Mitochondrial DNA Molecular Genetics Human Mitochondrial Diseases Immo E. Scheffler Professor of Biology University of California, San Diego 1 Mitochondria 2 cristae 3 1
Fig 12. Mitochondria of a heart muscle cell from a rabbit; cristae mitochondria are clearly seen; X 27,000 (from Masunaga, 1979) 4 A new paradigm of mitochondrial structure IE Scheffler 5 6 2
V Lehninger, Fig. 19-16 7 8 IMS MATRIX A. Matsuno-Yagi, 2001 9 3
Mitochondrial DNA Mitochondria were originally microorganisms that invaded another cell and established a symbiotic relationship with the host Mitochondria became subcellular organelles incapable of an independent existence 10 The mitochondrial genome in humans ~1/2% of the DNA in a mammalian cell is mitochondrial DNA Genome size: 16.5 kb; it is a circular genome completely sequenced in 1980's Hundreds to thousands of copies per somatic cell (hundreds of mitochondria) 11 Information content of the mt genome In vertebrates/mammals it codes for 2 ribosomal RNAs, 22 transfer RNAs, 13 proteins: 7 subunits of complex I (NADH dehydrogenase) 1 subunit of complex III (ubiquinol-cytochrome-c c oxidoreductase) 3 subunits of complex IV (cytochrome oxidase) 2 subunits of complex V (ATP synthase) 12 4
rrna genes complex III complex I trna genes Cytochrome oxidase (complex IV) ATP synthase (complex V) 13 Non-Mendelian inheritance 14 mtdna is inherited through the maternal lineage (1) 15 5
mtdna is inherited through the maternal lineage (2) 16 17 18 6
19 20 Translation Mitochondrial ribosomes, trnas Translation of codons differs from the universal genetic code Initiation factors Elongation factors Mitochondrial ribosomes, like bacterial ribosomes, are inhibited by chloramphenicol 21 7
Mutations in mtdna 22 Assorted myopathies and neuropathies observed clinically in recent decades: Neurological disorders Seizures (muscle jumps) Dementia with early onset Stroke-like symptoms Migraine headaches Retinopathy Partial blindness Hearing loss Ragged red fibers in muscle biopsies Weakness exacerbated by exercise Movement disorders 23 General features: 1. Broad spectrum of symptoms; variable in severity 2. Delayed onset with age 24 8
Is there a genetic basis? Yes, in many cases The pattern of inheritance is distinctly non-mendelian: There is maternal inheritance, or maternal transmission Conclusion: The mutation is in mtdna 25 Heteroplasmy Significant deletions are pathological Patients, to be alive, must have some capacity for oxidative phosphorylation They are always heteroplasmic A fraction of their mtdnas have the mutation/deletion; the other fraction is wild type The % heteroplasmy may determine the severity of the symptoms Heteroplasmy may vary from tissue to tissue 26 Point mutations Silent mutations (SNPs) of no consequence Point mutations with pathological consequences: Missense mutations in proteins Mutations in trnas or rrnas which alter the efficiency of protein synthesis May be homoplasmic with 100% mutated mtdna 27 9
If there are ~ a thousand copies of the wild type genome per cell, how can a single mutation in one such mtdna ever lead to a change in phenotype? How does heteroplasmy arise? How quickly does it change from one generation to the next? 28 No symptoms? 20% 10% 22% 70% 15% 25% 45% Affected individual Mild symptoms? 29 Trends in Genetics, 16:500-505 (2000) 30 10
A bottleneck can explain the variable heteroplasmy in offspring from a single female by a simple stochastic mechanism Trends in Genetics, 16:500-505 (2000) 31 There are an estimated 100,000-200,000 mtdna per oocyte - Assume that there is no mtdna replication (and biogenesis of mitochondria) for x zygotic divisions let x = 12 one cell (fertilized egg) 2 12 cells = 4096 cells 100,000 / 4096 = 24 mtdna/cell if x = 15 then one expects ~ 3 mtdna/cell This may be the BOTTLENECK 32 Examples of mitochondrial diseases 33 11
The Kearns-Sayre Syndrome mtdna has deletions of 2-7 kb Always heteroplasmic (45-75%) In a given individual: all deleted mtdnas have the same deletion Onset before the age 20 Progressive opthalmoplegia Pigmentary retinopathy Complete heart block Cerebellar ataxia 34 Familial Mitochondrial Encephalomyopathy (MERRF) myoclonic epilepsy with ragged-red muscle fibers symptoms and tests: VER: visual evoked response EEG: electroencephalograph p Mito. myop.: mitochondrial myopathy involving RRF Deafness ME: myoclonic epilepsy Dementia Hypoventilation trna lys : A-->G in T C loop 35 Leber's Hereditary Optic Neuropathy (LHON) The 11778 mutation changes an arginine to a histidine at amino acid 340 of subunit ND4 in complex I Defining feature: bilateral central vision loss The onset is typically delayed d until the late teens Mutations in several other subunits of complex I have also been associated with LHON In some cases more than one mitochondrial mutation may act synergistically 36 12
MELAS Mitochondrial Encephalomyopathy, Lactic acidosis, and Stroke-like episodes Similar to MERRF Seizures, dementia, recurrent headaches Often associated with point mutations in the trna leu at positions 3243 or 3771 37 Neuropathy, Ataxia, Retinitis Pigmentosa (NARP) A mutation in nucleotide 8993 (Leu-Arg) in subunit 6 of complex V (ATP synthase) Always heteroplasmic i.e. the mutation causes a severe (lethal) defect when homoplasmic 38 Construction of cybrids to study mitochondrial DNA mutations Use of human cells with no mtdna (created in tissue culture) ( rho-zero or ρ o human cells) Enucleated cells or synaptosomes from patients containing mitochondria but no nucleus Fuse these cells to make cybrids A normal nuclear genome is combined with mitochondria from different individuals with potential mtdna mutations One can then measure respiration and ATP production by the cells or mitochondria and compare with rates in normal cells/mitochondria 39 13
Study of xenomitochondrial cybrids Recipient cells: rho-zero or ρ o human cells have no mtdna; respiration-deficient Mitochondrial donor cells: enucleated primate cells 40 Primate evolution Nucleus and mitochondria are compatible Nucleus and mitochondria are incompatible New world monkeys Old world monkeys Lesser apes Gorilla Humans Chimpanzees Orangutans Dryopithecus Proconsul Sivapithecus 41 Cybrids as stem cells Published: New York Times, May 20, 2008 The House of Commons has defeated a bill that would have banned the creation of so-called hybrid embryos part human and part animal for medical research. That means scientists who obtain proper licenses will be allowed to create hybrid embryos by transferring DNA from human cells into animal eggs that have had most of their genetic information removed. The embryos would then be grown in a laboratory and their stem cells would be harvested for up to 14 days, after which they would be destroyed. Opponents call the process unethical and unnatural; supporters say the embryos could aid the understanding of genetic defects and diseases like Parkinson s Skeptics might add that such cells may suffer from a serious energy deficiency due to incompatible protein subunits encoded by the nucleus and mtdna 42 14
Nuclear mutations Many subunits of the ETC are encoded by nuclear genes A severe mutation in homozygous form that causes the complete loss of function would most likely be lethal Many patients with nuclear mutations have been found The mutations in most cases are missense mutations leading to partially active proteins, or to an inefficient assembly of a complex Individual complexes are typically affected 43 44 Mitochondrial DNA deletion syndrome Healthy parents produce live offspring who develop problems as they grow up: 1. Autosomal dominant progressive external opthalmoplegia exercise intolerance, hearing loss, major depression, cardiomyopathy (starting at 18-40 years) 2. Mitochondrial gastrointestinal encephalomyopathy Preliminary conclusion: mutations in enzymes that perturb nucleotide pools in mitochondria and affect mtdna replication (DNA polymerase γ, the helicase/primase TWINKLE?) 45 15
Mitochondrial DNA depletion syndrome Healthy parents produce live offspring who develop problems in very early childhood with inevitably fatal outcome It is found that in specific tissues (frequently liver or muscle) the mitochondrial DNA disappears Rare; no good pedigrees as yet No linkage analysis, but nuclear mutations suspected 46 Mitochondrial mutations and aging 47 O 2 Hydroxyl radical Superoxide Hydrogen peroxide ROS scavenger reactions 48 16
Damage to mtdna Mutations ROS Damage to proteins Damage to lipids in the mitochondrial membranes 49 Anti-oxidants Derived from the diet Ascorbic acid (vitamin C) Tocopherols (vitamin E) Carotenoids (precursor for vitamin A) Plant phenols (in chocolate and red wine) Coenzyme Q 50 Hieronymus Bosch Fountain of Youth 15 th century 51 17
Neurodegenerative diseases Alzheimer s Disease Parkinson s Disease Huntington s Disease They share some intriguing features: 1. Delayed (age-dependent) onset 2. Formation of insoluble aggregates in affected neuronal tissue/cells 52 Cellular garbage disposal The function of the proteasome requires ATP as an energy input Mitochondria are the major source of this ATP Perturbations in either system could interact synergistically to lead to the ultimate failure Genetic studies of familial human cases and with model systems such as fruitflies have identified genes/functions linking these two activities; however, much remains to be learned in the future 53 The metabolic syndrome Obesity Diabetes type II Cardiovascular disease etc. 54 18
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