Small extracellular vesicles (sEVs) mediate intercellular communication by transferring RNA, proteins, and lipids to recipient cells. These cargo molecules are selectively loaded into sEVs and mirror the physiological state of the donor cells. Given that sEVs can cross the blood–brain barrier and their composition can change in neurological disorders, the molecular signatures of sEVs in circulation can be potential disease biomarkers. Characterizing the molecular composition of sEVs from different cell types is an important first step in determining which donor cells contribute to the circulating sEVs. Cell culture supernatants from primary mouse cortical neurons and astrocytes were used to purify sEVs by differential ultracentrifugation and sEVs were characterized using nanoparticle tracking analysis, transmission electron microscopy and western blot. RNA sequencing was used to determine differential expression and loading patterns of miRNAs in sEVs released by primary neurons and astrocytes. Motif analysis was conducted on enriched miRNAs in sEVs and their respective donor cells. Sequencing total cellular RNA, and miRNAs from sEVs isolated from culture media of postnatal mouse cortical neurons and astrocytes revealed a distinct profile between sEVs and their corresponding cells. Though the total number of detected miRNAs in astrocytes was greater than neurons, neurons expressed more sEV-associated miRNAs than astrocytes. Only 20.7% of astrocytic miRNAs were loaded into sEVs, while 41.0% of neuronal miRNAs were loaded into sEVs, suggesting differences in the cellular sorting mechanisms. We identified short RNA sequence motifs, or EXOmotifs, on the miRNAs that were differentially loaded or excluded from sEVs. A sequence motif GUAC was enriched in astrocytic sEVs. miRNAs preferably retained in neurons or astrocytes had a similar RNA motif CACACA, suggesting a cell-type-independent mechanism to maintain cellular miRNAs. mRNAs of five RNA-binding proteins associated with passive or active RNA sorting into sEVs were differentially expressed between neurons and astrocytes, one of which, major vault protein was higher in astrocytes than in neurons and detected in astrocytic sEVs. Our studies suggest differences in RNA sorting into sEVs. These differences in miRNA signatures can be used for determining the cellular sources of sEVs altered in neurological disorders.

中文翻译：

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神经元和星形胶质细胞将差异RNA包装成小细胞外囊泡


小细胞外囊泡 (sEV) 通过将 RNA、蛋白质和脂质转移到受体细胞来介导细胞间通讯。这些货物分子被选择性地装载到 sEV 中并反映供体细胞的生理状态。鉴于 sEV 可以穿过血脑屏障，并且其组成可能在神经系统疾病中发生变化，因此循环中 sEV 的分子特征可能是潜在的疾病生物标志物。表征不同细胞类型 sEV 的分子组成是确定哪些供体细胞对循环 sEV 做出贡献的重要的第一步。来自原代小鼠皮质神经元和星形胶质细胞的细胞培养上清液用于通过差速超速离心纯化 sEV，并使用纳米颗粒跟踪分析、透射电子显微镜和蛋白质印迹来表征 sEV。 RNA 测序用于确定初级神经元和星形胶质细胞释放的 sEV 中 miRNA 的差异表达和负载模式。对 sEV 及其各自供体细胞中富集的 miRNA 进行基序分析。对从出生后小鼠皮质神经元和星形胶质细胞的培养基中分离出的 sEV 的总细胞 RNA 和 miRNA 进行测序，揭示了 sEV 与其相应细胞之间的独特特征。尽管星形胶质细胞中检测到的 miRNA 总数多于神经元，但神经元比星形胶质细胞表达更多的 sEV 相关 miRNA。只有 20.7% 的星形细胞 miRNA 被加载到 sEV 中，而 41.0% 的神经元 miRNA 被加载到 sEV 中，这表明细胞分选机制存在差异。我们在 sEV 中差异加载或排除的 miRNA 上鉴定了短 RNA 序列基序或 EXO 基序。序列基序 GUAC 在星形细胞 sEV 中富集。 优选保留在神经元或星形胶质细胞中的 miRNA 具有相似的 RNA 基序 CACACA，这表明维持细胞 miRNA 的细胞类型无关的机制。与被动或主动 RNA 分选到 sEV 相关的五种 RNA 结合蛋白的 mRNA 在神经元和星形胶质细胞之间存在差异表达，其中一种主要穹窿蛋白在星形胶质细胞中的表达高于神经元，并在星形胶质细胞 sEV 中检测到。我们的研究表明 sEV 中 RNA 分类存在差异。这些 miRNA 特征的差异可用于确定神经系统疾病中改变的 sEV 的细胞来源。