parkin: Biological Overview | Regulation | Developmental Biology | Effects of Mutation and Double Stranded RNAi | Evolutionary Homologs | References
Gene name - parkin
Cytological map position - 78C2
Function - enzyme
Symbol - park
FlyBase ID: FBgn0041100
Genetic map position -
Classification - RING-type E3 ubiquitin-protein ligase
Cellular location - cytoplasmic
Srivastav, S., Singh, S. K., Yadav, A. K. and Srikrishna, S. (2015) Folic acid supplementation ameliorates oxidative stress, metabolic functions and developmental anomalies in a novel fly model of Parkinson's disease. Neurochem Res [Epub ahead of print]. PubMed ID: 25963948
|Malik, B. R., Godena, V. K. and Whitworth, A. J. (2015). VPS35 pathogenic mutations confer no dominant toxicity but partial loss of function in Drosophila and genetically interact with parkin. Hum Mol Genet [Epub ahead of print]. PubMed ID: 26251041
Mutations in VPS35 (PARK17) cause autosomal dominant, late onset Parkinson's disease (PD). VPS35 forms a core component of the retromer complex that mediates the retrieval of membrane proteins from endosomes back to either the Golgi or plasma membrane. While aberrant endosomal protein sorting has been linked to several neurodegenerative diseases, the mechanisms by which VPS35 mutations and retromer function contribute to PD pathogenesis are not clear. To address this, transgenic Drosophila were generated that express variant forms of human VPS35 found in PD cases and the corresponding variants of the Drosophila ortholog. No evidence was found of dominant toxicity from any variant form including the pathogenic D620N mutation, even with aging. However, assessing the ability of Vps35 variants to rescue multiple vps35-mutant phenotypes, the D620N mutation was found to confer a partial loss of function. Recently, VPS35 has been linked to the formation of mitochondria-derived vesicles, which mediate the degradation of mitochondrial proteins and contribute to mitochondrial quality control. This process is also promoted by two other PD-lined genes parkin (PARK2) and PINK1 (PARK6). This study demonstrates that vps35 genetically interacts with parkin but interestingly not with pink1. Strikingly, Vps35 overexpression is able to rescue several parkin-mutant phenotypes. Together these findings provide in vivo evidence that the D620N mutation likely confers pathogenicity through a partial loss of function mechanism and that this may be linked to other known pathogenic mechanisms such as mitochondrial dysfunction.
|Huang, Z., Ren, S., Jiang, Y. and Wang, T. (2016). PINK1 and Parkin cooperatively protect neurons against constitutively active TRP channel-induced retinal degeneration in Drosophila. Cell Death Dis 7: e2179. PubMed ID: 27054334
Calcium has an important role in regulating numerous cellular activities. However, extremely high levels of intracellular calcium can lead to neurotoxicity, a process commonly associated with degenerative diseases. Despite the clear role of calcium cytotoxicity in mediating neuronal cell death in this context, the pathological mechanisms remain controversial. This study used a well-established Drosophila model of retinal degeneration, which involves the constitutively active TRPP365 channels, to study calcium-induced neurotoxicity. Disruption of mitochondrial function was found to be associated with the degenerative process. Further, increasing autophagy flux prevented cell death in TrpP365 mutant flies, and this depended on the PINK1/Parkin pathway. In addition, the retinal degeneration process was also suppressed by the coexpression of PINK1 and Parkin. These results provide genetic evidence that mitochondrial dysfunction has a key role in the pathology of cellular calcium neurotoxicity. In addition, the results demonstrated that maintaining mitochondrial homeostasis via PINK1/Parkin-dependent mitochondrial quality control can potentially alleviate cell death in a wide range of neurodegenerative diseases.
|Sung, H., Tandarich, L.C., Nguyen, K. and
Hollenbeck, P.J. (2016). Compartmentalized
regulation of Parkin-mediated mitochondrial quality control in the Drosophila
nervous system in vivo. J Neurosci 36: 7375-7391. PubMed ID: 27413149
In neurons, the normal distribution and selective removal of mitochondria are considered essential for maintaining the functions of the large asymmetric cell and its diverse compartments. Parkin, a E3 ubiquitin ligase associated with familial Parkinson's disease, has been implicated in mitochondrial dynamics and removal in cells including neurons. However, it is not clear how Parkin functions in mitochondrial turnover in vivo, or whether Parkin-dependent events of the mitochondrial life cycle occur in all neuronal compartments. Using the live Drosophila nervous system, this study investigated the involvement of Parkin in mitochondrial dynamics, distribution, morphology, and removal. Contrary to expectations, it was found that Parkin-deficient animals do not accumulate senescent mitochondria in their motor axons or neuromuscular junctions; instead, they contain far fewer axonal mitochondria, and these displayed normal motility behavior, morphology, and metabolic state. However, the loss of Parkin does produce abnormal tubular and reticular mitochondria restricted to the motor cell bodies. In addition, in contrast to drug-treated, immortalized cells in vitro, mature motor neurons rarely display Parkin-dependent mitophagy. These data indicate that the cell body is the focus of Parkin-dependent mitochondrial quality control in neurons, and argue that a selection process allows only healthy mitochondria to pass from cell bodies to axons, perhaps to limit the impact of mitochondrial dysfunction.
| Zhuang, N., Li, L., Chen, S. and Wang, T.
phosphorylation of PINK1 and Parkin is essential for mitochondrial
quality control. Cell Death Dis 7: e2501. PubMed ID: 27906179
Mitochondrial dysfunction has been linked to the pathogenesis of a large number of inherited diseases in humans, including Parkinson's disease, the second most common neurodegenerative disorder. The Parkinson's disease genes pink1 and parkin, which encode a mitochondrially targeted protein kinase, and an E3 ubiquitin ligase, respectively, participate in a key mitochondrial quality-control pathway that eliminates damaged mitochondria. This study established an in vivo PINK1/Parkin-induced photoreceptor neuron degeneration model in Drosophila with the aim of dissecting the PINK1/Parkin pathway in detail. Using LC-MS/MS analysis, Serine 346 was identified as the sole autophosphorylation site of Drosophila PINK1 and it was found that substitution of Serine 346 to Alanine completely abolishes the PINK1 autophosphorylation. Disruption of either PINK1 or Parkin phosphorylation impairs the PINK1/Parkin pathway, and the degeneration phenotype of photoreceptor neurons is obviously alleviated. Phosphorylation of PINK1 is not only required for the PINK1-mediated mitochondrial recruitment of Parkin but also induces its kinase activity toward Parkin. In contrast, phosphorylation of Parkin by PINK1 is dispensable for its translocation but required for its activation. Moreover, substitution with autophosphorylation-deficient PINK1 fails to rescue pink1 null mutant phenotypes. Taken together, these findings suggest that autophosphorylation of PINK1 is essential for the mitochondrial translocation of Parkin and for subsequent phosphorylation and activation of Parkin.
|Martinez, A., Lectez, B., Ramirez, J., Popp, O., Sutherland, J. D., Urbe, S., Dittmar, G., Clague, M. J. and Mayor, U. (2017). Quantitative proteomic analysis of Parkin substrates in Drosophila neurons. Mol Neurodegener 12(1): 29. PubMed ID: 28399880
Parkin (PARK2; see Drosophila Parkin) is an E3 ubiquitin ligase that is commonly mutated in Familial Parkinson's Disease (PD). In cell culture models, Parkin is recruited to acutely depolarised mitochondria by PINK1 (see Drosophila Pink1). PINK1 activates Parkin activity leading to ubiquitination of multiple proteins, which in turn promotes clearance of mitochondria by mitophagy. Many substrates have been identified using cell culture models in combination with depolarising drugs or proteasome inhibitors, but not in more physiological settings. This study utilized the recently introduced BioUb strategy to isolate ubiquitinated proteins in flies. Following Parkin Wild-Type (WT) and Parkin Ligase dead (LD) expression, mass spectrometry and stringent bioinformatics analysis identified those proteins differentially ubiquitinated, providing the first survey of steady state Parkin substrates using an in vivo model. An in vivo ubiquitination assay was used to validate one of those substrates in SH-SY5Y cells. This study identified 35 proteins that are more prominently ubiquitinated following Parkin over-expression. These include several mitochondrial proteins and a number of endosomal trafficking regulators such as v-ATPase sub-units, Syx5/STX5, Vps4. The retromer component, Vps35, another PD-associated gene that has recently been shown to interact genetically with parkin, was also identified. Importantly, Parkin-dependent ubiquitination of VPS35 was validated in human neuroblastoma cells. Collectively these results provide new leads to the possible physiological functions of Parkin activity that are not overtly biased by acute mitochondrial depolarisation.
|Zanon, A., et al. (2017). SLP-2 interacts with Parkin in mitochondria and prevents mitochondrial dysfunction in Parkin-deficient human iPSC-derived neurons and Drosophila. Hum Mol Genet. PubMed ID: 28379402
Mutations in the Parkin gene (PARK2; see Drosophila Parkin) have been linked to a recessive form of Parkinson's disease (PD) characterized by the loss of dopaminergic neurons in the substantia nigra. Deficiencies of mitochondrial respiratory chain complex I activity have been observed in the substantia nigra of PD patients, and loss of Parkin results in the reduction of complex I activity shown in various cell and animal models. Using co-immunoprecipitation and proximity ligation assays on endogenous proteins, this study demonstrates that Parkin interacts with mitochondrial Stomatin-like protein 2 (SLP-2), which also binds the mitochondrial lipid cardiolipin and functions in the assembly of respiratory chain proteins. SH-SY5Y cells with a stable knockdown of Parkin or SLP-2, as well as induced pluripotent stem cell-derived neurons from Parkin mutation carriers, showed decreased complex I activity and altered mitochondrial network morphology. Importantly, induced expression of SLP-2 corrected for these mitochondrial alterations caused by reduced Parkin function in these cells. In-vivo Drosophila studies showed a genetic interaction of Parkin and SLP-2, and further, tissue-specific or global overexpression of SLP-2 transgenes rescued parkin mutant phenotypes, in particular loss of dopaminergic neurons, mitochondrial network structure, reduced ATP production, and flight and motor dysfunction. The physical and genetic interaction between Parkin and SLP-2 and the compensatory potential of SLP-2 suggest a functional epistatic relationship to Parkin and a protective role of SLP-2 in neurons. This finding places further emphasis on the significance of Parkin for the maintenance of mitochondrial function in neurons and provides a novel target for therapeutic strategies.
|Cornelissen, T., Vilain, S., Vints, K., Gounko, N., Verstreken, P. and Vandenberghe, W. (2018). Deficiency of parkin and PINK1 impairs age-dependent mitophagy in Drosophila. Elife 7. PubMed ID: 29809156
Mutations in the genes for PINK1 and parkin cause Parkinson's disease. PINK1 and parkin cooperate in the selective autophagic degradation of damaged mitochondria (mitophagy) in cultured cells. However, evidence for their role in mitophagy in vivo is still scarce. This study generated a Drosophila model expressing the mitophagy probe mt-Keima. Using live mt-Keima imaging and correlative light and electron microscopy (CLEM) it was shown that mitophagy occurs in muscle cells and dopaminergic neurons in vivo, even in the absence of exogenous mitochondrial toxins. Mitophagy increases with aging, and this age-dependent rise is abrogated by PINK1 or parkin deficiency. Knockdown of the Drosophila homologues of the deubiquitinases USP15 and, to a lesser extent, USP30, rescues mitophagy in the parkin-deficient flies. These data demonstrate a crucial role for parkin and PINK1 in age-dependent mitophagy in Drosophila in vivo.
|Tan, K. L., Haelterman, N. A., Kwartler, C. S., Regalado, E. S., Lee, P. T., Nagarkar-Jaiswal, S., Guo, D. C., Duraine, L., Wangler, M. F., Bamshad, M. J., Nickerson, D. A., Lin, G., Milewicz, D. M. and Bellen, H. J. (2018). Ari-1 regulates myonuclear organization together with Parkin and is associated with aortic aneurysms. Dev Cell 45(2): 226-244.e228. PubMed ID: 29689197
Nuclei are actively positioned and anchored to the cytoskeleton via the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex. This study identified mutations in the Parkin-like E3 ubiquitin ligase Ariadne-1 (Ari-1) that affect the localization and distribution of LINC complex members in Drosophila. ari-1 mutants exhibit nuclear clustering and morphology defects in larval muscles. Ari-1 mono-ubiquitinates the core LINC complex member Koi. Surprisingly, functional redundancy was found between Parkin and Ari-1: increasing Parkin expression rescues ari-1 mutant phenotypes and vice versa. It was further shown that rare variants in the human homolog of ari-1 (ARIH1) are associated with thoracic aortic aneurysms and dissections, conditions resulting from smooth muscle cell (SMC) dysfunction. Human ARIH1 rescues fly ari-1 mutant phenotypes, whereas human variants found in patients fail to do so. In addition, SMCs obtained from patients display aberrant nuclear morphology. Hence, ARIH1 is critical in anchoring myonuclei to the cytoskeleton.
|Vincow, E. S., Thomas, R. E., Merrihew, G. E., Shulman, N. J., Bammler, T. K., MacDonald, J. W., MacCoss, M. J. and Pallanck, L. J. (2019). Autophagy accounts for approximately one-third of mitochondrial protein turnover and is protein selective. Autophagy: 1-14. PubMed ID: 30865561
The destruction of mitochondria through macroautophagy (autophagy) has been recognised as a major route of mitochondrial protein degradation. Autophagy was originally thought to degrade all mitochondrial proteins at the same rate, but recent work suggests that mitochondrial autophagy may be protein selective. To investigate these questions, a proteomics-based approach was used in the fruit fly Drosophila melanogaster, comparing mitochondrial protein turnover rates in autophagy-deficient Atg7 mutants and controls. ~35% of mitochondrial protein turnover occurred via autophagy. Similar analyses using parkin mutants revealed that parkin-dependent mitophagy accounted for ~25% of mitochondrial protein turnover, suggesting that most mitochondrial autophagy specifically eliminates dysfunctional mitochondria. The results were incompatible with uniform autophagic turnover of mitochondrial proteins and consistent with protein-selective autophagy. In particular, the autophagic turnover rates of individual mitochondrial proteins varied widely, and only a small amount of the variation could be attributed to tissue differences in mitochondrial composition and autophagy rate. Furthermore, analyses comparing autophagy-deficient and control human fibroblasts revealed diverse autophagy-dependent turnover rates even in homogeneous cells. In summary, this work indicates that autophagy acts selectively on mitochondrial proteins, and that most mitochondrial protein turnover occurs through non-autophagic processes.
Parkinson's disease (see Drosophila as a Model for Human Diseases: Parkinson's disease) is a common neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta and the accumulation of proteinaceous intraneuronal inclusions known as Lewy bodies. Little is known of the molecular mechanisms responsible for loss of dopaminergic neurons in PD; however, evidence suggests that environmental and genetic factors both play contributing roles. Mutation of a mammalian protein, Parkin, is associated with autosomal recessive juvenile parkinsonism (AR-JP). Parkin appears to be part of the cell's defense against damage caused by environmental insults (Steece-Collier, 2002). Parkin is an E3 ubiquitin ligase that degrades proteins with aberrant conformations (Imai, 2000).
To gain insight into the molecular mechanism responsible for selective cell death in AR-JP, a Drosophila model of this disorder has been created. Drosophila parkin null mutants exhibit reduced lifespan, locomotor defects, and male sterility. The locomotor defects derive from apoptotic cell death of muscle subsets, whereas the male sterile phenotype derives from a spermatid individualization defect at a late stage of spermatogenesis. Mitochondrial pathology is the earliest manifestation of muscle degeneration and a prominent characteristic of individualizing spermatids in parkin mutants. Drosophila parkin mutants also appear to have defects in a population of brain dopaminergic cells. Cells of the dorsomedial dopaminergic cell cluster reliably show shrinkage of the cell body and decreased tyrosine hydroxylase immunostaining in proximal dendrites in aged parkin mutants relative to controls. These results indicate that the tissue-specific phenotypes observed in Drosophila parkin mutants result from mitochondrial dysfunction, and as it does in mammals, mitochondrial dysfunction affects the dopaminergic neurons. These observations raise the possibility that similar mitochondrial impairment triggers the selective cell loss observed in AR-JP (Greene, 2003).
A second study in Drosophila analyses the effects of expression of a known mammalian substrates in Drosophila embryos. Panneuronal expression in Drosophila of Pael-R, a putative G protein-coupled transmembrane polypeptide, causes age-dependent selective degeneration of Drosophila dopaminergic (DA) neurons. Coexpression of Parkin degrades Pael-R and suppresses its toxicity, whereas interfering with endogenous Drosophila Parkin function promotes Pael-R accumulation and augments its toxicity. Furthermore, overexpression of Parkin can mitigate neuritic pathology induced by a second target of Parkin, alpha-Synuclein, and suppresses alpha-Synuclein toxicity. This study implicates Parkin as a central player in the molecular pathway of Parkinson's disease (PD) and suggests that manipulating Parkin expression may provide a novel avenue of PD therapy (Yang, 2003).
PD is the most common movement disorder and the second most common neurodegenerative diseases. PD patients suffer from rigidity, slowness of movement, tremor, and postural instability. The movement disorder in PD is largely due to the deficiency of brain dopamine content caused by the degeneration of DA neurons in the midbrain. Although the mechanism underlying the selective degeneration of DA neurons is still poorly understood, both exogenous environmental toxins and endogenous proteotoxins have been implicated in PD pathogenesis (Yang, 2003).
The molecular cloning of genes linked to familial forms of the disease has provided tremendous insights into the pathogenesis of PD. Missense mutations in the alpha-Synuclein (alpha-Syn) gene have been shown to be the cause of rare forms of autosomal dominant familial PD. alpha-Syn is an abundant brain protein enriched at presynaptic terminals. Mice with alpha-Syn gene deleted show increased striatal dopamine release but do not develop neurological phenotypes observed in PD. This suggests that familial alpha-Syn mutations may cause PD through a dominant gain-of-function mechanism. Significantly, wild-type alpha-Syn protein was found to be a major component of Lewy bodies, the proteinaceous aggregates found in PD and other diseases termed 'Synucleinopathies.' This suggests that the accumulation and aggregation of alpha-Syn is intimately involved in disease pathogenesis. Further support came from transgenic animal studies in which overexpression of wild-type and mutant forms of alpha-Syn in mouse and Drosophila led to alpha-Syn aggregate formation and neuronal dysfunction (Yang, 2003 and references therein).
Mutations in the parkin gene have been linked to AR-JP (Kitada, 1998). AR-JP patients develop the typical parkinsonian symptoms also as a result of loss of midbrain DA neurons. This usually occurs in the absence of Lewy body formation. Biochemical studies have shown that Parkin has E3 ubiquitin-protein ligase activity and that AR-JP-linked parkin mutations abolished this activity (Imai, 2000; Shimura, 2000; Zhang, 2000). Ubiquitin-protein ligases are components of the ubiquitin-proteasome pathway that degrades proteins with abnormal conformations. In this pathway, ubiquitin moiety is transferred to substrate proteins through ubiquitin-activating enzyme (E1), ubiquitin-carrier protein (E2), and ubiquitin ligase (E3). After a polyubiquitin chain is attached to the substrate protein, it is recognized by the 26S proteasome and targeted for degradation. Substrate specificity in this reaction is largely conferred by the interaction between E3 and the substrate (Yang, 2003).
A number of proteins have been identified as Parkin substrates in vitro. CDCrel-1, a synaptic vesicle-associated protein, has been shown to interact with and can be ubiquitinated by Parkin (Zhang, 2000). Synphilin-1, which was originally identified as a alpha-Syn-interacting protein, has also been shown to be a substrate of Parkin-mediated ubiquitination and degradation (Chung, 2001). It has not been determined whether CDCrel-1 or Synphilin-1 accumulates to higher levels in AR-JP patient brain as a result of loss of Parkin activity. Parkin has also been shown to promote the ubiquitination and degradation of an O-glycosylated form of alpha-Syn (alphaSp22) (Shimura, 2001). The abundance of alphaSP22 is very low in normal brain, but it was found at modest levels in AR-JP brains lacking Parkin activity. It is not known whether O-glycosylation is a strict prerequisite for alpha-Syn interaction with Parkin and its subsequent ubiquitination and whether these modifications play any significant roles in PD pathogenesis. It is possible that through Synphilin-1, Parkin and unmodified alpha-Syn could functionally interact in the disease process (Yang, 2003).
Parkin has recently been shown to ubiquitinate Pael-R, a putative G protein-coupled transmembrane polypeptide (Imai, 2001). When overexpressed in cultured cells, Pael-R tends to become unfolded and insoluble and induces endoplasmic reticulum (ER) stress. Parkin ubiquitinates Pael-R and promotes the degradation of the insoluble form of the protein. Moreover, the insoluble form of Pael-R has been found to accumulate in AR-JP patient brains (Imai, 2001). However, the causal relationship between Pael-R accumulation and AR-JP is not clear (Yang, 2003).
To test the functional significance of Parkin's biochemical interactions with Pael-R and alpha-Syn, transgenic flies expressing human Pael-R, Parkin, or alpha-Syn were used to analyze their in vivo relationships and their roles in the pathogenesis of PD. Panneuronal expression of Pael-R results in age-dependent selective degeneration of Drosophila DA neurons, suggesting that DA neurons are especially susceptible to Pael-R toxicity. Coexpression of human Parkin causes the degradation of Pael-R and suppresses the DA neuron degeneration phenotype, whereas interference of endogenous Drosophila parkin function promotes Pael-R accumulation and enhances Pael-R-induced neurotoxicity. These data provide strong in vivo evidence that Pael-R is a genuine substrate for Parkin and that the accumulation of Pael-R is a cause of DA neuron death in AR-JP. Interestingly, overexpression of Parkin can also suppress the toxicity of alpha-Syn, at least in part by mitigating alpha-Syn-induced neuritic pathology. This study implicates Parkin as a central player in the pathogenesis of different forms of PD (Yang, 2003).
The ability of overexpressed Parkin to suppress both Pael-R and alpha-Syn toxicity suggests that despite the difference in pathological manifestation between the recessive and dominant forms of PD, there is a common pathway of PD pathogenesis in which Parkin is a central player. The observation that Parkin is localized to Lewy bodies is consistent with this notion (Shimura, 2001; Schlossmacher, 2002). It is possible that Parkin is actively engaged in degrading abnormal alpha-Syn proteins in Lewy bodies. It is also possible that Parkin may be sequestered by alpha-Syn into inactive complexes, similar to the sequestration of transcription factor CBP by polyglutamine-repeat proteins into nuclear inclusions in Huntington's disease. This may interfere with Parkin's normal function in the ubiquitin/proteasome pathway and contribute to alpha-Syn toxicity. The latter scenario is consistent with the observation of accumulated ubiquitin immunostaining in alpha-Syn transgenic neurites and its disappearance after Parkin overexpression. A recent report has shown that overexpression of mutant alpha-Syn protein increases the sensitivity to proteasome inhibitors by decreasing proteasome function in mammalian cell culture, an effect that can be suppressed by overexpression of Parkin (Petrucelli, 2002). This is also consistent with the latter possibility. However, the observation of reduced neuritic alpha-Syn aggregates in Parkin-coexpressing flies is also consistent with the former possibility that Parkin directly acts on abnormal forms of alpha-Syn, which may constitute only a minor portion of total alpha-Syn protein in the cell. It is likely that both mechanisms may be involved in the disease process (Yang, 2003).
Previous studies have shown that in response to unfolded protein response (UPR), Parkin becomes upregulated at both the mRNA and protein level and that overexpression of Parkin can suppress UPR-induced cell death in cell culture models (Imai, 2000). Thus, Parkin may play a more general role in cellular quality control to protect neurons from unfolded protein-induced stress. It is therefore conceivable that overexpression of Parkin could also suppress neurotoxicity induced by unfolded proteins other than Pael-R and alpha-Syn. Given that overexpression of Parkin does not have obvious detrimental effects on the animal, manipulation of Parkin expression with small molecules may provide an effective strategy for PD therapy (Yang, 2003).
AR-JP has been placed into the group of neurodegenerative disorders characterized by the accumulation and aggregation of aberrant forms of proteins. This includes Amyotrophic Lateral Sclerosis, Alzheimer's disease, Huntington's disease, the late onset dominantly inherited forms of PD, and spongiform encephalopathies. In these various forms of neurodegenerative diseases, specific groups of neurons undergo degeneration as a result of the aggregation of distinct misfolded proteins. These proteins may cause cellular toxicity by affecting different aspects of neuronal physiology, from axonal transport to gene expression in the nucleus. In the case of Pael-R, abnormal forms of the protein may cause cellular toxicity through eliciting ER stress (Imai, 2001). Components of the ubiquitin-proteasome degradation pathway and molecular chaperones have been shown to associate with inclusions formed by these different proteins. This may reflect the cell's strategy to eliminate these misfolded proteins by repairing/refolding them with the molecular chaperones or degrading them through the ubiquitin-proteasome system (Yang, 2003).
The importance of the ubiquitin-proteasome system in neurodegeneration is highlighted by the association of familial forms of PD with mutations in Parkin and ubiquitin carboxy-terminal hydrolase L1 (UCHL-1), another enzyme involved in ubiquitin metabolism (Leroy, 1998), and the acceleration of spinocerebellar ataxia type 1 phenotype by loss-of-function of an E3 ubiquitin ligase in mice (Cummings, 1999). Hsp70 molecular chaperones have been implicated in disease processes by whole animal studies in Drosophila, that have shown that directed overexpression of Hsp70 attenuates whereas interference with endogenous chaperone activity exacerbates neurotoxicity associated with polyglutamine repeat-containing proteins and alpha-Syn. It remains to be determined whether Hsp70 molecular chaperones play significant roles in removing misfolded Pael-R protein. The fate of abnormal Pael-R protein depends on the relative kinetics of its interactions with the molecular chaperone pathway and the proteasome pathway (Imai, 2002). Unlike alpha-Syn, a small cytosolic protein, Pael-R is a large 7 transmembrane protein, the misfolded forms of which reside mainly in the ER. The Hsp70 class of molecular chaperones may have limited access to misfolded Pael-R or have limited ability to renature misfolded Pael-R proteins, and therefore the Parkin-mediated proteosome pathway may play a more dominant role in clearing them. Alternatively, other molecular chaperones may play more prominent roles in regulating the fate of misfolded Pael-R. One good candidate is the ER resident molecular chaperone BiP (GRP78), which associates with unfolded proteins in the ER and has been shown to be upregulated (Imai, 2000, 2001) during ER stress and in AR-JP patient brain (Yang, 2003).
One of the most intriguing features of neurodegenerative diseases is the cell type specificity of neuronal death. Previous studies suggested that the restricted tissue distribution of Pael-R might contribute to the selective degeneration of DA neurons in AR-JP (Imai, 2001). It was shown that Pael-R protein is widely expressed in the mouse brain, predominantly in oligodendrocytes. Its neuronal expression is restricted to a subset of neurons such as DA neurons in the substantia nigra and hippocampal neurons in the CA3 region. While the restricted tissue distribution of Pael-R may partially explain the selectivity of neuronal degeneration in AR-JP, this study shows that there are some features specific to the DA neurons that contribute to their particular vulnerability to Pael-R toxicity. It is possible that this selective vulnerability is due to a smaller capacity of DA neurons to handle Pael-R-induced ER stress. It is also possible that certain DA neuron-specific cofactors may contribute to Pael-R toxicity. Two risk factors, oxidative stress and dopamine, have been suggested to contribute to the selective toxicity of alpha-Syn toward DA neurons. DA neurons in the substantia nigra are known to produce excess reactive oxygen species such as superoxide anion, which in reaction with nitric oxide (NO) can generate the more potent oxidant peroxynitrite and cause damages to proteins and other macromolecules. It was also shown that dopamine can be ligated to alpha-Syn to form dopamine-alpha-Syn adducts. This adduct selectively inhibits the protofibril-to-fibril conversion and causes the accumulation of alpha-Syn protofibrils, which are the presumed toxic species. Future studies will test whether Pael-R is also modified by oxidative stress and dopamine and whether these modifications contribute to its toxicity (Yang, 2003 and references therein).
Many studies have shown that there is unanticipated conservation of signaling pathways, regulatory mechanisms, and cellular and physiological processes between flies and humans. The identification of Parkin as an important modulator of Pael-R and alpha-Syn toxicity suggests that fly models could be used to further understand the role of Parkin and its substrates in the pathogenesis of PD. The ability to perform facile genetic loss-of-function and overexpression screens in this system will allow the identification of new genes that can either attenuate or exacerbate disease phenotypes. Such studies will allow the delineation of the molecular pathways involved in the pathogenesis of PD and possibly other related neurodegenerative diseases and identify potential therapeutic drug targets (Yang, 2003).
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).
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).
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).
In the sequenced Drosophila genome, there is no clear fly homolog of human Pael-R. To address the role of Pael-R in causing neurodegeneration, the bipartite UAS-GAL4 system was used to target the expression of human Pael-R protein to different fly tissues and cell types. This system involves two transgenic lines, a UAS-Pael-R line, in which the human Pael-R cDNA is placed under the control of upstream activating sequence (UAS) for the yeast transcription factor GAL4, and a GAL4 line, which expresses GAL4 in a tissue- and cell type-specific pattern. In the progenies resulting from a cross between these two lines, Pael-R is expressed in the same tissue and cell type where GAL4 is expressed (Yang, 2003).
Multiple independent UAS-Pael-R transgenic lines were generated through P element-mediated germline transformation. To assess the levels of Pael-R expression in these different transgenic lines, these UAS-Pael-R lines were crossed to the scabrous-GAL4 line, a strong GAL4 driver line that directs transgene expression in the precursor cells and their differentiated progenies in the central and peripheral nervous system. Resulting transgenic embryos were examined for transgene expression by immunostaining with antibodies against Pael-R. Of ten independent transgenic lines analyzed, four give detectable expression of the Pael-R protein as determined by the immunostaining method. Two lines, a strong expression line and a moderate expression line, were chosen for subsequent studies (Yang, 2003).
To target the expression of Pael-R to DA neurons, the Ddc-GAL4 driver line, in which the expression of GAL4 is under the control of the DOPA decarboxylase gene promoter was used. Sections of aged control and transgenic fly brains were immunostained for tyrosine hydroxylase (TH), which specifically identifies DA neurons. Analysis was focused on the dorsomedial (DM) clusters of DA neurons, which have been well characterized and shown to preferentially degenerate in alpha-Syn transgenic flies (Feany, 2000; Auluck, 2002). The DM clusters are composed of approximately 18 DA neurons in wild-type adult fly brain. The two clusters are distributed in a bilaterally symmetric fashion with respect to the midline. The number of DA neurons in the DM clusters does not change significantly, even in 60-day-old senescent wild-type flies. In 40-day-old transgenic flies expressing Pael-R, however, it was observed that the number of DA neurons in the DM clusters was reduced compared to the controls. This reduction of DA neurons was correlated with the expression level of the Pael-R transgene, since the strong expression lines consistently showed a smaller number of DA neurons than the moderate expression line (Yang, 2003).
The loss of DA neurons could be caused by late onset neurodegeneration or early developmental abnormalities. To distinguish these possibilities, younger flies were examined. In newly eclosed transgenic flies (1-day-old) expressing Pael-R, the number of DA neurons in the DM clusters was similar to that observed in control flies. This suggests that the DA neurons are initially formed normally in the transgenic flies, but over time some of these neurons degenerate as a consequence of the overexpression of Pael-R protein (Yang, 2003).
The effects of expressing Pael-R ubiquitously in all the differentiated neurons of the fly nervous system were tested using the strong UAS-Pael-R line and the panneuronal driver elav-GAL4. Western blot analysis on adult fly heads showed that the amount of transgene-produced Pael-R protein per milligram of brain tissue in elav-GAL4 /UAS-Pael-R flies is 45%- 55% that of endogenous Pael-R protein in mouse brain. TH immunostaining revealed that, similar to that observed in Ddc-DAL4 driven transgenic flies, the number of DA neurons in the DM clusters is also reduced in elav-GAL4 driven transgenic flies (Yang, 2003).
Other neuronal types were examined to see if they are also affected after panneuronal expression of Pael-R. The monoclonal antibody 22C10, which recognizes the Futsch antigen, is highly expressed in the visual system as well as the central brain complex in adult flies. The staining of lamina and medulla neuropils by the 22C10 antibody was similar between the control and Pael-R transgenic flies. Overall morphology of the central brain was preserved in Pael-R transgenic flies, suggesting that ubiquitous expression of Pael-R in the brain does not cause widespread degeneration. Specific neuronal subtypes were examined. Immunostaining with the antibody against choline acetyltransferase, an enzyme in the biosynthetic pathway of acetylcholine, showed that the distribution and number of cholinergic neurons in major brain cholinergic centers were similar between control and Pael-R transgenics. Immunostaining of serotonergic neurons with anti-5HT antibody also did not reveal any obvious reduction of these neurons in major brain serotonergic centers. Although the possibility cannot be excluded that a small percentage of serotonergic or cholinergic neurons degenerate in Pael-R transgenic flies, this analysis indicates that there are no clusters of these neurons that are vulnerable to Pael-R toxicity like the DM clusters of DA neurons (Yang, 2003).
Whether the expression of Pael-R is toxic to cell types outside of the central nervous system was also examined. For this purpose, the UAS-Pael-R transgenic lines were crossed to GAL4 driver lines that have known expression patterns in non-CNS tissues. Previous studies showed that expression of many human disease-causing proteins such as polyglutamine repeat-containing proteins, tau, and alpha-Syn in fly eye caused defects in eye morphogenesis and retinal degeneration. In contrast, Pael-R expression in the eye has no phenotypic consequence . Pael-R expression in the eye was targeted to different stages of eye development using the elav-, sevenless-, gmr-, and eyeless-GAL4 lines. In all four cases, Pael-R-expressing flies had normal eye morphology and retina structure. Similarly, no phenotype was observed when the dpp-GAL4 and 24B-GAL4 lines were used to express Pael-R along the anterior-posterior boundary of imaginal discs and in muscle cells, respectively (Yang, 2003).
The only phenotype outside of the nervous system was observed when the apterous-GAL4 line was used to direct Pael-R expression. apterous-GAL4 is expressed in several larval imaginal discs including the dorsal mesothoracic and metathoracic discs. In newly eclosed flies from a cross between the apterous-GAL4 and the strong Pael-R lines, body fluids leaked from two spots located in a bilaterally symmetric fashion on the dorsal thorax at the junction between the notum and scutellum. The fluids quickly solidified and formed two dark patches adhering to the cuticle. This phenotype is presumably caused by the degeneration of the epithelial tissue or the underlying musculature at those sites (Yang, 2003).
Given the propensity of overexpressed Pael-R to become unfolded and insoluble and elicit ER stress in cell culture (Imai, 2001), it is possible that the degeneration phenotype observed in Pael-R transgenic flies is caused by the accumulation of abnormal and toxic forms of Pael-R protein. To test this possibility, it was asked if coexpression of human Parkin protein could promote the degradation of these aberrant proteins and therefore suppress Pael-R toxicity. In transgenic flies that coexpressed Pael-R and human Parkin in DA neurons under the control of the Ddc-GAL4 driver, it was observed that the number of DA neurons in the DM clusters is restored to near wild-type levels. Expression of the human Parkin protein alone using the Ddc-GAL4 driver did not affect the number of DA neurons in the DM clusters (Yang, 2003).
When human Parkin and Pael-R are coexpressed using the apterous-GAL4 driver, the thoracic toxicity of Pael-R is also suppressed and no dark patches form on the dorsal thorax. Expression of human Parkin alone using the apterous-GAL4 driver had no effect on thoracic morphology. These results indicate that coexpression of human Parkin suppresses Pael-R toxicity in two different cell types (Yang, 2003).
To gain further insights into the interaction between human Parkin and Pael-R at the molecular and cellular level, immunohistochemical analysis was performed of human Parkin and Pael-R proteins expressed in neural stem cell-derived DA neurons. Isolated Drosophila embryonic neuroblasts undergo stereotyped divisions when cultured in vitro. After being in culture for 12 hr, the neuroblast progenies exit the cell cycle and form clusters of differentiated neuronal or glial cell types. The neuronal progenies, which include an appropriate proportion of TH-positive DA neurons, are stable in culture for weeks. In cultured DA neurons, Pael-R is mainly localized to the cell surface, as indicated by its colocalization with Numb, a membrane protein. There is also a lower level of expression in neuronal processes. Human Parkin protein exhibits similar localization pattern in cultured DA neurons, but its localization to neuronal processes is more pronounced and appears punctate. Double labeling with Synaptotagmin suggests that the punctate staining of Parkin in neuronal processes represents its association with synaptic vesicles. This is consistent with earlier findings (Kubo, 2001) in mammalian cell culture (Yang, 2003).
The interaction between Pael-R and human Parkin was examined when they were coexpressed in postmitotic neurons under the control of the elav-GAL4 driver. In 1- or 2-day-old neuronal culture, Pael-R and Parkin showed colocalization, and in most neurons (135/154) the level of Pael-R protein was similar to that observed in neurons expressing Pael-R alone. In 6-day-old culture, however, the majority of these neurons (172/235) showed a significant reduction of Pael-R protein as judged by immunostaining. Interestingly, there was a significant portion (63/235) of neurons that still expressed normal levels of Pael-R protein in the presence of Parkin. Because the action of Parkin requires E1, E2, and possibly other cofactors, these neurons may miss certain cofactors required for Parkin-mediated degradation of Pael-R (Yang, 2003).
Whether DA neurons have the capacity to degrade Pael-R in the presence of human Parkin was tested. These two proteins were co-expressed in cultured DA neurons using the Ddc-GAL4 driver. Similar to that observed in elav-GAL4 neuronal culture described above, in younger cultures (1-2 days old) Pael-R and Parkin showed colocalization, and in most Ddc-GAL4-expressing neurons (27/31), the level of Pael-R was similar to that observed in neurons expressing Pael-R alone. But in older neuronal culture (4-6 days), Pael-R protein level was markedly reduced in most Ddc-GAL4-expressing neurons (21/29). In contrast, control neurons expressing Pael-R in the absence of Parkin showed robust Pael-R expression in 6-day-old culture. Thus, it is concluded that human Parkin promotes the degradation of Pael-R in selective groups of Drosophila neurons including DA neurons (Yang, 2003).
To demonstrate the in vivo relevance of these results, showing Parkin-mediated degradation of Pael-R in cell culture experiments, Western blot analysis was used to quantify Pael-R protein levels in transgenic flies with or without Parkin coexpression. Due to the small number of neurons expressing Ddc-GAL4 and the sensitivity of available Pael-R antibodies, Pael-R protein could not be detected in Ddc-Gal4 driven transgenic flies, even in the absence of Parkin. Therefore, elav-GAL4 driven transgenic flies, which express Pael-R protein in more cells, was used. In head extracts obtained from 30-day-old Pael-R transgenic flies driven by elav-GAL4, a significant reduction of Pael-R protein level was observed in the presence of coexpressed Parkin. In contrast, coexpression of Parkin has little effect on the level of a control GFP protein. Together, this immunohistochemical analysis on cultured neurons and Western blot analysis on adult fly brain demonstrate that Parkin promotes the degradation of Pael-R under physiological conditions (Yang, 2003).
Biochemical interactions between Parkin and Pael-R or an O-glycosylated form of alpha-Syn suggest that Parkin may be a common player in the proteasome pathway that regulates the metabolism of these two proteins. The detection of Parkin in Lewy bodies of Parkinson's disease and dementia with Lewy bodies (Shimura, 2001; Schlossmacher, 2002) further suggests that either Parkin is actively engaged in degrading abnormal alpha-Syn proteins or that Parkin is sequestered by alpha-Syn into inactive complexes in the aggregates. In either case, overexpression of Parkin would be predicted to enhance the degradation of abnormal alpha-Syn or to compensate for the reduction of Parkin activity caused by alpha-Syn sequestration and therefore suppress alpha-Syn toxicity. To test this hypothesis, alpha-Syn was coexpressed together with human Parkin in DA neurons. While the expression of wild-type or pathological forms of alpha-Syn (A30P or A53T) consistently caused a 50% reduction of DA neurons in the DM clusters in 20-day-old flies, this loss of DA neurons was suppressed when Parkin was coexpressed with alpha-Syn. Interestingly, unlike Parkin suppression of Pael-R toxicity, which is accompanied by a dramatic reduction of overall Pael-R protein level, immunohistochemical analysis shows that alpha-Syn level is comparable in transgenic flies with or without Parkin coexpression. This was further confirmed by Western blot analysis. Detailed examination of alpha-Syn distribution in DA neurons in the DM clusters reveals that while alpha-Syn forms numerous grain-like structures in the processes, there is a marked reduction of such structures in Parkin-coexpressing flies. Although it is not known whether all of these grain-like structures represent pathological forms of alpha-Syn, the suppression of alpha-Syn toxicity and concomitant reduction of these structures by Parkin coexpression suggest that at least some of them correspond to toxic forms of alpha-Syn. To further characterize the effect of Parkin overexpression on neuritic pathology induced by alpha-Syn, alpha-Syn transgenic fly brain was stained with anti-ubiquitin antibody, which can recognize Lewy bodies and Lewy neurites in human PD patient brain. In Ddc-GAL4 driven alpha-Syn transgenic fly brain, numerous ubiquitin-positive neurites could be detected. In Parkin-coexpressing flies, however, such abnormal neurites were almost completely eliminated. Therefore, mitigation of alpha-Syn toxicity by Parkin overexpression is associated with reduced alpha-Syn-induced neuritic pathology and reduced aggregation of alpha-Syn (Yang, 2003).
Parkinson's disease, a prevalent neurodegenerative disease, is characterized by the reduction of dopaminergic neurons resulting in the loss of motor control, resting tremor, the formation of neuronal inclusions and ultimately premature death. Two inherited forms of PD have been linked to mutations in the α-synuclein and parkin genes. The parkin protein functions as an ubiquitin ligase targeting specific proteins for degradation. Expression of human α-synuclein in Drosophila neurons recapitulates the loss of motor control, the development of neuronal inclusions, degeneration of dopaminergic neurons and the ommatidial array to provide an excellent genetic model of PD. To investigate the role of parkin, transgenic Drosophila were generated that conditionally express parkin under the control of the yeast UAS enhancer. While expression of parkin has little consequence, co-expression of parkin with α-synuclein in the dopaminergic neurons suppresses the α-synuclein-induced premature loss of climbing ability. In addition directed expression of parkin in the eye counteracts the α-synuclein-induced degeneration of the ommatidial array. These results show that parkin suppresses the PD-like symptoms observed in the α-synuclein-dependent Drosophila model of PD. It is concluded that the highly conserved parkin E3 ubiquitin ligase can suppress the damaging effects of human α-synuclein. These results are consistent with a role for parkin in targeting α-synuclein to the proteasome. If this relationship is conserved in humans, this suggests that up-regulation of parkin should suppress α-synucleinopathic PD. The development of therapies that regulate parkin activity may be crucial in the treatment of PD (Haywood, 2004; full text of article).
Parkinsons disease (PD) patients show a characteristic loss of motor control caused by the degeneration of dopaminergic neurons. Mutations in the genes that encode α-synuclein and parkin have been linked to inherited forms of this disease. The parkin protein functions as a ubiquitin ligase that targets proteins for degradation. Expression of isoforms of human α-synuclein in the Drosophila melanogaster nervous system forms the basis of an excellent genetic model that recapitulates phenotypic and behavioural features of PD. Using this model, the effect was analyzed of parkin co-expression on the climbing ability of aging flies, their life span, and their retinal degeneration. Co-expression of parkin can suppress phenotypes caused by expression of mutant α-synuclein. In the developing eye, parkin reduces retinal degeneration. When co-expressed in the dopaminergic neurons, the ability to climb is extended over time. If conserved in humans, it is suggested that upregulation of parkin may prove a method of suppression for PD induced by mutant forms of α-synuclein (Haywood, 2005).
These experiments demonstrate that the directed expression of parkin in the developing eye negates the retinal defects resulting from mutant α-synuclein expression. In addition, increased parkin expression in the dopaminergic neurons extends the climbing ability of aged flies that express mutant α-synuclein. This suggests that parkin can suppress the degeneration resulting from mutant α-synuclein expression in spite of the amino acid substitution present in the mutant form of α-synuclein that is thought to lead to a conformational change in the protein. Although the exact mechanism of phenotype suppression is not clear, it does indicate that mutant α-synuclein is likely a target of parkins ubiquitin-ligase activity. Co-immunoprecipitation studies have suggested that the parkin protein does not interact with or ubiquitinate unmodified α-synuclein but will ubiquitinate O-glycosylated α-synuclein (Haywood, 2005).
This study established the suppression of mutant α-synucleininduced retinal degeneration by the ectopic expression of parkin. Therefore, it is believed that mutant α-synuclein protein is modified in Drosophila in a manner that will enable it to be ubiquitinated by the parkin ubiquitin protein ligase then targeted to the proteasome for degradation. Transgenic Drosophila that express either the wild type or mutant form of α-synuclein in their central nervous systems, via the pan-neural elav-Gal4 transgene, have shown an age-dependent reduction in climbing ability when compared with control flies. Notably, flies that express mutant α-synuclein under the control of elav-Gal4 show a greater reduction in climbing ability. Although flies that express wild-type α-synuclein in their dopaminergic neurons show a marked premature loss of climbing ability, expression of mutant α-synuclein results in only a slight premature loss of the ability to climb. Regardless, over-expression by both of the parkin transgenes had the effect of extending the climbing ability of flies expressing A30Pα-synuclein when compared with the controls. Thus the premature loss of climbing ability arising from a mutant form of α-synuclein that is known to cause PD in humans was prevented by the directed expression of parkin (Haywood, 2005).
Over-expression of parkin suppresses the PD-like symptoms induced in Drosophila by wild-type and mutant α-synuclein with no apparent adverse consequences. It is suggested that the manipulation of the ubiquitin-proteasome degradation pathway in such a specific manner acts to remedy the toxicity of the accumulation of α-synuclein. Activation of parkin may be a viable treatment for PD caused by increased levels or mutant forms of α-synuclein and it is suggested that the selection of therapeutic strategies should be directed towards this end (Haywood, 2005).
Mutations in the parkin gene are a predominant cause of familial parkinsonism. Although initially described as a recessive disorder, emerging evidence suggest that single parkin mutations alone may confer increased susceptibility to Parkinson's disease. To better understand the effects of parkin mutations in vivo, transgenic Drosophila were generated overexpressing two human parkin missense mutants, R275W and G328E. Transgenic flies that overexpress R275W, but not wild-type or G328E, human parkin display an age-dependent degeneration of specific dopaminergic neuronal clusters and concomitant locomotor deficits that accelerate with age or in response to rotenone treatment. Furthermore, R275W mutant flies also exhibit prominent mitochondrial abnormalities in their flight muscles. Interestingly, these defects caused by the expression of human R275W parkin are highly similar to those triggered by the loss of endogenous parkin in parkin null flies. Together, these results provide the first in vivo evidence demonstrating that parkin R275W mutant expression mediates pathogenic outcomes and suggest the interesting possibility that select parkin mutations may directly exert neurotoxicity in vivo (Wang, 2007).
Although parkin-linked disease transmission is presumed to occur in a recessive manner, the expanding number of reports associating single parkin mutation with increased risk for PD has raised questions on the mode of disease transmission by parkin mutations. To date, several parkin mutations occurring on different regions of the protein have been found in heterozygous carriers. Interestingly, heterozygous PD cases harboring the R275W mutation are a recurrent theme in these studies. However, the identification of heterozygous parkin mutations remains controversial. Indeed, single parkin mutation cases are often overestimated in the literature. Importantly, single parkin mutations exist as bona fide heterozygous mutations (West, 2004). Another concern over heterozygous parkin mutation carriers is that they may occur incidentally. However, at least two recent case control studies of the parkin gene in early- and late-onset PD reveal the presence of several heterozygous parkin mutations, including R275W, that are not found in control individuals, thereby providing a direct association between heterozygous parkin mutations and increased susceptibility for PD. Notwithstanding the controversy surrounding the existence of single parkin mutations, whether and how heterozygous parkin condition affects dopaminergic neuronal survivability remains obscure (Wang, 2007).
The importance of functional parkin to dopaminergic neuronal survival is probably related to the multitude of neuroprotective roles it serves. Supporting this, it was found that flies overexpressing wild-type human parkin are more resistant to rotenone-induced degeneration and associated locomotor defects. Moreover, expression of wild-type human parkin in parkin null flies effectively prevents select dopaminergic neurons from degenerating. Although Ddc-GAL4-driven hparkin/- flies recorded similarly poor climbing scores to parkin null flies, this is likely attributable to the inability of Ddc-GAL4-driven hparkin expression to compensate for the widespread muscle pathology in parkin null flies. Notably, when driven by 24B-GAL4, wild-type but not R275W human parkin expression in parkin null flies significantly mitigates the mitochondrial phenotype of the latter. This is consistent with previous reports by others showing that expression of wild-type Drosophila parkin using 24B-GAL4 could rescue dparkin null phenotype. The apparent protection afforded by wild-type human parkin against dopaminergic neurodegeneration and mitochondrial pathology in parkin null flies suggests a certain degree of functional conservation between human and fly parkin (Wang, 2007).
Given its broad-spectrum neuroprotective roles, one could envisage that parkin haploinsufficiency could increase the risk of heterozygous parkin mutation carriers for PD. Consistent with this, it has bee demonstrated that age, mutations, and PD-linked stress could deplete the availability of soluble functional parkin in the brain (LaVoie, 2005; Sriram, 2005; Wang, 2005a; Wang, 2005b; Wong, 2007) and, as such, may underlie the progressive susceptibility of the brain to degeneration. Furthermore, a promoter variant of parkin associated with a lower expression of parkin occurs more commonly in PD cases than in controls. However, the possibility that dominant-negative mutations might account for a proportion of single parkin mutation-linked PD cases cannot be excluded. In particular, a recent family-based study conducted in Germany implicates the transmission of the R275W mutation as an autosomal dominant trait, thereby providing additional support for the notion that single R275W parkin mutation might be sufficient to cause disease (R. Krueger, personal communication to Wang, 2007). Supporting this, the current study demonstrates that the overexpression of parkin R275W in Drosophila is toxic to dopaminergic neuronal survival, albeit in select clusters. Furthermore, R275W expression in Drosophila flight muscles also promotes mitochondrial abnormalities. Thus, heterozygous parkin R275W expression in vivo may contribute to pathogenecity. Interestingly, these observations corroborate with a very recent study conducted by Sang (2007) who showed that expression of parkin T240R and Q311X mutants in Drosophila causes age-dependent, selective degeneration of DA neurons accompanied by progressive motor impairment. The dissimilar outcomes mediated by R275W and G328E mutants in flies may be related to their different properties, as observed in a previous in vitro study (Wang, 2005a; Wang, 2007 and references therein).
This study applied an unbiased confocal microscopy-based quantitative method to detect subtle neuronal loss in the fly brains. Similar to the observation made by Whitworth (2005) with parkin null mutants, comparable loss in the number of dopaminergic neurons within the PPL1 cluster was detected in parkin null flies at 20 d after eclosion. Furthermore, an obvious loss of dopaminergic neurons was observed in the PAM cluster of these flies. Importantly, R275W parkin-overexpressing flies show the same degeneration pattern as parkin null flies, and both these mutant fly species exhibit marked mitochondrial pathology, suggesting that heterozygous parkin mutations could result in similar outcomes to that arising from the overt loss of parkin function. It is noteworthy that flies expressing the R275W mutant also appear to be more susceptible to rotenone-induced neurotoxicity. Although accelerated neuronal loss is observed in all the dopaminergic clusters in the various fly strains after rotenone treatment, the PPL1 neurons in R275W are significantly more affected. Interestingly, the selective impact on neuronal integrity exerted by the expression of R275W in flies correlates well with their impaired climbing ability compared with age-matched control flies. Although previous studies have attributed compromised locomotory activity observed in parkin null flies primarily to defects in their musculature, the current results with Ddc-driven parkin R275W flies show that dopaminergic neurodegeneration in select clusters alone is sufficient to trigger climbing defects. Furthermore, the protection of similar clusters of neurons by the Ddc-driven expression of wild-type human parkin translates to better climbing scores. It is thus tempting to suggest a direct association between dopaminergic neurodegeneration and locomotory dysfunction in flies, a phenomenon that is seen in PD patients (Wang, 2007).
How the expression of R275W mutant influences the function of endogenous parkin remains to be elucidated, but its expression does not appear to aggravate the dopaminergic neurodegeneration process when expressed in parkin null flies. Although the latter is consistent with the R275W mutant parkin acting as a dominant-negative protein, the poorer climbing scores of R275W/- flies compared with R275W and parkin null flies is intriguing. Furthermore, the abnormal mitochondrial phenotype observed in the flight muscles of R275W mutant flies is qualitatively different from that seen in parkin null flies, suggesting that R275W parkin mutant may exert toxic effects in vivo via a different mechanism from that produced by the overt loss of parkin expression. As with all experiments involving the expression of transgenes in a heterologous system, the possibility cannot be excluded that the phenotypic characteristics exhibited by R275W mutant flies arise from neomorphic manifestations associated with the expression of a foreign gene. However, several observations made in this study would argue against R275W mutant acting as a neomorph. (1) Ddc-driven expression of R275W mutant in flies affects the same clusters of dopaminergic neurons as those in parkin null flies; (2) although the effects mediated by R275W and parkin null mutant on Drosophila flight muscle are not exactly identical, they nonetheless promote a similar outcome, i.e., mitochondrial abnormalities; (3) compared with the R275W mutant, the better-expressed parkin G328E mutant behaves as a relatively benign foreign protein in Drosophila. Importantly, when wild-type parkin-expressing flies were crossed with R275W mutant flies, it was found that their coexpression significantly mitigates the loss of PPL1 dopaminergic neurons observed in R275W flies. Although the exact mechanism of R275W mutant-mediated toxicity remains to be clarified, it is apparent that the R275W fly model recapitulates the essential symptomatic features of PD and thus represents an ideal in vivo model of parkin dysfunction. Given the demonstrated pathogenicity of R275W mutant parkin expression in flies, these results may also help explain the increased susceptibility of heterozygous parkin carriers to develop PD (Wang, 2007).
In summary, this study has provided important in vivo evidence demonstrating that select parkin mutations could generate marked neurotoxicity in vivo. Whether the neurotoxic effects mediated by these mutants could indeed account for a proportion of single parkin mutation-linked PD cases remains to be established. Additional study should help elucidate the pathogenic mechanism caused by these mutations (Wang, 2007).
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. 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. 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).
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. 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, 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).
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)Pod2 and ref(2)Pod3 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).
Loss-of-function mutations in PARK2, the gene encoding the E3 ubiquitin ligase Parkin, are the most frequent cause of recessive Parkinson's disease (PD). Parkin translocates from the cytosol to depolarized mitochondria, ubiquitinates outer mitochondrial membrane proteins and induces selective autophagy of the damaged mitochondria (mitophagy). This study show that Ubiquitin-specific protease 15 (USP15), a deubiquitinating enzyme (DUB) widely expressed in brain and other organs, opposes Parkin-mediated mitophagy, while a panel of other DUBs and a catalytically inactive version of USP15 do not. Moreover, knockdown of USP15 rescues the mitophagy defect of PD patient fibroblasts with PARK2 mutations and decreased Parkin levels. USP15 does not affect the ubiquitination status of Parkin or Parkin translocation to mitochondria, but counteracts Parkin-mediated mitochondrial ubiquitination. Knockdown of the DUB CG8334, the closest homolog of USP15 in Drosophila, largely rescues the mitochondrial and behavioural defects of parkin RNAi flies. These data identify USP15 as an antagonist of Parkin and suggest that USP15 inhibition could be a therapeutic strategy for PD cases caused by reduced Parkin levels (Cornelissen, 2014).
Northern blot analysis using poly(A)+ RNA from embryos, larvae, and adults detected parkin transcripts at all developmental stages with particularly high abundance in adults (Greene, 2003).
To generate a disruption of the parkin gene, a transposon mutagenesis screen was conducted by using a P element mapping close to parkin. This strategy yielded a single line, designated parkEP(3)LA1, bearing an insertion 71 bp upstream from the parkin start codon. To generate more severe alleles of parkin, the parkEP(3)LA1 insertion was mobilized with transposase under conditions favoring the creation of coincident deletions extending from the insertion locus. A large collection of deletion alleles were recovered from this screen, including several that remove all of the parkin coding sequence and thus represent null alleles of parkin. This work also yielded a chromosome bearing a precise excision of parkEP(3)LA1, which was maintained for use as a control chromosome (designated parkrvA) in these studies (Greene, 2003).
Flies bearing any of the parkin null alleles in trans to the Df(3L)Pc-MK deletion chromosome, which removes the parkin gene, are viable through the adult stage of development but exhibit a slight developmental delay, typically eclosing a day later than controls, and show significantly reduced longevity (P < 0.0001). parkin null flies have an average lifespan of 27 days, with none able to exceed 50 days of age, whereas flies bearing the parkrvA precise excision chromosome in trans to Df(3L)Pc-MK have a mean lifespan of 39 days and can survive up to 75 days (Greene, 2003).
Female parkin mutants are fertile and produce normal offspring, however, males are completely sterile. This finding permitted a screen of a collection of ~1,100 ethyl methanesulfonate-mutagenized homozygous viable male sterile lines for additional parkin mutations generated independently from deletion alleles. Sequencing the parkin gene from two of the mutants recovered from this screen revealed missense and premature stop codon mutations, verifying that the male sterile phenotype results from loss of parkin function (Greene, 2003).
Analysis of testes from homozygous or transheterozygous parkin mutants indicates that the male sterile phenotype derives from a late defect in spermatogenesis. Spermatogenesis appears to proceed normally in parkin mutants until the individualization stage, at which point a 64-cell germ-line cyst that normally separates into mature sperm cells fails to do so, resulting in an absence of mature sperm cells in the seminal vesicle. Ultrastructural analysis of developing spermatids in parkin mutants revealed structural irregularities in the sperm tails. Mature sperm tails usually consist of a flagellar axoneme, with a 9 + 2 arrangement of microtubules, and a specialized mitochondrial derivative known as the Nebenkern. Although the axoneme in parkin mutants appears normal, Nebenkern integrity is severely disrupted; some spermatids have multiple Nebenkern, whereas others have only an extremely diminished component. Additionally, the electron density of the Nebenkern outer matrix is significantly diminished with respect to wild type. These results suggest that defective Nebenkern formation and/or function may underlie the spermatid individualization failure of parkin mutants (Greene, 2003).
In addition to reduced longevity and male sterility, all of the parkin null alleles, as well as those recovered on the basis of the male sterile phenotype, confer a partially penetrant downturned wing phenotype as homozygotes or as transheterozygotes with the Df(3L)Pc-MK chromosome. The penetrance of this phenotype increases with age; ~40% of newly eclosed flies exhibit abnormal wing posture, whereas by 10 days of age more than 70% of the parkin mutants display this phenotype. This finding prompted the use of an assay of the locomotor ability of parkin mutants. These analyses revealed severe defects in both flight and climbing ability in parkin mutants. Both phenotypes were also manifest in parkin mutants with normal wing posture. The climbing decay rate was similar in parkin mutants and wild-type flies, indicating that parkin mutants begin adult life with a reduced climbing ability (Greene, 2003).
To address the origin of the locomotion defects in parkin mutants, the UAS/GAL4 system was used to express parkin in defined tissues. Two GAL4 lines that drive parkin expression in mesoderm were found to rescue the wing posture, flight, and climbing phenotypes of parkin mutants to wild-type or near wild-type levels, demonstrating that Parkin function is required in the musculature (Greene, 2003).
Histological analysis of the major flight muscles [the indirect flight muscles (IFMs)] from parkin mutants revealed severe disruption of muscle integrity, consistent with the role of muscle dysfunction in the parkin flight defect. Muscle integrity was almost completely restored in parkin mutants ectopically expressing parkin in muscles. Analysis of proboscis muscle from parkin mutants revealed pathology similar to that of the IFM, indicating that this phenotype is not specific to the flight muscles. However, the tergal depressor of trochanter muscle, involved in the jump response, and the larval body-wall muscles involved in larval locomotion are morphologically and functionally normal in parkin mutants, indicating that only a subset of muscles are affected by loss of parkin function (Greene, 2003).
Ultrastructural analysis of the IFM in 1- to 2-day-old parkin mutants revealed an overall decrease in the density of myofibrils, a broadening of the myofibril Z-line, and a shortening of the sarcomere length. However, the myofibril structural alterations were variable with some indistinguishable from those of control flies. By contrast, swollen mitochondria manifesting severe disruption and disintegration of the cristae were a uniform feature of the IFM in parkin mutants. Transgenic expression of parkin in the musculature restores the myofibril integrity and mitochondrial morphology. The temporal relationship between mitochondrial and myofibril pathology was investigated by analyzing IFM ultrastructure in parkin mutants at the pupal stage of development shortly after IFM formation. At 96 or 120 h after puparium formation the integrity of the myofibrils in parkin mutants was similar to controls, showing no signs of degeneration. The only detectable difference in IFM ultrastructure between parkin mutants and control animals at the pupal stage of development was a disintegration of the mitochondrial matrix in parkin mutants. These results demonstrate that mitochondrial pathology is an early indicator of muscle dysfunction and that the muscle pathology is degenerative in nature (Greene, 2003).
To determine whether muscle degeneration in parkin mutants proceeds through an apoptotic mechanism, the IFM in parkin mutants and age-matched control flies were subjected to terminal deoxynucleotidyltransferase-mediated dUTP end labeling (TUNEL) staining. At 96 and 120 h after puparium formation, no TUNEL staining was detected in parkin mutants and control flies. However, a dramatic increase in TUNEL-positive nuclei was observed in the IFM of 1-day-old adult parkin mutants relative to age-matched control flies, suggesting that the muscle mitochondrial defects ultimately result in cell death through an apoptotic mechanism (Greene, 2003).
To examine the role of parkin in the brain, sections were prepared from flies at 1, 10, and 30 days of age. Standard histologic analysis revealed appropriate development of the major brain centers. No obvious loss of neuropil integrity or cell cortical number was seen in aged parkin mutant flies compared with controls. Because dopaminergic neurons are a preferential target in AR-JP, brain sections were also immunostained for tyrosine hydroxylase. Dopaminergic neurons of the dorsomedial, dorsolateral, and anteromedial clusters and the medulla were assessed. No clear neuronal loss was observed in any of these cell groups. Tyrosine hydroxylase immunoreactive terminal density was also generally preserved. However, cells of the dorsomedial dopaminergic cell cluster reliably showed shrinkage of the cell body and decreased tyrosine hydroxylase immunostaining in proximal dendrites in aged parkin mutants relative to controls. No such changes were observed in other dopaminergic cell groups. The preferential effect on the dorsomedial cluster is intriguing given the enhanced toxicity of alpha-synuclein (Feany, 2000; Auluck, 2002), another protein implicated in familial PD, in this cluster of dopaminergic neurons (Greene, 2003).
An obvious difference between Drosophila parkin mutants and AR-JP concerns the tissues affected by loss of parkin function. Dopaminergic neurons in the substantia nigra appear to be the primary tissues affected in AR-JP individuals, whereas the most striking phenotypes in Drosophila parkin mutants derive from muscle and germ-line pathology. Nevertheless, the underlying molecular mechanisms responsible for pathology in these different tissues may be highly conserved. Indeed, ultrastructural examination of the male germ line and IFM in parkin mutants reveals mitochondrial defects as a common characteristic of pathology in these distinct tissue types. Although further work will be required to establish the relevance of mitochondrial pathology to the spermatid individualization defect, studies of IFM pathogenesis strongly indicate that mitochondrial pathology is a primary defect. Thus, these results suggest that the Drosophila parkin phenotypes derive from a common origin of mitochondrial dysfunction. There are a variety of cellular insults capable of producing the specific mitochondrial structural alterations observed in parkin mutants, and further work will be required to elucidate the mechanism by which loss of parkin function triggers mitochondrial pathology and ultimately cell death (Greene, 2003).
Although only a few of the factors contributing to the PD disorder have currently been identified in mammals, significant insight into the mechanism of neuronal death in PD has come from studies of the PD-inducing compound 1-methyl-4-phenylpyridinium (MPP+). MPP+ is a specific toxin of dopaminergic neurons that induces cell death by inhibiting mitochondrial complex I. This finding led to the identification of other mitochondrial complex I inhibitors that trigger death of dopaminergic neurons, and prompted studies of mitochondrial integrity in individuals with idiopathic PD. These studies revealed a correlation between PD and mitochondrial dysfunction, and together with the studies of mitochondrial toxins, provide strong support for mitochondrial dysfunction as a major component of PD (Greene, 2003).
The finding that mitochondrial pathology and apoptosis are prominent features of indirect flight muscle degeneration in Drosophila raises the possibility that similar mechanisms underlie dopaminergic neuron loss in AR-JP. Although previous studies have not addressed a role for parkin in mitochondrial integrity or apoptosis, a substantial body of evidence suggests that mitochondrial dysfunction and apoptosis are important factors underlying neurodegeneration in idiopathic PD. Thus, these findings provide a potential mechanistic link between AR-JP and the broader spectrum of idiopathic PD (Greene, 2003).
Panneuronal expression of Parkin substrate Pael-R in transgenic flies causes age-dependent selective degeneration of Drosophila dopaminergic (DA) neurons. The initial observation that in more than half of the Pael-R transgenic lines the protein is present at a low level undetectable by immunostaining suggests that there are mechanisms in Drosophila that limit its accumulation. Recent sequencing of the Drosophila genome has revealed a fly homolog of human Parkin. To test whether Drosophila parkin genetically interacts with the Pael-R transgene, the RNA interference (RNAi) technique was used to inhibit Drosophila parkin expression and examined the effect on Pael-R-induced DA neuron degeneration. To inhibit the expression of parkin in DA neurons of adult fly brain, UAS-IRParkin transgenic flies were generated that express double-stranded Parkin RNA as hairpin RNA from an inverted repeat of Parkin cDNA. Ubiquitous expression of hairpin Parkin RNA using a heat shock-GAL4 driver line resulted in a significant reduction of endogenous Parkin mRNA as determined by quantitative RT-PCR analysis (Yang, 2003).
When IRParkin and Pael-R transgenes were coexpressed in transgenic flies using the Ddc-GAL4 driver, a dosage-dependent acceleration of Pael-R-induced DA neuron degeneration was observed. Transgenic flies coexpressing Pael-R and two copies of the IRParkin transgene showed the degeneration phenotype when they were analyzed at 14 days of age. At this age, transgenic flies expressing Pael-R alone or coexpressing Pael-R and one copy of the IRParkin transgene showed a relatively normal number of DA neurons in the DM clusters. Furthermore, transgenic flies coexpressing Pael-R and two copies of IRParkin showed more severe DA degeneration phenotypes when analyzed at 30 days of age, with the average number of DA neurons in the DM clusters reduced by more than 50%. Thus, inhibition of endogenous Parkin function accelerates the kinetics and enhanced the severity of Pael-R-induced degeneration of DA neurons. In transgenic flies that express IRParkin alone, the number of DA neurons in the DM clusters is not significantly affected in 30-day-old flies. This indicates that activation of the RNAi pathway by itself is not deleterious to DA neurons. The lack of DA neuronal loss by Parkin RNAi alone may be due to the absence of toxic substrates such as Pael-R in Drosophila or that endogenous dParkin substrates may never accumulate to levels sufficient to kill DA neurons. However, since RNAi in IRParkin transgenic flies results in a reduction but not complete loss of Parkin transcripts, it is possible that the residual Parkin may still provide neuroprotective function. Generation of genetic null mutations in the dParkin locus could address this issue (Yang, 2003).
The acceleration of DA neuron degeneration after Parkin RNAi could be due to defects in the turnover or subcellular distribution of Pael-R protein. To investigate the mechanisms involved, Pael-R protein was analyzed by immunostaining cultured DA neurons that coexpressed IRParkin and Pael-R. Coexpression of IRParkin and Pael-R in DA neurons results in the accumulation of Pael-R to higher levels compared to expression of Pael-R alone. Some of the accumulated Pael-R protein was unevenly distributed. Thus, interfering with endogenous Parkin function results in increased stability and accumulation of Pael-R protein. This may be responsible for the acceleration and enhancement of Pael-R-induced DA neuron degeneration observed in Parkin RNAi transgenic flies (Yang, 2003).
Mutations in the gene parkin in humans (PARK2) are responsible for a large number of familial cases of autosomal-recessive Parkinson disease (PD). A Drosophila homolog of human PARK2 has been isolated and its expression and null phenotype have been characterized. parkin null flies have 30% lower mass than wild-type controls; this is in part accounted for by a reduced cell size and number. In addition, these flies are infertile, show significantly reduced longevity, and are unable to jump or fly. Rearing mutants on paraquat, which generates toxic free radicals in vivo, causes a further reduction in longevity. Furthermore, loss of parkin results in progressive degeneration of most indirect flight muscle (IFM) groups soon after eclosion, accompanied by apoptosis. However, parkin mutants have normal neuromuscular junction recordings during the third larval instar stage, suggesting that larval musculature is intact and that parkin is required only in pupal and adult muscle. parkin flies do not show an age-dependent dopaminergic neuron loss in the brain, even after aging adults for 3 weeks. Nevertheless, degeneration of IFMs demonstrates the importance of parkin in maintaining specific cell groups, perhaps those with a high-energy demand and the concomitant production of high levels of free radicals. parkin mutants will be a valuable model for future analysis of the mechanisms of cell and tissue degeneration (Pesah, 2004).
parkin mutant animals have reduced body and cell size at eclosion, suggesting possible defects in cell growth, proliferation and/or cell survival. This phenotype is particularly interesting given the interaction of human Parkin with cyclin E, an important regulator of cell cycle progression. The reduced body size of parkin mutants is similar to the phenotypes of insulin growth factor (IGF) receptor mutant flies and mice. Physiological effects of insulin in the brain are not limited to regulation of food intake and control of glucose uptake, but are also important in trophic actions on neurons and glial cells. Administration of the N-terminal tripeptide of IGF1 prevents loss of DA neurons after chemically (6-hydroxydopamine)-induced DA cell lesion in rats. In addition, IGF1 also protects against DA-induced neurotoxicity in vitro. These observations suggest parkin may play a role in the insulin signaling pathway during development or in adults (Pesah, 2004).
parkin mutant flies have increased sensitivity to paraquat toxicity. Paraquat, with its two N-methyl pyridinium moieties, is structurally similar to MPP(+), a toxic metabolite of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; when converted to the active toxin MPP+ by monoamine oxidase B it can induce parkinsonism in primates). In humans, toxins such as MPTP cause DA neuron-specific death in the substantia nigra because of production of oxygen radicals, resulting in Parkinsonian symptoms. Glutathione and superoxide dismutase (SOD) inactivate H2O2 and superoxide radicals, respectively, thereby reducing MPTP neurotoxicity in mice. Paraquat also causes high oxygen radical production and interferes with mitochondrial respiration, resulting in cell death. The effects of paraquat are not specific to DA cells and sensitivity to its toxicity reflects a general impairment of oxygen radical defense. Loss of Drosophila SOD also results in sensitivity to low doses of paraquat, reflecting a similar defect in oxygen radical defense. The results suggest that antioxidant defenses in parkin mutant flies are also impaired and that parkin may play a role in the oxidative stress response. One likely mechanism of Parkin function is to help rid the cell of specific proteins that are misfolded because of oxygen radical damage. This is consistent with the identification of human Parkin as an E3 ligase (Pesah, 2004 and references therein).
parkin mutant flies show a progressive apoptotic degeneration of indirect flight muscles (IFMs). Furthermore, this degeneration is accompanied by mitochondrial disintegration and loss of christae. Drosophila IFMs are groups of specialized muscle that are in a constant state of vibration. They require a high oxygen supply to sustain their respiratory activity, making this tissue especially susceptible to mitochondrial dysfunction. It is possible that the mitochondrial degeneration observed in parkin mutants results in increased susceptibility to oxygen radical damage because of the impairment of antioxidant defenses by mitochondria, culminating in cell death. The data from animal and tissue culture models of Parkinson disease suggest that mitochondrial dysfunction and oxygen radical damage are two crucial factors in the development of PD pathology. The data underscore the importance of the apoptotic pathway in DA cell loss. Oxidative stress such as that produced by MPTP can trigger apoptosis. Transgenic mice overexpressing the anti-apoptotic gene Bcl2 and mice null for the proapoptotic gene Bax are resistant to MPTP. Loss of parkin results in similar phenotypes in flies: increased sensitivity to oxygen radical stress and IFM apoptosis, suggesting that underlying mechanisms of cellular dysfunction may be similar between flies and humans (Pesah, 2004).
Individuals with Autosomal Recessive-Juvenile Parkinson's (AR-JP) exhibit a loss of DA neurons in the substantia nigra similar to that of idiopathic PD. Therefore, a similar phenotype in parkin mutant animals would be expected. Focus was placed on the dorsomedial cluster (DMC) neurons because they were shown to be affected in other Drosophila models of PD. However, no significant loss of DA neurons was observed at three weeks of age in parkin mutant animals compared with controls. In addition, no changes were seen in DA cell morphology in parkin mutants. Loss of mouse PARK2 also does not result in DA cell loss. However, increased extracellular DA concentration, abnormal neurophysiology and motor and cognitive behavioral deficits are observed in PARK2 mutant mice. By contrast, only motor deficits have been observed thus far in Drosophila. In another model of PD, overexpression of alpha-synuclein in mice does not result in DA cell loss but does cause DA neuron dysfunction. Similarly, in Alzheimer's disease mouse models employing overexpression of amyloid precursor protein, cognitive defects are present in the absence of cortical neuronal loss. The absence of cell loss in multiple models of neurodegenerative disease may reflect the shorter life span of flies and mice compared with humans. Although the mean onset of PD symptoms in AR-JP is the earliest of all genetically defined forms of Parkinson's disease, it is not clear how to compare fly, mouse and human life spans in this regard (Pesah, 2004).
Even though no DA loss is seen in parkin mutant animals, the mechanism of cell loss may be similar between humans and Drosophila. Neurons and muscle are two of the most energy-dependent tissues, and therefore have high numbers of mitochondria and are highly sensitive to mitochondrial insults. Although the mechanisms of cell death in IFMs and in DA neurons might be similar, it is possible that Drosophila IFMs are more sensitive than DA neurons to mitochondrial defects. The nature of this defect is still unclear; however, three mechanisms are likely. (1) It is possible that parkin functions in a trophic factor pathway that promotes cell survival, and in its absence cells become more susceptible to insults such as oxygen radicals. (2) parkin might be important in the stress response pathway, and in its absence the cell becomes more susceptible to various stimuli such as oxygen radical damage that triggers apoptosis. (3) It is possible that parkin is part of the cell death pathway, and its absence results in susceptibility to proapoptotic insults. Drosophila parkin mutants will serve as an invaluable model for understanding the biological role of parkin and may provide important clues concerning the molecular mechanisms of PD (Pesah, 2004).
Loss-of-function mutations of the parkin gene are a major cause of early-onset parkinsonism. To explore the mechanism by which loss of parkin function results in neurodegeneration, a genetic approach was used in Drosophila. Drosophila parkin mutants display degeneration of a subset of dopaminergic (DA) neurons in the brain. The neurodegenerative phenotype of parkin mutants is enhanced by loss-of-function mutations of the glutathione S-transferase S1 (GstS1) gene, which were identified in an unbiased genetic screen for genes that modify parkin phenotypes. Furthermore, overexpression of GstS1 in DA neurons suppresses neurodegeneration in parkin mutants. Given the previous evidence for altered glutathione metabolism and oxidative stress in sporadic Parkinson's disease (PD), these data suggest that the mechanism of DA neuron loss in Drosophila parkin mutants is similar to the mechanisms underlying sporadic PD. Moreover, these findings identify a potential therapeutic approach in treating PD (Whitworth, 2005; full text of article)
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).
Mutations in Pink1, a gene encoding a Ser/Thr kinase with a mitochondrial-targeting signal, are associated with Parkinsons 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 Pink1s 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).
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).
Mutations in human parkin have been identified in familial Parkinson's disease and in some sporadic cases. Expression of mutant but not wild-type human parkin in Drosophila causes age-dependent, selective degeneration of dopaminergic (DA) neurons accompanied by a progressive motor impairment. Overexpression or knockdown of the Drosophila vesicular monoamine transporter, which regulates cytosolic DA homeostasis, partially rescues or exacerbates, respectively, the degenerative phenotypes caused by mutant human parkin. These results support a model in which the vulnerability of DA neurons to parkin-induced neurotoxicity results from the interaction of mutant parkin with cytoplasmic dopamine (Sang, 2007; full text of article).
Most motor symptoms of PD are the result of progressive degeneration of DA neurons originating in the substantia nigra. The molecular pathways that lead to the death of this population of DA neurons are not known, and understanding the basis of selective mesencephalic DA neuron vulnerability may aid the rational design of therapeutics. Because all identified PD-linked genes are expressed ubiquitously in the CNS, it is unclear why such mutations give rise to selective pathology in the nigrostriatal system. It has been suggested that the pathogenesis of monogenic and perhaps idiopathic PD might involve proteins or neurochemicals that are particularly abundant in DA neurons. In vertebrates, nigral DA neurons are characterized by a distinct set of proteins that play a role in DA synthesis and metabolism, such as TH, Ddc, monoamine oxidase (MAO), and the plasma membrane dopamine transporter, as well as other proteins such as VMAT that may be differentially expressed. And of course, unlike other cells, DA neurons also store and release dopamine. The neurotoxic effects of dopamine may be mediated through general oxidative effects and perhaps more specific interactions with proteins implicated in familial PD. It has been observed that alpha-synuclein-induced neurotoxicity is observed in primary cultures of DA neurons but not in non-DA cortical neurons. Moreover, mutant forms of alpha-synuclein mutation form neurotoxic adducts with DA quinone. In addition, the oxidative effects of dopamine may increase protein nitrosylation, and parkin activity is altered by this modification both in vitro and in vivo (Sang, 2007).
Fruit flies use similar sets of genes for dopamine synthesis and transport, but they metabolize dopamine differently from mammals. Insects do not express MAO and are thought to use conjugation as the primary route for amine degradation. This difference may be related to the unique use of dopamine for the hardening and pigmentation of cuticles in insects and other arthropods. Indeed, the need to maintain adequate levels of cytosolic dopamine for these pathways may account for the somewhat surprising effects of Drosophila vesicular monoamine transporter (DVMAT) in total head concentrations of dopamine. Overexpression of DVMAT decreases total dopamine and inhibition of DVMAT using RNAi increases total tissue dopamine. Overexpression of mammalian VMAT2 in cultured cells decreases cytosolic dopamine, and decreased VMAT2 activity in mammals reduces total tissue dopamine. It is suggested that the cytoplasmic pool of dopamine may account for a larger proportion of total tissue dopamine in Drosophila, perhaps because of the requirement of dopamine for cuticle formation in other tissues. It is possible that this may render DA neurons in the fly particularly sensitive to neurotoxic mechanisms involving the conjugation of dopamine to cytoplasmic targets (Sang, 2007).
Based on previous findings in mammals, it is speculated that dopamine and/or oxidized derivatives might contribute to mutant parkin-induced degeneration. If so, mutant parkin phenotypes should be relatively specific for DA neurons and sensitive to modulation of cytoplasmic dopamine levels. Indeed, it was observed that DA but not cholinergic or histaminergic neurons degenerate in response to expression of mutant parkin. Interestingly, a more limited degenerative phenotype was observed in 5-HT neurons, consistent with the loss of other aminergic cell types in PD. DVMAT knockdown enhances mutant parkin phenotypes, suggesting that an increase in cytoplasmic dopamine increases the vulnerability of neurons to mutant parkin. Conversely, increasing DVMAT partially rescues pupal lethality and DA neuron degeneration, presumably by reducing cytoplasmic dopamine. These data therefore suggest that mutations in parkin either increase susceptibility of neurons to dopamine or its metabolites or that dopamine is permissive for the toxic effects of parkin. The Drosophila model, which provides robust behavioral and neuropathological phenotypes, suggests that some mutations may give rise to disease by dominant mechanisms. Moreover, this model may prove a useful addition to other genetically based vertebrate and invertebrate models aimed at understanding PD and identifying potential therapeutic targets (Sang, 2007).
Mutations in parkin and PTEN-induced kinase 1 (Pink1) lead to autosomal recessive forms of Parkinson's disease (PD). parkin and Pink1 encode a ubiquitin-protein ligase and a mitochondrially localized serine/threonine kinase, respectively. Recent studies have implicated Parkin and Pink1 in a common and evolutionarily conserved pathway for protecting mitochondrial integrity. To systematically identify novel components of the PD pathways, genetic background was generated that allowed a genome-wide F1 screen for modifiers of Drosophila parkin (park) and Pink1 mutant phenotype. From screening ~80% of the fly genome, a number of cytological regions were identified that interact with park and/or Pink1. Among them, four cytological regions were selected for identifying corresponding PD-interacting genes. By analyzing smaller deficiency chromosomes, available transgenic RNAi lines, and P-element insertions, five PD-interacting genes were identified. Among them, opa1 and drp1 have been previously implicated in the PD pathways, whereas debra (dbr), Pi3K21B and β4GalNAcTA are novel PD-interacting genes. This study took an unbiased genetic approach to systematically isolate modifiers of PD genes in Drosophila. Further study of novel PD-interacting genes will shed new light on the function of PD genes and help in the development of new therapeutic strategies for treating Parkinson's disease (Fernandes, 2008).
Among the three novel PD-interacting genes (i.e., debra, Pi3K21B, and β4GalNAcTA) isolated from the screen, debra (determiner of breaking down of Ci activator) (dbr) heterozygosity led to strong enhancement of the park-RNAi-induced wing phenotype. dbr encodes a novel zinc-binding protein of 1007 amino-acid residues. Cell culture studies showed that Dbr forms a complex with Slimb, a component of the SCF (Skpl, Cdc53 and F box) ubiquitin ligase complex, to mediate the polyubiquitination of the transcription factor Cubitus interruptus (Ci) and thus targets Ci into the lysosome for degradation. This raises the interesting possibility that Dbr functions together with Park in the ubiquitin-proteasome pathway for the control of protein quality. Reducing the dosage of dbr may thus increase the accumulation of toxic protein substrates, leading to the enhancement of the park phenotype. In this context, it is worth noting that a recent study showed that reducing the level of dbr also enhanced Ataxin3-induced neurodegeneration in Drosophila, which also resulted from accumulation of pathogenic proteins (Bilen, 2007). Additionally, since Dbr is a zinc-binding protein, Dbr may also play a role in regulating the level of intracellular zinc. Zinc dyshomeostasis has been shown to cause abnormalities in autophagy that are associated with Alzheimer's disease, Parkinson's disease, and Huntington's disease. Thus, it is possible that in addition to its interaction with Park in the ubiquitin-proteasome pathway, Dbr may interact with the PD pathway by regulating autophagy (Fernandes, 2008).
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. 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).
Parkinson's disease has been linked to altered mitochondrial function. Mutations in parkin (park), the Drosophila ortholog of a human gene that is responsible for many familial cases of Parkinson's disease, shorten life span, abolish fertility and disrupt mitochondrial structure. However, the role played by Park in mitochondrial function remains unclear. This study describe a novel Drosophila gene, clueless (clu), which encodes a highly conserved tetratricopeptide repeat protein that is related closely to the CluA protein of Dictyostelium, Clu1 of Saccharomyces cerevisiae and to similar proteins in diverse metazoan eukaryotes from Arabidopsis to humans. Like its orthologs, loss of Drosophila clu causes mitochondria to cluster within cells. Strong clu mutations resemble park mutations in their effects on mitochondrial function and the two genes interact genetically. Conversely, mitochondria in park homozygotes become highly clustered. It is proposed that Clu functions in a novel pathway that positions mitochondria within the cell based on their physiological state. Disruption of the Clu pathway may enhance oxidative damage, alter gene expression, cause mitochondria to cluster at microtubule plus ends, and lead eventually to mitochondrial failure (Cox, 2009).
Mitochondria are often positioned within cells by motor-dependent movement along microtubules. Clu protein appears to contact mitochondria, especially in the large Clu particles, many of which are located adjacent to microtubules. The current experiments suggest that clu-induced mitochondrial aggregates are caused by changes in microtubule-based mitochondrial transport. In both the Drosophila brain and ovary, mitochondria associate with transport complexes containing the adaptor protein Milt that are linked to both plus-end-directed motors such as Khc and minus-end-directed motors such as Dhc. During oogenesis, loss of Khc, or of one of the Milt isoforms that interacts with Khc, cause mitochondria to cluster in cellular regions that are rich in microtubule minus ends, presumably because countervailing movement toward plus ends has been lost. Strikingly, mitochondria in clu mutant ovaries accumulate at predicted sites of microtuble plus ends; these include the proximal region of GSCs. Thus, in some situations, clu mutations may act by interfering with minus-end-directed mitochondrial movement along microtubules. Whether Clu particles correspond to mitochondrial transport complexes that mediate these changes, or whether Clu acts indirectly on mitochondrial positioning could not be determined with certainty from these studies (Cox, 2009).
In other cells, such as nurse cells, clu mutations also cause pronounced mitochondrial clustering, but that clustering is not mimicked by mutations in genes encoding microtubule motor proteins or Milt. Other systems of mitochondrial localization may predominate in such cells but still be subject to regulation by the Clu pathway. For example, in plant cells, Clu was postulated to control mitochondrial localization by regulating the choice between microtubule-dependent and microfilament-dependent transport. In particular, the TPR domain of Clu was proposed to repress the interaction of mitochondria with microtubules by competing for binding with the TPR region of Kinesin light chain, thereby allowing transport along actin to predominate. The loss of this postulated interaction through clu mutation predicts that mitochondria would accumulate at microtubule minus ends in these cells. Although the default position where mitochondria accumulate in the absence of Clu might vary depending on the particular cell type and transport systems involved, the use of a Clu-dependent pathway to control mitochondrial subcellular location appears to be widespread (Cox, 2009).
The experiments show that mitochondria require Clu to maintain their subcellular location and structural integrity. In the presence of even a relatively mild clu mutation, transcripts from genes involved in mitochondrial function and in protection from oxidative damage are reduced. Consequently, it is proposed that the normal role of Clu is to function in a pathway that controls the location and activity of mitochondria within the cell. Frequently, mitochondria may need to move to a different subcellular location in order to maximize access to substrates or to mitigate damage from oxidative metabolism. For example, in neurons, microtubule-dependent mitochondrial transport is modulated based on the level of respiratory activity. Clu protein would participate in a pathway that senses the internal physiological state of individual mitochondria and transduces this information into homeostatic changes in their positions and metabolic activities. When the Clu pathway is impaired, mitochondria would not move or operate normally, and might consequently suffer damage. This scenario is consistent with the changes that were observed in the inner mitochondrial membranes of Clu mutants, with their reduced levels of mitochondrial enzymes and the observed changes in nuclear gene expression. The greater severity of clu mutations in Drosophila compared with Dictyostelium cluA and yeast clu1 might be because of an intrinsically greater requirement for dynamic mitochondrial positioning in the specialized cells of complex metazoans. This model may provide a rationale for the unexpected effects of microtubule inhibitors on mitochondrial function that have been reported recently (Cox, 2009).
A model illustrates how the clu and park pathway(s) might link microtubule-based mitochondrial transport, mitochondrial physiology and oxidative damage (See Model for clu and park function). Under normal conditions, the clu and park pathway(s) would sense the physiological state of mitochondria and activate appropriate levels of minus-end-directed mitochondrial movement along microtubules. For example, mitochondria that are low in respiratory substrates might move to cellular regions where these substrates are abundant. Mitochondria in need of repair might move close to the nucleus where appropriate repair genes would be induced. In conjunction with active plus-end-directed motors, the result would be that mitochondria move dynamically to locations throughout the cell that are appropriate to their physiological state. However, without functional clu and park pathway(s), or if the local level of toxic metabolic products exceeded a threshold, the mitochondria in question would cease minus-end-directed transport and undergo concerted plus-end-directed movement. In many cells, the major locus of microtubule minus ends is found near the nucleus so that plus-end-directed movement would increase the distance between reactive oxygen production and the nuclear DNA (Cox, 2009).
These studies have several implications for understanding Parkinson's disease. According to the model, the Clu pathway would contribute strongly to the ability of mitochondria to remain functional during aging, despite the high metabolic requirements and environmental stresses experienced by many tissues. This may be particularly important in neural cells such as those that are compromised in Parkinson's disease. In a cell whose Clu pathway is compromised, mitochondria would operate less efficiently, suffer more damage and wear out faster because they would spend more time in cellular locations that are not appropriate to their metabolic state. The number of mitochondria producing elevated levels of reactive oxygen species would rise, increasing reactive oxygen damage to the nucleus and other cellular components, thereby leading to greater cell death. Consequently, the level of Clu pathway function within an individual might influence their susceptibility to sporadic Parkinson's disease and to other late-onset neurological disorders (Cox, 2009).
Mitochondrial mutations may fall into two classes based on their phenotype and on their relationship to the Clu pathway. Some mutations, such as those affecting mitochondrial ribosomal proteins, cause bang sensitivity, but differ phenotypically from clu mutations. By contrast, mutations in park, pink1, rho7, mitochondrial ATP6, and mitochondrial cytochrome oxidase resemble clu mutations in causing general defects in movement, flight, muscle or nerve degeneration, male fertility, and longevity. The effects of this latter class of mutations may be too severe to be compensated for by Clu pathway operation, leading to mitochondrial mis-positioning and increased production of reactive oxygen species. Consistent with this, expression of anti-oxidants partially suppresses the effects of pink1 on neurodegeneration. Mutations in some of these genes, in addition to park, may also cause mitochondrial clustering and interact genetically with clu. Clearly, further studies of the Clu pathway will deepen understanding of how mitochondria are maintained in cells and why they sometimes become damaged with age (Cox, 2009).
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 interacte 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).
Mutations in parkin and LRRK2 together account for the majority of familial Parkinson's disease (PD) cases. Interestingly, recent evidence implicates the involvement of parkin and LRRK2 in mitochondrial homeostasis. Supporting this, this study shows by means of the Drosophila model system that, like parkin, LRRK2 mutations induce mitochondrial pathology in flies when expressed in their flight muscles, the toxic effects of which can be rescued by parkin coexpression. When expressed specifically in fly dopaminergic neurons, mutant LRRK2 results in the appearance of significantly enlarged mitochondria, a phenotype that can also be rescued by parkin coexpression. Importantly, this study found that epigallocatechin gallate (EGCG), a green tea-derived catechin, acts as a potent suppressor of dopaminergic and mitochondrial dysfunction in both mutant LRRK2 and parkin-null flies. Notably, the protective effects of EGCG are abolished when AMP-activated protein kinase (AMPK) is genetically inactivated, suggesting that EGCG-mediated neuroprotection requires AMPK. Consistent with this, direct pharmacological or genetic activation of AMPK reproduces EGCG's protective effects. Conversely, loss of AMPK activity exacerbates neuronal loss and associated phenotypes in parkin and LRRK mutant flies. Together, these results suggest the relevance of mitochondrial-associated pathway in LRRK2 and parkin-related pathogenesis, and that AMPK activation may represent a potential therapeutic strategy for these familial forms of PD (Ng, 2012).
AMPK is an evolutionarily conserved cellular energy sensor that is activated by ATP depletion or glucose starvation. When activated, AMPK switches the cell from an anabolic to a catabolic mode and, in so doing, helps to regulate cellular energy demands. It is noteworthy that LRRK2-mediated neurodegeneration has been reported to compromise neuronal energy homeostasis due to the chronic activation of protein translation, a highly energy-demanding process (Imai, 2008). Accordingly, overexpression of AMPK could restore the energy perturbation induced by mutant LRRK2 and preserve neuronal function. Another attractive explanation regarding AMPK-mediated neuroprotection came from recent studies demonstrating that AMPK, like parkin, can regulate mitophagy (Egan, 2011; Kim., 2011). Mitophagy induction in this case occurs specifically through AMPK-mediated phosphorylation of the autophagy initiator ATG1. Given this functional convergence between AMPK and parkin, it is tempting to speculate that mitophagy induction may represent a common denominator underlying their respective ability to rescue the phenotypes of LRRK2 G2019S-expressing flies. Thus, in the absence of the parkin or AMPK transgene, LRRK2 mutant expression could significantly retard the clearance of damaged mitochondria, which progressively accumulate with age. This may also explain why the mitochondrial phenotype it induces in the flight muscle is so reminiscent of that brought about by the loss of parkin function (Ng, 2012).
Parkinson's disease (PD) is characterized by movement disorders, including bradykinesia. Analysis of inherited, juvenile PD, identified several genes linked via a common pathway to mitochondrial dysfunction. This study has demonstrated that the larva of the Drosophila parkin mutant faithfully models the locomotory and metabolic defects of PD and is an excellent system for investigating their inter-relationship. parkin larvae displayed a marked bradykinesia that was caused by a reduction in both the frequency of peristalsis and speed of muscle contractions. Rescue experiments confirmed that this phenotype was due to a defect in the nervous system and not in the muscle. Furthermore, recordings of motoneuron activity in parkin larvae revealed reduced bursting and a striking reduction in evoked and miniature excitatory junction potentials, suggesting a neuronal deficit. This was supported by observations in parkin larvae that the resting potential was depolarized, oxygen consumption and ATP concentration were drastically reduced while lactate was increased. These findings suggest that neuronal mitochondrial respiration is severely compromised and there is a compensatory switch to glycolysis for energy production. parkin mutants also possessed overgrown neuromuscular synapses, indicative of oxidative stress, which could be rescued by overexpression of parkin or scavengers of reactive oxygen species (ROS). Surprisingly, scavengers of ROS did not rescue the resting membrane potential and locomotory phenotypes. It is therefore proposed that mitochondrial dysfunction in parkin mutants induces Parkinsonian bradykinesia via a neuronal energy deficit and resulting synaptic failure, rather than as a consequence of downstream oxidative stress (Vincent, 2012).
This study has found that the parkin gene knockout and hypomorph have reduced locomotion due to slower movement resembling bradykinesia, reduced synaptic potentials, decreased quantal size, more positive resting membrane potential (RMP), lower oxygen consumption, reduced ATP and increased lactate, along with overgrown synapses. The results provide a key description of the inter-related actions of a PD-related gene on the electrophysiology, anatomy and respiration of the most genetically tractable model organism, open to cellular and molecular analysis (Vincent, 2012).
Although several laboratories have generated parkin knockouts and hypomorphs, only adult phenotypes were reported previously. This is the first report of larval defects associated with mutations in this early-onset gene. Unlike previous studies, this study obtain few adult park25 or parkZ3678 homozygotes, so it is suspected that the severity of parkin phenotypes is affected by differences in food, possibly trace minerals that modulate oxidative stress (Vincent, 2012).
The key observation is that the neuronal parkin knockout produces a bradykinesia-like reduction in locomotion. Others have shown loss of mobility in geotaxic assays of adult flies following manipulation of PD-related genes in muscles. This study has gone further in showing that the larval deficit is neuronal, not muscular as slower peristalsis results from reduced activity in the motoneurons. This was shown in two ways: genetically and physiologically. Genetically, it was shown that (in the early stages of the degeneration) the nervous system is the main tissue affected by parkin as the locomotory and bradykinesia phenotypes can be rescued by neuronal expression of wild-type parkin. Physiologically, it was shown that both synaptic transmission and central pattern generator (CPG) activity are reduced. The reduction in CPG activity may result from reduced neurotransmission. The CPG may also be slowed by pumping deficits, as the low levels of ATP will increase the time taken to correct the ionic imbalance that builds up in neurons during each peristaltic wave. In addition, locomotion is also modulated by aminergic (dopamine, serotonin etc) neurons that depend on the Drosophila vesicular aminergic transporter (DVMAT). Mutations in this gene severely slow crawling. Since adult data show an interaction of DVMAT with parkin, so too, may larval bradykinesia result from parkin-induced disruption of aminergic transmission (Vincent, 2012).
The smaller EJPs and mEJPs in the parkin knockout indicate a reduction in synaptic transmission. This is most likely to result from reduced glutamate release—perhaps, as a result of problems with its transport into vesicles in an ATP-dependent manner. The recent suggestion that parkin may affect transmission through its interaction with endocytic proteins, e.g. endophilin-A is unlikely to explain the data: although parkin is known to function as a ubiquitin E3 ligase, failure of parkin to ubiquitinate and so degrade endophilin is unlikely to reduce synaptic transmission. A third possibility is that the motoneuron, like the muscle, has a more positive resting potential and this affects the dynamics of glutamate release. Finally, it is noted that the neuronal rescue did not fully restore the size of the EJP, so that a reduction in muscle impedance or post-synaptic ionic dynamics may also contribute to the reduction in synaptic transmission. Although the original descriptions of the phenotype of parkin null mice were divergent, more recent evidence indicates that these mice do have reduced dopamine release at striatal synapses. At the fly neuromuscular junction, manipulations of another PD-related gene, LRRK2, and its Drosophila homologue dLRRK, show complex effects on synaptic transmission. These include a reduction in synaptic transmitter release in the dLRRK knockout. However, the other effects of LRRK2 and parkin manipulations are dissimilar, indicating that these genes may not always be acting through a common cellular mechanism (Vincent, 2012).
The more positive RMP in parkin larvae is most likely to be due to a reduction in ATP synthesis, which requires oxygen. This was demonstrated using two techniques to record O2 consumption and by measuring ATP levels directly. A similar reduction in ATP production (by 58%) is reported in human fibroblasts from patients with parkin mutations. Low ATP will lead to reduced pumping of ions across the membrane and so a more positive RMP (although this may be counteracted by increased opening of ATP-sensitive potassium channels. Reports of Drosophila mutants with a more positive membrane potential are rare, but in one, the phosphoglycerate kinase mutant, nubian, this is due to reduced ATP levels. In the parkin knockout, it is predicted that neurons (as well as muscles) will also have a more positive membrane potential because the mutation is expressed globally (Vincent, 2012).
The reduction of oxygen consumption indicates severe mitochondrial dysfunction; not surprising in the light of the reports of deficient mitochondrial fission/fusion and mitochondrial swelling in a range of fly models of PD. The parkin larvae respond to this by increasing glycolysis, as indicated by the increase in lactate levels. Increased lactate has been reported in some PD patients, but not in others. This discrepancy might be due to a general increase in lactate (and changes in anaerobic metabolism) with age, and so the data, along with improvements in genotyping and other molecular techniques may make the role of glycolytic biomarkers in the progression of PD worth revisiting (Vincent, 2012).
Mitochondrial dysfunction will also induce oxidative stress. A key response to oxidative stress is the induction of autophagy, which causes increased growth of neuronal terminals. As with parkin, so also manipulations of a second PD-related gene, dLRRK cause overgrowth of the larval neuromuscular junction. It is noted that, in both transgenics, the overgrowth phenotype is effectively rescued by global, neuronal or muscle expression of wild-type protein. This shows a difference from the physiological and locomotor phenotypes, which were rescued by neuronal -- but not muscle -- expression of parkin (Vincent, 2012).
Excellent rescue was found of the neuronal overgrowth by relieving oxidative stress. Increased expression of cytosolic (catalase, superoxide dismutase 1) or mitochondrial (superoxide dismutase 2) enzymes that scavenge ROS are equally effective. Similarly, adult fly models of PD are rescued by overexpression of genes that reduce ROS, e.g. glutathione S-transferase or thioredoxin (Vincent, 2012).
However, over-expressing oxidative stress transgenes rescues only the neuronal overgrowth; the RMP and locomotor phenotype are not affected. (This is a major contrast to parkin expression, which rescues each of the oxidative stress, RMP and locomotor phenotypes.) Thus, expression of the ROS scavengers exposes fundamental physiological deficits, including the failure to maintain the RMP and the failure of the neuronal network of the CPG. In the same way, antioxidants like vitamin E have powerful cellular actions, but are not generally effective in the treatment of PD patients (Vincent, 2012).
In conclusion, this study found that parkin mutants recapitulate the bradykinesia symptoms of PD, and it is suggested that this is due to an energy deficit rather than oxidative stress. parkin-Dependent mitochondrial dysfunction reduces ATP levels and the ability of neurons to maintain an RMP. Oxidative stress has received considerable attention as a target for treating aging and neurodegenerative disorders; however, this study suggests that energy metabolism is a more important target for the treatment of PD (Vincent, 2012).
Studies of the familial Parkinson disease-related proteins PINK1 and Parkin have demonstrated that these factors promote the fragmentation and turnover of mitochondria following treatment of cultured cells with mitochondrial depolarizing agents. Whether PINK1 or Parkin influence mitochondrial quality control under normal physiological conditions in dopaminergic neurons, a principal cell type that degenerates in Parkinson disease, remains unclear. To address this matter, a method was developed to purify and characterize neural subtypes of interest from the adult Drosophila brain. Using this method, it was found that dopaminergic neurons from Drosophila parkin mutants accumulate enlarged, depolarized mitochondria, and that genetic perturbations that promote mitochondrial fragmentation and turnover rescue the mitochondrial depolarization and neurodegenerative phenotypes of parkin mutants. In contrast, cholinergic neurons from parkin mutants accumulate enlarged depolarized mitochondria to a lesser extent than dopaminergic neurons, suggesting that a higher rate of mitochondrial damage, or a deficiency in alternative mechanisms to repair or eliminate damaged mitochondria explains the selective vulnerability of dopaminergic neurons in Parkinson disease. This study validates key tenets of the model that PINK1 and Parkin promote the fragmentation and turnover of depolarized mitochondria in dopaminergic neurons. Moreover, the neural purification method provides a foundation to further explore the pathogenesis of Parkinson disease, and to address other neurobiological questions requiring the analysis of defined neural cell types (Burman, 2012).
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 (RetMEN2B) 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 RetMEN2B 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, RetMEN2B. 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 RetMEN2B, but interestingly one subunit was moderately downregulated in Pink1 mutants and upregulated by RetMEN2B, 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).
Parkinson's disease is a common neurodegenerative disease with complex clinical features. Autosomal recessive juvenile parkinsonism (AR-JP) maps to the long arm of chromosome 6 (6q25.2-q27) and is linked strongly to the markers D6S305 and D6S253; the former is deleted in one Japanese AR-JP patient. By positional cloning within this microdeletion, a complementary DNA clone has been isolated of 2,960 base pairs with a 1,395-base-pair open reading frame, encoding a protein of 465 amino acids with moderate similarity to ubiquitin at the amino terminus and a RING-finger motif at the carboxy terminus. The gene spans more than 500 kilobases and has 12 exons, five of which (exons 3-7) are deleted in the patient. Four other AR-JP patients from three unrelated families have a deletion affecting exon 4 alone. A 4.5-kilobase transcript that is expressed in many human tissues but is abundant in the brain, including the substantia nigra, is shorter in brain tissue from one of the groups of exon-4-deleted patients. Mutations in the newly identified gene appear to be responsible for the pathogenesis of AR-JP, and the protein product has therefore been named 'Parkin' (Kitada, 1998).
Autosomal recessive juvenile parkinsonism (AR-JP), one of the most common familial forms of Parkinson's disease, is characterized by selective dopaminergic neural cell death and the absence of the Lewy body, a cytoplasmic inclusion body consisting of aggregates of abnormally accumulated proteins. PARK2, mutations of which cause AR-JP, has been cloned but the function of the gene product, parkin, remains unknown. Parkin is involved in protein degradation as a ubiquitin-protein ligase collaborating with the ubiquitin-conjugating enzyme UbcH7; mutant parkins from AR-JP patients show loss of the ubiquitin-protein ligase activity. These findings indicate that accumulation of proteins that have yet to be identified causes a selective neural cell death without formation of Lewy bodies. These findings should enhance the exploration of the molecular mechanisms of neurodegeneration in Parkinson's disease as well as in other neurodegenerative diseases that are characterized by involvement of abnormal protein ubiquitination, including Alzheimer's disease, other tauopathies, CAG triplet repeat disorders and amyotrophic lateral sclerosis (Shimura, 2000).
To provide insight into the function of parkin, its intracellular distribution was examined in cultured cells. Parkin is localized in the trans-Golgi network and the secretory vesicles in U-373MG or SH-SY5Y cells by immunocytochemical analyses. In the subsequent subcellular fractionation studies of rat brain, it has been shown that parkin copurifies with synaptic vesicles (SVs) when low ionic conditions are used throughout the procedure. An immunoelectromicroscopic analysis indicates that parkin is present on the SV membrane. Parkin was readily released from SVs into the soluble phase by increasing ionic strength at neutral pH, but not by a non-ionic detergent. To elucidate its responsible region for membrane association, green fluorescent protein-tagged deletion mutants of parkin were transfected into COS-1 cells followed by subcellular fractionation. The ability of parkin to bind to the membranes resides in a broad region excluding the ubiquitin-like domain (Kubo, 2001).
Mutations in alpha-synuclein (alpha S) and parkin cause heritable forms of Parkinson's disease (PD). It was hypothesized that neuronal parkin, a known E3 ubiquitin ligase, facilitates the formation of Lewy bodies (LBs), a pathological hallmark of PD. Affinity-purified parkin antibodies label classical LBs in substantia nigra sections from four related human disorders: sporadic PD, inherited alphaS-linked PD, dementia with LBs (DLB), and LB-positive, parkin-linked PD. Anti-parkin antibodies also detected LBs in entorhinal and cingulate cortices from DLB brain and alphaS inclusions in sympathetic gangliocytes from sporadic PD. Double labeling with confocal microscopy of DLB midbrain sections has revealed that approximately 90% of anti-alpha S-reactive LBs are also detected by a parkin antibody to amino acids 342 to 353. Accordingly, parkin proteins, including the 53-kd mature isoform, are present in affinity-isolated LBs from DLB cortex. AlphaS and parkin co-localize within brainstem and cortical LBs. parkin appeared most enriched in cytosolic and postsynaptic fractions of adult rat brain, but also in purified, alpha S-rich presynaptic elements that additionally contained parkin's E2-binding partner, UbcH7. It is concluded that parkin and UbcH7 are present with alphaS in subcellular compartments of normal brain and that parkin frequently co-localizes with alpha S aggregates in the characteristic LB inclusions of PD and DLB. These results suggest that functional parkin proteins may be required during LB formation (Schlossmacher, 2002).
Parkin is a ubiquitin-protein isopeptide ligase (E3) involved in ubiquitin/proteasome-mediated protein degradation. Mutations in the parkin gene cause a loss-of-function and/or alter protein levels of parkin. As a result, the toxic build-up of parkin substrates is thought to lead to autosomal recessive juvenile parkinsonism. To identify a role for the ubiquitin-like domain (ULD) of parkin, a number of hemagglutinin (HA)-tagged parkin constructs were created using mutational and structural information. Western blotting and immunocytochemistry show a much stronger expression level for HA-parkin residues 77-465 (without ULD) than HA-parkin full-length (with ULD). The deletion of ULD in Drosophila parkin also causes a sharp increase in expression of the truncated form, suggesting that the function of the ULD of parkin is conserved across species. By progressive deletion analysis of parkin ULD, it was found that residues 1-6 of human parkin play a crucial role in controlling the expression levels of this gene. HA-parkin residues 77-465 show ubiquitination in vivo, demonstrating that the ULD is not critical for parkin auto-ubiquitination; ubiquitination seemed to cluster on the central domain of parkin (residues 77-313). These effects are specific for the ULD of parkin and not transfection-, toxic-, epitope tag-, and/or vector-dependent. Taken together, these data suggest that the 76 most NH(2)-terminal residues (ULD) dramatically regulate the protein levels of parkin (Finney, 2003).
Parkin, a product of the causative gene of autosomal-recessive juvenile parkinsonism (AR-JP), is a RING-type E3 ubiquitin ligase and has an amino-terminal ubiquitin-like (Ubl) domain. Although a single mutation that causes an Arg to Pro substitution at position 42 of the Ubl domain (the Arg 42 mutation) has been identified in AR-JP patients, the function of this domain is not clear. In this study, the three-dimensional structure has been determined of the Ubl domain of parkin by NMR, in particular by extensive use of backbone (15)N-(1)H residual dipolar-coupling data. Inspection of chemical-shift-perturbation data shows that the parkin Ubl domain binds the Rpn10 subunit of 26S proteasomes via the region of parkin that includes position 42. These findings suggest that the Arg 42 mutation induces a conformational change in the Rpn10-binding site of Ubl, resulting in impaired proteasomal binding of parkin, which could be the cause of AR-JP (Sakata, 2003).
Mutations of the parkin gene are the most frequent cause of early onset autosomal recessive parkinsonism. Inactivation of the parkin gene in mice results in motor and cognitive deficits, inhibition of amphetamine-induced dopamine release and inhibition of glutamate neurotransmission. The levels of dopamine are increased in the limbic brain areas of parkin mutant mice and there is a shift towards increased metabolism of dopamine by MAO. Although there was no evidence for a reduction of nigrostriatal dopamine neurons in the parkin mutant mice, the level of dopamine transporter protein was reduced in these animals, suggesting a decreased density of dopamine terminals, or adaptative changes in the nigrostriatal dopamine system. GSH levels were increased in the striatum and fetal mesencephalic neurons from parkin mutant mice, suggesting that a compensatory mechanism may protect dopamine neurons from neuronal death. These parkin mutant mice provide a valuable tool to better understand the preclinical deficits observed in patients with PD and to characterize the mechanisms leading to the degeneration of dopamine neurons that could provide new strategies for neuroprotection (Itier, 2003).
Loss-of-function mutations in parkin are the major cause of early-onset familial Parkinson's disease. To investigate the pathogenic mechanism by which loss of parkin function causes Parkinson's disease, a mouse model was generated bearing a germline disruption in parkin. Parkin/ mice are viable and exhibit grossly normal brain morphology. Quantitative in vivo microdialysis revealed an increase in extracellular dopamine concentration in the striatum of parkin/ mice. Intracellular recordings of medium-sized striatal spiny neurons show that greater currents are required to induce synaptic responses, suggesting a reduction in synaptic excitability in the absence of parkin. Furthermore, parkin/ mice exhibit deficits in behavioral paradigms sensitive to dysfunction of the nigrostriatal pathway. The number of dopaminergic neurons in the substantia nigra of parkin/ mice, however, is normal up to the age of 24 months, in contrast to the substantial loss of nigral neurons characteristic of Parkinson's disease. Steady-state levels of CDCrel-1, synphilin-1, and alpha-synuclein, which have all been identified as substrates of the E3 ubiquitin ligase activity of parkin, are unaltered in parkin/ brains. Together these findings provide the first evidence of a novel role for parkin in dopamine regulation and nigrostriatal function, and a non-essential role for parkin in the survival of nigral neurons in mice (Goldberg, 2003).
Mutations in the parkin gene underlie a familial form of Parkinson's disease known as autosomal recessive juvenile Parkinsonism (AR-JP). Dysfunction of parkin, a ubiquitin E3 ligase, has been implicated in the accumulation of ubiquitin proteasome system-destined substrates and eventually leads to cell death. However, regulation of parkin enzymatic activity is incompletely understood. This study investigated whether the ubiquitin E3 ligase activity of mammalian parkin could be regulated by neddylation. It was found that parkin could be a target of covalent modification with NEDD8, a ubiquitin-like posttranslational modifier. In addition, NEDD8 attachment caused an increase of parkin activity through the increased binding affinity for ubiquitin-conjugating E2 enzyme as well as the enhanced formation of the complex containing parkin and substrates. These findings point to the functional importance of NEDD8 and suggest that neddylation is one to the diverse modes of parkin regulation, potentially linking it to the pathogenesis of AR-JP (Um, 2012).
Cells maintain healthy mitochondria by degrading damaged mitochondria through mitophagy; defective mitophagy is linked to Parkinson's disease. This study reports that USP30, a deubiquitinase localized to mitochondria, antagonizes mitophagy driven by the ubiquitin ligase parkin (also known as PARK2) and protein kinase PINK1 (see Drosophila Pink1), which are encoded by two genes associated with Parkinson's disease. Parkin ubiquitinates and tags damaged mitochondria for clearance. Overexpression of USP30 removes ubiquitin attached by parkin onto damaged mitochondria and blocks parkin's ability to drive mitophagy, whereas reducing USP30 activity enhances mitochondrial degradation in neurons. Global ubiquitination site profiling identified multiple mitochondrial substrates oppositely regulated by parkin and USP30. Knockdown of USP30 (CG3016) rescues the defective mitophagy caused by pathogenic mutations in parkin and improves mitochondrial integrity in parkin- or PINK1-deficient flies. Knockdown of USP30 in dopaminergic neurons protects flies against paraquat toxicity in vivo, ameliorating defects in dopamine levels, motor function and organismal survival. Thus USP30 inhibition is potentially beneficial for Parkinson's disease by promoting mitochondrial clearance and quality control (Bingol, 2014).
Autosomal recessive juvenile parkinsonism (AR-JP) is caused by mutations in the parkin gene. Parkin protein is characterized by a ubiquitin-like domain at its NH(2)-terminus and two RING finger motifs and an IBR (in between RING fingers) at its COOH terminus (RING-IBR-RING). Parkin is a RING-type E3 ubiquitin-protein ligase that binds to E2 ubiquitin-conjugating enzymes, including UbcH7 and UbcH8, through its RING-IBR-RING motif. Moreover, unfolded protein stress induces up-regulation of both the mRNA and protein level of Parkin. Furthermore, overexpression of Parkin, but not a set of mutants without the E3 activity, specifically suppresses unfolded protein stress-induced cell death. These findings demonstrate that Parkin is an E3 enzyme and suggest that it is involved in the ubiquitination pathway for misfolded proteins derived from endoplasmic reticulum and contributes to protection from neurotoxicity induced by unfolded protein stresses (Imai, 2000).
Parkin gene mutations have been implicated in autosomal-recessive early-onset parkinsonism and lead to specific degeneration of dopaminergic neurons in midbrain. To investigate the role of Parkin in neuronal cell death, this protein was overproduced in PC12 cells in an inducible manner. In this cell line, neuronally differentiated by nerve growth factor, Parkin overproduction protected against cell death mediated by ceramide, but not by a variety of other cell death inducers (H(2)O(2), 4-hydroxynonenal, rotenone, 6-OHDA, tunicamycin, 2-mercaptoethanol and staurosporine). Protection was abrogated by the proteasome inhibitor epoxomicin and disease-causing variants, indicating that it was mediated by the E3 ubiquitin ligase activity of Parkin. Interestingly, Parkin acted by delaying mitochondrial swelling and subsequent cytochrome c release and caspase-3 activation observed in ceramide-mediated cell death. Subcellular fractionation demonstrated enrichment of Parkin in the mitochondrial fraction and its association with the outer mitochondrial membrane. Together, these results suggest that Parkin may promote the degradation of substrates localized in mitochondria and involved in the late mitochondrial phase of ceramide-mediated cell death. Loss of this function may underlie the degeneration of nigral dopaminergic neurons in patients with Parkin mutations (Darios, 2003).
A putative G protein-coupled transmembrane polypeptide, named Pael receptor, has been identified as an interacting protein with Parkin, a gene product responsible for autosomal recessive juvenile parkinsonism (AR-JP). When overexpressed in cells, this receptor tends to become unfolded, insoluble, and ubiquitinated in vivo. The insoluble Pael receptor leads to unfolded protein-induced cell death. Parkin specifically ubiquitinates this receptor in the presence of ubiquitin-conjugating enzymes resident in the endoplasmic reticulum and promotes the degradation of insoluble Pael receptor, resulting in suppression of the cell death induced by Pael receptor overexpression. Moreover, the insoluble form of Pael receptor accumulates in the brains of AR-JP patients. The unfolded Pael receptor is a substrate of Parkin, the accumulation of which may cause selective neuronal death in AR-JP (Imai, 2001).
Unfolded Pael receptor (Pael-R) is a substrate of the E3 ubiquitin ligase Parkin. Accumulation of Pael-R in the endoplasmic reticulum (ER) of dopaminergic neurons induces ER stress leading to neurodegeneration. CHIP, Hsp70, Parkin, and Pael-R formed a complex in vitro and in vivo. CHIP is a cytosolic U-box protein that interacts with Hsc70 through a set of tetratricorepeat motifs. The U-box represents a modified form of the ring-finger motif that is found in ubiquitin ligases and that defines the E4 family of polyubiquitination factors. The amount of CHIP in the complex is increased during ER stress. CHIP promotes the dissociation of Hsp70 from Parkin and Pael-R, thus facilitating Parkin-mediated Pael-R ubiquitination. Moreover, CHIP enhanced Parkin-mediates in vitro ubiquitination of Pael-R in the absence of Hsp70. Furthermore, CHIP enhances the ability of Parkin to inhibit cell death induced by Pael-R. Taken together, these results indicate that CHIP is a mammalian E4-like molecule that positively regulates Parkin E3 activity (Imai, 2002).
Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the progressive accumulation in selected neurons of protein inclusions containing alpha-synuclein and ubiquitin. Rare inherited forms of PD are caused by autosomal dominant mutations in alpha-synuclein or by autosomal recessive mutations in parkin, an E3 ubiquitin ligase. It was hypothesized that these two gene products interact functionally, namely, that parkin ubiquitinates alpha-synuclein normally and that this process is altered in autosomal recessive PD. A protein complex in normal human brain has been identified that includes parkin as the E3 ubiquitin ligase, UbcH7 as its associated E2 ubiquitin conjugating enzyme, and a new 22-kilodalton glycosylated form of alpha-synuclein (alphaSp22) as its substrate. In contrast to normal parkin, mutant parkin associated with autosomal recessive PD fails to bind alphaSp22. In an in vitro ubiquitination assay, alphaSp22 was modified by normal but not mutant parkin into polyubiquitinated, high molecular weight species. Accordingly, alphaSp22 accumulates in a non-ubiquitinated form in parkin-deficient PD brains. It is concluded that alphaSp22 is a substrate for parkin's ubiquitin ligase activity in normal human brain and that loss of parkin function causes pathological alphaSp22 accumulation. These findings demonstrate a critical biochemical reaction between the two PD-linked gene products and suggest that this reaction underlies the accumulation of ubiquitinated alpha-synuclein in conventional PD (Shimura, 2001).
One hypothesis for the etiology of Parkinson's disease (PD) is that subsets of neurons are vulnerable to a failure in proteasome-mediated protein turnover. Overexpression of mutant alpha-synuclein increases sensitivity to proteasome inhibitors by decreasing proteasome function. Overexpression of parkin decreases sensitivity to proteasome inhibitors in a manner dependent on parkin's ubiquitin-protein E3 ligase activity, and antisense knockdown of parkin increases sensitivity to proteasome inhibitors. Mutant alpha-synuclein also causes selective toxicity to catecholaminergic neurons in primary midbrain cultures, an effect that can be mimicked by the application of proteasome inhibitors. Parkin is capable of rescuing the toxic effects of mutant alpha-synuclein or proteasome inhibition in these cells. Therefore, parkin and alpha-synuclein are linked by common effects on a pathway associated with selective cell death in catecholaminergic neurons (Petrucelli, 2002).
Parkinson's disease is a common neurodegenerative disorder characterized by the loss of dopaminergic neurons and the presence of intracytoplasmic-ubiquitinated inclusions (Lewy bodies). Mutations in alpha-synuclein (A53T, A30P) and parkin cause familial Parkinson's disease. Both these proteins are found in Lewy bodies. The absence of Lewy bodies in patients with parkin mutations suggests that parkin might be required for the formation of Lewy bodies. parkin physically interacts with and ubiquitinates the alpha-synuclein-interacting protein, synphilin-1. Co-expression of alpha-synuclein, synphilin-1 and parkin result in the formation of Lewy-body-like ubiquitin-positive cytosolic inclusions. Familial-linked mutations in parkin disrupt the ubiquitination of synphilin-1 and the formation of the ubiquitin-positive inclusions. These results provide a molecular basis for the ubiquitination of Lewy-body-associated proteins and link parkin and alpha-synuclein in a common pathogenic mechanism through their interaction with synphilin-1 (Chung, 2002).
In addition to inhibiting the mitochondrial respiratory chain, toxins known to cause Parkinson's disease (PD), such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and rotenone, also strongly depolymerize microtubules and increase tubulin degradation. Microtubules are polymers of tubulin alpha/beta heterodimers, whose correct folding requires coordinated actions of cellular chaperonins and cofactors. Misfolded tubulin monomers are highly toxic and quickly degraded through a hitherto unknown mechanism. Parkin, a protein-ubiquitin E3 ligase linked to PD, tightly binds to microtubules in taxol-mediated microtubule coassembly assays. In lysates from the rat brain or transfected human embryonic kidney (HEK) 293 cells, alpha-tubulin and beta-tubulin strongly coimmunoprecipitate with parkin in the presence of colchicine, a condition in which tubulin exits as alpha/beta heterodimers. At the subcellular level, parkin exhibits punctate immunostaining along microtubules in rat brain sections, cultured primary neurons, glial cells, and cell lines. This pattern of subcellular localization is abolished in cells treated with the microtubule-depolymerizing drug colchicine. The binding between parkin and tubulin apparently leads to increased ubiquitination and accelerated degradation of alpha- and beta-tubulins in HEK293 cells. Similarly ubiquitinated tubulins are also observed in rat brain lysates. Furthermore, parkin mutants found in PD patients do not ubiquitinate or degrade either tubulin. Taken together, these results show that parkin is a novel tubulin-binding protein, as well as a microtubule-associated protein. Its ability to enhance the ubiquitination and degradation of misfolded tubulins may play a significant role in protecting neurons from toxins that cause PD (Ren, 2003).
Mutations of parkin, a protein-ubiquitin isopeptide ligase (E3), appear to be the most frequent cause of familial Parkinson's disease (PD). Parkin binds strongly to alpha/beta tubulin heterodimers and microtubules. The strong binding between parkin and tubulin, as well as that between parkin and microtubules, is mediated by three independent domains: linker, RING1, and RING2. These redundant strong interactions made it virtually impossible to separate parkin from microtubules by high concentrations of salt (3.8 m) or urea (0.5 m). Parkin co-purified with tubulin and was found in a highly purified tubulin preparation. Expression of either full-length parkin or any of its three microtubule-binding domains significantly attenuates colchicine-induced microtubule depolymerization. The abilities of parkin to bind to and stabilize microtubules are not affected by PD-linked mutations that abrogate its E3 ligase activity. Thus, the tubulin/microtubule-binding activity of parkin and its E3 ligase activity are independent. The strong binding between parkin and tubulin/microtubules through three redundant interaction domains may not only stabilize microtubules but also guarantee the anchorage of this E3 ligase on microtubules. Because many misfolded proteins are transported on microtubules, the localization of parkin on microtubules may provide an important environment for its E3 ligase activity toward misfolded substrates (Yang, 2005).
Mutations in parkin, which encodes a RING domain protein associated with ubiquitin ligase activity, lead to autosomal recessive Parkinson's disease characterized by midbrain dopamine neuron loss. Parkin functions in a multiprotein ubiquitin ligase complex that includes the F-box/WD repeat protein hSel-10 and Cullin-1. HSel-10 serves to target the parkin ubiquitin ligase activity to cyclin E, an hSel-10-interacting protein previously implicated in the regulation of neuronal apoptosis. Consistent with the notion that cyclin E is a substrate of the parkin ubiquitin ligase complex, parkin deficiency potentiates the accumulation of cyclin E in cultured postmitotic neurons exposed to the glutamatergic excitotoxin kainate and promotes their apoptosis. Furthermore, parkin overexpression attenuates the accumulation of cyclin E in toxin-treated primary neurons, including midbrain dopamine neurons, and protects them from apoptosis (Staropoli, 2003).
Parkin binds to the E2 ubiquitin-conjugating human enzyme 8 (UbcH8) through its C-terminal ring-finger. Parkin has ubiquitin-protein ligase activity in the presence of UbcH8. Parkin also ubiquitinates itself and promotes its own degradation. The synaptic vesicle-associated protein, CDCrel-1, interacts with Parkin through its ring-finger domains. Furthermore, Parkin ubiquitinates and promotes the degradation of CDCrel-1. Familial-linked mutations disrupt the ubiquitin-protein ligase function of Parkin and impair Parkin and CDCrel-1 degradation. These results suggest that Parkin functions as an E3 ubiquitin-protein ligase through its ring domains and that it may control protein levels via ubiquitination. The loss of Parkin's ubiquitin-protein ligase function in familial-linked mutations suggests that this may be the cause of familial autosomal recessive Parkinson's disease (Zhang, 2000).
Parkin, the most commonly mutated gene in familial Parkinson's disease, encodes an E3 ubiquitin ligase. A number of candidate substrates have been identified for parkin ubiquitin ligase action including CDCrel-1, o-glycosylated alpha-synuclein, Pael-R and synphilin-1. Parkin promotes the ubiquitination and degradation of an expanded polyglutamine protein. Overexpression of parkin reduces aggregation and cytotoxicity of an expanded polyglutamine ataxin-3 fragment. Parkin reduces proteasome impairment and caspase-12 activation induced by an expanded polyglutamine protein. Parkin forms a complex with the expanded polyglutamine protein, heat shock protein 70 (Hsp70) and the proteasome that may be important for the elimination of the expanded polyglutamine protein. Hsp70 enhances parkin binding and ubiquitination of expanded polyglutamine protein in vitro, suggesting that Hsp70 may help to recruit misfolded proteins as substrates for parkin E3 ubiquitin ligase activity. It is speculated that parkin may function to relieve ER stress by preserving proteasome activity in the presence of misfolded proteins. Loss of parkin function and the resulting proteasomal impairment may contribute to the accumulation of toxic aberrant proteins in neurodegenerative diseases including Parkinson's Disease (Tsai, 2003).
Lesions in the parkin gene cause early onset Parkinson's disease by a loss of dopaminergic neurons thus demonstrating a vital role for parkin in the survival of these neurons. Parkin is inactivated by caspase cleavage and the major cleavage site is after Asp 126. Caspases responsible for parkin cleavage were identified by several experimental paradigms. Transient coexpression of caspases and wild type parkin in HEK293 cells identified caspase-1, -3 and -8 as efficient inducers of parkin cleavage whereas caspase-2, -7, -9 and -11 do not induce cleavage. A D126A parkin mutation abrogates cleavage induced by caspase-1 and -8 but not by caspase-3. In anti-FAS treated Jurkat cells, parkin cleavage is inhibited by caspase inhibitors hFlip and CrmA but not by XIAP indicating that caspase-8 but not caspase-3 is responsible for the parkin cleavage in this model. Moreover, induction of apoptosis in caspase-3 deficient MCF7 cells, either by caspase-1 or -8 overexpression or by TNF-alpha treatment leads to parkin cleavage. These results demonstrate that caspase-1 and -8 can directly cleave parkin, and suggest that death receptor activation and inflammatory stress can cause loss of parkins ubiquitin ligase activity and thus cause accumulation of toxic parkin substrates and trigger dopaminergic cell death (Kahns, 2003).
In an effort to identify tumor suppressor gene(s) associated with the frequent loss of heterozygosity observed on chromosome 6q25-q27, a contig containing eight known genes, including the complete genomic structure of the Parkin gene, was constructed. Loss of heterozygosity (LOH) analysis of 40 malignant breast and ovarian tumors identified a common minimal region of loss, including two marker loci. Both loci exhibited the highest frequencies of loss of homozygosity in this study and are each located within the Parkin genomic structure. Whereas mutation analysis revealed no missense substitutions, expression of the Parkin gene appeared to be down-regulated or absent in the tumor biopsies and tumor cell lines examined. In addition, the identification of two truncating deletions in 3 of 20 ovarian tumor samples, as well as homozygous deletion of exon 2 in two lung adenocarcinoma cell lines, supports the hypothesis that hemizygous or homozygous deletions are responsible for the abnormal expression of Parkin in these samples. These data suggest that the loss of homozygosity observed at chromosome 6q25-q26 may contribute to the initiation and/or progression of cancer by inactivating or reducing the expression of the Parkin gene. Because Parkin maps to one of the most active common fragile sites in the human genome, it represents another example of a large tumor suppressor gene located at a common fragile site (Cesari, 2003).
Parkinson's disease (PD) is a neurodegenerative disorder characterized by degeneration of dopaminergic neurons in the substantia nigra. A locus for a rare familial form of PD has been mapped chromosome 1p36 (PARK6). Mutations in PINK1 (PTEN-induced kinase 1) are associated with PARK6. Two homozygous mutations have been identified affecting the PINK1 kinase domain in three consanguineous PARK6 families: a truncating nonsense mutation and a missense mutation at a highly conserved amino acid. Cell culture studies suggest that PINK1 is mitochondrially located and may exert a protective effect on the cell that is abrogated by the mutations, resulting in increased susceptibility to cellular stress. These data provide a direct molecular link between mitochondria and the pathogenesis of PD (Valente, 2004).
Several mutations in PTEN-induced putative kinase 1 (PINK1) gene have been reported to be associated with recessive parkinsonism. The encoded protein is predicted to be a Ser/Thr protein kinase targeted to mitochondria. Investigated in this study were the effects of mutations on PINK1 kinase activity in vitro and on expression levels and localization in mammalian cells. Two point mutations were examined: G309D, which was originally reported to be stable and properly localized in cells and L347P, which is of interest because it is present at an appreciable carrier frequency in the Philippines. Kinase activity was confirmed and artificial 'kinase-dead' mutants were produced that are stable but lack activity. The L347P mutation grossly destabilizes PINK1 and drastically reduces kinase activity, whereas G309D has much more modest effects on these parameters in vitro. This finding is in line with predictions based on homology modeling. The localization of PINK1 in transfected mammalian cells was examined by using constructs that were tagged with myc or GFP at either end of the protein. These results show that PINK1 is processed at the N terminus in a manner consistent with mitochondrial import, but the mature protein also exists in the cytosol. The physiological relevance of this observation is not yet clear, but it implies that a portion of PINK1 may be exported after processing in the mitochondria (Beilina, 2005).
Parkinson's disease (PD) is a progressive neurodegenerative illness associated with a selective loss of dopaminergic neurons in the nigrostriatal pathway of the brain. Despite the overall rarity of the familial forms of PD, the identification of single genes linked to the disease has yielded crucial insights into possible mechanisms of neurodegeneration. Recently, a putative mitochondrial kinase, PINK1, has been found mutated in an inherited form of parkinsonism. PINK1 mutations confer different autophosphorylation activity, which is regulated by the C-terminal portion of the protein. The mitochondrial localization of both wild-type and mutant PINK1 proteins has been unequivocally demonstrated and it has been proven that a short N-terminal part of PINK1 is sufficient for its mitochondrial targeting (Silvestri, 2005).
Mutations in the PTEN-induced kinase 1 (PINK1) gene have recently been implicated in autosomal recessive early onset Parkinson Disease. To investigate the role of PINK1 in neurodegeneration, human and murine neuronal cell lines were designed expressing either wild-type PINK1 or PINK1 bearing a mutation associated with Parkinson Disease. Under basal and staurosporine-induced conditions, the number of TUNEL-positive cells was lower in wild-type PINK1 expressing SH-SY5Y cells than in mock-transfected cells. This phenotype was due to a PINK1-mediated reduction in cytochrome c release from mitochondria, which prevents subsequent caspase-3 activation. Overexpression of wild-type PINK1 strongly reduced both basal and staurosporine-induced caspase 3 activity. Overexpression of wild-type PINK1 also reduced the levels of cleaved caspase-9, caspase-3, caspase-7, and activated poly(ADP-ribose) polymerase under both basal and staurosporine-induced conditions. In contrast, Parkinson disease-related mutations and a kinase-inactive mutation in PINK1 abrogate the protective effect of PINK1. Together, these results suggest that PINK1 reduces the basal neuronal pro-apoptotic activity and protects neurons from staurosporine-induced apoptosis. Loss of this protective function may therefore underlie the degeneration of nigral dopaminergic neurons in patients with PINK1 mutations (Petit, 2005).
The Parkinson's disease (PD) causative PINK1 gene encodes a mitochondrial protein kinase called PTEN-induced kinase 1 (PINK1). The autosomal recessive pattern of inheritance of PINK1 mutations suggests that PINK1 is neuroprotective and therefore loss of PINK1 function causes PD. Indeed, overexpression of PINK1 protects neuroblastoma cells from undergoing neurotoxin-induced apoptosis. As a protein kinase, PINK1 presumably exerts its neuroprotective effect by phosphorylating specific mitochondrial proteins and in turn modulating their functions. Towards elucidation of the neuroprotective mechanism of PINK1, the baculovirus-infected insect cell system was used to express the recombinant protein consisting of the PINK1 kinase domain either alone [PINK1(KD)] or with the PINK1 C-terminal tail [PINK1(KD+T)]. Both recombinant enzymes preferentially phosphorylate the artificial substrate histone H1 exclusively at serine and threonine residues, demonstrating that PINK1 is indeed a protein serine/threonine kinase. Introduction of the PD-associated mutations, G386A and G409V significantly reduces PINK1(KD) kinase activity. Since Gly-386 and Gly-409 reside in the conserved activation segment of the kinase domain, the results suggest that the activation segment is a regulatory switch governing PINK1 kinase activity. PINK1(KD+T) is approximately 6-fold more active than PINK1(KD). Thus, in addition to the activation segment, the C-terminal tail also contains regulatory motifs capable of governing PINK1 kinase activity. Finally, the availability of active recombinant PINK1 proteins permits future studies to search for mitochondrial proteins that are preferentially phosphorylated by PINK1. As these proteins are likely physiological substrates of PINK1, their identification will shed light on the mechanism of pathogenesis of PD (Sim, 2006).
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. 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).
Search PubMed for articles about Drosophila parkin
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Burman, J. L., Yu, S., Poole, A. C., Decal, R. B. and Pallanck, L. (2012). Analysis of neural subtypes reveals selective mitochondrial dysfunction in dopaminergic neurons from parkin mutants. Proc Natl Acad Sci U S A 109: 10438-10443. PubMed ID: 22691499
Cesari, R., et al. (2003). Parkin, a gene implicated in autosomal recessive juvenile parkinsonism, is a candidate tumor suppressor gene on chromosome 6q25-q27. Proc. Natl. Acad. Sci. 100(10): 5956-61. 12719539
Chung, K. K., et al. (2002). Parkin ubiquitinates the alpha-Synuclein -interacting protein, synphilin-1: implications for Lewy-body formation in Parkinson disease. Nat. Med. 7: 1144-1150. 11590439
Clark, I. E., et al. (2006). Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin. Nature 441(7097): 1162-6. 16672981
Cornelissen, T., Haddad, D., Wauters, F., Van Humbeeck, C., Mandemakers, W., Koentjoro, B., Sue, C., Gevaert, K., De Strooper, B., Verstreken, P. and Vandenberghe, W. (2014). The deubiquitinase USP15 antagonizes Parkin-mediated mitochondrial ubiquitination and mitophagy. Hum Mol Genet [Epub ahead of print]. PubMed ID: 24852371
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
Cummings, C. J., et al. (1999). Mutation of the E6-AP ubiquitin ligase reduces nuclear inclusion frequency while accelerating polyglutamine-induced pathology in SCA1 mice. Neuron 24: 879-892. 10624951
Darios, F., Corti, O., Lucking, C. B., Hampe, C., Muriel, M. P., Abbas, N., Gu, W. J., Hirsch, E. C., Rooney, T., Ruberg, M. and Brice, A. (2003). Parkin prevents mitochondrial swelling and cytochrome c release in mitochondria-dependent cell death. Hum. Mol. Genet. 12: 517-526. 12588799
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
Egan, D. F., et al. (2011) Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy. Science 331: 456-461. PubMed Citation: 21205641
Feany, M. B. and Bender, W. W. (2000). A Drosophila model of Parkinson's disease. Nature 404: 394-398. 10746727
Fernandes, C. and Rao, Y. (2011). Genome-wide screen for modifiers of Parkinson's disease genes in Drosophila. Mol Brain 4: 17. PubMed ID: 21504582
Finney, N., et al. (2003). The cellular protein level of Parkin is regulated by its ubiquitin-like domain. J. Biol. Chem. 278(18): 16054-8. 12621021
Goldberg, M. S., Fleming, S. M., Palacino, J. J., Cepeda, C., Lam, H. A., Bhatnagar, A., Meloni, E. G., Wu, N., Ackerson, L. C., Klapstein, G. J. et al. (2003). Parkin-deficient mice exhibit nigrostriatal deficits but not loss of dopaminergic neurons. J. Biol. Chem. 278: 43628-43635. 12930822
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Haywood, A. F. and Staveley, B. E. (2004). Parkin counteracts symptoms in a Drosophila model of Parkinson's disease. BMC Neurosci. 5: 14. 15090075
Haywood, A. F. and Staveley, B. E. (2005). Mutant alpha-synuclein-induced degeneration is reduced by parkin in a fly model of Parkinson's disease. Genome 49(5): 505-10. 16767175
Imai, Y., Soda, M. and Takahashi, R. (2000). Parkin suppresses unfolded protein stress-induced cell death through its E3 ubiquitin-protein ligase activity. J. Biol. Chem. 275: 35661-35664. 10973942
Imai, Y., Soda, M., Inoue, H., Hattori, N., Mizuno, Y. and Takahashi, R. (2001). An unfolded putative transmembrane polypeptide, which can lead to endoplasmic reticulum stress, is a substrate of Parkin. Cell 105: 891-902. 11439185
Imai, Y., Soda, M., Hatakeyama, S., Akagi, T., Hashikawa, T., Nakayama, K. I. and Takahashi, R. (2002). CHIP is associated with Parkin, a gene responsible for familial Parkinson's disease, and enhances its ubiquitin ligase activity. Mol. Cell 10(1): 55-67. 12150907
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 Citation: 18701920
Itier, J. M., Ibanez, P., Mena, M. A., Abbas, N., Cohen-Salmon, C., Bohme, G. A., Laville, M., Pratt, J., Corti, O., Pradier, L. et al. (2003). Parkin gene inactivation alters behaviour and dopamine neurotransmission in the mouse. Hum. Mol. Genet. 12: 2277-2291. 12915482
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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
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Lee, S. B., Kim, W., Lee, S. and Chung, J. (2007). Loss of LRRK2/PARK8 induces degeneration of dopaminergic neurons in Drosophila. Biochem. Biophys. Res. Commun. 358(2): 534-9. PubMed Citation: 17498648
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Petit, A., Kawarai, T., Paitel, E., Sanjo, N., Maj, M., Scheid, M., Chen, F., Gu, Y., Hasegawa, H. and Salehi-Rad, S., et al. (2005). Wild-type PINK1 prevents basal and induced neuronal apoptosis, a protective effect abrogated by Parkinson disease-related mutations. J. Biol. Chem. 280: 34025-34032. 16079129
Petrucelli, L., et al. (2002). Parkin protects against the toxicity associated with mutant alpha-synuclein: proteasome dysfunction selectively affects catecholaminergic neurons. Neuron 36: 1007-1019. 12495618
Poole, A. C., et al. (2010). The mitochondrial fusion-promoting factor mitofusin is a substrate of the PINK1/parkin pathway. PLoS One. 2010 Apr 7;5(4):e10054. PubMed Citation: 20383334
Ren, Y., Zhao, J. and Feng, J. (2003). Parkin binds to alpha/beta tubulin and increases their ubiquitination and degradation. J. Neurosci. 23(8): 3316-24. 12716939
Sakata, E., et al. (2003). Parkin binds the Rpn10 subunit of 26S proteasomes through its ubiquitin-like domain. EMBO Rep. 4(3): 301-6. 12634850
Sang, T. K., et al. (2007). A Drosophila model of mutant human parkin-induced toxicity demonstrates selective loss of dopaminergic neurons and dependence on cellular dopamine. J. Neurosci. 27(5): 981-92. Medline abstract: 17267552
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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
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Shimura, H., et al. (2001). Ubiquitination of a new form of alpha-Synuclein by Parkin from human brain: implications for Parkinson's disease. Science 293: 263-269. 11431533
Silvestri, L., Caputo, V., Bellacchio, E., Atorino, L., Dallapiccola, B., Valente, E. M. and Casari, G. (2005). Mitochondrial import and enzymatic activity of PINK1 mutants associated to recessive parkinsonism. Hum. Mol. Genet. 14: 3477-3492. 16207731
Sim, C. H., et al. (2006). C-terminal truncation and Parkinson's disease-associated mutations down-regulate the protein serine/threonine kinase activity of PTEN-induced kinase-1. Hum. Mol. Genet. 15(21): 3251-62. 17000703
Sriram, S. R., et al. (2005). Familial-associated mutations differentially disrupt the solubility, localization, binding and ubiquitination properties of parkin. Hum. Mol. Genet. 14: 2571-2586. PubMed citation: 16049031
Staropoli, J. F., McDermott, C., Martinat, C., Schulman, B., Demireva, E. and Abeliovich, A. (2003). Parkin is a component of an SCF-like ubiquitin ligase complex and protects postmitotic neurons from kainate excitotoxicity. Neuron 37(5): 735-49. 12628165
Steece-Collier, K., Maries, E. and Kordower, J. H. (2002). Etiology of Parkinson's disease: Genetics and environment revisited. Proc. Natl. Acad. Sci. 99: 13972-13974. 12391311
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date revised: 22 July 2015
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