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. 2014 Nov 20;10(11):e1004815.
doi: 10.1371/journal.pgen.1004815. eCollection 2014 Nov.

The Complex I Subunit NDUFA10 Selectively Rescues Drosophila pink1 Mutants Through a Mechanism Independent of Mitophagy

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Free PMC article

The Complex I Subunit NDUFA10 Selectively Rescues Drosophila pink1 Mutants Through a Mechanism Independent of Mitophagy

Joe H Pogson et al. PLoS Genet. .
Free PMC article

Abstract

Mutations in PINK1, a mitochondrially targeted serine/threonine kinase, cause autosomal recessive Parkinson's disease (PD). Substantial evidence indicates that PINK1 acts with another PD gene, parkin, to regulate mitochondrial morphology and mitophagy. However, loss of PINK1 also causes complex I (CI) deficiency, and has recently been suggested to regulate CI through phosphorylation of NDUFA10/ND42 subunit. To further explore the mechanisms by which PINK1 and Parkin influence mitochondrial integrity, we conducted a screen in Drosophila cells for genes that either phenocopy or suppress mitochondrial hyperfusion caused by pink1 RNAi. Among the genes recovered from this screen was ND42. In Drosophila pink1 mutants, transgenic overexpression of ND42 or its co-chaperone sicily was sufficient to restore CI activity and partially rescue several phenotypes including flight and climbing deficits and mitochondrial disruption in flight muscles. Here, the restoration of CI activity and partial rescue of locomotion does not appear to have a specific requirement for phosphorylation of ND42 at Ser-250. In contrast to pink1 mutants, overexpression of ND42 or sicily failed to rescue any Drosophila parkin mutant phenotypes. We also find that knockdown of the human homologue, NDUFA10, only minimally affecting CCCP-induced mitophagy, and overexpression of NDUFA10 fails to restore Parkin mitochondrial-translocation upon PINK1 loss. These results indicate that the in vivo rescue is due to restoring CI activity rather than promoting mitophagy. Our findings support the emerging view that PINK1 plays a role in regulating CI activity separate from its role with Parkin in mitophagy.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. A cell based RNAi screen to identify phenocopiers and suppressors of pink1 RNAi-induced mitochondrial hyperfusion.
(A) Schematic of the RNAi screen protocol (see Methods for details). (B) Representative images of Drosophila S2R+ cells for mitochondrial morphology following dsRNA treatment of the indicated genes, stained with MitoTracker Red and imaged live (top row). Fluorescence images are converted to binary (B&W) and inverted to clarify the mitochondrial morphology (bottom row). Numbers represent the designated ‘morphology score’: 1, fragmented; 2, wild type; 3, fused/tubular; 4, hyperfused/clumped. (C) Comparison of morphology score of screen library amplicons in WT and pink1 RNAi backgrounds. Solid-line box depicts those amplicons that phenocopy pink1 RNAi (box limits: mean ± s.d. pink1 control). Dashed-line box depicts amplicons which suppress pink1 RNAi-induced fusion back to WT morphology (box limits: mean ± s.d. DsRed control). Controls for fragmentation (Marf) and fusion (Drp1) are shown. Scale bar  = 10 µm.
Figure 2
Figure 2. ND42 RNAi but not other complex I subunits phenocopies pink1 RNAi.
(A) ND42 RNAi in Drosophila S2R+ cells stained with MitoTracker Red causes tubulation of the mitochondrial network, similar to pink1 RNAi. ND42 RNAi does not further perturb morphology in conjunction with pink1 RNAi. (B) Quantification of mitochondrial morphology as in A, scored in triplicate experiments. (C) RNAi of selected subunits of complex I or rotenone treatment do not phenocopy pink1 RNAi. (D) Quantification of morphology scored in triplicate experiments as in C. Histograms indicate mean ± s.d. of triplicate experiments. Inverted, binary images are shown below each fluorescence image to aid clarity of mitochondrial morphology. n>30 cells per experiment. Scale bar  = 10 µm.
Figure 3
Figure 3. ND42 or sicily overexpression can rescue pink1 mutant phenotypes in flies.
Overexpression of two ND42 transgenes, ND42 and ND42-HA, in a wild type background has no effect on climbing (A) or flight behavior (B). In pink1B9 mutants, climbing (C) and flight ability (D), normalized to control, is significantly rescued by overexpression ND42 or sicily. Histograms indicate mean ± s.e.m. (E) Transmission electron microscopy of flight muscle shows partial rescue of mitochondrial disruption. Scale bar  = 1 µm. Overexpression was driven by the ubiquitous driver da-GAL4. Control genotype is da-GAL4/+. Number of animals tested, n>50. * P<0.05, *** P<0.001, **** P<0.0001, One-way ANOVA with Bonferroni correction. Comparisons are with control (A, B) or pink1B9 mutants (C, D).
Figure 4
Figure 4. ND42 or sicily overexpression does not rescue parkin mutant phenotypes in flies.
In park25 mutants, climbing (A) and flight ability (B), normalized to control, is not rescued by ND42 or sicily overexpression. Histograms indicate mean ± s.e.m. (C) Transmission electron microscopy of flight muscle shows widespread disruption of mitochondrial integrity. Scale bar  = 1 µm. Overexpression was driven by the ubiquitous driver da-GAL4. Control genotype is da-GAL4/+. Number of animals tested, n>50. * P<0.05, *** P<0.001, **** P<0.0001, One-way ANOVA with Bonferroni correction. Comparisons are with pink1B9 mutants.
Figure 5
Figure 5. NDUFA10 knockdown slightly reduces CCCP-induced Parkin translocation and mitophagy.
(A) In HeLa cells stably transfected to express YFP-Parkin, before CCCP toxification (0 h) YFP-Parkin (green) has a diffuse cytoplasmic distribution in control (ctrl) siRNA treated cells. Following 4 h CCCP, YFP-Parkin co-localizes with mitochondria labeled with ATP5A immunostaining (red). PINK1 siRNA treatment almost completely abolishes YFP-Parkin translocation. (B) Quantification of YFP-Parkin translocation as in A. (C) Stably transfected HeLa cells expressing YFP-Parkin, before CCCP treatment (0 h, ctrl) have a normal (“High”) mitochondrial content. Following 24 h treatment with CCCP, a high proportion of control cells (ctrl) show complete degradation (“none”) or perinuclear aggregated (“low”) mitochondria, visualized by ATP5A immunostaining (red). PINK1 siRNA treatment almost completely abolishes mitophagy. (D) Quantification of mitochondrial content as in C. Histograms indicate mean ± s.d. of triplicate experiments. n>30 cells per experiment. Scale bar  = 20 µm. * P<0.05, ** P<0.01, *** P<0.001, Student's t-test compared with respective conditions in control siRNA.
Figure 6
Figure 6. NDUFA10/ND42 overexpression does not restore CCCP-induced Parkin translocation in the absence of pink1.
(A) Quantification of the percentage of cells showing mitochondrially localized Parkin following 4 hours CCCP treatment of HeLa cells stably expressing YFP-Parkin transfected with (B) control siRNA, (C) NDUFA10 siRNA, or (D) PINK1 siRNA treatment and transfection with NDUFA10 or fly ND42 expression constructs. Scale bar  = 20 µm. * P<0.05, *** P<0.001, One-way ANOVA with Bonferroni correction compared to respective control siRNA treatment.
Figure 7
Figure 7. ND42 and sicily overexpression can rescue complex I and ATP deficiencies in pink1 but not parkin mutant flies.
ND42 or sicily was overexpressed in (A, B) pink1B9 mutants or (C, D) park25 mutants. Charts show (A, C) the ratio of complex I to citrate synthase (CS) activity, and (B, D) relative ATP levels, normalized to control. Histograms indicate mean ± s.e.m. Overexpression was driven by the ubiquitous driver da-GAL4. Control genotype is da-GAL4/+. ** P<0.01, **** P<0.0001, One-way ANOVA with Bonferroni correction. Comparisons are with pink1B9 or park25 mutants, as appropriate.
Figure 8
Figure 8. Analysis of ND42 Ser-250 phospho-variant rescue of pink1 mutant climbing defect and complex I deficiency.
Transgenes from different sources, labeled ‘HB’ and ‘BDS’ , expressing wild type ND42 (WT), non-phosphorylatable (SA) or phospho-mimetic (SD) variants of Ser-250 were tested for rescue of climbing (A), flight (B) and complex I (C) deficiencies in pink1B9 mutants. For comparison, transgenic expression of the yeast complex I equivalent, NDI1, was also tested. Overexpression was driven by the ubiquitous driver da-GAL4. Control genotype is da-GAL4/+. * P<0.05, ** P<0.01, **** P<0.0001, One-way ANOVA with Bonferroni correction. Comparisons are with pink1B9 mutants unless otherwise shown.
Figure 9
Figure 9. Overexpression of parkin can rescue behavioral phenotypes in pink1 mutants but not complex I deficiency.
Analysis of (A) climbing and (B) flight ability in pink1B9 mutants overexpressing parkin. Charts show (C) the ratio of complex I to citrate synthase (CS) activity, and (D) relative ATP levels, normalized to control. Histograms indicate mean ± s.e.m. Overexpression was driven by the ubiquitous driver da-GAL4. Control genotype is da-GAL4/+. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001, One-way ANOVA with Bonferroni correction. Comparisons are with pink1B9 mutants.

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References

    1. Kitada T, Asakawa S, Hattori N, Matsumine H, Yamamura Y, et al. (1998) Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature 392: 605–608. - PubMed
    1. Valente EM, Abou-Sleiman PM, Caputo V, Muqit MM, Harvey K, et al. (2004) Hereditary early-onset Parkinson's disease caused by mutations in PINK1. Science 304: 1158–1160. - PubMed
    1. Clark IE, Dodson MW, Jiang C, Cao JH, Huh JR, et al. (2006) Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin. Nature 441: 1162–1166. - PubMed
    1. Flinn L, Mortiboys H, Volkmann K, Koster RW, Ingham PW, et al. (2009) Complex I deficiency and dopaminergic neuronal cell loss in parkin-deficient zebrafish (Danio rerio). Brain 132: 1613–1623. - PubMed
    1. Gautier CA, Kitada T, Shen J (2008) Loss of PINK1 causes mitochondrial functional defects and increased sensitivity to oxidative stress. Proc Natl Acad Sci U S A 105: 11364–11369. - PMC - PubMed

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