N-Terminally Truncated and Pyroglutamate-Modified Aβ Forms Are Measurable in Human Cerebrospinal Fluid and Are Potential Markers of Disease Progression in Alzheimer’s Disease

Abstract

Alzheimer’s disease (AD) is a pathology characterized by the accumulation in the brain of intracellular and extracellular amyloid-β (Aβ) aggregates, especially of Aβ1–40 and Aβ1–42 peptides. It is known that N-terminally truncated or modified Aβ forms also exist in AD brains and cerebrospinal fluid (CSF), and they play a key role in the pathogenesis of the disease. Herein, we developed an antibody-free method based on Solid-Phase Extraction and Electrospray Ionization Liquid Chromatography Mass Spectrometry for the identification and quantitation in human CSF of Aβ isoforms. In human CSF, we could detect and quantify a panel of 19 Aβ isoforms, including N-terminally truncated and pyroglutamate-modified forms, never quantified before in CSF. Among these, we identified novel N-terminally truncated Aβ species: four bound to copper and two phosphorylated forms, which were found to be the most common proteoforms in human CSF along with Aβ1–40, Aβ3–40, and AβpE11–42. We tested the newly developed and validated method in a pilot study on CSF from elderly individuals with subjective memory complaints (SMCs, n = 9), mild cognitive impairment (MCI, n = 18), and AD (n = 15); along with Aβ1–42, five N-terminally truncated forms (Aβ11–40, Aβ3–42, AβpE11–42, AβpE3–40, and Aβ4–40 Cu2+) are altered in AD/MCI. Thus, we demonstrated that N-terminally truncated and pyroglutamate-modified Aβ can be quantified in human CSF, and five of them, along with Aβ1–42, are potential markers of AD progression. The described method could represent a useful tool for patients’ stratification and monitoring. Moreover, the newly identified Aβ CSF species might represent new potential therapeutic targets.

Why the Ala-His-His Peptide Is an Appropriate Scaffold to Remove and Redox Silence Copper Ions from the Alzheimer’s-Related Aβ Peptide

“…In other words, if ATCUN-based peptides are aimed to be used against Cu(Aβ)-induced ROS formation, the rate of Cu II extraction from Aβ matters and, as we demonstrated, such a kinetic parameter is highly dependent on the exact sequence of the ATCUN peptide chosen. It is here interesting to note that, among the N-terminally truncated forms of Aβ recently put forward [ 92 , 93 , 94 , 95 , 96 ], several of them possess such H 2 N-Xxx-His (3N, Aβ 5–x ) [ 97 , 98 ], H 2 N-Xxx-Zzz-His (4N, Aβ 4–x , [ 85 , 86 ] Aβ 11–x [ 86 ]), and H 2 N-Xxx-His-His (hybrid 3N/4N, Aβ 12–x [ 99 ]) binding sites. Based on our results, the cleavage at position 12 leading to the hybrid 3N/4N binding site may be the most beneficial with respect to Cu(Aβ 1–x )-induced ROS formation.…”

Interactions of neurokinin B with copper(ii) ions and their potential biological consequences

“…3.5 fM, is more than sufficient to withstand the CNS conditions. The presence of Cu( ii ) complexes of Aβ 4–40/42 peptides 52 in the cerebrospinal fluid serves as a reference ( K d ∼ 30 fM). 53 The significant loss of the Cu( ii ) affinity of NKB upon its binding to SDS makes NKB a potential Cu( ii ) delivery agent for the hCtr1 membrane receptor.…”

N-terminally truncated Aβ4-x proteoforms and their relevance for Alzheimer’s pathophysiology

“…In spite of the numerous studies on the mechanisms contributing to the brain removal of Aβ, no available information addresses the potential pathways participating in the elimination of truncated forms of the peptide. The recent discovery of N-terminally truncated forms in plasma, together with the demonstration of Aβ4-x as well as pyroglutamate-modified AβpE3-x and AβpE11-x in CSF, points out the ability of these forms to cross the blood–brain and the brain-CSF barriers [ 22 , 100 , 101 ].…”

“…These structural changes support a contribution of pE-modified species to the disease process as suggested by their exacerbated neurotoxicity [ 12 – 15 ] and their abundance in AD brains compared to cognitively intact age-matched controls [ 16 – 20 ]. Additional N- and C-terminal truncations that significantly contribute to the heterogeneity and complexity of the Aβ profile in biological fluids and brain deposits have been increasingly identified in recent years [ 21 , 22 ]. These multiple degradation products are likely generated by the action of a number of Aβ-degrading enzymes, among which are neprilysin, insulin-degrading and endothelin-converting enzymes, plasmin, ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4), and matrix metalloproteases [ 23 – 29 ].…”