Lack of ABCA1 Considerably Decreases Brain ApoE Level and Increases Amyloid Deposition in APP23 Mice
2005; Elsevier BV; Volume: 280; Issue: 52 Linguagem: Inglês
10.1074/jbc.m504513200
ISSN1083-351X
AutoresRadosveta Koldamova, Matthias Staufenbiel, Iliya Lefterov,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoABCA1 (ATP-binding cassette transporter A1) is a major regulator of cholesterol efflux and high density lipoprotein (HDL) metabolism. Mutations in human ABCA1 cause severe HDL deficiencies characterized by the virtual absence of apoA-I and HDL and prevalent atherosclerosis. Recently, it has been reported that the lack of ABCA1 causes a significant reduction of apoE protein level in the brain of ABCA1 knock-out (ABCA1-/-) mice. ApoE isoforms strongly affect Alzheimer disease (AD) pathology and risk. To determine further the effect of ABCA1 on amyloid deposition, we used APP23 transgenic mice in which the human familial Swedish AD mutant is expressed only in neurons. We demonstrated that the targeted disruption of ABCA1 increases amyloid deposition in APP23 mice, and the effect is manifested by an increased level of Aβ immunoreactivity, as well as thioflavine S-positive plaques in brain parenchyma. We found that the lack of ABCA1 also considerably increased the level of cerebral amyloid angiopathy and exacerbated cerebral amyloid angiopathy-related microhemorrhage in APP23/ABCA1-/- mice. Remarkably, the elevation in parenchymal and vascular amyloid in APP23/ABCA1-/- mice was accompanied by a dramatic decrease in the level of soluble brain apoE, although insoluble apoE was not changed. The elevation of insoluble Aβ fraction in old APP23/ABCA1-/- mice, accompanied by a lack of changes in APP processing and soluble β-amyloid in young APP23/ABCA1-/- animals, supports the conclusion that the ABCA1 deficiency increases amyloid deposition. These results suggest that ABCA1 plays a role in the pathogenesis of parenchymal and cerebrovascular amyloid pathology and thus may be considered a therapeutic target in AD. ABCA1 (ATP-binding cassette transporter A1) is a major regulator of cholesterol efflux and high density lipoprotein (HDL) metabolism. Mutations in human ABCA1 cause severe HDL deficiencies characterized by the virtual absence of apoA-I and HDL and prevalent atherosclerosis. Recently, it has been reported that the lack of ABCA1 causes a significant reduction of apoE protein level in the brain of ABCA1 knock-out (ABCA1-/-) mice. ApoE isoforms strongly affect Alzheimer disease (AD) pathology and risk. To determine further the effect of ABCA1 on amyloid deposition, we used APP23 transgenic mice in which the human familial Swedish AD mutant is expressed only in neurons. We demonstrated that the targeted disruption of ABCA1 increases amyloid deposition in APP23 mice, and the effect is manifested by an increased level of Aβ immunoreactivity, as well as thioflavine S-positive plaques in brain parenchyma. We found that the lack of ABCA1 also considerably increased the level of cerebral amyloid angiopathy and exacerbated cerebral amyloid angiopathy-related microhemorrhage in APP23/ABCA1-/- mice. Remarkably, the elevation in parenchymal and vascular amyloid in APP23/ABCA1-/- mice was accompanied by a dramatic decrease in the level of soluble brain apoE, although insoluble apoE was not changed. The elevation of insoluble Aβ fraction in old APP23/ABCA1-/- mice, accompanied by a lack of changes in APP processing and soluble β-amyloid in young APP23/ABCA1-/- animals, supports the conclusion that the ABCA1 deficiency increases amyloid deposition. These results suggest that ABCA1 plays a role in the pathogenesis of parenchymal and cerebrovascular amyloid pathology and thus may be considered a therapeutic target in AD. The deposition of Aβ 3The abbreviations used are: Aββ-amyloidADAlzheimer diseaseAPPamyloid precursor proteinAPPflAPPfull-lengthsAPPsoluble APPhhumanCAAcerebral amyloid angiopathyCTFAPP carboxyl-terminal fragmentsHDLhigh density lipoproteinsThio-Sthioflavine SWBWestern blottingELISAenzyme-linked immunosorbent assayPBSphosphate-buffered salineCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidLOADlate onset ADLXRliver X receptorLRPlow density lipoprotein receptor-related protein.3The abbreviations used are: Aββ-amyloidADAlzheimer diseaseAPPamyloid precursor proteinAPPflAPPfull-lengthsAPPsoluble APPhhumanCAAcerebral amyloid angiopathyCTFAPP carboxyl-terminal fragmentsHDLhigh density lipoproteinsThio-Sthioflavine SWBWestern blottingELISAenzyme-linked immunosorbent assayPBSphosphate-buffered salineCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidLOADlate onset ADLXRliver X receptorLRPlow density lipoprotein receptor-related protein. in the brain parenchyma and vessels is a pathological hallmark of AD, and it is believed that Aβ plays a central role in the pathogenesis of the neuronal dysfunction and cognitive impairment during the progression of the disease. Unlike familial early onset forms of AD, which are caused by mutations in APP or presenilins, the cause of late onset AD (LOAD) remains unknown. The inheritance of the apoE ϵ4 allele (apoE4) is considered a strong and independent risk factor of AD and is associated with increased neuritic plaques and CAA (1Olichney J.M. Hansen L.A. Galasko D. Saitoh T. Hofstetter C.R. Katzman R. Thal L.J. Neurology. 1996; 47: 190-196Crossref PubMed Scopus (194) Google Scholar, 2Bertram L. Tanzi R.E. Pharmacol. Res. 2004; 50: 385-396Crossref PubMed Scopus (110) Google Scholar, 3Yamada M. J. Neurol. Sci. 2004; 226: 41-44Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). It has been reported that in APP transgenic mice the disruption of the apoE gene causes a dramatic reduction of parenchymal amyloid plaques and CAA (4Bales K.R. Verina T. Dodel R.C. Du Y. Altstiel L. Bender M. Hyslop P. Johnstone E.M. Little S.P. Cummins D.J. Piccardo P. Ghetti B. Paul S.M. Nat. Genet. 1997; 17: 263-264Crossref PubMed Scopus (696) Google Scholar, 5Bales K.R. Verina T. Cummins D.J. Du Y. Dodel R.C. Saura J. Fishman C.E. DeLong C.A. Piccardo P. Petegnief V. Ghetti B. Paul S.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 15233-15238Crossref PubMed Scopus (414) Google Scholar, 6Fryer J.D. Taylor J.W. DeMattos R.B. Bales K.R. Paul S.M. Parsadanian M. Holtzman D.M. J. Neurosci. 2003; 23: 7889-7896Crossref PubMed Google Scholar). β-amyloid Alzheimer disease amyloid precursor protein APPfull-length soluble APP human cerebral amyloid angiopathy APP carboxyl-terminal fragments high density lipoproteins thioflavine S Western blotting enzyme-linked immunosorbent assay phosphate-buffered saline 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid late onset AD liver X receptor low density lipoprotein receptor-related protein. β-amyloid Alzheimer disease amyloid precursor protein APPfull-length soluble APP human cerebral amyloid angiopathy APP carboxyl-terminal fragments high density lipoproteins thioflavine S Western blotting enzyme-linked immunosorbent assay phosphate-buffered saline 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid late onset AD liver X receptor low density lipoprotein receptor-related protein. Two independent groups have reported recently that the lack of ABCA1 causes >75% reduction of apoE protein level in the brain of ABCA1-/- mice (7Hirsch-Reinshagen V. Zhou S. Burgess B.L. Bernier L. McIsaac S.A. Chan J.Y. Tansley G.H. Cohn J.S. Hayden M.R. Wellington C.L. J. Biol. Chem. 2004; 279: 41197-41207Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 8Wahrle S.E. Jiang H. Parsadanian M. Legleiter J. Han X. Fryer J.D. Kowalewski T. Holtzman D.M. J. Biol. Chem. 2004; 279: 40987-40993Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). The decreased apoE level in the central nervous system was not related to apoE gene expression but likely was caused by increased metabolism of abnormally lipidated apoE containing lipoprotein (8Wahrle S.E. Jiang H. Parsadanian M. Legleiter J. Han X. Fryer J.D. Kowalewski T. Holtzman D.M. J. Biol. Chem. 2004; 279: 40987-40993Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). ABCA1 is a major regulator of cholesterol efflux, HDL metabolism, and reverse cholesterol transport (9Repa J.J. Turley S.D. Lobaccaro J.A. Medina J. Li L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1139) Google Scholar, 10Brewer Jr., H.B. Remaley A.T. Neufeld E.B. Basso F. Joyce C. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1755-1760Crossref PubMed Scopus (148) Google Scholar). Mutations in the ABCA1 gene cause severe HDL deficiencies, the most prominent of which is Tangier disease, which is characterized by the virtual absence of apoA-I and HDL, the accumulation of cholesterol in cells, and the prevalence of atherosclerosis (11Rust S. Rosier M. Funke H. Real J. Amoura Z. Piette J.C. Deleuze J.F. Brewer H.B. Duverger N. Denefle P. Assmann G. Nat. Genet. 1999; 22: 352-355Crossref PubMed Scopus (1249) Google Scholar, 12Brooks-Wilson A. Marcil M. Clee S.M. Zhang L.H. Roomp K. van Dam M. Yu L. Brewer C. Collins J.A. Molhuizen H.O. Loubser O. Ouelette B.F. Fichter K. Ashbourne-Excoffon K.J. Sensen C.W. Scherer S. Mott S. Denis M. Martindale D. Frohlich J. Morgan K. Koop B. Pimstone S. Kastelein J.J. Hayden M.R. Nat. Genet. 1999; 22: 336-345Crossref PubMed Scopus (1481) Google Scholar, 13Bodzioch M. Orso E. Klucken J. Langmann T. Bottcher A. Diederich W. Drobnik W. Barlage S. Buchler C. Porsch-Ozcurumez M. Kaminski W.E. Hahmann H.W. Oette K. Rothe G. Aslanidis C. Lackner K.J. Schmitz G. Nat. Genet. 1999; 22: 347-351Crossref PubMed Scopus (1328) Google Scholar). The transcriptional activation of ABCA1, and other genes involved in cholesterol metabolism, is controlled by nuclear liver X receptors α and β LXRα/β (14Tontonoz P. Mangelsdorf D.J. Mol. Endocrinol. 2003; 17: 985-993Crossref PubMed Scopus (519) Google Scholar, 15Wang N. Tall A.R. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1178-1184Crossref PubMed Scopus (215) Google Scholar). Target genes of LXRα/β have already been implicated in the control of APP proteolytic processing (16Koldamova R.P. Lefterov I.M. Ikonomovic M.D. Skoko J. Lefterov P.I. Isanski B.A. DeKosky S.T. Lazo J.S. J. Biol. Chem. 2003; 278: 13244-13256Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 17Sun Y. Yao J. Kim T.W. Tall A.R. J. Biol. Chem. 2003; 278: 27688-27694Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 18Brown III, J. Theisler C. Silberman S. Magnuson D. Gottardi-Littell N. Lee J.M. Yager D. Crowley J. Sambamurti K. Rahman M.M. Reiss A.B. Eckman C.B. Wolozin B. J. Biol. Chem. 2004; 279: 34674-34681Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). The effect was attributed primarily but not only to ABCA1. It was also reported that a set of genetic variants of ABCA1 modifies the risk for AD (19Katzov H. Chalmers K. Palmgren J. Andreasen N. Johansson B. Cairns N.J. Gatz M. Wilcock G.K. Love S. Pedersen N.L. Brookes A.J. Blennow K. Kehoe P.G. Prince J.A. Hum. Mutat. 2004; 23: 358-367Crossref PubMed Scopus (116) Google Scholar, 20Wollmer M.A. Streffer J.R. Lutjohann D. Tsolaki M. Iakovidou V. Hegi T. Pasch T. Jung H.H. Bergmann K. Nitsch R.M. Hock C. Papassotiropoulos A. Neurobiol. Aging. 2003; 24: 421-426Crossref PubMed Scopus (134) Google Scholar) in Scottish, Swedish, and English populations, although another study of American and UK populations found that ABCA1 variants do not appear to influence the risk of LOAD (21Li Y. Tacey K. Doil L. Luchene R.V. Garcia V. Rowland C. Schrodi S. Leong D. Lau K. Catanese J. Neurosci. Lett. 2004; 366: 268-271Crossref PubMed Scopus (51) Google Scholar). Experiments with any of those genetic variants or complex AD model systems (APP transgenic/ABCA1 knock-out animals for example) have not been reported, and so there is no definitive conclusion about the role of ABCA1 in AD. To determine further the effect of ABCA1 on amyloid deposition, we used APP23 transgenic mice in which APPsw is expressed only in the brain. These mice develop compact amyloid plaques in brain parenchyma and CAA, reminiscent of the pathological features of AD (22Van Dam D. Vloeberghs E. Abramowski D. Staufenbiel M. De Deyn P.P. CNS. Spectr. 2005; 10: 207-222Crossref PubMed Scopus (42) Google Scholar, 23Sturchler-Pierrat C. Abramowski D. Duke M. Wiederhold K.H. Mistl C. Rothacher S. Ledermann B. Burki K. Frey P. Paganetti P.A. Waridel C. Calhoun M.E. Jucker M. Probst A. Staufenbiel M. Sommer B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13287-13292Crossref PubMed Scopus (1234) Google Scholar, 24Sturchler-Pierrat C. Staufenbiel M. Ann. N. Y. Acad. Sci. 2000; 920: 134-139Crossref PubMed Scopus (127) Google Scholar). In this study APP23 mice were bred to ABCA1 knock-out mice, and Aβ-related pathology was evaluated in APP23 mice with intact (APP23/ABCA1+/+) or disrupted (APP23/ABCA1-/-) ABCA1 gene. Our results demonstrate that in APP23 mice the lack of ABCA1 increases Aβ deposition. Transgenic Mice—This study fully conformed to the guidelines outlined in the Guide for the Care and Use of Laboratory Animals from the United States Department of Health and Human Services and was approved by the University of Pittsburgh Institutional Animal Care and Use Committee. We used APP23 transgenic mice expressing human familial AD mutant APP751 with Swedish double mutation at positions 670/671 (APPK670N, M671L) (23Sturchler-Pierrat C. Abramowski D. Duke M. Wiederhold K.H. Mistl C. Rothacher S. Ledermann B. Burki K. Frey P. Paganetti P.A. Waridel C. Calhoun M.E. Jucker M. Probst A. Staufenbiel M. Sommer B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13287-13292Crossref PubMed Scopus (1234) Google Scholar). The expression of human APPsw is driven by the murine Thy-1 promoter and is restricted to neurons. APP23 (C57BL/6 background) were cross-bred to ABCA1+/- heterozygous mice (DBA/1-Abca1tm1Jdm/J; The Jackson Laboratory) to generate APP23/ABCA1+/- progeny (C57BL/6 × DBA/1). The progeny was identified by PCR, and APP23/ABCA1+/- mice were bred to ABCA1+/- littermates (F1, no APP transgene, mixed C57BL/6 × DBA/1 background) to yield APP23/ABCA1-/-, APP23/ABCA1+/-, and APP23/ABCA1+/+ bigenic littermate mice. All mice used in this study are hemizygous APP23. At weaning, a distortion in the Mendelian inheritance of the APP23/ABCA1-/- genotype was observed that was three times less than expected, suggesting a perinatal lethality of APP23/ABCA1-/- pups. APP23/ABCA1-/- mice appeared normal and weighed slightly less (statistically insignificant) compared with APP23/ABCA1+/+ littermates. APP23/ABCA1-/- developed similarly to APP23/ABCA1+/+, and no gross anatomical abnormalities were observed until 13 months of age. Animal Tissue Processing—Mice were anesthetized with pentobarbital (150 mg/kg, intraperitoneal) and perfused transcardially with 100 ml of PBS (0.1 m, pH 7.4). Brains were rapidly removed; the olfactory bulb and cerebellum were deleted and divided in hemibrains. One hemibrain was snap-frozen on dry ice and the other was drop-fixed in 4% phosphate-buffered paraformaldehyde at 4 °C for 48 h. Antibodies—Rabbit polyclonal anti-ABCA1 antibody was purchased from Novus Biologicals (Littleton, CO). The 6E10 monoclonal antibody (Signet, Dedham, MA) recognizes the first 17 amino acids of the Aβ peptide. 6E10 antibody was used for Western blotting (WB) to detect full-length human APP and sAPPα. Rabbit C8 polyclonal antibody (25Koldamova R.P. Lefterov I.M. Staufenbiel M. Wolfe D. Huang S. Glorioso J.C. Walter M. Roth M.G. Lazo J.S. J. Biol. Chem. 2005; 280: 4079-4088Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar) was used to detect CTF resulting from α- or β-secretase cleavages. Rabbit 869 antibody (25Koldamova R.P. Lefterov I.M. Staufenbiel M. Wolfe D. Huang S. Glorioso J.C. Walter M. Roth M.G. Lazo J.S. J. Biol. Chem. 2005; 280: 4079-4088Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar) was used to detect sAPPβ by WB. This antibody does not recognize full-length APP but recognizes the neoepitope generated after cleavage by β-secretase. Murine-specific apoE antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). TAU-5 antibody (NeoMarkers, Fremont, CA) is a phospho-independent antibody that recognizes total tau protein. Glyceraldehyde-3-phosphate dehydrogenase monoclonal antibody was purchased from Chemicon International (Temecula, CA). Secondary antibodies conjugated to horseradish peroxidase were from Jackson ImmunoResearch (West Grove, PA). Histology and Immunohistochemistry—HistoPrep (Fisher)-embedded hemibrains were cut in the coronal plane at 30 μm on a cryostat. Sections were selected 300 μm apart, starting from a randomly chosen section about 300 μm caudal from the first appearance of CA3 and the dentate gyrus. The immunostaining procedure was performed on floating sections as described by Matsuoka et al. (26Matsuoka Y. Picciano M. Malester B. LaFrancois J. Zehr C. Daeschner J.M. Olschowka J.A. Fonseca M.I. O'Banion M.K. Tenner A.J. Lemere C.A. Duff K. Am. J. Pathol. 2001; 158: 1345-1354Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Briefly, after washing in PBS containing 0.2% Triton X-100 (PBST), nonspecific binding was blocked by incubating the sections in PBST, 3% normal goat serum (Vector Laboratories) for 1 h before the incubation in biotinylated 6E10 at 4 °C overnight. Sections were then washed in PBST and incubated in ABC Elite reagent (Vector Laboratories), and immunoreactivity was visualized by using diaminobenzidene/nickel (DAB/Ni). The specificity of immunoreactivity was confirmed by the lack of signal when applying the same protocol on brain sections from nontransgenic wild type littermates. For thioflavine S (Thio-S) staining, floating sections were washed in PBST, mounted on Superfrost Plus slides (Fisher), coated with Vectabond (Vector Laboratories), and processed essentially as described previously (27Bussiere T. Bard F. Barbour R. Grajeda H. Guido T. Khan K. Schenk D. Games D. Seubert P. Buttini M. Am. J. Pathol. 2004; 165: 987-995Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Sections mounted on the slides were post-fixed in 10% formalin for 10 min, washed in PBS, incubated in 0.25% potassium permanganate for 10 min, treated with 2% potassium metabisulfite, 1% oxalic acid until they appeared white, and finally stained for 10 min in 0.015% Thio-S in 50% ethanol. After washing in 50% ethanol and water, the slides were dried and dipped into Histo-Clear before being coverslipped with Permount. All chemicals were from Sigma at the highest recommended grade of purity. Parenchymal and cerebrovascular amyloidoses were evaluated on the same sections but were recorded and presented separately. Microscopic examinations were carried out using Leica DM fluorescent microscope and images captured by CCD camera (DG 300F, Leica). For quantitative analysis, Aβ and amyloid deposition in the neocortex and hippocampus was defined as the percent area covered by Aβ immunoreactivity (% Aβ load) and Thio-S positivity (% Thio-S load), respectively. For each mouse, Aβ immunoreactivity or Thio-S-positive plaques were counted in 6–7 sections per hemibrain 300 μm apart at a magnification of 50. The percentage of area covered by Aβ-IR or Thio-S positivity was determined by examining the entire area of the section using IPLab 3.1 software. Cerebrovascular amyloidosis was evaluated on Thio-S-stained slides in pia, cortex, hippocampus, and thalamus, using the same approach and presented as % vascular amyloid. A qualitative rating scale of CAA severity, similar to that reported previously for APP23 mice was used (28Calhoun M.E. Burgermeister P. Phinney A.L. Stalder M. Tolnay M. Wiederhold K.H. Abramowski D. Sturchler-Pierrat C. Sommer B. Staufenbiel M. Jucker M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14088-14093Crossref PubMed Scopus (344) Google Scholar) as follows: severity grade 1, with amyloid confined to the vessel wall; severity grade 2, vascular amyloid with amyloid infiltrating the surrounding neuropil; and severity grade 3, dyshoric amyloid with amyloid deposition within the vessel wall and with a thick and complete amyloid coat around the vessel wall. A set of seven coronal sections/mouse equally spaced throughout the brain was evaluated, and Thio-S-positive vessel surface area was measured in each of four regions (pia, cortex, hippocampus, and thalamus). It should, however, be stated that because coronal sections were used in all cases, this value may be influenced by vessel orientation (e.g. if vessels that run perpendicular to the plane of section were more likely to contain fibrillar amyloid, the reported percentage would be an underestimate of the total affected surface area). Quantitation of Cerebral Hemorrhage—30-μm sections were mounted on microscopic slides, fixed for 10 min in 10% freshly prepared formalin, and processed according to Carson (29Carson F.L. Histotechnology: A Self-Instructional Text. 2nd Ed. ASCP, Chicago1996Google Scholar). The slides were well rinsed in distilled water, incubated in Perl's solution, equal parts of 2% potassium ferrocyanide and 2% hydrochloric acid, and rinsed again. The sections were counterstained with Nuclear Fast Red (Vector Laboratories) for 5 min, rinsed under running tap water for 5 min, and coverslipped. All sections were scanned using 5× objective and focal hemorrhages analyzed and counted at ×250. The numbers of Perl's Berlin blue-stained clusters of hemosiderin staining (or Prussian blue-positive sites) were counted on all sections, and the average number of sites per section was quantified. A set of six equally spaced sections throughout the neocortex, hippocampus, and thalamus were examined, and the number of positive profiles was determined and averaged to a per section value. Tissue Homogenizing and Western Blotting—The frozen hemibrains (only cortices and hippocampi) were homogenized in tissue homogenization buffer (THB) (250 mm sucrose, 20 mm Tris base, 1 mm EDTA, 1 mm EGTA, 1 ml per 150 mg of tissue) and protease inhibitors essentially as described before (25Koldamova R.P. Lefterov I.M. Staufenbiel M. Wolfe D. Huang S. Glorioso J.C. Walter M. Roth M.G. Lazo J.S. J. Biol. Chem. 2005; 280: 4079-4088Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). Protein extracts were prepared by 1:1 dilution of the initial homogenate with 2× RIPA buffer (10 mm Tris-HCl, pH 7.3, 1 mm MgCl2, and 0.25% SDS, 1% Triton X-100) in the presence of protease inhibitors (10 μg/ml leupeptin, 10 μg/ml aprotinin, and 10 μg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride) and sonication. WB for ABCA1, APPfl, and CTFα/β were made as described previously (25Koldamova R.P. Lefterov I.M. Staufenbiel M. Wolfe D. Huang S. Glorioso J.C. Walter M. Roth M.G. Lazo J.S. J. Biol. Chem. 2005; 280: 4079-4088Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). sAPPα and sAPPβ were determined by WB in soluble extracts prepared from brain homogenate using cold 0.4% diethylamine in 100 mm NaCl, centrifuged at 135,000 × g for 1 h at 4 °C, and neutralized by adding 0.5 m Tris-HCl, pH 6.8. Aβ ELISA—Soluble Aβ was extracted with 2× RIPA buffer and centrifuged at 135,000 × g. Insoluble Aβ was extracted from the resulting pellet using 70% ice-cold formic acid. The soluble and insoluble Aβ extracts were additionally diluted in EC buffer (20 mm sodium phosphate, 2 mm EDTA, 400 mm NaCl, 0.2% bovine serum albumin, 0.05% CHAPS, 0.4% Block Ace, 0.05% NaN3, pH 7.0), and Aβ1–40 and Aβ1–42 concentrations were measured by sandwich ELISA as described previously (25Koldamova R.P. Lefterov I.M. Staufenbiel M. Wolfe D. Huang S. Glorioso J.C. Walter M. Roth M.G. Lazo J.S. J. Biol. Chem. 2005; 280: 4079-4088Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). Briefly, ELISA for Aβ was performed using 6E10 as the capture antibody, and anti-Aβ40 (G2-10 monoclonal antibody) and anti-Aβ42 (G2-13 monoclonal antibody) monoclonal antibodies conjugated to horseradish peroxidase (Genetics Co., Schlieren, Switzerland) were used as the detection antibodies. The reaction was developed by TMB Microwell peroxidase substrate system (Kierkegaard & Perry Laboratories, Gaithersburg, MD). The final values of Aβ were based on Aβ1–40 and Aβ1–42 peptide standards (Bachem Biosciences, King of Prussia, PA). The amount of Aβ was normalized either to the total protein or to the expression of APPfl as measured by WB and was expressed as picomoles/mg. WB for insoluble Aβ was performed using 10 μl of formic acid-extracted Aβ. The solvent (formic acid) was evaporated, and the pellet was resuspended in 2× NuPAGE loading buffer. The proteins were resolved on 4–12% NuPAGE gels and transferred on the nitrocellulose membranes. Membranes were boiled for 5 min in PBS, and Aβtotal was detected with 1:1000 dilution of 6E10 antibody. WB for Soluble and Insoluble ApoE—The soluble apoE was extracted from brain homogenates using 2× RIPA buffer and sonication and was centrifuged at 135,000 × g. The insoluble pellet was dissolved in 70% formic acid, sonicated for 30 s, and centrifuged at 135,000 × g. WB for soluble and insoluble apoE were made using 30 μg of total protein. For insoluble apoE, formic acid was evaporated (as for Aβ WB), and the pellet was resuspended in 2× NuPAGE loading buffer. For soluble and insoluble apoE, the proteins were resolved on 4–12% NuPAGE gels and transferred on the nitrocellulose membranes. Insoluble apoE was detected with a 1:100 dilution of anti-mouse apoE antibody, and soluble apoE was detected with 1:500 dilution of the same antibody. To avoid stripping and re-probing of the membranes for a loading control, we used anti-tau antibody Tau5, which is phospho-independent and recognizes total tau (55–65 kDa). Statistical Analysis—Results are reported as means ± S.E. Statistical significance was determined by two-tailed Student's t test or Mann-Whitney test. We used Spearman correlations to determine the unadjusted relationships between continuous variables (GraphPad Prism software, Windows version 3.0, San Diego). ABCA1 Deficiency Does Not Affect the Expression of Human APP but Substantially Decreases Brain ApoE Level in APP23 Mice—To determine the effect of genetically engineered disruption of ABCA1 on Aβ deposition in APP23 transgenic mice, we generated APP23/ABCA1-/- animals. In this study we examined the following 13-month-old bigenic animals: APP23/ABCA1-/- (9 mice, 5 females and 4 males), APP23/ABCA1+/- (16 mice, 13 females and 3 males), and their wild type APP23/ABCA1+/+ littermates (14 mice, 7 females and 7 males). We first evaluated the expression of ABCA1 and full-length human amyloid precursor protein (hAPPfl) by Western blotting in aliquots of SDS extracts of cortices and hippocampi. As expected, the results proved that ABCA1 protein expression in APP23/ABCA1-/+ mice is reduced by 50% as compared with APP23/ABCA1+/+ and is absent in APP23/ABCA1-/- mice (Fig. 1A, upper panel). ABCA1 deletion did not influence human APPsw transgene expression (751 amino acids, "Swedish" isoform) (Fig. 1A, lower panel). Because ABCA1 deficiency as reported considerably decreases the amount of endogenous apoE protein in the brains of ABCA1-/- mice (7Hirsch-Reinshagen V. Zhou S. Burgess B.L. Bernier L. McIsaac S.A. Chan J.Y. Tansley G.H. Cohn J.S. Hayden M.R. Wellington C.L. J. Biol. Chem. 2004; 279: 41197-41207Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 8Wahrle S.E. Jiang H. Parsadanian M. Legleiter J. Han X. Fryer J.D. Kowalewski T. Holtzman D.M. J. Biol. Chem. 2004; 279: 40987-40993Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar), we determined the level of soluble (RIPA-extractable) apoE. In confirmation of the previous studies, we found that the level of soluble apoE in 13-month-old APP23/ABCA1-/- mice was reduced more than 70% when compared with APP23/ABCA1+/+ (Fig. 1B). The same decrease was observed in 3-month-old APP23/ABCA1-/- mice (data not shown). Lack of ABCA1 Increases the Level of Insoluble Aβ in 13-Month-old APP23 Mice—Next we examined whether the deficiency of ABCA1 could alter brain Aβ levels in APP23/ABCA1-/- mice. We measured levels of Aβ40 and Aβ42 in both RIPA-soluble and -insoluble brain homogenates of APP23/ABCA1-/- mice, and we compared the values to those in APP23/ABCA1+/+ and APP23/ABCA1-/+ mice. We found that in APP23/ABCA1-/- versus APP23/ABCA1+/+ mice, the lack of ABCA1 resulted in an increase of soluble (RIPA-extractable) Aβ40 and Aβ42, although the difference did not reach statistical significance (Fig. 2A). We also examined the level of insoluble (formic acid extractable) Aβ by WB using 6E10 antibody, which recognizes both Aβ40 and Aβ42 (referred as Aβtotal in Fig. 2B). The level of Aβtotal showed very high variability among all mice, with male mice (except one animal) having less Aβtotal than the female mice. Fig. 2B shows that insoluble Aβtotal in APP23/ABCA1-/- was increased more than 2-fold (p < 0.015 versus APP23/ABCA1+/+). The level of insoluble Aβ total in APP23/ABCA1-/+ mice was intermediate to that in APP23/ABCA1-/- and APP23/ABCA1+/+. To assess specifically the proportion of Aβ40 and Aβ42 in the increased insoluble Aβ, we evaluated their levels by ELISA. The results demonstrated a less than 2-fold increase of insoluble Aβ42 in APP23/ABCA1-/- mice (p < 0.035 versus APP23/ABCA1+/+) and a more than 2-fold increase of insoluble Aβ40 (p < 0.025 versus APP23/ABCA1+/+, Fig. 2C). We concluded that the disruption of ABCA1 in APP23 mice significantly increases the level of insoluble Aβ40 and Aβ42. To determine which of the two Aβ forms (soluble or insoluble) is predominantly increased in APP23/ABCA1-/-, we measured the percentage of soluble Aβ in the total Aβ (soluble plus insoluble). Fig. 2D demonstrates that when compared with APP23/ABCA1+/+ mice, the proportion of soluble Aβ40 in APP23/ABCA1-/- mice was considerably decreased, suggesting that more Aβ converts from the soluble to the insoluble state in the absence of ABCA1. The difference in the proportion of solubl
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