title: Abeta mediated diminution of MTT reduction--an artefact of single cell culture?
abstract: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) reduction assay is a frequently used and easily reproducible method to measure beta-amyloid (Abeta) toxicity in different types of single cell culture. To our knowledge, the influence of Abeta on MTT reduction has never been tested in more complex tissue. Initially, we reproduced the disturbed MTT reduction in neuron and astroglia primary cell cultures from rats as well as in the BV2 microglia cell line, utilizing four different Abeta species, namely freshly dissolved Abeta (25-35), fibrillar Abeta (1-40), oligomeric Abeta (1-42) and oligomeric Abeta (1-40). In contrast to the findings in single cell cultures, none of these Abeta species altered MTT reduction in rat organotypic hippocampal slice cultures (OHC). Moreover, application of Abeta to acutely isolated hippocampal slices from adult rats and in vivo intracerebroventricular injection of Abeta also did not influence the MTT reduction in the respective tissue. Failure of Abeta penetration into the tissue cannot explain the differences between single cells and the more complex brain tissue. Thus electrophysiological investigations disclosed an impairment of long-term potentiation (LTP) in the CA1 region of hippocampal slices from rat by application of oligomeric Abeta (1-40), but not by freshly dissolved Abeta (25-35) or fibrillar Abeta (1-40). In conclusion, the experiments revealed a glaring discrepancy between single cell cultures and complex brain tissue regarding the effect of different Abeta species on MTT reduction. Particularly, the differential effect of oligomeric versus other Abeta forms on LTP was not reflected in the MTT reduction assay. This may indicate that the Abeta oligomer effect on synaptic function reflected by LTP impairment precedes changes in formazane formation rate or that cells embedded in a more natural environment in the tissue are less susceptible to damage by Abeta, raising cautions against the consideration of single cell MTT reduction activity as a reliable assay in Alzheimer's drug discovery studies.
authors.affiliation: Project Group Neuropharmacology, Leibniz Institute for Neurobiology, Magdeburg, Germany. roenicke@zenit-magdeburg.de
authors.forename: Raik
authors.initials: R
authors.lastname: Rönicke
authors.forename: Anja
authors.initials: A
authors.lastname: Klemm
authors.forename: Jessica
authors.initials: J
authors.lastname: Meinhardt
authors.forename: Ulrich H
authors.initials: UH
authors.lastname: Schröder
authors.forename: Marcus
authors.initials: M
authors.lastname: Fändrich
authors.forename: Klaus G
authors.initials: KG
authors.lastname: Reymann
mesh_terms: D000544:Alzheimer Disease; D016229:Amyloid beta-Peptides / Q000737:chemistry*; D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D001921:Brain / Q000196:embryology; D004594:Electrophysiology; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism; D017774:Long-Term Potentiation; D008297:Male; D017628:Microglia / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013569:Synapses / Q000378:metabolism; D013778:Tetrazolium Salts / Q000494:pharmacology*; D013844:Thiazoles / Q000494:pharmacology*
chemical_list: D016229:Amyloid beta-Peptides; D013778:Tetrazolium Salts; D013844:Thiazoles; C022616:thiazolyl blue
references.citation: Neurobiol Aging. 2009 Mar;30(3):377-87
references.pmid: 17692997
references.citation: J Lipid Res. 2005 May;46(5):949-68
references.pmid: 15716586
references.citation: J Neurochem. 1995 Dec;65(6):2585-93
references.pmid: 7595555
references.citation: J Neurochem. 1997 May;68(5):1889-95
references.pmid: 9109514
references.citation: Neurosci Res. 1997 Oct;29(2):129-34
references.pmid: 9359461
references.citation: J Neurochem. 1997 Dec;69(6):2285-93
references.pmid: 9375659
references.citation: Proc Natl Acad Sci U S A. 1998 May 26;95(11):6448-53
references.pmid: 9600986
references.citation: Nat Med. 1998 Jul;4(7):827-31
references.pmid: 9662375
references.citation: Neurosci Res. 1998 Aug;31(4):295-305
references.pmid: 9809588
references.citation: Glia. 1999 Feb 15;25(4):390-403
references.pmid: 10028921
references.citation: Neuropharmacology. 2000 Jul 24;39(10):1779-87
references.pmid: 10884559
references.citation: J Neurosci Methods. 2000 Jun 30;99(1-2):1-7
references.pmid: 10936637
references.citation: Eur J Neurosci. 2001 Apr;13(7):1380-6
references.pmid: 11298798
references.citation: Brain Res. 2001 Jul 13;907(1-2):188-94
references.pmid: 11430902
references.citation: Biochem Soc Trans. 2002 Aug;30(4):552-7
references.pmid: 12196135
references.citation: Science. 2003 Apr 18;300(5618):486-9
references.pmid: 12702875
references.citation: Brain Res. 2003 Sep 19;985(1):98-107
references.pmid: 12957372
references.citation: Leuk Lymphoma. 2003 Nov;44(11):1957-62
references.pmid: 14738150
references.citation: Exp Neurol. 2003 Dec;184(2):923-9
references.pmid: 14769384
references.citation: J Cell Mol Med. 2004 Apr-Jun;8(2):241-8
references.pmid: 15256072
references.citation: Eur J Pharmacol. 2004 Oct 11;502(1-2):99-104
references.pmid: 15464094
references.citation: J Biol Chem. 1967 Nov 10;242(21):4987-93
references.pmid: 6058941
references.citation: J Cell Biol. 1981 Nov;91(2 Pt 1):584-8
references.pmid: 6796595
references.citation: Science. 1990 Oct 12;250(4978):279-82
references.pmid: 2218531
references.citation: Am J Pathol. 1993 Jan;142(1):17-24
references.pmid: 8424453
references.citation: Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1470-4
references.pmid: 8108433
references.citation: Drug Discov Today. 2005 Jul 15;10(14):993-1000
references.pmid: 16023058
references.citation: Protein Sci. 2005 Aug;14(8):2125-31
references.pmid: 15987892
references.citation: Curr Drug Targets CNS Neurol Disord. 2005 Aug;4(4):435-52
references.pmid: 16101559
references.citation: Brain Res. 2005 Sep 21;1056(2):158-67
references.pmid: 16125154
references.citation: Mol Genet Metab. 2005 Sep-Oct;86(1-2):188-99
references.pmid: 15963747
references.citation: Biochem Soc Trans. 2005 Nov;33(Pt 5):1087-90
references.pmid: 16246051
references.citation: J Neurochem. 2005 Nov;95(3):834-47
references.pmid: 16135089
references.citation: Exp Brain Res. 2006 Jan;168(1-2):11-24
references.pmid: 16175362
references.citation: Exp Mol Med. 2005 Dec 31;37(6):559-66
references.pmid: 16391517
references.citation: J Neurochem. 2006 Feb;96(4):917-23
references.pmid: 16412101
references.citation: Neurobiol Aging. 2006 Apr;27(4):570-5
references.pmid: 16481071
references.citation: FASEB J. 2006 Apr;20(6):714-6
references.pmid: 16473887
references.citation: Amyloid. 2006 Mar;13(1):6-12
references.pmid: 16690494
references.citation: FASEB J. 2006 Jun;20(8):1191-3
references.pmid: 16636106
references.citation: J Biol Chem. 2006 Jul 21;281(29):20315-25
references.pmid: 16714296
references.citation: J Neurosci Methods. 2006 Oct 15;157(1):32-8
references.pmid: 16682083
references.citation: Neuropharmacology. 2007 Jan;52(1):24-40
references.pmid: 16919684
references.citation: J Neurosci. 2007 Mar 14;27(11):2866-75
references.pmid: 17360908
references.citation: Eur J Pharmacol. 2007 Apr 30;561(1-3):85-90
references.pmid: 17320856
references.citation: J Neurochem. 2007 Jun;101(5):1172-84
references.pmid: 17286590
references.citation: J Pharmacol Sci. 2007 May;104(1):46-55
references.pmid: 17452809
references.citation: Exp Brain Res. 2007 Jun;179(4):621-30
references.pmid: 17171334
references.citation: Neuroscience. 2007 Jul 13;147(3):639-51
references.pmid: 17560726
references.citation: Cell Mol Life Sci. 2007 Aug;64(16):2066-78
references.pmid: 17530168
references.citation: Eur J Neurosci. 2007 Oct;26(8):2142-50
references.pmid: 17908170
references.citation: Biochem Soc Trans. 2007 Nov;35(Pt 5):1219-23
references.pmid: 17956317
references.citation: Proc Natl Acad Sci U S A. 2007 Dec 4;104(49):19232-7
references.pmid: 18042730
references.citation: J Nutr Health Aging. 2008 Jan;12(1):51S-7S
references.pmid: 18165846
references.citation: Biochemistry. 2008 Jan 8;47(1):50-9
references.pmid: 18078350
references.citation: FASEB J. 2008 May;22(5):1552-9
references.pmid: 18096814
references.citation: Prog Neuropsychopharmacol Biol Psychiatry. 1999 Apr;23(3):377-95
references.pmid: 10378224
references.citation: J Biol Chem. 1999 Sep 3;274(36):25945-52
references.pmid: 10464339
references.citation: Nat Neurosci. 2005 Jan;8(1):79-84
references.pmid: 15608634
references.citation: J Neurosci. 2005 Feb 2;25(5):1071-80
references.pmid: 15689542
references.citation: Eur J Pharmacol. 1995 Jan 16;288(2):125-30
references.pmid: 7720773