TNF-alpha-induced microglia activation requires miR-342: impact on NF-kB signaling and neurotoxicity
TNF-alpha-induced microglia activation requires miR-342: impact on NF-kB signaling and neurotoxicity
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TNF-α诱导的小胶质细胞活化需要miR-342：对NF-kB信号传导和神经毒性的影响
DOI：
10.1038/s41419-020-2626-6
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发表时间：
2020-06-02
影响因子：
9
通讯作者：
Almeida, Maria Ines
中科院分区：
文献类型：
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作者：
Bras, Joao Paulo;Bravo, Joana;Almeida, Maria Ines
越来越多的证据表明，由小胶质细胞过度激活引起的持续性神经炎与一些神经和精神疾病的发展和加重有关。在某些病理条件下，小胶质细胞产生高水平的细胞毒性和炎症介质，如肿瘤坏死因子-α(TNF-α)，可通过正反馈机制重新激活小胶质细胞。然而，可以有效靶向控制肿瘤坏死因子-α介导的小胶质细胞过度激活的特定分子介质尚未被发现。在这种背景下，我们的目标是鉴定新的肿瘤坏死因子-α介导的微RNA，并分析它们在小胶质细胞激活中的作用，以及探索它们对细胞与神经元通讯的影响。对肿瘤坏死因子-α刺激的原代大鼠小胶质细胞进行miRNA微阵列，然后进行RT-qPCR验证。通过体外功能获得和功能丧失分析以及蛋白质组学分析来分析miR-342在小胶质细胞激活中的作用。使用微流体系统将小胶质细胞与海马神经元进行共培养，以了解其对神经毒性的影响。原代培养的大鼠小胶质细胞经肿瘤坏死因子-α刺激后，NOS2、肿瘤坏死因子和IL-1b基因表达上调。此外，PH-NF-kB p65水平也升高。MiRNA微阵列分析和RT-qPCR验证表明，肿瘤坏死因子-α刺激诱导miR-342表达上调。有趣的是，在N9小胶质细胞中过表达miR-342足以通过抑制Bag-1激活NF-kB途径，导致TNF-α和IL-1β的分泌增加。相反，抑制miR-342可导致肿瘤坏死因子-α激活后这些细胞因子水平的显著下降。事实上，肿瘤坏死因子α刺激的小胶质细胞和miR-342过表达的小胶质细胞都极大地影响了神经元的活性。值得注意的是，在这些共同培养的上清中检测到亚硝酸盐水平的增加。在全球范围内，我们的发现表明miR-342是肿瘤坏死因子-α介导的小胶质细胞激活的关键介质，也是治疗小胶质细胞驱动的神经炎症的潜在靶点。
Growing evidences suggest that sustained neuroinflammation, caused by microglia overactivation, is implicated in the development and aggravation of several neurological and psychiatric disorders. In some pathological conditions, microglia produce increased levels of cytotoxic and inflammatory mediators, such as tumor necrosis factor alpha (TNF-alpha), which can reactivate microglia in a positive feedback mechanism. However, specific molecular mediators that can be effectively targeted to control TNF-alpha-mediated microglia overactivation, are yet to be uncovered. In this context, we aim to identify novel TNF-alpha-mediated micro(mi)RNAs and to dissect their roles in microglia activation, as well as to explore their impact on the cellular communication with neurons. A miRNA microarray, followed by RT-qPCR validation, was performed on TNF-alpha-stimulated primary rat microglia. Gain- and loss-of-function in vitro assays and proteomic analysis were used to dissect the role of miR-342 in microglia activation. Co-cultures of microglia with hippocampal neurons, using a microfluidic system, were performed to understand the impact on neurotoxicity. Stimulation of primary rat microglia with TNF-alpha led to an upregulation of Nos2, Tnf, and Il1b mRNAs. In addition, ph-NF-kB p65 levels were also increased. miRNA microarray analysis followed by RT-qPCR validation revealed that TNF-alpha stimulation induced the upregulation of miR-342. Interestingly, miR-342 overexpression in N9 microglia was sufficient to activate the NF-kB pathway by inhibiting BAG-1, leading to increased secretion of TNF-alpha and IL-1 beta. Conversely, miR-342 inhibition led to a strong decrease in the levels of these cytokines after TNF-alpha activation. In fact, both TNF-alpha-stimulated and miR-342-overexpressing microglia drastically affected neuron viability. Remarkably, increased levels of nitrites were detected in the supernatants of these co-cultures. Globally, our findings show that miR-342 is a crucial mediator of TNF-alpha-mediated microglia activation and a potential target to tackle microglia-driven neuroinflammation.