Riboflavin responsive mitochondrial myopathy is a ...

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Brautigam, C.A., Wynn, R.M., Chuang, J.L., Machius, M., Tomchick, D.R., Chuang, D.T., 2006. Structural insight into interactions between dihydrolipoamide ...
Mitochondrion 18 (2014) 49–57

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Riboflavin responsive mitochondrial myopathy is a new phenotype of dihydrolipoamide dehydrogenase deficiency. The chaperon-like effect of vitamin B2 Rosalba Carrozzo a,⁎, Alessandra Torraco a, Giuseppe Fiermonte b, Diego Martinelli c, Michela Di Nottia a, Teresa Rizza a, Angelo Vozza b, Daniela Verrigni a, Daria Diodato a, Giovanni Parisi b, Arianna Maiorana c, Cristiano Rizzo d, Ciro Leonardo Pierri b, Stefania Zucano b, Fiorella Piemonte a, Enrico Bertini a, Carlo Dionisi-Vici c,⁎⁎ a

Unit of Molecular Medicine for Neuromuscular and Neurodegenerative Diseases, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy Department of Biosciences, Biotechnologies and Biopharmaceutics, Laboratory of Biochemistry and Molecular Biology, University of Bari, Via E. Orabona 4, Bari, Italy Division of Metabolism, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy d Department of Laboratory Medicine, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy b c

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Article history: Received 27 May 2014 accepted 15 September 2014 Available online 22 September 2014 Keywords: Dihydrolipoamide dehydrogenase deficiency Mitochondrial myopathy Branched-chain amino acids α-Keto acids Riboflavin Chaperon

a b s t r a c t Dihydrolipoamide dehydrogenase (DLD, E3) is a flavoprotein common to pyruvate, α-ketoglutarate and branched-chain α-keto acid dehydrogenases. We found two novel DLD mutations (p.I40Lfs*4; p.G461E) in a 19 year-old patient with lactic acidosis and a complex amino- and organic aciduria consistent with DLD deficiency, manifesting progressive exertional fatigue. Muscle biopsy showed mitochondrial proliferation and lack of DLD cross-reacting material. Riboflavin supplementation determined the complete resolution of exercise intolerance with the partial restoration of the DLD protein and disappearance of mitochondrial proliferation in the muscle. Morphological and functional studies support the riboflavin chaperon-like role in stabilizing DLD protein with rescue of its expression in the muscle. © 2014 Elsevier B.V. and Mitochondria Research Society.

1. Introduction Mammalian dihydrolipoamide dehydrogenase (dihydrolipoamide: NAD+ oxidoreductase, DLD, also designed as lipoamide dehydrogenase/LAD, EC 1.8.1.4) is the common flavoprotein component of three mitochondrial multi-enzyme complexes: pyruvate dehydrogenase complex (PDHc), α-ketoglutarate dehydrogenase complex (KGDHc) and branched-chain α-keto acid dehydrogenase complex (BCKDHc). DLD is also defined as E3, because within these complexes it catalyzes the third stage of each reaction (Cameron et al., 2006; Patel and Roche, 1990). DLD is also a component of the glycine cleavage system, another mitochondrial enzyme complex, where it is known as protein L (Kikuchi and Hiraga, 1982). In the context of these four multi⁎ Correspondence to: R. Carrozzo, Unit of Molecular Medicine for Neuromuscular and Neurodegenerative Diseases, Bambino Gesù Children's Hospital, IRCCS, Piazza S. Onofrio, 4, 00165 Rome, Italy. Tel.: +39 06 68592102; fax: +39 06 68592024. ⁎⁎ Correspondence to: C. Dionisi-Vici, Division of Metabolism, Bambino Gesù Children's Hospital, IRCCS, Piazza S. Onofrio, 4, 00165 Rome, Italy. Tel.: + 39 06 68592275; fax: + 39 06 68592791. E-mail addresses: [email protected] (R. Carrozzo), [email protected] (C. Dionisi-Vici).

http://dx.doi.org/10.1016/j.mito.2014.09.006 1567-7249/© 2014 Elsevier B.V. and Mitochondria Research Society.

enzyme complexes, E3 utilizes dihydrolipoic acid and NAD+ to generate lipoic acid and NADH (Carothers et al., 1989). The active enzyme is a homodimer of 51 kDa subunits with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) (Hong et al., 1997). The DLD gene, located on chromosome 7q31–32, is approximately 20 kb long and contains 14 exons (Grafakou et al., 2003). Since the E3 is involved in different metabolic pathways within mitochondria, the clinical presentation of DLD deficiency (OMIM #246900) varies greatly, ranging from a severe neonatal presentation with hypoglycemia, ketoacidosis and encephalopathy or Leigh-like encephalopathy, psychomotor retardation and Reye-like syndrome to a milder presentation with exertional fatigue and myopathic features. The majority of patients become symptomatic within the first 1–2 years of age, and neonatal onset cases are associated with the worse prognosis; while some non-neonatal patients have been reported to survive into their second and third decades of age (Barak et al., 1998; Brassier et al., 2013; Quinonez et al., 2013; Quintana et al., 2010; Shaag et al., 1999). The wide clinical spectrum of DLD deficiency has also been attributed to the effects of different DLD mutations on the protein stability (Shaag et al., 1999) as well as on its ability to dimerize (Odièvre et al.,

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2005; Shany et al., 1999) or interact with other components of the three α-keto acid dehydrogenase complexes (Brautigam et al., 2006). E3 deficiency is often associated with an increased urinary excretion of α-keto acids, along with increased plasma levels of lactate and branched-chain amino acids leucine, valine and isoleucine. We report on the clinical, morphological, biochemical and molecular findings of a 19-year-old young man presenting with muscle weakness and pain, intermittent elevation of blood lactate, ketoacidosis and creatine kinase. The patient is compound heterozygous for two novel pathogenic mutations in the DLD gene, and he was strikingly responsive to riboflavin treatment with the complete reversion of myopathic features.

2. Materials and methods 2.1. Case report This 19 year-old male patient was the first born of healthy Italian parents, following a spontaneous miscarriage in his mother. Neonatal parameters, weight, length and head circumference were in the normal range at birth. Early psychomotor development was uneventful, but hepatomegaly and recurrent episodes of drowsiness with ketonuria were noticed in infancy. At age 3 y he started with an inability to climb stairs together with episodes of muscle pain, weakness, and perioral cyanosis during moderate physical activity. These episodes lasted up to 5 min, disappearing at rest, and they started again after muscle exercise. At age 4.5 y following a 17 h fasting he presented with an acute episode of lethargy, hypotonia, sweating and pallor. Biochemical evaluation at the emergency room revealed liver cytolysis (ASAT 199/ALAT 209 UI/L; nv b 40), mild acidosis (base excess −7.6) and massive ketonuria, with normal levels of blood ammonia and creatine kinase. Glucose and lactate in blood were not recorded at that time. Urinary organic acids (OAs) profile documented lactic aciduria and mild elevation of α-keto acids. Abdominal scan showed an enlarged and hyperechogenic liver. Brain CT scan and MRI were normal. A liver biopsy documented preserved architecture of liver parenchyma, swollen appearance of hepatocytes with centrally placed nuclei, pale and sometimes vacuolated cytoplasm, abundant PAS-positive cytoplasmic glycogen deposits, removed by diastase digestion, large and medium-sized drop steatosis. Around age 15 y he complained about episodes of myalgias and progressive exertional fatigue, together with numbness and weakness in the lower limb. Physical examination at age 17 y showed severe obesity ( IBW 177%, BMI 36.8), hepatomegaly, muscle weakness with limb girdle distribution and worsening of exertional fatigue with exercise-test interrupted after 1 min. Electromyography and nerve conduction studies were unremarkable. Routine biochemical examinations displayed intermittent hyperCKemia (up to 20×), increase of LDH (up to 10 ×), slight elevation of blood transaminases (ASAT 60/ALAT137), and gamma-GT (70–80 UI/L; nv b 40) along with mild hypercholesterolemia and hypertriglyceridemia. Metabolic investigations documented intermittent elevation of blood lactate (range 1.5–6.0 mmol/L; nv b 1.2) and a characteristic profile evocative of E3 deficiency, including persistent elevation of plasma branched-chain amino acids (valine peak 479 mmol/L, nv 150–320; isoleucine peak 146 mmol/L, nv 30–100 mmol/L; leucine peak 307 mmol/L, nv 50–190), detectable alloisoleucine (range 2–13 mmol/L; nv 0) and increased excretion of lactate, pyruvate, α-ketoglutarate, and branched-chain αhydroxy and α-keto acids (Supplementary Fig. 1). A first attempt of supplementation with thiamine and Vitamin B6 yielded no clinical effect. Subsequently, riboflavin was initiated at the dosage of 220 mg/ day which resulted after one year of treatment in the complete resolution of exercise intolerance, the normalization of blood lactate and of urinary organic acids, without significant changes of branched-chain amino acids in plasma.

2.2. Molecular investigations 2.2.1. Mutational analysis Genomic DNA was purified from blood and cultured skin fibroblasts using QIAamp DNA mini kit (QIAGEN, Valencia, CA, USA). The coding exons and exon–intron boundaries of nuclear-encoded DLD gene (GenBank accession # NM_000108) were PCR amplified using intronic primers (details of sequences and PCR conditions are available in Supplementary Table 1). PCR products were purified with Exo-Sap enzyme (USB, Cleveland, OH, USA). Mutation screening was performed by bidirectional sequencing using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) on an ABI3130xl automatic DNA Analyzer. Mutations were confirmed by resequencing a newly amplified PCR product. Segregation of the c.1382G N A and c.118+1GNT mutations were confirmed within the family. To assess the functional effect of the c.118+1GNT mutations, total RNA was extracted from cultured skin fibroblasts by TRIzol (Life Technologies) and reverse transcribed by using MultiScribe™ Reverse Transcriptase (Life Technologies). The region encompassing exons 1 to 3 was PCR-amplified using specific primers in the coding sequence and the resulting bands were gel purified using the MinElute Gel Extraction kit (QIAGEN, Valencia, CA, USA) and sequenced (Supplementary Table 1). DLD transcript stability was assayed by quantitative RT-PCR in an ABI PRISM 7500 Sequence Detection System (Life Technologies), using Power SYBR® Green I dye chemistry. DLD transcript expression was normalized to the level of GUSB, using the comparative Ct method outlined in Livak and Schmittgen (2001). 2.2.2. “In silico” analysis The pathogenicity of the human c.1382GNA mutation was predicted by using four bioinformatic tools based on heuristic methods: SIFT (http://sift.jcvi.org), PolyPhen-2 (http://genetics.bwh.harvard.edu/ pph2), SNPs&GO (http://snps-and-go.biocomp.unibo.it/snps-and-go), and MutPred (http://mutpred.mutdb.org). 2.2.3. Molecular modeling investigations Homo sapiens E3_WT protein sequence was obtained from the NCBI database. E3 orthologs from mammalia, fungi and plants were sampled by screening the Ref_Seq protein database by using blastp (http://blast. ncbi.nlm.nih.gov). A multiple sequence alignment with selected E3 subunit orthologs was obtained by using ClustalW (Persson, 2000). Modeler (Sánchez and Sali, 2000) was used to calculate structural models of human E3_G461E mutant protein by using as template the structure of human E3 (PDB_ID: 1ZMC), crystallized as a functional homodimer with cofactors FAD and NAD+ bound (Brautigam et al., 2005). Cofactors FAD and NAD+ were thus docked into the 3D model of the human E3_G461E mutant protein by using cofactor coordinates available in 1ZMC (Brautigam et al., 2005). Final models were examined in PyMOL (http://www.pymol.org/) and where side-chain packing led to clashes in the protein structure, alternative side-chain rotamers were evaluated. 2.3. Histochemical, biochemical and western blotting analyses in muscle tissue Quadriceps muscle and needle skin biopsies were performed after a written informed consensus. Isopentan frozen muscle sample was analyzed and cryostatic cross sections were processed according to standard histochemical procedures. A second muscle biopsy was performed one year later after treatment with riboflavin. Respiratory chain complex activities were measured in muscle homogenate using a reported spectrophotometric method (Bugiani et al., 2004). For electrophoresis in SDS polyacrylamide gels (SDS-PAGE), 40 μg of muscle homogenate and skin fibroblast mitochondria was loaded in a 12% denaturating gel. For immunoblot analysis, PVDF membranes

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were probed with monoclonal antibodies, purchased from Mitosciences (Eugene, OR, USA), which recognize the following proteins: complex I — 75 kDa subunit (NDUFS1); complex II — 70 kDa (SDH70); complex III — core 2 subunit (Core protein 2); complex IV — subunit IV (COXIV); and VDAC (porin) protein, the latter used as an internal control for equal loading. The specific antibodies for PDHc, the pyruvate dehydrogenase Antibody Cocktail (MSP02), and the anti-E3 antibody, were purchased from Mitosciences and Proteintech, respectively. Reactive bands were detected using the Lite Ablot Extend Long Lasting Chemiluminescent Substrate (Euroclone, Pero, Italy). Densitometry analysis was performed using the Quantity One Software (BioRad, Hercules, CA, USA). 2.4. Enzymatic activities in fibroblast mitochondria The PDHc, KGDHc and BCKDHc enzymatic activities were assayed essentially as previously described (Munujos et al., 1996; Nakai et al., 2000). Mitochondria (75 μg of protein) isolated from control and patient fibroblasts (Garcia et al., 2000) were solubilized with 0.05% (wt/vol) Triton X-100, and centrifuged at 10,000 ×g for 10 min at 4 °C. Supernatant was added to a mixture containing 100 mM Tris·HCl at pH 7.6, 4 mM CoA, 4 mM NAD+, 4 mM thiamine pyrophosphate and 40 μM rotenone. The assay was started with 8 mM α-ketoglutarate, 8 mM pyruvate or 12 mM α-ketoisocaproate and the formation of NADH was followed at 340 nm. Complex V activity (in the direction of ATP synthesis) was measured in fibroblast mitochondria of patient and age-matched controls, with pyruvate + malate, malate, or succinate as substrates, and using a reported spectrophotometric method (Rizza et al., 2009). 2.5. Cell treatment Cultured skin fibroblasts were treated using 5.3 μM and 50 μM riboflavin (Ribo, Sigma-Aldrich, USA) for 72 h; mitochondria obtained after treatment were used to measure PDHc, KGDHc, BCKDHc and mitochondrial complex V activities (as reported above). In addition, total cellular ATP content and ROS level were also assessed before and after 5.3 μM riboflavin treatment. 2.5.1. Cellular ATP content The content of cellular ATP was assayed luminometrically using the ATPLITE 1 STEP (PerkinElmer, Boston, MA, USA) according to the procedure recommended by the manufacturer and using 2 × 104 cells. Six different controls have been used in four different experiments, either in a regular medium as well as in a galactose-supplemented medium (5 mM). For patient, the experiments were run four times in a regular medium and four times in a galactose-supplemented medium. For either sample, each experiment was run three times at the same moment. Luminescence was measured using the EnSpire® Multimode Plate Readers (PerkinElmer, USA). 2.5.2. Reactive oxygen species measurements For intracellular ROS production, fibroblasts (2 × 104) were incubated with 15 μM H2DCFDA (Molecular Probes, Invitrogen, Paisley, UK) diluted in Krebs–Henseleit Modified buffer (Sigma-Aldrich, USA) for 45 min at 37 °C. After washing with the same buffer, the cells were incubated for 20 min with 1 mM H2O2. Fluorescence was measured at excitation and emission wavelengths of 490 and 525 nm, respectively, using the EnSpire® Multimode Plate Readers (PerkinElmer, USA). 2.6. Data and statistics Statistical analysis of the data was performed using Student's t-test. A p value b 0.05 (*) was considered significant, p b 0.005 (**) was considered highly significant, and p b 0.0005 (***) was considered extremely significant.

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3. Results 3.1. Molecular investigations Sequence analysis of coding exon and flanking intronic sequences revealed that the patient was compound heterozygous for two novel mutations in DLD, the c.118 + 1G N T was inherited from the mother, and the c.1382GN A [p.G461E] inherited from the father (Fig. 1A). PCR amplification of cDNA with oligonucleotide primers encompassing the region of the c.118+1GN T mutation showed a lower band in association to the wild type (Fig. 1B); in fact, the mutation produces a skipping of exon 2 and predicts a frameshift and the synthesis of a truncated protein [p.I40Lfs*4]. Quantitative RT-PCR did not show any variation in the amount of DLD transcript between control and patient when GUSB was used as reference gene (data not shown). The second mutation, c.1382G NA, affects a highly conserved residue, p.G461E (Fig. 2A), which is located in the interface domain of the homodimer (Fig. 2b) and predicted to be pathogenic by different software (SIFT, POLYPHEN, SNP&GO). It is therefore likely that this mutation diminishes the ability of E3 to homodimerize. In order to study the protein region affected by the mutation G461E, a 3D comparative model of the functional dimer was built and consists of a homodimer with NAD+ and FAD bound to each monomer. Both monomers consist in alternant α-helices and β-sheets. Mutated residues of each monomer are indicated together with the close ones involved in ionic interactions. In the human wild type E3 subunit the G461 side chain is located within a region involved in contacts with side-chains of residues from the close monomer (Fig. 2C). In particular G461 is adjacent to E462 that forms a salt bridge with R393 and both residues form hydrophilic interactions with the close Y359. It should be stressed that due to the two-fold symmetry in the human E3 homodimer, each of these contacts is present twice for every homodimer (Fig. 2D). Considering these results it appears that the substitution of the original G461 with a glutamic residue introduces a further negative charge, impairing local charge equilibrium. The novel missense changes were not detected in a large set of in-house ethnically-matched control chromosomes, in dbSNP (www.ncbi.nlm.nih.gov/projects/SNP/) and Exome Variant database (www.evs.gs.washington.edu/EVS/). Moreover, the novel p.G461E mutation resulted as damaging using bioinformatic tools based on heuristic methods for predicting pathogenic variants.

3.2. Western blotting, histochemical, and biochemical analyses in muscle tissue The novel DLD variants severely affected the amount of the corresponding protein product as demonstrated by western blotting analysis in the first muscle biopsy (−97%; Fig. 3A, Pt. 1st). Remarkably, western blotting in muscle biopsy performed after the patient was treated with riboflavin (Pt. 2nd), displays a recovery of the amount of E3 (Fig. 3A, Pt. 2nd). Moreover, the first muscle biopsy documented mitochondrial proliferation with clearly increased SDH and COX histochemical staining with the absence of COX negative fibers (Fig. 3B, panels A–B). In agreement with western blotting results, the second muscle biopsy performed after 1 year of riboflavin treatment showed a normalization of pathological signs (Fig. 3B, panels C–D). To assess whether riboflavin affected the amount of those complexes containing FAD as a cofactor, we evaluated the steady-state levels of some subunits of the respiratory chain complexes and the PDHc. In SDS-PAGE the steady-state levels of NDUFS1, SDH70, Core 2 and COX IV, belonging to complexes I, II, III and IV, respectively, were normally expressed (Supplementary Fig. 2a). Moreover, PDHc subunits (E2, E2/ 3bp, E1α) were also normal when compared to controls (Supplementary Fig. 2b).

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Fig. 1. Molecular analysis. A, Electropherogram encompassing the heterozygous c.118+1GNT and c.1382GNA mutations in DLD gene. The c.118+1GNT produces a skipping of exon 2 and predicts a frameshift and the synthesis of a truncated protein [p.I40Lfs*4]; this mutation was inherited from the mother. The c.1382GNA [p.G461E] mutation, located in exon 13, was present in the father. B, PCR and schematic representation of the patient's cDNA region encompassing the heterozygous c.118+1GNT mutation showing a skipping of exon 2.

The respiratory chain complexes activities did not show any significant differences with controls in both muscle biopsies, and also between themselves (data not shown). 3.3. Enzymatic activities in fibroblast mitochondria The enzymatic activities of PDHc, KGDHc and BCKDHc in isolated fibroblast mitochondria were significantly reduced between 60 and 80% (p b 0.0005) compared to controls (Fig. 4A). In the same sample the rate of ATP synthesis was found to be significantly reduced, with respect to the control mean values, when pyruvate + malate (−52%, p b 0.0005) and malate (− 34%, p b 0.0005) were used as substrates; while, normal rate values were obtained with succinate (Fig. 4B). 3.4. Cell treatment Considering the clinical improvement of the patient after riboflavin treatment, we supplemented cultured fibroblasts with this compound for 72 h, and in purified mitochondria PDHc, KGDHc and BCKDHCc activities were assayed. PDHc displayed an increased activity of 40% and 86%, after 5.3 μM and 50 μM riboflavin supplementation; KGDHc increased of 65% and 232%; BCKDHCc activity increased of 52% and 79%, respectively (Fig. 5A). No differences after riboflavin supplementation at any concentration used were observed in control mitochondria (Fig. 5A). The same treated mitochondria were used to measure complex V activity that significantly increased using pyruvate + malate, + 28% (p b 0.05) after supplementation of 5.3 μM riboflavin and

+46% (p b 0.0005) with 50 μM riboflavin, compared to untreated fibroblast mitochondria (Fig. 5B). An increasing trend was also evident using malate as substrate, +12% and +22%, respectively (Fig. 5B), although these values did not reach statistical significance. Mitochondria obtained from control skin fibroblasts did not show any differences after riboflavin supplementation at any concentration used and with either substrate (Fig. 5B). ATP content in patient's skin fibroblasts was slightly reduced with respect to controls in regular medium (data not shown) and reduced significantly in galactose medium (− 28%, p b 0.005) reflecting the lower efficiency of ATP production by OXPHOS (Fig. 6A). Then, we examined the effect of 5.3 μM riboflavin, which displayed a dramatic increase (+61%, p b 0.0005) in patient's fibroblasts specifically when cell cultures were grown in galactose medium (Fig. 6A). No effect was evident in control fibroblasts in similar condition. Moreover, riboflavin did not display any effect when fibroblasts, from both controls and patient, were grown in regular medium (data not shown). Considering intracellular ROS production we did not observe any significant variation at basal conditions with respect to controls (data not shown). When cell cultures were exposed to H2O2, ROS levels increased significantly only in the patient either compared to control fibroblasts in same conditions (+41%, p b 0.005; Fig. 6B) or compared to itself in regular medium (data not shown). Finally, treating fibroblasts with riboflavin showed no significant effects on ROS production at basal conditions (data not shown); in contrast, when mutant fibroblasts pretreated with riboflavin were exposed to cellular stress by adding H2O2, we noted a significant reduction on ROS levels compared

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Fig. 2. DLD_WT and DLD_ G461E 3D structural models. A, Amino acid alignment shows that the mutated glycine (G461E) is highly conserved in the DLD orthologs sampled from animalia, fungi and plants. Blocks of the same color show groups of the same or similar amino acids. The black arrowheads indicate the highly conserved glycine mutated in our patient; B, representative scheme of the DLD. Numbers denote amino acid numbering in protein, and arrows indicate the position of the reported mutations. Abbreviations: MTS, mitochondrial targeting sequence; FAD, FAD-binding domain; NAD+, NAD-binding domain; central, central domain; homodimer-interface, interface domain; C, overall structure of DLD. The 3D comparative model of the human DLD G461E homodimer is reported by cyan and green cartoon representation. The G461E mutations in the two monomers are represented by red spheres and indicated by black labels. NAD+ and FAD are reported in both monomers in yellow and magenta sticks indicated by black labels. D, Exploded views of the area containing the G461 (upper part) and the G461E (bottom part). Residues of the two DLD monomers interacting with G461 and G461E are labeled. H-bond interactions are reported by using black-thin dashed lines.

to the same cells in the presence of H2O2 alone (− 44%, p b 0.0005; Fig. 6B). No effect was evident in control fibroblasts in similar conditions.

4. Discussion DLD deficiency is a rare autosomal recessive metabolic disorder associated with variable phenotypic features. The metabolic profile of our patient was clearly consistent with E3 deficiency, showing the elevation of lactate and branched-chain amino acids in plasma (including alloisoleucine) with increased urinary excretion of α-keto acids. Differently from a recent paper reporting elevated plasma citrulline in DLD deficiency (Haviv et al., 2014), our patient had persistently normal levels of plasma citrulline (range 24–35 μmol/L, nv b 45). DLD-related enzymatic activities were decreased consistently in fibroblast mitochondria. The enzymatic activity of complex V, in the direction of ATP synthesis, was low using pyruvate + malate and malate as substrates as well. The few reported DLD deficient cases showing myopathic signs, presented with intermittent myoglobinuria, exertional fatigue, muscle weakness and ptosis in one case (Quintana et al., 2010). However, well defined myopathy associated to mitochondrial signs in muscle biopsy has never been reported so far. The presence of clear myopathic features in our patient prompted us to perform a muscle biopsy, which showed a characteristic picture of mitochondrial myopathy with clear mitochondrial proliferation at histochemistry. However, the entity of mitochondrial proliferation was not associated with a significant increase of citrate synthase activity in the muscle homogenate of the first muscle biopsy. Thus mitochondrial proliferation was moderate because

detectable only by muscle histology, and obviously normalized one year later under riboflavin supplementation. Sequencing of DLD led us to identify two novel mutations, the splice site c.118 + 1G NT, located at the end of mitochondrial targeting sequence and at the proximity of the FAD-binding domain, and the c.1382G NA mutation, which affects a highly conserved residue, p. G461E, located at the homodimer-interface. Most of the known disease-causing missense mutations occur at the level of three domains of the human DLD protein, the dimer interface, the active site, and the FAD and NAD+ binding sites (Supplementary Table 2). In agreement with other mutations described at the dimer interface (Brautigam et al., 2005), it appears that the G461E substitution found in our patient introduces a further negative charge, impairing local charge equilibrium. Thus the G461E mutation likely affects the homodimerization process of human E3 subunits, resulting in the lowering of human E3 activity, because both monomers participate to the disulfide-exchange sites of the homodimer, and most likely alter the protein turnover. The correct folding of the protein dimer depends also on the presence of NAD+ and FAD cofactors (Brautigam et al., 2005; Ciszak et al., 2006). It is therefore expected that a partial rescue of the mutant activity (i.e. in terms of protein turnover) could be obtained by increasing the concentration of cofactors (e.g. FAD) or of their precursors (e.g. riboflavin). This assumption is consistent with our results, showing both a recovery of DLD protein expression as well as the improvement of substrate oxidation depending on DLD-related enzymatic activities. Vitamins, that are essential nutrients acquired from food, are the precursors of different cofactors that represent essential ligands for a large series of metabolic enzymes. Ligands are very well known for

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Fig. 3. Effect of riboflavin treatment on muscle fibers. A, Western blotting analysis on SDS gel. Muscle homogenate from control (Ctrl), the first (Pt. 1st) and second (Pt. 2nd) patient's biopsies were separated by SDS-PAGE, and specific antibodies against DLD protein and porin (VDAC) were used. The mitochondrial protein porin was used as a control for equal loading. The quantity of DLD was found to be severely affected in both biopsies, although a recovery in the second muscle biopsy was evident. B, Histochemistry on muscle biopsy. Cytochrome c oxidase (a, c), and succinate dehydrogenase (b, d) of the first (a–b) and second muscle biopsy (c–d). Scale bar represents 20 μm. Asterisk underlined COX positive fibers with mitochondrial proliferation evident in the first biopsy, which disappear in the second biopsy after one-year riboflavin treatment.

their stabilizing effect on proteins, and the rationale of its successful use in many metabolic diseases may depend on their action as coenzymes or on a chaperone-like effect that increases protein stability and folding. An interesting feature of mutations associated with a phenotypic response to cofactor supplementation is their tendency to spread far from the binding site, suggesting that cofactors truly play a chaperone-like role, being able to compensate the structural defects induced by the gene mutation (Rodrigues et al., 2012). In recent years, there has been a renewal interest in studying the flavinylation process of fatty acid oxidation (FAO) enzymes and in finding correlations between genetic variants to the potential therapeutical use of riboflavin in patients with FAO disorders (Olsen et al., 2013). Henriques et al. (2009) demonstrated in a ETFβ mutant model that flavinylation has an effect on ETF stability, since the saturation of the FAD binding site significantly improved the stability of the apoprotein and rescued the destabilized forms. They suggest that in stress conditions, like high temperature or decreased FAD availability, ETF protein becomes deflavinylated and therefore destabilized. Moreover, external FAD increases the proteolytic stability, suggesting that an excess of FAD in the patient cells may increase the lifetime and availability of the active form of the protein. This aspect has been further elucidated by Cornelius et al. (2012) who demonstrated how certain ETF-QO variants associated to riboflavin responsive multiple acyl-CoA-dehydrogenase deficiency (RR-MADD), can cause mild folding defects sensitive to temperature and responsive to riboflavin supplementation, showing that

Fig. 4. Effect of G461E mutation on the enzymatic activity of the three mitochondrial dehydrogenases and ATP synthesis. A, PDH, KGDH and BCKDH complex assays. The PDHc, KGDHc and BCKDHc activities were assayed by measuring NADH formation, and using mitochondria extracted from control and patient's fibroblasts. The data represent the mean ± SEM of four independent experiments in duplicate. B, Spectrophotometric determination of complex V activity. Rate of ATP synthesis in mitochondria obtained from cultured fibroblasts was reduced of 52%, compared to the control mean, when pyruvate + malate were used as substrates to energize mitochondria. When malate was used the reduction was of 34%; whereas a normal value was obtained with succinate. The data represent the mean ± SD of three independent experiments in duplicate. ***: p b 0.0005.

riboflavin response derives from the ability of FAD to promote protein folding and stabilize the mature form of the protein. A riboflavin chaperon-like effect has also been hypothesized in patients with complex I defect harboring mutations in ACAD9, a gene coding for a riboflavin-dependent acyl-CoA dehydrogenase enzyme, and presenting with lactic acidosis and myopathy (Garone et al., 2013; Gerards et al., 2011). Indeed, a recent description on a patient with a different ACAD9 mutation showed the lack of response to riboflavin treatment probably because the new variant was closely to the FAD binding domain (Nouws et al., 2014). Riboflavin supplements most likely increase intra-mitochondrial FAD concentration favoring FAD binding, which is important for the catalytic activity of flavoproteins as well as for their folding, assembly and/or stability. These results pave the way for promising therapies, pointing on the raising of intra-mitochondrial cofactors, such as FAD, to compensate the decreased folding and catalytic capacity of certain enzymes due to mutations which predict to influence the FAD binding. In some DLD deficient patients, clinical and biochemical improvements using substrate and cofactor supplementations have been reported. Thiamine treatment was reported to be effective in two patients (Quintana et al., 2010; Shany et al., 1999), lipoic acid determined a good clinical response with dramatic improvement in lactic and pyruvic acidemia in an 8-month-old boy (Matalon et al., 1984). Hong et al. (2003) described the favorable outcome in a child with DLD deficiency,

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Fig. 5. A, Effect of fibroblasts riboflavin supplementation on PDHc, KGDHc and BCKDHc activities and ATP synthesis. The three complex activities were assayed using mitochondria obtained from cultured fibroblasts of the first biopsies without or with supplementation for 72 h of different amounts of riboflavin in the growing medium. PDHc activity increased of 40% and 86% in the presence of 5.3 and 50 μM of riboflavin, respectively. KGDHc increased of 65% and 232%; BCKDHc increased of 52% and 79%, respectively. The data represent the mean ± SEM of three independent experiments. B, Rate of ATP synthesis in mitochondria obtained from cultured fibroblasts after riboflavin supplementation. Complex V activity increased of 28% and 46%, using pyruvate + malate as a substrate, after supplementation of 5.3 μM and 50 μM riboflavin, respectively. When malate was used the activity of complex V increased of 12% and 22%. The data represent the mean ± SD of three independent experiments in duplicate for 5.3 μM riboflavin treatment, and two independent experiments after 50 mM riboflavin treatment. *: p b 0.05; **: p b 0.005; ***: p b 0.0005.

suffering from recurrent episodes of vomiting associated with encephalopathy from age 8 months, treated with a combination of riboflavin, biotin, coenzyme Q and carnitine, in contrast to the fatal outcome of his untreated siblings. In our patient, we first attempted thiamine supplementation however without beneficial effects. When switching to riboflavin supplementation, we observed a dramatic improvement of the clinical picture with the complete resolution of muscle weakness along with the improvement of metabolic abnormalities. While on riboflavin, the patient was able to strikingly change his physical skills regaining the ability of a regular sport activity. Accordingly, a muscle biopsy performed on riboflavin treatment clearly demonstrated the rescue of DLD protein levels along with the disappearance of histochemical mitochondrial proliferation highlighting the chaperon-like effect of riboflavin in our patient. Moreover, the beneficial effect of riboflavin was also evident quantifying the energy production, and particularly by monitoring the mitochondria complex V activity, as well as the total cellular ATP content. Finally, the administration of cofactors and/or their precursors also seems to favor the lowering of the oxidative damage in fibroblasts. In fact, the increased ROS production seen after the addition of H2O2 returned to basal levels when riboflavin was supplemented.

It is well known that superoxide anion radical and H2O2 are generated from at least ten distinct sites in the electron transport chain and associated pathways. Respiratory chain complexes I and III are usually reported as the principal producers (Quinlan et al., 2011; Quinlan et al., 2013), but many other mitochondrial enzymes, such as 2-oxoglutarate dehydrogenase (OGDH) (Bunik and Sievers, 2002), PDHc (Starkov et al., 2004), complex II (Quinlan et al., 2012), and glycerol 3-phosphate dehydrogenase (mGPDH) (Orr et al., 2012), can also reduce oxygen prematurely. Among these, α-KGDHc is not only a source but also one of the most sensitive targets of ROS in mitochondria (Starkov et al., 2004; Tretter and Adam-Vizi, 2004). ROS generation by α-KGDHc has been ascribed to the E3-subunit (Starkov et al., 2004), and disease causing mutations were recently shown to stimulate the ability of human DLD to produce superoxide radical and hydrogen peroxide in vitro (Ambrus et al., 2011). Moreover, DLD is known to possess a diaphorase activity that catalyzes the oxidation of NADH to NAD+ using different electron acceptors (Igamberdiev et al., 2004). In addition, the diaphorase activity, which leads to an increase in ROS production, is associated to the monomerization of the intact E3 component (Klyachko et al., 2005). An enhanced diaphorase activity, leading to localized ROS production and oxidative damage of E2-bound lipoic acid cofactor, was proved to

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Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.mito.2014.09.006. Acknowledgments This research was supported by grants from Telethon-Mitocon Project (Telethon GUP09004), and Ricerca Corrente (201302G002980) to EB. References

Fig. 6. A, Luminometric measurement of ATP and B, fluorimetric determination of ROS level in controls and patient's fibroblasts before and after supplementation of 5.3 μM riboflavin for 72 h. In galactose-supplemented medium the ATP content was reduced of 28% compared to controls in similar conditions. A significant increase was evident in patient's fibroblasts, after the addition of riboflavin (+61%). B, In the presence of H2O2 the level of ROS was significantly higher in patient compared to controls (+41%). When riboflavin was added, a dramatic reduction (−44%) of ROS level was evident. Data reported in a and b are represented as mean ± SD from three separated experiments performed in duplicate. Fibroblasts from controls and patient were grown in galactose-supplemented medium (Gal), and in medium supplemented with galactose and riboflavin (Gal + Ribo); moreover, cells were also incubated for 20 min with 1 mM H2O2. **: p b 0.005; ***: p b 0.0005.

increase the pathogenic mechanism of DLD interface domain mutations (Vaubel et al., 2011). All of these findings are in agreement with our results. In fact, considering that the mutation found in our patient destabilizes the homodimer this can shift the physiological function of E3 to diaphorase activity contributing to the increased ROS production. The demonstration that riboflavin treatment in fibroblasts of our patient lowered H2O2-induced ROS production may be therefore the consequence of a direct redox effect of riboflavin but it could also be due to its ability to correct the protein folding, reestablishing the dimerization state and thus shifting DLD from diaphorase activity to its physiological function. In conclusion we have reported on a new phenotype related to E3 deficiency consisting in a myopathy with weakness, fatigability and mitochondrial proliferation. This patient markedly improved with riboflavin supplementation and, very interestingly, his muscle biopsy, under riboflavin treatment, clearly showed a recovery of the DLD protein and normalization of morphology, supporting the evidence of a chaperonelike effect of riboflavin on the E3 mutant homodimer. Moreover, ROS production was clearly reduced in fibroblasts after riboflavin treatment, most likely as a consequence of its ability to correct the protein folding. Finally, since DLD deficiency is a potentially treatable mitochondrial disorder, a correct and early diagnosis is of utmost importance.

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