While >300 disease-causing variants have been identified in the mitochondrial DNA (mtDNA) polymerase γ, no mitochondrial phenotypes have been associated with POLRMT, the RNA polymerase responsible for transcription of the mitochondrial genome. Here, we characterise the clinical and molecular nature of POLRMT variants in eight individuals from seven unrelated families. Patients present with global developmental delay, hypotonia, short stature, and speech/intellectual disability in childhood; one subject displayed an indolent progressive external ophthalmoplegia phenotype. Massive parallel sequencing of all subjects identifies recessive and dominant variants in the POLRMT gene. Patient fibroblasts have a defect in mitochondrial mRNA synthesis, but no mtDNA deletions or copy number abnormalities. The in vitro characterisation of the recombinant POLRMT mutants reveals variable, but deleterious effects on mitochondrial transcription. Together, our in vivo and in vitro functional studies of POLRMT variants establish defective mitochondrial transcription as an important disease mechanism.

• MeSH
• dítě MeSH
• DNA řízené RNA-polymerasy chemie   genetika   MeSH
• dospělí MeSH
• fibroblasty metabolismus   patologie   MeSH
• genetická transkripce * MeSH
• kojenec MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• mitochondriální DNA genetika   MeSH
• mitochondrie genetika   MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mutace genetika   MeSH
• nemoci nervového systému genetika   patologie   MeSH
• oxidativní fosforylace MeSH
• podjednotky proteinů metabolismus   MeSH
• proteinové domény MeSH
• rodokmen MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

BACKGROUND: Effectiveness of L-asparaginase administration in acute lymphoblastic leukemia treatment is mirrored in the overall outcome of patients. Generally, leukemia patients differ in their sensitivity to L-asparaginase; however, the mechanism underlying their inter-individual differences is still not fully understood. We have previously shown that L-asparaginase rewires the biosynthetic and bioenergetic pathways of leukemia cells to activate both anti-leukemic and pro-survival processes. Herein, we investigated the relationship between the metabolic profile of leukemia cells and their sensitivity to currently used cytostatic drugs. METHODS: Altogether, 19 leukemia cell lines, primary leukemia cells from 26 patients and 2 healthy controls were used. Glycolytic function and mitochondrial respiration were measured using Seahorse Bioanalyzer. Sensitivity to cytostatics was measured using MTS assay and/or absolute count and flow cytometry. Mitochondrial membrane potential was determined as TMRE fluorescence. RESULTS: Using cell lines and primary patient samples we characterized the basal metabolic state of cells derived from different leukemia subtypes and assessed their sensitivity to cytostatic drugs. We found that leukemia cells cluster into distinct groups according to their metabolic profile. Lymphoid leukemia cell lines and patients sensitive to L-asparaginase clustered into the low glycolytic cluster. While lymphoid leukemia cells with lower sensitivity to L-asparaginase together with resistant normal mononuclear blood cells gathered into the high glycolytic cluster. Furthermore, we observed a correlation of specific metabolic parameters with the sensitivity to L-asparaginase. Greater ATP-linked respiration and lower basal mitochondrial membrane potential in cells significantly correlated with higher sensitivity to L-asparaginase. No such correlation was found in the other cytostatic drugs tested by us. CONCLUSIONS: These data support that cell metabolism plays a prominent role in the treatment effect of L-asparaginase. Based on these findings, leukemia patients with lower sensitivity to L-asparaginase with no specific genetic characterization could be identified by their metabolic profile.

• MeSH
• akutní lymfatická leukemie krev   farmakoterapie   patologie   MeSH
• antitumorózní látky farmakologie   terapeutické užití   MeSH
• asparaginasa farmakologie   terapeutické užití   MeSH
• biosyntetické dráhy účinky léků   MeSH
• chemorezistence MeSH
• dítě MeSH
• glykolýza účinky léků   MeSH
• kojenec MeSH
• kostní dřeň patologie   MeSH
• lidé MeSH
• membránový potenciál mitochondrií účinky léků   MeSH
• metabolom účinky léků   MeSH
• mitochondrie účinky léků   metabolismus   MeSH
• mladiství MeSH
• mladý dospělý MeSH
• nádorové buněčné linie MeSH
• oxidativní fosforylace účinky léků   MeSH
• předškolní dítě MeSH
• výsledek terapie MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

By determining the calcium retention capacity (CRC) of rat liver mitochondria, we confirmed and extended previous observations describing the activation of mitochondrial swelling by phosphate and tert-butyl hydroperoxide (t-BHP). Using CRC measurements, we showed that both phosphate and t-BHP decrease the extent of calcium accumulation required for the full mitochondrial permeability transition pore (MPTP) opening to 35 % of control values and to only 15 % when both phosphate and t-BHP are present in the medium. When changes in fluorescence were evaluated at higher resolution, we observed that in the presence of cyclosporine A fluorescence values return after each Ca(2+) addition to basal values obtained before the Ca(2+) addition. This indicates that the MPTP remains closed. However, in the absence of cyclosporine A, the basal fluorescence after each Ca(2+) addition continuously increased. This increase was potentiated both by phosphate and t-BHP until the moment when the concentration of intramitochondrial calcium required for the full opening of the MPTP was reached. We conclude that in the absence of cyclosporine A, the MPTP is slowly opened after each Ca(2+) addition and that this rate of opening can be modified by various factors such as the composition of the media and the experimental protocol used.

• MeSH
• fosfáty farmakologie   MeSH
• jaterní mitochondrie účinky léků   metabolismus   MeSH
• krysa rodu rattus MeSH
• potkani Wistar MeSH
• terc-butylhydroperoxid farmakologie   MeSH
• transportní proteiny mitochondriální membrány metabolismus   MeSH
• vápník metabolismus   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

l-asparaginase (ASNase), a key component in the treatment of childhood acute lymphoblastic leukemia (ALL), hydrolyzes plasma asparagine and glutamine and thereby disturbs metabolic homeostasis of leukemic cells. The efficacy of such therapeutic strategy will depend on the capacity of cancer cells to adapt to the metabolic challenge, which could relate to the activation of compensatory metabolic routes. Therefore, we studied the impact of ASNase on the main metabolic pathways in leukemic cells. Treating leukemic cells with ASNase increased fatty-acid oxidation (FAO) and cell respiration and inhibited glycolysis. FAO, together with the decrease in protein translation and pyrimidine synthesis, was positively regulated through inhibition of the RagB-mTORC1 pathway, whereas the effect on glycolysis was RagB-mTORC1 independent. As FAO has been suggested to have a pro-survival function in leukemic cells, we tested its contribution to cell survival following ASNase treatment. Pharmacological inhibition of FAO significantly increased the sensitivity of ALL cells to ASNase. Moreover, constitutive activation of the mammalian target of rapamycin pathway increased apoptosis in leukemic cells treated with ASNase, but did not increase FAO. Our study uncovers a novel therapeutic option based on the combination of ASNase and FAO inhibitors.

• MeSH
• akutní lymfatická leukemie farmakoterapie   metabolismus   patologie   MeSH
• asparaginasa terapeutické užití   MeSH
• autofagie účinky léků   MeSH
• lidé MeSH
• mastné kyseliny metabolismus   MeSH
• monomerní proteiny vázající GTP fyziologie   MeSH
• multiproteinové komplexy fyziologie   MeSH
• nádorové buněčné linie MeSH
• oxidace-redukce MeSH
• pyrimidiny biosyntéza   MeSH
• TOR serin-threoninkinasy fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

TMEM70, a 21-kDa protein localized in the inner mitochondrial membrane, has been shown to facilitate the biogenesis of mammalian F1Fo ATP synthase. Mutations of the TMEM70 gene represent the most frequent cause of isolated ATP synthase deficiency resulting in a severe mitochondrial disease presenting as neonatal encephalo-cardiomyopathy (OMIM 604273). To better understand the biological role of this factor, we generated Tmem70-deficient mice and found that the homozygous Tmem70-/- knockouts exhibited profound growth retardation and embryonic lethality at ∼9.5 days post coitum. Blue-Native electrophoresis demonstrated an isolated deficiency in fully assembled ATP synthase in the Tmem70-/- embryos (80% decrease) and a marked accumulation of F1 complexes indicative of impairment in ATP synthase biogenesis that was stalled at the early stage, following the formation of F1 oligomer. Consequently, a decrease in ADP-stimulated State 3 respiration, respiratory control ratio and ATP/ADP ratios, indicated compromised mitochondrial ATP production. Tmem70-/- embryos exhibited delayed development of the cardiovascular system and a disturbed heart mitochondrial ultrastructure, with concentric or irregular cristae structures. Tmem70+/- heterozygous mice were fully viable and displayed normal postnatal growth and development of the mitochondrial oxidative phosphorylation system. Nevertheless, they presented with mild deterioration of heart function. Our results demonstrated that Tmem70 knockout in the mouse results in embryonic lethality due to the lack of ATP synthase and impairment of mitochondrial energy provision. This is analogous to TMEM70 dysfunction in humans and verifies the crucial role of this factor in the biosynthesis and assembly of mammalian ATP synthase.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• homozygot MeSH
• kardiomyopatie metabolismus   MeSH
• membránové proteiny nedostatek   genetika   metabolismus   MeSH
• mitochondriální membrány metabolismus   MeSH
• mitochondriální nemoci genetika   metabolismus   MeSH
• mitochondriální proteiny nedostatek   genetika   metabolismus   MeSH
• mitochondriální protonové ATPasy biosyntéza   genetika   metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• mutace MeSH
• myši knockoutované MeSH
• myši MeSH
• oxidativní fosforylace MeSH
• těhotenství MeSH
• vrozené poruchy metabolismu metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH