The Nitric Oxide-Cyclic GMP Pathway Regulates FoxO and Alters Dopaminergic Neuron Survival in Drosophila

Activation of the forkhead box transcription factor FoxO is suggested to be involved in dopaminergic (DA) neurodegeneration in a Drosophila model of Parkinson's disease (PD), in which a PD gene product LRRK2 activates FoxO through phosphorylation. In the current study that combines Drosophila genetics and biochemical analysis, we show that cyclic guanosine monophosphate (cGMP)-dependent kinase II (cGKII) also phosphorylates FoxO at the same residue as LRRK2, and Drosophila orthologues of cGKII and LRRK2, DG2/For and dLRRK, respectively, enhance the neurotoxic activity of FoxO in an additive manner. Biochemical assays using mammalian cGKII and FoxO1 reveal that cGKII enhances the transcriptional activity of FoxO1 through phosphorylation of the FoxO1 S319 site in the same manner as LRRK2. A Drosophila FoxO mutant resistant to phosphorylation by DG2 and dLRRK (dFoxO S259A corresponding to human FoxO1 S319A) suppressed the neurotoxicity and improved motor dysfunction caused by co-expression of FoxO and DG2. Nitric oxide synthase (NOS) and soluble guanylyl cyclase (sGC) also increased FoxO's activity, whereas the administration of a NOS inhibitor L-NAME suppressed the loss of DA neurons in aged flies co-expressing FoxO and DG2. These results strongly suggest that the NO-FoxO axis contributes to DA neurodegeneration in LRRK2-linked PD

The Loss of PGAM5 Suppresses the Mitochondrial Degeneration Caused by Inactivation of PINK1 in Drosophila

PTEN-induced kinase 1 (PINK1), which is required for mitochondrial homeostasis, is a gene product responsible for early-onset Parkinson's disease (PD). Another early onset PD gene product, Parkin, has been suggested to function downstream of the PINK1 signalling pathway based on genetic studies in Drosophila. PINK1 is a serine/threonine kinase with a predicted mitochondrial target sequence and a probable transmembrane domain at the N-terminus, while Parkin is a RING-finger protein with ubiquitin-ligase (E3) activity. However, how PINK1 and Parkin regulate mitochondrial activity is largely unknown. To explore the molecular mechanism underlying the interaction between PINK1 and Parkin, we biochemically purified PINK1-binding proteins from human cultured cells and screened the genes encoding these binding proteins using Drosophila PINK1 (dPINK1) models to isolate a molecule(s) involved in the PINK1 pathology. Here we report that a PINK1-binding mitochondrial protein, PGAM5, modulates the PINK1 pathway. Loss of Drosophila PGAM5 (dPGAM5) can suppress the muscle degeneration, motor defects, and shorter lifespan that result from dPINK1 inactivation and that can be attributed to mitochondrial degeneration. However, dPGAM5 inactivation fails to modulate the phenotypes of parkin mutant flies. Conversely, ectopic expression of dPGAM5 exacerbated the dPINK1 and Drosophila parkin (dParkin) phenotypes. These results suggest that PGAM5 negatively regulates the PINK1 pathway related to maintenance of the mitochondria and, furthermore, that PGAM5 acts between PINK1 and Parkin, or functions independently of Parkin downstream of PINK1

蚊刺症患児の臨床的背景とtranilastの効果

一般小児科外来診療において,蚊刺により腫れて困っている患児がいることを多くの小児科医が経験しているが,これらの症状を呈する児の定義や治療は確立されていない.蚊によると考えられる虫刺症である蚊刺症の診断を考える上で,対象として保育園児童の蚊刺後の反応を比較検討し,蚊刺症と判断する基準について調査した.更に,蚊刺症と判断した症例に対するtranilast投与の効果についての評価を行った.当科および戸田中央総合病院小児アレルギー外来で蚊刺後の症状を主訴に蚊刺症として治療した児童35名を蚊刺症例群とし,荒川区内の2ヵ所の保育園児童128名を対照群とした.両群に蚊刺後の反応について,保護者に対しアンケート調査を行い比較検討した.また,蚊刺症例群にはtranilast投与の効果を5点満点とする評価の調査も併せて行った.蚊刺後の最も大きく腫れた直径は,対照群では中央値1.5cmに対し,蚊刺症例群では中央値3cmと蚊刺症例群の反応が大きい傾向であった.蚊刺後の腫脹期間は,対照群では中央値3日に対し,蚊刺症例群では中央値7日と長くなっていた.従って,蚊刺症例群の特徴としては,最も大きく腫れた直径が3cm以上で腫脹期間が7日以上であると考えられる.tranilast投与における蚊刺後の変化は,最も大きく腫れた直径の変化では,投与後は中央値0.5cmと減少し,腫脹期間の変化でも投与後は中央値2日と短縮した.tranilast投与の有効性の調査でも5点満点中4.25点と高い評価を認めた.このことから,蚊刺症に対するtranilast投与の効果が期待される.Many pediatricians often encounter children suffering from extreme swelling following mosquito bites. Our study attempted to make the definition of the mosquito-bite-induced condition. For the diagnosis of mosquito bites, 35 children served as the subjects of this study. They visited our department or the Pediatric Allergy Clinic of Toda Chuo General Hospital with major complaints of symptoms that ensued after being bitten by mosquitoes. Nursery school children who were bitten by mosquitoes served as controls and their responses were comparatively examined. The median maximum diameter of swelling after the mosquito bites is 1.5 cm and the swelling lasts about 3 days in the control group. In case of mosquito-bite-induced condition, the swelling after the mosquito bites exceeds 3 cm or more and lasts over 7 days. The effect of tranilast administration was evaluated on those cases judged to have been suffering from the mosquito-bite-induced conditions. After medication, the median maximum diameter and duration of swelling after mosquito bite was reduced. It was concluded from the results of our study that the reaction of the mosquito-bite-induced condition tended to be greater than the control group. Tranilast administration will be effective against the mosquito-bite-induced condition

蚊刺症患児の臨床的背景とtranilastの効果

一般小児科外来診療において,蚊刺により腫れて困っている患児がいることを多くの小児科医が経験しているが,これらの症状を呈する児の定義や治療は確立されていない.蚊によると考えられる虫刺症である蚊刺症の診断を考える上で,対象として保育園児童の蚊刺後の反応を比較検討し,蚊刺症と判断する基準について調査した.更に,蚊刺症と判断した症例に対するtranilast投与の効果についての評価を行った.当科および戸田中央総合病院小児アレルギー外来で蚊刺後の症状を主訴に蚊刺症として治療した児童35名を蚊刺症例群とし,荒川区内の2ヵ所の保育園児童128名を対照群とした.両群に蚊刺後の反応について,保護者に対しアンケート調査を行い比較検討した.また,蚊刺症例群にはtranilast投与の効果を5点満点とする評価の調査も併せて行った.蚊刺後の最も大きく腫れた直径は,対照群では中央値1.5cmに対し,蚊刺症例群では中央値3cmと蚊刺症例群の反応が大きい傾向であった.蚊刺後の腫脹期間は,対照群では中央値3日に対し,蚊刺症例群では中央値7日と長くなっていた.従って,蚊刺症例群の特徴としては,最も大きく腫れた直径が3cm以上で腫脹期間が7日以上であると考えられる.tranilast投与における蚊刺後の変化は,最も大きく腫れた直径の変化では,投与後は中央値0.5cmと減少し,腫脹期間の変化でも投与後は中央値2日と短縮した.tranilast投与の有効性の調査でも5点満点中4.25点と高い評価を認めた.このことから,蚊刺症に対するtranilast投与の効果が期待される.Many pediatricians often encounter children suffering from extreme swelling following mosquito bites. Our study attempted to make the definition of the mosquito-bite-induced condition. For the diagnosis of mosquito bites, 35 children served as the subjects of this study. They visited our department or the Pediatric Allergy Clinic of Toda Chuo General Hospital with major complaints of symptoms that ensued after being bitten by mosquitoes. Nursery school children who were bitten by mosquitoes served as controls and their responses were comparatively examined. The median maximum diameter of swelling after the mosquito bites is 1.5 cm and the swelling lasts about 3 days in the control group. In case of mosquito-bite-induced condition, the swelling after the mosquito bites exceeds 3 cm or more and lasts over 7 days. The effect of tranilast administration was evaluated on those cases judged to have been suffering from the mosquito-bite-induced conditions. After medication, the median maximum diameter and duration of swelling after mosquito bite was reduced. It was concluded from the results of our study that the reaction of the mosquito-bite-induced condition tended to be greater than the control group. Tranilast administration will be effective against the mosquito-bite-induced condition

Oncologic Outcomes of Laparoscopic Radical Hysterectomy Using the No-Look No-Touch Technique for Early Stage Cervical Cancer: A Propensity Score-Adjusted Analysis

We evaluated oncologic outcomes of laparoscopic radical hysterectomy using the no-look no-touch technique (NLNT). We analyzed patients with early stage (IA2, IB1, and IIA1, FIGO2008) cervical cancer treated between December 2014 and December 2019. The primary endpoint was disease-free survival (DFS). We compared the outcomes of the abdominal radical hysterectomy (ARH) and NLNT groups using a Cox model with inverse probability of treatment weighting (IPTW), according to propensity scores. We also evaluated NLNT’s non-inferiority to ARH using an evaluation of heterogeneity between the results of the Laparoscopic Approach to Cervical Cancer (LACC) trial and our study. ARH and NLNT were performed in 118 and 113 patients, respectively. The median follow-up duration was 3.2 years. After IPTW adjustment, the 3-year DFS rates (NLNT 92.4%; ARH 94.0%) and overall survival rates did not differ significantly between the groups. Furthermore, the 3-year DFS rates for patients with tumor sizes ≥ 2 cm in the NLNT (85.0%) and ARH (90.3%) groups did not differ significantly. No significant heterogeneity was observed between the LACC trial and our study (I2 = 60.5%, p = 0.111), although there was a trend toward a lower hazard ratio in our study. Laparoscopic radical hysterectomy using NLNT provides a favorable prognosis for early stage cervical cancer

NO signal is involved in FoxO-mediated neurodegeneration.

<p>(<b>A–D</b>) SEM images of the eyes of flies expressing the indicated genes. The genotypes are: <i>GMR-Gal4/UAS-dNOS</i> (<b>A</b>), <i>GMR-Gal4, UAS-dFoxO/UAS-dNOS</i> (<b>B</b>), <i>GMR-Gal4, UAS-dFoxO; UAS-sGCα99B^RNAi</i> (<b>C</b>), <i>GMR-Gal4, UAS-dFoxO; UAS-sGCβ100B^RNAi</i> (<b>D</b>), <i>GMR-Gal4, UAS-dFoxO/UAS-dNOS; UAS-sGCα99B^RNAi</i> (<b>E</b>), <i>GMR-Gal4, UAS-dFoxO/UAS-dNOS, DG2^k04703</i> (<b>F</b>).</p

cGKII stimulates FoxO-transcriptional activity.

<p>(<b>A, B</b>) cGKII and LRRK2 additively stimulate FoxO-transcriptional activity. FoxO-transcriptional activity was measured in extracts prepared from 293T cells transfected with the indicated plasmids and a plasmid for FoxO1, a FoxO reporter plasmid containing <i>Firefly</i> luciferase, and a plasmid for <i>Renilla</i> luciferase to monitor the transfection efficiency. The relative FoxO-transcriptional activity (<i>Firefly</i> luciferase activity) normalized to <i>Renilla</i> luciferase activity is presented. Data are presented as the mean ± SE for three independent experiments. β-galactosidase (Mock) served as a transfection control. (<b>C</b>) Introduction of the S319A (SA) mutation in FoxO1 reduced FoxO activity. Data are presented as the mean ± SE for three independent experiments. *, <i>p</i><0.05; **, <i>p</i><0.01. Co-transfection of kinase-dead forms of cGKII and LRRK2 also sitmulated FoxO (#, <i>p</i><0.05 <i>vs.</i> Control in <b>B</b>).</p

DG2 modulates FoxO <i>in vivo</i>.

<p>(<b>A</b>) dFoxO and the indicated transgenes were expressed in the <i>Drosophila</i> eyes using the <i>GMR-GAL4</i> driver. Extracts from brain tissues were subjected to western blot analysis. dFoxO-P; a phosphorylated form of dFoxO. (<b>B</b>) DG2 RNAi or GFP RNAi constructs were expressed in the <i>Drosophila</i> brain using the <i>elav-GAL4</i> driver. Western blot analysis for endogenous dFoxO was carried out as in (<b>A</b>). (<b>C</b>) <i>Drosophila</i> S2 cells were transfected with or without C-terminally Myc-tagged DG2 (DG2-Myc). Thirty-six hrs post transfection, cells were treated with or without 10 µM 8-Br-cGMP for 30 min. Cell lysate were then subjected to western blot analysis. (<b>D</b>) Human 293T cells were transfected with or without cGKII-Myc, and were treated with 8-Br-cGMP as in (<b>C</b>). Phosphorylation of the S319 site in endogenous FoxO1 was detected with phospho-specific antibody. (<b>E, F</b>) S2 cells expressing DG2-Myc (<b>E</b>) or DG1-Myc (<b>F</b>) were visualized with anti-Myc antibody (green), by counterstaining with DAPI (blue color). (<b>G–J</b>) HeLa cells expressing AKT-PH-GFP (green) along with cGKII-Myc (<b>G, H</b>) or cGKI-Myc (<b>I, J</b>) were visualized with anti-Myc antibody (red), by counterstaining with DAPI (blue color). AKT-PH-GFP was used for a marker protein of the plasma membrane <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030958#pone.0030958-Kwon2" target="_blank">[62]</a>. (<b>K</b>–<b>M</b>) Flp-In T-REx-293 cells harboring EGFP-FoxO1 gene were transiently transfected with cGKII-Myc, and EGFP-FoxO1 was induced with doxycycline. Enlarged views of the plasma membrane regions in cGKII-positive (Box1) and negative (Box2) cells are also shown in (<b>M</b>). Accumulation of FoxO1 along with cGKII in the plasma membrane is indicated by arrowheads. Scale bars = 5 µm for (<b>E, F</b>), 25 µm for (<b>G–J</b>) and 10 µm for (<b>K–M</b>).</p

Parkinson's disease-associated kinase PINK1 regulates Miro protein level and axonal transport of mitochondria.

Mutations in Pten-induced kinase 1 (PINK1) are linked to early-onset familial Parkinson's disease (FPD). PINK1 has previously been implicated in mitochondrial fission/fusion dynamics, quality control, and electron transport chain function. However, it is not clear how these processes are interconnected and whether they are sufficient to explain all aspects of PINK1 pathogenesis. Here we show that PINK1 also controls mitochondrial motility. In Drosophila, downregulation of dMiro or other components of the mitochondrial transport machinery rescued dPINK1 mutant phenotypes in the muscle and dopaminergic (DA) neurons, whereas dMiro overexpression alone caused DA neuron loss. dMiro protein level was increased in dPINK1 mutant but decreased in dPINK1 or dParkin overexpression conditions. In Drosophila larval motor neurons, overexpression of dPINK1 inhibited axonal mitochondria transport in both anterograde and retrograde directions, whereas dPINK1 knockdown promoted anterograde transport. In HeLa cells, overexpressed hPINK1 worked together with hParkin, another FPD gene, to regulate the ubiquitination and degradation of hMiro1 and hMiro2, apparently in a Ser-156 phosphorylation-independent manner. Also in HeLa cells, loss of hMiro promoted the perinuclear clustering of mitochondria and facilitated autophagy of damaged mitochondria, effects previously associated with activation of the PINK1/Parkin pathway. These newly identified functions of PINK1/Parkin and Miro in mitochondrial transport and mitophagy contribute to our understanding of the complex interplays in mitochondrial quality control that are critically involved in PD pathogenesis, and they may explain the peripheral neuropathy symptoms seen in some PD patients carrying particular PINK1 or Parkin mutations. Moreover, the different effects of loss of PINK1 function on Miro protein level in Drosophila and mouse cells may offer one explanation of the distinct phenotypic manifestations of PINK1 mutants in these two species