Abstract
Alzheimer’s disease (AD) is the most common form of age-related dementia, and the most urgent problem is that it is currently incurable. Amyloid-β (Aβ) peptide is believed to play a major role in the pathogenesis of AD. We previously reported that an Aβ N-terminal amino acid targeting monoclonal antibody (MAb), A8, inhibits Aβ fibril formation and has potential as an immunotherapy for AD based on a mouse model. To further study the underlying mechanisms, we tested our hypothesis that the single chain fragment variable (scFv) without the Fc fragment is capable of regulating either Aβ aggregation or disaggregation in vitro. Here, a model of cell-free Aβ “on-pathway” aggregation was established and identified using PCR, Western blot, ELISA, transmission electron microscopy (TEM) and thioflavin T (ThT) binding analyses. His-tagged A8 scFvs was cloned and solubly expressed in baculovirus. Our data demonstrated that the Ni-NTA agarose affinity-purified A8 scFv inhibited the forward reaction of “on-pathway” aggregation and Aβ fibril maturation. The effect of A8 scFv on Aβ fibrillogenesis was markedly more significant when administered at the start of the Aβ folding reaction. Furthermore, the results also showed that pre-formed Aβ fibrils could be disaggregated via incubation with purified A8 scFv, which suggested that A8 scFv is involved in the reverse reaction of Aβ aggregation. Therefore, A8 scFv was capable of both inhibiting fibrillogenesis and disaggregating matured fibrils. Our present study provides valuable insight into the regulators of ultrastructural dynamics of cell-free “on-pathway” Aβ aggregation and will assist in the development of therapeutic strategies for AD.
Highlights
Alzheimer’s disease (AD) is the most common and refractory neurodegenerative disease and has led to a huge socioeconomic and humanistic problem [1]
To treat the Aβ aggregation system with the single chain fragment variable (scFv) expressed in eukaryotic cells, we attempted to construct a rBacmid encoding anti-Aβ scFv in the baculovirus expression system in which the viruses were packaged in Sf9 cells
In this study, a scFv derived from an anti-Aβ monoclonal antibody (MAb) was successfully expressed in baculovirus and was shown to recognize Aβ42 and affect the regulation of the ultrastructural dynamics of on-pathway Aβ aggregation and disaggregation
Summary
Alzheimer’s disease (AD) is the most common and refractory neurodegenerative disease and has led to a huge socioeconomic and humanistic problem [1]. The pathological features of AD include progressive neuronal loss, the formation of beta amyloid (Aβ) plaques called senile plaques (SPs), and the accumulation of tau protein neurofibrillary tangles [2]. Inhibiton of Aβ Fibril Aggregation and Promotion of Disaggregation from amyloid β precursor protein (APP) through its cleavage by β- and γ- secretases in the amyloidogenesis pathway and is expressed normally and ubiquitously as a peptide consisting of 39–43 residues during the trafficking and turnover process [2,3]. During the process of fibrillogenesis, Aβ forms oligomers, protofibrils and fibrils that are deposited in the extracellular area of the brain as “amyloid plaques” [4]. Aβ fibrillogenesis may directly contribute to the initiation and progression of AD pathogenesis according to the “amyloid cascade hypothesis” [5], the mechanisms of AD pathogenesis are poorly understood. Studies investigating Aβ polymerization at the molecular level, the ultrastructural characteristics of Aβ fibrils, and the effects of endogenous and exogenous intervention in Aβ fibrillation will facilitate the design of Aβ aggregation inhibitors
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