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. 2020 Jan 21;10(1):828.
doi: 10.1038/s41598-020-57663-x.

Astrocyte Support for Oligodendrocyte Differentiation can be Conveyed via Extracellular Vesicles but Diminishes with Age

Cory M Willis  1 , Alexandra M Nicaise  2 , Ernesto R Bongarzone  3 , Maria Givogri  3 , Cory R Reiter  3 , Olivia Heintz  2 , Evan R Jellison  4 , Pearl A Sutter  2 , Gregg TeHennepe  5 , Guruprasad Ananda  5 , Anthony T Vella  4 , Stephen J Crocker  6
Affiliations

Astrocyte Support for Oligodendrocyte Differentiation can be Conveyed via Extracellular Vesicles but Diminishes with Age

Cory M Willis et al. Sci Rep. .

Abstract

The aging brain is associated with significant changes in physiology that alter the tissue microenvironment of the central nervous system (CNS). In the aged CNS, increased demyelination has been associated with astrocyte hypertrophy and aging has been implicated as a basis for these pathological changes. Aging tissues accumulate chronic cellular stress, which can lead to the development of a pro-inflammatory phenotype that can be associated with cellular senescence. Herein, we provide evidence that astrocytes aged in culture develop a spontaneous pro-inflammatory and senescence-like phenotype. We found that extracellular vesicles (EVs) from young astrocyte were sufficient to convey support for oligodendrocyte differentiation while this support was lost by EVs from aged astrocytes. Importantly, the negative influence of culture age on astrocytes, and their cognate EVs, could be countered by treatment with rapamycin. Comparative proteomic analysis of EVs from young and aged astrocytes revealed peptide repertoires unique to each age. Taken together, these findings provide new information on the contribution of EVs as potent mediators by which astrocytes can extert changing influence in either the disease or aged brain.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Astrocytes aged in vitro develop a senescence-like phenotype. (A) Analysis of mRNA expression for the senescence-associated genes p16INK4A, p21, p53, Il-6, Timp-1, and Mmp-3 by qPCR in young (white) and aged (purple) astrocyte cultures. (B) Expression of senescence-related genes p16INK4A, p21, and p53 following rapamycin treatment (25 nM/day, 72 h) Fold expression determined by normalization to expression in young astrocytes. Western blot analyses of (C) p21, (D) HMGB1, (E) TGFB1 and the intermediate filament protein (F) GFAP from young and aged astrocyte cell lysates. Densitometry (a.u.) for each factor was used to determine expression relative to β-actin. Representative immunocytochemistry for (G) p16INK4A and (H) p21 in young and aged astrocytes. Scale bar, 20 μm. (I) Representative SA-β-gal staining of young and aged astrocyte cultures, and (J) quantification of SA-β-gal staining in quadruplicate independent cultures. Scale bar, 20 μm. (I) Significance as indicated where: (A) **P < 0.01, ***P < 0.001, and ****P < 0.0001, Student’s t-test with Welch’s correction. (B) *P < 0.05, **P < 0.01, Student’s t-test with Welch’s correction. (C–F) *P < 0.05, **P < 0.01, and ***P < 0.001, Student’s t-test with Welch’s correction. (J) ****P < 0.0001, Student’s t-test with Welch’s correction. Values are expressed as mean ± SEM.
Figure 2
Figure 2
Identification and characterization of extracellular vesicles from young and aged astrocyte cultures. (A) Negative stain electron micrograph of EVs isolated from ACM of cultured astrocytes. (B) Electron micrographs of astrocyte-derived EVs in ACM verified by immunogold electron microscopy against the EV marker TSG101 and astrocyte marker GFAP. White arrowheads indicate 15 nm GFAP gold particles and black arrowheads indicate 10 nm TSG101 gold particles. Scale bar, 100 nm. Nanoparticle tracking analysis of ACM: particle concentration (C), particle size distribution (D), and mean particle size (E) in ACM of young (open circles) and aged astrocytes (filled circles). (F) Comparative table with particle concentration and mean particle size. Significance as indicated where: (C,E) P > 0.05, Student’s t-test with Welch’s correction. (D) P > 0.05, two-way ANOVA, Bonferroni’s post-hoc test). Values are expressed as mean ± SEM.
Figure 3
Figure 3
Extracellular vesicles from aged astrocytes do not support oligodendrocyte differentiation. (A) Experimental design used to test the effect of EVs isolated from ACM of young or aged astrocyte cultures on OPC maturation (rOPCs: rat OPCs). ACM from astrocytes was collected after 48 h, EVs isolated, and applied to rOPCs. Differentiation of OPCs was assayed after 48 h. (B) Representative images of mature oligodendrocytes (MBP+/OLIG2+) resulting from either young or aged EVs. Scale bar, 100 μm. Magnified panel scale bar, 20 μm. (C) Quantification of OL maturation following young and aged EV treatment. (D) Quantification of number of OLIG2+ OPCs under each treatment condition. n = 7–11 independent cultures per treatment. Significance as indicated where: (C) ****P < 0.001, one-way ANOVA, Tukey’s multiple comparisons test). yA-EVs = young astrocyte EVs; aA-EVs = aged astrocyte EVs. Values are expressed as mean ± SEM. Image of oligodendrocyte in Panel A from BioRender.com.
Figure 4
Figure 4
Negative effect of EVs from aged astrocytes is suppressed by treatment with rapamycin. (A) Experimental design to test whether pre-treatment of aged astrocytes with rapamycin (25 nM/day) for three days improved the ability of EVs to support OPC maturation (rOPCs, rat OPCs). ACM from rapamycin-treated astrocytes was collected after 48 h, EVs isolated and applied to rOPCs. Differentiation of OPCs was assayed after 48 h. (B) Representative images of mature oligodendrocytes (MBP+/OLIG2+) resulting from either aged or rapamycin-treated aged EVs. Scale bar, 100 μm. Magnified panel scale bar, 20 μm. (C) Quantification of OL maturation following aged and rapamycin-treated aged EV treatment. (D) No differences in number of OLIG2+ cells were observed from the varying treatments on the OPCs. n = 7–12 independent cultures per treatment. Significance as indicated where: (C) ****P < 0.001, one-way ANOVA, Tukey’s multiple comparisons test. aA-EVs = aged astrocyte EVs; aA-REVs = aged astrocyte rapamycin EVs. Values are expressed as mean ± SEM. Image of oligodendrocyte in Panel A from BioRender.com.
Figure 5
Figure 5
Proteomic analysis of young and aged astrocyte EV proteome. (A) Top 25 differentially expressed proteins (as listed by gene name) following quantitative analysis of protein abundance between young and aged astrocyte-derived EVs. (B) Differential expression and hierarchical cluster analysis between replicates using protein-wise exact tests. An FDR cutoff of 0.01 was used to identify significantly differentially expressed proteins.
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