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. 2011 Apr 13;31(15):5847-54.
doi: 10.1523/JNEUROSCI.4401-10.2011.

Naturally occurring autoantibodies against beta-amyloid: investigating their role in transgenic animal and in vitro models of Alzheimer's disease

Richard Dodel  1 , Karthikeyan Balakrishnan, Kathy Keyvani, Oliver Deuster, Frauke Neff, Luminita-Cornelia Andrei-Selmer, Stephan Röskam, Carsten Stüer, Yousef Al-Abed, Carmen Noelker, Monika Balzer-Geldsetzer, Wolfgang Oertel, Yansheng Du, Michael Bacher
Affiliations

Naturally occurring autoantibodies against beta-amyloid: investigating their role in transgenic animal and in vitro models of Alzheimer's disease

Richard Dodel et al. J Neurosci. .

Abstract

Alzheimer's disease (AD) is a neurodegenerative disorder primarily affecting regions of the brain responsible for higher cognitive functions. Immunization against β-amyloid (Aβ) in animal models of AD has been shown to be effective on the molecular level but also on the behavioral level. Recently, we reported naturally occurring autoantibodies against Aβ (NAbs-Aβ) being reduced in Alzheimer's disease patients. Here, we further investigated their physiological role: in epitope mapping studies, NAbs-Aβ recognized the mid-/C-terminal end of Aβ and preferentially bound to oligomers but failed to bind to monomers/fibrils. NAbs-Aβ were able to interfere with Aβ peptide toxicity, but NAbs-Aβ did not readily clear senile plaques although early fleecy-like plaques were reduced. Administration of NAbs-Aβ in transgenic mice improved the object location memory significantly, almost reaching performance levels of wild-type control mice. These findings suggest a novel physiological mechanism involving NAbs-Aβ to dispose of proteins or peptides that are prone to forming toxic aggregates.

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Figures

Figure 1.
Figure 1.
NAbs–Aβ were able to block the neurotoxic effect of oligomeric Aβ1-40 in neuroblastoma SH-SY5Y cells. SH-SY5Y cells (30,000 cells per well) were incubated alone or in the presence of oligomerized Aβ1-40 (prepared as described in Material and Methods). The viability of the cells was determined by the MTT assay. There was a concentration-dependent inhibition of Aβ-induced cell death in the presence of NAbs–Aβ. At a concentration of 15 μm NAbs–Aβ, an almost complete inhibition of Aβ-induced cell death was observed. Using the FT in a concentration up to 15 μm did not block Aβ-induced cytotoxicity. Values are expressed as percentage of control cultures for each experiment, and the data represent the mean ± SE of triplicate determinations from a representative experiment repeated at least three times with similar results.
Figure 2.
Figure 2.
Effect of NAbs–Aβ on plaque deposition and plaque load in transgenic APP TgCRND8 mice. A–D, Effect of NAbs–Aβ on plaque number (C) and plaque area (D) in old (age, 13 months) transgenic APP TgCRND8 mice. Transgenic mice were injected intraperitoneally once a week for the duration of 4 weeks with either FT after NAbs–Aβ purification as control (control, 200 μg; A; n = 10) or with a low (80 μg; C, D; n = 7) and high (200 μg; B–D; n = 8) dose of NAbs–Aβ, respectively. E–H, Effect of NAbs–Aβ on plaque number (G) and plaque area (H) in young transgenic mice (age, 4 months). Transgenic mice were injected intraperitoneally once a week for the duration of 4 weeks with either FT (flow-through depleted of NAbs–Aβ; 200 μg; control; E; n = 7) or a low (80 μg; G, H; n = 7) and high (200 μg; F–H; n = 8) dose of NAbs–Aβ, respectively. In old TgCRND8 mice, NAbs–Aβ did not affect the plaque load when compared with vehicle (FHT)-treated animals (B–D), whereas in young mice plaque number and plaque area was significantly suppressed after 4 weeks of treatment (F–H). The plaque density was significantly reduced after 4 weeks of both high (by 24%) and low (by 34%) dose treatment with NAbs–Aβ (p = 0.045 and 0.026, respectively; the difference between the low and the high dose in the respective figures was not significant). Furthermore, a reduction of plaque area by 47% was detected after high (p = 0.029) and low (p = 0.028) dose applications of NAbs–Aβ (H). Animals were killed 5 d after the last injection. Sagittal sections from FT- and NAbs–Aβ-treated young and old mice were stained for Aβ using anti-human Aβ monoclonal antibody 6F/3D. The average number (C, G) and area (D, H) of Aβ-immunoreactive deposits were quantified semiautomatically in the cortex and hippocampus (12 sections per brain were analyzed). Results were analyzed using a two-tailed Student's t test. Error bars represent the SD. I, J, Concentration of Aβ in CSF and plasma of APP transgenic mice after treatment with NAbs–Aβ. The concentration of Aβ in CSF (I) and plasma (J) was determined after the last treatment with NAbs–Aβ. There was an increase of the Aβ concentration in the plasma and a decrease of the Aβ concentration in CSF.
Figure 3.
Figure 3.
Immunohistochemistry to investigate the binding properties of the N-terminal monoclonal antibodies and NAbs–Aβ to amyloid deposits in transgenic APP23 mice and in brain samples of AD and human cerebral amyloid angiopathy. APP23 mice were used in this set of experiments because of their higher deposition of Aβ in vasculature. Similar results were detected with respect to amyloid deposition in TgCRND8 mice. A, Immunostaining of a brain sample from an APP23 mouse with NAbs–Aβ: no specific staining of the plaque (P) in the cortex is detectable. The inset represents the identical area with a positive staining using the 6F/3D monoclonal antibody. B, Immunostaining of a brain sample from an APP23 mouse with NAbs–Aβ: no specific staining of amyloid deposits in the vessel (V+) is detectable (the inset represents the identical area with positive staining using the 6F/3D monoclonal antibody). C, Immunostaining of a brain sample from an AD patient with NAbs–Aβ: no specific staining of a plaque (P) is detectable. The inset represents the identical area with positive staining using the 6F3D monoclonal antibody. D, Immunostaining of a brain sample from a cerebral amyloid angiopathy patient with NAbs–Aβ: no specific staining of amyloid deposits in the small vessels (V+) is visible, but there are some unspecific chromogen reactions with the Nissl substance. The inset represents the identical area with positive staining using the 6F3D monoclonal antibody.
Figure 4.
Figure 4.
A, Mapping of the Aβ epitope recognized by NAbs–Aβ using ELISA. Various peptides representing different parts of the Aβ sequence were tested for the avidity of NAbs–Aβ to bind in ELISA. The binding experiments showed an increased binding of the NAbs–Aβ toward the C-terminal region. We identified sequence AA28–40(42) as the major binding site. N-terminal regions of the Aβ peptide showed only low binding with the Aβ reversed sequence (Aβ40-1). The following peptides (with and without exchanged amino acids) were also investigated: Aβ1-40; Aβ1-42; Aβ1-12; Aβ1-15; Aβ10-28; Aβ20-38; Aβ20-31; Aβ25-40; Aβ26-42; Aβ28-42; Aβ30-42; Aβ1-40;G25A; Aβ1-40;S26A; Aβ1-40;G29I;G33I; Aβ1-42;G29I;G33I; Aβ1-40;D23K;K28D; Aβ1-40;G29I;G33I;A42I; Aβ1-40;M35A;V40A; Aβ1-40;I32A;M35A; Aβ1-40;I32A;V40A; Aβ1-40;I32A; Aβ28-42;A30G; Aβ28-42;G33A; Aβ28-42;M35A; Aβ28-42;V39C; Aβ28-42;I32A; Aβ28-42;I32A;M35A;V40A. B, Dot blots of various peptide samples of SEC fractions of Aβ were applied on nitrocellulose membrane (0.25 μg/3 μl spot). The mid-terminal antibody (MTA) directed against residues AA16–24 readily recognized monomeric and oligomeric forms of Aβ, whereas NAbs–Aβ only interacted with dimeric and trimeric forms but not monomeric forms. C–E, Immobilization and interaction analyses of monoclonal antibodies and NAbs–Aβ to Aβ1-40 and its different oligomeric forms were performed on a Biacore 2000 optical biosensor system. C, NAbs–Aβ preferentially bound to dimers, and also to trimers, but not to the monomeric form of Aβ. D, The on-rate of the monoclonal antibody MTA to Aβ was higher compared with the on-rate of NAbs–Aβ. E, There was a dose-dependent on-rate of the binding of NAbs–Aβ to Aβ1–40. OD, Optical density; RU, response units.
Figure 5.
Figure 5.
Neuropsychological testing in Tg2576 mice. Mean discrimination ratios for 14- to 16-month-old Tg2576 mice treated with either NAbs–Aβ or PBS. NAbs–Aβ-immunized Tg2576 mice but not PBS-treated Tg2576 mice showed a preference for exploring objects moved to a new location, indicated by a discrimination ratio above 0.5 (NAbs–Aβ group, n = 21; PBS group, n = 20; wild-type group, n = 20; ***p < 0.001). Error bars show SEM. WT, Wild type.
Figure 6.
Figure 6.
A, Currently known epitopes of the Aβ sequence. The NAbs–Aβ recognized a distinct C-terminal epitope compared with antibodies generated against the N terminus by active immunization in transgenic mice and promoted clearance of amyloid plaques (McLaurin et al., 2002). The T-cell activation sites and the B-cell epitope have been identified at AA14–34 and AA4–10, respectively (Monsonego et al., 2001). B, Assembly of Aβ as proposed by Petkova et al. (2002, 2005). Central Aβ1--40 molecules from the energy-minimized, five-chain system, viewed down the long axis of the fibril. Residues are color coded according to their side chains as hydrophobic (green), polar (magenta), positive (blue), or negative (red). C, Possible mode of lateral association for generating fibrils with greater mass-per-length and greater cross-sectional dimensions. Assembly of oligomers started at mid-terminal and C-terminal parts of the Aβ peptide. Therefore, active immunization with Aβ led to the generation of N-terminal antibodies because only the N-terminal part was available for T-cell interaction. Accordingly, only N-terminal antibodies were effective in removing plaques because only N-terminal parts were easily accessible to antibodies. In contrast, NAbs–Aβ recognized the dimeric form of Aβ and interfered with further assembly of monomers to oligomers. B and C are reproduced with permission from Proceedings of the National Academy of Sciences of the United States of America.
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