Ertan Mayatepek

Mitochondrial complex I deficiency: from organelle dysfunction to clinical disease

Mitochondria are essential for cellular bioenergetics by way of energy production in the form of ATP through the process of oxidative phosphorylation. This crucial task is executed by five multi-protein complexes of which mitochondrial NADH:ubiquinone oxidoreductase or complex I is the largest and most complicated one. During recent years, mutations in nuclear genes encoding structural subunits of complex I have been identified as a cause of devastating neurodegenerative disorders with onset in early childhood. Here, we present a comprehensive overview of clinical, biochemical and cell physiological information of 15 children with isolated, nuclear-encoded complex I deficiency, which was generated in a joint effort of clinical and fundamental research. Our findings point to a rather homogeneous clinical picture in these children and drastically illustrate the severity of the disease. In extensive live cell studies with patient-derived skin fibroblasts we uncovered important cell physiological aspects of complex I deficiency, which point to a central regulatory role of cellular reactive oxygen species production and altered mitochondrial membrane potential in the pathogenesis of the disorder. Moreover, we critically discuss possible interconnections between clinical signs and cellular pathology. Finally, our results indicate apparent differences to drug therapy on the cellular level, depending on the severity of the catalytic defect and identify modulators of cellular Ca(2+) homeostasis as new candidates in the therapy of complex I deficiency.

Large neutral amino acids block phenylalanine transport into brain tissue in patients with phenylketonuria

Large neutral amino acids (LNAAs), including phenylalanine (Phe), compete for transport across the blood-brain barrier (BBB) via the L-type amino acid carrier. Accordingly, elevated plasma Phe impairs brain uptake of other LNAAs in patients with phenylketonuria (PKU). Direct effects of elevated brain Phe and depleted LNAAs are probably major causes for disturbed brain development and function in PKU. Competition for the carrier might conversely be put to use to lower Phe influx when the plasma concentrations of all other LNAAs are increased. This hypothesis was tested by measuring brain Phe in patients with PKU by quantitative 1H magnetic resonance spectroscopy during an oral Phe challenge with and without additional supplementation with all other LNAAs. Baseline plasma Phe was approximately 1,000 micromol/l and brain Phe was approximately 250 micromol/l in both series. Without LNAA supplementation, brain Phe increased to approximately 400 micromol/l after the oral Phe load. Electroencephalogram (EEG) spectral analysis revealed acutely disturbed brain activity. With concurrent LNAA supplementation, Phe influx was completely blocked and there was no slowing of EEG activity. These results are relevant for further characterization of the LNAA carrier and of the pathophysiology underlying brain dysfunction in PKU and for treatment of patients with PKU, as brain function might be improved by continued LNAA supplementation.

Creatine deficiency syndrome caused by guanidinoacetate methyltransferase deficiency: Diagnostic tools for a new inborn error of metabolism

Hepatic guanidinoacetate methyltransferase deficiency induces a deficiency of creatine/phosphocreatine in muscle and brain and an accumulation of guanidinoacetic acid (GAA), the precursor of creatine. We describe a patient with this defect, a 4-year-old girl with a dystonic-dyskinetic syndrome in addition to developmental delay and therapy-resistant epilepsy. Several methods were used in the diagnosis of the disease: (1) the creatinine excretion in 24-hour urine was significantly lowered, whereas the creatinine concentration in plasma and in randomly collected urine was not strikingly different from control values; (2) the Sakaguchi staining reaction of guanidino compounds in random urine samples indicated an enhanced GAA excretion; (3) GAA excretion measured quantitatively by guanidino compound analysis using an amino acid analyzer was markedly elevated in random urine samples; (4) in vivo 1H magnetic resonance spectroscopy (MRS) revealed a strong depletion of creatine and an accumulation of GAA in brain; (5) in vivo phosphorus 31 MRS showed a strong decrease of the phosphocreatine resonance and a resonance identified as guanidinoacetate phosphate; and (6) in vitro 1H MRS showed an absence of creatine and creatinine resonances in cerebrospinal fluid and the occurrence of GAA in urine. For early detection of this disease, we recommend the Sakaguchi staining reaction of urine from patients with dystonic-dyskinetic syndrome, seizures, and psychomotor retardation. Positive results should result in further investigations including quantitative guanidino compound analysis and both in vivo and in vitro MRS. Although epilepsy was not affected by orally administered creatine (400 to 500 mg/kg per day), this treatment resulted in clinical improvement and an increase of creatine in cerebrospinal fluid and brain tissue.

Neurodegeneration in Methylmalonic Aciduria Involves Inhibition of Complex II and the Tricarboxylic Acid Cycle, and Synergistically Acting Excitotoxicity

Methylmalonic acidurias are biochemically characterized by an accumulation of methylmalonate (MMA) and alternative metabolites. There is growing evidence for basal ganglia degeneration in these patients. The pathomechanisms involved are still unknown, a contribution of toxic organic acids, in particular MMA, has been suggested. Here we report that MMA induces neuronal damage in cultures of embryonic rat striatal cells at a concentration range encountered in affected patients. MMA-induced cell damage was reduced by ionotropic glutamate receptor antagonists, antioxidants, and succinate. These results suggest the involvement of secondary excitotoxic mechanisms in MMA-induced cell damage. MMA has been implicated in inhibition of respiratory chain complex II. However, MMA failed to inhibit complex II activity in submitochondrial particles from bovine heart. To unravel the mechanism underlying neuronal MMA toxicity, we investigated the formation of intracellular metabolites in MMA-loaded striatal neurons. There was a time-dependent intracellular increase in malonate, an inhibitor of complex II, and 2-methylcitrate, a compound with multiple inhibitory effects on the tricarboxylic acid cycle, suggesting their putative implication in MMA neurotoxicity. We propose that neuropathogenesis of methylmalonic aciduria may involve an inhibition of complex II and the tricarboxylic acid cycle by accumulating toxic organic acids, and synergistic secondary excitotoxic mechanisms.

Physical Exercise–Induced Hypoglycemia Caused by Failed Silencing of Monocarboxylate Transporter 1 in Pancreatic β Cells

Exercise-induced hyperinsulinism (EIHI) is a dominantly inherited hypoglycemic disorder characterized by inappropriate insulin secretion during anaerobic exercise or on pyruvate load. We aimed to identify the molecular basis of this novel disorder of beta -cell regulation. EIHI mapped to chromosome 1 (LOD score 3.6) in a genome scan performed for two families with 10 EIHI-affected patients. Mutational analysis of the promoter of the SLC16A1 gene, which encodes monocarboxylate transporter 1 (MCT1), located under the linkage peak, revealed changes in all 13 identified patients with EIHI. Patient fibroblasts displayed abnormally high SLC16A1 transcript levels, although monocarboxylate transport activities were not changed in these cells, reflecting additional posttranscriptional control of MCT1 levels in extrapancreatic tissues. By contrast, when examined in beta cells, either of two SLC16A1 mutations identified in separate pedigrees resulted in increased protein binding to the corresponding promoter elements and marked (3- or 10-fold) transcriptional stimulation of SLC16A1 promoter-reporter constructs. These studies show that promoter-activating mutations in EIHI induce SLC16A1 expression in beta cells, where this gene is not usually transcribed, permitting pyruvate uptake and pyruvate-stimulated insulin release despite ensuing hypoglycemia. These findings describe a novel disease mechanism based on the failure of cell-specific transcriptional silencing of a gene that is highly expressed in other tissues.

Mild trimethylaminuria caused by common variants in FM03 gene

Mild to transient trimethylaminuria is caused by common variants in the FMO3 gene leading to greatly reduced enzyme activity in vivo. FMO3 deficiency may have clinical relevance well beyond unpleasant body odour.

Lack of creatine in muscle and brain in an adult with GAMT deficiency.

Guanidinoacetate methyltransferase deficiency, which so far has been exclusively detected in children, was diagnosed in a 26‐year‐old man. The full‐blown spectrum of clinical symptoms already had been present since infancy without progression of symptoms during adolescence. Cranial magnetic resonance imaging showed normal findings. Ophthalmological examination showed no retinal changes. Besides creatine deficiency in the brain, a distinct lack of phosphocreatine in skeletal muscle was proved by 31P magnetic resonance spectroscopy. Creatine substitution combined with a guanidinoacetate‐lowering diet introduced first at the age of 26 years was shown to be effective by an impressive improvement of epileptic seizures, mental capabilities, and general behavior and by normalization of the 31P spectrum in the skeletal muscle.

Conjugated Bilirubin Triggers Anemia by Inducing Erythrocyte Death

Hepatic failure is commonly associated with anemia, which may result from gastrointestinal bleeding, vitamin deficiency, or liver‐damaging diseases, such as infection and alcohol intoxication. At least in theory, anemia during hepatic failure may result from accelerated clearance of circulating erythrocytes. Here we show that bile duct ligation (BDL) in mice leads to severe anemia despite increased reticulocyte numbers. Bilirubin stimulated suicidal death of human erythrocytes. Mechanistically, bilirubin triggered rapid Ca2+ influx, sphingomyelinase activation, formation of ceramide, and subsequent translocation of phosphatidylserine to the erythrocyte surface. Consistent with our in vitro and in vivo findings, incubation of erythrocytes in serum from patients with liver disease induced suicidal death of erythrocytes in relation to their plasma bilirubin concentration. Consistently, patients with hyperbilirubinemia had significantly lower erythrocyte and significantly higher reticulocyte counts compared to patients with low bilirubin levels. Conclusion: Bilirubin triggers suicidal erythrocyte death, thus contributing to anemia during liver disease. (Hepatology 2015;61:275–284)

Treatment recommendations in long-chain fatty acid oxidation defects: consensus from a workshop

SummaryPublished data on treatment of fatty acid oxidation defects are scarce. Treatment recommendations have been developed on the basis of observations in 75 patients with long-chain fatty acid oxidation defects from 18 metabolic centres in Central Europe. Recommendations are based on expert practice and are suggested to be the basis for further multicentre prospective studies and the development of approved treatment guidelines. Considering that disease complications and prognosis differ between different disorders of long-chain fatty acid oxidation and also depend on the severity of the underlying enzyme deficiency, treatment recommendations have to be disease-specific and depend on individual disease severity. Disorders of the mitochondrial trifunctional protein are associated with the most severe clinical picture and require a strict fat-reduced and fat-modified (medium-chain triglyceride-supplemented) diet. Many patients still suffer acute life-threatening events or long-term neuropathic symptoms despite adequate treatment, and newborn screening has not significantly changed the prognosis for these severe phenotypes. Very long-chain acyl-CoA dehydrogenase deficiency recognized in neonatal screening, in contrast, frequently has a less severe disease course and dietary restrictions in many patients may be loosened. On the basis of the collected data, recommendations are given with regard to the fat and carbohydrate content of the diet, the maximal length of fasting periods and the use of l-carnitine in long-chain fatty acid oxidation defects.

NMDA receptor activation and respiratory chain complex V inhibition contribute to neurodegeneration in d‐2‐hydroxyglutaric aciduria

The inherited neurometabolic disease d‐2‐hydroxyglutaric aciduria is complicated by progressive neurodegeneration of vulnerable brain regions during infancy and early childhood, frequently presenting with hypotonia, epilepsy and psychomotor retardation. Here, we report that the pathogenetic role of the endogenously accumulating metabolite d‐2‐hydroxyglutarate (D‐2), which is structurally similar to the excitatory amino acid glutamate, is mediated by at least three mechanisms. (i) D‐2‐induced excitotoxic cell damage in primary neuronal cultures from chick and rat involved N‐methyl‐d‐aspartate (NMDA) receptor activation. Indeed, D‐2 activated recombinant NMDA receptors (NR1/NR2A, NR1/NR2B) but not recombinant alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole (AMPA) receptors in HEK293 cells. (ii) Fluorescence microscopy using fura‐2 as a calcium indicator and the oxidant‐sensitive dye dihydrorhodamine‐123 revealed that D‐2 disturbed intracellular calcium homeostasis and elicited the generation of reactive oxygen species. (iii) D‐2 reduced complex V (ATP synthase) activity of the mitochondrial respiratory chain, reflecting an impaired energy metabolism due to inhibition of ATP synthesis but without affecting the electron‐transferring complexes I–IV. Thus, D‐2 stimulates neurodegeneration by mechanisms well‐known for glutamate, NMDA or mitochondrial toxins. In conclusion, excitotoxicity contributes to the neuropathology of d‐2‐hydroxyglutaric aciduria, highlighting new neuroprotective strategies.