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• W2000769863 abstract "The two homologous presenilins are key factors for the generation of amyloid β-peptide (Aβ), since Alzheimer's disease (AD)-associated mutations enhance the production of the pathologically relevant 42-amino acid Aβ (Aβ42), and a gene knockout of presenilin-1 (PS1) significantly inhibits total Aβ production. Presenilins undergo proteolytic processing within the domain encoded by exon 9, a process that may be closely related to their biological and pathological activity. An AD-associated mutation within the PS1 gene deletes exon 9 (PS1Δexon9) due to a splicing error and results in the accumulation of the uncleaved full-length protein. We now demonstrate the unexpected finding that the pathological activity of PS1Δexon9 is independent of its lack to undergo proteolytic processing, but is rather due to a point mutation (S290C) occurring at the aberrant exon 8/10 splice junction. Mutagenizing the cysteine residue at position 290 to the original serine residue completely inhibits the pathological activity in regard to the elevated production of Aβ42. Like PS1Δexon9, the resulting presenilin variant (PS1Δexon9 C290S) accumulates as an uncleaved protein and fully replaces endogenous presenilin fragments. Moreover, PS1Δexon9 C290S exhibits a significantly increased biological activity in a highly sensitive in vivo assay as compared with the AD-associated mutation. Therefore not only the increased Aβ42 production but also the decreased biological function of PS1Δexon9 is due to a point mutation and independent of the lack of proteolytic processing. The two homologous presenilins are key factors for the generation of amyloid β-peptide (Aβ), since Alzheimer's disease (AD)-associated mutations enhance the production of the pathologically relevant 42-amino acid Aβ (Aβ42), and a gene knockout of presenilin-1 (PS1) significantly inhibits total Aβ production. Presenilins undergo proteolytic processing within the domain encoded by exon 9, a process that may be closely related to their biological and pathological activity. An AD-associated mutation within the PS1 gene deletes exon 9 (PS1Δexon9) due to a splicing error and results in the accumulation of the uncleaved full-length protein. We now demonstrate the unexpected finding that the pathological activity of PS1Δexon9 is independent of its lack to undergo proteolytic processing, but is rather due to a point mutation (S290C) occurring at the aberrant exon 8/10 splice junction. Mutagenizing the cysteine residue at position 290 to the original serine residue completely inhibits the pathological activity in regard to the elevated production of Aβ42. Like PS1Δexon9, the resulting presenilin variant (PS1Δexon9 C290S) accumulates as an uncleaved protein and fully replaces endogenous presenilin fragments. Moreover, PS1Δexon9 C290S exhibits a significantly increased biological activity in a highly sensitive in vivo assay as compared with the AD-associated mutation. Therefore not only the increased Aβ42 production but also the decreased biological function of PS1Δexon9 is due to a point mutation and independent of the lack of proteolytic processing. Alzheimer's disease amyloid β-peptide β amyloid precursor protein presenilin wild type polymerase chain reaction minimal essential medium enzyme-linked immunosorbent assay Early onset Alzheimer's disease (AD)1 can occur due to mutations within the genes encoding the β-amyloid precursor protein (βAPP) and the two presenilins, PS1 and PS2 (Refs. 1Sherrington R. Rogaev E.I. Liang Y. Rogaeva E.A. Levesque G. Ikeda M. Chi H. Lin C. Li G. Holman K. Tsuda T. Mar L. Foncin J.-F. Bruni A.C. Montesi M.P. Sorbi S. Rainero I. Pinessi L. Nee L. Chumakov I. Pollen D. Brookes A. Sanseau P. Polinsky R.J. Wasco W. da Silva H.A.R. Haines J.L. Pericak-Vance M.A. Tanzi R.E. Roses A.D. Fraser P.E. Rommens J.M. St. George-Hyslop P.H. Nature. 1995; 375: 754-760Crossref PubMed Scopus (3579) Google Scholar, 2Levy-Lahad E. Wasco W. Poorkaj P. Romano D.M. Oshima J. Pettingell W.H. Yu C. Jondro P.D. Schmidt S.D. Wang K. Crowley A.C. Fu Y.-H. Guenette S.Y. Galas D. Nemens E. Wijsman E.M. Bird T.D. Schellenberg G.D. Tanzi R.E. Science. 1995; 269: 973-977Crossref PubMed Scopus (2226) Google Scholar, 3Rogaev E.I. Sherrington R. Rogaeva E.A. Levesque G. Ikeda M. Liang Y. Chi H. Lin C. Holman K. Tsuda T. Mar L. Sorbi S. Nacmias B. Piacentini S. Amaducci L. Chumakov I. Cohen D. Lannfelt L. Fraser P.E. Rommens J.M. St. George-Hyslop P.H. Nature. 1995; 376: 775-778Crossref PubMed Scopus (1789) Google Scholar; summarized in Refs. 4Price D. Sisodia S. Annu. Rev. Neurosci. 1998; 21: 479-505Crossref PubMed Scopus (506) Google Scholar and 5Selkoe D.J. J. Biol. Chem. 1996; 271: 18295-18298Abstract Full Text Full Text PDF PubMed Scopus (758) Google Scholar). The mutations increase the generation of the 42-amino acid version of Aβ (Aβ42), which aggregates more readily (6Jarret J.T. Lansbury Jr., P.T. Cell. 1993; 73: 1055-1058Abstract Full Text PDF PubMed Scopus (1918) Google Scholar) and is therefore preferentially deposited in senile plaques (5Selkoe D.J. J. Biol. Chem. 1996; 271: 18295-18298Abstract Full Text Full Text PDF PubMed Scopus (758) Google Scholar). PS proteins are also involved in the physiological production of Aβ, since the lack of PS1 expression in PS1−/− mice results in a dramatically reduced Aβ production (7De Strooper B. Saftig P. Craessaerts K. Vanderstichele H. Guhde G. Annaert W. Von Figura K. Van Leuven F. Nature. 1998; 391: 387-390Crossref PubMed Scopus (1548) Google Scholar). All but one PS mutations are point mutations affecting conserved amino acids (4Price D. Sisodia S. Annu. Rev. Neurosci. 1998; 21: 479-505Crossref PubMed Scopus (506) Google Scholar, 5Selkoe D.J. J. Biol. Chem. 1996; 271: 18295-18298Abstract Full Text Full Text PDF PubMed Scopus (758) Google Scholar). However, due to a splicing error, the PS1Δexon9 mutation results in the deletion of the domain encoded by exon 9 (8Perez-Tur J. Froehlich S. Prihar G. Crook R. Baker M. Duff K. Wragg M. Busfield F. Lendon C. Clark R.F. Roques P. Fuldner R.A. Johnston J. Cowburn R. Forsell C. Axelman K. Lilius L. Houlden H. Karran E. Roberts G.W. Rossor M. Adams M.D. Hardy J. Goate A. Lannfelt L. Hutton M. Neuroreport. 1995; 7: 297-301Crossref PubMed Scopus (238) Google Scholar). This domain contains the cleavage site for proteolytic processing (9Thinakaran G. Borchelt D.R. Lee M.K. Slunt H.H. Spitzer L. Kim G. Ratovitsky T. Davenport F. Nordstedt C. Seeger M. Hardy J. Levey A.I. Gandy S.E. Jenkins N.A. Copeland N.G. Price D.L. Sisodia S.S. Neuron. 1996; 17: 181-190Abstract Full Text Full Text PDF PubMed Scopus (939) Google Scholar, 10Podlisny M. Citron M. Amarante P. Sherrington R. Xia W. Zhang J. Diehl T. Levesque G. Fraser P. Haass C. Koo E. Seubert P. St. George-Hyslop P. Teplow D. Selkoe D. Neurobiol. Dis. 1997; 3: 325-337Crossref PubMed Scopus (273) Google Scholar), and therefore PS1Δexon9 accumulates as an uncleaved protein (9Thinakaran G. Borchelt D.R. Lee M.K. Slunt H.H. Spitzer L. Kim G. Ratovitsky T. Davenport F. Nordstedt C. Seeger M. Hardy J. Levey A.I. Gandy S.E. Jenkins N.A. Copeland N.G. Price D.L. Sisodia S.S. Neuron. 1996; 17: 181-190Abstract Full Text Full Text PDF PubMed Scopus (939) Google Scholar). Since proteolytic processing is highly regulated (9Thinakaran G. Borchelt D.R. Lee M.K. Slunt H.H. Spitzer L. Kim G. Ratovitsky T. Davenport F. Nordstedt C. Seeger M. Hardy J. Levey A.I. Gandy S.E. Jenkins N.A. Copeland N.G. Price D.L. Sisodia S.S. Neuron. 1996; 17: 181-190Abstract Full Text Full Text PDF PubMed Scopus (939) Google Scholar, 11Thinakaran G. Harris C.L. Ratovitski T. Davenport F. Slunt H.H. Price D.L. Borchelt D.R. Sisodia S.S. J. Biol. Chem. 1997; 272: 28415-28422Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 12Steiner H. Capell A. Pesold B. Citron M. Kloetzel P.-M. Selkoe D. Romig H. Mendla K. Haass C. J. Biol. Chem. 1998; 273: 32322-32331Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar) and appears to be altered by PS mutations (Refs. 13Lee M.L. Borchelt D.R. Kim G. Thinakaran G. Slunt H. Ratovitski T. Martin L.J. Kittur A. Gandy S. Levey A. Jenkins N. Copeland N. Price D.L. Sisodia S. Nat. Med. 1997; 3: 756-760Crossref PubMed Scopus (130) Google Scholar, 14Mercken M. Takahashi H. Honda T. Sato K. Murayama M. Nakazato Y. Noguchi K. Imahori K. Takashima A. FEBS Lett. 1996; 389: 297-303Crossref PubMed Scopus (120) Google Scholar, 15Murayama O. Honda T. Mercken M. Neurosci. Lett. 1997; 229: 61-64Crossref PubMed Scopus (30) Google Scholar; summarized in Ref. 16Grünberg J. Capell A. Leimer U. Steiner B. Steiner H. Walter J. Haass C. Alzheimer's Res. 1997; 3: 253-259Google Scholar), the lack of proteolytic processing caused by the exon 9 deletion was expected to be responsible for its pathological activity. We now demonstrate the unexpected finding that the pathological function of PS1Δexon9 as well as its reduced biological activity is independent of its lack to undergo proteolytic processing, but is rather due to a point mutation (S290C) that is the result of the aberrant exon 8/10 splice junction. K293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 200 μg/ml G418 (to select for βAPP expression) and 200 μg/ml zeocin (to select for presenilin expression). K293 cells stably expressing PS1Δexon9 C290S were generated by transfection of K293 cells stably expressing βAPP containing the Swedish mutation (17Citron M. Oltersdorf T. Haass C. McConlogue A.Y. Seubert P. Vigo-Pelfrey C. Lieberburg I. Selkoe D.J. Nature. 1992; 360: 672-674Crossref PubMed Scopus (1534) Google Scholar). K293 cells stably transfected with Swedish βAPP695, wt PS1, and PS1Δexon9 were described previously (18Citron M. Westaway D. Xia W. Carlson G. Diehl T.S. Levesque G. Johnson-Wood K. Lee M. Seubert P. Davis A. Kholodenko D. Motter R. Sherrington R. Perry B. Yao H. Strome R. Lieberburg I. Rommens J. Kim S. Schenk D. Fraser P. St. George-Hyslop P. Selkoe D. Nat. Med. 1997; 3: 67-72Crossref PubMed Scopus (1161) Google Scholar). The cDNA encoding PS1Δexon9 C290S was constructed by mutagenizing the cysteine at codon 290 of the PS1Δexon9 cDNA (8Perez-Tur J. Froehlich S. Prihar G. Crook R. Baker M. Duff K. Wragg M. Busfield F. Lendon C. Clark R.F. Roques P. Fuldner R.A. Johnston J. Cowburn R. Forsell C. Axelman K. Lilius L. Houlden H. Karran E. Roberts G.W. Rossor M. Adams M.D. Hardy J. Goate A. Lannfelt L. Hutton M. Neuroreport. 1995; 7: 297-301Crossref PubMed Scopus (238) Google Scholar) to serine in a two-step PCR procedure. The following primers were designed: first PCR, PS1-187-F (5′-CCGAATTCAAGAAAGAACCTCAA-3′) and ΔE9-C290S-R (5′-GTGACTCCCTTTCTGTGGAGGAGTAAATGAGAGC-3′); second PCR, ΔE9-C290S-F (5′-GCTCTCATTTACTCCTCCACAGAAAGGGAGTCAC-3′) and PS1-STOP-R (5′-CGCCTCGAGGCAAATATGCTAGATATA-3′). After gel purification the PCR products were mixed and subjected to a final PCR with primers PS1-187-F and PS1-STOP-R. The resulting PCR product was digested with EcoRI/XhoI and cloned into the pcDNA3.1/Zeo(+) expression vector (Invitrogen). The cDNA was sequenced to verify successful mutagenesis. The polyclonal antibodies against amino acids 263–407 of PS1 (3027) and amino acids 297–356 of PS2 (3711) were described previously (19Walter J. Grünberg J. Capell A. Pesold B. Schindzielorz A. Citron M. Mendla K. St. George-Hyslop P. Multhaup G. Selkoe D.J. Haass C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5349-5354Crossref PubMed Scopus (101) Google Scholar, 20Walter J. Grünberg J. Schindzielorz A. Haass C. Biochemistry. 1998; 37: 5961-5967Crossref PubMed Scopus (57) Google Scholar). The monoclonal antibodies to the PS1 and PS2 loop were raised against fusion proteins containing amino acids 263–407 (BI.3D7) or 297–356 (BI.HF5C). Stably transfected K293 cell lines were grown to confluence in 10-cm dishes. After starvation for 1 h in 4 ml of methionine- and serum-free MEM (MEM supplemented with 1% l-glutamine and 1% penicillin/streptomycin) cells were metabolically labeled with 700 μCi of [35S]methionine (Promix, Amersham Pharmacia Biotech) in 4 ml of methionine- and serum-free MEM for 1 h. Cell extracts were prepared and subjected to immunoprecipitation of PS as described (12Steiner H. Capell A. Pesold B. Citron M. Kloetzel P.-M. Selkoe D. Romig H. Mendla K. Haass C. J. Biol. Chem. 1998; 273: 32322-32331Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). PS immunoprecipitates were solubilized in sample buffer containing 4 m urea for 10 min at 65 °C, separated on SDS-urea gels, and analyzed by fluorography (19Walter J. Grünberg J. Capell A. Pesold B. Schindzielorz A. Citron M. Mendla K. St. George-Hyslop P. Multhaup G. Selkoe D.J. Haass C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5349-5354Crossref PubMed Scopus (101) Google Scholar). Stably transfected K293 cell lines were grown to confluence. Cell extracts were prepared and subjected to immunoprecipitation as described (12Steiner H. Capell A. Pesold B. Citron M. Kloetzel P.-M. Selkoe D. Romig H. Mendla K. Haass C. J. Biol. Chem. 1998; 273: 32322-32331Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Following gel electrophoresis, immunoprecipitated proteins were identified by immunoblotting (12Steiner H. Capell A. Pesold B. Citron M. Kloetzel P.-M. Selkoe D. Romig H. Mendla K. Haass C. J. Biol. Chem. 1998; 273: 32322-32331Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Bound antibodies were detected by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech). Culture medium (2 ml) was collected from confluent K293 cells grown in six-well dishes for 24 h. The medium was assayed for Aβ40 and Aβ42 using a previously described ELISA assay (12Steiner H. Capell A. Pesold B. Citron M. Kloetzel P.-M. Selkoe D. Romig H. Mendla K. Haass C. J. Biol. Chem. 1998; 273: 32322-32331Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Aβ peptides were immunoprecipitated using antibody 6E10 (Senetek) according to established protocols (21Haass C. Schlossmacher M. Hung A.Y. Vigo-Pelfrey C. Mellon A. Ostaszewski B. Lieberburg I. Koo E. Schenk D. Teplow D. Selkoe D.J. Nature. 1992; 359: 322-325Crossref PubMed Scopus (1762) Google Scholar). Expression constructs were generated as described previously (22Baumeister R. Leimer U. Zweckbronner I. Jakubek C. Grünberg J. Haass C. Genes Funct. 1997; 1: 149-159Crossref PubMed Scopus (185) Google Scholar). Transgenic lines were established by microinjection of plasmid DNA mixtures into the C. elegans germ line to create extrachromosomal arrays as described previously (22Baumeister R. Leimer U. Zweckbronner I. Jakubek C. Grünberg J. Haass C. Genes Funct. 1997; 1: 149-159Crossref PubMed Scopus (185) Google Scholar). All plasmids used in this study were injected at a concentration of 20 ng/μl intosel-12(ar171) or sel-12(ar171)unc-1(e538) hermaphrodites along with ttx-3:GFP as a cotransformation marker. Successful transformation was monitored by the expression of GFP in the AIY interneurons of F1 and F2 generation animals. Four independent lines from the progeny of F2 generation animals were established. As the sel-12(ar171) animals never lay eggs (23Levitan D. Greenwald I. Nature. 1995; 377: 351-354Crossref PubMed Scopus (627) Google Scholar), rescue of the sel-12 defect can be quantified by scoring egg laying behavior in transgenic animals (22Baumeister R. Leimer U. Zweckbronner I. Jakubek C. Grünberg J. Haass C. Genes Funct. 1997; 1: 149-159Crossref PubMed Scopus (185) Google Scholar, 24Levitan D. Doyle T. Brousseau D. Lee M. Thinakaran G. Slunt H. Sisodia S. Greenwald I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14940-14944Crossref PubMed Scopus (343) Google Scholar). Consequently, for each transgenic line, we examined 50 transgenic animals for their ability to lay eggs and determined their brood size. The number of eggs laid by individual transgenic animals was counted and placed into four categories: “Egl+++,” robust egg laying, more than 30 eggs laid (wt phenotype); “Egl++,” 15–30 eggs laid; “Egl+,” 5–15 eggs laid; “Egl−,” no eggs laid. The PS1Δexon9 mutation changes a G to a T at the splice site for exon 9. It therefore destroys the minimal required consensus sequence for the splice acceptor site (8Perez-Tur J. Froehlich S. Prihar G. Crook R. Baker M. Duff K. Wragg M. Busfield F. Lendon C. Clark R.F. Roques P. Fuldner R.A. Johnston J. Cowburn R. Forsell C. Axelman K. Lilius L. Houlden H. Karran E. Roberts G.W. Rossor M. Adams M.D. Hardy J. Goate A. Lannfelt L. Hutton M. Neuroreport. 1995; 7: 297-301Crossref PubMed Scopus (238) Google Scholar), which then results in an aberrant deletion of the domain encoded by exon 9 (Ref. 8Perez-Tur J. Froehlich S. Prihar G. Crook R. Baker M. Duff K. Wragg M. Busfield F. Lendon C. Clark R.F. Roques P. Fuldner R.A. Johnston J. Cowburn R. Forsell C. Axelman K. Lilius L. Houlden H. Karran E. Roberts G.W. Rossor M. Adams M.D. Hardy J. Goate A. Lannfelt L. Hutton M. Neuroreport. 1995; 7: 297-301Crossref PubMed Scopus (238) Google Scholar; Fig.1 A). However, the mutation also changes codon 290 at the exon 8/10 splice junction from a serine to a cysteine (Ref. 8Perez-Tur J. Froehlich S. Prihar G. Crook R. Baker M. Duff K. Wragg M. Busfield F. Lendon C. Clark R.F. Roques P. Fuldner R.A. Johnston J. Cowburn R. Forsell C. Axelman K. Lilius L. Houlden H. Karran E. Roberts G.W. Rossor M. Adams M.D. Hardy J. Goate A. Lannfelt L. Hutton M. Neuroreport. 1995; 7: 297-301Crossref PubMed Scopus (238) Google Scholar; Fig. 1 A). We therefore have investigated if the single amino acid exchange or the deletion of the domain required for proteolytic processing is responsible for the pathological activity of the PS1Δexon9 mutation in regard to Aβ42 generation. For this purpose we mutagenized amino acid 290 of PS1Δexon9 to a serine (PS1Δexon9 C290S), thus correcting the point mutation (Fig. 1 A). cDNAs encoding PS1Δexon9 and PS1Δexon9 C290S were stably transfected into kidney 293 cells overexpressing βAPP containing the Swedish mutation. This cell line was used previously to determine the pathological effect of PS mutations on Aβ production (12Steiner H. Capell A. Pesold B. Citron M. Kloetzel P.-M. Selkoe D. Romig H. Mendla K. Haass C. J. Biol. Chem. 1998; 273: 32322-32331Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 18Citron M. Westaway D. Xia W. Carlson G. Diehl T.S. Levesque G. Johnson-Wood K. Lee M. Seubert P. Davis A. Kholodenko D. Motter R. Sherrington R. Perry B. Yao H. Strome R. Lieberburg I. Rommens J. Kim S. Schenk D. Fraser P. St. George-Hyslop P. Selkoe D. Nat. Med. 1997; 3: 67-72Crossref PubMed Scopus (1161) Google Scholar). Untransfected K293 cells expressing endogenous PS, as well as cell lines overexpressing PS1Δexon9 and PS1Δexon9 C290S were pulse-labeled with [35S]methionine for 1 h, and cell lysates were immunoprecipitated with antibody 3027 to the large loop of PS1. Both PS1Δexon9 and PS1Δexon9 C290S accumulated as full-length proteins (Fig. 1 B). As reported previously (9Thinakaran G. Borchelt D.R. Lee M.K. Slunt H.H. Spitzer L. Kim G. Ratovitsky T. Davenport F. Nordstedt C. Seeger M. Hardy J. Levey A.I. Gandy S.E. Jenkins N.A. Copeland N.G. Price D.L. Sisodia S.S. Neuron. 1996; 17: 181-190Abstract Full Text Full Text PDF PubMed Scopus (939) Google Scholar, 10Podlisny M. Citron M. Amarante P. Sherrington R. Xia W. Zhang J. Diehl T. Levesque G. Fraser P. Haass C. Koo E. Seubert P. St. George-Hyslop P. Teplow D. Selkoe D. Neurobiol. Dis. 1997; 3: 325-337Crossref PubMed Scopus (273) Google Scholar) very little endogenous full-length PS1 could be detected in the untransfected cell line. It has been shown before that overexpression of PS proteins results in the replacement of endogenous PS fragments and the accumulation of PS1Δexon9 prevents the formation of stable PS fragments (9Thinakaran G. Borchelt D.R. Lee M.K. Slunt H.H. Spitzer L. Kim G. Ratovitsky T. Davenport F. Nordstedt C. Seeger M. Hardy J. Levey A.I. Gandy S.E. Jenkins N.A. Copeland N.G. Price D.L. Sisodia S.S. Neuron. 1996; 17: 181-190Abstract Full Text Full Text PDF PubMed Scopus (939) Google Scholar, 11Thinakaran G. Harris C.L. Ratovitski T. Davenport F. Slunt H.H. Price D.L. Borchelt D.R. Sisodia S.S. J. Biol. Chem. 1997; 272: 28415-28422Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 12Steiner H. Capell A. Pesold B. Citron M. Kloetzel P.-M. Selkoe D. Romig H. Mendla K. Haass C. J. Biol. Chem. 1998; 273: 32322-32331Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). To prove that overexpression of PS1Δexon9 C290S still allows the reduction of endogenous PS fragments, cell lysates from unlabeled cells were immunoprecipitated with antibodies specific to the PS1 or PS2 loop domain (see “Experimental Procedures”), and PS fragments were identified with the PS1/PS2-specific monoclonal antibodies BI.3D7 and BI.HF5C (see “Experimental Procedures”). Whereas cell lines expressing endogenous presenilins produced PS1 as well as PS2 C-terminal fragments (Fig. 1, C and D), overexpression of PS1Δexon9 and PS1Δexon9 C290S inhibited the formation of endogenous PS1 and PS2 fragments (Fig. 1, C andD). This demonstrates that PS1Δexon9 C290S, like PS1Δexon9, fully replaced presenilins derived from the endogenous genes and also proves that PS1Δexon9 C290S does not undergo proteolytic processing but rather accumulates as an uncleaved full-length protein. In order to investigate the pathological activity of the mutant PS derivatives on Aβ production, control cells as well as cells overexpressing PS1Δexon9 and PS1Δexon9 C290S were metabolically labeled with [35S]methionine, and Aβ40 and Aβ42 peptides were immunoprecipitated from the conditioned medium using antibody 6E10. This antibody is raised to Aβ1–17 and therefore recognizes Aβ40 as well as Aβ42 (see “Experimental Procedures”). Isolated Aβ peptides were then separated on a previously described gel system, which allows the specific resolution of Aβ40 and Aβ42 (25Klafki H.-W. Wilfang J. Staufenbiel M. Anal. Biochem. 1996; 237: 24-29Crossref PubMed Scopus (98) Google Scholar). As shown in Fig.2A, cells overexpressing PS1Δexon9 secreted elevated levels of Aβ42 as compared with control cells. In contrast, the cell line stably transfected with PS1Δexon9 C290S produced significantly lower amounts of Aβ42 as cells expressing the Alzheimer's disease-associated PS1Δexon9 mutation. This suggests that the pathological activity of the PS1Δexon9 mutation is due to the point mutation generated at the aberrant splice junction. In order to quantitate Aβ42 and Aβ40 production, we used a previously described specific ELISA (12Steiner H. Capell A. Pesold B. Citron M. Kloetzel P.-M. Selkoe D. Romig H. Mendla K. Haass C. J. Biol. Chem. 1998; 273: 32322-32331Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). In this assay, two highly specific monoclonal antibodies are used for the discrimination of both peptide species (12Steiner H. Capell A. Pesold B. Citron M. Kloetzel P.-M. Selkoe D. Romig H. Mendla K. Haass C. J. Biol. Chem. 1998; 273: 32322-32331Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). As reported before (12Steiner H. Capell A. Pesold B. Citron M. Kloetzel P.-M. Selkoe D. Romig H. Mendla K. Haass C. J. Biol. Chem. 1998; 273: 32322-32331Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 18Citron M. Westaway D. Xia W. Carlson G. Diehl T.S. Levesque G. Johnson-Wood K. Lee M. Seubert P. Davis A. Kholodenko D. Motter R. Sherrington R. Perry B. Yao H. Strome R. Lieberburg I. Rommens J. Kim S. Schenk D. Fraser P. St. George-Hyslop P. Selkoe D. Nat. Med. 1997; 3: 67-72Crossref PubMed Scopus (1161) Google Scholar, 26Borchelt D. Thinakaran G. Eckman C. Lee M. Davenport F. Ratovitsky T. Prada C.-M. Kim G. Seekins S. Yager D. Slunt H. Wang R. Seeger M. Levey A. Gandy S. Copeland N. Jenkins N. Price D. Younkin S. Sisodia S.S. Neuron. 1996; 17: 1005-10013Abstract Full Text Full Text PDF PubMed Scopus (1338) Google Scholar), the PS1Δexon9 mutation results in an approximately 3-fold increase of the Aβ42/Aβtotal ratio (Fig. 2 B). In contrast, the cell line stably overexpressing PS1Δexon9 C290S showed no increased Aβ42/Aβtotal ratio (Fig. 2 B). Therefore using two different approaches we can show that the pathological activity of PS1Δexon9 is independent of its lack of proteolytic processing but is rather caused by the single amino acid change at the aberrant splice junction. Having demonstrated that PS1Δexon9 C290S is pathologically inactive, we also wanted to test if this protein is sufficient to rescue alin-12-mediated signaling defect that is a result of a mutant PS homologue (sel-12) in Caenorhabditis elegans (23Levitan D. Greenwald I. Nature. 1995; 377: 351-354Crossref PubMed Scopus (627) Google Scholar). As reported previously, transgenic expression of human presenilins rescues the phenotype caused by mutations ofsel-12 (22Baumeister R. Leimer U. Zweckbronner I. Jakubek C. Grünberg J. Haass C. Genes Funct. 1997; 1: 149-159Crossref PubMed Scopus (185) Google Scholar, 24Levitan D. Doyle T. Brousseau D. Lee M. Thinakaran G. Slunt H. Sisodia S. Greenwald I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14940-14944Crossref PubMed Scopus (343) Google Scholar). In contrast, transgenic expression of the PS1Δexon9 variant in C. elegans resulted in an incomplete rescue of the sel-12 mutant phenotype, as indicated by a significantly reduced brood size and reduced egg laying (Refs. 22Baumeister R. Leimer U. Zweckbronner I. Jakubek C. Grünberg J. Haass C. Genes Funct. 1997; 1: 149-159Crossref PubMed Scopus (185) Google Scholar and24Levitan D. Doyle T. Brousseau D. Lee M. Thinakaran G. Slunt H. Sisodia S. Greenwald I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14940-14944Crossref PubMed Scopus (343) Google Scholar; see also Table I). In order to assess the in vivo function of PS1Δexon9 C290S, we tested its ability to rescue the putative sel-12 null allelear171 (23Levitan D. Greenwald I. Nature. 1995; 377: 351-354Crossref PubMed Scopus (627) Google Scholar). Four independent transgenic strains expressing PS1Δexon9 C290S displayed robust egg laying (Table I). Based on both numbers of laid progeny and numbers of eggs in utero, the phenotype of these strains is almost indistinguishable of that of wild type worms (Table I). These results demonstrate that PS1Δexon9 C290S rescues the egg laying phenotype of the sel-12(ar171) mutant animals significantly better than PS1Δexon9 (Table I). Therefore, the reduced biological activity of PS1Δexon9 is also due to a single point mutation and completely independent of the lack of proteolytic processing.Table IRescue of the sel-12 egg laying defect by human PS genes expressed from the sel-12 promoterStrainTransgeneGenotypeEgg laying behavior++++++−N2Wild type50000BR1029Vectorsel-12(ar171)00050BR1083PS1Δexon9sel-12(ar171)311630BR1092PS1Δexon9 C290Ssel-12(ar171)49010BR1093PS1Δexon9 C290Ssel-12(ar171)47210BR1094PS1Δexon9 C290Ssel-12(ar171)47300BR1095PS1Δexon9 C290Ssel-12(ar171)49100For 50 transgenic animals each, the numbers of progeny were counted and grouped in the following categories: +++, over 30 progeny laid by individual animal; ++, 15–30 progeny laid; +, 5–15 progeny laid; −, no progeny laid. The sel-12(ar171) strains carried an additional unc-1(e538) marker that did not affect egg laying or rescuing frequency. It was backcrossed several times from the strain originally published (23Levitan D. Greenwald I. Nature. 1995; 377: 351-354Crossref PubMed Scopus (627) Google Scholar). Open table in a new tab For 50 transgenic animals each, the numbers of progeny were counted and grouped in the following categories: +++, over 30 progeny laid by individual animal; ++, 15–30 progeny laid; +, 5–15 progeny laid; −, no progeny laid. The sel-12(ar171) strains carried an additional unc-1(e538) marker that did not affect egg laying or rescuing frequency. It was backcrossed several times from the strain originally published (23Levitan D. Greenwald I. Nature. 1995; 377: 351-354Crossref PubMed Scopus (627) Google Scholar). Our results demonstrate that the pathological effect of the PS1Δexon9 mutation is independent of its lack of proteolytic cleavage and the deletion of the exon 9-encoded domain. Reverting the cysteine residue generated by the exon 8/10 splice junction at codon 290 back to its wt residue (serine) still inhibits its proteolytic processing, causes an accumulation of the uncleaved protein, and prevents formation of endogenous PS fragments. Although the artificially generated PS1Δexon9 C290S variant behaves like PS1Δexon9 in regard to the characteristic biochemical features described above, it does not allow pathological overproduction of Aβ42. Moreover, PS1Δexon9 C290S regains full biological activity in a very sensitive in vivoassay system. This demonstrates that not only the pathological overproduction of Aβ42 but also the reduced biological function is due to the single amino acid exchange. Therefore, similar to all other known PS mutations, the pathological effect of the PS1Δexon9 mutation is due to a rather subtle amino acid exchange at a single highly conserved codon whereas the large deletion of the complete domain encoded by exon 9 does not affect the biological and pathological function. However, consistent with our previous results (27Capell A. Grünberg J. Pesold B. Diehlmann A. Citron M. Nixon R. Beyreuther K. Selkoe D. Haass C. J. Biol. Chem. 1998; 273: 3205-3211Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar), we suggest that PS molecules lacking the domain encoded by exon 9 mimic a proteolytically processed and biologically active PS complex and therefore rescue the phenotype of the mutant nematode. Consequently, the rescuing activity of PS1Δexon9 is significantly enhanced when the pathologically relevant point mutation at codon 290 is reverted to the wt residue. It remains to be shown if the mutation at codon 290 is also pathologically active within the full-length protein like all other AD-associated point mutations. It may however be possible that the mutation only exhibits a pathological activity if it is aberrantly flanked by the domains encoded by exons 8 and 10. Structural changes that may specifically occur in the PS1Δexon9 molecule (12Steiner H. Capell A. Pesold B. Citron M. Kloetzel P.-M. Selkoe D. Romig H. Mendla K. Haass C. J. Biol. Chem. 1998; 273: 32322-32331Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 19Walter J. Grünberg J. Capell A. Pesold B. Schindzielorz A. Citron M. Mendla K. St. George-Hyslop P. Multhaup G. Selkoe D.J. Haass C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5349-5354Crossref PubMed Scopus (101) Google Scholar) might be responsible for the pathological activity of this very unusual mutation. Based on our results it would be interesting to investigate if the point mutations in the PS genes require the endoproteolytic cleavage for their pathological activity. Therefore artificial PS molecules should be generated containing an AD associated mutation as well as the smallest possible alteration of the sequence at the cleavage site which would inhibit PS processing. However, such mutations might be very difficult to generate since PS can be cleaved at multiple sites (10Podlisny M. Citron M. Amarante P. Sherrington R. Xia W. Zhang J. Diehl T. Levesque G. Fraser P. Haass C. Koo E. Seubert P. St. George-Hyslop P. Teplow D. Selkoe D. Neurobiol. Dis. 1997; 3: 325-337Crossref PubMed Scopus (273) Google Scholar,12Steiner H. Capell A. Pesold B. Citron M. Kloetzel P.-M. Selkoe D. Romig H. Mendla K. Haass C. J. Biol. Chem. 1998; 273: 32322-32331Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar) and PS molecules containing larger deletions at the cleavage sites might mimic a proteolytically processed PS molecule (27Capell A. Grünberg J. Pesold B. Diehlmann A. Citron M. Nixon R. Beyreuther K. Selkoe D. Haass C. J. Biol. Chem. 1998; 273: 3205-3211Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Note Added in Proof While this manuscript was in press, we found that expression of PS1Δexon9 C290S containing a FAD-associated mutation (M146L) results in elevated Aβ42 production, in further support of the findings presented here. We thank Roland Donhauser for injecting PS constructs into C. elegans and Drs. Bernard Lakowski and Helmut Jacobsen for critical discussion." @default.
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