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

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

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

Richness and diversity of microbial communities of the leaf litter of 4 plant species during 42 days incubation A: number of bands obtained from DGGE analysis of bacterial 16S rRNA gene PCR amplicons, B: Shannon’s index calculated from bacterial DGGE profiles, C: number of bands obtained from DGGE analysis of fungal 18S rRNA gene PCR amplicons, and D: Shannon’s index calculated from fungal DGGE profiles.

<p>The plant species <i>Baccaure ramiflora, Hevea brasiliensis, Pleioblastus amarus</i> and <i>Pometi tomentos</i> were denoted by Br, Hb, Pa and Pt, respectively.</p

DGGE profiles of fungal 18S rRNA gene amplicons derived from the leaf litter of the plant species A: <i>Baccaure ramiflora</i>, B: <i>Hevea brasiliensis</i>, C: <i>Pleioblastus amarus</i> and D: <i>Pometi tomentos</i>.

<p>The numbers of days of incubations are indicated. Each sampling day has two replicates of separate incubations denote by A and B. The arrows indicate the bands that have sequences affiliated to <i>Aspergillus</i> (day 1) and <i>Pleosporales</i> (day 42).</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

A Pilot Genome-Scale Profiling of DNA Methylation in Sporadic Pituitary Macroadenomas: Association with Tumor Invasion and Histopathological Subtype

<div><p>Pituitary adenomas (PAs) are neoplasms that may cause a variety of neurological and endocrine effects. Although known causal contributors include heredity, hormonal influence and somatic mutations, the pathophysiologic mechanisms driving tumorigenesis and invasion of sporadic PAs remain unknown. We hypothesized that alterations in DNA methylation are associated with PA invasion and histopathology subtype, and that genome-scale methylation analysis may complement current classification methods for sporadic PAs. Twenty-four surgically-resected sporadic PAs with varying histopathological subtypes were assigned dichotomized Knosp invasion scores and examined using genome-wide DNA methylation profiling and RNA sequencing. PA samples clustered into subgroups according to functional status. Compared with hormonally-active PAs, nonfunctional PAs exhibited global DNA hypermethylation (mean beta-value 0.47 versus 0.42, <i>P</i> = 0.005); the most significant site of differential DNA methylation was within the promoter region of the potassium voltage-gated channel <i>KCNAB2</i> (FDR = 5.11×10⁻¹⁰). Pathway analysis of promoter-associated CpGs showed that nonfunctional PAs are potentially associated with the ion-channel activity signal pathway. DNA hypermethylation tended to be negatively correlated with gene expression. DNA methylation analysis may be used to identify candidate genes involved in PA function and may potentially complement current standard immunostaining classification in sporadic PAs. DNA hypermethylation of <i>KCNAB2</i> and downstream ion-channel activity signal pathways may contribute to the endocrine-inactive status of nonfunctional PAs.</p></div

Gene expression.

<p>A, Linear regression analysis showed negative trend of DNA methylation and gene expression. B, Three overlapped significant genes of DNA methylation and gene expression when compared NFAs to FAs. C, gene <i>ODAM</i> expression in one FA subject of d0079 which secreted hormones of GH and TSH, <i>GAPDH</i> gene expression was used as control.</p

Top 30 significant genes with differential DNA methylation between NFAs and FAs.

a<p>Probe_CpG: the Illumina HM450 probe ID.</p>b<p>SD: standard deviation.</p>c<p>NFA-FA: the mean beta value subtractive difference between NFA tumors and FA tumors.</p>d<p>FDR: False Discovery Rate.</p>e<p>TSS200: 200 bp within transcription start site.</p>f<p>TSS1500: 1500 bp within the transcription start site.</p><p>* Promoter Associated.</p><p>Link to the whole information of the Probe_CpG: <a href="http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL13534" target="_blank">http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL13534</a>.</p