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The Journal of Neuroscience, April 7, 2004, 24(14):3592-3599; doi:10.1523/JNEUROSCI.5167-03.2004
 Previous Article | Next Article 
Neurobiology of Disease
Oligomerization of Alzheimer's  -Amyloid within Processes and Synapses of Cultured Neurons and Brain
Reisuke H. Takahashi,
Claudia G. Almeida,
Patrick F. Kearney,
Fangmin Yu,
Michael T. Lin,
Teresa A. Milner, and
Gunnar K. Gouras
Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, New York 10021
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Abstract
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Multiple lines of evidence implicate  -amyloid (A) in the pathogenesis of Alzheimer's disease (AD), but the mechanisms whereby A is involved remain unclear. Addition of A to the extracellular space can be neurotoxic. Intraneuronal A42 accumulation is also associated with neurodegeneration. We reported previously that in Tg2576 amyloid precursor protein mutant transgenic mice, brain A42 localized by immunoelectron microscopy to, and accumulated with aging in, the outer membranes of multivesicular bodies, especially in neuronal processes and synaptic compartments. We now demonstrate that primary neurons from Tg2576 mice recapitulate the in vivo localization and accumulation of A42 with time in culture. Furthermore, we demonstrate that A42 aggregates into oligomers within endosomal vesicles and along microtubules of neuronal processes, both in Tg2576 neurons with time in culture and in Tg2576 and human AD brain. These A42 oligomer accumulations are associated with pathological alterations within processes and synaptic compartments in Tg2576 mouse and human AD brains.
Key words: Alzheimer; amyloid; synapse; oligomer; transgenic; pathology
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Introduction
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Alzheimer's disease (AD) neuropathology is characterized by loss of synapses and neurons and by accumulation of senile plaques and neurofibrillary tangles. The -amyloid peptide (A) is the principal component of plaques and was further linked to AD by familial AD (FAD) mutations in the amyloid precursor protein (APP) near A cleavage sites (Selkoe, 2001 ; Golde, 2003). Transgenic mice harboring human APP with FAD mutations develop AD-like amyloidosis and synaptic, oxidative, physiological, and behavioral abnormalities (Holcomb et al., 1998; Chapman et al., 1999; Moechars et al., 1999; Pratico et al., 2001).
Extracellular addition of A to cultured neurons or injection into brain can be neurotoxic (Yankner et al., 1989; Geula et al., 1998). Evidence also indicates that accumulation of A42, the first A species found in plaques, occurs intracellularly (Echeverria and Cuello, 2002; Tabira et al., 2002) in AD (Gouras et al., 2000; D'Andrea et al., 2001), Down's syndrome (Gyure et al., 2001; Busciglio et al., 2002; Mori et al., 2002), and transgenic mice that develop A plaques (Wirths et al., 2001; Oddo et al., 2003; Sheng et al., 2003). Additionally, extracellular A42 can increase levels of intraneuronal A42 (Glabe, 2001). We demonstrated in Tg2576 transgenic mice that intraneuronal A42 accumulates with aging in the outer limiting membranes of multivesicular bodies (MVBs), especially in distal processes and synaptic compartments (Takahashi et al., 2002). Moreover, before and with the development of plaques, marked accumulation of A42 in MVBs was associated with subcellular morphological alterations within processes and synapses. Thus, understanding A biology within endosomal vesicles, such as MVBs, may be important in unraveling the mechanism of AD pathogenesis. In the present study, we set out to establish an in vitro primary neuronal culture system in which to investigate endosomal A accumulation. Analogous to increases of intracellular A42 reported by ELISA in NT2 cells with time in culture (Skovronsky et al., 1998), we now report intracellular accumulation of A42 within cultured primary neurons.
Increasing evidence indicates that the biophysical state of the most toxic form of A may be its protofibrillar rather than -pleated conformation (Selkoe, 2001; Klein, 2002). Levels of soluble, and potentially oligomeric A42, rather than plaques, are the best A correlate of cognitive dysfunction in AD (McLean et al., 1999; Naslund et al., 2000). In vitro evidence indicates that A monomers are converted to oligomers before aggregation into -pleated amyloid (Kirkitadze et al., 2002). It was reported that A oligomers are preferentially generated intracellularly rather than extracellularly (Walsh et al., 2000). We now present evidence that A42 oligomers arise especially within processes of cultured Tg2576 primary neurons with time in culture. Moreover, in Tg2576 and human AD brain, accumulation of A42 oligomers within neuronal processes and synaptic profiles is associated with pathological alterations. Both in vitro and in vivo we observe a redistribution of A42, as it aggregates from monomer to oligomer, from the outer membranes of endosomal vesicles– MVBs, where it normally resides, to the inner membranes of morphologically abnormal endosomal organelles and to microtubules. Our data further support the scenario that A42 aggregation occurs within neuronal processes and results in synaptic degeneration.
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Materials and Methods
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Antibodies. MBC42, kindly provided by Dr. Haruyasu Yamaguchi (Gunma University, Gunma, Japan), is a monoclonal antibody (mAb) to the C terminus of A42 and has been characterized previously (Takahashi et al., 2002). Results with MBC42 were additionally confirmed using a commercially available polyclonal anti-A42 antibody (Chemicon, Temecula, CA) [characterized in Kamal et al. (2001)]. A42 oligomers were detected using the rabbit polyclonal antibody, M16, kindly provided by Dr. Charles Glabe (University of California, Irvine, CA); M16 was raised against synthetic A1–42 and preferentially recognizes A42 oligomers on Western blot (Yang et al., 1995). For biochemical analyses, A, APP C-terminal fragments, and full-length APP were recognized by antibodies 4G8 (directed at A17–24; Signet Laboratories, Dedham, MA) and 6E10 (directed at A5–10; Signet Laboratories). Polyclonal rabbit Ab against lysobisphosphatidic acid (LBPA) was kindly provided by Dr. J. Gruenberg (University of Geneva, Geneva, Switzerland) (Kobayashi et al., 1998). Anti- -tubulin Ab was from Sigma (St. Louis, MO), and anti-neuron-specific nuclear protein (NeuN) Ab was from Chemicon.
Preparation of aggregated synthetic A42 peptides. Fibrillar A42 was made according to the method described by Fezoui et al. (2000). Briefly, lyophilized A42 synthetic peptides (American Peptide Company, Sunnyvale, CA) were dissolved in 20 mM NaOH, pH 10.5, at a concentration of 1 mg/ml, sonicated, and lyophilized. NaOH-treated A42 was dissolved in water at a concentration of 1 mg/ml and filtered through a 0.22 µm Ultrafree-MC filter (Millipore, Bedford, MA). A peptide (0.5 mg/ml) was buffered at a final concentration of 50 mM phosphate, 100 mM sodium chloride and incubated for 54 hr. Two hundred nanograms of aggregated A42 peptide were loaded in each lane for Western blot (see below).
Mice. Tg2576 mice harboring human APP with the Swedish 670/671 mutation (Hsiao et al., 1996) were used in this study, as described previously (Takahashi et al., 2002). All methods were approved by the Weill Medical College of Cornell University Institutional Animal Care and Use Committee and conformed to National Institutes of Health guidelines.
Human brain tissue. Human cortical brain tissue from neurologically normal controls (n = 2; ages 44 and 54) and subjects with AD (n = 2; ages 54 and 62) was obtained from the Department of Pathology, Weill Medical College of Cornell University, as a result of neurosurgical procedures unrelated to this study, as described previously (Takahashi et al., 2002).
Primary murine neuronal cultures. Primary neuronal cultures were derived from cerebral cortex and hippocampus of embryonic day (E) 16 Tg2576 or wild-type mice, as described previously (Gouras et al., 2000) with modifications. Cortices and hippocampi were incubated with 0.25% trypsin and then triturated in glass pipettes until neurons were dissociated. Neurons were plated in serum-free Neurobasal media with B27 supplement (Invitrogen, Gaithersburg, MD) and 2 mM L-glutamine on poly-D-lysine-treated (0.1 mg/ml; Sigma) 100 mm dishes. To maintain elevated levels of extracellular A, media was not changed. No differences in cell viability were detected by trypan blue staining or lactate dehydrogenase release between wild-type and transgenic neurons at 12 or 19 d in culture. Primary neurons are viable for >3–4 weeks under our culturing conditions. For immunofluorescence,  4 x 105 neurons were plated per well in six-well plates containing coverslips, and for immunoprecipitation–Western blot, 3.3 x 106 neurons were plated per 100-mm-diameter dish. Genotyping was performed on cerebellum from the same embryo.
Immunofluorescence microscopy. Immunofluorescence was performed on E16 primary neuronal cultures grown for 5, 12, or 19 d in vitro on poly-D-lysine-coated round glass coverslips (EM Science). Cultures were fixed for 20 min with 4% paraformaldehyde in PBS containing 0.12 M sucrose, permeabilized in 0.3% Triton X-100 in PBS for 5 min, and rinsed twice in PBS. The cells were preincubated in 10% BSA in PBS for 1 hr at 37°C, exposed to primary antibodies (diluted in 1% BSA in PBS) overnight at 4°C, and then rinsed in PBS and incubated with secondary antibodies for 1 hr at 37°C. MBC42 (1:150), M16 (1:1,000), LBPA (1:50), and -tubulin (1:300) were used as primary antibodies. Fluorescent secondary antibodies were Alexa 488 goat anti-mouse and Alexa 546 goat anti-rabbit (1:200; Molecular Probes, Eugene, OR). Immunoreactivity was captured with a Leica DM IRB microscope with a Hamamatsu digital camera or a Zeiss LSM 510 confocal microscope and analyzed using Meta-Morph software (Universal Imaging, West Chester, PA) for quantitative analysis. To keep neuronal cell numbers constant for comparisons of Tg2576 neurons at 12 versus 19 d in culture, several coverslips were cocultured in the same well. We randomly picked five different areas from a coverslip for analysis. Evaluation was done using a 20x objective on five different cell bodies and processes, and average intensities were measured in selected areas. The intensities of processes were divided by the intensities of cell bodies. The process-to-cell body intensity ratio in Tg2576 neurons was compared between 12 and 19 d in culture using Student's t test for statistical analysis. Data were expressed as the mean ± SEM, and the significance threshold was p < 0.05.
Immunogold electron microscopy. Mice were anesthetized with sodium pentobarbital (150 mg/kg, i.p.) and perfused via the ascending aorta with 3.75% acrolein (Polyscience, Warrington, PA) and 2% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. Human AD brain biopsy tissue was rapidly immersion-fixed with 1.875% acrolein and 2% paraformaldehyde in 0.1 M PB as described previously (Takahashi et al., 2002). Brain tissue was cut (40 µm thick) on a vibrating microtome and treated with 1% sodium borohydride, as described previously (Takahashi et al., 2002). For immunogold labeling, free-floating sections were labeled with MBC42 (1:50), M16 (1:100), or Chemicon A42 (1:50) antibodies by the immunogold–silver procedure of Chan et al. (1990). Sections were then incubated in secondary antibodies conjugated to 1 nm gold particles (Amersham Biosciences, Arlington Heights, IL) in 0.01% gelatin and 0.08% BSA in PBS. The conjugated gold particles were enhanced by treatment with silver solution (IntenSE, Amersham Biosciences). Sections were fixed in 2% osmium tetroxide in PB, embedded in EMBed 812, sectioned (65–76 nm thick), and counterstained with uranyl acetate and Reynolds' lead citrate (Milner et al., 1998). Final preparations were examined with a Philips CM10 electron microscope. Morphological terminology is consistent with Peters et al. (1991). Final 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).
For cell immunoelectron microscopy (EM), primary neurons were fixed for 45 min in 0.1% glutaraldehyde, 2% paraformaldehyde, and 0.005% saponin in 0.1 M PB at room temperature. Neurons were then rinsed in 0.1 M PB with 0.005% saponin and treated with 1% sodium borohydride. For immunogold labeling, the neurons were labeled with either MBC42 or M16 Ab by the immunogold–silver procedure as described above.
Immunoprecipitation–Western blot for A. Cultured primary neurons from Tg2576 mice were assayed for determination of A levels as described previously for cell lysates, using 4G8 for immunoprecipitation and 6E10 for blotting (Takahashi et al., 2002). To maintain equivalent neuronal numbers and density, equal numbers of dissociated Tg2576 neurons from a given mouse brain were cultured in three separate dishes, and dishes of each Tg2576 pup were cultured for 12 or 19 d and later compared with neurons derived from an equivalent set of cultures derived from different Tg2576 mouse pups. Neurons were lysed in 6% SDS, vortexed, and heated at 95°C for several minutes, followed by sonication. Equivalent amounts of protein, as determined by the Bradford technique (Bio-Rad Laboratories, Hercules, CA), were spun at 15,000 x g, and supernatants were adjusted to 190 mmol/l NaCl, 50 mmol/l Tris-HCl, pH 8.3, 6 mmol/l EDTA, and 2.5% Triton X-100 and incubated overnight with Ab 4G8 (Signet Laboratories), followed by addition of secondary rabbit anti-mouse Ab (Cappel, West Chester, PA) for 1 hr and then addition of protein A-Sepharose beads (Amersham Biosciences) for 2 hr (all at 4°C). Samples were run on 10–20% Tris-Tricine SDS PAGE gels followed by electrophoretic transfer to polyvinylidene-difluoride membrane (Millipore). Polyvinylidene-difluoride membranes were boiled in PBS for 5 min, followed by Western blotting with 6E10 for human A and visualization after enhanced chemiluminescence on Kodak X-OMAT AR5 film (Eastman-Kodak, Rochester, NY).
All experiments were repeated at least three times, and different primary cultures derived from at least three different embryos were averaged. For characterization of antibodies against A42, A42 synthetic peptides (America Peptide Company) were prepared as described above, and 200 ng was loaded into each lane. We used the following antibodies for blotting: MBC42 (1:300), 6E10 (1:1000), M16 (1:1000), anti--tubulin (1:1000), and anti-NeuN (1:1500) by a semidry blotting method. Intensity of bands was analyzed by Scion Image, followed by Student's t test for statistical analysis. Data were expressed as the mean ± SEM, and significance was assessed at p < 0.03.
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Results
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A42 immunofluorescence in cultured neurons has a vesicular pattern of staining
We previously used immunogold EM to localize A42 especially to the outer membranes of MVBs and smaller tubulovesicular organelles within the cytoplasm of neurons in normal, young mouse, rat, and human brains (Takahashi et al., 2002). Importantly, the specificity of our immuno-EM was confirmed by lack of labeling in APP knock-out mouse brain (Takahashi et al., 2002). In the present study, we used well characterized monoclonal antibodies against A42, which we had used previously for immuno-EM, for immunofluorescence microscopy of cultured primary neurons. We observed a vesicular pattern of A42 staining (Fig. 1A), consistent with A42 immunofluorescence in cultured Down's syndrome astrocytes (Busciglio et al., 2002) and neurons infected with Semliki Forest virus harboring human APP (Runz et al., 2002). We obtained analogous immunofluorescence using monoclonal (MBC42) or commercially available polyclonal anti-A42 antibodies (Chemicon) (Kamal et al., 2001). Colocalization with markers of subcellular organelles indicated significant overlap with markers of late endosomes, such as LBPA (Fig. 1A). LBPA has been reported previously to label late endosomal vesicles by immunofluorescence and immuno-EM (Kobayashi et al., 1998; Mobius et al., 2003).
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Figure 1. A42 within cultured neurons localizes to late endosomal vesicles and increases with time in culture, especially within processes. A, Confocal immunofluorescence microscopy of primary neurons at 19 d in culture. A42 fluorescence, with the Chemicon A42 Ab, partially localized with LBPA, which is known to be associated especially with late endosomes. Scale bar, 10 µm. B, Immunofluorescence of Tg2576 mouse primary neurons stained with mAb MBC42 against the C terminus of A42. Note the A42 increases in Tg2576 (Tg) neurons between 12 d (left) and 19 d (right) in culture. There is less A42 immunofluorescence in wild-type (WT) neurons cultured for 19 d (below). Scale bar, 50 µm. C, Tg2576 primary neurons: ratio of A42 immunofluorescence intensity in neuronal processes divided by that in cell bodies (processes/cell bodies) between 12 and 19 d in culture, with the ratio standardized to 1.0 for 12 d in culture. The A42 ratio of processes/cell bodies increased 1.65 ± 0.06-fold between 12 and 19 d in culture; p < 0.001. D, Representative images of -tubulin immunofluorescence in Tg2576 mouse primary neurons at 12 and 19 d in culture. There was no significant increase in-tubulin fluorescence, compared with that of A42 fluorescence (B), within processes between 12 and 19 d in culture. Scale bar, 50 µm. E, Cell body A42 detected by immunogold electron microscopy using Ab MBC42. Representative A42 in the outer membrane of a MVB, containing characteristic internal vesicles, in the cell body of a Tg2576 primary neuron at 21 d in culture. Scale bar, 500 nm. F, A42 immuno-EM of a neurite process. Representative image of A42 accumulation in outer membranes and rarely inner vesicles of MVBs in a neurite process of a Tg2576 primary neuron at 21 d in culture, detected by immunogold EM with MBC42. Note also an A42 gold particle at the plasma membrane (arrowhead). Scale bar, 500 nm.
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A42 accumulates and redistributes to neuronal processes within Tg2576 neurons with time in culture
To determine whether intraneuronal A42 increases with time in culture and to assess for changes in A42 distribution within neurons, we used immunofluorescence microscopy for A42 in Tg2576 neurons at 12 and 19 d in culture. As expected, neurons overexpressing the human APP mutant transgene had increased A42 immunofluorescence compared with wild-type neurons at 19 d in culture (Fig. 1B). Remarkably, we observed with time in culture a gradual shift of A42 immunofluorescence from cell bodies to neurites in Tg2576 neurons (Fig. 1B). Primary neurons at 19 d in culture had a more extensive network of processes than at 12 d. In addition, the number of processes was greater in "older" neurons, as was the intensity of A42 staining. The ratio of A42 fluorescence intensity per unit area of processes to that of cell bodies increased 1.65 ± 0.06-fold (p < 0.001) in Tg2576 neurons between 12 and 19 d in culture (Fig. 1C). Immunofluorescence for -tubulin confirmed an increase in the network of processes between 12 and 19 d in culture, but in contrast to A42, there was no significant increase in-tubulin immunofluorescence within processes (Fig. 1D). To determine the subcellular site(s) of these A42 increases in primary neurons, we used immuno-gold EM. A42 gold particles were most prominent in the outer membranes of endosomal vesicles, consistent with MVBs, in the cytoplasm of Tg2576 primary neurons cultured for 3 weeks, both in cell bodies (Fig. 1E) and within processes (Fig. 1F).
A accumulates in Tg2576 primary neurons with time in culture by Western blot
To confirm biochemically whether intraneuronal levels of A in Tg2576 cultured primary neurons increase with time in culture, we used immunoprecipitation using anti-A Ab 4G8 and Western blot using anti-A Ab 6E10. Immunoprecipitation–Western for human A demonstrated increases in levels of intracellular A with time in culture (Fig. 2A). Specifically, between 12 and 19 d in culture there was a 3.36 ± 0.51-fold increase (p < 0.03) in intracellular A levels by densitometry (Fig. 2B). There was a less pronounced 1.46 ± 0.29-fold increase, not reaching statistical significance (p = 0.19), in levels of full-length APP between 12 and 19 d in culture (Fig. 2B). There were no significant differences in levels of NeuN or -tubulin between 12 and 19 d in culture (Fig. 2A).
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Figure 2. Biochemical analysis of intraneuronal A accumulation in primary neurons with time in culture. A, Increased levels of A with time in culture in primary neurons from Tg2576 mouse by immunoprecipitation–Western blot. Equal amounts of 12- and 19-d-old cultured neuron cell lysates were immunoprecipitated with 4G8 and immunoblotted with 6E10 for determination of A and full-length APP levels. After immunoprecipitation with 4G8, supernatants were immunoblotted with antibodies against NeuN or -tubulin. B, A levels increased 3.36 ± 0.51-fold from 12 to 19 d in culture (p < 0.03). A slight increase in APP levels (1.46 ± 0.29-fold) from 12 to 19 d in culture was not statistically significant (p = 0.19). Data are expressed as mean ± SEM of three independent experiments. C, Aggregation-state specificity of antibodies against A42. Aggregated A42 (200 ng) was separated by gel electrophoresis, transferred to polyvinylidene-difluoride membrane, and blotted with MBC42, 6E10, and M16. MBC42 detected especially A42 monomers and some dimers but did not recognize oligomers. 6E10 recognized most aggregation states of A42, being most sensitive for monomers. M16 mainly recognized larger A42 oligomers, with relatively minor reactivity to A monomers.
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Aggregation state specificity of anti-A antibodies
Increasing numbers of reports are emphasizing the potentially critical role of A oligomers rather than A monomers or A fibrils in AD (Hardy and Selkoe, 2002; Klein, 2002). Because anti-A antibodies have selective affinities for the conformational state of A, a given Ab will not efficiently visualize the total pool of A. To characterize which conformations of A42 are recognized by the anti-A antibodies that we used, we blotted preaggregated synthetic A42 peptides with MBC42 and 6E10. MBC42 detected mainly A42 monomers and also some A42 dimers without detecting larger A42 oligomers (Fig. 2C, top). 6E10 detected monomeric A42 but also a wide variety of A42 oligomers (Fig. 2C, middle). To investigate changes in A42 oligomers we used Ab M16, which has been previously well characterized as binding A42 oligomers (Yang et al., 1995). We also found that M16 mainly detected oligomers of A42 (Fig. 2C, bottom).
A42 oligomerization within processes of Tg2576 neurons with time in culture
Because the monoclonal MBC42 predominantly detects A42 monomers (Fig. 1), we used the M16 Ab, which predominantly detects A42 oligomers, to more fully investigate A42 increases in Tg2576 neurons. We studied wild-type and Tg2576 neurons with time in culture by M16 immunofluorescence microscopy. M16 staining was not observed in wild-type mouse neurons at 5, 12, and 19 d in culture, or in primary neurons from Tg2576 mice cultured for 5 d (data not shown). In Tg2576 neurons at 12 d in culture, A42 oligomers were observed in cell bodies and rarely in processes (Fig. 3A, left column). In contrast, at 19 d in culture, A42 oligomers were rarely observed in cell bodies (Fig. 3A, arrows in top right panel) but were now numerous in neuronal processes, especially as punctate accumulations (Fig. 3A, arrow-heads in right column). The ratio of A42 oligomer intensity per area of processes compared with cell bodies increased 1.46 ± 0.09-fold between 12 and 19 d in culture (p < 0.01) (Fig. 3B).
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Figure 3. A42 oligomers accumulate within neuronal processes with time in culture. A, Increased oligomeric A42 (M16) immunofluorescence in processes of Tg2576 primary neurons with time in culture. Top panel, Localization of A42 in cell bodies (arrows) and processes (arrowheads) at 12 d (left) and 19 d (right) in culture. Bottom panel, Localization of A42 oligomers in neuronal processes. Scale bar, 10 µm. B, Ratio of oligomeric A42 immunofluorescence intensity in processes to that in cell bodies (processes/cell bodies) at 12 and 19 d in culture in Tg2576 primary neurons, with intensity ratio standardized as 1.0 at day 12 in culture. There was a 1.46 ± 0.09-fold increase in the process-to-cell body ratio of A42 oligomer fluorescence between 12 and 19 d in culture; p < 0.01. C, A42 oligomer accumulation in neuronal processes of Tg2576 neurons at 19 d in culture, especially within endosomal-like vesicles (thick arrows), and less prominently in their outer membranes (thin arrows). These processes are associated with morphological alterations, including fibrillar dark material (arrowheads) that appears associated with intravesicular A42 oligomers. Scale bar, 250 nm. D, A42 oligomers (arrows) also localized and accumulated along microtubules (arrowheads) within neuronal processes of Tg2576 neurons at 19 d in culture. Scale bar, 250 nm.
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To assess the subcellular localization of A42 oligomer increases in Tg2576 primary neurons with time in culture, we turned to immunogold EM using M16. In Tg2576 primary neurons at 19 d in culture, gold particles labeling A42 oligomers were found associated with amorphous floccular, fibrillar-appearing dark material within large endosomal-like organelles (Fig. 3C). We did not observe A42 oligomer labeling gold particles on normal-appearing endosomal organelles, as we had with antibodies directed mainly at A42 monomers (Fig. 1E,F). The darkened-appearing organelles containing the A42 oligomers may reflect altered or even "degenerating" endosomes, because they appear at times to contain MVB-characteristic smaller internal vesicles. A42 oligomer gold particles were mainly inside darkened endosomal vesicles, whereas A42 monomers were mainly on outer membranes (Fig. 1E,F). Interestingly, A42 oligomer immunogold labeling also prominently localized along microtubules within neuronal processes (Fig. 3D).
A42 oligomerization in Tg2576 mouse brain with aging
A previous report showed that microdeposits of A42 appear as early as 7–8 months in Tg2576 brain (Kawarabayashi et al., 2001). Although these microdeposits increase in number between 7 and 10 months, consistent initiation of AD-like plaque pathology begins at 11–13 months (Irizarry et al., 1997). In an attempt to observe the earliest stages of A42 aggregation and plaque formation, we labeled sections from 3-, 11-, and 20-month-old Tg2576 brains with the M16 Ab against A42 oligomers. We did not observe M16 immunolabeling of 11-month-old wild-type (Fig. 4A, top left panel) or 3-month-old Tg2576 mouse cerebral cortex (Fig. 4A, top right panel). In cortices from 11-month-old Tg2576 brains, M16 A42 oligomer staining was detected as microdeposits (Fig. 4A, bottom left panel; at higher magnification, Fig. 4B). At 20 months, A42 oligomer deposits were increased in number and size (Fig. 4A, bottom right panel). The early stage of A42 oligomer aggregation was then investigated in Tg2576 brain by immuno-EM using M16 immunoperoxidase labeling (Fig. 4C). In the absence of overt neuritic plaques, we observed intense A42 oligomer immunoreactivity in elongated patterns that could be consistent with degenerated neuronal processes (Fig. 4C). In the presence of amyloid plaques, M16 immunoreactivity, indicative of A42 oligomers, surrounded and was concentrated along the edges of the amyloid core at the center of plaques (Fig. 4D). The amyloid core was surrounded by swollen and distended M16 immunolabeled degenerating neuritic-appearing profiles (Fig. 4D, arrows).
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Figure 4. A42 oligomers accumulate in Tg2576 mouse brain. A, Low-magnification light microscopy images of M16 A42 oligomer immunoperoxidase labeling of wild-type (WT) and Tg2576 (Tg) mouse brains at different ages. A42 oligomers were not detected in 11-month-old wild-type (top left) or 3-month-old Tg2576 mouse brains (top right) but were faintly detected as dense micro-deposits (bottom left, arrow) in an 11-month-old Tg2576 mouse. By 20 months, M16 A42 oligomer staining showed increased number and size of microdeposits (bottom right) and also surrounded neuritic plaques. Scale bar, 50 µm. B, High-magnification light microscopic image of the rectangle in A (bottom left), showing a microdeposit (arrow) and even smaller A42 oligomer aggregation before plaques in an 11-month-old Tg2576 mouse (arrowhead). Scale bar, 30 µm. C, Immunoperoxidase EM of A42 oligomer aggregations distant from an obvious plaque. A42 oligomer immunoreactivity appears concentrated along processes (arrow), and structures around the aggregation sites are degenerating (vacuolated spaces, arrowheads). Scale bar, 1µm. D, Ultrastructural localization of A42 oligomers around a plaque. Marked accumulation of immunoperoxidase reaction product (appearing black; asterisks) representing A42 oligomers surrounds an unlabeled plaque core. Areas around the plaque core are degenerated and include degenerating neurites (thin arrow) and abnormal empty spaces (large arrow). For reference, more normal neurites distant from the plaque are also seen (plus signs). Scale bar, 1 µm.
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To better define the subcellular location(s) of A42 aggregation and oligomer accumulation we used immunogold EM with M16. Adjacent to the outer rim of A plaques staining with M16, we could detect dystrophic neurites containing M16 A oligomer gold particles (Fig. 5A,C). At times, these oligomer-containing neurites could be seen to contain subcellular organelles, such as mitochondria, although the cytoarchitecture invariably was abnormal in the presence of A oligomer labeling (Fig. 5A,C, for higher magnification of dystrophic A oligomer-containing neurites). Because M16 A oligomer labeling was associated with morphological–pathological alterations, adjacent nonpathological tissue is represented to underscore the fact that the surrounding normal tissue lacking M16 labeling demonstrates a normal subcellular cytoarchitecture (Fig. 5A,B; B is a higher magnification image of the normal cytoarchitecture seen in the rectangle in A). At high magnification even isolated areas of M16-labeled A42 oligomer aggregations distant from plaques were observed. We found A42 oligomer accumulations close to synaptic active zones (Fig. 5D). The normal microtubular network was absent in areas surrounding even isolated A42 oligomer accumulations (Fig. 5D). Distended-appearing processes with multiple M16 gold particles were observed that were markedly abnormal, with atypical electron-dense fibril-like material, large empty spaces, and lack of any well defined normal organelles, such as mitochondria (Fig. 5E).
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Figure 5. A oligomers by immunogold EM accumulate in dystrophic processes and synapses of Tg2576 mouse brain. A, A lower-magnification image of a degenerated, morphologically abnormal area around a plaque core (left) surrounded by normal-appearing neuropil (right). A degenerating neurite (empty arrow) is observed close to the plaque at the left edge of the figure. Normal dendrite (large asterisk) and a myelinated axon (small asterisk) are at the border of the normal and abnormal areas. A normal neuron is seen distant from the plaque (right); the rectangle within this normal-appearing neuron is enlarged in B, below. N, Nucleus. Scale bar, 1 µm. B, Normal organelles and cytoarchitecture can be seen in this higher-magnification image of the small rectangle within the neuron in A. G, Golgi apparatus; m, mitochondrion; N, nucleus. Scale bar, 250 nm. C, A higher-magnification image of an A oligomer containing degenerating neurites and abnormal empty spaces just around a plaque core (P). A oligomers (empty arrow) are associated with the plaque core, and degenerating neurites are associated with empty spaces. A relatively normal dendrite with an isolated M16 gold particle (large asterisk) and a myelinated axon (small asterisk) are observed distant from the plaque core. m, Mitochondrion. Scale bar, 500 nm. D, A cluster of gold particles representing A42 oligomers in a postsynaptic profile near an active zone of a synapse (top thin arrow). The plasma membranes of two postsynaptic profiles are indicated (large arrows). The cluster of A42 oligomers is surrounded by a clear area (empty arrowhead), in which the normal microtubular network (filled arrowheads) is absent. The lower synaptic profile, lacking A42 oligomers, shows a normal microtubular network throughout. Synaptic active zones are indicated by thin arrows. m, Mitochondrion. Scale bar, 250 nm. E, M16 immunogold EM of a Tg2576 neuritic process. Vertical section through a neuritic process in an 18-month-old Tg2576 mouse brain. Distant from a plaque, substantial A42 oligomer gold particles are associated with granular and fibril-like electron-dense material (arrowheads) in an abnormal pathological-appearing process with poorly defined cytoarchitecture, including empty spaces (empty arrowhead) and lacking any normal organelles, such as mitochondria. Arrows outline the plasma membrane of the neuritic process. Scale bar, 250 nm.
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A42 oligomer aggregation in human AD brain
A42 oligomers were not observed in normal, non-AD cortical biopsy tissue (data not shown). A42 oligomer accumulations, however, were observed in human AD cortical brain tissue, within abnormal processes and synaptic compartments, but not in more normal-appearing surrounding neuropil (Fig. 6A). Within dystrophic-appearing processes and synaptic compartments, A42 oligomers seemed at times to localize to tubulovesicular-like structures (Fig. 6B,C) or membranous profiles (Fig. 6B,D). Secondary to the altered cytoarchitecture associated with M16 labeling, these abnormal subcellular structures cannot be adequately classified as being related to endosomes, endoplasmic reticulum, or other organelles. A42 oligomer-containing synaptic compartments were markedly abnormal. They contained open spaces and poorly defined membrane fragments (Fig. 6B–D), and there was associated electron-dense fibril-like material (Fig. 6B). Figure 6D shows a markedly abnormal postsynaptic profile containing multiple A42 oligomer gold particles. The profile is devoid of any normal organelles and contains dark areas, consistent with degeneration. In addition, this abnormal postsynaptic profile synapses with an abnormally darkened presynaptic profile in the bottom left (Fig. 6D).
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Figure 6. A42 oligomerization within dystrophic processes of human AD brain. A, A42 oligomers (empty arrows) are observed in degenerating neurites and are not associated with more normal-appearing neuropil (middle area of image). A darkened, abnormal-appearing postsynaptic profile is also observed (plus sign). m, Mitochondrion. Scale bar, 1µm. B, A higher-power image of a degenerating neurite in A (A, right empty arrow). A42 oligomers (empty arrows) are associated with a tubulovesicular-like organelle (empty arrowhead) and granular fibril-like electron-dense material (filled arrowheads). Isolated A42 oligomers localize to the membrane of presynaptic profile (left empty arrow) associated with a darkened, degenerating-appearing postsynaptic profile (plus sign). m, Mitochondrion. C, Aggregated A42 oligomers (empty arrow) appear associated with a tubulovesicular-like organelle (empty arrowhead) in a postsynaptic compartment with an active zone (thin arrow). m, Mitochondrion. Scale bar, 300 nm. D, M16 A42 oligomers aggregate on tubular-like membranous structures within a disrupted, swollen dendrite containing abnormal dark profiles (asterisks), consistent with degeneration; no normal organelles can be found in this postsynaptic profile. Presynaptic profiles are seen on the right (arrow) and left (arrowhead) corners of the image; the left one is darkened, indicative of degeneration. Scale bar, 250 nm.
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Discussion
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We reported previously that MVBs are the major subcellular site of A42 accumulation within neurons and that accumulating A42 in MVBs within distal neuronal processes and synaptic profiles was associated with morphological alterations (Takahashi et al., 2002). We now provide data demonstrating that A42 accumulates also within processes of Tg2576 primary neurons in vitro with time in culture. These tissue culture results are consistent with our previous in vivo results in brains of Tg2576 mice with aging and human subjects with AD and extend these observations now to a cell culture system that can be manipulated experimentally. Moreover, we provide evidence that accumulating A42 within processes aggregates as A42 oligomers both in primary Tg2576 neurons with time in culture and in aging Tg2576 mouse brain and human AD brain.
Increasing evidence suggests that A42 oligomers are the most toxic form of A. We found that A42 oligomer accumulation occurred in processes and synaptic compartments. These processes and synaptic compartments were markedly abnormal, often lacking any normal cytoskeletal architecture or subcellular organelles. Because normal synaptic functioning requires these subcellular structures, these A42 oligomer accumulating processes and synaptic compartments are unlikely to allow for functional synapses. Synaptic dysfunction is the most established correlate of cognitive decline in AD (Masliah, 2001). Our results therefore provide direct evidence for the importance of A42 oligomers in the pathogenesis of AD.
Interestingly, M16 oligomers localized not only to the outer membranes of endosomal–MVB-like organelles, reminiscent of monomeric A42, but localized prominently inside darkened late endosomal-like organelles. The occurrence of early abnormalities in the endosomal–lysosomal system is well established in AD brain (Cataldo et al., 2000). Interestingly, late endosomal MVBs are increasingly being studied in the ubiquitin–proteasome system (UPS)-dependent recycling and degradation of various cellular receptors and proteins (Katzmann et al., 2002), including those involved in the regulation of synaptic plasticity (Ehlers, 2003). We hypothesized previously that intracellular A42 accumulation in AD may parallel a growing list of neurodegenerative diseases in which aberrant intracellular protein accumulation and impairment of the UPS may be involved. Interestingly, the translocation of EGF receptor–EGF complexes from the outer limiting membrane of MVBs to the inner vesicles was shown by immuno-EM to be proteasome dependent (Longva et al., 2002). Our observations associating A42 oligomer accumulation in the inner vesicles of darkened MVB-like organelles with neuritic pathology suggest that aberrant intraendosomal trafficking of A42 may be involved in the pathogenesis of AD. A better understanding of the cell biology and pathology of late endosomal A42 may be important in better understanding the early events in the pathogenesis of AD.
We also observed A42 oligomers not associated with vesicular organelles and appearing to reside along microtubules. It is well known that endosomal vesicles such as MVBs, which normally contain A42 in their outer membranes (Takahashi et al., 2002), are transported along microtubules. Vesicles containing APP, -site APP-cleaving enzyme, and presenilin were reported to be transported along microtubules in axons of peripheral nerve that can generate A (Kamal et al., 2001). The accumulation of A42 on microtubules provides an intracellular association between A and the microtubule-associated protein tau and thereby a more direct association for the two neuropathological hallmarks of AD, A plaques, and tau tangles. Of note, the A42 oligomers along microtubules were within the cytoplasm and outside the normal lipid bilayer environment for A. It was reported previously that transgenic mice overexpressing A intracellularly, but not transgenic mice overexpressing A extracellularly, demonstrate neurodegeneration (LaFerla et al., 1995). Furthermore, it was reported that A1–42, but not A1–40 or A42–1, is exquisitely neurotoxic when introduced into the cytosol of cultured primary neurons (Zhang et al., 2002).
The mechanisms by which extracellular A can modulate intracellular A42 remain unclear, as do the mechanisms by which A42 monomers accumulate in the outer membranes of MVBs, are translocated within endosomal vesicles as oligomers, and then are converted to amyloid-like electron-dense fibrils. A42 might gradually cause MVBs to degenerate as it oligomerizes. In support of this, A42 oligomers at times appear to be associated with vesicular organelles that sometimes even contain internal vesicles, characteristic of MVBs (Fig. 3C). Another possibility is that accumulating A42 monomers or oligomers on MVBs in processes impair vesicular transport of vital cargo by interfering with kinesin-based transport mechanisms. Release of A42 monomers or oligomers into the cytoplasm and subsequent aggregation on microtubules may also be critical determinants of neurotoxicity. The detailed mechanism by which neurite processes and synaptic compartments degenerate and the role of aggregated A42 in these events remain to be elucidated.
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Footnotes
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Received Nov 21, 2003;
revised February 23, 2004;
accepted February 28, 2004.
This work was supported by National Institutes of Health Grants HL18974 (T.A.M.), NS02037 (G.K.G.), and NS045677 (G.K.G.), Paul Beeson awards (M.T.L., G.K.G.), and the American Academy of Neurology Education and Research Foundation (G.K.G.). We appreciate the advice of Dr. Asao Hirano (Department of Pathology, Albert Einstein College of Medicine) and Dr. Frederick Maxfield (Department of Biochemistry, Weill Medical College of Cornell University).
Correspondence should be addressed to Dr. Gunnar K. Gouras, Laboratory of Alzheimer's Disease Neurobiology, Department of Neurology and Neuroscience, Weill Medical College of Cornell University, 525 East Sixty-Eighth Street, New York, NY 10021. E-mail: gkgouras{at}med.cornell.edu.
DOI:10.1523/JNEUROSCI.5167-03.2004
Copyright © 2004 Society for Neuroscience 0270-6474/04/243592-08$15.00/0
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Top
Abstract
Introduction
