The NEDD8-activating enzyme inhibitor MLN4924 reduces ischemic brain injury in mice

Blood–brain barrier (BBB) breakdown and inflammation occurring at the BBB have a key, mainly a deleterious role in the pathophysiology of ischemic stroke. Neddylation is a ubiquitylation-like pathway that is critical in various cellular functions by conjugating neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8) to target proteins. However, the roles of neddylation pathway in ischemic stroke remain elusive. Here, we report that NEDD8 conjugation increased during acute phase after ischemic stroke and was present in intravascular and intraparenchymal neutrophils. Inhibition of neddylation by MLN4924, also known as pevonedistat, inactivated cullin-RING E3 ligase (CRL), and reduced brain infarction and improved functional outcomes. MLN4924 treatment induced the accumulation of the CRL substrate neurofibromatosis 1 (NF1). By using virus-mediated NF1 silencing, we show that NF1 knockdown abolished MLN4924-dependent inhibition of neutrophil trafficking. These effects were mediated through activation of endothelial P-selectin and intercellular adhesion molecule-1 (ICAM-1), and blocking antibodies against P-selectin or anti–ICAM-1 antibodies reversed NF1 silencing-induced increase in neutrophil infiltration in MLN4924-treated mice. Furthermore, we found that NF1 silencing blocked MLN4924-afforded BBB protection and neuroprotection through activation of protein kinase C δ (PKCδ), myristoylated alanine-rich C-kinase substrate (MARCKS), and myosin light chain (MLC) in cerebral microvessels after ischemic stroke, and treatment of mice with the PKCδ inhibitor rottlerin reduced this increased BBB permeability. Our study demonstrated that increased neddylation promoted neutrophil trafficking and thus exacerbated injury of the BBB and stroke outcomes. We suggest that the neddylation inhibition may be beneficial in ischemic stroke.

Stroke continues to be a leading cause of death and the most frequent cause of disability in adults. Despite significant advances in decoding the pathophysiology of cerebral ischemia, therapeutic options for stroke are still limited. Ischemic injury to the brain rapidly triggers adhesion molecule expression on the activated endothelium (1), resulting in rolling, adhesion, and extravasation of blood-derived inflammatory cells (2). Infiltrating inflammatory cells, including neutrophils, result in irreversible impairment of blood–brain barrier (BBB) function and tissue damage through the release of reactive oxygen species (ROS), proteolytic enzymes, and proinflammatory mediators (3). However, our understanding of the links between BBB breakdown and peripheral neutrophils infiltrating the ischemic brain is still incomplete.Neddylation is the process of posttranslational protein modification by conjugating the ubiquitin-like protein, neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8), to target proteins (4). This process is catalyzed by NEDD8-activating enzyme E1 (NAE1 and UBA3), NEDD8-conjugating enzyme E2 (UBC12), and NEDD8 E3 ligase (5). The best characterized substrates of NEDD8 are cullins (6), which are scaffold proteins for the cullin-RING E3 ligase (CRL) (7). The conjugation of NEDD8 to cullins leads to the activation of CRL (8), which ubiquitinates a multitude of different proteins for targeted degradation (9). Recently, the neddylation pathway was reported to contribute to growth of a variety of cancer cells and inflammatory responses (10). In contrast, inhibition of neddylation by MLN4924, which is a small molecule inhibitor of NAE, suppressed tumor growth, reduced inflammation, and prevented atherogenesis (11–13). However, to the best of our knowledge, the role of neddylation in ischemic stroke has not been addressed so far.Using a mouse model of focal transient cerebral ischemia, we show that neddylation was up-regulated in the peri-infarct cortex after stroke and was expressed in neutrophils. Treatment with the neddylation inhibitor MLN4924 reduced brain infarction and improved neurological functions. We also demonstrated that MLN4924-afforded neuroprotection was mediated via anti-inflammatory and BBB-protective effects involving the accumulation of CRL substrate neurofibromatosis 1 (NF1).