PDF(Pubmed)
Abstract：
The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) is a deNEDDylase controlling ubiquitination activity of cullin-RING-E3 ligases (CRLs) and thus the levels of key cellular proteins. While the CSN and its catalytic subunit CSN5 have been extensively studied in cancer, its role in inflammatory and neurological diseases is less understood. Following verification that CSN5 is expressed in mouse and human brain, here we studied the role of the CSN in neuroinflammation and ischemic neuronal damage employing models of relevant brain-resident cell types, an ex vivo organotypic brain slice culture model, and the CRL NEDDylation state-modifying drugs MLN4924 and CSN5i-3, which mimic and inhibit, respectively, CSN5 deNEDDylase activity. Untargeted mass spectrometry-based proteomics revealed that MLN4924 and CSN5i-3 substantially alter the microglial proteome, including inflammation-related proteins. Applying these drugs and mimicking microglial and endothelial inflammation as well as ischemic neuronal stress by TNF and oxygen-glucose-deprivation/reoxygenation (OGD/RO) treatment, respectively, we could link CSN5/CSN-mediated cullin deNEDDylation to reduction of microglial inflammation, attenuated cerebral endothelial inflammation, improved barrier integrity, as well as protection from ischemic stress-induced neuronal cell death. Specifically, MLN4924 reduced phagocytic activity, motility, and inflammatory cytokine expression of microglial cells, and this was linked to inhibition of inflammation-induced NF-κB and Akt signaling. Inversely, Csn5 knockdown and CSN5i-3 increased NF-κB signaling. Moreover, MLN4924 abrogated TNF-induced NF-κB signaling in cerebral microvascular endothelial cells (hCMECs) and rescued hCMEC monolayers from OGD/RO-triggered barrier leakage, while CSN5i-3 exacerbated permeability. In an ex vivo organotypic brain slice model of ischemia/reperfusion stress, MLN4924 protected from neuronal death, while CSN5i-3 impaired neuronal survival. Neuronal damage was attributable to microglial activation and inflammatory cytokines, as indicated by microglial shape tracking and TNF-blocking experiments. Our results indicate a protective role of the CSN in neuroinflammation via brain-resident cell types involved in ischemic brain disease and implicate CSN activity-mimicking deNEDDylating drugs as potential therapeutics.
摘要：
组成型光形态发生9（COP9）信号体（CSN）是一种脱NEDDDase酶，控制cullin-RING-E3连接酶（CRL）的泛素化活性，从而控制关键细胞蛋白的水平。虽然CSN及其催化亚基CSN5在癌症中已被广泛研究，它在炎症和神经系统疾病中的作用知之甚少。在验证CSN5在小鼠和人脑中表达后，在这里，我们研究了CSN在神经炎症和缺血性神经元损伤中的作用，采用相关的脑驻留细胞类型的模型，离体器官型脑切片培养模型，和CRLNEDDDylation状态修饰药物MLN4924和CSN5i-3，它们模拟和抑制，分别,CSN5去NEDDDase活性。基于非靶向质谱的蛋白质组学显示，MLN4924和CSN5i-3实质上改变了小胶质细胞蛋白质组，包括炎症相关蛋白.应用这些药物并通过TNF和氧-葡萄糖剥夺/复氧（OGD/RO）治疗模拟小胶质细胞和内皮炎症以及缺血性神经元应激，分别,我们可以将CSN5/CSN介导的cullin去NEDD化与减少小胶质细胞炎症联系起来，减轻脑血管炎症，改进的屏障完整性，以及对缺血应激诱导的神经元细胞死亡的保护。具体来说,MLN4924降低吞噬活性，运动性,和小胶质细胞的炎症细胞因子表达，这与抑制炎症诱导的NF-κB和Akt信号有关。相反，Csn5敲低和CSN5i-3增加NF-κB信号传导。此外，MLN4924消除了脑微血管内皮细胞（hCMECs）中TNF诱导的NF-κB信号传导，并从OGD/RO触发的屏障渗漏中拯救了hCMEC单层，而CSN5i-3加剧了渗透性。在缺血/再灌注应激的离体器官型脑切片模型中，MLN4924保护免受神经元死亡，而CSN5i-3损害神经元存活。神经元损伤可归因于小胶质细胞活化和炎性细胞因子，如小胶质细胞形状追踪和TNF阻断实验所示。我们的结果表明，CSN通过参与缺血性脑疾病的大脑驻留细胞类型在神经炎症中具有保护作用，并暗示CSN活性模拟去NEDDylating药物作为潜在的治疗药物。
