Investigation of USP30 inhibition to enhance Parkin-mediated mitophagy: tools and approaches.

Eliona Tsefou,James M Staddon,Kunitoshi Takeda,Toru Watanabe,Robin Ketteler,Alison S Walker,Emily H Clark,Bianca Ramazio,Amy R Hicks,Christin Luft,Thomas Briston

doi:10.1042/bcj20210508

Abstract

Mitochondrial dysfunction is implicated in Parkinson disease (PD). Mutations in Parkin, an E3 ubiquitin ligase, can cause juvenile-onset Parkinsonism, probably through impairment of mitophagy. Inhibition of the de-ubiquitinating enzyme USP30 may counter this effect to enhance mitophagy. Using different tools and cellular approaches, we wanted to independently confirm this claimed role for USP30. Pharmacological characterisation of additional tool compounds that selectively inhibit USP30 are reported. The consequence of USP30 inhibition by these compounds, siRNA knockdown and overexpression of dominant-negative USP30 on the mitophagy pathway in different disease-relevant cellular models was explored. Knockdown and inhibition of USP30 showed increased p-Ser65-ubiquitin levels and mitophagy in neuronal cell models. Furthermore, patient-derived fibroblasts carrying pathogenic mutations in Parkin showed reduced p-Ser65-ubiquitin levels compared with wild-type cells, levels that could be restored using either USP30 inhibitor or dominant-negative USP30 expression. Our data provide additional support for USP30 inhibition as a regulator of the mitophagy pathway.

Highlights

Mitochondrial homeostasis is important for the survival of healthy cells
The association between mutations in PTEN-induced kinase 1 (PINK1) and Parkin and the development of Parkinson’s disease (PD), suggests that defective mitophagy and accumulation of damaged mitochondria are key factors involved in the aetiology of disease
In addition to genetic deficits implicated in mitophagy, mitochondrial dysfunction and reduced rates of mitophagy are evident in sporadic PD [48,49,50], again linking mitochondrial health and clearance processes to PD pathophysiology

Summary

Introduction

Mitochondrial homeostasis is important for the survival of healthy cells. Mitochondrial dysfunction has been linked to several diseases including neurodegenerative disorders such as Parkinson’s disease (PD) [1,2,3,4,5]. PD is a chronic, progressive neurodegenerative disease that has been linked mechanistically and genetically to alterations in mitochondrial homeostasis [1,6,7,8]. It is thought that damaged mitochondria accumulate in neuronal cells, leading to neurotoxicity. In PD the dopaminergic neurones in the substantia nigra display increased neurotoxicity accompanied by mitochondrial dysfunction, leading to the characteristic motor dysfunction [9]. It has been proposed that enhanced autophagic removal of damaged mitochondria by a process called mitophagy may be a therapeutic approach for the treatment of PD [5,10,11]

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