DOI: 10.5061/dryad.zw3r2287f
title: 7-Dehydrocholesterol-derived oxysterols cause neurogenic defects in Smith-Lemli-Opitz syndrome

description: Defective 3beta-hydroxysterol-delta7 -reductase (DHCR7) in the
 developmental disorder, Smith-Lemli-Opitz syndrome (SLOS), results in
 deficiency in cholesterol and accumulation of its precursor,
 7-dehydrocholesterol (7-DHC). Here, we show that loss of DHCR7 causes
 accumulation of 7-DHC-derived oxysterol metabolites, premature
 neurogenesis, and perturbation of neuronal localization in developing
 murine or human cortical neural precursors, both in vitro and in vivo. We
 found that a major oxysterol, 3b,5a-dihydroxycholest-7-en-6-one (DHCEO),
 mediates these effects by initiating crosstalk between glucocorticoid
 receptor (GR) and neurotrophin receptor kinase TrkB.  Either loss
 of DHCR7 or direct exposure to DHCEO causes hyperactivation of GR and TrkB
 and their downstream MEK-ERK-C/EBP signaling pathway in cortical neural
 precursors.  Moreover, direct inhibition of GR activation with an
 antagonist or inhibition of DHCEO accumulation with antioxidants rescues
 the premature neurogenesis phenotype caused by the loss of
 DHCR7.  These results suggest that GR could be a new therapeutic
 target against the neurological defects observed in SLOS.
description: SLOS (<em>DHCR7</em>-mutant) and wild-type human
 induced pluripotent stem cells (hiPSCs) were differentiated into human
 neural progenitor cells (hNPCs) using the STEMdiff SMADi Neural Induction
 kit (StemCell Technologies) according to the manufacturer’s protocol. Each
 genotype (wild-type and <em>DHCR7</em>-mutant) was prepared
 three separate replicates. Total RNA was isolated from hNPCs and was
 subject to RNA sequencing at Novogene (Chula Vista, California). Raw RNA
 sequencing reads in FASTQ format were mapped to the human genome using
 HISAT (https://ccb.jhu.edu/software/hisat/; Last accessed January 22, 2021), and format conversions were performed using Samtools. Cufflinks (http://cole-trapnell-lab.github.io/cufflinks/; Last accessed January 22, 2021) was used to estimate relative abundances of transcripts from each RNA sample. Cuffdiff, a module of Cufflinks, was then used to determine differentially expressed genes (DEGs) between WT and KO hNPCs. DEGs met the following criteria: adjusted <i>p</i> value &lt; .05 (corresponding to the allowed false discovery rate of 5%) and fold-change &gt; 1.5 between genotypes.  A two-way hierarchical clustering dendrogram (complete-linkage method, Euclidean distance scale) of DEGs was used to visualize biological variability among samples, generated by R software using the “pheatmap” package (https://cran.r-project.org/web/packages/pheatmap/). To elucidate biological functions of DEGs, the Core Analysis feature of Ingenuity Pathway Analysis (IPA®, Qiagen) was used to identify significantly enriched Diseases and Biological Functions related to the nervous system.  Network interactions among DEGs involved in the Biological Function “development of the central nervous system” were assessed using STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis, set at the highest confidence interaction score and only connected nodes displayed (https://string-db.org/cgi/input.pl?sessionId=xCahIfrzvltC; Last accessed January 22, 2021). Enriched KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways were identified among DEGs in the STRING network. Finally, a Venn diagram was generated to demonstrate the overlap between genes dysregulated in KO hNPCs and genes in the SFARI database, a collection of genes implicated in autism susceptibility (https://gene.sfari.org/; Last accessed January 22, 2021).