Accumulation of Intraneuronal β-Amyloid 42 Peptides Is Associated with Early Changes in Microtubule-Associated Protein 2 in Neurites and Synapses

Mice

Well-established Tg2576 mice with the human APP Swedish 670/671 mutation were used in this study. APP knockout mice were generously provided by Dr. Hui Zheng, Baylor College of Medicine, Houston, TX. Brain sections from Tg2576 and wild-type mice were analyzed at 2–3, 9–11, 17–18, and 26 months of age with at least n = 3 for quantification experiments and n = 2–3 for immunolabeling studies.

All mouse experiments were performed in strict compliance with the institutional guidelines of the Institutional Animal Care and Use Committee (IACUC) of Weill Cornell Medical College, New York, NY, USA, in accordance with National Institutes of Health guidelines. The protocol was approved by the Weill Cornell IACUC (Protocol Number #03039-162A). All perfusions were performed under deep isoflurane anesthesia followed by decapitation. All efforts were made to minimize suffering.

Antibodies

Aβ42 antibody AB5078P (Chemicon, Temecula, CA/Millipore, Billerica, MA) is a rabbit polyclonal antibody directed against the C-terminus of Aβ42 [32], [33]. This antibody had been biochemically characterized by Western blotting and mass spectrometry [32]. Antibody AB5078P was further reported to not cross-react to APP on Western blot by Agholme et al., [34]. In addition, we observed the same labeling pattern (predominately associated with MVBs and smaller vesicles but not with the Golgi apparatus) with this Aβ42 antibody AB5078P (Figure S1A) as we had with Aβ42 antibody MBC42, which we had characterized by Western blotting, immunohistochemistry and immuno-EM [13]. In contrast, immuno-EM labeling of APP indicates predominant localization to the Golgi apparatus [13], [35]. The AB5078P antibody recognizes monomers and to a lesser extent dimers and trimers (see Results). We note that Millipore ran out of this clone and that the currently available version of AB5078P is therefore not the same as the one we used in the present and prior studies, despite the use of the same clone number. M16 is a rabbit polyclonal antibody (kindly provided by Dr. Charles Glabe, University of California, Irvine, CA), raised against synthetic Aβ1–42; it preferentially recognizes insoluble HMW Aβ42 oligomers [13], [32], [33], [36], [37]. 6E10 (Signet Laboratories/COVANCE, Princeton, NJ) is a monoclonal antibody directed against residues 5–10 of Aβ peptides, and thus also recognizes full-length APP; it detects Aβ42 monomers and a wide variety of LMW and HMW Aβ42 oligomers [36]. Other antibodies used in this study: MAP2 (Sigma, St. Louis, MO) for dendrites, tau-1 (Chemicon/Millipore) for non-phosphorylated tau localized in axons.

Tissue preparation

Preparation of tissue sections from Tg2576 mice [38] was similar to that described previously [13], [38]. Mice were anesthetized with sodium pentobarbital (150 mg/kg, i.p.) and perfused via the ascending aorta with saline/heparin followed by 40 ml of 3.75% acrolein (Polyscience, Warrington, PA), 2% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. Tissue sections were cut on a vibratome (40 µm thick) and were kept in storage buffer composed of 30% sucrose and 30% ethylene glycol in PB at −20°C.

Immunolabeling

Immunolabeling for light microscopy was performed as previously described [36], [39]. Free-floating sections were incubated in primary antibodies (MAP2 1∶1000, tau-1 1∶500) for 24 h at room temperature and then for 24–48 h at 4°C. The sections were incubated in biotinylated horse anti-mouse immunoglobulin (IgG) secondary antibody (1∶400, Vector Laboratories, Burlingame, CA) for 30 min, followed by the peroxidase-avidin complex (Vectastain ABC kit, Vector) for 30 min. The secondary antibody was diluted in 0.1 M Tris-saline (pH 7.6) containing 0.1% bovine serum albumin (BSA). The reaction product with the ABC kit was visualized after incubation of sections with 3, 3′-diaminobenzidine (Aldrich Chemical, Milwaukee, WI) and hydrogen peroxide. The sections were observed using a system consisting of a Nikon Eclipse E600 microscope (Morrell Instrument Co., Melville, NY) equipped with a computer-controlled LEP BioPoint motorized stage (Ludl Electronic Products, Hawthorne, NY), a DEI-750 video camera (Optronics, Goleta, CA), and a Dell Dimension 4300 computer (Dell, Round Rock, TX).

For immunofluorescence, free-floating sections were first incubated in primary antibodies (Aβ42 1∶200, M16 1∶1000, MAP2 1∶1000, tau-1 1∶500) for 24 h at room temperature and then for 24–48 h at 4°C, followed by appropriate fluorescent secondary antibodies Alexa 488 goat anti-rabbit IgG (green) and Alexa 546 goat anti-mouse IgG (red) (Molecular Probes, Eugene, OR) for 1 h at 37°C. Images were taken using an Olympus IX70 microscope with a Hamamatsu digital camera.

Immunohistochemical analysis

For MAP2 quantification, MAP2 immuno-stained brain sections were viewed using a Nikon Eclipse E600 microscope with a 40× objective, and digital images were captured using the Stereo Investigator 4.35 software program (Microbrightfield, Williston, VT). Photomicrographs were prepared by adjusting levels, brightness and contrast in Adobe Photoshop 7.0. Images of areas from stratum radiatum (SR) of the hippocampal CA1 region were captured using NIH Image 1.63 (National Institutes of Health, Bethesda, MD). For quantitative densitometry, ten random areas from SR of the hippocampal CA1 region in Tg2576 and wild-type mice (n = 3, each) were analyzed. Immunoreactivity of MAP2 in the SR region was measured and indicated as arbitrary units with the mean gray value of 256 gray levels. To compensate for background staining between images, the average pixel density for 3 regions without any immunoreactivity on brain section was subtracted. Tissues from Tg2576 and wild-type mouse brain sections were processed together in the same crucibles. Optical density values were measured using NIH image. Net optical density values obtained after subtracting background values were indicated as arbitrary units.

Ratio of MAP2-immunoreactivity in arbitrary units in the SR region of Tg2576 mouse brains divided by that of wild-type mice were calculated at 2–3, 9–11, and 17–18 month-old mice. The ratio was standardized to 100% for MAP2-immunoreactivity arbitrary units of wild-type mice. A comparison was performed between 2–3 months and 9–11 months, 2–3 months and 17–18 months of age, respectively using Student's t test for statistical analysis. Data were expressed as the mean ±SEM, and the significance threshold was p<0.05.

Immuno-electron microscopy

Immuno-electron microscopy (EM) localization was performed as previously described [13], [38]. Free-floating sections for dual-labeling immuno-EM were incubated with Aβ42 (AB5078P 1∶50) and MAP2 (1∶1000) antibody and processed first for immunoperoxidase localization of MAP2 antibody as described above. Sections then were processed by the immunogold-silver method for localization of Aβ42 antibody. For this, sections were incubated with goat anti-rabbit IgG conjugated to 1 nm gold particles (AuroProbe One; Amersham Biosciences, Arlington Heights, IL) in 0.01% gelatin and 0.08% BSA in PBS, pH 7.4, for 2 hours at room temperature. Sections were rinsed in PBS, post-fixed in 2% glutaraldehyde in PBS for 10 minutes, and rinsed in PBS and 0.2 M sodium citrate buffer (pH 7.4). Conjugated gold particles were enhanced by treatment with silver solution (Amersham Biosciences). Sections were fixed in 2% osmium tetroxide in PB, dehydrated, embedded in EMBed 812, sectioned (65- to 76-nm thick), and counterstained with uranyl acetate and Reynolds' lead citrate [39]. Final preparations were examined using a Philips CM10 electron microscope. Illustrations were generated from a high-resolution digital imaging CCD camera system (Advanced Microscopy Techniques, Danvers, MA) and processed using Adobe Photoshop 7.0 (Adobe System, Mountain View, CA).

Aβ42 and MAP2 immunolabeled profiles were classified as previously described by Peters et al. [40]. Somata were identified by the presence of a nucleus. Dendrites contained regular microtubule arrays and mitochondria, and were usually postsynaptic to axon terminal profiles. Axon terminals had numerous small synaptic vesicles, often contacting other neuronal profiles, and had a minimal diameter greater than 0.2 µm. Astrocytic profiles were recognized by their tendency to conform to the boundaries of surrounding profiles, by the presence of glial filaments and/or by less microtubules.

Preparation and Western blotting of synthetic Aβ1–42 peptides

Aβ1–42 was aggregated according to the method described by Fezoui et al., [41]. Briefly, lyophilized, synthetic Aβ1–42 peptides (American Peptide Company, Sunnyvale, CA) were dissolved in 20 mM NaOH, pH 10.5, to a final concentration of 1 mg/ml, sonicated, and lyophilized. Peptide was re-dissolved in water at a concentration of 1 mg/ml and filtered through a 0.22 µm Ultrafree-MC filter (Millipore, Bedford, MA). Aβ1–42 peptide (0.5 mg/ml) was buffered with 50 mM PB, 100 mM NaCl and incubated for 54 h at room temperature. We previously used this method to characterize the specificity of Aβ antibodies to different aggregated forms of Aβ1–42 peptides [36]; this method shows reproducible ranges of Aβ1–42 peptides from monomers to HMV oligomers on Western blot. Five hundred nanograms of aggregated Aβ1–42 peptides were separated by electrophoresis in 10–20% Tris-Tricine sodium dodecyl sulfate (SDS) polyacrylamide gels (Invitrogen, Carlsbad, CA), transferred to polyvinylidene-difluoride membranes (Millipore), and blotted with Aβ42 (1∶300) or 6E10 (1∶1000) antibodies, followed by incubation with secondary-HRP antibodies and visualization after enhanced chemiluminescence (Amersham Biosciences).

Aβ42 antibody preferentially recognizes M/LMW Aβ42 peptides

We previously reported that immunoreactivity for the HMW Aβ42 oligomer-specific antibody M16 is absent in brains of wild-type mice at all ages, and in brains of Tg2576 mice until just prior to plaque formation, where it consistently was associated with structural pathology within neurites [36]. To characterize potential effects of Aβ42 peptide accumulation prior to HMW oligomerization, we sought an antibody that visualized M/LMW but not HMW Aβ42 oligomers. To analyze which conformations of Aβ42 peptides are recognized by the well-characterized Aβ42 antibody AB5078P [32], we immunoblotted synthetic Aβ1–42 peptides prepared to include monomers to oligomers [36]. By Western blotting, the Aβ42 antibody detected predominantly Aβ1–42 monomers (Figure 1A). Importantly, HMW Aβ1–42 oligomers were not detected. Modest band intensities of LMW Aβ1–42 dimers and trimers could be observed after longer exposure. In contrast, and as previously reported [36], [42], the Aβ domain/APP antibody 6E10 detected Aβ1–42 monomers and a wide variety of LMW and HMW oligomers (Figure 1A). We previously confirmed the specificity of the Aβ42 antibody (AB5078P) [43] by absence of immunofluorescence in cultured neurons derived from well-established APP knockout mice [44] compared to punctate/vesicular labeling in wild-type mice. Additionally, lack of appreciable labeling was seen in APP knockout mouse brains (Figure S1B). Despite these characterization studies, one cannot fully exclude that conformational specificity of Aβ antibodies may differ between Western blotting and immunohistochemistry.

M/LMW Aβ42 peptide accumulation and MAP2 reduction in dendrites of Tg2576 mouse brains with aging

Biochemical analyses have shown that Aβ42 peptides increase with aging in brains of Tg2576 mice even prior to plaque formation [45]. By immuno-EM, Aβ42 peptide accumulation was evident especially within distal neuronal processes and synaptic compartments with aging rather than in cell bodies [13]. However, which conformation of Aβ42 peptide induces the earliest pathological alterations and how Aβ42 peptide accumulation relates to pathology of microtubule-associated proteins remains unclear. Utilizing the well-defined anatomy of the hippocampus, we show in Tg2576 mice early and progressive M/LMW Aβ42 peptide accumulation with aging, especially in the stratum lacunosum-moleculare (SLM) of the hippocampal CA1 region (Figure 1B, upper panel). This region contains predominantly distal dendrites from CA1 pyramidal cells and their associated synaptic compartments. We next investigated M/LMW Aβ42 peptide accumulation in relation to the dendritic microtubule-associated protein, MAP2, in the hippocampus of Tg2576 mice with aging. Remarkably, as the immunoreactivity for Aβ42 peptides increased, that for MAP2 decreased in CA1 dendrites of Tg2576 mice with aging. This was especially noticeable in the SLM (Figure 1B). We also observed some decrease in the stratum radiatum (SR). Co-localization of Aβ42 and MAP2 in the SR region is much clearer with higher magnifications, although it is not as clear as in the SLM or PCL (data not shown). On the whole, MAP2 reduction is marked in the SLM, and is not as clear in the SR region. To confirm and quantify the decrease in MAP2-immunoreactivity in Tg2576 mice, sections of Tg2576 and wild-type mouse brains at different ages were labeled with MAP2 antibody by immunoperoxidase. Dendrites in the SR of the CA1 region of aging Tg2576 mice appeared sparser and of reduced caliber compared to age-matched wild-type mice, which was especially evident at 17–18 months of age (Figure 1C). This is consistent with several previous studies demonstrating reduced dendritic architecture in APP mutant transgenic mice [46]–[48]. Densitometric quantification showed a significant decline in MAP2-immunoreactivity in the SR of the CA1 region of Tg2576 mice as a function of aging (Figure 1D). Specifically, MAP2-immunoreactivity in the SR of CA1 proximal dendrites was 105±5.08% (n = 3; p = 0.444), 79.64±3.23% (n = 3; p = 0.034) and 69.69±0.25% (n = 3; p<0.001) that of wild-type mice at 2–3 months, 9–11 months and 17–18 months of age, respectively. Similar reductions in MAP2-immunoreactivity in Tg2576 compared to wild-type mouse hippocampi were also apparent in the region of CA1 terminal dendrites in the SLM (Figure S2). In contrast to the MAP2 reductions, we did not detect any appreciable changes in tubulin and actin (data not shown).

Ultrastructural analysis of M/LMW Aβ42 peptides and MAP2

Dual-labeling immuno-EM was used to analyze the subcellular pattern of M/LMW Aβ42 peptides and MAP2 in dendrites. In agreement with light microscopic results, we observed a correlation between increasing M/LMW Aβ42 peptide accumulation and reduced MAP2-immunoreactivity using EM (Figure 2). Postsynaptic compartments with no appreciable M/LMW Aβ42 peptide labeling showed strong MAP2 labeling; those with intermediate M/LMW Aβ42 peptide labeling showed intermediate MAP2 labeling; and those with strong M/LMW Aβ42 peptide labeling showed little MAP2 labeling (Figure 2A). Such images support that reduced MAP2 is not merely secondary to loss of dendrites, since less MAP2 labeling is seen in these still present M/LMW Aβ42 labeling dendrites and postsynaptic compartments. Near or adjacent to clearly dystrophic neurites, numerous M/LMW Aβ42 gold particles could be seen in dendrites and postsynaptic compartments that contained only slight MAP2-immunoreactivity (Figure 2B). At times, M/LMW Aβ42 gold particles were found to be directly associated with degenerated multivesicular bodies (MVBs) near fibril-like electron-dense material, which could represent early Aβ42 fibrils (Figure 2C, black thin arrow). We also observed marked accumulations of M/LMW Aβ42 peptides in degenerated dendrites or axon terminals lacking any clear cytoskeletal architecture (Figure 2D, black arrows). At the same time, normal appearing dendrites and postsynaptic compartments with strong immunoreactivity for MAP2 and little intracellular Aβ42 could occasionally be found even near to extracellular amyloid fibers representing an amyloid plaque (Figure 2D).

Lack of MAP2 at sites of HMW Aβ42 oligomers within dendrites

Figures 1 and 2 indicated that dendritic accumulation of M/LMW Aβ42 peptide is locally associated with decreases in MAP2. Co-localization of reducing MAP2 and M/LMW Aβ42 still occurs (Figure 1B merge, Figure 2A asterisk), particularly within large puncta in SR and stratum oriens (SO), which could represent focal sites of swollen, dystrophic neurites (Figure 3A, bottom). In contrast, there was no labeling of MAP2 within Aβ-labeled plaques or in a halo around plaques (Figure 3A, top). Since dendritic MAP2-immunoreactivity was reduced in the presence of M/LMW Aβ42 oligomers (an early stage of Aβ aggregation) and absent in plaques (a later stage of Aβ aggregation), we next investigated the relationship of MAP2 with HMW Aβ42 oligomers (an intermediate stage of Aβ aggregation) using the well-characterized antibody M16 [37]. We previously confirmed by Western blotting that M16 reacts preferentially with HMW Aβ1–42 oligomers and characterized the time course of M16- immunoreactivity in Tg2576 mice at different ages [36]. Immunoreactivity for M16 did not appear until just before plaques and by immuno-EM was invariably associated with localized subcellular pathology [36]. We now show that in neurites of Tg2576 brain sections co-stained with M16 and MAP2 antibodies, there was no co-localization of MAP2 with HMW Aβ42 oligomers (Figure 3B). There was no overlap of MAP2- immunoreactivity with thioflavin S staining, which detects β-pleated amyloid fibrils, either (Figure 3C). These data support that accumulation of early stage Aβ aggregates (M/LMW Aβ oligomers) is already locally associated with progressive cytoskeletal pathology, which is severe when later stage aggregates (HMW Aβ oligomers and Aβ fibrils) are present.

M/LMW Aβ42 peptides accumulate less in axons than in dendrites of Tg2576 mouse brains

Our data indicated that M/LMW Aβ42 peptide accumulation was associated with early alterations in the microtubule-associated protein MAP2 in dendrites. A hallmark of AD is the abnormal hyperphosphorylation of the microtubule-associated protein tau; unphosphorylated tau is also used as a marker for axons. We therefore next analyzed M/LMW Aβ42 oligomer and tau-immunoreactivities in Tg2576 mouse brains at 17–18 months, and 26 months of age. At 17–18 months, when M/LMW Aβ42-immunoreactivity accumulated markedly in dendrites, there was only modest M/LMW Aβ42 peptide co-localization with tau-1 antibody (specific for non-phosphrylated tau localized only in axons) in axons compared to dendrites (Figure S3, top, Figure 1B, bottom). In contrast, M/LMW Aβ42 peptide and tau-1 co-localization was less evident in wild-type mice (Figure S3, middle). In very old Tg2576 mice (26 months), with more marked M/LMW Aβ42 peptide accumulations, co-localization of M/LMW Aβ42 peptide and tau-1 became more evident (Figure S3, bottom), but not as apparent as it is for MAP2 (Figure 3A, bottom). These results indicate that M/LMW Aβ42 peptides accumulate with aging also in axons of Tg2576 mouse brains, although to a lesser extent than in dendrites. These results are consistent with our initial immuno-EM studies of Aβ42 peptide accumulation, which had been noted to occur in axons and dendrites, including their pre- and postsynaptic compartments, although it was most obvious within distal dendrites [13].