SUCLA2 mutations cause global protein succinylation contributing to the pathomechanism of a hereditary mitochondrial disease.

Mitochondrial acyl-coenzyme A species are emerging as important sources of protein modification and damage. Succinyl-CoA ligase (SCL) deficiency causes a mitochondrial encephalomyopathy of unknown pathomechanism. Here, we show that succinyl-CoA accumulates in cells derived from patients with recessive mutations in the tricarboxylic acid cycle (TCA) gene succinyl-CoA ligase subunit-ß (SUCLA2), causing global protein hyper-succinylation. Using mass spectrometry, we quantify nearly 1,000 protein succinylation sites on 366 proteins from patient-derived fibroblasts and myotubes. Interestingly, hyper-succinylated proteins are distributed across cellular compartments, and many are known targets of the (NAD+)-dependent desuccinylase SIRT5. To test the contribution of hyper-succinylation to disease progression, we develop a zebrafish model of the SCL deficiency and find that SIRT5 gain-of-function reduces global protein succinylation and improves survival. Thus, increased succinyl-CoA levels contribute to the pathology of SCL deficiency through post-translational modifications.

Pharmacologically targetable vulnerability in prostate cancer carrying RB1-SUCLA2 deletion.

RB1 gene is often homozygously deleted or mutated in prostate adenocarcinomas following acquirement of castration resistance and/or metastatic ability. We found that SUCLA2 gene is frequently involved in the deletion of the RB1 gene region in advanced prostate cancer. SUCLA2 constitutes the ß-subunit of succinate CoA ligase heterodimer that reversibly converts succinyl CoA into succinate. We sought the possibility that deletion of SUCLA2 gives rise to a metabolic vulnerability that could be targeted therapeutically. We found a significant metabolic shift in SUCLA2-deleted prostate cancer cells, including lower mitochondrial respiratory activity. By screening a number of libraries for compounds that induce cell death selectively in SUCLA2-deficient prostate cancer cells, we identified thymoquinone (2-isopropyl-5-methylbenzo-1,4-quinone) and PMA (phorbol-12-myristate-13-acetate) from a natural compound library. These findings indicate that the metabolic vulnerability in SUCLA2-deficient prostate cancer cells is pharmacologically targetable.

Clinical, Molecular, and Computational Analysis in two cases with mitochondrial encephalomyopathy associated with SUCLG1 mutation in a consanguineous family.

Deficiency of the mitochondrial enzyme succinyl COA ligase (SUCL) is associated with encephalomyopathic mtDNA depletion syndrome and methylmalonic aciduria. This disorder is caused by mutations in both SUCL subunits genes: SUCLG1 (α subnit) and SUCLA2 (ß subnit). We report here, two Tunisian patients belonging to a consanguineous family with mitochondrial encephalomyopathy, hearing loss, lactic acidosis, hypotonia, psychomotor retardation and methylmalonic aciduria. Mutational analysis of SUCLG1 gene showed, for the first time, the presence of c.41T > C in the exon 1 at homozygous state. In-silico analysis revealed that this mutation substitutes a conserved methionine residue to a threonine at position 14 (p.M14T) located at the SUCLG1 protein mitochondrial targeting sequence. Moreover, these analysis predicted that this mutation alter stability structure and mitochondrial translocation of the protein. In Addition, a decrease in mtDNA copy number was revealed by real time PCR in the peripheral blood leukocytes in the two patients compared with controls.

Loss of succinyl-CoA synthase ADP-forming ß subunit disrupts mtDNA stability and mitochondrial dynamics in neurons.

Succinyl Coenzyme A synthetase (SCS) is a key mitochondrial enzyme. Defected SCS ADP-forming ß subunit (SCS A-ß) is linked to lethal infantile Leigh or leigh-like syndrome. However, the impacts of SCS A-ß deficiency on mitochondria specifically in neurons have not yet been comprehensively investigated. Here, by down-regulating the expression levels of SCS A-ß in cultured mouse neurons, we have found that SCS A-ß deficiency induces severe mitochondrial dysfunction including lowered oxidative phosphorylation (OXPHOS) efficiency, increased mitochondrial superoxide production, and mtDNA depletion as well as aberrations of mitochondrial fusion and fission proteins, which eventually leads to neuronal stress. Our data also suggest that the deregulation of mitochondrial nucleoside diphosphate kinase (NDPK) together with defects in mitochondrial transcription factors including mitochondrial DNA pol γ and Twinkle contribute to SCS A-ß deficiency-mediated mtDNA instability. Furthermore, we have found that SCS A-ß deficiency has detrimental influence on neuronal mitochondrial dynamics. Put together, the results have furnished our knowledge on the pathogenesis of SCS A-ß deficiency-related mitochondrial diseases and revealed the vital role of SCS A-ß in maintaining neuronal mitochondrial quality control and neuronal physiology.

Succinyl-CoA synthetase (SUCLA2) deficiency in two siblings with impaired activity of other mitochondrial oxidative enzymes in skeletal muscle without mitochondrial DNA depletion.

Mutations in SUCLA2 result in succinyl-CoA ligase (ATP-forming) or succinyl-CoA synthetase (ADP-forming) (A-SCS) deficiency, a mitochondrial tricarboxylic acid cycle disorder. The phenotype associated with this gene defect is largely encephalomyopathy. We describe two siblings compound heterozygous for SUCLA2 mutations, c.985A>G (p.M329V) and c.920C>T (p.A307V), with parents confirmed as carriers of each mutation. We developed a new LC-MS/MS based enzyme assay to demonstrate the decreased SCS activity in the siblings with this unique genotype. Both siblings shared bilateral progressive hearing loss, encephalopathy, global developmental delay, generalized myopathy, and dystonia with choreoathetosis. Prior to diagnosis and because of lactic acidosis and low activity of muscle pyruvate dehydrogenase complex (PDC), sibling 1 (S1) was placed on dichloroacetate, while sibling 2 (S2) was on a ketogenic diet. S1 developed severe cyclic vomiting refractory to therapy, while S2 developed Leigh syndrome, severe GI dysmotility, intermittent anemia, hypogammaglobulinemia and eventually succumbed to his disorder. The mitochondrial DNA contents in skeletal muscle (SM) were normal in both siblings. Pyruvate dehydrogenase complex, ketoglutarate dehydrogenase complex, and several mitochondrial electron transport chain (ETC) activities were low or at the low end of the reference range in frozen SM from S1 and/or S2. In contrast, activities of PDC, other mitochondrial enzymes of pyruvate metabolism, ETC and, integrated oxidative phosphorylation, in skin fibroblasts were not significantly impaired. Although we show that propionyl-CoA inhibits PDC, it does not appear to account for decreased PDC activity in SM. A better understanding of the mechanisms of phenotypic variability and the etiology for tissue-specific secondary deficiencies of mitochondrial enzymes of oxidative metabolism, and independently mitochondrial DNA depletion (common in other cases of A-SCS deficiency), is needed given the implications for control of lactic acidosis and possible clinical management.

Knockdown of Sucla2 decreases the viability of mouse spermatocytes by inducing apoptosis through injury of the mitochondrial function of cells.

INTRODUCTION: Sucla2, a ß subunit of succinyl coenzyme A synthase, is located in the mitochondrial matrix. Sucla2 catalyzes the reversible synthesis of succinate and adenosine triphosphate (ATP) in the tricarboxylic acid cycle. Sucla2 expression was found to be correlated with the capacitation of boar spermatozoa. We have previously reported that Sucla2 was decreased in the testes of rats with spermatogenesis failure after exposure to endocrine disruptor BDE47. Yet, the expression model of Sucla2 in spermatogenesis and the function of Sucla2 in spermatogenic cells are still unclear. Our objective was to explore the localization of Sucla2 during mouse spermatogenesis and its function in the mouse spermatocyte line (GC2). MATERIAL AND METHODS: The localization of Sucla2 in the mouse testis was explored through immunohistochemistry (IHC). Sucla2 was knocked down in GC2 cells and its expression was detected by Western blot (WB) to verify the efficiency of the siRNA transfection. Mitochondrial membrane potential (MMP), apoptosis and ROS of GC2 were detected by flow cytometry. ATP production was measured by the luminometric method and the presence of Bcl2 of GC2 was detected by WB. RESULTS: Sucla2 is highly expressed in all germ cells but not in interstitial cells. Coarse Sucla2 signals are found in spermatocytes in stages VII­XII of mouse spermatogenesis. In GC2 cells, knockdown of Sucla2 decreased cell viability and MMP, induced apoptosis of GC2 cells, decreased ATP production, and Bcl2 expression, and increased ROS levels. CONCLUSIONS: Sucla2 is related to the developmental stages of mouse spermatogenesis. Knockdown of Sucla2 decreases the viability of mouse spermatocytes by inducing apoptosis via decreased mitochondrial function of the cells

Screen for abnormal mitochondrial phenotypes in mouse embryonic stem cells identifies a model for succinyl-CoA ligase deficiency and mtDNA depletion.

Mutations in subunits of succinyl-CoA synthetase/ligase (SCS), a component of the citric acid cycle, are associated with mitochondrial encephalomyopathy, elevation of methylmalonic acid (MMA), and mitochondrial DNA (mtDNA) depletion. A FACS-based retroviral-mediated gene trap mutagenesis screen in mouse embryonic stem (ES) cells for abnormal mitochondrial phenotypes identified a gene trap allele of Sucla2 (Sucla2(SAßgeo)), which was used to generate transgenic mice. Sucla2 encodes the ADP-specific ß-subunit isoform of SCS. Sucla2(SAßgeo) homozygotes exhibited recessive lethality, with most mutants dying late in gestation (e18.5). Mutant placenta and embryonic (e17.5) brain, heart and muscle showed varying degrees of mtDNA depletion (20-60%). However, there was no mtDNA depletion in mutant liver, where the gene is not normally expressed. Elevated levels of MMA were observed in embryonic brain. SCS-deficient mouse embryonic fibroblasts (MEFs) demonstrated a 50% reduction in mtDNA content compared with wild-type MEFs. The mtDNA depletion resulted in reduced steady state levels of mtDNA encoded proteins and multiple respiratory chain deficiencies. mtDNA content could be restored by reintroduction of Sucla2. This mouse model of SCS deficiency and mtDNA depletion promises to provide insights into the pathogenesis of mitochondrial diseases with mtDNA depletion and into the biology of mtDNA maintenance. In addition, this report demonstrates the power of a genetic screen that combines gene trap mutagenesis and FACS analysis in mouse ES cells to identify mitochondrial phenotypes and to develop animal models of mitochondrial dysfunction.

The severity of phenotype linked to SUCLG1 mutations could be correlated with residual amount of SUCLG1 protein.

BACKGROUND: Succinate-CoA ligase deficiency is responsible for encephalomyopathy with mitochondrial DNA depletion and mild methylmalonic aciduria. Mutations in SUCLA2, the gene encoding a ß subunit of succinate-CoA ligase, have been reported in 17 patients until now. Mutations in SUCLG1, encoding the α subunit of the enzyme, have been described in two pedigrees only. METHODS AND FINDINGS: In this study, two unrelated patients harbouring three novel pathogenic mutations in SUCLG1 were reported. The first patient had a severe disease at birth. He was compound heterozygous for a missense mutation (p.Pro170Arg) and a c.97+3G>C mutation, which leads to the complete skipping of exon 1 in a minigene expression system. The involvement of SUCLG1 was confirmed by western blot analysis, which showed absence of SUCLG1 protein in fibroblasts. The second patient has a milder phenotype, similar to that of patients with SUCLA2 mutations, and is still alive at 12 years of age. Western blot analysis showed some residual SUCLG1 protein in patient's fibroblasts. CONCLUSIONS: Our results suggest that SUCLG1 mutations that lead to complete absence of SUCLG1 protein are responsible for a very severe disorder with antenatal manifestations, whereas a SUCLA2-like phenotype is found in patients with residual SUCLG1 protein. Furthermore, it is shown that in the absence of SUCLG1 protein, no SUCLA2 protein is found in fibroblasts by western blot analysis. This result is consistent with a degradation of SUCLA2 when its heterodimer partner, SUCLG1, is absent.

Succinyl-CoA ligase deficiency: a mitochondrial hepatoencephalomyopathy.

This patient presented on the first day of life with pronounced lactic acidosis with an elevated lactate/pyruvate ratio. Urine organic acids showed Krebs cycle metabolites and mildly elevated methylmalonate and methylcitrate. The acylcarnitine profile showed elevated propionylcarnitine and succinylcarnitine. Amino acids showed elevated glutamic acid, glutamine, proline, and alanine. From the age 2 of mo on, she had elevated transaminases and intermittent episodes of liver failure. Liver biopsy showed steatosis and a decrease of mitochondrial DNA to 50% of control. She had bilateral sensorineural hearing loss. Over the course of the first 2 y of life, she developed a progressively severe myopathy with pronounced muscle weakness eventually leading to respiratory failure, Leigh disease, and recurrent hepatic failure. The hepatic symptoms and the metabolic parameters temporarily improved on treatment with aspartate, but neither muscle symptoms nor brain lesions improved. Laboratory testing revealed a deficiency of succinyl-CoA ligase enzyme activity and protein in fibroblasts because of a novel homozygous mutation in the SUCLG1 gene: c.40A>T (p.M14L). Functional analysis suggests that this methionine is more likely to function as the translation initiator methionine, explaining the pathogenic nature of the mutation. Succinyl-CoA ligase deficiency due to an SUCLG1 mutation is a new cause for mitochondrial hepatoencephalomyopathy.

Marked mitochondrial DNA depletion associated with a novel SUCLG1 gene mutation resulting in lethal neonatal acidosis, multi-organ failure, and interrupted aortic arch.

The aim of this study was to identify the causative genetic lesion in two apparently unrelated newborns having lethal lactic acidosis, multi-organ failure and congenital malformations including interrupted aortic arch, who exhibited mild methylmalonic aciduria, combined mitochondrial respiratory chain deficiency, and marked muscle mitochondrial DNA depletion. A novel mutation in the SUCLG1 gene was identified. Phenotype severity in Succinate-CoA ligase dysfunction appears to be more correlated to the muscle mtDNA content than to the tissue distribution of the heterodimer subunits. Prominent impairment of mitochondrial respiratory chain may result in deep ravages in developmental tissues leading to multiple organ failure and malformations.

New SUCLG1 patients expanding the phenotypic spectrum of this rare cause of mild methylmalonic aciduria.

Deficiencies in two subunits of the succinyl-coenzyme A synthetase (SCS) have been involved in patients with encephalomyopathy and mild methylmalonic aciduria (MMA). In this study, we described three new SUCLG1 patients and performed a meta-analysis of the literature. Our report enlarges the phenotypic spectrum of SUCLG1 mutations and confirms that a characteristic metabolic profile (presence of MMA and C4-DC carnitine in urines) and basal ganglia MRI lesions are the hallmarks of SCS defects. As mitochondrial DNA depletion in muscle is not a constant finding in SUCLG1 patients, this may suggest that diagnosis should not be based on it, but also that alternative physiopathological mechanisms may be considered to explain the combined respiratory chain deficiency observed in SCS patients.

Dystonia and deafness due to SUCLA2 defect; Clinical course and biochemical markers in 16 children.

Patients with SUCLA2 gene defects characteristically develop the trias of early hypotonia, progressive dystonia and sensori-neural deafness. We describe the clinical course and biochemical phenotype in 16 children from the Faroe Islands with a homozygous SUCLA2 splice site mutation. Elevated urinary 3-hydroxyisovaleric acid is a novel biochemical feature in patients. Progressive hearing loss, in combination with a characteristic metabolite profile (increased lactate, methylmalonic acid, C4-dicarboxylic carnitine, 3-hydroxyisovaleric acid) should lead the clinician to the correct diagnosis even in patients with only intermittent lactic acidemia. Direct SUCLA2 sequence analysis is suggested instead of an invasive muscle biopsy to obtain the diagnosis. Nutritional intervention may be considered in SUCLA2 patients.

Disorders caused by deficiency of succinate-CoA ligase.

Succinate-CoA ligase catalyses the reversible conversion of succinyl-CoA and ADP or GDP to succinate and ATP or GTP. It is a mitochondrial matrix enzyme and at least the ADP-forming enzyme is part of the Krebs cycle. The substrate specificity is determined by the beta subunit of succinate-CoA ligase, which is encoded by either SUCLA2 or SUCLG2. In patients with severe hypotonia, deafness and Leigh-like syndrome, mutations have been found in SUCLA2. Mutations have also been reported in SUCLG1, which encodes the alpha subunit found in both enzymes, in patients with severe infantile acidosis and lactic aciduria. Elevated methylmalonate and methylcitrate and severe mtDNA depletion were found in both disorders. The mtDNA depletion may be explained by the interaction of succinate-CoA ligase with nucleoside diphosphate kinase, which is involved in mitochondrial nucleotide metabolism.

Infantile mitochondrial disorders.

Mitochondrial disorders encompass any medical specialty and affect patients at any age. Likewise, the spectrum of clinical and genetic signatures of these disorders is ample, making a precise diagnosis difficult. We will report some of the major clinical phenotypes observed in infancy, their underlining molecular features, and will propose an approach to reach a more complete diagnosis.

Depletion of mtDNA: syndromes and genes.

Maintenance of mitochondrial DNA (mtDNA) requires the concerted activity of several nuclear-encoded factors that participate in its replication, being part of the mitochondrial replisome or ensuring the balanced supply of dNTPs to mitochondria. In the past decade, a growing number of syndromes associated with dysfunction due to tissue-specific depletion of mtDNA (MDS) have been reported. This article reviews the current knowledge of the genes responsible for these disorders, the impact of different mutations in the epidemiology of MDS and their role in the pathogenic mechanisms underlying the different clinical presentations.

Deficiency of the ADP-forming succinyl-CoA synthase activity is associated with encephalomyopathy and mitochondrial DNA depletion.

The mitochondrial DNA (mtDNA) depletion syndrome is a quantitative defect of mtDNA resulting from dysfunction of one of several nuclear-encoded factors responsible for maintenance of mitochondrial deoxyribonucleoside triphosphate (dNTP) pools or replication of mtDNA. Markedly decreased succinyl-CoA synthetase activity due to a deleterious mutation in SUCLA2, the gene encoding the beta subunit of the ADP-forming succinyl-CoA synthetase ligase, was found in muscle mitochondria of patients with encephalomyopathy and mtDNA depletion. Succinyl-CoA synthetase is invariably in a complex with mitochondrial nucleotide diphosphate kinase; hence, we propose that a defect in the last step of mitochondrial dNTP salvage is a novel cause of the mtDNA depletion syndrome.