Vascular Biology, Institute for Stroke and Dementia Research (ISD), LMU Klinikum, Ludwig-Maximilian-University (LMU) Munich, Feodor-Lynen-Straße 17, 81377, Munich, Germany.
Centre for Clinical Brain Sciences, The University of Edinburgh, Edinburgh, UK.
Cell Mol Life Sci. 2023 Aug 19;80(9):262. doi: 10.1007/s00018-023-04911-8.
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.
组成型光形态发生 9 (COP9) 信号体 (CSN) 是一种去 NEDDylase，控制着连接酶 (CRLs) 的泛素化活性，从而控制着关键细胞蛋白的水平。虽然 CSN 及其催化亚基 CSN5 在癌症中得到了广泛研究，但它在炎症和神经疾病中的作用知之甚少。在验证了 CSN5 在小鼠和人类大脑中表达后，我们在这里研究了 CSN 在神经炎症和缺血性神经元损伤中的作用，采用了相关脑驻留细胞类型的模型、离体器官型脑片培养模型以及 CRL NEDDylation 状态修饰药物 MLN4924 和 CSN5i-3，分别模拟和抑制 CSN5 的去 NEDDylase 活性。基于非靶向质谱的蛋白质组学分析表明，MLN4924 和 CSN5i-3 可显著改变小胶质细胞的蛋白质组，包括炎症相关蛋白。应用这些药物并模拟小胶质细胞和内皮细胞炎症以及 TNF 和氧葡萄糖剥夺/再灌注 (OGD/RO) 处理引起的缺血性神经元应激，我们可以将 CSN5/CSN 介导的连接酶去 NEDDylation 与小胶质细胞炎症的减少、脑内皮炎症的减弱、屏障完整性的改善以及对缺血性应激诱导的神经元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、迁移性和炎症细胞因子的表达，这与抑制炎症诱导的 NF-κB 和 Akt 信号通路有关。相反，Csn5 敲低和 CSN5i-3 增加了 NF-κB 信号通路。此外，MLN4924 阻断了 TNF 诱导的脑微血管内皮细胞 (hCMECs) 中的 NF-κB 信号通路，并挽救了 hCMEC 单层从 OGD/RO 触发的屏障渗漏，而 CSN5i-3 则加剧了通透性。在离体器官型脑片缺血/再灌注应激模型中，MLN4924 可防止神经元死亡，而 CSN5i-3 则损害神经元存活。神经元损伤归因于小胶质细胞的激活和炎症细胞因子，这是通过小胶质细胞形态跟踪和 TNF 阻断实验表明的。我们的结果表明，CSN 通过参与缺血性脑疾病的脑驻留细胞类型在神经炎症中发挥保护作用，并暗示 CSN 活性模拟去 NEDDylase 药物可能是潜在的治疗方法。
