Portal de Periódicos da CAPES

Observations in APP Bitransgenic Mice Suggest that Diffuse and Compact Plaques Form via Independent Processes in Alzheimer's Disease

2011; Elsevier BV; Volume: 178; Issue: 5 Linguagem: Inglês

10.1016/j.ajpath.2011.01.052

1525-2191

Anna Lord, Ola Philipson, Therése Klingstedt, Gunilla T. Westermark, Per Hammarström, K. Peter R. Nilsson, Lars Nilsson,

Trace Elements in Health

Studies of familial Alzheimer's disease suggest that misfolding and aggregation of amyloid-β (Aβ) peptides initiate the pathogenesis. The Arctic mutation of Aβ precursor protein (APP) results in AD, and Arctic Aβ is more prone to form Aβ protofibrils and extracellular deposits. Herein is demonstrated that the burden of diffuse Aβ deposits but not compact plaques is increased when tg-Swe mice are crossed with tg-ArcSwe mice synthesizing low levels of Arctic Aβ. The diffuse deposits in bitransgenic mice, which contain primarily wild-type Aβ42, accumulate in regions both with and without transgene expression. However, APP processing, when compared with tg-Swe, remains unchanged in young bitransgenic mice, whereas wild-type Aβ42 aggregation is accelerated and fibril architecture is altered in vitro and in vivo when a low level of Arctic Aβ42 is introduced. Thus, the increased number of diffuse deposits is likely due to physical interactions between Arctic Aβ and wild-type Aβ42. The selective increase of a single type of parenchymal Aβ deposit suggests that different pathways lead to formation of diffuse and compact plaques. These findings could have general implications for Alzheimer's disease pathogenesis and particular relevance to patients heterozygous for the Arctic APP mutation. Moreover, it further illustrates how Aβ neuropathologic features can be manipulated in vivo by mechanisms similar to those originally conceptualized in prion research. Studies of familial Alzheimer's disease suggest that misfolding and aggregation of amyloid-β (Aβ) peptides initiate the pathogenesis. The Arctic mutation of Aβ precursor protein (APP) results in AD, and Arctic Aβ is more prone to form Aβ protofibrils and extracellular deposits. Herein is demonstrated that the burden of diffuse Aβ deposits but not compact plaques is increased when tg-Swe mice are crossed with tg-ArcSwe mice synthesizing low levels of Arctic Aβ. The diffuse deposits in bitransgenic mice, which contain primarily wild-type Aβ42, accumulate in regions both with and without transgene expression. However, APP processing, when compared with tg-Swe, remains unchanged in young bitransgenic mice, whereas wild-type Aβ42 aggregation is accelerated and fibril architecture is altered in vitro and in vivo when a low level of Arctic Aβ42 is introduced. Thus, the increased number of diffuse deposits is likely due to physical interactions between Arctic Aβ and wild-type Aβ42. The selective increase of a single type of parenchymal Aβ deposit suggests that different pathways lead to formation of diffuse and compact plaques. These findings could have general implications for Alzheimer's disease pathogenesis and particular relevance to patients heterozygous for the Arctic APP mutation. Moreover, it further illustrates how Aβ neuropathologic features can be manipulated in vivo by mechanisms similar to those originally conceptualized in prion research. The cardinal histopathologic lesions in brains affected with Alzheimer's disease (AD) are neurofibrillary tangles and extracellular compact plaques. The plaques are composed of various amyloid-β (Aβ) peptides and stain with amyloid dyes such as Congo red. Aβ is generated via endoproteolysis of the amyloid precursor protein (APP). It aggregates and gives rise to compact plaques with an amyloid core, but also parenchymal diffuse Aβ deposits and cerebrovascular amyloid angiopathy in vessel walls. Discoveries of point mutations in the APP gene that caused familial AD provided major insights into the pathogenesis.1Hardy J. A hundred years of Alzheimer's disease research.Neuron. 2006; 52: 3-13Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar The Arctic mutation (E693G), located within the Aβ domain of APP, segregates with clinical AD.2Basun H. Bogdanovic N. Ingelsson M. Almkvist O. Näslund J. Axelman K. Bird T.D. Nochlin D. Schellenberg G.D. Wahlund L.O. Lannfelt L. Clinical and neuropathological features of the Arctic APP gene mutation causing early-onset Alzheimer disease.Arch Neurol. 2008; 65: 499-505Crossref PubMed Scopus (79) Google Scholar, 3Nilsberth C. Westlind-Danielsson A. Eckman C.B. Condron M.M. Axelman K. Forsell C. Stenh C. Luthman J. Teplow D.B. Younkin S.G. Näslund J. Lannfelt L. The “Arctic” APP mutation (E693G) causes Alzheimer's disease by enhanced Abeta protofibril formation.Nat Neurosci. 2001; 4: 887-893Crossref PubMed Scopus (887) Google Scholar Because Arctic Aβ (AβArc) is more prone to form protofibrils than is wild-type Aβ (Aβwt),3Nilsberth C. Westlind-Danielsson A. Eckman C.B. Condron M.M. Axelman K. Forsell C. Stenh C. Luthman J. Teplow D.B. Younkin S.G. Näslund J. Lannfelt L. The “Arctic” APP mutation (E693G) causes Alzheimer's disease by enhanced Abeta protofibril formation.Nat Neurosci. 2001; 4: 887-893Crossref PubMed Scopus (887) Google Scholar, 4Johansson A.S. Berglind-Dehlin F. Karlsson G. Edwards K. Gellerfors P. Lannfelt L. Physiochemical characterization of the Alzheimer's disease–related peptides A beta 1-42Arctic and A beta 1-42wt.FEBS J. 2006; 273: 2618-2630Crossref PubMed Scopus (80) Google Scholar this mutation was used in combination with the Swedish mutation (KM670/671NL) to develop an animal model rich in soluble oligomeric Aβ assemblies.5Lord A. Kalimo H. Eckman C. Zhang X.Q. Lannfelt L. Nilsson L.N. The Arctic Alzheimer mutation facilitates early intraneuronal Abeta aggregation and senile plaque formation in transgenic mice.Neurobiol Aging. 2006; 27: 67-77Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 6Stenh C. Englund H. Lord A. Johansson A.S. Almeida C.G. Gellerfors P. Greengard P. Gouras G.K. Lannfelt L. Nilsson L.N. Amyloid-beta oligomers are inefficiently measured by enzyme-linked immunosorbent assay.Ann Neurol. 2005; 58: 147-150Crossref PubMed Scopus (86) Google Scholar Several founder lines were generated; however, only the line with the highest APP expression (line B, Table 1) has been reported to date.5Lord A. Kalimo H. Eckman C. Zhang X.Q. Lannfelt L. Nilsson L.N. The Arctic Alzheimer mutation facilitates early intraneuronal Abeta aggregation and senile plaque formation in transgenic mice.Neurobiol Aging. 2006; 27: 67-77Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 7Englund H. Sehlin D. Johansson A.S. Nilsson L.N. Gellerfors P. Paulie S. Lannfelt L. Pettersson F.E. Sensitive ELISA detection of amyloid-beta protofibrils in biological samples.J Neurochem. 2007; 103: 334-345PubMed Google Scholar, 8Sahlin C. Lord A. Magnusson K. Englund H. Almeida C.G. Greengard P. Nyberg F. Gouras G.K. Lannfelt L. Nilsson L.N. The Arctic Alzheimer mutation favors intracellular amyloid-beta production by making amyloid precursor protein less available to alpha-secretase.J Neurochem. 2007; 101: 854-862Crossref PubMed Scopus (49) Google Scholar, 9Lord A. Englund H. Söderberg L. Tucker S. Clausen F. Hillered L. Gordon M. Morgan D. Lannfelt L. Pettersson F.E. Nilsson L.N. Amyloid-beta protofibril levels correlate with spatial learning in Arctic Alzheimer's disease transgenic mice.FEBS J. 2009; 276: 995-1006Crossref PubMed Scopus (70) Google Scholar Its neuropathologic phenotypes were compared in detail with those of a transgenic model harboring only the Swedish mutation (tg-Swe; line A, Table 1).10Philipson O. HammarströM P. Nilsson K.P. Portelius E. Olofsson T. Ingelsson M. Hyman B.T. Blennow K. Lannfelt L. Kalimo H. Nilsson L.N. A highly insoluble state of Abeta similar to that of Alzheimer's disease brain is found in Arctic APP transgenic mice.Neurobiol Aging. 2009; 30: 1393-1405Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar In other founder lines of tg-ArcSwe and tg-Swe, the synthesis of human APP and Aβ was low, and Aβ deposition was scarce or undetectable even in very old animals. Herein we cross-bred a tg-ArcSwe founder line with very scarce Aβ pathology with a plaque-depositing tg-Swe line, in which human APP was expressed at a high level. We deemed that the experiment would be interesting for the following reasons. First, amyloid formation in human brain and APP transgenic mice is perceived to be seeded; that is, rapid fibril formation is thought to be preceded by a slow and rate-limiting reaction in which a nucleus is formed.11Jarrett J.T. Lansbury Jr, P.T. Seeding “one-dimensional crystallization” of amyloid: a pathogenic mechanism in Alzheimer's disease and scrapie?.Cell. 1993; 73: 1055-1058Abstract Full Text PDF PubMed Scopus (1886) Google Scholar, 12Gajdusek D.C. Transmissible and nontransmissible dementias: distinction between primary cause and pathogenetic mechanisms in Alzheimer's disease and aging.Mt Sinai J Med. 1988; 55: 3-5PubMed Google Scholar Both in vitro and in vivo studies have clearly demonstrated that AβArc is more proto-fibrillogenic than Aβwt.4Johansson A.S. Berglind-Dehlin F. Karlsson G. Edwards K. Gellerfors P. Lannfelt L. Physiochemical characterization of the Alzheimer's disease–related peptides A beta 1-42Arctic and A beta 1-42wt.FEBS J. 2006; 273: 2618-2630Crossref PubMed Scopus (80) Google Scholar, 5Lord A. Kalimo H. Eckman C. Zhang X.Q. Lannfelt L. Nilsson L.N. The Arctic Alzheimer mutation facilitates early intraneuronal Abeta aggregation and senile plaque formation in transgenic mice.Neurobiol Aging. 2006; 27: 67-77Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 13Cheng I.H. Palop J.J. Esposito L.A. Bien-Ly N. Yan F. Mucke L. Aggressive amyloidosis in mice expressing human amyloid peptides with the Arctic mutation.Nat Med. 2004; 10: 1190-1192Crossref PubMed Scopus (109) Google Scholar It was hypothesized that simultaneous production of AβArc and Aβwt in bitransgenic mice would result in a prion-like aggregation mechanism14Prusiner S.B. Molecular biology of prion diseases.Science. 1991; 252: 1515-1522Crossref PubMed Scopus (1733) Google Scholar in which the more aggregation-prone AβArc would act as a β-sheet template and accelerate Aβwt fibrillization. Second, both AβArc and Aβwt are produced in patients heterozygous for the Arctic mutation, and the pathogenic effects of such a mixture of Aβ peptides, if any, are unknown. By comparing the neuropathologic features of bitransgenic mice (tg-ArcSwe and tg-Swe) with single transgenic tg-Swe mice, whether aggregation-prone AβArc regulates Aβwt aggregation and/or formation of amyloid cores in the brain could be assessed. It was observed that the mixture of AβArc and Aβwt in bitransgenic mice led to more Aβ42-immunoreactive (ir) diffuse deposits in areas with and without transgene expression in bitransgenic mice but did not lead to more compact plaques. These findings suggest that diffuse and compact plaques develop via independent processes.Table 1Primary Characteristics of Various APP-Transgenic LinesModelLineHuman APP expression+Murine APP expression=APP overexpression⁎APP expression compared with endogenous murine APP.Age at onset of plaque deposition (months)tg-SweA6.01Sevenfold APPSwe12tg-ArcSweB2.01Threefold APPArcSwe5–6tg-ArcSweD0.851Twofold APPArcSwe18tg-ArcSwe × tg-SweBitransgenic6.851∼Eightfold APP (APPArcSwe + APPSwe)12Nontransgenic01 APP expression compared with endogenous murine APP. Open table in a new tab The expression cassette used to generate transgenic models and the procedures of genotyping have been described.5Lord A. Kalimo H. Eckman C. Zhang X.Q. Lannfelt L. Nilsson L.N. The Arctic Alzheimer mutation facilitates early intraneuronal Abeta aggregation and senile plaque formation in transgenic mice.Neurobiol Aging. 2006; 27: 67-77Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar All mice were kept at the animal facility at Uppsala University and housed under standard conditions with a 12-hour light/dark cycle and with free access to food and water. The tg-ArcSwe mice primarily used in this study were derived from a founder line with a low level of transgene APP expression. The phenotypes of this founder line differ markedly from the high-expressor tg-ArcSwe model.5Lord A. Kalimo H. Eckman C. Zhang X.Q. Lannfelt L. Nilsson L.N. The Arctic Alzheimer mutation facilitates early intraneuronal Abeta aggregation and senile plaque formation in transgenic mice.Neurobiol Aging. 2006; 27: 67-77Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 10Philipson O. HammarströM P. Nilsson K.P. Portelius E. Olofsson T. Ingelsson M. Hyman B.T. Blennow K. Lannfelt L. Kalimo H. Nilsson L.N. A highly insoluble state of Abeta similar to that of Alzheimer's disease brain is found in Arctic APP transgenic mice.Neurobiol Aging. 2009; 30: 1393-1405Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar The various transgenic lines used in the study are given in Table 1. All mice were genotyped using PCR with primers located at 1638 to 1655 and 2299 to 2281 in Y00264, followed by digestion with MboII and separation of bands on 2% agarose gel. The PCR product from a tg-ArcSwe mouse was then cleaved into two bands (456 and 205 bp), that from a tg-Swe mouse into three bands (364, 205, and 92 bp), and that from a bitransgenic tg-ArcSwe and tg-Swe mouse harboring both transgenes into four bands (456, 364, 205, and 92 bp). All mice used in the study were genotyped at age 3 weeks (ear biopsy) and again after death (tail biopsy). The experiments were approved by an ethical committee and performed in compliance with national and local animal care and use guidelines (protocol C223/8). Mice were anesthetized using 0.4 mL tribromoethanol (Avertin), 25 mg/mL, and intracardially perfused with 0.9% (w/v) NaCl solution. Brain hemispheres were frozen on dry ice for biochemical analyses. Alternatively, they were either directly frozen in metylbutane at −25°C or immersed in 4% paraformaldehyde for 24 hours and used for immunohistochemical analyses. Fixed brain tissue was cryoprotected via sequential immersion in 10%, 20%, and 30% (w/v) sucrose for 24 hours. Coronal sections 25-μm thick were prepared with a sledge microtome and stored at 4°C in PBS with 10 mmol/L NaN3. Two sections per individual, approximately 500 μm apart (bregma −1.0 mm to −3.0 mm), were selected for quantitative immunostaining. Fixed tissue sections were incubated in 25 mmol/L prewarmed citrate buffer (pH 7.3) for 5 minutes at 85°C, rinsed in deionized water, and immersed in 70% formic acid for 5 minutes. Endogenous peroxidase activity was quenched with 0.3% dH2O2 in Dako block (DakoCytomation, Glostrup, Denmark) for 15 minutes, and sections were permeabilized with 0.4% Triton X-100. The sections were incubated with 1 μg/mL polyclonal Aβ40- or Aβ42-specific antibodies,15Näslund J. Haroutunian V. Mohs R. Davis K.L. Davies P. Greengard P. Buxbaum J.D. Correlation between elevated levels of amyloid beta-peptide in the brain and cognitive decline.JAMA. 2000; 283: 1571-1577Crossref PubMed Scopus (1096) Google Scholar monoclonal AβArc-specific antibodies (mab27 at 1 μg/mL9Lord A. Englund H. Söderberg L. Tucker S. Clausen F. Hillered L. Gordon M. Morgan D. Lannfelt L. Pettersson F.E. Nilsson L.N. Amyloid-beta protofibril levels correlate with spatial learning in Arctic Alzheimer's disease transgenic mice.FEBS J. 2009; 276: 995-1006Crossref PubMed Scopus (70) Google Scholar), 0.5 μg/mL N-terminal Aβ antibody 6E10 (Covance, Princeton, NJ) or 82E1 (IBL International GmbH, Hamburg, Germany), 0.2 μg/mL goat anti-mouse poprotein J (AF2747; R&D Systems, Inc., Minneapolis, MN), goat anti-apolipoprotein E (ApoE) (AB947, diluted 1:5000; Chemicon International Inc., Temecula, CA), or 0.5 μg/mL monoclonal anti-human heparan sulfate (H1890, against 10E4 epitope; US Biological, Swampscott, MA) in PBS-buffered saline solution with 0.1% Tween 20 at 4°C overnight. Biotinylated goat anti-rabbit, goat anti-mouse, or rabbit anti-goat antibodies (Vector Laboratories, Inc., Burlingame, CA) were applied for 30 minutes at room temperature. Sections were then incubated with streptavidin-coupled horseradish peroxidase (Mabtech AB, Nacka Strand, Sweden) for 30 minutes and developed using the Nova Red chromogen kit (Vector Laboratories, Inc.). For double staining with the Aβ42-specific antibody and either thioflavine T (ThT) or the luminescent conjugated oligothiophene (LCO) p-FTAA, the sections were treated as described, and a fluorescent secondary antibody [Alexa Fluor 594 goat anti-rabbit (Invitrogen Corp., Carlsbad, CA)] diluted 1:250 in PBS was used to visualize the Aβ immunostaining. The sections were washed in PBS and incubated with 3 μmol/L p-FTAA16Åslund A. Sigurdson C.J. Klingstedt T. Grathwohl S. Bolmont T. Dickstein D.L. Glimsdal E. Prokop S. Lindgren M. Konradsson P. Holtzman D.M. Hof P.R. Heppner F.L. Gandy S. Jucker M. Aguzzi A. Hammarström P. Nilsson K.P. Novel pentameric thiophene derivatives for in vitro and in vivo optical imaging of a plethora of protein aggregates in cerebral amyloidoses.ACS Chem Biol. 2009; 4: 673-684Crossref PubMed Scopus (232) Google Scholar in PBS or with 0.2% (w/v) ThT (Sigma-Aldrich Corp., St. Louis, MO) in PBS for 30 minutes with shaking at room temperature. After rinsing with PBS, the sections were washed in dH2O and mounted with Vectashield (Vector Laboratories, Inc.). Compact plaques were detected using Congo red (Sigma-Aldrich Corp.) as previously described.17Nilsson L.N. Bales K.R. DiCarlo G. Gordon M.N. Morgan D. Paul S.M. Potter H. Alpha-1-antichymotrypsin promotes beta-sheet amyloid plaque deposition in a transgenic mouse model of Alzheimer's disease.J Neurosci. 2001; 21: 1444-1451PubMed Google Scholar The burden of diffuse and compact deposits in the cerebral cortex was measured in two image fields per section at ×20 magnification. Images were captured using a Nikon microscope (DXM1200F; Nikon Instruments Inc., Melville, NY) equipped with a digital camera, converted to gray scale, and segmented with an autothreshold command (Image Pro-Plus; MediaCybernetics, Silver Spring, MD). Custom macros were used to measure the stained area of interest as a percentage of the total area. Frozen brain sections 10-μm thick from 18-month-old tg-ArcSwe (line B), tg-Swe (line A), and bitransgenic tg-ArcSwe (line D) and tg-Swe (line A) mice were fixed in absolute ethanol for 10 minutes and rehydrated in 50% ethanol followed by water. The sections were incubated in PBS for 10 minutes and stained with 3 μmol/L p-FTAA16Åslund A. Sigurdson C.J. Klingstedt T. Grathwohl S. Bolmont T. Dickstein D.L. Glimsdal E. Prokop S. Lindgren M. Konradsson P. Holtzman D.M. Hof P.R. Heppner F.L. Gandy S. Jucker M. Aguzzi A. Hammarström P. Nilsson K.P. Novel pentameric thiophene derivatives for in vitro and in vivo optical imaging of a plethora of protein aggregates in cerebral amyloidoses.ACS Chem Biol. 2009; 4: 673-684Crossref PubMed Scopus (232) Google Scholar in PBS for 30 minutes at room temperature. After rinsing with PBS, the sections were mounted with Dako fluorescence mounting medium (DakoCytomation A/S). The medium was allowed to solidify for 3 hours before the slide rims were sealed with nail polish. Spectra and fluorescence images were obtained using a fluorescence microscope (Zeiss Axioplan; Carl Zeiss Ltd., Jena, Germany) equipped with a SpectraCube (optical head) module (Applied Spectral Imaging, Ltd., Migdal Ha'Emek, Israel) using bandpass filters 405/30 (LP450), 470/40 (LP515), and 546/12 (LP590). p-FTAA bound to compact plaques (10 plaques per animal, three animals from each group) was excited at 405 and 546 nm. SpectraView 3.0 EXPO software (Applied Spectral Imaging, Ltd.) was used to merge the information from the double excitation and create full visible-light emission spectra (450 to 700 nm) for p-FTAA when combining those excitation wavelengths. Fluorescent spectral unmixing, which is a function within the program, was performed to separate the emitted light intensities according to wavelength. The emission spectrum for p-FTAA bound to compact plaques in tg-ArcSwe (line B) was used to define red emission, and the emission spectrum for p-FTAA bound to compact plaques in tg-Swe (line A) was used to define green emission. By performing spectral unmixing, the fraction of each defined spectrum could be determined for each compact plaque found in tg-ArcSwe (line B), tg-Swe (line A), and bitransgenic mice, respectively. For analysis of Aβ, multiphasic urea/SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis were performed. This gel system enables differentiation of Aβ of various lengths and also Aβwt and AβArc. Samples were subjected to electrophoresis using a 15% T, 5% C bicine-Tris separation gel [T, total concentration of acrylamide; C, bis-acrylamide cross-linker content (acrylamide/bis-acrylamide, 40%; Sigma-Aldrich Corp.)] containing 8 mol/L urea. They were run at 24 mA, 30 V. Gels and buffers have been described previously,18Wiltfang J. Arold N. Neuhoff V. A new multiphasic buffer system for sodium dodecyl sulfate–polyacrylamide gel electrophoresis of proteins and peptides with molecular masses 100,000–1000, and their detection with picomolar sensitivity.Electrophoresis. 1991; 12: 352-366Crossref PubMed Scopus (87) Google Scholar, 19Klafki H.W. Wiltfang J. Staufenbiel M. Electrophoretic separation of betaA4 peptides (1–40) and (1–42).Anal Biochem. 1996; 237: 24-29Crossref PubMed Scopus (98) Google Scholar with the distinction that in the present study, 8 mol/L urea was included in the stacking gel. For analysis of total brain Aβ, tissues at bregma −1.0 mm to −3.0 mm were used. Tissue sections (30 μm; bregma 1.0 mm to −0.1 mm) were dissected and analyzed for Aβ levels in striatum and cerebral cortex. Brain tissue was homogenized in a final concentration of 70% formic acid at a ratio of 1:10 (w/v) using a Dounce homogenizer, sonicated for 30 seconds, and centrifuged at 100,000g at 4°C for 60 minutes. The supernatants were stored at −80°C before they were analyzed with enzyme-linked immunosorbent assay (ELISA) and SDS-PAGE Western blot analysis. For analysis of APP levels, brain tissues (bregma −1.0 mm to −3.0 mm) from 2-month-old mice were homogenized in Tris-buffered saline solution [20 mmol/L Tris and 137 mmol/L NaCl (pH 7.6)] supplemented with 2% (w/v) SDS and complete protease inhibitor (Hoffman-La Roche AG, Basel, Switzerland), sonicated for 30 seconds, and centrifuged at 100,000g at 4°C for 60 minutes. Proteins in the supernatant were separated on 4% to 20% Tris-tricine gels (Invitrogen Corp.). Protein transfer and Western blot analysis were performed as previously described.5Lord A. Kalimo H. Eckman C. Zhang X.Q. Lannfelt L. Nilsson L.N. The Arctic Alzheimer mutation facilitates early intraneuronal Abeta aggregation and senile plaque formation in transgenic mice.Neurobiol Aging. 2006; 27: 67-77Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar Ninety-six–well plates were coated for 12 hours at 4°C with antibodies specific for Aβ40 or Aβ42 (200 ng per well) and used for measuring Aβ1-40 or Aβ1-42 levels. For the total Aβ assay, plates were coated with 100 ng per well of the N-terminal Aβ-specific antibody 82E1 (IBL International GmbH) in PBS [130 mmol/L NaCl, 7 mmol/L NaH2PO4, and 3 mmol/L Na2HPO4 (pH 7.4)] and blocked with 1% bovine serum albumin in phosphate-buffered NaCl, 0.15% Kathon (Rohm and Haas Co., Philadelphia, PA). Formic acid extracts of mouse brain were neutralized in 1 mol/L Tris (pH 10) and diluted in ELISA incubation buffer (PBS with 0.1% bovine serum albumin, 0.05% Tween 20). Samples or standards (recombinant wt/Arctic Aβ1-40 or 1-42) were added to the plates in triplicate and incubated for 2 hours at room temperature. Biotinylated mAb27, 0.5 μg/mL, with an epitope in the mid-domain of Arctic Aβ,9Lord A. Englund H. Söderberg L. Tucker S. Clausen F. Hillered L. Gordon M. Morgan D. Lannfelt L. Pettersson F.E. Nilsson L.N. Amyloid-beta protofibril levels correlate with spatial learning in Arctic Alzheimer's disease transgenic mice.FEBS J. 2009; 276: 995-1006Crossref PubMed Scopus (70) Google Scholar was used to measure total AβArc. Biotinylated 4G8, 0.5 μg/mL (Covance, Inc.), was used to detect total Aβ, and biotinylated mAb1C3, 0.5 μg/mL,7Englund H. Sehlin D. Johansson A.S. Nilsson L.N. Gellerfors P. Paulie S. Lannfelt L. Pettersson F.E. Sensitive ELISA detection of amyloid-beta protofibrils in biological samples.J Neurochem. 2007; 103: 334-345PubMed Google Scholar was used to quantify Aβ1-40 or Aβ1-42. Both biotinylated detection antibodies and streptavidin-coupled horseradish peroxidase (Mabtech AB), diluted 1:2000, were incubated for 1 hour in successive steps. K-blue aqueous (ANL-Produkter AB, älvsjö, Sweden) was used as horseradish peroxidase substrate, the reaction was stopped using 1 mol/L H2SO4, and the optical density was measured at 450 nm with a spectrophotometer (SpectraMax 190; Molecular Devices, Inc., Sunnyvale, CA). Wells were washed three times between each step in ELISA washing buffer (phosphate-buffered NaCl with 0.1% Tween 20 and 0.15% Kathon). The mAb158 ELISA7Englund H. Sehlin D. Johansson A.S. Nilsson L.N. Gellerfors P. Paulie S. Lannfelt L. Pettersson F.E. Sensitive ELISA detection of amyloid-beta protofibrils in biological samples.J Neurochem. 2007; 103: 334-345PubMed Google Scholar detects oligomers larger than 100 kDa, which is consistent with the original description of oligomeric assemblies of Aβ.20Walsh D.M. Lomakin A. Benedek G.B. Condron M.M. Teplow D.B. Amyloid beta-protein fibrillogenesis: detection of a protofibrillar intermediate.J Biol Chem. 1997; 272: 22364-22372Crossref PubMed Scopus (937) Google Scholar In brief, each well in 96-well plates was coated with 200 ng mAb158 in PBS overnight at 4°C and blocked with 1% bovine serum albumin. The samples were centrifuged at 17,900 × g for 5 minutes before incubation to ensure that they were devoid of any insoluble Aβ aggregates. Samples were added to plates in duplicate and incubated for 2 hours at room temperature. Biotinylated mAb158, 0.5 μg/mL, was added and incubated for 1 hour at room temperature, followed by incubation with streptaviden-coupled horseradish peroxidase for 1 hour at room temperature. K-blue aqueous (ANL-Produkter AB) horseradish peroxidase substrate was used, and the reaction was stopped using 1 mol/L H2SO4. After the blocking step, plates were washed three times between each step. All samples and antibodies were diluted in ELISA incubation buffer (PBS with 0.1% bovine serum albumin and 0.02% Tween 20). The standard was generated by incubating Aβ1-42wt (American Peptide Co., Inc., Sunnyvale, CA) in 50 μmol/L PBS for 30 minutes at 37°C, followed by centrifugation at 16,000 × g for 5 minutes. The supernatant was loaded on a Superdex 75 10/300 GL column, resulting in a single high-molecular-weight peak, which was collected. Recombinant Aβ1-42wt and Aβ1-42Arc (American Peptide Co., Inc.) were treated with 100% hexafluoroisopropanol (Sigma-Aldrich Corp.), reaching a final concentration of 1 mmol/L. Hexafluoroisopropanol was allowed to evaporate in a chemical fume hood, and Aβ was then solubilized as a 400-μmol/L stock solution in 10 mmol/L NaOH and stored at −80°C. Immediately before use, Aβ1-42wt and Aβ1-42Arc were diluted in PBS [50 mmol/L phosphate buffer and 100 mmol/L NaCl (pH 7.4)] to 20 μmol/L and added to wells of a nonbinding 96-well microtiter plate (Greiner Bio-One GmbH, Frickenhausen, Germany). ThT (Sigma Corp.) and p-FTAA16Åslund A. Sigurdson C.J. Klingstedt T. Grathwohl S. Bolmont T. Dickstein D.L. Glimsdal E. Prokop S. Lindgren M. Konradsson P. Holtzman D.M. Hof P.R. Heppner F.L. Gandy S. Jucker M. Aguzzi A. Hammarström P. Nilsson K.P. Novel pentameric thiophene derivatives for in vitro and in vivo optical imaging of a plethora of protein aggregates in cerebral amyloidoses.ACS Chem Biol. 2009; 4: 673-684Crossref PubMed Scopus (232) Google Scholar were prepared as 15-μmol/L stock solutions in PBS (pH 7.4) and added to the wells at a final concentration of 1.2 μmol/L. Plates were incubated at room temperature for up to 22 hours under agitation. Aβ aggregation was recorded with a fluorometer (Wallac Victor 2 multilabel counter; Perkin Elmer Inc., Waltham, MA) as described for ThT21LeVine III, H. Thioflavine T interaction with synthetic Alzheimer's disease beta-amyloid peptides: detection of amyloid aggregation in solution.Protein Sci. 1993; 2: 404-410Crossref PubMed Scopus (1896) Google Scholar and p-FTAA.16Åslund A. Sigurdson C.J. Klingstedt T. Grathwohl S. Bolmont T. Dickstein D.L. Glimsdal E. Prokop S. Lindgren M. Konradsson P. Holtzman D.M. Hof P.R. Heppner F.L. Gandy S. Jucker M. Aguzzi A. Hammarström P. Nilsson K.P. Novel pentameric thiophene derivatives for in vitro and in vivo optical imaging of a plethora of protein aggregates in cerebral amyloidoses.ACS Chem Biol. 2009; 4: 673-684Crossref PubMed Scopus (232) Google Scholar The wavelengths of excitation and emission were 440 and 485 nm, respectively. Experiments were performed in triplicate, and fluorescence from free ThT or p-FTAA, incubated without Aβ in parallel, was subtracted from the readings. The ultrastructure of Aβ aggregates was examined by collecting samples of Aβ-solutions, 20 μmol/L, from the kinetic experiment. Solutions of Aβ were diluted to 1 μmol/L in mH2O and spotted on grids coated with carbon-formvar. Excess fluid was removed after 30 seconds, and 2.5% uranyl acetate, 5 to 10 μL, was added to the grids, and again excess fluid was quickly removed. The negatively stained specimen were examined and photographed using an electron microscope (JEOL 1230; JEOL Ltd., Tokyo, Japan) at 100 kV. Data were analyzed using the Student's t-test or with one- or two-way analysis of variance followed by Tukey's multiple comparison post hoc test (GraphPad Software, Inc., San Diego, CA). Results were presented as values representing the group mean and standard error of the mean (SEM). The relationship between Aβ40-ir and Aβ42-ir burden was investigated