A kinetic aggregation assay allowing selective and sensitive amyloid-β quantification in cells and tissues.
The process of amyloid-β (Aβ) fibril formation is genetically and pathologically linked to Alzheimer's disease (AD). Thus, a selective and sensitive method for quantifying Aβ fibrils in complex biological samples allows a variety of hypotheses to be tested. Herein, we report the basis for a quantitative in vitro kinetic aggregation assay that detects seeding-competent Aβ aggregates in mammalian cell culture media, in Caenorhabditis elegans lysate, and in mouse brain homogenate. Sonicated, proteinase K-treated Aβ fibril-containing tissue homogenates or cell culture media were added to an initially monomeric Aβ(1-40) reporter peptide to seed an in vitro nucleated aggregation reaction. The reduction in the half-time (t(50)) of the amyloid growth phase is proportional to the quantity of seeding-competent Aβ aggregates present in the biological sample. An ion-exchange resin amyloid isolation strategy from complex biological samples is demonstrated as an alternative for improving the sensitivity and linearity of the kinetic aggregation assay.
Howard Hughes Medical Institute, Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, United States.
Deguo
D
Du
Amber N
AN
Murray
Ehud
E
Cohen
Hyun-Eui
HE
Kim
Ryan
R
Simkovsky
Andrew
A
Dillin
Jeffery W
JW
Kelly
D000682:Amyloid / Q000032:analysis* / Q000378:metabolism; D016229:Amyloid beta-Peptides / Q000032:analysis; D000818:Animals; D001923:Brain Chemistry; D017173:Caenorhabditis elegans / Q000737:chemistry*; D019286:Endopeptidase K / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D010446:Peptide Fragments / Q000032:analysis
D000682:Amyloid; D016229:Amyloid beta-Peptides; D010446:Peptide Fragments; C078592:amyloid beta-protein (1-40); C075222:amyloid beta-protein (1-42); D019286:Endopeptidase K
Nature. 2001 Jun 14;411(6839):810-3
11459061
Biochemistry. 2006 Feb 21;45(7):2234-42
16475811
J Struct Biol. 2000 Jun;130(2-3):280-9
10940232
Nature. 2003 Jan 9;421(6919):182-7
12483226
Methods Enzymol. 1997;289:44-67
9353717
Science. 2008 Feb 15;319(5865):916-9
18276881
Curr Opin Neurol. 1994 Aug;7(4):283-6
7952234
FEBS J. 2005 May;272(9):2231-6
15853808
Protein Sci. 2004 Jul;13(7):1933-8
15215533
Proc Natl Acad Sci U S A. 2007 Dec 26;104(52):20914-9
18096717
Proc Natl Acad Sci U S A. 2003 Apr 15;100(8):4837-42
12697936
Neurobiol Dis. 2008 Dec;32(3):412-9
18786637
Annu Rev Biochem. 1997;66:385-407
9242912
Methods Enzymol. 2006;413:326-44
17046404
Annu Rev Biochem. 2009;78:959-91
19298183
Curr Alzheimer Res. 2006 Dec;3(5):421-30
17168641
Methods Mol Biol. 2005;299:279-97
15980612
Brain. 2007 Oct;130(Pt 10):2607-15
17698496
Cell. 2009 Dec 11;139(6):1157-69
20005808
Acc Chem Res. 2005 Dec;38(12):911-21
16359163
Lab Invest. 1988 Apr;58(4):454-8
2965774
Clin Lymphoma Myeloma. 2006 Nov;7(3):182-5
17229332
Nat Methods. 2007 Aug;4(8):645-50
17643109
Science. 2002 Jul 19;297(5580):353-6
12130773
Science. 1991 Feb 8;251(4994):675-8
1899488
Biochemistry. 1998 Dec 22;37(51):17882-9
9922155
J Biol Chem. 2004 Apr 23;279(17):17490-9
14752113
Science. 2006 Mar 10;311(5766):1471-4
16469881
J Biol Chem. 2002 Oct 25;277(43):40810-5
12181315
Cell. 2005 Feb 25;120(4):545-55
15734686
Acta Neuropathol. 1994;87(6):554-67
7522386
Proc Natl Acad Sci U S A. 2005 Nov 29;102(48):17342-7
16293696
Biochemistry. 1993 May 11;32(18):4693-7
8490014
Science. 2003 Jan 31;299(5607):713-6
12560553
Science. 2006 Sep 15;313(5793):1604-10
16902091
Methods Enzymol. 1999;309:274-84
10507030
Biophys J. 2009 Feb 18;96(4):1529-36
19217869
J Mol Biol. 2004 Nov 26;344(3):757-67
15533443
Proc Natl Acad Sci U S A. 2004 Apr 6;101(14):4752-7
15034169
Proc Natl Acad Sci U S A. 2002 Feb 5;99(3):1485-90
11818542
Cell. 2005 Apr 8;121(1):73-85
15820680
Curr Neurol Neurosci Rep. 2002 Sep;2(5):457-64
12169227
Proc Natl Acad Sci U S A. 2009 Jul 7;106(27):11125-30
19549824
Amyloid. 1998 Jun;5(2):121-42
9686307
Methods Enzymol. 2006;412:285-99
17046664
Proc Natl Acad Sci U S A. 2008 Nov 25;105(47):18349-54
19015532
Proc Natl Acad Sci U S A. 2009 Nov 24;106(47):19813-8
19843697
PLoS Genet. 2009 Mar;5(3):e1000399
19266020
Science. 1992 Feb 7;255(5045):728-30
1738847
J Biol Chem. 1997 Jan 17;272(3):1870-5
8999874
Nature. 2003 Dec 18;426(6968):900-4
14685251
Biophys J. 2008 Jan 15;94(2):379-91
17890392
Ann Neurol. 2004 Mar;55(3):306-19
14991808