Mitochondria 2001 Meeting, San Diego, California, February 28–March 2, 2001

Mitochondrion 1 (2001) 87±116

www.elsevier.com/locate/mito

Abstracts

Mitochondria 2001 Meeting, San Diego, California, February 28±March 2, 2001 1 Molecular, biochemical, and histologic markers of oxidative damage in patients with Alzheimer's disease M.A. Smith Institute of Pathology, Case Western Reserve University, 2085 Adelbert Road, Cleveland, OH 44106, USA The ®nding that oxidative damage in Alzheimer's disease is limited to the neuronal cytoplasm suggesting that mitochondrial abnormalities might also be a part of the spectrum of chronic oxidative stress of Alzheimer's disease. In this study, we used in situhybridization to mtDNA, immunocytochemistry of cytochrome oxidase, and morphometry of electron micrographs of biopsy specimens to determine whether there are mitochondrial abnormalities in Alzheimer's disease and their relationship to oxidative damage marked by 8-hydroxyguanosine and nitrotyrosine. We found that the same neurons showing increased oxidative damage in Alzheimer's disease have a striking increase in mtDNA. Surprisingly, much of the mtDNA and cytochrome oxidase is found in the neuronal cytoplasm and in the case of mtDNA, the vacuoles associated with lipofuscin. Morphometric analysis showed that mitochondria are signi®cantly reduced in Alzheimer's disease while vacuoles associated with lipofuscin are signi®cantly increased. The relationship shown here between the site and extent of mitochondrial abnormalities and oxidative damage suggests an intimate association between these features in Alzheimer's disease. 2 Stimulation of brain, heart and skeletal muscle mitochondrial oxidative phosphorylation in aged rats after hydrosoluble coenzyme Q10 treatment A. Gvozdjakova, J. Kucharska, Z. Braunova Pharmacobiochemical Laboratory of the Medical Faculty, Comenius University, Bratislava, Slovak Republic Defects of the mitochondrial bioenergetics capabilities, coenzyme Q concentration and increased free oxygen radicals production participate in aging processes. Antioxidants ± such as coenzyme Q10 may play a role in the prevention of the aging degenPII: S 1567-724 9(01)00006-X

erative changes. The aim of this study was to investigate if the chronic effect of coenzyme Q10 treatment could improve mitochondrial respiratory chain function, ATP production and coenzyme Q concentration of brain, heart and skeletal muscle mitochondria damaged in aged rats. Methods: 19±22 month old rats (Wistar, male) were treated with hydrosoluble coenzyme Q10 (Q-Gel e, Tishcon Corp., USA) by intragastric tube over 4 weeks. The daily dose of CoQ10 was 200 mg/kg body weight. Mitochondrial oxidative phosphorylation and cytochrome oxidase activity were measured using a Clark oxygen electrode (Oxygraph Gilson, USA). CoQ10 and CoQ9 were determined by HPLC (LKB-Pharmacia, Sweden). Results: Different effects of CoQ10 on mitochondrial function of brain, heart and skeletal muscle in aged rats were proven. (1) In the brain, mitochondria were a signi®cantly increased function of the NAD- and FAD-part of the respiratory chain without increasing mitochondrial CoQ9 and CoQ10 concentrations. (2) In myocard mitochondria were stimulated in the FAD-part of the respiratory chain (without the NAD-part) with increased CoQ9 (6.6%) and CoQ10 (13.0%). (3) In skeletal muscle mitochondria were signi®cantly increased in the respiratory chain function with increased CoQ9 at 88.7%. In all organs there was increased mitochondrial cytochrome oxidase activity. Conclusion: Stimulation of mitochondrial oxidative phosphorylation after 4 weeks hydrosoluble CoQ10 treatment may be bene®cial in prevention of degenerative processes in aging. This study was supported by Grant Ministry of Education, Slovak Republic and Tishcon Corp., USA. 3 Detection of mitochondrial DNA damage and respiratory chain inhibition in yeast R.C. von Borstel 1, C. DeHaan 1, R.W. von Borstel 2, B. Barclay 3 1 Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9; 2Pro-Neuron, Inc., 16020 Industrial Drive, Gaithersburg, MD 20877, USA; 3Planet Biotechnologies, 14 Mission Avenue, St. Albert, Alberta, Canada T8N 1H4 Pharmaceutical compounds are not commonly tested for mito-

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chondrial damage effects. We have developed a rapid biological test in the yeast Saccharomyces cerevisiae to assay for drug-induced mitochondrial DNA damage and respiratory chain inhibition. One to three petri dishes are used for each assay. The substance to be tested is placed in a well in an agar-medium plate where the yeast cells have been spread. The substance diffuses into the agar, and, at the highest concentrations close to the well, the cells will exhibit the rho - phenotype if the agent is active. Then, one more plate is used to distinguish between DNA damage and respiratory chain inhibition for those agents. For the second assay, the cells that show the rho phenotype are replated in the absence of the agent. The cells that retain the rho - phenotype are evidence that the drug is a mitochondrial genotoxin, e.g. ethidium bromide and ¯uorocytosine are mitochondrial genotoxins. Cells that do not retain the rho - phenotype are evidence that the drug is a respiratory chain inhibitor, e.g. doxorubacin and thiabendoazole are respiratory chain inhibitors.

4 Late onset, subacute fulminant hepatic failure, renal failure, and coma resulting from valproic acid treatment and intercurrent infection in an adult with mitochondrial disease R.K. Naviaux 1, T. Hassanein 2, A.G. Roy 2, C. Behling 3, M.R. Gar®nkel 2, A. Precht 2, V. Pinto 2, K. Martinez 4, R.L. Curtis 5 1 The Mitochondrial and Metabolic Disease Center, Department of Medicine; 2Liver Transplant Program, Department of Surgery; 3 Department of Pathology; 4Department of Neurosciences; 5 Clinical Nutrition, University of California, San Diego School of Medicine, San Diego, CA, USA We describe the fatal case of valproic acid-induced, subacute fulminant hepatic failure in a 34-year-old female with mitochondrial disease. The patient was 60% heteroplasmic for the NARP mutation (T8993G) in mitochondrial DNA. She was treated for 9 months with valproic acid for partial complex seizures without evidence of liver toxicity. After 9 months, valproic acid therapy was stopped because it was ineffective in controlling the patient's seizures. Acute sinusitis complicated the last month of the patient's pre-admission course. Seventeen days after discontinuation of valproate therapy, the patient awoke with rapid breathing, in delirium, and was found to be in acute hepatic failure, acute renal failure, and severe metabolic acidosis. Her condition deteriorated to coma later in the day of admission. The patient was initially placed on the liver transplant list before a liver biopsy and her clinical course clari®ed the etiology of her hepatic injury. She suffered three cardiopulmonary arrests on the day of admission, and was in a coma for 10 days before awaking. Despite evidence of improving hepatic function, she died of sepsis on hospital day 20. We describe the clinical, biochemical, neurological, histopathologic, and ultrastructural features of valproic acid toxicity in the context of pre-existing mitochondrial disease. Conclusion: Valproic acid appears to unmask mitochondrial disease in patients with pre-existing mitochondrial dysfunction. If valproic acid must be used in the treatment of a patient with known mitochondrial disease, vigilant monitoring of liver function is required for at least 1±2 months following the discontinuation of therapy to rule out the possibility of late onset of

hepatocellular injury. In the context of fulminant hepatic failure, liver biopsy is essential to con®rm valproic acid liver injury and to rule out other etiologies, particularly in patients with mitochondrial disease.

5 Reactive oxygen species generation by mitochondria in pathophysiological models Y. Kushnareva, A. Andreyev, A.N. Murphy MitoKor, San Diego, CA, USA Reactive oxygen species (ROS) generation in mitochondria was measured using a new Amplex Red assay (Molecular Probes). The method is found to be suitable to detect low rates of ROS production in isolated heart and brain mitochondria oxidizing physiologically relevant NAD-linked substrates. Dependence of ROS generation on electron transport chain inhibition, Ca 21 load, mitochondrial permeability transition (MPT), and cytochrome c release was studied. These conditions are related to Parkinson's disease (Complex I defect) and different apoptotic cell death paradigms (MPT and cytochrome c release). (1) Under the experimental conditions used, Ca 21 uptake and/or MPT induction did not potentiate ROS generation in the presence of NAD-linked substrates. Moreover, in the presence of succinate, MPT was followed by a decrease in ROS production. The latter effect can be imitated by uncouplers of oxidative phosphorylation. (2) Complete inhibition of electron ¯ow distal to red-ox carriers of Complex I resulted in a dramatic increase in ROS production in the presence of NAD-linked substrates. In particular, myxothiazol, a known inhibitor of free radical formation in Complex III, stimulated ROS generation as did Complex I inhibitors rotenone and MPP 1. The data indicate that Complex I rather than Complex III is a primary site of ROS generation in both heart and brain mitochondria. Studies are in progress to elucidate the mechanisms of free radical formation in Complex I.

6 Expanding the spectrum of mitochondrial diseases: morbidity associated with low level heteroplasmy C.A. Bay 1, A. Goldstein 2, E.B. Sanders 3, L.-A. McDonald 3 1 Division of Medical Genetics, Department of Pediatrics, University of Pittsburgh School of Medicine; 2Division of Pediatric Neurology, Department of Pediatrics, University of Pittsburgh School of Medicine; 3Division of Medical Genetics, Children's Hospital of Pittsburgh, Pittsburgh, PA, USA Pedigree analysis of families with mtDNA point mutations and other disorders of energy metabolism suggests that family members have symptoms of systemic illness more often than would be expected by chance alone. The maternal grandmother of a boy with a known A3243G mutation and MELAS phenotype has been monitored. She considered herself well until age 58 when she noted the onset of intermittent abdominal pain. It was exacerbated by emotional distress, and relieved by Donnatal and dietary changes

Abstracts including small frequent meals and an increase in intake of fruits and vegetables. She was diagnosed with irritable bowel syndrome. At age 64 she was noted to have hypercholesterolemia (250 mg/dl, normal ,200), and was started on 10 mg/day of atorvastatin. After 4 months she developed progressive muscle pain and weakness, which abated after cessation of the medication. Her blood mtDNA mutation analysis was undetectable (,5%), but rectal biopsy samples were positive for the A3243G mutation with 14% heteroplasmy. Her 48-year-old male maternal line ®rst cousin, was on disability for chronic fatigue. He felt best with intake of infant formula. He was described as `gaunt'. Analysis of A3243G mtDNA mutation in blood was undetectable (,5%). Pedigree analysis of families with various mtDNA point mutations suggests that a subset of the following diseases might be due to low levels of heteroplasmy: irritable bowel syndrome, chronic fatigue syndrome, diabetes, deafness, mild hypercholesterolemia, and early myocardial infarction. They and other individuals with disorders of energy metabolism may have increased sensitivity to viral illnesses. Further, symptomatology may be due to compensatory mechanisms by the body to minimize the impact of energy insuf®ciency. We hypothesize that testing appropriate family members, and appropriate tissues, for low level heteroplasmy may diagnose these conditions, allow appropriate therapy, and minimize side effects related to medications.

7 Mitochondrial etiologies of pseudo-obstruction and dysmotility in children C. Bay, C. DiLorenzo, A. Goldstein, E. Sanders, L.-A. McDonald, M. DelVecchio, S. Shanske, M. Hirano University of Pittsburgh, Children's Hospital of Pittsburgh, Pittsburgh, PA; Columbia-Presbyterian Medical Center, New York, NY, USA Intestinal pseudo-obstruction and dysmotility are symptoms, which are a clue to a disease of mitochondrial function and/or energy metabolism. We hypothesize that genetic analysis of children with pseudo-obstruction and/or dysmotility may aid in the determination of the underlying mechanisms of pathogenesis in this group of patients, and that an understanding of pathogenesis may suggest potential treatment options. A series of children referred for diagnosis and management of pseudo-obstruction and/ or dysmotility are under evaluation. After gastroenterological determination that the child has evidence of dysmotility and/or pseudoobstruction, and consent for the study is obtained, a complete history and physical examination is performed. Pedigree analysis, in particular noting subtle symptoms suggesting a mitochondrial disease, is performed. Motility data and previous records are reviewed. Blood analysis of mtDNA mutation 3243 and thymidine phosphorylase activity is obtained. Testing results to date: blood for mtDNA 3243 mutation has been obtained in 20 participants. Thirteen are negative, and seven are pending. Blood for thymidine phosphorylase activity has been obtained on 19 participants. Thirteen are negative, and six samples are pending. Reviews of motility studies are in progress. A total of 21 patients are enrolled in the

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study to date. Pedigree analysis indicates two families consistent with a clinical diagnosis of MELAS, with positive maternal line inheritance. Record review indicates that at least two children have documented decreased activity of components of the electron transport chain, including Complex III de®ciency in one child, and Complex I and III de®ciency in a child with a family history consistent with MELAS. These data suggest that disorders of energy metabolism are an etiology in a subset of patients with pseudoobstruction and/or dysmotility, and that analysis of functional activity of components of the electron transport chain and pedigree analysis are indicated. They may be helpful in determining underlying pathogenesis, and potential treatments.

8 Brain Leigh-like lesions in children with Kearns±Sayre syndrome and widespread pleioplasmic mtDNA rearrangements E. Wilichowski, A. Borchert, A. Ohlenbusch, A. KohlschuÈtter, S. Schweitzer, A. von Moers, W. Voss, D. Das, F. Hanefeld Abteilung Paediatrie/Neuropaediatrie, Universitaets-Kinderklinik, Robert-Koch-Strasse 40, D-37075 Goettingen, Germany Introduction: Kearns±Sayre syndrome (KSS) is a mitochondrial encephalomyopathy characterized by onset before adulthood with external ophthalmoplegia, retinopathy, cerebellar dysfunction, deafness and heart block (`classical type'). In some patients, additional manifestations of various non-neuromuscular systems have been described (`multisystemic type'). Large-scale rearrangements (deletions, duplications) of the mitochondrial (mt)DNA are the genetic hallmark of KSS. Patients and methods: In a prospective study about the clinical, neuroradiological (CT/MRI/MRS), biochemical and genetic presentation of mitochondriopathies in childhood, we studied so far 26 patients with mtDNA rearrangements ranging from 3670 to 9485 bp. Tissue distribution and quanti®cation of the rearranged molecules (deletions, duplications) were determined. Fifteen patients show the multisystemic type (hypoparathyroidism [n ˆ 6], renal Fanconi syndrome [n ˆ 4], Bartter-like tubulopathy [n ˆ 1], diabetes mellitus [n ˆ 2], transient Pearson syndrome [n ˆ 2]). In all of them, pleioplasmic rearrangements (deletions 1 duplications) are present in all tissues studied. Results: Eight patients of this group (8±19 years) presented with a progressive neurological course including pyramidal and extrapyramidal movement disturbances and brain stem symptoms like strabism, nystagmus, swallowing abnormalities and somnolence leading to wheel chair-bound (n ˆ 8), tube feeding (n ˆ 4) and death (n ˆ 2). CT/MRI studies demonstrated symmetric lesions of the basal ganglia, thalamus and/or brain stem which are characteristic for those seen in Leigh syndrome. HMR spectroscopy of these Leigh-like alterations disclosed marked lactate accumulation and signs of progressive neuronal loss. Conclusion: In our study, widespread pleioplasmic mtDNA rearrangements are strongly associated with the multisystemic type of KSS and a poor prognosis. The progressive neurological course leading to immobility, brain stem dysfunction and early death is accompanied by the appearance of Leigh-like lesions. The presence of both, deleted and duplicated populations seems to be

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responsible for the ubiquitous tissue distribution of rearranged mtDNA and the severe biochemical abnormalities of oxidative phosphorylation especially in the brain where they are indistinguishable from those seen in Leigh syndrome.

9 Mitochondrial disease induced by Atorvastatin in a woman with A3243G mtDNA mutation identi®ed in colon biopsy samples but not in blood J.A. Shaver Jr. 1, A. Goldstein 2, C.A. Bay 3 1 University of Pittsburgh School of Medicine; 2Division of Pediatric Neurology, Department of Pediatrics, University of Pittsburgh School of Medicine; 3Division of Medical Genetics, Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Inhibitors of HMG CoA reductase (statins) are effective cholesterol-lowering agents, which inhibit the production of mevalonate. One consequence is reduced production of ubiquinone (Coenzyme Q10). It has been speculated that the side effects of statins, such as myopathy are attributable to the decreased level of CoQ10, and that statins might precipitate symptoms including myopathy in patients with underlying mitochondrial disease. A case of simvastatin associated rhabdomyolysis followed by MELAS syndrome is known. We report a family member of a known MELAS patient with subclinical mitochondrial disease that developed myopathy while receiving atorvastatin. A 64-year-old female with history positive for occasional abdominal discomfort and `pins and needles' sensation in toes was treated with atorvastatin (10 mg/day) for hypercholesterolemia. Concurrent medications included Celecoxib, Amitriptyline prn tingling, Vitamin C, Vitamin E, Prempro, and cetirizine. CBC, SGOT and SGPT were normal prior to and 8 weeks after commencing therapy. Approximately 3 months after starting atorvastatin, she developed weakness and muscle pain. Pain (3/10) originated in her calves, and over a period of 1 month progressed to her thighs and shoulders, intensifying in both degrees of weakness and pain (10/10). Pain was `dull and constant'. Fatigability and decreased muscle endurance was noted with daily activity. She stopped the statin medication simultaneously with Celecoxib after 4 months of therapy. Symptoms resolved within 3 weeks. Family history is positive for A3243G mutation in her daughter's son who was diagnosed because of typical MELAS symptomatology. Her brother had typical MELAS symptomatology. While her A3243G level in blood was undetectable (,5%), a colon biopsy was positive with 14% heteroplasmy. This suggests statins can trigger the transformation of a sub-clinical mitochondrial disorder into a clinically signi®cant state. Careful evaluation of mitochondrial function, mutational analysis with a high degree of sensitivity, and pedigree analysis is indicated in patients with statininduced manifestations.

10 Brain nitric oxide generation is neuroprotective in acute head injury J.C. Goodman, L. Cherian, R. Hlatky, Y. Furuya, R.M. Bryan, A.B. Valadka, C.S. Robertson Departments of Neurosurgery, Anesthesiology and Pathology, Baylor College of Medicine, Houston, TX, USA Nitric oxide (NO) metabolism has been studied extensively after cerebral ischemia, but few studies have been published regarding NO after traumatic brain injury. Studies examining inhibition or stimulation of nitric oxide synthetase (NOS) immediately after injury have shown both adverse and protective effects. The mitochondrion is the primary target of adverse effects of NO whereas the bene®cial effects of NO result from restoration of cerebral blood ¯ow (CBF) via vasodilatation. We examined NO metabolism in head injury in animals and man, and conclude that the bene®cial effects outweigh adverse effects. In rats subjected to controlled cortical impact, there is decreased CBF in the region impacted and subsequent evolution of a cortical contusion. Contusion volume is reduced by systemic administration of arginine, the precursor of NO, and volume is increased by administration of the NOS inhibitor, L-NAME. The protective effect of arginine is associated with restoration of regional CBF. Microdialysis studies measuring cerebral nitrate/nitrate levels as markers of NO as well as direct measurement by NO electrode, reveal that cortical impact is associated with an initial transient (5 min) elevation of NO followed by protracted (3 h) reduction. Arginine administration restores the NO levels to control values while L-NAME blunts the initial rise in NO and is associated with protracted low levels and poor outcome. We have also measured microdialysate nitrate/nitrite levels in head injured humans and have found that low NO levels are associated with poor outcome. These ®ndings suggest that restoration of CBF (and consequently mitochondrial function) by NO is more bene®cial than harmful effects of NO in acute head trauma. It must be recognized that head injury sets into motion a complex and evolving neurochemical cascade, and that the relative neuroprotective and neurotoxic effects of NO may change over time following injury. Supported by NIH NINDS PO1 NS27616.

11 A novel large-scale mitochondrial DNA deletion associated with multiple endocrine dysfunction, renal tubulopathy and deafness C. Bruno 1, P. Gandullia 2, F.M. Santorelli 3, M. Bado 1, C. Minetti 1, R. Gatti 2 1 Neuromuscular Diseases Unit, University of Genoa; 2III Division of Pediatrics, Gaslini Children's Hospital, Genoa; 3Division of Molecular Medicine, Bambino GesuÁ Children's Hospital, Rome, Italy Dysfunction of mitochondria can affect any organ system, but certain tissues, such as muscle, heart, and brain, are more susceptible to oxidative phosphorylation defects because of their high energy requirement. Endocrinological manifestations are frequent

Abstracts in mitocondrial diseases, especially in sporadic cases, and are often associated with large-scale deletions in mitochondrial DNA (mtDNA). However, endocrine dysfunctions rarely dominate the clinical picture in the majority of patients, in contrast with the predominant encephalomyopathy. We have identi®ed a novel 8.0 kb mtDNA deletion in muscle in a child with lactic acidosis, multiple endocrine dysfunction, progressive renal tubulopathy, bilateral cataract, and deafness. A 7-year-old boy presented at 2 years of age with failure to thrive, developmental regression and frequent episodes of vomiting. Subsequently he developed renal tubulopathy, bilateral cataract, and deafness. In addition he was found to have hypoparathyroidism and primary adrenal insuf®ciency. Neurological examination revealed mild hypotonia, weakness and bilateral ptosis. Histochemistry of skeletal muscle showed ragged-red and cytochrome c oxidative negative ®bers. Biochemical analysis of muscle homogenate revealed decreased Complex I and Complex IV enzyme activities. Molecular analysis using a PCR screening procedure revealed a 8.0 kb deletion of the mtDNA in muscle, the only tissue so far available for genetic studies. Direct sequence analysis of the junctional regions showed that the deletion was 8035 base pairs long, extending from nt 7485 to nt 15 519, and that was ¯anked by a perfect 10bp repeat. The proportion of deleted mtDNA, estimated by Southern blot analysis, was 60%. The uncommon association of hypoparathyroidism, primary adrenal insuf®ciency, renal tubulopathy, and deafness has never been previously reported in a patient with a mtDNA deletion. This observation further enlarges the clinical spectrum associated with mtDNA deletions, and reinforces the concept that endocrinopathies other than diabetes might dominate the clinical picture in mtDNA disorders.

12 Hearing impairment is common in various phenotypes of the mitochondrial DNA A3243G mutation M. Deschauer, T. MuÈller, S. Zierz Department of Neurology, Martin-Luther-UniversitaÈt HalleWittenberg, Ernst-Grube-Strasse 40, D-06097 Halle/Saale, Germany The aim of the study was to determine whether there are common symptoms within different phenotypes of the mitochondrial DNA A3243G mutation. Therefore a series of 52 adult patients with mitochondrial encephalomyopathies and their symptomatic relatives were screened for the A3243G mutation by restriction enzyme analysis. In addition to the clinical examination patients with the mutation were investigated by audiometry. The A3243G mutation was identi®ed in 16 patients (ten index patients and six symptomatic relatives). Six of sixteen patients presented with stroke-like episodes and met the classical criteria of MELAS syndrome, one of them had MELAS/MERRF overlap-syndrome. Two of sixteen patients presented with stroke-like episodes but did not meet classical criteria of MELAS. Predominant features of the eight other patients were: myopathy with hearing loss and diabetes (n ˆ 1), chronic progressive external ophthalmoplegia (n ˆ 1), diabetes

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with hearing loss (n ˆ 1), painful muscle stiffness with hearing loss (n ˆ 1), cardiomyopathy (n ˆ 1), diabetes (n ˆ 1), and hearing loss (n ˆ 2). In 11/16 patients hearing impairment was obvious on clinical examination. Additionally, all ®ve patients with normal hearing on clinical examination showed subclinical hearing loss. In four of them hearing loss was more pronounced than age-related hearing impairment, in one of them hearing loss could be agerelated likewise. In conclusion there was a wide variety of phenotypes representing the variable multisystemic involvement of the A3243G mutation. Only less than half of the patients presented with MELAS. Hearing impairment was the most common symptom, that was clinically or subclinically relevant in 15/16 (94%) of the patients.

13 Diagnosis of respiratory chain disorders ± the NCMD criteria N.I. Wolf, J.A.M. Smeitink UMC Nijmegen, Nijmegen Center for Mitochondrial Disorders (NCMD), Department of Paediatrics/Metabolic Disorders, Nijmegen, The Netherlands Disorders of the respiratory chain, or mitochondrial disorders, as they are often called, are among the most frequently encountered inborn errors of metabolism. In spite of enormous advances in the insight of genetic and cell biological mechanisms of those disorders, their diagnosis is still dif®cult from both the clinical and the biochemical point of view. In 1996, Walker et al. published a set of criteria as a ®rst attempt to reach consensus on when the diagnosis of a respiratory chain disorder should be made. Recently D. Thorburn and his group modi®ed these criteria for their use in paediatric patients. In order to offer reliable and reproducible criteria and thus to facilitate the diagnosis of a respiratory chain disorder especially in infants and children, we have developed a new classi®cation, the Nijmegen Center of Mitochondrial Disorders (NCMD) criteria, considering clinical, metabolic, imaging and histopathological features on the one side and results of biochemical investigation of fresh and frozen skeletal muscle on the other. In a pilot study, we tested those criteria in a group of 30 paediatric patients. Results obtained with the modi®ed Walker and the NCMD criteria are very similar with 17 patients being scored as de®nitely suffering from a respiratory chain disorder, whereas, if scored with the original Walker criteria, no patient reaches this category. Advantages of the NCMD classi®cation include the precise de®nition of clinical and metabolic items and the independent scoring of biochemical muscle investigations. Still, none of the three systems is currently able to distinguish between primary and secondary respiratory chain disturbances.

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14 Sensitive assay of mitochondrial dihydroorotate CoQ oxidoreductase using mass spectrometry T.P. Le, G.-C. Kwon, B.A. Barshop, R.K. Naviaux The Mitocohondrial and Metabolic Disease Center, University of California, San Diego, 200 W. Arbor Drive, San Diego, CA 921038467, USA Background: Dihydroorotate CoQ oxidoreductase (DHO-QO; E.C. 1.3.99.11) catalyzes the fourth step in de novo pyrimidine synthesis; the conversion of dihydroorotic acid to orotic acid. This enzyme is coupled to the mitochondrial electron transport chain by the requirement of oxidized CoQ for normal catalytic function. In vivostudies of this enzyme have been hampered by its low abundance and the relative insensitivity of conventional methods of detection. We report a novel assay for DHO-QO using gas chromatography and mass spectrometry (GC-MS). Methods: The assay was performed on mitochondria isolated from skeletal muscle biopsies of patients referred for suspicion of mitochondrial disease. Results: Kinetic analysis of skeletal muscle mitochondrial DHOQO showed a Km for dihydroorotate of 40 mM. Reaction progress was linear for at least 4 h at 378C. The assay was linear from 0.5 to 50 mg of mitochondrial protein. Detection sensitivity was 3.5 pmol of product produced in a typical 2 h reaction. The linear dynamic range was 3±30 000 Units (nmol/h per mg). Reproducibility (s/m) was 10% within-day and 18% between days. Application of this assay to a mixed population of 21 patients referred for evaluation of mitochondrial disease revealed a distribution with a range of 12± 78 U/mg. Conclusion: This assay provides a useful adjunct to current laboratory methods for the evaluation of patients with suspected mitochondrial disease.

15 A novel mutation (G14724A) in the mitochondrial tRNA (Glu) gene is associated with leukodystrophy and mitochondrial myopathy L. Vilarinho 1, C. Pereira 1, C. Barbot 2, C. Soares 3, C. Nogueira 1, A. Guimaraes 4, F.M. Santorelli 5 1 Medical Genetics Institute; 2Neuropediatrics Department of H.M.P.; 3Pediatrics Department of H.S. SebastiaÄo; 4 Neuropathology Unit of HGSA, Porto, Portugal; 5Medicine Molecolare Lab, Bambino de GesuÂ, Rome, Italy Clinical presentation in mtDNA-related disorders is heterogeneous and often initially misleading on the real nature of the genetic defect. We studied a 4-year-old-boy who carried a long standing diagnosis of undetermined leukodystrophy since age 3 years because of the presence of periventricular white matter hyperlucinecies by MRI. Careful clinical and neurophysiological examination revealed, however, an important proximal myopathy. A muscle biopsy showed a high percentage of ragged-red and cytochrome c oxidase negative ®bers suggestive of a defect in the mitochondrial respiratory chain. Biochemical study detected multiple enzyme de®ciencies. Residual activities of NADH-ubiquinone oxidoreduc-

tase (Complex I) and cytochrome c oxidase (Complex IV) were 46 and 23%, respectively. Molecular genetic studies ruled out the commonly encountered pathogenic mutations in the mtDNA. Combination of single strand conformation polymorphism and direct sequencing on an ABI 310 identi®ed a novel heteroplasmic mutation (G14724A) in the mitochondrial transfer RNA gene for glutamine (tRNA Glu). The mutation ful®lled consensus criteria for pathogenic mtDNA mutations, including assessment of the heteroplasmy which used a mispairing PCR method and employed the endonuclease RsaI. Adding to the plethora of syndromes associated with mtDNA mutations, our ®ndings also suggest that one should consider a mitochondrial disorder in cases of undetermined leukodystrophies.

16 Requirement of mitochondrial DNA for hydrogen-peroxideinduced apoptosis 1,2 H.K. Lee, 2Y.B. Lee, 2Y.K. Pak Department of Internal Medicine, College of Medicine, Seoul National University, Seoul 110-744; 2Division of Metabolic Diseases, Department of Biomedical Sciences, National Institute of Health, Seoul 122-701, South Korea Bcl-x, an integral membrane protein located mainly on the outer membrane of mitochondria prevents apoptosis in response to a variety of stimuli. Cytochrome c which is located in the mitochondrial intermembrane space is released to cytosol to initiate apoptosis. There may be a possible connection between mitochondrial function and apoptosis. We established a mitochondrial DNA (mtDNA) depleted SK-Hep1 human hepatoma cells (r o cells) and investigated the effect of hydrogen peroxide, one of reactive oxygen species (ROS), on apoptosis. A transient exposure of control SkHep1 cells (r 1 cells) to hydrogen peroxide (H2O2) induced apoptosis while r o cells did not. Treatment of H2O2 on r 1 cells decreased the protein levels of mitochondrial transcription factor A (mtTFA) and Bcl-x by 65 and 85%, respectively, and increased the speci®c activities of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, and succinate dehydrogenase (SDH), Complex II in electron transfer chain on mitochondria. In contrast, H2O2 failed to affect the mtTFA and Bcl-x protein levels and GAPDH and SDH activities in r o cells. These results indicated that H2O2-induced apoptosis required the functional mitochondria, mtDNA, and Bcl-x. It is possible that r o cells are resistant to H2O2 since mtDNA depletion repressed the normal mitochondrial functions, indicating that the suppression of mitochondrial function is not a prerequisite for apoptosis.

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Synthesis and distribution of pyruvate dehydrogenase in relation to cell cycle dependent changes in mitochondrial morphology D.H. Margineantu 1, R.M. Brown 3, G.K. Brown 3, A.H. Marcus 2, R.A. Capaldi 1 1 Institute of Molecular Biology, University of Oregon; 2Department of Chemistry and Materials Science Institute, University of Oregon, Eugene, OR 97403, USA; 3Genetics Laboratory, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK

Maternally-inherited dysautonomia: a novel, treatable syndrome often associated with heteroplasmy in the mtDNA control region R.G. Boles, S. Wong, A. Guedalia, S. Tran Le, M. Ito Division of Medical Genetics, Children's Hospital Los Angeles; Department of Pediatrics, University of Southern California School of Medicine, Los Angeles, CA; Department of Pediatrics, Children's Memorial Hospital, Chicago, IL, USA

Abnormalities of the shape and distribution of mitochondria have been for a long time associated with impaired function of this organelle. Dynamin-related proteins responsible for the maintenance of normal mitochondrial morphology are being characterized at a rapid pace and defects have been already related to human pathology. However, detailed information on the functional signi®cance of particular morphological states is still missing. The changes in morphology of mitochondria and the synthesis and (re)distribution of proteins in the organelle have been correlated using human primary ®broblast and osteosarcoma cell lines. Mitochondrial morphology was studied initially by ¯uorescence microscopy using MitoTracker Red. In and shortly after the S phase of the cell cycle, mitochondria were fragmented and perinuclear as shown by Aphidicolin treatment. In G1 phase, as trapped by removal of fetal calf serum, these fragmented mitochondria coalesced to from an extensive reticulum. The same cell cycle dependent changes in mitochondrial morphology were observed in cells in which green ¯uorescent protein (GFP) or red ¯uorescent protein (RFP) had been targeted to the organelle. Newly synthesized and incorporated protein was in fragmented mitochondria which converted later to the reticular form. Transient transfection did not alter the morphology changes. Newly synthesized, free GFP, RFP and GFP attached to the E1alpha subunit of pyruvate dehydrogenase (PDH) which appeared initially in only a proportion of the fragmented mitochondria, indicative of a heterogeneous distribution of protein entry sites in the mitochondrial mass. Both GFP and RFP ®lled up the reticulum rapidly as it reformed. The GFP-E1alpha chimera rescued a ®broblast cell line de®cient in E1alpha synthesis and it was almost all immunoprecipitated in intact PDH, con®rming assembly into the large 8 £ 10 6 Da complex. In comparison to free GFP or RFP, GFP labeled PDH redistributed very slowly after synthesis consistent with highly restricted movement of this large protein complex in the mitochondrial matrix.

Disorders of mitochondrial DNA (mtDNA) can result in maternal inheritance of a wide variety of clinical manifestations. Although critical for mtDNA replication and transcription, disease-associated base substitutions of the control region (mtDNA-CR) have not been previously reported. We report nine cases with a novel maternallyinherited syndrome characterized by intermittent episodes of migraine, cyclic vomiting, asymmetrical pain and/or swelling in the extremities, gut dysmotility and dysregulation of heart rate and body temperature. At least most of the clinical manifestations are explainable by dysautonomia and responded favorably in each case to speci®c treatment, including fasting avoidance and/or amitriptyline. In each family the mother is affected, as are most of the siblings. Additional manifestations in some individuals include seizures, depression, peripheral neuropathy, exercise intolerance and/or SIDS. Three cases have affected maternal half siblings. Urine organic acids, obtained while ill, demonstrate elevated Kreb cycle intermediates and/or ethylmalonate in all cases. In four families, rare/unique heteroplasmic mtDNA-CR variants were found, including two distinct point substitutions in two families, and length variants in the other two families. By contrast, mtDNA-CR point heteroplasmy was absent in 103 controls. The name, `Maternally-Inherited Dysautonomia Syndrome' is proposed. A primary defect of mtDNA is strongly suggested from the maternal inheritance of disease manifestations seen in all cases, and multiple mtDNA-CR variants seen in many. This syndrome is probably not very rare since our nine cases were ascertained from the practice of a single clinician. Diagnosis is important for purposes of treatment and accurate genetic counseling.

19 Mitochondrial DNA control region point heteroplasmy and associated clinical presentations in children with neuromuscular and/or multi-system dysfunction and an elevated lactate R.G. Boles, S. Tran Le, D. Chaudhari, T. Higashimoto, M. Ito Division of Medical Genetics, Children's Hospital Los Angeles; Department of Pediatrics, University of Southern California School of Medicine, Los Angeles, CA, USA Background: Children with idiopathic neuromuscular and/or multi-system disease and biochemical signs of energy depletion are frequently encountered in tertiary practice. Many of these children have affected matrilineal relatives yet mitochondrial DNA

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mutations are not found on standard analyses, suggesting the presence of novel mtDNA variants. Mutation in the mtDNA control region (mtDNA-CR) which interferes with replication or transcription is a plausible mechanism. Methods: Temporal temperature gradient gel electrophoresis (TTGE) was used to screen the entire mtDNA-CR in the blood of 67 children with idiopathic neuromuscular and/or multi-system disease and an elevated lactate from the practice of the ®rst author, and in 103 controls. Cases with abnormal ®ndings were further evaluated by sequencing. Results: Single nucleotide heteroplasmy was con®rmed in 9/67 patients and in 0/ 103 controls (P , 0.001). In an additional two patients, point heteroplasmy was highly suggested by TTGE but additional DNA was not available for sequencing. Although there is substantial variability among these 11 children, common manifestations include cardiomyopathy, fasting intolerance, migraine and dysautonomia. Intelligence was normal in one half. The identical mtDNA-CR heteroplasmic variants were present in the mothers and siblings in all cases tested, and maternal inheritance of disease is highly suggested by some of the family histories. Conclusions: mtDNA-CR point heteroplasmy is highly associated with disease and is common (one in six) among the children with idiopathic neuromuscular and/or multi-system dysfunction and an elevated lactate seen by one geneticist. Clinical manifestations vary, but are generally non-progressive. Several cases have responded favorably to speci®c therapy, especially the avoidance of fasting. Our ®ndings can be explained by several potential mechanisms.

20 The absence of VDAC1 does not alter the colocalization of mitochondria and MAP2 in mice ®broblasts K. An¯ous, W.J. Craigen Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Interaction between mitochondria and the cytoskeleton has previously been studied in vitro, and it has been reported that voltage-dependent anion channels (VDACs) constitute one of the binding sites for microtubule associated protein 2 (MAP2) (Linden and Karlsson, 1996). By using a commercial anti-MAPs antibody that stains mainly MAP2 and a mitochondrial marker dye MitoTracker Red, we studied by confocal microscopy MAP2 and mitochondria distribution in wild type and VDAC1-de®cient ®broblasts. In wild type ®broblasts, the ¯uorescence staining speci®c for MAP2 (FITC-green) and mitochondria (red) were both intense near the nucleus, and were overlapping in some areas (yellow). Near the plasma membrane, we observed a less intense staining corresponding to MAP2 (FITC-green). In the VDAC1-de®cient ®broblasts, we did not observe any signi®cant alteration in the colocalization or the intensity of MAP2 and mitochondria staining near the nucleus. Additionally, the overall staining pattern of cytoskeletal components such as tubulin and desmin was unchanged. These results suggest that VDAC1 does not play a prominent role in binding MAP2 to the mitochondrial outer membrane, at least in cultured cells. Use of ®broblasts de®cient in VDAC2 or VDAC3 or some combination of VDAC isoforms will give more insight into the

potential role of VDACs as binding sites for MAP2 or other cytoskeletal elements.

21 New methods for identifying and classifying mitochondrial disorders B.J. Hanson 1,M.F. Marusich 1,R.H. Triepels 2,R. Carrozzo 3, B. Schulenberg 1, R.A. Capaldi 1 1 University of Oregon, Eugene, OR, USA; 2Nijmegen Center for Mitochondrial Disorders, Nijmegen, The Netherlands; 3Ospedale Pediatrico `Bambino Gesu', Rome, Italy Disorders of mitochondrial energy metabolism occur in humans with a frequency of around one in 10 000 live births. Most are caused by dysfunction of one or more of the complexes of oxidative phosphorylation (OX PHOS). The OX PHOS complexes are comprised of an estimated 87 different structural proteins, 13 of which are encoded by mitochondrial DNA. In addition, there are numerous assembly and import factors required for the proper functioning of the OX PHOS system. Due to the large number of proteins involved in OX PHOS function, methods which help to identify and classify mitochondrial disorders are needed. We have produced a set of monoclonal antibodies which react with proteins from each of the OX PHOS complexes. We show that this set works well as an initial screen for identifying which complex is defective. This type of screen is relatively rapid, easily performed, and requires less sample than enzymatic assays. These antibodies have also been used in conjunction with sucrose gradient centrifugation to study the assembly state of the OX PHOS complexes in patient cell lines. We show that patients with defects in the same complex have different assembly pro®les depending on the protein that is mutated. Using this method, we have identi®ed one patient with a severe Complex I assembly problem which is a good candidate for having a defect in an as-yet unidenti®ed assembly factor. In addition, we show that patients with different SURF-1 mutations assemble different degrees of cytochrome coxidase which correlate to the amount of residual activity found in the cell lines. Furthermore, sucrose gradient fractions can be used for 2-D gel electrophoresis resulting in 3-D separation of mitochondrial proteins. This type of 3-D mitochondrial proteomics would allow patient cell lines to be compared to control samples for identi®cation of proteins with altered expression states.

22 Analysis of assembly of pyruvate dehydrogenase complex in normal human ®broblasts and PDH de®cient cell lines using anti-PDH monoclonal antibodies M.Y. Lib, M.F. Marusich, R.A. Capaldi University of Oregon, Eugene, OR 97403, USA Pyruvate dehydrogenase complex (PDH) is the pivotal enzyme of carbohydrate metabolism. The complex catalyses the irreversible oxidative decarboxylation of pyruvate, by producing acetyl-CoA,

Abstracts NADH and CO2. Such conditions as starvation, diabetes, sepsis, Alzheimer's disease as well as primary PDH de®ciencies are known to cause altered PDH activity. Monoclonal antibodies against different subunits of pyruvate dehydrogenase complex have now been raised in mice. We show that this set provides an important initial screen for PDH de®cient patients. One of the puri®ed monoclonal antibodies against the E2 subunit of PDH immunoprecipitates the entire PDH complex from bovine heart mitochondria, human heart mitochondria and from whole human ®broblasts. The polypeptide pro®le of the immunoprecipitate is similar to that of conventionally puri®ed PDH. This monoclonal antibody can be used to examine the assembly of PDH in normal human ®broblasts vs. patients' ®broblasts. The immunoprecipitated PDH can also be examined by 2-D gel electrophoresis. This allows identi®cation of coimmunoprecipitated polypeptides, which includes different PDH kinase isoforms. Moreover 2-D gel separation of immunoprecipitated PDH allows analysis of the phosphorylation state of the E1 alpha subunit.

23 sod2 de®cient mice show increased susceptibility to kainic acid excitotoxicity in vivo M.N. Patel, Y.-S. Ho, L.-P. Liang Departments of Medicine, National Jewish Medical and Research Center, University of Colorado Health Sciences Center, Denver, CO; Wayne State University, Detroit MI, USA We have previously demonstrated that systemic or local administration of kainic acid selectively inactivates mitochondrial aconitase, a superoxide-sensitive enzyme, at times preceding neuronal death (Neuroscience 101: 563, 2000). The objective of this study was to determine if mice de®cient in SOD2 showed increased susceptibility to kainic acid neurotoxicity. Kainic acid-induced behavioral seizures, mitochondrial aconitase inactivation (superoxide production) and hippocampal cell death were exacerbated in SOD2 heterozygote knockout mice. Kainic acid-induced mitochondrial aconitase inactivation and hippocampal neuronal loss were attenuated in mice overexpressing SOD2 or by Mn(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a catalytic antioxidant. These results support a role for mitochondrial superoxide production in hippocampal pathology produced by kainic acid. Supported by Parents Against Childhood Epilepsy and NIH RO1NS39587 to MNP.

24 Increased lung tumor formation in aged manganese superoxide dismutase (SOD2) heterozygous knockout mice B.J. Day 1, L.W. Velsor 1, D.C. Wallace 1 Department of Medicine, National Jewish Medical and Research Center, Denver, CO; 2Center for Molecular Medicine, Emory University School of Medicine, Atlanta, GA, USA The role of antioxidant enzymes as modulators of tumorigenicity

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remains controversial. However, several investigators have found that manganese superoxide dismutase (SOD2) possesses tumor suppressor activity. In support of this theory, we have found an increased frequency of bronchioloalveolar carcinomas in aged SOD2 tm1Rm1 heterozygous knockout mice. Aged SOD2 heterozygous knockout mice had less than 50% of lung SOD2 apoprotein expression than aged wild type mice. Lungs from SOD2 heterozygous knockout mice were examined from the ages ranging from 12 to 20 months of which 25% developed bronchioloalveolar carcinomas. These tumors were observed at only 8% in the wild type mice. In another group of aged SOD2 tm1Cje heterozygous mice on an outbred background (CD-1) we did not see an increased frequency of lung tumors but instead observed an accelerated lung ®brosis. Another frequent observation in both aged SOD2 heterozygous backgrounds was the presence of interstitial lymphocytic in®ltrates surrounding large vessels and airways that occurred in 75% of the SOD2 heterozygotes and only 50% in wild type mice. These lesions were always less intense in the wild type background than the SOD2 heterozygotes. A less frequent observation was bronchiolar hyperplasia that occurred in 25% of the SOD2 heterozygous mice and 17% in the wild type mice. In summary, the SOD2 heterozygous mice had an increased incidence of lung tumors along with other lung in¯ammatory changes. Decreased lung SOD2 expression appears to modulate lung tumorigenicity and in¯ammation in the aged mouse and is dependent upon the genetic background. Supported in part by grants from NIH HL59602 and HL31992 to BJD and AG13154 to DCW.

25 MEHMO (mental retardation, epileptic seizures, hypogenitalism, microcephaly, obesity): a new X-linked mitochondrial DNA depletion syndrome E. Leshinsky-Silver 1,2, R.K. Naviaux 3, A. Zinger 4, N. Bibi 5, V. Barash 6, M. Sadeh 7, D.Lev 2, T. Lerman- Sagie 2 1 Molecular Genetics Laboratory and Institute of Physiologic Hygiene, Wolfson Medical Center; 2The Mitochondrial Disease Clinic, Metabolic Neurogenetic Unit, Wolfson Medical Center, Holon, Israel; 3Biochemical Genetics and Metabolism, The Mitochondrial and Metabolic Disease Center, University of California, San Diego School of Medicine, San Diego, CA, USA 4 Genetic Institute, Kaplan Medical Center, Rechovot; 5Pediatric Intensive care Unit, Barzilai Medical Center, Ashkelon; 6 Biochemistry Department, Hadassah Medical Center, Jerusalem; 7 Department of Neurology, Wolfson Medical Center, Holon, Israel MEHMO is an X-linked disorder characterized by mental retardation, epileptic seizure hypogenitalism, microcephaly obesity and hypotonia. It was recently assigned to the locus Xp21.1±p22.13. We describe a child with MEHMO and lactic acidosis whose muscle biopsy revealed markedly reduced activities of complexes 1, 3, and 4 of the mitochondrial electron transport chain. Histological staining showed mitochondrial proliferation and lipid storage. Electron microscopy revealed abnormal and enlarged mitochondria with concentric cristae and electron dense bodies. Quantitative Southern blot analysis of the patient's DNA extracted from muscle biopsy

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using both mitochondrial and nuclear probes revealed mtDNA depletion. The residual mtDNA content was 30% of the normal. There was no evidence of mtDNA deletion, duplication, or rearrangement. These ®ndings suggested a de®ciency in one or more factors involved in the processing or control of mitochondrial DNA synthesis. This is the ®rst identi®cation of MEHMO as a mitochondrial disease and the ®rst example of an X-linked mitochondrial DNA depletion syndrome. MEHMO is one of a growing list of neurologic disorders associated with mtDNA depletion. Key words: MEHMO, mtDNA depletion, X-linked mental retardation.

26 Plasma lactic acid levels in mitochondrial disease: a good diagnostic or monitoring marker? A. Feigenbaum, P. Wu The Hospital for Sick Children and University of Toronto, Toronto, Canada Background: Traditionally, plasma lactate levels have been used as a screening test to diagnose mitochondrial disease. In addition, these levels have been used to monitor therapeutic interventions, dietary response, etc. Specialists in the ®eld recognize that normal lactate levels either in plasma, CSF, MRS brain or urine do not exclude the diagnosis. However, this has not been well described in texts or the literature and the message is not widely recognized by general practitioners including paediatricians, neurologists and other subspecialists who often see children and adults with possible mitochondrial disease. In addition, high lactate levels are often disregarded as spurious results, e.g. crying, poor venepuncture technique, secondary causes such as dehydration, cardiac or renal disease, etc. Methods: We retrospectively reviewed the records of 70 paediatric cases with con®rmed mitochondrial disease. We tabulated and graphed plasma lactate levels, pyruvate levels, MRS lactate levels where available, CSF lactate and urine lactate as well as indirect measures such as alanine levels. Results: The results will be shown in graphic form to illustrate the wide intra-individual and inter-individual variability of these levels over time. In addition we will illustrate the dif®culties in interpreting these results with respect to a general diagnosis of mitochondrial disease, the speci®c mitochondrial defect, and therapeutic interventions.

27 An analysis of 66 muscle biopsies for suspected mitochondrial myopathy F. Scaglia, J. Towbin, H. Vogel Departments of Pediatrics, Molecular and Human Genetics, and Pathology, Baylor College of Medicine. Houston, TX, USA A broad spectrum of clinical and laboratory abnormalities have been associated with mitochondrial encephalomyopathies. Certain respiratory chain complex de®ciencies or genetic defects may be associated with these abnormalities, but there is little evidence to

suggest whether they are predictive of a speci®c de®ciency. We analyzed clinical, laboratory, and pathological data in 66 muscle biopsies received for suspected mitochondrial disease that were subsequently tested for respiratory enzyme de®ciencies. The biopsies were received between 1995 and 2000. There were 31 females and 35 males, with ages ranging between 2 weeks and 24 years, although the median age was 18 months and 39% were under 12 months of age. Data was obtained with regard to the pattern of inheritance, developmental delay, abnormalities of tone or movement, seizures, cardiomyopathy, ophthalmic disease or deafness, lactic acidosis, CNS imaging abnormalities, histopathological evidence of mitochondrial proliferation, cytochrome c oxidase de®ciency by enzyme histochemistry, and ultrastructural abnormalities of mitochondria. Analysis of Complexes I±IV activities were determined along with citrate synthase as a measure of mitochondrial content. Mitochondrial proliferation, either by pathology or citrate synthase activity, was common, occurring in 16/23 (69%) with normal enzyme activities and in 25/43 (58%) with abnormal enzyme activities. Of the 43/66 with respiratory chain abnormalities, six groups were established according to type of complex de®ciency: Complex I (n ˆ 10), I±III, II±III consistent with coenzyme Q10 de®ciency (n ˆ 8), I 1 IV (n ˆ 3), II (n ˆ 4), IV (n ˆ 7), and combined I, III, and IV (n ˆ 11). Of the parameters studied, none showed a signi®cant correlation with a speci®c complex de®ciency. In this cohort of patients undergoing muscle biopsy, we conclude that mitochondrial proliferation is a common feature of pediatric neurological disease and infantile cardiomyopathy. Despite the association of certain clinical and pathological features with syndromes in mitochondrial disease, none appear to be predictive of a speci®c complex de®ciency.

28 Uptake and phosphorylation of thymidine and AZT in isolated heart mitochondria E.E. McKee, A.T. Bentley, J. Kraas, J. Gingerich South Bend Center for Medical Education/Indiana University School of Medicine, Notre Dame, IN 46556, USA Deoxyribonucleotide triphosphates are required for the replication of mitochondrial DNA and must be present in the mitochondrial matrix. However, not much is known about the form and manner in which these pools arise; nor, is much known concerning the effects of nucleoside analogs such as AZT (Zidovudane) on these processes. Since there appears to be deoxynucleoside and deoxynucleotide kinases expressed in the mitochondria matrix, it seems likely that deoxynucleosides are transported across the inner membrane and phosphorylated within the matrix to the triphospho form. We have studied the uptake and phosphorylation of [ 3H]thymidine or [ 3H]-AZT in isolated rat heart mitochondria as a function of time. The matrix and medium contents were rapidly separated by centrifugation through silicone oil and the labeled nucleosides and nucleotides were identi®ed and quantitated by high-pressure liquid chromatography (HPLC) analysis of both the matrix and the medium pools. Our results show that thymidine and AZT can be slowly transported into the mitochondrial matrix in a

Abstracts time and concentration dependent manner. The labeled thymidine is phosphorylated in the matrix to labeled TMP, TDP, and TTP, with the amount of TTP increasing in a time dependent manner. Labeled TDP and TTP were higher in the matrix, but were also found in the medium as a function of time. Labeled TMP was observed in the matrix only. AZT, was converted to AZT-MP as a function of time, but was not further phosphorylated. The AZT-MP formed appeared to accumulate in the matrix. Lastly, the presence of AZT did not inhibit uptake of labeled thymidine, but completely blocked the conversion of TMP to TDP and TTP. These results suggest that the mitochondrial toxicity associated with AZT may be related to the accumulation of AZT-MP in the matrix with depletion of TTP pools, rather than inhibition of mitochondrial DNA replication.

29 Biochemical and genetic characterization of complex I (NADHubiquinone oxidoreductase) mutants of Chinese hamster cells in tissue culture I.E. Schef¯er, N. Yadava, E.N. Smith Division of Biology, University of California, San Diego, La Jolla, CA 92093-0322, USA A serendipitous observation some time ago led to the ®rst characterization of a respiration-de®cient Chinese hamster mutant cell line in tissue culture. It has guided the design of an enrichment scheme for the isolation of additional mutant cell lines with severe defects in respiration. Among the collection of such mutants, several complementation groups were identi®ed with mutations affecting Complex I. The X-linked NDUFA1 gene encoding the MWFE protein represents one group. This is one of the ~28 proteins outside of the `core' de®ned by the prokaryotic proteins of Complex I. Several mutant alleles isolated independently in tissue culture are described that yield very low activities and show that the protein is essential for activity. A phylogenetic analysis of this highly conserved protein has directed attention to species-speci®c differences that make the primate MWFE protein inactive in hamster (rodent) cells. Based on such comparisons, mutant alleles made by site-directed mutagenesis, when expressed in a null mutant, yielded reduced Complex I activities, even though the mutant protein was assembled normally into the complex. These and other mutants promise to be valuable for structure±function analyses, especially in conjunction with a high-resolution structure that can be expected in the future.

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30 Measurement of NADH cytochrome c reductase activity in cultured ®broblasts: a rapid assay of clinical importance S. Raha, L. Johnstone, A. Tomoko Myint, J. Cameron, N. Mackay, B.H. Robinson Programme in Metabolics, Research Institute at the Hospital for Sick Children and the Department of Biochemistry, University of Toronto, Toronto, Canada De®ciencies in the NADH coenzyme Q oxidoreductase (Complex I) of the respiratory chain enzymes results in the development of severe lactic acidosis resulting in degeneration of neuronal, cardiac or muscle tissue. The clinical importance of understanding the mechanisms by which this complex operates and more importantly being able to demonstrate its role in disease is easily seen. In order to achieve this, it is necessary to accurately measure its activity. The usual method for the clinical assessment of Complex I de®ciency is to measure either the lactate to pyruvate ratio or to isolate mitochondria and measure the NADH cytochrome c reductase activity (Complex I 1 III). Although the latter is a more direct and ultimately more accurate method of evaluating Complex I activity, it usually requires at least ten plates of cultured ®broblasts. The growth of cultured ®broblasts and processing of multiple plates for each patient can prove to be a very costly process. In order to make the assessment of Complex I de®ciency a more cost effective clinical procedure we have developed a rapid assay that allows the determination of Complex I 1 III activity from one con¯uent plate of cultured ®broblasts. The procedure depletes the cell of cytosolic diaphorases, an enzyme which will utilize the NADH used to initiate Complex I activity and mask the rotenone sensitive Complex I activity. We have used extraction with digitonin to rapidly process a single plate of ®broblasts and measure the rotenone-sensitive Complex I 1 III activity. In addition, we have added bovine serum albumin to the assay buffer allowing for the accurate measurement of lower enzyme activities. In this presentation we will show that this method correlates well with other methods of diagnosing Complex I de®ciencies and is signi®cantly more rapid as compared to these techniques.

31 Mutation analysis of the X-linked pyruvate dehydrogenase E1a subunit (PDHA1) gene J.M. Cameron, V. Levandovskiy, A. Seyda, N. MacKay, B.H. Robinson Metabolism Programme, Hospital for Sick Children, Toronto, Ontario, Canada We have analyzed patients with de®cient or reduced PDH activities (pyruvate dehydrogenase complex de®ciency) for known or novel mutatuons by sequence analysis and determining the candidate gene regions for several patients with combined PDH de®ciencies. A mutation within exon 5 of the pyruvate dehydrogenase E1a subunit gene (PDHA1): nucleotide 491 A to G mutation (amino acid N164S) was identi®ed in patient 1 (male). This mutation has been

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previously identi®ed in males with PDH de®ciency. cDNA from the patient was ampli®ed by PCR and subcloned. Patient 2 (male) was shown to have a 214 C to T mutation (amino acid R72C) in cDNA, the mutation was con®rmed from 4/4 subclones from genomic DNA. Patient 3 (female) was shown to have a 947 C to T mutation (amino acid P316L) in 3/4 subclones from c DNA, and the mutation was con®rmed by sequencing from DNA. No mutations were detected in the E1a subunit of patients 4 and 5, and the PDHA1 gene could not be ampli®ed from patient 6 implying that the E1a subunit message was probably being degraded. We are currently examining other subunits and regulatory enzymes that could be defective, resulting in these patient's enzyme de®ciencies. We have also being examining patients with combined PDH de®ciencies, using the technique of microcell mediated single chromosome transfer and have successfully localized the candidate disease gene region to a region of 5 cM for one patient with multiple alpha-keto acid dehydrogenase de®ciency and defective respiratory chain components. We are currently using this technique on a number of other patients (7, 8 and 9). Patient 7 has moderate PDH de®ciency (25%) and an affected sibling. Patient 8 has partial PDH activity, high lactate/pyruvate ratio (L/P) and variably de®cient cytochrome oxidase. Patient 9 has partial PDH de®ciency, high L/P ratio, and partially de®cient cytochrome oxidase, cytrate synthase, succinate cytochrome C reductase and pyruvate carboxylase activities. We will report on the problem of whether the same disease gene region can be implicated in these patients, or if the cause is distinct.

32 Homozygosity of the G1541A mutation in SCO2 is associated with a novel phenotype of Leigh-like syndrome, neuropathy and late onset infantile hypertrophic cardiomyopathy M. Jaksch, P. Freisinger, R. Horvath, N. Horn, C. Macmillan, H. Lochmueller, E.A. Shoubridge Metabolic Disease Center Munich-Schwabing, Munich, Germany SCO2 encodes a mitochondrial inner membrane protein, thought to function as a copper transporter to cytochrome c oxidase (COX), the terminal enzyme of the respiratory chain. Mutations in SCO2 have been described in patients with COX de®ciency and fatal infantile hypertrophic cardiomyopathy, and encephalomyopathy, with death within the ®rst 6 months of life. All patients so far reported are compound heterozygotes for a missense mutation (G1541A) near the predicted CxxxC metal binding motif, however recent functional studies of the homologous mutation in yeast failed to demonstrate an effect on respiration. Here we report a 7 month and 10 month follow up study in two infants carrying a homozygous G1541A mutation. MRI and muscle morphology demonstrated an age-dependent progression of disease with predominant involvement of white matter, late appearance of basal ganglia lesions, neurogenic muscular atrophy and decreasing COX activity in one case. An increased copper uptake in the patient's ®broblasts clearly indicates an effect of the G1541A mutation on human copper metabolism. The observed tissue-speci®c effects of SCO2 mutations on mitochondrial respiration are re¯ected in different cell-lines of the patients: myoblasts exhibit a severe COX de®ciency whereas skin

®broblasts are affected to a minor extent only, thus representing human cell models for transfection and expression studies as well as for therapeutic approaches.

33 Endothelial dysfunction in patients with MELAS: role of oxidative stress in stroke Y. Koga 1, R. Fukiyama 1, H. Matsuoka 2, Y. Akita 1, T. Imaizumi 2, H. Kato 1 1 Department of Pediatrics and Child Health; 2Department of Internal Medicine III, Kurume University School of Medicine, Asah-machi, Kurume, Fukuoka 830-0011, Japan MELAS characterized by stroke before 20 years old, is a maternally-inherited mitochondrial multisystem disorder. Mitochondrial angiopathy demonstrating degenerative change with increased abnormal mitochondria in the endothelial cells of intramuscular small arteries and arterioles has been reported in many MELAS patients. However, the primary cause of the young MELAS stroke-like episodes, either mitochondrial cytopathy or angiopathy, or both is still controversial. Since abnormal mitochondria generates superoxide anion, we hypothesized that vascular complications in MELAS may be associated with endothelial dysfunction caused by oxidative stress. Nine patients were clinically, muscle-pathologically or genetically diagnosed as MELAS. Six patients have an A3243G mutation, one patient has a T3271C mutation in the mitochondrial tRNALeu (UUR) gene, and two patients their genetic abnormality has not been found. In this study, we examined ¯owmediated vasodilatation, as a non-invasive measure of endothelial function, and effects of an antioxidant, vitamin C in patients with MELAS. We analyzed the correlation between the amount of point mutation in the endothelial cell and the endothelial function by single-cell PCR analysis. We also studied the pharmacological effect on the clinical course, and biochemical parameters after administration of L-arginine to a patient in the acute phase of stroke on three separate occasions and on the functional aspects of the cerebral hemodynamics using single photon emission computed tomography (SPECT). Flow-mediated vasodilatation was signi®cantly less (10% of the age-matched controls) in MELAS patients. Endothelium-dependent vasodilatation induced by glyceryl trinitrate was also impaired. Vitamin C administration signi®cantly restored ¯ow-mediated dilation and glyceryl trinitrate-induced vasodilatation to near-normal levels in MELAS but did not affect them in controls. After the administration of L-arginine, all the symptoms of the patient suggesting the stroke-like episode were clinically improved. On SPECT using ECD, the intracranial hemodynamics were also improved in the ischemic area (in the left temporal lobe), but unchanged in the brain stem (thalamus). There are clear inverse correlations between the amount of point mutation and the capacity of endothelial dependent-vasodilatation in the endothelial cells. Our data demonstrated that angiopathy seen in MELAS involved abnormality in the capacity of vasodilatation in the endothelial system, which may play an important role in causing stroke-like episodes in this disorder.

Abstracts 34 The coding problem ± why no one can die of mitochondrial disease in America R.K. Naviaux The Mitochondrial and Metabolic Disease Center, University of California, San Diego, 200 West Arbor Drive, San Diego, CA 92103, USA The National Center for Health Statistics (NCHS) is maintained and operated by the Center for Disease Control (CDC) in Hyattsville, MD. The NCHS is responsible for collecting and analyzing data on health and disease in the US. A major goal of this work is to enable the US Congress to develop rational health care policies based on measured disease burdens, to identify areas of need, and to apportion research dollars according to that need. The words `mitochondria' or `mitochondrial' appear nowhere in the 456 pages of the NCHS most recent `state of the union' report, Health, United States, 2000, the 20 pages of the NCHS Programs and Activities report, or the 17 pages of the NCHS Current Legislative Authorities report. Death certi®cates and health insurance claims in the US are coded according to disease identi®cation numbers published in the International Classi®cation of Disease 9th Revision, Clinical Modi®cation (ICD-9-CM) that has been used from 1979 to 2000. Speci®c ICD-9 codes for the nearly 400 different mitochondrial disorders known are virtually absent. In most cases, only generic codes are available. Some of these generic codes used for mitochondrial disease include: `Acidosis, Lactic' 276.2; `Unspeci®ed disorder of metabolism' 277.9; `Other conditions due to chromosome anomalies' (which can be used for MELAS) 758.89; and `Late metabolic acidosis of the newborn' 775.7. One speci®c code is 330.8, `Other speci®ed degenerative diseases of the nervous system, Leigh disease'. The coding problem has been recognized by leaders in the Society for Inherited Metabolic Disorders (SIMD), and the American College of Medical Genetics (ACMG), but is yet to be successfully resolved at the level of national and international health care policy via rati®cation of new codes by the World Health Organization (WHO) for inclusion in the ICD-10-CM. The combined efforts of the UMDF, MMS, MRS, SIMD, and ACMG may be required to implement the proposed changes.

35 Identi®cation of the minimal inhibitory sequence of the mitochondrial F1-ATPase inhibitor protein using synthetic peptides E.S. Kornbrust 1, A.M. Bray 2, W.R. Sampson 2, H. Patsiouras 2, S.G. Glasco 1, C.M. Anderson 1 1 MitoKor, San Diego, CA 92121, USA; 2Mimotopes, Clayton Victoria 3168, Australia The endogenous mitochondrial ATPase inhibitor (IF1) is a small basic protein (~10 000 kDa) that regulates the activity of F1F0ATPase. This protein has previously been isolated from mitochondria of beef heart, rat liver, rat skeletal muscle, and yeast. The present study shows that IF1 is also present in human tissues, with

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highest expression observed in heart, muscle, and kidney. Using a series of truncation mutants and synthetic peptides derived from IF1, van Raaj et al. (Biochemistry 35: 15618±15625,1996) deduced the region from amino acids 14±47 to contain the minimal inhibitory sequence. To con®rm this deduction and to further re®ne the precise minimal inhibitory peptide, we synthesized the IF1 (14±47) peptide and systematically truncated residues from either end using Mimotopes PepSet technology which allows rapid synthesis and screening of peptide libraries for protein function studies. The inhibitory activities of the peptides were measured by spectrophotometric determination of oligomycin-sensitive ATPase activity in sub-mitochondrial particles from rat liver. The 39 peptides, ranging in length from 11 to 34 amino acids, differed greatly in activity. Twenty-three of the peptides comprised a set beginning with residue 14 as Nterminus, with sequential truncation of one C-terminal amino acid. Hence, the longest of the series was the 34-mer (14±47), and the shortest an 11-mer (14±24). Only the six longest peptides demonstrated inhibitory activity at the 1 mM screening concentration; the potency declined with decreasing length (e.g. EC50 for 14±47 ˆ 31 nM; EC50 for 14±46 ˆ 81 nM; EC50 for 14±45 ˆ 460 nM). None of the 16 20-mers derived from peptide 14±47 showed any inhibitory activity at 1 mM. In conclusion, we have con®rmed the minimal inhibitory sequence of the mitochondrial F1-ATPase inhibitor protein to consist of residues 14±47. The Multipin e peptide synthesis was a fast and effective tool to perform structure-activity analysis of the regulatory peptide.

36 Rescue of Complex I defects by use of the single-subunit NADHquinone (Q) oxidoreductase B.B. Seo, A. Matsuno-Yagi, T. Yagi The Scripps Research Institute, La Jolla, CA, USA It has been shown that defects of Complex I are involved in many human mitochondrial diseases. Our goal is to develop the incorporation of the yeast-type single-subunit NADH-Q oxidoreductase (Ndi1) into human mitochondria as a potential remedy for Complex I defects. Ndi1, which is encoded by the yeast NDI1 gene, is a versatile enzyme because it functions as a member of the respiratory chain, when introduced, in E. coli. Here we report that the Ndi1 enzyme can be functionally expressed in Chinese hamster cells (including the MWFE subunit-null mutant), HEK 293 cells, human 143B cells (including the ND4 subunit-null mutant), mouse MN9D cells and rat PC12 cells and that the Ndi1 protein is capable of compensating respiratory de®ciencies caused by defects in the host Complex I. Gene delivery was initially carried out by lipofection or calcium phosphate precipitation methods. More recently, we constructed a recombinant adeno-associated virus vector carrying NDI1 (rAAV-NDI1). With rAAV-NDI1, it was possible to achieve higher transduction ef®ciencies and to introduce the gene into non-proliferating cells. It was shown that the NDI1-transduced cells were able to grow in the presence of added Complex I inhibitor such as rotenone and 1-methyl-4-phenylpyridinium ion whereas control cells without the gene failed to survive. Expression of the Ndi1 protein in Complex I-de®cient mutant cells

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restored the NADH oxidase to the cells regardless of whether the defects are of nDNA origin or of mtDNA origin. Successful introduction of functional Ndi1 protein into mitochondria is a signi®cant step forward toward the clinical applications of this protein as a remedy of Complex I defects. It is highly likely that what was made possible with the yeast Ndi1 protein can be extended to other proteins that have to be targeted into mitochondria at the desired location with full activities.

37 The mtDNA ATPase6 T9176G mutation affects ATP output by blocking the proton pathway and results in Leigh syndrome A. Tessa 1, R. Carrozzo 1, R.A. Capaldi 2, F.M. Santorelli 1 1 Molecular Medicine Unit, Bambino Gesu Hospital, Rome, Italy; 2 Institute of Molecular Biology University of Oregon, Eugene, OR, USA MtDNA mutations are an important etiology of Leigh syndrome (LS), a heterogeneous group of neurodegenerative disorders associated with different defects of energy metabolism. The extent to which mtDNA alterations affect cellular production of ATP is largely unknown, however. We identi®ed a new mtDNA mutation (T9176G, change L217R) in the ATPase 6 gene in a child with LS. The mutation ful®lled accepted criteria for pathogenicity. The effects of the mutation in tissues and cells were investigated by using different approaches. ATPase activity was consistently normal. ATP synthesis rates were severely hampered. The residual activity was about 20% when we used succinate and negligible using malate as substrate to start the reaction. The morphology of ®broblast cell lines and the ability of their mitochondria to generate a valid membrane potential was monitored by ¯uorescence microscopy adding the dye JC-1. Results were consistent with a severe defect in ATP output. To gain insights into the pathogenesis, we modeled the L217R mutation in the a subunit of E. coli as the equivalent mutation L259R. ATP synthesis, hydrolysis, proton pumping and enzyme assembly were analyzed and compared with previously designed E. coli mutants (L259P, L207R and L207P) equivalent to the human mtDNA LS-associated T9176C, T8993G, and T8993C, respectively. In our hands, the L259R mutant showed practically absent proton pumping and ATP synthesis but it retained sensitivity to DCCD, speaking for a correct assembling of F1 and F0 moieties. Combining studies in human cells and in E. coli, it seems that the pathological effect of the T9176G is to inhibit activity of the holoenzyme probably by blocking the proton pathway. In recent models of ATP synthase function, this would argue for a block of the rotation of the c subunit ring.

38 Genetic counseling for mitochondrial disorders E.B. Sanders 1, L.C. McDonald 1, M.A. Del Vecchio 1, C.A. Bay 2 1 Division of Medical Genetics, Children's Hospital of Pittsburgh; 2 Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Mitochondrial disorders are being diagnosed with increased frequency. Although many genetic counseling issues are shared with traditional Mendelian disorders, mitochondrial disorders present a unique set of challenges. One of the most important roles of the genetic counselor is to obtain a very targeted and detailed pedigree to look for evidence of maternal line disease or a Mendelian inheritance pattern. It is important to check for the presence of subtle features in family members such as migraines, seizures, gastrointestinal complaints (irritable bowel syndrome/ constipation/diarrhea), hearing and/or vision loss, endocrine dysfunction (diabetes/hypothyroidism), lipomas, chronic fatigue and muscle weakness which could be a clue to the pattern of inheritance or even the speci®c disorder or point mutation. Genetic counselors are critical in tracking down records for the proband and family members and meticulously completing the paperwork so that testing is done properly. The diagnostic process is often a lengthy one, which can be extremely frustrating and emotionally draining for the family. If and when a diagnosis is made, the genetic counselor has the challenge of explaining dif®cult concepts such as heteroplasmy, oxidative phosphorylation, nuclear DNA, mitochondrial DNA, and nuclear-mitochondrial gene interactions. In addition, when a diagnosis of a maternally inherited mitochondrial disease is made in a proband, the same diagnosis may be made in other family members. As in other genetic disorders, the diagnosis is often accompanied by feelings of grief, despair and guilt. Providing psychological support can be a daunting task, even to an experienced genetic counselor. Individuals with mitochondrial diseases may bene®t tremendously from psychosocial counseling by a trained mental health provider. In summary, mitochondrial conditions present a unique challenge to genetic counselors and require signi®cant time and attention to detail to assist the team in the diagnostic process.

39 Disordered mitochondrial function in osteoarthritis K. Johnson 1, S. Ghosh 2, A. Murphy 2, R. Terkeltaub 1 1 VA Medical Center, University of California; 2MitoKor, San Diego, CA, USA Mitochondrial dysfunction has been implicated in certain neurodegenerative disorders of aging. Osteoarthritis (OA) is a highly prevalent cartilage degenerative disorder in aging. It has been noted that elevated production of nitric oxide (NO) by cartilage chondrocytes that occur in OA promotes matrix degeneration in the disease. We demonstrated that NO suppressed mitochondrial respiration and intracellular ATP in cultured chondrocytes,and provided evidence that mitochondrial ATP is essential for preser-

Abstracts ving normal chondrocyte function. Furthermore, we detected markedly impaired growth and depressed ATP concentrations in association with elevated NO production in knee cartilage chondrocytes from aged guinea pigs with spontaneous OA. We have developed screening assays that examine the ability of compounds to preserve mitochondrial function as well as growth potential and matrix synthetic activity of cultured chondrocytes exposed to micromolar amounts of the NO donor NOC-12 and the peroxynitrite donor Sin1, and to 10 ng/ml IL-1, an iNOS-inducing cytokine directly implicated in OA pathogenesis. These assays identi®ed MITO-2227 that at submicromolar concentrations markedly reduced chondrocyte ATP depletion and viability loss in response to Noc-12 and Sin-1, and concurrently suppressed the abilities of Noc-12, Sin-1 and IL-1 to attenuate collagen synthesis and increase matrix glycosaminoglycans degradation. We conclude that disordered mitochondrial respiration may be involved in the pathogenesis of OA, and that preservation of mitochondrial function has the potential to prevent the development of chondrocyte phenotypic features seen in cells from OA cartilage.

40 Oral pyrimidine treatment protects mice against striatal damage and behavioral impairment induced by 3nitropropionic acid J.A. Saydoff, L.S. Liu, R.W. von Borstel Pro-Neuron, Inc., Gaithersburg, MD 20877, USA De novo biosynthesis of uridine nucleotides is directly coupled to the respiratory chain via the mitochondrial enzyme dihydroorotate dehydrogenase. Symptoms of respiratory chain dysfunction in vivo may therefore involve pyrimidine de®cits. Oral administration of PN401 (triacetyluridine) delivers much higher levels of uridine to the circulation than oral administration of uridine itself. Huntington's disease (HD) involves progressive neuronal loss especially in the striatum, substantia nigra and thalamus in addition to weight loss. HD is associated with decreased activity of mitochondrial succinate dehydrogenase (Complex II). This enzyme can be inhibited in animals by i.p. 3-nitropropionic acid (3NP). Mice were treated daily with i.p. 3NP or saline and p.o. PN401 b.i.d. 4 g/kg or vehicle treatment began 1 day before the ®rst dose of 3NP. The vehicle 1 3NP group had neuronal damage detected by silver staining in the striatum, substantia nigra and/or thalamus in 80% of the mice with 38% mortality. The 3NP 1 PN401 group had reduced neuronal damage observed (13% of mice in only one area) and there was no mortality. In two subsequent studies, 3NP also induced weight loss and behavioral impairment in rotarod and activity measurements. PN401 signi®cantly ameliorated 3NP-induced weight loss and behavioral impairment in rotarod and activity measurements. Thus, oral PN401 treatment has neuroprotective effects in a model of mitochondrial dysfunction.

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41 Induction of the permeability transition contributes to mitochondrial injury and altered respiration during endotoxemia E.D. Crouser, M.W. Julian, D.R. Pfeiffer The Ohio State University, Columbus, OH, USA Altered O2 metabolism in systemic organs is a hallmark of organ failure during sepsis. Previous research suggests that mitochondrial injury is a major cause of impaired O2 metabolism during sepsis. Furthermore, ultrastructural analysis demonstrates dramatic mitochondrial swelling in systemic organs during acute sepsis, such as during the permeability transition (PT). Thus, we hypothesized that the PT is involved and that inhibition of the PT would protect against sepsis-induced injury. To test this hypothesis we used a well-established feline model of sepsis (IV LPS). In three experimental groups, LPS (3 mg/kg, IV; n ˆ 7), LPS (3 mg/kg) pretreated with cyclosporin A (CsA, 6 mg/kg, IV n ˆ 7) or control (vehicle; n ˆ 6), liver samples were obtained at 4 h and simultaneous evaluations of mitochondrial ultrastructure and respiratory function were carried out. Ultrastructural changes were most severe in the LPS treatment group as re¯ected by higher mitochondrial injury scores (2.8 ^ 0.2 vs. 1.3 ^ 0.2; LPS vs. control, P , 0.001), whereas, CsA pretreatment was partially protective (1.8 ^ .4, P , 0.5 compared to LPS alone). LPS treatment was also associated with decreased state 3 respiration, and an increase in state 4 respiration apparently re¯ecting partial uncoupling of mitochondrial O2 consumption from ATP production. The magnitude of structural injury correlated closely (r ˆ 20.97) to the extent of functional impairment. CsA pretreated animals had normal respiratory control in most cases, but some of the preparations exhibited reduced respiration under state 3, state 4 and uncoupled conditions. These rates were restored by adding exogenous cytochrome c. These ®ndings suggest that the permeability transition occurs during sepsis and contributes signi®cantly to impaired O2 metabolism. The results further indicate that the mitochondrial impairment is due, in part, to the release of cytochrome c. ALA grant RG-038-N, NIH grant 1 KO8 HLO4335-01 and AHA grant 0051013B.

42 Oxidation of mitochondrial respiratory chain Complex I substrates is reduced in lymphocytes from Alzheimer's disease patients J. Casademont, O. MiroÂ, P. Viedma, R. Blesa, F. Cardellach Internal Medicine and Neurology Departments, Hospital ClIÂnic, IDIBAPS and University of Barcelona, Villarroel 170, 08036 Barcelona, Spain Introduction: Several lines of evidence suggest that abnormalities in oxidative metabolism and speci®cally in mitochondria may play an important role in Alzheimer's disease (AD). Abnormalities have been found in brain tissues, as well as in non-neural cells such as ®broblasts and platelets. Among the multifactorial etiology of

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this disease, oxidative stress, acting as a by-product of the respiratory chain (RC), has been advocated to promote damage to mtDNA, proteins and membrane phospholipids, with further impairment of the respiratory machinery in a sort of catastrophic vicious cycle, leading to an increased neuronal susceptibility to cell death. Patient and methods: We studied oxygen consumption, activity of mitochondrial RC complexes and membrane lipid peroxidation in lymphocytes from 23 AD patients and 23 age-matched controls. Individual enzyme activity of Complexes II, III and IV were measured spectrophotometrically. Intact cell respiration and oxidative rates after addition of pyruvate-malate, glutamate-malate, succinate and G3P were assessed polarographically. Results were normalized by the activity of cytrate syntase. Lipid peroxidation of membranes was assessed measuring the loss of cis-parinaric acid ¯uorescence. Results: Oxidative rates with pyruvate-malate and glutamate-malate were decreased in AD patients (P ˆ 0.002 and 0.007, respectively). Conversely, oxidation of succinate, G3P, and the activities of Complexes II and III were normal, while the activity of Complex IV was low, but in the limit of statistical signi®cance. Lipid peroxidation of lymphocyte membranes was identical in AD and in controls. Discussion: Our ®ndings support the hypothesis that there is a generalized decreased oxidation of Complex I substrates in AD. The preserved lipid peroxidation in lymphocyte membranes indicates that, in non-neural tissues, an increased activity of antioxidant compensatory mechanisms may maintain lymphocytes in oxidative homeostatic balance, a circumstance that may not occur in neurons. Supported by Fundacio La Marato de TV3 2102/97 and FIS 00/ 927.

43 Non-radioactive high sensitivity heteroplasmy detection H.-G. Gao, M.L. Smith, R.K. Naviaux, E. Ferrer-Perez, A. Guttman, R.H. Haas The Mitochondrial and Metabolic Disease Center, University of California, San Diego, School of Medicine, 200 W. Arbor Drive, San Diego, CA 92103-8467, USA Aims/hypothesis: Detection of low percentage heteroplasmy remains a technical challenge in mitochondrial medicine. We have previously reported the use of high sensitivity radiolabled PCR analysis of the A3243G mutation allowing detection at better than 1% of heteroplasmy. We have applied an ultrathin layer electrophoresis (ULE) separation system with real time, ®ber optic bundle based laser induced ¯uorescence/avalanche photodiode detection to this problem. Methods: Using blood samples obtained from mothers of clinically affected MELAS patients we compared routine PCR with radiolabled PCR and with Hex labeled primer assays using ULE separation in a 1% enhanced agarose gel containing ethidium bromide. Radiolabeled and Hex labeled PCR assays were stopped after 15 PCR cycles in the linear phase of ampli®cation, allowing quantitative measurements of the percentage of mutation [1]. Results: The presence of a 1% heteroplasmic A3243G mutation was not visible by the standard PCR screening method. In fact, detection below 10% of mutation could not be achieved by

standard methods. The 1% level of mutation was readily detectable using the highly sensitive radioactive and Hex labeled ¯uorescent PCR assays. The ¯uorescent Hex labeled primer assay is fast and allows ready quantitation of low percentage heteroplasmy. Sensitivity is similar to radiolabled PCR with the advantages of speed and lack of radioactivity. Conclusions: Hex labeled PCR with ULE separation and laser photodiode detection is a fast and effective method for detection of low percentage heteroplasmy. This method is suitable for population screening for speci®c mutations such as the A3243G mutation responsible for 0.5±2% of diabetes. [1] Smith ML, Hua XY, Marsden DL et al., 1997. J Clin Endocrinol Metab 82:2826±2831.

44 Inactivation of OGG1 gene leads to increased frequency of mitochondrial deletions K.K. Singh, B. Sigala, H.A. Sikder, C. Schwimmer Johns Hopkins Oncology Center, Bunting-Blaustein Cancer Research Building, 1650 Orleans Street, Baltimore, MD 21231, USA A number of mitochondrial diseases are caused by mutations in mitochondrial DNA (mtDNA). However, the molecular mechanism(s) that are involved in mitochondrial mutagenesis is not clear. The OGG1 gene encodes a highly conserved DNA glycosylase between humans and yeast that repairs oxidized guanines in DNA. We have investigated the in vivo function of Ogg1 protein in yeast mitochondria. We demonstrate that inactivation of ogg1 leads to at least a 2-fold increase in production of spontaneous mitochondrial mutants compared with wild type. Using green ¯uorescent protein (GFP) we show that Ogg1p localizes to the mitochondria and the nucleus and contains distinct localization signals for these compartments. Our analysis shows that deletion of the ®rst 11 amino acids from the N-terminus abolishes the transport of GFPOgg1 fusion protein to the mitochondria. Removal of 76 amino acids from C-terminus that contains the bipartite nuclear localization signal (NLS) abolishes the GFP-Ogg1p transport to the nucleus. Interestingly, when both the MLS and the NLS were present, the protein localized to the cytoplasm. We provide evidence that both yeast and human Ogg1 proteins protect the mitochondrial genome from spontaneous as well as induced oxidative damage. Genetic analyses revealed that the combined inactivation of OGG1 and OGG2 (encoding an isoform of Ogg1 protein, also known as NTG1, eNdonuclease Three-like Glycosylase I) leads to suppression of spontaneously arising mutation in mitochondrial genome when compared with the ogg1 single mutant or the wild type. Together, these studies provide in vivo evidence for repair of oxidative lesions in mitochondrial genome by human and yeast Ogg1 proteins. Our study also identi®es Ogg2 as a suppressor of oxidative mutagenesis in mitochondria.

Abstracts 45 Alterations in gene expression in Leigh's patients with Complex I defects detected using the Mitochip DNA microarray V.F. Procaccio, K.W. Kerstann, S.H. Hosseini, P.Z. Golik, D.C. Wallace Center for Molecular Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA Leigh's syndrome can be caused by a variety of genetic defects including mutations in the nuclear encoded subunits of respiratory Complex I. Complex I or NADH dehydrogenase is composed of 43 polypeptides, seven encoded by the mitochondrial DNA (mtDNA) and the rest encoded by the nuclear DNA (nDNA). Analysis of selected nDNA genes from Leigh's patients with Complex I defects revealed that one patient was a compound heterozygote for two different missense mutations in the 23 kDa, Complex I, NDUFS8 subunit. This mutation resulted in a mild Complex I defect in the patient's lymphoblastoid cell line. To investigate the pathophysiology of the disease caused by these mutations, we examined changes in mitochondrially-related gene expression using our `Mitochip' DNA microarray. The Mitochip employed encompassed 590 human cDNAs derived from genes involved in energy metabolism, oxidative stress, and apoptosis. The mRNAs from patient and control lymphoblastoid cells were converted to cDNAs and differentially labeled with Cy3 and Cy5 dyes using reverse transcriptase. The labeled cDNAs were mixed and hybridized to polylysinecoated slides to which the cDNAs had been spotted. The spectra of the individual Mitochip spots were determined and the relative levels of patient and control mRNAs calculated. The resulting expression pro®les revealed that the patient's NDUFS8 missense mutations resulted in the down-regulation of both mtDNA (e.g. ND4, ND4L, and ND6) and nDNA (e.g. NDUFS5 and NDUFV2) Complex I genes. However, the mRNA levels of the NDUFS8 gene were not altered. These ®ndings suggest that gene expression pro®ling using the Mitochip may provide a powerful screening tool for mitochondrial disorders and may reveal new insights into mitochondrial disease pathophysiology.

46 Sunburnt mitochondrial DNA; patterns of UV-induced mitochondrial DNA damage in human skin M.A. Birch-Machin, K. Krishnan, A. Ray Department of Dermatology, Medical School, University of Newcastle, Newcastle upon Tyne, NE2 4HH, UK Ultraviolet radiation (UVR) is the major determinant of nonmelanoma skin cancer, which is the commonest cancer in many Caucasian populations. The principal aim of this study is the use of mitochondrial DNA (mtDNA) as a candidate biomarker of cumulative sun exposure in human skin. We have investigated: ² The direct effect of UVR on mtDNA. Using a south-western approach involving monoclonal antibodies against thymine dimers, we provide direct evidence for the presence of UV-

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induced damage in puri®ed mtDNA from human skin cells. ² The indirect consequences of UVR on mtDNA damage by looking at changes in the incidence of mtDNA; (i) deletions and (ii) duplications secondary to UV exposure. ² We investigated the incidence of the 4977-bp-common mtDNA deletion in 190 human skin samples. High levels (i.e. .2%) were signi®cantly associated with sun-exposed (27%, 27/100) compared to sun-protected sites (0%, (0/90) (Fisher's exact test, P , 0.0001). In addition we determined the spectrum of mtDNA deletions from the entire mitochondrial genome in 71 skin samples using a long PCR technique. There was a signi®cant increase in the number of deletions with increasing UVR exposure in the epidermis (Kruskal-Wallis test, P ˆ 0.0015). This observation was not confounded by age. ² We have used a `back to back' primer methodology to investigate the incidence of tandem duplications in the non-coding region with relation to sun-exposure in 32 age-matched, human skin samples. An increased incidence of duplications with increasing sun-exposure was observed, with duplications identi®ed in 10/24 but 0/10 samples from sun-exposed and sun-protected skin, respectively (Fisher's exact test, P ˆ 0.015). The sizes of the most frequent duplications were 200 and 260 bp. Interestingly these same samples also contained high levels (.2%) of the 4977-bp common mtDNA deletion. We conclude that mtDNA damage appears to be a good biomarker for exposure of skin to UVR.

47 Presence of mitochondrial proteins in extramitochondrial compartments: a newly emerging phenomenon of relevance to mitochondrial myopathies S. Sadacharan, R.S. Gupta Department of Biochemistry, McMaster University, Hamilton, Canada L8N 3Z5 The mitochondrial proteins are presently believed to reside and function only within the organelle. However, in recent years many examples have come to light where proteins originally identi®ed on the basis of extramitochondrial functions, upon characterization were found to be bona ®de mitochondrial proteins (Soltys and Gupta, 1999. TIBS, 24:174±177; Int Rev Cytol, 2000, 194:133± 196). This raises important questions concerning the cellular functions of mitochondrial proteins. To investigate this, we have examined by means of immunoelectron microscopy the subcellular localization in normal rat tissues of a number of well-characterized mitochondrial proteins which are encoded either by the nucleus (viz., cytochrome c oxidase subunit 6c, ATP synthase subunit, pyruvate dehydrogenase E2 subunit, cytochrome c, Hsp60, mitAspartate aminotransferase, mitHsp70, Cpn10, P32, TRAP-1), or by mitDNA (cytochrome c oxidase subunits I and II) using highly speci®c antibodies. In all tissues examined, these antibodies showed strong labeling of mitochondria, as was expected. In several tissues, the binding was seen exclusively within mitochondria. However, in a variety of tissues such as liver, acinar pancreas, anterior pituitary,

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spleen, salivary glands and kidney, these antibodies also showed strong and speci®c labeling of one or more of the following compartments ± pancreatic zymogen granules, growth hormone granules, secretory granules in islet cells, red blood cells and peroxisomes. In a number of cases, strong labeling was also observed at the cell surface and in nuclei. All of the observed labeling with these antibodies, both within mitochondria and in other compartments, was abolished upon adsorption of the antibodies with recombinant proteins or upon omitting the primary antibodies. The extramitochondrial localization of various proteins, particularly those encoded by mtDNA provides strong evidence that these proteins have reached these sites by exiting from mitochondria. These observations have important implications concerning the roles of mitochondria and mitochondrial-resident proteins in different diseases.

48 A population of Rett and Angelman syndrome patients has an associated electron transport chain de®ciency R.P. Saneto, B.H. Cohen, A. Frabotta, C.L. Hoppel The Cleveland Clinic Foundation; The Cleveland Veteran Affairs Medical Center, Cleveland, OH, USA Introduction: Mitochondrial cytopathies (MTC) can manifest with a variety of phenotypic expressions that depend on threshold effect, mutation type, and tissue expression. While investigating children for MTC, we found seven patients with bioenergetic disorders associated with Rett and Angelman syndromes. Patients and Methods: We evaluated seven children with unknown developmental disorders and epilepsy of suspected MTC. Based on clinical and laboratory screening, these patients underwent muscle biopsy for microscopy and determination of oxidative phosphorylation (OXPHOS) and/or electron transport chain (ETC) function. Results: Four children had the typical clinical manifestations of Rett syndrome. All of the children had ECT dysfunction and systemic lactic acidosis. Three patients had Complex III de®ciencies [4.1±9.2 mmole/min per gram wet weight; control (decylubinquinol-cytochrome c reductase) 19.4 ^ 6.3 mmole/min per gram wet weight). One patient had a Complex II de®ciency (0.2 mmole/min per gram wet weight; control (succinate dehydrogenase) 2.3 ^ 1.1 mmole/min per gram wet weight). Three of the children had the MeCP2 mutation. Three children had the clinical manifestations of Angelman syndrome. All of the children had the methylation pattern found in Angelman syndrome and systemic lactic acidosis. Two of the children had ECT dysfunction. One child had a Complex III de®ciency (2.7 mmole/min per gram wet weight) and one had a Complex I de®ciency (206 mmole/min per gram wet weight; control (rotenone sensitive NADH-cytochrome c reductase) 481 ^ 37 mmole/min per gram wet weight). One child had normal ETC function, but abnormal mitochondrion morphology by electron microscopy on skin ®broblast biopsy. Discussion: These patients presented with symptoms suggestive of a primary MTC and whose bioenergetic function as determined by polarogrpahy and/or spectrophotometry, or systemic lactic acidosis was abnormal. All eventually developed the clinical manifestations of Rett or Angelman syndrome. The early clinical manifestations of these syndromes can

often be so similar that distinguishing them from one another and other types of MTC can be challenging. Although the etiology of the mitochondrial dysfunction in both disorders remains unclear, the early clinical manifestations suggests that a primary or secondary disorder of energy production may be involved in both syndromes. Conclusion: Clinical acumen remains critical in obtaining the proper diagnosis. However, primary or secondary disorders of energy production may be more common than previously appreciated with both Rett and Angelman syndromes and possibly other common clinically characterized neurological disorders.

49 Autosomal dominant acute necrotizing encephalopathy associated with uncoupling of mitochondrial oxidative phosphorylation D. Neilson, S. Waniewski, R. Eiben, M. Wiznitzer, M. Varnes, C. Hoppel, M. Warman, D. Kerr Departments of Pediatrics, Genetics, and Medicine, Case Western Reserve University, DVA Medical Center, and MetroHealth Medical Center, Cleveland, OH, USA We have investigated clinical, radiographic, biochemical, and familial data from a large kindred with acute encephalopathy associated with febrile illnesses, including case presentations, pedigree, and metabolic analyses. Three-year-old, developmentally normal identical twins developed acute encephalopathy following a fever. Ammonia, blood lactate and pyruvate, urine organic acids, and plasma amino acids were normal. CSF lactate, pyruvate, and cell counts were normal. MRI of the brain demonstrated symmetric increased T2 and FLAIR weighted signal along the pons, midbrain, thalami, external capsule, and subinsular white matter in one case and included the medial temporal lobes and cerebellar hemispheres in the other. Oxidative phosphorylation analysis of intact mitochondria isolated from a muscle biopsy demonstrated an increase in state 4 oxidation rates and low respiratory control, indicating uncoupling. Both twins made remarkable virtually complete recoveries within 2 months. A six-generation pedigree reveals 16 individuals with similar acute encephalopathies. Some relatives recovered completely while others did not. Male-to-male transmission in a dominant pedigree demonstrates autosomal dominant inheritance. Unaffected obligate carriers prove incomplete penetrance. This family represents a previously undescribed, incompletely penetrant, autosomal dominant disorder with some similarities to other mitochondrial encephalopathies, but without the characteristic degenerative course.

Abstracts

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Hypertrophic cardiomyopathy due to a homoplasmic mitochondrial tRNA Ile mutation: studies in two families R.W. Taylor 1, M.M. Davidson 2, C. Giordano 2, G. d'Amati 3, C. Casali 3, M. Hirano 2, S. DiMauro 2, D.M. Turnbull 1 1 Department of Neurology, University of Newcastle upon Tyne, UK; 2Department of Neurology, College of Physicians and Surgeons of Columbia University, New York, USA; 3Istituto di Clinica delle Malattie Nervose e Mentali, Dipartimento di Medicina Sperimentale e Patologia, UniversitaÁ di Roma-La Sapienza, Rome, Italy

Reversal of paracrystalline inclusions in a patient with a G15497A missense mutation following creatine monohydrate supplementation M.A. Tarnopolsky 1, K. Chorneyko 2, D.K. Simon 3, D.R. Johns 3,4 1 Department of Neurology/Rehabilitation; 2Department of and Pathology, McMaster University, Hamilton, ON; 3Department of Neurology; 4Department of Opthalmology, Beth Israel Deaconess Medical Center, Boston, MA, USA.

Pathogenic point mutations within the mitochondrial genome (mtDNA) have been described in association with many clinical phenotypes. They occur in both protein-encoding genes and RNA (rRNA and tRNA) genes and are generally maternally inherited. With the exception of several well-characterized mutations associated with Leber hereditary optic neuropathy (LHON) and neurogenic weakness, ataxia with retinitis pigmentosa (NARP) which can occur in all mtDNA copies within an individual (homoplasmy), pathogenic mtDNA point mutations are present in a subset of mtDNA molecules (heteroplasmy). In this report, we present two independent families (one British, one Italian) presenting with hypertrophic cardiomyopathy in which a mitochondrial genetic defect was strongly suspected. In one family in which two siblings had died in the ®rst few years of life, histochemical analyses of affected heart tissue revealed a mosaic of COX reactivity, with .95% COX-negative cardiomyocytes in the left ventricle. This was associated with severe de®ciencies of Complexes I and IV. Surprisingly, studies of the skeletal muscle revealed normal histochemistry and biochemistry. Sequencing the entire mitochondrial genome of affected individuals from both families revealed an A4300G transition in the tRNA Ile gene, which was shown to be homoplasmic by PCR-RFLP analysis. Interestingly, this homoplasmic mutation was also present in blood samples taken from several maternal relatives. High resolution Northern blots demonstrated very low steady-state levels of the mature tRNA Ile in heart and skeletal muscle, and decreased levels in cultured myoblasts and ®broblasts. Transmitochondrial cybrids generated from the ®broblasts of one patient, however, did not exhibit a respiratory defect. Whilst the precise molecular mechanism by which this mutation causes disease remains unclear, a role for a synergistic, nuclearencoded factor cannot be excluded. Furthermore, these studies highlight the important role that homoplasmic tRNA substitutions may play in contributing to pathogenicity.

A hallmark of several of the mitochondrial cytopathies is the presence of paracrystalline inclusions in the mitochondria. These are composed of crystals of dimeric mitochondrial creatine kinase (mtCK). Creatine monohydrate has been shown to stabilize mtCK in the octameric form, which could allow for reversal or prevention of paracrystalline formation. We serendipitously found paracrystalline inclusions in a young male athlete who was involved in a study looking at the ultra-structure of intra-muscular lipid. His neurological examination was normal, yet he had a very high respiratory exchange ratio during exercise (seen in mitochondrial disorders). His mother had been diagnosed with seizures as a teenager, but had a normal neurological examination. Direct DNA sequencing of the mitochondrial genome revealed a novel missense mutation at G15497A of mtDNA that resulted in a glycine to serine conversion at a highly conserved site in the cytochrome b gene in both patients. No other potentially pathogenic mutations were identi®ed in the mtDNA sequencing. The patient was treated with creatine monohydrate (5 g/day) for a period of 5 weeks and had a repeat muscle biopsy of the ipsilateral vastus lateralisin which no paracrystalline inclusions were found and exercise RER was lower. He then discontinued the creatine supplementation for 3 months and had a repeat biopsy in the contralateral vastus lateralis showing early paracrystalline formation and electron densities in the mitochondria. Western blotting for mtCK protein did not show a reduction in total mtCK content. These observations suggest that paracrystalline inclusions in muscle are dynamic and may be reversed by creatine monohydrate supplementation, possibly by octameric stabilization. The potential long-term bene®ts of creatine monohydrate in mitochondrial disorders remains to be further elucidated. Supported by the Hamilton Health Sciences Corporation ± Department of Rehabilitation.

52 Inhibition of mitochondrial processing protease in vitro by protease inhibitors: a novel mechanism for mitochondrial dysfunction in HAART-associated lipodystrophy S.J. Zullo 1,B. Wei 2, A. Mukhopadhyay 2, L. Wood 3, H. Weiner 2 NIMH, Bethesda, MD; 2Purdue University, West Lafayette, IN; 3 NCI, Bethesda, MD, USA Background: Highly active antiretroviral therapy (HAART) can induce a lipodystrophy syndrome characterized by peripheral fat wasting and central adiposity, often associated with hyperlipidemia and insulin resistance. Indirect data suggest that mitochondrial

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dysfunction may play a role in this syndrome. While the mitochondrial toxicity due to nucleoside reverse-transcriptase inhibitors (NRTIs) is well described, the impact of protease inhibitors (PIs) on mitochondrial function is unknown. Many mitochondrial proteins encoded by the nuclear genome possess an amino-terminal leader peptide that directs the preprotein to the mitochondrial membrane. Mitochondrial processing protease (MPP) cleaves this leader sequence in the mitochondrial matrix. Lack of cleavage could result in non-functional mitochondrial proteins. Methods: We examined the effects of the PIs indinavir and nel®navir on yeast MPP, a recognized model for mammalian MPP. Radiolabelled in vitro transcribed and translated prealdehyde dehydrogenase (ALDH) was incubated with yeast mitochondria and increasing concentrations of PIs (0.1±5.0 mg/ml of drug). Controls were ALDH without mitochondria and ALDH with mitochondria but no drug. Following incubation, reactions were treated with proteinase K to destroy protein not imported into mitochondria. Additionally, the drugs were added to pure MPP to determine if this would prevent the processing of ALDH. Results: The ED50 for MPP inhibition was 2.5 mg/ml and 5 mg/ml for indinavir and nel®navir, respectively. MPP inhibition resulted in lack of cleavage of the mitochondiral leader peptide necessary to produce mature mitochondrial proteins. Inhibition of mitochondrial preprotein import occurred only at the highest concentration (5 mg/ml) of indinavir. The drugs also inhibited the pure protease. Conclusions: PIs commonly used as part of HAART in the treatment of HIV infection are capable of inhibiting MPP, impairing the cleavage of mitochondrial leader sequences and resulting in the delivery of mitochondrial preproteins rather than mature proteins to the mitochondrial matrix. These preproteins may impair mitochondrial function and contribute to the observed mitochondrial dysfunction implicated in HAART-associated lipodystrophy. Though the dose needed to cause inhibition was high, the drugs are administered in large doses to the patient and the inhibition could increase with time. It is not known if the drugs or an active metabolite can accumulate in the matrix space thus facilitating inhibition.

53 Differential bigenomic regulation of cytochrome oxidase subunit mRNAs in neurons M.T.T. Wong-Riley, C.Y Zhang. Department of Cell Biology, Neurobiology and Anatomy Medical College of Wisconsin, Milwaukee, WI 53226, USA Previous studies have shown that cytochrome oxidase can serve as a sensitive indicator of neuronal activity. Neuronal activity, in turn, differentially regulates the expression of mitochondrial- and nuclear-encoded subunits of this enzyme. We sought to determine if these changes were due to RNA synthesis rate or stability, or both. Primary neuronal cultures were treated with KCl to induce depolarizing activity. Five hours of treatment induced an up-regulation of subunits II (mitochondrial) and IV (nuclear) mRNAs. The relative rates of synthesis of these two mRNA species were also signi®cantly elevated. There was no statistically signi®cant change in the half-life of subunit II, which is around 84 min. However, the

half-life of subunit IV was increased from 50 to 102 min. Our results indicate that the mitochondrial subunit II mRNAs in neurons are regulated mainly at the transcriptional level, whereas the nuclear subunit IV mRNAs are regulated at both the synthetic and degradative levels. Both, however, are tightly controlled by neuronal activity. Supported by NIH R01 EY05439.

54 Mitochondrial elongation factor Tu is a target of phosphorylation in the ischemic myocardium H. He 1, M. Chen 1, N.K. Schef¯er 2, B.W. Gibson 2, L.L. Spremulli 3, R.A. Gottlieb 1 1 The Scripps Research Institute, La Jolla; 2University of California, San Francisco, CA; 3University of North Carolina, Chapel Hill, NC, USA The objective of this study was to identify the mitochondrial proteins that undergo changes in phosphorylation during global ischemia and reperfusion in the isolated rabbit heart. We also assessed whether the cardioprotective intervention of ischemic preconditioning affected mitochondrial protein phosphorylation. We established a reconstituted system using isolated mitochondria and cytosol from control or ischemic hearts. We found that phosphorylation of a 46 kDa protein on a serine residue was increased in ischemia and that phosphorylation was reduced in control or preconditioned hearts. Using 2D gel electrophoresis and mass spectrometry, we have identi®ed the 46 kDa protein as EF-Tumt. These data reveal that ischemia and preconditioning modulate the phosphorylation of EF-Tumt, and suggest that the mitochondrial protein synthesis machinery may be regulated by phosphorylation.

55 The catalytic activity of the respiratory chain-linked NADHubiquinone oxidoreductase (Complex I) in intact mitochondria V.G. Grivennikova 1, I.S. Krysova 1, L.E. Bakeeva 2, A.D. Vinogradov 1 1 Department of Biochemistry, School of Biology; 2A.N. Belozersky Institute for Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia A number of genetic diseases and pathological conditions are believed to be associated with the mitochondrial NADH-ubiquinone oxidoreductase (Complex I) de®ciency. The enzyme preparations with different degrees of resolution (detergent solubilized puri®ed enzyme, inside-out submitochondrial particles) show extremely complex steady-state and pre-steady-state kinetic properties [1]. The absence of a reliable catalytic assay procedure for Complex I in situ within intact mitochondria may lead to misconceptions and pitfalls concerning diagnosis of the enzyme de®ciency. To overcome this problem we developed a simple procedure for direct quantitation of Complex I activity in isolated mitochondria using channel-forming antibiotic alamethicin (Ala) for permeabilization

Abstracts of the inner mitochondrial membrane for externally added NADH [2]. Ala-treated mitochondria retain the matrix located proteins and catalyze the rotenone-sensitive oxidation of externally added NADH by quinone-acceptor at the rates expected if the enzyme active sites are freely accessible for the substrates. Electron microscopy shows that Ala treatment of tightly coupled rat heart mitochondria leads to considerable swelling of the matrix without disruption of the outer and inner mitochondrial membranes. The slow interconversion between active (A) and de-activated (D) enzyme forms, previously described for inside-out submitochondrial particles [1] is shown to be an intrinsic feature of Complex I in intact mitochondria. The A/D-transition affected by divalent cations is proposed to play a crucial role in recovery of the mitochondrial functions after exposure to 'non-working' conditions such as anoxia. [1] Vinogradov AD, 1998. Biochim Biophys Acta 1364:169±185. [2] Grivennikova VG, Kapustin AN, Vinogradov AD, 2001. J Biol Chem (in press). Supported by the Royal Swedish Academy of Science collaborative grant 12557 (to ADV).

56 Correction of renal tubular acidosis (RTA) in mitochondrial disease patients treated with triacetyluridine (PN401) R.K. Naviaux, K. McGowan, B.A. Barshop, W.L. Nyhan, R.H. Haas The Mitochondrial and Metabolic Disease Center (MMDC), University of California School of Medicine, 200 West Arbor Drive, San Diego, CA 92103-8467, USA Background: Patients with a variety of different mitochondrial disorders may be functionally de®cient in uridine. De novo pyrimidine biosynthesis is dependent on normal mitochondrial function and is coupled to the mitochondrial respiratory chain via ubiquinone and the enzyme dihydroorotate CoQ oxidoreductase (DHO-QO, EC 1.3.99.11), also known as dihydroorotate dehydrogenase (DHOD, EC 1.3.3.1). Cells with mitochondrial dysfunction in culture are known to be completely dependent on exogenous uridine for growth and survival because of a functional de®ciency in the activity of DHO-QO. Triacetyluridine (PN401; Pro-Neuron, Inc., Gaithersburg, MD) is a prodrug of uridine that is rapidly converted to uridine by non-speci®c esterases in the gastrointestinal epithelium after oral delivery. PN401 exhibits 5±10 times the bioavailability of unmodi®ed uridine, permitting the achievement of therapeutic blood levels of uridine with substantially less drug. Patients: Four patients with mitochondrial RTAs were studied. Mitochondrial RTAs often defy simple classi®cation as proximal (type II) or distal (type I) tubulopathies, as these patients are often mosaics and express features of both phenotypes leading to hyperchloremic (non-anion gap) metabolic acidoses. Patient 1 was a 2-year-old female with Leigh syndrome, lactic acidemia, and Complex I de®ciency who required 200 mEq/day of NaHCO3 to compensate for renal losses of alkali, and maintain serum bicarbonate levels above 20 mEq/l, 1 1 proteinuria, and signi®cant aminoaciduria with hydroxyprolinuria. Patient 2 was a 3-year-old female with Leigh syndrome, and Complex IV (COX) de®ciency, who also required 200 mEq/day of NaHCO3.

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Patient 3 was a 2-year-old male with Leigh syndrome, lactic acidemia, 1 1 proteinuria, and pyruvate dehydrogenase (PDH) de®ciency, who required up to 210 mEq/day. Patient 4 was an 11year-old male with 3-hydroxyisobutyric aciduria, lactic acidemia, and encephalomyopathy, who required 468 mEq/day. Methods: Blood and urine electrolytes, creatinine, pH, urinalysis, and venous blood gases were studied before and after treatment. Quantitative urine amino acids and organic acids were also obtained. Pre-enrollment doses of oral sodium bicarbonate were continued for the ®rst 3 days of PN401 treatment, then reduced weekly as tolerated to maintain serum bicarbonate above 20 mEq/l. Treatment: Patients received triacetyluridine (PN401) 2 g/m 2 PO TID. Results: Patient 1 experienced complete correction of her RTA within 24 h of treatment, maintaining a serum bicarbonate of more than 20 mEq/l without any further oral bicarbonate. She also had complete resolution of her hydroxyprolinuria within 2 weeks. Urinary bicarbonate losses in patient 2 were initially 99 mEq/l. The fractional excretion of bicarbonate (FEHCO3) was 9.3% prior to therapy. After 36 h of treatment with PN401, urinary bicarbonate losses were undetectable (,5 mEq/l). After 3 weeks of treatment, just 25% of the patient's preenrollment dose of bicarbonate was needed to maintain normal serum bicarbonate. Urinary bicarbonate losses in patient 3 were 59 mEq/l. The FEHCO3 was 10.0% prior to therapy. After 36 h of treatment with PN401, urinary bicarbonate losses were undetectable (,5 mEq/l). After 3 weeks of treatment the patient required just 10% of his previous dose of bicarbonate. Patient 4 has been treated for 1 week and has had a 35% reduction in his oral bicarbonate requirement. Conclusion: Renal tubular acidosis was corrected or dramatically improved in four out of four patients with mitochondrial disease treated with triacetyluridine (PN401).

57 Synthesis of mitochondrial DNA in permeabilized cells derived from patients with mitochondrial diseases and controls J. Poulton 1, C. Freeman Emmerson 1,2 1 Mitochondrial Genetics Group, University of Oxford Department of Paediatrics, John Radcliffe Hospital, Headington, OX3 9DU; 2 PDOP, Roche Products Ltd, 40 Broadwater Rd, Welwyn Garden City, UK The mechanisms that underlie the maintenance and increase of mutant mtDNA are central to our understanding of mitochondrial disease. We have therefore developed a technique based on saponin permeabilization that allows the study of mtDNA synthesis in intact cells. Permeabilisation of cells is used in established methods for nuclear processes. We have studied incorporation of radiolabelled DNA precursors into mitochondrial DNA in human cell lines, using aphidicolin to inhibit nuclear DNA replication. Total cell DNA is extracted, restriction digested and Southern blotted. A rate of synthesis can then be derived for each restriction band by estimating the incorporation of label relative steady state mtDNA (after probing with full length mtDNA). Where co-existing mutant and wild type mtDNA (heteroplasmy) can be distinguished using a restriction digest, their rates of synthesis can be compared within a single cell line.

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We investigated cybrid lines derived from two patients with MELAS. We demonstrated that the proportion of 3243G:C mutant mtDNA was reproducibly higher in newly synthesized than steady state mtDNA in one cybrid line derived form the rho zero 206 line (P ˆ 0.036) and the converse for the other cybrid line derived from the rho zero B2 line (P ˆ 0.006). These differences could re¯ect the relative replication rates of wild type and mutant mtDNA and are consistent with studies of mtDNA segregation in such lines. They may be attributable to (1) the nuclear background of the parent rho zero line, (2) differences in the mtDNA or (3) differences in the proportion of mutant mtDNA in the two lines. We are now adapting this technique to studying cell lines derived from patients with defects in mitochondrial biogenesis including mtDNA depletion and AdPEO and ®nd consistent alterations in the rate of mtDNA synthesis. This method will be useful in elucidating the pathophysiology of several types of mtDNA disease.

®laments. Certain intermediate ®lament-like proteins such as Mdm1p, are known to participate in mtDNA transmission. The mitochondrial processing peptidase functions not only in the removal of presequences, but has been implicated in the post-translational processing of mitochondrial polyproteins, similar to a number of viral proteases. The protovirus genome of 2500 BYA, is likely to have had a number of structurally and catalytically ¯exible features that have provided the founding genetic material for a number of modern protoctistal, plant, fungal, and animal cell virus genera. The viral origins of animal cell mtDNA described by the protovirus hypothesis provide an evolutionary explanation for why host defense cytokines inhibit mitochondrial DNA replication, transcription, and translation, and why pharmaceutical inhibitors of retrovirus and pararetrovirus infections regularly result in doselimiting mitochondrial toxicity in human therapeutic drug trials.

59 58 Is animal cell mitochondrial DNA the relic of a precambrian phage? A protovirus hypothesis R.K. Naviaux The Mitochondrial and Metabolic Disease Center, University of California, San Diego, 200 West Arbor Drive, San Diego, CA 92103, USA Bacteriophage infections of ®eld strains of bacteria living under oligotrophic conditions are endemic in present day microbial ecosystems. Most of the 37 bacterial genomes sequenced and annotated to date kwww.ncbi.nlm.nih.gov/PMGifs/Genomes/bact.htmll, contain evidence of past infection with phage. The size distributions of mitochondrial DNAs observed in the kingdoms Protoctista, Fungi, and Plants vary over 400 fold, from 5.9 to 2500 kb. This heterogeneity stands in striking contrast to the uniformity of mtDNA in the 4th eukaryotic kingdom, Animalia. Animal cell mtDNAs vary only 1.4 fold in size, from 13.8 to 19.5 kb. While the mtDNA of plants and certain members of Fungi and Protoctista may have evolved heterogeneously by the conventionally-taught method of sequential reduction of the protoendosymbiont genome to less than 1% of its original gene content, animal cell mtDNA is likely to have been built up from the molecular framework provided by an episomal Precambrian bacteriophage. This concept is called the protovirus hypothesis. Phylogenetic comparisons of animal cell mitochondrial DNA polymerase g show that it is more closely related to T5/T7-type bacteriophage DNA polymerases than to eubacterial DNA polymerase I. The animal cell mitochondrial RNA polymerase is a single-subunit polymerase, similar to T3/ T7-type phage RNA polymerases ± structurally distinct from the multi-subunit, eubacterial-like RNA polymerases of putative prototype animal cell mtDNA contained in Reclinomonas. The presence of two intergenic regions, spaced 2/3 around the genome is an architectural feature that most animal cell mtDNAs share with the genomes of ®lamentous bacteriophage. The hollow, ®ve-start helical geometry of bacterial sex pili and the capsid of ®lamentous bacteriophage is reminiscent of the ultrastructure of intermediate

Mitochondrial DNA replication and repair W.C. Copeland Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA The integrity of the mitochondrial genome is dependent on DNA replication and repair processes. Mutations and deletions in mitochondrial DNA are the cause of a wide range of mitochondrial diseases. These mutations occur either from exogenous damage, such as oxidative damage, or spontaneously during DNA replication. Human mitochondrial DNA is replicated by the two-subunit DNA polymerase gamma. This polymerase is composed of a 140 kDa subunit containing catalytic activity and a 55 kDa accessory subunit. The catalytic subunit contains DNA polymerase activity, 3 0 ±5 0 exonuclease proofreading activity, and 5 0 dRP lyase activity required for base excision repair. We have cloned, overexpressed, puri®ed, and characterized the human DNA polymerase gamma catalytic subunit, p140, and the p55 accessory subunit. The accessory subunit functions as a processivity factor and enhances DNA binding of the holoenzyme. Using our recombinant DNA polymerase gamma and accessory subunit, the contribution of mutations produced in mtDNA by the DNA polymerase is being studied. The human DNA polymerase gamma has high DNA synthesis ®delity at single base pairs but rather poor ®delity in homopolymeric runs. This suggests that homopolymeric runs in human mitochondrial DNA are potential hot spots for DNA replication errors. The accessory subunit lowers the ®delity of DNA synthesis in nearly all DNA substrates analyzed. As the only DNA polymerase in animal cell mitochondria, the DNA polymerase gamma must participate in DNA replication and DNA repair. The current understanding of mitochondrial DNA repair will be reviewed. For DNA adducts not repaired, the DNA polymerase gamma must deal with them by stalling at the adduct site or performing translesion synthesis, a potentially mutagenic event. The implication of this process and DNA polymerase gamma ®delity will be discussed.

Abstracts 60 Extramitochondrial localization of mitochondrial proteins: implications regarding mitochondrial functions and myopathies R.S. Gupta Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada L8N 3Z5 Mutations affecting mitochondrial components are responsible for a broad spectrum of genetic diseases including, cardiomyopathy, Leigh syndrome, Kearns±Sayre syndrome, MELAS, MERRF, deafness, encephalomyopathy, diabetes, myopathy, PEO, carotid body tumors, encephalopathy, myoglobinuria, sideroblastic anemia, splenic lymphoma, epilepsy, fatal infantile respiratory defects, etc. These disorders affect many different tissues and organs, often in a highly speci®c manner. Based on the known function of mitochondria in oxidative phosphorylation, it has proven dif®cult to logically explain how mutations in different components of the respiratory chain or mitochondrial protein synthesis, could lead to clinically highly speci®c and divergent phenotypes. Important insights bearing on these issues have unexpectedly come from studies, where researchers studying a variety of unrelated functions have serendipitously discovered that the proteins that they had been studying are actually mitochondrial proteins, although the functions affected are extramitochondrial. The examples of such proteins include: Hsp60, a protein originally identi®ed due to being altered in mutants resistant to antimitotic drugs; mHsp70, a protein independently identi®ed due to its involvement in cellular senescence and antigen presentation; Cpn10, a protein secreted in maternal serum which serves as an early pregnancy growth factor and exhibits immunosuppressive activity; m-aspartate aminotransferase, identi®ed and characterized as a protein involved in fatty acid binding and transport [1±3]. Recently, P32 and TRAP-1 proteins, which are involved in a variety of functions including cell surface receptors, signal transduction and neoplasia, have both been shown to be primarily mitochondrial matrix proteins [4,5]. To determine whether the involvement of mitochondrial proteins in extramitochondrial functions is restricted to only a few proteins, or whether it is a general phenomenon, we have undertaken to examine by means of immunoelectron microscopy the subcellular localization in normal rat tissues of a number of well-characterized mitochondrial proteins. Some of these proteins are encoded by the nucleus (viz., cytochrome c oxidase subunit 6c, ATP synthase subunit, pyruvate dehydrogenase E2 subunit, cytochrome c, Hsp60, maspartate aminotransferase, m-Hsp70, Cpn10, P32, TRAP-1), whereas the others (viz., cytochrome c oxidase subunits I and II) by the mtDNA. Highly speci®c antibodies to these proteins were employed in these studies to determine their subcellular localization. Results of these studies reveal that in all tissues examined, these antibodies showed strong labeling of mitochondria, as was expected. In several tissues, the presence of these protein was seen exclusively within mitochondria and no extramitochondrial labeling was observed. However, in a variety of tissues such as liver, acinar pancreas, anterior pituitary, spleen, salivary glands and kidney, these antibodies also showed strong and speci®c labeling of one or more of the following compartments ± pancreatic zymogen granules, growth hormone granules, secretory granules

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in islet cells, red blood cells and peroxisomes. In a number of cases, strong labeling was also observed at the cell surface and in the nucleus. All of the observed labeling with these antibodies, both within mitochondria and in other compartments, was abolished upon adsorption of the antibodies with recombinant proteins or upon omitting the primary antibodies, providing evidence of speci®city. It is surprising that all of the mitochondrial proteins that we have thus far examined are present in one or more extramitochondrial compartments, and no protein that is present exclusively in mitochondria in all of the tissues is found. The extramitochondrial localization of proteins encoded by mtDNA provides strong evidence that these proteins have reached these sites by exiting from mitochondria. The other mitochondrial proteins examined here are single protein products of single nuclear genes, and in some of these cases, evidence has been obtained that the primary translation products are initially imported into mitochondria and that the proteins found at extramitochondrial sites are derived from such proteins. Although the mechanisms responsible for the translocation of mitochondrial proteins to other destinations remains to be characterized, these results provide strong evidence that mitochondria and mitochondrial-resident proteins play an important role in diverse cellular processes and that their cellular functions are not restricted to within mitochondria. These observations are of much interest in terms of understanding the roles of mitochondria and mitochondrial-resident proteins in the etiology of different diseases. [1] Soltys BJ, Gupta RS, 2000. Int Rev Cytol 194:133±196. [2] Soltys BJ,Gupta RS, 1999. Trends Biochem Sci 24:174±177. [3] Cechetto JD, Soltys BJ, Gupta RS, 2000. J Histochem Cytochem 48:45±56. [4] Cechetto JD, Gupta RS, 2000. Exp Cell Res 260:30±39. [5] Soltys BJ, Kang D, Gupta RS, 2000. Cell Biol 114:245±255.

61 Mitochondrial DNA in fertility and development R. Jansen Sydney IVF and the University of Sydney, 4 O'Connell Street, Sydney 2000, Australia The mitochondrial genome is passed from one generation to the next by way of the egg cytoplasm, ordinarily without the opportunity of sexual recombination with the mitochondrial genome of the opposite sex. Mitochondria present in the midpiece of the spermatozoon are important functionally for the ¯agellar propulsion of sperm to the site of fertilization. Faulty mitochondria can cause male infertility. In virtually all animals studied, however, active steps are taken to minimize the risk of sperm mtDNA contaminating the embryonic mitochondrial genome. During spermatogenesis in mammals, mitochondrial DNA molecules per sperm cell are depleted to very low numbers (fewer than 50) and the mitochondria are tagged for later biochemical degradation by the fertilized egg. In human eggs, on the other hand, from the time of ®rst formation of the germ cells in the yolk sac in 2±3 week embryos through to ®nal oocyte maturity at ovulation, 15±50 years later, mitochondria

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and mtDNA circles increase from perhaps fewer than ten per germ cell to often more than a million. Between maturation of the egg as a secondary oocyte capable of fertilization and, 2 weeks later, the occurrence in the embryo of gastrulation (or formation of the de®nitive tissue layers), no new mtDNA is synthesized; yet mitochondrial function and mtDNA transcription are critically important during this time. If mtDNA is prevented from replicating in mice homozygous for a mutation of mitochondrial transcription factor A, embryonic development fails at a point that in humans would be considered an early miscarriage, or `blighted ovum'. Several conclusions may tentatively be drawn. First, the intergenerational sequence of (1) a constriction event in early embryogenesis during which mtDNA circles per egg fall to very low numbers (the `bottleneck'), (2) an explosive increase in the number of mtDNA circles, both per germ cell and in total among the entire germ cell population (an increase that starts soon after gastrulation), and (3) the constraint imposed by the competition for successful fertilization and propagation that then takes place between the millions of eggs formed, comprises the evolutionary mechanism that has conserved the integrity of the mitochondrial genome for more than 500 million years. Second, and more speculative, the slow deterioration that asexually reproducing genomes are nevertheless prone to over time (known as Muller's ratchet), might be further arrested by a ®nal testing of qualitative and/or quantitative mitochondrial genomic integrity during early embryogenesis ± a test that appears to be failed by more and more eggs as maternal age advances. Manifesting at ®rst as a rising incidence of karyotypically normal miscarriages, this transition to physiological sterility (a transition we have called `the oopause') is known clinically to affect women up to a decade before the menopause, when ovarian follicles and the eggs they contain are ®nally depleted. Experiments aimed at the `repair' of older eggs at fertilization in vitro using injections of cytoplasm from the eggs of younger women have been reported and children have been born. The safety of such cytoplasmic transfer with respect to the risks of mitochondrial disease as offspring grow older has not been established.

62 Cellular coenzyme Q10 redox poise and ROS generated therefrom constitute a major cell metabolic and gene regulatory system A.W Linnane Centre for Molecular Biology and Medicine, Epworth Medical Centre, Richmond, (Melbourne) Victoria, Australia Membrane potentials established by pH gradients and ion differentials are sub-cellular energy determinants and play a crucial role in the regulation of metabolism. ATP is synthesized by the mitochondria and chloroplasts utilizing proton motive force, with the key participation of coenzyme Q10 (or its analogues) in the generation of such force. Coenzyme Q10 is anecdotally associated with the successful treatment of a wide range of apparently unrelated conditions including heart disease, neuromuscular diseases, improved immune function, sports performance enhancement, among others.

Although the jury is still out, Coenzyme Q10 does indeed appear to have wide ranging therapeutic bene®ts; nonetheless, the rationale for the wide range and diversity of effects is at this time, far from clear. We have recently proposed [1] and herein additionally elaborate, the concept that a broad based cellular redox function may be suf®cient to encompass CoQ10's wide-ranging effects. We propose that CoQ10 plays a key role in poising the redox potential of a wide range of sub-cellular membrane compartments, resulting in the differential regulation of sub-cellular membrane activities; the different subcellular redox poises and their modulation would lead to signi®cant localized metabolic ¯uxes. Further, super oxide anion (giving rise to H2O2), generated by reactions involving coenzyme Q10 and its speci®c redox poise, play a major role in cellular regulation with H2O2 acting as a second messenger of Q10 expression in the regulation of gene expression. These concepts are derived from a consideration of a range of disparate data: ² Chloroplast transcription has been shown to be regulated by the relative oxidation/reduction level of plastoquinone [2]. It may be hypothesized that mitochondrial transcription is similarly regulated. ² Coenzyme Q10 through the Q cycle participates in the determination of mitochondrial membrane potential and in turn energy and mitochondrial substrate utilization. ² Nohl and Gille [3] have recently demonstrated the occurrence of a lysosomal coenzyme Q10 oxido-reductase system, which establishes a proton gradient across the membrane. Such a system will contribute to the regulation of metabolite movement in and out of the lysosome. ² Crane et al. [4] have extensively reported on the properties of a coenzyme Q10 NADH oxido-reductase enzyme complex in the plasma membrane, which again will contribute to redox potential poise and substrate movement. Further, Sun and Crane [5] have made a preliminary report on a coenzyme Q10 oxido-reductase localized in the Golgi membrane complex. We propose that further studies may show that the coenzyme Q10 located in other membrane systems re¯ects as yet undiscovered oxidoreductase systems (which will contribute to the determination of their individual membrane potentials). ² Protein conformations are in part determined by sulphydryl/ disulphide intraprotein crosslinks. Coenzyme Q10 functions as the H 1/e donor in this ±SH to S±S- conversion [6]. Enzymes in the cytoplasm function in the reduced ±SH state, while those of the sub-cellular compartments are active in the disulphide conformation. ² A key aspect of this proposal is that changes in redox poise would also participate in the anti-oxidant / pro-oxidant role of coenzyme Q10. Coenzyme Q10 acting as a pro-oxidant, continually gives rise to super oxide anion which SOD converts to H2O2. Hydrogen peroxide then functions as a second messenger re¯ecting Q10 redox to regulate gene expression [7,8]. In summary, we propose that two unrecognized functions of coenzyme Q10 are sub-cellular compartmentalized redox poise modulation and the generation of hydrogen peroxide as a second messenger contributing to the regulation of gene expression, cell

Abstracts differentiation and development. Small metabolic imbalances maintained over long periods of time can lead to systemic health crises. It follows from our proposition that the effects of coenzyme Q10 on cells and tissues will be wide ranging, not restricted to a limited number of pathways and with the capacity to modulate a range of tissue activities and disease processes by small intrinsic cell metabolic perturbations. Appropriate modulation of the processes by coenzyme Q10 administration can have favourable therapeutic outcomes. Each tissue/organ is de®ned and regulated by the expression of a speci®c sub-set of nuclear genes representing probably of the order of 8±12% of the total genome. Current studies in our laboratory are focused on the identi®cation of skeletal muscle gene sub-sets and the effect of coenzyme Q10 on gene expression and tissue development. Our hypothesis and the results to hand, in support of it, will be presented and discussed. [1] Linnane AW, Cellular redox activity of coenzyme Q10: effect of CoQ10 on human skeletal muscle. The Second Conference of the International Coenzyme Association; Frankfurt, Germany, 2000. [2] Pfannschmidt T, Nilsson A, Allen JF, 1999. Nature V397:625±628. [3] Gille L, Nohl H, 2000. Arch Biochem Biophys V375:347± 354. [4] Crane FL, Sun IL, Barr R, Morre DJ, 1984. Biomedical and Clinical Aspects of Coenzyme Q, in; Folkers K, Yamamura Y, Eds., Elsevier, Amsterdam, V4:77±85. [5] Sun IL, Crane FL, 1990. Oxidation Reduction at the Plasma Membrane, CRC Press: Boca Raton, FL, V1:257±280. [6]Glockshuber R, 1999. Nature V401:30±31. [7] Rusnak F, Reiter T, 2000. TIBS, V25:527±529. [8] Smith J, Ladi E, Mayer-ProÈschel M, Noble M, 2000. PNAS, V97:10032±10037.

63 Mutations in COX10, SCO1 and NDUFV1 genes in mitochondrial disorders P.B. Valnot, J.-C. von Kleist-Retzow, P. de Lonlay, S. Osmond, N. Gigarel, B. Mehaye, J.-P. Bonnefont, V. Cormier-Daire, A. RoÈtig, P. Rustin, A. Munnich HoÃpital Des Enfants-Malades, Medical Genetics Service, Unite INSERM U-393, Paris, France Isolated de®ciencies of cytochrome c oxidase (COX) activity represent a large proportion (28%) of respiratory chain de®ciencies encountered in childhood. They are disease-causing de®ciencies observed in a wide spectrum of clinical presentation. Mutations of SURF1 gene involved in COX assembly have been described in a sub-group of autosomal recessive COX±Leigh's disease. We have selected four consanguineous families with isolated COX de®ciency, but not SURF1 mutations, to possibly map the disease by linkage analysis. One family comprises eight healthy children and three affected children with de Toni±Fanconi±Debre syndrome and acute neurologic deterioration resembling Leigh syndrome, without clear evidence of muscle abnormality. Elevated cerebrospinal ¯uid lactate values were observed contrasting with

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normal blood lactate, and high 3-hydroxybutyrate/acetoacetate ratio with normal lactate/pyruvate ratio. By linkage analysis using the Perkin±Elmer Linkage Mapping Set consisting of about 400 markers with a mean genetic distance of 10 cM covering all autosomes, we identi®ed a homozygous locus on chromosome 17p11.3. Two genes encoding proteins involved in COX assembly, namely COX10 and SCO1, mapped in this region. For one of them, COX10, we identi®ed a homozygous mutation in the three affected children. The consanguineous parents were both heterozygous for the mutation, which was absent from 30 controls from the same ethnic group. Western blot analysis has already shown a subnormal assembly of the complex in mitochondria isolated from patients' COX de®cient ®broblasts, in agreement with the known function of COX 10 gene, a farnesyl transferase involved in the heme A synthesis. COX catalyzes electron transfer from cytochrome c to molecular oxygen and vectorial proton pumping across the inner mitochondrial membrane. Here we report on genetic linkage analysis in a multiplex family with an isolated COX defect causing neonatalonset hepatic failure and encephalopathy, and the subsequent identi®cation of mutations in a nuclear gene encoding a mitochondrial assembly protein SCO1. A genome-wide search allowed us to show that the four affected sibs, but not unaffected sibs, were haplo-identical at the D17S953-D17S1796 loci (17p13.1±q11.1). This region encompasses two genes, SCO1 and COX10, encoding proteins involved in COX assembly. Mutation analyses revealed that the four patients carried heterozygous SCO1 gene mutations. One allele, inherited from the father, harbored a frame shift mutation resulting in a premature stop codon and a highly unstable mRNA, which could not be detected by RT-PCR. A second mutation (C520T) was maternally inherited and changed a highly conserved proline into a leucine in the protein (P174L). SCO1 is involved in the insertion of copper into the cytochrome c oxidase and the mutated proline residue is believed to play a crucial role in the structure of the CXXXC copper-binding domain. The mutation was absent from 103 ethnically related controls. The clinical phenotype resulting from SCO1 mutation reported here markedly differs from that of mutations in the SURF1, COX10, or SCO2 genes, despite the fact that these genes are involved in COX assembly and/or maturation. The variable degree of organ involvement might re¯ect variable levels of the disease gene expression in different tissues. Finally, this result emphasizes the major role of assembly genes in the pathogenesis of COX de®ciency in human. NADH:ubiquinone oxidoreductase (Complex I) is the largest complex of the mitochondrial respiratory chain and this complex accounts for most cases of respiratory chain de®ciency in human. Only seven mitochondrial DNA genes but more than 35 nuclear genes encode Complex I. Among them, the NDUFV1 gene encodes the 51 kDa subunit which contains the NADH-, FMN- and Fe±Sbinding sites. In an attempt to elucidate the molecular bases of the respiratory chain disorders, we studied the NDUFV1 gene in a series of 36 patients with isolated Complex I de®ciency, after having excluded mitochondrial DNA mutations. The full length NDUFV1 mRNA extracted from cultured skin ®broblasts was reverse transcribed and tested by denaturing high performance liquid chromatography (DHPLC). Abnormal fragments were then directly sequenced. Six novel missense mutations were identi®ed in three patients presenting with Leigh's syndrome. All patients, born

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to non-consanguineous parents, were compound heterozygotes and the parents were heterozygous for the corresponding mutations. These mutations involved highly conserved amino acids. Most interestingly, 3/6 mutations altered the FMN-binding site of the 51 kDa subunit in two patients. Considering that the patients had normal respiratory chain activities in cultured ®broblasts, we hypothesize that the high concentration of FMN in the culture medium might have restored a normal Complex I activity. These results suggest that screening for NDUFV1 mutations is of great interest in patients with Complex I de®ciency, (even when normal respiratory chain enzyme activities in cultured ®broblasts is observed). In addition, they should prompt us to systematically put Complex I de®cient patients on a high ribo¯avin diet.

64 Human immunode®ciency virus infection, antiretroviral therapy and the mitochondrion D. Nolan Department of Clinical Immunology, Royal Perth Hospital, Perth, Australia Lactic acidosis and associated hepatic steatosis, neuropathy, myopathies, and more recently the `lipodystrophy syndrome' provide ongoing challenges in the clinical management of HIV infection, particularly in an era of effective long-term therapy and improved prognosis. Hence, elucidation of the associations between mitochondria, HIV and antiretroviral therapy continues to be of great importance. It has long been recognized that mitochondrial toxicities may accompany the long-term use of nucleoside analogue reverse transcriptase inhibitors (NRTIs) in the treatment of HIV infection. While these dideoxynucleoside compounds effectively suppress HIV replication by inhibiting the ability of viral reverse transcriptase, it is also well established that they can inhibit the activity of the unique mitochondrial DNA polymerase, polymerase gamma. The `pol-g ' hypothesis has evolved from this premise, providing a plausible explanation for the tissue-speci®c toxicities associated with select NRTIs. This model proposes that cellular toxicity occurs as a result of decreased mitochondrial DNA synthesis and subsequent depletion of mitochondrial DNA content, and that a given tissue's susceptibility to these effects is determined by its capacity to take up and then activate a given NRTI to its triphosphate form. Other factors may also be important determinants of mitochondrial toxicity associated with NRTI therapy, both in relation to its tissue speci®city as well as its expression, and the appreciation of these factors has often been aided by developments in the broader ®eld of mitochondrial research. In the context of the `pol-g ' hypothesis, there is an increasing awareness that intracellular handling of NRTIs and their derivatives, and mitochondrial targeting of these compounds, is an important determinant of toxicity. In addition, the recognition that polymerase gamma is a critical component of mitochondrial base excision repair has broadened the understanding of its biological activity, and suggests that inhibition of this high ®delity polymerase may induce qualitative as well as quantitative changes in mitochondrial DNA.

Mitochondrial dysfunction has also been implicated in the pathogenesis of HIV-associated immune suppression per se, with research indicating that mitochondrial derived pro-apoptotic signals are associated with viral protein products as well as intact HIV virus, and that incorporation of HIV into the mitochondrial DNA template may occur. The direct involvement of mitochondria in HIV pathogenicity implies that (1) HIV infection may be a co-factor in the pathogenesis of treatment-associated toxicities, and (2) that mitochondrial targeting of antiretroviral therapies (ART) may be intrinsic to their ef®cacy. The anti-apoptotic effects of HIV protease inhibitor therapy, which may in turn be due to reduced formation of pro-apoptotic HIV viral proteins, is a possible example of this effect. Mitochondrial effects of ART, therefore, are not necessarily deleterious. Mitochondrial components other than polymerase gamma may be involved in NRTI-induced toxicity. A number of researchers have identi®ed an association between the inner membrane ADP/ATP translocator and zidovudine, proposing that it may be involved in the pathogenesis of skeletal and/or cardiac myopathy induced by this therapy. Recent developments in research into myocarditis and cardiomyopathy, as well as an increasing understanding of the regulation of mitochondrial biogenesis, have supported the plausibility of this mechanism. The importance of appropriately regulating the mitochondrial nucleotide pool has also been the subject of intense interest, following the identi®cation of loss-of-function mutations in nuclear DNAencoded thymidine phosphorylase associated with mitochondrial DNA depletion and mutation in the MNGIE syndrome. The possibility that thymidine analogue NRTIs may cause dysregulated thymidine metabolism, particularly in post-mitotic tissue, and subsequent mitochondrial toxicity will be discussed.

65 Pathogenesis of primary disorders of mitochondrial DNA E. Schon Department of Neurology, Columbia University, New York, NY, USA There are two broad classes of pathogenic mutations in mitochondrial DNA (mtDNA): point mutations and large-scale rearrangements (duplications and/or deletions). While the discoveries of the last few years have transformed our understanding of mitochondrial diseases, almost all of our understanding has been con®ned to etiology. Pathogenesis, on the other hand, remains, on the whole, a mystery: although the quantitative aspects of mitochondrial genetics (heteroplasmy, mitotic segregation, and threshold effects) play a critical role in pathogenesis, we do not really know why a speci®c mutation causes a speci®c disease or constellation of symptoms. In spite of our ignorance, a few broad themes have emerged in the last few years that may allow us to begin to make headway on this problem. One is the observation that similar clinical presentations are sometimes associated with different mutations in different tRNAs in which the mutation is at the same position on the tRNA itself. A second is the observation that mutations causing `striatal necrosis syndromes' are located exclusively in a single gene,

Abstracts ATPase 6. A third is the relationship between large-scale deletions and duplications in the various disorders associated with mtDNA rearrangements. A fourth is the observation that some organ systems are on the whole unaffected in mitochondrial disease, even when they contain nearly homoplasmic levels of mutation. Given that, in the ®nal analysis, all mitochondrial diseases are presumably the result of either a partial or complete failure in oxidative energy production, it may be useful to focus on the synthesis and distribution of ATP, the ®nal common metabolite, within affected and unaffected cells. Such an analysis might eventually enable us to develop a `uni®ed theory' of mitochondrial pathogenesis.

66 Imaging and diagnosis J. Shoffner Children's Healthcare of Atlanta, Atlanta, GA, USA Oxidative phosphorylation (OXPHOS) diseases are a complex group of disorders with highly variable clinical manifestations. OXPHOS is the enzyme pathway responsible for producing approximately 90±95% of the ATP required by the body. Genes coding OXPHOS polypeptides and other important elements of this pathway are found in the nuclear DNA and the mitochondrial DNA (mtDNA). The mtDNA is located within the mitochondria rather than the cell nucleus and is maternally inherited. Since OXPHOS diseases are caused by both mutations in the mtDNA, complex diagnostic and genetic counseling issues arise. Imaging techniques are important components of patient diagnosis. The most important imaging modality in clinical practice is brain MRI. In order to illustrate the important role that neuroimaging plays in clinical practice cases that illustrate common MRI abnormalities will be presented. The role of these ®ndings on the diagnostic approach will be discussed. Finally, a brief overview of imaging strategies that provide insight into brain metabolism and their impact on mitochondrial disease diagnosis will be reviewed.

67 Therapeutics of mitochondrial DNA disease R.W. Taylor, D.M. Turnbull University of Newcastle-upon-Tyne, Newcastle-upon-Tyne, UK The incidence of mitochondrial DNA disease is at least one in 8000 in the population. In many patients there is a progressive disease with a severe clinical phenotype. At present there is no successful treatment, but a number of different agents have been tried. It is our aim to review both the available treatments and to consider therapeutic possibilities for the future. Pharmacological therapy of mitochondrial disorders A range of different pharmacological treatments has been tried for mtDNA defects. Perhaps the most used pharmacological agent is Coenzyme Q10 (CoQ10) (ubiquinone, ubidecarenone) which is a lipid-soluble, mobile component of the respiratory chain. Despite its widespread use, there is little evidence to support the effective-

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ness of this agent. Idebenone (2,3-dimethoxy-5-methy-6-(10hydroxy)-decyl-1,4-benzoquinone) is a synthetic analogue of CoQ10 which has been tried in Leber's hereditary optic neuropathy, MELAS and Friederich's ataxia. Other agents that have been tried include menadione, ribo¯avin, succinate, corticosteroids, carnitine, creatine and dichloroacetate. Currently there are clinical trials in progress with the latter agent in patients with MELAS. Supportive treatments Supportive care is a very important feature in patients with mtDNA disease. This includes appropriate management of diabetes and other endocrine disorders and the control of seizures using antiepileptic drugs. Ophthalmic splints or corrective surgery may be helpful to patients with progressive external ophthalmoplegia, and surgery can also improve the quality of life of patients with other ocular complications, such as cataracts. Patients with mitochondrial abnormalities can experience swallowing dif®culties, which may be alleviated by gastrostomy. Cochlear implantation may be of bene®t for patients with hearing loss, a common ®nding in mitochondrial defects and in particular MELAS. Timely placement of a pacemaker prevents heart block, and successful cardiac transplantation has been reported in patients with Kearns±Sayre syndrome and hypertrophic cardiomyopathy. What about mitochondrial gene therapy? Such are the shortcomings of the available biochemical and pharmacological therapies that genetic and other experimental strategies to treat patients with mitochondrial disease have been explored. A variety of different strategies have been tried all with very limited success. Attempts at complementing the mitochondrial DNA mutation has been tried in NARP. The recent report that human mitochondria are able to import yeast tRNA derivatives in vitro when supplemented with the required import-directing factors suggest that there exists the possibility for non-functional, mutant tRNAs to be replaced. Agents that manipulate mitochondrial DNA heteroplasmy include the use of oligomycin, a mitochondrial inhibitor of ATP synthesis. It has been shown that it is possible to signi®cantly increase the fraction of wild-type molecules in cells that harbour the T8933G NARP mutation under culture conditions that speci®cally select for the wild-type molecule. An approach with broader therapeutic applications is the development of molecules that bind to, and speci®cally inhibit the replication of, mutant mitochondrial DNAs. Induced muscle regeneration in mitochondrial disorders The replicative segregation of mitochondrial DNA has afforded another unique means of affecting mtDNA genotype in favour of the wild-type molecule in some myopathic patients. In some patients there is a high level of mutated mtDNA in mature muscle but very low or absent mutated mtDNA in the satellite cell population isolated from the same muscles. Using either bupivacaine or traumatic muscle injury to induce muscle necrosis, the regenerating muscle ®bres were shown to derive exclusively from the satellite cell population that harboured low or undetectable levels of mutant mitochondrial DNA and hence had normal biochemical activity.

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68 Mitochondrial genes in degenerative disease and aging D.C. Wallace Emory University School of Medicine, Atlanta, GA 30322, USA It is becoming increasingly apparent that mitochondrial dysfunction is a central factor in degenerative diseases and aging. Mitochondrial physiology: One of the primary functions of the mitochondrion is the generation of cellular energy by the process of oxidative phosporylation (OXPHOS). OXPHOS encompasses the electron transport chain (ETC) which oxidizes hydrocarbons with oxygen and harnesses the energy to create an electrochemical gradient (Dc ) across the mitochondrial inner membrane. This Dc is utilized by the ATP synthase (Complex V) to convert ADP 1 Pi to ATP. ADP and ATP are exchanged across the inner membrane by the adenine nucleotide translocator (ANT). As a toxic by-product of OXPHOS, electrons from the ETC can be transferred directly to O2 to give superoxide anion (O2z2). Mitochondrial O2z2 is converted to H2O2 by Mn superoxide dismutase (MnSOD), and H2O2 is converted to H2O by glutathione peroxidase (GPx1). The mitochondria are the primary initiators of apoptosis, mediated by the mitochondrial permeability transition pore (mtPTP). Increased mitochondrial Ca 21 or ROS and/or decreased Dc or ATP tend to activate the mtPTP to initiate apoptosis (Wallace, 1999). Mitochondrial diseases: Mitochondrial diseases have been associated with both mtDNA and nuclear DNA (nDNA) mutations. Examples of mtDNA diseases are Leber's hereditary optic neuropathy (LHON) and/or dystonia caused by a missense mutation at nucleotide pair (np) 14459 ND6 gene (Jun et al., 1994, 1996) and myoclonic epilepsy and ragged red ®ber (MERRF) disease caused by a protein synthesis mutation in the tRNA Lys gene at np 8344 (Shoffner et al. 1990, Wallace et al. 1988). An example of a mitochondrial disease caused by a nDNA mutation is autosome dominant progressive external ophthalmoplegia (AdPEO). In some cases this disease can be caused by mutations in the ANT1 gene which destabilizes the mtDNA and results in the accumulation of multiple mtDNA rearrangements in muscle (Kaukonen et al., 2000). Mitochondrial function also declines with age in the post-mitotic tissues in association with the accumulation of mtDNA rearrangement and base substitution mutations. Somatic mtDNA rearrangement mutations accumulate in human skeletal muscle, heart and brain (Corral-Debrinski et al., 1991, 1992a,b, 1994; Horton et al., 1995; Melov et al., 1995), and in mouse heart and brain (Melov et al., 1997). The somatic mtDNA T414G control region mutation identi®ed in ®broblasts derived from older subjects (Michikawa et al., 1999) was found to accumulate in human skeletal muscle, though not brain (Murdock et al., 2000). However, variants affecting the number of Cs a homopolymeric run in the control region were cloned from a human brain by fusing brain synaptosomes to cultured mtDNA-de®cient (r o) cells (Trounce et al., 2000). Hence, aging is associated with the accumulation of somatic mtDNA mutation. Mouse models and the pathophysiology of mitochondrial disease: To investigate the pathophysiology of mitochondrial disease, deleterious mitochondrial mutations have been introduced

into mouse mtDNA and nDNA. The deleterious mtDNA mutation imparts resistance to the mitochondrial ribosomal inhibitor chloramphenicol (CAP). The nDNA mutations have inactivated the heart-muscle isoform of the ANT (Ant1), the GPx1, and the MnSOD. The CAP R mtDNA mutation is the result of a T to C transition at np 2433 in the mtDNA 16S rRNA and causes partial respiratory de®ciency. This mutation has been introduced into the mouse germline by fusing CAPR cytoplasts from mouse 501-1 cells to female embryonic stem (ES) cells, previously cured of their resident mtDNAs by treatment with rhodamine-6G (Levy et al., 1999). Chimeric mice generated from these CAP R ES cells developed bilateral nuclear cataracts, vacuolated retinal epithelia, and hamatonatous growths of the optic nerve head. Breading of female chimeric animals has permitted the transmission and the CAP R mtDNAs to offspring in both the heteroplasmic and homoplasmic states. All CAP R neonates showed growth retardation and died in the neonatal period of mitochondrial myopathy and dilated cardiomyopathgy (Sligh et al., 2000) To determine the importance of energy de®ciency in mitochondrial disease, we genetically-inactivation the mouse Ant1 nDNA gene. Mice have two ANTs: ANT1, which is expressed at high levels in heart and skeletal muscle; and ANT2, which is expressed in all tissues except the skeletal muscle (Levy et al., 2000). As a consequence, Ant1 knockout mice derive almost no skeletal muscle ATP from mitochondria, reduced heart ATP from mitochondria, but normal liver ATP from mitochondria. Moreover, the ANT1-de®cient mice develop lactic acidosis, mitochondrial myopathy with sever fatigability and a hypertrophied cardiomyopathy that progresses to dilated cardiomyopathy (Graham et al., 1997). Inhibition of ADP/ATP exchange results in the hyper-polarization of Dc causing inhibition of the ETC and the generation of excess O2z2. Consequently, mitochondria H2O2 production is elevated in heart, skeletal muscle, and brain. This increased ROS production is associated with the induction of MnSOD and GPx1, and an increase in mtDNA rearrangements (Esposito et al., 1999). The proliferation of mitochondria in the ANT-de®cient skeletal muscle is similar to that seen in mitochondrial disease patients where mtDNA and nDNA mitochondrial genes are coordinately induced (Heddi et al., 1999). Applying differential display (DD) (Murdock et al., 1999) and hybridization to our 452 cDNA mitochondrial microarray (the Mitochip), we found that the up-regulation of mitochondrial genes is associated with the induction of genes for ROS detoxi®cation and apoptosis. To determine the importance of mitochondrial ROS toxicity in mitochondrial disease, we have genetically inactivated the GPx1 gene. Mice lacking GPx1 have increased mitochondrial H2O2 production, a 20% reduction in weight, and a one-third reduction in the respiratory control ratio and the mitochondrial power output (Esposito et al., 2000). Genetic inactivation of MnSOD (Sod2) should increase mitochondrial O2z2 production. Homozygous mutant animals have a complete MnSOD-de®ciency and die at approximately 8 days of age of a dilated cardiomyopathy with lipid accumulation in the liver (Li et al., 1995). This is associated with the inactivation of mitochondria the iron-sulfur center containing enzymes including Complex I, II and mitochondrial aconitase (Melov et al., 1999).

Abstracts Heterozygous animals have a 50% reduction in MnSOD which results in a life-long reduction in Dc , reduced ADP-stimulated respiration, increased endogenous respiration, and a marked sensitization of the mtPTP. In older animals, this leads to a wave of apoptosis which removes cells with the greatest mitochondrial damage, but which causes tissue and organ failure due to cell loss (Kokoszka et al., 2001). Hence, mitochondrial ROS damage is an integral component of the normal aging process. Catalytic anti-oxidant mimetics such as MnTBAP can ameliorate the toxic effects of O2z2. Treatment of MnSOD-de®cient newborn mice can rescue the cardiac defect, extending the mean life span to about 18 days. However, MnTBAP does not cross the blood±brain barrier, and the mice subsequently develop movement disorders and die of a spongiform encephalopathy (Melov et al., 1997). Treatment with other SOD mimetics (EUK8 and EUK134), which do cross the blood±brain barrier, further protects the animals. The relevance of mitochondrial oxidative stress to longevity was demonstrated by growing C. elegans in EUK134. The mev-1 mutant of C. elegans alters a Complex II subunit, increasing mitochondrial ROS production and shortening life-span. Growth of mev-1 in EUK134 restores normal life-span. Furthermore, growth of wild type C. elegans in the drug extends the lifespan by 50% (Melov et al., 2000). Therefore, mitochondrial ROS toxicity has been linked to longevity. These data suggest that degenerative diseases and aging are both caused by mitochondrial decline, with the age-of-onset of symptoms being determined by the individual's inherited bioenergetic genotype and initial energetic capacity. As the individuals ages, somatic mtDNA mutations caused by oxidative damage accumulate, eroding his mitochondrial function. If the individual starts with a high energetic capacity, then many years are required before energy production is suf®ciently impaired to cause energetic failure and cell loss due to apoptosis. However, if the individual inherits an mitochondrial defect, then he starts at a lower energetic capacity and crosses energetic expression thresholds prematurely resulting in cell loss by apoptosis and tissue decline (Wallace, 1999) Corral-Debrinski M, Stepien G, Shoffner JM, Lott MT, Kanter K, Wallace DC, 1991. JAMA 266:1812±1816. Corral-Debrinski M, Horton T, Lott MT, Shoffner JM, Beal MF, Wallace DC, 1992a. Nat Genet 2:324±329. Corral-Debrinski M, Shoffner JM, Lott MT, Wallace DC, 1992b. Mutat Res 275:169±180. Corral-Debrinski M, Horton T, Lott MT, Shoffner JM, McKee AC, Beal MF, Graham BH et al., 1994. Genomics 23:471±476. Esposito LA, Melov S, Panov A, Cottrell BA, Wallace DC, 1999. Proc Natl Acad Sci USA 96:4820±4825. Esposito LA, Kokoszka JE, Waymire KG, Cottrell B, MacGregor GR, Wallace DC, 2000. Free Rad Biol Med 28:754±766. Graham B, Waymire K, Cottrell B, Trounce IA, MacGregor GR, Wallace DC, 1997. Nat Genet 16:226±234. Heddi A, Stepien G, Benke PJ, Wallace DC, 1999. J Biol Chem 274:22968±22976. Horton TM, Graham BH, Corral-Debrinski M, Shoffner JM, Kaufman AE, Beal BF, Wallace DC, 1995. Neurology 45:1879± 1883. Jun AS, Brown MD, Wallace DC, 1994. Proc Natl Acad Sci USA 91:6206±6210.

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Jun AS, Trounce IA, Brown MD, Shoffner JM, Wallace DC, 1996. Mol Cell Biol 16:771±777. Kaukonen J, Juselius JK, Tiranti V, Kyttala A, Zeviani M, Comi GP, Keranen S et al., 2000. Science 289:782±785. Kokoszka JE, Coskun P, Esposito L, Wallace DC, 2001. Proc Natl Acad Sci USA, February 27, 2001 Edition. Levy SE, Waymire KG, Kim YL, MacGregor GR, Wallace DC, 1999. Transgen Res 8:137±145. Levy SE, Chen Y-S, Graham BH, Wallace DC, 2000. Gene 254:57±66. Li Y, Huang TT, Carlson EJ, Melov S, Ursell PC, Olson JL, Noble LJ et al., 1995. Nat Genet 11:376±381. Melov S, Shoffner JM, Kaufman A, Wallace DC, 1995. Nucl Acids Res 23:4122±4126. Melov S, Hinerfeld D, Esposito L, Wallace DC, 1997. Nucl Acids Res 25:974±982. Melov S, Coskun P, Patel M, Tunistra R, Cottrell B, Jun AS, Zastawny TH et al., 1999. Proc Natl Acad Sci USA 96:846±851. Melov S, Ravenscroft J, Malik S, Gill MS, Walker DW, Clayton PE, Wallace DC et al., 2000. Science 289:1567±1569. Michikawa Y, Mazzucchelli F, Bresolin N, Scarlato G, Attardi G, 1999. Science 286:774±779. Murdock D, Boone BE, Esposito L, Wallace DC, 1999. J Biol Chem 274:14429±14433. Murdock DG, Christacos NC, Wallace DC, 2000. Nucl Acids Res 28:4350±4355. Shoffner JM, Lott MT, Lezza AM, Seibel P, Ballinger SW, Wallace DC, 1990. Cell 61:931±937. Sligh JE, Levy SE, Waymire KG, Allard P, Dillehay DL, Nusinowitz S, Heckenlively JR et al., 2000. Proc Natl Acad Sci USA 97:14461±14466. Trounce I, Schmiedel J, Yen HC, Hosseini S, Brown MD, Olson JJ, Wallace DC, 2000. Nucl Acids Res 28:2164±2170. Wallace DC, Zheng X, Lott MT, Shoffner JM, Hodge JA, Kelley RI, Epstein CM et al., 1988. Cell 55:601±610. Wallace DC, 1999. Science 283:1482±1488.

69 Mechanisms of mitochondrial division and inheritance M.P. Yaffe University of California, San Diego, Division of Biology, 0347 Section of Cell and Developmental Biology, La Jolla, CA 920930347, USA Mitochondria proliferate by growth and division of preexisting mitochondria. Growth of mitochondria occurs through the local synthesis of proteins and lipids and via the import of proteins and lipids synthesized outside the organelle. These processes have been described in great detail, but insights into the mechanisms by which mitochondria are divided and distributed to daughter cells prior to cytokinesis are only now emerging. One approach to the study of molecular mechanisms that mediate mitochondrial inheritance and changes in mitochondrial morphology has been the isolation of Saccharomyces cerevisiae mutants displaying defects in mitochondrial shape and distribution. Molecular and microscopic analysis of

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these mutants has led to identi®cation of some of the cellular components that mediate mitochondrial behavior. These components include Mdm1p, an intermediate ®lament-like protein of the cytosol, and three proteins of the mitochondrial outer membrane, Mdm10p, Mdm12p, and Mmm1p. Analysis of these mutants has also revealed an essential role for ubiquitination in mitochondrial inheritance. Genetic and molecular analysis of another mutant, mdm17, has led to the identi®cation of three components that act

at the mitochondrial outer membrane to mediate the ®ssion of mitochondrial tubules. Additional studies using the yeast Schizosaccharomyces pombe are revealing components important for the alignment of mitochondria along microtubules. These studies should lead to an understanding of mechanism by which mitochondrial dynamics are coordinated with cell division and differentiation.