The Development of Mitochondria Medicine

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Karolinska Institutet, June of 2004.
The Development of Mitochondria Medicine
Rolf Luft
I am grateful for the invitation to talk here today. My contributions to this field go back to 1959-60, a time when cellular particles were not known to be involved in human Pathology. Indeed, human mitochondria had not even been isolated and studied.
What we today call "mitochondrial medicine" is only a part of the whole panorama of diseases based on disordered mitochondrial function. A major group is due to defects related to fuel transport and metabolism in the mitochondria. The diseases in this group are well established and extensively reviewed.
In any event, having removed this group from the discussion, we can focus on respiratory chain diseases witch have been linked to defects either in mitochondrial DNA itself or in nuclear DNA encoding for mitochondrial proteins.
It is interesting to note that the first cell organelle ever to be linked to a human disease was the mitochondrion. In 1959-62,1 and my coworkers presented evidence for mitochondrial dysfunction in a patient. This started a revolution in chemical pathology according to Albert Lehninger. Also of interest in the context of this symposium is, that this discovery emanated from, an observation in a patient: a 27-year old woman with the highest oxygen consumption ever recorded. Involvement of the thyroid gland was excluded.
The question then was raised: What regulates oxygen consumption in the body? The idea of involvement of the mitochondria came to me when I looked into textbooks of biochemistry where, almost exclusively, studies on rat liver mitochondria were the basis for views of energy metabolism. I suggested that some error m the coupling of oxidation to phosphorylation might explain the patient's enormous oxygen consumption.
Our studies began with developing methods for isolation of skeletal muscle mitochondria in rat and then in man. The very first experiment on muscle mitochondria from the patient and from a control subject clearly demonstrated the functional defect:
1. a nearly maximal rate of respiration in the presence of substrate alone without addition of ADP+Pi but almost normal phosphorylafing efficiency in the presence of ADP + Pi
2. the mitochondria exhibited high ATPase activity which was only slight1y stimulated by the uncoupler, 2,4-dinitrophenol
3. these were features of "loosely coupled "respiration - deficient respiratory control with partially maintained ability to synthesize ATP - and accounted fully for all symptoms of the patient
4. electron microscopy revealed striking structural abnormalities, a marked density of cristae and a large accumulation of mitochondria of highly variable size in the perinuclear zone of the muscle cells, etc
5. all enzymes and cofactors measured were present, big thc level of coenzyme Q was relatively low and cytochrome oxidase high; the content of RNA was high and no uncoupling agent was found in muscle homogenates.
These were the essential findings derived from a mass of data. In the beginning of the 1960es, we proposed that the lack of respiratory control might be due to enhanced proliferation of mitochondria with the formation of a component necessary for maintaining tight coupling between respiration and phosphorylation having failed to keep, pace with the proliferation. Somewhat later - when Peter Mitchell had arrived on the scene - we suggested that a short-circuit of the flow of protons in the inner membranes had occurred, partly inhibiting ATP production but preserving electron transport. Further advances in the elucidafion of the defective mechanism in Luft's disease will have to wait until another patient appears in addition to the two described thus far.
In the past months, Sjóstrand from the University of California has offered another explanation for the pathogenesis of Luft's disease. His starting point is the structural modification of the cristae in the mitochondria of our patient assuming a marked zig-zag formation. Accordingly, leakage of protons from these cristae would ensue, impairing the coupling of respiration and ATP synthesis. And Sjostrand continues- "The structural damage is conceived of to be caused by a genetic defect preventing proper aggregation of the enzyme molecules in the cristae. Lust’s disease may therefore the first known disease caused primarily by a structural disorder at the molecular level." Again - we have to await a third patient with Luft's disease to test this proposal.
Luft's disease was described in toto M_ 1962. Remarkably, almost eight years passed before a few other patients were reported with aberrations of mitochondrial function, although with quite different symptoms. In the meantime, in 1964, without attracting special attention, the discovery of specific mitochondrial DNA was made. And not until 1988 did we witness a new revolution in mitochondrial pathophysiology with the report of an association of different sporadic encephalomyopathies with large deletions of mtDNA. This group of diseases - genetic mitochondrial disorders due to mutations in mtDNA and dysfunction in electron transport - has expanded dramatically during the past 10 years. There are now more than 50 different pathogenic mtDNA base substitution mutations and hundreds of mtDNA rearrangement mutations (deletions and insertions) linked to human disease. In addition, several lines of evidence suggest that mitochondrial dysfunction may play a role in the ageing process and in some of our most common age-related disorders, e.g., heart failure, some neurodegenerative diseases and diabetes mellitus.
It is also now well established that synthesis of some mitochondrial proteins are regulated by mtDNA. But most of these proteins are encoded by the nuclear genome, translated on cytoplasm ribosomes and imported into the mitochondrion As fig. 7 shows, proteins targeted for Mitochondria usually have a nútochondrial amino terminal leader peptide, which is cleaved after import into the organelle. However, at present, the majority of mitochondrial diseases with established genetic cause are based on mutations of mtDNA and not nuclear DNA.
In the last few years, we few witnessed a tremendous development of mitochondrial genetics. Large rearrangements of mtDNA often occur spontaneously, whereas point mutations usually are maternally inherited.
 
Replication of mtDNA is not coordinated with the cell cycle and, therefore, random segregation of mutated mtDNA occurs. Furthermore, somatic mutagenesis may cause accumulation of mutations in postmitotic tissues which possibly, could contribute to, e.g., the normal aging process or the development of some common chronic disease. as already mentioned. In this connection, the key role of mtDNA heteroplasmy is most interesting. The threshold at which symptoms arise in relationship to the energy demand of a particular tissue is a comer stone in the development of the diseases. Heart muscle, skeletal muscle in action, the central nervous system, liver and kidney and endocrine glands, because of their high energy demands, are particularly sensitive to alterations in oxidative phosphorylation.
Pathogenic mutations in nuclear genes regulating oxidative phosphorylation are gradually being recognized. The first one was reported in 1995 in two siblings with Leigh syndrome and a complex II deficiency, and a few other reports have followed. Of special interest are recent reports of the neurodegenerative disorders, Friedrich's ata2da, Wilsons's disease and autosomal spastic paraplegia probably being of mitochondrial origin. Friedrich's ataxia is caused by mutations in the gene frataxin, which regulates mitochondrial iron transport. Lack of frataxin damages the Fe-S-dependent respiratory chain complexes I, II and III and aconitase. Autosomal recessive hereditary spastic paraplegia involves mutations of the paraplegia gene. Wilson's disease I shall omit for the present.
An intricate problem that we will have to live with for a long time is the possible involvementof mitochondrial dysfunction in ageing and age-related diseases. It is true that several lines of correlative and genetic data implicate mitochondrial dysfunction in the naturally occurring process of ageing. In 1986, Miquel and Fleming presented the "oxygen radical-mitochondrial injury hypothesis of Aging". It states that the raised respiration of differentiated cells wall increase the production of radical oxygen species to levels exceeding detox" defenses.
This will impair the cell's capacity to regenerate mitochondria, and lead to progressive deterioration of ATP production. A decline in the respiratory chaincapacity has been reported with increasing ago, particularly of complex I and IV, associated with increased oxidative damage to mtDNA. However, some studies have not corroborated such findings. This lack of correlation between age and reduced phosphorylation capacity is not surprising, if one considers dm low levels of accumulated mutations observed in old people in comparison with the levels required for respiratory chain dysfunction in established mitochondrial dysfunction.
We may look with some expectations at new studies using in vitro cultured cells to elucidate the impact of mitochondrial dysfunction on ageing. One study on mitochondria-mediated transformation of human cells lacking mtDNA reported a possible relationship. Some have shown that accumulation of nuclear rather than mtDNA mutations may be responsible for the age-related reduction of oxidative phosphorylation. This is only the beginning of an interesting new avenue to pursue.
This I wrote a week ago. Two days later, I received a copy of a paper in Science, in which Professor Giuseppe Attardi and his coworkers report on "aging dependent large accumulation of point mutations in the human mtDNA control region for replication". I suppose that Professor Attardi will discuss his findings this afternoon. A connection between mitochondrial dysfunction and diabetes mellitus has been discussed for some time. The starting point was, first, the increased appearance of diabetes in patients with defined mtDNA mutations; second, the direct evidence for mtDNA involvement in pedigrees, with maternally transmitted diabetes and deafness and, third, diabetes in a family with sensory-neural deafness associated with the common MELAS mutation (A3243G) in the tRNA [Leu(uuR)]. Again except for these findings, diabetes and mitochondrial dysfunction remains an enigma.
For several years, a possible involvement of mitochondrial dysfunction in neurodegenerative disorders has been debated. Circumstantial evidence suggests that there is deficient oxidative phosphorylation and increased oxidative damage in Parkinson's disease and possibly in other age-related neurodegenerative disorders, e.g.. Alzheimer's disease and inherited amyotrophic lateral sclerosis. To list some of the findings: a deficiency in complex I and increased oxidative damage in the substantial Nigra of Parkinson patients; the effect of the neurotoxin MPTP on dopamìnic neurons in mammals; the increased risk of certain mtDNA polymorphism for the development of Parkinson's disease. Limited availability of human brain tissue hampers these studies. We can foresee new approaches in this field with the further development of transgenic animal models, where energy production has been deleted in different regions of the brain
Over all, we have today no established treatment of mitochondrial dìsorders. (1), coenzyme Q, with its dual role as electron transporter in the respiratory chain and as scavenger molecule, has, been tried extensively. Some impressive data concern its effect on symptoms in NMAS and Kearms-Sayre syndromes. (2), peptide nucleic acids (PNA) are synthetic polyamid nucleic acids which can be designed to be complementary to short mtDNA sequences harboring point mutations or deletion breakpoints. PNA's have been used as antigens probes to selectively inhibit the replication of mutated mtDNA - with some success in vìtro. The obstacle to its uptake by mitochondria may be overcome by a leader peptide to the PNA molecule. (3), a third approach that has been attempted is by, first, inducing a localized necrosis in a small muscle region in a patient with mitochondrial myopathy and, then letting muscle segments regenerated from stem cells take over function. Surprisingly, the regenerated muscle segment completely change the genotype. These "new" muscle cells contain very low levels of mutated mtDNA with a resulting normal respiratory chain function.
Certainly, we are looking forward to gene therapy, the ultimate treatment. It would replace or repair the defective gene and lead to permanent cure. However, it is currently impossible to introduce genes into mitochondria of in vitro cultured cells. Short pieces of nucleic acids can be attached to protein leader sequences and imported into isolated mitochondria. But a low frequency of recombination in mammalian mitochondria is a serious drawback.
During the last 10 years, we have witnessed the enormous growth of mitochondrial medicine. New investigative techiniques have continuously been adopted. The results have widened our knowledge of the intricate mechanísms involved in the chemical pathology of the increasing number of diseases with mitochondrial involvement. If I had to select new developments brightning the outlook for the future, they would include the use of animal models mitocodrial diseases. New insights into the pathophysiology of such disorders have already been obtained from such models. Let me mention a few:
1. An animal model for mitochondrial diseases was generated by targeted inactivation of the nuclear-encoded anti-gene, providing a model for ATP deficiency. Homozygous knockout animals are viable, but exhibit increased levels of blood lactate,- mitochondrial myopathy and hypertrophic cardiomyopathy with mitochondrial proliferation.
2, Disruption of the mìtochondríal transcriptor factor A (Tfam) gene demonstrated that this nuclear-encoded protein is necessary for mtDNA maintenance in vivo. Homozygous Tfam knockouts die in midgestation. The mutant embryos lack mtDNA and have a severe respiratory chain deficiency with massive accumulation of morphologically abnormal mitochondria. Recently, the ere-lox P conditional knockout strategy was used to selectively disrupt Tfam in heart and skeletal muscle. These mutant animals developed mitochmdrial cardiomyopathy with features similar to the finding mtDNA deletion díseases.
3. A novel method for producing animal models of mitochondrial myopathy may provide a valuable model for treatment trials. Human myoblasts containing different pathopenic mtDNA mutations were emplanted into irradiated muscle of severe combined immnunodeficiency mice. Injected myoblasts expressed human muscle markers and were also innervated.
4. Recent observations using animal models suggest the possibility of introducing mtDNA mutations into the animal germline. This may result in a better understanding of how mtDNA mutations affect energy production, and will provide insights into the role such defects may play in the pathophysiology of mitochondrìal disorders.
It has been a privilege to follow the development of the field of mitochondríal medicine since its infancy - and even before . I thank you for your attention.