N-Truncated Aβ2-X Starting with Position Two in Sporadic Alzheimer’s Disease Cases and Two Alzheimer Mouse Models

Abstract

According to the modified amyloid hypothesis, the key event in the pathogenesis of Alzheimer's disease (AD) is the deposition of neurotoxic amyloid β-peptides (Aβs) in plaques and cerebral blood vessels. Additionally to full-length peptides, a great diversity of N-truncated Aβ variants is derived from the larger amyloid-β protein precursor (AβPP). Vast evidence suggests that Aβx-42 isoforms play an important role in triggering neurodegeneration due to their high abundance, amyloidogenic propensity and toxicity. Although N-truncated Aβ peptides and Aβx-42 species appear to be the crucial players in AD etiology, the Aβ2-X isoforms did not receive much attention yet. The present study is the first to show immunohistochemical evidence of Aβ2-X in cases of AD and its distribution in AβPP/PS1KI and 5XFAD transgenic mouse models using a novel antibody pAB77 that has been developed using Aβ2-14 as antigen. Positive plaques and congophilic amyloid angiopathy (CAA) were observed in AD cases and in both mouse models. While in AD cases, abundant CAA and less pronounced plaque pathology was evident, the two mouse models showed predominantly extracellular Aβ deposits and minor CAA staining. Western blotting and a capillary isoelectric focusing immunoassay demonstrated the high specificity of the antibody pAb77 against Aβ-variants starting with the N-terminal Alanine-2.

N-truncated Aβ4–x peptides in sporadic Alzheimer’s disease cases and transgenic Alzheimer mouse models

“…Furthermore, elongated amyloidogenic Aβ 1–43 peptides have recently been reported [ 6 ]. Similar heterogeneity has been observed at the Aβ N-terminus, and several N-terminally truncated Aβ forms, including Aβ 2–x [ 7 ], Aβ pE3–x [ 8 , 9 ], Aβ 5–x [ 10 , 11 ], and Aβ pE11–x [ 9 , 12 , 13 ], have been demonstrated in parenchymal or vascular deposits of human AD brains by immunohistochemical methods. An even more complex pattern of Aβ peptides has been reported by the use of mass spectrometry [ 14 – 17 ], which detected further variants such as Aβ 7–42 , Aβ 8–42 , or Aβ 9–42 .…”

“…For SDS-PAGE analysis, stock solutions of synthetic Aβ 1–40 , Aβ 2–40 , Aβ 3–40 , pyroglutamate Aβ 3–40 (Aβ pE3–40 ), and Aβ 4–40 were prepared in sample buffer (0.36 M bis-tris, 0.16 M bicine, 15% wt/vol sucrose, 1% wt/vol SDS, and 0.0075% wt/vol bromophenol blue) and stored at −80 °C. The Aβ peptides were further diluted in sample buffer, and aliquots containing 0.1, 1, and 10 ng of peptide per lane were separated by urea-bicine/bis-tris/Tris/sulfate SDS-PAGE and analyzed by immunoblotting as previously described [ 7 ]. The blotting membranes were blocked in 2% electrochemiluminescence (ECL) advance blocking agent (GE Healthcare Life Sciences, Little Chalfont, UK) in PBS with 0.1% Tween 20 (PBS-T) for at least 4 h at room temperature (RT) and probed with the primary antibody 029-2 (1:300 in PBS-T) at 4 °C overnight.…”

N-Terminally Truncated Aß Peptide Variants in Alzheimer’s Disease

“…an Aβ 2-x -specific polyclonal antibody confirmed the presence of Aβ 2-x peptides in both parenchymal and vascular deposits of sporadic AD cases as well as transgenic mouse models such as APP/PS1KI or 5XFAD (37). As the sequence of fulllength Aβ starts with an Asp residue in position 1, it has been suggested that proteolysis of Aβ 1-x peptides by the exopeptidase aminopeptidase A, which releases Glu and Asp residues from the N-termini of proteins, could result in the generation of Aβ 2-x species (38).…”

Deposition of C-terminally truncated Aβ species Aβ37 and Aβ39 in Alzheimer’s disease and transgenic mouse models

“…One-dimensional urea-Bicine/Bis-Tris/Tris/sulfate SDS-PAGE (1D-Aβ-PAGE) was carried out as described previously [ 45 ]. Protein extracts were prepared from 12-month-old WT and 5XFAD mouse brain hemispheres by sequential extraction with Tris-buffered saline (TBS, 120 mM NaCl, 50 mM Tris, pH7.5) and 2 % sodium dodecyl sulfate (SDS).…”

“…Following centrifugation for 20 min at 17000 x g, the resulting pellet was sonified in 2 % SDS and centrifuged for 20 min at 17000 x g. The protein concentrations of the supernatants were determined with the Roti-Quant protein assay (Carl Roth). Aβ peptide immunoprecipitation was carried out with mAb6E10 (Covance) as previously described [ 18 , 45 ]. For Western-immunoblot analysis, samples with a protein concentration of 2 mg/mL were prepared in electrophoresis sample buffer (final composition: 0.36 M Bistris, 0.16 M bicine, 15 % (w/v) sucrose, 1 % (w/v) SDS, 0,0075 % bromophenol blue).…”