Loss of Microglial Parkin Inhibits Necroptosis and Contributes to Neuroinflammation
Loss of Microglial Parkin Inhibits Necroptosis and Contributes to Neuroinflammation
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DOI：
10.1007/s12035-018-1264-9
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发表时间：
2019-04-01
影响因子：
5.1
通讯作者：
Pereira Rodrigues, Cecilia Maria
中科院分区：
文献类型：
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作者：
Antunes Dionisio, Pedro Eloi;Oliveira, Sara Rodrigues;Pereira Rodrigues, Cecilia Maria
Parkin是一种参与帕金森病（PD）的E3泛素连接酶。坏死性凋亡是一种受调节的细胞死亡形式，依赖于受体相互作用蛋白1（RIP 1）和3（RIP 3）。重要的是，帕金参与了可以改变炎症和坏死性凋亡的泛素化事件。在这里，我们研究了帕金如何影响小胶质细胞的功能。BV-2小神经胶质细胞与zVAD. fviv（zVAD）的孵育诱导高水平的细胞死亡和活力丧失，而N9小神经胶质细胞和原代小神经胶质细胞需要进一步的刺激。重要的是，Necrostatin-1（Nec-1），RIP 1激酶活性的抑制剂，废除细胞死亡，从而暗示RIP 1依赖性坏死性凋亡的细胞死亡。细胞死亡的特征在于坏死体组装，如通过RIP 1/RIP 3在不溶性组分中的螯合和MLKL磷酸化所确定的，这些都被Nec-1消除。此外，坏死性凋亡诱导条件导致TNF-分泌，这可能反过来有助于自分泌坏死性凋亡激活。有趣的是，parkin敲低保护BV-2细胞免于zVAD诱导的坏死性凋亡，这可能取决于在siRNA-PARK 2转染的细胞中检测到的更高的RIP 1泛素化水平。这种作用与炎症无关，因为用沉默的parkin对BV-2和原代小胶质细胞进行促炎刺激导致更强的促炎基因表达，这与zVAD暴露的BV-2细胞的观察结果相反。LPS介导的炎症因NF-B/JNK过度活化而加剧。最后，在任何条件下都没有检测到线粒体ROS产生的改变，从而排除了parkin在线粒体自噬中的作用。总之，在这里，我们揭示了帕金可能有未被怀疑的作用，小胶质细胞通过调节泛素化。帕金丢失加剧炎症并促进活化的小胶质细胞的存活，从而导致慢性神经炎症。
Parkin is an E3 ubiquitin ligase involved in Parkinson's disease (PD). Necroptosis is a regulated form of cell death that depends on receptor interacting protein 1 (RIP1) and 3 (RIP3). Importantly, parkin has been implicated in ubiquitination events that can alter inflammation and necroptosis. Here, we investigated how parkin influences microglial function. Incubation of BV-2 microglial cells with zVAD.fmk (zVAD) induced high levels of cell death and viability loss, while N9 microglial cells and primary microglia required further stimuli. Importantly, necrostatin-1 (Nec-1), an inhibitor of RIP1 kinase activity, abrogated cell death, thus implicating RIP1-dependent necroptosis in cell death. Cell death was characterized by necrosome assembly, as determined by sequestration of RIP1/RIP3 in insoluble fractions and by MLKL phosphorylation, which were all abolished by Nec-1. Also, necroptosis-inducing conditions led to TNF- secretion, which may in turn contribute to autocrine necroptosis activation. Interestingly, parkin knockdown protected BV-2 cells from zVAD-induced necroptosis, which may depend on the higher RIP1 ubiquitination levels detected in siRNA-PARK2 transfected cells. This effect was independent of inflammation, since pro-inflammatory stimulation of BV-2 and primary microglia with silenced parkin resulted in stronger pro-inflammatory gene expression, an opposite observation from zVAD-exposed BV-2 cells. LPS-mediated inflammation was exacerbated by NF-B/JNK over-activation. Finally, no alterations in mitochondrial ROS production were detected in any condition, thereby excluding the role of parkin in mitophagy. In conclusion, here, we reveal that parkin may have unsuspected roles in microglia by modulating ubiquitination. Parkin loss exacerbates inflammation and promotes survival of activated microglia, thus contributing to chronic neuroinflammation.