Jacques Grassi

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTEnzyme immunoassays of eicosanoids using acetylcholine esterase as label: an alternative to radioimmunoassayP. Pradelles, J. Grassi, and J. MacloufCite this: Anal. Chem. 1985, 57, 7, 1170–1173Publication Date (Print):June 1, 1985Publication History Published online1 May 2002Published inissue 1 June 1985https://pubs.acs.org/doi/10.1021/ac00284a003https://doi.org/10.1021/ac00284a003research-articleACS PublicationsRequest reuse permissionsArticle Views731Altmetric-Citations549LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

Prion diseases are transmissible neurodegenerative disorders affecting humans and animals for which no therapeutic or prophylactic regimens exist. During the last three years several studies have shown that anti-PrP monoclonal antibodies (mAbs) can antagonize prion propagation in vitro and in vivo, but the mechanisms of inhibition are not known so far. To identify the most powerful mAbs and characterize more precisely the therapeutic effect of anti-PrP antibodies, we have screened 145 different mAbs produced in our laboratory for their capacity to cure cells constitutively expressing PrPSc. Our results confirm for a very large series of antibodies that mAbs recognizing cell-surface native PrPc can efficiently clean and definitively cure infected cells. Antibodies having a cleaning effect are directed against linear epitopes located in at least four different regions of PrP, suggesting an epitope-independent inhibition mechanism. The consequence of antibody binding is the sequestration of PrPc at the cell surface, an increase of PrPc levels recovered in cell culture medium, and an internalization of antibodies. Taken together these data suggest that the cleaning process is more likely due to a global effect on the PrP trafficking and/or transconformation process. Two antibodies, Sha31 and BAR236, show an IC50 of 0.6 nM, thus appearing 10-fold more efficient than previous antibodies described in the literature. Finally, five co-treatments were also tested, and only one of them, described previously (SAF34 + SAF61), lowered PrPSc levels in the cells synergistically.

Cyclooxygenase (Cox) exists in two forms in human endothelial cells (HUVEC). We have raised antibodies that recognize the sequence of the carboxyl-terminal portion of the human Cox-2 (C)-NASSSRSGLD-DINPTVLLK. Cyclooxygenase activity of HUVEC challenged with interleukin 1 alpha or a phorbol ester increased in parallel with the mass of a protein doublet analyzed by Western blot using antibodies directed against the Cox-2 peptide; a monoclonal antibody directed against Cox-1 showed a small change in protein mass. A 35S-labeled protein doublet with a molecular mass of approximately 70,000 daltons was immunoprecipitated with the anti-Cox-2 antiserum in L-[35S] methionine-labeled cells stimulated with interleukin 1 alpha. This protein was not recovered by pretreating the antiserum with the Cox-2 peptide before immunoprecipitation. A minor variation in 35S-immunoprecipitated protein was obtained with the polyclonal anti-Cox-1 antibody. Both immunoprecipitated Cox-1 and Cox-2 possessed cyclooxygenase activity that was inhibited by flurbiprofen. Endoglycosidase H treatment of immunoprecipitated Cox-2 proteins caused a decline in the apparent molecular size similar to that observed with immunoprecipitated Cox-1 or sheep cyclooxygenase but did not suppress the doublet. These results show by direct protein measurement that HUVEC synthesize the novel Cox-2 under appropriate stimulation, with little changes of Cox-1.

We showed previously that PrPc undergoes constitutive and phorbol ester-regulated cleavage inside the 106–126 toxic domain of the protein, leading to the production of a fragment referred to as N1. Here we show by a pharmacological approach thato-phenanthroline, a general zinc-metalloprotease inhibitors, as well as BB3103 and TAPI, the inhibitors of metalloenzymes ADAM10 (Adisintegrinand metalloprotease); and TACE,tumor necrosis factorα-converting enzyme; ADAM17), respectively, drastically reduce N1 formation. We set up stable human embryonic kidney 293 transfectants overexpressing human ADAM10 and TACE, and we demonstrate that ADAM10 contributes to constitutive N1 production whereas TACE mainly participates in regulated N1 formation. Furthermore, constitutive N1 secretion is drastically reduced in fibroblasts deficient for ADAM10 whereas phorbol 12,13-dibutyrate-regulated N1 production is fully abolished in TACE-deficient cells. Altogether, our data demonstrate for the first time that disintegrins could participate in the catabolism of glycosyl phosphoinositide-anchored proteins such as PrPc. Second, our study identifies ADAM10 and ADAM17 as the protease candidates responsible for normal cleavage of PrPc. Therefore, these disintegrins could be seen as putative cellular targets of a therapeutic strategy aimed at increasing normal PrPcbreakdown and thereby depleting cells of the putative 106–126 “toxic” domain of PrPc. We showed previously that PrPc undergoes constitutive and phorbol ester-regulated cleavage inside the 106–126 toxic domain of the protein, leading to the production of a fragment referred to as N1. Here we show by a pharmacological approach thato-phenanthroline, a general zinc-metalloprotease inhibitors, as well as BB3103 and TAPI, the inhibitors of metalloenzymes ADAM10 (Adisintegrinand metalloprotease); and TACE,tumor necrosis factorα-converting enzyme; ADAM17), respectively, drastically reduce N1 formation. We set up stable human embryonic kidney 293 transfectants overexpressing human ADAM10 and TACE, and we demonstrate that ADAM10 contributes to constitutive N1 production whereas TACE mainly participates in regulated N1 formation. Furthermore, constitutive N1 secretion is drastically reduced in fibroblasts deficient for ADAM10 whereas phorbol 12,13-dibutyrate-regulated N1 production is fully abolished in TACE-deficient cells. Altogether, our data demonstrate for the first time that disintegrins could participate in the catabolism of glycosyl phosphoinositide-anchored proteins such as PrPc. Second, our study identifies ADAM10 and ADAM17 as the protease candidates responsible for normal cleavage of PrPc. Therefore, these disintegrins could be seen as putative cellular targets of a therapeutic strategy aimed at increasing normal PrPcbreakdown and thereby depleting cells of the putative 106–126 “toxic” domain of PrPc. human embryonic kidney phorbol 12,13-dibutyrate β-amyloid precursor protein secreted α-secretase-derived βAPP Spongiform encephalopathies are neurodegenerative diseases that are characterized by the cerebral deposition of a 33–35-kDa protein called prion (1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5130) Google Scholar). It is thought that the prion-associated pathology occurs when normal prion, referred to as cellular prion or PrPc, is converted into an insoluble and highly protease-resistant protein particle called PrPres or scrapie (PrPsc) (2Ghetti B. Piccardo P. Frangione B. Bugiani O. Giaccone G. Young K. Prelli F. Farlow M.R. Dlouhy S.R. Tagliavini F. Brain Pathol. 1996; 6: 127-145Crossref PubMed Scopus (172) Google Scholar). Prion diseases, which can be of sporadic or genetic origins, all led to fatal issues (1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5130) Google Scholar). Although normal and pathogenic PrP have the same primary structures, it appears that they could undergo distinct post-transductional events (2Ghetti B. Piccardo P. Frangione B. Bugiani O. Giaccone G. Young K. Prelli F. Farlow M.R. Dlouhy S.R. Tagliavini F. Brain Pathol. 1996; 6: 127-145Crossref PubMed Scopus (172) Google Scholar). Among them, several lines of evidence indicate that PrP is targeted by distinct proteolytic activities as was shown in normal and Creutzfeldt-Jakob-affected brains (3Chen 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). Thus, the normal cleavage appears to occur at the 110/111–112 peptide bond (leading to a fragment referred to as N1; see Fig. 1A) whereas the pathological breakdown is located more N-terminally at the 90–91 site (3Chen 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). This leftward shift leads to the preservation of the 106–126 sequence domain of PrP, which behaves as the toxic “core” of the protein (4Forloni G. Angeretti N. Chiesa R. Monzani E. Salmona M. Bugiani O. Tagliavini F. Nature. 1993; 362: 543-546Crossref PubMed Scopus (896) Google Scholar, 5Brown D.R. Trends Neurosci. 2001; 24: 85-90Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Normal cleavage could be seen as a means to deplete the protein of its potential pathogenicity. Thus, the nature of the proteases involved in the “normal” cleavage of PrPc and the putative up-regulators of such a process are of considerable interest. We demonstrated recently that in human HEK2931 cells, as well as in murine TSM1 neurons, normal PrPc was cleaved constitutively (6Vincent 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). As reported previously, the secreted fragment has a molecular mass of about 11.5 kDa and is labeled by SAF32 and 8G8 but not by PRI308 (see Fig. 1B), which recognizes an epitope overlapping the 111–112 bond (see “Materials and Methods”). Therefore, both molecular mass and immunological characterization indicate that the secreted fragment corresponds to the N1 product generated upon proteolytic attack of PrPc at the 111–112 peptide bond. This hydrolysis could be up-regulated by several effectors of the protein kinase C pathway but not by protein kinase A agonists (6Vincent 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). Here we identified the proteases involved in both constitutive and regulated hydrolytic pathways by combined pharmacological, transfection, and knockout analyses. SAF32 raised against the 79–92 residues of PrP and all other monoclonal antibodies appearing in Fig. 1 have been characterized previously (7Demart S. Fournier J.G. Creminon C. Frobert Y. Lamoury F. Marce D. Lasmezas C. Dormont D. Grassi J. Deslys J.P. Biochem. Biophys. Res. Commun. 1999; 265: 652-657Crossref PubMed Scopus (126) Google Scholar). The rabbit polyclonal AL45 directed against TACE was described previously (8Zhang Y. Jiang J. Black R.A. Baumann G. Frank S.J. Endocrinology. 2000; 141: 4342-4348Crossref PubMed Scopus (117) Google Scholar). ADAM10 was detected with a polyclonal antibody from Euromedex. Phorbol 12,13-dibutyrate (PDBu),o-phenanthroline, pepstatin, E64, and 4-(2-Aminoethyl)benzenesulfonyl-fluoride were from Sigma. BB3103 (hydroxamic acid-based zinc metalloprotease inhibitor) was kindly provided by British Biotech, and TAPI (a tumor necrosis factor α-converting enzyme inhibitor) was kindly supplied by Immunex. HEK293 cells were cultured as described previously (6Vincent 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). HEK293 cells overexpressing ADAM10 or TACE were obtained after transfection of 2 μg of ADAM10 and TACE cDNA with DAC30 reagent (Eurogentec). Positive clones were identified by Western blot analysis by means of the above anti-TACE- and -ADAM10-specific polyclonal antibodies. Cells were maintained at 37 °C in 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum containing penicillin (100 units/ml−1), streptomycin (50 mg/ml−1), and geneticin (0.5 mg/ml−1). Mouse embryonic fibroblasts (wild-type, ADAM10−/−, and TACE−/−) were cultured at 37 °C in 5% CO2 in 50% Dulbecco's modified Eagle's medium/50% Ham's F-12 containing 5% fetal calf serum containing penicillin (100 units/ml−1) and streptomycin (50 mg/ml−1). HEK293 cells grown in 35-mm dishes were washed twice with phosphate-buffered saline and resuspended in 500 μl of lysis buffer (10 mm Tris/HCl, pH 7.5, 150 mm NaCl, 0.5% Triton X-100, 0.5% deoxycholate, 5 mm EDTA) in the presence of a protease inhibitor mixture (Sigma) and then 50 μg of protein were subjected to SDS-polyacrylamide gel electrophoresis on an 8% Tris/glycine gel. Proteins were transferred onto nitrocellulose membrane (2 h, 100 V) and incubated overnight at 4 °C with AL45 or anti-ADAM10 antibodies (dilution 1/1000 in phosphate-buffered saline/0.05% Tween/5% milk). Bound antibodies were detected using a goat anti-rabbit peroxydase-conjugated secondary antibody (dilution 1/5000) (Amersham Pharmacia Biotech), and immunological complexes were revealed with enhanced chemiluminescence as described (6Vincent 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). Identification and immunological characterization of N1 was reported previously (6Vincent 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). Briefly, cells cultured in 35-mm dishes were washed twice with phosphate-buffered saline and incubated for 8 h at 37 °C in the absence (control) or in the presence of various pharmacological agents in 1 ml of serum-depleted Dulbecco's modified Eagle's medium. Media were collected, immunoprecipitated, and identified by Western blot analysis with SAF32 (see Ref. 6Vincent 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 for details). Corresponding cells were lysed, and PrPc was detected by Western blot analysis as reported (6Vincent 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). Statistical analyses were performed with Prism software (Graphpad Software, San Diego, CA) using the unpaired t test for pairwise comparisons. All results are expressed as means ± S.E. values, and statistical significance corresponds to a p value <0.05. We established that among a series of classical inhibitors that target distinct classes of proteases, only o-phenanthroline, a zinc-metalloprotease-blocking agent, was able to drastically (and to a similar extent) reduce N1 production by TSM1 and HEK293 cells whereas serine, thiol, and acidic protease inhibitors were ineffective (Fig.2A). To identify putative metalloenzymes involved in N1 production, we examined the effect of TAPI and BB3103. These inhibitors have been shown to block ADAM10 (adisintegrin andmetalloprotease) and TACE (9Middelhoven P.J. Ager A. Roos D. Verhoeven A.J. FEBS Lett. 1997; 414: 14-18Crossref PubMed Scopus (54) Google Scholar, 10Black R.A. Rauch C.T. Kozlosky C.J. Peschon J. Slack J.L. Wolfson M.F. Castner B.J. Stocking K.L. Reddy P. Srinivasan S. Nelson N. Boiani N. Schosley K.A. Gerhart M. Davis R. Fitzner J.N. Johnson R.S. Paxton R.S. March C.J. Cerretti D.P. Nature. 1997; 385: 729-733Crossref PubMed Scopus (2705) Google Scholar). These metalloenzymes are responsible for the “shedding” of various transmembrane proteins (11Massague J. Pandiella A. Annu. Rev. Biochem. 1993; 62: 515-541Crossref PubMed Scopus (600) Google Scholar, 12Hooper N.M. Karran E.H. Turner A.J. Biochem. J. 1997; 321: 265-279Crossref PubMed Scopus (560) Google Scholar) and have been shown to contribute to the constitutive and protein kinase C-regulated α-secretase cleavage of the β-amyloid precursor protein in various cell lines including HEK293 cells (13Lammich S. Kojro E. Postina R. Gilbert S. Pfeiffer R. Jasionowski M. Haass C. Fahrenholz F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3922-3927Crossref PubMed Scopus (983) Google Scholar, 14Buxbaum J.D. Liu K.N. Luo Y. Slack J.L. Stocking K.L. Peschon J. Johnson R.S. Castner B.J. Cerretti D.P. Black R.A. J. Biol. Chem. 1998; 273: 27765-27767Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar, 15Lopez-Perez E. Zhang Y. Frank S.J. Creemers J. Seidah N. Checler F. J. Neurochem. 2001; 76: 1532-1539Crossref PubMed Scopus (120) Google Scholar). Both TAPI and BB3103 significantly reduce the production of N1 in TSM1 and HEK293 cells (Fig. 2B). It is noteworthy that a 10 μm concentration of TAPI and BB3103 diminishes constitutive N1 production to a same extent but does not totally abolish its formation (Fig. 2C, from four independent experiments). Furthermore, we established that TAPI and BB3103 did not produce an additive effect on constitutive N1 production, indicating that the two inhibitors likely block an identical protease involved in basal N1 production (not shown). Interestingly, TAPI-and BB3103-mediated reduction of N1 is very similar to that achieved by means of o-phenanthroline (Fig. 2, A–C). To delineate the respective contribution of ADAM10 and TACE in N1 formation, we set up stably transfected HEK293 cells overexpressing these enzymes (Fig. 3A). The PDBu-sensitive N1 formation was drastically enhanced in TACE-expressing cells but not in ADAM10 transfectants (Fig. 3, B andC). By contrast, ADAM 10 slightly increases constitutive production of N1 (not shown). The contribution of ADAM10 and TACE in the constitutive and PDBu-regulated N1 formation, respectively, was further examined by the selective depletion of their genes. First, we verified that wild-type mice embryonic fibroblasts also exhibit both constitutive and protein kinase C-regulated N1 formation (Fig.4A). As expected, TACE−/− and ADAM10−/− fibroblasts did not display TACE and ADAM10 immunoreactivities (Fig. 4B). The deficiency of ADAM10 gene led to a mean 51% reduction of constitutive N1 formation without altering the extent of the responsiveness to phorbol esters (Fig. 4,C and E). This reduction was consistently observed with three distinct clones (clone 3, 44 ± 7% of inhibition, n = 4; clone 7, 70 ± 4,n = 4; clone 40, 48 ± 11, n = 6). Conversely, TACE gene disruption fully abolishes the phorbol ester-stimulated N1 production without significantly affecting N1 constitutive production (Fig. 4, D and F). Transfection analysis and gene disruption clearly demonstrated that the PDBu-regulated pathway of normal PrPc is fully ascribable to TACE. By contrast, it appears that ADAM10 only partially contributes to the constitutive N1 formation as underlined by the 50% inhibition of N1 recovery observed in ADAM10−/− fibroblasts. This extent of inhibition (about 50%) matches that observed with the disintegrin inhibitors TAPI and BB3103 (Fig. 2). That TAPI- and BB3103-sensitive constitutive N1 formation is because of ADAM10 appears likely, but one cannot exclude the possibility that another disintegrin(s) also contribute to residual N1 production. In this context, it should be noted that Schlöndorff et al. (16Schlöndorff J. Lum L. Blobel C.P. J. Biol. Chem. 2001; 276: 14665-14674Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) described recently two shedding proteases that, based on pharmacological and biochemical properties, appear distinct from TACE and ADAM10 and that could be detected in mouse embryonic fibroblasts. It is noticeable that shedding enzymes have been characterized as proteolytic activities involved in the release of extracellular domains of various transmembrane proteins (11Massague J. Pandiella A. Annu. Rev. Biochem. 1993; 62: 515-541Crossref PubMed Scopus (600) Google Scholar, 12Hooper N.M. Karran E.H. Turner A.J. Biochem. J. 1997; 321: 265-279Crossref PubMed Scopus (560) Google Scholar). Our study is, to our knowledge, the first demonstration of the involvement of disintegrins in the cleavage of a glycosylphosphatidylinositol-anchored proteins. The parallel between the physiological cleavage occurring on the β-amyloid precursor protein (βAPP) and PrPc is extremely interesting. Thus, βAPP undergoes a normal cleavage by an activity referred to as α-secretase (for review see Ref. 17Checler F. J. Neurochem. 1995; 65: 1431-1444Crossref PubMed Scopus (421) Google Scholar). This processing leads to the secretion of sAPPα, an N-terminal fragment exhibiting neuroprotective and cytotrophic properties (18Mattson M.P. Physiol. Rev. 1997; 77: 1081-1132Crossref PubMed Scopus (877) Google Scholar). Several studies indicated that sAPPα production could also be constitutive or regulated in a protein kinase C-dependent manner (for review see Ref. 17Checler F. J. Neurochem. 1995; 65: 1431-1444Crossref PubMed Scopus (421) Google Scholar). Interestingly, several studies reported on a major involvement of disintegrins in the α-secretase cleavage of βAPP. Thus, ADAM10 appears to contribute to the constitutive sAPPα production in various cell lines (13Lammich S. Kojro E. Postina R. Gilbert S. Pfeiffer R. Jasionowski M. Haass C. Fahrenholz F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3922-3927Crossref PubMed Scopus (983) Google Scholar, 14Buxbaum J.D. Liu K.N. Luo Y. Slack J.L. Stocking K.L. Peschon J. Johnson R.S. Castner B.J. Cerretti D.P. Black R.A. J. Biol. Chem. 1998; 273: 27765-27767Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar, 15Lopez-Perez E. Zhang Y. Frank S.J. Creemers J. Seidah N. Checler F. J. Neurochem. 2001; 76: 1532-1539Crossref PubMed Scopus (120) Google Scholar) whereas TACE is predominantly responsible for PDBu-regulated α-secretase cleavage (14Buxbaum J.D. Liu K.N. Luo Y. Slack J.L. Stocking K.L. Peschon J. Johnson R.S. Castner B.J. Cerretti D.P. Black R.A. J. Biol. Chem. 1998; 273: 27765-27767Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar). In both βAPP and PrPc catabolisms, it appears that an additional and yet unidentified activity also participates to the constitutive production of either sAPPα (15Lopez-Perez E. Zhang Y. Frank S.J. Creemers J. Seidah N. Checler F. J. Neurochem. 2001; 76: 1532-1539Crossref PubMed Scopus (120) Google Scholar) or N1 (present work). Overall, the above observations indicate that both βAPP and PrPc undergo constitutive and protein kinase C-dependent normal cleavage because of two disintegrins, ADAM10 and TACE, mainly responsible for the basal and regulated breakdowns, respectively. ADAM10 and TACE cleavages occur at the 110–111/112 bond,i.e. inside the 106–126 domain that has been suggested to bear the toxic potential of the protein. Here again, it is striking to note that α-secretase, when targeting βAPP, cleaves inside a sequence domain corresponding to the β-amyloid peptide, the “pathogenic” component of senile plaques invading the cortical areas of Alzheimer's disease-affected brains (19Selkoe D.J. Annu. Rev. Neurosci. 1994; 17: 489-517Crossref PubMed Scopus (828) Google Scholar). Interestingly, it was shown that the enhancement of α-secretase cleavage by protein kinase C activation in mice engineered to overproduce the Aβ peptide led to a 50% inhibition of Aβ load in mouse brain (20Savage M. Trusko S.P. Howland D.S. Pinsker L.R. Mistretta S. Reaume A.G. Greenberg B.D. Siman R. Scott R.W. J. Neurosci. 1998; 18: 1743-1752Crossref PubMed Google Scholar). Therefore, ADAM10 and TACE could theoretically be seen as potential therapeutic targets, and increasing their activity could be seen as a means to deplete cells from the 106–126 toxic core borne by PrPc. Endogenous PrPc is thought to be necessary for infectivity of pathogenic inoculates in mice (1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5130) Google Scholar, 21Blättler T. Brandner S. Raeber A.J. Klein M.A. Volgtländer T. Weissmann C. Aguzzi A. Nature. 1997; 389: 69-73Crossref PubMed Scopus (244) Google Scholar). Infectious inoculates are innocuous in mice in which the PrP gene has been knocked out. Whether infectivity is also reduced in mice overexpressing ADAM10 or TACE because of reduced endogenous content of PrPc is currently being examined. TAPI, TACE cDNA, and TACE−/− fibroblasts were generously provided by Dr. R. Black (Immunex), and BB3103 was a kind gift from British Biotech. ADAM10 cDNA was a kind gift from Dr. C. Lunn (Sherring Plough). We are indebted to Drs. Y. Zhang and S. Franck for providing anti-TACE antibodies.