Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Sep 14;8(9):1087.
doi: 10.3390/cells8091087.

The Anti-Apoptotic Effect of ASC-Exosomes in an In Vitro ALS Model and Their Proteomic Analysis

Roberta Bonafede  1 , Jessica Brandi  2 , Marcello Manfredi  3   4 , Ilaria Scambi  5 , Lorenzo Schiaffino  6 , Flavia Merigo  7 , Ermanna Turano  8 , Bruno Bonetti  9 , Emilio Marengo  10   11 , Daniela Cecconi  12 , Raffaella Mariotti  13
Affiliations

The Anti-Apoptotic Effect of ASC-Exosomes in an In Vitro ALS Model and Their Proteomic Analysis

Roberta Bonafede et al. Cells. .

Abstract

Stem cell therapy represents a promising approach in the treatment of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). The beneficial effect of stem cells is exerted by paracrine mediators, as exosomes, suggesting a possible potential use of these extracellular vesicles as non-cell based therapy. We demonstrated that exosomes isolated from adipose stem cells (ASC) display a neuroprotective role in an in vitro model of ALS. Moreover, the internalization of ASC-exosomes by the cells was shown and the molecules and the mechanisms by which exosomes could exert their beneficial effect were addressed. We performed for the first time a comprehensive proteomic analysis of exosomes derived from murine ASC. We identified a total of 189 proteins and the shotgun proteomics analysis revealed that the exosomal proteins are mainly involved in cell adhesion and negative regulation of the apoptotic process. We correlated the protein content to the anti-apoptotic effect of exosomes observing a downregulation of pro-apoptotic proteins Bax and cleaved caspase-3 and upregulation of anti-apoptotic protein Bcl-2 α, in an in vitro model of ALS after cell treatment with exosomes. Overall, this study shows the neuroprotective effect of ASC-exosomes after their internalization and their global protein profile, that could be useful to understand how exosomes act, demonstrating that they can be employed as therapy in neurodegenerative diseases.

Keywords: amyotrophic lateral sclerosis; apoptosis; extracellular vesicles; mesenchymal stem cells; proteomic profiling; therapy.

PubMed Disclaimer

Conflict of interest statement

The authors declare that no conflicts of interest.

Figures

Figure 1
Figure 1
TEM and western blot analysis of adipose stem cells (ASC)-exosomes. Electron microscopy shows vesicles with characteristic morphology and size of exosomes. Scale bar, 100 nm (A). The blots show western blot detection of the expression of HSP70 (70 kDa), CD9 (25 kDa) and CD81 (26 kDa) in exosomes (EXO); ASC lysates (ASC) was used as positive control (B).
Figure 2
Figure 2
Gene ontology (GO) enrichment of the ASC-exosomes identified proteins according to Database for Annotation, Visualization and Integrated Discovery (DAVID) functional annotation. The top 10 enriched biological process (A) molecular function (B) and cellular component (C) are reported. The percentage represents the portion of the genes encoding the proteins with the corresponding gene ontology biological processes (GOBPs), gene ontology molecular functions (GOMFs) or gene ontology cellular components (GOCCs) in the ASC-exosomes proteins.
Figure 3
Figure 3
Cytoscape based ClueGo/CluePedia pathway analysis and visualization. Enriched pathways were obtained from the Kyoto Encyclopaedia of Genes and Genome (KEGG) database. Terms are grouped based on shared genes (kappa score) showed in different colors. The size of nodes indicated the degree of significance. The most significant term defined the name of the group.
Figure 4
Figure 4
Protein network of identified ASC-exosomes proteins. Schematic view of known and predicted protein interactions according to the STRING database (v. 10). Each node represents a protein, and each edge represents an interaction. Only interactions with the medium confidence score (0.4) are shown. Interactions include physical and functional associations, showing the evidence view.
Figure 5
Figure 5
Western blot analysis of phospho-Akt and SOD1 expression in ASC-exosomes and ASC. The blots show western blot detection of the expression of phospho-Akt (60 kDa) (A) and SOD1 (16 kDa) (B) in exosomes (EXO); ASC lysates were used as positive control. Amido Black staining was used as total loading control (C).
Figure 6
Figure 6
Zymogram assay of SOD1 protein. The assay shows that exosomes (EXO) contain the active form of SOD1 protein. The ASC and deprived ASC (ASC-) were used as the positive control (A). Comassie blue staining was used as total loading control (B).
Figure 7
Figure 7
Acridine orange/propidium iodide (AO/PI) double staining on NSC-34(G93A) cells. Apoptotic and live cells were visualized after AO/PI double staining. The green fluorescence staining by AO indicate live cells, while orange/red fluorescence indicate the PI staining that bound to DNA after damaged membranes. The image shows cells in a basal condition (no cell death was detected and nucleus is uniformly distributed), cells after H2O2 treatment (the nucleus is located in bias and apoptosis-associated changes of cell membranes can be detected, indicating a process of disintegration) and cells after treatment with H2O2 and exosomes (H2O2 + EXO) in which a rescue of cells from death is detected, with an increase in cell viability compared to cells after H2O2 treatment Magnification 20× (A). The graph shows the percentage of cell viability of NSC-34(G93A) cells in basal condition and after H2O2 and ASC-exosomes treatment (H2O2 + EXO). Cell viability significant increased after ASC-exosomes treatment. One-way ANOVA and Bonferroni post-hoc analysis were performed between all the experimental conditions (*** p < 0.001) (B).
Figure 8
Figure 8
TEM images of cells treated with exosomes-ultra-small superparamagnetic iron oxide nanoparticles (USPIO). TEM images showed no damaged cell after ASC-exosomes treatment; magnification 4400×, scale bar 1 µm (A). In (B) note a representative image of phospholipidic membrane structure contained high electron-density particles, whose dimension are attributable to USPIO nanoparticles used to label ASC-exosomes; magnification 46,000×, scale bar 100 nm. In (C), a higher magnification of the section squared in (B) is shown; magnification 140,000×, scale bar 50 nm.
Figure 9
Figure 9
Expression profile of apoptotic markers in NSC-34(G93A) cells. The blots show western blot analysis of Cleaved Caspase 3 (A), Bax (B) and Bcl-2 α (C) proteins performed in NSC-34(G93A) cells (used as control, CNTR), NSC-34 (G93A) cells treated with H2O2 and NSC-34 (G93A) cells treated with H2O2 and exosomes (EXO). Amido Black staining was used as total loading control (D).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Kiernan M.C., Vucic S., Cheah B.C., Turner M.R., Eisen A., Hardiman O., Burrell J.R., Zoing M.C. Amyotrophic lateral sclerosis. Lancet. 2011;377:942–955. doi: 10.1016/S0140-6736(10)61156-7. - DOI - PubMed
    1. Lewis C.M., Suzuki M. Therapeutic applications of mesenchymal stem cells for amyotrophic lateral sclerosis. Stem Cell Res. Ther. 2014;5:32. doi: 10.1186/scrt421. - DOI - PMC - PubMed
    1. Meamar R., Nasr-Esfahani M.H., Mousavi S.A., Basiri K. Stem cell therapy in amyotrophic lateral sclerosis. J. Clin. Neurosci. 2013;20:1659–1663. doi: 10.1016/j.jocn.2013.04.024. - DOI - PubMed
    1. Bonafede R., Mariotti R. ALS Pathogenesis and Therapeutic Approaches: The Role of Mesenchymal Stem Cells and Extracellular Vesicles. Front. Cell Neurosci. 2017;11:80. doi: 10.3389/fncel.2017.00080. - DOI - PMC - PubMed
    1. Marconi S., Bonaconsa M., Scambi I., Squintani G.M., Rui W., Turano E., Ungaro D., D’Agostino S., Barbieri F., Angiari S., et al. Systemic treatment with Adipose-Derived Mesenchymal Stem Cells ameliorates clinical and pathological features in the Amyotrophic Lateral Sclerosis Murine Model. Neuroscience. 2013;248:333–343. doi: 10.1016/j.neuroscience.2013.05.034. - DOI - PubMed

Publication types

Substances