s Parkinson's Disease

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Parkinson's Disease - A disease of mitochondrial dysfunction
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Drosophila genes associated with Parkinson's disease
mask
milton
miro
ND-42
parkin
Pink1
Rab11
sicily
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Autophagy
Gaucher's disease
Mitochondria and mitochondrial function
Neuromuscular junction
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Relevant 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

Abstract
Alpha-synuclein (αSyn) plays a central role in the pathogenesis of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Recent multicenter genetic studies have revealed that mutations in the glucocerebrosidase 1 (GBA1) gene, which are responsible for Gaucher's disease, are strong risk factors for PD and DLB. However, the mechanistic link between the functional loss of glucocerebrosidase (GCase) and the toxicity of αSyn in vivo is not fully understood. This study employed Drosophila models to examine the effect of GCase deficiency on the neurotoxicity of αSyn and its molecular mechanism. Behavioral and histological analyses show that knockdown of the Drosophila homolog of GBA1 (dGBA1) exacerbates the locomotor dysfunction, loss of dopaminergic neurons and retinal degeneration of αSyn-expressing flies. This phenotypic aggravation is associated with the accumulation of proteinase K (PK)-resistant αSyn, rather than with changes in the total amount of αSyn, raising the possibility that glucosylceramide (GlcCer), a substrate of GCase, accelerates the misfolding of αSyn. Indeed, in vitro experiments reveal that GlcCer directly promotes the conversion of recombinant αSyn into the PK-resistant form, representing a toxic conformational change. Similar to dGBA1 knockdown, knockdown of the Drosophila homolog of β-galactosidase (β-Gal) also aggravates locomotor dysfunction of the αSyn flies, and its substrate GM1 ganglioside accelerates the formation of PK-resistant αSyn. These findings suggest that the functional loss of GCase or β-Gal promotes the toxic conversion of αSyn via aberrant interactions between αSyn and their substrate glycolipids, leading to the aggravation of αSyn-mediated neurodegeneration (Suzuki, 2015).

Highlights

  • GCase deficiency increases αSyn toxicity in Drosophila.
  • GCase deficiency accelerates the accumulation of PK-resistant αSyn.
  • Glucosylceramide promotes the formation of PK-resistant αSyn.
  • β-Gal deficiency increases αSyn toxicity in flies and GM1 ganglioside promotes the formation of PK-resistant αSyn.

Discussion
This study investigated the molecular mechanisms underlying the effect of GCase deficiency on αSyn toxicity. It was demonstrated that loss of GCase function exacerbates αSyn neurotoxicity in vivo and that this aggravation is associated with the accelerated accumulation of PK-resistant αSyn. In addition, GlcCer, which  accumulates in the brain of dGBA1 knockdown flies, directly promotes the formation of PK-resistant αSyn, suggesting that the accumulation of GlcCer by GCase deficiency promotes the toxic conversion of αSyn, leading to exacerbation of its neurotoxicity (Suzuki, 2015).

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).

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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

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

Highlights

  • Overexpression of PINK1 or Parkin in rat hippocampal axons decreases mitochondrial movement.
  • PINK1 functions upstream of Parkin to inhibit mitochondrial motility.
  • PINK1 functions upstream of Parkin to inhibit mitochondrial motility in Drosophila larval axons.
  • The PINK1/Parkin pathway induces Miro degradation and releases kinesin from mitochondria.
  • Mitochondrial depolarization causes PINK1 and Parkin to associate with Miro and cause Miro degradation.
  • PINK1 phosphorylates Miro.
  • Ser156 of human Miro mediates the effects of PINK1 and Parkin expression.

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

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

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

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

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

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

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Liang, Z., Chan, H. Y. E., Lee, M. M. and Chan, M. K. (2021). A SUMO1-derived peptide targeting SUMO-interacting motif inhibits alpha-synuclein aggregation. Cell Chem Biol. PubMed ID: 33444530

Abstract

The accumulation of α-synuclein amyloid fibrils in the brain is linked to Parkinson's disease and other synucleinopathies. The intermediate species in the early aggregation phase of α-synuclein are involved in the emergence of amyloid toxicity and considered to be the most neurotoxic. The N-terminal region flanking the non-amyloid-β component domain of α-synuclein has been implicated in modulating its aggregation. This study reports the development of a SUMO1-derived peptide inhibitor (SUMO1(15-55); see Drosophila Sumo), which targets two SUMO-interacting motifs (SIMs) within this aggregation-regulating region and suppresses α-synuclein aggregation. Molecular modeling, site-directed mutagenesis, and binding studies are used to elucidate the mode of interaction, namely, via the binding of either of the two SIM sequences on α-synuclein to a putative hydrophobic binding groove on SUMO1(15-55). Subsequent studies show that SUMO1(15-55) also reduces α-synuclein-induced cytotoxicity in cell-based and Drosophila disease models (Liang, 2021).

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Jewett, K. A., Thomas, R. E., Phan, C. Q., Lin, B., Milstein, G., Yu, S., Bettcher, L. F., Neto, F. C., Djukovic, D., Raftery, D., Pallanck, L. J. and Davis, M. Y. (2021). Glucocerebrosidase reduces the spread of protein aggregation in a Drosophila melanogaster model of neurodegeneration by regulating proteins trafficked by extracellular vesicles. PLoS Genet 17(2): e1008859. PubMed ID: 33539341

Abstract

Abnormal protein aggregation within neurons is a key pathologic feature of Parkinson's disease (PD). The spread of brain protein aggregates is associated with clinical disease progression, but how this occurs remains unclear. Mutations in glucosidase, beta acid 1 (GBA), which encodes glucocerebrosidase (GCase), are the most penetrant common genetic risk factor for PD and dementia with Lewy bodies and associate with faster disease progression. To explore how GBA mutations influence pathogenesis, Drosophila model of GBA deficiency (Gba1b) was created that manifests neurodegeneration and accelerated protein aggregation. Proteomic analysis of Gba1b mutants revealed dysregulation of proteins involved in extracellular vesicle (EV) biology, and altered protein composition of EVs was found from Gba1b mutants. Accordingly, it was hypothesized that GBA may influence pathogenic protein aggregate spread via EVs. It was found that accumulation of ubiquitinated proteins and Ref(2)P, Drosophila homologue of mammalian p62, were reduced in muscle and brain tissue of Gba1b flies by ectopic expression of wildtype GCase in muscle. Neuronal GCase expression also rescued protein aggregation both cell-autonomously in brain and non-cell-autonomously in muscle. Muscle-specific GBA expression reduced the elevated levels of EV-intrinsic proteins and Ref(2)P found in EVs from Gba1b flies. Perturbing EV biogenesis through neutral sphingomyelinase (nSMase), an enzyme important for EV release and ceramide metabolism, enhanced protein aggregation when knocked down in muscle, but did not modify Gba1b mutant protein aggregation when knocked down in neurons. Lipidomic analysis of nSMase knockdown on ceramide and glucosylceramide levels suggested that Gba1b mutant protein aggregation may depend on relative depletion of specific ceramide species often enriched in EVs. Finally, ectopically expressed GCase was identified within isolated EVs. Together, these findings suggest that GCase deficiency promotes accelerated protein aggregate spread between cells and tissues via dysregulated EVs, and EV-mediated trafficking of GCase may partially account for the reduction in aggregate spread (Jewett, 2021).

Many genetic influences of PD have now been identified, and much work has been focused on how these genes lead to protein aggregation through mechanisms such as protein misfolding and autophagy defects. However, none of these genes have been implicated in cell-to-cell spread of pathogenic protein aggregates, which closely correlates with clinical disease progression. Proteomic analysis and non-cell-autonomous rescue of protein aggregation in Gba1b mutants has led to the hypothesize that GBA mutations may influence the rate of propagation of protein aggregates between neurons. This work suggests a link between GBA mutations and faster spread of intracellular protein aggregates via a novel EV-mediated mechanism, possibly explaining the recent clinical finding that GBA mutations accelerate the progression of clinical disease. Using a Drosophila model of GBA deficiency that manifests accelerated protein aggregation, this study found that expressing WT GCase in specific tissues of a GBA-deficient fly can not only rescue protein aggregation cell-autonomously and in distant tissues, but also rescue alterations in protein cargo observed in EVs isolated from Gba1b mutant hemolymph. Interestingly, ectopically expressed WT GCase itself was found within EVs of GBA-deficient flies, suggesting that the non-cell-autonomous rescue due to GCase expression is mediated by both reduction in aggregated proteins in EVs and trafficking of GCase via EVs to distant cells and tissues. Perturbing EV biogenesis through decreased expression of ESCRT-independent nSMase affected protein aggregation in local tissues in a tissue-dependent manner, and further decreased a subset of Cer species already reduced in Gba1b mutants. Interestingly, this subset of Cer species is known to be enriched in EV membranes. Together, these findings suggest that mutations in GBA result in the accelerated spread of protein aggregates through changes in cellular lipid composition and dysregulation of proteins trafficked by EVs (Jewett, 2021).

Although the model of GBA mutations promoting spread of protein aggregates via EVs is novel, the idea that proteostasis can be maintained in a non-cell-autonomous fashion is well supported in the literature. For example, in C. elegans, misfolded α-synuclein accumulating in endo-lysosomal vesicles was found to be transmitted from muscle to the hypodermis, a nearby tissue, for degradation. It is possible that a non-cell-autonomous mechanism is necessary because certain tissues may be more efficient in reducing protein aggregation. This has been previously described, where overexpression of FOXO in Drosophila muscle decreased aging-related protein aggregates in muscle as well as brain and other distant tissues, but FOXO overexpression in adipose tissue was unable to prevent protein aggregation in muscle. In the curren model, overexpressing dGba1b in Drosophila muscle or neuronal tissue prevented accumulation of protein aggregates throughout the organism, however overexpression of WT GCase in midgut and fat body did not significantly reduce protein aggregation in the brain. These discrepancies could be due to tissue-specific biogenesis of EVs, which could depend on factors such as metabolic rate or endovesicular trafficking. Although dGba1b is expressed in all tissues, a second homologue of human GBA1, dGba1a, is expressed only in the midgut. Gba1b mutants retain ~25% expression of dGba1a. Deficiency of dGba1a was found to extend lifespan and does not result in significant accumulation of GlcCer, suggesting that there can be significant tissue-specific differences in function for GCase that could influence EV biogenesis (Jewett, 2021).

The unexpected results from perturbation of EV biogenesis suggest that the EV-mediated regulation of protein aggregation is tissue-specific and complex. Because an increase in EV-intrinsic proteins and alteration of protein cargo were observed in Gba1b mutants, it is anticipated that genetic perturbations decreasing the biogenesis of EVs might rescue protein aggregation non-cell-autonomously by reducing the production of dysregulated EVs. However, decreased expression of ESCRT-independent nSMase in muscle did not rescue protein aggregation in heads, suggesting that a tissue-specific decrease in biogenesis of dysregulated EVs is not sufficient to reduce protein aggregation in the rest of the organism, and the cargo of EVs may need to be corrected to reduce spread of protein aggregation. In contrast, decreased expression of nSMase in the nervous system had no effect on protein aggregation in the head. This difference in outcome in perturbation of EV biogenesis in muscle and neurons could be due to cell-specific compensatory mechanisms or intrinsic metabolic demands and solicits further investigation (Jewett, 2021).

A possible explanation for why decreased muscle expression of nSMase enhanced cell-autonomous protein aggregation and EV protein cargo alterations is that both GCase and nSMase enzymatically produce Cer. If GCase-deficient phenotypes are dependent on a relative reduction in Cer, decreased nSMase expression could exacerbate Gba1b mutant phenotypes. Indeed, lipidomic analysis of alterations in Cer metabolism due to nSMase knockdown revealed a further decrease in a subset of Cer species that were already significantly decreased in Gba1b mutants compared to controls. The further reduction in Cer species due to nSMase knockdown correlates with enhancement of cell-autonomous protein aggregation and EV cargo alterations, suggesting that accelerated protein aggregation in Gba1b mutants is mediated by Cer deficiency rather than GlcCer accumulation, as nSMase knockdown had a much more modest effect on the significantly increased levels of GlcCer species in Gba1b mutants compared to controls (Jewett, 2021).

Cer has been implicated in the composition and biogenesis of EVs, and nSMase knockdown further altered EV cargo in Gba1b mutants, suggesting that decreased Cer levels may directly influence EV biogenesis in Gba1b mutants. However, Cer species were not globally decreased, suggesting that the regulation of Cer metabolism is complex and may be more dependent on specific Cer species. Interestingly, only 1 of the 9 Cer species significantly increased in Gba1b mutants versus controls had a monounsaturated fatty acyl group, while all 5 of the Cer species significantly decreased in Gba1b mutants versus controls had a monounsaturated fatty acyl group, suggesting GBA influences the metabolism of specific subset of Cer species that may be implicated in Gba1b mutant phenotypes. This subset of Cer species is enriched in species with long chain monounsaturated fatty acyl chains. Interestingly, lipids with monounsaturated fatty acyl groups are an abundant component in mammalian exosome membranes. Investigating the alterations in lipid composition of EVs resulting from GCase deficiency and nSMase knockdown will be important in elucidating the role of Cer metabolism in Gba1b mutant phenotypes (Jewett, 2021).

This work suggests that GCase deficiency influences EV biogenesis to promote faster propagation of pathogenic protein aggregates throughout the tissues of an organism, which may be a compensatory response to cell-autonomous lysosomal stress. In the initial characterization of the Drosophila GBA-deficient model, accelerated insoluble ubiquitinated protein aggregates, accumulation of Ref(2)P, and oligomerization of ectopically expressed human α-synuclein in was found Gba1b mutants, suggesting an impairment in lysosomal degradation. A similar GBA-deficient Drosophila model also found evidence of lysosomal dysfunction, including enlarged lysosomes in GBA-deficient brains. However, proteomic analysis of Gba1b mutants did not support a profound impairment in autophagy, but instead suggested dysregulation of EVs with altered protein cargo which could be suppressed locally with knockdown of genes encoding ESCRT machinery important for EV biogenesis. Based on these results, it is believed that the initial observations of increased insoluble ubiquitinated proteins and Ref(2)P in Gba1b mutants are due to lysosomal stress. One possible explanation for the proteomic findings is that there may be a compensatory increase in EV biogenesis and packaging of autophagy substrates within EVs for discard outside of the cell in Gba1b mutants. Such an increase may have prevented detection od defects in autophagy. Upregulation of EV biogenesis may be cell-autonomously neuroprotective in the setting of lysosomal stress, particularly in aggregation-prone neurodegenerative diseases such as PD. It was recently demonstrated in a human neuronal cell culture model of PD that inhibiting macroautophagy protects against α-synuclein-induced cell death by promoting the release of α-synuclein-containing EVs. However, it remains possible that upregulating EV biogenesis may relieve lysosomal stress within cells containing aggregate-prone proteins, while simultaneously promoting the spread of protein aggregates between cells and throughout the organism (Jewett, 2021).

This work suggests a novel mechanism for GBA in reducing the spread of pathogenic protein aggregation from cell-to-cell via regulation of EV protein cargo, but many key questions remain. To better understand the progression of neurodegenerative diseases, it is important to uncover the mechanisms by which GCase deficiency alters EV protein content and biogenesis, identify the specific changes in EVs facilitating propagation of pathogenic protein aggregates, and determine how these changes influence recipient cells internalizing dysregulated EVs. GCase is a critical enzyme in ceramide metabolism, hydrolyzing glucosylceramide into glucose and ceramide. Ceramides are a key component of EV membranes, and alterations in ceramide metabolism due to GCase deficiency may directly influence EV biogenesis and protein cargo trafficked via EVs. Further studies using this Drosophila model and mammalian cell culture models should better elucidate how GCase deficiency alters the protein cargo of EVs to induce propagation of pathogenic protein aggregates, as well as whether endogenous GCase is enzymatically functional when trafficked to distant tissues via EVs. Understanding this mechanism could have broad implications in understanding the pathogenesis of aggregate-prone neurodegenerative diseases and reveal new therapeutic targets to slow or halt disease progression (Jewett, 2021).

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

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

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

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

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

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

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

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

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

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

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

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Li, Y., Chen, W. and Wang, D. (2023). Promotion of mitochondrial fragmentation suppresses the formation of mitochondrial spherical compartmentation in PINK1(B9)Drosophila melanogaster. Biochem Biophys Res Commun 676: 48-57. PubMed ID: 37481943

Mitochondria undergo structural changes reflective of functional statuses. Ultrastructural characterizing of mitochondria is valuable for understanding mitochondrial dysfunction in various pathological conditions. PINK1, a Parkinson's disease (PD) associated gene, plays key roles in maintaining mitochondrial function and integrity. In Drosophila melanogaster, deficiency of PINK1 results in PD-like pathologies due to mitochondrial abnormalities. This study reports the existence of a new type of mitochondrial-membrane deformity, mitochondrial spherical compartmentation (MSC), caused by PINK1 deficiency in Drosophila. The MSC is a three-dimensional spheroid-like mitochondrial membrane structure encompassing nonselective contents. Upregulation of dDrp1, downregulation of dMarf, and upregulation of dArgK1-A-all resulting in mitochondrial fragmentation-were able to suppress the formation of MSC. Furthermore, arginine kinase, only when localizing to the vicinity of mitochondria, induced mitochondrial fragmentation and reversed the MSC phenotype. In summary, this study demonstrates that loss of dPINK1 leads to the formation of mitochondrial-membrane deformity MSC, which responds to mitochondrial dynamics. In addition, these data suggest a new perspective of how phosphagen energy-buffer system might regulate mitochondrial dynamics (Li, 2023).

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Moehlman, A. T., Kanfer, G. and Youle, R. J. (2023). Loss of STING in parkin mutant flies supresses muscle defects and mitochondria damage. PLoS Genet 19(7): e1010828. PubMed ID: Parkinson's Disease (PD) remains unresolved. In the model organism Drosophila melanogaster, loss of the early-onset PD gene parkin (the ortholog of human PRKN) results in impaired climbing ability, damage to the indirect flight muscles, and mitochondrial fragmentation with swelling. These stressed mitochondria have been proposed to activate innate immune pathways through release of damage associated molecular patterns (DAMPs). Parkin-mediated mitophagy is hypothesized to supress mitochondrial damage and subsequent activation of the cGAS/STING innate immunity pathway, but the relevance of this interaction in the fly remains unresolved. Using a combination of genetics, immunoassays, and RNA sequencing, this study investigated a potential role for STING in the onset of parkin-null phenotypes. The findings demonstrate that loss of Drosophila STING in flies rescues the thorax muscle defects and the climbing ability of parkin-/- mutants. loss of STING also supresses the disrupted mitochondrial morphology in parkin-/- flight muscles, suggesting unexpected feedback of STING on mitochondria integrity or activation of a compensatory mitochondrial pathway. In the animals lacking both parkin and sting, PINK1 is activated and cell death pathways are surpressed. These findings support a unique, non-canonical role for Drosophila STING in the cellular and organismal response to mitochondria stress (Moehlman, 2023).

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Maor, G., Dubreuil, R. R. and Feany, M. B. (2023). α-synuclein promotes neuronal dysfunction and death by disrupting the binding of ankyrin to β spectrin. bioRxiv. PubMed ID: 37333277

α-synuclein plays a key role in the pathogenesis of Parkinson's disease and related disorders, but critical interacting partners and molecular mechanisms mediating neurotoxicity are incompletely understood. This study shows that α-synuclein binds directly to β-spectrin. Using males and females in a Drosophila model of α-synuclein-related disorders this study demonstrated that β-spectrin is critical for α-synuclein neurotoxicity. Further, the ankyrin binding domain of β-spectrin is required for α-synuclein binding and neurotoxicity. A key plasma membrane target of ankyrin, Na (+) /K (+) ATPase, is mislocalized when human α-synuclein is expressed in Drosophila. Accordingly, membrane potential is depolarized in α-synuclein transgenic fly brains. The same pathway was examined in human neurons, and it was found that Parkinson's disease patient-derived neurons with a triplication of the α-synuclein locus show disruption of the spectrin cytoskeleton, mislocalization of ankyrin and Na (+) /K (+) ATPase, and membrane potential depolarization. These findings define a specific molecular mechanism by which elevated levels of α-synuclein in Parkinson's disease and related α-synucleinopathies leads to neuronal dysfunction and death.

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Rai, P., Ratnaparkhi, A. and Roy, J. K (2023). Rab11 rescues muscle degeneration and synaptic morphology in the park(13)/+ Parkinson model of Drosophila melanogaster. Brain Res 1816: 148442. PubMed ID: 37302569

Mutation in parkin and pink1 is associated with Parkinson's disease (PD), the most common movement disorder characterized by muscular dysfunction. In a previous study, it was observed that Rab11, a member of the small Ras GTPase family, regulates the mitophagy pathway mediated by Parkin and Pink1 in the larval brain of the Drosophila PD model. sThis study describes that the expression and interaction of Rab11 in the PD model of Drosophila is highly conserved across different phylogenic groups. The loss of function in these two proteins, i.e., Parkin and Pink1, leads to mitochondrial aggregation. Rab11 loss of function results in muscle degeneration, movement disorder and synaptic morphological defects. Overexpression of Rab11 in park13 heterozygous mutant improves muscle and synaptic organization by reducing mitochondrial aggregations and improving cytoskeleton structural organization. The functional relationship was also shown between Rab11 and Brp, a pre-synaptic scaffolding protein, required for synaptic neurotransmission. Using park13 heterozygous mutant and pink1RNAi lines, this study showed reduced expression of Brp and consequently, there were synaptic dysfunctions including impaired synaptic transmission, decreased bouton size, increase in the bouton numbers, and the length of axonal innervations at the larval neuromuscular junction (NMJ). These synaptic alterations were rescued with the over-expression of Rab11 in the park13 heterozygous mutants. In conclusion, this work emphasizes the importance of Rab11 in rescuing muscle degeneration, movement dysfunction and synaptic morphology by preserving mitochondrial function in the PD model of Drosophila (Rai, 2023).

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Hardy, R. E., Chung, I., Yu, Y., Loh, S. H. Y., Morone, N., Soleilhavoup, C., Travaglio, M., Serreli, R., Panman, L., Cain, K., Hirst, J., Martins, L. M., MacFarlane, M. and Pryde, K. R. (2023). The antipsychotic medications aripiprazole, brexpiprazole and cariprazine are off-target respiratory chain complex I inhibitors. Biol Direct 18(1): 43. PubMed ID: 37528429

Antipsychotic drugs are the mainstay of treatment for schizophrenia and provide adjunct therapies for other prevalent psychiatric conditions, including bipolar disorder and major depressive disorder. However, they also induce debilitating extrapyramidal syndromes (EPS), such as Parkinsonism, in a significant minority of patients. The majority of antipsychotic drugs function as dopamine receptor antagonists in the brain while the most recent 'third'-generation, such as aripiprazole, act as partial agonists. Despite showing good clinical efficacy, these newer agents are still associated with EPS in ~ 5 to 15% of patients. However, it is not fully understood how these movement disorders develop. This study combined clinically-relevant drug concentrations with mutliscale model systems to show that aripiprazole and its primary active metabolite induce mitochondrial toxicity inducing robust declines in cellular ATP and viability. Aripiprazole, brexpiprazole and cariprazine were shown to directly inhibit respiratory complex I through its ubiquinone-binding channel. Importantly, all three drugs induced mitochondrial toxicity in primary embryonic mouse neurons, with greater bioenergetic inhibition in ventral midbrain neurons than forebrain neurons. Finally, chronic feeding with aripiprazole resulted in structural damage to mitochondria in the brain and thoracic muscle of adult Drosophila melanogaster consistent with locomotor dysfunction. Taken together, this study showed show that antipsychotic drugs acting as partial dopamine receptor agonists exhibit off-target mitochondrial liabilities targeting complex I.

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Rasheed, M. Z., Khatoon, R., Talat, F., Alam, M. M., Tabassum, H. and Parvez, S. (2023). Melatonin Mitigates Rotenone-Induced Oxidative Stress and Mitochondrial Dysfunction in the Drosophila melanogaster Model of Parkinson's Disease-like Symptoms. ACS Omega 8(8): 7279-7288. PubMed ID: 36872990

Parkinson's disease (PD) is the second most common neurodegenerative disorder; however, its etiology remains elusive. Antioxidants are considered to be a promising approach for decelerating neurodegenerative disease progression owing to extensive examination of the relationship between oxidative stress and neurodegenerative diseases. This study investigated the therapeutic effect of melatonin against rotenone-induced toxicity in the Drosophila model of PD. The 3-5 day old flies were divided into four groups: control, melatonin alone, melatonin and rotenone, and rotenone alone groups. According to their respective groups, flies were exposed to a diet containing rotenone and melatonin for 7 days. Melatonin was found to significantly reduced the mortality and climbing ability of Drosophila because of its antioxidative potency. It alleviated the expression of Bcl 2, tyrosine hydroxylase (TH), NADH dehydrogenase, mitochondrial membrane potential, and mitochondrial bioenergetics and decreased caspase 3 expression in the Drosophila model of rotenone-induced PD-like symptoms. These results indicate the neuromodulatory effect of melatonin, and that it is likely modulated against rotenone-induced neurotoxicity by suppressing oxidative stress and mitochondrial dysfunctions.

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Maitra, U., Conger, J., Owens, M. M. M. and Ciesla, L. (2023). Predicting structural features of selected flavonoids responsible for neuroprotection in a Drosophila model of Parkinson's disease. Neurotoxicology 96: 1-12. PubMed ID: 36822376

Nature-derived bioactive compounds have emerged as promising candidates for the prevention and treatment of diverse chronic illnesses, including neurodegenerative diseases. However, the exact molecular mechanisms underlying their neuroprotective effects remain unclear. Most studies focus solely on the antioxidant activities of natural products which translate to poor outcome in clinical trials. Current therapies against neurodegeneration only provide symptomatic relief, thereby underscoring the need for novel strategies to combat disease onset and progression. This study has employed an environmental toxin-induced Drosophila Parkinson's disease (PD) model as an inexpensive in vivo screening platform to explore the neuroprotective potential of selected dietary flavonoids. A specific group of flavonoids known as flavones displaying protection against paraquat (PQ)-induced neurodegenerative phenotypes was indentified involving reduced survival, mobility defects, and enhanced oxidative stress. Interestingly, the other groups of investigated flavonoids, namely, the flavonones and flavonols failed to provide protection indicating a requirement of specific structural features that confer protection against PQ-mediated neurotoxicity in Drosophila. Based on this screen, the neuroprotective flavones lack a functional group substitution at the C3 and contain α,β-unsaturated carbonyl group. Furthermore, flavones-mediated neuroprotection is not solely dependent on antioxidant properties through nuclear factor erythroid 2-related factor 2 (Nrf2) but also requires regulation of the immune deficiency (IMD) pathway involving NFκB and the negative regulator poor Imd response upon knock-in (Pirk). These data have identified specific structural features of selected flavonoids that provide neuroprotection against environmental toxin-induced PD pathogenesis that can be explored for novel therapeutic interventions (Maitra, 2023).

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Di Leva, F., Filosi, M., Oyston, L., Silvestri, E., Picard, A., Lavdas, A. A., Lobbestael, E., Baekelandt, V., Neely, G. G., Pramstaller, P. P., Hicks, A. A. and Corti, C. (2023). Increased Levels of the Parkinson's Disease-Associated Gene ITPKB Correlate with Higher Expression Levels of alpha-Synuclein, Independent of Mutation Status. Int J Mol Sci 24(3). PubMed ID: 36768321

Autosomal dominant mutations in the gene encoding α-synuclein (SNCA) were the first to be linked with hereditary Parkinson's disease (PD). Duplication and triplication of SNCA has been observed in PD patients, together with mutations at the N-terminal of the protein, among which A30P and A53T influence the formation of fibrils. By overexpressing human α-synuclein in the neuronal system of Drosophila, this study functionally validated the ability of IP3K2, an ortholog of the GWAS identified risk gene, Inositol-trisphosphate 3-kinase B (ITPKB), to modulate α-synuclein toxicity in vivo. ITPKB mRNA and protein levels were also increased in SK-N-SH cells overexpressing wild-type α-synuclein, A53T or A30P mutants. Kinase overexpression was detected in the cytoplasmic and in the nuclear compartments in all α-synuclein cell types. By quantifying mRNAs in the cortex of PD patients, higher levels of ITPKB mRNA were observed when SNCA was expressed more (p < 0.05), compared to controls. A positive correlation was also observed between SNCA and ITPKB expression in the cortex of patients, which was not seen in the controls. This observation was replicated in a public dataset. These data, generated in SK-N-SH cells and in cortex from PD patients, show that the expression of α-synuclein and ITPKB is correlated in pathological situations (Di Leva, 2023).

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Huang, Y., Wen, D., Yuan, Y. and Chen, W. (2023). Gene Set Enrichment Analysis and Genetic Experiment Reveal Changes in Cell Signaling Pathways Induced by alpha-Synuclein Overexpression. Biomedicines 11(2). PubMed ID: 36830800

Abnormal accumulation of alpha synuclein (α-Syn) in sporadic and familial Parkinson's disease (PD) may be a key step in its pathogenesis. In this study, the expression matrix of the GSE95427 dataset after α-Syn overexpression in human glioma cell line H4 was obtained from the GEO database. The Gene Set Enrichment Analysis (GSEA) method was used to reanalyze this dataset to evaluate the possible functions of åalpha;-Syn. The results showed that the tumor necrosis factor alpha (TNF-α) signal was significantly activated in α-Syn-overexpressing cells, and oxidative phosphorylation signal, extracellular matrix signal, cell cycle related signal and fatty acid metabolism signal were significantly inhibited. Moreover, the alpha;-Syn-expressing transgenic Drosophila model of Parkinson's disease and knocked-down eiger, a TNF superfamily ligand homologue, indicated that the TNF-α pathway plays a role in the common pathogenesis of synucleinopathies. This analysis based on GSEA data provides more clues for a better understanding of α-Syn function (Huang, 2023).

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Jacquemyn, J., Kuenen, S., Swerts, J., Pavie, B., Vijayan, V., Kilic, A., Chabot, D., Wang, Y. C., Schoovaerts, N., Corthout, N. and Verstreken, P. (2023). Parkinsonism mutations in DNAJC6 cause lipid defects and neurodegeneration that are rescued by Synj1. CNPJ Parkinsons Dis 9(1): 19. PubMed ID: 36739293

Recent evidence links dysfunctional lipid metabolism to the pathogenesis of Parkinson's disease, but the mechanisms are not resolved. This study generated a new Drosophila knock-in model of DNAJC6/Auxilin and found that the pathogenic mutation causes synaptic dysfunction, neurological defects and neurodegeneration, as well as specific lipid metabolism alterations. In these mutants, membrane lipids containing long-chain polyunsaturated fatty acids, including phosphatidylinositol lipid species that are key for synaptic vesicle recycling and organelle function, are reduced. Overexpression of another protein mutated in Parkinson's disease, Synaptojanin-1, known to bind and metabolize specific phosphoinositides, rescues the DNAJC6/Auxilin lipid alterations, the neuronal function defects and neurodegeneration. This work reveals a functional relation between two proteins mutated in Parkinsonism and implicates deregulated phosphoinositide metabolism in the maintenance of neuronal integrity and neuronal survival (Jacquemyn, 2023).

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Popovic, R., Mukherjee, A., Leal, N. S., Morris, L., Yu, Y., Loh, S. H. Y. and Miguel Martins, L. (2023). Blocking dPerk in the intestine suppresses neurodegeneration in a Drosophila model of Parkinson's disease. Cell Death Dis 14(3): 206. PubMed ID: 36949073

Parkinson's disease (PD) is characterised by selective death of dopaminergic (DA) neurons in the midbrain and motor function impairment. Gastrointestinal issues often precede motor deficits in PD, indicating that the gut-brain axis is involved in the pathogenesis of this disease. The features of PD include both mitochondrial dysfunction and activation of the unfolded protein response (UPR) in the endoplasmic reticulum (ER). PINK1 is a mitochondrial kinase involved in the recycling of defective mitochondria, and PINK1 mutations cause early-onset PD. Like PD patients, pink1 mutant Drosophila show degeneration of DA neurons and intestinal dysfunction. These mutant flies also lack vital proteins due to sustained activation of the kinase R-like endoplasmic reticulum kinase (dPerk), a kinase that induces the UPR. This study investigated the role of dPerk in intestinal dysfunction. Intestinal expression of dPerk impairs mitochondrial function, induces cell death, and decreases lifespan. This study found that suppressing dPerk in the intestine of pink1-mutant flies rescues intestinal cell death and is neuroprotective. It is concluded that in a fly model of PD, blocking gut-brain transmission of UPR-mediated toxicity, is neuroprotective (Popovic, 2023).

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Li, W., Pan, X., Li, M., Ling, L., Zhang, M., Liu, Z., Zhang, K., Guo, J. and Wang, H. (2023). Impact of age on the rotenone-induced sporadic Parkinson's disease model using Drosophila melanogaster. Neurosci Lett 805: 137187. PubMed ID: 36921666

Rotenone, a naturally occurring toxin, has been used to induce sporadic Parkinson's disease (PD) in Drosophila melanogaster for decades. However, the age of flies varies considerably between studies in this model. To investigate the impact of age on the rotenone-induced PD model, male flies were collected at the age of 1, 5, 7, and 10 days post-eclosion, respectively. Then, flies were immediately exposed to a feeding medium supplemented with 250 μM rotenone for seven days. The motor ability of Drosophila was detected by negative geotaxis assay, and the number of dopamine (DA) neurons and tyrosine hydroxylase (TH) expression levels were evaluated. The results showed that both the motor deficits and mortality increased with age. The flies older than five days showed typical PD features, including the loss of DA neurons, decreased TH expression levels, and decreased locomotive ability. However, 1-day-old flies displayed an unstable motor deficit and little TH expression changes after seven days of rotenone exposure. Lastly, after 7 days of exposure to rotenone, the death rate of flies rapidly increased with increasing starting age. The death rates of 1-, 5-, 7-, and 10-days old flies were 10.0%, 22.8%, 41.5%, and 50.4%, respectively. The findings of this study suggest that age is a crucial factor impacting the Drosophila PD model. This information provides a reference for the age selection to use this model for future studies (Li, 202).

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Chaves, N. S. G., Janner, D. E., Poetini, M. R., Fernandes, E. J., de Almeida, F. P., Musachio, E. A. S., Reginaldo, J. C., Dahleh, M. M. M., de Carvalho, A. S., Leimann, F. V., Gonçalves, O. H., Ramborger, B. P., Roehrs, R., Prigol, M. and Guerra, G. P. (2023). β-carotene-loaded nanoparticles protect against neuromotor damage, oxidative stress, and dopamine deficits in a model of Parkinson's disease in Drosophila melanogaster. Comp Biochem Physiol C Toxicol Pharmacol 268: 109615. PubMed ID: 36940893

Abstract

β-carotene-loaded nanoparticles improves absorption by increasing bioavailability. The Drosophila melanogaster model of Parkinson's disease must be helpful in investigating potential neuroprotective effects. Four groups of four-day-old flies were exposed to: (1) control; (2) diet containing rotenone (500 &mu,M); (3) &bets;-carotene-loaded nanoparticles (20 μM); (4) β-carotene-loaded nanoparticles and rotenone for 7 days. Then, the percentage of survival, geotaxis tests, open field, aversive phototaxis and food consumption were evaluated. At the end of the behaviors, the analyses of the levels of reactive species (ROS), thiobarbituric acid reactive substances (TBARS), catalase (CAT) and superoxide dismutase (SOD) activity was carried out, as well as an evaluation of the levels of dopamine and acetylcholinesterase (AChE) activity, in the head of flies. Nanoparticles loaded with β-carotene were able to improve motor function, memory, survival and also restored the oxidative stress indicators (CAT, SOD, ROS and TBARS), dopamine levels, AChE activity after exposure to rotenone. Overall, nanoparticles loaded with β-carotene showed significant neuroprotective effect against damage induced by the Parkinson-like disease model, emerging as a possible treatment. Overall, β-carotene-loaded nanoparticles presented significant neuroprotective effect against damage induced by model of Parkinson-like disease, emerging as a possible treatment (Chaves, 2023).

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Ciampelli, C., Galleri, G., Puggioni, S., Fais, M., Iannotta, L., Galioto, M., Becciu, M., Greggio, E., Bernardoni, R., Crosio, C. and Iaccarino, C. (2023). Inhibition of the Exocyst Complex Attenuates the LRRK2 Pathological Effects. Int J Mol Sci 24(16). PubMed ID: 37628835

Abstract

Pathological mutations in leucine-rich repeat kinase 2 (LRRK2) gene are the major genetic cause of Parkinson's disease (PD). Multiple lines of evidence link LRRK2 to the control of vesicle dynamics through phosphorylation of a subset of RAB proteins. However, the molecular mechanisms underlying these processes are not fully elucidated. Previous work has demonstrated that LRRK2 increases the exocyst complex assembly by Sec8 interaction, one of the eight members of the exocyst complex, and that Sec8 over-expression mitigates the LRRK2 pathological effect in PC12 cells. This study extended this analysis using LRRK2 drosophila models and show that the LRRK2-dependent exocyst complex assembly increase is downstream of RAB phosphorylation. Moreover, exocyst complex inhibition rescues mutant LRRK2 pathogenic phenotype in cellular and Drosophila models. Finally, prolonged exocyst inhibition leads to a significant reduction in the LRRK2 protein level, overall supporting the role of the exocyst complex in the LRRK2 pathway. Taken together, this study suggests that modulation of the exocyst complex may represent a novel therapeutic target for PD (Ciampelli, 2023).

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Rasheed, M. Z., Khatoon, R., Talat, F., Alam, M. M., Tabassum, H. and Parvez, S. (2023). Melatonin Mitigates Rotenone-Induced Oxidative Stress and Mitochondrial Dysfunction in the Drosophila melanogaster Model of Parkinson's Disease-like Symptoms. ACS Omega 8(8): 7279-7288. PubMed ID: 36872990

Abstract
Parkinson's disease (PD) is the second most common neurodegenerative disorder; however, its etiology remains elusive. Antioxidants are considered to be a promising approach for decelerating neurodegenerative disease progression owing to extensive examination of the relationship between oxidative stress and neurodegenerative diseases. This study investigated the therapeutic effect of melatonin against rotenone-induced toxicity in the Drosophila model of PD. The 3-5 day old flies were divided into four groups: control, melatonin alone, melatonin and rotenone, and rotenone alone groups. According to their respective groups, flies were exposed to a diet containing rotenone and melatonin for 7 days. Melatonin was found to significantly reduced the mortality and climbing ability of Drosophila because of its antioxidative potency. It alleviated the expression of Bcl 2, tyrosine hydroxylase (TH), NADH dehydrogenase, mitochondrial membrane potential, and mitochondrial bioenergetics and decreased caspase 3 expression in the Drosophila model of rotenone-induced PD-like symptoms. These results indicate the neuromodulatory effect of melatonin, and that it is likely modulated against rotenone-induced neurotoxicity by suppressing oxidative stress and mitochondrial dysfunctions.

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Maitra, U., Conger, J., Owens, M. M. M. and Ciesla, L. (2023). Predicting structural features of selected flavonoids responsible for neuroprotection in a Drosophila model of Parkinson's disease. Neurotoxicology 96: 1-12. PubMed ID: 36822376

Abstract
Nature-derived bioactive compounds have emerged as promising candidates for the prevention and treatment of diverse chronic illnesses, including neurodegenerative diseases. However, the exact molecular mechanisms underlying their neuroprotective effects remain unclear. Most studies focus solely on the antioxidant activities of natural products which translate to poor outcome in clinical trials. Current therapies against neurodegeneration only provide symptomatic relief, thereby underscoring the need for novel strategies to combat disease onset and progression. This study has employed an environmental toxin-induced Drosophila Parkinson's disease (PD) model as an inexpensive in vivo screening platform to explore the neuroprotective potential of selected dietary flavonoids. A specific group of flavonoids known as flavones displaying protection against paraquat (PQ)-induced neurodegenerative phenotypes was indentified involving reduced survival, mobility defects, and enhanced oxidative stress. Interestingly, the other groups of investigated flavonoids, namely, the flavonones and flavonols failed to provide protection indicating a requirement of specific structural features that confer protection against PQ-mediated neurotoxicity in Drosophila. Based on this screen, the neuroprotective flavones lack a functional group substitution at the C3 and contain α,β-unsaturated carbonyl group. Furthermore, flavones-mediated neuroprotection is not solely dependent on antioxidant properties through nuclear factor erythroid 2-related factor 2 (Nrf2) but also requires regulation of the immune deficiency (IMD) pathway involving NFκB and the negative regulator poor Imd response upon knock-in (Pirk). These data have identified specific structural features of selected flavonoids that provide neuroprotection against environmental toxin-induced PD pathogenesis that can be explored for novel therapeutic interventions.

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Kang, K. H., Han, J. E., Kim, H., Kim, S., Hong, Y. B., Yun, J., Nam, S. H., Choi, B. O. and Koh, H. (2023). PINK1 and Parkin Ameliorate the Loss of Motor Activity and Mitochondrial Dysfunction Induced by Peripheral Neuropathy-Associated HSPB8 Mutants in Drosophila Models. Biomedicines 11(3). PubMed ID: 36979812

Abstract
Charcot-Marie-Tooth disease (CMT) is a group of inherited peripheral nerve disorders characterized by progressive muscle weakness and atrophy, sensory loss, foot deformities and steppage gait. Missense mutations in the gene encoding the small heat shock protein HSPB8 (HSP22) have been associated with hereditary neuropathies, including CMT. HSPB8 is a member of the small heat shock protein family sharing a highly conserved α-crystallin domain that is critical to its chaperone activity. This study modeled HSPB8 mutant-induced neuropathies in Drosophila. The overexpression of human HSPB8 mutants in Drosophila neurons produced no significant defect in fly development but led to a partial reduction in fly lifespan. Although these HSPB8 mutant genes failed to induce sensory abnormalities, they reduced the motor activity of flies and the mitochondrial functions in fly neuronal tissue. The motor defects and mitochondrial dysfunction were successfully restored by PINK1 and parkin, which are Parkinson's disease-associated genes that have critical roles in maintaining mitochondrial function and integrity. Consistently, kinetin riboside, a small molecule amplifying PINK1 activity, also rescued the loss of motor activity in the HSPB8 mutant model.

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Li, W., Pan, X., Li, M., Ling, L., Zhang, M., Liu, Z., Zhang, K., Guo, J. and Wang, H. (2023). Impact of age on the rotenone-induced sporadic Parkinson's disease model using Drosophila melanogaster. A Neurosci Lett 805: 137187. PubMed ID: 36921666

Abstract
Rotenone, a naturally occurring toxin, has been used to induce sporadic Parkinson's disease (PD) in Drosophila melanogaster for decades. However, the age of flies varies considerably between studies in this model. To investigate the impact of age on the rotenone-induced PD model, male flies were collected at the age of 1, 5, 7, and 10 days post-eclosion, respectively. Then, flies were immediately exposed to a feeding medium supplemented with 250 μM rotenone for seven days. The motor ability of Drosophila was detected by negative geotaxis assay, and the number of dopamine (DA) neurons and tyrosine hydroxylase (TH) expression levels were evaluated. The results showed that both the motor deficits and mortality increased with age. The flies older than five days showed typical PD features, including the loss of DA neurons, decreased TH expression levels, and decreased locomotive ability. However, 1-day-old flies displayed an unstable motor deficit and little TH expression changes after seven days of rotenone exposure. Lastly, after 7 days of exposure to rotenone, the death rate of flies rapidly increased with increasing starting age. The death rates of 1-, 5-, 7-, and 10-days old flies were 10.0%, 22.8%, 41.5%, and 50.4%, respectively. The findings of this study suggest that age is a crucial factor impacting the Drosophila PD model. This information provides a reference for the age selection to use this model for future studies.

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Popovic, R., Mukherjee, A., Leal, N. S., Morris, L., Yu, Y., Loh, S. H. Y. and Miguel Martins, L. (2023). Blocking dPerk in the intestine suppresses neurodegeneration in a Drosophila model of Parkinson's disease. Cell Death Dis 14(3): 206. PubMed ID: 36949073

Abstract
Parkinson's disease (PD) is characterised by selective death of dopaminergic (DA) neurons in the midbrain and motor function impairment. Gastrointestinal issues often precede motor deficits in PD, indicating that the gut-brain axis is involved in the pathogenesis of this disease. The features of PD include both mitochondrial dysfunction and activation of the unfolded protein response (UPR) in the endoplasmic reticulum (ER). PINK1 is a mitochondrial kinase involved in the recycling of defective mitochondria, and PINK1 mutations cause early-onset PD. Like PD patients, pink1 mutant Drosophila show degeneration of DA neurons and intestinal dysfunction. These mutant flies also lack vital proteins due to sustained activation of the kinase R-like endoplasmic reticulum kinase (dPerk), a kinase that induces the UPR. This study investigated the role of dPerk in intestinal dysfunction. Intestinal expression of dPerk impairs mitochondrial function, induces cell death, and decreases lifespan. This study found that suppressing dPerk in the intestine of pink1-mutant flies rescues intestinal cell death and is neuroprotective. It is concluded that in a fly model of PD, blocking gut-brain transmission of UPR-mediated toxicity, is neuroprotective.

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Chaves, N. S. G., Janner, D. E., Poetini, M. R., Fernandes, E. J., de Almeida, F. P., Musachio, E. A. S., Reginaldo, J. C., Dahleh, M. M. M., de Carvalho, A. S., Leimann, F. V., Gonçalves, O. H., Ramborger, B. P., Roehrs, R., Prigol, M. and Guerra, G. P. (2023). β-carotene-loaded nanoparticles protect against neuromotor damage, oxidative stress, and dopamine deficits in a model of Parkinson's disease in Drosophila melanogaster. Comp Biochem Physiol C Toxicol Pharmacol 268: 109615. PubMed ID: 36940893

Abstract
β-carotene-loaded nanoparticles improves absorption by increasing bioavailability. The Drosophila melanogaster model of Parkinson's disease must be helpful in investigating potential neuroprotective effects. Four groups of four-day-old flies were exposed to: (1) control; (2) diet containing rotenone (500 &mu,M); (3) &bets;-carotene-loaded nanoparticles (20 μM); (4) β-carotene-loaded nanoparticles and rotenone for 7 days. Then, the percentage of survival, geotaxis tests, open field, aversive phototaxis and food consumption were evaluated. At the end of the behaviors, the analyses of the levels of reactive species (ROS), thiobarbituric acid reactive substances (TBARS), catalase (CAT) and superoxide dismutase (SOD) activity was carried out, as well as an evaluation of the levels of dopamine and acetylcholinesterase (AChE) activity, in the head of flies. Nanoparticles loaded with β-carotene were able to improve motor function, memory, survival and also restored the oxidative stress indicators (CAT, SOD, ROS and TBARS), dopamine levels, AChE activity after exposure to rotenone. Overall, nanoparticles loaded with β-carotene showed significant neuroprotective effect against damage induced by the Parkinson-like disease model, emerging as a possible treatment. Overall, β-carotene-loaded nanoparticles presented significant neuroprotective effect against damage induced by model of Parkinson-like disease, emerging as a possible treatment.

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Rosado-Ramos, R., Pocas, G. M., Marques, D., Foito, A., D, M. S., Lopes-da-Silva, M., Goncalves, L. G., Menezes, R., Ottens, M., Stewart, D., Ibanez de Opakua, A., Zweckstetter, M., Seabra, M. C., Mendes, C. S., Outeiro, T. F., Domingos, P. M. and Santos, C. N. (2023). Genipin prevents alpha-synuclein aggregation and toxicity by affecting endocytosis, metabolism and lipid storage. Nat Commun 14(1): 1918. PubMed ID: 37024503

Abstract
Parkinson's Disease (PD) is a common neurodegenerative disorder affecting millions of people worldwide for which there are only symptomatic therapies. Small molecules able to target key pathological processes in PD have emerged as interesting options for modifying disease progression. It has been previously shown that a (poly)phenol-enriched fraction (PEF) of Corema album L. leaf extract modulates central events in PD pathogenesis, namely α-synuclein (αSyn) toxicity, aggregation and clearance. PEF was now subjected to a bio-guided fractionation with the aim of identifying the critical bioactive compound. Genipin, an iridoid, which relieves αSyn toxicity and aggregation was identified. Furthermore, genipin promotes metabolic alterations and modulates lipid storage and endocytosis. Importantly, genipin was able to prevent the motor deficits caused by the overexpression of αSyn in a Drosophila melanogaster model of PD. These findings widens the possibility for the exploitation of genipin for PD therapeutics.

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O'Hanlon, M. E., Tweedy, C., Scialo, F., Bass, R., Sanz, A. and Smulders-Srinivasan, T. K. (2022). Mitochondrial electron transport chain defects modify Parkinson's disease phenotypes in a Drosophila model. Neurobiol Dis 171: 105803. PubMed ID: 35764292

Abstract
Defects in Mitochondria have been implicated in Parkinson's disease (PD). A targeted heterozygous enhancer/suppressor screen was performed using Drosophila mutations within mitochondrial electron transport chain (ETC) genes against a null PD mutation in parkin. The interactions were assessed by climbing assays at 2-5 days as an indicator of motor function. A strong enhancer mutation in COX5A was examined further for L-dopa rescue, oxygen consumption, mitochondrial content, and reactive oxygen species. A later timepoint of 16-20 days was also investigated for both COX5A and a suppressor mutation in cyclope. Mutations in individual genes for subunits within the mitochondrial respiratory complexes have interactions with parkin, while others do not, irrespective of complex. One intriguing mutation in a complex IV subunit (cyclope) shows a suppressor rescue effect at early time points, improving the gross motor defects caused by the PD mutation, providing a strong candidate for drug discovery. Most mutations, however, show varying degrees of enhancement or slight suppression of the PD phenotypes. Thus, individual mitochondrial mutations within different oxidative phosphorylation complexes have different interactions with PD with regard to degree and direction. Upon further investigation of the strongest enhancer (COX5A), the mechanism by which these interactions occur initially does not appear to be based on defects in ATP production, but rather may be related to increased levels of reactive oxygen species. This work highlights some key subunits potentially involved in mechanisms underlying PD pathogenesis, implicating ETC complexes other than complex I in PD.

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Xue, J., Zhu, Y., Wei, L., Huang, H., Li, G., Huang, W., Zhu, H. and Duan, R. (2022). Loss of Drosophila NUS1 results in cholesterol accumulation and Parkinson's disease-related neurodegeneration. Faseb j 36(7): e22411. PubMed ID: 35695805

Abstract

NgBR is the Nogo-B receptor, encoded by NUS1 gene. As NgBR contains a C-terminal domain that is similar to cis-isoprenyltransferase (cis-IPTase), NgBR was speculated to stabilize nascent Niemann-Pick type C 2 (NPC2) to facilitate cholesterol transport out of lysosomes. Mutations in the NUS1 were known as risk factors for Parkinson's disease (PD). In a previous study, it was shown that knockdown of Drosophila NUS1 orthologous gene tango14 causes decreased climbing ability, loss of dopaminergic neurons, and decreased dopamine contents. In this study, tango14 mutant flies were generated with a mutation in the C-terminal enzyme activity region using CRISPR/Cas9. tango14 mutant showed a reduced lifespan with locomotive defects and cholesterol accumulation in Malpighian tubules and brains, especially in dopaminergic neurons. Multilamellar bodies were found in tango14 mutants using electron microscopy. Neurodegenerative-related brain vacuolization was also detected in tango14 knockdown flies in an age-dependent manner. In addition, tango14 knockdown increased α-synuclein (α-syn) neurotoxicity in α-syn-overexpressing flies, with decreased locomotive activities, dopamine contents, and the numbers of dopaminergic neurons in aging flies. Thus, these observations suggest a role of NUS1, the ortholog of tango14, in PD-related pathogenesis.

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Neves, P. F. R., Milanesi, B. B., Paz, L. V., de Miranda Monteiro, V. A. C., Neves, L. T., da Veiga, L. C., da Silva, R. B., Sulzbach, J. H., Knijkik, G. P., de Revoredo Ribeiro, E. C., de Souza Silva, E. L., Vieira, M. Q., Bagatini, P. B., Wieck, A., Mestriner, R. G. and Xavier, L. L. (2022). Age-related tolerance to paraquat-induced parkinsonism in Drosophila melanogaster. Toxicol Lett 361: 43-53. PubMed ID: 35367327

Abstract

Paraquat (PQ) is a widely used herbicide that can cross the dopaminergic neuronal membrane, accumulate in mitochondria and damage complex I of the electron transport chain, leading to neuronal death. In Drosophila melanogaster, PQ exposure leads to the development of parkinsonism and is a classical model for studying Parkinson's Disease (PD). Muscle mitochondrial dysfunction, affecting survival and locomotion, is described in familial PD in D. melanogaster mutants. However, no study has shown the effects of PQ-induced parkinsonism in D. melanogaster regarding muscle ultrastructure and locomotor behavior at different ages. Thus, this study evaluated survival, locomotion, and morphological parameters of mitochondria and myofibrils using transmission electron microscopy in 2 and 15-day-old D. melanogaster, treated with different PQ doses: control, 10, 50, 100, 150, and 200 mM. PQ100mM presented 100% lethality in 15-day-old D. melanogaster, while in 2-day-old animals PQ150mM produced 20% lethality. Bradykinesia was only observed in 15-day-old D. melanogaster treated with PQ10 mM and PQ50 mM. However, these results are unlikely to be associated with changes to morphology. Taken together, these data indicate pathophysiological differences between PQ-induced parkinsonism and familial parkinsonism in D. melanogaster (resultant from gene mutations), demonstrating for the first time a differential susceptibility to PQ in two developmental stages.

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O'Hanlon, M. E., Tweedy, C., Scialo, F., Bass, R., Sanz, A. and Smulders-Srinivasan, T. K. (2022). Mitochondrial electron transport chain defects modify Parkinson's disease phenotypes in a Drosophila model. Faseb j 36(8): e22432. Neurobiol Dis 171: 105803. PubMed ID: 35764292

Abstract

Mitochondrial defects have been implicated in Parkinson's disease (PD) More evidence of mitochondrial involvement arose when many of the genes whose mutations caused inherited PD were discovered to be subcellularly localized to mitochondria or have mitochondrial functions. The aim of this study was to better understand mitochondrial dysfunction in PD by evaluating mitochondrial respiratory complex mutations in a Drosophila model of PD. This study conducted a targeted heterozygous enhancer/suppressor screen using Drosophila mutations within mitochondrial electron transport chain (ETC) genes against a null PD mutation in parkin. The interactions were assessed by climbing assays at 2-5 days as an indicator of motor function. A strong enhancer mutation in COX5A was examined further for L-dopa rescue, oxygen consumption, mitochondrial content, and reactive oxygen species. A later timepoint of 16-20 days was also investigated for both COX5A and a suppressor mutation in cyclope. Mutations in individual genes for subunits within the mitochondrial respiratory complexes were found to have interactions with parkin, while others do not, irrespective of complex. One intriguing mutation in a complex IV subunit (cyclope) shows a suppressor rescue effect at early time points, improving the gross motor defects caused by the PD mutation, providing a strong candidate for drug discovery. Most mutations, however, show varying degrees of enhancement or slight suppression of the PD phenotypes. Thus, individual mitochondrial mutations within different oxidative phosphorylation complexes have different interactions with PD with regard to degree and direction. Upon further investigation of the strongest enhancer (COX5A), the mechanism by which these interactions occur initially does not appear to be based on defects in ATP production, but rather may be related to increased levels of reactive oxygen species. This work highlights some key subunits potentially involved in mechanisms underlying PD pathogenesis, implicating ETC complexes other than complex I in PD.

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Fevga, C., Tesson, C., Mascaro, A. C., ..., Mandemakers, W., Brice, A. and Bonifati, V. (2022). PTPA variants and impaired PP2A activity in early-onset parkinsonism with intellectual disability. Brain. PubMed ID: 36073231

Abstract

The protein phosphatase 2A complex (PP2A), the major Ser/Thr phosphatase in the brain, is involved in a number of signaling pathways and functions, including the regulation of crucial proteins for neurodegeneration, such as alpha-synuclein, tau, and LRRK2. This study reports the identification of variants in the PTPA/PPP2R4 gene, encoding a major PP2A activator, in two families with early-onset parkinsonism and intellectual disability. Functional studies were performed on the disease-associated variants in cultured cells and knock-down of ptpa in Drosophila melanogaster. A homozygous PTPA variant, c.893T > G (p.Met298Arg), was identified in patients from a South African family with early-onset parkinsonism and intellectual disability. Screening of a large series of additional families yielded a second homozygous variant, c.512C > A (p.Ala171Asp), was identified in a Libyan family with a similar phenotype. Both variants co-segregate with disease in the respective families. The affected subjects display juvenile-onset parkinsonism and intellectual disability. The motor symptoms were responsive to treatment with levodopa and deep brain stimulation of the subthalamic nucleus. In overexpression studies, both the PTPA p.Ala171Asp and p.Met298Arg variants were associated with decreased PTPA RNA stability and decreased PTPA protein levels; the p.Ala171Asp variant additionally displayed decreased PTPA protein stability. Crucially, expression of both variants was associated with decreased PP2A complex levels and impaired PP2A phosphatase activation. PTPA ortholog knock-down in Drosophila neurons induced a significant impairment of locomotion in the climbing test. This defect was age-dependent and fully reversed by L-DOPA treatment. It is conclude that bi-allelic missense PTPA variants associated with impaired activation of the PP2A phosphatase cause autosomal recessive early-onset parkinsonism with intellectual disability. Thee findings might also provide new insights for understanding the role of the PP2A complex in the pathogenesis of more common forms of neurodegeneration.

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Zhou, Z. D., Saw, W. T., Ho, P. G. H., Zhang, Z. W., Zeng, L., Chang, Y. Y., Sun, A. X. Y., Ma, D. R., Wang, H. Y., Zhou, L., Lim, K. L. and Tan, E. K. (2022). The role of tyrosine hydroxylase-dopamine pathway in Parkinson's disease pathogenesis. Cell Mol Life Sci 79(12): 599. PubMed ID: 36409355

Abstract

Parkinson's disease (PD) is characterized by selective and progressive dopamine (DA) neuron loss in the substantia nigra and other brain regions, with the presence of Lewy body formation. Most PD cases are sporadic, whereas monogenic forms of PD have been linked to multiple genes, including Leucine kinase repeat 2 (LRRK2) and PTEN-induced kinase 1 (PINK1), two protein kinase genes involved in multiple signaling pathways. There is increasing evidence to suggest that endogenous DA and DA-dependent neurodegeneration have a pathophysiologic role in sporadic and familial PD. This study generated patient-derived dopaminergic neurons and human midbrain-like organoids (hMLOs), transgenic (TG) mouse and Drosophila models, expressing both mutant and wild-type (WT) LRRK2 and PINK1. Using these models,the effect of LRRK2 and PINK1 on tyrosine hydroxylase (TH)-DA pathway was studied. PD-linked LRRK2 mutations were able to modulate TH-DA pathway, resulting in up-regulation of DA early in the disease which subsequently led to neurodegeneration. The LRRK2-induced DA toxicity and degeneration were abrogated by wild-type (WT) PINK1 (but not PINK1 mutations), and early treatment with a clinical-grade drug, α-methyl-L-tyrosine (α-MT), a TH inhibitor, was able to reverse the pathologies in human neurons and TG Drosophila models. Opposing effects between LRRK2 and PINK1 on TH expression were also identified, suggesting that functional balance between these two genes may regulate the TH-DA pathway. These findings highlight the vital role of the TH-DA pathway in PD pathogenesis. LRRK2 and PINK1 have opposing effects on the TH-DA pathway, and its balance affects DA neuron survival. LRRK2 or PINK1 mutations can disrupt this balance, promoting DA neuron demise. These findings provide support for potential clinical trials using TH-DA pathway inhibitors in early or prodromic PD.

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Tibashailwa, N., Stephano, F., Shadrack, D. M., Munissi, J. J. E. and Nyandoro, S. S. (2022). Neuroprotective potential of cinnamoyl derivatives against Parkinson's disease indicators in Drosophila melanogaster and in silico models. Neurotoxicology. PubMed ID: 36410467

Abstract

Parkinson's disease (PD) is a movement disorder resulting from the loss of dopaminergic neurons over time. While there is no cure for PD, available conventional therapies aid to manage the motor symptoms. Natural products (NPs) derived from plants are among the most potent alternative therapies for PD. This study explored the neuroprotective potential of selected cinnamoyl derivatives namely toussaintine A (1), E-toussaintine E (2), asperphenamate (3) and julocrotine (4) against PD indicators using rotenone-challenged Drosophila melanogaster and in silico models. The compounds were first assessed for their toxicity preceding treatment experiments. Adult flies (aged 1-4 days) were exposed to varying concentrations of the compounds for 7 days. During the experiment, the mortality of flies was observed, and the lethal concentration (LC(50)) of each tested compound was determined. The LC(50) values were found to be 50.1, 55.6, 513.5, and 101.0μM for compounds 1, 2, 3, and 4, respectively. For seven days, flies were exposed to 500μM of rotenone and co-fed with a chosen dose of 40μM of each test compound in the diet. Using a negative geotaxis test, rotenone-challenged flies exhibited compromised climbing ability in comparison to control flies, the condition that was reversed by the action of studied compounds. Rotenone exposure also elevated malondialdehyde levels in the brain tissues, as measured by lipid peroxidation, when compared to control flies. In flies exposed to rotenone and co-fed with the compounds, this effect was lessened. In flies exposed to rotenone, mRNA levels of antioxidant enzymes such as superoxide dismutase and catalase were raised but were normalized in flies treated with the investigated compounds. Moreover, in-silico studies examined the inhibitory ability of compounds 1 - 4 against selected PD molecular targets, revealing the strong power of toussaintine A (1) against Adenosine receptor 2 (A2AR) and monoamine oxidase B. Thus, theser findings suggest that cinnamoyl derivatives have neuroprotective potential via reducing the oxidative burden and improving locomotor ability after toxin invectives. In particular, compound 1 at lower doses can simultaneously be a potential inhibitor of A2AR and an anti-oxidative mediator in the development of anti-PD agents.

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Inoshita, T., Liu, J. Y., Taniguchi, D., Ishii, R., Shiba-Fukushima, K., Hattori, N. and Imai, Y. (2022). Parkinson disease-associated Leucine-rich repeat kinase regulates UNC-104-dependent axonal transport of Arl8-positive vesicles in Drosophila. iScience 25(12): 105476. PubMed ID: 36404922

Abstract

Some Parkinson's disease (PD)-causative/risk genes, including the PD-associated kinase leucine-rich repeat kinase 2 (LRRK2), are involved in membrane dynamics. Although LRRK2 and other PD-associated genes are believed to regulate synaptic functions, axonal transport, and endolysosomal activity, it remains unclear whether a common pathological pathway exists. This study reports that the loss of Lrrk, an ortholog of human LRRK2, leads to the accumulation of the lysosome-related organelle regulator, Arl8 along with dense core vesicles at the most distal boutons of the neuron terminals in Drosophila. Moreover, the inactivation of a small GTPase Rab3 and altered Auxilin activity phenocopied Arl8 accumulation. The accumulation of Arl8-positive vesicles is UNC-104-dependent and modulated by PD-associated genes, Auxilin, VPS35, RME-8, and INPP5F, indicating that VPS35, RME-8, and INPP5F are upstream regulators of Lrrk. These results indicate that certain PD-related genes, along with LRRK2, drive precise neuroaxonal transport of dense core vesicles.

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Liu, W., Lim, K. L. and Tan, E. K. (2022). Intestine-derived α-synuclein initiates and aggravates pathogenesis of Parkinson's disease in Drosophila. Transl Neurodegener 11(1): 44. PubMed ID: 36253844

Abstract

Aberrant aggregation of α-synuclein (α-syn) is a key pathological feature of Parkinson's disease (PD), but the precise role of intestinal α-syn in the progression of PD is unclear. In a number of genetic Drosophila models of PD, α-syn was frequently ectopically expressed in the neural system to investigate the pathobiology. This study investigated the potential role of intestinal α-syn in PD pathogenesis using a Drosophila model. Human α-syn was overexpressed in Drosophila guts, and life span, survival, immunofluorescence and climbing were evaluated. Immunofluorescence, Western blotting and reactive oxygen species (ROS) staining were performed to assess the effects of intestinal α-syn on intestinal dysplasia. High-throughput RNA and 16S rRNA gene sequencing, quantitative RT-PCR, immunofluorescence, and ROS staining were performed to determine the underlying molecular mechanism. It was found that the midgut α-syn alone recapitulated many phenotypic and pathological features of PD, including impaired life span, loss of dopaminergic neurons, and progressive motor defects. The intestine-derived α-syn disrupted intestinal homeostasis and accelerated the onset of intestinal ageing. Moreover, intestinal expression of α-syn induced dysbiosis, while microbiome depletion was efficient to restore intestinal homeostasis and ameliorate the progression of PD. Intestinal α-syn triggered ROS, and eventually led to the activation of the dual oxidase (DUOX)-ROS-Jun N-terminal Kinase (JNK) pathway. In addition, α-syn from both the gut and the brain synergized to accelerate the progression of PD. The intestinal expression of α-syn recapitulates many phenotypic and pathologic features of PD, and induces dysbiosis that aggravates the pathology through the DUOX-ROS-JNK pathway in Drosophila. These findings provide new insights into the role of intestinal α-syn in PD pathophysiology (Liu, 2022).

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Ayajuddin, M., Phom, L., Koza, Z., Modi, P., Das, A., Chaurasia, R., Thepa, A., Jamir, N., Neikha, K. and Yenisetti, S. C. (2022). Adult health and transition stage-specific rotenone-mediated Drosophila model of Parkinson's disease: Impact on late-onset neurodegenerative disease models. Front Mol Neurosci 15: 896183. PubMed ID: 36017079

Abstract

Parkinson's disease (PD) affects almost 1% of the population worldwide over the age of 50 years. Exposure to environmental toxins like paraquat and rotenone is a risk factor for sporadic PD which constitutes 95% of total cases. Herbicide rotenone has been shown to cause Parkinsonian symptoms in multiple animal models. Drosophila is an excellent model organism for studying neurodegenerative diseases (NDD) including PD. The aging process is characterized by differential expression of genes during different life stages. Hence it is necessary to develop life-stage-matched animal models for late-onset human disease(s) such as PD. Such animal models are critical for understanding the pathophysiology of age-related disease progression and important to understand if a genotropic drug/nutraceutical can be effective during late stages. With this idea, an adult life stage-specific (health and transition phase, during which late-onset NDDs such as PD sets in) rotenone-mediated Drosophila model of idiopathic PD was developed. Drosophila is susceptible to rotenone in dose-time dependent manner. Rotenone-mediated fly model of sporadic PD exhibits mobility defects (independent of mortality), inhibited mitochondrial complex I activity, dopaminergic (DAergic) neuronal dysfunction (no loss of DAergic neuronal number; however, reduction in rate-limiting enzyme tyrosine hydroxylase (TH) synthesis), and alteration in levels of dopamine (DA) and its metabolites; 3,4-Dihydroxyphenylacetic acid (DOPAC) and Homovanilic acid (HVA) in brain-specific fashion. These PD-linked behaviors and brain-specific phenotypes denote the robustness of the present fly model of PD. This novel model will be of great help to decipher life stage-specific genetic targets of small molecule mediated DAergic neuroprotection; understanding of which is critical for formulating therapeutic strategies for PD.

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Rai, P. and Roy, J. K. (2022). Rab11 regulates mitophagy signaling pathway of Parkin and Pink1 in the Drosophila model of Parkinson's disease. Biochem Biophys Res Commun 626: 175-186. PubMed ID: 35994827

Abstract

Parkinson's disease (PD) is a common neurodegenerative disorder caused by the loss of dopaminergic neurons in the substantia nigra. The pathophysiology of this disease is the formation of the Lewy body, mostly consisting of alpha-synuclein and dysfunctional mitochondria. There are two common PD-associated genes, Pink1 (encoding a mitochondrial ser/thr kinase) and Parkin (encoding cytosolic E3-ubiquitin ligase), involved in the mitochondrial quality control pathway. They assist in removing damaged mitochondria via selective autophagy (mitophagy) which if unchecked, results in the formation of protein aggregates in the cytoplasm. The role of Rab11, a small Ras-like GTPase associated with recycling endosomes, in PD is still unclear. The present study used the PD model of Drosophila melanogaster and found that Rab11 has a crucial role in the regulation of mitochondrial quality control and endo-lysosomal pathways in association with Parkin and Pink1 and Rab11 acting downstream of Parkin. Additionally, overexpression of Rab11 in parkin mutant rescued the mitochondrial impairment, suggesting the therapeutic potential of Rab11 in PD pathogenesis.

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Sanz, F. J., Solana-Manrique, C., Lilao-Garzon, J., Brito-Casillas, Y., Muñoz-Descalzo, S. and Paricio, N. (2022). Exploring the link between Parkinson's disease and type 2 diabetes mellitus in Drosophila. Faseb j 36(8): e22432. PubMed ID: 35766235

Abstract

Parkinson's disease (PD) is the second most common neurodegenerative disease. Diabetes mellitus (DM) is a metabolic disease characterized by high levels of glucose in blood. Recent epidemiological studies have highlighted the link between both diseases; it is even considered that DM might be a risk factor for PD. To further investigate the likely relation of these diseases, a Drosophila PD model was used based on inactivation of the DJ-1β gene (ortholog of human DJ-1), and diet-induced Drosophila and mouse type 2 DM (T2DM) models, together with human neuron-like cells. T2DM models were obtained by feeding flies with a high sugar-containing medium, and mice with a high fat diet. The results showed that both fly models exhibit common phenotypes such as alterations in carbohydrate homeostasis, mitochondrial dysfunction or motor defects, among others. In addition, it was demonstrated that T2DM might be a risk factor of developing PD since the diet-induced fly and mouse T2DM models present DA neuron dysfunction, a hallmark of PD. This study also confirmed that neurodegeneration is caused by increased glucose levels, which has detrimental effects in human neuron-like cells by triggering apoptosis and leading to cell death. Besides, the observed phenotypes were exacerbated in DJ-1β mutants cultured in the high sugar medium, indicating that DJ-1 might have a role in carbohydrate homeostasis. Finally, it was confirmed that metformin, an antidiabetic drug, is a potential candidate for PD treatment and that it could prevent PD onset in T2DM model flies. This result supports antidiabetic compounds as promising PD therapeutics.

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Fellgett, A., Middleton, C. A., Munns, J., Ugbode, C., Jaciuch, D., Wilson, L., Chawla, S. and Elliott, C. J. H. (2021). Multiple Pathways of LRRK2-G2019S/Rab10 Interaction in Dopaminergic Neurons. J Parkinsons Dis. PubMed ID: 34250948

Abstract

Inherited mutations in the LRRK2 protein are the common causes of Parkinson's disease, but the mechanisms by which increased kinase activity of mutant LRRK2 leads to pathological events remain to be determined. In vitro assays (heterologous cell culture, phospho-protein mass spectrometry) suggest that several Rab proteins might be directly phosphorylated by LRRK2-G2019S. An in vivo screen of Rab expression in dopaminergic neurons in young adult Drosophila demonstrated a strong genetic interaction between LRRK2-G2019S and Rab10. To determine if Rab10 is necessary for LRRK2-induced pathophysiological responses in the neurons that control movement, vision, circadian activity, and memory. These four systems were chosen because they are modulated by dopaminergic neurons in both humans and flies. LRRK2-G2019S was expressed in Drosophila dopaminergic neurons and the effects of Rab10 depletion on Proboscis Extension, retinal neurophysiology, circadian activity pattern ('sleep'), and courtship memory determined in aged flies. Rab10 loss-of-function rescued LRRK2-G2019S induced bradykinesia and retinal signaling deficits. Rab10 knock-down, however, did not rescue the marked sleep phenotype which results from dopaminergic LRRK2-G2019S. Courtship memory is not affected by LRRK2, but is markedly improved by Rab10 depletion. Anatomically, both LRRK2-G2019S and Rab10 are seen in the cytoplasm and at the synaptic endings of dopaminergic neurons. It is concluded that, in Drosophila dopaminergic neurons, Rab10 is involved in some, but not all, LRRK2-induced behavioral deficits. Therefore, variations in Rab expression may contribute to susceptibility of different dopaminergic nuclei to neurodegeneration seen in people with Parkinson's disease (Fellgett, 2021).

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Pallos, J., Jeng, S., McWeeney, S. and Martin, I. (2015). Dopamine neuron-specific LRRK2 G2019S effects on gene expression revealed by translatome profiling. Neurobiol Dis: 105390. PubMed ID: 33984508

Abstract

Leucine-rich repeat kinase 2 (LRRK2) mutations are the most common genetic cause of late-onset autosomal dominant Parkinson's disease. The pathogenic G2019S mutation enhances LRRK2 kinase activity and induces neurodegeneration in C. elegans, Drosophila and rodent models through unclear mechanisms. Gene expression profiling has the potential to provide detailed insight into the biological pathways modulated by LRRK2 kinase activity in vivo. Prior studies have surveyed the effects of LRRK2 G2019S on genome-wide mRNA expression in complex brain tissues with high cellular heterogeneity, limiting their power to detect more restricted gene expression changes occurring in a cell type-specific manner. This study used translating ribosome affinity purification (TRAP) coupled to RNA-seq to profile dopamine neuron-specific gene expression changes caused by LRRK2 G2019S in the Drosophila CNS. A modest number of genes were differentially expressed in the presence of mutant LRRK2 that represent a broad range of molecular functions including DNA repair (RfC3), mRNA metabolism and translation (Ddx1 and lin-28), calcium homeostasis (MCU), and other categories (Ugt37c1, disp, l(1)G0196, CG6602, CG1126 and CG11068). Further analysis on a subset of these genes revealed that LRRK2 G2019S did not alter their expression across the whole brain, consistent with dopamine neuron-specific effects uncovered by the TRAP approach that may offer insight into the neurodegenerative process. This is the first study to profile the effects of LRRK2 G2019S on DA neuron gene expression in vivo. Beyond providing a set of differentially expressed gene candidates relevant to LRRK2, this study demonstrates the effective use of TRAP to perform high-resolution assessment of dopamine neuron gene expression for the study of PD (Pallos, 2021).

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

Abstract

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).

Discussion

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).

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Ham, S. J., Lee, D., Xu, W. J., Cho, E., Choi, S., Min, S., Park, S. and Chung, J. (2021). (2021). Loss of UCHL1 rescues the defects related to Parkinson's disease by suppressing glycolysis. Sci Adv 7(28). PubMed ID: 34244144

The role of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1; also called PARK5) in the pathogenesis of Parkinson's disease (PD) has been controversial. This study finds that the loss of UCHL1 destabilizes pyruvate kinase (PKM) and mitigates the PD-related phenotypes induced by PTEN-induced kinase 1 (PINK1) or Parkin loss-of-function mutations in Drosophila and mammalian cells. In UCHL1 knockout cells, cellular pyruvate production and ATP levels are diminished, and the activity of AMP-activated protein kinase (AMPK) is highly induced. Consequently, the activated AMPK promotes the mitophagy mediated by Unc-51-like kinase 1 (ULK1) and FUN14 domain-containing 1 (FUNDC1), which underlies the effects of UCHL1 deficiency in rescuing PD-related defects. Furthermore, this study identified tripartite motif-containing 63 (TRIM63) as a previously unknown E3 ligase of PKM and demonstrate its antagonistic interaction with UCHL1 to regulate PD-related pathologies. These results suggest that UCHL1 is an integrative factor for connecting glycolysis and PD pathology (Ham, 2021).

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Inoue, E., Suzuki, T., Shimizu, Y., Sudo, K., Kawasaki, H. and Ishida, N. (2021). Saffron ameliorated motor symptoms, short life span and retinal degeneration in Parkinson's disease fly models. Gene 799: 145811. PubMed ID: 34224829

Parkinson's disease (PD) is a common neurodegenerative disorder with motor symptoms linked to the loss of dopaminergic neurons in the brain. α-Synuclein is an aggregation-prone neural protein that plays a role in the pathogenesis of PD. In a previous paper, it was found that saffron; the stigma of Crocus sativus Linne (Iridaceae), and its constituents (crocin and crocetin) suppressed aggregation of α-synuclein and promoted the dissociation of α-synuclein fibrils in vitro. This study investigated the effect of dietary saffron and its constituent, crocetin, in vivo on a fly PD model overexpressing several mutant α-synuclein in a tissue-specific manner. Saffron and crocetin significantly suppressed the decrease of climbing ability in the Drosophila overexpressing A30P (A30P fly PD model) or G51D (G51D fly PD model) mutated α-synuclein in neurons. Saffron and crocetin extended the life span in the G51D fly PD model. Saffron suppressed the rough-eyed phenotype and the dispersion of the size histogram of the ocular long axis in the eye of A30P fly PD model. Saffron had a cytoprotective effect on a human neuronal cell line with α-synuclein fibrils. These data showed that saffron and its constituent crocetin have protective effects on the progression of PD disease in animals in vivo and suggest that saffron and crocetin can be used to treat PD (Inoue, 2021).

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Sarkar, S., Olsen, A. L., Sygnecka, K., Lohr, K. M. and Feany, M. B. (2021). alpha-synuclein impairs autophagosome maturation through abnormal actin stabilization. PLoS Genet 17(2): e1009359. PubMed ID: 33556113

Vesicular trafficking defects, particularly those in the autophagolysosomal system, have been strongly implicated in the pathogenesis of Parkinson's disease and related α-synucleinopathies. However, mechanisms mediating dysfunction of membrane trafficking remain incompletely understood. Using a Drosophila model of α-synuclein neurotoxicity with widespread and robust pathology, this study found that human α-synuclein expression impairs autophagic flux in aging adult neurons. Genetic destabilization of the actin cytoskeleton rescues F-actin accumulation, promotes autophagosome clearance, normalizes the autophagolysosomal system, and rescues neurotoxicity in α-synuclein transgenic animals through an Arp2/3 dependent mechanism. Similarly, mitophagosomes accumulate in human α-synuclein-expressing neurons, and reversal of excessive actin stabilization promotes both clearance of these abnormal mitochondria-containing organelles and rescue of mitochondrial dysfunction. These results suggest that Arp2/3 dependent actin cytoskeleton stabilization mediates autophagic and mitophagic dysfunction and implicate failure of autophagosome maturation as a pathological mechanism in Parkinson's disease and related α-synucleinopathies (Sarkar, 2021).

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Reiszadeh S. J., Ramesh, S. R., Finkelstein, D. I. and Haddadi, M. (2020). alpha-Synuclein E46K Mutation and Involvement of Oxidative Stress in a Drosophila Model of Parkinson's Disease. Parkinsons Dis 2021: 6621507. PubMed ID: 34285796

Parkinson's disease (PD) is an age-associated neurodegenerative condition in which some genetic variants are known to increase disease susceptibility on interaction with environmental factors inducing oxidative stress. Different mutations in the SNCA gene (synuclein α) are reported as the major genetic contributors to PD. E46K mutation pathogenicity has not been investigated as intensive as other SNCA gene mutations including A30P and A53T. In this study, based on the GAL4-UAS binary genetic tool, transgenic Drosophila melanogaster flies expressing wild-type and E46K-mutated copies of the human SNCA gene were constructed. Overexpression of human α-synuclein in the central nervous system of these transgenic flies led to disorganized ommatidia structures and loss of dopaminergic neurons. E46K α-synuclein caused remarkable climbing defects, reduced survivorship, higher ethanol sensitivity, and increased PQ-mediated mortality. A noticeable decline in activity of catalase and superoxide dismutase enzymes besides considerable increase in the levels of lipid peroxidation and reactive oxygen species was observed in head capsule homogenates of α-synuclein-expressing flies, which indicates obvious involvement of oxidative stress as a causal factor in SNCA (E46K) neurotoxicity. In all the investigations, E46K copy of the SNCA gene was found to impose more severe defects when compared to wild-type SNCA. It can be concluded that the constructed Drosophila models developed PD-like symptoms that facilitate comparative studies of molecular and cellular pathways implicated in the pathogenicity of different α-synuclein mutations (Reiszadeh, 2021).

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Sarkar, S., Murphy, M. A., Dammer, E. B., Olsen, A. L., Rangaraju, S., Fraenkel, E. and Feany, M. B. (2020). Comparative proteomic analysis highlights metabolic dysfunction in α-synucleinopathy. NPJ Parkinsons Dis 6(1): 40. PubMed ID: 33311497

The synaptic protein α-synuclein is linked through genetics and neuropathology to the pathogenesis of Parkinson's disease and related disorders. However, the mechanisms by which α-synuclein influences disease onset and progression are incompletely understood. To identify pathogenic pathways and therapeutic targets proteomic analysis was performed in a highly penetrant new Drosophila model of α-synucleinopathy. 476 significantly upregulated and 563 significantly downregulated proteins were identified in heads from α-synucleinopathy model flies compared to controls. Multiple complementary analyses was used to identify and prioritize genes and pathways within the large set of differentially expressed proteins for functional studies. Gene Ontology enrichment analysis was performed, the proteomic changes were integrated with human Parkinson's disease genetic studies, and the α-synucleinopathy proteome was compared with that of tauopathy model flies, which are relevant to Alzheimer's disease and related disorders. These approaches identified GTP cyclohydrolase (GCH1) and folate metabolism as candidate mediators of α-synuclein neurotoxicity. In functional validation studies, it was found that the knockdown of Drosophila Gch1 enhanced locomotor deficits in α-synuclein transgenic flies, while folate supplementation protected from α-synuclein toxicity. This integrative analysis suggested that mitochondrial dysfunction was a common downstream mediator of neurodegeneration. Accordingly, Gch1 knockdown enhanced metabolic dysfunction in α-synuclein transgenic fly brains while folate supplementation partially normalized brain bioenergetics. An integrative approach was outlined and implemented to identify and validate potential therapeutic pathways using comparative proteomics and genetics and capitalizing on the facile genetic and pharmacological tools available in Drosophila (Sarkar, 2020).

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Liu, Q., Bautista-Gomez, J., Higgins, D. A., Yu, J. and Xiong, Y. (2021). Dysregulation of the AP2M1 phosphorylation cycle by LRRK2 impairs endocytosis and leads to dopaminergic neurodegeneration. Sci Signal 14(693). PubMed ID: 34315807

Mutations in the kinase LRRK2 and impaired endocytic trafficking are both implicated in the pathogenesis of Parkinson's disease (PD). Expression of the PD-associated LRRK2 mutant in mouse dopaminergic neurons was shown to disrupt clathrin-mediated endocytic trafficking. This study explored the molecular mechanism linking LRRK2 to endocytosis and found that LRRK2 bound to and phosphorylated the μ2 subunit of the adaptor protein AP2 (AP2M1), a core component of the clathrin-mediated endocytic machinery. Analysis of human SH-SY5Y cells and mouse neurons and tissues revealed that loss of LRRK2 abundance or kinase function resulted in decreased phosphorylation of AP2M1, which is required for the initial formation of clathrin-coated vesicles (CCVs). In contrast, overexpression of LRRK2 or expression of a Parkinson's disease-associated gain-of-function mutant LRRK2 (G2019S) inhibited the uncoating of AP2M1 from CCVs at later stages and prevented new cycles of CCV formation. Thus, the abundance and activity of LRRK2 must be calibrated to ensure proper endocytosis. Dysregulated phosphorylation of AP2M1 from the brain but not thyroid tissues of LRRK2 knockout and G2019S-knockin mice suggests a tissue-specific regulatory mechanism of endocytosis. Furthermore, this study found that LRRK2-dependent phosphorylation of AP2M1 mediated dopaminergic neurodegeneration in a Drosophila model of PD. Together, these findings provide a mechanistic link between LRRK2, AP2, and endocytosis in the pathogenesis of PD.

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Xie, J., Chen, S., Bopassa, J. C. and Banerjee, S. (2021). Drosophila tubulin polymerization promoting protein mutants reveal pathological correlates relevant to human Parkinson's disease. Sci Rep. 11(1):13614. PubMed ID: 34193896

Parkinson's disease (PD) is a progressive neurodegenerative disorder with no known cure. PD is characterized by locomotion deficits, nigrostriatal dopaminergic neuronal loss, mitochondrial dysfunctions and formation of α-Synuclein aggregates. A well-conserved and less understood family of Tubulin Polymerization Promoting Proteins (TPPP) is also implicated in PD and related disorders, where TPPP exists in pathological aggregates in neurons in patient brains. However, there are no in vivo studies on mammalian TPPP to understand the genetics and neuropathology linking TPPP aggregation or neurotoxicity to PD. The only Drosophila homolog of human TPPP is named Ringmaker (Ringer). This study reports that adult ringer mutants display progressive locomotor disabilities, reduced lifespan and neurodegeneration. Importantly, the findings reveal that Ringer is associated with mitochondria and ringer mutants have mitochondrial structural damage and dysfunctions. Adult ringer mutants also display progressive loss of dopaminergic neurons. Together, these phenotypes of ringer mutants recapitulate some of the salient features of human PD patients, thus allowing utilization of ringer mutants as a fly model relevant to PD, and further exploration of its genetic and molecular underpinnings to gain insights into the role of human TPPP in PD (Xie, 2021)

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Terriente-Felix, A., Wilson, E. L. and Whitworth, A. J. (2020). Drosophila phosphatidylinositol-4 kinase fwd promotes mitochondrial fission and can suppress Pink1/parkin phenotypes. PLoS Genet 16(10): e1008844. PubMed ID: 33085661

Balanced mitochondrial fission and fusion play an important role in shaping and distributing mitochondria, as well as contributing to mitochondrial homeostasis and adaptation to stress. In particular, mitochondrial fission is required to facilitate degradation of damaged or dysfunctional units via mitophagy. Two Parkinson's disease factors, PINK1 and Parkin, are considered key mediators of damage-induced mitophagy, and promoting mitochondrial fission is sufficient to suppress the pathological phenotypes in Drosophila Pink1/parkin mutants. Additional factors were sought that impinge on mitochondrial dynamics and which may also suppress Pink1/parkin phenotypes. The Drosophila phosphatidylinositol 4-kinase IIIβ homologue, Four wheel drive (Fwd), promotes mitochondrial fission downstream of the pro-fission factor Drp1. Previously described only as male sterile, this study identified several new phenotypes in fwd mutants, including locomotor deficits and shortened lifespan, which are accompanied by mitochondrial dysfunction. Finally, fwd overexpression can suppress locomotor deficits and mitochondrial disruption in Pink1/parkin mutants, consistent with its function in promoting mitochondrial fission. Together these results shed light on the complex mechanisms of mitochondrial fission and further underscore the potential of modulating mitochondrial fission/fusion dynamics in the context of neurodegeneration (Terriente-Felix, 2020).

Mitochondria are dynamic organelles that are transported to the extremities of the cell and frequently undergo fusion and fission events that influence their size, branching and degradation. Many of the core components of the mitochondrial fission and fusion machineries have been well characterised. There include the pro-fusion factors Mfn1/2 and Opa1, and pro-fission factors Drp1 and Mff. Maintaining an appropriate balance of fission and fusion, as well as transport dynamics, is crucial for cellular health and survival as mutations in many of the core components cause severe neurological conditions in humans and model organisms. Recently, a role for phosphatidylinositol 4-phosphate [PI(4)P] in mitochondrial fission has been elucidated in cultured cells (Nagashima, 2020; Science 367(6484): 1366-1371), but the in vivo consequences have not yet been described (Terriente-Felix, 2020).

The mitochondrial fission/fusion cycle has been linked to the selective removal of damaged mitochondria through the process of autophagy (termed mitophagy), in which defective mitochondria are engulfed into autophagosomes and degraded by lysosomes. Two genes that have been firmly linked to the mitophagy process are PINK1 and PRKN. Mutations in these genes cause autosomal-recessive juvenile parkinsonism, associated with degeneration of midbrain dopaminergic neurons and motor impairments, among other symptoms and pathologies. Studies from a wide variety of model systems have shown various degrees of mitochondrial dysfunction associated with mutation of PINK1/PRKN homologues including disrupted fission/fusion. Drosophila have proven to be a fruitful model for investigating the function of the conserved homologues Pink1 and parkin, with these mutants exhibiting robust mitochondrial disruption and neuromuscular phenotypes. Importantly, several studies have shown that the pathological consequences of loss of Pink1 or parkin can be largely suppressed by genetic manipulations that increase mitochondrial fission or reduce fusion (Terriente-Felix, 2020).

To identify genes involved in mitochondrial quality control and homeostasis, an RNAi screen was performed in Drosophila S2 cells to identify kinases and phosphatases that phenocopy or suppress hyperfused mitochondria caused by loss of Pink1 (Pogson, 2014). This study identified the phosphatidylinositol 4-kinase IIIβ homologue, four wheel drive (fwd), whose knockdown phenocopied Pink1 RNAi, resulting in excess mitochondrial fusion. Drosophila mutant for fwd have been reported to be viable but male sterile due to incomplete cytokinesis during spermatogenesis. While muscle-specific knockdown has shown to impact neuromuscular junction formation (Forrest, 2013), no other organismal phenotypes or mitochondrial involvement have been described to date. Thus, this study sought to better understand the role of Fwd in mitochondrial homeostasis (Terriente-Felix, 2020).

This study has characterised fwd mutants for organismal phenotypes associated with Pink1/parkindysfunction and analysed the impact on mitochondrial form and function. Genetic interactions were investigated between fwd and Pink1/parkin, as well as with mitochondrial fission/fusion factors. It was found that loss of fwd inhibited mitochondrial function, causing increased mitochondrial length and branching, and decreased respiratory capacity. These effects were associated with shortened lifespan and dramatically reduced locomotor ability, similar to Pink1 and parkin mutants. Furthermore, fwd overexpression was sufficient to significantly suppress Pink1/parkin mutant locomotor deficits and mitochondrial phenotypes. Interestingly, it was found that the mitochondrial and locomotion phenotypes in fwd mutants can be rescued by loss of pro-fusion factors Marf and Opa1, but the pro-fission activity of Drp1 appears to require fwd. These results support a role for fwd in regulating mitochondrial morphology, specifically in facilitating mitochondrial fission, and further substantiate the important contribution of aberrant mitochondrial fission/fusion dynamics in Pink1/parkinphenotypes (Terriente-Felix, 2020).

Previous work identified fwd as a gene whose knockdown induces mitochondrial hyperfusion in cultured cells, similar to loss of Pink1. This study has validated that the genetic loss or knockdown of fwd also causes excess mitochondrial fusion in neuronal cells in vivo, leading to increased mitochondrial length and branching. As mitochondrial fission/fusion dynamics have been shown to be important for mitochondrial homeostasis, it is not surprising that this also has an impact on respiration at the organismal level and on organismal fitness and vitality. While fwd mutants have mainly been characterised for their male sterility phenotype, this study describes new organismal phenotypes associated with loss of fwd: profound locomotor deficits and shortened lifespan. Interestingly, while the data reveal a stronger requirement for fwd in the nervous system compared to the musculature to maintain normal motor behaviour, fwd is required in muscle for neuromuscular junction formation. Furthermore, consistent with the observations on lifespan, Fwd overexpression has previously been shown to confer increased lifespan. Thus, Fwd clearly has a more widespread role in organismal vitality than previously appreciated (Terriente-Felix, 2020).

The robust locomotor phenotype allowed a test of the genetic relationship between fwd and core components of the mitochondrial fission/fusion machinery. Given the excess mitochondrial fusion upon loss of fwd, suppression of the organismal phenotypes by reduction of fusion factors Marf and Opa1 was expected. However, it was surprising that overexpression of the fission factor Drp1 was unable to ameliorate organismal phenotypes or even the increased mitochondrial length, though it was able to revert the increased branching caused by loss of fwd. These results suggested that Drp1 requires Fwd to drive mitochondrial fission. Consistent with this, Drp1 overexpression was no longer able to rescue Pink1/parkin mutant phenotypes in the absence of fwd. These genetic experiments strongly hint at a functional link between Drp1 and Fwd but do not illuminate the molecular mechanism underpinning it. Fwd is the Drosophila homologue of phosphatidylinositol 4-kinase IIIβ [PI(4)KB], which mediates the phosphorylation of phosphatidylinositol to generate phosphatidylinositol 4-phosphate [PI(4)P]. PI(4)P is one of the most abundant phosphoinositides, which is usually concentrated in the trans-Golgi network; thus, the mechanism by which PI(4)P may influence mitochondrial dynamics is not immediately obvious. However, while this manuscript was in preparation, Godi, 1999 (Nat Cell Biol 1(5): 280-287) reported that Golgi-derived PI(4)P-containing vesicles were required for the final stages of mitochondrial fission (Nagashima, 2020; Science 367(6484): 1366-1371). In that study, the authors found that loss of PI(4)KIIIβ led to hyperfusion and increased branching of the mitochondrial network, consistent with what was observed in this study. Moreover, Nagashima described that while Drp1 was still recruited, it was unable to fully execute the scission event, although the reason is unclear, leading to extended mitochondrial constriction sites. Genetic evidence that the action of Drp1 requires Fwd is consistent with these findings, and provides an in vivo validation of Nagashima's results. Currently, it is unclear why Drp1 overexpression was able to revert the increased branching caused by loss of fwd but the mechanisms of branch formations are not well understood. It is interesting to note that while Nagashima suggest a universal role for PI(4)P in mitochondrial fission, the current in vivo analysis reveals that while fwd affected mitochondrial morphology in the nervous system, it appeared to have a much more limited role in the musculature. These tissue-specific requirements were borne out in the strong locomotor deficits caused by neuronal loss of fwd but much less so by knockdown in muscles. Clearly, further work is required to better understand the complexities of regulated fission/fusion events in different cell contexts in vivo (Terriente-Felix, 2020).

A key role of mitochondrial fission/fusion dynamics is in contributing to a quality control mechanism of mitochondrial sorting to eliminate dysfunctional units via mitophagy. A substantial body of evidence from cellular models indicates that mammalian PINK1 and Parkin act to promote damage-induced mitophagy, and some in vivo evidence from Drosophila also supports this. However, the precise nature of PINK1/Parkin-mediated mitochondrial turnover in vivo is debated with contradictory results emerging. Nevertheless, interventions to combat the decline in mitochondrial homeostasis remain a key challenge to combatting PINK1/PRKN related pathologies. One mechanism that seems to provide substantial benefit in physiological contexts is through augmenting mitochondrial fission, which presumably facilitates the flux of damaged mitochondrial components towards turnover. This study, provide further evidence that augmenting a pro-fission pathway is beneficial against Pink1 and parkin dysfunction. As phosphoinositides can be interconverted by the action of multiple enzymes that may be druggable, these findings suggest another potential route towards a therapeutic intervention (Terriente-Felix, 2020).

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Agostini, F., Bubacco, L., Chakrabarti, S. and Bisaglia, M. (2023). α-Synuclein Toxicity in Drosophila melanogaster Is Enhanced by the Presence of Iron: Implications for Parkinson's Disease. Antioxidants (Basel) 12(2). PubMed ID: 36829820

Abstract
Parkinson's disease (PD) is the second most common neurodegenerative disorder, characterized by the preferential loss of dopaminergic neurons and by the accumulation of intracellular inclusions mainly composed of α-synuclein (&alpha-Syn). While the etiopathogenesis of the disorder is still elusive, recent experimental evidence supports the involvement of ferroptosis, an iron-dependent cell death pathway, in the pathogenesis of PD. Using different ferroptosis inducers and inhibitors, this study evaluated, in vivo, the involvement of iron in the α-Syn-mediated toxicity. Using a Drosophila melanogaster model of PD based on the selective over-expression of α-Syn within dopaminergic neurons, this study demonstrated that the over-expression of α-Syn promotes the accumulation of protein aggregates, which is accompanied by dopaminergic neurodegeneration, locomotor impairment, and lifespan reduction. These pathological phenotypes were further exacerbated by reduced intracellular levels of glutathione or increased concentrations of iron. Coherently, both the use of an iron chelator and the presence of the antioxidant compound N-acetylcysteine exerted protective effects. Overall, these results support the involvement of ferroptosis in the α-Syn-mediated toxicity.

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Koza, Z., Ayajuddin, M., Das, A., Chaurasia, R., Phom, L. and Yenisetti, S. C. (2023). Sexual dysfunction precedes motor defects, dopaminergic neuronal degeneration, and impaired dopamine metabolism: Insights from Drosophila model of Parkinson's disease. Front Neurosci 17: 1143793. PubMed ID: 37025374

Abstract
Sexual dysfunction (SD) is one of the most common non-motor symptoms of Parkinson's disease (PD) and remains the most neglected, under-reported, and under-recognized aspect of PD. Studies have shown that Dopamine (DA) in the hypothalamus plays a role in regulating sexual behavior. But the detailed mechanism of SD in PD is not known. Drosophila melanogaster shares several genes and signaling pathways with humans which makes it an ideal model for the study of a neurodegenerative disorder such as PD. Courtship behavior of Drosophila is one such behavior that is closely related to human sexual behavior and so plays an important role in understanding sexual behavior in diseased conditions as well. In the present study, a sporadic SD model of PD using Drosophila was developed and SD phenotype was observed based on abnormalities in courtship behavior markers. The Drosophila SD model was developed in such a way that at the window of neurotoxin paraquat (PQ) treatment [PQ is considered a crucial risk factor for PD due to its structural similarity with 1-methyl-4-phenyl pyridinium (MPP+), the active form of PD-inducing agent, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)], it does not exhibit mobility defects but shows SD. The whole brain tyrosine hydroxylase immunostaining showed no observable dopaminergic (DAergic) degeneration (number of DA neurons and fluorescence intensity of fluorescently labeled secondary antibodies that target anti-TH primary antibody) of the SD model. Similarly, there was no significant depletion of brain DA and its metabolite levels (HVA and DOPAC) as determined using HPLC-ECD (High-Performance Liquid Chromatography using Electrochemical Detector). The present study illustrates that the traits associated with courtship and sexual activity provide sensitive markers at the earlier stage of PD onset. This PQ-induced SD fly model throws an opportunity to decipher the molecular basis of SD under PD conditions and to screen nutraceuticals/potential therapeutic molecules to rescue SD phenotype and further to DAergic neuroprotection.

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Dumitrescu, E., Copeland, J. M. and Venton, B. J. (2023). Parkin Knockdown Modulates Dopamine Release in the Central Complex, but Not the Mushroom Body Heel, of Aging Drosophila. ACS Chem Neurosci 14(2): 198-208. PubMed ID: 36576890

Abstract
Parkinson's disease (PD) is characterized by progressive degeneration of dopaminergic neurons leading to reduced locomotion. Mutations of parkin gene in Drosophila produce the same phenotypes as vertebrate models, but the effect of parkin knockdown on dopamine release is not known. This study reports age-dependent, spatial variation of dopamine release in the brain of parkin-RNAi adult Drosophila. Dopamine was repetitively stimulated by local application of acetylcholine and quantified by fast-scan cyclic voltammetry in the central complex or mushroom body heel. In the central complex, the main area controlling locomotor function, dopamine release is maintained for repeated stimulations in aged control flies, but lower concentrations of dopamine are released in the central complex of aged parkin-RNAi flies. In the mushroom body heel, the dopamine release decrease in older parkin-RNAi flies is similar to controls. There is not significant dopaminergic neuronal loss even in older parkin knockdown flies, which indicates that the changes in stimulated dopamine release are due to alterations of neuronal function. In young parkin-RNAi flies, locomotion is inhibited by 30%, while in older parkin-RNAi flies it is inhibited by 85%. Overall, stimulated dopamine release is modulated by Parkin in an age and brain region dependent manner. Correlating the functional state of the dopaminergic system with behavioral phenotypes provides unique insights into the PD mechanism. Drosophila can be used to study dopamine functionality in PD, elucidate how genetics influence dopamine, and test potential therapies to maintain dopamine release.

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Baisgaard, A. E., Koldby, K. M., Kristensen, T. N., Nyegaard, M. and Rohde, P. D. (2023). Functionally Validating Evolutionary Conserved Risk Genes for Parkinson's Disease in Drosophila melanogaster. Insects 14(2). PubMed ID: 36835737

Abstract
Parkinson's disease (PD) is a heterogeneous and complex neurodegenerative disorder and large-scale genetic studies have identified >130 genes associated with PD. Although genomic studies have been decisive for understanding of the genetic contributions underlying PD, these associations remain as statistical associations. Lack of functional validation limits the biological interpretation; however, it is labour extensive, expensive, and time consuming. Therefore, the ideal biological system for functionally validating genetic findings must be simple. This study's aim was to assess systematically evolutionary conserved PD-associated genes using Drosophila melanogaster. From a literature review, a total of 136 genes have found to be associated with PD in GWAS studies, of which 11 are strongly evolutionary conserved between Homo sapiens and D. melanogaster. By ubiquitous gene expression knockdown of the PD-genes in D. melanogaster, the flies' escape response was investigated by assessing their negative geotaxis response, a phenotype that has previously been used to investigate PD in D. melanogaster. Gene expression knockdown was successful in 9/11 lines, and phenotypic consequences were observed in 8/9 lines. The results provide evidence that genetically modifying expression levels of PD genes in D. melanogaster caused reduced climbing ability of the flies, potentially supporting their role in dysfunctional locomotion, a hallmark of PD.

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Wang, X., Rimal, S., Tantray, I., Geng, J., Bhurtel, S., Khaket, T. P., Li, W., Han, Z. and Lu, B. (2022). Prevention of ribosome collision-induced neuromuscular degeneration by SARS CoV-2-encoded Nsp1. Proc Natl Acad Sci U S A 119(42): e2202322119. PubMed ID: 36170200

Abstract
An overarching goal of aging and age-related neurodegenerative disease research is to discover effective therapeutic strategies applicable to a broad spectrum of neurodegenerative diseases. Little is known about the extent to which targetable pathogenic mechanisms are shared among these seemingly diverse diseases. Translational control is critical for maintaining proteostasis during aging. Gaining control of the translation machinery is also crucial in the battle between viruses and their hosts. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing COVID-19 pandemic. This study shows that overexpression of SARS-CoV-2-encoded nonstructural protein 1 (Nsp1) robustly rescued neuromuscular degeneration and behavioral phenotypes in Drosophila models of Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. These diseases share a common mechanism: the accumulation of aberrant protein species due to the stalling and collision of translating ribosomes, leading to proteostasis failure. Genetic and biochemical analyses revealed that Nsp1 acted in a multipronged manner to resolve collided ribosomes, abort stalled translation, and remove faulty translation products causative of disease in these models, at least in part through the ribosome recycling factor ABCE1, ribosome-associated quality-control factors, autophagy, and AKT signaling. Nsp1 exhibited exquisite specificity in its action, as it did not modify other neurodegenerative conditions not known to be associated with ribosome stalling. These findings uncover a previously unrecognized mechanism of Nsp1 in manipulating host translation, which can be leveraged for combating age-related neurodegenerative diseases that are affecting millions of people worldwide and currently without effective treatment.

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Baisgaard, A. E., Koldby, K. M., Kristensen, T. N., Nyegaard, M. and Rohde, P. D. (2023). Functionally Validating Evolutionary Conserved Risk Genes for Parkinson's Disease in Drosophila melanogaster. Insects 14(2). PubMed ID: 36835737
Summary:
Neuronal activity causes use-dependent decline in protein function. However, it is unclear how this is coupled to local quality control mechanisms. This study shows in Drosophila that the endocytic protein Endophilin-A (EndoA) connects activity-induced calcium influx to synaptic autophagy and neuronal survival in a Parkinson disease-relevant fashion. Mutations in the disordered loop, including a Parkinson disease-risk mutation, render EndoA insensitive to neuronal stimulation and affect protein dynamics: when EndoA is more flexible, its mobility in membrane nanodomains increases, making it available for autophagosome formation. Conversely, when EndoA is more rigid, its mobility reduces, blocking stimulation-induced autophagy. Balanced stimulation-induced autophagy is required for dopagminergic neuron survival, and a variant in the human ENDOA1 disordered loop conferring risk to Parkinson disease also blocks nanodomain protein mobility and autophagy both in vivo and in human-induced dopaminergic neurons. Thus, this study revealed a mechanism that neurons use to connect neuronal activity to local autophagy and that is critical for neuronal survival.

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Baisgaard, A. E., Koldby, K. M., Kristensen, T. N., Nyegaard, M. and Rohde, P. D. (2023). Functionally Validating Evolutionary Conserved Risk Genes for Parkinson's Disease in Drosophila melanogaster. Insects 14(2). PubMed ID: 36835737

Abstract
Parkinson's disease (PD) is a heterogeneous and complex neurodegenerative disorder and large-scale genetic studies have identified >130 genes associated with PD. Although genomic studies have been decisive for understanding of the genetic contributions underlying PD, these associations remain as statistical associations. Lack of functional validation limits the biological interpretation; however, it is labour extensive, expensive, and time consuming. Therefore, the ideal biological system for functionally validating genetic findings must be simple. This study's aim was to assess systematically evolutionary conserved PD-associated genes using Drosophila melanogaster. From a literature review, a total of 136 genes have found to be associated with PD in GWAS studies, of which 11 are strongly evolutionary conserved between Homo sapiens and D. melanogaster. By ubiquitous gene expression knockdown of the PD-genes in D. melanogaster, the flies' escape response was investigated by assessing their negative geotaxis response, a phenotype that has previously been used to investigate PD in D. melanogaster. Gene expression knockdown was successful in 9/11 lines, and phenotypic consequences were observed in 8/9 lines. The results provide evidence that genetically modifying expression levels of PD genes in D. melanogaster caused reduced climbing ability of the flies, potentially supporting their role in dysfunctional locomotion, a hallmark of PD.

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Solana-Manrique, C., Sanz, F. J., Torregrosa, I., Palomino-Schatzlein, M., Hernandez-Oliver, C., Pineda-Lucena, A. and Paricio, N. (2022). Metabolic Alterations in a Drosophila Model of Parkinson's Disease Based on DJ-1 Deficiency. Cells 11(3). PubMed ID: 35159141

Abstract
Parkinson's disease (PD) is the second-most common neurodegenerative disorder, whose physiopathology is still unclear. Moreover, there is an urgent need to discover new biomarkers and therapeutic targets to facilitate its diagnosis and treatment. Previous studies performed in PD models and samples from PD patients already demonstrated that metabolic alterations are associated with this disease. In this context, the aim of this study is to provide a better understanding of metabolic disturbances underlying PD pathogenesis. To achieve this goal, a Drosophila PD model was used based on inactivation of the DJ-1β gene (ortholog of human DJ-1). Metabolomic analyses were performed in 1-day-old and 15-day-old DJ-1β mutants and control flies using (1)H nuclear magnetic resonance spectroscopy, combined with expression and enzymatic activity assays of proteins implicated in altered pathways. The results showed that the PD model flies exhibited protein metabolism alterations, a shift from the tricarboxylic acid cycle to glycolytic pathway to obtain ATP, together with an increase in the expression of some urea cycle enzymes. Thus, these metabolic changes could contribute to PD pathogenesis and might constitute possible therapeutic targets and/or biomarkers for this disease.

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Poetini, M. R., Musachio, E. A. S., Araujo, S. M., Bortolotto, V. C., Meichtry, L. B., Silva, N. C., Janner, D. E., La Rosa Novo, D., Mesko, M. F., Roehrs, R., Ramborger, B. P. and Prigol, M. (2022). Improvement of non-motor and motor behavioral alterations associated with Parkinson-like disease in Drosophila melanogaster: Comparative effects of treatments with hesperidin and L-dopa. Neurotoxicology 89: 174-183. PubMed ID: 35167856

Abstract
Non-motor alterations such as anxiety and memory deficit may represent early indications of Parkinson's disease (PD), and therapeutic strategies that reduce non-motor alterations are promising alternatives for the treatment. Therefore, the search for natural compounds that act on motor and non-motor complications is highly relevant. In this sense, this study has demonstrated the role of hesperidin (Hsd) as a citrus flavonoid and its pharmacological properties as an antioxidant and neuroprotective agent. The objective was to evaluate Hsd in developing motor and non-motor alterations in a Drosophila melanogaster model of Parkinson-like disease induced by iron (Fe) exposure. The flies were divided into six groups: control, Hsd (10 μM), L-dopa (positive control, 1 mM), Fe (1 mM), Fe + Hsd, and Fe + L-dopa. Motor coordination tests, memory assessment through aversive phototaxy, and anxiety-like behaviors characterized in flies, such as grooming and aggressiveness, were performed. The Hsd attenuated motor and non-motor alterations, such as motor coordination, memory deficits and anxiety-like behaviors, attenuated monoaminergic deficits, and lowered Fe levels in the head of flies. In addition, Hsd prolonged the life of the flies, thereby standing out from the L-dopa-treated group. Thus, Hsd can protect the dopaminergic system from insults caused by Fe, preventing non-motor alterations in PD; Hsd also reduced Fe levels in the flies' heads, suggesting that iron chelation may represent an important mechanism of action, in addition to its antioxidant action.

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Akinade, T. C., Babatunde, O. O., Adedara, A. O., Adeyemi, O. E., Otenaike, T. A., Ashaolu, O. P., Johnson, T. O., Terriente-Felix, A., Whitworth, A. J. and Abolaji, A. O. (2022). Protective capacity of carotenoid trans-astaxanthin in rotenone-induced toxicity in Drosophila melanogaster. Sci Rep 12(1): 4594. PubMed ID: 35301354

Abstract
Trans-astaxanthin (TA), a keto-carotenoid found in aquatic invertebrates, possesses anti-oxidative and anti-inflammatory activities. Rotenone is used to induce oxidative stress-mediated Parkinson's disease (PD) in animals. Probes were performed to see if TA would protect against rotenone-induced toxicity in Drosophila melanogaster. Trans-astaxanthin and rotenone were separately orally exposed to flies in the diet to evaluate longevity and survival rates, respectively. Consequently, the ameliorative actions of TA on rotenone -induced toxicity was evaluated in Drosophila after 7 days' exposure. Additionally, molecular docking of TA was performed against selected pro-inflammatory protein targets. It was observed that TA increased the lifespan of D. melanogaster by 36.36%. Moreover, TA ameliorated rotenone-mediated inhibition of Catalase, Glutathione-S-transferase and Acetylcholinesterase activities, and depletion of Total Thiols and Non-Protein Thiols contents. Trans-astaxanthin prevented behavioural dysfunction and accumulation of Hydrogen Peroxide, Malondialdehyde, Protein Carbonyls and Nitric Oxide in D. melanogaster. Trans-astaxanthin showed higher docking scores against the pro-inflammatory protein targets evaluated than the standard inhibitors. Conclusively, the structural features of TA might have contributed to its protective actions against rotenone-induced toxicity.

Chaouhan, H. S., Li, X., Sun, K. T., Wang, I. K., Yu, T. M., Yu, S. H., Chen, K. B., Lin, W. Y. and Li, C. Y. (2022). Calycosin Alleviates Paraquat-Induced Neurodegeneration by Improving Mitochondrial Functions and Regulating Autophagy in a Drosophila Model of Parkinson's Disease. Antioxidants (Basel) 11(2). PubMed ID: 35204105

Abstract
Parkinson's disease (PD) is the second most common age-related neurodegenerative disorder with limited clinical treatments. The occurrence of PD includes both genetic and environmental toxins, such as the pesticides paraquat (PQ), as major contributors to PD pathology in both invertebrate and mammalian models. Calycosin, an isoflavone phytoestrogen, has multiple pharmacological properties, including neuroprotective activity. However, the paucity of information regarding the neuroprotective potential of calycosin on PQ-induced neurodegeneration led to an exploration of whether calycosin can mitigate PD-like phenotypes and the underlying molecular mechanisms. A PQ-induced PD model in Drosophila was used as a cost-effective in vivo screening platform to investigate the neuroprotective efficacy of natural compounds on PD. Calycosin showed a protective role in preventing dopaminergic (DA) neuronal cell death in PQ-exposed Canton S flies. Calycosin-fed PQ-exposed flies exhibit significant resistance against PQ-induced mortality and locomotor deficits in terms of reduced oxidative stress, loss of DA neurons, the depletion of dopamine content, and phosphorylated JNK-caspase-3 levels. Additionally, mechanistic studies show that calycosin administration improves PQ-induced mitochondrial dysfunction and stimulates mitophagy and general autophagy with reduced pS6K and p4EBP1 levels, suggestive of a maintained energy balance between anabolic and catabolic processes, resulting in the inhibition of neuronal cell death. Collectively, this study substantiates the protective effect of calycosin against PQ-induced neurodegeneration by improving DA neurons' survival and reducing apoptosis, likely via autophagy induction, and it is implicated as a novel therapeutic application against toxin-induced PD pathogenesis.

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Heremans, I. P., Caligiore, F., Gerin, I., Bury, M., Lutz, M., Graff, J., Stroobant, V., Vertommen, D., Teleman, A. A., Van Schaftingen, E. and Bommer, G. T. (2022). Parkinson's disease protein PARK7 prevents metabolite and protein damage caused by a glycolytic metabolite. Proc Natl Acad Sci U S A 119(4). PubMed ID: 35046029

Abstract
Cells are continuously exposed to potentially dangerous compounds. Progressive accumulation of damage is suspected to contribute to neurodegenerative diseases and aging, but the molecular identity of the damage remains largely unknown. This study reports that PARK7, an enzyme mutated in hereditary Parkinson's disease, prevents damage of proteins and metabolites caused by a metabolite of glycolysis. The glycolytic metabolite 1,3-bisphosphoglycerate (1,3-BPG) spontaneously forms a novel reactive intermediate that avidly reacts with amino groups. PARK7 acts by destroying this intermediate, thereby preventing the formation of proteins and metabolites with glycerate and phosphoglycerate modifications on amino groups. As a consequence, inactivation of PARK7 [or its orthologs (DJ-1α in Drosophila)] in human cell lines, mouse brain, and Drosophila melanogaster leads to the accumulation of these damaged compounds, most of which have not been described before. This work demonstrates that PARK7 function represents a highly conserved strategy to prevent damage in cells that metabolize carbohydrates. This represents a fundamental link between metabolism and a type of cellular damage that might contribute to the development of Parkinson's disease.

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Adedara, A. O., Babalola, A. D., Stephano, F., Awogbindin, I. O., Olopade, J. O., Rocha, J. B. T., Whitworth, A. J. and Abolaji, A. O. (2022). An assessment of the rescue action of resveratrol in parkin loss of function-induced oxidative stress in Drosophila melanogaster. Sci Rep 12(1): 3922. PubMed ID: 35273283

Abstract
Loss-of-function mutations in parkin is associated with onset of juvenile Parkinson's disease (PD). Resveratrol is a polyphenolic stilbene with neuroprotective activity. This study evaluated the rescue action of resveratrol in parkin mutant D. melanogaster. The control flies (w1118) received diet-containing 2% ethanol (vehicle), while the PD flies received diets-containing resveratrol (15, 30 and 60 mg/kg diet) for 21 days to assess survival rate. Consequently, similar treatments were carried out for 10 days to evaluate locomotor activity, oxidative stress and antioxidant markers. mRNA levels were determined of Superoxide dismutase 1 (Sod1, an antioxidant gene) and ple, which encodes tyrosine hydroxylase, the rate-limiting step in dopamine synthesis. The data showed that resveratrol improved survival rate and climbing activity of PD flies compared to untreated PD flies. Additionally, resveratrol protected against decreased activities of acetylcholinesterase and catalase and levels of non-protein thiols and total thiols displayed by PD flies. Moreover, resveratrol mitigated against parkin mutant-induced accumulations of hydrogen peroxide, nitric oxide and malondialdehyde. Resveratrol attenuated downregulation of ple and Sod1 and reduction in mitochondrial fluorescence intensity displayed by PD flies. Overall, resveratrol alleviated oxidative stress and locomotor deficit associated with parkin loss-of-function mutation and therefore might be useful for the management of PD.

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Hernandez-Diaz, S., Ghimire, S., Sanchez-Mirasierra, I., Montecinos-Oliva, C., Swerts, J., Kuenen, S., Verstreken, P. and Soukup, S. F. (2022). Endophilin-B regulates autophagy during synapse development and neurodegeneration. Neurobiol Dis 163: 105595. PubMed ID: 34933093

Abstract
Synapses are critical for neuronal communication and brain function. To maintain neuronal homeostasis, synapses rely on autophagy. Autophagic alterations cause neurodegeneration and synaptic dysfunction is a feature in neurodegenerative diseases. In Parkinson's disease (PD), where the loss of synapses precedes dopaminergic neuron loss, various PD-causative proteins are involved in the regulation of autophagy. So far only a few factors regulating autophagy at the synapse have been identified and the molecular mechanisms underlying autophagy at the synapse is only partially understood. this study describes Endophilin-B (EndoB) as a novel player in the regulation of synaptic autophagy in health and disease. EndoB is required for autophagosome biogenesis at the synapse, whereas the loss of EndoB blocks the autophagy induction promoted by the PD mutation LRRK2(G2019S). EndoB is required to prevent neuronal loss. Moreover, loss of EndoB in the Drosophila visual system leads to an increase in synaptic contacts between photoreceptor terminals and their post-synaptic synapses. These data confirm the role of autophagy in synaptic contact formation and neuronal survival.

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Girard, V., Jollivet, F., Knittelfelder, O., Celle, M., Arsac, J. N., Chatelain, G., Van den Brink, D. M., Baron, T., Shevchenko, A., Kuhnlein, R. P., Davoust, N. and Mollereau, B. (2021). Abnormal accumulation of lipid droplets in neurons induces the conversion of alpha-Synuclein to proteolytic resistant forms in a Drosophila model of Parkinson's disease. PLoS Genet 17(11): e1009921. PubMed ID: 34788284 Parkinson's disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein (αSyn) aggregation and associated with abnormalities in lipid metabolism. The accumulation of lipids in cytoplasmic organelles called lipid droplets (LDs) was observed in cellular models of PD. To investigate the pathophysiological consequences of interactions between αSyn and proteins that regulate the homeostasis of LDs, a transgenic Drosophila model of PD was used in which human αSyn is specifically expressed in photoreceptor neurons. It was first found that overexpression of the LD-coating proteins Perilipin 1 or 2 (dPlin1/2), which limit the access of lipases to LDs, markedly increased triacylglyclerol (TG) loaded LDs in neurons. However, dPlin-induced-LDs in neurons are independent of lipid anabolic, and catabolic enzymes, indicating that alternative mechanisms regulate neuronal LD homeostasis. Interestingly, the accumulation of LDs induced by various LD proteins (dPlin1, dPlin2, CG7900 or KlarsichtLD-BD) was synergistically amplified by the co-expression of αSyn, which localized to LDs in both Drosophila photoreceptor neurons and in human neuroblastoma cells. Finally, the accumulation of LDs increased the resistance of αSyn to proteolytic digestion, a characteristic of αSyn aggregation in human neurons. It is proposed that αSyn cooperates with LD proteins to inhibit lipolysis and that binding of αSyn to LDs contributes to the pathogenic misfolding and aggregation of αSyn in neurons (Girard, 2021).

This study has investigated the mechanisms that regulate LD homeostasis in neurons, the contribution of αSyn to LD homeostasis, and whether αSyn-LD binding influences the pathogenic potential of αSyn. Expression of the LD proteins, dPlin1 and dPlin2, CG7900 or of the LD-binding domain of Klarsicht increased LD accumulation in Drosophila photoreceptor neurons and that this phenotype was amplified by co-expressing the PD-associated protein αSyn. Transfected and endogenous αSyn co-localized with PLINs on the LD surface in human neuroblastoma cells, as demonstrated by confocal microscopy and PLA assays. Neuronal accumulation of LDs was not dependent on the canonical enzymes of TG synthesis (Mdy, dFatp), Bmm/dATGL-dependent lipolysis or lipophagy inhibition. One possible explanation for LD accumulation is that LD proteins inhibit an unknown lipase in Drosophila photoreceptor neurons. Finally, it was observed that LD accumulation in photoreceptor neurons was associated with increased resistance of αSyn to proteinase K digestion, suggesting that LD accumulation might promote αSyn misfolding, an important step in the progression towards PD. Thus, this study has uncovered a potential novel role for LDs in the pathogenicity of αSyn in PD (Girard, 2021).

Understanding of the mechanisms of LD homeostasis in neurons under physiological or pathological conditions is far from complete. Neurons predominantly synthesize ATP through aerobic metabolism of glucose, rather than through FA β-oxidation, which likely explains the relative scarcity of LDs in neurons compared with glial cells. This study used the Drosophila adult retina that is composed of photoreceptor neurons and glial cells to explore the mechanism regulating LD homeostasis in the nervous system. The canonical mechanisms regulating TG turnover and LD formation are dependent on evolutionary conserved regulators of lipogenesis and lipolysis in the fly adipose tissue, called fat body, or in other non-fat cells, such as glial cells. Indeed, it has been shown that de novo TG-synthesis enzymes Dgat1/Mdy and dFatp, are required for LD biogenesis in the fat body and glial cells. This is in contrast to dPlin-induced neuronal accumulation of LDs (this study), which occurs through a mechanism, independent of Mdy- and dFatp-mediated de novo TG synthesis. One possibility is that LD biogenesis depends on Dgat2 in neurons. However, the fact that there are three Dgat2 paralogs encoded by the fly genome and that no triple mutant is available, precluded its functional analysis in the current study (Girard, 2021).

The evolutionarily conserved and canonical TG lipase Bmm, otholog of mammalian adipose triglyceride lipase (ATGL) regulates lipolysis in the fat body. This study shows that Bmm regulates LD abundance in glial cells but not in photoreceptor neurons. Interestingly, in both bmm-mutant Drosophila (this study) and ATGL-mutant mice, neurons do not accumulate LDs. This suggests the existence of an unknown and possibly cell type specific lipase regulating the degradation of LDs in neurons. This is supported by the fact that the overexpression of dPlins proteins, which are known inhibitors of lipolysis, promotes LD accumulation in photoreceptor neurons. In further support of a neuron-specific TG lipase, the human hereditary spastic paraplegia gene DDHD2, a member of the iPLA1/PAPLA1 family, was proposed to be the main lipase regulating TG metabolism in the mammalian brain. A recent study, showed that Bmm plays a role in the somatic cells of the gonad and in neurons to regulate systemic TG breakdown. It was also suggested that Bmm may play a role in regulating LD turnover in neurons, although this was not directly tested in this study. The results using bmm knock-down and bmm mutants do not support a role of Bmm in the regulation of LD accumulation in photoreceptor neurons. However, the possibility cannot be excluded that Bmm would be required in a subpopulation of neurons to regulate LD content but this would require further analyses. Finally, the possibility cannot be excluded that the overexpression of LD proteins, such as dPlins but also CG7900 or the Klarsicht lipid-binding domain promotes LD accumulation by shielding and stabilizing LDs rather than limiting the access of lipases to LDs. Indeed, stabilization of LDs could well be an ancestral function of PLINs, as reported for yeast and Drosophila adipose tissue. Thus, inhibiting lipolysis and/or stabilizing LDs, allows the formation of LDs, which would be otherwise actively degraded in photoreceptor neurons. This opens avenues to further study LD homeostasis but also their pathophysiological role in diseases of the nervous system (Girard, 2021).

Earlier studies have observed the accumulation of LDs in cellular models of PD. For example, LDs form in SH-SY5Y cells exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a dopaminergic neurotoxin prodrug that causes PD-like symptoms in animal and cellular models. In addition, studies in yeast, rat dopaminergic neurons, and human induced pluripotent stem cells have proposed that αSyn expression induces lipid dysregulation and LD accumulation, but the underlying mechanisms remained unclear. Low levels of αSyn accumulation were hypothezised to perturb lipid homeostasis by enhancing unsaturated FA synthesis and the subsequent accumulation of DGs and TGs. The present study showed that αSyn expression alone did not enhance the accumulation of LDs but instead required concomitant overexpression of a LD protein. Moreover, αSyn expression alone had no effect on DG, TG, or LD content in Drosophila photoreceptor neurons, which indicates that αSyn-induced LDs are not driven by increased TG biosynthesis in this cellular context. Instead, the fact that endogenous αSyn and PLIN3 proteins co-localized at the LD surface in human neuroblastoma cells, suggests that LD-associated αSyn have a direct physiological function in promoting neutral lipid accumulation by inhibiting lipolysis. This hypothesis is supported by experiments in HeLa cells transfected with αSyn, loaded with fatty acids, in which the overexpression of αSyn protects LDs from lipolysis (Girard, 2021).

The results show that LDs contribute to αSyn conversion to proteinase K resistant forms, which indicates that LDs may be involved in the progression of PD pathology. This is an apparent discrepancy with the results in a previous study, in which LDs protect from lipotoxicity cells expressing αSyn. That study used cellular models including yeast cells, and rat cortical neuron primary cultures exposed or not to oleic acid. In such cellular context, it was proposed that αSyn induces the accumulation of toxic diacylglycerol (DG), which is subsequently converted to TG and sequestered into LDs. LDs are thus protective by allowing the sequestration of toxic lipids. In the fly retina study, αSyn expression did not induce TG accumulation. In the Drosophila nervous system, toxic DG may not reach sufficient level to promote photoreceptor toxicity. Interestingly, this difference allowed study of the binding of αSyn to LD and examine their contribution to pathological conversion of αSyn. Indeed, the results suggest an alternative but not mutually exclusive role for LDs in promoting αSyn misfolding and conversion to a proteinase K-resistant form. The increased LD surface could provide a physical platform for αSyn deposition and conversion. In support of this hypothesis, it was previously proposed that αSyn aggregation is facilitated in the presence of synthetic phospholipid vesicles. Thus, the current results point to a direct role of LDs on αSyn resistance to proteinase K digestion (Girard, 2021).

This study showed that the accumulation of LD proteins, such as dPlins, is a prerequisite for the increased LD accumulation induced by αSyn in neurons. This raises the possibility that some physiological or pathological conditions will favor the expression and/or accumulation of LD proteins, which triggers the neuronal accumulation of LDs. Interestingly, it was proposed that age-dependent accumulation of fat and dPlin2 is dependent on the histone deacetylase (HDAC6) in Drosophila. Moreover, an accumulation of LD-containing cells (lipid-laden cells), associated with PLIN2 expression, was observed in meningeal, cortical and neurogenic brain regions of the aging mice. Finally, a recent expression study on all human perlipin proteins (PLIN1-5), found that PLIN2 accumulates, particularly in neurons, in brains of old subjects and of patients with Alzheimer disease. As an alternative putative mechanism regulating LD level, it was shown that targeted degradation of PLIN2 and PLIN3 occurs by chaperone-mediated autophagy (CMA). Thus, in aging tissue with decreased HDAC6 or reduced basal CMA, the accumulation of PLINs may initiate LD accumulation, hence favoring αSyn-induced LD production. In this study, mutations in the central autophagy gene Atg8 did not lead to LD accumulation in Drosophila retina. Thus a more systematic analysis will be required to identify the proteolytic mechanisms regulating dPlins degradation and LD accumulation in the aged Drosophila nervous system (Girard, 2021).

Based on a combination of the current results and these observations, a model is proposed of LD homeostasis in healthy and diseased neurons. In healthy neurons, relatively few LDs are detected due to a combination of low basal rate of TG synthesis, active lipolysis and limited LD shielding capacity. In pathological conditions such as PD, possibly in combination with an age-dependent ectopic fat accumulation and Plin proteins increased expression, αSyn and Plins could cooperate to limit lipolysis and promote the accumulation of LDs in neurons. This could set a vicious cycle in which αSyn enhances Plin-dependent LD stabilization, which, in turn, would increase αSyn conversion to a proteinase K-resistant form, culminating in αSyn aggregation and formation of cytoplasmic inclusion bodies. Collectively, these results raise the possibility that αSyn binding to LDs could be an important step in the pathogenesis of PD (Girard, 2021).

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Hung, Y. C., Huang, K. L., Chen, P. L., Li, J. L., Lu, S. H., Chang, J. C., Lin, H. Y., Lo, W. C., Huang, S. Y., Lee, T. T., Lin, T. Y., Imai, Y., Hattori, N., Liu, C. S., Tsai, S. Y., Chen, C. H., Lin, C. H. and Chan, C. C. (2021). UQCRC1 engages cytochrome c for neuronal apoptotic cell death. Cell Rep 36(12): 109729. PubMed ID: 34551295

Human ubiquinol-cytochrome c reductase core protein 1 (UQCRC1) is an evolutionarily conserved core subunit of mitochondrial respiratory chain complex III. This study recently identified the disease-associated variants of UQCRC1 from patients with familial parkinsonism, but its function remains unclear. This study investigates the endogenous function of UQCRC1 in the human neuronal cell line and the Drosophila nervous system. Flies with neuronal knockdown of uqcrc1 exhibit age-dependent parkinsonism-resembling defects, including dopaminergic neuron reduction and locomotor decline, and are ameliorated by UQCRC1 expression. Lethality of uqcrc1-KO is also rescued by neuronally expressing UQCRC1, but not the disease-causing variant, providing a platform to discern the pathogenicity of this mutation. Furthermore, UQCRC1 associates with the apoptosis trigger cytochrome c (cyt-c), and uqcrc1 deficiency increases Cyt-c in the cytoplasmic fraction and activates the caspase cascade. Depleting cyt-c or expression of the anti-apoptotic p35 ameliorates uqcrc1-mediated neurodegeneration. The findings identified a role for UQCRC1 in regulating cyt-c-induced apoptosis (Hung, 2021).

Parker-Character, J., Hager, D. R., Call, T. B., Pickup, Z. S., Turnbull, S. A., Marshman, E. M., Korch, S. B., Chaston, J. M. and Call, G. B. (2021). An altered microbiome in a Parkinson's disease model Drosophila melanogaster has a negative effect on development. Sci Rep 11(1): 23635. PubMed ID: 34880269

Abstract
Parkinson's disease (PD) is the second most common neurodegenerative disease, besides Alzheimer's Disease, characterized by multiple symptoms, including the well-known motor dysfunctions. It is well-established that there are differences in the fecal microbiota composition between Parkinson's disease (PD) patients and control populations, but the mechanisms underlying these differences are not yet fully understood. To begin to close the gap between description and mechanism, the relationship between the microbiota and PD was studied in a model organism, Drosophila melanogaster. First, fecal transfers were performed with a D. melanogaster model of PD that had a mutation in the parkin (park25) gene. Results indicate that the PD model feces had a negative effect on both pupation and eclosion in both control and park25 flies, with a greater effect in PD model flies. Analysis of the microbiota composition revealed differences between the control and park25 flies, consistent with many human studies. Conversely, gnotobiotic treatment of axenic embryos with feces-derived bacterial cultures did not affect eclosure. It is speculated that this result might be due to similarities in bacterial prevalence between mutant and control feces. Further, a bacteria-potentiated impact on mutant and control fly phenotypes was confirmed by measuring eclosure rate in park25 flies that were mono-associated with members of the fly microbiota. Both the fecal transfer and the mono-association results indicate a host genotype-microbiota interaction. Overall, this study concludes functional effects of the fly microbiota on PD model flies, providing support to the developing body of knowledge regarding the influence of the microbiota on PD.

Ulgherait, M., Midoun, A. M., Park, S. J., Gatto, J. A., Tener, S. J., Siewert, J., Klickstein, N., Canman, J. C., Ja, W. W. and Shirasu-Hiza, M. (2021). Circadian autophagy drives iTRF-mediated longevity. Nature 598(7880): 353-358. PubMed ID: 34588695

Abstract
Time-restricted feeding (TRF) has recently gained interest as a potential anti-ageing treatment for organisms from Drosophila to humans. TRF restricts food intake to specific hours of the day. Because TRF controls the timing of feeding, rather than nutrient or caloric content, TRF has been hypothesized to depend on circadian-regulated functions; the underlying molecular mechanisms of its effects remain unclear. To exploit the genetic tools and well-characterized ageing markers of Drosophila, this study developed an intermittent TRF (iTRF) dietary regimen that robustly extended fly lifespan and delayed the onset of ageing markers in the muscles and gut. iTRF enhanced circadian-regulated transcription, and iTRF-mediated lifespan extension required both circadian regulation and autophagy, a conserved longevity pathway. Night-specific induction of autophagy was both necessary and sufficient to extend lifespan on an ad libitum diet and also prevented further iTRF-mediated lifespan extension. By contrast, day-specific induction of autophagy did not extend lifespan. Thus, these results identify circadian-regulated autophagy as a critical contributor to iTRF-mediated health benefits in Drosophila. Because both circadian regulation and autophagy are highly conserved processes in human ageing, this work highlights the possibility that behavioural or pharmaceutical interventions that stimulate circadian-regulated autophagy might provide people with similar health benefits, such as delayed ageing and lifespan extension.

Li, J., Lim, R. G., Kaye, J. A., Dardov, V., Coyne, A. N., Wu, J., Milani, P., Cheng, A., Thompson, T. G., Ornelas, L., Frank, A., Adam, M., Banuelos, M. G., Casale, M., Cox, V., Escalante-Chong, R., Daigle, J. G., Gomez, E., Hayes, L., Holewenski, R., Lei, S., Lenail, A., Lima, L., Mandefro, B., Matlock, A., Panther, L., Patel-Murray, N. L., Pham, J., Ramamoorthy, D., Sachs, K., Shelley, B., Stocksdale, J., Trost, H., Wilhelm, M., Venkatraman, V., Wassie, B. T., Wyman, S., Yang, S., Van Eyk, J. E., Lloyd, T. E., Finkbeiner, S., Fraenkel, E., Rothstein, J. D., Sareen, D., Svendsen, C. N. and Thompson, L. M. (2021). An integrated multi-omic analysis of iPSC-derived motor neurons from C9ORF72 ALS patients. iScience 24(11): 103221. PubMed ID: 34746695

Abstract
Neurodegenerative diseases are challenging for systems biology because of the lack of reliable animal models or patient samples at early disease stages. Induced pluripotent stem cells (iPSCs) could address these challenges. This study investigated DNA, RNA, epigenetics, and proteins in iPSC-derived motor neurons from patients with ALS carrying hexanucleotide expansions in C9ORF72. Using integrative computational methods combining all omics datasets, this study identified novel and known dysregulated pathways. A C9ORF72 Drosophila model was used to distinguish pathways contributing to disease phenotypes from compensatory ones, and alterations in some pathways were confirmed in postmortem spinal cord tissue of patients with ALS. A different differentiation protocol was used to derive a separate set of C9ORF72 and control motor neurons. Many individual -omics differed by protocol, but some core dysregulated pathways were consistent. This strategy of analyzing patient-specific neurons provides disease-related outcomes with small numbers of heterogeneous lines and reduces variation from single-omics to elucidate network-based signatures.

Manivannan, S. N., Roovers, J., Smal, N., Myers, C. T., Turkdogan, D., Roelens, F., Kanca, O., Chung, H. L., Scholz, T., Hermann, K., Bierhals, T., Caglayan, H. S., Stamberger, H., Mefford, H., de Jonghe, P., Yamamoto, S., Weckhuysen, S. and Bellen, H. J. (2021). De novo FZR1 loss-of-function variants cause developmental and epileptic encephalopathies. Brain. PubMed ID: 34788397

Abstract
FZR1, which encodes the Cdh1 subunit of the Anaphase Promoting Complex, plays an important role in neurodevelopment by regulating the cell cycle and by its multiple post-mitotic functions in neurons. In this study, evaluation of 250 unrelated patients with developmental and epileptic encephalopathies and a connection on GeneMatcher led to the identification of three de novo missense variants in FZR1. Functional studies in Drosophila were performed using three different mutant alleles of the Drosophila homolog of FZR1 fzr. All three individuals carrying de novo variants in FZR1 had childhood onset generalized epilepsy, intellectual disability, mild ataxia and normal head circumference. Two individuals were diagnosed with the developmental and epileptic encephalopathy subtype Myoclonic Atonic Epilepsy. Functional evidence is provided that the missense variants are loss-of-function alleles using Drosophila neurodevelopment assays. Using three fly mutant alleles of the Drosophila homolog fzr and overexpression studies, it was shown that patient variants can affect proper neurodevelopment. This study consolidates the relationship between FZR1 and developmental epileptic encephalopathy, and expands the associated phenotype. It is concluded that heterozygous loss-of-function of FZR1 leads to developmental epileptic encephalopathies associated with a spectrum of neonatal to childhood onset seizure types, developmental delay and mild ataxia. In summary, this approach of targeted sequencing using novel gene candidates and functional testing in Drosophila will help solve undiagnosed myoclonic atonic epilepsy or developmental epileptic encephalopathy cases.

Pant, C., Chakrabarti, M., Mendonza, J. J., Ganganna, B., Pabbaraja, S. and Pal Bhadra, M. (2021). Aza-Flavanone Diminishes Parkinsonism in the Drosophila melanogaster Parkin Mutant. ACS Chem Neurosci. PubMed ID: 34763419

Abstract
Parkinson's disease is a chronic and progressive neurodegenerative disease, induced by slow and progressive death of the dopaminergic (DA) neurons from the midbrain region called substantia nigra (SNc) leading to difficulty in locomotion. At present, very few potential therapeutic drugs are available for treatment, necessitating an urgent need for development. In this current study, the parkin transgenic Drosophila melanogaster model that induces selective loss in dopaminergic neurons and impairment of locomotory functions has been used to see the effect of the aza-flavanone molecule. D. melanogaster serves as an amazing in vivo model making valuable contribution in the development of promising treatment strategies. In-silico study showed spontaneous binding of this molecule to the D2 receptor making it a potential dopamine agonist. PARKIN protein is well conserved, and it has been reported that Drosophila PARKIN is 42% identical to human PARKIN. Interestingly, this molecule enhances the motor coordination and survivability rate of the transgenic flies along with an increase in expression of the master regulator of Dopamine synthesis, that is, tyrosine hydroxylase (TH), in the substantia nigra region of the fly brain. Moreover, it plays a significant effect on mitochondrial health and biogenesis via modulation of a conserved mitochondrial protein PHB2. Therefore, this molecule could lead to the development of an effective therapeutic approach for the treatment of PD.

Kowada, R., Kodani, A., Ida, H., Yamaguchi, M., Lee, I. S., Okada, Y. and Yoshida, H. (2021). The function of Scox in glial cells is essential for locomotive ability in Drosophila. Sci Rep 11(1): 21207. PubMed ID: 34707123

Abstract
Synthesis of cytochrome c oxidase (Scox) is a Drosophila homolog of human SCO2 encoding a metallochaperone that transports copper to cytochrome c, and is an essential protein for the assembly of cytochrome c oxidase in the mitochondrial respiratory chain complex. SCO2 is highly conserved in a wide variety of species across prokaryotes and eukaryotes, and mutations in SCO2 are known to cause mitochondrial diseases such as fatal infantile cardioencephalomyopathy, Leigh syndrome, and Charcot-Marie-Tooth disease, a neurodegenerative disorder. These diseases have a common symptom of locomotive dysfunction. However, the mechanisms of their pathogenesis remain unknown, and no fundamental medications or therapies have been established for these diseases. This study demonstrated that the glial cell-specific knockdown of Scox perturbs the mitochondrial morphology and function, and locomotive behavior in Drosophila. In addition, the morphology and function of synapses were impaired in the glial cell-specific Scox knockdown. Furthermore, Scox knockdown in ensheathing glia, one type of glial cell in Drosophila, resulted in larval and adult locomotive dysfunction. This study suggests that the impairment of Scox in glial cells in the Drosophila CNS mimics the pathological phenotypes observed by mutations in the SCO2 gene in humans.

Licata, N. V., Cristofani, R., Salomonsson, S., Wilson, K. M., Kempthorne, L., Vaizoglu, D., D'Agostino, V. G., Pollini, D., Loffredo, R., Pancher, M., Adami, V., Bellosta, P., Ratti, A., Viero, G., Quattrone, A., Isaacs, A. M., Poletti, A. and Provenzani, A. (2021). C9orf72 ALS/FTD dipeptide repeat protein levels are reduced by small molecules that inhibit PKA or enhance protein degradation. EMBO J: e105026. PubMed ID: 34791698

Abstract
Intronic GGGGCC (G4C2) hexanucleotide repeat expansion within the human C9orf72 gene represents the most common cause of familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of repeat-containing C9orf72 RNA results in the production of neurotoxic dipeptide-repeat proteins (DPRs). This study developed a high-throughput drug screen for the identification of positive and negative modulators of DPR levels. HSP90 inhibitor geldanamycin and aldosterone antagonist spironolactone were found to reduce DPR levels by promoting protein degradation via the proteasome and autophagy pathways respectively. Surprisingly, cAMP-elevating compounds boosting protein kinase A (PKA) activity increased DPR levels. Inhibition of PKA activity, by both pharmacological and genetic approaches, reduced DPR levels in cells and rescued pathological phenotypes in a Drosophila model of C9ALS/FTD. Moreover, knockdown of PKA-catalytic subunits correlated with reduced translation efficiency of DPRs, while the PKA inhibitor H89 reduced endogenous DPR levels in C9ALS/FTD patient-derived iPSC motor neurons. Together, these results suggest new and druggable pathways modulating DPR levels in C9ALS/FTD.

Lee, H., Lee, J. J., Park, N. Y., Dubey, S. K., Kim, T., Ruan, K., Lim, S. B., Park, S. H., Ha, S., Kovlyagina, I., Kim, K. T., Kim, S., Oh, Y., Kim, H., Kang, S. U., Song, M. R., Lloyd, T. E., Maragakis, N. J., Hong, Y. B., Eoh, H. and Lee, G. (2021). Multi-omic analysis of selectively vulnerable motor neuron subtypes implicates altered lipid metabolism in ALS. Nat Neurosci. PubMed ID: 34782793

Abstract
Amyotrophic lateral sclerosis (ALS) is a devastating disorder in which motor neurons degenerate, the causes of which remain unclear. In particular, the basis for selective vulnerability of spinal motor neurons (sMNs) and resistance of ocular motor neurons to degeneration in ALS has yet to be elucidated. This study applied comparative multi-omics analysis of human induced pluripotent stem cell-derived sMNs and ocular motor neurons to identify shared metabolic perturbations in inherited and sporadic ALS sMNs, revealing dysregulation in lipid metabolism and its related genes. Targeted metabolomics studies confirmed such findings in sMNs of 17 ALS (SOD1, C9ORF72, TDP43 (TARDBP) and sporadic) human induced pluripotent stem cell lines, identifying elevated levels of arachidonic acid. Pharmacological reduction of arachidonic acid levels was sufficient to reverse ALS-related phenotypes in both human sMNs and in vivo in Drosophila and SOD1(G93A) mouse models. Collectively, these findings pinpoint a catalytic step of lipid metabolism as a potential therapeutic target for ALS.

Rivera, M. J., Contreras, A., Nguyen, L. T., Eldon, E. D. and Klig, L. S. (2021). Regulated inositol synthesis is critical for balanced metabolism and development in Drosophila melanogaster. Biol Open 10(10). PubMed ID: 34710213

Abstract
Myo-inositol is a precursor of the membrane phospholipid, phosphatidylinositol (PI). It is involved in many essential cellular processes including signal transduction, energy metabolism, endoplasmic reticulum stress, and osmoregulation. Inositol is synthesized from glucose-6-phosphate by myo-inositol-3-phosphate synthase (MIPSp). The Drosophila melanogaster Inos gene encodes MIPSp. Abnormalities in myo-inositol metabolism have been implicated in type 2 diabetes, cancer, and neurodegenerative disorders. Obesity and high blood (hemolymph) glucose are two hallmarks of diabetes, which can be induced in Drosophila melanogaster third-instar larvae by high-sucrose diets. This study shows that dietary inositol reduces the obese-like and high-hemolymph glucose phenotypes of third-instar larvae fed high-sucrose diets. Furthermore, this study demonstrates Inos mRNA regulation by dietary inositol; when more inositol is provided there is less Inos mRNA. Third-instar larvae with dysregulated high levels of Inos mRNA and MIPSp show dramatic reductions of the obese-like and high-hemolymph glucose phenotypes. These strains, however, also display developmental defects and pupal lethality. The few individuals that eclose die within two days with striking defects: structural alterations of the wings and legs, and heads lacking proboscises. This study is an exciting extension of the use of Drosophila melanogaster as a model organism for exploring the junction of development and metabolism.

Scharenbrock, A. R., Katzenberger, R. J., Fischer, M. C., Ganetzky, B. and Wassarman, D. A. (2021). Beta-blockers reduce intestinal permeability and early mortality following traumatic brain injury in Drosophila. MicroPubl Biol 2021. PubMed ID: 34723144

Abstract
Traumatic brain injury (TBI) frequently leads to non-neurological consequences such as intestinal permeability. The beta-blocker drug labetalol, which inhibits binding of catecholamine neurotransmitters to adrenergic receptors, reduces intestinal permeability in a rat TBI model. Using a Drosophila melanogaster TBI model, previous studies found a strong positive correlation between intestinal permeability and mortality within 24 hours of TBI in a standard laboratory line (w1118) and across genetically diverse inbred lines from the Drosophila Genetic Reference Panel (DGRP). This study reports that feeding injured w1118 flies the beta-blockers labetalol and metoprolol reduced intestinal permeability and mortality. Additionally, metoprolol reduced intestinal permeability when 18 DGRP fly lines were analyzed in aggregate, but neither beta-blocker affected mortality. These data indicate that the mechanism underlying disruption of the intestinal barrier by adrenergic signaling following TBI is conserved between humans and flies and that mortality following TBI in flies is not strictly dependent on disruption of the intestinal barrier. Thus, the fly TBI model is useful for shedding light on the mechanism and consequences of adrenergic signaling between the brain and intestine following TBI in humans.

Wang, Y. and Westermark, G. T. (2021). The Amyloid Forming Peptides Islet Amyloid Polypeptide and Amyloid beta Interact at the Molecular Level. Int J Mol Sci 22(20). PubMed ID: 34681811

Abstract
Epidemiological studies support a connection between the two common disorders, type-2 diabetes and Alzheimer's disease. Both conditions have local amyloid formation in their pathogenesis, and cross-seeding between islet amyloid polypeptide (IAPP) and amyloid β (Aβ) could constitute the link. The bimolecular fluorescence complementation (BiFC) assay was used to investigate the occurrence of heterologous interactions between IAPP and Aβ and to compare the potential toxic effects of IAPP/Aβ, IAPP/IAPP, and Aβ/Aβ expression in living cells. Microscopy was used to confirm the fluorescence and determine the lysosomal, mitochondrial areas and mitochondrial membrane potential, and a FACS analysis was used to determine ROS production and the role for autophagy. Drosophila melanogaster expressing IAPP and Aβ was used to study their co-deposition and effects on longevity. Co-expression of IAPP and Aβ resulted in fluorophore reconstitution to the same extent as determined for homologous IAPP/IAPP or Aβ/Aβ expression. The BiFC(+)/BiFC(-) ratio of lysosomal area calculations increased in transfected cells independent of the vector combinations, while only Aβ/Aβ expression increased mitochondrial membrane potential. Expression combinations containing Aβ were necessary for the formation of a congophilic amyloid. In Drosophila melanogaster expressing IAPP/Aβ, co-deposition of the amyloid-forming peptides caused reduced longevity. The BiFC results confirmed a heterologous interaction between IAPP and Aβ, while co-deposits in the brain of Drosophila suggest mixed amyloid aggregates.

Yamazoe, T., Nakahara, Y., Katsube, H. and Inoue, Y. H. (2021). Expression of Human Mutant Preproinsulins Induced Unfolded Protein Response, Gadd45 Expression, JAK-STAT Activation, and Growth Inhibition in Drosophila. Int J Mol Sci 22(21). PubMed ID: 34769468

Abstract
Mutations in the insulin gene (INS) are frequently associated with human permanent neonatal diabetes mellitus. However, the mechanisms underlying the onset of this genetic disease is not sufficiently decoded. This study induced expression of two types of human mutant INSs in Drosophila using its ectopic expression system and investigated the resultant responses in development. Expression of the wild-type preproinsulin in the insulin-producing cells (IPCs) throughout the larval stage led to a stimulation of the overall and wing growth. However, ectopic expression of human mutant preproinsulins, hINS(C96Y) and hINS(LB15YB16delinsH), neither of which secreted from the β-cells, could not stimulate the Drosophila growth. Furthermore, neither of the mutant polypeptides induced caspase activation leading to apoptosis. Instead, they induced expression of several markers indicating the activation of unfolded protein response, such as ER stress-dependent Xbp1 mRNA splicing and ER chaperone induction. The mutant polypeptides were found to induce the expression of Growth arrest and DNA-damage-inducible 45 (Gadd45) in imaginal disc cells. ER stress induced by hINS(C96Y) also activated the JAK-STAT signaling, involved in inflammatory responses. Collectively, it is speculated that the diabetes-like growth defects appeared as a consequence of the human mutant preproinsulin expression was involved in dysfunction of the IPCs, rather than apoptosis.

Yap, Z. Y., Efthymiou, S., Seiffert, S., ..., Houlden, H. and Yoon, W. H. (2021). Bi-allelic variants in OGDHL cause a neurodevelopmental spectrum disease featuring epilepsy, hearing loss, visual impairment, and ataxia. Am J Hum Genet. PubMed ID: 34800363

Abstract
The 2-oxoglutarate dehydrogenase-like (OGDHL) protein is a rate-limiting enzyme in the Krebs cycle that plays a pivotal role in mitochondrial metabolism. OGDHL expression is restricted mainly to the brain in humans. This study reports nine individuals from eight unrelated families carrying bi-allelic variants in OGDHL with a range of neurological and neurodevelopmental phenotypes including epilepsy, hearing loss, visual impairment, gait ataxia, microcephaly, and hypoplastic corpus callosum. The variants include three homozygous missense variants (p.Pro852Ala, p.Arg244Trp, and p.Arg299Gly), three compound heterozygous single-nucleotide variants (p.Arg673Gln/p.Val488Val, p.Phe734Ser/p.Ala327Val, and p.Trp220Cys/p.Asp491Val), one homozygous frameshift variant (p.Cys553Leufs(∗)16), and one homozygous stop-gain variant (p.Arg440Ter). To support the pathogenicity of the variants, a novel CRISPR-Cas9-mediated tissue-specific knockout was developed with cDNA rescue system for dOgdh, the Drosophila ortholog of human OGDHL. Pan-neuronal knockout of dOgdh led to developmental lethality as well as defects in Krebs cycle metabolism, which was fully rescued by expression of wild-type dOgdh. Studies using the Drosophila system indicate that p.Arg673Gln, p.Phe734Ser, and p.Arg299Gly are severe loss-of-function alleles, leading to developmental lethality, whereas p.Pro852Ala, p.Ala327Val, p.Trp220Cys, p.Asp491Val, and p.Arg244Trp are hypomorphic alleles, causing behavioral defects. Transcript analysis from fibroblasts obtained from the individual carrying the synonymous variant (c.1464T>C [p.Val488Val]) in family 2 showed that the synonymous variant affects splicing of exon 11 in OGDHL. Human neuronal cells with OGDHL knockout exhibited defects in mitochondrial respiration, indicating the essential role of OGDHL in mitochondrial metabolism in humans. Together, these data establish that the bi-allelic variants in OGDHL are pathogenic, leading to a Mendelian neurodevelopmental disease in humans.

Xiao, G., Zhao, M., Liu, Z., Du, F. and Zhou, B. (2021). Zinc antagonizes iron-regulation of tyrosine hydroxylase activity and dopamine production in Drosophila melanogaster. BMC Biol 19(1): 236. PubMed ID: 34732185

Abstract
Dopamine (DA) is a neurotransmitter that plays roles in movement, cognition, attention, and reward responses, and deficient DA signaling is associated with the progression of a number of neurological diseases, such as Parkinson's disease. Due to its critical functions, DA expression levels in the brain are tightly controlled, with one important and rate-limiting step in its biosynthetic pathway being catalyzed by tyrosine hydroxylase (TH), an enzyme that uses iron ion (Fe(2+)) as a cofactor. A role for metal ions has additionally been associated with the etiology of Parkinson's disease. However, the way dopamine synthesis is regulated in vivo or whether regulation of metal ion levels is a component of DA synthesis is not fully understood. This study analyzed the role of Catsup, the Drosophila ortholog of the mammalian zinc transporter SLC39A7 (ZIP7), in regulating dopamine levels. Catsup was found to be a functional zinc transporter that regulates intracellular zinc distribution between the ER/Golgi and the cytosol. Loss-of-function of Catsup leads to increased DA levels, and the increased dopamine production was shown to be due to a reduction in zinc levels in the cytosol. Zinc ion (Zn(2+)) negatively regulates dopamine synthesis through direct inhibition of TH activity, by antagonizing Fe(2+) binding to TH, thus rendering the enzyme ineffective or non-functional. These findings uncovered a previously unknown mechanism underlying the control of cellular dopamine expression, with normal levels of dopamine synthesis being maintained through a balance between Fe(2+) and Zn(2+) ions. The findings also provide support for metal modulation as a possible therapeutic strategy in the treatment of Parkinson's disease and other dopamine-related diseases.

AYazar, V., Kang, S. U., Ha, S., Dawson, V. L. and Dawson, T. M. (2021). Integrative genome-wide analysis of dopaminergic neuron-specific PARIS expression in Drosophila dissects recognition of multiple PPAR-γ associated gene regulation. Sci Rep 11(1): 21500. PubMed ID: 34728675

Abstract
The transcriptional repressor called parkin interacting substrate (PARIS; ZNF746) was initially identified as a novel co-substrate of parkin and PINK1 that leads to Parkinson's disease (PD) by disrupting mitochondrial biogenesis through peroxisome proliferator-activated receptor gamma (PPARγ) coactivator -1α (PGC-1α) suppression. Since its initial discovery, growing evidence has linked PARIS to defective mitochondrial biogenesis observed in PD pathogenesis. This study employed conditional translating ribosome affinity purification (TRAP) followed by RNA sequencing (TRAP-seq) for transcriptome profiling of DA neurons in transgenic Drosophila lines expressing human PARIS wild type (WT) or mutant (C571A). The results demonstrated that PPARγ functions as a master regulator of PARIS-induced molecular changes at the transcriptome level, confirming that PARIS acts primarily on PGC-1α to lead to neurodegeneration in PD. Moreover, this study identified that PARIS actively modulates expression of PPARγ target genes by physically binding to the promoter regions. Together, this work revealed how PARIS drives adverse effects on modulation of PPAR-γ associated gene clusters in DA neurons.

Cheng, X., Xie, M., Luo, L., Tian, Y., Yu, G., Wu, Q., Fan, X., Yang, D., Mao, X., Gaur, U. and Yang, M. (2022). Inhibitor GSK690693 extends Drosophila lifespan via reduce AKT signaling pathway. Mech Ageing Dev 202: 111633. PubMed ID: 35065134

Abstract
Aging is a process involving physiological changes that lead to the decline of biological functions of various tissues and organs of the body. Therefore, it is crucial to find anti-aging drugs that can intervene with the changes induced because of aging and slow down the degeneration of the biological functions. Among many signaling pathways linked with aging and aging-related diseases, PI3K-AKT signaling pathway has attracted major attention in aging biology. This paper demonstrates that AKT inhibitor GSK690693 can extend lifespan in Drosophila irrespective of start of the treatment from the beginning of life or the mid-life. Effect of GSK690693 for lifespan extension has been primarily related to the improvements in oxidative resistance, intestinal integrity and increased autophagy, but not in physical activity or starvation resistance. Furthermore, GSK690693 treatment reduced the activation of AKT and ERK, consequently activating FOXO, GSK-3β and apoptosis to modulate longevity of flies. Remarkably, GSK690693 did not induce hyperglycemia after treatment. The results indicate that GSK690693 may become an effective compound for anti-aging intervention.

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De Lazzari, F., Agostini, F., Plotegher, N., Sandre, M., Greggio, E., Megighian, A., Bubacco, L., Sandrelli, F., Whitworth, A. J. and Bisaglia, M. (2023). DJ-1 promotes energy balance by regulating both mitochondrial and autophagic homeostasis. Neurobiol Dis 176: 105941. PubMed ID: 36473592

Abstract
The protein DJ-1 is mutated in rare familial forms of recessive Parkinson's disease and in parkinsonism accompanied by amyotrophic lateral sclerosis symptoms and dementia. DJ-1 is considered a multitasking protein able to confer protection under various conditions of stress. However, the precise cellular function still remains elusive. In the present work, fruit flies lacking the expression of the DJ-1 homolog dj-1β were assessed as compared to control aged-matched individuals. Behavioral evaluations included lifespan, locomotion in an open field arena, sensitivity to oxidative insults, and resistance to starvation. Molecular analyses were carried out by analyzing the mitochondrial morphology and functionality, and the autophagic response. It was demonstrated that dj-1β null mutant flies are hypoactive and display higher sensitivity to oxidative insults and food deprivation. Analysis of mitochondrial homeostasis revealed that loss of dj-1β leads to larger and more circular mitochondria, characterized by impaired complex-I-linked respiration while preserving ATP production capacity. Additionally, dj-1β null mutant flies present an impaired autophagic response, which is suppressed by treatment with the antioxidant molecule N-Acetyl-L-Cysteine. Overall, these data point to a mechanism whereby DJ-1 plays a critical role in the maintenance of energy homeostasis, by sustaining mitochondrial homeostasis and affecting the autophagic flux through the maintenance of the cellular redox state. In light of the involvement of DJ-1 in neurodegenerative diseases and considering that neurons are highly energy-demanding cells, particularly sensitive to redox stress, this study sheds light on a key role of DJ-1 in the maintenance of cellular homeostasis.

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Agostini, F., Bubacco, L., Chakrabarti, S. and Bisaglia, M. (2023). α-Synuclein Toxicity in Drosophila melanogaster Is Enhanced by the Presence of Iron: Implications for Parkinson's Disease. Antioxidants (Basel) 12(2). PubMed ID: 36829820

Abstract
Parkinson's disease (PD) is the second most common neurodegenerative disorder, characterized by the preferential loss of dopaminergic neurons and by the accumulation of intracellular inclusions mainly composed of α-synuclein (&alpha-Syn). While the etiopathogenesis of the disorder is still elusive, recent experimental evidence supports the involvement of ferroptosis, an iron-dependent cell death pathway, in the pathogenesis of PD. Using different ferroptosis inducers and inhibitors, this study evaluated, in vivo, the involvement of iron in the α-Syn-mediated toxicity. Using a Drosophila melanogaster model of PD based on the selective over-expression of α-Syn within dopaminergic neurons, this study demonstrated that the over-expression of α-Syn promotes the accumulation of protein aggregates, which is accompanied by dopaminergic neurodegeneration, locomotor impairment, and lifespan reduction. These pathological phenotypes were further exacerbated by reduced intracellular levels of glutathione or increased concentrations of iron. Coherently, both the use of an iron chelator and the presence of the antioxidant compound N-acetylcysteine exerted protective effects. Overall, these results support the involvement of ferroptosis in the α-Syn-mediated toxicity.

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Carvajal-Oliveros, A., Dominguez-Baleon, C., Sanchez-Diaz, I., Zambrano-Tipan, D., Hernandez-Vargas, R., Campusano, J. M., Narváez-Padilla, V. and Reynaud, E. (2023). Parkinsonian phenotypes induced by Synphilin-1 expression are differentially contributed by serotonergic and dopaminergic circuits and suppressed by nicotine treatment. PLoS One 18(3): e0282348. PubMed ID: 36857384

Abstract
Synphilin-1 is a protein encoded by the human SNCAIP gene whose function has yet to be fully understood. However, it has been linked to familial Parkinson's disease (PD). Synphilin-1 is a major component of the Lewy bodies found in neurons in the substantia nigra pars compacta of PD patients. Synphilin-1 expression in serotonergic and/or dopaminergic neurons of Drosophila melanogaster induces neurodegeneration, as well as motor and non-motor PD like symptoms. This work examined the contribution of the serotonergic and dopaminergic circuits in the development of PD-like phenotypes. It was found that olfactory and visual symptoms are majorly contributed by the Parkinson's disease (PD). serotonergic system, and that motor symptoms and reduction in survival are mainly contributed by the dopaminergic system. Chronic nicotine treatment was able to suppress several of these symptoms. These results indicate that both the serotonergic and dopaminergic systems contribute to different aspects of PD symptomatology and that nicotine has beneficial effects on specific symptoms.

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Agostini, F., Bubacco, L., Chakrabarti, S. and Bisaglia, M. (2023). α-Synuclein Toxicity in Drosophila melanogaster Is Enhanced by the Presence of Iron: Implications for Parkinson's Disease. Antioxidants (Basel) 12(2). PubMed ID: 36829820

Abstract
Parkinson's disease (PD) is the second most common neurodegenerative disorder, characterized by the preferential loss of dopaminergic neurons and by the accumulation of intracellular inclusions mainly composed of α-synuclein (&alpha-Syn). While the etiopathogenesis of the disorder is still elusive, recent experimental evidence supports the involvement of ferroptosis, an iron-dependent cell death pathway, in the pathogenesis of PD. Using different ferroptosis inducers and inhibitors, this study evaluated, in vivo, the involvement of iron in the α-Syn-mediated toxicity. Using a Drosophila melanogaster model of PD based on the selective over-expression of α-Syn within dopaminergic neurons, this study demonstrated that the over-expression of α-Syn promotes the accumulation of protein aggregates, which is accompanied by dopaminergic neurodegeneration, locomotor impairment, and lifespan reduction. These pathological phenotypes were further exacerbated by reduced intracellular levels of glutathione or increased concentrations of iron. Coherently, both the use of an iron chelator and the presence of the antioxidant compound N-acetylcysteine exerted protective effects. Overall, these results support the involvement of ferroptosis in the α-Syn-mediated toxicity.

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Usher, J. L., Sanchez-Martinez, A., Terriente-Felix, A., Chen, P. L., Lee, J. J., Chen, C. H. and Whitworth, A. J. (2022). Parkin drives pS65-Ub turnover independently of canonical autophagy in Drosophila. EMBO Rep: e202153552. PubMed ID: 36250243

Abstract
Parkinson's disease-related proteins, PINK1 and Parkin, act in a common pathway to maintain mitochondrial quality control. While the PINK1-Parkin pathway can promote autophagic mitochondrial turnover (mitophagy) following mitochondrial toxification in cell culture, alternative quality control pathways are suggested. To analyse the mechanisms by which the PINK1-Parkin pathway operates in vivo, methods were developed to detect Ser65-phosphorylated ubiquitin (pS65-Ub) in Drosophila. Exposure to the oxidant paraquat led to robust, Pink1-dependent pS65-Ub production, while pS65-Ub accumulates in unstimulated parkin-null flies, consistent with blocked degradation. Additionally, it was shown that pS65-Ub specifically accumulates on disrupted mitochondria in vivo. Depletion of the core autophagy proteins Atg1, Atg5 and Atg8a did not cause pS65-Ub accumulation to the same extent as loss of parkin, and overexpression of parkin promoted turnover of both basal and paraquat-induced pS65-Ub in an Atg5-null background. Thus, this study has established that pS65-Ub immunodetection can be used to analyse Pink1-Parkin function in vivo as an alternative to reporter constructs. Moreover, the findings suggest that the Pink1-Parkin pathway can promote mitochondrial turnover independently of canonical autophagy in vivo.

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Wang, X., Rimal, S., Tantray, I., Geng, J., Bhurtel, S., Khaket, T. P., Li, W., Han, Z. and Lu, B. (2022). Prevention of ribosome collision-induced neuromuscular degeneration by SARS CoV-2-encoded Nsp1. Proc Natl Acad Sci U S A 119(42): e2202322119. PubMed ID: 36170200

Abstract
An overarching goal of aging and age-related neurodegenerative disease research is to discover effective therapeutic strategies applicable to a broad spectrum of neurodegenerative diseases. Little is known about the extent to which targetable pathogenic mechanisms are shared among these seemingly diverse diseases. Translational control is critical for maintaining proteostasis during aging. Gaining control of the translation machinery is also crucial in the battle between viruses and their hosts. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing COVID-19 pandemic. This study shows that overexpression of SARS-CoV-2-encoded nonstructural protein 1 (Nsp1) robustly rescued neuromuscular degeneration and behavioral phenotypes in Drosophila models of Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. These diseases share a common mechanism: the accumulation of aberrant protein species due to the stalling and collision of translating ribosomes, leading to proteostasis failure. Genetic and biochemical analyses revealed that Nsp1 acted in a multipronged manner to resolve collided ribosomes, abort stalled translation, and remove faulty translation products causative of disease in these models, at least in part through the ribosome recycling factor ABCE1, ribosome-associated quality-control factors, autophagy, and AKT signaling. Nsp1 exhibited exquisite specificity in its action, as it did not modify other neurodegenerative conditions not known to be associated with ribosome stalling. These findings uncover a previously unrecognized mechanism of Nsp1 in manipulating host translation, which can be leveraged for combating age-related neurodegenerative diseases that are affecting millions of people worldwide and currently without effective treatment.

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Li, J., Amoh, B. K., McCormick, E., Tarkunde, A., Zhu, K. F., Perez, A., Mair, M., Moore, J., Shulman, J. M., Al-Ramahi, I. and Botas, J. (2023). Integration of transcriptome-wide association study with neuronal dysfunction assays provides functional genomics evidence for Parkinson's disease genes. Hum Mol Genet 32(4): 685-695. PubMed ID: 36173927

Abstract
Genome-wide association studies (GWAS) have markedly advanced understanding of the genetics of Parkinson's disease (PD), but they currently do not account for the full heritability of PD. This study presents an integrative approach that combines transcriptome-wide association study (TWAS) with high-throughput neuronal dysfunction analyses in Drosophila to discover and validate candidate PD genes. 160 candidate genes were identified whose misexpression is associated with PD risk via TWAS. Candidates were validated using orthogonal in silico methods and found to be functionally related to PD-associated pathways (i.e. endolysosome). These TWAS-predicted transcriptomic alterations were mimicked in a Drosophila PD model, and it was discovered that 50 candidates can modulate α-Synuclein (α-Syn)-induced neurodegeneration, allowing new genes to be nominated in previously known PD loci. This study also uncovered additional novel PD candidate genes within GWAS suggestive loci (e.g. TTC19, ADORA2B, LZTS3, NRBP1, HN1L), which are also supported by clinical and functional evidence. These findings deepen understanding of PD, and support applying the integrative approach to other complex trait disorders.

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Maor, G., Dubreuil, R. R. and Feany, M. B. (2023). α-synuclein promotes neuronal dysfunction and death by disrupting the binding of ankyrin to β-spectrin. J Neurosci. PubMed ID: 36653193

Abstract
α-synuclein plays a key role in the pathogenesis of Parkinson's disease and related disorders, but critical interacting partners and molecular mechanisms mediating neurotoxicity are incompletely understood. This study shows that α-synuclein binds directly to β-spectrin. Using males and females in a Drosophila model of α-synuclein-related disorders this study demonstrated that β-spectrin is critical for α-synuclein neurotoxicity. Further, the ankyrin binding domain of β-spectrin is required for α-synuclein binding and neurotoxicity. A key plasma membrane target of ankyrin, Na(+)/K(+) ATPase, is mislocalized when human α-synuclein is expressed in Drosophila. Accordingly, membrane potential is depolarized in α-synuclein transgenic fly brains. The same pathway was examined in human neurons and it was found that Parkinson's disease patient-derived neurons with a triplication of the α-synuclein locus show disruption of the spectrin cytoskeleton, mislocalization of ankyrin and Na(+)/K(+) ATPase, and membrane potential depolarization. These findings define a specific molecular mechanism by which elevated levels of α-synuclein in Parkinson's disease and related α-synucleinopathies leads to neuronal dysfunction and death.

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De Lazzari, F., Agostini, F., Plotegher, N., Sandre, M., Greggio, E., Megighian, A., Bubacco, L., Sandrelli, F., Whitworth, A. J. and Bisaglia, M. (2023). DJ-1 promotes energy balance by regulating both mitochondrial and autophagic homeostasis. Neurobiol Dis 176: 105941. PubMed ID: 36473592

Abstract
The protein DJ-1 is mutated in rare familial forms of recessive Parkinson's disease and in parkinsonism accompanied by amyotrophic lateral sclerosis symptoms and dementia. DJ-1 is considered a multitasking protein able to confer protection under various conditions of stress. However, the precise cellular function still remains elusive. In the present work, fruit flies lacking the expression of the DJ-1 homolog dj-1β were assessed as compared to control aged-matched individuals. Behavioral evaluations included lifespan, locomotion in an open field arena, sensitivity to oxidative insults, and resistance to starvation. Molecular analyses were carried out by analyzing the mitochondrial morphology and functionality, and the autophagic response. It was demonstrated that dj-1β null mutant flies are hypoactive and display higher sensitivity to oxidative insults and food deprivation. Analysis of mitochondrial homeostasis revealed that loss of dj-1β leads to larger and more circular mitochondria, characterized by impaired complex-I-linked respiration while preserving ATP production capacity. Additionally, dj-1β null mutant flies present an impaired autophagic response, which is suppressed by treatment with the antioxidant molecule N-Acetyl-L-Cysteine. Overall, these data point to a mechanism whereby DJ-1 plays a critical role in the maintenance of energy homeostasis, by sustaining mitochondrial homeostasis and affecting the autophagic flux through the maintenance of the cellular redox state. In light of the involvement of DJ-1 in neurodegenerative diseases and considering that neurons are highly energy-demanding cells, particularly sensitive to redox stress, this study sheds light on a key role of DJ-1 in the maintenance of cellular homeostasis.

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Dhanushkodi, N. R., Abul Khair, S. B., Ardah, M. T. and Haque, M. E. (2023). ATP13A2 Gene Silencing in Drosophila Affects Autophagic Degradation of A53T Mutant α-Synuclein. Int J Mol Sci 24(2). PubMed ID: 36675288

Abstract
Mutations in ATP13A2 (PARK9), an autophagy-related protein, cause Kufor-Rakeb syndrome, an autosomal recessive, juvenile-onset form of parkinsonism. α-Synuclein (α-syn) is a presynaptic neuronal protein that forms toxic aggregates in Parkinson's disease (PD). α-syn aggregation and autophagic flux in ATP13A2-knockdown occurs Drosophila expressing either wild-type (WT) or mutant α-syn. Dopaminergic (DA) neuron loss was studied by confocal microscopy. Sleep and circadian activity were evaluated in young and old flies using a Drosophila activity monitor. Thirty-day-old ATP13A2-RNAi A53T-α-syn flies had increased Triton-insoluble α-syn levels, compared to control A53T-α-syn flies without ATP13A2-RNAi. Whole-brain staining revealed significantly fewer dopaminergic (DA) neurons in the PPL2 cluster of 30-day-old ATP13A2-RNAi flies expressing WT-, A30P-, and A53T-α-syn than in that of controls. In ATP13A2-RNAi A53T-α-syn flies, autophagic flux was decreased, as indicated by increased accumulation of Ref(2)P, the Drosophila p62 homologue. ATP13A2 silencing decreased total locomotor activity in young, and enhanced sleep features, similar to PD (decreasing bout length), in old flies expressing A53T-α-syn. ATP13A2 silencing also altered the circadian locomotor activity of A30P- and A53T-α-syn flies. Thus, ATP13A2 may play a role in the autophagic degradation of A53T-α-syn.

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Dumitrescu, E., Copeland, J. M. and Venton, B. J. (2023). Parkin Knockdown Modulates Dopamine Release in the Central Complex, but Not the Mushroom Body Heel, of Aging Drosophila. ACS Chem Neurosci 14(2): 198-208. PubMed ID: 36576890

Abstract
Parkinson's disease (PD) is characterized by progressive degeneration of dopaminergic neurons leading to reduced locomotion. Mutations of parkin gene in Drosophila produce the same phenotypes as vertebrate models, but the effect of parkin knockdown on dopamine release is not known. This study reports age-dependent, spatial variation of dopamine release in the brain of parkin-RNAi adult Drosophila. Dopamine was repetitively stimulated by local application of acetylcholine and quantified by fast-scan cyclic voltammetry in the central complex or mushroom body heel. In the central complex, the main area controlling locomotor function, dopamine release is maintained for repeated stimulations in aged control flies, but lower concentrations of dopamine are released in the central complex of aged parkin-RNAi flies. In the mushroom body heel, the dopamine release decrease in older parkin-RNAi flies is similar to controls. There is not significant dopaminergic neuronal loss even in older parkin knockdown flies, which indicates that the changes in stimulated dopamine release are due to alterations of neuronal function. In young parkin-RNAi flies, locomotion is inhibited by 30%, while in older parkin-RNAi flies it is inhibited by 85%. Overall, stimulated dopamine release is modulated by Parkin in an age and brain region dependent manner. Correlating the functional state of the dopaminergic system with behavioral phenotypes provides unique insights into the PD mechanism. Drosophila can be used to study dopamine functionality in PD, elucidate how genetics influence dopamine, and test potential therapies to maintain dopamine release.

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Ren, M., Yang, Y., Heng, K. H. Y., Ng, L. Y., Chong, C. Y., Ng, Y. T., Gorur-Shandilya, S., Lee, R. M. Q., Lim, K. L., Zhang, J. and Koh, T. W. (2022). MED13 and glycolysis are conserved modifiers of alpha-synuclein-associated neurodegeneration. Cell Rep 41(12): 111852. PubMed ID: 36543134

Abstract
alpha-Synuclein (α-syn) is important in synucleinopathies such as Parkinson's disease (PD). While genome-wide association studies (GWASs) of synucleinopathies have identified many risk loci, the underlying genes have not been shown for most loci. Using Drosophila, 3,471 mutant chromosomes were screened for genetic modifiers of α-synuclein and 12 genes were identified. Eleven modifiers have human orthologs associated with diseases, including MED13 and CDC27, which lie within PD GWAS loci. Drosophila Skd/Med13 and glycolytic enzymes are co-upregulated by α-syn-associated neurodegeneration. While elevated α-syn compromises mitochondrial function, co-expressing skd/Med13 RNAi and α-syn synergistically increase the ratio of oxidized-to-reduced glutathione. The resulting neurodegeneration can be suppressed by overexpressing a glycolytic enzyme or treatment with deferoxamine, suggesting that compensatory glycolysis is neuroprotective. In addition, the functional relationship between α-synuclein, MED13, and glycolytic enzymes is conserved between flies and mice. It is proposed that hypoxia-inducible factor and MED13 are part of a druggable pathway for PD.

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Ren, M., Yang, Y., Heng, K. H. Y., Ng, L. Y., Chong, C. Y., Ng, Y. T., Gorur-Shandilya, S., Lee, R. M. Q., Lim, K. L., Zhang, J. and Koh, T. W. (2022). MED13 and glycolysis are conserved modifiers of alpha-synuclein-associated neurodegeneration. Cell Rep 41(12): 111852. PubMed ID: 36543134

Abstract
alpha-Synuclein (α-syn) is important in synucleinopathies such as Parkinson's disease (PD). While genome-wide association studies (GWASs) of synucleinopathies have identified many risk loci, the underlying genes have not been shown for most loci. Using Drosophila, 3,471 mutant chromosomes were screened for genetic modifiers of α-synuclein and 12 genes were identified. Eleven modifiers have human orthologs associated with diseases, including MED13 and CDC27, which lie within PD GWAS loci. Drosophila Skd/Med13 and glycolytic enzymes are co-upregulated by α-syn-associated neurodegeneration. While elevated α-syn compromises mitochondrial function, co-expressing skd/Med13 RNAi and α-syn synergistically increase the ratio of oxidized-to-reduced glutathione. The resulting neurodegeneration can be suppressed by overexpressing a glycolytic enzyme or treatment with deferoxamine, suggesting that compensatory glycolysis is neuroprotective. In addition, the functional relationship between α-synuclein, MED13, and glycolytic enzymes is conserved between flies and mice. It is proposed that hypoxia-inducible factor and MED13 are part of a druggable pathway for PD.

Cortot, J., Farine, J. P., Ferveur, J. F. and Everaerts, C. (2022). Aging-Related Variation of Cuticular Hydrocarbons in Wild Type and Variant Drosophila melanogaster. J Chem Ecol. PubMed ID: 35022940

Abstract
The cuticle of all insects is covered with hydrocarbons which have multiple functions. Cuticular hydrocarbons (CHCs) basically serve to protect insects against environmental harm and reduce dehydration. In many species, some CHCs also act as pheromones. CHCs have been intensively studied in Drosophila species and more especially in D. melanogaster. In this species, flies produce about 40 CHCs forming a complex sex- and species-specific bouquet. The quantitative and qualitative pattern of the CHC bouquet was characterized during the first days of adult life but remains unexplored in aging flies. This study characterized CHCs during the whole-or a large period of-adult life in males and females of several wild type and transgenic lines. Both types of lines included standard and variant CHC profiles. Some of the genotypes tested in this study showed very dramatic and unexpected aging-related variation based on their early days' profile. This study provides a concrete dataset to better understand the mechanisms underlying the establishment and maintenance of CHCs on the fly cuticle. It could be useful to determine physiological parameters, including age and response to climate variation, in insects collected in the wild.

De Groef, S., Wilms, T., Balmand, S., Calevro, F. and Callaerts, P. (2021). Sexual Dimorphism in Metabolic Responses to Western Diet in Drosophila melanogaster. Biomolecules 12(1). PubMed ID: 35053181

Abstract
Obesity is a chronic disease affecting millions of people worldwide. The fruit fly (Drosophila melanogaster) is an interesting research model to study metabolic and transcriptomic responses to obesogenic diets. However, the sex-specific differences in these responses are still understudied and perhaps underestimated. This study exposed adult male and female Dahomey fruit flies to a standard diet supplemented with sugar, fat, or a combination of both. The exposure to a diet supplemented with 10% sugar and 10% fat efficiently induced an increase in the lipid content in flies, a hallmark for obesity. This increase in lipid content was more prominent in males, while females displayed significant changes in glycogen content. A strong effect of the diets on the ovarian size and number of mature oocytes was also present in females exposed to diets supplemented with fat and a combination of fat and sugar. In both males and females, fat body morphology changed and was associated with an increase in lipid content of fat cells in response to the diets. The expression of metabolism-related genes also displayed a strong sexually dimorphic response under normal condi-tions and in response to sugar and/or fat-supplemented diets. This study shows that the exposure of adult fruit flies to an obesogenic diet containing both sugar and fat allowed studying sexual dimorphism in metabolism and the expression of genes regulating metabolism.

Casale, A. M., Liguori, F., Ansaloni, F., Cappucci, U., Finaurini, S., Spirito, G., Persichetti, F., Sanges, R., Gustincich, S. and Piacentini, L. (2022). Transposable element activation promotes neurodegeneration in a Drosophila model of Huntington's disease. iScience 25(1): 103702. PubMed ID: 35036881

Abstract
Huntington's disease (HD) is an autosomal dominant disorder with progressive motor dysfunction and cognitive decline. The disease is caused by a CAG repeat expansion in the IT15 gene, which elongates a polyglutamine stretch of the HD protein, Huntingtin. No therapeutic treatments are available, and new pharmacological targets are needed. Retrotransposons are transposable elements (TEs) that represent 40% and 30% of the human and Drosophila genomes and replicate through an RNA intermediate. Mounting evidence suggests that mammalian TEs are active during neurogenesis and may be involved in diseases of the nervous system. This study shows that TE expression and mobilization are increased in a Drosophila melanogaster HD model. By inhibiting TE mobilization with Reverse Transcriptase inhibitors, polyQ-dependent eye neurodegeneration and genome instability in larval brains are rescued and fly lifespan is increased. These results suggest that TE activation may be involved in polyQ-induced neurotoxicity and a potential pharmacological target.

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Kim, Y. W., Al-Ramahi, I., Koire, A., Wilson, S. J., Konecki, D. M., Mota, S., Soleimani, S., Botas, J. and Lichtarge, O. (2020). Harnessing the paradoxical phenotypes of APOE epsilon2 and APOE epsilon4 to identify genetic modifiers in Alzheimer's disease. Alzheimers Dement. PubMed ID: 33576571

Abstract
The strongest genetic risk factor for idiopathic late-onset Alzheimer's disease (LOAD) is apolipoprotein E (APOE) ε4, while the APOE ε2 allele is protective. However, there are paradoxical APOE ε4 carriers who remain disease-free and APOE ε2 carriers with LOAD. Exomes of healthy APOE ε4 carriers and APOE ε2 Alzheimer's disease (AD) patients were compared, prioritizing coding variants based on their predicted functional impact; 216 genes were identified with differential mutational load between these two populations. These candidate genes were significantly dysregulated in LOAD brains, and many modulated tau- or β42-induced neurodegeneration in Drosophila. Variants in these genes were associated with AD risk, even in APOE ε3 homozygotes, showing robust predictive power for risk stratification. Network analyses revealed involvement of candidate genes in brain cell type-specific pathways including synaptic biology, dendritic spine pruning and inflammation. These potential modifiers of LOAD may constitute novel biomarkers, provide potential therapeutic intervention avenues, and support applying this approach as larger whole exome sequencing cohorts become available.

Gabrawy, M. M., Khosravian, N., Morcos, G. S., Morozova, T. V., Jezek, M., Walston, J. D., Huang, W., Abadir, P. M. and Leips, J. (2022). Genome-Wide Analysis in Drosophila Reveals the Genetic Basis of Variation in Age-Specific Physical Performance and Response to ACE Inhibition. Genes (Basel) 13(1). PubMed ID: 35052483

Abstract
Despite impressive results in restoring physical performance in rodent models, treatment with renin-angiotensin system (RAS) inhibitors, such as Lisinopril, have highly mixed results in humans, likely, in part, due to genetic variation in human populations. To date, the genetic determinants of responses to drugs, such as RAS inhibitors, remain unknown. Given the complexity of the relationship between physical traits and genetic background, genomic studies which predict genotype- and age-specific responses to drug treatments in humans or vertebrate animals are difficult. Using 126 genetically distinct lines of Drosophila melanogaster, this study tested the effects of Lisinopril on age-specific climbing speed and endurance. The data show that functional response and sensitivity to Lisinopril treatment ranges from significant protection against physical decline to increased weakness depending on genotype and age. Furthermore, genome-wide analyses led to identification of evolutionarily conserved genes in the WNT signaling pathway as being significantly associated with variations in physical performance traits and sensitivity to Lisinopril treatment. Genetic knockdown of genes in the WNT signaling pathway, Axin, frizzled, nemo, and wingless, diminished or abolished the effects of Lisinopril treatment on climbing speed traits. These results implicate these genes as contributors to the genotype- and age-specific effects of Lisinopril treatment and because they have orthologs in humans, they are potential therapeutic targets for improvement of resiliency. This approach should be widely applicable for identifying genomic variants that predict age- and sex-dependent responses to any type of pharmaceutical treatment.

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Yazar, V., Kang, S. U., Ha, S., Dawson, V. L. and Dawson, T. M. (2021). Integrative genome-wide analysis of dopaminergic neuron-specific PARIS expression in Drosophila dissects recognition of multiple PPAR-γ associated gene regulation. Sci Rep 11(1): 21500. PubMed ID: 34728675

Abstract
The transcriptional repressor called parkin interacting substrate (PARIS; ZNF746) was initially identified as a novel co-substrate of parkin and PINK1 that leads to Parkinson's disease (PD) by disrupting mitochondrial biogenesis through peroxisome proliferator-activated receptor gamma (PPARγ) coactivator -1α (PGC-1α) suppression. Since its initial discovery, growing evidence has linked PARIS to defective mitochondrial biogenesis observed in PD pathogenesis. This study employed conditional translating ribosome affinity purification (TRAP) followed by RNA sequencing (TRAP-seq) for transcriptome profiling of DA neurons in transgenic Drosophila lines expressing human PARIS wild type (WT) or mutant (C571A). The results demonstrated that PPARγ functions as a master regulator of PARIS-induced molecular changes at the transcriptome level, confirming that PARIS acts primarily on PGC-1α to lead to neurodegeneration in PD. Moreover, this study identified that PARIS actively modulates expression of PPARγ target genes by physically binding to the promoter regions. Together, this work revealed how PARIS drives adverse effects on modulation of PPAR-γ associated gene clusters in DA neurons.

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Xiao, G., Zhao, M., Liu, Z., Du, F. and Zhou, B. (2021). Zinc antagonizes iron-regulation of tyrosine hydroxylase activity and dopamine production in Drosophila melanogaster. BMC Biol 19(1): 236. PubMed ID: 34732185

Abstract
Dopamine (DA) is a neurotransmitter that plays roles in movement, cognition, attention, and reward responses, and deficient DA signaling is associated with the progression of a number of neurological diseases, such as Parkinson's disease. Due to its critical functions, DA expression levels in the brain are tightly controlled, with one important and rate-limiting step in its biosynthetic pathway being catalyzed by tyrosine hydroxylase (TH), an enzyme that uses iron ion (Fe(2+)) as a cofactor. A role for metal ions has additionally been associated with the etiology of Parkinson's disease. However, the way dopamine synthesis is regulated in vivo or whether regulation of metal ion levels is a component of DA synthesis is not fully understood. This study analyzed the role of Catsup, the Drosophila ortholog of the mammalian zinc transporter SLC39A7 (ZIP7), in regulating dopamine levels. Catsup was found to be a functional zinc transporter that regulates intracellular zinc distribution between the ER/Golgi and the cytosol. Loss-of-function of Catsup leads to increased DA levels, and the increased dopamine production was shown to be due to a reduction in zinc levels in the cytosol. Zinc ion (Zn(2+)) negatively regulates dopamine synthesis through direct inhibition of TH activity, by antagonizing Fe(2+) binding to TH, thus rendering the enzyme ineffective or non-functional. These findings uncovered a previously unknown mechanism underlying the control of cellular dopamine expression, with normal levels of dopamine synthesis being maintained through a balance between Fe(2+) and Zn(2+) ions. The findings also provide support for metal modulation as a possible therapeutic strategy in the treatment of Parkinson's disease and other dopamine-related diseases.

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Pant, C., Chakrabarti, M., Mendonza, J. J., Ganganna, B., Pabbaraja, S. and Pal Bhadra, M. (2021). Aza-Flavanone Diminishes Parkinsonism in the Drosophila melanogaster Parkin Mutant. ACS Chem Neurosci. PubMed ID: 34763419

Abstract
Parkinson's disease is a chronic and progressive neurodegenerative disease, induced by slow and progressive death of the dopaminergic (DA) neurons from the midbrain region called substantia nigra (SNc) leading to difficulty in locomotion. At present, very few potential therapeutic drugs are available for treatment, necessitating an urgent need for development. In this current study, the parkin transgenic Drosophila melanogaster model that induces selective loss in dopaminergic neurons and impairment of locomotory functions has been used to see the effect of the aza-flavanone molecule. D. melanogaster serves as an amazing in vivo model making valuable contribution in the development of promising treatment strategies. In-silico study showed spontaneous binding of this molecule to the D2 receptor making it a potential dopamine agonist. PARKIN protein is well conserved, and it has been reported that Drosophila PARKIN is 42% identical to human PARKIN. Interestingly, this molecule enhances the motor coordination and survivability rate of the transgenic flies along with an increase in expression of the master regulator of Dopamine synthesis, that is, tyrosine hydroxylase (TH), in the substantia nigra region of the fly brain. Moreover, it plays a significant effect on mitochondrial health and biogenesis via modulation of a conserved mitochondrial protein PHB2. Therefore, this molecule could lead to the development of an effective therapeutic approach for the treatment of PD.

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Hung, Y. C., Huang, K. L., Chen, P. L., Li, J. L., Lu, S. H., Chang, J. C., Lin, H. Y., Lo, W. C., Huang, S. Y., Lee, T. T., Lin, T. Y., Imai, Y., Hattori, N., Liu, C. S., Tsai, S. Y., Chen, C. H., Lin, C. H. and Chan, C. C. (2021). UQCRC1 engages cytochrome c for neuronal apoptotic cell death. Cell Rep 36(12): 109729. PubMed ID: 34551295

Human ubiquinol-cytochrome c reductase core protein 1 (UQCRC1) is an evolutionarily conserved core subunit of mitochondrial respiratory chain complex III. This study recently identified the disease-associated variants of UQCRC1 from patients with familial parkinsonism, but its function remains unclear. This study investigates the endogenous function of UQCRC1 in the human neuronal cell line and the Drosophila nervous system. Flies with neuronal knockdown of uqcrc1 exhibit age-dependent parkinsonism-resembling defects, including dopaminergic neuron reduction and locomotor decline, and are ameliorated by UQCRC1 expression. Lethality of uqcrc1-KO is also rescued by neuronally expressing UQCRC1, but not the disease-causing variant, providing a platform to discern the pathogenicity of this mutation. Furthermore, UQCRC1 associates with the apoptosis trigger cytochrome c (cyt-c), and uqcrc1 deficiency increases Cyt-c in the cytoplasmic fraction and activates the caspase cascade. Depleting cyt-c or expression of the anti-apoptotic p35 ameliorates uqcrc1-mediated neurodegeneration. The findings identified a role for UQCRC1 in regulating cyt-c-induced apoptosis (Hung, 2021).

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Solana-Manrique, C., Sanz, F. J., Ripolles, E., Bano, M. C., Torres, J., Munoz-Soriano, V. and Paricio, N. (2020). Enhanced activity of glycolytic enzymes in Drosophila and human cell models of Parkinson's disease based on DJ-1 deficiency. Free Radic Biol Med. PubMed ID: 32726690

Parkinson's disease (PD) is a neurodegenerative debilitating disorder characterized by progressive disturbances in motor, autonomic and psychiatric functions. One of the genes involved in familial forms of the disease is DJ-1, whose mutations cause early-onset PD. Besides, it has been shown that an over-oxidized and inactive form of the DJ-1 protein is found in brains of sporadic PD patients. Interestingly, the DJ-1 protein plays an important role in cellular defense against oxidative stress and also participates in mitochondrial homeostasis. Flies mutant for the DJ-1β gene, the Drosophila ortholog of human DJ-1, exhibited disease-related phenotypes such as motor defects, increased reactive oxygen species production and high levels of protein carbonylation. The present study demonstrated that DJ-1β mutants also show a significant increase in the activity of several regulatory glycolytic enzymes. Similar results were obtained in DJ-1-deficient SH-SY5Y neuroblastoma cells, thus suggesting that loss of DJ-1 function leads to an increase in the glycolytic rate. In such a scenario, an enhancement of the glycolytic pathway could be a protective mechanism to decrease ROS production by restoring ATP levels, which are decreased due to mitochondrial dysfunction. The results also show that meclizine and dimethyl fumarate, two FDA-approved compounds with different clinical applications, are able to attenuate PD-related phenotypes in both models. Moreover, it was found that they may exert their beneficial effect by increasing glycolysis through the activation of key glycolytic enzymes. Taken together, these results are consistent with the idea that increasing glycolysis could be a potential disease-modifying strategy for PD, as recently suggested. Besides, they also support further evaluation and potential repurposing of meclizine and dimethyl fumarate as modulators of energy metabolism for neuroprotection in PD.

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Fernandez-Cruz, I., Sanchez-Diaz, I., Narvaez-Padilla, V. and Reynaud, E. (2020). Rpt2 proteasome subunit reduction causes Parkinson's disease like symptoms in Drosophila, IBRO Rep 9: 65-77. PubMed ID: 32715147

The dysfunction of the proteasome-ubiquitin system is commonly reported in several neurodegenerative diseases. Post mortem samples of brains of patients with Parkinson´s disease present cytoplasmic inclusions that are rich in proteins such as ubiquitin, Tau, and α-synuclein. In Parkinson´s disease, a specific reduction of some of the proteasome subunits has also been reported. However, the specific role of the different proteasome subunits in dopaminergic neuron degeneration has not been thoroughly explored. In this work, the Gal4/UAS system was used to test fourteen Drosophila melanogaster RNAi lines from the Bloomington Drosophila Stock Center. Each of these lines targets a different proteasome subunit. To identify the strains that were able to induce neurodegeneration, the expression of these lines was driven to the eye, and they were catagorized as a function of the extent of neurodegeneration that they induced. The targeted proteasomal subunits are conserved in mammals and therefore may be relevant to study proteasome related diseases. The RNAi line among the regulatory subunits with the most penetrant phenotype targeted the proteasomal subunit Rpt2 and its phenotypes were further characterized. Rpt2 knockdown in the Drosophila central nervous system reduced the activity of the proteasome, augmented the amount of insoluble ubiquitinated protein, and elicited motor and non-motor phenotypes that were similar to the ones found in Drosophila and other models for Parkinson's disease. When Rpt2 is silenced pan-neurally, third instar larvae have locomotion dysfunctions and die during pupation. Larval lethality was avoided using the Gal80-Gal4 system to induce the expression of the Rpt2 RNAi to dopaminergic neurons only after pupation. The reduction of Rpt2 in adult dopaminergic neurons causes reduced survival, hyperactivity, neurodegeneration, and sleep loss; probably recapitulating some of the sleep disorders that Parkinson's disease patients have before the appearance of locomotion disorders (Fernandez-Cruz, 2020).

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Ingles-Prieto, A., Furthmann, N., Crossman, S. H., Tichy, A. M., Hoyer, N., Petersen, M., Zheden, V., Biebl, J., Reichhart, E., Gyoergy, A., Siekhaus, D. E., Soba, P., Winklhofer, K. F. and Janovjak, H. (2021). Optogenetic delivery of trophic signals in a genetic model of Parkinson's disease. PLoS Genet 17(4): e1009479. PubMed ID: 33857132

Optogenetics has been harnessed to shed new mechanistic light on current and future therapeutic strategies. This has been to date achieved by the regulation of ion flow and electrical signals in neuronal cells and neural circuits that are known to be affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and are implicated in degenerative disorders, has never been demonstrated in an animal model of disease. This study reengineered the human and Drosophila melanogaster REarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1B9 flies that exhibit loss of PTEN-induced putative kinase 1 (PINK1), a kinase associated with familial Parkinson's disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRET via the PI3K/NF-kappaB pathway. These results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to inspire novel strategies towards a spatio-temporal regulation of tissue repair.

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Arsac, J. N., Sedru, M., Dartiguelongue, M., Vulin, J., Davoust, N., Baron, T. and Mollereau, B. (2021). Chronic Exposure to Paraquat Induces Alpha-Synuclein Pathogenic Modifications in Drosophila. Int J Mol Sci 22(21). PubMed ID: 34769043

Parkinson's disease (PD) is characterized by the progressive accumulation of neuronal intracellular aggregates largely composed of α-Synuclein (αSyn) protein. The process of αSyn aggregation is induced during aging and enhanced by environmental stresses, such as the exposure to pesticides. Paraquat (PQ) is an herbicide which has been widely used in agriculture and associated with PD. PQ is known to cause an increased oxidative stress in exposed individuals but the consequences of such stress on αSyn conformation remains poorly understood. To study Syn pathogenic modifications in response to PQ, Drosophila expressing human αSyn were exposedto a chronic PQ protocol. It was first shown that PQ exposure and αSyn expression synergistically induced fly mortality. The exposure to PQ was also associated with increased levels of total and phosphorylated forms of αSyn in the Drosophila brain. Interestingly, PQ increased the detection of soluble αSyn in highly denaturating buffer but did not increase αSyn resistance to proteinase K digestion. These results suggest that PQ induces the accumulation of toxic soluble and misfolded forms of αSyn but that these toxic forms do not form fibrils or aggregates that are detected by the proteinase K assay. Collectively, these results demonstrate that Drosophila can be used to study the effect of PQ or other environmental neurotoxins on Syn driven pathology (Arsac, 2021).

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Olsen, A. L. and Feany, M. B. (2021). Parkinson's disease risk genes act in glia to control neuronal alpha-synuclein toxicity. Neurobiol Dis 159: 105482. PubMed ID: 34390834 Idiopathic Parkinson's disease is the second most common neurodegenerative disease and is estimated to be approximately 30% heritable. Genome wide association studies have revealed numerous loci associated with risk of development of Parkinson's disease. The majority of genes identified in these studies are expressed in glia at either similar or greater levels than their expression in neurons, suggesting that glia may play a role in Parkinson's disease pathogenesis. The role of individual glial risk genes in Parkinson's disease development or progression is unknown, however. It was hypothesized that some Parkinson's disease risk genes exert their effects through glia. A Drosophila model of α-synucleinopathy was developed in which gene expression can be individually expressed in neurons and glia. Human wild type α-synuclein is expressed in all neurons, and these flies develop the hallmarks of Parkinson's disease, including motor impairment, death of dopaminergic and other neurons, and α-synuclein aggregation. In these flies, a candidate genetic screen was performed, using RNAi to knockdown 14 well-validated Parkinson's disease risk genes in glia, and the effect on locomotion was measured in order to identify glial modifiers of the &alpha-synuclein phenotype. Four modifiers were identified: aux, Lrrk, Ric, and Vps13, orthologs of the human genes GAK, LRRK2, RIT2, and VPS13C, respectively. Knockdown of each gene exacerbated neurodegeneration as measured by total and dopaminergic neuron loss. Knockdown of each modifier also increased α-synuclein oligomerization. These results suggest that some Parkinson's disease risk genes exert their effects in glia and that glia can influence neuronal α-synuclein proteostasis in a non-cell-autonomous fashion. Further, this study provides proof of concept that this novel Drosophila α-synucleinopathy model can be used to study glial modifier genes, paving the way for future large unbiased screens to identify novel glial risk factors that contribute to PD risk and progression.

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

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

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Krzystek, T. J., Banerjee, R., Thurston, L., Huang, J., Swinter, K., Rahman, S. N., Falzone, T. L. and Gunawardena, S. (2021). Differential mitochondrial roles for alpha-synuclein in DRP1-dependent fission and PINK1/Parkin-mediated oxidation. Cell Death Dis 12(9): 796. PubMed ID: 34404758

Abstract
Mitochondria are highly dynamic organelles with strict quality control processes that maintain cellular homeostasis. Within axons, coordinated cycles of fission-fusion mediated by dynamin related GTPase protein (DRP1) and mitofusins (MFN), together with regulated motility of healthy mitochondria anterogradely and damaged/oxidized mitochondria retrogradely, control mitochondrial shape, distribution and size. This study has isolated the mechanistic role of α-syn in mitochondrial homeostasis in vivo in a humanized Drosophila model of Parkinson's disease (PD). It was shown that excess α-syn causes fragmented mitochondria, which persists with either truncation of the C-terminus (α(1-120)) or deletion of the NAC region (α(ΔNAC)). Using in vivo oxidation reporters Mito-roGFP2-ORP1/GRX1 and MitoTimer, it was found that α-mediated fragments were oxidized/damaged, but α(1-120)-induced fragments were healthy, suggesting that the C-terminus is required for oxidation. α-mediated oxidized fragments showed biased retrograde motility, but α(1-120)-mediated healthy fragments did not, demonstrating that the C-terminus likely mediates the retrograde motility of oxidized mitochondria. Depletion/inhibition or excess DRP1-rescued α-syn-mediated fragmentation, oxidation, and the biased retrograde motility, indicating that DRP1-mediated fragmentation is likely upstream of oxidation and motility changes. Further, excess PINK/Parkin, two PD-associated proteins that function to coordinate mitochondrial turnover via induction of selective mitophagy, rescued α-syn-mediated membrane depolarization, oxidation and cell death in a C-terminus-dependent manner, suggesting a functional interaction between &alpha-syn and PINK/Parkin. Taken together, these findings identify distinct roles for α-syn in mitochondrial homeostasis, highlighting a previously unknown pathogenic pathway for the initiation of PD.

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Guo, Q., Wang, B., Wang, X., Smith, W. W., Zhu, Y. and Liu, Z. (2021). Activation of Nrf2 in Astrocytes Suppressed PD-Like Phenotypes via Antioxidant and Autophagy Pathways in Rat and Drosophila Models. Cells 10(8). PubMed ID: 34440619

Abstract
The oxidative-stress-induced impairment of autophagy plays a critical role in the pathogenesis of Parkinson's disease (PD). This study investigated whether the alteration of Nrf2 in astrocytes protected against 6-OHDA (6-hydroxydopamine)- and rotenone-induced PD-like phenotypes, using 6-OHDA-induced rat PD and rotenone-induced Drosophila PD models. In the PD rat model, Nrf2 expression was significantly higher in astrocytes than in neurons. CDDO-Me (CDDO methyl ester, an Nrf2 inducer) administration attenuated PD-like neurodegeneration mainly through Nrf2 activation in astrocytes by activating the antioxidant signaling pathway and enhancing autophagy in the substantia nigra and striatum. In the PD Drosophila model, the overexpression of Nrf2 in glial cells displayed more protective effects than such overexpression in neurons. Increased Nrf2 expression in glial cells significantly reduced oxidative stress and enhanced autophagy in the brain tissue. The administration of the Nrf2 inhibitor ML385 reduced the neuroprotective effect of Nrf2 through the inhibition of the antioxidant signaling pathway and autophagy pathway. The autophagy inhibitor 3-MA partially reduced the neuroprotective effect of Nrf2 through the inhibition of the autophagy pathway, but not the antioxidant signaling pathway. Moreover, Nrf2 knockdown caused neurodegeneration in flies. Treatment with CDDO-Me attenuated the Nrf2-knockdown-induced degeneration in the flies through the activation of the antioxidant signaling pathway and increased autophagy. An autophagy inducer, rapamycin, partially rescued the neurodegeneration in Nrf2-knockdown Drosophila by enhancing autophagy. These results indicate that the activation of the Nrf2-linked signaling pathways in glial cells plays an important neuroprotective role in PD models.

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Putz, S. M., Kram, J., Rauh, E., Kaiser, S., Toews, R., Lueningschroer-Wang, Y., Rieger, D. and Raabe, T. (2021). Loss of p21-activated kinase Mbt/PAK4 causes Parkinson-like phenotypes in Drosophila. Dis Model Mech 14(6). PubMed ID: 34125184

Abstract
Parkinson's disease (PD) provokes bradykinesia, resting tremor, rigidity and postural instability, and also non-motor symptoms such as depression, anxiety, sleep and cognitive impairments. Similar phenotypes can be induced in Drosophila melanogaster through modification of PD-relevant genes or the administration of PD-inducing toxins. Recent studies correlated deregulation of human p21-activated kinase 4 (PAK4) with PD, leaving open the question of a causative relationship of mutations in this gene for manifestation of PD symptoms. To determine whether flies lacking the PAK4 homolog Mushroom bodies tiny (Mbt) show PD-like phenotypes, a variety of PD criteria was tested. This study demonstrated that mbt mutant flies show PD-like phenotypes including age-dependent movement deficits, reduced life expectancy and fragmented sleep. They also react to a stressful situation with higher immobility, indicating an influence of Mbt on emotional behavior. Loss of Mbt function has a negative effect on the number of dopaminergic protocerebral anterior medial (PAM) neurons, most likely caused by a proliferation defect of neural progenitors. The age-dependent movement deficits are not accompanied by a corresponding further loss of PAM neurons. Previous studies highlighted the importance of a small PAM subgroup for age-dependent PD motor impairments. This study shows that impaired motor skills are caused by a lack of Mbt in this PAM subgroup. In addition, a broader re-expression of Mbt in PAM neurons improves life expectancy. Conversely, selective Mbt knockout in the same cells shortens lifespan. It is concluded that mutations in Mbt/PAK4 can play a causative role in the development of PD phenotypes.

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Bhat, S., Guthrie, D. A., Kasture, A., El-Kasaby, A., Cao, J., Bonifazi, A., Ku, T., Giancola, J. B., Hummel, T., Freissmuth, M. and Newman, A. H. (2021). Tropane-Based Ibogaine Analog Rescues Folding-Deficient Serotonin and Dopamine Transporters. ACS Pharmacol Transl Sci 4(2): 503-516. PubMed ID: 33860180

Abstract

Missense mutations that give rise to protein misfolding are rare, but collectively, defective protein folding diseases are consequential. Folding deficiencies are amenable to pharmacological correction (pharmacochaperoning), but the underlying mechanisms remain enigmatic. Ibogaine and its active metabolite noribogaine correct folding defects in the dopamine transporter (DAT), but they rescue only a very limited number of folding-deficient DAT mutant proteins, which give rise to infantile Parkinsonism and dystonia. In this study, a series of analogs was generated by reconfiguring the complex ibogaine ring system and exploring the structural requirements for binding to wild-type transporters, as well as for rescuing two equivalent synthetic folding-deficient mutants, SERT-PG(601,602)AA and DAT-PG(584,585)AA. The most active tropane-based analog (9b) was also an effective pharmacochaperone in vivo in Drosophila harboring the DAT-PG(584,585)AA mutation and rescued 6 out of 13 disease-associated human DAT mutant proteins in vitro. Hence, a novel lead pharmacochaperone has been identified that demonstrates medication development potential for patients harboring DAT mutations.

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Buck, S. A., Steinkellner, T., Aslanoglou, D., Villeneuve, M., Bhatte, S. H., Childers, V. C., Rubin, S. A., De Miranda, B. R., O'Leary, E. I., Neureiter, E. G., Fogle, K. J., Palladino, M. J., Logan, R. W., Glausier, J. R., Fish, K. N., Lewis, D. A., Greenamyre, J. T., McCabe, B. D., Cheetham, C. E. J., Hnasko, T. S. and Freyberg, Z. (2021).Vesicular glutamate transporter modulates sex differences in dopamine neuron vulnerability to age-related neurodegeneration.. Aging Cell: e13365. PubMed ID: 33909313

Abstract

Age is the greatest risk factor for Parkinson's disease (PD) which causes progressive loss of dopamine (DA) neurons, with males at greater risk than females. Intriguingly, some DA neurons are more resilient to degeneration than others. Increasing evidence suggests that vesicular glutamate transporter (VGLUT) expression in DA neurons plays a role in this selective vulnerability. We investigated the role of DA neuron VGLUT in sex- and age-related differences in DA neuron vulnerability using the genetically tractable Drosophila model. This study found sex differences in age-related DA neurodegeneration and its associated locomotor behavior, where males exhibit significantly greater decreases in both DA neuron number and locomotion during aging compared with females. Dynamic changes in DA neuron VGLUT expression mediate these age- and sex-related differences, as a potential compensatory mechanism for diminished DA neurotransmission during aging. Importantly, female Drosophila possess higher levels of VGLUT expression in DA neurons compared with males, and this finding is conserved across flies, rodents, and humans. Moreover, this study showed that diminishing VGLUT expression in DA neurons eliminates females' greater resilience to DA neuron loss across aging. This offers a new mechanism for sex differences in selective DA neuron vulnerability to age-related DA neurodegeneration. Finally, in mice, this study showed that the ability of DA neurons to achieve optimal control over VGLUT expression is essential for DA neuron survival. These findings lay the groundwork for the manipulation of DA neuron VGLUT expression as a novel therapeutic strategy to boost DA neuron resilience to age- and PD-related neurodegeneration.

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Yamaguchi, A., Ishikawa, K. I., Inoshita, T., Shiba-Fukushima, K., Saiki, S., Hatano, T., Mori, A., Oji, Y., Okuzumi, A., Li, Y., Funayama, M., Imai, Y., Hattori, N. and Akamatsu, W. (2020). Identifying Therapeutic Agents for Amelioration of Mitochondrial Clearance Disorder in Neurons of Familial Parkinson Disease. Stem Cell Reports 14(6): 1060-1075. PubMed ID: 32470327

Abstract

Parkinson disease (PD) is a neurodegenerative disorder caused by the progressive loss of midbrain dopaminergic neurons, and mitochondrial dysfunction is involved in its pathogenesis. This study aimed to establish an imaging-based, semi-automatic, high-throughput system for the quantitative detection of disease-specific phenotypes in dopaminergic neurons from induced pluripotent stem cells (iPSCs) derived from patients with familial PD having Parkin or PINK1 mutations, which exhibit abnormal mitochondrial homeostasis. The proposed system recapitulates the deficiency of mitochondrial clearance, ROS accumulation, and increasing apoptosis in these familial PD-derived neurons. 320 compounds were screened for their ability to ameliorate multiple phenotypes, and four candidate drugs were identified. Some of these drugs improved the locomotion defects and reduced ATP production caused by PINK1 inactivation in Drosophila and were effective for idiopathic PD-derived neurons with impaired mitochondrial clearance. These findings suggest that the proposed high-throughput system has potential for identifying effective drugs for familial and idiopathic PD (Yamaguchi, 2020).

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Xie, J., Chen, S., Bopassa, J. C. and Banerjee, S. (2021).Drosophila tubulin polymerization promoting protein mutants reveal pathological correlates relevant to human Parkinson's disease. Sci Rep. 11(1):13614. PubMed ID: 34193896

Abstract
Parkinson's disease (PD) is a progressive neurodegenerative disorder with no known cure. PD is characterized by locomotion deficits, nigrostriatal dopaminergic neuronal loss, mitochondrial dysfunctions and formation of α-Synuclein aggregates. A well-conserved and less understood family of Tubulin Polymerization Promoting Proteins (TPPP) is also implicated in PD and related disorders, where TPPP exists in pathological aggregates in neurons in patient brains. However, there are no in vivo studies on mammalian TPPP to understand the genetics and neuropathology linking TPPP aggregation or neurotoxicity to PD. The only Drosophila homolog of human TPPP is named Ringmaker (Ringer). This study reports that adult ringer mutants display progressive locomotor disabilities, reduced lifespan and neurodegeneration. Importantly, the findings reveal that Ringer is associated with mitochondria and ringer mutants have mitochondrial structural damage and dysfunctions. Adult ringer mutants also display progressive loss of dopaminergic neurons. Together, these phenotypes of ringer mutants recapitulate some of the salient features of human PD patients, thus allowing utilization of ringer mutants as a fly model relevant to PD, and further exploration of its genetic and molecular underpinnings to gain insights into the role of human TPPP in PD.

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Carvajal-Oliveros, A., Domínguez-Baleon, C., Zarate, R. V., Campusano, J. M., Narvaez-Padilla, V. and Reynaud, E. (2021). Nicotine suppresses Parkinson's disease like phenotypes induced by Synphilin-1 overexpression in Drosophila melanogaster by increasing tyrosine hydroxylase and dopamine levels. Sci Rep 11(1): 9579. PubMed ID: 33953275

It has been observed that there is a lower Parkinson's disease (PD) incidence in tobacco users. Nicotine is a cholinergic agonist and is the principal psychoactive compound in tobacco linked to cigarette addiction. Different studies have shown that nicotine has beneficial effects on sporadic and genetic models of PD. This work evaluated nicotine's protective effect in a Drosophila melanogaster model for PD where Synphilin-1 (Sph-1) is expressed in dopaminergic neurons. Nicotine has a moderate effect on dopaminergic neuron survival that becomes more evident as flies age. Nicotine is beneficial on fly survival and motility increasing tyrosine hydroxylase and dopamine levels, suggesting that cholinergic agonists may promote survival and metabolic function of the dopaminergic neurons that express Sph-1. The Sph-1 expressing fly is a good model for the study of early-onset phenotypes such as olfaction loss one of the main non-motor symptom related to PD. Thd data suggest that nicotine is an interesting therapeutic molecule whose properties should be explored in future research on the phenotypic modulators of the disease and for the development of new treatments.

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Qiao, J. D. and Mao, Y. L. (2020). Knockout of PINK1 altered the neural connectivity of Drosophila dopamine PPM3 neurons at input and output sites. Invert Neurosci 20(3): 11. PubMed ID: 32766952

Abstract

Impairment of the dopamine system is the main cause of Parkinson disease (PD). PTEN-induced kinase 1 (PINK1) is possibly involved in pathogenesis of PD. However, its role in dopaminergic neurons has not been fully established yet. In the present investigation, the PINK1 knockout Drosophila model to explore the role of PINK1 in dopaminergic neurons. Electrophysiological and behavioral tests indicated that PINK1 elimination enhances the neural transmission from the presynaptic part of dopaminergic neurons in the protocerebral posterior medial region 3 (PPM3) to PPM3 neurons (which are homologous to those in the substantia nigra in humans). Firing properties of the action potential in PPM3 neurons were also altered in the PINK1 knockout genotypes. Abnormal motor ability was also observed in these PINK1 knockout animals. These results indicate that knockout of PINK1 could alter both the input and output properties of PPM3 neurons (Qiao, 2020).

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Wan, Z., Xu, J., Huang, Y., Zhai, Y., Ma, Z., Zhou, B. and Cao, Z. (2020). Elevating bioavailable iron levels in mitochondria suppresses the defective phenotypes caused by PINK1 loss-of-function in Drosophila melanogaster. Biochem Biophys Res Commun 532(2): 285-291. PubMed ID: 32873392

Abstract

Parkinson's disease (PD) is the second most common progressive neurodegenerative disease, which is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Iron deposit was found in the SNpc of PD patients and animal models, however, the mechanisms involved in disturbed iron metabolism remain unknown. Identifying the relationship between iron metabolism and PD is important for finding new therapeutic strategies. This study found that transgenic overexpression (OE) of Drosophila mitoferrin (dmfrn) or knockdown of Fer3HCH significantly mitigated the reduced mitochondrial aconitase activity, abnormal wing posture, flight deficits and mitochondrial morphology defects associated with PINK1 loss-of-function (LOF). Further work demonstrated that dmfrn OE or Fer3HCH knockdown significantly rescued the impaired mitochondrial respiration in PINK1 LOF flies, indicating that dmfrn or Fer3HCH may rescue PINK1 LOF phenotypes through elevating mitochondrial bioavailable iron levels to promote mitochondrial respiration (Wan, 2020).

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Rahul, Naz, F., Jyoti, S. and Siddique, Y. H. (2020). Effect of kaempferol on the transgenic Drosophila model of Parkinson's disease. Sci Rep 10(1): 13793. PubMed ID: 32796885

Abstract

The present study was aimed to study the effect of kaempferol, on the transgenic Drosophila model of Parkinson's disease. Kaempferol was added in the diet at final concentration of 10, 20, 30 and 40 µM and the effect was studied on various cognitive and oxidative stress markers. The results of the study showed that kaempferol, delayed the loss of climbing ability as well as the activity of PD flies in a dose dependent manner compared to unexposed PD flies. A dose-dependent reduction in oxidative stress markers was also observed. Histopathological examination of fly brains using anti-tyrosine hydroxylase immunostaining has revealed a significant dose-dependent increase in the expression of tyrosine hydroxylase in PD flies exposed to kaempferol. Molecular docking results revealed that kaempferol binds to human alpha synuclein at specific sites that might results in the inhibition of alpha synuclein aggregation and prevents the formation of Lewy bodies (Rahul, 2020).

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Blosser, J. A., Podolsky, E. and Lee, D. (2020). L-DOPA-Induced Dyskinesia in a Genetic Drosophila Model of Parkinson's Disease. Exp Neurobiol 29(4): 273-284. PubMed ID: 32921640

Abstract

Motor symptoms in Parkinson's disease (PD) are directly related to the reduction of a neurotransmitter dopamine. Therefore, its precursor L-DOPA became the gold standard for PD treatment. However, chronic use of L-DOPA causes uncontrollable, involuntary movements, called L-DOPA-induced dyskinesia (LID) in the majority of PD patients. LID is complicated and very difficult to manage. Current rodent and non-human primate models have been developed to study LID mainly using neurotoxins. Therefore, it is necessary to develop a LID animal model with defects in genetic factors causing PD in order to study the relation between LID and PD genes such as α-synuclein. This study first showed that a low concentration of L-DOPA (100 μM) rescues locomotion defects (i.e., speed, angular velocity, pause time) in Drosophila larvae expressing human mutant α-synuclein (A53T). This A53T larval model of PD was used to further examine dyskinetic behaviors. High concentrations of L-DOPA (5 or 10 mM) causes hyperactivity such as body bending behavior (BBB) in A53T larva, which resembles axial dyskinesia in rodents. Using ImageJ plugins and other third party software, dyskinetic BBB has been accurately and efficiently quantified. Further, a dopamine agonist pramipexole (PRX) partially rescues BBB caused by high L-DOPA. This Drosophila genetic LID model will provide an important experimental platform to examine molecular and cellular mechanisms underlying LID, to study the role of PD causing genes in the development of LID, and to identify potential targets to slow/reverse LID pathology (Blosser, 2020).

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Sun, L., Zhang, J., Chen, W., Chen, Y., Zhang, X., Yang, M., Xiao, M., Ma, F., Yao, Y., Ye, M., Zhang, Z., Chen, K., Chen, F., Ren, Y., Ni, S., Zhang, X., Yan, Z., Sun, Z. R., Zhou, H. M., Yang, H., Xie, S., Haque, M. E., Huang, K. and Yang, Y. (2020). Attenuation of epigenetic regulator SMARCA4 and ERK-ETS signaling suppresses aging-related dopaminergic degeneration. Aging Cell 19(9): e13210. PubMed ID: 32749068

Abstract

How complex interactions of genetic, environmental factors and aging jointly contribute to dopaminergic degeneration in Parkinson's disease (PD) is largely unclear. This study applied frequent gene co-expression analysis on human patient substantia nigra-specific microarray datasets to identify potential novel disease-related genes. In vivo Drosophila studies validated two of 32 candidate genes, a chromatin-remodeling factor SMARCA4 and a biliverdin reductase BLVRA. Inhibition of SMARCA4 was able to prevent aging-dependent dopaminergic degeneration not only caused by overexpression of BLVRA but also in four most common Drosophila PD models. Furthermore, down-regulation of SMARCA4 specifically in the dopaminergic neurons prevented shortening of life span caused by α-synuclein and LRRK2. Mechanistically, aberrant SMARCA4 and BLVRA converged on elevated ERK-ETS activity, attenuation of which by either genetic or pharmacological manipulation effectively suppressed dopaminergic degeneration in Drosophila in vivo. Down-regulation of SMARCA4 or drug inhibition of MEK/ERK also mitigated mitochondrial defects in PINK1 (a PD-associated gene)-deficient human cells. These findings underscore the important role of epigenetic regulators and implicate a common signaling axis for therapeutic intervention in normal aging and a broad range of age-related disorders including PD (Sun, 2020).

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Chauhan, N., Shrivastava, N. K., Agrawal, N. and Shakarad, M. N. (2020). Wnt2 overexpression protects against PINK1 mutant induced mitochondrial dysfunction and oxidative stress. Wing patterning in faster developing Drosophila is associated with high ecdysone titer and wingless expression. Mech Dev: 103626. PubMed ID: 32526278

Abstract
'Developmental robustness' is the ability of biological systems to maintain a stable phenotype despite genetic, environmental or physiological perturbations. In holometabolous insects, accurate patterning and development is guaranteed by alignment of final gene expression patterns in tissues at specific developmental stage such as molting and pupariation, irrespective of individual rate of development. In the present study, faster developing Drosophila melanogaster populations were used that show reduction of ~22% in egg to adult development time. Flies from the faster developing population exhibit phenotype constancy, although significantly small in size. The reduction in development time in faster developing flies is possibly due to coordination between higher ecdysteroid release and higher expression of developmental genes. The two together might be ensuring appropriate pattern formation and early exit at each development stage in the populations selected for faster pre-adult development compared to their ancestral controls. This study reports that apart from plasticity in the rate of pattern progression, alteration in the level of gene expression may be responsible for pattern integrity even under reduced development time.

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Xia, S. R., Wen, X. Y., Fan, X. L., Chen, X. R., Wei, Z. W., Li, Q. H. and Sun, L. (2020). Wnt2 overexpression protects against PINK1 mutant induced mitochondrial dysfunction and oxidative stress. Mol Med Rep 21(6): 2633-2641. PubMed ID: 32323790

Abstract

The PTEN induced putative kinase 1 (PINK1) mutation is the second most common cause of autosomal recessive adolescent Parkinson's disease (PD). Furthermore, mitochondrial disorders and oxidative stress are important mechanisms in the pathogenesis of PD. Numerous members of the Wnt family have been found to be associated with neurodegenerative diseases. Therefore, the present study investigated the role of the Wnt2 gene in PINK1B9 transgenic flies, which is a PD model, and its underlying mechanism. It was identified that overexpression of Wnt2 reduced the abnormality rate of PD transgenic Drosophila and improved their flight ability, while other intervention groups had no significant effect. Furthermore, an increase in ATP concentration normalized mitochondrial morphology, and increased the mRNA expression levels of NADHubiquinone oxidoreductase chain 1 (ND1), ND42, ND75, succinate dehydrogenase complex subunits B, Cytochrome b and Cyclooxygenase 1, which are associated with Wnt2 overexpression. Moreover, overexpression of Wnt2 in PD transgenic Drosophila resulted in the downregulation of reactive oxygen species and malondialdehyde production, and increased manganese superoxide dismutase (MnSOD), while glutathione was not significantly affected. It was found that overexpression of Wnt2 did not alter the protein expression of betacatenin in PINK1B9 transgenic Drosophila, but did increase the expression levels of PPARG coactivator 1alpha (PGC1alpha) and forkhead box subgroup O (FOXO). Collectively, the present results indicated that the Wnt2 gene may have a protective effect on PD PINK1B9 transgenic Drosophila. Thus, it was speculated that the reduction of oxidative stress and the restoration of mitochondrial function via Wnt2 overexpression may be related to the PGC1alpha/FOXO/MnSOD signaling pathway in PINK1 mutant transgenic Drosophila (Xia, 2020).

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Shiba-Fukushima, K., Inoshita, T., Sano, O., Iwata, H., Ishikawa, K. I., Okano, H., Akamatsu, W., Imai, Y. and Hattori, N. (2020). A Cell-Based High-Throughput Screening Identified Two Compounds that Enhance PINK1-Parkin Signaling. iScience 23(5): 101048. PubMed ID: 32335362

Abstract

Early-onset Parkinson's disease-associated PINK1-Parkin signaling maintains mitochondrial health. Therapeutic approaches for enhancing PINK1-Parkin signaling present a potential strategy for treating various diseases caused by mitochondrial dysfunction. This study reports two chemical enhancers of PINK1-Parkin signaling, identified using a robust cell-based high-throughput screening system. These small molecules, T0466 and T0467, activate Parkin mitochondrial translocation in dopaminergic neurons and myoblasts at low doses that do not induce mitochondrial accumulation of PINK1. Moreover, both compounds reduce unfolded mitochondrial protein levels, presumably through enhanced PINK1-Parkin signaling. These molecules also mitigate the locomotion defect, reduced ATP production, and disturbed mitochondrial Ca(2+) response in the muscles along with the mitochondrial aggregation in dopaminergic neurons through reduced PINK1 activity in Drosophila. These results suggested that T0466 and T0467 may hold promise as therapeutic reagents in Parkinson's disease and related disorders (Shiba-Fukushima, 2020).

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Petridi, S., Middleton, C. A., Ugbode, C., Fellgett, A., Covill, L. and Elliott, C. J. H. (2020). In Vivo Visual Screen for Dopaminergic Rab <--> LRRK2-G2019S Interactions in Drosophila Discriminates Rab10 from Rab3. G3 (Bethesda). PubMed ID: 32321836

Abstract

LRRK2 mutations cause Parkinson's, but the molecular link from increased kinase activity to pathological neurodegeneration remains undetermined. Previous in vitro assays indicate that LRRK2 substrates include at least 8 Rab GTPases. This hypothesis was examined in vivo in a functional, electroretinogram screen, expressing each Rab with/without LRRK2-G2019S in selected Drosophila dopaminergic neurons. The screen discriminated Rab10 from Rab3. The strongest Rab/LRRK2-G2019S interaction is with Rab10; the weakest with Rab3. Rab10 is expressed in a different set of dopaminergic neurons from Rab3. Thus, anatomical and physiological patterns of Rab10 are related. It is concluded that Rab10 is a valid substrate of LRRK2 in dopaminergic neurons in vivo. It is proposed that variations in Rab expression contribute to differences in the rate of neurodegeneration recorded in different dopaminergic nuclei in Parkinson's (Petridi, 2020).

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Xu, Y., Xie, M., Xue, J., Xiang, L., Li, Y., Xiao, J., Xiao, G. and Wang, H. L. (2020). EGCG ameliorates neuronal and behavioral defects by remodeling gut microbiota and TotM expression in Drosophila models of Parkinson's disease. Faseb j. PubMed ID: 32157731

Abstract

Parkinson's disease (PD) is the second most common neurodegenerative disease. Eigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, is known to exert a beneficial effect on PD patients. Although some mechanisms were suggested to underlie this intervention, it remains unknown if the EGCG-mediated protection was achieved by remodeling gut microbiota. In the present study, 0.1 mM or 0.5 mM EGCG was administered to the Drosophila melanogaster with PINK1 (PTEN induced putative kinase 1) mutations, a prototype PD model, and their behavioral performances, as well as neuronal/mitochondrial morphology (only for 0.5 mM EGCG treatment) were determined. According to the results, the mutant PINK1(B9) flies exhibited dopaminergic, survival, and behavioral deficits, which were rescued by EGCG supplementation. Meanwhile, EGCG resulted in profound changes in gut microbial compositions in PINK1(B9) flies, restoring the abundance of a set of bacteria. Notably, EGCG protection was blunted when gut microbiota was disrupted by antibiotics. Four bacterial strains were isolated from fly guts and the supplementation of individual Lactobacillus plantarum or Acetobacter pomorum strain exacerbated the neuronal and behavioral dysfunction of PD flies, which could not be rescued by EGCG. Transcriptomic analysis identified TotM as the central gene responding to EGCG or microbial manipulations. Genetic ablation of TotM blocked the recovery activity of EGCG, suggesting that EGCG-mediated protection warrants TotM. Apart from familial form, EGCG was also potent in improving sporadic PD symptoms induced by rotenone treatment, wherein gut microbiota shared regulatory roles. Together, these results suggest the relevance of the gut microbiota-TotM pathway in EGCG-mediated neuroprotection, providing insight into indirect mechanisms underlying nutritional intervention of Parkinson's disease (Xu, 2020).

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Pirooznia, S. K., Yuan, C., Khan, M. R., Karuppagounder, S. S., Wang, L., Xiong, Y., Kang, S. U., Lee, Y., Dawson, V. L. and Dawson, T. M. (2020). PARIS induced defects in mitochondrial biogenesis drive dopamine neuron loss under conditions of parkin or PINK1 deficiency. Mol Neurodegener 15(1): 17. PubMed ID: 32138754

Abstract

Mutations in PINK1 and parkin cause autosomal recessive Parkinson's disease (PD). Evidence placing PINK1 and parkin in common pathways regulating multiple aspects of mitochondrial quality control is burgeoning. However, compelling evidence to causatively link specific PINK1/parkin dependent mitochondrial pathways to dopamine neuron degeneration in PD is lacking. This study examined how PINK1/parkin mediated regulation of the pathogenic substrate PARIS impacts dopaminergic mitochondrial network homeostasis and neuronal survival in Drosophila. The UAS-Gal4 system was employed for cell-type specific expression of the various transgenes. Effects on dopamine neuronal survival and function were assessed by anti-TH immunostaining and negative geotaxis assays. Defects in mitochondrial biogenesis were shown to drive adult onset progressive loss of dopamine neurons and motor deficits in Drosophila models of PINK1 or parkin insufficiency. Such defects result from PARIS dependent repression of dopaminergic PGC-1alpha and its downstream transcription factors NRF1 and TFAM that cooperatively promote mitochondrial biogenesis. Dopaminergic accumulation of human or Drosophila PARIS recapitulates these neurodegenerative phenotypes that are effectively reversed by PINK1, parkin or PGC-1alpha overexpression in vivo. PARIS is the only co-substrate of PINK1 and parkin to specifically accumulate in the DA neurons and cause neurodegeneration and locomotor defects stemming from disrupted dopamine signaling. These findings identify a highly conserved role for PINK1 and parkin in regulating mitochondrial biogenesis and promoting mitochondrial health via the PARIS/ PGC-1alpha axis. The Drosophila models described in this study effectively recapitulate the cardinal PD phenotypes and thus will facilitate identification of novel regulators of mitochondrial biogenesis for physiologically relevant therapeutic interventions (Pirooznia, 2020).

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Casu, M. A., Mocci, I., Isola, R., Pisanu, A., Boi, L., Mulas, G., Greig, N. H., Setzu, M. D. and Carta, A. R. (2020). Neuroprotection by the Immunomodulatory Drug Pomalidomide in the Drosophila LRRK2(WD40) Genetic Model of Parkinson's Disease. Front Aging Neurosci 12: 31. PubMed ID: 32116655

Abstract

The search for new disease-modifying drugs for Parkinson's disease (PD) is a slow and highly expensive process, and the repurposing of drugs already approved for different medical indications is becoming a compelling alternative option for researchers. Genetic variables represent a predisposing factor to the disease and mutations in leucine-rich repeat kinase 2 (LRRK2) locus have been correlated to late-onset autosomal-dominant PD. The common fruit fly Drosophila melanogaster carrying the mutation LRRK2 loss-of-function in the WD40 domain (LRRK2(WD40)), is a simple in vivo model of PD and is a valid tool to first evaluate novel therapeutic approaches to the disease. Recent studies have suggested a neuroprotective activity of immunomodulatory agents in PD models. In this study the immunomodulatory drug Pomalidomide (POM), a Thalidomide derivative, was examined in the Drosophila LRRK2(WD40) genetic model of PD. Mutant and wild type flies received increasing POM doses (1, 0.5, 0.25 mM) through their diet from day 1 post eclosion, until postnatal day (PN) 7 or 14, when POM's actions were evaluated by quantifying changes in climbing behavior as a measure of motor performance, the number of brain dopaminergic neurons and T-bars, mitochondria integrity. LRRK2(WD40) flies displayed a spontaneous age-related impairment of climbing activity, and POM significantly and dose-dependently improved climbing performance both at PN 7 and PN 14. LRRK2(WD40) fly motor disability was underpinned by a progressive loss of dopaminergic neurons in posterior clusters of the protocerebrum, which are involved in the control of locomotion, by a low number of T-bars density in the presynaptic bouton active zones. POM treatment fully rescued the cell loss in all posterior clusters at PN 7 and PN 14 and significantly increased the T-bars density. Moreover, several damaged mitochondria with dilated cristae were observed in LRRK2(WD40) flies treated with vehicle but not following POM. This study demonstrates the neuroprotective activity of the immunomodulatory agent POM in a genetic model of PD. POM is an FDA-approved clinically available and well-tolerated drug used for the treatment of multiple myeloma. If further validated in mammalian models of PD, POM could rapidly be clinically tested in humans (Casu, 2020).

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Ham, S. J., Lee, D., Yoo, H., Jun, K., Shin, H. and Chung, J. (2020). Decision between mitophagy and apoptosis by Parkin via VDAC1 ubiquitination. Proc Natl Acad Sci U S A. PubMed ID: 32047033

Abstract

VDAC1 is a critical substrate of Parkin responsible for the regulation of mitophagy and apoptosis. This study demonstrates that VDAC1 can be either mono- or polyubiquitinated by Parkin in a PINK1-dependent manner. VDAC1 deficient with polyubiquitination (VDAC1 Poly-KR) hampers mitophagy, but VDAC1 deficient with monoubiquitination (VDAC1 K274R) promotes apoptosis by augmenting the mitochondrial calcium uptake through the mitochondrial calcium uniporter (MCU) channel. The transgenic flies expressing Drosophila Porin K273R, corresponding to human VDAC1 K274R, show Parkinson disease (PD)-related phenotypes including locomotive dysfunction and degenerated dopaminergic neurons, which are relieved by suppressing MCU and mitochondrial calcium uptake. To further confirm the relevance of these findings in PD, a missense mutation of Parkin was discovered in PD patients, T415N, which lacks the ability to induce VDAC1 monoubiquitination but still maintains polyubiquitination. Interestingly, Drosophila Parkin T433N, corresponding to human Parkin T415N, fails to rescue the PD-related phenotypes of Parkin-null flies. Taken together, these results suggest that VDAC1 monoubiquitination plays important roles in the pathologies of PD by controlling apoptosis (Ham, 2020).

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Lee, J. J., Andreazza, S. and Whitworth, A. J. (2020). The STING pathway does not contribute to behavioural or mitochondrial phenotypes in Drosophila Pink1/parkin or mtDNA mutator models. Sci Rep 10(1): 2693. PubMed ID: 32060339

Abstract

Mutations in PINK1 and Parkin/PRKN cause the degeneration of dopaminergic neurons in familial forms of Parkinson's disease but the precise pathogenic mechanisms are unknown. The PINK1/Parkin pathway has been described to play a central role in mitochondrial homeostasis by signalling the targeted destruction of damaged mitochondria, however, how disrupting this process leads to neuronal death was unclear until recently. An elegant study in mice revealed that the loss of Pink1 or Prkn coupled with an additional mitochondrial stress resulted in the aberrant activation of the innate immune signalling, mediated via the cGAS/Relish, was insufficient to suppress the behavioural deficits or mitochondria disruption in the Pink1/parkin mutants. Thus, it is concluded that phenotypes associated with loss of Pink1/parkin are not universally due to aberrant activation of the STING pathway (Lee, 2020).

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Diana, A., Collu, M., Casu, M. A., Mocci, I., Aguilar-Santelises, M. and Setzu, M. D. (2020). Improvements of motor performances in the Drosophila LRRK2 loss-of-function model of Parkinson's disease: Effects of dialyzed leucocyte Extracts from Human Serum. Brain Sci 10(1). PubMed ID: 31947539

Abstract

Within neurodegenerative syndromes, Parkinson's disease (PD) is typically associated with its locomotor defects, sleep disturbances and related dopaminergic (DA) neuron loss. The fruit fly, Drosophila melanogaster, with leucine-rich repeat kinase 2 mutants (LRRK2) loss-of-function in the WD40 domain, provides mechanistic insights into corresponding human behaviour, possibly disclosing some physiopathologic features of PD in both genetic and sporadic forms. Moreover, several data support the boosting impact of innate and adaptive immunity pathways for driving the progression of PD. In this context, human dialyzable leukocyte extracts (DLE) have been extensively used to transfer antigen-specific information that influences the activity of various immune components, including inflammatory cytokines. Hence, the main goal of this study was to ascertain the therapeutic potential of DLE from male and female donors on D. melanogaster LRRK2 loss-of-function, as compared to D. melanogaster wild-type (WT), in terms of rescuing physiological parameters, such as motor and climbing activities, which are severely compromised in the mutant flies. Finally, in search of the anatomical structures responsible for restored functions in parkinsonian-like mutant flies, this study found a topographical correlation between improvement of locomotor performances and an increased number of dopaminergic neurons in selective areas of LRRK2 mutant brains (Diana, 2020).

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Imai, Y., Inoshita, T., Meng, H., Shiba-Fukushima, K., Hara, K. Y., Sawamura, N. and Hattori, N. (2019). Light-driven activation of mitochondrial proton-motive force improves motor behaviors in a Drosophila model of Parkinson's disease. Commun Biol 2: 424. PubMed ID: 31799427

Abstract

Mitochondrial degeneration is considered one of the major causes of Parkinson's disease (PD). Improved mitochondrial functions are expected to be a promising therapeutic strategy for PD. This study introduced a light-driven proton transporter, Delta-rhodopsin (dR), to Drosophila mitochondria, where the mitochondrial proton-motive force (Deltap) and mitochondrial membrane potential are maintained in a light-dependent manner. The loss of the PD-associated mitochondrial gene CHCHD2 resulted in reduced ATP production, enhanced mitochondrial peroxide production and lower Ca(2+)-buffering activity in dopaminergic (DA) terminals in flies. These cellular defects were improved by the light-dependent activation of mitochondrion-targeted dR (mito-dR). Moreover, mito-dR reversed the pathology caused by the CHCHD2 deficiency to suppress alpha-synuclein aggregation, DA neuronal loss, and elevated lipid peroxidation in brain tissue, improving motor behaviors. This study suggests the enhancement of Deltap by mito-dR as a therapeutic mechanism that ameliorates neurodegeneration by protecting mitochondrial functions (Imai, 2019).

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Chung, H. J., Islam, M. S., Rahman, M. M. and Hong, S. T. (2019).Neuroprotective function of Omi to alpha-synuclein-induced neurotoxicity. Neurobiol Dis: 104706. PubMed ID: 31837423

Abstract
The main pathological hallmark of Parkinson's disease (PD) is the presence of Lewy bodies, which mainly consist of aggregated alpha-synuclein. Based on the neurotoxicity of oligomeric alpha-synuclein and its significance in the aetiology of PD, there has been decades of effort to elucidate an enzyme specifically degrading oligomeric alpha-synuclein. This study reports an enzyme, Omi, which specifically recognizes and precisely degrades oligomeric alpha-synuclein but not monomeric alpha-synuclein. After enzymatic and functional analyses of Omi in in vitro, an in vivo assay system of dual gene interaction was developed in Drosophila to investigate further the etiological role of Omi in PD. Pan-neuronal expression of Omi rescued Parkinsonism in a Drosophila model of PD, while Knockout of Omi exacerbated Parkinsonism. Expression of Omi counteracted the alpha-synuclein-induced retinal degeneration, providing additional evidence for Omi's protective role oligomeric alpha-synuclein. This work reports identification of the catabolic pathway of oligomeric alpha-synuclein as well as showing how Omi functions as the key molecule in the recognition and degradation of toxic oligomeric alpha-synuclein, a possible cause of neurodegeneration in PD, without affecting monomeric alpha-synuclein which is a native essential molecule for the normal function of neurons.

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Yedlapudi, D., Xu, L., Luo, D., Marsh, G. B., Todi, S. V. and Dutta, A. K. (2019). Targeting alpha synuclein and amyloid beta by a multifunctional, brain-penetrant dopamine D2/D3 agonist D-520: Potential therapeutic application in Parkinson's disease with dementia. Sci Rep 9(1): 19648. PubMed ID: 31873106

Abstract
A significant number of people with Parkinson's disease (PD) develop dementia in addition to cognitive dysfunction and are diagnosed as PD with dementia (PDD). This is characterized by cortical and limbic alpha synuclein (alpha-syn) accumulation, and high levels of diffuse amyloid beta (Abeta) plaques in the striatum and neocortical areas. In this regard, this study evaluated the effect of a brain-penetrant, novel multifunctional dopamine D2/D3 agonist, D-520 on the inhibition of Abeta aggregation and disintegration of alpha-syn and Abeta aggregates in vitro using purified proteins and in a cell culture model that produces intracellular Abeta-induced toxicity. The effect of D-520 was evaluated in a Drosophila model of Abeta1-42 toxicity. It is reported that D-520 inhibits the formation of Abeta aggregates in vitro and promotes the disaggregation of both alpha-syn and Abeta aggregates. Finally, in an in vivo Drosophila model of Abeta1-42 dependent toxicity, D-520 exhibited efficacy by rescuing fly eyes from retinal degeneration caused by Abeta toxicity. These data indicate the potential therapeutic applicability of D-520 in addressing motor dysfunction and neuroprotection in PD and PDD, as well as attenuating dementia in people with PDD.

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Sim, J. P. L., Ziyin, W., Basil, A. H., Lin, S., Chen, Z., Zhang, C., Zeng, L., Cai, Y. and Lim, K. L. (2019). Identification of PP2A and S6 kinase as modifiers of Leucine-rich repeat kinase-induced neurotoxicity. Neuromolecular Med. PubMed ID: 31664682

Abstract

Mutations in LRRK2 are currently recognized as the most common monogenetic cause of Parkinsonism. The elevation of kinase activity of LRRK2 that frequently accompanies its mutations is widely thought to contribute to its toxicity. Accordingly, many groups have developed LRRK2-specific kinase inhibitors as a potential therapeutic strategy. Given that protein phosphorylation is a reversible event, this study sought to elucidate the phosphatase(s) that can reverse LRRK2-mediated phosphorylation, with the view that targeting this phosphatase(s) may similarly be beneficial. Using an unbiased RNAi phosphatase screen conducted in a Drosophila LRRK2 model, PP2A was identified as a genetic modulator of LRRK2-induced neurotoxicity. Further, ribosomal S6 kinase (S6K), a target of PP2A, was also identified as a novel regulator of LRRK2 function. Finally, modulation of PP2A or S6K activities were shown to ameliorate LRRK2-associated disease phenotype in Drosophila.

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Man Anh, H., Linh, D. M., My Dung, V. and Thi Phuong Thao, D. (2019). Evaluating dose- and time-dependent effects of vitamin C treatment on a Parkinson's disease fly model. Parkinsons Dis 2019: 9720546. PubMed ID: 30719278

Abstract

Parkinson's disease (PD) is a common neurodegenerative disorder and characterized by progressive locomotive defects and loss of dopaminergic neurons (DA neuron). Currently, there is no potent therapy to cure PD, and the medications merely support to control the symptoms. It is difficult to develop an effective treatment, since the PD onset mechanism of PD is still unclear. Oxidative stress is considered as a major cause of neurodegenerative diseases, and there is increasing evidence for the association between PD and oxidative stress. Therefore, antioxidant treatment may be a promising therapy for PD. Drosophila with knockdown of dUCH, a homolog of UCH-L1 which is a PD-related gene, exhibited PD-like phenotypes including progressive locomotive impairments and DA neuron degeneration. Moreover, knockdown of dUCH led to elevated level of ROS. Thus, dUCH knockdown flies can be used as a model for screening of potential antioxidants for treating PD. Previous studies demonstrated that curcumin at 1 mM and vitamin C at 0.5 mM could improve PD-like phenotypes induced by this knockdown. With the purpose of further investigating the efficiency of vitamin C in PD treatment, dUCH knockdown Drosophila model was used to examine the dose- and time-dependent effects of vitamin C on PD-like phenotypes. The results showed that although vitamin C exerted neuroprotective effects, high doses of vitamin C and long-term treatment with this antioxidant also resulted in side effects on physiology. It is suggested that dose-dependent effects of vitamin C should be considered when used for treating PD (Man Anh, 2019).

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Aggarwal, A., Reichert, H. and VijayRaghavan, K. (2019). A locomotor assay reveals deficits in heterozygous Parkinson's disease model and proprioceptive mutants in adult Drosophila. Proc Natl Acad Sci U S A. PubMed ID: 31748267

Abstract

Severe locomotor impairment is a common phenotype of neurodegenerative disorders such as Parkinson's disease (PD). Drosophila models of PD, studied for more than a decade, have helped in understanding the interaction between various genetic factors, such as parkin and PINK1, in this disease. To characterize locomotor behavioral phenotypes for these genes, fly climbing assays have been widely used. While these simple current assays for locomotor defects in Drosophila mutants measure some locomotor phenotypes well, it is possible that detection of subtle changes in behavior is important to understand the manifestation of locomotor disorders. This study introduces a climbing behavior assay which provides such fine-scale behavioral data and tests this proposition for the Drosophila model. This inexpensive, fully automated assay was used to quantitatively characterize the climbing behavior at high parametric resolution in 3 contexts. First, wild-type flies were characterized, and a hitherto unknown sexual dimorphism in climbing behavior was uncovered. Second, climbing behavior was studied of heterozygous mutants of genes implicated in the fly PD model, and previously unreported prominent locomotor defects were revealed in some of these heterozygous fly lines. Finally, locomotor defects were studied in a homozygous proprioceptory mutation (Trp-gamma1) known to affect fine motor control in Drosophila. Moreover, aberrant geotactic behavior was identified in Trp-gamma1 mutants, thereby opening up a finer assay for geotaxis and its genetic basis. This assay is therefore a cost-effective, general tool for measuring locomotor behaviors of wild-type and mutant flies in fine detail and can reveal subtle motor defects (Aggarwal, 2019).

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Mori, A., Hatano, T., Inoshita, T., Shiba-Fukushima, K., Koinuma, T., Meng, H., Kubo, S. I., Spratt, S., Cui, C., Yamashita, C., Miki, Y., Yamamoto, K., Hirabayashi, T., Murakami, M., Takahashi, Y., Shindou, H., Nonaka, T., Hasegawa, M., Okuzumi, A., Imai, Y. and Hattori, N. (2019). Parkinson's disease-associated iPLA2-VIA/PLA2G6 regulates neuronal functions and alpha-synuclein stability through membrane remodeling. Proc Natl Acad Sci U S A 116(41): 20689-20699. PubMed ID: 31548400

Abstract

Mutations in the iPLA2-VIA/PLA2G6 gene are responsible for PARK14-linked Parkinson's disease (PD) with alpha-synucleinopathy. However, it is unclear how iPLA2-VIA mutations lead to alpha-synuclein (alpha-Syn) aggregation and dopaminergic (DA) neurodegeneration. This study reports that iPLA2-VIA-deficient Drosophila exhibits defects in neurotransmission during early developmental stages and progressive cell loss throughout the brain, including degeneration of the DA neurons. Lipid analysis of brain tissues reveals that the acyl-chain length of phospholipids is shortened by iPLA2-VIA loss, which causes endoplasmic reticulum (ER) stress through membrane lipid disequilibrium. The introduction of wild-type human iPLA2-VIA or the mitochondria-ER contact site-resident protein C19orf12 in iPLA2-VIA-deficient flies rescues the phenotypes associated with altered lipid composition, ER stress, and DA neurodegeneration, whereas the introduction of a disease-associated missense mutant, iPLA2-VIA A80T, fails to suppress these phenotypes. The acceleration of alpha-Syn aggregation by iPLA2-VIA loss is suppressed by the administration of linoleic acid, correcting the brain lipid composition. These findings suggest that membrane remodeling by iPLA2-VIA is required for the survival of DA neurons and alpha-Syn stability (Mori, 2019).

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Ding, Y., Kong, D., Zhou, T., Yang, N. D., Xin, C., Xu, J., Wang, Q., Zhang, H., Wu, Q., Lu, X., Lim, K., Ma, B., Zhang, C., Li, L. and Huang, W. (2019). alpha-Arbutin protects against Parkinson's disease-associated mitochondrial dysfunction in vitro and in vivo. Neuromolecular Med. PubMed ID: 31401719

Abstract

Parkinson's disease (PD), the most common neurodegenerative movement disorder, is characterized by the progressive loss of dopaminergic neurons in substantia nigra. The underlying mechanisms of PD pathogenesis have not been fully illustrated and currently PD remains incurable. Accumulating evidences suggest that mitochondrial dysfunction plays pivotal role in the dopaminergic neuronal death. Therefore, discovery of novel and safe agent for rescuing mitochondrial dysfunction would benefit PD treatment. This study demonstrated that alpha-Arbutin (Arb), a natural polyphenol extracted from Ericaceae species, displayed significant protective effect on the rotenone (Rot)-induced mitochondrial dysfunction and apoptosis of human neuroblastoma cell (SH-SY5Y). It was further found that the neuroprotective effect of Arb was associated with ameliorating oxidative stress, stabilizing of mitochondrial membrane potential, and enhancing adenosine triphosphate production. To investigate the underlying mechanism, the AMP-activated protein kinase and autophagy pathway was checked, were found to be involved in the neuroprotection of Arb. Moreover, the protective effect of Arb was explored in Drosophila PD model, and Arb was found to rescue parkin deficiency-induced motor function disability and mitochondrial abnormality of Drosophila. Taken together, this study demonstrated that Arb got excellent neuroprotective effect on PD models both in vitro and in vivo and Arb might serve as a potent therapeutic agent for the treatment of PD (DingY, 2019).

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Wu, S., Tan, K. J., Govindarajan, L. N., Stewart, J. C., Gu, L., Ho, J. W. H., Katarya, M., Wong, B. H., Tan, E. K., Li, D., Claridge-Chang, A., Libedinsky, C., Cheng, L. and Aw, S. S. (2019). Fully automated leg tracking of Drosophila neurodegeneration models reveals distinct conserved movement signatures. PLoS Biol 17(6): e3000346. PubMed ID: 31246996

Abstract

Some neurodegenerative diseases, like Parkinsons Disease (PD) and Spinocerebellar ataxia 3 (SCA3), are associated with distinct, altered gait and tremor movements that are reflective of the underlying disease etiology. Drosophila melanogaster models of neurodegeneration have led to understanding of the molecular mechanisms of disease. However, it is unknown whether specific gait and tremor dysfunctions also occur in fly disease mutants. To answer this question, a machine-learning image-analysis program, Feature Learning-based LImb segmentation and Tracking (FLLIT), was developed that automatically tracks leg claw positions of freely moving flies recorded on high-speed video, producing a series of gait measurements. Notably, unlike other machine-learning methods, FLLIT generates its own training sets and does not require user-annotated images for learning. Using FLLIT, high-throughput and high-resolution analysis of gait and tremor features were carried out in Drosophila neurodegeneration mutants for the first time. Fly models of PD and SCA3 exhibited markedly different walking gait and tremor signatures, which recapitulated characteristics of the respective human diseases. Selective expression of mutant SCA3 in dopaminergic neurons led to a gait signature that more closely resembled those of PD flies. This suggests that the behavioral phenotype depends on the neurons affected rather than the specific nature of the mutation. Different mutations produced tremors in distinct leg pairs, indicating that different motor circuits were affected. Using this approach, fly models can be used to dissect the neurogenetic mechanisms that underlie movement disorders (Wu, 2019).

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Doktor, B., Damulewicz, M. and Pyza, E. (2019). Effects of MUL1 and PARKIN on the circadian clock, brain and behaviour in Drosophila Parkinson's disease models. BMC Neurosci 20(1): 24. PubMed ID: 31138137

Abstract

Mutants which carry mutations in genes encoding mitochondrial ligases MUL1 and PARKIN are convenient Drosophila models of Parkinson's disease (PD). In several studies it has been shown that in Parkinson's disease sleep disturbance occurs, which may be the result of a disturbed circadian clock. This study found that the ROS level was higher, while the anti-oxidant enzyme SOD1 level was lower in mul1(A6) and park(1) mutants than in the white mutant used as a control. Moreover, mutations of both ligases affected circadian rhythms and the clock. The expression of clock genes per, tim and clock and the level of PER protein were changed in the mutants. Moreover, expression of ATG5, an autophagy protein also involved in circadian rhythm regulation, was decreased in the brain and in PDF-immunoreactive large ventral lateral clock neurons. The observed changes in the molecular clock resulted in a longer period of locomotor activity rhythm, increased total activity and shorter sleep at night. Finally, the lack of both ligases led to decreased longevity and climbing ability of the flies. It is concluded that all of the changes observed in the brains of these Drosophila models of PD, in which mitochondrial ligases MUL1 and PARKIN do not function, may explain the mechanisms of some neurological and behavioural symptoms of PD (Doktor, 2019).

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Sakai, R., Suzuki, M., Ueyama, M., Takeuchi, T., Minakawa, E. N., Hayakawa, H., Baba, K., Mochizuki, H. and Nagai, Y. (2019). E46K mutant alpha-synuclein is more degradation resistant and exhibits greater toxic effects than wild-type alpha-synuclein in Drosophila models of Parkinson's disease. PLoS One 14(6): e0218261. PubMed ID: 31242217

Abstract

Alpha-synuclein (alphaSyn) plays key roles in the pathogenesis of Parkinson's disease (PD). The mechanisms underlying the variance in the clinical phenotypes of familial PD caused by missense mutations in the alphaSyn gene remain elusive. This study established novel Drosophila models expressing either wild-type (WT) alphaSyn or one of five alphaSyn mutants (A30P, E46K, H50Q, G51D, and A53T) using site-specific transgenesis, which express transgenes at equivalent levels. Expression of either WT or mutant alphaSyn in the compound eyes by the GMR-GAL4 driver caused mild rough eye phenotypes with no obvious difference among the mutants. Upon pan-neuronal expression by the nSyb-GAL4 driver, these alphaSyn-expressing flies showed a progressive decline in locomotor function. Notably, it was found that E46K, H50Q, G51D, and A53T alphaSyn-expressing flies showed earlier onset of locomotor dysfunction than WT alphaSyn-expressing flies, suggesting their enhanced toxic effects. Whereas mRNA levels of WT and mutant alphaSyn were almost equivalent, it was found that protein expression levels of E46K alphaSyn were higher than those of WT alphaSyn. In vivo chase experiments using the drug-inducible GMR-GeneSwitch driver demonstrated that degradation of E46K alphaSyn protein was significantly slower than WT alphaSyn protein, indicating that the E46K alphaSyn mutant gains resistance to degradation in vivo. It is therefore conclude that the novel site-specific transgenic fly models expressing either WT or mutant alphaSyn are useful to explore the mechanisms by which different alphaSyn mutants gain toxic functions in vivo (Sakai, 2019).

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Cackovic, J., Gutierrez-Luke, S., Call, G. B., Juba, A., O'Brien, S., Jun, C. H. and Buhlman, L. M. (2018). Vulnerable parkin loss-of-function Drosophila dopaminergic neurons have advanced mitochondrial aging, mitochondrial network loss and transiently reduced autophagosome recruitment. Front Cell Neurosci 12: 39. PubMed ID: 29497364

Abstract

Selective degeneration of substantia nigra dopaminergic (DA) neurons is a hallmark pathology of familial Parkinson's disease (PD). While the mechanism of degeneration is elusive, abnormalities in mitochondrial function and turnover are strongly implicated. An Autosomal Recessive-Juvenile Parkinsonism (AR-JP) Drosophila melanogaster model exhibits DA neurodegeneration as well as aberrant mitochondrial dynamics and function. Disruptions in mitophagy have been observed in parkin loss-of-function models, and changes in mitochondrial respiration have been reported in patient fibroblasts. Whether loss of parkin causes selective DA neurodegeneration in vivo as a result of lost or decreased mitophagy is unknown. This study employs the use of fluorescent constructs expressed in Drosophila DA neurons that are functionally homologous to those of the mammalian substantia nigra. Evidence is provided that degenerating DA neurons in parkin loss-of-function mutant flies have advanced mitochondrial aging and that mitochondrial networks are fragmented and contain swollen organelles. This study also found that mitophagy initiation is decreased in park (Drosophila parkin/PARK2 ortholog) homozygous mutants, but autophagosome formation is unaffected, and mitochondrial network volumes are decreased. As the fly ages, autophagosome recruitment becomes similar to control, while mitochondria continue to show signs of damage, and climbing deficits persist. Interestingly, aberrant mitochondrial morphology, aging and mitophagy initiation were not observed in DA neurons that do not degenerate. These results suggest that parkin is important for mitochondrial homeostasis in vulnerable Drosophila DA neurons, and that loss of parkin-mediated mitophagy may play a role in degeneration of relevant DA neurons or motor deficits in this model (Cackovic, 2018).

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Ran, D., Xie, B., Gan, Z., Sun, X., Gu, H. and Yang, J. (2018). Melatonin attenuates hLRRK2-induced long-term memory deficit in a Drosophila model of Parkinson's disease. Biomed Rep 9(3): 221-226. PubMed ID: 30271597

Abstract

As the most common genetic cause of Parkinson's disease (PD), the role of human leucine-rich repeat kinase 2 (hLRRK2) in the efficacy of PD treatment is a focus of study. A previous study demonstrated that mushroom body (MB) expression of hLRRK2 in Drosophila could recapitulate the clinical feature of sleep disturbances observed in PD patients, and melatonin (MT) treatment could attenuate the hLRRK2-induced sleep disorders and synaptic dysfunction, suggesting the therapeutic potential of MT in PD patients carrying hLRRK2 mutations; however, no further study into the impacts on memory deficit was conducted. Therefore, in the current paper, the study of the effects of MT on hLRRK2 flies was continued, to determine its potential role in the improvement of memory deficit in PD. To achieve this, the Drosophila learning and memory phases, including short- and long-term memory, were recorded; furthermore, the effect of MT on calcium channel activity during neurotransmission was detected using electrophysiology patch clamp recordings. It was demonstrated that MT treatment reversed hLRRK2-induced long-term memory deficits in Drosophila; furthermore, MT reduced MB calcium channel activities. These findings suggest that MT may exerts therapeutic effects on the long-term memory of PD patients via calcium channel modulation, thus providing indication of its potential to maintain cognitive function in PD patients (Ran, 2018).

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Himmelberg, M. M., West, R. J. H., Elliott, C. J. H. and Wade, A. R. (2018). Abnormal visual gain control and excitotoxicity in early-onset Parkinson's disease Drosophila models. J Neurophysiol. 119(3):957-970 PubMed ID: 29142100

Abstract

The excitotoxic theory of Parkinson's disease (PD) hypothesises that a pathophysiological degeneration of dopaminergic neurons stems from neural hyperactivity at early stages of disease, leading to mitochondrial stress and cell death. Recent research has harnessed the visual system of Drosophila PD models to probe this hypothesis. This study investigated whether abnormal visual sensitivity and excitotoxicity occur in early-onset PD Drosophila models DJ-1Delta72, DJ1-Delta93, and PINK15. An electroretinogram was used to record steady state visually evoked potentials driven by temporal contrast stimuli. At 1 day of age, all early-onset PD mutants had a twofold increase in response amplitudes when compared to w- controls. Further, excitotoxicity was found to occur in older early-onset PD models after increased neural demand is applied via visual stimulation. In an additional analysis, a linear discriminant analysis was used to test whether there were subtle variations in neural gain control that could be used to classify Drosophila into their correct age and genotype. The discriminant analysis was highly accurate, classifying Drosophila into their correct genotypic class at all age groups at 50-70% accuracy (20% chance baseline). Differences in cellular processes link to subtle alterations in neural network operation in young flies, all of which lead to the same pathogenic outcome. These data are the first to demonstrate abnormal gain control and excitotoxicity in early-onset PD Drosophila mutants. It is concluded that early-onset PD mutations may be linked to more sensitive neuronal signalling in prodromal animals that may cause the expression of PD symptomologies later in life (Himmelberg, 2017).

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Valadas, J. S., Esposito, G., Vandekerkhove, D., Miskiewicz, K., Deaulmerie, L., Raitano, S., Seibler, P., Klein, C. and Verstreken, P. (2018). ER lipid defects in neuropeptidergic neurons impair sleep patterns in Parkinson's disease. Neuron 98(6): 1155-1169.e1156. PubMed ID: 29887339

Abstract

Parkinson's disease patients report disturbed sleep patterns long before motor dysfunction. In parkin and pink1 models, this study has identified circadian rhythm and sleep pattern defects and has mapped these to specific neuropeptidergic neurons in fly models and in hypothalamic neurons differentiated from patient induced pluripotent stem cells (iPSCs). Parkin and Pink1 control the clearance of mitochondria by protein ubiquitination. Although major defects were not observed in mitochondria of mutant neuropeptidergic neurons, excess of endoplasmic reticulum-mitochondrial contacts was found. These excessive contact sites cause abnormal lipid trafficking that depletes phosphatidylserine from the endoplasmic reticulum (ER) and disrupts the production of neuropeptide-containing vesicles. Feeding mutant animals phosphatidylserine rescues neuropeptidergic vesicle production and acutely restores normal sleep patterns in mutant animals. Hence, sleep patterns and circadian disturbances in Parkinson's disease models are explained by excessive ER-mitochondrial contacts, and blocking their formation or increasing phosphatidylserine levels rescues the defects in vivo (Valadas, 2018).

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Prasad, V., Wasser, Y., Hans, F., Goswami, A., Katona, I., Outeiro, T. F., Kahle, P. J., Schulz, J. B. and Voigt, A. (2018). Monitoring alpha-synuclein multimerization in vivo. Faseb j: fj201800148RRR. PubMed ID: 30252534

Abstract

The pathophysiology of Parkinson's disease is characterized by the abnormal accumulation of alpha-synuclein (alpha-Syn), eventually resulting in the formation of Lewy bodies and neurites in surviving neurons in the brain. Although alpha-Syn aggregation has been extensively studied in vitro, there is limited in vivo knowledge on alpha-Syn aggregation. This study used the powerful genetics of Drosophila melanogaster and developed an in vivo assay to monitor alpha-Syn accumulation by using a bimolecular fluorescence complementation assay. Both genetic and pharmacologic manipulations affected alpha-Syn accumulation. Interestingly, it was also found that alterations in the cellular protein degradation mechanisms strongly influenced alpha-Syn accumulation. Administration of compounds identified as risk factors for Parkinson's disease, such as rotenone or heavy metal ions, had only mild or even no impact on alpha-Syn accumulation in vivo. Finally, this study showed that increasing phosphorylation of alpha-Syn at serine 129 enhances the accumulation and toxicity of alpha-Syn. Altogether, this study establishes a novel model to study alpha-Syn accumulation and illustrates the complexity of manipulating proteostasis in vivo (Prasad, 2018).

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Lee, K. S., Huh, S., Lee, S., Wu, Z., Kim, A. K., Kang, H. Y. and Lu, B. (2018). Altered ER-mitochondria contact impacts mitochondria calcium homeostasis and contributes to neurodegeneration in vivo in disease models. Proc Natl Acad Sci U S A. PubMed ID: 30185553

Abstract

Calcium (Ca(2+)) homeostasis is essential for neuronal function and survival. Altered Ca(2+) homeostasis has been consistently observed in neurological diseases. How Ca(2+) homeostasis is achieved in various cellular compartments of disease-relevant cell types is not well understood. This study shows in Drosophila Parkinson's disease (PD) models that Ca(2+) transport from the endoplasmic reticulum (ER) to mitochondria through the ER-mitochondria contact site (ERMCS) critically regulates mitochondrial Ca(2+) (mito-Ca(2+)) homeostasis in dopaminergic (DA) neurons, and that the PD-associated PINK1 protein modulates this process. In PINK1 mutant DA neurons, the ERMCS is strengthened and mito-Ca(2+) level is elevated, resulting in mitochondrial enlargement and neuronal death. Miro, a well-characterized component of the mitochondrial trafficking machinery, mediates the effects of PINK1 on mito-Ca(2+) and mitochondrial morphology, apparently in a transport-independent manner. Miro overexpression mimics PINK1 loss-of-function effect, whereas inhibition of Miro or components of the ERMCS, or pharmacological modulation of ERMCS function, rescued PINK1 mutant phenotypes. Mito-Ca(2+) homeostasis is also altered in the LRRK2-G2019S model of PD and the PAR-1/MARK model of neurodegeneration, and genetic or pharmacological restoration of mito-Ca(2+) level is beneficial in these models. Our results highlight the importance of mito-Ca(2+) homeostasis maintained by Miro and the ERMCS to mitochondrial physiology and neuronal integrity. Targeting this mito-Ca(2+) homeostasis pathway holds promise for a therapeutic strategy for neurodegenerative diseases (Lee, 2018).

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Issa, A. R., Sun, J., Petitgas, C., Mesquita, A., Dulac, A., Robin, M., Mollereau, B., Jenny, A., Cherif-Zahar, B. and Birman, S. (2018). The lysosomal membrane protein LAMP2A promotes autophagic flux and prevents SNCA-induced Parkinson disease-like symptoms in the Drosophila brain. Autophagy. PubMed ID: 29989488

Abstract

The autophagy-lysosome pathway plays a fundamental role in the clearance of aggregated proteins and protection against cellular stress and neurodegenerative conditions. Alterations in autophagy processes, including macroautophagy and chaperone-mediated autophagy (CMA), have been described in Parkinson disease (PD). CMA is a selective autophagic process that depends on LAMP2A (Lysosomal associated membrane protein 2A), a mammal and bird-specific membrane glycoprotein that translocates cytosolic proteins containing a KFERQ-like peptide motif across the lysosomal membrane. Drosophila reportedly lack CMA and use endosomal microautophagy (eMI) as an alternative selective autophagic process. This study reports that neuronal expression of human LAMP2A protected Drosophila against starvation and oxidative stress, and delayed locomotor decline in aging flies without extending their lifespan. LAMP2A also prevented the progressive locomotor and oxidative defects induced by neuronal expression of PD-associated human SNCA (synuclein alpha) with alanine-to-proline mutation at position 30 (SNCA(A30P)). LAMP2A expression stimulated selective autophagy in the adult brain and not in the larval fat body. Noteworthy, neurally expressed LAMP2A markedly upregulated levels of Drosophila Atg5, a key macroautophagy initiation protein, and of the Atg5-containing complex, and that it increased the density of Atg8a/LC3-positive puncta, which reflects the formation of autophagosomes. Furthermore, LAMP2A efficiently prevented accumulation of the autophagy defect marker Ref(2)P/p62 in the adult brain under acute oxidative stress. These results indicate that LAMP2A can promote autophagosome formation and potentiate autophagic flux in the Drosophila brain, leading to enhanced stress resistance and neuroprotection (Issa, 2018).

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Basso, V., Marchesan, E., Peggion, C., Chakraborty, J., von Stockum, S., Giacomello, M., Ottolini, D., Debattisti, V., Caicci, F., Tasca, E., Pegoraro, V., Angelini, C., Antonini, A., Bertoli, A., Brini, M. and Ziviani, E. (2018). Regulation of ER-mitochondria contacts by Parkin via Mfn2. Pharmacol Res. PubMed ID: 30219582

Abstract

Parkin, an E3 ubiquitin ligase and a Parkinson's disease (PD) related gene, translocates to impaired mitochondria and drives their elimination via autophagy, a process known as mitophagy. Mitochondrial pro-fusion protein Mitofusins (Mfn1 and Mfn2) were found to be a target for Parkin mediated ubiquitination. Mfns are transmembrane GTPase embedded in the outer membrane of mitochondria, which are required on adjacent mitochondria to mediate fusion. In mammals, Mfn2 also forms complexes that are capable of tethering mitochondria to endoplasmic reticulum (ER), a structural feature essential for mitochondrial energy metabolism, calcium (Ca(2+)) transfer between the organelles and Ca(2+) dependent cell death. Despite its fundamental physiological role, the molecular mechanisms that control ER-mitochondria cross talk are obscure. Ubiquitination has recently emerged as a powerful tool to modulate protein function, via regulation of protein subcellular localization and protein ability to interact with other proteins. Ubiquitination is also a reversible mechanism, which can be actively controlled by opposing ubiquitination-deubiquitination events. This work found that in Parkin deficient cells and parkin mutant human fibroblasts, the tether between ER and mitochondria is decreased. The site of Parkin dependent ubiquitination was identified, and it was shown that the non-ubiquitinatable Mfn2 mutant fails to restore ER-mitochondria physical and functional interaction. Finally, advantage was taken of an established in vivo model of PD to demonstrate that manipulation of ER-mitochondria tethering by expressing an ER-mitochondria synthetic linker is sufficient to rescue the locomotor deficit associated to an in vivo Drosophila model of PD (Basso, 2018).

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Kim, E. Y., Kang, K. H. and Koh, H. (2018). Cyclophilin 1 (Cyp1) mutation ameliorates oxidative stress-induced defects in a Drosophila DJ-1 null mutant. Biochem Biophys Res Commun. PubMed ID: 30297105

Abstract

Drosophila cyclophilin 1 (Cyp1) is a structural and functional homolog of mammalian cyclophilin D (CypD), a unique mitochondrial cyclophilin (Cyp) that regulates the inner mitochondrial membrane permeability transition and cell survival under cellular stresses such as oxidative damage. This study generated and characterized a Drosophila Cyp1 mutant. Cyp1 mutant flies successfully developed into adults and showed no significant defects in mitochondrial morphology, function, and content. However, oxidative damage significantly decreased in Cyp1 mutant flies, and inhibition of Cyp1 expression substantially increased the survival under various oxidative stress paradigms. Moreover, Cyp1 mutation successfully ameliorated survival rates, locomotor activity, and dopaminergic neuron quantity in a Drosophila DJ-1 mutant under oxidative stress, further confirming the protective role of Cyp1 mutation against oxidative stress. In conclusion, these results suggest Cyp1 and its human homolog CypD as putative molecular targets for the treatment of DJ-1 deficiency-associated diseases, including Parkinson's disease (Kim, 2018).

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Cunningham, P. C., Waldeck, K., Ganetzky, B. and Babcock, D. T. (2018) (2018). Neurodegeneration and locomotor dysfunction in Drosophila scarlet mutants. J Cell Sci. PubMed ID: 30154211

Abstract

Parkinson's Disease (PD) is characterized by the loss of dopaminergic neurons, resulting in progressive locomotor dysfunction. Identification of genes required for the maintenance of these neurons should help to identify potential therapeutic targets. However, little is known regarding the factors that render dopaminergic neurons selectively vulnerable to PD. This study shows that Drosophila melanogaster scarlet mutants exhibit an age-dependent progressive loss of dopaminergic neurons, along with subsequent locomotor defects and a shortened lifespan. Knockdown of Scarlet specifically within dopaminergic neurons is sufficient to produce this neurodegeneration, demonstrating a unique role for Scarlet beyond its well-characterized role in eye pigmentation. Both genetic and pharmacological manipulation of the kynurenine pathway rescued loss of dopaminergic neurons by promoting synthesis of the free radical scavenger Kynurenic Acid (KYNA) and limiting the production of the free radical generator 3-hydroxykynurenine (3-HK). Finally, this study showed that expression of wild-type Scarlet is neuroprotective in a model of PD, suggesting that manipulating kynurenine metabolism may be a potential therapeutic option in treating PD (Cunningham, 2018).

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Zhang, S., Feng, R., Li, Y., Gan, L., Zhou, F., Meng, S., Li, Q. and Wen, T. (2018). Degradation of alpha-synuclein by dendritic cell factor 1 delays neurodegeneration and extends lifespan in Drosophila. Neurobiol Aging 67: 67-74. PubMed ID: 29649746

Abstract

Parkinson's disease (PD) is a common neurodegenerative disease associated with the progressive loss of dopaminergic neurons in the substantia nigra. Proteinaceous depositions of alpha-synuclein (alpha-syn) and its mutations, A30P and A53T, are one important characteristic of PD. However, little is known about their aggregation and degradation mechanisms. Dendritic cell factor 1 (DCF1) is a membrane protein that plays important roles in nerve development in mouse. This study aimed to show that DCF1 overexpression in a PD Drosophila model significantly ameliorates impaired locomotor behavior in third instar larvae and normalizes neuromuscular junction growth. Furthermore, climbing ability also significantly increased in adult PD Drosophila. More importantly, the lifespan dramatically extended by an average of approximately 23%, and surprisingly, DCF1 could prevent alpha-syn-induced dopaminergic neuron loss by aggregating alpha-syn in the dorsomedial region of Drosophila. Mechanistically, it was confirmed that DCF1 could degrade alpha-syn both in vivo and in vitro. These findings revealed an important role of DCF1 in PD process and may provide new potential strategies for developing drugs to treat neurodegenerative diseases (Zhang, 2018).

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Saridaki, T., Nippold, M., Dinter, E., Roos, A., Diederichs, L., Fensky, L., Schulz, J. B. and Falkenburger, B. H. (2018). FYCO1 mediates clearance of alpha-synuclein aggregates through a Rab7-dependent mechanism. J Neurochem. Pubmed ID: 29747217

Abstract

Parkinson disease can be caused by mutations in the alpha-synuclein gene and is characterized by aggregates of alpha-synuclein protein. Overexpression of the small GTPase Rab7 (see Drosophila Rab7) can induce clearance of alpha-synuclein aggregates. This study investigated which Rab7 effectors mediate this effect. To model Parkinson disease the pathogenic A53T mutant of alpha-synuclein was expressed in HEK293T cells and Drosophila melanogaster. The Rab7 effectors FYVE and coiled-coil domain-containing protein 1 (FYCO1) and Rab-interacting lysosomal protein (RILP) were investigated. FYCO1-EGFP was found to decorate vesicles containing alpha-synuclein. RILP-EGFP also decorated vesicular structures, but they did not contain alpha-synuclein. FYCO1 overexpression reduced the number of cells with alpha-synuclein aggregates, defined as visible particles of EGFP-tagged alpha-synuclein, whereas RILP did not. FYCO1 but not RILP reduced the amount of alpha-synuclein protein as assayed by western blot, increased the disappearance of alpha-synuclein aggregates in time-lapse microscopy, and decreased alpha-synuclein-induced toxicity assayed by the Trypan blue assay. siRNA-mediated knockdown of FYCO1 but not RILP reduced Rab7 induced aggregate clearance. Collectively, these findings indicate that FYCO1 and not RILP mediates Rab7 induced aggregate clearance. Electron microscopic analysis and insertion of lysosomal membranes into the plasma membrane indicate that FYCO1 could lead to secretion of alpha-synuclein aggregates. Extracellular alpha-synuclein as assayed by ELISA was, however, not increased with FYCO1. Coexpression of FYCO1 in the fly model decreased alpha-synuclein aggregates as shown by the filter trap assay and rescued the locomotor deficit resulting from neuronal A53T-alpha-synuclein expression. This latter finding confirms that a pathway involving Rab7 and FYCO1 stimulates degradation of alpha-synuclein and could be beneficial in patients with Parkinson disease (Saridaki, 2018).

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Hussain, A., Pooryasin, A., Zhang, M., Loschek, L. F., La Fortezza, M., Friedrich, A. B., Blais, C. M., Ucpunar, H. K., Yepez, V. A., Lehmann, M., Gompel, N., Gagneur, J., Sigrist, S. J. and Grunwald Kadow, I. C. (2018). Inhibition of oxidative stress in cholinergic projection neurons fully rescues aging-associated olfactory circuit degeneration in Drosophila. Elife 7. PubMed ID: 29345616

Abstract

Loss of the sense of smell is among the first signs of natural aging and neurodegenerative diseases such as Alzheimer's and Parkinson's. Cellular and molecular mechanisms promoting this smell loss are not understood. This study shows that Drosophila melanogaster also loses olfaction before vision with age. Within the olfactory circuit, cholinergic projection neurons show a reduced odor response accompanied by a defect in axonal integrity and reduction in synaptic marker proteins. Using behavioral functional screening, this study pinpoints that expression of the mitochondrial reactive oxygen scavenger SOD2 in cholinergic projection neurons is necessary and sufficient to prevent smell degeneration in aging flies. Together, these data suggest that oxidative stress induced axonal degeneration in a single class of neurons drives the functional decline of an entire neural network and the behavior it controls. Given the important role of the cholinergic system in neurodegeneration, the fly olfactory system could be a useful model for the identification of drug targets (Hussain, 2018).

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Ordonez, D. G., Lee, M. K. and Feany, M. B.. (2018). alpha-synuclein induces mitochondrial dysfunction through spectrin and the actin cytoskeleton. Neuron 97(1): 108-124.e106. PubMed ID: 29249285

Abstract

Genetics and neuropathology strongly link alpha-synuclein aggregation and neurotoxicity to the pathogenesis of Parkinson's disease and related alpha-synucleinopathies. This study describes a new Drosophila model of alpha-synucleinopathy based on widespread expression of wild-type human alpha-synuclein, which shows robust neurodegeneration, early-onset locomotor deficits, and abundant alpha-synuclein aggregation. Results of forward genetic screening and genetic analysis were used in this new model to demonstrate that alpha-synuclein expression promotes reorganization of the actin filament network and consequent mitochondrial dysfunction through altered Drp1 localization. Similar changes are present in a mouse alpha-synucleinopathy model and in postmortem brain tissue from patients with alpha-synucleinopathy. Importantly, evidence is provided that the interaction of alpha-synuclein with spectrin initiates pathological alteration of the actin cytoskeleton and downstream neurotoxicity. These findings suggest new therapeutic approaches for alpha-synuclein induced neurodegeneration (Ordonez, 2018).

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Steinkellner, T., Zell, V., Farino, Z. J., Sonders, M. S., Villeneuve, M., Freyberg, R. J., Przedborski, S., Lu, W., Freyberg, Z. and Hnasko, T. S. (2018).Role for VGLUT2 in selective vulnerability of midbrain dopamine neurons. J Clin Invest 128(2): 774-788. PubMed ID: 29337309

Abstract

Parkinson's disease is characterized by the loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). DA neurons in the ventral tegmental area are more resistant to this degeneration than those in the SNc, though the mechanisms for selective resistance or vulnerability remain poorly understood. A key to elucidating these processes may lie within the subset of DA neurons that corelease glutamate and express the vesicular glutamate transporter VGLUT2. This study addressed the potential relationship between VGLUT expression and DA neuronal vulnerability by overexpressing VGLUT in DA neurons of flies and mice. In Drosophila, VGLUT overexpression led to loss of select DA neuron populations. Similarly, expression of VGLUT2 specifically in murine SNc DA neurons led to neuronal loss and Parkinsonian behaviors. Other neuronal cell types showed no such sensitivity, suggesting that DA neurons are distinctively vulnerable to VGLUT2 expression. Additionally, most DA neurons expressed VGLUT2 during development, and coexpression of VGLUT2 with DA markers increased following injury in the adult. Finally, conditional deletion of VGLUT2 made DA neurons more susceptible to Parkinsonian neurotoxins. These data suggest that the balance of VGLUT2 expression is a crucial determinant of DA neuron survival. Ultimately, manipulation of this VGLUT2-dependent process may represent an avenue for therapeutic development (Steinkellner, 2018).

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Chen, A. Y. and Tully, T. (2018). A stress-enhanced model for discovery of disease-modifying gene: Ece1-suppresses the toxicity of alpha-synuclein A30P. Neurobiol Dis 114: 153-163. PubMed ID: 29524599

Abstract

Parkinson's disease (PD) is a progressive motor neurodegenerative disorder, characterized by a selective loss of dopaminergic neurons in the substantia nigra. The complexity of disease etiology includes both genetic and environmental factors. Human alpha-Synuclein A30P (A30P) is a mutant gene identified in early onset PD and has been shown to result selective dopamine neuron loss in transgenic A30P flies and mice. Paraquat (PQ) is an herbicide and an oxidative stress generator, linked to sporadic PD. To identify disease modifier genes, two independently-duplicated experiments were performed of microarray, capturing genome-wide transcriptional changes in A30P flies, chronically fed with PQ-contaminated food. It was hypothesized that the best time point of identifying a disease modifier gene is at time when flies showed maximal combined toxicity of A30P transgene and PQ treatment during an early stage of disease and that effective disease modifiers gene are those showing transcriptional changes to oxidative stress in A30P expressing and not in wild type animals. Fly Neprilysin3 (Nep3) is one identified gene that is highly conserved. Its mouse and human homolog is endothelin-converting enzyme-1 (Ece1). To investigate the neuroprotective effect of Ece1, NS1 cells and mouse midbrain neurons expressing A30P, treated with or without PQ were used. ECE1 expression protected against A30P toxicity on cell viability, on neurite outgrowth and ameliorated A30P accumulation in vitro. Expression of ECE1 in vivo suppressed dopamine neuron loss and alleviated the corresponding motor deficits in mice with A30P-expression. This study leverages a new approach to identify disease modifier genes using a stress-enhanced PD animal model (Chen, 2018).

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Stephano, F., Nolte, S., Hoffmann, J., El-Kholy, S., von Frieling, J., Bruchhaus, I., Fink, C. and Roeder, T. (2018). Impaired Wnt signaling in dopamine containing neurons is associated with pathogenesis in a rotenone triggered Drosophila Parkinson's disease model. Sci Rep 8(1): 2372. PubMed ID: 29403026

Abstract

Parkinson's disease, which is the one of the most common neurodegenerative movement disorder, is characterized by a progressive loss of dopamine containing neurons. The mechanisms underlying disease initiation and development are not well understood and causative therapies are currently not available. A Drosophila model was used to elucidate the molecular processes during early stages of Parkinson's disease. To induce Parkinson's disease-like phenotypes, flies were treated with the pesticide rotenone, and dopamine producing neurons of animals were isolated that were at an early disease stage. Transcriptomic analyses revealed that gene ontologies associated with regulation of cell death and neuronal functions were significantly enriched. Moreover, the activities of the MAPK/EGFR- and TGF-β signaling pathways were enhanced, while the Wnt pathway was dampened. In order to evaluate the role of Wnt signaling for survival of dopaminergic neurons in the disease model, the reduced Wnt signaling activity was rescued by ectopic overexpression of armadillo/β-catenin. This intervention rescued the rotenone induced movement impairments in the Drosophila model. Taken together, this initial study showed a highly relevant role of Wnt signaling for dopamine producing neurons during pathogenesis in Parkinson's disease and it implies that interfering with this pathway might by a suitable therapeutic option for the future (Stephano, 2018).

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Tran, H. H., Dang, S. N. A., Nguyen, T. T., Huynh, A. M., Dao, L. M., Kamei, K., Yamaguchi, M. and Dang, T. T. P. (2018). Drosophila Ubiquitin C-Terminal Hydrolase Knockdown Model of Parkinson's Disease. Sci Rep 8(1): 4468. PubMed ID: 29535397

Abstract

Parkinson's disease (PD) is the second most common neurodegenerative disorder worldwide. Many factors have been shown to contribute to its pathogenesis including genetic and environmental factors. Ubiquitin C-terminal hydrolase L1 (UCHL1) is also known to be involved in the pathogenesis of PD. The study of UCHL1 mas modeled in Drosophila melanogaster and investigated its functions in PD. The specific knockdown of the Drosophila ortholog of UCHL1 (dUCH) in dopaminergic neurons (DA neurons) led to the underdevelopment and/or degeneration of these neurons, specifically in DL1 DA neuron cluster in the larval brain lobe and PPM2, PPM3, PPL2ab, and VUM DA neuron clusters in the adult brain. These defects were followed by a shortage of dopamine in the brain, which subsequently resulted in locomotor dysfunction. The degeneration of DA neurons in dUCH knockdown adult brain, which occurred progressively and severely during the course of aging, mimics the epidemiology of PD. DA neuron and locomotor defects were rescued when dUCH knockdown flies were treated with vitamin C, a well-known antioxidant. These results suggest that dUCH knockdown fly is a promising model for studying the pathogenesis and epidemiology of PD as well as the screening of potential antioxidants for PD therapeutics (Tran, 2018).

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Lavoy, S., Chittoor-Vinod, V. G., Chow, C. Y. and Martin, I. (2018). Genetic Modifiers of Neurodegeneration in a Drosophila Model of Parkinson's Disease. Genetics. PubMed ID: 29907646

Abstract

Disease phenotypes can be highly variable among individuals with the same pathogenic mutation. There is increasing evidence that background genetic variation is a strong driver of disease variability in addition to the influence of environment. To understand the genotype-phenotype relationship that determines the expressivity of a pathogenic mutation, a large number of backgrounds must be studied. This can be efficiently achieved using model organism collections such as the Drosophila Genetic Reference Panel (DGRP). This study used the DGRP to assess the variability of locomotor dysfunction in a LRRK2 G2019S Drosophila melanogaster model of Parkinson's disease. Substantial variability was found in the LRRK2 G2019S locomotor phenotype in different DGRP backgrounds. A genome-wide association study for candidate genetic modifiers reveals 177 genes that drive wide phenotypic variation, including 19 top association genes. Genes involved in the outgrowth and regulation of neuronal projections are enriched in these candidate modifiers. RNAi functional testing of the top association and neuronal projection-related genes reveals that pros, pbl, ct and CG33506 significantly modify age-related dopamine neuron loss and associated locomotor dysfunction in the Drosophila LRRK2 G2019S model. These results demonstrate how natural genetic variation can be used as a powerful tool to identify genes that modify disease-related phenotypes. This study reports novel candidate modifier genes for LRRK2 G2019S that may be used to interrogate the link between LRRK2, neurite regulation and neuronal degeneration in Parkinson's disease (Lavoy, 2018).

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Mohite, G. M., Dwivedi, S., Das, S., Kumar, R., Paluri, S., Mehra, S., Ruhela, N., S, A., Jha, N. N. and Maji, S. K. Parkinson's disease associated alpha-synuclein familial mutants promote dopaminergic neuronal death in Drosophila melanogaster. ACS Chem Neurosci. PubMed ID: 29906099

Abstract

alpha-Synuclein (alpha-Syn) aggregation and amyloid formation are associated with loss of dopaminergic neurons in Parkinson's disease (PD). In addition, familial mutations in alpha-Syn are shown to be one of the definite causes of PD. Familial PD associated alpha-Syn G51D, H50Q and E46K mutations were extensively studied using the Drosophila model system. The data showed that flies expressing alpha-Syn familial mutants have a shorter lifespan and exhibit more climbing defects compared to wild-type (WT) flies in an age-dependent manner. The immuno-fluorescence studies of the brain from the old flies showed more dopaminergic neuronal cell death in all mutants compared to WT. This adverse effect of alpha-Syn familial mutations highly correlated with the sustained population of oligomer production/ retention in mutant flies. Furthermore, this was supported by in vitro studies, where significantly higher amount of oligomer was observed in mutants compared to WT. The data suggest that the sustained population of oligomer formation/ retention could be a major cause of cell death by alpha-Syn familial mutants (Mohite, 2018).

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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

Abstract

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).

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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

Abstract

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).

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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

Abstract

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).

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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

Abstract

Parkin (PARK2; see Drosophila Parkin) is an E3 ubiquitin ligase that is commonly mutated in Familial Parkinson's Disease (PD). In cell culture models, Parkin is recruited to acutely depolarised mitochondria by PINK1 (see Drosophila Pink1). PINK1 activates Parkin activity leading to ubiquitination of multiple proteins, which in turn promotes clearance of mitochondria by mitophagy. Many substrates have been identified using cell culture models in combination with depolarising drugs or proteasome inhibitors, but not in more physiological settings. This study utilized the recently introduced BioUb strategy to isolate ubiquitinated proteins in flies. Following Parkin Wild-Type (WT) and Parkin Ligase dead (LD) expression, mass spectrometry and stringent bioinformatics analysis identified those proteins differentially ubiquitinated, providing the first survey of steady state Parkin substrates using an in vivo model. An in vivo ubiquitination assay was used to validate one of those substrates in SH-SY5Y cells. This study identified 35 proteins that are more prominently ubiquitinated following Parkin over-expression. These include several mitochondrial proteins and a number of endosomal trafficking regulators such as v-ATPase sub-units, Syx5/STX5, Vps4. The retromer component, Vps35, another PD-associated gene that has recently been shown to interact genetically with parkin, was also identified. Importantly, Parkin-dependent ubiquitination of VPS35 was validated in human neuroblastoma cells. Collectively these results provide new leads to the possible physiological functions of Parkin activity that are not overtly biased by acute mitochondrial depolarisation (Martinez, 2017).

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Bardai, F. H., Ordonez, D. G., Bailey, R. M., Hamm, M., Lewis, J. and Feany, M. B. (2018). Lrrk promotes tau neurotoxicity through dysregulation of actin and mitochondrial dynamics. PLoS Biol 16(12): e2006265. PubMed ID: 30571694

Abstract

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson disease. Genetics and neuropathology link Parkinson disease with the microtubule-binding protein tau, but the mechanism of action of LRRK2 mutations and the molecular connection between tau and Parkinson disease are unclear. This study investigated the interaction of LRRK and tau in Drosophila and mouse models of tauopathy. Either increasing or decreasing the level of fly Lrrk enhances tau neurotoxicity, which is further exacerbated by expressing Lrrk with dominantly acting Parkinson disease-associated mutations. At the cellular level, altering Lrrk expression promotes tau neurotoxicity via excess stabilization of filamentous actin (F-actin) and subsequent mislocalization of the critical mitochondrial fission protein dynamin-1-like protein (Drp1). Biochemically, monomeric LRRK2 exhibits actin-severing activity, which is reduced as increasing concentrations of wild-type LRRK2, or expression of mutant forms of LRRK2 promote oligomerization of the protein. Overall, these findings provide a potential mechanistic basis for a dominant negative mechanism in LRRK2-mediated Parkinson disease, suggest a common molecular pathway with other familial forms of Parkinson disease linked to abnormalities of mitochondrial dynamics and quality control, and raise the possibility of new therapeutic approaches to Parkinson disease and related disorders (Bardai, 2018).

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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

Abstract

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).

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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

Abstract

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).

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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

Abstract

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).

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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

Abstract

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).

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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

Abstract

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).

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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

Abstract

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).

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Khair, S. B. A., Dhanushkodi, N. R., Ardah, M. T., Chen, W., Yang, Y. and Haque, M. E. (2018). Silencing of Glucocerebrosidase gene in Drosophila enhances the aggregation of Parkinson's disease associated α-Synuclein mutant A53T and affects locomotor activity. Front Neurosci 12: 81. PubMed ID: 29503608

Abstract

Mutations in glucocerebrosidase (GBA), a lysosomal enzyme are the most common genetic risk factor for developing Parkinson's disease (PD). This study examined how reduced GCase activity affects α-synuclein (α-syn) and its mutants (A30P and A53T) aggregation, neurodegeneration, sleep and locomotor behavior in a fly model of PD. Drosophila were developed with GBA gene knockdown (RNAi) (with reduced GCase activity) that simultaneously expresses either wildtype (WT) or mutants such as A30P or A53T α-syn. Western blot and confocal microscopy were performed to study the α-syn aggregation and neurodegeneration in these flies. Sleep and locomotor activity of those flies were also studied using Drosophila activity monitor (DAM) system. Western blot analysis showed that GBA RNAi A53T α-syn flies (30 days old) had an increased level of Triton insoluble synuclein (that corresponds to α-syn aggregates) compared to corresponding A53T flies without GBA RNAi (control), while mRNA expression of α-syn remained unchanged. Confocal imaging of whole brain staining of 30 days old Drosophila showed a statistically significant decrease in neuron numbers in PPL1 cluster in flies expressing α-syn WT, A30P and A53T in the presence GBA RNAi compared to corresponding control. Staining with conformation specific antibody for α-syn aggregates showed an increased number of neurons staining for α-syn aggregates in A53T fly brain with GBA RNAi compared to control A53T flies, thus confirming the protein analysis finding that under decreased GBA enzyme activity, mutant A53T aggregates more than the control A53T without GBA silencing. Sleep analysis revealed decreased total activity in GBA silenced flies expressing mutant A53T compared to both A53T control flies and GBA RNAi flies without synuclein expression. It is concluded that in A53T flies with reduced GCase activity, there is increased α-syn aggregation and dopamine (DA) neuronal loss. This study demonstrates that reduced GCase activity both in the context of heterozygous GBA1 mutation associated with PD and in old age, contribute to increased aggregation of mutant α-syn A53T and exacerbates the phenotype in a fly model of PD (Abul Khair, 2018).

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Tas, D., Stickley, L., Miozzo, F., Koch, R., Loncle, N., Sabado, V., Gnagi, B. and Nagoshi, E. (2018). Parallel roles of transcription factors dFOXO and FER2 in the development and maintenance of dopaminergic neurons. PLoS Genet 14(3): e1007271. PubMed ID: 29529025

Abstract

Forkhead box (FOXO) proteins are evolutionarily conserved, stress-responsive transcription factors (TFs) that can promote or counteract cell death. Mutations in FOXO genes are implicated in numerous pathologies, including age-dependent neurodegenerative disorders, such as Parkinson's disease (PD). However, the complex regulation and downstream mechanisms of FOXOs present a challenge in understanding their roles in the pathogenesis of PD. This study investigate the involvement of FOXO in the death of dopaminergic (DA) neurons, the key pathological feature of PD, in Drosophila. dFOXO null mutants exhibit a selective loss of DA neurons in the subgroup crucial for locomotion, the protocerebral anterior medial (PAM) cluster, during development as well as in adulthood. PAM neuron-targeted adult-restricted knockdown demonstrates that dFOXO in adult PAM neurons tissue-autonomously promotes neuronal survival during aging. dFOXO and the bHLH-TF 48-related-2 (FER2) act in parallel to protect PAM neurons from different forms of cellular stress. Remarkably, however, dFOXO and FER2 share common downstream processes leading to the regulation of autophagy and mitochondrial morphology. Thus, overexpression of one can rescue the loss of function of the other. These results indicate a role of dFOXO in neuroprotection and highlight the notion that multiple genetic and environmental factors interact to increase the risk of DA neuron degeneration and the development of PD (Tas, 2018).

This study demonstrates that dFOXO is tissue-autonomously required for the maintenance of DA neurons in the PAM cluster during aging. Evidence is presented that dFOXO and FER2 act in parallel pathways to protect PAM neurons from different forms of cellular stress. However, dFOXO and FER2 partly share downstream pathways leading to the control of autophagy and mitochondrial morphology. Thus, overexpression of one can rescue the loss of function of the other. These results highlight the notion that multiple genetic and environmental risk factors interact and affect DA neuron survival. Importantly, genome-wide association studies (GWAS) and functional studies in mammals implicated FOXO family TFs, including FOXO1, FOXO3, FOXA1 and FOXA2, in the maintenance of DA neurons and in PD. The current results are in accordance with these studies and further suggest that dfoxo loss of function offers a valuable tool to study the pathogenesis of sporadic PD (Tas, 2018).

In mammals, although constitutive activation of FOXO3 induces loss of DA neurons in the SN, the expression of a dominant negative FOXO3 causes oxidative damage that leads to DA neuron loss. Nevertheless, both the dominant-negative form and mild activation of FOXO3 are neuroprotective in mice overexpressing α-Synuclein. Thus, FOXO3 can be protective or detrimental to DA neurons in the substantia nigra (SN) depending on its activity levels and genetic background. Likewise, in Drosophila, previous studies have shown paradoxical roles for dFOXO in the survival of DA neurons in various PD models. dFOXO overexpression has been shown to ameliorates mitochondrial abnormality and protects DA neurons in Pink1 null mutants. Conversely, dFOXO mediates the death of DA neurons by inducing apoptosis in DJ-1β loss-of-function mutants and in flies overexpressing dLRRK (Tas, 2018).

The apparent paradox concerning the role of FOXOs suggests that the activity of FOXO factors should be tightly regulated in order to exert neuroprotective function, i.e., activity levels of FOXO factors that are too high or too low are both detrimental to DA neurons. Alternatively, the differences in the reagents and experimental conditions used to examine the role of FOXOs in prior studies may have contributed to the differences in the interpretation of the results. A number of previous experiments in Drosophila studies mentioned above used global overexpression of dFOXO and tissues other than DA neurons, such as eyes, muscles and wings, were mainly analyzed to evaluate its effect. Furthermore, for dfoxo loss of function experiments, these studies used dfoxo21 or dfoxo25, which contain nucleotide transversions resulting in premature stop codons but nevertheless are not null alleles (Tas, 2018).

The present study used a genuine null allele of dfoxo, dfoxoΔ94, to examine whether endogenous dFOXO is protective or detrimental to DA neurons. The results demonstrating that dFOXO is protective to DA neurons in the PAM cluster under basal conditions are in accordance with a previous report. Curiously, however, that study observed the loss of DA neurons in the DL1 (dorso lateral 1) cluster, which corresponds to the PPL1 cluster in the nomenclature for adult DA neurons. Additionally, by PAM neuron-targeted constitutive and adult-restricted dfoxo RNAi, the current study shows that dFOXO expression within adult PAM neurons is required for the maintenance of PAM neurons in aged flies (Tas, 2018).

Overexpression of dfoxo in PAM neurons prevents the developmental impairment and age-dependent loss of PAM neurons in Fer22 mutants. Conversely, Fer2 overexpression ameliorates the effect of dfoxoΔ94 mutation on the development and maintenance of PAM neurons. Since Fer2 and dfoxo do not transcriptionally regulate each other, the reciprocal rescue suggests that their downstream mechanisms partly overlap. In line with this interpretation, this study showed that autophagy and mitochondrial morphology are commonly impaired in PAM neurons of dfoxoΔ94 and Fer22 mutants (Tas, 2018).

Mounting evidence indicates that FOXO factors regulate autophagy by controlling the expression of Atg genes in flies and mammals. FOXOs also regulate factors controlling mitophagy and mitochondrial remodeling in mammals. Therefore, dFOXO may regulate autophagy and mitophagy in PAM neurons, although dysregulation in autophagy and mitochondrial morphology in dfoxoΔ94 could be secondary effects of cellular damage. Uncovering genetic pathways downstream of dFOXO and FER2 and how they intersect will yield valuable information, especially because the current results suggest that targeted overexpression of dfoxo or Fer2 in DA neurons may confer protection against DA neuron demise in various genetic models of PD (Tas, 2018).

Consistent with the known role of PAM neurons in controlling locomotion, startle-induced climbing ability in dfoxoΔ94 and Fer22 mutants is significantly improved by the expression of dfoxo with R58E02-GAL4. However, R58E02>dfoxo does not rescue the shortened lifespan of dfoxoΔ94 and even further reduces the lifespan of Fer22. Therefore, neuroprotective role of dFOXO is independent of its role in longevity regulation. Many fly models of PD show lifespan shortening, which is likely caused by the systemic effect of mitochondrial impairment and/or elevated oxidative stress levels rather than DA neuron demise. Lifespan shortening of dfoxoΔ94 and Fer22 mutants may be similarly attributed to the impairment in mitochondrial biology or (in the case of Fer22) oxidative stress regulation in cells other than PAM neurons (Tas, 2018).

Given that mitochondrial dysfunction and oxidative stress are tightly linked and both implicated in neurodegeneration, it is surprising that no evidence was found that PAM degeneration in dfoxoΔ94 is associated with chronic or acute oxidative stress, unlike Fer22 mutants. The results also show no evidence that amino acid intake during adulthood is relevant for survival of PAM neurons. Then, how is dFOXO signaling activated during adulthood to promote PAM neuron survival in aged flies? Aging is associated with loss of proteostasis and FOXOs play a key role in cellular proteostasis. Consistent with the findings in other tissues, autophagy levels in PAM neurons decrease with age, and this is accelerated in dfoxoΔ94. Thus, age-dependent decrease in basal activity of autophagy might be an intracellular stress signal that leads to the activation of dFOXO in PAM neurons (Tas, 2018).

This study reveals an unexpected crosstalk between two pathways mediated by two TFs, dFOXO and FER2, in the development and maintenance of DA neurons in the PAM cluster. Importantly, both genes are also required for the proper development of PAM neurons. This is in line with the fact that several mammalian TFs required for DA neuron development play critical roles in the maintenance of adult midbrain DA neurons. dFOXO homologs FOXA1 and A2 fall within this category, suggesting that TFs having dual roles in the development and maintenance of DA neurons is an evolutionarily conserved mechanism of neuroprotection. Furthermore, the data suggest that loss of dfoxo expression before adulthood has lasting detrimental effect on the survival of PAM neurons in aging flies, which may be partly regulated non-cell-autonomously by dFOXO in the larval fat body or in other tissues. In conclusion, this study provides a starting point to investigate TF networks underlying the link between aberrant neural development and neurodegeneration, which will present new opportunities to better understand the etiology of sporadic PD (Tas, 2018).

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Davis, M. Y., Trinh, K., Thomas, R. E., Yu, S., Germanos, A. A., Whitley, B. N., Sardi, S. P., Montine, T. J. and Pallanck, L. J. (2016). Glucocerebrosidase Deficiency in Drosophila Results in alpha-Synuclein-Independent Protein Aggregation and Neurodegeneration. PLoS Genet 12(3): e1005944. PubMed ID: 27019408

Abstract

Mutations in the glucosidase, beta, acid (GBA1) gene cause Gaucher's disease, and are the most common genetic risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB) excluding variants of low penetrance. Because alpha-synuclein-containing neuronal aggregates are a defining feature of PD and DLB, it is widely believed that mutations in GBA1 act by enhancing alpha-synuclein toxicity. To explore this hypothesis, the Drosophila GBA1 homolog, dGBA1b, was deleted, and the phenotypes of dGBA1b mutants were compared in the presence and absence of alpha-synuclein expression. Homozygous dGBA1b mutants exhibit shortened lifespan, locomotor and memory deficits, neurodegeneration, and dramatically increased accumulation of ubiquitinated protein aggregates that are normally degraded through an autophagic mechanism. Ectopic expression of human alpha-synuclein in dGBA1b mutants resulted in a mild enhancement of dopaminergic neuron loss and increased alpha-synuclein aggregation relative to controls. However, alpha-synuclein expression did not substantially enhance other dGBA1b mutant phenotypes. These findings indicate that dGBA1b plays an important role in the metabolism of protein aggregates, but that the deleterious consequences of mutations in dGBA1b are largely independent of alpha-synuclein. Future work with dGBA1b mutants should reveal the mechanism by which mutations in dGBA1b lead to accumulation of protein aggregates, and the potential influence of this protein aggregation on neuronal integrity (Davis, 2016).

To gain insight into the molecular mechanisms underlying PD, DLB and neuronopathic forms of GD, a Drosophila model of glucocerebrosidase deficiency was developed. Glucocerebrosidase deficiency in Drosophila results in shortened lifespan, a variety of age-dependent behavioral phenotypes, neurodegeneration and the accumulation of insoluble proteins that are normally degraded through an autophagic mechanism. While these phenotypes are reminiscent of α-synucleinopathies, glucocerebrosidase deficiency only mildly influenced the neuronal toxicity and aggregation of α-synuclein, and ectopic expression of α-synuclein did not significantly enhance the glucocerebrosidase deficient phenotypes. Together, these findings indicate that the pathological consequences of glucocerebrosidase deficiency in Drosophila are largely independent of α-synuclein, and that glucocerebrosidase deficiency is the major contributor to pathology in diseases associated with GBA1 mutations (Davis, 2016).

The finding that α-synuclein is not a central participant in the pathogenesis associated with glucocerebrosidase deficiency is consistent with recent studies in two different fish species. The inverse correlation between glucocerebrosidase activity and α-synuclein aggregation in Drosophila is also consistent with previous studies in rodent models, vertebrate cell culture, post-mortem brain tissues from PD patients, and a recent study in Drosophila. Although it was only possible to observe an influence of glucocerebrosidase deficiency on the aggregation of the p.A53T variant of α-synuclein, this finding may simply reflect the fact that this variant is more aggregation-prone, thus allowing increased sensitivity to detect aggregation. However, this work showing the dGBA1b gene is the predominant Drosophila GBA1 homolog expressed in the fly head contrasts with a recent report showing that neuronal inactivation of the Drosophila dGBA1a gene exacerbated the toxicity of α-synuclein in dopaminergic neurons and in the fly eye (Suzuki, 2015). Additional studies will be required to fully address the role of dGBA1a, which appears to remain largely functional in the available mutant, and to definitively rule out a role for the CG31413 gene situated between dGBA1a and dGBA1b on the phenotypes of GBA1ΔTT homozygotes (Davis, 2016).

Although this work indicates that glucocerebrosidase deficiency has little influence on the toxicity of α-synuclein, the association of GBA1 mutations with PD and DLB frequently involves heterozygous carriers of GBA1 missense alleles. This finding has led to the suggestion that the GBA1 mutations act through a dominant toxic gain-of-function mechanism to cause PD and DLB, perhaps by seeding α-synuclein aggregates via a prion-like mechanism. Because this work involved a putative null allele of dGBA1b, this potential model of pathogenesis could not be addressed. Previous work also indicates that ectopic expression of human α-synuclein in Drosophila confers only mild phenotypic consequences, so it is also possible that the influence of glucocerebrosidase deficiency on α-synuclein toxicity is not readily evident in Drosophila. While these potential confounds are fully acknowledged, several compelling observations suggest that a loss-of-function mechanism best explains the influence of GBA1 mutations on PD and DLB. For example, many different GBA1 mutations are associated with α-synucleinopathies, including putative null alleles, and the molecular severity of a GBA1 allele correlates with the risk of developing an α-synucleinopathy in heterozygous carriers. Perhaps the strongest evidence for a loss-of-function mechanism is the finding that individuals with biallelic GBA1 mutations have a substantially elevated risk for developing PD relative to heterozygous GBA1 mutation carriers. Together these findings offer support for the relevance of animal models bearing null alleles of the GBA1 gene, including this fly model of glucocerebrosidase deficiency, on understanding of the influence of GBA1 mutations in PD and DLB (Davis, 2016).

The glucocerebrosidase deficient fly model should be a valuable tool in future work aimed at understanding the mechanisms underlying the neurodegenerative diseases associated with mutations in GBA1. Although glucocerebrosidase deficiency does not result in dopaminergic neuron degeneration in Drosophila, this finding does not necessarily challenge the utility of the fly model to understand the role of glucocerebrosidase deficiency in PD. Previous work has established that mutational inactivation of Drosophila homologs of genes involved in heritable forms of PD often results in phenotypes that appear discordant with those seen in humans. For example, null mutations of the PINK1 or parkin genes in Drosophila result in dramatic muscle degeneration and germ line defects that are not evident in humans bearing null mutations in these genes. However, substantial insight into the roles of PINK1 and Parkin in mitochondrial quality control derived directly from studies of PINK1 and Parkin in the Drosophila musculature and germ line. It is anticipated that similarly important insight into the mechanisms underlying neuronopathic GD, PD and DLB will come from studies of the phenotypes of the fly model of glucocerebrosidase deficiency (Davis, 2016).

This work suggests at least two general mechanisms by which glucocerebrosidase deficiency triggers neuropathology. First, glucocerebrosidase deficiency may impair autophagy, resulting in increased protein aggregation. This work in GBA1b mutant flies showing accumulation of Ref(2)P, HMW α-synuclein aggregates, and protein aggregates that are normally degraded through an autophagic mechanism supports this model. Glucocerebrosidase is an important lysosomal enzyme in lipid metabolism, and a deficiency in this enzyme could influence lysosome membrane fluidity, vesicular dynamics, and the biogenesis of lysosomes. These effects could impair the trafficking of misfolded proteins to the lysosome and/or fusion of autophagic vacuoles. As no decrease was observed in Cathepsin D activity in GBA1b mutant flies, lysosomal function may not be impaired by glucocerebrosidase deficiency. Alternatively, glucocerebrosidase deficiency may promote the formation of protein aggregates, rather than impair their degradation. Lipid composition has been shown to influence the kinetics of formation of protein aggregates and α-synuclein fibrilization, suggesting that an alteration in lipid composition resulting from glucocerebrosidase deficiency could accelerate the accumulation of protein aggregates. These aggregates might subsequently seed further aggregation in a prion-like mechanism. In support of this model, lipid composition has been shown to affect the kinetics of amyloid-β aggregation, and recent studies suggest that non-autonomous spreading of α-synuclein fibrils may contribute to PD pathogenesis. While it remains unclear whether the increased protein aggregates that were observed in GBA1b mutant flies are due to impaired degradation or accelerated formation of misfolded proteins, α-synuclein expression did not enhance the abundance of protein aggregates, arguing against an additive influence of α-synuclein on protein aggregation metabolism. Future experiments will be required to distinguish between these models and reveal the underlying mechanism of GBA1-mediated accumulation of protein aggregates (Davis, 2016).

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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

Abstract

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).

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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

Abstract

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).

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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

Abstract

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).

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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

Abstract

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).

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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

Abstract

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).

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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

Abstract

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).

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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

Abstract

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).

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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: 1-9. PubMed ID: 27936908

Abstract

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).

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M'Angale, P. G. and Staveley, B. E. (2016). The Bcl-2 homologue Buffy rescues alpha-synuclein-induced Parkinson disease-like phenotypes in Drosophila. BMC Neurosci 17: 24. PubMed ID: 27192974

Abstract

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).

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Celardo, I., Lehmann, S., Costa, A.C., Loh, S.H. and Miguel Martins, L.. (2017). dATF4 regulation of mitochondrial folate-mediated one-carbon metabolism is neuroprotective. Cell Death Differ [Epub ahead of print]. PubMed ID: 28211874

Abstract
Neurons rely on mitochondria as their preferred source of energy. Mutations in PINK1 and PARKIN cause neuronal death in early-onset Parkinson's disease (PD), thought to be due to mitochondrial dysfunction. In Drosophila pink1 and parkin mutants, mitochondrial defects lead to the compensatory upregulation of the mitochondrial one-carbon cycle metabolism genes by an unknown mechanism. This study uncovers that this branch is triggered by the activating transcription factor 4 (ATF4). ATF4 regulates the expression of one-carbon metabolism genes SHMT2 and NMDMC as a protective response to mitochondrial toxicity. Suppressing Shmt2 or Nmdmc causes motor impairment and mitochondrial defects in flies. Epistatic analyses show that suppressing the upregulation of Shmt2 or Nmdmc deteriorates the phenotype of pink1 or parkin mutants. Conversely, the genetic enhancement of these one-carbon metabolism genes in pink1 or parkin mutants is neuroprotective. The study concludes that mitochondrial dysfunction caused by mutations in the Pink1/Parkin pathway engages ATF4-dependent activation of one-carbon metabolism as a protective response. These findings show a central contribution of ATF4 signalling to PD that may represent a new therapeutic strategy (Celardo, 2017).

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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

Abstract
Parkinson's disease (PD) is a common neurodegenerative disorder that exhibits motor and non-motor symptoms, as well as pathological hallmarks, including dopaminergic (DA) neuron death and formation of alpha-synuclein (alpha-Syn) Lewy bodies. Cyclin-G-associated kinase (GAK), a PD susceptibility gene identified through genome-wide association studies (GWAS), is a ubiquitous serine/threonine kinase involved in clathrin uncoating (see Drosophila Clathrin heavy chain), though its PD-related function remains elusive. This study implicates the Drosophila GAK homolog, auxilin (aux), in a broad spectrum of parkinsonian-like symptoms. Downregulating aux expression leads to progressive loss of climbing ability, decreased lifespan, and age-dependent DA neuron death similar to alpha-Syn overexpression. Reduced aux expression further enhances and accelerates alpha-Syn-mediated DA neuron loss. Flies with reduced aux expression are more sensitive to the toxin paraquat, suggesting that genetic and environmental factors intertwine. Taken together, these findings decipher a pivotal role for GAK/aux and suggest mechanisms underlying PD (Song, 2017).

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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

Abstract
F-box proteins act as the protein recognition component of the Skp-Cul-F-box class of ubiquitin ligases. Two members of a gene sub-family encoding these proteins, FBXO7 and FBXO32, have been implicated in the onset and progression of degenerative disease. FBXO7 is responsible for rare genetic forms of Parkinson disease, while FBXO32 has been implicated in muscle wasting. The third gene in this family, FBXO9, is related to growth signaling, but the role of this gene in degenerative disease pathways has not been thoroughly investigated. Characterizing the putative Drosophila melanogaster homologue of this gene, CG5961, enables modeling and analysis of the consequence of targeted alteration of gene function and the effects on the overall health of the organism. Comparison of the protein domains of Homo sapiens FBXO9 and the putative D. melanogaster homologue CG5961 revealed a high degree of conservation between the protein domains. Directed expression of CG5961 (via CG5961EP) and inhibition of CG5961 (through a stable RNAi transgene) in the developing D. melanogaster eye caused abnormalities in adult structures (ommatidia and inter-ommatidial bristles). Directed expression of either CG5961 or CG5961-RNAi in the dopaminergic neurons led to a reduced lifespan compared to that in lacZ controls. Protein structures of CG5961 and FBXO9 are highly similar, and the effects of altered expression of CG5961 in neuron-rich tissues was studied. The results suggest that CG5961 activity is necessary for the proper formation of neuronal tissue and that targeted alteration of gene expression in dopaminergic neurons leads to a reduced lifespan (Merzetti, 2016).

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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

Abstract

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).

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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

Abstract
Gaucher disease (GD) results from mutations in the acid beta-glucocerebrosidase (GCase) encoding gene, GBA, which leads to accumulation of glucosylceramides. GD patients and carriers of GD mutations have a significantly higher propensity to develop Parkinson disease (PD) in comparison to the non-GD population. This study used Drosophila to show that development of PD in carriers of GD mutations results from the presence of mutant GBA alleles. Drosophila has two GBA orthologs (CG31148 and CG31414), each of which has a minos insertion, which creates C-terminal deletion in the encoded GCase. Flies double heterozygous for the endogenous mutant GBA orthologs presented Unfolded Protein Response (UPR) and developed parkinsonian signs, manifested by death of dopaminergic cells, defective locomotion and a shorter life span. Transgenic flies carrying the mutant human N370S, L444P and the 84GG variants were established. UPR activation and development of parkinsonian signs could be recapitulated in flies expressing these three mutant variants.UPR and parkinsonian signs could be partially rescued by growing the double heterozygous flies, or flies expressing the N370S or the L444P human mutant GCase variants, in the presence of the pharmacological chaperone ambroxol, which binds and removes mutant GCase from the ER. However flies expressing the 84GG mutant, that does not express mature GCase, did not exhibit rescue by ambroxol. These results strongly suggest that the presence of a mutant GBA allele in dopaminergic cells leads to ER stress and to their death, and contributes to development of Parkinson disease (Maor, 2016).

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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

Abstract
Parkinson disease is a debilitating and incurable neurodegenerative disorder, affecting approximately 1-2% of people over sixty-five years old. Oxidative damage is considered to play a central role in the progression of Parkinson disease and strong evidence links chronic exposure to the pesticide paraquat with the incidence of the disease, most probably through the generation of oxidative damage. This work demonstrated in human SH-SY5Y neuroblastoma cells the beneficial role of superoxide dismutase (SOD) enzymes against paraquat-induced toxicity, as well as the therapeutic potential of the SOD-mimetic compound M40403. Having verified the beneficial effects of superoxide dismutation in cells, the effects were then evaluated using Drosophila melanogaster as in vivo model. Besides protecting against the oxidative damage induced by paraquat treatment, these data demonstrated that in Drosophila M40403 was able to compensate for the loss of endogenous SOD enzymes, acting both at a cytosolic and mitochondrial level. Because previous clinical trials have indicated that the M40403 molecule is well tolerated in humans, this study may have important implication for the treatment of Parkinson disease (Filograna, 2016).

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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

Abstract
The co-enzyme nicotinamide adenine dinucleotide (NAD+) is an essential co-factor for cellular energy generation in mitochondria as well as for DNA repair mechanisms in the cell nucleus involving NAD+-consuming poly (ADP-ribose) polymerases (PARPs). Mitochondrial function is compromised in animal models of Parkinson's disease (PD) associated with Parkin mutations. This study uncovered alterations in NAD+ salvage metabolism in Drosophila parkin mutants. A dietary supplementation with the NAD+ precursor nicotinamide rescues mitochondrial function and is neuroprotective. Further, by mutating Parp in parkin mutants, it was shown that this increases levels of NAD+ and its salvage metabolites. This also rescues mitochondrial function and suppresses dopaminergic neurodegeneration. It is concluded that strategies to enhance NAD+ levels by administration of dietary precursors or the inhibition of NAD+-dependent enzymes, such as PARP, that compete with mitochondria for NAD+ could be used to delay neuronal death associated with mitochondrial dysfunction (Lehmann, 2016).

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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

Abstract
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).

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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

Abstract
Common neurodegenerative proteinopathies, such as Alzheimer's disease (AD) and Parkinson's disease (PD), are characterized by the misfolding and aggregation of toxic protein species, including the amyloid β (Aβ) peptide, microtubule-associated protein Tau (Tau), and alpha-synuclein (αSyn) protein. These factors also show toxicity in Drosophila. Using standardized conditions and medium-throughput assays, this study expressed human Tau, Aβ or αSyn selectively in neurons of the adult Drosophila retina and monitored age-dependent changes in both structure and function, based on tissue histology and recordings of the electroretinogram (ERG), respectively. Each protein was found to cause a unique profile of neurodegenerative pathology. Strikingly, expression of Tau leads to progressive loss of ERG responses whereas retinal architecture and neuronal numbers are largely preserved. By contrast, Aβ induces modest, age-dependent neuronal loss without degrading the retinal ERG. αSyn expression is characterized by marked retinal vacuolar change, progress photoreceptor cell death, and delayed-onset but modest ERG changes. Surprisingly, Tau and αSyn each cause prominent but distinct synaptotoxic profiles, including disorganization or enlargement of photoreceptor terminals, respectively. These findings suggest that Drosophila may be useful for revealing determinants of neuronal dysfunction that precede cell loss, including synaptic changes, in the adult nervous system (Chouhan, 2016). 

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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

Abstract
Spirulina (Arthrospira platensis) is a cyanobacterium (blue-green alga) consumed by humans and other animals because of its nutritional values and pharmacological properties. Apart from high protein contents, it also contains high levels of antioxidant and anti-inflammatory compounds, such as carotenoids, β-carotene, phycocyanin, and phycocyanobilin, indicating its possible pharmaco-therapeutic utility. In the present study using DJ-1βΔ93 flies, a Parkinson's disease model in Drosophila, this study has demonstrated the therapeutic effect of spirulina and its active component C-phycocyanin (C-PC) in the improvement of lifespan and locomotor behavior. The findings indicate that dietary supplementation of spirulina significantly improves the lifespan and locomotor activity of paraquat-fed DJ-1βΔ93 flies. Furthermore, supplementation of spirulina and C-PC individually and independently reduced the cellular stress marked by deregulating the expression of heat shock protein 70 and Jun-N-terminal kinase signaling in DJ-1βΔ93 flies. A significant decrease in superoxide dismutase and catalase activities in spirulina-fed DJ-1βΔ93 flies tends to indicate the involvement of antioxidant properties associated with spirulina in the modulation of stress-induced signaling and improvement in lifespan and locomotor activity in Drosophila DJ-1βΔ93 flies. These results suggest that antioxidant boosting properties of spirulina can be used as a nutritional supplement for improving the lifespan and locomotor behavior in Parkinson's disease (Kumar. 2017).

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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

Abstract
Parkinson's disease can be caused by mutations in the α-synuclein gene and is characterized by aggregates of α-synuclein protein. Aggregates are degraded by the autophago-lysosomal pathway. Since Rab7 has been shown to regulate trafficking of late endosomes and autophagosomes, it was hypothesized that overexpressing Rab7 might be beneficial in Parkinson's disease. To test this hypothesis this study expressed the pathogenic A53T mutant of α-synuclein in HEK293 cells and Drosophila. In HEK293 cells, EGFP-Rab7 decorated vesicles contain α-synuclein. Rab7 overexpression reduced the percentage of cells with α-synuclein particles and the amount of α-synuclein protein. Clearance of α-synuclein is explained by the increased occurrence of acidified α-synuclein vesicles with Rab7 overexpression, presumably representing autolysosomes. In the fly model, Rab7 rescued the locomotor deficit induced by neuronal expression of A53T-α-synuclein. Rab7 might be involved in the biogenesis of protective, autophagosome-like organelles in dopaminergic neurons. Taken together, Rab7 increased the clearance of α-synuclein aggregates, reduced cell death, and rescued the phenotype in a fly model of Parkinson's disease. These findings indicate that Rab7 is rate-limiting for aggregate clearance, and that Rab7 activation may offer a therapeutic strategy for Parkinson's disease (Dinter, 2016).

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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

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

Highlights

  • Ser182, Ser324, and Thr325 in DMiro mediate PINK1/Parkin-dependent degradation.
  • Generation of a fly model expressing DMiroS182A,S324A,T325A in a DMiro null background.
  • DMiroS182A,S324A,T325A or loss of PINK1 increases mitochondrial movement.
  • DMiroS182A,S324A,T325A or loss of PINK1 causes synaptic overgrowth at NMJs.
  • DMiroS182A,S324A,T325A causes DA neurodegeneration in adult brains.

Discussion
Although several phosphorylation substrates of the mitochondrial Ser/Thr kinase PINK1 have been identified, the precise functional consequences of PINK1-mediated phosphorylation in vivo remain unclear. This study reports that in Drosophila PINK1 may inhibit mitochondrial movement and synaptic growth at larval NMJs, and protect DA neurons in adult brains, by phosphorylating the atypical GTPase DMiro (Tsai, 2014).

Drosophila is a robust genetic and cellular tool for modeling human neurodegenerative diseases. Loss of PINK1 in Drosophila mimics many aspects of PD pathology, including a severe loss of 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).

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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

Abstract
Parkinson's disease (PD) is characterized by progressive dopaminergic neuronal loss and the formation of abnormal protein aggregates, referred to as Lewy bodies (LBs). PINK1 is a serine/threonine protein kinase that protects cells from stress-induced mitochondrial dysfunction. PINK1 gene mutations cause one form of autosomal recessive early-onset PD. Transglutaminase 2 (TG2) is an intracellular protein cross-linking enzyme that has an important role in LB formation during PD pathogenesis. This study identifies PINK1 as a novel TG2 binding partner and shows that PINK1 stabilizes the half-life of TG2 via inhibition of TG2 ubiquitination and subsequent proteasomal degradation. PINK1 affects TG2 stability in a kinase-dependent manner. In addition, PINK1 directly phosphorylates TG2 in carbonyl cyanide m-chlorophenyl hydrazine-induced mitochondrial damaged states, thereby enhancing TG2 accumulation and intracellular protein cross-linking products. The study further confirms the functional link between upstream PINK1 and downstream TG2 in Drosophila melanogaster. These data suggest that PINK1 positively regulates TG2 activity, which may be closely associated with aggresome formation in neuronal cells (Min, 2015).

Highlights

  • Catalytic core, barrel-1, and barrel-2 domains of TG2 interact with the PINK1 kinase domain.
  • PINK1 increases TG2 protein stability.
  • PINK1 phosphorylates TG2 under carbonyl cyanide m-Chlorophenyl hydrazine treatment and under resting conditions.
  • PINK1 Inhibits TG2 Ubiquitination.
  • PINK1-mediated TG2 phosphorylation enhances intracellular protein cross-linking product formation.
  • Drosophila PINK1 genetically interacts with the Drosophila TG2 homolog.

Discussion
The dPINK1 gene (CG4523) has a 52% similarity to human PINK1. The dPINK1 knockout flies show abnormal symptoms that are also observed in PD patients, such as indirect flight muscle and dopaminergic neuronal degeneration accompanied by locomotive defects. Furthermore, dPINK1 overexpression in flies causes disorganization of the ommatidial array and rough eye phenotypes. Moreover, hPINK1 overexpression results in flies with increased life span upon treatment with paraquat and H2O2. Additionally, overexpression of both PINK1 and PARKIN generate a severe rough eye phenotype, which significantly exceeds that of PINK1 or PARKIN overexpression alone. These genes act in a common pathway to maintain mitochondrial integrity and regulate both muscle and dopaminergic neurons. Fly dPINK1 mutants show muscle and dopaminergic neuronal degeneration. These mutants are also sensitive to multiple stresses and have reduced life spans and ATP levels. dPINK1 knockdown with RNAi also induces dopaminergic neuronal loss and ommatidial degeneration. These earlier studies indicate that dPINK1 plays an important role in the regulation of neuronal survival (Min, 2015).

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).

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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 

Abstract
Mitochondrial dysfunction plays important roles in Parkinson's disease (PD) and the degradation of the damaged mitochondria by the mitochondria quality control system is important for dopaminergic (DA) neuronal survival. BNIP3L/Nix is a mitochondrial outer membrane protein that is required for the selective clearance of mitochondria. This study found that the mitochondrial protein BNIP3L acts downstream of the PINK1/PARK2 pathway to induce mitophagy. BNIP3L is a substrate of PARK2 to drive PARK2-mediated mitophagy. The ubiquitination of BNIP3L by PARK2 recruits NBR1 to mitochondria, thereby targeting mitochondria for degradation. BNIP3L rescues mitochondrial defects in pink1 mutant Drosophila but not in park mutant Drosophila, indicating that the clearance of mitochondria induced by BNIP3L depends on the presence of PARK2. In cells intoxicated with mitochondrial complex I inhibitors rotenone, 6-OHDA or MPP(+), the disrupted mitochondria are not appropriately eliminated by mitophagy due to the improper degradation of BNIP3L. Thus, BNIP3L, as a substrate of PARK2, promotes mitophagy in the PINK1/PARK2 pathway associated with PD pathogenesis (Gao, 2015).

Highlights

  • BNIP3L acts at downstream of the PINK1/PARK2 pathway.
  • BNIP3L acts as a mitochondrial substrate of PARK2.
  • The autophagy adaptor NBR1 promotes the BNIP3L-mediated mitophagy.
  • Inhibition of mitochondria complex I activity induces BNIP3L degradation and interferes with mitophagy.

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

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

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

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

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Kim, H., Yang, J., Kim, M. J., Choi, S., Chung, J. R., Kim, J. M., Yoo, Y. H., Chung, J. and Koh, H. (2016). Tumor Necrosis Factor Receptor-associated Protein 1 (TRAP1) mutation and TRAP1 inhibitor Gamitrinib-triphenylphosphonium (G-TPP) induce a Forkhead Box O (FOXO)-dependent cell protective signal from Mitochondria. J Biol Chem 291(4): 1841-1853. PubMed ID: 26631731

Abstract

TRAP1 (tumor necrosis factor receptor-associated protein 1), a mitochondrial Hsp90 family chaperone, has been identified as a critical regulator of cell survival and bioenergetics in tumor cells. To discover novel signaling networks regulated by TRAP1, Drosophila TRAP1 mutants were generated. The mutants successfully developed into adults and produced fertile progeny, showing that TRAP1 is dispensable in development and reproduction. Surprisingly, mutation or knockdown of TRAP1 markedly enhanced Drosophila survival under oxidative stress. Moreover, TRAP1 mutation ameliorated mitochondrial dysfunction and dopaminergic (DA) neuron loss induced by deletion of a familial Parkinson disease gene PINK1 (Pten-induced kinase 1) in Drosophila. Gamitrinib-triphenylphosphonium, a mitochondria-targeted Hsp90 inhibitor that increases cell death in HeLa and MCF7 cells, consistently inhibited cell death induced by oxidative stress and mitochondrial dysfunction induced by PINK1 mutation in mouse embryonic fibroblast cells and DA cell models such as SH-SY5Y and SN4741 cells. Additionally, gamitrinib-triphenylphosphonium also suppressed the defective locomotive activity and DA neuron loss in Drosophila PINK1 null mutants. In further genetic analyses, enhanced expression of Thor, a downstream target gene of transcription factor FOXO, was shown in TRAP1 mutants. Furthermore, deletion of FOXO almost nullified the protective roles of TRAP1 mutation against oxidative stress and PINK1 mutation. These results strongly suggest that inhibition of the mitochondrial chaperone TRAP1 generates a retrograde cell protective signal from mitochondria to the nucleus in a FOXO-dependent manner (Kim, 2016).

This study generated and characterized Drosophila TRAP1 mutants. TRAP1 mutants successfully developed into adults and showed no significant defects in their life span. Although TRAP1 has been regarded as a mitochondrial protective protein, no meaningful defects were observed in mitochondrial morphology, ATP level, and mtDNA content in 3- or 30-day-old TRAP1 mutants. However, under treatment of free radical inducers, such as rotenone or paraquat, loss of TRAP1 significantly increased survival rate. Moreover, TRAP1 mutation ameliorated oxidative stress sensitivity, mitochondrial dysfunction, and DA neuronal loss in Drosophila PINK1 null mutants (Figs. 3 and 4). Consistent with these fruit fly data, TRAP1 KO mice showed reduced age-associated tissue degeneration with activated oxidative chain complex activities (Kim, 2016).

These genetic data were fully supported by the following pharmacological analyses. G-TPP inhibited cell death and restored the decreased mitochondrial membrane potential in various paraquat-treated mammalian cells, such as MEFs and DA neuron model cell lines. Moreover, G-TPP treatment ameliorated decreased motor activity and DA neuron degeneration in Drosophila PINK1 null mutants and rescued mitochondrial dysfunction in PINK1 null MEFs. Overall, genetic and pharmacological data clearly demonstrated that TRAP1 inhibition can induce resistance against oxidative stress and rescue PINK1 null defects in both Drosophila and mammalian systems (Kim, 2016).

These results raised important questions: How does TRAP1 suppression induce oxidative stress resistance although it increases ROS levels? What is the molecular mechanism underlying the cell protection induced by TRAP1 inhibition? ROS has been regarded as detrimental to many biological processes. However, recent reports showed that ROS can activate beneficial signals especially from mitochondria. When Schulz (2007) restricted glucose availability in Caenorhabditis elegans, life span extension and oxidative stress resistance accompanied by increased ROS production. Pretreatment of anti-oxidants, such as NAC, inhibited elevated expression of cell protective enzymes in glucose-restricted worms and subsequently blocked the extension of life span and the resistance against oxidative stress. Consistently, in TRAP mutants, NAC treatment suppressed the enhancement in survival on paraquat-containing media, suggesting that ROS generated by TRAP1 mutation is not detrimental but beneficial as shown in previous studies. Then what makes ROS beneficial? Types of ROS from mitochondria are observed in long-lived C. elegans mutants. Mitochondrial superoxide, which was also detected in dihydroethidium staining of TRAP1 mutants, was critical to the life span extension induced by several mitochondrial protein mutations. In other analyses, low doses of paraquat, which generate various types of ROS from mitochondria, successfully prolonged life span, whereas higher concentrations shortened it. These results suggest that certain types or amounts of ROS are critical to its beneficial roles and indicate that TRAP1 down-regulation potentially induces appropriate types or amounts of ROS for cell protection (Kim, 2016).

In genetic analyses to find a molecular link between TRAP1 inhibition and cell protection, FOXO loss of function nullified the oxidative stress resistance induced by TRAP1 mutation or TRAP1 inhibition. Consistently, loss of function mutations of FOXO reaggravated the rescued phenotypes of PINK1 null mutants by TRAP1 mutation or G-TPP treatment and also suppressed TRAP1 mutation-induced gene expression of Thor, a FOXO target gene that has a critical role in mitochondrial protection and oxidative stress resistance. It was also observed that TRAP1 inhibition requires FOXO transcription factors to induce cell protection against oxidative stress in mammalian cells. These data consistently demonstrated that FOXO transcription factors mediate cell protection and survival signal induced by TRAP1 inhibition. Moreover, NAC suppressed the enhanced Thor expression and the resistance against oxidative stress in TRAP1 mutants, suggesting that ROS generated by TRAP1-inhibited mitochondria induces FOXO-mediated gene expression to protect cells and animals from oxidative stress and PINK1 mutation (Kim, 2016).

It was observed that G-TPP successfully protects various mammalian cells, such as NIH 3T3, MEF, SH-SY5Y, and SN4741, from oxidative stress. Contrarily, it also potentiated oxidative stress-induced cell toxicity in HeLa and MCF7 cells that are very sensitive to TRAP1 inhibitors. In biochemical analyses, G-TPP caused toxicity in cells with elevated expression of TRAP1, whereas G-TPP protects cells expressing TRAP1 at relatively low levels. These results raise a possibility that TRAP1 expression level reflects different cellular contexts such as amounts of stress on mitochondria. In cells under heavy mitochondrial stress, TRAP1 is overexpressed to protect mitochondria. In this case, TRAP1 inhibition by G-TPP treatment abruptly stops mitochondrial protection mechanisms and subsequently induces cell death. However, it is possible that, in cells not mainly dependent on TRAP1-mediated protection, TRAP1 is weakly expressed, and its inhibition can generate weak and beneficial mitochondrial stress to induce cell protective signals. Testing this hypothesis and finding the molecular mechanism underlying the correlation between TRAP1 expression levels and the sensitivity to G-TPP will be future topics (Kim, 2016).

This report has shown that genetic and pharmacological inhibition of TRAP1 protects cells from oxidative stress and mitochondrial dysfunction. Furthermore, they can generate a compensatory retrograde signal from mitochondria, also known as mitohormesis, to up-regulate cell protective gene expression. These unexpected results raise the possibility that TRAP1 inhibitors developed for anti-cancer therapy might be used to treat human pathology induced by mitochondrial disorders, including PD (Kim, 2016).

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

Abstract

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).

Discussion

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).

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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

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

Highlights

  • An RNAi screen for suppressors or phenocopiers of pink1 RNAi-induced mitochondrial fusion.
  • ND42 knockdown specifically phenocopies pink1 RNAi induced mitochondrial hyperfusion.
  • ND42 overexpression can rescue pink1 but not parkin mutants.
  • NDUFA10 minimally affects Parkin translocation and mitophagy.
  • ND42 overexpression restores CI activity in pink1 mutants.
  • Analysis of ND42 Ser-250 phospho-variants in the rescue of pink1 mutant locomotion and CI activity.
  • Overexpression of parkin rescues pink1 mutants but does not restore CI activity.

Discussion

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).

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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

Abstract
PTEN-induced kinase 1 (Pink1) and ubiquitin E3 ligase Parkin function in a linear pathway to maintain healthy mitochondria via regulating mitochondrial clearance and trafficking. Mutations in the two enzymes cause the familial form of Parkinson's disease (PD) in humans, as well as accumulation of defective mitochondria and cellular degeneration in flies. This study shows that loss of function of a scaffolding protein Mask, also known as ANKHD1 (Ankyrin repeats and KH domain containing protein 1) in humans, rescues the behavioral, anatomical and cellular defects caused by pink1 or parkin mutations in a cell-autonomous manner. Moreover, similar rescue can also be achieved if Mask knock-down is induced in parkin adult flies when the mitochondrial dystrophy is already manifested. It was found that Mask genetically interacts with Parkin to modulate mitochondrial morphology and negatively regulates the recruitment of Parkin to mitochondria. Also, loss of Mask activity promotes co-localization of the autophagosome marker with mitochondria in developing larval muscle, and that an intact autophagy pathway is required for the rescue of parkin mutant defects by mask loss of function. Together, these data strongly suggest that Mask/ANKHD1 activity can be inhibited in a tissue- and timely-controlled fashion to restore mitochondrial integrity under PD-linked pathological conditions (Zhu, 2015).

Highlights

  • Loss-of-function of mask, a novel parkin interactor, suppresses anatomic and behavioral defects of both pink1 and parkin mutant flies.
  • Knocking down Mask suppresses mitochondrial defects in pink1 and parkin mutants cell-autonomously.
  • Knocking down Mask at later adult stage rescues parkin mutant defects in mitochondrial morphology and function.
  • Mask regulates mitochondrial morphology and distribution.
  • Mask antagonizes the function of Parkin in regulating mitochondrial morphology.
  • Mask may function as a selective inhibitor of mitophagy.

Discussion

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).

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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

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

Highlights

  • pink1 mutants exhibit mitochondrial and individualization defects in spermatids.
  • pink1 mutants undergo apoptotic muscle degeneration and fragmentation of mitochondrial cristae.
  • pink1 mutants are sensitive to multiple stresses, and have reduced lifespan and ATP levels.
  • Fly pink1 is functionally conserved with human PINK1, and acts upstream of parkin.

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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

Abstract
Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal-dominant Parkinsonism with pleomorphic pathology including deposits of aggregated protein and neuronal degeneration. The pathogenesis of LRRK2-linked PD is not fully understood. Using co-immunoprecipitation, this study found that LRRK2 interacted with synphilin-1, a cytoplasmic protein that interacts with α-synuclein and has implications in PD pathogenesis. LRRK2 interacted with the N-terminus of synphilin-1 whereas synphilin-1 predominantly interacted with the C-terminus of LRRK2, including kinase domain. Co-expression of synphilin-1 with LRRK2 increased LRRK2-induced cytoplasmic aggregation in cultured cells. Moreover, synphilin-1 also attenuated mutant LRRK2-induced toxicity and reduced LRRK2 kinase activity in cultured cells. Knockdown of synphilin-1 by siRNA enhanced LRRK2 neuronal toxicity. In vivo Drosophila studies, coexpression of synphilin-1 and mutant G2019S-LRRK2 in double transgenic Drosophila increased survival and improved locomotor activity. Expression of synphilin-1 protects against G2019S-LRRK2-induced dopamine neuron loss and reduced LRRK2 phosphorylation in double transgenic fly brains. These findings demonstrate that synphilin-1 attenuates mutant LRRK2-induced PD-like phenotypes and plays a neural protective role (Liu, 2016).

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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

Abstract
A central pathological hallmark of Parkinson's disease (PD) is the presence of proteinaceous depositions known as Lewy bodies, which consist largely of the protein α-synuclein (aSyn). Mutations, multiplications and polymorphisms in the gene encoding aSyn are associated with familial forms of PD and susceptibility to idiopathic PD. Alterations in aSyn impair neuronal vesicle formation/transport, and likely contribute to PD pathogenesis by neuronal dysfunction and degeneration. aSyn is functionally associated with several Rab family GTPases, which perform various roles in vesicle trafficking. This study explores the role of the endosomal recycling factor Rab11 in the pathogenesis of PD using Drosophila models of aSyn toxicity. It was found that aSyn induces synaptic potentiation at the larval neuromuscular junction by increasing synaptic vesicle (SV) size, and that these alterations are reversed by Rab11 overexpression. Furthermore, Rab11 decreases aSyn aggregation and ameliorates several aSyn-dependent phenotypes in both larvae and adult fruit flies, including locomotor activity, degeneration of dopaminergic neurons and shortened lifespan. This work emphasizes the importance of Rab11 in the modulation of SV size and consequent enhancement of synaptic function and suggests that targeting Rab11 activity could have a therapeutic value in PD (Breda, 2015).

Highlights

  • Rab11 normalizes aSyn-dependent potentiation of synaptic transmission at the Drosophila larval neuromuscular junction.
  • Rab11 ameliorates aSyn synaptic defects by restoration of synaptic vesicle size.
  • Rab11 ameliorates aSyn-dependent larval crawling impairments.
  • Rab11 reverses the loss of dopaminergic neurons in adult Drosophila brains.
  • Rab11 ameliorates disease-relevant phenotypes in adult flies expressing aSyn.

Discussion
Although the exact function of aSyn is not fully understood, some studies suggest that it is linked to synaptic transmission. This is emphasized by its localization to pre-synaptic termini and its involvement in several steps of the neurotransmission machinery. The overexpression of aSyn in murine dopaminergic neurons leads to inhibition of TH activity with a consequent reduction in dopamine synthesis. Moreover, aSyn association with the distal reserve pool of SVs and the SNARE complex indicate a physiological role of aSyn in the recycling of SVs and their fusion with the plasma membrane. aSyn was also found to interact with several Rab GTPase proteins, which are highly conserved regulators of intracellular membrane trafficking between organelles. aSyn associates with Rab3a, Rab5 and Rab8 in dementia with LB patients and mouse models, and an interaction between endocytosed aSyn and Rab11a was observed in mammalian cells. Notably, Rab1 and Rab8a overexpression reverses aSyn-dependent impairment of ER–Golgi transport in yeast, fruit flies and Caenorhabditis elegans (Breda, 2015).

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).

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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

Abstract
Mitochondria are key regulators of cellular homeostasis, and mitochondrial dysfunction is strongly linked to neurodegenerative diseases, including Alzheimer's and Parkinson's. Mitochondria communicate their bioenergetic status to the cell via mitochondrial retrograde signaling. To investigate the role of mitochondrial retrograde signaling in neurons, mitochondrial dysfunction was induced in the Drosophila nervous system. Neuronal mitochondrial dysfunction causes reduced viability, defects in neuronal function, decreased redox potential, and reduced numbers of presynaptic mitochondria and active zones. Neuronal mitochondrial dysfunction stimulates a retrograde signaling response that controls the expression of several hundred nuclear genes. Drosophila hypoxia inducible factor alpha (HIFalpha) ortholog Similar (Sima) regulates the expression of several of these retrograde genes, suggesting that Sima mediates mitochondrial retrograde signaling. Remarkably, knockdown of Sima restores neuronal function without affecting the primary mitochondrial defect, demonstrating that mitochondrial retrograde signaling is partly responsible for neuronal dysfunction. Sima knockdown also restores function in a Drosophila model of the mitochondrial disease Leigh syndrome and in a Drosophila model of familial Parkinson's disease. Thus, mitochondrial retrograde signaling regulates neuronal activity and can be manipulated to enhance neuronal function, despite mitochondrial impairment (Cagin, 2015).

Discussion

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).

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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

Abstract

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).

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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

Abstract
Parkinson's disease is a movement disorder characterized by degeneration of dopaminergic neurons in the substantia nigra pars compacta. Dopaminergic neuronal loss also occurs in Drosophila melanogaster upon directed expression of α-synuclein, a protein implicated in the pathogenesis of Parkinson's disease and a major component of proteinaceous Lewy bodies. This study reports that directed expression of the molecular chaperone Hsp70 prevents dopaminergic neuronal loss associated with α-synuclein in Drosophila and that interference with endogenous chaperone activity accelerates α-synuclein toxicity. Furthermore, Lewy bodies in human postmortem tissue immunostain for molecular chaperones, also suggesting that chaperones may play a role in Parkinson's disease progression (Auluck, 2002).

Highlights

  • Hsp70 protects against α-synuclein–induced dopaminergic neuronal degeneration.
  • Hsp70 does not alter the appearance of α-synuclein inclusions in the Drosophila brain.
  • Inhibition of constitutive Hsp70 activity enhances α-synuclein–induced loss of dopaminergic neurons in the dorsomedial clusters.
  • Lesions from tissue of human patients with Parkinson's disease, and other synucleinopathies, are immunopositive for the molecular chaperone Hsp70 and co-chaperone Hsp40.

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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

Abstract
As our society ages, neurodegenerative disorders like Parkinson`s disease (PD) are increasing in pandemic proportions. While mechanistic understanding of PD is advancing, a treatment with well tolerable drugs is still elusive. This study shows that administration of the naturally occurring polyamine spermidine, which declines continuously during aging in various species, alleviates a series of PD-related degenerative processes in the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, two established model systems for PD pathology. In the fruit fly, simple feeding with spermidine inhibits loss of climbing activity and early organismal death upon heterologous expression of human α-synuclein, which is thought to be the principal toxic trigger of PD. Also, administration of spermidine rescues α-synuclein-induced loss of dopaminergic neurons, a hallmark of PD, in nematodes. Alleviation of PD-related neurodegeneration by spermidine is accompanied by induction of autophagy, suggesting that this cytoprotective process may be responsible for the beneficial effects of spermidine administration (Büttner, 2014).

Highlights

  • Spermidine prevents ?Syn-induced motor dysfunction and death of D. melanogaster.
  • Spermidine reduces ?Syn neurotoxicity in C. elegans and induces autophagy.

Discussion

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).

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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 

Abstract
One of the most important questions in Parkinson’s disease (PD) regards the selective vulnerability of a specific population of dopaminergic (DA) neurons. Recent reports identify Ca2+ channel as a potential source of this vulnerability. This work uses a Drosophila primary neuronal cell culture system as an in vitro PD model to explore the role of Ca2+ homeostasis in DA neurodegeneration and protection. It was shown that the Ca2+ chelator EGTA is neuroprotective against a PD toxin MPP+ (40 μM). Genetic tools available in Drosophila were used to manipulate Ca2+ channel activity and it was found that DA neurons lacking functional Ca2+ channels (i.e., cacophony mutants) are inherently protected against MPP+ toxicity. Furthermore, overexpression of wild type Ca2+ channels in DA neurons blocks the rescue effect of a D2 agonist quinpirole on DA neurodegeneration. These findings support the idea that Ca2+ is a source of vulnerability for DA neurons and that the modulation of Ca2+ levels in DA neurons could be a potential neuroprotective treatment (Wiemerslage, 2015).

Highlights

  • Ca2+ chelation is neuroprotective to DA neurons.
  • Ca2+ channel mutants are inherently protected from MPP+ treatment.
  • Overexpression of wild type Ca2+ channels prevents rescue of DA neurodegeneration by D2R agonist.

Discussion
The findings from this study's experiments with the Ca2+chelator EGTA and the Ca2+ channel mutant support the hypothesis that Ca2+ dysregulation is a source of vulnerability in DA neurons. EGTA rescues DA neurodegeneration induced by a PD toxin MPP+. Also, cultures with mutant Ca2+ channels are inherently protected from MPP+ toxicity. Furthermore, overexpression of wild type Ca2+ channels specifically in DA neurons prevents the rescue by a D2 agonist quinpirole – a drug that normally prevents PD-like neurodegeneration (Wiemerslage, 2015).

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).

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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

Abstract
Mutations of VPS35, a component of the retromer complex have been associated with late onset familial Parkinson’s disease. The D620N mutation in VPS35 appears to be most prevalent, however, P316S was found in two cases within the same family and a control, whereas L774M was identified in 6 cases and 1 control. In vivo evidence of their pathogenicity is lacking. This study investigated the in vivo effects of P316S, D620N and L774M using Drosophila as a model. By generating transgenic human VPS35-expressing mutations in flies, it was demonstrated that VPS35 D620N transgenic flies lead to late-onset loss of TH-positive DA neurons, poor mobility, shortened lifespans and increased sensitivity to rotenone, a PD-linked environmental toxin, with some of these phenotypes observed for P316S but not in L774M transgenic flies. The study concludes that D620N, and to a smaller extent P316S, are associated with pathogenicity in PD (Wang, 2014).

Highlights

  • VSP35 D620N promotes dopaminergic (DA) neurodegeneration and loss of locomotor activity.
  • Rotenone treatment further exacerbates the pathogenicity of D620N VPS35 variant in DA neuron degeneration.

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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

Abstract
The fruit fly Drosophila melanogaster has long been used as a model organism for human diseases, including Parkinson?s disease (PD). Its short lifespan, simple maintenance, and the widespread availability of genetic tools allow researchers to study disease mechanisms as well as potential drug therapies. Many different PD models have already been developed, including ones utilizing mutated α-Syn and chronic exposure to rotenone. However, few animal models have been used to study interaction between the PD causing factors. This study developed a new model of PD for use in the larval stage in order to study interaction between genetic and environmental factors. First, the 3rd instar larvae (90–94 hours after egg laying) expressing a mutated form of human α-Syn (A53T) in dopaminergic (DA) neurons were video-taped and quantified for locomotion (e.g. crawling pattern and speed) using ImageJ software. A53T mutant larvae show locomotion deficits and also loss of DA neurons in age-dependent manner. Similarly, larvae chronically exposed to rotenone (10 μM in food) show age-dependent decline in locomotion accompanied by loss of DA neurons. It was further shown that combining the two models, by exposing A53T mutant larvae to rotenone, causes a much more severe PD phenotype (i.e. locomotor deficit). These findings exhibit the interaction between genetic and environmental factors underlying development of PD symptoms. The model developed in this study can be used to further study mechanisms underlying the interaction between genes and different environmental PD factors, as well as to explore potential therapies for PD treatment (Varga, 2014).

Highlights

  • Mutant human α-Synuclein causes locomotor deficits and age-dependent degeneration of dopaminergic neurons in Drosophila larvae.
  • Rotenone exposure causes concentration-dependent locomotor deficits and breakdown of dopaminergic neurons.
  • Expression of mutant α-Synuclein and exposure to rotenone do not cause olfactory defects.

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Reviews

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

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More in IF

Transcriptional regulation of Msh homologs and potential role in Parkinson's therapy

Ubiquitin conjugating enzymes: interactions with ubiquitin protein ligases with discussion on role in Parkinson's disease

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Recent Updates

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

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Date revised: 4 December 2018

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