2. Academic Validation
  3. Targeting mitochondrial deubiquitinase USP30 to induce mitophagy in heteroplasmic mitochondrial diseases

Targeting mitochondrial deubiquitinase USP30 to induce mitophagy in heteroplasmic mitochondrial diseases

  • Pharmacol Rep. 2026 Apr;78(2):519-534. doi: 10.1007/s43440-026-00829-7.
Brígida R Pinho 1 2,  Vasco Martins 1 2,  Anitta R Chacko 3,  Célia Nogueira 4,  Michael R Duchen 3,  Jorge M A Oliveira 5 6 7
Affiliations

Affiliations

  • 1 UCIBIO Applied Molecular Biosciences Unit, Mitochondria and Neurobiology Lab, Faculdade de Farmácia, Universidade do Porto, Porto, 4500-313, Portugal.
  • 2 Associate Laboratory i4HB Institute for Health and Bioeconomy, Faculdade de Farmácia, Universidade do Porto, Porto, 4500-313, Portugal.
  • 3 Department of Cell and Developmental Biology, University College London, London, UK.
  • 4 Department of Human Genetics, National Institute of Health Doutor Ricardo Jorge, Porto, Portugal.
  • 5 UCIBIO Applied Molecular Biosciences Unit, Mitochondria and Neurobiology Lab, Faculdade de Farmácia, Universidade do Porto, Porto, 4500-313, Portugal. [email protected].
  • 6 Associate Laboratory i4HB Institute for Health and Bioeconomy, Faculdade de Farmácia, Universidade do Porto, Porto, 4500-313, Portugal. [email protected].
  • 7 Departamento de Ciências do Medicamento, Laboratório de Farmacologia, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, Porto, 4050-313, Portugal. [email protected].
PMID: 41587019 DOI: 10.1007/s43440-026-00829-7
Abstract

BACKGROUND: Mitochondrial DNA (mtDNA) diseases are heterogeneous and lack effective treatments. Their severity correlates with mutant mtDNA load. Mitophagy degrades dysfunctional mitochondria, contributing to a healthy mitochondrial pool. USP30, a mitochondrial Deubiquitinase, limits Mitophagy by removing the ubiquitin tagging mitochondria for degradation. We investigated whether inhibiting USP30 could enhance Mitophagy and reduce mutant mtDNA load in a heteroplasmic mitochondrial disease. METHODS: Cybrids cells harboring mutant m.8993T > G mtDNA - common cause of NARP syndrome and maternally inherited Leigh syndrome (MILS) - were treated with USP30 Inhibitor MF-094 under glycolytic and Oxidative Phosphorylation conditions. On-target activity of MF-094 was assessed by mitochondrial ubiquitination (western-blot) and mitolysosome formation (microscopy). The mutation’s effects were investigated on cell proliferation and metabolism (respirometry and ATP levels). The impact of MF-094 on mutant mtDNA load and mtDNA copy number was quantified by PCR. RESULTS: Comparing with control cells (0% mutant mtDNA), cells with mutant mtDNA exhibited reduced proliferation and ATP levels under Oxidative Phosphorylation conditions; and reduced oxygen consumption, increased extracellular acidification, and sustained resazurin metabolism after mitochondrial inhibition under glycolytic conditions. MF-094 induced Mitophagy via increased mitolysosome formation. Mechanistically, MF-094 showed on-target effects, increasing mitochondrial ubiquitination. However, chronic treatment (3–6 weeks) evoked only a small (5%) non-significant reduction in mutant mtDNA load. CONCLUSIONS: Despite inducing Mitophagy, the USP30 Inhibitor MF-094 showed little potential to manage m.8993T > G related diseases, as it did not significantly reduce the load of this NARP/MILS causing mtDNA mutation. These results highlight the complexity of mutant mtDNA management and the need for innovative strategies for these disorders.

Keywords

ATP synthase; Autophagy; Cybrid; Mitochondrial DNA; Mitochondrion; Ubiquitin.

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