FBXO7/ntc and USP30 antagonistically set the ubiquitination threshold for basal mitophagy and provide a target for Pink1 phosphorylation in vivo

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Abstract

Functional analyses of genes linked to heritable forms of Parkinson’s disease (PD) have revealed fundamental insights into the biological processes underpinning pathogenic mechanisms. Mutations in PARK15/FBXO7 cause autosomal recessive PD and FBXO7 has been shown to regulate mitochondrial homeostasis. We investigated the extent to which FBXO7 and its Drosophila orthologue, ntc, share functional homology and explored its role in mitophagy in vivo. We show that ntc mutants partially phenocopy Pink1 and parkin mutants and ntc overexpression supresses parkin phenotypes. Furthermore, ntc can modulate basal mitophagy in a Pink1- and parkin-independent manner by promoting the ubiquitination of mitochondrial proteins, a mechanism that is opposed by the deubiquitinase USP30. This basal ubiquitination serves as the substrate for Pink1-mediated phosphorylation that triggers stress-induced mitophagy. We propose that FBXO7/ntc works in equilibrium with USP30 to provide a checkpoint for mitochondrial quality control in basal conditions in vivo and presents a new avenue for therapeutic approaches.

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    Reply to the reviewers

    1. General Statements

    We thank all the reviewers for their time and effort in the peer-review process. We appreciate the positive reflections on the study and the feedback comments which were well thought-out and articulated. Considering these comments has led us to deeper reflections on the conceptualization of the mechanisms at play, and we hope that our responses here and revisions of the manuscript have improved the presentation of the data and our interpretation of these complex matters. As a result, we have now incorporated a new supplementary figure 5 and present a new model figure with the corresponding comments in the text.

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  2. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #3

    Evidence, reproducibility and clarity

    In this manuscript, Sanchez-Martinez et al characterise the role of nutracker (ntc), the presumed Drosophila orthologue of human FBXO7 (whose gene is mutated in autosomal recessive PD), in mitophagy and phenotypes associated with neurodegeneration in flies (climbing index, dopaminergic neuron loss, rough eye phenotype, and others). FBXO7 (human) has been previously shown to restore parkin (not Pink1) phenotypes and mitochondrial morphology in Drosophila and implicated in Pink1-parkin mitophagy, however the role of ntc in basal mitophagy and its genetic interaction with USP30 has not been previously reported. Key findings include: …

  3. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    In this ms. Sanchez-Martinez and colleagues study the role of the ub ligase FBXO7, in regulating mitophagy - highlighting that mutations in FBXO7 associate with Parkinson's disease and defects in mitochondrial homeostasis. Using the fly as model, they carry out a series of expts. investigating ntc (Drosophila ortholog of FBX07) demonstrating that it can functionally rescue Parkin but not PINK1 deficiency. Expanding on this, they propose a model whereby ntc/FBX07 regulates basal mitophagy and also acts as a priming Ub-ligase for Parkin mediated mitophagy, finding that the dub USP30 counteracts these ntc function. Overall the data are …

  4. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons


    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript Sanchez-Martinez et al investigated the function of ntc, a Drosophila homologue of FBXO7. The mechanisms by which mutations in this protein cause autosomal recessive PD are poorly understood. The protein has previously been implicated in PINK1/Parkin mitophagy however the mechanistic detail is lacking. The data presented here provide an important insight into the molecular functions of ntc as well as mitophagy in vivo in general. Ntc was found to promote ubiquitination of mitochondrial proteins which is counteracted by USP30. The basal ubiquitination regulated by these two enzymes is proposed to act as a …