Functional Impacts of <em>NRXN1</em> Knockdown on Neurodevelopment in Stem Cell Models

<div><p>Exonic deletions in <i>NRXN1</i> have been associated with several neurodevelopmental disorders, including autism, schizophrenia and developmental delay. However, the molecular mechanism by which <i>NRXN1</i> deletions impact neurodevelopment remains unclear. Here we used human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) as models to investigate the functional impacts of <i>NRXN1</i> knockdown. We first generated hiPSCs from skin fibroblasts and differentiated them into neural stem cells (NSCs). We reduced <i>NRXN1</i> expression in NSCs via a controlled shRNAmir-based knockdown system during differentiation, and monitored the transcriptome alteration by RNA-Seq and quantitative PCR at several time points. Interestingly, half reduction of <i>NRXN1</i> expression resulted in changes of expression levels for the cell adhesion pathway (20 genes, P = 2.8×10⁻⁶) and neuron differentiation pathway (13 genes, P = 2.1×10⁻⁴), implicating that single-gene perturbation can impact biological networks important for neurodevelopment. Furthermore, astrocyte marker GFAP was significantly reduced in a time dependent manner that correlated with <i>NRXN1</i> reduction. This observation was reproduced in both hiPSCs and hESCs. In summary, based on <i>in vitro</i> models, <i>NRXN1</i> deletions impact several biological processes during neurodevelopment, including synaptic adhesion and neuron differentiation. Our study highlights the utility of stem cell models in understanding the functional roles of copy number variations (CNVs) in conferring susceptibility to neurodevelopmental diseases.</p> </div

α-<i>NRXN1</i> knockdown block astrocytes differentiation in time-dependent manner.

<p><b>A.</b> and <b>B.</b> shRNAmir knockdown of α-<i>NRXN1</i> in H9 (A) and iPS (B) have >50% knockdown efficiency in a time-dependent manner, and block the astrocytes differentiation in a time-dependent manner, without influencing neuronal differentiation. <i>sh2</i>: shRNAmir clone V2THS_68983; <i>sh3</i>: shRNAmir clone V2THS_246996.</p

The hiPSCs are fully pluripotent.

<p><b>A.</b> immunocytochemistry and alkaline phosphatase (ALP) staining for pluripotent markers. Nuclear markers: <i>Oct 4</i> and <i>Nanog</i>; Surface markers: <i>SSEA-4, Tra-1-60</i>. <b>B</b>. qPCR for various pluripotent genes indicates that hiPSCs are very similar to hESCs H9 in terms of gene expression levels. <b>C. </b><i>in vivo</i> differentiation of hiPSCs to three germ layers. Ectoderm marker: <i>TUJ-1</i>; Mesoderm marker: <i>SMA</i>; Endoderm marker: <i>AFP</i>. <b>D.</b> hiPSCs can form teratoma in mouse containing derivatives of all three embryonic germ layers (ectoderm, mesoderm, and endoderm), shown by histopathology staining.</p

A network of known protein-protein interactions that includes all first-degree neighbors (direct interaction partners) and second-degree neighbors (interaction partners with first-degree neighbors) of <i>NRXN1</i>.

<p>(A) First degree neighbors are plotted surrounding <i>NRXN1</i>, while second-degree neighbors are plotted in the outer circle. Genes with differential expression P-values less than 0.05 are colored by their fold change values (red: down-regulated, green: up-regulated), with higher color intensity indicating higher fold changes. B, a zoomed-in view of the portion of the network surrounding <i>NRXN1</i> (black square in panel A). Multiple genes that directly interact with <i>NRXN1</i> are down-regulated as a result of <i>NRXN1</i> knockdown.</p

Neural stem cells (NSCs) derived from human embryonic stem cells H9 and hiPS maintain differentiation potential.

<p><b>A.</b> NESTIN staining indicates that close to 100% positive NSCs are derived from H9 and hiPS. <b>B.</b> qPCR showed that hESCs (H9) and iPS highly express pluripotency markers <i>Oct4, Nanog</i> and <i>Sox2</i>, yet NSCs highly express NSCs markers <i>Pax6</i> and <i>Nestin</i>. <b>C.</b> H9 and hiPS derived NSCs can differentiate into both neural and glial lineage as stained by neuron marker <i>TUJ-1</i>, astrocyte marker <i>GFAP</i> and oligodendrocyte marker <i>Olig2</i>. <b>D.</b> H9 and <b>E.</b> iPS derived NSCs differentiated in time-dependent manner, with predicated gene expression pattern. <i>w</i>, abbr. of week.</p

Smek promotes corticogenesis through regulating Mbd3’s stability and Mbd3/NuRD complex recruitment to genes associated with neurogenesis

<div><p>The fate of neural progenitor cells (NPCs) during corticogenesis is determined by a complex interplay of genetic or epigenetic components, but the underlying mechanism is incompletely understood. Here, we demonstrate that Suppressor of Mek null (Smek) interact with methyl-CpG–binding domain 3 (Mbd3) and the complex plays a critical role in self-renewal and neuronal differentiation of NPCs. We found that Smek promotes Mbd3 polyubiquitylation and degradation, blocking recruitment of the repressive Mbd3/nucleosome remodeling and deacetylase (NuRD) complex at the neurogenesis-associated gene loci, and, as a consequence, increasing acetyl histone H3 activity and cortical neurogenesis. Furthermore, overexpression of Mbd3 significantly blocked neuronal differentiation of NPCs, and Mbd3 depletion rescued neurogenesis defects seen in <i>Smek1/2</i> knockout mice. These results reveal a novel molecular mechanism underlying Smek/Mbd3/NuRD axis-mediated control of NPCs’ self-renewal and neuronal differentiation during mammalian corticogenesis.</p></div

Effect of methyl-CpG–binding domain protein 3 (Mbd3) knockdown on neuronal gene expression and promoter occupancy over the course of differentiation of <i>Suppressor of Mek null double knockout</i> (<i>Smek dKO</i>) neural progenitor cells (NPCs).

<p>(A) <i>Wild-type (WT)</i> or <i>Smek1/2 dKO</i> NPCs were electroporated with either control pLKO3G-shScramble or pLKO3G-shMbd3 lentiviral vector and grown for 2 d in N2 medium with basic fibroblast growth factor (bFGF). qPCR analysis was performed to detect indicated mRNAs (<i>n</i> = 3 or 6). (B) Mbd3 knockdown in <i>Smek1/2 dKO</i> NPCs decreases Mbd3 occupancy of <i>Dlx1</i>, <i>Tlx3</i>, <i>NeuroD1</i>, <i>Ascl1</i>, and <i>Lbx1</i> promoters but not that of <i>Gfap</i>, as determined by chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) (<i>n</i> = 3). (C) Immunostaining to detect Tuj1 (red) and enhanced green fluorescent protein (EGFP) (green) expression. Nuclear staining is shown by 4',6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 100 μm. Red arrows indicate Tuj1/EGFP double-positive cells, and white arrows indicate EGFP-positive cells. (D) Quantification of panel C. All data are presented as average ± SD. <i>t</i> test analysis was performed to calculate significance (*<i>p</i> < 0.05, **<i>p</i> < 0.005; not significant (ns), <i>p</i> > 0.05). All individual quantification data underlying panels A, B, and D can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001220#pbio.2001220.s018" target="_blank">S2 Data</a>.</p

Both endogenous and overexpressed methyl-CpG–binding domain protein 3 (Mbd3) are degraded as neural progenitor cells (NPCs) differentiate.

<p>(A) Immunostaining to detect Mbd3 expression in <i>wild-type</i> (<i>WT</i>), <i>Smek1 knockout</i> (<i>S1-KO</i>), <i>Smek2 knockout</i> (<i>S2-KO</i>) and <i>Smek1/2 double knockout</i> (<i>S1/2-dKO</i>) cells. Nuclear staining is shown by 4',6-diamidino-2-phenylindole (DAPI) (blue). Mbd3 is shown in green. Scale bar, 50 μm. (Lower) Quantification of Fig 3A. Scale bar, 50 μm. Diff. (d), differentiation days in vitro. (B) Western blot of Mbd3 in <i>WT</i> or <i>Smek knockout</i> NPCs that are under differentiation for 0, 1, or 3 d (<i>n</i> = 3). α-tubulin was used as an internal loading control. Arrows indicate multiple Mbd3 isoforms around 30–40 kDa (upper, Mbd3A; middle, Mbd3B; bottom, Mbd3C). (C) Quantification of Fig 3B. (D) Reverse transcription PCR (RT-PCR) analysis of <i>Mbd3</i> and <i>Gapdh</i> transcript levels at indicated days (d) of differentiation (<i>n</i> = 2). (E) Quantification of data shown in Fig 3D. (F) (left panel) Immunoblot (IB) analysis of Mbd3 and α-tubulin in cells treated with cycloheximide (CHX) for indicated times (h, hours) with or without MG132 in NPCs (<i>n</i> = 3) or 293T cells (<i>n</i> = 5). (Right panel) Quantification of band intensity in Fig 3F, left panel using the ImageJ software. (G) NPCs were treated with MG132 for 6 h, harvested, and the lysates were immunoblotted with anti-Mbd3, anti–α-tubulin and anti–β-catenin antibodies. β-catenin was used as a positive control (<i>n</i> = 2). Quantification of band intensity in Fig 3G, bottom panel using the ImageJ software. (H) Ubiquitylation of overexpressed Mbd3 in NPCs (<i>n</i> > 3). (I) Ubiquitylation of overexpressed Mbd3 in HEK293T transfected with Flag-Mbd3 and HA-Ub expression vectors and treated 1 d later with MG132 were detected by immunoprecipitation with Flag antibody followed by immunoblotting with HA antibody (upper panel) (<i>n</i> > 2). Levels of each protein in the whole cell lysate are shown with western blot. (J) Same as panels H and I except that endogenous Mbd3 polyubiquitylation was shown (<i>n</i> = 3). Values correspond to the average ± SD. Statistical <i>t</i> test analysis was performed to calculate significance (*<i>p</i> < 0.05, **<i>p</i> < 0.005, ***<i>p</i> < 0.0005; not significant (ns), <i>p</i> > 0.05). All quantification data underlying panels A, C, E, F, and G can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001220#pbio.2001220.s018" target="_blank">S2 Data</a>.</p

Boulogne news and Boulogne gazette : published every tuesday

23 septembre 18571857/09/23 (N953).Appartient à l’ensemble documentaire : IledeFr1Appartient à l’ensemble documentaire : NordPdeC

Suppressor of Mek null (Smek) and methyl-CpG–binding domain protein 3 (Mbd3) colocalize on neuronal gene promoters.

<p>(A) Smek1-chromatin immunoprecipitation sequencing (ChIP-seq) analysis in NPCs. Distribution of Smek1-binding peaks in NPCs. Proximal promoter regions are defined as sequences within 3 kb of the transcription starting site (TSS) of annotated genes. (B) Gene ontology (GO) analysis of annotated genes at Smek1-binding sites. (C) Smek1-binding peaks in NPCs in differentiation genes such as <i>Dlx1</i>, <i>Tlx3</i>, <i>NeuroD1</i>, <i>Ascl1</i>, and <i>Lbx1</i>. (D) ChIP-quantitative PCR (qPCR) analysis of Mbd3 occupancy at a Smek-binding locus in undifferentiated or differentiated conditions in <i>WT</i> (<i>n</i> = 3) and <i>Smek1/2 dKO</i> (<i>n</i> = 3) NPCs. (E) ChIP-qPCR analysis of Smek1 and Mbd3 occupancy at a Smek-binding locus in 0.2 or 2 d differentiated conditions in NPCs knocked down by shScramble (<i>n</i> = 3) and shMbd3 (<i>n</i> = 3) NPCs. (F) ChIP-qPCR analysis of HDAC1, HDAC2, MTA1, and acetyl histone H3 occupancy at a Smek-binding locus in undifferentiated or differentiated conditions in <i>WT</i> (<i>n</i> = 3) and <i>Smek1/2 dKO</i> (<i>n</i> = 3) NPCs. In panels D–F, immunoglobulin G (IgG) ChIP served as a negative control. Values are normalized to input control and represent average ± SD. <i>t</i> test analysis was performed to calculate the statistical significance (*<i>p</i> < 0.05, **<i>p</i> < 0.005). The ChIP-seq dataset for panels A–C can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001220#pbio.2001220.s017" target="_blank">S1 Data</a> and all individual quantification data for panels D–F can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001220#pbio.2001220.s018" target="_blank">S2 Data</a>.</p