Prion diseases are characterized by the conversion of the normal cellular prion protein (PrPC), a glycoprotein that is anchored to the cell membrane by a glycosylphosphatidylinositol moiety, into an isoform that is protease-resistant (PrPres) and pathogenic. In inherited prion diseases, mutations in the prion protein (PrPM) engender the conversion of PrPM into PrPres. We developed a cell model of Gerstmann-Sträussler-Scheinker disease, a neurodegenerative condition characterized by PrPM-containing amyloid deposits and neuronal loss, by expressing the Gerstmann-Sträussler-Scheinker haplotype Q217R-129V in human neuroblastoma cells. By comparison to PrPC, this genotype results in the following alterations of PrPM: 1) expression of an aberrant form lacking the glycosylphosphatidylinositol anchor, 2) increased aggregation and protease resistance, and 3) impaired transport to the cell surface. Most of these alterations are temperature-sensitive, indicating that they are due to misfolding of PrPM. Prion diseases are characterized by the conversion of the normal cellular prion protein (PrPC), a glycoprotein that is anchored to the cell membrane by a glycosylphosphatidylinositol moiety, into an isoform that is protease-resistant (PrPres) and pathogenic. In inherited prion diseases, mutations in the prion protein (PrPM) engender the conversion of PrPM into PrPres. We developed a cell model of Gerstmann-Sträussler-Scheinker disease, a neurodegenerative condition characterized by PrPM-containing amyloid deposits and neuronal loss, by expressing the Gerstmann-Sträussler-Scheinker haplotype Q217R-129V in human neuroblastoma cells. By comparison to PrPC, this genotype results in the following alterations of PrPM: 1) expression of an aberrant form lacking the glycosylphosphatidylinositol anchor, 2) increased aggregation and protease resistance, and 3) impaired transport to the cell surface. Most of these alterations are temperature-sensitive, indicating that they are due to misfolding of PrPM. Prion diseases include inherited and sporadic forms as well as forms that are acquired by infection (1Parchi P. Petersen R.B. Gambetti P. Semin. Virol. 1996; 7: 1-7Crossref Scopus (2) Google Scholar). They are commonly associated with three major phenotypes referred to as: 1) Creutzfeldt-Jakob disease (CJD), 1The abbreviations used are: CJD, Creutzfeldt-Jakob disease; GSS, Gerstmann-Sträussler-Scheinker; GPI, glycosylphosphatidylinositol; FFI, fatal familial insomnia; ER, endoplasmic reticulum; PI-PLC, phosphatidylinositol-specific phospholipase-C; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; RITC, rhodamine isothyocyanate; PK, proteinase K; PNGase,N-glycosidase-F; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-glycine; Endo-H, endoglycosidase H. 2) fatal familial insomnia (FFI), and 3) Gerstmann-Sträussler-Scheinker disease (GSS) (1Parchi P. Petersen R.B. Gambetti P. Semin. Virol. 1996; 7: 1-7Crossref Scopus (2) Google Scholar). All prion diseases are believed to share the same basic pathogenic mechanism that involves the conversion of a normal protein called cellular prion protein (PrPC) into a form that is partially resistant to proteases (PrPres) and is infectious (2Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4611-4614Crossref PubMed Scopus (65) Google Scholar). PrPC, a glycoprotein encoded by a gene (PRNP) located on human chromosome 20 (3Prusiner S.B. DeArmond S.J. Annu. Rev. Neurosci. 1994; 17: 311-319Crossref PubMed Scopus (160) Google Scholar), undergoes the non-obligatory addition of one or two N-linked oligosaccharide chains leading to the expression of three glycoforms containing none, one, or two glycans (4Haraguchi T. Fisher S. Olofsson S. Endo T. Groth D. Tarantino A. Borchelt D.R. Teplow D. Hood L. Burlingame A. Lycke E. Kobata A. Prusiner S.B. Arch. Biochem. Biophys. 1989; 274: 1-13Crossref PubMed Scopus (186) Google Scholar, 5Bolton D.C. Mayer R.K. Prusiner S.B. J. Virol. 1985; 53: 596-606Crossref PubMed Google Scholar, 6Caughey B. Race R.E. Ernst D. Buchmeier M.J. Chesebro B. J. Virol. 1989; 63: 175-181Crossref PubMed Google Scholar) and the linking of a glycosylphosphatidylinositol (GPI) anchor at the C terminus (7Stahl N. Baldwin M.A. Burlingame A.L. Prusiner S.B. Biochemistry. 1990; 29: 8879-8884Crossref PubMed Scopus (145) Google Scholar, 8Stahl N. Borchelt D.R. Hsiao K. Prusiner S.B. Cell. 1987; 51: 229-240Abstract Full Text PDF PubMed Scopus (913) Google Scholar). PrPC is transported to the plasma membrane through the secretory pathway and is then reinternalized and cleaved in the endocytic compartment to generate N-terminal and C-terminal fragments, the latter being returned to the cell surface (9Harris D.A. Huber M.T. van Dijken P. Shyng S.L. Chait B.T. Wang R. Biochemistry. 1993; 32: 1009-1016Crossref PubMed Scopus (189) Google Scholar, 10Shyng S.L. Huber M.T. Harris D.A. J. Biol. Chem. 1993; 268: 15922-15928Abstract Full Text PDF PubMed Google Scholar). The conversion of PrPC to PrPres entails the switch from a predominantly α-helical to a β-sheet conformation resulting in a form that, beside being protease-resistant and infectious, is insoluble in non-ionic detergents, has the propensity to aggregate, and can polymerize into amyloid fibrils (2Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4611-4614Crossref PubMed Scopus (65) Google Scholar). In sporadic prion diseases, PrPres is thought to form initially as the result of a spontaneous stochastic event, and the conversion process would then be maintained autocatalytically by the endogenous PrPres. In inherited prion diseases, it has been hypothesized that the mutant PrP (PrPM) spontaneously converts into PrPres (11DeArmond S.J. Prusiner S.B. Am. J. Pathol. 1995; 146: 785-811PubMed Google Scholar). A distinctive feature of inherited prion diseases is the phenotypic heterogeneity (1Parchi P. Petersen R.B. Gambetti P. Semin. Virol. 1996; 7: 1-7Crossref Scopus (2) Google Scholar). One determinant of this heterogeneity is thePRNP codon 129, the site of a common methionine (129M)/valine (129V) polymorphism (12Goldfarb L.G. Brown P. Goldgaber D. Am. J. Hum. Genect. 1989; 45 (abstr.): 189Google Scholar). Therefore, the PRNPdeterminant of the phenotype in inherited prion diseases is the haplotype established by the pathogenic mutation and codon 129. The Q217R-129V haplotype, which segregates with GSS, is associated with a phenotype characterized by prominent PrP deposits, often with the characteristics of amyloid, which contain PrPres forms of all sizes but especially N-terminal and C-terminal truncated forms (13Tagliavini F. Prelli F. Porro M. Rossi G. Giaccone G. Farlow M.R. Dloughy S.R. Ghetti B. Bugiani O. Frangione B. Cell. 1994; 79: 695-703Abstract Full Text PDF PubMed Scopus (137) Google Scholar). To understand the individual steps involved in the pathogenesis of inherited prion diseases, we have generated several transfected cell models using a human neuroblastoma cell system to characterize PrPM. In this study we report on the metabolism of the PrPM expressed in cells transfected with the Q217R-129VPRNP construct. We observed complex changes in the biogenesis of PrPM which include the following: 1) expression of aberrant PrPM forms, some of which lack the GPI anchor and are retained in the endoplasmic reticulum (ER)-cis Golgi compartment; 2) presence of aggregated forms that exhibit decreased protease sensitivity in intracellular compartments and fail to reach the plasma membrane; and 3) underrepresentation of PrPM at the cell surface. Remarkably, these changes largely revert at reduced temperature, suggesting that abnormal folding of PrPM plays a major role in the pathogenesis of this GSS variant. The human neuroblastoma cell line M17 was obtained from Dr. J. Biedler (Memorial Sloan-Kettering Cancer Center, New York); Opti-MEM, fetal bovine serum, penicillin-streptomycin, methionine and cysteine-free DMEM, and Lipofectin from was from and from was from and from protein was from was to M.A. D.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar) or by T. maintained at in with fetal and in a containing of transfected cells with neuroblastoma cells expressing normal or mutant PrP generated as R.B. P. S.L. Gambetti P. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). cells maintained in containing at on cells. all cells and used at The following to to human PrP a that an on human PrP New in R. M. R. H. J. Virol. 1987; PubMed Google to human PrP L. Brown P. P. Petersen R.B. J. F. P. P. Ghetti B. L.G. D.C. E. Gambetti P. L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google of and A. In a cells used of protein was used from cells expressing normal or mutant with to the using bovine as a To PrP in cellular cells with and in a containing and in Tris-buffered 20 containing of and phenylmethylsulfonyl was by at and the protein in the was with of at by and to at at containing in containing and 20 at and with or in normal and bovine as Teplow P. Gambetti P. L. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). with the to and and on by To the of was at with a and using One To PrP in the cells with as and the PrP was and as in with and with in at The was and at at to cell in the with of at and by and the PrP was by as or PrP with of and in and with in or in at with of at in and by of cell surface cells to on with containing bovine and with at then three with and with an at with PBS, cells in and on a in containing was by the of by the of cells. intracellular cells as and in All was at with as with and the cells with with containing serum, by in then with by with or by The cells in PBS, and observed using a In a cells in used with and in the same at or with in with or at or as The cells and in with methionine and was the of and the cells at the of the the was and the cells with by in PBS, containing of and cell and with in the presence of bovine and with of protein and with of and protein was by in and by PrP by at in of 20 and containing protease and by The was at in a at The was and the was in of in the and with at in and as proteinase cells as in the of protease and with proteinase or The was by the addition of PMSF, and the and cell with and at with in as N. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). was with in The cells with PBS, as and by PrP in the was as by and to PrP by with cellular by the cell at with cellular in the and in the several with and by in A. E. Proc. Natl. Acad. Sci. U. S. A. 1989; PubMed Scopus Google Scholar). and by and the of PrP in was by with or and C of the form by using and in the presence of to a the normal and aberrant form on and the by and in 20 at The gel and at by with at The gel was by of the in a into a at 20 The protein was at with or in as L. Brown P. P. Petersen R.B. J. F. P. P. Ghetti B. L.G. D.C. E. Gambetti P. L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar). The resulting on H. G. Biochem. 1987; PubMed Scopus Google Scholar) and by of the with the to PrP three to the three PrP glycoforms as a of with two glycans an of with one complex and a of the form 1) B. Race R.E. Ernst D. Buchmeier M.J. Chesebro B. J. Virol. 1989; 63: 175-181Crossref PubMed Google Scholar). the and forms with the form at with an that at 20 the N-terminal truncated PrP as in a study Teplow P. Gambetti P. L. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). is prominent in with the terminus to PrP two at and 1) which are N-terminal truncated forms of the and and lack the Teplow P. Gambetti P. L. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). two are prominent following The N-terminal truncated form is it is by the it is to at with the truncated and forms following the following to PrPM with the a prominent of in addition to the three glycoforms in PrPC 3) The is to that of PrPC, that the of form at with the that from PrPC in three as 1) the truncated form is by of 2) truncated forms at and and 3) the PrPM form is by the that the of PrPM and PrPC are PrPM of but the of the glycoforms and of the PrPM The and glycoforms and of the PrPM and are from the of PrPC that are 20 and The form of the PrPM. surface PrPC was with phosphatidylinositol-specific C an that GPI of the protein with the or the same forms as in cell all three and truncated PrPC forms The of cell surface PrPM by is 45 that of PrPC, and the and truncated forms and the form are The underrepresentation of PrPM at the cell surface be due to to of aggregation or aberrant with the plasma membrane S. Harris D.A. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Harris D.A. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). Therefore, we cell surface expression of PrPC and PrPM by and PrPC and PrPM at the cell surface and 1) by and by with or 2) with following cell and by The results obtained with of these are to obtained with and a of the surface PrPM of The PrPC and PrPM on the plasma membrane by using with PrPC, the of PrPM is reduced on by the underrepresentation of PrPM with results in a of the associated with PrPC and of that associated with PrPM these that of PrPM and PrPC are in the intracellular PrPM is at the plasma with impaired transport to that cell In PrPM is at two of and Therefore, to the and transport of a of PrP with a with three that are common to PrPC and PrPM. The two the of the and forms and and exhibit gel from of the forms they contain The with the form and B. Race R.E. Ernst D. Buchmeier M.J. Chesebro B. J. Virol. 1989; 63: 175-181Crossref PubMed Google Scholar, R.B. P. S.L. Gambetti P. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). PrPM two forms at and and a of the glycans the and glycoforms that observed in the and form is in PrPC and the and of the and forms are in PrPC and the of PrPC and PrPM are a and from to PrPC and PrPM in PrPM the of PrPC a The form is and the this form of the the which is a of the intracellular PrPM with the that the truncated forms are prominent in PrPM in the PrPC or of The PrP from the plasma membrane by PI-PLC, and with the at the in PrPC and PrPM cells and The of PrPM is that of PrPC of the PrPM and PrPC, 3) and the form of PrP is at the surface of the mutant cells all three glycoforms are well in the cells of the PrPC with the a in the of the and forms with to the forms with the that these result from the of PrP (9Harris D.A. Huber M.T. van Dijken P. Shyng S.L. Chait B.T. Wang R. Biochemistry. 1993; 32: 1009-1016Crossref PubMed Scopus (189) Google Scholar, A. Borchelt D.R. D. Prusiner S.B. Biol. Cell. PubMed Scopus Google Scholar). In in PrPM the to PrPC and the forms are transported to the surface in The the PrPC and PrPM and that intracellular PrPM is and transported in to the cell that PrPM and is retained in an intracellular as an aggregate, the mutant protein be by would the observed of PrPM in the Therefore, we the site of intracellular and the of intracellular PrPM is of and surface PrPM and PrPC was following with non-ionic and to aggregated from and and by A and PrPC is of a of the is The is to the of the form being In cleaved and surface PrPM are the of the all indicating that aggregation of the in a compartment to M. S. Annu. Rev. Biochem. 1985; PubMed Scopus Google Scholar). To aggregated PrPM be cell and insoluble of PrPC and PrPM to and the resulting was PrP All PrPC and all of the are by a of insoluble PrPM in the the the in PrPM observed in is due to the aggregated PrPM was by in S. Harris D.A. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), the of aggregation be The presence of proteinase PrPM was by PrPC and PrPM cell with of at with that of the PrPM is PrPC is and and All PrPM forms, and the forms, are in the All the forms a in as in prion diseases with to PrP and to cellular we observed that the of intracellular PrPC is with a of the Golgi The of intracellular with the Golgi PrPC and with an and with a of the compartment The of the a and a and that it in the Golgi M. S. Annu. Rev. Biochem. 1985; PubMed Scopus Google Scholar). the form at on following and with the N-terminal to PrP and and suggesting that it the C-terminal GPI and the GPI anchor K. N. T. Biochem. J. 1996; PubMed Scopus Google Scholar). To this and cells with and and 2) or and which the GPI In to the and forms of PrPC and the form is the of the GPI anchor The of the C-terminal GPI is by obtained from and the lack of of the form with the The as well as the and forms are the aberrant forms that are in the PrPM cells A and the the form is to and in the M. S. Annu. Rev. Biochem. 1985; PubMed Scopus Google Scholar). with but with to the or C The and the forms are by the and are that the aggregation of the mutant protein is associated with an aberrant conformation was obtained cells at a to folding of J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. M.J. PubMed Scopus Google Scholar). at this that and of intracellular PrPM is to that of PrPC to of and from that at the aberrant and forms the from the plasma membrane with the and forms that are at of PrP that the of PrPC and PrPM is at at PrPM PrPC and of cells to that PrPM in the form at the and forms the the form and the role of glycans in protein folding and that folding in the of that following a to PrPM predominantly with the Golgi as PrPC with The complex changes in the biogenesis of PrPM by the Q217R-129V genotype that a mutation the metabolism of human PrPM in as was the mutation R.B. P. S.L. Gambetti P. J. Biol. 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In the form to be retained in the it The and characteristics of the aberrant PrPM forms is the of the and the is an or an truncated the in the The and be generated by the aberrant of a The mutation the the of which aggregate, a PrPM is transported to the plasma membrane and the to is to the of the of PrPM and glycoforms are from that of the of with the cell membrane at is the of PrPC and is largely by the the intracellular of PrP are and all the glycoforms are in the PrPM C A and The underrepresentation of the and at the cell in the truncated forms are PrPM in intracellular the form to in the PrPM forms in intracellular by to and with of the of this study is that the of aggregated PrPM is reduced following the of mutant cells at that aggregation of PrPM is the result of folding in the J. Biol. Chem. Full Text PDF PubMed Google Scholar). A has been observed on a mutant with of J. M.J. PubMed Scopus Google Scholar). has have been to the of on S. J. Biol. 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Cell. 1994; 79: 695-703Abstract Full Text PDF PubMed Scopus (137) Google Scholar). In the common PrP of a of and fragments, the latter being the result of (13Tagliavini F. Prelli F. Porro M. Rossi G. Giaccone G. Farlow M.R. Dloughy S.R. Ghetti B. Bugiani O. Frangione B. Cell. 1994; 79: 695-703Abstract Full Text PDF PubMed Scopus (137) Google Scholar). The that the pathogenesis of the disease, the changes are by the of in as well as by the expression of aberrant PrPM The aberrant truncated forms of PrPM are of an abnormal N-terminal that contain the that has been to be G. N. R. E. M. Bugiani O. F. 1993; PubMed Scopus Google Scholar). the cell model and the human disease in two major as 1) the of to of the protease-resistant PrPM in the mutant cells is at and 2) the transfected cells lack the PrPM in the the and the of cells in the of the PrPM in the inherited prion diseases D.R. B. 1996; PubMed Scopus Google Scholar). 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