Antisense oligonucleotides targeting ubiquitin specific peptidase USP30 enhance degradation of TOM20 following acute acetaminophen injury
Andrew F. Powers1, Jennifer Hoffmann1, Sheri L. Booten1, Shuling Guo1, Brett P. Monia1 and Mariam Aghajan1
1Ionis Pharmaceuticals, Carlsbad, CA 92010
*studies were funded by Ionis Pharmaceuticals
Mitochondria are exquisitely dynamic organelles that perform essential cellular biosynthetic reactions including production of ATP, amino acids, nucleotides, iron-sulfur clusters and various metabolic intermediates. Perturbations in mitochondrial homeostasis activate the Pink1-Parkin system, which functions to ubiquitylate outer membrane proteins leading to degradation of damaged mitochondria by mitophagy. Recently, it was shown that USP30 directly opposes the activity of Parkin by removing ubiquitin chains from mitochondrial protein TOM20, and that knockdown of USP30 improves mitochondrial integrity in vitro and in vivo by increasing mitophagy. Here we tested the hypothesis that antisense oligonucleotide (ASO)-mediated knockdown of USP30, which specifically modifies USP30 expression through RNase H-mediated degradation of USP30 mRNA, enhances turnover of TOM20 following acute mitochondrial injury. ASOs targeting USP30 elicited robust knockdown (>90%) in mouse hepatocellular carcinoma cells stably expressing Parkin. We challenged cells overnight with the mitochondrial depolarizing agent CCCP and observed that loss of USP30 significantly increased disappearance of TOM20 as revealed by immunofluorescence microscopy. To determine if USP30 knockdown has similar effects following acute mitochondrial injury in vivo, we dosed C57bl/6 mice subcutaneously with ASO for 2 weeks and then challenged with 300mg/kg acetaminophen (APAP) for 6h. We observed greater than 80% reduction in liver USP30 following ASO treatment, with no significant effect on autophagy status or TOM20 abundance when mice were challenged with PBS. Mice treated with USP30 ASOs plus APAP exhibited a significant increase in liver autophagy, where p62 protein was robustly depleted and LC3B lipidation was enhanced. In addition, western blots for TOM20 revealed a significant decrease following treatment with USP30 ASOs plus APAP. These results suggest that ASO knockdown of USP30 enhances degradation of damaged mitochondria via p62 and TOM20, and highlight the therapeutic potential of USP30 ASOs in treating conditions associated with deficits in mitochondrial function.
