Sanna Marjavaara

FGF-21 as a biomarker for muscle-manifesting mitochondrial respiratory chain deficiencies: a diagnostic study

BACKGROUND Muscle biopsy is the gold standard for diagnosis of mitochondrial disorders because of the lack of sensitive biomarkers in serum. Fibroblast growth factor 21 (FGF-21) is a growth factor with regulatory roles in lipid metabolism and the starvation response, and concentrations are raised in skeletal muscle and serum in mice with mitochondrial respiratory chain deficiencies. We investigated in a retrospective diagnostic study whether FGF-21 could be a biomarker for human mitochondrial disorders. METHODS We assessed samples from adults and children with mitochondrial disorders or non-mitochondrial neurological disorders (disease controls) from seven study centres in Europe and the USA, and recruited healthy volunteers (healthy controls), matched for age where possible, from the same centres. We used ELISA to measure FGF-21 concentrations in serum or plasma samples (abnormal values were defined as >200 pg/mL). We compared these concentrations with values for lactate, pyruvate, lactate-to-pyruvate ratio, and creatine kinase in serum or plasma and calculated sensitivity, specificity, and positive and negative predictive values for all biomarkers. FINDINGS We analysed serum or plasma from 67 patients (41 adults and 26 children) with mitochondrial disorders, 34 disease controls (22 adults and 12 children), and 74 healthy controls. Mean FGF-21 concentrations in serum were 820 (SD 1151) pg/mL in adult and 1983 (1550) pg/mL in child patients with respiratory chain deficiencies and 76 (58) pg/mL in healthy controls. FGF-21 concentrations were high in patients with mitochondrial disorders affecting skeletal muscle but not in disease controls, including those with dystrophies. In patients with abnormal FGF-21 concentrations in serum, the odds ratio of having a muscle-manifesting mitochondrial disease was 132·0 (95% CI 38·7-450·3). For the identification of muscle-manifesting mitochondrial disease, the sensitivity was 92·3% (95% CI 81·5-97·9%) and specificity was 91·7% (84·8-96·1%). The positive and negative predictive values for FGF-21 were 84·2% (95% CI 72·1-92·5%) and 96·1 (90·4-98·9%). The accuracy of FGF-21 to correctly identify muscle-manifesting respiratory chain disorders was better than that for all conventional biomarkers. The area under the receiver-operating-characteristic curve for FGF-21 was 0·95; by comparison, the values for other biomarkers were 0·83 lactate (p=0·037, 0·83 for pyruvate (p=0·015), 0·72 for the lactate-to-pyruvate ratio (p=0·0002), and 0·77 for creatine kinase (p=0·013). INTERPRETATION Measurement of FGF-21 concentrations in serum identified primary muscle-manifesting respiratory chain deficiencies in adults and children and might be feasible as a first-line diagnostic test for these disorders to reduce the need for muscle biopsy. FUNDING Sigrid Jusélius Foundation, Jane and Aatos Erkko Foundation, Molecular Medicine Institute of Finland, University of Helsinki, Helsinki University Central Hospital, Academy of Finland, Novo Nordisk, Arvo and Lea Ylppö Foundation.

Infantile-onset spinocerebellar ataxia and mitochondrial recessive ataxia syndrome are associated with neuronal complex I defect and mtDNA depletion

Infantile-onset spinocerebellar ataxia (IOSCA) is a severe neurodegenerative disorder caused by the recessive mutation in PEO1, leading to an Y508C change in the mitochondrial helicase Twinkle, in its helicase domain. However, no mitochondrial dysfunction has been found in this disease. We studied here the consequences of IOSCA for the central nervous system, as well as the in vitro performance of the IOSCA mutant protein. The results of the mtDNA analyses were compared to findings in a similar juvenile or adult-onset ataxia syndrome, mitochondrial recessive ataxia syndrome (MIRAS), caused by the W748S mutation in the mitochondrial DNA polymerase (POLG). We show here that IOSCA brain does not harbor mtDNA deletions or increased amount of mtDNA point mutations, whereas MIRAS brain shows multiple deletions of mtDNA. However, IOSCA, and to a lesser extent also MIRAS, show mtDNA depletion in the brain and the liver. In both diseases, especially large neurons show respiratory chain complex I (CI) deficiency, but also CIV is decreased in IOSCA. Helicase activity, hexamerization and nucleoid structure of the IOSCA mutant were, however, unaffected. The lack of in vitro helicase defect or cell culture phenotype suggest that Twinkle-Y508C dysfunction affects mtDNA maintenance in a highly context and cell-type specific manner. Our results indicate that IOSCA is a new member of the mitochondrial DNA depletion syndromes.

Deficiency of the INCL protein Ppt1 results in changes in ectopic F1-ATP synthase and altered cholesterol metabolism

Infantile neuronal ceroid lipofuscinosis (INCL) is a severe neurodegenerative disease caused by deficiency of palmitoyl protein thioesterase 1 (PPT1). INCL results in dramatic loss of thalamocortical neurons, but the disease mechanism has remained elusive. In the present work we describe the first interaction partner of PPT1, the F(1)-complex of the mitochondrial ATP synthase, by co-purification and in vitro-binding assays. In addition to mitochondria, subunits of F(1)-complex have been reported to localize in the plasma membrane, and to be capable of acting as receptors for various ligands such as apolipoprotein A-1. We verified here the plasma membrane localization of F(1)-subunits on mouse primary neurons and fibroblasts by cell surface biotinylation and TIRF-microscopy. To gain further insight into the Ppt1-mediated properties of the F(1)-complex, we utilized the Ppt1-deficient Ppt1(Delta ex4) mice. While no changes in the mitochondrial function could be detected in the brain of the Ppt1(Delta ex4) mice, the levels of F(1)-subunits alpha and beta on the plasma membrane were specifically increased in the Ppt1(Delta ex4) neurons. Significant changes were also detected in the apolipoprotein A-I uptake by the Ppt1(Delta ex4) neurons and the serum lipid composition in the Ppt1(Delta ex4) mice. These data indicate neuron-specific changes for F(1)-complex in the Ppt1-deficient cells and give clues for a possible link between lipid metabolism and neurodegeneration in INCL.

DARS2 mutations in mitochondrial leucoencephalopathy and multiple sclerosis

Background Leucoencephalopathy with brain stem and spinal cord involvement and high brain lactate (LBSL) was first defined by characteristic magnetic resonance imaging and spectroscopic findings. The clinical features include childhood or juvenile onset slowly progressive ataxia, spasticity, and dorsal column dysfunction, occasionally accompanied by learning difficulties. Mutations in DARS2, encoding mitochondrial aspartyl-tRNA synthetase, were recently shown to cause LBSL. The signs and symptoms show some overlap with the most common leucoencephalopathy of young adults, multiple sclerosis (MS). Objective To clarify the molecular background of LBSL patients in Finland, and to look for DARS2 mutations in a group of MS patients. Methods Clinical evaluation of LBSL patients, DARS2 sequencing and haplotype analysis, and carrier frequency determination in Finland. Results All eight LBSL patients were compound heterozygotes for DARS2 mutations: all carried R76SfsX5 change, seven had M134_K165del, and one had C152F change. Axonal neuropathy was found in five of the eight patients. The carrier frequencies of the R76SfsX5 and M134_K165del mutations were 1:95 and 1:380, respectively. All patients shared common European haplotypes, suggestive of common European LBSL ancestors. No enrichment of the two common DARS2 mutations was found in 321 MS patients. Conclusion All LBSL patients were compound heterozygotes, which suggests that DARS2 mutation homozygosity may be lethal or manifest as a different phenotype. The authors show here that despite identical mutations the clinical picture was quite variable in the patients. Axonal neuropathy was an important feature of LBSL. DARS2 mutations cause childhood-to-adolescence onset leucoencephalopathy, but they do not seem to be associated with MS.

Characterization of complex III deficiency and liver dysfunction in GRACILE syndrome caused by a BCS1L mutation.

A homozygous mutation in the complex III chaperone BCS1L causes GRACILE syndrome (intrauterine growth restriction, aminoaciduria, cholestasis, hepatic iron overload, lactacidosis). In control and patient fibroblasts we localized BCS1L in inner mitochondrial membranes. In patient liver, kidney, and heart BCS1L and Rieske protein levels, as well as the amount and activity of complex III, were decreased. Major histopathology was found in kidney and liver with cirrhosis and iron deposition, but of iron-related proteins only ferritin levels were high. In placenta from a GRACILE fetus, the ferrooxidases ceruloplasmin and hephaestin were upregulated suggesting association between iron overload and placental dysfunction.

Protein synthesis and assembly in mitochondrial disorders.

Human mitochondrial DNA (mtDNA) codes for 13 polypeptides which constitute the central core of the oxidative phosphorylation (OXPHOS) complexes. The machinery for mitochondrial protein synthesis has a dual origin: a full set of tRNAs, as well as the 12S and 16S rRNAs are encoded in the mitochondrial genome, while most factors necessary for translation are encoded by nuclear genes. The mitochondrial translation apparatus is highly specialized in expressing membrane proteins, and couples the synthesis of proteins to the insertion into the mitochondrial inner membrane. In recent years it has become clear that defects of mitochondrial translation and protein assembly cause several mitochondrial disorders. Since direct studies on protein synthesis in human mitochondria are still a relatively difficult task, we owe our current knowledge of this field to the large amount of genetic and biochemical studies performed in the yeast Saccharomyces cerevisiae. These studies have allowed the identification of several genes involved in mitochondrial protein synthesis and assembly, and have provided insights into the conserved mechanisms of mitochondrial gene expression. In the present review we will discuss the most recent advances in the understanding of the mechanisms and factors that govern mammalian mitochondrial translation/protein insertion, as well as known pathologies associated with them.

Fatal neonatal lactic acidosis caused by a novel de novo mitochondrial G7453A tRNA-Serine (UCN) mutation

Introduction:Heteroplasmic mitochondrial DNA (mtDNA) mutations are an important cause of childhood disorders, but the role of homoplasmic mtDNA mutations in severe neonatal manifestations is not well understood.Methods:The following were performed: full mtDNA sequencing for mutation detection, blue-native protein analysis of autopsy-derived tissues to detect respiratory chain (RC) deficiency, light and electron microscopy for morphologic analysis, and northern blot and computational modeling to study the effect of mtDNA mutations on transfer RNA (tRNA) stability.Results:We describe data from a patient with fatal neonatal lactic acidosis caused by a novel homoplasmic mutation at a highly conserved nucleotide G7453A within the tRNASer (UCN) in mtDNA. The patient’s heart, skeletal muscle, brain, and liver showed severe combined complex I and IV (CI and CIV) deficiencies, accompanied by severe depletion of mature tRNASer (UCN). The mutation was absent in the patient’s mother and in a placental sample from a subsequent pregnancy of the mother, suggesting a de novo mutation.Discussion:We conclude that the G7453A mutation of mtDNA manifests with exceptional severity as compared with other tRNASer (UCN) mutations, typically associated with sensorineural deafness. De novo homoplasmic mtDNA tRNA-mutations should be considered as a cause of fatal neonatal lactic acidosis.

A mouse model of mitochondrial complex III dysfunction induced by myxothiazol.

Myxothiazol is a respiratory chain complex III (CIII) inhibitor that binds to the ubiquinol oxidation site Qo of CIII. It blocks electron transfer from ubiquinol to cytochrome b and thus inhibits CIII activity. It has been utilized as a tool in studies of respiratory chain function in in vitro and cell culture models. We developed a mouse model of biochemically induced and reversible CIII inhibition using myxothiazol. We administered myxothiazol intraperitoneally at a dose of 0.56 mg/kg to C57Bl/J6 mice every 24 h and assessed CIII activity, histology, lipid content, supercomplex formation, and gene expression in the livers of the mice. A reversible CIII activity decrease to 50% of control value occurred at 2 h post-injection. At 74 h only minor histological changes in the liver were found, supercomplex formation was preserved and no significant changes in the expression of genes indicating hepatotoxicity or inflammation were found. Thus, myxothiazol-induced CIII inhibition can be induced in mice for four days in a row without overt hepatotoxicity or lethality. This model could be utilized in further studies of respiratory chain function and pharmacological approaches to mitochondrial hepatopathies.