The Role of Neutrophil Proteins on the Amyloid Beta-RAGE Axis

We would like to thank Dr. Arthur Owora, previously a Research Biostatistician of the Department of Pharmaceutical Sciences, University of Oklahoma Health Sciences Center, for his assistance on the statistical analysis performed in this study. We thank Dr. Sixia Chen of the Department of Biostatistics and Epidemiogy, University of Oklahoma Health Sciences Center, for his additional input on the statistical analysis. We thank the Laboratory for Molecular Biology and Cytometry Research at the University of Oklahoma Health Sciences Center for the use of the Core Facility which allowed us to perform the MALDI-TOF MS and MS/MS experiments. GM-0111 was provided as a gift by Dr. Justin Savage, GlycoMira Therapeutics, Inc.We previously showed an elevated expression of the neutrophil protein, cationic antimicrobial protein of 37kDa (CAP37), in brains of patients with Alzheimer’s disease (AD), suggesting that CAP37 could be involved in AD pathogenesis. The first step in determining how CAP37 might contribute to AD pathogenesis was to identify the receptor through which it induces cell responses. To identify a putative receptor, we performed GAMMA analysis to determine genes that positively correlated with CAP37 in terms of expression. Positive correlations with ligands for the receptor for advanced glycation end products (RAGE) were observed. Additionally, CAP37 expression positively correlated with two other neutrophil proteins, neutrophil elastase and cathepsin G. Enzyme-linked immunosorbent assays (ELISAs) demonstrated an interaction between CAP37, neutrophil elastase, and cathepsin G with RAGE. Amyloid beta 1–42 (Aβ1–42), a known RAGE ligand, accumulates in AD brains and interacts with RAGE, contributing to Aβ1–42 neurotoxicity. We questioned whether the binding of CAP37, neutrophil elastase and/or cathepsin G to RAGE could interfere with Aβ1–42 binding to RAGE. Using ELISAs, we determined that CAP37 and neutrophil elastase inhibited binding of Aβ1–42 to RAGE, and this effect was reversed by protease inhibitors in the case of neutrophil elastase. Since neutrophil elastase and cathepsin G have enzymatic activity, mass spectrometry was performed to determine the proteolytic activity of all three neutrophil proteins on Aβ1–42. All three neutrophil proteins bound to Aβ1–42 with different affinities and cleaved Aβ1–42 with different kinetics and substrate specificities. We posit that these neutrophil proteins could modulate neurotoxicity in AD by cleaving Aβ1–42 and influencing the Aβ1–42 –RAGE interaction. Further studies will be required to determine the biological significance of these effects and their relevance in neurodegenerative diseases such as AD. Our findings identify a novel area of study that underscores the importance of neutrophils and neutrophil proteins in neuroinflammatory diseases such as AD.Yeshttp://www.plosone.org/static/editorial#pee

Selecting Multitarget Peptides for Alzheimer’s Disease

Alzheimer’s disease (AD) is a multifactorial disease with a complex pathogenesis. Developing multitarget drugs could be a powerful strategy to impact the progressive loss of cognitive functions in this disease. The purpose of this study is to select a multitarget lead peptide candidate among a series of peptide variants derived from the neutrophil granule protein cathepsin G. We screened eight peptide candidates using the following criteria: (1) Inhibition and reversion of amyloid beta (Aβ) oligomers, quantified using an enzyme-linked immunosorbent assay (ELISA); (2) direct binding of peptide candidates to the human receptor for advanced glycation end-products (RAGE), the Toll-like receptor 4 (TLR4) and the S100 calcium-binding protein A9 (S100A9), quantified by ELISA; (3) protection against Aβ oligomer-induced neuronal cell death, using trypan blue to measure cell death in a murine neuronal cell line; (4) inhibition of TLR4 activation by S100A9, using a human TLR4 reporter cell line. We selected a 27-mer lead peptide that fulfilled these four criteria. This lead peptide is a privileged structure that displays inherent multitarget activity. This peptide is expected to significantly impact cognitive decline in mouse models of Alzheimer’s disease, by targeting both neuroinflammation and neurodegeneration

Neutrophil proteins inhibit Aβ_1–42 binding to RAGE.

<p>(a) Graph shows binding of Aβ_1–42 to RAGE when CAP37 was added simultaneously to wells at increasing concentrations in the presence (inverted triangles) or absence (squares) of protease inhibitors. An IC₅₀ value of 1.28 μM was computed for inhibition of Aβ_1–42 binding to RAGE by CAP37. Two separate Kruskal-Wallis tests were performed for Aβ_1–42 binding in the presence and absence of protease inhibitors. A Dunn’s multiple comparisons test was performed for each separate Kruskal-Wallis test to compare binding of Aβ_1–42 to RAGE in the presence of increasing concentrations of CAP37. Data are mean ± SEM of results. *p<0.05, **p<0.01. (b) Graph shows binding of Aβ_1–42 to RAGE when neutrophil elastase was added simultaneously to wells at increasing concentrations in the presence (inverted triangles) or absence (squares) of protease inhibitors. An IC₅₀ value of 4.85 nM was computed for inhibition of Aβ_1–42 binding to RAGE by neutrophil elastase in the absence of protease inhibitors. Two separate Kruskal-Wallis tests were performed for Aβ_1–42 binding in the presence and absence of protease inhibitors. A Dunn’s multiple comparisons test was performed for each separate Kruskal-Wallis test to compare binding of Aβ_1–42 to RAGE in the presence of increasing concentrations of neutrophil elastase. No inhibition occurred in the presence of protease inhibitors. Data are mean ± SEM of results. *p<0.05.</p

RAGE binding to Aβ_1–42, CAP37, neutrophil elastase, and cathepsin G.

<p>RAGE binding to Aβ_1–42, CAP37, neutrophil elastase, and cathepsin G.</p

Proposed model for the modulation of RAGE signaling by CAP37, neutrophil elastase, and cathepsin G.

<p>(a) CAP37, neutrophil elastase (NE), and/or cathepsin G (CG) may act as RAGE agonists to induce a cell signaling cascade that leads to activation of the transcription factor NF-κB. Activation of NF-κB could then stimulate the production of pro-inflammatory cytokines and pro-oxidants including reactive oxygen species (ROS) and nitric oxide (NO) as well as RAGE itself. In this way, the neutrophil proteins would likely act as foes in the course of chronic inflammatory disease such as AD. (b) CAP37, neutrophil elastase, and cathepsin G may prevent the Aβ_1-42-RAGE interaction and the corresponding NF-κB signaling cascade. In this way, the neutrophil proteins would act as friends to prevent chronic neuroinflammation driven by Aβ_1–42 activation of RAGE.</p

Neutrophil elastase rapidly cleaves Aβ_1–42.

<p>(a) MALDI-TOF spectra of Aβ_1–42 incubated alone (red) or with neutrophil elastase (green) at t≈0.25 min. Peaks are labeled with mass-to-charge ratios (m/z), and mass is in Daltons (Da). A peak of 4500 Da, which represents intact Aβ_1–42, appears in all Aβ_1–42 spectra. Two fragment peaks at 3616 Da and 3505 Da are already present after incubation with neutrophil elastase at t≈0.25 min. Neutrophil elastase incubated alone is shown in blue, and no peak at 4500 Da is exhibited by neutrophil elastase. (b) MALDI-TOF spectra at t = 60 min. Note the reduced size of the full Aβ_1–42 peak and the peaks at 3616 Da and 3605 Da and the presence of 5 additional fragment peaks at 2877 Da, 2168 Da, 2069 Da, 1425 Da, and 1355 Da when Aβ_1–42 incubated with neutrophil elastase (green spectra). (c) Graph shows % relative peak intensity of Aβ_1–42 and its fragments (intact Aβ_1–42 + Aβ_1–42 fragments) analyzed at t≈ 0.25 min, t = 5 min, t = 15 min, t = 30 min, and t = 60 min. Data are representative of 3 independent experiments.</p