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

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

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