Research topics and interests
Disorders of mitochondrial energy provision belong to the most frequent and serious metabolic diseases for which no treatment is available. Genetic defects of mitochondrial oxidative phosphorylation constitute a large and heterogeneous group of mitochondrial diseases which predominantly affect tissues with high energy demands, ranging from severe encephalomyopathies of early infancy and childhood to a variety of senescence degenerative diseases. Etiopathogenetic mechanisms include "energetic insufficiency" as well as activated production of reactive oxygen species by mitochondrial respiratory chain which trigger apoptoic processes. As the mitochondrial bioenergetic machinery is the product of two genomes, the underlying molecular defects can be caused by mutations either in mitochondrial or in nuclear DNA. The main scientific interest of the Department are the molecular-genetic basis of mammalian bioenergetics and etiopathogenesis of human mitochondrial diseases. Specifically we are interested in biogenesis and function of energy-converting enzymes and transport proteins of the inner mitochondrial membrane that are involved in ATP and heat production.

Previous studies and scientific partners
Previous research performed by the Department since 1990 has been focused on control mechanisms and factors (catecholamines, thyroid hormones, cytokines) involved in the regulation at transcriptional and translational levels of the synthesis of the inner mitochondrial membrane proteins (ATP synthase, UCP1, cytochrome c oxidase) during cell differentiation, ontogenetic development and adaptive processes. In these studies thermogenic brown adipose tissue and derived cell cultures often served as advantageous experimental models, for the high dynamics of mitochondrial biogenesis, unique tissue specificity and high responsiveness to various hormones and regulatory factors. The following main results have been obtained.

The stimulatory effect of thyroid hormone on rodent brown adipose tissue has been shown to depend on the presence of active type II thyroxine 5-'deiodinase in brown adipose tissue. We found that biosynthesis of thyroxine 5'-deiodinase can be highly stimulated by catecholamines via ß3-adrenergic receptors. Studies in collaboration with the Institute for Mother and Child in Prague further demonstrated that the same enzyme is present in human brown adipose tissue where its activity develops during the last trimester of gestation, in parallel with the amount of UCP1, the key component of brown adipose tissue thermogenesis.

Attempts to investigate the potential involvement of brown adipose tissue in pyrogenic responses showed high expression of IL-l and IL-6 genes for pyrogenic cytokines in brown fat, which can be activated by LPS, pyrogenic cytokines, as well as by catecholamines. Multifactorial regulation of IL-1 and IL-6 production as well as the observed nuclear localisation of IL1 in differentiated adipocytes indicated a potential role of brown adipose tissue in attenuation of pyrogenic signals during febrile states.
Probably the most important finding of this period was related to the control of mitochondrial biogenesis. The regulation of ATPase biosynthesis has been shown to be completely independent from the regulation of other oxidative phosphorylation enzymes and we have further demonstrated that transcriptional control of only one of 16 subunits constituting the enzyme, the protonophoric subunit c can regulate the amount of ATPase during ontogeny and differentiation of brown adipose tissue. The selective control included two P1 and P2 isogenes encoding subunit c and their expression correlated also with the specific content of ATPase in other mammalian tissues, indicating a general importance of this mechanism. The main partner in these studies was the Oncology Institute, SAV, Bratislava (the late S. Kužela) and the Wenner-Gren Institute, Stockholm University (B. Cannon and U. Andersson).

Aims and strategy of research since 1995
During the last years our interest became much more oriented toward the mechanisms of mitochondrial biogenesis and biosynthesis of oxidative phosphorylation complexes with major focus on pathological defects of mitochondrial energy provision and human mitochondrial diseases. Mitochondrial oxidative phosphorylation defects are often caused by inherited or sporadic mutations of mitochondrial DNA but also by mutations in a large number of nuclear genes, many of which are still unknown and have to be identified. Human genetic defects thus represent unique models for understanding the biogenesis of energyconverting components of mammalian mitochondria.

In order to facilitate this type of research the Department has developed close and efficient collaboration with researchers and clinicians at the Paediatric Department and Institute for Inherited Metabolic Disorders at the 1st Medical Faculty, Charles University, and, as a result, a new diagnostic Laboratory for mitochondrial diseases was established. Within a few years and with the help of research funds mainly obtained from the Ministry of Health and the Grant Agency of the Czech Republic essential modern molecular genetic, biochemical and functional methods could be introduced and applied in specialised diagnostic protocols and several research projects on molecular etiopathogenesis of mitochondrial disorders, developmental changes of human mitochondrial enzymes and regulatory mechanisms and components essential for biosynthesis and of oxidative phosphorylation complexes. Part of these studies are related to possible application of the new knowledge to prenatal diagnostics and genetic counselling.
This arrangement makes it possible to cover all levels of investigation from gene to patient and studies performed are increasingly based on the involvement of pregraduate and postgradute students. Very important for recent studies has been collaboration with several foreign partners, including the University of Amsterdam (L.Nijtmans, C. Van den Bogert), LKA Salzburg (W. Sperl), University of Innsbruck (E. Gnaiger), Royal Free and University College Medical School, London (J.W. Taanman), University of Lyon (C. Godinot) and McGill University, Montreal (E. Shoubridge). Since 2000, the Department as well as the partner laboratories at the 1st Medical Faculty became associated with the Center for Integrated Genomics.

Recent results
1. Heteroplasmic T8993G mutation in the mtDNA gene for ATPase subunit a, which causes fatal encephalomyopathy (Leigh syndrome) has been shown to alter the biosynthesis and stability of the ATPase complex which forms incomplete assembly intermediates, resulting in a pronounced decrease of ATP synthesis. With the help of selective inhibition of mitochondrial protein synthesis by doxycyclin it was possible to simulate subunit a defect in vitro and show that subunit a enters the enzyme at the latest stage of assembly.
2. We have identified a new type of mitochondrial disease which is due to a selective defect of ATP synthase. The nuclear origin of the defect has been demonstrated with the aid of genetic complementation in mitochondrial cybrids. Methionine labelling experiments in fibroblasts and cybrids further showed that the low content of ATPase is caused by decreased biosynthesis of the enzyme due to impaired assembly of the ATPase complex.
3. A new cytofluorometric method using tetramethyl rhodamine methzl ester has been developed for sensitive analysis of mitochondrial membrane potential Dy in cultured cells and used for studies of functional manifestation of mtDNA 8334 mutation in tRNALys (MERF). Changes of Dy in cybrid clones harbouring different proportions of normal and mutated mtDNA revealed a nonlinear relationship between mtDNA heteroplasmy and energisation of the mitochondrial membrane, indicating that 25 % of normal mtDNA is sufficient to rescue the defect.
4. A complex approach to prenatal diagnostics of cytochrome c oxidase defects has been developed and used for prenatal diagnosis in two families. The approach is based on identification of the genetic origin of the defect by mitochondrial cybrids and combination of functional analysis of cultured amniocytes (activity of oxidative phosphorylation enzymes, ATP synthesis), with quantification of oxidative phosphorylation antigens (BN and 2D PAGE, WB analysis).
5. Two new mammalian cytosolic proteins called BARB1 and BAR2 (130 and 80 kDa) have been identified that selectively interact with 3´untranslated region of b-F1ATPase mRNA. BARB1 is a novel poly(A)binding protein and it achieves its mRNA selectivity through interaction with BARB2, that binds through a 22bp element with a uridylate core. The complex between these two proteins may be involved in posttranscriptional regulation of gene expression.

Focus of future studies
1. Mutations of mtDNA and nuclear genes encoding subunits of ATP synthase and respiratory chain enzymes  the role of nuclear background in transmission and segregation of mtDNA mutations
2. Complementation of mitochondrial defects of nuclear origin by monochromosomal transfer.
3. The role of glycerophosphate dehydrogenase in mitochondrial production of reactive oxygen species.
4. Candidate genes and putative assembly factors for human oxidative phosphorylation proteins.
5. Posttranscriptional events in biosynthesis of oxidative phosphorylation enzymes.

Publications