School of Medicine, Department of Neuroscience and Experimental Therapeutics, Medical Research and Education, Texas A&M University Health Science Center, Building 8447 Riverside Parkway, Bryan, TX, 77807-3260, USA.
Women's Health in Neuroscience, Texas A&M University Health Science Center, Bryan, TX, USA.
Biol Sex Differ. 2023 Apr 15;14(1):19. doi: 10.1186/s13293-023-00503-0.
Prenatal alcohol (ethanol) exposure (PAE) results in brain growth restriction, in part, by reprogramming self-renewal and maturation of fetal neural stem cells (NSCs) during neurogenesis. We recently showed that ethanol resulted in enrichment of both proteins and pro-maturation microRNAs in sub-200-nm-sized extracellular vesicles (EVs) secreted by fetal NSCs. Moreover, EVs secreted by ethanol-exposed NSCs exhibited diminished efficacy in controlling NSC metabolism and maturation. Here we tested the hypothesis that ethanol may also influence the packaging of RNAs into EVs from cell-of-origin NSCs.
Sex-specified fetal murine iso-cortical neuroepithelia from three separate pregnancies were maintained ex vivo, as neurosphere cultures to model the early neurogenic niche. EVs were isolated by ultracentrifugation from NSCs exposed to a dose range of ethanol. RNA from paired EV and cell-of-origin NSC samples was processed for ribosomal RNA-depleted RNA sequencing. Differential expression analysis and exploratory weighted gene co-expression network analysis (WGCNA) identified candidate genes and gene networks that were drivers of alterations to the transcriptome of EVs relative to cells.
The RNA content of EVs differed significantly from cell-of-origin NSCs. Biological sex contributed to unique transcriptome variance in EV samples, where > 75% of the most variant transcripts were also sex-variant in EVs but not in cell-of-origin NSCs. WGCNA analysis also identified sex-dependent enrichment of pathways, including dopamine receptor binding and ectoderm formation in female EVs and cell-substrate adhesion in male EVs, with the top significant DEGs from differential analysis of overall individual gene expressions, i.e., Arhgap15, enriched in female EVs, and Cenpa, enriched in male EVs, also serving as WCGNA hub genes of sex-biased EV WGCNA clusters. In addition to the baseline RNA content differences, ethanol exposure resulted in a significant dose-dependent change in transcript expression in both EVs and cell-of-origin NSCs that predominantly altered sex-invariant RNAs. Moreover, at the highest dose, ~ 73% of significantly altered RNAs were enriched in EVs, but depleted in NSCs.
The EV transcriptome is distinctly different from, and more sex-variant than, the transcriptome of cell-of-origin NSCs. Ethanol, a common teratogen, results in dose-dependent sorting of RNA transcripts from NSCs to EVs which may reprogram the EV-mediated endocrine environment during neurogenesis.
产前酒精（乙醇）暴露（PAE）部分通过在神经发生过程中重新编程胎儿神经干细胞（NSC）的自我更新和成熟，导致大脑生长受限。我们最近表明，乙醇导致 200nm 以下大小的胎儿 NSC 分泌的细胞外囊泡（EV）中的蛋白质和促成熟 microRNA 富集。此外，乙醇暴露的 NSC 分泌的 EV 控制 NSC 代谢和成熟的功效降低。在这里，我们测试了这样一个假设，即乙醇也可能影响源自原始细胞的 NSC 中 RNA 包装到 EV 中。
从三个单独的妊娠中分离出性别的胎儿鼠皮质神经上皮细胞，在体外作为神经球培养物，以模拟早期神经发生龛。通过超速离心从暴露于乙醇剂量范围内的 NSC 中分离 EV。对配对的 EV 和原始 NSC 样本的 RNA 进行核糖体 RNA 耗尽 RNA 测序处理。差异表达分析和探索性加权基因共表达网络分析（WGCNA）鉴定了候选基因和基因网络，这些基因和基因网络是 EV 中转录组相对于细胞发生改变的驱动因素。
EV 的 RNA 含量与原始 NSC 有显著差异。生物性别导致 EV 样本中转录组的独特变异，其中 >75%的最变异转录本在 EV 中也是性别变异的，但在原始 NSC 中则不是。WGCNA 分析还鉴定了性别依赖的途径富集，包括女性 EV 中的多巴胺受体结合和外胚层形成，以及男性 EV 中的细胞-基质粘附，从整体个体基因表达差异分析的 top 显著 DEGs，即 Arhgap15，在女性 EV 中富集，Cenpa，在男性 EV 中富集，也作为性别偏向 EV WGCNA 聚类的 WCGNA 枢纽基因。除了基线 RNA 含量差异外，乙醇暴露还导致 EV 和原始 NSC 中转录表达的显著剂量依赖性变化，主要改变性别变异的 RNA。此外，在最高剂量下， >73%的显著改变的 RNA 富集在 EV 中，但在 NSCs 中耗尽。
EV 的转录组与原始 NSC 的转录组明显不同，且性别变异更大。乙醇是一种常见的致畸剂，导致 NSC 中 RNA 转录本的剂量依赖性分拣到 EV 中，这可能会重新编程神经发生过程中的 EV 介导的内分泌环境。
