Ethanol Exposure Increases miR-140 in Extracellular Vesicles: Implications for Fetal Neural Stem Cell Proliferation and Maturation

Abstract

Background: Neural stem cells (NSCs) generate most of the neurons of the adult brain in humans, during the mid-first through second-trimester period. This critical neurogenic window is particularly vulnerable to prenatal alcohol exposure, which can result in diminished brain growth. Previous studies showed that ethanol (EtOH) exposure does not kill NSCs, but, rather, results in their depletion by influencing cell cycle kinetics and promoting aberrant maturation, in part, by altering NSC expression of key neurogenic miRNAs. NSCs reside in a complex microenvironment rich in extracellular vesicles, shown to traffic miRNA cargo between cells.

Methods: We profiled the miRNA content of extracellular vesicles from control and EtOH-exposed ex vivo neurosphere cultures of fetal NSCs. We subsequently examined the effects of one EtOH-sensitive miRNA, miR-140-3p, on NSC growth, survival, and maturation.

Results: EtOH exposure significantly elevates levels of a subset of miRNAs in secreted extracellular vesicles. Overexpression of one of these elevated miRNAs, miR-140-3p, and its passenger strand relative, miR-140-5p, significantly increased the proportion of S-phase cells while decreasing the proportion of G₀ /G₁ cells compared to controls. In contrast, while miR-140-3p knockdown had minimal effects on the proportion of cells in each phase of the cell cycle, knockdown of miR-140-5p significantly decreased the proportion of cells in G₂ /M phase. Furthermore, miR-140-3p overexpression, during mitogen-withdrawal-induced NSC differentiation, favors astroglial maturation at the expense of neural and oligodendrocyte differentiation.

Conclusions: Collectively, the dysregulated miRNA content of extracellular vesicles following EtOH exposure may result in aberrant neural progenitor cell growth and maturation, explaining brain growth deficits associated with prenatal alcohol exposure.

Keywords: Exosomes; Extracellular Vesicles; Fetal Alcohol; Neural Stem Cells; microRNA.

Figure 1. NSCs are Abundant Producers of EVs

1A) Immunoblot of CD63 expression in EVs and NSCs. Differences in CD63 molecular weight between EV and NSC fraction are due to polyethylene glycol in the Exoquick-TC™ preparation. 1B) TEM image of EVs immunolabelled with CD63. Yellow circles indicate puncta with CD63 labelling. 1C) Nanosight image of EVs derived from NSCs Mkr=molecular weight marker

Figure 2. Ethanol Alters the miRNA Profile in NSC-derived EVs

2A) Volcano plot of miRNA expression in EVs from control and ethanol treated NSCs. X-axis depicts Log₂ transformed fold change of Ethanol/Control miRNA expression. Orange data points above the dotted line represent miRNAs significantly affected by ethanol exposure. The red dot signifies miR-140–3p and the blue dot signifies miR-140–5p. n=5 samples per group 2B) MTT absorbance values following control, miR-140–3p, and miR-310a-3p overexpression in NSCs. n=10 samples per group. 2C) Relative abundance of miR-140–3p (red-dotted line) and miR-140–5p (blue-dotted line) compared to other miRNAs within NSCs (top) and NSC-derived EVs (bottom). *p<0.05, ***p<0.001 by Student’s t-test

Figure 3. miR-140–3p and miR-140–5p Regulate the Rate of DNA Synthesis

3A) EdU Intensity of NSCs following miR-140–3p or miR-140–5p overexpression 3B) EdU Intensity of NSCs following miR-140–3p or miR-140–5p inhibition n=5 samples per group, ^#p<0.05, ^###p<0.001 by ANOVA. *p<0.05, ***p<0.001 by Dunnett’s post-hoc.

Figure 4. miR-140 Influences Cell Cycle Dynamics

Proportion of cells in S, G₀/G_1, or G₂/M phase of the cell cycle following miR-140–3p and miR-140–5p overexpression (4A) or inhibition (4B). Representative flow cytometry images are displayed on the right panel. n=5 samples per group, ^#p<0.05, ^##p<0.01 by ANOVA. *p<0.05, ***p<0.001 by Dunnett’s post-hoc.

Figure 5. miR-140 Does Not Influence Apoptotic Cell Death

Quantification of caspase 3/7 activity following miR-140–3p and miR-140–5p overexpression (4A) or inhibition (4B). n=5 samples per group, n.s. indicates no significance.

Figure 6. miR-140–3p Expression Decreases During NSC differentiation

6A) Photomicrographs of a neurosphere over a 3-day mitogen-withdrawal-induced differentiation paradigm. Inset images depict the bipolar morphological appearance and immunolabeling with anti-neurofilament antibody of maturing, migratory neurons. Arrow heads demarcate axonal processes of bipolar neurons and arrows indicate a neurofilament labeled axon. 6B) miR-140–3p and 6C) mir-140–5p expression in NSCs over a three-day mitogen-withdrawal-induced differentiation paradigm n=15 samples per group, ^##p<0.01 by ANOVA, **p<0.01 by Dunnett’s post-hoc. D0, D1, and D3 indicate days 0, 1, and 3 of differentiation, respectively.

Figure 7. miR-140–3p Influences NSC Differentiation

Expression of 7A) GFAP, 7B) Glast, 7C) DCX, 7D) NeUN, 7E) PDGFRα, 7F) Olig2, and 7G) Nestin in NSCs following miR-140–3p overexpression in a three-day mitogen-withdrawal-induced differentiation paradigm. n=5 samples per group, ⁺⁺p<0.01, ⁺⁺⁺p<0.001, ⁺⁺⁺⁺p<0.0001 interaction effect between differentiation and miR-140–3p overexpression, ANOVA. **p<0.01, ***p<0.001, ****p<0.0001 by Sidak’s post-hoc. D0, D1, and D3 indicate days 0, 1, and 3 of differentiation, respectively.

Figure 8. miR-140–5p Influences NSC Differentiation

Expression of 8A) GFAP, 8B) Glast, 8C) DCX, 8D) NeUN, 8E) PDGFRα, 8F) Olig2, and 8G) Nestin in NSCs following miR-140–5p overexpression in a three-day mitogen-withdrawal-induced differentiation paradigm. n=5 samples per group, ⁺⁺p<0.01, ⁺⁺⁺p<0.001, ⁺⁺⁺⁺p<0.0001 interaction effect between differentiation and miR-140–5p overexpression, ANOVA. ^#p<0.05 main effect of miR-140–5p overexpression, ANOVA. **p<0.01, ***p<0.001, ****p<0.0001 by Sidak’s post-hoc. D0, D1, and D3 indicate days 0, 1, and 3 of differentiation, respectively.

Figure 9. miR-140 host gene expression is correlated with glial markers

Correlation plot of WWP2, the mir-140 host gene (miR-140 HG), with different markers of neural and glial maturation.