Drosophila Porin/VDAC Affects Mitochondrial Morphology

Voltage-dependent anion channel (VDAC) has been suggested to be a mediator of mitochondrial-dependent cell death induced by Ca2+ overload, oxidative stress and Bax-Bid activation. To confirm this hypothesis in vivo, we generated and characterized Drosophila VDAC (porin) mutants and found that Porin is not required for mitochondrial apoptosis, which is consistent with the previous mouse studies. We also reported a novel physiological role of Porin. Loss of porin resulted in locomotive defects and male sterility. Intriguingly, porin mutants exhibited elongated mitochondria in indirect flight muscle, whereas Porin overexpression produced fragmented mitochondria. Through genetic analysis with the components of mitochondrial fission and fusion, we found that the elongated mitochondria phenotype in porin mutants were suppressed by increased mitochondrial fission, but enhanced by increased mitochondrial fusion. Furthermore, increased mitochondrial fission by Drp1 expression suppressed the flight defects in the porin mutants. Collectively, our study showed that loss of Drosophila Porin results in mitochondrial morphological defects and suggested that the defective mitochondrial function by Porin deficiency affects the mitochondrial remodeling process

PINK1 alleviates thermal hypersensitivity in a paclitaxel-induced Drosophila model of peripheral neuropathy

Paclitaxel is a representative anticancer drug that induces chemotherapy-induced peripheral neuropathy (CIPN), a common side effect that limits many anticancer chemotherapies. Although PINK1, a key mediator of mitochondrial quality control, has been shown to protect neuronal cells from various toxic treatments, the role of PINK1 in CIPN has not been investigated. Here, we examined the effect of PINK1 expression on CIPN using a recently established paclitaxel-induced peripheral neuropathy model in Drosophila larvae. We found that the class IV dendritic arborization (C4da) sensory neuron-specific expression of PINK1 significantly ameliorated the paclitaxel-induced thermal hyperalgesia phenotype. In contrast, knockdown of PINK1 resulted in an increase in thermal hypersensitivity, suggesting a critical role for PINK1 in sensory neuron-mediated thermal nociceptive sensitivity. Interestingly, analysis of the C4da neuron morphology suggests that PINK1 expression alleviates paclitaxel-induced thermal hypersensitivity by means other than preventing alterations in sensory dendrites in C4da neurons. We found that paclitaxel induces mitochondrial dysfunction in C4da neurons and that PINK1 expression suppressed the paclitaxel-induced increase in mitophagy in C4da neurons. These results suggest that PINK1 mitigates paclitaxel-induced sensory dendrite alterations and restores mitochondrial homeostasis in C4da neurons and that improvement in mitochondrial quality control could be a promising strategy for the treatment of CIPN. © 2020 Kim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.TRU

Inhibition of ERK-MAP kinase signaling by RSK during Drosophila development

Although p90 ribosomal S6 kinase (RSK) is known as an important downstream effector of the ribosomal protein S6 kinase/extracellular signal-regulated kinase (Ras/ERK) pathway, its endogenous role, and precise molecular function remain unclear. Using gain-of-function and null mutants of RSK, its physiological role was successfully characterized in Drosophila. Surprisingly, RSK-null mutants were viable, but exhibited developmental abnormalities related to an enhanced ERK-dependent cellular differentiation such as ectopic photoreceptor- and vein-cell formation. Conversely, overexpression of RSK dramatically suppressed the ERK-dependent differentiation, which was further augmented by mutations in the Ras/ERK pathway. Consistent with these physiological phenotypes, RSK negatively regulated ERK-mediated developmental processes and gene expressions by blocking the nuclear localization of ERK in a kinase activity-independent manner. In addition, we further demonstrated that the RSK-dependent inhibition of ERK nuclear migration is mediated by the physical association between ERK and RSK. Collectively, our study reveals a novel regulatory mechanism of the Ras/ERK pathway by RSK, which negatively regulates ERK activity by acting as a cytoplasmic anchor in Drosophila

Isocitrate protects <i>DJ-1</i> null dopaminergic cells from oxidative stress through NADP⁺-dependent isocitrate dehydrogenase (IDH)

<div><p><i>DJ-1</i> is one of the causative genes for early onset familiar Parkinson’s disease (PD) and is also considered to influence the pathogenesis of sporadic PD. DJ-1 has various physiological functions which converge on controlling intracellular reactive oxygen species (ROS) levels. In RNA-sequencing analyses searching for novel anti-oxidant genes downstream of DJ-1, a gene encoding NADP⁺-dependent isocitrate dehydrogenase (IDH), which converts isocitrate into α-ketoglutarate, was detected. Loss of <i>IDH</i> induced hyper-sensitivity to oxidative stress accompanying age-dependent mitochondrial defects and dopaminergic (DA) neuron degeneration in <i>Drosophila</i>, indicating its critical roles in maintaining mitochondrial integrity and DA neuron survival. Further genetic analysis suggested that DJ-1 controls IDH gene expression through nuclear factor-E2-related factor2 (Nrf2). Using <i>Drosophila</i> and mammalian DA models, we found that IDH suppresses intracellular and mitochondrial ROS level and subsequent DA neuron loss downstream of DJ-1. Consistently, trimethyl isocitrate (TIC), a cell permeable isocitrate, protected mammalian <i>DJ-1</i> null DA cells from oxidative stress in an IDH-dependent manner. These results suggest that isocitrate and its derivatives are novel treatments for PD associated with <i>DJ-1</i> dysfunction.</p></div

IDH inhibits oxidative stress-induced cell death in <i>DJ-1</i> null mammalian DA cells.

<p>(A) Comparison of <i>IDH1</i> mRNA expression levels in wild type (<i>WT</i>) and <i>DJ-1</i> null (<i>DJ-1</i>^<i>-/-</i>) SN4741 cells under control (CON) or H₂O₂ (H₂O₂) treatment (n = 3). (B) <i>IDH2</i> mRNA expression level change was analyzed in <i>DJ-1</i> null SN4741 cells under control (CON) or H₂O₂ (H₂O₂) treatment, with scramble (-) or Keap1 siRNA (+) (n = 3). (C) Cell viability of wild type (<i>WT</i>), <i>DJ-1</i> null (<i>DJ-1</i>^<i>-/-</i>), IDH1-overexpressing <i>DJ-1</i> null (<i>IDH1 DJ-1</i>^<i>-/-</i>) and IDH2-overexpressing <i>DJ-1</i> null (<i>IDH2 DJ-1</i>^<i>-/-</i>) SN4741 cells under 1.5 mM H₂O₂ treatment for 6 h. Cell viability was measured by MTT assay. The inset shows immunoblot of FLAG-tagged IDH1 and IDH2. β–tubulin as a loading control. (D) Propidium iodide and annexin V FITC staining of H₂O₂-treated SN4741 cells. (E) Necrotic cell death rates (n = 3). (F) Flow cytometric analysis of CM-H₂DCFDA-stained SN4741 cells. Gray-filled area: <i>WT</i> or <i>DJ-1</i> null SN4741 cells without H₂O₂ treatment; Red line: H₂O_2-treated SN4741 cells; Green line: H₂O_2-treated IDH1-overexpressing SN4741 cells; Blue line: H₂O_2-treated IDH2-overexpressing SN4741 cells. (G) Fold change in the mean of fluorescence intensity (MFI) of CM-H₂DCFDA in flow cytometric analysis (n = 3). (H) Flow cytometric analysis of MitoSOX-stained SN4741 cells. Gray-filled area: <i>WT</i> or <i>DJ-1</i> null SN4741 cells without H₂O₂ treatment; Red line: H₂O₂-treated SN4741 cells; Green line: H₂O₂-treated IDH1-overexpressing SN4741 cells; Blue line: H₂O₂-treated IDH2-overexpressing SN4741 cells. (I) Fold change in the mean of fluorescence intensity (MFI) of MitoSOX in flow cytometric analysis (n = 3). (J) Cell viability of MitoTEMPO-treated SN4741 cells under H₂O₂ treatment. Cell viability was measured by MTT assays (n = 3). Data information: Significance was determined by one-way ANOVA with Sidak correction [**, P<0.01; ***, P<0.001; NS, not significant (P>0.05)]. Error bars indicate SD.</p

IDH mRNA expression is induced by DJ-1 through Nrf2 pathway.

<p>(A-C) Comparison of <i>IDHm1</i> (A), <i>IDHm2</i> (B), or <i>IDHc</i> (C) mRNA expression levels in the whole body of wild type (<i>WT</i>), <i>DJ-1β</i> null mutants (<i>DJ-1β</i>^<i>ex54</i>) and <i>DJ-1β</i> null mutants with a heterozygous <i>Keap1</i> mutation (<i>DJ-1β</i>^<i>ex54</i> <i>Keap1</i>^<i>EY5/+</i>) under control (CON) or rotenone treatment (Rotenone) (n≥3). (D) Survival curves of wild types (<i>WT</i>), <i>DJ-1β</i> null mutants (<i>DJ-1β</i>^<i>ex54</i>), <i>Keap1</i> heterozygous mutants (<i>Keap1</i>^<i>EY5/+</i>) and <i>DJ-1β</i> null mutants with a heterozygous <i>Keap1</i> mutation (<i>DJ-1β</i>^<i>ex54</i> <i>Keap1</i>^<i>EY5/+</i>) under rotenone treatment (log-rank test: <i>DJ-1β</i>^<i>ex54</i> VS <i>WT</i> & <i>DJ-1β</i>^<i>ex54</i> VS <i>DJ-1β</i>^<i>ex54</i> <i>Keap1</i>^<i>EY5/+</i>: P<0.001, <i>Keap1</i>^<i>EY5/+</i> VS <i>DJ-1β</i>^<i>ex54</i> <i>Keap1</i>^<i>EY5/+</i>: P = 0.9896, n = 120 for <i>WT</i> and <i>DJ-1β</i>^<i>ex54</i>; n = 150 for <i>Keap1</i>^<i>EY5/+</i>; n = 140 for <i>DJ-1β</i>^<i>ex54</i> <i>Keap1</i>^<i>EY5/+</i>) (E) Survival curves of adult flies under H₂O₂ treatment (log-rank test: <i>DJ-1β</i>^<i>ex54</i> VS <i>WT</i> & <i>DJ-1β</i>^<i>ex54</i> VS <i>DJ-1β</i>^<i>ex54</i> <i>Keap1</i>^<i>EY5/+</i>: P<0.001, <i>Keap1</i>^<i>EY5/+</i> VS <i>DJ-1β</i>^<i>ex54</i> <i>Keap1</i>^<i>EY5/+</i>: P = 0.1613, n = 109 for <i>WT</i>; n = 119 for <i>DJ-1β</i>^<i>ex54</i>; n = 100 for <i>Keap1</i>^<i>EY5/+</i>; n = 120 for <i>DJ-1β</i>^<i>ex54</i> <i>Keap1</i>^<i>EY5/+</i>). All life span assays were carried out at 25°C and were repeated at least twice. (F) Comparison of <i>IDHm1</i> mRNA expression levels in the whole body of <i>hs-GAL4</i> control flies (<i>hs</i>), CncC-overexpressing files (<i>hs>CncC</i>), CncC and Keap1-overexpressing flies (<i>hs>CncC Keap1</i>) and CncC, Keap1 and DJ-1β-overexpressing flies (<i>hs>CncC Keap1 DJ-1β</i>) (n≥3). (G) Comparison of <i>IDHc</i> mRNA expression levels in the whole body of flies (n≥3). (H-I) Confocal images (H) and graphs (I) of the average number of DA neurons within DL1 and DM clusters of the adult brains from 6-day-old <i>elav-GAL4</i> control flies (<i>elav</i>), <i>DJ-1β</i> null mutants (<i>elav DJ-1β</i>^<i>ex54</i>) and CncC-overexpressing <i>DJ-1β</i> null mutants (<i>elav>CncC DJ-1β</i>^<i>ex54</i>) after rotenone treatments. DA neurons were stained with anti-TH antibody (green). (n = 30 for each genotype). Scale bars: 20 μm. (J-K) Confocal images (J) and graphs (K) of the average number of DA neurons within DL1 and DM clusters of the adult brains of the 6-day-old flies after H₂O₂ treatments. DA neurons were stained with anti-TH antibody (green). (n = 27 for <i>elav</i>; n = 30 for other genotypes). Scale bars: 20 μm. (L) Comparison of luciferase activity in control (Con) or CncC-transfected (CncC) S2 cells (n = 3). The reporter plasmid with wild type (<i>WT</i>) or ARE site-mutated (Mut) <i>IDH</i> promoter was co-transfected to quantitatively measure activation of each promoter by CncC transcription factor. The construction of <i>IDH</i> reporters were described in Materials and Methods and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006975#pgen.1006975.s002" target="_blank">S2C Fig</a>. Data information: Significance was determined by one-way ANOVA with Sidak correction [*, P<0.05; **, P<0.01; ***, P<0.001; NS, not significant (P>0.05)]. Error bars indicate SD.</p

Schematic models of the protective effects of IDH on DA neurons against oxidative stress in <i>DJ-1</i> null background.

<p>Schematic models of the protective effects of IDH on DA neurons against oxidative stress in <i>DJ-1</i> null background.</p

List of differentially expressed genes in <i>DJ-1β</i> null flies on rotenone-containing media compared to wild type controls.

<p>List of differentially expressed genes in <i>DJ-1β</i> null flies on rotenone-containing media compared to wild type controls.</p