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Brain Advance Access originally published online on June 23, 2003
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Brain, Vol. 126, No. 9, 1935-1939, September 2003
© 2003 Guarantors of Brain
doi: 10.1093/brain/awg191

Human anti-ß-amyloid antibodies block ß-amyloid fibril formation and prevent ß-amyloid-induced neurotoxicity

Yansheng Du1, Xing Wei1, Richard Dodel2, Norbert Sommer3, Harald Hampel4, Feng Gao1, Zhizhong Ma1, Liming Zhao1, Wolfgang H. Oertel and Martin Farlow1

1 Department of Neurology, Indiana University School of Medicine, Indianapolis, IN, 2 Department of Neurology, Friedrich-Wilhelms-University, Bonn, 3 Department of Neurology, Philipps University, Marburg and 4 Department of Psychiatry, Ludwig-Maximilian University, Munich, Germany

Correspondence to: Yansheng Du, PhD, Department of Neurology, School of Medicine, Indiana University,975 W. Walnut Street IB 457, Indianapolis, IN 46202, USA E-mail: ydu{at}iupui.edu

Received November 25, 2002. Revised March 31, 2003. Accepted April 7, 2003.


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 Material and methods
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 References
 
The accumulation of ß-amyloid (Aß) in neuritic plaques is thought to be causative for the progression of Alzheimer’s disease (AD). Recently, both active immunization and passive administration of Aß antibodies dramatically attenuated amyloid plaque deposition, neuritic dystrophy, astrogliosis and behaviour deficits in transgenic animals. In addition, we and others have found that titres of naturally occurring anti-Aß antibodies in the CSF of AD patients are significantly lower than those in age-matched controls. Treatment with intravenous immunoglobulins (a preparation that contained anti-Aß antibodies) significantly lowered CSF levels of Aß in non-demented patients. In this study, anti-Aß antibodies were isolated from immunoglobulin preparations and these anti-Aß antibodies strongly block fibril formation or disrupt formation of fibrilar structures. Furthermore, these antibodies almost completely prevented neurotoxicity of Aß. In contrast, immunoglobulins depleted of anti-Aß antibodies had little effect on Aß fibril formation or protection of neuronal cells. This study supports the findings that human anti-Aß antibodies may interfere with the pathogenesis of AD by more than one mechanism, and administration of polyclonal human anti-Aß antibodies isolated from plasma is a potential therapeutic agent to prevent or slow down disease progression.

Keywords: Alzheimer’s disease; ß-amyloid; neurotoxicity; immunotherapy; Aß antibodies

Abbreviations: Aß = ß-amyloid; AD = Alzheimer’s disease; ELISA = enzyme-linked immunosorbent assay;Ig = immunoglobulin; IVIG = intravenous immunoglobulins; ThT = thioflavin T


   Introduction
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 Introduction
Material and methods
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Cortical atrophy, neuronal loss, region-specific amyloid deposition, neuritic plaques and neurofibrillary tangles are key neuropathological features in the brain of Alzheimer’s disease (AD) patients (Selkoe, 1994Go). The accumulation of ß-amyloid (Aß, a 39–42 amino acid proteolytic product of the amyloid precursor protein in neuritic plaques is thought to be causative for progression of the disease (Kang et al., 1987Go). Aß is normally produced by cells and can be detected as a circulating peptide in the plasma and CSF of healthy humans (Haass et al., 1992Go). In AD, it has been postulated that increased production and/or a decreased metabolism/clearance of Aß may be primary events that lead to amyloid plaque deposition and subsequently to the cascade of other neuropathological changes associated with the disease. In vitro studies using synthetic Aß peptide(s) have shown that neurotoxicity is dependent on Aß being fibrillar and predominantly present in a ß-pleated sheet conformation (Beyreuther and Masters, 1997Go).

Schenk and colleagues and others investigated alterations in the deposition of Aß in amyloid precursor protein (V717F) transgenic mice following immunization with pre-aggregated Aß1–42 and passive administration of antibodies raised against Aß (Schenk et al., 1999Go; Bard et al., 2000Go). Both active immunization and passive administration of Aß antibodies attenuated amyloid plaque deposition, neuritic dystrophy, astrogliosis and behaviour deficits in transgenic animals (Schenk et al., 1999Go; Bard et al., 2000Go; Morgan et al., 2000Go; DeMattos et al., 2001Go, 2002Go; Dodart et al., 2002Go). In these studies, increased titres of mouse anti-human Aß antibody were necessary for the observed reduction in plaque burden to occur. These findings raise the possibility that formation and clearance of an Aß:antibody complex may decrease brain Aß deposition, either following antibody generation within the CNS or by peripheral antibody transport across the blood–brain barrier (Wisniewski and Sigurdsson, 2002Go; Dodel et al., 2003Go). Recently, we and others have found that titres of naturally occurring anti-Aß antibodies in the CSF of AD patients are significantly lower than those in age-matched controls (Du et al., 2001Go; Weksler et al., 2002Go). Our data, in addition to results from experiments with transgenic mice, suggest that an impaired or reduced ability to generate antibodies specific against Aß may be one mechanism that contributes to the pathogenesis of AD. Our hypothesis has also been supported by our investigations into changes in Aß levels in individuals who were treated with intravenous immunoglobulin (IVIG) preparation (a preparation that contained anti-Aß antibodies). Treatment with IVIG increased both CSF and serum levels of anti-Aß antibodies and significantly lowered CSF levels of Aß, possibly by facilitating transport of Aß from the CSF to the serum (Dodel et al., 2002Go). It has been demonstrated previously that a specific monoclonal antibody raised against the N-terminal region of Aß can disaggregate Aß fibril formations and prevent their toxic effects on PC12 cells (Frenkel et al., 2000Go). We therefore tested whether purified human anti-Aß antibodies, which we have recently isolated from human IVIG, may have the same effects as these mouse monoclonal antibodies with respect to preventing Aß fibril formation and neurotoxicity of Aß.


   Material and methods
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 Material and methods
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Purification of Aß antibody
The column was packed with NHS-activated Sepharose 4B (Pharmacia Biotech, Piscataway, NJ, USA) labelled with Aß1–40 (0.6 mg/ml drained Sepharose), and was equilibrated and washed with phosphate-buffered saline (PBS) (pH 7.4). After passing purified human plasma immunoglobulin G (IgG) (Octapharma, Langnfeld, Germany) through the column, fractions were eluted with the elution buffer (50 mM glycine and 150 mM NaCl at pH 2.5) and tested by using an Aß antibody enzyme-linked immunosorbent assay (ELISA) (Du et al., 2001Go).

Aß antibody ELISA
Ninety-six-well ELISA plates were coated with Aß1–40, which was dissolved in coating buffer (1.7 mM NaH2PO4, 98 mM Na2HPO4, 0.05% sodium azide, pH 7.4). After incubation of plates with a blocking buffer (0.25% casein in PBS, 0.05% sodium azide, pH 7.4), samples were loaded overnight at 4°C. Biotinylated reporter antibody, monoclonal antihuman IgG (cross-reacted with mouse IgG; Sigma Chemical Co., St Louis, MO, USA), was successively incubated for 1 h at room temperature. Anti-biotin antibody conjugated with horseradish peroxidase was added for 1 h at room temperature and colour substrate, TMP, was added and the plates read on a plate reader (Bio-Rad 3550; Bio-Rad, Hercules, CA, USA) at 450 nm (Du et al., 2001Go).

Characterization of Aß antibodies
The purified antibodies were tested for complement binding using a modification of the commercial complement assay by Virion (Munich, Germany) using the stock antibody at 1 : 1000 (and lower) and the Aß peptide at 1 µg/ml.

The IgG subclasses of the purified antibody samples were measured by nephelometry. To test for polyclonality of the purified Aß antibodies, western blotting was performed.

Fluorometric experiments
Fluorometry was performed as described previously (Naiki et al., 1989Go). Synthetic Aß was incubated with or without purified Aß antibodies in PBS buffer at 37°C overnight. The samples were added to 50 mM glycine pH 9.2, 2 µM thioflavin T (ThT) (Sigma) at a final volume of 2 ml. Fluorescence was measured spectrophotometrically at excitation and emission wavelengths of 435 and 485 nm, respectively. Samples were run in triplicate and plotted as mean ± SD. Fluorometric experiments were perfomed as described previously (Du et al., 1998Go).

Primary rat neuronal culture and neurotoxicity assays
Rat cortical cells were prepared from 18-day-old Sprague–Dawley rat fetuses as described previously (Du et al., 1998Go). Briefly, embryonic day 18 rat cortex cells were prepared and seeded into 24-well polyethylenimine-coated culture plates at a cell density of 5 x 105 cells/well, in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum for 2 days, and then maintained in Neurobasal culture medium with B27 serum substitute (Invitrogen, Carlsbad, CA, USA) for another 5 days before treatment. Aß was incubated in PBS overnight at 37°C in the absence or presence of purified human Aß antibodies in vitro and was then added to cells at a concentration of 50 µM. After exposure of the cells with these incubates for 72 h, 100 µl of the media was removed and tested for release of lactate dehydrogenase levels with a standard 340 nm lactate dehydrogenase assay (Sigma).


   Results
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 Summary
 Introduction
 Material and methods
 Results
Discussion
 References
 
We first isolated human anti-Aß antibodies from IVIG by using an affinity column coated with Aß1–40. We found that these adherent antibodies had a strong anti-Aß signal using ELISA as compared with pass-through IgG. There was a >200-fold difference in the fluid containing anti-Aß antibodies as compared with pass-through IgG (Fig. 1). The distribution of the different IgG subclasses in the Aß antibody sample were as follows: IgG1, 63.8% (83.3 mg/ml); IgG2, 19.9% (26 mg/ml); IgG3, 9% (11.79 mg/ml); IgG4, 7.3% (9.57 mg/ml). Thus, the IgG subclasses of these antibodies are completely normally distributed like commercial intravenous IgG products. Furthermore, we found that these antibodies have a low affinity for complement fixing and are of polyclonal origin.



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  		Fig. 1 Analysis of Aß binding by purified anti-Aß antibodies in an ELISA assay. The purified anti-Aß antibodies (0.07 µM) and pass-through IgG (0.07 µM) were added to the Aß1–40-coated wells. Bound antibodies were detected with horse radish peroxidase-conjugated secondary antibodies. Purified anti-Aß antibodies showed a strong signal compared with pass-through IgG. AA = anti-Aß antibodies; PA = pass-through IgG.

 
We next investigated whether purified human anti-Aß antibodies blocked or disaggregated Aß fibril formation by using ThT reagent, which binds specifically to fibrillar structures. Human anti-Aß antibodies (0.07 µM) incubated with fresh Aß (50 µM) or preformed Aß fibrils (50 µM) strongly blocked fibril formation or disrupted formation of fibril structures, as evidenced by a substantial decrease in ThT fluorescence (Figs 2 and 3). In contrast, the pass-through IgG had little effect on fibril formation or structure (P = not significant). Furthermore, we applied in vitro tests, using cultured rat hippocampal neurons, to test whether human anti-Aß antibodies (0.07 µM) would exhibit a neuroprotective effect towards cultured neurons following exposure to toxic concentrations of Aß25–35 and Aß1–40 (50 µM). As shown in Fig. 4, human anti-Aß antibodies almost completely prevented neurotoxicity of Aß. In contrast, immunoglobulins depleted of anti-Aß antibodies (pass-through IgG) had little effect on protecting neuronal cells.





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  		Fig. 2 Purified anti-Aß antibodies inhibit Aß25–35 fibril formation and disaggregate preformed Aß25–35 fibres. Co-incubation of purified anti-Aß antibodies (0.07 µM) with (A) 50 µM Aß25–35, (B) 50 µM Aß1–40 or (C) 50 µM Aß1–42 in PBS inhibits Aß fibril formation as measured by ThT staining. The fluorescence of the ThT assay is proportional to fibrillar Aß and was used to assess fibril morphology. Purified anti-Aß antibodies significantly inhibited Aß fibril formation.

 




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  		Fig. 3 Purified anti-Aß antibodies inhibit Aß25–35 fibril formation and disaggregate preformed Aß25–35 fibres. The fibrillar state of preformed (A) 50 µM Aß25–35, (B) 50 µM Aß1–40 or (C) 50 µM Aß1–42 in PBS was measured with or without incubation with antibodies overnight. Purified anti-Aß antibodies disaggregated preformed Aß fibres. Samples were run in triplicate and plotted as the mean ± SD (***P < 0.001, **P < 0.01, *P < 0.05 compared with Aß only, one-way ANOVA). Ab = Aß; AA = anti-Aß antibodies; PA = pass-through IgG; N.S. = not significant.

 



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  		Fig. 4 Effects of anti-Aß antibodies on Aß-induced neurotoxicity. Exposure of fetal rat hippocampal neurons to (A) 50 µM Aß25–35 or (B) Aß1–40 resulted in a reduction in neuronal survival during a 72 h incubation period. Purified anti-Aß antibodies (0.07 µM) significantly attenuated Aß-induced neuronal death. Both MTT agent assay (bar) and lactate dehydrogenase assay (line or bar) were used to estimate cell death. The data represent the mean ± SD of triplicate determinations from a representative experiment repeated at least three times with similar results (***P < 0.001, *P < 0.05, compared with Aß only, one-way ANOVA). C = untreated cultures; Ab = Aß; AA = anti-Aß antibodies; PA = pass-through IgG.

 

   Discussion
Top
 Summary
 Introduction
 Material and methods
 Results
 Discussion
References
 
We have identified specific anti-Aß antibodies (IgG) in both the serum and the CSF from non-immunized humans that may act in an immune-mediated Aß clearance pathway (Du et al., 2001Go; Dodel et al., 2002Go). In an earlier study, human antibodies reactive with Aß were isolated and cloned from human B-cell lines from AD patients; however, the role of these antibodies in AD pathogenesis remained unclear (Iwata et al., 2000Go). In our previous study, we and others have detected a significant difference in the amount of Aß antibodies in AD patients compared with controls (Du et al., 2001Go; Weksler et al., 2002Go), and have found that treatment with IVIG results in a significant decrease of total Aß as well as Aß1–42 in the CSF compared with baseline. In addition, mean Aß antibody concentration increased in the CSF (Dodel et al., 2002Go). These findings suggest that human Aß antibodies are able to lower the CSF Aß concentration, which may reduce Aß deposition in brain. Most recently, immunization or administration of Aß antibody was shown to reduce memory impairment in amyloid precursor protein transgenic mice (Bard et al., 2000Go; Morgan et al., 2000Go; DeMattos et al., 2001Go; Dodart et al., 2002Go) and mouse monoclonal antibodies were shown to block Aß fibril formation and toxicity (McLaurin et al., 2002Go). We therefore investigated whether or not purified human Aß antibodies had similar impacts on Aß fibril formation and toxicity.

Our results suggest that human anti-Aß antibodies isolated from the plasma block Aß fibril formation and prevent Aß-induced neurotoxicity. In addition to the clearence of Aß, these two mechanisms may interfere with plaque formation as well as preventing loss of neuronal function in AD. Interestingly, purified anti-Aß antibodies can disaggregate both preformed Aß1–40 as well as active truncated Aß25–35, and they also block both peptide-induced neurotoxicity, suggesting these antibody fractions include antibodies against not just the N-terminal of Aß, but also the middle site of Aß (Bard et al., 2003Go). When testing preparations from different vendors, we did not find a batch-to-batch variation of intravenous IgG preparations in respect to anti-Aß fibril formation and anti-Aß-induced neurotoxicity. Our data therefore provide further support for the hypothesis that human anti-Aß antibodies may interfere with AD pathogenesis by more than one mechanism. Furthermore, these antibodies are polyclonal and do not bind complement. Whether or not they complex with Aß and trigger a local inflammatory reaction to induce cerebral haemorrhage or meningoencephalitis in humans remains to be determined (Pfeifer et al., 2002Go; Nicoll et al., 2003Go). However, in our small clinical trials on both AD and non-AD patients, we did not observe such an effect (Dodel et al., 2002Go; R.Dodel, H.Hampel, C.Depboylo, S.Lin, F.Gao, S.Schock, unpublished data on IVIG treatment in six AD patients). All these data suggest that administration of polyclonal human anti-Aß antibodies isolated from plasma is a potential therapeutic agent to prevent or slow down AD progression. The therapeutic efficacy as well as practical clinical utility of these effects and/or mechanisms, however, remain to be determined.


   References
Top
 Summary
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Bard F, Cannon C, Barbour R, Burke RL, Games D, Grajeda H, et al. Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat Med 2000; 6: 916–9.[CrossRef][Web of Science][Medline]

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DeMattos RB, Bales KR, Cummins DJ, Dodart JC, Paul SM, Holtzman DM. Peripheral anti-A beta antibody alters CNS and plasma A beta clearance and decreases brain A beta burden in a mouse model of Alzheimer’s disease. Proc Natl Acad Sci USA 2001; 98: 8850–5.[Abstract/Free Full Text]

DeMattos RB, Bales KR, Cummins DJ, Paul SM, Holtzman DM. Brain to plasma amyloid-beta efflux: a measure of brain amyloid burden in a mouse model of Alzheimer’s disease. Science 2002; 295: 2264–7.[Abstract/Free Full Text]

Dodart JC, Bales KR, Gannon KS, Greene SJ, DeMattos RB, Mathis C, et al. Immunization reverses memory deficits without reducing brain Abeta burden in Alzheimer’s disease model. Nat Neurosci 2002; 5: 452–7.[Web of Science][Medline]

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