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MiRNA Profiling in Plasma Neural-Derived Small Extracellular Vesicles from Patients with Alzheimer’s Disease

Serpente, Maria, Fenoglio, Chiara, D’Anca, Marianna, Arcaro, Marina, Sorrentino, Federica, Visconte, Caterina, Arighi, Andrea, Fumagalli, Giorgio G., Porretti, Laura, Cattaneo, Alessandra, Ciani, Miriam, Zanardini, Roberta, Benussi, Luisa, Ghidoni, Roberta, Scarpini, Elio, Galimberti, Daniela·2020·Cells
DOI 10.3390/cells9061443PMC PMC7349735
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Abstract

Small extracellular vesicles (EVs) are able to pass from the central nervous system (CNS) into peripheral blood and contain molecule markers of their parental origin. The aim of our study was to isolate and characterize total and neural-derived small EVs (NDEVs) and their micro RNA (miRNA) cargo in Alzheimer’s disease (AD) patients. Small NDEVs were isolated from plasma in a population consisting of 40 AD patients and 40 healthy subjects (CTRLs) using high throughput Advanced TaqMan miRNA OpenArrays®, which enables the simultaneous determination of 754 miRNAs. MiR-23a-3p, miR-223-3p, miR-100-3p and miR-190-5p showed a significant dysregulation in small NDEVs from AD patients as compared with controls (1.16 ± 0.49 versus 7.54 ± 2.5, p = 0.026; 9.32 ± 2.27 versus 0.66 ± 0.18, p <0.0001; 0.069 ± 0.01 versus 0.5 ± 0.1, p < 0.0001 and 2.9 ± 1.2 versus 1.93 ± 0.9, p < 0.05, respectively). A further validation analysis confirmed that miR-23a-3p, miR-223-3p and miR-190a-5p levels in small NDEVs from AD patients were significantly upregulated as compared with controls (p = 0.008; p = 0.016; p = 0.003, respectively) whereas miR-100-3p levels were significantly downregulated (p = 0.008). This is the first study that carries out the comparison between total plasma small EV population and NDEVs, demonstrating the presence of a specific AD NDEV miRNA signature.

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Figure 1 Nanoparticle tracking analysis (NTA). Representative outcomes of total small extracellular vesicles (EVs) suspension and its negative control are reported.
Figure 1 Nanoparticle tracking analysis (NTA). Representative outcomes of total small extracellular vesicles (EVs) suspension and its negative control are reported.CC BY · PubMed Central
Figure 2 Total and neural-derived extracellular vesicles (NDEVs) enriched fractions from plasma. A and B. Flow cytometry analysis dot-plot. Panel (a) represents the negative control, (b) the neural-derived EVs from plasma of one AD patient. The EVs are stained with the fluorophore FITC. In Panel (a) there is an absence of fluorescent emission FITC (green peak), which instead is present in the positive sample panel (b). The same result is shown in the dot plot. Indeed, in the negative control the bead-Ab system is located in the quadrant Q3, in the absence of FITC fluorescence, while in the sample the signal shifts in the Q4 quadrant, where the FITC fluorescence is positive. C, D, E, F TEM analysis. Ultrastructural examination of fractions enriched in EVs (c) CD81-positive, (d) of neural derivation (L1CAM positive). TEM images showed that EVs were oval or oval or bowl-shaped capsuled. No substantial difference in the morphology of EVs isolated from fresh samples (e) compared with frozen ones (f). Western blot analysis. L1CAM, CD9, VPS35, TSG101 markers were all detected in the NDEVs enriched fraction of plasma samples (g).
Figure 2 Total and neural-derived extracellular vesicles (NDEVs) enriched fractions from plasma. A and B. Flow cytometry analysis dot-plot. Panel (a) represents the negative control, (b) the neural-derived EVs from plasma of one AD patient. The EVs are stained with the fluorophore FITC. In Panel (a) there is an absence of fluorescent emission FITC (green peak), which instead is present in the positive sample panel (b). The same result is shown in the dot plot. Indeed, in the negative control the bead-Ab system is located in the quadrant Q3, in the absence of FITC fluorescence, while in the sample the signal shifts in the Q4 quadrant, where the FITC fluorescence is positive. C, D, E, F TEM analysis. Ultrastructural examination of fractions enriched in EVs (c) CD81-positive, (d) of neural derivation (L1CAM positive). TEM images showed that EVs were oval or oval or bowl-shaped capsuled. No substantial difference in the morphology of EVs isolated from fresh samples (e) compared with frozen ones (f). Western blot analysis. L1CAM, CD9, VPS35, TSG101 markers were all detected in the NDEVs enriched fraction of plasma samples (g).CC BY · PubMed Central
Figure 3 (a) Hierarchical clustering of samples based on microRNA (miRNA) profiles observed in total plasma EVs from Alzheimer’s disease (AD) patients and control subjects (CTRLs). The most variant (according to the coefficient of variation) miRNAs were used for clustering analysis (Pearson correlation, average linkage). Rows are centered and unit variance scaling is applied to normalized expression values. Color indicates relative up- (red) or downregulation for each miRNA (row). (b) Principle component analysis based on the most variant miRNAs in total plasma EVs.
Figure 3 (a) Hierarchical clustering of samples based on microRNA (miRNA) profiles observed in total plasma EVs from Alzheimer’s disease (AD) patients and control subjects (CTRLs). The most variant (according to the coefficient of variation) miRNAs were used for clustering analysis (Pearson correlation, average linkage). Rows are centered and unit variance scaling is applied to normalized expression values. Color indicates relative up- (red) or downregulation for each miRNA (row). (b) Principle component analysis based on the most variant miRNAs in total plasma EVs.CC BY · PubMed Central
Figure 4 (a) Hierarchical clustering of samples based on microRNA (miRNA) profiles observed in total plasma EVs from AD patients and CTRLs. The most variant (according to coefficient of variation) miRNAs detected in NDEVs were used for clustering analysis (Pearson correlation, average linkage). Rows are centered and unit variance scaling is applied to normalized expression values. Color indicates relative up- (red) or downregulation for each miRNA (row). (b) Principle component analysis based on the most variant miRNAs in NDEVs.
Figure 4 (a) Hierarchical clustering of samples based on microRNA (miRNA) profiles observed in total plasma EVs from AD patients and CTRLs. The most variant (according to coefficient of variation) miRNAs detected in NDEVs were used for clustering analysis (Pearson correlation, average linkage). Rows are centered and unit variance scaling is applied to normalized expression values. Color indicates relative up- (red) or downregulation for each miRNA (row). (b) Principle component analysis based on the most variant miRNAs in NDEVs.CC BY · PubMed Central
Figure 5 (a) The Venn diagram shows the distribution of the most dysregulated miRNAs (> or < 2.5 -fold compared to controls) detected in both subpopulations of EVs. (b) Relative quantification of statistically deregulated miRNAs in AD patients (AD n = 20 and CTRLs n = 20; miR-23a-3p * p = 0.026; miR-223-3p *** p < 0.0001; miR-100-3p *** p < 0.0001) as compared with CTRLs.
Figure 5 (a) The Venn diagram shows the distribution of the most dysregulated miRNAs (> or < 2.5 -fold compared to controls) detected in both subpopulations of EVs. (b) Relative quantification of statistically deregulated miRNAs in AD patients (AD n = 20 and CTRLs n = 20; miR-23a-3p * p = 0.026; miR-223-3p *** p < 0.0001; miR-100-3p *** p < 0.0001) as compared with CTRLs.CC BY · PubMed Central
Figure 6 RNase protection assay. The miR-223-3p, miR-190-5p and miR-23a-3p were quantified by q-PCR in total and neural-derived plasma EVs. EVs were treated with or without RNase I and/or Triton X-100 (n = 4).
Figure 6 RNase protection assay. The miR-223-3p, miR-190-5p and miR-23a-3p were quantified by q-PCR in total and neural-derived plasma EVs. EVs were treated with or without RNase I and/or Triton X-100 (n = 4).CC BY · PubMed Central
Figure 7 Relative expression of best dysregulated miRNAs tested in NDEVs from the remaining 20 AD patients and 20 CTRLs (miR-23a-3p ** p = 0.008; miR223-3 * p = 0.016; miR-190a-5p ** p = 0.003; miR-100-3p ** p = 0.008).
Figure 7 Relative expression of best dysregulated miRNAs tested in NDEVs from the remaining 20 AD patients and 20 CTRLs (miR-23a-3p ** p = 0.008; miR223-3 * p = 0.016; miR-190a-5p ** p = 0.003; miR-100-3p ** p = 0.008).CC BY · PubMed Central
Figure 8 The miRNA targets and pathways prediction. (a) A 3-set Venn diagram overlap of mRNA targets statistically deregulated in NDEVs in AD patients (miR-23a-3p, miR-223-3p, miR-100-3p. and miR-190a-5p. (b) Heatmaps of significant pathways for statistically significant NDEV miRNAs, predicted by DIANA-miRPath (v.3.0). Pathways are depicted on the x-axis and miRNAs on the y-axis. The color code represents the log (p-value), with the most significant predicted miRNA-pathway interactions in red.
Figure 8 The miRNA targets and pathways prediction. (a) A 3-set Venn diagram overlap of mRNA targets statistically deregulated in NDEVs in AD patients (miR-23a-3p, miR-223-3p, miR-100-3p. and miR-190a-5p. (b) Heatmaps of significant pathways for statistically significant NDEV miRNAs, predicted by DIANA-miRPath (v.3.0). Pathways are depicted on the x-axis and miRNAs on the y-axis. The color code represents the log (p-value), with the most significant predicted miRNA-pathway interactions in red.CC BY · PubMed Central
Figure 9 (a) Heatmaps of significant pathways predicted by DIANA-miRPath (v.3.0) for specific miRNAs detected in NDEVs. Pathways are depicted on the x-axis and miRNAs on the y-axis. The color code represents the log (p-value), with the most significant predicted miRNA-pathway interactions in red. (b) Tissue site of expression determined using FunRich v3.0 (http://funrich.org/). The percentage of targets showing regional enrichment (blue bar) and the significance of regional enrichment (red bar). Top x-axis is –log10 (p-value) of regional enrichment; bottom x-axis is the percentage of targets overlapping with the region.
Figure 9 (a) Heatmaps of significant pathways predicted by DIANA-miRPath (v.3.0) for specific miRNAs detected in NDEVs. Pathways are depicted on the x-axis and miRNAs on the y-axis. The color code represents the log (p-value), with the most significant predicted miRNA-pathway interactions in red. (b) Tissue site of expression determined using FunRich v3.0 (http://funrich.org/). The percentage of targets showing regional enrichment (blue bar) and the significance of regional enrichment (red bar). Top x-axis is –log10 (p-value) of regional enrichment; bottom x-axis is the percentage of targets overlapping with the region.CC BY · PubMed Central

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Metadata

Authors
Serpente, M., Fenoglio, C., D’Anca, M., Arcaro, M., Sorrentino, F., Visconte, C., Arighi, A., Fumagalli, G. G., Porretti, L., Cattaneo, A., Ciani, M., Zanardini, R., Benussi, L., Ghidoni, R., Scarpini, E., & Galimberti, D.
Affiliations
  • Department of Pathophysiology and Transplantation, Dino Ferrari Center, University of Milan, 20122 Milan, Italy
  • Neurodegenerative Diseases Unit, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, 20122 Milan, Italy
  • Flow Cytometry Service, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 20122 Milan, Italy
  • Department of Transfusion Medicine and Haematology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 20122 Milan, Italy
  • Molecular Markers Laboratory, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, 25125 Brescia, Italy
ORCID
Venue
Cells
Publisher
MDPI AG
Published
2020-06-10
Volume
9
Issue
6
Pages
1443
ISSN
2073-4409
DOI
10.3390/cells9061443
PMC
PMC7349735
Cited by
116
References
4
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
Ministero della Salute (RF-2018-12365333, NET- 2011-02346784)

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CoChat. (2026). Citation check: MiRNA Profiling in Plasma Neural-Derived Small Extracellular Vesicles from Patients with Alzheimer’s Disease. https://app.cochat.ai/papers/10.3390/cells9061443
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Serpente, M., Fenoglio, C., D’Anca, M., Arcaro, M., Sorrentino, F., Visconte, C., Arighi, A., Fumagalli, G. G., Porretti, L., Cattaneo, A., Ciani, M., Zanardini, R., Benussi, L., Ghidoni, R., Scarpini, E., & Galimberti, D. (2020). MiRNA Profiling in Plasma Neural-Derived Small Extracellular Vesicles from Patients with Alzheimer’s Disease. Cells. https://doi.org/10.3390/cells9061443