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However, we did not detect mitolysosomes in flight muscle. Surprisingly, in Pink1 or parkin null flies, we did not observe any substantial impact on basal mitophagy. Our findings provide evidence that Pink1 and parkin are not essential for bulk basal mitophagy in Drosophila. They also emphasize that mechanisms underpinning basal mitophagy remain largely obscure.
Mitochondria are essential organelles that perform many critical metabolic functions but are also a major source of damaging reactive oxygen species and harbor proapoptotic factors. Multiple homeostatic processes operate to maintain mitochondrial integrity; however, terminally damaged organelles are degraded through the process of targeted mitochondrial autophagy mitophagy to prevent potentially catastrophic consequences.
Such homeostatic mechanisms are particularly important for postmitotic, energetically demanding tissues such as nerves and muscles. In brief, upon loss of mitochondrial membrane potential as occurs in damaged or dysfunctional mitochondria, PINK1 accumulates on the outer mitochondrial membrane OMM and initiates the mitophagy signal by phosphorylating both ubiquitin and parkin.
This process acts as a feed-forward mechanism ultimately decorating the OMM with phosphoubiquitin chains that are then recognized by ubiquitin adaptor proteins leading to the engulfment of the depolarized mitochondria by autophagosomes.
As a pathogenic mechanism, failure in mitophagy offers an attractive explanation for multiple, longstanding observations that implicate mitochondrial defects in the pathogenicity of PD, such as systemic mitochondrial complex I deficits and high levels of mitochondrial DNA mutations in PD patients Schapira et al.
In addition, the unique physiological characteristics of substantia nigra neurons, such as their extensive arborization, myriad synaptic connections, and continuous pacemaking activity, place an extreme demand on mitochondrial function to meet their high energy and calcium buffering requirements. This may explain the selective vulnerability of these neurons to loss of mitochondrial integrity Sulzer and Surmeier, However, relatively little is known about mitophagy under physiological conditions in vivo Cummins and Gotz, ; Rodger et al.
One of the limitations to studying mitophagy in vivo has been the paucity of suitable reporters. Recently, two in vivo mitophagy reporter models have been described in mice Sun et al. Both systems exploit pH-sensitive properties of mKeima and GFP, respectively, to enable the differential labeling of mitochondria in the acidic microenvironment of the lysosome as a proxy endpoint readout. Initial studies on these two reporter lines have revealed a surprisingly widespread and heterogeneous distribution of basal mitophagy, but the involvement of PINK1 and parkin has not been addressed.
We have generated Drosophila melanogaster lines expressing these mitophagy reporters to provide the first global view of the prevalence of mitophagy across the organism and to determine the relative contribution of Pink1 and parkin to basal mitophagy.
In contrast to most mammalian models, loss of Drosophila Pink1 or parkin leads to robust phenotypes in locomotor activity and dopaminergic DA neuron loss Greene et al. We find that although basal mitophagy is widespread in many tissues in Drosophila , the incidence of mitolysosomes, and hence, basal mitophagy, is unaffected by loss of Pink1 or parkin. Hence, we propose that Pink1 and parkin are largely dispensable for basal mitophagy and have mitophagy-independent functions in neuromuscular tissues in Drosophila.
Multiple lines were established and immunoblotting confirmed comparable expression between lines Fig. S1, A and B. Expression of these reporters in all lines appeared benign and did not noticeably affect development or viability. To ensure that the expression of these reporters did not overtly interfere with normal cellular functions in vivo, particularly in the sensitive neuromuscular systems of interest here, we analyzed locomotor activity in flies expressing high levels of the mito-QC or mt-Keima in all tissues and observed no significant impact Fig.
S1, C—F. Nevertheless, we also sought to verify these conditions in our Drosophila lines. As expected, we observed substantial colocalization of mito-QC and mt-Keima with the mitochondrial protein ATP5A in two tissues highly amenable for mitochondrial imaging analysis, larval epidermal cells and adult flight muscle Fig.
Interestingly, the outer membrane targeted mito-QC shows a nonuniform distribution across the network in epidermal cells, which reflects the dynamic and heterogeneous nature of the mitochondrial network.
Validation of mito-QC and mt-Keima mitophagy reporters. A—C Immunohistochemical and confocal imaging analysis of larval epidermal cells A and C and adult flight muscle B. Fluorescence corresponding to neutral pH and acidic pH are shown in green and red, respectively. LysoTracker is shown in white. Similarly, for mt-Keima, the fluorescence spectrum shifts to reflect an increased signal from excitation at nm when under more acidic conditions Fig. To further validate these puncta as mitolysosomes, we assessed their formation in tissue lacking the canonical autophagy pathway generated by knockdown of the key autophagy factor Atg5.
Moreover, we sought to demonstrate that these reporters were sensitive enough to reveal induced mitophagy. Although sustained treatment with mitochondrial depolarizing agents, such as carbonyl cyanide 3-chlorophenylhydrazone, was not feasible in vivo, it was previously reported that iron chelation by deferiprone induced mitophagy reported by mito-QC Allen et al.
Feeding animals deferiprone, we observed an increase in mitolysosomes reported by mito-QC and mt-Keima Fig. Collectively, these findings validate the mitophagy signal of mito-QC and mt-Keima as faithful reporters of mitolysosomes in Drosophila , similarly to previously characterized mouse models Sun et al.
We next determined the prevalence of mitolysosomes under basal conditions in multiple tissues. In larvae, we analyzed epidermal cells, because they have an elaborate mitochondrial morphology, and the ventral ganglion, a major portion of the central nervous system CNS. Here, we directly compared the mitolysosome signal reported by mito-QC and mt-Keima. Microscopic analysis revealed that mitolysosomes are abundant under basal conditions in both epidermal cells and CNS Fig. In contrast, mitolysosomes were almost undetectable in larval body wall muscles unpublished data.
Importantly, mito-QC and mt-Keima revealed a very similar abundance and distribution of mitolysosomes, substantiating their utility as equivalent mitophagy reporters. Comparison of mito-QC and mt-Keima mitophagy reporters in larval tissues. During this stage of analysis, we found that the mt-Keima signal, which was already markedly weaker than that generated by mito-QC, was rapidly bleached upon extended exposure or repeated scanning. Repeated imaging was necessary to achieve adequate penetration into complex tissue and for z-stack 3D reconstruction for mitolysosome quantification.
For this reason, the subsequent analyses mainly focused on analyzing mito-QC. We next analyzed adult tissues where postmitotic, highly energetic tissues would be expected to accumulate more mitochondrial damage and therefore likely undergo more mitophagy.
Again, we observed abundant mitolysosomes, widespread in a medial region of the posterior protocerebrum adult brain; Fig. The amount of mitophagy signal did not markedly increase with ageing. Mitolysosome analysis with mito-QC in adult tissues. Confocal microscopy analysis of mito-QC reporter in WT adult brain 2 d old , DA neurons at 2 and 30 d old, flight muscle 2 d old , and preadult pupal flight muscle, as indicated.
Quantification of mitolysosomes is shown in Figs. Somewhat surprisingly, but consistent with observations in larvae, we did not observe any appreciable mitolysosomes in 2-d-old flight muscle Fig. This is notable for two reasons. First, adult flight muscle in particular has extremely abundant mitochondria necessary to power flight. Second, Pink1 and parkin mutant Drosophila display robust degeneration of flight muscles. It could be reasonably assumed that this degeneration results from a failure in mitophagy induction.
The lack of mitophagy signal is especially surprising given the abundant mitochondria and apparent abundant lysosomes in this tissue Demontis and Perrimon, We reasoned that analyzing adult tissue could possibly have missed a critical period when mitophagy is active and required for muscle integrity, such as when the tissue is being formed in development. However, we also could not detect mitolysosomes at these earlier time points Fig.
Collectively, from these observations, we surmise that mitophagy is abundant in various tissues during development and in adult flies, especially in neuronal tissue, but is not detectable in muscle tissue at either stage. Mammalian PINK1 and parkin have been described to play important roles in toxin-induced mitophagy; however, the impact of PINK1 and parkin on basal mitophagy in vivo has not been extensively studied.
To address this, we combined our mitophagy reporters with well-characterized Pink1 and parkin Drosophila mutants. Analyzing the previously described tissues where mitolysosomes were abundant, we found that mitophagy was not dramatically reduced in genetic null mutants for Pink1 Fig. To quantify the degree of mitophagy we applied a semiautomated quantification analysis to segment 3D images to identify and quantify bona fide mitolysosomes Fig.
Quantitative analyses revealed there was no statistical difference in number of mitolysosomes between WT and Pink1 or parkin mutant animals for most cell types, except for larval CNS, where the difference in Pink1 mutants just reached significance Figs. To further verify this unexpected lack of effect, we qualitatively assessed mt-Keima in Pink1 mutants, and we also observed no gross difference in mitophagy signal Fig.
Basal mitophagy is minimally affected in Pink1 mutants. Confocal microscopy analysis of mito-QC reporter in Pink1 B9 mutant larval epidermis and CNS, adult brain 2 d old , DA neurons at 2 and 30 d old, and adult flight muscle 2 d old , as indicated. Quantification of WT control samples is the same as shown in Figs.
Basal mitophagy is minimally affected in parkin mutants. Confocal microscopy analysis of mito-QC reporter in park 25 larval epidermis and CNS, DA neurons at 2 and 30 d old, and adult flight muscle 2 d old , as indicated.
However, we again observed no mitophagy signal in Pink1 or parkin mutants Figs. Thus, collectively these data indicate that loss of Pink1 or parkin does not substantially influence basal mitophagy in neuromuscular tissues. The notion that PINK1 and parkin cooperate to mediate bulk degradation of mitochondria has become a dominant concept in the field of PD pathogenesis.
We have made use of two previously devised mitophagy reporter constructs to visualize mitophagy in fly models for the first time.
Our observations reveal that basal mitophagy is highly prevalent in multiple Drosophila tissues; however, this mitophagic process is essentially unaffected by null mutations in Pink1 or parkin.
Importantly, while this work was under review, a complementary study was published reporting the effect on the mito-QC reporter system in Pink1 knockout mice McWilliams et al.
This study reached essentially the same conclusion as our study that basal mitophagy is unaffected by loss of PINK1 in most tissues. Together, our complementary studies analyzing independent mitophagy reporters in multiple mutant models provide strong evidence that the minimal impact of PINK1 and parkin on basal mitophagy is likely a conserved phenomenon.
Several alternative explanations can be considered. One clear possibility is that another mode of mitophagy can be induced as a compensatory mechanism. Indeed, there is some precedence for this as it was recently reported that MUL1 acts redundantly alongside parkin Yun et al. Moreover, although germline knockout of murine PINK1 and parkin has been shown to have very mild phenotypes Lee et al. One possibility that would fit this scenario is induction of an alternative mitophagy pathway or up-regulation of other mitochondrial quality-control processes.
Clearly, more needs to be learned about the complex regulatory mechanisms governing mitochondrial turnover under physiological conditions, and our Drosophila model offers an excellent system to genetically address these mechanisms. Indeed, whether the observed basal mitophagy is operating as a quality control mechanism or some other homeostatic process, such as maintenance of mitochondrial levels, is unknown.
We have not yet systematically assessed the kinetics of these mitophagy reporters, but we note that the observed mitolysosomes have not been detectably dynamic within the range 10—15 min of live imaging. Our primary motivation was to analyze physiological, basal mitophagy but it may be informative to assess the feasibility of monitoring stress- or toxin-induced mitophagy in vivo. It is possible that loss of parkin or Pink1 may slow the kinetics of this accelerated process but the timescales involved 10—24 h in cell culture may still be too challenging for in vivo studies.
A more targeted stimulus such as light induced reactive oxygen species generation may be more amenable Yang and Yang, ; Ashrafi et al. That selective degradation of mitochondrial components may also occur in vivo is supported by compelling evidence using a mass spectrometric method to analyze isotope labeled mitochondrial protein turnover in flies Vincow et al.
In light of this, it is clear that further work is needed to define the conditions in which PINK1 and parkin promote mitochondrial homeostasis. The mitophagy reporter lines described here provide new model systems for studying physiological mitophagy and can be potentially used for screening for novel regulators.
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