Activation of Ca2+ phosphatase Calcineurin regulates Parkin translocation to mitochondria and mitophagy in flies

Abstract

Selective removal of dysfunctional mitochondria via autophagy is crucial for the maintenance of cellular homeostasis. This event is initiated by the translocation of the E3 ubiquitin ligase Parkin to damaged mitochondria, and it requires the Serine/Threonine-protein kinase PINK1. In a coordinated set of events, PINK1 operates upstream of Parkin in a linear pathway that leads to the phosphorylation of Parkin, Ubiquitin, and Parkin mitochondrial substrates, to promote ubiquitination of outer mitochondrial membrane proteins. Ubiquitin-decorated mitochondria are selectively recruiting autophagy receptors, which are required to terminate the organelle via autophagy. In this work, we show a previously uncharacterized molecular pathway that correlates the activation of the Ca²⁺-dependent phosphatase Calcineurin to Parkin translocation and Parkin-dependent mitophagy. Calcineurin downregulation or genetic inhibition prevents Parkin translocation to CCCP-treated mitochondria and impairs stress-induced mitophagy, whereas Calcineurin activation promotes Parkin mitochondrial recruitment and basal mitophagy. Calcineurin interacts with Parkin, and promotes Parkin translocation in the absence of PINK1, but requires PINK1 expression to execute mitophagy in MEF cells. Genetic activation of Calcineurin in vivo boosts basal mitophagy in neurons and corrects locomotor dysfunction and mitochondrial respiratory defects of a Drosophila model of impaired mitochondrial functions. Our study identifies Calcineurin as a novel key player in the regulation of Parkin translocation and mitophagy.

Fig. 1. CCCP promotes Parkin translocation to mitochondria and lysosomes.

A Representative confocal images of MEF cells transfected with mCherry-Parkin and mito-YFP for 2 days before being treated with DMSO or 10 μM CCCP for 3 h. The panels on the right show enlarged merged views of the boxed areas. B Quantification of A. Graph bar shows mean ± SEM of percentage of cells with mCherry-Parkin on mitochondria for at least ≥ 300 cells per biological replicate. Student’s t-test (n = 9-10; p < 0.0001). C Quantification of A by using Squassh. The graph bars show mean ± SEM of Squassh colocalization coefficient for at least ≥ 50 images per biological replicate. 0=no colocalization, 1 = perfect colocalization. Student’s t-test (n = 5; p < 0.0001). D Representative confocal images of MEF cells transfected with mCherry-Parkin and LAMP1GFP for 2 days and treated with 10 μM CCCP for 3 h. E Quantification of D by using Squassh. The graph bars show mean ± SEM of Squassh colocalization coefficient for at least ≥ 50 images per biological replicate. Student’s t-test (n = 3; p < 0.05). F Representative confocal images of MEF cells transfected with mCherry-Parkin and with Rab5GFP for 2 days and treated with 10 μM CCCP for 3 h. G Quantification of F by using Squassh. The graph bars show mean ± SEM of Squassh colocalization coefficient for at least ≥ 50 images per biological replicate. Student’s t-test (n = 3; p < 0.05).

Fig. 2. Parkin translocation to mitochondria is regulated by Calcineurin.

A Representative confocal images of MEF cells transfected with mCherry-Parkin, mito-YFP and dominant negative CaN (ΔCnA^H151Q) or the empty vector (EV) for 2 days before being treated with DMSO or 10 μM CCCP for 3 h. B Quantification of A. Graph bar shows mean ± SEM of percentage of cells with mCherry-Parkin on mitochondria for at least ≥ 300 cells per biological replicate. Two-way ANOVA followed by Tukey’s multiple comparison test (n = 3-9; p < 0.0001). C Quantification of A using Squassh. The graph bars show mean ± SEM of Squassh colocalization coefficient for at least ≥ 50 images per biological replicate. 0 = no colocalization, 1 = perfect colocalization. Two-way ANOVA followed by Tukey’s multiple comparison test (n = 4-5; p < 0.001). D Representative confocal images of MEF cells transfected with mCherry-Parkin, mito-YFP in which CaN was downregulated, and relative control. E Quantification of D. Graph bar shows mean ± SEM of percentage of cells with mCherry-Parkin on mitochondria for at least ≥ 300 cells per biological replicate. Two-way ANOVA followed by Tukey’s multiple comparison test (n = 3; p < 0.01). F Quantification of D using Squassh. The graph bar shows mean ± SEM of Squassh colocalization coefficient for at least ≥ 50 images per biological replicate. 0=no colocalization, 1 = perfect colocalization. At least 3 independent experiments were performed. Two-way ANOVA followed by Tukey’s multiple comparison test (n = 3; p < 0.01).

Fig. 3. Parkin translocation is induced by Calcineurin in the absence of PINK1.

A Representative confocal images of PINK1 KO MEF cells transfected with mCherry-Parkin, mito-YFP and with empty vector (EV) or constitutively active CaN (ΔCnA). B Quantification of A Graph bar shows mean ± SEM of percentage of cells with mCherry-Parkin on mitochondria for at least ≥ 300 cells per biological replicate. Two-way ANOVA followed by Tukey’s multiple comparison test (n = 3-4; p < 0.001). C Quantification of A using Squassh. The graph bars show mean ± SEM of Squassh colocalization coefficient for at least ≥ 50 images per biological replicate. 0 = no colocalization, 1 = perfect colocalization. At least 3 independent experiments were performed. Two-way ANOVA followed by Tukey’s multiple comparison test (n = 3-4; p < 0.001). D Representative confocal images of PINK1 KO MEF cells transfected with mCherry-ParkinS65E, UbS65E, mito-YFP and with empty vector (EV) or dominant negative CaN (ΔCnA^H151Q). E Quantification of D Graph bar shows mean ± SEM of percentage of cells with mCherry-Parkin on mitochondria for at least ≥ 300 cells per biological replicate. Two-way ANOVA followed by Tukey’s multiple comparison test (n = 4; p < 0.0001). F Quantification of D using Squassh. The graph bars show mean ± SEM of Squassh colocalization coefficient for at least ≥ 50 images per biological replicate. 0 = no colocalization, 1 = perfect colocalization. Two-way ANOVA followed by Tukey’s multiple comparison test (n = 4; p < 0.0001). G Parkin thermal stability assay. WT MEFs expressing constitutive active CaN (ΔCnA) or empty vector (EV) were suspended in PBS and snap-freezed in liquid nitrogen before being aliquoted into a PCR strip and incubated at the indicated temperature for 3 min. The lysates were centrifugated at high speed and the soluble fraction was loaded into SDS-PAGE gel. Representative Western blotting analysis for Parkin stability is shown. H Densitometric analysis of G. Student’s t-test (n = 4; p < 0.05). I Parkin thermal stability assay. PINK1 KO MEFs expressing constitutive active CaN (ΔCnA) or empty vector (EV) were suspended in PBS and snap-freezed in liquid nitrogen before being aliquoted into a PCR strip and incubated at the indicated temperature for 3 min. The lysates were centrifugated at high speed and the soluble fraction was loaded into SDS-PAGE gel. Representative Western blotting analysis for Parkin stability is shown. J Densitometric analysis of I. Student’s t-test (n = 6; p < 0.05).

Fig. 4. Parkin translocation induced by Calcineurin is Miro1-dependent.

A Representative confocal images of Ctrl siRNA MEF cells transfected with mCherry-Parkin, mito-YFP and with empty vector (EV) or constitutively active CaN (ΔCnA) for 2 days before being treated with DMSO or 10 μM CCCP for 3 h. B Quantification of A using Squassh. The graph bars show mean ± SEM of Squassh colocalization coefficient for at least ≥ 10 images per biological replicate. 0 = no colocalization, 1 = perfect colocalization. Two-way ANOVA followed by Holm-Sidak multiple comparison test (n = 3; p < 0.01). C Representative confocal images of Miro1 siRNA MEF cells transfected with mCherry-Parkin, mito-YFP and with empty vector (EV) or constitutively active CaN (ΔCnA) for 2 days before being treated with DMSO or 10 μM CCCP for 3 h. D Quantification of C using Squassh. The graph bars show mean ± SEM of Squassh colocalization coefficient for at least ≥ 10 images per biological replicate. 0=no colocalization, 1 = perfect colocalization. Two-way ANOVA followed by Holm-Sidak multiple comparison test (n = 3; p < 0.01).

Fig. 5. Calcineurin interacts with Parkin.

A In vitro interaction assay: schematic representation. Affinity-purified recombinant His-tagged Parkin is produced from bacteria, and coupled to a His-affinity resin to generate Parkin-His-resin. The resin is incubated with protein lysate extracted from cells expressing Flag-tagged CaN. The resin is washed to remove nonspecifically adhering proteins, and Imidazole is used to elute the complexes from the resin. The obtained eluate is separated by SDS-PAGE and analyzed by immunoblotting. B In vitro interaction assay. His-tagged Parkin or His-tagged MEF2D coupled to a His-affinity resin are incubated with cell lysate obtained from MEFs transfected with CaN-Flag or USP14-Flag, as indicated. The eluate is separated by SDS-PAGE and analyzed by immunoblotting using anti-Flag, anti-Parkin and anti-His antibodies (FT = flow through; EL=eluate). C Representative images of Proximity Ligation Assay (PLA) performed on HeLa cells transfected with Parkin-mCherry and CaN-FLAG (PPP3CB-FLAG). After fixation, cells were processed for investigating Parkin proximity interactions by incubating with anti-Parkin antibody and anti-PPP3CB antibody, and corresponding secondary antibodies, or secondary antibodies alone as negative control. CaN and Parkin interactions are represented by green dots. As negative biological control for PLA we used anti-Parkin antibody and anti-GRASP65 antibody in YPF-Parkin expressing HeLa cells. As positive control for PLA, we used anti-GRASP65 antibody and anti-GM130 antibody. GRASP65 is a peripheral membrane protein that resides in the cis-Golgi apparatus, and interacts with GM130 (also expressed in the cis-Golgi network) but not with Parkin. GRASP65 and GM130 interactions are represented by red dots. White squares contain higher-magnification images. D Quantification of C. Graph bar shows mean ± SEM of PLA dots per cell for at least ≥ 350 cells per biological replicate. Student’s t-test (n = 3-4; p < 0.05). E HEK 293 T cells were treated with DMSO or CCCP-2 h and subjected to immunoprecipitation (IP) of Parkin using mouse anti-Parkin antibody or anti-mouse IgG as negative control. Western Blot analysis was performed with rabbit anti-CaN antibody or mouse anti-Parkin antibody on the pulled-down samples. Inputs represent 5% of the protein lysates and IP eluate 100% of the protein lysates. Mouse IgG TrueBlot® ULTRA enabled detection of Parkin band, without hindrance by interfering immunoprecipitating IgG heavy chains.

Fig. 6. Parkin translocation is induced by Calcineurin independently of Drp1 mitochondrial recruitment and activity.

A Representative confocal images of wild type MEF cells transfected with mCherry-Parkin and YFP-Drp1 or YFP-Drp1 S637A plus dominant negative CaN (ΔCnA^H151Q) for 2 days before being treated with DMSO or 10 μM CCCP for 3 h. B Quantification of A. Graph bar shows mean ± SEM of percentage of cells with mCherry-Parkin puncta for ≥ 300 cells per biological replicate. Two-way ANOVA followed by Tukey’s multiple comparison test (n = 3). C Representative confocal images of wild type MEF cells transfected with mCherry-Parkin, mito-YFP and constitutive active CaN (ΔCnA) plus Drp1 or Drp1 K38A for 2 days before being treated with DMSO or 10 μM CCCP for 3 h. D Quantification of C. Graph bar shows mean ± SEM of percentage of cells with mCherry-Parkin on mitochondria for ≥ 300 cells per biological replicate. Two-way ANOVA followed by Tukey’s multiple comparison test (n = 3).

Fig. 7. Calcineurin is required for CCCP-induced mitophagy.

A Western blot analysis of protein lysates extracted from MEFs expressing empty vector (EV) or dominant negative CaN (ΔCnA^H151Q). Cells were treated with 10 μM CCCP for the indicated time. B Quantification of A. Line chart shows mean ± SEM of ATP5A protein level normalized to Actin. Student’s t-test (n = 3; p < 0.01). C Western blot analysis of protein lysates extracted from MEFs downregulating Calcineurin and control. Cells were treated with 10 μM CCCP for the indicated time. D Quantification of C. Line chart shows mean ± SEM of ATP5A protein level normalized to Actin. Student’s t-test (n = 5; p < 0.05). E Representative confocal images of cells transfected with LC3GFP and MitoKate plus dominant negative CaN (ΔCnA^H151Q) or empty vector (EV). F Representative confocal images of cells transfected with LC3GFP and MitoKate in CaN downregulating condition and matching control. G Quantification of E using Squassh. Two-way ANOVA followed by Tukey’s multiple comparison test (n = 3; p < 0.05). H Quantification of F using Squassh. Two-way ANOVA followed by Tukey’s multiple comparison test (n = 3; p < 0.05). I mt-Keima analysis in CaN downregulation condition upon CCCP treatment. mt-Keima demonstrates a greater than 4-fold change in ratiometric fluorescence in CCCP-treated cells, which was blunted upon CaN downregulation. Two-way ANOVA followed by Tukey’s multiple comparison test (n = 3; p < 0.001).

Fig. 8. Calcineurin is required for CCCP-induced mitophagy.

A Representative image of mt-Keima expressing cells acquired on confocal microscope Operetta High-Content Imaging system. MEFs were transfected with constitutive active CaN (ΔCnA) or empty vector (EV). B mt-Keima analysis in MEFs expressing constitutive active CaN (ΔCnA) and relative control (EV). Two-way ANOVA followed by Tukey’s multiple comparison test (n = 6; p < 0.01). C Representative electron microscope images showing the presence of autophagosomes with mitochondrial-like structures inside in MEFs expressing constitutive active CaN (ΔCnA) and control (EV). 24 h CCCP-treated cells were used as positive control. Mitochondrial-like structures were identified as described in Chakraborty et al. [130]. D Bar graph represents mean ± SEM of the number of autophagosomes and autolysosomes per cell. At least 60 cells per biological replicate were analyzed from each group. Student’s t-test (n = 3-4; p < 0.01).

Fig. 9. Calcineurin requires PINK1 to induce mitophagy in MEFs.

A Representative Western blot analysis of protein lysates extracted from PINK1 WT and PINK1 KO MEFs. Cells of the indicated genotype were transfected with constitutive active CaN (ΔCnA) or empty vector (EV). Graph bars shows mean ± SEM of indicated mitochondrial protein level normalized to Actin. Student’s t-test (n = 8-11; *p < 0.05; **p < 0.01). B Representative confocal images of cells of the indicated genotype transfected with constitutive active CaN (ΔCnA) or empty vector (EV). Cells were pretreated with proteasome inhibitor MG132 before being fixed, permeabilized and incubated with the indicated primary antibodies and corresponding fluorophore-conjugated secondary antibodies. C Quantification of B using Squassh. Two-way ANOVA followed by Tukey’s multiple comparison test (n = 4; p < 0.05). D Representative confocal images of MEFs transfected with constitutive active CaN (ΔCnA) or empty vector (EV), and with the GFP-TAB2 NZF sensor for K63-linked ubiquitin chain, and mitoRFP to visualize the mitochondrial network. In one group 10μM CCCP was used as a positive control to trigger Parkin translocation and Parkin-dependent ubiquitination of mitochondria. E Quantification of D. Graph bars shows the co-localisation of k63 linked ubiquitin probe (GFP-TAB2 NZF) with mitochondria. Co-localisation index was analysed with Coloc2 plugin (Fiji). tM2 = fraction of GFP TAB2 NZF co-localising with mitochondria (0 = null, 1 = 100%). F Representative Western blot analysis of protein lysates extracted from PINK1 WT and PINK1 KO MEFs, and incubated with the indicated antibodies, after being immunoprecipiated with HisPur Ni-NTA Magnetic Beads (Thermoscientific). Cells of the indicated genotype were transfected with His-Ubiquitin and constitutive active CaN (ΔCnA) or empty vector (EV). Cells were pretreated with proteasome inhibitor MG132 for 4 h before being collected.

Fig. 10. Constitutive active Calcineurin corrects locomotor ability and mitochondrial function of PINK1 KO flies.

A Schematic representation of the climbing assay. 10 flies were put into a tube in a dark room. A light was put on the top of the tube. After tapping the flies at the bottom of the tube, the number of flies that successfully climbed above the 6-cm mark after 10 s was recorded. B Graph bar shows mean ± SEM of the climbing performance of flies of the indicated genotype. One-way ANOVA followed by Sidak’s multiple comparison test (n = 5; p ≤ 0.001). C Graph bar shows mean ± SEM of the climbing rescue (arbitrary unit) of PINK1 KO flies (PINK1B9) overexpressing Parkin and treated as indicated for 48 h with DMSO or FK506. Student’s t-test (n = 5; p ≤ 0.01). D Graph bar shows mean ± SEM of the climbing performance of PINK1 KO flies (PINK1B9) upon expression of constitutive active calcineurin (CanA-14F), and treated as indicated for 48 h with DMSO or FK506. Two-way ANOVA followed by Sidak’s multiple comparison test (n = 5; p ≤ 0.001). E Representative confocal microscopy images of neurons in the ventral nerve chord of third instar larvae. Fluorescence corresponding to neutral pH (GFP) and acidic pH (mCherry) are shown. Mitolysosomes are counted as GFP-negative/mCherry-positive (red-only) puncta. The mitophagy mask generated by the mito-QC Counter allows visualization of the quantified mitophagy areas [129]. F Quantification of total number of mitolysosomes per cell using the mito-QC Counter [129] (Fiji). At least 40 cells were analyzed per animal. One-way ANOVA followed by Sidak’s test for multiple comparisons (n = 6; p ≤ 0.01). G Quantitative analysis of respiratory fitness of mitochondria from adult flies of the indicated genotype. Graph shows respiratory control ratio (RCR) calculated as described in Materials and Methods. One-way ANOVA followed by Sidak’s test for multiple comparison test (n = 10–11, p ≤ 0.01). H Quantitative analysis of respiratory fitness of mitochondria from adult flies of the indicated genotype. Graph bar shows ADP-stimulated respiration calculated as described in Materials and Methods. One-way ANOVA followed by Sidak’s test for multiple comparison test (n = 10-11, p ≤ 0.01).

Fig. 11. Schematic representation of the pathway regulating Parkin translocation and mitophagy induced by Calcineurin.

Mitochondrial membrane potential drives PINK1 import into healthy mitochondria through the TOM and TIM complexes. Once on the IMM, PINK1 gets cleaved by MPP and PARL and eventually degraded by the ubiquitin-proteasome system [45, 131]. In this scenario, CaN is not active and Parkin is kept in the cytosol. Mitochondria depolarization induced by CCCP is followed by cytosolic Ca²⁺ rise [56, 132], which activates CaN [133]. Activated CaN promotes mitochondrial recruitment of Drp1 and Drp1-dependent mitochondrial fission by dephosphorylating Drp1 [51]. Activated CaN also promotes dephosphorylation of TFEB to induce its activation and the expression of autophagy and lysosomal genes [57]. In parallel, CaN interacts with Parkin and promotes Parkin translocation to mitochondria in a PINK1-independent fashion. The interaction between CaN and Parkin might be direct or via a binding partner.