Pathologic and Therapeutic Implications for the Cell Biology of Parkin

Abstract

Mutations in the E3 ligase parkin are the most common cause of autosomal recessive Parkinson's disease (PD), but it is believed that parkin dysfunction may also contribute to idiopathic PD. Since its discovery, parkin has been implicated in supporting multiple neuroprotective pathways, many revolving around the maintenance of mitochondrial health quality control and governance of cell survival. Recent advances across the structure, biochemistry, and cell biology of parkin have provided great insights into the etiology of parkin-linked and idiopathic PD and may ultimately generate novel therapeutic strategies to slow or halt disease progression. This review describes the various pathways in which parkin acts and the mechanisms by which parkin may be targeted for therapeutic intervention.

Introduction

Of all neurodegenerative disorders, Parkinson's disease (PD) is the second most prevalent affecting about 9.5 per 1,000 of the population aged at least 65 or older (Dauer and Przedborski 2003, Hirtz, Thurman et al. 2007). Since the first identification of a monogenic, inherited form of PD in 1997, researchers have made great strides in elucidating the biochemical pathways that underlie this disease. Yet the precise etiologies of inherited and sporadic PD still remain unknown. Patients present with a wide range of pathological symptoms that include bradykinesia, resting tremor, postural instability and rigidity (Wirdefeldt, Adami et al. 2011). The classic pathological manifestations of the disease include the loss of dopaminergic neurons in the substantia nigra pars compacta and the accumulation of intracellular cytoplasmic aggregates called Lewy bodies (Forno 1996). However, it is now appreciated that both the pathology and symptomology extend far beyond the nigrostriatal system (Hornykiewicz and Kish 1987, Kupsky, Grimes et al. 1987, Braak, Ghebremedhin et al. 2004, Langston 2006, Jain 2011). Unfortunately, available treatments to date are mostly geared towards the movement disturbances while the non-nigral features of PD are less well addressed.

The majority of PD cases are idiopathic, but about 5-10% of the cases have a known underlying genetic basis (Dawson and Dawson 2003). Leucine-rich repeat kinase 2 (LRRK2) and α-synuclein are associated with autosomal dominant forms of PD, whereas parkin, PINK1 (PTEN-induced kinase 1), DJ-1 and ATP13a2 are associated with autosomal recessive forms. Of these, mutations in the gene encoding parkin are the most common cause of autosomal recessive PD, comprising about 50% of all recessive forms of the disease (Lucking, Durr et al. 2000). While the biochemical function of some proteins implicated in PD are not known (e.g. α-synuclein, DJ-1, LRRK2), parkin is a well-established ubiquitin E3 ligase whose crystal structure was recently solved (Riley, Lougheed et al. 2013, Trempe, Sauve et al. 2013, Wauer and Komander 2013). The wealth of information surrounding parkin structure and function, and particularly the strides made in the last two years, make parkin biology an attractive target for therapeutic intervention in sporadic PD, where wild-type parkin expression is preserved but may be functionally compromised or amenable to facilitation. In this review, we will detail the various molecular pathways affected by parkin, as well as the possible upstream and downstream interactors that could be targeted not only for PD, but also for other neurodegenerative diseases.

Parkin and Parkinsonism

Over a decade and a half ago, parkin was identified by the investigation of a chromosomal deletion in Japanese patients diagnosed with autosomal recessive-juvenile Parkinson's disease (AR-JP) (Matsumine, Saito et al. 1997, Kitada, Asakawa et al. 1998). Patients diagnosed with AR-JP exhibited the same characteristic symptoms as those with typical late-onset PD and were L-DOPA responsive, but postmortem analyses revealed the surprising lack of expected Lewy bodies (Takahashi, Ohama et al. 1994, Mori, Kondo et al. 1998, Yokochi 2000). Later studies, however, did report the presence of Lewy bodies in the substantia nigra and the locus coeruleus of patients with compound heterozygous parkin mutations (Farrer, Chan et al. 2001, Pramstaller, Schlossmacher et al. 2005, Sharp, Marder et al. 2014). Patients with AR-JP possessed large deletions in chromosome 6 within in the coding region of parkin (Matsumine, Saito et al. 1997, Kitada, Asakawa et al. 1998). However, later studies revealed that parkin mutations were found not only in early-onset AR-JP, but also in late-onset PD (Oliveira, Scott et al. 2003). Following the discovery of this genetic disruption, it was shown that the gene product was a RING domain E3 ubiquitin ligase (Shimura, Hattori et al. 2000, Zhang, Gao et al. 2000). Parkin is a large (1.3Mb) gene that is translated to a 465 amino-acid protein that is expressed in various tissues including the heart, testis and skeletal muscle. It is also abundantly expressed in the brain, especially the substantia nigra (Kitada, Asakawa et al. 1998, Huynh, Dy et al. 2001). There have been over 200 mutations in parkin identified in patients which span all domains of the protein (Corti, Lesage et al. 2011) and include point mutations, exon rearrangements and small deletions (Mata, Lockhart et al. 2004). Given that some of the disease-associated mutations abrogate translation of a functional protein, it has been presumed that the more subtle missense mutations would likewise cause loss-of-function, and some experimental evidence would support this assertion (Henn, Gostner et al. 2005, Sriram, Li et al. 2005, Schlehe, Lutz et al. 2008, Bosco, LaVoie et al. 2011). However, not all the mutations may directly affect E3 ligase activity, as it has been argued that some point mutations lead to decreased solubility and the propensity for aggregation of the protein (Henn, Gostner et al. 2005, Schlehe, Lutz et al. 2008), which may be particularly true of the truncated mutants.

New Insights from Parkin Structure

Parkin is a multi-domain protein, belonging to a class of RING domain E3 ligases. Parkin is unusual in that it has two RING domains, an inverted RBR (RING-InbetweenRING-RING) domain and an ubiquitin-like motif (Ubl) within its N-terminus (Sakata, Yamaguchi et al. 2003). It also contains a unique parkin-specific domain (UPD) containing the RING0 domain (Kahle, Leimer et al. 2000, Hristova, Beasley et al. 2009). The crystal structure of parkin was recently solved by multiple groups (Riley, Lougheed et al. 2013, Trempe, Sauve et al. 2013, Wauer and Komander 2013), and these impressive efforts have provided valuable insights into the behavior of parkin in the cell (Dove and Klevit 2013). These structures agreed in finding that parkin exists natively in an auto-inhibited state; the N-terminal region of parkin is folded over the RING1 and RING2 domains, occluding the active site, which requires a conformational change in order to execute an ubiquitination reaction (Riley, Lougheed et al. 2013, Trempe, Sauve et al. 2013). The structure also reveals how specific mutations in parkin affect its enzymatic activity and its folding capacity. Interestingly, even though PD-associated parkin mutations are widespread and not confined to any particular domain of the protein, they can be categorized according to predicted effects on zinc binding and protein folding, catalytic efficiency, or association with E2s, substrates, or cofactors (Trempe, Sauve et al. 2013).

Solubility: Role for parkin deficiency in sporadic PD?

Parkin contains 35 cysteine residues, making up almost 8% of the protein, which is high when considered against the proteomic average of 2% (Bosco, LaVoie et al. 2011, Dove and Klevit 2013). This characteristic is likely at least partially responsible for the protein's susceptibility to stress-induced aggregation or misfolding (Bosco, LaVoie et al. 2011). Studies have also shown that various PD-linked stressors including oxidative, nitrosative and dopamine stresses altered parkin structure, making it more insoluble (Winklhofer, Henn et al. 2003, LaVoie, Ostaszewski et al. 2005, Wang, Tan et al. 2005, Meng, Yao et al. 2011). Perhaps consistent with these in vitro studies, there is an age-dependent decrease in parkin solubility in human brain (Pawlyk, Giasson et al. 2003). Parkin solubility was also found to be decreased in brain tissue from sporadic PD and Diffuse Lewy Body disease patients compared to otherwise healthy controls (LaVoie, Ostaszewski et al. 2005, Wang, Ko et al. 2005, Kawahara, Hashimoto et al. 2008, Lonskaya, Desforges et al. 2013), as well as in the blood of PD patients (Vinish, Prabhakar et al. 2010). Interestingly, soluble parkin levels were also significantly decreased in cortices from post-mortem Alzheimer's disease (AD) patients, compared to healthy controls (Lonskaya, Shekoyan et al. 2013). This study also reported that insoluble parkin co-localized with intracellular amyloid beta. Collectively, these findings suggest that stress-induced or ageing-dependent decreases in soluble, active parkin in the brain may serve as a biochemical phenocopy of loss-of-function mutations in the protein, and contribute to risk of idiopathic PD.

Animal models

Several in vivo models have been generated to date to understand the molecular pathways affected due to loss of a functional parkin protein. In C. elegans, knocking out the parkin homolog led to increased susceptibility to apoptosis (Ved, Saha et al. 2005). Parkin-null Drosophila exhibited severe mitochondrial defects and spontaneous apoptosis in muscle, and rendered the male knockout flies sterile (Greene, Whitworth et al. 2003). Moreover, genetic interaction studies indicated that both parkin and PINK1 proteins were participants of the same pathway with PINK1 upstream of parkin, as exogenous parkin expression could partially rescue PINK1 knockout phenotype, but no rescue of the parkin phenotype was observed with PINK1 expression (Clark, Dodson et al. 2006, Park, Lee et al. 2006). Similar to Drosophila, iPSC-derived neurons generated from isolated dermal fibroblasts from human patients with homozygous PARK2 mutations displayed abnormal mitochondrial morphology, increased density of the abnormal mitochondria, and aberrant mitochondrial clearance (Imaizumi, Okada et al. 2012), implicating parkin in maintaining mitochondrial health in human neurons.

Despite the intriguing phenotype of the parkin-null fly, parkin knockout mice do not display the severe mitochondrial defects expected in brain and do not display a conspicuous parkinsonian phenotype (Goldberg, Fleming et al. 2003, Perez and Palmiter 2005). Though, neurons obtained from parkin KO mice did display susceptibility to apoptotic stress, consistent with the pro-apoptotic phenotype in fly (Johnson, Berger et al. 2012, Muller-Rischart, Pilsl et al. 2013, Charan, Johnson et al. 2014). Thus, there is a notable disparity across animal models that would indicate that the biochemical pathways influenced by parkin perhaps express some species specificity, or that compensatory redundancies may exist in some organisms but not others.

Functions attributed to Parkin in PD

Multiple mitochondrial functions have been ascribed to parkin that can collectively be categorized under mitochondrial quality control and integrity, suggesting a broad role for parkin in mitochondrial health and cell survival. It is worth noting that at rest, parkin is predominantly localized within the cytosol and may regulate many of these mitochondrial processes from a distance, whereas the relocalization of parkin to the mitochondria is an important step in its role in mitochondrial turnover.

Mitochondrial clearance via mitophagy

Autophagy is an essential and highly regulated intracellular mechanism that allows for the clearance of misfolded, mutated proteins as well as entire organelles by their sequestration and degradation in autophagolysosomal compartments (Mizushima 2007). Since parkin is an E3 ligase, earlier studies indicated that parkin might be involved in the ubiquitination of substrates in order to target them for classic degradation via the ubiquitin-proteasome system (UPS) (Tanaka, Suzuki et al. 2001). Later, parkin was also shown to play a role in a more direct mechanism of facilitating mitochondrial clearance via autophagy, termed mitophagy (Narendra, Tanaka et al. 2008). Upon stress-inducing mitochondrial depolarization by the uncoupling agent CCCP (carbonyl cyanide m-chlorophenyl hydrazone), another PD-related gene PINK1 stabilizes on the mitochondrial membrane, acting as a beacon for parkin to translocate to the mitochondria (Narendra, Jin et al. 2010). Recently, it was shown that phosphorylation of parkin by PINK1, as well as by PINK1-dependent phosphorylation of ubiquitin, was required to activate its E3 ligase activity (Kane, Lazarou et al. 2014, Kazlauskaite, Kondapalli et al. 2014, Koyano, Okatsu et al. 2014, Ordureau, Sarraf et al. 2014). Upon this translocation, parkin initiates ubiquitination of the outer mitochondrial membranes and recruitment of the proteasome, thus marking them for UPS and autophagic destruction (Narendra, Tanaka et al. 2008, Chan, Salazar et al. 2011, Yoshii, Kishi et al. 2011). This was confirmed by the rapid recruitment of LC3 and the LC3-binding adapter protein p62 at the mitochondria (Narendra, Jin et al. 2010, Yang and Yang 2013, Ashrafi, Schlehe et al. 2014). Several outer mitochondrial membrane proteins such as TOM20 (translocase of outer membrane 20), VDAC (voltage-dependent anion channel) and Bcl-2 have been shown to be directly associated with parkin (Chen, Gao et al. 2010, Chan, Salazar et al. 2011). A component of the autophagic machinery, Ambra1 (activating molecule in Beclin1-regulated autophagy) was also identified to interact with parkin and promote mitochondrial clearance (Van Humbeeck, Cornelissen et al. 2011). A robust, large-scale analysis of all the parkin ubiquitylome during mitophagy identified 1654 proteins that ubiquitinated at the mitochondria (Sarraf, Raman et al. 2013).

Though mitophagy has been routinely studied in immortalized cell-lines, parkin-dependent mitophagy in neurons remains less well understood. There has been a large disparity across studies assessing endogenous neuronal parkin translocation to the mitochondria upon mitochondrial depolarization (Grenier, McLelland et al. 2013). Several factors such as bioenenergetics and culture conditions are thought to affect the process of mitophagy (Van Laar, Arnold et al. 2011, Yao, Gandhi et al. 2011, Amadoro, Corsetti et al. 2014). Studies in our lab have also demonstrated that parkin-mediated mitophagy was a variable process, and depended on multiple factors, including cell-type, level of parkin expression, and the resolution of the techniques used to assess translocation and mitophagy (Charan, Johnson et al. 2014). Notably, using high-resolution microscopy aided by microfluidics, parkin-dependent mitophagy could be readily observed in neuronal axons (Ashrafi, Schlehe et al. 2014), perhaps providing the most convincing demonstration of neuronal mitophagy to date (Lu 2014). Thus, PINK1 and parkin-dependent mitophagy is one of the mechanisms by which parkin maintains mitochondrial quality control, but how defects in this process contribute to selective cell loss and PD pathology remains to be fully understood.

Regulation of Apoptosis

Several functions of parkin implicate coordinated functions of both parkin and PINK1, frequently requiring the stabilization of PINK1 at the mitochondria. However, the anti-apoptotic function of parkin would appear to be PINK1-independent. The inhibition of cytochrome c release and subsequent apoptosis by parkin is not associated with a stabilization of PINK1 nor the translocation of parkin to the mitochondria (Berger, Cortese et al. 2009) that occurs only under depolarized conditions (Narendra and Youle 2011), providing a clear distinction from the events preceding mitochondrial turnover (Johnson, Charan et al. 2012). The neuroprotective role of parkin has been widely documented since the cloning of the gene. Parkin has been shown to prevent cell death under several different conditions such as ER stress (Bouman, Schlierf et al. 2011), proteotoxic stress (Imai, Soda et al. 2000), dopamine stress (Jiang, Ren et al. 2004), mitochondrial toxins (Rosen, Veereshwarayya et al. 2006, Henn, Bouman et al. 2007), apoptotic stress (Darios, Corti et al. 2003, Berger, Cortese et al. 2009, Johnson, Berger et al. 2012, Charan, Johnson et al. 2014), increased Wnt signaling (Rawal, Corti et al. 2009) as well as alpha-synuclein toxicity (Petrucelli, O'Farrell et al. 2002, Lo Bianco, Schneider et al. 2004). One of the mechanisms by which parkin is neuroprotective is through inhibition of cytochrome c release from the mitochondria (Darios, Corti et al. 2003, Berger, Cortese et al. 2009). Our group demonstrated the parkin-mediated prevention of Bax translocation from the cytoplasm to the mitochondria, and the regulation of this individual parkin substrate was able to fully account for the control over cytochrome c release and cell death (Johnson, Berger et al. 2012). Further studies, including additional work from our group and others have since confirmed the ubiquitination (Johnson, Berger et al. 2012, Sarraf, Raman et al. 2013) and regulation (Muller-Rischart, Pilsl et al. 2013, Charan, Johnson et al. 2014) of Bax by parkin, and that parkin is neuroprotective under apoptotic stress conditions (Muller-Rischart, Pilsl et al. 2013, Charan, Johnson et al. 2014).

Ectopic expression of parkin has been shown to be protective in many different cell-types, suggesting that the anti-apoptotic role of parkin could be applied universally. Moreover, from a functional point-of view, the PINK1-dependent and PINK1-independent pathways of parkin are interlinked and may not be mutually exclusive (Johnson, Charan et al. 2012). Indeed, our group has shown that HEK293 (human embryonic kidney 293) cells overexpressing parkin could undergo CCCP-induced mitophagy as well as robust parkin-dependent ubiquitination of Bax with comparable efficiency (Charan, Johnson et al. 2014), negating the likelihood that these respective functions are cell-type dependent.

Parkin and the Bcl-2 family

In line with evidence of parkin-dependent regulation of cytochrome c release and apoptosis, it is not surprising that parkin has been shown to interact with several members of the Bcl2 family. The Bcl2 family plays a critical role in maintaining the delicate balance between survival and programmed cell death. Parkin was shown to interact with anti-apoptotic Bcl-2 protein to modulate autophagy (Chen, Gao et al. 2010), as well as interact with pro-apoptotic Bax to regulate cytochrome c release induced apoptosis (Johnson, Berger et al. 2012). Parkin also interacts with regulatory members of the Bcl2 family such as Nix, an outer mitochondrial membrane protein that is a BH3 domain-only member of the Bcl-2 family (Ding, Ni et al. 2010). Nix is thought to play a role in facilitating parkin recruitment to the mitochondria to initiate mitochondrial clearance (Ding, Ni et al. 2010). Another BH3 domain-only protein Bnip3 was also shown to play a role in the mitochondrial recruitment of parkin and induce mitophagy (Lee, Lee et al. 2011), and recently argued to be a substrate of parkin (Gao, Chen et al. 2015). Overall, this suggests a very close partnership between parkin and the Bcl2 family members in the maintenance of cellular health. Indeed, our group showed that swapping the BH3 domain of the substrate Bax with the BH3 domain of a non-substrate, Bcl-xl, led to a loss of recognition of Bax by parkin (Charan, Johnson et al. 2014). Collectively, these data suggest a critical role for the BH3 domain to facilitate the association of parkin and Bcl2 proteins, and that the precise conformation of each BH3 domain might be a determinant for binding.

Mitochondrial Morphology by Fission/Fusion

The interplay between mitochondrial fission and fusion plays a critical role in the metabolic behavior of mitochondria (Liesa and Shirihai 2013) and is likely highly important for the isolation and clearance of unhealthy mitochondria (Detmer and Chan 2007, Molina, Wikstrom et al. 2009, Exner, Lutz et al. 2012). Perturbation of the fusion and fission equilibrium has been implicated in PD (Santos and Cardoso 2012, Van Laar and Berman 2013) as well as several other neurodegenerative disorders such as AD, amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD) (Chaturvedi and Flint Beal 2013). Fusion is largely regulated by outer mitochondrial membrane proteins such as OPA1 (optic atrophy 1), mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2) (Chen, Detmer et al. 2003, Cipolat, Martins de Brito et al. 2004). Fission on the other hand, is mediated by the GTPases Drp1 that relocalizes to the tip of mitochondria from the cytoplasm, and Fis1 at the mitochondria, causing the pinching off of the mitochondrion to form two daughter mitochondria (Smirnova, Griparic et al. 2001, James, Parone et al. 2003, Loson, Song et al. 2013). Interestingly, the parkin substrate Bax has also been implicated in the fission/fusion relationship as it is found along fission sites during apoptosis (Karbowski, Lee et al. 2002) but has a pro-fusion functionality that can be divorced from its apoptotic function (Suen, Norris et al. 2008, Whelan, Konstantinidis et al. 2012). Several studies suggest PINK1 and Parkin play a regulatory role in the mitochondrial fusion/fission pathway (Lutz, Exner et al. 2009, Park, Lee et al. 2009, Poole, Thomas et al. 2010). Mfn2 was shown to be a substrate of both PINK1 and parkin (Poole, Thomas et al. 2010, Chan, Salazar et al. 2011, Chen and Dorn 2013). Phosphorylation of Mfn2 by PINK1 mediated parkin recruitment and subsequent ubiquitination in cardiac mitochondria of Drosophila, initiating its autophagic clearance (Chen and Dorn 2013). Moreover, parkin has been reported to regulate the translocation of Drp1 to the mitochondria to promote fission (Lutz, Exner et al. 2009) as well as ubiquitinate it to promote its degradation via the proteasome (Wang, Song et al. 2011).

As stated earlier, parkin-deficient flies displayed swollen and dysfunctional mitochondria (Greene, Whitworth et al. 2003), which may indicate a defect in the fission pathway. Thus, it was hypothesized that the PINK1-parkin pathway promotes the fission pathway and inhibits the fusion pathway in order to facilitate unhealthy mitochondrial clearance by mitophagy in Drosophila (Deng, Dodson et al. 2008). In contrast, in mammalian models knockdown of parkin led to increased fragmentation, suggesting an increase in the fission pathway along with a decrease in mitochondrial membrane potential (Lutz, Exner et al. 2009). This discrepancy across species in the role of parkin during fission/fusion is still unclear, however, several explanations including as bioenergetics, tissue-specificity and temporal differences may be at play (Pilsl and Winklhofer 2012).

Transcriptional Regulation

Though parkin is an E3 ligase with the primary function of ubiquitin transfer to substrates, there have been a few studies that implicate parkin more broadly in transcriptional regulation of proteins. A transcriptional repressor of PGC-1alpha (PPAR gamma coactivator-1alpha) named PARIS (PARkin Interacting Substrate) was identified as a substrate of parkin E3 ligase activity (Shin, Ko et al. 2011). Parkin was also identified as a transcriptional repressor of the transcription factor p53 (da Costa, Sunyach et al. 2009), and suggested to be regulated downstream of p53 (Zhang, Lin et al. 2011), implicating a dual role in tumor suppression. Interestingly, presenilin-1 and presenilin-2, proteins implicated in familial AD and downstream targets of p53, were also identified to be transcriptional targets of parkin activity (Duplan, Sevalle et al. 2013). In some cases, the transcriptional regulation by parkin was found to be independent of its E3 ligase activity, and by a direct physical interaction with the promoter (da Costa, Sunyach et al. 2009, Alves da Costa and Checler 2012, Duplan, Sevalle et al. 2013). The precise mechanism of this regulation remains unknown and further study is required to discover other transcriptional targets of parkin and understand this potential aspect of parkin function.

Universal role of parkin

Cardiac health

Parkin KO mice do not display spontaneous loss of dopaminergic neurons nor show deficits in motor activity. However, studies have shown that parkin KO mice have several cardiac deficits. Parkin deficient mice have decreased recovery of cardiac contractility after sepsis activation, and impaired mitophagy after myocardial infarction (Kubli, Quinsay et al. 2013, Kubli, Zhang et al. 2013, Piquereau, Godin et al. 2013). This resulted in decreased survival and larger infarct sizes when compared to wild type controls. Proteins implicated in interacting with parkin also impinge upon cardiac health. Deficiency of the parkin substrate Mfn2 in the cardiomyocytes of both Drosophila and mice led to mitochondrial dysfunction and cardiomyopathy (Chen and Dorn 2013). Loss of the parkin interacting protein Nix/BNIP3 in the heart also caused accumulation of dysfunctional mitochondria and cardiac dysfunction (Dorn 2010). Moreover, dysregulation of mitochondrial fission through Drp1 inhibition in rat hearts protected them against ischemia and reduced cell death (Ong, Subrayan et al. 2010). Therefore, the cell biology of parkin, and its homeostatic functions clearly exert important physiological influence outside the brain. Interestingly, there is a significantly increased incidence of cardiomyopathy in aged patients with PD as compared to non-PD patients (Zesiewicz, Strom et al. 2004). Though cardiac sympathetic denervation is more common in PD patients, this study did not account for factors that may bias the results, such as PD medications thought to contribute to heart disease and more routine doctor visits that would provide early diagnosis.

Role in cancer

Though at the opposite ends of the spectrum in terms of cellular health, there have been many epidemiological studies linking parkin with altered risks for cancer (Checler and Alves da Costa 2014, Xu, Lin et al. 2014). Parkin was thought to act as a tumor-suppressor gene because of its location in chromosome 6, implicated as a hotspot in several human cancers (Garber 2010), and deletions in the PARK2 locus and parkin mutations were found in a wide variety of tumors (Cesari, Martin et al. 2003, Veeriah, Taylor et al. 2010). Recently, a vast study of about 5000 tumors showed that deletions in parkin were the most commonly occurring deletions, comprising of almost 30% of all tumors studied (Bartek and Hodny 2014, Gong, Zack et al. 2014). On the surface, these results do seem counterintuitive to the anti-apoptotic and mitochondrial quality control functions of parkin. However, this study speculated that parkin plays a role in regulating the cell cycle, and that loss of parkin function leads to perturbations in the cell cycle pathway by the accumulation of one of its substrates, Cyclin E (Staropoli, McDermott et al. 2003), allowing cells to continually divide (Veeriah, Morris et al. 2010, Gong, Zack et al. 2014). However, another study found no significant correlation between parkin mutation carriers and cancer history when compared to non-carriers (Alcalay, Clark et al. 2012). Therefore, the link between the tumor-suppressor properties of parkin and the neuroprotective properties of parkin warrant further investigation.

Role in other diseases

The balance between programmed cell-death and mitochondrial homeostasis needs to be highly regulated and any perturbation along this axis can have adverse effects on cell survival. Unsurprisingly, given its role in both these pathways, parkin has been linked to several other disorders such as AD (Burns, Zhang et al. 2009), autism (Glessner, Wang et al. 2009), multiple sclerosis (Witte, Bol et al. 2009), leprosy (Mira, Alcais et al. 2004) and inclusion body myositis (Rosen, Veereshwarayya et al. 2006). Accumulation of dysfunctional mitochondria and increases in cytochrome c release and apoptosis were linked to disrupted parkin function in diabetic kidney disease (DKD) (Zhan, Usman et al. 2014). It was also shown that parkin has a role to play in the normal production and secretion of insulin by pancreatic beta cells, defects in which cause diabetes mellitus (Jin, Kim et al. 2014). Interestingly, the viral infection by Hepatitis C also uses the parkin-dependent mitophagy pathway to prevent apoptosis and increase the life of infection (Kim, Syed et al. 2014).

A few parkin substrates belong to signaling pathways involved in regulation of critical functions ranging from endocytosis to inflammation. These include Eps15 (epidermal growth factor receptor substrate 15), involved in EGFR signaling and trafficking (Fallon, Belanger et al. 2006), beta-catenin, a component of the Wnt pathway that regulates cell proliferation (Rawal, Corti et al. 2009), and TRAF2 (TNF-receptor associated factor 2) and NEMO (NF-κB essential modifier), members of the NF-κB pathway that has a central role in development and immunity (Henn, Bouman et al. 2007, Muller-Rischart, Pilsl et al. 2013). Parkin function is also implicated in cardiac recovery after sepsis (Piquereau, Godin et al. 2013), and during neuroinflammation (Frank-Cannon, Tran et al. 2008, Tran, Nguyen et al. 2011). Thus, the integrity of parkin function is critical for the regulation of various cellular functions, defects in which can promote pathology.

Parkin targeted therapy for PD and other neurodegenerative diseases

As the life expectancy of our society dramatically increases, the burden of PD rises considerably. It is estimated that by the year 2030, the number of individuals above the age of 50 affected with PD in the world would rise to between 8.7 and 9.3 million (Dorsey, Constantinescu et al. 2007). Thus, it is imperative to find and implement successful therapeutic strategies in order to develop new treatments for this disorder. Moreover, protein aggregation due to defects in protein clearance and mitochondrial disturbances are central features in many neurodegenerative diseases (Bossy-Wetzel, Schwarzenbacher et al. 2004, Ross and Poirier 2004, Chen and Chan 2009, Bosco, LaVoie et al. 2011, Chaturvedi and Flint Beal 2013), thus targeting parkin-dependent survival pathways and mitochondrial quality control could potentially be therapeutically relevant far beyond PD. We outline a few of the possible mechanisms by which parkin function could be augmented or activated to therapeutic advantage.

Targeting mitochondrial health

Maintenance of mitochondrial health in the cell is proving to be a very crucial component in the preservation of neuronal function. Oxidative stress has been long thought to be one of the contributing factors in mitochondrial dysfunction and in the pathogenesis of PD. Studies have shown that exposure to oxidative stress causes decreased solubility of parkin, impairing its function (Bosco, LaVoie et al. 2011). To date, there have been several compounds generated and studied that reduce oxidative stress and improve mitochondrial function, and some of them such as creatine and CoQ10 are already being tested in clinical trials (Chaturvedi and Beal 2013, Jin, Kanthasamy et al. 2014), albeit with not much success reported. A different aspect of reducing oxidative stress in PD models also being studied is targeting activation of the Nrf2 pathway using synthetic triterpenoids (Kaidery, Banerjee et al. 2013). Nrf2 is a transcription factor that regulates the antioxidant response element (ARE) and confers cellular protective from oxidative insult (Lee, Shih et al. 2003). This targeted activation of the Nrf2 pathway would be a promising approach for the amelioration of stress-induced pathologies. Other molecules such as resveratrol and uric acid have also been implicated in the prevention of oxidative stress and promoting mitochondrial and cellular health in PD (Davis, Grandinetti et al. 1996, Cipriani, Chen et al. 2010, Chen, Burdett et al. 2013, Ferretta, Gaballo et al. 2014).

Another approach could be to target PINK1 since PINK1 and parkin are components of the same pathway regulating the turnover of damaged mitochondria, and the stabilization of PINK1 on the mitochondria is an essential step for the recruitment of parkin in order to carry out its ubiquitination function (Narendra, Jin et al. 2010). A recent study found that an ATP analog, kinetin triphosphate, increased PINK1 stabilization at the mitochondria, thus promoting higher parkin recruitment and decreased cell death (Hertz, Berthet et al. 2013). This would be an interesting methodology to find small molecules that would facilitate parkin recruitment to the mitochondria, thus increasing the efficiency of clearance of damaged mitochondria in idiopathic patients in whom parkin expression is preserved. Moreover, the recent discovery of the requirement of PINK1 to phosphorylate ubiquitin in order to activate parkin (Kane, Lazarou et al. 2014, Kazlauskaite, Kondapalli et al. 2014, Koyano, Okatsu et al. 2014) also highlights the benefits of targeting PINK1. Augmenting the PINK1 kinase activity could in turn promote phosphorylation of ubiquitin and parkin and increase its activity.

There are several post-translational changes that are thought to inactivate parkin. Modifications such as nitrosylation (Chung, Thomas et al. 2004, Yao, Gu et al. 2004), methamphetamine induced conjugation of 4-hydroxy-2-nonenal (4-HNE) to parkin (Moszczynska and Yamamoto 2011) and the irreversible dopamine-induced covalent modifications (LaVoie, Ostaszewski et al. 2005) are known to affect parkin activity. Reduction of these post-translational modifications with drugs such as melatonin (Reiter, Korkmaz et al. 2008) and taurine (Askwith, Zeng et al. 2012) are thought to reduce nitrosative stress, and Vitamin E implicated in inhibiting parkin changes (Moszczynska and Yamamoto 2011) could be further explored to support parkin activity.

Reducing parkin auto-inhibition: Catalytically active parkin

The crystal structure of parkin confirmed that under basal conditions, parkin lies in an auto-inhibited state (Riley, Lougheed et al. 2013, Trempe, Sauve et al. 2013, Wauer and Komander 2013). Since parkin overexpression during aging was shown to protect for proteotoxic stress, improve mitochondrial health and increase life-span (Rana, Rera et al. 2013), there would be a certain advantage to increase parkin activity during aging. Screening for small molecules that would bind and induce a conformational change in parkin to expose its catalytic domain would mimic overexpression of parkin and promote mitophagic turnover of damaged mitochondria and prevent cell death. Even modest allosteric modulators could be of potential benefit.

Phosphorylation of parkin appears to be a prerequisite for its ubiquitination activity. Parkin is phosphorylated by the mitochondrial kinase PINK1 at Ser65 (Kondapalli, Kazlauskaite et al. 2012) (Shiba-Fukushima, Imai et al. 2012, Iguchi, Kujuro et al. 2013), however, other serines have also been identified as phosphorylation sites in parkin (Yamamoto, Friedlein et al. 2005, Rubio de la Torre, Luzon-Toro et al. 2009). Phosphorylation of parkin by other kinases at other sites may also be part of a greater network of proteins regulation of its ligase activity (Yamamoto, Friedlein et al. 2005, Rubio de la Torre, Luzon-Toro et al. 2009). Given that cytosolic parkin has been implicated in virtually all non-mitophagic functions and substrates of parkin, there are likely other kinases that play a role in parkin activation. Understanding the precise role that kinases play in the activation and possibly repression of parkin E3 ligase activity will be essential to its pharmacological manipulation.

DUBs

Ubiquitination and clearance of proteins via the UPS is highly regulated and many mechanisms have evolved to keep protein destruction in check. One of the ways protein turnover is regulated is through deubiquitinating enzymes. Deubiquitinases (DUBs) are enzymes that remove ubiquitin molecules from proteins marked for degradation, thus reversing ubiquitination and removing the signal for subsequent degradation of the target protein. Recently, several studies were published assessing the role of DUBs associated with parkin function. USP15 and USP30 were identified as playing a role in parkin-mediated mitophagy (Bingol, Tea et al. 2014, Cornelissen, Haddad et al. 2014, Cunningham, Baughman et al. 2015). These studies reported that the overexpression of the respective DUB prevented parkin-mediated mitophagy and mitochondrial clearance. Bingol et al showed that knocking down USP30 enhanced mitochondrial clearance and promoted parkin-mediated mitophagy, while Cornelissen and colleagues showed that knockdown of the Drosophila homolog of USP15 rescued the mitochondrial and behavioral defects in parkin null flies. The fact that a DUB could rescue a parkin null fly, suggests compensatory E3 ligases in this model organism. Another deubiquitinating enzyme USP8/UBPY was discovered to play a critical role in parkin-mediated mitophagy preferentially removing the K6-linked ubiquitin chains from parkin, affecting its translocation, and subsequent degradation of substrates via mitophagy (Durcan, Tang et al. 2014).

Thus, one could hypothesize that targeting these DUBs to inhibit their activity would enhance parkin function and facilitate removal of unhealthy mitochondria and promote cell survival. Indeed, an earlier study showed that a small molecule inhibitor of another DUB, Usp14, enhanced proteasomal function and promoted protein turnover (Lee, Lee et al. 2010), confirming the drugability of these proteases and the proof-of-concept of their potential efficacy. The discovery of these parkin associated DUBs proffer an exciting opportunity to promote mitochondrial clearance and reduce accumulation of toxic substrates.

Compensatory E3 ligase

Transfer of an ubiquitin moiety to a protein is a multi-step process requiring the functions of an ubiquitin activating (E1), ubiquitin conjugating (E2) and the ubiquitin ligase (E3) enzymes, which confer the substrate specificity of this cascade. A recent curated database showed that human cells contain 2 E1s, 29 E2s and 563 E3 enzymes (Bhowmick, Pancsa et al. 2013). This system of ubiquitination resembles a pyramidal structure, enabling a highly regulated process and conferring functional diversity and complexity (Bhowmick, Pancsa et al. 2013). There have been multiple E2 enzymes thought to be associated with parkin. The enzymes UbcH7 and UbcH8 as well as the ER-associated E2s Ubc6 and Ubc7 have all been identified as binding partners (Imai, Soda et al. 2000, Shimura, Hattori et al. 2000, Zhang, Gao et al. 2000, Imai, Soda et al. 2001, Shimura, Schlossmacher et al. 2001). Thus, these E3 ligases may have unique or shared substrates within the cell, making it interesting to postulate having an E3 ligase that could compensate for the lack of parkin function. Since PD is primarily an aging disorder, one can hypothesize that one E3 ligase may compensate for another masking symptoms early in life, and the capacity to compensate for parkin deficiency may decrease with age. This observation could also account for the lack of a parkinsonian phenotype in parkin KO mice, since parkin function might be uniquely redundant with another E3 ligase in mice. Future studies, especially in mice, to study the E3 ligases compensating for lack of parkin function would be exciting to target it and generate redundancy to some degree in humans.

Conclusion

Current treatments for PD primarily involve only the management of motor symptoms, but do not address disease progression or the numerous non-nigral features of the disorder. Parkin plays an integral role within the cell and the wealth of information we now have regarding parkin biology and biochemistry far exceeds what we now know about many other neurologic disease related genes. Importantly, the far majority of PD patients have intact expression of a functional parkin protein and given the numerous homeostatic functions implicated for this protein, activating parkin may represent a disease modifying approach. The solved crystal structure of parkin, recent links between phosphorylation and parkin activation, and the characterization of antagonistic DUBs that can likewise be pharmacologically targeted, all pave the ground forward for translational efforts to stimulate parkin biology and tap into a potential endogenous reservoir of neuroprotection in the human brain.

Highlights.

• Parkin biology is relevant to the etiology of familial and idiopathic PD

• There is a critical need to understand the full consequences of parkin deficiency

• Parkin regulates complex pathways of mitochondrial quality control and cell death

• Players in parkin biology may evolve to novel targets for therapeutic intervention