Mitochondrial turnover is required for proper cellular function. Both mitochondrial biogenesis and mitophagy are impaired in several degenerative and age-related diseases. The search for mitophagy activators recently emerged as a new therapeutical approach; however, there is a lack in suitable tools to follow mitochondrial turnover in a high-throughput manner. We demonstrate that the fluorescent protein, MitoTimer, is a reliable and robust probe to follow mitochondrial turnover. The screening of 15 000 small molecules led us to two chemically-related benzothiophenes that stimulate basal mitophagy in the beta-cell line, INS1. Enhancing basal mitophagy was associated with improved mitochondrial function, higher Complex I activity and Complex II and III expressions in INS1 cells, as well as better insulin secretion performance in mouse islets. The possibility of further enhancing mitophagy in the absence of mitochondrial stressors points to the existence of a ‘basal mitophagy spare capacity'. To this end, we found two small molecules that can be used as models to better understand the physiological regulation of mitophagy.

Introduction

Mitochondrial quality control (mtQC) reflects all cellular processes that ensure the maintenance of the pool of healthy and functional mitochondria, which is critical to meet the cellular demands of energy, calcium buffering, metabolic flux and control of apoptosis. At one end of mtQC, there is the synthesis of new mitochondria through biogenesis, and at the other end, their elimination through selective autophagy, mitophagy [1–4].

The balance between these two opposing forces determines the mitochondrial turnover rate, allowing for a constant, tissue-specific, mitochondrial mass, which can be challenged by physiological demands [5]. Physical exercise and calorie restriction, for example, enhance mitochondrial biogenesis, increasing net mitochondrial volume [6–9], while mitophagy is triggered in some cell types during developmental stages [10], in response to oxygen deprivation and as a consequence of mitochondrial damage [5].

During aging, there is a decline in mitochondriogenesis and mitophagy. While reduced biogenesis compromises the energy availability for the cell, reduced ability to eliminate dysfunctional mitochondria has deleterious effects, since these organelles have declined dynamics and trafficking and are more prone to generate reactive oxygen species (ROS). Oxidative damage to biomolecules is largely related to aging and cell death. Long-lived cells such as neurons, cardiomyocytes and beta-cells exhibit very low or non-existent replication capacity, and are as such particularly vulnerable to autophagy and mitophagy impairment and to excessive mitochondrial ROS formation [11].

Boosting autophagy has been shown to be beneficial in animal models of neurodegenerative [12–14] and cardiac diseases [15], hepatic fibrosis [16], diabetes [17], and cancer [18], and small-molecule enhancers of autophagy are increasingly being tested with promising results [18–20]. General stimulation of autophagy may help to revert mitophagy impairment when the impairment results from a compromise in the autophagic machinery (lysosome/autophagosome axis) [21]. However, stimulating autophagy per se does not address mitophagy-specific pathways. It is widely accepted that mitophagy is a selective event [3,1], with its own regulators, and mitophagy-targeted tools for high-throughput screening are being pursued [12].

To date, genome-wide screens based upon the activation of Pink1/Parkin were developed to better understand the activators of this regulatory axis [22–25]. However, a broader assay for mitochondrial turnover is required to identify small molecules with different targets, and potentially unveil new mitophagy regulatory pathways, in a high-throughput manner. While Pink1 and Parkin are strongly implicated in stress-induced mitophagy, very few studies have focused on physiological mitophagy [26] and fewer on basal mitophagy [27,28].

MitoTimer was previously engineered, by adding a mitochondrial-targeting sequence to the Timer protein [29,30], a mutant of Ds-Red, which emits green fluorescence when newly translated, with the emission shifting over time to red, a property which allows MitoTimer to be used to follow mitochondrial turnover (Figure 1), as demonstrated by us and others [29–33].

MitoTimer model.

Figure 1.
MitoTimer model.

MitoTimer, translated as a green fluorescent protein, is imported to newly synthesized mitochondria. Over time, the fluorescence shifts from green to red. MitoTimer red/green ratio is an established tool to follow mitochondrial aging and turnover [30]. MitoTimer red/green ratio is directly proportional to the aging of the mitochondria and inversely proportional to mitophagy and mitochondrial biogenesis.

Figure 1.
MitoTimer model.

MitoTimer, translated as a green fluorescent protein, is imported to newly synthesized mitochondria. Over time, the fluorescence shifts from green to red. MitoTimer red/green ratio is an established tool to follow mitochondrial aging and turnover [30]. MitoTimer red/green ratio is directly proportional to the aging of the mitochondria and inversely proportional to mitophagy and mitochondrial biogenesis.

Screening 15 000 small molecules in immortalized beta-cells INS1 stably expressing MitoTimer led to the discovery of two chemically-related benzothiophene derivatives (SPB08007 and MWP00839) that were able to increase basal mitochondrial turnover through enhanced mitophagy without triggering loss of mitochondrial membrane potential (ψm). Enhanced turnover promoted by SPB08007 resulted in a pool of healthier mitochondria with improved mitochondrial function, Complex I activity, and higher Complex II and III content. In mouse islets, insulin secretion capacity was enhanced due to lower basal secretion, an index associated with islet health.

Methodology

Mitotimer expressing INS1 cell generation, cell culture and treatments

MitoTimer-reporter was cloned into the pHAGE2 lenti-vector under the full-length EF1-alpha promoter to obtain constitutive expression. For the high-content screen (HCS), INS1 cells were infected with lentivirus carrying mitochondrial complex-I-targeted-MitoTimer plasmid. Highly expressing cells were selected by high-speed fluorescence-activated cell sorter (SY3200 cell sorter, Synergy, iCyt) and kept by the addition of puromycin (1 mg.l−1) in the cell media. Cells were thawed before each experiment, and MitoTimer expression checked through fluorescence.

INS1 cells were cultured with 100 IU/ml penicillin/streptomycin in RPMI media (12 mM glucose, 10% fetal bovine serum, 1 mM pyruvic acid, 10 mM Hepes, 2 mM glutamine and 0.1% beta-mercaptoethanol) at 37°C and 5% CO2.

High-content screening

To test the effects of different known modulators of autophagy and mitophagy on MitoTimer red/green ratio, and therefore determine the best control to use in the HCS accordingly to the estimated z-factor [34], INS1 cells expressing MitoTimer were treated for 18 h with: urolithin A 50 µM, bafilomycin A 200 nM, chloroquine 20 µM, and rapamycin 100 nM and imaged in two wavelengths (FITC — excitation 482/35 nM, emission 520/35 nm and TRITC — excitation 543/22 nm, emission 593/40 nm) under an Operetta PerkinElmer microscope system with 40× lens. HCS assay was developed at the Drug Discovery Unit, INCPM, Weizmann Institute of Science. The screening of 15 000 compounds (LOPAC — pharmacologically active compounds, Maybridge — diverse library and Analyticon) was performed in INS1 cells stably expressing MitoTimer under EF-1-alpha promoter. Briefly, cells were treated with 10 µM of each compound for 18 h and imaged under two wavelengths (FITC — excitation 482/35 nM, emission 520/35 nm and TRITC — excitation 543/22 nm, emission 593/40 nm) under ImageXpress Micro High-Content Imaging System/Molecular Devices. Rapamycin 100 nM was used as a control. In each cell plate, the neutral (DMSO) and negative control (Rapamycin) were repeated in 16 replicates while the treatments were performed in duplicates. Each plate was replicated and only hits found in the two independent plates were considered. Two fields of each well were analyzed, which allowed the analysis of 40–80 cells per well. MitoTimer red/green ratio for every cell was done in MetaXpress. To establish the hits which affect mitochondrial turnover due to mitochondrial clearance, four criteria were followed based on the calculated z-score (z-score ≥ 0.5) relative to the control (DMSO treated cells): (1) reduced MitoTimer red/green ratio; (2) MitoTimer red/green ratio reduction due to effect on the red readout; (3) green readout not increased more than 0.2 P-value from vehicle and (4) treatments which resulted in massive cell death were excluded. The compounds which affected MitoTimer red/green ratio in a dose–response, ranging from 10 to 50 µM, were classified as a hit.

The ‘screenability' or robustness of a high-throughput screen (HTS) can be expressed as a z′ factor [34], which is calculated as follow:

Estimated z-factor (z′) was calculated by 
z=1(3σc+σs)|μcμs|,
where σ and µ are standard deviation and mean, respectively, of control (c) and sample (s).

mCherry-GFP-Fis1101–152 determination of mitophagy

INS1 cells were seeded into four compartment CELLVIEW™ glass bottom cell culture dishes at a density of 2 × 104 cells/compartment. After 48 h cells were transduced with an adenoviral construct encoding the fluorescent mitophagy reporter mCherry-GFP-Fis1101–152 for 24 h. A media change containing the indicated treatment concentration or 0.1% DMSO as vehicle control was performed 8 h before the imaging session. Imaging was performed using a 63X Plan Neofluar objective and the Airyscan module of a Zeiss LSM880 confocal microscope. Twenty-five visual fields containing 59–85 cells in total were imaged per condition. The experiment was repeated three times independently. Image analysis was performed with FIJI ImageJ 1.51p and CellProfiler 2.2.0 rev ac0529e. Briefly, individual cells were cropped and the background was subtracted using a median filter assisted processing method [35]. Ratios of red/green fluorescence channels were computed and mitophagy positive structures were recognized based on a lower ratio cut-off of 2.5 of red over green. Mitophagy events per cell were determined by the following formula: 
Mitophagy events per cell=Total area of mitophagy positive structures per cellAverage area of one mitophagy structure.
Data were normalized to DMSO controls to account for day to day variation. Statistical analysis was performed using GraphPad Prism 7.02. Significant treatment differences were determined using One-way ANOVA and Tukey post-hoc analysis. Values of P < 0.05 (*) were considered significant.

Mitotracker green fluorescence, anion radical superoxide and mitochondrial membrane potential

INS1 cells were seeded into wells of a 96-wells glass bottom cell culture plate (CELLSTAR) at a density of 2 × 104 cells/well. To assess mitochondrial mass and anion radical superoxide, INS1 cells were treated with SPB08007, MWP00839 (25 µM) or DMSO 0.1% and after 18 h stained with Mitosox 500 nM for 30 min or 10 min, respectively, washed and imaged in the same conditions. Integrated fluorescence of each field was analyzed in Image J after background subtraction and threshold (IsoData) application. Twenty-four fields of each condition were analyzed in each experiment.

To assess mitochondrial membrane potential, INS1 cells were plated in each compartment of a quadrant imaging dish (Greiner Bio-One International) at a density of 1.2 × 105 cells/well. After 48 h, cells were incubated RPMI-1640 containing 25 µM of compound A or vehicle (0.1% DMSO). After 18 h, the cells were stained in RPMI-1640 media containing 200 nM mitotracker green (MTG) and 15 nM of tetramethylrhodamine, ethyl ester (TMRE); cells were then washed three times and finally kept in RPMI-1640 containing TMRE but lacking MTG. Live-Imaging was performed using a 63X Plan Neofluar objective and the Airyscan module of a Zeiss LSM880 confocal microscope. The mitochondrial membrane potential was analyzed by TMRE fluorescence corrected by MTG, using the Fiji/ImageJ software. One-way ANOVA and Tukey's multiple comparison test were used for statistical analysis; P-values ≤ 0.05 (*) were considered significantly different.

Cellular oxygen consumption

An hour before oxygen consumption measurements, cell media was replaced by assay media (2 mM glucose, 0.8 mM Mg2+, 1.8 mM Ca2+, 143 mM NaCl, 5.4 mM KCl, 0.91 mM, NaH2PO4 and 15 mg/ml Phenol red) for 120 min at 37°C (no CO2) before loading into the Seahorse Bioscience XF24 extracellular analyzer [36]. The ports of the cartridge containing the oxygen probes were loaded with the compounds to be injected during the assay (50 μl/port) and the cartridge was calibrated.

Basal respiration was recorded for 30 min, at ∼5 min intervals until system stabilization. Glucose was injected at a final concentration of 12 mM and glucose-stimulated respiration was recorded for ∼15 min. FCCP (Carbonyl cyanide-4-phenylhydrazone) was used at final concentration of 4 μM and injected with sodium pyruvate (Sigma) at a final concentration of 5 mM. Oligomycin and antimycin were used at final concentrations of 2 and 4 μM, respectively. All respiratory modulators were used at ideal concentrations titrated during preliminary experiments (not shown) and oxygen consumption rates were recorded for up to 15 min due to the toxicity of these compounds. OCR typical chart is displayed as OCR percentage of basal respiration. All the OCR values were subtracted from the lowest antimycin OCR value.

Complex I activity assay

Mitochondrial complex I enzyme activity was measured using Abcam's complex I enzyme activity microplate assay Kit (ab109721) by following the oxidation of reduced nicotinamide adenine dinucleotide (NADH) to oxidized NAD+ and the simultaneous reduction in a dye which leads to increased absorbance at 450 nm.

Cell viability

Viability was measured using an XTT kit (Biological Industries), as recommended by the manufacturer. Thirty thousand cells were seeded in a flat 96-well plate. To each well, 100 µl of growth media was added. The cells were incubated in a 5% CO2 incubator at 37°C. Cells were used to assay proliferation after 24–96 h. Each test included a blank containing complete medium without cells as a background control. To prepare a reaction solution sufficient for one plate (96 wells), 0.1 ml activation solution was added to 5 ml XTT reagent. Fifty microliters of the reaction solution was added to each well and the plate was incubated in an incubator for 2–24 h. Following the incubation, the plate was shook gently to evenly distribute the dye in the wells and the absorbance of the samples was measured against a background control with a spectrophotometer (ELISA reader) at a wavelength of 480 nm. To determine non-specific readings we used a wavelength of 650 nm which we subtracted from the 480 nm measurement. The average absorbance of the blank control wells was finally subtracted from that of the other wells. For all the experiment concerning cell viability, a kit of XTT reagent was used (Biological Industries).

Cell treatments and mitochondrial extraction

INS1 cells were treated with SBP08007 or MWP00839 25 µM for 24 h. Mitochondrial and cytosolic fractions from INS1 cells were separated by using the Mitochondria Isolation Kit for Cultured Cells (Pierce). INS1 cells were lysed in a hypotonic buffer (10 mM NaCl, 1.5 mM MgCl2 and 10 mM Tris–HCl, pH 7.5), and mitochondria were extracted in a Dounce homogenizer in mitochondrial buffer (1 mM EDTA, 210 mM mannitol, 70 mM sucrose and 5 mM Tris–HCl, pH 7.5), followed by centrifugation at 1300×g for 10 min at 4°C. The supernatant was further centrifuged at 17 000×g for 15 min at 4°C to pellet the mitochondria. The crude mitochondrial fraction was resuspended for washing and centrifuged at 17 000×g for 15 min at 4°C. The pellets were collected as the mitochondrial fraction.

Western blot

Cell lysates were diluted in Laemmli sample buffer (100 mM Tris–HCl, 2% SDS, 10% glycerol, 0.1% bromophenol blue) containing 5% β-mercaptoethanol. After heating at 95°C for 5 min, proteins were separated by SDS–PAGE previously added of Stain Free reagent (Bio-Rad), and transferred onto PVDF membranes. Membranes were blocked with 5% non-fat milk and detection of individual proteins was carried out by blotting with a specific primary antibody against ubiquitin (Calbiochem 1 : 1000). Chemiluminescence detection using a secondary peroxidase-linked anti-rabbit (Calbiochem; 1 : 10 000) and a detection system from Pierce KLP (Rockford, IL, U.S.A.) was performed. Stain-free total protein labeling was imaged in Image Lab 6.0 Software (Bio-Rad), which was used for quantification of the mitochondria and normalization to total protein staining [37].

Islet isolation

Islets were isolated from DBA/2 mice, as described in [38]. Briefly, pancreata of anesthetized mice were infused with collagenase (1 mg/ml, type XI, Sigma–Aldrich, Rehovot, Israel), excised and incubated for 30 min at 37°C. The digested tissue was vortexed, filtered and washed in HBSS (Biological Industries) containing 0.5% BSA (Sigma). The pellet was resuspended in RPMI medium 1640 supplemented with 10% FCS, 50 units/ml penicillin and 50 μg/ml streptomycin (all from Biological Industries). Islets were collected on a 100-μm cell strainer (BD, Falcon) and hand-picked using stereoscope (Zeiss, Oberkochen, Germany).

Glucose stimulation insulin secretion assay

After 24-h pretreatment with SPB08007 25 µM, islets were washed twice with PBS (Biological Industries, Bet-Haemek, Israel). For basal insulin secretion, the islets were incubated with Krebs-Ringer bicarbonate (KRB) buffer supplemented with 0.5% BSA and 2 mM glucose for 30 min at 37°C and 5% CO2. For stimulated insulin secretion, the buffer was replaced with KRB buffer supplemented with 0.5% BSA and 16 mM glucose and the islets were incubated for additional 60 min. Supernatant insulin content was measured by ELISA (Merck Milipore, Burlington, MA).

Results and discussion

Mitotimer optimization to follow mitochondrial turnover in a high-content screen

The aging of the mitochondrial network can be followed through the fluorescence switch of MitoTimer from green to red over time. Newly synthesized protein is incorporated into newly formed mitochondria [29]. Where MitoTimer expression is inducible, for example under a doxycycline promoter, a pulse of doxycycline generates an initially green mitochondrial network which, after 16 h, becomes a mix of green and red fluorescence (observable as yellow and orange) and is seen as completely red after 48 h [30].

We developed a system in which MitoTimer is stably expressed under the EF-1-alpha promoter. In this system MitoTimer is continuously expressed; basal rates of synthesis and degradation of mitochondria, and therefore MitoTimer, are steady, resulting in a constant red/green ratio. The ratiometric analysis corrects for differences in expression levels and gives a more robust index than the comparison of individual integrated fluorescence intensities [31].

Manipulating mitochondrial turnover alters MitoTimer red/green ratio, in a manner inversely proportional to mitochondrial biogenesis and mitophagy [29,31,33]. Therefore, green and red fluorescence intensities must be analyzed individually to conclude which events are impacting the ratio. As summarized by Trudeau et al. [31] changes to MitoTimer green levels reflect protein incorporation into the mitochondria, mainly due to mitochondrial biogenesis or import, while changes in MitoTimer red fluorescence indicate changes in the degradation rate of the protein, if integrated green fluorescence intensity is not altered between experimental groups. Where green and red MitoTimer fluorescence intensities are affected, the ratio may not reflect changes of mitochondrial turnover.

The length of the experiment was adjusted so that conditions affecting green MitoTimer levels did not result in changes in red MitoTimer levels. Since every green protein becomes red after 18–20 h, all cell treatments were performed under 18 h, thereby separating the effects on MitoTimer red from MitoTimer green. This is particularly important in mitophagy studies since, in most cases, mitochondrial biogenesis increases in parallel to mitophagy [31].

We tested three classes of compounds as controls to establish MitoTimer red/green ratio as an index to follow mitochondrial turnover in a HCS aiming to find the best control: (1) Urolithin A, a natural compound recently discovered as a stimulator of mitophagy [39]; (2) inhibitors of macroautophagy, bafilomycin A and chloroquine [40] and (3) rapamycin, a classical mTOR (mechanistic target of rapamycin) inhibitor which stimulates macroautophagy in most cell types [14]. Urolithin A significantly reduced MitoTimer red/green ratio in INS1 cells (Supplementary Figure S1A,B), due to reduction in red fluorescence, indicating enhanced mitochondrial clearance. Preventing lysosome acidification and fusion with autophagosome as a result of bafilomycin A1 treatment [41] enhanced the population of red mitochondria, and therefore, increased red/green MitoTimer ratio; the same was observed when neutralizing lysosome acidity with chloroquine (Supplementary Figure S1C). We previously demonstrated that inhibiting these last steps of autophagy affects MitoTimer red/green ratio readout in MEF cells [30]. Rapamycin increased MitoTimer red/green ratio by 50% (Supplementary Figure S1C), due to an accumulation of red fluorescence over time, resulting in a time-dependent increase in red/green ratio (Supplementary Figure S1D,E).

Although macroautophagy is required for the complete elimination of mitochondria and its inhibition certainly impairs basal mitophagy in some cell types [42,43], there is no evidence the stimulation of macroautophagy in the absence of mitochondrial stress can increase mitophagy, a selective event which requires loss of ψm and segregation of the damage units from the network [3,1]. In fact, Gomes et al. [44] have demonstrated that autophagy activation by nutrient starvation impairs mitophagy due to mitochondrial elongation and inability to segregate, and rapamycin was shown to reproduce the same phenotype regarding mitochondrial shape as starvation.

Rapamycin effect on MitoTimer red/green ratio was the strongest among all controls tested, and provided an average prime z-factor (z′) relative to vehicle, DMSO (dimethyl sulfoxide) of 0.4 indicating a robust response, therefore, rapamycin was used as a neutral control in every cell plate in parallel to neutral control DMSO. Briefly, z′ expresses how effectively the assay separates the positive and negative control values, based on means and standard deviations of both controls (full formula on methodology section), and it indicates the ‘screenability' of a HTS. The closer to 1 the z′ is, the more robust the assay is [34].

z′-score was calculated for the experimental conditions; in this case, it determines how far a testing molecule is from the control, indicating the quality of the assay and providing a cut-off to establish hits in a HCS/HTS. In the present HCS, we focused on the search for molecules that enhance mitochondrial turnover (lower red/green MitoTimer ratio, with reduced red MitoTimer levels). To determine that a small molecule is a hit, red/green MitoTimer ratio and red fluorescence reduction z′-score was set to be higher than 0.5 from DMSO; and green fluorescence and cell number variation of less than 0.5 z-score from DMSO control (Figure 2).

Hits finding criteria.

Figure 2.
Hits finding criteria.

Flow chart of the criteria used to establish a given compound as a ‘hit' which improves mitochondrial clearance.

Figure 2.
Hits finding criteria.

Flow chart of the criteria used to establish a given compound as a ‘hit' which improves mitochondrial clearance.

Identification of two chemically-related compounds that stimulate basal mitochondrial turnover through mitophagy

The HCS steps are summarized in Figure 3. Briefly, after establishing the best expression system which allowed a steady-state red/green MitoTimer ratio in the controls for the screening, INS1 cells were plated in 384-well plates, and in each well, 10 µM of a given compound were added for 16–18 h and live-cell imaging was performed using FITC (excitation 482/35 nm, emission 520/35 nm) and TRITC (excitation 543/22 nm, emission 593/40 nm) channels. The small molecules were chosen from three libraries: LOPAC — pharmacologically active compounds (1280 compounds), Maybridge — diverse library (9920 compounds) and Analyticon (3840 compounds). Individual cells were identified using the red channel, and red and green MitoTimer fluorescence were analyzed and ratio determined per cell. In the Hit finder software (GeneData), shown in Supplementary Figure S2, the small molecules which impacted mitochondrial turnover based on the chosen parameters (Figure 2) were identified. From 15 000 compounds, 47 reduced the red/green MitoTimer ratio due to a decrease in red MitoTimer levels (Figure 4A). The molecules that acted in a dose–response manner (4, 0.027%) were classified as the final hits. Two molecules passed our criteria, both benzothiophene derivatives from the Maybridge library, SPB08007 and MWP00839, are structurally similar (Figure 4B). These hits were further investigated.

High-content screening development.

Figure 3.
High-content screening development.

Steps used to perform the high-content screening for small molecules that improve mitochondrial clearance based on the Mitotimer model.

Figure 3.
High-content screening development.

Steps used to perform the high-content screening for small molecules that improve mitochondrial clearance based on the Mitotimer model.

High-content screening results.

Figure 4.
High-content screening results.

(A) Final hits number. (B) Chemical structures of the two hits identified as mitochondrial turnover stimulators: SPB08007 and MWP00839, from Maybridge library. (C) Representative images of Mitotimer fluorescence in INS1 cells treated compound SPB08007 or MWP00839 50 µM for 18 h. Images were acquired with an ImageXpress Micro High-Content Imaging System/Molecular devices microscope. (D,E) Dose–response effects of compound SPB08007 and MWP00839, respectively, on Mitotimer red/green ratio (expressed as % variation from the DMSO controls, Mean ± SEM).

Figure 4.
High-content screening results.

(A) Final hits number. (B) Chemical structures of the two hits identified as mitochondrial turnover stimulators: SPB08007 and MWP00839, from Maybridge library. (C) Representative images of Mitotimer fluorescence in INS1 cells treated compound SPB08007 or MWP00839 50 µM for 18 h. Images were acquired with an ImageXpress Micro High-Content Imaging System/Molecular devices microscope. (D,E) Dose–response effects of compound SPB08007 and MWP00839, respectively, on Mitotimer red/green ratio (expressed as % variation from the DMSO controls, Mean ± SEM).

SPB08007 and MWP00839 at 10 µM reduced red/green MitoTimer ratio (Figure 4C,D), due to decreased red MitoTimer fluorescence intensity. These results indicate a higher mitochondrial turnover rate due to mitochondrial clearance. The maximum reduction on the ratio index was 40% and 60%, respectively, at 50 µM dose (Figure 4C–E), which also reflected an increase in MitoTimer green readout. Mitophagy and mitochondrial biogenesis have been reported to be activated in parallel [45] and at the highest concentrations, SPB08007 and MWP00839 are likely stimulating the formation of new mitochondria as well.

The impact of the hits on mitochondrial turnover was further confirmed by a mitophagy-specific assay, a protein with a tandem mCherry-GFP tag attached to the localization signal of the protein FIS1 at the OMM. Mitochondria of cells expressing this protein fluoresce red and green. When mitochondria enter the lysosomes, the GFP fluorescence is quenched, the remaining red fluorescence is seen as red puncta, representing the undergoing mitophagy events [46].

INS1 cells treatment with SPB08007 and MWP00839 significantly increased the number of acidic puncta (Figure 5A,B). Interestingly, the formation of acidic puncta reached a peak at 8 h (Figure 5B). In cells treated with MWP00839, the number of mitophagy events was sustained at 18 h, whereas in cells treated with SPB08007 mitophagy returned to control levels at 18 h, suggesting basal mitophagy can be stimulated, but is tightly regulated (Figure 5A). Basal mitophagy is currently being explored and, contrary to stress-induced mitophagy, was unveiled to be independent of PINK1 in most mammalian and Drosophila melanogaster tissues [27,28]. These findings further confirm higher mitochondrial clearance upon treatment with SPB08007 and MWP00839. At the dose used for most of the assays, the compounds conserved the cellular viability, and SPB08007 effects were comparable to DMSO control (Supplementary Figure S3).

Hits SPB08007 and MWP00839 effects on mitophagy confirmed through other methods.

Figure 5.
Hits SPB08007 and MWP00839 effects on mitophagy confirmed through other methods.

(A) Co-localization of mCherry-GFP-FIS1101-152 in INS1 cells after 8 h treatment with compounds SPB08007, MWP00839 (25 µM) or DMSO 0.1%. Cells were imaged using a 63× Plan Neofluar objective and the Airyscan module of a Zeiss LSM880 confocal microscope. (B) Mitophagy events per cell defined by the total area of mCherry-GFP colocalized structures per cell/average area of one mitophagy structure. Data are normalized by DMSO and expressed as Mean ± SEM from three independent experiments (30–40 cells were analyzed in each experiment). Values of *P < 0.05 vs. DMSO (One-way ANOVA and Tukey post-hoc analysis).

Figure 5.
Hits SPB08007 and MWP00839 effects on mitophagy confirmed through other methods.

(A) Co-localization of mCherry-GFP-FIS1101-152 in INS1 cells after 8 h treatment with compounds SPB08007, MWP00839 (25 µM) or DMSO 0.1%. Cells were imaged using a 63× Plan Neofluar objective and the Airyscan module of a Zeiss LSM880 confocal microscope. (B) Mitophagy events per cell defined by the total area of mCherry-GFP colocalized structures per cell/average area of one mitophagy structure. Data are normalized by DMSO and expressed as Mean ± SEM from three independent experiments (30–40 cells were analyzed in each experiment). Values of *P < 0.05 vs. DMSO (One-way ANOVA and Tukey post-hoc analysis).

The degradation rate of mitochondrial proteins varies between them and differs from mitochondria as a whole [47], therefore, following specific mitochondrial proteins is a limited approach to infer changes in mitochondrial mass or turnover. Nonetheless, C-II protein SDHB (succinate dehydrogenase complex iron sulfur subunit B, Complex II) and C-III protein UQCRC2 (Cytochrome b-c1 complex subunit 2) were increased by 2 and 1.5 fold, respectively, (Supplementary Figure S4), similarly to what was observed by Ryu et al. [39] following stimulation of mitophagy in C2C12 myotubes with Urolithin A. CI — NDFUB8 levels (Supplementary Figure S4A,D) were unaffected by SPB08007.

SPB08007 improves mitochondrial efficiency and Complex I activity

SPB08007 increased basal (Figure 6A,B), glucose-stimulated (Figure 6A,C) and maximum (Figure 6A,E) mitochondrial oxygen consumption rates (mtOCRs), while conserving mitochondrial coupling (oligomycin-sensitive respiration), resulting in higher ATP-linked oxygen consumption (Figure 6A,D). In the absence of an effect on mitochondrial coupling or increased mitochondrial mass, higher basal respiration is normally a response to a higher energy demand [4], which can be triggered by mitophagy/biogenesis events and by other unknown cellular processes activated by this compound. The spare respiratory capacity (increase in mtOCR caused by the uncoupler FCCP) relative to basal respiration was unchanged by SPB08007, despite higher absolute values of mtOCR (Figure 6A,E), which indicates that mitochondria are using more of their respiratory reserve compared with the control, probably due to a higher energetic demand.

SPB08007 increases mitochondrial oxygen consumption rates (mtOCR), but not spare capacity.

Figure 6.
SPB08007 increases mitochondrial oxygen consumption rates (mtOCR), but not spare capacity.

INS1 cells were treated with SPB08007 (25 µM) or DMSO (0.1%) for 48 h. (A) Representative mitochondrial oxygen consumption rates (OCR) of INS1 cells under basal condition (2 mM glucose) and after the subsequent addition of 10 mM glucose, 4 µM oligomicin, 5 µM FCCP and 2 µM antimycin., (B) basal mtOCR (last measurement after glucose addition), (C) glucose-stimulated mtOCR (last measurement after glucose addition), (D) ATP-linked mtOCR (glucose-stimulated mtOCR minus oligomycin-insensitive-mtOCR) and (E) maximal mtOCR (highest OCR after FCCP addition), respectively. (F) Mitochondrial spare capacity (% of maximum mtOCR relative to basal). OCR values in the presence of antimycin (non-mitochondrial respiration) were subtracted from all quantifications. Data expressed as Mean ± SEM from four independent experiments. *P < 0.05 vs. DMSO (Unpaired t-test).

Figure 6.
SPB08007 increases mitochondrial oxygen consumption rates (mtOCR), but not spare capacity.

INS1 cells were treated with SPB08007 (25 µM) or DMSO (0.1%) for 48 h. (A) Representative mitochondrial oxygen consumption rates (OCR) of INS1 cells under basal condition (2 mM glucose) and after the subsequent addition of 10 mM glucose, 4 µM oligomicin, 5 µM FCCP and 2 µM antimycin., (B) basal mtOCR (last measurement after glucose addition), (C) glucose-stimulated mtOCR (last measurement after glucose addition), (D) ATP-linked mtOCR (glucose-stimulated mtOCR minus oligomycin-insensitive-mtOCR) and (E) maximal mtOCR (highest OCR after FCCP addition), respectively. (F) Mitochondrial spare capacity (% of maximum mtOCR relative to basal). OCR values in the presence of antimycin (non-mitochondrial respiration) were subtracted from all quantifications. Data expressed as Mean ± SEM from four independent experiments. *P < 0.05 vs. DMSO (Unpaired t-test).

The higher mitochondrial efficiency was associated with a two-and-a-half-fold increase in mitochondrial Complex I activity (Figure 7A,B), which contributes to a faster flow of electrons (Figure 7). The ψm was not reduced by SPB08007 (Figure 8A,B), strongly indicating this drug's mechanism of action is not through loss of ψm. Indeed, anion radical superoxide formation, measured by Mitosox, was unaffected (Figure 8C,D). Thus, unlike most known mitophagy activators [39,48,49], SPB08007 is not causing mitochondrial stress or uncoupling, similarly to P-62-mediated mitophagy inducer (PMI), a compound described to induce mitophagy independently of Parkin and without lowering the mitochondrial membrane potential [50].

SPB08007 increases mitochondrial complex I activity.

Figure 7.
SPB08007 increases mitochondrial complex I activity.

(A) Representative traces of NADH oxidation by mitochondria from INS1 cells previously treated with 25 µM SPB08007 or 0.1% DMSO. (B) Complex I activity expressed in OD/min/mg of protein. Data expressed as Mean ± SEM from three experiments. P = 0.0368 vs. DMSO control (Unpaired t-test).

Figure 7.
SPB08007 increases mitochondrial complex I activity.

(A) Representative traces of NADH oxidation by mitochondria from INS1 cells previously treated with 25 µM SPB08007 or 0.1% DMSO. (B) Complex I activity expressed in OD/min/mg of protein. Data expressed as Mean ± SEM from three experiments. P = 0.0368 vs. DMSO control (Unpaired t-test).

SPB08007 increases mitochondrial membrane potential, without affecting anion radical superoxide formation.

Figure 8.
SPB08007 increases mitochondrial membrane potential, without affecting anion radical superoxide formation.

INS1 cells were treated for 24 h with SPB08007 (25 µM) or DMSO (0.1%). Mitochondrial membrane potential was analyzed cell by cell through TMRE staining corrected by mitotracker green (MTG). Cells were imaged using a 100× Plan Neofluar objective and the Airyscan module of a Zeiss LSM880 confocal microscope. (A) Representative images of TMRE and MTG co-localization. (B) TMRE/MTG, relative to DMSO. (C) Representative images of Mitosox staining, acquired using a 40× lens of a PerkinElmer Operetta high-content imaging system microscope; oligomycin (4 µM) was used as positive control. (D) Mitosox Integrated Fluorescence quantification. Each experiment was repeated twice, 59–85 cells of each group were analyzed in each experiment for TMRE/MTG, and 48 fields were analyzed in each experiment for Mitosox analysis. Data expressed as Mean ± SEM. *P < 0.05 vs. DMSO (paired t-test); ***P < 0.05 vs. DMSO and SPB08007 (One-way ANOVA and Tukey post-hoc analysis).

Figure 8.
SPB08007 increases mitochondrial membrane potential, without affecting anion radical superoxide formation.

INS1 cells were treated for 24 h with SPB08007 (25 µM) or DMSO (0.1%). Mitochondrial membrane potential was analyzed cell by cell through TMRE staining corrected by mitotracker green (MTG). Cells were imaged using a 100× Plan Neofluar objective and the Airyscan module of a Zeiss LSM880 confocal microscope. (A) Representative images of TMRE and MTG co-localization. (B) TMRE/MTG, relative to DMSO. (C) Representative images of Mitosox staining, acquired using a 40× lens of a PerkinElmer Operetta high-content imaging system microscope; oligomycin (4 µM) was used as positive control. (D) Mitosox Integrated Fluorescence quantification. Each experiment was repeated twice, 59–85 cells of each group were analyzed in each experiment for TMRE/MTG, and 48 fields were analyzed in each experiment for Mitosox analysis. Data expressed as Mean ± SEM. *P < 0.05 vs. DMSO (paired t-test); ***P < 0.05 vs. DMSO and SPB08007 (One-way ANOVA and Tukey post-hoc analysis).

In fact, the ψm was slightly, but significantly, raised by this drug, without parallel changes in mitochondrial morphology (Supplementary Figure S5). The ongoing basal mitophagy activation is likely eliminating the mitochondrial subpopulation with lower potential, as expected, increasing the pool of coupled mitochondria, with a higher average TMRE/MTG ratio (Figure 8A,B). The improved CI activity, without changes in CI protein levels, may also reflect the resulting concentration of ‘young mitochondria' (less damaged), as indicated by lower MitoTimer red/green ratio (Figure 4). Interestingly, mitochondrial proteins isolated from INS1 cells previously treated with SPB08007, were found to be less ubiquitinated (Supplementary Figure S6), suggesting SPB08007 is not causing mitochondrial ubiquitination, but is rather improving the elimination of organelles with higher ubiquitin content.

SPB08007 decreases basal insulin secretion

Elevated basal insulin secretion under fasting conditions together with insufficient stimulated insulin release is an important hallmark of type 2 diabetes [51]. In vitro, high basal insulin secretion has been linked to mitochondrial dysfunction, for example in response to glucolipotoxicity [52] and PINK1 deficiency [53]. SPB08007 significantly reduced insulin secretion at 2 mM glucose (basal), while maintaining the levels of glucose-stimulated secretion (16 mM of glucose) (Figure 9A), resulting in a higher secretion fold in response to glucose (Figure 9B), which reflects a better metabolic coupling [54,55]. We previously found similar results in mouse islets treated with serum of calorie-restricted animals [56], an in vitro model associated with extended cellular lifespan and improved mitochondrial function [57,58].

SPB08007 improves the glucose-stimulated insulin secretion fold in mouse islets.

Figure 9.
SPB08007 improves the glucose-stimulated insulin secretion fold in mouse islets.

(A) Mouse islets were pre-incubated with SPB08007 (25 µM) or DMSO (0.1%) for 24 h. Islets were adapted to KRB media containing glucose 2 mM for 1 h, followed by media exchange containing glucose 2 mM (basal) or 12 mM (stimulated). After 1 h, Insulin released in the media was measured by ELISA. (B) Insulin secretion fold (glucose stimulated/basal secretion). Data expressed as Mean ± SEM from three independent experiments (each experiment was performed in four replicates with six islets per replicate). *P < 0.05 vs. DMSO basal (unpaired t-test). ***P < 0.001 vs. DMSO (unpaired t-test).

Figure 9.
SPB08007 improves the glucose-stimulated insulin secretion fold in mouse islets.

(A) Mouse islets were pre-incubated with SPB08007 (25 µM) or DMSO (0.1%) for 24 h. Islets were adapted to KRB media containing glucose 2 mM for 1 h, followed by media exchange containing glucose 2 mM (basal) or 12 mM (stimulated). After 1 h, Insulin released in the media was measured by ELISA. (B) Insulin secretion fold (glucose stimulated/basal secretion). Data expressed as Mean ± SEM from three independent experiments (each experiment was performed in four replicates with six islets per replicate). *P < 0.05 vs. DMSO basal (unpaired t-test). ***P < 0.001 vs. DMSO (unpaired t-test).

Overall, by establishing MitoTimer as a feasible tool to follow mitochondrial turnover in a HTS/HCS, and focusing on mitochondrial clearance, we found two chemically-related small molecules that enhance basal mitophagy without causing mitochondrial stress. The existence of a ‘spare basal mitophagy' which can be activated in a physiological context presents an exciting, new model for studying mitophagy and suggests that novel regulators of this pathway could be viable targets for future drug development.

Abbreviations

     
  • CI

    mitochondrial complex I

  •  
  • CII

    mitochondrial complex II

  •  
  • CIII

    mitochondrial complex III

  •  
  • DMSO

    dimethyl sulfoxide

  •  
  • FCCP

    carbonyl cyanide-4-phenylhydrazone

  •  
  • HCS

    high-content screening

  •  
  • HTS

    high-throughput screen

  •  
  • KRB

    Krebs-Ringer bicarbonate

  •  
  • MTG

    mitotracker green

  •  
  • mtOCR

    mitochondrial oxygen consumption rate

  •  
  • mTOR

    mechanistic target of rapamycin

  •  
  • mtQC

    mitochondrial quality control

  •  
  • NDUFB8

    NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8

  •  
  • OCR

    oxygen consumption rate

  •  
  • OXPHOS

    oxidative phosphorylation

  •  
  • PINK1

    PTEN induced putative kinase 1

  •  
  • ROS

    reactive oxygen species

  •  
  • SDHB

    succinate dehydrogenase complex iron sulfur subunit B

  •  
  • TMRE

    tetramethylrhodamine, ethyl ester

  •  
  • UQCRC2

    cytochrome b-c1 complex subunit 2

  •  
  • z′

    z-prime factor

  •  
  • ψm

    mitochondrial membrane potential

Authors Contribution

F.M.C., N.K., A.P., B.M.B., D.W., E.A., Y.R., R.G. and G.L. designed, conducted the experiments and analyzed the data. F.M.C., G.L., A.P., D.W. and O.S. wrote the manuscript. O.S., H.B. and E.C.L. supervised and supported the work.

Funding

This work was financed by the National Institute for Biotechnology in the Negev.

Acknowledgements

Prof. Assaf Rudich and Prof. Varda Shoshan-Barmatz from the Ben Gurion University in the Negev for the scientific input; Dr. Yulia Beckman and Dr. Alon Zila for the technical support and Ms. Priscilla Vassão for the artistic help. FMC had a fellowship from the National Institute for Biotechnology in the Negev (NIBN) from 2014 to 2017.

Competing Interests

F.M.C., G.L. and O.S. are co-inventors in a patent filled by this institute. A.P. and O.S. are co-founders of Enspire Bio.

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Supplementary data