Mitochondrial Case Studies

Underlying Mechanisms and Diagnosis

Russell Saneto, Sumit Parikh, Bruce H Cohen

• Publisher: Academic Press
• Year: 2015
• Print ISBN: 9780128008775
• eBook ISBN: 9780128011492
• Topic: Biowissenschaften
• Subtopic: Immunologie

Mitochondrial Case Studies: Underlying Mechanisms and Diagnosis offers the science behind mitochondrial disease with a case studies approach. Since mitochondrial diseases are diverse and influenced by genetic, environmental, and social-economic factors, this publication will help students, physicians, scientists, health care students, and families recognize and accurately diagnose mitochondrial disease and learn about potential treatments. - Reviews case studies as a helpful teaching tool to increase awareness and improve diagnosis - Provides information on underlying mechanisms of mitochondrial disease - Includes basic mitochondrial dysfunction research through patient case studies to best illustrate the entire disease process

Chapter 1

Introduction

Mitochondrial Medicine

Bruce H. Cohen Northeast Ohio Medical University, Rootstown, OH, USA The NeuroDevelopmental Science Center and Divison of Neurology, Department of Pediatrics, Children’s Hospital and Medical Center of Akron, Akron, OH, USA

Abstract

Fifty years has passed since the first description of a case with illness caused by mitochondrial dysfunction was published. Mitochondrial diseases occur primarily as a result of mutations occurring in genes that encode the approximate 1100–1500 genes responsible for mitochondrial structure, maintenance, and function. The mitochondria is the source of the vast majority of energy production within the cell, extracting energy from the molecular bonds within proteins, carbohydrates, and fat by way of a series of biochemical couples resulting in proton translocation across an impermeable membrane, and then capturing that energy as the protons flow back through the membrane resulting in the condensation of inorganic phosphate onto adenosine diphosphate to form adenosine triphosphate. In this process, oxygen serves as a sink for chemical reduction to water. Although mitochondrial disease is believed to be due to lack of adequate energy production, the additional functions of the mitochondria suggest that, in part, altered free radical production may also play a role in illness. The clinical aspects of mitochondrial dysfunction are varied, and diagnosing mitochondrial disease involves identifying the clinical phenotype and ordering confirmatory biochemical and/or genetic tests. This introduction will review the history of mitochondrial disease and serve to introduce the basic structure and function of the mitochondria.

Keywords

Electron transport chain; Heteroplasmy; Mitochondria; mtDNA; Respiratory chain

Introduction

Although case descriptions of cases with mitochondrial dysfunction had been described in the literature for decades before Luft’s report in 1962, the underlying mitochondrial pathophysiology was not documented until the description of a 30-year-old woman with an illness present since childhood. Not only did this case represent the first illness linked to mitochondrial dysfunction, it led to the now-established concept of an illness caused by dysfunction of an organelle. In the ensuing half of a century, a remarkable story unfolded, which has included the elucidation of a new tiny but essential fragment of human DNA within the mitochondria, the interactions of this small piece of DNA with the DNA contained in the nucleus, the advances in laboratory medicine allowing measurement of the enzymatic and polarographic function of the respiratory chain (RC), the descriptions of dozens of phenotypic disorders linked to the biochemical derangements within the mitochondria, and the discovery of the myriad of genetic mutations linked to human disease.

Many of the clinical diseases were initially associated with a descriptor that was defined by histologic features, typically seen in muscle. The initial microscopic findings included the description of a nonspecific light microscopy histochemical finding of the ragged red fiber, described in 1963 by Engel and coworkers, followed 2 years later by Shy and Gonatas’ report of ultrastructural (electron microscopic) mitochondrial findings. For example, case’s illnesses were defined as ragged red fiber myopathy. Over time, these visual features were supported by complementary enzymatic dysfunction such as the techniques described to measure pyruvate dehydrogenase complex (1970, Blass et al.), complex III (1970, Spiro et al.), carnitine palmitotransferase (1973, DiMauro et al.), and complex IV (1977, Willems et al.). Again, a case’s illness was often defined by the associated biochemical defect such as cytochrome c oxidase deficiency. The term mitochondrial encephalomyopathy was coined by Yehuda Shapira in 1977. Although both microscopy and enzymology could be performed in many tissues, for well over three decades, the muscle biopsy became the primary method of detection and confirmation of mitochondrial disease. The advent of molecular genetic diagnostics allowed for some cases to achieve a diagnosis without a muscle biopsy, but not until the full mitochondrial DNA (mtDNA) genome could be sequenced quickly and the nuclear DNA (nDNA) genes were being discovered (both occurring in the 2007–2008 era) did the molecular genetic technology become part of the day-to-day diagnostic evaluation. Finally, the application of massive parallel sequencing technology (so-called Next-Gen sequencing) allowed for detection of mutations in both known and putative gene candidates.

Overview of Mitochondrial Structure and Function

The mitochondria are a complex organelle responsible for the vast majority of cellular energy production. The final process in energy metabolism involves the phosphorylation of adenosine diphosphate (ADP) to adenosine triphosphate (ATP). ATP can be thought of the energy currency for almost all cellular functions, where hydrolysis of ATP releases energy along with ADP and P_i, which are returned to the mitochondria to again be re-phosphorylated into ATP. About 1500 enzymatic and structural proteins are necessary for normal mitochondrial function, and most of the genes that encode for these proteins reside in the nDNA. Pathogenic mutations in these genes may be passed along from parent to child with the same Mendelian and non-Mendelian rules that apply for other nuclear genes. As with other single nuclear gene disorders, de novo mutations may arise and cause disease. Mitochondria are unique organelles as they contain a small piece of circular DNA, which is referred to as mtDNA, discovered by Nass and Nass in 1963. Human mtDNA contains 37 genes encoding the translational machinery (2 rRNAs and 22 tRNAs) and 13 enzymatic proteins that make up only a portion of the electron transport chain (ETC; also known as the respiratory chain). These 13 proteins include subunits of complex I (7 proteins), complex III (1 protein), complex IV (3 proteins), and complex V (2 proteins). Unlike the nDNA, mutations in mtDNA are inherited by maternal transmission, meaning that mutations are passed from the mitochondria contained in the oocyte to all offspring. There is no paternal contribution of mtDNA to the offspring. The mitochondria contained along the midshaft of the spermatozoa either do not enter the oocyte or are immediately destroyed by proteases on the inner wall of the oocyte. Most pathogenic mutations present in the germline of the mother will be inherited or passed on to all children. Another feature of mtDNA is the concept of heteroplasmy. With nDNA, aside from notable exceptions that are beyond the scope of this discussion, one allele is inherited from each parent, so there are three possible states with respect to pathogenic inheritance: mutant homozygote, wild homozygote, or heterozygote (and hemizygote for X-linked disorders). Mutations in mtDNA tend not to be all or none, meaning that mutations occur in a variable percentage, the concept known as heteroplasmy. A mutation may occur in only a few percentage of mtDNA copies (low-level mutant heteroplasmy) or a large percentage of mtDNA (high-level mutant heteroplasmy). Aside from mutations causing Leber hereditary optic neuropathy (LHON), in which the mutations occur with 100% homoplasmy, most of the mtDNA mutations occur with variable levels of heteroplasmy. Higher percentages of mutant heteroplasmy are generally associated with earlier disease onset and more severe presentations. Because there is variability in mutant heteroplasmy in the mature oocyte, each offspring may have, on average, a different percent of mutant heteroplasmy than the mother. Furthermore, the mutations may segregate differently during embryogenesis, thus resulting in different ultimate organ dysfunction later in life.

The physical structure of the mitochondria is constantly changing in vivo. The organelle is composed of an outer mitochondrial membrane (OMM) and a heavily infolded inner mitochondrial membrane (IMM). One major property of the IMM that is not found in any other cellular membrane is the presence of the phospholipid cardiolipin. Unlike the other phospholipids that are composed of two hydrocarbon tails, cardiolipin has four, and this structure likely is responsible for the complex tight curves in the membrane that allow for the infolding. Proper cardiolipin content is also necessary to allow the proper stoichiometry and supercomplex structure of the ETC elements. There are contact points riveting the IMM and OMM containing the complex group of proteins that form the voltage-dependent anion channel and mitochondrial permeability transition pore. This structure is responsible for several critical functions that include the adenosine nucleotide transporter, which exchanges ATP exchange for ADP, as well as components for calcium exchange and apoptosis.

Embedded within the IMM are the five multicomplex proteins referred to as the RC or ETC. It is the reducing equivalents derived from the tricarboxylic acid cycle and fatty acid beta-oxidation (nicotinamide adenine dinucleotide and flavin adenine dinucleotide) that donate electrons into complexes I and II, and they supply the electrical force required to pump protons from the matrix into the intermembrane space. The intermembrane space has a small volume and functions to store the electrochemical capacitance. The protons can flow back into the matrix through a pore in complex V, which results in the physical movement of a molecular rotor, condensing a phosphate moiety onto a molecule of ADP, to form ATP. This electrochemical charge not only results in ATP formation by complex V, but also is an affector and effector of calcium regulation, and, ultimately, the most critical factor initiating intrinsic apoptosis if this potential is lost. Coenzyme Q10 is a mobile electron carrier, which shuttles electrons in a redox cycle between complexes I and III and complexes II and III. Cytochrome c shuttles electrons in a redox reaction between complex III and complex IV. In addition to its function as a proton pump, complex IV is the site in the ETC where molecular oxygen is reduced tetravalently to water. The process of proton translocation across the IMM, the chemical reduction of molecular oxygen to water, and condensation of phosphate onto ADP to form ATP is termed oxidative phosphorylation.

The mitochondrial matrix is the volume within the IMM and contains hundreds of enzymes necessary to the tricarboxylic acid cycle, the urea cycle, the functional cascade of proteins necessary for apoptosis, the synthesis of a number of amino acids, the breakdown of long-chain fatty acids, and the oxidation of eight amino acids. Also found within the matrix of the individual mitochondria are 2–10 copies of mtDNA.

The structure of the mitochondria depends on the specific cell type and if the cell is living or preserved. Within fixed cells, the individual mitochondria assume a cylindrical shape about 1 μm in length. It is estimated that most fixed cells contain 1000 or so mitochondria. In the living cell, the mitochondrial structure differs significantly between individual tissues, but it generally forms a syncytial network that is constantly changing shape. As part of this dynamic structure, both budding formations (created by mitochondrial fission) and reorganization of separate mitochondria (mitochondrial fusion) occur constantly. Failure of mitochondrial fission and fusion is known to cause human disease.

The mtDNA is contained within the matrix. It is a circular molecule with 16,569 nucleotide pairs, which contain 37 genes. These genes encode for 13 structural proteins that are contained in complexes I, III, and IV as well as the 22 transfer RNAs and two ribosomal RNAs required for mtDNA translation. The translational components (mt-rRNA and mt-tRNA) differ structurally from those in the nucleus of the cell and are structurally similar to that of the bacteria, the ancestral forerunner to the mitochondria. Some of the protein coding language of the mtDNA differs from its nuclear counterparts as well. The remainder of the mitochondrial structure and enzymes are encoded by nDNA. This includes all the other subunits of the ET...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• Contributors
• Preface
• Chapter 1. Introduction: Mitochondrial Medicine
• Part I. Mitochondrial DNA Encoded Diseases
• Part II. Nuclear Encoded Diseases
• Index