COP9信号体（CSN）是一种deNEDDylase，可控制cullin-RING-E3连接酶（CRLs）的泛素化活性，从而调节关键细胞蛋白的水平。尽管CSN及其催化亚基CSN5在癌症中已有广泛研究，但其在炎症和神经系统疾病中的作用尚不清楚。通过验证CSN5在小鼠和人脑中的表达，我们研究了CSN在神经炎症和缺血性神经细胞损伤中的作用，采用与相关的脑细胞类型、离体器官培养模型以及模拟和抑制CSN5 deNEDDylase活性的CRL NEDDylation状态调节药物MLN4924和CSN5i-3。无靶向基于质谱的蛋白质组学研究发现，MLN4924和CSN5i-3显著改变了小胶质细胞的蛋白质组，包括炎症相关蛋白。通过应用这些药物模拟小胶质细胞和内皮细胞炎症以及通过TNF和缺氧葡萄糖剥离/复氧（OGD/RO）处理模拟缺血性神经细胞压力，我们能够将CSN5/CSN介导的cullin去neddylation与小胶质炎症减少、脑血管内皮炎症减轻、屏障完整性改善以及对缺血性压力引起的神经细胞死亡的保护联系起来。具体而言，MLN4924降低了小胶质细胞的吞噬活性、运动性和炎症细胞因子的表达，这与炎症诱导的NF-κB和Akt信号的抑制有关。相反，Csn5沉默和CSN5i-3增加了NF-κB信号。此外，MLN4924消除了脑微血管内皮细胞（hCMECs）中TNF诱导的NF-κB信号，并挽救了hCMEC单层在OGD/RO诱导的屏障渗漏下的完整性，而CSN5i-3则加重了渗透性。在缺血/复灌压力的离体器官培养脑片模型中，MLN4924保护了神经细胞的存活，而CSN5i-3则损害了神经细胞的存活。神经细胞损伤归因于小胶质激活和炎性细胞因子，这一点可以通过小胶质细胞形状跟踪和TNF阻断实验来证明。我们的结果表明，CSN在神经炎症中的保护作用通过与缺血性脑疾病相关的神经系统细胞类型进行调节，并且暗示CSN活性模拟去neddylation药物可能是潜在的治疗药物。©2023. 作者（们）。

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.© 2023. The Author(s).