UC Irvine UC Irvine Electronic Theses and Dissertations Title Mitochondrial Metabolism and Morphology in Mitochondrial Disease States Permalink https://escholarship.org/uc/item/7ss5250z Author Simon, Mariella T. Publication Date 2016 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, IRVINE Mitochondrial Metabolism and Morphology in Mitochondrial Disease States DISSERTATION submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY In Biological Sciences by Mariella Theresa Simon Dissertation Committee: Professor Susanne Rafelski, Chair Professor Jose Abdenur, Co-Chair Professor Michelle Digman Professor Cristina Kenney Professor Grant MacGregor Professor Ali Mortazavi 2016 Chapter 2 © 2015 Plos BYCC Chapter 3 © 2013 Springer Chapter 4 © 2016 Elsevier All other materials © 2016 Mariella Theresa Simon DEDICATION To those individuals who believe(d) that the suffering of mitochondrial disease patients (young and old) is real and not made up or self-inflicted. For those who did this long before modern science could explain the underlying mechanisms. For all their pain that we cannot see that is real. “Die Elementarkörnchen der Zellen, welche noch heute ihre analogen Vertreter in den primären Mikroorganismen haben und welche seit jenen Perioden in den Zellen existieren, vermögen nicht mehr sebständige Lebewesen zu werden.” “The elementary granules of cells, which still have their analogs in primary micro- organisms and which exist since these time periods inside of cells, are no longer able to exist independently.” Richard Altman 1890 ii TABLE OF CONTENTS LIST OF FIGURES v LIST OF TABLES vii LIST OF ABBREVIATIONS viii ACKNOWLEDGEMENTS xi CURRICULUM VITAE xiv ABSTRACT OF DISSERTATION xix CHAPTER 1: Introduction 1.1 Principles of Mitochondrial Medicine 1 1.2 Age related mitochondrial disease phenotypes 13 1.3 Exercise as treatment for mitochondrial disease 33 CHAPTER 2: Mutations of Human NARS2, Encoding the Mitochondrial 35 Asparaginyl-tRNA Synthetase, Cause Nonsyndromic Deafness and Leigh Syndrome 2.1 Introduction 36 2.2 Results 38 2.3 Discussion 69 2.4 Materials and Methods 76 2.5 Acknowledgements 85 2.6 Contributions 85 CHAPTER 3: Abnormalities in Glycogen Metabolism in a Patient With Alpers’ Syndrome Presenting with Hypoglycemia 86 3.1 Introduction 87 3.2 Results 88 3.3 Discussion 94 3.4 Acknowledgements 98 3.5 Contributions 98 CHAPTER 4 Mutations in TFAM, Encoding Mitochondrial Transcription Factor A, Cause Neonatal Liver Failure Associated with mtDNA Depletion 99 iii 4.1 Introduction 100 4.2 Materials and Methods 103 4.3 Results 108 4.4 Discussion 120 4.5 Acknowledgements 124 4.6 Contributions 125 CHAPTER 5 Activation of a Cryptic Splice Site in the Mitochondrial Elongation Factor GFM1 Causes Combined OXPHOS Deficiency in Two Brothers with Microcephaly, Developmental Delay and Seizure Disorder 126 5.1 Introduction 127 5.2 Materials and Methods 129 5.3 Results 132 5.4 Discussion 139 5.5 Conclusion 143 5.6 Acknowledgements 143 5.7 Contributions 143 CHAPTER 6 Novel Mutations in the Substrate Binding Domain of the Mitochondrial Matrix Protease Lonp1 are a Cause of Mitochondrial Disease 149 6.1 Introduction 150 6.2 Results 155 6.3 Discussion 189 6.4 Materials and Methods 196 6.5 Acknowledgements 200 6.6 Contributions 200 CHAPTER 7 Discussion 200 7.1 The success of the “bedside to bench and back to bedside” 201 paradigm requires collaborative and multidisciplinary approaches 7.2 Emerging methods in mitochondrial medicine 204 7.3 Tissue specificity - a conundrum of mitochondrial disease 209 WEB RESOURCES 227 REFERENCES 228 iv LIST OF FIGURES Figure 1.1 The Cycle of Bedside to Bench and Back Figure 1.2 Electron microscopy of muscle from Luft’s patient Figure 1.3 Cartoon of a mitochondrion with its basic structure of outer and inner membrane, intermembrane space and matrix Figure 2.1 NARS2 mutations identified in two unrelated families Figure S2.1 Western blot and blue native gel analyses for patient fibroblasts and transmitochondrial cybrids Figure S2.2 Audiograms of individuals V.4 and V.8 from family PKDF406 Figure S2.3 Segregation and Sanger sequencing of mt-tRNACys A5793G Figure S2.4 Sequencing chromatograms Figure 2.2 Expression of Nars2 in mouse inner ear and brain Figure 2.3 NARS2 structure and molecular modeling Figure 2.4 NARS2 homodimerization and RNA level: effect of the p.Val213Phe and p.Asn381Ser mutations Figure S2.5 Interaction between HA- and GFP-tags Figure S2.6 Impact of the p.Val213Phe and p.Asn381Ser mutations on the NARS2 expression level in vitro Figure S2.7 Effect of the p.Val213Phe and p.Asn381Ser mutations on NARS2 localization Figure S2.8 Aminoacylation assays for mitochondrial tRNAAsn Figure S2.9 SDS-PAGE and western blot analysis of lentiviral transduction and expression of NARS2 Figure 2.5 Analysis of the impact of the NARS2 mutations on mitochondrial functions Figure 3.1 Diffusion-weighted images (MRI #2 and #5) Figure 4.1 Molecular analysis confirms segregation with disease of recessive TFAM p.Pro178Leu mutation Figure 4.2 Liver biopsy reveals cirrhosis and cholestasis v Figure 4.3 Skeletal muscle reveals findings consistent with mitochondrial myopathy Figure 4.4 Molecular and biochemical studies in primary fibroblasts Figure 4.5 Characterization of respiratory chain function in fibroblasts Figure 4.6 Nucleoid morphology is disrupted Figure 5.1 Mutations in patient 1 and 2 seggregate with disease Figure 5.2 Mutations in GFM1 are associated with abnormal splicing and result in decreased levels of GFM1 protein and abnormalities in complexes of the ETC Figure 6.1 LonpP1 mutations cluster in the AAA domain Figure 6.2 Modeling of patient mutations onto LonP protein structure Figure 6.3 Seahorse mitostress profile allows for assessment of spare respiratory capacity Figure 6.4 Patient cell lines have decreased spare respiratory capacity depending on availability of pyruvate Figure 6.5 Patient cell lines have decreased spare respiratory capacity depending on carbon source in response to addition of Antibiotic/Antimycotic Figure 6.6 Extracellular Acidification Rate (ECAR) is differently modulated in patient 1 and 2 Figure 6.7 Western blot for LonP1 and it’s targets reveals low levels of LonP1 for patient 1 Figure 6.8 Relative mtDNA copy number is modulated by the use of antibiotics in cell culture Figure 6.9 Large aggregated nucleoids are observed for patient 1 via immunohistochemistry in glucose media without antibiotic Figure 7.1 Overview of functional studies prompted by delineation of novel gene variants Figure 7.2 Impaired astrocytic glycogen metabolism may contribute to basal ganglia disorders Figure 7.3 Immunohistochemistry of Astrocytes shows mitochondria in parts of PAP vi LIST OF TABLES Table S2.1 Abnormal levels of urine organic acids of patient II.1 Table S2.2 Mitochondrial respiratory chain complex activities of muscle homogenate Table 2.1 Clinical findings of affected PKDF406 family members Table S2.3 Summary of Exome sequencing analysis for LS06 Table S2.4a Summary of Exome sequencing analysis for PKDF406 Table S2.4b Primer sequences used to amplify and sequence human NARS2 coding exons Table S2.5 Primer sequences used to amplify mouse Nars2 Table S2.6 Predicted effect of p.Val213Phe and p.Asn381Ser missense mutations on NARS2 Table 2.2 Summary of the known aminoacyl tRNA-synthetase genes associated with deafness Table S2.7 Summary of sequencing statistic for LS06 Table 3.1 Fasting study #1 Table 3.2 Fasting study #2 Table 4.1 Clinical Manifestations of hepatocerebral mitochondrisal DNA depletion syndromes Table 4.2 Mitochondrial respiratory chain enzyme analysis (ETC) - Skeletal muscle Table S5.1 Clinical phenotypes associated with mutations in GFM1 Table 6.1 Electron transport chain studies for muscle homogenate Table 6.2 Oxphos analysis for patient 1 Table 6.3 Comparison of phenotype for patient 1 and 2 vs CODAS syndrome Table 6.4 Insilico pathogenicity prediction for LonP1 variants Table 6.5 Summary of main Seahorse Study findings contrasted with mtDNA copy number vii LIST OF ABBREVIATIONS Auditory brainstem response (AABR) Abnormal carbohydrate deficient transferrin (CDT) Adenosine triphosphate (ATP) Adult onset polyglucosan body disease (APBD) Alanine aminotransferase (ALT) Alzheimer’s disease (AD) Amyloid beta (Aβ) Antibiotic-antimycotic (Anti-Anti) ATPase associated with diverse cellular activities (AAA) Basal respiration (BR) Base pair (bp) Beta-2 microglobulin (B2M) Beta-hydroxybutyrate (b-OH-Butyrate) Biotine-thiamine-responsive basal ganglia disease (BBGD) Blue native gel analysis (BNG) Cerebral spinal fluid (CSF) Charcot Marie Tooth disease (CMT) Clinical exome sequencing (CES) Combined respiratory deficiency (COXPD) Creatine Kinase (CK) Cybrids (cytoplasmic hybrids) Cytochrome C (CYCS) Cytochrome c oxidase subunit IV isoform 1 (COX4-1) Cytochrome oxidase (COX) 2,6-dichlorophenolindophenol (DCPIP) Deoxyguanosine kinase (DGUOK) Detergent compatible (DC) E. coli lon protease (EcLon) Echocardiogram (Echo) Electrocardiogram (EKG) Electroencephalography (EEG) Electron microscopy (EM) Electron transport chain (ETC) Endoplasmic reticulum (ER) Endoplasmic reticulum unfolded protein response (UPR)ER Enzyme carbamoyl phosphate synthetase I (CPS1) Exome aggregation consortium (ExAC) Exome variant server (EVS) Extracellular acidification rate (ECAR) Fibroblast (f) Fluid-attenuated inversion recovery (FLAIR) Free fatty acids (FFA) Fresh frozen plasma (FFP) viii Glutathione