- Email: [email protected]
Genetically and metabolically corrected pluripotent stem cells from patients with mtDNA disease
S38
Abstracts
Body of Abstract: We have been studying the metabolic adaptations of neurons with cytochrome c oxidase (complex IV) deficiency in our mice model of mitochondrial encephalopathy created by the conditional ablation of COX10 in neurons. COX10 is a farnesyl transferase that participates in heme a biosynthesis and is indispensable for the proper assembly and function of CIV. In the COX10 knockout (KO) mice, the onset of the mitochondrial defect at about 2 months of age does not coincide with the first signs of neuronal cell death at 4 months. Different brain regions are differentially affected by the mitochondrial dysfunction, been cingulate cortex, hippocampus and piriform cortex the most vulnerable brain regions in this mouse model. As the disease progresses (8 months) the COX10 KO mice shows a massive neuronal degeneration extending to all the cortical regions that eventually leads to a premature death at 10–12 months of age. The onset of the mitochondrial defect in neurons triggered an adaptive metabolic reprograming in the COX10 KO characterized by increased glucose uptake, glycolytic and pentose phosphate intermediates, and elevated enzymatic activity of hexokinase and pyruvate kinase (glycolytic enzymes) at 3 months of age. The metabolic changes likely compensated for the mitochondrial dysfunction since ATP levels in 3 months KO where similar to control values and CIV activity is already decreased to 50% of controls in tissue homogenates. The metabolic remodeling seems to be driven by post-translational modifications rather than by significant changes at the level of gene expression. We found that the changes in the metabolism correlated with changes in the activity/phosphorylation state of GSK3 (glycogen synthase kinase). GSK3 is known to regulate metabolism and cell survival. At the onset of the mitochondrial defect GSK3 was inactive/phosphorylated in the KO mouse, however at 3 months it switched to an active/dephosphorylated state presumably leading to neuronal death. In this study, we are investigating if the pharmacological inhibition of GSK3 to mimic the adaptive metabolic response naturally occurring in the COX10 KO will promote neuronal survival. Preliminary data with small number of mice showed decreased tunel positive cells in the hippocampus of mice treated with GSK3 specific inhibitor. We are currently testing the effect of different doses of the inhibitor on neuronal survival. Our preliminary data suggests that modulation of GSK3 activity can result beneficial for the treatment of mitochondrial encephalopathies and other neurodegenerative diseases associated with mitochondrial dysfunction. doi:10.1016/j.mito.2015.07.105
Abstract 101 Genetically and metabolically corrected pluripotent stem cells from patients with mtDNA disease Presenter: Amy Koski Amy Koskia,b, Hong Maa,b, Clifford D.L. Folmesc, Jun Wud, Robert Moreye, Sergio Mora-Castillae, Alejandro Ocampod, Li Mad, Joanna Poultonf, Xinjian Wangg, Riffat Ahmeda,b, Eunju Kanga,b, Yeonmi Leea,b, Tomonari Hayamaa,b, Ying Lia,b, Crystal Van Dykena,b, Nuria Marti Gutierreza,b, Rebecca Tippner-Hedgesa,b, Nargiz Mitalipova,b, Paula Amatoh, Don P. Wolfa,b, Taosheng Huangg, Andre Terzicc, Louise C. Laurente, Juan Carlos Izpisua Belmonted, Shoukhrat Mitalipova,b a Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 S.W. Bond Ave., Portland, OR 97239, USA b Division of Reproductive & Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, 505 N.W. 185th Ave., Beaverton, OR 97006, USA c Center for Regenerative Medicine and Department of Medicine, Division of Cardiovascular Diseases, Mayo Clinic, Rochester, MN 55905, USA d Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
e Department of Reproductive Medicine, University of California, San Diego, Sanford Consortium for Regenerative Medicine, 2880 Torrey Pines Scenic Drive, La Jolla, CA 92037, USA f Department of Obstetrics and Gynaecology, John Radcliffe Hospital, University of Oxford, Headington, Oxford OX3 9DU, UK g Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA h Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Oregon Health & Science University, 3181 Southwest Sam Jackson Park Road, Portland, OR 97239, USA
Abstract: Mitochondria play a major role in energy production via oxidative phosphorylation, which is dependent upon the expression of critical genes encoded by mitochondrial DNA (mtDNA). Inherited mtDNA mutations cause fatal or severely debilitating disorders in patients for whom there is no cure. Clinical manifestations of mtDNA disease vary based on specific mutation and ratio of mutant to wild type mtDNA within each cell (heteroplasmy). To explore the feasibility of generating genetically corrected pluripotent stem cells for future regenerative applications, we generated a panel of ten isogenic induced pluripotent stem cell (iPSC) lines from patients with MELAS (3243ANG) and Leigh disease (8993TNG and 13513GNA) mutations. iPSC lines containing exclusively wild type mtDNA were recovered from all three patients due to spontaneous segregation of heteroplasmic mtDNA. In contrast, all iPSC lines derived from a Leigh disease patient with homoplasmic (8993TNG) mutation carried exclusively mutant mtDNA. Somatic cell nuclear transfer (SCNT) enabled the replacement of the mutant mtDNA with wild type donor mtDNA and generated genetically corrected nuclear transfer embryonic stem cells (NT-ESCs). In contrast to the impaired oxygen consumption and ATP production observed in cells containing mutant mtDNA, genetic correction in NT-ESCs and iPSCs rescued metabolic function to levels observed in wild type controls. We conclude that iPSC and SCNT based reprogramming offer complementary strategies to generate genetically and functionally corrected pluripotent stem cells from patients with heteroplasmic and homoplasmic mtDNA disease. doi:10.1016/j.mito.2015.07.106
Abstract 102 Going on a diagnostic journey Presenter: Mary Elizabeth Parker Mary Elizabeth Parker Texas State University, Austin, TX, USA Abstract: Differential diagnosis of a pediatric patient with multisystem involvement and a complex presentation is a challenge in health care. Patient phenotypes do not always follow familiar patterns. The disease process may be dynamic causing different manifestations at different times. In order to recognize a disorder clinically, signs and symptoms (clinical indicators) need to cluster in a manner that consolidates a definition, some in the context of the lifespan. If a disorder is undefined based on clinical presentation, further diagnostic testing is indicated. To address differential diagnosis in patients with complex multisystem and metabolic disorders that have not been well-defined to date, it is important to review the established methods of differential diagnosis for other pediatric disorders in order to develop a decision-making tool. A tool to guide healthcare professionals in accurately diagnosing or making appropriate referrals of clients with undiagnosed disorders, particularly children, would be beneficial. In this work the operational definition of undiagnosed is a phenotype (presentation) that does not correlate with any known disease entity clinically via laboratory or other diagnostic means. While various algorithms, descriptions, and clinical pathways are available for known
Abstracts
Body of Abstract: We have been studying the metabolic adaptations of neurons with cytochrome c oxidase (complex IV) deficiency in our mice model of mitochondrial encephalopathy created by the conditional ablation of COX10 in neurons. COX10 is a farnesyl transferase that participates in heme a biosynthesis and is indispensable for the proper assembly and function of CIV. In the COX10 knockout (KO) mice, the onset of the mitochondrial defect at about 2 months of age does not coincide with the first signs of neuronal cell death at 4 months. Different brain regions are differentially affected by the mitochondrial dysfunction, been cingulate cortex, hippocampus and piriform cortex the most vulnerable brain regions in this mouse model. As the disease progresses (8 months) the COX10 KO mice shows a massive neuronal degeneration extending to all the cortical regions that eventually leads to a premature death at 10–12 months of age. The onset of the mitochondrial defect in neurons triggered an adaptive metabolic reprograming in the COX10 KO characterized by increased glucose uptake, glycolytic and pentose phosphate intermediates, and elevated enzymatic activity of hexokinase and pyruvate kinase (glycolytic enzymes) at 3 months of age. The metabolic changes likely compensated for the mitochondrial dysfunction since ATP levels in 3 months KO where similar to control values and CIV activity is already decreased to 50% of controls in tissue homogenates. The metabolic remodeling seems to be driven by post-translational modifications rather than by significant changes at the level of gene expression. We found that the changes in the metabolism correlated with changes in the activity/phosphorylation state of GSK3 (glycogen synthase kinase). GSK3 is known to regulate metabolism and cell survival. At the onset of the mitochondrial defect GSK3 was inactive/phosphorylated in the KO mouse, however at 3 months it switched to an active/dephosphorylated state presumably leading to neuronal death. In this study, we are investigating if the pharmacological inhibition of GSK3 to mimic the adaptive metabolic response naturally occurring in the COX10 KO will promote neuronal survival. Preliminary data with small number of mice showed decreased tunel positive cells in the hippocampus of mice treated with GSK3 specific inhibitor. We are currently testing the effect of different doses of the inhibitor on neuronal survival. Our preliminary data suggests that modulation of GSK3 activity can result beneficial for the treatment of mitochondrial encephalopathies and other neurodegenerative diseases associated with mitochondrial dysfunction. doi:10.1016/j.mito.2015.07.105
Abstract 101 Genetically and metabolically corrected pluripotent stem cells from patients with mtDNA disease Presenter: Amy Koski Amy Koskia,b, Hong Maa,b, Clifford D.L. Folmesc, Jun Wud, Robert Moreye, Sergio Mora-Castillae, Alejandro Ocampod, Li Mad, Joanna Poultonf, Xinjian Wangg, Riffat Ahmeda,b, Eunju Kanga,b, Yeonmi Leea,b, Tomonari Hayamaa,b, Ying Lia,b, Crystal Van Dykena,b, Nuria Marti Gutierreza,b, Rebecca Tippner-Hedgesa,b, Nargiz Mitalipova,b, Paula Amatoh, Don P. Wolfa,b, Taosheng Huangg, Andre Terzicc, Louise C. Laurente, Juan Carlos Izpisua Belmonted, Shoukhrat Mitalipova,b a Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 S.W. Bond Ave., Portland, OR 97239, USA b Division of Reproductive & Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, 505 N.W. 185th Ave., Beaverton, OR 97006, USA c Center for Regenerative Medicine and Department of Medicine, Division of Cardiovascular Diseases, Mayo Clinic, Rochester, MN 55905, USA d Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
e Department of Reproductive Medicine, University of California, San Diego, Sanford Consortium for Regenerative Medicine, 2880 Torrey Pines Scenic Drive, La Jolla, CA 92037, USA f Department of Obstetrics and Gynaecology, John Radcliffe Hospital, University of Oxford, Headington, Oxford OX3 9DU, UK g Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA h Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Oregon Health & Science University, 3181 Southwest Sam Jackson Park Road, Portland, OR 97239, USA
Abstract: Mitochondria play a major role in energy production via oxidative phosphorylation, which is dependent upon the expression of critical genes encoded by mitochondrial DNA (mtDNA). Inherited mtDNA mutations cause fatal or severely debilitating disorders in patients for whom there is no cure. Clinical manifestations of mtDNA disease vary based on specific mutation and ratio of mutant to wild type mtDNA within each cell (heteroplasmy). To explore the feasibility of generating genetically corrected pluripotent stem cells for future regenerative applications, we generated a panel of ten isogenic induced pluripotent stem cell (iPSC) lines from patients with MELAS (3243ANG) and Leigh disease (8993TNG and 13513GNA) mutations. iPSC lines containing exclusively wild type mtDNA were recovered from all three patients due to spontaneous segregation of heteroplasmic mtDNA. In contrast, all iPSC lines derived from a Leigh disease patient with homoplasmic (8993TNG) mutation carried exclusively mutant mtDNA. Somatic cell nuclear transfer (SCNT) enabled the replacement of the mutant mtDNA with wild type donor mtDNA and generated genetically corrected nuclear transfer embryonic stem cells (NT-ESCs). In contrast to the impaired oxygen consumption and ATP production observed in cells containing mutant mtDNA, genetic correction in NT-ESCs and iPSCs rescued metabolic function to levels observed in wild type controls. We conclude that iPSC and SCNT based reprogramming offer complementary strategies to generate genetically and functionally corrected pluripotent stem cells from patients with heteroplasmic and homoplasmic mtDNA disease. doi:10.1016/j.mito.2015.07.106
Abstract 102 Going on a diagnostic journey Presenter: Mary Elizabeth Parker Mary Elizabeth Parker Texas State University, Austin, TX, USA Abstract: Differential diagnosis of a pediatric patient with multisystem involvement and a complex presentation is a challenge in health care. Patient phenotypes do not always follow familiar patterns. The disease process may be dynamic causing different manifestations at different times. In order to recognize a disorder clinically, signs and symptoms (clinical indicators) need to cluster in a manner that consolidates a definition, some in the context of the lifespan. If a disorder is undefined based on clinical presentation, further diagnostic testing is indicated. To address differential diagnosis in patients with complex multisystem and metabolic disorders that have not been well-defined to date, it is important to review the established methods of differential diagnosis for other pediatric disorders in order to develop a decision-making tool. A tool to guide healthcare professionals in accurately diagnosing or making appropriate referrals of clients with undiagnosed disorders, particularly children, would be beneficial. In this work the operational definition of undiagnosed is a phenotype (presentation) that does not correlate with any known disease entity clinically via laboratory or other diagnostic means. While various algorithms, descriptions, and clinical pathways are available for known