Mitochondrial disorders in children: toward development of small‐molecule treatment strategies
2016; Springer Nature; Volume: 8; Issue: 4 Linguagem: Inglês
10.15252/emmm.201506131
ISSN1757-4684
AutoresWerner J.H. Koopman, Julien Beyrath, Cheuk‐Wing Fung, Saskia Koene, Richard J. Rodenburg, Peter H.G.M. Willems, Jan Smeitink,
Tópico(s)ATP Synthase and ATPases Research
ResumoReview8 March 2016Open Access Mitochondrial disorders in children: toward development of small-molecule treatment strategies Werner JH Koopman Werner JH Koopman Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands Centre for Systems Biology and Bioenergetics, Radboud University Medical Center, Nijmegen, The Netherlands Search for more papers by this author Julien Beyrath Julien Beyrath Khondrion BV, Nijmegen, The Netherlands Search for more papers by this author Cheuk-Wing Fung Cheuk-Wing Fung Department of Pediatrics, Radboud Center for Mitochondrial Medicine, Radboud University Medical Center, Nijmegen, The Netherlands Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, Queen Mary Hospital, University of Hong Kong, Hong Kong Search for more papers by this author Saskia Koene Saskia Koene Department of Pediatrics, Radboud Center for Mitochondrial Medicine, Radboud University Medical Center, Nijmegen, The Netherlands Search for more papers by this author Richard J Rodenburg Richard J Rodenburg Department of Pediatrics, Radboud Center for Mitochondrial Medicine, Radboud University Medical Center, Nijmegen, The Netherlands Search for more papers by this author Peter HGM Willems Peter HGM Willems Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands Centre for Systems Biology and Bioenergetics, Radboud University Medical Center, Nijmegen, The Netherlands Search for more papers by this author Jan AM Smeitink Corresponding Author Jan AM Smeitink Centre for Systems Biology and Bioenergetics, Radboud University Medical Center, Nijmegen, The Netherlands Khondrion BV, Nijmegen, The Netherlands Department of Pediatrics, Radboud Center for Mitochondrial Medicine, Radboud University Medical Center, Nijmegen, The Netherlands Search for more papers by this author Werner JH Koopman Werner JH Koopman Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands Centre for Systems Biology and Bioenergetics, Radboud University Medical Center, Nijmegen, The Netherlands Search for more papers by this author Julien Beyrath Julien Beyrath Khondrion BV, Nijmegen, The Netherlands Search for more papers by this author Cheuk-Wing Fung Cheuk-Wing Fung Department of Pediatrics, Radboud Center for Mitochondrial Medicine, Radboud University Medical Center, Nijmegen, The Netherlands Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, Queen Mary Hospital, University of Hong Kong, Hong Kong Search for more papers by this author Saskia Koene Saskia Koene Department of Pediatrics, Radboud Center for Mitochondrial Medicine, Radboud University Medical Center, Nijmegen, The Netherlands Search for more papers by this author Richard J Rodenburg Richard J Rodenburg Department of Pediatrics, Radboud Center for Mitochondrial Medicine, Radboud University Medical Center, Nijmegen, The Netherlands Search for more papers by this author Peter HGM Willems Peter HGM Willems Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands Centre for Systems Biology and Bioenergetics, Radboud University Medical Center, Nijmegen, The Netherlands Search for more papers by this author Jan AM Smeitink Corresponding Author Jan AM Smeitink Centre for Systems Biology and Bioenergetics, Radboud University Medical Center, Nijmegen, The Netherlands Khondrion BV, Nijmegen, The Netherlands Department of Pediatrics, Radboud Center for Mitochondrial Medicine, Radboud University Medical Center, Nijmegen, The Netherlands Search for more papers by this author Author Information Werner JH Koopman1,2, Julien Beyrath3, Cheuk-Wing Fung4,5, Saskia Koene4, Richard J Rodenburg4, Peter HGM Willems1,2 and Jan AM Smeitink 2,3,4 1Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands 2Centre for Systems Biology and Bioenergetics, Radboud University Medical Center, Nijmegen, The Netherlands 3Khondrion BV, Nijmegen, The Netherlands 4Department of Pediatrics, Radboud Center for Mitochondrial Medicine, Radboud University Medical Center, Nijmegen, The Netherlands 5Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, Queen Mary Hospital, University of Hong Kong, Hong Kong *Corresponding author. Tel: +31 24 3614430; E-mail: [email protected] EMBO Mol Med (2016)8:311-327https://doi.org/10.15252/emmm.201506131 See the Glossary for abbreviations used in this article. See also: A Suomalainen (October 2015) PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract This review presents our current understanding of the pathophysiology and potential treatment strategies with respect to mitochondrial disease in children. We focus on pathologies due to mutations in nuclear DNA-encoded structural and assembly factors of the mitochondrial oxidative phosphorylation (OXPHOS) system, with a particular emphasis on isolated mitochondrial complex I deficiency. Following a brief introduction into mitochondrial disease and OXPHOS function, an overview is provided of the diagnostic process in children with mitochondrial disorders. This includes the impact of whole-exome sequencing and relevance of cellular complementation studies. Next, we briefly present how OXPHOS mutations can affect cellular parameters, primarily based on studies in patient-derived fibroblasts, and how this information can be used for the rational design of small-molecule treatment strategies. Finally, we discuss clinical trial design and provide an overview of small molecules that are currently being developed for treatment of mitochondrial disease. Glossary Blue Native-PAGE A polyacrylamide gel-based electrophoresis technique that allows separation of protein complexes in their native state. It is often used for diagnostic and research purposes regarding the mitochondrial oxidative phosphorylation system. Complementation study A test to study and confirm the pathogenicity of a mutation based on a phenotypic screen. Often such a study is carried out by introducing the wild-type gene in a patient (-derived) cell line and assessing the reversal of the biological consequences of the mutated gene. The latter could involve normalization of a depolarized mitochondrial membrane potential, reversal of aberrant mitochondrial structure, or restoration of an enzymatic deficiency. Clinical trial A crucial part of the drug development process consisting of 4 phases. During a clinical trial (phase 1 to 3), the safety, pharmacokinetics, pharmacodynamics, and effectiveness of a compound are assessed in healthy volunteers and patient cohorts. Phase 4 consists of post-market surveillance. Leigh syndrome A severe pediatric syndrome, first described by Dennis Leigh in 1951, which is caused by either mutations in mitochondrial or nuclear DNA. LHON Leber's hereditary optic neuropathy. A disorder that causes maternally inherited blindness. LHON is frequently caused by mutations in subunits of mitochondrial complex I, encoded by the mitochondrial DNA. MELAS Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes. A classical mitochondrial disorder primarily caused by the m.3243A>G mutation in the mitochondrial DNA. MRI Magnetic resonance imaging, a non-invasive technique that uses magnetic fields and radio waves to visualize different parts of the human body. It is often applied during the diagnostic phase when a mitochondrial disorder is suspected for analysis of anatomical and physiological changes. Outcome measures Measures to the study the outcome of an intervention strategy such as the effect of small-molecule treatment during a clinical trial. Whole-exome sequencing A technique that allows sequencing of all protein-coding genes within the genome. The latter is known as the exome and constitutes only a small part (∼1%) of the total genome. Introduction Mitochondria are semi-autonomous organelles that are present in the cytosol of virtually all cells. They consist of a double-membrane system that envelops the mitochondrial matrix compartment. Functionally, mitochondria are key players in cellular ATP production, fatty acid oxidation, heme biosynthesis, apoptosis induction, heat generation, and calcium homeostasis. Mitochondrial diseases are an expanding group of disorders of which the first signs and symptoms can become apparent from prenatal development to late adulthood (Koopman et al, 2012, 2013; Vafai & Mootha, 2012; Chinnery, 2015; Lightowlers et al, 2015). These disorders can be defined as “primary” (i.e., arising from a mutation in one of the genes encoding a mitochondria-localized protein) or “secondary” (i.e., arising from an external influence on mitochondria). The latter include off-target effects of cholesterol-lowering statin drugs (e.g., Schirris et al, 2015) and defects in the normal breakdown of branched-chain amino acids as in propionic acidurias, which cause severe combined respiratory chain deficiencies (e.g., Schwab et al, 2006). Here, we primarily focus on mitochondrial diseases that (i) clinically manifest themselves before the age of 18 years and (ii) arise from mutations in nuclear DNA (nDNA)-encoded structural proteins or assembly factors of the mitochondrial oxidative phosphorylation (OXPHOS) system. The mitochondrial OXPHOS system is embedded in the mitochondrial inner membrane (MIM) and represents the final step in the conversion of nutrients to energy by catalyzing the generation of ATP (Fig 1A). This process is carried out by the combined action of the mitochondrial electron transport chain (ETC) complexes (Fig 1B) and the ATP-producing FoF1-ATPase (complex V; Fig 1C). The ETC consists of four multiprotein complexes (complex I to complex IV). Complex I and complex II abstract electrons from reduced nicotinamide adenine dinucleotide (NADH) and reduced flavin adenine dinucleotide (FADH2), respectively. Subsequently, these electrons are donated to the electron carrier coenzyme Q10, which transports them to complex III. From thereon, electrons are transferred to the electron carrier cytochrome c and transported to complex IV. At the latter complex, the electrons are donated to molecular oxygen (O2) to form water. The energy released by the electron transport is used to drive trans-MIM proton (H+) efflux from the mitochondrial matrix by complexes I, III, and IV, thereby creating an inward-directed proton-motive force (PMF). The latter consists of a chemical (ΔpH) and an electrical component (Δψ; Fig 1C), and is used by complex V to generate ATP by chemiosmotic coupling (Mitchell, 1961). In addition, ΔpH and/or Δψ are essential in sustaining virtually all other mitochondrial functions like the import of pre-proteins from the cytosol and ion/metabolite exchange (Fig 1C). In normal healthy cells (Fig 1A; red), cellular ATP is predominantly generated through subsequent metabolic reactions of the glycolysis pathway (cytosol), the pyruvate dehydrogenase complex (PDHC, mitochondrial matrix), the tricarboxylic acid (TCA) cycle (mitochondrial matrix), and the OXPHOS system (MIM). This ATP is used to fuel energy-consuming cellular processes. Figure 1. Glycolytic and mitochondrial ATP production, the electron transport chain, and oxidative phosphorylation(A) Glucose (Glc) and glutamine (Gln) enter the cell via dedicated transporters. In the cytosol, Glc is converted in the glycolysis pathway into pyruvate (Pyr), which is transported to the mitochondrial matrix by the mitochondrial Pyr carrier (MPC). Alternatively, Pyr can be converted into lactate (Lac) by the action of lactate dehydrogenase (LDH). Inside the mitochondrial matrix, Pyr is converted in to acetyl coenzyme A (acetyl-CoA; not shown) by pyruvate dehydrogenase (PDH) to enter the tricarboxylic acid (TCA) cycle. The latter supplies the oxidative phosphorylation system (OXPHOS) with substrates in the form of reduced nicotinamide adenine dinucleotide (NADH) and reduced flavin adenine dinucleotide (FADH2). In addition, Gln can enter the mitochondrial matrix where it is converted by glutaminase into glutamate (not shown), a TCA cycle substrate. Also fatty acids can enter the mitochondrial matrix and enter the TCA cycle following their conversion into acetyl-CoA (not shown). In healthy cells, the conversion of Glc into Pyr and its further metabolic conversion by the TCA and OXPHOS system constitute the major pathway for ATP generation (marked in red). (B) The mitochondrial electron transport chain (ETC) consists of 4 multisubunit protein complexes (complex I to IV) that are embedded in the mitochondrial inner membrane (MIM). Electrons are donated by NADH (at complex I) and FADH2 (at complex II) to coenzyme Q10 (Q), which transports them to complex III. From thereon, electrons are transported to complex IV by cytochrome c (c) where they are donated to molecular oxygen (O2). Although not discussed here, in addition to the ETC complexes also other proteins can provide coenzyme Q10 and cytochrome c with electrons (red boxes) in a tissue-dependent manner. During electron transport, energy is liberated and used expel protons (H+) from the mitochondrial matrix intro the inter-membrane space (IMS) between the MIM and mitochondrial outer membrane (MOM). As a consequence, the mitochondrial matrix displays an increased pH and the MIM has a highly negative-inside membrane potential (Δψ). (C) Together, the pH (ΔpH) and potential difference (Δψ) across the MIM determine the magnitude of the proton-motive force (PMF), which is used by the FoF1-ATPase (complex V) to drive mitochondrial ATP production from inorganic phosphate (Pi) and ADP. In addition to ATP generation, virtually all other mitochondrial processes including ion exchange and pre-protein import require a proper ΔpH and/or Δψ. The magnitude of the PMF not only depends on the combined action of the ETC and complex V (i.e., the oxidative phosphorylation system; OXPHOS) but also is affected by other electrogenic systems. These include uncoupling proteins (UCPs), the Pi transporter (PiT), and the adenine nucleotide translocator (ANT). This figure was compiled based on (Koopman et al, 2010; Valsecchi et al, 2010; Liemburg-Apers et al, 2011; Koopman et al, 2012, 2013 and Liemburg-Apers et al, 2015). Download figure Download PowerPoint Diagnosing OXPHOS disease in children The five complexes of the mitochondrial OXPHOS system are assembled from 92 distinct proteins, requiring the assistance of at least 37 nDNA-encoded assembly factors (Nouws et al, 2012; Koopman et al, 2013). Each OXPHOS complex except complex II is of bi-genomic origin and contains subunits encoded by mitochondrial DNA (mtDNA) and nDNA (Koopman et al, 2013). Complex I is built up from 44 subunits (7 mtDNA- and 37 nDNA-encoded), complex II of 4 subunits, complex III of 11 subunits (1 mtDNA, 10 nDNA), complex IV of 14 subunits (3 mtDNA, 11 nDNA), and complex V of 19 subunits (2 mtDNA, 17 nDNA). As mentioned in the introduction, we here primarily focus on mitochondrial diseases arising from mutations in nDNA-encoded structural OXPHOS subunits and assembly factors (Fig 2). Since the first description by Luft of a patient with exercise intolerance and hyperthermia (Luft et al, 1962), the clinical spectrum of diseases associated with OXPHOS malfunction has tremendously broadened. In our opinion, a mitochondrial disease should be considered in any patient presenting with an unexplained mono- or multisystemic disease, whether progressive or not. Applying this relatively broad definition has important consequences for the diagnostic approach in children, which is truly challenging given the general lack of awareness within the broader medical community, the relatively low incidence of mitochondrial disease, and the complex genotype–phenotype relationship typical of these inborn errors (Smeitink, 2003). It is also important to increase awareness for mitochondrial disorders to shorten the potentially long delays between appearance of the first symptoms and diagnostic analysis and confirmation of the defect. Regarding the various clinical presentations of mitochondrial OXPHOS disease, detailed reviews have been presented elsewhere (Kisler et al, 2010; Koene & Smeitink, 2011; Lake et al, 2016). The majority of children suffering from mitochondrial OXPHOS disease lack a specific syndromic appearance and some do not display increased blood lactate levels (Triepels et al, 1999). In case of neurodegenerative diseases, mutations in OXPHOS subunits (mtDNA- or nDNA-encoded) and assembly factors have, for instance, been associated with (Koopman et al, 2013): Leigh (-like) syndrome, leukoencephalopathy, MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) syndrome, NARP (neuropathy, ataxia, and retinitis pigmentosa), Parkinsonism/MELAS, and (susceptibility) modification of PD (Parkinson disease), and AD (Alzheimer's disease). Figure 2. Currently identified mutations in nDNA-encoded OXPHOS subunits and assembly factors causing mitochondrial disease in childrenOXPHOS structural subunits in which a pathological mutation was reported are depicted in red for each complex. Assembly factors are indicated (in blue). The data in this figure were compiled from (Koopman et al, 2012, 2013; Mayr et al, 2015; Ostergaard et al, 2015; van Rahden et al, 2015). Download figure Download PowerPoint The diagnostic process When clinical signs and symptoms suggest a mitochondrial disorder, the following general strategy is carried out: (i) a detailed analysis of the medical/family history to discriminate between maternal (mtDNA) and Mendelian (nDNA) inheritance, (ii) evaluation of mitochondrial biomarkers like lactate, pyruvate, alanine, fibroblast growth factor 21 (FGF21; Suomalainen et al, 2011) and growth and differentiation factor 15 in blood (GDF15; Yatsuga et al, 2015) or the excretion of TCA cycle intermediates in urine, and (iii) detailed investigations of affected organs and tissues by medical specialists like neurologists, ophthalmologists, cardiologists, and/or by imaging techniques such as echocardiography or magnetic resonance imaging (MRI) of brain, heart, and skeletal muscle, and by neurophysiological studies (such as obtaining an electroencephalogram; EEG). The decision to perform a skeletal muscle biopsy for histopathological, immunohistochemical, and/or enzymological analysis is taken based on the “Bernier” or “Wolf/Smeitink” mitochondrial disease criteria (MDC; Bernier et al, 2002; Wolf & Smeitink, 2002), which might include biochemical (e.g., ATP production rate) and general criteria (e.g., clinical presentation) to establish the probability of a mitochondrial disease. Biochemical analysis usually includes spectrophotometric activity analysis of individual OXPHOS complexes. Blue Native-PAGE, which can be used to assess the relative amount of fully assembled OXPHOS complexes, can also be a useful tool (e.g., van Rahden et al, 2015). In addition to muscle tissue, enzymes are often examined in cultured skin fibroblasts. In combination with a muscle biopsy, both positive and negative fibroblast results are informative with respect to predicting the underlying genetic defect. Genetic analysis can be carried out either by traditional candidate gene sequencing, or by more comprehensive approaches including direct (whole) exome sequencing (in which all coding regions in the genome are sequenced) or whole-genome sequencing, often followed by functional complementation analysis in patient-derived cells. The impact of whole-exome sequencing The implementation of whole-exome sequencing as a routine molecular diagnostic test for mitochondrial disorders has resulted in the identification of numerous variants in many different genes (DaRe et al, 2013; Lieber et al, 2013; Ohtake et al, 2014; Taylor et al, 2014; Wortmann et al, 2015). Genetic variants include known pathogenic mutations, the clinical/biochemical phenotype of which matches those described in the literature and public databases. In this case, the diagnosis is clear and no further functional tests are necessary. When an unknown gene variant is encountered, there are several possibilities: (i) the variant occurs in a gene that is associated with a known disease and interferes with the function of the gene product; again, this means that no additional functional tests are necessary, (ii) the variant results in an amino acid change at the protein level or possibly interferes with the expression of a disease-associated gene and/or the splicing of its mRNA; this means that the consequences of the variant are not unambiguously predictable and a functional test can help to establish the diagnosis, (iii) the variant occurs in a gene of unknown function or a gene not previously associated with disease; this means that the diagnosis requires a functional confirmation of the defect. In general, mitochondrial disorders display a poor correlation between genotype and phenotype. This further emphasizes the importance of functional validation of genetic variants (Daud et al, 2015). For genes known to be mutated in OXPHOS disease, the possibility to perform relatively low-cost diagnostic exome sequencing using DNA extracted from blood samples has certainly sped up and facilitated the diagnostic process (Wortmann et al, 2015). However, it is important to realize that when a negative result is obtained, this does not conclusively rule out the presence of a mitochondrial disease. This is caused by the fact that: (i) the exome sequencing technique displays non-optimal coverage meaning that mutations in non-covered parts of the exome cannot be excluded, (ii) even a whole exome covers only approximately 1% of the whole genome, (iii) the pathophysiological effects of many polymorphisms are unknown, and (iv) the number of mutated genes demonstrated to be involved in mitochondrial disease is continuously increasing. As an example of the latter, we demonstrated that a patient with intellectual disability and multisystemic problems displayed an isolated enzymatic complex IV deficiency caused by a pathological mutation in the centrosomal protein of 89 kDa (CEP89), previously not associated with mitochondrial dysfunction (van Bon et al, 2013). As a consequence, it becomes increasingly difficult to classify mitochondrial diseases in children based upon their phenotypic presentation. Therefore, the number of patients that will be correctly diagnosed with a mitochondrial (OXPHOS) disease will be underestimated. A possible solution to this problem could be that patients for whom an obvious explanation for their symptomatology cannot be established are referred to specialized (inter)national centers of excellence (e.g., the Radboud Center for Mitochondrial Medicine in the Netherlands), which can decide on an appropriate cost- and time-effective diagnostic strategy. The relevance of complementation studies Genetic complementation studies are carried out by introducing the wild-type DNA of the mutated gene into patient-derived cells (usually skin fibroblasts). DNA transduction by lentiviral transmission is an efficient way to achieve genetic complementation (e.g., Jonckheere et al, 2011). Although it is relatively time-consuming, this procedure yields a population of stably complemented cells. Transient transfections can also be used, for instance, using an insect virus (baculovirus) system (e.g., Kirby et al, 2004; Hoefs et al, 2008). In this approach, the protein to-be-overexpressed can be tagged with a fluorescent protein (e.g., Hoefs et al, 2008), or be part of an IRES (internal ribosome entry site)-containing construct (e.g., Bouabe et al, 2008). Using the IRES strategy allows simultaneous expression of the protein and a fluorescent marker protein without them being physically attached to each other. Although with a transient transfection protocol, not all cells are necessarily transfected, it can be advantageous when a microscopic readout is used since it allows side-by-side comparison of transfected (complemented) and non-transfected (non-complemented) patient cells. Depending on the nature of the mutation, functional confirmation of pathogenicity can obtained using a relatively simple readout like enzymatic activity (Haack et al, 2012; Ngu et al, 2012; Jonckheere et al, 2013; Szklarczyk et al, 2013) or protein expression levels (assessed by Western blotting; Hoefs et al, 2008). More advanced strategies include comparative analysis of control, patient, and complemented patient primary fibroblasts with respect to mitochondrial morphology (Koopman et al, 2005; Jonckheere et al, 2011) and Δψ (Hoefs et al, 2008). Unfortunately, functional studies of the genetic defect are often lacking in published studies, especially when a large number of patients have been analyzed. Although large-scale complementation and other functional studies may be practically challenging, they should be carried out to prevent detected variants prematurely or erroneously ending up in pathogenic mutation databases. For example, a recent study described two novel mutations in SCO2 (a complex IV assembly gene) in patients with early-onset myopia (Jiang et al, 2014). Although the authors did state that additional studies are needed to prove the pathogenicity of these (and other) mutations, the latter are nevertheless listed in the Human Gene Mutation Database (HGMD) as “disease-causing” ( www.hgmd.cf.ac.uk). This is not to criticize mutation databases as these are extremely valuable to clinical practice, but to illustrate that clinicians and laboratory specialists should remain critical about the information provided by these databases. Similarly, in publications dealing with genetic variants, incorrect or incomplete information can have important clinical implications for individual patients. Cellular pathophysiology of OXPHOS dysfunction During the last 15 years, we have strongly advocated the use of living cells (in addition to cell homogenates, isolated mitochondria, and fixed tissue samples), to study the pathophysiology of mitochondrial dysfunction in general and OXPHOS mutations in particular (e.g., Smeitink et al, 2004; Koopman et al, 2010, 2012, 2013; Willems et al, 2015). Live-cell analysis is crucial since mitochondrial and cellular functions are tightly interconnected. This is illustrated by the following observations: (i) mitochondrial morphology and function are altered by isolation procedures, (ii) the cellular environment is required to provide substrates to mitochondria and the OXPHOS system, (iii) mitochondria exchange ions and metabolites (e.g., ADP, Pi and ATP) with the cytosol, and (iv) mitochondrial and cellular functioning are linked and controlled by (retrograde) signaling pathways (e.g., Palmieri, 2008; Koopman et al, 2010; Picard et al, 2011; Szabo & Zoratti, 2014). In principle, a mutation in an OXPHOS protein-encoding gene can reduce the catalytic activity of the OXPHOS complex, its protein levels, or both. In case of isolated complex I deficiency, the reduction in the levels of fully assembled complex I in primary patient fibroblasts was linked to a proportional decrease in complex I residual activity (e.g., Valsecchi et al, 2010). This suggests that mutations in nDNA-encoded complex I subunits reduce the biosynthesis of fully active complex I and/or induce complex I destabilization (i.e., an “expression defect”). Only in a single case (i.e., a patient carrying an Asp446Asn mutation in the NDUFS2 gene of complex I), a reduced complex I activity was not paralleled by a lower level of fully assembled complex I (i.e., a “catalytic defect”; Ngu et al, 2012). Quantitative live-cell microscopy of primary fibroblasts from 14 control subjects and 24 children with isolated complex I deficiency revealed that patient cells displayed a less negative Δψ (i.e., “depolarization”), increased mitochondrial NADH levels, elevated reactive oxygen (ROS) levels, aberrations in mitochondrial morphology, and disturbed cytosolic and mitochondrial Ca2+ and ATP handling (Koopman et al, 2005, 2007, 2008; Verkaart et al, 2007a; Golubitzky et al, 2011; Valsecchi et al, 2010; Roestenberg et al, 2012; Leman et al, 2015). Interestingly, several of the above aberrations were also observed in fibroblasts carrying mutations in other OXPHOS complexes and assembly factors (e.g., Szklarczyk et al, 2013), in mouse cells with genetic complex I deficiency (e.g., Kruse et al, 2008; Valsecchi et al, 2012, 2013), in cells treated with OXPHOS inhibitors (e.g., Forkink et al, 2014, 2015; Distelmaier et al, 2015; Schöckel et al, 2015), and in cells with mutations in non-OXPHOS mitochondrial proteins (e.g., Mortiboys et al, 2008; Heeman et al, 2011; Hoffmann et al, 2012). This suggests that, depending on the cellular context and experimental conditions, the observed aberrations in primary skin fibroblasts of patients with inherited complex I deficiency reflect the “general” consequences typical of mitochondrial dysfunction. Correlation analysis suggested that NADH and ROS levels increased as a direct consequence of the reduced expression of fully active complex I (Verkaart et al, 2007a,b). Moreover, patient fibroblasts with a moderate reduction in complex I activity displayed a much smaller increase in ROS levels than fibroblasts with a large reduction in complex I activity (Koopman et al, 2007). In addition, mitochondrial mor
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