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

Abstract

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.

DGCR8 Promotes Neural Progenitor Expansion and Represses Neurogenesis in the Mouse Embryonic Neocortex

“…Taken together, this evidence indicates that DGCR8 promotes cortical NPC self-renewal and represses their differentiation in vivo , possibly by a cell-autonomous function. Our results are consistent with previous observations in mouse ESCs (Wang et al, 2007 ; Cirera-Salinas et al, 2017a , b ) and NPCs in vitro (Liu et al, 2017 ). Of note, overexpression of DGCR8 at later developmental stages (i.e., when upper cortical layer neurons are generated) selectively promotes expansion of BPs (Figure 5 ), opening intriguing perspectives for a DGCR8-dependent control in the radial neocortex enlargement in evolution, which reflects a striking increase in BP population and upper cortical layers size (Fietz and Huttner, 2011 ; Reillo et al, 2011 ; Shitamukai et al, 2011 ; Wang et al, 2011 ; Borrell and Reillo, 2012 ; Hevner and Haydar, 2012 ; Kelava et al, 2012 ; Betizeau et al, 2013 ; LaMonica et al, 2013 ).…”

Characterization of iPSCs derived from low grade gliomas revealed early regional chromosomal amplifications during gliomagenesis

“…Collected cells were washed twice with serumless DMEM/F12 (300×G, 7 minutes, 4°C), counted in the presence of 0.04% trypan blue to enumerate viable cells. Dissociated LGG cells were cultured in cell culture plates coated with poly-L-ornithine (Sigma-Aldrich) and laminin (EMD-Millipore) in LGG medium (Neurobasal-A medium containing 1% N-2, 2% vitamin A-free B-27, 2 mM L-Glutamine [Thermo Fisher], 20 ng/ml basic FGF [Stemgent], and 20 ng/ml human EGF [Cell Signaling]) as described [15,16]. Medium was exchanged as needed, and cells grew adherently to the bottom of flasks.…”

“…For immunostaining, iPSCs were fixed in 4% PFA, blocked in Protein Block (Agilent), stained with primary antibodies at 4 °C overnight and secondary antibodies at room temperature for 1 hour as described [15,25]. Primary antibodies used were OCT4 (sc-5279, Santa Cruz Biotech), SOX2 (561469, BD Biosciences), and NANOG (AF1977, R&D Systems).…”

Elevated p53 Activities Restrict Differentiation Potential of MicroRNA-Deficient Pluripotent Stem Cells

“…Immunostaining was performed as described previously ( Liu et al., 2017 ). In brief, EBs were fixed in 4% paraformaldehyde, blocked in Protein Block (Dako), and incubated with the appropriate primary antibodies overnight at 4°C and secondary antibodies for 1 hr at room temperature.…”

“…Cells were analyzed on a BD Fortessa flow cytometer. Apoptosis analysis was performed as described previously ( Liu et al., 2017 ). In brief, ESCs were treated with the indicated concentration of nutlin-3a or 50 ng/mL NCS (Sigma-Aldrich) for 24 hr.…”