InteractiveFly: GeneBrief

PTEN-induced putative kinase 1 : Biological Overview | References

BIOLOGICAL OVERVIEW

Mutations in PTEN-induced kinase 1 (PINK1), a mitochondrial Ser/Thr kinase, cause an autosomal recessive form of Parkinson's disease (see Drosophila as a Model for Human Diseases: Parkinson's disease), PARK6. To investigate the mechanism of PINK1 pathogenesis, the Drosophila Pink1 knockout (KO) model was used. In mitochondria isolated from Pink1-KO flies, mitochondrial respiration driven by the electron transport chain (ETC) is significantly reduced. This reduction is the result of a decrease in ETC complex I and IV enzymatic activity. As a consequence, Pink1-KO flies also display a reduced mitochondrial ATP synthesis. Because mitochondrial dynamics is important for mitochondrial function and Pink1-KO flies have defects in mitochondrial fission, whether fission machinery deficits underlie the bioenergetic defect in Pink1- KO flies was investigated. It was found that the bioenergetic defects in the Pink1-KO can be ameliorated by expression of Drp1, a key molecule in mitochondrial fission. Further investigation of the ETC complex integrity in wild type, Pink1-KO, PInk1-KO/Drp1 transgenic, or Drp1 transgenic flies indicates that the reduced ETC complex activity is likely derived from a defect in the ETC complex assembly, which can be partially rescued by increasing mitochondrial fission. Taken together, these results suggest a unique pathogenic mechanism of PINK1 PD: The loss of PINK1 impairs mitochondrial fission, which causes defective assembly of the ETC complexes, leading to abnormal bioenergetics (Liu, 2011).

Ample evidence indicates that mitochondrial dysfunction plays a pivotal role in the development of Parkinson's disease (PD). A 30%-40% reduction of mitochondrial electron transport chain (ETC) complex I activity has been observed in the postmortem brains of idiopathic PD patients. 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) and rotenone, inhibitors of ETC complex I, induce clinical and pathological manifestations that recapitulate cardinal PD symptoms in humans and in animal models, supporting the hypothesis that mitochondrial bioenergetic defects contribute to PD pathogenesis (Liu, 2011).

The significance of mitochondrial dysfunction in the development of PD was further strengthened by the discovery of PINK1 as the causal gene of PARK6. PINK1 encodes a mitochondrial kinase (Valente, 2004), but its physiological role remains to be elucidated. Reduction or loss of PINK1 causes bioenergetic deficits that include loss of membrane potential, calcium buffering, ATP synthesis rate, and respiration in cell culture systems (Wood-Kaczmar, 2008; Liu, 2009; Gegg, 2009; Sandebring, 2009). In Drosophila and mouse PINK1-KOs, decreased ETC complex I mediated respiration and ATP content have been reported (Gautier, 2008; Gispert, 2009; Morais, 2009). ETC complex assembly depends on inner mitochondrial membrane integrity, which is maintained by fusion and fission processes. Fusion and fission regulate the number, size, and morphology of mitochondria in a dynamic manner, and perturbing these processes could affect membrane stability (Chan, 2006). Several key molecules that regulate these delicate processes have been identified: Dynamin-like GTPase (Drp1) is a key molecule in fission, whereas mitofusin (Mfn) and optic atrophy 1 (Opa1) play a major role in fusion. Mutations in Mfn 2 and Opa1 result in Charcot-Marie-Tooth neuropathy and autosomal dominant optical atrophy, respectively (Liu, 2011 and references therein).

Pink1-KO flies have deficits in mitochondrial fission and abnormal mitochondrial morphology, which can be alleviated by one additional copy of Drp1 (Yang, 2008; Poole, 2008; Deng; 2008), indicating that the fission machinery contributes to the mitochondrial pathology in flies. This study reports that the fission machinery defect contributes to bioenergetic deficits in the Pink1-KO flies by impairing ETC complex assembly. This finding raised the possibility that these biochemical and mitochondrial dysfunctions in Pink1 flies are also the basis of human PD patient pathogenesis. It is therefore important to validate these in human patients (Liu, 2011).

This paper describes two unique PINK1 pathogenic mechanisms: (1) Loss of PINK1 causes a defective assembly of the oxidative phosphorylation (OXPHOS) complexes, which leads to impaired mitochondrial OXPHOS. (2) Enhancing mitochondrial fission ameliorates OXPHOS machinery assembly and mitochondrial functional defects (Liu, 2011).

Loss of Pink1 leads to impaired ETC complex IV function, and previous observations were confirmed that Pink1-KOs also have ETC complex I defects (Liu, 2009; Gispert, 2009; Morais, 2009). These data provide strong support to the notion that mitochondrial OXPHOS defects are integral parts of PD pathogenesis. It is important to note that deficits in ETC complex I were reported in human PD patients, as well as in pharmacological mouse models of PD, and deficits in ETC complex IV were found in an α-synuclein transgenic mouse model of PD. Most importantly, the current findings reveal that defective OXPHOS complex assembly underlies the impaired OXPHOS function. It will be interesting to investigate whether this mechanism is also true in human patients with PINK1 mutations (Liu, 2011).

This study further revealed that OXPHOS complex assembly and function is modulated by mitochondrial dynamics and involves the fission machinery. In this case, Drp1 partially rescued the deficits of OXPHOS assembly and function. It is possible that Drp1 promotes mitochondrial fission, which contributes to 'regenerating' functional competent mitochondria through redistribution of mitochondrial DNA, RNA, and proteins. OXPHOS complex assembly could be improved by a 'rejuvenated' population of mitochondria in the Pink1-KO/Drp1. Alternatively, Drp1 could achieve its beneficial effect by promoting the clearance of damaged mitochondria through processes such as autophagy and mitophagy. This possibility is supported by recent evidence that Pink1 was implicated in the clearance of damaged mitochondria (Dagda, 2009; Narendra; 2010; Geisler, 2010; Michiorri, 2010; Vives-Bauza, 2010). It will be interesting to investigate how PINK1 affects mitophagy in vivo, and how mitochondrial dynamics influences mitophagy. Based on the current data, a working hypothesis is proposed involving a bifurcated pathway for PINK1 pathogenesis as follows: the direct path may involve reduced or lack of phosphorylation of OXPHOS complex subunits by PINK1, which impairs OXHOS complex assembly and function. The indirect path may involve Drp1 and other intermediates that are important for mitochondrial membrane stability and dynamics, thereby affecting OXPHOS complex assembly and function. With the assays and system this study established, these pathways can now be dissected in greater molecular details, especially with the power of Drosophila genetics. Furthermore, if these findings are validated in human PD patients, the knowledge of this aspect of pathogenesis is important for designing therapeutic strategies (Liu, 2011).

PINK1-mediated phosphorylation of Miro inhibits synaptic growth and protects dopaminergic neurons. in Drosophila

Mutations in the mitochondrial Ser/Thr kinase PINK1 cause Parkinson's disease. One of the substrates of PINK1 is the outer mitochondrial membrane protein Miro, which regulates mitochondrial transport. This study uncovered novel physiological functions of PINK1-mediated phosphorylation of Miro, using Drosophila as a model. Endogenous Drosophila Miro (DMiro) was replaced with transgenically expressed wildtype, or mutant DMiro predicted to resist PINK1-mediated phosphorylation. The expression of phospho-resistant DMiro in a DMiro null mutant background was found to phenocopy a subset of phenotypes of PINK1 null. Specifically, phospho-resistant DMiro increased mitochondrial movement and synaptic growth at larval neuromuscular junctions, and decreased the number of dopaminergic neurons in adult brains. Therefore, PINK1 may inhibit synaptic growth and protect dopaminergic neurons by phosphorylating DMiro. Furthermore, muscle degeneration, swollen mitochondria and locomotor defects found in PINK1 null flies were not observed in phospho-resistant DMiro flies. Thus, this study established an in vivo platform to define functional consequences of PINK1-mediated phosphorylation of its substrates (Tsai, 2014).

Mutations in the Ser/Thr kinase PINK1 (PTEN-induced Putative Kinase 1) cause Parkinson's disease (PD), one of the most common neurodegenerative disorders. Emerging evidence suggests that PINK1 functions upstream of another PD-associated protein, the E3 ubiquitin ligase Parkin, to clear damaged mitochondria via mitophagy. How PINK1 primes cytosolic Parkin for mitophagy remains unclear, although PINK1-mediated phosphorylation of Parkin or ubiquitin may be involved. Previous work has shown that PINK1, in cooperation with Parkin, also regulates mitochondrial trafficking by controlling turn-over of Miro (Wang, 2011), an outer mitochondrial membrane (OMM) protein that anchors the kinesin and dynein motors to mitochondria. Work in cultured cells has demonstrated that mitochondrial depolarization or damage stabilizes PINK1 on the OMM. Concomitantly, PINK1 phosphorylates Miro, which then activates proteasomal degradation of Miro in a Parkin-dependent manner and arrests mitochondrial transport. This may serve as a critical step in quarantining damaged mitochondria prior to their degradation via mitophagy. However, the physiological significance of PINK1-mediated phosphorylation of Miro in vivo has not yet been determined (Tsai, 2014).

Recent studies have shown that Mitofusin, another OMM protein, is also a common substrate for both PINK1 and Parkin. Mitofusin facilitates mitochondrial fusion, and mitochondrial damage rapidly degrades Mitofusion causing mitochondria to fragment prior to mitophagy. PINK1 also phosphorylates the anti-apoptotic protein Bcl-xL on the OMM of depolarized mitochondria, not to regulate mitophagy, but to prevent cell death. In addition to the PINK1 OMM substrates Miro, Mitofusin and Bcl-xL, PINK1 mediates phosphorylation of the mitochondrial chaperon TRAP1 and the Serine protease HtrA2, which are both located in the mitochondrial inter-membrane space. This wide range of the potential substrates of PINK1 suggests that it may have multiple cellular functions (Tsai, 2014).

The consensus target sequence for phosphorylation by PINK1 has remained elusive. To date, Miro is the only PINK1 mitochondrial substrate whose phosphorylation residues have been determined (Wang, 2011). Two Drosophila Miro (DMiro) peptides with a high degree of similarity to the human sequence were identified as potential targets of PINK1-mediated phosphorylation in vitro. This study has determined the critical role of PINK1 phosphorylation sites on DMiro for maintaining neuronal homeostasis and protecting dopaminergic (DA) neurons in vivo (Tsai, 2014). <>Although several phosphorylation substrates of the mitochondrial Ser/Thr kinase PINK1 have been identified, the precise functional consequences of PINK1-mediated phosphorylation in vivo remain unclear. This study report that in Drosophila PINK1 may inhibit mitochondrial movement and synaptic growth at larval NMJs, and protect DA neurons in adult brains, by phosphorylating the atypical GTPase DMiro (Tsai, 2014).

Drosophila is a robust genetic and cellular tool for modeling human neurodegenerative diseases. Loss of PINK1 in Drosophila mimics many aspects of PD pathology, including a severe loss of DA neurons, which is a hallmark of PD. However, few of the molecular and cellular mechanisms underlying the behavioral and cellular phenotypes of PINK1 null mutant flies have been clearly defined. This study identifies that DMiro^{S182A,S324A,T325A}, which is predicted to resist PINK1-mediated phosphorylation, causes increased mitochondrial movement, synaptic overgrowth, and loss of DA neurons. All three of these defects are also observed in PINK1 null mutant flies. Hence, this work suggests that Miro is a crucial substrate for causing these phenotypes by mutant PINK1. This study opens a new door to fully dissect PINK1 functions by studying its individual substrates. Since PINK1-related hereditary PD shares symptomatic and pathological similarities with the majority of idiopathic PD, such work will advance understanding of the cellular and molecular underpinnings of PD's destructive path (Tsai, 2014).

Extensive studies using cell cultures have established a critical role for PINK1 in damage-induced mitophagy. PINK1/Parkin-dependent regulation of mitochondrial transport by controlling Miro protein levels on mitochondria is likely a key step prior to initiating mitophagy in cultured neurons. This study show that PINK1-mediated phosphorylation of DMiro is required for normal mitochondrial movement in axon terminals, synaptic growth, and the neuroprotection of DA neurons. Importantly, loss of PINK1-mediated phosphorylation of DMiro has no significant effect on the mitochondrial membrane potential, excluding the possibility that the observed phenotypic effects are due to an impairment of mitophagy and an accumulation of damaged mitochondria. Accordingly, under these conditions PINK1-mediated phosphorylation of DMiro may not be required for mitophagy. However, this does not necessarily contradict its mitophagic role; rather, this represents circumstances under which its mitophagic role is dispensable. It is tempting to speculate that an efficient regulation of mitophagy is more critical in aging neurons (Tsai, 2014).

These studies identify a conserved site in human and Drosophila Miro, MiroSer156/DMiroSer182, to be a main residue for PINK1-mediated phosphorylation. Additional conserved sites were found in DMiro that may have a cooperative role. Future studies determining their functions in mammalian systems are warranted to confirm if a similar regulatory mechanism is at play. This study suggests that these PINK1 phosphorylation sites in DMiro are not absolutely required for the subsequent Parkin-dependent degradation of DMiro, because when harsh treatment of mitochondrial uncoupler CCCP is applied, the phospho-resistant DMiro^{S182A,S324A,T325A} is degraded. The failure of DMiro^{S182A,S324A,T325A} to prevent degradation under this condition might be due to PINK1-mediated phosphorylation on other sites that promote DMiro degradation, or due to activation of additional mechanisms. In two recent studies, Miro^S156A is significantly degraded by co-expression of PINK1 and Parkin in addition to CCCP treatment in Hela cells, or by overexpression of Parkin together with Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, another mitochondrial uncoupler) treatment in SH-SY5Y cells; whereas in a previous study, Miro^S156A is resistant to degradation when only PINK1 or Parkin is individually expressed in HEK293T cells (Wang, 2011). This again suggests that if the PINK1/Parkin pathway is overwhelmingly activated, mutating the few known PINK1-mediated phosphorylation residues in Miro is not sufficient to prevent its degradation (Tsai, 2014).

Why is mitochondrial motility increased in "DMiro^null, da > DMiro^{S182A,S324A,T325A}"? DMiro^{S182A,S324A,T325A} is resistant to PINK1/Parkin-mediated degradation, which may lead to more DMiro^{S182A,S324A,T325A} accumulation on mitochondria. Unexpectedly, DMiro^{S182A,S324A,T325A} protein level in "DMiro^null, da > DMiro^{S182A,S324A,T325A}" is not significantly upregulated as compared with DMiro^wildtype in "DMiro^null, da > DMiro^wildtype" using fly whole body lysates. It is likely that PINK1/Parkin-dependent degradation of Miro only occurs in certain cell types, at certain subcellular locations, on certain populations of mitochondria, or under certain circumstances, and thus it is hard to detect a dramatic change using whole body lysates or without overexpression of PINK1/Parkin. Future mechanistic study is needed to test these hypotheses, such as detecting Miro subcellular localization and expression levels in different cell types, in different developmental stages, and with different mitochondrial stresses (Tsai, 2014).

This work highlights the importance of a precise control of mitochondrial movement for neuronal health. Anterograde mitochondrial transport in axons is mediated by a conserved motor/adaptor complex, which includes the motor kinesin heavy chain (KHC), the adaptor protein Milton and the mitochondrial membrane anchor Miro. In the current model, Miro binds to Milton, which in turn binds to KHC recruiting mitochondria to the motors and microtubules. In addition to the transmembrane domain inserted into the OMM, Miro features a pair of EF-hands and two GTPase domains. Miro was also recently found to be a substrate of the Ser/Thr kinase PINK1 and of the E3 ubiquitin ligase Parkin, both mutated in PD. Thus, mitochondrial transport can be regulated by multiple signals upstream of Miro and the motor complex maintaining energy and Ca²⁺ homeostasis in neuronal processes and terminals. For example, loss of PINK1-mediated phosphorylation of DMiro increases local mitochondrial movement at NMJs. In turn, this may disrupt synaptic homeostasis leading to synaptic overgrowth by mechanisms yet to be identified. Similarly, the loss of DA neurons in adult brains could well be a consequence of impaired synaptic homeostasis together with an accumulation of dysfunctional mitochondria. Local signals that regulate mitochondrial transport through Miro must be crucial to supporting neuronal functions. This study elucidates a fundamental biological mechanism demanded by a healthy neuron (Tsai, 2014).

PINK1 and Parkin regulate IP(3)R-mediated ER calcium release

Although defects in intracellular calcium homeostasis are known to play a role in the pathogenesis of Parkinson's disease (PD), the underlying molecular mechanisms remain unclear. This study shows that loss of PTEN-induced kinase 1 (PINK1) and Parkin leads to dysregulation of inositol 1,4,5-trisphosphate receptor (IP(3)R) activity, robustly increasing ER calcium release. In addition, CDGSH iron sulfur domain 1 (CISD1, also known as mitoNEET) functions were identifed downstream of Parkin to directly control IP(3)R. Both genetic and pharmacologic suppression of CISD1 and its Drosophila homolog CISD (also known as Dosmit) restore the increased ER calcium release in PINK1 and Parkin null mammalian cells and flies, respectively, demonstrating the evolutionarily conserved regulatory mechanism of intracellular calcium homeostasis by the PINK1-Parkin pathway. More importantly, suppression of CISD in PINK1 and Parkin null flies rescues PD-related phenotypes including defective locomotor activity and dopaminergic neuronal degeneration. Based on these data, it is proposed that the regulation of ER calcium release by PINK1 and Parkin through CISD1 and IP(3)R is a feasible target for treating PD pathogenesis (Ham, 2023).

This study provides a new insight into the mechanistic connection between the dysregulation of intracellular calcium homeostasis and PD pathogenesis induced by PINK1 or Parkin deficiency. PINK1 or Parkin KO mammalian cells exhibit increased IP3R activity, leading to increased ER calcium release and cytosolic calcium levels. CISD1, a substrate of Parkin, directly controls IP3R activity and ER calcium release, indicating that PINK1, Parkin, CISD1, and IP3R all function in the same essential pathway that regulates ER and cytosolic calcium homeostasis. Loss of CISD or treatment with the CISD inhibitor pioglitazone restores the elevated ER calcium release in PINK1 and Parkin null flies and fully rescues their PD-related phenotypes. Taken together, the increased IP3R activity and ER calcium release caused by PINK1 and Parkin deficiency are key to PD pathogenesis, all of which can be rescued by suppression of CISD1 activity (Ham, 2023).

In humans, there are three isoforms of CISD, CISD1, CISD2 (also known as ERIS, Miner1, NAF-1, WFS2, and ZCD2), and CISD3 (also called as MiNT). Among the three isoforms, CISD1 and CISD2 contain a single CDGSH domain and a transmembrane domain that facilitates their anchoring to the outer membrane of mitochondria and the ER, respectively. These two isoforms form homodimers within their respective organelles. CISD3 functions as a monomer and contains two CDGSH domains. CISD3 localizes specifically to the mitochondrial matrix. However, no isoforms exist in Drosophila CISD and this single protein shows sequence similarity with both human CISD1 and CISD2. Isoform CISD1 was selected for the experiment, as CISD1 is a much better substrate for Parkin E3 ligase compared to CISD2. Furthermore, it is well known that Parkin localizes to the mitochondria upon its activation and subsequently, ubiquitinates mitochondrial protein substrates. When subcellular localization of human CISD1/2 and Drosophila CISD proteins were observed, human CISD1 and Drosophila CISD were localized in the mitochondria; however, human CISD2 was localized in the ER. Considering these points, human CISD1 was selected as the mammalian counterpart of Drosophila CISD and the experiments were performed accordingly. Interestingly, it has been demonstrated previously that CISD2 is required for BCL2 to suppress IP3R activity. Thus, this study on the regulatory mechanism of IP3R activity through CISD1 is distinct from the earlier study on the regulation of IP3R activity by CISD2. Despite the structural and functional similarities between CISD1 and CISD2, the two proteins have distinct subcellular localizations and are involved in roles independent of one another, due to their interactions with different proteins. Overall, the results of the prior and current studies suggest that both CISD1 and CISD2 are modulators of IP3R activity, but they do so via their unique mechanisms that are distinctive of each other. (Ham, 2023).

Previous studies reported that CISD1 is involved in iron homeostasis and the downregulation of CISD1 causes iron accumulation and ROS production in mitochondria. In light of these effects, ROS levels were measured in PINK1 and Parkin WT or KO mammalian cells and Drosophila, and an increase was confirmed in ROS levels in PINK1 and Parkin KO MEF cells and Drosophila. An increase was also observed in ROS levels in CISD KD and KO flies, compared to control flies. Interestingly, CISD1/CISD KD or KO in PINK1 and Parkin KO cells and Drosophila resulted in similar ROS levels compared to PINK1 and Parkin KO cells and Drosophila. Furthermore, the increased ROS levels in PINK1 or Parkin KO cells and Drosophila were not restored when CISD1/CISD was knocked down or deleted. These results implicated that the rise in ROS levels induced by loss of CISD1/CISD is not directly involved in the rescue of PD phenotypes, which was observed in CISD1/CISD loss-of-function experiments (Ham, 2023).

The Fe-S binding capability of CISD1 may play a role in its interactions with IP3R. CISD1 has been reported to interact with several proteins, including CISD2, VDAC1, and transferrin receptor (TfR). CISD249, VDAC115,50, and TfR51 proteins have been shown to interact with each other, and this interaction has been implicated in the regulation of iron homeostasis, redox signaling, and Fe-S cluster synthesis in the mitochondria. However, whether the Fe-S binding motif of CISD1 plays an essential role in protein-protein interactions is unclear. Whether the functions of CISD1 related to Fe-S binding are important to regulate IP3R activity were tested, and it was identified that the cysteine 74 residue in the Fe-S binding motif (in the CDGSH domain) of CISD1 is critical for the interaction with IP3R1. However, it was also confirmed that the Fe-S binding motif of CISD1 binds with IP3R despite C72A substitution and CDGSH pentapeptide deletion mutations. Thus, it was postulate that the structural change in the Fe-S binding motif of CISD1 does not affect the binding between CISD1 and IP3R and that pioglitazone reduces the binding of CISD1 with IP3R regardless of the stability of Fe-S binding. Altogether, the Fe-S binding ability of CISD1 is not directly related to regulating IP3R activity (Ham, 2023).

Flies with either CISD RNAi or CISD KO exhibited lower ER calcium release and cytosolic calcium levels compared to mef2-GAL4 control flies. Upon crossing with CISD RNAi or CISD KO flies, PINK1 or Parkin null flies displayed a greater reduction in ER calcium release and cytosolic calcium levels than mef2-GAL4 control flies. This observation can be explained by the varying amounts of endogenous CISD in the flies. Notably, CISD RNAi flies exhibited significantly lower endogenous CISD amounts compared to the control flies, while PINK1 or Parkin KO flies presented higher levels. The elevated amount of endogenous CISD protein in PINK1 or Parkin KO flies contributes to the increased ER calcium release observed in these flies, while the reduced endogenous CISD protein in CISD RNAi or CISD KO flies results in a more significant decrease in ER calcium release compared to the control flies. Moreover, flies resulting from the crossing of PINK1/Parkin KO with CISD RNAi/CISD KO demonstrated lower endogenous CISD levels compared to the control flies, leading to a larger reduction in ER calcium release or cytosolic calcium levels. Collectively, these findings proposed that ER calcium release and cytosolic calcium levels are modulated proportionally to the amount of endogenous CISD protein present (Ham, 2023).

Defects in ER calcium homeostasis can also have profound effects on other organelles through physical contact sites, including the ER-mitochondria interconnections known as Mitochondria-associated membranes (MAMs). MAMs are enriched with the MCU complex in the inner mitochondrial membrane and IP3R on the ER membrane. MCU and IP3R are coupled via the glucose-regulated protein 75 (Grp75), which links IP3R to the VDAC1 on the outer mitochondrial membrane, establishing connections that allow calcium exchange between the ER and mitochondria. Interestingly, previous studies show that inhibition of MCU or VDAC1 partially rescues the PD phenotypes of PINK1- and Parkin-deficient flies, suggesting that the disruption of MAMs may alleviate PD pathogenesis. Previous studies also report that the level of MAM contacts was increased in cultured human fibroblasts from PD patients carrying PINK1 or Parkin pathogenic mutations and PINK1 and Parkin null mutant flies60. In addition, our present results demonstrate that CISD1 directly binds to and regulates IP3R activity, and CISD1 is localized at MAMs and the mitochondrial outer membrane12. These data therefore suggest that CISD1 and the PINK1-Parkin pathway are crucial for the formation and maintenance of MAM structure and ER-mitochondrial calcium transduction, which in turn are critical for mitochondria-related physiology and pathologic phenotypes including calcium-dependent metabolic changes, ROS production, mitophagy, mitochondrial permeability transition, and apoptosis (Ham, 2023).

Through extensive studies, it is understood that loss of PINK1 or Parkin impairs mitophagy and that defective mitophagy is one of the potential contributing factors to the onset of PD. Furthermore, a recent study has shown increased mitophagy in thoraces and neurons of CISD KO or KD Drosophila, and the reduced mitophagy in PINK1 or Parkin KO flies was alleviated by crossing them with CISD KO or KD flies. In the current study, it was observed that loss of CISD1/CISD reduced the elevated cytosolic calcium levels observed in PINK1 or Parkin KO cells and Drosophila. Intracellular calcium signaling is an important factor in mitophagy regulation. Nix, also known as BCL2 interacting protein 3 like (BNIP3L), exhibits biological activity at both the mitochondria and the ER. At the mitochondria, Nix functions as a selective autophagy receptor, facilitating the recruitment of LC3B71. In muscle, during a mitophagy response, Nix promotes ER-dependent calcium signaling to activate the mitochondrial fission regulator dynamin-related protein 1 (DRP1), indicating the contribution of Nix to mitophagy. During hypoxia, mitochondrial Lon protein promotes FUNDC1-ULK1-mediated mitophagy at the MAMs, which depends on its binding with mitochondrial Na+/Ca2+ exchanger (NCLX). This interaction stabilizes the FUNDC1-ULK1 at the MAMs and initiates the mitophagy by regulating calcium levels between the mitochondria and cytosol. This process occurs independently of PINK1 and Parkin. Furthermore, other calcium-sensitive proteins and pathways may also contribute to PINK1-Parkin-independent mitophagy. For example, CaMKII-AMP-activated protein kinase (AMPK) pathway has been implicated in the regulation of mitophagy. Activation of AMPK by CaMKII can promote mitophagy by phosphorylating and activating proteins involved in autophagy initiation. This suggests the possibility that mitophagy could be activated by the decreased cytosolic calcium levels in CISD1/CISD KO or KD cells and Drosophila. Collectively, the regulation of mitophagy by CISD1/CISD holds the potential to alleviate PD pathogenesis caused by loss of PINK1 or Parkin. However, further investigation is required to unravel the molecular mechanism underlying mitophagy regulation by CISD1 and its interplay with intracellular calcium signaling (Ham, 2023).

Although degeneration of DA neurons is known to occur in PD, how such selective neurodegeneration occurs remains unknown. The current results show that DA neuronal loss and locomotor impairments in PINK1 and Parkin KO flies can be rescued by adjusting ER calcium release, suggesting that ER and mitochondrial calcium dysregulation may cause selective DA neuronal death. Intracellular calcium signaling in DA neurons is extremely fine-tuned as it controls many cellular processes including gene transcription, membrane excitability, dopamine neurotransmitter secretion, and synaptic plasticity. Furthermore, energy production in neurons is tightly regulated by ER and mitochondrial calcium. DA neurons promote mitochondria calcium influx from the ER to stimulate OXPHOS and the production of ATP. This bioenergetic control system is costly, as enhancing OXPHOS in the absence of strong ATP demand leads to mitochondrial hyperpolarization, retrograde electron flux through the electron transport chain, and increased production of ROS. Therefore, continuous dysregulation of calcium homeostasis in DA neurons along with exposure to risk factors (i.e., aging, mitochondrial toxins, mutations) may selectively induce metabolic stress and mitochondrial damages, leaving DA neurons more vulnerable than other neuronal populations to death (Ham, 2023).

While the importance of calcium regulation in PD pathogenesis has been recognized, previous trials of calcium-related drugs had failed to improve symptoms in PD patients. This study proposes that pioglitazone, a thiazolidinedione (TZD) and antidiabetic drug, can alleviate PD pathogenesis. Though previous clinical studies have reported mixed results on the effectiveness of pioglitazone against PD, the results clearly demonstrate that feeding pioglitazone to flies rescues PD-related phenotypes induced by PINK1 or Parkin deficiency. In addition, pioglitazone treatment reverses the increased ER calcium release and cytosolic calcium levels in PINK1 and Parkin KO MEF cells. This study thus established that pioglitazone can specifically protect PD pathogenesis caused by dysregulation of intracellular calcium homeostasis, calling for future clinical studies of pioglitazone and its analogs to be conducted specifically on PD patients that harbor PINK1 or Parkin mutations (Ham, 2023).

Roles of PINK1 in regulation of systemic growth inhibition induced by mutations of PTEN in Drosophila

The maintenance of mitochondrial homeostasis requires PTEN-induced kinase 1 (PINK1)-dependent mitophagy, and mutations in PINK1 are associated with Parkinson's disease (PD). PINK1 is also downregulated in tumor cells with PTEN mutations. However, there is limited information concerning the role of PINK1 in tissue growth and tumorigenesis. This study shows that the loss of pink1 caused multiple growth defects independent of its pathological target, Parkin. Moreover, knocking down pink1 in muscle cells induced hyperglycemia and limited systemic organismal growth by the induction of Imaginal morphogenesis protein-Late 2 (ImpL2). Similarly, disrupting PTEN activity in multiple tissues impaired systemic growth by reducing pink1 expression, resembling wasting-like syndrome in cancer patients. Furthermore, the re-expression of PINK1 fully rescued defects in carbohydrate metabolism and systemic growth induced by the tissue-specific pten mutations. These data suggest a function for PINK1 in regulating systemic growth in Drosophila and shed light on its role in wasting in the context of PTEN mutations (Han, 2021).

This study reports that PINK1 regulated systemic growth in a non-autonomous manner. The loss of PINK1 in muscle cells specifically upregulated the insulin/IGF signaling antagonist ImpL2, thus inhibiting organ growth by dysregulating carbohydrate metabolism and insulin signaling. Furthermore, strong genetic evidence is provided that PINK1 activity is dispensable for cell growth but absolutely required for the induction of tissue wasting following the knockdown of the tumor suppressor gene PTEN. This effect is mediated through systemic insulin signaling. In sharp contrast to wild-type cells, PTEN mutant cells retain high levels of membrane PIP3 independent of InR/PI3K, thus maintaining high cellular insulin signaling and a high rate of cell growth despite low circulatory insulin activities. Thus, mutations in PTEN enhance cell growth by maintaining high levels of insulin signaling, but they also inhibit the growth of other tissues in a non-autonomous manner through the transcriptional repression of PINK1 (Han, 2021).

Although induced by the tumor suppressor PTEN, PINK1 does not inhibit cell growth in a number of cancer cell lines. Instead, PINK1 has been shown to promote cell growth and proliferation in various models. This study demonstrated that in the fly fat body, wing, and eye, the loss of PINK1 impaired cell growth. This is the opposite of PTEN, which leads to overgrowth when mutated. Moreover, despite the fact that the expression of PINK1 is reduced by mutations in pten, ectopic expression of PINK1 does not affect the growth of either wild-type or pten mutant cells, indicating that PINK1 may not simply function as a regulator of cellular growth, particularly in pten-associated tumors. Interestingly, knocking down pten or pink1 in specific tissues reduces the insulin signaling and cell growth of peripheral tissue and leads to hyperglycemia and weight loss. Moreover, re-expression of PINK1 completely rescued the insulin resistance and systemic growth inhibition caused by tissue-specific mutations of pten. Supporting these findings, pten mutant clones have been shown to reduce the insulin signaling and growth potential of surrounding tissues. Furthermore, this study found that the secreted insulin/IGF antagonist impl2 is highly expressed upon the loss of both pink1 and pten and is reduced to wild-type levels by the re-expression of PINK1. Importantly, the systemic growth defects resulting from the tissue-specific knockdown of pten or pink1 were completely rescued by reducing impl2 to wild-type levels. These data suggest that reducing PINK1 expression may mediated cachexia and systemic metabolic dysfunction induced by tumors associated with PTEN mutations (Han, 2021).

Much like PTEN mutations, tumors induced in the adult Drosophila midgut by expressing the oncogene Yorkie (Yki) in intestine stem cells, or by transplanting tumors that express RasV12 in cells mutant for scribble, also secrete high levels of ImpL2, thereby reducing systemic insulin/IGF signaling and inducing organ wasting. It was established that the overexpression of ImpL2 in specific tissues in flies led to wasting in surrounding tissues. ImpL2 is a major mediator that is both necessary and sufficient for wasting. Together with the current results, these data suggest that a common mechanism underlying tumor cachexia may be the disruption of systemic insulin/IGF signaling that then results in hyperglycemia, insulin resistance, and organ wasting. There are seven IGF-binding proteins (IGFBPs) in mammals, which are dysregulated in different kinds of tumors. In the context of cancer, analyses of IGFBPs have largely focused on their effects on tumor growth. Recently, IGFBP-3 was found to be upregulated in human pancreatic cancer, supporting a potential role for insulin-binding peptides in the induction of tumor cachexia. Moreover, IGFBP-7 expression is associated with tumor growth and functions as a tumor suppressor in the colon, rectum, breast, and thyroid by either directly inducing apoptosis or inhibiting tumor growth via the deregulation of p16, p21, p53, and ERK signaling. In glioblastoma, however, IGFBP-7 plays an oncogenic role and stimulates tumor cell proliferation. It is possible that besides functions in tumor cells, IGFBP-7 may play a role in tumor cachexia as ImpL2 in flies (Han, 2021).

The InR/PI3K cascades in tumor cells with expression of Yki and RasV12 may also be shut down by reduced circulatory insulin/IGF signaling. This suggests that other mechanisms of organ wasting may exist. It has recently been demonstrated that Yki-associated tumors produce the epidermal growth factor receptor (EGFR) ligand, Vein, to trigger autonomous EGFR signaling and produce the platelet-derived growth factor (PDGF)- and vascular endothelial growth factor (VEGF)-related factor 1 (Pvf1) ligand to non-autonomously activate PVR (PDGF and VEGF receptor-related) signaling and wasting in peripheral cells. For PTEN mutant tumors, PI3K/AKT signals remain high regardless of circulatory ImpL2 level. This is explained by the function of PTEN as a negative regulator of AKT downstream of InR/PI3K. The re-expression of impl2 completely abolished the systemic growth defects without affecting the overgrowth of pten mutant cells. Therefore, it is suggested that the induction of ImpL2 expression by mutations in pten through PINK1 is both necessary and sufficient for peripheral organ wasting. The results, together with previous studies, highlight the importance of dissecting metabolic responses to cancer. The focus of cancer research is gradually expanding, from cancer cells to the tumor microenvironment to the system as a whole. In addition, it will be interesting to test whether some malignant tumors that harbor PTEN and/or PINK1 mutations (e.g., glioblastoma) display the elevated expression of IGFBPs, which alter systemic metabolism in a manner similar to Drosophila. Furthermore, these findings may provide a new strategy for treating such tumors (Han, 2021).

PTEN is endowed with both lipid and protein phosphatase activities. Because PTEN functionally antagonizes the lipid kinase PI3K, the depletion of PTEN results in high levels of the lipid second messenger PIP3, resulting in the increased membrane recruitment and activation of Akt. This leads to enhanced cellular growth, proliferation, and survival. PINK1 promotes cell growth and prevents cell death through its cytosolic downstream targets, such as mTORC2 and p53. However, PINK1 has also been shown to function inside mitochondria to promote the activity of complex I of the mitochondrial respiratory chain, and a recent study using three-dimensional (3D) structured illumination super-resolution microscopy suggested that under physiological conditions, PINK1 localizes to the cristae membrane and intracristae space. Moreover, endogenous PINK1 localizes to both cytosolic and mitochondrial fractions. Consistent with the localization of PINK1 to the IMM, this study found that targeting PINK1 to the IM fully restored complex I activity and rescued the growth defects of pink1 mutants. By contrast, OMM-localized PINK1 did not affect growth associated with pink1 mutation, but completely rescued muscle degeneration phenotypes of pink1 mutants associated with the loss of mitophagy. These data suggest that PINK1 activity is required inside mitochondria but not in the cytosol to promote complex I activity and cell growth. As a protein kinase, PINK1 kinase activity is required to maintain complex I activity in mitochondria, and PINK1 kinase activity in cytosol protects neurons. Inside mitochondria, PINK1 directly phosphorylates the NDUFA10/ND42 subunit to promote complex I activity. However, the overexpression of both wild-type and phosphorylation-mimic ND42 is not able to suppress the defective growth of pink1 mutants, indicating that multiple physiological targets of PINK1 may exist. It has been recently reported that PINK1 could phosphorylate the IMM protein MIC60 to promote complex I assembly and maintain oxidative phosphorylation. Nevertheless, PINK1 promotes growth when inside the mitochondria and protects cells against stress by initiating mitophagy when anchored to the OMM (Han, 2021).

Similar to knocking down pink1 in muscle cells, the disruption of complex I activity by muscle-specific knockdown of NDUFS1/ND75 reduces insulin signaling non-autonomously by inducing ImpL2 expression. Although the mechanisms are unclear, this work, together with the observations of other groups, strongly implies that the knockdown of pink1 disrupts the function of complex I, thus upregulating the expression of ImpL2 and inhibiting cell growth non-autonomously. These findings describe key functional associations between PINK1 and PTEN in tumorgenesis and systemic growth. This study shows that impaired PINK1-associated mitochondrial function plays a key role in wasting-like syndrome and reveal that protecting mitochondrial function in cancer patients may provide tremendous benefit (Han, 2021).

Ret rescues mitochondrial morphology and muscle degeneration of Drosophila Pink1 mutants

Parkinson's disease (PD)-associated Pink1 and Parkin proteins are believed to function in a common pathway controlling mitochondrial clearance and trafficking. Glial cell line-derived neurotrophic factor (GDNF) and its signaling receptor Ret are neuroprotective in toxin-based animal models of PD. However, the mechanism by which GDNF/Ret protects cells from degenerating remains unclear. This study investigated whether the Drosophila homolog of Ret can rescue Pink1 and park mutant phenotypes. It was shown that signaling active version of Ret (Ret^MEN2B) rescues muscle degeneration, disintegration of mitochondria and ATP content of Pink1 mutants. Interestingly, corresponding phenotypes of park mutants were not rescued, suggesting that the phenotypes of Pink1 and park mutants have partially different origins. In human neuroblastoma cells, GDNF treatment rescues morphological defects of PINK1 knockdown, without inducing mitophagy or Parkin recruitment. GDNF also rescues bioenergetic deficits of PINK knockdown cells. Furthermore, overexpression of Ret^MEN2B significantly improves electron transport chain complex I function in Pink1 mutant Drosophila. These results provide a novel mechanism underlying Ret-mediated cell protection in a situation relevant for human PD (Klein, 2014).

The receptor tyrosine kinase Ret is already known to be required for long-term survival of nigral dopamine neurons in mice, and stimulation with its ligand GDNF protects dopamine neurons from cell death in a variety of toxin-based rodent and primate models of PD. The present work found that a signaling-active version of the Drosophila homolog of Ret suppresses degeneration of muscle tissue and mitochondrial abnormalities in Pink1 mutants. Interestingly, park mutants were not rescued. In human SH-SY5Y cells, stimulation of endogenous Ret by GDNF rescued both morphological and bioenergetic defects of mitochondria in PINK1-depleted cells. Pink1 and Parkin were previously shown to interact genetically in Drosophila in what was proposed to be a linear pathway, and a significant body of work has described how Pink1 and Parkin function to initiate mitophagy of impaired mitochondria, and arrest of mitochondrial trafficking. However, in the cell culture model of this study, Ret signaling did not induce mitophagy or Parkin recruitment, arguing that Ret rescues PINK1 deficits independently of Parkin. A recent study demonstrated that Pink1 mutants in contrast to park mutants have decreased function of complex I of the electron transport chain, suggesting that Pink1 is required for maintaining efficient complex I enzymatic activity and that this function is upstream of mitochondrial remodeling. This study found that Ret rescued both the impairment of complex I activity, and partially the mitochondrial morphology in Pink1 mutants, suggesting that complex I is a target of Ret signaling. Previous studies of complex I inhibition or genetic depletion have shown mild morphological impairments in Drosophila muscle, contrary to the stronger phenotype of Pink1 mutants. Therefore, it was somewhat unexpected that restoring complex I activity would be sufficient to rescue also morphological defects. One interpretation is that the Pink1 mutant morphological phenotype is more severe due to a synergistic effect of deficits in remodeling/mitophagy and complex I activity, which in this study was partially rescued. Another possibility is that Ret signaling not only targets complex I, but also morphology in a Parkin-independent manner (Klein, 2014).

Extrapolated to mammalian models, the results suggest a novel mechanism by which the GDNF family of neurotrophic factors may promote survival of dopamine neurons in PD. Several of the mammalian models where the neuroprotective effects of GDNF treatment were initially discovered, were in fact models of mitochondrial dysfunction, either directly via complex I inhibition by MPTP treatment. In light of the current findings, it would be interesting to investigate whether or not GDNF improves complex I activity in these model systems. GDNF has been tested in models of α-synuclein overexpression, a pathology that is not known to cause complex I deficiency, but did not show any neuroprotective effects, fitting with the current hypothesis (Klein, 2014).

The current findings support recent evidence showing that Pink1 has an important function related to complex I activity, which is independent of its function in recruiting Parkin to the outer mitochondrial membrane upon loss of membrane potential. This model is consistent with a partial rescue of Pink1 deficiencies, e.g., by either overexpressing Parkin or the yeast complex I equivalent NADH dehydrogenase, or, in the current work, Ret^MEN2B. In addition, the current findings are consistent with a recent study showing that Pink1-deficient flies but not Parkin-deficient flies can be rescued by TRAP1, which also seems to have beneficial effects on complex I activity (Klein, 2014).

The pathways by which Ret signaling targets complex I and rescues Pink1 mutants requires further investigation. Also, the mechanism by which Pink1 regulates complex I remains elusive, it may regulate for example gene expression, phosphorylation status or assembly. Gene expression analysis showed that most subunits are unchanged by Ret^MEN2B, but interestingly one subunit was moderately downregulated in Pink1 mutants and upregulated by Ret^MEN2B, which may improve function. However, the possibility cannot be excluded that Ret signaling targets complex I, and perhaps other metabolic components, by different means (Klein, 2014).

Brain-derived neurotrophic factor (BDNF) protects mouse cortical neurons against drug-induced excitotoxicity, an effect that was blocked by the complex I inhibitor Rotenone and a MEK1/2 inhibitor, suggesting that BDNF signaling via the Ras/Erk pathway can regulate complex I function (Markham, 2012). The signaling properties and functions of Drosophila Ret are not characterized in great detail, but it is structurally homologous to mammalian Ret and can, to some extent, activate the same signaling pathways (Abrescia, 2005). Mammalian Ret on the other hand, has been extensively characterized and is known to activate a number of downstream signaling pathways including Ras/ERK, phosphoinositol-3 kinase (PI3K)/Akt, phospholipase C-gamma (PLCγ), Janus kinase (JAK)/STAT, and ERK5, several of which have pro-survival effects, most notably the PI3K/Akt pathway (Sariola, 2003; Pascual, 2011). Recent studies of Pink1 and park mutant Drosophila have indicated that PI3K/Akt signaling or components downstream of this pathway rather exacerbates Pink1 and park mutant phenotypes, making it an unlikely candidate for rescue (Klein, 2014).

Additional studies are required to elucidate the details by which Pink1 and Ret regulate complex I activity, and whether this finding is transferrable to mammalian models. In summary, this work shows that Ret signaling can rescue phenotypes of Pink1 mutants by restoring mitochondrial respiration and specifically complex I function, and thereby suggests a potential novel mechanism underlying GDNF‐mediated protection in mammalian PD models. In the future, screening of PD patients for complex I deficiencies and subjecting specifically those individuals to GDNF treatment may provide a new therapeutic strategy (Klein, 2014).

Clueless, a protein required for mitochondrial function, interacts with the PINK1-Parkin complex in Drosophila

Loss of mitochondrial function often leads to neurodegeneration and is thought to be one of the underlying causes of neurodegenerative diseases such as Parkinson's disease. However, the precise events linking mitochondrial dysfunction to neuronal death remain elusive. PTEN-induced putative kinase 1 (PINK1) and Parkin (Park), either of which, when mutated, are responsible for early-onset PD, mark individual mitochondria for destruction at the mitochondrial outer membrane. The specific molecular pathways that regulate signaling between the nucleus and mitochondria to sense mitochondrial dysfunction under normal physiological conditions are not well understood. This study shows that Drosophila Clueless (Clu), a highly conserved protein required for normal mitochondrial function, can associate with Translocase of the outer membrane (TOM) 20, Porin and PINK1, and is thus located at the mitochondrial outer membrane. Previous studies have found that clu genetically interacts with park in Drosophila female germ cells. This study shows that clu also genetically interacts with PINK1, and epistasis analysis places clu downstream of PINK1 and upstream of park. In addition, Clu forms a complex with PINK1 and Park, further supporting that Clu links mitochondrial function with the PINK1-Park pathway. Lack of Clu causes PINK1 and Park to interact with each other, and clu mutants have decreased mitochondrial protein levels, suggesting that Clu can act as a negative regulator of the PINK1-Park pathway. Taken together, these results suggest that Clu directly modulates mitochondrial function, and that Clu's function contributes to the PINK1-Park pathway of mitochondrial quality control (Sen, 2015).

Mitochondrial function is intimately linked to cellular health. These organelles provide the majority of ATP for the cell in addition to being the sites for major metabolic pathways such as fatty acid β-oxidation and heme biosynthesis. In addition, mitochondria are crucial for apoptosis, and they can irreparably damage the cell via oxidation when their biochemistry is abnormally altered. Given these many roles, tissues and cell types with high energy demands, such as neurons, are particularly sensitive to changes in mitochondrial function. This is also true for germ cell mitochondria because mitochondria are inherited maternally from the egg's cytoplasm and are thus the sole source of energy for the newly developing embryo (Sen, 2015).

Mitochondrial biology is complex owing to the dynamic nature of the organelle and the fact that most of the proteins required for function are encoded in the nucleus. In addition to the metabolites they provide, mitochondria undergo regulated fission, fusion and transport along microtubules. Because mitochondria cannot be made de novo, and tend to accumulate oxidative damage due to their biochemistry, they are subject to organelle and protein quality-control measures that involve mitochondrial and cytoplasmic proteases, as well as a specialized organelle-specific autophagy called mitophagy. However, the specific molecular signaling pathways between the nucleus and mitochondria that are used to sense which individual mitochondria are damaged during normal cellular homeostasis in vivo are not well understood (Sen, 2015). This study used the Drosophila ovary to identify genes regulating mitochondrial function and have characterized mitochondrial dynamics during Drosophila oogenesis. Germ cells contain large numbers of mitochondria that can be visualized at the single organelle level, making this system useful for studying genes that control mitochondrial function (Sen, 2015).

The gene clueless (clu) is crucial for mitochondrial localization in germ cells. Clu has homologs in many different species, and shows 53% amino acid identity to the human homolog, CLUH. The molecular role of Clu is not known. The yeast homolog, Clu1p, was found to interact with the eukaryotic initiation factor 3 (eIF3) complex in yeast and bind mRNA; however, the significance of this is not clear. CLUH has also been shown to bind mRNA. Flies mutant for clu are weak, uncoordinated, short-lived, and male and female sterile (Cox, 2009). Lack of Clu causes a sharp decrease in ATP, increased mitochondrial oxidative damage and changes in mitochondrial ultrastructure (Cox, 2009; Sen, 2013). Levels of Clu protein are homogeneously high in the cytoplasm and it is also found in large mitochondrially-associated particles. Although Clu clearly has an effect on mitochondria function, whether this is direct or indirect has not yet been established (Sen, 2015).

Parkin (Park), an E3 ubiquitin ligase, acts with PTEN-induced putative kinase 1 (PINK1) to target mitochondria for mitophagy. clu genetically interacts with park, and Clu particles are absent in park mutants, indicating that Clu might play a role in Park's mechanism (Cox, 2009; Sen, 2013). park and PINK1 have been identified as genes that, when mutated, cause early-onset forms of Parkinson's disease. Upon mitochondrial depolarization, PINK1 is stabilized on the mitochondrial outer membrane, recruiting Park, which then goes on to ubiquitinate many surface proteins, thus marking and targeting that mitochondrion for mitophagy. Before their biochemical interaction was recognized, PINK1 was placed upstream of park in a genetic pathway in Drosophila. Understanding Park and PINK1's role in mitochondrial quality control has shed light on the neurodegeneration underlying Parkinson's disease (Sen, 2015).

This study shows that Clu's mitochondrial role is well conserved, because the human homolog, CLUH, can rescue the fly mutant. Clu peripherally associates with mitochondria because it forms a complex with the mitochondrial outer-membrane proteins Porin and Translocase of the outer membrane (TOM) 20, supporting that the loss of mitochondrial function caused by lack of Clu is a direct effect. In addition, this study found that clu genetically interacts with PINK1 and, using epistasis, clu was placed upstream of park, but downstream of PINK1. Clu forms a complex with PINK1, and is able to interact with Park after the mitochondrial membrane potential is disrupted. Finally, lack of Clu causes PINK1 and Park to interact with each other, as well as causing a decrease in mitochondrial proteins, which suggests that Clu negatively regulates PINK1-Park function. Taken together, these data identify Clu as a mitochondrially-associated protein that plays a direct role in maintaining mitochondrial function and that binds TOM20, and support a role for Clu linking mitochondrial function to the PINK1-Park pathway (Sen, 2015).

Drosophila Clu is a large, highly conserved protein that shares its Clu and TPR domains with its human homolog, CLUH. Expressing CLUH in flies that are mutant for clu rescues the mutant phenotypes; thus, the human protein can use the fly machinery to fulfill the role of Clu. To date, all the evidence supports the idea that Clu has a role in mitochondrial function; however, it has been unclear how direct it is. In this study, using IPs, it was shown that Clu can associate with three proteins located on the mitochondrial outer membrane, TOM20, Porin and PINK1. Thus, Clu is not only a cytoplasmic protein, but can also be a peripherally associated mitochondrial protein, supporting the idea that this highly conserved protein directly affects mitochondrial function (Sen, 2015).

clu mutants share many phenotypes with park and PINK1 mutant flies, including flight muscle defects and sterility. Mitochondria are also mislocalized in PINK1 mutant germ cells, similarly to park mutants, and form large knotted clumps that include circularized mitochondria, which is consistent with increased fusion events. Mitochondria in clu mutant germ cells, on the other hand, do not show any signs of changes in fission or fusion (Cox, 2009). clu also genetically interacts with PINK1 and park, with double heterozygotes having clumped mitochondria in germ cells and a loss of Clu particles, and double knockdown of clu with PINK1 or park in flight muscle causing an increase in abnormal wing posture (Cox, 2009). Park functions in a pathway with PINK1 to elicit a mitophagic response, and overexpressing park can rescue PINK1 phenotypes in Drosophila. Using S2R+ cells and clu RNAi knockdown, this study found that overexpressing Park, but not PINK1, causes mitochondria to disperse. In adult flies, overexpressing full-length clu rescues the abnormal wing phenotype as well as mitochondrial phenotypes of PINK1 mutants, and overexpressing full-length clu or CLUH in PINK1, but not park, mutants rescues their thoracic indentation. These results place clu upstream of park, but downstream of PINK1. PINK1 stabilization on the mitochondrial outer membrane signals for Park to translocate to the organelle and subsequently ubiquitinate different proteins on the mitochondrial surface. Thus, it is somewhat surprising in Drosophila that loss of PINK1 can be rescued by increased amounts of Park, and suggests that there might be additional roles that Park plays in the cell. These data support the idea that an excess of Park overcomes deficits in mitochondrial function because it can rescue a loss of Clu as well. Mitochondrial clumping seems to be one of the responses to mitochondrial damage, in this system and in human tissue culture cells; thus, the dispersal upon Park overexpression in clu-RNAi-treated S2R+ cells is likely a sign of better mitochondrial health (Sen, 2015).

This study shows that Clu reciprocally immunoprecipitates with overexpressed PINK1 under normal cell culture conditions. PINK1 has been shown to directly bind TOM20, and Clu can also form a complex with TOM20, suggesting that all three proteins are found in close proximity at the mitochondrial membrane. Clu still immunoprecipitates with PINK1 when PINK1 is no longer targeted to the mitochondrial outer membrane (PINK1ΔMTS). This result indicates that Clu forms a complex with PINK1 independent of TOM20 or any other mitochondrial outer membrane proteins. Under normal conditions, PINK1 degradation happens so quickly that there are undetectable levels found at the outer mitochondrial membrane. Therefore, how is it possible that Clu is found in a complex with PINK1 in the absence of mitochondrial damage? It is likely that overexpressed PINK1 overwhelms the normal degradation process, thus becoming aberrantly stabilized at the outer mitochondrial membrane. Alternatively, it is possible that low levels of mitochondrial damage could account for the PINK1 being stabilized at the outer membrane, and then being able to interact with Clu (Sen, 2015).

Mitophagy ultimately leads to mitochondrial degradation in the lysosome. Currently, the literature involving Park and PINK1 uses mitochondrial protein levels as a read-out of mitophagy. However, recent data shows that different mitochondrial proteins have different half-lives, likely depending on what type of protein quality-control mechanism they use. Recent papers have examined protein half-life and found that Drosophila and yeast mitochondrial proteins, particularly those of Complex I in the case of flies, have increased half-lives when mitophagy proteins are missing. In addition, mitochondrial protein quality control does not always require destruction of the entire mitochondrion, but can selectively destroy certain proteins. For the mitochondrial proteins examined, all were greatly reduced in clu and PINK1 mutants, but not substantially altered in park mutants. This suggests that the turnover of the mitochondrial proteinsexamined is more sensitive to the absence of clu and PINK1 than park. This study found that Park and PINK1 form a complex in the absence of Clu. Thus, Clu is not necessary for this interaction, and loss of Clu causes a PINK1-Park interaction. This, plus the fact that Clu can be found at the outer mitochondrial membrane in a complex with both PINK1 and Park, suggests that Clu can influence mitochondrial quality or function, perhaps by regulating mitochondrial protein levels (Sen, 2015).

Yeast Clu1p was identified as a component of the eukaryotic initiation factor 3 (eIF3) complex and as an mRNA-binding protein. From IP and mass spectrometry data of the current study, there evidence that Clu can associate with the ribosome as well. Although CCCP is commonly used to force mitophagy and mitochondrial protein turnover, this treatment might not mimic the more subtle damage and changes mitochondria likely face in vivo. Mitochondrial protein import, for example, requires an intact mitochondrial membrane potential. Given the curent data, it is possible that Clu could function in co-translational import of proteins, as well as act as a sensor to couple PINK1-Park complex activation to how well protein import occurs. This would help explain why this study found that loss of Clu triggers a PINK1-Park interaction. In addition, Park and PINK1 directly interact with Porin and TOM20, respectively, placing them and Clu at the same place at the outer mitochondrial membrane. Recently, CLUH has been found to bind mRNAs for nuclear-encoded mitochondrial proteins, supporting a potential role in co-translational import. Further experiments are required to understand the precise relationship between Clu, TOM20, PINK1 and Park (Sen, 2015).

Mitochondria clearly undergo targeted destruction and require robust quality-control mechanisms, which are very active areas of investigation. PINK1 and Park's molecular mechanisms are particularly relevant to Parkinson's disease, given that inherited mutations in PARK2 and PINK1 can cause early-onset Parkinsonism. The molecular mechanisms that control mitophagy are becoming increasingly complex, involving membrane and cell biology; however, to date, the field has yet to visualize and understand the role of basal mitophagy levels in vivo. In the future, studying mitochondria and Clu function in Drosophila germ cells could lead to a better understand the role of mitochondrial protein turnover and quality control in the normal life cycle of tissues (Sen, 2015).

Vitamin K2 is a mitochondrial electron carrier that rescues pink1 deficiency

Human UBIAD1 localizes to mitochondria and converts vitamin K₁ to vitamin K₂. Vitamin K₂ is best known as a cofactor in blood coagulation, but in bacteria it is a membrane-bound electron carrier. Whether vitamin K₂ exerts a similar carrier function in eukaryotic cells is unknown. This study identified Drosophila UBIAD1/Heix as a modifier of pink1, a gene mutated in Parkinson's disease that affects mitochondrial function. Vitamin K₂ was found to be necessary and sufficient to transfer electrons in Drosophila mitochondria. Heix mutants showed severe mitochondrial defects that are rescued by vitamin K₂, and, similar to ubiquinone, vitamin K₂ transferred electrons in Drosophila mitochondria, resulting in more efficient adenosine triphosphate (ATP) production. Thus, mitochondrial dysfunction was rescued by vitamin K₂ that serves as a mitochondrial electron carrier, helping to maintain normal ATP production (Vos, 2012).

PINK1 and Parkin target Miro for phosphorylation and degradation to arrest mitochondrial motility

Cells keep their energy balance and avoid oxidative stress by regulating mitochondrial movement, distribution, and clearance. This study reports that two Parkinson's disease (PD) proteins, the Ser/Thr kinase PINK1 and ubiquitin ligase Parkin, participate in this regulation by arresting mitochondrial movement. PINK1 phosphorylates Miro, a component of the primary motor/adaptor complex that anchors kinesin to the mitochondrial surface. The phosphorylation of Miro activates proteasomal degradation of Miro in a Parkin-dependent manner. Removal of Miro from the mitochondrion also detaches kinesin from its surface. By preventing mitochondrial movement, the PINK1/Parkin pathway may quarantine damaged mitochondria prior to their clearance. PINK1 was shown to act upstream of Parkin, but the mechanism corresponding to this relationship has been unknown. This study proposes that PINK1 phosphorylation of substrates triggers the subsequent action of Parkin and the proteasome (Wang, 2011).

Although genetic and cell biological data have placed PINK1 upstream of Parkin in a pathway that regulates mitochondrial morphology and degradation, the relationship of the two enzymes has been obscure. One model proposes that Parkin is a PINK1 substrate activated by phosphorylation, but others have failed to find this phosphorylation. Findings in this study indicate an alternative model: PINK1 and Parkin bind to the same target and its phosphorylation by PINK1 allows Parkin, presumably acting as an ubiquitin-ligase, to designate that protein for removal from the mitochondrial membrane and proteasomal degradation. Indeed, hMiro1 and hMiro2 were shown to be among a list of proteins down-regulated by Parkin overexpression and CCCP. The ability of Parkin to bring about Miro degradation is consistent with its ability to ubiquitinate mitofusin and thereby to cause its degradation through the sequential action of p97/VPC and the proteasome. Interestingly, Miro and mitofusin interact with one another and their shared interaction with Parkin suggests coordinated regulation (Wang, 2011).

Mitochondrial motility is especially critical to neurons where it may take days for a mitochondrion to move between the cell body and a distant axonal or dendritic ending. The need for mitochondria to undergo turnover, as well as their redistribution to balance changes in local energy demand, make mitochondrial movement an important on-going and regulated process. The mitochondrion-specific adaptor proteins, Miro and Milton, are control points for this motility. Damaged mitochondria in cell lines selectively recruit Parkin and are in turn targeted for mitophagy. In contrast to an earlier report, it was found that this recruitment also occurs in axons; when highly expressed, YFP-Parkin is observed on mitochondria without depolarization (consistent with its ability to arrest mitochondrial motility upon overexpression), but with lower expression levels it is recruited to mitochondria by treatment with Antimycin A. Parkin recruitment is initiated by the depolarization-induced stabilization of PINK1 on the mitochondrial surface and PINK1 is also upstream of Parkin in regulating mitochondrial morphology. This relationship also holds for mitochondrial motility. PINK1 arrests mitochondrial motility in wildtype but not Parkin−/− mice or Parkin RNAi flies. Mitochondrial depolarization with CCCP causes the degradation of Miro in a Parkin-dependent manner. Similarly, PINK1 expression causes the degradation of Miro in Parkin expressing cells, but not in Parkin-lacking HeLa cells (Wang, 2011).

In previous genetic studies of PINK1 and Parkin, differences are noted between mice and Drosophila. Drosophila loss of function mutants exhibit profound defects in mitochondrial morphology that are seen in knockout mice only when neurons are additionally stressed. Differences were also observed in this study between Drosophila and murine models. In both, PINK1 or Parkin overexpression arrests mitochondria and in both Parkin is required downstream of PINK1. However, in Drosophila neurons, RNAi knockdown of PINK1 or Parkin increases mitochondrial motility whereas differences of motility in murine Parkin−/− neurons are not statistically significant. These differences may reflect a difference in how the species employ the pathway: in mammals, it may be strictly reserved for the response to mitochondrial depolarization whereas in the fly, whose short lifespan may make mitochondrial damage less critical, it may contribute to the ongoing turnover of proteins that participate in mitochondrial dynamics (Wang, 2011)

The ability of Parkin overexpression to alter mitochondrial motility in the presence of PINK1 RNAi or mitochondrial morphology in a PINK1 null background indicates that, although PINK1 can stimulate Parkin function, Parkin can act independently as well. Results from this study do not show if Parkin is effective because of residual PINK1 in the RNAi-expressing cells, because other kinases can also activate Miro as a Parkin substrate, or because elevated levels of Parkin can lead to Miro degradation even in the absence of a phosphorylation. Thus, PINK1 is likely to enhance Parkin function but probably is not required (Wang, 2011).

The observation that two PD-associated genes encode regulators of mitochondrial motility is consistent with other findings linking misregulation of mitochondrial dynamics to neurodegeneration. Changes in mitochondrial distribution, transport, and dynamics are implicated in Charcot-Marie-Tooth, Amyotrophic Lateral Sclerosis, Alzheimer’s and Huntington’s diseases. These findings underscore the importance of mitochondrial dynamics for supplying distal regions with sufficient energy and Ca2+-buffering capacity, compensating for changes in energy demand, refreshing older mitochondria through fusion with newly-synthesized mitochondria, and clearing damaged mitochondria (Wang, 2011).

Clarification of the relationship of PINK1 and Parkin supports the view that PD is a mitochondrial disorder. In the etiology of PD, the regulation of Miro levels may be significant. Either through a specific sorting pathway or as a consequence of the random reassortment of mitochondrial proteins that occur with repeated fusion and fission, some organelles or fragments of the organelle will arise in which the burden of dysfunctional proteins is sufficient to compromise the membrane potential. The resulting stabilization of PINK1 on the surface and targeting of Miro, mitofusin, and other proteins for Parkin action and degradation, will bring about the sequestration and eventual engulfment of that dysfunctional organelle. Sequestration and mitophagy thereby prevent further cellular damage due to reactive oxygen species and enable the cellular complement of mitochondria to be replenished by healthier organelles. The greater the stresses on mitochondria, the more acute the need for this clearance pathway. The heightened sensitivity of the dopaminergic neurons in the substantia nigra to disruption of this ubiquitous pathway may therefore reflect exceptional challenges for mitochondria in these cells. Those stresses may include the susceptibility of dopamine to oxidation and high rates of Ca2+ influx. When this quality control mechanism is defective in patients carrying mutations in either gene, damaged mitochondria will retain Miro and mitofusin, and therefore may move about in the neuron and, through fusion reactions, reintroduce damaged components to otherwise healthy organelles rather than undergo mitophagy (Wang, 2011).

Discovery of catalytically active orthologues of the Parkinson's disease kinase PINK1: analysis of substrate specificity and impact of mutations

Missense mutations of the phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) gene cause autosomal-recessive Parkinson's disease. To date, little is known about the intrinsic catalytic properties of PINK1 since the human enzyme displays such low kinase activity in vitro. In contrast to mammalian PINK1, insect orthologues of PINK1 from Drosophila melanogaster (dPINK1), Tribolium castaneum (TcPINK1) and Pediculus humanus corporis (PhcPINK1) are active as judged by their ability to phosphorylate the generic substrate myelin basic protein. The most active orthologue, TcPINK1, was exploited to assess its substrate specificity and elaborated a peptide substrate (PINKtide, KKWIpYRRSPRRR) that can be employed to quantify PINK1 kinase activity. Analysis of PINKtide variants reveal that PINK1 phosphorylates serine or threonine, but not tyrosine, and it was shown that PINK1 exhibits a preference for a proline at the +1 position relative to the phosphorylation site. This study has investigated the effect of Parkinson's disease-associated PINK1 missense mutations and found that nearly all those located within the kinase domain, as well as the C-terminal non-catalytic region, markedly suppress kinase activity. This emphasizes the crucial importance of PINK1 kinase activity in preventing the development of Parkinson's disease. These findings will aid future studies aimed at understanding how the activity of PINK1 is regulated and the identification of physiological substrates (Woodroof, 2011).

DJ-1 is critical for mitochondrial function and rescues PINK1 loss of function

Mutations or deletions in PARKIN/PARK2, PINK1/PARK6, and DJ-1/PARK7 lead to autosomal recessive parkinsonism. In Drosophila, deletions in parkin and pink1 result in swollen and dysfunctional mitochondria in energy-demanding tissues. The relationship between DJ-1 and mitochondria, however, remains unclear. This study reports that Drosophila and mouse mutants in DJ-1 show compromised mitochondrial function with age. Flies deleted for DJ-1α and DJ-1β manifest similar defects as pink1 and parkin mutants: male sterility, shortened lifespan, and reduced climbing ability. Poorly coupled mitochondria were found in vitro and reduced ATP levels in fly and mouse DJ-1 mutants. Surprisingly, up-regulation of DJ-1 can ameliorate pink1, but not parkin, mutants in Drosophila; cysteine C104 (analogous to C106 in human) is critical for this rescue, implicating the oxidative functions of DJ-1 in this property. These results suggest that DJ-1 is important for proper mitochondrial function and acts downstream of, or in parallel to, pink1. These findings link DJ-1, pink1, and parkin to mitochondrial integrity and provide the foundation for therapeutics that link bioenergetics and parkinsonism (Hao, 2010).

Parkinson's disease (PD) is the most common movement disorder and the second most common neurodegenerative disease. Clinically, it is characterized by resting tremor, rigidity, bradykinesia, gait abnormality, and slow movement. PD patients show severe dopaminergic neuron loss, resulting in a decrease of striatal dopamine levels responsible for the motor features. Age is the most potent risk factor for PD, but other contributing factors include exposure to environmental toxins like paraquat and rotenone. Although predominantly idiopathic, genetic mutations account for ≈10% of cases. Studies of genes responsible for familial parkinsonism/PD are yielding critical insight into mechanisms shared by sporadic and familial disease (Hao, 2010).

Mutations in DJ-1/PARK7, PINK1/PARK6, and PARKIN/PARK2 lead to autosomal recessive parkinsonism. Properties of DJ-1 suggest that it may be at a compelling intersection for several risk factors in PD, including genetics, oxidative stress, environmental factors, and age. First, DJ-1 gene mutations lead to early-onset autosomal recessive parkinsonism (Bonifati, 2003). Second, the DJ-1 protein is sensitive to oxidative stress and may act as a redox-responsive molecular chaperone that can prevent protein misfolding (Shendelman, 2004). Third, tolerance toward paraquat in animals is mediated, in part, through modifications of DJ-1 protein (Meulener, 2006). Finally, age induces the same modifications of the DJ-1 protein as environmental toxins (Meulener, 2006). Mice mutant for DJ-1 show dopamine reuptake dysfunction (Goldberg, 2005) and have increased sensitivity to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine (MPTP) (Kim, 2005). Two DJ-1 orthologs (DJ-1a and DJ-1b) exist in Drosophila, and when deleted, flies have decreased climbing ability (Park, 2005) and increased sensitivity to H₂O₂, paraquat, and rotenone (Meulener, 2005). Taken together, these data indicate that the DJ-1 protein responds to key risk factors for PD, including age and toxins (Hao, 2010).

Parkin is an E3 ubiquitin ligase whose mutations account for the majority of autosomal recessive parkinsonism, and Parkin may be important for mitophagy. The second most common cause of early-onset parkinsonism is mutations in PINK1, a kinase localized to mitochondria that may be involved in mitochondrial fission (Valente, 2004). In Drosophila, loss of function of either pink1 or parkin leads to male sterility and abnormal wing posture (Greene, 2003; Clark, 2006; Park, 2006). Moreover, up-regulation of parkin rescues pink1 mutation, thus placing the two genes in the same genetic pathway, with parkin downstream of pink1 (Clark, 2006; Park, 2006). Electron microscopic analysis of pink1 and parkin null mutants shows swollen mitochondria in flight muscle and testes, suggesting that these genes are important for mitochondrial integrity. Interestingly, epidemiological and other studies have linked complex I inhibitors, such as rotenone, paraquat, and 1-methyl-4-phenylpridinium (MPTP metabolite), to parkinsonism in humans. Given that DJ-1 is also localized to mitochondria, and that mutations in DJ-1 lead to parkinsonism, these results raise the question of whether there are links between DJ-1 and mitochondrial function (Hao, 2010).

The link between DJ-1 and mitochondria, however, has been circumstantial and limited to cell culture studies. This study demonstrates that DJ-1 loss of function leads to mitochondrial dysfunction in an age-dependent manner in both fly and mouse. Genetic evidence is provided that DJ-1 interacts with the pink1/parkin pathway, because DJ-1 up-regulation can compensate for reduction of pink1 activity. Consistent with important interactions between DJ-1 and pink1/parkin, up-regulation of either pink1 or parkin is deleterious in a DJ-1 mutant background. These findings indicate that DJ-1 and pink1/parkin fall into two parallel pathways whose function critically impacts mitochondrial activity (Hao, 2010).

Double knockout (DKO) mutant flies showed several classic defects that reflect mitochondrial dysfunction. First, DKO flies have an overall reduced fitness (shortened lifespan), consistent with mitochondrial involvement in reduced lifespan and accelerated aging in worms, flies, and mice. Second, DKO flies have defects in spermatogenesis reflective of disrupted mitochondrial function. In the fly, mitochondrial mutants are often infertile because germ cell maturation requires both energy and mitochondrial morphological changes. Similarly, DJ-1 protein levels correlate with infertility in pharmacologically treated male rats that leads to reduced glycolytic enzyme activities in the sperm. Third, DKO flies showed age-dependent declines in both climbing activity and ATP level. The age-dependent onset of these deficiencies is consistent with the notion that nonlethal mitochondrial mutants can compensate for mitochondrial inefficiency until older ages, when more pressure is exerted on the system. These data show that DJ-1 DKO mitochondria have a lower RCR, suggesting that the mitochondria can function, albeit at lower capacity and with lower reserve capacity. Over time, DKO cells may no longer effectively compensate, and defects manifest. These data indicate that DJ-1 activity is important for proper mitochondrial function over time (Hao, 2010).

As in fly DJ-1 mutants, mitochondrial dysfunction was observed in DJ-1 knockout mice skeletal muscle. Although no dysfunction was seen in mitochondria isolated from mouse brain, muscle is one of the high energy demanding tissues. These data agree with previous findings that DJ-1 knockout mice do not have a significant change in lifespan or reduction in dopamine levels with age. The age-dependent reduction in rotorod endurance in DJ-1 knockout mice is consistent with findings showing specific age-dependent impairments in endurance in DJ-1 knockout mice. Interestingly, previous studies shown no change was seen in neuromuscular junction or muscle by histology in DJ-1 knockout mice. The changes in mitochondrial function and efficiency observed in this study can explain the loss of endurance. Alteration in muscle-related activities was not observed in another study of DJ-1 null mice, suggesting that genetic background may affect the presentation of this impairment. It is also intriguing that murine muscle seems to be more affected than brain, in that ATP reductions were detected in mouse muscle but not brain in the current study. It is possible that, unlike humans, the energy demand of murine muscle, like that of Drosophila, is higher than that of brain, thus the consequence of mitochondrial dysfunction in the absence of DJ-1 manifests first in the muscle. Taken together, it is suggested that loss of DJ-1 activity may lead to age-dependent mitochondrial dysfunction in tissues with high energy demands (Hao, 2010).

This study presents evidence that DJ-1 loss of function causes similar mitochondrial defects in aged Drosophila. Moreover, it was also shown that DJ-1 can rescue pink1, although not parkin, loss. DJ-1a does not ameliorate all pink1 defects: DJ-1a could not rescue the pink1 mutant infertility. The selective rescue of pink1 mutants by DJ-1 places DJ-1 downstream of pink1. The findings that DJ-1 cannot rescue parkin mutants, and that parkin cannot rescue DKO mutants, suggest that DJ-1 may not be directly downstream of pink1 (neither in between pink1 and parkin nor downstream of parkin). It is proposed that DJ-1 defines a pathway parallel to that of pink1/parkin. It is likely that there is partial convergence/overlap downstream between the pathways, given the common effects. It cannot, however, be ruled out that DJ-1 selectively rescues a target downstream of pink1 pathway that is responsible for the effects on thoracic mitochondria. It is interesting that Omi/HtrA2 has also been suggested to partially rescue pink1 mutants in Drosophila in a pathway that is similarly independent of parkin (Hao, 2010).

These data indicate a critical balance of activities between the DJ-1 and pink1/parkin pathways, because up-regulation of either pink1 or parkin leads to lethality in DKO flies. In accord to the finding that up-regulation of pink1 leads to a deleterious Drosophila eye effect in a wild-type background, the current data further suggest that in DKO, a background sensitized for mitochondrial dysfunction, an increase in pink1 levels simply causes lethality. The ability of DJ-1 to respond to oxidative stress through C104 modification or as an atypical peroxidase fits nicely with the hypothesis that DJ-1 is important for proper mitochondrial function. However, it should be noted that C104 residue of DJ-1 might sense oxidative stress independent of the proposed peroxidase function. As such, when DJ-1 activity is compromised, cells accumulate more oxidative stress and mitochondrial dysfunction with age. pink1 deletion may also lead to increased oxidative stress that can be partially reduced through up-regulation of DJ-1. In line with this, when the ability of DJ-1b to respond to oxidative stress was mitigated by mutation of C104, but not C45, rescue of pink1^B9 wing posture was abrogated. Interestingly, DJ-1b with either C104A or C45A also failed to rescue pink1^B9 fertility phenotype. Taken together, these data argue that DJ-1 rescue of pink1^B9 mutants requires its ability to respond and protect against oxidative stress in specific tissues (Hao, 2010).

Although previous studies suggest that DJ-1 cannot rescue pink1 mutants (Yang, 2006), several important differences exist between those data and the current findings: first, the method of pink1 reduction (RNAi vs. gene deletion in the present study); and second, the method by which DJ-1 was up-regulated (muscle-specific vs. ubiquitous driver in the present study). Moreover, when assessing rescue, a quantitative measurement was used because the wing effect was not 100% penetrant in pink1^B9 mutants. This approach allowed assessment of genes that partially rescue pink1 mutants. Additionally, this study found that the expression level of DJ-1 transgenes seems to be critical for rescue, with higher levels of expression being less likely to rescue. These findings indicate that DJ-1 rescues the pink1 mutant in an expression level–dependent manner. Understanding the detailed mechanism by which DJ-1 rescue of pink1 is so critically dose dependent may provide key insight into the intersection of the pathways (Hao, 2010).

Mitochondria play a crucial role in neurodegenerative diseases. The current findings strongly indicate that mitochondrial dysfunction plays an important role in inherited forms of early-onset parkinsonism, as well as in sporadic disease, which may involve similar genetic players and pathways. These findings highlight the value of defining when mitochondrial function decline occurs relative to disease onset, and the central role of mitochondria as a target in PD therapeutic research (Hao, 2010).

The mitochondrial fusion-promoting factor mitofusin is a substrate of the PINK1/parkin pathway

Loss-of-function mutations in the PINK1 or parkin genes result in recessive heritable forms of parkinsonism. Genetic studies of Drosophila orthologs of PINK1 and parkin indicate that PINK1, a mitochondrially targeted serine/threonine kinase, acts upstream of Parkin, a cytosolic ubiquitin-protein ligase, to promote mitochondrial fragmentation, although the molecular mechanisms by which the PINK1/Parkin pathway promotes mitochondrial fragmentation are unknown. This study tested the hypothesis that PINK1 and Parkin promote mitochondrial fragmentation by targeting core components of the mitochondrial morphogenesis machinery for ubiquitination. The steady-state abundance of the mitochondrial fusion-promoting factor Drosophila Mitofusin (Mitochondrial assembly regulatory factor, Marf or dMfn) is inversely correlated with the activity of PINK1 and Parkin in Drosophila. dMfn is ubiquitinated in a PINK1- and Parkin-dependent fashion and dMfn co-immunoprecipitates with Parkin. By contrast, perturbations of PINK1 or Parkin did not influence the steady-state abundance of the mitochondrial fission-promoting factor Drp1 or the mitochondrial fusion-promoting factor Opa1, or the subcellular distribution of Drp1. These findings suggest that dMfn is a direct substrate of the PINK1/Parkin pathway and that the mitochondrial morphological alterations and tissue degeneration phenotypes that derive from mutations in PINK1 and parkin result at least in part from reduced ubiquitin-mediated turnover of dMfn (Poole, 2010).

In previous work, it has been shown that genetic manipulations that promote mitochondrial fragmentation, including increased drp1 gene dosage and decreased opa1 or dmfn gene dosage, dramatically suppress the PINK1 and parkin mutant phenotypes in Drosophila. These findings, coupled with previous work demonstrating that PINK1 acts upstream of Parkin in a common pathway, led to a hypothesis that PINK1 and Parkin influence mitochondrial integrity by regulating core components of the mitochondrial morphogenesis machinery through ubiquitination (Poole, 2008). The current results provide direct support for this hypothesis by demonstrating that dMfn is ubiquitinated in a PINK1- and Parkin-dependent fashion and that the steady-state abundance of dMfn is increased in PINK1 and parkin mutants and decreased in PINK1- and Parkin-overexpressing flies. These findings suggest a model in which PINK1 phosphorylates either dMfn or Parkin and thereby increases the efficiency with which Parkin is able to ubiquitinate dMfn. The subsequent ubiquitin-mediated turnover of dMfn would then inhibit mitochondrial fusion, and thus promote mitochondrial fragmentation. While the current findings were in review, another study primarily using cultured Drosophila S2 cells (Ziviani, 2010) also reported that dMfn is a substrate of the PINK1/Parkin pathway, thus providing additional support for these conclusions (Poole, 2010).

The finding that the PINK1/Parkin pathway promotes mitochondrial fragmentation led to a proposal that this pathway may act to segregate damaged portions of the mitochondrial reticulum for turnover through an autophagic mechanism (Poole, 2008). Several recent studies provide compelling support for this hypothesis by demonstrating that treatment of cultured vertebrate cells with mitochondrial damaging agents triggers PINK1 to selectively recruit Parkin to damaged mitochondria, where Parkin acts to promote the autophagic turnover of these mitochondria, presumably by ubiquitinating particular mitochondrial targets (Narendra, 2010; Geisler, 2010; Narenda, 2008; Vives-Bauza, 2009). These studies, together with the current findings raise the possibility that the selective Parkin-mediated ubiquitination and subsequent degradation of dMfn on damaged portions of the mitochondrial reticulum, coupled with ongoing mitochondrial fission serves to sequester the mitochondrial damage to small fusion-incompetent mitochondria that are subsequently eliminated through autophagy. However, the size of ubiquitinated dMfn suggests that it is triply ubiquitinated and previous work indicates that a chain of four or more ubiquitins is required for efficient targeting to the proteasome. Thus, alternative interpretations of the findings, although not mutually exclusive, are that ubiquitination of dMfn inactivates the fusion-promoting activity of dMfn, or serves as a tag marking the damaged mitochondria for destruction by autophagy. The finding that the ubiquitination of a peroxisomal surface protein is sufficient to signal the autophagic degradation of this organelle is consistent with the latter model. Experiments are currently underway to distinguish these possibilities (Poole, 2010).

While a model in which the PINK1/Parkin pathway promotes mitochondrial fragmentation through the ubiquitination of dMfn is completely consistent with previous work on PINK1 and Parkin in Drosophila, recent findings from vertebrate cell culture studies challenge this model. In particular, several of the studies of PINK1 in vertebrate systems have found that reduced PINK1 activity results in mitochondrial fragmentation, suggesting that PINK1 may promote mitochondrial fusion—exactly the opposite of the conclusion drawn from studies of the PINK1/Parkin pathway in flies. While additional work will be required to resolve these apparent conflicts, it is important to point out that the findings from studies of PINK1 and Parkin in flies have involved tissues that are profoundly affected by loss of PINK1 and Parkin activity, whereas the tissue sources of the cells that have been used in at least some of the conflicting vertebrate studies are largely unaffected by mutations in PINK1 and parkin. Thus, a possible explanation for these apparently discordant findings is that the mitochondrial fragmentation resulting from reduced PINK1 activity that has been observed in vertebrate systems involves a compensatory induction of mitochondrial fragmentation in these cells, which perhaps also explains their relative insensitivity to the loss of PINK1 activity. In potential support of this model is the finding that while the mitochondrial fragmentation seen in PINK1-deficient vertebrate cells can be rescued by inactivating Drp1, this manipulation enhances the cell death associated with PINK1 deficiency, a finding that is entirely consistent with work in flies. Future work should resolve these apparent conflicts and further clarify the influence of PINK1- and Parkin-dependent ubiquitination of dMfn on mitochondrial integrity (Poole, 2010).

Drosophila parkin requires PINK1 for mitochondrial translocation and ubiquitinates mitofusin

Loss of the E3 ubiquitin ligase Parkin causes early onset Parkinson's disease, a neurodegenerative disorder of unknown etiology. Parkin has been linked to multiple cellular processes including protein degradation, mitochondrial homeostasis, and autophagy; however, its precise role in pathogenesis is unclear. Recent evidence suggests that Parkin is recruited to damaged mitochondria, possibly affecting mitochondrial fission and/or fusion, to mediate their autophagic turnover. The precise mechanism of recruitment and the ubiquitination target are unclear. This study shows in Drosophila cells that PINK1 is required to recruit Parkin to dysfunctional mitochondria and promote their degradation. Furthermore, PINK1 and Parkin mediate the ubiquitination of the profusion factor Mfn on the outer surface of mitochondria. Loss of Drosophila PINK1 or parkin causes an increase in Mfn abundance in vivo and concomitant elongation of mitochondria. These findings provide a molecular mechanism by which the PINK1/Parkin pathway affects mitochondrial fission/fusion as suggested by previous genetic interaction studies. It is hypothesized that Mfn ubiquitination may provide a mechanism by which terminally damaged mitochondria are labeled and sequestered for degradation by autophagy (Ziviani, 2010).

Maintenance of mitochondrial homeostasis appears to be an important function of the PINK1/Parkin pathway in multiple model systems and is likely a key factor in mediating neurodegeneration. Recent studies have begun to shed light on the potential mechanism by which this pathway maintains a healthy mitochondrial population. Emerging evidence indicates that PINK1 is required to recruit Parkin to damaged or dysfunctional mitochondria, whereupon it promotes mitophagy (Geisler, 2010; Narendra, 2010; Vives-Bauza, 2010). Regulated mitochondrial fission and fusion events are thought to contribute to a quality control mechanism to help 'sort out' terminally damaged mitochondria for degradation. Importantly, PINK1 and parkin have previously been shown to genetically interact with components of the mitochondrial fission/fusion machinery and to affect mitochondrial morphology; however, the molecular mechanisms are not known. This study provides further evidence that PINK1 is required for Parkin translocation to damaged mitochondria and that this pathway affects mitochondrial morphology. Evidence is also provided that the PINK1/Parkin pathway promotes the ubiquitination and regulates the levels of the profusion protein Mfn, thus providing a potential molecular mechanism by which PINK1/Parkin may modulate mitochondrial dynamics (Ziviani, 2010).

Consistent with recent reports, this study found that the translocation of Parkin to damaged mitochondria and their subsequent autophagy is dependent on PINK1. However, the molecular mechanisms that promote Parkin's recruitment to mitochondria are still unclear. PINK1's kinase activity, but not mitochondrial localization, appears to be necessary for Parkin translocation. Because PINK1 can be found extramitochondrially and may directly phosphorylate Parkin, this may be a mechanism to stimulate its translocation. Alternatively, it may phosphorylate a Parkin substrate, e.g., Mfn, and thereby provide a recruitment signal. Interestingly, this study found that loss of Mfn reduces but does not eliminate Parkin translocation. Recent evidence indicates that Parkin also ubiqutinates VDAC on the outer mitochondrial surface (Geisler, 2010), suggesting that there may be multiple recruitment substrates. Although further work is required to elucidate these mechanisms, these findings suggest a molecular basis for the genetic hierarchy in which PINK1 acts upstream of Parkin (Ziviani, 2010).

To understand the role of Parkin translocation, this study took a candidate approach to identifying putative substrates. Because the function of Parkin and PINK1 has been linked with mitochondrial dynamics, key components of the mitochondrial fission and fusion machinery were surveyed for ubiquitin modification. Mfn, which localizes to the outer surface of mitochondria, was found to be ubiquitinated in a PINK1/Parkin-dependent manner and accumulates upon loss of PINK1 or parkin. Interestingly, the ubiquitinated isoforms do not show a typical ubiquitination 'ladder' but instead appear to reflect a pattern of one and three or four ubiquitin adducts. Although it remains to be shown that Parkin directly mediates this ubiquitination, there is evidence that Parkin can mediate monoubiquitination and K27 and K63 linkages. These modes of ubiquitination are not typically linked to proteasome degradation, and there is growing speculation that important pathogenic functions of Parkin may be proteasome independent (Ziviani, 2010).

Numerous elegant studies have demonstrated that the mitochondrial network is extremely dynamic and responds rapidly and reversibly to many physiological changes including potentially toxic challenges such as oxidative stress and calcium flux. Although mitochondrial remodeling can contribute to promoting cell death, it can also act in a protective manner by contributing to a quality control process that likely involves degradation by autophagy/lysosomes. Recent work has reported observations that, following a fission event, regulated fusion of daughter mitochondria can determine whether they rejoin the network or are sequestered for degradation. Refusion appears to be dependent upon the recovery of mitochondrial membrane potential after division and likely represents a mechanism to sort out terminally dysfunctional mitochondria. Because Mitofusins mediate the tethering and fusion of mitochondria via homo- and heterotypic interaction of their HR2 domains, it is hypothesized that Parkin-mediated Mfn ubiquitination may interfere with intermolecular interactions preventing fusion. Alternatively, Mfn ubiquitination may lead to a selective removal of Mfn from damaged mitochondria and thus reduce the refusion capacity of those mitochondria. Consistent with this, it was found that loss of parkin or PINK1, and hence loss of ubiquitination, leads to increased Mfn levels and mitochondrial elongation, presumably due to excess fusion. Thus, Mfn ubiquitination may provide a signal that simultaneously prevents the refusion of terminally damaged mitochondria and labels them for safe degradation by autophagy (Ziviani, 2010).

It is reasonable to suppose that under normal conditions the majority of mitochondria are relatively healthy, and thus mitochondrial turnover is an infrequent event. This is supported by the observation that Complex Vα levels are not significantly altered by decreased mitophagy. However, this rationale implies that Mfn accumulates and is selectively ubiquitinated on mitochondria targeted for degradation although this remains to be shown. Interestingly, the current findings provide a molecular mechanism that can explain the previously reported genetic interactions between PINK1 and parkin and the fission/fusion factors - in particular, that promoting mitochondrial fragmentation by overexpression of Drp1 or by reduction of Mfn and Opa1 is able to partially suppress the locomotor deficits, muscle degeneration, and mitochondrial abnormalities. Together these findings suggest that aberrant accumulation of Mfn may mediate the loss of mitochondrial homeostasis caused by loss of PINK1 or parkin. Although further work will be needed to determine whether this contributes to PD pathogenesis, these results support the emerging hypothesis that the PINK1/Parkin pathway acts to regulate the safe degradation of terminally damaged mitochondria as a quality control mechanism (Ziviani, 2010).

Drosophila ref(2)P is required for the parkin-mediated suppression of mitochondrial dysfunction in pink1 mutants

Autophagy is a critical regulator of organellar homeostasis, particularly of mitochondria. Upon the loss of membrane potential, dysfunctional mitochondria are selectively removed by autophagy through recruitment of the E3 ligase Parkin by the PTEN-induced kinase 1 (PINK1) and subsequent ubiquitination of mitochondrial membrane proteins. Mammalian sequestrome-1 (p62/SQSTM1) is an autophagy adaptor, which has been proposed to shuttle ubiquitinated cargo for autophagic degradation downstream of Parkin. This study shows that loss of ref(2)P, the Drosophila orthologue of mammalian P62, results in abnormalities, including mitochondrial defects and an accumulation of mitochondrial DNA with heteroplasmic mutations, correlated with locomotor defects. Furthermore, expression of Ref(2)P is able to ameliorate the defects caused by loss of Pink1, and this depends on the presence of functional Parkin. Finally, both the PB1 and UBA domains of Ref(2)P are crucial for mitochondrial clustering. It is concluded that Ref(2)P is a crucial downstream effector of a pathway involving Pink1 and Parkin and is responsible for the maintenance of a viable pool of cellular mitochondria by promoting their aggregation and autophagic clearance (de Castro, 2013).

Mitochondrial dysfunction has been strongly associated with different neurodegenerative diseases, such as PD. Cells within complex multicellular organisms have developed quality-control mechanisms to cope with the many challenges that are constantly imposed on mitochondria and to suppress the accumulation of dysfunctional organelles. This study provides evidence that the Drosophila orthologue of p62, Ref(2)P is an important component of the Pink1/Parkin quality-control pathway (de Castro, 2013).

ref(2)P mutant flies exhibited several pathological and functional phenotypes reminiscent of those observed in the pink1 or parkin mutants. They exhibited the following: mitochondrial abnormalities of the sperm cells; defective locomotor activity; and a shorter lifespan. These defective phenotypes are more profound in the ref(2)P mutants lacking the UBA domain, suggesting that the ability of Ref(2)P to bind ubiquitinated targets is required for mitochondrial integrity and function. Despite the observed mitochondrial defects in the ref(2)P^od2 and ref(2)P^od3 mutants, no global alterations were seen in mitochondrial mass or function, suggesting that mitochondrial dysfunction in the ref(2)P mutants is not as pronounced as that observed in pink1 or parkin mutant flies. The observed decrease in motor performance from an early age suggests that any mitochondrial defects in ref(2)P mutants might preferentially affect tissues with high energetic demand such as the skeletal muscles and spermatids. It is therefore possible that such defects are undetectable in the respirometry assays, as these are performed using mitochondria derived from whole flies. There have been a number of reports of mtDNA point mutations associated with neurodegenerative diseases such as PD. This study shows that defects in ref(2)P, the single Drosophila P62 orthologue, result in an increase in mtDNA heteroplasmy. It is therefore conceivable that defects in mitophagy might contribute to neurodegenerative diseases such as PD by increasing the load of deleterious mtDNA mutations, leading eventually to increased mitochondrial dysfunction and an impairment of neuronal function (de Castro, 2013).

ref(2)P mutants exhibited a marked sensitivity to rotenone, an organic pesticide that directly targets respiration by inhibiting mitochondrial complex I. These mutants also showed a lower sensitivity to paraquat, an herbicide widely used in agriculture that has been linked to PD. Paraquat increases oxidative stress, whereas rotenone causes mitochondrial dysfunction; however, both processes are linked and both pesticides affect these mechanisms. Paraquat does not directly target mitochondria. In cells, it undergoes redox cycling in vivo to produce superoxide-free radicals that can damage not only these organelles but also other cellular components. It was therefore reasoned that, within this context, the Ref(2)P-dependent mitophagy might be particularly important in suppressing damage caused by PD-linked toxins whose mechanism of action directly targets mitochondrial function such as rotenone (de Castro, 2013).

It is noted that expression of ref(2)P is capable of suppressing pink1 but not parkin mutant phenotypes. This finding indicates that Ref(2)P exerts a protective effect downstream of Pink1 but requires the presence of functional Parkin. This is compatible with a model in which Ref(2)P recognises mitochondrial substrates ubiquitinated by Parkin and therefore, in the absence of Parkin, is incapable of recognising ubiquitin-decorated mitochondria and targeting them for autophagy. Parkin failed to restore the mitochondrial function of pink1 mutant flies in the absence of functional Ref(2)P, supporting the notion that Ref(2)P is a critical downstream effector of Parkin (de Castro, 2013).

Mutations in ref(2)P suppressed mitochondrial aggregation in the pink1 mutant flies. In mammalian cells, p62 has been suggested to mediate the aggregation of dysfunctional mitochondria into tight clusters, thereby minimising the surface area exposed to other cellular components. This study shows that this function of p62 is conserved in Drosophila and that Ref(2)P coordinates mitochondrial clustering through its PB1 and UBA domains. Reducing the surface area of impaired mitochondria within the cell by mitochondrial clustering may help minimise the uptake of respiratory substrates and limit the spread of mitochondrial ROS to other cellular compartments. Additionally, the clustering of the dysfunctional mitochondria could be beneficial to subcellular compartments with high-energy requirements, such as neuronal synapses, by preventing damaged mitochondria from being transported at the expense of bioenergetically active mitochondria. Alternatively, as p62 clustering of ubiquitinated substrates has been shown to cause cell death in the absence of its autophagic degradation, it is possible that, in Drosophila, Ref(2)P functions as a sensor of defective mitophagy quality control, triggering cell death when the removal of Parkin-ubiquitinated mitochondria is insufficient. This scenario could explain the suppression of the pink1 mutant phenotypes by ref(2)P expression (de Castro, 2013).

Finally, both Parkin and p62 are important regulators of mitophagy. Parkin is responsible for the autophagic elimination of damaged mitochondrial units. p62, on the other hand, binds directly to the autophagy protein LC3 and is believed to serve as an autophagy receptor for ubiquitinated protein aggregates as well as peroxisomes and intracellular bacteria. The data provide robust genetic evidence that inhibiting autophagy through mutations in Drosophila atg1 prevents both Parkin and Ref(2)P from exerting their protective effects on mitochondria (de Castro, 2013).

These data indicate that enhancing the autophagy pathway improved mitochondrial function in a Drosophila model of mitochondrial dysfunction, suggesting this pathway and clearance of damaged mitochondria as a potential therapeutic target in PD pathogenesis. This opens a promising avenue of exploring the role of autophagy-inducing agents in the prevention and treatment of neurodegenerative diseases, such as PD associated with mitochondrial dysfunction (de Castro, 2013).

Leucine-Rich Repeat Kinase 2 interacts with Parkin, DJ-1 and PINK-1 in a Drosophila melanogaster model of Parkinson's disease

Mutations in the LRRK2 gene are the most common genetic cause of familial Parkinson's disease (PD). However, its physiological and pathological functions are unknown. Therefore, several independent Drosophila lines were generated carrying WT or mutant human LRRK2 (mutations in kinase, COR or LRR domains, resp.). Ectopic expression of WT or mutant LRRK2 in dopaminergic neurons caused their significant loss accompanied by complex age-dependent changes in locomotor activity. Overall, the ubiquitous expression of LRRK2 increased lifespan and fertility of the flies. However, these flies were more sensitive to rotenone. LRRK2 expression in the eye exacerbated retinal degeneration. Importantly, in double transgenic flies, various indices of the eye and dopaminergic survival were modified in a complex fashion by a concomitant expression of PINK1, DJ-1 or Parkin. This evidence suggests a genetic interaction between these PD-relevant genes (Venderova, 2009).

These results are of significance because of the following: (1) hLRRK2 fly models were generated using several independent hLRRK2 mutant lines which support a pro-death role of LRRK2 in dopaminergic neurons. (2) LRRK2 flies have multiple surprising phenotypes not expected of a protein with pro-death function. This includes a complex and nonlinear behavioral phenotype, as well as increased basal lifespan. (3) These transgenic flies show increased sensitivity to rotenone, a commonly used pesticide and a complex I inhibitor that increases a production of reactive oxygen species, both in terms of lifespan and dopaminergic loss, suggesting a potentially important relationship between environment and genetics. (4) Finally, this study shows a complex interesting genetic interaction between LRRK2 and the recessive PD genes (Venderova, 2009).

Recently, two papers have described the effects of expression of the fly orthologue of hLRRK2, dLRRK, with conflicting results. Lee (2007) shows no loss of dopaminergic neurons or deficits in climbing ability. In contrast, Imai (2008) shows loss of dopamine and of dopaminergic neurons accompanied by behavioral deficits. Because hLRRK2 and dLRRK exhibit only 38%-44% similarity in their domains, and because questions have been raised as to whether dLRRK is a true orthologue of hLRRK2, it is important to assess the effect of hLRRK2 expression. Accordingly, independent lines of WT and mutant hLRRK2-expressing Drosophila were developed and characterized. These flies display no overt developmental defects, notably a lack of nervous system pathology. This is perhaps unexpected given the association of LRRK2 with axonal development and outgrowth (MacLeod, 2006). Thus, subtle effects on nervous system integrity cannot be ruled out at this point. Clearly, however, the results indicate that expression of any of the human or other LRRK2 mutants result in loss of dopaminergic neurons. These results are consistent with the notion that LRRK2 expression results in selective dopaminergic loss in Drosophila without overt effects on other neuronal subpopulations. A similar degree of loss of dopaminergic neurons has been observed in all clusters for both WT and kinase domain mutant of hLRRK2. Taken together with the current evaluation of WT and three independent LRRK2 mutants, these data strongly support a pro-death role for ectopic LRRK2 expression, at least in Drosophila (Venderova, 2009).

The effects of LRRK2 expression on locomotor behavior are complex. After an expected initial deterioration in performance compared with control (that correlates with loss of DA neurons), all the transgenic lines outperformed the control flies at later time points. While the earlier diminution of activity is consistent with that reported by others with dLRRK expression or expression of the human G2019S mutant, the results suggest that the consequences of dopaminergic loss may be quite complex at later time points. Clearly, at these points, behavior does not correlate with dopaminergic loss. However, it is speculated that this effect reflects a dopaminergic or non-dopaminergic compensatory mechanism resulting from loss of dopaminergic neurons. Consistent with this, it is known that mice treated with the dopaminergic toxin MPTP exhibit an increase in dopamine turnover. This may reflect a mechanism by which the surviving dopaminergic terminals compensate for a decrease in the neuronal population. In fact, numerous reports have suggested that under certain conditions, mice may display increased locomotor activity upon dopaminergic toxin MPTP treatment. Similarly, it is proposed that the later increase in locomotor activity observed in flies may be a compensatory response to loss of a subset of dopaminergic terminals (Venderova, 2009).

The results also show some surprising results when it comes to basal lifespan of hLRRK2 transgenic flies. For example, under room temperature conditions with ubiquitous expression, most transgenic hLRRK2 lines showed increased lifespan compared with controls. Interestingly, in support of the data, dLRRK loss-of-function mutants have a slightly shorter lifespan (Wang, 2008). This suggests that LRKK2, in addition to its pro-death function as it relates to dopaminergic neurons, may possess properties which are protective. It is important to note that these results contrast with a recently published paper (Liu, 2008) which shows a shortened lifespan of flies expressing WT or kinase mutant of hLRRK2. The reason for this discrepancy is unclear. However, the flies in the current study were grown under less-crowded conditions than previously reported and that the control flies in the aforementioned report showed significantly shorter lifespan than the controls in the current study. Finally, it is important to note that specific neuronal expression of mutant LRRK2 (in contrast to ubiquitous expression of LRRK2 or expression of WT LRRK2 in neurons) did not promote differences in lifespan at any temperature. However, LRRK2 mutant expression in TH positive neurons still affected climbing behavior in a complex pattern. It is, therefore, unlikely that the observed behavioral differences are due to alterations in relative lifespan (Venderova, 2009).

Due to the relatively low lifetime penetrance of LRRK2 mutations, it is likely that environmental factors play an important role in the etiology of familial PD. Rotenone is a commonly used pesticide and a complex I inhibitor that increases a production of reactive oxygen species. It has been used to model PD in rodents and in Drosophila. A chronic paradigm was used with lower doses of rotenone that would more realistically mimic a possible exposure to environmental toxins. Hence, the maximum survival of control flies in this experiment was relatively long, over 2 months. All hLRRK2-expressing lines were significantly more sensitive to rotenone than controls. These results are consistent with the notion that mutations in other PD genes, such as DJ-1 and Parkin, also render cells more sensitive to a variety of external stressors. Moreover, rotenone-treated hLRRK2 flies exhibited the greatest degree of dopaminergic loss, compared with both rotenone-treated controls, or vehicle-treated hLRRK2 expressing flies. Taken together, these results point to a potentially important interaction between environmental factors, such as rotenone, and genetic makeup in the control of loss of dopaminergic neurons (Venderova, 2009).

The reasons why LRRK2 expression increases basal lifespan while increasing susceptibility to exogenous environmental stress are unclear. LRKK2 has recently been shown to regulate responses to oxidative stress through phosphorylating 4E-BP. 4E-BP, in its un-phosphorylated state, acts as a brake on a cap-dependent translation mediated by eIF4E. Clearly, the regulation of this pathway (and protein translation) has a large number of consequences depending upon the circumstances. Overexpression of dLRRK has been linked to oxidative stress via this pathway (Imai, 2009). Interestingly, some authors noted that low levels of oxidative stress result in increased longevity. It is possible, for example, that overexpression of hLRRK2 may result in such an increase under low stress (basal) conditions, but reduce longevity when confronted with higher levels of environmental stressors. Further studies are required to explore these possibilities (Venderova, 2009).

The transgenic flies showed a complex eye phenotype, including glossy and rough surface with necrotic lesions, pigmentation loss, holes, disorganization and/or loss of interommatidial bristles and disorganization of the ommatidial array. This phenotype allowed for analysis of the interaction of LRRK2 with other known PD genes. Strong evidence is presented that the three recessive PD genes interact with LRRK2. However, the genetic interactions are not straightforward. The fact that they do not always follow what one would expect (e.g. that overexpression of PINK1 is protective) highlights the complexity of the matter. Just as one example, PINK1 (as well as Parkin or DJ-1) clearly present a relatively straightforward interaction with LRRK2 when it comes to the formation of black lesions. In most cases, expression of PINK1 leads to a reduction in black lesions while loss of PINK1 exacerbates these black lesions. This would strongly implicate a protective role of PINK1 in black lesions formation with respect to LRRK2. However, when one looks at other parameters, such as bristle loss, PINK1 expression in fact exacerbates the LRRK2 phenotype. It seems that the right dose of (or balance between) LRRK2, PINK1, DJ-1 and Parkin is crucial for cell survival. In the case of PINK1, this might make sense considering growing evidence of the importance of PINK1 in mitochondrial dynamics and quality control. In this case, too much PINK1 activity might have a deleterious effect, similar perhaps to loss of function. This observation also adds a level of complexity to the understanding of the protective role of PINK1. It is proposed that the direction of the interaction (suppression versus enhancement of the phenotype) depends on several other factors, especially the parameter/cell type studied (Venderova, 2009).

LRRK2 impacts a subset of signaling pathways common to these PD genes, although the biochemical underpinnings of the interaction between LRRK2 and the other Parkinson's genes are unknown. For example, DJ-1 has been shown to modulate the PI3 kinase/AKT pathway in flies, an upstream branch of mTOR pathway which regulates 4E-BP. In addition, Parkin has been shown to interact with LRRK2 in mammalian cells in vitro. It is important to emphasize that only certain hLRRK2 mutations affect the different parameters analyzed and/or genetically interact with hPINK1, hParkin or hDJ-1. The reason for this is unclear but may relate to potentially different signaling pathways affected by different mutants (Venderova, 2009).

In conclusion, this study reports an hLRRK2 fly model of PD and identified PINK1, Parkin and DJ-1 as LRRK2 interactors. This demonstrates that this model is suitable for a suppressor/enhancer screening (Venderova, 2009).

Silent information regulator 2 (Sir2) and Forkhead box O (FOXO) complement mitochondrial dysfunction and dopaminergic neuron loss in Drosophila PTEN-induced kinase 1 (PINK1) null mutant

PTEN-induced kinase 1 (PINK1), which is associated with early onset Parkinson disease, encodes a serine-threonine kinase that is critical for maintaining mitochondrial function. Moreover, another Parkinson disease-linked gene, parkin, functions downstream of PINK1 in protecting mitochondria and dopaminergic (DA) neuron. In a fly genetic screening, knockdown of Sir2 blocked PINK1 overexpression-induced phenotypes. Consistently, ectopic expression of Sir2 successfully rescued mitochondrial defects in PINK1 null mutants, but unexpectedly, failed in parkin mutants. In further genetic analyses, deletion of FOXO nullified the Sir2-induced mitochondrial restoration in PINK1 null mutants. Moreover, overexpression of FOXO or its downstream target gene such as SOD2 or Thor markedly ameliorated PINK1 loss-of-function defects, suggesting that FOXO mediates the mitochondrial protecting signal induced by Sir2. Consistent with its mitochondria-protecting role, Sir2 expression prevented the DA neuron loss of PINK1 null mutants in a FOXO-dependent manner. Loss of Sir2 or FOXO induced DA neuron degeneration, which is very similar to that of PINK1 null mutants. Furthermore, PINK1 deletion had no deleterious effect on the DA neuron loss in Sir2 or FOXO mutants, supporting the idea that Sir2, FOXO, and PINK1 protect DA neuron in a common pathway. Overall, these results strongly support the role of Sir2 and FOXO in preventing mitochondrial dysfunction and DA neuron loss, further suggesting that Sir2 and FOXO function downstream of PINK1 and independently of Parkin (Koh, 2012).

From these findings, the following model is proposed for PINK1-mediated mitochondrial protection. To protect mitochondria, PINK1 translocates Parkin to mitochondria and activates its E3 ubiquitin ligase activity. In mitochondria, Parkin ubiquitinates mitochondrial proteins such as voltage-dependent anion channel 1 (VDAC1) and mitofusin (Mfn) to regulate mitochondrial remodeling process. In addition to the direct action in mitochondria, PINK1 transduces signals to the cytosol and activates Sir2. Sir2 deacetylates FOXO and induces the FOXO-dependent transcription of mitochondrial protective genes including SOD2 and Thor in the nucleus. The expressed proteins locate to the cytosol or mitochondria and play their roles such as scavenging harmful reactive oxygen species (ROS) and enhancing production of mitochondrial proteins. Through the direct regulation of mitochondrial protein turnover and the induction of mitochondrial protective gene expression, PINK1 can efficiently protect cells from mitochondrial damages (Koh, 2012).

Roles of PINK1, mTORC2, and mitochondria in preserving brain tumor-forming stem cells in a noncanonical Notch signaling pathway

The self-renewal versus differentiation choice of Drosophila and mammalian neural stem cells (NSCs) requires Notch (N) signaling. How N regulates NSC behavior is not well understood. This study shows that canonical N signaling cooperates with a noncanonical N signaling pathway to mediate N-directed NSC regulation. In the noncanonical pathway, N interacts with PTEN-induced kinase 1 (PINK1) to influence mitochondrial function, activating mechanistic target of rapamycin complex 2 (mTORC2)/AKT signaling. Importantly, attenuating noncanonical N signaling preferentially impairs the maintenance of Drosophila and human cancer stem cell-like tumor-forming cells. These results emphasize the importance of mitochondria to N and NSC biology, with important implications for diseases associated with aberrant N signaling (Lee, 2013).

These results uncover a novel mechanism of N in regulating NSC self-renewal and maintenance through a noncanonical signaling pathway involving PINK1, mTORC2, and AKT. A central feature of this noncanonical N signaling pathway is specific mitochondrial roles of N in regulating respiratory chain complex (RCC) function through direct interactions with PINK1 and select RCC subunits and in activating mTORC2. N could act through a number of possible mechanisms, such as facilitating the import or assembly of RCC components, as suggested by its interaction with complex I subunits, or directing the quality control of mitochondria, as has been implicated for PINK1. Future studies will determine the exact domains of N involved in PINK1 interaction and whether noncanonical N signaling is ligand-dependent. Although the exact mechanism remains to be determined, the results will help in understanding earlier observations in Drosophila that mutations in N affected mitochondrial respiration and data from mammalian systems implicating N in mitochondrial and metabolic regulation. The physiological significance of this newly defined noncanonical N pathway is also underscored by the phenotypes of various diseases associated with N dysregulation. For example, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a disease caused by mutations in NOTCH3, is associated with mitochondrial impairment. On the other hand, GOF mutations of Notch1 are implicated in over half of human T-cell acute lymphoblastic leukemia (T-ALL), in which a pathogenic role of mTORC2 has been proposed, although how N impinges on mTORC2 in this setting is unknown. Mammalian N has been shown to act through AKT and mitochondria to promote T-cell survival, although the mechanism is distinct from the one uncovered in this study. Finally, mTORC2 was shown to be required for the self-renewal and maintenance of cancer stem cells but dispensable in normal stem cells. The findings that cancer stem cell-like brain tumor-forming cells are particularly dependent on the noncanonical N pathway in flies and humans identify the newly discovered noncanonical N signaling pathway as a potential target for disease intervention (Lee, 2013).

Reduction of protein translation and activation of autophagy protect against PINK1 pathogenesis in Drosophila melanogaster

Mutations in PINK1 and Parkin cause familial, early onset Parkinson's disease. In Drosophila, PINK1 and Parkin mutants show similar phenotypes, such as swollen and dysfunctional mitochondria, muscle degeneration, energy depletion, and dopaminergic (DA) neuron loss. PINK1 and Parkin have been shown to genetically interact with the mitochondrial fusion/fission pathway, and PINK1 and Parkin have been proposed to form a mitochondrial quality control system that involves mitophagy. However, the in vivo relationships among PINK1/Parkin function, mitochondrial fission/fusion, and autophagy remain unclear; and other cellular events critical for PINK1 pathogenesis remain to be identified. This study shows that PINK1 genetically interact with the protein translation pathway. Enhanced translation through S6K activation significantly exacerbates PINK1 mutant phenotypes, whereas reduction of translation shows suppression. Induction of autophagy by Atg1 overexpression also rescues PINK1 mutant phenotypes, even in the presence of activated S6K. Downregulation of translation and activation of autophagy are already manifested in PINK1 mutant, suggesting that they represent compensatory cellular responses to mitochondrial dysfunction caused by PINK1 inactivation, presumably serving to conserve energy. Interestingly, the enhanced PINK1 mutant phenotype in the presence of activated S6K can be fully rescued by Parkin, apparently in an autophagy-independent manner. These results reveal complex cellular responses to PINK1 inactivation and suggest novel therapeutic strategies through manipulation of the compensatory responses (Liu, 2010; full text of article)

Previously, PINK1 and Parkin have been suggested to interact with mitochondrial fusion/fission machinery and the autophagy pathway. This study found that PINK1 also genetically interacts with the protein translation pathway. Increased global protein translation with S6K or eIF4E over expression (OE) exacerbates PINK1 mutant phenotypes, while decreased translation has the opposite effects. Overexpression of constitutively active S6Ks dramatically enhances muscle and DA neuron degeneration in PINK1 mutant flies, which can be mitigated by the co-expression of RpS6 RNAi or RpS9 RNAi, supporting that the TOR/S6K pathway modifies PINK1 mutant phenotypes through regulating global translation. Recently, it has been reported that pathogenic leucine-rich repeat kinase 2 (LRRK2), which represents the most frequent molecular lesions found in Parkinson's disease, promotes 4E-BP phosphorylation, resulting in increased eIF4E-mediated translation, enhanced sensitivity to oxidative stress, and DA neuron loss. Taken together, these results support the idea that deregulated protein translation is generally involved in the pathogenesis of Parkinson's disease (Liu, 2010).

Deregulated translation affects Parkinson's disease pathogenesis most likely at the level of energy metabolism, since protein translation is a very energy-consuming process, of which ribosomal biogenesis is the most costly, consuming approximately 80% of the energy in proliferating cells. This study shows that forced upregulation of ribosomal biogenesis in the fly muscle by the overexpression of constitutively active S6K is well tolerated in WT flies; however, such manipulation in PINK1 RNAi flies completely abolishes their flight ability, depletes ATP in the muscle and enhances muscle and DA neuron degeneration. The tolerance of increased protein translation by wild type flies is probably due to the existence of an intact mitochondrial quality control system containing PINK1 and Parkin, which can either eliminate damaged mitochondria generated during elevated energy production or minimize damages caused by increased ROS generated during energy production. However, in PINK1 or Parkin mutants that lack a functional mitochondrial quality control system, increased protein translation and the corresponding energy demand will translate into increased ROS generation, accumulation of dysfunctional mitochondria, and eventual energy depletion and tissue degeneration. Since downregulation of translation through knockdown of S6K, RpS6, or RpS9 is beneficial to PINK1 mutant flies, and S6K activity is already tuned down in PINK1 mutant flies, reduction of translation likely represents one of the cellular compensatory responses to the energy deficit caused by mitochondrial dysfunction in PINK1 mutants. Interestingly, partial reduction of S6K activity prolonges fly lifespan, whereas increased S6K activity has the opposite effects on longevity. The effects of S6K on animal lifespan and PINK1 mutant phenotypes can both be explained by the energy metabolism hypothesis and they offer a tantalizing link between aging and the pathogenesis of Parkinson's disease (Liu, 2010).

Supporting the energy metabolism model, it was shown that downregulation of protein translation by knocking down positive regulators of translation (S6K, RpS6, RpS9) or overexpressing a negative regulator (4E-BP) could rescue PINK1 mutant phenotypes. These manipulations presumably act by preserving cellular energy and reducing the workload and ROS production of mitochondria. Previously, 4E-BP OE was suggested to rescue PINK1 mutant phenotype by upregulating Cap-independent translation of stress related genes, including antioxidant genes, and boosting antioxidant gene activity has been suggested as a therapeutic strategy in the PINK1 and Parkin models of Parkinson's disease. This study found that although overexpression of antioxidant genes, such as Catalase, GTPx-1, SOD and GstS1, all showed some degree of rescue of PINK1 mutant phenotypes, their effects were in general weaker than that of Atg1 OE, Parkin OE, or Marf RNAi, particularly in the PINK1 RNAi/S6K-TE OE background. These data suggest that increasing autophagy and mitochondrial fission might be better choices to combat PINK1-related Parkinson's disease (Liu, 2010).

Autophagy is a conserved cellular process through which cytoplasmic content or defective intracellular organelles can be eliminated or recycled. Although autophagy is usually induced under adverse conditions to provide means for survival, basal level of autophagy in the cell is just as critical to the physiological health of the organism, since defects in autophagy are frequently associated with cancer, neurodegeneration, and aging. The induction of autophagy leads to the de novo formation of double membrane structure called isolation membrane, which expands to form a sealed compartment named autophagosome that will engulf materials destined for degradation. The large size of mitochondria likely poses a challenge for the autophagy machinery, as engulfment of an entire mitochondrion requires a significant amount of building materials for autophagosome formation. This is especially the case in PINK1 mutant where dysfunctional mitochondria becomes grossly swollen or aggregated. Previously, it has been shown that increased mitochondrial fission or Parkin OE could efficiently rescue the enlarged mitochondria phenotype in PINK1 mutants. The rescuing effect by increased mitochondrial fission could be due to the fact that it decreases mitochondrial size and makes it easier for the autophagosome to engulf the entire mitochondrion during mitophagy. In addition, increased mitochondrial fission could facilitate the segregation of the healthy part of a mitochondrion from the unhealthy part, thus enhancing the selective elimination of dysfunctional mitochondria through mitophagy. Supporting the mitophagy model, Parkin has been proposed to promote the efficient removal of damaged mitochondria by selectively ubiquitinating proteins on damaged mitochondria. A key prediction of the mitophagy model is that the protective effects of Parkin OE and increased mitochondrial fission as in the case of Marf RNAi will depend on the autophagy pathway. Surprisingly, this study found that blocking autophagy through Atg1 RNAi or Atg18 RNAi failed to block Parkin OE or Marf RNAi's rescuing abilities in PINK1 mutant, although Atg18 RNAi was effective in blocking the rescuing ability of Atg1 OE. This result suggests that the rescuing effect of Parkin OE or Marf RNAi is not entirely dependent on autophagy, and that other processes are likely involved. For example, Parkin has been suggested to promote mitochondrial biogenesis and regulate protein translation. Further studies are needed to elucidate the exact molecular functions of Parkin that are critically involved in mitochondrial function and tissue maintenance in vivo (Liu, 2010).

Given the well-established catabolic role of autophagy in degrading cytoplasmic contents, it helps recycle nutrients and provide energy source needed for survival under harsh conditions. In PINK1 mutants that suffer energy deficit due to mitochondrial dysfunction, induction of autophagy would present as a compensatory response to cope with the limited energy supply. Indeed, this study found that basal autophagy is induced in PINK1 mutant, and further increase of autophagy through Atg1 OE protects against PINK1 pathogenesis. Thus, decreased translation and increased autophagy both represent compensatory responses in PINK1 mutant flies, and further augmentation of these responses can effectively protect against the toxic effects of PINK1 inactivation. A previous study in cultured mammalian cells also indicated that autophagy is induced in response to PINK1 inactivation. Thus, the in vivo compensatory responses revealed in this study are likely relevant to PINK1 pathogenesis in mammals. Pharmacological interventions that promote these responses offer potential new treatment strategies for Parkinson's disease (Liu, 2010).

PINK1-Parkin Pathway Activity Is Regulated by Degradation of PINK1 in the Mitochondrial Matrix

Loss-of-function mutations in PINK1 , which encodes a mitochondrially targeted serine/threonine kinase, result in an early-onset heritable form of Parkinson's disease. Previous work has shown that PINK1 is constitutively degraded in healthy cells, but selectively accumulates on the surface of depolarized mitochondria, thereby initiating their autophagic degradation. Although PINK1 is known to be a cleavage target of several mitochondrial proteases, whether these proteases account for the constitutive degradation of PINK1 in healthy mitochondria remains unclear. To explore the mechanism by which PINK1 is degraded, a screen was performed for mitochondrial proteases that influence PINK1 abundance in the fruit fly Drosophila melanogaster. Genetic perturbations targeting the matrix-localized protease Lon were found to cause dramatic accumulation of processed PINK1 species in several mitochondrial compartments, including the matrix. Knockdown of Lon did not decrease mitochondrial membrane potential or trigger activation of the mitochondrial unfolded protein stress response (UPRmt), indicating that PINK1 accumulation in Lon-deficient animals is not a secondary consequence of mitochondrial depolarization or the UPRmt. Moreover, the influence of Lon on PINK1 abundance was highly specific, as Lon inactivation had little or no effect on the abundance of other mitochondrial proteins. Further studies indicated that the processed forms of PINK1 that accumulate upon Lon inactivation are capable of activating the PINK1-Parkin pathway in vivo. These findings thus suggest that Lon plays an essential role in regulating the PINK1-Parkin pathway by promoting the degradation of PINK1 in the matrix of healthy mitochondria (Thomas, 2014).

The Complex I subunit NDUFA10 selectively rescues Drosophila pink1 mutants through a mechanism independent of mitophagy

Mutations in PINK1, a mitochondrially targeted serine/threonine kinase, cause autosomal recessive Parkinson's disease (PD). Substantial evidence indicates that PINK1 acts with another PD gene, parkin, to regulate mitochondrial morphology and mitophagy. However, loss of PINK1 also causes complex I (CI) deficiency, and has recently been suggested to regulate CI through phosphorylation of NDUFA10/ND42 subunit. To further explore the mechanisms by which PINK1 and Parkin influence mitochondrial integrity, a screen was conducted in Drosophila cells for genes that either phenocopy or suppress mitochondrial hyperfusion caused by pink1 RNAi. Among the genes recovered from this screen was ND42. In Drosophila pink1 mutants, transgenic overexpression of ND42 or its co-chaperone sicily was sufficient to restore CI activity and partially rescue several phenotypes including flight and climbing deficits and mitochondrial disruption in flight muscles. Here, the restoration of CI activity and partial rescue of locomotion does not appear to have a specific requirement for phosphorylation of ND42 at Ser-250. In contrast to pink1 mutants, overexpression of ND42 or sicily failed to rescue any Drosophila parkin mutant phenotypes. It was also found that knockdown of the human homologue, NDUFA10, only minimally affecting CCCP-induced mitophagy, and overexpression of NDUFA10 fails to restore Parkin mitochondrial-translocation upon PINK1 loss. These results indicate that the in vivo rescue is due to restoring CI activity rather than promoting mitophagy. These findings support the emerging view that PINK1 plays a role in regulating CI activity separate from its role with Parkin in mitophagy (Pogson, 2014: PubMed).

Mitochondrial dysfunction is associated with the pathogenesis of PD. In autophagic degradation system, ubiquitination of the substrates provides a signal recognized by autophagic adaptors. As PARK2 is an E3 ligase and it ubiquitinates multiple substrates, its mitochondrial substrate is important for mitochondrial ubiquitination, a process critical for mitophagy. It was shown earlier that PARK2 mediates ubiquitination of some mitochondrial proteins. Most of them are associated with mitochondrial fusion–fission cycle, such as mitofusin 1/2 (Mfn1/2), dynamin-related protein 1 (Drp1), Miro and voltage-dependent anion channel 1 (VDAC1). Mfn1 and Mfn2 are important proteins involved in mitochondrial fusion–fission cycle. As the substrates of PARK2, the degradation of Mfn1 and Mfn2 are driven by PARK2, to prevent the damaged mitochondria to be fused to healthy mitochondria, and to promote mitochondrial fission at mitophagy initiation step. Recently, Mfn2 was identified as a PARK2 receptor to mediate PARK2 recruitment to damaged mitochondria, but as a receptor, its mediated substrates are still unclear. Miro is also a mitochondrial out membrane protein for axonal transport of mitochondria and identified as a mitochondrial substrate of PARK2 for the proteasome degradation. Miro degradation prevents mitochondrial movement and may initiate mitophagy induction. VDAC1 is a PARK2 substrate that may be recognized by p62 to mediate mitophagy, and it was also reported that the major role of VDAC1 in mitophagy is to mediate PARK2 recruitment onto mitochondria (Gao, 2015).

BNIP3L is a mitochondrial protein important for a selective autophagic degradation of mitochondria during reticulocytes maturation, and BNIP3L−/− mice exhibit mitochondrial retention in their reticulocytes. Using genetic assays in Drosophila, it was found that overexpression of BNIP3L can rescue the phenotype of mitochondrial dysfunction in pink1 mutant fly, but not in park mutant fly. In cultured cells, BNIP3L induces mitophagy in PARK2 wild-type cells but not in PARK2-deficient HeLa cells. Importantly, the direct interactions between PARK2 and BNIP3L and the enhancement of BNIP3L ubiquitiniation by PARK2 are observed. Moreover, the interactions between BNIP3L and PARK2, and the ubiquitination of BNIP3L are significantly increased when PARK2 is translocated to mitochondria, suggesting that BNIP3L is a substrate of PARK2 on mitochondria. These findings are of help to understand the mitochondrial phenotype rescue in Drosophila in genetic assays. As PARK2 acts downstream of PINK1 and overexpression of PARK2 rescues the phenotype of pink1 mutant fly, it suggests that some other factors may also affect PARK2 recruitment to damaged mitochondria in vivo but the unknown factors are not effective as PINK1 so that the phenotype of pink1 mutant fly is only rescued with the presence of extensive PARK2. It was also found that BNIP3L is a substrate of PARK2 and its function definitely depends on PAKR2. Thus, overexpression of BNIP3L rescues the phenotype of pink1 mutant fly because of the presence of PARK2 but overexpression of BNIP3L fails to rescue the phenotype in the absence of PARK2. These findings provide evidence that BNIP3L is a downstream factor of the PINK1/PARK2 pathway and that BNIP3L strictly depends on PARK2 to induce mitophagy (Gao, 2015).

Although DA neurons possess an intact PINK1/PARK2/BNIP3L pathway to cope with the disrupted mitochondria in most sporadic PD patients, increased levels of disrupted mitochondria with reduced complex I activity have been detected in PD brains. Rotenone and MPTP that inhibit complex I activity are causative factors for PD. In this study, it was observed that cells with reduced mitochondrial complex I activity induced by rotenone, MPP+ or 6-OHDA present a significant degradation of BNIP3L and that the BNIP3L-mediated mitochondrial degradation pathway is disrupted, thereby resulting in a retention of the damaged mitochondria. Interestingly, BNIP3L is degraded after the usage of mitochondrial complex I inhibitors, which will not be blocked by both lysosomal and protease inhibitors. As the lysosomal inhibitor blocks mitophagy; and the proteasomal inhibitor blocks the proteasomal system as well as mitophagy because of an inhibition of mitofusin I and II degradation, a process necessary for the initiation of mitophagy, the degradation of BNIP3L is unlikely caused by the proteasome or mitophagy. It is highly possible that it is processed by unknown proteases that are activated under mitochondrial complex I inhibitor treatment. Together with previous findings by other investigators, showing that PINK1 or PARK2 mutants interfere with mitophagy, this study suggests that the degradation of BNIP3L caused by complex I inhibition factors results in BNIP3L-inability to clear the damaged mitochondria. Thus, these findings also provide a mechanistic explanation why the existing PINK1/PARK2 pathway fails to clear the damaged mitochondria caused by complex I inhibitors in PD (Gao, 2015).

In summary, this study identifies that BNIP3L is a substrate of PARK2 on mitochondria. The BNIP3L ubiquitination induced by mitochondria-located PARK2 recruits the NBR1 to mitochondria to target the mitochondria for degradation. However, the environmental toxins that induce BNIP3L degradation can disrupt the PINK1/PARK2/BNIP3L-mediated mitophagy and cause an accumulation of damaged mitochondria, leading to the injury of DA neurons and occurrence of the disease (Gao, 2015).

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

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. This study shows 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. It was found that Mask genetically interacts with Parkin to modulate mitochondrial morphology and negatively regulates the recruitment of Parkin to mitochondria. Also, 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, these 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 (Zhu, 2015).

Recent studies suggest that PINK1 activates Parkin E3 ubiquitin ligase activity by phosphorylating both Parkin and ubiquitin, and that PINK1 recruits Parkin to the damaged mitochondrial membrane, where Parkin ubiquitinates a pool of outer mitochondrial membrane proteins and promotes mitophagy. 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 (Zhu, 2015).

Flies carrying pink1 or parkin mutations show severe mitochondrial morphological and functional defects in multiple tissues as well as age-dependent  dopaminergic (DA) dysfunction, 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. However, manipulation of mitochondrial dynamics causes the opposite effect on loss of parkin or pink1 function in mammalian cells, 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, the CI subunit NDUFA10, the GDNF receptor Ret, Sicily, dNK or Trap1 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 (Zhu, 2015).

This study shows 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. It also shows that such a rescue requires the presence of a functional autophagy pathway. Although tissue- and temporal-specific knock-down of Mask was performed with mainly one mask RNAi line, the mask loss-of-function analysis with mask genetic mutants and another independent RNAi line support the same notion that Mask dynamically regulates mitochondrial morphology. Together, these 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 show that inhibition of deubiquitinases USP30 and USP15 enhances mitochondrial clearance and quality control, and rescues mitochondrial impairment caused by pink1 or parkin mutations (Zhu, 2015).

It was found 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 demonstrates that Mask physically interacts with the Hippo effector Yorkie, and functions as an essential cofactor of Yorkie in promoting downstream target-gene expression. Interestingly, the Yorkie pathway was also shown to regulate mitochondrial structure and function during fly development. Together, these findings bring up an intriguing possibility that Mask and Yorkie together regulate mitochondrial size during development and disease. It was also shown that reducing Mask activity at the relatively progressed stage of parkin-dependent muscle degeneration mitigates the mitochondrial defects and impairs muscle function, indicating that the human Mask homolog ANKHD1 may serve as a potential therapeutic target for treating PD caused by pink1/parkin mutations (Zhu, 2015).

The complex I subunit NDUFA10 selectively rescues Drosophila pink1 mutants through a mechanism independent of mitophagy

Mutations in PINK1, a mitochondrially targeted serine/threonine kinase, cause autosomal recessive Parkinson's disease (PD). Substantial evidence indicates that PINK1 acts with another PD gene, parkin, to regulate mitochondrial morphology and mitophagy. However, loss of PINK1 also causes complex I (CI) deficiency, and was recently suggested to regulate CI through phosphorylation of NDUFA10/ND42 subunit. To further explore the mechanisms by which PINK1 and Parkin influence mitochondrial integrity, this study conducted a screen in Drosophila cells for genes that either phenocopy or suppress mitochondrial hyperfusion caused by pink1 RNAi. Among the genes recovered from this screen is ND42. In Drosophila pink1 mutants, transgenic overexpression of NADH dehydrogenase (ubiquinone) 42 kDa subunit (ND42) or its co-chaperone severe impairment of CI with lengthened youth (sicily) is sufficient to restore CI activity and partially rescue several phenotypes including flight and climbing deficits and mitochondrial disruption in flight muscles. Here, the restoration of CI activity and partial rescue of locomotion does not appear to have a specific requirement for phosphorylation of ND42 at Ser-250. In contrast to pink1 mutants, overexpression of ND42 or sicily fails to rescue any Drosophila parkin mutant phenotypes. It was also found that knockdown of the human homologue, NDUFA10, only minimally affects CCCP-induced mitophagy, and overexpression of NDUFA10 fails to restore Parkin mitochondrial-translocation upon PINK1 loss. These results indicate that the in vivo rescue is due to restoring CI activity rather than promoting mitophagy. These findings support the emerging view that PINK1 plays a role in regulating CI activity separate from its role with Parkin in mitophagy (Pogson, 2014).

PINK1 and Parkin have long been genetically linked in a common pathway that promotes mitochondrial homeostasis at least partly by directing the autophagic degradation of dysfunctional mitochondria as a mechanism of mitochondrial quality control. While this model potentially explains the occurrence of CI deficiency, oxidative stress, calcium dysregulation and elevated mtDNA mutations seen in patient tissues, and the age-related onset of PD, other models have been proposed to explain the pathological consequences of PINK1 and Parkin deficiency. Moreover, many mechanistic details by which the PINK1-Parkin pathway functions remain unexplained. To address these matters, this study conducted an RNAi screen to identify genes whose loss-of-function either phenocopies or suppresses a pink1 RNAi phenotype. A number of genes were identified that fulfill these criteria; the study then focused on ND42/NDUFA10 given the extensive literature implicating CI deficiency in PD pathogenesis and the fact that CI deficiency was previously reported in PINK1 mutant models and patient samples (Pogson, 2014).

Loss of ND42/NDUFA10 phenocopies the effect of pink1 loss on mitochondrial morphology in Drosophila cells, and ND42 overexpression rescues the pink1 mutant phenotypes. However, NDUFA10 knockdown causes only modest effects on mitophagy, supporting a separate link between CI and PINK1 function. The simplest interpretation of these findings is that PINK1 normally regulates ND42/NDUFA10 abundance or activity through direct phosphorylation. Indeed, it was recently reported that NDUFA10 lacks phosphorylation at Ser-250 in Pink1-/- cells, although it remains to be determined whether PINK1 directly or indirectly regulates NDUFA10 phosphorylation. Moreover, it was reported that expression of a phospho-mimetic version of ND42/NDUFA10 specifically rescues phenotypes in multiple PINK1 deficient systems, while an S250A mutant version of ND42/NDUFA10 that is incapable of being phosphorylated is unable to confer rescue. Consistent with this, it was found that at equivalent expression levels, the phospho-mimetic (SD) provides a slightly better phenotypic rescue than the other variants, and likewise promotes a higher CI activity. Nevertheless, it is also shown that the non-phosphorylatable S250A version is still able to restore CI activity and significantly rescue the climbing deficit in pink1 mutant flies (Pogson, 2014).

While further studies are needed to clarify the functional relationship between PINK1 and NDUFA10 in the regulation of CI, findings from this study provide further support to the mounting evidence that many manipulations that promote CI activity – overexpression of NDUFA10, sicily, heix, Ret, dNK, TRAP1 and NDI1, or treatment with vitamin K, deoxynucleosides or folic acid – can rescue pink1 mutants, suggesting a more general defect underlies CI deficiency in loss of pink1. The study hypothesizes that the loss of CI activity in pink1 mutants may be due to a general de-stabilization of CI. Assembly is a particular challenge for such a large, multi-subunit complex and occurs in a stepwise process that is highly regulated by many factors. Even its association with other ETC complexes in supercomplexes affects CI's stability. There is evidence for reduced complex stability in pink1 mutants, though this may not be specific to CI. One possibility is that PINK1 influences CI stability by directly promoting the assembly of CI, which may be regulated by NDUFA10 (Pogson, 2014).

These findings also further support that the mechanism by which PINK1 influences CI activity appears to be separable from its well-characterized role in mitophagy, since, in agreement with some studies but in contrast to others, a clear evidence of CI deficiency in parkin mutant flies was not found. Moreover, it is unexpected to find that overexpression of parkin does not rescue the CI deficiency in pink1 mutants, because substantial previous work has shown that parkin overexpression rescues all of the other pink1 phenotypes, and because a prediction of the PINK1-parkin mitophagy pathway is that activation would trigger the selective removal of mitochondria deficient in CI activity. This suggests that CI deficiency alone cannot fully account for adult locomotor phenotypes seen in pink1 mutants. Further studies are needed to clarify full spectrum of cellular defects in pink1 and parkin mutants and their relative importance to the pathologic mechanism (Pogson, 2014).

Rapamycin activation of 4E-BP prevents parkinsonian dopaminergic neuron loss

Mutations in PINK1 and parkin cause autosomal recessive parkinsonism, a neurodegenerative disorder characterized by the loss of dopaminergic neurons. To highlight potential therapeutic pathways, this study identified factors that genetically interact with parkin/PINK1. Overexpression of the translation inhibitor 4E-BP can suppress all pathologic phenotypes including degeneration of dopaminergic neurons in Drosophila. 4E-BP is activated in vivo by the TOR inhibitor rapamycin, which can potently suppress pathology in PINK1/parkin mutants. Rapamycin also ameliorates mitochondrial defects in cells from parkin-mutant patients. Recently, 4E-BP was shown to be inhibited by the most common cause of parkinsonism, dominant mutations in LRRK2. This study further shows that loss of the Drosophila LRRK2 homolog activates 4E-BP and is also able to suppress PINK1/parkin pathology. Thus, in conjunction with recent findings these results suggest that pharmacologic stimulation of 4E-BP activity may represent a viable therapeutic approach for multiple forms of parkinsonism (Tain, 2009).

This study used Drosophila as a model system to uncover genetic suppressors in order to understand the pathogenic mechanisms and to highlight putative therapeutic pathways for PD. Thor, the sole Drosophila homolog of mammalian 4E-BP1, has been identified as a genetic modifier of parkin. In the present study, the genetic interaction of Thor with parkin and PINK1 was investigated. While loss-of-function mutations in Thor dramatically decrease parkin and PINK1 mutant viability, overexpression of 4E-BP is able to suppress PINK1 and parkin mutant phenotypes, including degeneration of dopaminergic neurons. These results suggest that 4E-BP acts to mediate or promote a survival response implemented upon loss of parkin or PINK1 (Tain, 2009).

4E-BP1 is an inhibitor of 5' cap-dependent protein translation, which is known to play an important role in cellular response to changes in environmental conditions such as altered nutrient levels and various physiological stresses. It has been demonstrated that Drosophila 4E-BP is important for survival under a wide variety of stresses including starvation, oxidative stress, unfolded protein stress and immune challenge. Such a response pathway represents a likely target for possible manipulation by therapeutics. Fenetic evidence supports this idea, hence, this study attempted to validate whether this represented a viable therapeutic target (Tain, 2009).

4E-BP activity is regulated post-translationally by the TOR signaling pathway. Activated TOR hyper-phosphorylates 4E-BP inhibiting it leading to promotion of 5' cap-dependent translation. Rapamycin is a small molecule inhibitor of TOR signaling and has been shown to lead to 4E-BP hypo-phosphorylation. Genetic evidence suggested that administration of rapamycin to parkin/PINK1 mutants should relieve 4E-BP inhibition and confer a protective effect. Exposing mutant animals to rapamycin during development caused an increase in hypo-phosphorylated 4E-BP and, remarkably, was sufficient to suppress all pathologic phenotypes, including muscle degeneration, mitochondrial defects and locomotor ability. Continued administration of rapamycin during aging also completely suppressed progressive degeneration of dopaminergic neurons (Tain, 2009).

To validate this pathway as a viable target for therapy, the studies were extended to human tissue. There is growing evidence that mitochondrial dysfunction is a key pathologic event across the spectrum of parkinsonism. Mitochondrial defects have been demonstrated in a number of cell lines derived from patients with parkin mutations. This study shows that rapamycin is also capable of ameliorating mitochondrial bioenergetic and morphological defects in parkin-deficient PD patient cell lines. Thus, the results provide strong support for the proposition that modulating 4E-BP mediated translation by pharmaceuticals such as rapamycin can be efficacious in vivo and is relevant to human pathophysiology (Tain, 2009).

TOR signaling regulates a number of downstream effectors other than 4E-BP, for example, up-regulation of S6 kinase promoting protein synthesis and cell proliferation, and down-regulation of autophagy likely through inhibition of ATG1. The coordinated regulation of these pathways serves to optimize cellular activity in response to vital changes such as nutrient availability and environmental stresses. Stimulation of autophagy under nutrient-deprived conditions is a survival mechanism that recycles essential metabolic components, but this mechanism also promotes the degradation of aggregated or misfolded proteins. Thus, the potential therapeutic effects of rapamycin have been widely promoted as a strategy to combat a number of neurodegenerative diseases including PD primarily for its perceived role in promoting autophagic clearance of aggregated proteins. However, recent studies have provided compelling evidence that the pro-survival effects of rapamycin can be mediated in the absence of autophagy by reducing protein translation. This study has demonstrated that genetic ablation of 4E-BP is sufficient to completely abrogate any beneficial effects of rapamycin in vivo while inhibiting Atg5, a key mediator of autophagy, does not diminish the efficacy of rapamycin-mediated protection. Together, these results indicate that in this instance the major protective effects of rapamycin treatment are mediated through regulated protein translation, with little or no contribution from autophagy (Tain, 2009).

A switch from cap-dependent to cap-independent translation is likely to effect widespread changes in the proteome, particularly the induction of pro-survival factors including chaperones, anti-oxidants and detoxifying enzymes. In support of this, it was shown that transgenic or rapamycin-induced 4E-BP activation leads to increased protein levels of GstS1, a major detoxification enzyme in Drosophila. Interestingly, it was previously shown that transgenic overexpression of Drosophila GstS1 is able to suppress dopaminergic neuron loss in parkin mutants. Elucidating the global changes in response to 4E-BP activation will be crucial to understanding the exact molecular mechanisms of neuro-protection but currently remains unresolved (Tain, 2009).

The potential importance of 4E-BP modulation as a therapeutic target is underscored by recent findings that report the most common genetic cause of PD, dominant mutations in LRRK2, inhibit 4E-BP function through direct phosphorylation (Imai, 2008). Expression of these mutations causes disruption of dopaminergic neurons in Drosophila and mouse, however, in striking similarity to the current results, overexpression of 4E-BP can circumvent the pathogenic effects of mutant LRRK2 and prevent neurodegeneration (Imai, 2008) in Drosophila. This study shows that loss of Drosophila LRRK leads to activation of 4E-BP and can suppress pathology in PINK1 and parkin mutants. These data further support a link between LRRK2 and 4E-BP activity and a common cause of PD. Thus, the results indicate that promoting 4E-BP activity may be beneficial in preventing neurodegeneration in multiple forms of parkinsonism. Since 4E-BP activity can be manipulated by small molecule inhibitors such as rapamycin, this pathway represents a viable therapeutic target. It will be particularly interesting to determine whether rapamycin is efficacious in ameliorating pathologic phenotypes in the recently reported LRRK2 transgenic mouse model, but further studies will be necessary to determine whether pharmacologic modulation of 4E-BP function is therapeutically relevant in all forms of parkinsonism including sporadic PD (Tain, 2009).

The Parkinson's disease genes pink1 and parkin promote mitochondrial fission and/or inhibit fusion in Drosophila

Mutations in PTEN-induced kinase 1 (pink1) or parkin cause autosomal-recessive and some sporadic forms of Parkinson's disease. pink1 acts upstream of parkin in a common genetic pathway to regulate mitochondrial integrity in Drosophila. Mitochondrial morphology is maintained by a dynamic balance between the opposing actions of mitochondrial fusion, controlled by Mitofusin (mfn) and Optic atrophy 1 (opa1), and mitochondrial fission, controlled by drp1. This study explored interactions between pink1/parkin and the mitochondrial fusion/fission machinery. Muscle-specific knockdown of the fly homologue of Mfn (Marf) or opa1, or overexpression of drp1, results in significant mitochondrial fragmentation. Mfn-knockdown flies also display altered cristae morphology. Interestingly, knockdown of Mfn or opa1 or overexpression of drp1, rescues the phenotypes of muscle degeneration, cell death, and mitochondrial abnormalities in pink1 or parkin mutants. In the male germline, genetic interactions were observed between pink1 and the testes-specific mfn homologue fuzzy onion, and between pink1 and drp1. These data suggest that the pink1/parkin pathway promotes mitochondrial fission and/or inhibits fusion by negatively regulating mfn and opa1 function, and/or positively regulating drp1. However, pink1 and parkin mutant flies show distinct mitochondrial phenotypes from drp1 mutant flies, and flies carrying a heterozygous mutation in drp1 enhance the pink1-null phenotype, resulting in lethality. These results suggest that pink1 and parkin are likely not core components of the drp1-mediated mitochondrial fission machinery. Modification of fusion and fission may represent a novel therapeutic strategy for Parkinson's disease (Deng, 2008; full text of article).

Pink1 regulates mitochondrial dynamics through interaction with the fission/fusion machinery

Mitochondria form dynamic tubular networks that undergo frequent morphological changes through fission and fusion, the imbalance of which can affect cell survival in general and impact synaptic transmission and plasticity in neurons in particular. Some core components of the mitochondrial fission/fusion machinery, including the dynamin-like GTPases Drp1, Mitofusin, Opa1, and the Drp1-interacting protein Fis1, have been identified. How the fission and fusion processes are regulated under normal conditions and the extent to which defects in mitochondrial fission/fusion are involved in various disease conditions are poorly understood. Mitochondrial malfunction tends to cause diseases with brain and skeletal muscle manifestations and has been implicated in neurodegenerative diseases such as Parkinson's disease (PD). Whether abnormal mitochondrial fission or fusion plays a role in PD pathogenesis has not been shown. This study shows that Pink1, a mitochondria-targeted Ser/Thr kinase linked to familial PD, genetically interacts with the mitochondrial fission/fusion machinery and modulates mitochondrial dynamics. Genetic manipulations that promote mitochondrial fission suppress Drosophila Pink1 mutant phenotypes in indirect flight muscle and dopamine neurons, whereas decreased fission has opposite effects. In Drosophila and mammalian cells, overexpression of Pink1 promotes mitochondrial fission, whereas inhibition of Pink1 leads to excessive fusion. These genetic interaction results suggest that Fis1 may act in-between Pink1 and Drp1 in controlling mitochondrial fission. These results reveal a cell biological role for Pink1 and establish mitochondrial fission/fusion as a paradigm for PD research. Compounds that modulate mitochondrial fission/fusion could have therapeutic value in PD intervention (Yang, 2008; full text of article).

The PINK1/Parkin pathway regulates mitochondrial morphology

Loss-of-function mutations in the PTEN-induced kinase 1 (PINK1) or parkin genes, which encode a mitochondrially localized serine/threonine kinase and a ubiquitin-protein ligase, respectively, result in recessive familial forms of Parkinsonism. Genetic studies in Drosophila indicate that PINK1 acts upstream of Parkin in a common pathway that influences mitochondrial integrity in a subset of tissues, including flight muscle and dopaminergic neurons. The mechanism by which PINK1 and Parkin influence mitochondrial integrity is currently unknown, although mutations in the PINK1 and parkin genes result in enlarged or swollen mitochondria, suggesting a possible regulatory role for the PINK1/Parkin pathway in mitochondrial morphology. To address this hypothesis, the influence of genetic alterations affecting the machinery that governs mitochondrial morphology was examined on the PINK1 and parkin mutant phenotypes. Heterozygous loss-of-function mutations of drp1, which encodes a key mitochondrial fission-promoting component, are largely lethal in a PINK1 or parkin mutant background. Conversely, the flight muscle degeneration and mitochondrial morphological alterations that result from mutations in PINK1 and parkin are strongly suppressed by increased drp1 gene dosage and by heterozygous loss-of-function mutations affecting the mitochondrial fusion-promoting factors OPA1 and Mfn2. Finally, it was found that an eye phenotype associated with increased PINK1/Parkin pathway activity is suppressed by perturbations that reduce mitochondrial fission and enhanced by perturbations that reduce mitochondrial fusion. These studies suggest that the PINK1/Parkin pathway promotes mitochondrial fission and that the loss of mitochondrial and tissue integrity in PINK1 and parkin mutants derives from reduced mitochondrial fission (Poole, 2008; full text of article).

Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by the degeneration of dopaminergic neurons in the substantia nigra. Mitochondrial dysfunction has been implicated as an important trigger for Parkinson's disease-like pathogenesis because exposure to environmental mitochondrial toxins leads to Parkinson's disease-like pathology. Recently, multiple genes mediating familial forms of Parkinson's disease have been identified, including PTEN-induced kinase 1 (PINK1 ; PARK6 ) and parkin (PARK2 ), which are also associated with sporadic forms of Parkinson's disease. PINK1 encodes a putative serine/threonine kinase with a mitochondrial targeting sequence. So far, no in vivo studies have been reported for pink1 in any model system. This study shows that removal of Drosophila PINK1 homologue (CG4523; hereafter called pink1) function results in male sterility, apoptotic muscle degeneration, defects in mitochondrial morphology and increased sensitivity to multiple stresses including oxidative stress. Pink1 localizes to mitochondria, and mitochondrial cristae are fragmented in pink1 mutants. Expression of human PINK1 in the Drosophila testes restores male fertility and normal mitochondrial morphology in a portion of pink1 mutants, demonstrating functional conservation between human and Drosophila Pink1. Loss of Drosophila parkin shows phenotypes similar to loss of pink1 function. Notably, overexpression of parkin rescues the male sterility and mitochondrial morphology defects of pink1 mutants, whereas double mutants removing both pink1 and parkin function show muscle phenotypes identical to those observed in either mutant alone. These observations suggest that pink1 and parkin function, at least in part, in the same pathway, with pink1 functioning upstream of parkin. The role of the pink1-parkin pathway in regulating mitochondrial function underscores the importance of mitochondrial dysfunction as a central mechanism of Parkinson's disease pathogenesis (Clark, 2006; full text of article).

Mitochondrial pathology and muscle and dopaminergic neuron degeneration caused by inactivation of Drosophila Pink1 is rescued by Parkin

Mutations in Pink1, a gene encoding a Ser/Thr kinase with a mitochondrial-targeting signal, are associated with Parkinson's disease (PD), the most common movement disorder characterized by selective loss of dopaminergic neurons. The mechanism by which loss of Pink1 leads to neurodegeneration is not understood. This study shows that inhibition of Drosophila Pink1 (dPink1) function results in energy depletion, shortened lifespan, and degeneration of select indirect flight muscles and dopaminergic neurons. The muscle pathology was preceded by mitochondrial enlargement and disintegration. These phenotypes could be rescued by the wild type but not the pathogenic C-terminal deleted form of human Pink1 (hPink1). The muscle and dopaminergic phenotypes associated with dPink1 inactivation show similarity to that seen in parkin mutant flies and could be suppressed by the overexpression of Parkin but not DJ-1. Consistent with the genetic rescue results, , in dPink1 RNA interference (RNAi) animals, the level of Parkin protein is significantly reduced. Together, these results implicate Pink1 and Parkin in a common pathway that regulates mitochondrial physiology and cell survival in Drosophila (Yang, 2006).

What could be the molecular mechanism underlying the degeneration phenotypes induced by Pink1 dysfunction? At lease three possibilities could be envisioned. First, Pink1 may regulate energy metabolism. When Pink1 function is compromised, tissues that have the greatest demand for energy, presumably the IFM and dopaminergic neurons, may become particularly vulnerable. The observation that inhibition of Pink1 leads to ATP depletion is consistent with this possibility. The second possibility is that Pink1 may be normally required to guard against the mitochondrial pathway of apoptotic cell death, as suggested earlier. In this regard, it is interesting to note that Parkin has also been shown to prevent mitochondrial swelling and cytochrome c release in mitochondria-dependent cell death. Thus, Parkin and Pink1 may participate in a common pathway that protects cells against mitochondria-dependent cell death induced by toxic insults. The abnormal mitochondrial morphology associated with Parkin and Pink1 inactivation also suggests the third possibility that they may play fundamental roles in regulating mitochondrial biogenesis or mitochondrial dynamics, such as mitochondrial fusion or fission events. A connection between aberrant mitochondrial fission/fusion and neurodegeneration has been appreciated before. Further studies are needed to distinguish among these possibilities (Yang, 2006).

in vivo rescue studies clearly showed that the C terminus of hPink1 is required for hPink1 to rescue dPink1 RNAi phenotypes. Thus, although the C-terminal deleted form of Pink1 may have higher in vitro kinase activity in terms of autophosphorylation, it is incapable of providing the full spectrum of Pink1's biological activity. It is possible that C-terminal deletion may affect the binding of Pink1 to its substrates or other cofactors. Alternatively, deletion of Pink1 C terminus may contribute to disease pathogenesis by causing deregulation of Pink1 kinase activity (Yang, 2006).

The in vivo biochemical study showed that in dPink1 RNAi animals the level of dParkin is significantly reduced. This result provides one explanation of why Pink1 and Parkin mutants give very similar mutant phenotypes and why Parkin overexpression can rescue dPink1 RNAi phenotypes. It further supports the notion that Parkin acts downstream of Pink1 in a common pathway. The biochemical mechanism by which Pink1 regulates Parkin protein level requires further investigation. In summary, the mitochondrial pathology and IFM and dopaminergic neuron degeneration phenotypes observed in dPink1 RNAi animals and the clear genetic interaction between Pink1 and Parkin suggest that further genetic analysis of the cellular pathway involving Pink1 and Parkin will reveal fundamental mechanisms governing mitochondrial and cellular maintenance. Such mechanisms will likely be applicable to mammalian systems (Yang, 2006).

Antioxidants protect PINK1-dependent dopaminergic neurons in Drosophila

Parkinson's disease (PD) is the most frequent neurodegenerative movement disorder. Mutations in the PINK1 gene are linked to the autosomal recessive early onset familial form of PD. The physiological function of PINK1 and pathological abnormality of PD-associated PINK1 mutants are largely unknown. Inactivation of Drosophila PINK1 (dPINK1) using RNAi results in progressive loss of dopaminergic neurons and in ommatidial degeneration of the compound eye, which is rescued by expression of human PINK1 (hPINK1). Expression of human SOD1 suppresses neurodegeneration induced by dPINK1 inactivation. Moreover, treatment of dPINK1 RNAi flies with the antioxidants SOD and vitamin E significantly inhibits ommatidial degeneration. Thus, dPINK1 plays an essential role in maintaining neuronal survival by preventing neurons from undergoing oxidative stress, thereby suggesting a potential mechanism by which a reduction in PINK1 function leads to PD-associated neurodegeneration (Wang, 2006; full text of article).

Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by parkin

Autosomal recessive juvenile parkinsonism (AR-JP) is an early-onset form of Parkinson's disease characterized by motor disturbances and dopaminergic neurodegeneration. To address its underlying molecular pathogenesis, loss-of-function mutants of Drosophila PTEN-induced putative kinase 1 (PINK1), a novel AR-JP-linked gene (Valente, 2004), were generated and characterized. This study shows that PINK1 mutants exhibit indirect flight muscle and dopaminergic neuronal degeneration accompanied by locomotive defects. Furthermore, transmission electron microscopy analysis and a rescue experiment with Drosophila Bcl-2 demonstrated that mitochondrial dysfunction accounts for the degenerative changes in all phenotypes of PINK1 mutants. Notably, it was also found that PINK1 mutants share marked phenotypic similarities with parkin mutants. Transgenic expression of Parkin markedly ameliorated all PINK1 loss-of-function phenotypes, but not vice versa, suggesting that Parkin functions downstream of PINK1. Taken together, genetic evidence clearly establishes that Parkin and PINK1 act in a common pathway in maintaining mitochondrial integrity and function in both muscles and dopaminergic neurons (Park, 2006; full text of article).

The ubiquitination of PINK1 is restricted to its mature 52-kDa form

Along with Parkin, PINK1 plays a critical role in maintaining mitochondrial quality control. Although PINK1 is expressed constitutively, its level is kept low in healthy mitochondria by polyubiquitination and ensuing proteasomal degradation of its mature, 52 kDa, form. This study shows that the target of PINK1 polyubiquitination is the mature form and is mediated by ubiquitination of a conserved lysine at position 137. Notably, the full-length protein also contains Lys-137 but is not ubiquitinated. On the basis of these data, it is proposed that cleavage of full-length PINK1 at Phe-104 disrupts the major hydrophobic membrane-spanning domain in the protein, inducing a conformation change in the resultant mature form that exposes Lys-137 to the cytosol for subsequent modification by the ubiquitination machinery. Thus, the balance between the full-length and mature PINK1 allows its levels to be regulated via ubiquitination of the mature form and ensures that PINK1 functions as a mitochondrial quality control factor (Liu, 2017). Zeng, Q., et al. (2018). The bHLH protein Nulp1 is essential for femur development via acting as a cofactor in Wnt signaling in Drosophila. Curr Mol Med 17(7): 509-517. PubMed ID: 29437009
Summary:

Search PubMed for articles about Drosophila Pink1

Bonifati, V, et al. (2003), Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism. Science 299: 256-259. PubMed ID: 12446870

Chan, D. C. (2006). Mitochondria: Dynamic organelles in disease, aging, and development. Cell 125: 1241-1252. PubMed ID: 16814712

Clark, I. E., et al. (2006). Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin. Nature 441(7097): 1162-6. PubMed ID: 16672981

Sen, A., Kalvakuri, S., Bodmer, R. and Cox, R. T. (2015). Clueless, a protein required for mitochondrial function, interacts with the PINK1-Parkin complex in Drosophila. Dis Model Mech 8: 577-589. PubMed ID: 26035866

Sen, A., Damm, V. T. and Cox, R. T. (2013). Drosophila clueless is highly expressed in larval neuroblasts, affects mitochondrial localization and suppresses mitochondrial oxidative damage. PLoS One 8: e54283. PubMed ID: 23342118

Cox, R. T. and Spradling, A. C. (2009). Clueless, a conserved Drosophila gene required for mitochondrial subcellular localization, interacts genetically with parkin. Dis Model Mech 2: 490-499. PubMed ID: 19638420

Cox, R. T. and Spradling, A. C. (2009). Clueless, a conserved Drosophila gene required for mitochondrial subcellular localization, interacts genetically with parkin. Dis Model Mech 2: 490-499. PubMed ID: 19638420

Dagda, R. K., et al. (2009). Loss of PINK1 function promotes mitophagy through effects on oxidative stress and mitochondrial fission. J. Biol. Chem. 284: 13843-13855. PubMed ID: 19279012

de Castro, I. P., Costa, A. C., Celardo, I., Tufi, R., Dinsdale, D., Loh, S. H. and Martins, L. M. (2013). Drosophila ref(2)P is required for the parkin-mediated suppression of mitochondrial dysfunction in pink1 mutants. Cell Death Dis 4: e873. PubMed ID: 24157867

Deng, H., Dodson, M. W., Huang, H. and Guo, M. (2008). The Parkinson's disease genes pink1 and parkin promote mitochondrial fission and/or inhibit fusion in Drosophila. Proc. Natl. Acad. Sci. 105: 14503-14508. PubMed ID: 18799731

Gao, F., Chen, D., Si, J., Hu, Q., Qin, Z., Fang, M. and Wang, G. (2015). The mitochondrial protein BNIP3L is the substrate of PARK2 and mediates mitophagy in PINK1/PARK2 pathway. Hum Mol Genet 24: 2528-2538. PubMed 

Gautier, C. A., Kitada, T. and Shen, J. (2008). Loss of PINK1 causes mitochondrial functional defects and increased sensitivity to oxidative stress. Proc. Natl. Acad. Sci. 105: 11364-11369. PubMed ID: 18687901

Gegg, M. E., Cooper, J. M., Schapira, A. H. V. and Taanman, J.-W. (2009). Silencing of PINK1 expression affects mitochondrial DNA and oxidative phosphorylation in dopaminergic cells. PLoS ONE 4: e4756. PubMed ID: 19270741

Geisler, S., et al. (2010). PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nat. Cell Biol. 12: 119-131. PubMed ID: 2009841

Gispert, S., et al. (2009). Parkinson phenotype in aged PINK1-deficient mice is accompanied by progressive mitochondrial dysfunction in absence of neurodegeneration. PLoS ONE 4:.e5777. PubMed ID: 19492057

Goldberg, M. S., et al. (2005). Nigrostriatal dopaminergic deficits and hypokinesia caused by inactivation of the familial Parkinsonism-linked gene DJ-1. Neuron 45: 489-496. PubMed ID: 15721235

Greene, J. C., et al. (2003). Mitochondrial pathology and apoptotic muscle degeneration in Drosophila parkin mutants. Proc. Natl. Acad. Sci. 100: 4078-4083. PubMed ID: 12642658

Ham, S. J., Yoo, H., Woo, D., Lee, D. H., Park, K. S. and Chung, J. (2023). PINK1 and Parkin regulate IP(3)R-mediated ER calcium release. Nat Commun 14(1): 5202. PubMed ID: 37626046

Han, Y., Zhuang, N. and Wang, T. (2021). Roles of PINK1 in regulation of systemic growth inhibition induced by mutations of PTEN in Drosophila. Cell Rep 34(12): 108875. PubMed ID: 33761355

Hao, L. Y., Giasson, B. I. and Bonini, N. M. (2010). DJ-1 is critical for mitochondrial function and rescues PINK1 loss of function. Proc. Natl. Acad. Sci. 107(21): 9747-52. PubMed ID: 20457924

Imai, Y., et al. (2008). Phosphorylation of 4E-BP by LRRK2 affects the maintenance of dopaminergic neurons in Drosophila. EMBO J. 27: 2432-2443. PubMed ID: 18701920

Kim, R. H., et al. (2005). Hypersensitivity of DJ-1-deficient mice to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine (MPTP) and oxidative stress. Proc. Natl. Acad. Sci. 102: 5215-5220. PubMed ID: 15784737

Klein, P., Muller-Rischart, A. K., Motori, E., Schonbauer, C., Schnorrer, F., Winklhofer, K. F. and Klein, R. (2014). Ret rescues mitochondrial morphology and muscle degeneration of Drosophila Pink1 mutants. EMBO J 33: 341-355. PubMed ID: 24473149

Koh, H., et al. (2012). Silent information regulator 2 (Sir2) and Forkhead box O (FOXO) complement mitochondrial dysfunction and dopaminergic neuron loss in Drosophila PTEN-induced kinase 1 (PINK1) null mutant. J. Biol. Chem. 287(16): 12750-8. PubMed ID: 22378780

Lee, K. S., Wu, Z., Song, Y., Mitra, S. S., Feroze, A. H., Cheshier, S. H. and Lu, B. (2013). Roles of PINK1, mTORC2, and mitochondria in preserving brain tumor-forming stem cells in a noncanonical Notch signaling pathway. Genes Dev 27: 2642-2647. PubMed ID: 24352421

Liu, S. and Lu, B. (2010). Reduction of protein translation and activation of autophagy protect against PINK1 pathogenesis in Drosophila melanogaster. PLoS Genet. 6(12): e1001237. PubMed ID: 21151574

Liu, W., et al. (2009). PINK1 defect causes mitochondrial dysfunction, proteasomal deficit and alpha-synuclein aggregation in cell culture models of Parkinson's disease. PLoS ONE 4: e4597. PubMed ID: 19242547

Liu, W., et al. (2011). Pink1 regulates the oxidative phosphorylation machinery via mitochondrial fission. Proc. Natl. Acad. Sci. 108(31): 12920-4. PubMed ID: 21768365

Liu, Y., Guardia-Laguarta, C., Yin, J., Erdjument-Bromage, H., Martin, B., James, M., Jiang, X. and Przedborski, S. (2017). The ubiquitination of PINK1 is restricted to its mature 52-kDa form. Cell Rep 20(1): 30-39. PubMed ID: 28683321

Liu, Z., et al. (2008). A Drosophila model for LRRK2-linked parkinsonism. Proc. Natl. Acad. Sci. 105: 2693-2698. PubMed ID: 18258746

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date revised: 18 February 2024

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