Exacerbation of cerebral amyloid angiopathy-associated microhemorrhage in amyloid precursor protein transgenic mice by immunotherapy is dependent on antibody recognition of deposited forms of amyloid beta

Abstract

Passive immunization with an antibody directed against the N terminus of amyloid beta (Abeta) has recently been reported to exacerbate cerebral amyloid angiopathy (CAA)-related microhemorrhage in a transgenic animal model. Although the mechanism responsible for the deleterious interaction is unclear, a direct binding event may be required. We characterized the binding properties of several monoclonal anti-Abeta antibodies to deposited Abeta in brain parenchyma and CAA. Biochemical analyses demonstrated that the 3D6 and 10D5, two N-terminally directed antibodies, bound with high affinity to deposited forms of Abeta, whereas 266, a central domain antibody, lacked affinity for deposited Abeta. To determine whether 266 or 3D6 would exacerbate CAA-associated microhemorrhage, we treated aged PDAPP mice with either antibody for 6 weeks. We observed an increase in both the incidence and severity of CAA-associated microhemorrhage when PDAPP transgenic mice were treated with the N-terminally directed 3D6 antibody, whereas mice treated with 266 were unaffected. These results may have important implications for future immune-based therapeutic strategies for Alzheimer's disease.

Figures

Figure 1.

Aβ42:Aβ40 ratio varies with age in brain and cerebral vessels. Accumulation of Aβ peptides in whole brain or isolated microvessels from PDAPP mice was measured by ELISA at multiple ages (3-12 mice per group; error bars represent SE). The data are presented as picograms of Aβ per milligram of wet weight of brain (A) or vessels (B). Data demonstrate robust accumulation of Aβ42 in parenchyma (A) with up to 95% total Aβ being 1-42 by 15 months (C). In contrast, isolated vessels with CAA (B, D) show selective enrichment of Aβ40, resulting in equivalent amounts of both species by 25 months (D).

Figure 2.

Thioflavin S staining of brain sections and cerebral vessels to identify amyloid deposits. Thioflavin S staining of 50 μm floating sections from a 21-month-old PDAPP mouse brain (A, B) and isolated cerebral vessels from a 25-month-old PDAPP mouse (C-F) is shown. Note the areas of intense thioflavin S staining (yellow color) in the isolated vessels that appear to be localized on the outside of the vessels (brain side) (D-F). C, A 40× bright-field view of isolated vessels demonstrating the integrity of the vessel preparation.

Figure 3.

Antibody affinity for deposited Aβ by immunostaining in PDAPP sections. Immunostaining was performed on formalin-fixed brain sections from a 21-month-old PDAPP mouse. N-terminal domain antibodies 3D6 (C) and 10D5 (D) were able to bind deposited Aβ, whereas mid-domain antibody 266 (E) was indistinguishable from biotinylated isotype control (B) or no primary antibody (A). Floating sections were stained with biotinylated antibodies at a concentration of 3.0 μg/ml. Levels of biotinylation were similar among all antibodies (data not shown).

Figure 4.

Antibody affinity for deposited Aβ by immunostaining in human Alzheimer brain sections. Immunostaining was performed on nonfixed cryostat-cut brain sections from an Alzheimer brain. Sections were stained with the biotinylated antibodies at a concentration of 3.0 μg/ml. N-terminal domain antibodies 3D6 (C) and 10D5 (D) were able to bind deposited Aβ, whereas mid-domain antibody 266 (E) was indistinguishable from biotinylated isotype control (B) or no primary antibody (A).

Figure 5.

Anti-Aβ antibody affinity for CAA in vitro. Microvessels were isolated and pooled from 29-month-old PDAPP mice. Aliquots of vessels were incubated with the biotinylated antibodies under physiological conditions. A, ELISAs specific for biotinylated (b) antibodies show that 10D5 and 3D6, two N-terminal antibodies, bound with high affinity to the amyloid bearing vessels, whereas 266, a mid-domain anti-Aβ antibody, lacked specific binding and was similar to the IgG control. B, Aβ ELISA demonstrated that equivalent levels of amyloid were present in the vessel aliquots among the different treatments. C, Normalized to the biotinylated 266 values, the N-terminal antibodies b10D5 and b3D6 bound to the amyloid bearing vessels ∼50- and 100-fold higher, respectively.

Figure 6.

Anti-Aβ antibody affinity for CAA in vivo. To test whether antibodies will cross the BBB and bind to CAA-bearing vessels in vivo, PDAPP mice (25 months of age) were injected intravenously with 100 μg of biotinylated (b) antibody and killed 24 h later. Cerebral vessels were isolated and independently analyzed for the accumulation of biotinylated antibodies (3 animals per antibody). Similar to the in vitro analyses, the biotinylated 3D6 accumulated to a larger degree than the other N-terminally directed antibody, 10D5. The levels of 266 and control IgG were below the level of detection for the assay.

Figure 7.

Histology and cytochemistry of brain sections from aged PDAPP mice given 266 or 3D6 for 6 weeks. DAB-EP staining for hemosiderin indicated increased numbers of positive cells in the meninges adjacent to the neocortex in a 3D6-treated mouse (B) compared with an age-matched IgG1-treated control (A). Meningeal microhemorrhages in 3D6-treated mice often surrounded meningeal vessels and extended into adjacent neocortex as indicated by H&E (D) and DAB-EP (E) staining. G, H, Focal hemorrhage in the neocortex in a 3D6-treated mouse as indicated by H&E (G) and DAB-EP staining (H). C, F, Extensive hemorrhage adjacent to the hippocampus in a 3D6-treated mouse as indicated by H&E (C) and DAB-EP (F) staining. Scale bars: A, C-G, 100 μm; B, H, 10 μm.

Figure 8.

Mean severity index of hemosiderin staining in the meninges. Mean severity index of hemosiderin staining in the meninges indicated an approximately twofold increase in 3D6-treated mice compared with IgG-treated control mice (*p < 0.05).