The Caenorhabditis elegans Aβ1–42 Model of Alzheimer Disease Predominantly Expresses Aβ3–42

Abstract

Transgenic expression of human amyloid β (Aβ) peptide in body wall muscle cells of Caenorhabditis elegans has been used to better understand aspects of Alzheimer disease (AD). In human aging and AD, Aβ undergoes post-translational changes including covalent modifications, truncations, and oligomerization. Amino truncated Aβ is increasingly recognized as potentially contributing to AD pathogenesis. Here we describe surface-enhanced laser desorption ionization-time of flight mass spectrometry mass spectrometry of Aβ peptide in established transgenic C. elegans lines. Surprisingly, the Aβ being expressed is not full-length 1–42 (amino acids) as expected but rather a 3–42 truncation product. In vitro analysis demonstrates that Aβ3–42 self-aggregates like Aβ1–42, but more rapidly, and forms fibrillar structures. Similarly, Aβ3–42 is also the more potent initiator of Aβ1–40 aggregation. Seeded aggregation via Aβ3–42 is further enhanced via co-incubation with the transition metal Cu(II). Although unexpected, the C. elegans model of Aβ expression can now be co-opted to study the proteotoxic effects and processing of Aβ3–42. Transgenic expression of human amyloid β (Aβ) peptide in body wall muscle cells of Caenorhabditis elegans has been used to better understand aspects of Alzheimer disease (AD). In human aging and AD, Aβ undergoes post-translational changes including covalent modifications, truncations, and oligomerization. Amino truncated Aβ is increasingly recognized as potentially contributing to AD pathogenesis. Here we describe surface-enhanced laser desorption ionization-time of flight mass spectrometry mass spectrometry of Aβ peptide in established transgenic C. elegans lines. Surprisingly, the Aβ being expressed is not full-length 1–42 (amino acids) as expected but rather a 3–42 truncation product. In vitro analysis demonstrates that Aβ3–42 self-aggregates like Aβ1–42, but more rapidly, and forms fibrillar structures. Similarly, Aβ3–42 is also the more potent initiator of Aβ1–40 aggregation. Seeded aggregation via Aβ3–42 is further enhanced via co-incubation with the transition metal Cu(II). Although unexpected, the C. elegans model of Aβ expression can now be co-opted to study the proteotoxic effects and processing of Aβ3–42. Numerous studies support a role for aggregating Aβ 3The abbreviations used are: Aβamyloid βADAlzheimer diseaseSELDI-TOF MSsurface-enhanced laser desorption ionization-time of flight mass spectrometryQCglutaminyl cyclaseMES2-(N-morpholino) ethanesulfonic acidBicineN,N-bis(2-hydroxyethyl)glycineThTthioflavin-TpEpyroglutamateEMelectron micrograph. 3The abbreviations used are: Aβamyloid βADAlzheimer diseaseSELDI-TOF MSsurface-enhanced laser desorption ionization-time of flight mass spectrometryQCglutaminyl cyclaseMES2-(N-morpholino) ethanesulfonic acidBicineN,N-bis(2-hydroxyethyl)glycineThTthioflavin-TpEpyroglutamateEMelectron micrograph. in mediating the toxicity that underlies AD (1Hardy J. Selkoe D.J. Science. 2002; 297: 353-356Crossref PubMed Scopus (10863) Google Scholar, 2Tanzi R.E. Bertram L. Cell. 2005; 120: 545-555Abstract Full Text Full Text PDF PubMed Scopus (1481) Google Scholar). 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Francke M. Kehlen A. Holzer M. Hutter-Paier B. Prokesch M. Windisch M. Jagla W. Schlenzig D. Lindner C. Rudolph T. Reuter G. Cynis H. Montag D. Demuth H.U. Rossner S. Nat. Med. 2008; 14: 1106-1111Crossref PubMed Scopus (289) Google Scholar, 18Schilling S. Appl T. Hoffmann T. Cynis H. Schulz K. Jagla W. Friedrich D. Wermann M. Buchholz M. Heiser U. von Hörsten S. Demuth H.U. J. Neurochem. 2008; 106: 1225-1236Crossref PubMed Scopus (63) Google Scholar). Aβ1–42 itself cannot be cyclized by QC to Aβ3(pE)-42 (19Shirotani K. Tsubuki S. Lee H.J. Maruyama K. Saido T.C. Neurosci. Lett. 2002; 327: 25-28Crossref PubMed Scopus (21) Google Scholar), unlike Aβ that commences with an N-terminal glutamic acid-residue (e.g. Aβ3–42 and Aβ11–42) (20Schilling S. Hoffmann T. Manhart S. Hoffmann M. Demuth H.U. FEBS Lett. 2004; 563: 191-196Crossref PubMed Scopus (139) Google Scholar). QC has broad expression in mammalian brain (21Pohl T. Zimmer M. Mugele K. Spiess J. Proc. Natl. Acad. Sci. 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Mizusawa H. Shoji S. Kanazawa I. J. Biol. Chem. 1994; 269: 32721-32724Abstract Full Text PDF PubMed Google Scholar); however, the process that generates Aβ3–42 is unknown. Currently there are no reported animal models of Aβ3–42 expression. Advances in surface-enhanced laser desorption ionization-time of flight mass spectrometry (SELDI-TOF MS) analysis now facilitate accurate identification of particular Aβ species. Using this technology, we examined well characterized C. elegans transgenic models of AD that develop amyloid aggregates (25Link C.D. Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 9368-9372Crossref PubMed Scopus (508) Google Scholar, 26Fay D.S. Fluet A. Johnson C.J. Link C.D. J. Neurochem. 1998; 71: 1616-1625Crossref PubMed Scopus (143) Google Scholar) to see whether the human Aβ they express is post-translationally modified. The strains N2, wild type; CL2006, dvIs2(pCL12(unc-54:hu-Aβ 1–42) + pRF4) (25Link C.D. Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 9368-9372Crossref PubMed Scopus (508) Google Scholar); CL2120, dvIs14(pCL12(unc-54:hu-Aβ 1–42) + mtl-2:gfp); and CL2122, dvIs15(mtl-2:gfp) (26Fay D.S. Fluet A. Johnson C.J. Link C.D. J. Neurochem. 1998; 71: 1616-1625Crossref PubMed Scopus (143) Google Scholar) were provided by the Caenorhabditis Genetics Center. All strains were cultured at 20 °C on 8P/22Na medium (27Bianchi L. Driscoll M. Wormbook. 2006; (The C. elegans Research Community, ed) doi/10.1895/wormbook.1.122.1Google Scholar), and then at the first day of adulthood (4 days old), were aged at 25 °C as indicated. Human Aβ1–40, Aβ1–42, and Aβ3–42 peptides were synthesized by the W. M. Keck Laboratory (Yale University, New Haven, CT). Peptides were dissolved in 60 mm NaOH at room temperature and then diluted to 1 mg/ml in distilled H2O and 10× PBS (PBS is defined as 50 mm sodium phosphate, 2.7 mm KCl, 137 mm NaCl, pH 7.4) at a volume ratio of 2:7:1. Preparations were sonicated for 10 min in a water bath and then centrifuged at 13,500 × g for 10 min at 4 °C; the supernatant was then filtered through 0.2-μm filters (Supor, PALL) and kept on ice for immediate use. Peptide concentration was determined by measuring the absorbance value at 214 nm and applying the molar extinction coefficient values of 91,460 m−1 cm−1 for Aβ1–40 and Aβ3–42 and 94,530 m−1 cm−1 for Aβ1–42. Molar extinction coefficients were determined using amino acid analysis (Australian Proteome Analysis Facility Ltd.) and UV spectrometry (Lambda 25 UV-visible, PerkinElmer Life Sciences). Synchronized 4-day-old C. elegans adults were filtered (to remove eggs and larvae) through 40-μm nylon mesh (BD Biosciences) prior to sonication in chilled TBS (100 mm Tris-Cl and 150 mm NaCl, pH 8.0). After homogenization with a probe sonicator, lysates were clarified by centrifugation (13,500 × g for 5 min), and then the supernatant was removed and then kept on ice for immediate use or stored at −20 °C for subsequent analysis. For synthetic Aβ1–42 and Aβ3–42 standards, 40 pmol was analyzed. SELDI-TOF MS was performed using our established protocol (28Adlard P.A. Cherny R.A. Finkelstein D.I. Gautier E. Robb E. Cortes M. Volitakis I. Liu X. Smith J.P. Perez K. Laughton K. Li Q.X. Charman S.A. Nicolazzo J.A. Wilkins S. Deleva K. Lynch T. Kok G. Ritchie C.W. Tanzi R.E. Cappai R. Masters C.L. Barnham K.J. Bush A.I. Neuron. 2008; 59: 43-55Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar). Immunocapture used affinity-purified W0-2 (epitope: Aβ 5–8) (29Ida N. Hartmann T. Pantel J. Schröder J. Zerfass R. Förstl H. Sandbrink R. Masters C.L. Beyreuther K. J. Biol. 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Link of and of the Barnham of and Bush for and of this In we the of the of the of for to this All strains were provided by the Caenorhabditis Genetics by the U. S. of for Research with