genes associated with Parkinson's disease
Mitochondria and mitochondrial function
studies of Parkinson's disease
Suzuki, M., Fujikake, N., Takeuchi, T., Kohyama-Koganeya, A., Nakajima, K., Hirabayashi, Y., Wada, K. and Nagai, Y. (2015). Glucocerebrosidase deficiency accelerates the accumulation of proteinase K-resistant α-synuclein and aggravates neurodegeneration in a Drosophila model of Parkinson's disease. Hum Mol Genet 24: 6675-6686. PubMed ID: 26362253
The phenotypic aggravation by GCase deficiency in αSyn flies was associated with the accumulation of PK-resistant αSyn, rather than with changes in the total amount of αSyn, suggesting that the production of this PK-resistant αSyn species might play a key role in the neurotoxicity. Although the toxicity of PK-resistant αSyn was not directly demonstrated, there was a tight association between the neurotoxicity of αSyn and its PK resistance. PK-resistant αSyn oligomers that are formed as an intermediate conformer in the course of in vitro αSyn fibrillization have been shown to cause oxidative stress in primary neurons at much higher levels than non-PK-resistant oligomers. It has been shown that two kinds of αSyn fibrils exhibiting different vulnerabilities to PK digestion can be isolated from repetitive seeded fibrillization, and the αSyn strain more resistant to PK digestion is more toxic to neurons. In addition, αSyn fibril strains produced using different buffers show different vulnerabilities to PK digestion, and their toxicities are associated with their resistance to PK digestion. Interestingly, αSyn fibrils with different levels of PK resistance have different structures, cross-seeding abilities and propagation properties both in vitro and in vivo, all of which are reminiscent of the properties of prions. Therefore, it is possible that the accelerated formation of PK-resistant αSyn that was observed in the GCase-deficient flies represents the ‘prion-like conversion’ of αSyn and that this toxic species leads to phenotypic aggravation by promoting the prion-like seeding and propagation of αSyn (Suzuki, 2015).
The idea that αSyn is degraded in lysosomes has led to several studies on the basis of the hypothesis that loss of GCase activity compromises the αSyn-degrading function of lysosomes, resulting in αSyn accumulation. Several groups have demonstrated that decreased GCase activity results in increased amounts of αSyn, using cultured neurons, human iPSC-derived neurons from GBA1 mutation carriers and mice treated with a GCase inhibitor. In contrast, two other groups have reported that GCase activity does not correlate with the amount of αSyn in neuronal cells, whereas the expression of a mutant GCase that maintains its enzyme activity increases the amount of αSyn, favoring a gain-of-function mechanism in the pathogenesis of GBA1-associated PD. In the fly model, the amount of total αSyn was not significantly increased by GCase deficiency, despite the phenotypic aggravation. However, a recent study using PD model mice with a GBA1 mutation has shown that the total amount of αSyn in the brain lysates is not increased, but the rate of αSyn degradation assessed by pulse-chase experiments is decreased in primary neurons from the same mice. Thus, the possibility that αSyn degradation is compromised by lysosomal dysfunction can not be completely excluded, even though changes in the total amount of αSyn are not detected (Suzuki, 2015).
In addition to the fly model experiments, it was demonstrated by in vitro experiments that GlcCer directly promotes the formation of PK-resistant αSyn, as a mechanism for the increased accumulation of PK-resistant αSyn in the dGBA1a-RNAi flies. These results are consistent with a previous report showing a direct effect of GlcCer on the stability of αSyn oligomers. Moreover, a significant increase in αSyn dimers by the incubation of αSyn with GlcCer-containing liposomes was also found, which is consistent with the finding that the amount of αSyn dimers is significantly increased in GD patients. It was also shown that β-Gal knockdown exacerbates the locomotor dysfunction of αSyn flies, and GM1 directly promotes the PK resistance of αSyn, supporting the hypothesis that aberrant interactions of αSyn with glycolipids trigger the toxic conversion of αSyn, resulting in increased neurotoxicity in vivo. It has been demonstrated that GM1 specifically binds to αSyn and induces its oligomerization, thereby inhibiting its fibrillation. Interestingly, a recent report shows that iPSC-derived neurons from GBA1-associated PD patients exhibit not only decreased GCase activity, but also decreased β-Gal activity, which can be rescued by zinc-finger nuclease-mediated gene correction, implying a crosstalk between GCase and β-Gal activities. Taken together, it is possible that a loss of β-Gal activity also contributes to the acceleration of αSyn toxicity in GBA1-associated PD. It is noted that the direct binding of GM1 to the amyloid β protein also triggers its toxic conversion, implying a common or similar role of glycolipids in the conversion of neurodegenerative disease-related proteins from their non-toxic to toxic forms (Suzuki, 2015).
Then, where in a cell does the accumulated GlcCer interact with αSyn to convert it into a PK-resistant form? One possibility is that αSyn is transported into lysosomes via macroautophagy or chaperone-mediated autophagy, where it interacts with accumulated GlcCer. Then, GlcCer-associated αSyn is secreted from the cells, taken up by itself or by the surrounding cells and accumulates in the cytosol. The other possibility is that the accumulated GlcCer in the lysosome leaks into the cytosol and interacts with αSyn in the cytosol, as the leakage of GlcCer into the cytosol has been reported in both GD patients and GD model mice. There have been no reports to date of the level of GlcCer in the brain of PD patients with a GBA1 mutation. However, in iPSC-derived neurons from two PD patients with a heterozygous GBA1 mutation (RecNcil/wt and N370S/wt), which causes an approximately 50% decrease in GCase activity, the amount of GlcCer has been reported to be about 2-fold higher than that of isogenic gene-corrected iPSC-derived neurons. Furthermore, GlcCer has been reported to accumulate in the brains of GD patients, in which GCase activity decreases (by 80–90%). GCase activity has also been found to be moderately decreased in the brains of GBA1 mutant carrier PD patients (58% decrease in the substantia nigra). Collectively, these data suggest that GlcCer accumulates in the brains of GBA1 mutation carrier PD patients (Suzuki, 2015).
This study focused on the loss-of-function aspect of GBA1 mutations, but there is another possibility arguing the gain-of-function toxicity of mutant GCase, because most mutant GCases are prone to misfold in the endoplasmic reticulum (ER). Human skin fibroblasts derived from GD patients and carriers are reported to induce the unfolded protein response, which is also observed in Drosophila models of GD expressing human mutant GCase. Ambroxol, a potential pharmacological chaperone for mutant GCase, has been shown to ameliorate both ER stress and the phenotypes of these Drosophila models. Interestingly, ambroxol treatment also suppresses the misfolding of mutant GCase, subsequently resulting in an enhancement of cellular GCase activity. Therefore, chemical chaperone therapy can be expected to exert beneficial effects against GD, via the amelioration of both the gain-of-function aspect through ER stress and the loss-of-function aspect through decreased GCase activity. As ER stress has been suggested to be involved in the neurodegeneration that occurs in PD, the synergistic effects of chemical chaperone therapy would also be effective for GBA1-associated PD patients, through the suppression of both ER stress and the toxic conversion of αSyn by GlcCer accumulation (Suzuki, 2015).
Wang, X., Winter, D., Ashrafi, G., Schlehe, J., Wong, Y.L., Selkoe, D., Rice, S., Steen, J., LaVoie, M..J and Schwarz, T.L. (2011). PINK1 and Parkin target Miro for phosphorylation and degradation to arrest mitochondrial motility. Cell 147: 893-906. PubMed ID: 22078885
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).
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
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. 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. Lack of Clu causes a sharp decrease in ATP, increased mitochondrial oxidative damage and changes in mitochondrial ultrastructure. 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. 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 tetratricopeptide repeat (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 showed 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. 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. 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. The 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).
Bardai, F. H., Wang, L., Mutreja, Y., Yenjerla, M., Gamblin, T. C. and Feany, M. B. (2017). A conserved cytoskeletal signaling cascade mediates neurotoxicity of FTDP-17 tau mutations in vivo. J Neurosci. PubMed ID: 29138281
The microtubule binding protein tau is strongly implicated in multiple neurodegenerative disorders, including frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), which is caused by mutations in tau. In vitro, FTDP-17 mutant versions of tau can reduce microtubule binding and increase aggregation of tau, but the mechanism by which these mutations promote disease in vivo is not clear. This study took a combined biochemical and in vivo modeling approach to define functional properties of tau driving neurotoxicity in vivo. Wild type human tau and five FTDP-17 mutant forms of tau were expressed in Drosophila using a site-directed insertion strategy to ensure equivalent levels of expression. Multiple markers of neurodegeneration and neurotoxicity were analyzed in transgenic animals, including analysis of both males and females. FTDP-17 mutations act to enhance phosphorylation of tau and thus promote neurotoxicity in an in vivo setting. Further, it was demonstrated that phosphorylation-dependent excess stabilization of the actin cytoskeleton is a key phosphorylation-dependent mediator of the toxicity of wild type tau, and of all the FTDP-17 mutants tested. Finally, it was shown that important downstream pathways, including autophagy and the unfolded protein response, are co-regulated with neurotoxicity and actin cytoskeletal stabilization in brains of flies expressing wild type human and various FTDP-17 tau mutants, supporting a conserved mechanism of neurotoxicity of wild type tau and FTDP-17 mutant tau in disease pathogenesis (Bardai, 2017).
Molina-Mateo, D., Fuenzalida-Uribe, N., Hidalgo, S., Molina-Fernandez, C., Abarca, J., Zarate, R. V., Escandon, M., Figueroa, R., Tevy, M. F. and Campusano, J. M. (2017). Characterization of a presymptomatic stage in a Drosophila Parkinson's disease model: Unveiling dopaminergic compensatory mechanisms. Biochim Biophys Acta [Epub ahead of print]. PubMed ID: 28716706
Parkinson disease (PD) is a degenerative disorder characterized by several motor symptoms including shaking, rigidity, slow movement and difficult walking, which has been associated to the death of nigro-striatal dopaminergic neurons. >90% of PD patients also present olfactory dysfunction. Although the molecular mechanisms responsible for this disease are not clear, hereditary PD is linked to mutations in specific genes, including the PTEN-induced putative kinase 1 (PINK1). This work provides a thorough temporal description of the behavioral effects induced by a mutation in the PINK1 gene in adult Drosophila. The data suggests that the motor deficits associated to PD are fully revealed only by the third week of age. However, olfactory dysfunction is detected as early as the first week of age. Immunofluorescence and neurochemical data is provided that led to a proposal that compensatory changes occur in this Drosophila model for PD. These compensatory changes are associated to specific components of the dopaminergic system: the biosynthetic enzymes, Tyrosine hydroxylase and Dopa decarboxylase, and the Dopamine transporter, a plasma membrane protein involved in maintaining dopamine extracellular levels at physiologically relevant levels. Thus, these data help define presymptomatic and symptomatic phases in this PD animal model, and that compensatory changes occur in the dopaminergic neurons in the presymptomatic stage (Molina-Mateo, 2017).
Chen, J., Xue, J., Ruan, J., Zhao, J., Tang, B. and Duan, R. (2017). Drosophila CHIP protects against mitochondrial dysfunction by acting downstream of Pink1 in parallel with Parkin. FASEB J. PubMed ID: 28778978
Mitochondrial kinase PTEN-induced putative kinase 1 (PINK1) and E3 ubiquitin ligase Parkin function in a common pathway to regulate mitochondrial homeostasis contributing to the pathogenesis of Parkinson disease. The carboxyl terminus of Hsc70-interacting protein (CHIP) acts as a heat shock protein 70/heat shock protein 90 cochaperone to mediate protein folding or as an E3 ubiquitin ligase to target proteins for degradation. In this study, overexpression of Drosophila CHIP suppressed a range of Pink1 mutant phenotypes in flies, including abnormal wing posture, thoracic indentation, locomotion defects, muscle degeneration, and loss of dopaminergic neurons. Mitochondrial defects of Pink1 mutant, such as excessive fusion, reduced ATP content, and crista disorganization, were rescued by CHIP but not its ligase-dead mutants. Similar phenotypes and mitochondrial impairment were ameliorated in Parkin mutant flies by wild-type CHIP. Inactivation of CHIP with null fly mutants resulted in mitochondrial defects, such as reduced thoracic ATP content at 3 d old, decreased thoracic mitochondrial DNA content, and defective mitochondrial morphology at 60 d old. CHIP mutants did not exacerbate the phenotypes of Pink1 mutant flies but markedly shortened the life span of Parkin mutant flies. These results indicate that CHIP is involved in mitochondrial integrity and may act downstream of Pink1 in parallel with Parkin (Chen, 2017).
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, ALiX/PDCD6IP and 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 (Martinez, 2017).
Lee, B. I., Suh, Y. S., Chung, Y. J., Yu, K. and Park, C. B. (2017). Shedding light on Alzheimer's beta-amyloidosis: Photosensitized methylene blue inhibits self-assembly of beta-amyloid peptides and disintegrates their aggregates. Sci Rep 7(1): 7523. PubMed ID: 28790398
Abnormal aggregation of β-amyloid (Aβ) peptides is a major hallmark of Alzheimer's disease (AD). In spite of numerous attempts to prevent the β-amyloidosis, no effective drugs for treating AD have been developed to date. Among many candidate chemicals, methylene blue (MB) has proved its therapeutic potential for AD in a number of in vitro and in vivo studies; but the result of recent clinical trials performed with MB and its derivative was negative. With the aid of multiple photochemical analyses, this study first reports that photoexcited MB molecules can block Aβ42 aggregation in vitro. Furthermore, an in vivo study using Drosophila AD model demonstrates that photoexcited MB is highly effective in suppressing synaptic toxicity, resulting in a reduced damage to the neuromuscular junction (NMJ), an enhanced locomotion, and decreased vacuole in the brain. The hindrance effect is attributed to Aβ42 oxidation by singlet oxygen generated from photoexcited MB. Finally, this study shows that photoexcited MB possess a capability to disaggregate the pre-existing Aβ42 aggregates and reduce Aβ-induced cytotoxicity. This work suggests that light illumination can provide an opportunity to boost the efficacies of MB toward photodynamic therapy of AD in future (Lee, 2017).
Molina-Mateo, D., Fuenzalida-Uribe, N., Hidalgo, S., Molina-Fernandez, C., Abarca, J., Zarate, R. V., Escandon, M., Figueroa, R., Tevy, M. F. and Campusano, J. M. (2017). Characterization of a presymptomatic stage in a Drosophila Parkinson's disease model: Unveiling dopaminergic compensatory mechanisms. Biochim Biophys Acta [Epub ahead of print]. PubMed ID: 28716706
Parkinson disease (PD) is a degenerative disorder characterized by several motor symptoms including shaking, rigidity, slow movement and difficult walking, which has been associated to the death of nigro-striatal dopaminergic neurons. Although the molecular mechanisms responsible for this disease are not clear, hereditary PD is linked to mutations in specific genes, including the PTEN-induced putative kinase 1 (PINK1). This work provides a thorough temporal description of the behavioral effects induced by a mutation in the PINK1 gene in adult Drosophila. The data suggests that the motor deficits associated to PD are fully revealed only by the third week of age. However, olfactory dysfunction is detected as early as the first week of age. Immunofluorescence and neurochemical data is provided that leads to the idea that compensatory changes occur in this Drosophila model for PD. These compensatory changes are associated to specific components of the dopaminergic system: the biosynthetic enzymes, Tyrosine hydroxylase and Dopa decarboxylase, and the Dopamine transporter, a plasma membrane protein involved in maintaining dopamine extracellular levels at physiologically relevant levels. Thus, behavioral, immunofluorescence and neurochemical data help define for the first time presymptomatic and symptomatic phases in this PD animal model, and that compensatory changes occur in the dopaminergic neurons in the presymptomatic stage (Molina-Mateo, 2017).
Inoshita, T., Arano, T., Hosaka, Y., Meng, H., Umezaki, Y., Kosugi, S., Morimoto, T., Koike, M., Chang, H. Y., Imai, Y. and Hattori, N. (2017). Vps35 in cooperation with LRRK2 regulates synaptic vesicle endocytosis through the endosomal pathway in Drosophila. Hum Mol Genet [Epub ahead of print]. PubMed ID: 28482024
Mutations of the retromer component Vps35 and endosomal kinase LRRK2 are linked to autosomal dominant forms of familial Parkinson's disease (PD). However, the physiological and pathological roles of Vps35 and LRRK2 in neuronal functions are poorly understood. This study demonstrated that the loss of Drosophila Vps35 (dVps35) affects synaptic vesicle. recycling, dopaminergic synaptic release and sleep behavior associated with dopaminergic activity, which is rescued by the expression of wild-type dVps35 but not the PD-associated mutant dVps35 D647N. Drosophila LRRK2 dLRRK together with Rab5 and Rab11 is also implicated in synaptic vesicle recycling, and the manipulation of these activities improves the Vps35 synaptic phenotypes. These findings indicate that defects of synaptic vesicle recycling in which two late-onset PD genes, Vps35 and LRRK2, are involved could be key aspects of PD etiology (Inoshita, 2017).
Julienne, H., Buhl, E., Leslie, D. S. and Hodge, J. J. L. (2017). Drosophila PINK1 and parkin loss-of-function mutants display a range of non-motor Parkinson's disease phenotypes. Neurobiol Dis 104: 15-23. PubMed ID: 28435104
Parkinson's disease (PD) is more commonly associated with its motor symptoms and the related degeneration of dopamine (DA) neurons. However, PD patients also display a wide range of non-motor symptoms, including memory deficits and disruptions of their sleep-wake cycles. These have a large impact on their quality of life, but their etiology is poorly understood. The fruit fly Drosophila has already been successfully used to model PD, and has been used extensively to study relevant non-motor behaviours in other contexts, but little attention has yet been paid to modelling non-motor symptoms of PD in this genetically tractable organism. This study examined memory performance and circadian rhythms in flies with loss-of-function mutations in two PD genes: PINK1 and parkin. Learning and memory abnormalities were found in both mutant genotypes, as well as a weakening of circadian rhythms that is underpinned by electrophysiological changes in clock neurons. This study paves the way for further work that may help us understand the mechanisms underlying these neglected aspects of PD, thus identifying new targets for treatments to address these non-motor problems specifically and perhaps even to halt disease progression in its prodromal phase (Julienne, 2017).
Alexopoulou, Z., Lang, J., Perrett, R. M., Elschami, M., Hurry, M. E., Kim, H. T., Mazaraki, D., Szabo, A., Kessler, B. M., Goldberg, A. L., Ansorge, O., Fulga, T. A. and Tofaris, G. K. (2016). Deubiquitinase Usp8 regulates alpha-synuclein clearance and modifies its toxicity in Lewy body disease. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 27444016
In Parkinson disease, misfolded α-synuclein accumulates, often in a ubiquitinated form, in neuronal inclusions termed Lewy bodies. An important outstanding question is whether ubiquitination in Lewy bodies is directly relevant to alpha-synuclein trafficking or turnover and Parkinson's pathogenesis. By comparative analysis in human postmortem brains, it was found that ubiquitin immunoreactivity in Lewy bodies is largely due to K63-linked ubiquitin chains and markedly reduced in the substantia nigra compared with the neocortex. The ubiquitin staining in cells with Lewy bodies inversely correlated with the content and pathological localization of the deubiquitinase Usp8. Usp8 interacted and partly colocalized with alpha-synuclein in endosomal membranes and, both in cells and after purification, it deubiquitinated K63-linked chains on alpha-synuclein. Knockdown of Usp8 in the Drosophila eye reduced alpha-synuclein levels and α-synuclein-induced eye toxicity. Accordingly, in human cells, Usp8 knockdown increased the lysosomal degradation of α-synuclein. In the dopaminergic neurons of the Drosophila model, unlike knockdown of other deubiquitinases, Usp8 protected from α-synuclein-induced locomotor deficits and cell loss. These findings strongly suggest that removal of K63-linked ubiquitin chains on α-synuclein by Usp8 is a critical mechanism that reduces its lysosomal degradation in dopaminergic neurons and may contribute to α-synuclein accumulation in Lewy body disease (Alexopoulou, 2016).
Merzetti, E.M., Dolomount, L.A. and Staveley, B.E. (2016). The FBXO7 homologue nutcracker and binding partner PI31 in Drosophila melanogaster models of Parkinson's disease Genome [Epub ahead of print]. PubMed ID: 27936908
Parkinsonian-pyramidal syndrome (PPS) is an early onset form of Parkinson's disease (PD) that shows degeneration of the extrapyramidal region of the brain to result in a severe form of PD. The toxic protein build-up has been implicated in the onset of PPS. Protein removal is mediated by an intracellular proteasome complex: an E3 ubiquitin ligase, the targeting component, is essential for function. FBXO7 encodes the F-box component of the SCF E3 ubiquitin ligase linked to familial forms of PPS. The Drosophila melanogaster homologue nutcracker (ntc) and a binding partner, PI31, have been shown to be active in proteasome function. This study shows that altered expression of either ntc or PI31 in dopaminergic neurons leads to a decrease in longevity and locomotor ability, phenotypes both associated with models of PD. Furthermore, expression of ntc-RNAi in an established α-synuclein-dependent model of PD rescues the phenotypes of diminished longevity and locomotor control (Merzetti, 2016).
M'Angale, P.G. and Staveley, B.E. (2016). Bcl-2 homologue Debcl enhances α-synuclein-induced phenotypes in Drosophila. PeerJ 4: e2461. PubMed ID: 27672511
The common hallmark for both sporadic and familial forms of Parkinson disease (PD) is mitochondrial dysfunction. Mammals have at least twenty proapoptotic and antiapoptotic Bcl-2 family members, in contrast, only two Bcl-2 family genes have been identified in Drosophila melanogaster, the proapoptotic mitochondrial localized Debcl and the antiapoptotic Buffy. The expression of the human transgene α-synuclein, a gene that is strongly associated with inherited forms of PD, in dopaminergic neurons (DA) of Drosophila, results in loss of neurons and locomotor dysfunction to model PD in flies. The altered expression of Debcl in the DA neurons and neuron-rich eye and along with the expression of α-synuclein offers an opportunity to highlight the role of Debcl in mitochondrial-dependent neuronal degeneration and death. The directed overexpression of Debcl using the Ddc-Gal4 transgene in the DA of Drosophila results in flies with severely decreased survival and a premature age-dependent loss in climbing ability. The inhibition of Debcl results in enhanced survival and improved climbing ability whereas the overexpression of Debcl in the α-synuclein-induced Drosophila model of PD results in more severe phenotypes. In addition, the co-expression of Debcl along with Buffy partially counteracts the Debcl-induced phenotypes, to improve the lifespan and the associated loss of locomotor ability observed. In complementary experiments, the overexpression of Debcl along with the expression of α-synuclein in the eye, enhances the eye ablation that results from the overexpression of Debcl. The co-expression of Buffy along with Debcl overexpression results in the rescue of the moderate developmental eye defects. The co-expression of Buffy along with inhibition of Debcl partially restores the eye to a roughened eye phenotype. Taken all together these results clarify on the role for Debcl in neurodegenerative disorders (M'Angale, 2016).
Wiemerslage, L., Ismael, S. and Lee, D.(2016). Early alterations of mitochondrial morphology in dopaminergic neurons from Parkinson's disease-like pathology and time-dependent neuroprotection with D2 receptor activation. Mitochondrion [Epub ahead of print]. PubMed ID: 27423787
Neuroprotection, to prevent vulnerable cell populations from dying, is perhaps the main strategy for treating Parkinson's disease (PD). Yet in clinical practice, therapy is introduced after the disease is well established and many neurons have already disappeared, while experimentally, treatment is typically added at the same time that PD pathology is instigated. This study uses an already established Drosophila melanogaster model of PD to test for early markers of neurodegeneration and if those markers are reversible following neuroprotective treatment. Specifically, primary neuronal cultures were treated with the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), and neuritic, dopaminergic mitochondria were tracked over time, observing a fragmenting change in their morphology before cell death. A neuroprotective treatment (quinpirole, a D2 receptor agonist) was added at different timepoints to determine if the changes in mitochondrial morphology are reversible. Neuroprotective treatment must be added concomitantly to prevent changes in mitochondrial morphology and subsequent cell death. This work further supports Drosophila's use as a model organism and mitochondria's use as a biomarker for neurodegenerative disease. But mainly, this work highlights an import factor for experiments in neuroprotection - time of treatment. These results highlight the problem that current neuroprotective treatments for PD may not be used the same way that they are tested experimentally (Wiemerslage, 2016).
M'Angale, P. G. and Staveley, B. E. (2017). Bax-inhibitor-1 knockdown phenotypes are suppressed by Buffy and exacerbate degeneration in a Drosophila model of Parkinson disease. PeerJ 5: e2974. PubMed ID: 28243526
Bax inhibitor-1 (BI-1) is an evolutionarily conserved cytoprotective transmembrane protein that acts as a suppressor of Bax-induced apoptosis by regulation of endoplasmic reticulum stress-induced cell death. BI-1 was knocked down in the sensitive dopa decarboxylase (Ddc) expressing neurons of Drosophila to investigate its neuroprotective functions. BI-1-induced phenotypes were rescied by co-expression with the pro-survival Buffy, and the effect of BI-1 knockdown on the neurodegenerative alpha-synuclein-induced Parkinson disease (PD) model was determined. Knockdown of BI-1 was achieved under the direction of the Ddc-Gal4 transgene and resulted in shortened lifespan and precocious loss of locomotor ability. Co-expression of Buffy with BI-1-RNAi resulted in suppression of the reduced lifespan and impaired climbing ability. Expression of human alpha-synuclein in Drosophila dopaminergic neurons results in neuronal degeneration, accompanied by the age-dependent loss in climbing ability. It is concluded that knockdown of BI-1 in the dopaminergic neurons of Drosophila results in a shortened lifespan and premature loss in climbing ability, phenotypes that appear to be strongly associated with models of PD in Drosophila, and which are suppressed upon overexpression of Buffy and worsened by co-expression with alpha-synuclein. This suggests that BI-1 is neuroprotective and its knockdown can be counteracted by the overexpression of the pro-survival Bcl-2 homologue (M'Angale, 2017).
Sanchez-Martinez, A., Beavan, M., Gegg, M. E., Chau, K. Y., Whitworth, A. J. and Schapira, A. H. (2016). Parkinson disease-linked GBA mutation effects reversed by molecular chaperones in human cell and fly models. Sci Rep 6: 31380. PubMed ID: 27539639
GBA gene mutations are the greatest cause of Parkinson disease (PD). GBA encodes the lysosomal enzyme glucocerebrosidase (GCase) but the mechanisms by which loss of GCase contributes to PD remain unclear. Inhibition of autophagy and the generation of endoplasmic reticulum (ER) stress are both implicated. Mutant GCase can unfold in the ER and be degraded via the unfolded protein response, activating ER stress and reducing lysosomal GCase. Small molecule chaperones that cross the blood brain barrier help mutant GCase refold and traffic correctly to lysosomes are putative treatments for PD. This study treated fibroblast cells from PD patients with heterozygous GBA mutations and Drosophila expressing human wild-type, N370S and L444P GBA with the molecular chaperones ambroxol and isofagomine. Both chaperones increased GCase levels and activity, but also GBA mRNA, in control and mutant GBA fibroblasts. Expression of mutated GBA in Drosophila resulted in dopaminergic neuronal loss, a progressive locomotor defect, abnormal aggregates in the ER and increased levels of the ER stress reporter Xbp1-EGFP. Treatment with both chaperones lowered ER stress and prevented the loss of motor function, providing proof of principle that small molecule chaperones can reverse mutant GBA-mediated ER stress in vivo and might prove effective for treating PD (Sanchez-Martinez, 2016).
Lehmann, S., Loh, S. H. and Martins, L. M. (2016). Enhancing NAD+ salvage metabolism is neuroprotective in a PINK1 model of Parkinson's disease. Biol Open [Epub ahead of print]. PubMed ID: 28011627
Familial forms of Parkinson's disease (PD) caused by mutations in PINK1 are linked to mitochondrial impairment. Defective mitochondria are also found in Drosophila models of PD with pink1 mutations. The co-enzyme nicotinamide adenine dinucleotide (NAD+) is essential for both generating energy in mitochondria and nuclear DNA repair through NAD+-consuming poly(ADP-ribose) polymerases (PARPs). This study found alterations in NAD+ salvage metabolism in Drosophila pink1 mutants and showed that a diet supplemented with the NAD+ precursor nicotinamide rescued mitochondrial defects and protected neurons from degeneration. Additionally, a mutation of Parp improved mitochondrial function and was neuroprotective in the pink1 mutants. It is concluded that enhancing the availability of NAD+ by either the use of a diet supplemented with NAD+ precursors or the inhibition of NAD+-dependent enzymes, such as PARPs, which compete with mitochondria for NAD+ is a viable approach to preventing neurotoxicity associated with mitochondrial defects (Lehmann, 2016).
Yedlapudi, D., Joshi, G. S., Luo, D., Todi, S. V. and Dutta, A. K. (2016). nhibition of α-synuclein aggregation by multifunctional dopamine agonists assessed by a novel in vitro assay and an in vivo Drosophila synucleinopathy model. Sci Rep 6: 38510. PubMed ID: 27917933
Aggregation of α synuclein (α-syn) leading to dopaminergic neuronal death has been recognized as one of the main pathogenic factors in the initiation and progression of Parkinson's disease (PD). Consequently, α-syn has been targeted for the development of therapeutics for PD. This study developed a novel assay to screen compounds with α-syn modulating properties by mimicking recent findings from in vivo animal studies involving intrastriatal administration of pre-formed fibrils in mice, resulting in increased α-syn pathology accompanying the formation of Lewy-body (LB) type inclusions. In vitro generated α-syn pre-formed fibrils induce seeding of α-syn monomers to produce aggregates in a dose-and time-dependent manner under static conditions in vitro. These aggregates were toxic towards rat pheochromocytoma cells (PC12). Multifunctional dopamine agonists D-519 and D-520 exhibited significant neuroprotection in this assay, while their parent molecules did not. The neuroprotective properties of these compounds were further evaluated in a Drosophila model of synucleinopathy. Both of the compounds showed protective properties in fly eyes against the toxicity caused by α-syn. Thus, the in vitro results on modulation of aggregation and toxicity of α-syn by a novel assay were further validated with the in vivo experiments (Yedlapudi, 2016).
Sun, X., et al. Melatonin attenuates hLRRK2-induced sleep disturbances and synaptic dysfunction in a Drosophila model of Parkinson's disease(2016). Mol Med Rep [Epub ahead of print]. PubMed ID: 26985725
Sleep problems are the most common non-motor symptoms in Parkinson's disease (PD), and are more difficult to treat than the motor symptoms. In the current study, the role of human leucine-rich repeat kinase 2 (hLRRK2), the most common genetic cause of PD, was investigated with regards to sleep problems, and the therapeutic potential of melatonin in hLRRK2-associated sleep problems was explored in Drosophila. hLRRK2 was selectively expressed in the mushroom bodies (MBs) in Drosophila and sleep patterns were measured using the Drosophila Activity Monitoring System. MB expression of hLRRK2 resulted in sleep problems, presynaptic dysfunction as evidenced by reduced miniature excitatory postsynaptic current (mEPSC) and excitatory postsynaptic potential (EPSP) frequency, and excessive synaptic plasticity such as increased axon bouton density. Treatment with melatonin at 4 mM significantly attenuated the sleep problems and rescued the reduction in mEPSC and EPSP frequency in the hLRRK2 transgenic flies. The present study demonstrates that MB expression of hLRRK2 in flies recapitulates the clinical features of the sleep disturbances in PD, and that melatonin attenuates hLRRK2-induced sleep disorders and synaptic dysfunction, suggesting the therapeutic potential of melatonin in PD patients carrying LRRK2 mutations (Sun, 2016).
Sun, X., et al.Quintero-Espinosa, D., Jimenez-Del-Rio, M. and Velez-Pardo, C.(2016). Knockdown transgenic Lrrk Drosophila resists paraquat-induced locomotor impairment and neurodegeneration: A therapeutic strategy for Parkinson's disease. Brain Res 1657:253-261. PubMed ID: 28041945
Leucine-rich repeat kinase 2 (LRRK2) has been linked to familial and sporadic Parkinson's disease. However, it is still unresolved whether LRRK2 in dopaminergic (DAergic) neurons may or may not aggravate the phenotype. This study demonstrate that knocking down (KD) the Lrrk gene by RNAi in DAergic neurons untreated or treated with paraquat (PQ) neither affected the number of DAergic clusters, tyrosine hydroxylase (TH) protein levels, lifespan nor locomotor activity when compared to control (i.e. TH/+) flies. KD transgenic Lrrk flies dramatically increased locomotor activity in presence of TH enzyme inhibitor α-methyl-para-tyrosine (aMT), whereas no effect on lifespan was observed in both fly lines. Most importantly, KD Lrrk flies had reduced lipid peroxidation (LPO) index alone or in presence of PQ and the antioxidant minocycline (MC, 0.5 mM). Taken together, these findings suggest that Lrrk appears unessential for the viability of DAergic neurons in D. melanogaster. Moreover, Lrrk might negatively regulate homeostatic levels of dopamine, thereby dramatically increasing locomotor activity, extending lifespan, and reducing oxidative stress (OS). These data also indicate that reduced expression of Lrrk in the DAergic neurons of transgenic TH>Lrrk-RNAi/+ flies conferred PQ resistance and absence of neurodegeneration. The findings support the notion that reduced/suppressed LRRK2 expression might delay or prevent motor symptoms and/or frank Parkinsonism in individuals at risk to suffer autosomal dominant Parkinsonism (AD-P) by blocking OS-induced neurodegenerative processes in the DAergic neurons (Quintero-Espinosa, 2016).27936908
Parkinsonian-pyramidal syndrome (PPS) is an early onset form of Parkinson's disease (PD) that shows degeneration of the extrapyramidal region of the brain to result in a severe form of PD. The toxic protein build-up has been implicated in the onset of PPS. Protein removal is mediated by an intracellular proteasome complex: an E3 ubiquitin ligase, the targeting component, is essential for function. FBXO7 encodes the F-box component of the SCF E3 ubiquitin ligase linked to familial forms of PPS. The Drosophila melanogaster homologue nutcracker (ntc) and a binding partner, Proteasome inhibitor 31 kDa (PI31), have been shown to be active in proteasome function. This study shows that altered expression of either ntc or PI31 in dopaminergic neurons leads to a decrease in longevity and locomotor ability, phenotypes both associated with models of PD. Furthermore, expression of ntc-RNAi in an established alpha-synuclein-dependent model of PD rescues the phenotypes of diminished longevity and locomotor control (Merzetti, 2016).27192974
Only two Bcl-2 family genes have been found in Drosophila melanogaster including the pro-cell survival, human Bok-related orthologue, Buffy. The directed expression of alpha-synuclein, a gene contributing to inherited forms of Parkinson disease (PD), in the dopaminergic neurons (DA) of flies provides a robust model of PD complete with the loss of neurons and accompanying motor defects. This study altered the expression of Buffy in the dopamine producing neurons and in the developing neuron-rich eye, with and without the expression of alpha-synuclein. To alter the expression of Buffy in the dopaminergic neurons of Drosophila. The directed expression of Buffy in the dopamine producing neurons, via aDdc-Gal4 transgene, resulted in flies with increased climbing ability and enhanced survival, while the inhibition of Buffy in the dopaminergic neurons reduced climbing ability over time prematurely, similar to the phenotype observed in the alpha-synuclein-induced Drosophila model of PD. Subsequently, the expression of Buffy was altered in the alpha-synuclein-induced Drosophila model of PD. Analysis revealed that Buffy acted to rescue the associated loss of locomotor ability observed in the alpha-synuclein-induced model of PD, while Buffy RNA interference resulted in an enhanced alpha-synuclein-induced loss of climbing ability. In complementary experiments the overexpression of Buffy in the developing eye suppressed the mild rough eye phenotype that results from Gal4 expression and from alpha-synuclein expression. When Buffy is inhibited the roughened eye phenotype is enhanced. It is concluded that the inhibition of Buffy in DA neurons produces a novel model of PD in Drosophila. The directed expression of Buffy in DA neurons provides protection and counteracts the alpha-synuclein-induced Parkinson disease-like phenotypes. Taken all together this demonstrates a role for Buffy, a Bcl-2 pro-cell survival gene, in neuroprotection (M'Angale, 2016).28211874
Song, L., He, Y., Ou, J., Zhao, Y., Li, R., Cheng, J., Lin, C. H. and Ho, M. S. (2017). Auxilin underlies progressive locomotor deficits and dopaminergic neuron loss in a Drosophila model of Parkinson's disease. Cell Rep 18(5): 1132-1143. PubMed ID: 28147270
Merzetti, E. M. and Staveley, B. E. (2016). Altered expression of CG5961, a putative Drosophila melanogaster homologue of FBXO9, provides a new model of Parkinson disease. Genet Mol Res 15. PubMed ID: 27173356
Shiba-Fukushima, K., Ishikawa, K. I., Inoshita, T., Izawa, N., Takanashi, M., Sato, S., Onodera, O., Akamatsu, W., Okano, H., Imai, Y. and Hattori, N. (2017). Evidence that phosphorylated ubiquitin signaling is involved in the etiology of Parkinson's disease. Hum Mol Genet [Epub ahead of print]. PubMed ID: 28541509
The ubiquitin (Ub) kinase PINK1 and the E3 Ub ligase Parkin, two gene products associated with young-onset Parkinson's disease (PD), participate in mitochondrial quality control. The phosphorylation of mitochondrial polyUb by PINK1, which is activated in a mitochondrial membrane potential (DeltaPsim)-dependent manner, facilitates the mitochondrial translocation and concomitant enzymatic activation of Parkin, leading to the clearance of phospho-polyUb-tagged mitochondria via mitophagy. Thus, Ub phosphorylation is a key event in PINK1-Parkin-mediated mitophagy. This study examined the role of phospho-Ub signaling in the pathogenesis of PD using fly PD models, human brain tissue and dopaminergic neurons derived from induced pluripotent stem cells (iPSCs) containing Parkin or PINK1 mutations, as well as normal controls. Phospho-Ub signaling was shown to be highly conserved between humans and Drosophila, and phospho-Ub signaling and the relocation of axonal mitochondria upon DeltaPsim reduction are indeed compromised in human dopaminergic neurons containing Parkin or PINK1 mutations. Moreover, phospho-Ub signaling is prominent in tyrosine hydroxylase-positive neurons compared with tyrosine hydroxylase-negative neurons, suggesting that PINK1-Parkin signaling is more required for dopaminergic neurons. These results shed light on the particular vulnerability of dopaminergic neurons to mitochondrial stress (Shiba-Fukushima, 2017).
Maor, G., Cabasso, O., Krivoruk, O., Rodriguez, J., Steller, H., Segal, D. and Horowitz, M. (2016). The contribution of mutant GBA to the development of parkinson disease in Drosophila. Hum Mol Genet [Epub ahead of print]. PubMed ID: 27162249
Filograna, R., et al. (2016). SOD-mimetic M40403 is protective in cell and fly models of paraquat toxicity: Implications for Parkinson disease. J Biol Chem. PubMed ID: 26953346
Lehmann, S., Costa, A. C., Celardo, I., Loh, S. H. and Martins, L. M. (2016). Parp mutations protect against mitochondrial dysfunction and neurodegeneration in a PARKIN model of Parkinson's disease. Cell Death Dis 7: e2166. PubMed ID: 27031963
Lehmann, S., Loh, S. H. and Martins, L. M. (2016). Enhancing NAD+ salvage metabolism is neuroprotective in a PINK1 model of Parkinson's disease. Biol Open 6(2):141-147. PubMed ID: 28011627
Chouhan, A.K., Guo, C., Hsieh, Y.C., Ye, H., Senturk, M., Zuo, Z., Li, Y., Chatterjee, S., Botas, J., Jackson, G.R., Bellen, H.J. and Shulman, J.M. (2016). Uncoupling neuronal death and dysfunction in Drosophila models of neurodegenerative disease. Acta Neuropathol Commun 4: 62. PubMed ID: 27338814
Kumar, A., Christian, P. K., Panchal, K., Guruprasad, B. R. and Tiwari, A. K. (2017). Supplementation of spirulina (Arthrospira platensis) improves lifespan and locomotor activity in paraquat-sensitive DJ-1βΔ93 flies, a Parkinson's disease model in Drosophila melanogaster. J Diet Suppl: 1-16. PubMed ID: 28166438
Dinter, E., Saridaki, T., Nippold, M., Plum, S., Diederichs, L., Komnig, D., Fensky, L., May, C., Marcus, K., Voigt, A., Schulz, J. B. and Falkenburger, B. H.(2016). Rab7 induces clearance of α-synuclein aggregates. J Neurochem 138(5):758-74. PubMed ID: 27333324
Tsai, P.I., Course, M.M., Lovas, J.R., Hsieh, C.H., Babic, M., Zinsmaier, K.E. and Wang, X. (2014). PINK1-mediated phosphorylation of Miro inhibits synaptic growth and protects dopaminergic neurons in Drosophila. Sci Rep 4: 6962. PubMed ID: 25376463
Drosophila is a robust genetic and cellular tool for modeling human neurodegenerative diseases. Loss of PINK1 in Drosophila mimics many aspects of PD pathology, including a severe loss of dopaminergic (DA) neurons, which is a hallmark of PD. However, only a few of the molecular and cellular mechanisms underlying the behavioral and cellular phenotypes of PINK1 null mutant flies have been clearly defined. This study identifies that DMiroS182A,S324A,T325A, which is predicted to resist PINK1-mediated phosphorylation, causes increased mitochondrial movement, synaptic overgrowth, and loss of DA neurons. All three of these defects are also observed in PINK1 null mutant flies. These observations suggests that Miro is a crucial substrate for causing these phenotypes by mutant PINK1 and open a new door to fully dissect PINK1 functions by studying its individual substrates. Since PINK1-related hereditary PD shares symptomatic and pathological similarities with the majority of idiopathic PD, such work will advance our understanding of the cellular and molecular underpinnings of PD's destructive path (Tsai, 2014).
Extensive studies using cell cultures have established a critical role for PINK1 in damage-induced mitophagy. PINK1/Parkin-dependent regulation of mitochondrial transport by controlling Miro protein levels on mitochondria is likely a key step prior to initiating mitophagy in cultured neurons. In this study, it was shown that PINK1-mediated phosphorylation of DMiro is required for normal mitochondrial movement in axon terminals, synaptic growth, and the neuroprotection of DA neurons. Importantly, loss of PINK1-mediated phosphorylation of DMiro has no significant effect on the mitochondrial membrane potential, excluding the possibility that the observed phenotypic effects are due to an impairment of mitophagy and an accumulation of damaged mitochondria. Accordingly, under these conditions PINK1-mediated phosphorylation of DMiro may not be required for mitophagy. However, this does not necessarily contradict its mitophagic role; rather, this represents circumstances under which its mitophagic role is dispensable. It is tempting to speculate that an efficient regulation of mitophagy is more critical in aging neurons (Tsai, 2014).
A conserved site in human and Drosophila Miro, MiroSer156/DMiroSer182, was identified to be the main residue for PINK1-mediated phosphorylation. Additional conserved sites in DMiro were also found that may have a cooperative role. Future studies determining their functions in mammalian systems are warranted to confirm if a similar regulatory mechanism is at play. The study suggests that these PINK1 phosphorylation sites in DMiro are not absolutely required for the subsequent Parkin-dependent degradation of DMiro, because when harsh treatment of CCCP is applied, the phospho-resistant DMiroS182A,S324A,T325A is degraded. The failure of DMiroS182A,S324A,T325A to prevent degradation under this condition might be due to PINK1-mediated phosphorylation on other sites that promote DMiro degradation, or due to activation of additional mechanisms. In two recent studies, MiroS156A was found to be significantly degraded by co-expression of PINK1 and Parkin in addition to CCCP treatment in Hela cells, or by overexpression of Parkin together with Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, another mitochondrial uncoupler) treatment in SH-SY5Y cells; whereas in another study, MiroS156A was found to be resistant to degradation when only PINK1 or Parkin is individually expressed in HEK293T cells. This again suggests that if the PINK1/Parkin pathway is overwhelmingly activated, mutating the few known PINK1-mediated phosphorylation residues in Miro is not sufficient to prevent its degradation (Tsai, 2014).
Why is mitochondrial motility increased in “DMironull, da > DMiroS182A,S324A,T325A”? DMiroS182A,S324A,T325A is resistant to PINK1/Parkin-mediated degradation, which may lead to more DMiroS182A,S324A,T325A accumulation on mitochondria. Unexpectedly, DMiroS182A,S324A,T325A protein level in “DMironull, da > DMiroS182A,S324A,T325A” is not significantly upregulated as compared with DMirowildtype in “DMironull, da > DMirowildtype” using fly whole body lysates. It is likely that PINK1/Parkin-dependent degradation of Miro only occurs in certain cell types, at certain subcellular locations, on certain populations of mitochondria, or under certain circumstances, and thus it is hard to detect a dramatic change using whole body lysates or without overexpression of PINK1/Parkin. Future mechanistic study is needed to test these hypotheses, such as detecting Miro subcellular localization and expression levels in different cell types, in different developmental stages, and with different mitochondrial stresses (Tsai, 2014).
This study highlights the importance of a precise control of mitochondrial movement for neuronal health. Anterograde mitochondrial transport in axons is mediated by a conserved motor/adaptor complex, which includes the motor kinesin heavy chain (KHC), the adaptor protein milton and the mitochondrial membrane anchor Miro. In the current model, Miro binds to milton, which in turn binds to KHC recruiting mitochondria to the motors and microtubules. In addition to the transmembrane domain inserted into the OMM, Miro features a pair of EF-hands and two GTPase domains. Miro was also recently found to be a substrate of the Ser/Thr kinase PINK14 and of the E3 ubiquitin ligase Parkin, both mutated in PD. Thus, mitochondrial transport can be regulated by multiple signals upstream of Miro and the motor complex maintaining energy and Ca2+ homeostasis in neuronal processes and terminals. For example, loss of PINK1-mediated phosphorylation of DMiro increases local mitochondrial movement at NMJs. In turn, this may disrupt synaptic homeostasis leading to synaptic overgrowth by mechanisms yet to be identified. Similarly, the loss of DA neurons in adult brains could well be a consequence of impaired synaptic homeostasis together with an accumulation of dysfunctional mitochondria. Local signals that regulate mitochondrial transport through Miro must be crucial to supporting neuronal functions. This study elucidates a fundamental biological mechanism demanded by a healthy neuron (Tsai, 2014).
Min, B., Kwon, Y.C., Choe, K.M. and Chung, K.C. (2015). PINK1 phosphorylates transglutaminase 2 and blocks its proteasomal degradation. J Neurosci Res 93: 722-735. PubMed ID: 25557247
From these findings, the GMR-GAL4 system was utilized to determine whether there is a genetic interaction between dPINK1 and dTG-a. Genetic analyses of dPINK1 and dTG-a in Drosophila reveals that they act together in a common pathway. Transgenic flies with dPINK1 overexpression produce more severe fly eye formation defects than flies overexpressing dTG-a. In addition, coexpression of dPINK1 and dTG-a in flies results in the loss of bristles and more severe disorganization of the ommatidial array compared with that observed in the eyes of flies overexpressing each gene alone. In agreement with in vitro data of this study, results from the fly model further demonstrate that dPINK1 lies upstream of dTG-a. Taken together, the study demonstrates biochemical interaction and functional link between PINK1 and TG2. Also, PINK1-mediated TG2 activation plays a role in LB formation and PD progression. Further studies examining this interaction would help to advance the current understanding of PD pathogenesis (Min, 2015).
Gao, F., Chen, D., Si, J., Hu, Q., Qin, Z., Fang, M. and Wang, G. (2015). The mitochondrial protein BNIP3L is the substrate of PARK2 and mediates mitophagy in PINK1/PARK2 pathway. Hum Mol Genet 24: 2528-2538. PubMed ID: 25612572
BNIP3L is a mitochondrial protein important for a selective autophagic degradation of mitochondria during reticulocytes maturation, and BNIP3L−/− mice exhibit mitochondrial retention in their reticulocytes. Using genetic assays in Drosophila, it was found that overexpression of BNIP3L can rescue the phenotype of mitochondrial dysfunction in pink1 mutant fly, but not in park mutant fly. In cultured cells, BNIP3L induces mitophagy in PARK2 wild-type cells but not in PARK2-deficient HeLa cells. Importantly, the direct interactions between PARK2 and BNIP3L and the enhancement of BNIP3L ubiquitiniation by PARK2 are observed. Moreover, the interactions between BNIP3L and PARK2, and the ubiquitination of BNIP3L are significantly increased when PARK2 is translocated to mitochondria, suggesting that BNIP3L is a substrate of PARK2 on mitochondria. These findings are of help to understand the mitochondrial phenotype rescue in Drosophila in genetic assays. As PARK2 acts downstream of PINK1 and overexpression of PARK2 rescues the phenotype of pink1 mutant fly, it suggests that some other factors may also affect PARK2 recruitment to damaged mitochondria in vivo but the unknown factors are not effective as PINK1 so that the phenotype of pink1 mutant fly is only rescued with the presence of extensive PARK2. It was also found that BNIP3L is a substrate of PARK2 and its function definitely depends on PAKR2. Thus, overexpression of BNIP3L rescues the phenotype of pink1 mutant fly because of the presence of PARK2 but overexpression of BNIP3L fails to rescue the phenotype in the absence of PARK2. These findings provide evidence that BNIP3L is a downstream factor of the PINK1/PARK2 pathway and that BNIP3L strictly depends on PARK2 to induce mitophagy (Gao, 2015).
Although DA neurons possess an intact PINK1/PARK2/BNIP3L pathway to cope with the disrupted mitochondria in most sporadic PD patients, increased levels of disrupted mitochondria with reduced complex I activity have been detected in PD brains. Rotenone and MPTP that inhibit complex I activity are causative factors for PD. In this study, it was observed that cells with reduced mitochondrial complex I activity induced by rotenone, MPP+ or 6-OHDA present a significant degradation of BNIP3L and that the BNIP3L-mediated mitochondrial degradation pathway is disrupted, thereby resulting in a retention of the damaged mitochondria. Interestingly, BNIP3L is degraded after the usage of mitochondrial complex I inhibitors, which will not be blocked by both lysosomal and protease inhibitors. As the lysosomal inhibitor blocks mitophagy; and the proteasomal inhibitor blocks the proteasomal system as well as mitophagy because of an inhibition of mitofusin I and II degradation, a process necessary for the initiation of mitophagy, the degradation of BNIP3L is unlikely caused by the proteasome or mitophagy. It is highly possible that it is processed by unknown proteases that are activated under mitochondrial complex I inhibitor treatment. Together with previous findings by other investigators, showing that PINK1 or PARK2 mutants interfere with mitophagy, this study suggests that the degradation of BNIP3L caused by complex I inhibition factors results in BNIP3L-inability to clear the damaged mitochondria. Thus, these findings also provide a mechanistic explanation why the existing PINK1/PARK2 pathway fails to clear the damaged mitochondria caused by complex I inhibitors in PD (Gao, 2015).
In summary, this study identifies that BNIP3L is a substrate of PARK2 on mitochondria. The BNIP3L ubiquitination induced by mitochondria-located PARK2 recruits the NBR1 to mitochondria to target the mitochondria for degradation. However, the environmental toxins that induce BNIP3L degradation can disrupt the PINK1/PARK2/BNIP3L-mediated mitophagy and cause an accumulation of damaged mitochondria, leading to the injury of DA neurons and occurrence of the disease (Gao, 2015).
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
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).
Pogson, J.H., Ivatt, R.M., Sanchez-Martinez, A., Tufi, R., Wilson, E., Mortiboys, H and Whitworth, A.J. (2014). The complex I subunit NDUFA10 selectively rescues Drosophila pink1 mutants through a mechanism independent of mitophagy. PLoS Genet 10: e1004815. PubMed ID: 25412178
PINK1 and Parkin have long been genetically linked in a common pathway that promotes mitochondrial homeostasis at least partly by directing the autophagic degradation of dysfunctional mitochondria as a mechanism of mitochondrial quality control. While this model potentially explains the occurrence of CI deficiency, oxidative stress, calcium dysregulation and elevated mtDNA mutations seen in patient tissues, and the age-related onset of PD, other models have been proposed to explain the pathological consequences of PINK1 and Parkin deficiency. Moreover, many mechanistic details by which the PINK1-Parkin pathway functions remain unexplained. To address these matters, this study conducted an RNAi screen to identify genes whose loss-of-function either phenocopies or suppresses a pink1 RNAi phenotype. A number of genes were identified that fulfill these criteria; the study then focused on ND42/NDUFA10 given the extensive literature implicating CI deficiency in PD pathogenesis and the fact that CI deficiency was previously reported in PINK1 mutant models and patient samples (Pogson, 2014).
Loss of ND42/NDUFA10 phenocopies the effect of pink1 loss on mitochondrial morphology in Drosophila cells, and ND42 overexpression rescues the pink1 mutant phenotypes. However, NDUFA10 knockdown causes only modest effects on mitophagy, supporting a separate link between CI and PINK1 function. The simplest interpretation of these findings is that PINK1 normally regulates ND42/NDUFA10 abundance or activity through direct phosphorylation. Indeed, it was recently reported that NDUFA10 lacks phosphorylation at Ser-250 in Pink1-/- cells, although it remains to be determined whether PINK1 directly or indirectly regulates NDUFA10 phosphorylation. Moreover, it was reported that expression of a phospho-mimetic version of ND42/NDUFA10 specifically rescues phenotypes in multiple PINK1 deficient systems, while an S250A mutant version of ND42/NDUFA10 that is incapable of being phosphorylated is unable to confer rescue. Consistent with this, it was found that at equivalent expression levels, the phospho-mimetic (SD) provides a slightly better phenotypic rescue than the other variants, and likewise promotes a higher CI activity. Nevertheless, it is also shown that the non-phosphorylatable S250A version is still able to restore CI activity and significantly rescue the climbing deficit in pink1 mutant flies (Pogson, 2014).
While further studies are needed to clarify the functional relationship between PINK1 and NDUFA10 in the regulation of CI, findings from this study provide further support to the mounting evidence that many manipulations that promote CI activity – overexpression of NDUFA10, sicily, heix, Ret, dNK, TRAP1 and NDI1, or treatment with vitamin K, deoxynucleosides or folic acid – can rescue pink1 mutants, suggesting a more general defect underlies CI deficiency in loss of pink1. The study hypothesizes that the loss of CI activity in pink1 mutants may be due to a general de-stabilization of CI. Assembly is a particular challenge for such a large, multi-subunit complex and occurs in a stepwise process that is highly regulated by many factors. Even its association with other ETC complexes in supercomplexes affects CI's stability. There is evidence for reduced complex stability in pink1 mutants, though this may not be specific to CI. One possibility is that PINK1 influences CI stability by directly promoting the assembly of CI, which may be regulated by NDUFA10 (Pogson, 2014).
These findings also further support that the mechanism by which PINK1 influences CI activity appears to be separable from its well-characterized role in mitophagy, since, in agreement with some studies but in contrast to others, a clear evidence of CI deficiency in parkin mutant flies was not found. Moreover, it is unexpected to find that overexpression of parkin does not rescue the CI deficiency in pink1 mutants, because substantial previous work has shown that parkin overexpression rescues all of the other pink1 phenotypes, and because a prediction of the PINK1-parkin mitophagy pathway is that activation would trigger the selective removal of mitochondria deficient in CI activity. This suggests that CI deficiency alone cannot fully account for adult locomotor phenotypes seen in pink1 mutants. Further studies are needed to clarify full spectrum of cellular defects in pink1 and parkin mutants and their relative importance to the pathologic mechanism (Pogson, 2014).
Zhu, M., Li, X., Tian, X. and Wu, C. (2015). Mask loss-of-function rescues mitochondrial impairment and muscle degeneration of Drosophila pink1 and parkin mutants. Hum Mol Genet 24: 3272-3285. PubMed ID: 25743185
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).
Clark, I.E., Dodson, M.W., Jiang, C., Cao, J.H., Huh, J.R., Seol, J.H., Yoo, S.J., Hay, B.A. and Guo, M. (2006). Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin. Nature 441: 1162-1166. PubMed ID: 16672981
Liu, J., Li, T., Thomas, J. M., Pei, Z., Jiang, H., Engelender, S., Ross, C. A. and Smith, W. W. (2016). Synphilin-1 attenuates mutant LRRK2-induced neurodegeneration in Parkinson's disease models. Hum Mol Genet. PubMed ID: 26744328
Breda, C., Nugent, M.L., Estranero, J.G., Kyriacou, C.P., Outeiro, T.F., Steinert, J.R. and Giorgini, F. (2015). Rab11 modulates α-synuclein-mediated defects in synaptic transmission and behaviour. Hum Mol Genet 24: 1077-1091. PubMed ID: 25305083
Synaptic dysfunction occurs in several human neurodegenerative diseases prior to neuronal loss and the manifestation of deficient behaviours. For this reason, this study first investigated the effect of aSyn toxicity in Drosophila larvae. It was found that synaptic transmission is altered at the NMJ, and that this correlates with an enlargement of SVs. These physiological effects have a negative consequence on larval crawling behaviour when aSyn is expressed either in the motorneurons or in the dopaminergic neurons. Locomotor dysfunction is maintained through to adults, as evidenced by decreased climbing at all post-eclosion ages tested. Finally, the survival of flies expressing pan-neuronal aSyn is diminished compared with the controls. Strikingly, for all these aSyn-mediated abnormalities, overexpression of Drosophila Rab11 significantly ameliorates these mutant phenotypes (Breda, 2015).
It was earlier demonstrated that Rab11 similarly rectifies several phenotypes in a fruit fly model of HD, including compromised spontaneous miniature and evoked transmission and SV size, although these effects are in the inverse direction to those observed in the current study with aSyn flies. The ability of Rab11 to normalize SV size in both experimental paradigms indicates that it plays a critical role in homeostasis of SV size. Indeed, several Rab (-interacting) proteins have been suggested to act as mediators of synaptic homeostasis. Supporting the observations in this study, aSyn overexpression in primary mouse neurons causes an enlargement in SVs. It was further found that the increased amplitude of miniature events correlates with larger SV diameters (Breda, 2015).
How does aSyn expression lead to SV enlargement? A possible explanation can be the propensity of aSyn to interact with Rab5. Rab5 plays a key role in preventing the fusion of SVs with each other—known as homotypic fusions. Therefore, by sequestering or negatively interacting with Rab5, aSyn may lead to increased fusion between SVs resulting in their enlargement. Alternatively, aSyn may function as a scaffold protein attracting and promoting the fusion between multiple vesicles. This is in agreement with the observation that the expression of aSyn in yeast causes vesicle accumulation. aSyn was also shown to interact with clathrin signalling and clathrin together with its adaptor proteins is involved in regulating vesicular size. This could point towards a possible regulation of synaptic transmission via aSyn. The possibility that the aSyn-dependent increases of SVs, miniature events and evoked release occur by promoting the assembly of the SNARE complex, with its role in fusion mechanisms with the plasma membrane, should also be considered (Breda, 2015).
It is evident from this study that aSyn perturbs—and that simultaneous Rab11 overexpression restores—vesicle trafficking. In this regard, Rab3—a GTPase localized to SVs—was earlier implicated in synaptic homeostasis. Given the ubiquitous expression of Rab11 and its localization to synaptic boutons, it is possible that Rab11 performs a similar function at the synapse. In this scenario, aSyn would perturb the fine balance in synaptic transmission by interfering directly with Rab11 or with some effectors that regulate its function. The overexpression of Rab11 would thereby compensate this dysfunction, shifting the system to a more ‘normal’ state. Recent work indicates that aSyn interacts with the GTP/GDP-binding pocket of Rab8a, abrogating its function. It is thus possible that aSyn may have a similar negative impact on the inactive–active state of Rab11. Enhanced Rab11 activity could also modulate the endosomal recycling rate in aSyn flies and thereby alter delivery of vesicle-required proteins to the membrane. Notably, Rab11 has been shown to interact with the ε subunit of the vacuolar type H+-ATPase, and a possible enhanced interaction with the vesicular H+-ATPase could alter vesicular parameters and rescue aSyn-dependent defects (Breda, 2015).
aSyn present in vesicles has an increased propensity to aggregate compared with its cytosolic counterpart. Thus, aSyn inclusion formation due to accumulation of defective vesicles may serve as an initial signal for enhancing further aggregation processes. This aSyn cluster may then sequester several proteins—including Rab11—depleting cellular factors involved in the diverse physiological processes. Interaction between aSyn and Rab11 has been demonstrated in parallel work conducted in a human cell model of PD by co-immunoprecipitation and co-localization studies. Importantly, Rab11 expression was found to decrease the number of aSyn inclusions and cellular toxicity, likely due to enhanced aSyn secretion from the cells, which may explain the alterations in aSyn localization observed at the larval NMJ due to Rab11 overexpression. This study's findings from aged Drosophila heads support this role for Rab11 in the clearance of toxic insoluble aSyn, and further show that its protective properties depends on its activity, as the Rab11 dominant-negative variant fails to rescue any of the phenotypes assessed. Although the mechanisms are not fully understood, aSyn can actively be secreted from neurons via the endocytic pathway, exosomes and the ER/Golgi-to-plasma membrane secretory pathway (Breda, 2015).
Cagin, U., Duncan, O. F., Gatt, A. P., Dionne, M. S., Sweeney, S. T. and Bateman, J. M. (2015). Mitochondrial retrograde signaling regulates neuronal function. Proc Natl Acad Sci U S A 112: E6000-6009. PubMed ID: 26489648
The human brain constitutes approximately 2% of body weight but consumes 20% of available oxygen because of its high energy demand. Mitochondria are abundant in neurons and generate the majority of cellular ATP through the action of the mitochondrial ATP synthase complex. Mitochondrial disorders are one of the most common inherited disorders of metabolism and have diverse symptoms, but tissues with a high metabolic demand, such as the nervous system, are frequently affected. The primary insult in all mitochondrial diseases is to mitochondrial function, but the etiology of these diseases is highly pleiotropic. This phenomenon is poorly understood, but suggests that the cellular response to mitochondrial dysfunction may be complex and vary between cell types and tissues (Cagin, 2015).
Mitochondrial retrograde signaling is defined as the cellular response to changes in the functional state of mitochondria. Mitochondrial retrograde signaling enables communication of information about changes in processes such as mitochondrial bioenergetic state and redox potential to the rest of the cell and is thus a key mechanism in cellular homeostasis. The best characterized retrograde responses involve mitochondrial dysfunction eliciting changes in nuclear gene transcription. In yeast, mitochondrial dysfunction causes changes in the expression of genes involved in supplying mitochondria with oxaloacetate and acetyl CoA, the precursors of α-ketoglutarate and glutamate, to compensate for failure of the tricarboxylic acid (TCA) cycle (Cagin, 2015).
In proliferating mammalian cell models, mitochondrial retrograde signaling is more diverse and involves increases in cytosolic-free Ca2+, leading to activation of Ca2+-responsive calcineurin, causing the up-regulation of genes controlling Ca2+ storage and transport. In addition to mitochondrial diseases, alterations in mitochondrial function are also associated with late onset neurodegenerative diseases such as Alzheimer's and Parkinson's. Thus, the neuronal response to mitochondrial function may be altered in these diseases and contribute to disease progression. However, neuronal-specific mitochondrial retrograde signaling is poorly understood and its role in neuronal homeostasis is completely unknown (Cagin, 2015).
This study has developed a neuronal-specific model of mitochondrial dysfunction in Drosophila and used this to characterize mitochondrial retrograde signaling in vivo. Retrograde signaling is shown to regulate neuronal function and can be manipulated to alleviate the effects of mitochondrial dysfunction in neurons (Cagin, 2015).
This study shows that the Drosophila HIFα ortholog Sima is potentially a key regulator of the mitochondrial retrograde response in the nervous system and that knockdown of Sima dramatically improves neuronal function in this and other models of mitochondrial dysfunction. Surprisingly, Sima activity in part causes the dysfunction of neurons containing defective mitochondria. Previous studies of Drosophila mutants in the regulatory and catalytic subunits of the mitochondrial DNA polymerase Polγ have demonstrated that loss of mtDNA replication in Drosophila causes mtDNA loss, reduced neuronal stem cell proliferation, and developmental lethality. To avoid the pleiotropic effects of using homozygous mutant animals, this study developed a neuronal-specific model of mitochondrial dysfunction. The phenotypes resulting from TFAM overexpression and expression of a mitochondrially targeted restriction enzyme were characterized, and both of these tools were used to model neuronal-specific mitochondrial dysfunction (Cagin, 2015).
Overexpression of mitochondrial transcription factor A (TFAM) results in mitochondrial dysfunction caused by inhibition of mitochondrial gene expression, rather than an alteration in mtDNA copy number. Overexpression of TFAM has been shown to have different effects depending on the cell type, model system, or ratio of TFAM protein to mtDNA copy number. The current results are consistent with in vitro studies and overexpression of human TFAM in mice and human cells, which have shown that excess TFAM results in the suppression of mitochondrial gene transcription. Ubiquitous expression of mitoXhoI causes early developmental lethality and that, although there was no significant mtDNA loss, the majority of mtDNA was linearized. Given that mtDNA is transcribed as two polycistronic mRNAs, a double-stranded break in coxI would block the transcription of the majority of mitochondrially encoded genes, resulting in severe mitochondrial dysfunction (Cagin, 2015).
Using a Drosophila motor neuron model, mitochondrial dysfunction was found to cause a reduction in the number of active zones, loss of synaptic mitochondria, and locomotor defects. Mitochondrial dysfunction caused by overexpression of PINK1 or Parkin decreases the rate of mitochondrial transport in vitro and in vivo. Furthermore, a recent study using KillerRed demonstrated that local mitochondrial damage results in mitophagy in axons. Therefore, the acute loss of synaptic mitochondria in the current model may result from defects in mitochondrial transport and/or mitophagy (Cagin, 2015).
Previous studies in mice have examined the effects of neuronal mitochondrial dysfunction by using mitoPstI expression, or targeted knockout of TFAM. Knockout of TFAM specifically in mouse dopaminergic neurons (the 'MitoPark' mouse model) causes progressive loss of motor function, intraneuronal inclusions, and eventual neuronal cell death. Interestingly, cell body mitochondria are enlarged and fragmented and striatal mitochondria are reduced in number and size in MitoPark dopaminergic neurons, suggesting that the effects of neuronal mitochondrial dysfunction are conserved in Drosophila and mammals. Larvae mutant for the mitochondrial fission gene drp1 have fused axonal mitochondria and almost completely lack mitochondria at the NMJ, similar to motor neurons overexpressing TFAM or expressing mitoXhoI (Cagin, 2015).
Adult drp1 mutant flies also have severe behavioral defects. Synaptic reserve pool vesicle mobilization is inhibited in drp1 mutant larvae because of the lack of ATP to power the myosin ATPase required for reserve pool tethering and release. Reserve pool vesicle mobilization is likely to be similarly affected in TFAM overexpressing or mitoXhoI-expressing motor neurons, which would result in locomotor defects in these animals (Cagin, 2015).
Interestingly, expression of the Arctic form of β-amyloid1-42 (Aβ) in Drosophila giant fiber neurons also leads to the depletion of synaptic mitochondria and decreased synaptic vesicles. Synaptic loss and alterations in neuronal mitochondrial morphology have also been observed in postmortem tissue from Alzheimer's disease patients. The parallels between these phenotypes and those in the current model suggest a common underlying mechanism (Cagin, 2015).
Using microarray analysis, this study found that mitochondrial dysfunction in neurons regulates the expression of hundreds of nuclear genes. The Drosophila CNS contains different neuronal subtypes, and glial cells, so the results of the microarray are heterogeneous, representing the pooled response to mitochondrial dysfunction throughout the CNS. Mitochondrial dysfunction was phenotypically characterized in motor neurons, but not all of the genes identified from the microarrays are expressed in motor neurons, e.g., Ilp3. The specific genes that are regulated differ depending on whether mitochondrial dysfunction results from TFAM overexpression or knockdown of ATPsynCF6. However, a core group of approximately 140 genes are similarly regulated in both conditions (Cagin, 2015).
Yeast mutants in different components of the TCA cycle result in differing retrograde responses and comparison of somatic cell hybrids (cybrids) carrying the A3243G mtDNA mutation with cybrids completely lacking mtDNA (ρ0 cells) showed overlapping but distinct gene expression profiles. Moreover, another study comparing cybrids with increasing levels of the A3243G mtDNA mutation showed markedly different alterations in nuclear gene expression, depending on the severity of mitochondrial dysfunction (Cagin, 2015).
Taken together, these data suggest that the cellular response to mitochondrial dysfunction is not uniform and adapts to the specific defect and severity of the phenotype. Therapeutic strategies targeting mitochondrial dysfunction in human disease may therefore need to be tailored to the specific mitochondrial insult. Concomitant with the current findings, previous studies have shown that in yeast, Drosophila, and mammalian-proliferating cells, retrograde signaling activates the expression of hypoxic/glycolytic genes and the insulin-like growth factor-1 receptor pathway to compensate for mitochondrial dysfunction. Rtg1 and Rtg3, the transcription factors that coordinate the mitochondrial retrograde response in yeast, are not conserved in metazoans. In mammalian proliferating cellular models, the retrograde response activates the transcription factors nuclear factor of activated T cells (NFAT), CAAT/enhancer binding protein δ (C/EBPδ), cAMP-responsive element binding protein (CREB), and an IκBβ-dependent nuclear factor κB (NFκB) c-Rel/p50. Whether these transcription factors regulate mitochondrial retrograde signaling in the mammalian nervous system is not known (Cagin, 2015).
HIFα/Sima is a direct regulator of LDH expression in flies and mammals, and this study found that Sima also regulates the expression of two other retrograde response genes, Thor and Ilp3, in the Drosophila nervous system. Importantly, Sima is required for the increase in Thor expression in response to mitochondrial dysfunction. Sima has been strongly implicated as a key regulator of mitochondrial retrograde signaling in Drosophila S2 cells knocked down for the gene encoding subunit Va of complex IV. sima, Impl3, and Thor expression were all increased in this model, and there is a significant overlap with the genes regulated in the current model (Cagin, 2015).
These data support the possibility that the Drosophila HIFα ortholog Sima is a key transcriptional regulator of neuronal mitochondrial retrograde signaling. HIFα is stabilized in hypoxia through the action of prolyl hydroxylases and this mechanism was thought to require ROS, but HIFα stabilization may in fact be ROS independent. In mammalian cells carrying the mtDNA A1555G mutation in the 12S rRNA gene, mitochondrial retrograde signaling has been shown to be activated by increased ROS, acting through AMPK and the transcription factor E2F1 to regulate nuclear gene expression. In the Drosophila eye, loss of the complex IV subunit cytochrome c oxidase Va (CoVa) causes decreased ROS. However, retrograde signaling upon loss of CoVa was not mediated by decreased ROS, but by increased AMP activating AMPK. Similarly, the small decrease in redox potential in neurons in response to mitochondrial dysfunction in the current model makes it unlikely that ROS are the mediator of the retrograde signal. Moreover, HIFα physically interacts with several transcriptional regulators including the Drosophila and mammalian estrogen-related receptor and Smad3, as well as its heterodimeric binding partner HIFβ, to regulate gene expression. Mitochondrial retrograde signaling may modulate these or other unidentified HIFα interactors and, thus, control HIF target gene expression without directly regulating HIFα (Cagin, 2015).
In cancer cell models, mitochondrial dysfunction promotes cell proliferation, increased tumourigenicity, invasiveness, and the epithelial-to-mesenchymal transition via retrograde signaling. In these models, inhibition of retrograde signaling prevents these tumourigenic phenotypes. Neuronal mitochondrial dysfunction in the current model causes a cellular response, resulting in a severe deficit in neuronal function. This response may have evolved to protect neurons, through decreased translation and increased glycolysis, from the short-term loss of mitochondrial function. Over longer periods, however, this response may be counterproductive because it results in decreased neuronal activity and locomotor function. Inhibition of neuronal mitochondrial retrograde signaling, through knockdown of Sima, dramatically improves neuronal function. Thus, mitochondrial retrograde signaling contributes to neuronal pathology and can be modified to improve the functional state of the neuron (Cagin, 2015).
Importantly, this intervention works without altering the primary mitochondrial defect. Knockdown of Sima not only abrogates the acute defects in neuronal function, but also suppresses the reduced lifespan caused by neuronal mitochondrial damage. The benefits of reduced Sima expression therefore extend throughout life. In addition to TFAM overexpression, this study also shows that Sima knockdown in neurons rescues a Drosophila model of the mitochondrial disease Leigh syndrome. However, Sima knockdown does not rescue the lethality caused by a temperature-sensitive mutation in coxI (Cagin, 2015).
Mitochondrial diseases are complex, and mutations in different COX assembly factors cause varying levels of COX deficiency in different tissues. The increasing number of Drosophila models of mitochondrial dysfunction will help to unravel the mechanisms underlying the varied pathology of mitochondrial diseases. Ubiquitous knockdown of Sima also partially restores the climbing ability of parkin mutant flies. The ability of reduced Sima expression to rescue both mitochondrial dysfunction and Parkinson's disease models reinforces the link between mitochondrial deficiency and Parkinson's and suggests that retrograde signaling may be a therapeutic target in Parkinson's disease. HIF1α inhibitors are in clinical trials for lymphoma and so, if the current findings can be replicated in mammalian models, HIF1α inhibitors may be candidates for repurposing to treat mitochondrial diseases and neurodegenerative diseases associated with mitochondrial dysfunction, such as Parkinson's disease (Cagin, 2015).
Sanz, F. J., Solana-Manrique, C., Munoz-Soriano, V., Calap-Quintana, P., Molto, M. D. and Paricio, N. (2017). Identification of potential therapeutic compounds for Parkinson's disease using Drosophila and human cell models. Free Radic Biol Med 108: 683-691. PubMed ID: 28455141
Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease. It is caused by a loss of dopaminergic neurons in the substantia nigra pars compacta, leading to a decrease in dopamine levels in the striatum and thus producing movement impairment. Major physiological causes of neurodegeneration in PD are oxidative stress (OS) and mitochondrial dysfunction; these pathophysiological changes can be caused by both genetic and environmental factors. Although most PD cases are sporadic, it has been shown that 5-10% of them are familial forms caused by mutations in certain genes. One of these genes is the DJ-1 oncogene (PARK7), which is involved in an early-onset recessive PD form. Currently, PD is an incurable disease for which existing therapies are not sufficiently effective to counteract or delay the progression of the disease. Therefore, the discovery of alternative drugs for the treatment of PD is essential. This study used a Drosophila PD model to identify candidate compounds with therapeutic potential for this disease. These flies carry a loss-of-function mutation in the DJ-1β gene, the Drosophila ortholog of human DJ-1, and show locomotor defects reflected by a reduced climbing ability. A pilot modifier chemical screen was performed, and several candidate compounds were identified based on their ability to improve locomotor activity of PD model flies. Some of them were also able to reduce OS levels in these flies. To validate the compounds identified in the Drosophila screen, a human cell PD model was generated by knocking down DJ-1 function in SH-SY5Y neuroblastoma cells. The results showed that some of the compounds were also able to increase the viability of the DJ-1-deficient cells subjected to OS, thus supporting the use of Drosophila for PD drug discovery. Interestingly, some of them have been previously proposed as alternative therapies for PD or tested in clinical trials and others are first suggested in this study as potential drugs for the treatment of this disease (Sanz, 2017).
Auluck, P.K., Chan, H.Y., Trojanowski, J.Q., Lee, V.M. and Bonini, N.M. (2002). Chaperone suppression of α-synuclein toxicity in a Drosophila model for Parkinson's disease. Science 295: 865-868. PubMed ID: 11823645
Büttner, S., Broeskamp, F., Sommer, C., Markaki, M., Habernig, L., Alavian-Ghavanini, A., Carmona-Gutierrez, D., Eisenberg, T., Michael, E., Kroemer, G., Tavernarakis, N., Sigrist, S.J. and Madeo, F. (2014). Spermidine protects against α-synuclein neurotoxicity. Cell Cycle 13: 3903-3908. PubMed ID: 25483063
Deregulation of autophagy has emerged as culprit in diverse neurodegenerative processes. An accumulation of autophagosomes was reported in post-mortem brain tissue from AD, PD, and HD patients as well as in diverse cell culture, fly and mouse models of these diseases. This phenotype most probably results from lysosomal depletion and defective lysosomal clearance. Several genetic factors implicated in the pathology of PD, including αSyn, the leucine-rich repeat kinase LRRK2, the ubiquitin ligase parkin or the PTEN-induced putative kinase PINK1, were shown to differentially influence autophagic processes. Vice versa, modulation of autophagy alters the cellular consequences of these factors’ toxic gain-of-function or loss-of-function, though exact mechanisms remain yet to be elucidated (Büttner, 2014).
In mouse models for AD and PD, overexpression of the pro-autophagic regulator Beclin-1 (Atg6) ameliorates signs of neurodegeneration, and mice conditionally lacking neuronal Atg5 or Atg7 display severe behavior and motor deficits, abundant cellular protein inclusions, and neurodegeneration in several brain regions. These results affirm the importance of basal autophagy to maintain neuronal homeostasis. Accordingly, the pharmacological induction of autophagy is thought to represent a potential strategy to ameliorate neurodegenerative demise. Treatment with rapamycin, for example, which induces autophagy via inactivation of mTOR, was demonstrated to be neuroprotective in several cell culture and animal models of PD, HD and AD. Nevertheless, though autophagy was demonstrated to be a pro-survival process in most studies, and genetic or chemical induction of autophagy generally provides neuroprotection, excessive autophagy has been suggested to contribute to non-apoptotic neuronal cell death. For instance, neurodegeneration after brain injury can be prevented by conditional Atg7 deficiency in mice, and Beclin-1 silencing or chemical inhibition of autophagy protects from cell death in cell culture models of AD. Similarly, pharmacological induction of autophagy via rapamycin turns out to be neurotoxic in alternative scenarios than those where protection is observed. These data indicate that a tightly controlled balance of autophagic processes is mandatory for neuronal survival (Büttner, 2014).
The data presented in this study shows that spermidine-mediated neuroprotection in both D. melanogaster and C. elegans models for αSyn-toxicity is accompanied by an induction of autophagy. Even though the causal involvement of autophagic processes in this protection remains to be elucidated, these results are in line with studies reporting an impairment of autophagy upon high gene doses of αSyn and enhanced neuroprotection upon induction of autophagy by pharmacological and genetic means such as treatment with resveratrol, trehalose, metformin, or rapamycin or overexpression of Beclin-1 or TFEB, the major transcriptional regulator of the autophagic pathway. Altogether these studies place the fine-tuning of autophagy regulation rather than autophagy itself, which as a degradative process is not intrinsically protective, at the core of neuronal viability during PD (Büttner, 2014).
Wiemerslage, L. and Lee, D. (2015). Role of Drosophila calcium channel cacophony in dopaminergic neurodegeneration and neuroprotection. Neurosci Lett 584: 342-346. PubMed ID: 25445363
By manipulating Ca2+ levels either by chelation with EGTA or exclusion via null mutation of a Drosophila Ca2+ channel, cacophony (cac), this study was able to prevent an increase in intracellular Ca2+ concentration in their experiments. EGTA treatment decreases intracellular Ca2+ levels by chelating extracellular Ca2+ and thus, disrupting the concentration gradient of Ca2+ across the cell membrane. In the Ca2+ channel mutant, less Ca2+ enters into the cell during depolarization as cac codes for a major voltage-gated Ca2+ channel in Drosophila. Thus, cells in both conditions should be less susceptible to excitotoxicity due to reduced Ca2+ influxes. Otherwise, excessive Ca2+ enters the cell and is buffered by the mitochondria, and ROS increases. In cultures treated with either EGTA or with mutant Ca2+ channels, DA neurons are protected from MPP+-induced degeneration. Thus, treatments that decrease the intracellular Ca2+ concentration are protective to DA neurons, possibly due to reduced ROS. Indeed, several studies have found a variety of Ca2+ signaling modulations (e.g., a Ca2+ channel blocker fendiline) that are neuroprotective against PD-like insults (Wiemerslage, 2015).
Rodent studies show that L-type channels confer vulnerability in DA neurons in PD. Interestingly, cac is known to possibly code for N, P, and/or Q type while Drosophila DmcaD is homologous to L-type Ca2+ channels. However, results from this study confirm that cac is the major type of Ca2+ channels in the Drosophila cultured neurons as cac mutation results in more than 50% reduction of Ca2+ currents and thus, neuroprotective against MPP+ toxicity. Therefore, the exact Ca2+ channel type may not be a crucial factor as EGTA chelation also rescues DA neurons from MPP+ toxicity. Further, it was shown earlier inhibition of a T-type Ca2+ channel is neuroprotective and that these T-type Ca2+ channels could also be a source of vulnerability in DA neurons (Wiemerslage, 2015).
A puzzling finding is the difference in the number of DA neurons between control groups of different experiments (e.g. TH-GFP versus cac mutant). In these cultures, the typical number of DA neurons is about four DA neurons per 1000 cells. But the cac mutants (cacHC129) and balancer chromosome control (ActGFP) both give a much lower ratio of around one DA neuron per 1000 cells. This difference is attributed to inherent properties within the mutant lines that affect the rates of differentiation and survival of the DA neurons. Indeed, Ca2+ signaling (or at very least – electrical activity which may involve Ca2+ influx) seems involved in the differentiation of DA neurons (Wiemerslage, 2015).
Lastly, it was shown that overexpression of Ca2+ channels removes the neuroprotective effect of the D2 agonist quinpirole. This is likely due to an increase in susceptibility to excitotoxic signaling by increased Ca2+ influx. Previously, it was shown that blocking action potentials can have a neuroprotective effect on DA neurons, and also that the D2 agonist quinpirole has an inhibitory effect on DA neurons by reducing the cellular excitability. Thus, overexpression of wild type cacophony Ca+2 channels can increase intracellular Ca2+ levels and compromise the inhibitory effect of quinpirole. Questions still remain about the signaling pathway between dopamine D2 receptors (D2Rs) and Ca2+ signaling. D2R agonists activate Gαi subunits in Drosophila, which inhibit the production of cAMP. Several transgenic lines are available in Drosophila that manipulate G-protein subunits and cAMP signaling/production. Future studies will examine how intracellular Ca2+ modulates cell death signaling in DA neurons compared to other cell types. One could test intracellular events that modulate the rescue effect of PD therapies using calcium dyes (e.g., Fluo-4 or Fura2) or genetically encoded Ca2+ indicators (GECIs) such as GCaMP – providing direct evidence on intracellular Ca2+ levels for DA neurodegeneration and its rescue. Additionally, a specific GECI tagged to the mitochondrial matrix can be used to examine Ca2+ levels specifically in mitochondria following PD-like treatments and neuroprotective agents such D2 agonists (Wiemerslage, 2015).
Wang, H.S., Toh, J., Ho, P., Tio, M., Zhao, Y. and Tan, E.K. (2014). In vivo evidence of pathogenicity of VPS35 mutations in the Drosophila. Mol Brain 2014 7: 73. PubMed ID: 25288323
Varga, S.J., Qi, C., Podolsky, E. and Lee, D. (2014). A new Drosophila model to study the interaction between genetic and environmental factors in Parkinson's disease. Brain Res 1583: 277-286. PubMed ID: 25130663
West, R.J., Furmston, R., Williams, C.A. and Elliott, C.J. (2015). Neurophysiology of Drosophila models of Parkinson's disease. Parkinsons Dis 2015: 381281. PubMed ID: 25960916
Transcriptional regulation of Msh homologs and potential role in Parkinson's therapy
conjugating enzymes: interactions with ubiquitin protein
ligases with discussion on role in Parkinson's disease
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
Wang, B., Liu, Q., Shan, H., Xia, C. and Liu, Z. (2015). Nrf2 inducer and cncC overexpression attenuates neurodegeneration due to α-synuclein in Drosophila. Biochem Cell Biol 93: 351-358. PubMed ID: 26008822
Kim, M., Semple, I., Kim, B., Kiers, A., Nam, S., Park, H.W., Park, H., Ro, S.H., Kim, J.S., Juhász, G and Lee, J.H. (2015). Drosophila Gyf/GRB10 interacting GYF protein is an autophagy regulator that controls neuron and muscle homeostasis. Autophagy 11: 1358-1372. PubMed ID: 26086452
Zhang, S., Xie, J., Xia, Y., Yu, S., Gu, Z., Feng, R., Luo, G., Wang, D., Wang, K., Jiang, M., Cheng, X., Huang, H., Zhang, W. and Wen, T. (2015). LK6/Mnk2a is a new kinase of alpha synuclein phosphorylation mediating neurodegeneration. Sci Rep 5: 12564. PubMed ID: 26220523
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
West, R.J., Elliott, C.J. and Wade, A.R. (2015). Classification of Parkinson's disease genotypes in Drosophila using spatiotemporal profiling of vision. Sci Rep. 5: 16933. PubMed ID: 26362253
Araujo, S.M., de Paula, M.T., Poetini, M.R., Meichtry, L., Bortolotto, V.C., Zarzecki, M.S., Jesse, C.R. and Prigol, M. (2015). Effectiveness of γ-oryzanol in reducing neuromotor deficits, dopamine depletion and oxidative stress in a Drosophila melanogaster model of Parkinson's disease induced by rotenone. Neurotoxicology [Epub ahead of print]. PubMed ID: 26366809
Kong, Y., Liang, X., Liu, L., Zhang, D., Wan, C., Gan, Z. and Yuan, L. (2015). High throughput sequencing identifies microRNAs mediating α-synuclein toxicity by targeting neuroactive-ligand receptor interaction pathway in early stage of Drosophila Parkinson's disease model. PLoS One 10: e0137432. PubMed ID: 26361355
Date revised: 18 Dec 2015
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