The subventricular zone (SVZ) is the largest neurogenic niche in the

The subventricular zone (SVZ) is the largest neurogenic niche in the adult mammalian brain. Sox9 knockdown leads to increased neuron formation. Thus, miR-124 mediated repression of Sox9 is important for progression along the SVZ stem cell lineage to neurons. Introduction The subventricular zone (SVZ) is the largest germinal region in the adult mammalian brain Evofosfamide and harbours stem cells that generate olfactory bulb interneurons. The neural stem cells within this neurogenic niche are specialized astrocytes (Type B cells) that give rise to rapidly dividing transit amplifying cells (Type C cells) 1. The majority of these cells then generate neuroblasts (Type A cells) that migrate along the rostral migratory stream (RMS) and differentiate into granule and periglomerular interneurons in the olfactory bulb 1. A small number of oligodendrocytes are also generated by the adult SVZ 2, 3. Although much has been elucidated about the identity and lineage of SVZ stem cells, the regulatory mechanisms underlying stem cell self-renewal and differentiation are still largely unknown. MicroRNAs (miRNAs) are small non-coding RNAs that are emerging as key post-transcriptional regulators implicated in developmental and disease processes 4, 5. miRNAs largely act as repressors of gene expression either by guiding the cleavage of their target mRNAs or by inhibiting their translation 4, 6. Their ability to potentially regulate large numbers of target genes simultaneously suggests that they may be important sculptors of transcriptional networks. As such, they are attractive candidates to regulate stem cell lineage progression. We have identified several miRNAs expressed at different stages of the SVZ stem cell lineage, one of which is miR-124, the most abundant miRNA in the adult brain 7. We therefore investigated the role of miR-124 in the adult SVZ neurogenic niche. Previous work has shown that over-expression of miR-124 in HeLa cells shifts their mRNA profile towards a brain enriched pattern 8, whereas blocking miR-124 in cultured neurons leads to the up-regulation of non-neuronal transcripts 9. Based on over-expression in cell lines and in embryonic stem cells, miR-124 has been proposed to mediate neuronal differentiation 10, 11. This is achieved in part via targeting PTBP1, a repressor of neuron-specific splicing 11 and SCP1, a component of the REST transcription repressor IKK-gamma (phospho-Ser85) antibody complex 12. However, probing the role of miR-124 has been challenging. Two studies in the developing chick spinal cord investigating the role of miR-124 had differing findings. One reported that miR-124 has no effect on neuronal differentiation 13 whereas the other reported that miR-124 has modest effects on promoting neurogenesis 12. Thus, the role of miR-124 in neurogenesis is still unclear. Here we uncover a novel role for miR-124 in regulating the temporal progression of neurogenesis in the adult SVZ. miR-124 is first up-regulated at the transition between transit amplifying cells and neuroblasts, and further increases as neuroblasts exit the cell cycle. Blocking miR-124 maintains SVZ cells as dividing precursors, whereas ectopic expression of miR-124 promotes precocious neuronal differentiation. Knockdown of endogenous miR-124 during regeneration leads to the formation of hyperplasias and a delay in neuronal regeneration. We identify and as miR-124 targets. We show that is an important physiological target of miR-124 in SVZ neuroblasts where mRNA is present but not protein. Sox9 over-expression in SVZ cells abolishes the production of neurons. In contrast, Sox9 knockdown leads to increased neurogenesis and decreased glial formation. Thus, the protein levels of Sox9 must be down-regulated for neuronal differentiation and are tightly controlled at the post-transcriptional level by miR-124 as cells progress along the SVZ lineage. Results miR-124 is expressed by neuroblasts in the adult SVZ niche To determine the spatial and cell-type specific distribution of miR-124 in the adult SVZ neural stem cell niche, we performed hybridization using DIG-labeled RNA probes targeting the mature form of miR-124. miR-124 was expressed at low levels in both the SVZ and RMS (Fig. 1aCd) and was greatly up-regulated in mature granule and periglomerular neurons in the olfactory bulb (Fig. 1b, e) at levels seen in mature neurons throughout the brain (Fig. Evofosfamide 1h, Supplementary Fig. 1a). In contrast, miR-124 was not expressed in oligodendrocytes or astrocytes (data not shown). To distinguish which cell types in the adult SVZ express Evofosfamide miR-124, we performed hybridization in combination with immunostaining for markers expressed at different stages of the SVZ lineage (Fig. 1f). miR-124 was not expressed at the earliest stages of the lineage in brain lipid binding protein (BLBP+) SVZ astrocytes or in dividing doublecortin-negative (DCX?) transit amplifying cells (Fig. 1g, h) but was expressed in DCX+ neuroblasts.

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