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Mitochondria in Biology and Medicine
Mitochondrion 12 (2012) 472–476
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Review
Mitochondria in Biology and Medicine Claus Desler, Lene Juel Rasmussen ⁎ Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, DK-2200 Copenhagen, Denmark
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Article history: Received 9 May 2012 Accepted 25 June 2012 Available online 30 June 2012 Keywords: Conference Mitochondria Mitochondrial disease Cancer Mitochondrial haplogroups
a b s t r a c t Ever since the first diagnosis of a mitochondrial disease in 1959 (Ernster et al., 1959), the interest for mitochondrial cytopathies has continued to increase. Originally it was believed that the condition was very rare and primarily effected high-energy requiring tissues resulting in a select few pathologies (Luft, 1994). Since 1959, the understanding of mitochondrial cytopathies has evolved immensely and mitochondrial cytopathies are now known to be the largest group of metabolic diseases and to be resulting in a wide variety of pathologies. “Mitochondria in Biology and Medicine” was the title of the first annual conference of Society of Mitochondrial Research and Medicine — India. The conference was organized by A. S. Sreedhar, Keshav Singh and Kumarasamy Thangaraj, and was held at The Centre for Cellular and Molecular Biology (CCMB) Hyderabad, India, during 9–10 December 2011. The conference featured talks from internationally renowned scientists within the field of mitochondrial research and offered both students and fellow researchers a comprehensive update to the newest research within the field. This paper summarizes key outcomes of the presentations. © 2012 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
Contents 1. Mitochondrial dysfunction in aging and tumorigenesis . . . . . . . . . 2. Mitochondria as a target for anticancer therapies . . . . . . . . . . . . 3. Mitochondrial haplogroups as migration markers and as determinants for 4. Pathologies resulting from mitochondrial dysfunctions . . . . . . . . . 5. Diagnosis of mitochondrial dysfunction in the clinic. . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Mitochondrial dysfunction in aging and tumorigenesis During aging of an organism, and as a result of mitochondrial dysfunctions, the efficiency of oxidative phosphorylation will decline. This is in part a result of a lowering of mitochondrial DNA (mtDNA) content the resultant decline of ATP levels in tissues with high energy demand such as brain, muscle and heart is a major contributory factor to the symptoms of aging (reviewed in Desler et al., 2012). A correction of mitochondrial function therefore has the potential to restore the efficiency of oxidative phosphorylation and prevent age related disorders. Samit Adhya from Genetic Engineering Laboratory at the Indian Institute of Chemical Biology, Kolkata, India has developed a carrier complex derived from a protozoal mitochondrial tRNA import complex (Jash and ⁎ Corresponding author at: Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Building 18.1, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark. Tel.: +45 35326717. E-mail address: [email protected] (L.J. Rasmussen).
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Adhya, 2011). This carrier is able to transport polycistronic RNA up to 6.7 kb in length into the mitochondria (Mahato et al., 2011). Within the mitochondria the RNA is processed to yield functional mRNA and tRNA and translation is demonstrated. In a human hepatocarcinoma cell line harboring multiple mtDNA deletions, mitochondrial respiration was restored using several carrier complexes with polycistronic RNA encoding mitochondrial subunits of the electron transport chain. In mice, RNAs labeled with different fluorescent tags were injected into the tail vein in a complex with the carrier. As a result, tagged RNA was demonstrated to localize to mitochondria of different organs of the mouse. When introducing polycistronic RNA encoding mitochondrial subunits into middle aged rats, the treatment could enhance the respiratory capacity of the skeletal muscle underlining the clinical potential of the carrier system. A recurring theme of several of the presentations regarded the link between dysfunctional mitochondria and tumorigenesis. Keshav K. Singh of the Department of Genetics School of Medicine at the University of Alabama at Birmingham, USA opened his lecture by reminding
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his audience that mitochondrial dysfunction is a hallmark of cancer. Alterations of mtDNA have been correlated with tumor progression and have been reported in a variety of cancers including ovarian, thyroid, salivary, kidney, liver, lung, colon, gastric, brain, bladder, head and neck, and breast cancers (Modica-Napolitano and Singh, 2002, 2004; Penta et al., 2001). The laboratory of Keshav Singh has been using cell cultures devoid of mitochondrial DNA (rho 0 cells) to model whether a depletion of mtDNA can lead to tumorigenesis. The biological relevance of rho 0 cells has been demonstrated in different tissue where they can appear in a mosaic together with cells containing mtDNA (Fayet et al., 2002; Taylor et al., 2003). When comparing rho0 cell lines with their parental mtDNA containing cell lines it has been possible to demonstrate that deletion of mtDNA can induce the production of mitochondrial reactive oxygen species (ROS) and lead to epigenetic changes in the nucleus (Smiraglia et al., 2008). Concurrently, it has in vitro been demonstrated that rho0 cells display a more aggressive tumorigenic phenotype when compared to parental cell lines in respect to anchorage independence and ability to migrate (Singh et al., 2005). As a result it has been shown that growth of rho0 tumors in nude mice were much more aggressive than corresponding growth of tumors with mtDNA (Petros et al., 2005; Shidara et al., 2005). These results have prompted Keshav K. Singh to propose the existence of a mitocheckpoint that monitors the functional state of the mitochondria and aid cells in repairing damaged mitochondria by affecting several nuclear encoded factors. If the mitochondria are severely damaged, the mitocheckpoint can trigger apoptosis. However, if the damage to mitochondria is continuous or persistent the mitocheckpoint may fail leading to genomic instability and cancer. Mutations of the mitochondrial genome as well as a decrease of the mtDNA copy number of breast, liver, lung and colorectal cancers are associated with a poorer prognosis for the patients suffering the cancer (Lièvre et al., 2005; Matsuyama et al., 2003; Yamada et al., 2006; Yu et al., 2007). Concurrently it has been demonstrated that mutations in the mitochondrial polymerase gamma results in a depletion of mtDNA (Kujoth et al., 2005; Trifunovic et al., 2004). Vanniarajan Ayyasamy from Aravind Medical Research Foundation in Madurai, India reported that mutations in the gene encoding the polymerase gamma were present in 12 out of 19 breast tumor samples. In correspondence, the content of mtDNA was decreased in these breast tumors indicating an important role for the polymerase in breast cancer. This correlation was further verified with the construction of cell lines expressing a mutated version of polymerase gamma where it was demonstrated that expression of the polymerase caused a decrease of mtDNA and increased the affected cells invasion abilities (Singh et al., 2009). Ayyasamy ended his presentation with a study where the occurrence of CAG repeats in the polymerase gamma gene in African American woman was determined and correlated with breast cancer. He concluded that the study suggested the possibility of an increased risk of breast cancer in woman with minor CAG repeat variants, but no statistical significant difference could be established with the number of subjects available for the study. Traditionally, the relationship between dysfunctional mitochondria and tumor prognosis and progression has been explained by the detrimental effects of mitochondrial produced ROS. Even though Lene Juel Rasmussen from Center for Healthy Aging at University of Copenhagen, Denemak does not dispute the importance of ROS, she pointed out that the response of dysfunctional mitochondria affects the cells in more ways than just with the overproduction of ROS (Desler et al., 2010). Complete depletion of mtDNA shuts down the mitochondrial respiration and subsequent mitochondrial production of ROS, nevertheless the mutation rate of rho0 yeast is still higher than parental yeast with mitochondrial DNA (Rasmussen et al., 2003). Similarly human rho0 cells have been demonstrated to have a decreased chromosomal stability (Desler et al., 2007). Data presented demonstrated that the cellular dNTP levels of both rho0 yeast and human cells were decreased as a
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result of mitochondrial dysfunction. By itself, a dNTP imbalance is mutagenic for both mtDNA and nuclear DNA (Bebenek and Kunkel, 1990; Meuth, 1989), however preliminary work presented also suggested that imbalanced dNTP pools affect the DNA response induced by alkylating agents. Mitochondrial DNA mutations are common in a broad range of cancers, not just gastric cancer, and it has been suggested that they play a role in the onset of cancer. There is evidence that H. pylori infection induces mutations in the mtDNA. Lene Juel Rasmussen's group showed that H. pylori infection of gastric cells caused mutations to appear in specific regions of the mtDNA and that this was dependent on the H. pylori virulence factors CagA and VacA (Machado et al., 2009). 2. Mitochondria as a target for anticancer therapies Utilizing the mitochondria to promote apoptosis in cancer cells is a very attractive endpoint when developing cancer therapeutics. Dhyan Chandra from the Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York, USA has investigated the molecular mechanism behind how resveratrol induces apoptosis in cancer cells. Through a series of elegant knock-down experiments, Dhyan Chandra and his group have demonstrated that resveratrol induces Bax oligomerization and activation within the cytosol in a process mediated by the X-linked inhibitor of apoptosis protein (XIAP) (Gogada et al., 2011). Activated Bax thereafter translocates to the mitochondria allowing release of cytochrome c, caspase activation and resulting apoptosis. Most importantly, it was demonstrated that p53 was not directly involved in this process, suggesting that resveratrol can induce apoptosis in cancer cells that lack p53 activity or harbor mutant p53. Silibinin is a dietary agent found in artichoke and milk thistle. It is a molecule that has attracted the attention of Rana P. Singh of School of Life Sciences at Central University of Gujarat, Ahmadabad, India due to its anticancer properties and very low toxicity. Mice who had been xenografted with the human bladder cell tumor RT4 cells and feed with silibinin for a period of 12 weeks displayed significant inhibitory effects on tumor growth when compared to control. The amount of apoptotic events in the tumor tissue was correspondingly 3–4 fold increased in mice fed with silibinin. The silibinin mediated apoptosis was in vitro mediated through activation of p53 and caspase activation. As presented, the p53 activation by sibilinin is mediated via the ATM–Chk-2 pathway, which in turn activates caspase 2, in part, via the JNK1/2 kinases and initiates a caspase–cascade activation for mitochondrial apoptosis. Heat shock proteins are a class of functionally related proteins that function as molecular chaperones. Heat shock protein 90 (Hsp90) has been identified as a potential biomarker to target cancer and the ansamycin antibiotic 17AAG is currently under clinical evaluation as an anticancer drug targeting Hsp90 (Vishal et al., 2011). A. S. Sreedhar from the Centre for Cellular and Molecular Biology in Hyderabad has demonstrated the importance for Hsp90 for correct functioning of the mitochondria. Inhibition of Hsp90 by 17AAG has been demonstrated to induce mitochondrial dysfunction including ex vivo mitochondrial swelling, a change in mitochondrial membrane potential, mitochondrial elongation and vacuolization (Vishal et al., 2011). Proteomic analysis revealed that 17AAG treatment resulted in 61% reduction of mitochondrial proteins compared to control. The effect of an inhibition of Hsp90 by 17AAG on the mitochondrial integrity can therefore offer an important explanation to the efficiency of 17AAG as an anticancer drug. Mitochondrial targeted nitroxides are compounds that due to their lipophilic properties can pass biological membranes and due to their cationic properties are preferentially concentrated in the mitochondria of treated cells (Murphy and Smith, 2007). B. Kalyanaraman of the Free Radical Research Center at Medical College of Wisconsin,
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USA has investigated the mitochondrial targeted nitroxide Mitocarboxy proxyl (Mito-CP) which have properties mimicking superoxide dismutase and thereby have the potential to inhibit mitochondrial produced ROS. It has been demonstrated that the production of mitochondrial ROS is essential for Kras-induced anchorage independent growth through the ERK MAPK signaling pathway (Weinberg and Chandel, 2009). According to Kalyananraman, inhibiting both the glycolytic production of ATP and mitochondrial production of ROS by treatment with 2-deoxy-D-glucose (2-DG) and Mito-CP respectively would primarily target cancer cells and inhibit their growth and spreading. Accordingly, when co-treating with these two compounds, a synergistic inhibitory effect was seen in treated cancer cells and mouse tumors (Cheng et al., 2012). Kalyanaraman also discussed how Mito-CP could be used when cisplatin is used as a chemotherapeutic. It is known that cisplatin is nephrotoxic through a mechanism that presumably is related to ROS damage to the mitochondria. When co-treating cisplatin with Mito-CP it was possible to attenuate the cisplatin-induced nephrotoxicity (Mukhopadhyay et al., 2012). Cervical cancer is the most common cancer of woman in developing countries. Although much effort has been made to identify molecular markers for screening of cervical cancers, none have so far been identified. Kapeattu Satyamoorthy of Division of Biotechnology, Manipal Life Sciences Centre, Manipal University, India, have sequenced the entire mtDNA from non-malignant and malignant cervical samples. It was possible to identify 30 novel variations and additional 20 alterations in the samples that previously been associated with different diseases. Furthermore, mitochondrial microsatellite instability was demonstrated for the D-loop region. Differences for mitochondria were also demonstrated for malignant cervical cancer samples when compared to non-malignant samples. These include decrease of mitochondrial mass, copy number and mitochondrial membrane potential. The result obtained, suggest that the changes that occur in mitochondria during cellular transformation can function as molecular markers and be used in the risk assessment of cervical cancer. An aberrant expression of certain mitochondrial proteins like Bcl-2 as well as non-mitochondrial molecular markers can serve as predictors of therapy efficacy when treating patients suffering colorectal cancer according to Upender Manne of Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, USA. However, his major point is that this only applies for a subset of patients depending on a variety of factors, including tumor location, pathologic stage, and race/ethnicity of the patient. In his talk, Upender Manne, therefore, promoted a more personalized strategy where molecular markers for colon cancer should be investigated for cancers in different anatomic locations, at different stages of progression while also considering the race/ethnicity of the patient. Such strategies would have the potential of reducing the personal and socioeconomic burden of cancer. The prognosis for patients with oral squamous cell carcinoma remains poor in spite of advances in therapy of many other malignancies. Ravi Mehrotra of Moti Lal Nehru College, Allabad, India, stressed the importance of early diagnosis and highlighted the urgent need to devise critical diagnostic tools for early detection of oral dysplasia and malignancy that are practical, noninvasive and can be performed in an out-patient setup. He continued his presentation by critically reviewing diagnostic tests available today, including brush biopsy, toluidine blue staining, autoflorescence, salivary proteomics, DNA analysis and spectroscopy. 3. Mitochondrial haplogroups as migration markers and as determinants for mitochondrial function and disease K. Thangaraj from the Centre for Cellular and Molecular Biology, Hyderabad, India began his presentation by summarizing the properties of mtDNA that distinguishes mtDNA from nuclear DNA. These properties include maternal inheritance and the absence of recombination amongst
others. These properties allow the molecule to be used in investigation of the molecular basis of several diseases in forensic sciences but also in studies in origin and migration. Thangaraj continued by explaining that the Indian population historically was very admixed due to groupings based on caste, tribe, religion and language. This admixture allows the correlation between different specific Indian mtDNA haplotypes and rare genetic diseases only affecting a subgroup of the Indian population (Dhandapany et al., 2009; Rani et al., 2010). Furthermore, by comparing different Indian mtDNA haplogroups, it has been possible to portrait the ancient migration from Africa to India (Reich et al., 2009). According to a hypothesis by Wallace et al. (Ruiz-Pesini et al., 2004) the haplotype of the mtDNA is a determinant of the efficiency of oxidative phosphorylation. The mitochondrial membrane potential is primarily utilized to synthesize ATP, however the potential can also be lost as heat through uncoupling of the mitochondrial membrane. The magnitude of this loss is thus dependent on the mtDNA haplotype. According to this hypothesis, certain haplogroups produce more heat by uncoupling than other and are therefore better suited to live in climates with extreme cold (Ruiz-Pesini et al., 2004). As this hypothesis in part can explain why certain ethnicities are more disposed than other to a range of metabolic diseases including diabetes and obesity, Erich Gnaiger from D. Swarovski Research Laboratory in Austria wanted to test this hypothesis. For a period of 6-weeks 16 Danish participants crossed the Greenland icecap on cross-country skies. Immediately before and after the crossing, muscle biopsies were taken from skeletal muscle of the arm and leg from the Danish subjects and from Inuit hunters representing two different mtDNA haplogroups. From the biopsies, the mitochondrial respiratory capacity was measured using the Oroboros Oxygraph (Oroboros Instruments) and compared. It was found that the Danish test-subjects had an adaptive response to the intense exercise in the arctic, however it was also found that mitochondrial coupling control was tightly conserved across the Danish and the Inuit mtDNA haplogroups. From this experiment it was therefore not possible to prove the hypothesis set forth by Wallace and co-workers. However, as Erich Gnaiger concluded, the Danish haplogroup can also have been influenced by cold selection due to repeated continental glaciations. 4. Pathologies resulting from mitochondrial dysfunctions Today mitochondrial diseases can be very difficult to diagnose. To improve upon this, Daniel Lieber from the Department of Systems Biology at Harvard University, Boston, USA has worked on a method to sequence the entire mtDNA and the exons of ~ 1600 nuclear genes encoding proteins localized to the mitochondrion and thereby be able to characterize the entire mitochondrial exome (MitoExome) (Calvo et al., 2012; Lieber et al., 2012). DNA was extracted from the blood or lymphoblasts of 75 patients believed to be suffering from mitochondrial diseases along with 371 controls. According to the work presented by Daniel Lieber 45% of patients suffering from severe, infantile cases of a mitochondrial disease were demonstrated to have recessive genes within the MitoExome, while recessive genes were found in 12% of patients suffering from moderate mitochondrial diseases and only 4% of healthy controls. Even though not all patients could have their disease diagnosed using this approach, it opens the possibility for further development and in the future better and faster diagnosis of mitochondrial patients to be used in the clinic. Approximately 30-40% of men in the reproductive age suffer from qualitative or quantitative defects in sperm production as reviewed in (Shamsi et al., 2008). Mutation and depletion of mtDNA in sperm is known to result in sperm dysfunction and infertility. Rima Dada from Department of Anatomy, AIIMS, New Delhi, India has investigated Indian males suffering from idiopathic infertility and reported a significant increase of ROS levels in the infertile (Venkatesh et al., 2009). The increased levels of ROS can both cause and be caused by
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mutations in mtDNA and correspondingly she reported an increase of mtDNA mutations in the infertile. However, the increase in ROS levels can also be caused by other factors, and accordingly it was demonstrated that the antioxidant capacity of the seminal plasma of the infertile cases was significantly lower. The increased ROS levels were found to correlate with the DNA fragmentation and telomere shortening of sperm cells. Rima Dada concluded her presentation by promoting more research into the effects of supplementing antioxidants in the attempt to promote conception. ROS is an essential component of the innate immune response against intracellular bacteria. The contribution of ROS generated via the phagosomal NADPH oxidase machinery is established (Lambeth, 2004), however the involvement of mitochondrial produced ROS in the innate immune response remains unclear. Sankar Ghosh of Department of Microbiology and Immunology, Columbia University, New York, USA, has demonstrated how the engagement of a subset of the Toll-like receptors augments generation of mitochondrial ROS (West et al., 2011). The response includes translocation of the Toll-like receptor TRAF6 to the mitochondria where it interacts with and ubiquitinates ECSIT, which is implicated in the assembly of the mitochondrial electron transport chain (Vogel et al., 2007), resulting in an increased generation of mitochondrial ROS. ECSIT and TRAF6 depleted macrophages are significantly impaired in their ability to kill intracellular bacteria. Furthermore, expressing catalase in mitochondria also impaired bacterial killing. These results implicate TRAF6 and ECSIT in a novel pathway linking innate immune signaling to mitochondrial generation of ROS (West et al., 2011). The primary product of lipid peroxidation is the reactive and toxic aldehyde 4-hydroxynonenal (4HNE). The aldehyde can be produced as a result of mitochondrial ROS reacting with the mitochondrial and plasma membrane. Aldehyde dehydrogenases (ALDH) are a group of enzymes that are involved in detoxification and removal of reactive aldehydes. Correspondingly, it has been shown that activation of ALDH 2, in rats prior to an ischemic event, reduced the infarct size by 60% likely by inhibiting the formation of cytotoxic aldehydes and thereby attenuating the oxidative stress caused by the event (Chen et al., 2008). As presented by Suresh S. Palaniyandi of the Division of Hypertension and Vascular Research at the Henry Ford Hospital, Michigan, USA, the diabetic heart is more prone to myocardial infarct and heart failure. Furthermore, the diabetic heart is demonstrated to express reduced levels of ALDH (Hamblin et al., 2007). This prompted Palaniyandi to hypothesize that an upregulation of ALDH may reduce diabetes induced cardiac damage analogous to its effect in ischemia. ALDH can be activated by the compound Alda-1 (Chen et al., 2008). As presented he has found that treating cardiomyocytes with high levels of glucose is demonstrated to reduce the levels of ALDH. This effect was attenuated when treating with Alda-1 and as a result apoptosis induced by the high glucose content was prevented in the cardiomyocytes. In mouse models of diabetes, treatment with Alda-1 resulted in an increase in ALDH activity after 12 weeks of treatment and attenuate myocardial fibrosis in the mice. As ALDH activity is low and 4HNE levels are high in human diabetic heart samples, this provides an opportunity for ALDH activators to be used as potential therapeutic agents for diabetes mediated cardiac complications. Besides nuclear genes, mutations in mitochondrial genes can also be involved in the etiology and pathophysiology of hearing loss. Madhu Khullar from Department of Experimental Medicine and Biotechnology, Chandigarh, India focused in her presentation on the specific form of hearing loss known as non-syndromic sensorineural hearing loss (NSHL), where 1–2% of cases are believed to be caused by mutations in mtDNA. The mtDNA mutations believed to be involved in the condition are predominantly genes encoding 12S rRNA and tRNAser involved in translation of mtDNA. When Madhu Khullar investigated subjects from the North Indian population suffering from the condition, she could reveal novel mutations in the genes encoding 12S rRNA and 16S rRNA. This novelty suggests that ethnic variations play a role for prevalence and nature for NSHL.
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Mitochondria play essential roles in neuronal function at synapses. By using C. elegans as a model system, Sandhya P. Koushika from National Centre for Biological Sciences, Bengaluru, India, hae established that the mitochondrial distribution are non-random in neurons. This clearly indicates that the process is regulated. By using microtubule mutants she was able to demonstrate that the distribution was altered revealing microtubules as important mediators of mitochondrial distribution. The distribution of mitochondria along the neuronal process co-relates with the ability of the organism to respond to repeated touch. This suggests that axonal mitochondria may play important roles in neuronal function. 5. Diagnosis of mitochondrial dysfunction in the clinic The first annual conference for the Society of Mitochondrial Research and Medicine, was rounded off with three exceptional talks on the topic of clinical diagnosis of mitochondrial diseases. N. Gayathri from National Institute of Mental Health and Neurosciences, Bengaluru, India presented the battery of histochemical stains and electron microscopy used on skeletal muscle biopsies to diagnose mitochondrial disorders. A. K. Meena from Nizam's Institute of Medical Sciences located in Hyderabad, India presented examples of patients with mitochondrial diseases in a clinical application and the tools available to diagnose these diseases. These tools included investigation of creatine kinase, lactate and glucose in the blood, histochemistry of muscle biopsies, PCR and RFLP analysis of mtDNA, sequencing of mtDNA and electron microscopy of mitochondria. Challa Sundaram from Nizam's Institute of Medical Sciences, Hyderabad, India introduced the eye as a high energy requiring organ often affected by mitochondrial dysfunction. The most common ocular manifestation of a mitochondrial disease is chronic progressive external opthalmoplegia (CPEO), and even though there is no cure for the disease it is important to differentiate the disease from other diseases with similar clinical features including Grave's disease and myasthenia gravis. Sundaram demonstrated that muscle biopsies can be utilized for the diagnosis of CPEO in the form of ragged red fibers and COX negative fibers (Sundaram et al., 2011). The first mitochondrial disease was diagnosed a little more than 50 years ago. In the meantime major breakthroughs have been made within this field, with the promise of even more to be made with an immense contribution to research fields covering aging, tumorigenesis, neuronal disorders, heart diseases, diabetes and many others. With this first annual conference of the Indian Society of Mitochondrial Research and Medicine, it was clearly demonstrated that the region is at the absolute forefront of mitochondrial research. At the same time, the conference established common ground where outstanding researchers within the field can exchange ideas, initiate collaborations and pave the road for many future breakthroughs within the field of mitochondrial research. Acknowledgements We are thankful to Branden Hopkinson for critical reading of manuscript. This work was supported by a grant from Nordea-fonden. References Bebenek, K., Kunkel, T.A., 1990. Frameshift errors initiated by nucleotide misincorporation. Proc. Natl. Acad. Sci. U. S. A. 87, 4946–4950. Calvo, S.E., Compton, A.G., Hershman, S.G., Lim, S.C., Lieber, D.S., Tucker, E.J., Laskowski, A., Garone, C., Liu, S., Jaffe, D.B., Christodoulou, J., Fletcher, J.M., Bruno, D.L., Goldblatt, J., Dimauro, S., Thorburn, D.R., Mootha, V.K., 2012. Molecular diagnosis of infantile mitochondrial disease with targeted next-generation sequencing. Sci. Transl. Med. 4, 118ra10. Chen, C.-H., Budas, G.R., Churchill, E.N., Disatnik, M.-H., Hurley, T.D., Mochly-Rosen, D., 2008. Activation of aldehyde dehydrogenase-2 reduces ischemic damage to the heart. Science 321, 1493–1495. Cheng, G., Zielonka, J., Dranka, B.P., McAllister, D., Mackinnon, A.C., Joseph, J., Kalyanaraman, B., 2012. Mitochondria targeted drugs synergize with 2-deoxyglucose to trigger breast cancer cell death. Cancer Res. 72, 2634–2644.
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Penta, J.S., Johnson, F.M., Wachsman, J.T., Copeland, W.C., 2001. Mitochondrial DNA in human malignancy. Mutat. Res. 488, 119–133. Petros, J.A., Baumann, A.K., Ruiz-Pesini, E., Amin, M.B., Sun, C.Q., Hall, J., Lim, S., Issa, M.M., Flanders, W.D., Hosseini, S.H., Marshall, F.F., Wallace, D.C., 2005. mtDNA mutations increase tumorigenicity in prostate cancer. Proc. Natl. Acad. Sci. U. S. A. 102, 719–724. Rani, D.S., Dhandapany, P.S., Nallari, P., Govindaraj, P., Singh, L., Thangaraj, K., 2010. Mitochondrial DNA haplogroup “R” is associated with Noonan syndrome of south India. Mitochondrion 10, 166–173. Rasmussen, A.K., Chatterjee, A., Rasmussen, L.J., Singh, K.K., 2003. Mitochondria-mediated nuclear mutator phenotype in Saccharomyces cerevisiae. Nucleic Acids Res. 31, 3909–3917. Reich, D., Thangaraj, K., Patterson, N., Price, A.L., Singh, L., 2009. Reconstructing Indian population history. Nature 461, 489–494. Ruiz-Pesini, E., Mishmar, D., Brandon, M., Procaccio, V., Wallace, D.C., 2004. Effects of purifying and adaptive selection on regional variation in human mtDNA. Science 303, 223–226. Shamsi, M.B., Kumar, R., Bhatt, A., Bamezai, R.N.K., Kumar, R., Gupta, N.P., Das, T.K., Dada, R., 2008. Mitochondrial DNA mutations in etiopathogenesis of male infertility. Indian J. Urol. 24, 150–154. Shidara, Y., Yamagata, K., Kanamori, T., Nakano, K., Kwong, J.Q., Manfredi, G., Oda, H., Ohta, S., 2005. Positive contribution of pathogenic mutations in the mitochondrial genome to the promotion of cancer by prevention from apoptosis. Cancer Res. 65, 1655–1663. Singh, K.K., Kulawiec, M., Still, I., Desouki, M.M., Geradts, J., Matsui, S., 2005. Inter-genomic cross talk between mitochondria and the nucleus plays an important role in tumorigenesis. Gene 354, 140–146. Singh, K.K., Ayyasamy, V., Owens, K.M., Koul, M.S., Vujcic, M., 2009. Mutations in mitochondrial DNA polymerase-gamma promote breast tumorigenesis. J. Hum. Genet. 54, 516–524. Smiraglia, D.J., Kulawiec, M., Bistulfi, G.L., Gupta, S.G., Singh, K.K., 2008. A novel role for mitochondria in regulating epigenetic modification in the nucleus. Cancer Biol. Ther. 7, 1182–1190. Sundaram, C., Meena, A.K., Uppin, M.S., Govindaraj, P., Vanniarajan, A., Thangaraj, K., Kaul, S., Kekunnaya, R., Murthy, J.M.K., 2011. Contribution of muscle biopsy and genetics to the diagnosis of chronic progressive external opthalmoplegia of mitochondrial origin. J. Clin. Neurosci. 18, 535–538. Taylor, R.W., Barron, M.J., Borthwick, G.M., Gospel, A., Chinnery, P.F., Samuels, D.C., Taylor, G.A., Plusa, S.M., Needham, S.J., Greaves, L.C., Kirkwood, T.B., Turnbull, D.M., 2003. Mitochondrial DNA mutations in human colonic crypt stem cells. J. Clin. Invest. 112, 1351–1360. Trifunovic, A., Wredenberg, A., Falkenberg, M., Spelbrink, J.N., Rovio, A.T., Bruder, C.E., Bohlooly-Y, M., Gidlöf, S., Oldfors, A., Wibom, R., Törnell, J., Jacobs, H.T., Larsson, N.G., 2004. Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature 429, 417–423. Venkatesh, S., Riyaz, A.M., Shamsi, M.B., Kumar, R., Gupta, N.P., Mittal, S., Malhotra, N., Sharma, R.K., Agarwal, A., Dada, R., 2009. Clinical significance of reactive oxygen species in semen of infertile Indian men. Andrologia 41, 251–256. Vishal, C., Kumar, J.U., Veera Brahmendra Swamy, C., Nandini, R., Srinivas, G., Kumaresan, R., Shashi, S., Sreedhar, A.S., 2011. Repercussion of mitochondria deformity induced by anti-Hsp90 drug 17AAG in human tumor cells. Drug Target Insights 5, 11–32. Vogel, R.O., Janssen, R.J.R.J., van den Brand, M.A.M., Dieteren, C.E.J., Verkaart, S., Koopman, W.J.H., Willems, P.H.G.M., Pluk, W., van den Heuvel, L.P.W.J., Smeitink, J.A.M., Nijtmans, L.G.J., 2007. Cytosolic signaling protein Ecsit also localizes to mitochondria where it interacts with chaperone NDUFAF1 and functions in complex I assembly. Genes Dev. 21, 615–624. Weinberg, F., Chandel, N.S., 2009. Reactive oxygen species-dependent signaling regulates cancer. Cell. Mol. Life Sci. 66, 3663–3673. West, A.P., Brodsky, I.E., Rahner, C., Woo, D.K., Erdjument-Bromage, H., Tempst, P., Walsh, M.C., Choi, Y., Shadel, G.S., Ghosh, S., 2011. TLR signalling augments macrophage bactericidal activity through mitochondrial ROS. Nature 472, 476–480. Yamada, S., Nomoto, S., Fujii, T., Kaneko, T., Takeda, S., Inoue, S., Kanazumi, N., Nakao, A., 2006. Correlation between copy number of mitochondrial DNA and clinicopathologic parameters of hepatocellular carcinoma. Eur. J. Surg. Oncol. 32, 303–307. Yu, M., Zhou, Y., Shi, Y., Ning, L., Yang, Y., Wei, X., Zhang, N., Hao, X., Niu, R., 2007. Reduced mitochondrial DNA copy number is correlated with tumor progression and prognosis in Chinese breast cancer patients. IUBMB Life 59, 450–457.
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Review
Mitochondria in Biology and Medicine Claus Desler, Lene Juel Rasmussen ⁎ Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, DK-2200 Copenhagen, Denmark
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Article history: Received 9 May 2012 Accepted 25 June 2012 Available online 30 June 2012 Keywords: Conference Mitochondria Mitochondrial disease Cancer Mitochondrial haplogroups
a b s t r a c t Ever since the first diagnosis of a mitochondrial disease in 1959 (Ernster et al., 1959), the interest for mitochondrial cytopathies has continued to increase. Originally it was believed that the condition was very rare and primarily effected high-energy requiring tissues resulting in a select few pathologies (Luft, 1994). Since 1959, the understanding of mitochondrial cytopathies has evolved immensely and mitochondrial cytopathies are now known to be the largest group of metabolic diseases and to be resulting in a wide variety of pathologies. “Mitochondria in Biology and Medicine” was the title of the first annual conference of Society of Mitochondrial Research and Medicine — India. The conference was organized by A. S. Sreedhar, Keshav Singh and Kumarasamy Thangaraj, and was held at The Centre for Cellular and Molecular Biology (CCMB) Hyderabad, India, during 9–10 December 2011. The conference featured talks from internationally renowned scientists within the field of mitochondrial research and offered both students and fellow researchers a comprehensive update to the newest research within the field. This paper summarizes key outcomes of the presentations. © 2012 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
Contents 1. Mitochondrial dysfunction in aging and tumorigenesis . . . . . . . . . 2. Mitochondria as a target for anticancer therapies . . . . . . . . . . . . 3. Mitochondrial haplogroups as migration markers and as determinants for 4. Pathologies resulting from mitochondrial dysfunctions . . . . . . . . . 5. Diagnosis of mitochondrial dysfunction in the clinic. . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Mitochondrial dysfunction in aging and tumorigenesis During aging of an organism, and as a result of mitochondrial dysfunctions, the efficiency of oxidative phosphorylation will decline. This is in part a result of a lowering of mitochondrial DNA (mtDNA) content the resultant decline of ATP levels in tissues with high energy demand such as brain, muscle and heart is a major contributory factor to the symptoms of aging (reviewed in Desler et al., 2012). A correction of mitochondrial function therefore has the potential to restore the efficiency of oxidative phosphorylation and prevent age related disorders. Samit Adhya from Genetic Engineering Laboratory at the Indian Institute of Chemical Biology, Kolkata, India has developed a carrier complex derived from a protozoal mitochondrial tRNA import complex (Jash and ⁎ Corresponding author at: Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Building 18.1, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark. Tel.: +45 35326717. E-mail address: [email protected] (L.J. Rasmussen).
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Adhya, 2011). This carrier is able to transport polycistronic RNA up to 6.7 kb in length into the mitochondria (Mahato et al., 2011). Within the mitochondria the RNA is processed to yield functional mRNA and tRNA and translation is demonstrated. In a human hepatocarcinoma cell line harboring multiple mtDNA deletions, mitochondrial respiration was restored using several carrier complexes with polycistronic RNA encoding mitochondrial subunits of the electron transport chain. In mice, RNAs labeled with different fluorescent tags were injected into the tail vein in a complex with the carrier. As a result, tagged RNA was demonstrated to localize to mitochondria of different organs of the mouse. When introducing polycistronic RNA encoding mitochondrial subunits into middle aged rats, the treatment could enhance the respiratory capacity of the skeletal muscle underlining the clinical potential of the carrier system. A recurring theme of several of the presentations regarded the link between dysfunctional mitochondria and tumorigenesis. Keshav K. Singh of the Department of Genetics School of Medicine at the University of Alabama at Birmingham, USA opened his lecture by reminding
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his audience that mitochondrial dysfunction is a hallmark of cancer. Alterations of mtDNA have been correlated with tumor progression and have been reported in a variety of cancers including ovarian, thyroid, salivary, kidney, liver, lung, colon, gastric, brain, bladder, head and neck, and breast cancers (Modica-Napolitano and Singh, 2002, 2004; Penta et al., 2001). The laboratory of Keshav Singh has been using cell cultures devoid of mitochondrial DNA (rho 0 cells) to model whether a depletion of mtDNA can lead to tumorigenesis. The biological relevance of rho 0 cells has been demonstrated in different tissue where they can appear in a mosaic together with cells containing mtDNA (Fayet et al., 2002; Taylor et al., 2003). When comparing rho0 cell lines with their parental mtDNA containing cell lines it has been possible to demonstrate that deletion of mtDNA can induce the production of mitochondrial reactive oxygen species (ROS) and lead to epigenetic changes in the nucleus (Smiraglia et al., 2008). Concurrently, it has in vitro been demonstrated that rho0 cells display a more aggressive tumorigenic phenotype when compared to parental cell lines in respect to anchorage independence and ability to migrate (Singh et al., 2005). As a result it has been shown that growth of rho0 tumors in nude mice were much more aggressive than corresponding growth of tumors with mtDNA (Petros et al., 2005; Shidara et al., 2005). These results have prompted Keshav K. Singh to propose the existence of a mitocheckpoint that monitors the functional state of the mitochondria and aid cells in repairing damaged mitochondria by affecting several nuclear encoded factors. If the mitochondria are severely damaged, the mitocheckpoint can trigger apoptosis. However, if the damage to mitochondria is continuous or persistent the mitocheckpoint may fail leading to genomic instability and cancer. Mutations of the mitochondrial genome as well as a decrease of the mtDNA copy number of breast, liver, lung and colorectal cancers are associated with a poorer prognosis for the patients suffering the cancer (Lièvre et al., 2005; Matsuyama et al., 2003; Yamada et al., 2006; Yu et al., 2007). Concurrently it has been demonstrated that mutations in the mitochondrial polymerase gamma results in a depletion of mtDNA (Kujoth et al., 2005; Trifunovic et al., 2004). Vanniarajan Ayyasamy from Aravind Medical Research Foundation in Madurai, India reported that mutations in the gene encoding the polymerase gamma were present in 12 out of 19 breast tumor samples. In correspondence, the content of mtDNA was decreased in these breast tumors indicating an important role for the polymerase in breast cancer. This correlation was further verified with the construction of cell lines expressing a mutated version of polymerase gamma where it was demonstrated that expression of the polymerase caused a decrease of mtDNA and increased the affected cells invasion abilities (Singh et al., 2009). Ayyasamy ended his presentation with a study where the occurrence of CAG repeats in the polymerase gamma gene in African American woman was determined and correlated with breast cancer. He concluded that the study suggested the possibility of an increased risk of breast cancer in woman with minor CAG repeat variants, but no statistical significant difference could be established with the number of subjects available for the study. Traditionally, the relationship between dysfunctional mitochondria and tumor prognosis and progression has been explained by the detrimental effects of mitochondrial produced ROS. Even though Lene Juel Rasmussen from Center for Healthy Aging at University of Copenhagen, Denemak does not dispute the importance of ROS, she pointed out that the response of dysfunctional mitochondria affects the cells in more ways than just with the overproduction of ROS (Desler et al., 2010). Complete depletion of mtDNA shuts down the mitochondrial respiration and subsequent mitochondrial production of ROS, nevertheless the mutation rate of rho0 yeast is still higher than parental yeast with mitochondrial DNA (Rasmussen et al., 2003). Similarly human rho0 cells have been demonstrated to have a decreased chromosomal stability (Desler et al., 2007). Data presented demonstrated that the cellular dNTP levels of both rho0 yeast and human cells were decreased as a
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result of mitochondrial dysfunction. By itself, a dNTP imbalance is mutagenic for both mtDNA and nuclear DNA (Bebenek and Kunkel, 1990; Meuth, 1989), however preliminary work presented also suggested that imbalanced dNTP pools affect the DNA response induced by alkylating agents. Mitochondrial DNA mutations are common in a broad range of cancers, not just gastric cancer, and it has been suggested that they play a role in the onset of cancer. There is evidence that H. pylori infection induces mutations in the mtDNA. Lene Juel Rasmussen's group showed that H. pylori infection of gastric cells caused mutations to appear in specific regions of the mtDNA and that this was dependent on the H. pylori virulence factors CagA and VacA (Machado et al., 2009). 2. Mitochondria as a target for anticancer therapies Utilizing the mitochondria to promote apoptosis in cancer cells is a very attractive endpoint when developing cancer therapeutics. Dhyan Chandra from the Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York, USA has investigated the molecular mechanism behind how resveratrol induces apoptosis in cancer cells. Through a series of elegant knock-down experiments, Dhyan Chandra and his group have demonstrated that resveratrol induces Bax oligomerization and activation within the cytosol in a process mediated by the X-linked inhibitor of apoptosis protein (XIAP) (Gogada et al., 2011). Activated Bax thereafter translocates to the mitochondria allowing release of cytochrome c, caspase activation and resulting apoptosis. Most importantly, it was demonstrated that p53 was not directly involved in this process, suggesting that resveratrol can induce apoptosis in cancer cells that lack p53 activity or harbor mutant p53. Silibinin is a dietary agent found in artichoke and milk thistle. It is a molecule that has attracted the attention of Rana P. Singh of School of Life Sciences at Central University of Gujarat, Ahmadabad, India due to its anticancer properties and very low toxicity. Mice who had been xenografted with the human bladder cell tumor RT4 cells and feed with silibinin for a period of 12 weeks displayed significant inhibitory effects on tumor growth when compared to control. The amount of apoptotic events in the tumor tissue was correspondingly 3–4 fold increased in mice fed with silibinin. The silibinin mediated apoptosis was in vitro mediated through activation of p53 and caspase activation. As presented, the p53 activation by sibilinin is mediated via the ATM–Chk-2 pathway, which in turn activates caspase 2, in part, via the JNK1/2 kinases and initiates a caspase–cascade activation for mitochondrial apoptosis. Heat shock proteins are a class of functionally related proteins that function as molecular chaperones. Heat shock protein 90 (Hsp90) has been identified as a potential biomarker to target cancer and the ansamycin antibiotic 17AAG is currently under clinical evaluation as an anticancer drug targeting Hsp90 (Vishal et al., 2011). A. S. Sreedhar from the Centre for Cellular and Molecular Biology in Hyderabad has demonstrated the importance for Hsp90 for correct functioning of the mitochondria. Inhibition of Hsp90 by 17AAG has been demonstrated to induce mitochondrial dysfunction including ex vivo mitochondrial swelling, a change in mitochondrial membrane potential, mitochondrial elongation and vacuolization (Vishal et al., 2011). Proteomic analysis revealed that 17AAG treatment resulted in 61% reduction of mitochondrial proteins compared to control. The effect of an inhibition of Hsp90 by 17AAG on the mitochondrial integrity can therefore offer an important explanation to the efficiency of 17AAG as an anticancer drug. Mitochondrial targeted nitroxides are compounds that due to their lipophilic properties can pass biological membranes and due to their cationic properties are preferentially concentrated in the mitochondria of treated cells (Murphy and Smith, 2007). B. Kalyanaraman of the Free Radical Research Center at Medical College of Wisconsin,
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USA has investigated the mitochondrial targeted nitroxide Mitocarboxy proxyl (Mito-CP) which have properties mimicking superoxide dismutase and thereby have the potential to inhibit mitochondrial produced ROS. It has been demonstrated that the production of mitochondrial ROS is essential for Kras-induced anchorage independent growth through the ERK MAPK signaling pathway (Weinberg and Chandel, 2009). According to Kalyananraman, inhibiting both the glycolytic production of ATP and mitochondrial production of ROS by treatment with 2-deoxy-D-glucose (2-DG) and Mito-CP respectively would primarily target cancer cells and inhibit their growth and spreading. Accordingly, when co-treating with these two compounds, a synergistic inhibitory effect was seen in treated cancer cells and mouse tumors (Cheng et al., 2012). Kalyanaraman also discussed how Mito-CP could be used when cisplatin is used as a chemotherapeutic. It is known that cisplatin is nephrotoxic through a mechanism that presumably is related to ROS damage to the mitochondria. When co-treating cisplatin with Mito-CP it was possible to attenuate the cisplatin-induced nephrotoxicity (Mukhopadhyay et al., 2012). Cervical cancer is the most common cancer of woman in developing countries. Although much effort has been made to identify molecular markers for screening of cervical cancers, none have so far been identified. Kapeattu Satyamoorthy of Division of Biotechnology, Manipal Life Sciences Centre, Manipal University, India, have sequenced the entire mtDNA from non-malignant and malignant cervical samples. It was possible to identify 30 novel variations and additional 20 alterations in the samples that previously been associated with different diseases. Furthermore, mitochondrial microsatellite instability was demonstrated for the D-loop region. Differences for mitochondria were also demonstrated for malignant cervical cancer samples when compared to non-malignant samples. These include decrease of mitochondrial mass, copy number and mitochondrial membrane potential. The result obtained, suggest that the changes that occur in mitochondria during cellular transformation can function as molecular markers and be used in the risk assessment of cervical cancer. An aberrant expression of certain mitochondrial proteins like Bcl-2 as well as non-mitochondrial molecular markers can serve as predictors of therapy efficacy when treating patients suffering colorectal cancer according to Upender Manne of Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, USA. However, his major point is that this only applies for a subset of patients depending on a variety of factors, including tumor location, pathologic stage, and race/ethnicity of the patient. In his talk, Upender Manne, therefore, promoted a more personalized strategy where molecular markers for colon cancer should be investigated for cancers in different anatomic locations, at different stages of progression while also considering the race/ethnicity of the patient. Such strategies would have the potential of reducing the personal and socioeconomic burden of cancer. The prognosis for patients with oral squamous cell carcinoma remains poor in spite of advances in therapy of many other malignancies. Ravi Mehrotra of Moti Lal Nehru College, Allabad, India, stressed the importance of early diagnosis and highlighted the urgent need to devise critical diagnostic tools for early detection of oral dysplasia and malignancy that are practical, noninvasive and can be performed in an out-patient setup. He continued his presentation by critically reviewing diagnostic tests available today, including brush biopsy, toluidine blue staining, autoflorescence, salivary proteomics, DNA analysis and spectroscopy. 3. Mitochondrial haplogroups as migration markers and as determinants for mitochondrial function and disease K. Thangaraj from the Centre for Cellular and Molecular Biology, Hyderabad, India began his presentation by summarizing the properties of mtDNA that distinguishes mtDNA from nuclear DNA. These properties include maternal inheritance and the absence of recombination amongst
others. These properties allow the molecule to be used in investigation of the molecular basis of several diseases in forensic sciences but also in studies in origin and migration. Thangaraj continued by explaining that the Indian population historically was very admixed due to groupings based on caste, tribe, religion and language. This admixture allows the correlation between different specific Indian mtDNA haplotypes and rare genetic diseases only affecting a subgroup of the Indian population (Dhandapany et al., 2009; Rani et al., 2010). Furthermore, by comparing different Indian mtDNA haplogroups, it has been possible to portrait the ancient migration from Africa to India (Reich et al., 2009). According to a hypothesis by Wallace et al. (Ruiz-Pesini et al., 2004) the haplotype of the mtDNA is a determinant of the efficiency of oxidative phosphorylation. The mitochondrial membrane potential is primarily utilized to synthesize ATP, however the potential can also be lost as heat through uncoupling of the mitochondrial membrane. The magnitude of this loss is thus dependent on the mtDNA haplotype. According to this hypothesis, certain haplogroups produce more heat by uncoupling than other and are therefore better suited to live in climates with extreme cold (Ruiz-Pesini et al., 2004). As this hypothesis in part can explain why certain ethnicities are more disposed than other to a range of metabolic diseases including diabetes and obesity, Erich Gnaiger from D. Swarovski Research Laboratory in Austria wanted to test this hypothesis. For a period of 6-weeks 16 Danish participants crossed the Greenland icecap on cross-country skies. Immediately before and after the crossing, muscle biopsies were taken from skeletal muscle of the arm and leg from the Danish subjects and from Inuit hunters representing two different mtDNA haplogroups. From the biopsies, the mitochondrial respiratory capacity was measured using the Oroboros Oxygraph (Oroboros Instruments) and compared. It was found that the Danish test-subjects had an adaptive response to the intense exercise in the arctic, however it was also found that mitochondrial coupling control was tightly conserved across the Danish and the Inuit mtDNA haplogroups. From this experiment it was therefore not possible to prove the hypothesis set forth by Wallace and co-workers. However, as Erich Gnaiger concluded, the Danish haplogroup can also have been influenced by cold selection due to repeated continental glaciations. 4. Pathologies resulting from mitochondrial dysfunctions Today mitochondrial diseases can be very difficult to diagnose. To improve upon this, Daniel Lieber from the Department of Systems Biology at Harvard University, Boston, USA has worked on a method to sequence the entire mtDNA and the exons of ~ 1600 nuclear genes encoding proteins localized to the mitochondrion and thereby be able to characterize the entire mitochondrial exome (MitoExome) (Calvo et al., 2012; Lieber et al., 2012). DNA was extracted from the blood or lymphoblasts of 75 patients believed to be suffering from mitochondrial diseases along with 371 controls. According to the work presented by Daniel Lieber 45% of patients suffering from severe, infantile cases of a mitochondrial disease were demonstrated to have recessive genes within the MitoExome, while recessive genes were found in 12% of patients suffering from moderate mitochondrial diseases and only 4% of healthy controls. Even though not all patients could have their disease diagnosed using this approach, it opens the possibility for further development and in the future better and faster diagnosis of mitochondrial patients to be used in the clinic. Approximately 30-40% of men in the reproductive age suffer from qualitative or quantitative defects in sperm production as reviewed in (Shamsi et al., 2008). Mutation and depletion of mtDNA in sperm is known to result in sperm dysfunction and infertility. Rima Dada from Department of Anatomy, AIIMS, New Delhi, India has investigated Indian males suffering from idiopathic infertility and reported a significant increase of ROS levels in the infertile (Venkatesh et al., 2009). The increased levels of ROS can both cause and be caused by
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mutations in mtDNA and correspondingly she reported an increase of mtDNA mutations in the infertile. However, the increase in ROS levels can also be caused by other factors, and accordingly it was demonstrated that the antioxidant capacity of the seminal plasma of the infertile cases was significantly lower. The increased ROS levels were found to correlate with the DNA fragmentation and telomere shortening of sperm cells. Rima Dada concluded her presentation by promoting more research into the effects of supplementing antioxidants in the attempt to promote conception. ROS is an essential component of the innate immune response against intracellular bacteria. The contribution of ROS generated via the phagosomal NADPH oxidase machinery is established (Lambeth, 2004), however the involvement of mitochondrial produced ROS in the innate immune response remains unclear. Sankar Ghosh of Department of Microbiology and Immunology, Columbia University, New York, USA, has demonstrated how the engagement of a subset of the Toll-like receptors augments generation of mitochondrial ROS (West et al., 2011). The response includes translocation of the Toll-like receptor TRAF6 to the mitochondria where it interacts with and ubiquitinates ECSIT, which is implicated in the assembly of the mitochondrial electron transport chain (Vogel et al., 2007), resulting in an increased generation of mitochondrial ROS. ECSIT and TRAF6 depleted macrophages are significantly impaired in their ability to kill intracellular bacteria. Furthermore, expressing catalase in mitochondria also impaired bacterial killing. These results implicate TRAF6 and ECSIT in a novel pathway linking innate immune signaling to mitochondrial generation of ROS (West et al., 2011). The primary product of lipid peroxidation is the reactive and toxic aldehyde 4-hydroxynonenal (4HNE). The aldehyde can be produced as a result of mitochondrial ROS reacting with the mitochondrial and plasma membrane. Aldehyde dehydrogenases (ALDH) are a group of enzymes that are involved in detoxification and removal of reactive aldehydes. Correspondingly, it has been shown that activation of ALDH 2, in rats prior to an ischemic event, reduced the infarct size by 60% likely by inhibiting the formation of cytotoxic aldehydes and thereby attenuating the oxidative stress caused by the event (Chen et al., 2008). As presented by Suresh S. Palaniyandi of the Division of Hypertension and Vascular Research at the Henry Ford Hospital, Michigan, USA, the diabetic heart is more prone to myocardial infarct and heart failure. Furthermore, the diabetic heart is demonstrated to express reduced levels of ALDH (Hamblin et al., 2007). This prompted Palaniyandi to hypothesize that an upregulation of ALDH may reduce diabetes induced cardiac damage analogous to its effect in ischemia. ALDH can be activated by the compound Alda-1 (Chen et al., 2008). As presented he has found that treating cardiomyocytes with high levels of glucose is demonstrated to reduce the levels of ALDH. This effect was attenuated when treating with Alda-1 and as a result apoptosis induced by the high glucose content was prevented in the cardiomyocytes. In mouse models of diabetes, treatment with Alda-1 resulted in an increase in ALDH activity after 12 weeks of treatment and attenuate myocardial fibrosis in the mice. As ALDH activity is low and 4HNE levels are high in human diabetic heart samples, this provides an opportunity for ALDH activators to be used as potential therapeutic agents for diabetes mediated cardiac complications. Besides nuclear genes, mutations in mitochondrial genes can also be involved in the etiology and pathophysiology of hearing loss. Madhu Khullar from Department of Experimental Medicine and Biotechnology, Chandigarh, India focused in her presentation on the specific form of hearing loss known as non-syndromic sensorineural hearing loss (NSHL), where 1–2% of cases are believed to be caused by mutations in mtDNA. The mtDNA mutations believed to be involved in the condition are predominantly genes encoding 12S rRNA and tRNAser involved in translation of mtDNA. When Madhu Khullar investigated subjects from the North Indian population suffering from the condition, she could reveal novel mutations in the genes encoding 12S rRNA and 16S rRNA. This novelty suggests that ethnic variations play a role for prevalence and nature for NSHL.
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Mitochondria play essential roles in neuronal function at synapses. By using C. elegans as a model system, Sandhya P. Koushika from National Centre for Biological Sciences, Bengaluru, India, hae established that the mitochondrial distribution are non-random in neurons. This clearly indicates that the process is regulated. By using microtubule mutants she was able to demonstrate that the distribution was altered revealing microtubules as important mediators of mitochondrial distribution. The distribution of mitochondria along the neuronal process co-relates with the ability of the organism to respond to repeated touch. This suggests that axonal mitochondria may play important roles in neuronal function. 5. Diagnosis of mitochondrial dysfunction in the clinic The first annual conference for the Society of Mitochondrial Research and Medicine, was rounded off with three exceptional talks on the topic of clinical diagnosis of mitochondrial diseases. N. Gayathri from National Institute of Mental Health and Neurosciences, Bengaluru, India presented the battery of histochemical stains and electron microscopy used on skeletal muscle biopsies to diagnose mitochondrial disorders. A. K. Meena from Nizam's Institute of Medical Sciences located in Hyderabad, India presented examples of patients with mitochondrial diseases in a clinical application and the tools available to diagnose these diseases. These tools included investigation of creatine kinase, lactate and glucose in the blood, histochemistry of muscle biopsies, PCR and RFLP analysis of mtDNA, sequencing of mtDNA and electron microscopy of mitochondria. Challa Sundaram from Nizam's Institute of Medical Sciences, Hyderabad, India introduced the eye as a high energy requiring organ often affected by mitochondrial dysfunction. The most common ocular manifestation of a mitochondrial disease is chronic progressive external opthalmoplegia (CPEO), and even though there is no cure for the disease it is important to differentiate the disease from other diseases with similar clinical features including Grave's disease and myasthenia gravis. Sundaram demonstrated that muscle biopsies can be utilized for the diagnosis of CPEO in the form of ragged red fibers and COX negative fibers (Sundaram et al., 2011). The first mitochondrial disease was diagnosed a little more than 50 years ago. In the meantime major breakthroughs have been made within this field, with the promise of even more to be made with an immense contribution to research fields covering aging, tumorigenesis, neuronal disorders, heart diseases, diabetes and many others. With this first annual conference of the Indian Society of Mitochondrial Research and Medicine, it was clearly demonstrated that the region is at the absolute forefront of mitochondrial research. At the same time, the conference established common ground where outstanding researchers within the field can exchange ideas, initiate collaborations and pave the road for many future breakthroughs within the field of mitochondrial research. Acknowledgements We are thankful to Branden Hopkinson for critical reading of manuscript. This work was supported by a grant from Nordea-fonden. References Bebenek, K., Kunkel, T.A., 1990. Frameshift errors initiated by nucleotide misincorporation. Proc. Natl. Acad. Sci. U. S. A. 87, 4946–4950. Calvo, S.E., Compton, A.G., Hershman, S.G., Lim, S.C., Lieber, D.S., Tucker, E.J., Laskowski, A., Garone, C., Liu, S., Jaffe, D.B., Christodoulou, J., Fletcher, J.M., Bruno, D.L., Goldblatt, J., Dimauro, S., Thorburn, D.R., Mootha, V.K., 2012. Molecular diagnosis of infantile mitochondrial disease with targeted next-generation sequencing. Sci. Transl. Med. 4, 118ra10. Chen, C.-H., Budas, G.R., Churchill, E.N., Disatnik, M.-H., Hurley, T.D., Mochly-Rosen, D., 2008. Activation of aldehyde dehydrogenase-2 reduces ischemic damage to the heart. Science 321, 1493–1495. Cheng, G., Zielonka, J., Dranka, B.P., McAllister, D., Mackinnon, A.C., Joseph, J., Kalyanaraman, B., 2012. Mitochondria targeted drugs synergize with 2-deoxyglucose to trigger breast cancer cell death. Cancer Res. 72, 2634–2644.
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