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A Naturally Occurring C-terminal Fragment of the Prion Protein (PrP) Delays Disease and Acts as a Dominant-negative Inhibitor of PrPSc Formation

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A Naturally Occurring C-terminal Fragment of the Prion Protein (PrP) Delays Disease and Acts as a Dominant-negative Inhibitor of PrPSc Formation
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  ANaturallyOccurringC-terminalFragmentofthePrionProtein(PrP)DelaysDiseaseandActsasaDominant-negativeInhibitorofPrP Sc Formation * Receivedforpublication,July25,2011,andinrevisedform,October19,2011  Published,JBCPapersinPress,October24,2011,DOI10.1074/jbc.M111.286195 LauraWestergard ‡ ,JessieA.Turnbaugh § ,andDavidA.Harris §1 Fromthe ‡ DepartmentofCellBiologyandPhysiology,WashingtonUniversitySchoolofMedicine,St.Louis,Missouri63110andthe § DepartmentofBiochemistry,BostonUniversitySchoolofMedicine,Boston, Massachusetts 02118 Background:  C1 is the main physiological cleavage fragment of PrP, but its role in disease is unknown. Results: C1isnottoxicwhenexpressedinmiceanddelaystheonsetofdiseaseandPrP Sc formationwhenco-expressedwithWTPrP. Conclusion:  C1 is a dominant-negative inhibitor of PrP Sc formation. Significance:  Modulation of C1 cleavage may represent a therapeutic strategy for combating PrP Sc infection. Thecellularprionprotein(PrP C )undergoesconstitutivepro-teolytic cleavage between residues 111/112 to yield a solubleN-terminal fragment (N1) and a membrane-anchored C-termi-nal fragment (C1). The C1 fragment represents the major pro-teolytic fragment of PrP C in brain and several cell types. Toexplore the role of C1 in prion disease, we generated Tg(C1)transgenicmiceexpressingthisfragment(PrP(  23–111))inthepresence and absence of endogenous PrP. In contrast to severalother N-terminally deleted forms of PrP, the C1 fragment doesnot cause a spontaneous neurological disease in the absence of endogenous PrP. Tg(C1) mice inoculated with scrapie prionsremain healthy and do not accumulate protease-resistant PrP,demonstratingthatC1isnotasubstrateforconversiontoPrP Sc (the disease-associated isoform). Interestingly, Tg(C1) mice co-expressing C1 along with wild-type PrP (either endogenous orencoded by a second transgene) become ill after scrapie inocu-lation, but with a dramatically delayed time course compared with mice lacking C1. In addition, accumulation of PrP Sc  wasmarkedly slowed in these animals. Similar effects were pro-duced by a shorter C-terminal fragment of PrP(  23–134).These results demonstrate that C1 acts as dominant-negativeinhibitor of PrP Sc formation and accumulation of neurotoxicforms of PrP. Thus, C1, a naturally occurring fragment of PrP C ,might play a modulatory role during the course of prion dis-eases. In addition, enhancing production of C1, or exogenously administering this fragment, represents a potential therapeuticstrategy for the treatment of prion diseases. Transmissible spongiform encephalopathies such as Creutz-feldt-Jakob disease and bovine spongiform encephalopathy arefatal neurodegenerative disorders whose pathology is associ-ated with propagation of prions, novel infectious agents whosetransmission is based on changes in protein conformationrather than inheritance of nucleic acid sequence (1). Prionpropagation depends on conversion of an endogenous cellularglycoprotein(PrP C ) 2 intoanaggregated,protease-resistantiso-form (PrP Sc ) that is rich in   -sheet structure (1–4). PrP C issynthesizedonendoplasmicreticulum-attachedribosomesandtransits the secretory pathway to the cell surface, where mostmoleculesareattachedtotheouterleafletofthelipidbilayerviaa C-terminal glycosylphosphatidylinositol (GPI) anchor (5).Most of the protein resides in lipid rafts on the plasma mem-brane,althoughsomemoleculesareconstitutivelyendocytosed via clathrin-coated pits and are then recycled back to the cellsurface (6–9).After its synthesis, PrP C is known to undergo proteolyticprocessing in at least three sites. One cleavage (sometimesreferred to as the  -cleavage) occurs between residues 111/112to yield a soluble N-terminal fragment called N1 and a GPI-anchored, C-terminal fragment called C1 (10–13). The N1/C1cleavage occurs constitutively in 10–50% of the molecules, butit can be stimulated by activators of protein kinase C (10,13–15). There is disagreement about the cellular site and pro-teases responsible for the   -cleavage, with endosomal/lyso-somalcompartments,latecompartmentsofthesecretorypath-way, and the cell surface (mediated by a disintegrin andmetalloproteases (ADAMs)) having all been suggested (6,15–18).Asecondcleavageoccursbetweenresidues89/90,gen-eratingasolubleN2fragmentandaGPI-anchoredC2fragment(10). This so-called   -cleavage occurs at low levels under nor-mal conditions, possibly catalyzed by reactive oxygen speciesactingoncell-surfacePrP,butitisenhancedduringgenerationofPrP Sc (10,19–21).Athirdcleavage,catalyzedbymembersof the ADAM protease family, occurs near the site of GPI anchorattachment (residue 230), shedding most of the polypeptidechain into the extracellular medium (11, 22, 23). Additionalproteolyticcleavagesmayalsooccuratlowlevels(24).Thepro-teolytic fragments generated by these different cleavage reac-tions may have a role in the physiological functions of PrP C , *  Thisworkwassupported,inwholeorinpart,byNationalInstitutesofHealthGrants NS40975, NS052526, NS056376, and NS065244 (to D. A. H.). 1  Towhomcorrespondenceshouldbeaddressed:Dept.ofBiochemistry,Bos-ton University School of Medicine, 72 East Concord St., K225, Boston, MA02118. Tel.: 617-638-4362; Fax: 617-638-5339; E-mail: daharris@bu.edu. 2  The abbreviations used are: PrP C , cellular prion protein; GPI, glycosylphos-phatidylinositol; PrP, prion protein; GFAP, glial fibrillary acidic protein;PNGase, peptide: N  -glycosidase; p.i., post-inoculation.  THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 51, pp. 44234–44242, December 23, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. 44234  JOURNAL OF BIOLOGICAL CHEMISTRY   VOLUME 286•NUMBER 51• DECEMBER23,2011   b  y g u e  s  t   onA pr i  l  2  0  ,2  0 1  6 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om   such as protection against oxidative stress (20), although thisremains unclear in part because of uncertainty about the nor-mal biological role of PrP C .C1 is quantitatively the major proteolytic fragment of PrPpresent in brain and many cells types (10–12, 20, 25). In thisstudy, we sought to investigate the role of C1 by the creation of transgenic mice that express this fragment in the presence andabsence of endogenous PrP. We demonstrate that although C1itself is not inherently toxic nor convertible to PrP Sc after inoc-ulationofTg(C1)animals,itactsasapotentdominant-negativeinhibitor, significantly delaying scrapie illness and decreasingPrP Sc production from wild-type PrP. These results indicatethattheC1cleavageproductactsasaphysiologicallygeneratedinhibitor of prion propagation, and therefore increasing pro-duction of this fragment represents a potential therapeuticstrategy for treatment of prion diseases. EXPERIMENTALPROCEDURES ConstructionofTransgenicMice —AcDNAencodingmurineC1(  23–111)wasgeneratedbyPCRamplification.Thefollowingprimers were used: 5   (5  -TCCGAAAGCTTCTCGAGGC-CGCCACCATGGCGAACCTTGGCTACTGGCTGCTGGC-CCTCTTTGTGACTATGTGGACTGATGTCGGCCTCTG-CAGGCCCATGATCCATTTTGGC-3  ) and 3   (5  -CGGAC-TCTAGACTCGAGTCATCATCCCACGATCAGGAAGAT-3  ).CloningofacDNAencodingPrP(  23–134)hasbeendescribedelsewhere (30). The 5   primers contain HindIII and XhoIrestrictionsitesalongwithaKozakconsensussequence.The3  primer incorporated XhoI and XbaI sites for the initial cloninginto the pcDNA 3.1(  ) Hygro plasmid and subsequent inser-tion into the transgenic vector. The resulting PCR product wasdigested with HindIII and XbaI and cloned into pcDNA 3.1(  )Hygro (Invitrogen).To create the transgenic mouse vector, a fragment encodingthe C1 sequence was released from the pcDNA 3.1(  )Hygro/C1 plasmid by digestion with XhoI and ligated into theXhoI site of MoPrP.Xho (26). Colony PCR was performed toselect clones containing the insert in the correct orientation,using the following primers: P1 (5  -AACCGAGCTGAAGCA-TTCTGCC-3  ) and P4 (5  -CACGAGAAATGCGAAGGAAC-AAGC-3  ) (27). The transgene was released from the recombi-nant plasmid by NotI restriction digestion, purified on GFXPCRDNAcolumns(GEHealthcare),andinjectedintothepro-nuclei of fertilized eggs from mice on the C57BL6  CBA back-ground. Transgenic founders were bred initially to Tga20  /  mice on a C57BL6/CBA/129 background (obtained from theEuropean Mouse Mutant Archive (EMMA)) and were thenback-crossed to  Prn-p 0/0 mice on a pure C57BL6 background(from EMMA).Genotyping of transgenic mice was performed by PCR anal- ysisoftailDNApreparedusingthePuregeneDNAisolationkit(Gentra Systems, Minneapolis, MN). Genotyping was per-formed using primers P1 and P4 (27). These primers amplify both the C1 and Tga20 transgenes, which can be distinguishedfrom each other by size. P2 (5  -CTTCAGCCTAAATACTGG-GCAC-3  ) and P4 (5  -CACGAGAAATGCGAAGGAACAAGC-3  )primers(27)wereusedtoamplifythe  Prn-p allele.AllTg(C1)miceusedinthisstudywereheterozygousfortheC1transgene( i.e.  Tg(C1  /  )).  Histology —Animals were perfused transcardially with 4%paraformaldehyde, after which brains were removed and post-fixed in the same solution. Paraffin sections of brain werestained with hematoxylin and eosin or GFAP as described pre- viously (28).  Biochemical Procedures —10% w/v brain homogenates weregenerated by mechanical dissociation of single hemispheresusingplasticpestles(SouthJerseyPrecisionToolandMoldInc.,Vineland, NJ) in phosphate-buffered saline (PBS) containing aproteaseinhibitormixture(RocheAppliedScience).Fordegly-cosylation,20  gofproteinwastreatedwithPNGaseFfor3hat37 °C. Western blots were performed using anti-PrP antibody 6H4 or D18 (kind gift of Dennis Burton (Scripps Institute, LaJolla, CA)).ForproteinaseKtreatment,10%w/vbrainhomogenatespre-pared as above were centrifuged for 5 min at 2,300   g  . Thesupernatant was diluted 1:10 in lysis buffer (10 m M  Tris-HCl,pH 7.4, 0.5% Triton X-100, 0.5% sodium deoxycholate) andincubated with 20   g/ml of proteinase K (PK) at 37 °C for 1 hprior to Western blotting with anti-PrP antibody 6H4. RESULTS Creation of Transgenic Mice Expressing the C1 Fragment of  PrP  —To investigate the properties of the C-terminal cleavageproduct, C1, in the absence of full-length PrP or the N1 frag-ment, we generated transgenic mice (designated Tg(C1)) thatexpress a PrP molecule deleted for residues 23–111 (Fig. 1  A )undercontrolofamodified  Prn-p promoter(26).Aftercleavageof the N-terminal signal peptide (residues 1–22) and attach-ment of the C-terminal GPI anchor (at residue 230) duringbiosynthesis, the transgenically encoded protein was predictedto correspond to the C1 fragment that is endogenously gener-ated from full-length PrP by proteolytic cleavage.Founderswerebredontothe  Prn-p 0/0 geneticbackgroundtoobtainTg(C1)/  Prn-p 0/0 micethatexpressedtheC1fragmentintheabsenceofanyWTPrP.Offivetransgene-positivefoundersobtained, three transmitted the transgene to their progeny.WesternblotanalysisofbrainhomogenatesrevealeddetectablePrP in only one of the lines (Fig. 1  B ). Quantification with theOdyssey infrared imaging system revealed that this lineexpressed the C1 protein at a level  7 times that of WT PrP in  Prn-p   /   mice and intermediate between the levels of WT PrPinTga20  /  andTga  /0 mice(datanotshown,seeFig.1  B ).Thetransgenically encoded protein exhibited three different glyco-forms, analogous to WT PrP (Fig. 1  B ,  lane 7  ). When sampleswere treated with PNGase to remove  N  -linked oligosaccha-rides, the protein migrated at the same position as the C1 frag-ment generated endogenously in the brains of   Prn-p   /   andTga20 mice (Fig. 1  B ,  filled arrowhead  ).We observed Tg(C1)  /Prn-p 0/0 mice for signs of spontaneousillness. We noted no clinical symptoms or increased mortality for up to 1 year (Fig. 2  A ,  pink line/squares ). In contrast,Tg(F35)/  Prn-p 0/0 mice expressing PrP deleted for residues32–134 at even lower levels (  2 times) (29) showed neurolog-ical symptoms, including ataxia, kyphosis, hyper-activity, hindlimb paralysis, and tail clasp by 30.21    2.6 days, and suc- NaturallyOccurringInhibitorofPrP  Sc  Formation DECEMBER23,2011• VOLUME 286•NUMBER 51  JOURNAL OF BIOLOGICAL CHEMISTRY   44235   b  y g u e  s  t   onA pr i  l  2  0  ,2  0 1  6 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om   cumbed by 100 days (Fig. 2  A ,  red line/crosses ). The brains of Tg(C1)/  Prn-p 0/0 animals showed no histological abnormalitiesat 1 year, in contrast to dramatic cerebellar degeneration inTg(F35) mice (Fig. 2  B ). Tg(C1) mice on the  Prn-p  /0 and  Prn- p  /  genetic backgrounds were also clinically and neurohisto-logically normal (data not shown). Tg(C1)MiceAreResistanttoInfectionwithScrapieandCan-not Propagate PrP  Sc —Tg(C1)/  Prn-p 0/0 mice were inoculatedintracerebrally with the RML strain of scrapie and observed forclinicalsymptoms.Animalsremainedhealthyforatleast1yearafter inoculation and showed no increase in mortality, similarto  Prn-p 0/0 lackingtheC1transgene(Fig.3  A ,  greenline/squares and  blue line/diamonds ). In contrast, scrapie-inoculated  Prn- p   /   mice became terminally ill at 171.8  16.2 days (Fig. 3  A , red line/crosses ). The brains of inoculated Tg(C1)/  Prn-p 0/0 mice did not display any histological abnormalities based onhematoxylin/eosinoranti-GFAPstaining,similartoinoculated  Prn-p 0/0 mice (Fig. 3  B ). We did not detect protease-resistantPrP in brain homogenates prepared from inoculated Tg(C1)/  Prn-p 0/0 mice (Fig. 3 C  ,  lane 4 ), although homogenates frominfected  Prn-p   /   animalsdisplayedthecharacteristicPrP(27–30) fragment (Fig. 3 C  ,  lane 2 ). As expected, no protease-resis-tant PrP was detected in the brains of inoculated  Prn-p 0/0 mice(data not shown). C1 Inhibits Disease Progression and PrP  Sc  Accumulation in Mice Co-expressing WT PrP  —The observation that Tg(C1)/  Prn-p 0/0 miceareresistant to scrapieinfectionand cannot pro-duce PrP Sc led us to wonder whether C1 might act as a domi-nant-negative inhibitor of prion propagation in miceco-expressing WT PrP encoded either by the endogenous  Prn-p  gene or the Tga20 transgene. To test this hypothesis, weintracerebrally inoculated Tg(C1)/  Prn-p  /  and Tg(C1)/Tga20  /0 mice with RML scrapie. We observed that survivaltime was significantly longer in these mice than in the corre-sponding  Prn-p   /   and Tga20  /0 mice that lacked the C1transgene (Fig. 4  A ). The mean survival time in Tg(C1)/  Prn- p  /  mice was 229.3  18.5 days compared with 171.8  16.2days in  Prn-p   /   mice and 112.1    10.9 days in Tg(C1)/Tga20  /0 mice compared with 76.9    6.2 in Tga20  /0 mice.Importantly, we found that co-expression of mutant PrP withwild type did not impact expression levels of either protein anddid not change WT PrP cleavage (Fig. 4  B ).We also analyzed the effect of the C1 transgene on scrapie-induced pathology, in particular the extent of spongiformchangeandastrogliosis.AlthoughsurvivaltimeswerelongerinTg(C1)/  Prn-p  /  and Tg(C1)/Tga20  /0 mice, at the terminalstageofdiseasethedegreeofspongiformchangeinthecerebel-lum,hippocampus,andbrainstemintheseanimalswascompa- FIGURE 1.  Schematic illustration of PrP constructs, and analysis of PrPexpression in transgenic mice.  A,  schematic of wild-type (WT), C1 (  23–111), and PrP(  23–134). Structural domains of PrP are indicated by the col-ored blocks:  SS,  signal sequence ( blue );  PBD,  polybasic domain (  yellow  );  OR, octapeptide repeats ( orange );  CC,  charged cluster ( red  );  HD,  hydrophobicdomain ( green );  GPI,  GPI attachment signal (  purple ). The  dotted lines  indicatedeletedregions. B, Westernblotanalysisofproteinexpression.Brainsamplesfrom mice of the indicated genotypes were normalized for total protein,treated with or without PNGase F to remove  N  -linked oligosaccharides(  and  lanes ,respectively),andsubjectedtoWesternblottingwithanti-PrPantibody 6H4.  Filled   and  open arrowheads  to the left of   lane 2  indicate thepositions of cleavage products C1 and C2, respectively. The upper band in lane8 ( asterisk  )representsresidual,mono-glycosylatedC1thatwasnotcom-pletelyshiftedbytreatmentwithPNGase.MolecularsizemarkersaregiveninkDa.FIGURE 2. Tg(C1)miceonthe Prn-p 0/0 backgrounddonotdevelopspon-taneous neurological illness.  A,  survival was monitored in mice of thefollowing genotypes, with the number of animals indicated in parentheses: Tg(C1)/ Prn-p 0/0 (10), Prn-p   /0 (8), Prn-p 0/0 (7),andTg(F35)/ Prn-p 0/0 (12). B, cer-ebellar sections from 365 day-old mice of the indicated genotypes werestained with hematoxylin and eosin.  Scale bar   (applicable to all panels) is1 mm. NaturallyOccurringInhibitorofPrP  Sc  Formation 44236  JOURNAL OF BIOLOGICAL CHEMISTRY   VOLUME 286•NUMBER 51• DECEMBER23,2011   b  y g u e  s  t   onA pr i  l  2  0  ,2  0 1  6 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om   rable with that seen in  Prn-p   /   and Tga20  /0 mice, respec-tively(Fig.5,  A –  H  ,anddatanotshown).GFAPstainingwasalsosimilar in terminally ill Tg(C1)/  Prn-p  /  and  Prn-p   /   mice(Fig. 5,  I   and  J  ). Interestingly, however, GFAP staining wasmuchmoreintenseinTg(C1)/Tga20  /0 micethaninTga20  /0 mice at the terminal stage (Fig. 5,  K   and  L ). Uninfected animalsof all genotypes lacked detectable GFAP staining (data notshown).TodeterminetheeffectofC1expressiononaccumulationof PrP Sc , brain homogenates were treated with PK, and theamount of protease-resistant PrP was analyzed by Westernblotting. Brains were collected from Tg(C1)/  Prn-p  /  mice attwo different time points as follows: during the pre-symptom-atic stage (180 days post-inoculation (p.i.)) and at the terminalstage (250 days p.i.). At the earlier time point, there was sub-stantially less PK-resistant PrP in Tg(C1)/  Prn-p  /  mice thanin terminally ill  Prn-p   /   mice at 152 days p.i. (Fig. 6  A ). How-ever, by the time Tg(C1)/  Prn-p  /  mice reached the terminalphase, PrP Sc had accumulated to a level comparable with thatseen in terminally ill  Prn-p   /   mice (Fig. 6  B ). This result indi-cates that expression of C1 slows, but does not prevent, accu-mulation of PrP Sc , which eventually reaches levels seen in micelacking the C1 transgene.Interestingly,thepresenceofC1hadanevenmoreprofoundeffect on accumulation of PrP Sc in Tga20  /0 mice. Even at theterminalstage,Tg(C1)/Tga20  /0 mice(120daysp.i.)containedsignificantly less PrP Sc in their brains than terminally illTga20  /0 mice (80 days p.i.) (Fig. 6 C  ). Despite this dramaticreduction in the amount of PrP Sc , the brains of terminally illTg(C1)/Tga20  /0 micestillcontainedinfectiousscrapieprions,as demonstrated by the ability of brain samples to transmitdisease to Tga20  /  indicator mice. Incubation times after FIGURE3. Scrapie-inoculatedTg(C1)/ Prn-p 0/0 micedonotdevelopclinicalillnessorhistopathology,anddonotaccumulateprotease-resistantPrP.  A, survivalwasmonitoredinmiceofthefollowinggenotypesafterRMLinoc-ulation, with the number of animals indicated in parentheses:  Prn-p 0/0 (9), Tg(C1)/ Prn-p 0/0 (8), Prn-p   /   (8). B, sectionsfromthecerebellum(  panels1 and 2 ) or hippocampus (  panels 3  and  4 ) from 365 day-old mice of the indicatedgenotypes were stained with hematoxylin and eosin or anti-GFAP antibody,respectively.  Insets  show hippocampal sections stained with DAPI to revealcell nuclei.  Scale bar   in  panel 4  (applicable to all panels) is 1 mm.  C,  Westernblottingforprotease-resistantPrP.Brainhomogenatescontainingequivalentamounts of protein from mice of the indicated genotypes were treated withorwithout20  g/mlproteinaseKfor1hat37 °C(  and  lanes ,respectively),and were subjected to Western blotting with anti-PrP antibody 6H4.FIGURE4. C1prolongssurvivaltimeinmiceexpressingWTPrP.  A, survivalwas monitored in mice of the following genotypes after scrapie inoculation,with the number of animals indicated in parentheses:  Prn-p   /  (8), Tg(C1)/ Prn-p   /  (8), Tga20   /0 (16), and Tg(C1)/Tga20   /0 (8). Significant differenceswere found between the following groups as determined by an unpairedstudent’s  t   test: Tg(C1)/ Prn- P   /  versus Prn-P    /  (  p    0.0001); Tg(C1)/ Tga20   /0 versus  Tga20   /0 (  p    0.0001).  B,  brain homogenates containingequal amounts of total protein from mice of the indicated genotypes weretreated with or without PNGase F to remove  N  -linked oligosaccharides(  and  lanes ,respectively),andsubjectedtoWesternblottingwithanti-PrPantibody D18.  Filled   and  open arrowheads  indicate the positions of cleavageproducts C1 and C2, respectively. Molecular size markers are given in kDa. NaturallyOccurringInhibitorofPrP  Sc  Formation DECEMBER23,2011• VOLUME 286•NUMBER 51  JOURNAL OF BIOLOGICAL CHEMISTRY   44237   b  y g u e  s  t   onA pr i  l  2  0  ,2  0 1  6 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om   inoculation of Tga20  /  mice with 1% brain homogenatesderived from Tg(C1)/Tga20  /0 mice were indistinguishablefrom those for homogenates derived from Tga20  /0 mice lack-ingtheC1transgene(62.3  5.3 versus 61.6  6.7days,respec-tively,  p  0.7,byStudent’s t  test).Moreover,therecipientmiceaccumulated similar amounts of protease-resistant PrP in theirbrains after inoculation with both sets of samples (data notshown). This result implies that, although Tg(C1)/Tga20  /0 mice accumulate greatly reduced levels of PrP Sc in their brains,these animals accumulate infectious prions with the ability topropagate in Tga20  /  host mice expressing WT PrP.  A Shorter C-terminal Fragment of PrP Also Acts as Domi-nant-negative Inhibitor  —We created Tg(  23–134) mice thatsynthesize a form of PrP that, after biosynthetic processing,correspondstoresidues135–230.Thisfragmentlacksahydro-phobic domain (residues 112–134) that is present at the N ter-minus of the C1 fragment (  23–111) (Fig. 1  A ). Tg(  23–134)mice remain healthy and, unlike Tg(F35) mice expressingPrP(  32–134),donotdevelopspontaneousneurologicalillness(30).To see if the shorter C-terminal fragment produced inTg(  23–134) mice was capable of sustaining prion propaga-tion, we inoculated two lines, L and H, that express the trun-cated protein at 0.2 and 1   levels, respectively, with RMLscrapie prions. Like Tg(C1) mice, Tg(  23–134)/  Prn-p 0/0 mice did not exhibit any clinical illness for   1 year afterinoculation (Fig. 7  A ,  pink line/squares  and  green line/trian- gles ). Moreover, the brains of inoculated animals did notdisplay any abnormalities by hematoxylin/eosin staining(Fig. 7  B ) or GFAP histochemistry (data not shown) and didnot contain any PK-resistant PrP by Western blotting (datanot shown).TodeterminewhetherPrP(  23–134)hasaninhibitoryeffecton scrapie propagation similar to that of C1, we inoculatedTg(  23–134)/  Prn-p  /  andTg(  23–134)/Tga20  /0 micewithRMLprions.Inbothkindsofmice,thetimetoterminaldiseasewasincreasedbythepresenceofPrP(  23–134),comparedwiththecorrespondingmicelackingthetruncatedprotein(Fig.8  A ).The survival time in Tg(  23–134H)/  Prn-p  /  mice was184.5  3.5 days compared with 171.8  16.2 days in  Prn-p   /   mice and 93.9  8.1 days in Tg(  23–134H)/Tga20  /0 mice or83  3.8 days in Tg(  23–134L)/Tga20  /0 mice compared with76.9  6.2 in Tga20  /0 mice. Expression of   23–134 in the H(1  ) line led to a statistically significant increase in survivaltime in both  Prn-p   /   and Tga20  /0 mice, although this pro-longation was not as dramatic as for C1, possibly because the FIGURE5. EffectofC1onscrapie-inducedpathologyinterminallyillmiceexpressingWTPrP. Miceoftheindicatedgenotypesweretakenforhistologicalanalyses at the terminal stage of illness, with the age at sacrifice given in parentheses: Tg(C1)/ Prn-p   /   (233d),  Prn-p   /   (158d), Tg(C1)/Tga20   /0 (120d), Tga20   /0 (65d). Sections from the cerebellum were stained with hematoxylin and eosin (  A–H  ) and sections from the hippocampus with anti-GFAP antibody( I–L ).Areaswithinthecerebellarwhitematteroutlinedbytheboxesin  A–D areshownathighermagnificationin E–H  . Scalebars, 1m M (  A–D and I–L )and50  m( E–H  ). NaturallyOccurringInhibitorofPrP  Sc  Formation 44238  JOURNAL OF BIOLOGICAL CHEMISTRY   VOLUME 286•NUMBER 51• DECEMBER23,2011   b  y g u e  s  t   onA pr i  l  2  0  ,2  0 1  6 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om 
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