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Extracellular Vesicles of Alzheimer’s Disease Patients as a Biomarker for Disease Progression

Molecular Neurobiology volume 57, pages 4156–4169 (2020)Cite this article

Abstract

Alzheimer’s disease (AD) is a progressive neurodegenerative brain pathology and the most common form of dementia. Evidence suggests that extracellular vesicles (EVs) containing cytokines and microRNA are involved in inflammation regulation. The current study aimed to explore a potential impact of AD patients’ EVs on disease progression. Blood samples were collected after obtaining signed informed consent (No. 0462-14-RMB) from 42 AD patients at three stages of disease severity and from 19 healthy controls (HC). EV size and concentration were studied by nanotracking analysis. EV membrane antigens were defined by flow cytometry and Western blot; EV protein contents were screened by protein array; the miRNA content was screened by nanostring technology and validated by RT-PCR. HC and AD patients’ EVs consisted of a mixture of small (< 100 nm) and larger vesicles. The myelin oligodendrocyte glycoprotein (MOG) expression on EVs correlated with disease severity. EVs of patients with moderate and severe AD had significantly higher levels of MOG, compared with mild AD patients. Levels of EVs expressing the axonal glycoprotein CD171 were significantly higher in severe AD patients than in HC. Increase in endothelial EVs was observed in AD patients. An above twofold increase was found in the content of inflammatory cytokines and > 50% decrease in growth factors in AD patients’ EVs compared with HC-EVs. Levels of let-7g-5p, miR126-3p, miR142-3p, miR-146a-5p, and mir223-3p correlated with disease severity. Neural damage, specific miRNA downregulation, and inflammatory cytokine upregulation, found in patients’ EVs, might be used as a biomarker reflecting AD severity.

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Author notes

  1. Benjamin Brenner and Judith Aharon-Peretz contributed equally to this work.

Affiliations

  1. Tel Aviv Sourasky Medical Center, Tel-Aviv, Israel

    Anat Aharon & Rawan Sayed Ahmad

  2. Department of Hematology and Bone Marrow Transplantation, Rambam Health Care Campus, Haifa, Israel

    Anat Aharon, Annie Sabbach & Benjamin Brenner

  3. Bruce Rappaport Faculty of Medicine, Technion - Israel Institute of Technology, Haifa, Israel

    Anat Aharon, Benjamin Brenner & Judith Aharon-Peretz

  4. Cognitive Neurology Unit, Rambam Health Care Campus, Haifa, Israel

    Polina Spector, Nizar Horrany & Judith Aharon-Peretz

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  1. Anat Aharon
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Correspondence to Anat Aharon.

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The study was approved by the Institutional Review Board of the Rambam Health Care Campus, Haifa, Israel (Approval No. 0462-14-RMB). The study participants were recruited after signing an informed consent form

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Electronic Supplementary Material

Western blot EV-pellet (isolated from 300 μl PPP) was loaded and separated on 12% acrylamide gel and then transferred to immune blot-PVDF membrane (Bio-Rad, Herculs CA, USA). This was followed by immunoblotting with the appropriate antibodies: mouse monoclonal anti human-CD81 and CD63 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or with anti-calnexin (abcam). After incubation with the primary antibody, the membranes were washed and incubated with horseradish peroxidase (HRP) conjugated secondary antibodies (Cell Signaling Technology, Massachusetts, USA). (Santa Cruz). Then, a chemiluminescence kit (EZ-ECL, Biological industrial, Israel) was used to detect the fluorescence. The western blot (WB) assay results were quantified using myECL™ Imager and analyzed using MyImageAnalysis Software (both from Thermo Fisher Scientific, Waltham, MA USA).

ESM 1

Representative gels images with molecular weight marker (MWM) and representative samples of each study cohort (a). and graphs summarizing the bend density of CD63 (c2) and CD81 (c3). (JPG 595 kb)

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Aharon, A., Spector, P., Ahmad, R.S. et al. Extracellular Vesicles of Alzheimer’s Disease Patients as a Biomarker for Disease Progression. Mol Neurobiol 57, 4156–4169 (2020). https://doi.org/10.1007/s12035-020-02013-1

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Keywords

  • Alzheimer’s disease (AD)
  • Extracellular vesicles (EVs)
  • Cytokines
  • MicroRNA (miRNA)