Defects in mitochondrial axonal transport and membrane potential without increased reactive oxygen species production in a Drosophila model of Friedreich ataxia.

Friedreich ataxia, a neurodegenerative disorder resulting from frataxin deficiency, is thought to involve progressive cellular damage from oxidative stress. In Drosophila larvae with reduced frataxin expression (DfhIR), we evaluated possible mechanisms of cellular neuropathology by quantifying mitochondrial axonal transport, membrane potential (MMP), and reactive oxygen species (ROS) production in the DfhIR versus wild-type nervous system throughout development. Although dying-back neuropathy in DfhIR larvae did not occur until late third instar, reduced MMP was already apparent at second instar in the cell bodies, axons and neuromuscular junctions (NMJs) of segmental nerves. Defects in axonal transport of mitochondria appeared late in development in distal nerve of DfhIR larvae, with retrograde movement preferentially affected. As a result, by late third instar the neuromuscular junctions (NMJs) of DfhIR larvae accumulated a higher density of mitochondria, many of which were depolarized. Notably, increased ROS production was not detected in any neuronal region or developmental stage in DfhIR larvae. However, when challenged with antimycin A, neurons did respond with a larger increase in ROS. We propose that pathology in the frataxin-deficient nervous system involves decreased MMP and ATP production followed by failures of mitochondrial transport and NMJ function.

Mutations in the mitochondrial genome confer resistance of cancer cells to anticancer drugs.

The majority of cancer cells harbor homoplasmic somatic mutations in the mitochondrial genome. We show here that mutations in mitochondrial DNA (mtDNA) are responsible for anticancer drug tolerance. We constructed several trans-mitochondrial hybrids (cybrids) with mtDNA derived from human pancreas cancer cell lines CFPAC-1 and CAPAN-2 as well as from healthy individuals. These cybrids contained the different mitochondrial genomes with the common nuclear background. We compared the mutant and wild-type cybrids for resistance against an apoptosis-inducing reagent and anticancer drugs by exposing the cybrids to staurosporine, 5-fluorouracil, and cisplatin in vitro, and found that all mutant cybrids were more resistant to the apoptosis-inducing and anticancer drugs than wild-type cybrids. Next, we transplanted mutant and wild-type cybrids into nude mice to generate tumors. Tumors derived from mutant cybrids were more resistant than those from wild-type cybrids in suppressing tumor growth and inducing massive apoptosis when 5-fluorouracil and cisplatin were administered. To confirm the tolerance of mutant cybrids to anticancer drugs, we transplanted a mixture of mutant and wild-type cybrids at a 1:1 ratio into nude mice and examined the effect by the drugs on the drift of the ratio of mutant and wild-type mtDNA. The mutant mtDNA showed better survival, indicating that mutant cybrids were more resistant to the anticancer drugs. Thus, we propose that mutations in the mitochondrial genome are potential targets for prognosis in the administration of anticancer drugs to cancer patients.

Protection of hepatic cells from apoptosis induced by ischemia/reperfusion injury by protein therapeutics.

AIM: Apoptosis is involved in hepatic ischemia/reperfusion injury. The protein FNK, constructed from an anti-apoptotic protein Bcl-x(L), exhibits the stronger anticell death activity. We evaluated the effect of FNK on apoptosis after hepatic ischemia and reperfusion, using FNK-overexpressing transgenic mice and the HIV/Tat protein-transduction-domain (PTD) that mediates the introduction of FNK into cells when fused with FNK (PTD-FNK). METHODS: Mice were given hepatic ischemic insult for 90 min followed by reperfusion for 3 h. FNK overexpression was determined by immunohistochemistry and Western blot. PTD-FNK was intraperitoneally injected into wild mice 3 h before the insult. Liver injury was determined by the caspase activation, DNA fragmentation, and hematoxylin-eosin and terminal deoxynucleotidyl transferase-mediated dUTP- digoxigenin nick-end labelling (TUNEL) stainings. RESULTS: In FNK-transgenic mice, FNK overexpression inhibited the activation of caspase 3/caspase 3-like activity and DNA fragmentation caused by the injury. In wild mice preinjected with PTD-FNK, PTD-FNK significantly inhibited the caspase activation and DNA fragmentation, reduced the area of liver vacuolization, and protected hepatic cells surrounding blood vessels, irrespective of central or portal veins, from the ischemia/reperfusion damage. CONCLUSIONS: FNK inhibits apoptotic death due to the ischemia/reperfusion injury. Our results provide the reasonable expectation of therapeutic protein PTD-FNK for clinical applications, such as transplantation, to protect against ischemia/reperfusion injury.

Positive contribution of pathogenic mutations in the mitochondrial genome to the promotion of cancer by prevention from apoptosis.

The role of mitochondrial dysfunction in cancer has been a subject of great interest and much ongoing investigation. Although most cancer cells harbor somatic mutations in mitochondrial DNA (mtDNA), the question of whether such mutations contribute to the promotion of carcinomas remains unsolved. Here we used trans-mitochondrial hybrids (cybrids) containing a common HeLa nucleus and mtDNA of interest to compare the role of mtDNA against the common nuclear background. We constructed cybrids with or without a homoplasmic pathogenic point mutation at nucleotide position 8,993 or 9,176 in the mtDNA ATP synthase subunit 6 gene (MTATP6) derived from patients with mitochondrial encephalomyopathy. When the cybrids were transplanted into nude mice, the MTATP6 mutations conferred an advantage in the early stage of tumor growth. The mutant cybrids also increased faster than wild type in culture. To complement the mtDNA mutations, we transfected a wild-type nuclear version of MTATP, whose codons were converted to the universal genetic codes containing a mitochondrial target sequence, into the nucleus of cybrids carrying mutant MTATP6. The restoration of MTATP slowed down the growth of tumor in transplantation. Conversely, expression of a mutant nuclear version of MTATP6 in the wild-type cybrids declined respiration and accelerated the tumor growth. These findings showed that the advantage in tumor growth depended upon the MTATP6 function but was not due to secondary nuclear mutations caused by the mutant mitochondria. Because apoptosis occurred less frequently in the mutant versus wild-type cybrids in cultures and tumors, the pathogenic mtDNA mutations seem to promote tumors by preventing apoptosis.

MIDAS/GPP34, a nuclear gene product, regulates total mitochondrial mass in response to mitochondrial dysfunction.

To investigate the regulatory system in mitochondrial biogenesis involving crosstalk between the mitochondria and nucleus, we found a factor named MIDAS (mitochondrial DNA absence sensitive factor) whose expression was enhanced by the absence of mitochondrial DNA (mtDNA). In patients with mitochondrial diseases, MIDAS expression was increased only in dysfunctional muscle fibers. A majority of MIDAS localized to mitochondria with a small fraction in the Golgi apparatus in HeLa cells. To investigate the function of MIDAS, we stably transfected HeLa cells with an expression vector carrying MIDAS cDNA or siRNA. Cells expressing the MIDAS protein and the siRNA constitutively showed an increase and decrease in the total mass of mitochondria, respectively, accompanying the regulation of a mitochondria-specific phospholipid, cardiolipin. In contrast, amounts of the mitochondrial DNA, RNA and proteins did not depend upon MIDAS. Thus, MIDAS is involved in the regulation of mitochondrial lipids, leading to increases of total mitochondrial mass in response to mitochondrial dysfunction.