Taraka R. Donti

Mutations in the Mitochondrial Methionyl-tRNA Synthetase Cause a Neurodegenerative Phenotype in Flies and a Recessive Ataxia (ARSAL) in Humans

The study of Drosophila neurodegenerative mutants combined with genetic and biochemical analyses lead to the identification of multiple complex mutations in 60 patients with a novel form of ataxia/leukoencephalopathy.

Fatty Acid Oxidation-Driven Src Links Mitochondrial Energy Reprogramming and Oncogenic Properties in Triple-Negative Breast Cancer

Transmitochondrial cybrids and multiple OMICs approaches were used to understand mitochondrial reprogramming and mitochondria-regulated cancer pathways in triple-negative breast cancer (TNBC). Analysis of cybrids and established breast cancer (BC) cell lines showed that metastatic TNBC maintains high levels of ATP through fatty acid β oxidation (FAO) and activates Src oncoprotein through autophosphorylation at Y419. Manipulation of FAO including the knocking down of carnitine palmitoyltransferase-1A (CPT1) and 2 (CPT2), the rate-limiting proteins of FAO, and analysis of patient-derived xenograft models confirmed the role of mitochondrial FAO in Src activation and metastasis. Analysis of TCGA and other independent BC clinical data further reaffirmed the role of mitochondrial FAO and CPT genes in Src regulation and their significance in BC metastasis.

Mitochondrial fusion but not fission regulates larval growth and synaptic development through steroid hormone production.

Mitochondrial fusion and fission affect the distribution and quality control of mitochondria. We show that Marf (Mitochondrial associated regulatory factor), is required for mitochondrial fusion and transport in long axons. Moreover, loss of Marf leads to a severe depletion of mitochondria in neuromuscular junctions (NMJs). Marf mutants also fail to maintain proper synaptic transmission at NMJs upon repetitive stimulation, similar to Drp1 fission mutants. However, unlike Drp1, loss of Marf leads to NMJ morphology defects and extended larval lifespan. Marf is required to form contacts between the endoplasmic reticulum and/or lipid droplets (LDs) and for proper storage of cholesterol and ecdysone synthesis in ring glands. Interestingly, human Mitofusin-2 rescues the loss of LD but both Mitofusin-1 and Mitofusin-2 are required for steroid-hormone synthesis. Our data show that Marf and Mitofusins share an evolutionarily conserved role in mitochondrial transport, cholesterol ester storage and steroid-hormone synthesis. DOI: http://dx.doi.org/10.7554/eLife.03558.001

Mitochondrial myopathy, lactic acidosis, and sideroblastic anemia (MLASA) plus associated with a novel de novo mutation (m.8969G>A) in the mitochondrial encoded ATP6 gene

Mitochondrial myopathy, lactic acidosis and sideroblastic anemia (MLASA) is a rare mitochondrial disorder that has previously been associated with mutations in PUS1 and YARS2. In the present report, we describe a 6-year old male with an MLASA plus phenotype. This patient had features of MLASA in the setting of developmental delay, sensorineural hearing loss, epilepsy, agenesis of the corpus callosum, failure to thrive, and stroke-like episodes. Sequencing of the mitochondrial genome identified a novel de novo, heteroplasmic mutation in the mitochondrial DNA (mtDNA) encoded ATP6 gene (m.8969G>A, p.S148N). Whole exome sequencing did not identify mutations or variants in PUS1 or YARS2 or any known nuclear genes that could affect mitochondrial function and explain this phenotype. Studies of fibroblasts derived from the patient revealed a decrease in oligomycin-sensitive respiration, a finding which is consistent with a complex V defect. Thus, this mutation in MT-ATP6 may represent the first mtDNA point mutation associated with the MLASA phenotype.

Impaired Mitochondrial Energy Production Causes Light-Induced Photoreceptor Degeneration Independent of Oxidative Stress

Two insults often underlie a variety of eye diseases including glaucoma, optic atrophy, and retinal degeneration—defects in mitochondrial function and aberrant Rhodopsin trafficking. Although mitochondrial defects are often associated with oxidative stress, they have not been linked to Rhodopsin trafficking. In an unbiased forward genetic screen designed to isolate mutations that cause photoreceptor degeneration, we identified mutations in a nuclear-encoded mitochondrial gene, ppr, a homolog of human LRPPRC. We found that ppr is required for protection against light-induced degeneration. Its function is essential to maintain membrane depolarization of the photoreceptors upon repetitive light exposure, and an impaired phototransduction cascade in ppr mutants results in excessive Rhodopsin1 endocytosis. Moreover, loss of ppr results in a reduction in mitochondrial RNAs, reduced electron transport chain activity, and reduced ATP levels. Oxidative stress, however, is not induced. We propose that the reduced ATP level in ppr mutants underlies the phototransduction defect, leading to increased Rhodopsin1 endocytosis during light exposure, causing photoreceptor degeneration independent of oxidative stress. This hypothesis is bolstered by characterization of two other genes isolated in the screen, pyruvate dehydrogenase and citrate synthase. Their loss also causes a light-induced degeneration, excessive Rhodopsin1 endocytosis and reduced ATP without concurrent oxidative stress, unlike many other mutations in mitochondrial genes that are associated with elevated oxidative stress and light-independent photoreceptor demise.

Aromatic L-amino acid decarboxylase deficiency diagnosed by clinical metabolomic profiling of plasma

Aromatic L-amino acid decarboxylase (AADC) deficiency is an inborn error of metabolism affecting the biosynthesis of serotonin, dopamine, and catecholamines. We report a case of AADC deficiency that was detected using the Global MAPS platform. This is a novel platform that allows for parallel clinical testing of hundreds of metabolites in a single plasma specimen. It uses a state-of-the-art mass spectrometry platform, and the resulting spectra are compared against a library of ~2500 metabolites. Our patient is now a 4 year old boy initially seen at 11 months of age for developmental delay and hypotonia. Multiple tests had not yielded a diagnosis until exome sequencing revealed compound heterozygous variants of uncertain significance (VUS), c.286G>A (p.G96R) and c.260C>T (p.P87L) in the DDC gene, causal for AADC deficiency. CSF neurotransmitter analysis confirmed the diagnosis with elevated 3-methoxytyrosine (3-O-methyldopa). Metabolomic profiling was performed on plasma and revealed marked elevation in 3-methoxytyrosine (Z-score +6.1) consistent with the diagnosis of AADC deficiency. These results demonstrate that the Global MAPS platform is able to diagnose AADC deficiency from plasma. In summary, we report a novel and less invasive approach to diagnose AADC deficiency using plasma metabolomic profiling.

Chronic Oral l-Carnitine Supplementation Drives Marked Plasma TMAO Elevations in Patients with Organic Acidemias Despite Dietary Meat Restrictions

Recent studies have implicated trimethylamine N-oxide (TMAO) in atherosclerosis, raising concern about L-carnitine, a common supplement for patients with inborn errors of metabolism (IEMs) and a TMAO precursor metabolized, in part, by intestinal microbes. Dietary meat restriction attenuates carnitine-to-TMAO conversion, suggesting that TMAO production may not occur in meat-restricted individuals taking supplemental L-carnitine, but this has not been tested. Here, we mine a metabolomic dataset to assess TMAO levels in patients with diverse IEMs, including organic acidemias. These data were correlated with clinical information and confirmed using a quantitative TMAO assay. Marked plasma TMAO elevations were detected in patients treated with supplemental L-carnitine, including those on a meat-free diet. On average, patients with an organic acidemia had ~45-fold elevated [TMAO], as compared to the reference population. This effect was mitigated by metronidazole therapy lasting 7 days each month. Collectively, our data show that TMAO production occurs at high levels in patients with IEMs receiving oral L-carnitine. Further studies are needed to determine the long-term safety and efficacy of chronic oral L-carnitine supplementation and whether suppression or circumvention of intestinal bacteria may improve L-carnitine therapy.

Diagnosis of adenylosuccinate lyase deficiency by metabolomic profiling in plasma reveals a phenotypic spectrum.

Adenylosuccinate lyase (ADSL) deficiency is a rare autosomal recessive neurometabolic disorder that presents with a broad-spectrum of neurological and physiological symptoms. The ADSL gene produces an enzyme with binary molecular roles in de novo purine synthesis and purine nucleotide recycling. The biochemical phenotype of ADSL deficiency, accumulation of SAICAr and succinyladenosine (S-Ado) in biofluids of affected individuals, serves as the traditional target for diagnosis with targeted quantitative urine purine analysis employed as the predominate method of detection. In this study, we report the diagnosis of ADSL deficiency using an alternative method, untargeted metabolomic profiling, an analytical scheme capable of generating semi-quantitative z-score values for over 1000 unique compounds in a single analysis of a specimen. Using this method to analyze plasma, we diagnosed ADSL deficiency in four patients and confirmed these findings with targeted quantitative biochemical analysis and molecular genetic testing. ADSL deficiency is part of a large a group of neurometabolic disorders, with a wide range of severity and sharing a broad differential diagnosis. This phenotypic similarity among these many inborn errors of metabolism (IEMs) has classically stood as a hurdle in their initial diagnosis and subsequent treatment. The findings presented here demonstrate the clinical utility of metabolomic profiling in the diagnosis of ADSL deficiency and highlights the potential of this technology in the diagnostic evaluation of individuals with neurologic phenotypes.

Functional cellular analyses reveal energy metabolism defect and mitochondrial DNA depletion in a case of mitochondrial aconitase deficiency.

Defects in the tricarboxylic acid cycle (TCA) are associated with a spectrum of neurological phenotypes that are often difficult to diagnose and manage. Whole-exome sequencing (WES) led to a rapid expansion of diagnostic capabilities in such disorders and facilitated a better understanding of disease pathogenesis, although functional characterization remains a bottleneck to the interpretation of potential pathological variants. We report a 2-year-old boy of Afro-Caribbean ancestry, who presented with neuromuscular symptoms without significant abnormalities on routine diagnostic evaluation. WES revealed compound heterozygous missense variants of uncertain significance in mitochondrial aconitase (ACO2), which encodes the TCA enzyme ACO2. Pathogenic variants in ACO2 have been described in a handful of families as the cause of infantile cerebellar-retinal degeneration syndrome. Using biochemical and cellular assays in patient fibroblasts, we found that ACO2 expression was quantitatively normal, but ACO2 enzyme activity was <20% of that observed in control cells. We also observed a deficiency in cellular respiration and, for the first time, demonstrate evidence of mitochondrial DNA depletion and altered expression of some TCA components and electron transport chain subunits. The observed cellular defects were completely restored with ACO2 gene rescue. Our findings demonstrate the pathogenicity of two VUS in ACO2, provide novel mechanistic insights to TCA disturbances in ACO2 deficiency, and implicate mitochondrial DNA depletion in the pathogenesis of this recently described disorder.

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

ABSTRACT 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 (Sucla2SAβgeo), which was used to generate transgenic mice. Sucla2 encodes the ADP-specific β-subunit isoform of SCS. Sucla2SAβgeo 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.