Systemic Catabolism of Alzheimer's Aβ40 and Aβ42
2004; Elsevier BV; Volume: 279; Issue: 44 Linguagem: Inglês
10.1074/jbc.m407668200
ISSN1083-351X
AutoresJorge Ghiso, Marcos Shayo, Miguel Calero, Douglas Ng, Yasushi Tomidokoro, Sam Gandy, Agueda Rostagno, Blas Frangione,
Tópico(s)Aluminum toxicity and tolerance in plants and animals
ResumoTo better understand the physiologic excretion and/or catabolism of circulating peripheral amyloid β (Aβ), we labeled human Aβ40 (monomeric, with predominant unordered structure) and Aβ42 (mixture of monomers and oligomers in ∼50:50 ratio, rich in β-sheet conformation) with either Na125I or 125I-tyramine cellobiose, also known as the cell-trapping ligand procedure, testing their blood clearance and organ uptake in B6SJLF1/J mice. Irrespective of the labeling protocol, the peptide conformation, and the degree of oligomerization, both Aβ40 and Aβ42 showed a short half-life of 2.5–3.0 min. The liver was the major organ responsible for plasma clearance, accounting for >60% of the peptide uptake, followed by the kidney. In vivo, hepatocytes captured >90% of the radiolabeled peptides which, after endocytosis, were preferentially catabolized and excreted into the bile. Biliary excretion of intact as well as partially degraded Aβ species became obviously relevant at doses above 10 μg. The use of biotin-labeled Aβ allowed the visualization of the interaction with HepG2 cells in culture, whereas competitive inhibition experiments with unlabeled Aβ demonstrated the specificity of the binding. The capability of the liver to uptake, catabolize, and excrete large doses of Aβ, several orders of magnitude above its physiologic concentration, may explain not only the femtomolar plasma levels of Aβ but the little fluctuation observed with age and disease stages. To better understand the physiologic excretion and/or catabolism of circulating peripheral amyloid β (Aβ), we labeled human Aβ40 (monomeric, with predominant unordered structure) and Aβ42 (mixture of monomers and oligomers in ∼50:50 ratio, rich in β-sheet conformation) with either Na125I or 125I-tyramine cellobiose, also known as the cell-trapping ligand procedure, testing their blood clearance and organ uptake in B6SJLF1/J mice. Irrespective of the labeling protocol, the peptide conformation, and the degree of oligomerization, both Aβ40 and Aβ42 showed a short half-life of 2.5–3.0 min. The liver was the major organ responsible for plasma clearance, accounting for >60% of the peptide uptake, followed by the kidney. In vivo, hepatocytes captured >90% of the radiolabeled peptides which, after endocytosis, were preferentially catabolized and excreted into the bile. Biliary excretion of intact as well as partially degraded Aβ species became obviously relevant at doses above 10 μg. The use of biotin-labeled Aβ allowed the visualization of the interaction with HepG2 cells in culture, whereas competitive inhibition experiments with unlabeled Aβ demonstrated the specificity of the binding. The capability of the liver to uptake, catabolize, and excrete large doses of Aβ, several orders of magnitude above its physiologic concentration, may explain not only the femtomolar plasma levels of Aβ but the little fluctuation observed with age and disease stages. Alzheimer's disease (AD) 1The abbreviations used are: AD, Alzheimer's disease; Aβ, amyloid β; sAβ, soluble form of Aβ; APP, amyloid precursor protein; TC, tyramine-cellobiose; LRP-1, low density lipoprotein receptor-related protein-1; LRP-2, low density lipoprotein receptor-related protein-2; TTR, transthyretin; SAP, serum amyloid P component; HPLC, high performance liquid chromatography; RP-HPLC, reverse phase-HPLC; MALDI-TOF, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry; CRP, C-reactive protein; SEC-R, serpin-enzyme complex receptor; Me2SO, dimethyl sulfoxide; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl]glycine; CAPS, 3-(cyclohexylamino)propanesulfonic acid; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; MS, mass spectrometry; PBS, phosphate-buffered saline.1The abbreviations used are: AD, Alzheimer's disease; Aβ, amyloid β; sAβ, soluble form of Aβ; APP, amyloid precursor protein; TC, tyramine-cellobiose; LRP-1, low density lipoprotein receptor-related protein-1; LRP-2, low density lipoprotein receptor-related protein-2; TTR, transthyretin; SAP, serum amyloid P component; HPLC, high performance liquid chromatography; RP-HPLC, reverse phase-HPLC; MALDI-TOF, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry; CRP, C-reactive protein; SEC-R, serpin-enzyme complex receptor; Me2SO, dimethyl sulfoxide; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl]glycine; CAPS, 3-(cyclohexylamino)propanesulfonic acid; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; MS, mass spectrometry; PBS, phosphate-buffered saline. is the most frequent type of amyloidosis in humans and the commonest form of clinical dementia. Extracellular Aβ amyloid deposits in the form of amyloid plaques and cerebral amyloid angiopathy as well as intraneuronal neurofibrillary tangles co-exist in the brain parenchyma, being the cognitive areas the most severely affected. Aβ, a 39–42-amino acid-long peptide of unknown biological function, is an internal processing product of a larger type I transmembrane precursor molecule, APP, codified by a single multiexonic gene located on chromosome 21 (reviewed in Ref. 1Ghiso J. Frangione B. Adv. Drug Delivery Rev. 2002; 54: 1539-1551Crossref PubMed Scopus (153) Google Scholar). A soluble form of Aβ (sAβ) is present in the biological fluids of both normal individuals and AD patients as well as in cytosolic soluble fractions of normal, AD, and Down's syndrome brain homogenates (2Seubert P. Vigo-Pelfrey C. Esch F. Lee M. Dovey H. Davis D. Sinha S. Schlossmacher M. Whaley J. Swilndlehurst C. McCormack R. Wolfert R. Selkoe D. Lieberberg I. Schenk D.B. 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Zaccheo D. Dagna-Bricarelli F. Scartezzini P. Bertolini S. Mann D.M. Tabaton M. Gambetti P. Nat. Med. 1996; 2: 93-95Crossref PubMed Scopus (320) Google Scholar). Although the primary structures of deposited Aβ and sAβ are indistinguishable, the circulating peptide is predominantly 40 residues long, whereas sAβ42, the major species in parenchymal deposits, is only a minor component of the circulating pool. To the present, it is not clear whether circulating sAβs reflect systemic production, brain clearance, or both. The blood-brain barrier has the capability to modulate sAβ brain uptake and clearance by controlling the uptake of circulating sAβ, either in its free form or bound to its transport apolipoproteins, as well as the elimination of brain-derived Aβ via transport-mediated clearance mechanisms (reviewed in Ref. 8Zlokovic B. Adv. Drug Delivery Rev. 2002; 54: 1553-1559Crossref PubMed Scopus (53) Google Scholar). Experimentally determined transport rates indicate that the receptor for advance glycation end products mediates the influx of free Aβ into the brain (9Mackic J.B. Stins M. McComb J.G. Calero M. Ghiso J. Kim K.S. Yan S.D. Stern D. Schmidt A.M. Frangione B. Zlokovic B. J. Clin. Investig. 1998; 102: 734-743Crossref PubMed Scopus (215) Google Scholar), whereas low density lipoprotein receptor-related protein 2 (LRP-2, also known as gp330 or megalin) is the receptor involved in the uptake of Aβ-apoJ complexes (10Zlokovic B. Martel C. Matsubara E. McCombo J.G. Zheng G. McCluskey R.T. Frangione B. Ghiso J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4229-4234Crossref PubMed Scopus (376) Google Scholar). In contrast, brain clearance of Aβ at the blood-brain barrier occurs through different receptors, it is largely mediated by LRP-1 and modulated by the LRP-1 ligands apoE and α2-macroglobulin (11Shibata M. Yamada S. Kumar S.R. Calero M. Bading J. Frangione B. Holtzman D.M. Miller C.A. Strickland D.K. Ghiso J. Zlokovic B. J. Clin. Investig. 2000; 106: 1489-1499Crossref PubMed Scopus (1106) Google Scholar). Different proteases (i.e. neprilysin, endothelin-converting enzyme, angiotensin-converting enzyme, plasmin, and insulin-degrading enzyme) have been implicated in proteolysis-related clearance of Aβ from the central nervous system (12Carson J.A. Turner A.J. J. Neurochem. 2002; 81: 1-8Crossref PubMed Scopus (229) Google Scholar, 13Morelli L. Llovera R. Ibemdahl S. Castaño E.M. Neurochem. Int. 2002; 27: 1387-1399Crossref Scopus (26) Google Scholar, 14Farris W. Mansourian S. Leissring M.A. Eckman E.A. Bertram L. Eckman C.B. Tanzi R.E. Selkoe D. Am. J. Pathol. 2004; 164: 1425-1434Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 15Morelli L. Llovera R. Gonzalez S.A. Affranchino J.L. Prelli F. Frangione B. Ghiso J. Castaño E.M. J. Biol. Chem. 2003; 278: 23221-23226Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), although their final contribution to the mechanisms of Aβ homeostasis still remains unclear. The systemic, physiologic sAβ excretion/catabolism mechanisms are poorly understood. Clearance experiments in rats showed that after infusion of radiolabeled Aβ peptides into the lateral ventricle, 40% of the injected radioactivity was present in the blood and urine and taken up by the liver and the kidneys in as little as 3.5 min, indicating not only a fast clearance mechanism but also the involvement of systemic organs in the excretion and/or catabolism (16Ghersi-Egea J.-F. Gorevic P. Ghiso J. Frangione B. Patlak C.S. Fenstermacher J. J. Neurochem. 1996; 67: 880-883Crossref PubMed Scopus (203) Google Scholar). The high urinary levels of radioactive tracer compared with those in plasma suggested a key role for the renal clearance. In contrast, immunoprecipitation and mass spectrometry analysis of urine from normal individuals demonstrated the presence of intact sAβ40 at very low levels, accounting for only a minute fraction of the total circulating pool (17Ghiso J. Calero M. Matsubara E. Governale S. Chuba J. Beavis R. Wisniewski T. Frangione B. FEBS Lett. 1997; 408: 105-108Crossref PubMed Scopus (70) Google Scholar), thereby suggesting a different metabolic/excretory pathway. We investigated the excretion/catabolism of human Aβ40 and Aβ42 species in mice using both iodinated peptides and peptides labeled with an intracellularly trapped ligand procedure based on the use of 125I-tyramine-cellobiose (TC), a sugar adduct that is not degraded by mammalian cells and therefore accumulates in the organs involved in the uptake (18Pittman R.C. Carew T.E. Glass C.K. Green S.R. Taylor C.A. Attie A.D. Biochem. J. 1983; 212: 791-800Crossref PubMed Scopus (222) Google Scholar). This procedure has been widely used to determine sites of catabolism of many proteins, including some related to amyloid diseases, i.e. transthyretin (TTR) (19Makover A. Moriwaki H. Ramakrishnan R. Saraiva M.J.M. Blaner W.S. Goodman D.S. J. Biol. Chem. 1988; 263: 8598-8603Abstract Full Text PDF PubMed Google Scholar) or serum amyloid P component (SAP). Our results indicate that sAβ peptides have a short life in circulation, the liver being the major organ and the hepatocytes the main cell type responsible for their uptake and degradation/excretion. The findings are confirmed in vitro by using hepatocytes in culture and validated in vivo, analyzing Aβ degradation species in the bile of mice injected with human Aβ. Aβ-(1–40) (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV) and Aβ-(1–42) (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA), homologous to residues 672–711 and 672–713, respectively, of human Aβ precursor protein APP770 as well as a derivative of Aβ-(1–40) bearing a single biotin molecule at the N-terminal aspartate residue, were all synthesized at the W. M. Keck Facility at Yale University using N-tert-butyloxycarbonyl chemistry, purified by reverse phase-high performance liquid chromatography (RP-HPLC), their molecular masses corroborated by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and their concentration assessed by amino acid analysis. Both peptides were supplied as a single component eluted from a reverse phase C4 column with experimental molecular masses of 4329.1 Da for Aβ40 (expected, 4329.9 Da) and 4513.6 Da for Aβ42 (expected, 4514.1 Da). Peptides KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY (37 residues, homologous to human amylin) and KMQQNGYENPTYKGGEQMQN (20 amino acids, homologous to the C terminus of APP) were used as unrelated controls for specificity. Peptides were initially dissolved in either Me2SO or distilled deionized water. The peptides were briefly sonicated, diluted to their final concentration in 50 mm Tris-HCl, 150 mm NaCl, pH 7.4 (TBS), centrifuged at 14,000 × g for 10 min, sterilized using a 0.22-μm pore-size filter (Millex GP; Millipore Co., Bedford, MA), and either immediately used or stored at –80 °C. If Me2SO was used for solubilization, its final concentration in the sample never represented >1% of the total volume. Peptides were analyzed by amino acid sequence and MALDI-TOF mass spectrometry and structurally characterized via size exclusion chromatography and CD spectroscopy. The integrity of their biological activity was assessed through their binding interaction with apolipoprotein J (apoJ). The degree of oligomerization of the various peptides was measured by size exclusion chromatography using a Superdex 75 10/300 GL column (Amersham Biosciences) equilibrated in TBS, pH 7.4, at a flow rate of 0.5 ml/min. The effluent was monitored by absorbance at 220 nm, and fractions were collected accordingly. Aliquots of ∼200 ng were immediately lyophilized, re-dissolved in sample buffer, and separated on 16.5% Tris-Tricine SDS-polyacrylamide gels. The aliquots were then analyzed by Western blot after transfer onto polyvinylidene difluoride membranes (Immobilon P, Millipore Corp, Bedford, MA) employing 10 mm CAPS buffer (Sigma), pH 11.0, containing 10% methanol. After blocking for 1 h at 37 °C with 3% nonfat dry milk in TBS containing 0.1% Tween 20 (TBS-T), the electrotransferred material was incubated with a mixture of mouse monoclonal anti-human Aβ antibodies 6E10 and 4G8 (Signet, Dedham, MA; 1:5000 in TBS-T) followed by affinity-purified horseradish peroxidase-conjugated goat anti-mouse Ig (BioSource International; 1:5000 in TBS-T). Fluorograms were developed with enhanced chemiluminescence reagents (ECL, Amersham Biosciences) and exposed to Hyperfilm ECL (Amersham Biosciences). CD spectroscopy was used to estimate the predominant secondary structure of the different peptides. Spectra in the far-UV light (195–260 nm) were recorded at 24 °C with a Jasco J-720 spectropolarimeter (Jasco Corp.; Tokyo, Japan), using a cell path of 0.1 cm and a protein concentration of 0.15 mg/ml. Results were expressed in terms of mean residue ellipticity (degree·cm2·dmol–1). The dissociation constants of apoJ interaction with native and iodinated Aβ peptides were estimated by solid phase enzyme-linked immunosorbent assay using Aβ40- or Aβ42-coated (400 ng/100 μl/well) microtiter plates as described (20Calero M. Tokuda T. Rostagno A. Kumar A. Zlokovic B. Frangione B. Ghiso J. Biochem. J. 1999; 344: 375-383Crossref PubMed Scopus (82) Google Scholar). After blocking with 0.3% gelatin and 3% polyvinylpyrrolidone-40 in TBS-T, increasing concentrations of apoJ (0–20 nm) were incubated with the Aβ-coated wells. Bound apoJ was detected with mouse monoclonal G7 (anti-apoJ/clusterin,1:2000; Quidel Corp., San Diego, CA), followed by horseradish peroxidase-conjugated goat F(ab′)2 anti-mouse antiserum (1:4000, Amersham Biosciences). The reaction was developed for 15 min with 3,3′,5,5′-tetramethylbenzidine peroxidase substrate (Bio-Rad), stopped with 2.5 n H2SO4, and quantitated at 450 nm on a 7520 Microplate Reader (Cambridge Technology, Watertown, MA). Nonlinear regression analysis and estimation of dissociation constants were assessed with the aid of GraphPad Prism version 4.0 (GraphPad, San Diego, CA). Radiolabeling and Characterization of Aβ-iodinated Species—Ten micrograms of the Aβ peptides were labeled with 2 mCi of Na[125I] (Amersham Biosciences) using either lactoperoxidase (Sigma) or N-chlorobenzenesulfonamide (IODO-BEADS, Pierce), according to the respective manufacturer's instructions and standard protocols. Labeled Aβ40 was separated in a C4 narrow bore column (Vydac, The Separations Group, Hesperia, CA) using a 30-min 25–40% linear gradient of acetonitrile in 0.05% trifluoroacetic acid and a flow rate of 200 μl/min. Labeled Aβ42 was also separated in a C4 narrow bore column (Vydac) using a 40-min 30–42% linear gradient of acetonitrile in 0.05% trifluoroacetic acid and a flow rate of 200 μl/min. In both cases, specific activities were in the range of 80–100 μCi/μg, and radioactivity was >95% trichloroacetic acid-precipitable. The iodinated Aβ species and the degree of Met-35 oxidation were assessed through MALDI-TOF MS analysis of the HPLC peaks obtained in parallel Aβ-labeling experiments employing Na[127I] (Sigma) instead of Na[125I] under identical experimental conditions. The MS analysis was performed at the New York University Protein Analysis Facility using α-cyano-4-hydroxycinnamic acid (Sigma) matrix on a Micromass TofSpec-2E MALDI-TOF mass spectrometer in linear mode using standard instrument settings. Because sAβ species in the circulation consist mainly of nonoxidized species, to better reproduce physiological conditions, only those peaks containing iodinated nonoxidized Aβ were used for the uptake experiments. The use of lactoperoxidase consistently rendered lower amounts of undesirable oxidized peptides while maintaining specific activities at similar levels as IODO-BEADS that, in turn, translated in a better yield of nonoxidized, radioiodinated peptides. CD spectroscopy and binding to apoJ were used to demonstrate the lack of structural and functional changes in the Aβ peptides following iodination. The degree of oligomerization of radiolabeled peptides was assessed by Western blot as described above. Two proteins of different molecular masses and well known half-lives in rodents, human IgG (EMD Biosciences, San Diego, CA) and SAP (Sigma), were also radioiodinated as above and used as controls for the pharmacokinetic parameters described below. Their specific activities were in the range of 50–70 μCi/μg with >95% trichloroacetic acid precipitability in both cases. Identification of Targeted Amino Acid Residues in the Iodination Procedure—To identify the iodinated amino acid residues, Aβ-(1–40) and Aβ-(1–42) were labeled with Na[127I], the nonoxidized species separated by RP-HPLC identically as above, the resulting peaks subjected to separate proteolytic degradations with three different enzymes (trypsin, endoproteinase Asp-N, and endoproteinase Glu-C), and the subsequent proteolytic fragments analyzed by MALDI-TOF mass spectrometry to assess iodine incorporation. Each of the HPLC-purified, nonoxidized, Na[127I]-labeled Aβ40 peaks (2–5 μg) was dissolved in 5 μl of distilled water, with equal volumes of either 100 mm Tris-HCl buffer, pH 8, 100 mm phosphate buffer, pH 8, or 100 mm ammonium carbonate buffer, pH 8, and incubated for 3 h at 37 °C with either modified trypsin (Roche Applied Science; 1:25 w/w), endoproteinase Asp-N (Roche Applied Science; 1:50 w/w), or endoproteinase Glu-C (Roche Applied Science; 1:25 w/w), respectively. Digests were desalted using Zip-Tip C18 micro RP column (Millipore) as recommended by the manufacturer, utilizing 90% acetonitrile, 0.1% trifluoroacetic acid (v/v) for elution prior to MS analysis. To assess iodine incorporation in Aβ42 peptides, identical procedures were followed with the only difference that because RP-HPLC separation of iodinated and nonoxidized species did not result in well delineated separate peaks, as shown under “Results,” the nonoxidized pool was subjected to proteolytic degradation. The resulting peptides were further analyzed by MALDI-TOF-mass spectrometry. The labeled 125I-TC adduct was prepared as described before (18Pittman R.C. Carew T.E. Glass C.K. Green S.R. Taylor C.A. Attie A.D. Biochem. J. 1983; 212: 791-800Crossref PubMed Scopus (222) Google Scholar) by scaling down the reagents 50 times. In brief, 240 μmol each of cellobiose, tyramine, and NaBH3CN were allowed to react for 6 days at room temperature in 0.2 m sodium phosphate buffer, pH 7.5. Following adjustment of the pH to 5.5, the reaction products were separated on a cation exchange column (0.6 × 18 cm AG-50W, Bio-Rad), and the TC adducts were eluted with 0.5 m NH4OH. The adduct, further purified in a silicic acid column (0.7 × 26 cm), was eluted with butanol/acetic acid/water (7:1:2) after free tyramine at the end of the column. The purified TC was freeze-dried and stored at –20 °C until used. Approximately 10 nmol (∼4.8 μg) were radioiodinated with Na[125I] (3 mCi) for 30 min at room temperature in a N-chlorobenzene sulfonamide-coated tube (10 μg; IODO-GEN; Pierce), and the reaction was stopped by transferring the solution to a tube containing sodium metabisulfite and sodium iodine. After labeling, the 125I-TC adduct was activated with cyanuric chloride in acetone (1.8 μg/20 μl) and sodium hydroxide (5 μl of 4 mm) for 20 s, and the reaction was quenched by the addition of acetic acid (3 μl of 10 mm). The activated 125I-TC adduct was immediately bound to the corresponding Aβ peptides by addition to 50 μg of each of the peptides (1 mg/ml in 0.1 m phosphate buffer, pH 7.5) and incubation for 1 h at room temperature. The resulting 125I-TC-labeled peptides were separated by RP-HPLC in a Vydac C4 column using the same linear gradients as above. Specific activities of the labeled TC peptides after purification (typically 20 μCi/μg) were calculated from the integration of the peak areas obtained at 220 nm, and the radioactivity counts were obtained from aliquots of the respective pools. Experiments were performed on 8–12-week-old male B6SJLF1/J mice, (Jackson Laboratories, Bar Harbor, ME) following approval by the Institutional Animal Care and Use Committee at New York University. Typically, groups of 6–8 animals were anesthetized with Isoflurane, and the left jugular vein was isolated. Each group received a bolus intravenous injection of 2 × 107 cpm of either 125I-Aβ40, 125I-Aβ42, 125I-TC Aβ40, or 125I-TC Aβ42 peptides in 200 μl of lactate Ringer's solution (106 mm NaCl, 1.5 mm CaCl2, 24 mm sodium lactate, 4 mm potassium lactate, pH 7) containing 1% ovalbumin. Blood samples were collected from the tail vein at 0.5, 1.5, 5, 15, 30, 60, and 120 min using heparinized capillary tubes (Fisher). To assess the integrity of the peptides, a plasma aliquot at each time point was cold-precipitated with 12% trichloroacetic acid in lactate Ringer's containing 1% bovine serum albumin. Counts were assessed in a scintillation counter (LS6500; Beckman Instruments, Fullerton, CA), and total as well as trichloroacetic acid-precipitable counts/min were recorded. Results were expressed as remaining counts/min (percentage of initial counts/min) at each bleed time, with the 0.5-min time point considered as 100%. The half-lives of the radiolabeled peptides were calculated from the clearance curves generated by using a standard exponential decay equation in GraphPad Prism, and pharmacokinetic parameters for the IgG and SAP standard proteins were estimated by using noncompartmental pharmacokinetics data analysis from PK Solutions Software (Montrose, CO) following a bi-exponential equation. To assess organ distribution, groups of 6–8 animals received an intravenous injection of 2 × 107 cpm of either 125I-Aβ40, 125I-Aβ42, 125I-TC Aβ40, or 125I-TC Aβ42, as above, and were sacrificed by decapitation at 120 min. All organs, carcass, urine, feces, and bile were collected and weighed; the radioactivity was assessed, and the resulting counts/min were normalized per g of wet tissue. Parenchymal and nonparenchymal liver cells were isolated according to well established protocols (21Seglen P.O. Methods Toxicol. 1993; 1: 231-243Google Scholar, 22Friedman S.L. Methods Toxicol. 1993; 1: 292-310Google Scholar). Briefly isoflurane-anesthetized mice were intravenously bolus-injected with 2 × 107 cpm of either 125I-TC Aβ40 or 125I-TC Aβ42 in 200 μl of lactate Ringer's containing 1% ovalbumin, as described above. After 10 min, the liver was exposed and an intravenous catheter (Angiocath, BD Biosciences) inserted in the portal vein. The iliolumbar vein was then sectioned, and the liver was perfused at 37 °C with perfusion buffer (142 mm NaCl, 6.7 mm KCl, 10 mm HEPES, 6 mm NaOH, pH 7.4) at a flow rate of 2 ml/min until the organ was blanched (typically, about 10 min). The liver was subsequently removed from the carcass and further perfused at 37 °C for another 10 min with 0.05% w/v collagenase (type I, Sigma) in 4.8 mm CaCl2, 68.4 mm NaCl, 6.7 mm KCl, 100 mm HEPES, 66 mm NaOH, pH 7.4. After excision, the liver was placed in 20 ml of ice-cold suspension buffer (68.4 mm NaCl, 5.3 mm KCl, 1.1 mm KH2PO4, 0.7 mm Na2SO4, 30.2 mm HEPES, 3.0 mm TES, 36.3 mm Tricine, 52.5 mm NaOH, 1.2 mm CaCl2, and 0.6 mm MgCl2, pH 7.4) and gently raked to release the cells. The resulting cell suspension was filtered through a 100-μm nylon mesh to remove connective tissue debris and cell clumps, incubated on a tilting platform at 37 °C for 30 min, cooled in an ice-water bath, and centrifuged at 20 × g (Beckman J-6B) for 2 min at 4 °C. The resulting supernatant was used for further isolation of nonparenchymal cells, and the pellet, enriched in hepatocytes, was resuspended in ice-cold wash buffer (perfusion buffer supplemented with 1.2 mm CaCl2) and further depleted in nonparenchymal cells by four subsequent cycles of low speed centrifugation steps, as above. For the isolation of nonparenchymal cells, all supernatants were pooled, centrifuged at 500 × g for 7 min at 4 °C, resuspended in 2 ml of modified Eagle's medium (Sigma), overlaid on top of a discontinuous 6, 8, 12, and 15% arabinogalactan (Sigma) gradient, and centrifuged at 25 °C for 25 min at 49,000 × g in a Beckman XL ultracentrifuge using a SW-40 rotor (Beckman Coulter). Distribution of the radioactive tracer was assessed in each fraction, and cell morphology was evaluated after cytospin centrifugation (Shandon Cytospin Cytocentrifuge, Thermo Electron Co., Woburn, MA) by Sudan IV and Harris hematoxylin stainings. Human hepatoma HepG2 cells (ATCC, Manassas, VA) were cultured in growth media (Eagle's minimum essential medium with 2 mm l-glutamine and Earle's balanced salt solution adjusted to contain 1.5 g/liter sodium bicarbonate, 0.1 mm nonessential amino acids, and 1.0 mm sodium pyruvate (ATCC)), supplemented with 10% fetal bovine serum (Sigma) and penicillin/streptomycin (Sigma), as recommended by ATCC. For maintenance, cells were subcultured at a ratio 1:4 with medium replacement every 3 days. For binding experiments, cells were seeded onto Lab-Tek 4-well glass chamber slides (Nalge Nunc International, Naperville, IL) at a concentration of 7.5 × 104 cells in 0.5 ml of growth media per chamber and cultured for 48 h until 60–70% confluence. Cells were washed three times with ice-cold Dulbecco's PBS and maintained on ice during the subsequent experiment. Biotin-labeled Aβ40 was dissolved in distilled deionized water, briefly sonicated, centrifuged at 14,000 × g for 10 min, sterilized using a 0.22-μm pore filter, diluted to 10 μm in culture medium depleted of fetal bovine serum, and allowed to interact with the HepG2 cells for 2 h at 4 °C. After incubation, cells were washed three times with ice-cold PBS and fixed for 10 min with 4% paraformaldehyde in PBS before incubation with fluorescein-conjugated streptavidin (BioSource; 1:500) for 30 min. Following three PBS washes, nuclear DNA was counterstained with 4′-6-diamidino-2-phenylindole (0.5 μg/ml in Vectashield; Vector Laboratories, Inc., Burlingame, CA). Samples were visualized in an Olympus BX51 epifluorescence microscope, and images were acquired and analyzed with the aid of a digital imaging analyzer using Cytovision software version 2.7 (Applied Imaging Co., Santa Clara, CA). The specificity of HepG2-Aβ40 interaction was assessed by competitive inhibition of biotin-labeled Aβ40 binding by 10-fold molar excess of either unlabeled Aβ40 or unrelated peptides amylin and APP-(751–770). Preparation of Anti-Aβ-coated Paramagnetic Beads—Fifty microliters of goat anti-mouse IgG-coated paramagnetic beads (Dynabeads M-280; Dynal Biotech, Lake Success, NY) were allowed to interact with 3 μg each of anti-Aβ mouse monoclonal antibodies 4G8 and 6E10 (recognizing segments 1–16 and 17–24 of Aβ, respectively) for 2 h at room temperature followed by 16 h at 4 °C under constant end-to-end rotation, and subsequently were washed/blocked with PBS containing 0.1% bovine serum albumin. Aβ Immunoprecipitation in Bile and Urine Samples—Increasing amounts of nonradiolabeled human Aβ40 (0, 1, 10, or 100 μg) in 200 μl of lactate Ringer's solution were bolus-injected intravenously into different sets of mice (n = 2 for 0 μg, n = 8 for 1 μg, n = 7 for 10 μg, and n = 7 for 100 μg Aβ injection). The gallbladders and urinary bladders were removed 5, 15, or 30 min after peptide injection. Following washing out of blood, bile and urine
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