Neddylation inhibition ameliorates steatosis in NAFLD by boosting hepatic fatty acid oxidation via the DEPTOR-mTOR axis

Abstract

Objective Neddylation is a druggable and reversible ubiquitin-like post-translational modification upregulated in many diseases, including liver fibrosis, hepatocellular carcinoma, and more recently, non-alcoholic fatty liver disease (NAFLD). Herein, we propose to address the effects of neddylation inhibition and the underlying mechanisms in pre-clinical models of NAFLD. Methods Hepatic neddylation measured by immunohistochemical analysis and NEDD8 serum levels measured by ELISA assay were evaluated in NAFLD clinical and pre-clinical samples. The effects of neddylation inhibition by using a pharmacological small inhibitor, MLN4924, or molecular approaches were assessed in isolated mouse hepatocytes and pre-clinical mouse models of diet-induced NAFLD, male adult C57BL/6 mice, and the AlfpCre transgenic mice infected with AAV-DIO- shNedd8. Results Neddylation inhibition reduced lipid accumulation in oleic acid-stimulated mouse primary hepatocytes and ameliorated liver steatosis, preventing lipid peroxidation and inflammation in the mouse models of diet-induced NAFLD. Under these conditions, increased Deptor levels and the concomitant repression of mTOR signaling were associated with augmented fatty acid oxidation and reduced lipid content. Moreover, Deptor silencing in isolated mouse hepatocytes abolished the anti-steatotic effects mediated by neddylation inhibition. Finally, serum NEDD8 levels correlated with hepatic neddylation during the disease progression in the clinical and pre-clinical models Conclusions Overall, the upregulation of Deptor, driven by neddylation inhibition, is proposed as a novel effective target and therapeutic approach to tackle NAFLD.

Neddylation tunes peripheral blood mononuclear cells immune response in COVID-19 patients

“…Based on these premises, we described the importance of neddylation, a reversible PTM, for immune activation in COVID-19 patients and the potential efficacy of MLN4924, the pharmacological inhibitor of neddylation. We have recently described that neddylated proteins appear in the serum of patients with liver pathology, which is associated with disease progression [ 30 ]. High levels of proteins in the blood could be a sign of chronic infection or inflammation [ 31 ].…”

“…The protocol for protein isolation and western blotting was described in Serrano-Maciá et al, 2021 [ 30 ]. Protein gels were transferred into nitrocellulose blotting membranes.…”

“…Healthy control cells from each donor were analyzed in independent duplicates for a better readout of the control group. Protein was extracted following previous data [ 30 ]. Briefly, protein was extracted using 7 M urea, 2 M thiourea, 4% CHAPS.…”

Emerging therapeutic potential of adeno-associated virus-mediated gene therapy in liver fibrosis

“…reported that hepatic neddylation was induced in clinical and preclinical non-alcoholic fatty liver disease (NAFLD), suggested by increased NEDD8 expression levels. 90 Notably, specific knockdown of NEDD8 in hepatocytes could be achieved by injecting AAV-DIO-shNedd8 into Alfp-Cre transgenic mice, which efficiently reduced neddylation levels and lipid accumulation in the livers of mice induced by a high-fat diet with choline deficiency, thereby inhibiting the development of NAFLD to NASH. 90 …”

“… 90 Notably, specific knockdown of NEDD8 in hepatocytes could be achieved by injecting AAV-DIO-shNedd8 into Alfp-Cre transgenic mice, which efficiently reduced neddylation levels and lipid accumulation in the livers of mice induced by a high-fat diet with choline deficiency, thereby inhibiting the development of NAFLD to NASH. 90 …”