Elsevier

Neurobiology of Aging

Volume 33, Issue 7, July 2012, Pages 1320-1328
Neurobiology of Aging

Regular paper
Nanobody specific for oligomeric beta-amyloid stabilizes nontoxic form

https://doi.org/10.1016/j.neurobiolaging.2010.09.020Get rights and content

Abstract

While accumulation and deposition of beta amyloid (Aβ) is a primary pathological feature of Alzheimer's disease (AD), increasing evidence has implicated small, soluble oligomeric aggregates of Aβ as the neurotoxic species in AD. Reagents that specifically recognize oligomeric morphologies of Aβ have potential diagnostic and therapeutic value. Using a novel biopanning technique that combines phage display technology and atomic force microscopy, we isolated the nanobody E1 against oligomeric Aβ. Here we show that E1 specifically recognizes a small oligomeric Aβ aggregate species distinct from the species recognized by the A4 nanobody previously reported by our group. While E1, like A4, blocks assembly of Aβ into larger oligomeric and fibrillar forms and prevents any Aβ induced toxicity toward neuronal cells, it does so by binding a small Aβ oligomeric species, directing its assembly toward a stable nontoxic conformation. The E1 nanobody selectively recognizes naturally occurring Aβ aggregates produced in human AD brain tissue indicating that a variety of morphologically distinct Aβ aggregate forms occur naturally and that a stable low-n nontoxic Aβ form exists that does not readily aggregate into larger forms. Because E1 catalyses the formation of a stable nontoxic low-n Aβ species it has potential value as a therapeutic reagent for AD which can be used in combination with other therapeutic approaches.

Introduction

Alzheimer's disease (AD) is a common neurodegenerative disorder of the elderly, affecting more than 5 million people in the USA alone at an annual estimated cost of 100 billion dollars per year (Rafii and Aisen, 2009). AD is characterized by the presence of extracellular amyloid deposits and intracellular neurofibrillary tangles resulting in a progressive loss of cognitive abilities and dementia (Hardy and Selkoe, 2002). The primary constituent of the amyloid plaques is the beta-amyloid peptide (Aβ) (Glenner and Wong, 1984).

The amyloid cascade hypothesis of AD suggests that progressive aggregation of Aβ is the critical event leading to progressive neurodegeneration and eventually AD (Hardy and Higgins, 1992, Selkoe, 1991). Increasing evidence suggests that soluble oligomeric species are the critical toxic components in AD toxicity (Haass and Selkoe, 2007, Klein et al., 2001). Sodium dodecyl sulfate (SDS)-stable low-n oligomers of Aβ block hippocampal long term potentiation and cause impairment of short term memory in animals (Lambert et al., 1998, Wang et al., 2002). Soluble Aβ oligomers extracted from Alzheimer's disease brains were also shown to potently impair synapse structure (Shankar et al., 2008).

Numerous strategies to control Aβ production and aggregation and to decrease Aβ-induced toxicity in animal models of AD have been studied (Wolfe, 2002). Both active and passive immunotherapies targeting Aβ have shown promise in animal models of AD (Lambert et al., 2001, Lambert et al., 2007, Lee et al., 2006). Clinical trials using active immunization with aggregated Aβ also showed promise, as patients with higher antibody titers to Aβ showed a slower rate of cognitive decline than the patients who did not develop antibodies (Hock et al., 2003), however, the trials were halted due to incidences of meningioencephalitis (Birmingham and Frantz, 2002, Orgogozo et al., 2003, Schenk, 2002).

Passive immunization using antibody fragments lacking the Fc region that do not initiate an immune response have advantages over active immunization strategies for a range of in vivo applications due to their small size, defined specificity, lower cross-reactivity due to the absence of constant regions, and their short residence time in the system (Adams et al., 1993, Colcher et al., 1990, Huston et al., 1993, Milenic et al., 1991, Yokota et al., 1992). Selective targeting of Aβ using antibody fragments or nanobodies such as single chain antibody variable domains (scFv) has been shown to significantly retard amyloid deposition in the brains of rats (Levites et al., 2006). Because oligomeric forms of Aβ induce toxicity and memory loss in AD, isolation of nanobodies that can selectively target specific Aβ oligomeric morphologies and block neurotoxicity have potential value as diagnostics and therapeutics for AD. Stabilization of an Aβ form that does not aggregate into higher, more toxic morphologies has been suggested as a valuable therapeutic option for AD (Ono et al., 2009).

Recently, we identified an anti-oligomer nanobody, A4, isolated by an atomic force microscope (AFM)-based biopanning technique that inhibited aggregation and extracellular toxicity of Aβ in vitro (Zameer et al., 2008). Here we describe a second anti-oligomeric nanobody, E1, that binds a smaller earlier stage oligomeric form of Aβ than the previously reported A4, indicating that multiple morphologically distinct oligomeric Aβ forms can naturally occur. Similar to A4, E1 does not bind monomeric or fibrillar Aβ forms and inhibits Aβ aggregation and fibril formation, however the E1 nanobody stabilizes formation of a small nontoxic low-n Aβ species, whereas A4 binds a larger oligomeric form. The E1 nanobody reacts with oligomeric Aβ contained in brain tissue from human AD brains but not age-matched nondemented brain tissue. When administered extracellularly, the E1 nanobody decreases intrinsic toxicity in SH-SY5Y neuroblastoma cells. Because E1 stabilizes a low-n oligomeric Aβ form, but does not react with monomeric, larger oligomeric, or fibrillar forms, E1 has potential value as an early diagnostic tool to detect low-n Aβ aggregates, as well as in therapeutic applications to neutralize Aβ induced toxicity.

Section snippets

Isolation of E1 nanobody

E1 was isolated against oligomeric Aβ from a diverse phage displayed scFv library using a novel AFM-based biopanning technology as previously described (Zameer et al., 2008). E1 plasmid DNA from an E. coli TG1 stock clone was isolated and then transformed into the nonsuppressor E. coli HB2151 cell line for production of soluble nanobody.

Expression and purification of soluble nanobody

E1 and A4 nanobodies were produced and purified essentially as previously described (Zameer et al., 2008). The supernatant and cell lysate from a 1 L culture

Expression and purification of soluble nanobody

E1 was previously isolated by AFM-based biopanning against oligomeric Aβ and based on preliminary phage enzyme-linked immunosorbent assay (ELISA) results bound to oligomeric but not monomeric or fibrillar Aβ (Zameer et al., 2008). To further study E1 specificity, soluble protein was produced from the nonsuppressor HB2151 host cells, and purified to homogeneity as indicated by a single protein band with a molecular mass of 29 kDa, corresponding to expression of a full-length scFv on both sodium

Discussion

Aggregation and deposition of Aβ play a critical role in neurodegeneration in AD and numerous therapeutic strategies targeting Aβ are being actively pursued including clearance of existing aggregates from the brain, decreasing the production of Aβ, inhibiting the initial conformational change of Aβ to its pathological β-pleated sheet form, and blocking the formation of Aβ oligomers (LeVine, 2002, LeVine, 2007, Look et al., 2007). While these different strategies have shown promise in cell,

Disclosure statement

There are no conflicts of interest with this report.

Acknowledgements

This work was supported by grants from the American Health Assistance Foundation, the Arizona Biomedical Research Commission, and the Arizona Department of Health Services for the Arizona Alzheimer's Consortium. We are grateful to the Sun Health Research Institute Brain Donation Program of Sun City, Arizona for the provision of human brain tissue. The Brain Donation Program is supported by the National Institute on Aging (P30 AG19610 Arizona Alzheimer's Disease Core Center), the Arizona

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