In vitro supplementation with deoxynucleoside monophosphates rescues mitochondrial DNA depletion

Abstract

Mitochondrial DNA depletion syndromes are a genetically heterogeneous group of often severe diseases, characterized by reduced cellular mitochondrial DNA content. Investigation of potential therapeutic strategies for mitochondrial DNA depletion syndromes will be dependent on good model systems. We have previously suggested that myotubes may be the optimal model system for such studies. Here we firstly validate this technique in a diverse range of cells of patients with mitochondrial DNA depletion syndromes, showing contrasting effects in cell lines from genetically and phenotypically differing patients. Secondly, we developed a putative therapeutic approach using variable combinations of deoxynucleoside monophosphates in different types of mitochondrial DNA depletion syndromes, showing near normalization of mitochondrial DNA content in many cases. Furthermore, we used nucleoside reverse transcriptase inhibitors to precisely titrate mtDNA depletion in vitro. In this manner we can unmask a physiological defect in mitochondrial depletion syndrome cell lines which is also ameliorated by deoxynucleoside monophosphate supplementation. Finally, we have extended this model to study fibroblasts after myogenic transdifferentiation by MyoD transfection, which similar to primary myotubes also showed deoxynucleoside monophosphate responsive mitochondrial DNA depletion in vitro, thus providing a more convenient method for deriving future models of mitochondrial DNA depletion. Our results suggest that using different combinations of deoxynucleoside monophosphates depending on the primary gene defect and molecular mechanism may be a possible therapeutic approach for many patients with mitochondrial DNA depletion syndromes and is worthy of further clinical investigation.

Copyright © 2012 Elsevier Inc. All rights reserved.

Figures

Fig. 1

(A) Human myotubes (MT) with POLG (p1, p2) deficiency showed a significant (p<0.05) increase of the mtDNA copy number reaching normal levels when supplemented with 200 μM dNMP mix or with 400 μM dNMP mix compared with unsupplemented myotubes of the same genotype. Remarkably, myotubes derived from a patient (p2) with a very mild clinical phenotype showed a higher original mtDNA copy number, than myotubes derived from a patient (p1) with a severe childhood onset fatal Alpers syndrome. No effect of dNMP supplementation was detected in control myotubes. A significant difference (p<0.05) between unsupplemented and supplemented myotubes of the same genotype is indicated by a star. Error bars indicate standard deviation (n=3). MB (myoblasts). (B) Myogenic transdifferentiation of four fibroblast lines by MyoD gene transfer resulted in 6 days in multinucleated myotubes and an increase of mtDNA copy number in both patients (p7, p8) and in a control (c5) cell line. Supplementation with 400 μM dNMP mix led to a significant (p<0.05) increase of mtDNA/nDNA ratio in all patient cells (p6, p7, p8) compared to untreated cells, but not in the control cell line. A significant difference (p<0.05) between unsupplemented and supplemented myotubes of the same genotype is indicated by a star. Error bars indicate standard deviation (n=3). (C) Myogenic transdifferentiation after adenovirus-mediated MyoD gene transfer was confirmed by immunofluorescence of the two myoblast markers desmin and actinin.

Fig. 2

Myoblasts (MB) derived from a patient (p5) with RRM2B deficiency had significantly lower mtDNA copy number compared to control cells, and differentiation to myotubes (MT) did not lead to a further decrease of mtDNA copy number as compared to myoblasts. There were no changes in mtDNA copy number observed after supplementation with 400 μM dNMP mix during differentiation and fusion into myotubes. A significant difference (p<0.05) between patient and control cells is indicated by a star. Error bars indicate standard deviation.

Fig. 3

(A) Treatment with abacavir resulted in apparent increase in relative mtDNA copy number. Error bars indicate standard deviation. MB myoblasts, MT myotubes. (B) Beta-dystroglycan staining revealed a seriously reduced fusion capacity and decreased cell division in cells treated with abacavir, indicating that abacavir treated cells showed different morphology compared to untreated myotubes due to decreased cell division and a decreased fusion, thus explaining the apparent increase in cellular mtDNA copy number.

Fig. 4

(A) The incubation with didanosine (300 μM) and stavudine (300 μM) respectively led to a significant (pPOLG (p1, p2) and DGUOK deficiency (p3, p4). Stavudine caused a milder decrease of the mtDNA copy number compared to didanosine. A significant difference (p<0.05) between untreated and treated myotubes of the same genotype is indicated by a star. Error bars indicate standard deviation. (B) Incubation with lower concentrations (5 μM and 50 μM) of didanosine after 3 days led to a decrease of the mtDNA copy number in myoblasts from patients with DGUOK deficiency (p3, p4) and a control cell line (c1). A significant difference (p<0.05) between untreated and treated myoblasts of the same genotype is indicated by a star. Error bars indicate standard deviation. MB myoblasts. (C) When dAMP/dGMP (400 μM) was added to didanosine-supplemented media (5 μM and 50 μM), mtDNA copy numbers were restored (p<0.05) in all cell lines. A significant difference (p<0.05) between unsupplemented and supplemented myotubes of the same genotype is indicated by a star. Error bars indicate standard deviation.

Fig. 5

(A) Incubation with 50 μM didanosine for 9 days led to a severe decrease of COX activity in myotubes (p3) derived from a patient with DGUOK deficiency and to a lesser extent in control cells (c3).(B) Lane 1: myoblasts; lane 2: myotubes; lane 3: myotubes + didanosine; lane 4: myotubes + didanosine + dAMP/dGMP. Primary human myoblasts (lane 1) from a patient with DGUOK deficiency (p3) and one control cell line (c3) showed a low specific band for cytochrome c oxidase (COX, complex IV) subunit II (COX II) (~20 kDa) compared to myotubes. Treatment with didanosine (50 μM) led to a decreased intensity of COX II in patient myotubes, but remained normal in control cells. Supplementation with 400 μM dAMP/dGMP resulted in a partial COX II rescue.