MicroRNA (miRNA) expression in the brain is altered in neurodegenerative diseases. Recent studies demonstrated that selected miRNAs conventionally regulating gene expression at the post-transcriptional level can act extracellularly as signaling molecules. The identity of miRNA species serving as membrane receptor ligands involved in neuronal apoptosis in the central nervous system (CNS), as well as the miRNAs’ sequence and structure required for this mode of action remained largely unresolved. Using a microarray-based screening approach we analyzed apoptotic cortical neurons of C56BL/6 mice and their supernatant with respect to alterations in miRNA expression/presence. HEK-Blue Toll-like receptor (TLR) 7/8 reporter cells, primary microglia and macrophages derived from human and mouse were employed to test the potential of the identified miRNAs released from apoptotic neurons to serve as signaling molecules for the RNA-sensing receptors. Biophysical and bioinformatical approaches, as well as immunoassays and sequential microscopy were used to analyze the interaction between candidate miRNA and TLR. Immunocytochemical and -histochemical analyses of murine CNS cultures and adult mice intrathecally injected with miRNAs, respectively, were performed to evaluate the impact of miRNA-induced TLR activation on neuronal survival and microglial activation. We identified a specific pattern of miRNAs released from apoptotic cortical neurons that activate TLR7 and/or TLR8, depending on sequence and species. Exposure of microglia and macrophages to certain miRNA classes released from apoptotic neurons resulted in the sequence-specific production of distinct cytokines/chemokines and increased phagocytic activity. Out of those miRNAs miR-100-5p and miR-298-5p, which have consistently been linked to neurodegenerative diseases, entered microglia, located to their endosomes, and directly bound to human TLR8. The miRNA-TLR interaction required novel sequence features, but no specific structure formation of mature miRNA. As a consequence of miR-100-5p- and miR-298-5p-induced TLR activation, cortical neurons underwent cell-autonomous apoptosis. Presence of miR-100-5p and miR-298-5p in cerebrospinal fluid led to neurodegeneration and microglial accumulation in the murine cerebral cortex through TLR7 signaling. Our data demonstrate that specific miRNAs are released from apoptotic cortical neurons, serve as endogenous TLR7/8 ligands, and thereby trigger further neuronal apoptosis in the CNS. Our findings underline the recently discovered role of miRNAs as extracellular signaling molecules, particularly in the context of neurodegeneration.

中文翻译：

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凋亡皮质神经元释放的 microRNA-100-5p 和 microRNA-298-5p 是导致神经退行性变的内源性 Toll 样受体 7/8 配体

大脑中的 MicroRNA (miRNA) 表达在神经退行性疾病中发生了改变。最近的研究表明，通常在转录后水平调节基因表达的选定 miRNA 可以在细胞外充当信号分子。作为涉及中枢神经系统 (CNS) 中神经元凋亡的膜受体配体的 miRNA 物种的身份，以及这种作用模式所需的 miRNA 序列和结构在很大程度上仍未得到解决。使用基于微阵列的筛选方法，我们分析了 C56BL/6 小鼠的凋亡皮质神经元及其上清液中 miRNA 表达/存在的变化。HEK-Blue Toll 样受体 (TLR) 7/8 报告细胞，来自人和小鼠的原代小胶质细胞和巨噬细胞被用来测试从凋亡神经元释放的已鉴定 miRNA 作为 RNA 感应受体的信号分子的潜力。生物物理和生物信息学方法，以及免疫测定和序列显微镜被用来分析候选 miRNA 和 TLR 之间的相互作用。分别对小鼠 CNS 培养物和鞘内注射 miRNA 的成年小鼠进行免疫细胞化学和组织化学分析，以评估 miRNA 诱导的 TLR 活化对神经元存活和小胶质细胞活化的影响。我们确定了从凋亡皮质神经元释放的特定模式的 miRNA，其激活 TLR7 和/或 TLR8，具体取决于序列和物种。小胶质细胞和巨噬细胞暴露于凋亡神经元释放的某些 miRNA 类别导致不同细胞因子/趋化因子的序列特异性产生和吞噬活性增加。在这些 miRNA 中，一直与神经退行性疾病相关的 miR-100-5p 和 miR-298-5p 进入小胶质细胞，位于其内体，并直接与人类 TLR8 结合。miRNA-TLR相互作用需要新的序列特征，但成熟miRNA没有特定的结构形成。由于 miR-100-5p 和 miR-298-5p 诱导的 TLR 激活，皮质神经元经历了细胞自主凋亡。脑脊液中 miR-100-5p 和 miR-298-5p 的存在通过 TLR7 信号传导导致小鼠大脑皮层中的神经变性和小胶质细胞积聚。我们的数据表明，特定的 miRNA 从凋亡的皮层神经元中释放出来，作为内源性 TLR7/8 配体，从而引发 CNS 中进一步的神经元凋亡。我们的研究结果强调了最近发现的 miRNA 作为细胞外信号分子的作用，特别是在神经退行性变的情况下。