Subcellular topography of neuronal Abeta peptide in APPxPS1 transgenic mice - PubMed (original) (raw)

Subcellular topography of neuronal Abeta peptide in APPxPS1 transgenic mice

Dominique Langui et al. Am J Pathol. 2004 Nov.

Abstract

In transgenic mice expressing human mutant beta-amyloid precursor protein (APP) and mutant presenilin-1 (PS1), Abeta antibodies labeled granules, about 1 microm in diameter, in the perikaryon of neurons clustered in the isocortex, hippocampus, amygdala, thalamus, and brainstem. The granules were present before the onset of Abeta deposits; their number increased up to 9 months and decreased in 15-month-old animals. They were immunostained by antibodies against Abeta 40, Abeta 42, and APP C-terminal region. In double immunofluorescence experiments, the intracellular Abeta co-localized with lysosome markers and less frequently with MG160, a Golgi marker. Abeta 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, Abeta, APP-C terminal, cathepsin D, and flotillin-1 epitopes were found in the lumen of multivesicular bodies. This study shows that Abeta 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 Abeta peptide in the extracellular space.

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Figures

Figure 1

Figure 1

Maps of the extracellular Aβ deposits and Aβ-positive granules. The extracellular deposits of Aβ peptide (left) and the neurons containing Aβ-positive granules (right) were manually mapped using a dedicated software (Explora Nova, La Rochelle). Each dot is an Aβ deposit (left) or a granule containing neurone (right) A: 2 months; B: 5 months; C: 9 months; D: 11 months; E: 15 months. Bar, 1 mm.

Figure 2

Figure 2

Immunoperoxidase Aβ8–17 immunostaining of Thy-1 double APP751SLxPS1M146L transgenic mouse (A, B, and C) and Thy-1 APP751SL single transgenic (D) mouse brain sections. The mice were 9 months (A, B, and C) and 5 months old (D). A: Distribution of Aβ immunoreactive deposits. Or, stratum oriens; Pyr, stratum pyramidale; Rad, stratum radiatum. The rectangle in A is enlarged in B. B: Dentate gyrus corresponding to rectangle in A, numerous plaques are visible (the arrow points to one of them). C: Intraneuronal granules (arrow) close to a plaque (arrowhead) in APPxPS1 transgenic animal. D: Intraneuronal granules (arrow) in an APP single transgenic animal. Bars: A, 0,5 mm; B, 50 μm; C, 15 μm; D, 10 μm. E: Brain homogenates from one wild-type mouse (lanes 1, 3, 5, and 7) and one Thy-1 double APP751SLxPS1M146L transgenic mouse (lanes 2, 4, 6, and 8) were analyzed by immunoblot using antibodies against APP Cter 705–751 (lanes 1 and 2), APP Cter 737–751 (lanes 3 and 4), Aβ8–17 (lanes 5 and 6), and APP Nter66–81 (lanes 7 and 8). Two bands (approximate molecular weight, 120 and 110 kd band, arrows) were detected by the antibodies to APP Cter and the antibody to APP Nter (22C11). These bands correspond to the full-length APP. The 120 kd visible in the transgenic animal corresponds to the human APP transgene. The antibody against Aβ8–17 did not cross-react with the full-length APP, either native or transgenic (lanes 5 and 6).

Figure 3

Figure 3

Double immunofluorescence examined with laser confocal microscope. Labeling with the Aβ peptide antibody is shown in red. Labeling with the other antibodies is shown in green. See Table 2 for details concerning the antibodies. Lamp2: lysosomal-associated membrane protein 2. Flotillin-1: a marker of raft and of multivesicular bodies. APP Cter, C terminus of the amyloid precursor protein (antibody raised against APP705–751); APP Nter, N terminus of the amyloid precursor protein (antibody raised against APP66–81). In H and I, arrows point to intraneuronal granules that are immunolabeled with both anti-Aβ8–17 and APP Cter (APP705–751) antibodies. In K and L, arrows point to intraneuronal granules, which are only labeled with anti-Aβ antibody (Aβ1–40 polyclonal, Chemicon) and not with the APP Nter antibody. Bars: A to F, 5 μm; G to L, 10 μm.

Figure 4

Figure 4

Double immunofluorescence examined with laser confocal microscope. Antibodies labeling organelles markers (A, D, G, and J) and Aβ8–17 (B, E, H, and K) are visualized respectively in red and in green. See Table 2 for details concerning the antibodies. The pictures on the right (C, F, I, and L) are merged images of the green and red signals; yellow indicates co-localization. Reticulum, endoplasmic reticulum labeled by Bip/GRP78; Golgi, Golgi apparatus labeled by MG160; Endosome, labeling by the antibody directed against EEA1 (early endosome autoantigen I). Lysosomes, organelles labeled by an antibody against cathepsin D, ie, lysosomes and multivesicular bodies having merged with lysosomes. Bars: A to C, 5 μm; D to L, 10 μm.

Figure 5

Figure 5

Proportion (%) of the total volume of intracellular Aβ peptide co-localized with the organelle marker. The Aβ antibody is the monoclonal antibody 6FD3, directed against amino acids 8–17 of the peptide (Dako). See Table 2 for details concerning the antibodies.

Figure 6

Figure 6

Electron microscopy. A: Conventional electron microscopy picture showing the appearance of intracellular granule (arrow); N, nucleus; R, endoplasmic reticulum. B to H: Immunoelectron microscopy. B: Labeling of an intraneuronal granule with anti-Aβ17–31 (E50,46). Gold particles are located in the lumenal vesicles (arrow). C: Double immunolabeling of intraneuronal granule with anti-Aβ17–31 (20-nm gold particle, arrowhead) and anti-flotillin (10-nm gold particle, arrow). D: Double immunolabeling of intraneuronal granule with anti-Aβ8–17 (10-nm gold particle, arrow) and anti-cathepsin D (20-nm gold particle, arrowhead). E: Double immunolabeling of a multivesicular body–lysosome with anti-APP Cter (20-nm gold particle, arrowhead) and anti-flotillin (10-nm gold particle, arrow). F: Labeling of an Aβ deposit with the anti-Aβ17–31 antibody decorating amyloid fibrils (arrowhead). The content of an extracellular vesicle is also labeled (arrow). (G) Labeling of Aβ deposit with the anti-cathepsin D antibody showing gold particles associated with amyloid fibrils (arrowhead) or between them (arrow). H: Double immunolabeling of an Aβ deposit with anti-Aβ17–31 (20-nm gold particle, arrowhead) and anti-flotillin (10-nm gold particle, arrow). Note that 10-nm gold particles are located in between the typical amyloid fibrils. Bars A to C and E to H, 400 nm; D, 250 nm.

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References

    1. Glenner GG, Wong CW. Alzheimer’s disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun. 1984;120:885–890. - PubMed
    1. Masters CL, Simms G, Weinman NA, Multhaup G, McDonald BL, Beyreuther K. Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc Natl Acad Sci USA. 1985;82:4245–4249. - PMC - PubMed
    1. Kang J, Lemaire HG, Unterbeck A, Salbaum JM, Masters CL, Grzeschik KH, Multhaup G, Beyreuther K, Muller-Hill B. The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell-surface receptor. Nature. 1987;325:733–736. - PubMed
    1. Selkoe DJ. Alzheimer’s disease: genes, proteins, and therapy. Physiol Rev. 2001;81:741–766. - PubMed
    1. Gouras GK, Xu H, Jovanovic JN, Buxbaum JD, Wang R, Greengard P, Relkin NR, Gandy S. Generation and regulation of beta-amyloid peptide variants by neurons. J Neurochem. 1998;71:1920–1925. - PubMed

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