Canonical microRNAs Enable Differentiation, Protect Against DNA Damage, and Promote Cholesterol Biosynthesis in Neural Stem Cells

Neural stem cells (NSCs) have the capacity to differentiate into neurons, astrocytes, and oligodendrocytes, and therefore represent a promising donor tissue source for treating neurodegenerative diseases and repairing injuries of the nervous system. However, it remains unclear how canonical microRNAs (miRNAs), the subset of miRNAs requiring the Drosha-Dgcr8 microprocessor and the type III RNase Dicer for biogenesis, regulate NSCs. In this study, we established and characterized Dgcr8^-/- NSCs from conditionally Dgcr8-disrupted mouse embryonic brain. RNA-seq analysis demonstrated that disruption of Dgcr8 in NSCs causes a complete loss of canonical miRNAs and an accumulation of pri-miRNAs. Dgcr8^-/- NSCs can be stably propagated in vitro, but progress through the cell cycle at reduced rates. When induced for differentiation, Dgcr8^-/- NSCs failed to differentiate into neurons, astrocytes, or oligodendrocytes under permissive conditions. Compared to Dgcr8^+/- NSCs, Dgcr8^-/- NSCs exhibit significantly increased DNA damage. Comparative RNA-seq analysis and gene set enrichment analysis (GSEA) revealed that Dgcr8^-/- NSCs significantly downregulate genes associated with neuronal differentiation, cell cycle progression, DNA replication, protein translation, and DNA damage repair. Furthermore, we discovered that Dgcr8^-/- NSCs significantly downregulate genes responsible for cholesterol biosynthesis and demonstrated that Dgcr8^-/- NSCs contain lower levels of cholesterol. Together, our data demonstrate that canonical miRNAs play essential roles in enabling lineage specification, protecting DNA against damage, and promoting cholesterol biosynthesis in NSCs.