Transcriptional States and microRNA Regulation of Adult Neural Stem Cells

Adult neural stem cells are specialized astrocytes that generate neurons in restricted regions of the mammalian brain. The largest neurogenic region is the ventricular-subventricular zone, which lines the lateral ventricles and generates olfactory bulb neurons. Stem cell astrocytes give rise to new neurons in both homeostatic and regenerative conditions, suggesting that they can potentially be harnessed for regenerating the brain after injury, stroke, or neurodegenerative disease. Previous work has shown that stem cell astrocytes exist in both quiescent and activated states, but due to a lack of markers, it was not feasible to purify them. Using a novel fluorescence activated cell sorting (FACS) strategy that allows quiescent neural stem cells (qNSCs) and activated neural stem cells (aNSCs) to be purified for the first time, we performed transcriptome profiling to illuminate the molecular pathways active in each population. This analysis revealed that qNSCs are enriched in signaling pathways, especially G-protein coupled receptors, as well as for adhesion molecules, which facilitate interactions with the niche. qNSCs and aNSCs utilize different metabolic pathways. qNSCs are enriched for lipid and glycolytic metabolism, while aNSCs are enriched for DNA, RNA, and protein metabolism. Many receptors and ligands are reciprocally distributed between qNSCs and aNSCs, suggesting that they may regulate each other. Finally, comparison of the transcriptomes of qNSCs and aNSCs with their counterparts in other organs revealed that pathways underlying stem cell quiescence are shared across diverse tissues. A key step in recruiting adult neural stem cells for brain repair is to define the molecular pathways regulating their switch from a quiescent to an activated state. MicroRNAs are small non-coding RNAs that simultaneously target hundreds of mRNAs for degradation and translational repression. MicroRNAs have been implicated in stem cell self-renewal and differentiation. However, their role in adult neural stem cell activation is unknown. We performed miRNA profiling of FACS-purified quiescent and activated adult neural stem cells to define their miRNA signatures. Bioinformatic analysis identified the miR-17~92 cluster as highly upregulated in activated (actively dividing) stem cells in comparison to their quiescent counterparts. Conditional deletion of the miR-17~92 cluster in FACS purified neural stem cells in vitro reduced adult neural stem cell activation, proliferation, and self-renewal. In addition, miR-17~92 deletion led to a selective decrease in neuronal differentiation. Using an in vivo conditional deletion model, we showed that loss of miR-17~92 led to an increase in the proportion of GFAP+ cells and decrease in MCM2+ cells, as well as decreased neurogenesis. Finally, I identify Sphingosine 1 phosphate receptor 1 (S1pr1) as a computationally predicted target of the miR-17~92 cluster. S1pr1 is highly enriched in quiescent neural stem cells. Treatment of quiescent neural stem cells with S1P, the ligand for S1PR1, reduced their activation and proliferation. In vivo deletion of miR-17~92 lead to an increase in S1PR1+ cells, even among MCM2+ cells. Together, these data reveal that the miR-17~92 cluster is a key regulator of adult neural stem cell activation from the quiescent state and subsequent proliferation.