Cellular Pathophysiological Consequences of BCS1L Mutations

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Cellular pathophysiological consequences of BCS1L mutations in mitochondrial complex III enzyme deficiency Maria Moran, Lorena Marín Buera, Maria Carmen Gil, Henry Rivera, Alberto Blazquez, Sara Seneca, Maria Vazquez Lopez, Joaquin Arenas, Miguel A Martin, Cristina Ugalde

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Maria Moran, Lorena Marín Buera, Maria Carmen Gil, Henry Rivera, Alberto Blazquez, et al.. Cel- lular pathophysiological consequences of BCS1L mutations in mitochondrial complex III enzyme deficiency. Human Mutation, Wiley, 2010, 31 (8), pp.930. 10.1002/humu.21294. hal-00552400

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Cellular pathophysiological consequences of BCS1L mutations in mitochondrial complex III enzyme deficiency

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Journal: Human Mutation

Manuscript ID: humu-2010-0090.R1

Wiley - Manuscript type: Research Article

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Complete List of Authors: Moran, Maria; Hospital Universitario 12 de Octubre, Centro de Investigacion Marín Buera, Lorena; Hospital Universitario 12 de Octubre, Centro de Investigacion Gil, Maria Carmen; Hospital Universitario 12 de Octubre, Centro de Investigacion Rivera, Henry; Hospital Universitario 12 de Octubre, Centro de Investigacion Blazquez, Alberto; Hospital Universitario 12 de Octubre, Centro de Investigacion Seneca, Sara; AZ-VUB, Center of Medical Genetics Vazquez Lopez, Maria; Hospital General Universitario Gregorio Marañón, Sección de Neuropediatría Arenas, Joaquin; Hospital Universitario 12 de Octubre, Centro de Investigación; Hospital Universitario 12 de Octubre, Centro de Investigacion Martin, Miguel; Hospital Universitario 12 de Octubre, Centro de Investigación; Hospital Universitario 12 de Octubre, Centro de Investigacion Ugalde, Cristina; Hospital Universitario 12 de Octubre, Centro de Investigacion

Mitochondria, Respiratory chain , Complex III deficiency, BCS1L Key Words: mutations

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Morán et al. 1 1 2 3 Cellular pathophysiological consequences of BCS1L mutations in mitochondrial 4 5 6 complex III enzyme deficiency 7 8 María Morán, 1,2,* Lorena Marín-Buera, 1,2,* M. Carmen Gil-Borlado, 1,2 Henry Rivera, 1,2 9 10 Alberto Blázquez, 1,2 Sara Seneca, 3 María Vázquez-López,4 Joaquín Arenas, 1,2 Miguel 11 12 1,2 1,2 13 A. Martín, and Cristina Ugalde 14 15 16 17 18 * These authors equally contributed to this work 19 20 For Peer Review 21 22 1Centro de Investigación, Hospital Universitario 12 de Octubre, Madrid, Spain; 2Centro 23 24 25 de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), U723, Spain; 26 27 3Center of Medical Genetics, AZ-VUB, Brussels, Belgium; and 4Sección de 28 29 Neuropediatría, Hospital General Universitario Gregorio Marañón, Madrid, Spain. 30 31 32 33 34 Corresponding author: Dr. Cristina Ugalde, Centro de Investigación, Hospital 35 36 Universitario 12 de Octubre. Avda. de Córdoba s/n. 28041 Madrid. Phone: +34 91 390 37 38 39 8763, FAX: +34 91 390 8544, e-mail: [email protected] 40 41 42 43 44 Abbreviated title: Role of mutated BCS1L in complex III deficiency 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Morán et al. 2 1 2 3 ABSTRACT 4 5 6 Mutations in BCS1L, an assembly factor that facilitates the insertion of the 7 8 catalytic Rieske Iron-Sulphur subunit into respiratory chain complex III, result in a wide 9 10 variety of clinical phenotypes that range from the relatively mild Björnstad syndrome, to 11 12 13 the severe GRACILE syndrome. To better understand the pathophysiological 14 15 consequences of such mutations, we studied fibroblasts from six complex III-deficient 16 17 18 patients harbouring mutations in the BCS1L gene. Cells from patients with the most 19 20 severe clinical phenotypesFor exhibited Peer slow growth Review rates in glucose medium, variable 21 22 combined enzyme deficiencies and assembly defects of respiratory chain complexes I, 23 24 25 III and IV, increased H2O2 levels, unbalanced expression of the cellular antioxidant 26 27 defences, and apoptotic cell death induction. Besides, all patients showed cytosolic 28 29 accumulation of the BCS1L protein, suggestive of an impaired mitochondrial import, 30 31 32 assembly or stability defects of the BCS1L complex, fragmentation of the mitochondrial 33 34 networks, and decreased MFN2 protein levels. The observed structural alterations were 35 36 independent of the respiratory chain function and ROS production. Our results provide 37 38 39 new insights into the role of pathogenic BCS1L mutations in mitochondrial function and 40 41 dynamics. 42 43 44 45 46 KEYWORDS: Mitochondria; respiratory chain complex III deficiency; BCS1L 47 48 mutations. 49 50 51 52 53 54 55 56 57 58 59 60

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Morán et al. 3 1 2 3 INTRODUCTION 4 5 6 Mitochondrial respiratory chain complex III (CIII, ubiquinol-cytochrome c 7 8 oxidoreductase or cytochrome bc1 complex, E.C.1.10.2.2) catalyzes the transfer of 9 10 electrons from reduced coenzyme Q to cytochrome c with a concomitant translocation 11 12 13 of protons across the inner membrane (Baum et al., 1967). The purified bovine complex 14 15 is a ~450 kDa symmetric homodimer (Iwata 1998; Xia et al., 1997). Each monomer is 16 17 18 composed of eight structural subunits, and three catalytic subunits: cytochrome b, the 19 20 only subunit encodedFor in the mitochondrial Peer genome, Review cytochrome c1, and the Rieske Iron- 21 22 Sulphur Protein (RISP). 23 24 25 Mitochondrial complex III enzyme deficiency (MIM #124000 ) is a relatively 26 27 infrequent diagnosed defect of the oxidative phosphorylation (OXPHOS) system (Benit 28 29 30 et al., 2009). The vast majority of nuclear mutations leading to complex III deficiency 31 32 have been reported in the BCS1L gene (MIM #603647), which encodes a mitochondrial 33 34 inner membrane protein necessary for the correct biogenesis of respiratory chain 35 36 37 complex III (Cruciat et al., 1999; Fernandez-Vizarra et al., 2007; Fernandez-Vizarra et 38 39 al., 2009; Nobrega et al., 1992). BCS1L mutations lead to three main clinical 40 41 ) 42 phenotypes (Ramos-Arroyo et al., 2009): i) Björnstad syndrome (MIM #262000 , an 43 44 autosomal recessive disorder characterized by sensorineural hearing loss and pili torti 45 46 ( 47 (Hinson et al., 2007); ii) GRACILE syndrome MIM #603358), a Finnish-heritage 48 49 disease caused by the homozygous p.S78G mutation (Fellman 2002; Visapaa et al., 50 51 2002), which is characterized by fetal growth retardation, aminoaciduria, cholestasis, 52 53 54 iron overload, lactic acidosis, and early death; and iii) complex III deficiency in 55 56 neonates or infants presenting with encephalopathy, alone or in combination with 57 58 visceral involvement (Blazquez et al., 2009; de Lonlay et al., 2001; De Meirleir et al., 59 60 2003; Fernandez-Vizarra et al., 2007; Gil-Borlado et al., 2009).

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Morán et al. 4 1 2 3 Human BCS1L protein consists of 419 aminoacids, and shares functional 4 5 6 homology and 50% identity with yeast Bcs1p (Nobrega et al., 1992; Petruzzella et al., 7 8 1998) . In yeast, Bcs1p acts as an ATP-dependent chaperone, maintaining pre-complex 9 10 III in a competent state for the subsequent assembly of the Rieske and Qcr10 subunits 11 12 13 (Cruciat et al., 1999). The absence of Bcs1p prevents the assembly of Rieske within 14 15 complex III and leads to a complete respiratory deficiency. Bcs1p spans three different 16 17 18 domains: i) the N-terminal domain comprises aminoacid residues 1 to 126, which 19 20 contain all the requiredFor information Peer for the mitochondrialReview targeting and sorting of the 21 22 protein (Folsch et al., 1996; Petruzzella et al., 1998); ii) a Bcs1p-specific domain 23 24 25 contains aminoacid residues that are important for Bcs1p activity and stability (Nouet et 26 27 al., 2009); and iii) the C-terminal region contains a ~200 aminoacids domain that 28 29 encompasses two motifs corresponding to nucleotide binding sites, a characteristic 30 31 32 feature of the AAA (for ATPases associated with various cellular activities) protein 33 34 superfamily (Frickey and Lupas, 2004). 35 36 Pathogenic mutations have been described in the three different domains of 37 38 39 human BCS1L, although the pathophysiological mechanisms that contribute to the 40 41 clinical manifestations of the disease remain poorly understood. In some BCS1L - 42 43 44 mutated patients presenting with Björnstad syndrome or complex III deficiency, an 45 46 assembly defect of the RISP subunit led to a mitochondrial complex III enzyme defect 47 48 (Blazquez et al., 2009; Fernandez-Vizarra et al., 2007; Gil-Borlado et al., 2009; Hinson 49 50 51 et al., 2007). However, in patients presenting with the characteristic BCS1L mutation 52 53 c.232A>G (p.S78G) leading to GRACILE syndrome, complex III activity was 54 55 essentially normal despite low BCS1L levels (Fellman et al., 2008). Iron overload has 56 57 58 been reported in some patients with BCS1L mutations leading to GRACILE syndrome 59 60 or complex III deficiency, but not Björnstad Syndrome, suggesting a second hypothetic

role for BCS1L in iron metabolism (De Meirleir et al., 2003; Visapaa et al., 2002). It

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Morán et al. 5 1 2 3 has also been proposed that the pathophysiology of the BCS1L mutations might be 4 5 6 regulated by reactive oxygen species (ROS) production in a mutation and disease- 7 8 dependent manner (Hinson et al., 2007). In human cells, BCS1L knockdown caused the 9 10 disassembly of the mitochondrial supercomplexes, and led to morphological alterations 11 12 13 of the mitochondrial network (Tamai et al., 2008). Besides, it caused the down 14 15 regulation of LETM1, a mitochondrial Ca 2+/H + antiporter that is implicated in the 16 17 18 human Wolf-Hirschhorn syndrome (Jiang et al., 2009; Tamai et al., 2008). 19 20 In order to unveilFor the cellPeer biological consequencesReview of BCS1L defects, we have 21 22 performed a comparative series of cellular and biochemical studies in skin fibroblasts 23 24 25 from six complex III-deficient patients harbouring mutations in the BCS1L gene. Our 26 27 results establish a correlation between specific cellular pathophysiological effects of 28 29 such mutations and the severity of the clinical manifestations of the disease, and provide 30 31 32 new insights into the role of BCS1L in mitochondrial function and dynamics. 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Morán et al. 6 1 2 3 PATIENTS, MATERIALS AND METHODS 4 5 6 Patients 7 8 Patient 1 was an 11 days old female, born to parents who were first cousins, 9 10 whose body weight, height and head circumference were below 3 rd percentile. Clinical 11 12 13 examination showed hypotonia, weak reflexes, and mild hepatomegaly. Blood 14 15 chemistry revealed marked lactic acidosis (6.8 mmol/L, normal <2.0), elevation of liver 16 17 18 enzymes and alanine levels, decreased serum albumin, elevated ferritin levels, and 19 20 decreased total ironFor binding capacity Peer and transferrin Review levels. Organic acids analysis in 21 22 urine displayed prominent lactic aciduria, and mild increase in 2-OH butyric, 3-OH 23 24 25 butyric, fumaric, succinic, and 4-OH phenyl-lactic acid levels. There was also 26 27 generalized aminoaciduria, glucosuria and hyperphosphaturia consistent with renal 28 29 Fanconi syndrome. A mitochondrial disease was suspected, and a treatment with 30 31 32 coenzyme Q, carnitine, vitamins and cofactors was initiated that failed to correct 33 34 acidosis. The BCS1L gene was sequenced based on some key described clinical 35 36 features, such as lactic acidosis, hepatopathy, iron overload, and tubulopathy, together 37 38 39 with neurological signs, which are often found in patients with mutations in this gene 40 41 (de Lonlay et al., 2001; De Meirleir et al., 2003); therefore; no muscle biopsy was 42 43 44 performed. Peritoneal dialysis was unsuccessful because of the presence of severe 45 46 peritonitis, which was treated with intravenous antibiotics. Although enteral nutrition 47 48 led to weight gain and sodium bicarbonate therapy managed eventually to control the 49 50 51 metabolic acidosis, she died at 7 months of age by a Pseudomonas sepsis . 52 53 Patient 5, a girl of 19 months of age born to non-consanguineous parents, 54 55 presented with fever, seizures, progressive deterioration of consciousness, and 56 57 58 respiratory dysfunction. Neurological examination showed hypertonia, hyperintense 59 60 deep tendon reflexes, and an extensor response in the plantar reflex. Laboratory data

showed a dramatic increase in the serum levels of AST and ALT (>6000 IU/L), and

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Morán et al. 7 1 2 3 increased serum lactate (2.9 mmol/L, normal <2.0). Brain CT scanning showed bilateral 4 5 6 hypodensities in brainstem, thalamus, and basal ganglia. Brain MRI displayed supra and 7 8 infratentorial symmetric bilateral white matter lesions, as well as bilateral T2-weighted 9 10 hyperintense signals in the thalamus. Clinical examination at age 5 years revealed 11 12 13 hypertonia with hypereflexia and spasticity in the upper and lower limbs. 14 15 Patients 2, 3, 4 and 6 were reported before (Blazquez et al., 2009; De Meirleir et 16 17 18 al., 2003; Gil-Borlado et al., 2009). A summary of clinical, genetic, and biochemical 19 20 data from the six complexFor III-deficient Peer patients Review is shown in Table 1. 21 22 23 Enzyme activities of mitochondrial respiratory chain complexes 24 25 Mitochondrial respiratory chain enzyme activities (Table 2) were performed in 26 27 28 cultured skin fibroblasts according to established methods (Blazquez et al., 2009). 29 30 Genomic DNA sequence analysis 31 32 Total DNA was extracted from fibroblasts by standard methods. Sequencing of 33 34 35 the mitochondrial MT-CYB gene (MIM #516020 ), which encodes the complex III 36 37 cytochrome b subunit, excluded mutations in this gene. Overlapping fragments of the 38 39 entire BCS1L genomic region, which included all exons, introns, and the 5’ and 3’ UTR 40 41 42 regions, were sequenced on an ABI Prism System 3130xL Genetic Analyzer (Applied 43 44 Biosystems), and compared with the GenBank reference sequence for the BCS1L gene 45 46 (NG_008018.1). The mutations were confirmed by PCR-RFLP analyses, and four 47 48 49 hundred alleles of healthy ethnically-matched controls were screened for the mutations. 50 51 Fibroblast cultures and cell growth analysis 52 53 54 Cultured skin fibroblasts from patients and controls were plated in 4.5 g/l 55 56 glucose-containing Dulbecco´s modified Eagle´s Medium (DMEM) (Invitrogen) 57 58 supplemented with 10% fetal bovine serum (FBS) and 100 IU/ml penicillin and 100 59 60 IU/ml streptomycin. Mycoplasma testing was performed by DAPI staining and

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Morán et al. 8 1 2 3 epifluorescence microscopy in fibroblasts, which yielded negative results. Cells were 4 5 6 balanced for passage number. Multiple series of P10 plates were seeded each with a 7 8 constant number of cells (5x10 5) and cell growth was assessed in normal glucose 9 10 medium. Triplicate plates were trypsinized and counted daily for 4 days. 11 12 13 SDS-PAGE analysis 14 15 Whole cell homogenates were prepared by resuspending 2x10 6 cells in 125 µl 16 17 18 PBS containing 2% (w/v) n-dodecyl ß-D-maltoside. Following 15 min incubation on 19 20 ice, homogenates wereFor centrifuged Peer (30 min, 12000Review g, 4°C). The supernatant was mixed 21 22 with an equal volume of Tricine sample buffer (Biorad) containing 2% (v/v) 2- 23 24 25 mercaptoethanol. The mixture was kept at room temperature for 60 min. Proteins were 26 27 separated on 10 % polyacrylamide gels and transferred to PROTRAN ® nitrocellulose 28 29 membranes (Whatman GmbH). 30 31 32 Cellular fractionation 33 34 Approximately 4x10 6 fibroblasts were harvested, washed in PBS, resuspended in 35 36 an appropriate isotonic buffer (0.25 M sucrose, 10 mM Tris-HCl pH 7.8, 0.2 mM 37 38 TM 39 EDTA, and 0.1 mM PMSF) and homogenized using ReadyPrep Minigrinders 40 41 (Biorad). Unbroken cells and nuclei were pelleted by centrifugation at 1000 g for 10 42 43 44 min. Supernatants were centrifuged at 10000 g for 25 min. The resulting supernatant 45 46 was isolated as the cytoplasmic fraction and subject to Western-blot analysis. 47 48 Assessment of assembly and/or stability of mitochondrial OXPHOS complexes 49 50 51 Two-dimensional blue native gel electrophoresis (2D-BN/SDS-PAGE) and 52 53 mitochondrial complex I in gel activity assay were performed with digitonin-enriched 54 55 mitochondrial fractions from the six patients´ fibroblasts and up to five different control 56 57 58 fibroblasts, as described elsewhere (Nijtmans et al., 2002). 59 60 Antibodies

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Morán et al. 9 1 2 3 Western-blot analyses were performed with antibodies raised against the 4 5 6 following proteins: polyclonal BCS1L (ProteinTech Group); monoclonal BCS1L 7 8 (Abnova); mitochondrial complex I NDUFA9 and NDUFS3 subunits, complex II 9 10 SDHA subunit, complex III Rieske Iron-Sulphur protein (RISP) and core 2 protein, and 11 12 13 complex IV COX2 and COX5A subunits (Molecular Probes); DRP1 and OPA1 (BD 14 15 Biosciences); MFN2 (Abnova); gluthatione peroxidase (GPx, Assay Designs); 16 17 18 gluthatione reductase (GR, Abcam); MnSOD, catalase (CAT), and β-actin (Sigma). 19 20 Peroxidase-conjugatedFor anti-mouse Peer or anti-rabbit Review IgGs (Molecular Probes) were used as 21 22 secondary antibodies. The signal was detected with ECL ® plus (Amersham 23 24 25 Biosciences). Band densities were evaluated by densitometric scanning (Image J). 26 27 Indirect Immunofluorescence 28 29 30 Cells were fixed with 4% paraformaldehyde for 15 min and permeabilized for 15 31 32 min with 0.1% Triton X-100. Cells were incubated in blocking buffer containing 10% 33 34 goat serum for 1h at room temperature. The mouse monoclonal anti-complex V α 35 36 37 subunit (MitoSciences) was used as a primary antibody. Texas Red-conjugated anti- 38 39 mouse (MitoSciences) was used as a secondary antibody. After appropriate rinsing, 40 41 cover slips were mounted in ProLong Gold antifade reagent (Molecular Probes) on 42 43 44 glass slides, and cells were viewed with a Zeiss LSM 510 Meta confocal microscope 45 46 and a 63x planapochromat objective. 47 48 Analysis of intracellular ROS in live cells by confocal microscopy 49 50 51 Cultured skin fibroblasts grown on cover slips were loaded with 5 M 2’, 7’- 52 53 dichlorofluorescein diacetate (DCF-DA, Sigma) for 20 min in the dark in a CO 2 54 55 56 incubator. After loading, the dye was removed and cells were washed twice with PBS 57 58 before image collection with a Zeiss LSM 510 Meta confocal microscope. The image 59 60 collection was carried out with a 20x lens plus a 2x digital zoom using the 488 nm

excitation laser line.

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Morán et al. 10 1 2 3 Measurement of intracellular ROS production by Flow Cytometry 4 5 6 Cells were grown until 65% confluence, trypsinized and resuspended in fresh 7 8 medium. Cell suspensions were incubated for 30 min at 37 ºC in a CO 2 incubator with 5 9 10 M DCF-DA. To measure the intracellular H O levels, the DCF-DA fluorescence was 11 2 2 12 13 excited by a 15 mW laser tuned to 488 nm and the emitted fluorescence was measured 14 15 in a FACscan Becton Dickinson flow cytometer. Data acquisition and analysis was 16 17 18 performed using the Cell Quest program (Becton Dickinson). 19 20 Cell death quantificationFor Peer Review 21 22 Apoptotic cells were quantified in skin fibroblasts with the In Situ Cell Death 23 24 25 Detection kit, Fluorescein (Boehringer Mannheim) – based on the TUNEL method – 26 27 and by Cell Death Detection ELISA PLUS assay (Roche), used to quantify DNA 28 29 fragmentation in fibroblast cells undergoing apoptosis, according to the manufacturers´ 30 31 32 protocols. 33 34 Data Analysis 35 36 All determinations were performed at least in triplicate. Mitochondrial 37 38 39 morphology was determined as follows: the proportion of cells with essentially 40 41 elongated filamentous mitochondria (% of elongated), with an intermediate 42 43 44 mitochondrial morphology (% of intermediate), or with only punctuate mitochondria (% 45 46 of punctuate) was determined in five different controls and each of the patients´ 47 48 fibroblast cell lines by three independent examiners (Guillery et al., 2008). To measure 49 50 51 mitochondrial diameter (width), the Image J software was used after setting the scale in 52 53 the images according to the objective employed to obtain the photographs; results were 54 55 presented as mean (±SD) values of 200 determinations for each cell line (Moran et al., 56 57 58 2010). One-way ANOVA analysis was used for statistics. When a significant P-value 59 60 was obtained, the Scheffé test was employed post hoc to determine differences between

groups. A level of P<0.05 was selected to indicate statistical significance.

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Morán et al. 11 1 2 3 RESULTS 4 5 6 Clinical, genetic and biochemical features of the BCS1L patients. 7 8 9 We studied six patients, in five of whom (P2-P6) a major respiratory chain 10 11 complex III defect was previously identified either in muscle homogenates or 12 13 fibroblasts (Blazquez et al., 2009; De Meirleir et al., 2003; Gil-Borlado et al., 2009). We 14 15 16 first excluded mutations in the mitochondrial MT-CYB gene, which encodes the 17 18 complex III cytochrome b subunit. 19 20 Sequencing Forof the entire Peer BCS1L genomic Review sequence and PCR-RFLP analyses in 21 22 23 fibroblasts DNA revealed the presence of mutations in all patients (Figure 1A, Table 1). 24 25 Patient P1 was homozygous for the reported c.296C>T (p.P99L) mutation (de Lonlay et 26 27 28 al., 2001), and patients P2 and P3, who were siblings, were compound heterozygous for 29 30 the c.133C>T (p.R45C) and c.166C>T (p.R56X) mutations (De Meirleir et al., 2003). 31 32 These three patients, who died before 8 months of age, displayed all the classic 33 34 35 symptoms of BCS1L -associated complex III deficiency except for signs of 36 37 encephalopathy, which were only present in patients P1 and P2. They presented with 38 39 lactic acidosis, hepatopathy, hypotonia, and iron overload, usually associated with 40 41 42 GRACILE syndrome. In addition, patients P1 and P2 had renal dysfunction, and 43 44 patients P2 and P3 exhibited hypoglycemia. Patient P1 showed a combined defect of 45 46 respiratory chain complexes I, III, and IV in fibroblasts, and patient P2 showed a 47 48 49 combined defect of respiratory chain complexes I and III (Table 2). Due to the poor 50 51 growth of the fibroblasts from patient P3, we could not get enough amount of sample 52 53 54 for the spectrophotometric activity measurements. A respiratory chain complex III 55 56 deficiency was previously shown in fibroblasts from this patient (De Meirleir et al., 57 58 2003), although it could not be determined whether it was a single or a combined 59 60 enzyme defect because complex I activity was not measured. Patient P4 was compound

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Morán et al. 12 1 2 3 heterozygous for the mutations c.166C>T (p.R56X), g.1181A>G and g.1164C>G (Gil- 4 5 6 Borlado et al., 2009), and showed a combined defect of respiratory chain complexes III 7 8 and IV in fibroblasts (Table 2). Patient P6 was homozygous for the c.148A>T (p.T50A) 9 10 mutation, and had a single complex III defect in fibroblasts (Blazquez et al., 2009). 11 12 13 Both patients presented with hepatopathy, lactic acidosis, and hypoglycemia, but patient 14 15 P4 presented a worse disease progression leading to death before 1 year of age. Patient 16 17 18 P5 constitutes a particular case. This patient presented with lactic acidosis, hepatopathy, 19 20 encephalopathy, andFor a single complexPeer III defect Review in muscle, which was not observed in 21 22 fibroblasts (Table 2). She harboured the previously reported p.R184C mutation in one 23 24 25 allele (Fernandez-Vizarra et al., 2007; Hinson et al., 2007), and also carried a 26 27 homozygous g.1892A>G genetic variant that, like the previously reported g.1433T>A 28 29 mutation (Visapaa et al., 2002), lies within an intronic region in the 5’ upstream 30 31 32 regulatory sequence of BCS1L . Due to the lack of muscle sample, reverse transcriptase 33 34 PCR analysis of BCS1L cDNA was performed in fibroblasts from patient P5, which 35 36 excluded missplicing due to a putative deep intronic mutation missed by standard 37 38 39 sequencing (data not shown). Similarly, real-time PCR analysis excluded alterations in 40 41 BCS1L transcript levels (data not shown). These results suggest that the g.1892A>G 42 43 44 polymorphism is not pathogenic. No other nucleotide variations were found after 45 46 sequencing the whole BCS1L genomic region, suggesting that the p.R184C change 47 48 might behave as a manifesting heterozygous mutation. In support of this observation, 49 50 51 the p.R184C mutation has been always reported as compound heterozygous in several 52 53 patients presenting with either complex III deficiency or Björnstad syndrome 54 55 (Fernandez-Vizarra et al., 2007; Hinson et al., 2007). Neurological signs were observed 56 57 58 in patients P1, P2, P5 and P6, which could be related to the possible morphogenetic role 59 60 of BCS1L in the formation of neural tube structures (Kotarsky et al., 2007). In patient

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Morán et al. 13 1 2 3 P3 these neurological alterations were not apparent, maybe due to the fact that she died 4 5 6 soon after birth (Table 1). 7 8 Growth capabilities of BCS1L mutated fibroblasts. 9 10 In normal glucose medium, growth rates were reduced in all patients’ fibroblasts 11 12 13 compared to controls, and revealed substantial differences among the mutant cell lines 14 15 investigated (Figure 1B). Fibroblasts from patient P5 showed a moderate growth rate, 16 17 18 whereas those from patient P6 showed a slightly less efficient growth capacity. 19 20 Fibroblasts from patientsFor P1, P2,Peer and P4 exhibited Review similar growth rates, slower than 21 22 those from patients P5 and P6. Cell growth was severely compromised in fibroblasts 23 24 25 from patient P3, which presented the slowest cell growth rate. 26 27 Effect of the BCS1L mutations on the expression, subcellular location and 28 29 assembly of the BCS1L protein. 30 31 32 To examine the steady-state expression levels of the BCS1L protein and 33 34 35 respiratory chain subunits in mutant fibroblasts, whole cell lysates were analyzed by 36 37 dodecylsulfate-polyacrylamide gel electrophoresis (SDS–PAGE) followed by Western- 38 39 blot. As observed in Figure 2A, BCS1L expression was similar in patients´ and control 40 41 42 fibroblasts. However, the levels of the complex III Rieske Iron-Sulphur protein (RISP) 43 44 showed prominent differences among patients. A ~40-60 % reduction in RISP 45 46 expression was observed in patients P1, P2 and P3, while in the remainder three patients 47 48 49 protein levels were similar to controls. In agreement with previous observations this 50 51 reduction was specific, as the amount of other complex III subunits (such as Core2) was 52 53 54 comparable between patients and controls (Fernandez-Vizarra et al., 2007; Hinson et al., 55 56 2007). The steady-state levels of other respiratory chain subunits, such as complex I 57 58 NDUFA9, complex IV COX5A or complex II SDHA, were comparable between 59 60 patients and controls.

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Morán et al. 14 1 2 3 In our patients, most mutations were located within the mitochondrial targeting 4 5 6 and sorting domains of the BCS1L protein (Figure 1A). To investigate the cellular 7 8 localization of BCS1L, we performed subcellular fractionations of control and mutant 9 10 fibroblasts, and the cytoplasmic fractions were subject to Western-blot analysis. As 11 12 13 observed in Figure 2B, BCS1L was absent in the cytosolic fractions of control 14 15 fibroblasts, while it accumulated at high levels in the cytoplasmic fractions from 16 17 18 patients P2, P3, P4, and P6, and mildly accumulated in patients P1 and P5. 19 20 Mitochondrial contaminationFor wasPeer excluded withReview an antibody against the complex IV 21 22 COX2 subunit. These results suggest that the mitochondrial import of BCS1L might be 23 24 25 hampered in all patients. 26 27 Next, mitochondrial-enriched fractions were separated by blue native gel 28 29 electrophoresis (BN-PAGE) to evaluate both the intramitochondrial BCS1L steady-state 30 31 32 levels, and the physical state of the BCS1L complex in mutant fibroblasts (Figure 2C). 33 34 As expected, Western-blot analysis with an antibody against the BCS1L protein showed 35 36 a marked reduction in the amount of the ~400 kDa BCS1L complex in fibroblasts from 37 38 39 patients P2, P3, P4, and P6 relative to controls, and normal levels in patients P1 and P5. 40 41 Additionally, a substantial accumulation of two lower subcomplexes of ~200 kDa and 42 43 44 ~100 kDa was observed in patients P1-P5. These signals could correspond to either 45 46 BCS1L complex assembly intermediates or breakdown products that accumulate due to 47 48 possible structural alterations in the mutated BCS1L proteins. These results were 49 50 51 confirmed by two-dimensional blue native gel electrophoresis (2D-BN/SDS-PAGE) of 52 53 the mitochondrial native complexes (Supplemental Figure S1). Controls predominantly 54 55 showed the fully-assembled BCS1L complex (~400 kDa) with absence of lower 56 57 58 subcomplexes (Supplemental Figure S1, upper panel), whereas all patients’ samples 59 60 showed either reduced levels of the fully-assembled BCS1L complex or an

accumulation of lower molecular weight subcomplexes. These results indicate that the

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Morán et al. 15 1 2 3 assembly and/or stability of the mitochondrial BCS1L complex are also severely 4 5 6 affected in all patients. 7 8 Physical state of the mitochondrial respiratory chain complexes in BCS1L mutated 9 10 fibroblasts. 11 12 13 To evaluate the physical state of the respiratory chain complexes in our BCS1L 14 15 16 mutant fibroblasts, a duplicate BN-PAGE gel was blotted and further analyzed by 17 18 Western-blot using antibodies raised against different OXPHOS subunits. When an 19 20 antibody against theFor complex IIIPeer RISP subunit Review was used, decreased steady-state levels 21 22 23 of the ~450 kDa complex III dimer were found in fibroblasts from patients P1-P3 24 25 (Figure 2D, third panel). Additionally, the RISP subunit accumulated in a ~300 kDa 26 27 28 subcomplex in patients P1 and P4. This accumulation product was absent in the control 29 30 samples, and did not co-localize with any of the BCS1L complexes. Complex III 31 32 expression levels were normal in patients P5 and P6. Therefore, our results indicate a 33 34 35 complex III assembly or stability defect in patients P1-P4, which correlated with the 36 37 complex III enzyme activities shown in Table 2. Incubation with an antibody that 38 39 recognizes the complex I NDUFS3 subunit (Figure 2D, second panel) revealed 40 41 42 decreased steady-state levels of fully-assembled complex I in patients P1, P2 and P3, 43 44 but normal complex I levels in the remaining patients. In agreement with the 45 46 spectrophotometric respiratory chain enzyme measurements in Table 2, a complex I in- 47 48 49 gel activity (IGA) assay (Figure 2D, upper panel) also showed low complex I activities 50 51 in patients P1, P2 and P3 compared to controls. Patient P5 also displayed a slight 52 53 54 reduction in complex I activity. This reduction neither correlated with the previous 55 56 biochemical activity measurements nor with the normal expression levels of complex I, 57 58 raising doubts about its functional significance. Patients P4 and P6 showed normal 59 60 complex I enzyme activities. Western blot analysis using an antibody against complex

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Morán et al. 16 1 2 3 IV COX5A subunit revealed slightly decreased steady-state levels of this complex in 4 5 6 patients P1 and P4 (Figure 2D, fourth panel), which also correlated with the 7 8 corresponding cytochrome c oxidase activities. Finally, the levels of respiratory chain 9 10 complex II were comparable between all patients and controls (Figure 2D, fifth panel). 11 12 13 The signals from the blots were quantified, and the complex I/complex II, complex 14 15 III/complex II, and complex IV/complex II ratios were used as numerical values for the 16 17 18 expression levels of fully-assembled complexes I, III and IV, respectively 19 20 (Supplemental FigureFor S2). This Peer whole data setReview confirmed a combined assembly/stability 21 22 defect of the respiratory chain in patients P1 to P4. 23 24 25 Structural alterations of the mitochondrial network. 26 27 To assess the mitochondrial morphology of our BCS1L mutant fibroblasts, we 28 29 performed confocal microscopy studies by immunofluorescence using an antibody 30 31 32 against the ATP synthase (complex V) α-subunit (Figure 3A). As expected, control 33 34 fibroblasts displayed a majority of highly elongated branched mitochondrial filaments, 35 36 37 and the presence of some shorter tubules in a minority of control cells (Benard and 38 39 Rossignol, 2008; Guillery et al., 2008). Conversely, all patients´ fibroblasts showed 40 41 abnormal mitochondrial morphologies in glucose medium, with predominantly 42 43 44 fragmented phenotypes along with the accumulation of perinuclear short entangled 45 46 tubules, punctuate mitochondria and ring structures (Figures 3B and 3C). The most 47 48 severe structural alterations were observed in fibroblasts from patients P2 and P3, which 49 50 51 in general displayed a punctuate or an intermediate mitochondrial morphology with 52 53 many short mitochondrial tubules, and a low number of elongated branched 54 55 56 mitochondria (Figure 3C). The mitochondrial transversal section (width) was also 57 58 measured in our control and mutant fibroblasts, since this is the most constant 59 60 dimension of the organelle, and variations in this parameter reflect alterations of the

mitochondrial morphology (Moran et al., 2010; Santamaria et al., 2006). As observed in

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Morán et al. 17 1 2 3 Figure 3D, all patients´ fibroblasts exhibited a significant increase in mitochondrial 4 5 6 diameter (one-way ANOVA main effect: P<0.001), supporting our previous 7 8 observations. 9 10 We also analyzed the expression of proteins that regulate the fusion-fission 11 12 13 events by Western-blot using whole cell lysates from control and BCS1L mutant 14 15 fibroblasts (Figure 4A). The steady-state levels of the DRP1 and OPA1 proteins, 16 17 18 involved in mitochondrial fission and fusion, respectively, were comparable between 19 20 mutant and control Forfibroblasts. Peer Mild decreased Review expression levels of the MFN2 fusion 21 22 protein were found in all mutants. The signals corresponding to MFN2 expression were 23 24 25 quantified, and the densitometric values were normalized with the β-actin antibody, 26 27 used as an internal loading control (Figure 4B). 28 29 BCS1L 30 ROS levels and antioxidant defences in mutated fibroblasts. 31 32 To analyze the intracellular ROS levels, we first used confocal microscopy in 33 34 35 control and mutant live cells incubated with DCF-DA, widely used to determine the 36 37 intracellular levels of ROS, mainly oxygen peroxide (H 2O2) (Figure 5A). Experiments 38 39 showed increased H O levels in patients P1, P2, P3 and P4 compared to controls, while 40 2 2 41 42 these levels remained within control values in patients P5-P6. The H2O2 levels were 43 44 next quantified by flow cytometry (Figure 5B), which confirmed a significant increase 45 46 47 of H 2O2 production in patients P1-P4, a not significant increase in patient P5, and 48 49 normal H 2O2 levels in patient P6. 50 51 52 Next, we analyzed the steady-state expression levels of the cellular antioxidant 53 54 enzymes by Western-blot using whole cell lysates from control and BCS1L mutant 55 56 57 fibroblasts (Figure 5C). The signals from triplicate blots were quantified by 58 59 densitometry, normalized for the expression levels of β -actin, and then expressed as the 60 percentage of control cells (Figure 5D). The steady-state levels of mitochondrial

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Morán et al. 18 1 2 3 manganese superoxide dismutase (MnSOD) were increased in patients P1, P3, P4 and 4 5 6 P6, while patients P2 and P5 showed a normal expression of this enzyme. The catalase 7 8 (CAT) steady-state levels were upregulated in patient P2, and downregulated in patient 9 10 P3, while its expression was normal in the remaining patients. Another ROS scavenging 11 12 13 enzyme, glutathione peroxidase (GPx), showed mildly increased steady-state levels in 14 15 patients P1, P2 and P3, and a normal expression in the other patients. Finally, the 16 17 18 glutathione reductase (GR) expression levels were only increased in patient P1. These 19 20 results show a generalFor overexpression Peer of the Reviewantioxidant defences in all patients, except 21 22 P5, compared to the controls. 23 24 BCS1L 25 Analysis of apoptotic cell death induction in mutated fibroblasts. 26 27 Mitochondrial fragmentation and excessive ROS production have both been 28 29 widely associated with programmed cell death (Suen et al., 2008; Valko et al., 2007). 30 31 32 The possible effect of BCS1L mutations on cell death induction was evaluated by using 33 34 an epifluorescence microscopy TUNEL assay (Figure 6A). Increased amounts of 35 36 apoptotic nuclei were observed in fibroblasts from patients P1, P2 and P3, but 37 38 39 unexpectedly, not in patient P4. Patients P5 and P6 also presented a similar number of 40 41 apoptotic nuclei relative to the controls (Figure 6B). Accordingly, increased nuclear 42 43 44 DNA fragmentation was confirmed in fibroblasts from patients P1, P2 and P3 (Figure 45 46 6C), suggesting an effect of their specific BCS1L mutations on cell death induction. 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Morán et al. 19 1 2 3 DISCUSSION 4 5 6 To better understand the pathophysiology of BCS1L mutations, we have 7 8 characterized cultured skin fibroblasts from six complex III-deficient patients with 9 10 defects in this putative complex III assembly factor. Based in our data, mutations in the 11 12 13 BCS1L gene affect mitochondrial function at least at four levels: i) by altering the 14 15 mitochondrial import and assembly of the BCS1L protein, thus hampering the 16 17 18 biosynthetic pathway and activity of the BCS1L complex; ii) by destabilizing the RISP 19 20 subunit; iii) by hinderingFor the assemblyPeer or stability Review of respiratory complexes I, III and 21 22 IV, which in turn would lead to increased ROS production and cell death; and iv) by 23 24 25 inducing structural alterations in the mitochondrial network morphology. 26 27 Although no clear correlation could be established between the location of the 28 29 mutations and the type of clinical presentation, the severity of the clinical phenotypes 30 31 32 apparently correlated with the functional alterations observed in fibroblasts. In fact, in 33 34 patients P1-P4, who consistently showed onset of symptoms at birth and early death, we 35 36 found severe delays in cell growth rates, enzyme deficiencies and assembly defects of 37 38 39 respiratory chain complexes I, III and IV, increased production of reactive oxygen 40 41 species and altered expression of the ROS scavenging enzymes. Furthermore, cells from 42 43 44 patients P1-P3 displayed a higher tendency to undergo apoptosis than controls and 45 46 fibroblasts from the remainder patients. These alterations were evident in cells grown in 47 48 glucose medium, in contrast with other studies in which the induction of metabolic 49 50 51 stress was necessary in order to enhance the pathophysiological phenotypes of 52 53 fibroblasts with OXPHOS defects (Guillery et al., 2008; Zanna et al., 2008). Although 54 55 an initial limitation of this study was the fibroblast culture system, which uses a cell 56 57 58 type that is probably less vulnerable to BCS1L defects than neurons and renal tubule 59 60 cells that are affected in patients, our results highlight the severity of the respiratory

chain dysfunction observed in these cells. Patients P5 and P6 presented relatively more

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Morán et al. 20 1 2 3 benign clinical courses, as evidenced by their later onset of symptoms and longer 4 5 6 survival (i.e., they are still alive at ages 4-5 years). Accordingly, the fibroblasts from 7 8 these patients displayed the best cell growth curves in glucose medium, mild or even 9 10 normal enzyme activities and assembly of the mitochondrial respiratory chain 11 12 13 complexes, normal ROS production, and they did not undergo apoptosis. 14 15 In control cells BCS1L is imported into mitochondria, where it possibly interacts 16 17 18 with other proteins forming a ~400 kDa complex whose functional significance remains 19 20 to be elucidated (Fernandez-VizarraFor Peer et al., 2009). Review In all patients´ fibroblasts, defects in 21 22 the assembly or stability of the BCS1L complex were found in the form of accumulated 23 24 25 low molecular weight subcomplexes that could be caused by structural alterations in the 26 27 mutated BCS1L protein. Additionally, the assembly defects were partially due to a 28 29 stalled import of the BCS1L protein, which would decrease the steady-state levels of the 30 31 32 intramitochondrial BCS1L complex. Nearly all mutations were located at the N- 33 34 terminal domain of BCS1L, which contains all the required information for the 35 36 mitochondrial targeting and sorting of the protein (Folsch et al., 1996; Stan et al., 2003). 37 38 39 This region holds a single transmembrane domain that is essential for anchoring the 40 41 BCS1L protein into the inner membrane. The transmembrane domain is followed by an 42 43 44 amphipathic α-helix that acts as an internal targeting signal. Immediately C-terminal 45 46 there is an import-auxiliary region that, together with the amphipathic segment, is 47 48 essential for the interaction with the TOM mitochondrial import machinery and the 49 50 51 intramitochondrial sorting of BCS1L. The p.P99L mutation found in patient P1 52 53 probably modifies the tertiary structure of the BCS1L protein immediately after the 54 55 56 mitochondrial auxiliary import domain (Folsch et al., 1996; Petruzzella et al., 1998; 57 58 Stan et al., 2003). Patients P2 and P3 harboured the p.R56X nonsense mutation, which 59 60 creates a premature stop codon in the membrane translocation domain, together with the

p.R45C mutation, which changes a highly conserved aminoacid residue within the

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Morán et al. 21 1 2 3 amphipathic α-helix containing the internal targeting sequence (De Meirleir et al., 4 5 6 2003). Patient P4 also harboured the p.R56X nonsense mutation, plus the g.1181A>G 7 8 and g.1164C>G mutations in the 5’UTR region of BCS1L mRNA that affected its 9 10 11 stability (Gil-Borlado et al., 2009). By quantitative RT-PCR experiments, we previously 12 13 demonstrated that out of the total amount of BCS1L transcripts expressed in the 14 15 patient´s fibroblasts, ~15% of these harboured the p.R56X nonsense mutation, while the 16 17 18 remainder transcripts (~85%) carried the 5’UTR mutations. The fact that the mRNA 19 20 harbouring the p.R56XFor mutation Peer was expressed Review at low levels suggested the expression 21 22 of a truncated BCS1L protein. In this regard, the import-auxiliary region is partially lost 23 24 25 in the 56-aminoacid BCS1L truncated form. In yeast, this region plays an essential role 26 27 in the translocation of Bcs1p across the outer membrane, and a precursor lacking this 28 29 30 region cannot be properly inserted into the inner membrane (Folsch et al., 1996). One 31 32 possibility would be that in patients P2-P4, the BCS1L truncated form could compete 33 34 with the BCS1L longer form, encoded by the second allele, for either its interaction 35 36 37 with the TOM complex or its internalization into the inner mitochondrial membrane, 38 39 impeding its proper import and intramitochondrial sorting. The p.T50A mutation found 40 41 in patient P6 changes a mildly conserved aminoacid residue located at the beginning of 42 43 44 the import auxiliary region (Blazquez et al., 2009) that also seems to affect the import of 45 46 the BCS1L protein into the organelle. Interestingly, in patient P5 both the import and 47 48 assembly of the BCS1L protein were mildly hampered, probably by effect of the 49 50 51 p.R184C mutation, although the effect of a second unidentified mutation should also be 52 53 considered. Studies in yeast Bcs1p support the importance of aminoacid residues close 54 55 56 to the equivalent R184 position for Bcs1p activity and stability (Nouet et al., 2009). 57 58 In human cells, BCS1L knockdown caused the disassembly of the mitochondrial 59 60 respiratory chain (Tamai et al., 2008). Accordingly, the steady-state levels of

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Morán et al. 22 1 2 3 mitochondrial complex III were severely decreased in our first three patients, who 4 5 6 exhibited reduced expression levels of the complex III RISP subunit, and curiously, 7 8 presented with iron overload. It might be possible that the RISP subunit that cannot be 9 10 incorporated into complex III could undergo a rapid degradation. Another interesting 11 12 13 possibility would be that BCS1L could be involved in the maturation of the Rieske 14 15 subunit, i.e. through the incorporation of the iron sulphur cluster into the RISP protein. 16 17 18 The observed reduction in complex III levels was accompanied by a mild decrease in 19 20 the steady-state levelsFor of respiratory Peer chain complex Review I, probably as a result of the lack of 21 22 stability of mitochondrial complex III (Acin-Perez et al., 2004). Moreover, decreased 23 24 25 levels of mitochondrial complex IV were observed in two patients, suggesting a 26 27 secondary role of BCS1L in complex IV maintenance. In agreement with this 28 29 observation, other authors reported patients with BCS1L mutations who presented with 30 31 32 combined deficiencies of respiratory chain complexes III and IV (Fernandez-Vizarra et 33 34 al., 2007). Fibroblasts from patients P5 and P6, who showed either normal respiratory 35 36 chain activities or a mild single complex III enzyme defect, respectively, exhibited 37 38 39 normal assembly patterns of the respiratory chain complexes despite lower expression 40 41 levels of the BCS1L complex. A similar observation was reported in Finnish patients 42 43 44 with GRACILE Syndrome (Fellman et al., 2008). 45 46 Currently there are many unresolved questions concerning the intricate 47 48 relationships between mitochondrial function and dynamics, ROS generation and 49 50 51 apoptosis. In this regard, it has been previously proposed that the respiratory chain 52 53 modulates apoptosis possibly through the generation of reactive oxygen species (Kwong 54 55 et al., 2007), and that in this process the mitochondrial fusion and fission machineries 56 57 58 are involved (Suen et al., 2008). Increased ROS production was previously shown in 59 60 lymphoblasts from patients with BCS1L mutations associated with Björnstad Syndrome

or complex III enzyme deficiency (Hinson et al., 2007). In our mutant cell lines, only

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Morán et al. 23 1 2 3 those mutations that affected more severely the mitochondrial respiratory chain function 4 5 6 led to an overproduction of reactive oxygen species (patients P1-P4). Cells probably 7 8 tried to compensate the increased accumulation of H2O2 by up regulating the expression 9 10 of their antioxidant defences. However, the increased expression of the scavenging 11 12 13 enzymes was probably not sufficient to compensate the high H2O2 levels in patients P1- 14 15 P4. In fibroblasts from patient P5, the mutation was probably not causing an abnormal 16 17 18 ROS generation because the respiratory chain enzyme activities were normal. Although 19 20 cells from patient P6For exhibited Peer a mild single Reviewcomplex III defect, suggesting some ROS 21 22 generation through this complex, the high expression of the superoxide dismutase in this 23 24 25 patient was probably sufficient to maintain ROS at normal levels. Our data thus support 26 27 a direct link between defects in mitochondrial respiratory chain activities and ROS 28 29 production, and agree with previous studies in which a direct correlation between the 30 31 32 severity of OXPHOS defects and ROS production was observed (Verkaart et al., 2007). 33 34 Conversely, in a recent work we showed normal ROS levels in fibroblasts from severely 35 36 CI-deficient patients (Moran et al., 2010). These conflicting results may indicate that the 37 38 39 impact on ROS production of mutations affecting OXPHOS complexes is not simple. 40 41 Probably the effect of each mutation would depend on how it alters: i) the 42 43 44 function/conformation of the affected protein, ii) the interactions with other respiratory 45 46 chain components, and iii) as a result, the electron flux trough the whole respiratory 47 48 chain. Therefore, not only the complex affected but also the type of mutation and the 49 50 51 genetic background of each patient would play a role on mitochondrial ROS production. 52 53 As expected, cells from patients P1-P3 displayed a higher tendency to undergo 54 55 apoptosis than those from patients P4-P6. However, no clear correlation could be 56 57 58 established between the rise in ROS levels and cell death, since in fibroblasts from 59 60 patient P4, H2O2 was generated at high levels with no detectable cell death induction.

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Morán et al. 24 1 2 3 Earlier studies also suggested a role for BCS1L in the maintenance of 4 5 6 mitochondrial morphology (Tamai et al., 2008). In support of these observations, all our 7 8 BCS1L mutated fibroblasts presented fragmentation of the mitochondrial network, 9 10 which could be partially amplified by the downregulated expression of the MFN2 11 12 13 protein. However, it is equally plausible that MFN2 is degraded because of the defect in 14 15 mitochondrial function, thus further studies are necessary in order to elucidate this 16 17 18 issue. Interestingly, these structural alterations neither correlated with the expression 19 20 levels/defective activitiesFor of thePeer mitochondrial Review respiratory chain complexes, nor with the 21 22 increased ROS generation found in some cell lines. These data contrast with other 23 24 25 studies where a clear correlation between the severity of respiratory chain complex I 26 27 defects and a fragmented disposition of the mitochondrial network was documented 28 29 (Koopman et al., 2005), and suggest that marked changes in mitochondrial morphology 30 31 32 may occur independently from the mitochondrial respiratory chain function and ROS 33 34 production. 35 36 Future functional studies based in the development of animal models, such as a 37 38 39 conditional BCS1L knock-out mouse, not yet published, or a battery of knock-in mice 40 41 harbouring different pathogenic mutations found in humans, will be of outmost 42 43 44 importance to elucidate the precise mechanisms that interconnect these processes. These 45 46 studies will probably allow to understand the different clinical manifestations of 47 48 BCS1L-associated disorders, i.e., why the Björnstad Syndrome is so mild compared 49 50 51 with GRACILE Syndrome or with complex III deficiency, or to demonstrate that the 52 53 phenotypic variability of BCS1L mutations really depends on the nature of the mutation 54 55 and the tissue-specific expression of the gene, a hypothesis that has been widely 56 57 58 proposed (Hinson et al., 2007; Kotarsky et al., 2007; Ramos-Arroyo et al., 2009). This 59 60 will contribute to a deeper understanding of mitochondrial diseases, and consequently,

will improve the search of molecular targets to develop future therapeutic approaches.

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Morán et al. 25 1 2 3 ACKNOWLEDGEMENTS 4 5 6 We acknowledge Pilar del Hoyo for her excellent technical support. This work 7 8 was supported by Instituto de Salud Carlos III (grant numbers PI05-0379 and PI08-0021 9 10 to C.U., and PI06-0547 to M.A.M.). 11 12 13 14 15 16 17 18 19 20 For Peer Review 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Morán et al. 26 1 2 3 REFERENCES 4 5 6 Acin-Perez R, Bayona-Bafaluy MP, Fernandez-Silva P, Moreno-Loshuertos R, Perez- 7 8 Martos A, Bruno C, Moraes CT, Enriquez JA. 2004. Respiratory complex III is 9 10 required to maintain complex I in mammalian mitochondria. Mol Cell 13:805- 11 12 13 815. 14 15 Baum H, Rieske JS, Silman HI, Lipton SH. 1967. On the Mechanism of Electron 16 17 18 Transfer in Complex III of the Electron Transfer Chain. Proc Natl Acad Sci 19 20 USA 57:798-805.For Peer Review 21 22 Benard G, Rossignol R. 2008. Ultrastructure of the mitochondrion and its bearing on 23 24 25 function and bioenergetics. Antioxid Redox Signal 10:1313-1342. 26 27 Benit P, Lebon S, Rustin P. 2009. Respiratory-chain diseases related to complex III 28 29 deficiency. Biochim Biophys Acta 1793:181-185. 30 31 32 Blazquez A, Gil-Borlado MC, Moran M, Verdu A, Cazorla-Calleja MR, Martin MA, 33 34 Arenas J, Ugalde C. 2009. Infantile mitochondrial encephalomyopathy with 35 36 unusual phenotype caused by a novel BCS1L mutation in an isolated complex 37 38 39 III-deficient patient. Neuromuscul Disord 19:143-146. 40 41 Cruciat CM, Hell K, Folsch H, Neupert W, Stuart RA. 1999. Bcs1p, an AAA-family 42 43 44 member, is a chaperone for the assembly of the cytochrome bc(1) complex. 45 46 EMBO J 18:5226-5233. 47 48 de Lonlay P, Valnot I, Barrientos A, Gorbatyuk M, Tzagoloff A, Taanman JW, 49 50 51 Benayoun E, Chretien D, Kadhom N, Lombes A, de Baulny HO, Niaudet P, 52 53 Munnich A, Rustin P, Rötig A. 2001. A mutant mitochondrial respiratory chain 54 55 assembly protein causes complex III deficiency in patients with tubulopathy, 56 57 58 encephalopathy and liver failure. Nat Genet 29:57-60. 59 60 De Meirleir L, Seneca S, Damis E, Sepulchre B, Hoorens A, Gerlo E, Garcia Silva MT,

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Morán et al. 30 1 2 3 Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. 2007. Free radicals 4 5 6 and antioxidants in normal physiological functions and human disease. Int J 7 8 Biochem Cell Biol 39:44-84. 9 10 Verkaart S, Koopman WJ, van Emst-de Vries SE, Nijtmans LG, van den Heuvel LW, 11 12 13 Smeitink JA, Willems PH. 2007. Superoxide production is inversely related to 14 15 complex I activity in inherited complex I deficiency. Biochim Biophys Acta 16 17 18 1772:373-381. 19 20 Visapaa I, Fellman ForV, Vesa J, PeerDasvarma A, HuttonReview JL, Kumar V, Payne GS, Makarow 21 22 M, Van Coster R, Taylor RW, Turnbull DM, Suomalainen A, Peltonen L. 2002. 23 24 25 GRACILE syndrome, a lethal metabolic disorder with iron overload, is caused 26 27 by a point mutation in BCS1L. Am J Hum Genet 71:863-876. 28 29 Xia D, Yu CA, Kim H, Xia JZ, Kachurin AM, Zhang L, Yu L, Deisenhofer J. 1997. 30 31 32 Crystal structure of the cytochrome bc1 complex from bovine heart 33 34 mitochondria. Science 277:60-66. 35 36 Zanna C, Ghelli A, Porcelli AM, Karbowski M, Youle RJ, Schimpf S, Wissinger B, 37 38 39 Pinti M, Cossarizza A, Vidoni S, Valentino ML, Rugolo M, Carelli V. 2008. 40 41 OPA1 mutations associated with dominant optic atrophy impair oxidative 42 43 44 phosphorylation and mitochondrial fusion. Brain 131:352-367. 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Morán et al. 31 1 2 3 FIGURE LEGENDS 4 5 6 Figure 1. (A) Schematic representation of the BCS1L cDNA (NM_004328.4 7 8 Genbank reference sequence), slightly modified from (Fernandez-Vizarra et al., 2007). 9 10 Aminoacid or nucleotide changes found in the patients (P1 to P6) are shown with 11 12 13 arrows. Non-coding exons are indicated as -1 and -2. Coding exons representative of the 14 15 BCS1L open-reading frame are indicated as 1 to 7. The numbered bar indicates the 16 17 18 aminoacid position along the protein sequence. The ATG +1 translation initiation site is 19 20 indicated in grey withFor an arrow. Peer The BCS1L Reviewfunctional domains, deduced by homology 21 22 with yeast bcs1, are indicated as follows: TMD, transmembrane domain; MTS, 23 24 25 mitochondrial targeting sequence; IAS, import auxiliary sequence; BCS1L, activity and 26 27 stability domain; AAA-ATPase, AAA-protein family specific ATPase domain. (B) 28 29 Effect of the BCS1L mutations on cell growth. Cells were plated on P10 plates at 5x10 5 30 31 32 cells per plate in glucose-containing media, and counted on a daily basis for 4 days. 33 34 Each data point represents the mean ± SD of values obtained from each cell line tested 35 36 37 at least in triplicate. C1-C2, control fibroblasts. P1-P6, patients’ fibroblasts. 38 39 Figure 2. (A) Western-blot analysis of the BCS1L protein and mitochondrial 40 41 respiratory chain subunits. Samples corresponding to 30 g protein from whole 42 43 44 fibroblast lysates were run through 10% SDS–PAGE gels and subsequently blotted. The 45 46 levels of the BCS1L protein and the different respiratory chain subunits were detected 47 48 49 using antibodies against the indicated proteins. The signal corresponding to β-actin was 50 51 used for normalization. (B) Subcellular fractionations were performed in control and 52 53 54 patients´ fibroblasts, and 15 g protein from cytosolic fractions were subject to 55 56 Western-blot analysis. (C) To evaluate the expression and assembly of the 57 58 intramitochondrial BCS1L protein, BN-PAGE analysis was performed with 59 60 mitochondrial-enriched fractions from control and BCS1L mutated fibroblasts, and

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Morán et al. 32 1 2 3 followed by Western-blot analysis using an antibody against BCS1L. The approximate 4 5 6 molecular masses of three detected BCS1L complexes are indicated. (D) The assembly 7 8 of the mitochondrial respiratory chain complexes was analyzed with 40 g of digitonin- 9 10 11 isolated mitochondria on 4-15% blue native gels. A complex I in-gel activity (IGA) 12 13 assay is shown in the upper panel. Duplicate gels were subject to Western-blot analysis 14 15 using antibodies against the following OXPHOS subunits: complex I (CI) NDUFS3, 16 17 18 complex III (CIII) Rieske Iron-Sulfur protein (RISP), complex IV (CIV) subunit 19 20 COX5A, and complexFor II (CII) Peer SDHA subunit. Review C1-C3, controls. P1-P6, patients. 21 22 Figure 3. Mitochondrial morphology in control and BCS1L mutated fibroblasts 23 24 25 grown in normal glucose media. (A) The mitochondrial morphology of fixed cells was 26 27 analyzed by immunofluorescence using an anti-complex V α subunit antibody. C1-C2, 28 29 BCS1L 30 controls. P1-P6, mutated fibroblasts. Scale bar = 20µm. (B) Different shapes of 31 32 the mitochondrial network. (a, b) depict controls with the classic filamentous 33 34 mitochondrial network. The most numerous shapes found in patients´ fibroblasts 35 36 37 included dense entangled perinuclear mitochondrial networks (c, d), intermediate shapes 38 39 with elongated and short mitochondrial tubules (e, f), circular structures (g), small 40 41 tubules with short branches (h, i, j), vesicles (k), and long fragmented mitochondria (l). 42 43 44 (C) The proportion of cells with essentially elongated filamentous mitochondria (% of 45 46 elongated), with an intermediate mitochondrial morphology (% of intermediate), or with 47 48 only punctuate mitochondria (% of punctuate) was determined. Data are presented as 49 50 51 mean ± SD of the analysis of >400 cells from each patient and five controls. (D) 52 53 Mitochondrial diameter in mutant fibroblasts grown in normal glucose medium. The 54 55 56 results shown are the means ± SD of at least 200 determinations from each cell line. 57 58 One-way ANOVA analysis: significant main effect (P<0.001) was found. Scheffé test 59 60 post hoc : *(P<0.01), significantly different from controls.

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Morán et al. 33 1 2 3 Figure 4. Effect of the BCS1L mutations on the expression of proteins involved 4 5 6 in mitochondrial dynamics. (A) 30 g of total protein from fibroblast lysates were run 7 8 through 10% SDS–PAGE gels and immunoblotted using specific antibodies against the 9 10 11 indicated proteins. C1-C2, controls. P1-P6, patients´ fibroblasts. (B) Relative MFN2 12 13 expression levels. The signals from four independent blots were quantified, and 14 15 normalized for the signal corresponding to β-actin. Data are presented as mean ± SD of 16 17 18 the analysis of individual patients´ fibroblasts and 5 different controls (indicated as C). 19 20 (a.u.), arbitrary units.For Peer Review 21 22 23 Figure 5. Increased ROS production and altered expression of the cellular 24 25 antioxidant defences in the BCS1L mutated fibroblasts. Hydrogen peroxide levels were 26 27 analyzed in live cells (A) by confocal microscopy, or (B) by flow cytometry, using 28 29 30 DCF-DA as a probe. C-, control without DCF-DA incubation. C, control incubated with 31 32 DCF-DA. P1-P6, patients´ fibroblasts incubated with DCF-DA. (C) Unbalanced 33 34 expression of the cellular antioxidant defences. 30 g of total protein from cell lysates 35 36 37 were run through 10% SDS–PAGE gels and immunoblotted using the indicated 38 39 antibodies. MnSOD, mitochondrial superoxide dismutase. CAT, catalase. GPx, 40 41 42 gluthatione peroxidase. GR, gluthatione reductase. C1, C2, controls. P1-P6, patients. 43 44 (D) The signals from three independent blots were quantified, and the densitometric 45 46 values normalized with the anti-β-actin antibody. (a.u.), arbitrary units. Data are 47 48 49 presented as mean ± SD of the analysis of individual patients´ fibroblasts and 5 different 50 51 controls. 52 53 54 Figure 6. Effect of the BCS1L mutations on cell death. (A) Increased apoptotic 55 56 cell death was observed in fibroblasts from patients P1, P2, and P3 by epifluorescence 57 58 microscopy TUNEL assay. C, control. C+, positive control after 5 min incubation with 59 60 12,5 g/ml DNAse I. (B) Apoptotic nuclei were quantified and the data are expressed as

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Morán et al. 34 1 2 3 the mean ± SD of the analysis of ~500 cells from each patient and 5 different controls. 4 5 6 (C) The relative DNA fragmentation was quantified using the Cell Death Detection 7 8 ELISA PLUS assay (Roche). 9 10 11 12 13 14 15 16 17 18 19 20 For Peer Review 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 P1 P2 P3 P4 P5 P6 2 3 Sex Female Male Female Male Female Male 4 Age at onset Birth Birth Birth Birth 19 months 6 months 5 6 Age at death 7 months 3 months 3 weeks 11 months Alive (5 years) Alive (4 years) 7 Fetal growth retardation + + + - - + 8 Lactic acidosis + + + + + + 9 Tubulopathy + + - + - - 10 Hepatopathy + + + + + + 11 Hypoglycemia 12 - + + + - + 13 Failure to thrive + For + Peer - Review + + + 14 Encephalopathy - + - - + - 15 Hypotonia + + + + - + 16 Seizures + - - - + - 17 18 Microcephaly + - - + - + 19 Other symptoms Ptosis Anemia Spasticity in the upper Abnormal subcutaneous 20 and lower limbs fat distribution, hypertrichosis, 21 dysmorphic features, mild 22 sensorineural hearing loss 23 Symmetric bilateral 24 Delayed white matter lesions, Brain MRI n.d. Normal Normal Normal 25 myelination bilateral hyperintense 26 signals in thalamus 27 Laboratory data 28 Iron overload yes yes yes no n.d. no 29 30 % CI Activity n.d. Normal n.d. n.d. Normal Normal 31 % CIII Activity n.d. 64% 39 %* 30 % 38% 26% 32 % CIV Activity n.d. Normal Normal* Normal Normal Normal 33 [p.R56X] + 34 BCS1L mutations [p.P99L]+ [p.P99L] [p.R56X]+[p.R45C] [p.R56X]+[p.R45C] [p.R184C]+[?] [p.T50A]+[ p.T50A] [g.1181A>G, g.1164C>G] 35 36 37 Table 1. Clinical, biochemical and genetic features of the BCS1L -mutated patients. Respiratory chain enzyme activities (in muscle homogenates, except *in fibroblasts) are 38 expressed as the percentages of the residual enzyme activities relative to mean control values. CI, complex I. CIII, complex III. CIV, complex IV. n.d., not determined. 39 40 41 42 43 44 45 John Wiley & Sons, Inc. 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Human Mutation Page 36 of 44

1 2 3 Table 2. Residual activities of the mitochondrial respiratory chain enzymes in cultured skin fibroblasts 4 5 derived from controls and the index patients. 6 7 8 9 Complex I Complex II Complex III Complex IV CS 10 11 Controls range (n=11) 26-50 10-18 28-87 52-105 60-160 12 13 Controls mean±SD 37±10 14±3 60±27 85±26 125±44 14 15 P1 21.4 16.4 17.5 50.7 85 16 17 P2 24.0 16.7 17.8 62.2 103 18 19 P3 n.d. n.d. n.d. n.d. n.d. 20 For Peer Review 21 P4 29.4 9.3 18.5 44.1 65 22 23 P5 30.0 11.7 36.2 76.1 101 24 25 P6 27.1 11.2 27.1 59.5 105 26 27 28 29 30 Enzyme activities measured in our laboratory are expressed as nmol·min -1·mg -1 protein. Abnormal values 31 32 are indicated in bold characters. Enzyme activities of fibroblasts from patient P3 were previously 33 34 measured in (De Meirleir, et al., 2003), and are indicated in Table 1. CS, citrate synthase. n.d., not 35 36 determined. 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 209x297mm (600 x 600 DPI) 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 38 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 195x230mm (600 x 600 DPI) 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 39 of 44 Human Mutation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 189x262mm (600 x 600 DPI) 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 40 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 209x297mm (600 x 600 DPI) 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 41 of 44 Human Mutation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 For Peer Review 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 Supplemental Figure S1. For the separation of the individual components from the 49 50 native BCS1L complexes, 10% tricine SDS-gels were run in the second dimension, 51 52 blotted and incubated with the BCS1L antibody. The ~100 kDa and ~200 kDa 53 54 55 subcomplexes that accumulate in the patients are indicated as BCS1La and BCS1Lb, 56 57 respectively. BCS1Lc corresponds to the fully-assembled BCS1L complex. Arrows 58 59 indicate the directions of the first (1D) and second (2D) dimensions, respectively. 60

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 For Peer Review 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Supplemental Figure S2. The signals obtained from the IGA assays and Western-blots 42 43 44 corresponding to the respiratory chain complexes from three independent blue native 45 46 blots were quantified, the densitometric values were normalized for the expression 47 48 levels of mitochondrial complex II, and expressed as the percentage of the control 49 50 51 values. (a.u.), arbitrary units. Data are presented as mean ± SD of the analysis of 52 53 individual patients´ fibroblasts (P1-P6) and 2 different controls (C). 54 55 56 57 58 59 60

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