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Genetic analysis of mitochondrial protein misfolding in Drosophila melanogaster

Protein misfolding has a key role in several neurological disorders including Parkinson's disease. Although a clear mechanism for such proteinopathic diseases is well established when aggregated proteins accumulate in the cytosol, cell nucleus, endoplasmic reticulum and extracellular space, little is known about the role of protein aggregation in the mitochondria. Here we show that mutations in both human and fly PINK1 result in higher levels of misfolded components of respiratory complexes and increase in markers of the mitochondrial unfolded protein response. Through the development of a genetic model of mitochondrial protein misfolding employing Drosophila melanogaster, we show that the in vivo accumulation of an unfolded protein in mitochondria results in the activation of AMP-activated protein kinase-dependent autophagy and phenocopies of pink1 and parkin mutants. Parkin expression acts to clear mitochondria with enhanced levels of misfolded proteins by promoting their autophagic degradation in vivo, and refractory to Sigma P (ref(2)P), the Drosophila orthologue of mammalian p62, is a critical downstream effector of this quality control pathway. We show that in flies, a pathway involving pink1, parkin and ref(2)P has a role in the maintenance of a viable pool of cellular mitochondria by promoting organellar quality control.

Aberrant protein folding is linked to several neurological disorders including Parkinson's disease (PD). Such proteinopathic diseases are often linked to the formation of intracellular aggregates of toxic proteins in the cytosol, cell nucleus, endoplasmic reticulum and extracellular space (reviewed in Rubinsztein 1 ). Additionally, the deterioration of functional mitochondria is thought to have important consequences for the function and survival of neurons, and has therefore been proposed to have a causative role in neurological disorders such as PD (reviewed in de Castro et al. 2 ). In spite of the evidence linking both protein aggregation and mitochondrial dysfunction to PD, surprisingly little is known whether mitochondrial protein aggregates can also have a significant role in neurodegenerative diseases.
Recently, the PD-associated kinase PINK1 was identified as an important modulator of the mitochondrial serine protease HtrA2 activity. 3 Deletion of HtrA2 in mice results in the accumulation of unfolded proteins in the mitochondria, indicating that protein aggregation in this organelle may contribute to the advancement of neurodegenerative diseases. 4 Additionally, the ubiquitin ligase Parkin has been shown to act in organellar quality control, to promote the autophagy of damaged mitochondria 5 through a PINK1 recruitment mechanism. 6 Thus, PINK1 seems to act as an upstream modulator of both molecular and organellar quality control pathways via HtrA2 and Parkin, respectively.
Through the analysis of post-mortem brains from PD patients carrying PINK1 mutations that affect the phosphorylation status of the serine protease HtrA2, we detected the presence of enhanced levels of misfolded components of mitochondrial respiratory complexes as well as an increase in the levels of the mitochondrial HSP-60, a marker of the activation of the mitochondrial unfolded protein response (UPR mt ) in the nematode Caenorhabditis elegans (reviewed in Broadle and Hartl 7 ). These results indicate that the accumulation of protein aggregates in the mitochondria might be detrimental to mitochondrial function and led us to develop an in vivo model for the selective accumulation of misfolded proteins in this organelle.
To dissect the in vivo consequences of mitochondrial protein aggregation, we make use of the fruit fly, Drosophila melanogaster. Here, we show that Drosophila mutants in pink1 and parkin display higher levels of misfolded components of mitochondrial respiratory complexes and an increase in the levels of the mitochondrial HSP-60 (reviewed in Broadle and Hartl 7 ). Using a novel genetic model of mitochondrial protein misfolding, we show that the in vivo accumulation of an unfolded protein causes generalised mitochondrial dysfunction and accompanied by engagement of autophagy in an AMP-activated protein kinase (AMPK)-dependent manner.
This genetic model of mitochondrial protein misfolding phenocopies pink1 and parkin mutants, suggesting that a compromise on mitochondrial protein quality control might be related to mitochondrial dysfunction caused by the loss of Pink1 or Parkin. Our study also suggests that Parkin expression acts to clear defective mitochondria with high levels of unfolded proteins by promoting their autophagic degradation in vivo, and refractory to Sigma P (ref(2)P), the Drosophila orthologue of mammalian p62, is a critical downstream effector of this quality control pathway.

Results
A novel Drosophila model of mitochondrial protein misfolding. To determine whether PINK1 dysfunction in humans is linked to protein conformational stress, we analysed the levels of misfolded mitochondrial respiratory components in brain samples from deceased PD patients, using an approach designed to investigate mitochondrial protein misfolding in HtrA2 mutant mice. 4 In this analysis, we included PD patients harbouring heterozygous PINK1 mutations as well as idiopathic disease subjects (IPD) with no identified mutations in this gene. This revealed that patients carrying either the Y431H or the C575R mutations have significant levels of unfolded mitochondrial respiratory complexes ( Figure 1a). Interestingly, levels of human HSP-60 positively correlate with the degree of misfolded respiratory complexes in these patients, suggesting that in humans protein conformational stress in the mitochondria is potentially linked to the activation of the UPR mt . Notably, we have previously reported that patients carrying either the Y431H or the C575R mutations have decreased levels of phospho-HtrA2, and are therefore likely to have defective activation of the mitochondrial serine protease HtrA2, a protein implicated in mitochondrial stress response. 3 To determine whether Drosophila mutants of either pink1 or parkin mimicked the accumulation of unfolded proteins in mitochondria observed in PD patients, we analysed the levels of insoluble respiratory complexes in these flies. We observed enrichment of several respiratory complex subunits in both mutants (Figures 1b and c), suggesting that these proteins are misfolded in mitochondria with defective Pink1 or Parkin.
Comparatively, little is known about the consequences of protein misfolding in mitochondria in vivo as no genetic models are available to address this issue. To directly determine the significance of protein misfolding in this organelle, we generated a genetic model of mitochondrial protein misfolding in Drosophila using a mutant form of the mitochondrial matrix protein, ornithine transcarbamilase (OTC) that accumulates in an unfolded state and that was first used to characterise the UPR mt in mammalian cells. 8 Using the UAS/GAL4 9 system, we generated transgenic flies expressing the correctly folded version of this protein (OTC) as a control or the unfolded form (dOTC). After confirming that    Protein conformational stress in mitochondria has been shown to lead to activation of the UPR mt characterised by the induction of the mitochondrial chaperones hsp-60 and hsp-6 in the nematode Caenorhabditis elegans. 10 We observed a significant upregulation of the mRNA levels for hsp-60 as well as hsc-70-5, the Drosophila orthologue of C. elegans hsp-6, in flies where dOTC was expressed ubiquitously using the da-GAL4 driver; as well as in pink1 and parkin mutant animals ( Figure 1d). This was accompanied by a significant induction of the protein levels of mitochondrial HSP-60 in dOTC, pink1 and parkin mutants but not for the cytosolic HSP-70 (Figure 1e), supporting the view that accumulation of unfolded proteins in the mitochondrial compartment and loss of pink1 or parkin results in the selective upregulation of target genes encoding mitochondrial stress proteins in flies. We have also detected an upregulation of HSP-60 in flies treated with Paraquat, a pesticide linked to PD by epidemiological studies, known to result in high levels of reactive oxygen species (ROS) and consistent with its damaging effect on mitochondrial proteins (Supplementary Figure S1C).
We then crossed dOTC flies with genetic reporters of both the cytosolic ubiquitin-proteasome system (UPS) and the endoplasmic reticulum unfolded-protein response (UPR ER ). These experiments failed to reveal any impairment of the UPS or activation of the UPR ER in flies expressing dOTC (Supplementary Figure S2), further confirming the specificity of UPR mt activation by protein conformational stress in Drosophila.
We next assayed mitochondrial activity in flies expressing dOTC and controls by measuring state III respiration, and detected a significant decrease in oxygen consumption Strikingly, this mitochondrial loss was also observed in pink1 mutants. 11 Taken together, these results suggest that excessive protein misfolding leads to compromised mitochondrial function and a loss of mitochondrial mass.
Accumulation of misfolded proteins in mitochondria phenocopies pink1 and parkin mutant flies. As we detected UPR mt activation in flies ubiquitously expressing dOTC and in parkin and pink1 mutant flies (Figure 1d), we decided to further investigate the phenotypic consequences of dOTC expression. We began to observe an abnormal (held up) wing posture in 10-15% of the flies expressing dOTC at 3 days after eclosion (Figures 3a and b); therefore, we choose to analyse the indirect flight muscles of these animals. Expression of dOTC resulted in the formation of mitochondrial aggregates in the indirect flight muscles (Figures 3c and d), and ultrastructural analysis showed a highly disorganised muscle structure, vacuolisation and fragmentation of mitochondrial cristae (Figures 3e-h). Flies expressing dOTC also displayed an age-dependent impaired climbing ability, suggesting a locomotor deficit ( Figure 3i) and reduced overall survival ( Figure 3j). We were able to detect a significant impairment in mitochondrial function in dOTCexpressing flies prior to any overt pathological changes, suggesting that a compromise in mitochondrial function lies at the apex of the detected pathologies. Interestingly, the phenotypes observed in this fly model of the UPR mt were similar to those of parkin and pink1 mutants, [11][12][13][14] supporting  Mitochondrial dysfunction induced by protein conformational stress leads to mitochondrial fragmentation and the induction of autophagy. Recently, fission has been shown to mediate the clearance of damaged mitochondria by autophagy, 15 a recycling programme for impaired and defective cellular components. We confirmed that OTC and dOTC were correctly targeted to mitochondria, by co-expressing each protein with a mitochondrial-targeted GFP reporter in larval tissues using the en-GAL4 driver.
Interestingly, we noted a significant degree of fragmentation of the mitochondrial network in the larval wing disc expressing dOTC (Figures 4a-c), suggesting an imbalance towards fission, and therefore we decided to test whether autophagy was being activated in these transgenic animals.
In order to study autophagy, we made use of a germline driver nosGAL4 to express GFP-LC3 in larvae. By western blot analysis in adult flies, we showed that dOTC-induced mitochondrial dysfunction was accompanied by accumulation of the autophagy marker LC3-II (Figure 4d).  (Figure 4h). To test whether AMPK is a key mediator of dOTC-induced autophagic activation, we downregulated the expression of this kinase using RNA interference (Figure 4i). Upon partial loss of AMPK and of its kinase activity, dOTC expression failed to trigger the accumulation of the autophagic marker, LC3-II (Figure 4j). This suggests that AMPK can have a role in the activation of autophagy as a protective mechanism that ensures both the removal of defective mitochondria as well as some recycling of macromolecules required for anabolic processes.
Parkin-mediated enhancement of autophagy suppressed mitochondrial dysfunction induced by protein conformational stress. Recently, it has been proposed that Pink1 and Parkin might be involved in a common pathway controlling mitochondrial morphology, function and degradation. Several groups have reported that loss of pink1 or parkin in Drosophila leads to very similar mitochondrial phenotypes, and although pink1 is unable to rescue the defects seen in parkin mutants, overexpression of parkin rescues the mitochondrial pathology seen in pink1 mutants, indicating that pink1 acts genetically upstream of parkin. 11,12 In mammalian cells, following recruitment by PINK1, Parkin can translocate to damaged mitochondria upon loss of the mitochondrial membrane potential (Dcm) and act to mediate their removal via a mechanism involving autophagy. 5,6 Thus, we decided to determine the effects of enhanced parkin expression in flies expressing dOTC. We first observed that the climbing defects and abnormal mitochondrial morphology observed in these flies were markedly suppressed by parkin expression (Figure 5a and Supplementary Figures  S4A-C, respectively). Ultrastructural analysis of the indirect flight muscles also revealed a marked recovery of tissue structure and mitochondrial integrity (Figures 5d and e), and moreover, respiration defects and ATP levels were partially rescued by parkin expression (Supplementary Figures  S4D-F), suggesting that Parkin can compensate for protein conformational stress in mitochondria. We proceeded to analyse autophagy by comparing the levels of LC3-II in these transgenic flies. Parkin expression was sufficient to induce autophagy to a degree similar to that observed in control flies in which autophagy was induced by starvation ( Figure 5b). Interestingly, co-expression of dOTC and Parkin led to an increased accumulation of LC3-II, suggesting that Parkin can enhance autophagy upon mitochondrial stress as a protective  (Figure 5b). To confirm the requirement of autophagy in this Parkin-mediated protective effect, we tested the epistatic effects of a mutation in the autophagy-regulating kinase, atg1. Parkin suppression of the dOTC-induced climbing defects, tissue structure and mitochondrial integrity were absent in atg1 mutants (Figures 5c, f and g), suggesting that autophagy is required for Parkin-mediated rescue of flies expressing dOTC.
Drosophila ref (2)P is required for parkin-mediated suppression of mitochondrial dysfunction induced by protein conformational stress. Parkin encodes a multifunctional E3 ubiquitin ligase that mediates its own ubiquitination and that of other target proteins. 16 Mitochondrial translocation of Parkin involves the formation of polyubiquitin chains on specific mitochondrial proteins such as VDAC1 and Mitofusin, and this process seems to be involved in Parkin-dependent mitophagy. 17,18 The mammalian protein p62/SQSTM1 is an autophagy receptor that can interact with ubiquitin conjugated to a target and LC3/GABARAP on the autophagosome (reviewed in Kirkin et al. 19 ). It has recently been reported that p62/ SQSTM1 is critical for PINK1/Parkin-mediated mitophagy. 17 Thus, we determined whether p62/SQSTM1 could act as an adaptor protein in the final step of the mitochondrial autophagy. Co-immunoprecipitation experiments in mammalian cells revealed a strong interaction between p62 and Parkin ( Figure 6a). Interestingly, removal of the ubiquitin-like domain (UBL) from Parkin had no effect on the formation of Parkin-p62 complexes.
As Parkin and p62 seemed to physically interact, we next tested whether the single Drosophila p62 orthologue, ref(2)P, CG10360, could act genetically downstream of parkin to promote the autophagic clearance of damaged mitochondria. Ref(2)P contains three domains involved in protein-protein interaction: a PB1 domain involved in multimerisation, a ZZ zinc finger and a ubiquitin-associated domain (UBA). Drosophila ref(2)P od2 mutants lack the PB1 domain whereas ref(2)P od3 mutants lack the UBA domain. 20,21 Both mutants are viable but completely male sterile. 21 Interestingly, dysfunction in both the Pink1 and Parkin pathways is linked to male sterility because of defective mitochondria. 12,13 Analysis of the indirect flight muscles in ref(2)P od2 and ref(2)P od3 mutants revealed vacuolisation of mitochondrial cristae in both mutants (Figure 6b), suggesting that defects in ref (2)P are linked to mitochondrial dysfunction.
To determine the role of ref(2)P in the clearance of damaged mitochondria, we tested its requirement in parkin-mediated suppression of the phenotypes observed in dOTC-expressing flies. Parkin was unable to suppress the climbing deficits observed in dOTC flies carrying ref(2)P mutations (Figure 6c). Additionally, Parkin also failed to suppress the mitochondrial loss, addressed through the analysis of the levels of mitochondrial complex I, caused by dOTC in ref (2)

Discussion
Late onset neurodegenerative diseases like PD are associated with the formation of toxic protein aggregates. It has been proposed that in amyotrophic lateral sclerosis (ALS), the toxicity of ALS-linked SOD1 mutations results from the selective accumulation and aggregation of SOD1 mutant protein in spinal cord mitochondria, 22,23 indicating that mitochondrial protein misfolding may contribute to neurodegeneration in humans. Here, we have performed an in vivo characterisation of a mitochondrial stress-responsive pathway likely to respond to protein misfolding in this organelle.
We have determined that defects in Pink1 and Parkin are associated with protein misfolding of components of the respiratory complexes in mitochondria. By generating and characterising an in vivo model of mitochondrial protein misfolding, we obtained evidence that this defect is closely linked to the phenotypic changes observed in the PD-related Drosophila mutants, pink1 and parkin. Importantly, our data suggests that ref(2)P has a critical role in mediating autophagy of defective mitochondria, suggesting that this protein might be a critical effector in an organellar quality control pathway that acts when molecular quality control mechanisms in mitochondria are overwhelmed by excessive protein misfolding. PD is characterised by a selective loss of dopaminergic neurons in the substantia nigra. We targeted the expression of dOTC to dopaminergic neurons and failed to detect any loss of dopaminergic neurons in dOTC-expressing flies (Supplementary Figure S1F). It is noteworthy that there are conflicting reports concerning the role of Drosophila pink1 in dopaminergic neurodegeneration. 11,12 It is therefore difficult to conclude whether dopaminergic neurons in flies show enhanced sensitivity to mitochondrial dysfunction as a consequence of protein misfolding. Comparatively, little is known about the contribution of protein aggregation in the mitochondria to age-related neurodegenerative diseases such as PD. Interestingly, it has recently been shown that in the multicellular organism Caenorhabditis elegans, widespread protein insolubility and aggregation are inherent parts of aging and are likely to influence total lifespan and the onset of neurodegenerative diseases. 24 Our data is compatible with a model where UPR mt activation might be an early marker of mitochondrial dysfunction in neurodegenerative diseases. As mitochondrial dysfunction was reported to be linked to other neurodegenerative disorders such as Alzheimer's disease and Huntington's disease (reviewed in de Castro et al. 2 ), it would be important to determine whether the activation of the UPR mt is also  associated with these conditions. Nevertheless, given the technical challenges with obtaining robust data on transcriptional changes derived from the analysis of post-mortem human brains, it is likely that model organisms like Drosophila where these diseases can be initially modelled, could provide important clues regarding the link between UPR mt activation and neurodegenerative diseases in general. AMPK is a major sensor of the cellular AMP:ATP ratio and is activated by metabolic stresses that inhibit ATP production (reviewed in Hardie 25 ). We observed an enhanced phosphorylation of both the energy-sensing kinase AMPK and its downstream target acetyl-CoA carboxylase (ACC) upon dOTC expression in flies. It is tempting to propose that, upon mitochondrial dysfunction caused by protein misfolding, AMPK senses a loss of ATP and has a protective role by activating autophagy and ensuring both the removal of defective mitochondria as well as some recycling of macromolecules required for anabolic processes. It is therefore plausible that enhancing AMPK activity might have a positive role to promote efficient mitochondrial quality control.
It is interesting that parkin expression led to an almost complete recovery of mitochondrial integrity in flies expressing dOTC (Supplementary Figures S4A-E). We reason that besides clearing defective mitochondria through autophagy, Parkin can possibly promote the replacement of defective mitochondria with viable organelles. Mammalian PARKIN has been recently shown to indirectly regulate the function of PGC-1a, a transcriptional co-activator of genes involved in mitochondrial biogenesis. 26 This regulation involves the ubiquitination of the PGC-1a transcriptional repressor PARIS (ZNF746). It is attractive to propose that in Drosophila, the recovery of viable mitochondria promoted by Parkin involves not only the efficient clearance of defective organelles through autophagy but also an enhancement of PGC-1a activity through the regulation of a putative Drosophila orthologue of PARIS.
Our data suggest that a pathway involving pink1, parkin and ref(2)P has an important role in the maintenance of a viable pool of mitochondria, possibly by ensuring that mitochondria containing excessive levels of unfolded proteins are selectively recycled via autophagy. In humans, mitochondria are the major suppliers of energy to the brain. Neurons have low glycolytic rates, 27 and therefore rely heavily on mitochondria for energy production. Mitochondria are also capable of buffering cytoplasmic calcium, and thus have a role in the regulation of intracellular calcium dynamics. It is therefore possible that defects in mitochondrial quality control have major implications for both neuronal survival and efficient synaptic transmission.
Although highly speculative, it is tempting to suggest that enhancing pathways that promote autophagy in humans might delay age-related diseases by promoting a healthy pool of viable mitochondria in neuronal cells, sustaining energy demands and efficiently buffering intracellular calcium levels.