Journal of Molecular Biology
Volume 384, Issue 4, 26 December 2008, Pages 917-928
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Anti-oligomeric Aβ Single-chain Variable Domain Antibody Blocks Aβ-induced Toxicity Against Human Neuroblastoma Cells

https://doi.org/10.1016/j.jmb.2008.09.068Get rights and content

Abstract

The Amyloid-β (Aβ) peptide is a major component of the amyloid plaques associated with Alzheimer's disease (AD). Recent studies suggest that the most toxic forms of Aβ are small, soluble oligomeric aggregates. Here, we report the isolation and characterization of a single-chain variable domain (scFv) antibody isolated against oligomeric Aβ using a protocol developed in our laboratory that combines phage display technology and atomic force microscopy (AFM). Starting with a randomized, single framework phage display library, after three rounds of selection against oligomeric Aβ, we identified an scFv that bound oligomeric Aβ specifically, but not monomeric or fibrillar forms. The anti-oligomeric scFv inhibits Aβ aggregation and toxicity, and reduces the toxicity of preformed oligomeric Aβ towards human neuroblastoma cells. When used to probe samples of human brain tissue, the scFv reacted with AD tissue but not a healthy control or Parkinson's disease brain samples. The anti-oligomeric Aβ scFv therefore has potential therapeutic and diagnostic applications in specifically targeting or identifying the toxic morphologies of Aβ in AD brains.

Introduction

Alzheimer's disease (AD) patients show a progressive decline in memory and cognitive functions. Pathologically AD brains contain extracellular plaques composed mainly of the amyloid peptide and intracellular neurofibrillary tangles composed primarily of the cytoskeletal protein tau.1 The relationship between brain lesions and disease progression is still controversial.2, 3 The early findings from cell biology, genetics and pathology helped in formulation of the amyloid cascade hypothesis, which states that either overproduction of amyloid-β (Aβ) or failure to clear this peptide eventually leads to AD.4 Central to the amyloid cascade hypothesis was the assumption that neuronal death in AD is a consequence of insoluble deposits of large amyloid fibrils. Indeed, some of the earlier studies clearly demonstrated the need for Aβ aggregation to induce toxicity,5, 6 and it was assumed that, since amyloid fibrils were present, these insoluble aggregates caused toxicity. This contradicted earlier studies showing only a weak correlation between the severity of dementia and the density of fibrillar amyloid plaques.7, 8, 9 Studies in transgenic models showed similarly that recovery of memory loss following passive immunization with antibodies against Aβ did not reduce amyloid plaque burden in these animals,10, 11 indicating that while memory loss was Aβ-dependent it was not dependent on amyloid plaque burden. Other studies showed that ApoJ could block assembly of Aβ into fibrillar aggregates; however, the mixture was still toxic to a PC12 cell line12,13 due to the presence of small globular proteins, referred to as Aβ-derived diffusible ligands (ADDLs).14

Analysis of samples extracted from frontal cortex and putamen in PBS from AD brain indicated variable proportions of monomers, dimers and trimers of Aβ,15 and numerous reports of SDS-stable dimers and trimers in the soluble fraction of human brain and in extracts of amyloid plaques15, 16, 17, 18 suggested that SDS-stable low-n oligomers of Aβ are the fundamental units of amyloid deposits. SDS-stable low-n oligomers (dimers, trimers and tetramers) were shown to block hippocampal LTP in rats, an effect that was reversed by anti-Aβ antibody, while pre-incubation with insulin-degrading enzyme to deplete monomers and not oligomers did not prevent the inhibition of LTP.19 In a related study, these low-n oligomers also caused impairment of short-term memory in animals, one of the earliest symptom associated with AD.20 Recently, it has been shown that these same oligomers can affect dendritic morphology in neuronal cells by causing synaptic losses.21 Together, these studies clearly suggest that low-n Aβ oligomers may be important mediators in synaptic dysfunction in early AD. The presence of a higher molecular mass dodecameric Aβ form was correlated with spatial memory performance in a Tg2576 APP transgenic mouse model;22 however, the same Tg2576 mice showed other impairments long before the first apparent detection of dodecamer species.23, 24 Therefore, while the dodecamer form may account for the spatial memory impairment in Tg2576 mice, other impairments are likely caused by other lower-n oligomers.25 Various non-fibrillar Aβ preparations can alter neuronal architecture, and cause changes in axonal transport.26, 27, 28, 29, 30 Therefore, the various studies using transgenic mouse models, human brain, synthetic Aβ peptide, and Aβ containing cell culture medium show multiple forms of non-fibrillar Aβ can cause toxicity in a variety of different ways.

Immunotherapy using both active and passive immunization regimens against Aβ has shown promise as an effective therapeutic strategy in treating AD. Active immunization strategies in transgenic mice models of AD showed a reduction in Aβ deposits in the brain,31, 32 and improvement in memory and learning deficits,33, 34 Human clinical trials were initiated on the basis of the initial success reported in animal models; however, Phase 2 trials were halted when several patients developed meningoencephalitis.31, 35, 36 Nonetheless, the follow-up reports of these trials showed some promise, as they revealed that patients with higher antibody titers to plaques showed a slower rate of cognitive decline than the patients who did not develop antibodies.37, Passive immunization of PDAPP and other transgenic mouse models38, 39, 40 showed improvement from memory deficits without reducing the plaque burden in the brain. Reduction in memory deficits without changing the amyloid plaque burden may reflect the fact that the antibodies reduce the amount of small, soluble oligomeric species by various mechanisms.

Single-chain variable domain (ScFv) antibodies represent an alternative therapeutic approach that can avoid the inflammatory responses seen with conventional antibodies. We showed earlier that scFvs produced against both the full-length peptide and a fragment of Aβ were able to inhibit Aβ-induced toxicity towards neuronal cells.41, 42. We recently developed a novel atomic force microscopy (AFM)-based biopanning protocol that allowed us to isolate scFv antibody fragments that specifically recognize either oligomeric or fibrillar α-synuclein.43, 44 Here, we used a similar method to produce scFv antibodies against oligomeric Aβ. We demonstrate by ELISA and dot blot analysis that the scFv binds only to oligomeric and not monomeric or fibrillar Aβ forms. The scFv was able to inhibit Aβ42 aggregation and fibril formation, as well as reduce oligomer-induced toxicity towards human neuroblastoma SH-SY5Y cells. The scFv also reacted with oligomeric Aβ aggregates contained in human AD brain tissue, but not healthy brain or Parkinson's disease (PD) brain tissue.

Section snippets

Biopanning against oligomeric Aβ

Oligomeric Aβ42 was deposited on mica and incubated with the Tomlinson I and J libraries. Before panning, the aggregate morphology of Aβ was verified by AFM (Fig. 1). Eluted phage titers showed an increase after each of the three rounds of panning, indicating successful enrichment. Polyclonal phage ELISA against oligomeric Aβ42 (Fig. 1b) also showed an increase in binding after each round of panning. Eluted phage from round 3 was used to infect Escherichia coli TG1, and 48 individual clones

Discussion

There is substantial and compelling evidence to suggest that oligomeric forms of Aβ are the pathologically significant and stable species in AD.14, 19, 45, 46, 47 This evidence has led to two significant modifications in the original amyloid cascade hypothesis:48 (1) early memory loss is due to synaptic dysfunction and not necessarily caused by neuronal death; and (2) synaptic failure is attributed to oligomeric, and not fibrillar, Aβ. The hypothesis that Aβ oligomers cause most toxicity in AD

Phage display scFv library

The Tomlinson I and J phage display scFv libraries were obtained from the MRC Laboratory of Molecular Biology and the MRC Center for Protein Engineering (Cambridge, UK). Each library comprises over 100 × 106 different scFv fragments cloned in an ampicillin-resistant phagemid vector and transformed into TG1 E. coli cells. ScFv fragments comprise a single polypeptide with the VH and VL domains attached to each other by a flexible glycine-serine linker.

Production of Aβ Oligomers and fibrils

Aβ40 and Aβ42 (Biosource, USA) were dissolved

Acknowledgements

This work was supported by grants from the Arizona Department of Health Services for the Arizona Alzheimer's Consortium and the Arizona Biomedical Research Commission and the American Health Assistance Foundation. We thank Min Wang for technical help with AFM, and Philip Schulz for technical assistance.

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