Increased dNTP pools rescue mtDNA depletion in human POLG‐deficient fibroblasts

Abstract

Polymerase γ catalytic subunit (POLG) gene encodes the enzyme responsible for mitochondrial DNA (mtDNA) synthesis. Mutations affecting POLG are the most prevalent cause of mitochondrial disease because of defective mtDNA replication and lead to a wide spectrum of clinical phenotypes characterized by mtDNA deletions or depletion. Enhancing mitochondrial deoxyribonucleoside triphosphate (dNTP) synthesis effectively rescues mtDNA depletion in different models of defective mtDNA maintenance due to dNTP insufficiency. In this study, we studied mtDNA copy number recovery rates following ethidium bromide‐forced depletion in quiescent fibroblasts from patients harboring mutations in different domains of POLG. Whereas control cells spontaneously recovered initial mtDNA levels, POLG‐deficient cells experienced a more severe depletion and could not repopulate mtDNA. However, activation of deoxyribonucleoside (dN) salvage by supplementation with dNs plus erythro‐9‐(2‐hydroxy‐3‐nonyl) adenine (inhibitor of deoxyadenosine degradation) led to increased mitochondrial dNTP pools and promoted mtDNA repopulation in all tested POLG‐mutant cells independently of their specific genetic defect. The treatment did not compromise POLG fidelity because no increase in multiple deletions or point mutations was detected. Our study suggests that physiologic dNTP concentration limits the mtDNA replication rate. We thus propose that increasing mitochondrial dNTP availability could be of therapeutic interest for POLG deficiency and other conditions in which mtDNA maintenance is challenged.—Blázquez‐Bermejo, C., Carreño‐Gago, L., Molina‐Granada, D., Aguirre, J., Ramon, J., Torres‐Torronteras, J., Cabrera‐Pérez, R., Martín, M. Á., Domínguez‐González, C., de la Cruz, X., Lombès, A., García‐Arumí, E., Martí, R., Cámara, Y. Increased dNTP pools rescue mtDNA depletion in human POLG‐deficient fibroblasts. FASEB J. 33, 7168–7179 (2019). http://www.fasebj.org

Defects in mtDNA replication challenge nuclear genome stability through nucleotide depletion and provide a unifying mechanism for mouse progerias

“…Supplementation of exogenous nucleosides was not sufficient to rescue the growth defects nor to decrease the genomic DNA breaks in mutator cells (Extended Data Fig. 4), in line with a recent report of adenosine deaminase activity preventing successful supplementation of cells with extracellular nucleosides 23 . We found expression of several nucleotidases to be increased in mutator cells, despite the wholecellular dNTP depletion (Extended Data Fig.…”

Therapy Prospects for Mitochondrial DNA Maintenance Disorders

“…Interestingly, similar positive effects of dN supplementation on mtDNA replication have been observed in vitro and in vivo for mutations in genes not directly related with dNTP metabolism, such as POLG and MPV17 [ 110 , 111 , 181 ]. This is particularly significant for the case of patients with mutations in POLG , as this is the most prevalent gene among all those causing MDDS.…”

“…The main reason that this enzyme has been claimed to have a role in dNTP homeostasis relies on the observation that dNTP addition rescues mtDNA depletion, as observed in ABAT -deficient cultured cells [ 44 ]. In fact, dNs are the actual molecules that enter the cell after dNTP addition to the cell culture [ 92 ], and the enhancement of mtDNA replication by intracellular dN-stimulated dNTP synthesis has been observed for deficiencies other than those directly related with dNTP synthesis [ 110 , 111 ]. In fact, increased dNTP availability may increase mtDNA copy number even in fully functional cells [ 112 ].…”

Clinical, pathological and genetic spectrum in 89 cases of mitochondrial progressive external ophthalmoplegia

“…We suggest that reaching a definite genetic diagnosis is essential for patients with mitochondrial PEO since it allows proper genetic counselling and precision medicine in the near future; in this regard, patients with TK2 or POLG mutations could benefit from treatment with nucleosides 18 19…”