1. Introduction
Loss of protein and organelle homeostasis is well documented during aging. However, whereas physiological decline in proteostasis is expected in older adults, this seems to be more severe and pathologically relevant in age-related neurodegenerative disorders, such as Parkinson’s disease (PD), Alzheimer’s disease (AD), Huntington’s disease (HD) and amyothrophic lateral sclerosis (ALS) [
1].
When it comes to familiar PD in particular, the molecular link between deficient mechanisms of proteostasis and disease onset is more evident, in that two genes mutated in familiar forms of PD, Ser/Thr kinase PINK1 and E3-ubiquitin ligase Parkin, operate as key regulators of mitochondrial degradation. Under normal conditions, PINK1 levels are maintained low: the protein is imported into mitochondria through its mitochondrial targeting sequence, and it is processed by the matrix processing peptidase (MPP) and the presenilins-associated rhomboid-like (PARL) protease. Cleaved PINK1 is retrotranslocated to the cytosol and rapidly degraded by the proteasome. When mitochondria depolarize following mitochondrial damage, PINK1 fails to be imported into mitochondria and its cleavage is reduced. The protein accumulates on the outer mitochondrial membrane (OMM), and its stabilization leads to an increase in its kinase activity and autophosphorylation. PINK1 recruits cytosolic Parkin to the mitochondria by phosphorylating ubiquitin on serine 65 (Ser65) and Parkin. Parkin phosphorylation, which also occurs at Ser65, leads to the release of Parkin auto-inhibited conformation and promotes its interaction with phospho-ubiquitin. Activated Parkin polyubiquitinates itself and multiple substrates on the OMM, including VDAC, TOM20, FIS1, Miro and mitochondrial pro-fusion proteins Mfn1, Mfn2 and Marf (fly homologue of Mfn1/2). The ubiquitin chains formed on the proteins of the OMM serve as substrates for the kinase activity of PINK1, which in turn recruits more Parkin, leading to a feed-forward loop that culminates with the recruitment of autophagy receptors p62, Optineurin (OPTN) and nuclear dot protein 52 kDa (NDP52). These receptors interact with mitochondria via their ubiquitin-binding domain and with the autophagosome via their LC3-interacting region (LIR) motif, ensuring the targeting of mitochondria to the forming phagophore [
2,
3,
4]. 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. These alternative E3 ubiquitin ligases often operate on the same mitochondrial targets that are ubiquitinated by Parkin, as is the case for E3 ubiquitin ligase MUL1, which shares the same substrate (Mfn) with Parkin [
5]. This evidence suggests that Parkin is not indispensable for mitophagy but rather acts to amplify PINK1 signal.
Besides ubiquitin-mediated mitophagy, mitochondrial removal can also be promoted by autophagy receptors, such as NIX, BNIP3, FUNDC1, Cardiolipin and Prohibitin 2 (PHB2). Mitophagy receptors can recruit autophagosomes to mitochondria in a PINK1/Parkin independent fashion [
6], indicating that PINK1/Parkin-independent mitophagy pathways also exist, which can presumably be enhanced to ameliorate mitochondrial quality control in their absence.
In the quest of potential enhancers of Parkin-independent mitophagy pathways, deubiquitinating enzymes (DUBs) are interesting candidates for their activity on the ubiquitination status of proteins. In this respect, ubiquitin-specific protease USP8 is a compelling target for its reported role in the modulation of autophagy and mitophagy and its physiologically relevant catalytic and non-catalytic (scaffolding) activities. In particular, USP8 represents a typical multidomain DUB that exerts several functions: it deubiquitinates the epidermal growth factor receptor (EGFR) on the plasma membrane and prevents its degradation by the endosome–lysosome pathway, a process known as receptor down-regulation. As a result, USP8 activity enhances the stability of EGFR (an essential regulator of proliferation and differentiation), whereas USP8 inhibition promotes EGFR down-regulation. This is consistent with the antitumorigenic effects of USP8 inhibition that have been reported in several cancer models [
7,
8]. Another direct target of USP8 deubiquitinating activity is EPG5, an autophagy regulator that mediates autophagosome/lysosome fusion. EPG5 maintains a high autophagic flux to support ESCs stemness. USP8 deubiquitinates EPG5, an event that is required for EPG5-LC3 interaction, and plays an essential role in EPG5-dependent autophagy in the maintenance of ESCs stemness [
9]. USP8 also plays a critical role in the development and homeostasis of T cells, in that specific inactivation of USP8 in T cells affects thymocyte maturation via specific activation of genes controlled by the transcription factor Foxo. As a consequence, specific ablation of USP8 in T cells profoundly affects the homeostasis and development of the immune system [
10].
Besides the ubiquitin-specific protease activity, USP8 plays an essential role as a scaffolding protein that is connected to the endosomal trafficking and transport [
11]. In particular, USP8 harbors an N-terminal microtubule interacting and transport (MIT) domain and two atypical central SH3-binding motifs (SH3BMs) that flank a 14-3-3 protein-binding motif (14-3-3BM). The MIT domain interacts with charged multivesicular body proteins (CHMP), components of the endosomal sorting complexes required for transport III (ESCRT-III), whereas the SH3BM interacts with the signal transducing adaptor molecule (STAM), which is a part of the ESCRT-0 complex [
12].
As it can be inferred from these essential catalytic and non-catalytic functions of USP8 the expression of this DUB is an absolute requirement for proper tissue development and differentiation and for endosome sorting and trafficking. Not surprisingly, USP8 KO is embryonically lethal in mammals and flies, whereas mutations that enhance USP8 catalytic activity causes Cushing disease in humans by sustaining EGFR signaling.
Of particular relevance for this work, USP8 activity has been recently linked to autophagy and mitophagy, although with contrasting results. 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. 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. USP8 is also directly connected to Parkin-mediated mitophagy by controlling the removal of K6-linked ubiquitin from Parkin. Stabilization of ubiquitin moieties on the Parkin molecule by USP8 knockdown does not seem to correlate to an increase in Parkin degradation. On the contrary, Parkin levels increase when USP8 is downregulated, whereas CCCP-induced Parkin recruitment is delayed, as well as mitophagy [
17].
The observations that USP8 down-regulation might inhibit the autophagic flux and mitophagy points to a potential aggravating effect for USP8 reduction in models in which accumulation of misfolded proteins and aberrant mitochondria is implicated. Nevertheless, many publications indicate that USP8 down-regulation is protective in models that can benefit from enhanced proteostasis. In particular, USP8 knockdown decreases β-secretase levels and Aβ production in an in vitro model of AD [
18]. 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]. 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]. In these models of neurodegeneration, in vitro and in vivo downregulation of USP8 correlated with decreased levels of mitochondrial fusion protein Marf/MFN and normalized Marf/MFN levels of PINK1 KO flies that are pathologically elevated.
In this work, we wanted to extend the work that has been performed and explore the effect of USP8 down-regulation in neurons in the context of autophagy and mitophagy, using
D. melanogaster as a model organism. We subsequently investigated the effect of USP8 inhibition using the potent inhibitor DUBs-IN-2 [
21] in mammalian cells, particularly in iNeurons generated from human embryonic stem cells (hESCs) [
22]. 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.
2. Materials and Methods
2.1. Fly Strains and Husbandry
Drosophila were raised under standard conditions at 25 °C with a 12 h:12 h light:dark cycle, on agar, cornmeal, yeast food. w
1118 (BDSC_5905), UAS-GFP-mCherry-Atg8a (BDSC_37749) and nSybGAL4 (BDSC_51635) fly lines were obtained from the Bloomington Drosophila Stock Center. The UAS-USP8
GD1285 RNAi and UAS Marf RNAi (VDRC 105261) lines were obtained from the VDRC Stock Center. The USP8
−/+ line was kindly provided by Satoshi Goto [
23]. The lines park
25, UAS-mito-QC and Act5cGAL4 were generated previously [
24,
25]. For larval experiments, L3 wandering larvae were selected based on their phenotypes.
2.2. Larvae Dissection and Fixation
Larval brain dissections were performed in PBS and fixed in 4% formaldehyde, pH 7.0 for 20 min. Subsequently, brains were washed in PBS and mounted in Mowiol® 4–88 (Sigma-Aldrich, 81381, St. Louis, MO, USA). Samples were dissected in the morning or in the afternoon and imaged in the afternoon of the same day or the following morning, respectively.
2.3. Microscopy and Image Analysis
Fluorescence microscopy imaging was performed using a Zeiss LSM 900 confocal microscope equipped with 100× Plan Apochromat (oil immersion, NA 1.4) objective lenses at 2× digital zoom. Z-stacks were acquired at 0.5 µm steps. For each larval brain, two images of different areas were taken. In the graphs, each data point represents one brain. For both autophagy and mitophagy analyses, samples were imaged via sequential excitations (488 nm, green; 561 nm, red). Laser power and gain settings were adjusted depending on the fluorophore but were maintained across samples. For quantification, maximum intensity projections were created and analyzed using Fiji (ImageJ 2.9.0) software. For autolysosomes quantification, the number of mCherry-only puncta was quantified using the mQC-counter plugin [
26], maintaining the same parameters across samples (radius for smoothing images = 1; ratio threshold = 0.8; red channel thresh: stdDev above mean = 1). To quantify autophagosomes (yellow dots), the green and red channels were threshold (mean intensity + 3 × StdDev and mean intensity + 2 × StdDev, respectively). Objects present in both the mCherry and GFP masks were counted. For mitolysosome quantification, the number of mCherry-only puncta was quantified using the mQC-counter plugin [
26], maintaining the same parameters across samples (radius for smoothing images = 1; ratio threshold = 1; red channel thresh: stdDev above mean = 1).
2.4. Electron Microscopy
Thoraces were prepared from 3-day-old adult flies and fixed O.N. in 2% paraformaldehyde and 2.5% glutaraldehyde. After rinsing in 0.1 M cacodylate buffer with 1% tannic acid, samples were postfixed in 1:1 2% OsO4 and 0.2 M cacodylate buffer for 1 h. Samples were rinsed, dehydrated in an ethanol series and embedded by using Epon. Ultrathin sections were examined using a transmission electron microscope.
2.5. S2R+ Cell Culture
D. melanogaster S2R+ cells were cultured in Schneider’s Drosophila medium (Biowest, Nuaillé, France) supplemented with 10% heat-inactivated fetal calf serum. Cells were maintained at 25 °C and passaged routinely.
2.6. Gene Silencing
Drosophila dsRNA probes were prepared using MEGA script kit (Ambion, Waltham, MA, USA) following the manufacturer’s instructions. The CG5798/USP8 dsRNA probe was acquired from the Sheffield RNAi Screening Facility.
2.7. S2R+ Transfection, Cell Imaging Acquisition and Processing
A total of 2 × 105 S2R+ cells were plated in a 24-well plate and transfected with 1 μg DNA, 1.5 μL Effectene (QIAGEN), 1.5 μL enhancer and 20 μL EC buffer 1 day after plating following the manufacturer’s instructions. When required, copper sulfate solution was added to the cells to induce plasmid expression. The following plasmids were used: UAS-mt-Keima, actin-GAL4, mito-dsRed and Parkin-GFP. For USP8 down-regulation, cells were treated with control or USP8 dsRNA probes (ctrl RNAi, Usp8 RNAi). For the Parkin recruitment experiment, after plating, S2R+ cells were treated with either 10 μM CCCP (Sigma-Aldrich) (treated cells) or equal amount of DMSO (control cells) for the indicated amount of time. Cells were collected for the experiments 72 h after transfection.
For imaging acquisition and processing, S2R+ cells were plated on 24 mm round glass coverslips and co-transfected with the indicated plasmids (Parkin-GFP, mito-DsRed, LC3-RFP) and/or dsRNA probes (ctrl RNAi, Usp8 RNAi) for 48–72 h before imaging. Images were acquired using a UPlanSApo 60×/1.35 NA objective (iMIC Adromeda, TILL Photonics, Pleasanton, CA, USA) upon excitation with 561 and 488 nm lasers. Parkin translocation was evaluated by counting the number of cells with Parkin puncta on mitochondria. Autophagosome–mitochondria interaction was evaluated by measuring the percentage of LC3 that co-localized with mitochondria by Mander’s coefficient of co-localization using ImageJ JACoP (“Just Another Colocalization Plug in”), following 3D volume rendered reconstruction of 40 z-axis images, separated by 0.2 μm (software: ImageJ, plug in: volume and JACoP).
2.8. Flow Cytometry
A total of 72 h after transfection, S2R+ cells were gently washed with PBS and collected in 300 μL HBSS + HEPES for flow cytometry. mt-Keima expressing cells were analysed by flow cytometry (BD FACSAria sorter, San Jose, CA, USA) to measure mitophagy levels in control cells (ctrl RNAi) or cells with altered USP8 expression (Usp8 RNAi), following an established protocol [
12]. Briefly, cells were analysed with a flow cytometer (BD FACSAria™) equipped with 405 nm and 561 nm lasers. Cells were simultaneously excited with a violet laser (405 nm), with emission detected at 610 ± 10 nm with a BV605 detector, and with a yellow-green laser (561 nm), with emission detected at 610 ± 10 nm by a PE-CF594 detector. mt-Keima-positive cells were gated based on their ratio of emission at PE-CF594/BV605 in a “high” or “low” gate. The proportion of mitophagic cells was represented by the percentage of cells in the “high” gate among the mt-Keima-positive population.
2.9. Thermal Stability Assay
A total of 1 × 106 cells were plated onto 10 cm Petri dishes and treated after 24 h with dsRNA probes (ctrl RNAi or Usp8 RNAi). Next, cells were resuspended in PBS, snap-frozen in liquid nitrogen and thawed 4 times. The solution was aliquoted into a PCR strip and incubated at the indicated temperature for 3 min. The lysates were centrifuged at 16,000× g for 30 min at 4 °C. The soluble fraction was loaded onto SDS-PAGE gel.
2.10. Protein Extraction
Cells were collected in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 1% NP-40, 0.25% sodium deoxycholate and 1 mM EDTA in distilled water, adjusted pH to 7.4) with freshly added protease inhibitor cocktail (PIC) and incubated on ice for 30 min before being centrifuged at maximum speed at 4 °C for 15 min. The protein concentrations of the samples was determined using the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA). 2-Mercaptoethanol (Sigma-Aldrich) was mixed into the samples and the proteins were then denatured at 95 °C for 5 min.
2.11. Western Blot
Western blots were performed using ExpressPlus PAGE Gel 4–12% or 4–20% (GenScript, Piscataway, NJ, USA). Proteins were transferred to PVDF membranes (MERCK-Millipore) using the Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA) following manufacturer’s instructions. Membranes were incubated with indicated antibodies and imaged with ImageQuant LAS4000. Band densiometry quantification was performed using ImageJ software. The following antibodies were used: anti-Actin (1:1000; Chemicon MAB1501, Tokyo, Japan), α-ATP5A (1:4000, Abcam ab14748, Cambridge, MA, USA), α-Cyclophilin D (1:500, Abcam ab110324), α-TOM20 (1:1000, Santa Cruz sc-11415, Dallas, TX, USA) and α-VDAC (1:1000, Abcam ab15895). Canonical secondary antibodies used were sheep anti-mouse or donkey anti-rabbit HRP (GE Healthcare, Chicago, IL, USA). Immunoreactivity was visualized with Immobilon Forte Western HRP substrate (Millipore, Burlington, MA, USA).
2.12. Isolation and Identification of Ubiquitin Modifications by Mass Spectrometry
To identify the full repertoire of USP8 targets, protein lysates extracted from 200 CTR (Act5cGAL4/+) or USP8 KD (Act5cGAL4/+; UAS USP8 RNAi/+) flies were subjected to immunoaffinity isolation and mass spectrometry analysis to enrich and identify K-GG peptides from digested protein lysates as previously described [
13]. Fly lysates were prepared in lysis buffer (9 M urea, 20 mM HEPES pH 8.0, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate and 1 mM β-glycerophosphate) by brief sonication on ice. Protein samples (20 mg) were reduced at 55 °C for 30 min in 4.1 mM DTT, cooled 10 min on ice and alkylated with 9.1 mM iodoacetamide for 15 min at room temperature in the dark. Samples were diluted 3-fold with 20 mM HEPES pH 8.0 and digested in 10 μg mL
−1 trypsin-TPCK (Promega, Madison, WI, USA) overnight at room temperature. Following digestion, trifluoroacetic acid (TFA) was added to a final concentration of 1% to acidify the peptides before desalting on a Sep-PakC18 cartridge (Waters). Peptides were eluted from the cartridge in 40% acetonitrile and 0.1% TFA, flash frozen and lyophilized for 48 h. Dry peptides were gently resuspended in 1.4 mL 1× immunoaffinity purification (IAP) buffer (Cell Signaling Technology, Danvers, MA, USA) and cleared by centrifugation for 5 min at 10,000×
g rcf at 4 °C. Precoupled anti-KGG beads (Cell Signaling Technology) were washed in 1× IAP buffer before contacting the digested peptides. Immunoaffinity enrichment was performed for 2 h at 4 °C. Beads were washed 2× with IAP buffer and 4× with PBS before 2× elution of peptides in 0.15% TFA for 10 min each at room temperature.
2.13. LC-Chip-MS/MS Analysis
Chromatographic separation was performed on a chip including a 160 nL trapping column and a 150 mm length and 75 μm internal diameter analytical column, both packed with a Zorbax 300SB 5 μm C18 phase (Agilent Technologies, Waldbronn, Germany). The mobile phase was composed of H2O/FA (100:0.1, v/v) (A) ACN/H2O/FA (90:10:0.1, v/v/v) and (B) degassed by ultrasonication for 15 min before use. Analytical process was performed in two steps: First, the sample was loaded on the trapping column during an isocratic enrichment phase using the capillary pump delivering a mobile phase in isocratic mode composed of H2O/ACN/FA (97:3:0.1, v/v/v) at a flow rate of 4 µL/min. A flush volume of 6 µL was used to remove unretained components. Then, after valve switching, a gradient elution phase in backflush mode was performed through the enrichment and analytical columns using the nanopump. The analysis was performed using a gradient starting at 3% B that linearly ramped up to 45% B in 30 min at a flow rate of 300 nL/min and then up to 95% B in 5 min. The column was then rinsed with 95% B for 5 min before returning to 3% B. Ten column volumes were used for re-equilibration prior to the next injection. The total analysis time was 43 min for each run. All the experiments were carried out with an 8 µL sample injection volume. During the analysis, the injection needle was thoroughly rinsed three times from the inside and the outside with a mix of ACN/H2O/TFA (60:40:0.1, v/v/v) commanded by an injection program set in the injector parameters. The identifications were performed using an electrospray MS-MS using a 6340 series ion trap mass spectrometer (Agilent Technologies). The collision energy was set automatically depending on the mass of the precursor ion. Each MS full scan was followed by MS/MS scans of the six most intense precursor ions detected in the MS scan (exclusion time: 1 min). The results were subsequently introduced into the database for protein identification searches using Spectrum Mill (Agilent Technologies). All searches were carried out with “Drosophila melanogaster” as taxonomy in the NCBInr database and 0.5 Da of tolerance on MS/MS fragments. The search parameters allowed fixed modifications for cysteine (carboxyamidomethylation) and variable modifications for methionine (oxidation) and for lysine (ubiquitination). Two missed cleavages were allowed. VML score displays the VML (variable modification localization) score of the modification selected, which is the difference in score between equivalent identified sequences with different variable modification localizations. A VML score of >1.1 indicates confident localization. A score of 1 implies that there is a distinguishing ion of b or y ion type and 0.1 means that when unassigned, the peak is 10% of the intensity of the base peak.
2.14. Generation of Stable Mitophagic Flux Reporters hESC Lines and Differentiation
H9 hESCs (WiCell Institute) were cultured in TeSR™-E8™ medium (StemCell Technologies, Vancouver, BC, Canada) on Matrigel-coated tissue culture plates with daily medium change. Cells were passaged every 4–5 days with 0.5 mM EDTA in DMEM/F12 (Sigma). For introduction of TRE3G-NGN2 into the AAVS1 site, a donor plasmid pAAVS1-TRE3G-NGN2 was generated by replacing the EGFP sequence with an N-terminal flag-tagged human NGN2 cDNA sequence in plasmid pAAVS1-TRE3G-EGFP (Addgene plasmid # 52343, Watertown, MA, USA). A total of 5 μg of pAAVS1-TRE3G-NGN2, 2.5 mg hCas9 (Addgene plasmid # 41815) and 2.5 mg gRNA_AAVS1-T2 (Addgene plasmid # 41818) were electroporated into 1 × 10
6 H9 cells. The cells were treated with 0.25 mg/mL puromycin for 7 days and surviving colonies were expanded and subjected to genotyping. H9 hESC harboring the mitochondrial matrix mCherry-GFP flux reporter were generated by transfection of 1 × 10
5 cells with 1 μg pAC150-PiggyBac-matrix-mCherry-eGFPXL [
24] and 1 μg pCMV-HypBAC-PiggyBac-Helper (Sanger Institute) in conjunction with the transfection reagent FuGENE HD (Promega, Madison, WI, USA). The cells were selected and maintained in TeSR™-E8™ medium supplemented with 200 mg/mL hygromycin; hygromycin was kept in the medium during differentiation to iNeurons. For H9 hESC conversion into iNeurons, cells were treated with Accutase (Thermo Fisher Scientific, Waltham, MA, USA) and plated on Matrigel-coated tissue plates in DMEM/F12 supplemented with 1× N2, 1× NEAA (Thermo Fisher Scientific), human brain-derived neurotrophic factor (BDNF, 10 ng/mL, PeproTech, Waltham, MA, USA), human neurotrophin-3 (NT-3, 10 ng/L, PeproTech), human recombinant laminin (0.2 mg/mL, Life Technologies, Waltham, MA, USA), Y-27632 (10 mM, PeproTech) and doxycycline (2 mg/mL, Sigma-Aldrich) on Day 0. On Day 1, Y-27632 was withdrawn. On Day 2, the medium was replaced with neurobasal medium supplemented with 1× B27 and 1× Glutamax (Thermo Fisher Scientific) containing BDNF, NT-3 and 2 mg/mL doxycycline. Starting on Day 4, half of the medium was replaced every other day thereafter. On Day 7, the cells were treated with Accutase (Thermo Fisher Scientific) and plated on Matrigel-coated tissue plates. Doxycycline was withdrawn on Day 10. Treatments and experiments were performed between days 11 and 13.
2.15. Statistical Analysis
Statistical analyses were performed using GraphPad Prism 8 software. Data are represented as box plots (min to max, all data points showed) or as mean ± SEM. Statistical significance was measured by an unpaired t-test, one-way or two-way ANOVA or Kruskal–Wallis nonparametric test followed by ad hoc multiple comparison test. p-Values are indicated in the figure legend. Data information: n = number of biological replicates; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.
4. Discussion
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. Whereas a physiological decline in proteostasis is expected in aged individuals, in age-associated neurodegenerative conditions this drop seems to be pathologically exacerbated [
1]. What are the reason for this? In the quest for the potential regulators of proteostasis that might be affected in neurodegenerative conditions, deubiquitinating enzymes (DUBs) are interesting candidates for their fine-tuning activity on the ubiquitination statuses of proteins. DUBs are proteases that counteract ubiquitination by cleaving ubiquitin moieties from proteins. 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 implicated. One interesting DUB in this context is the ubiquitin-specific protease USP8, as its knockdown or pharmacological inhibition is protective in different models of neurodegeneration. 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]. USP8 down-regulation also protects from α-synuclein-induced toxicity in an α-synuclein fly model of PD [
19], and its down-regulation or pharmacological inhibition ameliorates the phenotype of PINK1 and Parkin KO flies [
20]. Interestingly, USP8 is highly expressed in the brain and specifically in dopaminergic neurons. Moreover, its levels seem to be inversely correlated with the extent of Lewy Body (LB) ubiquitination in post-mortem brains of PD patients [
19]. This evidence indicates a protective effect of USP8 down-regulation, which might depend on its proteostatic activity or other activities correlated to USP8 pleiotropic functions.
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.
Thus, in this work we wanted to explore the possibility of a proteostatic effect of USP8 down-regulation in neurons.
We started by taking an unbiased approach to determine the repertoire of USP8 substrates and identify signaling pathways that are specifically altered upon USP8 down-regulation. To this aim, we generated fly lines stably down-regulating USP8 and performed a mass spectrometry (MS)-based analysis from protein lysates extracted from WT and USP8-down-regulating flies and subjected them to immunoaffinity isolation to enrich K-GG peptides [
38]. The method is based on the identification of di-glycine (GG) ubiquitin remnants that are left on lysine (K) residues after trypsinization and allows identifying ubiquitinated fragments, which should be specifically enriched in USP8-down-regulating conditions. Flies are an ideal model to do so because, as opposed to mice in which transcription and/or translation of the intact allele does compensates for the loss of one gene copy, USP8 down-regulation or hemizygosity in the fly exhibits reduced protein levels.
Gene ontology analysis on the ubiquitinated proteins identified signaling pathways regulating tissue differentiation (dorsoventral axis formation, Hegdehog signaling pathway, Foxo signaling pathway). Surprisingly, mitophagy was among the ten highest scoring KEGG pathways that came out from this analysis. Of particular relevance for us, among the hits that scored a significant VML index were Marf, fly ortologue of mitochondrial pro-fusion protein Mitofusin and Porin/VDAC. Both proteins are key regulators of mitochondrial quality control, in that Parkin-dependent ubiquitination of Marf/Mfn or VDAC signals selected mitochondria for degradation. Based on these results, the next step was to investigate the potential autophagic and mitophagic effect of USP8 down-regulation by taking advantage of fluorescent probes that allow measuring the autophagic and mitophagic flux in the drosophila brain in combination with fly genetics. These approaches allowed identifying a mitophagic effect of USP8 down-regulation, which was clearly detectable in vivo in the fly brain and also in neurons of human origin.
Interestingly, USP8 down-regulation promoted basal mitophagy in a Parkin-independent fashion (
Figure 2D,E), whereas it inhibited Parkin mitochondrial translocation and mitophagy under stress condition (
Supplementary Figure S3). The latter was in agreement with a previous study showing that USP8-dependent deubiquitination of Parkin is required for Parkin recruitment and activation following CCCP-induced mitochondrial stress [
17]. Thus, USP8 seems to play a role in both Parkin-independent (basal) mitophagy and stress-induced mitophagy triggered by mitochondrial depolarization. The molecular mechanism of mitophagy induction under basal conditions is unknown. 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. 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. Marf/Mfn is also target of E3 ubiquitin ligases other than Parkin, for example MUL1 [
5]. It is possible that MUL1 operates as the E3 ubiquitin ligase regulating the ubiquitination of Marf/Mfn and mitophagy, in opposition to USP8. Further studies are required to clearly dissect the molecular mechanism of mitophagy induction triggered by USP8 down-regulation and its physiological importance.
Of particular relevance for a potential therapeutic application of USP8 down-regulation, potent and highly specific inhibitors of USP8 are available, which were generated based on the USP8 crystal structure. The best inhibitors at present were developed as derivatives of 9-oxo-9H-indeno[1,2-b]pyrazine-2,3-dicarbonitrile [
21]. Detailed pharmacokinetic data and dosing regimes are available. These compounds (DUBs-IN-2 and DUBs-IN-1) have an IC
50 value in the range of 200 nM and are highly specific for USP8 (e.g., IC
50 value of >100 μM for Usp7). 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]. 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]. Thus, important prerequisites for compound optimization and drug development exist for USP8 and can be readily exploited in aged-associated neurodegenerative disease models.
In summary, in this work we show that we can enhance autophagy and mitophagy by down-regulating USP8, a DUB that is upregulated in age-related neurodegenerative conditions [
19,
42]. 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]. Among the proteostatic mechanisms that might have therapeutic implications for the treatment of neurodegenerative conditions, mitophagy plays a crucial role. 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]. 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.
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.