In vitro supplementation with dAMP/dGMP leads to partial restoration of mtDNA levels in mitochondrial depletion syndromes

Abstract

Mitochondrial DNA depletion syndrome, a frequent cause of childhood (hepato)encephalomyopathies, is defined as a reduction of mitochondrial DNA copy number related to nuclear DNA. It was previously shown that mtDNA depletion can be prevented by dAMP/dGMP supplementation in deoxyguanosine kinase-deficient fibroblasts. We investigated myotubes of patients diagnosed with mtDNA depletion carrying pathogenic mutations in DGUOK, POLG1 (Alpers syndrome) and TYMP. Differentiating myotubes of all patients and controls were supplemented with different doses of dAMP/dGMP or dAMP/dGMP/dCMP in TYMP deficiency, and analysed for mtDNA/nDNA ratio and for cytochrome c oxidase (COX) activity. Serum deprivation and myotube formation triggered a decrease in mtDNA copy number in DGUOK or POLG1 deficient myotubes, but not in TYMP deficiency and healthy controls. Supplementation with dAMP/dGMP leads to a significant and reproducible rescue of mtDNA depletion in DGUOK deficiency. POLG1 deficient myotubes also showed a mild, not significant increase in mtDNA copy number. MtDNA depletion did not result in deficient COX staining in DGUOK and POLG1-deficient myotubes. Treatment with ethidium bromide resulted in very severe depletion and absence of COX staining in all cell types, and no recovery was observed after supplementation with dAMP/dGMP. We show that supplementation with dAMP/dGMP increases mtDNA copy number significantly in DGUOK deficient myotubes and, leads to a mild, non-significant improvement of mtDNA depletion in POLG1 deficiency. No adverse effect on mtDNA copy number was observed on high-dose supplementation in vitro. Further studies are needed to determine possible therapeutic implications of dAMP/dGMP supplementation for DGUOK deficiency in vivo.

Recessive deoxyguanosine kinase deficiency causes juvenile onset mitochondrial myopathy

“…This decrease in mtDNA copy number is consistent with the mtDNA deficiency that was shown in patient samples from muscle tissue. Supplementation with dAMP and dGMP now causes a significant 250% increase in mtDNA copy number (paired t-test value of 0.035) also consistent with that previously published in DGUOK deficient myoblasts [21].…”

Overexpression of DNA Polymerase Zeta Reduces the Mitochondrial Mutability Caused by Pathological Mutations in DNA Polymerase Gamma in Yeast

“…It has been reported that expression of a yeast mitochondrial repair/recombination enzyme, Cce1, the cruciform-cutting endonuclease, in human cells harboring partially duplicated mtDNA induced the appearance of wt mtDNA, thus demonstrating that expression of a yeast protein could be beneficial for mtDNA stability in mammalian cells [78]. Such a genetic rescue could be associated to a treatment with dNTPs, which seems to be beneficial for the detrimental effects caused by specific mutations in human POLG : the processivity defect of human DNA polymerase gamma harboring Y955C mutation was overcome by increasing the dATP or dTTP concentration during in vitro synthesis of mtDNA fragments [65] and the treatment with dAMP and dGMP leads to a slight increase in the levels of mtDNA in myoblasts deriving from an Alpers' patient harboring mutations A467T and K1191N in POLG [79].…”

A Review Comparing Deoxyribonucleoside Triphosphate (dNTP) Concentrations in the Mitochondrial and Cytoplasmic Compartments of Normal and Transformed Cells

“…In fibroblasts from patients with TK2 deficiency, mitochondrial dTTP, dCTP, and dATP pools were all decreased while dGTP was slightly increased in one patient and slightly decreased in another patient [36]. The fact that externally supplied deoxyribonucleosides and deoxyribonucleoside monophosphates are able to rescue mtDNA depletion provides further evidence that limited substrate availability can cause mtDNA depletion [35, 37, 38]. Experiments from cellular and animal models have also reported disruptions in mitochondrial dNTP pool homeostasis [39-41].…”