Mask loss-of-function rescues mitochondrial impairment and muscle degeneration of Drosophila pink1 and parkin mutants

Abstract

PTEN-induced kinase 1 (Pink1) and ubiquitin E3 ligase Parkin function in a linear pathway to maintain healthy mitochondria via regulating mitochondrial clearance and trafficking. Mutations in the two enzymes cause the familial form of Parkinson's disease (PD) in humans, as well as accumulation of defective mitochondria and cellular degeneration in flies. Here, we show that loss of function of a scaffolding protein Mask, also known as ANKHD1 (Ankyrin repeats and KH domain containing protein 1) in humans, rescues the behavioral, anatomical and cellular defects caused by pink1 or parkin mutations in a cell-autonomous manner. Moreover, similar rescue can also be achieved if Mask knock-down is induced in parkin adult flies when the mitochondrial dystrophy is already manifested. We found that Mask genetically interacts with Parkin to modulate mitochondrial morphology and negatively regulates the recruitment of Parkin to mitochondria. We also provide evidence that loss of Mask activity promotes co-localization of the autophagosome marker with mitochondria in developing larval muscle, and that an intact autophagy pathway is required for the rescue of parkin mutant defects by mask loss of function. Together, our data strongly suggest that Mask/ANKHD1 activity can be inhibited in a tissue- and timely-controlled fashion to restore mitochondrial integrity under PD-linked pathological conditions.

Introduction

Recent studies of two autosomal recessive Parkinson's disease (PD) genes, pink1 and parkin, have accumulated compelling evidence that Parkin promotes degradation of dysfunctional mitochondria by autophagy (mitophagy) in a PINK1-dependent manner, suggesting that mitochondrial defects observed in familial PD may be the result of impaired mitochondrial quality control (1–8). In addition to causing pink1- and parkin-linked PD, mitochondrial dysfunction is also associated with sporadic PD and is sufficient to cause parkinsonism (9,10). Toxins that inhibit mitochondrial complex I (CI) activity or introduce excessive oxidative stress have been shown to cause dopaminergic (DA) neuronal loss in humans as well as primate and rodent PD animal models (11). Mice that lack a mitochondrial protein TFAM in midbrain DA neurons develop progressive Parkinsonism (12). Moreover, patients with sporadic PD show impaired mitochondrial electron transport chain activity in their brains, platelets and skeletal muscles (13–16). However, the primary causes and consequences of mitochondrial dysfunction in sporadic PD are not fully understood, which limits the development of strategies to restore the mitochondrial integrity in the diseased cells. One way to overcome this hurdle is to identify mechanisms that work in concert with the PINK1–Parkin pathway to enhance the clearance of damaged mitochondria and promote mitochondrial homeostasis in the affected cells.

Research using the Drosophila model has provided much insight on the cell biological defects associated with PD. The in vivo function of Pink1 was first uncovered in flies showing that Pink1 acts upstream of Parkin to maintain normal mitochondrial function and quality control (6,17,18). Further genetic studies in flies also revealed important roles of the Pink1/Parkin pathway in regulating mitochondrial clearance, dynamics and axonal transport as well as respiratory chain turnover (19–23). Using the Dorsophila model system, here we show that Drosophila Mask interacts with Parkin to regulate mitochondrial morphology.

We identified Drosophila Mask as one of the potential Parkin-interaction proteins. Mask is a highly conserved large ankyrin-repeat-and-KH-domain-containing protein that was originally identified in an enhancer screen as a component of receptor tyrosine kinase downstream signaling (24). Mask plays essential roles in mitotic cells during development as a regulator of cell proliferation (24), a major component of centrosome and nuclear matrix (25,26) and a co-transcription factor of the Hippo pathway (27,28). Also, its human homolog ANKHD1 expresses at relatively higher levels in acute leukemia cells (29) and multiple myeloma cells (30). However, its function in post-mitotic cells including neurons and muscle cells is largely unknown. Here, we show that mask genetically interacts with pink1 and parkin, and that mask loss-of-function suppresses pink1 and parkin mutant phenotypes in flies. We found that Mask may function as a novel negative regulator of mitophagy during development. Such a suppressing function of Mask can be reduced in PD-related pathological tissues to promote the clearance of damaged mitochondria, and by doing so restore mitochondrial integrity.

Results

Loss-of-function of mask, a novel parkin interactor, suppresses anatomic and behavioral defects of both pink1 and parkin mutant flies

To identify Parkin-interacting proteins in Drosophila, we first generated a UAS-Parkin-TAP transgene and confirmed its functionality in rescuing parkin mutant defects (data not shown). We then performed Tandem Affinity Purification experiments using adult fly heads that express Parkin-TAP, and identified Drosophila Mask as one of the potential Parkin-interacting proteins (Supplementary Material, Fig. S1). To determine the genetic interaction and epistasis between parkin and mask, we tested whether loss of mask function can modulate pink1 and parkin mutant phenotypes that were previously reported in flies. The mask null (mask^10.22/Df) mutants die during the late larval stage with a smaller body size, which may attribute to the function of mask in regulating cell growth (24,27,28). However, both mask heterozygous (mask^10.22/+) mutants and hypomorphic P-element-insertion (EY13048) (mask^P/10.22) mutants, which showed reduced Mask protein levels (about ∼35 and 70% of the wild-type level, respectively) (Fig. 1A and B), are viable to adulthood with no apparent developmental defects. Interestingly, these partial-loss-of-function mask mutations suppressed the thorax indentation phenotype of both pink1 and parkin mutant flies in a dose-dependent manner (Fig. 1C and D). Histological and electron microscopic analysis of the adult indirect flight muscles revealed substantial suppression of muscular and mitochondrial degeneration in pink1 and parkin mutants by the mask hypomorphic mutations (Fig. 1E). Such suppressions at the anatomic and cellular levels were also reflected in both functional and behavioral levels. The reduced level of total ATP in parkin mutant adult flies was significantly rescued in parkin-mask double mutant adult flies (Fig. 1F). Both pink1:mask and parkin:mask double mutants showed significantly improved flight ability compared with pink1 and parkin singular mutants, respectively, again in a mask-dose-dependent manner (Fig. 1G). These data suggest that partial loss of mask function genetically suppresses defects in mitochondrial morphology and function caused by impaired PINK1–Parkin pathway.

Figure 1.

Loss-of-function of mask suppresses anatomic and behavioral defects of both pink1 and parkin mutant flies. (A) Western blot analysis of endogenous Mask protein levels in larval muscles of wild type, mask null (mask^10.22/Df), heterozygous (mask^10.22/+) and hypomorphic (mask^P/10.22) mutants. (B) Quantification of endogenous Mask protein levels normalized to corresponding β-Tubulin levels, shown as percentage of the wild-type level. (C) Collapsed-thorax phenotypes (white arrows) of pink1 and parkin mutants were significantly rescued by mask partial loss-of-function. (D) Percentage of collapsed-thorax phenotype. (E) Top panels: longitudinal sections of the thoraces. Middle and lower panels: TEM analysis of indirect flight muscle of 3-day-old flies. Scale bars were indicated. (F) Quantification of total ATP levels in wild-type, parkin, parkin/mask or mask mutants. The relative ATP levels obtained by ATP assays were normalized to wild type and presented as percentages to the relative ATP level in wild-type adults. (G) Quantification of flight ability. ^#P = 0.07, *P< 0.001, **P < 0.005, ***P < 0.05.

As described above, previous studies on the highly conserved Drosophila orthologs of PINK1 and parkin demonstrated that mutations in the two genes cause accumulations of morphologically enlarged or swollen mitochondria, especially in tissues that demand high output of mitochondrial function such as flight muscles and sperm cells (2,6,7,17,22). Here, we demonstrate that such mitochondrial defects in pink1 or parkin mutants are also manifest in Drosophila larval muscles. Using Mito-GFP to label mitochondria, we found that pink1 or parkin mutant third-instar (L3) larvae showed enlarged or swollen mitochondria in muscles throughout the body wall (Supplementary Material, Fig. S2). Such defects were cell autonomous since muscle expression of transgenic PINK1 and Parkin each rescued the mitochondrial phenotypes in pink1 and parkin mutants, respectively (Supplementary Material, Fig. S2). In agreement with the known epistasis between PINK1 and Parkin, muscle expression of Parkin also rescued pink1 mutant phenotype (Supplementary Material, Fig. S2). Consistently, our genetic analysis showed that mask loss-of-function is able to suppress the mitochondrial defects caused by loss of pink1 or parkin functions in larval muscles (Supplementary Material, Fig. S2). Therefore, the larval muscle is another valid system to study mitochondrial quality control regulated by the pink1–parkin pathway and its modulators, especially those whose loss-of-function cause lethality during late larval and/or pupal stages.

Knocking down Mask suppresses mitochondrial defects in pink1 and parkin mutants cell-autonomously

We next utilized tissue-specific knock-down of mask using the UAS/Gal4 system to understand the cell-autonomy of the mask-mediated suppression of mitochondrial defects in pink1 or parkin mutants. Two TRiP mask RNAi transgenes, both inserted at the same genomic locus, were each analyzed for their abilities to knock down endogenous Mask. Larval muscle expressing P{TriP.JF01147} showed no significant change in Mask protein levels, while muscle expressing P{TriP.HMS01045} showed ∼85% reduction of Mask protein levels (Fig. 2A and B). We found that although larvae expressing P{TriP.HMS01045} in muscle show roughly the same body size and enter pupal stage at about the same time as those expressing either no RNAi or P{TriP.JF01147}, they die at late pupal stage, suggesting essential roles of mask in completing metamorphosis. Because P{TriP.JF01147} is non-functional in reducing Mask protein levels and does not cause mask loss-of-function phenotype, this RNAi line was used as a valid control for the insertion site of mask RNAi P{TriP.HMS01045}.

Figure 2.

Cell autonomous knockdown of Mask in muscles and dapamnergic neurons suppresses pink1/parkin mutant mitochondrial defects. (A) Western blot analysis of endogenous Mask protein levels in the larval muscles expressing control RNAi (TRiP.JF01147) and mask RNAi (TRiP.HMS01045). (B) Quantification of endogenous Mask protein levels normalized to corresponding β-Tubulin levels. (C) Representative single-layer confocal images of larval muscle in wild-type larval muscles expressing Mito-GFP and control RNAi; parkin or pink1 mutant larval muscles expressing Mito-GFP and either the control or the mask RNAi. Each inset shows a high-magnification image of the boxed area. Scale bars, white 10 µm; orange 3 µm. (D) Representative confocal images of a cluster of DA neurons expressing Mito-GFP in wild type, or in parkin mutant adult brains co-expressing the control or the mask RNAi. Scale bar, 5 µm. (E) Wild-type flies expressing GeneSwitch(GS)-MHC-Gal4, Mito-GFP and Control RNAi, and parkin mutants expressing GS-MHC-Gal4, Mito-GFP and either the control RNAi, or the mask RNAi were collected right after eclosion and raised in fly food containing RU486. Five days later, their indirect fight muscles were analyzed by Phalloidin staining and Mito-GFP auto-fluorescence as shown by the representative single-layer confocal images. Scale bar, 10 µm. (F) Quantification of the area of Mito-GFP aggregation relative to muscle area. (G) Quantification of the flight ability of 5-day-old parkin mutant adult flies expressing GS-MHC-Gal4, Mito-GFP and either control or mask RNAi. (H) Lifespan of parkin mutant adult flies expressing GS-MHC-Gal4, Mito-GFP and either control or mask RNAi. (Note for G and H: before eclosion, animals were raised in food without RU486; after eclosion, animals were raised in food with RU486). *P < 0.001.

When we drove muscle expression of the UAS-RNAis (with 24B-Gal4) in the pink1 or parkin mutant background, we found that mask RNAi, but not the control RNAi, suppressed the phenotype of swollen mitochondria in both pink1 and parkin mutant larval muscles (Fig. 2C). Similar results were observed when another muscle-specific driver MHC-Gal4 was used to express the UAS-RNAis (data not shown), suggesting that knocking down Mask in larval muscle suppresses the muscular mitochondrial phenotype of pink1 and parkin mutants. Consistent with these observations, muscle expression of another independent mask RNAi (VDRC: 103411) also significantly suppressed swollen-mitochondria phenotype (Supplementary Material, Fig. S3). We next tested whether reducing Mask in adult tissues suppresses parkin mutant mitochondrial defects. Parkin mutant flies show similar swollen-mitochondria phenotype in DA neurons. This phenotype was substantially suppressed by expression of the mask RNAi, but not the control RNAi, specifically in DA neurons (Fig. 2D). To test whether reducing mask activity in adult muscle is also capable of suppressing parkin mutant defects, we took advantage of the GeneSwitch-MHC-Gal4 (GS-MHC) (31), a drug-inducible muscle-specific driver, which allows the RNAi to be expressed at adulthood and thus bypasses pupal lethality caused by constitutive muscle-specific knockdown of mask. Freshly eclosed parkin mutant flies carrying GS-MHC and control or Mask RNAi were raised on food with RU486, a drug that activates GeneSwitch and triggers muscle expression of the RNAis. Five days later, we found that flies with Mask RNAi showed almost no mitochondrial aggregation in the indirect flight muscles, compared with the flies with control RNAi (Fig. 2E and F). This rescue of muscle mitochondrial morphology was also accompanied by a recovery of flight muscle function indicated by better flight ability of Mask RNAi flies than control RNAi flies (Fig. 2G). Interestingly, the lifespan of parkin mutant flies with mask RNAi expression in adult muscles was also significantly increased compared with that of control RNAi expression (Fig. 2H). Together these data suggest that mask loss-of-function cell-autonomously suppresses the mitochondrial morphological defects and part of the functional deficit induced by impaired Pink1–Parkin pathway.

Knocking down Mask at later adult stage rescues parkin mutant defects in mitochondrial morphology and function

The inducible RNAi system described above also allowed us to further test the ability of mask knock-down to suppress the parkin mutant mitochondrial defects at stages when these defects have already manifested and advanced. To do this, we raised freshly eclosed parkin mutant adult flies (carrying GS-MHC and UAS-mask RNAi transgenes) on regular food for 5 days, which allowed for substantial progression of swollen mitochondria in their indirect flight muscles (Fig. 3A). We then transferred half of the flies to RU486-containing food to induce muscle expression of the mask RNAi, leaving the other half on regular food as control. Another 5 days later, the RU486-fed flies showed a markedly reduced number of swollen mitochondria in their indirect flight muscles compared with time-matched control flies (Fig. 3A and B). This rescue of mitochondrial morphology was also accompanied with better muscle function as shown by significantly increased flight ability in the RU486-treated group of flies (Fig. 3C). These data suggest that reducing Mask activity in later adulthood can dynamically clear damaged mitochondria in parkin mutants, and by doing so, partially restores cellular function.

Figure 3.

Knocking down Mask at later adult stage mitigates parkin mutant defects in mitochondrial morphology and function. (A) Five-day-old parkin mutant flies expressing GS-MHC-Gal4, Mito-GFP and the mask RNAi (raised in food without RU486) showed substantial mitochondrial morphological defects. These flies were divided into two groups: one group was raised in food containing RU486, and the other without. Five days later, the swollen-mitochondria defects in the parkin mutants were significantly suppressed in the RU486 treatment group, but not in the control group. The panel on the right shows a high-magnification image of the boxed area in the left panel. Scale bars, white 10 µm; orange 5 µm. (B) Quantification of the area of swollen Mito-GFP relative to muscle area. (C) Quantification of flight ability. *P < 0.001, **P < 0.05, ^#P > 0.5.

Mask regulates mitochondrial morphology and distribution

To understand how reducing Mask activity mitigates pink1/parkin mutant mitochondrial defects, we first analyzed the mitochondrial morphology of mask loss of function. Lateral views of wild-type larval body wall muscles revealed that the majority of mitochondria form a layer on top of the muscle actin filaments toward the lumen (Fig. 4A–A″). Mask protein is expressed in wild-type larval muscle at a very modest level (Fig. 4A′ and B′). Both the endogenous and muscle-expressed transgenic Mask proteins are localized at the same layer as the mitochondria (Fig. 4A′ and C′). Within this layer, however, Mask proteins are close to but not co-localized with mitochondria (Fig. 4A–A″ and C–C″). Complete loss of Mask proteins resulted in largely fragmented and dispersed mitochondria in multiple orientations: top views of the mitochondrial layer showed much reduced connectivity of mitochondrial network compared with that in wild-type larval muscle (Fig. 4B); lateral views of the muscular/mitochondrial layers revealed that a large number of spherical mitochondria are no longer restricted at the top layer, but mis-localized inside the actin filament layer (Fig. 4B and B″, brackets). Expressing the UAS-Mask transgene in the mask mutant larval muscle rescued mitochondrial connectivity as well as distribution defects (Fig. 4C–C″). In fact, muscle expression of the UAS-Mask transgene in mask mutant and wild-type background displayed a similar effect on mitochondrial morphology—a more densely tangled mitochondrial network than wild type (data not shown)—probably due to the high-level expression of the Mask transgene driven by the Gal4/UAS system (Fig. 4D). Muscle expression of UAS-Mask also rescued the smaller body size and larval lethality of mask null mutants, however the animals failed to survive through late pupal stage (data not shown). Muscle-specific knockdown of mask using the mask RNAi caused very similar mitochondrial phenotypes as the mask null mutants (Fig. 4E). Collectively, these data suggest that Mask inhibits mitochondrial discreteness or promotes mitochondrial connectivity in a cell-autonomous fashion.

Figure 4.

Loss of mask function alters mitochondrial morphology and distribution. (A–C″) Slides view of Z-stack confocal images of the body wall muscle from wild type (A–A″), mask null (B–B″), mask null with muscle expression of UAS-Mask transgene (C–C″) larvae. Larvae were co-stained with Phalloidin (for actin), anti-ATP5α (for mitochondria) and anti-Mask antibodies, and single channel as well as merged channels were presented as indicated. Both top views (central panel) and lateral views (bottom and right panels) were shown. Scale bar, 10 µm. Arrowheads mark the lumen side and asterisks mark the epidermis side of larval body wall muscles. Brackets in the lateral images indicate the muscle actin filament layer. Note massive mis-localized mitochondria in this layer only in mask mutant muscles. (D) Western blot analysis of protein expression levels of endogenous and transgenic UAS-Mask in the larval muscles. (E) Representative confocal images of muscle mitochondria (Mito-GFP) in wild type, Drp1^1/2, 24B-Gal4-driven mask RNAi and Drp1^1/2 plus 24B-Gal4-driven mask RNAi.

The loss-of-function phenotype of mask led us to test whether Mask inhibits mitochondrial fission. The GTPase Dynamin-related protein 1(Drp1) is required for mitochondrial fission (32). Drosophila drp1 mutants alone showed slightly enlarged mitochondria in larval muscle (Fig. 4E). However, loss of drp1 function did not modify the mitochondrial morphological defects of muscle knockdown of Mask (Fig. 4E), suggesting that Mask regulates mitochondrial morphology and distribution through a mechanism that is likely independent of the Drp1-mediated fission process.

Mask antagonizes the function of Parkin in regulating mitochondrial morphology

Studies in Drosophila have shown that overexpression of Parkin in adult flies results in mitochondrial fragmentation in indirect flight muscle (i.e. smaller and rounder mitochondria) (33). Similar results were also observed in mammalian cells expressing exogenous Parkin proteins (34–36). We generated a UAS-mCherry-Parkin transgene that is capable of rescuing parkin mutant phenotype (data not shown). Consistent with the results in adult flight muscle, expressing the mCherry-Parkin transgene in larval muscle led to similar changes in mitochondrial morphology: mitochondria were more disconnected and spherical, compared with the tubular and connected mitochondria in wild-type animals (Fig. 5A). The mitochondrial morphological change caused by Parkin gain-of-function is reminiscent of the mask loss-of-function phenotype (Fig. 4). Given that mask loss-of-function suppresses parkin loss-of-function phenotype, we next tested whether down- and up-regulation of Mask activity modulate the effects of Parkin gain-of-function. Co-expressing the UAS-mask RNAi with UAS-mCherry-Parkin greatly enhanced the mitochondrial morphological phenotype, in that the mitochondria became even smaller, rounder and were almost completely separated from each other. On the other hand, co-expressing the UAS-Mask transgene with UAS-mCherry-Parkin remarkably suppressed the mCherry-Parkin-induced changes in mitochondrial morphology, in that the mitochondria became highly tangled and connected (Fig. 5A). In addition, knocking down mask activity promoted translocation of mCherry-Parkin to mitochondria (Fig. 5A and B). Together, these data suggest that Mask antagonizes the action of Parkin in regulating mitochondrial morphology.

Figure 5.

Mask antagonizes the function of Parkin in regulating mitochondrial morphology. (A) Representative single-layer confocal images of wild-type larvae expressing 24B-Gal4-driven UAS-mCherry-Parkin, Mito-GFP together with the control RNAi, the mask RNAi or UAS-Mask, co-stained with anti-GFP and anti-DsRed antibodies. Each inset shows a high-magnification image of the boxed area. Scale bars, white 10 µm; orange 3 µm. (B) Quantification of average area of co-localization of mCherry-ATG8 and Mito-GFP puncta, shown as percentage of the total muscle area (see method for more details). **P < 0.05.

Mask may function as a selective inhibitor of mitophagy

The fact that mask loss-of-function promotes clearance of damaged mitochondria caused by pink1/parkin mutations, and enhances mitochondrial localization of Parkin led us to hypothesize that one normal function of mask is to inhibit mitophagy. To directly test this hypothesis, we next expressed mCherry-tagged ATG8 (an autophagosome marker) and Mito-GFP in larval muscle and analyzed the co-localization of mCherry-ATG8 and Mito-GFP to assay mitophagy. In Drosophila, basal autophagy is developmentally upregulated in fat body from mid-L3 stage (∼103 h after egg laying) by ecdysone-mediated signaling (37,38). Because such ecdysone signaling could also elevate autophagy in mid-L3 stage larval muscle, which may interfere with our analysis of mitophagy, we chose early-L3 stage larvae (∼93 h after egg laying) for our assay. mCherry-ATG8 co-expressed with control RNAi showed homogenous distribution in larval muscle; however, mCherry-ATG8 co-expressed with mask RNAi showed punctate expression pattern and distribution (Fig. 6A). The puncta formation was accompanied by an increase of full-length mCherry-ATG8 protein level in mask knock-down larval muscle, when compared with the control (Fig. 6A). Co-staining of mCherry and Mito-GFP showed that mask knock-down larvae contain mCherry-ATG8 puncta either surrounding Mito-GFP puncta or co-localizing with Mito-GFP puncta, possibly indicating different stages of mitophagy (Fig. 6A, inset). Quantification showed a striking elevation of mCherry-ATG8/Mito-GFP co-localization in mask knock-down larval muscle, as compared with the control (Fig. 6B). These data suggest that reducing Mask activity in larval muscle may promote mitophagy. A functional autophagy pathway is required for mitophagy as mutations in most core autophagy components block mitophagy to various degrees (39). If loss of mask function rescues pink1/parkin defects via promoting mitophagy, then inhibition of mitophagy would block the beneficial effect of mask loss-of-function. Indeed, when we introduced a heterozygous atg18 mutant into the system, the rescue of parkin mutant thorax indentation by partial loss of mask function was completely abolished (Fig. 6C).

Figure 6.

Mask functions as a selective mitophagy inhibitor. (A) Representative single-layer confocal images of confocal images of wild type larvae expressing 24B-Gal4-driven UAS-mCherry-ATG8, Mito-GFP together with the control RNAi or mask RNAi, co-stained with anti-GFP and anti-DsRed antibodies. The inset shows a high-magnification image of the boxed area. Scale bars, white 5 µm; orange 2 µm. (B) Quantification of average number of puncta that show co-localization of mCherry-ATG8 and Mito-GFP, normalized to muscle area (see Materials and Methods for more details). (C) Percentage of flies with collapsed thorax in parkin, parkin plus atg18^KG03090/+, parkin plus mask^10.220/+ and parkin plus mask^10.220/+plus atg18^KG03090/+. (D) Western blot analysis of mCherry-ATG8 in larval muscles expressing 24B-Gal4-driven UAS-mCherry-ATG8 together with the control RNAi, the mask RNAi or UAS-Mask, at 93 h after egg laying. The arrow points to free mCherry. (E) Quantification of the levels of full-length mCherry-ATG8 and free mCherry as readout of autophagic flux. Intensities of protein levels were normalized to β-Tubulin, and were shown relative to control. *P < 0.001. n.s., not significant.

Mitophagy represents a specific form of autophagy. We next tested whether Mask-mediated inhibition is specific to mitophagy or general to all forms of autophagy. The free mCherry degraded from mCherry-ATG8 can be followed by western blot to assay autophagic flux (40). We found that there is no significant change in the autophagic flux among control RNAi, mask RNAi and UAS-Mask expressing larval muscle by comparing either absolute levels of free mCherry or relative levels of free mCherry to full-length mCherry-ATG8 among the three samples (Fig. 6D and E). These data suggest that the basal autophagy levels are not affected by down- and up-regulation of Mask activity, and that Mask may not be a general autophagy inhibitor, but instead a selective mitophagy inhibitor.

Discussion

Recent studies suggest that PINK1 activates Parkin E3 ubiquitin ligase activity by phosphorylating both Parkin and ubiquitin (41–44), and that PINK1 recruits Parkin to the damaged mitochondrial membrane, where Parkin ubiquitinates a pool of outer mitochondrial membrane proteins and promotes mitophagy (9,45). These data suggest that mitochondrial dysfunction observed in PD may be the result of compromised mitochondrial quality control mechanisms. Therefore, understanding the pathways of mitochondrial quality control holds the key to unravelling the pathogenesis of PD and other disorders associated with mitochondrial dysfunction.

Flies carrying pink1 or parkin mutations show severe mitochondrial morphological and functional defects in multiple tissues as well as age-dependent DA dysfunction (46,47), making it a great genetic model to study mechanisms of mitochondrial homeostasis. Using this model system, previous studies in Drosophila have identified a number of pathways that can be manipulated to rescue the parkin and/or pink1 mutant phenotype. First, increasing mitochondrial fission or decreasing fusion rescues the phenotypes of muscle degeneration and mitochondrial abnormalities in pink1 or parkin mutants (22,23,48). However, manipulation of mitochondrial dynamics causes the opposite effect on loss of parkin or pink1 function in mammalian cells (49–51), indicating that Pink1 and Parkin may regulate mitochondrial dynamics in a context-dependent manner. Second, promoting mitochondrial electron transport chain CI activity by overexpressing a yeast NADH dehydrogenase (52), the CI subunit NDUFA10 (53), the GDNF receptor Ret (54), Sicily (55), dNK (56) or Trap1 (57) rescue pink1 mutant mitochondrial defects without affecting parkin mutant phenotypes, suggesting a distinct role of Pink1 in regulating CI activity in addition to its role in Parkin-mediated mitophagy. Here, we showed that a highly conserved scaffolding protein Mask, whose normal function is to regulate mitochondrial morphology and selectively inhibit mitophagy, can be targeted in a tissue- and temporal-specific manner to suppress both pink1 and parkin mutant defects in Drosophila. We also showed that such a rescue requires the presence of a functional autophagy pathway. Although our tissue- and temporal-specific knock-down of Mask was performed with mainly one mask RNAi line, our mask loss-of-function analysis with mask genetic mutants and another independent RNAi line supported the same notion that Mask dynamically regulates mitochondrial morphology. Together, our data suggest that enhancing mitochondrial quality control may serve as a common approach to mitigate mitochondrial dysfunction caused by PD-linked genetic mutations. Consistent with this notion, recent studies showed that inhibition of deubiquitinases USP30 and USP15 enhances mitochondrial clearance and quality control, and rescues mitochondrial impairment caused by pink1 or parkin mutations (47,58).

Our data indicate that loss of mask function enhances the formation of autophagosome surrounding mitochondria. However, the increase of mCherry-ATG8 did not result in significant increase of free mCherry, suggesting the flux of autophagic degradation is not affected. Further studies are required to elucidate the molecular details by which Mask regulates mitochondrial morphology and function. Recent studies on the connection between Mask and the Hippo pathway demonstrated that Mask physically interacts with the Hippo effector Yorkie, and functions as an essential cofactor of Yorkie in promoting downstream target-gene expression (27,28). Interestingly, the Yorkie pathway was also shown to regulate mitochondrial structure and function during fly development (59). Together, these findings brought up an intriguing possibility that Mask and Yorkie together regulate mitochondrial size during development and disease.

Our data show that reducing Mask activity at the relatively progressed stage of parkin-dependent muscle degeneration mitigates the mitochondrial defects and impaired muscle function, indicating that the human Mask homolog ANKHD1 may serve as a potential therapeutic target for treating PD caused by pink1/parkin mutations. It will be interesting to validate whether our findings are transferrable to mammalian models.

Materials and Methods

Tandem-affinity-purification procedure

To purify Parkin-associated protein complex, we collected ∼30 000 adult fly heads expressing Parkin-CTAP in neurons (BG380-Gal4; UAS-Parkin-CTAP). TAP purification was performed as previously described (60,61).

Drosophila strains and genetics

Flies were maintained at 25°C on standard food except for the RU486 administration. The following strains were used in this study: parkin^Δ21(7), parkin²⁵ and UAS-Parkin (2), pink1^B9 and UAS-PINK1(6), mask^10.22(24), Drp1¹ and Drp1² (62), UAS-mcherry-ATG8 (From Dr Neufeld), BG380-Gal4 (neuron specific) (63), MHC-GeneSwitch-Gal4 (MHC-GS-Gal4) (31), mask^Df317, mask^EY13048 (P-element insertion), UAS-control-RNAi (P{TRiP.JF01147}), UAS-mask-RNAi (P{TRiP.HMS01045}), 24B-GAL4 (muscle-specific), TH-GAL4 (Dopaminergic-neuron-specific), UAS-Mito-GFP and atg18^KG03090 from the Bloomington stock center and UAS-mask-RNAi (v103411) from Vienna Drosophila RNAi Center.

The RU486-GeneSwitch system was administrated according to Osterwalder et al. (31). Adult flies at a specified age were transferred to fly food containing 200 µg/ml RU486 to induce gene expression at specified adult stage.

Transgenic constructs

A full-length parkin cDNA (DGRC clone LD33094) was obtained and the coding sequence was cloned to a pUAST-CTAP vector (derivates from a TAP construct provided by EUROSCARF, Germany) (61,64) or pUAST-mCherry vector (a gift from Richard Daniels, unpublished) to generate UAS-parkin-CTAP or UAS-mcherry-Pakrin constructs, respectively. To generate a full-length mask cDNA, a cDNA clone (LD31436) which covers base pair 2695–9316 of mask ORF was obtained from DGRC. To obtain the 5′ end (1–2695) and 3′ end (9316–12006) cDNA fragments of mask, reverse transcription and subsequent PCR reactions were performed using mRNA purified from adult fly heads and mask gene-specific primers. These three cDNA fragments were ligated together sequentially using two unique restriction sites HindIII and BamHI to obtain a full-length mask cDNA. This cDNA was then cloned into the pUAST vector to generate a pUAST-UAS-Mask construct. All transgenic fly lines were generated by BestGene Inc. (Chino Hills, CA, USA).

Western blots

Western blots were performed according to standard procedures. The following primary antibodies were used: rabbit anti-Mask (1 : 2000) (24), Rat anti-MTS (65), mouse anti-mCherry antibody (1 : 1000, 1C51, NBP1–96752 Novus Biologicals) and mouse anti-β-Tubulin (1 : 1000, E7) monoclonal antibody from Developmental Studies Hybridoma Bank. All secondary antibodies were used at 1 : 10 000. Data were collected using Luminescent Image Analyzer LAS-3000 (FUJIFILM) and quantified using Multi Gage (FUJIFILM).

Immunocytochemistry

Larval muscle were dissected in ice-cold PBS and fixed in 4% PFA for 30 min. The fixed tissues were stained following standard procedures. Rabbit anti-MASK antibody (24) was pre-absorbed with mask mutant tissues, and then used at 1 : 1000. The other primary antibodies used were: mouse anti-GFP and rabbit anti-GFP antibody (A11122, Invitrogen, Carlsbad, CA, USA) at 1 : 1000, anti-ATP5α (ab14748, Abcam) at 1 : 1000, rabbit anti-mCherry at 1 : 1000 (632496, Clontech). For analysis of mitochondrial morphology in DA neurons, brains were dissected from adult heads in ice-cold PBS, fixed in 4% PFA for 30 min and washed in 0.1% PTX. Subsequently, brains were stained with mouse anti-GFP (1 : 1000) and rabbit anti-TH (1 : 1000, AB152, Millipore). The following secondary antibodies (from Jackson ImmunoResearch) were used: Cy3 conjugated goat anti-rabbit IgG at 1 : 1000, Dylight 488 conjugated anti-mouse IgG at 1 : 1000.

For direct visualization of mito-GFP in adult indirect flight muscle, notums of adult flies were dissected, fixed in 4% PFA, some were stained with Phalloidin and indirect muscle fibers were then isolated and mounted in Vectashield (Vector Lab).

Confocal imaging and analysis

Single-layer or z-stack confocal images were captured on a Nikon (Tokyo, Japan) C1 confocal microscope. Images shown in the same figure were acquired using the same gain from samples that had been simultaneously fixed and stained. For larval muscle mitochondrial analysis all images were acquired at muscle 6 in segments A2–A3. For adult DA neurons, images were acquired at cluster PPL1. Quantification of swollen-mitochondria phenotype at adult indirect flight muscles was done using imaging software NIS-Elements (Nikon). Random areas in the adult indirect muscles were selected, areas that show swollen Mito-GFP signals were measured and summed up. For each data point, the total area of swollen Mito-GFP was divided by the corresponding total muscle area to measure the severity of the mitochondrial morphological defect. Co-localization of mCherry-Parkin and Mito-GFP, as well as mitophagy at larval muscle mitochondria were quantified using imaging software NIS-Elements (Nikon). For analysis of mitophagy, larvae were dissected at stage ∼93 h after egg laying. Areas of mCherry-ATG8-positive punctae in the single-layer-scanned images were selected and average mean intensities of mCherry-ATG8 and Mito-GFP for each punctate area were simultaneously acquired. An arbitrary threshold line for Mito-GFP mean intensity was determined for all the whole data set. The number of mCherry-ATG8-positive punctae that also have Mito-GFP signal over this threshold were counted and recorded for each genotype.

Electron microscopy

Transmission electron microscopy analysis of indirect flight muscle was performed following standard protocol (23). Thoraxes of 3-day-old adult flies were fixed in paraformaldehyde/glutaraldehyde, postfixed in osmium tetroxide, dehydrated and embedded in Epon. Tissue sections 1.5 µm thick were stained with Toluidine Blue. Sections 80 nm thick were stained with uranyl acetate and lead citrate and examined using a JEM-1400 transmission electron microscope (LSU Socolofsky Microscopy Center).

Thoracic indentation phenotype analysis

To quantify the frequency of thoracic indentations, individual flies were examined 3 days after eclosion to determine whether there were indentations in the cuticle of the thorax. Images of thoracic indentation were taken from adult flies using Nikon AZ-100 microscope.

Analysis of longevity and flight ability

For lifespan analysis, 1-day-old flies were collected and divided into 20 female or male flies per vial and raised on standard or RU486 fly food at 25°C. All flies were flipped into fresh vials every 2 days. The number of surviving flies was recorded during the time of flipping. The same experiments were performed six times for each genotype. For flight analysis, 10 flies (1-day old, five males and five females) were placed in each vial, flipped into fresh food daily and raised at 25°C for 3 days. Flight assays were performed as described by Poole et al. (48). Flies were gently tapped through a funnel placed 30 cm above a 14 cm-diameter Petri dish containing mineral oil. The number of flies that landed in the mineral oil were recorded.

ATP assays

The ATP assays were performed as previous described (56,66). For each genotype, three male and three female flies (1–2 days old) were homogenized for the luminescence-based ATP assay (CellTiter-Glo Luminescent Cell Viability Assay, Promega, Fitchburg, WI, USA). The relative ATP levels were calculated by dividing the luminescence by the total protein concentration (determined by the Bradford method), normalized to wild type and presented as percentages to the relative ATP level in wild-type adults.

Statistical analysis

Statistical analysis was performed and graphs were generated in Origin (Origin Lab, Northampton, MA, USA). Each sample was compared with other samples in the group (more than two) using ANOVA, or with the other sample in a group of two using t-test. All histograms are shown as mean ± SEM. The n numbers of each statistical analysis are the following: for Figures 1B, 1F, 2B, 4D and 6E, independent WB experiments were repeated four times for statistical analysis; for Figure 1D, n = 7, 8, 6, 10, 9, 6, 7, 9, respectively; for Figure 1G, n = 5, 5, 8, 9, 5, 6, 8, respectively; for Figure 2F, n = 6, 6, 7, respectively; for Figure 2G, n = 6, 7, respectively; For Figure 3B, n = 5 for each sample; For Figure 3C, n = 12, 16, respectively; For Figure 5B, n = 3 for each sample; for Figure 6B, n = 12, 8, respectively; for Figure 6C, n = 191, 74, 300, 289, respectively.

Supplementary Material

Supplementary Material is available at HMG online.

Funding

This work is supported by a NIH/NINDS grant (NS070962) and a NIH/NIGMS COBRE pilot grant (GM103340) to C.W.