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Cited by 19Autophagy in Disease and TherapyParkinson's Disease Mechanisms and TreatmentsUbiquitin and proteasome pathways

The DOI matches its Crossref record; 43 of the 43 references it cites were found in a registry or an index; none of the 43 sources its claims cite is checked for support yet.

USP8 Down-Regulation Promotes Parkin-Independent Mitophagy in the Drosophila Brain and in Human Neurons

Open access
Mauri, Sofia, Bernardo, Greta, Martinez, Aitor, Favaro, Mariavittoria, Trevisan, Marta, Cobraiville, Gael, Fillet, Marianne, Caicci, Federico, Whitworth, Alexander J., Ziviani, Elena·2023·Cells
DOI 10.3390/cells12081143PMC PMC10136645
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Abstract

Stress-induced mitophagy, a tightly regulated process that targets dysfunctional mitochondria for autophagy-dependent degradation, mainly relies on two proteins, PINK1 and Parkin, which genes are mutated in some forms of familiar Parkinson’s Disease (PD). Upon mitochondrial damage, the protein kinase PINK1 accumulates on the organelle surface where it controls the recruitment of the E3-ubiquitin ligase Parkin. On mitochondria, Parkin ubiquitinates a subset of mitochondrial-resident proteins located on the outer mitochondrial membrane, leading to the recruitment of downstream cytosolic autophagic adaptors and subsequent autophagosome formation. Importantly, PINK1/Parkin-independent mitophagy pathways also exist that can be counteracted by specific deubiquitinating enzymes (DUBs). Down-regulation of these specific DUBs can presumably enhance basal mitophagy and be beneficial in models in which the accumulation of defective mitochondria is implicated. Among these DUBs, USP8 is an interesting target because of its role in the endosomal pathway and autophagy and its beneficial effects, when inhibited, in models of neurodegeneration. Based on this, we evaluated autophagy and mitophagy levels when USP8 activity is altered. We used genetic approaches in D. melanogaster to measure autophagy and mitophagy in vivo and complementary in vitro approaches to investigate the molecular pathway that regulates mitophagy via USP8. We found an inverse correlation between basal mitophagy and USP8 levels, in that down-regulation of USP8 correlates with increased Parkin-independent mitophagy. These results suggest the existence of a yet uncharacterized mitophagic pathway that is inhibited by USP8.

Overview

AI snapshot

Written from the paper’s full text.

TL;DR

USP8 down-regulation or inhibition increases basal autophagy and Parkin-independent mitophagy in Drosophila neurons and in human iNeurons. The authors propose that this mechanism may explain the protective effects of USP8 reduction in neurodegeneration models.

Key findings

  • Mass spectrometry of WT vs USP8 knockdown flies identified 1149 vs 940 modified peptides with VML score >1.0; 254 peptides were ubiquitinated only in USP8-down-regulating flies and 347 only in WT. Mitophagy was among the top KEGG pathways, with Marf/Mitofusin and Porin/VDAC among hits.
  • In Drosophila larval brain, USP8 down-regulation increased autolysosomes without changing autophagosome number, and TEM showed more autophagic vesicles in thoracic muscle; this also occurred in Parkin KO flies.
  • Basal mitophagy was increased in USP8-down-regulating fly brains: WT showed on average 4–5 mitolysosomes per cell, with a significant increase upon USP8 down-regulation, including in Parkin KO background. Mitochondrial aspect ratio decreased, indicating increased fission.
  • In S2R+ cells, USP8 knockdown increased LC3–mitochondria colocalization and mt-Keima 561/405 ratio; TOM20, VDAC, CyPD and CV were only slightly decreased. Parkin was more thermally stable but did not translocate to mitochondria even with CCCP, and stress-induced mitophagy was reduced.
  • In human H9 hESC-derived iNeurons, the USP8 inhibitor DUBs-IN-2 (0.5–1 μM/24–48 h) enhanced basal mitophagy comparably to 0.5 μM antimycin/oligomycin.

Methods

  • Drosophila genetics: pan-neuronal nSybGAL4-driven GFP-mCherry-Atg8a and mito-QC reporters in WT, USP8 RNAi, USP8−/+, and Parkin KO backgrounds; larval brain confocal imaging and thoracic muscle TEM.
  • S2R+ cell assays: USP8 dsRNA knockdown, LC3-RFP/MitoTracker imaging, mt-Keima flow cytometry, Western blotting of mitochondrial proteins, Parkin thermal stability, and CCCP-induced Parkin recruitment.
  • Ubiquitin MS: K-GG immunoaffinity enrichment from 200 WT and USP8 knockdown flies followed by LC-chip-MS/MS; human iNeurons generated from H9 hESCs by doxycycline-inducible NGN2 expression and treated with DUBs-IN-2.

Limitations

  • The paper states that the molecular mechanism of basal mitophagy induction by USP8 down-regulation is unknown and that further studies are required to dissect this mechanism and its physiological importance.
  • The text makes evident that the MS hits, including Marf/Mfn, are candidate USP8 targets rather than direct proof of deubiquitination, and the modest decreases in mitochondrial proteins may be compensated by mitochondrial biogenesis.
  • The human neuron findings rely on pharmacological USP8 inhibition with DUBs-IN-2 rather than genetic USP8 loss, and no in vivo human data are presented.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.3390/cells12081143

Cites

43 of 43 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Verification

Claims the paper makes

0 of 43 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    In WT flies, basal mitophagy levels average four to five mitolysosomes per cell.

    “In WT flies, we observed on average four to five mitolysosomes per cell, whereas in the brains of USP8-down-regulating flies, data analysis showed a significant increase in the number of mitolysosomes, indicative of enhanced mitophagy in this condition (Figure 2B,C).”
    Quote in the paperNot a substantial claim
  • Findingown data

    Down-regulation of USP8 correlates with increased Parkin-independent mitophagy.

    “We found an inverse correlation between basal mitophagy and USP8 levels, in that down-regulation of USP8 correlates with increased Parkin-independent mitophagy.”
    • Also stated in: § 3.1. Description of Experimental Results · hedged
      “Importantly, this effect was also induced in a Parkin KO background, indicating that the mitophagic effect of USP8 down-regulation is Parkin independent (Figure 2D,E).”
    Quote in the paperSubstantial claim
  • Findingown data

    In brains of USP8-down-regulating flies, there is a significant increase in the number of mitolysosomes.

    “In WT flies, we observed on average four to five mitolysosomes per cell, whereas in the brains of USP8-down-regulating flies, data analysis showed a significant increase in the number of mitolysosomes, indicative of enhanced mitophagy in this condition (Figure 2B,C).”
    Quote in the paperSubstantial claim
  • Findingown data

    USP8 down-regulation increases autophagy levels in Parkin KO flies.

    “Importantly, USP8 down-regulation enhanced the autophagic flux in the larval brain of Parkin KO flies (Figure 1G–I).”
    • Also stated in: § 3.1. Description of Experimental Results · plain
      “In Parkin KO flies, USP8 down-regulation also increases autophagy levels, supporting the hypothesis of a protective effect of USP8 down-regulation that depends on autophagy.”
    Quote in the paperSubstantial claim
  • Findingown data

    USP8 down-regulation enhances mitophagy in flies.

    “In WT flies, we observed on average four to five mitolysosomes per cell, whereas in the brains of USP8-down-regulating flies, data analysis showed a significant increase in the number of mitolysosomes, indicative of enhanced mitophagy in this condition (Figure 2B,C).”
    • Also stated in: § 3.1. Description of Experimental Results · hedged
      “Importantly, this effect was also induced in a Parkin KO background, indicating that the mitophagic effect of USP8 down-regulation is Parkin independent (Figure 2D,E).”
    Quote in the paperSubstantial claim
  • It may state two separate claims as one. 31
  • Probably not a substantial claim. 26
  • The quote only partly states this claim. 1

  • It may state two separate claims as one.

    Findingown data

    In WT larval brain, genetic down-regulation of USP8 resulted in an increased number of autolysosomes, whereas the number of autophagosomes did not change.

    “In the larval brain of WT flies, genetic down-regulation of USP8 (both RNAi and USP8−/+) (Supplementary Figure S1A) resulted in an increased number of autolysosomes, whereas the number of autophagosomes did not change (Figure 1B–D).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Among the identified peptides, 254 are ubiquitinated only in USP8 down-regulating flies and 347 only in WT flies.

    “Among these, we identified 254 peptides that are ubiquitinated in USP8 down-regulating flies only (Supplementary Excel File S2) and 347 that are only ubiquitinated in WT flies (Supplementary Excel File S3).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    USP8 down-regulation enhances autophagy and mitophagy in flies and in neurons of human origin.

    “We found that USP8 down-regulation enhances autophagy and mitophagy in flies and in neurons of human origin, providing a mechanistic explanation for the protective effect of USP8 reduction observed in several models of neurodegeneration.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Released neurotransmitter molecules are visible at synaptic clefts and detectable levels of NMDA-R are expressed.

    “Released neurotransmitter molecules are visible at synaptic clefts (Supplementary Figure S4G) and detectable levels of NMDA-R are expressed, which we assessed by Western blotting analysis (Supplementary Figure S4H).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim.

    Findingown data

    Mitophagy was among the ten highest scoring KEGG pathways from the analysis.

    “Surprisingly, mitophagy was among the ten highest scoring KEGG pathways that came out from this analysis.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingcitation

    Mitochondrial respiration and Complex I activity are not affected in USP8-down-regulating conditions.

    “our previous study showed that mitochondrial respiration and Complex I activity are not affected in USP8-down-regulating conditions [20].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [20] Corra, 2019, Inhibition of the deubiquitinase USP8 corrects a Drosophila PINK1 model of mitochondria dysfunction, Life Sci. Alliance source

  • It may state two separate claims as one.

    Findingown data

    The proteostatic (autophagic/mitophagic) effect of USP8 down-regulation occurs under basal conditions and in a Parkin KO background.

    “However, our in vivo results indicate that the proteostatic (autophagic/mitophagic) effect of USP8 down-regulation occurs under basal conditions and in Parkin KO background.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingcitation

    USP8-deficient flies did not show any particular detrimental phenotypes and mitochondrial functionality remained intact.

    “Importantly, USP8-deficient flies did not show any particular detrimental phenotypes and mitochondrial functionality remained intact [20].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [20] Corra, 2019, Inhibition of the deubiquitinase USP8 corrects a Drosophila PINK1 model of mitochondria dysfunction, Life Sci. Alliance source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingown data

    After 4 days of differentiation, iNeurons start to develop a clear neuronal network.

    “After 4 days of differentiation, the cells start to develop a clear neuronal network (Supplementary Figure S4A)”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    USP8 pharmacological inhibition by DUBs-IN-2 (0.5-1 μM/24-48 h) enhances basal mitophagy in human iNeurons, comparable to 0.5 μM antimycin/oligomycin treatment.

    “In these neurons of human origin, we found that USP8 pharmacological inhibition by DUBs-IN-2 (0.5–1 μM/24–48 h) enhances basal mitophagy, an effect that was comparable to 0.5 μM antimycin/oligomycin (AO) treatment, which we used as positive control for mitophagy induction (Figure 4C).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    In USP8 down-regulating conditions, basal mitophagy is enhanced, whereas stress-induced mitophagy triggered by CCCP (10 μM/20 h) is reduced.

    “In this condition, basal mitophagy is enhanced (Figure 3D), whereas stress-induced mitophagy triggered by CCCP (10 μM/20 h) is reduced (Supplementary Figure S3E,F).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Immunoblotting confirmed that stem cell marker OCT3/4 is only present until day 2 of differentiation, whereas βIII-Tubulin gradually increased until day 14 upon induction.

    “Immunoblotting experiments confirmed that stem cell marker OCT3/4 is only present until day 2 of differentiation, whereas the expression of neuronal marker βIII-Tubulin gradually increased until day 14 upon induction (Supplementary Figure S4D).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingown data

    EM images of iNeurons after 14 days of differentiation show neuronal cells with distinguishable neuronal soma, axon hillock and axonal and dendritic projections.

    “Our representative electron microscopy (EM) images of iNeurons after 14 days of differentiation show neuronal cells with distinguishable neuronal soma, axon hillock and axonal and dendritic projections (Supplementary Figure S4E,F).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    At the end of differentiation (14 days), iNeurons exhibit expression of typical neuronal markers MAP2 and βIII-tubulin and lose pluripotency markers OCT4 and sox2.

    “and at the end of the differentiation process (14 days), iNeurons exhibit the expression of the typical neuronal markers MAP2 and βIII-tubulin and lose pluripotency markers OCT4 and sox2 (Supplementary Figure S4B).”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Interpretationcitation

    The decreased mitochondrial aspect ratio in USP8-down-regulating flies supports the previous observation that USP8-down-regulating S2R+ cells display fragmented mitochondria.

    “These results fully support what we previously observed in USP8-down-regulating S2R+ cells that displayed fragmented mitochondria [20].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [20] Corra, 2019, Inhibition of the deubiquitinase USP8 corrects a Drosophila PINK1 model of mitochondria dysfunction, Life Sci. Alliance source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    USP8 is directly connected to Parkin-mediated mitophagy by controlling the removal of K6-linked ubiquitin from Parkin.

    “USP8 is also directly connected to Parkin-mediated mitophagy by controlling the removal of K6-linked ubiquitin from Parkin.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [17] Durcan, 2014, USP8 regulates mitophagy by removing K6-linked ubiquitin conjugates from parkin, EMBO J. source

  • Probably not a substantial claim.

    Premisecitation

    USP8 loss of function in D. melanogaster leads to the accumulation of autophagosomes due to a blockade of the autophagic flux.

    “In particular, USP8 loss of function in D. melanogaster leads to the accumulation of autophagosomes due to a blockade of the autophagic flux [13,14], whereas in HeLa cells [13] and HEK293T cells [15], USP8 knockdown enhances the autophagic flux.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [13] Jacomin, A.C., Bescond, A., Soleilhac, E., Gallet, B., Schoehn, G., Fauvarque, M.O., and Taillebourg, E. (2015). The Deubiquitinating Enzyme UBPY Is Required for Lysosomal Biogenesis and Productive Autophagy in Drosophila. PLoS ONE, 10. source
    • cites [14] Jacomin, A.C., Fauvarque, M.O., and Taillebourg, E. (2016). A functional endosomal pathway is necessary for lysosome biogenesis in Drosophila. BMC Cell Biol., 17. source

  • It may state two separate claims as one.

    Premisecitation

    Parkin is not indispensable for mitophagy but rather acts to amplify PINK1 signal.

    “This evidence suggests that Parkin is not indispensable for mitophagy but rather acts to amplify PINK1 signal.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [5] Yun, 2014, MUL1 acts in parallel to the PINK1/parkin pathway in regulating mitofusin and compensates for loss of PINK1/parkin, eLife source

  • It may state two separate claims as one.

    Premisecitation

    USP8 down-regulation or pharmacological inhibition ameliorates the phenotype of PINK1 and Parkin KO flies by preventing neurodegeneration and rescues mitochondrial defects, lifespan and locomotor dysfunction.

    “We also previously demonstrated that USP8 down-regulation or its pharmacological inhibition ameliorates the phenotype of PINK1 and Parkin KO flies by preventing neurodegeneration and rescues mitochondrial defects, lifespan and locomotor dysfunction in these flies [20].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [20] Corra, 2019, Inhibition of the deubiquitinase USP8 corrects a Drosophila PINK1 model of mitochondria dysfunction, Life Sci. Alliance source

  • It may state two separate claims as one.

    Premisecitation

    USP8 knockdown leads to increased lysosomal degradation of α-synuclein and protects from α-synuclein-induced toxicity and cell loss in an α-synuclein fly model of PD.

    “USP8 knockdown also leads to increased lysosomal degradation of α-synuclein, and it protects from α-synuclein-induced toxicity and cell loss in an α-synuclein fly model of PD [19].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [19] Alexopoulou, 2016, Deubiquitinase Usp8 regulates alpha-synuclein clearance and modifies its toxicity in Lewy body disease, Proc. Natl. Acad. Sci. USA source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    USP8 knockdown decreases β-secretase levels and Aβ production in an in vitro model of AD.

    “In particular, USP8 knockdown decreases β-secretase levels and Aβ production in an in vitro model of AD [18].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [18] Yeates, 2016, The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation, J. Biol. Chem. source

  • Probably not a substantial claim.

    Premisecitation

    USP8 negatively regulates autophagy by deubiquitinating the autophagy factors TRAF6, BECN1 and p62.

    “More recently, it was reported that USP8 negatively regulates autophagy by deubiquitinating the autophagy factors TRAF6, BECN1 and p62 [16], supporting the hypothesis that USP8 reduction can be used to promote autophagy.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [16] Kim, 2022, USP8 regulates liver cancer progression via the inhibition of TRAF6-mediated signal for NF-kappaB activation and autophagy induction by TLR4, Transl. Oncol. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Other E3-ubiquitin ligases such as MUL1, SMURF1 and Gp78 have been proposed to ubiquitinate mitochondrial proteins and promote mitophagy in a Parkin-independent fashion.

    “Importantly, in addition to Parkin, other E3-ubiquitin ligases, such as MUL1, SMURF1 and Gp78, have been proposed to ubiquitinate mitochondrial proteins and promote mitophagy in a Parkin-independent fashion.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [5] Yun, 2014, MUL1 acts in parallel to the PINK1/parkin pathway in regulating mitofusin and compensates for loss of PINK1/parkin, eLife source

  • It may state two separate claims as one.

    Interpretationreasoning

    Our work provides a mechanistic explanation for the protective effect of USP8 down-regulation via enhancement of mitophagy and lays the basis for further development of studies targeting DUBs, USP8 in particular, in neurodegenerative conditions.

    “Our work provides a mechanistic explanation for the protective effect of USP8 down-regulation that is via enhancement of mitophagy and lays the basis for further development of studies targeting DUBs (USP8 in particular) in neurodegenerative conditions.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationown data

    Our MS data suggests that Marf/Mfn is a possible target for USP8, providing a mechanistic insight into the molecular mechanism of mitophagy induction, which might be via a USP8 effect on the ubiquitination levels of Marf/Mfn.

    “Nevertheless, our MS data suggests that Marf/Mfn is a possible target for USP8 (Supplementary Excel File S2), thus providing a mechanistic insight into the molecular mechanism of mitophagy induction, which might be via a USP8 effect on the ubiquitination levels of Marf/Mfn.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Marf/Mfn and VDAC are key regulators of mitochondrial quality control, in that Parkin-dependent ubiquitination of Marf/Mfn or VDAC signals selected mitochondria for degradation.

  • Probably not a substantial claim.

    Premisecitation

    One of the best-characterized ubiquitin-dependent mitophagy pathways depends on activation of the E3-ubiquitin ligase Parkin.

    “As previously discussed, one of the best-characterized ubiquitin-dependent mitophagy pathways depends on the activation of E3-ubiquitin ligase Parkin [32].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [32] Narendra, 2008, Parkin is recruited selectively to impaired mitochondria and promotes their autophagy, J. Cell Biol. source

  • It may state two separate claims as one.

    Premisecitation

    USP8 down-regulation or pharmacological inhibition ameliorates the phenotype of PINK1 and Parkin KO flies.

    “and its down-regulation or pharmacological inhibition ameliorates the phenotype of PINK1 and Parkin KO flies [20].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [20] Corra, 2019, Inhibition of the deubiquitinase USP8 corrects a Drosophila PINK1 model of mitochondria dysfunction, Life Sci. Alliance source

  • It may state two separate claims as one.

    Premisecitation

    DUBs-IN-2 seems to be well tolerated in vivo in rodents, and it has been safely used to treat gastric cancer in mice.

    “DUBs-IN-2 seems to be well tolerated in vivo in rodents, and it has been safely used to treat gastric cancer in mice [41].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [41] Sun, 2020, USP8 Inhibitor Suppresses HER-2 Positive Gastric Cancer Cell Proliferation and Metastasis via the PI3K/AKT Signaling Pathway, OncoTargets Ther. source

  • It may state two separate claims as one.

    Premisecitation

    USP8-deficient cells do not display defects in mitochondrial respiration, nor loss of mitochondrial membrane potential to trigger PINK1/Parkin activation.

    “Our previous studies showed that USP8-deficient cells do not display any defects in mitochondrial respiration, nor loss of mitochondrial membrane potential to trigger PINK1/Parkin activation [20].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [20] Corra, 2019, Inhibition of the deubiquitinase USP8 corrects a Drosophila PINK1 model of mitochondria dysfunction, Life Sci. Alliance source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Interpretationreasoning

    Mitochondrial degradation induced by USP8 down-regulation may be compensated for by productive mitochondrial biogenesis.

    “Because the reduction in mitochondrial content is quite modest, and USP8 downregulating cells do not display any defects in mitochondrial respiration [20], it is possible that mitochondrial degradation induced by USP8 down-regulation is compensated for by productive mitochondrial biogenesis.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [20] Corra, 2019, Inhibition of the deubiquitinase USP8 corrects a Drosophila PINK1 model of mitochondria dysfunction, Life Sci. Alliance source
    • The authors state this as an assumption: If mitochondria are degraded and this is not compensated by productive mitochondrial biogenesis, mitochondrial loss should be reflected by a decrease in the levels of mitochondrial resident proteins.

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    Marf/Mfn is a mitochondrial fusion protein with pleiotropic functions that is also a target of Parkin-dependent ubiquitination upon stress-induced mitophagy.

    “Marf/Mfn is a mitochondrial fusion protein with pleiotropic functions [39,40] that is also a target of Parkin-dependent ubiquitination upon stress-induced mitophagy.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [39] Scorrano, 2008, Mitofusin 2 tethers endoplasmic reticulum to mitochondria, Nature source
    • cites [40] Debattisti, 2014, Reduction of endoplasmic reticulum stress attenuates the defects caused by Drosophila mitofusin depletion, J. Cell Biol. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    The best USP8 inhibitors at present were developed as derivatives of 9-oxo-9H-indeno[1,2-b]pyrazine-2,3-dicarbonitrile.

    “The best inhibitors at present were developed as derivatives of 9-oxo-9H-indeno[1,2-b]pyrazine-2,3-dicarbonitrile [21].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [21] Colombo, 2010, Synthesis and biological evaluation of 9-oxo-9H-indeno[1,2-b]pyrazine-2,3-dicarbonitrile analogues as potential inhibitors of deubiquitinating enzymes, ChemMedChem source

  • Probably not a substantial claim. It may be too vague to check against evidence.

    Interpretationreasoning

    Approaches that enhance mitochondrial quality control, such as mitophagy, might be beneficial to degrade dysfunctional mitochondria as sources of potentially toxic compounds.

    “In this scenario, approaches that enhance mitochondrial quality control, such as mitophagy, might be beneficial to degrade dysfunctional mitochondria as sources of potentially toxic compounds.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    Overexpression of Ngn2 and Sox11 promotes differentiation of primary fibroblasts into cholinergic neurons, whereas it inhibits GABAergic neuronal differentiation.

    “It is known that overexpression of Ngn2 and Sox11 (another transcription factor involved in neuronal induction during embryonic development) promote the differentiation of primary fibroblasts into cholinergic neurons, whereas they inhibits GABAergic neuronal differentiation [35].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [35] Roybon, 2010, GABAergic differentiation induced by Mash1 is compromised by the bHLH proteins Neurogenin2, NeuroD1, and NeuroD2, Cereb. Cortex source

  • It may state two separate claims as one.

    Premisecitation

    Both inhibitors kill HCT116 colon cancer cells and PC-3 prostate cancer cells, and DUBs-IN-2 has been used to diminish tumorigenesis in breast cancer and in corticotroph tumor cells.

    “Both inhibitors kill HCT116 colon cancer cells and PC-3 prostate cancer cells, and DUBs-IN-2 has been used to diminish tumorigenesis in breast cancer [7] and in corticotroph tumor cells [8].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [7] Shin, 2020, Deubiquitylation and stabilization of Notch1 intracellular domain by ubiquitin-specific protease 8 enhance tumorigenesis in breast cancer, Cell Death Differ. source
    • cites [8] Kageyama, 2020, Ubiquitin-specific protease 8 inhibitor suppresses adrenocorticotropic hormone production and corticotroph tumor cell proliferation, Endocr. J. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    One proposed underlying mechanism of neurodegeneration includes alterations in mitochondrial function and increased oxidative stress that can affect the proteostatic capacity of the cell.

    “Indeed, one proposed underlying mechanism of neurodegeneration includes alterations in mitochondrial function and increased oxidative stress that can affect the proteostatic capacity of the cell [43].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [43] Muqit, 2006, Expanding insights of mitochondrial dysfunction in Parkinson’s disease, Nat. Rev. Neurosci. source

  • It may state two separate claims as one.

    Premisecitation

    USP8 knockdown decreases Amyloid β (Aβ) production in an in vitro model of AD, presumably by promoting lysosome-dependent degradation of β-secretase.

    “USP8 knockdown decreases Amyloid β (Aβ) production in an in vitro model of AD, presumably by promoting lysosome-dependent degradation of β-secretase, the enzyme involved in amyloid precursor protein (APP) processing [18].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [18] Yeates, 2016, The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation, J. Biol. Chem. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    In age-associated neurodegenerative conditions, the drop in proteostasis seems to be pathologically exacerbated.

    “Whereas a physiological decline in proteostasis is expected in aged individuals, in age-associated neurodegenerative conditions this drop seems to be pathologically exacerbated [1].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1] Kaushik, 2015, Proteostasis and aging, Nat. Med. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Interpretationreasoning

    It is possible that MUL1 operates as the E3 ubiquitin ligase regulating the ubiquitination of Marf/Mfn and mitophagy, in opposition to USP8.

    “It is possible that MUL1 operates as the E3 ubiquitin ligase regulating the ubiquitination of Marf/Mfn and mitophagy, in opposition to USP8.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. It may state two separate claims as one.

    Interpretationreasoning

    Specific DUB down-regulation can presumably enhance protein degradation, autophagy and basal mitophagy and be beneficial in neurodegenerative diseases in which accumulation of misfolded proteins and aberrant mitochondria is implicated.

    “Given that one of the main functions of ubiquitination is to promote protein degradation, as well as bridging the ubiquitin proteasome system (UPS) to autophagy and mitophagy, specific DUB down-regulation can presumably enhance protein degradation, autophagy and basal mitophagy and be beneficial in neurodegenerative diseases in which accumulation of misfolded proteins and aberrant mitochondria is”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Ngn2 regulates the commitment of neural progenitors to neuronal fate during development.

    “Ngn2 regulates the commitment of neural progenitors to neuronal fate during development [33] and induces early postnatal astroglia into neurons [34].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [33] Sun, 2001, Neurogenin promotes neurogenesis and inhibits glial differentiation by independent mechanisms, Cell source

  • It may state two separate claims as one.

    Premisecitation

    These iNeurons express glutamatergic synaptic proteins vGLUT1, PSD95 and SYN1 and excitatory synaptic function when in co-culture with mouse glial cells.

    “These iNeurons express glutamatergic synaptic proteins such as vesicular glutamate transporter 1 (vGLUT1), postsynaptic density-95 (PSD95) and synapsin1 (SYN1) and excitatory synaptic function when in co-culture with mouse glial cells [22].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [22] Zhang, 2013, Rapid single-step induction of functional neurons from human pluripotent stem cells, Neuron source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Loss of proteostasis is well documented during aging, in part as a consequence of progressive physiological decline in the proteolytic activity of the ubiquitin-proteasome and lysosome-autophagy systems.

    “Loss of proteostasis is well documented during aging, in part as a consequence of the progressive physiological decline in the proteolytic activity of two major degradative systems: the ubiquitin–proteasome and the lysosome–autophagy systems.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1] Kaushik, 2015, Proteostasis and aging, Nat. Med. source

  • Probably not a substantial claim.

    Premisenone given

    The consequences of USP8 manipulation in post-mitotic, long-lived cells such as neurons are poorly defined.

    “Whereas the functions of USP8 have been extensively explored in the context of EGFR endocytosis in different cell types and in the regulation of stem cell proliferation and self-renewal in stem cells, the consequences of USP8 manipulation in post-mitotic, long-lived cells such as neurons is poorly defined.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationown data

    Basal mitophagy is induced by USP8 down-regulation and the mitophagic effect under basal conditions is Parkin independent.

    “These results support the hypothesis that basal mitophagy is induced by USP8 down-regulation and that the mitophagic effect of USP8 down-regulation under basal conditions is Parkin independent.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. The paper may give no reason why it holds.

    Interpretationreasoning

    The mitophagic effect of USP8 down-regulation might not necessarily translate into an obvious decrease in mitochondrial mass.

    “In this scenario, the mitophagic effect of USP8 down-regulation might not necessarily translate into an obvious decrease in mitochondrial mass.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    Ngn2 expression in hESCs produces an excitatory layer 2/3 cortical neuron that exhibits AMPA-receptor-dependent spontaneous synaptic activity and a relatively smaller NMDA-receptor-mediated synaptic current.

    “Ngn2 expression in hESCs produces an excitatory layer2/3 cortical neuron that exhibits AMPA-receptor-dependent spontaneous synaptic activity and a relatively smaller NMDA-receptor-mediated synaptic current [22].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [22] Zhang, 2013, Rapid single-step induction of functional neurons from human pluripotent stem cells, Neuron source

  • It may state two separate claims as one.

    Interpretationown data

    These cells fully develop as neurons and seem to make functional synapses.

    “Thus, these cells fully develop as neurons and seem to make functional synapsis.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Premisecitation

    DUBs-IN-2 and DUBs-IN-1 have an IC50 value in the range of 200 nM and are highly specific for USP8, with an IC50 value of >100 μM for Usp7.

    “These compounds (DUBs-IN-2 and DUBs-IN-1) have an IC50 value in the range of 200 nM and are highly specific for USP8 (e.g., IC50 value of >100 μM for Usp7).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    Many studies have shown that promoting autophagy increases lifespan and rescues the pathological phenotype of animal models of neurodegeneration.

    “Many studies have shown that promoting autophagy increases lifespan and rescues the pathological phenotype of animal models of neurodegeneration, supporting the hypothesis of a protective effect of enhanced proteostasis to prevent neuronal loss [1].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1] Kaushik, 2015, Proteostasis and aging, Nat. Med. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Marf/Mfn is also a target of E3 ubiquitin ligases other than Parkin, for example MUL1.

    “Marf/Mfn is also target of E3 ubiquitin ligases other than Parkin, for example MUL1 [5].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [5] Yun, 2014, MUL1 acts in parallel to the PINK1/parkin pathway in regulating mitofusin and compensates for loss of PINK1/parkin, eLife source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Ngn2 induces early postnatal astroglia into neurons.

    “Ngn2 regulates the commitment of neural progenitors to neuronal fate during development [33] and induces early postnatal astroglia into neurons [34].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [34] Heinrich, C., Blum, R., Gascon, S., Masserdotti, G., Tripathi, P., Sanchez, R., Tiedt, S., Schroeder, T., Gotz, M., and Berninger, B. (2010). Directing astroglia from the cerebral cortex into subtype specific functional neurons. PLoS Biol., 8. source
52 of 52

  • The authors state this as an assumption.

    If mitochondria are degraded and this is not compensated by productive mitochondrial biogenesis, mitochondrial loss should be reflected by a decrease in the levels of mitochondrial resident proteins.

    “The assumption here is that if mitochondria are degraded and this is not compensated by productive mitochondrial biogenesis, mitochondrial loss should be reflected by a decrease in the levels of these proteins.”

  • The paper relies on this without support.

    Mitochondrial fission promotes uncoupled respiration as a means to reduce oxidative stress.

    “Mitochondrial fission is known to promote uncoupled respiration as a means to reduce oxidative stress, which, when elevated, triggers mitochondrial quality control mechanisms to remove damaged mitochondrial components.”

  • The paper relies on this without support.

    USP8 is highly expressed in the brain and specifically in dopaminergic neurons.

    “Interestingly, USP8 is highly expressed in the brain and specifically in dopaminergic neurons.”

  • The paper relies on this without support.

    Marf/Mfn and VDAC are key regulators of mitochondrial quality control, in that Parkin-dependent ubiquitination of Marf/Mfn or VDAC signals selected mitochondria for degradation.

    “Both proteins are key regulators of mitochondrial quality control, in that Parkin-dependent ubiquitination of Marf/Mfn or VDAC signals selected mitochondria for degradation.”
4 of 4

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Conflicting excitation wavelengths for the mt-Keima ratio

    Minor

    The results describe the mt-Keima measurement as a 561/405 ratio, while the Figure 3 caption calls it a 561/458 nm ratio. These give different excitation wavelengths for the same reported measurement.

  • Conflicting localization of the mtx-QCXL reporter

    Minor

    Figure 4 describes the same mtx-QCXL reporter as targeted to the inner mitochondrial membrane in panel A and to the mitochondrial matrix in panel B.

2 of 2

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Metadata

Authors
Mauri, S., Bernardo, G., Martinez, A., Favaro, M., Trevisan, M., Cobraiville, G., Fillet, M., Caicci, F., Whitworth, A. J., & Ziviani, E.
Affiliations
  • University of Padua (IT)
  • University of Cambridge (GB)
  • Laboratory for the Analysis of Medicines, Center for Interdisciplinary Research on Medicines (CIRM), Quartier Hopital, University of Liege, Avenue Hippocrate 15, 4000 Liege, Belgium
  • University of Liège (BE)
Corresponding author
Ziviani, Elena
ORCID
Venue
Cells
Publisher
MDPI AG
Published
2023-04-13
Volume
12
Issue
8
Pages
1143
ISSN
2073-4409 (ISSN-L)
DOI
10.3390/cells12081143
PMC
PMC10136645
OpenAlex
W4365448823
Cited by
19
References
43
Citations by year
2026: 6 · 2025: 9 · 2024: 2 · 2023: 2
FWCI
1.92
Type
journal article
Language
en
Open access
Licence
Locations
  • Cells (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • ORBi (University of Liège) (submittedVersion, other-oa)
  • Apollo (University of Cambridge) (submittedVersion, cc-by)
  • Padua Research Archive (University of Padova) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • M.J. Fox foundation (MJFF-019788, MC_UU_00028/6)
  • AriSLA (“mitoALS”) (MJFF-019788, MC_UU_00028/6)
  • Medical Research Council (MJFF-019788, MC_UU_00028/6)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Autophagy in Disease and Therapy
  • Health Sciences › Medicine › Clinical Neurology › Parkinson's Disease Mechanisms and Treatments
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
Keywords
USP8, PINK1, Parkin, deubiquitinase, autophagy, Parkinson's disease, mitochondrial quality control, Drosophila melanogaster brain, human neurons, neurodegeneration
MeSH terms
PTEN-Induced Putative Kinase, Animals, Brain, Drosophila, Drosophila melanogaster, Humans, Neurons, Endopeptidases, Protein Kinases, Down-Regulation, Protein Serine-Threonine Kinases, Drosophila Proteins, Ubiquitin Thiolesterase, Ubiquitin-Protein Ligases, Endosomal Sorting Complexes Required for Transport, Mitophagy

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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

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