Abstract
The mechanisms of amyloid-β (Aβ)-degradation and clearance in Alzheimer’s disease (AD) pathogenesis have been relatively little studied. Short Aβ-fragments form by enzymatic cleavage and alternate amyloid-beta precursor protein (APP)-processing. Here we characterized a novel polyclonal Aβ-antibody raised against an Aβ mid-domain and used it to investigate microglial Aβ-uptake in situ by microscopy at the light- and ultrastructural levels. The rabbit Aβ-mid-domain antibody (ab338), raised against the mid-domain amino acids 21–34 (Aβ21–34), was characterized with biochemical and histological techniques. To identify the epitope in Aβ recognized by ab338, solid phase and solution binding data were compared with peptide folding scores as calculated with the Tango software. The ab338 antibody displayed high average affinity (KD: 6.2 × 10−10 M) and showed preference for C-terminal truncated Aβ-peptides ending at amino acid 34 and Aβ-mid domain peptides with high scores of β-turn structure. In transgenic APP-mouse brain, ab338 labelled amyloid plaques and detected Aβ-fragments in microglia at the ultra- and light microscopic levels. This reinforces a role of microglia/macrophages in Aβ-clearance in vivo. The ab338 antibody might be a valuable tool to study Aβ-clearance by microglial uptake and Aβ-mid-domain peptides generated by enzymatic degradation and alternate production.
Highlights
Alzheimer’s disease (AD), the major cause of dementia[1], presents with cerebral region-specific neuropathological lesions; extracellular amyloid-β (Aβ) plaques and intracellular neurofibrillary tangles (NFTs)[2]
To examine the ab[338] epitope the antigen Aβ21–34, as well as Aß-peptides non- and partly overlapping with Aβ21–34, and scrambled Aβ1–42 were used as coat in an indirect enzyme-linked immunosorbent assay (ELISA)
The data are presented on a relative scale with binding to the Aβ21–34 peptide defined as 100% and vehicle-coat as 0%
Summary
Alzheimer’s disease (AD), the major cause of dementia[1], presents with cerebral region-specific neuropathological lesions; extracellular amyloid-β (Aβ) plaques and intracellular neurofibrillary tangles (NFTs)[2]. The β-site APP cleaving enzyme 1 (BACE1 known as Asp2) has been identified as the β-secretase[10,11]. Following serial BACE1 and γ-secretase cleavage, Aβ1–38, Aβ1–40 and Aβ1–42 (Aβ38, Aβ40, Aβ42) are released, and shorter and longer peptides like Aβ1–37, Aβ1–39 and Aβ1–43 since γ-secretase cuts by complex enzymatic m echanisms[9]. Initial α-site cleavage in the Aβ mid-region (aa 16–17)[16] generates N-truncated peptides, precluding aggregation, and amyloidogenic APP-processing due to BACE1 and subsequent γ-secretase cleavage. While Aβ-peptides are released upon synaptic activity[19,20], Aβ-clearance mechanisms include drainage with exit across the blood brain barrier[21,22] and enzymatic breakdown by multiple enzymes. At intra- and extracellular localizations these enzymes can by cleavage reduce the potential of Aβ-peptides to aggregate. The inclusion of multiple familial AD mutations in such mouse models may artificially affect Aβ-composition and downstream responses, including microglial activation
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