SREBF1 links lipogenesis to mitophagy and sporadic Parkinson disease

Mitochondrial quality control has an impact on many diseases, but intense research has focused on the action of 2 genes linked to heritable forms of Parkinson disease (PD), PINK1 and PARK2/parkin, which act in a common pathway to promote mitophagy. However, criticism has been raised that little evidence links this mechanism to sporadic PD. To gain a greater insight into the mechanisms of PINK1-PARK2 mediated mitophagy, we undertook a genome-wide RNAi screen in Drosophila and human cell models. Strikingly, we discovered several components of the lipogenesis pathway, including SREBF1, playing a conserved role in mitophagy. Our results suggest that lipids influence the stabilization of PINK1 during the initiation of mitophagy. Importantly, SREBF1 has previously been identified as a risk locus for sporadic PD, and thus implicates aberrant mitophagy as contributing to sporadic PD. Our findings suggest a role for lipid synthesis in PINK1-PARK2 mediated mitophagy, and propose a mechanistic link between familial and sporadic PD, supporting a common etiology.     

Glutathione S-transferase omega suppresses the defective phenotypes caused by PINK1 loss-of-function in Drosophila

Loss-of-function mutation of the PTEN-induced kinase 1 (PINK1) gene is a common cause of early-onset Parkinson’s disease (PD). Glutathione S-transferase omega (GSTO) is a phase II detoxification enzyme that conjugates targets to glutathione, and has recently been implicated in parkin-associated PD. In this study, we found Drosophila GstO2 to be a novel genetic suppressor of the PINK1 loss-of-function mutant. We show that GstO2A expression is reduced in PINK1 mutants. Moreover, the upregulation of GstO2A restores muscle degeneration and dopaminergic neuron loss in PINK1 mutants. Given the previous data of a reduced expression of GstO2A and decreased glutathionylation of ATP synthase β subunit in parkin or PINK1 mutants, these results suggest that the function of GstO2 is regulated by the PINK1/parkin pathway and that GstO2 also has a protective role in PINK1-associated PD.     

The deubiquitinating enzyme DUBAI stabilizes DIAP1 to suppress Drosophila apoptosis

Deubiquitinating enzymes (DUBs) counteract ubiquitin ligases to modulate the ubiquitination and stability of target signaling molecules. In Drosophila, the ubiquitin–proteasome system has a key role in the regulation of apoptosis, most notably, by controlling the abundance of the central apoptotic regulator DIAP1. Although the mechanism underlying DIAP1 ubiquitination has been extensively studied, the precise role of DUB(s) in controlling DIAP1 activity has not been fully investigated. Here we report the identification of a DIAP1-directed DUB using two complementary approaches. First, a panel of putative Drosophila DUBs was expressed in S2 cells to determine whether DIAP1 could be stabilized, despite treatment with death-inducing stimuli that would induce DIAP1 degradation. In addition, RNAi fly lines were used to detect modifiers of DIAP1 antagonist-induced cell death in the developing eye. Together, these approaches identified a previously uncharacterized protein encoded by CG8830, which we named DeUBiquitinating-Apoptotic-Inhibitor (DUBAI), as a novel DUB capable of preserving DIAP1 to dampen Drosophila apoptosis. DUBAI interacts with DIAP1 in S2 cells, and the putative active site of its DUB domain (C367) is required to rescue DIAP1 levels following apoptotic stimuli. DUBAI, therefore, represents a novel locus of apoptotic regulation in Drosophila, antagonizing cell death signals that would otherwise result in DIAP1 degradation.     

S-(4-Nitrophenacyl)glutathione is a specific substrate for glutathione transferase omega 1-1

Glutathione transferase omega 1-1 (GSTO1-1) catalyzes the biotransformation of arsenic and is implicated as a factor influencing the age-at-onset of Alzheimer’s disease and the posttranslational activation of interleukin 1β (IL-1β). Investigation of the biological role of GSTO1-1 variants has been hampered by the lack of a specific assay for GSTO1-1 activity in tissue samples that contain other GSTs and other enzymes with similar catalytic specificities. Previous studies (P. G. Board and M. W. Anders, Chem. Res. Toxicol. 20 (2007) 149-154) have shown that GSTO1-1 catalyzes the reduction of S-(phenacyl)glutathiones to acetophenones. A new substrate, S-(4-nitrophenacyl)glutathione (4NPG), has been prepared and found to have a high turnover with GSTO1-1 but negligible activity with GSTO2-2 and other members of the glutathione transferase superfamily. A spectrophotometric assay with 4NPG as a substrate has been used to determine GSTO1-1 activity in several human breast cancer cell lines and in mouse liver and brain tissues.     

Drosophila as a model for antiviral immunity

The fruit fly Drosophila melanogaster has been successfully used to study numerous biological processes including immune response. Flies are naturally infected with more than twenty RNA viruses making it a valid model organism to study host-pathogen interactions during viral infections. The Drosophila antiviral immunity includes RNA interference, activation of the JAK/STAT and other signaling cascades and other mechanisms such as autophagy and interactions with other microorganisms. Here we review Drosophila as an immunological research model as well as recent advances in the field of Drosophila antiviral immunity.     

Drosophila Trap1 protects against mitochondrial dysfunction in a PINK1/parkin model of Parkinson's disease

Mitochondrial dysfunction caused by protein aggregation has been shown to have an important role in neurological diseases, such as Parkinson''s disease (PD). Mitochondria have evolved at least two levels of defence mechanisms that ensure their integrity and the viability of their host cell. First, molecular quality control, through the upregulation of mitochondrial chaperones and proteases, guarantees the clearance of damaged proteins. Second, organellar quality control ensures the clearance of defective mitochondria through their selective autophagy. Studies in Drosophila have highlighted mitochondrial dysfunction linked with the loss of the PTEN-induced putative kinase 1 (PINK1) as a mechanism of PD pathogenesis. The mitochondrial chaperone TNF receptor-associated protein 1 (TRAP1) was recently reported to be a cellular substrate for the PINK1 kinase. Here, we characterise Drosophila Trap1 null mutants and describe the genetic analysis of Trap1 function with Pink1 and parkin. We show that loss of Trap1 results in a decrease in mitochondrial function and increased sensitivity to stress, and that its upregulation in neurons of Pink1 mutant rescues mitochondrial impairment. Additionally, the expression of Trap1 was able to partially rescue mitochondrial impairment in parkin mutant flies; and conversely, expression of parkin rescued mitochondrial impairment in Trap1 mutants. We conclude that Trap1 works downstream of Pink1 and in parallel with parkin in Drosophila, and that enhancing its function may ameliorate mitochondrial dysfunction and rescue neurodegeneration in PD.     

Drosophila as a model system to unravel the layers of innate immunity to infection

Innate immunity relies entirely upon germ-line encoded receptors, signalling components and effector molecules for the recognition and elimination of invading pathogens. The fruit fly Drosophila melanogaster with its powerful collection of genetic and genomic tools has been the model of choice to develop ideas about innate immunity and host-pathogen interactions. Here, we review current research in the field, encompassing all layers of defence from the role of the microbiota to systemic immune activation, and attempt to speculate on future directions and open questions.     

Inactivation of Pink1 gene in vivo sensitizes dopamine-producing neurons to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and can be rescued by autosomal recessive Parkinson disease genes, Parkin or DJ-1

Mutations in the mitochondrial PTEN-induced kinase 1 (Pink1) gene have been linked to Parkinson disease (PD). Recent reports including our own indicated that ectopic Pink1 expression is protective against toxic insult in vitro, suggesting a potential role for endogenous Pink1 in mediating survival. However, the role of endogenous Pink1 in survival, particularly in vivo, is unclear. To address this critical question, we examined whether down-regulation of Pink1 affects dopaminergic neuron loss following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the adult mouse. Two model systems were utilized: virally delivered shRNA-mediated knockdown of Pink1 and germ line-deficient mice. In both instances, loss of Pink1 generated significant sensitivity to damage induced by systemic MPTP treatment. This sensitivity was associated with greater loss of dopaminergic neurons in the Substantia Nigra pars compacta and terminal dopamine fiber density in the striatum region. Importantly, we also show that viral mediated expression of two other recessive PD-linked familial genes, DJ-1 and Parkin, can protect dopaminergic neurons even in the absence of Pink1. This evidence not only provides strong evidence for the role of endogenous Pink1 in neuronal survival, but also supports a role of DJ-1 and Parkin acting parallel or downstream of endogenous Pink1 to mediate survival in a mammalian in vivo context.     

Interaction between Sleep and Metabolism in Drosophila with Altered Octopamine Signaling

Sleep length and metabolic dysfunction are correlated, but the causal relationship between these processes is unclear. Octopamine promotes wakefulness in the fly by acting through the insulin-producing cells (IPCs) in the fly brain. To determine if insulin signaling mediates the effects of octopamine on sleep:wake behavior, we assayed flies in which insulin signaling activity was genetically altered. We found that increasing insulin signaling does not promote wake, nor does insulin appear to mediate the wake-promoting effects of octopamine. Octopamine also affects metabolism in invertebrate species, including, as we show here, Drosophila melanogaster. Triglycerides are decreased in mutants with compromised octopamine signaling and elevated in flies with increased activity of octopaminergic neurons. Interestingly, this effect is mediated at least partially by insulin, suggesting that effects of octopamine on metabolism are independent of its effects on sleep. We further investigated the relative contribution of metabolic and sleep phenotypes to the starvation response of flies with altered octopamine signaling. Hyperactivity (indicative of foraging) induced by starvation was elevated in octopamine receptor mutants, despite their high propensity for sleep, indicating that their metabolic state dictates their behavioral response under these conditions. Moreover, flies with increased octopamine signaling do not suppress sleep in response to starvation, even though they are normally hyper-aroused, most likely because of their high triglyceride levels. Together, these data suggest that observed correlations between sleep and metabolic phenotypes can result from shared molecular pathways rather than causality, and environmental conditions can lead to the dominance of one phenotype over the other.     

Specific myosin heavy chain mutations suppress troponin I defects in Drosophila muscles

We show that specific mutations in the head of the thick filament molecule myosin heavy chain prevent a degenerative muscle syndrome resulting from the hdp2 mutation in the thin filament protein troponin I. One mutation deletes eight residues from the actin binding loop of myosin, while a second affects a residue at the base of this loop. Two other mutations affect amino acids near the site of nucleotide entry and exit in the motor domain. We document the degree of phenotypic rescue each suppressor permits and show that other point mutations in myosin, as well as null mutations, fail to suppress the hdp2 phenotype. We discuss mechanisms by which the hdp2 phenotypes are suppressed and conclude that the specific residues we identified in myosin are important in regulating thick and thin filament interactions. This in vivo approach to dissecting the contractile cycle defines novel molecular processes that may be difficult to uncover by biochemical and structural analysis. Our study illustrates how expression of genetic defects are dependent upon genetic background, and therefore could have implications for understanding gene interactions in human disease.     

Quantitative mapping of reversible mitochondrial Complex I cysteine oxidation in a Parkinson disease mouse model

Differential cysteine oxidation within mitochondrial Complex I has been quantified in an in vivo oxidative stress model of Parkinson disease. We developed a strategy that incorporates rapid and efficient immunoaffinity purification of Complex I followed by differential alkylation and quantitative detection using sensitive mass spectrometry techniques. This method allowed us to quantify the reversible cysteine oxidation status of 34 distinct cysteine residues out of a total 130 present in murine Complex I. Six Complex I cysteine residues were found to display an increase in oxidation relative to controls in brains from mice undergoing in vivo glutathione depletion. Three of these residues were found to reside within iron-sulfur clusters of Complex I, suggesting that their redox state may affect electron transport function.     

Neuronal necrosis and spreading death in a Drosophila genetic model

Brain ischemia often results in neuronal necrosis, which may spread death to neighboring cells. However, the molecular events of neuronal necrosis and the mechanisms of this spreading death are poorly understood due to the limited genetic tools available for deciphering complicated responses in mammalian brains. Here, we engineered a Drosophila model of necrosis in a sub-population of neurons by expressing a leaky cation channel in the Drosophila eye. Expression of this channel caused necrosis in defined neurons as well as extensive spreading of cell death. Jun N-terminal kinase (JNK)-mediated, caspase-independent apoptosis was the primary mechanism of cell death in neurons, while caspase-dependent apoptosis was primarily involved in non-neuronal cell death. Furthermore, the JNK activation in surrounding neurons was triggered by reactive oxygen species (ROS) and Eiger (Drosophila tumor necrosis factor α (TNFα)) released from necrotic neurons. Because the Eiger/ROS/JNK signaling was also required for cell death induced by hypoxia and oxidative stress, our fly model of spreading death may be similar to brain ischemia in mammals. We performed large-scale genetic screens to search for novel genes functioning in necrosis and/or spreading death, from which we identified several classes of genes. Among them, Rho-associated kinase (ROCK) had been reported as a promising drug target for stroke treatment with undefined mechanisms. Our data indicate that ROCK and the related trafficking pathway genes regulate neuronal necrosis. We propose the suppression of the function of the trafficking system, ROS and cytokines, such as TNFα, as translational applications targeting necrosis and spreading death.     

Characterization of PINK1 (PTEN-induced Putative Kinase 1) Mutations Associated with Parkinson Disease in Mammalian Cells and Drosophila

Mutations in PINK1 (PTEN-induced putative kinase 1) are tightly linked to autosomal recessive Parkinson disease (PD). Although more than 50 mutations in PINK1 have been discovered, the role of these mutations in PD pathogenesis remains poorly understood. Here, we characterized 17 representative PINK1 pathogenic mutations in both mammalian cells and Drosophila. These mutations did not affect the typical cleavage patterns and subcellular localization of PINK1 under both normal and damaged mitochondria conditions in mammalian cells. However, PINK1 mutations in the kinase domain failed to translocate Parkin to mitochondria and to induce mitochondrial aggregation. Consistent with the mammalian data, Drosophila PINK1 mutants with mutations in the kinase domain (G426D and L464P) did not genetically interact with Parkin. Furthermore, PINK1-null flies expressing the transgenic G426D mutant displayed defective phenotypes with increasing age, whereas L464P mutant-expressing flies exhibited the phenotypes at an earlier age. Collectively, these results strongly support the hypothesis that the kinase activity of PINK1 is essential for its function and for regulating downstream Parkin functions in mitochondria. We believe that this study provides the basis for understanding the molecular and physiological functions of various PINK1 mutations and provides insights into the pathogenic mechanisms of PINK1-linked PD.     

Drosophila as a model for unfolded protein response research

Endoplasmic Reticulum (ER) is an organelle where most secretory and membrane proteins are synthesized, folded, and undergo further maturation. As numerous conditions can perturb such ER function, eukaryotic cells are equipped with responsive signaling pathways, widely referred to as the Unfolded Protein Response (UPR). Chronic conditions of ER stress that cannot be fully resolved by UPR, or conditions that impair UPR signaling itself, are associated with many metabolic and degenerative diseases. In recent years, Drosophila has been actively employed to study such connections between UPR and disease. Notably, the UPR pathways are largely conserved between Drosophila and humans, and the mediating genes are essential for development in both organisms, indicating their requirement to resolve inherent stress. By now, many Drosophila mutations are known to impose stress in the ER, and a number of these appear similar to those that underlie human diseases. In addition, studies have employed the strategy of overexpressing human mutations in Drosophila tissues to perform genetic modifier screens. The fact that the basic UPR pathways are conserved, together with the availability of many human disease models in this organism, makes Drosophila a powerful tool for studying human disease mechanisms. [BMB Reports 2015; 48(8): 445-453]     

Dopamine in Drosophila: setting arousal thresholds in a miniature brain

In mammals, the neurotransmitter dopamine (DA) modulates a variety of behaviours, although DA function is mostly associated with motor control and reward. In insects such as the fruitfly, Drosophila melanogaster, DA also modulates a wide array of behaviours, ranging from sleep and locomotion to courtship and learning. How can a single molecule play so many different roles? Adaptive changes within the DA system, anatomical specificity of action and effects on a variety of behaviours highlight the remarkable versatility of this neurotransmitter. Recent genetic and pharmacological manipulations of DA signalling in Drosophila have launched a surfeit of stories—each arguing for modulation of some aspect of the fly''s waking (and sleeping) life. Although these stories often seem distinct and unrelated, there are some unifying themes underlying DA function and arousal states in this insect model. One of the central roles played by DA may involve perceptual suppression, a necessary component of both sleep and selective attention.