A seed for Alzheimer amyloid in the brain

Abstract

A fundamental question about the early pathogenesis of Alzheimer's disease (AD) concerns how toxic aggregates of amyloid beta protein (Abeta) are formed from its nontoxic soluble form. We hypothesized previously that GM1 ganglioside-bound Abeta (GAbeta) is involved in the process. We now examined this possibility using a novel monoclonal antibody raised against GAbeta purified from an AD brain. Here, we report that GAbeta has a conformation distinct from that of soluble Abeta and initiates Abeta aggregation by acting as a seed. Furthermore, GAbeta generation in the brain was validated by both immunohistochemical and immunoprecipitation studies. These results imply a mechanism underlying the onset of AD and suggest that an endogenous seed can be a target of therapeutic strategy.

Figure 1.

Amyloid fibril formation from soluble Aβ in the absence or presence of GM1-containing liposomes. a, Kinetics of Aβ fibrillogenesis using Aβ (Aβ40 and Aβ42) solutions, with or without removing undissolved peptide aggregates, which can act as preexisting seeds. Aβ solutions were incubated at 50 μm and 37°C. Open and filled circles indicate ThT fluorescence intensities of Aβ42 solutions without and with removing undissolved peptide aggregates, respectively. Open and filled squares indicate ThT fluorescence intensities of Aβ40 solutions without and with removing undissolved peptide aggregates, respectively. b, Kinetics of Aβ fibrillogenesis. Aβ (Aβ40 and Aβ42) solutions, after removal of undissolved peptide aggregates, were incubated at 50 μm and 37°C in the presence of GM1-containing liposomes (filled circles) or GM1-lacking liposomes (plus signs) or were incubated in the absence of liposomes (open circles). The GM1-containing liposomes alone were also incubated in the absence of Aβ (triangles). The fluorescence intensity of thioflavin T was obtained by excluding background activity at 0 hr. Inset, Semilogarithmic plot of the difference, A – F(t), versus incubation time (0–24 hr). F(t) represents the increase in fluorescence intensity as a function of time in the case of Aβ incubated with GM1-containing liposomes, and A is tentatively determined as F (infinity). Linear regression and correlation coefficient values were calculated (r = 0.997). F(t) is described by a differential equation: F′ (t) = B – CF(t). c, Electron micrographs of the Aβ40 solutions incubated at 50 μm and 37°C for 24 hr with GM1-containing liposomes (left and right top panels) or without liposomes (right bottom panel). The liposomes are indicated by asterisks. Scale bars, 50 nm. d, Westernblot of Aβ40 solutions incubated at 50 μm and 37°C for 24 hr in the presence or absence of GM1-containing liposomes. The incubated Aβ solutions were centrifuged at 100,000 × g for 15 min. Ten nanograms of Aβ in the supernatant were subjected to SDS-PAGE (4–20%) after glutaraldehyde treatment. The Aβ in the gel was detected by Westernblotting using BAN052. Aβ oligomers were detected in the Aβ solution incubated in the presence of GM1-containing liposomes but not detected in the incubation mixture containing Aβ alone.

Figure 2.

Viability of neurons treated with Aβ40 incubated in the presence of GM1 ganglioside. Phase-contrast and calsein AM–ethidium homodimer-stained photographs of cultured neurons were taken after treatment with TBS (a, b), Aβ40 (c, d), GM1-lacking liposomes (e, f), GM1-lacking liposomes plus Aβ40 (g, h), GM1-containing liposomes (i, j), and GM1-containing liposomes plus Aβ40 (k, l). m, Viable cells stained with calcein AM were counted. The data represent means ± SE for triplicate samples. *p < 0.003 versus other treatments. Scale bars, 20 μm.

Figure 3.

Characterization of the binding specificity of 4396C. a, Immunoelectron micrographs of liposomes. GM1-containing and GM1-lacking liposomes were subjected to immunoelectron microscopy of 4396C or isotype-matched control IgG staining after incubation with soluble Aβ40. GM1(+), GM1-containing liposomes; GM1(–), GM1-lacking liposomes. Scale bar, 50 nm. b, Quantitative assay of binding of 4396C to liposomes. GM1-containing liposomes were incubated with 4396C (diamonds) or isotype-matched control IgG (filled squares) after their mixing with soluble Aβ40 at indicated concentrations. GM1-lacking liposomes were also incubated with 4396C (triangles) or isotype-matched control IgG (× symbols). Ab, Antibody. c, Dot blot analysis. Left, Liposomes carrying GAβ (GAβ40), Aβ40, and GM1 in amounts equal to those contained in blotted liposomes (300 and 600 ng of Aβ40; 2 and 4 μg of GM1) were blotted. The blots were incubated with 4396C, BAN052, HRP-conjugated CTX, or isotype-matched control IgG. Right, Liposomes carrying GAβ (GAβ40 and GAβ42), prepared using Aβ40 or Aβ42, and Aβ40 and Aβ42 in amounts equal to those contained in GAβ40 and GAβ42 (600 ng of each peptide) were blotted. The blots were incubated with 4396C. d, Inhibition of amyloid fibril formation from soluble Aβ40 by 4396C. Left, Soluble Aβ40 was incubated with GM1-containing liposomes in the absence (filled squares) or presence of an antibody (4396C or 4G8). The molar ratios of 4396C to soluble Aβ40 were 0.3:50 (triangles), 1.3:50 (circles), and 4:50 (open squares) and that of 4G8 to Aβ40 was 4:50 (diamonds). Right, Soluble Aβ40 was incubated with preformed Aβ40 fibrils in the absence of an antibody (filled squares) or in the presence of 4396C. The molar ratios of 4396C to soluble Aβ40 were 0.3:50 (triangles), 1.3:50 (circles), and 4:50 (open squares). e, Immunoelectron micrographs of preformed Aβ40 fibrils. Aβ40 fibrils were formed by the extension reaction of Aβ40 seeds (10 μg/ml) with seed-free Aβ40 (50 μm), as described in Materials and Methods, and subjected to immunoelectron microscopy of 4396C, 4G8, or isotype-matched control IgG staining. Scale bar, 50 nm.

Figure 4.

Immunohistochemistry of GAβ in sections of human brains. a, Immunostaining of serial sections of the cerebral cortex of an AD brain fixed in Kryofix and pretreated with SDS. Neurons (arrowheads) were immunostained by 4396C but not by 4G8, whereas plaques (arrows) were immunostained by 4G8 but not by 4396C. The asterisks indicate the same blood vessel in the serial sections. Scale bar, 50 μm. b, Immunostaining by 4396C of sections of cerebral cortices of DS (left) and control (right) brains fixed in Kryofix and pretreated with SDS (DS, 47 years old; control, 65 years old). Scale bar, 50 μm.

Figure 5.

Immunohistochemistry and immunoprecipitation of GAβ in sections of nonhuman primate brains. a, Immunostaining by 4396C of sections of the cerebral cortices of primate brains, which were fixed in paraformaldehyde, from animals of different ages. Scale bar, 50 μm. Inset, Higher magnification. Scale bar, 20 μm. b, Double immunostaining of sections of the cerebral cortex of a 36-year-old primate brain, which was fixed in paraformaldehyde, after the blocking of autofluorescence by pretreatment with Sudan Black B. Colocalization of immunostaining by 4396C and that by BAN052 or CTX is shown in the merged image. Scale bar, 25 μm. c, Immunoprecipitation of GAβ by 4396C from cerebral cortices of primates at different ages. Immunoprecipitates were blotted and reacted with BAN052 or HRP-conjugated CTX.