Epitope and isotype specificities of antibodies to beta -amyloid peptide for protection against Alzheimer's disease-like neuropathology

Abstract

Transgenic PDAPP mice, which express a disease-linked isoform of the human amyloid precursor protein, exhibit CNS pathology that is similar to Alzheimer's disease. In an age-dependent fashion, the mice develop plaques containing beta-amyloid peptide (Abeta) and exhibit neuronal dystrophy and synaptic loss. It has been shown in previous studies that pathology can be prevented and even reversed by immunization of the mice with the Abeta peptide. Similar protection could be achieved by passive administration of some but not all monoclonal antibodies against Abeta. In the current studies we sought to define the optimal antibody response for reducing neuropathology. Immune sera with reactivity against different Abeta epitopes and monoclonal antibodies with different isotypes were examined for efficacy both ex vivo and in vivo. The studies showed that: (i) of the purified or elicited antibodies tested, only antibodies against the N-terminal regions of Abeta were able to invoke plaque clearance; (ii) plaque binding correlated with a clearance response and neuronal protection, whereas the ability of antibodies to capture soluble Abeta was not necessarily correlated with efficacy; (iii) the isotype of the antibody dramatically influenced the degree of plaque clearance and neuronal protection; (iv) high affinity of the antibody for Fc receptors on microglial cells seemed more important than high affinity for Abeta itself; and (v) complement activation was not required for plaque clearance. These results indicate that antibody Fc-mediated plaque clearance is a highly efficient and effective process for protection against neuropathology in an animal model of Alzheimer's disease.

Figures

Figure 1

Antibodies produced by immunization with N-terminal fragments of Aβ bind to amyloid plaques. (a) Peptides encompassing various domains of Aβ1–42 (synthesized contiguous to T cell epitope derived from ovalbumin) were used to immunize PDAPP mice. A reverse mer, Aβ5–1, was used as a negative control. (b) ELISA titers against aggregated Aβ1–42 were significantly higher over the length of the study in the Aβ5–11 and Aβ15–24 groups than in the Aβ1–5 group (1:14,457, P < 0.01, and 1:12,257, P < 0.05 vs. 1:3,647, respectively; ANOVA followed by post hoc Tukey's test). (c) Unfixed cryostat sections from untreated PDAPP mouse brain were exposed to the sera of mice immunized with Aβ5–1, Aβ3–9, Aβ5–11, or Aβ15–24 (titers normalized to 1:1,000 for staining). Antibodies to Aβ15–24 did not bind to amyloid plaques. (Scale bar, 500 μm.)

Figure 2

Capture of soluble Aβ1–42 by antibodies is not associated with reduced amyloid burden or neuritic pathology. (a) Sera from mice immunized with fragments of Aβ were examined for their ability to capture radiolabeled soluble Aβ1–42 in an RIA. Sera from all animals immunized with Aβ15–24 were able to capture soluble Aβ1–42 (one serum sample had a titer higher than 1:1,350, and a precise titer was not determined) compared with 27% of those in the Aβ1–5 group and 3% of the Aβ3–9 group. Amyloid burden (b) and neuritic pathology (c) were evaluated with image analysis by a blinded microscopist. Values are expressed as a percentage of the mean of the Aβ5–1 group (negative control reverse mer peptide). The Aβ5–11 group was evaluated at a separate sitting from the other groups but in conjunction with the same negative control group as an internal reference (second Aβ5–1* set, on the right). Amyloid burden was reduced significantly in the Aβ1–5, Aβ3–9, and Aβ5–11 groups (P < 0.001). The bars represent median values, and the dashed horizontal line indicates the control level. Neuritic burden was reduced significantly in the Aβ3–9 and Aβ5–11 groups (P < 0.05). Neither endpoint was altered significantly by immunization with the Aβ15–24 group. Statistical analysis was performed with square-root transformation (to normalize nonparametric distributions) and analyzed with ANOVA. A Dunnett's test then was used to compare the multiple groups Aβ1–5, Aβ3–9, and Aβ15–24 with their Aβ5–1 control and Mann–Whitney for the Aβ5–11 group with its corresponding Aβ5–1* control.

Figure 3

Comparing antibodies against Aβ3–7 in the ex vivo assay, IgG2a antibodies clear β amyloid plaque more efficiently that either the IgG1 or IgG2b isotypes. Murine primary microglial cells were cultured with unfixed cryostat sections of PDAPP mouse brain in the presence of antibodies of different isotypes directed against Aβ3–7. Irrelevant IgG1, IgG2a, and IgG2b antibodies were used as the respective isotype-matched negative controls. After 24 h of incubation, the total level of Aβ remaining in the cultures was measured by ELISA. The two anti-Aβ IgG2a antibodies reduced Aβ levels in the cultures (69% for 2C1 and 73% for 12B4; P < 0.001) more efficiently than the IgG2b isotype antibodies (48% for 12A11, P < 0.05, and 59% for 3A3, P < 0.001). The anti-Aβ IgG1 antibodies did not significantly reduce Aβ levels. Data were analyzed with ANOVA followed by a post hoc Dunnett's test.

Figure 4

Anti-Aβ IgG2a antibodies reduced AD-like neuropathology more efficiently than other isotypes in vivo. PDAPP mice received weekly i.p. injections of antibodies starting at 12 months of age for 6 months. (a) Total Aβ levels shown for individual mice sorted by treatment group (n = 30). The bars represent median values, and the dashed horizontal line indicates the control level. Although Aβ levels were reduced significantly in all antibody groups (P < 0.001 vs. PBS; ANOVA followed by post hoc Dunnett's test), the IgG2a groups exhibited the highest degree of clearance. (b) The percentage of frontal cortex occupied by neuritic dystrophy was determined by image analysis. The different groups within this experiment were analyzed in two sets by using the same PBS group as an internal standard (PBS-1 and PBS-2). PBS-1 was the control for the 6C6, 10D5, 12B4, and 12A11 groups, and PBS-2 was the control for the 2C1 and 3A3 groups. To compare the groups, values for individual animals are expressed as a percentage of the mean of their respective PBS control group (set at 100%). The bars represent median values, and the dashed horizontal line indicates the control level. Neuritic dystrophy was reduced significantly only by the IgG2a isotype antibodies (12B4, P < 0.05, and 2C1, P < 0.001; ANOVA followed by post hoc Dunnett's test). (c) Dystrophic neurites were labeled with the amyloid precursor protein-specific antibody 8E5, and were found in association with plaques. Relative to PBS control, the neuritic pathology was reduced significantly in animals treated with 12B4 but not 10D5. (Scale bar, 250 μm.)