard S. Ermonval M. Chebassier C. Laplanche J.L. Lehmann S. Launay J.M. Kellermann O. Science. 2000; 289: 1925-1928Crossref PubMed Scopus (672) Google Scholar, 15Brown D.R. Schulz-Schaeffer W.J. Schmidt B. Kretzschmar H.A. Exp. Neurol. 1997; 146: 104-112Crossref PubMed Scopus (389) Google Scholar, 16Brown D.R. Wong B.-S. Hafiz F. Clive C. Haswell S.J. Jones I.M. Biochem. J. 1999; 344: 1-5Crossref PubMed Scopus (487) Google Scholar). Although controversial, PrP-null mice are also found to be impaired in long term potentiation (17Collinge J. Whittington M.A. Sidle K.C.L. Smith C.J. Palmer M.S. Clarke A.R. Jefferys J.G.R. Nature. 1994; 370: 295-297Crossref PubMed Scopus (684) Google Scholar, 18Lledo P.M. Tremblay P. DeArmond S. Prusiner S. Nicoll R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2403-2407Crossref PubMed Scopus (190) Google Scholar, 19Herms J.W. Kretzchmar H. Titz S. Keller B. Eur. J. Neurosci. 1995; 7: 2508-2512Crossref PubMed Scopus (77) Google Scholar). In addition, it has been shown that PrP-null neuronal cell lines are more susceptible to serum deprivation-induced cell death and that Bcl-2 overexpression can attenuate the sensitivity of PrP-null neuronal cell lines to serum deprivation (20Kuwahara C. Takeuchi A. 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 (372) Google Scholar). Kurschner and Morgan (21Kurschner C. Morgan J. Mol. Brain Res. 1996; 37: 249-258Crossref PubMed Google Scholar, 22Kurschner C. Morgan J. Mol. Brain Res. 1995; 30: 165-168Crossref PubMed Scopus (150) Google Scholar) reported that yeast PrP fusion proteins interact with Bcl-2. Furthermore, four identical N-terminal PrP octapeptide repeats (OR) that are highly conserved in evolution share limited similarity with the Bcl-2 homology domain 2 (BH2) of Bcl-2 proteins (23LeBlanc A. Wang S.S. Handbook of the Aging Brain. Academic Press, NY1998: 202-214Google Scholar, 24Yin X.-M. Oltvai Z.N. Korsmeyer S.J. Nature. 1994; 369: 321-323Crossref PubMed Scopus (1212) Google Scholar). Bcl-2 proteins are central to the regulation of cell death, and the BH2 domain is crucial to the anti-apoptotic function of Bcl-2 and its interaction with the pro-apoptotic Bax protein (24Yin X.-M. Oltvai Z.N. Korsmeyer S.J. Nature. 1994; 369: 321-323Crossref PubMed Scopus (1212) Google Scholar, 25Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4755) Google Scholar). Based on these features of PrP, we hypothesized that similar to Bcl-2 family members, PrP may play a role in the regulation of neuronal apoptosis. In the present study, we have investigated the role of PrP in neuronal survival against the pro-apoptotic Bax protein. Human Bcl-2 cDNA was a kind gift from Dr. Walter Nishioka (Vical Inc. San Diego, CA). PrP and PrP-D178N and -T183A DNAs were PCR-amplified as described (26Nitrini R. Rosemberg S. Passos-Bueno M.R. Lughetti P. Papadopoulos M. Carrilho P.E. Caramelli P. Albrecht S. Zatz M. LeBlanc A.C. Ann. Neurol. 1997; 42: 138-146Crossref PubMed Scopus (100) Google Scholar, 27Medori R. Tritschler H.J. LeBlanc A. Villare F. Manetto V. Chen H.Y. Xue R. Leal S. Montagna P. Cortelli P. Tinuper P. Avoni P. Mochi M. Baruzzi A. Hauw J. Lugaresi E. Autilio-Gambetti L. Gambetti P. N. Engl. J. Med. 1992; 326: 444-449Crossref PubMed Scopus (500) Google Scholar). Bax-α cDNA was PCR-amplified from human neuron cDNA. cDNAs were cloned into pBluescript KSII (pBSK−; Stratagene) or pCep4β (Invitrogen). The APP695 construct has been described elsewhere (28LeBlanc A.C. Gambetti P. Biochem. Biophys. Res. Commun. 1994; 204: 1371-1380Crossref PubMed Scopus (24) Google Scholar). The PrP antisense construct (PrPAS) was made by cloning the entire PrP coding region in the reverse orientation in theBamHI/NotI sites of pCep4β. Deletion of the OR region was performed by PCR amplification of the two regions flanking the ORs using the following primers. To generate the 5′-fragment: forward (PDG2), 5′-TACTGAGAATTCGCAGTCCATTATGGCGAACCTTGGCTGCTGG-3′ and reverse (P3−), 5′-ACCACCGCCCTGAGG-3′; to generate the 3′-fragment: forward (P4+), 5′-ACCCACAGTCAGTGG-3′ and reverse (PDG1), 5′-GTACTGAGGATCCTCCTCATCCCACTATCAGGAAGA-3′. The two fragments were then blunt-end ligated and cloned in the proper orientation in pBSK− and pCep4β. Deletion of the GPI anchor signal was generated by PCR using PDG2 (forward) and reverse 5′-TCCGGATCCCTATCCTCTCTGG-3′ primers. R155 PrP antiserum against residues 36–56 was raised in our laboratory. Human primary neurons were cultured as described (29LeBlanc A.C. J. Neurosci. 1995; 15: 7837-7846Crossref PubMed Google Scholar) and microinjected with 25 pl containing 0.75 pg of DNA and 2.5 pg of dextran Texas Red (DTR) in phosphate-buffered saline (30Zhang Y. Goodyer C. LeBlanc A. J. Neurosci. 2000; 20: 8384-8389Crossref PubMed Google Scholar). Each injection was done on 200 neurons in at least three independent neuronal preparations. The cells were fixed in 4% paraformaldehyde, 4% sucrose in phosphate-buffered saline. Cell death was assessed using the in situ cell detection kit (Roche Molecular Biochemicals) according to the manufacturer's instructions. The cells were read blindly on coded slides. The percentage of cell death was determined as the number of TUNEL and DTR double-positive over the total number of DTR-microinjected neurons. Neurons were fixed in 4% paraformaldehyde, 4% sucrose 24 h after microinjection of the antisense PrP construct. The cells were permeabilized with 0.1% Triton X-100, blocked with 10% fetal goat serum, incubated with R155 (1:100), and detected with goat anti-rabbit IgAll-FITC. Cells were grown to 70–80% confluency in 6-well plates and transfected with 3 µg of plasmid DNA using the LipofectAMINE 2000 reagent (Life Technologies, Inc.) according to the manufacturer's instructions. Transient transfectants were assessed for protein expression at 48 h post-transfection. Stable cell lines for each construct were also studied. Cells were metabolically labeled for 4 h using 100 µCi/ml [35S]methionine. Cells were lysed in Nonidet P-40 lysis buffer, and PrP was immunoprecipitated in radioimmune precipitation buffer using anti-PrP R155 (31Harlow E. D. L. Using Antibodies. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1999: 223-265Google Scholar). The immune complexes were separated on Tris-Tricine gels and subjected to autoradiography. Human primary neurons were preincubated for 1 h in the presence or absence of 5 µg/ml brefeldin A or 5 µmmonensin (Sigma), microinjected as described above, and then incubated in the absence or presence of either 5 µg/ml brefeldin A or 5 µm monensin for 12 h. The cells were fixed at the indicated times, and cell death was assessed as described above. The effect of brefeldin A on prion maturation and secretion was studied in neuronal cultures incubated in serum- and methionine-free media in the presence of 5 µg/ml brefeldin A for 1 h and then incubated in 100 µCi/ml of [35S]methionine for 1, 3, and 12 h. Immunoprecipitations were done with a 1:100 dilution of polyclonal anti-PrP antisera R155 as previously described (29LeBlanc A.C. J. Neurosci. 1995; 15: 7837-7846Crossref PubMed Google Scholar). The significance of variance was analyzed with ANOVA followed by post-hoc Dunnett's or Scheffé's test. A p < 0.05 was taken as a significant difference. PrP, Bax, and Bcl-2 are normally expressed in human neurons. However, Bax is not pro-apoptotic unless it is induced through insult or overexpression. We chose to induce Bax-mediated cell death by microinjection of Bax cDNA because cell death can then be directly attributed to Bax overexpression. The role of PrP and Bcl-2 against Bax-mediated cell death was then assessed by co-microinjecting the cDNAs encoding Bcl-2 and PrP with the Bax cDNA in human primary neurons in culture. The cDNAs were expressed under the cytomegalovirus promoter of the episomal Cep4β construct (32Yates J.L. Warren N. Sugden B. Nature. 1985; 313: 812-815Crossref PubMed Scopus (975) Google Scholar). Bax is known to cause apoptosis in a number of neuronal cells (33Shindler K.S. Latham C.B. Roth K.A. J. Neurosci. 1997; 17: 3112-3119Crossref PubMed Google Scholar, 34Putcha G.V. Deshmukh M. Johnson E.M. J. Neurosci. 1999; 19: 7476-7485Crossref PubMed Google Scholar). Injection of Bax cDNA, but not vector pCep4β, Bcl-2, or PrP cDNAs, also induces a rapid cell death in 90% of these human primary neurons within 48 h (Fig.1A). Co-injection of Bax cDNA with either PrP or Bcl-2 cDNA protects against Bax-mediated cell death. The triple injection of PrP, Bcl-2, and Bax does not further enhance protection. In contrast, microinjection of a cDNA encoding amyloid precursor protein (APP) does not prevent Bax-mediated cell death. These results show that PrP and Bcl-2 can both efficiently prevent Bax-mediated cell death in human neurons. To test if the neuroprotective function of PrP is merely a consequence of overexpression, endogenous PrP expression was inhibited by injecting neurons with a PrPAS cDNA Cep4β episomal construct. This construct has previously been used successfully to inhibit high levels of APP expression (35LeBlanc A. Kovacs D. Chen H. Villare F. Tykocinski M. Autilio-Gambetti L. Gambetti P. J. Neurosci. Res. 1992; 31: 635-645Crossref PubMed Scopus (90) Google Scholar). The antisense construct did not induce cell death in the absence of Bax cDNA (Fig. 1B). However, the antisense cDNA enhanced Bax-mediated neuronal cell death at 12 and 24 h of injection. We confirmed that PrP expression decreases in the PrPAS-microinjected neurons by immunocytochemistry (Fig. 1B, inset). Therefore, endogenous PrP can protect to some extent against Bax-mediated cell death. The antisense study also indicates that the loss of PrP expression is not a problem for these neurons unless Bax pro-apoptotic properties have been launched. The PrP octapeptide repeat region plays an important role against oxidative stress (15Brown D.R. Schulz-Schaeffer W.J. Schmidt B. Kretzschmar H.A. Exp. Neurol. 1997; 146: 104-112Crossref PubMed Scopus (389) Google Scholar, 16Brown D.R. Wong B.-S. Hafiz F. Clive C. Haswell S.J. Jones I.M. Biochem. J. 1999; 344: 1-5Crossref PubMed Scopus (487) Google Scholar). In addition, an increase in the number of OR in PrP leads to disease in human and mice (36Owen F. Poulter M. Shah T. Collinge J. Lofthouse R. Baker H. Ridley R. McVey J. Crow T.J. Brain Res. Mol. Brain Res. 1990; 7: 273-276Crossref PubMed Scopus (109) Google Scholar, 37Chiesa R. Drisaldi B. Quaglio E. Migheli A. Piccardo P. Ghetti B. Harris D.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5574-5579Crossref PubMed Scopus (137) Google Scholar). To determine whether PrP anti-Bax function also requires the octapeptide repeat region, we tested the neuroprotective ability of PrP lacking the four OR with similarity to BH2 (PrPΔOR) against Bax. PrPΔOR abolishes the neuroprotective function of PrP (Fig. 2A). The lack of function of PrPΔOR cDNA construct could be caused by the absence of expression of this mutant PrP. Alternatively, the protein could be expressed but unstable. Because neurons are microinjected, we can only assess the expression of the exogenous PrP by immunocytochemistry. However, neurons express considerable levels of PrP, and it is therefore impossible to test the amount of proteins expressed from the Cep4β constructs in the microinjected neurons. Assuming that recombinant PrP is expressed similarly in neurons and other eukaryotic cells, we verified the expression level of PrP in eukaryotic cells by transfecting erythroleukemia K562 and neuroblastoma M17, two cell lines that lack endogenous expression of PrP. The transfected cells were selected for hygromycin resistance and PrP expression verified by immunoprecipitation of PrP from protein extracts of [35S]methionine metabolically labeled cells. We find that PrPΔOR is expressed at levels comparable with those of wild type PrP (Fig. 2B). Therefore, the loss of function of PrP is likely not a consequence of low PrPΔOR expression levels but rather the result of the disruption of the OR region. To assess the location of the neuroprotective PrP, PrP- and Bax-microinjected neurons were treated with the Golgi-disaggregating agent, BFA or monensin, an ionophore that prevents trafficking of secreted proteins past the cis-Golgi (38Dinter A. Berger E.G. Histochem. Cell Biol. 1998; 109: 571-590Crossref PubMed Scopus (316) Google Scholar). BFA and monensin did not alter Bax-mediated cell death but completely inhibited the neuroprotective function of PrP against Bax (Fig. 2C). The action of BFA and monensin on PrP maturation was confirmed by radiolabeling and immunoprecipitating PrP (Fig.2D). In the absence of BFA or monensin, three PrP protein forms were immunoprecipitated. The higher molecular weight form becomes more abundant with time of labeling indicating that this protein represents the mature glycosylated form of PrP. In BFA, only two lower molecular weight proteins were immunoprecipitated, indicating the unglycosylated and immature glycosylated PrP expected of proteins retained in the endoplasmic reticulum. The immature PrP accumulates in BFA-treated neurons with time, and after 12 h of labeling represents the most abundant form. However, the mature form of PrP is not seen in BFA at 12 h indicating that BFA has effectively prevented the trafficking of PrP into the Golgi apparatus. Monensin has an effect on PrP that is similar to that of BFA. No mature protein is observed even after 12 h of labeling. However, monensin also reduces the level of PrP expression raising the additional possibility that with monensin treatment, low expression of PrP cannot compensate for the pro-apoptotic effect of Bax overexpression. In normal neurons, a small amount of PrP is immunoprecipitated from secreted medium after 12 h of labeling. However, this released PrP is not observed in BFA- or monensin-treated neurons. These results indicate that trafficking past the cis-Golgi is required for the neuroprotective function of PrP. In most cells, the majority of PrP exists as a GPI-anchored cell surface protein while Bax is a cytosolic protein with a C-terminal hydrophobic membrane anchor (8Borchelt D.R. Scott M. Taraboulos A. Stahl N. Prusiner S.B. J. Cell Biol. 1990; 110: 743-752Crossref PubMed Scopus (435) Google Scholar, 39Hsu Y. Wolter K. Youle R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3668-3672Crossref PubMed Scopus (1021) Google Scholar). To determine whether the GPI-anchored PrP mediates the neuroprotective function through signal transduction (14Mouillet-Richard S. Ermonval M. Chebassier C. Laplanche J.L. Lehmann S. Launay J.M. Kellermann O. Science. 2000; 289: 1925-1928Crossref PubMed Scopus (672) Google Scholar), we compared the neuroprotective ability of a PrP mutant lacking the GPI-anchor signal peptide sequence (PrPΔGPI)versus wild type PrP against Bax-induced neuronal apoptosis. PrPΔGPI is expressed at much lower levels than wild type PrP but is also secreted in the medium (Fig. 2B). However, PrPΔGPI is still neuroprotective (Fig. 2E) indicating that the cell surface GPI-anchored PrP is not necessary for PrP's neuroprotective function. We additionally tested if C-terminal mutations of PrP associated with prion diseases altered the neuroprotective ability of PrP against Bax-mediated cell death (Fig.3C). Whereas the T183A Familial Atypical Spongiform Encephalopathy (FASE) mutation (26Nitrini R. Rosemberg S. Passos-Bueno M.R. Lughetti P. Papadopoulos M. Carrilho P.E. Caramelli P. Albrecht S. Zatz M. LeBlanc A.C. Ann. Neurol. 1997; 42: 138-146Crossref PubMed Scopus (100) Google Scholar) partially inhibits PrP function, the D178N Fatal Familial Insomnia (FFI) mutation (27Medori R. Tritschler H.J. LeBlanc A. Villare F. Manetto V. Chen H.Y. Xue R. Leal S. Montagna P. Cortelli P. Tinuper P. Avoni P. Mochi M. Baruzzi A. Hauw J. Lugaresi E. Autilio-Gambetti L. Gambetti P. N. Engl. J. Med. 1992; 326: 444-449Crossref PubMed Scopus (500) Google Scholar) completely abolishes the PrP neuroprotective function against Bax. These results indicate that the loss of function of PrP may be involved in the pathophysiology of these two diseases. In the present manuscript, we show an unprecedented function for prion protein against Bax-mediated neuronal cell death. Whereas the infectious nature of PrP is extensively studied, little is known about its normal cellular function. Kuwahara et al. (20Kuwahara C. Takeuchi A. 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 (372) Google Scholar) have shown that neuronal cell lines derived from PrP-null mice were more susceptible to serum deprivation and could be rescued by Bcl-2 or PrP. However, the mechanism by which PrP protected these cells was not proposed. Here, we show that PrP can prevent Bax-mediated cell death to levels equal to the neuroprotective function of Bcl-2. The neuroprotective effect of PrP is strong and prevents almost all Bax-microinjected neurons from cell death. It has previously been demonstrated that PrP protects neurons against oxidative stress through the octapeptide repeats (16Brown D.R. Wong B.-S. Hafiz F. Clive C. Haswell S.J. Jones I.M. Biochem. J. 1999; 344: 1-5Crossref PubMed Scopus (487) Google Scholar). Similarly, we find that deletion of the octapeptide repeats eliminates the neuroprotective function of PrP against Bax. However, PrP protects only 30% of cells against oxidative stress (15Brown D.R. Schulz-Schaeffer W.J. Schmidt B. Kretzschmar H.A. Exp. Neurol. 1997; 146: 104-112Crossref PubMed Scopus (389) Google Scholar, 40White A.R. Collins S.J. Maher F. Jobling M.F. Stewart L.R. Thyer J.M. Beyreuther K. Masters C.L. Cappai R. Am. J. Pathol. 1999; 155: 1723-1730Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar) 2A. LeBlanc, Y. Bounhar, and Y. Zhang, unpublished observations. compared with almost 100% protection against Bax. The neuroprotective function of normal PrP against Bax is surprising and unexpected. Generally, negative functions have been attributed to the PrP. However, a natural neuroprotective function is reasonable. First, PrP is highly expressed in some neurons and therefore would be expected to have a beneficial function. Second, the presence of endogenous PrP appears to be neuroprotective in vivo. For example, the presence of endogenous mouse PrP allows the production of hamster scrapie PrP in GFAP-hamster PrP transgenic mice infected with the hamster prion strain, 263K. In addition, transmission of disease is obtained from these mice. However, theprnp+/+ mice do not develop the neurodegenerative disorder observed in mouseprnp-null/GFAP-hamster PrP transgenic mice (41Raeber A.J. Race R.E. Brandner S. Priola S.A. Sailer A. Bessen R.A. Mucke L. Manson J. Aguzzi A. Oldstone M.B. Weissmann C. Chesebro B. EMBO J. 1997; 16: 6057-6065Crossref PubMed Scopus (188) Google Scholar). Although reduced titers (10–100-fold) of the PrP proteinase K-resistant form and infectivity in the mouse prnp+/+/transgenic hamster PrP could explain the lack of neurotoxicity, an alternative possibility is that the endogenous PrP is neuroprotective against the presence of the scrapie isoform of the PrP. PrP also protects against the cytotoxic effects of Doppel in Purkinge neurons (42Nishida N. Tremblay P. Sugimoto T. Shigematsu K. Shirabe S. Petromilli C. Erpel S.P. Nakaoke R. Atarashi R. Houtani T. Torchia M. Sakaguchi S. DeArmond S.J. Prusiner S.B. Katamine S. Lab. Invest. 1999; 79: 689-697PubMed Google Scholar). Bax is a powerful executioner of neurons (33Shindler K.S. Latham C.B. Roth K.A. J. Neurosci. 1997; 17: 3112-3119Crossref PubMed Google Scholar, 34Putcha G.V. Deshmukh M. Johnson E.M. J. Neurosci. 1999; 19: 7476-7485Crossref PubMed Google Scholar). We find that this is also true in these human primary neurons. Although microinjection of recombinant active caspases leads to a slow death of the human primary neurons (30Zhang Y. Goodyer C. LeBlanc A. J. Neurosci. 2000; 20: 8384-8389Crossref PubMed Google Scholar), Bax induces TUNEL-positive cell death in almost 70% of cells within 12 h (Fig. 1B). Therefore, PrP may be a natural strong protector against a major neuronal pro-apoptotic protein. Our finding that the GPI anchor is not required for PrP's anti-Bax function is also quite surprising. It is well established that the cell surface GPI-anchored PrP is the most abundant isoform of PrP in most cells. The results of the BFA experiment show that the neuroprotective form of PrP requires post-cis-Golgi modification or trafficking for function. However, with deletion of the GPI sequence, which abolishes the possibility of the GPI anchoring of PrP, the function is retained. Therefore, we conclude that the cell surface GPI-anchored PrP is not necessarily required for the neuroprotective function of PrP against Bax and that either a secreted or transmembrane form of PrP can embody the neuroprotective function. Interestingly, we find that the FFI and FASE PrP mutations undergo a loss of function against Bax-mediated cell death. Whether this is because of altered protein conformation or improper trafficking will need to be determined. However, these results suggest a possible mechanism to account for the extensive neuronal loss in these two neurodegenerative diseases. The unusual transmissible features of prion diseases has led to research where the major emphasis is on the “disease-causing” forms of PrP. Therefore, our finding that PrP is actually a strong neuroprotective agent against the major pro-apoptotic Bax protein is counterintuitive. We require further work in order to decipher the underlying molecular mechanism of PrP function against Bax. However, this study shows an interesting twist regarding the function of the already quite fascinating prion protein. We thank Jennifer Hammond, Beverly Akerman, and Megan Blacker for technical assistance and Dr. Walter Nishioka for the human Bcl-2 cDNA.