Biogenesis of Mitochondrial Complex I in Health and Disease

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Biogenesis of mitochondrial complex I in health and disease

Biogenesis of mitochondrial complex I in health and disease Thesis Radboud University Nijmegen with a summary in Dutch

ISBN 978-90-9023316-1

Cover pictures by Felix Distelmaier: TMRM fluorescent mitochondria of patient fibroblasts Cover design by Rolf Janssen

Printed by PrintPartners Ipskamp, Enschede, The Netherlands

Biogenesis of mitochondrial complex I in health and disease

Een wetenschappelijke proeve op het gebied van de Medische Wetenschappen

Proefschrift

ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de rector magnificus prof. mr. S.C.J.J. Kortmann, volgens besluit van het College van Decanen in het openbaar te verdedigen op

donderdag 2 oktober 2008 om 13:30 uur precies

door

Rolf Jacques Roy Johan Janssen

geboren op 20 maart 1976 te Nijmegen Promotor: prof. dr. J.A.M. Smeitink

Copromotores: dr. L.G.J. Nijtmans dr. L.P.W.J. van den Heuvel

Manuscriptcommissie: prof. dr. B. Wieringa, voorzitter prof. dr. R.E. Brock prof. dr. R. de Groot prof. dr. G.J.M. Pruijn prof. dr. R.J.A. Wanders (AMC, Universiteit van Amsterdam)

The publication of this thesis was financially supported by the Department of Pediatrics of the Radboud University Nijmegen Medical Centre and by sigma-tau BV, Utrecht.

Table of Contents

List of abbreviations 7

Chapter I 11 General introduction – Aim, objectives and outline of the thesis

Chapter II 19 Genetic defects in the oxidative phosphorylation (OXPHOS) system – A review Adapted from: Expert Review of Molecular Diagnostics 2004, Volume 4, Number 2, Page 143-156

Chapter III 53 Mitochondrial complex I: structure, function and pathology – A review Journal of Inherited Metabolic Disease 2006, Volume 29, Number 4, Page 499-515

Chapter IV 89 CIA30 complex I assembly factor: a candidate for human complex I deficiency? Human Genetics 2002, Volume 110, Number 3, Page 264-270

Chapter V 105 Human mitochondrial complex I assembly is mediated by NDUFAF1 FEBS Journal 2005, Volume 272, Number 20, Page 5317-5326

Chapter VI 127 Differences in assembly or stability of complex I and other mitochondrial OXPHOS complexes in inherited complex I deficiency Human Molecular Genetics 2004, Volume 13, Number 6, Page 659-667

Chapter VII 149 Contiguous gene deletion of ELOVL7, ERCC8, and NDUFAF2 in a patient with progressive encephalomyopathy Submitted

Chapter VIII 177 General discussion – Conclusions and future perspectives

Summary 201

Samenvatting 205

Dankwoord 209

Curriculum vitae 211

List of publications 213

List of abbreviations

1D one-dimensional 2D two-dimensional 3D three-dimensional ACP acyl carrier protein AD Alzheimer disease ADP adenosine 5‘-diphosphate ad/arPEO autosomal dominant/autosomal recessive progressive external ophthalmoplegia ATP adenosine 5‘-triphosphate BN blue native bp base pair CI-V complex I-V cAMP 3‘,5‘-cyclic adenosine monophosphate cDNA complementary DNA CIA complex I intermediate associated protein CNS central nervous system CoA coenzyme A COX cytochrome c oxidase CPEO chronic progressive external ophthalmoplegia CS citrate synthase CSA/B Cockayne syndrome type A/B CSF cerebrospinal fluid cyt cytochrome DDM n-dodecyl- -D-maltoside dNTP deoxyribonucleoside triphosphate ECL enhanced chemiluminescence ELOVL elongation of very long-chain fatty acids EM electron microscopy EPR electron paramagnetic resonance ER endoplasmic reticulum ESI-MS electrospray ionization mass spectrometry EST expressed sequence tag + FADH2/FAD flavin adenine dinucleotide – reduced/oxidized FeS iron-sulfur FILA fatal infantile lactic acidosis FMN flavin mononucleotide GFP green fluorescent protein GG-NER global genome nucleotide excision repair

GRACILE growth retardation, aminoaciduria, cholestasis, iron overload, lactic acidosis and early death GTP guanosine 5‘-triphosphate HCEM hypertrophic cardiomyopathy and encephalomyopathy HD Huntington disease HEK human embryonic kidney HEt hydroethidine HPLC high-pressure liquid chromatography HSP hereditary spastic paraplegia IEF isoelectric focusing IGA in-gel activity IgG immunoglobulin G IVS intervening sequence (intron) kDa kilo Dalton KSS Kearns-Sayre syndrome LDAO lauryldimethylamine oxide (N,N-dimethyldodecylamine N-oxide) LHON Leber hereditary optic neuropathy LS Leigh syndrome MDS mtDNA depletion syndrome MELAS mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes MERRF myoclonus epilepsy and ragged-red fibers MILS maternally inherited Leigh syndrome MNGIE mitochondrial neurogastrointestinal encephalomyopathy mRNA messenger RNA mtDNA mitochondrial DNA MTE multiple tissue expression MTS mitochondrial targeting sequence MTS Mohr-Tranebjaerg syndrome NAD(P)H/NAD(P)+ nicotinamide adenine dinucleotide (phosphate) – reduced/oxidized NARP neuropathy, ataxia and retinitis pigmentosa ND NADH dehydrogenase nDNA nuclear DNA NDUFA- NADH dehydrogenase ubiquinone α-subcomplex protein NDUFAF- NADH dehydrogenase ubiquinone assembly factor NDUFB- NADH dehydrogenase ubiquinone β-subcomplex protein NDUFC- NADH dehydrogenase ubiquinone subcomplex unknown protein NDUFS- NADH dehydrogenase ubiquinone iron-sulfur protein NDUFV- NADH dehydrogenase ubiquinone flavoprotein OMIM online mendelian inheritance in man OXPHOS oxidative phosphorylation PAGE polyacrylamide gel electrophoresis PCR polymerase chain reaction

PD Parkinson disease PDHc pyruvate dehydrogenase complex Q ubiquinone

QH2 ubiquinol RNAi RNA interference ROS reactive oxygen species rRNA ribosomal RNA RT-PCR reverse transcriptase-polymerase chain reaction SDS sodium dodecyl sulfate SEM standard error of the mean siRNA small interfering RNA SNP single nucleotide polymorphism TAP tandem affinity purification TCA tricarboxylic acid TC-NER transcription-coupled nucleotide excision repair TMRM tetramethyl rhodamine methyl ester tRNA transfer RNA UEM unspecified encephalomyopathy UV ultraviolet VLCFA very long-chain fatty acid WB western blotting

Chapter I

General Introduction - Aim, Objectives and Outline of the Thesis -

Chapter I

General introduction

Oxidative phosphorylation (OXPHOS) has a prominent role in energy metabolism of the cell. Embedded in the lipid bilayer of the mitochondrial inner membrane, the OXPHOS system is the final biochemical pathway in energy production. The OXPHOS system comprises five membrane-bound multiprotein complexes and two mobile electron carriers—ubiquinone and cytochrome c—that transfer electrons from NADH and succinate to molecular oxygen. While transferring electrons, complexes I, III and IV function as redox pumps using redox energy to translocate protons across the membrane into the intermembrane space. The resulting proton gradient constitutes a membrane potential that serves as the driving force for ATP production by complex V (Fig 1).

Figure 1. The OXPHOS system. At complex I, one of two entry points of the OXPHOS system, NADH is oxidized and electrons are transferred to ubiquinone (Q), while protons (H+) are pumped across the mitochondrial inner membrane. Complex II, the second entry point, transfers electrons from succinate into the system, using ubiquinone as an electron acceptor. Subsequently, electrons are transferred from ubiquinone to complex III, and via the water-soluble electron carrier cytochrome c (cyt c), which shuttles between complex III and IV, to complex IV. Complex IV transfers electrons to molecular oxygen (O2), forming water (H2O). Simultaneously, the proton gradient build by complexes I, III, and IV is used as a driving force by complex V, channeling protons back into the matrix, to convert ADP and inorganic phosphate

(Pi) into ATP. At the bottom, the total number of subunits, as well as the number of subunits encoded by the nuclear (nDNA) and mitochondrial (mtDNA) DNA, are depicted for all five complexes and the total OXPHOS system.

Chapter I

The aim of this thesis is to expand our understanding of the biogenesis and functioning of complex I of the OXPHOS system in health and disease. To date, mutations have been found in the genes encoding numerous structural components of complex I, however, a significant portion of cases of complex I deficiency cannot be explained by mutations in these constituents. Alternative candidates for complex I deficiency are the proteins that guide assembly of the constituents into a functional complex. The main objectives are 1) identification of new genes that are involved in the assembly process of complex I and that are responsible for complex I deficiency; and 2) elucidation of the underlying pathological mechanisms. Biogenesis and functioning of the OXPHOS system is dependent on the finely tuned interaction between the nuclear and the mitochondrial genomes, both contributing to a complex build-up that consists of 89 subunits in total. This suggests that disturbances of the integrity of the system can be caused by numerous genetic defects and can result in a variety of metabolic and biochemical alterations. Consequently, OXPHOS deficiencies manifest in a wide variety of clinical symptoms, presenting from infancy to late adulthood and affecting many different organs and tissues, either in an organ-specific or multisystemic form (Fig 2). Furthermore, OXPHOS deficiencies can either be isolated, with one complex affected, or combined, with two or more complexes affected. Four different types of inheritance contribute to the heterogeneity of OXPHOS disorders: autosomal recessive or dominant, X- linked and maternal or mitochondrial. The broad clinical spectrum at which OXPHOS deficiencies manifest and their underlying extended genetic background will be reviewed in Chapter II. Diseases caused by disturbances of the OXPHOS system are amongst the most frequent inborn errors of metabolism, with an incidence of 1 in 5,000 live births. Isolated complex I deficiency is the most common cause of OXPHOS disorders. Complex I, or NADH:ubiquinone oxidoreductase, represents the main entry point of the OXPHOS system, as it initiates electron transfer by oxidizing NADH. Mammalian complex I consists of 45 subunits of which seven are encoded by the mitochondrial DNA (mtDNA) and 38 by the nuclear genome. Being accountable for a heterogeneous group of clinical phenotypes, the primary defects underlying complex I deficiency are genetically heterogeneous as well. Mutations have been found in each of the seven mtDNA-encoded subunits, resulting in either single-organ phenotypes, as Leber hereditary optic neuropathy (LHON), or multisystemic syndromes, e.g., mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS). For mtDNA mutations presentation of the disease often occurs in late childhood or adolescence and there is a great variability in clinical course and intrafamilial presentation. So far, mutations have been found in twelve of the nuclear genes encoding structural components of complex I. Clinical phenotypes caused by mutations in

General introduction

Figure 2. Organs and systems affected in OXPHOS disorders. OXPHOS disorders manifest in a great variety of symptoms in many different organs and systems of the body. This diagram depicts the most common symptoms that can occur in different combinations among patients.

nuclear-encoded subunits generally present in infancy and early childhood. Nine of those genes (NDUFA1, NDUFA2, NDUFS1, NDUFS3, NDUFS4, NDUFS6, NDUFS7, NDUFS8 and NDUFV1) are mainly associated with brain and brainstem disorders, mostly Leigh(-like) syndrome or leukoencephalopathy, while three others (NDUFA11, NDUFS2 and NDUFV2) are exclusively associated with hypertrophic cardiomyopathy and encephalomyopathy. The pathology of complex I deficiency as well as the structure, function and biogenesis of mitochondrial complex I will be reviewed in Chapter III.

Chapter I

In addition to 45 structural components, probably numerous, yet unknown, nuclear factors are needed for proper assembly and functioning of complex I, as has been reported extensively for complex IV. Identification of these assembly factors will open a new field of possible candidates for complex I deficiency. To date, two characterized human complex I assembly chaperones have been proved responsible for complex I deficiency. The identification of the first human complex I assembly factor, NDUFAF1, as the human homologue of the Neurospora crassa assembly chaperone CIA30 will be described in Chapter IV. Further characterization of the function of NDUFAF1 in the assembly process of complex I and the investigation of its involvement in complex I deficiency will be described in Chapter V. Mitochondrial complex I assembly is an intricate process, which is nowadays believed to proceed through a series of distinct assembly intermediates by combining evolutionarily conserved functional modules in a semi-sequential manner. Disturbances of the process by, for instance, mutation of subunits can lead to accumulation and/or depletion of several intermediate assemblies, providing so-called assembly profiles. The effects of mutations in different complex I subunits on complex I assembly/stability will be described in Chapter VI. A second (complex I) assembly factor, NDUFAF2, has been found responsible for complex I deficiency in a unique patient with a contiguous gene deletion, which will be described in Chapter VII. Besides the disturbance of ATP production, complex I deficiency exhibits additional cell biological consequences. Increased production of reactive oxygen species (ROS) may have deleterious effects on mtDNA, protein complexes and membrane integrity. Alterations in mitochondrial morphology, i.e. branching or fragmentation, changes in membrane potential and mitochondrial NAD(P)H levels are other examples of disturbances of mitochondrial physiology. Some downstream alterations in mitochondrial physiology as a consequence of complex I deficiency will be described in Chapter VII. Finally, Chapter VIII will summarize the established findings and will give an overview of the eminent role for complex I assembly in complex I deficiency. It will also shine a light on the directions future research will take concerning complex I deficiency.

General introduction

Chapter II

Genetic defects in the oxidative phosphorylation (OXPHOS) system - A review -

Rolf J.R.J. Janssen, Lambert P.W.J. van den Heuvel and Jan A.M. Smeitink

Nijmegen Center for Mitochondrial Disorders, Laboratory of Pediatrics and Neurology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

Adapted from Expert Review of Molecular Diagnostics 2004, Volume 4, Number 2, Page 143-156

Chapter II

Genetic defects in the OXPHOS system

Abstract The oxidative phosphorylation (OXPHOS) system consists of five multiprotein complexes and two mobile electron carriers embedded in the lipid bilayer of the mitochondrial inner membrane. With the exception of complex II and the mobile carriers, the other parts of the OXPHOS system are under dual genetic control. Due to this bigenomic control, the inheritance of OXPHOS system defects is either maternal, in the case of mitochondrial DNA mutations, autosomal or X-linked, in the case of nuclear gene defects. In this review, our current genetic understanding of OXPHOS system enzyme deficiencies will be summarized, and future directions that the field might take to unravel so-far genetically unresolved OXPHOS system enzyme deficiencies will be described, with special emphasis on complex I biogenesis.

Chapter II

Introduction Deficiencies of the oxidative phosphorylation (OXPHOS) system are phenotypically as well as genetically heterogeneous. One of the most common mitochondrial disorders is Leigh syndrome. Leigh syndrome is an early-onset progressive neurodegenerative disorder and involves encephalopathy with or without lactic acidemia, occasionally complicated by cardiomyopathy or a multisystemic presentation[1]. Clinical signs such as motor and/or intellectual retardation, as well as other neurological symptoms including ataxia, dystonia, hypotonia and optic atrophy, are frequently encountered[2]. Patients with strongly suggestive clinical and histochemical features but with an atypical or noninvestigated neuropathology are referred to as Leigh-like[2,3]. Combinations of mitochondrial DNA (mtDNA) and nuclear DNA mutations, tissue specificity and environmental factors determine the onset and course of the disease. Most patients die a few years after first clinical manifestation. Besides Leigh and Leigh-like syndrome, many other genetic disorders represent deficiency of the OXPHOS system. The underlying biochemical defect can either be isolated, with one OXPHOS complex affected, or combined, with two or more OXPHOS complexes affected, in a tissue- specific manner. Four different types of inheritance contribute to the heterogeneity of OXPHOS disorders: autosomal recessive or dominant, X-linked and maternal or mitochondrial. As isolated complex I deficiency is most frequently encountered[4], complex I will be the main focus of this review. Since the OXPHOS system is under genetic control of two interacting genomes, mutations causing OXPHOS deficiency can either be found in mitochondrial or nuclear genes.

Mitochondrial genes involved in OXPHOS deficiency The particular genetic characteristics of mtDNA—maternal inheritance, polyplasmy, heteroplasmy, the threshold effect and mitotic segregation—are the features that distinguish disorders due to mtDNA defects, which follow the mitochondrial mode of inheritance, from disorders due to nuclear gene defects following mendelian inheritance. These characteristics also give rise to a broad spectrum of clinical features affecting different organs and tissues that can vary from one individual to another. Since mtDNA is maternally inherited, mtDNA mutations are only transmitted from mothers to their progeny. Each cell harbors thousands of identical copies of mtDNA, which is known as polyplasmy, with up to ten molecules per mitochondrion. Heteroplasmy occurs when wild type and mutant mtDNA coexists in cells and tissues. When the percentage of wild type mtDNA drops below a certain value, the OXPHOS system will be disturbed and clinical manifestation of the disease will take place, i.e., the threshold effect. Thresholds for onset of symptoms are tissue specific and are lower for tissues with a high demand for OXPHOS metabolism, such as brain, heart, skeletal

Genetic defects in the OXPHOS system muscle, retina, renal tubules and the endocrine glands[5]. The degree of heteroplasmy can also change during cell division since redistribution of mitochondria occurs randomly, i.e., mitotic segregation[5,6]. Thereby, mutation loads can surpass or drop below a certain (pathogenic) threshold, altering the phenotype of that particular cell line or tissue. These rules of mitochondrial genetics explain the wide variety of symptoms related to age and tissue among patients bearing the same mutated gene or even the same specific mutation. Different mtDNA mutations may lead to similar clinical phenotypes, as illustrated in the case of Leber hereditary optic neuropathy (LHON). Primary mutations leading to LHON have been assigned to ND1, ND4 and ND6, and secondary or intermediate mutations have been reported for ND1, ND2, ND4, ND4L, ND5, CYB, COI, COII, COIII and ATP6. A T8993G mutation in the ATPase 6 gene is the common mutation for neuropathy, ataxia and retinitis pigmentosa (NARP) syndrome[7], and this specific mutation has also been associated with maternally inherited Leigh syndrome (MILS), provided that percentages of heteroplasmy exceed 95% of mutant mtDNA in affected tissues (e.g., fibroblasts, muscle, nerve and brain)[8]. Most mutations in the cytochrome b gene are related to progressive exercise intolerance[9-11], although some have been reported in cases of cardiomyopathy[12], LHON[13,14] and the Parkinsonism/mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS) overlap syndrome[15]. The genes for the mtDNA-encoded subunits of cytochrome c oxidase (COX) are, besides LHON[16,17], also associated with sideroblastic anemia (COI)[18], isolated motor neuron disease (COI)[19], multisystem mitochondrial disorders (COI and COII)[20,21] and recurrent myoglobinuria (COI and COIII)[22,23]. Due to the unique genetic code and the separate machinery for mitochondrial translation, not only mutations in the protein-encoding genes, which encode subunits of the OXPHOS complexes, but also the genes encoding transfer RNA (tRNA) and ribosomal RNA (rRNA) can, when mutated, lead to OXPHOS deficiency. Since instead of one protein, mitochondrial protein synthesis in general is disturbed in these cases, deficiencies of all OXPHOS complexes, except complex II, can occur. Mutations in the tRNA Leu(UUR) gene are often associated with MELAS[24], another genetic heterogeneous syndrome that can be caused by mutations in ND1, ND5, ND6, COIII and tRNA Lys, Gln, Val and Phe. The tRNA Leu(UUR) mutations, on the contrary, may also give rise to cardiomyopathy[25], mitochondrial myopathy[26], chronic progressive external ophthalmoplegia (CPEO)[27] and diabetes and deafness[28]. Mutations in the tRNA Lys gene are the underlying defect in almost all cases of myoclonus epilepsy with ragged-red fibers (MERRF) syndrome[29,30], but they can also lead to other syndromes. Mutations in the small (12S) rRNA gene cause nonsyndromic sensorineural deafness most often[31] and 16S rRNA mutations have been associated with cardiomyopathy[32] and with Rett syndrome[33].

Chapter II

In addition to the maternally inherited specific mutations, sporadic rearrangements of mtDNA can occur. These deletions and duplications of mtDNA fragments probably originate in the germ line, although a few maternally inherited cases have been described[34]. Depending on when in oogenesis the deletion occurred, either all three germ layers can bear the deleted mtDNA, resulting in multisystemic Kearns-Sayre syndrome (KSS), or segregation to the hematopoietic lineage, causing Pearson‘s syndrome, or segregation to the muscle, leading to CPEO, can take place[5]. Table 1 summarizes the most frequently encountered mtDNA defects, the biochemical consequences of these mutations, as well as the associated clinical presentations.

Nuclear genes involved in OXPHOS deficiency Nuclear genes responsible for disturbance of the OXPHOS system can be categorized on a structural and functional level: genes encoding structural components of the OXPHOS system; genes encoding assembly factors of the OXPHOS complexes; genes involved in biogenesis of the OXPHOS system; genes involved in maintenance of mtDNA; genes involved in biogenesis and maintenance of the mitochondrial membranes; and genes associated with secondary defects of the OXPHOS system (Tab 2).

Genes encoding structural components of the OXPHOS system Complex I—Isolated complex I (or NADH:ubiquinone oxidoreductase; EC 1.6.5.3) deficiency is one of the most frequent disturbances of the OXPHOS system and frequently follows an autosomal recessive mode of inheritance[4]. Mutations have been identified in twelve of the 38 nuclear genes encoding structural subunits of complex I. Seven of them, NDUFV1, NDUFV2, NDUFS1, NDUFS2, NDUFS3, NDUFS7 and NDUFS8, encode so-called essential or core subunits and are all thought to be involved in the configuration of the iron–sulfur clusters. Mutations in these genes apparently affect intramolecular electron transfer. Five other genes, NDUFA1, NDUFA2, NDUFA11, NDUFS6 and NDUFS4, encode accessory subunits. Clinical presentation, in the majority of cases, involves Leigh or Leigh-like syndrome (NDUFS1, NDUFS3, NDUFS4, NDUFS7, NDUFS8, NDUFA1 and NDUFA2)[96-102] or leukodystrophy (NDUFS1 and NDUFV1)[97,103], although mutations in the NDUFS2, NDUFV2 and NDUFA11 genes have specifically been associated with hypertrophic cardiomyopathy and encephalomyopathy[104-106]. Complex II—Complex II (or succinate:ubiquinone oxidoreductase; EC 1.3.5.1) consists of four nuclear DNA-encoded subunits and functions both as an enzyme complex of the OXPHOS system and as an essential enzyme of the tricarboxylic acid (TCA) cycle. The peripheral arm consists of a flavoprotein, where the active site containing covalently bound 24

Genetic defects in the OXPHOS system

Table 1. Mitochondrial genes involved in oxidative phosphorylation (OXPHOS) deficiency

Affected biochemical Gene (mutation) Main clinical presentation Ref. mechanism

Subunits of the OXPHOS system

MTND1 (G3460A) OXPHOS complex I LHON [35] Adult-onset dystonia, spasticity and core-type MTND1 (A3796G) OXPHOS complex I [36] myopathy MTND1 (G3697A, G3946A, OXPHOS complex I MELAS [37] T3949C) MTND2 (G5244A) OXPHOS complex I LHON [13]

MTND2 (T4681C) OXPHOS complex I Leigh syndrome [38]

MTND3 (T10158C) OXPHOS complex I Leigh syndrome [39] Progressive epilepsy, stroke-like episodes, optic MTND3 (T10191C) OXPHOS complex I [40] atrophy and cognitive decline MTND3 (G10197A) OXPHOS complex I Leigh syndrome and dystonia [41]

MTND4 (C11777A) OXPHOS complex I Leigh syndrome [42]

MTND4 (G11778A) OXPHOS complex I LHON [35]

MTND4 (G11832A) OXPHOS complex I Exercise intolerance [43]

MTND4L (T10663C) OXPHOS complex I LHON [44]

MTND5 (T12706C) OXPHOS complex I Leigh syndrome [45]

MTND5 (A13084T) OXPHOS complex I Leigh-MELAS overlap syndrome [46]

MTND5 (G13513A) OXPHOS complex I MELAS [47]

MTND5 (G13513A) OXPHOS complex I Leigh-like syndrome [48]

MTND5 (A13514G) OXPHOS complex I MELAS-like [49]

MTND5 (G13708A) OXPHOS complex I LHON [13]

MTND6 (G14453A) OXPHOS complex I MELAS [50]

MTND6 (G14459A) OXPHOS complex I LHON and dystonia [51]

MTND6 (G14459A) OXPHOS complex I Leigh syndrome [52] MTND6 (C14482G, C14482A, OXPHOS complex I LHON [35,53,54] T14484C, A14495G) MTCYB (del14787-14790) OXPHOS complex III Parkinsonism/MELAS overlap syndrome [15]

MTCYB (G15059A) OXPHOS complex III Exercise muscle intolerance and myoglobinuria [9] Exercise intolerance and mitochondrial MTCYB (G15242A) OXPHOS complex III [11] encephalomyopathy MTCYB (G15257A) OXPHOS complex III LHON [13,14]

MTCYB (C15452A) OXPHOS complex III Ischemic cardiomyopathy [12]

MTCYB (G15615A) OXPHOS complex III Exercise muscle intolerance [10]

MTCO1 (5-bp del 5‘UTR) OXPHOS complex IV Isolated motor neuron disease [19]

MTCO1 (T6721C, T6742C) OXPHOS complex IV Sideroblastic anemia [18]

Chapter II

MTCO1 (G6930A) OXPHOS complex IV Multisystem mitochondrial disorder [20]

MTCO1 (G7444A) OXPHOS complex IV LHON [16]

MTCO1 (G5920A) OXPHOS complex IV Recurrent myoglobinuria [22]

MTCO2 (G7598A) OXPHOS complex IV LHON [17]

MTCO2 (T7671A) OXPHOS complex IV Proximal myopathy and lactic acidosis [55]

MTCO2 (G7706A) OXPHOS complex IV Alpers-Huttenlocher-like syndrome [56]

MTCO2 (G7896A) OXPHOS complex IV Multisystem mitochondrial disorder [21]

MTCO2 (del8042AT) OXPHOS complex IV Severe lactic acidosis and hepatic involvement [57]

MTCO3 (15-bp del) OXPHOS complex IV Recurrent myoglobinuria [23]

MTCO3 (ins9537C) OXPHOS complex IV Leigh-like syndrome [58]

MTCO3 (G9804A, G9438A) OXPHOS complex IV LHON [59]

MTCO3 (T9957C) OXPHOS complex IV MELAS [60]

MTATP6 (T8993G) OXPHOS complex V NARP [7]

MTATP6 (T8993G) OXPHOS complex V MILS [8]

MTATP6 (T9101C) OXPHOS complex V LHON [61] Apical hypertrophic cardiomyopathy and MTATP8 (G8529A) OXPHOS complex V [62] neuropathy Transfer RNAs MTTL1 Leu(UUR) (A3243G, Mitochondrial translation MELAS [24] T3271C, T3291C) MTTL1 Leu(UUR) (A3243G) Mitochondrial translation MERRF/MELAS overlap syndrome [63]

MTTL1 Leu(UUR) (A3243G) Mitochondrial translation Diabetes and deafness [28]

MTTL1 Leu(UUR) (A3243G) Mitochondrial translation CPEO; Kearns-Sayre syndrome [27] Cardiomyopathy and/or mitochondrial MTTL1 Leu(UUR) (C3303T) Mitochondrial translation [25] myopathy MTTL1 Leu(UUR) (T3250C, Mitochondrial translation Mitochondrial myopathy [26] A3302G) MTTL2 Leu(CUN) (A12320G) Mitochondrial translation Mitochondrial myopathy [64]

MTTL2 Leu(CUN) (T12297C) Mitochondrial translation Dilated cardiomyopathy [65]

MTTL2 Leu(CUN) (G12315A) Mitochondrial translation CPEO [66]

MTTL2 Leu(CUN) (G12301A) Mitochondrial translation Sideroblastic anemia [67]

MTTK Lys (A8344G, A8296G) Mitochondrial translation MERRF [29,30]

MTTK Lys (T8316C) Mitochondrial translation MELAS [68] Cardiomyopathy and hearing loss; Leigh MTTK Lys (G8363A) Mitochondrial translation [69] syndrome MTTK Lys (G8342A) Mitochondrial translation CPEO [70]

MTTI Ile (A4300G) Mitochondrial translation Hypertrophic cardiomyopathy [71] Mitochondrial myopathy, ataxia and MTTI Ile (A4267G) Mitochondrial translation [72] sensorineural deafness MTTI Ile (T4285C) Mitochondrial translation CPEO [73]

Genetic defects in the OXPHOS system

MTTS1 Ser(UCN) (A7445G) Mitochondrial translation Sensorineural deafness [74]

MTTS1 Ser(UCN) (T7512C) Mitochondrial translation MERFF/MELAS overlap syndrome [75]

MTTS2 Ser(AGY) (C12258A) Mitochondrial translation Diabetes and deafness [28]

MTTA Ala (T5628C) Mitochondrial translation CPEO [76] Hypertrophic cardiomyopathy; MTTC Cys (A5814G) Mitochondrial translation [77] encephalomyopathy MTTD Asp (A7543G) Mitochondrial translation Myoclonic epilepsy [78] Mitochondrial myopathy and lactic acidosis, MTTE Glu (T14687C) Mitochondrial translation [79] retinopathy and respiratory failure MTTE Glu (T14709C) Mitochondrial translation Mitochondrial myopathy and diabetes mellitus [80]

MTTF Phe (G583A) Mitochondrial translation MELAS [81]

MTTF Phe (T618C) Mitochondrial translation Mitochondrial myopathy [82] Hypertrophic cardiomyopathy; MTTG Gly (T9997C) Mitochondrial translation [83] encephalomyopathy MTTM Met (T4409C) Mitochondrial translation Mitochondrial myopathy [84]

MTTN Asn (G5703A) Mitochondrial translation CPEO [85]

MTTP Pro (G15990A) Mitochondrial translation Mitochondrial myopathy [85]

MTTQ Gln (ins4370A) Mitochondrial translation Myopathy [87]

MTTQ Gln (G4332A) Mitochondrial translation MELAS [88]

MTTT Thr (G15915A) Mitochondrial translation Mitochondrial encephalomyopathy [89]

MTTV Val (G1642A) Mitochondrial translation MELAS [90]

MTTV Val (G1644C) Mitochondrial translation Leigh syndrome [91]

MTTW Trp (ins5537T) Mitochondrial translation Leigh syndrome [92]

MTTY Tyr (G5877A) Mitochondrial translation CPEO [93]

Ribosomal RNAs

MTRNR1 12S rRNA (A1555G) Mitochondrial translation Nonsyndromic sensorineural hearing loss [31]

MTRNR1 12S rRNA (A1555G) Mitochondrial translation Mitochondrial cardiomyopathy [94] Sensorineural hearing loss, parkinsonism and MTRNR1 12S rRNA (T1095C) Mitochondrial translation [95] neuropathy MELAS, diabetes mellitus, hyperthyroidism and MTRNR2 16S rRNA (C3093G) Mitochondrial translation [32] cardiomyopathy MTRNR2 16S rRNA (C2835T) Mitochondrial translation Rett syndrome [33]

LHON: Leber hereditary optic neuropathy; MELAS: mitochondrial encephalopathy with lactic acidosis and stroke-like episodes; NARP: neuropathy ataxia and retinitis pigmentosa; MILS: maternally inherited Leigh syndrome; MERRF: myoclonus epilepsy with ragged-red fibers; CPEO: chronic progressive external ophthalmoplegia

FAD is located, and an iron–sulfur protein, and is anchored to the membrane by two transmembrane proteins. A mutation in the flavoprotein encoding the SDHA gene of a patient with Leigh syndrome was the first mutation identified in a nuclear gene causing

Chapter II

OXPHOS deficiency[107]. On the contrary, mutations in the genes encoding the iron–sulfur protein, SDHB, and the anchor proteins, SDHC and SDHD, manifest in a phenotypically different manner; they cause hereditary paragangliomas and/or pheochromocytomas[108-111]. Complex III—Complex III (or ubiquinol:cytochrome c oxidoreductase; EC 1.10.2.2) transfers electrons from the ubiquinone pool to cytochrome c and is composed of eleven subunits, of which ten are nuclear DNA-encoded. Recently, the first mutation in a nuclear DNA-encoded subunit of complex III was reported: the ubiquinol–cytochrome c reductase binding protein (UQCRB or QP-C) gene[112]. Whereas complex III deficiency is often associated with multisystemic and tissue-specific disorders, such as (encephalo)myopathy or cardiomyopathy[9-12,15,113], in this case no psychomotor retardation or neurological impairment has been observed, although laboratory investigations revealed hypoglycemia and lactic acidemia. Complex IV & V—To date, no mutations have been reported in the structural nuclear components of complex IV (or cytochrome c oxidase; COX; EC 1.9.3.1) or complex V (or

F0F1-ATPase; EC 3.6.3.14). Most syndromes with isolated complex IV deficiency are caused by mutations in genes encoding proteins involved in the proper assembly of this 13-subunit rich complex.

Genes encoding assembly factors of the OXPHOS complexes Assembly factors are proteins that mediate the assembly process of subunits and intermediate complexes into fully assembled OXPHOS complexes and can have chaperone- like functions. Over the years, a few assembly factors have been identified for complex I and III and many for complex IV. Two characterized human complex I assembly factors have been proved responsible for complex I deficiency. In a patient presenting with an early-onset progressive encephalopathy resembling vanishing white matter disease, a null mutation was found in the NDUFAF2 gene, encoding human complex I assembly factor 2 (also known as human B17.2L), which is a paralogue of subunit NDUFA12 of the peripheral arm of complex I[114]. A mutation in the gene encoding a second complex I assembly factor, NDUFAF1, has been associated with cardioencephalomyopathy[115]. Recently, a third putative complex I assembly factor encoded by the C6ORF66 gene has been reported carrying pathogenic mutations in six patients with mitochondrial encephalomyopathy or antenatal cardiomyopathy[116]. The only known assembly factor gene responsible for isolated complex III deficiency is BCS1L[117], encoding a mitochondrial inner membrane protein participating as a chaperone in assembly of the Rieske iron–sulfur subunit of complex III[118,119]. Mutations in BCS1L have been associated with neonatal proximal tubulopathy, hepatic involvement and encephalopathy[120], with a lethal metabolic disorder with iron overload, named GRACILE

Genetic defects in the OXPHOS system

(growth retardation, aminoaciduria, cholestasis, iron overload, lactic acidosis and early death) syndrome[120], and recently with Björnstad syndrome[121]. Complex IV is composed of 13 structural subunits, three of which are encoded by the mtDNA and form the catalytic core of the enzyme. The other ten are encoded by the nucleus. A large number of accessory factors are necessary for the assembly and maintenance of the active holoenzyme complex[122], and several mutations in these factors leading to complex IV deficiency have been described. The first gene encoding an assembly factor known to be responsible for COX deficiency was the SURF-1 gene. SURF-1 is needed for maintenance of COX activity and probably involved in the early stages of COX assembly[1,122,123]. Leigh syndrome is the most common clinical manifestation observed in SURF-1 patients and is associated with loss of function mutations resulting in undetectable protein levels of SURF-1[124,125]. SURF-1 mutations have also been described in non-Leigh phenotypes, e.g., isolated leukodystrophy has also been described[126,127]. Two other COX assembly genes, SCO1 and SCO2, encode copper ion binding proteins that facilitate the insertion of copper into the COX subunits I and II and the subsequent incorporation of subunits I and II into the holoenzyme. Mutations in SCO1 and SCO2, which are more frequently encountered, correlate with hypertrophic cardiomyopathy and encephalopathy[128,129], combined with hepatic failure in a case of a SCO1 patient[130]. The COX10 and COX15 genes are involved in the synthesis of heme A, a prosthetic group of COX. Mutations in these genes result in a clinical manifestation comparable with that of SCO2[131,132]. Mutations in the LRPPRC gene cause Leigh syndrome French Canadian variant (LSFC), a variant of Leigh syndrome with additional hepatic involvement[133]. In LSFC besides the brain, only the liver exhibits marked reduction in COX activity. LRP130, which is encoded by the LRPPRC gene, is a component of a complex that affects numerous processes related to energy homeostasis[134].

For F0F1-ATPase, complex V of the OXPHOS system, the only pathogenic mutation in a nuclear gene encoding an assembly factor (or subunits of the complex itself), so far has been found in the ATP12 gene, in a patient presenting with mitochondrial encephalomyopathy with lactic acidosis and hepatic involvement[135].

Genes involved in the biogenesis of the OXPHOS system OXPHOS deficiencies can also be due to defects in biogenesis of the OXPHOS system, i.e., defects in transcription of mtDNA, or defects in translation of mitochondrial messenger RNA (mRNA). The latter defects can occur in the case of defective components of the mitochondrial ribosome or the mitochondrial translation system. Mutations in the genes of two ribosomal proteins, MRPS16 and MRPS22, have been detected in cases of fatal neonatal lactic acidosis, and hypertrophic cardiomyopathy and tubulopathy, respectively[136,137].

Chapter II

Mutations in mitochondrial translation elongation factors EFG1, EFTu and EFTs have been encountered in cases of encephalo(myo)pathy with (EFG1)[137] or without (EFTs)[139] hepatic involvement, leukodystrophy (EFTu)[140], but also in a case of hypertrophic cardiomyopathy (EFTs)[139]. Another component of the mitochondrial translation machinery, pseudouridine synthase 1 (PUS1), is responsible for post-transcriptional pseudouridylation of specific mitochondrial tRNAs and mutations in the PUS1 gene are specific for mitochondrial myopathy and sideroblastic anemia (MLASA)[141]. Genes involved in biosynthesis of ubiquinone—one of the two mobile electron carriers of the OXPHOS system—include COQ2, PDSS1, and PDSS2, which form another group of genes, when mutated, leading to OXPHOS disorders[142-144].

Genes involved in mtDNA maintenance Mutations in nuclear genes involved in mtDNA maintenance cause disorders that clinically resemble those caused by mtDNA mutations, i.e., a primary nuclear gene defect causes secondary mtDNA loss or multiple deletion formation but follows a mendelian inheritance pattern[145]. The most frequently described disorder is autosomal dominant progressive external ophthalmoplegia (adPEO), for which four different disease genes have been identified: ANT1, encoding the adenine nucleotide translocator or ADP/ATP translocator[146]; POLG1 and POLG2, encoding catalytic subunit A and accessory subunit B, respectively, of the mtDNA-specific polymerase gamma[147,148,150]; and PEO1, encoding the Twinkle protein, a mtDNA helicase[151-153]. Mutations following a recessive mode of inheritance (arPEO) have also been reported for POLG1[149]. Other frequently encountered clinical phenotypes observed with POLG1 mutations are Alpers syndrome and Alpers- Huttenlocher syndrome[154,155]. While mtDNA polymerase gamma and Twinkle are directly involved in mtDNA replication, another group of genes is involved in the mitochondrial deoxyribonucleoside triphosphate (dNTP) salvage pathway, which supplies the mitochondria with dNTP pools needed for mtDNA synthesis. Besides defective mtDNA replication, disturbance of the dNTP pools can also cause mitochondrial DNA depletion syndrome (MDS). MDS is a clinically heterogeneous group of autosomal recessive disorders characterized by tissue-specific reduction in mtDNA copy number. Affected tissues show both decreased activity of the mtDNA-encoded OXPHOS complexes (I, III, IV and V) and mtDNA depletion. Several tissue-specific forms can be distinguished. The genes encoding the salvage-pathway enzymes thymidine kinase 2 (TK2) and deoxyguanosine kinase (DGUOK) have specifically been associated with a myopathic form and a hepatocerebral form of MDS, respectively[156-158]. A third enzyme involved in the regulation of the mitochondrial dNTP pool is thymidine phosphorylase (TP). Deficiency of TP causes multiple mtDNA deletions and/or mtDNA depletion leading to mitochondrial neurogastrointestinal

Genetic defects in the OXPHOS system encephalomyopathy (MNGIE)[159,160]. Two other genes, SUCLA2 and SUCLG1, encoding the β- and α-subunit, respectively, of the TCA-cycle enzyme succinate-coenzyme A ligase (SUCL) have been associated with a encephalomyopathic form and fatal infantile lactic acidosis (FILA) associated with mtDNA depletion, respectively[161,162]. It is hypothesized that mtDNA depletion is caused by decreased mitochondrial nucleoside diphosphate kinase (NDPK) activity resulting from the inability of NDPK to form a complex with SUCL[162]. NDPKs are protein kinases that are involved in de novo synthesis of dNTPs. Mutations in the MPV17 gene, of which the function remains unclear, have also been associated with an early-onset hepatocerebral form of MDS[163]. Recently, yet another gene, RRM2B, encoding the cytosolic tumor suppressor p53-inducible ribonucleotide reductase small subunit (p53R2), has been associated with mtDNA depletion, which implies that p53R2 has a crucial role in dNTP supply for mtDNA synthesis. Mutations in the RRM2B gene presented with encephalopathy and renal failure[164]. The link between the genetic defects of enzymes of the mitochondrial dNTP salvage pathway and mtDNA depletion strongly suggests the involvement of the mitochondrial dNTP pool in the pathogenesis of the MDS and underscores the importance of maintaining balanced mitochondrial dNTP pools[157]. The identification and interpretation of novel gene defects underlying these diseases will provide further understanding of the intergenomic communication between the nuclear and mitochondrial genomes[145,159].

Genes involved in biogenesis and maintenance of the mitochondrial membranes Besides defects of genes directly involved in biogenesis of the OXPHOS system, the system might also become disturbed by other factors affecting its functioning, such as alterations in the lipid composition of the mitochondrial inner membrane. In Barth syndrome, an X-linked cardiac and skeletal mitochondrial myopathy, disturbance of cardiolipin metabolism has been observed. Mutations of the TAZ gene, encoding tafazzin, a phospholipid acyltransferase involved in acyl-specific remodeling of cardiolipin (the specific mitochondrial phospholipid), may be responsible for the alterations of mitochondrial lipids leading to changes in mitochondrial architecture and function[165-167]. Several mitochondrial enzymes that are involved in the formation and maintenance of the mitochondrial network, called dynamin-related GTPases, have recently been implicated in mitochondrial disease. Two of them mediate the mitochondrial fusion process: optic atrophy 1 protein (OPA1), which is located in the intermembrane space, is required for fusion of the mitochondrial inner membranes and cristae. The OPA1 gene has been identified as the gene responsible for autosomal dominant optic atrophy (DOA), the most common form of inherited optic neuropathy characterized by early-onset visual impairment[168,169]. Mitofusin 2 (MFN2), which is embedded in the mitochondrial outer

Chapter II membrane, mediates the tethering of mitochondria with each other during the fusion process. Mutations in the MFN2 gene are the cause of Charcot-Marie-Tooth disease type 2A2 (CMT2A2), a peripheral neuropathy characterized by axonal degeneration[170], and CMT type 6 (CMT6) also hereditary motor and sensory neuropathy type VI (HMSN VI)[171]. A third GTPase, called dynamin-like protein 1 (DLP1) or dynamin-related protein 1 (DRP1), is required for mitochondrial fission and mutations in the DLP1 gene have been encountered in a case of microcephaly, optic atrophy and metabolic aberrations[172].

Genes associated with secondary defects of the OXPHOS system OXPHOS deficiencies also occur as secondary defects, i.e., as a consequence of defective related mechanisms, for instance the import of nuclear DNA-encoded mitochondrial proteins (e.g., OXPHOS components) into the mitochondrion. A mutation in the gene for subunit 8a of the translocase of the inner mitochondrial membrane, TIMM8A or DDP1, causes human deafness dystonia syndrome, also known as Mohr-Tranebjaerg syndrome (MTS), a progressive neurodegenerative disorder[173,174]. TIMM8A belongs to a family of evolutionarily conserved proteins and is organized in a complex together with the TIM13 protein in the mitochondrial intermembrane space. This complex mediates the import and insertion of nuclear DNA-encoded hydrophobic membrane proteins into the mitochondrial inner membrane. Although the involvement of Ddp1 in the import of mitochondrial proteins implies that the underlying defect of the MTS is a defect in mitochondrial OXPHOS and phenotypes associated with the systemic OXPHOS defects overlap with clinical symptoms of MTS[175], enzyme activities of the OXPHOS complexes show normal values in muscle biopsy. Therefore, the specific mitochondrial dysfunction in MTS remains to be clarified[176]. Two genes encoding mitochondrial solute carriers have been implicated in mitochondrial disease. The SLC25A19 gene or DNC encodes a mitochondrial thiamine pyrophosphate (ThPP) transporter and the SLC19A2 gene or THTR1 encodes a plasma membrane thiamine

(or vitamin B1) transporter. Mutations in the SLC25A19 gene lead to lack of mitochondrial ThPP—a coenzyme of the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complexes—and concomitant reduction of energy produced by oxidative metabolism, which is the cause of Amish lethal microcephaly (MCPHA) and alpha-ketoglutaric aciduria[177,178]. Mutations in the SLC19A2 gene cause thiamine-responsive megaloblastic anemia with diabetes mellitus and progressive sensorineural deafness (TRMA), also known as Rogers syndrome. TRMA is an early-onset autosomal recessive disorder with a severe deficiency of pyruvate dehydrogenase and complex I in muscle tissue that responds to thiamine treatment[179-181]. Friedreich ataxia is an autosomal recessive disorder that is predominantly caused by the abnormal expansion of a GAA repeat in the first intron of the FXN gene, which encodes the

Genetic defects in the OXPHOS system mitochondrial protein frataxin[182]. Frataxin is a component of the human iron–sulfur cluster assembly machinery and plays a role in the maturation of both mitochondrial and cytosolic iron–sulfur proteins[183]. The lack of frataxin appears to result in oxidative damage to the highly sensitive iron–sulfur protein complexes I, II, III of the OXPHOS system and the TCA cycle-enzyme aconitase and should be related to mitochondrial iron accumulation[184]. Although significant reductions in the activities of these enzymes are present in the heart[185], the deficiencies of the OXPHOS system are presumed to be secondary. Hereditary spastic paraplegia (HSP) is a genetically heterogeneous neurodegenerative disorder characterized by axonal degeneration and presents with progressive weakness and spasticity of the lower limbs. In addition, muscle biopsies from the most severely affected HSP patients showed histological evidence of OXPHOS defects, including ragged-red fibers and COX-negative fibers, whereas biochemical analysis revealed reduced complex I activity[186]. The spastic paraplegia gene, SPG7, encodes a subunit of the m-AAA protease termed paraplegin, which has both metallopeptidase and chaperone-like activities at the mitochondrial inner membrane. The m-AAA protease has a dual role in protein quality control and ribosome assembly in mitochondria. With mitochondrial ribosomal protein Mrpl32 as a proteolytic substrate, a crucial function for the control of mitochondrial protein synthesis is assigned to the m-AAA protease[187]. Patients with mutations in SPG7, expressing a secondary OXPHOS defect, represent one of several subtypes of HSP. Ethylmalonic encephalopathy (EE) is a devastating infantile metabolic disorder affecting the brain, gastrointestinal tract, and peripheral vessels, and is characterized by high levels of ethylmalonic acid in body fluids and decreased cytochrome c oxidase activity in skeletal muscle. EE is specifically caused by mutations in the ETHE1 gene, which encodes a mitochondrial matrix protein of unknown function[188]. However, the severe consequences of its malfunctioning indicate an important role in mitochondrial homeostasis and energy metabolism.

Expert opinion Future directions of research in OXPHOS deficiency As the number of genes known to be involved in the biogenesis of the OXPHOS system is still growing, mapping and identification of responsible genes for OXPHOS deficiency becomes more laborious. Therefore, it is essential to first categorize the biochemical defects before further characterization of the genetic defects can take place. Traditional approaches to the diagnosis of mitochondrial disorders have been based on enzymatic activity assays, which are time consuming and often require large patient samples. Capaldi‘s group developed an immunocytochemical assay to aid in the detection of mitochondrial disorders

Chapter II

Table 2. Nuclear genes involved in oxidative phosphorylation (OXPHOS) deficiency

Gene (protein Affected biochemical Main clinical presentation Ref. name) mechanism

Subunits of the OXPHOS system

NDUFS1 OXPHOS complex I Leigh syndrome and leukodystrophy [97] Hypertrophic cardiomyopathy and NDUFS2 OXPHOS complex I [104] encephalomyopathy NDUFS3 OXPHOS complex I Leigh syndrome [98]

NDUFS4 OXPHOS complex I Leigh syndrome [96, 189]

NDUFS4 OXPHOS complex I Leigh-like syndrome [190,191]

NDUFS6 OXPHOS complex I Unspecified mitochondrial encephalomyopathy [192]

NDUFS7 OXPHOS complex I Leigh syndrome [99]

NDUFS8 OXPHOS complex I Leigh syndrome [100]

NDUFV1 OXPHOS complex I Leukodystrophy and myoclonic epilepsy [97, 103] Hypertrophic cardiomyopathy and NDUFV2 OXPHOS complex I [105] encephalomyopathy NDUFA1 OXPHOS complex I Leigh syndrome [101]

NDUFA1 OXPHOS complex I Myoclonic epilepsy and developmental delay [101]

NDUFA2 OXPHOS complex I Leigh syndrome [102]

NDUFA8, NDUFS2 OXPHOS complex I Mitochondrial encephalopathy [193]

NDUFA11 OXPHOS complex I Encephalocardiomyopathy [106]

NDUFA11 OXPHOS complex I FILA [106]

SDHA OXPHOS complex II Leigh syndrome [107]

SDHA OXPHOS complex II Late-onset optic atrophy, ataxia and myopathy [194] Hereditary paraganglioma/ pheochromocytoma SDHB OXPHOS complex II [108,109] (PGL4) SDHC OXPHOS complex II Hereditary paraganglioma (PGL3) [110] Hereditary paraganglioma/ pheochromocytoma SDHD OXPHOS complex II [111] (PGL1) UQCRB OXPHOS complex III Hypoglycemia and lactic academia [112]

Assembly factors of the OXPHOS complexes Hypertrophic cardiomyopathy and NDUFAF1 OXPHOS complex I [115] encephalomyopathy NDUFAF2 OXPHOS complex I Leukoencephalopathy [114] Mitochondrial encephalopathy and hepatic NDUFAF2 OXPHOS complex I [195] involvement C6ORF66 OXPHOS complex I Mitochondrial encephalomyopathy [116]

C6ORF66 OXPHOS complex I Antenatal cardiomyopathy [116] Neonatal proximal tubulopathy, hepatic involvement BCS1L OXPHOS complex III [117] and encephalopathy BCS1L OXPHOS complex III GRACILE syndrome [120]

Genetic defects in the OXPHOS system

BCS1L OXPHOS complex III Björnstad syndrome [121]

SURF-1 OXPHOS complex IV Leigh syndrome [124,125]

SURF-1 OXPHOS complex IV Leukodystrophy [126] Villous atrophy, hypertrichosis and mild neurological SURF-1 OXPHOS complex IV [127] involvement SCO1 OXPHOS complex IV Neonatal-onset hepatic failure and encephalopathy [129]

SCO2 OXPHOS complex IV Encephalopathy and hypertrophic cardiomyopathy [128,129] Mitochondrial encephalopathy, hypotonia ataxia and COX10 OXPHOS complex IV [131] seizures COX15 OXPHOS complex IV Fatal infantile hypertrophic cardiomyopathy [132]

LRPPRC OXPHOS complex IV Leigh syndrome French Canadian [133] Lactic acidosis, mitochondrial encephalomyopathy ATP12 OXPHOS complex V [135] and hepatic involvement OXPHOS system biogenesis

MRPS16 Mitochondrial translation Agenesis of corpus callosum, dysmorphism and FILA [136]

MRPS22 Mitochondrial translation Hypertrophic cardiomyopathy and tubulopathy [137]

EFG1 (GFM1) Mitochondrial translation Hepatoencephalopathy [138]

EFTs (TSFM) Mitochondrial translation Mitochondrial encephalomyopathy [139]

EFTs (TSFM) Mitochondrial translation Hypertrophic cardiomyopathy [139]

EFTu (TUFM) Mitochondrial translation Macrocystic leukodystrophy with micropolygyria [140]

PUS1 Mitochondrial translation MLASA [141]

Ubiquinone (CoQ10) COQ2 Encephalomyopathy and nephropathy [142] biosynthesis Ubiquinone (CoQ10) PDSS1 Encephaloneuropathy and deafness [143] biosynthesis Ubiquinone (CoQ10) PDSS2 Leigh syndrome and nephropathy [144] biosynthesis Mitochondrial DNA maintenance

POLG1 (polymerase γA) mtDNA replication adPEO; arPEO [147-149]

POLG1 (polymerase γA) mtDNA replication Alpers syndrome [154]

POLG1 (polymerase γA) mtDNA replication Alpers-Huttenlocher syndrome [155]

POLG2 (polymerase γB) mtDNA replication adPEO [150]

PEO1 (twinkle) mtDNA replication adPEO [151-153]

PEO1 (twinkle) mtDNA replication IOSCA [196]

PEO1 (twinkle) mtDNA replication MDS - hepatocerebral form [197] Mitochondrial dNTP salvage ANT1 adPEO [146] pathway Mitochondrial dNTP salvage TK2 MDS - myopathic form [156] pathway Mitochondrial dNTP salvage DGUOK MDS - hepatocerebral form [157,158] pathway Mitochondrial dNTP salvage ECGF1 or TP MDS - MNGIE [159,160] pathway Mitochondrial dNTP salvage SUCLA2 MDS - encephalomyopathic form [161] pathway 35

Chapter II

Mitochondrial dNTP salvage SUCLG1 MDS - FILA [162] pathway Mitochondrial dNTP salvage MPV17 MDS - hepatocerebral form [163] pathway Mitochondrial dNTP salvage RRM2B MDS - nephrocerebral form [164] pathway Mitochondrial membrane biogenesis and maintenance

TAZ (tafazzin) Cardiolipin metabolism Barth syndrome [165,166] Mitochondrial inner OPA1 Autosomal dominant optic atrophy [168,169] membrane maintenance MFN2 Mitochondrial fusion CMT2A2 [170]

MFN2 Mitochondrial fusion CMT6 or HMSN VI [171] Microcephaly, optic atrophy and metabolic DPL1 or DRP1 Mitochondrial fission [172] aberrations Secondary defects of the OXPHOS system Mitochondrial protein Deafness dystonia syndrome or Mohr-Tranebjaerg TIMM8A [173,174] import syndrome SLC25A19 or DNC or Mitochondrial ThPP Amish lethal microcephaly (MCPHA) [177,178] TPC transport THTR1 or SLC19A2 Thiamine transport TRMA or Rogers syndrome [179-181] Iron-sulfur cluster FXN (frataxin) Friedreich ataxia [182] biogenesis Mitochondrial protein SPG7 (paraplegin) processing/quality control Hereditary spastic paraplegia [186] and ribosome assembly ETHE1 unknown Ethylmalonic encephalopathy [188]

FILA: fatal infantile lactic acidosis; GRACILE: growth retardation, aminoaciduria, cholestasis, iron overload, lactic acidosis and early death; MLASA: myopathy, lactic acidosis and sideroblastic anemia; adPEO: autosomal dominant progressive external ophthalmoplegia; arPEO: autosomal recessive PEO; IOSCA: infantile onset spinocerebellar ataxia; MDS: mtDNA depletion syndrome; MNGIE: mitochondrial neurogastrointestinal encephalomyopathy; CMT2A2: Charcot-Marie-Tooth disease type 2A2; CMT6: CMT type 6; HMSN VI: hereditary motor and sensory neuropathy type VI; ThPP: thiamine pyrophosphate; TRMA: thiamine-responsive megaloblastic anemia

by identifying the defective complex using monoclonal antibodies to subunits of each of the OXPHOS complexes[198]. Both mtDNA- and nuclear DNA-encoded defects can be distinguished. This test could be performed quickly and uses less sample material. Different strategies are used for elucidation of nuclear gene defects in biogenesis of the OXPHOS system. With the chromosome transfer technique, chromosomes or parts of chromosomes can be identified which can complement the unknown defective (responsible) gene. In order to identify the chromosome harboring the mutated gene, microcell-mediated transfer of human chromosome (fragments) into OXPHOS-deficient cultured patient fibroblasts is performed. Functional complementation of the mitochondrial defect can be established with a complementation test based on restoration of the complex enzyme activity or the complex assembly[125,199]. In the past this technique has revealed the SURF-1 gene as a mutational hotspot for Leigh syndrome with COX deficiency[125].

Genetic defects in the OXPHOS system

Five-year view As it is the largest of the five OXPHOS complexes, deficiency of complex I is a major contributor to the total of deficiencies of the OXPHOS system. With a total of 45 subunits, complex I is the most complicated multisubunit protein of the mitochondrial inner membrane[200-202]. Over the years, bovine heart complex I has been studied extensively by the Walker group[203]. With the almost complete characterization of all structural subunits enabling mutational analysis in complex I-deficient patients, the focus on complex I research, with regard to the pathogenesis of complex I deficiency, has now shifted towards the biogenesis of complex I and especially the understanding of the assembly process of the complex, which is still a black box. One human gene, CIA30 (currently known as NDUFAF1), which encodes a nonstructural protein and is involved in assembly of human complex I, has been characterized[204]. The gene product CIA30 is a homologue of a yeast protein found to be transiently associated with a 350-kDa complex I intermediate[205]. Another yeast complex I intermediate-associated protein, CIA84, has not been identified in humans. As for complex IV, for which many assembly factors are known at present, more assembly factors are expected to be identified for complex I in the near future. The availability of an increasing number of antibodies directed against subunits of complex I significantly contributes to the elucidation of the composition of subcomplexes and will eventually lead to a greater understanding of the assembly process. More and more techniques are becoming available to characterize different intermediate subcomplexes in subsequent steps of the assembly process. Capaldi‘s group is developing a complex I immunocapture assay analogous to a recently developed complex V assay[206], in which fully assembled and functionally active complex I, as well as partially assembled subcomplexes, can be purified and measured in terms of activity. Walker‘s group distinguished three major subcomplexes that can be obtained by treatment of intact complex I from bovine heart mitochondria with mildly chaotropic detergents[200]. Recently, Antonicka and coworkers proposed a model in which seven distinct intermediate subcomplexes can be identified[207]. With the characterization of the intermediate subcomplexes and subsequent exploration of the assembly process, the understanding of complex I biogenesis will become clearer in the near future, thereby opening doors to the identification of new (assembly) factors involved in the process and increasing the number of candidate genes for complex I deficiency.

Chapter II

Key issues • Deficiencies of the oxidative phosphorylation (OXPHOS) system are phenotypically and genetically heterogeneous. • Since the OXPHOS system is under genetic control by two interacting genomes, mutations causing OXPHOS deficiency can either be found in the mitochondrial DNA or nuclear genes. • Due to the unique genetic code and the separate machinery for mitochondrial translation, mutations in the protein-encoding genes, encoding subunits of the OXPHOS complexes, as well as mutations in the genes encoding transfer RNA and ribosomal RNA, can lead to OXPHOS deficiency. • Nuclear genes responsible for disturbance of the OXPHOS system can be categorized on a structural and functional level: genes encoding structural components of the OXPHOS system; genes encoding assembly factors of the OXPHOS complexes; genes involved in the biogenesis of the OXPHOS system; genes involved in the maintenance of mitochondrial DNA; genes involved in biogenesis and maintenance of the mitochondrial membranes; and genes associated with secondary defects of the OXPHOS system. • Isolated complex I deficiency is the most common disturbance of the OXPHOS system and frequently follows an autosomal recessive mode of inheritance. Mutations have been identified in twelve nuclear genes encoding structural subunits of complex I: NDUFV1-2, NDUFS1-4, NDUFS6-8 and NDUFA1-2, NDUFA11, and three genes encoding (putative) complex I assembly chaperones: NDUFAF1, NDUFAF2 and C6ORF66. • With the exploration of the assembly process, the understanding of complex I biogenesis will become more clear in the near future, opening doors to the identification of new (assembly) factors involved in the process and increasing the number of candidate genes for complex I deficiency.

Acknowledgements Part of this work has been made possible by a grant from The Netherlands Organization for Scientific Research (NWO, number 901-03-156).

Genetic defects in the OXPHOS system

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MA, Quattrone A, Battaloglu E, Polyakov AV, Timmerman V, Schroder JM, Vance JM (2004) Mutations in the mitochondrial GTPase mitofusin 2 cause Charcot-Marie-Tooth neuropathy type 2A. Nat Genet 36(5):449-451 Erratum in: Nat Genet (2004) 36(6):660 171. Zuchner S, De Jonghe P, Jordanova A, Claeys KG, Guergueltcheva V, Cherninkova S, Hamilton SR, Van Stavern G, Krajewski KM, Stajich J, Tournev I, Verhoeven K, Langerhorst CT, de Visser M, Baas F, Bird T, Timmerman V, Shy M, Vance JM (2006) Axonal neuropathy with optic atrophy is caused by mutations in mitofusin 2. Ann Neurol 59(2):276-281 172. Waterham HR, Koster J, van Roermund CW, Mooyer PA, Wanders RJ, Leonard JV (2007) A lethal defect of mitochondrial and peroxisomal fission. N Engl J Med 356(17):1736-1741 173. Koehler CM, Leuenberger D, Merchant S, Renold A, Junne T, Schatz G (1999) Human deafness dystonia syndrome is a mitochondrial disease. Proc. Natl. Acad. Sci. USA 96(5):2141-2146 174. Roesch K, Curran SP, Tranebjaerg L, Koehler CM (2002) Human deafness dystonia syndrome is caused by a defect in assembly of the DDP1/TIMM8a-TIMM13 complex. Hum Mol Genet 11(5):477- 486 175. Wallace DC, Murdock DG (1999) Mitochondria and dystonia: the movement disorder connection? Proc Natl Acad Sci U S A 96(5):1817-1819 176. Binder J, Hofmann S, Kreisel S, Wöhrle JC, Bäzner H, Krauss JK, Hennerici MG, Bauer MF (2003) Clinical and molecular findings in a patient with a novel mutation in the deafness-dystonia peptide (DDP1) gene. Brain 126(Pt 8):1814-1820 177. Rosenberg MJ, Agarwala R, Bouffard G, Davis J, Fiermonte G, Hilliard MS, Koch T, Kalikin LM, Makalowska I, Morton DH, Petty EM, Weber JL, Palmieri F, Kelley RI, Schäffer AA, Biesecker LG (2002) Mutant deoxynucleotide carrier is associated with congenital microcephaly. Nat Genet 32(1):175-179 178. Lindhurst MJ, Fiermonte G, Song S, Struys E, De Leonardis F, Schwartzberg PL, Chen A, Castegna A, Verhoeven N, Mathews CK, Palmieri F, Biesecker LG (2006) Knockout of Slc25a19 causes mitochondrial thiamine pyrophosphate depletion, embryonic lethality, CNS malformations, and anemia. Proc Natl Acad Sci U S A 103(43):15927-15932 179. Labay V, Raz T, Baron D, Mandel H, Williams H, Barrett T, Szargel R, McDonald L, Shalata A, Nosaka K, Gregory S, Cohen N (1999) Mutations in SLC19A2 cause thiamine-responsive megaloblastic anaemia associated with diabetes mellitus and deafness. Nat Genet 22(3):300-304 180. Scharfe C, Hauschild M, Klopstock T, Janssen AJ, Heidemann PH, Meitinger T, Jaksch M (2000) A novel mutation in the thiamine responsive megaloblastic anaemia gene SLC19A2 in a patient with deficiency of respiratory chain complex I. J Med Genet 37(9):669-673 181. Diaz GA, Banikazemi M, Oishi K, Desnick RJ, Gelb BD (1999) Mutations in a new gene encoding a thiamine transporter cause thiamine-responsive megaloblastic anaemia syndrome. Nat Genet 22(3):309-312 182. Campuzano V, Montermini L, Moltò MD, Pianese L, Cossée M, Cavalcanti F, Monros E, Rodius F, Duclos F, Monticelli A, Zara F, Cañizares J, Koutnikova H, Bidichandani SI, Gellera C, Brice A, Trouillas P, De Michele G, Filla A, De Frutos R, Palau F, Patel PI, Di Donato S, Mandel JL, Cocozza S, Koenig M, Pandolfo M (1996) Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion. Science 271(5254):1423-1427 183. Stehling O, Elsässer HP, Brückel B, Mühlenhoff U, Lill R (2004) Iron-sulfur protein maturation in human cells: evidence for a function of frataxin. Hum Mol Genet 13(23):3007-3015 184. Rotig A, Sidi D, Munnich A, Rustin P (2002) Molecular insights into Friedreich's ataxia and antioxidant-based therapies. Trends Mol Med 8(5):221-224 Review 185. Bradley JL, Blake JC, Chamberlain S, Thomas PK, Cooper JM, Schapira AH (2000) Clinical, biochemical and molecular genetic correlations in Friedreich's ataxia. Hum Mol Genet 9(2):275- 282 186. Casari G, De Fusco M, Ciarmatori S, Zeviani M, Mora M, Fernandez P, De Michele G, Filla A, Cocozza S, Marconi R, Dürr A, Fontaine B, Ballabio A (1998) Spastic paraplegia and OXPHOS impairment caused by mutations in paraplegin, a nuclear-encoded mitochondrial metalloprotease. Cell 93(6):973-983

Chapter II

187. Nolden M, Ehses S, Koppen M, Bernacchia A, Rugarli EI, Langer T (2005) The m-AAA protease defective in hereditary spastic paraplegia controls ribosome assembly in mitochondria. Cell 123(2):277-289 188. Tiranti V, D'Adamo P, Briem E, Ferrari G, Mineri R, Lamantea E, Mandel H, Balestri P, Garcia- Silva MT, Vollmer B, Rinaldo P, Hahn SH, Leonard J, Rahman S, Dionisi-Vici C, Garavaglia B, Gasparini P, Zeviani M (2004) Ethylmalonic encephalopathy is caused by mutations in ETHE1, a gene encoding a mitochondrial matrix protein. Am J Hum Genet 74(2):239-252 189. Bénit P, Steffann J, Lebon S, Chretien D, Kadhom N, de Lonlay P, Goldenberg A, Dumez Y, Dommergues M, Rustin P, Munnich A, Rötig A (2003) Genotyping microsatellite DNA markers at putative disease loci in inbred/multiplex families with respiratory chain complex I deficiency allows rapid identification of a novel nonsense mutation (IVS1nt -1) in the NDUFS4 gene in Leigh syndrome. Hum Genet 112(5-6):563-566 190. Budde SM, van den Heuvel LP, Janssen AJ, Smeets RJ, Buskens CA, DeMeirleir L, Van Coster R, Baethmann M, Voit T, Trijbels JM, Smeitink JA (2000) Combined enzymatic complex I and III deficiency associated with mutations in the nuclear encoded NDUFS4 gene. Biochem Biophys Res Commun 275(1):63-68 191. Petruzzella V, Vergari R, Puzziferri I, Boffoli D, Lamantea E, Zeviani M, Papa S (2001) A nonsense mutation in the NDUFS4 gene encoding the 18 kDa (AQDQ) subunit of complex I abolishes assembly and activity of the complex in a patient with Leigh-like syndrome. Hum Mol Genet 10(5):529-535 192. Kirby DM, Salemi R, Sugiana C, Ohtake A, Parry L, Bell KM, Kirk EP, Boneh A, Taylor RW, Dahl HH, Ryan MT, Thorburn DR (2004) NDUFS6 mutations are a novel cause of lethal neonatal mitochondrial complex I deficiency. J Clin Invest 114(6):837-45 193. Bugiani M, Invernizzi F, Alberio S, Briem E, Lamantea E, Carrara F, Moroni I, Farina L, Spada M, Donati MA, Uziel G, Zeviani M (2004) Clinical and molecular findings in children with complex I deficiency. Biochim Biophys Acta 1659(2-3):136-147 194. Birch-Machin MA, Taylor RW, Cochran B, Ackrell BA, Turnbull DM (2000) Late-onset optic atrophy, ataxia, and myopathy associated with a mutation of a complex II gene. Ann Neurol 48(3):330-335 195. Janssen RJ, Distelmaier F, Smeets RJ, Wijnhoven T, Østergaard E, Jaspers NG, Raams A, Kemp S, Rodenburg RJ, Willems PH, van den Heuvel LP, Smeitink JA, Nijtmans LG (2008) Contiguous gene deletion of ELOVL7, ERCC8, and NDUFAF2 in a patient with progressive encephalomyopathy. Submitted 196. Nikali K, Suomalainen A, Saharinen J, Kuokkanen M, Spelbrink JN, Lönnqvist T, Peltonen L (2005) Infantile onset spinocerebellar ataxia is caused by recessive mutations in mitochondrial proteins Twinkle and Twinky. Hum Mol Genet 14(20):2981-2990 197. Hakonen AH, Isohanni P, Paetau A, Herva R, Suomalainen A, Lonnqvist T (2007) Recessive Twinkle mutations in early onset encephalopathy with mtDNA depletion. Brain 130(Pt 11):3032- 3040 198. Hanson BJ, Capaldi RA, Marusich MF, Sherwood SW (2002) An immunocytochemical approach to detection of mitochondrial disorders. J Histochem Cytochem 50(10):1281-1288 199. De Lonlay P, Mugnier C, Sanlaville D, Chantrel-Groussard K, Bénit P, Lebon S, Chrétien D, Kadhom N, Saker S, Gyapay G, Romana S, Weissenbach J, Munnich A, Rustin P, Rötig A (2002) Cell complementation using Genebridge 4 human:rodent hybrids for physical mapping of novel mitochondrial respiratory chain deficiency genes. Hum Mol Genet (26):73-81 200. Carroll J, Fearnley IM, Shannon RJ, Hirst J, Walker JE (2003) Analysis of the subunit composition of complex I from bovine heart mitochondria. Mol Cell Proteomics (2):117-126 201. Carroll J, Shannon RJ, Fearnley IM, Walker JE, Hirst J (2002) Definition of the nuclear encoded protein composition of bovine heart mitochondrial complex I. Identification of two new subunits. J Biol Chem 277(52):50311-50317 202. Carroll J, Fearnley IM, Skehel JM, Shannon RJ, Hirst J, Walker JE (2006) Bovine complex I is a complex of 45 different subunits. J Biol Chem 281(43):32724-32727

Genetic defects in the OXPHOS system

203. Hirst J, Carroll J, Fearnley IM, Shannon RJ, Walker JE (2003) The nuclear encoded subunits of complex I from bovine heart mitochondria. Biochim Biophys Acta 1604(3):135-150 Review 204. Janssen R, Smeitink J, Smeets R, van Den Heuvel L (2002) CIA30 complex I assembly factor: a candidate for human complex I deficiency? Hum Genet (3):264-270 205. Kuffner R, Rohr A, Schmiede A, Krull C, Schulte U (1998) Involvement of two novel chaperones in the assembly of mitochondrial NADH:Ubiquinone oxidoreductase (complex I). J Mol Biol 283(2):409-417 206. Aggeler R, Coons J, Taylor SW, Ghosh SS, Garcia JJ, Capaldi RA, Marusich MF (2002) A functionally active human F1F0 ATPase can be purified by immunocapture from heart tissue and fibroblast cell lines. Subunit structure and activity studies. J Biol Chem 277(37):33906-33912 207. Antonicka H, Ogilvie I, Taivassalo T, Anitori RP, Haller RG, Vissing J, Kennaway NG, Shoubridge EA (2003) Identification and characterization of a common set of complex I assembly intermediates in mitochondria from patients with complex I deficiency. J Biol Chem 278(44):43081-43088

Chapter III

Mitochondrial complex I: structure, function and pathology - A review -

Rolf J.R.J. Janssen, Leo G.J. Nijtmans, Lambert P.W.J. van den Heuvel, and Jan A.M. Smeitink

Nijmegen Center for Mitochondrial Disorders, Laboratory of Pediatrics and Neurology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

Journal of Inherited Metabolic Disease 2006, Volume 29, Number 4, Page 499-515

Chapter III

Mitochondrial complex I

Abstract Oxidative phosphorylation (OXPHOS) has a prominent role in energy metabolism of the cell. Being under bigenomic control, correct biogenesis and functioning of the OXPHOS system is dependent on the finely tuned interaction between the nuclear and the mitochondrial genome. This suggests that disturbances of the system can be caused by numerous genetic defects and can result in a variety of metabolic and biochemical alterations. Consequently, OXPHOS deficiencies manifest as a broad clinical spectrum. Complex I, the biggest and most complicated enzyme complex of the OXPHOS system, has been subjected to thorough investigation in recent years. Significant progress has been made in the field of structure, composition, assembly and pathology. Important gains in the understanding of the Goliath of the OXPHOS system are: exposing the electron transfer mechanism and solving the crystal structure of the peripheral arm, characterization of almost all subunits and some of their functions, and creating models to elucidate the assembly process with concomitant identification of assembly chaperones. Unraveling the intricate mechanisms underlying the functioning of this membrane-bound enzyme complex in health and disease will pave the way for developing adequate diagnostic procedures and advanced therapeutic treatment strategies.

Chapter III

Introduction Embedded in the lipid bilayer of the mitochondrial inner membrane, the oxidative phosphorylation (OXPHOS) system is the final biochemical pathway in energy production of the cell. Five multisubunit enzyme complexes, together with two mobile electron carriers ubiquinone (Q) and cytochrome c (cyt c) constitute the mitochondrial OXPHOS system. By transferring electrons from complex to complex, via Q and cyt c, complexes I, III and IV function as redox pumps using redox energy to translocate protons across the membrane. As a result of the build-up of a proton gradient, a membrane potential is created. At the end of the chain, complex IV reduces the final electron acceptor molecular oxygen to water, while complex V, or F1F0-ATP synthase, uses the membrane potential as a driving force to produce ATP.

Subcellular functioning of complex I in the oxidative phosphorylation system NADH:ubiquinone oxidoreductase (complex I; EC 1.6.5.3), the first and largest (and most complicated) of the five complexes, is one of the two entry points of the OXPHOS system, succinate:ubiquinone oxidoreductase (complex II; EC 1.3.5.1) being the other. It initiates electron transfer by oxidizing NADH and using the lipid-soluble ubiquinone as the electron acceptor. In an exergonic redox reaction, following upon binding of NADH to complex I, the transfer of two electrons from NADH to ubiquinone in the mitochondrial inner membrane is coupled to the translocation of four protons from the negative matrix side to the positive side of the intermembrane space:

NADH + H+ + Q + 4H+matrix NAD+ + QH2 + 4H+intermembrane space

The two electrons provided by the oxidation of NADH are transferred to the primary electron acceptor of complex I, a noncovalently bound flavin mononucleotide (FMN). Subsequently, electrons are transduced through redox centers represented by eight iron- sulfur (FeS) clusters two binuclear, N1a and N1b, and six tetranuclear clusters, N2, N3-

N6a, N6b to the final acceptor ubiquinone (Q), which then is reduced to ubiquinol (QH2). Bacterial complex I (e.g. complex I of Escherichia coli, Thermus thermophilus, and others), referred to as NDH-1, has a ninth, tetranuclear, iron-sulfur cluster, N7, which is not part of the conserved electron transfer pathway from FMN to ubiquinone. The redox reactions are tightly linked to the pumping of protons across the mitochondrial inner membrane; the transfer of two electrons is coupled to the translocation of four protons. Because of its complexity and the lack of available high-resolution crystallographic three-dimensional (3D) structures, little is known about the mechanisms underlying enzymatic functioning of mitochondrial complex I. Unlike for the other OXPHOS complexes, 56

Mitochondrial complex I high-resolution 3D structures have not been solved for complex I yet. It has been generally accepted from low-resolution 3D structures revealed by electron microscopy[1,2] that the mitochondrial as well as the bacterial complex I have an ‗L‘-shaped or ‗boot‘ structure, as has been demonstrated for the enzyme of Neurospora crassa[3], Yarrowia lipolytica[4], Escherichia coli[5] and bovine heart mitochondria[1]. This structure is constituted of two arms that are perpendicular to each other[6]: a hydrophobic arm embedded in the lipid membrane and a hydrophilic, peripheral arm protruding into the matrix or bacterial cytoplasm. Sazanov and colleagues provided the first structural information of bacterial complex I in a native membrane environment by showing that intact complex I is L-shaped in a lipid bilayer, and confirmed this for the active enzyme in solution[7]. This supports the view of an L-shaped conformation in vivo and contradicts the view of a ‗horseshoe‘ conformation (the two complex arms arranged side by side) of the active enzyme in vivo proposed by Bottcher et al.[8]. It is proposed that the latter conformation is mistaken for dimers of complex I with a horseshoe-like appearance by lack of, or by inadequately staining of peripheral arms[7]. In a recent study, Hirst and coworkers underlined the importance of native lipids for the stabilization as well as the activation of complex I, by developing a method of preparing fully active complex I from bovine heart mitochondria[9]. Different classes of interactions between phospholipids and complex I have been defined: tightly bound cardiolipin may be required for structural integrity of the complex (but also a functional role has not been ruled out), while the amounts of the more weakly bound phosphatidylcholine and phosphatidylethanolamine are determinant for catalytic activity[10]. An important step in elucidating the enzymatic mechanisms of energy transduction in complex I has been made recently by the group of Sazanov. The atomic structure of the hydrophilic peripheral arm, including the arrangement of iron-sulfur clusters, has been determined for complex I from Thermus thermophilus with the use of x-ray crystallography[11,12]. Seven clusters are arranged in a continuous chain connecting the two catalytic sites of the enzyme. The first cluster in the chain is the tetranuclear cluster N3 located in the 51-kDa metalloprotein (NDUFV1),1 which also contains the NADH-binding site and the primary electron acceptor FMN. Clusters N1b, N4, N5 and the distal tetranuclear cluster N7 are located in the 75-kDa subunit (NDUFS1). Clusters N6a and N6b are located in

1For consistency, the following rules have been applied concerning the subunit nomenclature: the bovine nomenclature is used (with the human nomenclature in parentheses) when referring to subunits of bovine complex I, or homologous subunits of T. thermophilus, or N. crassa. The human nomenclature is used (with the bovine nomenclature in parentheses) when referring to subunits of the human enzyme or all eukaryotic homologues in general. 57

Chapter III the TYKY subunit (NDUFS8) in the connecting domain, close to the interface with the membrane arm. The last cluster in the chain is likely to be N2, which is in the PSST subunit (NDUFS7), and it has been suggested that it reduces ubiquinone at the interface with the membrane domain. The likely pathway of electron transfer from FMN to ubiquinone follows the shortest route: FMN-N3-N1b-N4-N5-N6a-N6b-N2-Q. The binuclear cluster N1a, which is in the 24-kDa subunit (NDUFV2), is unlikely to participate directly in the electron transfer from FMN to ubiquinone. N1a can accept electrons from FMN but cannot pass them directly to the nearest cluster of the main redox chain. A role as antioxidant has been suggested for cluster N1a: by accepting electrons from the more exposed FMN, excessive generation of reactive oxygen species (ROS) can be prevented[12]. The mechanisms of proton pumping by complex I discussed in literature are based on two main models: direct (redox-driven) and indirect (conformation-driven) coupling between electron transfer and proton translocation. In the first model, referred to as the Q-cycle mechanism, it is assumed that the quinone-binding sites and proton translocation components, located in the membrane, are in close contact with the redox centers in the peripheral arm, allowing direct interaction. In the second model, the redox centers of the peripheral arm are coupled to distal proton translocating components that are located at the end of the membrane arm, through long-range conformational changes[13]. A combination of both mechanisms has also been discussed. In the latter situation, two modules are suggested to participate in proton pumping: the hydrogenase module acts as a redox-driven proton pump and the transporter module acts as a conformation-driven proton pump. This implies that complex I contains two energy-coupling sites[14,15]. Recently, a novel complex I-proton pumping mechanism has been proposed by Ohnishi and Salerno based on redox-driven conformational changes of the quinone-binding site[16]. Two distinct ubiquinone-binding sites were assigned within the membrane domain of complex I.

The subunits of mitochondrial complex I Evolutionary conservation and subunit composition of mammalian complex I Bacterial complex I, or NDH-1, consists of 14 different subunits, which all have homologues in the mitochondrial or eukaryotic enzyme. These 14 mitochondrial homologues, referred to as ‗core subunits‘, are essential for catalysis of electron transfer from NADH to ubiquinone and for generation of the membrane potential, and are therefore together considered as the ‗minimal‘ constitution of the enzyme. Because of its minimal subunit composition, NDH-1 can be used as a model for the more complex mammalian enzyme. Half of the core subunits are encoded by the nuclear genome and half by the mitochondrial genome, with both genomes representing subunits of distinct parts of the complex. The seven nuclear DNA- encoded core subunits (NDUFS1-3, NDUFS7-8, NDUFV1-2) are the most conserved among

Mitochondrial complex I the eukaryotic subunits. They constitute the peripheral domain, comprising all redox centers, while the seven subunits of the mitochondrial genome (ND1-6, ND4L) form the membrane domain[9]. Because of extensive analyses over many years of the subunit composition of complex I from bovine heart mitochondria, it has become the best-characterized eukaryotic complex I[9,17-21]. To date, identification of all structural components of mammalian complex I is assumed to be completed and has resulted in a total number of 45 subunits that have all been assigned to well-characterized genes. Homologues of all bovine complex I subunits have been identified in human complex I, and the human genes have been identified by homology with the bovine sequences. Because of high sequence identity between bovine and human subunits, the bovine enzyme can be adopted as a model for human complex I. Assuming there is one copy of each subunit present in complex I, its molecular mass is 980 kDa[9].

The mitochondrial DNA-encoded subunits With the exception of complex II and the mobile electron carriers, the OXPHOS system is subjected to dual genetic control, i.e. the OXPHOS constituents are encoded by two physically separate genomes: the mitochondrial and the nuclear genomes. The mitochondrial DNA (mtDNA) codes for 13 polypeptides, i.e. structural components for complex I (7), complex III (1), complex IV (3) and complex V (2), two ribosomal RNAs, and 22 transfer RNAs. The seven mtDNA-encoded subunits of complex I, ND1-6 and ND4L, are all hydrophobic and bear a number of transmembrane helices. Together with ~20 integral membrane proteins in total, they form the membrane arm of the enzyme, contributing >60 transmembrane segments[22]. The subunits ND2, ND4, and ND5 seem to originate from one common ancestor. From sequence comparisons it has been suggested that they are related to subunits of K+ or Na+/H+ antiporters, which makes them good candidates for components of the proton translocation machinery[15]. Because of its quinone-binding site, subunit ND1 is proposed to be involved in ubiquinone binding.

The nuclear DNA-encoded subunits Thirty-eight of the 45 subunits of mammalian complex I are encoded by the nuclear genome. Eighteen subunits have N-terminal precursor mitochondrial targeting sequences (MTSs) that are cleaved upon import into the mitochondrial inner membrane or the matrix (Tab 1). The 20 remaining characterized subunits lack precursor import sequences and are somehow imported into the mitochondria by internal signals within the mature protein. Post- translational modifications of individual subunits have been identified by electrospray ionization mass spectrometry (ESI-MS). Fourteen subunits have post-translationally

Chapter III

Table 1. The subunits of human complex I Post-translational Bovine E. coli Predicted mass Human gene Subcomplex a modification N. crassa homologued Biochemical features (EPR signal) homologue homologue mature proteing (N-terminal)b ND1 ND1 I - ND1 NuoH 35.7 ND2 ND2 I - ND2 NuoN 39.0 ND3 ND3 I - ND3 NuoA 13.2 ND4 ND4 I - ND4 NuoM 51.6 ND4L ND4L I - ND4L NuoK 10.7 ND5 ND5 I - ND5 NuoL 67.0 ND6 ND6 I - ND6 NuoJ 18.7 NDUFV1 51 kDa I MTS 1-20 51 kDa NuoF 48.6 FMN; [4Fe-4S] (N3) NDUFV2 24 kDa I MTS 1-32 24 kDa NuoE 23.7 [2Fe-2S] (N1a) NDUFV3 10 kDa I MTS 1-34 - 8.4 NDUFS1 75 kDa I MTS 1-23 78 kDa NuoG 77.0 [2Fe-2S] (N1b); 2 x [4Fe-4S] (N4, N5) NDUFS2 49 kDa I MTS 1-33 49 kDa NuoCDf 49.1 NDUFS3 30 kDa I MTS 1-36 30.4 kDa NuoCDf 26.4 NDUFS4 18 kDa (AQDQ) I MTS 1-42 21 kDa 15.4 NDUFS5 15 kDa I Met predicted orthologuee 12.4 NDUFS6 13 kDa I MTS 1-28 28.7 kDa 10.7 NDUFS7 PSST I MTS 1-38 19.3 kDa NuoB 19.8 [4Fe-4S] (N2) NDUFS8 TYKY I MTS 1-34 21.3c kDa NuoI 20.3 2 x [4Fe-4S] (N6a, N6b) NDUFA1 MWFE I - 9.8 kDa 8.0 NDUFA2 B8 I Met; N-acetyl 10.5 kDa 10.8 NDUFA3 B9 I Met; N-acetyl 9.3 kDa 9.3 NDUFA4 MLRQ nd - - 9.4 NDUFA5 B13 I Met; N-acetyl 29.9 kDa 13.3 NDUFA6 B14 I Met; N-acetyl 14.8 kDa 15.0 NDUFA7 B14.5a I Met; N-acetyl - 12.4 NDUFA8 PGIV I Met 20.8 kDa 20.0 60

Mitochondrial complex I

NDUFA9 39 kDa I MTS 1-35 40 kDa 38.9 NADPH NDUFA10 42 kDa I (loosely) MTS 1-35 - 37.1 NDUFA11 B14.7 I Met; N-acetyl 21.3b kDa 14.9 NDUFA12 B17.2 I N-acetyl 13.4 kDa 17.1 NDUFA13 B16.6 I Met; N-acetyl predicted orthologuee 16.6 NDUFB1 MNLL I Met - 7.0 NDUFB2 AGGG I MTS 1-33 predicted orthologuee 8.6 NDUFB3 B12 I Met; N-acetylc - 11.3 His-methylated NDUFB4 B15 I & I Met; N-acetyl predicted orthologuee 15.1 NDUFB5 SGDH I MTS 1-46 - 17.0 NDUFB6 B17 I Met; N-acetyl - 15.4 NDUFB7 B18 I Met; N-myristoyl predicted orthologuee 16.3 NDUFB8 ASHI I MTS 1-28 19 kDa 18.8 NDUFB9 B22 I Met; N-acetyl predicted orthologuee 21.7 NDUFB10 PDSW I Met 12.3 kDa 20.7 NDUFB11 ESSS I MTS 1-29 predicted orthologuee 17.3 NDUFAB1 SDAP I & I MTS 1-68 9.6 kDa ACP 10.2 Ser-phosphopantetheine NDUFC1 KFYI I MTS 1-27 - 5.9 NDUFC2 B14.5b I N-acetylc - 14.2 21.3 kDa 21.3a kDa 20.9 kDa 17.8 kDa a Subunit distribution over subcomplexes according to reanalysis of the resolution of bovine complex I into subcomplexes by Carroll et al.[21] b MTS: cleavage of mitochondrial targeting sequence (number of amino acids from human sequences acquired from the NCBI protein database: www.ncbi.nlm.nih.gov); Met: cleavage of N-terminal methionine; N-acetyl: acetylation of N-terminal residue; N-myristoyl: myristoylation of N-terminal residue c Subunits B14.5b and B12 are partially acetylated d Adopted from Videira and Duarte[23] e Predicted orthologue by Gabaldon et al.[24] f In E. coli both subunits are fused g Predicted masses of the human mature proteins in kDa adopted from Murray et al.[25]. NDUFC1, ND4L, and ND6 were not detected by Murray et al.[25] 61

Chapter III modified N-termini, the so-called B-subunits of the bovine enzyme (Tab 1). Modifications mostly concern acetylation of the first residue after removal of the N-terminal methionine, although for some subunits acetylation of the initiator methionine occurs. In one case, the B18 subunit (NDUFB7), myristoylation occurs instead of acetylation. Six subunits either with or without N-terminal methionine removal remain unmodified[26]. The function of N- terminal acetylation of complex I subunits is not known, although a role in post-translational transfer of the respective subunits into mitochondria is proposed. The mirystoyl group of the B18 subunit, which is part of the membrane arm, has been assumed to bind the subunit to the mitochondrial inner membrane[26]. Besides the permanent N-terminal modifications, several other types of post-translational modifications have been identified in the extensively analyzed bovine and human mitochondrial complex I. In addition to the incorporation of eight iron-sulfur clusters, a few other post-translational modifications occur in complex I subunits. Methylation of several histidine residues takes place in the B12 subunit (NDUFB3), and the SDAP subunit (NDUFAB1) carries a phosphopantetheine prosthetic group[26]. In contrast to N-acetylation, protein methylation is reversible by demethylases, and methylation-demethylation reactions are thought to have regulatory functions[26]. The phosphopantetheine cofactor is important for the function of the protein in a soluble state, in type II mitochondrial fatty acid synthesis, but its significance in the complex I-bound state is less clear[27]. In addition, several complex I subunits are known to undergo additional post-translational phosphorylation, for instance the 39-kDa bovine subunit (NDUFA9) and an 18-kDa subunit of complex I. Reversible phosphorylation of complex I subunits has been implicated in regulation of enzymatic activity and possibly affects electron transfer and superoxide production[28]. The 39-kDa subunit of complex I was phosphorylated at a tyrosine residue and putative tyrosine-phosphorylated subunits for the other complexes have been identified. From these findings it has been hypothesized that tyrosine kinases and phosphatases are involved in the regulation of oxidative phosphorylation[29]. The phosphorylated 18-kDa subunit of complex I was assigned by Papa and colleagues as the AQDQ subunit (NDUFS4) and they demonstrated that cAMP-dependent phosphorylation of the 18-kDa subunit can regulate complex I activity and overall respiratory activity[30,31]. Later, by a combination of Edman sequencing and mass spectrometric analysis, Walker and colleagues identified the phosphorylated 18-kDa subunit as the ESSS subunit (NDUFB11) with the phosphorylation site at serine-20, and demonstrated that it is was not the AQDQ subunit (NDUFS4) that was phosphorylated[32]. They also found a second phosphorylated protein, the MWFE subunit (NDUFA1), with a phosphorylation site at serine-55. Both sites conform to the standard motifs for cAMP-dependent phosphorylation sites[33]. A third phosphorylated subunit is the bovine 42-kDa subunit (NDUFA10), located in the hydrophobic membrane arm[34]. In

Mitochondrial complex I contrast to the phosphorylation sites in the ESSS and MWFE subunits, serine-59 of the 42- kDa subunit appeared to be phosphorylated when isolated directly from mitochondria, i.e. without prior kinase treatment[28].

Topology of complex I Over the years, the group of Walker obtained considerable information about the subunit topology of bovine complex I. Extensive analyses of the intact complex and various subcomplexes from bovine heart mitochondria have played an important role in defining the subunit composition of complex I, and resulted in the localization of the individual subunits to structurally and functionally distinct parts of the complex (Fig 1). With mildly chaotropic detergents (LDAO), intact complex I can be resolved into four subcomplexes: I , I , I , and I [20,21,36]. Separation of the subunits has been carried out by three independent methods (1D SDS-PAGE, 2D IEF/SDS-PAGE, and reversed-phase HPLC) and subsequent analysis of the individual subunits has been done by tryptic peptide mass fingerprinting and tandem mass spectrometry[21]. Subcomplex I consists of the hydrophilic peripheral arm plus part of the hydrophobic membrane arm, as subcomplex I contains the main part of the membrane arm. However, some subunits are not found in either subcomplex I or I and can together be considered as subcomplex I . Under slightly different conditions subcomplex I dissociates to produce subcomplex I representing the hydrophilic or peripheral arm. I contains 15 subunits including the seven nuclear DNA-encoded core subunits carrying all redox cofactors: the FMN and eight iron-sulfur clusters (Fig 1). These subunits, which are present in apparently stoichiometric amounts[9], are predominantly hydrophilic; only one subunit, B16.6 (NDUFA13), carries a transmembrane helix. Subcomplex I consists of the subunits of I and nine additional subunits in apparently stoichiometric amounts. Containing several transmembrane helices, these subunits are likely to anchor subcomplex I to the inner membrane. Since the subcomplex I B14.7 subunit (NDUFA11) is also predicted to contain transmembrane helices and is detected in trace amounts in subcomplex I , it can be assumed that the B14.7 subunit together with the B16.6 subunit is located in the small connection between the peripheral arm and the membrane arm of complex I. The 42- kDa subunit is weakly associated with the complex and probably bound to subcomplex I . Subcomplex I , consisting of twelve subunits in apparently stoichiometric amounts (and a thirteenth, the B14.5b subunit, in substoichiometric amounts), contains most transmembrane helices of all subcomplexes and constitutes the major part of the membrane arm. Two subunits, the SDAP (NDUFAB1) and B15 subunits (NDUFB4), are present in both subcomplexes I and I in substoichiometric amounts, and are probably located at the boundary between the two subcomplexes[21]. The MLRQ subunit (NDUFA4) was not

Chapter III

Figure 1. Subunit composition of mammalian complex I. Subunits have been grouped by the subcomplex they have been identified in, after complex I fractionation according to Walker and coworkers[21]. The human subunit nomenclature is shown in blue, the bovine subunit nomenclature is shown in grey. Subunits that have been reported harboring pathogenic mutations causing complex I deficiency are depicted with an *. (Figure adapted from Nijtmans et al.[35]).

detected in any of the subcomplexes; its presence in complex I is proposed to be dependent on the presence of cardiolipin. The remaining five subunits, KFYI (NDUFC1), ND1, ND2, ND3, and ND4L reside in subcomplex I . Subunits ND1 and ND2 are grouped together, as are subunits ND4 and ND5. Together with the PSST subunit (NDUFS7), carrier of iron-sulfur cluster N2, which is generally assumed to be the immediate electron donor for ubiquinone, and the 49-kDa subunit (NDUFS2), ND1 has implicated in the formation of the ubiquinone binding pocket. With the N2 cluster placed in or close to the centre of the connecting domain, ND1 and proton translocating subunit ND2 are therefore supposed to be located near the thin stalk of the complex, connecting the peripheral and membrane arms[37]. In contrast, the other suspected proton translocating subunits, ND4 and ND5, are believed to be located at the distal part of the membrane domain[2], as has been proved for E. coli complex I[13]. These findings are

Mitochondrial complex I indicative of a combined, i.e. redox-driven and conformational-driven, mechanism of energy transduction. For the human enzyme, Murray and colleagues identified 42 homologues of the 45 bovine heart complex I subunits by mass spectrometry analysis of complex I from human heart mitochondria isolated by means of an immunocapture method[25]. Keeping in mind that all characterized bovine genes encoding complex I subunits have homologues in the human genome (Tab 1; Fig 1), together with the high degree of subunit homology between the bovine and the human species, the bovine enzyme can be regarded as a bona fide model for human complex I.

Function of individual subunits While for the 14 core subunits of complex I, containing all redox centers and substrate- binding sites, primary roles in electron transfer and proton translocation have been established, the functions of the remaining 31 accessory or supernumerary subunits is less understood. They may have general roles in preventing ROS generation and protection against oxidative damage by shielding redox groups from reaction with oxygen, in binding of the complex to the membrane, and in the stabilization of the enzyme by forming a scaffold around the core subunits[9,38]. Also more specific functions in the regulation of activity or the assembly of other subunits into the holocomplex have been implied. However, some subunits are known to carry out an additional function besides their role in energy transduction. They are involved in distinct biochemical processes in the same or even another subcompartment of the cell. Others show homology to protein families suggesting specific functions that have yet to be substantiated by experimental evidence (see below)[9]. Two nucleotide-binding sites, one for NADH and one for NADPH, have been established in mitochondrial complex I. The first, NADH-binding site, is found in the 51-kDa core subunit (NDUFV1), which also binds the FMN, providing the conversion of the 2-electron donor NADH to the 1-electron transferring iron-sulfur clusters[39]. Together with the 24-kDa subunit (NDUFV2) and the 10-kDa subunit (NDUFV3), the 51-kDa subunit forms the flavoprotein part of the enzyme. The other nucleotide-binding site, NADPH is assigned to the 39-kDa accessory subunit (NDUFA9). This subunit shows a considerably higher sequence homology between mammals and fungi than most of the accessory subunits. The conserved nucleotide-binding site of the 39-kDa subunit is characteristic of a heterogeneous family of reductases/isomerases, all biosynthetic enzymes with NADH or NADPH cofactors[38]. In N. crassa complex I, the tightly bound NADPH is shielded from the other redox groups, not participating in the electron transfer chain. Interestingly, lack of the subunit leads to loss of complex I activity[38].

Chapter III

Analogous to known interactions between members of the reductase/isomerase family, and acyl carrier proteins (ACPs), it has been hypothesized that the 39-kDa subunit may form a ‗biosynthetic module‘ with the SDAP subunit (NDUFAB1), another highly conserved accessory subunit with a putative biosynthetic function[9]. The bovine SDAP subunit and its N. crassa homologue are ACPs with a phosphopantetheine prosthetic group and are closely related to the ACPs involved in fatty acid biosynthesis in bacteria and chloroplasts. A possible role for the ACP/SDAP subunit of complex I in mitochondrial de novo fatty acid synthesis, elongation, or desaturation has been suggested. Recently, it was found that most of the SDAP subunit is present as a soluble protein in the mitochondrial matrix of bovine heart mitochondria, and thus available to carry the intermediates of the type II fatty acid synthesis. Its significance as a complex I-bound subunit, however, is less clear[27]. The MWFE subunit (NDUFA1), being an integral membrane protein of the I subcomplex, is probably located in the membrane domain close to the stalk of the complex, and has a key function in complex I assembly. It has been suggested that presence of the MWFE protein is required for the synthesis of the mtDNA-encoded ND subunits and their incorporation into the complex[40,41]. Since MWFE has been identified as one of the complex I subunits that can be phosphorylated, functional activation upon phosphorylation can be speculated. The NDUFA13 subunit (B16.6), the only hydrophobic subunit of the peripheral domain, is also known as GRIM-19, for gene associated with retinoid-interferon-induced mortality 19 protein, and is the product of a cell death regulatory gene induced by interferon- and retinoic acid which promotes apoptosis[42]. By biochemical and cellular studies, a dual function for GRIM-19 has been implicated: one involved in the OXPHOS system as a structural component of complex I, the other playing a key role in initiation of apoptosis[43,44]. The question arises whether GRIM-19 is involved in independent cellular processes in different subcellular compartments, or whether GRIM-19 is the next link connecting mitochondria and apoptosis[9]. The nuclear DNA-encoded subunit B14.7 (NDUFA11) shows homology to Tim proteins (Tim17, Tim22 and Tim23) of the translocase of the inner membrane 17/22 (TIM17/22) family, which are involved in protein import from the cytosol into the mitochondrial matrix and inner membrane[20,24]. A related protein import mechanism may be operating for proteins synthesized in the mitochondrial matrix. It has therefore been proposed that the B14.7 subunit may be involved in the incorporation of the mtDNA-encoded ND subunits into the mitochondrial inner membrane. Two other subunits, NDUFA2 (B8) and NDUFA10 (42 kDa), have shown homology to mitochondrial ribosomal proteins L43 and S25, and to mitochondrial thymidine kinase 2 (TK2), respectively[24], suggesting a possible connection between complex I and mitochondrial protein synthesis that has to be subjected to thorough functional analysis.

Mitochondrial complex I

Homology studies like these can provide useful clues in determining the functions of the many accessory subunits of mitochondrial complex I.

The assembly process of mitochondrial complex I The Neurospora crassa model The complex I assembly process has been studied in detail in the aerobic fungus Neurospora crassa[45]. The hydrophilic peripheral arm and the hydrophobic membrane arm are formed independently. A small (~200 kDa) and a large (~350 kDa) intermediate membrane-bound subcomplex are joined together to form the membrane arm[46]. In N. crassa mutants obtained by gene disruption, the lack of single subunits causes a complete block in the assembly pathway. In some mutants of a subunit of the peripheral arm, a complex I lacking only that single subunit is assembled (51 kDa; 39 kDa; 24 kDa). In most cases, however, owing to loss of one of its subunits, the peripheral arm remains unstable, leading to accumulation of only the membrane arm. In its turn, lacking a subunit of the membrane arm, the peripheral arm is still assembled, and accumulates together with the small and large assembly intermediate complexes of the membrane arm. One subunit of the peripheral arm, the acyl carrier protein (SDAP) represents an exception. Loss of this subunit not only prevents peripheral arm assembly but also disturbs formation of the membrane arm[47,48]. In N. crassa, two complex I intermediate associated proteins, CIA30 and CIA84, play an essential role in the assembly of the membrane arm. Since they are transiently associated with the large membrane arm assembly intermediate and are not part of holocomplex I, both are thought to function as chaperones. Travelling back and forth between a pool of monomers and the assembly intermediates the CIA proteins participate in many assembly cycles[46]. Loss of either CIA protein will prevent the formation of the large membrane arm assembly intermediate, leading to accumulation of the small assembly intermediate and the peripheral arm. Functioning as chaperones, it has been suggested that they might maintain the assembly-competent state of the large membrane arm intermediate, preventing aggregation and misfolding[49].

The assembly process of mammalian complex I Several recent studies of disturbed assembly processes, investigating the accumulation of assembly intermediates, have provided some insights in the assembly process of mammalian, and especially human, complex I[50,51]. Some subunits are shown to be essential for the assembly of others into the complex. For instance, the hydrophobic subunits ND4 and ND6 have been shown to be essential for the assembly of other ND subunits into the complex[52-54]. Loss of the ND4 subunit will prevent the formation of the complex I 67

Chapter III membrane arm[55] and mutation of subunits ND6 and ND1 will lead to a severe reduction of holocomplex I levels[56]. Subunit ND2 is also needed for the assembly and/or stability of complex I. In a patient with a mutation in ND2, complex I assembly is compromised, leading to the formation of subcomplexes without NADH dehydrogenase activity[50]. In contrast, subunit ND5 is not essential for assembly of the other ND subunits into the complex, and loss of ND5 leads only to a lower efficiency of assembly or to instability of the membrane arm. ND5 is likely to be located at the periphery of the membrane arm, and therefore may be the last of the ND subunits to be assembled into the complex[55]. Nevertheless, since synthesis of ND5 is rate limiting, ND5 is essential for complex I activity[52]. In addition, loss of subunit ND3 will also lead to a relatively normal assembly profile[57]. The MWFE (NDUFA1) subunit also has a key role in the process of complex I assembly. With the development of a conditional complex I assembly system in Chinese hamster fibroblasts, Scheffler and coworkers showed that the insertion and stabilization of some ND subunits is dependent on the presence of MWFE and some other nuclear DNA-encoded subunits. The simultaneous insertion of the ND subunits and MWFE into the membrane and interaction with some other nuclear DNA-encoded subunits, resulting in the formation of an assembly intermediate, appears to be a crucial step in assembly of mammalian complex I[41]. Loss of one or all seven of the ND subunits encoded by the mitochondrial genome, in contrast, has no effect on the protein levels of the nuclear DNA-encoded complex I subunits of the peripheral arm[55]. The majority of the subunits of the peripheral arm are most likely already associated in an assembly intermediate subcomplex[22]. The PSST subunit, which is absent in this subcomplex, forms an exception. The localization of the PSST subunit near the membrane domain may explain its dependence on the presence of these integral membrane proteins, i.e. the ND subunits. Several accumulated subcomplexes contained the NDUFS2 (49 kDa), NDUFS3 (30 kDa) and NDUFS8 (TYKY) subunits, which are thought to constitute the complex I connecting module between the membrane arm and the peripheral arm[22]. Lack of the TYKY subunit (NDUFS8), another subunit of the peripheral domain that is in close contact with the PSST subunit, also reduces the levels of subunits NDUFV1 (51 kDa), NDUFA10 (42 kDa), and NDUFA9 (39 kDa). The suggested interaction with these subunits underlines the proposed function of subunit NDUFS8 in assembly or stability of complex I, specifically in connecting the membrane and peripheral domains of complex I[58,59]. Recently, this has been confirmed in a patient with mutations in the NDUFS8 gene, for which assembly of the complex I holoenzyme was prevented almost completely[60].

The human complex I assembly pathway In 2003, a possible pathway for human complex I assembly was proposed[50] that differs significantly from the N. crassa model[46]: many more assembly intermediates are

Mitochondrial complex I encountered in the human pathway. For the human enzyme, at least seven distinct intermediate subcomplexes are identified, as was observed in a cohort of four patients with complex I deficiency. Some of the subcomplexes contain both peripheral and membrane arm subunits, suggesting that the peripheral and membrane arms are not assembled in separate, independent pathways[50]. Since different genetic defects underlying complex I deficiency reveal a common set of subcomplexes, these subcomplexes most likely represent intermediate complexes of the assembly pathway. Similar intermediate subcomplexes were observed in an additional study of patients with mutations in nuclear DNA-encoded subunits[60]. Nevertheless, we must take into account that the accumulation of lower- molecular weight subcomplexes in complex I-deficient patients could be due not only to an assembly defect but also to disturbance of stability of the complex[60]. Accumulation of an additional intermediate subcomplex of approximately 800 kDa (referred to as ~830 kDa by Antonicka et al.[50]) has been observed in patients with a mutation in the NDUFS4 subunit[31], indicative of defects in complex I assembly or stability. The existence of this relatively large subcomplex of ~800 kDa, suggests that NDUFS4 will be incorporated at a late stage in the assembly process[60]. It contains subunits of the peripheral domain, NDUFS3, NDUFS5, and NDUFA9, but shows no NADH dehydrogenase activity in a complex I in-gel activity assay. Moreover, this intermediate subcomplex has particularly been found in patients with mutations in the NDUFS4 and NDUFV1 genes, but not in other complex I- deficient patients or normal controls[50]. The accumulation of an approximately 750-kDa intermediate subcomplex observed in patients with mutations in the peripheral arm subunit NDUFS6[57] possibly resembles the assembly profile of the NDUFS4 and NDUFV1 patients. Therefore, NDUFS6 is also likely to play an essential role in complex I assembly or stability[57]. Regarding the distribution of eleven studied subunits over the seven distinguishable subcomplexes, the following groups of subunits can be considered to follow similar assembly routes: NDUFS2, NDUFS3, and NDUFA9; NDUFV2, NDUFS4, and NDUFS7; ND1 and NDUFA2; and NDUFS5 and NDUFA6. Combining these findings, the following assembly pathway has been proposed. First, subunits of the peripheral arm, including the 49-, 39-, and 30-kDa subunits, are assembled together and associated with some other subunits of the peripheral arm, resulting in a second assembly intermediate. Second, the partially assembled peripheral arm is coupled to several subunits of the membrane arm, including ND1, to form a membrane-bound complex. Subsequently, a subcomplex including the PSST, 24-, and 18- kDa subunits is associated with the peripheral arm; and finally the membrane arm is completed[50,61]. The second assumption is in conflict with the semi-sequential human complex I assembly pathway proposed by Ugalde and colleagues[51], which states that the membrane arm subunits ND1, ND6, and B17, are assembled into a ~700-kDa membrane

Chapter III arm subcomplex before association with a ~600-kDa peripheral arm subcomplex takes place. This model more or less resembles the N. crassa model, for which it is suggested that complex I follows a modular assembly pathway, i.e. complex I is formed by combining three different evolutionarily conserved modules: an NADH-dehydrogenase module, a hydrogenase module, and a transporter module[14,62].

Involvement of assembly chaperones For at least half of the patients with a complex I deficiency caused by disturbed assembly of the holocomplex, the primary defect cannot be assigned to the structural components of the complex. While assembly chaperones have been described for the other complexes of the OXPHOS system, proteins required for assembly of mammalian complex I long remained unknown. However, four years ago, the human homologue of the N. crassa CIA30, NDUFAF1, was characterized[63] and recently its functioning in complex I assembly has been investigated[64]. Reduction in the amount of NDUFAF1, achieved by means of RNA interference, leads to a reduced amount of fully assembled complex I and complex I activity, whereas overexpression of NDUFAF1 leads to an increase of fully assembled complex I. Furthermore, NDUFAF1 associates with two high-molecular weight assembly intermediate complexes of approximately 600 kDa and 700 kDa. Expression of NDUFAF1 has been investigated in a conditional complex I assembly system, comprising inhibition and recovery of mitochondrial protein synthesis, and in patients with a disturbed complex I assembly, caused by mutations in one of the subunits. When the assembly of the membrane arm is either disturbed (as shown for a patient with an ND5 mutation) or induced (in the conditional complex I assembly system), NDUFAF1 becomes more predominant in the ~700-kDa complex. Therefore, it has been concluded that NDUFAF1 is an important protein for the assembly and/or stability of human complex I, and might act as a chaperone by preventing misfolding or degradation of assembly intermediates[64]. The predicted human homologue of the other assembly chaperone of N. crassa complex I, CIA84, which has been identified by means of comparative genomics (PTCD1, GenBank accession number: NM_015545)[24], is still awaiting functional characterization. A third assembly chaperone for mitochondrial complex I has also been identified and characterized recently[65]. By comparing genomes of yeast species that do (aerobic yeasts) and do not (fermentative yeasts) express complex I, several candidate genes for complex I assembly factors were selected. Among them is the NDUFAF2 gene that encodes complex I assembly factor 2 (also known as B17.2L for B17.2-Like protein), a paralogue of the small subunit of the peripheral arm NDUFA12 (B17.2). Mutation of the NDUFAF2 gene leads to complex I assembly defects, as both the amount of holocomplex I and the catalytic activity are compromised, and assembly intermediates of 380 and 480 kDa are accumulated. The

Mitochondrial complex I

NDUFAF2 protein associates specifically with the accumulating 830-kDa intermediate subcomplex observed in patients with mutations in the NDUFS4 and NDUFV1 genes. The 830-kDa subcomplex, co-immunoprecipitated by anti-NDUFAF2 antibody, contained subunits of both the peripheral (NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFV1) and membrane (ND1) arms. While a chaperone function in the late stages of complex I assembly has been proposed, further functioning of the protein needs to be investigated. The same protein has been studied earlier, under the name mimitin, for myc-induced mitochondrial protein, and has been assigned to function in myc-dependent cell proliferation in tumor cell lines[66]. Another mitochondrial protein interacting with complex I subcomplexes was detected by Bourges and colleagues. In human cells lacking ND subunits, mitochondrial prohibitin was detected in a subcomplex containing subunits NDUFS2 (49 kDa), NDUFS3 (30 kDa) and NDUFS8 (TYKY), which are thought to constitute the complex I connecting module between the membrane arm and the peripheral arm[55]. Prohibitin is a mitochondrial inner membrane protein, which is known in yeast to interact with complex IV subunits to prevent their proteolysis by m-AAA proteases. By stabilizing newly synthesized mitochondrial translation products, prohibitin is now proposed to act as a chaperone not only for complex IV assembly[67] but also for complex I assembly[55].

Supercomplex formation The arrangement of the OXPHOS complexes into higher supramolecular structures, called supercomplexes, seems to be well established in various organisms, as bacteria[68], lower eukaryotes and mammals[69]. By the use of different solubilization detergents (e.g. lauryl maltoside, digitonin, Triton X-100) supercomplexes with varying stoichiometries have been demonstrated in bacterial as well as mitochondrial membranes[70]. Supercomplexes comprising complexes I, III and IV are also called respirasomes. Supercomplexes containing monomeric complex I, dimeric complex III, and up to four complexes IV (I1 III2 IV0-4) have been reported for bovine mitochondria. In bovine mitochondria nearly all complex I associates with dimeric complex III, while association of complex IV seems to be more detergent-sensitive[70]. The largest of the supercomplexes,

I1III2IV4, has been proposed to constitute a complete respirasome[69]. Schägger proposed a model for the mammalian OXPHOS system consisting of two supercomplexes with and without complex I (I1III2IV4 and III2IV4) accompanied by monomeric complex II and dimeric complex V in a 2:1:3:3 ratio[70]. The functional significance of supercomplex formation can be supported by the following characteristics: channeling of the substrates ubiquinol and cytochrome c, preventing

Chapter III competition from other enzymes; catalytic enhancement as a result of reduction of the diffusion time of substrates; sequestration of the reactive intermediate ubisemiquinone, preventing the generation of the pathogenic superoxide by reaction with oxygen[69]; and stabilization of the individual OXPHOS complexes[71,72]. Supercomplex formation is essential for the assembly and/or stability of complex I. For instance, decreased amounts of membrane-bound complex I have been shown for Paracoccus denitrificans mutants lacking complexes III and IV, suggesting a decreased stability of complex I when not assembled into a supercomplex[68]. For human mitochondria it has also been established that complex I is stabilized by the association with dimeric complex III in a supercomplex. Primary genetic defects affecting complex III assembly, e.g. mutations in the mtDNA cytochrome b gene, result in secondary complex I deficiency by failing to form supercomplexes[71]. Since complex I assembly is not compromised, it has been concluded that the stability of complex I is severely affected. Additionally, cytochrome b mutations affecting complex III activity, while not disturbing complex III assembly, have no effect on complex I and result in an isolated complex III deficiency[71]. There are also examples of mutations in nuclear genes encoding the QP-C subunit of complex III[73] and the complex III assembly factor BCS1L[74] that disturb complex III assembly and present with a combined complex I + III deficiency, supporting the significance of supercomplex formation for complex I stability. Conversely, primary complex I defects affecting the stability of complex III have also been reported for patients with mutations in the NDUFS2 and NDUFS4 genes[60,75]. It has to be further investigated whether the physical interaction between complexes I and III is mediated through these subunits NDUFS2 and NDUFS4.

Clinical manifestation and genetic heterogeneity Mitochondrial disorders, or more specifically diseases caused by disturbances in the mitochondrial OXPHOS system, are the most frequent inborn errors of metabolism, with an incidence of 1 in 5,000 live births[76,77]. Owing to dual genetic control, with both nuclear and mitochondrial genomes contributing to the complexity of the OXPHOS system (which consists of 89 structural components in total), defects in oxidative phosphorylation result in a wide variety of clinical phenotypes, presenting from infancy to late adulthood. OXPHOS disorders can involve many different organs and tissues, in an organ-specific or multisystemic form. However, tissues with a high energy demand, such as brain, heart and skeletal muscle, are more dependent on OXPHOS, and therefore more vulnerable to defects than others. Heredity patterns can vary from maternal, autosomal dominant or recessive, to X-linked and sporadic. Especially for mtDNA mutations, there is no clear genotype-

Mitochondrial complex I

phenotype relation: different mtDNA mutations either in the same or in different mitochondrial genes may lead to similar clinical phenotypes, whereas the same mutations or different mutations in the same gene may result in several distinct phenotypes. Clinical manifestation of pathogenic mutations in the mitochondrial DNA is often associated with onset in late childhood or adulthood, although some cases of mtDNA mutations with an early onset are described. Variability in clinical course and intrafamilial presentation are important characteristics of mtDNA mutations[78]. Particular features of mitochondrial genetics contributing to the heterogeneous nature of OXPHOS disorders are maternal inheritance, heteroplasmy and the threshold effect, and mitotic segregation. Mitochondria are exclusively maternally inherited, so primary mtDNA mutations and their respective phenotypes are transmitted only from mothers to their progeny. Since hundreds or thousands of copies of mtDNA are present in each cell (called polyplasmy), different populations of wild-type and mutant mtDNA can occur in cells and tissues, a feature known as heteroplasmy. The threshold for a pathogenic mtDNA mutation is the minimum percentage of mutant mtDNA that is needed for biochemical expression and clinical manifestation of the disease. The threshold of a certain mutation can vary between tissues, in relation to their dependence on oxidative metabolism. We speak of mitotic segregation when, as a consequence of the redistribution of mitochondria, the degree of heteroplasmy changes during cell division. As a result, the pathogenic threshold can be crossed, altering the phenotype of that particular cell line or tissue. Among the group of OXPHOS disorders, isolated complex I deficiency is the most common cause of OXPHOS dysfunction[78-80]. Although complex I deficiency is the responsible biochemical defect for a heterogeneous group of diseases, some distinction can be made. Clinical phenotypes of complex I deficiency caused by mutations in nuclear DNA-encoded subunits generally present in infancy and early childhood, while presentation of the disease due to mutations in the mtDNA often occurs in late childhood or adolescence. Among the latter group there is a great variability in clinical course and intrafamilial presentation. mtDNA mutations have been identified in each of the seven ND genes, expressing various clinical symptoms, either restricted to a single organ or tissue, e.g. Leber hereditary optic neuropathy (LHON; MIM 535000), or presenting in a multisystemic manner, e.g. mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS; MIM 540000). LHON is a common cause of central vision loss in young adults. It is a maternally inherited disorder characterized by bilateral, usually sequential, optic neuropathy, presenting in late adolescence or early adulthood. In some patients a more widespread neuromuscular disease is seen with cardiac involvement or encephalopathic features, particularly dystonia[81,82]. Co- occurrence of multiple sclerosis-like white-matter lesions has also been reported[83]. Three

Chapter III common point mutations are responsible for the majority of the LHON cases; these are G3460A in ND1, G11778A in ND4, and T14484C in ND6. A few other mutations associated with LHON have been described in the ND2, ND5[84], and ND4L[85] genes, but the ND1 and ND6 genes are considered as mutational hot spots for LHON[86,87]. The combination of LHON and dystonia has also been assigned to mutations in ND6[88]. MELAS syndrome is probably the most common mitochondrial encephalomyopathy, and is characterized by mental retardation, psychomotor deterioration, migraine, seizures, convulsions and lactic acidosis in serum or cerebrospinal fluid (CSF). Presentation is usually during childhood or adolescence and in some cases deafness and diabetes can develop at a later stage. Mutations have been found in the ND1[56], ND5[89], and ND6[90] genes. The ND5 gene appeared to be a hot spot for mutations associated with MELAS, but also for several overlap syndromes. Many MELAS overlap syndromes have been described with features typical of other mitochondrial diseases, such as cardiomyopathy, myoclonus and ragged-red fibers, and ataxia[81]. For instance, LHON/MELAS[91], MELAS/MERRF[92], and even Leigh/MELAS[93] overlap syndromes, all associated with ND5 mutations, have been reported[94]. LHON/MELAS overlap syndrome associated with mutations in ND1 has also been reported[95].

Nuclear genes associated with complex I deficiency When isolated complex I deficiency presents in the neonatal period, infancy or early childhood, most probably the genetic defect must be sought in the nuclear genes. Also mendelian inheritance patterns indicate nuclear gene defects, mostly being autosomal recessive. A diversity of clinical phenotypes has been described to occur in complex I- deficient patients, which can be classified into five main clinical phenotypes: severe neonatal lactic acidosis, also known as fatal infantile lactic acidosis (FILA), Leigh syndrome, neonatal cardiomyopathy with lactic acidosis, leukodystrophy with macrocephaly, and hepatopathy with renal tubulopathy[78,96]. Finally, there is a group of unspecified mitochondrial encephalomyopathies that present a combination of neuromuscular symptoms that could not be assigned to a specific phenotype. The most common clinical phenotype associated with early-onset complex I deficiency is Leigh syndrome, first described as subacute necrotizing encephalomyelopathy[97]. Leigh syndrome is an early-onset, progressive neurological disorder characterized by motor and intellectual developmental delay, signs of brainstem and basal ganglia involvement, and increased lactate levels in blood and/or CSF[98]. Further clinical features are optic atrophy, ophthalmoparesis, hypotonia, ataxia and dystonia[76]. Mutations causing complex I deficiency associated with Leigh syndrome have been found in seven nuclear genes encoding complex I subunits: NDUFA1[99], NDUFA2[100], NDUFS1[101,102], NDUFS3[103], NDUFS4[104],

Mitochondrial complex I

NDUFS7[105], and NDUFS8[58,106]. Mutations in NDUFS4 have also been described for Leigh- like syndrome[75,107,108]. Whereas most cases of Leigh syndrome are caused by autosomal recessive nuclear gene defects, recently an increasing number of cases of maternally inherited Leigh syndrome (MILS) have been reported, associated with mutations in the mitochondrial complex I genes: ND2[109], ND3[110,111], ND4[112], ND5[113,114], and ND6[54,112,115]. Severe neonatal lactic acidosis, also known as FILA, is characterized by extremely high lactate levels in blood and CSF and high lactate/pyruvate ratios, resulting in severe metabolic crisis with secondary cardiac and respiratory failure[78,112]. Just recently, the first mutation in a gene encoding a complex I subunit, NDUFA11, causing FILA has been described[116]. Leukoencephalopathy or leukodystrophy with macrocephaly is an early-onset progressive neurological disorder characterized by progressive cerebral white-matter deterioration accompanied by enlargement of the head. Mutations have been described for the complex I genes NDUFV1[101,117] and NDUFS1[101,112]. Neonatal cardiomyopathy with lactic acidosis comprises early-onset, progressive hypertrophic cardiomyopathy, possibly accompanied by prominent neurological symptoms such as hypotonia, psychomotor retardation, microcephaly and cataract. Hypertrophic cardiomyopathy and encephalopathy has been associated with mutations in the complex I genes NDUFS2[118], NDUFV2[119] as well as NDUFA11[116]. Two cases of other, unspecified, mitochondrial encephalomyopathies have been described recently. Kirby and colleagues reported two cases diagnosed as lethal infantile mitochondrial disease in patients presenting with hypotonia, lethargy, seizures and a persistent lactic acidosis[57]. The responsible pathogenic mutations were located in the NDUFS6 gene, causing a complex I assembly or stability defect. Bugiani and colleagues described another interesting case of mitochondrial encephalopathy in a patient presenting with severe neonatal hypotonia, dysmorphic features, epilepsy and signs of brainstem involvement, associated with a markedly reduced residual complex I activity in muscle[112]. Interestingly, two heterozygous missense mutations were found in two different genes: NDUFS2 (paternally inherited) and NDUFA8 (maternally inherited), indicating digenic inheritance. It has been suggested that the pathogenesis of the biochemical defect could be the consequence of two dominant-negative mutations in different genes. In conclusion, complex I deficiency-causing mutations have been found in twelve nuclear genes encoding complex I subunits. Mutations in nine of these genes, i.e. NDUFA1, NDUFA2, NDUFS1, NDUFS3, NDUFS4, NDUFS6, NDUFS7, NDUFS8 and NDUFV1, cause brain and brainstem disorders, mostly Leigh(-like) syndrome or leukoencephalopathy, while mutations in the NDUFS2, NDUFV2 and NDUFA11 genes have exclusively been linked to hypertrophic cardiomyopathy and encephalomyopathy[116,118,119]. Although some mutations

Chapter III have a great share in a certain clinical phenotype, a genotype-phenotype relation is not clearly apparent. Expanding our knowledge of complex I biogenesis and functioning will aid in finding an explanation for the clinical variability and underlying genetic heterogeneity of complex I deficiency. In addition, probably numerous, yet unknown, nuclear factors are needed for proper assembly and functioning of complex I. Identification of these assembly factors will open a new field of possible candidates for complex I deficiency. The first mutation in a complex I assembly factor gene was found in the NDUFAF2 gene in a complex I-deficient patient with early-onset progressive encephalopathy. The clinical presentation resembles that of leukoencephalopathy with vanishing white matter[65]. Later, pathogenic mutations have been found in the NDUFAF1 gene of a patient presenting with cardioencephalomyopathy[120] and in the C6ORF66 gene, encoding a third putative complex I assembly factor, in six patients with mitochondrial encephalomyopathy or antenatal cardiomyopathy[121].

Neurodegeneration and secondary complex I deficiency Recently, complex I deficiency has been clearly associated with the pathogenesis of many devastating neurodegenerative disorders, including Parkinson disease[122,123]. It has become clear that the OXPHOS system may be impaired by mutation of many more mitochondrial and even non-mitochondrial proteins, or may be disturbed as a secondary effect of other biochemical defects of intracellular metabolism[124]. OXPHOS defects caused by genes encoding non-OXPHOS mitochondrial proteins, e.g. frataxin and paraplegin, are responsible for Friedreich ataxia and hereditary spastic paraparesis, respectively. Friedreich ataxia is an autosomal recessive neurodegenerative disease characterized by progressive ataxia, neuropathy, skeletal abnormalities and cardiomyopathy. Mutations in the nuclear gene encoding frataxin, which is involved in iron homeostasis in mitochondria, result in severe deficiencies of iron-sulfur cluster-containing complexes I-III and of the Krebs cycle enzyme aconitase[124]. Hereditary spastic paraparesis (HSP) is characterized by progressive spasticity and weakness of the lower limbs, possibly accompanied by neurological signs as retinopathy, optic atrophy, ataxia, dementia, and mental retardation. One of the genes responsible for HSP is the gene encoding paraplegin, an m-AAA metalloprotease, which has a proteolytic and chaperone-like function in the folding process of mitochondrial inner membrane proteins. Mutations in this gene have been associated with a reduced complex I activity and an increased sensitivity to oxidative stress, resulting in an autosomal recessive form of HSP[125]. Recently, it has been demonstrated in both yeast and mouse models that the m-AAA protease is directly involved in the proteolytic control of the mitochondrial ribosome assembly process by processing MrpL32, a subunit of the large ribosomal particle. Affecting mitochondrial protein synthesis, impairment of m-

Mitochondrial complex I

AAA protease activity has been proposed to not only disturb the assembly of complex I but also that of the other components of the OXPHOS system[126]. In Huntington disease (HD) and motor neuron disease (or amyotrophic lateral sclerosis), OXPHOS deficiency, and complex I deficiency in particular, is secondary to free radical generation, which is induced by mutations in a gene encoding a non-mitochondrial protein[124]. In Huntington disease, the responsible mutations are in a nuclear gene encoding a non-mitochondrial protein of unknown function, named huntingtin. In a HD patient with severe complex I deficiency, multiple mtDNA deletions were found indicating the involvement of OXPHOS defects and oxidative damage[127]. Similar complex I and other complex deficiencies secondary to oxidative damage by ROS have been implicated in Wilson disease[124]. For Parkinson disease (PD) it is known that accumulation of oxidative damage caused by reactive oxygen and nitrogen species, particularly to complex I, is responsible for the degeneration of dopaminergic neurons of the substantia nigra[25]. Recently, it has been shown that reduced complex I function in PD brain mitochondria arises from oxidation of its catalytic subunits from internal processes, rather than from external oxidative stress, and correlates with complex I misassembly[122]. A possible explanation may be provided by two other recent studies[128,129]. They independently linked clonal expansion of somatic mtDNA mutations, resulting in high levels of mtDNA deletions in individual cells, to the observed OXPHOS deficiency, specifically in neurons of the substantia nigra. Determining the primary defect underlying the mtDNA deletions in PD brain remains complicated. The high oxidative capacity and increased oxidative stress of the substantia nigra neurons may contribute to the accumulation of mtDNA deletions and impairment of the OXPHOS system, but also impaired mtDNA replication has been proposed as a possible cause[128]. However, the question whether additional mtDNA-[130] or nuclear DNA-encoded abnormalities in combination with environmental factors are required to cause dopaminergic neuronal loss needs to be addressed[82,112]. Furthermore, complex I deficiency has been implicated in Alzheimer disease (AD), Down syndrome (DS), schizophrenia and bipolar disorder[131]. A reduction in the protein levels of complex I subunits encoding genes NDUFV2 and NDUFS1 has been established in several brain regions from patients with Down syndrome and Alzheimer disease[132]; however, the relationship of the defect to the pathogenesis of the disease is not known. Mutations in the mtDNA ND1 gene[133] and polymorphisms in the promoter region of the nuclear NDUFV2 gene[134,135] associated with complex I deficiency, have both been suggested as genetic risk factors for bipolar disorder.

Chapter III

Concluding remarks Mitochondrial complex I is one of the most complicated enzymes of the eukaryotic cell. Forty-five subunits originating from two separate genomes and a number of cofactors constitute a membrane-bound enzyme complex that operates within a large respirasome structure. Owing to its bigenomic control, deficiency of complex I will lead to a broad spectrum of clinical symptoms and biochemical changes in various tissues. Many devastating neurodegenerative disorders have been associated with complex I deficiency, emphasizing the need to elucidate the molecular mechanisms underlying the functioning of complex I in energy transduction. The characterization of the 45 genes encoding complex I subunits has been an important first step in determining the structure of complex I. Establishing the subunit composition of complex I from bovine and human heart mitochondria and the assignment of the cofactors to their respective subunits has been important for our knowledge of its enzymatic function. Determining the arrangement of the iron-sulfur clusters with the X-ray crystallographic structure of the peripheral domain of complex I from T. thermophilus has been a further important step, providing more insight in the electron transfer mechanism and the subunits involved. When additional crystallographic structures of the membrane domain and holocomplex I become available, the coupling mechanism between electron transfer and proton translocation will be clarified. Characterization of the functions of individual subunits will also be needed to gain insight in the enzymatic function of complex I. The presence of some subunits raised questions about their function in complex I and their link to other biochemical processes. For instance, the NDUFA13 subunit (GRIM-19) provides a link between complex I and apoptotic cell death, and the NDUFAB1 subunit (SDAP), the acyl carrier protein, has been suggested to be involved in mitochondrial fatty acid synthesis. However, the functions of most of the accessory subunits remain to be explored. Although, all the structural components of complex I have been identified, little is known about the biogenesis of complex I and how its interaction with other complexes of the OXPHOS system is regulated. The first models of the human complex I assembly pathway are a step in the right direction towards solving the assembly process and need to be further investigated. Analysis of the complex I assembly status of patients with a complex I deficiency will provide more information on the mechanisms of complex I assembly and/or stabilization and on the consequences in a disturbed situation. In at least half of the patients, complex I deficiency is caused by disturbance of assembly of the holocomplex; however, little is known about the chaperones that guide the assembly process. The recent identification of the first complex I assembly chaperone associated with complex I deficiency is the beginning of exploring a new direction in the pathogenesis of complex I

Mitochondrial complex I deficiency. In a comprehensive comparative genomics analysis of the evolution of eukaryotic complex I, several paralogous groups resulting from gene duplications were detected for some complex I subunits. These paralogues of complex I subunits may have retained their functional interaction with complex I and may now be considered as candidates for complex I assembly factors[24]. In the future, more complex I-related proteins will be identified by means of a bioinformatics approach comparing whole genomes. Elucidating the mechanisms underlying the biogenesis and functioning of complex I and expanding our knowledge of the pathogenesis of complex I deficiency will be of utmost importance for performing adequate diagnostics and the development of future therapeutic treatment.

Acknowledgements Part of this work was supported by The Netherlands Organization for Scientific Research (NWO; grant number 901-03-156), the Prinses Beatrix Fonds and the European Union's Sixth Framework Programme for Research, Priority 1 ―Life Sciences, Genomics and Biotechnology for Health‖, contract number LSHM-CT-2004-503116‖.

Chapter III

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Mitochondrial complex I

57. Kirby DM, Salemi R, Sugiana C, Ohtake A, Parry L, Bell KM, Kirk EP, Boneh A, Taylor RW, Dahl HH, Ryan MT, Thorburn DR (2004) NDUFS6 mutations are a novel cause of lethal neonatal mitochondrial complex I deficiency. J Clin Invest 114(6):837-845 58. Procaccio V, Wallace DC (2004) Late-onset Leigh syndrome in a patient with mitochondrial complex I NDUFS8 mutations. Neurology 62(10):1899-1901 59. Chevallet M, Dupuis A, Lunardi J, van Belzen R, Albracht SP, Issartel JP (1997) The NuoI subunit of the Rhodobacter capsulatus respiratory Complex I (equivalent to the bovine TYKY subunit) is required for proper assembly of the membraneous and peripheral domains of the enzyme. Eur J Biochem 250(2):451-458 60. Ugalde C, Janssen RJ, van den Heuvel LP, Smeitink JA, Nijtmans LG (2004) Differences in assembly or stability of complex I and other mitochondrial OXPHOS complexes in inherited complex I deficiency. Hum Mol Genet 13(6):659-667 61. Vogel R, Nijtmans L, Ugalde C, van den Heuvel L, Smeitink J (2004) Complex I assembly: a puzzling problem. Curr Opin Neurol 17(2):179-186 62. Videira A (1998) Complex I from the fungus Neurospora crassa. Biochim Biophys Acta 1364(2):89- 100 Review 63. Janssen R, Smeitink J, Smeets R, van den Heuvel L (2002) CIA30 complex I assembly factor: a candidate for human complex I deficiency? Hum Genet 110(3):264-270 64. Vogel RO, Janssen RJ, Ugalde C, Grovenstein M, Huijbens RJ, Visch HJ, van den Heuvel LP, Willems PH, Zeviani M, Smeitink JA, Nijtmans LG (2005) Human mitochondrial complex I assembly is mediated by NDUFAF1. FEBS J 272(20):5317-5326 65. Ogilvie I, Kennaway NG, Shoubridge EA (2005) A molecular chaperone for mitochondrial complex I assembly is mutated in a progressive encephalopathy. J Clin Invest 115(10):2784-2792 66. Tsuneoka M, Teye K, Arima N, Soejima M, Otera H, Ohashi K, Koga Y, Fujita H, Shirouzu K, Kimura H, Koda Y (2005) A novel Myc-target gene, mimitin, that is involved in cell proliferation of esophageal squamous cell carcinoma. J Biol Chem 280(20):19977-19985 67. Nijtmans LG, de Jong L, Artal Sanz M, Coates PJ, Berden JA, Back JW, Muijsers AO, van der Spek H, Grivell LA (2000) Prohibitins act as a membrane-bound chaperone for the stabilisation of mitochondrial proteins. EMBO J 19(11):2444-2451 68. Stroh A, Anderka O, Pfeiffer K, Yagi T, Finel M, Ludwig B, Schägger H (2004) Assembly of respiratory complexes I, III, and IV into NADH oxidase supercomplex stabilises complex I in Paracoccus denitrificans. J Biol Chem 279(6):5000-5007 69. Schägger H, Pfeiffer K. (2000) Supercomplexes in the respiratory chains of yeast and mammalian mitochondria. EMBO J 19(8):1777-1783 70. Schägger H (2002) Respiratory chain supercomplexes of mitochondria and bacteria. Biochim Biophys Acta 1555(1-3):154-159 71. Acín-Pérez R, Bayona-Bafaluy MP, Fernández-Silva P, Moreno-Loshuertos R, Pérez-Martos A, Bruno C, Moraes CT, Enríquez JA (2004) Respiratory complex III is required to maintain complex I in mammalian mitochondria. Mol Cell 13(6):805-815 72. Schägger H, de Coo R, Bauer MF, Hofmann S, Godinot C, Brandt U (2004) Significance of respirasomes for the assembly/stability of human respiratory chain complex I. J Biol Chem 279(35):36349-36353 73. Haut S, Brivet M, Touati G, Rustin P, Lebon S, Garcia-Cazorla A, Saudubray JM, Boutron A, Legrand A, Slama A (2003) A deletion in the human QP-C gene causes a complex III deficiency resulting in hypoglycaemia and lactic acidosis. Hum Genet 113(2):118-122 74. de Lonlay P, Valnot I, Barrientos A, Gorbatyuk M, Tzagoloff A, Taanman JW, Benayoun E, Chrétien D, Kadhom N, Lombès A, de Baulny HO, Niaudet P, Munnich A, Rustin P, Rötig A (2001) A mutant mitochondrial respiratory chain assembly protein causes complex III deficiency in patients with tubulopathy, encephalopathy and liver failure. Nat Genet 29(1):57-60 75. Budde SM, van den Heuvel LP, Janssen AJ, Smeets RJ, Buskens CA, De Meirleir L, Van Coster R, Baethmann M, Voit T, Trijbels JM, Smeitink JA (2000) Combined enzymatic complex I and III deficiency associated with mutations in the nuclear-encoded NDUFS4 gene. Biochem Biophys Res Commun 275(1):63-68 83

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76. Smeitink J, van den Heuvel L, DiMauro S (2001) The genetics and pathology of oxidative phosphorylation. Nat Rev Genet 2(5):342-352 77. Smeitink J, Zeviani M, Turnbull DM, Jacobs HT (2006) Mitochondrial medicine: a metabolic perspective on the pathology of oxidative phosphorylation disorders. Cell Metab 3(1):9-13 78. Loeffen JL, Smeitink JA, Trijbels JM, Janssen AJ, Triepels RH, Sengers RC, van den Heuvel LP (2000) Isolated complex I deficiency in children: clinical, biochemical and genetic aspects. Hum Mutat 15(2):123-134 Review 79. Scaglia F, Towbin JA, Craigen WJ, Belmont JW, Smith EO, Neish SR, Ware SM, Hunter JV, Fernbach SD, Vladutiu GD, Wong LJ, Vogel H (2004) Clinical spectrum, morbidity, and mortality in 113 pediatric patients with mitochondrial disease. Pediatrics 114(4):925-931 80. Smeitink J, Sengers R, Trijbels F, van den Heuvel L (2001) Human NADH:ubiquinone oxidoreductase. J Bioenerg Biomembr 33(3):259-266 81. Schapira AH, Cock HR (1999) Mitochondrial myopathies and encephalomyopathies. Eur J Clin Invest 29(10):886-898 Review 82. Leonard JV, Schapira AH (2000) Mitochondrial respiratory chain disorders I: mitochondrial DNA defects. Lancet 355(9200):299-304 Review 83. Kovács GG, Höftberger R, Majtényi K, Horváth R, Barsi P, Komoly S, Lassmann H, Budka H, Jakab G (2005) Neuropathology of white matter disease in Leber's hereditary optic neuropathy. Brain 128(Pt 1):35-41 84. Brown MD, Voljavec AS, Lott MT, Torroni A, Yang CC, Wallace DC (1992) Mitochondrial DNA complex I and III mutations associated with Leber's Hereditary Optic Neuropathy. Genetics 130(1):163-173 85. Brown MD, Starikovskaya E, Derbeneva O, Hosseini S, Allen JC, Mikhailovskaya IE, Sukernik RI, Wallace DC (2002) The role of mtDNA background in disease expression: a new primary LHON mutation associated with Western Eurasian haplogroup J. Hum Genet 110(2):130-138 86. Valentino ML, Barboni P, Ghelli A, Bucchi L, Rengo C, Achilli A, Torroni A, Lugaresi A, Lodi R, Barbiroli B, Dotti M, Federico A, Baruzzi A, Carelli V (2004) The ND1 gene of complex I is a mutational hot spot for Leber's hereditary optic neuropathy. Ann Neurol 56(5):631-641 87. Chinnery PF, Brown DT, Andrews RM, Singh-Kler R, Riordan-Eva P, Lindley J, Applegarth DA, Turnbull DM, Howell N (2001) The mitochondrial ND6 gene is a hot spot for mutations that cause Leber's hereditary optic neuropathy. Brain 124(Pt 1):209-218 88. Jun AS, Trounce IA, Brown MD, Shoffner JM, Wallace DC (1996) Use of transmitochondrial cybrids to assign a complex I defect to the mitochondrial DNA-encoded NADH dehydrogenase subunit 6 gene mutation at nucleotide pair 14459 that causes Leber hereditary optic neuropathy and dystonia. Mol Cell Biol 16(3):771-777 89. Santorelli FM, Tanji K, Kulikova R, Shanske S, Vilarinho L, Hays AP, DiMauro S (1997) Identification of a novel mutation in the mtDNA ND5 gene associated with MELAS. Biochem Biophys Res Commun 238(2):326-328 90. Ravn K, Wibrand F, Hansen FJ, Horn N, Rosenberg T, Schwartz M (2001) An mtDNA mutation, 14453G-->A, in the NADH dehydrogenase subunit 6 associated with severe MELAS syndrome. Eur J Hum Genet 9(10):805-809 91. Pulkes T, Eunson L, Patterson V, Siddiqui A, Wood NW, Nelson IP, Morgan-Hughes JA, Hanna MG (1999) The mitochondrial DNA G13513A transition in ND5 is associated with a LHON/MELAS overlap syndrome and may be a frequent cause of MELAS. Ann Neurol 46(6):916-919 92. Naini AB, Lu J, Kaufmann P, Bernstein RA, Mancuso M, Bonilla E, Hirano M, DiMauro S (2005) Novel mitochondrial DNA ND5 mutation in a patient with clinical features of MELAS and MERRF. Arch Neurol 62(3):473-476 93. Crimi M, Galbiati S, Moroni I, Bordoni A, Perini MP, Lamantea E, Sciacco M, Zeviani M, Biunno I, Moggio M, Scarlato G, Comi GP (2003) A missense mutation in the mitochondrial ND5 gene associated with a Leigh-MELAS overlap syndrome. Neurology 60(11):1857-1861 94. DiMauro S, Hirano M (2005) Mitochondrial encephalomyopathies: an update. Neuromuscul Disord 15(4):276-286

Mitochondrial complex I

95. Blakely EL, de Silva R, King A, Schwarzer V, Harrower T, Dawidek G, Turnbull DM, Taylor RW (2005) LHON/MELAS overlap syndrome associated with a mitochondrial MTND1 gene mutation. Eur J Hum Genet 13(5):623-627 96. Leigh D (1951) Subacute necrotizing encephalomyelopathy in an infant. J Neurol Neurosurg Psychiatry 14(3):216-221 97. Pitkanen S, Feigenbaum A, Laframboise R, Robinson BH (1996) NADH-coenzyme Q reductase (complex I) deficiency: heterogeneity in phenotype and biochemical findings. J Inherit Metab Dis 19(5):675-686 98. Rahman S, Blok RB, Dahl HH, Danks DM, Kirby DM, Chow CW, Christodoulou J, Thorburn DR (1996) Leigh syndrome: clinical features and biochemical and DNA abnormalities. Ann Neurol 39(3):343-351 99. Fernandez-Moreira D, Ugalde C, Smeets R, Rodenburg RJ, Lopez-Laso E, Ruiz-Falco ML, Briones P, Martin MA, Smeitink JA, Arenas J (2007) X-linked NDUFA1 gene mutations associated with mitochondrial encephalomyopathy. Ann Neurol 61(1):73-83 100. Hoefs SJ, Dieteren CE, Distelmaier F, Janssen RJ, Epplen A, Swarts HG, Forkink M, Rodenburg RJ, Nijtmans LG, Willems PH, Smeitink JA, van den Heuvel LP (2008) NDUFA2 complex I mutation leads to Leigh disease. Am J Hum Genet 82(6):1306-1351 101. Bénit P, Chretien D, Kadhom N, de Lonlay-Debeney P, Cormier-Daire V, Cabral A, Peudenier S, Rustin P, Munnich A, Rötig A (2001) Large-scale deletion and point mutations of the nuclear NDUFV1 and NDUFS1 genes in mitochondrial complex I deficiency. Am J Hum Genet 68(6):1344- 1352 102. Martín MA, Blázquez A, Gutierrez-Solana LG, Fernández-Moreira D, Briones P, Andreu AL, Garesse R, Campos Y, Arenas J (2005) Leigh syndrome associated with mitochondrial complex I deficiency due to a novel mutation in the NDUFS1 gene. Arch Neurol 62(4):659-661 103. Benit P, Slama A, Cartault F, Giurgea I, Chretien D, Lebon S, Marsac C, Munnich A, Rotig A, Rustin P (2004) Mutant NDUFS3 subunit of mitochondrial complex I causes Leigh syndrome. J Med Genet 41(1):14-17 104. Bénit P, Steffann J, Lebon S, Chretien D, Kadhom N, de Lonlay P, Goldenberg A, Dumez Y, Dommergues M, Rustin P, Munnich A, Rötig A (2003) Genotyping microsatellite DNA markers at putative disease loci in inbred/multiplex families with respiratory chain complex I deficiency allows rapid identification of a novel nonsense mutation (IVS1nt -1) in the NDUFS4 gene in Leigh syndrome. Hum Genet 112(5-6):563-566 105. Triepels RH, van den Heuvel LP, Loeffen JL, Buskens CA, Smeets RJ, Rubio Gozalbo ME, Budde SM, Mariman EC, Wijburg FA, Barth PG, Trijbels JM, Smeitink JA (1999) Leigh syndrome associated with a mutation in the NDUFS7 (PSST) nuclear encoded subunit of complex I. Ann Neurol 45(6):787-790 106. Loeffen J, Smeitink J, Triepels R, Smeets R, Schuelke M, Sengers R, Trijbels F, Hamel B, Mullaart R, van den Heuvel L (1998) The first nuclear-encoded complex I mutation in a patient with Leigh syndrome. Am J Hum Genet 63(6):1598-1608 107. Van den Heuvel L, Ruitenbeek W, Smeets R, Gelman-Kohan Z, Elpeleg O, Loeffen J, Trijbels F, Mariman E, de Bruijn D, Smeitink J (1998) Demonstration of a new pathogenic mutation in human complex I deficiency: a 5-bp duplication in the nuclear gene encoding the 18-kD (AQDQ) subunit. Am J Hum Genet 62(2):262-268 108. Petruzzella V, Vergari R, Puzziferri I, Boffoli D, Lamantea E, Zeviani M, Papa S (2001) A nonsense mutation in the NDUFS4 gene encoding the 18 kDa (AQDQ) subunit of complex I abolishes assembly and activity of the complex in a patient with Leigh-like syndrome. Hum Mol Genet 10(5):529-535 109. Ugalde C, Hinttala R, Timal S, Smeets R, Rodenburg RJ, Uusimaa J, van den Heuvel LP, Nijtmans LG, Majamaa K, Smeitink JA (2007) Mutated ND2 impairs mitochondrial complex I assembly and leads to Leigh Syndrome. Mol Genet Metab 90(1):10-14 110. Leshinsky-Silver E, Lev D, Tzofi-Berman Z, Cohen S, Saada A, Yanoov-Sharav M, Gilad E, Lerman- Sagie T (2005) Fulminant neurological deterioration in a neonate with Leigh syndrome due to a maternally transmitted missense mutation in the mitochondrial ND3 gene. Biochem Biophys Res Commun 334(2):582-587 85

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111. Crimi M, Papadimitriou A, Galbiati S, Palamidou P, Fortunato F, Bordoni A, Papandreou U, Papadimitriou D, Hadjigeorgiou GM, Drogari E, Bresolin N, Comi GP (2004) A new mitochondrial DNA mutation in ND3 gene causing severe Leigh syndrome with early lethality. Pediatr Res 55(5):842-846 112. Bugiani M, Invernizzi F, Alberio S, Briem E, Lamantea E, Carrara F, Moroni I, Farina L, Spada M, Donati MA, Uziel G, Zeviani M (2004) Clinical and molecular findings in children with complex I deficiency. Biochim Biophys Acta 1659(2-3):136-147 113. Kirby DM, Boneh A, Chow CW, Ohtake A, Ryan MT, Thyagarajan D, Thorburn DR (2003) Low mutant load of mitochondrial DNA G13513A mutation can cause Leigh's disease. Ann Neurol 54(4):473-478 114. Taylor RW, Morris AA, Hutchinson M, Turnbull DM (2002) Leigh disease associated with a novel mitochondrial DNA ND5 mutation. Eur J Hum Genet 10(2):141-144 115. Kirby DM, Kahler SG, Freckmann ML, Reddihough D, Thorburn DR (2000) Leigh disease caused by the mitochondrial DNA G14459A mutation in unrelated families. Ann Neurol 48(1):102-104 116. Berger I, Hershkovitz E, Shaag A, Edvardson S, Saada A, Elpeleg O (2008) Mitochondrial complex I deficiency caused by a deleterious NDUFA11 mutation. Ann Neurol 63(3):405-408 117. Schuelke M, Smeitink J, Mariman E, Loeffen J, Plecko B, Trijbels F, Stöckler-Ipsiroglu S, van den Heuvel L (1999) Mutant NDUFV1 subunit of mitochondrial complex I causes leukodystrophy and myoclonic epilepsy. Nat Genet 21(3):260-261 118. Loeffen J, Elpeleg O, Smeitink J, Smeets R, Stöckler-Ipsiroglu S, Mandel H, Sengers R, Trijbels F, van den Heuvel L (2001) Mutations in the complex I NDUFS2 gene of patients with cardiomyopathy and encephalomyopathy. Ann Neurol 49(2):195-201 119. Bénit P, Beugnot R, Chretien D, Giurgea I, De Lonlay-Debeney P, Issartel JP, Corral-Debrinski M, Kerscher S, Rustin P, Rötig A, Munnich A (2003) Mutant NDUFV2 subunit of mitochondrial complex I causes early onset hypertrophic cardiomyopathy and encephalopathy. Hum Mutat 21(6):582-586 120. Dunning CJ, McKenzie M, Sugiana C, Lazarou M, Silke J, Connelly A, Fletcher JM, Kirby DM, Thorburn DR, Ryan MT (2007) Human CIA30 is involved in the early assembly of mitochondrial complex I and mutations in its gene cause disease. EMBO J 26(13):3227-3237 121. Saada A, Edvardson S, Rapoport M, Shaag A, Amry K, Miller C, Lorberboum-Galski H, Elpeleg O (2008) C6ORF66 is an assembly factor of mitochondrial complex I. Am J Hum Genet 82(1):32-38 122. Keeney P, Xie J, Capaldi RA, Bennett Jr JP (2006) Parkinson's disease brain mitochondrial complex I has oxidatively damaged subunits and is functionally impaired and misassembled. J Neurosci 26(19):5256-5264 123. Greenamyre JT, Sherer TB, Betarbet R, Panov AV (2001) Complex I and Parkinson's disease. IUBMB Life 52:(3-5)135-141 124. Schapira AH (2002) Primary and secondary defects of the mitochondrial respiratory chain. J Inherit Metab Dis 25(3):207-214 Review 125. Atorino L, Silvestri L, Koppen M, Cassina L, Ballabio A, Marconi R, Langer T, Casari G (2003) Loss of m-AAA protease in mitochondria causes complex I deficiency and increased sensitivity to oxidative stress in hereditary spastic paraplegia. J Cell Biol 163(4):777-787 126. Nolden M, Ehses S, Koppen M, Bernacchia A, Rugarli EI, Langer T (2005) The m-AAA protease defective in hereditary spastic paraplegia controls ribosome assembly in mitochondria. Cell 123(2):277-289 127. Arenas J, Campos Y, Ribacoba R (1998) Complex I defect in muscle from patients with Huntington's disease. Ann Neurol 43(3):397-400 128. Bender A, Krishnan KJ, Morris CM, Taylor GA, Reeve AK, Perry RH, Jaros E, Hersheson JS, Betts J, Klopstock T, Taylor RW, Turnbull DM (2006) High levels of mitochondrial DNA deletions in substantia nigra neurons in aging and Parkinson disease. Nat Genet 38(5):515-517. 129. Kraytsberg Y, Kudryavtseva E, McKee AC, Geula C, Kowall NW, Khrapko K (2006) Mitochondrial DNA deletions are abundant and cause functional impairment in aged human substantia nigra neurons. Nat Genet 38(5):518-520

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130. Parker WD Jr, Parks JK (2005) Mitochondrial ND5 mutations in idiopathic Parkinson's disease. Biochem Biophys Res Commun 326(3):667-669 131. Karry R, Klein E, Ben Shachar D (2004) Mitochondrial complex I subunits expression is altered in schizophrenia: a postmortem study. Biol Psychiatry 55(7):676-684 132. Kim SH, Vlkolinsky R, Cairns N, Fountoulakis M, Lubec G (2001) The reduction of NADH ubiquinone oxidoreductase 24- and 75-kDa subunits in brains of patients with Down syndrome and Alzheimer's disease. Life Sci 68(24):2741-2750 133. Munakata K, Tanaka M, Mori K, Washizuka S, Yoneda M, Tajima O, Akiyama T, Nanko S, Kunugi H, Tadokoro K, Ozaki N, Inada T, Sakamoto K, Fukunaga T, Iijima Y, Iwata N, Tatsumi M, Yamada K, Yoshikawa T, Kato T (2004) Mitochondrial DNA 3644T-->C mutation associated with bipolar disorder. Genomics 84(6):1041-1050 134. Washizuka S, Kakiuchi C, Mori K, Kunugi H, Tajima O, Akiyama T, Nanko S, Kato T (2003) Association of mitochondrial complex I subunit gene NDUFV2 at 18p11 with bipolar disorder. Am J Med Genet B Neuropsychiatr Genet 120(1):72-78 135. Washizuka S, Iwamoto K, Kazuno AA, Kakiuchi C, Mori K, Kametani M, Yamada K, Kunugi H, Tajima O, Akiyama T, Nanko S, Yoshikawa T, Kato T (2004) Association of mitochondrial complex I subunit gene NDUFV2 at 18p11 with bipolar disorder in Japanese and the National Institute of Mental Health pedigrees. Biol Psychiatry 56(7):483-489

Chapter IV

CIA30 complex I assembly factor: a candidate for human complex I deficiency?

Rolf J.R.J. Janssen, Jan A.M. Smeitink, Roel J.P. Smeets, and Lambert P.W.J. van den Heuvel

Nijmegen Center for Mitochondrial Disorders, Laboratory of Pediatrics and Neurology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

Human Genetics 2002, Volume 110, Number 3, Page 264-270

Chapter IV

CIA30 complex I assembly factor

Abstract The human mitochondrial NADH:ubiquinone oxidoreductase (complex I), the first complex of the oxidative phosphorylation system, is composed of at least 42 subunits. Little is known about the assembly process of these subunits into the mature complex. Recently, two proteins in Neurospora crassa have been found to be involved in the assembly of complex I. These proteins are not constituent parts of the mature complex but are associated with smaller intermediate complexes of the assembly process and have a chaperone-like function. We have characterized the human homologue of one of these two complex I intermediate associated proteins, named CIA30, and show that expression of the human CIA30 protein2 is ubiquitous with a slightly higher expression in various heart tissues, kidney, lung and liver. As deletion of the Neurospora crassa CIA genes results in severe disruption of the assembly process, human CIA30 can be considered as a candidate gene related to complex I deficiency. Thirteen patients with an isolated complex I deficiency, but who were ruled out for mutations in the mtDNA and the 35 nuclear genes of the complex, were subjected to mutational analysis of the gene coding for the human CIA30 protein. Four new single nucleotide polymorphisms (SNPs) were detected but no functional mutation was found.

2 Human CIA30 is currently known as NDUFAF1, complex I assembly factor 1 91

Chapter IV

Introduction Isolated complex I deficiency is the most frequently encountered defect of disturbances of the mitochondrial oxidative phosphorylation (OXPHOS) system[1,2]. Human complex I, NADH:ubiquinone oxidoreductase, is a multiprotein complex known to be composed of (at least) 42 subunits, seven of which are encoded by mitochondrial DNA (mtDNA). Based on homology with the bovine complex, it is assumed that the human complex is L-shaped, partly located in the mitochondrial inner membrane and partly protruding into the matrix[3]. The main function of the complex is the transportation of electrons from NADH to ubiquinone, simultaneously accompanied by the transfer of protons across the mitochondrial inner membrane to the intermembrane space. Complex I deficiency has been related to mutations in genes of mtDNA-encoded subunits[1,4- 6], mtDNA-encoded tRNAs[1,7] and nuclear DNA-encoded subunits[1,8-12]. In a group of 24 patients with an isolated complex I deficiency in skeletal muscle tissue and cultured skin fibroblasts, eleven patients were screened positive for nuclear encoded defects of complex I subunits[1,8,19]. As no mitochondrial or nuclear DNA mutations were found in the remaining 13 patients, candidates should be sought among additional genes encoding proteins required for the assembly of a functional complex I. Recently, in collaboration with Capaldi and colleagues, we have reported a new approach for detecting and characterizing complex I deficiencies[20]. By using monoclonal antibodies directed against several complex I subunits in conjunction with western blotting and sucrose gradient studies, differences in assembly patterns can be distinguished[20]. The aberrant patterns are indicative of a possible defect in a complex I assembly factor. To date, little is known about the assembly process of the subunits into the mature complex and no evidence of a complex-I-specific assembly protein has been found in humans as yet. Küffner et al.[21] have reported the involvement of two chaperone proteins in the assembly of complex I of the fungus Neurospora crassa. During the assembly process, these complex I intermediate associated proteins (CIA30 and CIA84) are transiently associated with a complex I assembly intermediate[21]. Since the correct assembly is a prerequisite for a functional complex, genes encoding human homologues of these two proteins are logical candidates for complex I deficiency. Here, we report the genomic sequence of the human homologue of CIA30 together with homologues of mouse, Caenorhabditis elegans, Drosophila melanogaster and Aspergillus nidulans, the tissue distribution of gene expression, and a mutational detection study.

CIA30 complex I assembly factor

Patients, materials and methods Patient group Our patient study group consisted of 13 children with an isolated decreased activity of complex I in skeletal muscle tissue and cultured skin fibroblasts (male:female ratio of 11:2). The clinical characteristics of these patients were described previously by Loeffen et al.[1,13,15]. In previous studies, no mtDNA rearrangements (deletions, insertions or duplications), common mtDNA point mutations or mutations in any of the 35 nuclear genes and the seven mitochondrial ND genes of complex I were found in these patients.

Elucidation of the human CIA30 genomic sequence The protein and nucleotide databases of the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov) were subjected to a basic local alignment search tool (BLAST) search with the cDNA and amino acid sequence of the Neurospora crassa CIA30 gene (accession numbers: AJ001726 and CAA04954). Human Genome BLAST was performed with the discovered human cDNA sequence.

Tissue distribution of human CIA30 expression A human multiple tissue expression (MTE) array (CLONTECH Laboratories), carrying poly A+ RNA from 72 different human tissues, was hybridized with a [α-32P]-dCTP labeled cDNA probe encompassing the human CIA30 open reading frame at 65°C for 24 h by using standard hybridization solutions according to manufacturer‘s protocol. Subsequently, the MTE Array was analyzed by autoradiography.

Mutational detection Total RNA isolated from cultured skin fibroblasts was used for first-strand cDNA synthesis with SuperScript II RNase H- reverse transcriptase (Gibco BRL Life Technologies). A total volume of 40 µl reaction mixture, containing 50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2, 0.01 M dithiothreitole, 1.25 mM dNTP mix, 0.625 µg oligo(dT), 0.625 µg random hexamer primers and 60 U RNasin ribonuclease inhibitor (oligo(dT), primers and inhibitor, all from Promega), was incubated at 42°C for 1 h. The reaction was stopped by heating at 96°C for 5 min, followed by direct cooling on ice. Amplification of the human CIA30 cDNA sequence was carried out in three overlapping polymerase chain reaction (PCR) fragments covering the entire open reading frame by using sense and antisense primers mentioned in Table 1. Primers were constructed based on the cDNA sequence of an unknown human protein from NCBI GenBank (accession numbers: BC000780 and AAH00780), a result of the BLAST search.

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Table 1. Primers used for PCR on human CIA30 cDNA

Primer name Primer sequence (5′ 3′) Location 3’end Sense primer 1 CGTCAGTGAAACACTTAGAGC cDNA position 264 Antisense primer 1 CGGTCCTCTCCAATGATCC cDNA position 666 Sense primer 2 GAAGCAATATACCATTTTAGGC cDNA position 646 Antisense primer 2 GATATTCACCATCCAAGGCC cDNA position 1010 Sense primer 3 AGATGTCTTACGATTGGTCCC cDNA position 950 Antisense primer 3 TATCATTGTAGATGCTAGTACCC cDNA position 1378

The following PCR conditions were used: 35 cycles of 30 s at 92°C, 30 s at 54°C (fragments 1 and 2) or 57.5°C (fragment 3) and 1 min at 72°C. The cycles were preceded by an initial denaturation step of 2 min at 92°C and were followed by a final extension of 7 min at 72°C. PCR fragments were separated on a 1.5% agarose gel and bands were made visible by ethidium bromide staining. After purification, PCR products were cycle-sequenced, by using the same primers mentioned above, and analyzed on an automated ABI Prism 377 DNA sequencer (Applied Biosystems), according to the manufacturer's protocol.

Results Characterization of the human CIA30 gene A BLAST search of the NCBI protein and nucleotide databases with the amino acid sequence of the Neurospora crassa CIA30 gene revealed a putative human CIA30 homologue, i.e. a sequence encoding an unknown human hypothetical protein (CGI-65, accession numbers: BC000780 and AAH00780) with an E value of 1*e-08. Subsequently, a Human Genome BLAST with this sequence led to the localization of the entire gene on a genomic contig (NT_010194) corresponding to chromosome 15. The human CIA30 gene has been localized to chromosome 15q13.3 (LOC51103) and encompasses ~15 kb. The gene is composed of five exons and has a coding sequence of 984 bp with the translation initiation codon located in the second exon. A TATA-box sequence at position -39 to -36 and a CG-box sequence at position -54 to -49, calculated backwards from the transcription initiation site, are present in the promoter region (Fig 1). Approximately 1 kb of the promoter region has been examined for sequence motifs suggesting the binding of transcription factors. Several potential sites for known transcription factors have been identified, including a site for nuclear respiratory factor 2 (NRF-2) at position -100 to -91[22]. A large number of CpG residues can be observed around the transcriptional start site.

CIA30 complex I assembly factor

-1000 acacacctgt aatcccagct acttgggagg ctgaggcagg agaattgctc caacccagga gtcggaggtt gcagatcgcg ccactgcact acagcctggg caacagagtg agactccgtc tcaaaaaaaa aaagtgcttt aagagaggtg tgcacaaggt acaaacagat gccctgagca actggcgacc gtgtggggcg -800 agactcggga agggctggta ggaggggctg ctgtccaggt gcgtggtggt cctcaagaat tcatgggggc aattccagca gaatcggcct tttgaggcct caggcagttc ttgtggggac cgaggcgaca gataaggaag gaagggatac aggcgcgtta gcaataactg ggttctattt gaaagacgca gggggccggg -600 cttggtggct cgcgcctgta atctcagcac tttgggaggc cgaggcgggt ggatcacgag gtcaggagat cgagatcatc ccggctaaca tggtgaaacc ccgtctctac taaaaataca aaaattagtc gggcatgatg gcgcgcacct gtaatcccag ctactcggga ggctgaggca ggagaatcgc ttgaatctgg -400 gaggcagagg ttgtagtgag ccgagatcgc gccactgcac tccagcctgg gcgacagagc gagaatccgt ctcaaaaaaa aaaaaaaaaa aaaagacgca gggatggagt ttgccgcctg aaaggataca ggaaggagag gcgaaaaact tatctgaatt ctcactgctt ccttacaact ggcgaccaag tggaccaaaa -200 acctccaaaa acctcagttg gctgggctac ggggtccttg ctgtccttcc tgggcttctg tactcggccg gaactttggt gttctgacgc cttgtttggc -100 atcggaaggg aaaagcagat ggacctatac gggtaaagtg gcttctgggc ggaaggtaca ctataggctc ggggaggtaa gcggcggcag gccggcggtt +1 ggtgtgtccc gggtgtgggg aggcgacaga gccctggcac ttgagggttg agggggcctc cccagcgcgg cgaaccgtct agcctccgga ggccaggccg 101 tgagtgcggg aggtatacgc caaggcggaa gaattttgcc actcactacc tgtgtgacct cgggtaaatt agccttggaa cgtcagtttc ttcgtctcta 201 taattgaaat aataatagta cctctctcag gattgttgtg agccgtcagt gaaacactta gagcagtttc tggcacatgg taaggcatgg >>>IVS1<<< 280 ctgttatagg tagaattggg ctatttgctg aagcttcttg gtggcccttg ctagcccagg aagaaactta cattttgatt tttttgtacc ATGGCTTTGG M A L 3 371 TTCACAAATT GCTGCGTGGT ACTTATTTTC TCAGAAAATT CTCTAAGCCA ACTTCTGCCT TGTATCCATT TTTGGGTATT CGCTTTGCAG AGTATTCCAG V H K L L R G T Y F L R K F S K P T S A L Y P F L G I R F A E Y S S 37 471 TAGTCTTCAG AAACCAGTGG CTTCTCCTGG CAAAGCCTCC TCACAGAGGA AGACTGAAGG GGATTTGCAA GGAGATCACC AGAAAGAAGT TGCTTTGGAT S L Q K P V A S P G K A S S Q R K T E G D L Q G D H Q K E V A L D 70 571 ATAACTTCTT CTGAGGAGAA GCCTGATGTT AGTTTCGATA AAGCAATTAG AGATGAAGCA ATATACCATT TTAGGCTTTT GAAGGATGAA ATTGTGGATC I T S S E E K P D V S F D K A I R D E A I Y H F R L L K D E I V D 103 671 ATTGGAGAGG ACCGGAAGGC CACCCTCTGC ATGAGGTCTT GCTGGAACAA GCCAAGGTTG TCTGGCAATT CCGGGGGAAA GAAGATTTGG ATAAGTGGAC H W R G P E G H P L H E V L L E Q A K V V W Q F R G K E D L D K W T 137 771 AGTGACTTCT GATAAGACGA TTGGAGGCAG AAGTGAAGTG TTTTTGAAAA TGGGCAAGAA TAACCAAAGT GCACTGCTAT ATGGAACTCT GAGCTCTGAG V T S D K T I G G R S E V F L K M G K N N Q S A L L Y G T L S S E 170 871 GCGCCTCAGG ACGGGGAGTC TACCCGAAGT GGGTACTGTG CAATGATATC CAGGATTCCA AGGgtaggtg aggcccagga >>>IVS2<<< ttcaatatct A P Q D G E S T R S G Y C A M I S R I P R 191

934 tagGGTGCTT TTGAGAGGAA GATGTCTTAC GATTGGTCCC AGTTCAATAC TCTGTATCTC CGTGTACGTG GGGATGGTCG GCCTTGGATG GTGAATATCA G A F E R K M S Y D W S Q F N T L Y L R V R G D G R P W M V N I 223 1031 AGGAGGACAC AGATTTCTTC CAGAGGACGA ATCAGATGTA TAGTTACTTC ATGTTCACCC GCGGGGGACC CTACTGGCAG GAGGTCAAGg taacagcata K E D T D F F Q R T N Q M Y S Y F M F T R G G P Y W Q E V K 253

1120 >>>IVS3<<< ttctttcagA TTCCTTTTTC CAAATTTTTC TTCTCTAATC GAGGAAGAAT CCGGGATGTT CAGCATGAGC TTCCGCTTGA TAAGgtaaca I P F S K F F F S N R G R I R D V Q H E L P L D K 278

1195 tattcctgta >>>IVS4<<< tattttaaat ttagATCTCT TCTATAGGAT TCACCTTGGC TGATAAAGTG GATGGTCCAT TCTTCCTGGA GATAGATTTT I S S I G F T L A D K V D G P F F L E I D F 300 1261 ATTGGCGTGT TTACTGATCC AGCTCATACA GAAGAATTTG CCTATGAAAA TTCTCCAGAG CTTAACCCAA GGCTTTTTAA ATAAagatca tatggtagtt I G V F T D P A H T E E F A Y E N S P E L N P R L F K 327 1361 ttgttttact aatctaaggg tactagcatc tacaatgata tagacaaaat aaaatatttc tttaatggca tccaac 95

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Figure 1. Genomic and deduced amino acid sequence of human CIA30 (page 95). The genomic sequence of human CIA30 including 1 kb of promoter region, five exons of 279, 654, 186, 75 and 242 bp in length, respectively, and the exon-intron boundaries of the intervening sequences (IVSs). The lengths of IVS1, IVS2, IVS3 and IVS4 are 5002, 1416, 6336 and 854 bp, respectively (not shown). IVSs, TATA box (position -39), CG box (position -54), NRF-2 site (position -100) and poly A signal (position 1408) are depicted in bold, CG elements are underlined. The deduced amino acid sequence is placed directly underneath the coding sequence, which is depicted in capitals. cDNA numbering is depicted left, amino acid numbering right.

Chromosome 19 bears a putative pseudogene of human CIA30 (localization 19p12), which consists of the coding sequence of the gene not interrupted by any intervening sequences and which is 90% identical to the CIA30 gene.

Primary structure of the human CIA30 protein The protein is composed of 327 amino acids and has a predicted (N-terminal) mitochondrial targeting sequence composed of the first 24 amino acids of which six are positively charged and five are hydroxylated residues. This leader sequence has a probability of export to mitochondria of 0.9838 (MITOPROT: prediction of mitochondrial targeting sequences; MITOP database; http://www.mips.biochem.mpg.de/proj/medgen/mitop/). The protein including the leader sequence has a calculated molecular mass of 37.7 kDa. A search for protein patterns and domains at the PROSITE database of the ExPASy proteomics server (http://www.expasy.org/tools/scnpsite.html) shows the presence of several often-occurring patterns. Among them are two possible cAMP- and cGMP-dependent protein kinase phosphorylation sites: an RKFS amino acid sequence located in the leader sequence at amino acid positions 15-18 and an RKMS amino acid sequence located at amino acid positions 196-199.

Homology studies A BLAST search of protein and nucleotide databases revealed putative CIA30 homologues (accession numbers given in parentheses) of Mus musculus (AK010328, BAB26855), Caenorhabditis elegans (CEC50B8, CAB01129), Drosophila melanogaster (AC007892, AAF56928), and the known Aspergillus nidulans CiaA gene (AF236661, AAK14054). The CIA30 protein is moderately conserved among species and exhibits the highest homology in the C-terminal part of the protein. A multiple alignment of the CIA30 homologues together with the human and Neurospora crassa protein is depicted in Figure 2.

CIA30 complex I assembly factor

Homo sapiens Mus musculus Caenorhabditis elegans Drosophila melanogaster Neurospora crassa Aspergillus nidulans

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Figure 2. Comparison of CIA30 homologues (page 97). Alignment of CIA30 homologues of Homo sapiens, Mus musculus, Caenorhabditis elegans, Drosophila melanogaster, Neurospora crassa and Aspergillus nidulans. Identical amino acids in all six organisms are shaded darkly, identical amino acids in four or five of the six organisms are shaded lightly.

Tissue distribution of human CIA30 expression To obtain more information concerning the expression of human CIA30 in a wide range of human tissues, a human MTE array was used for screening with radioactively labeled CIA30 cDNA. All human tissues revealed expression of human CIA30 mRNA. Dots with a slightly higher intensity were seen for various heart tissues (left and right atrium and ventricle and interventricular septum), kidney, lung and liver, indicative of a higher expression of CIA30 mRNA in these tissues (Fig 3). High intensity dots of hybridization with human DNA can be explained by the presence of a pseudogene. Thus, expression of the human CIA30 protein is ubiquitous with a slightly higher expression in various heart tissues, kidney, lung and liver.

Mutational analysis of the human CIA30 gene Direct sequencing of the human CIA30 open reading frame of 13 patients with an isolated complex I deficiency in skeletal muscle tissue and cultured skin fibroblasts and who were ruled out for mutations in all 42 nuclear and mitochondrial genes of the complex and common mtDNA alterations revealed five heterozygous nucleotide substitutions, four of which resulted in amino acid substitutions (Tab 2). One of them, the R9H substitution, is a known polymorphism recorded in the NCBI SNP database (Reference SNP Id: rs1899). Since the other four nucleotide substitutions frequently occur in expressed sequence tags (ESTs), they should be characterized as polymorphisms or silent mutations.

Table 2. Polymorphisms of human CIA30

Nucleotide substitutiona Amino acid substitution Reference SNP Id

G386A R9H rs1899 G452T R31L - G886A E176 K - G1269A V303 V - C1301G A314G - acDNA numbering

CIA30 complex I assembly factor

Figure 3. Tissue distribution of human CIA30 expression. Autoradiogram of the human MTE array hybridized with the radiolabeled CIA30 cDNA probe. The key to the type and position of poly A+ RNA and control DNA dotted onto the membrane is depicted beneath the autoradiogram.

Chapter IV

Discussion Isolated complex I deficiency is the most frequently observed OXPHOS disorder. Genetic causes have been found in mtDNA-encoded tRNAs[1,7], in genes of mtDNA-encoded subunits[1,4-6] and in nuclear DNA-encoded subunits of complex I[1,8-12]. In contrast to, for example, OXPHOS complex IV[23,24], no defects of the assembly of complex I have been described as yet. Complex I consists of a membrane arm and a matrix arm that assemble independently in separate pathways[25]. Assembly of the membrane arm requires two intermediates, called the large and small membrane arm assembly intermediates. In Neurospora crassa, two proteins are transiently associated with the large membrane arm assembly intermediate but are not associated with the other assembly intermediates or the fully assembled complex[21]. Deletion of the genes encoding these proteins, i.e., CIA30 and CIA84, results in the disruption of the assembly process by prevention of the formation of the large membrane arm intermediate[21]. Neurospora crassa CIA30 and CIA84 function as specific chaperones in the formation of complex I. We present the genomic sequence of the first human complex I assembly factor, a homologue of Neurospora crassa CIA30. The putative mitochondrial targeting sequence and the presence of a NRF-2 site indicate that the gene product is presumably a mitochondrial protein. NRF-2 has been shown to contribute to the transcriptional regulation of a number of nuclear genes that code for subunits of respiratory chain complexes, including cytochrome c oxidase, and for other mitochondrial proteins, such as the β-subunit of ATP synthase and the mitochondrial transcription factor[22,26]. Expression of the human CIA30 protein is ubiquitous with a slightly higher expression in various heart tissues, kidney, lung and liver. This finding and the high occurrence of CpG elements around the transcription initiation start, which could indicate the presence of a CpG island, suggest that CIA30 is a housekeeping gene, as are most genes of the OXPHOS system. In contrast to mouse with which human CIA30 is nearly 80% identical, there is a moderate level of homology between human CIA30 and homologues of Caenorhabditis elegans, Drosophila melanogaster, Neurospora crassa and Aspergillus nidulans. No large stretches of conservation have been identified but some residues are conserved among all six species. The primary structure of the CIA30 protein shows no similarity to known proteins and CIA30 cannot be classified as a member of one of the chaperone families, which makes it plausible that CIA30 is a single-target chaperone specific for complex I[21]. Further investigations are needed to elucidate the role of the putative phosphorylation sites of CIA30. A cAMP-dependent protein kinase phosphorylation site, located on the 18-kDa subunit of complex I, has been shown to be involved in the activation of the complex[27].

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Subjection of our patient group of 13 patients with isolated complex I deficiency in skeletal muscle tissue and cultured skin fibroblasts to mutational analysis of the human CIA30 gene revealed four new DNA alterations resulting in three amino acid substitutions. Since none of these substitutions concerned highly conserved amino acid residues, and since the corresponding nucleotide alterations occurred in ESTs, no pathogenic consequences can be ascribed to any of them. Although no pathogenic mutations in the human CIA30 gene were found in 13 patients of this investigation, this number is too low to allow any speculation about the importance of human CIA30 in relation to isolated complex I deficiency. In our patient group, candidate genes for isolated complex I deficiency should be sought among other, as yet uncharacterized genes encoding complex I specific assembly factors. A human (or other organism's) homologue(s) of Neurospora crassa CIA84, for instance, has not been identified to date. Recently, Triepels et al.[20] have described a promising method for selecting patients by the examination of complex I assembly profiles. Western blotting with the use of a set of monoclonal antibodies directed against various subunits of complex I in conjunction with sucrose gradient studies can reveal aberrant assembly patterns suggesting a possible defect in a complex I assembly factor. One option for tracking down the defective gene is the chromosome transfer technique. Since most of our patients are male, the X-chromosome might be the carrier of a defective gene.

Acknowledgements This work was supported by The Netherlands Organization for Scientific Research (NWO; number 901-03-156).

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References

1. Loeffen JL, Smeitink JA, Trijbels JM, Janssen AJ, Triepels RH, Sengers RC, van den Heuvel LP (2000) Isolated complex I deficiency in children: clinical, biochemical and genetic aspects. Hum Mutat 15(2):123-134 Review 2. Smeitink J, van den Heuvel L, DiMauro S (2001) The genetics and pathology of oxidative phosphorylation. Nat Rev Genet 2(5):342-352 Review 3. Walker JE (1992) The NADH:ubiquinone oxidoreductase (complex I) of respiratory chains. Q Rev Biophys 25(3):253-324 Review 4. Howell N, Kubacka I, Xu M, McCullough DA (1991) Leber hereditary optic neuropathy: involvement of the mitochondrial ND1 gene and evidence for an intragenic suppressor mutation. Am J Hum Genet 48(5):935-942 5. Majander A, Huoponen K, Savontaus ML, Nikoskelainen E, Wikstrom M (1991) Electron transfer properties of NADH:ubiquinone reductase in the ND1/3460 and the ND4/11778 mutations of the Leber hereditary optic neuroretinopathy (LHON). FEBS Lett 292(1-2):289-292 6. Jun AS, Brown MD, Wallace DC (1994) A mitochondrial DNA mutation at nucleotide pair 14459 of the NADH dehydrogenase subunit 6 gene associated with maternally inherited Leber hereditary optic neuropathy and dystonia. Proc Natl Acad Sci USA 91(13):6206-6210 7. James AM, Wei YH, Pang CY, Murphy MP (1996) Altered mitochondrial function in fibroblasts containing MELAS or MERRF mitochondrial DNA mutations. Biochem J 318(Pt 2):401-407 8. Loeffen J, Smeitink J, Triepels R, Smeets R, Schuelke M, Sengers R, Trijbels F, Hamel B, Mullaart R, van den Heuvel L (1998) The first nuclear-encoded complex I mutation in a patient with Leigh syndrome. Am J Hum Genet 63(6):1598-1608 9. Benit P, Chretien D, Kadhom N, de Lonlay-Debeney P, Cormier-Daire V, Cabral A, Peudenier S, Rustin P, Munnich A, Rotig A (2001) Large-scale deletion and point mutations of the nuclear NDUFV1 and NDUFS1 genes in mitochondrial complex I deficiency. Am J Hum Genet 68(6):1344- 1352 10. Triepels RH, van den Heuvel LP, Loeffen JL, Buskens CA, Smeets RJ, Rubio Gozalbo ME, Budde SM, Mariman EC, Wijburg FA, Barth PG, Trijbels JM, Smeitink JA (1999) Leigh syndrome associated with a mutation in the NDUFS7 (PSST) nuclear encoded subunit of complex I. Ann Neurol 45(6):787-790 11. Van den Heuvel L, Ruitenbeek W, Smeets R, Gelman-Kohan Z, Elpeleg O, Loeffen J, Trijbels F, Mariman E, Bruijn D de, Smeitink J (1998) Demonstration of a new pathogenic mutation in human complex I deficiency: a 5-bp duplication in the nuclear gene encoding the 18-kD (AQDQ) subunit. Am J Hum Genet 62(2):262-268 12. Schuelke M, Smeitink J, Mariman E, Loeffen J, Plecko B, Trijbels F, Stockler-Ipsiroglu S, van den Heuvel L (1999) Mutant NDUFV1 subunit of mitochondrial complex I causes leukodystrophy and myoclonic epilepsy. Nat Genet 21(3):260-261 13. Loeffen J, Smeets R, Smeitink J, Ruitenbeek W, Janssen A, Mariman E, Sengers R, Trijbels F, van den Heuvel L (1998) The X-chromosomal NDUFA1 gene of complex I in mitochondrial encephalomyopathies: tissue expression and mutation detection. J Inherit Metab Dis 21(3):210- 215 14. Loeffen JL, Triepels RH, van den Heuvel LP, Schuelke M, Buskens CA, Smeets RJ, Trijbels JM, Smeitink JA (1998) cDNA of eight nuclear encoded subunits of NADH:ubiquinone oxidoreductase: human complex I cDNA characterization completed. Biochem Biophys Res Commun 253(2):415- 422 15. Loeffen J, Smeets R, Smeitink J, Triepels R, Sengers R, Trijbels F, van den Heuvel L (1999) The human NADH:ubiquinone oxidoreductase NDUFS5 (15 kDa) subunit: cDNA cloning, chromosomal localization, tissue distribution and the absence of mutations in isolated complex I-deficient patients. J Inherit Metab Dis 22(1):19-28 16. Triepels R, van den Heuvel L, Loeffen J, Smeets R, Trijbels F, Smeitink J (1998) The nuclear- encoded human NADH:ubiquinone oxidoreductase NDUFA8 subunit: cDNA cloning, chromosomal localization, tissue distribution, and mutation detection in complex-I-deficient patients. Hum Genet 103(5):557-563 102

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17. Triepels R, Smeitink J, Loeffen J, Smeets R, Buskens C, Trijbels F, van den Heuvel L (1999) The human nuclear-encoded acyl carrier subunit (NDUFAB1) of the mitochondrial complex I in human pathology. J Inherit Metab Dis 22(2):163-173 18. Schuelke M, Loeffen J, Mariman E, Smeitink J, van den Heuvel L (1998) Cloning of the human mitochondrial 51 kDa subunit (NDUFV1) reveals a 100% antisense homology of its 3'UTR with the 5'UTR of the gamma-interferon inducible protein (IP-30) precursor: is this a link between mitochondrial myopathy and inflammation? Biochem Biophys Res Commun 254(2):599-606 19. Smeitink J, Loeffen J, Smeets R, Triepels R, Ruitenbeek W, Trijbels F, van den Heuvel L (1998) Molecular characterization and mutational analysis of the human B17 subunit of the mitochondrial respiratory chain complex I. Hum Genet 103(2):245-250 20. Triepels RH, Hanson BJ, van den Heuvel LP, Sundell L, Marusich MF, Smeitink JA, Capaldi RA (2001) Human complex I defects can be resolved by monoclonal antibody analysis into distinct subunit assembly patterns. J Biol Chem 276(12):8892-8897 21. Küffner R, Rohr A, Schmiede A, Krull C, Schulte U (1998) Involvement of two novel chaperones in the assembly of mitochondrial NADH:ubiquinone oxidoreductase (complex I). J Mol Biol 283(2):409-417 22. Virbasius JV, Virbasius CA, Scarpulla RC (1993) Identity of GABP with NRF-2, a multisubunit activator of cytochrome oxidase expression, reveals a cellular role for an ETS domain activator of viral promoters. Genes Dev 7(3):380-392 23. Tiranti V, Hoertnagel K, Carrozzo R, Galimberti C, Munaro M, Granatiero M, Zelante L, Gasparini P, Marzella R, Rocchi M, Bayona-Bafaluy MP, Enriquez JA, Uziel G, Bertini E, Dionisi-Vici C, Franco B, Meitinger T, Zeviani M (1998) Mutations of SURF-1 in Leigh disease associated with cytochrome c oxidase deficiency. Am J Hum Genet 63(6):1609-1621 24. Zhu Z, Yao J, Johns T, Fu K, De Bie I, Macmillan C, Cuthbert AP, Newbold RF, Wang J, Chevrette M, Brown GK, Brown RM, Shoubridge EA (1998) SURF1, encoding a factor involved in the biogenesis of cytochrome c oxidase, is mutated in Leigh syndrome. Nat Genet 20(4):337-343 25. Tuschen G, Sackmann U, Nehls U, Haiker H, Buse G, Weiss H (1990) Assembly of NADH:ubiquinone reductase (complex I) in Neurospora mitochondria. Independent pathways of nuclear-encoded and mitochondrially encoded subunits. J Mol Biol 213(4):845-857 26. Guo A, Nie F, Wong-Riley M (2000) Human nuclear respiratory factor 2 alpha subunit cDNA: isolation, subcloning, sequencing, and in situ hybridization of transcripts in normal and monocularly deprived macaque visual system. J Comp Neurol 417(2):221-232 27. Papa S, Scacco S, Sardanelli AM, Vergari R, Papa F, Budde S, van den Heuvel L, Smeitink J (2001) Mutation in the NDUFS4 gene of complex I abolishes cAMP-dependent activation of the complex in a child with fatal neurological syndrome. FEBS Lett 489(2-3):259-262

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Human mitochondrial complex I assembly is mediated by NDUFAF1

Rutger O. Vogel1*, Rolf J.R.J. Janssen1*, Cristina Ugalde1,2, Melissa Grovenstein1, Richard J. Huijbens1, Henk-Jan Visch3, Lambert P.W.J. van den Heuvel1, Peter H. Willems3, Massimo Zeviani4, Jan A.M. Smeitink1 and Leo G.J. Nijtmans1

1) Nijmegen Center for Mitochondrial Disorders, Laboratory of Pediatrics and Neurology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands 2) Centro de Investigacion, Hospital Universitario 12 de Octubre, Madrid, Spain 3) Department of Biochemistry, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands 4) Unit of Molecular Neurogenetics, National Institute of Neurology C. Besta, Milan, Italy * Joint first authorship

FEBS Journal 2005, Volume 272, Number 20, Page 5317-5326

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Abstract Complex I (NADH:ubiquinone oxidoreductase) is the largest multiprotein enzyme of the oxidative phosphorylation system. Its assembly in human cells is poorly understood and no proteins assisting this process have yet been described. A good candidate is NDUFAF1, the human homologue of Neurospora crassa complex I chaperone CIA30. Here, we demonstrate that NDUFAF1 is a mitochondrial protein that is involved in the complex I assembly process. Modulating the intramitochondrial amount of NDUFAF1 by knocking down its expression using RNA interference leads to a reduced amount and activity of complex I. NDUFAF1 is associated with two complexes of 600 and 700 kDa in size of which the relative distribution is altered in two complex I-deficient patients. Analysis of NDUFAF1 expression in a conditional complex I assembly system shows that the 700-kDa complex may represent a key step in the complex I assembly process. Based on these data, we propose that NDUFAF1 is an important protein for the assembly/stability of complex I.

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Introduction The failure to assemble a properly functioning complex I (NADH:ubiquinone oxidoreductase, EC 1.6.5.3) results in complex I deficiency. This is a major contributor to mitochondrial disease and frequently results in early childhood death[1]. As complex IV (cytochrome c oxidase) deficiency is frequently caused by mutations in an assembly factor[2], it is likely that a similar situation holds for complex I. However, in contrast to the 15 assembly chaperones already found for complex IV, so far no assembly proteins have been described for mammalian complex I. Given the complexity of the enzyme, many assembly factors are likely to be needed and still await detection. We have described a candidate gene, NDUFAF1, which has 28% homology with Neurospora crassa complex I assembly chaperone CIA30[3]. The present study is the first to investigate the possible role of NDUFAF1 in the assembly of complex I in human cells. Complex I is the first of five multiprotein complexes which together constitute the oxidative phosphorylation system. In this system NADH is oxidized by complex I, after which electrons are transferred via electron carriers (ubiquinone and cytochrome c) and via complexes III and IV to the final electron acceptor molecular oxygen. The energy of this transfer is used to translocate protons across the mitochondrial inner membrane. The thus generated proton gradient is used by complex V (ATP synthase) to generate ATP. Mammalian complex I is an L-shaped structure consisting of at least 45 subunits[4-6] of which seven are encoded by the mitochondrial genome. The complex can be subdivided into three functionally distinct fragments. The NADH dehydrogenase segment includes the redox cofactor flavin mononucleotide, which is involved in the oxidation of the NADH substrate. The hydrogenase part contains several iron-sulfur clusters which are involved in electron transfer to the electron transporter ubiquinone. The membrane-bound transporter part of complex I is involved in proton translocation. Whether and how electron transport and proton translocation are coupled is yet uncertain[7]. Assembly of complex I is an intricate process which has been studied in several organisms[8- 10]. The most extended investigation of complex I assembly was performed for the fungus Neurospora crassa[11,12]. In the proposed model, the hydrophilic peripheral arm and the hydrophobic membrane arm of complex I are assembled independently before being joined together. The search for a more detailed description of the assembly pathway has recently resulted in the publication of the first models for human complex I assembly[13,14]. Both models differ considerably, illustrating the fact that the complex I assembly pathway is far from solved. The model we describe shows considerable homology to N. crassa complex I assembly. We propose that, analogous to the situation in N. crassa, complex I is assembled semi-sequentially: discrete functional modules are assembled independently and are joined in several steps to form a peripheral arm and a membrane arm assembly intermediate. In

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Human complex I assembly is mediated by NDUFAF1 more detail, this entails the formation of peripheral arm assembly intermediate D (600 kDa) from intermediates H (80 kDa), G (150 kDa) and F (250 kDa) and the formation of membrane arm assembly intermediate C (700 kDa) from the combination of membrane proteins with intermediate E (400 kDa). Peripheral arm intermediate D consists of a core or highly conserved hydrophilic subunits (such as the 49-kDa, 39-kDa and 30-kDa subunits), while membrane arm intermediate C consists of highly conserved hydrophobic subunits (such as ND1 and ND6). These two key intermediates are combined to form assembly intermediate B (950 kDa) and finally holocomplex I (A, 1 MDa)[14]. Two candidate assembly proteins for complex I were found in N. crassa: complex I intermediate associated proteins CIA30 and CIA84[15]. Knockouts of the cia genes in N. crassa resulted in membrane arm subunit knockout phenotypes. The CIA proteins are thought to chaperone the combination of the small and large membrane arm intermediates of complex I via binding to the large membrane arm intermediate. The binding of CIA84 is transient as the protein cycles between a bound and unbound state. Immunoprecipitations using a CIA84 antibody have resulted in the identification of associated subunits in the membrane arm[15]. So far nothing is known about the possible involvement of CIA30 homologue NDUFAF1 in human complex I assembly. In this paper, we demonstrate that NDUFAF1 acts as an assembly protein for complex I in human cells, in line with the proposed function of its homologue CIA30 in the fungus N. crassa.

Materials and methods Generating the NDUFAF1 antibody To produce antibodies against NDUFAF1, two oligopeptides were selected. The first peptide corresponds to the mid-portion of the NDUFAF1 sequence, amino acids 43V-57G; the second peptide corresponds to the C-terminus of the protein, amino acids 314F-327K. A mixture of both peptides was coupled to keyhole limpet hemocyanin, serving as an immunogenic carrier. Rabbits were immunized with an injection of the antigen-carrier conjugate, followed by three subsequent boosters, one every three weeks. The two antisera were collected from a final bleeding and tested for specific detection of the NDUFAF1 protein by western blotting. Both antisera displayed comparable specificity.

Cell cultures JM109 E. coli cells (Promega, Madison, WI, USA) were cultured in Luria Burtani (LB) medium and the appropriate antibiotic was added to the medium. 143B206 ρ0 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Biowhitaker, Walkersville, MD, 109

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USA) supplemented with 5% (v/v) fetal bovine serum (FBS), antibiotics, 1 mM uridine and 100 µg/mL bromodeoxyuridine. HeLa cells and 143B osteosarcoma cells were cultured in DMEM supplemented with 10% (v/v) FBS, penicillin and streptomycin (Gibco, Paisley, UK). HEK293 T-REx™ cells without the TO/NDUFAF1 construct were cultured in the same medium to which 5 µg/mL blasticidin (Invitrogen, Carlsbad, CA, USA) was added to maintain the repressor construct. To HEK293 T-REx™ cells containing the TO/NDUFAF1 construct additionally 300 µg/mL Zeocin™ (Invitrogen) was added to maintain the inducible construct. siRNA transfection For transfection, HeLa cells were plated in DMEM supplemented with 10% (v/v) FBS (without antibiotic) in 24-well plates with a cell density of 1.5*104 cells per well. The next day, cells were transfected with siRNA duplex (control: Cyclophilin B (Dharmacon, Lafayette, CO, USA), NDUFAF1#1 antisense strand: 5‘-ACUAACAUCAGGCUUCUCCdTdT- 3‘, NDUFAF1#2 antisense strand: 5‘-UAACUAUACAUCUGAUUCGdTdT-3‘) in 3 µL oligofectamine (Invitrogen) to achieve a final concentration of 10 nM siRNA per well. Cells were incubated at 37 °C in a CO2 incubator for 48-72 h until they were ready to assay for protein knockdown or to perform a second transfection.

Cellular fractionation

Approximately 5*106 HEK293 cells were harvested, washed in NaCl/Pi, resuspended in an appropriate isotonic buffer [0.25 M sucrose, 5 mM Tris/HCl, pH 7.5, and 0.1 mM phenylmethylsulfonylfluoride (PMSF)] and homogenized using a glass Teflon homogenizer. Unbroken cells and nuclei were pelleted by centrifugation at 600 g for 15 min. Supernatants were centrifuged at 10,000 g for 25 min. The resulting supernatant was isolated as the cytoplasmic fraction and the mitochondrial pellet was washed once with the isotonic buffer containing 1 mM EDTA, pH 8.

Creating the inducible NDUFAF1 construct and transfection into HEK293 T-REx™ cells A PCR of total cell cDNA was performed to specifically amplify NDUFAF1 cDNA using Native Pfu DNA polymerase (Stratagene, La Jolla, CA, USA). Primer sequences are (5‘ 3‘): NDUFAF1 forward: 5‘-CGCGGAATTCATGGCTTTGGTTCACAAATTGC-3‘, NDUFAF1 reverse: 5‘-CGCGTCTAGATTTAAAAAGCCTTGGGTTAAGCTC-3‘. The amplified fragment and pcDNA4/TO/myc-His A (Invitrogen) (TO) were digested with EcoRI and XbaI restriction enzymes (Gibco, Paisley, UK) and subsequently ligated using T4 DNA Ligase (Invitrogen) and transformed into JM109 competent E. coli cells (Promega). Clones containing the plasmid were obtained by kanamycin (Sigma Aldrich, St Louis, MO, 110

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USA) selection and were cultured to obtain sufficient construct for transfection. After sequence verification, transfection of the construct was performed on HEK293 T-REx™ cells (Invitrogen), which stably express the tetracycline repressor operon, using the Superfect® Transfection Reagent (Qiagen). Cells were grown to a confluency of 60-70% in a 6-well plate and cells in each well were transfected by adding 2 µg of the construct according to the protocols described in the Superfect® Transfection Reagent Handbook (Qiagen). Stable clones were obtained by culturing under selective pressure of 300 µg/mL Zeocin™ (Invitrogen). HEK293 T-REx™ clones containing the TO/NDUFAF1 construct were induced for expression of the transgene by adding 0.1-1 µg/mL doxycycline to the growth medium for the times indicated in the results section.

Immunofluorescence assay A culture of a TO/NDUFAF1 containing HEK293 T-REx™ clone was seeded onto a coverslip in a 24-wells plate and grown overnight to achieve a confluency of 50% on the day of the assay. Cells were prestained with Mitotracker Red (Molecular Probes, Eugene, OR, USA) prior to paraformaldehyde fixation. For fluorescence imaging, cell preparates were attached to the stage of an inverted microscope (Axiovert 200 M, Carl Zeiss, Jena, Germany) equipped with a 63×/1.25 NA Plan NeoFluar objective (Carl Zeiss). Alexa Fluor® 488 and Mitotracker Red were excited at 488 nm and 570 nm, respectively, using a monochromator (Polychrome IV, TILL Photonics, Gräfelfing, Germany). Fluorescence emission light was either directed by a 505DRLPXR dichroic mirror (Omega Optical Inc., Brattleboro, VT, USA) through a 535AF26 emission filter (Alexa Fluor® 488) or by a 595DRLP dichroic mirror through a 645AF75 emission filter (Mitotracker Red) onto a CoolSNAP HQ monochrome CCD-camera (Roper Scientific, Vianen, The Netherlands). All hardware was controlled with METAFLUOR 6.0 software (Universal Imaging Corporation, Downingtown, PA, USA) running on a PC equipped with 1 Gb RAM running WINDOWS XP Professional.

Digitonin isolation and solubilization of mitochondria Approximately 1*106 trypsin-harvested cells were pelleted and resuspended in 100 µL of cold

NaCl/Pi to which 100 µL of 4% digitonin (w/v) was added to achieve a final concentration of 2% (w/v) digitonin. This sample was shortly vortexed and incubated on ice for 10 min to solubilize cell membranes. After this, 1 mL of cold NaCl/Pi was added and the sample was centrifuged at 10,000 g for 10 min at 4 °C to obtain a mitochondria-enriched organelle pellet. To remove traces of digitonin, this pellet was washed twice with 1 mL of cold NaCl/Pi. Mitochondrial proteins were solubilized by the addition of 100 µL of AC/BT (1.5 M aminocaproic acid, 75 mM bis-tris pH 7.0) and 20 µL of 10% (w/v) n-dodecyl-β-D-maltoside

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Chapter V and incubation on ice for 10 min. The solubilized proteins are retained in the supernatant after centrifugation at 10,000 g for 25 min at 4 °C, which was used for further analysis.

Gel electrophoresis and in-gel activity assays The protein concentration for blue native polyacrylamide gel electrophoresis (BN-PAGE) and SDS-PAGE was determined in the n-dodecyl-β-D-maltoside solubilized supernatants before adding coomassie blue containing sample buffer, using a MicroBCA protein assay kit (Pierce). Blue native 5-15% gradient gels were loaded with 40 µg of digitonin-isolated mitochondria and, after electrophoresis, were further processed for western blotting, second dimension 10% SDS-PAGE or in-gel activity assays as described earlier[18]. SDS-PAGE analysis was performed by loading 40 µg of protein per lane on 10% SDS-PAGE gels as described before[14].

Blotting and detection Blue native and SDS gels were blotted onto PROTRAN® nitrocellulose transfer membrane (Schleicher and Schuell BioScience) according to standard procedures. Primary antibodies used are against the following proteins: NDUFA9 (39 kDa; Molecular Probes), ND1 (a gift from A. Lombes, INSERM, Paris, France), Complex II 70-kDa subunit, COXII (both Molecular Probes), GADPH, porin, HSP70 (all Abcam, Cambridge, UK), c-myc (Invitrogen) and NDUFAF1 (our laboratory). Secondary antibodies used are swine anti-rabbit IgG horseradish peroxidase (SWARPO), goat anti-rabbit IgG horseradish peroxidase (GARPO) and goat anti-mouse IgG horseradish peroxidase (GAMPO) (Molecular Probes). Signal detection was performed using the ECL® plus Western Blotting Detection System (Amersham Biosciences). After ECL, the blots were exposed to X-OMAT UV film (KODAK). ECL signals were quantified using the IMAGEPRO-PLUS 4.1 image analysis software (Media Cybernetics, Silver Spring, MD, USA).

Results NDUFAF1 localizes in the mitochondrion To analyze the subcellular localization of NDUFAF1 we harvested HEK293 cells at 90% confluency, prepared protein samples of total cell lysate, isolated mitochondria and the cytoplasmic fraction and performed SDS polyacrylamide gel electrophoresis (PAGE). A western blot of this SDS gel was incubated with antibodies against NDUFAF1, a cytoplasmic marker (GAPDH), a marker for the mitochondrial inner membrane (COXII), a marker for the mitochondrial outer membrane (porin) and a marker for both cytoplasm and the

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Human complex I assembly is mediated by NDUFAF1 mitochondrial matrix (HSP70) (Fig 1). NDUFAF1 is clearly more abundant in isolated mitochondria than in the total cell protein extract and is absent in the cytoplasmic protein extract. These data show a specific localization of NDUFAF1 in the mitochondrion.

Figure 1. Western blot analysis of cellular fractionation of HEK293 cells. An SDS-PAGE (10%) gel was loaded with 40 μg protein per lane as follows: lane 1, total cell protein extract; lane 2, cytoplasmic protein extract; lane 3, mitochondrial protein extract. The western blot of this gel was treated with antibodies against NDUFAF1, GAPDH (cytoplasmic marker), COXII and porin (mitochondrial markers) and HSP70 (loading control).

To confirm this result and to use another antibody for localization studies, we decided to create a construct in which the NDUFAF1 gene was tagged with an immunogenic epitope.

We cloned NDUFAF1 into an inducible vector in-frame with a c-myc and His6 tag and transfected this construct into a human embryonic kidney cell line (HEK293), which stably expresses the tetracycline repressor gene. Stable clones were selected and induction of protein expression was tested using western blot analysis. Digitonin-isolated mitochondria were obtained from cultures of an inducible clone after 0, 1, 2, 4, 24, 48 and 72 h of induction with 0.1 µg/mL of doxycycline and analyzed by western blotting (Fig 2). This concentration of doxycyline does not interfere with mitochondrial protein translation[14]. Using both the NDUFAF1 and the c-myc antibodies both the endogenous and the induced (slightly larger than the endogenous protein due to the C-terminal tags) NDUFAF1 could be observed after induction. To investigate the cellular localization of the NDUFAF1-c-myc fusion protein we used a monoclonal c-myc antibody in an immunohistochemical experiment, further incubated with a secondary antibody to which a fluorescent Alexa probe was coupled. The overlay of the Alexa dye staining with mitochondrial control Mitotracker Red shows that induced NDUFAF1 indeed migrates to the mitochondrion (Fig 3). Biochemical fractionation of the NDUFAF1-c-myc expressing cells indicates that this fusion protein also exclusively localizes in mitochondria (results not shown).

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Figure 2. Western blot analysis of induction of NDUFAF1 in HEK293 cells. An SDS-PAGE (10%) gel was loaded with 40 μg protein per lane of a mitochondrial protein extract of an inducible HEK293 clone and blotted. Cells were induced with 0.1 μg/mL of doxycycline for 0 (control), 1, 2, 4, 24, 48 and 72 h. Antibodies used are against NDUFAF1 and complex I membrane arm subunit ND1. Induced and endogenous NDUFAF1 and ND1 signals are indicated on the right. Quantification of the ECL signals is shown in the histogram below, each bar corresponding to the lane above. The signal is expressed as percentage of the 0 h time point signal for both endogenous NDUFAF1 (white bars) and induced NDUFAF1 (black bars).

NDUFAF1 knock down reduces the complex I level and activity Based on its homology with alleged complex I chaperone CIA30 in N. crassa, NDUFAF1 is an interesting candidate assembly protein for human complex I. The possible involvement of NDUFAF1 in complex I assembly/stability was investigated by performing RNA interference experiments. To knock down NDUFAF1 protein expression, two different small interfering RNA (siRNA) oligonucleotides were designed for targeting NDUFAF1 mRNA. Both siRNAs displayed similar knock down effects. Figure 4 shows a transfection of 48 h, followed by consecutive transfections of 48, 72 or 96 h with 10 nM of siRNA #2 (data for siRNA #1 not shown). RNA interference effects are analyzed for NDUFAF1 protein (Fig 4A), fully assembled complex I (Fig 4B) and complex I in-gel activity (Fig 4C). NDUFAF1 expression can be knocked down to less than 30% of the control signal (Fig 4A, lanes 4 and 5). This leads to a 40% decrease of fully assembled complex I (Fig 4B, lane 5). Complex I activity is

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Figure 3. Immunofluorescence mitochondrial localization of induced NDUFAF1. Cells that are induced for NDUFAF1 expression by 1 μg/mL of doxycycline for 24 h were treated with either anti-c-myc IgG coupled to Alexa Fluor 488 to verify the presence of induced NDUFAF1 (top panel) or mitochondrial control Mitotracker Red (middle panel). The bottom panel shows an overlay of the two signals.

compromised as well, as can be seen by the 50% decrease of signal in Figure 4C (lane 5). Interestingly, longer second incubations with siRNA lead to a greater inhibitory effect. Control transfections with an siRNA targeting cyclophilin B performed under the same

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Chapter V circumstances did not lead to a reduction of the NDUFAF1 signal, or to a reduction in the amount of fully assembled complex I (data not shown). This analysis shows that knockdown of NDUFAF1 protein leads to a decrease in the amount and activity of fully assembled complex I. Conversely, preliminary evidence suggests that overexpression of NDUFAF1 leads to an increase in the expression of complex I (data not shown).

NDUFAF1 is present in two high-molecular weight protein complexes To investigate whether NDUFAF1 is present in high-molecular weight protein complexes, its expression pattern was analyzed by means of two-dimensional blue native/SDS-PAGE (2D BN/SDS-PAGE) and western blotting. Control HEK293 cells were harvested at 90% confluency and lysates of digitonin-isolated mitochondria were run on 2D BN/SDS gels before blotting and antibody detection (Fig 5). Two NDUFAF1-containing high-molecular weight complexes of about 600 and 700 kDa can be observed (Fig 5, complexes 2 and 1, respectively). As a size reference, complex I (1 MDa) is shown by using an antibody against the NDUFA9 (39 kDa) subunit of complex I. The expression pattern of NDUFAF1 when it is overexpressed was analyzed by using the doxycycline inducible expression system in combination with 2D BN/SDS-PAGE. After 4 h of induction (Fig 6), induced NDUFAF1 can be seen to migrate from its monomeric form towards the complexes of 600 and 700 kDa, confirming the data observed for endogenous NDUFAF1 in the control situation.

NDUFAF1 expression pattern changes in patients with mutations leading to complex I assembly defects Our complex I assembly model[14] confirms the separate assembly of membrane and peripheral arms described in previous assembly models in N. crassa. A clue about the stage at which NDUFAF1 may operate in this human model can be obtained by screening for the NDUFAF1 expression pattern in complex I-deficient patients. For this study, we have used fibroblasts from an NDUFS8 (TYKY) patient (L. van den Heuvel, manuscript in preparation) and an ND5 patient cybrid cell line with 90% of heteroplasmy (D393G)[19] as representatives of both a peripheral arm and a membrane arm subunit mutation. Cells were harvested to obtain mitochondrial lysates which were run on 2D BN/SDS gels for western blotting and antibody incubation (Fig 7). The NDUFS8 (TYKY) patient cell line is represented in Figure 7A and the ND5 cybrid cell line is represented in Figure 7B. For both patients, complex I expression analysis using NDUFA9 (39 kDa) antibody reveals a decrease in the amount of holocomplex I (represented by Figure 7A, see also Ugalde et al.[14] for detailed description of the composition of complex I assembly intermediates). Less NDUFAF1 is observed in the control fibroblast cell line used for the NDUFS8 (TYKY) patient compared to the cybrid control cell line, and the 700-kDa complex seems absent (Fig 7A,

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Figure 4. RNA interference of NDUFAF1 in HeLa cells. A, B and E, HeLa cells were transiently transfected twice for 48 and 48 h, 48 and 72 h, and 48 and 96 h, respectively. Quantification of the signals is represented in the histograms below each panel. Each bar corresponds to the lane above it, representing the percentage of signal compared to the ‘untreated signal’, corrected for the loading control signal (CoII). A. Western blot of SDS gel. B. Western blot of blue native gel. C. In-gel activity assay. For these panels: lane 1, untreated cells; lane 2, mock transfection (no siRNA); lane 3, transfection with NDUFAF1 siRNA #2 for 48 and 48 h, consecutively; lane 4, transfection with NDUFAF1 siRNA #2 for 48 and 72 h, consecutively; lane 5, transfection with NDUFAF1 siRNA #2 for 48 and 96 h, consecutively. Antibodies used are against NDUFAF1, NDUFA9 (complex I) and CoII-70 kDa (complex II, loading control). In-gel activity results are indicated with IGA, western blot results are indicated with WB.

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Figure 5. 2D BN/SDS-PAGE expression analysis of a mitochondrial protein extract from HEK293 cells. 40 µg protein per lane was loaded on a blue native 5-15% gradient gel (1st dimension) followed by 2nd dimension separation using SDS- PAGE (10%) gels and western blotting. Antibodies used are against NDUFAF1 and NDUFA9 (39 kDa), indicated at the left of each panel. CI refers to fully assembled complex I (complex A in Ugalde et al.[14]). Numbers 1 and 2 refer to NDUFAF1 complexes of 700 and 600 kDa, respectively.

Figure 6. 2D BN/SDS-PAGE expression analysis of mitochondrial protein extracts from HEK293 control cells and an NDUFAF1 inducible HEK293 clone. The inducible clone was induced for NDUFAF1 expression for 1, 2 and 4 h with 1 μg/mL of doxycycline. 40 μg protein per lane was loaded on a blue native 5-15% gradient gel (1st dimension) followed by 2nd dimension separation using SDS-PAGE (10%) gels and western blotting. Anti-NDUFAF1 antibody was used for immunoblot detection. NDUFAF1 containing intermediates are indicated with 1 (700 kDa) and 2 (600 kDa). Induced and endogenously expressed NDUFAF1 are indicated on the right with ‘induced’ and ‘endogenous’, respectively, to differentiate between the endogenous protein and the slightly larger tagged induced protein.

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NDUFAF1 panels). This difference in expression intensity is observed more often and appears to be cell type dependent[14]. Irrespective of this, what can clearly be seen is the strong increase of NDUFAF1 in the 600-kDa complex in the patient compared to the control. So, interestingly, complex I and its assembly intermediates are less abundant whereas more NDUFAF1 is present in the 600-kDa complex. Additionally, the ND5 patient displays a different relative distribution of NDUFAF1 between the 600- and 700-kDa complexes compared to the control (Fig 7A, complexes 2 and 1). NDUFAF1 is more prominently present at 700 kDa in the 90% cybrid compared to the control. As the observed effects are the consequence of complex I membrane arm subunit mutation, this allows the possibility that NDUFAF1 is involved in membrane arm assembly.

NDUFAF1 expression pattern changes in a conditional complex I assembly system To further investigate this, we used a conditional complex I assembly system. A high concentration (10 µg/mL or more) of doxycycline results in the inhibition of mitochondrial protein synthesis. Releasing this inhibition allows investigation of the complex I assembly process and has recently been used in our group to establish a human complex I assembly model[14]. Using this system in combination with 2D BN/SDS-PAGE analysis of NDUFAF1 expression allows investigation of the possible involvement of NDUFAF1 with complex I membrane arm assembly intermediates (Fig 7C). The ratio of the 600- and 700-kDa complexes (Fig 7C, complexes 2 and 1, respectively), shows a remarkable change after the resumption of mitochondrial protein synthesis. In control 143B osteosarcoma cells, NDUFAF1 is predominantly present in the 600-kDa complex (Fig 7C, complex 2). After five days of inhibition of mitochondrial protein synthesis (Fig 7C, t = 0 h), a minor amount of NDUFAF1 is still present in this complex. At 12 h after release of inhibition, NDUFAF1 becomes more predominant in the 700-kDa complex (Fig 7C, complex 1) up to 48 h after release of inhibition. After this time, the amount of NDUFAF1 in the 700-kDa complex decreases and finally returns to the wild-type state in the 600-kDa complex after five days. It seems that NDUFAF1 appears in the 700-kDa complex while complex I assembly proceeds and releases when the control complex I amount is assembled (Fig 7C, complex A). These kinetics are not displayed for the 600-kDa NDUFAF1 complex, which is still present after 5 days of doxycycline inhibition and increases gradually while complex I assembly proceeds (Fig 7C, complex 2). The expression kinetics described above support the notion that when complex I membrane arm assembly is either disturbed or induced, NDUFAF1 becomes more abundant in the 700-kDa complex.

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Figure 7. NDUFAF1 expression in two complex I-deficient patients (A and B) and in a conditional complex I assembly system (C). Mitochondrial pellets were solubilized and 40 µg of protein was loaded onto a blue native 5-15% gradient gel. Expression profiles were analyzed by 2D BN/SDS-PAGE, western blotting and antibody incubation. Antibodies used are against NDUFAF1 and NDUFA9 (39 kDa). Assembly stages described in[14] are indicated at the top by A, B and D. NDUFAF1 containing complexes are indicated by 1 (700 kDa) and 2 (600 kDa). A. Expression analysis of control 143B osteosarcoma cells and a complex I peripheral arm subunit patient cell line [NDUFS8 mutation, (L. van den Heuvel, unpublished data)]. B. Expression analysis of control cybrids (cybrid control) and a complex I membrane arm subunit patient cell line (ND5 mutation D393G)[19] with heteroplasmy level of 90%. C. Expression analysis of

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mitochondrial protein extracts from 143B osteosarcoma cells inhibited for mitochondrial protein synthesis by treatment with 15 μg/mL of doxycycline for 5 days. Samples were taken at 0, 3, 6, 12, 24, 48 h and 5 days after release of doxycycline inhibition. Control (143B osteosarcoma cells) panel is at the top.

Discussion Studying complex I assembly factors will not only aid the development of an accurate assembly pathway but will also contribute to the elucidation of the molecular mechanism responsible for many of the genetically unexplained complex I deficiencies. Analogous to the numerous complex III and IV assembly proteins which are not part of the structural framework of these complexes, it is to be expected that such proteins also exist for complex I. However, so far only two candidate complex I assembly factors were found in the fungus Neurospora crassa: CIA30 and CIA84[15]. The human homologue of CIA30, named NDUFAF1, could be identified[3], but has remained unstudied since its discovery. The mitochondrial localization of NDUFAF1 shown by cellular fractionation (Fig 1) and immunofluorescence microscopy (Fig 3) is consistent with the in silico prediction of an N- terminal mitochondrial targeting sequence and the distribution pattern of expression in different tissues[3]. Our findings support the role of NDUFAF1 in the regulation of assembly/stability of human complex I. Firstly, the knockdown of NDUFAF1 expression by RNA interference shows that the protein is required for correct complex I assembly and/or stabilization (Fig 4). After knockdown of NDUFAF1, the amount of fully assembled complex I is reduced and enzymatic activity is impaired. Surprisingly, no accumulation of assembly intermediates is observed in the NDUFAF1 RNAi experiments on 2D BN/SDS gels, suggesting that NDUFAF1 may be involved in the stabilization of these intermediates rather than in active combination of assembly intermediates (R. Janssen, unpublished results). Secondly, prolonged overexpression of NDUFAF1 in HEK293 cells leads to an increased amount of fully assembled complex I. NDUFAF1 occurs in two high-molecular weight protein complexes of 600 and 700 kDa. Its expression in two complex I-deficient patient cell lines differs greatly when either membrane arm or peripheral arm assembly is compromised by mutation (Fig 7A,B). Mutation in membrane subunit ND5 results in a relative increase of the 700-kDa NDUFAF1 complex very similar to the shift in the conditional assembly system. Peripheral arm subunit NDUFS8 mutation results in a completely different expression profile. While in this patient complex I and its assembly intermediates are diminished, more NDUFAF1 is present at 600 kDa. This indicates that despite the comigration of this complex with peripheral arm assembly intermediate D[14], they are different complexes.

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Additional support for the involvement of NDUFAF1 in complex I assembly/stability comes from analysis of its 2D BN/SDS assembly profiles in a conditional complex I assembly system (Fig 7C). Absence of NDUFAF1 from the 700-kDa complex after inhibition of assembly shows that association of NDUFAF1 with the complex is hampered. As after doxycycline inhibition of mitochondrial protein synthesis no mitochondrial protein can be produced, this suggests that NDUFAF1 is bound to a mtDNA-encoded protein. The transient shift of NDUFAF1 to the 700-kDa complex after assembly resumes, suggests that NDUFAF1 is required in this complex while assembly proceeds. This is supported by the fact that when after five days the complex I amount is restored, the amount of associated NDUFAF1 in this complex is also reduced to control levels. The 700-kDa NDUFAF1 complex may therefore represent an important step in the process of complex I assembly. The model for complex I assembly in humans proposed by our group supports the idea that complex I assembly occurs in a modular fashion, and is largely compatible with the N. crassa model for complex I assembly[14]. In this system, complex I is assembled via the combination of preassembled evolutionarily conserved modules like the bricks of a Lego system. In more detail, this entails that the membrane arm appears to be assembled in several steps by combining a small and large intermediate. The peripheral arm is assembled independently and is joined to the complete membrane arm in a later stage. Based on the results presented in this paper, we propose that NDUFAF1 modulates this process. An active role in assembly may serve to ease the combination of assembly intermediates while, alternatively, a stabilizing or scaffolding role may serve to prevent misfolding or degradation of assembly intermediates. This function for NDUFAF1 is in line with the proposed mechanism described for the N. crassa homologue CIA30[15]. In this study CIA30 was suggested to aid the combination of the small and large membrane arm intermediates in complex I assembly by exclusive binding to the large membrane arm intermediate[12,16]. Knockout of the cia genes in N. crassa resulted in a block in complex I assembly, characterized by the absence of the large membrane arm intermediate and the accumulation of the small membrane arm intermediate and the peripheral arm[15]. The acquired data do not conflict with this idea. Both disturbance of membrane arm assembly (ND5 cybrid) and pressurizing the assembly system by releasing doxycycline inhibition of mitochondrial translation result in accumulation of the 700-kDa NDUFAF1 complex. However, to ascertain direct involvement in complex I membrane arm assembly, the exact composition of the NDUFAF1 intermediates is a prerequisite. In addition, the observed changes in NDUFAF1 assembly status in complex I-deficient patients can be indicative for the possible gene defect and we are currently investigating more patients to test this.

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It is quite conceivable that, analogous to NDUFAF1, many more proteins are involved in the stabilization of complex I assembly intermediates. A recent example is the possible function of apoptosis-inducing factor (AIF) in intramitochondrial assembly/maintenance of respiratory chain complexes in a mouse model system[17]. Future investigation of the exact composition of complex I assembly intermediates will be a great step forward in studying the function of NDUFAF1 and finding new complex I assembly chaperones.

Acknowledgements We thank Dr A. Lombes (Inserm, Paris, France) for providing, the anti-ND1 and anti-ND6 IgGs. This work was supported by the ‗Prinses Beatrix Fonds‘ to J.S. and L.v.d.H. (grant number 02-0104) and the European Union's Sixth Framework Programme for Research, Priority 1 ‗Life Sciences, Genomics and Biotechnology for Health‘ (contract number LSHM- CT-2004-503116). The Netherlands Organization for Scientific Research supported L.N. with a ‗Vernieuwingsimpuls‘ grant. C.U. is recipient of a research contract from Instituto de Salud Carlos III (ISC III CP04/00011).

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References

1. Smeitink J, van den Heuvel L, DiMauro S (2001) The genetics and pathology of oxidative phosphorylation. Nat Rev Gene 2(5):342-352 2. Barrientos A, Barros MH, Valnot I, Rotig A, Rustin P, Tzagoloff A (2002) Cytochrome oxidase in health and disease. Gene 286(1):53-63 3. Janssen R, Smeitink J, Smeets R, van den Heuvel L (2002) CIA30 complex I assembly factor: a candidate for human complex I deficiency? Hum Genet 110(3):264-270 4. Grigorieff N (1999) Structure of the respiratory NADH: ubiquinone oxidoreductase (complex I). Curr Opin Struct Biol 9(4):476-483 5. Carroll J, Fearnley IM, Shannon RJ, Hirst J, Walker JE (2003) Analysis of the subunit composition of complex I from bovine heart mitochondria. Mol Cell Proteomics 2(2):117-126 6. Hirst J, Carroll J, Fearnley IM, Shannon RJ, Walker JE (2003) The nuclear encoded subunits of complex I from bovine heart mitochondria. Biochim Biophys Acta 1604(3):135-150 7. Vinogradov AD (2001) Respiratory complex I: structure, redox components, and possible mechanisms of energy transduction. Biochemistry 66(10):1086-1097 8. Cardol P, Matagne RF, Remacle C (2002) Impact of mutations affecting ND mitochondria-encoded subunits on the activity and assembly of complex I in Chlamydomonas. Implication for the structural organization of the enzyme. J Mol Biol 319(5):1211-1221 9. Stroh A, Anderka O, Pfeiffer K, Yagi T, Finel M, Ludwig B, Schagger H (2004) Assembly of respiratory complexes I, III, and IV into NADH oxidase supercomplex stabilizes complex I in Paracoccus denitrificans. J Biol Chem 279(6):5000-5007 10. Yadava N, Houchens T, Potluri P, Scheffler IE (2004) Development and characterization of a conditional mitochondrial complex I assembly system. J Biol Chem 279(13):12406-12413 11. Videira A (1998) Complex I from the fungus Neurospora crassa. Biochim Biophys Acta 1364:89- 100 12. Schulte U (2001) Biogenesis of respiratory complex I. J Bioenerg Biomembr 33(3):205-212 13. Antonicka H, Ogilvie I, Taivassalo T, Anitori RP, Haller RG, Vissing J, Kennaway NG, Shoubridge EA (2003) Identification and characterization of a common set of complex I assembly intermediates in mitochondria from patients with complex I deficiency. J Biol Chem 278(44):43081-43088 14. Ugalde C, Vogel R, Huijbens R, van den Heuvel B, Smeitink J, Nijtmans L (2004) Human mitochondrial complex I assembles through the combination of evolutionary conserved modules: a framework to interpret complex I deficiencies. Hum Mol Genet 13(20):2461-2472 15. Kuffner R, Rohr A, Schmiede A, Krull C, Schulte U (1998) Involvement of two novel chaperones in the assembly of mitochondrial NADH:Ubiquinone oxidoreductase (complex I). J Mol Biol 283(2):409-417 16. Duarte M, Videira A (2000) Respiratory chain complex I is essential for sexual development in Neurospora and binding of iron sulfur clusters are required for enzyme assembly. Genetics 156(2):607-615 17. Vahsen N, Cande C, Briere JJ, Benit P, Joza N, Larochette N, Mastroberardino PG, Pequignot MO, Casares N, Lazar V, Feraud O, Debili N, Wissing S, Engelhardt S, Madeo F, Piacentini M, Penninger JM, Schagger H, Rustin P, Kroemer G (2004) AIF deficiency compromises oxidative phosphorylation. EMBO J 23(23):4679-4689 18. Nijtmans LG, Henderson NS, Holt IJ (2002) Blue native electrophoresis to study mitochondrial and other protein complexes. Methods 26(4):327-334 19. Corona P, Antozzi C, Carrara F, D'Incerti L, Lamantea E, Tiranti V, Zeviani M (2001) A novel mtDNA mutation in the ND5 subunit of complex I in two MELAS patients. Ann Neurol 49(1):106- 110

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Human complex I assembly is mediated by NDUFAF1

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Differences in assembly or stability of complex I and other mitochondrial OXPHOS complexes in inherited complex I deficiency

Cristina Ugalde*, Rolf J.R.J. Janssen*, Lambert P.W.J. van den Heuvel, Jan A.M. Smeitink and Leo G.J. Nijtmans

Nijmegen Center for Mitochondrial Disorders, Laboratory of Pediatrics and Neurology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands * Joint first authorship

Human Molecular Genetics 2004, Volume 13, Number 6, Page 659-667

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Differences in assembly or stability of complex I

Abstract NADH:ubiquinone oxidoreductase (complex I) deficiency is amongst the most encountered defects of the mitochondrial oxidative phosphorylation (OXPHOS) system and is associated with a wide variety of clinical signs and symptoms. Mutations in complex I nuclear structural genes are the most common cause of isolated complex I enzyme deficiencies. The cell biological consequences of such mutations are poorly understood. In this paper we have used blue native polyacrylamide gel electrophoresis in order to study how different nuclear mutations affect the integrity of mitochondrial OXPHOS complexes in fibroblasts from 15 complex I-deficient patients. Our results show an important decrease in the levels of intact complex I in patients harboring mutations in nuclear-encoded complex I subunits, indicating that complex I assembly and/or stability is compromised. Different patterns of low-molecular weight subcomplexes are present in these patients, suggesting that the formation of the peripheral arm is affected at an early assembly stage. Mutations in complex I genes can also affect the stability of other mitochondrial complexes, with a specific decrease of fully assembled complex III in patients with mutations in NDUFS2 and NDUFS4. We have extended this analysis to patients with an isolated complex I deficiency in which no mutations in structural subunits have been found. In this group, we can discriminate between complex I assembly and catalytic defects attending to the fact whether there is a correlation between assembly/activity levels or not. This will help us to point more selectively to candidate genes for pathogenic mutations that could lead to an isolated complex I defect.

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Introduction Mitochondrial diseases are a heterogeneous group of multisystemic disorders mostly caused by the dysfunction of one or more enzyme complexes of the oxidative phosphorylation (OXPHOS) system. NADH:ubiquinone oxidoreductase (complex I) deficiency is a frequently diagnosed defect of the mitochondrial OXPHOS system generally associated with severe metabolic disorders of childhood, like progressive encephalomyopathy, leukodystrophy or Leigh syndrome. Patients show a wide spectrum of clinical presentations and many die very young because of a fatal dysfunction of tissues with high metabolic energy rates[1]. Mammalian complex I is a ~980-kDa holoenzyme that consists of two functional parts: a catalytic peripheral arm that protrudes into the matrix, and a membrane arm for proton translocation, which is embedded in the mitochondrial inner membrane (Fig 1). Complex I is made up of at least 39 different structural proteins encoded by the nuclear genome and seven subunits encoded by the mitochondrial DNA[2]. By using mildly chaotropic detergents the complex has been resolved into various subcomplexes, each containing a different subunit composition[2]. Although in some cases mitochondrial DNA mutations have been described as causing a complex I deficiency, they only account for a small percentage of the total number of complex I-deficient patients. In the majority of these patients mutations in nuclear genes are expected. The identification and genetic characterization of 45 human complex I nuclear structural genes has been completed[1,2]. As a result of an extensive mutation detection program at our center in a research cohort of 24 patients presenting with an isolated complex I deficiency, pathogenic mutations were found in five complex I nuclear genes: NDUFS2[3], NDUFS4[4,5], NDUFS7[6], NDUFS8[7] and NDUFV1[8]. Later, other groups detected new mutations in NDUFS4[9,10], NDUFS1[11], NDUFV1[11] and NDUFV2[12,13]. Except for NDUFS4, all the pathogenic mutations are located in the structural core subunits that are highly conserved from bacteria to mammals[14]. In the remaining patients no mutations have been identified after screening all complex I structural genes, thus the genetic defect leading to an isolated complex I deficiency must be found in nuclear genes encoding regulatory proteins of complex I biogenesis or activity. The exact mechanisms by which these mutations affect the function of mitochondrial complex I remain unclear. Studies in Yarrowia lipolytica have shown that mutations in the genes encoding subunits PSST and TYKY (NDUFS7 and NDUFS8 homologues, respectively) affected electron transport by altering complex I in vitro kinetics, resulting in a catalytically impaired complex I[15]. Similarly, mutational analyses of the yeast NDUFS2 homologue revealed its central role (together with PSST and TYKY) in the catalytic core for ubiquinone reduction[16]. A 5-bp duplication in the NDUFS4 gene destroys a cAMP-dependent consensus phosphorylation site, abolishing complex I activity in a Leigh syndrome patient[17]. It has been recently demonstrated that patients with genetic alterations in NDUFS4 show low levels of mitochondrial complex I

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Differences in assembly or stability of complex I together with an accumulation of an intermediate subcomplex[18], suggesting defects in complex I assembly or stability. To better characterize complex I deficiencies, we used western blotting in combination with sucrose gradient centrifugation in our patient cohort[19]. Differences found in the relative subunit composition of complex I in these patients seemed to depend on the gene mutated, suggesting that distinct pathogenic mutations within the same gene could lead to a similar assembly defect. Furthermore, a severe disturbance in complex I assembly was confirmed in one patient harboring an unknown nuclear mutation[19].

Figure 1. Approximate position of mitochondrial complex I nuclear-encoded core subunits plus others participating in the present study. Highlighted are the subunits in which mutations have been analyzed. Subcomplexes Iλ, Iβ and Iγ are indicated.

In order to study how these mutations affect the integrity of mitochondrial OXPHOS complexes, we have now extended the biochemical studies by using blue native polyacrylamide gel electrophoresis (BN-PAGE)[20]. Here we report our results in cultured skin fibroblasts from 15 complex I-deficient patients harboring mutations in complex I- related nuclear genes (Tab 1). Six patients with known mutations in complex I subunits show low relative amounts of fully assembled complex I that correlate with low levels of complex I activity detected in-gel, suggesting that complex I assembly or stability is compromised. By two-dimensional BN-PAGE, different patterns of complex I sub-products are detected in

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Table 1. Clinical, biochemical and genetic characteristics of the investigated complex I-deficient patients

Patient Gene Fraction Mutation Biochemistry CI act. (%) Other act. (%) Phenotype Ref.

P1 NDUFS8 Iλ P79L/R102H 2x4Fe-4S clusters 69a All normal Leigh syndrome [7] P2 NDUFS7 Iλ V122M 4Fe-4S cluster (N2) 68a All normal Leigh syndrome [6] P3 NDUFS2 Iλ P229Q H+ translocation/Q-binding 36a All normal HCEM [3] P4 NDUFS2 Iλ R228Q H+ translocation/Q-binding 39a All normal HCEM [3] P5 NDUFS4 Iλ W97X cAMP dep. phosphorylation 59a/47b CIII 98a/87b Leigh-like syndrome [5] P6 NDUFS4 Iλ R106X cAMP dep. phosphorylation 36a/30b CIII 100a/88b Leigh syndrome [21] P7 unknown unknown 45a All normal FILA P8 unknown unknown 39a All normal UEM P9 unknown unknown 60a All normal FILA P10 unknown unknown 85a All normal Leigh syndrome P11 unknown unknown 36a All normal Leigh-like syndrome P12 unknown unknown 63a All normal FILA [47] P13 unknown unknown 73a All normal PCA P14 unknown unknown 42a All normal Leigh syndrome P15 unknown unknown 27a All normal Leigh syndrome Enzymatic activities (act.) were measured in triplicate in at least two independent patient-derived samples. The values for the residual complex I (CI) and complex III (CIII) activities are given as a percentage of the lowest control value (set as 100%) after a) normalization by COX or b) normalization by the matrix enzyme citrate synthase. Q, ubiquinone; HCEM, hypertrophic cardiomyopathy and encephalomyopathy; FILA, fatal infantile lactic acidosis; UEM, unspecified encephalomyopathy; PCA, progressive cerebellar ataxia.

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Differences in assembly or stability of complex I patients with different mutated genes. Two patients harboring unknown nuclear mutations present such low complex I activity/assembly levels, suggesting that the genetic defect should be found in a new complex I subunit or assembly factor. The remainder seven patients show almost normal levels of the fully assembled holoenzyme with low complex I activities, compatible with a catalytic defect in which a mutation could be found in a factor participating in the activation of complex I. Finally, we have analyzed how the different mutations in complex I genes affect the stability of other OXPHOS complexes, demonstrating a specific decrease of intact mitochondrial complex III in patients with mutations in NDUFS2 and NDUFS4.

Patients, materials and methods Patients Table 1 summarizes the genetic, clinical and biochemical features of the patients‘ fibroblasts analyzed in this study. Patients and mutations were previously reported[3,5,6,7,21,24,47].

Cell cultures Fibroblasts were obtained from skin biopsies and cultured in M199 medium (Life Technologies) supplemented with 10% fetal calf serum (FCS) and 100 IU/ml penicillin and 100 IU/ml streptomycin.

Complex I assembly and in-gel activity studies One- and two-dimensional BN-PAGE and mitochondrial complex I in-gel activity assays were performed with digitonin-isolated mitochondria from patients‘ fibroblasts as previously described[48]. Proteins were transferred to a PROTRAN® nitrocellulose membrane (Schleicher and Schuell). Western blotting was performed using primary antibodies raised against complex I subunits NDUFA9, NDUFS3 and NDUFS5, against core protein 2 and COXI subunits from mitochondrial complexes III and IV, respectively, and against the α- subunit of F0F1-ATPase (Molecular Probes). Peroxidase-conjugated anti-mouse immunoglobulins (Molecular Probes) were used as secondary antibodies. The signal was detected by using ECL® plus reagents (Amersham Biosciences) and the quantification of the blots was performed using ImagePro-Plus 4.1 image analysis software (Media Cybernetics, Silver Spring, MD, USA).

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Results The integrity of human mitochondrial complex I is altered by mutations in nuclear- encoded subunits Mitochondria isolated from cultured skin fibroblasts of complex I-deficient patients (Tab 1) in which mutations have been described in subunits NDUFS2[3], NDUFS4[5,21], NDUFS7[6] and NDUFS8[7] were run under native conditions in a BN-PAGE system and subjected to a complex I in-gel activity assay (Fig 2A). As expected, low complex I activities were confirmed in all the patient samples when compared with control levels. A second activity below 440 kDa is normally observed in all samples tested, which include rho zero cells (unpublished data), and it could correspond to other NADH dehydrogenases present in mitochondria. A duplicate gel was blotted and incubated with antibodies raised against the complex I subunits NDUFA9 and NDUFS3 (Fig 2B and C, respectively). Decreased levels of fully assembled complex I were observed in all cases, which correlate with the low complex I activities found in the patients. These results suggest that disturbances in the assembly pathway or in the stability of mitochondrial complex I are the direct cause of the decreased activity levels. This effect seemed to be more pronounced in two patients with mutations in NDUFS8 (patient 1) and NDUFS2 (patient 3). Moreover, patients 5 and 6 carrying mutations in the NDUFS4 gene accumulated high levels of a lower-molecular weight subcomplex (~800 kDa, gray arrow) that showed no complex I activity in Figure 2A, as has been recently described[18]. The same product was observed in three additional patients with different mutations in NDUFS4 (data not shown). Other subcomplexes containing subunit NDUFS3 were commonly present in patients and control samples (Fig 2C, small black arrows). Surprisingly, patient 3 (P229Q mutation in NDUFS2) repeatedly showed a band at a position higher than complex I (Fig 2C, dashed arrow). It also appeared in the NDUFS4 patients 5 and 6 under different electrophoretic conditions (data not shown). In patient 3, this band was more intense than the one corresponding to the fully assembled complex (estimated ratio 2:1) and it could correspond to the presence of mitochondrial supercomplexes I1III2 or

I1III2IV1[22]. To check this possibility and, at the same time, analyze the expression levels of native complexes III and IV in the different patients, the same blot was incubated with antibodies against core protein 2 (complex III) and COXI (complex IV) subunits (Fig 2E and D, respectively). Presence of complexes III or IV was not detected at the high-molecular weight range, suggesting that the upper band would contain only complex I, maybe representing an inactive conformation of this complex (it does not show NADH dehydrogenase activity in the in-gel activity assay). Further studies are necessary to fully confirm this hypothesis.

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Figure 2. BN-PAGE analysis of mitochondrial complexes I, III and IV in fibroblasts from patients with characterized mutations in nuclear-encoded complex I subunits. Mitochondrial particles were isolated as described in ‘Patients, materials and methods’ and 30 µg protein was analyzed on a blue native 5-15% gradient gel for the separation of multisubunit complexes. A. In-gel activity assay (IGA) of mitochondrial complex I confirming a decrease in all the activities of patients compared to control samples. A non-specific NADH dehydrogenase activity below 440 kDa is indicated on the left with an arrowhead. B. A second gel was run in duplicate and western blot analysis was performed using antibodies against complex I subunits NDUFA9 and (C) NDUFS3, or (D) against complex IV subunit COXI and NDUFA9 or (E) complex III core protein 2. A high-molecular weight band containing NDUFS3 in patient 3 is indicated by a dashed arrow. The accumulation of the typical 800-kDa subcomplex observed in NDUFS4 patients (patients 5 and 6) is indicated by a gray arrow. Lower subcomplexes that are common to control and patient samples are indicated by small black arrows. CI, fully assembled complex I (~980 kDa). CIII, mitochondrial complex III dimer (~600 kDa). CIV, mitochondrial complex IV (~200 kDa). CIII+CIV indicates the presence of a supercomplex containing both complexes that is frequently observed in BN-PAGE analysis. The positions in the gel of the ferritin monomer (440 kDa) and dimer (880 kDa) used as molecular mass standards are indicated on the left.

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Mutations in NDUFS2 and NDUFS4 subunits affect the stability of mitochondrial complex III When we compared the ratios between native mitochondrial complexes, it appeared that the levels of the fully assembled complex III dimer (~600 kDa) were clearly decreased in the patients harboring different mutations in NDUFS2 (patients 3 and 4) and NDUFS4 (patients 5 and 6), were slightly decreased by the mutation in NDUFS7 (patient 2), but not affected by the mutation in NDUFS8 (Fig 2E). This result supports the idea of a physical interaction between both complexes. In agreement with this, a combined complex I+III deficiency has been described for three patients carrying mutations in the NDUFS4 gene[5,21]. A similar reduction in the levels of complex III was observed in two out of three additional patients harboring different mutations in NDUFS4, making it a total of four patients. All these patients harbor missense mutations leading to premature stop codons in the central part of the protein. In the remainder patient with normal assembled complex III[4], the mutation is located very near to the carboxy terminus of the protein, suggesting that this region is important for the activity of complex I but not relevant for the stability of mitochondrial complex III (Ugalde et al., manuscript in preparation). Because all the patients showed normal cytochrome c oxidase (COX) enzymatic activities and the enzymatic values for complex I were calculated relative to COX (Tab 1), the antibody against COXI subunit was also used to normalize for the expression levels of mitochondrial complex IV (Fig 2D). Fully assembled COX levels were slightly increased in patients 4, 5 and 6 when compared with the control, but this is probably due to differences in sample loading in this particular experiment. The signals from the blots were quantified and the complex I/complex IV and complex III/complex IV ratios were used as numerical values for the expression levels of fully assembled complex I and III, respectively (Tab 2). An apparent lack of correlation between the residual complex I enzymatic activities shown in Table 1 and the amount of fully assembled complex I quantified in Table 2 occurred in some patients. For instance, patients 1 and 2 presented a similar complex I activity but the differences in the amount of complex I calculated in Table 2 were bigger (7 and 50%, respectively). These differences might come from the mitochondrial extraction method used in each assay. For the biochemical activity measurements, differential centrifugation in glucose gradient was used, whereas for the BN- PAGE assay, digitonin extraction was used. Although digitonin is considered a mild detergent, it could enhance the disruption of the complex and the loss of complex I activity under circumstances that would promote the loss of stability of the complex (such as certain mutations in complex I subunits). If this is the case, this treatment does not seem to affect the levels of fully assembled complex I in control fibroblasts.

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Reversibly, patients with mutations in NDUFS2 and NDUFS4 showed a relatively important decrease of assembled complex III levels with very limited or no complex III enzymatic defect. Possibly the reduction in the levels of fully assembled complex III is not sufficient to reach the threshold necessary to provoke a marked decrease on complex III activity, as it has been shown in a parkinsonism-related complex III deficiency[23].

Table 2. Numerical values for the expression levels of mitochondrial complexes I, III and IV in patients with known mutations in nuclear DNA-encoded subunits

Patient CI CIII CIV CI/CIV CIII/CIV

C 100 100 100 1.00 1.00 P1 7 ± 1 100 ± 4 95 ± 4 0.07 1.05 P2 50 ± 2 87 ± 3 105 ± 7 0.48 0.83 P3 10 ± 0 59 ± 3 103 ± 5 0.10 0.57 P4 24 ± 2 55 ± 6 136 ± 8 0.18 0.40 P5 24 ± 4 64 ± 5 125 ± 7 0.19 0.51 P6 38 ± 4 73 ± 6 121 ± 5 0.31 0.60 To calculate the expression levels of complexes I and III, three independent blue native blots were quantified and the values normalized to those obtained from complex IV. Values below the normal expression range are highlighted in bold.

Distinct patterns of low-molecular weight subcomplexes in patients with mutations in nuclear structural genes To study in more detail how mutations in nuclear genes affected complex I assembly or stability, mitochondrial native (sub)complexes were separated in a second dimension SDS- PAGE system, blotted and incubated with antibodies against three complex I subunits: NDUFS3, localized in the peripheral arm, and NDUFS5 and NDUFA9, localized in the peripheral and membrane arm boundary (Fig 3). Three different effects were observed: (i) the repeated presence of lower amounts of fully assembled complex I in patients‘ fibroblasts relative to controls; (ii) different patterns of low-molecular weight subcomplexes that contain the NDUFS3 subunit; and (iii) similar accumulation of an ~800-kDa subcomplex in patients with different mutated genes. These results confirm that the assembly and/or stability of mitochondrial complex I is severely affected by these mutations. The most dramatic effects were observed in the two patients with a heterozygous P79L/R102H mutation in the NDUFS8 subunit and with a homozygous P229Q mutation in NDUFS2 (data not shown), where the signals for complex I subunits were almost undetectable. In all the patients NDUFS3-containing subcomplexes below 450 kDa accumulated, probably

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Figure 3. Different patterns of complex I subcomplexes are observed in fibroblasts from patients with known mutations by two-dimensional BN/SDS-PAGE. Mitoplasts were extracted and 40 µg protein separated on a blue native 5-15% gradient gel. For the separation of individual subunits a SDS-PAGE (10%) gel was run in the second dimension. Gels were blotted. For the positioning of complex I (~980 kDa) primary antibodies against complex I subunits NDUFA9 (39 kDa), NDUFS3 (30 kDa) and NDUFS5 (15 kDa) were used in

control and patients’ fibroblasts. An antibody against the α-subunit of F0F1-ATPase was used as

a reference molecular weight marker for the relative position of complex V (~750 kDa) and F1-

ATPase subcomplex (~450 kDa), depicted as V and F1 respectively. CI indicates the relative position of fully assembled complex I. Lower-molecular weight subcomplexes are indicated by vertical arrows. The directions of the first (1D) and second (2D) dimension are shown.

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Differences in assembly or stability of complex I indicating that the formation of the peripheral arm is interrupted at very early stages of the assembly process. Subunits NDUFS5 and NDUFA9 were mostly present in the ~800-kDa subcomplex, suggesting their incorporation at a later stage of complex I assembly. According to the results obtained in the first dimension, the accumulation of this late subcomplex is especially abundant in patients harboring mutations in the NDUFS4 gene (Fig 3, lower panel).

Differences in catalytic versus assembly defects in patients harboring unknown mutations in nuclear genes The same analysis was extended to nine fibroblast cell lines from patients in which mutations have been discarded in nuclear and mitochondrial complex I structural genes[24]. Again, a complex I deficiency was confirmed in all the patients by a complex I in-gel activity assay (Fig 4A). A complex I assembly/activity correlation was only found in patients 11 and 15 (Fig 4B). In these cases the levels of fully assembled complex I were very low and the levels of complex I activity were almost undetectable, suggesting an assembly defect as the primary cause of the decreased complex I activity. A lower-molecular weight subcomplex also accumulated in patient 15 (Fig 4B, gray arrow). Remarkably, patient 11 is the same in which an assembly defect was previously described by other means[19]. These data indicate that these two patients are good candidates for carrying mutations in a yet unknown complex I subunit or assembly factor. In the rest of this second group of patients complex I activities were low but the levels of fully assembled complex I were normal, so the impairment of complex I activity is most probably due to a catalytic defect. The pattern and expression levels of native complexes III (Fig 4C) and IV (data not shown) were also analyzed, but no significant differences were detected between patients and controls.

Discussion Complex I deficiency comprises a number of clinically heterogeneous disorders of energy metabolism of which Leigh (or Leigh-like) syndrome is the most commonly diagnosed. Unlike complex IV-deficient Leigh syndrome patients, in whom mutations have been mostly found in complex IV assembly factors such as SURF1 or SCO2, all mutations responsible for complex I deficiencies detected so far are located in complex I structural genes[25,26]. Patients with mutations in the complex I nuclear-encoded subunits NDUFS4, NDUFS7 and NDUFS8 plus, in our hands, 50% of the patients in whom no mutations have been found yet, present with this devastating disorder (Tab 1). Mutations found in the NDUFS2 gene have been specifically associated with hypertrophic cardiomyopathy and encephalomyopathy[3].

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Figure 4. Mitochondrial complex I catalytic versus assembly defects can be distinguished in fibroblasts from isolated complex I-deficient patients by BN- PAGE analysis. Mitochondria were isolated and analyzed as described in Figure 2. A. Complex I in-gel activity assay of mitochondrial complex I. The unspecific NADH dehydrogenase activity below 440 kDa is indicated with arrowheads. B. Western blotting analysis of native complex I with antibodies against complex I subunit NDUFA9. The presence of a lower-molecular weight subcomplex is indicated with a gray arrow. C. Same analysis with an antibody against mitochondrial complex III core protein 2. The top band indicated as CI is a residual band from incomplete stripping of the membrane after probing for complex I. CI, CIII and CIV are mitochondrial complexes I, III and IV, respectively. The positions of the ferritin monomer (440 kDa) and dimer (880 kDa) used as molecular mass standards are indicated on the left.

Despite genetic and clinical differences, in these patients there seems to be a common pathogenic mechanism leading to complex I or IV deficiencies that would involve a problem in the proper assembly or stabilization of the mitochondrial OXPHOS complexes. This in turn would lead to a decrease of OXPHOS activities and to structural changes in the

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Differences in assembly or stability of complex I mitochondrial inner membrane that subsequently could induce proton leakage, accumulation of toxic reactive oxygen species and release of apoptosis-inducing factors. According to this hypothesis, it has been recently demonstrated that complex I-deficient Leigh syndrome patients with genetic alterations in NDUFS4 or in the mitochondrial ND6 gene show low levels of native mitochondrial complex I together with an accumulation of lower intermediate assemblies[18,27]. In rodent cells, complex I assembly and activity are affected by the lack of the NDUFA1-homologue subunit, MWFE[28]. In Neurospora crassa, complex I nuclear-encoded subunits play a central role of in the assembly and function of the holoenzyme[29]. In this paper we show that, in six complex I-deficient patients where mutations in nuclear subunits have been found, complex I assembly and/or stability are severely compromised. Decreased levels of fully assembled complex I correlate with the low complex I activities found in the patients, suggesting an assembly/stability defect as the primary pathogenic mechanism. To explain the causes we must take into account different factors, like the position and conservation of the mutations, the localization of the mutated subunits within the complex and the individual role of each subunit within the overall function of complex I. All the mutations are found in highly conserved amino acid positions during evolution, but maybe some positions are functionally more relevant than others and provoke a more dramatic complex I defect. All the mutations analyzed are located in subunits forming the peripheral arm of the complex, therefore these mutations are expected to affect the formation and catalytic activity of this specific fragment. Little is known about the function of the so-called complex I accessory subunits, but studies mainly performed in N. crassa and Y. lipolytica have shed light on the precise role of the core subunits. The most evident complex I defect was found in patients with P79L/R102H amino acid changes in NDUFS8 and a P229Q substitution in NDUFS2, in whom native complex I was almost undetectable. The homologues of NDUFS7 and NDUFS2 have been described in Y. lipolytica to form the catalytic core for ubiquinone binding, a process in which NDUFS8 is also involved[16]. Reconstruction in Y. lipolytica of the human pathogenic mutations found in the NDUFS7 and NDUFS8 genes did not lead to an impaired complex I assembly but only to a complex I catalytic defect[15]. The same occurred with mutations in highly conserved residues of the NDUFS7 and NDUFS2 homologues[30,31]. However, disruption of any of these three subunits in N. crassa showed impaired assembly of the peripheral arm, accumulation of the membrane arm and a consequent catalytic defect[32-34]. The differences found in complex I defects between our complex I-deficient patients and the yeast model give evidence of the necessary precaution that must be taken when mimicking human physiological conditions in lower species. All our patients with mutations in NDUFS4 also showed a marked decrease in the levels of fully assembled complex I together with high levels of a defective subcomplex of ~800 kDa not present in controls, as already described[18]. Additionally, the presence of

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Chapter VI lower bands between ~100-450 kDa containing complex I NDUFS3 subunit was detected both in control and all patient samples. These subcomplexes possibly correspond to assembly intermediates from the pathway recently proposed for complex I assembly[35]. In a second dimension, distinct patterns of lower-molecular weight subcomplexes were clearly observed in patients carrying mutations in different complex I subunits. The low amounts of fully assembled complex I and the relative accumulation of NDUFS3-containing subcomplexes below 450 kDa suggest that the formation of the peripheral arm is interrupted at very early stages of the assembly process. This result is in agreement with the complex I assembly model in which the NDUFS3 subunit would form part of assembly intermediates since the beginning of the process[35]. Because of the malformation of the peripheral arm, the membrane arm might have problems getting incorporated and form the final assembled holoenzyme. Then subunits NDUFS5 and NDUFA9, present in the peripheral and membrane arm boundary region, would get accumulated at a later stage of assembly forming, together with the peripheral arm, an incomplete intermediate of ~800 kDa. This is especially noticeable in patients with mutations in the NDUFS4 gene. The two mutations analyzed here lead to a truncated version of NDUFS4. It has been previously reported in other NDUFS4 patients that this intermediate contains a number of complex I subunits including NDUFS3, NDUFS5 and NDUFA9, but not NDUFS4 itself[18]. These results suggest that NDUFS4 would be incorporated at this late stage in the assembly process, as has been reported[35], and that the lack of NDUFS4 would hamper the next step in the formation of fully assembled complex I. The possibility also exists that the accumulation of lower- molecular weight subcomplexes in these patients could not be only due to an assembly defect or to a lower rate of incorporation of the different subunits into the complex, but also to a lower complex I stability and its consequent disruption. Additional experiments must be performed in order to reach definitive conclusions. In the remainder complex I-deficient patients harboring unknown mutations, catalytic versus assembly (or stability) defects have been easily distinguished by BN-PAGE. Two patients show a similar complex I assembly defect as established in the patients carrying mutations in nuclear-encoded subunits. Mutations in complex I subunits have been discarded in all 45 complex I subunits. These two patients are candidates to carry mutations in a complex I assembly factor. Our identification of the human homologue of a N. crassa complex I assembly protein, CIA30, proves the existence of such factors[36]. Unfortunately, no disease-causing mutations have been found in any of these genes yet. In the rest of this second group of patients the levels of fully assembled complex I are normal. The impairment in complex I activity cannot be explained by a defect in complex I assembly but to a pure catalytic defect, suggesting a different pathogenic mechanism leading to an isolated complex I deficiency. In these cases, mutations should be found in nuclear genes that regulate crucial

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Differences in assembly or stability of complex I steps in the activation of mitochondrial complex I. Examples of such candidate genes could be found in proteins involved in the phosphorylation of complex I subunits that could participate in the complex I activation/inactivation transition process, like mitochondrial- specific isoforms of cAMP dependent kinases or phosphatases[37,38]. In support of this, it has been shown in different species that mitochondrial complex I reversibly alternates between an active and a de-activated conformation that probably depends on the cell respiratory status[39-41]. Finally, how these mutations affect the stability of native mitochondrial complexes III and IV in our patient group has been analyzed. In different experiments, the steady-state levels of fully assembled complex IV did not vary substantially in these patients when compared with controls. However, the levels of the native complex III dimer were clearly decreased in the patients harboring mutations in NDUFS2 and NDUFS4, but not significantly altered by the mutations in the NDUFS7 or NDUFS8 genes, or in any patient with unknown mutations in nuclear genes. This finding is in agreement with the combined complex I+III deficiency described for three patients carrying mutations in the NDUFS4 gene[5,21]. Reversibly, a specific mutation in cytochrome b has been found to lead to a combined complex III+I deficiency in a patient with progressive exercise intolerance[42]. These results suggest a physical interaction between complexes I and III that could be mediated through these subunits. Experimental evidence supports this idea. The association of the mitochondrial respiratory chain complexes into higher supramolecular structures called supercomplexes seems to be well established in different organisms, including mammals[43]. In Y. lipolytica, the ubiquinone-binding 49-kDa subunit (homologue of NDUFS2) seems to be located in an external position near the tip of the peripheral arm[44]. By cross-linking studies, a direct interaction between the homologues of NDUFS2 and NDUFS4 (49- and 18-kDa subunits, respectively) has been observed in bovine[45]. It would explain the similar phenotypes found in patients with mutations in these two genes. In these patients carrying mutations in NDUFS2 or NDUFS4, the decreased stability of mitochondrial complex III is accompanied by a slight or no reduction of complex III activity. This could be explained by internal compensatory activation mechanisms or by the fact that a loss of bigger amounts of assembled complex III could be necessary before getting a considerable decrease of complex III activity, as has been suggested in cybrid clones harboring increasing amounts of mutant cytochrome b[23]. Finally, the first report demonstrating that complex I mutations interfere with the function or assembly of other OXPHOS complexes has been recently described in a Caenorhabditis elegans model[46]. Summarizing, by BN-PAGE we have been able to differentiate catalytic versus assembly/stability defects in complex I-deficient patients and to study the effects of these mutations on the stability of other mitochondrial OXPHOS complexes. Using this technique will not only expand the cell biological knowledge of the

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Chapter VI mitochondrial energy metabolism, but it will also be useful as an additional tool to improve the pre- and postnatal diagnostic possibilities in patients with a mitochondrial complex I deficiency.

Acknowledgements We thank Richard Huijbens for technical support. This study was partly supported by the ‗Prinses Beatrix Fonds‘ to J.S. and L.v.d.H. (grant numbers 98-0108 and 02-0104). The Netherlands Organization for Scientific Research supported L.N. with a ‗Vernieuwingsimpuls‘ grant, and R.J. and J.S. (grant number 901-03-156). At the time of this work C.U. was recipient of a European Union Marie Curie Fellowship (proposal number MCFI-2000-02003).

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References

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17. Papa S, Scacco S, Sardanelli AM, Vergari R, Papa F, Budde S, van den Heuvel L, Smeitink J (2001) Mutation in the NDUFS4 gene of complex I abolishes cAMP-dependent activation of the complex in a child with fatal neurological syndrome. FEBS Lett 489(2-3):259-262 18. Scacco S, Petruzzella V, Budde S, Vergari R, Tamborra R, Panelli D, van den Heuvel LP, Smeitink JA, Papa S (2003) Pathological mutations of the human NDUFS4 gene of the 18-kDa (AQDQ) subunit of complex I affect the expression of the protein and the assembly and function of the complex. J Biol Chem 278(45):44161-44167 19. Triepels RH, Hanson BJ, van den Heuvel LP, Sundell L, Marusich MF, Smeitink JA, Capaldi RA (2001) Human complex I defects can be resolved by monoclonal antibody analysis into distinct subunit assembly patterns. J Biol Chem 276(12):8892-8897 20. Schägger H, von Jagow G (1991) Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 199(2):223-231 21. Budde SM, van den Heuvel LP, Smeets RJ, Skladal D, Mayr JA, Boelen C, Petruzzella V, Papa S, Smeitink JA (2003) Clinical heterogeneity in patients with mutations in the NDUFS4 gene of mitochondrial complex I. J Inherit Metab Dis 26(8):813-815 22. Schägger H, Pfeiffer K (2000) Supercomplexes in the respiratory chains of yeast and mammalian mitochondria. EMBO J 19(8):1777-1783 23. Rana M, de Coo I, Diaz F, Smeets H, Moraes CT (2000) An out-of-frame cytochrome b gene deletion from a patient with parkinsonism is associated with impaired complex III assembly and an increase in free radical production. Ann Neurol 48(5):774-781 24. Loeffen JL, Smeitink JA, Trijbels JM, Janssen AJ, Triepels RH, Sengers RC, van den Heuvel LP (2000) Isolated complex I deficiency in children: clinical, biochemical and genetic aspects. Hum Mutat 15(2):123-134 25. Smeitink JA, Loeffen JL, Triepels RH, Smeets RJ, Trijbels JM, van den Heuvel LP (1998) Nuclear genes of human complex I of the mitochondrial electron transport chain: state of the art. Hum Mol Genet 7(10):1573-1579 26. Schon EA, Manfredi G (2003) Neuronal degeneration and mitochondrial dysfunction. J Clin Invest 111(3):303-312 27. Ugalde C, Triepels RH, Coenen MJ, van den Heuvel LP, Smeets R, Uusimaa J, Briones P, Campistol J, Majamaa K, Smeitink JA, Nijtmans LG (2003) Impaired complex I assembly in a Leigh syndrome patient with a novel missense mutation in the ND6 gene. Ann Neurol 54(5):665- 669 28. Yadava N, Potluri P, Smith EN, Bisevac A, Scheffler IE (2002) Species-specific and mutant MWFE proteins. Their effect on the assembly of a functional mammalian mitochondrial complex I. J Biol Chem 277(24):21221-21230 29. Videira A, Duarte M (2002) From NADH to ubiquinone in Neurospora mitochondria. Biochim Biophys Acta 1555(1-3):187-191 30. Ahlers PM, Zwicker K, Kerscher S, Brandt U (2000) Function of conserved acidic residues in the PSST homologue of complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica. J Biol Chem 275(31):23577-23582 31. Kashani-Poor N, Zwicker K, Kerscher S, Brandt U (2001) A central functional role for the 49-kDa subunit within the catalytic core of mitochondrial complex I. J Biol Chem 276(26):24082-24087 32. Schulte U, Weiss H (1995) Generation and characterization of NADH: ubiquinone oxidoreductase mutants in Neurospora crassa. Methods Enzymol 260:3-14 33. Duarte M, Videira A (2000) Respiratory chain complex I is essential for sexual development in Neurospora and binding of iron sulfur clusters are required for enzyme assembly. Genetics 156(2):607-615 34. Duarte M, Pópulo H, Videira A, Friedrich T, Schulte U (2002) Disruption of iron-sulphur cluster N2 from NADH: ubiquinone oxidoreductase by site-directed mutagenesis. Biochem J 364(Pt 3):833-839 35. Antonicka H, Ogilvie I, Taivassalo T, Anitori RP, Haller RG, Vissing J, Kennaway NG, Shoubridge EA (2003) Identification and characterization of a common set of complex I assembly

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intermediates in mitochondria from patients with complex I deficiency. J Biol Chem 278(44):43081-43088 36. Janssen R, Smeitink J, Smeets R, van den Heuvel L (2002) CIA30 complex I assembly factor: a candidate for human complex I deficiency? Hum Genet 110(3):264-270 37. Papa S (2002) The NDUFS4 nuclear gene of complex I of mitochondria and the cAMP cascade. Biochim Biophys Acta 1555(1-3):147-153 38. Schulenberg B, Aggeler R, Beechem JM, Capaldi RA, Patton WF (2003) Analysis of steady-state protein phosphorylation in mitochondria using a novel fluorescent phosphosensor dye. J Biol Chem 278(29):27251-27255 39. Grivennikova VG, Kapustin AN, Vinogradov AD (2001) Catalytic activity of NADH-ubiquinone oxidoreductase (complex I) in intact mitochondria. evidence for the slow active/inactive transition. J Biol Chem 276(12):9038-9044 40. Böttcher B, Scheide D, Hesterberg M, Nagel-Steger L, Friedrich T (2002) A novel, enzymatically active conformation of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I). J Biol Chem 277(20):17970-17977 41. Grivennikova VG, Serebryanaya DV, Isakova EP, Belozerskaya TA, Vinogradov AD (2003) The transition between active and de-activated forms of NADH:ubiquinone oxidoreductase (Complex I) in the mitochondrial membrane of Neurospora crassa. Biochem J 369(Pt 3):619-626 42. Lamantea E, Carrara F, Mariotti C, Morandi L, Tiranti V, Zeviani M (2002) A novel nonsense mutation (Q352X) in the mitochondrial cytochrome b gene associated with a combined deficiency of complexes I and III. Neuromusc Disord 12(1):49-52 43. Schägger H (2002) Respiratory chain supercomplexes of mitochondria and bacteria. Biochim Biophys Acta 1555(1-3):154-159 44. Zickermann V, Bostina M, Hunte C, Ruiz T, Radermacher M, Brandt U (2003) Functional implications from an unexpected position of the 49-kDa subunit of NADH:ubiquinone oxidoreductase. J Biol Chem 278(31):29072-29078 45. Yamaguchi M, Hatefi Y (1993) Mitochondrial NADH:ubiquinone oxidoreductase (complex I): proximity of the subunits of the flavoprotein and the iron-sulfur protein subcomplexes. Biochemistry 32(8):1935-1939 46. Grad LI, Lemire BD (2004) Mitochondrial complex I mutations in Caenorhabditis elegans produce cytochrome c oxidase deficiency, oxidative stress, and vitamin-responsive lactic acidosis. Hum Mol Genet 13(3):303-314 47. Bentlage HA, Wendel U, Schägger H, ter Laak HJ, Janssen AJ, Trijbels JM (1996) Lethal infantile mitochondrial disease with isolated complex I deficiency in fibroblasts but with combined complex I and IV deficiencies in muscle. Neurology 47(1):243-248 48. Nijtmans LG, Henderson NS, Holt IJ (2002) Blue Native electrophoresis to study mitochondrial and other protein complexes. Methods 26(4):327-334

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Contiguous gene deletion of ELOVL7, ERCC8, and NDUFAF2 in a patient with progressive encephalomyopathy

Rolf J.R.J. Janssen1, Felix Distelmaier2, Roel J.P. Smeets1, Tessa Wijnhoven2, Elsebet Østergaard3, Nicolaas G.J. Jaspers4, Anja Raams4, Stephan Kemp5, Richard J.T. Rodenburg1, Peter H.G.M. Willems2, Lambert P.W.J. van den Heuvel1, Jan A.M. Smeitink1, and Leo G.J. Nijtmans1

1) Nijmegen Center for Mitochondrial Disorders at the Department of Pediatrics, Laboratory of Pediatrics and Neurology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands 2) Department of Membrane Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands 3) Department of Clinical Genetics, National University Hospital Rigshospitalet, Copenhagen, Denmark 4) Department of Genetics, Erasmus Medical Centre, Rotterdam, The Netherlands 5) Laboratory Genetic Metabolic Diseases, Academic Medical Center/Emma Children‘s Hospital, Amsterdam, The Netherlands

Submitted

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Abstract Amongst mitochondrial disorders contiguous gene syndromes are rarely encountered. In this report, we describe a patient presenting with an apparently mitochondrial encephalomyopathy accompanied by several abnormal dysmorphic features, which is caused by a homozygous triple gene deletion on chromosome 5. The deletion encompasses the NDUFAF2, ERCC8, and ELOVL7 genes, encoding complex I assembly factor 2 (also known as human B17.2L), a protein of the transcription-coupled nucleotide excision repair (TC- NER) machinery, and a putative elongase of very long-chain fatty acid synthesis, respectively. This is the first chromosomal microdeletion associated with a mitochondrial phenotype and isolated complex I deficiency. Besides, mutations of the ELOVL7 gene have not been implicated in human disease before. Patient fibroblasts demonstrated disturbance of complex I assembly and defective TC-NER. Due to the low expression level of ELOVL7 in human fibroblasts, changes in fatty acid synthesis could not be assessed in the patient‘s fibroblasts. Furthermore, downstream alterations in mitochondrial physiology have been established, e.g., depolarization of mitochondrial membrane potential, increased mitochondrial NAD(P)H levels and increased intracellular superoxide production. By means of a complementation assay, wild-type NDUFAF2 has been demonstrated to not only fully restore complex I activity and protein amount in our patient‘s fibroblasts, but also to reinstate normal mitochondrial physiology.

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Introduction Contiguous gene syndromes are rare disorders characterized by apparently unrelated clinical signs and symptoms, which are caused by deletion of two or more genes that are adjacent to one another on a chromosome. Each of the individual genes involved may give rise to distinct clinical and biochemical features. Numerous examples of such syndromes have been described in literature, e.g., Prader-Willi/Angelman syndrome[1,2] (MIM 176270, 105830), Williams-Beuren syndrome[3] (MIM 194050), Miller-Dieker lissencephaly syndrome[4] (MIM 247200), DiGeorge syndrome[5] (MIM 188400), and Smith-Magenis syndrome[6] (MIM 182290). Because of certain characteristic signs and symptoms, some of these syndromes have been described as clinical entities prior to establishment of their chromosomal etiology. Investigation of these disorders on the molecular level offers unique insights into the function of affected genes and their relation to the disease phenotype. Disturbances of the oxidative phosphorylation (OXPHOS) system are the cause of a broad spectrum of multisystem disorders that are often referred to as mitochondrial encephalomyopathies because of the prominent involvement of the nervous system and striated muscle. With an incidence of 1 in 5,000 live births, OXPHOS disorders are amongst the most frequent inborn errors of metabolism[7,8]. The OXPHOS system comprises five membrane-bound multi-protein complexes and two mobile electron carriers that transfer electrons from NADH and succinate to molecular oxygen. Simultaneously, protons are translocated across the mitochondrial inner membrane; thereby creating a membrane potential, which is the driving force of ATP production. Isolated complex I deficiency is the most common cause of OXPHOS disorders. Complex I, or NADH:ubiquinone oxidoreductase, represents the main entry point of the OXPHOS system, as it initiates electron transfer by oxidizing NADH. Mammalian complex I consists of 45 subunits of which seven are encoded by the mitochondrial DNA (mtDNA) and 38 by the nuclear genome[9]. Being accountable for a heterogeneous group of clinical phenotypes, the primary defects underlying complex I deficiency are genetically heterogeneous as well. Mutations have been found in each of the seven mtDNA-encoded subunits, resulting in either single-organ phenotypes, as Leber hereditary optic neuropathy (LHON; MIM 535000), or multisystemic syndromes, e.g., mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS; MIM 540000). Furthermore, mutations have been found in twelve of the nuclear genes encoding structural components of complex I. Nine of those genes are mainly associated with brain and brainstem disorders, mostly Leigh(-like) syndrome or leukoencephalopathy, while three others are exclusively associated with hypertrophic cardiomyopathy and encephalomyopathy[10-12]. In addition to 45 structural components, probably numerous, yet unknown, nuclear factors are needed for proper assembly and functioning of complex I, as has been reported

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Contiguous gene deletion of ELOVL7, ERCC8, and NDUFAF2 extensively for complex IV. The intricate assembly process of mitochondrial complex I is starting to unravel with the characterization of the first few known complex I assembly factors, supported by the recent identification of patients with corresponding disease causing genotypes. Identification of these assembly factors will open a new field of possible candidates for complex I deficiency. To date, two characterized human complex I assembly chaperones have been proved responsible for complex I deficiency. Ogilvie et al. reported the first mutation in an assembly chaperone: a patient presenting with an early-onset progressive encephalopathy resembling vanishing white matter disease carried a null mutation in the NDUFAF2 gene (GenBank, NM_174889), encoding complex I assembly factor 2 (also known as chaperone B17.2L for B17.2-like protein) [13]. Dunning et al. described a complex I-deficient patient with cardioencephalomyopathy caused by two compound heterozygous missense mutations in the gene encoding complex I assembly factor 1, NDUFAF1 (also known as CIA30; GenBank, NM_016013) [14]. More recently, a mutation in a putative third assembly chaperone, a mitochondrial protein encoded by the C6ORF66 gene (GenBank, NM_014165), has been detected in a consanguineous family of complex I- deficient patients presenting with infantile mitochondrial encephalomyopathy[15]. Contiguous gene syndromes characterized by a ‗mitochondrial phenotype‘ have been rarely reported[16]. A possible explanation is that disturbances of the integrity of the OXPHOS system result in a broad range of clinical manifestations presenting from infancy to late adulthood and affecting many different organs and tissues and therefore, characteristic constellations of symptoms and anomalies may be under-recognized. In addition, mutations in nuclear structural genes of complex I are usually associated with very severe clinical phenotypes (e.g. Leigh syndrome or fatal infantile lactic acidosis), resulting in early death, mostly within the first five years of life, and accordingly, the involvement of other genes with less severe clinical manifestations may be overshadowed[17]. To date, no contiguous gene syndromes associated with isolated complex I deficiency are known. In this report, we describe a patient presenting with an apparently mitochondrial encephalomyopathy accompanied by several abnormal dysmorphic features, which is caused by a homozygous triple gene deletion on chromosome 5. The deletion encompasses the NDUFAF2 gene encoding complex I assembly factor 2, the ERCC8 gene (GenBank, NM_000082), involved in the transcription-coupled nucleotide excision repair (TC-NER) pathway, and ELOVL7 (GenBank, NM_024930), a member of the elongase (ELOVL: elongation of very long-chain fatty acids) gene family. We investigated the consequences of the lack of chaperone NDUFAF2 for the biogenesis and functioning of the OXPHOS system and established downstream alterations in mitochondrial physiology. Moreover, we confirmed Cockayne syndrome type A (CSA; MIM 216400) deficiency, as a result of the ERCC8 deletion, by analysis of the NER system. Unfortunately, due to the low expression level of ELOVL7 in

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Chapter VII human fibroblasts, specific changes in endogenous fatty acid levels as a consequence of the ELOVL7 deletion could not be assessed in the patient‘s fibroblasts.

Patients, materials and methods Case report The girl was the second child of healthy consanguineous parents (first cousins). Family history was unremarkable (one older sister and two younger brothers not affected). Prenatal ultrasound screening revealed intrauterine growth retardation. The patient was born at 38 weeks of gestation by caesarean section. Birth weight was 1.815 kg (~0.5 kg <3rd percentile), length 45 cm (~1 cm <3rd percentile) and head circumference 31 cm (~0.5 cm <3rd percentile). APGAR scores were 6/8/10. Postpartally, the girl appeared dysmature and developed respiratory distress. Apart from microcephaly, several abnormal features including long philtrum, micrognathia, low-set ears, and unusually dry skin were noted. Laboratory investigations revealed elevated levels of lactate (2.5-3.4 mmol/L; normal range 0.6-1.8) and alanine in blood (1046 µmol/L; normal range <350). Urine screening was, apart from increased alanine excretion, normal. Cranial ultrasound and a computed tomography scan of the brain revealed no obvious pathology. Abdominal ultrasound demonstrated hepatomegaly. The further clinical course of the patient was characterized by severe developmental delay, failure to thrive, progressive microcephaly, myoclonic epilepsy, pyramidal signs, and spasticity. The girl repeatedly developed aspiration pneumonias and finally died at the age of 14 months due to cardiorespiratory failure. Electron microscopy (EM) studies of liver tissue revealed abnormally swollen mitochondria and inclusions of granula in the endoplasmic reticulum, possibly accumulations of lipids. Histological evaluation of muscle tissue showed few small atrophic fibers, but was otherwise normal. No ragged-red fibers or cytochrome c oxidase-negative fibers were seen. However, also in this tissue, ultrastructural studies by EM demonstrated swollen mitochondria.

Cell culture and sample preparation Patient and healthy control fibroblasts were obtained from skin biopsies and cultured to confluence in medium 199 with Earle's salt (M199; Invitrogen) supplemented with 10% (v/v) fetal bovine serum (Greiner Bio-One) and 100 IU/mL penicillin and 100 µg/mL streptomycin (Invitrogen) in a humidified atmosphere of 95% air and 5% CO2 at 37 C. For microscopy analysis, cells were seeded on glass coverslips (Ø 24 mm) and cultured to ~70% confluence[3] in a humidified atmosphere (95% air, 5% CO2) at 37 C. Cells were harvested with 0.25% (w/v) trypsine (Becton Dickinson), resuspended in PBS (Invitrogen) and mitochondria-enriched fractions were obtained by incubation with 2% (w/v) digitonin 154

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(Calbiochem) on ice for 10 min, followed by centrifugation at 10,000 g and 4 °C for 10 min and washing with PBS, twice. Mitochondrial fractions were resuspended in 1.75 M aminocaproic acid (Fluka) and 75 mM bis-tris (Fluka) at pH 7.0, and (OXPHOS) protein complexes were solubilized using 2% (w/v) of n-dodecyl- -D-maltoside (DDM; Sigma- Aldrich) during incubation on ice for 10 min, followed by centrifugation and washing at the same conditions described above. The protein concentration was determined using the Micro BCA protein assay kit (Pierce Biotechnology).

SDS and blue native polyacrylamide gel electrophoresis and western blotting SDS and blue native polyacrylamide gel electrophoresis (BN-PAGE) was performed as described previously[18]. Twenty micrograms of mitochondrial protein fractions were loaded on 10% polyacrylamide gels for SDS-PAGE and 40 µg of mitochondrial protein fractions on 5-15% polyacrylamide gradient gels for BN-PAGE. For second dimension SDS-PAGE, first dimension BN-PAGE lanes were excised and placed on top of a 10% polyacrylamide SDS gel and run as described previously[18]. SDS and BN gels were subjected to western blotting using nitrocellulose membranes (Schleicher and Schuell). Subsequently, immunodetection was performed using monoclonal or polyclonal antibodies directed against complex I subunits NDUFA9 (Invitrogen), NDUFB6 (MitoSciences), NDUFS2 (Prof B. Robinson, Toronto), NDUFS3 (Invitrogen), and ND1 (Dr A. Lombes, Paris), complex II subunit SDHA (Invitrogen), complex III subunit Core2 (Invitrogen), complex IV subunit COXII (Invitrogen), and complex I assembly chaperones NDUFAF1 (our laboratory)[19] and NDUFAF2 (Prof M. Tsuneoka, Kurume). Peroxidase-conjugated anti-mouse and anti-rabbit IgGs (Invitrogen) were used as secondary antibodies. Signals were generated by means of ECL plus (Amersham Biosciences) and autoradiography.

Complex I in-gel activity assay Complex I in-gel activity was detected as NADH:nitrotetrazolium blue oxidoreductase activity by incubation of BN gels in the presence of 0.15 mM NADH (Roche), 3 mM nitrotetrazolium blue chloride (NTB; Sigma-Aldrich) and 2 mM (2.5 mg/mL) Tris-HCl (Sigma-Aldrich) at pH 7.4, for 1 h protected from light.

RNA isolation, PCR amplification and DNA sequencing Patient and control total DNA and mRNA was isolated from fibroblasts and blood samples according to standard procedures. cDNA was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) of mRNA according to manufacturer‘s protocol (Promega) and was subjected to PCR amplification using a primer set encompassing the total NDUFAF2 mRNA 155

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sequence (GenBank, NM_174889; forward (5‘ 3‘) CTGGAGCATTACCCCTACTGCG, reverse (5‘ 3‘) TAGTCACATCCATATACATGAAAAG). PCR products were cycle-sequenced using the ABI Prism BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems) and analyzed by a 3130xl Genetic Analyzer (Applied Biosystems), according to the manufacturer‘s protocol.

Cloning of baculoviral constructs and generation of recombinant baculoviruses The cDNA sequences of the coding region of the NDUFAF2 gene and of the mitochondrial targeting sequence of the gene encoding subunit VIII of cytochrome c oxidase (COX8) were cloned into a baculovirus vector (pFastBacTMDual vector (Invitrogen) modified for protein expression in mammalian cells by Visch et al.[20]) behind the cytomegalovirus (CMV) promoter and adjacent to a C-terminal GFP-tag sequence. The obtained baculoviral vector constructs were used to generate infectious recombinant baculoviruses by site-specific transposon-mediated insertion into a baculovirus genome (bacmid) propagated in Escherichia coli cells, as described by Luckow et al.[21]. Isolated recombinant bacmids were used to infect Spodoptera frugiperda 9 (Sf9) insect cells for amplification of viruses.

Transduction of complex I-deficient fibroblasts with baculoviral constructs Fibroblasts were grown to ~70% confluence in 25-cm2 tissue culture flasks (NUNC) before they were transduced with the appropriate baculovirus (virus titer: 3 * 107) at a multiplicity of infection of ~10 by incubating in a total volume of 1.6 mL M199 medium for 3 h at 37 °C. After incubation, the virus-containing medium was discarded and replaced by normal growth medium (M199). The next day 5 mM sodium butyrate (Sigma-Aldrich) was added to the medium to enhance protein expression and 3 days after transduction, cells were harvested.

Fluorescence microscopy of TMRM, NAD(P)H and HEt oxidation products For quantitative analysis of tetramethyl rhodamine methyl ester (TMRM; Invitrogen), NAD(P)H and hydroethidine (HEt; Molecular Probes) oxidation product fluorescence, coverslips were mounted in an incubation chamber and placed on the stage of an inverted microscope (Axiovert 200M, Carl Zeiss) equipped with a 40×/1.3 NA (used for HEt and NAD(P)H measurements) and a 63×/1.25 NA (for TMRM measurements) Plan NeoFluar objective (Carl Zeiss). All experiments were performed at 37 C during which cells were maintained in HEPES-Tris medium (132 mM NaCl, 4.2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5.5 mM D-glucose and 10 mM HEPES at pH 7.4). Fluorescence of TMRM, NAD(P)H and HEt oxidation products was exited using a monochromator (Polychrome IV, TILL Photonics) and images were obtained using a CoolSNAP HQ monochrome CCD-camera 156

Contiguous gene deletion of ELOVL7, ERCC8, and NDUFAF2

(Roper Scientific Photometrics). Hardware was controlled with Metafluor 6.0 software (Universal Imaging Corporation). Quantification of TMRM fluorescence Mitochondrial TMRM fluorescence was analyzed according to a novel protocol[22,23]. In brief, fibroblasts were incubated in medium 199 containing 100 nM TMRM for 25 min at 37 C. After loading, cells were washed with PBS. Fibroblasts were imaged using 63×/1.25 NA Plan NeoFluar objective (Carl Zeiss) and TMRM was excited at 540 nm with an exposure time of 100 ms. Fluorescence light was directed by a 560DRLP dichroic mirror (Omega Optical Inc) through a 565ALP emission filter (Omega Optical Inc). The acquired images were processed to generate a mitochondria-specific mask. The latter was superimposed on the original image to allow quantification of TMRM fluorescence exclusively in the mitochondrial structures. Quantification of intracellular NAD(P)H autofluorescence Measurement of intracellular NAD(P)H autofluorescence was carried out according to a protocol described previously[23,24]. Briefly, fibroblasts were imaged using a 40×/1.3 NA Plan NeoFluar objective (Carl Zeiss). The cells were excited at 360 nm and fluorescence emission light was directed by a 415DCLP dichroic mirror (Omega Optical Inc) through a 510WB40 emission filter (Omega Optical Inc). Routinely, 10 fields of view were analyzed per coverslip using an image capturing time of 1s. Quantification of superoxide production Fluorescence of HEt oxidation products was analyzed as described previously[25]. In brief, fibroblasts were incubated in HEPES-Tris medium containing 10 μM HEt for 10 min at 37 C. The reaction was stopped by washing of the cells with PBS. Cells were excited at 490 nm, using a 40×/1.3 NA Plan NeoFluar objective (Carl Zeiss), and fluorescence emission light was directed by a 525DRLP dichroic mirror (Omega Optical Inc) through a 565ALP emission filter (Omega Optical Inc). Routinely, 10 fields of view were analyzed per coverslip using an acquisition time of 100 ms. Quantitative image analysis of TMRM, NAD(P)H and HEt measurements was performed with Metamorph 6.0 (Universal Imaging Corporation) and Image Pro Plus 5.1 (Media Cybernetics). In each experiment, the average value obtained with the corresponding control cell line was set at 100%, to which all other values were related. Numerical results were visualized using Origin Pro 7.5 software (Originlabs) and values from multiple experiments were expressed as means ± SEM (standard error of the mean). Statistical significance (Bonferroni corrected) was assessed using Student‘s t-test.

Nucleotide excision repair assays Transcription-coupled NER (TC-NER) and global genome NER (GG-NER) activities of cultured fibroblasts after exposure to germicidal UV-C rays were assayed using standard autoradiographic methods described elsewhere[26] and expressed as a percentage of the

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Chapter VII activities of UV-exposed normal control cells. Overall cell survival was routinely assayed by global incorporation of tritiated thymidine 4 days after exposure to graded UV doses[27].

Measurement of very long-chain fatty acid levels Total cellular fatty acids were analyzed using the electrospray ionization mass spectrometry (ESI-MS) method described previously[28].

Results Enzyme activity measurements of OXPHOS complexes The enzyme activities of all five OXPHOS complexes were determined by spectroscopy measurements in patient fibroblasts and frozen muscle tissue. Complex I activity was significantly impaired in fibroblasts as well as frozen muscle tissue (data not shown), whereas all other complexes (CII, CIII, CIV, and CV) showed normal values. The residual complex I activity in the patient‘s fibroblasts is 45% of the lowest control value (Tab 1). Enzyme activities of all other complexes are within the reference ranges. Additional complex I activity measurements in liver demonstrated a severe complex I deficiency (data not shown).

Table 1. OXPHOS enzyme activities in fibroblasts

Enzyme activitiesa Activity (Reference range)

Complex I 49 (110-260) Complex II 871 (536-1027) Complex III 2170 (1270-2620) Complex II+III (Succ:cyt c oxidoreductase; SCC) 189 (160-440) Complex IV (Cytochrome c oxidase) 801 (680-1190) Complex V (ATP synthase) 421 (209-935) Citrate synthase 250 (144-257)

a Enzyme activities are expressed in mU/mU COX except for complex IV, which is expressed in mU/mU CS, and citrate synthase (CS), which is expressed in mU/mg protein. COX and CS levels were normal in the patient. Enzymatic activities were measured in triplicate in at least two independent patient-derived samples.

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Complex I deficiency confirmed by decreased amounts of fully assembled complex I To investigate the complex I deficiency on protein level mitoplast fractions of the patient‘s fibroblasts were subjected to blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by either an in-gel activity assay (IGA), or western blotting and immunodetection using antibodies directed against complexes I, II, and III (Fig 1A). The patient‘s fibroblasts showed a severe decrease in both complex I activity and protein amount compared to two control fibroblasts cell lines, whereas normal control levels were shown for complexes II and III. Subsequently, SDS-PAGE analysis of the mitoplast fractions was performed to examine the protein levels of several complex I subunits and assembly chaperones using monoclonal and polyclonal antibodies directed against several OXPHOS proteins (Fig 1B). A significant decrease in the protein levels of complex I subunits NDUFS3, NDUFA9, and NDUFB6, as well as complex I assembly factor NDUFAF1, was shown for the patient compared to normal control fibroblasts, whereas subunit NDUFS2 showed a milder decrease. Remarkably, no protein at all was detected for the complex I assembly factor NDUFAF2 in the patient fibroblasts. In contrast, normal NDUFAF2 protein levels were observed for two complex I- deficient patients with unknown mutations. In addition, all patients showed normal levels of subunit SDHA of complex II.

A B

Figure 1. BN- and SDS-PAGE analysis of patient and control fibroblasts. A. 1D BN-PAGE shows a decrease in fully assembled complex I activity and protein amount for the patient (P(del)) in duplicate, compared to control fibroblasts (C1 and C2). Subunits against which the antibodies are used are depicted in between brackets. B. SDS-PAGE shows decreased levels of complex I subunits in the patient and a total lack of assembly chaperone NDUFAF2. Two complex I- deficient patients with unknown mutations are also shown (lanes 2 and 3). CI, II, III: complexes I, II, III; IGA: in-gel activity assay

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Mutational analysis In the past, extensive mutational analysis performed on the patient i.e., screening for mtDNA rearrangements (deletions, insertions or duplications) and common mtDNA point mutations and screening of all 38 nuclear genes and seven mitochondrial ND genes encoding the 45 complex I subunits for mutations did not reveal the molecular defect. Because of lack of detection of NDUFAF2 protein, we investigated the corresponding NDUFAF2 mRNA sequence. Noticeably, analysis of PCR amplification of cDNA derived from the patient‘s fibroblasts, did not reveal a PCR-product for the NDUFAF2 sequence, suggesting a destabilized mRNA, a promoter mutation, or a gene deletion. Subsequently, PCR amplification of the patient‘s genomic DNA failed to retrieve the NDUFAF2 gene, indicating a (total) gene deletion. Further genomic analysis of the region surrounding the NDUFAF2 gene led to detection of a homozygous deletion of ~450 kb on chromosome 5. The location was refined using chromosome map markers at 5q12.1 ranging from nucleotide 60,082,769 to 60,530,221 (UCSC Genome Browser, March 2006 assembly). The deletion encompasses three known genes, i.e., ELOVL7 (GenBank, NM_024930), ERCC8 (GenBank, NM_000082), and NDUFAF2 (GenBank, NM_174889). Screening of genomic DNA isolated from blood samples of the patient‘s relatives revealed that both parents, two brothers and a sister are heterozygous for the deletion.

Complementation of the complex I defect with wild-type NDUFAF2 To prove the pathogenicity of the NDUFAF2 deletion, functional complementation of the complex I defect was carried out by means of baculoviral transduction of patient and healthy control fibroblasts with constructs expressing either GFP-tagged NDUFAF2 protein or COX8 leader peptide fused to GFP. Three days after transduction mitoplast fractions were subjected to BN-PAGE followed by an in-gel activity assay or western blotting and immunodetection (Fig 2A). NDUFAF2-GFP transduced patient cells showed a marked increase in complex I activity and protein amount when compared to untransduced and COX8-GFP transduced patient cells. Complex I activity as well as protein amount was restored to normal control levels, whereas control cells themselves showed no changes upon NDUFAF2-GFP or COX8-GFP transduction. In addition, protein levels of complexes II and III remained unaffected in both patient and healthy control cells. To examine the specificity of complementation, two other complex I-deficient patient cell lines, bearing mutations in the NDUFS4 and NDUFS7 subunits, were subjected to the complementation assay (Fig 2A). The NDUFS4 patient, who totally lacks fully assembled complex I—detectable by BN-PAGE analysis—in fibroblasts, did not show any changes in holocomplex I levels and activity after NDUFAF2-GFP transduction. Also the level of the 830-kDa subcomplex of complex I, which is normally observed in patients with mutations in

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Figure 2. Complementation of the complex I defect with wild-type NDUFAF2. A. BN-PAGE analysis of transduction of patient and control fibroblasts with baculoviral constructs encoding either wild-type GFP-tagged NDUFAF2, or GFP targeted to mitochondria by the COX8 leader peptide. Complex I activity as well as protein amount have specifically been restored to normal control levels for our patient (P(del)) upon NDUFAF2 transduction, when compared to transduction with COX8-GFP. Control fibroblasts (C1 and C2) show no changes in complex I activity and protein amount after transduction with wild-type NDUFAF2. The complex I defect in patients with mutations in the NDUFS4 (P(S4)) and NDUFS7 (P(S7)) subunits cannot be complemented by wild-type NDUFAF2. Levels of complexes II and III remain unaffected upon transduction in each cell line. Subunits against which the antibodies are used are depicted in between brackets. B. SDS-PAGE analysis of expression of NDUFAF2-GFP and COX8-GFP constructs in patient and control fibroblasts using polyclonal anti-EGFP antibody. untr: untransduced; AF2: NDUFAF2-GFP transduced; COX8: COX8-GFP transduced; CI, II, III: complexes I, II, III; 830kD: 830-kDa subcomplex of complex I; IGA: in-gel activity assay

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Chapter VII the NDUFS4 subunit, did not change upon NDUFAF2-GFP transduction. The NDUFS7 patient, who bears very low amounts of holocomplex I in fibroblasts and likewise activity, remained also unaffected by NDUFAF2-GFP transduction. The inability to complement complex I deficiency caused by other mutations confirms the specificity of NDUFAF2 complementation for our patient.

Complex I assembly profile More insight in the patient‘s complex I assembly profile can be achieved by subjecting first dimension BN-PAGE-separated complexes to second dimension SDS-PAGE (2D BN/SDS- PAGE) followed by western blotting and immunodetection with antibodies against several complex I subunits and assembly chaperone NDUFAF1 (Fig 3). Disturbance of complex I assembly in the patient was demonstrated by accumulation of low-molecular weight complex I subcomplexes, containing complex I subunits NDUFS2, NDUFS3 (sub 2-5), and ND1 (sub 4,5), which correspond to complex I assembly intermediates 2, 3, 4, and 5 of the model for human mitochondrial complex I assembly proposed by Vogel et al.[29]. The accumulation of a high-molecular weight subcomplex containing subunits NDUFB6 and ND1 was also more pronounced in the patient when compared to control fibroblasts. This subcomplex corresponds to the ‗appearing subcomplex‘ a1 of the model. In addition, there was a decrease in NDUFAF1 associated with the ~500-kDa complex, which was described before by Vogel et al. in complex I-deficient patients with mutations in structural complex I subunits[30]. As expected, no changes in complex IV levels were observed after incubation with anti-COXII antibody.

Lack of NDUFAF2 alters mitochondrial physiology and function To investigate the effects of the complex I deficiency on mitochondrial physiology, several intracellular and mitochondrial parameters were measured in control and patient fibroblasts. Subsequently, we investigated whether these alterations could be restored by transduction with NDUFAF2-GFP. As an estimate of mitochondrial membrane potential, TMRM fluorescence intensity was measured in living cells using a recently developed method[22,23] (Fig 4A). The accumulation of the lipophilic fluorescent cation TMRM in the mitochondrial matrix follows the Nernst equation and, therefore, its fluorescence intensity is a sensitive readout of mitochondrial membrane potential[23]. Membrane potential was reduced in our patient‘s fibroblasts by 9% (± 1% SEM) compared to control fibroblasts. This reduction in fluorescence corresponds to an estimated depolarization of 17.5 mV. A 7% (± 1.2% SEM) reduction was measured for the NDUFS7 patient cells. Transduction with NDUFAF2-GFP almost completely restored membrane potential in our patient, whereas no significant effect could be observed for the

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Figure 3. Complex I assembly profile of patient and control fibroblasts. 2D BN/SDS-PAGE shows accumulation of low-molecular weight subcomplexes 1-5 in the patient’s fibroblasts (P(del)) and a decrease in the NDUFAF1-containing complex, compared to control fibroblasts (C2)). Subcomplexes have been numbered according to the human complex I assembly pathway model described by Vogel et al.[29]. Accumulation of ‘appearing subcomplex 1’ (a1) is also more pronounced for the patient. Normal levels of COXII demonstrate undisturbed complex IV assembly.

complex I-deficient NDUFS7 patient. Besides depolarization of mitochondrial membrane potential, increase in NAD(P)H levels is another hallmark of complex I dysfunction, as a consequence of decreased NADH oxidation by complex I. Mitochondrial NAD(P)H levels of patient and control fibroblasts were measured as NAD(P)H autofluorescence intensity[24] (Fig 4B). Mitochondrial NAD(P)H levels were significantly increased in the patient, to an average of 180% (± 11.7% SEM) of normal control levels, and dropped to an average of 118% (± 4.2% SEM) after transduction with NDUFAF2-GFP. Since the NDUFS7 patient did not present with elevated mitochondrial NAD(P)H levels, fibroblasts of a complex I-deficient patient with a mutation in the NDUFS2 subunit were used for comparison. The average NAD(P)H level of 125% (± 3.6% SEM) in this NDUFS2 patient did not change upon NDUFAF2-GFP transduction.

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Figure 4. Measurement of mitochondrial membrane potential, mitochondrial NAD(P)H levels and intracellular superoxide levels. Measurements were performed on patient and control fibroblasts that remained untransduced or had been transduced with the NDUFAF2-GFP baculoviral construct. Measurements of all transduced and untransduced patient cells have been normalized to the average values of the corresponding transduced and untransduced control cells, respectively. A. Mitochondrial membrane potential of patient (P(del), n=68; P(S7), n=31) and control (C1, n=58) fibroblasts was estimated from the average measured TMRM fluorescence pixel intensity. Wild-type NDUFAF2 almost completely restores membrane potential to normal control levels (C1, n=55) in our patient (P(del), n=87), whereas no significant effect can be observed for the complex I-deficient NDUFS7 patient (P(S7), n=82). B. Mitochondrial NAD(P)H levels of patient (P(del), n=34; P(S2), n=57) and control (C1, n=57) fibroblasts were measured as average NAD(P)H autofluorescence intensity. Mitochondrial NAD(P)H levels have significantly been reduced in our patient (P(del), n=84) upon NDUFAF2-GFP transduction, but have not been changed for an NDUFS2 patient (P(S2), n=59). Control: C1, n=44. C. Intracellular superoxide levels of patient (P(del), n=130; P(S7), n=89) and control fibroblasts (C1, n=388) were measured as rates of hydroethidine (HEt) oxidation. The level of superoxide production has significantly been reduced in our patient (P(del), n=145) upon NDUFAF2-GFP transduction, but has not been changed for the NDUFS7 patient (P(S7), n=157). Control: C1, n=215. P values < 0.01 (**) and < 0.001 (***) were considered significantly different from corresponding control.

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A third common feature of fibroblasts harboring complex I deficiency is superoxide production. To investigate intracellular superoxide levels, oxidation rates of hydroethidine (HEt) were determined by quantification of its fluorescent oxidation products[25] (Fig 4C). The patient‘s fibroblasts demonstrated remarkably high superoxide levels, i.e., an average of 354% (± 11.5% SEM) of control levels, compared to 167% (± 6.6% SEM) for the NDUFS7 patient. After transduction with NDUFAF2-GFP, superoxide levels reduced significantly to an average of 127% (± 4.3% SEM) in our patient, whereas no changes could be observed for the NDUFS7 patient. Taken together, complementation of the observed cell physiological consequences with wild- type NDUFAF2 is specific for our complex I-deficient patient with deletion of the NDUFAF2 gene, since no recovery is observed for the complex I-deficient patients with mutations in the NDUFS7 and NDUFS2 subunits. Thus, these data underscore the view that NDUFAF2 is required for normal complex I functioning and mitochondrial physiology.

Investigation of the nucleotide excision repair mechanism The ERCC8 gene is essential for transcription-coupled nucleotide excision repair (TC-NER) of UV-induced DNA damage and is typically defective in patients with Cockayne syndrome belonging to complementation group A[31,32] (CSA; MIM 216400). Since the ERCC8 gene is located in the deleted area, the patient‘s fibroblasts were subjected to NER analysis. Patient fibroblasts displayed lowered TC-NER and normal global genome (GG) NER activities compared to normal controls fibroblasts after exposure to UV radiation (Fig 5A), indicating defective TC-NER but proficient GG-NER. Analysis of cellular survival after exposure to increasing UV doses revealed a UV sensitivity of our patient´s cells that is characteristic of CSA patients (Fig 5B). Moreover, the TC-NER defect could be fully restored by cell fusion with Cockayne syndrome type B (CSB) patient fibroblasts (defective in the ERCC6 gene), but not with cells from a CSA patient (data not shown). These repair parameters in combination with the genetic complementation analysis unequivocally demonstrate an ERCC8/CSA defect in our patient‘s fibroblasts.

Analysis of fatty acid levels Proteins belonging to the ELOVL (elongation of very long-chain fatty acids) family are required for the first reaction in the elongation cycle of very long-chain fatty acid (VLCFA) synthesis[33,34]. To examine the effect of the deletion of the ELOVL7 gene on fatty acid biosynthesis, endogenous levels of saturated (C14:0–C28:0), mono-unsaturated (C18:1– C28:1) and poly-unsaturated (omega-3 and omega-6 series) fatty acids were determined in fibroblasts of our patient, of two healthy control cell lines, and of four complex I-deficient patient cell lines with mutations in the NDUFAF2, NDUFS4 (two cell lines) and NDUFS7

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Chapter VII genes. Unfortunately, no differences could be observed in fatty acid levels between patient and control fibroblasts. To establish whether this could be due to a low expression level of ELOVL7 in human fibroblasts, we performed PCR analysis using mRNA derived from a neuroblastoma cell line (SK-N-BE(2)c) as a positive control. This revealed that ELOVL7 is highly expressed in SK-N-BE(2)c cells, but not in human primary fibroblasts (unpublished data S. Kemp).

Figure 5. NER analysis of patient and control fibroblasts after exposure to UV radiation. A. NER activities of our patient have been expressed as percentages of UV-exposed normal control cells tested in the same experiment. Global genome NER (GG) was measured autoradiographically as ‘unscheduled DNA synthesis’ and transcription-coupled NER (TC) by recovery of overall RNA synthesis after UV exposure as described elsewhere[31]. B. Overall cell survival after exposure to graded UV doses[32]. Proliferative activity of patient and control fibroblasts after UV exposure has been expressed as a percentage of unirradiated cells. The UV-survival curve of our patient matches the curve of a known CSA patient. NER: nucleotide excision repair

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Discussion We describe a patient, presenting with an apparently mitochondrial encephalomyopathy that is caused by a homozygous triple gene deletion encompassing the genes NDUFAF2, ERCC8, and ELOVL7. This is the first homozygous chromosomal microdeletion associated with a mitochondrial phenotype and isolated complex I deficiency, and the fourth patient found lacking the complex I assembly factor NDUFAF2[13,35]. The ERCC8 gene encodes a protein involved in DNA repair and mutations of the gene cause Cockayne syndrome type A. The ELOVL7 gene encodes a putative elongase of VLCFA synthesis and has not been linked with pathogenic mutations and/or deletions before. We investigated the consequences of deletion of each gene for the molecular mechanisms of cellular metabolism in which they exert their functions.

NDUFAF2 Considering the alleged mitochondria-involved clinical manifestation, disturbance of the OXPHOS system substantiated by enzyme activity measurements revealing an isolated complex I deficiency is believed to play a major role in the pathology of our patient. We investigated the effects of the null mutation of chaperone NDUFAF2 in our patient on the assembly of the OXPHOS system and especially complex I assembly. The total lack of NDUFAF2 protein in our patient leads to the accumulation of assembly intermediates 2 to 5 of our human complex I assembly pathway model[29]. The assembly intermediates contain both peripheral arm and membrane arm subunits, i.e., NDUFS2 and NDUFS3 in 2-5 and ND1 in 4 and 5, respectively. To prove that the disturbance of complex I assembly is due to the lack of chaperone NDUFAF2 and not to the lack of the other two proteins (ERCC8 and ELOVL7), complementation of the complex I defect by wild-type NDUFAF2 has been established. NDUFAF2-GFP recombinant protein fully complemented the complex I deficiency in our patient by restoring fully assembled complex I protein amount and complex I in-gel activity to normal control levels. Contrarily, two complex I-deficient patient cell lines with mutations in the NDUFS4 and NDUFS7 subunits could not be complemented by NDUFAF2-GFP, underlining its specificity for our NDUFAF2 mutated patient. Furthermore, we investigated the cell physiological consequences of complex I dysfunction due to lack of NDUFAF2 in our patient. Several intracellular and mitochondrial parameters indicated impaired mitochondrial function and disturbed mitochondrial physiology, e.g., increased intracellular superoxide and mitochondrial NAD(P)H levels, and decreased mitochondrial membrane potential. Interestingly, all observed physiological alterations could be restored (in our patient) upon complementation with wild-type NDUFAF2, while no recovery was observed for the complex I-deficient patients with mutations in the 167

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NDUFS7 and NDUFS2 subunits. Hereby, the complementation assay proves the pathogenicity of the NDUFAF2 deletion for normal complex I functioning and mitochondrial physiology in our patient. Since there is still a small amount of fully assembled active complex I present in the patient‘s fibroblasts, complex I assembly is not entirely prevented by the total lack of NDUFAF2 protein. This means that chaperone NDUFAF2 probably facilitates efficient assembly, possibly by catalyzing a step in the process or by stabilizing an assembly intermediate, as has been argued before by Ogilvie et al.[13]. To elucidate the function of NDUFAF2 in complex I assembly we have to focus on its association with an 830-kDa subcomplex of complex I, which is accumulating in patient fibroblasts harboring mutations in the NDUFS4, NDUFS6 and NDUFV1 subunits[13,36,37] and possibly also the NDUFS1 subunit[38]. Transduction experiments of fibroblasts with the NDUFAF2-GFP construct revealed the presence of NDUFAF2 associated with the 830-kDa subcomplex not only in a disturbed state of complex I assembly (i.e., in patient cells with a mutation in of one of the above mentioned subunits), but also in a healthy state of complex I assembly (unpublished data R. Janssen). Besides, NDUFAF2 does not associate with smaller (<830 kDa) subcomplexes (low- molecular weight assembly intermediates) in patient and control fibroblasts under normal conditions, nor under inhibition of mitochondrial protein synthesis (by chloramphenicol), i.e., when small, nuclear encoded subunit-containing subcomplexes accumulate[29] (unpublished data R. Janssen). This confirms the observations of the in vitro import and assembly assays performed by Lazarou et al.[39], with radiolabeled NDUFAF2 strictly associating with the 830-kDa subcomplex (named ‗~800-kDa subassembly‘). Together, these findings strongly suggest a role for NDUFAF2 restricted to the late stages of complex I assembly, while absence of NDUFAF2 results in stalling of the process at the stage of assembly intermediate 5 of the assembly model as is demonstrated by the accumulation of intermediates 4 and 5 in our patient‘s fibroblasts. It can be argued that NDUFAF2 associates with the 830-kDa subcomplex, facilitates the incorporation of the NADH dehydrogenase module of complex I, i.e., the flavoprotein part (subunits NDUFV1-3) and some additional subunits of the peripheral arm of the complex (e.g., NDUFS4, NDUFS6, and possibly NDUFS1)[13,39], and releases the complex when its fully assembled state is reached.

ERCC8 In mammalian cells, various DNA repair systems exist in order to cope with attacks of endogenous and environmental genotoxic agents on their genomes. One of them is the nucleotide excision repair (NER) pathway, a complex system that removes chemically and UV-induced helix-distorting lesions. The transcription-coupled repair subpathway of the NER (TC-NER) is a mechanism that specifically removes transcription-blocking lesions from

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Contiguous gene deletion of ELOVL7, ERCC8, and NDUFAF2 the transcribed strands of actively transcribed genes[31,32]. In contrast, global genome NER (GG-NER) removes lesions in the entire genome. Cockayne syndrome type A, a rare autosomal recessive disorder characterized by UV sensitivity, severe neurological abnormalities, and progeriod symptoms, is caused by mutations in the ERCC8 gene, which encodes the Cockayne syndrome type A protein (CSA) that is part of the core of the TC-NER machinery[40]. Cockayne syndrome (CS) is primarily characterized by mild cutaneous photosensitivity, postnatal growth failure, progressive neurological dysfunction, and symptoms reminiscent of segmental accelerated ageing. This progressive disorder generally manifests at 1-2 years of age and results in death at puberty or early adulthood[40,41]. In addition to this classic CS, called type I, there is a less common early-onset form of CS, called type II, with more severe symptoms that already present at birth, with a concomitant shortened life expectancy (average 2 years). The genetic basis underlying the type I and II differences has not been elucidated and is independent of the severity of the TC-NER deficiency. In CS patients, a wide range of additional severe physical and mental manifestations develop, including microcephaly, retinal degeneration, deafness, and skeletal abnormalities such as osteoporosis and a bird-like face[40,41]. In our patient‘s fibroblasts we unequivocally demonstrated the presence of the distinct repair defect of CSA patients. Indeed, some of her symptoms, such as microcephaly, reduced birth weight, facial dysmorphology, and dry skin, may well be compatible with CS type II. However, we cannot dismiss the possibility that the patient‘s early demise at 14 months prevented any clear typical signs associated with the more common CS type I to become manifest. Finally, the option of an interaction between CS and the phenotypes associated with the other gene defects remains fully open.

ELOVL7 In mammals, fatty acids consisting of up to 16 carbons in length, which are synthesized by the cytosolic fatty acid synthase (FAS) complex, as well as fatty acids taken up from the diet, are further elongated into long-chain fatty acids containing up to 20 carbon atoms, and/or into very long-chain fatty acids (VLCFAs) with over 20 carbon atoms[34]. Synthesis of VLCFAs takes place at the cytoplasmic side of the endoplasmic reticulum (ER) and involves four successive enzymatic steps, which are carried out by individual membrane-bound proteins. The first regulatory reaction step in the elongation cycle, the condensation of a fatty acyl-CoA and malonyl-CoA, is the rate-limiting step, and is performed by members of the ELOVL protein family. To date, seven enzymes, elongases termed ELOVL1-7, have been identified in mammals and are suggested to perform the condensation reaction in the elongation cycle[34]. Noteworthily, the substrate specificity for all of the seven ELOVL

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Chapter VII proteins has not been determined yet, and mutations of the ELOVL7 gene have not been reported before. To examine its involvement in VLCFA elongation, and to examine the consequences of lack of ELOVL7 for fatty acid metabolism, we performed analysis of fatty acid biosynthesis in our patient. Unfortunately, no significant differences in endogenous fatty acid levels could be observed when compared to the levels of controls and other complex I-deficient patients (with single mutations in NDUFAF2 or subunits NDUFS4 and NDUFS7). The low expression level of the ELOVL7 gene in fibroblasts, compared to the other elongases, can be an explanation for these results. Reminding the tissue specificity of the ELOVL family members, it might also be possible that ELOVL7 does not have a function in skin fibroblasts, and exerts its function and consequently displays its defect in other tissues. Therefore, investigation of other tissues will be needed to assign a function in fatty acid synthesis to ELOVL7. For instance, the apparent lipid inclusions observed in the ER of a liver biopsy of the patient could be indicative of disturbance of fatty acid metabolism, and thus of involvement of the ELOVL7 deletion in the pathology. This hypothesis is supported by the observation of Ikeda et al., that ELOVL7 has a preference for interaction with 3-hydroxyacyl- CoA dehydratase 3 (HACD3), another enzyme of the elongation cycle, which exhibits a remarkably high expression in liver[42]. Additionally, the hepatomegaly established in our patient was not observed in the NDUFAF2 patients reported before, who all are assumed to have wild-type ELOVL7 and had normal liver function[13,35].

In conclusion, we present here a unique case of deletion of three genes with distinct functions, in which all three possibly contribute to the pathology of the patient. Involvement on a cell pathological level has been proven for NDUFAF2 and ERCC8 by complementing the defects in their distinct biochemical pathways, whereas involvement of the ELOVL7 deletion can be presumed. Moreover, we argue that the mitochondrial defect is the severest of all in this constellation, which is nicely sustained by the reinstatement of normal mitochondrial physiology in living cells upon complementation of the genetic defect. From a clinical perspective, it is quite plausible that the most severe disease condition with the earliest onset and the fastest progression will overshadow the clinical phenotypes of less severity and with a later onset emerging from the other gene defects. On the other hand, having a clinical phenotype diverging reasonably from the phenotype presented by patients with solely NDUFAF2 mutations, argues for a contribution of the other deleted genes to the condition of the patient. Considering the broad clinical spectrum of mitochondrial disorders and the discrepancy in symptoms amongst patients with the same gene defects, it is plausible that additional gene defects may be present next to the main mitochondrial defect and influence the clinical outcome as is illustrated by this case of a contiguous gene deletion.

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Acknowledgements The authors would like to thank Makato Tsuneoka (Kurume University School of Medicine, Kurume) for providing anti-NDUFAF2 antibody. This work has been supported by The Netherlands Organization for Scientific Research (NWO; grant numbers 901-03-156 and 016.086.328), the European Leukodystrophy Association (ELA: 2006-031I4), and by the European Union's Sixth Framework Programme for Research, Priority 1 ―Life Sciences, Genomics and Biotechnology for Health‖ (contract number LSHM-CT-2004-503116, entitled EUMITOCOMBAT).

Web Resources GenBank, http://www.ncbi.nlm.nih.gov/GenBank Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim UCSC Genome Browser, March 2006 assembly, http://genome.ucsc.edu/cgi-bin/hgGateway

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References

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exchange restores agonist-induced ATP production and Ca2+ handling in human complex I deficiency. J Biol Chem 279(39):40328-40336 21. Luckow VA, Lee SC, Barry GF, Olins PO (1993) Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. J Virol 67(8):4566-4579 22. Distelmaier F, Koopman WJ, Testa ER, de Jong AS, Swarts HG, Mayatepek E, Smeitink JA, Willems PH (2008) Life cell quantification of mitochondrial membrane potential at the single organelle level. Cytometry A 73(2):129-138 23. Komen JC, Distelmaier F, Koopman WJ, Wanders RJ, Smeitink J, Willems PH (2007) Phytanic acid impairs mitochondrial respiration through protonophoric action. Cell Mol Life Sci 64(24):3271-3281 24. Verkaart S, Koopman WJ, Cheek J, van Emst-de Vries SE, van den Heuvel LW, Smeitink JA, Willems PH (2007) Mitochondrial and cytosolic thiol redox state are not detectably altered in isolated human NADH:ubiquinone oxidoreductase deficiency. Biochim Biophys Acta 1772(9):1041- 1051 25. Verkaart S, Koopman WJ, van Emst-de Vries SE, Nijtmans LG, van den Heuvel LW, Smeitink JA, Willems PH (2007) Superoxide production is inversely related to complex I activity in inherited complex I deficiency. Biochim Biophys Acta 1772(3):373-381 26. Jaspers NG, Raams A, Silengo MC, Wijgers N, Niedernhofer LJ, Robinson AR, Giglia-Mari G, Hoogstraten D, Kleijer WJ, Hoeijmakers JH, Vermeulen W (2007) First reported patient with human ERCC1 deficiency has cerebro-oculo-facio-skeletal syndrome with a mild defect in nucleotide excision repair and severe developmental failure. Am J Hum Genet 80(3):457-466 27. Jaspers NG, Raams A, Kelner MJ, Ng JM, Yamashita YM, Takeda S, McMorris TC, Hoeijmakers JH (2002) Anti-tumour compounds illudin S and Irofulven induce DNA lesions ignored by global repair and exclusively processed by transcription- and replication-coupled repair pathways. DNA Repair 1(12):1027-1038 28. Valianpour F, Selhorst JJ, van Lint LE, van Gennip AH, Wanders RJ, Kemp S (2003) Analysis of very long-chain fatty acids using electrospray ionization mass spectrometry. Mol Genet Metab 79(3):189-196 29. Vogel RO, Dieteren CE, van den Heuvel LP, Willems PH, Smeitink JA, Koopman WJ, Nijtmans LG (2007) Identification of mitochondrial complex I assembly intermediates by tracing tagged NDUFS3 demonstrates the entry point of mitochondrial subunits. J Biol Chem 282(10):7582-7590 30. Vogel RO, van den Brand MA, Rodenburg RJ, van den Heuvel LP, Tsuneoka M, Smeitink JA, Nijtmans LG (2007) Investigation of the complex I assembly chaperones B17.2L and NDUFAF1 in a cohort of CI deficient patients. Mol Genet Metab 91(2):176-182 31. Troelstra C, van Gool A, de Wit J, Vermeulen W, Bootsma D, Hoeijmakers JH (1992) ERCC6, a member of a subfamily of putative helicases, is involved in Cockayne's syndrome and preferential repair of active genes. Cell 71(6):939-953 32. De Waard H, de Wit J, Andressoo JO, van Oostrom CT, Riis B, Weimann A, Poulsen HE, van Steeg H, Hoeijmakers JH, van der Horst GT (2004) Different effects of CSA and CSB deficiency on sensitivity to oxidative DNA damage. Mol Cell Biol 24(18):7941-7948 33. Denic V, Weissman JS (2007) A molecular caliper mechanism for determining very long-chain fatty acid length. Cell 130(4):663-677 34. Jakobsson A, Westerberg R, Jacobsson A (2006) Fatty acid elongases in mammals: their regulation and roles in metabolism. Prog Lipid Res 45(3):237-249 35. Barghuti F, Elian K, Gomori JM, Shaag A, Edvardson S, Saada A, Elpeleg O (2008) The unique neuroradiology of complex I deficiency due to NDUFA12L defect. Mol Genet Metab 94(1):78-82 36. Ugalde C, Janssen RJ, van den Heuvel LP, Smeitink JA, Nijtmans LG (2004) Differences in assembly or stability of complex I and other mitochondrial OXPHOS complexes in inherited complex I deficiency. Hum Mol Genet 13(6):659-667 37. Kirby DM, Salemi R, Sugiana C, Ohtake A, Parry L, Bell KM, Kirk EP, Boneh A, Taylor RW, Dahl HH, Ryan MT, Thorburn DR (2004) NDUFS6 mutations are a novel cause of lethal neonatal mitochondrial complex I deficiency. J Clin Invest 114(6):837-845

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38. Iuso A, Scacco S, Piccoli C, Bellomo F, Petruzzella V, Trentadue R, Minuto M, Ripoli M, Capitanio N, Zeviani M, Papa S (2006) Dysfunctions of cellular oxidative metabolism in patients with mutations in the NDUFS1 and NDUFS4 genes of complex I. J Biol Chem 281(15):10374-10380 39. Lazarou M, McKenzie M, Ohtake A, Thorburn DR, Ryan MT (2007) Analysis of the assembly profiles for mitochondrial- and nuclear-DNA-encoded subunits into complex I. Mol Cell Biol 27(12):4228-4237 40. Nance MA, Berry SA (1992) Cockayne syndrome: review of 140 cases. Am J Med Genet 42(1):68-84 Review 41. Andressoo JO, Hoeijmakers JH (2005) Transcription-coupled repair and premature ageing. Mutat Res 577(1-2):179-194 Review 42. Ikeda M, Kanao Y, Yamanaka M, Sakuraba H, Mizutani Y, Igarashi Y, Kihara A (2008) Characterization of four mammalian 3-hydroxyacyl-CoA dehydratases involved in very long-chain fatty acid synthesis. FEBS Lett 582(16):2435-2440

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General Discussion - Conclusions and Future Perspectives -

Chapter VIII

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General discussion

Defects underlying human complex I deficiency Cohort of patients with isolated complex I deficiency Isolated complex I deficiency is the most common cause among OXPHOS disorders. Although late-onset (late childhood or adolescence) diseases haven been encountered, clinical presentation of isolated complex I deficiency usually starts directly after birth or in early childhood and comprises a great variety of signs and symptoms in many different organs. Organs with a high energy demand, e.g., the brain, heart, skeletal muscles, kidneys and the liver, are the most affected ones. Complex I failure often leads to severe multisystem disorders that result in early death[1-4]. Unfortunately, besides a couple of positive therapeutic efforts in some individual patients, rational treatment strategies for complex I deficiency are hardly available. This emphasizes the importance of prenatal diagnosis of the disease and subsequent genetic counseling. The most commonly applied prenatal diagnostic test is biochemical analysis of enzymatic activities of the OXPHOS system in native chorionic villi. However, a negative test, i.e. measurement of normal complex I activity in chorionic villi, does not necessarily mean that a complex I deficiency in the neonate can be excluded for the full 100%. For prenatal diagnosis in the case of mtDNA mutations there are two concerns[5]. One, the mutation load in chorionic villi does not necessarily correspond to that of other fetal tissues, and two, the mutation load measured in prenatal samples may shift in utero or after birth due to mitotic segregation. If the nuclear genetic defect in the patient is known, more specific and accurate prenatal diagnosis can be established by means of mutational analysis at the nuclear DNA level. Therefore, characterization of the genetic defects causing complex I deficiency, will not only expand our understanding of the pathological mechanisms underlying OXPHOS disease, but will also improve the prenatal diagnostic possibilities. For this purpose, several years ago, within the Nijmegen Center for Mitochondrial Disorders (NCMD), we started investigating a defined cohort of 27 patients from 24 families with established isolated complex I deficiency (i.e. sustaining normal functioning of the other four OXPHOS complexes) in muscle tissue and in cultured skin fibroblasts. At first, mutational analysis of the genes expressing the structural components of complex I was started. After characterization of the genes corresponding to the 38 nuclear DNA-encoded subunits of complex I, mutational screening yielded 15 patients (from 13 families) with molecular defects in one of the 38 nuclear or seven mitochondrial ND genes: in NDUFS2 in three families[6], NDUFS4 in three families[7,8], NDUFS7 in one family[9], NDUFS8 in one family[10], NDUFV1 in three families[11], ND2 in one family[12] and in ND6 in one family[13]. Additional screening for common point mutations and large rearrangements (e.g., deletions, insertions, and duplications) of the mtDNA completed the screening of structural components of complex I. At this point, the patient cohort had been diminished to twelve complex I-

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Chapter VIII deficient patients (from eleven families) with unidentified genetic defects. Numerous (yet unidentified) external factors involved in complex I biogenesis, maintenance or functioning were possible candidates for carrying the defects responsible for the complex I deficiencies in our remaining patient cohort. The characterization of the human genes encoding the nuclear subunits of complex I and the identification of the first mutations in these genes was the starting point for nuclear mutational screening of complex I-deficient patients in many research centers all over the world yielding molecular defects in NDUFA1[14], NDUFA2[15], NDUFA11[16], NDUFS1[17], NDUFS3[18], NDUFS6[19] and NDUFV2[20].

Chaperones of complex I assembly possible candidates for complex I deficiency One group of candidates comprises the assembly chaperones, proteins involved in the guidance of the build-up of a complex out of 45 individual subunits. Analogous to cytochrome c oxidase (COX), or complex IV, for which many assembly factors have been described, comparable factors could be expected for complex I assembly, too. Many of these factors are known to be involved in the etiology of COX deficiencies. However, at that time the only two complex I assembly chaperones that had been described, had been found in the fungus Neurospora crassa: the complex I intermediate associated proteins CIA30 and CIA84. We identified the first human complex I assembly factor, a homologue of the CIA30 protein, NDUFAF1, and screened our patient cohort for mutations in its gene, which has been described in Chapter IV. Four new single nucleotide polymorphisms (SNPs) were detected but no pathogenic mutation was found, declining NDUFAF1 as a candidate gene for complex I deficiency in our original patient cohort. Recently, the first mutation in the NDUFAF1 gene causing complex I deficiency has been reported[21]. Two independent studies based on comparative genomics analysis brought to light a putative second human complex I assembly chaperone, NDUFAF2 (known before as B17.2L), which was consequently posed as a candidate for complex I deficiency[22,23]. The NDUFAF2 gene that has been arisen from a duplication event, encodes a paralogue of complex I subunit NDUFA12 (B17.2 in bovine nomenclature). Ogilvie et al.[23] reported the first mutation in this gene being responsible for complex I deficiency. Screening of our patient cohort resulted in one patient with isolated complex I deficiency lacking the NDUFAF2 gene, which has been described in Chapter VII. A homozygous (total) gene deletion of the NDUFAF2 gene appeared to cause the complex I deficiency in our patient, as has been proven with a baculoviral complementation assay using wild-type NDUFAF2 cDNA. A third putative complex I assembly factor with pathogenic mutations causing complex I deficiency has been reported recently[24]. Noteworthily, the gene, C6ORF66, encodes a calmodulin-binding protein (HRPAP20) that has previously been shown to increase breast cancer cell invasion[25]. Despite the many genes

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General discussion that are currently known to cause complex I deficiency, mutations in these genes have not been found in the remainder of our patient cohort. Other genes encoding proteins that are associated with complex I functioning or biogenesis such as PTCD1 (CIA84 homologue), Ecsit, HtrA2/Omi and a couple of yet uncharacterized complex I-related proteins derived from the comparative genomics analysis studies mentioned earlier[22] have been subjected to mutational analysis without retrieving any pathogenic mutations (R. Janssen unpublished results). Thus, further exploring of candidate genes in the field of complex I biogenesis will be needed for this patient cohort.

Survey of mitochondrial complex I assembly

Differences in assembly profiles implications for the complex I assembly process Mitochondrial complex I assembly is an intricate process, which is currently believed to proceed through a series of distinct assembly intermediates by combining evolutionarily conserved functional modules. Disturbances of the process by, for instance, mutation of subunits can lead to accumulation and/or depletion of several intermediate subassemblies, providing so-called assembly profiles. To interpret these assembly profiles we must take into account the following factors: the nature of the mutation (missense, nonsense, neutral) and conservation of the mutated residues within the subunit, the localization of the mutated subunit within the complex and the direct interactions with other subunits. A model for the topology of the subunits of mammalian complex I based on the fractionation of bovine complex I using mildly chaotropic detergents[26] has been depicted in Figure 1. From investigation of assembly profiles of six complex I-deficient patients with mutations in nuclear DNA-encoded subunits (NDUFS2, NDUFS4, NDUFS7, NDUFS8) it appeared that complex I assembly and/or stability has severely been compromised (Chapter VI). Decreased levels of fully assembled complex I correlate with the low enzymatic complex I activities a finding that has later been confirmed by a follow-up study[27]. These results suggest that disturbances in the assembly pathway or the stability of complex I are the direct cause of the decreased complex I activity in these patients. Besides lower amounts of fully assembled complex I, different patterns of low-molecular weight subcomplexes containing the NDUFS3 subunit occur (Chapter VI). Regarding the relative accumulation of NDUFS3-containing subcomplexes in the low- molecular weight range (~100–450 kDa), it seems that, when the NDUFS4 subunit is mutated, assembly is stalled at a certain point, but part of the peripheral arm can still be formed. In addition, absence of (most of) these subcomplexes would indicate that the

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Figure 1. A model for the topology of the subunits of mammalian complex I. With mildly chaotropic detergents, intact bovine complex I has been resolved into various subcomplexes that have subsequently been subjected to extensive analysis[26]. Subcomplex I represents the peripheral arm of the complex, subcomplex I consists of subcomplex I plus part of the membrane arm and subcomplex I forms the major part of the membrane arm. Subunits that do not associate with the I or I subcomplexes are grouped as the I ‘subcomplex’, a fraction for which it is not clear whether it represents a true fragment of complex I. Some subunits associate with two subcomplexes (I /I ), for others it is not exactly clear which subcomplex they associate with (I /I ). The nuclear DNA-encoded subunits are named according to the human nomenclature and preceded by the prefix ‘NDUF’.

formation of the peripheral arm is interrupted at very early stages of the assembly process, when the NDUFS2, NDUFS7 and NDUFS8 subunits are mutated. In patients with mutations in the NDUFS4 subunit, an additional subcomplex of ~800 kDa, that shows no in-gel activity, accumulates instead of fully assembled complex I (Chapter VI). This indicates that NDUFS4 is incorporated at a late stage in the assembly process and that lack of NDUFS4 would hamper the final step in the assembly of complex I. Two

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General discussion subunits located in the peripheral and membrane arm boundary region, NDUFA9 and NDUFS5, associate with the ~800-kDa subcomplex (Chapter VI). The NDUFS5 subunit is likely to be incorporated at a late stage of assembly since it is absent in low-molecular weight subcomplexes and it did not co-precipitate with NDUFAF1-containing assembly intermediates[21]. For the NDUFA9 subunit, however, it might as well still be possible to assemble at early stages in the assembly process, as has been proposed by Antonicka et al.[28] and Lazarou et al.[29]. Since NDUFA9 is located in the peripheral and membrane arm boundary region, lack of detection of NDUFA9 in low-molecular weight subcomplexes (Chapter VI) might be due to its loose association with assembly intermediates[26].

Elucidating the pathway of de novo complex I assembly The subcomplexes observed in the patients fit well within the recently published complex I assembly model[30] that described low-molecular weight assembly intermediate complexes, which the NDUFS3 subunit is part of. According to this model, six distinct assembly intermediate complexes containing the NDUFS3 subunit could be distinguished by means of an inducible NDUFS3-green fluorescent protein (GFP) expression system in human embryonic kidney (HEK293) cells. Assembly intermediates could be identified when they reappeared after the release of chloramphenicol inhibition of mitochondrial translation, i.e., when synthesis of the mtDNA-encoded ND subunits is resumed. A shift in NDUFS3-GFP signal from the smaller subcomplexes 2 and 3 towards the larger subcomplexes 4–6 and fully assembled complex I indicated that the smaller subcomplexes (2 and 3) rather represent products of assembly than of instability and/or degradation. Furthermore, it could be concluded that the presence of ND subunits is prerequisite to subsequent formation of the larger assembly intermediates (4–6) and fully assembled complex I. Analogously, the NDUFS3-containing subcomplexes accumulating in the patients (Chapter VI) likely represent true intermediates of assembly rather than instability or degradation products, as well. In conclusion, the observation of accumulated subcomplexes in complex I-deficient patients supports the hypothesis that de novo complex I assembly proceeds through a series of distinct assembly intermediates. The association of the NDUFS2 subunit with the same six NDUFS3-containing subcomplexes[30] implies that both subunits are assembled at an early stage in the process and that they occur in close contact of each other. Together with subunits NDUFS7 and NDUFS8 they are part of the hydrogenase module that is believed to be formed at the beginning of the assembly process[31] and to proceed up to subcomplex 3 of the assembly model[30]. The next step is the incorporation of preassembled membrane modules containing the mtDNA-encoded subunits, with ND1 being the first to be assembled, together with more nuclear DNA-encoded subunits first NDUFA13 and others, then NDUFA1, NDUFA2,

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NDUFA6, NDUFA9, NDUFB6 and others[30,31]. Finally, the NADH dehydrogenase module, containing NDUFV1–3, NDUFS4 and NDUFS6 (and NDUFS1), is incorporated to complete complex I assembly (Chapter VII)[23,29,31]. Mentioned steps are illustrated in an adapted model of the pathway of de novo complex I assembly, which is depicted in Figure 2.

Figure 2. A model of the human complex I assembly pathway. It is hypothesized that de novo complex I assembly proceeds through a series of distinct assembly intermediates by combining evolutionarily conserved functional modules. Several assembly chaperones (NDUFAF1, NDUFAF2, Ecsit) associate with the intermediate complexes while facilitating specific steps during the assembly process and dissociate from the complex when its fully assembled state is obtained. Adapted from Vogel et al.[30].

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An alternative route for nuclear DNA-encoded subunits to assemble Recently, Lazarou et al.[29] hypothesized a novel complementary mechanism of complex I biogenesis involving exchange of preexisting subunits with newly imported ones via a dynamic process to maintain complex I homeostasis. Besides following the route of de novo complex I assembly (which is the case after release of inhibition of mitochondrial translation) incorporation of newly synthesized nuclear DNA-encoded subunits directly into preexisting assembly intermediates or the fully assembled complex takes place. Since most mtDNA-encoded complex I subunits are highly hydrophobic they are cotranslationally inserted into the mitochondrial inner membrane, mediated by chaperones such as Oxa1l, Letm1 and Mrpl45[32,33], where they assemble via intermediate membrane modules[28,29,31]. In contrast, many nuclear DNA-encoded subunits are not hydrophobic and appear capable of direct incorporation into the holocomplex or assembly intermediate complexes. Lazarou et al.[29] investigated the assembly of newly imported (nuclear DNA-encoded) subunits in mitochondria by means of radioactive labeling and chase assays. Subunits NDUFV3, NDUFS4 and NDUFS6 all appeared to assemble directly into complex I and their absence in intermediate assemblies indicates that they are incorporated at a late stage of complex I assembly[29]. They are required for the assembly of NDUFV1 and NDUFV2, and therewith, for the stabilization of the NADH dehydrogenase part that is located at the tip of the peripheral arm. A number of subunits, such as NDUFB8, NDUFS1, NDUFS2, NDUFS7, and NDUFA9 also integrate into additional subcomplexes in the ~480-kDa and ~550-kDa ranges[29], which correspond with assembly intermediates of the complex I assembly models mentioned before[28,31]. Conclusively, the dynamic process of exchange of newly imported subunits with ones in preexisting complexes may enable a mechanism to aid in the turnover of subunits, some of which are prone to oxidative damage[29] a thought that becomes interesting in the light of the recently observed accumulation of oxidatively damaged complex I subunits in the brains of patients with Parkinson disease[34].

Assembly chaperones NDUFAF1, NDUFAF2 and Ecsit facilitate the assembly process The biogenesis of the OXPHOS system comprises a complex series of processes. Expression of the mtDNA-encoded subunits, expression and import of the nuclear-DNA encoded subunits, addition of prosthetic groups, insertion of the subunits into the mitochondrial inner membrane, assembly of the holocomplexes, and the final formation of supercomplexes are all processes that are tightly regulated by the actions of numerous ancillary proteins[35]. Although the list of proteins known to be involved in these processes is growing rapidly, many more factors are expected to be identified. For instance, assembly of complex IV (of the OXPHOS system) is known to proceed through a series of intermediates that involve a

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Chapter VIII number of chaperones[36]. Complex IV consists of only thirteen subunits but requires more than fifteen different components for its coordinated assembly into a mature complex[35]. Complex I assembly, on the contrary, involves 45 subunits that are believed to assemble in a similar fashion involving many more ancillary factors. However, so far, four proteins are suggested to be involved in human complex I assembly and/or stabilization exerting chaperone-like functions, i.e., NDUFAF1 (CIA30), NDUFAF2 (B17.2L), Ecsit and C6ORF66.

NDUFAF1 Knockdown of NDUFAF1 expression by means of RNA interference results in reduced amounts of fully assembled complex I and impaired enzymatic activity, supporting a role for NDUFAF1 in the regulation of assembly and/or stabilization of human complex I (Chapter V). However, the absence of accumulating assembly intermediates suggests that NDUFAF1 may be involved in the stabilization rather than in active combination of assembly intermediates. NDUFAF1 associates with two high-molecular weight protein complexes of approximately 600 and 700 kDa (Chapter V). The ~700-kDa complex seems to accumulate when membrane arm assembly is hampered, e.g., by mutation of a subunit of the membrane arm (patient with an ND5 mutation). Likewise, levels of the ~600-kDa complex increase when peripheral arm assembly is hampered (i.e. in a patient with an NDUFS8 mutation). The NDUFAF1-associated ~700-kDa complex disappears when mitochondrial translation is inhibited and depletion of mtDNA-encoded (ND) subunits occurs. The transient reappearance of the complex after release of inhibition of mitochondrial protein synthesis suggests that i) NDUFAF1 binds to a mtDNA-encoded (ND) subunit either directly or indirectly present in the ~700-kDa complex, and that ii) NDUFAF1 is required by this complex to proceed with assembly (Chapter V). A role for NDUFAF1 in assembly of (part of) the membrane arm corresponds with the function that was initially proposed for its counterpart CIA30 in Neurospora crassa[37]. Representing either an active role in assemble to facilitate the combination of assembly intermediates, or a stabilizing role to prevent degradation of assembly intermediates, remains subject to debate. The sizes of the NDUFAF1 complexes of ~600 kDa and ~700 kDa have recently been clarified as being in the ~500–850-kDa range[27], which is also in concordance with the NDUFAF1-associated complexes described by Dunning et al.[21]. By means of co- immunoprecipitation studies and immunodepletion analysis they found that NDUFAF1 associates transiently with assembly intermediate complexes of ~460 and ~830 kDa that contain mtDNA-encoded subunits ND1, ND2 and ND3 of the membrane arm, as well as various nuclear DNA-encoded subunits (including NDUFA6, NDUFA9, NDUFB6, NDUFS3 and NDUFS7). Analysis of newly translated mtDNA-encoded subunits in a patient with an NDUFAF1 mutation, revealed that ND2 was absent, while ND1 appeared to stall transiently in an ~400-kDa subcomplex, before disappearing completely, too[21]. These findings correspond with the noticed absence of accumulating assembly intermediates after

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General discussion knockdown of NDUFAF1 by RNA interference (Chapter V). It seems that lack of NDUFAF1 leads to loss of the ~460-kDa assembly intermediate that contains ND2, thereby preventing combination with an assembly intermediate containing ND1 and the concomitant formation of the ~830-kDa subcomplex, and hence assembly of the holocomplex[21]. Taken together, NDUFAF1 is probably involved in the early assembly or stabilization of a subcomplex of the membrane arm containing ND2 and ND3 enabling association with an assembly intermediate containing ND1, which leads to the formation of an ~830-kDa intermediate subcomplex. Finalizing assembly of the peripheral arm would initiate its release from the complex[21]. The role of chaperone NDUFAF1 has been integrated in the model of complex I assembly depicted in Figure 2. NDUFAF2 Lack of putative assembly chaperone NDUFAF2 leads to disturbance of complex I assembly, demonstrated by reduced amounts and activity of complex I and accumulation of low-molecular weight complex I subcomplexes, corresponding with assembly intermediates 2–5 of the model for human complex I assembly (Chapter VII). NDUFAF2 has exclusively been found associated with an ~830-kDa subcomplex of complex I (Chapter VII)[23,27,29]. This ~830-kDa subcomplex (or ~800-kDa subcomplex), mentioned earlier as a late assembly intermediate of complex I, specifically accumulates in patients with mutations in the subunits NDUFS4, NDUFS6 and NDUFV1 and appears to lack subunits of the tip of the peripheral arm, i.e. NDUFV1–3, NDUFS4 and NDUFS6 (Chapter VI)[19,23,29]. For these patients increased protein levels of NDUFAF2 have been observed[27]. Transduction experiments of fibroblasts with a baculoviral NDUFAF2-GFP construct revealed the presence of NDUFAF2 associated with the 830-kDa subcomplex not only in a disturbed state of complex I assembly (i.e., in patient cells with a mutation in of one of the above mentioned subunits), but also in a healthy state of complex I assembly (Fig 3, R. Janssen unpublished results). This supports the view that the 830-kDa subcomplex accumulating in patients is not an artifact of destabilized complex I but in fact represents a stalled intermediate of complex I assembly, as is proposed by Lazarou et al.[29]. To re- evaluate the possibility of NDUFAF2 association with low-molecular weight assembly intermediates, mitochondrial protein translation was inhibited by chloramphenicol treatment of fibroblasts from an NDUFS4 patient and a healthy control, prior to NDUFAF2- GFP transduction, to consequently accumulate NDUFS3-containing low-molecular weight subcomplexes[30]. Directly after release of inhibition and concurrent resumption of complex I assembly, NDUFAF2 associated with the accumulating 830-kDa subcomplex in the patient cells, whereas no association with low-molecular weight assembly intermediates was observed (R. Janssen unpublished results). These findings strongly suggest a role for NDUFAF2 restricted to the late stages of complex I assembly.

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Regarding the lack of in-gel activity of the 830-kDa subcomplex (Chapter VI) and the subunits it is missing, it can be hypothesized that NDUFAF2 associates with the ~830-kDa assembly intermediate, facilitates the incorporation of the NADH dehydrogenase module of complex I or at least an essential part of its functional structure and releases the complex when its fully assembled state is reached. The role of chaperone NDUFAF2 has been integrated in the model of complex I assembly depicted in Figure 2.

Figure 3. Association of NDUFAF2 with an assembly intermediate complex. NDUFS4 patient (P(S4)) and healthy control fibroblasts (C2) were transduced with baculoviral constructs NDUFAF2-GFP (AF2) and COX8-GFP (COX8), and subsequently subjected to BN-PAGE analysis and western blotting. Using an anti-EGFP polyclonal antibody, association of NDUFAF2-GFP with the 830-kDa subcomplex of complex I is clearly detected in the NDUFS4 patient cells (left panel). For comparison, the same blot was incubated with anti-NDUFA9 antibody to show fully assembled complex I in control cells and the 830- kDa subcomplex in the NDUFS4 patient (right panel). A closer look at a longer exposure of the blot (see enlargement) reveals the presence of the 830-kDa subcomplex in control cells as well (indicated by the arrow), albeit weakly detectable. At the bottom of the blot unbound NDUFAF2-GFP and COX8-GFP is detected. untr: untransduced; AF2: NDUFAF2-GFP transduced; COX8: COX8-GFP transduced; CI: fully assembled complex I; 830kD-sub: 830- kDa subcomplex of complex I

Ecsit Recently, a third putative assembly chaperone, named Ecsit, has been found interacting with NDUFAF1, by means of tandem affinity purification (TAP) of mitochondrial lysates of HEK293 cells expressing TAP-tagged NDUFAF1 protein[38]. In previous studies, Ecsit has been described as a cytosolic signaling protein essential for inflammatory response

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(the Toll pathway of innate immunity) and the BMP (bone morphogenetic protein) pathway of embryonic development[39,40]. Ecsit associates with the high-molecular weight NDUFAF1- containing complexes in the range of ~500–850 kDa. Protein knockdown of Ecsit established by RNA interference resulted in specifically disturbed complex I assembly and/or stability and accumulation of NDUFS3-containing assembly intermediate complexes in the range of ~500 kDa[38]. Furthermore, Ecsit knockdown resulted in severely decreased NDUFAF1 protein levels and corresponding NDUFAF1 complexes in the range of ~500–850 kDa, indicating a chaperone-like function for Ecsit in stabilizing these complexes. It can be concluded that Ecsit is required for correct complex I assembly and/or stability and may represent a link between mitochondrial functioning, immune response and embryonic development. C6ORF66 The C6ORF66 gene, encoding a calmodulin-binding protein (HRPAP20), has been reported as another putative complex I assembly chaperone[24]. In two complex I- deficient patients carrying C6ORF66 mutations, pathogenicity was proven as complex I activity was restored by complementation with wild-type C6ORF66 cDNA. The protein awaits functional characterization yet.

Structural dependence of complex I on supercomplex formation It has been argued that fully assembled complex III is required to maintain complex I, since loss of assembled complex III has been found to lead to reduced levels of complex I[41]. Disturbed assembly of complex III due to loss of the mtDNA-encoded subunit cytochrome b causes a severe reduction in the level of fully assembled complex I. Although complex I was assembled initially, its stability was severely hampered. In addition, if complex III activity is impaired, for instance, by complex III-specific inhibitor antimycin A, complex III assembly remains unaffected and normal complex I levels are observed[41]. Conversely, physical absence of complex I seems not to have drastic influences on complex III assembly and/or stability. However, complex I deficiency combined with a moderate complex III deficiency has been observed for patients with mutations in the complex I subunits NDUFS2 and NDUFS4[8]. Protein levels of complex III are clearly lowered for NDUFS2 patients, while several NDUFS4 patients show a slight decrease in protein levels of assembled complex III, as well as in spectrophotometrically measured enzymatic activities (Chapter VI). Two explanations can be argued, one, complex I naturally occurs in the form of supramolecular structures, named supercomplexes, which are composed of several OXPHOS complexes. When these supercomplexes can not be formed by the physical absence of complex III, complex I is susceptible to degradation. And two, complex III actively participates in the assembly process of complex I by an yet undetermined mechanism[41]. The complex III dimer may act as a scaffold to facilitate complex I assembly, possibly by

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Chapter VIII stabilizing unassembled structures[29]. Counting in favor of the latter explanation, is the observation that complex I chaperone NDUFAF2 associates with a supercomplex, consisting of the complex III dimer and the ~830-kDa subcomplex of complex I, which is also accumulating in patients with mutation in the NDUFS4, NDUFS6 and NDUFV1 subunits (R. Janssen unpublished results)[23,29]. The same association has transiently been detected in mitochondria of healthy control fibroblasts[29]. These findings imply that complex I does not need to be fully assembled before it associates with other complexes into supercomplexes and that these complexes (i.e. the complex III dimer) may even participate in the assembly process itself. Complex IV has also been implicated in stabilization of complex I, and may be needed for complex I assembly and/or stability in a similar way as complex III[42].

Intracellular consequences of complex I deficiency Indicators of mitochondrial disease To elucidate the pathology of complex I deficiency it is important to examine its biological consequences at a cellular level. When a key component of the OXPHOS system, e.g. complex I, is missing or defective, electron transfer is hampered and ATP production is impaired. Disturbance of the OXPHOS system evokes a series of metabolic alterations that result in a variety of clinical symptoms. An important intracellular alteration is the change in mitochondrial membrane potential. A complex I defect can hamper proton transport across the mitochondrial inner membrane, which as a result gets depolarized. Defective complex I will also affect the NADH oxidation rate leading to an excess of mitochondrial NADH and a lack of NAD+. The increased NADH/NAD+ ratio delivers feedback inhibition of the TCA cycle and the pyruvate dehydrogenase complex (PDHc), resulting in an increase in mitochondrial pyruvate levels. Subsequently, excess of pyruvate is converted in the cytoplasm either into lactate by lactate dehydrogenase (LDH) or into alanine by transamination. As a final consequence elevated levels of lactate, pyruvate and alanine occur in body fluids, i.e. in blood, urine and frequently also in cerebrospinal fluid (CSF)[43]. Many patients suffering from mitochondrial disease have lactic acidemia (i.e. elevated blood lactate levels), which is not only an indicator of an OXPHOS defect, but the lactate itself can have deleterious effects. Another consequence of complex I deficiency is the generation of free radicals, superoxide in particular. Decreased complex I activity as well as decreased amounts of fully assembled complex I correlates with increased intracellular superoxide levels, as has been established in a cohort of 21 patients with isolated complex I deficiency[44]. As also known for cultured skin fibroblasts treated with rotenone, increased superoxide production in complex I- deficient patient fibroblasts is a primary consequence of (partial) inhibition of complex I activity[45]. Although superoxide and its derived reactive oxygen species (ROS) play

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General discussion important roles in a variety of signaling processes at physiological concentrations[46], they have deleterious effects for the cell at pathological concentrations. How, in fact, high ROS levels exert influence on the integrity and functioning of the OXPHOS system or mitochondria in general, remains subject of debate. Complex I deficiency can also influence mitochondrial morphology. It is hypothesized that complex I, when assembled with other the OXPHOS complexes in supramolecular structures, contributes to the unique shaping of the mitochondrial inner membrane and cristae[47]. Loss of fully assembled complex I could therefore lead to disturbance of mitochondrial inner membrane integrity and thereby change total mitochondrial morphology. These changes in mitochondrial morphology could finally induce apoptosis.

Cell physiological changes in a complicated case of complex I deficiency In Chapter VII the cell physiological consequences of complex I dysfunction due to lack of assembly chaperone NDUFAF2 have been described. The patient with the contiguous gene deletion, which lacks NDUFAF2, exhibits extraordinarily high cellular superoxide levels in fibroblasts compared to other complex I-deficient patients. The patient‘s superoxide level extended up to 354% of normal control levels, which correlates inversely with the low amount of fully assembled complex I observed in its fibroblasts. By comparison, cellular superoxide levels measured in the 21 complex I-deficient patients (with or without established mutations) ranged from 104% to 353% of normal control levels, whereas levels for 13 patients with established mutations in complex I subunits encoded by the nuclear DNA ranged from 104% to 295% of normal control levels[44]. Nonetheless, the high superoxide level and low amount of fully assembled complex I in the patient‘s fibroblasts satisfy the inverse relationship between superoxide production and amount of fully assembled complex I in fibroblasts of patients with isolated complex I deficiency, which has been reported by Verkaart et al.[44]. Changes in mitochondrial membrane potential, 91% of normal control levels, and NAD(P)H levels, 180% of normal control levels, are comparable to other complex I-deficient patients, i.e., ~92% of normal control levels for mitochondrial membrane potential[48] and a range from 112% to 191% of normal control levels for NAD(P)H oxidation[49]. Mitochondrial parameters as superoxide production and membrane potential are regarded as key indicators of mitochondrial health and metabolic activity. Accurate determination of these parameters in living cells is of great importance in addressing many research questions[48].

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Future perspectives Currently, the list of genes responsible for human complex I deficiency is still growing, now including all genes encoding ‗core subunits‘, continuing with the genes encoding ‗accessory subunits‘ and starting with the genes encoding assembly factors (e.g., NDUFAF1, NDUFAF2, and C6ORF66). The intricate process of complex I assembly is starting to unravel with the characterization of the first few known complex I assembly factors, as NDUFAF1, NDUFAF2 and Ecsit, supported by the current identification of patients with corresponding disease causing genotypes. This will open new avenues for identification of assembly factors and possibly explain many more yet unsolved cases of isolated complex I deficiency. Since NDUFAF1 and NDUFAF2 were found while associating with assembly intermediate complexes, examination of the composition of these subcomplexes, for instance by means of mass spectrometry, would present a worthwhile approach to identify new chaperones of complex I assembly. Characterizing additional functions of the accessory subunits and assembly chaperones of complex I is an interesting course to explore functional relationships with other intra- and extramitochondrial metabolic pathways. To illustrate, the NDUFA13 subunit, which is also known as GRIM-19, was originally identified as a critical regulatory protein for interferon- beta and retinoic acid-induced cell death, providing a link with the mitochondrial apoptosis pathway[50]. In line with this, apoptosis-inducing factor (AIF) is suspected of having a role in the biogenesis and/or maintenance of complex I[51] and subunit NDUFS1 is recognized as a critical, first substrate for cleavage upon caspase activation during execution of apoptosis[52]. All these findings are posing complex I as a participant in apoptosis. Furthermore, there is credible evidence that the NDUFAB1 subunit functions as a soluble acyl carrier protein in the mitochondrial matrix, providing a link with mitochondrial type II fatty acid synthesis[53,54]. Close connections with the mitochondrial translation and import pathways have been suggested through subunits NDUFA2 and NDUFA11 that share a high degree of homology with, respectively, components of the mitochondrial ribosome, MRPL43 and MRPS25[22], and the mitochondrial protein import machinery, Tim proteins 17, 22 and 23[55]. Finally, assembly chaperone Ecsit may embody the link between complex I and pathways of the immune response. Mitochondrial parameters as membrane potential, mitochondrial morphology and superoxide production are regarded as key indicators of mitochondrial health and metabolic activity. Accurate determination of these parameters in living cells is besides gaining insight in its molecular background, of great importance for elucidation of the pathology of OXPHOS disorders.

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At present, a remedy for OXPHOS disorders, and complex I deficiency in particular, is not on hand. Many clinical trials of treatment with a diversity of supplements have been performed in the past with varying success. Incidental successes have been established with administration of creatine providing energy and modestly increasing muscle strength and with supplementation of antioxidants, in the form of ‗cocktails‘ of vitamins or cofactors such as ubiquinone, idebenone, riboflavin, lipoic acid, thiamine, vitamin E and vitamin K. Antioxidants may scavenge harmful free radicals and/or enhance the efficacy of the OXPHOS system. Orally administered ubiquinone (Q) can be taken up by mitochondria, but only to a limited extent. MitoQ, a lipophilic triphenylphosphonium (TPP+) cation conjugated to ubiquinone, is a compound that selectively targets ubiquinone to mitochondria[56]. When the ubiquinone is reduced to its active ubiquinol redox form it can protect mitochondria against oxidative damage, after which it is recycled back to its active form by the mitochondrial respiratory chain[56]. In the past, some cases of mitochondrial myopathy caused by complex I deficiency have shown normalization of complex I activity along with clear clinical improvement upon prolonged riboflavin therapy[57-59]. Administration of metabolites and cofactors is especially important in disorders due to primary deficiencies of specific compounds, such as carnitine and coenzyme Q (ubiquinone). Unfortunately, most studies on the effects of supplements have produced mixed results, because of divergent responses to the supplements among patients. The heterogeneous nature of (the molecular defects underlying) OXPHOS disorders is a likely explanation for the divergent responsiveness among patients with mitochondrial disease. Genetic-based strategies are more promising treatment approaches that are currently being developed for patients with mtDNA defects. One such procedure is ‗gene shifting‘, i.e., decreasing the ratio of mutant to wild-type mitochondrial genomes, which is applied in patients with a pure mitochondrial myopathy. This can be achieved in various ways[5]. First, satellite cells with low levels of mutated mtDNA are stimulated to proliferate after which they are fused with mature muscle fibers, reducing the overall proportion of mutant mtDNA to sub-threshold levels[60,61]. Second, inhibition of replication of mutant mtDNA by selective hybridization of peptide nucleic acids (PNAs) favors propagation of wild-type mtDNA and causes the proportion of mutant mtDNA to drop below the pathological threshold[62]. Third, import of specific restriction endonucleases can selectively degrade mutant mtDNA[63]. Fourth, inducing mitochondrial fusion can lead to reorganization of the heterogeneous distribution of mutant mtDNAs among mitochondria, thereby dropping the proportion of mutant mtDNA below the pathological threshold. Other experimental treatments concerning ‗gene therapy‘ that are currently under development include allotopic expression of mitochondrial genes transferring a wild-type version of a mutated mitochondrial gene to the nucleus[64,65] and xenotopic

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expression expressing cognate proteins from mitochondrial or nuclear genes from other organisms. Nuclear transfer is a technique that can be used to prevent the transmission of mtDNA disease. The strategy involves the transfer of nuclear DNA from an oocyte with mutant mtDNA to an enucleated oocyte from a female with normal mtDNA. The transmitochondrial oocyte can be fertilized in vitro and implanted in the uterus[66]. In conclusion, with our expanding knowledge of the OXPHOS system, and mitochondrial function in general, it more and more tends to appear in the center of a complex constellation of biochemical pathways, serving (almost) every cell of the body. This, once again, emphasizes the importance of finding a cure for mitochondrial disease.

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54. Cronan JE, Fearnley IM, Walker JE (2005) Mammalian mitochondria contain a soluble acyl carrier protein. FEBS Lett 579(21):4892-4896 55. Carroll J, Shannon RJ, Fearnley IM, Walker JE, Hirst J (2002) Definition of the nuclear encoded protein composition of bovine heart mitochondrial complex I. Identification of two new subunits. J Biol Chem 277(52):50311-50317 56. Cochemé HM, Kelso GF, James AM, Ross MF, Trnka J, Mahendiran T, Asin-Cayuela J, Blaikie FH, Manas AR, Porteous CM, Adlam VJ, Smith RA, Murphy MP (2007) Mitochondrial targeting of quinones: therapeutic implications. Mitochondrion 7 Suppl:S94-102 Review 57. Bernsen PL, Gabreëls FJ, Ruitenbeek W, Sengers RC, Stadhouders AM, Renier WO (1991) Successful treatment of pure myopathy, associated with complex I deficiency, with riboflavin and carnitine. Arch Neurol 48(3):334-338 58. Bernsen PL, Gabreëls FJ, Ruitenbeek W, Hamburger HL (1993) Treatment of complex I deficiency with riboflavin. J Neurol Sci 118(2):181-187 59. Ogle RF, Christodoulou J, Fagan E, Blok RB, Kirby DM, Seller KL, Dahl HH, Thorburn DR (1997) Mitochondrial myopathy with tRNA(Leu(UUR)) mutation and complex I deficiency responsive to riboflavin. J Pediatr 130(1):138-145 60. Clark KM, Bindoff LA, Lightowlers RN, Andrews RM, Griffiths PG, Johnson MA, Brierley EJ, Turnbull DM. (1997) Reversal of a mitochondrial DNA defect in human skeletal muscle. Nat Genet 16(3):222-224 61. Taivassalo T, Fu K, Johns T, Arnold D, Karpati G, Shoubridge EA (1999) Gene shifting: a novel therapy for mitochondrial myopathy. Hum Mol Genet 8(6):1047-1052. 62. Taylor RW, Chinnery PF, Turnbull DM, Lightowlers RN (1997) Selective inhibition of mutant human mitochondrial DNA replication in vitro by peptide nucleic acids. Nat Genet 15(2):212-215 63. Tanaka M, Borgeld HJ, Zhang J, Muramatsu S, Gong JS, Yoneda M, Maruyama W, Naoi M, Ibi T, Sahashi K, Shamoto M, Fuku N, Kurata M, Yamada Y, Nishizawa K, Akao Y, Ohishi N, Miyabayashi S, Umemoto H, Muramatsu T, Furukawa K, Kikuchi A, Nakano I, Ozawa K, Yagi K (2002) Gene therapy for mitochondrial disease by delivering restriction endonuclease SmaI into mitochondria. J Biomed Sci 9(6 Pt 1):534-541 64. Manfredi G, Fu J, Ojaimi J, Sadlock JE, Kwong JQ, Guy J, Schon EA (2002) Rescue of a deficiency in ATP synthesis by transfer of MTATP6, a mitochondrial DNA-encoded gene, to the nucleus. Nat Genet 30(4):394-399 65. Guy J, Qi X, Pallotti F, Schon EA, Manfredi G, Carelli V, Martinuzzi A, Hauswirth WW, Lewin AS (2002) Rescue of a mitochondrial deficiency causing Leber Hereditary Optic Neuropathy. Ann Neurol 52(5):534-542 66. Gardner JL, Craven L, Turnbull DM, Taylor RW (2007) Experimental strategies towards treating mitochondrial DNA disorders. Biosci Rep 27(1-3):139-150 Review

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General discussion

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Summary

200

Summary

Oxidative phosphorylation (OXPHOS) is the final biochemical pathway of energy (ATP) production in the cell. Defects of the OXPHOS system are amongst the most frequent inborn errors of metabolism and result in many, often devastating diseases affecting different organs and tissues. The OXPHOS system consists of five multiprotein complexes that are built out of 89 structural protein components, which are encoded by both the mitochondrial and nuclear DNA. Correct biogenesis and functioning of the OXPHOS system is dependent on the finely tuned interaction between the nuclear and the mitochondrial genomes. As a consequence, disturbances of the system can be caused by numerous genetic defects and can manifest in a variety of metabolic alterations that result in a plethora of clinical symptoms. In Chapter II we give an overview of the many genetic defects underlying OXPHOS deficiencies and discuss how the four different types of inheritance autosomal recessive or dominant, X-linked and maternal or mitochondrial contribute to the heterogeneity of OXPHOS disease and possibly can be supportive of linking clinical diagnosis to molecular defects. The most common cause of OXPHOS disease is isolated complex I deficiency. Complex I is the largest and most complicated of five complexes and represents the main entrance of the OXPHOS pathway. Structure, function and biogenesis of mitochondrial complex I is reviewed in Chapter III, as well as the pathology of complex I deficiency. Complex I deficiency is the underlying defect of a heterogeneous group of clinical phenotypes, comprising single-organ conditions as well as multisystemic syndromes. Mutations responsible for complex I deficiency can be found in many genes encoding structural subunits (encoded by the mitochondrial as well as the nuclear DNA), assembly factors, or proteins otherwise involved in the biogenesis or functioning of complex I, or of its cofactors (e.g. FMN). Additionally, complex I deficiency occurs as in many devastating neurodegenerative disorders, such as Parkinson disease and Alzheimer disease. The biogenesis of complex I is an intricate process of assembly of 45 individual subunits into a membrane-bound multiprotein structure. This process is suspected of being facilitated by the action of several (yet to be characterized) assembly chaperones. In Chapter IV we report the identification of the first human complex I assembly factor, NDUFAF1, which is the human homologue of assembly chaperone CIA30 of the fungus Neurospora crassa. We present the genomic sequence of the NDUFAF1 gene and demonstrate that expression of the NDUFAF1 protein is ubiquitous with a slightly higher expression in heart, kidney, lung and liver. Mutational analysis of the NDUFAF1 gene in 13 patients with an isolated complex I deficiency resulted in the detection of four new single nucleotide polymorphisms (SNPs), but 201

Summary no functional mutations were found. Nevertheless, we propose the NDUFAF1 gene as a candidate for complex I deficiency. Characterization of the function of NDUFAF1 in the assembly process of complex I and the investigation of its involvement in complex I deficiency is described in Chapter V. By means of subcellular fractionation and immunofluorescence microscopy, mitochondrial localization of NDUFAF1 is demonstrated, which is consistent with an in silico prediction of an N-terminal mitochondrial targeting sequence. Knockdown of NDUFAF1 expression established by RNA interference leads to reduced amounts of complex I and impaired enzymatic activity. Furthermore, we demonstrate that NDUFAF1 associates with two high-molecular weight complexes, one of which (the larger) seems to accumulate when membrane arm assembly is hampered, and disappears when mtDNA-encoded subunits are depleted by inhibition of mitochondrial translation. This NDUFAF1-containing complex transiently reappears when assembly resumes. These findings suggest that NDUFAF1 facilitates assembly and/or stability of complex I, possibly through associating with an assembly intermediate complex by binding to a mtDNA-encoded subunit. The assembly process of mitochondrial complex I is currently believed to proceed through a series of distinct assembly intermediates by combining evolutionarily conserved functional modules. Disturbances of the process by, for instance, mutation of subunits can lead to accumulation and/or depletion of several assembly intermediate complexes, as a result of which so-called assembly profiles can be constituted. In Chapter VI we describe how mutations in different complex I subunits affect the integrity of complex I and interpret the assembly profiles it brings forth. In six patients with complex I deficiency caused by mutations in nuclear DNA-encoded subunits (NDUFS2, NDUFS4, NDUFS7, NDUFS8) we show reduced levels of fully assembled complex I and complex I in-gel activity that correlate with the low spectrophotometrically measured complex I enzyme activities. These results suggest that disturbance of the assembly process or the stability of complex I, caused by mutation of the mentioned subunits, is the direct cause of the decreased complex I activities in these patients. However, some patients with unknown mutations show no correlation between their low complex I activities and the levels of the fully assembled complex I, which can be indicative of a catalytic defect. Mutations may occur in genes involved in the activation of complex I or in the synthesis one of its cofactors (FMN, FeS clusters). In the patients with mutations in the nuclear DNA-encoded subunits accumulation of low- molecular weight subcomplexes containing the NDUFS3 subunit occurs in different patterns. These NDUFS3-containing subcomplexes fit well within a recently proposed model of complex I assembly and likely represent true intermediates of assembly rather than instability or degradation products. In patients with mutations in the NDUFS4 subunit, an additional subcomplex of ~830 kDa (~800 kDa in Chapter VI), that shows no in-gel activity,

202

Summary accumulates instead of fully assembled complex I. This suggests that NDUFS4 is incorporated at a late stage in the assembly process. In addition to the complex I deficiency, a moderate complex III deficiency has been observed for patients with NDUFS2 and NDUFS4 mutations. In Chapter VII a unique case of complex I deficiency is described. A patient presenting with an apparently mitochondrial encephalomyopathy carries a homozygous triple gene deletion on chromosome 5 encompassing the NDUFAF2, ERCC8, and ELOVL7 genes. The genes encode the human complex I assembly factor 2, also known as B17.2L, a protein of the nucleotide excision repair (NER) machinery, and a putative elongase of very long-chain fatty acid (VLCFA) synthesis, respectively. The patient‘s fibroblasts demonstrate a NER defect that is characteristic of Cockayne syndrome type A and that can unequivocally be ascribed to the deletion of the ERCC8 gene. Unfortunately, due to the low expression level of ELOVL7 in human fibroblasts, specific changes in endogenous fatty acid levels as a consequence of the ELOVL7 deletion could not be assessed in the patient‘s fibroblasts. By use of a baculoviral complementation assay with wild-type NDUFAF2, the loss of the NDUFAF2 gene has been proven to cause the complex I deficiency. Lack of NDUFAF2 protein leads to disturbance of complex I assembly, which is demonstrated by reduced amounts and activity of complex I and accumulation of small assembly intermediates. Furthermore, investigation of cell physiological consequences reveals increased intracellular superoxide and mitochondrial NAD(P)H levels, and decreased mitochondrial membrane potential. All observed cell physiological alterations are restored upon complementation with wild-type NDUFAF2. Chaperone NDUFAF2 exclusively associates with the ~830-kDa subcomplex (~800 kDa in Chapter VI) that accumulates specifically in patients with mutations in the subunits located in the tip of the peripheral arm (NDUFS4, NDUFS6, NDUFV1) and that corresponds to a late assembly intermediate of complex I. It can be hypothesized that NDUFAF2 associates with the ~830-kDa assembly intermediate, facilitates the incorporation of the NADH dehydrogenase module of complex I and releases the complex when its fully assembled state is reached. Finally, the stepwise, de novo, assembly of complex I is discussed in Chapter VIII. The proposed roles of assembly chaperones NDUFAF1 and NDUFAF2 are integrated in a revised model of the complex I assembly pathway. The alternative assembly mechanism of dynamic exchange of subunits is reflected shortly, as is the role of a third putative assembly chaperone, named Ecsit, and the importance of complex III for complex I assembly. To understand the pathology of complex I deficiency it is important to not only elucidate the molecular background, but also to examine its physiological consequences at a cellular level. Mitochondrial parameters as membrane potential, mitochondrial morphology and superoxide production are regarded as key indicators of mitochondrial health and metabolic

203

Summary activity. In conclusion, elucidating the mechanisms underlying the biogenesis and functioning of complex I and expanding our knowledge of the pathogenesis of complex I deficiency will be of utmost importance for performing accurate diagnostics and ultimately, for development of future therapeutic treatment.

204

Samenvatting

Voor het dagelijks functioneren van ons lichaam is energie nodig, veel energie! Deze energie wordt geproduceerd in de vorm van ATP in de vele ‗mini-energiecentrales‘, de mitochondriën, die zich in vrijwel elke cel van ons lichaam bevinden. Oxidatieve fosforylering (OXFOS) is de laatste biochemische stap in het productieproces van ATP. In het kort houdt dit in dat oxidatie van energierijke producten van de stofwisseling (metabolieten) gevolgd door een serie biochemische reacties, er toe leidt dat zuurstof wordt gereduceerd tot water en ADP wordt omgezet in het energierijke ATP. Dit proces wordt uitgevoerd door vijf eiwitcomplexen die zich in de binnenmembraan van de mitochondriën bevinden. Deze eiwitcomplexen vormen samen met twee elektronendragers (ubiquinon en cytochroom c) het OXFOS systeem en zijn opgebouwd uit 89 verschillende eiwitcomponenten (of subunits). Het DNA dat de code (ook wel gen genoemd) bevat voor de aanmaak van deze eiwitten bevindt zich voornamelijk in de celkern. Daarnaast hebben de mitochondriën een eigen DNA molecuul dat voor enkele van deze eiwitten codeert. Door een fout in het DNA, een mutatie genoemd, wordt een defect eiwit aangemaakt dat op zijn beurt een defect in het OXFOS systeem kan veroorzaken. Een defect OXFOS systeem kan vervolgens tot een verstoring van het ATP-productieproces leiden met uiteindelijk een energietekort tot gevolg. Defecten van het OXFOS systeem, OXFOS deficiënties, behoren tot de meest voorkomende aangeboren stofwisselingsstoornissen en komen tot uiting in de vorm van vaak zeer ernstige ziekten, die veelal een vroege dood inleiden. Vooral organen die een hoog energieverbruik hebben, zoals de spieren en de hersenen, zijn aangedaan, maar ook afwijkingen aan de ogen, de lever, de nieren en het spijsverteringsstelsel kunnen voorkomen. Een correcte opbouw en het goed functioneren van het OXFOS systeem is afhankelijk van de nauwkeurig afgestemde samenwerking tussen het mitochondriële en het kern-DNA. Derhalve kunnen verstoringen van het systeem veroorzaakt worden door tal van genetische defecten die een reeks van metabole en biochemische veranderingen in de cel teweeg kunnen brengen en die in een diversiteit aan klinische symptomen tot uiting kunnen komen. In Hoofdstuk II wordt een overzicht gegeven van de vele genetische defecten die aan de basis liggen van OXFOS deficiënties en wordt bediscussieerd hoe de vier verschillende typen van overerving autosomaal recessief of dominant, X-gebonden en maternaal of mitochondrieel bijdragen aan de diversiteit van OXFOS ziekten en mogelijk een handvat kunnen bieden voor het koppelen van een klinische diagnose aan een moleculair defect. De meest voorkomende oorzaak van OXFOS ziekten is geïsoleerde complex I deficiëntie. Complex I is de grootste en meest gecompliceerde van de vijf eiwitcomplexen en belichaamt 205

Samenvatting de belangrijkste toegangspoort van het OXFOS proces. Zowel de structuur, de functie en de vorming van mitochondrieel complex I, alsook de pathologie van complex I deficiëntie, worden besproken in Hoofdstuk III. Complex I deficiëntie is het achterliggende defect van een heterogene groep klinische fenotypen die bestaat uit zowel één-orgaan aandoeningen als multisystemische syndromen. Mutaties die complex I deficiëntie veroorzaken, kunnen gevonden worden in de genen die coderen voor subunits (gecodeerd door zowel het mitochondriële als het kern-DNA), assemblage factoren of eiwitten die op andere wijze betrokken zijn bij de opbouw of het functioneren van complex I of zijn cofactoren (FMN). Complex I deficiëntie komt ook voor in vele, ernstige neurodegeneratieve aandoeningen, zoals de ziekten van Parkinson en Alzheimer. De vorming van complex I is een gecompliceerd proces van assemblage van 45 individuele subunits tot één membraangebonden eiwitstructuur. Dit proces wordt waarschijnlijk gecoördineerd en vergemakkelijkt door de tussenkomst van enkele (nog te karakteriseren) assemblage chaperonnes. In Hoofdstuk IV wordt de identificatie van de eerste humane assemblage factor van complex I, NDUFAF1, gerapporteerd. NDUFAF1 is de humane homoloog van assemblage chaperonne CIA30 van de schimmel Neurospora crassa. Naast het presenteren van de genomische sequentie van het NDUFAF1 gen wordt getoond dat expressie van het NDUFAF1 eiwit in alle weefsels plaats vindt, met een enigszins hogere expressie in het hart, de nieren, de longen en de lever. Mutatieanalyse van het NDUFAF1 gen in dertien patiënten met geïsoleerde complex I deficiëntie resulteerde in de detectie van vier nieuwe single nucleotide polymorfismen (SNPs), maar functionele mutaties werden niet gevonden. Toch kan NDUFAF1 beschouwd worden als een kandidaat gen voor complex I deficiëntie. De karakterisering van de functie van NDUFAF1 in het assemblage proces van complex I wordt beschreven in Hoofdstuk V. Met behulp van subcellulaire fractionering en immuunfluorescentie microscopie wordt mitochondriële lokalisering van NDUFAF1 aangetoond, wat consistent is met een in silico voorspelling van de aanwezigheid van een N- terminale mitochondriële targeting sequentie. Knockdown van NDUFAF1 expressie, gecreëerd d.m.v. RNA interference, leidt tot een verminderde hoeveelheid complex I en verlaagde enzymactiviteit. Verder wordt getoond dat NDUFAF1 associeert met complexen van hoogmoleculair gewicht, waarvan één (de grootste) lijkt te accumuleren als membraanarm assemblage verhinderd wordt en verdwijnt als depletie van mitochondrieel DNA-gecodeerde subunits plaats vindt, t.g.v. remming van mitochondriële translatie. Dit NDUFAF1-bevattende complex komt tijdelijk opnieuw op wanneer assemblage zich hervat. Aan de hand van deze bevindingen kunnen we veronderstellen dat NDUFAF1 de assemblage en/of de stabiliteit van complex I bevordert mogelijk door te associëren met een assemblage intermediair via binding met een mitochondrieel DNA-gecodeerde subunit.

206

Samenvatting

De huidige opvatting is dat het assemblage proces van mitochondrieel complex I via een aantal duidelijk onderscheidbare assemblage intermediairen verloopt door evolutionair geconserveerde functionele modules te combineren. Verstoringen van het proces, door bijvoorbeeld mutatie van subunits, kan leiden tot accumulatie en/of depletie van enkele assemblage intermediairen, waardoor zogenaamde assemblage profielen kunnen worden samengesteld. Hoofdstuk VI beschrijft hoe mutaties in verschillende complex I subunits de integriteit van complex I beïnvloeden en beschouwt de hieruit voortvloeiende assemblage profielen. Zes bestudeerde complex I-deficiënte patiënten met mutaties in kern-DNA- gecodeerde subunits (NDUFS2, NDUFS4, NDUFS7, NDUFS8) vertonen verminderde hoeveelheden volledig geassembleerd complex I en verlaagde in gel complex I activiteiten, die correleren met de lage, spectrofotometrisch gemeten complex I enzymactiviteiten. Deze resultaten veronderstellen dat verstoring van het assemblage proces of de stabiliteit van complex I, veroorzaakt door mutatie van genoemde subunits, het directe gevolg is van de verlaagde complex I activiteiten in deze patiënten. Echter, sommige patiënten met onbekende mutaties vertonen geen correlatie tussen complex I activiteit en de hoeveelheid volledig geassembleerd complex I, wat mogelijk duidt op een katalytisch defect. Mutaties die hiervoor verantwoordelijk zijn, kunnen gezocht worden in genen die betrokken zijn bij de activering van complex I of bij de synthese van één van zijn cofactoren (FMN, FeS clusters). In patiënten met mutaties in de kern-DNA-gecodeerde subunits vindt accumulatie van subunit NDUFS3-bevattende subcomplexen van laagmoleculair gewicht plaats in verschillende patronen. Deze subunit NDUFS3-bevattende subcomplexen passen goed binnen een nieuw model van complex I assemblage en belichamen waarschijnlijk ‗ware‘ assemblage intermediairen en niet instabiliteits- of afbraakproducten. In patiënten met mutaties in de NDUFS4 subunit accumuleert een additioneel subcomplex van ~830 kDa (~800 kDa in Hoofdstuk VI), dat geen in gel activiteit vertoont, in plaats van volledig geassembleerd complex I. Dit suggereert dat de NDUFS4 subunit in een laat stadium van het assemblage proces wordt geïncorporeerd in het complex. Voor patiënten met NDUFS2 en NDUFS4 mutaties is naast complex I deficiëntie een gematigde complex III deficiëntie waargenomen. In Hoofdstuk VII wordt een unieke casus van complex I deficiëntie behandeld. Een patiënt met een ogenschijnlijk mitochondriële encefalomyopathie draagt een homozygote drievoudige gendeletie op chromosoom 5, die de NDUFAF2, ERCC8 en ELOVL7 genen omvat. Deze genen coderen respectievelijk voor de humane complex I assemblage factor 2 (ook bekend onder de naam B17.2L), een eiwit van de nucleotide excision repair (NER) machinerie en een elongase van de zeer-lange-keten-vetzuursynthese. De patiëntfibroblasten vertonen een NER defect dat karakteristiek is voor Cockayne syndroom type A en dat ontegenzeggelijk kan worden toegeschreven aan de deletie van het ERCC8 gen. Als gevolg

207

Samenvatting van het lage expressie niveau van ELOVL7 in humane fibroblasten kunnen er geen specifieke veranderingen in de endogene vetzuurniveaus worden vastgesteld in de fibroblasten van de patiënt. Hierdoor kan er (nog) geen specifiek defect in de vetzuursynthese worden toegeschreven aan de deletie van het ELOVL7 gen. Met een baculovirale complementatietest met gebruik van wildtype NDUFAF2 is aangetoond dat de complex I deficiëntie wordt veroorzaakt door verlies van het NDUFAF2 gen. Gebrek aan het NDUFAF2 eiwit leidt tot verstoring van complex I assemblage, dat aangeduid wordt door een verlaagde hoeveelheid en activiteit van complex I en accumulatie van assemblage intermediairen. Bovendien laat onderzoek naar de celfysiologische consequenties zien dat intracellulaire superoxide productie en mitochondriële NAD(P)H niveaus verhoogd zijn en dat de mitochondriële membraan potentiaal verlaagd is. Alle waargenomen celfysiologische veranderingen worden hersteld na complementatie met wildtype NDUFAF2. Chaperonne NDUFAF2 associeert uitsluitend met het ~830-kDa subcomplex (~800 kDa in Hoofdstuk VI), dat specifiek accumuleert in patiënten met mutaties in de subunits die zich in de top van de perifere arm bevinden (NDUFS4, NDUFS6, NDUFV1) en dat correspondeert met een late assemblage intermediair van complex I. We kunnen veronderstellen dat NDUFAF2 associeert met de ~830 kDa-assemblage intermediair, vervolgens de incorporatie van de NADH-dehydrogenase module bevordert en het complex pas verlaat als de volledig geassembleerde vorm bereikt is. Ten slotte wordt de stapsgewijze de novo assemblage van complex I bediscussieerd in Hoofdstuk VIII. De vermeende rollen in het assemblage proces voor chaperonnes NDUFAF1 en NDUFAF2 worden geïntegreerd in een herzien model van de complex I assemblage route. Het alternatieve assemblage mechanisme van dynamische uitwisseling van subunits wordt kort behandeld, alsook de rol van een derde assemblage chaperonne, genaamd Ecsit, en de invloed van complex III op complex I assemblage. Om de pathologie van complex I deficiëntie te begrijpen is het van belang niet alleen de moleculaire basis in kaart te brengen, maar ook de fysiologische consequenties op cellulair niveau te onderzoeken. Mitochondriële parameters als membraan potentiaal, mitochondriële morfologie en superoxide productie worden gezien als graadmeter voor mitochondriële gesteldheid en metabole activiteit. Tot besluit, het ophelderen van de mechanismen die schuilgaan achter de opbouw en het functioneren van complex I, en het uitbreiden van onze kennis van de pathogenese van complex I deficiëntie, zal van uiterst belang zijn voor het uitvoeren van accurate diagnostiek, en uiteindelijk, voor de ontwikkeling van toekomstige therapeutische behandeling.

208

Dankwoord

Voor al die mensen familie, vrienden en collega‘s die zich regelmatig afvroegen, al dan niet hardop: ―…maar wanneer gaat hij dan eindelijk eens promoveren?‖ Het is nu zover! Proefschrift afgerond! Vele mensen hebben in meer of mindere mate een steentje bijgedragen aan de totstandkoming van dit proefschrift. Bij dezen wil ik hen allen hiervoor bedanken, te beginnen bij mijn promotor Jan Smeitink.

Jan, ten eerste bedankt voor het geduld dat je hebt gehad met het afronden van dit proefschrift! Maar bovenal wil ik je bedanken voor de kans die je mij hebt geboden onderzoek te kunnen doen in een omgeving waarin zowel onderzoek en diagnostiek, als lab en kliniek bij elkaar komen en elkaar ondersteunen en aanvullen waar mogelijk. Hierdoor wordt het onderzoek direct in een klinische context geplaatst, wat motiverend en inspirerend werkt. Ook ben ik je dankbaar voor het aanwenden van je buitenlandse contacten, waardoor het voor mij mogelijk was te kunnen werken op The Scripps Research Institute in La Jolla, Californië en aan de Universiteit van Oregon in Eugene, Oregon, en zo eens te ervaren hoe het eraan toe gaat in andere onderzoeksgroepen. Fijn was het ook dat ik mijn onderzoek kon voortzetten binnen het EUmitocombat project waar het belang van samenwerking, zowel binnen de muren van het UMC St Radboud als over de grens, voor de voortgang van het onderzoek eens temeer duidelijk werd.

Ten tweede wil ik mijn beide copromotoren Leo Nijtmans en Bert van den Heuvel bedanken. Leo, jouw komst naar Nijmegen was een impuls voor de hele groep, waardoor het complex I assemblage onderzoek flink uitbreidde en een nieuwe richting kreeg. Het is altijd prettig samenwerken met jou geweest. Bert, bedankt voor de sturende begeleiding in het begin van mijn project en je kritische kijk op en ondersteunende commentaar bij de totstandkoming van mijn artikelen.

Mijn beide paranimfen, Rutger en Cindy, wil ik bedanken voor het aantreden als paranimf en voor de prettige samenwerking de afgelopen jaren binnen het ‗complex I assemblage groepje‘. Ook tijdens de congressen was het erg gezellig met jullie. Rutger, jouw bijdrage is ook in dit boekje terug te vinden, bedankt daarvoor. Cindy, jij bent zeker goed op weg naar het voltooien van je eigen promotie. Ik wens je daar heel veel succes bij!

Mariël, Maria Antonietta, Melissa, Sharita, Hans en Murtada wil ik bedanken voor de prettige samenwerking binnen het ‗complex I assemblage groepje‘ en binnen het

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Dankwoord

EUmitocombat project. Cristina, muchas gracias voor jouw bijdrage die ook terug te vinden is in dit boekje en voor de leuke, altijd door Spaanse gerechten en tapas gekenmerkte feestjes bij jou thuis. Richard H., bedankt voor je hulp tijdens mijn soms erg breed opgezette blue native-experimenten en voor je ‗zaag- en timmeractiviteiten‘ tijdens de vele verhuizingen van werkplek.

Van de ‗DNA-groep‘ wil ik Roel, Edwin en Heleen bedanken voor het inspringen bij het ‗sequencen‘, waarin met name Roel een belangrijk deel voor zijn rekening heeft genomen. De ‗weefselkweek‘ en ‗spiergroep‘ wil ik bedanken voor hun ondersteuning van mijn onderzoek door het aanleveren van de patiëntencellen en het uitvoeren van enzymactiviteitsmetingen. De (oud-)collega‘s van de afdeling Biochemie, Felix, Herman, Tessa, Werner, Peter, Sjoerd en Henk-Jan, bedankt voor de prettige samenwerking en jullie bijdrage aan mijn onderzoek.

Furthermore, I would like to thank Prof. Takao Yagi, his wife Dr. Akemi Matsuno-Yagi and Dr. Byoung Boo Seo, as well as Prof. Rod Capaldi, Devin, James, and Rodrigue, for giving me a warm welcome into their research groups during my stays in La Jolla, California and Eugene, Oregon, respectively. You turned my stays abroad, at the lab as well as outside the lab, into a pleasant experience. Thanks! I‘d also like to thank Prof. Immo Scheffler for his knowledgeable collaboration during his sabbatical stay at our lab.

De vele collega‘s en oud-collega‘s (en dat zijn er door de jaren heen heel wat geweest) van het lab LKN al was het dan Kindergeneeskunde en Neurologie, oudbouw en nieuwbouw, of P en Q wil ik bedanken voor de prettige samenwerking op het lab en op de schrijfplekken van de verschillende kantoorruimten c.q. –tuinen, die ik de afgelopen jaren met m.n. Sandra, Henkjan en Ivon gedeeld heb. Maar vooral ook iedereen bedankt voor de vele leuke etentjes, dagjes-uit en andere activiteiten die we met het lab gedaan hebben! Joyce, bedankt voor de relativerende gesprekken aan het einde van de werkdag tijdens onze fietstochten terug naar ‗Nijmegen-Noord‘. Als ik ook maar iets van jouw niet-aflatende positieve instelling heb weten over te nemen is dat al pure winst. En voor alle medepromovendi die nog volop in het promotietraject zitten: heel veel succes daarbij!

Als laatste wil ik mijn ouders en zussen bedanken voor alle niet-werkgerelateerde steun en voor het weten wanneer juist niet te vragen naar de voortgang van mijn proefschrift of naar de datum van mijn promotie.

Tot slot, zonder de bereidwilligheid en medewerking van patiëntjes en hun ouders zou onderzoek doen een stuk moeilijker zijn. Bedankt!

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Curriculum Vitae

Rolf Janssen wordt geboren op 20 maart 1976 te Nijmegen. Tijdens zijn middelbareschoolperiode doorloopt hij het VWO op het Elshofcollege in Nijmegen, dat hij in mei 1994 afrondt met het behalen van het diploma. In september 1994 begint hij de opleiding scheikunde aan de Katholieke Universiteit Nijmegen (nu Radboud Universiteit Nijmegen). Tijdens deze opleiding volgt hij een tweetal stages, te beginnen met de stage klinische chemie, van september 1997 tot mei 1998, op de afdeling Kindergeneeskunde en Neurologie van het UMC St Radboud o.l.v. prof. dr. R. Wevers, waar hij onderzoek doet naar de genetische defecten van de ziekte van Brody en tyrosine hydroxylase deficiëntie. Van september 1998 tot augustus 1999 volgt de stage biochemie op de afdeling Biochemie van de Faculteit der Natuurwetenschappen, Wiskunde en Informatica o.l.v. prof. dr. W. van Venrooij, met als onderwerp de eiwitstructuur van de humane RNase MRP endoribonuclease. Na het behalen van het doctoraal diploma in maart 2000, begint hij in april 2000 aan het promotieonderzoek ―NADH:ubiquinone oxidoreductase: molecular defects and remedy‖, als onderdeel van het Nijmegen Centrum voor Mitochondriële Ziekten, op de afdeling Laboratorium Kindergeneeskunde en Neurologie o.l.v. prof. dr. J. Smeitink en dr. L. van den Heuvel en later dr. L. Nijtmans, wat resulteert in het vervaardigen van dit proefschrift. Een gedeelte van zijn promotieonderzoek voert hij uit tijdens twee werkbezoeken aan de laboratoria van prof. dr. T. Yagi aan The Scripps Research Institute in La Jolla, Californië (januari – maart 2001) en prof. dr. R. Capaldi aan de Universiteit van Oregon in Eugene, Oregon (juli 2002), in de Verenigde Staten van Amerika. Van juli 2004 tot juli 2008 zet hij als postdoc in dezelfde onderzoeksgroep het onderzoek voort onder de titel ―Assembly of complex I in health and disease‖ dat onderdeel vormt van het door de Europese Unie gefinancierde EUmitocombat project, een samenwerkingsverband van 21 Europese onderzoeksgroepen.

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List of publications

Janssen RJRJ, Distelmaier F, Smeets RJP, Wijnhoven T, Østergaard E, Jaspers NGJ, Raams A, Kemp S, Rodenburg RJT, Willems PHGM, van den Heuvel LPWJ, Smeitink JAM, and Nijtmans LGJ (2008) Contiguous gene deletion of ELOVL7, ERCC8, and NDUFAF2 in a patient with progressive encephalomyopathy. Submitted

Hoefs SJ, Dieteren CEJ, Distelmaier F, Janssen RJRJ, Epplen A, Swarts HG, Forkink M, Rodenburg RJT, Nijtmans LGJ, Willems PHGM, Smeitink JAM, and van den Heuvel LPWJ (2008) NDUFA2 complex I mutation leads to Leigh disease. Am J Hum Genet 2008 Jun; 82(6): 1306-1351

Vogel RO, Janssen RJRJ, van den Brand MAM, Dieteren CEJ, Verkaart S, Koopman WJH, Willems PHGM, Pluk W, van den Heuvel LPWJ, Smeitink JAM, and Nijtmans LGJ (2007) Cytosolic signaling protein Ecsit also localizes to mitochondria where it interacts with chaperone NDUFAF1 and functions in complex I assembly. Genes Dev 2007 Mar; 21(5): 615- 624

Janssen RJRJ, Nijtmans LGJ, van den Heuvel LPWJ, and Smeitink JAM (2006) Mitochondrial complex I: structure, function and pathology. J Inherit Metab Dis 2006 Aug; 29(4): 499-515 Review

Vogel RO*, Janssen RJRJ*, Ugalde C, Grovenstein M, Huijbens RJ, Visch HJ, van den Heuvel LPWJ, Willems PHGM, Smeitink JAM, and Nijtmans LGJ (2005) Human mitochondrial complex I assembly is mediated by NDUFAF1. FEBS J 2005 Oct; 272(20): 5317-5326

Janssen RJRJ, van den Heuvel LPWJ, and Smeitink JAM (2004) Genetic defects in the oxidative phosphorylation (OXPHOS) system. Expert Rev Mol Diagn 2004 Mar; 4(2): 143- 156 Review

Ugalde C*, Janssen RJRJ*, van den Heuvel LPWJ, Smeitink JAM, and Nijtmans LGJ (2004) Differences in assembly or stability of complex I and other mitochondrial OXPHOS complexes in inherited complex I deficiency. Hum Mol Genet 2004 Mar; 13(6): 659-667

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Janssen RJRJ, Smeitink JAM, Smeets RJP, and van den Heuvel LPWJ (2002) CIA30 complex I assembly factor: a candidate for human complex I deficiency? Hum Genet 2002 Mar; 110(3): 264-270

Van Eenennaam H, van der Heijden A, Janssen RJRJ, van Venrooij WJ, and Pruijn GJM (2001) Basic domains target protein subunits of the RNase MRP complex to the nucleolus independently of complex association. Mol Biol Cell 2001 Nov; 12(11): 3680-3689

Janssen RJRJ, Wevers RA, Häussler M, Luyten JA, Steenbergen-Spanjers GC, Hoffmann GF, Nagatsu T, and van den Heuvel LPWJ (2000) A branch site mutation leading to aberrant splicing of the human tyrosine hydroxylase gene in a child with a severe extrapyramidal movement disorder. Ann Hum Genet 2000 Sep; 64(Pt 5): 375-382

* Joint first authorship

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