Research Article: Human cerebrospinal fluid 6E10-immunoreactive protein species contain amyloid precursor protein fragments

Date Published: February 28, 2019

Publisher: Public Library of Science

Author(s): Marianne K. O. Grant, Maureen Handoko, Malgorzata Rozga, Gunnar Brinkmalm, Erik Portelius, Kaj Blennow, Karen H. Ashe, Kathleen R. Zahs, Peng Liu, Stephen D. Ginsberg.


In a previous study, we reported that levels of two types of protein species—a type of ~55-kDa species and a type of ~15-kDa species—are elevated in the lumbar cerebrospinal fluid (CSF) of cognitively intact elderly individuals who are at risk for Alzheimer’s disease (AD). These species are immunoreactive to the monoclonal antibody 6E10, which is directed against amino acids 6–10 of amyloid-β (Aβ), and their levels correlate with levels of total tau and tau phosphorylated at Thr181. In this study, we investigated the molecular composition of these AD-related proteins using immunoprecipitation (IP)/Western blotting coupled with IP/mass spectrometry. We show that canonical Aβ1-40/42 peptides, together with amyloid-β precursor protein (APP) fragments located N-terminally of Aβ, are present in the ~55-kDa, 6E10-immunoreactive species. We demonstrate that APP fragments located N-terminally of Aβ, plus the N-terminal region of Aβ, are present in the ~15-kDa, 6E10-immunoreactive species. These findings add to the catalog of AD-related Aβ/APP species found in CSF and should motivate further study to determine whether these species may serve as biomarkers of disease progression.

Partial Text

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that is pathologically characterized by extracellular amyloid plaques, intracellular neurofibrillary tangles and neuronal loss in selected brain regions. Amyloid-β (Aβ) peptides, the main component of plaques, are generated from sequential processing of amyloid precursor protein (APP) by β- and γ- secretase with Aβ1–40 being most abundant, while Aβ peptides ending at position 42 (Aβ42) are most hydrophobic and the major component of amyloid plaques [1]. The 1992 finding that Aβ peptides could be secreted to the cerebrospinal fluid (CSF) [2] stimulated more than two decades of study to determine whether CSF Aβ could serve as a biomarker to diagnose AD or monitor disease progression—research that is still ongoing. Using enzyme-linked immunosorbent assay (ELISA) to measure Aβ42, Motter et al. showed a significant reduction in peptide levels in CSF of AD patients [3], which has been reproduced later in multiple studies (for review, see for example [4, 5]). Aβ40, the most abundant Aβ variant present in CSF [6], shows similar levels in AD patients and age-matched cognitively normal individuals [7]. Since levels of Aβ40 can be used to balance inter-individual variations in total Aβ production, the use of the Aβ42/Aβ40 ratio has been reported to improve diagnostic accuracy [8, 9]; however, the reduction in CSF Aβ42 also occurs in patients with neurodegenerative disorders other than AD [10–12]. Although combining CSF Aβ biomarkers with amyloid neuroimaging biomarkers (e.g., PiB amyloid positron emission tomography) and other CSF biomarkers (e.g., total tau (t-tau) and phosphorylated tau (p-tau)) has proven to be useful for AD diagnosis, other molecular biomarkers are needed to enhance the sensitivity and specificity of diagnosis of various neurodegenerative disorders as well as to differentiate clinical phenotypes of AD.

The goal of this study was to further characterize 6E10-immunoreactive species that we had previously reported to be elevated in the CSF of elderly individuals at risk for AD [23]. Using IP/WB coupled with IP/MS, we show that canonical Aβ1-40/42 peptides, together with APP fragments located N-terminally of Aβ, are found in the protein species that migrate at ~55 kDa. We also show that APP fragments located N-terminally of Aβ, plus the N-terminal region of Aβ, are present in the ~15-kDa species.




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