Molecular dysfunction associated with the human mitochondrial 3302A>G mutation in the MTTL1 (mt-tRNA(Leu(UUR))) gene

The gene encoding mt-tRNA(Leu(UUR)), MT-TL1, is a hotspot for pathogenic mtDNA mutations. Amongst the first to be described was the 3302A>G transition which resulted in a substantial accumulation in patient muscle of RNA19, an unprocessed RNA intermediate including mt-16S rRNA, mt-tRNA(Leu(UUR)) and MTND1. We have now been able to further assess the molecular aetiology associated with 3302A>G in transmitochondrial cybrids. Increased steady-state levels of RNA19 was confirmed, although not to the levels previously reported in muscle. This data was consistent with an increase in RNA19 stability. The mutation resulted in decreased mt-tRNA(Leu(UUR)) levels, but its stability was unchanged, consistent with a defect in RNA19 processing responsible for low tRNA levels. A partial defect in aminoacylation was also identified, potentially caused by an alteration in tRNA structure. These deficiencies lead to a severe defect in respiration in the transmitochondrial cybrids, consistent with the profound mitochondrial disorder originally associated with this mutation

Mitochondrial DNA mutations affect calcium handling in differentiated neurons

Mutations in the mitochondrial genome are associated with a wide range of neurological symptoms, but many aspects of the basic neuronal pathology are not understood. One candidate mechanism, given the well-established role of mitochondria in calcium buffering, is a deficit in neuronal calcium homoeostasis. We therefore examined calcium responses in the neurons derived from various ‘cybrid’ embryonic stem cell lines carrying different mitochondrial DNA mutations. Brief (∼50 ms), focal glutamatergic stimuli induced a transient rise in intracellular calcium concentration, which was visualized by bulk loading the cells with the calcium dye, Oregon Green BAPTA-1. Calcium entered the neurons through N-methyl-d-aspartic acid and voltage-gated calcium channels, as has been described in many other neuronal classes. Intriguingly, while mitochondrial mutations did not affect the calcium transient in response to single glutamatergic stimuli, they did alter the responses to repeated stimuli, with each successive calcium transient decaying ever more slowly in mitochondrial mutant cell lines. A train of stimuli thus caused intracellular calcium in these cells to be significantly elevated for many tens of seconds. These results suggest that calcium-handling deficits are likely to contribute to the pathological phenotype seen in patients with mitochondrial DNA mutations

The Variability of the Harlequin Mouse Phenotype Resembles that of Human Mitochondrial-Complex I-Deficiency Syndromes

Background: Despite the considerable progress made in understanding the molecular bases of mitochondrial diseases, no effective treatments have been developed to date. Faithful animal models would be extremely helpful for designing such treatments. We showed previously that the Harlequin mouse phenotype was due to a specific mitochondrial complex I deficiency resulting from the loss of the Apoptosis Inducing Factor (Aif) protein. Methodology/Principal Findings: Here, we conducted a detailed evaluation of the Harlequin mouse phenotype, including the biochemical abnormalities in various tissues. We observed highly variable disease expression considering both severity and time course progression. In each tissue, abnormalities correlated with the residual amount of the respiratory chain complex I 20 kDa subunit, rather than with residual Aif protein. Antioxidant enzyme activities were normal except in skeletal muscle, where they were moderately elevated. Conclusions/Significance: Thus, the Harlequin mouse phenotype appears to result from mitochondrial respiratory chain complex I deficiency. Its features resemble those of human complex I deficiency syndromes. The Harlequin mouse hold

Mouse Studies to Shape Clinical Trials for Mitochondrial Diseases: High Fat Diet in Harlequin Mice

BACKGROUND: Therapeutic options in human mitochondrial oxidative phosphorylation (OXPHOS) diseases have been poorly evaluated mostly because of the scarcity of cohorts and the inter-individual variability of disease progression. Thus, while a high fat diet (HFD) is often recommended, data regarding efficacy are limited. Our objectives were 1) to determine our ability to evaluate therapeutic options in the Harlequin OXPHOS complex I (CI)-deficient mice, in the context of a mitochondrial disease with human hallmarks and 2) to assess the effects of a HFD. METHODS AND FINDINGS: Before launching long and expensive animal studies, we showed that palmitate afforded long-term death-protection in 3 CI-mutant human fibroblasts cell lines. We next demonstrated that using the Harlequin mouse, it was possible to draw solid conclusions on the efficacy of a 5-month-HFD on neurodegenerative symptoms. Moreover, we could identify a group of highly responsive animals, echoing the high variability of the disease progression in Harlequin mice. CONCLUSIONS: These results suggest that a reduced number of patients with identical genetic disease should be sufficient to reach firm conclusions as far as the potential existence of responders and non responders is recognized. They also positively prefigure HFD-trials in OXPHOS-deficient patients

Bezielle Selectively Targets Mitochondria of Cancer Cells to Inhibit Glycolysis and OXPHOS

Bezielle (BZL101) is a candidate oral drug that has shown promising efficacy and excellent safety in the early phase clinical trials for advanced breast cancer. Bezielle is an aqueous extract from the herb Scutellaria barbata. We have reported previously that Bezielle was selectively cytotoxic to cancer cells while sparing non-transformed cells. In tumor, but not in non-transformed cells, Bezielle induced generation of ROS and severe DNA damage followed by hyperactivation of PARP, depletion of the cellular ATP and NAD, and inhibition of glycolysis. We show here that tumor cells' mitochondria are the primary source of reactive oxygen species induced by Bezielle. Treatment with Bezielle induces progressively higher levels of mitochondrial superoxide as well as peroxide-type ROS. Inhibition of mitochondrial respiration prevents generation of both types of ROS and protects cells from Bezielle-induced death. In addition to glycolysis, Bezielle inhibits oxidative phosphorylation in tumor cells and depletes mitochondrial reserve capacity depriving cells of the ability to produce ATP. Tumor cells lacking functional mitochondria maintain glycolytic activity in presence of Bezielle thus supporting the hypothesis that mitochondria are the primary target of Bezielle. The metabolic effects of Bezielle towards normal cells are not significant, in agreement with the low levels of oxidative damage that Bezielle inflicts on them. Bezielle is therefore a drug that selectively targets cancer cell mitochondria, and is distinguished from other such drugs by its ability to induce not only inhibition of OXPHOS but also of glycolysis. This study provides a better understanding of the mechanism of Bezielle's cytotoxicity, and the basis of its selectivity towards cancer cells

Neonatal presentation of ventricular tachycardia and a Reye-like syndrome episode associated with disturbed mitochondrial energy metabolism

BACKGROUND: Hyperammonemia, hypoglycemia, hepatopathy, and ventricular tachycardia are common presenting features of carnitine-acylcarnitine translocase deficiency (Mendelian Inheritance in Man database: *212138), a mitochondrial fatty acid oxidation disorder with a lethal prognosis. These features have not been identified as the presenting features of mitochondrial cytopathy in the neonatal period. CASE PRESENTATION: We describe an atypical presentation of mitochondrial cytopathy in a 2 day-old neonate. She presented with a Reye-like syndrome episode, premature ventricular contractions and ventricular tachycardia. Initial laboratory evaluation exhibited a large amount of 3-methylglutaconic acid on urine organic acid analysis, mild orotic aciduria and a nonspecific abnormal acylcarnitine profile. The evaluation for carnitine-acylcarnitine translocase deficiency and other fatty acid oxidation disorders was negative. The patient later developed a hypertrophic cardiomyopathy and continued to be affected by recurrent Reye-like syndrome episodes triggered by infections. A muscle biopsy exhibited signs of a mitochondrial cytopathy. During the course of her disease, her Reye-like syndrome episodes have subsided; however, cardiomyopathy has persisted along with fatigue and exercise intolerance. CONCLUSIONS: This case illustrates that, in the neonatal period, hyperammonemia and ventricular tachycardia may be the presenting features of a lethal carnitine-acylcarnitine translocase deficiency or of a mitochondrial cytopathy, associated with a milder clinical course. This association broadens the spectrum of presenting phenotypes observed in patients with disturbed mitochondrial energy metabolism. Also, the presence of 3-methylglutaconic aciduria suggests mitochondrial dysfunction and mild orotic aciduria could potentially be used as a marker of mitochondrial disease

Renal involvement in mitochondrial cytopathies

Mitochondrial cytopathies constitute a group of rare diseases that are characterized by their frequent multisystemic involvement, extreme variability of phenotype and complex genetics. In children, renal involvement is frequent and probably underestimated. The most frequent renal symptom is a tubular defect that, in most severe forms, corresponds to a complete De Toni-Debré-Fanconi syndrome. Incomplete proximal tubular defects and other tubular diseases have also been reported. In rare cases, patients present with chronic tubulo-interstitial nephritis or cystic renal diseases. Finally, a group of patients develop primarily a glomerular disease. These patients correspond to sporadic case reports or can be classified into two major defects, namely 3243 A>G tRNALEU mutations and coenzyme Q10 biosynthesis defects. The latter group is particularly important because it represents the only treatable renal mitochondrial defect. In this Educational Review, the principal characteristics of these diseases and the main diagnostic approaches are summarized

Region-Specific Expression of Mitochondrial Complex I Genes during Murine Brain Development

Mutations in the nuclear encoded subunits of mitochondrial complex I (NADH:ubiquinone oxidoreductase) may cause circumscribed cerebral lesions ranging from degeneration of the striatal and brainstem gray matter (Leigh syndrome) to leukodystrophy. We hypothesized that such pattern of regional pathology might be due to local differences in the dependence on complex I function. Using in situ hybridization we investigated the relative expression of 33 nuclear encoded complex I subunits in different brain regions of the mouse at E11.5, E17.5, P1, P11, P28 and adult (12 weeks). With respect to timing and relative intensity of complex I gene expression we found a highly variant pattern in different regions during development. High average expression levels were detected in periods of intense neurogenesis. In cerebellar Purkinje and in hippocampal CA1/CA3 pyramidal neurons we found a second even higher peak during the period of synaptogenesis and maturation. The extraordinary dependence of these structures on complex I gene expression during synaptogenesis is in accord with our recent findings that gamma oscillations – known to be associated with higher cognitive functions of the mammalian brain – strongly depend on the complex I activity. However, with the exception of the mesencephalon, we detected only average complex I expression levels in the striatum and basal ganglia, which does not explain the exquisite vulnerability of these structures in mitochondrial disorders

Metabolic phenotype of methylmalonic acidemia in mice and humans: the role of skeletal muscle

<p>Abstract</p> <p>Background</p> <p>Mutations in methylmalonyl-CoA mutase cause methylmalonic acidemia, a common organic aciduria. Current treatment regimens rely on dietary management and, in severely affected patients, liver or combined liver-kidney transplantation. For undetermined reasons, transplantation does not correct the biochemical phenotype.</p> <p>Methods</p> <p>To study the metabolic disturbances seen in this disorder, we have created a murine model with a null allele at the methylmalonyl-CoA mutase locus and correlated the results observed in the knock-out mice to patient data. To gain insight into the origin and magnitude of methylmalonic acid (MMA) production in humans with methylmalonyl-CoA mutase deficiency, we evaluated two methylmalonic acidemia patients who had received different variants of combined liver-kidney transplants, one with a complete liver replacement-kidney transplant and the other with an auxiliary liver graft-kidney transplant, and compared their metabolite production to four untransplanted patients with intact renal function.</p> <p>Results</p> <p>Enzymatic, Western and Northern analyses demonstrated that the targeted allele was null and correctable by lentiviral complementation. Metabolite studies defined the magnitude and tempo of plasma MMA concentrations in the mice. Before a fatal metabolic crisis developed in the first 24–48 hours, the methylmalonic acid content per gram wet-weight was massively elevated in the skeletal muscle as well as the kidneys, liver and brain. Near the end of life, extreme elevations in tissue MMA were present primarily in the liver. The transplant patients studied when well and on dietary therapy, displayed massive elevations of MMA in the plasma and urine, comparable to the levels seen in the untransplanted patients with similar enzymatic phenotypes and dietary regimens.</p> <p>Conclusion</p> <p>The combined observations from the murine metabolite studies and patient investigations indicate that during homeostasis, a large portion of circulating MMA has an extra-heptorenal origin and likely derives from the skeletal muscle. Our studies suggest that modulating skeletal muscle metabolism may represent a strategy to increase metabolic capacity in methylmalonic acidemia as well as other organic acidurias. This mouse model will be useful for further investigations exploring disease mechanisms and therapeutic interventions in methylmalonic acidemia, a devastating disorder of intermediary metabolism.</p