Projects Funded by the UMDF

(click link for project description)

David C Chan, California Institute of Technology, "Understanding the role of mitochondrial fusion in mitochondrial myopathies"
Joseph A. Garcia, University of Texas SW Medical Center at Dallas, "The hypoxia sensing transcription factor EPAS1/HIF-2a is a novel mitochondrial disease candidate in mice and man"
Ramon Marti, Institut de Recerca de I'Hospital de la Santa Creu i Sant Pau in Barcelona, Spain, "Restoration of thymidine phosphorylase activity in MNGIE patients through platelets infusion"
Miriam H. Meisler, University of Michigan, "The nuclear-encoded gene OMI and mitochondrial disease"
Vamsi K. Mootha, Broad Institute, Massachusetts Institute of Technology, "Genomic Approaches to Human Cytochrome c Oxidase Deficiency"
Brian Robinson, Metabolism Research Program, Hospital for Sick Children, "Drug development for the regulation of respiratory chain components in mitochondria"
Stefan Strack, University of Iowa, Carver College of Medicine, "Protein phosphatase 2A in mitochondrial function and disease"
Volkmar Weissig, Northeastern University, Department of Pharmaceutical Sciences, "Development of a method for transforming mitochondria in living mammalian cells with exogenous DNA"
Immo Scheffler, Ph.D., University of California, San Diego, "Application of RNA interference in the study of NADH-ubiquinone oxidoreductase (complex I) assembly in mammalian mitochondria"
Mikhail Alexeyev, Ph.D., University of South Alabama, "Selective Elimination of Defective Mitochondrial Genomes as an Approach to the Reversal of NARP and MILS Syndromes, Heritable Mitochondrial Disorders"
Matthew Freeman, Ph.D., Laboratory of Molecular Biology - M.R.C., Cambridge, UK, "Role of Rhomboid Proteolysis in Optic Atrophy"
Koji Okamoto, Ph.D., University of Utah, "Molecular Basis of Mitochondrial Membrane Dynamics: a New Paradigm of Human Disease"
Bernard Lemire, Ph.D., University of Alberta, "The Use of the Yeast CYB2 Gene As Therapy for Complex I Mutations in a C. elegans Model System"
Giovanni Manfredi, MD, Ph.D., Weill Medical College of Cornell University, "MtDNA complementation and recombination in mitochondrial disorders"
Philip Schwartz, Ph.D., Children's Hospital of Orange County, "Electrophysiologic Properties of Neural Stem Cells from Patients with Mitochondrial Disease"
Yidong Bai, Ph.D., University of Texas Health Science Center at San Antonio, "Exploiting the potential of yeast NDI1 gene in the therapy of diseases linked with mtDNA"
Tanja Taivassalo, Ph.D., Institute for Exercise & Environmental Medicine, Dallas, Texas, "Exercise-induced mitochondrial gene shifting: Resistance training as a therapy for sporadic mtDNA mutations"
Jose Hernandez-Yago, Ph.D., Institute for Cell Research, Valencia, Spain, "Transport diseases in mitochondria: Full screening of DNA alterations in human genes encoding TOMM and TIMM complexes in patients with mitochondrial diseases"
Min-Xin Guan, Ph.D., Children's Hospital Medical Center, Cincinnati, OH, "Biochemical Basis for Maternally Inherited Deafness"
Brian Robinson, Ph.D., The Hospital for Sick Children, Toronto, Ontario, "Efficacy of prenatal diagnosis of mitochondrial diseases."
Edwin Kirk, M.D., Sydney Children's Hospital, Randwick, NSW, "Complex I: The role of nuclear genes in disorders of childhood due to mitochondrial Complex I deficiency."
Dikoma Shungu, Ph.D., Columbia University, New York, NY, "Quantitative In Vivo 1H Magnetic Resonance Spectroscopic Imaging of Cerebral Lactate as a Screening Test for Mitochondrial Disorders"
George Perry, M.D., Case Western Reserve University, Cleveland, OH, "Is oxidative damage a result of metabolic abnormalities in Alzheimer disease?"
Cecilia Giulivi, Ph.D., University of Minnesota, Duluth, "Characterization of Mitochondrial Nitric-Oxide Synthase"
Brian Robinson, Ph.D., The Hospital for Sick Children, Toronto, Ontario, "Efficacy of Prenatal Diagnosis of Mitochondrial Diseases"
John Shoffner, M.D., Horizon Molecular Laboratory, Norcross, GA, "Gene mutations in Leigh's Disease"
Carolyn Bay, M.D., Children's Hospital of Pittsburgh, "Mitochondrial etiologies of pseudoobstruction and dysmotility in children"
Richard Boles, M.D., Children's Hospital of Los Angeles, "Search for Pathogenic Mitochondrial DNA Mutations Using Temporal Temperature Gradient Gel Electrophoresis (TTGE)"

1. David C Chan, California Institute of Technology, "Understanding the role of mitochondrial fusion in mitochondrial myopathies"

Dr. Chanâ€™s lab will investigate the formation of cell organelles called mitochondria that are responsible for energy production in human cells. Human cells contain several kinds of organelles, each of which has specific jobs. Mitochondria are especially complex machines made from a variety of proteins. The genetic code for the manufacture of most of these proteins resides in the DNA of the cellâ€™s nucleus. Mitochondria are unique among organelles, however, because the nucleus does not contain all of the genetic information needed to make them. Some of this information resides in the mitochondriaâ€™s own DNA.

Dr. Chanâ€™s lab will breed lab mice that have undergone tissue-specific deactivation of factors needed for mitochondrial fusion and will study the effects that this has on skeletal and cardiac myopathies. Instead of being made from scratch, new mitochondria always develop from pre-existing ones, with constant replenishing of the necessary proteins as directed by both the nuclear DNA and mitochondrial DNA. Interestingly, at any point in time variable numbers of mitochondria may fuse together to form elongated structures, or longer structures may fragment into the smaller bean-shaped structures often depicted in biology textbooks. The rate at which these events, division and fusion, occur is under the control of a specific group of enzymes called GTPases that reside in mitochondria. Some mitochondrial diseases are caused by defective mtDNA and fusion may actually protect the mitochondria from following the incorrect code of defective mtDNA.

Dr. Chan plans to investigate the role of three GTPases required for the fusion of mammalian mitochondria. Previous research has shown that mouse embryos lacking these will die early in their development. He will produce lab mice that have undergone deactivation of the GTPases needed for mitochondrial fusion, but only in their skeletal muscle and heart muscle. Because human mitochondrial diseases often affect function of these two types of muscle, this research will provide insight into the role that fusion of mitochondria plays in progression of the disease.

Prepared by Steven G. Bassett, Ph.D.
Seton Hill University
Greensburg, PA

2. Joseph A. Garcia, University of Texas SW Medical Center at Dallas, "The hypoxia sensing transcription factor EPAS1/HIF-2a is a novel mitochondrial disease candidate in mice and man"

Dr. Garciaâ€™s lab will screen cells obtained from children with mitochondrial disease for the presence of an abnormal protein that has lost the ability to stimulate the production of anti-oxidant enzymes. Mitochondria are energy-producing organelles contained in most human cells. They contain a series of molecules that collectively are called the respiratory chain. The respiratory chain is the cellular site where most of the energy is derived from foods that we eat. Adequate amounts of oxygen must be available for the chain to function properly and produce the energy-rich molecule ATP. While we might consider only the benefits of getting enough oxygen as we take each breath, most of us are not aware of the potential problems if the oxygen is not quickly used in the mitochondria for energy production. Mitochondrial disease results from a variety of problems with the energy-producing machinery of the patientâ€™s mitochondria. Abnormal mitochondria are unable to reliably and efficiently produce energy and will not use all of the oxygen that is available to them. The unused oxygen may accumulate to the point where it could begin to damage the mitochondria. This is because the presence of excess oxygen in the cells leads to the production of a variety of chemicals known collectively as reactive oxygen species (ROS). Free radicals and other types of ROS can remove electrons from the atoms that make up our cells. Extensive exposure to ROS can lead to oxidative stress, causing extensive damage to any tissues made of cells that contain abnormal mitochondria.

Cells possess several means of protecting themselves against damage from ROS, including antioxidant enzymes. The synthesis of these protective enzymes is regulated by the cellular protein EPAS1/HIF-2a (EH), which responds to oxidative stress by activating specific genes. Upon receiving a signal from EH, these genes direct the production of antioxidant enzymes that will then minimize ROS damage. Dr. Garciaâ€™s lab will screen cells that have been collected from 200 children with mitochondrial disease for the presence of mutated genes that provide the code for the EH regulatory protein. They will then insert these coding errors into cultured cells in the lab to determine the specific nature of the dysfunction associated with the protein.

Dr. Garcia will also work with mice to further understand the regulation of antioxidant enzymes. Loss of EH in mice results in increased levels of ROS and causes them to develop a condition that is similar to mitochondrial disease in humans. This similarity provides an excellent animal model for study of the disease. Dr. Garcia will work both with mice that lack EH and with normal control mice, comparing the capacity of their skeletal muscle cells to produce the energy-containing molecule ATP.

Prepared by Steven G. Bassett, Ph.D.
Seton Hill University
Greensburg, PA

3. Ramon Marti, Institut de Recerca de I'Hospital de la Santa Creu i Sant Pau in Barcelona, Spain, "Restoration of thymidine phosphorylase activity in MNGIE patients through platelets infusion"

Dr. Martiâ€™s lab is investigating a rare disease that affects the digestive system. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a disease of the mitochondria, cell organelles that are responsible for energy production. The severely disrupted digestive function associated with MNGIE typically leads to death in the early adult years. Mitochondria are one example of the complex machines, called organelles, that are found in the human cell. These organelles are made from a variety of proteins. The genetic code required for the manufacture of most these proteins resides in the DNA of the cellâ€™s nucleus. Mitochondria are unique among organelles, however, in that the nucleus does not contain all of the genetic information needed to make them. Some of this information resides in the mitochondriaâ€™s own DNA. In order to replicate their own DNA, mitochondria require the activity of an enzyme called thymidine phosphorylase. Unfortunately, a mutation in the gene that provides the genetic code for thymidine phosphorylase causes the production of an abnormal version of the enzyme. The resulting buildup of thymidine, which normally is phosphorylated during the replication of mitochondrial DNA, has a toxic effect on mitochondria and over time will severely diminish the energy-producing capacity of the cells in which these damaged organelles reside.

Platelets, blood cells involved in clotting, contain a number of enzymes and biologically active compounds and are especially rich in thymidine phosphorylase. Dr. Marti plans to infuse MNGIE patients with platelets obtained from healthy blood donors. The expected outcome is that the plasma levels of toxic thymidine in the MNGIE patients will be significantly reduced. Patients will receive platelet infusions every week for one month, during which plasma thymidine levels will be measured and any changes in clinical status will be monitored.

Prepared by Steven G. Bassett, Ph.D.
Seton Hill University
Greensburg, PA

4. Miriam H. Meisler, University of Michigan, "The nuclear-encoded gene OMI and mitochondrial disease"

Dr. Meisler has been conducting research with mice that possess a mutation causing abnormal mitochondrial function. Cells contain several kinds of machines called organelles, each of which has specific functions. The organelles under study in Dr. Meislerâ€™s lab are the mitochondria, especially complex cell organelles that are responsible for energy production. The mutated mouse gene is called OMI and it causes the affected mice to develop a neuromuscular disease that results in severely uncoordinated movement and wasting of muscles. There are a number of diseases that cause degeneration of the nervous system and the muscles that it controls. The changes experienced by the mice with the mutated OMI gene are a direct result of the inability of their abnormal mitochondria to provide adequate energy.

Building upon her experience with this mouse disease model, Dr. Meisler has designed a study in which she will screen 300 patients with inherited mitochondrial disease for OMI mutations similar to those found in mice. Mutated DNA sequences that occur in the mitochondrial disease patients, but not in healthy controls, will be tested for their involvement in the development of mouse neurodegenerative disease. This will be accomplished by microinjection of the candidate DNA sequences into fertilized eggs that would normally develop into mice with the fatal disease.

Prepared by Steven G. Bassett, Ph.D.
Seton Hill University
Greensburg, PA

5. Vamsi K. Mootha, Broad Institute, Massachusetts Institute of Technology, "Genomic Approaches to Human Cytochrome c Oxidase Deficiency"

Dr. Moothaâ€™s research team will identify genes that code for factors responsible for directing the synthesis of an important component of the mitochondrion. Mitochondria are energy-producing organelles contained in most human cells. They possess a series of molecules that are called collectively the respiratory chain. The respiratory chain is the cellular site where most of the energy is derived from foods that we eat to synthesize the energy-rich molecule ATP. The complex known as "cytochrome c oxidase" is an important component of the respiratory chain. Various assembly factors are required to direct the manufacture of new cytochrome oxidase complexes in the mitochondria.

Researchers have identified five human assembly factor mutations that can lead to abnormally functioning cytochrome c oxidase and diminished production of ATP by the mitochondria. Dr. Mootha has recognized the need for a comprehensive inventory of the nuclear encoded genes that are required for proper cytochrome c oxidase assembly and function. Computer analysis of data made available by the Human Genome Project can be used to perform a search for all human DNA containing the genetic code for cytochrome c oxidase assembly factors. Once specific DNA sequences have been identified as potential candidates, the research team will validate these sequences through the use of RNA interference in cultured cells. This procedure prevents the cells from expressing that particular DNA sequence. Subsequent biochemical assays of these cells will measure any changes in mitochondrial function that resulted from blocking expression of the DNA. Information gained from this research will increase our understanding of normal mitochondria function, providing insights into what goes awry when mitochondrial disease develops.

Prepared by Steven G. Bassett, Ph.D.
Seton Hill University
Greensburg, PA

6. Brian Robinson, Metabolism Research Program, Hospital for Sick Children, "Drug development for the regulation of respiratory chain components in mitochondria"

Dr. Robinsonâ€™s research team will screen a large number of chemicals from a family of compounds that show promise for stimulating the production of mitochondrial respiratory proteins. Mitochondria are energy-producing organelles contained in most human cells. They possess a series of energy-producing molecules that collectively are called the respiratory chain and are responsible for synthesizing the energy-rich molecule ATP. Some mitochondrial diseases result from deficiencies of certain respiratory enzymes that are part of the chain. But these defects are rarely total, with some capacity for ATP production remaining. Dr. Robinsonâ€™s goal is to look for drugs that could be used to enhance whatever residual activity is present in abnormal mitochondria, so as to increase overall energy production in mitochondrial disease patients.

Prior discovery of two nuclear transcription factors that increase the rate at which mitochondrial respiratory chain proteins are synthesized has led to Dr. Robinsonâ€™s suggestion that drugs could be developed to stimulate the activity of these factors. Such stimulation is indeed what happens in response to exercise. Dr. Robinson has access to a large chemical library of heterocyclic compounds that may stimulate the transcription factors in the absence of exercise. He plans to conduct a systematic screening of these chemicals to identify ones that show promise for stimulating the production of mitochondrial respiratory proteins.

Prepared by Steven G. Bassett, Ph.D.
Seton Hill University
Greensburg, PA

7. Stefan Strack, University of Iowa, Carver College of Medicine, "Protein phosphatase 2A in mitochondrial function and disease"

Dr. Strackâ€™s laboratory is interested in conducting basic research that could lead to the development of new treatments for mitochondrial disease. Mitochondria are energy-producing organelles contained in most human cells and can cause specific disease conditions when they are damaged. Dr. Strack will develop methods of blocking the activity of a mitochondrial regulatory protein that normally renders brain cells vulnerable to toxin-induced degeneration. His ultimate goal is to find a means of rescuing the cells from the effects of the toxins. His research group is interested in neurodegenerative diseases such as Huntingtonâ€™s chorea and Parkinsonâ€™s disease.

Previous research identified a regulatory protein that is localized to mitochondria in cells of the brain. The protein renders cultured brain cells vulnerable to damage by toxins. If the protein is inhibited, then resistance to the toxins is conferred upon the cells. In his proposal, Dr. Strack suggested that development of drugs against the protein may provide an effective treatment for mitochondrial disease. He will use genetically-engineered viruses to place inhibitors specific to the protein into the brains of rats, followed by testing for resistance to neurotoxins that normally poison the brain. Finding effective means of rescuing the cells from the effects of the toxins could provide insight into prevention of the nervous system degeneration that is typical of some mitochondrial diseases.

Prepared by Steven G. Bassett, Ph.D.
Seton Hill University
Greensburg, PA

8. Volkmar Weissig, Northeastern University, Department of Pharmaceutical Sciences, "Development of a method for transforming mitochondria in living mammalian cells with exogenous DNA"

Dr. Weissigâ€™s research group is interested in developing a means of replacing defective DNA sequences in mitochondria with the correct sequences that are required for normal function. Mitochondria are complex energy-producing cell organelles that are made from a variety of proteins. The genetic code for the manufacture of most of these proteins resides in the DNA of the cellâ€™s nucleus. Mitochondria are unique among organelles in that the nucleus does not contain all of the genetic information needed to make them. Some of this information resides in the mitochondriaâ€™s own DNA. If a defect is present in the mitochondrial DNA (mtDNA), then it will keep the mitochondria from making the normal proteins needed for energy production. Many mitochondrial diseases due to mtDNA defects develop into serious, ultimately fatal neuromuscular disorders. Gene therapy as an approach to treatment of these diseases holds great promise.

Dr. Weissig plans to develop and perfect a mechanism for transporting normal DNA through the interior of cells to the mitochondria, with subsequent mitochondrial uptake of the DNA. Researchers are also developing gene therapy approaches to the treatment of diseases such as muscular dystrophy. This is more straightforward than treatment of mitochondrial disease because it is easier to insert the correct DNA sequence into the nucleus of cell. Extra challenges are associated with inserting the correct DNA sequence into mitochondria. Development of a reliable method for insertion of DNA into mitochondria would be a significant step in treatment of often fatal mitochondrial diseases.

Prepared by Steven G. Bassett, Ph.D.
Seton Hill University
Greensburg, PA

9. Immo Scheffler, Ph.D., University of California, San Diego, "Application of RNA interference in the study of NADH-ubiquinone oxidoreductase (complex I) assembly in mammalian mitochondria"

Amount of Award: $100,000
Award Date: 2003

Project Summary: Small interfering RNAs (siRNAs) can nowadays be readily produced in many cells including mammalian cells. RNA interference (RNAi) has become a novel and powerful method to knock down the expression of specific genes in such cells, an alternative to the classical approach utilizing mutations. With the complete knowledge of many of the genomes, any gene can in principle become a target, and hence any protein of interest can be eliminated from a cell to study the physiological and pathological consequences.

Our interest is focused on a large complex in the inner mitochondrial membrane that is essential for respiration (complex I, NADH-quinone oxidoreductase). The complex has a total of 45 subunits, of which 38 are encoded by nuclear genes, and 7 are encoded by mitochondrial genes. Thus, 38 genes are potentially subject to manipulation by RNAi. A fundamental issue is that 14 orthologous proteins in bacteria can perform a very similar function, and the challenge is to elucidate the role of over 30 additional subunits that have been added to the complex in mammalian mitochondria in the course of evolution. RNAi-based technology will for the first time permit a systematic examination of the role of each of these nuclear-encoded subunits in the assembly, stability, activity and regulation of complex I. The insights gained can be applied to the diagnosis and understanding of mitochondrial diseases, particularly the growing class of such diseases resulting from a (partial) complex I deficiency. Partial deficiencies can arise either from a reduced specific activity of the complex or from lower levels due to assembly defects. In the longer term, the technology can be adapted to perform such experiments in whole animals such as a mouse, with the prospect of developing good animal models for the study of the broader physiological and pathological aspects of these diseases.

10. Mikhail Alexeyev, Ph.D., University of South Alabama, "Selective Elimination of Defective Mitochondrial Genomes as an Approach to the Reversal of NARP and MILS Syndromes, Heritable Mitochondrial Disorders"

Amount of Award: $100,000
Award Date: 2003

Project Summary: The NARP and MILS syndromes are devastating disorders caused by T8993G mutation in mitochondrial DNA. There exists a compelling evidence that this mutation is the most commonly identified mtDNA mutation in children. Clinically, NARP is characterized by sensory neuropathy, cerebellar ataxia, retinitis pigmentosa, dementia, seizures and developmental delay. MILS, on the other hand, is usually associated with more severe manifestations and early onset (4 to 5 months). Patients with NARP and MILS syndromes are typically heteroplasmic, which means that their cells have both normal and mutant mitochondrial DNA. The difference in the clinical severity is largely due to the mutant load (i.e. % of mutant mtDNAs in every given cell). The life expectancy and quality of life in patients is believed to inversely correlate with mutant load. Thus, patients with MILS have very high mutant loads, typically >90% mutant mtDNA. NARP is usually associated with intermediate mutant loads of 60 to 80%, and mutant loads less than 60% are generally asymptomatic. Therefore, it appears that lowerlng the mutant load below the 60% threshold should be sufficient to render both NARP and MILS patients asymptomatic.

Amazingly, two bacteria, Serratia marcescens and Xanthomonas malvacearum, produce enzymes that can selectively recognize and destroy mutant, but not normal mitochondrial genomes with 99.95% efficiency in a test tube. Therefore, the central idea of this application is to achieve therapeutic effect through targeting of bacterial enzymes to mitochondria. If successful, this approach may allow, for the first time ever, to revert, rather than to just treat, the underlying mitochondrial disease. Our calculations indicate that even if bacterial enzymes will be only 1 % as efficient in mitochondria as they are in a test tube, they still should be able to bring mutant DNA content down below 60% threshold in a patient with 93% mutant mitochondrial DNA. Preliminary data indicate viability of the proposed approach. Thus, the focus of this proposal is to further experimental procedures to target therapeutic enzymes to mitochondria.

11. Matthew Freeman, Ph.D., Laboratory of Molecular Biology - M.R.C., Cambridge, UK, "Role of Rhomboid Proteolysis in Optic Atrophy"

Amount of Award: $90,000
Award Date: 2003

Project Summary: Dominant optic atrophy is a genetic disease that causes early childhood blindness with prevalence as high as 1: 10,000 and is the result of defects in a protein called OPA1. OPA1 is a protein that is responsible for maintaining the proper structure and function of the mitochondria, a vital part of the every cell. For OPA1 to function it needs to be cut at the correct place and the correct time by another protein in the cell. We have recently characterized the protein, called Rhomboid, which cuts OPA. We believe that this may be an important step in describing the molecular mechanism of this disease. By using the mouse as a model organism we hope to determine the link between OP A and Rhomboid regulation of mitochondrial function and human optic atrophy.

12. Koji Okamoto, Ph.D., University of Utah, "Molecular Basis of Mitochondrial Membrane Dynamics: a New Paradigm of Human Disease"

Amount of Award: $83,400
Award Date: 2003

Project Summary: Mitochondria are dynamic organelles that change size and shape in order to optimize their energy production and supply for cellular functions. Loss of normal mitochondrial morphology results in a variety of human diseases including neurological disorders and some types of cancer. Changes in mitochondrial morphology are also associated with programmed cell death and aging in humans. Although recent findings suggest that deregulation and loss of mitochondrial fusion may contribute to defects associated with mitochondrial diseases, very little is known about the mechanisms that regulate mitochondrial fusion. To understand pathogenesis leading to such diseases, regulatory molecules for mitochondrial fusion must be identified and characterized.

The transmembrane GTPase, called Fzo1 in yeast, is essential for mitochondrial fusion in both yeast and humans. Recent analyses in yeast suggested that GTP hydrolysis by Fzo1 is required for mitochondrial fusion. Regardless of which fusion step requires GTP hydrolysis by Fzo1, it seems very likely that a GTPase-activating protein (GAP) plays a crucial role in modulating mitochondrial fusion. To understand how the GTP-driven molecular device regulates mitochondrial membrane fusion, we will identify a GAP that stimulates GTP hydrolysis by Fzo1 in yeast. We will then analyze this GAP by a combination of biochemical, cell biological and genetic approaches. Moreover, we will investigate the GAP-mediated stimulation of the Fzo1 GTPase activity in vitro.

Since Fzo1 is an evolutionarily conserved GTPase, it is also likely that yeast Fzo1 GAP has a human homologue. What we learn about the mechanism of mitochondrial fusion in budding yeast will be directly relevant to the mechanism of mitochondrial fusion in humans. The proposed project will help determine how the Fzo1 GTPase cycle controls mitochondrial fusion, allowing researchers to investigate the role of regulated fusion and mitochondrial fragmentation during apoptosis. In addition, our research will provide insight into molecular bases for mitochondrial fusion that could contribute to the future development of gene therapies based on inter- mitochondrial complementation. Finally, this study will promote establishment of animal models for understanding pathogenesis of human diseases associated with mitochondrial fusion defects.

13. Bernard Lemire, Ph.D., University of Alberta, "The Use of the Yeast CYB2 Gene As Therapy for Complex I Mutations in a C. elegans Model System"

Amount of Award: $76,780
Award Date: 2003

Project Summary: The mitochondrial respiratory chain (MRC) is the major source of energy for most cells and tissues in the human body. It captures energy from the food we eat by catalyzing the transfer of electrons from NADH, which is derived from that food, to the oxygen we breathe. When MRC function is impaired, NADH accumulates and is diverted towards the formation of lactic acid resulting in lactic acidosis. This condition can produce malaise, weakness, exercise intolerance, and vomiting. It may also contribute to the long-term progression of and developmental delays associated with mitochondrial diseases by modulating the expression of genes related to energy metabolism.

We propose to investigate the use of a yeast enzyme called cytochrome b2 that will directly act to reduce the levels of both lactic acid and NADH. We have isolated and studied a series of complex I MRC mutants that mimic known human mutations in the nematode, Caenorhabditis elegans. The nematode is a very simple animal with an MRC that closely resembles its human counterpart and serves as a sophisticated genetic model system. We will introduce the DNA encoding cytochrome b2 into each of the mutants and evaluate how the yeast protein affects animal fitness as measured by fertility , motility , lifespan, and levels of lactic acid. These results will address the contribution of lactic acidosis to mitochondrial diseases and may lead to the development of a new therapy. Our results will lead to a better understanding of the fundamental biological processes surrounding mitochondrial energy production in normal and in disease states.

14. Giovanni Manfredi, MD, Ph.D., Weill Medical College of Cornell University, "MtDNA complementation and recombination in mitochondrial disorders"

Amount of Award: $50,000.00
Award Date: 2003

Project Summary: Mutations in the mitochondrial DNA (mtDNA) cause mitochondrial diseases. MtDNA is maternally inherited. Thus, mutations in the mtDNA typically result in family pedigrees exhibiting maternal inheritance, i.e. the disease should pass only through females, and essentially all the children inherit the mtDNA mutation. However, despite this simple way of inheritance, the manifestation of mtDNA-related diseases may be very variable even within the same family. Often, in patients with mitochondrial diseases associated with mtDNA mutations, normal and mutated mtDNAs may coexist within cells and tissues, a condition known as heteroplasmy. An important, but still not well understood, concept in the genetics of mitochondrial diseases is that of complementation in heteroplasmic cells. It is the subject of debate whether in human mitochondria functional complementation between mtDNA molecules is an efficient process or even whether it occurs at all. This is a very important question because if mutant mtDNA molecules function as independent units, unable to interact across different organelles, the protective effect of normal molecules coexisting with the mutated ones (heteroplasmy) would be rather limited. Moreover, the consequences of randomly occurring new mtDNA mutations, for example acquired during aging, would be more severe in the absence of efficient complementation between normal and mutated mtDNA molecules. This is especially relevant for patients that already harbor an inherited pathogenic mtDNA mutation, in whom the occurrence of acquired mutations may precipitate the clinical phenotype. To address these issues, we have generated a hybrid cell culture model from the fusion of two human cell lines with identical nuclear DNA, but each with a distinct mutation in the mtDNA. This model allows us to study complementation among mutated mtDNAs in a controlled system. Our goal is to better understand the mechanisms underlying mitochondrial complementation, because we believe that this may contribute to the identification of novel tools for the treatment of these diseases.

15. Philip Schwartz, Ph.D., Children's Hospital of Orange County, "Electrophysiologic Properties of Neural Stem Cells from Patients with Mitochondrial Disease"

Amount of Award: $81,574
Award Date: 2002

Project Summary: One of the more prevalent and debilitating features of mitochondrial disease is seizures. Seizures result from uncontrolled electrical activity within the brain. This altered electrical activity may result from changes in any number of basic properties of neurons, the functional cellular units of the brain. Unfortunately, our scientific understanding of the electrical changes in these cells is very incomplete as there has been no way to study neurons with mitochondrial disease while these cells are alive and functioning. Our current understanding of these changes comes only from inferences made from studying EEGs, the responses of patients to various drugs, and the brains of patients that have died with mitochondrial disease.

Recently, my colleagues and I have shown that we can harvest living neural stem cells from the post-mortem human brain, that is from the brain after death of the individual. The same cannot be done for neurons as neurons die very rapidly after death of the individual. Neural stem cells do not. Neural stem cells are immature brain cells that can, in the laboratory, be coaxed into becoming mature neurons. By harvesting neural stem cells from patients that have died with mitochondrial disease and growing these cells in the laboratory, we have produced a living, functioning, brain cell preparation with which we can now study the electrical changes associated with these cells in these same patients.

In the work described in this proposal, therefore, neural stem cells will be harvested from patients with mitochondrial disease and from patients with no neurologic disease (for comparison). This will be done in collaboration with the National Human Neural Stem Cell Resource which I direct. The cells will be grown up in my laboratory and coaxed into becoming electrically active neurons. The electrical properties of these cells, under a variety of conditions, will then be studied in detail. Importantly, we will study not only the basic electrical properties of these cells but also their responses to certain nutrients and drugs so that we may identify better potential anti-seizure treatments for patients with mitochondrial disease.

16. Yidong Bai, Ph.D., University of Texas Health Science Center at San Antonio, "Exploiting the potential of yeast NDI1 gene in the therapy of diseases linked with mtDNA"

Amount of Award: $66,000
Award Date: 2002

Project Summary: The mitochondrial complex I, is the largest and least understood component of the energy producing system, consisting at least 43 subunits. Mutations in genes encoding subunits of complex I are associated with diseases, in particular the various forms of Leber's hereditary optic neuropathy (LHON) that cause blindness. Impaired complex I activities have also been reported to be related to other neurodegenerative diseases and aging. The ability to repair mutations in mitochondrial complex I via gene therapy holds the promise of treatment of mitochondrial diseases such as LHON. In contrast to the multisubunit complex I enzyme found in mammalian cells, the corresponding enzyme in yeast, NDI1, is a single polipeptide. Previously, we showed that the yeast NDI1 is active in a human cell line with an artificial l mutation in mitochondrial DNA (mtDNA) ND4 gene. In this study we plan to further determine if the NDI1 gene can rescue mitochondrial function in cells carrying an mtDNA mutation that causes LHON in humans. More detail analysis of the activities of NDI1 gene in mammalian cells will also be performed. Finally we will initiate the production of transgenic mice expressing a complex I mutation transfected with NDI1 gene. The success of this study will significantly advance our endeavor to use gene therapy for the treatment of mitochondrial disease caused by complex I deficiency.

17. Tanja Taivassalo, Ph.D., Institute for Exercise & Environmental Medicine, Dallas, Texas, "Exercise-induced mitochondrial gene shifting: Resistance training as a therapy for sporadic mtDNA mutations"

Amount of Award: $61,389
Award Date: 2002

Project Summary: A clinical research trial is proposed to determine the efficacy of a novel approach to therapy for selective patients with mitochondrial disorders. The approach is based on the concept of "mitochondrial gene shifting" through exercise training, where resistance exercise is used to shift a patient's own normal mitochondrial genes from muscle precursor cells into their existing skeletal muscle which contains high levels of abnormal mitochondria and mitochondrial DNA. This has the potential to reverse the accumulation of abnormal mitochondrial DNA that occurs over time within skeletal muscle, restore a more normal function of muscle mitochondria, and thereby increase the energy-generating capacity of the cell. Also, normal adaptive increases in muscle strength associated with resistance training would be expected to further improve overall functional and physical capacity in patients, particularly in those demonstrating muscle weakness in addition to decreased muscle endurance. Due to the non-invasive nature and practicality of this approach, we believe resistance exercise training offers an imminent realistic advance in therapy and will be of functional significance in the quality of life of patients affected with mitochondrial myopathies.

18. Jose Hernandez-Yago, Ph.D., Institute for Cell Research, Valencia, Spain, "Transport diseases in mitochondria: Full screening of DNA alterations in human genes encoding TOMM and TIMM complexes in patients with mitochondrial diseases"

Amount of Award: $41,085
Award Date: 2002

Project Summary: The term "mitochondrial diseases" encompasses a heterogeneous group of disorders in which a primary mitochondrial dysfunction is suspected or proven by morphologic, genetic or biochemical criteria. The dual genetic control of mitochondrial proteins and the complex mechanisms needed for their synthesis, transport and correct assembly explain why a variety of genetic errors can cause mitochondrial diseases.

Many mitochondrial diseases suggest dysfunction in the transport processes of mitochondrial proteins from the cytosol to their mitochondrial compartments. We propose in this project the complete screening of the human genes encoding all the subunits of the mitochondrial protein transport machinery (TOMM and TIMM complexes) in patients with mitochondrial diseases as well as in the general population. The finding that the Mohr-Tranebjaerg syndrome-a mitochondrial disease that is a recessive degenerative syndrome characterized by postlingual progressive sensorineural deafness as the first presenting symptom in early childhood, followed by progressive dystonia, spasticity, dysphagia, mental deterioration, paranoia and cortical blindness- is due to defects in the hTimm8a gene, is a significant example of the severe consequences resulting from mitochondrial protein transport dysfunction.

In our laboratory we use a high throughput technology to screen DNA alterations that makes viable the screening of the ca. 100 exons of the approximately 15 subunits of TOMM and TIMM complexes. It is noteworthy that these exons are short enough to be amplified as a whole, using flanking intron primers.

19. Min-Xin Guan, Ph.D., Children's Hospital Medical Center, Cincinnati, OH, "Biochemical Basis for Maternally Inherited Deafness"

Amount of Award: $33,000
Award Date: 2001

Project Summary: Hearing loss is the most frequent sensory disorder. One in 1000 children is born deaf, an equal number lose their hearing by adulthood and half the population experience significant hearing impairment by the age of 65 years. Deafness can be due to genetic or environmental causes or a combination of both. Despite the recent progress in molecular characterization of deafness, the biochemical and molecular pathogenic mechanisms underlying the maternally inherited deafness remain poorly understood.

Recent results of genetic studies showed that an African-American fanilly with maternally inherited nonsyndromic hearing loss have been associated with the mitochondrial T7 511 C mutation in the tRNA Ser(UCN) gene, which is commonly related to deafness. To elucidate the pathogenic mechanism of this mutation, we have constructed a disease cell model by transferring mitochondria from lymphoblastoid cell lines derived from deaf individuals with mtDNA mutations or from controls lacking mutations, into human mtDNA-less (pO) cells. This application proposes two aims: 1). These transmitochondrial cell lines will be analyzed for the presence and severity of mitochondrial dysfunction associated with mtDNA mutations. 2). To study if over-expression of human mitochondrial Seryl-tRNA synthetase in these transmitochondrial cell lines can suppress the biochemical phenotype associated with T7511C mutation.

Success of this project will enhance a better understanding of pathogenic mechanisms of matemally inherited deafness, lead to the future therapies directed toward specific underlying abnormalities, and the development of animal models to test them.

20. Brian Robinson, Ph.D., The Hospital for Sick Children, Toronto, Ontario, "Efficacy of prenatal diagnosis of mitochondrial diseases."

Amount of Award: $15,000.00
Award Date: 2001

Project Summary: Prenatal diagnosis of mitochondrial diseases by examination of biochemical metabolite or enzyme profiles is undoubtedly possible, at least for some types of disease. We propose that the biochemical abnormalities in fibroblasts of affected children can be documented and used to carry out prenatal diagnosis in amniotic cell cultures. While we have been collecting data from prenatal diagnosis of nuclear-encoded mitochondrial diseases for a number of years, this data set is far from complete. We have had one year of funding from UMDF to continue this project, one more year of funding should allow us to complete our studies and make some conclusions. This project will be funded to see if this hypothesis can be verified for a number of mitochondrial diseases to include cytochrome oxidase deficiency, complex I deficiency and pyruvate dehydrogenase complex deficiency.

21. Edwin Kirk, M.D., Sydney Children's Hospital, Randwick, NSW, "Complex I: The role of nuclear genes in disorders of childhood due to mitochondrial Complex I deficiency."

Amount of Award: $12,000
Award Date: 2001

Project Summary: Complex I is a collection of proteins located in the mitochondria of the cell. These proteins work together to drive the first steps in the production of energy. Children with Complex I deficiency, often suffer devastating consequences such as deterioration of brain, muscle and other organ function. They often die in the first years of life. Complex I deficiency is a genetic disorder, but in most cases, the underlying genetic fault is not known. There are at least 42 different protein building blocks which combine to make Complex I, each of which is coded for by a different gene. Because there are so many possible genes involved, finding the gene fault in a child with Complex I deficiency has been a very difficult task. Recently, however, a group of scientists in the Netherlands looked at 8 complex I genes which seem to be particularly important (because they have changed the least during evolution). They found gene faults (mutations) in a total of 5 out of 20 families. We have samples from 40 children with proven complex I deficiency available for this study. This is a unique resource and represents all children in Australia with confirmed Complex I deficiency. We plan to search for mutations in the 8 important genes mentioned above, in the 40 families for whom we have DNA samples. If a mutation is found in a family, then the child's medical history will be reviewed. We will seek to identify patterns in the types of problems experienced by children with faults in each gene, to try to guide searches for faulty genes in future patients. If the faulty gene in a family is identified, it will enable us to provide answers for the family about the cause of the condition and how it is inherited in their family. For some, this information may also provide the option of testing future pregnancies to find out whether the baby is affected.

22. Dikoma Shungu, Ph.D., Columbia University, New York, NY, "Quantitative In Vivo 1H Magnetic Resonance Spectroscopic Imaging of Cerebral Lactate as a Screening Test for Mitochondrial Disorders"

Amount of Award: $36,719
Award Date: 2000

Project Summary: This research will seek to develop a test for screening patients suspected of having a mitochondrial disease. The test will be noninvasive in that it will be based on using magnetic resonance spectroscopic imaging (MRSI)â€”a technique that is nearly identical to standard MRI, except that it can measure levels of various biochemicals in the human bodyâ€”to precisely quantify levels of lactic acid in the cerebrospinal fluid (CSF) of suspected patients. We believe this to be worthwhile because studies we have conducted over the past 5 years on many patients with mitochondrial diseases have shown that a large proportion of these patients have measurable levels of lactic acid in their CSF, whereas control individuals did not. If further refined, this technique for measuring lactate in the CSF may prove to be a very useful tool for screening patients with mitochondrial diseases, and helping assess the degree of severity of the disease and its progression in future clinical trials of promising therapies.

23. George Perry, M.D., Case Western Reserve University, Cleveland, OH, "Is oxidative damage a result of metabolic abnormalities in Alzheimer disease?"

Amount of Award: $18,281 Award Date: 2000 Project Summary: Evidence continues to accumulate that increased oxidative damage is important in Alzheimer disease (AD) and may be the biochemical basis of the increased incidence of AD with aging. In published studies, we have shown oxidative damage affects every neuron in susceptible neuronal populations in cases of AD, but not in normal people. In unpublished studies, we show that the only abnormality of vulnerable neurons in AD is changes in mitochondria. This proposal will specifically investigate the idea that oxidative damage in AD begins as mitochondrial abnormalities. To address this issue, we will 1) determine the relationship between oxidative damage, mitochondrial abnormalities and the pathology of the disease; and 2) determine whether some of the relationships noted in Aim 1 for AD hold true in Down syndrome. Completion of these aims will help to define the relationship of oxidative damage to mitochondrial abnormalities.

24. Cecilia Giulivi, Ph.D., University of Minnesota, Duluth, "Characterization of Mitochondrial Nitric-Oxide Synthase"

Amount of Award: $31,137
Award Date: 1999

Project Summary: Expansion of the field of mitochondrial disease can be anticipated in several directions. First, into age-related degenerative diseases; second, into areas connected with new concepts in mitochondrial biochemistry and physiology. Of great interest are thorough studies aiming at a better understanding of the processes underlying mitochondrial defects. In this context, the recent finding of an enzyme, i.e., mitochondrial nitric-oxide synthase, that generates nitric-oxide, well-known modulator of important biological processes such as immunity, vasodilation, and neurotransmission, offers a new perspective in mitochondrial bioenergetics. We reported that the production of nitric-oxide by mitochondria modulates the synthesis of ATP; this latter compound constitutes the currency that supports cellular work. Considering the important role that nitric-oxide might have on ATP generation, this project is aimed at identifying the nitric-oxide synthase in different tissues, providing basis for future studies that will address strategies for attempts at the treatment of mitochondrial diseases associated with a defective enzymatic expression.

25. Brian Robinson, Ph.D., The Hospital for Sick Children, Toronto, Ontario, "Efficacy of Prenatal Diagnosis of Mitochondrial Diseases"

Amount of Award: $8,863
Award Date: 1999

Project Summary: Prenatal diagnosis of mitochondrial diseases by examination of biochemical metabolite or enzyme profiles is undoubtedly possible, at least for some types of disease. We propose that the biochemical abnormalities in fibroblasts of affected children can be documented and used to carry out prenatal diagnosis in amniotic cell cultures. While we have been collecting data from prenatal diagnosis of nuclear-encoded mitochondrial diseases for a number of years under a small program funded by the US March of Dimes, this data set is far from complete. This project will be funded to see if this hypothesis can be verified for a number of mitochondrial diseases to include cytochrome oxidase deficiency, Complex I deficiency and pyruvate dehydrogenase complex deficiency.

26. John Shoffner, M.D., Horizon Molecular Laboratory, Norcross, GA, "Gene mutations in Leigh's Disease"

Amount of Award: $30,000
Award Date: 1998

Project Summary: Leighâ€™s disease is a disorder in which degeneration in the basal ganglia structures, brainstem, and cerebellum cause considerable morbidity and mortality. Although most patients with Leigh's disease harbor defects in oxidative phosphorylation, mtDNA mutations are identified in less than 20% of cases when screening for known mtDNA mutations is performed. From 66 cases with Leigh's disease, we will select cases and test for mitochondrial DNA mutations based on the clinical features and biochemistry results. If the selected patients do not harbor mtDNA mutations, we will begin looking at specific types of mutations involving complex I genes that are found in the nuclear DNA.

27. Carolyn Bay, M.D., Children's Hospital of Pittsburgh, "Mitochondrial etiologies of pseudoobstruction and dysmotility in children"

Amount of Award: $5,000
Award Date: 1998

Project Summary: Approximately 75-100 children with serious, and chronic intestinal symptoms called pseudoobstruction and/or dysmotility, will participate in this 2 year study of potential mitochondrial etiologies. In addition to their pediatric gastrointestinal evaluation, the children will be evaluated from a genetic and medical genetic laboratory perspective. The purpose is to provide an explanation for the child's symptoms, and to determine how frequently rnitochondrial disorders are the underlying reason for the child's severe gastrointestinal problems. This will be done by a careful pediatric history, including a family tree, and physical examination. All children will have DNA isolated, and we will test for the mtDNA 3243 mutation. Then we will determine if additional mitochondrial testing is indicated. If agreed to by the family and the gastroenterologists, we will arrange for additional testing of other mitochondrial disorders. By testing the most frequently associated mitochondrial abnormalities known to cause these specific gastrointestinal problems of dysmotility and pseudoobstruction, we hope to learn the prevalence of mitochondrial disease in this group of pediatric patients. This information will provide additional insight into the spectrum of gastrointestinal problems that a child with a mitochondrial disorder might experience. We hope that an accurate diagnosis will provide further insight into improved treatment strategies for children experiencing these symptoms.

28. Richard Boles, M.D., Children's Hospital of Los Angeles, "Search for Pathogenic Mitochondrial DNA Mutations Using Temporal Temperature Gradient Gel Electrophoresis (TTGE)"

Amount of Award: $30,000
Award Date: 1997

Project Summary: Mutations of mitochondrial DNA (mtDNA) have been found in children with a wide variety of different disease manifestations and, due to its high mutation rate, are believed to be the underlying cause in a sizable fraction of children with mitochondrial disorders. Past efforts to identify mtDNA mutations in children have been hampered by the size of the mtDNA and the difficulty in distinguishing disease causing mutations from the numerous normal sequence variations found in mtDNA ('polymorphisms'). Most, if not all, children with mtDNA mutations have coexisting normal and mutant mtDNA ('heteroplasmy'), suggesting that a heteroplasmy detection method might be an effective method to screen populations for mtDNA disorders. Polymorphisms are almost always homoplasmic (single type of mtDNA).

A new mutation detection method, TTGE, was adapted in our laboratory for use with mtDNA. TTGE is rapid, inexpensive and very sensitive with 100% of known mtDNA mutations detected to date. Heteroplasmy (mutation) was clearly distinguishable from polymorphisms. At present we have used this method to scan 1/3 of the mtDNA, and we plan to extend the covered area to screen all of the mtDNA for mutation. After this we plan to use this technique to screen specific populations, including children with mitochondrial disorders, mental retardation, epilepsy, sudden infant death, etc. for mtDNA mutations. Our preliminary data in screening only 10% of the mtDNA sequence in 100 patients with suspected mitochondrial disease revealed 7 cases of heteroplasmy (mutation). This data demonstrates the power of TTGE to detect mtDNA mutations and suggests that mtDNA disorders are far more common than previously demonstrated. TTGE has great potential as a clinical screening test for general usage in patients with suspected mitochondrial disease. Detection of a mtDNA mutation could benefit families by allowing for presymptomatic and prenatal diagnosis in addition to providing a definitive diagnosis and more accurate genetic counseling.