miR-124 regulates adult neurogenesis in the subventricular zone stem cell niche

Abstract

The subventricular zone (SVZ) is the largest neurogenic niche in the adult mammalian brain. We found that the brain-enriched microRNA miR-124 is an important regulator of the temporal progression of adult neurogenesis in mice. Knockdown of endogenous miR-124 maintained purified SVZ stem cells as dividing precursors, whereas ectopic expression led to precocious and increased neuron formation. Furthermore, blocking miR-124 function during regeneration led to hyperplasias, followed by a delayed burst of neurogenesis. We identified the SRY-box transcription factor Sox9 as being a physiological target of miR-124 at the transition from the transit amplifying cell to the neuroblast stage. Sox9 overexpression abolished neuronal differentiation, whereas Sox9 knockdown led to increased neuron formation. Thus miR-124-mediated repression of Sox9 is important for progression along the SVZ stem cell lineage to neurons.

Figure 1. miR-124 expression in the adult SVZ niche

(a–e) In situ hybridization of miR-124 in the adult mouse brain. (a) Sagittal schema showing SVZ (red), which extends along the entire lateral ventricle. Image in (b) corresponds to area in box in (a). miR-124 is expressed at low levels in the SVZ (arrow) and RMS (arrowhead) and is up-regulated in the olfactory bulb (OB, asterisk). Other labeled cells are differentiated neurons (see Supplementary Fig. 1a). (c–e) miR-124 signal in coronal sections corresponding to areas shown in boxes in SVZ (c, arrow), RMS (d, arrowhead) and OB (e). miR-124 is not detected in the corpus callosum (c, CC) or other white matter fiber tracts. Scale bar, 100µm. (f) Schema showing the SVZ lineage and markers expressed at each stage. (g, h) In situ hybridization for miR-124 combined with immunostaining in SVZ coronal sections. CC is at top and striatum is at bottom. Right panels show in situ signal alone. (g) miR-124 is expressed in DCX⁺ neuroblasts (red, arrow) but not in BLBP⁺ astrocytes (green, white arrowheads). Dashed lines outline the lateral ventricle. (h) miR-124 is expressed in DCX⁺ neuroblasts (green) and in mature NeuN⁺ neurons (blue, black arrowheads) but is not detected in Ki67⁺DCX⁻ transit amplifying cells (red, white arrowheads). Scale bar, 20µm. (i) Quantitative RT-PCR for miR-124 on FACS purified SVZ populations. Data represent mean ± s.e.m. normalized to 5S rRNA from five independent experiments. Asterisks, p<0.05, two-tailed paired Students-t test.

Figure 2. Knockdown of miR-124 in vitro

(a–c) Representative micrographs of colonies from SVZ stem cells co-cultured on rat astrocyte monolayers in the presence of penetratin (pen)-conjugated antisense 2’OMe-RNAs against miR-124 (AS-124) or miR-194 (AS-194). M2/M6 (green) distinguishes mouse cells on immunonegative rat cells. More BrdU⁺ cells (blue) and fewer neurons (TuJ1, red) are present in AS-124 treated cultures. BF, bright field. Scale bar, 50 µm. (d) Quantification of total neurons (TuJ1⁺BrdU⁻), dividing cells (BrdU⁺) and other cells (TuJ1⁻BrdU⁻). (e) Colony composition of neurons (TuJ1⁺BrdU⁻), neuroblasts (TuJ1⁺BrdU⁺), dividing cells (BrdU⁺) and other cells (TuJ1⁻BrdU⁻) within individual colonies. AS-124 treatment decreases the proportion of neurons (black) derived from single SVZ stem cells by maintaining them as dividing neuroblasts (dark gray) and transit amplifying cells (light gray). (f) Quantification of neurosphere (NS) formation of unsorted and FACS purified cells. Histogram shows ratio to untreated culture. (g) Quantification of survival of FACS purified neuroblasts. All data represent mean ± s.e.m. from at least three independent experiments. Asterisks, p<0.05, two-tailed paired Students-t test.

Figure 3. Ectopic expression of miR-124 in vitro

(a) Schema shows the retroviral construct for miR-124 over-expression. The mouse mir-124–3 genomic locus (hairpin) with ~125 bp flanking sequences was cloned into the murine stem cell virus (MSCV) backbone under the control of the H1 promoter. The expression of enhanced GFP was driven by a second promoter (PGK promoter) placed in the opposite orientation to the miRNA expression cassette. (b) Sequence of mutated miR-124 in the retroviral construct (RV-124mt). Six nucleotides (bold) of the 5’ seed region (box) were mutated. (c) Quantitative RT-PCR of miR-124 levels induced by retroviral infection in neurospheres. Neurospheres transduced by RV-124 ectopically express miR-124 to the physiological levels present in purified neuroblasts (mCD24 population). (d) Quantification of GFP⁺ neurons (TuJ1⁺BrdU⁻) dividing cells (BrdU⁺) and other cells (TuJ1⁻BrdU⁻) after miR-124 over-expression. (e) Colony composition of neurons (TuJ1⁺BrdU⁻, black), neuroblasts (TuJ1⁺BrdU⁺, dark gray), dividing cells (BrdU⁺, light gray) and other cells (TuJ1⁻BrdU⁻, white) within individual colonies after RV-124 over-expression. All data represent mean ± s.e.m. from six independent experiments. Asterisks, p<0.05, two-tailed paired Students-t test.

Figure 4. miR-124 over-expression and knockdown in vivo

(a–c,e–g) Representative micrographs of SVZ whole mount preparations three days after retrovirus infection. GFP⁺ dividing cells (arrows) and migratory neuroblasts (in chains, TuJ1⁺ or DCX⁺, red) were present after RV-GFP (a), RV-GFP +AS-124 (b), RV-124mt (e) and RV-124 (f) infection. (c,g) are higher magnification view of boxes in b and f, respectively. Lower panels show split channel of Ki67 immunostaining. AS-124 maintains SVZ precursors as clusters of dividing cells (c, arrowheads) whereas RV-124 induces cell cycle exit and neuronal differentiation (g, arrowheads indicate post-mitotic migrating neurons). Scale bar, 50 µm. (d, h) Quantification of total neurons (TuJ⁺Ki67⁻ or DCX⁺Ki67⁻), dividing cells (Ki67⁺) and other cells (Ki67⁻DCX⁻) derived from infected SVZ precursors after miR-124 knockdown (d) and RV-124 over-expression (h). Data represent mean ± s.e.m from six whole mounts. Asterisks, p<0.05, two-tailed paired Students t-test. A total of n=1260 (RV-GFP with saline), n=492 (RV-GFP with penetratin-only), n=477 (RV-GFP with AS-124), n=895 (RV-124), and n=938 (RV-124mt) retrovirally transduced cells were counted.

Figure 5. miR-124 knockdown delays SVZ regeneration

(a) Timeline of experiments. Ara-C filled mini-osmotic pumps were implanted for 6 days and replaced by AS-124 or penetratin-only filled pumps for an additional 5 or 7 days. (b–q) SVZ whole mount preparations after five (b–i) or seven (j–q) days of regeneration immunostained for BLBP (b,f,j,n), Ki67 (c,g,k,o), TuJ1 (d,h,l,p) and DCX (e,i,m,q). At 5 days, more dividing cells and small hyperplasias (g, arrowheads) and very few neuroblasts (h,i) are present in AS-124 treated brains. At seven days, a large number of TuJ1⁺ and DCX⁺ neuroblasts suddenly appear (p,q).

Figure 6. Sox9 is a miR-124 target

(a) Luciferase reporter assays for DLX2, JAG1 and SOX9, three miR-124 targets. Histogram showing ratio of Renilla to Firefly luciferase activity normalized to empty vector transfected cells (baseline). Data represent mean ± 2x s.e.m. from six independent experiments. Asterisks, p<0.01, two-tailed paired Student-t test. (b) Schema of dlx-2, jagged1 and sox9 transcripts and miR-124 target sites (red). Lines underneath depict region of 3’UTR cloned into luciferase reporter vectors. (c–f) miR-124 over-expression in neonatal astrocytes. Immunostaining for Sox9 (blue, c,e), Vimentin (red, c,d) and GFP (green, c). Sox9 protein is down-regulated by miR-124 over-expression (c, e, arrow) as compared to untransfected cells (c–e, arrowheads), shown in corresponding fluorescence intensity plot (f, red is bright, blue is dim). (g–i) miR-124 over-expression in undifferentiated adult SVZ adherent cultures. Immunostaining for Sox9 (red), Nestin (blue) and GFP (green). Sox9 protein is down-regulated by miR-124 over-expression (i, arrows) but not by controls (g,h, arrowheads). (j–m) Sox9 immunostaining in coronal sections of adult mouse SVZ. Sox9 (red, upper and lower panels) is expressed by GFP⁺ SVZ astrocytes in hGFAP-GFP mice (j), by a subset of EGFR⁺ transit amplifying cells (k, green) and mCD24⁺ ependymal cells (m, green). DCX⁺ neuroblasts (l, green, outlined) lack Sox9 protein. (n–p) In situ hybridization of Sox9 (black signal) combined with immunostaining for DCX (green) and NeuN (red). (o,p) High magnification view of box in (n) showing sox9 mRNA (arrow, p) in a DCX⁺ neuroblast (arrow, o). Scale bars, 20 µm.

Figure 7. Effect of miR-124 knockdown on Sox9 protein levels

(a,b) Immunostaining of TuJ1 (green), Sox9 (red) and BrdU (blue) in 5 DIV co-cultures. Right panels show Sox9 channel. In untreated cultures, BrdU⁺ dividing neuroblasts express low or undetectable levels of Sox9 protein (a). AS-124 treatment up-regulates SOX9 protein expression in dividing neuroblasts (b, arrows). Note that rat astrocyte monolayer underneath also expresses Sox9 (a,b, asterisks). (c–e) Immunostaining of DCX (green) and Sox9 (red) in coronal sections of the adult mouse brain. Right panels show Sox9 channel in untreated, penetratin-only (pen) and AS-124 infused brain. After 6 days of AS-124 infusion, Sox9 protein was ectopically expressed by migrating DCX⁺ neuroblasts in the RMS (e, outlined by dashed lines). Scale bars, 20 µm.

Figure 8. Sox9 knockdown induces neuronal differentiation

(a) Schema of the Sox9 retroviral construct (RV-Sox9). Sox9 open reading frame (ORF) and the reporter gene (mCherry) are under control of the CMV promoter and are transcribed as bicistronic transcripts. The internal ribosomal entry sites (IRES) allow the expression of mCherry. (b–g) Representative micrographs of retrovirally transduced SVZ adherent cultures after 7 days of differentiation following EGF withdrawal. Immunostaining for GFAP (blue), TuJ1 (red) and reporter genes (GFP or mCherry, green, except for e, mCherry in red). RV-124 induces neuron production at the expense of astrocyte formation (b) as compared to RV-124 mt controls (c). In contrast, Sox9 over-expression eliminates neurogenesis and maintains infected cells as GFAP⁺ astrocytes (d). Sox9 lacking a 3’UTR dominates the gliogenesis phenotype and is not rescued by miR-124 over-expression (e). Sox9 knockdown by RV-shSox9-C (g) increases neuronal differentiation as compared to controls (f) and resembles the phenotype of RV-124 transduced cells. Scale bar, 20 µm. (h,i) Quantification of astrocytes (GFAP⁺) and neurons (TuJ1⁺) present after retroviral infection. Data represent mean ± s.e.m. from at least three independent experiments. Asterisks, p<0.01, two-tailed paired Students-t test. The percentages of both astrocytes and neurons in RV-124, RV-Sox9, and RV-124/RV-Sox9 are statistically significant as compared to the uninfected and RV-124mt controls (h). The percentage of neurons in RV-shSox9-C is significantly higher as compared to RV-shRan and RV-shSox9-A (i).