Impact of amyloid β aggregate maturation on antibody treatment in APP23 mice

Abstract

Introduction: The deposition of the amyloid β protein (Aβ) in the brain is a hallmark of Alzheimer's disease (AD). Removal of Aβ by Aβ-antibody treatment has been developed as a potential treatment strategy against AD. First clinical trials showed neither a stop nor a reduction of disease progression. Recently, we have shown that the formation of soluble and insoluble Aβ aggregates in the human brain follows a hierarchical sequence of three biochemical maturation stages (B-Aβ stages). To test the impact of the B-Aβ stage on Aβ immunotherapy, we treated transgenic mice expressing human amyloid precursor protein (APP) carrying the Swedish mutation (KM670/671NL; APP23) with the Aβ-antibody β1 or phosphate-buffered saline (PBS) beginning 1) at 3 months, before the onset of dendrite degeneration and plaque deposition, and 2) at 7 months, after the start of Aβ plaque deposition and dendrite degeneration.

Results: At 5 months of age, first Aβ aggregates in APP23 brain consisted of non-modified Aβ (representing B-Aβ stage 1) whereas mature Aβ-aggregates containing N-terminal truncated, pyroglutamate-modified AβN3pE and phosphorylated Aβ (representing B-Aβ stage 3) were found at 11 months of age in both β1- and PBS-treated animals. Protective effects on commissural neurons with highly ramified dendritic trees were observed only in 3-month-old β1-treated animals sacrificed at 5 months. When treatment started at 7 months of age, no differences in the numbers of healthy commissural neurons were observed between β1- and PBS-treated APP23 mice sacrificed with 11 months.

Conclusions: Aβ antibody treatment was capable of protecting neurons from dendritic degeneration as long as Aβ aggregation was absent or represented B-Aβ stage 1 but had no protective or curative effect in later stages with mature Aβ aggregates (B-Aβ stage 3). These data indicate that the maturation stage of Aβ aggregates has impact on potential treatment effects in APP23 mice.

Fig. 1

Degeneration of commissural neurons with a highly ramified dendritic tree: Effects of β1 antibody treatment. a, b Layer III type 1 commissural neurons in the frontocentral cortex traced with DiI. The detectable type 1 commissural neurons did not show significant differences between PBS- and β1-treated APP23 mice even at 5 months of age. c The number of the type 1 commissural neurons in 5-month-old β1 treated mice was higher than that in PBS-treated animals whereas no such differences were observed for type 2 and 3 commissural neurons. d No differences in the numbers of DiI-traced type 1–3 commissural neurons were seen among 11-month-old PBS- and β1-treated APP23 mice. Graphs represent mean values (symbols) and standard errors (whiskers), * p < 0.05 (Statistical analysis in Additional file 3: Table S3). Calibration bar in b (valid for a, b) = 37.5 μm

Fig. 2

Aβ plaque pathology in the frontocentral cortex of APP23 mice: Effects of β1 antibody treatment. Aβ plaque pathology in PBS- and β1-treated APP23 mice. The staining pattern in 11-month-old mice as well as the plaque loads at 5 and 11 months of age did not differ between PBS- and anti-Aβ (β1) treated animals when staining the plaques with antibodies raised against Aβ₄₂ (a–c), Aβ₄₀ (d–f), Aβ_N3pE (g–i), and pAβ (j–l). Graphs c, f, i, and l represent mean values (symbols) and standard errors (whiskers). (Statistical analysis see Additional file 3: Table S3). Calibration bar in a (valid for a, b, d, e, g, h, j, k) = 280 μm

Fig. 3

Epitope masking effects of β1 antibody treatment in the frontocentral cortex of APP23 mice. Epitope modification after anti-Aβ (β1) antibody treatment in 11-month-old APP23 mice. Although an antibody raised against Aβ_1–17 (6E10) exhibited similar levels of Aβ plaques (a, b - arrows) as provided by the respective Aβ (6E10) plaque load (c) the β1 antibody used for treatment detected less plaques in the treated animals than in the PBS-controls (d, e - arrows) as confirmed by a significantly lowered β1 plaque load (f). Less plaques were also detected with B10AP antibody fragments detecting protofibril/ fibril-specific epitopes (g, h - arrows) with respective changes in the B10AP plaque load (i). Graphs c, f, and i represent mean values (symbols) and standard errors (whiskers), * p < 0.05 (Statistical analysis in Additional file 3: Table S3). Calibration bar in a (valid for a, b, d, e, g, h) = 280 μm

Fig. 4

Soluble, dispersible, membrane-associated, and plaque-associated Aβ in APP23 mice: Effects of β1 antibody treatment. Semiquantitative comparison of Aβ, Aβ_N3pE, and pAβ in the soluble, dispersible, membrane-associated, and plaque-associated fraction of brain homogenates of PBS- and β1-treated 5- and 11-month-old APP23 mice received by quantification of western blots displayed in Additional file 8: Figure S5. No differences between PBS- and β1-treated animals except for plaque-associated Aβ in 5-month-old mice: β1-treated animals exhibited slightly more non-modified plaque-associated Aβ than non-treated mice. Aβ_N3pE and pAβ were not detected in brain homogenates of 5-month-old APP23 mice but in the dispersible, membrane-associated, and plaque-associated fraction of 11-month-old mice without differences in relation to the treatment. Graphs represent mean values (white symbols 5-month-old mice; black symbols 11-month-old mice) and standard errors (whiskers). (* p < 0.05; detailed Statistical analysis in Additional file 3: Table S3)

Fig. 5

Soluble and dispersible (insoluble) Aβ fibrils/protofibrils and non-fibrillar oligomers in APP23 mice: Effects of β1 antibody treatment. Semiquantitative analysis of western blots from soluble and dispersible non-fibrillar oligomers immunoprecipitated with A11 and protofibrils and fibrils immunoprecipitated with B10AP from the respective brain homogenate fractions in 5- and 11-month-old β1- and PBS-treated APP23 mice. 5-month-old β1-treated mice exhibited soluble Aβ-oligomers, protofibrils and fibrils that were not seen in PBS-treated controls (* p < 0.05; detailed statistical analysis in Additional file 3: Table S3). Although dispersible Aβ oligomers, protofibrils and fibrils occurred in 5-month-old APP23 mice as well there were no differences between β1- and PBS-treated mice. Aβ_N3pE and pAβ were not seen in 5-month-old mice. No differences in soluble and dispersible Aβ aggregates were observed between 11-month-old β1- and PBS-treated mice. Aβ_N3pE and pAβ were not detectable in soluble oligomers, protofibrils and fibrils as well as in dispersible oligomers at both ages whereas dispersible fibrils and protofibrils exhibited both Aβ_N3pE and pAβ. Graphs represent mean values (white symbols 5-month-old mice; black symbols 11-month-old mice) and standard errors (whiskers). (Full blots Additional file 9: Figure S6; Statistical analysis Additional file 3: Table S3)

Fig. 6

Immunoglobulin-bound Aβ detected in oligomers, protofibrils and fibrils in APP23 mice: Effects of β1 antibody treatment. Semiquantitative analysis of western blots after immunoprecipitation of immunoglobulin (antibody)-bound Aβ by precipitation of antibodies with protein G-coated magnetic beads. Subsequent western blot analysis with anti-Aβ_1–17 revealed antibody-bound Aβ in the dispersible fraction of both β1- and PBS-treated mice at both ages. In 5-month-old β1-treated APP23 mice antibody-bound Aβ was found in the soluble fraction whereas no antibody-bound Aβ was precipitated in PBS-treated animals (* p < 0.05). At 11-months of age both, β1 and PBS-treated animals exhibited antibody bound soluble Aβ in similar amounts. Antibody-bound Aβ_N3pE was not observed whereas 11-month-old (but not 5-month-old) APP23 mice showed similar amounts of dispersible antibody-bound pAβ. No soluble antibody-bound pAβ was seen. Graphs represent mean values (white symbols 5-month-old mice; black symbols 11-month-old mice) and standard errors (whiskers). (Full blots Additional file 10: Figure S7; Statistical analysis in Additional file 3: Table S3)

Fig. 7

Serum Aβ immunoprecipitated by protein-G bound antibodies in APP23 mice: Effects of β1 antibody treatment. Semiquantitative assessments of western blot analysis of blood serum for Aβ and immunoprecipitation of intrinsic serum antibodies by incubation with protein G-coated magnetic beads and with subsequent western blotting for Aβ. Aβ was only seen in 5-month-old β1-treated APP23 mice after antibody-immunoprecipitation and detection with anti-Aβ_1–17 (6E10) (* p < 0.05). Aβ_N3pE and pAβ were not found in these precipitates. PBS-treated and 5-month-old mice did not exhibit detectable amounts of Aβ. Graphs represent mean values (white symbols 5-month-old mice; black symbols 11-month-old mice) and standard errors (whiskers). (Full blots Additional file 11: Figure S8; Statistical analysis in Additional file 3: Table S3)

Fig. 8

Effects of β1 antibody treatment in APP23 mice. Schematic representation of anti-Aβ (β1) treatment effects when applied before the onset of neurodegeneration and Aβ plaque deposition (3–5 months) and when provided with prevalent pathology (7–11 months). Anti-Aβ antibody treatment protected type 1 commissural neurons (Type 1) from dendritic degeneration accumulated antibody-bound oligomers, fibrils and protofibrils in the soluble fraction containing Aβ. These effects were seen in B-Aβ stage 1. No positive effect on Type 1 commissural neurons was found when β1-treatment was performed from 7 to 11 months of age. Moreover, plaque loads and biochemically detectable amounts of soluble, dispersible, membrane-associated, and plaque-associated Aβ were similar in β1- and PBS-treated APP23 mice. Aβ aggregation at 11 months of age corresponded to B-Aβ stage 3, i.e. fully mature, AD-related Aβ aggregates. Here, β1-treatment caused epitope modifications of Aβ aggregates resulting in less β1- and B10AP-positive plaques in treated mice. Moreover, at 11 months of age presence of antibody-bound non-modified Aβ in the serum was visible, whereas antibody-bound modified Aβ_N3pE and pAβ were not seen in the blood plasma