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.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Mpv17 ablation in CFW mice causes liver specific mtDNA depletion and deficiency of all respiratory chain complexes that contain mtDNA-encoded subunits.

(A-C) Mpv17 expression (A), mtDNA copy number (B) and steady state levels of OXPHOS subunits (C) in the liver, kidney and brain of wild-type (WT, Mpv17+/+) and knockout (KO, Mpv17-/-) mice. (B) Quantification of mtDNA in WT and KO mice. Data are expressed as mean ± SEM of n = 6. (Student test, *** P<0.001, NS, not significant, p>0.05). (D) BN-PAGE analysis of OXPHOS complexes in the liver of WT and KO mice. Sup-C, supercomplexes, Sub-C, subcomplexes of the OXPHOS system. Vdac levels were used as a loading control on a 12% SDS-PAGE gel.

Fig 2. Mpv17-/- mouse liver mitochondria have significantly reduced levels of two precursors of DNA synthesis, dGTP and dTTP.

The abundance of dNTPs was determined by means of an extension assay after extraction of nucleotides from liver (A), kidney (B) or brain (C) of Mpv17+/+ and Mpv17-/- mice (see Methods). All mice (n = 5) were sacrificed between 8 and 10 weeks of age. P values were obtained using Mann-Whitney test (* P<0.05, NS, not significant P>0.05).

Fig 3. Mpv17 ablation results in a marked increase of mtDNA replication intermediates.

Analysis of mtDNA replication intermediates in the liver of WT and Mpv17-/- mice. Mitochondrial DNAs from six Mpv17-/- and six control livers were isolated, digested with BclI and fractionated by 2D-AGE and blot hybridized to a probe to the major non-coding region (NCR) of the murine mitochondrial genome.

Fig 4. MPV17 is upregulated in non-dividing cells and quiescence induces mtDNA depletion in MPV17-deficient human fibroblasts, unless supplemented with deoxynucleosides.

(A) Steady state levels of MPV17 protein in proliferating and quiescent control fibroblasts. (B) Relative mtDNA levels in proliferating control (black) and MPV17 mutant fibroblasts (gray). (C) MtDNA quantification in quiescent fibroblasts. The mtDNA amount is expressed relative to the mean of the controls. (D) mtDNA copy number of quiescent control and MPV17-mutant fibroblasts. Data are expressed as mean ± SEM of n = 8. (Student’s t test: ***P<0.001). (E) Mitochondrial dNTP levels in quiescent control and MPV17-mutant fibroblasts. dATP levels were disregarded owing to the low values obtained for the controls. dCTP and dGTP were measured in 3 independent experiments in 3 patients and 3 control cell lines. *** P<0.001—Mann-Whitney test. (F) Relative mtDNA copy number in quiescent fibroblasts supplemented with deoxynucleosides. Quiescent fibroblasts were cultured for 14 days in the absence or presence of 50 μM or 100 μM deoxyadenosine (AdR), deoxycytosine (CdR), deoxyguanosine (GdR), deoxythymidine (TdR) alone or in the different combinations as indicated. The amount of mtDNA is expressed relative to its amount in proliferating cells (Student’s t test: *P<0.05; **P<0.01; ***P<0.001); C1 and C2, control fibroblasts; P1-P5, fibroblasts derived from patients with pathological mutations in MPV17 (S2 Table).

Fig 5. Deoxynucleoside supplementation enables full mtDNA recovery in MPV17-deficient fibroblasts after drug-induced transient depletion.

(A) Slow recovery of mtDNA copy number in quiescent MPV17-mutant fibroblasts after depletion with ethidium bromide (EB). Two control (black) and four MPV17 patient-derived cell lines (red) were tested in two or three independent experiments. (Student’s t test: ***P<0.001) (B) MtDNA recovery in control (black) and MPV17-deficient fibroblasts (red) with or without deoxynucleoside supplementation (open and closed circles, respectively) of 100 μM AdR, CdR, and GdR. Data are expressed as mean ± SEM of two control and three MPV17 patient-derived cell lines, tested in two independent experiments (Student’s t test: *P<0.05). (C) MtDNA copy number was monitored via Q-PCR during and after ethidium bromide (EB)-induced mtDNA depletion (as per Fig 5B). C1 –control (black) and P4 MPV17-deficient fibroblast (red), with or without (open and closed circles, respectively) 50 μM of the indicated deoxynucleosides.

Fig 6. The abundance of the mitochondrial deoxynucleotide (di- and tri- phosphate) transporters is increased in the absence of MPV17.

Representative immunoblot of the equilibrative nucleoside transporter (ENT1) in (A) control and MPV17-mutant fibroblasts in dividing and quiescent cells, and (B) in the liver of wild-type (WT) and knockout (KO) mice. (C) Steady state levels of the mitochondrial deoxynucleotide transporters 1 and 2 (PNC1 and PNC2) in control and MPV17 deficient fibroblasts in proliferating and quiescent conditions. (D) Pnc1 and Pnc2 steady state levels in the liver, of type (WT) and knockout (KO) mice. Vinculin (VCL), and Tom20 were used as loading control.

Fig 7. MPV17 loss of function affects the purine branch of mitochondrial salvage pathway.

Representative immunoblot thymidine kinase 2 (TK2) in (A) control and MPV17-mutant fibroblasts in dividing and quiescent cells, and (B) in the liver of wild-type (WT) and knockout (KO) mice. (C) Steady state levels of adenylate kinase 2 and 3 (AK2 and AK3) and Deoxyguanosine Kinase (Dguok) in the liver of wild-type (WT) and knockout (KO) mice. The arrow indicates the Dguok isoform downregulated in KO mouse liver. The samples were from 2 month-old mice unless indicated. (D) AK2, AK3 and DGUOK steady state levels in control and MPV17 mutant fibroblasts in proliferating and quiescent cells. Vinculin, GAPDH, and Tom20 were used as loading control.