Mitochondrial DNA Depletion and Thymidine Phosphate Pool Dynamics in a Cellular Model of Mitochondrial Neurogastrointestinal Encephalomyopathy

Abstract

Mitochondrial (mt) neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease associated with depletion, deletions, and point mutations of mtDNA. Patients lack a functional thymidine phosphorylase and their plasma contains high concentrations of thymidine and deoxyuridine; elevation of the corresponding triphosphates probably impairs normal mtDNA replication and repair. To study metabolic events leading to MNGIE we used as model systems skin and lung fibroblasts cultured in the presence of thymidine and/or deoxyuridine at concentrations close to those in the plasma of the patients, a more than 100-fold excess relative to controls. The two deoxynucleosides increased the mt and cytosolic dTTP pools of skin fibroblasts almost 2-fold in cycling cells and 8-fold in quiescent cells. During up to a two-month incubation of quiescent fibroblasts with thymidine (but not with deoxyuridine), mtDNA decreased to ϳ50% without showing deletions or point mutations. When we removed thymidine, but maintained the quiescent state, mtDNA recovered rapidly. With thymidine in the medium, the dTTP pool of quiescent cells turned over rapidly at a rate depending on the concentration of thymidine, due to increased degradation and resynthesis of dTMP in a substrate (‫؍‬futile) cycle between thymidine kinase and 5-deoxyribonucleotidase. The cycle limited the expansion of the dTTP pool at the expense of ATP hydrolysis. We propose that the substrate cycle represents a regulatory mechanism to protect cells from harmful increases of dTTP. Thus MNGIE patients may increase their consumption of ATP to counteract an unlimited expansion of the dTTP pool caused by circulating thymidine. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)2 is an autosomal recessive human mitochondrial (mt) disease, caused by the defective function of the cytosolic enzyme thymidine phosphorylase (1) and is associated with depletion (2), multiple deletions (2, 3), and point mutations of mtDNA (4). Thymidine phosphorylase converts the pyrimidine deoxynucleosides thymidine and deoxyuridine to free bases and deoxyribose 1-phosphate. Human blood from healthy individuals contains Ͻ0.05 M of the two deoxynucleosides, whereas both thymidine and deoxyuridine are found at 10 -20 M concentrations in plasma from MNGIE patients (5, 6). It has been proposed that this large increase in circulating deoxynucleosides results in a similar increase in the intracellular concentration of their triphosphates disrupting the normal balance of the 4 dNTPs and thereby interfering with the replication of mtDNA (4, 6). Experimental evidence supports this hypothesis. Addition of thymidine to the medium of cultured, rapidly growing cells lead to an increase in the dTTP pool and a rapid cessation of nuclear (and probably also mt) DNA replication caused by a depletion of the dCTP pool (7). Depletion of dCTP, as well as the concomitant increase of dGTP and dATP were secondary to the allosteric effects of the large dTTP pool on the enzyme ribonucleotide reductase. In that...

Mitochondrial Thymidine Kinase and the Enzymatic Network Regulating Thymidine Triphosphate Pools in Cultured Human Cells

“…4B). These results concern total cellular dTTP in quiescent fibroblasts, but we obtained similar results when we separated the mt dTTP pool, substantiating previous data (7,18).…”

“…At physiological low concentrations, thymidine contributes to the maintenance of the dTTP pool through salvage by TK2. On the other hand, salvage of excess circulating thymidine caused by TP deficiency disrupts the regulation of the dTTP pool and causes mtDNA abnormalities (18,19).…”

“…The two enzymes have substrate specificities that allow for the phosphorylation of all four deoxyribonucleosides required for DNA synthesis. Isotope-flow experiments with radioactive deoxyribonucleosides indicate that the intramitochondrial salvage pathway is constitutively active (7,18). Indeed, until the discovery of the pathology linked to p53R2 deficiency, it was considered the main provider of dNTPs for mtDNA replication in quiescent and differentiated cells where the cytosolic pool is very low.…”

Gastrointestinal Dysmotility in Mitochondrial Neurogastrointestinal Encephalomyopathy Is Caused by Mitochondrial DNA Depletion

“…10 In contrast, the load of deleted mtDNA molecules may be reduced in replicating cells because of negative selection. 23 In summary, our study demonstrates that severe depletion of mtDNA is the most striking molecular defect in smooth muscle of the GI tract and vascular wall of MNGIE patients. The depletion of mtDNA correlates with histopathological abnormalities and is likely due to toxic levels of dThd and dUrd disrupting mitochondrial nucleotide pool, as confirmed by in vitro studies.…”

“…The depletion of mtDNA correlates with histopathological abnormalities and is likely due to toxic levels of dThd and dUrd disrupting mitochondrial nucleotide pool, as confirmed by in vitro studies. 12,23 The mechanism for this effect remains unknown.…”