Subcellular Topography of Neuronal Aβ Peptide in APPxPS1 Transgenic Mice
2004; Elsevier BV; Volume: 165; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)63405-0
ISSN1525-2191
AutoresDominique Langui, Nadège Girardot, Khalid Hamid El Hachimi, Bernadette Allinquant, Véronique Blanchard, Laurent Pradier, Charles Duyckaerts,
Tópico(s)Cellular transport and secretion
ResumoIn transgenic mice expressing human mutant β-amyloid precursor protein (APP) and mutant presenilin-1 (PS1), Aβ antibodies labeled granules, about 1 μm in diameter, in the perikaryon of neurons clustered in the isocortex, hippocampus, amygdala, thalamus, and brainstem. The granules were present before the onset of Aβ deposits; their number increased up to 9 months and decreased in 15-month-old animals. They were immunostained by antibodies against Aβ 40, Aβ 42, and APP C-terminal region. In double immunofluorescence experiments, the intracellular Aβ co-localized with lysosome markers and less frequently with MG160, a Golgi marker. Aβ accumulation correlated with an increased volume of lysosomes and Golgi apparatus, while the volume of endoplasmic reticulum and early endosomes did not change. Some granules were immunolabeled with an antibody against flotillin-1, a raft marker. At electron microscopy, Aβ, APP-C terminal, cathepsin D, and flotillin-1 epitopes were found in the lumen of multivesicular bodies. This study shows that Aβ peptide and APP C-terminal region accumulate in multivesicular bodies containing lysosomal enzymes, while APP N-terminus is excluded from them. Multivesicular bodies could secondarily liberate their content in the extracellular space as suggested by the association of cathepsin D with Aβ peptide in the extracellular space. In transgenic mice expressing human mutant β-amyloid precursor protein (APP) and mutant presenilin-1 (PS1), Aβ antibodies labeled granules, about 1 μm in diameter, in the perikaryon of neurons clustered in the isocortex, hippocampus, amygdala, thalamus, and brainstem. The granules were present before the onset of Aβ deposits; their number increased up to 9 months and decreased in 15-month-old animals. They were immunostained by antibodies against Aβ 40, Aβ 42, and APP C-terminal region. In double immunofluorescence experiments, the intracellular Aβ co-localized with lysosome markers and less frequently with MG160, a Golgi marker. Aβ accumulation correlated with an increased volume of lysosomes and Golgi apparatus, while the volume of endoplasmic reticulum and early endosomes did not change. Some granules were immunolabeled with an antibody against flotillin-1, a raft marker. At electron microscopy, Aβ, APP-C terminal, cathepsin D, and flotillin-1 epitopes were found in the lumen of multivesicular bodies. This study shows that Aβ peptide and APP C-terminal region accumulate in multivesicular bodies containing lysosomal enzymes, while APP N-terminus is excluded from them. Multivesicular bodies could secondarily liberate their content in the extracellular space as suggested by the association of cathepsin D with Aβ peptide in the extracellular space. 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We investigated brain samples from transgenic mice expressing human-mutated APP (APP751 with both the Swedish and the London mutation) and human PS1 bearing the M146L mutation.36Blanchard V Moussaoui S Czech C Touchet N Bonici B Planche M Canton T Jedidi I Gohin M Wirths O Bayer TA Langui D Duyckaerts C Tremp G Pradier L Time sequence of maturation of dystrophic neurites associated with Abeta deposits in APP/PS1 transgenic mice.Exp Neurol. 2003; 184: 247-263Crossref PubMed Scopus (249) Google Scholar Double immunofluorescent labeling with a combination of antibodies against Aβ, APP, and different organelle markers were examined with confocal microscopy and the volume occupied by the various organelles was evaluated. The intracellular localization of the Aβ peptide was determined at the ultrastructural level by immunogold electron microscopy. Generation and characterization of single APP751 with the Swedish and London mutation (APP751SL) and double APP751SLxPS1M146L transgenic mice were described previously.34Wirths O Multhaup G Czech C Feldmann N Blanchard V Tremp G Beyreuther K Pradier L Bayer TA Intraneuronal APP/A beta trafficking and plaque formation in beta-amyloid precursor protein and presenilin-1 transgenic mice.Brain Pathol. 2002; 12: 275-286Crossref PubMed Scopus (114) Google Scholar, 35Czech C Delaere P Macq AF Reibaud M Dreisler S Touchet N Schombert B Mazadier M Mercken L Theisen M Pradier L Octave JN Beyreuther K Tremp G Proteolytical processing of mutated human amyloid precursor protein in transgenic mice.Brain Res Mol Brain Res. 1997; 47: 108-116Crossref PubMed Scopus (32) Google Scholar, 36Blanchard V Moussaoui S Czech C Touchet N Bonici B Planche M Canton T Jedidi I Gohin M Wirths O Bayer TA Langui D Duyckaerts C Tremp G Pradier L Time sequence of maturation of dystrophic neurites associated with Abeta deposits in APP/PS1 transgenic mice.Exp Neurol. 2003; 184: 247-263Crossref PubMed Scopus (249) Google Scholar, 37Schmitz C Rutten BP Pielen A Schafer S Wirths O Tremp G Czech C Blanchard V Multhaup G Rezaie P Korr H Steinbusch HW Pradier L Bayer TA Hippocampal neuron loss exceeds amyloid plaque load in a transgenic mouse model of Alzheimer's disease.Am J Pathol. 2004; 164: 1495-1502Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar In these animals, APP is expressed at a high level in all cortical neurons under the control of the Thy-1 promoter. Human PS1 with the M146L mutation is expressed under the control of the HMG-CoA reductase promoter. The level of amyloid load was found to be quite reproducible at a given age; the coefficient of error between animals is below 10%.36Blanchard V Moussaoui S Czech C Touchet N Bonici B Planche M Canton T Jedidi I Gohin M Wirths O Bayer TA Langui D Duyckaerts C Tremp G Pradier L Time sequence of maturation of dystrophic neurites associated with Abeta deposits in APP/PS1 transgenic mice.Exp Neurol. 2003; 184: 247-263Crossref PubMed Scopus (249) Google Scholar PS1 single transgenic mice, which do not develop amyloid plaques, and wild-type animals were also examined. Intracytoplasmic Aβ accumulation was searched for in 24 APPxPS1 mice, five APP single transgenic, 13 PS1 single transgenic mice, and six wild-type animals of the same genetic background (C57Bl6) (Table 1). Animals were handled by Transgenic Services Department Charles River Laboratories France, and handled according to the French guidelines for animal care.Table 1Genotype, Age, and Number of AnimalsGenotype Age (months)WTPS1APPAPPxPS121324522226247229233511112224151Total613524 Open table in a new tab Animals (4 wild-type mice, 13 PS1, 4 APP, and 22 APPxPS1 transgenic mice from 2 to 15 months of age, Table 1) were sacrificed by intracardial perfusion with 4% paraformaldehyde (PFA) in phosphate- buffered saline (PBS), pH 7.4. Brains were removed and post-fixed for 1 hour in PFA at 4°C. Coronal sections (2-mm thick) were made, sectioned with a freezing microtome or embedded in paraffin. For electron microscopy, three additional 9-month-old animals (one APP, one APPxPS1, and one wild-type) were used. Post-fixation in 2.5% glutaraldehyde for 24 hours was added to the previous protocol. For Western blot analysis, crude brain homogenates were used. Snap-frozen cerebral hemispheres from one wild-type and one APPxPS1 (9- month-old animals) were weighed and homogenized on ice in 10 volumes (w/v) of buffer containing 0.32 mol/L sucrose, 4 mmol/L Tris-HCl, pH 7.4 and a protein inhibitor cocktail (Complete, Roche Diagnostics GmbH, Mannheim, Germany). Protein concentration was determined by BCA protein assay (Pierce, Rockford, IL). Proteins from crude brain homogenates (10 μg) were mixed with Laemmli sample buffer, then separated by electrophoresis on 12% Tris-HCl polyacrylamide gel. Proteins were transferred onto a nitrocellulose membrane (0.45 μm, Amersham, France), blocked with 5% (w/v) nonfat dry milk in TBST (50 mmol/L Tris-HCl (pH 8.1), 150 mmol/L NaCl, 0.05% (v/v) Tween 20), and incubated overnight at 4°C with the primary antibody. The following primary antibodies were applied to the membrane (Table 2): anti-APP Nter 1:2000, anti-APP Cter (737–751) 1:1000, anti-APP Cter (705–751) 1:3000, and anti-Aβ8–17 1:500 in 5% milk TBST. Binding of the primary antibody was detected with a horseradish peroxidase-conjugated secondary antibody at a 1:5000 dilution followed by the enhanced chemiluminescence detection system (ECL, Amersham, France) according to the manufacturer's instructions.Table 2AntibodiesAntibodyDilutionImmunogenSourceLabelingAPP-Cter rabbit polyclonal1/2000Synthetic peptide conjugated to KLH corresponding to aa 705–751 of the huAPP751B. Allinquant, INSERM U573, Paris, FranceC-terminal region of APPAPP-Cter goat polyclonal1/100Synthetic peptide conjugated to BSA, corresponding to aa 737–751 of the huAPP751Abcam, Cambridge, UK #2083C-terminal region of APPAPP Nter mouse monoclonal (clone 22C11)1/200purified recombinant Alzheimer precursor APP695 fusion proteinChemicon International, distributed by Euromedex, Souffelweyersheim, FranceN-terminal region of APP (aa 66–81)β-amyloid (Aβ) mouse monoclonal (clone 6F/D3)1/200KLH-aa 8–17 from human Aβ peptideDako Corporation, Glostrup, DenmarkAβ peptide 40 & 42β-amyloid (Aβ) rabbit polyclonal1/100Synthetic β-amyloid peptide 1–40 conjugated to BSAChemicon International, Temecula, CA, USAAβ peptide 40 & 42β-amyloid (Aβ) E50 Rabbit polyclonal1/100Aa 17–31 human Aβ peptideH. Akiyama, Institute of Psychiatry, Tokyo, JapanAβ peptide 40 & 42β-amyloid (Aβ) FCA 18 rabbit polyclonal1/500KLH-aa 1–8 from human Aβ peptide KLH-DAEFRHDS-CysF. Checler Sofia Antipolis, Nice, FranceN terminal part of Aββ-amyloid (Aβ) FCA 3340 rabbit polyclonal1/50Aa 33–40 human Aβ peptide KLH-Cys-GLMVGGVVF. Checler Sofia Antipolis, Nice, FranceAβ 40β-amyloid (Aβ) FCA 3542 rabbit polyclonal1/50Aa 35–42 human Aβ peptide KLH-Cys-MVGGVVIAF. Checler Sofia Antipolis, Nice, FranceAβ 42Bip (Grp78) rabbit polyclonal1/200KLH-aa 645–654 rat Grp78StressGen Biotechnologies Corporation, VictoriaEndoplasmic reticulumGolgi rabbit polyclonal1/1000MG-160 sialoglycoproteinN. Gonatas, University of Pennsylvania, Philadelphia, USAGolgi apparatusCathepsin D rabbit polyclonal1/1000Human liver active cathepsin DDako Corporation, Glostrup, DenmarkEnriched in lysosome lumenLAMP2 rabbit polyclonal1/200aa 1–207 of human Lamp2Santa Cruz, Santa Cruz, USALysosome membraneEarly endosomal antigen 1 rabbit polyclonal1/250human EEA1 aa 1391–1410Affinity Bioreagents, Golden, USAEarly endosomesCox 2 rabbit polyclonal1/500subunit 2 of human mitochondrial complex IVA. Lombès, Inserm U153, ParisMitochondriaFlotillin-1 mouse monoclonal1/1000aa 312–428 (C-terminal) of mouse flotillin-1Translab, Erembodegem, BelgiumProtein enriched in raftGFAP rabbit polyclonal1/500GFAP from cow spinal cordDako Corporation, Glostrup, DenmarkAstrocytesUbiquitin rabbit polyclonal1/500Ubiquitin isolated from cow erythrocytesDako Corporation, Glostrup, DenmarkUbiquitinated proteinsSNAP25 mouse monoclonal, (clone SMI 81)1/5000synaptosome-associated proteinSternberger, Lutherville, USASynapses Open table in a new tab Hematoxylin-eosin (H&E) and Bodian silver stains were routinely performed. To demonstrate the amyloid nature of the deposits, sections were stained with Congo red and thioflavin S. Immunostaining was performed on 5-μm thick paraffin sections. Sections were de-paraffinized in xylene, re-hydrated through ethanol (100%, 90%, 80%, and 70%) and finally brought to water. They were microwaved twice at 400 W in citrate buffer 0.01 mol/L, pH 6.0 for 10 minutes. When immunoperoxidase was used, endogenous peroxidase activity was quenched in a TBST solution containing 0.3% H2O2 and 20% methanol. Non-specific binding was blocked by incubating sections for 20 minutes in 2% bovine serum albumin in TBST. Appropriate dilutions of primary antibodies (Table 2) were then applied overnight in a humidified chamber at room temperature. Monoclonal antibody 22C11 against APP (APP66–81) is commercially available. It does not cross-react with the Aβ sequence.38Hoffmann J Twiesselmann C Kummer MP Romagnoli P Herzog V A possible role for the Alzheimer amyloid precursor protein in the regulation of epidermal basal cell proliferation.Eur J Cell Biol. 2000; 79: 905-914Crossref PubMed Scopus (68) Google Scholar, 39Hilbich C Monning U Grund C Masters CL Beyreuther K Amyloid-like properties of peptides flanking the epitope of amyloid precursor protein-specific monoclonal antibody 22C11.J Biol Chem. 1993; 268: 26571-26577Abstract Full Text PDF PubMed Google Scholar Rabbit polyclonal antibody against the C-terminal region of APP (generous gift of B. Allinquant, Inserm U573, Centre Paul Broca, Paris, France) was generated in the rabbit using the intracellular portion corresponding to amino acids (aa) 705–751. This APP fragment does not include the Aβ peptide sequence. Another polyclonal antibody against the C-terminal APP region encompassing aa 737–751 made in the goat was purchased (Abcam). This antibody does not recognize the Aβ sequence. E50 (generous gift of H. Akiyama, Institute of Psychiatry, Tokyo, Japan) is a polyclonal antibody raised against aa 17–31 (Aβ17–31) of human Aβ peptide. FCA 1–8 (generous gift of F. Checler, Institut de Pharmcologie Moleculaire et Cellulaire, Valbonne Sophia-Antipolis, France) recognizes the N-terminal portion of Aβ (aa 1–8), while, FCA40 and FCA42 (generous gifts of F. Checler) are specific for Aβ C terminus (including the COOH group), ending respectively at aa 40 and 42.40Barelli H Lebeau A Vizzavona J Delaere P Chevallier N Drouot C Marambaud P Ancolio K Buxbaum JD Khorkova O Heroux J Sahasrabudhe S Martinez J Warter JM Mohr M Checler F Characterization of new polyclonal antibodies specific for 40 and 42 amino acid-long amyloid beta peptides: their use to examine the cell biology of presenilins and the immunohistochemistry of sporadic Alzheimer's disease and cerebral amyloid angiopathy cases.Mol Med. 1997; 3: 695-707Crossref PubMed Google Scholar The antibodies against organelles included a rabbit anti-serum against MG160 (a Golgi sialoglycoprotein), which labels Golgi cis- and medial cis-ternae41Croul S Mezitis SG Stieber A Chen YJ Gonatas JO Goud B Gonatas NK Immunocytochemical visualization of the Golgi apparatus in several species, including human, and tissues with an antiserum against MG-160, a sialoglycoprotein of rat Golgi apparatus.J Histochem Cytochem. 1990; 38: 957-963Crossref PubMed Scopus (78) Google Scholar (generous gift of N. Gonatas, University of Pennsylvania, Philadelphia, PA, USA) and a rabbit anti-serum against Cox2, a mitochondrial enzyme (generous gift of A. Lombès, Inserm U573, Paris, France).42Possekel S Lombes A Ogier de Baulny H Cheval MA Fardeau M Kadenbach B Romero NB Immunohistochemical analysis of muscle cytochrome c oxidase deficiency in children.Histochem Cell Biol. 1995; 103: 59-68Crossref PubMed Scopus (24) Google Scholar The GRP78 antibody recognizes a KDEL sequence causing retention in the ER. The antibody against cathepsin D labels the lysosomes in which it is concentrated. EEA1 (early endosome antigen 1), a 162-kd autoantigen associated with subacute cutaneous systemic lupus erythematosus, localizes to early endosomes through the zinc-binding motif FYVE and interacts with Rab-5.43Stenmark H Aasland R Toh BH D'Arrigo A Endosomal localization of the autoantigen EEA1 is mediated by a zinc-binding FYVE finger.J Biol Chem. 1996; 271: 24048-24054Crossref PubMed Scopus (377) Google Scholar (Table 2). The appropriate biotinylated secondary antibody was applied for 30 minutes followed by 30 minutes incubation with streptavidine-peroxidase complex. The horseradish peroxidase activity was revealed with 3–3′-diaminobenzidine (DAB). The ABC-system and the peroxidase substrate were used according to the manufacturer's instructions (Chemmate K500111, Dako Corporation, Glostrup). Primary antibodies were omitted in control sections. Double immunofluorescence, on sections from 5- and 9-month-old animals, made use of antibodies from different species (one mouse monoclonal and one rabbit polyclonal). The secondary antibodies were directly linked with fluorescein isothiocyanate (FITC), carbocyanin 2 or 3 (Cy2 or Cy3). In some experiments, an intermediate step of streptavidin-biotin amplification was applied (Table 3). Slides were examined with a Leica confocal microscope (TCS), equipped with a krypton-argon laser (excitation wavelengths at 488, 568, and 647 nm).Table 3Secondary Antibodies and Fluorochromes for ImmunofluorescenceFluorochromeSpecificitySpeciesAbsorption (nm)Emission (nm)DilutionOriginCy3Anti-rabbitGoat5525701/400Jackson ImmunoResearch LaboratoriesCy2Streptavidin conjugated (used with biotinylated anti-mouse)492510Streptavidin CY2:1/400 (biotinylated anti-mouse:1/200)Streptavidin CY2, Jackson ImmunoResearch Laboratories; Biotinylated anti-mouse, AmershamFITCAnti-rabbitGoat4925201/400Jackson ImmunoResearch LaboratoriesCy3Anti-mouseGoat5525701/400Jackson ImmunoResearch Laboratories Open table in a new tab To visualize the distribution of Aβ-positive intraneuronal granules and amyloid deposits, a series of sagittal section cut at 25-μm thickness was obtained at ages 2, 5, 9, 11, and 15 months. They were immunostained with anti-Aβ8–17. The maps were drawn with the following apparatus, furnished by Explora Nova (La Rochelle, France) a camera, plugged in the phototube of a microscope, transmitted the microscopical image to a video s
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