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Esophageal squamous cell carcinoma (ESCC) is an aggressive cancer with late-stage detection and poor prognosis. This emphasizes the need to identify new markers for early diagnosis and treatment. Altered mitochondrial genome (mtDNA) content in primary tumors correlates with poor patient prognosis. Here we used three-dimensional (3D) organoids of esophageal epithelial cells (EECs) from the MPV17-/- mouse model of mtDNA depletion to investigate the contribution of reduced mtDNA content in ESCC oncogenicity. To test if mtDNA defects are a contributing factor in ESCC, we used oncogenic stimuli such as ESCC carcinogen 4-nitroquinoline oxide (4-NQO) treatment, or expressing p53R175H oncogenic driver mutation. We observed that EECs and 3D-organoids with mtDNA depletion had cellular, morphological and genetic alterations typical of an oncogenic transition. Furthermore, mitochondrial dysfunction induced cellular transformation is accompanied by elevated mitochondrial fission protein, DRP1 and pharmacologic inhibition of mitochondrial fission by mDivi-1 in the MPV17-/- organoids reversed the phenotype to that of normal EEC organoids. Our studies show that mtDNA copy number depletion, activates a mitochondrial retrograde response, potentiates telomere defects, and increases the oncogenic susceptibility towards ESCC. Furthermore, mtDNA depletion driven cellular plasticity is mediated via altered mitochondrial fission-fusion dynamics.

Mitochondria are dynamic organelles frequently undergoing fission and fusion events to maintain their integrity, bioenergetics and spatial distribution, which is fundamental to the processes of cell survival. Disruption in mitochondrial dynamics plays a role in cancer. Therefore, proteins involved in regulating mitochondrial dynamics are potential targets for treatment. mDIVI1 is an inhibitor of the mitochondrial fission protein DRP1, which induces i) mitochondrial oxidative stress and ii) effectively reduces mitochondrial metabolism. We show here that mDIVI1 is able to inhibit 3D tumorsphere forming capacity, cell migration and stemness-related signalling in breast cancer cells, indicating that mDIVI1 can potentially be used for the therapeutic elimination of cancer stem cells (CSCs).

// Jia Wang 1 , Yuchen Xie 2 , Xiaobin Bai 1 , Ning Wang 1 , Hai Yu 1, 2 , Zhong Deng 1, 2 , Minxue Lian 1 , Shuo Yu 2 , Hao Liu 1 , Wanfu Xie 1 and Maode Wang 1 1 Department of Neurosurgery, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi 710061, China 2 School of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, China Correspondence to: Wanfu Xie, email: wanfu67@aliyun.com Maode Wang, email: maodewang@163.com Keywords: glioma stem-like cells; glioblastoma; TTK; MTFR2 Received: June 15, 2017 Accepted: November 21, 2017 Published: December 11, 2017 ABSTRACT Accumulating evidence has proved that glioma stem-like cells (GSCs) are responsible for tumorigenesis, treatment resistance, and subsequent tumor recurrence in glioblastoma (GBM). In this study, we identified dual specificity protein kinase TTK (TTK) as the most up-regulated and differentially expressed kinase encoding genes in GSCs. Functionally, TTK was essential for in vitro clonogenicity and in vivo tumor propagation in GSCs. Clinically, TTK expression was highly enriched in GBM, moreover, was inversely correlated with a poor prognosis in GBM patients. Mechanistically, mitochondrial fission regulator 2 (MTFR2) was identified as one of the most correlated genes to TTK and transcriptionally regulated TTK expression via activation of TTK promoter. Collectively, MTFR2-dependent regulation of TTK plays a key role in maintaining GSCs in GBM and is a potential novel druggable target for GBM.

It has been reported that isosteviol, a widely known sweeteners, can protect against myocardial ischemia-reperfusion (IR) injury in isolated guinea pig heart. Here, we aim to confirm the cardioprotective effect of its sodium salt, isosteviol sodium (STVNa), against IR injury and its potential molecular mechanism in H9c2 cardiomyocytes. STVNa significantly improved cell viability, restored mitochondrial membrane potential, decreased cellular reactive oxygen species generation, and inhibited cell apoptosis. Furthermore, STVNa treatment changed the morphology of mitochondria from fragmented, discontinuous forms to normal elongated, tubular forms. Cyto-immunofluorescence and western blot analysis revealed that STVNa inhibited mitochondrial fission proteins dynamin-related protein 1 (Drp1), and mitochondrial fission 1 (Fis1), thus plays a key role in cardioprotection. These findings, for the first time, suggest that STVNa can protect against myocardial IR injury through reverse mitochondrial fission.

// Wei Xu 1, 2, * , Hongwei Yu 3, * , Ruixue Ma 1 , Lina Ma 1 , Qiushuang Liu 1 , Huitong Shan 1 , Chengyu Wu 1 , Rong Zhang 1 , Yuhong Zhou 1 and Hongli Shan 1 1 Department of Pharmacology (The State-Province Key Laboratories of Biomedicine Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin Medical University, Harbin 150081, China 2 Department of Cardiology, The Second Affiliated Hospital of Harbin Medical University, Harbin 150081, China 3 Department of Histoembryology, Harbin Medical University, Harbin 150081, China * These authors have contributed equally to this work Correspondence to: Hongli Shan, email: shanhongli@ems.hrbmu.edu.cn Yuhong Zhou, email: zhoulinder@163.com Keywords: apelin, cardiomyocyte apoptosis, mitochondrial fission, hypoxia, myocardial infarction Received: July 19, 2017 Accepted: September 21, 2017 Published: October 10, 2017 ABSTRACT It is known that dynamin-related protein 1 (Drp1)-mediated mitochondrial fission plays an important role in ischemic injury of myocardial infarction (MI). Apelin, an endogenous ligand for Apelin receptor, acts as a key modulator of cardiovascular diseases. Here, we examined the effects of Apelin on MI injury and underlying mechanisms. Adult male C57BL/6J mice were treated with Apelin for 4 weeks and then subjected coronary artery ligation (LAD) to induce MI and the protective effects of Apelin on MI injury were evaluated at 6 h post LAD. Mitochondrial fission was significantly increased in MI as evidenced by enhanced expression of phosphorylated Drp1 (p-Drp1 ser 616 ) without affecting total Drp-1 level and degenerative transformation of mitochondria into short rods as typical fission. Apelin markedly inhibited p-Drp1 ser 616 and preserved mitochondrial morphology in MI. Similar effects of Apelin were consistently observed in primary cultured cardiomyocytes under hypoxia. Apelin decreased hypoxia-induced cardiomyocyte apoptosis as evidenced by decreased TUNEL-positive cells and preserved mitochondrial membrane potential (MMP). Apelin decreased Bax/Bcl-2 ratio and limited the release of cytochrome C and activation of caspase-9 and caspase-3 both in vivo and in vitro . Finally, Apelin diminished the infarct size and normalized the impaired cardiac function as indicated by rescuing of the decreased ejection faction and fractional shortening in MI mice. In conclusion, Apelin prevented mitochondrial fission by inhibiting p-Drp1 Ser616 , which prevents loss of MMP and inhibits the mitochondria-mediated apoptosis. These results indicate that the inhibition of Drp-1 activation by Apelin is a novel mechanism of cardioprotection against MI injury.

MicroRNA-21 acts as an oncogene by promoting cell proliferation and migration, whereas inhibiting apoptosis in majority of cancers. MicroRNA-21 is upregulated in human keloid fibroblasts. We hypothesized that microRNA-21 may contribute to pathogenesis of keloid fibroblasts. First, enhanced miR-21 but reduced mitochondrial-mediated apoptosis observed in keloid tissues indicated its importance in keloids development. Second, upregulation of microRNA-21 induced a decrease in the ratio of BAX to BCL-2 and suppressed mitochondrial fission in keloid fibroblasts. Third, by attenuating the decline in cellular mitochondrial membrane potential, overexpression of miR-21 suppressed cytochrome c release to the cytoplasm, followed by a decrease in the activity of intracellular caspase-9 and caspase-3, suggesting that mitochondrial-mediated proapoptotic pathway was impaired. Simultaneously, intracellular reactive oxygen species were decreased, indicating microRNA-21 undermined oxidative stress. This phenotype was reversed by miR-21 inhibition. Therefore, our study demonstrates that inhibition of microRNA-21 induces mitochondrial-mediated apoptosis in keloid fibroblasts, proposing microRNA-21 as a potential therapeutic target in keloid fibroblasts.

// Zhuyun Chen 1, * , Xiaofei An 2, * , Xi Liu 1, * , Jia Qi 3 , Dafa Ding 4 , Min Zhao 5 , Suyan Duan 1 , Zhimin Huang 1 , Chengning Zhang 1 , Lin Wu 1 , Bo Zhang 1 , Aihua Zhang 5 , Yanggang Yuan 1 and Changying Xing 1 1 Department of Nephrology, The First Affiliated Hospital of Nanjing Medical University, Nanjing Medical University, Nanjing, China 2 Department of Endocrinology, Jiangsu Province Hospital of Chinese Medicine, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, China 3 Department of Pharmacy, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, China 4 Department of Endocrinology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing Medical University, Nanjing, China 5 Department of Nephrology, Nanjing Children’s Hospital, Nanjing Medical University, Nanjing, China * These authors have contributed equally to this work Correspondence to: Yanggang Yuan, email: ygyuan@njmu.edu.cn Changying Xing, email: cyxing62@126.com Keywords: hyperoside, adriamycin, mitochondrial fission, podocyte injury Received: July 12, 2017 Accepted: August 27, 2017 Published: September 28, 2017 ABSTRACT Podocyte injury underlies many forms of glomerular diseases. Our previous study showed that hyperoside, a naturally occurring flavonoid, could decrease albuminuria at the early stage of diabetic nephropathy by ameliorating renal damage and podocyte injury. However, its protective mechanism against podocyte injury is unknown. A previous study demonstrated that hyperoside might inhibit amyloid β-protein-induced neurotoxicity by suppressing mitochondrial dysfunction. Both mitochondrial dysfunction and its upstream determinant mitochondrial fission were closely related to podocyte injury. Thus, in the current study, we tested the effect of hyperoside on mitochondrial dysfunction and mitochondrial fission in adriamycin (ADR)-induced podocyte injury. In the mice model of ADR-induced nephropathy, hyperoside treatment inhibited ADR-induced albuminuria and podocyte injury. Meanwhile, hyperoside also blocked ADR-induced mitochondrial dysfunction and mitochondrial fission. Consistently, in cultured human podocytes, hyperoside suppressed ADR-induced podocyte injury, mitochondrial dysfunction and mitochondrial fission. All these results indicated that hyperoside might inhibit ADR-induced mitochondrial dysfunction and podocyte injury through suppressing mitochondrial fission both in vivo and in vitro . The underlying mechanisms which we revealed support the therapeutic effects of hyperoside for a broad range of glomerular diseases.

// Hideaki Tsuyoshi 1, 3, 4 , Vincent Kam Wai Wong 2 , Yu Han 2 , Makoto Orisaka 4 , Yoshio Yoshida 4 and Benjamin K. Tsang 1, 2, 3 1 Department of Obstetrics & Gynecology, Cellular & Molecular Medicine, Interdisciplinary School of Health Sciences, University of Ottawa, Ottawa, Ontario, Canada 2 State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Avenida Wai Long, Taipa, Macau, China 3 Chronic Disease Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada 4 Department of Obstetrics & Gynecology, University of Fukui, Fukui, Japan Correspondence to: Benjamin K. Tsang, email: btsang@ohri.ca Yoshio Yoshida, email: yyoshida@u-fukui.ac.jp Keywords: ovarian cancer, chemoresistance, mitochondrial dynamics, G2/M arrest, Ssd Received: March 20, 2017 Accepted: July 19, 2017 Published: September 16, 2017 ABSTRACT Cisplatin (CDDP) and its derivatives are first line anti-cancer drugs for ovarian cancer (OVCA). However, chemoresistance due to high incidence of p53 mutations leads to poor clinical prognosis. Saikosaponin-d (Ssd), a saponin from a herbal plant extract, has been shown to induce cell death and sensitize chemoresistant cells to chemotherapeutic agents. Here, we demonstrated that Ssd sensitized chemoresistant OVCA cells with either p53-wt, -mutant and -null to CDDP. The action of Ssd appears to be through induction of mitochondrial fragmentation and G2/M arrest. Ssd is mediated via calcium signaling, up-regulation of the mitochondrial fission proteins Dynamin-related protein 1 (Drp1) and optic atrophy 1 (Opa1), and loss in mitochondrial membrane potential (MMP). Moreover, in the presence of CDDP, Ssd also down-regulates protein phosphatase magnesium-dependent 1 D (PPM1D) and increases the phosphorylation of checkpoint protein kinases (Chk) 1, cell division cycle 25c (Cdc25c) and Cyclin dependent kinase 1 (Cdk1). Our findings suggest that Ssd could sensitize OVCA to CDDP independent of the p53 status through multiple signaling pathways. They support the notion that Ssd may be a novel adjuvant for the treatment of chemoresistant OVCA.

// Zhuo Yang 1 , ZiYi Feng 2 , JiaHui Gu 1 , XinHui Li 1 , QianZhe Dong 3 , KuiRan Liu 1 , Yan Li 1 and Ling OuYang 1 1 Department of Obstetrics and Gynecology, Shengjing Hospital Affiliated to China Medical University, Heping District, Shenyang, Liaoning, People’s Republic of China 2 China Medical University, Shenbei New District, Shenyang, Liaoning, People’s Republic of China 3 Department of Pathology, China Medical University, Shenbei New District, Shenyang, Liaoning, People’s Republic of China Correspondence to: Zhuo Yang, email: yangz@sj-hospital.org Keywords: ovarian cancer, miR-488, Six1, mitochondrial dynamics, Drp1 Abbreviations: CCK8: Cell Counting Kit-8; ROS: reactive oxygen species Received: April 18, 2017 Accepted: July 12, 2017 Published: September 15, 2017 ABSTRACT Dysregulation of miR-488 has been implicated in several human cancers. In this study, we aim to explore its expression and biological function in ovarian cancers. We found miR-488 expression was downregulated in ovarian cancer tissues. Using CCK8 and colony formation assay showed that miR-488 inhibited SKOV3 cell proliferation and colony formation, with downregulation of cyclin D1 and cyclin E protein. While miR-488 inhibitor promoted OVCAR3 cell growth and colony formation. Cell viability and Annexin V/PI staining showed that miR-488 downregulated cell survival and increased apoptosis rate when treated with cisplatin and paclitaxel. Further experiments using MitoTracker and JC-1 staining indicated that miR-488 regulated mitochondrial fission/fusion balance and inhibited mitochondrial membrane potential, with p-Drp1, Drp1 and Fis1 downregulation. Luciferase reporter assay showed that Six1 is a target of miR-488. We also found a negative association between Six1 and miR-488 in ovarian cancer tissues. In addition, Six1 overexpression induced mitochondrial fission and increased mitochondrial potential, with upregulation of Drp1 signaling. Six1 depletion showed the opposite effects. Restoration of Six1 in SKOV3 cells rescued decreased p-Drp1 and Drp1 expression induced by miR-488 mimic. Six1 plasmid also reversed the effects of miR-488 on chemoresistance and apoptosis. Taken together, the present study showed that, by targeting Six1, miR-488 inhibits chemoresistance of ovarian cancer cells through regulation of mitochondrial function.

// Chiao-Yin Sun 1, 2, 3 , Mei-Ling Cheng 3, 4, 5, 6 , Heng-Chih Pan 1, 2 , Jia-Hung Lee 1, 7 and Chin-Chan Lee 1, 3 1 Department of Nephrology, Chang Gung Memorial Hospital, Keelung, Taiwan 2 Kidney Research Center, Chang Gung Memorial Hospital, Taoyuan, Taiwan 3 School of Medicine, Chang Gung University, Taoyuan, Taiwan 4 Metabolomics Core Laboratory, Healthy Aging Research Center, Chang Gung University, Taoyuan, Taiwan 5 Clinical Phenome Center, Chang Gung Memorial Hospital, Taoyuan, Taiwan 6 Department of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan 7 Medical Research Center, Chang Gung Memorial Hospital, Keelung, Taiwan Correspondence to: Chiao-Yin Sun, email: fish3970@gmail.com Keywords: uremic toxins, metabolic stress, mitochondrial fusion, mitochondrial mass, mitophagy Received: May 02, 2017 Accepted: July 29, 2017 Published: September 08, 2017 ABSTRACT Protein-bound uremic toxins, indoxyl sulfate and p -cresol sulfate, increase oxidative stress and adversely affect chronic kidney disease progression and cardiovascular complications. In this study, we examined whether mitochondria are the target of indoxyl sulfate and p -cresol sulfate intoxication in vivo and in vitro . The kidneys of 10-week-old male B-6 mice with ½-nephrectomy treated with indoxyl sulfate and p -cresol sulfate were used for the animal study. Cultured human renal tubular cells were used for the in vitro study. Our results indicated that indoxyl sulfate and p -cresol sulfate impaired aerobic and anaerobic metabolism in vivo and in vitro . Indoxyl sulfate and p -cresol sulfate caused mitochondrial fission by modulating the expression of mitochondrial fission–fusion proteins. Mitochondrial dysfunction and impaired biogenesis could be protected by treatment with antioxidants. The in vitro study also demonstrated that indoxyl sulfate and p -cresol sulfate reduced mitochondrial mass by activating autophagic machinery. In summary, our study suggests that mitochondrial injury is one of the major pathological mechanisms for uremic intoxication, which is related to chronic kidney disease and its complications.

Acrolein (Acr), a highly reactive unsaturated aldehyde, can cause various lung diseases including asthma, chronic obstructive pulmonary disease (COPD), and lung cancer. We have found that Acr can damage not only genomic DNA but also DNA repair proteins causing repair dysfunction and enhancing cells’ mutational susceptibility. While these effects may account for Acr lung carcinogenicity, the mechanisms by which Acr induces lung diseases other than cancer are unclear. In this study, we found that Acr induces damages in mitochondrial DNA (mtDNA), inhibits mitochondrial bioenergetics, and alters mtDNA copy number in human lung epithelial cells and fibroblasts. Furthermore, Acr induces mitochondrial fission which is followed by autophagy/ mitophagy and Acr-induced DNA damages can trigger apoptosis. However, the autophagy/ mitophagy process does not change the level of Acr-induced mtDNA damages and apoptosis. We propose that Acr-induced mtDNA damages trigger loss of mtDNA via mitochondrial fission and mitophagy. These processes and mitochondria dysfunction induced by Acr are causes that lead to lung diseases.

// Xu Zhou 1, * , Hui-Ying Wang 1, * , Bin Wu 1, * , Cai-Yi Cheng 1 , Wei Xiao 2 , Zhen-Zhong Wang 2 , Yu-Yu Yang 1 , Ping Li 1 and Hua Yang 1 1 State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, China 2 State Key Laboratory of New-tech for Chinese Medicine Pharmaceutical Process, Jiangsu Kanion Pharmaceutical Co., Ltd., Lianyungang, China * These authors have contributed equally to this work Correspondence to: Ping Li, email: liping2004@126.com Hua Yang, email: 104yang104@163.com Keywords: ginkgolide K, mitochondrial fission, mitochondrial permeability transition pore, neuron apoptosis, ischemic stroke Received: December 22, 2016 Accepted: April 27, 2017 Published: May 18, 2017 ABSTRACT Ginkgolide K (GK) belongs to the ginkgolide family of natural compounds found in Ginkgo biloba leaves, which have been used for centuries to treat cerebrovascular and cardiovascular diseases. We evaluated the protective effects of GK against neuronal apoptosis by assessing its ability to sustain mitochondrial integrity and function. Co-immunoprecipitation showed that Drp1 binding to GSK-3β was increased after an oxygen-glucose deprivation/reperfusion (OGD/R) insult in cultured neuroblastoma cells. This induced Drp1 and GSK-3β translocation to mitochondria and mitochondrial dysfunction, which was attenuated by GK. GK also reduced mitochondrial fission by increasing Drp1 phosphorylation at Ser637 and inhibiting mitochondrial Drp1 recruitment. In addition, GK exposure induced GSK-3β phosphorylation at Ser9 and enhanced the interaction between adenine nucleotide translocator (ANT) and p-GSK-3β. This interaction suppressed the interaction between ANT and cyclophilin D (CypD), which inhibited mitochondrial permeability transition pore (mPTP) opening. Similarly, suppression of mitochondrial fission by Mdivi-1 also inhibited GSK-3β-induced mPTP opening. Treating mice with GK prevented GSK-3β and Drp1 translocation to mitochondria and attenuated mitochondrial dysfunction after middle cerebral artery occlusion. We therefore propose that by inhibiting mitochondrial fission and attenuating mPTP opening, GK exerts neuroprotective effects that mitigate or prevent neuronal damage secondary to ischemic stroke.

One of the severe limitations of chemotherapy is the development of drug resistance. However, the mechanisms underlying chemotherapy resistance remain to be elucidated. Mitochondrial mediated apoptosis is a form of cell death induced by chemotherapy. Several chemotherapeutic agents have been shown to induce mitochondrial fission, and finally activate the apoptosis cascade in various cancer cells. Here, we report that the mitochondrial membrane protein 18 (MTP18) induced mitochondrial fragmentation in gastric cancer cells under doxorubicin (DOX) exposure. Upon over-expression of MTP18, a sub-cytotoxic dose of DOX could sensitize a significant number of cells to undergo mitochondrial fission and subsequent apoptosis. These findings suggest that MTP18 can enhance the sensitivity of gastric cancer cells to DOX. Mechanistically, we found that MTP18 enriched dynamic-related protein 1 (DRP1) accumulation in mitochondria and it was responsible for mediating DOX-induced signaling required for mitochondrial fission. Intriguingly, MTP18 expression was downregulated during DOX treatment. Thus, down-regulation of MTP18 expression could be one of the resistance factors interfering with DOX-induced apoptosis in gastric cancer cells.

// Hongchao Gou 1, * , Mingqiu Zhao 1, * , Hailuan Xu 1 , Jin Yuan 1 , Wencheng He 1 , Mengjiao Zhu 1 , Hongxing Ding 1 , Lin Yi 1 and Jinding Chen 1 1 College of Veterinary Medicine, South China Agricultural University, Guangzhou, People’s Republic of China * These authors have contributed equally to this work Correspondence to: Jinding Chen, email: jdchen@scau.edu.cn Keywords: classical swine fever virus (CSFV), mitochondrial fission, mitophagy, apoptosis Received: August 17, 2016 Accepted: March 17, 2017 Published: April 11, 2017 ABSTRACT Classical swine fever virus (CSFV), which causes typical clinical characteristics in piglets, including hemorrhagic syndrome and immunosuppression, is linked to hepatitis C and dengue virus. Oxidative stress and a reduced mitochondrial transmembrane potential are disturbed in CSFV-infected cells. The balance of mitochondrial dynamics is essential for cellular homeostasis. In this study, we offer the first evidence that CSFV induces mitochondrial fission and mitophagy to inhibit host cell apoptosis for persistent infection. The formation of mitophagosomes and decline in mitochondrial mass relevant to mitophagy were detected in CSFV-infected cells. CSFV infection increased the expression and mitochondrial translocation of Pink and Parkin. Upon activation of the PINK1 and Parkin pathways, Mitofusin 2 (MFN2), a mitochondrial fusion mediator, was ubiquitinated and degraded in CSFV-infected cells. Mitophagosomes and mitophagolysosomes induced by CSFV were, respectively, observed by the colocalization of LC3-associated mitochondria with Parkin or lysosomes. In addition, a sensitive dual fluorescence reporter (mito-mRFP-EGFP) was utilized to analyze the delivery of mitophagosomes to lysosomes. Mitochondrial fission caused by CSFV infection was further determined by mitochondrial fragmentation and Drp1 translocation into mitochondria using a confocal microscope. The preservation of mitochondrial proteins, upregulated apoptotic signals and decline of viral replication resulting from the silencing of Drp1 and Parkin in CSFV-infected cells suggested that CSFV induced mitochondrial fission and mitophagy to enhance cell survival and viral persistence. Our data for mitochondrial fission and selective mitophagy in CSFV-infected cells reveal a unique view of the pathogenesis of CSFV infection and provide new avenues for the development of antiviral strategies.

// Chuanyan Zhao 1, * , Zhuyun Chen 1, * , Jia Qi 2 , Suyan Duan 1 , Zhimin Huang 1 , Chengning Zhang 1 , Lin Wu 1 , Ming Zeng 1 , Bo Zhang 1 , Ningning Wang 1 , Huijuan Mao 1 , Aihua Zhang 3, 4 , Changying Xing 1 , Yanggang Yuan 1 1 Department of Nephrology, the First Affiliated Hospital of Nanjing Medical University, Nanjing Medical University, Nanjing, China 2 Department of Pharmacy, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, China 3 Department of Nephrology, Nanjing Children's Hospital, Nanjing Medical University, Nanjing, China 4 Institute of Pediatrics, Nanjing Medical University, Nanjing, China * These authors are contributed equally to this work Correspondence to: Yanggang Yuan, email: ygyuan@njmu.edu.cn Changying Xing, email: cyxing1962@163.com Keywords: Drp1, mitophagy, mitochondrial dysfunction, mitochondrial fission, cisplatin Received: July 10, 2016 Accepted: February 07, 2017 Published: February 18, 2017 ABSTRACT Cisplatin chemotherapy often causes acute kidney injury (AKI) in cancer patients. There is increasing evidence that mitochondrial dysfunction plays an important role in cisplatin-induced nephrotoxicity. Degradation of damaged mitochondria is carried out by mitophagy. Although mitophagy is considered of particular importance in protecting against AKI, little is known of the precise role of mitophagy and its molecular mechanisms during cisplatin-induced nephrotoxicity. Also, evidence that activation of mitophagy improved mitochondrial function is lacking. Furthermore, several evidences have shown that mitochondrial fission coordinates with mitophagy. The aim of this study was to investigate whether activation of mitophagy protects against mitochondrial dysfunction and renal proximal tubular cells injury during cisplatin treatment. The effect of mitochondrial fission on mitophagy was also investigated. In cultured human renal proximal tubular cells, we observed that 3-methyladenine, a pharmacological inhibitor of autophagy, blocked mitophagy and exacerbated cisplatin-induced mitochondrial dysfunction and cells injury. In contrast, autophagy activator rapamycin enhanced mitophagy and protected against the harmful effects of cisplatin on mitochondrial function and cells viability. Suppression of mitochondrial fission by knockdown of its main regulator dynamin-related protein-1 (Drp1) decreased cisplatin-induced mitophagy. Meanwhile, Drp1 suppression protected against cisplatin-induced cells injury by inhibiting mitochondrial dysfunction. Our results provide evidence that Drp1-depedent mitophagy has potential as renoprotective targets for the treatment of cisplatin-induced AKI.

// Guo-Bing Li 1, 2, * , Ruo-Qiu Fu 1, * , Han-Ming Shen 3 , Jing Zhou 1 , Xiao-Ye Hu 1 , Yan-Xia Liu 1 , Yu-Nong Li 1 , Hong-Wei Zhang 1 , Xin Liu 1 , Yan-Hao Zhang 1 , Cheng Huang 4 , Rong Zhang 2 , Ning Gao 1 1 Department of Pharmacognosy, College of Pharmacy, 3rd Military Medical University, Chongqing, China 2 Department of Pharmacy, The Second Affiliated Hospital of Third Military Medical University, Chongqing, China 3 Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Republic of Singapore 4 Drug Discovery Lab, School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai, China * These authors have contributed equally to this work Correspondence to: Ning Gao, email: gaoning59@163.com Rong Zhang, email: xqpharmacylab@126.com Keywords: polyphyllin I, DRP1, mitochondrial fission, mitophagy, PINK1 Received: May 17, 2016 Accepted: December 12, 2016 Published: January 02, 2017 ABSTRACT The molecular mechanisms underlying the anti-breast cancer effects of polyphyllin I, a natural compound extracted from Paris polyphylla rhizomes, are not fully understood. In the present study, we found that polyphyllin I induces mitochondrial translocation of DRP1 by dephosphorylating DRP1 at Ser637, leading to mitochondrial fission, cytochrome c release from mitochondria into the cytosol and, ultimately apoptosis. Polyphyllin I also increased the stabilization of full-length PINK1 at the mitochondrial surface, leading to the recruitment of PARK2, P62, ubiquitin, and LC3B-II to mitochondria and culminating in mitophagy. PINK1 knockdown markedly suppressed polyphyllin I-induced mitophagy and enhanced polyphyllin I-induced, DRP1-dependent mitochondrial fission and apoptosis. Furthermore, suppression of DRP1 by mdivi-1 or shRNA inhibited PINK1 knockdown/polyphyllin I-induced mitochondrial fragmentation and apoptosis, suggesting that PINK1 depletion leads to excessive fission and, subsequently, mitochondrial fragmentation. An in vivo study confirmed that polyphyllin I greatly inhibited tumor growth and induced apoptosis in MDA-MB-231 xenografts, and these effects were enhanced by PINK1 knockdown. These data describe the mechanism by which PINK1 contributes to polyphyllin I-induced mitophagy and apoptosis and suggest that polyphyllin I may be an effective drug for breast cancer treatment.

// Lei Zhan 1 , Haiyan Cao 2 , Gang Wang 2 , Yinghua Lyu 2 , Xiacheng Sun 2 , Jiaze An 3 , Zhenbiao Wu 4 , Qichao Huang 2 , Bingrong Liu 1 , Jinliang Xing 2 1 Department of Gastroenterology, Second Affiliated Hospital of Harbin Medical University, Harbin, 150086, China 2 State Key Laboratory of Cancer Biology and Experimental Teaching Center of Basic Medicine, Fourth Military Medical University, Xi’an, 710032, China 3 Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an, 710032, China 4 Department of Clinical Immunology, Xijing Hospital, Fourth Military Medical University, Xi’an, 710032, China Correspondence to: Bingrong Liu, email: bingrongliu@qq.com Jinliang Xing, email: xingjl@fmmu.edu.cn Keywords: mitochondrial dynamics, cell proliferation, liver cancer, p53, NF-κB Received: April 18, 2016 Accepted: July 28, 2016 Published: August 17, 2016 ABSTRACT Mitochondria are highly dynamic and undergo constant fusion and fission that are essential for maintaining physiological functions of cells. Recently, we have reported that increased mitochondrial fission promotes autophagy and apoptosis resistance in hepatocellular carcinoma (HCC) cell through ROS-mediated coordinated regulation of NF-κB and p53 pathways. However, little is known about the roles of mitochondrial dynamics in HCC cell proliferation, another key feature of cancer cells. In this study, we systematically investigated the functional role of mitochondrial fission in the regulation of HCC cell proliferation. Furthermore, the underlying molecular mechanisms were deeply explored. We found that, increased mitochondrial fission by forced expression of Drp1 promoted the proliferation of HCC cells both in vitro and in vivo mainly by facilitating G1/S phase transition of cell cycle. Whereas, Drp1 knockdown or treatment with mitochondrial division inhibitor-1 induced significant G1 phase arrest in HCC cells and reduced tumor growth in the xenotransplantation model. We further demonstrated that the proliferation-promoting role of Drp1-mediated mitochondrial fission was mediated via p53/p21 and NF-κB/cyclins pathways. Moreover, the crosstalk between p53 and NF-κB pathways was proved to be involved in the regulation of mitochondrial fission-mediated cell proliferation. In conclusion, our findings demonstrate that Drp1-mediated mitochondrial fission plays a critical role in the regulation of cell cycle progression and HCC cell proliferation. Thus, targeting Drp1-dependent mitochondrial fission may provide a novel strategy for suppressing tumor growth of HCC.

Fanconi anemia (FA) is a rare genetic disorder associated with bone-marrow failure, genome instability and cancer predisposition. Recently, we and others have demonstrated dysfunctional mitochondria with morphological alterations in FA cells accompanied by high reactive oxygen species (ROS) levels. Mitochondrial morphology is regulated by continuous fusion and fission events and the misbalance between these two is often accompanied by autophagy. Here, we provide evidence of impaired autophagy in FA. We demonstrate that FA cells have increased number of autophagic (presumably mitophagic) events and accumulate dysfunctional mitochondria due to an impaired ability to degrade them. Moreover, mitochondrial fission accompanied by oxidative stress (OS) is a prerequisite condition for mitophagy in FA and blocking this pathway may release autophagic machinery to clear dysfunctional mitochondria.

// Shuaifeng Li 1 , Jiaxing Wang 1 , Ao Zhou 1 , Faheem Ahmed Khan 1 , Lin Hu 1 and Shujun Zhang 1 1 Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan, China Correspondence to: Shujun Zhang, email: // Keywords : PRRSV; mitochondrial fission; mitophagy; apoptosis; Pathology Section Received : January 30, 2016 Accepted : July 09, 2016 Published : July 24, 2016 Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) causes acute mitochondrial dysfunction by elevating the level of reactive oxygen species. Mitochondrial dynamics and mitophagy are essential for the maintenance of mitochondrial homeostasis. Here we show that PRRSV infection stimulated mitochondrial fission and mitophagy to attenuate apoptosis in Marc145 cells. PRRSV infection induced the expression of Drp1, enhanced phosphorylation of Drp1 at Ser616 and its subsequent translocation to mitochondria. Furthermore, PRRSV infection increased the expression of PINK1 and Parkin and also stimulated the recruitment of Parkin to mitochondria. In addition, a sensitive dual fluorescence vector expressing mito-mRFP-EGFP targeted mitochondria was employed to observe the complete mitophagy by delivering dysfunctional mitochondria to lysosome for degradation. Interfering the expression of Drp1 and or Parkin suppressed PRRSV replication. More importantly, silencing of Drp1 or Parkin caused significant elevation in apoptotic signaling. These results suggest that PRRSV infection stimulates mitochondrial fission and mitophagy to facilitate virus replication most probably by attenuating apoptosis.

The ERCC8/CSA gene encodes a WD-40 repeat protein (CSA) that is part of a E3-ubiquitin ligase/COP9 signalosome complex. When mutated, CSA causes the Cockayne Syndrome group A (CS-A), a rare recessive progeroid disorder characterized by sun sensitivity and neurodevelopmental abnormalities. CS-A cells features include ROS hyperproduction, accumulation of oxidative genome damage, mitochondrial dysfunction and increased apoptosis that may contribute to the neurodegenerative process. In this study, we show that CSA localizes to mitochondria and specifically interacts with the mitochondrial fission protein dynamin-related protein (DRP1) that is hyperactivated when CSA is defective. Increased fission is not counterbalanced by increased mitophagy in CS-A cells thus leading to accumulation of fragmented mitochondria. However, when mitochondria are challenged with the mitochondrial toxin carbonyl cyanide m-chloro phenyl hydrazine, CS-A fibroblasts undergo mitophagy as efficiently as normal fibroblasts, suggesting that this process remains targetable to get rid of damaged mitochondria. Indeed, when basal mitophagy was potentiated by overexpressing Parkin in CSA deficient cells, a significant rescue of the dysfunctional mitochondrial phenotype was observed. Importantly, Parkin overexpression not only reactivates basal mitophagy, but plays also an anti-apoptotic role by significantly reducing the translocation of Bax at mitochondria in CS-A cells. These findings provide new mechanistic insights into the role of CSA in mitochondrial maintenance and might open new perspectives for therapeutic approaches.

Differentiation-inducing factor (DIF) defines a group of chlorinated hexaphenones that orchestrate stalk-cell differentiation in the slime mold Dictyostelium discoideum (DD). DIF-1 and 3 have also been reported to have tumor inhibiting properties; however, the mechanisms that underlie the effects of these compounds remain poorly defined. Herein, we show that DIF-3 rapidly triggers Ca2+ release and a loss of mitochondrial membrane potential (MMP) in the absence of cytochrome c and Smac release and without caspase activation. Consistently with these findings, we also detected no evidence of apoptosis in cells treated with DIF-3 but instead found that this compound induced autophagy. In addition, DIF-3 promoted mitochondrial fission in K562 and HeLa cells, as assessed by electron and confocal microscopy analysis. Importantly, DIF-3 mediated the phosphorylation and redistribution of dynamin-related protein 1 (DRP1) from the cytoplasmic to the microsomal fraction of K562 cells. Pharmacological inhibition or siRNA silencing of DRP1 not only inhibited mitochondrial fission but also protected K562 cells from DIF-3-mediated cell death. Furthermore, DIF-3 potently inhibited the growth of imatinib-sensitive and imatinib-resistant K562 cells. It also inhibited tumor formation in athymic mice engrafted with an imatinib-resistant CML cell line. Finally, DIF-3 exhibited a clear selectivity toward CD34+ leukemic cells from CML patients, compared with CD34− cells. In conclusion, we show that the potent anti-leukemic effect of DIF-3 is mediated through the induction of mitochondrial fission and caspase-independent cell death. Our findings may have important therapeutic implications, especially in the treatment of tumors that exhibit defects in apoptosis regulation.

Non-thermal atmospheric gas plasma (AGP) exhibits cytotoxicity against malignant cells with minimal cytotoxicity toward normal cells. However, the mechanisms of its tumor-selective cytotoxicity remain unclear. Here we report that AGP-activated medium increases caspase-independent cell death and mitochondrial network collapse in a panel of human cancer cells, but not in non-transformed cells. AGP irradiation stimulated reactive oxygen species (ROS) generation in AGP-activated medium, and in turn the resulting stable ROS, most likely hydrogen peroxide (H2O2), activated intracellular ROS generation and mitochondrial ROS (mROS) accumulation. Culture in AGP-activated medium resulted in cell death and excessive mitochondrial fragmentation and clustering, and these responses were inhibited by ROS scavengers. AGP-activated medium also increased dynamin-related protein 1-dependent mitochondrial fission in a tumor-specific manner, and H2O2 administration showed similar effects. Moreover, the vulnerability of tumor cells to mitochondrial network collapse appeared to result from their higher sensitivity to mROS accumulation induced by AGP-activated medium or H2O2. The present findings expand our previous observations on death receptor-mediated tumor-selective cell killing and reinforce the importance of mitochondrial network remodeling as a powerful target for tumor-selective cancer treatment.

Intrinsic apoptosis eliminates cells with damaged DNA and cells with dysregulated expression of oncogene. PGAM5, a member of the phosphoglycerate mutase family, has two splicing variants: PGAM5L (the long form) and PGAM5S (the short form). It has been well established that PGAM5 is at the convergent point of multiple necrosis pathways. However, the role of PGAM5 in intrinsic apoptosis is still controversial. Here we report that the PGAM5L, but not PGAM5S is a prerequisite for the activation of Bax and dephosphorylation of Drp1 in arenobufagin and staurosporine induced intrinsic apoptosis. Knockdown of PGAM5L inhibits the translocation of Bax to the mitochondria and reduces mitochondrial fission. The interaction between PGAM5L and Drp1 was observed in both arenobufagin and staurosporine treated HCT116 cells, but not in HCT116 Bax(-/-) cells. Bax transfection rescues the formation of the triplex in both arenobufagin and staurosporine stimulated HCT116 Bax(-/-) cells. Arenobufagin shows remarkable anti-cancer effects both in orthotropic and heterotropic CRC models and demonstrates less toxic effects as compared with that of cisplatin. Bax-PGAM5L-Drp1 complex is detected in arenobufagin and staurosporine treated CRC cells in vitro and in arenobufagin and cisplatin treated tumor in vivo as well. In summary, our results demonstrate that Bax-PGAM5L-Drp1 complex is required for intrinsic apoptosis execution.

// Ponarulselvam Sekar 1 , Duen-Yi Huang 2 , Shwu-Fen Chang 1 and Wan-Wan Lin 1, 2 1 Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan 2 Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan Correspondence to: Wan-Wan Lin, email: wwllaura1119@ntu.edu.tw Keywords: P2X7; ATP; acidification; mitochondrial respiration; mitochondrial fission Received: April 20, 2017 Accepted: January 24, 2018 Published: January 29, 2018 ABSTRACT Extracellular adenosine 5′-triphosphate (ATP) is a damage-associated molecular pattern and contributes to inflammation associated diseases including cancer. Extracellular acidosis is a novel danger signal in the inflammatory sites, where it can modulate inflammation, immunity and tumor growth. Extracellular acidification was shown to inhibit P2X7-mediated channel currents, while it remains unknown how acidification and P2X7 together affect cellular responses. Here, we treated BV-2 microglial cells with ATP in a short period (

Cofilin is a member of the actin-depolymerizing factor (ADF) family protein, which plays an essential role in regulation of the mitochondrial apoptosis. It remains unclear how cofilin regulates the mitochondrial apoptosis. Here, we report for the first time that natural compound 4-methylthiobutyl isothiocyanate (erucin) found in consumable cruciferous vegetables induces mitochondrial fission and apoptosis in human breast cancer cells through the mitochondrial translocation of cofilin. Importantly, cofilin regulates erucin-induced mitochondrial fission by interacting with dynamin-related protein (Drp1). Knockdown of cofilin or Drp1 markedly reduced erucin-mediated mitochondrial translocation and interaction of cofilin and Drp1, mitochondrial fission, and apoptosis. Only dephosphorylated cofilin (Ser 3) and Drp1 (Ser 637) are translocated to the mitochondria. Cofilin S3E and Drp1 S637D mutants, which mimick the phosphorylated forms, suppressed mitochondrial translocation, fission, and apoptosis. Moreover, both dephosphorylation and mitochondrial translocation of cofilin and Drp1 are dependent on ROCK1 activation. In vivo findings confirmed that erucin-mediated inhibition of tumor growth in a breast cancer cell xenograft mouse model is associated with the mitochondrial translocation of cofilin and Drp1, fission and apoptosis. Our study reveals a novel role of cofilin in regulation of mitochondrial fission and suggests erucin as a potential drug for treatment of breast cancer.

// Chia-Woei Wang 1, 2 , Wei-Hsuan Hsu 3 , Chen-Jei Tai 1, 2, 4, 5 1 Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei 11042, Taiwan 2 Department of Obstetrics and Gynecology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 11042, Taiwan 3 Biochemical Process Technology Department, Center of Excellence for Drug Development, Biomedical Technology and Device Research Laboratories, Industrial Technology Research Institute, Hsinchu 30068, Taiwan 4 Department of Traditional Chinese Medicine, Department of Internal Medicine, Taipei Medical University Hospital, Taipei 11042, Taiwan 5 Traditional Herbal Medicine Research Center, Taipei Medical University Hospital, Taipei 11042, Taiwan Correspondence to: Chen-Jei Tai, email: chenjtai@tmu.edu.tw Keywords: cordycepin, mitochondrial fusion, mitochondrial fission, estrogen-related receptor (ERR)-α, antimigration Received: September 16, 2016 Accepted: November 23, 2016 Published: December 09, 2016 ABSTRACT Cordycepin (3′-deoxyadenosine) is a compound for antitumor, which has been found to exert antiangiogenic, antimetastatic, and antiproliferative effects, as well as inducing apoptosis. However, the association between cancer metastasis and mitochondrial activity in cordycepin-treated ovarian carcinoma cells remains unclear. The 50 and 100 μM of cordycepin inhibits mitochondrial fusion and induces mitochondrial fission, respectively. These suggested that cordycepin showed the down-regulation of mitochondrial function and limitation of energy production. Because of activation of mitochondria and generation of energy are needed in cancer cell migration/invasion. After 24 h treatment, cordycepin suppresses epithelial–mesenchymal transition and migration in ovarian carcinoma cells through inhibiting estrogen-related receptor (ERR)-α. The ERRα is a co-transcription factor for gene expressions associated with mitochondrial fusion. Our results indicate that cordycepin suppresses metastasis and migration of ovarian carcinoma cells via inhibiting mitochondrial activity in non-toxic concentrations, and cordycepin has potential benefits in ovarian cancer therapy.

// Deepak Kumar Tanwar 1 , Danitra J. Parker 2 , Priyanka Gupta 2 , Brian Spurlock 2 , Ronald D. Alvarez 3 , Malay Kumar Basu 1 , Kasturi Mitra 2 1 Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA 2 Department of Genetics, University of Alabama at Birmingham, Birmingham, AL, USA 3 Department of Obstetrics and Gynecology, University of Alabama at Birmingham, Birmingham, AL, USA Correspondence to: Kasturi Mitra, email: kasturi@uab.edu Malay Kumar Basu, email: malay@uab.edu Keywords: Drp1, mitochondria, cell cycle, cancer, genome analyses Received: March 15, 2016 Accepted: June 30, 2016 Published: August 04, 2016 ABSTRACT Mitochondrial metabolic reprogramming is a hallmark of tumorigenesis. Although mitochondrial function can impact cell cycle regulation it has been an understudied area in cancer research. Our study highlights a specific involvement of mitochondria in cell cycle regulation across cancer types. The mitochondrial fission process, which is regulated at the core by Drp1, impacts various cellular functions. Drp1 has been implicated in various cancer types with no common mechanism reported. Our Drp1-directed large-scale analyses of the publically available cancer genomes reveal a robust correlation of Drp1 with cell-cycle genes in 29 of the 31 cancer types examined. Hypothesis driven investigation on epithelial ovarian cancer (EOC) revealed that Drp1 co-expresses specifically with the cell-cycle module responsible for mitotic transition. Repression of Drp1 in EOC cells can specifically attenuate mitotic transition, establishing a potential casual role of Drp1 in mitotic transition. Interestingly, Drp1-Cell-Cycle co-expression module is specifically detected in primary epithelial ovarian tumors that robustly responded to chemotherapy, suggesting that Drp1 driven mitosis may underlie chemo-sensitivity of the primary tumors. Analyses of matched primary and relapsed EOC samples revealed a Drp1-based-gene-expression-signature that could identify patients with poor survival probabilities from their primary tumors. Our results imply that around 60% of platinum-sensitive EOC patients undergoing relapse show poor survival, potentially due to further activation of a mitochondria driven cell-cycle regime in their recurrent disease. We speculate that this patient group could possibly benefit from mitochondria directed therapies that are being currently evaluated at various levels, thus enabling targeted or personalized therapy based cancer management.

The inner membrane of mitochondria accommodates the oxidative phosphorylation complexes that are composed of subunits encoded and synthesized in two different cellular compartments, with most of the subunits expressed from nuclear DNA. Interestingly, mitochondrial gene expression provides only a handful of proteins that nevertheless represent the catalytic core subunits of the oxidative phosphorylation machinery, rendering mitochondrial translation a crucial process for cellular metabolism. Because mitochondria developed from a prokaryotic ancestor it has long been assumed that mitochondrial gene expression might employ similar mechanisms as found in bacteria. However, recent work has revealed many novel organelle-specific aspects that probably evolved to facilitate and optimize gene expression in the organelle. Mitochondria contain a full genetic system with DNA, mitochondrial ribosomes and all factors required to: A) replicate and transcribe the DNA, B) mature and turnover mRNAs, tRNAs and catalytic RNA, and C) assemble mitochondrial ribosomes. Moreover, mitochondria-specific ribosomes are responsible for the synthesis of the hydrophobic core subunits of the oxidative phosphorylation system. How all these factors are organized in time and space and coordinated with nuclear gene expression is a busy area of mitochondrial research. Indeed, recent work shows that the mitochondrial gene expression machinery is much higher organized than previously assumed. Studies in the mammalian system showed that factors involved in RNA metabolism and ribosome assembly are present in distinct foci, termed RNA granules, that are found in close proximity to the mtDNA [1–3, 5]. These assemblies are thought to connect transcription and maturation of the RNA with downstream processes like ribosome assembly, tRNA modifications and mRNA maturation. The organization of mitochondrial gene expression might extend far beyond these RNA granules. By developing a system allowing the purification of intact mitochondrial ribosomes, we could demonstrate that mitochondrial ribosomes in yeast form large expressosome-like assemblies that gather almost all factors involved in posttranscriptional steps of gene expression that we termed MIOREX complexes [4]. Some of those complexes further co-localize with the mitochondrial nucleoid thereby consolidating all steps of gene expression (nucleoid-MIOREX). This higher organization of mitochondrial ribosomes might has evolved to bring all necessary factors in close proximity, making the whole process of gene expression faster and more efficient. Hence, the close coupling of transcription, mRNA maturation and translation with oxidative phosphorylation complex assembly allows the channeled transport of mRNAs like in a construction-line. This RNA channeling on the ribosome represents a completely novel form of translation organization. However, it is still an intriguing question how the two types of MIOREX complexes are functionally connected and what specific role either of the complexes has. One hypothesis is that the peripheral-MIOREX complexes might help to coordinate translation with oxidative phosphorylation complex assembly in a system where gene expression is not regulated at the level of transcription. We and others have recently identified feedback loops operating in the mitochondrial matrix that connect translation of key respiratory chain subunits with the efficiency of their assembly [6, 7]. In cases where assembly is blocked, translation of these mRNAs is reduced, and stimulated in case the mitochondrially encoded subunits are properly assembled. This elegant system directly monitors the efficiency of assembly to coordinate mitochondrial gene expression with the influx of nuclear encoded subunits allowing the cell to optimally invest resources. The careful organization and channeled transfer of mRNAs in the MIOREX complexes reflects a common theme in mitochondrial biology: Many processes are organized in large assemblies making subsequent biochemical reactions more efficient. Surely, it will be a long, challenging and exciting way to unravel the unique features that mitochondria evolved to express a handful of genes and to learn which other features mitochondria acquired in their journey from a prokaryotic endosymbiont to a “modern” organelle. Because most mitochondrial diseases arise from aberrant mitochondrial gene expression, insights into this fascinating system will allow to develop new therapeutic strategies for these often fatal diseases.

// Ting-Fang Che 1 , Ching-Wen Lin 2 , Yi-Ying Wu 5 , Yu-Ju Chen 6 , Chia-Li Han 7 , Yih-leong Chang 8 , Chen-Tu Wu 8 , Tzu-Hung Hsiao 9 , Tse-Ming Hong 5, * , Pan-Chyr Yang 1, 2, 3, 4, * 1 Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei 100, Taiwan 2 Institute of Biomedical Science, Academia Sinica, Taipei 115, Taiwan 3 NTU Center for Genomic Medicine, College of Medicine, National Taiwan University, Taipei 100, Taiwan 4 Department of Internal Medicine, College of Medicine, National Taiwan University, Taipei 100, Taiwan 5 Institute of Clinical Medicine, College of Medicine, National Cheng-Kung University, Tainan 701, Taiwan 6 Institute of Chemistry, Academia Sinica 115, Taipei, Taiwan 7 Master Program for Clinical Pharmacogenomics and Pharmacoproteomics, School of Pharmacy, Taipei Medical University 110, Taipei, Taiwan 8 Department of Pathology and Graduate Institute of Pathology, College of Medicine, National Taiwan University, Taipei 100, Taiwan 9 Department of Medical Research, Taichung Veterans General Hospital, Taichung 407, Taiwan * These authors have contributed equally to this work Correspondence to: Pan-Chyr Yang, e-mail: pcyang@ntu.edu.tw Tse-Ming Hong, e-mail: tmhong@mail.ncku.edu.tw Keywords: cancer metastasis, EGFR, mitochondria dynamics Received: May 19, 2015 Accepted: October 06, 2015 Published: October 19, 2015 ABSTRACT Dysfunction of the mitochondria is well-known for being associated with cancer progression. In the present study, we analyzed the mitochondria proteomics of lung cancer cell lines with different invasion abilities and found that EGFR is highly expressed in the mitochondria of highly invasive non-small-cell lung cancer (NSCLC) cells. EGF induces the mitochondrial translocation of EGFR; further, it leads to mitochondrial fission and redistribution in the lamellipodia, upregulates cellular ATP production, and enhances motility in vitro and in vivo . Moreover, EGFR can regulate mitochondrial dynamics by interacting with Mfn1 and disturbing Mfn1 polymerization. Overexpression of Mfn1 reverses the phenotypes resulting from EGFR mitochondrial translocation. We show that the mitochondrial EGFR expressions are higher in paired samples of the metastatic lymph node as compared with primary lung tumor and are inversely correlated with the overall survival in NSCLC patients. Therefore, our results demonstrate that besides the canonical role of EGFR as a receptor tyrosine, the mitochondrial translocation of EGFR may enhance cancer invasion and metastasis through regulating mitochondria dynamics.

// Yoshihiro Suzuki-Karasaki 1,2 , Kyoko Fujiwara 2,3 , Kosuke Saito 2,3 , Miki Suzuki-Karasaki 4 , Toyoko Ochiai 4 and Masayoshi Soma 2,3 1 Division of Physiology, Department of Biomedical Sciences, Nihon University School of Medicine, Tokyo, Japan 2 Innovative Therapy Research Group, Nihon University Research Institute of Medical Science, Tokyo, Japan 3 Division of General Medicine, Department of Internal Medicine, Nihon University School of Medicine, Tokyo, Japan 4 Department of Dermatology, Nihon University Surugadai Hospital, Tokyo, Japan Correspondence to: Yoshihiro Suzuki-Karasaki, email: // Keywords : TRAIL, mitochondrial fragmentation, depolarization, reactive oxygen species, tumor-selective killing Received : February 09, 2015 Accepted : May 13, 2015 Published : May 25, 2015 Abstract Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) is a promising anticancer drug due to its tumor-selective cytotoxicity. Here we report that TRAIL exhibits distinct effects on the mitochondrial networks in malignant cells and normal cells. Live-cell imaging revealed that multiple human cancer cell lines and normal cells exhibited two different modes of mitochondrial responses in response to TRAIL and death receptor agonists. Mitochondria within tumor cells became fragmented into punctate and clustered in response to toxic stimuli. The mitochondrial fragmentation was observed at 4 h, then became more pronounced over time, and associated with apoptotic cell death. In contrast, mitochondria within normal cells such as melanocytes and fibroblasts became only modestly truncated, even when they were treated with toxic stimuli. Although TRAIL activated dynamin-related protein 1 (Drp1)-dependent mitochondrial fission, inhibition of this process by Drp1 knockdown or with the Drp1 inhibitor mdivi-1, potentiated TRAIL-induced apoptosis, mitochondrial fragmentation, and clustering. Moreover, mitochondrial reactive oxygen species (ROS)-mediated depolarization accelerated mitochondrial network abnormalities in tumor cells, but not in normal cells, and TRAIL caused higher levels of mitochondrial ROS accumulation and depolarization in malignant cells than in normal cells. Our findings suggest that tumor cells are more prone than normal cells to oxidative stress and depolarization, thereby being more vulnerable to mitochondrial network abnormalities and that this vulnerability may be relevant to the tumor-targeting killing by TRAIL.

Twenty years of research and advances in pathophysiology have not yet translated into effective therapeutic tools for improving survival after an acute kidney injury (AKI) episode. AKI is a disease process characterised by rapidly decreasing renal excretory function, with accumulation of nitrogen metabolism and other waste products. AKI is a major public health concern with a rapidly increasing prevalence and unacceptably high mortality rate, claiming 1.7 million lives each year [1]. AKI is characterized by renal tubular damage, for which a plausible key mediator, amenable to pharmacological manipulation, has not been identified yet. Due to their high-energy demand, tubular cells are rich in mitochondria, and alterations in these organelles have been recognized as a hallmark in the initiation and progression of the disease [2]. Increased production of reactive oxygen species (ROS) and decreased antioxidant defences, make mitochondria particularly susceptible to injury [2]. These are highly mobile organelles that exist in dynamic networks whose function relies on complex molecular machinery, finely tuned and balanced between fission― accompanied by mitochondrial membrane depolarization and oxidative damage―and fusion, more commonly leading to energy production. Mitochondrial fission is catalyzed by Dynamin Related Protein 1 (Drp1), which is recruited from the cytosol to the outer mitochondrial membrane by Mitochondria fission factor (Mff) to form spirals to sever both membranes of the organelle. Conversely, mitochondria join through the process of fusion, which is mediated by the coordinated activities of Mitofusins (Mfn) and Optic atrophy 1 (Opa1) in the outer and inner mitochondrial membrane, respectively. Fission and fusion may also serve as an important quality control mechanism for preserving the functional integrity of the mitochondrial network. Fusion helps to ease physiological stress levels by mixing the contents of partially damaged mitochondria as a form of complementation, while fission is needed to remove mitochondria from the network when the organelles become damaged and membrane potential depolarization occurs across the inner membrane. Depolarized mitochondria are substrates for the translocation of several proteins, such as PTEN-induced Putative Kinase 1 (PINK1), which tags the organelle for elimination through the mitophagic machinery. Ample evidence indicates that, in experimental models of AKI, mitochondrial fission predominates and organelle impairment precedes renal dysfunction [3]. Mitochondrial vitality appears to be maintained by Sirtuin 3 (SIRT3), which belongs to an evolutionarily conserved family of seven NAD+-dependent deacetylases, three of which (SIRT3-5) are mainly localized in the mitochondrion [4]. In previous studies we demonstrated that elevated renal SIRT3 expression reduces ROS and ameliorates mitochondria dynamics that translate into the longevity phenotype in mice deficient for angiotensin II type 1 receptor [5]. Whether renal SIRT3 could be a master regulator of injury and repair in AKI represented a logical extension of these studies. Cisplatin was used in mice to induce AKI that, by affecting proximal tubular cells, leads to increased generation of ROS and changes in mitochondrial dynamics [6]. These abnormalities were associated with an 80% reduction in the renal expression of SIRT3 mRNA and protein. In an attempt to pharmacologically manipulate the system, AICAR, an activator of the SIRT3 upstream signal AMPK, or the antioxidant agent acetyl-L-carnitine (ALCAR), were used. Both compounds improved renal function and ameliorated tubular injury in cisplatin-induced AKI mice. The renoprotective effect was accompanied by the upregulation of PGC-1α, a crucial mitochondrial biogenesis regulator able to promote SIRT3 gene expression, and NAMPT, the rate-limiting enzyme in the biosynthesis of NAD+, ultimately leading to restoration of SIRT3 expression/activity. At the ultrastructural level, AICAR treatment tipped mitochondrial dynamics towards fusion, as it preserved mitochondrial fragmentation induced by the chemotherapic and reduced expression of the mitochondrial fission mediator Drp1 in the organelles of AKI mice. In vitro, in tubular cells SIRT3 overexpression promoted mitochondrial fusion by limiting Drp1 recruitment through the downregulation of Mff and by upregulating Opa1. Moreover, the overexpression of SIRT3 promoted mitochondrial integrity by counteracting the cisplatin-dependent mitochondrial membrane depolarization and translocation of PINK1 on the organelle membrane. The biological and clinical relevance of SIRT3 in AKI was further documented in SIRT3−/− mice. In these animals, cisplatin induced earlier and remarkably more severe renal dysfunction and, unlike their WT littermates, all SIRT3-deficient mice died within 7 days. SIRT3-deficient animals were fully resistant to the therapeutic effects of both AICAR and ALCAR, which, conversely, were consistently beneficial to SIRT3-competent mice. Taken together, our results indicate that SIRT3 is a crucial regulator of mitochondrial functional integrity and a non-redundant player in tubular injury and repair in AKI. Given the high prevalence of mitochondrial dysfunction in many chronic diseases, such as metabolic syndrome, premature ageing and cancer, along with the profound impact SIRT3 has on mitochondrial function and metabolism, the obvious translational consequence of these in vivo and in vitro studies will be to look for potential SIRT3-specific activating compounds, which the current pharmacopeia is remarkably lacking. Recent work has provided evidence that honokiol is capable of blocking cardiac hypertrophy by increasing SIRT3 activity [7], paving the way for disclosing novel SIRT3 activators. Now is the time for academia and industry to jointly tackle this challenge, offering hope to patients who still die of conditions that lack effective treatment.

Mitochondria are organelles that host many metabolic processes, including the generation of energy in the form of adenosine triphosphate (ATP) through oxidative phosphorylation. They contain their own genome, however, the vast majority of mitochondrial proteins are encoded in the nucleus. Nuclei sense changes in mitochondrial functioning via retrograde signaling pathways, resulting in modified gene expression to restore homeostasis. These pathways can be triggered by altered ATP synthesis, increased levels of unfolded or damaged proteins, or changes in the production of reactive oxygen species (ROS), a major by-product of the electron transport chain. Indeed, inhibition of electron transport activity leads to enhanced ROS levels and, somewhat paradoxically, promotes increased lifespan in a range of model organisms. The complex relationship between mitochondrial function, ROS and aging has been a topic of intense research for many years, with particular focus on the retrograde signaling pathways that act as the conduit of information between mitochondria and nuclei [1]. We have uncovered a novel nuclear role for the respiratory enzyme CLK-1 in maintaining mitochondrial function by regulating the expression of genes that promote ROS homeostasis and dampen the activity of a key stress pathway, the mitochondrial unfolded response (UPRmt) [2]. Importantly, CLK-1 localizes to nuclei dependent upon mitochondrial ROS production, indicating that it acts as a barometer of respiratory activity that can directly mediate retrograde signaling to the nucleus (Figure ​(Figure1).1). CLK-1 (also called COQ7) is a diiron containing monooxygenase that catalyzes the hydroxylation of 5-demethoxyubiquinone, a critical step in the biosynthesis of the mitochondrial electron transport chain cofactor ubiquinone. C. elegans clk-1 null mutants and heterozygous mice display defective oxidative phosphorylation, increased ROS levels and, similar to many other respiration mutants, have extended lifespans [3]. It had been assumed that this longevity phenotype was solely due to CLK-1 acting in mitochondria. However, the expression of a CLK-1 mutant that specifically localizes to nuclei partially suppressed the extended lifespan of clk-1 null C. elegans, indicating that the nuclear form of CLK- 1 independently affects longevity [2]. As elevated ROS levels and activation of the UPRmt are reported to promote longevity, their suppression by nuclear CLK-1 would be consistent with its role in limiting lifespan. Figure 1 Nuclear CLK-1 regulates mitochondrial homeostasis and longevity We propose that CLK-1 functions as a rheostat to maintain ROS homeostasis and keep stress-responsive pathways in check. Elevated ROS produced by mitochondria direct a pool of CLK-1 to the nucleus where it regulates the expression of genes that decrease ROS levels and limit activation of the UPRmt. Conversely, decreased ROS lead to a reduction in nuclear CLK-1 and relieve its effects on gene expression allowing ROS levels to recover and the UPRmt to return to basal levels, thus maintaining homeostasis (Figure ​(Figure1).1). CLK-1 therefore coordinates ROS responses with the UPRmt, a pathway that protects against the accumulation of unfolded or misfolded proteins within mitochondria. This suggests that distinct mitochondrial signals can be integrated at the level of nuclear gene expression to provide a unified response. Important questions concerning the dual mitochondrial and nuclear functions of CLK-1 remain unanswered. Firstly, how do changes in ROS levels lead to the nuclear accumulation of CLK-1? The mitochondrial and nuclear forms of CLK-1 are distinguishable; the mitochondrial form is cleaved at the N-terminus by a protease following import while the nuclear form is the full-length protein, indicating that it has not been imported into mitochondria prior to its translocation to the nucleus. Interestingly, mutation of the mitochondrial targeting sequence in CLK-1 results in its nuclear accumulation independently of ROS levels [2]. This would be consistent with a mechanism by which ROS inhibit mitochondrial import of CLK-1, but whether this occurs by blocking mitochondrial targeting of CLK-1 or by preventing import through either the outer or inner mitochondrial membranes is unclear. Interestingly, a transcription factor component of the C. elegans UPRmt, the pathway suppressed by nuclear CLK-1, may be similarly regulated. ATFS-1 is targeted to mitochondria and rapidly degraded in the absence of UPRmt induction, while activation of the UPRmt leads to impaired import of newly synthesized ATFS-1 and its redirection to the nucleus where it regulates the expression of genes that restore mitochondrial proteostasis [4]. Thus, the inhibition of mitochondrial import in response to changes in mitochondrial function may be a common mechanism of regulating retrograde signaling to the nucleus. A second unresolved issue is the mechanism by which CLK-1 regulates gene expression. This is independent of its role in ubiquinone biosynthesis [2]. CLK-1 is a relatively small protein (around 20kDa) that does not contain obvious elements associated with DNA binding or transcriptional activation/suppression. However, it associates with many genomic loci suggesting that it may directly regulate transcription [2]. An interesting possibility is that the enzymatic activity of CLK-1 modifies transcriptional regulatory factors or chromatin. There is precedent for mitochondrial enzymes regulating nuclear gene expression, including the yeast arginine biosynthesis enzyme Arg5,6 [5] and the pyruvate dehydrogenase complex [6]. Furthermore, many mitochondrial proteins have been detected in nuclei although their roles remain uncharacterized [7]. Our study provides a novel example of how distinct forms of a respiratory enzyme can be separately targeted to mitochondria or nuclei to regulate different cellular processes. This research contributes to our understanding of the diverse signaling mechanisms controlling mitochondrial function and ROS homeostasis that can affect aging and disease.

Mitochondria form a highly dynamic network driven by opposing scission and fusion events. DRP1 is an essential modulator of mitochondrial fission and dynamics within mammalian cells. Its fission activity is regulated by posttranslational modifications such as activating phosphorylation at serine 616. DRP1 activity has recently been implicated as being dysregulated in numerous human disorders such as cancer and neurodegenerative diseases. Here we describe the development of a cell-based screening assay to detect DRP1 activation. We utilized this to undertake focused compound library screening and identified potent modulators that affected DRP1 activity including ICG-001, which is described as WNT/β-catenin signaling inhibitor. Our findings elucidate novel details about ICG-001's mechanism of action (MOA) in mediating anti-proliferative activity. We show ICG-001 both inhibits mitochondrial fission and activates an early endoplasmic reticulum (ER) stress response to induce cell death in susceptible colorectal cancer cell lines.

Mitochondrial function declines with age and is associated with age-related disorders and cell death. In the retina this is critical as photoreceptor energy demands are the greatest in the body and aged cell loss large (~30%). But mitochondria can fuse or divide to accommodate changing demands. We explore ageing mitochondrial dynamics in young (1 month) and old (12 months) mouse retina, investigating changes in mitochondrial fission (Fis1) and fusion (Opa1) proteins, cytochrome C oxidase (COX III), which reflects mitochondrial metabolic status, and heat shock protein 60 (Hsp60) that is a mitochondrial chaperon for protein folding.Western blots showed each protein declined with age. However, within this, immunostaining revealed increases of around 50% in Fis1 and Opa1 in photoreceptor inner segments (IS). Electron microscope analysis revealed mitochondrial fragmentation with age and marked changes in morphology in IS, consistent with elevated dynamics. COX III declined by approximately 30% in IS, but Hsp60 reductions were around 80% in the outer plexiform layer.Our results are consistent with declining mitochondrial metabolism. But also with increased photoreceptor mitochondrial dynamics that differ from other retinal regions, perhaps reflecting attempts to maintain function. These changes are the platform for age related photoreceptor loss initiated after 12 months.