| Abstract: |
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a severe human disease caused by mutations in TYMP, the gene encoding thymidine phosphorylase (TP). It belongs to a broader group of disorders characterized by a pronounced reduction in mitochondrial DNA (mtDNA) copy number in one or more tissues. In most cases, these disorders are caused by mutations in genes involved in deoxyribonucleoside triphosphate (dNTP) metabolism. It is generally accepted that imbalances in mitochondrial dNTP pools resulting from these mutations interfere with mtDNA replication. Nonetheless, the precise mechanistic details of this effect, in particular, how an excess of a given dNTP (e. g. , imbalanced dTTP excess observed in TP deficiency) might lead to mtDNA depletion, remain largely unclear. Using an in organello replication experimental model with isolated murine liver mitochondria, we observed that overloads of dATP, dGTP, or dCTP did not reduce the mtDNA replication rate. In contrast, an excess of dTTP decreased mtDNA synthesis, but this effect was due to secondary dCTP depletion rather than to the dTTP excess in itself. This was confirmed in human cultured cells, demonstrating that our conclusions do not depend on the experimental model. Our results demonstrate that the mtDNA replication rate is unaffected by an excess of any of the 4 separate dNTPs and is limited by the availability of the dNTP present at the lowest concentration. Therefore, the availability of dNTP is the key factor that leads to mtDNA depletion rather than dNTP imbalances. These results provide the first test of the mechanism that accounts for mtDNA depletion in MNGIE and provide evidence that limited dNTP availability is the common cause of mtDNA depletion due to impaired anabolic or catabolic dNTP pathways. Thus, therapy approaches focusing on restoring the deficient substrates should be explored. Mitochondria are subcellular organelles that constitute the main energy supply within the cell. They contain their own DNA, which should be continuously replicated to ensure the correct mitochondrial function. Several mitochondrial diseases are caused by genetic defects that compromise this replication and result in mitochondrial DNA depletion. In most cases, these genetic defects block the synthesis of dATP, dGTP, dCTP, and dTTP, the 4 nucleotides needed for mitochondrial DNA replication. However, for one of these disorders (mitochondrial neurogastrointestinal encephalomyopathy, MNGIE), the biochemical pathways needed to synthesize them are intact, but degradation of dTTP is genetically blocked, leading to dTTP accumulation. We investigated the biochemical mechanisms through which the dTTP excess leads to mitochondrial DNA depletion in MNGIE, and we found that the delay of mitochondrial DNA replication rate observed when dTTP is in excess is not caused by this excess in itself. Instead, the dTTP overload produces a secondary dCTP depletion that actually delays mitochondrial DNA replication. Therefore, the common factor accounting for mitochondrial DNA depletion in these disorders is the limited availability of one or more nucleotides. This indicates that strategies to provide nucleotides to patients' mitochondria should be explored as a possible treatment for these fatal disorders. |
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(Hospital Universitari Vall d'Hebron. Institut de Recerca)
