What have we learned from Drosophila models of Parkinson’s disease?

SECTION I

Animal models of PD

A. Bjorklund and M. A. Cenci (Eds.)

Progress in Brain Research, Vol. 184

ISSN: 0079-6123

Copyright � 2010 Elsevier B.V. All rights reserved.

CHAPTER 1

What have we learned from Drosophila models of Parkinson’s disease? Ming Guo
Department of Neurology, Department of Molecular and Medical Pharmacology, Brain Research Institute,

David Geffen School of Medicine, Los Angeles, CA, USA

Abstract: Parkinson’s disease (PD) is characterized clinically by motor symptoms such as resting tremor, slowness of movement, rigidity, and postural instability, and pathologically by the degeneration of multiple neuronal types, including, most notably, dopaminergic (DA) neurons in the substantia nigra. Current medical treatment for PD focuses on dopamine replacement, but dopamine replacement ultimately fails and has little effect on a variety of dopamine-independent symptoms both within and outside the nervous system. To develop new therapies, we need to aim at alleviating widespread cellular defects in addition to those focusing on DA neuronal survival. Recent observations in Drosophila have provided important insights into the cellular basis of PD pathogenesis through the demonstration that two genes associated with familial forms of PD, pink1 and parkin, function in a common pathway. In this pathway, pink1 functions upstream of parkin to regulate mitochondrial fission/fusion dynamics and normal mitochondrial function. Subsequent observations in both fly and mammalian systems show that these proteins are important for sensing mitochondrial damage and recruiting damaged mitochondria to the quality control machinery for subsequent removal. This chapter reviews these findings, as well as studies of DJ­ 1 and Omi/HtrA2, two additional genes associated with PD that have also been implicated to regulate mitochondrial function. The chapter ends by discussing how Drosophila can be used to probe further the functions of pink1 and parkin and the regulation of mitochondrial quality more generally. In addition to PD, defects in mitochondrial function are associated with normal aging and with many diseases of aging. Thus, insights gained from the studies of mitochondrial dynamics and quality control in Drosophila are likely to be of general significance. Keywords: Parkinson’s disease; Drosophila; PINK1; parkin; mitochondria; dopaminergic neurons; animal model; mitochondrial fusion and fission; mitophagy


Corresponding author. Tel.: þ1-310-2069406; E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)84001-4

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Parkinson’s disease is a disorder involving more than dopaminergic neuronal loss Parkinson’s disease (PD) is the second most common neurodegenerative disorder affecting 5% of people over the age of 80. N and no treatments can definitively halt the progression of the disease. Thus, understanding the disease mechanisms and identifying new therapeutic strategies are crucial for treating patients with PD. Clinical features of PD include “motor symptoms” such as resting tre­ mor, slowness of movement, rigidity, and postural instability. PD is characterized pathologically by the degeneration of multiple neuronal types including, most notably, dopaminergic (DA) neurons in the substantia nigra of the midbrain (Dauer and Przed­ borski, 2003). The mainstay of current medical treatment for PD is dopamine replacement. How­ ever, this treatment becomes less effective over time and is often associated with intolerable side effects. In addition, PD patients also present with a variety of non-motor symptoms (Simuni and Sethi, 2008). These include including dementia, which occurs in one third of patients, psychiatric symptoms, such as depression, anxiety, obsession, and sleep disruption, and symptoms outside of the nervous system, including skin lesions and musculoskeletal abnorm­ alities. Some of these non-motor symptoms may be more debilitating than the motor impairment, but they do not usually respond to dopamine replace­ ment. In addition, pathology of many non-DA neu­ rons, including olfactory and brain stem neurons, predates that of DA neurons (Braak et al., 2003). In short, PD is a multi-system disease affecting more than DA neurons. Therefore, therapies targeted to DA neurons or their targets (such as dopamine replacement, cell transplantation, and deep brain stimulation) can provide some therapeutic benefit to patients, particularly with respect to the motor symptoms. However, a true cure requires that we develop therapies that target the underlying cellular defects. A prerequisite for this work is that we understand the pathogenesis of PD at the cellular and molecular levels. As will be described below, mitochondrial dysfunction, which has

consequences in multiple tissues, is crucial for pathogenesis in at least some forms of PD.

Familial forms of PD Although once believed to be solely an environ­ mental disease, over the past decade mutations in five genes have been definitively shown to med­ iate familial forms of PD. Mutations in PARKIN (PARK2) (Kitada et al., 1998), DJ-1 (PARK7) (Bonifati et al., 2003), and PTEN-induced kinase 1 (PINK1, PARK6) (Valente et al., 2004) are asso­ ciated with autosomal recessive forms of PD, while mutations in a-Synuclein (PARK1 (Poly­ meropoulos et al., 1997) and PARK4 (Singleton et al., 2003)) and Leucine-rich repeat kinase 2 (LRRK2)/Dardarin (PARK8) (Paisan-Ruiz et al., 2004; Zimprich et al., 2004) are associated with autosomal dominant forms of the disease. Muta­ tions in ATP13A2 (PARK9), which encodes a lysosomal ATPase, have been found in an atypi­ cal, autosomal recessive parkinsonism (Ramirez et al., 2006); however, the clinical manifestations, of this disease are quite distinct from PD (Schnei­ der et al., 2010). The “PARK” here refers to genetic loci that have been identified from family linkage studies for PD (Hardy et al., 2009). Together, these single gene-mediated, Mendelian forms of the disease represent about 10–15% of all PD cases. The clinical features of at least some of these familial forms of PD bear significant similar­ ity to those of sporadic forms. Thus, the hope is that studies of familial forms of the disease will also provide insights into the more prevalent sporadic forms. Formal nomenclature has utilized the term PD for sporadic PD and parkinsonism for genetic forms of PD. This is largely based on the fact that the cause of PD was previously thought to be non-genetic and solely environmental, a notion that is clearly no longer believed to be true (Hardy et al., 2006). It is also probable that as research advances, more genes that mediate Mendelian forms of PD and multigenic forms of PD will be identified. Thus, to simplify, this

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chapter uses “PD” as an overall term for both sporadic and familial forms of PD. Drosophila as a model system to study PD The identification of genes that mediate familial PD provides an unprecedented opportunity to understand the pathogenesis of the disease. Stu­ dies of in vivo functions of these PD genes in model systems have been instrumental in under­ standing PD pathogenesis. Among the various model organisms, Drosophila melanogaster has emerged as an especially effective tool to study PD genes. Drosophila has been used extensively for investigating complex biological processes, such as cell death (Hay and Guo, 2003, 2006; Hay et al., 2004), and complex behaviors such as circadian rhythms, learning and memory, sleep, and aggression. The accumulated studies from over a century have left the modern fly field with powerful molecular genetic tools (Adams and Sekelsky, 2002; St Johnston, 2002; Venken and Bellen, 2005). Drosophila also has a compact gen­ ome size (1/30th of the human genome), limited genetic redundancy, and a short generation time (10 days). The complete sequence of the Droso­ phila genome (Adams et al., 2000) has revealed that 77% of human disease genes are conserved in the fly (Bier, 2005; Rubin et al., 2000). These features make flies an excellent model system in which to study the function of disease genes including those involved in neurodegenerative dis­ eases (Lessing and Bonini, 2009) and in which to dissect genetic pathways related to these disease genes. The adult brain of Drosophila contains clusters of DA neurons (Nassel and Elekes, 1992) and these neurons degenerate when flies are fed rote­ none (Coulom and Birman, 2004), a complex I inhibitor that also triggers DA neuronal degenera­ tion in mammals (Bove et al., 2005; Sherer et al., 2003). Among the genes that mediate familial PD, only alpha-synuclein does not have a homolog in Drosophila. Nevertheless, expression of human

wild-type and PD-causing mutant forms of alpha­ synuclein in Drosophila results in DA neuronal loss (Feany and Bender, 2000; Trinh et al., 2008). In this chapter, we will mainly focus on Droso­ phila homologs of genes associated with recessive forms of PD: PINK1, parkin, and DJ-1 as they relate to mitochondrial function. We will also emphasize the implications that these studies in Drosophila have for advancing our understanding of PD in clinical settings. It is important to note that although DA neurons do show slight degen­ eration in some Drosophila models of PD (Trinh et al., 2008; Whitworth et al., 2005), the signifi­ cance of Drosophila models of PD involves much more than DA neuron pathology. Flies show defects in multiple systems, reminiscent of the multiple system involvement in PD patients. It is the cellular basis of these defects that provides insight into the pathogenesis of PD. Pink1 and parkin function in a common pathway to regulate mitochondrial integrity The PARK2 locus encodes Parkin, which has RING-finger motifs and E3 ubiquitin ligase activity in in vitro assays. Shortly after it was cloned, a major hypothesis was that loss of parkin resulted in the aberrant accumulation of toxic proteins, perhaps as a result of a failure of the ubiquitin– proteasome system to degrade substrates of Parkin ubiquitination. Consistent with this hypothesis, Parkin catalyzes K48-mediated polyubiquitination, which targets substrates for proteasomal degrada­ tion, and multiple proteins have been shown to interact with Parkin and are ubiquitinated in a Parkin-dependent manner in vitro. In some cases, overexpression of parkin can suppress toxicity asso­ ciated with overexpression of the potential target of Parkin ubiquitination. However, few of these sub­ strates have been shown to accumulate in vivo, in PARK2 patients, or parkin knockout mice, leaving their significance unclear, though clearly worthy of further investigation (reviewed in Dawson and Dawson, 2010; West et al., 2007).

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Parkin also catalyzes monoubiquitination and K63-linked polyubiquitination (reviewed in West et al., 2007), as well as a very recently described K27-linked ubiquitination (Geisler et al., 2009). Monoubiquitination and K63-linked polyubiquiti­ nation can influence cellular processes such as signal transduction, transcriptional regulation, and protein and membrane trafficking without promoting substrate degradation (Mukhopadhyay and Riezman, 2007). Together with the fact that overexpression of parkin protects from death asso­ ciated with proteasome inhibition (Chung et al., 2004; Petrucelli et al., 2002), the above observa­ tions suggest that at least some important compo­ nents of Parkin’s neuroprotective activity when overexpressed—which may or may not be the same as those lost when parkin is absent—involve ubiquitin-dependent processes other than K48­ linked ubiquitination and proteasome-dependent protein degradation. Important clues to the endogenous functions of parkin have come from studies of Drosophila parkin mutants. Flies lacking parkin exhibit dramatic mitochondrial defects—swollen mitochondria that have severely fragmented cristae—in several energy-intensive tissues, including the male germline and adult flight muscle (Greene et al., 2003; Pesah et al., 2004). The flight muscles ultimately die and their death shows features of apoptosis (Greene et al., 2003). Flies lacking parkin also display a small but significant degeneration of a subset of DA neurons (Whitworth et al., 2005). These studies in Drosophila provided the first in vivo indication that parkin regulates mitochon­ drial integrity. Subsequently, it was reported that although severe defects in mitochondrial morphol­ ogy are not observed in parkin knockout mice, these animals do display mitochondrial functional defects including reduced mitochondrial respira­ tory activity (Palacino et al., 2004). Key studies that strengthen the idea that parkin regulates mitochondrial function have come from studies of pink1, the Drosophila homolog of PINK1, and its interaction with parkin. pink1 encodes a protein with a mitochondrial targeting

sequence and a serine–threonine kinase domain. We and others reported that a large fraction of Pink1 is localized to mitochondria (Clark et al., 2006) and Drosophila lacking pink1 show pheno­ types very similar to those of flies lacking parkin: mutants are viable but exhibit increased stress sensitivity and mitochondrial morphological defects in testes and muscle (Clark et al., 2006; Park et al., 2006; Yang et al., 2006). pink1 mutants also show reduced ATP levels and mitochondrial DNA (mtDNA) content (Clark et al., 2006; Park et al., 2006; Yang et al., 2006). Flies lacking endo­ genous pink1 function but expressing PD-asso­ ciated mutant forms of pink1, either by overexpression (Yang et al., 2006) or under the control of the endogenous pink1 promoter (Yun et al., 2008), show phenotypes similar to those of pink1 null mutants, consistent with PINK1-asso­ ciated disease being the result of loss of function. As in parkin mutant flies, mitochondria in pink1 mutant flight muscle are swollen with fragmented cristae and these cells ultimately undergo apopto­ tic death (Clark et al., 2006; Park et al., 2006; Yang et al., 2006). Mitochondria within DA neurons in pink1 mutants also display aberrant morphology and there is a small but statistically significant loss of a subset of these neurons with age (Park et al., 2006; Yang et al., 2006). Mitochondrial dysfunction and oxidative stress were originally implicated in PD pathogenesis in the 1980s, based on the findings that exposure to the environmental toxin, 1-methyl-4-phenyl­ 1,2,3,6-tetrahydropyridine (MPTP), which inhibits mitochondrial respiration and promotes produc­ tion of reactive oxygen species (ROS), causes loss of DA neurons in humans and primates (Bove et al., 2005; Langston et al., 1983). It is important to note, however, that while human exposure to mitochondrial toxins kills DA neurons, resulting in a PD-like syndrome with motor symptoms, there is little effect on other tissues. In contrast, the findings that pink1 and parkin regulate mito­ chondrial integrity in multiple tissues implicate mitochondrial dysfunction as the central mechan­ ism for PD pathogenesis in both DA neurons and

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non-DA tissues. Studies of the PINK1/PARKIN pathway thus have the potential to uncover new therapies targeting the cellular defects that cause cell loss in PD, going beyond the current treat­ ment strategies of dopamine and DA neuron replacement. The similar phenotypes observed in pink1 and parkin null mutants suggested that the two genes might function in a common pathway. Indeed, parkin overexpression in flies suppresses all pink1 mutant phenotypes tested (Clark et al., 2006; Park et al., 2006; Yang et al., 2006), while pink1 overexpression does not compensate for loss of parkin function (Clark et al., 2006; Park et al., 2006). Furthermore, double mutants lacking both pink1 and parkin have phenotypes identical to, rather than stronger than, either single mutant (Clark et al., 2006; Park et al., 2006; Yang et al., 2006). In addition, several groups have reported that Parkin and Pink1 can physically interact in at least some contexts (Kim et al., 2008; Xiong et al., 2009). Together, these observations provide com­ pelling in vivo evidence that pink1 and parkin act in a linear pathway that affects mitochondrial function, with parkin functioning downstream of pink1. Several lines of evidence suggest that these observations on pink1 and parkin function in flies are relevant to humans. First, PD patients who harbor mutations in PINK1 or PARKIN are clinically indistinguishable (Ibanez et al., 2006) and mice lacking both pink1 and parkin show phenotypes no worse than those of the single mutants (Kitada et al., 2009), consistent with the hypothesis that these genes function in a common genetic pathway. Second, expression of human PINK1 (Clark et al., 2006; Yang et al., 2006) or PARKIN in Drosophila suppresses phenotypes caused by loss of function of pink1 or parkin, respectively, suggesting that the human and fly proteins are functionally conserved. Third, recent studies of pink1 knockout mice, which do not show DA neuron loss or severe defects in mito­ chondrial morphology (though see below for other evidence of effects on mitochondrial

morphology), nonetheless show reduced respira­ tory activity (Gautier et al., 2008; Morais et al., 2009). In particular, both mouse and Drosophila pink1 mutants show defects in complex I activity (Morais et al., 2009). Finally, pathological changes and defects in mitochondrial respiration have been detected in peripheral tissues from patients with PINK1 (Hoepken et al., 2006) or PARKIN (Muftuoglu et al., 2004) mutations. The pink1/parkin pathway promotes mitochondrial fission and/or inhibits fusion How do pink1 and parkin regulate mitochondrial function? Examination of pink1 and parkin mutant phenotypes in the Drosophila male germline pro­ vided an important clue that these proteins regulate mitochondrial fission/fusion dynamics. During Dro­ sophila spermatogenesis, mitochondria undergo sig­ nificant morphological changes (Fuller, 1993). Early spermatids undergo mitochondrial aggregation and fusion, creating a spherical structure known as the nebenkern, which is composed of two intertwined mitochondria (Fuller, 1993). During subsequent spermatid elongation, the nebenkern unfurls, yielding two mitochondrial derivatives that are maintained throughout subsequent stages of sper­ matogenesis. In both pink1 and parkin mutants, however, only one mitochondrial derivative is seen, suggesting a defect in mitochondrial fission or an overabundance of fusion (Deng et al., 2008). Mitochondria are continually undergoing cycles of fission and fusion. This allows mitochondria to change shape and share components. Mitochon­ drial dynamics also plays an important role in facil­ itating recruitment of mitochondria to specific cellular compartments such as synapses where ATP or Ca2þ buffering demands are high. Mito­ chondrial fusion is promoted by mitofusin (mfn), which is required for outer membrane fusion, and Optic atrophy 1 (Opa-1), which is essential for inner membrane fusion. Mitochondrial fission is pro­ moted by dynamin-related protein 1 (Drp1), a pre­ dominantly cytoplasmic protein that is recruited to

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mitochondria during fission. The recruitment of Drp1 may involve a mitochondrial outer membrane protein Fis1 (reviewed in Chen and Chan, 2009). In addition to the spermatid morphology defects seen in pink1 or parkin mutants, several other lines of evidence support the hypothesis that the pink1/parkin pathway regulates mitochondrial dynamics. First, mitochondria in pink1 or parkin mutants are clumped in large aggregates in both DA neurons and flight muscle. They are also swollen and have disrupted cristae. Second, these cellular defects in mitochondrial morphology, as well as other defects such as the degeneration of flight muscle, cell death, locomotion defects, and a decrease in dopamine levels in fly heads, can be suppressed by increasing the expression of the pro-fission molecules drp1 or fis1 and/or decreas­ ing levels of the pro-fusion molecules mitofusin or opa1 (Deng et al., 2008; Park et al., 2009; Poole et al., 2008; Yang et al., 2008). Third, heterozyg­ osity for drp1 is lethal in a pink1 mutant back­ ground, consistent with the idea that pink1 and drp1 work in the same direction to promote fission (Deng et al., 2008; Poole et al., 2008). That said, it is important to note that the phenotypes asso­ ciated with loss of pink1 or parkin, and loss of drp1, are distinct (Deng et al., 2008), indicating that Pink1 and Parkin are not core components of the fission machinery, but instead regulators of the process. Since both cellular defects and organismal defects can be rescued by manipulating mitochon­ drial dynamics, manipulation of mitochondrial dynamics provides a novel therapeutic strategy. Observations that point to roles of pink1 and parkin in regulating mitochondrial morphology have also been obtained in mammalian systems. However, in contrast to the story in Drosophila, which is consistent across cell types and labs, in mammalian systems various effects have been observed. Enlarged mitochondria have been observed in pink1 striatal neurons (Gautier et al., 2008) and in COS7 cells in which pink1 was silenced using RNAi. In the latter system, this phenotype was suppressed by fis1 or drp1 overexpression, as in Drosophila (Yang et al., 2008).

However, others have observed that loss of pink1 results in fission, with decreased levels of drp1 resulting in suppression (Dagda et al., 2009; Exner et al., 2007; Lutz et al., 2009; Sandebring et al., 2009). The reasons for these differences are not clear but they are undoubtedly interesting and important. Screens in Caenorhabditis elegans have shown that disruption of many genes leads to changes in mitochondrial morphology, including fragmentation or elongation, indicating that mor­ phology is very sensitive to a variety of signaling pathways and physiological states (Ichishita et al., 2008). Thus, it is likely that the final mitochondrial morphology phenotype observed in any particular cell type, particularly with respect to the presence or absence of pink1/parkin, will depend on many variables. In any case, what is most important is not the specific morphology observed but the functional state of the mitochondrial population and the mechanisms by which this is influenced by pink1 and parkin. Recent observations in mam­ mals and flies detailed below have provided a number of important insights. Pink1 and parkin promotes mitophagy In a seminal work, Youle and colleagues showed that in mammalian cells Parkin is recruited to mito­ chondria whose inner membrane has been depo­ larized (an outcome common to multiple forms of mitochondrial damage) or that have been treated with the herbicide paraquat, an inducer of com­ plex-1-dependent reactive oxygen species (ROS) (Narendra et al., 2008). Recruitment of Parkin was followed by removal of these (damaged) mito­ chondria through a specialized form of autophagy known as mitophagy, in which mitochondria are specifically degraded following engulfment by autophagosomes (reviewed in Goldman et al., 2010). Recent findings suggest that mitophagy is intimately linked with changes in mitochondrial size and shape brought about through fission and fusion (reviewed in Hyde et al., 2010). Mitophagy requires both the loss of fusion and the presence of

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fission. Importantly, decreased mitophagy results in the accumulation of oxidized proteins and decreased cellular respiration, strongly suggesting that the end result of this process is the selective removal of damaged mitochondria (Twig et al., 2008). Mitophagy, but not recruitment of Parkin to depolarized mitochondria, requires Drp1, indi­ cating that Parkin-dependent mitophagy requires fission (Narendra et al., 2008). Work in flies and mammals further demon­ strates that recruitment of Parkin to mitochondria depends on Pink1 (Geisler et al., 2009; Narendra et al., 2009; Vives-Bauza et al., 2010; Ziviani et al., 2010). Parkin fails to be recruited to depolarized mitochondria in cells lacking pink1 and in some but not all pink1 disease mutant backgrounds. Pink1’s ability to recruit Parkin requires Pink1 kinase activity but how kinase activity serves to recruit Parkin is unclear. In flies, early work sug­ gested a role for Pink1-dependent phosphoryla­ tion of Parkin as important for recruitment (Kim et al., 2008). But in mammals, while Pink1 and Parkin appear to localize near each other, there is no evidence that they bind each other, that Pink1 phosphorylates Parkin, or that Parkin ubi­ quitinates Pink1 (Vives-Bauza et al., 2010). How does Pink1 promote the recruitment of Parkin specifically to damaged mitochondria? Pink1 protein levels are specifically upregulated on damaged mitochondria (Narendra et al., 2009; Vives-Bauza et al., 2010; Ziviani et al., 2010). Pink1 is a membrane protein with its C-terminus facing the cytoplasm (Zhou et al., 2008). In healthy mito­ chondria, Pink1 is constitutively cleaved, releasing its C-terminal kinase domain into the cytoplasm where it is degraded in a proteasome-dependent manner. In damaged mitochondria that have lost their membrane potential, cleavage decreases and full-length Pink1 remains anchored to the mem­ brane (Narendra et al., 2009). Mitochondrial anchorage is all that is required, because tethering of Pink1 to the mitochondrial membrane through other methods is sufficient to recruit Parkin (Narendra et al., 2009). As expected, based on these observations, overexpression of pink1 in a

wild-type background, but not a parkin mutant background, is also sufficient to promote Parkin recruitment (Vives-Bauza et al., 2010; Ziviani et al., 2010). In cultured Drosophila cells, recruit­ ment of Parkin results in the ubiquitination and removal of Mitofusin, while loss of pink1 and parkin results in accumulation of Mitofusin (Ziviani et al., 2010). Ubiquitination of Mitofusin may func­ tion to prevent outer mitochondrial membrane fusion, thus facilitating the segregation and isola­ tion of damaged mitochondria. Ubiquitinated Mitofusin may also serve as a signal for mitophagy, a role also suggested for ubiquitinated voltagedependent anion channel 1 (VDAC1), which is generated in a parkin-dependent manner in response to mitochondrial damage in mammalian cells (Geisler et al., 2009). The fate of VDAC1 has not been examined in Drosophila. Interestingly, loss of mitofusin in Drosophila, and VDAC1 in mammalian cells, results in decreased recruitment of Parkin to mitochondria, suggesting that these proteins may be involved in recruitment as well. The protease that cleaves Pink in response to stress remains to be identified. In Drosophila, the Rhom­ boid-7 protease is required for Pink1 cleavage, though it remains to be shown that this cleavage activity is regulated by mitochondrial stress (Whit­ worth et al., 2008). The mammalian ortholog, PARL, is not required for damage-dependent Pink1 cleavage (Narendra et al., 2009). While these findings are intriguing, it is important to note that the recruitment of Parkin to mitochon­ dria has been carried out in cells lines (Ziviani et al., 2010). It remains to be shown that recruit­ ment of Parkin occurs in vivo in tissues that show phenotypes when pink1/parkin are removed and that this is associated with mitophagy. The findings that the pink1/parkin pathway reg­ ulates mitochondrial dynamics and mitophagy suggest an exciting model in which a failure of mitochondrial quality control lies at the heart of PD pathogenesis. In this model when mitochon­ dria undergo damage, Pink1 senses the damage, becomes stabilized, and recruits Parkin specifi­ cally to the damaged mitochondria. Parkin then

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mediates degradation of Mitofusin, promoting mitochondrial fission and mitophagy to remove these damaged mitochondria. In PINK1/parkin­ mediated PD, damaged mitochondria fail to be cleared, thus resulting in posing significant risk of damage to cells. Are there other components in the pink1/parkin pathway? Are there any other factors that function in the pink1/parkin pathway? Mutations in DJ-1 cause autosomal recessive forms of PD (Bonifati et al., 2003). DJ-1 has been suggested to function through various mechanisms, including as a tran­ scriptional coactivator, a protease, and a molecu­ lar chaperone (Dodson and Guo, 2007; Kahle et al., 2009), but exactly how DJ-1 exerts its pro­ tective effects remains unclear. Studies in both cell culture and animal models have demonstrated that DJ-1 deficiency increases sensitivity to cell death induced by oxidative stress, whereas overexpression is protective (Dodson and Guo, 2007; Kahle et al., 2009). These and other observations have suggested a model in which DJ-1 senses the redox state of the cell, as a result of the modifica­ tion of cysteines, and under oxidative conditions is activated to exert protective effects. Recent cellbased studies have reported physical interactions of DJ-1 with Pink1 (Tang et al., 2006) and with Parkin (Moore et al., 2005; Xiong et al., 2009), and that DJ-1 can serve as a substrate for Parkin­ dependent ubiquitination (Olzmann et al., 2007). In addition, loss of DJ-1 is associated with defects in mitochondrial integrity and respiration in mam­ malian cells (Krebiehl et al., 2010). In Drosophila, flies lacking DJ-1 (deletions) are viable, with their most prominent phenotype being increased sensitivity to oxidative stress as assayed by reduced survival upon paraquat or rotenone feeding (Menzies et al., 2005; Meulener et al., 2005, 2006). In one study, DJ-1 was proposed to promote health through regulation of phosphati­ dyl-inositol 3-kinase and AKT (Yang et al., 2005).

However, these studies utilized RNAi to silence expression of DJ-1, which also resulted in lethality (Yang et al., 2005). Since deletion of the DJ-1 coding regions or silencing of DJ-1 expression using other RNAi constructs results in viable flies (Meulener et al., 2005; M. Guo, unpublished obser­ vations), it remains possible that some of the observed interactions between DJ-1 and other genes represent off-targeting effects of DJ-1 RNAi, an issue that should be further explored. In any case, overexpression of DJ-1 fails to rescue pink1 mutant muscle phenotypes (Yang et al., 2006) or male sterility due to lack of pink1 or parkin (M. Guo, unpublished observations). Therefore, there is currently no in vivo support in Drosophila for the idea that DJ-1 acts in the same genetic pathway as pink1/parkin. Omi/HtrA2 encodes a mitochondrially localized serine protease (Vande Walle et al., 2008) and has been suggested to function downstream of PINK1 in a common pathway (Plun-Favreau et al., 2007) based on the findings that mammalian Omi/HtrA2 binds Pink1 and that the phosphorylation of Omi/ HtrA2 is dependent on Pink1 in mammalian cells (Plun-Favreau et al., 2007). One group has also reported the presence of mutations/polymorphisms in Omi/HtrA2 in sporadic PD patients (Strauss et al., 2005). Overexpression of Omi/HtrA2 pro­ motes apoptosis (Vande Walle et al., 2008) but loss of Omi/HtrA2 does not result in less apopto­ sis, though it does result in some loss of non-DA neurons in the striatum (Jones et al., 2003; Martins et al., 2004; Rathke-Hartlieb et al., 2002). Does Omi/HtrA2 function as a downstream tar­ get of pink1 in vivo? An overexpression-based genetic interaction is observed between pink1 and omi (Whitworth et al., 2008; Yun et al., 2008). How­ ever, in contrast to pink1 mutants, omi null mutants have normal mitochondrial morphology in both muscle and testes, and a normal number of DA neurons (Yun et al., 2008). In addition, extensive loss-of-function-based genetic interaction studies fail to provide any in vivo evidence supporting the hypothesis that omi functions in the same pathway as pink1, either upstream or downstream,

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to either positively or negatively regulate pink1. They also do not provide any clear evidence that omi acts in a parallel manner to regulate mitochon­ drial morphology (Yun et al., 2008). These loss-of­ function-based analyses are more relevant to PD than are omi overexpression-based analyses because the reported Omi/HtrA2 mutations asso­ ciated with PD are proposed to represent loss-of­ function or dominant-negative mutations (Strauss et al., 2005). In addition, mutant forms of the Dro­ sophila Omi/HtrA2 analogous to the reported dis­ ease form in sporadic PD patients, as well as a phosphorylation incompetent mutation of a serine residue thought to be the direct phosphorylation target of Pink1 (Plun-Favreau et al., 2007) retain significant, if not full, Omi/HtrA2 function in vivo (Yun et al., 2008). Thus, loss-of-function studies strongly suggest that omi does not play an essential role in regulating mitochondrial integrity in the pink1/parkin pathway (Yun et al., 2008). This con­ clusion is supported by recent work that found no association between mutations in Omi/HtrA1 and PD and work showing that the PD-associated muta­ tion in Omi/HtrA2 occur with comparable fre­ quency in unaffected populations, suggesting they represent simple polymorphisms (Ross et al., 2008; Simon-Sanchez and Singleton, 2008). Finally, it has recently been reported that overexpression of 4E-BP, which suppresses capdependent translation when hypo-phosphorylated, suppresses muscle degeneration, DA neuronal degeneration, and mitochondrial integrity pheno­ types seen in pink1 or parkin mutants (Tain et al., 2009). Consistent with these findings, feeding flies rapamycin, which inhibits Target of Rapamycin (TOR) thereby activating 4E-BP, also suppresses pink1/parkin phenotypes (Tain et al., 2009). These findings implicate cap-dependent translational con­ trol of unknown targets as regulators of the pink1/ parkin pathway or pathways that can compensate for their absence. The targets of translational reg­ ulation in this context are unknown. It is worth noting that activation of 4E-BP in the context of life span extension in flies results in increased translation of a number of mitochondrial proteins,

suggesting these as obvious targets for future char­ acterization (Zid et al., 2009). Contributions of Drosophila and future directions Recent findings of the pink1/parkin pathway raise many interesting questions. What regulates the stabilization of Pink1? What is the identity of the protease that cleaves Pink1 and how is its activity regulated? How does Pink1 regulate Parkin activity and localization? How does mutation of the Pink1 kinase domain disrupt Parkin recruitment? How does Parkin recruitment serve to promote mito­ phagy? Ubiquitination-dependent recruitment of the mitophagy machinery (Geisler et al., 2010), directed movement of damaged mitochondria to the site of autophagy (Vives-Bauza et al., 2010), and disruption of mitochondrial fusion (Tain et al., 2009) have all been suggested; do other mechanisms exist as well? Work in mammals has identified independent pathways that promote mitophagy (Zhang and Ney, 2009). In cells that lack func­ tional pink1 and/or parkin, are there other ways of inducing mitophagy? Can cellular physiology be manipulated so as to decrease the consequences of losing pink1 and/or parkin signaling in order to slow down or prevent mitochondrial damage from occurring? Finally, do pink1/parkin have mito­ phagy-independent roles in regulating mitochon­ drial and/or cellular physiology? The advantages of studying the pink1/parkin pathway in Drosophila are three-fold: (1) Loss of pink1 or parkin in the fly results in robust pheno­ types at the organismal level (held-up wings, male sterility, muscle degeneration, locomotion defects) as well as the subcellular level (mitochondrial morphology) in multiple tissues. Importantly, these phenotypes are present in young adults. Thus, they can be examined (and scored for sup­ pression or enhancement) without having to carry out prolonged aging studies. In contrast, studies of knockouts of parkin and pink1 in mice have not shown DA neuronal loss and yielded subtle phe­ notypes (Fleming et al., 2005; Gautier et al., 2008;

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Goldberg et al., 2003; Itier et al., 2003; Kitada et al., 2007; Palacino et al., 2004; Perez and Palmi­ ter, 2005; Von Coelln et al., 2004; ), which are not suitable for screens for genetic modifiers. In short, Drosophila provides robust genetic and cell biolo­ gical readouts for mitochondrial function and integ­ rity. (2) The epistatic studies of genes that mediate familial PD, which allow one to order the action of genes in a pathway, are stringent, yet rapid and straightforward to carry out in flies. (3) Unbiased forward genetic screens and compound screens for regulators of the pink1/parkin pathway, or path­ ways that can compensate for their loss, are straightforward to carry out in flies. Given that defects in mitochondria accumulate in normal aging as well as in PD, identifying multiple ways of activating mitophagy may be generally useful therapeutically in many diseases of aging. In short, Drosophila provides an indispensable and unique opportunity to contribute to the PD field. Finally, it is worth discussing the opportunities provided by the pink1 and parkin phenotypes of male sterility. These phenotypes are associated with defects in mitochondrial morphology but these phe­ notypes are only partly suppressed (the morpholo­ gical defects but not the fertility) by expression of drp1 or silencing of mitofusin. Importantly, pink1 and parkin are in this context involved in a devel­ opmental transition, not a stress response. This suggests that they may respond to different signals and could implement outcomes different from those in somatic cells. Given the evolutionary conservation of the pink1/parkin pathway, it is reasonable to propose that similar unexplored functions for these proteins may also exist in mam­ mals. These functions may also be related to those involved in protecting from PD. In summary, work in Drosophila has provided key insights in understanding PD. Flies provided the first in vivo evidence that pink1 and parkin regulate mitochondrial integrity. The stringent epistatic studies provided compelling evidence that pink1 and parkin function in a common genetic pathway, with pink1 positively regulating parkin. In contrast, loss-of-function-based studies

have not provided in vivo support for DJ-1 or Omi/HtrA2 as components of the pink1/parkin pathway. In addition, studies in Drosophila also suggest that the pink/parkin pathway promotes mitochondrial fission and/or inhibits fusion. These studies provide the first demonstration that manip­ ulation of mitochondrial dynamics can suppress the pink1/parkin phenotypes both at the cellular level (mitochondrial integrity) and at the organismal level (muscle degeneration, dopamine levels, and locomotion), thereby providing novel therapeutic targets. More recent work in cultured cells demon­ strates that pink1 and parkin regulate mitophagy in Drosophila, paralleling work in mammals, suggest­ ing the exciting possibility that failure of mitophagy, and thus mitochondrial quality control, underlies the pathogenesis of PINK1/parkin-mediated PD. The unique attributes of Drosophila (robust phe­ notypes, straightforward genetics providing oppor­ tunities for genome-wide genetic screens and drug screens) suggest that Drosophila studies will con­ tinue to move the field forward. These studies may help identify novel therapies for PD and potentially other aging-related neurodegenerative disorders.

Acknowledgments The author is grateful to Bruce A. Hay and the Guo lab members for discussions, and Bruce A. Hay and Mark Dodson for comments on the manuscript. This work is supported by grants and funds from National Institute of Health (R01, K02, P01), the Glenn Family Foundation, the Alfred P. Sloan Foundation, the Esther A. and Joseph Klingenstein Fellowship, and the McKnight Foun­ dation of Neuroscience to M.G.

Abbreviations PD pink1 Drp1 Opa-1

Parkinson’s disease PTEN-induced kinase 1 Dynamin-related protein 1 Optic atrophy 1

13

DA mtDNA ROS RNAi VDAC1

Dopaminergic Mitochondrial DNA Reactive oxygen species RNA interference Voltage-dependent anion channel 1

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