Identification of Novel APP/Aβ Isoforms in Human Cerebrospinal Fluid

Abstract

Background: Aggregation of β-amyloid (Aβ) into oligomers and plaques is the central pathogenic mechanism in Alzheimer’s disease (AD). Aβ is produced from the amyloid precursor protein (APP) by β- and γ-secretases, whereas, in the nonamyloidogenic pathway, α-secretase cleaves within the Aβ sequence, and thus precludes Aβ formation. A lot of research has focused on Aβ production and the neurotoxic 42-amino-acid form of Aβ (Aβ1–42), while less is known about the nonamyloidogenic pathway and how Aβ is degraded. Objective: To study the Aβ metabolism in man by searching for novel Aβ peptides in cerebrospinal fluid (CSF). Methods: Immunoprecipitation, using an anti-Aβ antibody, 6E10, was combined with either matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or nanoflow liquid chromatography and tandem mass spectrometry. Results: We identified 12 truncated APP/Aβ peptides in the CSF, all of which end at amino acid 15 in the Aβ sequence, i.e. 1 amino acid before the proposed α-secretase site. Of these 12 APP/Aβ peptides, 11 are novel peptides and start N-terminally of the β-secretase site. The most abundant APP/Aβ peptide starts 25 amino acids before the β-secretase site, APP/Aβ (–25 to 15), and had a concentration of approximately 80 pg/ml. The identity of all the APP/Aβ peptides was verified in a cohort of AD patients and controls. A first pilot study also showed that the intensity of several APP/Aβ peaks in CSF was higher in AD cases than in controls. Conclusion: These data suggest an enzymatic activity that cleaves the precursor protein in a specific manner that may reflect a novel metabolic pathway for APP and Aβ.

Metalloprotease Meprin β Generates Nontoxic N-terminal Amyloid Precursor Protein Fragments in Vivo

“…Signals between 40 and 35 kDa were detected that are in the range of the cleavage sites identified by TAILS. We show that the meprin ␤-derived N-APP11 fragment identified by the 22C11 antibody and further analyzed by N-terminal sequencing is consistent with a peptide detected in human brain fluids of both AD patients and a control cohort (44). Based on mass spectrometry, this study led to the identification of six novel N-APP fragments starting with the mature N terminus and ending with various amino acids (119, 121-124, and 126).…”

“…Sequencing of this proteolytic APP695 fragment by Edman degradation revealed indeed the mature N terminus starting with the sequence LEVP. This, in combination with the identified cleavage site by degradomics (Ser 124 /Asp 125 ), is the exact peptide sequence recently detected in human brain lysates (44,45).…”

“…Independent from the amyloid hypothesis, this finding might prove a novel enzymatic mechanism, important for the physiological turnover of APP. Indeed, in our degradomics approach, we could identify meprin ␤-specific cleavage between Ser 124 and Asp 125 revealing the exact C terminus of the 11-kDa N-APP fragment observed in vivo (44). Interestingly, the authors could not detect the DR6 ligand in the cerebrospinal fluid (CSF), described by Nikolaev et al (23).…”

Site-specific characterization of threonine, serine, and tyrosine glycosylations of amyloid precursor protein/amyloid β-peptides in human cerebrospinal fluid

“…Analysis of fragment spectra (MS 2 ) induced by collision-induced dissociation (CID) confirmed the sequence of Aβ1-15, 1-16, 1-17, 1-40, and 1-42 (21,24). By specifically searching for glycosidic fragmentation patterns in the MS 2 and MS 3 (second generation fragment) spectra we were able to identify O-glycosylated Aβ1-15, 1-16, 1-17, 1-18, 1-19, and 1-20 (Aβ1-X series) as well as Aβ−3-15, 4-15, 4-17, and 5-17 peptides, also included in the Aβ1-X series, with an additional mass and structure corresponding to a Neu5AcHexðNeu5AcÞHexNAc-O-ðSA 2 Þ glycan within each of the peptides (Fig.…”

The 7th Leonard Berg Symposium, Part 2

“…Also at ICAD, Zetterberg presented his group's detection in CSF of AD patients of a set of truncated Aβ forms, as well as a set of AβPP fragments, both of which point to the existence of a new, yet-to-bedefined cleavage sequence of AβPP [26][27][28]. Beyond AβPP and Aβ species, a wealth of potential markers are being discovered.…”