extfieldsofstudy: Medicine Biology

text:
Atg1-mediated autophagy suppresses tissue degeneration in pink1/parkin mutants by promoting mitochondrial fission in Drosophila

Mitochondrial dysfunction is considered a hallmark of multiple neurodegenerative diseases, including Parkinson’s disease (PD). The PD familial genes pink1 and parkin function in a conserved pathway that regulates mitochondrial function, including dynamics (fusion and fission). Mammalian cell culture studies suggested that the pink1/parkin pathway promotes mitophagy (mitochondrial autophagy). Mitophagy through mitochondrial fission and autolysosomal recycling was considered a quality control system at the organelle level. Whether defects in this quality control machinery lead to pathogenesis in vivo in PD remains elusive. Here, we found that elevating autophagy by atg1 overexpression can significantly rescue mitochondrial defects and apoptotic cell death in pink1 and parkin mutants in Drosophila. Surprisingly, the rescue effect relied both on the autophagy–lysosome machinery and on drp1, a mitochondrial fission molecule. We further showed that Atg1 promotes mitochondrial fission by posttranscriptional increase in the Drp1 protein level. In contrast, increasing fission (by drp1 overexpression) or inhibiting fusion (by knocking down mitofusin [mfn]) rescues pink1 mutants when lysosomal or proteasomal machinery is impaired. Taken together, our results identified Atg1 as a dual-function node that controls mitochondrial quality by promoting mitochondria fission and autophagy, which makes it a potential therapeutic target for treatment of mitochondrial dysfunction–related diseases, including PD.

INTRODUCTION
Accumulating evidence indicates that mitochondrial dysfunction might have a causative role in Parkinson's disease (PD) pathogenesis (Schapira, 2008). For instance, PD patients have reduced complex I activity, and mitochondrial toxins, such as MPTP and rotenone, can induce acute parkinsonism (Schapira, 1993). Moreover, mutations in the PD-related genes PINK1 and PARKIN cause mitochondrial dysfunction. We and others have previously reported that Drosophila pink1 and parkin genes function in the same genetic pathway, with pink1 acting upstream of parkin, to regulate mitochondrial morphology and tissue maintenance of indirect flight muscles (hereafter referred to as "muscles") and dopaminergic (DA) neurons (Clark et al., 2006;Park et al., 2006;Yang et al., 2006Yang et al., , 2008Deng et al., 2008;Poole et al., 2008). Studies employing mammalian cell culture and other animal models revealed that the regulation of mitochondrial integrity and function by the pink1/parkin pathway is well conserved (Palacino et al., 2004;Exner et al., 2007;Gautier et al., 2008;Flinn et al., 2009;Lutz et al., 2009;Yu et al., 2011).
Mitochondria are dynamic organelles that continually undergo fusion and fission. Dynamin-related protein 1 (Drp1) is a cytosolic GTPase that can assemble around the mitochondrial outer membrane and trigger mitochondrial fission. The GTPases Mitofusion (Mfn1 and Mfn2) and the optical atrophy 1 (Opa1) mediate fusion of the outer and inner membranes, respectively (Chen and Chan, 2009;Westermann, 2010). Mitochondrial dynamics has been linked to multiple mitochondrial functions, including respiratory capacity, apoptosis, nutritional status, and mitochondrial quality control (Tatsuta and Langer, 2008;Youle and van der Bliek, 2012;Ashrafi and Schwarz, 2013). It was implied that fusion of mitochondria favors their functional repair, while fission leads to elimination of the irreversible damaged organelles (Tatsuta and Langer, 2008;Youle and van der Bliek, 2012). In line with this model, Twig et al. (2008) showed in cell culture that mitochondrial fission generated two heterogeneous mitochondria in terms of mitochondrial membrane potential. Hyperpolarized mitochondria can readily fuse with other mitochondria and reenter the network, while severely depolarized ones lose fusion ability and are subsequently degraded by autophagy.
Autophagy mediates the lysosome-dependent turnover of macromolecules and organelles. Macroautophagy is the most extensively studied form of autophagy, involving formation of double membrane vesicles, known as autophagosomes. The (macro) autophagy process consists of the following three major steps: autophagosome formation, maturation through fusion with multivesicle bodies (MVBs)/lysosomes, and degradation of the content within the lysosomes (He and Klionsky, 2009;Chen and Klionsky, 2011). Autophagosome formation is controlled by a series of autophagy-related proteins (ATGs). In all eukaryotes, autophagy is induced via the autophagy-related gene 1 (Atg1) complex (Mizushima, 2010). Expansion of the autophagosome membrane requires two distinct sets of ubiquitin-like protein conjugation systems, Atg8 and Atg5-Atg12. Both systems share one E1-like protein, Atg7 (He and Klionsky, 2009;Mizushima, 2010). A second step is autophagosome maturation. Fusion of autophagosomes and endosomes with lysosomes have converging steps and share common components. For instance, Rab7, a small GTPase, is essential for maturation of autophagosomes/endosomes (Jager et al., 2004;Hyttinen et al., 2013). Components of the Class C vacuolar protein sorting (Vps-C) complex, such as Carnation (Car) and Vps16, are required for both endosomal trafficking and autophagosome maturation in Drosophila (Sevrioukov et al., 1999;Sriram et al., 2003). The final step is degradation of contents in the autolysosome by lysosomes. The acidic environment inside the lysosome is crucial for the degradation process. The low pH (∼4-5) is maintained by ATP-dependent proton pumps, such as vacuole-ATPases (V-ATPases). Inhibition of V-ATPase function causes the failure of acidification of several intracellular compartments, including endosomes and lysosomes. V-ATPases are highly conserved large multisubunit complexes composed of a peripheral domain (V1) responsible for ATP hydrolysis and an integral domain (V0) that carries out the proton transport (Nelson, 2003). VhaAC39 encodes one of the two V-ATPase V0 d subunits in Drosophila. Loss of the VhaAC39 gene causes loss of acidic compartments and deregulation of endocytosis (Yan et al., 2009).
Although generally considered a nonselective process, autophagy can selectively degrade organelles. Recent evidence overwhelmingly indicates that, in mammalian cell lines, Pink1 and Parkin participate in selective degradation of damaged mitochondria by autophagy (Matsuda et al., 2010;Narendra et al., 2010). In brief, Parkin, an E3 ubiquitin ligase, is recruited by Pink1, a mitochondrial targeted serine-threonine kinase, which was stabilized on chemically uncoupled mitochondria. Ubiquitination of substrates on the outer membranes of mitochondria, including Mfn1, Mfn2 and Miro, facilitates the segregation of severely damaged mitochondrial and halts mitochondrial motility (Chen and Dorn, 2013;Wang et al., 2011). Finally, these depolarized mitochondria are removed by proteasome system (UPS) and autophagy machinery, which are recruited by parkin (Chan et al., 2011). The canonical ATG pathway is involved in the removal process, as LC3, P62, and ATG5 are required . Clearly, the pink1/parkin mediated mitophagy process has been established in cell lines, and it is proposed to play an important role in mitochondrial quality control at the organelle level. However, the physiological significance of mitophagy in terms of regulating pink1/ parkin associated pathogenesis is still elusive (Whitworth and Pallanck, 2017).
Here, we found mitochondrial autophagy to be beneficial; however, it is dispensable for cell survival and tissue maintenance in pink1/parkin mutants in Drosophila when mitochondrial fission is simultaneously enhanced.

RESULTS
Overexpression of Atg1 can rescue pink1/parkin null mitochondrial defects and muscle degeneration Pink1 705 mutants, a null allele of pink1 (Clark et al., 2006;Deng et al., 2008) (pink1 5 in short and hereafter), show severe mitochondrial defects in muscles. Wild-type mitochondria are of regular shape and align between the muscle fibers, as indicated by mitochondrial targeted mito::GFP ( Figure 1A). Pink1 5 muscles are filled with aberrant clumps of intense mito::GFP signals ( Figure 1A). Under transmission electron microcopy (TEM), the mitochondrial cristae in wildtype muscles are densely packed, while pink1 5 mitochondria are swollen with broken cristae ( Figure 1B). Mitochondrial defects in pink1 5 were also observed by toluidine blue staining: mitochondria in wild-type muscles were densely stained and dark, while mitochondria of pink1 5 mutants were swollen and faint ( Figure 1C). Owing to severe mitochondrial dysfunction, the muscles in pink1 5 mutants degenerate, as indicated by age-dependent accumulation of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive nuclei and irregular indentation of the external thorax ( Figure 1, D and E).
Autophagy is the major route for mitochondrial clearance. In Drosophila, overexpression of Atg1 alone is sufficient to increase autophagy (Scott et al., 2007;Chang and Neufeld, 2009). We sought to test whether enhancing autophagy by overexpression of Atg1 can rescue pink1/parkin muscle degeneration.
Overexpressing Atg1 by panmuscle drivers, Mef2-Gal4 or Mhc-Gal4 using the binary UAS-GAL4 system (Brand and Perrimon, 1993), was lethal (unpublished data). We therefore induced Atg1 overexpression (Atg1 OE ) using IFM-Gal4, which is an indirect flight muscle-specific driver (Yun et al., 2014). Atg1 OE muscles had significantly smaller mitochondria than the wild type, and no obvious muscle degeneration was observed in young adults (2-4 d old; Figure 1, A and F). Interestingly, mitochondrial defects in pink1 5 mutants, such as mito::GFP clumps and swollen and broken cristae, were rescued by Atg1 OE (Figure 1, A, B, and F). This rescuing effect was confirmed by antibody staining against mitochondrial complex V subunit (Supplemental Figure S1, A-D). Muscle degeneration in pink1 5 mutants, as indicated by TUNEL-positive nuclei and thoracic indentation, was suppressed by Atg1 OE (Figure 1, D, E, and G). Furthermore, Atg1 OE also rescued mitochondrial defects and muscle degeneration in parkin RNAi flies (Figure 1. H, I, L, and M). Overexpressing a kinase-inactive form of Atg1 (UAS::Atg1 K38Q ) failed to rescue pink1 5 defects (Figure 1, J-M), suggesting that the rescuing effect of Atg1 depends on its kinase activity.

Functional autophagy machinery is required for phenotypic rescue mediated by Atg1 overexpression
The mitochondrial morphology in pink1 5 muscles correlated closely with cell viability: in viable cells (TUNEL-negative), mitochondrial were elongated and formed big clumps, while in degenerating muscles (TUNEL-positive), the mitochondria were substantially fragmented (Supplemental Figure S2, A-C). We sought to examine autophagy activity in wild-type and pink1 5 muscles. LysoTracker stains acidic cellular compartments, including lysosomes. No obvious Ly-soTracker-positive vesicle colocalized with mito::GFP in wild-type muscles (Figure 2A), while pink1 5 muscles showed significant increases of LysoTracker staining in both number and size ( Figure 2, B and C). Interestingly, in those degenerating pink1 5 muscles, around 30% of the LysoTracker-positive vesicles colocalized with mito::GFP ( Figure 2, B and D). In both cases, a subset of mito::GFP was adjacent to LysoTracker-positive vesicles (Figure 2, B and C). Colocalization of mitoGFP with LysoTracker implied either blockage or enhanced mitochondrial autophagy (Klionsky et al., 2008); however, LysoTracker was found inside the mitochondria, often with faint mito::GFP (arrows in Figure 2B), indicating that mitochondria are degrading inside lysosomes.
To further distinguish between these two possibilities, we introduced an autophagy reporter, mCherry-GFP-Atg8, in which GFP but not mCherry is quenched inside acid lysosomes (Mauvezin et al., 2015). Significantly more GFP + Cherry + and GFP -Cherry + positive vesicles were observed in pink1 5 mutants, indicating augmented autophagy ( Figure 2F and Supplemental Figure S5). Overexpression of Atg1 (Atg1 OE ) induced autophagy in pink1 5 mutants, as indicated To test whether autophagy is required for the rescuing effect of Atg1 OE on pink1 5 mutants, components of the autophagy pathway were inhibited in the pink1 5 ; Atg1 OE background. Atg7 is essential for autophagy induction, and flies lacking Atg7 are viable, although autophagy was severely impaired . As expected, null mutants of atg7 d14/d77 blocked the Atg1 OE -rescuing effect in pink1 5 muscles ( Figure 2G and Supplemental Figure S5). Rab7 (YFPfused Rab7 under UAS control) expressed in muscles readily colocalizes with LysoTracker, indicating that Rab7 is involved in endolysosomal trafficking (Supplemental Figure S6A). Indeed, knockdown of FIGURE 2: Functional autophagy machinery is required for Atg1 overexpression-mediated rescuing effect on pink1 mutants. (A, B) Acidic compartments (stained by LysoTracker in red) and mitochondria (mito::GFP in green) in freshly dissected muscles. Arrows point to potential colocalization of mito::GFP and LysoTracker. "Viable (-)" muscles in pink1 mutants have large mitoGFP clumps and are always TUNEL-negative. In "degenerating (+)" muscles, mito::GFP clumps are replaced by "fragmented" mitochondria and are always TUNEL-positive (see also Supplemental Figure S2 and main text for details). (C) Quantification of LysoTracker-positive vesicles with diameter greater than 0.2 µm in muscles. (D) Percentage of LysoTracker-positive vesicles showing colocalization with mito::GFP in pink1 5 muscles. In both C and D, pink1 5 (-) means viable muscle, while pink1 5 (+) means degenerating muscles. At least 20 muscles from 5-6 male flies were counted. ***p < 0.001, *p < 0.05 (unpaired t test). (E) Mitochondrial autophagy activity was quantified based on localization of LysoTracker and mitoGFP. Number of events counted for each genotype is labeled under the chart. (F) Autophagic activity was measured based on the genetic reporter UASmCherry-GFP-Atg8. mCherry + GFP -, mCherry + GFP + , and mCherry -GFP + were measured separately. Events counted for each condition are indicated. (G) Autophagy is required for Atg1 OE rescuing effect on pink1 mutants. Mitochondrial size was normalized with controls. At least 100 mitochondria from more than 10 animals were counted and analyzed for each genotype. For E, F, G, one-way analysis of variance (ANOVA) was performed for statistical analysis. SEM and p value are shown: ***p < 0.001, **p < 0.01, *p < 0.05.
Rab7 or components of the Vps-C complex resulted in accumulated aberrant large LysoTracker-positive structures in muscles (Supplemental Figure S6B; knockdown efficiency of RNA interference [RNAi] lines used in this study is shown in Supplemental Figure S7) and blockage of autophagy in pink1 5 muscles (Figure 2, E-G, and Supplemental Figures S3-S5). Although knockdown of Rab7 or components of the Vps-C complex did not change mitochondrial morphology (Supplemental Figure S6C), they abrogated the rescue effect of Atg1 OE , as mito::GFP aggregates reappeared in pink1 5 muscles ( Figure 2G and Supplemental Figure S5). Inhibiting lysosomal acidification by VhaAC39a RNAi was also able to block the rescue effect of Atg1 OE ( Figure 2G and Supplemental Figure S5). These results indicated that the autolysosomal pathway is required for Atg1 OE -mediated mitochondrial clearance in pink1 5 muscles.
Atg1 overexpression rescues pink1 5 pathogenesis by promoting mitochondrial fission As indicated above, mitochondria are significantly smaller in Atg1 OE muscles, suggesting that Atg1 can regulate mitochondrial dynamics. Because mitochondrial fission is essential for maintaining mitochondria integrity and cell survival in pink1 5 muscles (Deng et al., 2008), we propose that mitochondrial dynamics is also required for the rescuing effect of Atg1 OE . Mitochondrial fission was inhibited by knocking down Drp1 (Supplemental Figure S8). Consistent with previous results (Deng et al., 2008), we did not observe mito::GFP clumps or TUNEL-positive nuclei in Drp1 RNAi muscles, although mitochondria were slightly fused (Figure 3, A-D, and Supplemental Figure S8). However, knocking down Drp1 in a pink1 5 background exacerbated the mitochondrial morphological defects and the percentage of TUNEL-positive cell death ( Figure 3D). Surprisingly, Atg1 OE no longer rescues pink1 5 in the absence of Drp1, as mito-GFP clumps and TUNEL-positive nuclei reappeared (Figure 3, A-D). Also, the "fragmented" mitochondria in Atg1 OE were significantly restored by Drp1 RNAi (Figure 4A and Supplemental Figure S9). On the other hand, knockdown of Atg1 in muscles resulted in the accumulation of more mitochondria with irregular shapes, increased volume and size ( Figure 4A and Supplemental Figure S9). Intriguingly, these irregularly elongated mitochondria in Atg1 RNAi muscles were fully suppressed by Drp1 overexpression ( Figure 4A and Supplemental Figure S9). These results further indicated that Atg1 regulates mitochondrial dynamics.
We next tested whether Atg1 can regulate core components of the mitochondrial machinery. A mild increase in drp1 transcripts was found in Atg1 OE muscles by RT-qPCR ( Figure 4B). However, the Drp1 protein level increased two-to threefold in Atg1 OE muscles, as observed by anti-HA staining of Drp1-HA (HA-tagged Drp1 under an endogenous promoter; Figure 4, C and D). Significant increases of Drp1-HA foci were also found in Atg1 OE muscles ( Figure 4E). Taken together, these results indicate that Atg1 promotes mitochondrial fission at least partially through Drp1 and that drp1 is required for the Atg1 OE rescue effect on pink1 5 muscle degeneration. Drp1 overexpression or mfn knockdown rescues pink1 5 mitochondrial abnormality and muscle degeneration when autophagy and proteasome are impaired We sought to test whether enhancing fission can rescue pink1 5 pathogenesis when the degradation systems (proteasome/autophagy) are impaired. Lipidation of Atg8 (a homologue of LC3) is commonly used as a marker for autophagosomes (Kabeya et al., 2000;Klionsky et al., 2008). In young wild-type muscles (3-5 d old), Atg8::mCherry positive dots are smaller than mitochondria (<0.1 µm in diameter vs. 0.2-2 µm for mitochondria), and they rarely colocalized with mito::GFP (0/200 dots in muscles from 10 different flies; Supplemental Figures S3 and S10A). Knocking down Atg1 in muscles suppressed Atg8::mCherry positive punctae in wild-type muscles (Supplemental Figure S10, A and B). Interestingly, although no signs of degeneration occurred in young Atg1 RNAi muscles, Atg1 RNAi accelerated the appearance of TUNEL positive muscles in pink1 5 mutants ( Figure 5, A and B), suggesting that autophagy is beneficial for cell survival. Strikingly, pink1 5associated defects, such as mitochondrial abnormalities and TUNEL-positive cell death, were fully suppressed by overexpressing drp1 in an Atg1 RNAi background ( Figure 5, A and B).
Age-dependent muscle degeneration in pink1 5 was evaluated by TUNEL staining of hemithoraces. A significant increase of TUNEL-positive muscles was observed with age in pink1 5 mutants (55% positive in 4-d-old vs. 80% in 20-d-old animals; Figure 5C). However, Drp1 OE sustains the rescue effect in 20-d-old pink1 5 mutant muscles when Atg1 is simultaneously inhibited ( Figure 5C).
In Atg7 d14/d77 transheterozygous mutant muscles, the mitochondria were largely normal and there was no obvious cell death ( Figure 5A). Muscle degeneration in pink1 5 worsened in an Atg7 d14/d77 background, as indicated by increasing TUNEL-positive muscles ( Figure 5B). Interestingly, Drp1 OE also rescued pink1 5 muscles in the absence of Atg7 ( Figure 5, A and B). Knocking down genes for downstream autophagy processes, such as Rab7 and Vha39AC, also failed to block the rescue effect of Drp1 OE on pink1 5 muscle pathogenesis (unpublished data).
Inhibiting the proteasome activity by overexpressing DTS-7 (dominant temperature-sensitive mutation of the proteasome β2 subunit; Schweisguth, 1999;Belote and Fortier, 2002) and exposing the animals to the restrictive temperature (29°C) produced large ubiquitin-positive inclusion in muscles (Supplemental Figure S10, C and D). This indicated that DTS-7 can efficiently suppress proteasome activity. We found that DTS-7 OE deteriorated pink1 5 muscle defects; however, it still failed to block the rescue effect of Drp1 OE (Figure 5, A and B).
Drp1 overexpression rescues mitochondrial abnormalities and dopaminergic neuron loss in pink1 RNAi brain when autophagy and the proteasome are defective Progressive loss of dopaminergic (DA) neurons is one of the hallmarks of PD. Pink1 and parkin mutants also experienced severe mitochon- Muscles from at least 10 hemithoraces of each genotype are dissected and stained by TUNEL assay. (C) Age-dependent increasing of TUNEL-positive muscles in pink1 5 mutants rescued by Drp1 overexpression when autophagy was inhibited. Age of flies was included in the chart. Indirect flight muscles are multinucleated and the nuclei in pink1 5 muscle are synchronized, that is, either all negative or all positive in terms of TUNEL staining. Student's t test for statistics. *p < 0.05 and ***p < 0.001. drial defects and dopaminergic neuron loss (Park et al., 2006;Yang et al., 2006Yang et al., , 2008Poole et al., 2008;Yun et al., 2008Yun et al., , 2014. Mitochondria in DA neurons are normally elongated, but form clumps in a pink1 RNAi background, as indicated by mito::GFP driven by a DA neuron-specific driver, tyrosine hydroxylase (TH) GAL4 (Figure 6, A and  B). pink1 RNAi flies encountered slight but significant loss of DA neurons, especially in the protocerebral posterior lateral 1 (PPL1) cluster (Park et al., 2006;Poole et al., 2008;Yang et al., 2006; Figure 6C).
We sought to test the physiological relationship of autophagy and mitochondrial dynamics to DA neuron viability in pink1 RNAi flies. First, autophagic activity was examined in DA neurons. A few Atg8::mCherry-positive autophagosomes were observed in these neurons. Similarly to those found in muscles, these punctae barely colocalized with mito::GFP. Also, these Atg8::mCherry positive dots were substantially blocked by Atg1 RNAi in DA neurons, indicating that Atg1 RNAi suppresses autophagy efficiently in DA neurons. DA neurons with Drp1 OE have comparable Atg8-positive vesicles. On the other hand, DA neurons with pink1 RNAi had significantly larger, fused Atg8-positive vesicles, and these vesicles partially associated with mito::GFP ( Figure 6A).
Atg1 RNAi in DA neurons had no obvious defects in mitochondrial morphology, and the cell numbers in the PPL1 cluster were comparable to those in the controls. However, the mito::GFP clumps and cell loss in PPL1 cluster in pink1 RNAi flies were further exacerbated by Atg1 RNAi in DA neurons ( Figure 6, B and C). Drp1 overexpression fully rescues mito::GFP clumps and degeneration in DA neurons of pink1 RNAi flies. Moreover, the rescuing effects were sustained in the Atg1 RNAi background (Figure 6, B and C). Similarly, inhibiting fusion by knocking down mfn could also significantly rescue mito::GFP clumps in pink1 RNAi flies, even when atg1 RNAi was silenced simultaneously ( Figure 6, B and C).
These results suggested that, in contrast to the degradative processes, enhancing fission or inhibiting fusion is essential for cell survival in pink1/parkin mutants.

DISCUSSION
Recent studies of mammalian cell lines implicated that pink1/parkinmediated mitophagy is crucial for mitochondrial quality control. In brief, Pink1 is stabilized on heavily uncoupled mitochondria and recruits Parkin. Ubiquitination of mitochondrial outer membrane proteins facilitates subsequent turnover of the mitochondria by proteolysis and autophagy. However, the physiological contribution of mitophagy to pink1/parkin null-mediated pathogenesis is elusive.
Autophagy is a catabolic process that helps maintain energy balance by recycling cellular compartments. Our data indicated that enhancing autophagy was generally beneficial for flies lacking pink1/parkin, which is consistent with a recent finding (Liu and Lu, 2010). However, we were not able to recapitulate the mitophagy FIGURE 6: Overexpressing Drp1 rescues mitochondrial defects and dopaminergic (DA) neuron loss in pink1 5 mutants when autophagy or proteasome is impaired. (A) Autophagosomes in DA neurons were visualized by UAS::Atg8::mCherry under the driver TH-Gal4, where mitochondria are labeled by mito::GFP in green. A' is the mitoGFP channel in gray. (B) Genetic analysis of mitochondrial morphology in DA neurons is represented by TH-Gal4 > mitoGFP in gray. (C) Quantification of DA neuron cell number in PPL1 clusters. DA neurons were labeled by THGal4 > CD8::GFP. PPL1 regions of 10 brains for each genotype were counted. Average standard error is shown and statistics were performed by Student's t test. *p < 0.05, **p < 0.01. process in Drosophila. First, under physiological conditions, there are only a few autophagosomes (marked by Atg8::mCherry) in these tissues, and these dots were never colocalized with mitochondria (labeled with mito::GFP). Second, mitochondrial uncoupling, either by feeding the flies with CCCP (carbonyl cyanide m-chlorophenyl hydrazine) or by genetically overexpressing hUCP2 (Fridell et al., 2005), failed to increase the colocalization of Atg8::mCherry and mito::GFP (0/300 dots in muscles from 12 different flies; unpublished data). Finally, no obvious Pink1 stabilization or Parkin recruitment was observed after mitochondrial uncoupling (Supplemental Figure S12, A-F). Consistent with our results, a recent paper also failed to detect a role of pink1/parkin in mitophagy in Drosophila (Lee et al., 2018).
Why was mitophagy not observed in Drosophila muscles? It might be simply because mitophagy happens rarely, or occurs extremely fast or slowly, and these events failed to be detected in the current experimental settings. Indeed, Parkin recruitment to uncoupled mitochondria is substantially different in fibroblasts from that in neurons in terms of kinetics and extent (4 h after CCCP treatment, 70% recruitment in neurons; 2 h, 100% recruitment in fibroblasts; Seibler et al., 2011;Van Laar et al., 2011). Alternatively, mitophagy might only become prominent after extreme mitochondrial insults. Here, we found mitochondrial depolarization only after prolonged high-dose CCCP feeding (Supplemental Figure S12, G and H). It is quite possible that the buffering capacity of mitochondria in different tissues may vary; delivery method and timing may also alter the feeding effect of CCCP in vivo. In fact, pink1/parkinmediated mitophagy relies heavily on severe mitochondrial uncoupling, such as FCCP/ CCCP. How it compares with physiological mitochondrial dysfunction is unknown (de Vries et al., 2012;Grenier et al., 2013). In fact, in a mouse model deficient for the mitochondrial transcription factor A, no parkin recruitment to mitochondria was observed in the brain (Sterky et al., 2011). It was proposed that both mitochondrial dynamics and mitochondrial autophagy contribute to mitochondrial quality control (Twig and Shirihai, 2011;McCoy and Cookson, 2012;Ashrafi and Schwarz, 2013): mitochondrial fission facilitates the segregation of severely damaged mitochondria, which are then selectively eaten up by autophagy machinery. Mitochondrial fission facilitates mitophagy, since blocking fission by a dominant negative form of Drp1 impairs the mitophagy process (Dagda et al., 2009;Frank et al., 2012;Rana et al., 2017). Pink1 and parkin are required in mitochondrial quality control, since they regulate both processes. If these two processes are physiologically linked, we would assume that both processes would be essential for pink1/ parkin null related pathogenesis.
However, we found here that increasing fission or inhibiting fusion can still rescue pink1/parkin muscle degeneration when the autophagy or proteasome activity was hampered. Hence, we propose that, although clearance of severely damaged mitochondria by autophagy is beneficial, a subset of healthy mitochondria segregated by mitochondrial fission was sufficient to maintain cellular health and tissue survival in pink1/parkin mutants (Figure 7). Atg1 is a highly conserved Ser/Thr kinase that plays an essential role in autophagy. Substrates of this kinase identified so far, such as Atg13 and Atg9, are components of the autophagy pathway (Chang and Neufeld, 2009;Papinski et al., 2014). Here we found that Atg1 can promote mitochondrial fission at least partially through Drp1. How does Atg1 regulate Drp1 protein levels? Posttranscriptional modifications, such as phosphorylation, can modulate Drp1 activity and hence mitochondrial morphology. Thus, Atg1 might directly or indirectly modify Drp1 posttranscriptionally.
Atg1 activity is tightly regulated by the nutrition-sensing mTOR pathway (Chang and Neufeld, 2009;Mizushima, 2010). Mitochondrial dynamics and mitochondrial metabolic activity are also regulated by nutrition status (Liesa and Shirihai, 2013). Our results suggested that Atg1 might coordinate mitochondrial dynamics and autophagy activity to cope with the nutritional status of cells (Figure 7). The dual role of Atg1 in mitochondrial dynamics and autophagy makes it a hotspot for mitochondrial quality control.
FIGURE 7: Dual beneficial roles of Atg1 in pink1/parkin pathogenesis. In pink1/parkin mutants, mitochondria are dysfunctional (colored in yellow). Mitochondrial fission helps to segregate "severely" damaged mitochondria (red ones) and is crucial for mitochondrial quality control and cell survival in pink1/parkin mutants. Overexpressing Atg1 is beneficial at least in two ways. On one hand, it promotes fission through Drp1, which would help to pinch off the "damaged" portion of "bad" mitochondria (red); on the other hand, it accelerates autophagic degradation of severely damaged mitochondria. When degradation machinery is impaired, "healthier" mitochondria (green) generated by elevated fission are sufficient for cell survival.
RNA isolation, cDNA synthesis, and RT-qPCR RNA was isolated from thorax by Trizol. cDNA synthesis was performed using a combination of Oligo-dT and random hexamer priming by the Clontech RNA to cDNA EcoDry Premix Kit. Quantitative RT-PCR was performed using the BioRad iTaq Fast Sybr Green enzyme mix, 10-µl reactions in triplicate, on a Roche Light Cycler 480. Standard curves were generated for targets and normalized with the control gene Actin-5C. t test and SEM. were performed for statistical analysis. ***: p value < 0.001. Primers used in this study:

Immunofluorescence and confocal microscopy
For muscles, thoraces were dissected and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS). After thoraces were washed three times in PBS, muscle fibers were isolated and stained with rhodamine phalloidin (Invitrogen; 1:1000) in PBS + 1% Triton X-100. For antibody staining, muscle fibers were permeabilized and blocked in PBS + 0.1% Triton X-100 and incubated in primary and secondary antibodies diluted in PBS + 1% bovine serum albumin (BSA). The following primary antibodies were used: chicken anti-HA (Millipore, Billerica, CA), mouse anti-ATP Synthase (Mitosciences, Eugene, OR), and mouse anti-myc (Sigma, MO). For DA neurons, brains of 3-d-old male flies were dissected and fixed in 4% paraformaldehyde in PBS. All images were taken on a Zeiss LSM5 confocal microscope.

TUNEL assay and quantification
Thoraces from adult male flies were dissected and fixed in 4% paraformaldehyde in PBS. Muscle fibers were isolated and subsequently permeabilized and blocked in T-TBS-3% BSA (T-TBS: 0.1% Triton X-100, 50 mM Tris-Cl [pH 7.4], and 188 mM NaCl). After blocking, TUNEL staining was carried out using an In-Situ Cell Death Detection Kit from Roche. Percentages of TUNEL-positive muscles are quantified based on muscle fibers from at least 10 flies in each genotype. For TUNEL-positive muscles quantification, hemithoraces were dissected and prepared as previously reported (Schonbauer et al., 2011). After fixation and permeabilization, TUNEL assays were performed. At least 50-60 muscles from 10 thoraces were examined for each genotype.

Mitochondrial size measurement
Mitochondrial in muscles of indicated genotypes were labeled with mitochondrial-targeted GFP (mitoGFP) and imaged by confocal microscopy under the same setting.
Individual mitochondria were then circled out and the volume of ROI was measured with ImageJ. At least 100 mitochondria from 10 animals were analyzed. t tests were performed for statistical analysis.

Analysis of dopaminergic neurons
Fly brains were dissected from 25-d-old male flies. Dopaminergic neurons were labeled with anti-tyrosine hydroxylase staining. Brains were imaged by confocal microscopy and PPL1 cluster neurons were counted per brain hemisphere. Data were analyzed by Prism 5 software from at least 10 brains for each genotype.
Embedding, sections, toluidine blue staining, and TEM Thoraces from young male flies were dissected, fixed in paraformaldehyde/glutaraldehyde, postfixed in osmium tetraoxide, dehydrated in ethanol, and embedded in Epon. After polymerization of Epon, blocks were cut to generate 1.5 µm-thick sections using a glass knife, or 80 nm-thick sections using a diamond knife on a microtome (Leica, Germany). Toluidine blue was used to stain 1.5 µmthick tissue sections. Thin sections (80 nm thick) were stained with uranyl acetate and lead citrate and then examined using a JEOL 100C transmission electron microscope (UCLA Brain Research Institute Electron Microscopy Facility). At least six thoraces were examined in each sample.

Drosophila lysate preparation and Western blotting
Thoraces from adult flies were homogenized in RIPA buffer containing protease inhibitors (Roche). Total protein concentration was measured using a Bradford assay kit (Bio-Rad, Hercules, CA), and the same amount of protein was loaded onto SDS-polyacrylamide gels. The following primary antibodies were used for Western blots: mouse anti-HA (Millipore) and rabbit anti-Actin (Sigma).

LysoTracker and TMRE staining
Muscles was freshly dissected in PBS and incubated in a dark chamber within which PBS contains 1/1000 diluted LysoTracker Red DND-99 (Life Technologies) or 1 µM TMRE (dissolved in 100% ethanol, from Molecular Probes) for 30 min. After brief rinsing and washing in PBS, muscles were mounted in PBS and immediately imaged under microscopy.