MPV17 Loss Causes Deoxynucleotide Insufficiency and Slow DNA Replication in Mitochondria

Abstract

MPV17 is a mitochondrial inner membrane protein whose dysfunction causes mitochondrial DNA abnormalities and disease by an unknown mechanism. Perturbations of deoxynucleoside triphosphate (dNTP) pools are a recognized cause of mitochondrial genomic instability; therefore, we determined DNA copy number and dNTP levels in mitochondria of two models of MPV17 deficiency. In Mpv17 ablated mice, liver mitochondria showed substantial decreases in the levels of dGTP and dTTP and severe mitochondrial DNA depletion, whereas the dNTP pool was not significantly altered in kidney and brain mitochondria that had near normal levels of DNA. The shortage of mitochondrial dNTPs in Mpv17-/- liver slows the DNA replication in the organelle, as evidenced by the elevated level of replication intermediates. Quiescent fibroblasts of MPV17-mutant patients recapitulate key features of the primary affected tissue of the Mpv17-/- mice, displaying virtual absence of the protein, decreased dNTP levels and mitochondrial DNA depletion. Notably, the mitochondrial DNA loss in the patients’ quiescent fibroblasts was prevented and rescued by deoxynucleoside supplementation. Thus, our study establishes dNTP insufficiency in the mitochondria as the cause of mitochondrial DNA depletion in MPV17 deficiency, and identifies deoxynucleoside supplementation as a potential therapeutic strategy for MPV17-related disease. Moreover, changes in the expression of factors involved in mitochondrial deoxynucleotide homeostasis indicate a remodeling of nucleotide metabolism in MPV17 disease models, which suggests mitochondria lacking functional MPV17 have a restricted purine mitochondrial salvage pathway.

MPV17-related mitochondrial DNA maintenance defect: New cases and review of clinical, biochemical, and molecular aspects

“…Study of fibroblasts obtained from individuals with MPV17 deficiency showed similar results with decreased mitochondrial deoxynucleotides and mtDNA depletion. Additionally, the mtDNA loss in these fibroblasts was prevented and rescued by deoxynucleoside supplementation (Dalla Rosa et al., ). This study established deoxynucleotide insufficiency in the mitochondria as the cause of mtDNA depletion in MPV17 deficiency (Dalla Rosa et al., ).…”

“…Studying mitochondria from Mpv17 deficient mice liver and fibroblasts obtained from individuals with MPV17 deficiency showed decreased mitochondrial nucleotides and severe mtDNA depletion. Interestingly, nucleoside supplementation to these fibroblasts was able to prevent and rescue the mtDNA loss; therefore, nucleoside supplementation can be a potential therapeutic strategy for MPV17 ‐related mtDNA maintenance defect (Dalla Rosa et al., ).…”

Ribonucleotides in mitochondrial DNA

“…For instance, fibroblasts from patients with defects in mitochondrial dNTP metabolism show altered frequencies of the individual rNMPs . Furthermore, mice defective in the mitochondrial inner membrane protein MPV17 that is implicated in mitochondrial disease exhibit decreased dGTP and dTTP pools in the liver and increased frequency of rGMP embedded in mtDNA . However, the correlation between rNTP/dNTP ratios and mtDNA rNMP frequency in mammals might not be as clear‐cut as in budding yeast.…”

Identification of a novel heterozygous guanosine monophosphate reductase (GMPR) variant in a patient with a late‐onset disorder of mitochondrial DNA maintenance

“…dNTPs were extracted from total homogenates or mitochondrial pellets and dNTP pools were measured by the polymerase-based method as described previously. 36 2.10 | Quantitative and qualitative assessments of mtDNA in proliferating and quiescent cells DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen), according to the manufacturer instructions. Quantification of relative mtDNA copy number in proliferating and quiescent cells was performed as described previously.…”