7-Dehydrocholesterol-derived oxysterols cause neurogenic defects in Smith-Lemli-Opitz syndrome

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.

Methods
SLOS (DHCR7-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 DHCR7-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 p value < .05 (corresponding to the allowed false discovery rate of 5%) and fold-change > 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).

发育障碍疾病史密斯-莱米-奥皮茨综合征（Smith-Lemli-Opitz syndrome, SLOS）中，3β-羟固醇Δ7-还原酶（3beta-hydroxysterol-delta7-reductase, DHCR7）缺陷会导致胆固醇缺乏，并使其前体7-脱氢胆固醇（7-dehydrocholesterol, 7-DHC）蓄积。本研究证实，DHCR7缺失会造成7-DHC衍生氧固醇代谢物蓄积、神经发生提前，以及在体内外的小鼠和人皮质神经前体细胞中引发神经元定位紊乱。我们发现，主要氧固醇3b,5a-二羟胆甾-7-烯-6-酮（3b,5a-dihydroxycholest-7-en-6-one, DHCEO）通过介导糖皮质激素受体（glucocorticoid receptor, GR）与神经营养因子受体激酶TrkB（neurotrophin receptor kinase TrkB）之间的交叉对话，引发上述效应。无论是DHCR7缺失，还是直接暴露于DHCEO，都会使皮质神经前体细胞中的GR与TrkB及其下游MEK-ERK-C/EBP信号通路过度激活。此外，使用拮抗剂直接抑制GR激活，或通过抗氧化剂阻断DHCEO蓄积，均可挽救DHCR7缺失引发的提前神经发生表型。上述结果表明，GR可作为SLOS患者神经系统缺陷的新型治疗靶点。

方法
本研究使用STEMdiff SMADi神经诱导试剂盒（StemCell Technologies），按照厂商说明书将SLOS（DHCR7突变型）与野生型人诱导多能干细胞（human induced pluripotent stem cells, hiPSCs）诱导分化为人神经前体细胞（neural progenitor cells, hNPCs），每种基因型（野生型与DHCR7突变型）均设置3次独立生物学重复。从hNPCs中提取总RNA，并在Novogene公司（美国加利福尼亚州丘拉维斯塔）进行RNA测序。将FASTQ格式的原始RNA测序读数通过HISAT（https://ccb.jhu.edu/software/hisat/; 最后访问时间：2021年1月22日）比对至人类基因组，并使用Samtools进行格式转换。使用Cufflinks（http://cole-trapnell-lab.github.io/cufflinks/; 最后访问时间：2021年1月22日）估算每个RNA样本的转录本相对丰度，随后通过Cufflinks模块Cuffdiff鉴定野生型与敲除型hNPCs之间的差异表达基因（differentially expressed genes, DEGs）。DEGs需满足以下标准：校正P值<0.05（对应5%的允许错误发现率），且基因型间折叠变化>1.5。使用R软件结合pheatmap包（https://cran.r-project.org/web/packages/pheatmap/）构建DEGs的双向层次聚类树（完全连接法、欧氏距离标尺），以可视化样本间的生物学异质性。为阐明DEGs的生物学功能，使用Ingenuity通路分析（Ingenuity Pathway Analysis, IPA®, Qiagen）的核心分析功能，筛选与神经系统相关的显著富集疾病与生物学功能。使用STRING基因/蛋白质相互作用检索工具（Search Tool for the Retrieval of Interacting Genes/Proteins, STRING）评估参与“中枢神经系统发育”生物学功能的DEGs之间的网络相互作用，设置为最高置信度相互作用评分，仅显示相连节点（https://string-db.org/cgi/input.pl?sessionId=xCahIfrzvltC; 最后访问时间：2021年1月22日）。在STRING网络的DEGs中筛选富集的京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路。最后，绘制韦恩图以展示敲除型hNPCs中失调基因与SFARI数据库（自闭症易感基因集合，https://gene.sfari.org/; 最后访问时间：2021年1月22日）中基因的重叠情况。