Mitochondrial Deoxyguanosine Kinase Regulates NAD+ Biogenesis Independent of Mitochondria Complex I Activity

Overexpression of DGUOK promotes mitochondria oxidative phosphorylation and lung adenocarcinoma progression. However, the role and mechanism of DGUOK in regulation of mitochondria function and lung cancer progression still poorly understood. Here we demonstrated that DGUOK regulated NAD+ biogenesis. Depletion of the DGUOK significantly decreased NAD+ level. Furthermore, knockout of the DGUOK considerably reduced expression of the NMNAT2, a key molecule controlling NAD+ synthesis, at both mRNA and protein levels. Ectopic expression of the NMNAT2 abrogated the effect of knockdown of DGUOK on NAD+. Notably, this regulation is independent of DGUOK -mediated mitochondria complex I activity. We also showed that NMNAT2 was highly expressed in lung adenocarcinoma and negatively correlated with the patient overall survival. Our study suggested that DGUOK regulates NAD+ in a NMNAT2 dependent manner and DGUOK-NMNAT2-NAD+ axis could be a potential therapeutic target in lung adenocarcinoma.

PO-225 The Effects on Mitochondria of Exercise Intervention PINK1 RNAi Drosophila Model of Parkinson's Disease Progression

Objective This research were using PINK1 RNAi drosophila model as object to investigate the effects of the 3 weeks duration exercise intervention drosophila model of Parkinson’s disease progression. Including mitochondria gather situation and mitochondria complex I activity and climbing activity and wing posture. Trying to prove that Exercise-training can help with the Parkinson disease’s symptom, and also to find out the mechanism of the result. Methods Two genotype were used in this research, the normal type VAS-mtGFPo Mhc/Mhc and the Parkinson type VAS-mtGFP/Cyo Mhc-RNAi/TM2 and all with fluorescent labeling mitochondria by GFP. The exercise-training last for 3 weeks, 5 times per week, we separated the Parkinson type in 4 groups by different training time: 0min per day, 30min per day, 60min per day and 90min per day. The healthy type was control group. We measured the climbing ability and the wings posture every week, also used confocal microscope to watch the aggregation of mitochondria of the chest muscle every week. After 3 weeks training, we grinded all the drosophila to get the mitochondria, and measured the activity of mitochondria complex I. At last, we compared every index of each group with T-text by using SPSS 17.0 and the significance level as the criterion. Results 1）The climbing ability: No significant difference found in first week, there was a significant difference between 0min group and 30min group in second week and there was a very significant difference between 0min group and 30min group in third week. （P＜0.01）.2) Wing posture: There was a very significant difference between 0min group and 30min group in third week. （P＜0.01）. 3) Activity of mitochondria complex I: Because of the small sample of this experiment, we can’t get a appropriate concentration of mitochondria, but we still got some changes between 30min and 0min. 4) The pictures of mitochondria by confocal microscope: The 30min group has the least aggregations of the mitochondria but a lot aggregations were found in other pictures of other group. Conclusions 1)The suitable Exercise-training can improve the climbing ability and the account of wing posture and extend the life span of drosophila model of Parkinson’s disease. 2) The suitable Exercise-training can relieve the situation of mitochondrial gathering and improve the activity of mitochondrial complex I.

Downregulation of DJ-1 Fails to Protect Mitochondrial Complex I Subunit NDUFS3 in the Testes and Contributes to the Asthenozoospermia

Asthenozoospermia (AS), an important cause of male infertility, is characterized by reduced sperm motility. Among the aetiologies of AS, inflammation seems to be the main cause. DJ-1, a conserved protein product of thePARK7gene, is associated with male infertility and plays a role in oxidative stress and inflammation. Although our previous studies showed that a reduction in DJ-1 was accompanied by mitochondrial dysfunction in the sperm of patients with AS, the specific mechanism underlying this association remained unclear. In this study, we found that compared to the patients without AS, the expression of mitochondrial protein nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) Fe-S protein 3 (NDUFS3) was also significantly decreased in the sperm of patients with AS. Similarly, decreased expression of DJ-1 and NDUFS3 and reduced mitochondria complex I activity were evident in a rat model of AS. Moreover, we showed that the interaction between DJ-1 and NDUFS3 in rat testes was weakened by ORN treatment. These results suggest that the impaired mitochondrial activity could be due to the broken interaction between DJ-1 and NDUFS3 and that downregulation of DJ-1 in sperm and testes contributes to AS pathogenesis.

Neuroprotective Effect of Ginkgolide B on Bupivacaine-Induced Apoptosis in SH-SY5Y Cells

Local anesthetics are used routinely and effectively. However, many are also known to activate neurotoxic pathways. We tested the neuroprotective efficacy of ginkgolide B (GB), an active component of Ginkgo biloba, against ROS-mediated neurotoxicity caused by the local anesthetic bupivacaine. SH-SY5Y cells were treated with different concentrations of bupivacaine alone or following preincubation with GB. Pretreatment with GB increased SH-SY5Y cell viability and attenuated intracellular ROS accumulation, apoptosis, mitochondrial dysfunction, and ER stress. GB suppressed bupivacaine-induced mitochondrial depolarization and mitochondria complex I and III inhibition and increased cleaved caspase-3 and Htra2 expression, which was strongly indicative of activation of mitochondria-dependent apoptosis with concomitantly enhanced expressions of Grp78, caspase-12 mRNA, protein, and ER stress. GB also improved ultrastructural changes indicative of mitochondrial and ER damage induced by bupivacaine. These results implicate bupivacaine-induced ROS-dependent mitochondria, ER dysfunction, and apoptosis, which can be attenuated by GB through its antioxidant property.

The membrane domain of complex I is not assembled in the stopper mutant E35 of Neurospora

The assembly of mitochondrial NADH : ubiquinone oxidoreductase (complex I) was studied in the E35 stopper mutant of Neurospora crassa at different times during growth in liquid media. Assembly of complex I as well as of its membrane domain is impaired in this strain throughout the growth period. Nevertheless, a structure that resembles the peripheral arm of the enzyme is still formed in the mitochondria of this mutant. The absence of the membrane domain of complex I in E35 can be attributed to the specific deletion of the mitochondrial ND2 and ND3 subunits of the enzyme.Key words: mitochondria, complex I, stopper mutants, Neurospora crassa.