pevonedistat and Inflammation

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.

Topics: Animals; Brain; COP9 Signalosome Complex; Cullin Proteins; Cytokines; Endothelial Cells; Humans; Inflammation; Mice; Neuroinflammatory Diseases; NF-kappa B

Topics: Apoptosis; beta-Transducin Repeat-Containing Proteins; Cell Line, Tumor; Cycloheximide; Cyclopentanes; Humans; Inflammation; Liver Neoplasms; NF-KappaB Inhibitor alpha; Pyrimidines; RNA, Messenger; Ubiquitin-Activating Enzymes; Ubiquitins; Xenograft Model Antitumor Assays

Topics: Alcoholism; Animals; Central Nervous System Depressants; Central Nervous System Sensitization; Cullin Proteins; Cyclopentanes; Disease Models, Animal; Enzyme Inhibitors; Ethanol; Hyperalgesia; Inflammation; Locomotion; Male; Mice; Mice, Inbred BALB C; NEDD8 Protein; Pyrimidines

RhoGTPases regulate cytoskeletal dynamics, migration and cell-cell adhesion in endothelial cells. Besides regulation at the level of guanine nucleotide binding, they also undergo post-translational modifications, for example ubiquitination. RhoGTPases are ubiquitinated by Cullin RING ligases which are in turn regulated by neddylation. Previously we showed that inhibition of Cullin RING ligase activity by the neddylation inhibitor MLN4924 is detrimental for endothelial barrier function, due to accumulation of RhoB and the consequent induction of contractility. Here we analyzed the effect of pharmacological activation of Cullin RING ligases on endothelial barrier integrity in vitro and in vivo. CSN5i-3 induced endothelial barrier disruption and increased macromolecule leakage in vitro and in vivo. Mechanistically, CSN5i-3 strongly induced the expression and activation of RhoB and to lesser extent of RhoA in endothelial cells, which enhanced cell contraction. Elevated expression of RhoGTPases was a consequence of activation of the NF-κB pathway. In line with this notion, CSN5i-3 treatment decreased IκBα expression and increased NF-κB-mediated ICAM-1 expression and consequent adhesion of neutrophils to endothelial cells. This study shows that sustained neddylation of Cullin RING-ligases leads to activation the NF-κB pathway in endothelial cells, elevated expression of RhoGTPases, Rho/ROCK-dependent activation of MLC and disruption of the endothelial barrier.

Topics: Animals; COP9 Signalosome Complex; Cyclopentanes; Endothelium, Vascular; Gene Expression Regulation; Green Fluorescent Proteins; HEK293 Cells; Human Umbilical Vein Endothelial Cells; Humans; Inflammation; Intercellular Adhesion Molecule-1; Intracellular Signaling Peptides and Proteins; Neutrophils; NF-kappa B p50 Subunit; NF-KappaB Inhibitor alpha; Peptide Hydrolases; Pyrimidines; rho-Associated Kinases; rhoA GTP-Binding Protein; rhoB GTP-Binding Protein; Ubiquitin; Up-Regulation; Zebrafish