A Cell-Based High-Throughput Screening Identified Two Compounds that Enhance PINK1-Parkin Signaling

ATP production was moderately stimulated by lower concentrations (0.1-0.6 μM) of both T0466 and T0467 at 24 h.

“ATP production was moderately stimulated by lower concentrations (0.1–0.6 μM) of both cpds at 24 h, whereas treatment with 1 μM T0466 mildly reduced ATP production at 24 and 48 h (Figure S4B).”

There was no evidence that T0466 and T0467 directly bind to Parkin.

“However, these cpds failed to stimulate Parkin E3 activity in vitro (data not shown), and there was no evidence that the cpds directly bind to Parkin (Figure S2C).”

Mild degradation of Mfn1 was observed in the presence of Parkin.

“Mild degradation of Mn1 was also observed in the presence of Parkin.”

GFP-MLKL was localized in the cytosol with occasional punctate signals.

“GFP-MLKL was localized in the cytosol with occasional punctate signals.”

Treatment with 20 μM T0467 for 3 h caused GFP-Parkin translocation to mitochondria in approximately 21% of HeLa/GFP-Parkin cells.

“When HeLa/GFP-Parkin cells were treated with 5 μM T0466 or 20 μM T0467 for 3 h, GTP-Parkin was translocated to the mitochondria in approximately 44% and 21% of cells, respectively (Figure 2B).”

T0466 and T0467 failed to stimulate Parkin E3 activity in vitro.

“However, these cpds failed to stimulate Parkin E3 activity in vitro (data not shown), and there was no evidence that the cpds directly bind to Parkin (Figure S2C).”

T0466 does not affect MIC60 phosphorylation by PKA.

“In vitro kinase assays showed that T0466 does not affect MIC60 phosphorylation by PKA (Figure S3E).”

T0466 treatment did not affect TBK1 activation in HeLa cells.

“T0466 treatment did not affect TBK1 activation in HeLa cells (Figure S3D).”

At concentrations that barely affected ΔΨm in HeLa cells (2 μM T0466, 3 μM T0467), ΔOTC degradation was Parkin-dependent.

“Using the cpds at concentrations that barely affected ΔΨm in HeLa cells (i.e., 2 μM for T0466 and 3 μM for T0467), we confirmed the Parkin dependence of ΔOTC degradation (Figure 2E).”

Treatment with 5 μM T0466 for 3 h caused GFP-Parkin translocation to mitochondria in approximately 44% of HeLa/GFP-Parkin cells.

“When HeLa/GFP-Parkin cells were treated with 5 μM T0466 or 20 μM T0467 for 3 h, GTP-Parkin was translocated to the mitochondria in approximately 44% and 21% of cells, respectively (Figure 2B).”

MLKL overexpression did not affect the mitophagy time course.

“Moreover, MLKL overexpression did not affect the mitophagy time course, suggesting that MLKL is unlikely to be a T0466 target in PINK1-Parkin signaling (Figure S3B).”

The NL reporter responded in the presence of Parkin following treatment with over 10 nM of valinomycin.

“The NL reporter responded in the presence of Parkin following treatment with over 10 nM of val.”

T0466 and T0467 did not elevate PINK1 levels.

“Consistent with these observations, these cpds did not elevate PINK1 levels, indicating that the molecular targets of these cpds are likely to be a protein other than PINK1.”

Treatment with 5 μM T0467 for 8 h did not affect OPA1 processing and PINK1 levels.

“Treatment with more than 3 μM of T0466 for 8 h caused a small increase in OPA1 processing and low levels of PINK1 accumulation (Figure 3A, lanes 4 and 5), whereas 5 μM T0467 did not affect OPA1 processing and PINK1 levels (Figure 3A, lane 8).”

In vitro kinase assays indicated that T0466 neither activated nor inhibited PINK1 kinase activity in the presence or absence of ATP.

“In vitro kinase assays indicated that T0466 neither activated nor inhibited PINK1 kinase activity in the presence or absence of ATP (Figure S3C).”

Treatments with T0466 and T0467 at concentrations of 0.1-1 μM did not show any cell toxicity by 48 h.

“Treatments with T0466 and T0467 at concentrations of 0.1–1 μM did not show any cell toxicity by 48 h (Figure S4A).”

Higher T0466 concentrations reduced ΔΨm.

“Monitoring of OPA1 processing revealed that higher T0466 concentrations reduced ΔΨm.”

Two compounds, T0466 and T0467, were obtained as drug candidates for PINK1-Parkin signaling activation.

“Two cpds, T0466 and T0467, were obtained as drug candidates for PINK1-Parkin signaling activation (Figures 1E and 1F).”

Treatment with 1 μM T0466 mildly reduced ATP production at 24 and 48 h.

“ATP production was moderately stimulated by lower concentrations (0.1–0.6 μM) of both cpds at 24 h, whereas treatment with 1 μM T0466 mildly reduced ATP production at 24 and 48 h (Figure S4B).”

T0467 induced the removal of ΔOTC without OPA1 processing.

“Moreover, T0467 induced the removal of ΔOTC without OPA1 processing, suggesting that these cpds facilitate Parkin activation independently of ΔΨm status.”

Over 12 μM T0467 was required for Parkin translocation.

“Over 5 μM of T0466 sufficiently stimulated the mitochondrial translocation of GFP-Parkin 3–8 h after treatment, whereas over 12 μM of T0467 was required for Parkin translocation (Figures 2A, S2A, and S2B).”

Simultaneous treatment with kinetin triphosphate (KTP) did not enhance T0466 or T0467 efficacy.

“Simultaneous treatment with a previously characterized PINK1 activation molecule, kinetin triphosphate (KTP), did not enhance T0466 or T0467 efficacy (Figure S2A) (Hertz et al., 2013).”

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The subcellular localization of GFP-MLKL was not altered by T0466 or by mitochondrial uncoupling treatment.

“The subcellular localization of GFP-MLKL was not altered by T0466 or by mitochondrial uncoupling treatment (Figure S3A).”

Over 5 μM T0466 sufficiently stimulated mitochondrial translocation of GFP-Parkin 3-8 h after treatment.

“Over 5 μM of T0466 sufficiently stimulated the mitochondrial translocation of GFP-Parkin 3–8 h after treatment, whereas over 12 μM of T0467 was required for Parkin translocation (Figures 2A, S2A, and S2B).”

OPA1 processing status revealed that T0466 affected ΔΨm at concentrations >1 μM in dopaminergic neurons and HeLa cells.

“OPA1 processing status revealed that T0466 affected ΔΨm at concentrations >1 μM in dopaminergic neurons and HeLa cells.”

T0466 and T0467 mitigate mitochondrial aggregation in dopaminergic neurons through reduced PINK1 activity in Drosophila.

“These molecules also mitigate the locomotion defect, reduced ATP production, and disturbed mitochondrial Ca2+ response in the muscles along with the mitochondrial aggregation in dopaminergic neurons through reduced PINK1 activity in Drosophila.”

PINK1 levels at T0466 concentrations ≥1 μM were comparable with those of healthy controls.

“However, PINK1 levels at T0466 concentrations ≥1 μM were comparable with those of healthy controls.”

T0466 and T0467 are chemical enhancers of PINK1-Parkin signaling.

“We report two chemical enhancers of PINK1-Parkin signaling, identified using a robust cell-based high-throughput screening system.”

Mitochondrial aggregation of body-wall muscles caused by PINK1 inactivation is markedly improved by T0467 and KTP.

“Mitochondrial aggregation of body-wall muscles by PINK1 inactivation was partially ameliorated by T0466 treatment and markedly improved by T0467 and KTP (Figure 4D).”

In PINK1 knockdown flies, aggregated or irregularly aligned mitochondria are observed in body-wall muscles.

“However, in PINK1 knockdown flies, aggregated or irregularly aligned mitochondria were observed (Figure 4D).”

All cpds examined mitigated the PINK1 inactivation-mediated mitochondrial morphological defects.

“All cpds examined mitigated the PINK1 inactivation-mediated larval locomotion defects and mitochondrial morphological defects and reduced ATP production.”

Sequential electrostimulation of motor neuron nerves in muscle-specific PINK1 knockdown flies causes a gradual increase in GCaMP intensity baseline during five consecutive stimulations.

“Sequential electrostimulation of motor neuron nerves in muscle-specific PINK1 knockdown flies caused a gradual increase in GCaMP intensity baseline during five consecutive stimulations, indicating a delay in mitochondrial Ca2+ efflux due to mitochondrial dysfunction (Figures 5A and S5C).”

T0466 and T0467 significantly improved the locomotion defects in PINK1 knockdown larvae.

“T0466 and T0467 significantly improved the locomotion defects in PINK1 knockdown larvae (Figure 4B).”

In normal control flies (LacZ RNAi, DMSO), mitochondria of body-wall muscles are aligned alongside the myofibrils.

“In normal control flies (LacZ RNAi, DMSO), the mitochondria of body-wall muscles were aligned alongside the myofibrils.”

Mitochondrial aggregates due to PINK1 inactivation are suppressed by T0466 and T0467.

“Mitochondrial aggregates due to PINK1 inactivation are suppressed by T0466 and T0467.”

Mitochondrial aggregation in larval body-wall muscles caused by PINK1 inactivation was improved after administration of T0466, T0467, or KTP.

“Mitochondrial aggregation in larval body-wall muscles caused by PINK1 inactivation was improved after the administration of T0466, T0467, or KTP.”

T0467 suppressed the altered mitochondrial Ca2+ response caused by reduced PINK1 expression.

“Moreover, T0467 suppressed the altered mitochondrial Ca2+ response caused by reduced PINK1 expression.”

Mitochondrial translocation of GFP-Parkin was observed in myoblasts at higher concentrations (>2.5 μM T0466 and >5 μM T0467) than those required in dopaminergic neurons.

“Mitochondrial translocation of GFP-Parkin was also observed in myoblasts at higher concentrations (>2.5 μM and >5 μM for T0466 and T0467, respectively) than those required in dopaminergic neurons (Figure 3D).”

These two cpds reduced the levels of the mitochondrial unfolded protein ΔOTC without ΔΨm reduction-induced PINK1 accumulation.

“Moreover, these two cpds reduced the levels of the mitochondrial unfolded protein ΔOTC without ΔΨm reduction-induced PINK1 accumulation.”

Treatment with T0467 and KTP at concentrations most effectual in motor behavior analyses significantly improves the delay in mitochondrial Ca2+ decay after the stimulation-mediated Ca2+ spike.

“Treatment with T0467 and KTP at concentrations most effectual in motor behavior analyses significantly improved the delay in mitochondrial Ca2+ decay after the stimulation-mediated Ca2+ spike, suggesting that T0467 and KTP alleviate PINK1 inactivation-induced mitochondrial dysfunction (Figures 5A and 5B).”

• Also stated in: § T0466 and T0467 Mitigate Phenotypes Caused by Reduced PINK1 Activity in Drosophila · plain

  “Reduced PINK1 activity-induced delay in mitochondrial Ca2+ efflux is improved by T0467 and KTP.”

• Also stated in: § Discussion · plain

  “Additionally, T0467 and KTP improved the mitochondrial Ca2+ response in Drosophila larval muscles.”

T0466 and T0467 reduce unfolded mitochondrial protein levels.

“Moreover, both compounds reduce unfolded mitochondrial protein levels, presumably through enhanced PINK1-Parkin signaling.”

T0466 and T0467 did not show obvious toxicity in Drosophila at concentrations <50 μM.

“T0466 and T0467 did not show obvious toxicity in Drosophila at concentrations <50 μM.”

Inactivation of PINK1 in larval muscles reduced the velocity of locomotion to approximately 50% of that of control LacZ knockdown flies.

“Inactivation of PINK1 in the larval muscles affected crawling activity and reduced the velocity of locomotion to approximately 50% of that of control LacZ knockdown flies (Figure 4B).”

All cpds examined reduced ATP production.

“All cpds examined mitigated the PINK1 inactivation-mediated larval locomotion defects and mitochondrial morphological defects and reduced ATP production.”

ATP production in PINK1 knockdown larvae was approximately 50% of that of LacZ knockdown flies.

“ATP production in PINK1 knockdown larvae was approximately 50% of that of LacZ knockdown flies and improved following T0466, T0467, and KTP administration (Figure 4C).”

Treatment with both cpds suppressed mitochondrial aggregates of larval dopaminergic neurons caused by PINK1 inactivation.

“Although Drosophila has an evolutionarily conserved blood-brain barrier (BBB), treatment with both cpds suppressed mitochondrial aggregates of larval dopaminergic neurons caused by PINK1 inactivation, suggesting that these drugs go through at least the Drosophila BBB (Figures 5C–5E) (Davis et al., 2019, Hindle et al., 2017, Limmer et al., 2014, Mayer et al., 2009, Zhang et al., 2018).”

All cpds examined mitigated the PINK1 inactivation-mediated larval locomotion defects.

“All cpds examined mitigated the PINK1 inactivation-mediated larval locomotion defects and mitochondrial morphological defects and reduced ATP production.”

ATP production in PINK1 knockdown larvae improved following T0466, T0467, and KTP administration.

“ATP production in PINK1 knockdown larvae was approximately 50% of that of LacZ knockdown flies and improved following T0466, T0467, and KTP administration (Figure 4C).”

Mitochondrial aggregation of body-wall muscles caused by PINK1 inactivation is partially ameliorated by T0466 treatment.

“Mitochondrial aggregation of body-wall muscles by PINK1 inactivation was partially ameliorated by T0466 treatment and markedly improved by T0467 and KTP (Figure 4D).”

Using this approach, we isolated two candidate cpds.

“Using this approach, we isolated two candidate cpds.”

T0466 does not modulate PINK1, TBK1, and PKA kinase activities.

“These results suggest that T0466 does not modulate PINK1, TBK1, and PKA kinase activities.”

The NL-Mfn1 system worked using cell suspensions with sensitivity comparable to that measured using adherent cells.

“The NL-Mfn1 system also worked using cell suspensions, the sensitivity of which was comparable with that measured using adherent cells (Figure S1).”

The NL-Mfn1 screening system reliably detected Mfn1 degradation only in the presence of Parkin.

“The NL-Mfn1 screening system reliably detected Mfn1 degradation only in the presence of Parkin.”

• Also stated in: § Development of a Cell-Based HTS System for PINK1-Parkin Activation Drugs · plain

  “This was observed by reduced NL activity in cells grown at two different densities, with a Z′-factor value of 0.50–0.57 (Figure 1B).”

PINK1-Parkin signaling maintains mitochondrial health.

“Early-onset Parkinson's disease-associated PINK1-Parkin signaling maintains mitochondrial health.”

• Also stated in: § Introduction · plain

  “Drosophila and mammalian cell studies revealed that PINK1 and Parkin have roles in mitochondrial quality control (Clark et al., 2006, Matsuda et al., 2010, Narendra et al., 2010, Park et al., 2006, Yang et al., 2006).”

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PINK1 and Parkin maintain dopaminergic neuron survival through correcting the dysfunctional mitochondrial pool.

“Subsequent studies using animal models for accelerated mitochondrial genomic error accumulation (Pickrell et al., 2015) and for mitochondrial stress through unfolded proteins (Pimenta de Castro et al., 2012) support the notion that PINK1 and Parkin maintain dopaminergic neuron survival through correcting the dysfunctional mitochondrial pool.”

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T0466 and T0467 facilitate Parkin activation independently of ΔΨm status.

“Moreover, T0467 induced the removal of ΔOTC without OPA1 processing, suggesting that these cpds facilitate Parkin activation independently of ΔΨm status.”

Mitofusin 1 (Mfn1) is rapidly degraded by active Parkin in association with mitochondrial depolarization-dependent PINK1 activation.

“Mitofusin family member, Mitofusin 1 (Mfn1), is rapidly degraded by active Parkin in association with mitochondrial depolarization-dependent PINK1 activation (Shiba-Fukushima et al., 2012).”

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MLKL is unlikely to be a T0466 target in PINK1-Parkin signaling.

“Moreover, MLKL overexpression did not affect the mitophagy time course, suggesting that MLKL is unlikely to be a T0466 target in PINK1-Parkin signaling (Figure S3B).”

The molecular targets of T0466 and T0467 are likely to be a protein other than PINK1.

“Consistent with these observations, these cpds did not elevate PINK1 levels, indicating that the molecular targets of these cpds are likely to be a protein other than PINK1.”

PINK1 is constitutively degraded by mitochondrial proteases and the ubiquitin-proteasome pathway in a mitochondrial membrane potential (ΔΨm)-dependent manner.

“The PINK1 mitochondrial serine/threonine protein kinase is constitutively degraded by a combination of mitochondrial proteases and the ubiquitin-proteasome pathway in a mitochondrial membrane potential (ΔΨm)-dependent manner (Jin et al., 2010).”

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The cell-based HTS for PINK1-Parkin signaling described here is robust.

“Here, we describe a robust cell-based HTS for PINK1-Parkin signaling.”

PINK1-Parkin activity is higher in dopaminergic neurons than in other cells differentiated from the same iPSC lines.

“Previously, we reported that PINK1-Parkin activity is higher in dopaminergic neurons than in other cells differentiated from the same iPSC lines (Shiba-Fukushima et al., 2017).”

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The cpds identified in this study might be especially effective in dopaminergic neurons affected in PD.

“Thus, the cpds identified in this study might be especially effective in dopaminergic neurons affected in PD.”

T0466 and T0467 do not appear to act directly on PINK1.

“In contrast, T0466 and T0467 do not appear to act directly on PINK1 and have unique potential for application as therapeutic reagents in diseases associated with mitochondrial degeneration.”

This study did not determine the molecular targets of these cpds.

“Although these cpds are effective in mammalian cells and Drosophila in the presence of PINK1, this study did not determine the molecular targets of these cpds.”

Drosophila has an evolutionarily conserved blood-brain barrier (BBB).

“Although Drosophila has an evolutionarily conserved blood-brain barrier (BBB), treatment with both cpds suppressed mitochondrial aggregates of larval dopaminergic neurons caused by PINK1 inactivation, suggesting that these drugs go through at least the Drosophila BBB (Figures 5C–5E) (Davis et al., 2019, Hindle et al., 2017, Limmer et al., 2014, Mayer et al., 2009, Zhang et al., 2018).”

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Parkin activated by T0466 or T0467 treatment was translocated to mitochondria in dopaminergic neurons.

“GFP-Parkin relocation from the whole cytosol to the punctate structures in dopaminergic neuron cultures was observed after treatment with 1 μM T0466 or 2.5 μM T0467 for 8 h, and the Parkin foci were overlapped with TOM20 signals, suggesting that Parkin activated by the drug treatment was translocated to the mitochondria (Figures 3B and 3C).”

These drugs (T0466 and T0467) go through at least the Drosophila BBB.

“Although Drosophila has an evolutionarily conserved blood-brain barrier (BBB), treatment with both cpds suppressed mitochondrial aggregates of larval dopaminergic neurons caused by PINK1 inactivation, suggesting that these drugs go through at least the Drosophila BBB (Figures 5C–5E) (Davis et al., 2019, Hindle et al., 2017, Limmer et al., 2014, Mayer et al., 2009, Zhang et al., 2018).”

Unlike rodent PINK1 or Parkin models, Drosophila models exhibit obvious mitochondrial degeneration.

“Unlike rodent PINK1 or Parkin models, Drosophila models exhibit obvious mitochondrial degeneration (Clark et al., 2006, Park et al., 2006, Yang et al., 2006).”

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The gradual increase in GCaMP intensity baseline in PINK1 knockdown flies indicates a delay in mitochondrial Ca2+ efflux due to mitochondrial dysfunction.

“Sequential electrostimulation of motor neuron nerves in muscle-specific PINK1 knockdown flies caused a gradual increase in GCaMP intensity baseline during five consecutive stimulations, indicating a delay in mitochondrial Ca2+ efflux due to mitochondrial dysfunction (Figures 5A and S5C).”