PARK15/FBXO7 is dispensable for PINK1/Parkin mitophagy in iNeurons and HeLa cell systems

The protein kinase PINK1 and ubiquitin ligase Parkin promote removal of damaged mitochondria via a feed-forward mechanism involving ubiquitin (Ub) phosphorylation, Parkin activation, and ubiquitylation of mitochondrial outer membrane proteins to support recruitment of mitophagy receptors. The ubiquitin ligase substrate receptor FBXO7/PARK15 is mutated in an early-onset parkinsonian-pyramidal syndrome. Previous studies have proposed a role for FBXO7 in promoting Parkin-dependent mitophagy. Here, we systematically examine the involvement of FBXO7 in depolarization-dependent mitophagy in the well-established HeLa and induced-neurons cell systems. We find that FBXO7-/- cells have no demonstrable defect in: 1) kinetics of pUb accumulation, 2) pUb puncta on mitochondria by super-resolution imaging, 3) recruitment of Parkin and autophagy machinery to damaged mitochondria, 4) mitophagic flux, and 5) mitochondrial clearance as quantified by global proteomics. Moreover, global proteomics of neurogenesis in the absence of FBXO7 reveals no obvious alterations in mitochondria or other organelles. These results argue against a general role for FBXO7 in Parkin-dependent mitophagy and point to the need for additional studies to define how FBXO7 mutations promote parkinsonian-pyramidal syndrome.


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Organelle quality control underlies cellular health and is often defective in disease 38 and pathological states. Arguably the best understood such quality control pathway is  ligase, which marks damaged mitochondria for elimination (Narendra et al, 2008). Parkin 51 and PINK1 are both mutated in early-onset recessive forms of Parkinson's disease and 52 understanding how these enzymes work has been a major focus of the field (Ng et al, 2021; In healthy mitochondria, PINK1 is imported into the mitochondrial translocon and 55 rapidly processed for degradation Yamano & Youle, 2013). In response to 56 mitochondrial damage -such as depolarization, accumulation of mitochondrial misfolded 57 proteins, or defects in mitochondrial fusion -PINK1 is stabilized on the mitochondrial outer 117 To test if FBXO7 is a pivotal amplifier of the PINK1/Parkin pathway, we employed 118 CRISPR-Cas9-based gene editing to generated knockouts for FBXO7 in HeLa HFT/TO-119 PRKN cells  and confirmed insertion of a frameshift mutation by

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Similarly, we assessed depolarization-dependent pUb accumulation in the iNeuron 151 system. control or FBXO7 -/-ES cells were converted to day 12 iNeurons and depolarized 152 with AO in duplicate ( Figure 1C). pUb was detected by immunoblotting at 6 and 24 h and 153 was indistinguishable between WT and two independent FBXO7 -/clones ( Figure 1D).

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Taken together, these data do not support a universal role for FBXO7 in promoting the 155 earliest step in mitophagy signalling -PINK1-dependent Ub phosphorylation -in the HeLa

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Depolarization by AO led to the expected mitochondrial fragmentation and clustering, thus 208 causing an increase in local HSP60 signal and a right-shift in mtIntDen (Supplemental 209 Figure 4C, top panel). This shift was also observed to a similar extend in FBXO7 -/cells.

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Thus, both control and FBXO7 -/cells robustly respond to AO-induced mitophagy. Next, we 211 tested if Parkin intensities are increasing on mitochondria on an "organelle object" level. As          Figure 4C). If this response is a compensatory mechanism of the knockout cell line to 351 cope with cytotoxic insults is an intriguing idea. Last, we examined whether FBXO7 -/is 352 required for the maintenance of the mitochondrial organelle pool during differentiation.

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Previous work has shown significant metabolic rewiring during the differentiation from hESC 354 to iNeurons, including a switch from glycolysis to oxidative phosphorylation as the cells main 355 energy source (Ordureau et al., 2021). Since this switch is accompanied by increased

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All details and catalogue numbers can be found in the Materials

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Cloning and plasmid generation

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Cell culture, generation of lentiviral stable cell lines.

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Stable cell lines were generated using lentivirus generated from HEK293T cells.

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Chemiluminescence and colorimetric images were acquired using a BioRad ChemiDoc MP

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The sample was vacuum centrifuged to near dryness, and following reconstitution in 1% FA,

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The Supplemental Data

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Image quantification was performed in ImageJ/FiJi using custom-written batch-macros.

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In brief, raw confocal images of mitochondrial targeted mt-mKeima XL were divided  Table (Supplemental Table S9).

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Fixed-cell microscopy -general acquisition parameters

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Immunofluorescently labelled Hela or iNeurons (antibodies indicated in figures and figure   953 legends and details in Materials    Table (Supplemental Table 9).

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Microscopy-based mtDNA turnover measurements in HeLa and iNeurons