The synthetic peptide PrP-(106–126) has previously been shown to be neurotoxic. Here, for the first time, we report that it induces apoptosis in the human neuroblastoma cell line SH-SY5Y. The earliest detectable apoptotic event in this system is the rapid depolarization of mitochondrial membranes, occurring immediately upon treatment of cells with PrP-(106–126). Subsequent to this, cytochrome c release and caspase activation were observed. Caspase inhibitors demonstrated that while the peptide activates caspases they are not an absolute requirement for apoptosis. Parallel to caspase activation, PrP-(106–126) was also observed to trigger a rise in intracellular calcium through release of mitochondrial calcium stores. This leads to the activation of calpains, another family of proteases. A calpain inhibitor demonstrated that while calpains are activated by the peptide they also are not an absolute requirement for apoptosis. Interestingly a combination of caspase and calpain inhibitors significantly inhibited apoptosis. This illustrates alternative pathways leading to apoptosis via caspases and calpains and that blocking both pathways is required to inhibit apoptosis. These results implicate the mitochondrion as a primary site of action of PrP-(106–126). The synthetic peptide PrP-(106–126) has previously been shown to be neurotoxic. Here, for the first time, we report that it induces apoptosis in the human neuroblastoma cell line SH-SY5Y. The earliest detectable apoptotic event in this system is the rapid depolarization of mitochondrial membranes, occurring immediately upon treatment of cells with PrP-(106–126). Subsequent to this, cytochrome c release and caspase activation were observed. Caspase inhibitors demonstrated that while the peptide activates caspases they are not an absolute requirement for apoptosis. Parallel to caspase activation, PrP-(106–126) was also observed to trigger a rise in intracellular calcium through release of mitochondrial calcium stores. This leads to the activation of calpains, another family of proteases. A calpain inhibitor demonstrated that while calpains are activated by the peptide they also are not an absolute requirement for apoptosis. Interestingly a combination of caspase and calpain inhibitors significantly inhibited apoptosis. This illustrates alternative pathways leading to apoptosis via caspases and calpains and that blocking both pathways is required to inhibit apoptosis. These results implicate the mitochondrion as a primary site of action of PrP-(106–126). scrapie isoform of prion protein native cellular prion protein permeability transition change in mitochondrial membrane potential scrambled phosphate-buffered saline N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide propidium iodide 5,5′,6–6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolecarbocyanine iodide 530/30 band pass filter 585/42 band pass filter 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis- (acetoxymethyl ester) Prion-related encephalopathies are a family of neurodegenerative disorders including conditions such as scrapie in sheep, bovine spongiform encephalopathy in cattle, and Creutzfeldt-Jakob disease and Gerstmann-Straussler-Scheinker syndrome among others in humans. They are characterized by vacuolation of the neuropil, neuronal loss, and gliosis (1Masters C.L. Richardson Jr., E.P. Brain. 1978; 101: 333-344Crossref PubMed Scopus (309) Google Scholar). In many cases this is also accompanied by the extracellular accumulation of the scrapie isoform (PrPSC)1 of the normal cellular prion protein (PrPC), which can aggregate into fibrils in the extracellular matrix (2Mikol J. Biomed. Pharmacother. 1999; 53: 19-26Crossref PubMed Scopus (11) Google Scholar). PrPSC is widely believed to be the infectious agent of these diseases (3Prusiner S.B. Science. 1991; 252: 1515-1522Crossref PubMed Scopus (1749) Google Scholar), and the formation of PrPSC is thought to be via a post-translational conformational change by which PrPC complexes with PrPSC to yield two molecules of PrPSC (4Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5167) Google Scholar). PrPC is a cell surface protein mainly expressed in the neuronal and glial cells of the central nervous system. The exact function of PrPC remains unknown, although recent studies have implicated it in copper metabolism (5Pauly P.C. Harris D.A. J. Biol. Chem. 1998; 273: 33107-33110Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar, 6Ruiz F.H. Silva E. Inestrosa N.C. Biochem. Biophys. Res. Commun. 2000; 269: 491-495Crossref PubMed Scopus (75) Google Scholar) and signal transduction (7Mouillet-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). Its expression is necessary for the pathogenesis of the spongiform encephalopathies (8Bueler H. Aguzzi A. Sailer A. Greiner R.A. Autenried P. Aguet M. Weissmann C. Cell. 1993; 73: 1339-1347Abstract Full Text PDF PubMed Scopus (1814) Google Scholar). Apoptosis is a physiologically important cellular suicide pathway, which has also been implicated in a number of pathological conditions (9Gorman A.M. McGowan A. O'Neill C. Cotter T. J. Neurol. Sci. 1996; 139: 45-52Abstract Full Text PDF PubMed Scopus (149) Google Scholar). There is some evidence to indicate that the mechanism of neuronal cell death in prion diseases is apoptosis as apoptotic neurons have been observed in the brain of scrapie-infected sheep (10Fairbairn D.W. Carnahan K.G. Thwaits R.N. Grigsby R.V. Holyoak G.R. O'Neill K.L. FEMS Microbiol. Lett. 1994; 115: 341-346Crossref PubMed Scopus (90) Google Scholar), the brain and retinae of mice infected with the 79A strain of scrapie (11Giese A. Groschup M.H. Hess B. Kretzschmar H.A. Brain Pathol. 1995; 5: 213-221Crossref PubMed Scopus (146) Google Scholar), and the brain of human Creutzfeldt-Jakob disease patients (12Gray F. Chretien F. Adle-Biassette H. Dorandeu A. Ereau T. Delisle M.B. Kopp N. Ironside J.W. Vital C. J. Neuropathol. Exp. Neurol. 1999; 58: 321-328Crossref PubMed Scopus (147) Google Scholar). A synthetic peptide corresponding to residues 106–126 of human PrP (PrP-(106–126)) has previously been found to induce apoptosis in primary rat hippocampal cultures (13Forloni G. Angeretti N. Chiesa R. Monzani E. Salmona M. Bugiani O. Tagliavini F. Nature. 1993; 362: 543-546Crossref PubMed Scopus (896) Google Scholar), primary mouse cerebellar cultures (14Brown D.R. Schmidt B. Kretzschmar H.A. Nature. 1996; 380: 345-347Crossref PubMed Scopus (499) Google Scholar), the rat pituitary clonal cell line GH3 (15Florio T. Thellung S. Amico C. Robello M. Salmona M. Bugiani O. Tagliavini F. Forloni G. Schettini G. J. Neurosci. Res. 1998; 54: 341-352Crossref PubMed Scopus (79) Google Scholar), and more recently in mouse retinae in vivo (16Ettaiche M. Pichot R. Vincent J.P. Chabry J. J. Biol. Chem. 2000; 275: 36487-36490Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). PrP-(106–126) is one of a number of peptides corresponding to sequences within fragments of amyloid proteins isolated from the brains of patients suffering from Gerstmann-Straussler-Scheinker syndrome (17Tagliavini F. Prelli F. Ghiso J. Bugiani O. Serban D. Prusiner S.B. Farlow M.R. Ghetti B. Frangione B. EMBO J. 1991; 10: 513-519Crossref PubMed Scopus (171) Google Scholar). It retains the ability of PrPSC to aggregate into amyloid-like fibrils and the tendency to adopt a mostly β-sheet structure (18Tagliavini F. Prelli F. Verga L. Giaccone G. Sarma R. Gorevic P. Ghetti B. Passerini F. Ghibaudi E. Forloni G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9678-9682Crossref PubMed Scopus (235) Google Scholar). Residues 106–126 of PrP constitute a region maintained in all the PrP isoforms that have been found to accumulate in the brains of patients suffering from prion diseases. Under normal physiological conditions a catabolic pathway also leads to cleavage of this region of the prion protein at residues 110 and 111 (19Chen 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). The importance of the 106–126 sequence of the prion protein makes it a useful model for the in vitro study of prion-induced cell death. In this study we determine, for the first time, the effect of PrP-(106–126) in human neuroblastoma cells in vitro and demonstrate its ability to induce apoptosis. Furthermore, we establish the mitochondrion as a target of PrP-(106–126) action and show that the peptide activates two distinct biochemical pathways involving caspases and calpains, which both result in apoptosis. PrP-(106–126) (KTNMKHMAGAAAAGAVVGGLG) and PrP-(106–126) scrambled (scr) (AVHTGLGAMAALNMVVGGAAGL) were synthesized and purified by MWG Biotech (Milton Keynes, UK). Peptides were dissolved in sterile phosphate-buffered saline (PBS) to a concentration of 1 mm before use and were freshly prepared before each experiment. The inhibitors z-VAD-fmk (Enzymes Systems Products, Livermore, CA) and calpeptin (Sigma) were added to cells to The of peptide was by M.H. J. H. C. M. Biochem. J. 2000; PubMed Scopus Google Scholar). of peptides in were added to with (Sigma) to a concentration of were at for and was a with and of and were prepared in The human neuroblastoma cell line was in with and were maintained at in a cells were maintained in with and for cells were was by the of (Sigma) to a of cells with was added to each to a concentration of and for at were at for The was by of the and of of to each to the the were at a were in of was by that and propidium iodide with cells were in and with and for at were by a UK). was through a 530/30 band pass filter to and through a 585/42 band pass filter to in is of apoptosis before an in of is observed. was isolated and by the of A. D.R. Biol. 1995; PubMed Scopus Google Scholar). were in mm 1 mm 1 1 mm mm mm and 1 of protein inhibitor 1 1 and 1 and for were at for The was into and protein were a protein was by of and to a were and the The were mouse and c were and with for at S. A. C. A. Lett. PubMed Scopus Google Scholar). membrane potential was by a was through a the of which in number as the mitochondrial membrane were in of in for and in 1 of mm were in of primary for 1 at and in 1 of was and to the in each of was were for 1 in and in 1 of were in of and was a through in the is of caspase with an and with were as were in and and in of mm mm for the were at for to the membrane The was to a and the membrane was in of mm mm A protein was both and an of protein was into each of a of each of Systems was and the of each was to with mm 1 mm mm in were at for the of the was a with and of and were in and in of of was added to each were in of each was into of a were at for in and a with and of and The of was by the in at to H. in Scholar). was as of change of in intracellular calcium concentration were by cells with for to and of a was through a and an in is of an in calcium were in 1 of 1 mm and bovine in of and were at for at and the was to a The was at for at The was in of 1 bovine mm and 1 at for at and in of 1 bovine the was at for at and in c was by c The was as the PrP-(106–126) has previously been to induce cell death as a result of its ability to (13Forloni G. Angeretti N. Chiesa R. Monzani E. Salmona M. Bugiani O. Tagliavini F. Nature. 1993; 362: 543-546Crossref PubMed Scopus (896) Google Scholar, J. A. N.C. 1996; 5: PubMed Scopus Google Scholar). The of the peptide in this study was by of a in the of protein PrP-(106–126) was to aggregate immediately at prepared at in and it its The scrambled of the peptide PrP-(106–126) was observed to induce cell death in a as by 1 is to a by mitochondrial which as the cell of this is an of cell The mechanism of cell death by PrP-(106–126) was shown to be apoptosis by 1 to which from the to the of the cell membrane apoptosis D.R. J. Exp. 1995; PubMed Scopus Google Scholar). Apoptosis be as as treatment of the cell were were with and The of cells in the cells 1 in cells in the This is the apoptotic The in to the of membrane permeability and The of the in each is in the The mechanism of cell death was to be via apoptosis by the of upon of isolated from peptide cells The cleavage of into fragments is a biochemical of apoptosis in a number of Cotter The Scholar). were observed upon treatment with A number of apoptotic are observed to treatment of cells with characterized by a of potential has been found to be a central event in many cases of apoptosis Cotter 1998; Scopus Google Scholar). of the of by an in of to of within of treatment with the is at mitochondrial membrane depolarization is observed to immediately upon treatment of the cells with PrP-(106–126) in is as a function of the within this cytochrome c is from the into the and is detectable by the of an a a protein of the mitochondrial that is apoptosis membrane which leads to caspase The caspases are a family of for the cleavage of a number of apoptosis C.L. G. Res. 2000; Google Scholar). these apoptotic at apoptosis is not detectable by In of the rapid of PrP-(106–126) the we the family of These are important of of both and and are to at the of the mitochondrion D.A. J. 1998; PubMed Scopus Google Scholar, S. J. S. J.M. S. S. F. J. 1998; PubMed Google Scholar, M. N. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). the intracellular of an cells were with PrP-(106–126) a treatment with PrP-(106–126) was a of protein as with the change in the of also the of the peptide family and In to was observed with we found change in the intracellular of in to PrP-(106–126). a caspase cleavage site was previously to be in A. N.C. R. M.B. J.M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), the effect of a caspase inhibitor previously to was The of by PrP-(106–126) was found to be inhibited in cells that were with with change in A concentration of was shown to as by the the of caspases the protein of the in the of PrP-(106–126). The treatment of cells with not inhibit mitochondrial depolarization this event of caspase activation and not inhibit apoptosis blocking that caspases are not necessary for PrP-(106–126) to induce cell death. caspases found to be for apoptosis in this another mechanism be at the as this was the site of the earliest observed effect of we the of to of mitochondrial function as this is the primary site of intracellular has been shown to have a in a number of apoptotic S. McGowan Cotter Biochem. 1995; PubMed Scopus Google Scholar, C. S. 1996; Google Scholar). the intracellular of and as as the of and not also at the and the of by the in at to in to PrP-(106–126). not as a also in the of the intracellular and is the primary mechanism of the in to PrP-(106–126). and were as as both these induce in The peptide was not found to the cells to death by a not function of the mitochondrion is in the of intracellular calcium we PrP-(106–126) be an effect through the of calcium the we demonstrated an intracellular rise in calcium by an in in to PrP-(106–126) The is and calcium a by This was in the of a calcium to show that the of the calcium was intracellular not an intracellular calcium was found to inhibit this in calcium the intracellular of the of the calcium rise The two calcium in the cell are the and the which was calcium into the in this system we which a rapid release of calcium stores. treatment of cells with an in calcium was observed the that its cells were with and a in calcium was observed This that PrP-(106–126) calcium from a site the The the of calcium in the cell to for the rise in calcium the of calpains in to the rise in intracellular The calpains are a of that have also been implicated in apoptosis. They can in and with the cell membrane the N. Acad. Sci. 1999; PubMed Scopus Google Scholar, T. T. H. Neurosci. Lett. 2000; PubMed Scopus Google Scholar, B. M. K.L. J. 1999; 73: PubMed Google Scholar). of cells with PrP-(106–126) an in calpain was in both the cells in and membrane as a calpain of cells with the calpain inhibitor calpeptin was found to inhibit the of the calpains by PrP-(106–126) not cell death. cells were with a combination of and apoptosis by PrP-(106–126) was significantly inhibited The of the PrP-(106–126) peptide in a human neuronal cell line has not been demonstrated expression of PrPC has been to be a of PrP-(106–126) (14Brown D.R. Schmidt B. Kretzschmar H.A. Nature. 1996; 380: 345-347Crossref PubMed Scopus (499) Google Scholar). we observed the of PrPC in the cell line upon not In some the of PrP-(106–126) has been found to the of neuronal cells with (14Brown D.R. Schmidt B. Kretzschmar H.A. 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In we have for the first time, that PrP-(106–126) induces apoptosis in a human neuronal cell The site of action to be the mitochondrion membrane depolarization and calcium release This is through an with the which is to be the of the mitochondrion the apoptotic can be through the action of caspases calpains, and apoptosis can be by the of
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