Lewkowicz Przemysław, Cwiklińska Hanna, Mycko Marcin P, Cichalewska Maria, Domowicz Małgorzata, Lewkowicz Natalia, Jurewicz Anna, Selmaj Krzysztof W
Department of Neurology, Laboratory of Neuroimmunology, Medical University of Lodz, 92-213 Lodz, Poland.
Department of Neurology, Laboratory of Neuroimmunology, Medical University of Lodz, 92-213 Lodz, Poland
J Neurosci. 2015 May 13;35(19):7521-37. doi: 10.1523/JNEUROSCI.4794-14.2015.
MicroRNAs (miRNAs) associate with Argonaute (Ago), GW182, and FXR1 proteins to form RNA-induced silencing complexes (RISCs). RISCs represent a critical checkpoint in the regulation and bioavailability of miRNAs. Recent studies have revealed dysregulation of miRNAs in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE); however, the function of RISCs in EAE and MS is largely unknown. Here, we examined the expression of Ago, GW182, and FXR1 in CNS tissue, oligodendrocytes (OLs), brain-infiltrating T lymphocytes, and CD3(+)splenocytes (SCs) of EAE mic, and found that global RISC protein levels were significantly dysregulated. Specifically, Ago2 and FXR1 levels were decreased in OLs and brain-infiltrating T cells in EAE mice. Accordingly, assembly of Ago2/GW182/FXR1 complexes in EAE brain tissues was disrupted, as confirmed by immunoprecipitation experiments. In parallel with alterations in RISC complex content in OLs, we found downregulation of miRNAs essential for differentiation and survival of OLs and myelin synthesis. In brain-infiltrating T lymphocytes, aberrant RISC formation contributed to miRNA-dependent proinflammatory helper T-cell polarization. In CD3(+) SCs, we found increased expression of both Ago2 and FXR1 in EAE compared with nonimmunized mice. Therefore, our results demonstrate a gradient in expression of miRNA between primary activated T cells in the periphery and polarized CNS-infiltrating T cells. These results suggest that, in polarized autoreactive effector T cells, miRNA synthesis is inhibited in response to dysregulated RISC assembly, allowing these cells to maintain a highly specific proinflammatory program. Therefore, our findings may provide a mechanism that leads to miRNA dysregulation in EAE/MS.
微小RNA（miRNA）与AGO、GW182和FXR1蛋白结合形成RNA诱导沉默复合体（RISC）。RISC是miRNA调控和生物利用度的关键检查点。最近的研究揭示了miRNA在多发性硬化症（MS）及其动物模型实验性自身免疫性脑脊髓炎（EAE）中的失调；然而，RISC在EAE和MS中的功能在很大程度上尚不清楚。在这里，我们检测了EAE小鼠中枢神经系统组织、少突胶质细胞（OL）、脑浸润性T淋巴细胞和CD3(+)脾细胞（SC）中AGO、GW182和FXR1的表达，发现整体RISC蛋白水平显著失调。具体而言，EAE小鼠的OL和脑浸润性T细胞中AGO2和FXR1水平降低。相应地，免疫沉淀实验证实EAE脑组织中AGO2/GW182/FXR1复合体的组装被破坏。与OL中RISC复合体含量的变化同时，我们发现对OL分化、存活和髓鞘合成至关重要的miRNA下调。在脑浸润性T淋巴细胞中，异常的RISC形成导致了miRNA依赖性促炎辅助性T细胞极化。在CD3(+)SC中，我们发现与未免疫小鼠相比，EAE中AGO2和FXR1的表达均增加。因此，我们的结果表明在外周初级活化T细胞和极化的中枢神经系统浸润性T细胞之间miRNA表达存在梯度。这些结果表明，在极化的自身反应性效应T细胞中，miRNA合成因失调的RISC组装而受到抑制，使这些细胞能够维持高度特异性的促炎程序。因此，我们的发现可能提供了一种导致EAE/MS中miRNA失调的机制。
