1.  Introduction

Mitochondria are essential cell organelles in the cytoplasm which have a double-membrane. They are thought to have arisen about 1.5 billion years ago and to originate from a symbiotic association between oxidative bacteria and glycolytic proto-eukaryotic cells [Margulis, 1974]. “Modern” mitochondria retain a number of features that reflect their endosymbiotic origin. These include the double membrane structure and a bacteria-like circular mitochondrial genome with mitochondria-specific transcription, translation, and protein assembly systems [Margulis, 1974; Gray et al., 1999; Lopez et al., 2002].

Mitochondria are made up of two highly specialized membrane systems. These are the inner and the outer membranes. In the center of the mitochondrion and between the membranes there are two aqueous compartments: the matrix and the inter-membrane space [Frey et al., 2000]. The two membrane systems contain carrier proteins and channels that regulate the exchange of substrates between the compartments. The inner membrane is especially rich in proteins, e.g. the high molecular weight multi-protein-complexes of the respiratory chain are located at the inner mitochondrial membrane. The total number of different proteins or polypeptides making up a mitochondrion is estimated to be around 1000 [Lopez et al., 2002].

Mitochondria serve many important functions for the cell. These are the oxidative ATP-production, the degradation of fatty acids, the modulation of intracellular calcium homeostasis and a major role in cell signaling and apoptosis, as well as biosynthesis (e.g. heme-groups, nucleotides, and amino acids) and degradation (e.g. urea cycle) of metabolites [Lopez et al., 2002]. Below I describe the functions of the mitochondria shortly:

The oxidative phosphorylation takes place in the mitochondrion and is the main pathway of oxidative ATP-production in animals, plants and many forms of microbial life (e.g. yeast). One mole ATP hydrolyzes into one mole ADP and inorganic phosphate with concomitant release of 3054 Joules. This free energy can be made available to all cellular compartments that take up ATP. Most mammalian cells rely on the ATP produced this way for survival and anabolism [Grossman et al., 1996]. The respiratory chain-oxidative phosphorylation system consists of five multi-subunit enzyme complexes [Smeitink et al., 2001]. Mitochondrial complexes I, III and IV function as proton pumps to generate an electro-chemical gradient across the inner membrane. This proton gradient is then utilized by the ATP-synthase (complex V) to generate ATP from ADP and inorganic phosphate.

The carnitine-dependent transport of fatty acids and their β–oxidation is another important metabolic pathway located in the mitochondrion. Most of the fatty acids to be oxidized for energy production by intra-mitochondrial β-oxidation have to be transported from the cytosol into the mitochondrion. For transport, the fatty acids are first esterified with Coenzyme A (CoA) for “activation”, and are then coupled to carnitine to transverse the mitochondrial dou[page 2↓]ble membrane. All enzymes of the β-oxidation are mitochondrial enzymes [Stryer, 1995; Kerner et al., 2000]. Acetyl-CoA, NADH, and FADH₂, which are generated in each round of fatty acid oxidation, will later be channeled either into the citric acid cycle or directly into the respiratory chain to produce ATP.

The citric acid cycle, also named the “Krebs’ cycle” or “tricarboxylic acid cycle”, is located in the mitochondrion too. This is the final common pathway for different metabolites such as carbohydrates, fatty acids and amino acids. The details of this cycle are shown in Fig. 1-1. The compounds with a high redox-potential [reduced nicotinamide-adenine-dinucleotide (NADH) and reduced flavin-adenine-dinucleotide (FADH₂)], which are generated in this cycle, are later delivered to the respiratory chain of the mitochondrion in order to generate ATP.

The urea cycle has a role in the degradation of amino acids. It is partially located in the mitochondria of liver cells. In this pathway ammonia is detoxified, which is a by-product of amino acid catabolism. The cycle comprises four reactions and enzyme systems. The first reaction, the formation of citrulline from ammonia and ornithine, takes place in the matrix of the mitochondrion. Citrulline is then exported from the mitochondrion to the cytosol, where the other steps of the urea cycle take place [Krebs et al., 1932; Katunuma et al., 1966]. The details of this cycle are shown in Fig. 1-2.

Heme, which is needed as a prosthetic group in several important proteins such as hemoglobin, myoglobin and cytochrome C, is partly synthesized in the mitochondrion. The condensation of succinyl-CoA and glycine to δ-aminolevulic acid is the key-step of the heme-synthesis and takes place in the mitochondrion. δ-Aminolevulic acid is then delivered into the cytosol where coproporphyrinogen III is formed after a series of reactions. This molecule later returns into the mitochondrion to be converted into heme. The details of this process are depicted in Fig. 1-3.

In recent years mitochondria have been discovered to be able to initiate apoptosis by the release of several mediators like cytochrome c and apoptosis-inducing factor. These mediators activate the caspase family proteases which result in apoptosis [Osiewacz, 1997; Green et al., 1998].

Beyond that there are still other biochemical pathways located in the mitochondrion such as pathways for iron metabolism and for calcium signaling. Recent findings also indicate that mitochondria appear to be responsible for functional age-related impairments of human tissues and organs [Osiewacz, 2002] and may influence cellular mechanisms and pathways located in the cytosol such as insulin secretion [Green et al., 1998].

Each mitochondrion contains up to 10 copies of mitochondrial DNA (mtDNA). The mtDNA, which was completely sequenced in 1981 [Anderson et al., 1981], is a 16.56 kbp circular and double-stranded molecule. It encodes 13 polypeptides, 12S and 16S rRNA and 22 transfer-RNAs. All of these products are essential for the formation of a functional mitochondrion. All 13 polypeptides encoded by the mtDNA are components of the respiratory chain complexes. However, the total number of polypeptide subunits of all five mitochondrial respiratory complexes exceeds 88 [Lestienne, 1992; “Neuromuscular Disease Center” (see list of internet sites)]. Four of five enzyme complexes of the respiratory chain-oxidative phosphorylation system are encoded by both the nuclear DNAand the mtDNA. Only complex II (succinate: ubiquinone oxidoreductase; SDH) is made up exclusively of four nuclear encoded polypeptides. Seven of the 43 subunits of complex I (NADH: ubiquinone oxidoreductase), one of the eleven subunits of complex III (ubiquinol: cytochrome c oxidoreductase), three subunits of 13 subunits of complex IV (cytochrome c oxidase; COX), and two membrane components of complex V (adenosine triphosphate (ATP) synthase) are encoded by the mtDNA [Pesole et al., 2000].

The genetics of vertebrate mtDNA is characterized by these unique features:

• Maternal inheritance : This means that only the mtDNA of the oocyte can be transmitted to the offspring [Giles et al ., 1980]. With very rare exceptions the sperm mtDNA does not contribute to the fetus [Gustafson et al. , 2002].
• Heteroplasmy and threshold effect : The term heteroplasmy means that two populations of mtDNA - the wild type and the mutation type - coexist in an individual, in an organ or even in a single cell. Since deleterious mtDNA-mutations usually affect only parts of the mtDNA copies, the disease phenotype will only be expressed if the number of the mutant gene copies surpasses a certain threshold.
• High mutation rate : mtDNA is thought to be vulnerable due to its compact structure, its lack of histone protection, its insufficient repair mechanisms and its exposure to reactive oxygen species generated along the respiratory chain. This vulnerability results in a high mutation rate, about 10-20 fold higher than that of the nuclear DNA [Osiewacz, 1997; Zeviani et al., 1998; DiMauro, 2000].

Therefore, in inherited mitochondrial diseases the genetic defect might reside in the mitochondrial DNA or in the nuclear DNA. For example, in the former case the inheritance pattern [page 5↓]is maternal, while it might be autosomal or X-chromosomal recessive or autosomal dominant in the later case.

Traditionally, the term “mitochondrial disorders” describes defects in the energy-generating apparatus of the mitochondrion, i.e. the respiratory chain coupled to the oxidative phosphorylation [Bauer et al., 1999]. Mitochondrial disorders comprise a heterogeneous group of clinical phenotypes, which can result from mutations in the mtDNA, the nuclear DNA or both. Abnormalities of the electron transport and the oxidative phosphorylation system are probably the most common causes of mitochondrial disorders [Schapira et al., 1999]. However, mitochondrial diseases can also result from defects in metabolic pathways located only partially in the mitochondria (e.g. the pyruvate-dehydrogenase-complex deficiency). Mitochondrial disorders may manifest themselves at any time of life, from infancy to late adulthood. They may affect virtually any tissue either alone or in combination. Tissues with high energy-requirements such as heart, muscles, brain, kidney and endocrine organs are most commonly affected [Lopez, 2002].

The first mitochondrial disease that was understood at the molecular level was Leber’s hereditary optic neuropathy (LHON) with a mutation in a mtDNA encoded subunit of complex I [Wallace et al., 1988] and the Kearns-Sayre syndrome with a large deletion in the mtDNA [Holt et al., 1988]. The current classification of mitochondrial disorders is based on the kind and the location of the genetic defect (mtDNA versus nuclear DNA).

• Large-scale duplications or deletions of the mtDNA : three main clinical syndromes are associated with large-scale rearrangements of the mtDNA. They are :
  
  Kearns-Sayre syndrome (OMIM 530000): this is a mitochondrial encephalomyopathy defined by the triad of progressive external ophthalmoplegia (PEO), pigmentary retinopathy and conduction block of the heart plus either the increase of cerebral spinal fluid protein (above 100 mg/dl) or cerebellar ataxia.
  CPEO : this is a syndrome with chronic progressive external ophthalmoplegia that manifests itself mostly in adult patients. Since the etiology is not homogenous, several gene defects could lead to CPEO, such as mtDNA-deletions (OMIM 157640) or mutations in nuclear genes [POLG (OMIM 174763) and ANT1 (OMIM 103220 )].
  Pearson syndrome (OMIM 557000): also termed Pearson’s bone marrow-pancreas syndrome, it is a rare disorder of early infancy. It is characterized by sideroblastic anemia with pancytopenia and exocrine pancreatic insufficiency.
  
  
  Additionally, large-scale rearrangements of mtDNA were occasionally reported in patients with hypoparathyroidism, growth hormone deficiency and infertility [Folgero et al ., 1993; Wilichowski et al. , 1997; Boles et al . 1998]. Somatic mtDNA deletions have also been detected in various tumors [Polyak et al., 1998; Leonard et al., 2000a]. [page 6↓]
• Point mutations of the mtDNA : can be subdivided into missense mutations that affect (A) the rRNA or tRNA-genes and that (B) one of the 13 protein-encoding genes .
  
  A) tRNA- and rRNA-mutations have a global effect on mitochondrial protein synthesis. Until now, approximately 69 different mutations in 18 out of the 22 tRNA-genes of the mtDNA have been reported [MITOMAP]. Some of the mutations are associated with neurological syndromes such as the mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (=MELAS syndrome; caused by a tRNA^Leu(UUR) mutation; OMIM 540000) and myoclonic epilepsy with ragged-red fibres (=MERRF syndrome; caused by a tRNA^Lys mutation; OMIM 545000). Patients with mtDNA-mutations have a wide phenotypic variability. The “classic” 3243A>G point mutation in the mitochondrial encoded tRNA^Leu(UUR) , which is known mainly as the “MELAS-mutation” might also cause other clinical symptoms such as cardiomyopathy, CPEO, myoclonic epilepsy and maternally inherited diabetes with deafness. On the other hand, the MELAS syndrome can also be caused by several other nucleotide exchanges within mitochondria encoded tRNA-genes, such as mutations at the mtDNA-nucleotides 3252, 3256, 3271, and 3291 [MITOMAP].
  
  B) Point-mutations of the mtDNA that affect genes which encode polypeptides. These mutations may cause:
  
  Leber’s hereditary optic neuropathy (LHON ; OMIM 535000): it is characterized by bilateral, acute or sub-acute loss of central vision due to optic atrophy. A total of 17 mtDNA-mutations is known to be associated with LHON [Wallace et al., 1999]. However, the primary LHON mutations affect subunits of complex I.
  Neuropathy, ataxia, and retinitis pigmentosa (NARP ; OMIM 551500): this maternally inherited, adult-onset syndrome is caused by a point mutation at position 8993 in the mtDNA-encoded ATP synthase 6 subunit gene.

The second group of mitochondrial disorders is due to mutations in nuclear genes. These mutations may affect structural subunits of the respiratory chain, their assembly, the replication of the mtDNA and the transport of polypeptides through the mitochondrial double membrane [Zeviani et al., 1999; Leonard et al., 2000b; Sue et al., 2000; Orth et al., 2001]. These gene defects can be grouped as follows:

• Mutations in structural genes: The most common mitochondrial disorder of this group is Leigh syndrome , i.e. infantile sub-acute necrotizing encephalomyelopathy that is thought to be caused by a severe failure of energy production in the developing brain. Several different defects of mitochondrial enzyme complexes including pyruvate dehydrogenase complex (PDHc) and respiratory chain complexes I, II, IV, and V can lead to Leigh syndrome. Other diseases such as hereditary spastic paraplegia (progressive weakness and spasticity of the lower limbs; OMIM 602783) with mutations in the PARAPLEGIN -gene also fall into this group.
• Mutation in assembly genes: SURF1, SCO1 and2 and COX10 are assembly proteins of complex IV [Tiranti et al ., 1998; Petruzzella et al ., 1998; Papadopoulou et al ., 1999]. Mutations in these genes can lead to Leigh syndrome and in some cases to hypertrophic cardiomyopathy .
• Mutations in genes involved in mitochondrial nucleotide metabolism: Mutations in these genes (ANT1, TP, TWINKLE) disturb the mtDNA-replication leading to multiple deletions [page 7↓]of the mtDNA. Such patients suffer from a mitochondrial neurogastrointestinal encephalomyopathy (MNGIE; OMIM 603041) or the autosomal dominant progressive external ophthalmoplegia (adPEO; OMIM 103220).
• Mutations in genes involved in mitochondrial iron hemostasis: Friedreich’s ataxia , an autosomal recessive disease with cerebral ataxia, peripheral neuropathy and hypertrophic cardiomyopathy (OMIM 229300) is due to the deficiency of frataxin, which is a mitochondrial protein functioning in the iron-metabolism. Additionally the X-linked sideroblastic anaemia with ataxia (OMIM 301310) that is caused by the defects of the ABC7 -gene (ATP-binding cassette, transporter 7) also belongs to this group.
• Mutations in transmembrane transport proteins: The only known disease of this type is the X-linked deafness-dystonia (Mohr-Tranebjaerg) syndrome that is caused by a mutation of a mitochondrial protein (TIM8) that functions as a transporter for peptides through the mitochondrial double membrane.

The diagnosis of mitochondrial disorders has to rely on the sum of clinical, morphological, biochemical, and molecular genetic investigations since there is no explicit relation between genotype and phenotype. Atypical clinical pictures can be observed quite frequently in mitochondrial disorders. With the exception of typical syndromes like MELAS or MERRF, histological studies of muscle biopsy specimens are usually recommended in suspected cases. Characteristic changes include the presence of paracrystalline mitochondrial inclusions, mitochondria with abnormal size and shape, ragged-red-fibres (RRFs) in muscle, fat deposits and histochemically focal enzyme deficiencies (e.g. patchy COX-deficiency or SDH-deficiency) [Zeviani et al., 1998; Parker, 2000]. In most cases, however, biochemical analysis have to be performed in order to formulate a diagnosis [Letellier et al., 2000]. Using enzymatic tests, the activities of pyruvate dehydrogenase complex (PDHc), carnitine-palmitoyl-transferase and all complexes of the respiratory chain-oxidative phosphorylation system can be determined in muscle homogenate. Single enzyme activities can also be measured in cultured fibroblasts and in blood cells (lymphocytes and platelets). But only the molecular genetic analysis can verify the diagnosis of a mitochondrial disorder. In the case of a maternal inheritance pattern the investigations will focus on the analysis of the mtDNA. Otherwise, the biochemical results may narrow possible candidate genes to screen for mutations. For example, in the case of an isolated complex I deficiency, one would at first sequence the structural subunits of complex I in which mutations have been described before.

It is estimated that the mitochondrial proteome consists of approximately 1000 distinct proteins [Lopez et al., 2002]. With the exception of 13 proteins, which are encoded by the mtDNA, most mitochondrial proteins are encoded by nuclear genes, including most of the mitochondrial OXPHOS proteins, the metabolic enzymes, the DNA and RNA polymerases, the ribosomal proteins, and the mtDNA regulatory factors [Grivell et al., 1988; Wallace, 1999]. These proteins are synthesized at the encoplasmatic reticulum and are later imported into the mitochondrion. Fig. 1-4depicts this principle of the transportation of the preproteins through the double membrane. Before being transported into the mitochondrion, proteins are synthesized as preproteins, i.e. precursors that contain transit sequences either as amino-terminal targeting pre-sequences, or as targeting and sorting information sequences within the mature proteins. The cytosolic preproteins are imported through the translocases of the outer membrane (TOM) when their targeting information is recognized by the receptors of TOM. They are then sorted either directly to the outer membrane, the inter-membrane space or to the [page 8↓]translocases of the inner-membrane (TIM). Preproteins with a typical amino-terminal targeting sequence engage the TIM17/TIM23 complex that guides preproteins into the matrix. In the matrix the targeting sequencesare removed by the matrix-processing-protease, and the remaining polypeptide chains are folded by chaperones into mature proteins. Preproteins, which lack a targeting sequence, engage with the TIM22 complex to be inserted into the inner membrane [Millar et al., 1994; Shore et al ., 1995; Hanson et al., 1996; Koehler, 2000].

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