**2. Genetics and pathology of OXPHOS**

## **2.1 Genetics of OXPHOS system**

The OXPHOS system is composed of more than 80 different proteins, 13 of which are encoded by the mitochondrial DNA (mtDNA) and the others by the nuclear genome (nDNA) (Chinnery & Turnbull, 2001; Wallace, 1992). There are seven mtDNA-encoded subunits in complex I, one in complex III, three in complex IV and two in complex V. Complex II consists solely of nDNA-encoded subunits.

The structure of human mitochondrial DNA (mtDNA) was reported ≈ 30 years ago (Clayton & Vinograd, 1967). The transcription products of mtDNA include 2 ribosomal RNA species (12S and 16S rRNA), 13 messenger RNAs and 22 transfer RNAs. Replication of mtDNA occurs independently from cell cycle phase and from replication of nuclear DNA. Mitochondrial DNA is present in 103–104 identical copies in each cell, with the exception of sperm and mature oocytes, in which mtDNA copy numbers are ≈ 102 and ≈ 105, respectively. In general, there are believed to be two to ten copies of DNA per mitochondrion. The sequences of mtDNAs from unrelated individuals in human populations typically differ by about 0.3 %. Most individuals, however, have a single mtDNA sequence variant in all their cells (homoplasmy). mtDNA transmission occurred exclusively through the maternal lineage.

Almost all of the nDNA-encoded OXPHOS subunits have been characterized at the cDNA level and several at the genomic level in humans. In general, the chromosomal distribution of the genes seems to be random, and expression of most gene products is ubiquitous but predominates in tissues or organs with a high energy demand.

Richard Scarpulla and co-workers have provided important insight into the regulatory mechanisms that are involved in the transcriptional control of OXPHOS genes (Gugneja et al., 1996; Huo & Scarpulla, 1999; Wu et al., 1999). They identified the nuclear respiratory factors NRF1 and NRF2, which act on overlapping subsets of nuclear genes that are involved in the biogenesis of the respiratory chain. Recent mammalian studies have identified PGC1 as a crucial regulator of cardiac mitochondrial number and function in response to energy demand (Lehman et al., 2000). Analysis of the expression pattern of OXPHOS genes revealed that their regulation might also be exerted post-transcriptionally (Di Liegro et al., 2000).

#### **2.2 OXPHOS diseases**

30 Bioenergetics

matrix through complex V, ADP and Pi are bound, condensed and released as ATP. With Complex V, the ETC complexes constitute the OXPHOS system. The OXPHOS system generates the vast majority of cellular ATP during oxidative metabolism. Some of the ATP is used for the mitochondrion's own needs, but most of it is transported outside the organelle by the adenine nucleotide translocator (ANT) and used for diverse cell functions (Hatefi,

Fig. 1. Schematic view of of mitochondrial OXPHOS system (A), and. mitochondrial Ca2+ influx and efflux mechanisms (B). Large size arrows indicate the interplay between

The OXPHOS system is composed of more than 80 different proteins, 13 of which are encoded by the mitochondrial DNA (mtDNA) and the others by the nuclear genome (nDNA) (Chinnery & Turnbull, 2001; Wallace, 1992). There are seven mtDNA-encoded subunits in complex I, one in complex III, three in complex IV and two in complex V.

The structure of human mitochondrial DNA (mtDNA) was reported ≈ 30 years ago (Clayton & Vinograd, 1967). The transcription products of mtDNA include 2 ribosomal RNA species (12S and 16S rRNA), 13 messenger RNAs and 22 transfer RNAs. Replication of mtDNA occurs independently from cell cycle phase and from replication of nuclear DNA. Mitochondrial DNA is present in 103–104 identical copies in each cell, with the exception of

1985; Saraste, 1999) (Figure 1 A).

mitochondrial Ca2+ signaling and OXPHOS.

**2. Genetics and pathology of OXPHOS** 

Complex II consists solely of nDNA-encoded subunits.

**2.1 Genetics of OXPHOS system** 

Among the inborn errors of metabolism, mitochondrial disorders are the most frequent with an estimated incidence of at least 1 in 10,000 births (reviewed in (Smeitink et al., 2001)). Although the term mitochondrial disorder is very broad, it usually refers to diseases that are caused by disturbances in the OXPHOS system. After the first description, ≈ 40 years ago, of a patient with "loose coupling" — a defect in the coupling between mitochondrial respiration and phosphorylation — by Luft and collaborators (Luft et al., 1962), thousands of patients have been diagnosed by measurement of OXPHOS-system enzyme activities. The great complexity of the OXPHOS system, which consists of proteins, some encoded by the mitochondrial genome and others by the nuclear genome, may explain the wide variety of clinical phenotypes that are associated with genetic defects in oxidative phosphorylation. Disease-causing defects can occur in a single OXPHOS complex (isolated deficiency) or multiple complexes at the same time (combined deficiency). OXPHOS diseases give rise to a variety of clinical manifestations, particularly in organs and tissues with high-energy demand such as brain (encephalopathies), heart (cardiomyopathies), skeletal muscle (myopathies) and liver (hepatopathies) (reviewed in (Finsterer, 2006a, 2006b; Schaefer et al., 2004)).

We have also to consider the presence of fundamental differences between mitochondrial genetics and Mendelian genetics when studying human OXPHOS diseases. These differences are linked to maternal inheritance of mtDNA, polyplasmy, heteroplasmy and the threshold effect, whereby a critical number of mutated mtDNAs must be present for the OXPHOS system to malfunction (Wallace, 2005).

One of the frequent OXPHOS disorders is Leigh Syndrome (OMIM 256000), an early-onset progressive neurodegenerative disorder, leading to death mostly within a few years after the onset of the symptoms. This disorder is characterized by lesions of necrosis and capillary proliferation in variable regions of the central nervous system. Clinical signs and symptoms comprise muscular hypotonia, developmental delay, abnormal eye movements, seizures, respiratory irregularities and failure to thrive. Other mitochondrial disorders caused by

Mitochondrial Calcium Signalling: Role in Oxidative Phosphorylation Diseases 33

 Pathogenic mtDNA proteins synthesis mutations can also result in multisystem disorders with wide range of symptoms. The most common mtDNA protein synthesis mutation is A3243G in the tRNA(Leu). This mutation is linked to a variety of clinical symptoms. When present at relatively low level (10%-30%) in the blood, the patient may manifest only type II diabetes. By contrast, when the mutation is present in > 70% of the mtDNA, it causes more severe symptoms including short stature, cardiomyopathy, Chronic Progressive External Ophthalmoplegia (CPEO; OMIM157640)

Nuclear DNA mutations linked to OXPHOS diseases includes defects in structural OXPHOS genes, faulty inter-genomic communication, and defects in OXPHOS assembly, homeostasis and import. Most nuclear gene mutations affect various protein subunits of complex I and

The first structural OXPHOS-gene mutation was reported in two sisters with Leigh syndrome and isolated complex II deficiency (Bourgeron et al., 1995). The pathogenic mutation was in the gene that encodes the flavoprotein: SDHA (succinate dehydrogenase subunit A). Subsequently, another family was found to have mutations in this subunit (Parfait et al., 2000). Very interestingly, two groups independently reported mutations of the complex II subunit D and C genes in hereditary paraganglioma — usually benign, vascularized tumours in the head and in the neck (Baysal et al., 2000; Niemann & Muller, 2000). This work has uncovered a new and surprising association between mitochondrial defects and carcinogenesis. Genetic characterization of Complex I deficiency in a patient with a Leigh-like presentation revealed a 5-base-pair (bp) duplication in NDUFS4 (NADH dehydrogenase (ubiquinone) Fe–S protein 4) that destroys the consensus phosphorylation site in the gene product and extends the length of the protein by 14 amino acids (van den Heuvel et al., 1998). Further studies have revealed that this duplication abolishes cyclic-AMP-dependent phosphorylation of NDUFS4, thereby impairing activation of the complex. Further complex I mutations have been identified and ≈ 40% of complex I deficiencies in children, in which the defect is detected in cultured skin fibroblasts, can now be explained

OXPHOS defects caused by defective interplay between the mitochondrial and nuclear genomes have also been described. The clinical features of the Mitochondrial Neuro-GastroIntestinal Encephalomyopathy syndrome (MNGIE) include ophthalmoparesis, peripheral neuropathy, leucoencephalopathy and gastrointestinal symptoms (chronic diarrhea and intestinal dysmotility). Muscle biopsy shows ragged red fibers (RRFs) and COX-negative fibers and either partial isolated complex IV deficiency or combined OXPHOS-complex deficiencies (Hirano et al., 1994). Mitochondrial DNA analysis in this

encephalomyopathies (Bruno et al., 1999; Comi et al., 1998).

and MELAS (Goto et al., 1990; van den Ouweland et al., 1994).

**2.2.2 Nuclear DNA mutations linked to OXPHOS diseases** 

by mutations in structural nuclear genes (Loeffen et al., 1998, 2000).

complex II.

males. Most LHON patients harbor one of three point mutations that affect mtDNA complex I, or the NADH:ubiquinone oxidoreductase (ND) genes: G3460A in ND1, G11778A in ND4 and T14484C in ND6. Patrick Chinnery and colleagues showed that the mitochondrial ND6 gene is a hot spot for LHON mutations and suggested that the ND6 gene should be sequenced in all LHON patients who do not harbour one of the three common LHON mutations (Chinnery et al., 2001). Rare nonsense or frameshift mutants in Cytochrome oxydase subunit I (COI) have been associated with

OXPHOS defects include Mitochondrial Encephalopathy, Lactic Acidosis and Stroke-like episodes (MELAS; OMIM 540000), Myoclonic Epilepsy with Ragged Red Fibers (MERRF; OMIM 545000), Neurogenic weakness, Ataxia, Retinitis Pigmentosa/Maternally Inherited Leigh Syndrome (NARP/MILS; OMIM 516060), Leber's Hereditary Optic Neuropathy (LHON; OMIM 535000), and Mohr–Tranebjaerg syndrome (a.k.a. Deafness Dystonia Syndrome; OMIM 304700).

Because of the genetic complexity of the energy-generating system, many other diseases have been shown to be associated with defect in mitochondrial function (DiMauro & Moraes, 1993; DiMauro & Schon, 2003). For example, there is increasing evidence that inherited OXPHOS dysfunction is also implicated in diabetes, age-related neurodegenerative diseases, such as Parkinson's, Alzheimer and Huntington's diseases, and various forms of cancers (Shoubridge, 2001; Zeviani & Carelli, 2007).

### **2.2.1 Mitochondrial DNA mutations linked to OXPHOS diseases**

The complexity of mitochondrial DNA mutations linked to OXPHOS diseases is that one mutation can cause a broad spectrum of clinical manifestations. Conversely, different mutations can be associated with the same clinical phenotype. Specific phenotypes include forms of blindness, deafness, movement disorders, dementia, cardiovascular diseases, muscle weakness, renal dysfunction, and endocrine disorders including diabetes. In the past 20 years, more than 100 point mutations and innumerable rearrangements have been associated with human mitochondrial diseases. In this context, it is worth mentioning, however, that we still lack comprehensive and unbiased epidemiological data about the frequency of known mtDNA mutations. Although tRNA genes as a whole represent ≈ 10% of the mtDNA, mutations in these genes account for ≈ 75% of mtDNA-related diseases.

We can identify three categories of pathogenic mtDNA mutations: rearrangement mutations, polypeptide gene missense mutations, and protein synthesis (rRNA and tRNA) gene mutations (reviewed in (Wallace, 2005)).


OXPHOS defects include Mitochondrial Encephalopathy, Lactic Acidosis and Stroke-like episodes (MELAS; OMIM 540000), Myoclonic Epilepsy with Ragged Red Fibers (MERRF; OMIM 545000), Neurogenic weakness, Ataxia, Retinitis Pigmentosa/Maternally Inherited Leigh Syndrome (NARP/MILS; OMIM 516060), Leber's Hereditary Optic Neuropathy (LHON; OMIM 535000), and Mohr–Tranebjaerg syndrome (a.k.a. Deafness Dystonia

Because of the genetic complexity of the energy-generating system, many other diseases have been shown to be associated with defect in mitochondrial function (DiMauro & Moraes, 1993; DiMauro & Schon, 2003). For example, there is increasing evidence that inherited OXPHOS dysfunction is also implicated in diabetes, age-related neurodegenerative diseases, such as Parkinson's, Alzheimer and Huntington's diseases, and

The complexity of mitochondrial DNA mutations linked to OXPHOS diseases is that one mutation can cause a broad spectrum of clinical manifestations. Conversely, different mutations can be associated with the same clinical phenotype. Specific phenotypes include forms of blindness, deafness, movement disorders, dementia, cardiovascular diseases, muscle weakness, renal dysfunction, and endocrine disorders including diabetes. In the past 20 years, more than 100 point mutations and innumerable rearrangements have been associated with human mitochondrial diseases. In this context, it is worth mentioning, however, that we still lack comprehensive and unbiased epidemiological data about the frequency of known mtDNA mutations. Although tRNA genes as a whole represent ≈ 10% of the mtDNA, mutations in these genes account for ≈ 75% of mtDNA-related diseases. We can identify three categories of pathogenic mtDNA mutations: rearrangement mutations, polypeptide gene missense mutations, and protein synthesis (rRNA and tRNA)

 Rearrangement mutations of mtDNA can be either inherited or spontaneous. Inherited mtDNA rearrangements are primarily insertions. The first inherited insertion mutation to be identified caused maternally inherited diabetes and deafness (Ballinger et al., 1992, 1994). Spontaneous mtDNA deletions result in a related spectrum of symptoms, irrespective of the position of the deletion end points. This is because virtually all deletions remove at least one tRNA and thus inhibit protein synthesis (Moraes et al., 1989). Thus the nature and severity of the mtDNA deletion rearrangement is not a consequence of the nature of the rearrangement, but rather of the tissue distribution of

 Missense mutations in mtDNA polypeptide genes can also result in an array of clinical manifestations. Three relatively frequently observed point mutations are A3243G in the tRNA(Leu)(UUR) gene, A8344G in the tRNA(Lys) gene and T8993G in the ATPase 6 gene and are associated with NARP when present at lower percentage of mutants or with lethal Leigh syndrome when present at higher percentage of mutants (Holt et al., 1990; Tatuch et al., 1992). Mutations have also been identified in mtDNA genes that encode proteins of the OXPHOS system, such as the cytochrome b gene and the mitochondrial complex I genes. A prominent example of the latter group of mtDNA protein-coding gene mutations is LHON, which is a common cause of subacute bilateral optic neuropathy that usually presents in early adult life and that predominantly affects

various forms of cancers (Shoubridge, 2001; Zeviani & Carelli, 2007).

**2.2.1 Mitochondrial DNA mutations linked to OXPHOS diseases** 

gene mutations (reviewed in (Wallace, 2005)).

the rearranged mtDNAs.

Syndrome; OMIM 304700).

males. Most LHON patients harbor one of three point mutations that affect mtDNA complex I, or the NADH:ubiquinone oxidoreductase (ND) genes: G3460A in ND1, G11778A in ND4 and T14484C in ND6. Patrick Chinnery and colleagues showed that the mitochondrial ND6 gene is a hot spot for LHON mutations and suggested that the ND6 gene should be sequenced in all LHON patients who do not harbour one of the three common LHON mutations (Chinnery et al., 2001). Rare nonsense or frameshift mutants in Cytochrome oxydase subunit I (COI) have been associated with encephalomyopathies (Bruno et al., 1999; Comi et al., 1998).

 Pathogenic mtDNA proteins synthesis mutations can also result in multisystem disorders with wide range of symptoms. The most common mtDNA protein synthesis mutation is A3243G in the tRNA(Leu). This mutation is linked to a variety of clinical symptoms. When present at relatively low level (10%-30%) in the blood, the patient may manifest only type II diabetes. By contrast, when the mutation is present in > 70% of the mtDNA, it causes more severe symptoms including short stature, cardiomyopathy, Chronic Progressive External Ophthalmoplegia (CPEO; OMIM157640) and MELAS (Goto et al., 1990; van den Ouweland et al., 1994).

#### **2.2.2 Nuclear DNA mutations linked to OXPHOS diseases**

Nuclear DNA mutations linked to OXPHOS diseases includes defects in structural OXPHOS genes, faulty inter-genomic communication, and defects in OXPHOS assembly, homeostasis and import. Most nuclear gene mutations affect various protein subunits of complex I and complex II.

The first structural OXPHOS-gene mutation was reported in two sisters with Leigh syndrome and isolated complex II deficiency (Bourgeron et al., 1995). The pathogenic mutation was in the gene that encodes the flavoprotein: SDHA (succinate dehydrogenase subunit A). Subsequently, another family was found to have mutations in this subunit (Parfait et al., 2000). Very interestingly, two groups independently reported mutations of the complex II subunit D and C genes in hereditary paraganglioma — usually benign, vascularized tumours in the head and in the neck (Baysal et al., 2000; Niemann & Muller, 2000). This work has uncovered a new and surprising association between mitochondrial defects and carcinogenesis. Genetic characterization of Complex I deficiency in a patient with a Leigh-like presentation revealed a 5-base-pair (bp) duplication in NDUFS4 (NADH dehydrogenase (ubiquinone) Fe–S protein 4) that destroys the consensus phosphorylation site in the gene product and extends the length of the protein by 14 amino acids (van den Heuvel et al., 1998). Further studies have revealed that this duplication abolishes cyclic-AMP-dependent phosphorylation of NDUFS4, thereby impairing activation of the complex. Further complex I mutations have been identified and ≈ 40% of complex I deficiencies in children, in which the defect is detected in cultured skin fibroblasts, can now be explained by mutations in structural nuclear genes (Loeffen et al., 1998, 2000).

OXPHOS defects caused by defective interplay between the mitochondrial and nuclear genomes have also been described. The clinical features of the Mitochondrial Neuro-GastroIntestinal Encephalomyopathy syndrome (MNGIE) include ophthalmoparesis, peripheral neuropathy, leucoencephalopathy and gastrointestinal symptoms (chronic diarrhea and intestinal dysmotility). Muscle biopsy shows ragged red fibers (RRFs) and COX-negative fibers and either partial isolated complex IV deficiency or combined OXPHOS-complex deficiencies (Hirano et al., 1994). Mitochondrial DNA analysis in this

Mitochondrial Calcium Signalling: Role in Oxidative Phosphorylation Diseases 35

proteins that are involved in intermembrane protein transport in mitochondria. Therefore, the DDP syndrome should be considered as the first example of a new group of mitochondrial import diseases (Koehler et al., 1999). Finally, OPA1 is caused by defects in a dynamin-related protein that is targeted to mitochondria and might exert its function in mitochondrial biogenesis and in stabilization of mitochondrial membrane complexes

Cybrids, or "cytoplasmic hybrids," are cultured cells manipulated to contain introduced mitochondrial DNA (mtDNA). Cybrids have been a central tool to unravel effects of mtDNA mutations in OXPHOS diseases. In this way, the nuclear genetic complement is held constant so that observed effects on OXPHOS can be linked to the introduced mtDNA. The cybrids are produced by first treating mitochondrial donor cells with cytochalasin B to weaken the cytoskeleton, before subjecting the cells to a centrifugal force, either as attached cells or in suspension. The dense nuclei are extruded, leaving plasma membrane-bound "cytoplasts" containing cell cytoplasm and organelles, including mitochondria. These cytoplasts are then fused with a nuclear donor cell line. The first mammalian cultured cell phenotype identified to segregate with mtDNA was in human (HeLa) cells, where mtDNA imparted resistance to the antibiotic chloramphenicol (Spolsky & Eisenstadt, 1972). Several other mtDNA‐linked drug‐resistant phenotypes were identified in mammalian cells in the 1970s and 1980s, including resistance to the complex III inhibitors antimycin and myxothiazol (Howell & Gilbert, 1988) and to the complex I inhibitor rotenone (Bai & Attardi, 1998). The development of robust DNA‐sequencing methods leads to the identification of single‐base substitutions in the 16S rRNA gene of the mtDNA of independently derived yeast, mouse, and human chloramphenicol cell lines (Blanc & Dujon, 1980; Kearsey & Craig, 1981). These pioneering studies were in turn followed by identification of the first cytochrome b mutants

(Howell & Gilbert, 1988) and more recently ND5, ND6, and COI mutants.

The second cellular OXPHOS model corresponds to the isolation of a human cell line without mtDNA (called Rho0 cells). Employing an approach first used in yeast (Slonimski et al., 1968), cells were incubated with low levels of the drug ethidium bromide, which intercalates DNA. Low levels of the drug selectively inhibits the gamma‐DNA polymerase responsible for mtDNA replication, and with ongoing cell division, the mtDNAs are ''diluted'' to the point where clones can be isolated without detectable organelle genomes. King and Attardi (King & Attardi, 1989) also discovered the absolute requirement for pyruvate gained by these cells and confirmed the previous observation from Paul Desjardins and collaborators (Desjardins et al., 1985) that mtDNA‐less cells also required added uridine for growth. This allowed a selection regime to be used after cytoplast– Rho0 cell fusion so that unfused Rho0 cells could be eliminated and cybrids selected with the use of an appropriate nuclear drug‐resistant marker (King & Attardi, 1989). Apart from their value in cybrid experiments, such Rho0 cells represent a unique research tool by themselves. They are a surprising reminder that OXPHOS is dispensable, at least for some differentiated mammalian cell types. In this instance, ATP production is 100% from glycolysis, so the cells acidify culture media very rapidly by producing large quantities of lactate. They retain functional mitochondria (except lacking OXPHOS), which show a transmembrane potential

(Delettre et al., 2000).

**3.1 Cybrids and Rho<sup>0</sup>**

**3. Models to study OXPHOS diseases** 

 **cells** 

autosomal recessive syndrome showed mtDNA deletions, depletion, or both. The MNGIE locus was mapped to chromosome 22q13.32-qter, a region that contains the thymidine phosphorylase (TP) gene (gene symbol ECGF1). Studies on patients showed that TP activity was markedly decreased. Ichizo Nishino and collaborators found various homozygous as well as compound heterozygous ECGF1 mutations in the genomic DNA of MNGIE patients (Nishino et al., 1999). The precise mechanism by which TP deficiency leads to mtDNA rearrangements have still to be explained, but imbalance of the mitochondrial nucleotide pool is likely to have a role. Autosomal dominant Progressive External Ophtalmogia (adPEO) is an adult-onset mitochondrial disorder that is characterized by progressive external ophthalmoplegia and variable additional features, including exercise intolerance, ataxia, depression, hypogonadism, hearing deficit, peripheral neuropathy and cataract (Zeviani et al., 1990). Some patients carry mtDNA deletions, although the disease is inherited in an autosomal fashion. Of the two autosomal loci for this disorder, the 4q-adPEO locus includes the gene for the heart and skeletal muscle isoform of the ANT1. Kaukonen and collaborators (Kaukonen et al., 2000) identified two heterozygous missense mutations in this gene in several families and in one sporadic patient with adPEO.

Enzyme complex I and IV deficiencies are by far the most frequently observed abnormalities of the OXPHOS system. In sharp contrast to isolated complex I deficiencies, no mutations have been found as yet in the ten nuclear genes that encode the structural proteins of complex IV (Adams et al., 1997). The discovery of mutations in a nuclear assembly gene that is associated with COX deficiency resulted from chromosomal transfer experiments. This approach identified mutations in the SURF1 gene in patients with COX-deficient Leigh syndrome (Tiranti et al., 1998; Zhu et al., 1998). SURF1 is part of a cluster of unrelated housekeeping genes and is the only gene of this cluster that is known or believed to be involved in COX assembly (Tiranti et al., 1999). Nuclear gene defects that are associated with isolated complex III or complex V deficiencies have not yet been discovered. In recent years, four inherited neurodegenerative diseases, Friedreich ataxia, hereditary spastic paraplegia, human DDP syndrom (deafness/dystonia peptide) and dominant optic atrophy (OPA1) have also been shown to be mitochondrial disorders that are caused by nuclear DNA mutations in the genes for frataxin, paraplegin, DDP and OPA1, respectively. Mitochondria obtained from heart biopsies of Friedreich ataxia patients disclosed specific defects in the citric-acid cycle enzyme aconitase, and complex I–III activities (Rotig et al., 1997). The causative Friedreich ataxia protein, frataxin, has an essential role in mitochondrial iron homeostasis, and Friedreich ataxia can therefore be considered as an OXPHOS homeostasis defect. Muscle biopsies from the autosomal recessive form of patients with hereditary spastic paraplegia revealed histochemical signs of a mitochondrial disorder, namely RRFs, COX-negative fibers and succinate dehydrogenase-positive hyperintense fibers (Casari et al., 1998). Linkage and subsequent mutation analysis revealed large deletions in a gene dubbed paraplegin (Casari et al., 1998). Owing to the homology with a yeast mitochondrial ATPase with both proteolytic and chaperone-like activities, it has been suggested that this form of hereditary spastic paraplegia could be a neurodegenerative disorder due to OXPHOS deficiency, attributing a putative function in the assembly or import of respiratory chain subunits or cofactors to paraplegin (Di Donato, 2000). The DDP syndrome, an X-linked recessive disorder also known as the Mohr–Tranebjaerg syndrome, is associated with a novel defect in mitochondrial protein import (Koehler et al., 1999). The defective gene is homologous to the yeast protein Tim8, which belongs to a family of proteins that are involved in intermembrane protein transport in mitochondria. Therefore, the DDP syndrome should be considered as the first example of a new group of mitochondrial import diseases (Koehler et al., 1999). Finally, OPA1 is caused by defects in a dynamin-related protein that is targeted to mitochondria and might exert its function in mitochondrial biogenesis and in stabilization of mitochondrial membrane complexes (Delettre et al., 2000).
