Altered Glycosylated PrP Proteins Can Have Different Neuronal Trafficking in Brain but Do Not Acquire Scrapie-like Properties
2005; Elsevier BV; Volume: 280; Issue: 52 Linguagem: Inglês
10.1074/jbc.m509557200
ISSN1083-351X
AutoresEnrico Cancellotti, Frances K. Wiseman, Nadia L. Tuzi, Herbert Baybutt, Paul Monaghan, L Aitchison, Jennifer Simpson, Jean Manson,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoN-Linked glycans have been shown to have an important role in the cell biology of a variety of cell surface glycoproteins, including PrP protein. It has been suggested that glycosylation of PrP can influence the susceptibility to transmissible spongiform encephalopathy and determine the characteristics of the many different strains observed in this particular type of disease. To understand the role of carbohydrates in influencing the PrP maturation, stability, and cell biology, we have produced and analyzed gene-targeted murine models expressing differentially glycosylated PrP. Transgenic mice carrying the PrP substitution threonine for asparagine 180 (G1) or threonine for asparagine 196 (G2) or both mutations combined (G3), which eliminate the first, second, and both glycosylation sites, respectively, have been generated by double replacement gene targeting. An in vivo analysis of altered PrP has been carried out in transgenic mouse brains, and our data show that the lack of glycans does not influence PrP maturation and stability. The presence of one chain of sugar is sufficient for the trafficking to the cell membrane, whereas the unglycosylated PrP localization is mainly intracellular. However, this altered cellular localization of PrP does not lead to any overt phenotype in the G3 transgenic mice. Most importantly, we found that, in vivo, unglycosylated PrP does not acquire the characteristics of the aberrant pathogenic form (PrPSc), as was previously reported using in vitro models. N-Linked glycans have been shown to have an important role in the cell biology of a variety of cell surface glycoproteins, including PrP protein. It has been suggested that glycosylation of PrP can influence the susceptibility to transmissible spongiform encephalopathy and determine the characteristics of the many different strains observed in this particular type of disease. To understand the role of carbohydrates in influencing the PrP maturation, stability, and cell biology, we have produced and analyzed gene-targeted murine models expressing differentially glycosylated PrP. Transgenic mice carrying the PrP substitution threonine for asparagine 180 (G1) or threonine for asparagine 196 (G2) or both mutations combined (G3), which eliminate the first, second, and both glycosylation sites, respectively, have been generated by double replacement gene targeting. An in vivo analysis of altered PrP has been carried out in transgenic mouse brains, and our data show that the lack of glycans does not influence PrP maturation and stability. The presence of one chain of sugar is sufficient for the trafficking to the cell membrane, whereas the unglycosylated PrP localization is mainly intracellular. However, this altered cellular localization of PrP does not lead to any overt phenotype in the G3 transgenic mice. Most importantly, we found that, in vivo, unglycosylated PrP does not acquire the characteristics of the aberrant pathogenic form (PrPSc), as was previously reported using in vitro models. Glycoproteins are subject to a number of post-translational modifications as they pass through the secretory pathway. During polypeptide chain synthesis, N-glycosylation is initiated by the transfer of core glycans to target asparagines. Processing of core glycans into the complex type is then achieved in the endoplasmic reticulum (ER) 4The abbreviations used are: ERendoplasmic reticulumBSAbovine serum albuminESembryonic stemFFIfatal familial insomniaGPIglycosylphosphatidylinositolPBSphosphate-buffered salinePIPLCphosphatidylinositol phospholipase CPKproteinase KTSEtransmissible spongiform encephalopathyPMSFphenylmethylsulfonyl fluoride. and Golgi apparatus compartments (1Helenius A. Aebi M. Science. 2001; 291: 2364-2369Crossref PubMed Scopus (1986) Google Scholar). Protein-attached glycans have been shown to have a wide range of biological functions, most notably stabilization of protein structure and cellular trafficking (2Lowe J.B. Marth J.D. Annu. Rev. Biochem. 2003; 72: 643-691Crossref PubMed Scopus (530) Google Scholar). endoplasmic reticulum bovine serum albumin embryonic stem fatal familial insomnia glycosylphosphatidylinositol phosphate-buffered saline phosphatidylinositol phospholipase C proteinase K transmissible spongiform encephalopathy phenylmethylsulfonyl fluoride. PrP is a glycoprotein attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor (3Bolton D.C. Meyer R.K. Prusiner S.B. J. Virol. 1985; 53: 596-606Crossref PubMed Google Scholar, 4Stahl N. Borchelt D.R. Hsiao K. Prusiner S.B. Cell. 1987; 51: 229-240Abstract Full Text PDF PubMed Scopus (908) Google Scholar, 5Harris D.A. Br. Med. Bull. 2003; 66: 71-85Crossref PubMed Scopus (142) Google Scholar). Whereas its normal function has yet to be defined, expression of PrP is essential for the development of transmissible spongiform encephalopathy (TSE) or prion disease (6Bueler H. Aguzzi A. Sailer A. Greiner R.A. Autenried P. Aguet M. Weissmann C. Cell. 1993; 73: 1339-1347Abstract Full Text PDF PubMed Scopus (1811) Google Scholar, 7Manson J.C. Clarke A.R. Hooper M.L. Aitchison L. McConnell I. Hope J. Mol. Neurobiol. 1994; 8: 121-127Crossref PubMed Scopus (496) Google Scholar). The TSEs are a group of fatal neurodegenerative diseases that can be sporadic, inherited, or acquired by infection. TSE diseases include scrapie of sheep and goats, bovine spongiform encephalopathy in cattle, and a number of human forms of the disease such as Creutzfeldt-Jackob disease, variant Creutzfeldt-Jackob disease linked with bovine spongiform encephalopathy, Gerstmann-Straussler-Scheinker syndrome, Kuru, and fatal familial insomnia (FFI) (8Will R.G. Br. Med. Bull. 2003; 66: 255-265Crossref PubMed Scopus (195) Google Scholar, 9Gambetti P. Kong Q. Zou W. Parchi P. Chen S.G. Br. Med. Bull. 2003; 66: 213-239Crossref PubMed Scopus (421) Google Scholar, 10Sigurdson C.J. Miller M.W. Br. Med. Bull. 2003; 66: 199-212Crossref PubMed Scopus (89) Google Scholar). A central event in all prion diseases appears to be a conformational modification of the normal cellular prion protein (PrPC) from a soluble form with a predominant α-helical conformation to the pathogenic form (PrPSc) that is aggregated, rich in β-sheets, partially resistant to proteinase K digestion, and insoluble in nondenaturing detergents (11Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5155) Google Scholar). PrP contains two N-glycan attachment sequences (NXT) at amino acids 180 and 196 in mice. These sites are variably glycosylated in vivo such that un-, mono-, and diglycosylated glycotypes are observed (12Stimson E. Hope J. Chong A. Burlingame A.L. Biochemistry. 1999; 38: 4885-4895Crossref PubMed Scopus (142) Google Scholar, 13Rudd P.M. Endo T. Colominas C. Groth D. Wheeler S.F. Harvey D.J. Wormald M.R. Serban H. Prusiner S.B. Kobata A. Dwek R.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13044-13049Crossref PubMed Scopus (246) Google Scholar). The biological significance of each of the glycotypes of PrP (un-, mono-, and diglycosylated) is unknown. Both N-glycosylation sites are conserved in the PrP gene (Prnp) from all species, suggesting that N-glycans play an important role in the protein function (14van Rheede T. Smolenaars M.M. Madsen O. de Jong W.W. Mol. Biol. Evol. 2003; 20: 111-121Crossref PubMed Scopus (117) Google Scholar). A number of reports have shown that the lack of sugars can induce the PrPC to PrPSc transition in vitro, suggesting that perturbations in glycosylation may contribute to the development of disease, destabilizing PrP structure and allowing it to acquire spontaneously PrP-like properties (15Kocisko D.A. Come J.H. Priola S.A. Chesebro B. Raymond G.J. Lansbury P.T. Caughey B. Nature. 1994; 370: 471-474Crossref PubMed Scopus (791) Google Scholar, 16Lehmann S. Harris D.A. J. Biol. 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Cayetano J. Rogers M. Groth D. Torchia M. Tremblay P. Scott M.R. Cohen F.E. Prusiner S.B. Neuron. 1997; 19: 1337-1348Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 22Neuendorf E. Weber A. Saalmuller A. Schatzl H. Reifenberg K. Pfaff E. Groschup M.H. J. Biol. Chem. 2004; 279: 53306-53316Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). To investigate the in vivo effect of glycosylation on PrP biochemical properties and its cellular biology, we have developed a gene-targeted transgenic model in which the host Prnp is replaced by a modified Prnp transgene in the correct genomic location (23Manson J.C. Tuzi N.L. Exp. Rev. Mol. Med. 2001; http://www.expertreviews.org/01002952h.htmPubMed Google Scholar). This model represents a valid tool to analyze the effect of mutations of the host Prnp in TSE susceptibility, since the Prnp gene expression is controlled by the normal regulatory elements of endogenous PrP (24Moore R.C. Hope J. McBride P.A. McConnell I. Selfridge J. Melton D.W. Manson J.C. Nat. Genet. 1998; 18: 118-125Crossref PubMed Scopus (169) Google Scholar, 25Manson J.C. Jamieson E. Baybutt H. Tuzi N.L. Barron R. McConnell I. Somerville R. Ironside J. Will R. Sy M.S. Melton D.W. Hope J. Bostock C. EMBO J. 1999; 18: 6855-6864Crossref PubMed Scopus (203) Google Scholar, 26Barron R.M. Thomson V. Jamieson E. Melton D.W. Ironside J. Will R. Manson J.C. EMBO J. 2001; 20: 5070-5078Crossref PubMed Scopus (108) Google Scholar, 27Barron R.M. Thomson V. King D. Shaw J. Melton D.W. Manson J.C. J. Gen. Virol. 2003; 84: 3165-3172Crossref PubMed Scopus (28) Google Scholar, 28Tuzi N.L. Clarke A.R. Bradford B. Aitchison L. Thomson V. Manson J.C. Genesis. 2004; 40: 1-6Crossref PubMed Scopus (13) Google Scholar). Three transgenic lines have been generated, each containing a point mutation in the Prnp gene eliminating the first, second, or both of the glycosylation sites: N180T (G1), N196T (G2), and N180T-N196T (G3). Using these mice, we have investigated whether the lack of glycans can alter the expression level of the PrP protein, its conformation and intracellular localization, and its ability to acquire the biochemical characteristics of the pathogenic form. We report here that whereas glycans appear to control the cellular location of PrP, the presence of sugars does not dramatically change the biology of PrP, and there is no evidence of PrPSc-like properties in either mono- or unglycosylated PrP. The results reported here are important in determining the physiological function of PrP glycoforms and in understanding their role in the infectious and pathogenic process of TSEs. Mouse monoclonal antibody 8H4, epitope (residues 145–220) binding is independent of the N-linked glycosylation, because it reacts with both recombinant PrP and all native glycoforms (29Zanusso G. Liu D. Ferrari S. Hegyi I. Yin X. Aguzzi A. Hornemann S. Liemann S. Glockshuber R. Manson J.C. Brown P. Petersen R.B. Gambetti P. Sy M.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8812-8816Crossref PubMed Scopus (176) Google Scholar). 7A12 (epitope 90–140) is a mouse monoclonal anti-PrP antibody (30Li R. Liu T. Wong B.S. Pan T. Morillas M. Swietnicki W. O'Rourke K. Gambetti P. Surewicz W.K. Sy M.S. J. Mol. Biol. 2000; 301: 567-573Crossref PubMed Scopus (106) Google Scholar). FH11 is a mouse monoclonal antibody that binds the N-terminal region of PrP and is used extensively in enzyme-linked immunosorbent assays (31Foster J.D. Wilson M. Hunter N. Vet. Rec. 1996; 139: 512-515Crossref PubMed Scopus (58) Google Scholar). AG4 is a mouse monoclonal antibody with epitope recognition between residues 31 and 51, with a further area of binding between amino acids 147 and 163. 1B3 and 1A8 are both rabbit polyclonal antibodies against PrP. Rat monoclonal anti-tubulin antibody (Abcam) has been used as loading control in Western blot experiments. Alexa Fluor ® 488 and Alexa Fluor ® 568 are IgG-labeled with fluorescent dye (Molecular Probes, Inc., Eugene, OR). Rabbit anti-cow glial fibrillary acidic protein (DAKO) is an antibody recognizing a specific astrocytic marker. The endoplasmic reticulum marker anti-ERp60 is raised in rabbit against porcine ERp60 peptide PIIQEEKPKKKKKAQEDL in the C terminus of the protein (32Rouiller I. Brookes S.M. Hyatt A.D. Windsor M. Wileman T. J. Virol. 1998; 72: 2373-2387Crossref PubMed Google Scholar). The Golgi marker 23C rat monoclonal, IgG2c, clone 23c was raised in rats against recombinant mouse TCP-1α, C-terminal half (33Willison K. Lewis V. Zuckerman K.S. Cordell J. Dean C. Miller K. Lyon M.F. Marsh M. Cell. 1989; 57: 621-632Abstract Full Text PDF PubMed Scopus (63) Google Scholar). PrP codon 180 and 196 alterations were introduced into HM-1 embryonic stem (ES) cells. Briefly, a gene-targeting vector was constructed using isogenic 129/Ola Prnpa DNA from a HM-1 genomic library in λ DASH II (Stratagene). The PrP codon 180 and 196 alterations were introduced into a 1.1-kb XmaIII-EcoRI exon 3 fragment containing the open reading frame by the Kunkel method (34Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4558) Google Scholar). This was ligated with the 5′ and 3′ homologous sequences derived from a 7.8-kb BamHI-EcoRV genomic clone spanning 129/OlaPrnp exon 3. A LoxPneomycin/thymidine kinase-selectable cassette (provided by Alan Clarke, University of Cardiff, UK) was ligated into a unique SalI site 1600 bases downstream of exon 3 in the pBluescript plasmid (Stratagene). The pBluescript vector previously had its SalI site removed, so this was a unique site in the targeting vector. The open reading frame encoding PrP in the targeting vector was sequenced at each step in the cloning procedure to confirm the presence of the alterations and the absence of any other cloning artifacts. Culture conditions for the ES cell line HM-1 have been described previously (35Selfridge J. Pow A.M. McWhir J. Magin T.M. Melton D.W. Somat. Cell. Mol. Genet. 1992; 18: 325-336Crossref PubMed Scopus (92) Google Scholar). HM-1 cells (5 × 107) were electroporated using a gene pulser (Bio-Rad) at 800 V and 3 millifarads with 250 μg of linearized targeting vector DNA in 0.8 ml of Hepes-phosphate-buffered saline, pH 7.05. Cells were rested for 15 min and plated at 5 × 105/10-cm plate. 24–48 h after electroporation, G418 selection medium was added. Medium was changed every 2–3 days, and colonies were selected for PCR screening 15 days after electroporation. 107 targeted HM-1 cells in 0.8 ml of serum-free growth medium were electroporated with 25 μg of the plasmid pCre2 (provided by Alan Clarke). Two pulses of 230 V, 500 microfarads were given. Cells were rested for 15 min and plated at 104 cell/10-cm plate. On day 6 after electroporation, 2 mm gancylovir was added to the growth medium. Colonies were picked and screened on day 15. Half of the cells from surviving colonies were used to prepare DNA for all PCR analyses Detection of Homologous Recombination Events—A 1600-bp PCR product was synthesized between the neomycin/thymidine kinase cassette and a site outside the targeting vector. The reaction-specific oligonucleotides are LoxP, situated immediately upstream of the 3′ loxP site (TCGATCGACTAGAGCTTGCGGA), and 3′Map1, located 200 bases 3′ to the EcoRV site (CTAAGTGACCTAGGCACATGTC). The cycle conditions were 3 min at 94 °C and then 35 cycles of 1 min at 94 °C, 1 min at 60 °C, and 2 min at 72 °C and then 10 min at 72 °C (GeneAmp 9700; PerkinElmer Life Sciences). Those positive for the selection cassette were then analyzed for the glycosylation mutation using the mismatch-specific PCR reaction described below for genotyping. Removal of the Selectable Marker—The removal of the selectable marker left one LoxP site. This is screened for using oligonucleotides 5′ and 3′ to the PrP gene SalI site NLTVitro creA (AGAACAGGTCTGACCACACTGGTT) and NLTVitro creB (AATGGTTAAACTTTCGTTAAGGAT). Wild type PrP alleles will give a PCR product of 242 bp, whereas those containing a loxP site will be 342 bp. Sites containing an unexcised neomycin/thymidine kinase cassette would be over 5 kb. The cycle conditions were 3 min at 94 °C and then 30 cycles of 45 s at 94 °C, 45 s at 60 °C, and 45 s at 72 °C and then 10 min at 72 °C. Targeted ES cells were used to generate chimeric mice as described previously (25Manson J.C. Jamieson E. Baybutt H. Tuzi N.L. Barron R. McConnell I. Somerville R. Ironside J. Will R. Sy M.S. Melton D.W. Hope J. Bostock C. EMBO J. 1999; 18: 6855-6864Crossref PubMed Scopus (203) Google Scholar) to obtain G1 and G2 heterozygous mice expressing mono- and unglycosylated PrP and G3 heterozygous mice expressing unglycosylated PrP. Heterozygous mice were bred to produce an inbred homozygous line. 129/Ola mice were used as wild type controls, since the had been generated on a 129/Ola background. NPU PrP–/– mice (7Manson J.C. Clarke A.R. Hooper M.L. Aitchison L. McConnell I. Hope J. Mol. Neurobiol. 1994; 8: 121-127Crossref PubMed Scopus (496) Google Scholar) were used as negative controls in all of the experiments performed. G1 and G2 mutant alleles were detected using a mismatch PCR technique. An oligonucleotide mixture was used at 1 pmol that contained a forward oligonucleotide 9910 (AACCTCAAGCATGTGGCAGGGGCTGCGGCAGCTGG), a reverse oligonucleotide 9912 (TCAGTGCCAGGGGTATTAGCCTATGGGGGACACAG), and a mutant-specific or wild type oligonucleotide (also in the reverse orientation) in a ratio of 20:1:20. The reaction-specific oligonucleotides are G1-mut (GCTGCTTGATGGTGATAG), G1-WT (GCTGCTTGATGGTGATAT), G2-mut (CATCGGTCTCGGTGAAGG), and G2-WT (CATCGGTCTCGGTGAAGT) (mutated nucleotides are in boldface type). The cycle conditions were 3 min at 94 °C and then 35 cycles of 50 s at 94 °C, 30 s at 60 °C, and 1 min at 72 °C and then 10 min at 72 °C. All other reaction components were those recommended by the supplier (Invitrogen). Genomic DNA was prepared using a Puregene Isolation kit (Gentra Systems). DNA (15 mg/reaction) was digested with restriction enzymes and then separated on a 1% agarose gel and blotted to Hybond-N nylon membrane (Amersham Biosciences). Hybridization was performed using ULTAhyb solution (Ambion) using a 700-bp EcoRV-BamHI fragment (3′ probe) and an 884-bp PCR product as probes. Following stringent wash procedures (0.1× SSC at 65 °C), the blots were exposed to x-ray film for 2 days. Total RNA was isolated using RNAzolTM B (Biogenesis) based on the guanidinium thiocyanate/phenol/chloroform extraction method (36Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63196) Google Scholar). A 20-μg aliquot of total RNA was separated on a 1.0% agarose-formaldehyde denaturing gel, transferred to Hybond N (Amersham Biosciences), and probed with a 32P-labeled 936-bp KpnI-EcoRI fragment from exon 3 of Prnp. Mice were killed by cervical dislocation, and brains were removed, flash frozen in liquid nitrogen, and then stored at –70 °C until required. Half or whole brains were weighed and mechanically homogenized from frozen in nine volumes of ice-cold Nonidet P-40 lysis buffer (1% Nonidet 40, 0.5% sodium deoxycholate, 150 mm NaCl, 50 mm Tris, pH 7.5) with the addition of phenylmethylsulfonyl fluoride (PMSF) (final concentration 1 μm; Sigma) to prevent protein degradation by endogenous proteases. The homogenate was centrifuged at 8000 rpm for 10 min to remove debris. Total protein was denatured in 1× Novex Tris/glycine SDS sample buffer (Invitrogen) and 1× NuPage sample-reducing agent (Invitrogen) for 30 min at 95 °C. Proteins were separated by electrophoresis at 125 V through a Novex precast Tris/glycine gel (12 or 14% acrylamide, Tris/glycine; Invitrogen). Proteins in the acrylamide gel were transferred to polyvinylidene difluoride membrane at 25 V (125 A/gel) using a semidry transfer blotter (Bio-Rad) in 1× transfer solution (48 mm Tris, 39 mm glycine, 0.375% SDS, 20% methanol). Total brain proteins (5% brain homogenate, Nonidet P-40 lysis buffer, 10 mm phenylmethylsulfonyl fluoride) were denatured in 1× glycoprotein denaturing buffer (0.5% SDS, 1% β-mercaptoethanol; New England Biolabs) at 100 °C for 10 min prior to incubation with Peptide N-glycosidase F (30,000 units/ml; New England Biolabs) in 1% Nonidet 40 (New England Biolabs) and 1× G7 reaction buffer (50 mm NaPO4; New England Biolabs) at 37 °C for 2–4 h. The reaction was terminated by freezing at –20 °C or SDS denaturation. Mouse brain homogenates (10%) were prepared in ice-cold Nonidet P-40 buffer. Each homogenate was then split into two aliquots, one treated with proteinase K (PK; Roche Applied Science) and one not. In order to assess the sensitivity to enzyme digestion, wild type, G1, G2, and G3 brain homogenates were each treated with varying concentrations of PK: 20, 10, and 5 μg/ml at 37 °C for 1 h. A milder treatment was also carried out, incubating the samples with PK (20 μg/ml) at 4 °C for 1 h. The samples were then analyzed by Western blotting using 8H4 or 7A12 monoclonal antibodies for PrP detection. The method is an adaptation of that of Barnard et al. (37Barnard G. Helmick B. Madden S. Gilbourne C. Patel R. Luminescence. 2000; 15: 357-362Crossref PubMed Scopus (30) Google Scholar). PrPC was extracted from brain homogenate (10– 1 tissue, Nonidet P-40 lysis buffer, 10 mm phenylmethylsulfonyl fluoride) by mechanical homogenization in 1 m guanidine hydrochloride (25 mm Tris, 1 m guanidine hydrochloride (Sigma); 0.5% Triton X-100 (Sigma)). This was then diluted in DELFIA ® assay buffer (Tris-buffered saline with bovine serum albumin (BSA), bovine γ-globulins, Tween 40, diethylenetiaminepentacetic acid (PerkinElmer), leading to a final concentration equivalent to of 10 mg/ml original tissue. Capture antibodies FH11 (1:200) or AG4 (1:200) were bound to 96-well plates by overnight incubation at 4 °C. Wells were blocked with 2% bovine serum albumin (Roche Applied Science) in sterile 1× PBS (Oxoid) with 3 m NaN3, for 1 h shaking at room temperature. The plate was then incubated with samples and standards, shaken at room temperature for 1 h, and then incubated with europium (Eu3+)-labeled detector antibodies 7A12 (FH11 or AG4 captures) or 8H4 (1:3000) (FH11 capture only). DELFIA ® enhancement solution was added to the samples to facilitate the formation of Eu-(2-NTA)3(TOPO)2–3. After 5 min of shaking at room temperature, Eu3+ emission (615 nm) was calculated using a time-resolved technique. Between each step, the plate was washed in 1× DELFIA ® wash concentrate (TBST; PerkinElmer Life Sciences) using the DELFIA ® automatic plate washer (Wallace). The program WorkOut was used to analyze absorbance from standard and samples and to produce the standard curve (based on a linear model of emission). PrPC was extracted from brain homogenate (10–1 tissue; Nonidet P-40 lysis buffer; 1 mm phenylmethylsulfonyl fluoride) by mechanical homogenization in 1 m guanidine hydrochloride (25 mm Tris, 1 m guanidine hydrochloride (Sigma); 0.5% Triton X-100 (Sigma)). This was then diluted in DELFIA ® assay buffer, leading to a final concentration equivalent to of 10 mg/ml of original tissue. Proteins insoluble in 1 m guanidine hydrochloride (PrPSc) were separated from those that were soluble (PrPC) by centrifugation at 13,000 rpm for 10 min. The resultant pellet was resuspended in 6 m guanidine hydrochloride prior to dilution in DELFIA ® assay buffer, to a concentration equivalent to 10 mg/ml original tissue. Measurement of the sample concentration was then performed as described above (DELFIA ® analysis). Mouse brain homogenates (10%) were prepared by homogenizing in ice-cold PBS containing 10 mm phenylmethylsulfonyl fluoride. Each sample was centrifuged at 13,000 rpm for 5 min at room temperature. The supernatant was collected and centrifuged at 25,000 rpm for 10 min at 4 °C. The pellets were resuspended in 500 μl of cold PBS, and then each sample was split. One half was treated with phosphatidylinositol phospholipase C (PIPLC; 0.5 units/ml; Sigma), whereas the second remained untreated. The samples were incubated for 10 h at 4 °C. Samples were then centrifuged at 13,000 rpm for 15 min at 4 °C. The supernatant (membrane-released fraction) and the pellet (membrane-associated fraction) were analyzed by Western blotting using the monoclonal antibody 8H4. A group of 10 homozygous G3 mice were monitored up to 850 days and compared with a group of wild type mice. The animals were age- and sex-matched. Animals were monitored constantly by a group of independent observers for any neurodegenerative signs. Between 800 and 900 days, mice were culled. Brains were retained; half of the brain was fixed for standard lesion profiling and plaque analysis, and the other half was flash frozen for biochemical analysis. Brain Sections were hematoxylin and eosin-stained and scored for vacuolar degeneration on a scale of 0 to 5 in nine standard gray matter areas and three standard white matter areas as described previously (38Fraser H. Dickinson A.G. Nature. 1967; 216: 1310-1311Crossref PubMed Scopus (81) Google Scholar). Sections were immunostained using standard procedures. Briefly, sections were blocked with normal goat serum and probed overnight with polyclonal antibody anti-glial fibrillary acidic protein at a dilution of 1:400. A parallel panel of sections was also probed with normal mouse serum as a control. Antibody binding was detected with biotinylated goat anti-rabbit secondary antibody (Jackson) and the Vectastain Elite ABC Kit (Vector Laboratories). Reaction products were visualized with diaminobenzidine, and sections were lightly counterstained with hematoxylin. Pictures were taken using a Nikon Eclipse E800 microscope. Mouse brains were fixed in periodate/lysine/paraformaldehyde or in a paraformaldehyde/glutaraldehyde mix (4% paraformaldehyde, 0.025% glutaraldehyde) for 4 h. Brain sections were cut at a thickness of 70 μm using a vibrating microtome (Leica). The sections were permeabilized for 1 h in PBS, 0.1% Triton at room temperature before blocking overnight at room temperature in PBS, 0.5% BSA in a humid chamber. Sections were blocked for a further 1 h at 37°C in Mouse On Mouse Ig blocking reagent/PBS when mouse monoclonal antibodies were used (Vector Laboratories). After blocking, the tissues were incubated at 37 °C with primary antibody (diluted in PBS/protein concentrate when mouse monoclonals were used; Vector Laboratories) for 90 min (39Monaghan P. Watson P.R. Cook H. Scott L. Wallis T.S. Robertson D. J. Microsc. 2001; 203: 223-226Crossref PubMed Scopus (15) Google Scholar). To get a specific signal, several anti-PrP antibodies were used at different concentrations: 8H4 (1:1000 and 1:2000), AG4 (1:1000 and 1:2000), 1B3 (1:800, 1:1000, and 1:2000), and 1A8 (1:1000 and 1:2000). After extensive washes in a calcium/magnesium-free PBS solution, the sections were incubated at 37 °C with the secondary antibody goat anti-mouse Alexa Fluor ® 488 conjugate diluted 1:200 in PBS/BSA for 90 min. The samples were then washed with a calcium/magnesium-free PBS solution for 20 min and then stained with 4′,6-diamidino-2-phenylindole nuclear marker (1:10,000; Molecular Probes) for 30 min at room temperature. After extensive washes in ultrapure water, the sections were mounted for microscopic analysis. Co-localization experiments were carried out using the same basic method as above with some modifications. For ERp60/8H4 (1:400 and 1:500), the primary antibodies were mixed in PBS/BSA so that the tissue was incubated with them simultaneously. Similarly, the secondary antibodies, Alexa 488-conjugated goat anti-mouse antibodies and Alexa 568-conjugated goat anti-rabbit, were simultaneously incubated. Controls for cross-reactivity were used, and none was detected. For 23C/8H4, cross-reactivity was detected; to eliminate it, a sequential staining method was used. Sections were first incubated with 8H4 (1:500) diluted in PBS/protein concentrate (Vector Laboratories), washed in PBS, and then incubated with Alexa 488-conjugated goat anti-mouse using a method identical to that for single 8H4 labeling. Sections were then washed 10 times in PBS before being blocked for 1 h in MOM Ig-blocking reagent (Vector Laboratories). Block was removed by washing for 10 min in PBS/protein concentrate before incubation of the section with 23C (1:50) in PBS/protein concentrate. The sections were then washed and incubated with Alexa 568-conjugated goat anti-rat. The sections were washed, stained with 4′,6-diamidino-2-phenylindole, and mounted for analysis. Sections were imaged with a Leica TCS SP2 laser-scanning confocal microscope. Construction of Gene-targeted Mice with Altered N-Linked Glycosylation of PrP—Using the Cre-loxP recombination and gene-targeting approaches, three inbred lines of transgenic mice with alterations in the N-linked glycosylation consensus sites Asn-Xaa-Thr were generated. Gene targeting was used to alter the Asn residue to Thr at 180 (N180T), 196 (N196T), or both 180 and 196 (N180T/N196T) (Fig. 1) in the HM-1 ES cell line. The positive ES clones were subsequent
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