Mitochondrion 12 (2012) 57–65 Contents lists available at ScienceDirect Mitochondrion j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m i t o Review Pharmacological targeting of mitochondrial complex I deficiency: The cellular level and beyond Peggy Roestenberg a, b, 1, Ganesh R. Manjeri a, b, 1, Federica Valsecchi a, b, Jan A.M. Smeitink b, Peter H.G.M. Willems a, Werner J.H. Koopman a,⁎ a b Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands Department of Pediatrics, Nijmegen Centre of Mitochondrial Disorders, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands a r t i c l e i n f o Article history: Received 1 November 2010 Received in revised form 20 January 2011 Accepted 25 June 2011 Available online 2 July 2011 Keywords: Fibroblast Calcium CGP37165 ROS Trolox mitoQ a b s t r a c t Complex I (CI) represents a major entry point of electrons in the mitochondrial electron transport chain (ETC). It consists of 45 different subunits, encoded by the mitochondrial (mtDNA) and nuclear DNA (nDNA). In humans, mutations in nDNA-encoded subunits cause severe neurodegenerative disorders like Leigh Syndrome with onset in early childhood. The pathophysiological mechanism of these disorders is still poorly understood. Here we summarize the current knowledge concerning the consequences of nDNA-encoded CI mutations in patient-derived cells, present mouse models for human CI deficiency, and discuss potential treatment strategies for CI deficiency. © 2011 Elsevier B.V. and Mitochondria Research Society. All rights reserved. Contents 1. 2. 3. 4. 5. Mitochondrial complex I . . . . . . . . . . . . . . . . . . . . . . . . . . . Isolated complex I deficiency in humans. . . . . . . . . . . . . . . . . . . . Cellular consequences of isolated complex I deficiency in patient skin fibroblasts 3.1. Complex I biogenesis and degradation . . . . . . . . . . . . . . . . . 3.2. Functional impairment of mitochondria and adaptation in patient cells . . . 3.3. Other key cellular features of the patient cell phenotype. . . . . . . . . Mouse models of isolated complex I deficiency . . . . . . . . . . . . . . . . 4.1. The WB NDUFS4 −/− mouse . . . . . . . . . . . . . . . . . . . . . . 4.2. The Nes NDUFS4 −/− and PC NDUFS4 −/− mouse . . . . . . . . . . . . . 4.3. The CWB NDUFS4 −/− mouse . . . . . . . . . . . . . . . . . . . . . . 4.4. The NDUFS4-PM mouse . . . . . . . . . . . . . . . . . . . . . . . . Pharmacological targeting of complex I deficiency . . . . . . . . . . . . . . . 5.1. The antioxidant Trolox. . . . . . . . . . . . . . . . . . . . . . . . . 5.2. The antioxidant vitamin E . . . . . . . . . . . . . . . . . . . . . . . 5.3. The antioxidant vitamin C . . . . . . . . . . . . . . . . . . . . . . . 5.4. Mitochondria-targeted drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 58 58 59 59 59 59 59 60 60 60 60 60 61 61 61 Abbreviations: AMPK, AMP-activated protein kinase; CAT, catalase; CI, complex I; CL, cardiolipin; COX, cytochrome c oxidase; ER, endoplasmic reticulum; ERCa, ER Ca2+ content; ETC, electron transport chain; FAD, flavin adenine dinucleotide; FCS, fluorescence correlation spectroscopy; FMN, flavin mononucleotide; GPx, glutathione peroxidase; HZ, heterozygous; IMS, inter membrane space; KO, knockout; MERFF, Myoclonic Epilepsy with Ragged Red Fibers; MIM, mitochondrial inner membrane; MT, mutant; MnSOD, manganese superoxide dismutase; mtDNA, mitochondrial DNA; nDNA, nuclear DNA; OXPHOS, oxidative phosphorylation; PGC-1α, PPAR γ co-activator 1α; PMF, proton-motive force; PPAR γ, peroxisome proliferator-activated receptor γ; ROS, reactive oxygen species; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase; Tfam, mtDNA transcription factor A; TPP, tri-phenyl phosphonium; UCP, uncoupling proteins; WT, wild type; Δψ, mitochondrial membrane potential. ⁎ Corresponding author at: 286 Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, P.O. Box 9101, NL-6500 HB Nijmegen, The Netherlands. Tel.: + 31 24 3614589; fax: + 31 24 3616413. E-mail address: w.koopman@ncmls.ru.nl (W.J.H. Koopman). 1 These authors share the first authorship. 1567-7249/$ – see front matter © 2011 Elsevier B.V. and Mitochondria Research Society. All rights reserved. doi:10.1016/j.mito.2011.06.011 58 P. Roestenberg et al. / Mitochondrion 12 (2012) 57–65 5.4.1. TPP compounds . . . . . . . . . . . . . . . 5.4.2. Sk-compounds . . . . . . . . . . . . . . . 5.4.3. SS-peptides . . . . . . . . . . . . . . . . . 5.5. The benzothiazepine CGP37157 . . . . . . . . . . . . 5.6. Riboflavin . . . . . . . . . . . . . . . . . . . . . . 5.7. Peroxisome proliferator-activated receptor γ coactivator 5.8. Stabilization of CI . . . . . . . . . . . . . . . . . . 5.9. Nutritional intervention: the ketogenic diet . . . . . . 6. Summary and conclusions. . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1α . . . . . . . . . . 1. Mitochondrial complex I Complex I (CI or NADH:ubiquinone oxidoreductase; EC 1.6.5.3) is a L-shaped multi-subunit enzyme that is assembled in the mitochondrial matrix and embedded in the mitochondrial inner membrane (MIM; Dieteren et al., 2008; Vogel et al., 2007). In humans, CI consists of 45 different subunits, seven of which (the ND subunits) are encoded by the mitochondrial DNA (mtDNA) and the remainder by the nuclear (nDNA) genome. CI constitutes an entry point of electrons in the electron transport chain (ETC), which further consists of three other complexes (CII, CIII and CIV). CI oxidizes NADH to NAD+ and transports the electrons obtained from this reaction to coenzyme Q10 (ubiquinone). The energy released by this transport is used to expel protons from the mitochondrial matrix to the inter membrane space (IMS). A mechanistic model explaining the coupling between CI electron and H+ transport has been presented recently (Efremov et al., 2010; Hunte et al., 2010). CI contributes to the proton-motive force (PMF) required to drive various mitochondrial functions including ATP production by the FoF1-ATP-synthase (CV) (Koopman et al., 2010). Together, the ETC and CV constitute the oxidative phosphorylation (OXPHOS) system. Only 14 evolutionary conserved CI ‘core’ subunits are required to carry out the catalytic function of CI, seven encoded by the mtDNA (ND1, ND2, ND3, ND4, ND4L, ND5, ND6) and seven by the nDNA (NDUFV1, NDUFV2, NDUFS1, NDUFS2, NDUFS3, NDUFS7, NDUFS8). The function of the remaining 31 ‘accessory’ subunits, which are all nDNA-encoded, remains largely unknown but it is likely that they play a role in the biogenesis, stabilization and/or regulation of CI (Brandt, 2006; Koopman et al., 2010; Valsecchi et al., 2010). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 61 61 62 62 62 62 62 63 63 63 life and then suffer from a rapidly progressing and fatal disease phenotype (Distelmaier et al., 2009a; Valsecchi et al., 2010). This means that muscle biopsy material for research purposes is scarce. For this reason, we studied the cellular consequences of CI deficiency in primary patient-derived skin fibroblasts, which are more readily available. The flat morphology of these cells makes them ideally suited for (automated) quantitative live-cell microscopy analysis following introduction of chemical and/or protein-based reporter molecules (Distelmaier et al., 2008; Forkink et al., 2010; Koopman et al., 2005b, 2006a,b; Mortiboys et al., 2008; Willems et al., 2009a,b). Importantly, measurements need to be carried out at sub-confluent cell densities to prevent changes induced by cell–cell contacts. Moreover, use of primary cells with a finite lifespan like fibroblasts requires that passage numbers (PN) are kept low and matched between different 2. Isolated complex I deficiency in humans In humans, CI deficiency can be caused by: (i) mutations in mtDNAencoded subunits of CI, (ii) mutations in nDNA-encoded subunits of CI, or (iii) mutations in nDNA-encoded CI assembly/stabilization factors. In this review we focus on nDNA-encoded mutations in CI subunits. Mutations in such subunits are generally autosomal recessively inherited, meaning that both parents have to be heterozygous carriers. Currently, disease-causing mutations have been described for the following nDNA-encoded subunits: NDUFV1, NDUFV2, NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS6, NDUFS7, NDUFS8, NDUFA1, NDUFA2 and NDUFA11 (see Distelmaier et al., 2009a; Koene et al., 2011; Valsecchi et al., 2010 and the references therein). Patients with a defect in a nDNA-encoded CI structural gene display basal ganglia and/or brainstem lesions, respiratory abnormalities, muscular hypotonia, failure to thrive, seizures, and lactic acidemia. Detailed information about the clinical aspects of these diseases is provided elsewhere (Distelmaier et al., 2009a). 3. Cellular consequences of isolated complex I deficiency in patient skin fibroblasts Most children with isolated CI deficiency due to mutations in nDNA-encoded CI subunits develop symptoms during the first year of Fig. 1. Consequences of nDNA-encoded mutations in CI. The total amount of fully assembled and active CI within the cell (a) is determined by the balance between CI biogenesis (b) and degradation (c). Mutations in nDNA-encoded CI subunits (d) lead to functional impairment of CI (e) and induce changes in cellular physiology (f, h). If these changes are not properly counterbalanced by adaptive responses (j), including alterations in mitochondrial morphology and glycolysis upregulation (k), cell death can occur (g). This is likely reflected by the fact that fibroblasts from patients with isolated CI deficiency display increased ROS levels but no increased cellular lipid peroxidation or changes in thiol redox state (i). This figure is based upon experimental data obtained in fibroblasts of patients with nDNA-encoded isolated CI deficiency. Primary and possible adaptive pathways are indicated in red and green, respectively. Abbreviations: Δψ, mitochondrial membrane potential; NADH, reduced nicotinamide adenine dinucleotide; NADPH, reduced nicotinamide adenine dinucleotide phosphate; nDNA, nuclear DNA; and ROS, reactive oxygen species. P. Roestenberg et al. / Mitochondrion 12 (2012) 57–65 cell lines and experiments (Ghneim and Al-Sheikh, 2010). Further advantages and disadvantages of the fibroblast model system are discussed in more detail elsewhere (Koopman et al., 2005b, 2007a, 2010; Valsecchi et al., 2010; Willems et al., 2008). The cell biological consequences of isolated CI deficiency in skin fibroblasts from patients with nDNA-encoded CI mutations are summarized below. 3.1. Complex I biogenesis and degradation The total amount of fully assembled and active CI (Fig. 1a), depends on the balance between CI biogenesis (Fig. 1b) and CI degradation (Fig. 1c). Mutations in nDNA-encoded CI subunits (Fig. 1d) can lead to: (i) a reduced level of fully-assembled CI (e.g. NDUFS2, NDUFS7, or NDUFS8 mutations), (ii) a complete absence of fully-assembled CI and presence of an 830-kDa subcomplex (NDUFS4 mutations), or (iii) a combination of the two (e.g. NDUFS1, NDUFV1 mutations; Valsecchi et al., 2010). It is currently unclear whether these mutations hamper CI biogenesis, stimulate destabilization/breakdown of the fully-assembled CI or a combination of the two. 3.2. Functional impairment of mitochondria and adaptation in patient cells CI malfunction leads to functional impairment of mitochondria (Fig. 1e) and induction of downstream cellular effects (Fig. 1f). In this sense, mitochondria in patient fibroblasts display a partially depolarized mitochondrial membrane potential (Δψ; Fig. 1h; Distelmaier et al., 2009b; Visch et al., 2004). It is expected that mitochondrial impairment triggers an adaptive response (Fig. 1j), which might stimulate CI biogenesis, reduce CI degradation, or mitigate the cellular consequences of CI deficiency. Using fluorescence correlation spectroscopy (FCS) analysis of single intracellular mitochondria, it was found that protein diffusion in the mitochondrial matrix of patient-derived skin fibroblasts was faster than in control cells (Koopman et al., 2008a). Similar results were obtained in control fibroblasts, treated with a low concentration (100 nM, 72 h) of the CI-inhibitor rotenone (Koopman et al., 2007b). Faster diffusion occurred without changes in mitochondrial ultrastructure, suggesting that the viscosity of the mitochondrial membrane solvent decreases in CI-deficient fibroblasts. This is compatible with an adaptive switch toward a (more) glycolytic mode of energy production in patient cells (Fig. 1k), which might further reduce the detrimental consequences of CI malfunction by providing an alternative source of ATP. The latter might relate to the fact that cell death (Fig. 1g) was not detected in cultures of patient fibroblasts as well as control fibroblasts cultured with a low (100 nM, 72 h) concentration of rotenone (Koopman et al., 2005a,b). Possibly the extent of adaptation depends on the degree of CI deficiency and/or a threshold value of CI deficiency needs to be exceeded in order to trigger adaptation. 3.3. Other key cellular features of the patient cell phenotype In addition to Δψ depolarization, patient cells displayed increased NAD(P)H autofluorescence (Verkaart et al., 2007) and increased reactive oxygen species (ROS) levels (Koopman et al., 2007a; Verkaart et al., 2007). However, no changes in the cytosolic and mitochondrial thiol redox status and extent of cellular lipid peroxidation were detected in CI deficient patient fibroblasts (Verkaart et al., 2007). This suggests that oxidative stress is absent or only minor in patient fibroblasts, possibly due to adaptive up regulation of antioxidant systems (Fig. 1i). Quantification of mitochondrial morphology in a cohort of 14 patient fibroblast cell lines (see Koopman et al., 2007a and Willems et al., 2009a for data analysis and representative images of mitochondrial morphologies) revealed two distinct ‘classes’ of patient fibroblasts, one in which the cells mainly contained short circular ‘fragmented’ mitochondria (class I) and one in which the cells displayed a normal ‘filamentous’ mitochondrial morphology (class II). 59 Although all patient cells displayed a reduced CI activity and increased ROS levels, CI activity was significantly lower and ROS levels were significantly higher in class I cells, suggesting that extent of reduction in CI activity and ensuing increase in ROS levels are linked to mitochondrial fragmentation. The latter might further increase the extent of mitochondrial dysfunction (Chen et al., 2005). Conversely, filamentous mitochondria might ‘protect’ against the consequences of increased ROS levels by antioxidant and/or ROS-induced damage sharing (Fig. 1j; Koopman et al., 2005a,b, 2007a). If the latter is the case, reversal of mitochondrial fragmentation by stimulation of mitochondrial fusion or inhibition of mitochondrial fission might be beneficial in class I patient fibroblast. It is currently unclear whether alterations in ROS levels are upstream or downstream of the mitochondrial morphology changes. CI deficiency also induced a variable but significant decrease in the amount of ionic calcium (Ca 2+) stored in the endoplasmic reticulum (ER) in 7 out of 12 patient cell lines (Valsecchi et al., 2009; Visch et al., 2004, 2005). We provided evidence that this was due to a reduced fuelling of ER Ca 2+ pumps (sarco/endoplasmic reticulum Ca2+-ATPase or SERCA pumps) by ATP from closely apposed mitochondria (Willems et al., 2008). As a consequence of the reduced ER Ca2+ content (ERCa), hormone stimulation of the patient cells led to the following aberrations: (i) a cytosolic Ca 2+ signal with a reduced peak amplitude, (ii) a mitochondrial Ca2+ signal with a reduced peak amplitude, (iii) a mitochondrial ATP signal with a reduced peak amplitude, and (iv) a slower ATP-dependent Ca2+ removal from the cytosol. This demonstrates that CI deficiency disturbs the coupling between hormone stimulation, cytosolic and mitochondrial Ca2+ signaling, mitochondrial Ca2+-stimulated ATP generation and fueling of cytosolic Ca2+ pumps by mitochondrial ATP (Fig. 1h). 4. Mouse models of isolated complex I deficiency To better understand the consequences of isolated CI deficiency and effects of mitigating compounds with respect to toxicity, pharmacokinetics and therapeutic potential, suitable animal models are required. Many mouse models have been described for mitochondrial disorders and these have been comprehensively reviewed elsewhere (e.g. Bénit et al., 2010; Koene et al., 2011; Oliveira et al., 2010; Pinkert and Trounce, 2007; Russell et al., 2005; Tyynismaa and Suomalainen, 2009; Vempati et al., 2008; Wallace and Fan, 2009). Here we will focus on five mouse models that were specifically designed to mimic isolated CI deficiency in humans, which all involve the NDUFS4 gene. This gene constitutes a mutational hotspot in humans and encodes an 18-kDa accessory subunit of CI. The first 4 mouse models are all based on excision of exon 2 of the NDUFS4 gene. These models are: (i) a whole-body knockout (“the WB NDUFS4 −/− mouse”; Kruse et al., 2008), (ii) a neuron- and glia-specific knockout (“the Nes NDUFS4 −/− mouse”; Quintana et al., 2010), (iii) a Purkinje cell specific knockout (“the PC NDUFS4 −/− mouse"; Quintana et al., 2010) and (iv) a conditional whole-body knockout (“the CWB NDUFS4 −/− mouse”; Quintana et al., 2010). Additionally, (v) a mouse model harboring a point mutation in the NDUFS4 gene (“the NDUFS4-PM mouse”; Ingraham et al., 2009) has been developed. More detailed information about these models is provided below. 4.1. The WB NDUFS4 −/− mouse The WB NDUFS4 −/− knockout (KO) mice were generated by deletion of exon 2 which resulted in a frame shift mutation and undetectable levels of the NDUFS4 protein. Prior to day P30 the behavior of the KO mice was similar to control wild type (WT) mice. At 3 weeks of age the KO mice were already smaller than their WT and heterozygous (HZ) littermates. Most of the KO animals lost their body hair, which grew back during the next hair-growth cycle. Between P35–P50, the KO animals stopped gaining weight and even displayed 60 P. Roestenberg et al. / Mitochondrion 12 (2012) 57–65 weight loss. This was accompanied by worsening ataxia, stopped grooming and, finally, death from a fatal encephalomyopathy. Further phenotypic features of the KO mice include a retarded growth rate, a decreased body temperature (2 °C), cataracts, blindness, hearing loss, loss of motor skills and lethargy. KO animals displayed significantly higher serum lactate levels than WT mice. Resting ATP demand, phosphorylation capacity and resting O2 consumption in muscle, as determined by 31P-NMR spectroscopy, were similar in KO and WT mice. In muscle tissue mitochondrial ultrastructure appeared normal although large subsarcolemmal clusters of mitochondria were present in the soleus but not in the extensor digitorum longus muscle fibers of KO mice. Furthermore, a marked decrease in NADH:ubiquinone oxidoreductase (CI) activity was observed in KO while cytochrome c oxidase (COX) activity was similar to WT. Progressive neuronal deterioration and gliosis were observed in specific brain areas of the KO mice. These observations corresponded to behavioral changes during disease advance, with early involvement of the olfactory bulb, cerebellum, and vestibular nuclei (Quintana et al., 2010). Neurons, particularly in those brain regions, showed aberrant mitochondrial morphology. Electron microscopy (EM) analysis revealed that mitochondria were swollen and/or displayed a compact cristae structure. A similar analysis of a ‘class I’ and ‘class II’ patient cell line displaying fragmented and filamentous morphology, respectively (see Section 3.3), did not reveal any changes in mitochondrial ultrastructure (Koopman et al., 2008a). This suggests that the ultrastructural changes in the fibroblasts are prevented by an adaptive response and/or that the cellular consequences of CI deficiency are cell-type specific. Activation of caspase 8, but not caspase 9, in the affected brain regions of the WB NDUFS4 −/− animal was concluded to implicate the initiation of the extrinsic apoptotic pathway. The limited caspase 3 activation and the predominance of ultrastructural features of necrotic cell death suggest a switch from apoptosis to necrosis in affected neurons. From these data it was suggested that dysfunctional CI in specific brain regions results in progressive glial activation that on its turn promotes neuronal death and ultimately mortality of the mouse. CI activity in submitochondrial particles from liver of KO mice was undetectable using spectrophotometric assays. However, CI-driven O2 consumption in intact liver cells was about half that of WT. Native gel electrophoresis revealed reduced levels of intact CI. In our laboratory blue native gel electrophoresis has been performed on different tissues of the KO mice. Instead of fully assembled CI an inactive 830-kDa subcomplex was observed. A similarly sized 830-kDa CI subcomplex has been reported in patients with NDUFS4 mutations (Ogilvie et al., 2005, Papa et al., 2009; Ugalde et al., 2004; Valsecchi et al., 2010; Vogel et al., 2007). Taken together, these findings suggest that when the NDUFS4 subunit is absent, CI fails to assemble properly or is instable. 4.2. The Nes NDUFS4 −/− and PC NDUFS4 −/− mouse Two different brain-specific NDUFS4 −/− mice have been generated (Quintana et al., 2010). Mice that brain-specifically lack NDUFS4 were generated by crossing NDUFS4 lox/lox mice with mice expressing Cre recombinase from the Nestin locus. The phenotype of the resulting “Nes NDUFS4 −/−” mice generally resembled that of the WB NDUFS4 −/− mice including retarded growth, loss of motor ability, breathing abnormalities, and a maximum life span of approximately 7 weeks. As both the WB NDUFS4 −/− and the Nes NDUFS4 −/− models show cell death of Purkinje cells in the lobules of the cerebellum, also a Purkinje cell selective NDUFS4 KO (PC NDUFS4 −/−) was generated. These mice only manifested mild behavioral and neuropathological abnormalities. Combined with the results obtained in the WB NDUFS4 −/− and Nes NDUFS4 −/− models, this suggests that death of Purkinje cells in the Nes NDUFS4 −/− animals is likely a secondary effect. This might be due to dysregulation of the cerebellar circuitry or hypersensitivity to hypoxia (Quintana et al., 2010). 4.3. The CWB NDUFS4 −/− mouse Conditional knockout of NDUFS4 was achieved by crossing NDUFS4 lox/lox mice with mice bearing an inducible Cre recombinase gene. The latter gene can be induced in most cells by tamoxifen (Quintana et al., 2010). Tamoxifen treatment at day ~ P60 induced a large decrease in the abundance of NDUFS4 protein in most brain regions. Effects in other tissues are not reported thus far. Seven months after tamoxifen treatment the mice displayed a phenotype that resembled the early stage of WB NDUFS4 −/− mice. This rather mild phenotype, also in comparison to Nes NDUFS4 −/− mice, might be explained by incomplete or absent recombination in some brain regions, despite intensive tamoxifen treatment. Alternatively, complete absence of NDUFS4 during animal development might have more severe consequences than (partial) NDUFS4 knockout at a later stage of development. 4.4. The NDUFS4-PM mouse This mouse model was generated by introducing a point mutation in the NDUFS4 gene, resulting in loss of the last 10–15 amino acids of the last exon of the protein (Ingraham et al., 2009). Interestingly, HZ mice showed expression and even integration of the mutant (MT) NDUFS4 protein in the CI holocomplex. In heart mitochondria, the amount of WT NDUFS4 protein present in the CI holocomplex was 2.5fold higher than that of the MT NDUFS4 subunit. However, the presence of the latter led to a significant reduction in CI activity, lower CI-mediated O2 consumption, and increased lactate concentrations in organs of HZ mice. The clinical phenotype of the NDUFS4-PM HZ mice was not reported. In sharp contrast to the other four mouse models, homozygote NDUFS4-PM mice were not viable and their presence in the early embryonic state (bE9) could not be demonstrated. This suggests that in homozygous animals the presence of a mutated NDUFS4 protein leads to a much more severe phenotype than complete absence of NDUFS4. 5. Pharmacological targeting of complex I deficiency In vivo treatment of CI deficiency is tremendously challenging because a possible beneficial drug effect also depends on the way of administration, concentration, (bio)distribution, turnover and clearance. In addition, (long term) drug effectiveness and toxicity have to be considered. In recent years, pharmacological targeting of mitochondrial dysfunction has been extensively reviewed (Finsterer and Segall, 2010; Hersh, 2010; Jin et al., 2010; Moncada, 2010; Valsecchi et al., 2010; Wallace et al., 2010; Wenz et al., 2010). In our research we have successfully applied an antioxidant (Trolox) and a benzothiazepine (CGP37157), to mitigate several cellular consequences of CI deficiency. Below we summarize our results with these compounds (Section 5.1, Section 5.5) and also briefly present several other strategies that might proof useful for mitigation of CI deficiency in patient cells and mouse models. 5.1. The antioxidant Trolox Our data suggests that preventing Δψ depolarization may normalize ERCa and, as a consequence, the aberrations in cytosolic Ca 2+ and mitochondrial Ca 2+ /ATP signals during hormone-stimulation (Willems et al., 2008). Given the fact that patient cells also displayed increased ROS levels, we investigated the effect of the vitamin E derived, water soluble, antioxidant Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). In a first study, we observed that Trolox (500 μM, 72 h) effectively reduced cellular ROS and increased the expression of fully assembled CI both in control and patient cells (Koopman et al., 2008b). A follow up study (Distelmaier et al., 2009b) revealed that Trolox fully normalized the depolarized Δψ, as well as the P. Roestenberg et al. / Mitochondrion 12 (2012) 57–65 aberrations in cytosolic Ca 2+ and mitochondrial Ca2+/ATP signaling during hormone-stimulation. We presented a mechanism (Valsecchi et al., 2010), in which CI deficiency hampers CI function and thereby directly results in Δψ depolarization. Alternatively, the elevated ROS in patient cells might induce activation of mitochondrial uncoupling proteins (UCP) leading to Δψ depolarization and hampered local ATP supply to SERCAs. The latter would then prevent proper SERCAmediated calcium uptake, leading to a reduced ERCa. Alternatively, ROS can directly inhibit the SERCAs, also leading to reduced ERCa. In this system, Trolox would inhibit UCP activation and/or SERCA inhibition and thereby restore Δψ and ERCa. Studies concerning the mode-ofaction of Trolox are currently being performed in our laboratory. 5.2. The antioxidant vitamin E Chronic oral administration of vitamin E (α-tocopherol) prevented the loss of mitochondrial function and reduced ROS-induced damage in aging mice and was able to reduce the aging-induced inhibition brain CI activity in mice in vivo (Navarro et al., 2005; Navarro and Boveris, 2010). These beneficial effects were accompanied by an increased lifespan, better neurological performance and higher exploratory activity of the animals. Importantly, the levels of αtocopherol in mouse brain increased from 11.5 to 26.2 nmol/g brain, showing that it was able to cross the blood brain barrier. In humans there is still controversy on the use of vitamin E supplementation (Navarro et al., 2005; Navarro and Boveris, 2010). For instance, a meta-analysis claimed that vitamin E supplementation increases human mortality (Bjelakovic et al., 2007). However, these results are challenged by clinical evidence that vitamin E supplements are safe at high dosages (Hathcock et al., 2005), and by the reported effects of vitamins E and C in the reduction of the prevalence/incidence of Alzheimer disease in an elderly population (Zandi et al., 2004). 5.3. The antioxidant vitamin C Vitamin C, or ascorbate, is well known for its antioxidant capacity. This vitamin may also serve as a first line of defense against the oxidative injury to the cell because in comparison to coenzyme Q10 and vitamin E, ascorbate is the first antioxidant to be consumed by free radicals and is not synthesized by human fibroblasts (Niki, 1991). These cells, however, do have the ability to transport the extracellular ascorbate by a Na +-dependent carrier-mediated system (Welch et al., 1993). In human skin fibroblasts from patients with respiratory chain deficiencies, ascorbate was able to reduce superoxide production while increasing respiratory chain function (Sharma and Mongan, 2001). In aging human fibroblasts in vitro, ascorbate prevented the age-dependent decline in respiratory chain activities (Ghneim and AlSheikh, 2010). This positive effect is compatible with the fact that ascorbate can neutralize hydroxyl and peroxyl species, is able to reduce lipid peroxidation, and increases O2 utilization and mtDNA transcription rates (Ghneim and Al-Sheikh, 2010; Phillips et al., 1999). 5.4. Mitochondria-targeted drugs Currently, there are several strategies that allow targeting of chemical compounds to the mitochondrial matrix in living cells. These include targeted antioxidants and mitochondria-targeted peptides (see: Forkink et al., 2010; Valsecchi et al., 2010 and the references therein). In the following three subsections we discuss tri-phenyl phosphonium (TPP) compounds, ‘Skulachev’ (Sk) compounds and Szeto-Schiller (SS) peptides in more detail. 5.4.1. TPP compounds One group of compounds which specifically accumulate in mitochondria consists of antioxidants that are covalently coupled to a tri-phenyl-phosphonium (TPP) cation. These molecules pass 61 phospholipid bilayers without requiring a specific uptake mechanism and, because of their positive charge, preferentially accumulate (500– 1000 fold) within mitochondria in a Δψ-dependent manner (Murphy and Smith, 2007). The latter property also could be a potential drawback of TPP compounds because they accumulate less in depolarized mitochondria. In this way, the intra-mitochondrial TPP concentration might not reach sufficiently high levels. In theory, introducing positively charged TPP molecules into the mitochondrial matrix might even induce further Δψ-depolarization and additional impairment of mitochondrial function. A well-studied member of the TPP family is “MitoQ” (i.e. mitoquinone; a derivative of coenzyme Q10). Given its relatively large hydrophobicity, MitoQ is preferentially adsorbed to the matrixfacing leaflet of the MIM, with the TPP moiety at the membrane surface at the level of the fatty acid carbonyls and the alkyl chain and ubiquinol moiety inserted into the hydrophobic core of the lipid bilayer (Murphy and Smith, 2007). In healthy fibroblasts treated with rotenone (100 nM, 72 h), MitoQ prevented rotenone-induced mitochondrial elongation and lipid peroxidation but did not block rotenone-induced ROS production (Koopman et al., 2005a). This is compatible with MitoQ being localized inside the MIM and thereby preventing oxidation of mitochondrial lipids. In this sense our results provide evidence that the Δψ-dependent accumulation, as well as the depolarizing effect of MitoQ accumulation do not prevent MitoQ action. Use of MitoQ in mouse models as well as patients in vivo, revealed that its use is safe in vivo, even during long term use. Administration in the drinking water of mice resulted in measurable levels of MitoQ in all tissues, including the brain, although the concentrations differed between organs (Rodriguez-Cuenca et al., 2010). This illustrates that MitoQ is able to cross the blood-brain barrier which is an important hurdle in therapies for CI deficiency. Furthermore, MitoQ was excreted in the urine and bile as unchanged MitoQ or with sulfation or glucuronidation of the quinol ring with no indication of other metabolites (Li et al., 2007; Ross et al., 2008). In addition to MitoQ also other antioxidant variants like MitoVitE, MitoTEMPOL and MitoPBN were developed (Murphy and Smith, 2007). The detailed in vivo characteristics of these compounds and their effects on CI deficiency require further investigation. 5.4.2. Sk-compounds Skulachev et al., developed another group of interesting mitochondria-targeted compounds. These SKQs consist of a TPP-linked plastoquinone moiety (Skulachev et al., 2009). When compared to MitoQ, SKQs displayed a higher permeability in a variety of in vitro, ex vivo and in vivo models. SKQs possess a strong antioxidant activity at relatively low (nM) concentrations (Antonenko et al., 2008). When used at higher (μM) concentrations SKQs were shown to act as prooxidants. Applied in vivo, SKQs displayed beneficial effects during hydrogen peroxide- and ischemia-induced heart arrhythmia, heart infarction, kidney ischemia and stroke (Bakeeva et al., 2008). Currently, no data of SkQs in relation to CI deficiency are available. However, it would be interesting to determine whether SkQs are able to influence the downstream effects of CI deficiency. 5.4.3. SS-peptides A third group of mitochondria-targeted antioxidants consists of the so called Szeto-Schiller (SS) peptides. This is a novel class of small cell permeable peptide antioxidants that target to mitochondria in a Δψ-independent manner. The structural motif of these peptides centers on alternating aromatic residues and basic amino acids (Szeto, 2006). SS-peptides are small and relatively easy to synthesize, readily soluble in water, and resistant to peptidase degradation. Besides, they display a high blood-brain barrier permeability and a relative long elimination half-life in sheep and rats, which allows their testing in neurological disease models (Szeto, 2006). In cell experiments, SS-31 accumulated 5000-fold in the mitochondrial fraction. SS-31 was 62 P. Roestenberg et al. / Mitochondrion 12 (2012) 57–65 localized close to the site(s) of mitochondrial ROS production and protected against mitochondrial oxidative damage and further ROS production (Zhao et al., 2004). SS-31 prevented Δψ depolarization, reduced cellular ROS and inhibited apoptosis in neurons treated with the pro-oxidant tert-butyl-hydroperoxide (Zhao et al., 2005). The effects of SS-peptides in CI deficiency models are currently unknown. 5.5. The benzothiazepine CGP37157 CGP37157, a benzothiazepine, is an inhibitor of mitochondrial Na+/Ca2+ exchange (Cox and Matlib, 1993). CGP37157 normalized aberrant mitochondrial Ca 2+ handling during hormone stimulation of cybrid cells carrying the tRNALys mutation associated with MERRF syndrome (Myoclonic Epilepsy with Ragged Red Fibers; Brini et al., 1999). To directly interfere with aberrant mitochondrial Ca 2+ signaling, we investigated the effect of CGP37157 in fibroblasts from patients with isolated CI deficiency (Visch et al., 2004). Short-term pre-treatment with CGP37157 (1 μM, 2 min) fully normalized the amplitude of the hormone-induced mitochondrial Ca2+ signal, without altering this parameter in healthy fibroblasts. Also the reduced maximal [ATP] in the mitochondrial matrix and cytosol were fully normalized by CGP37157 treatment. The effect of CGP37157 was independent of the presence of extracellular Ca2+, excluding a stimulatory effect on Ca2+ entry across the plasma membrane. These findings suggest that the mitochondrial Na+/Ca2+ exchanger is a potential target for drugs aiming to restore or improve Ca 2+-stimulated mitochondrial ATP synthesis in CI deficiency. 5.6. Riboflavin Riboflavin, or vitamin B2, is a precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which function as cofactors in CI and CII, respectively. In vitro, riboflavin treatment of CIdeficient patient fibroblasts virtually normalized the rate of ATP production (Bar-Meir et al., 2001). In vivo, riboflavin supplementation, with or without co-administration of carnitine, has been reported to be effective in a number of patients with CI deficiency. Primary effects included improvement of muscle tone, exercise capacity/tolerance and serum lactate levels, although the results of the treatment substantially differed between different studies (reviewed by Marriage et al., 2003). A mechanism was proposed in which riboflavin inhibits the proteolytic breakdown of CI, leading to an increase in CI enzymatic activity (Gold and Cohen, 2001; Marriage et al., 2003; Vergani et al., 1999). Dosages of riboflavin for treatment of OXPHOS disorders ranged between 9 and 300 mg/day without observation of adverse effects (Marriage et al., 2003). Although the results obtained with riboflavin vary, they clearly demonstrate that in some patients with CI deficiency, riboflavin alone or in combination with other supplements is beneficial (Marriage et al., 2003; Panetta et al., 2004). 5.7. Peroxisome proliferator-activated receptor γ coactivator 1α The peroxisome proliferator-activated receptor γ (PPAR γ) coactivator 1α (PGC-1α), is a transcriptional factor regulator that stimulates transcription of genes involved in cellular energy metabolism and also can display antioxidant activity (Scarpulla, 2008; Wenz, 2009). PGC-1α can influence mitochondrial biogenesis and function by modulating transcription of the mtDNA transcription factor A (Tfam) gene (Wu et al., 1999) Several ways to therapeutically modulate PGC-1α have been reported. Bezafibrate is a widely used drug acting as a pan (i.e. α, δ and γ) PPAR agonist. In this way it increases the activity of CI and other ETC enzymes (complex III and complex IV) in healthy and OXPHOSdeficient cells through PGC-1α upregulation (Bastin et al., 2008). In COX10 KO mice displaying progressive myopathy, bezafibrate stimulated OXPHOS gene expression in skeletal muscle and induced a global increase in oxidative metabolism (Wenz et al., 2008). Resveratrol is a polyphenolic phytoalexin that is abundantly present in grapes, peanuts and red wine. Resveratrol stimulates sirtuin activity and thereby transcription of nNDA-encoded genes involved in energy metabolism (Shin et al., 2009; Ungvari et al., 2009). It was demonstrated that resveratrol-induced activation of the sirtuin Sirt1, and its effector PGC-1α, alter mitochondrial number and function (Lagouge et al., 2006). Dietary delivery of resveratrol increased mitochondrial abundance and aerobic capacity in cultured endothelial cells and mice (Baur et al., 2006; Csiszar et al., 2009; Lagouge et al., 2006; Robb et al., 2008). Besides effects on PGC1α, also effects of resveratrol on the cellular antioxidant system have been observed. Both dietary and subcutaneous resveratrol delivery methods were capable of altering the activities of the key antioxidant enzymes glutathione peroxidase (GPx), catalase (CAT) and (mitochondrial) manganese superoxide dismutase (MnSOD), and increasing mitochondrial content in heart, brain and liver (Robb et al., 2008). A double blind healthy volunteer study showed that frequent administration of resveratrol is well tolerated, but results in low plasma concentrations despite a 4-hr oral administration regime (Almeida et al., 2009). As far as we know, no data are currently available on the side effects of continuous resveratrol administration. PGC-1α levels in skeletal muscle can also be stimulated by endurance exercise via a mechanism involving repetitive changes in cytosolic Ca 2+ concentrations and AMP-activated protein kinase (AMPK) activation (Benton et al., 2008; Ojuka, 2004; Wu et al., 2002). It was suggested that, in healthy subjects, endurance exercise increases skeletal muscle oxidative capacity by improving the metabolic flux through CI (Daussin et al., 2008). Endurance exercise also improved the physiological and biochemical features in muscles of patients with mitochondrial disease due to mtDNA mutations (Taivassalo and Haller, 2004, 2005). 5.8. Stabilization of CI Observations in patient and mouse cells show that physical interaction of CI with CIII, but not enzymatic activity of CIII, stabilizes CI (Acín-Pérez et al., 2004; Fernandez-Vizarra et al., 2007). Even a partial CI (i.e. the CI 830 kDa subassembly, see Section 4.1) can associate with CIII into supercomplexes (Lazarou et al., 2007; Ogilvie et al., 2005). Another important factor for structural integrity and function of CI, other OXPHOS complexes, and other mitochondrial membrane-embedded proteins, is the MIM lipid cardiolipin (CL; Houtkooper and Vaz, 2008; Schlame et al., 2000; Sharpley et al., 2006). CL is very sensitive to peroxidation by mitochondrial ROS and the latter was demonstrated to affect CI activity in bovine heart and rat heart/liver mitochondria (Petrosillo et al., 2009). This means that prevention of mitochondrial ROS production and/or ensuing CL peroxidation might be a good strategy to increase CI stability in patient cells as well as in vivo. 5.9. Nutritional intervention: the ketogenic diet Nutritional approaches like a ketogenic diet may also be useful. Ketogenic diets have a high fat content, are adequate in protein and low in carbohydrates. During the regime of a ketogenic diet, ketone bodies are used as a substitute for glucose to generate energy for the brain. Ketone bodies are a more efficient source of energy per unit of oxygen than glucose (Veech et al., 2001). Evidence has been provided that a ketogenic diet induces a coordinated upregulation of mitochondrial genes involved in energy metabolism and might stimulate mitochondrial biogenesis as well (Yudkoff et al., 2001). This suggests that the capacity of neurons to withstand metabolic challenges might be increased by a ketogenic diet thereby providing neuroprotection in neurodegenerative disorders. P. Roestenberg et al. / Mitochondrion 12 (2012) 57–65 6. Summary and conclusions Currently, no effective treatment for CI deficiency is available. Safe in vivo gene therapy for nuclear-encoded types of CI deficiency is still not feasible. In vitro, pharmacological treatment was able to ameliorate certain parameters of mitochondrial function in CI deficient patient cells. However, in vivo targeting of CI deficiency is a much larger challenge. So far, only data from other in vivo models involving mitochondrial dysfunction are available. The recent development of the first, nuclear-encoded, knockout mouse models for CI deficiency, and of novel strategies for mitochondrial targeting of pharmaceuticals, creates excellent opportunities for evaluation of the effectiveness of pharmacological treatment of CI deficient patients in the future. Acknowledgments This work was supported by a grant of the ‘Prinses Beatrix Fonds’ (No: OP-05-04) and by the CSBR (Centres for Systems Biology Research) initiative from the Netherlands Organisation for Scientific Research (NWO; No: CSBR09/013V). We apologize to those authors whose articles we were unable to cite because of space limitations. References Acín-Pérez, R., Bayona-Bafaluy, M.P., Fernández-Silva, P., Moreno-Loshuertos, R., Pérez-Martos, A., Bruno, C., Moraes, C.T., Enríquez, J.A., 2004. Respiratory complex III is required to maintain complex I in mammalian mitochondria. Mol. Cell. 13, 805–815. 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