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 .

INTRODUCTION

Mitochondrial DNA depletion syndrome (MDS) is defined as a reduction of mitochondrial DNA (mtDNA) copy number in different tissues, leading to insufficient synthesis of respiratory chain (RC) complexes I, III, IV and V ( 1–3 ). MDS is a frequent cause of severe childhood (hepato)encephalomyopathies and responsible for ∼50% of combined-RC deficiencies in childhood ( 4 ). The maintenance of mtDNA requires several nuclear-encoded factors participating in the replication, in the balanced supply of dNTPs to mitochondria or being part of the mitochondrial replisome ( 5 ). The identification of several human disease genes coding for enzymes or proteins involved in nucleotide metabolism underscores the importance of understanding the nature and sources of mtDNA precursor pools ( 3 ).

Mutations of eight nuclear genes ( DGUOK, MPV17, POLG1, TYMP, TK2, SUCLA2, SUCLG1, RRM2B ), most of them involved in the synthesis or maintenance of mitochondrial nucleotide pools, were identified in ∼60% of all MDS cases implying further genetic heterogeneity ( 3 , 4 ). Mitochondrial depletion may initially affect single organs, typically skeletal muscle or liver, and later spread to other tissues causing severe hepatoencephalopathy or encephalomyopathy ( 4 , 5 ).

Thymidine phosphorylase ( TYMP ) defects are associated with leukodystrophy and gastrointestinal disorder ( 6 ). Mutations in deoxyguanosine kinase ( DGUOK , dGK ) ( 7 ), MPV17 ( 8 ) and polymerase gamma (POLG1) ( 9 ) were described in the hepatocerebral form of MDS, whereas thymidine kinase 2 ( TK2) mutations are associated with myopathy and CK elevation ( 10 ). Myopathy in various combinations with hypotonia, tubulopathy, seizures, respiratory distress, diarrhea and lactic acidosis and a very severe depletion of mtDNA occur in children carrying mutations in the RRM2B gene ( 11 ). Some children may have a milder course and longer survival ( 12 ). Mutations in two further Krebs-cycle enzymes, the ADP-forming succinyl-CoA synthase ( SUCLA2) ( 13 ) and the alpha subunit of succinate-coenzyme A ligase ( SUCLG1 ) ( 14 ), were identified in patients with mtDNA depletion and encephalomyopathy. Patients carrying mutations in SUCLA2 are frequently affected by mild methylmalonic aciduria, Leigh-like encephalomyopathy, dystonia and deafness ( 15 , 16 ).

The dNTP pools for the nuclear genome are synthesized primarily in the cytosol and presumably pass into the nucleus passively through the nuclear pore complex ( 17 ). The provision of dNTPs to the mitochondrial replisome is a more complex process and regulated in concert with the cell cycle. The earliest indication that precursor pools serving mitochondria are distinct from the nuclear precursor pools came in 1976, when Bogenhausen and Clayton ( 18 ) showed that in cultured mouse cells treated with antimetabolites, mtDNA replication proceeds almost undisturbed, whereas nuclear replication was strongly inhibited. The incubation of rat liver mitochondria with exogeneous dNTPs led to the incorporation of nucleotides into mtDNA showing that deoxyribonucleotides synthesized in the cytosol are transported to mitochondria ( 19 , 20 ). Later studies reported that the nucleotide transport of monophosphates is independent of the ATP/ADP carrier ( 21 ) and a two-directional communication between the mitochondrial and cytosolic dNTP pools exists and guanine deoxynucleotides easily enter mitochondria from the cytosol ( 22 ). The same studies showed that in the absence of DNA synthesis, the size of the dGTP pool is maintained by increased degradation of dGMP with excretion of deoxyguanosine into the medium, avoiding the accumulation of dGMP in toxic amounts ( 22 ).

Mitochondrial dNTPs are formed by the salvage of deoxyribonucleosides catalyzed by four different deoxynucleoside kinases ( 23 , 24 ). Two of them, thymidine kinase 1 and deoxycytidine kinase, are localized in the cytosol and their products must enter mitochondria to become available for mtDNA synthesis. The other two enzymes, TK2 and dGK, are localized inside mitochondria ( 22 ). The substrate specificity of TK2 permits the phosphorylation of thymidine and deoxycytidine, and that of dGK the phosphorylation of deoxyguanosine and deoxyadenosine ( 25 , 26 ).

It was previously shown that mtDNA synthesis occurs during the S-phase in dGK deficient fibroblasts, in contrast to control fibroblasts where mtDNA synthesis is cell cycle independent ( 27 , 28 ). When cycling is inhibited by serum deprivation and cells are in a resting state, the mtDNA content in dGK deficient fibroblasts drops considerably, yet remains stable in control fibroblasts. Furthermore, the decline in mtDNA content in resting, dGK deficient cells can be prevented by dGMP and dAMP supplementation, suggesting that substrate limitation triggers mtDNA depletion in dGK deficient cells. On the basis of these findings, we performed in vitro studies on cultured human myoblasts and myotubes of patients with pathogenic mutations in DGUOK, POLG1 and TYMP . Muscle cells of patients with POLG1 and TYMP mutations served as a disease controls in comparison with DGUOK deficient cells and were also supplemented with dAMP/dGMP. To compensate the excessive levels of thymidine in TYMP defect cells, we also performed supplementation with a combination of dAMP/dGMP/dCMP.

RESULTS

Human primary myotubes carrying mutations in DGUOK and POLG1 show mtDNA depletion

Myotubes of patients with DGUOK (p1, p2) and POLG1 (p3) deficiency but not with TYMP mutations (p4) showed a highly significant ( P < 0.01) decrease of mtDNA copy numbers if compared with control myotubes (Fig.  1 A). POLG1 mutant cells were from a patient with Alpers syndrome and mtDNA depletion. The mtDNA/nDNA ratio progressively decreased during differentiation and reached the lowest level on day 5, when virtually all cells had differentiated into myotubes (Fig.  1 B). Myotubes were kept in culture for 6 days.

Figure 1.

( A ) Human myotubes (MT) with DGUOK (p1, p2) and POLG1 (p3) deficiency, but not myotubes with TYMP mutations (p4) and not control myotubes (c1, c2) showed a significant ( P < 0.01) decrease of mtDNA copy numbers after 7 days of differentiation, if compared with myoblasts (MB). All cultures were carried out in three separate wells, and each well was tested by quantitative PCR in triplicate. A significant difference ( P < 0.01) between myoblasts and myotubes of the same genotype is indicated by a star. Error bars indicate standard deviation, n -values show the number of wells, investigated for each cell type. ( B ) The mtDNA/nDNA ratio was progressively decreasing over time in DGUOK (data not shown) and POLG1 deficiency (p3) but not in controls during differentiation from myoblasts (MB) into myotubes (MT) and reached the lowest level on day 5, when virtually all cells had differentiated. A significant difference ( P < 0.01) between myoblasts and myotubes of the same genotype is indicated by a star. Error bars indicate standard deviation, n -values show the number of wells, investigated for each cell type.

Figure 1.

( A ) Human myotubes (MT) with DGUOK (p1, p2) and POLG1 (p3) deficiency, but not myotubes with TYMP mutations (p4) and not control myotubes (c1, c2) showed a significant ( P < 0.01) decrease of mtDNA copy numbers after 7 days of differentiation, if compared with myoblasts (MB). All cultures were carried out in three separate wells, and each well was tested by quantitative PCR in triplicate. A significant difference ( P < 0.01) between myoblasts and myotubes of the same genotype is indicated by a star. Error bars indicate standard deviation, n -values show the number of wells, investigated for each cell type. ( B ) The mtDNA/nDNA ratio was progressively decreasing over time in DGUOK (data not shown) and POLG1 deficiency (p3) but not in controls during differentiation from myoblasts (MB) into myotubes (MT) and reached the lowest level on day 5, when virtually all cells had differentiated. A significant difference ( P < 0.01) between myoblasts and myotubes of the same genotype is indicated by a star. Error bars indicate standard deviation, n -values show the number of wells, investigated for each cell type.

On the basis of these results, we performed supplementation studies of DGUOK and POLG1 deficient myotubes showing mtDNA depletion with different concentrations and combinations of nucleotides and tested their effect on mtDNA copy number. DGUOK deficient cells of two-independent patients (p1, p2) showed a highly significant increase ( P < 0.01) in mtDNA copy number reaching normal levels after supplementation with dAMP/dGMP, and there was a mild, non-significant ( P > 0.05) increase of mtDNA/nDNA also in control myotubes. This effect was dose-dependent. The highest mtDNA copy number was reached on supplementation with a combination of 200–400 µ m dAMP/dGMP, while higher concentrations did not lead to a further increase (Fig.  2 A). Substitution with dAMP or dGMP alone had less effect, showing that DGUOK deficiency possibly causes a defect in the biosynthesis of both purine nucleotides (data not shown). In contrast, no increase of mtDNA copy number was detected after supplementation with different concentrations of uridine (data not shown).

Figure 2.

( A ) Two independent DGUOK deficient cell lines (p1, p2) showed a highly significant ( P < 0.01) increase in mtDNA copy number reaching normal levels when supplemented with 200 µ m dAMP/dGMP or with 400 µ m dAMP/dGMP compared with unsupplemented myotubes of the same genotype. This effect was dose-dependent. The mtDNA copy number, reached on supplementation with 400 µ m dAMP/dGMP did not increase further with higher doses. A significant difference ( P < 0.01) between unsupplemented and supplemented myotubes of the same genotype is indicated by a star. Error bars indicate standard deviation, n -values show the number of wells, investigated for each cell type. ( B ) The increase in mtDNA copy numbers was significant ( P < 0.01) in DGUOK deficient cells (p1, p2) on supplementation with 200 dAMP/dGMP and on supplementation with 400 µ m dAMP/dGMP compared with unsupplemented myotubes. In cells with mutations in POLG1 (p3) and TYMP (p4) and in controls a similar, but milder increase of mtDNA copy number was observed after dAMP/dGMP supplementation which did not reach statistical significance ( P > 0.05). A significant difference ( P < 0.01) between unsupplemented and supplemented myotubes of the same genotype is indicated by a star. Error bars indicate standard deviation, n -values show the number of wells, investigated for each cell type.

Figure 2.

( A ) Two independent DGUOK deficient cell lines (p1, p2) showed a highly significant ( P < 0.01) increase in mtDNA copy number reaching normal levels when supplemented with 200 µ m dAMP/dGMP or with 400 µ m dAMP/dGMP compared with unsupplemented myotubes of the same genotype. This effect was dose-dependent. The mtDNA copy number, reached on supplementation with 400 µ m dAMP/dGMP did not increase further with higher doses. A significant difference ( P < 0.01) between unsupplemented and supplemented myotubes of the same genotype is indicated by a star. Error bars indicate standard deviation, n -values show the number of wells, investigated for each cell type. ( B ) The increase in mtDNA copy numbers was significant ( P < 0.01) in DGUOK deficient cells (p1, p2) on supplementation with 200 dAMP/dGMP and on supplementation with 400 µ m dAMP/dGMP compared with unsupplemented myotubes. In cells with mutations in POLG1 (p3) and TYMP (p4) and in controls a similar, but milder increase of mtDNA copy number was observed after dAMP/dGMP supplementation which did not reach statistical significance ( P > 0.05). A significant difference ( P < 0.01) between unsupplemented and supplemented myotubes of the same genotype is indicated by a star. Error bars indicate standard deviation, n -values show the number of wells, investigated for each cell type.

In cells with mutations in POLG1 and TYMP a similar, but milder effect of dAMP/dGMP supplementation was observed (Fig.  2 B). These results were not statistically significant ( P > 0.05). Uridine administration did not show any effect.

In MNGIE caused by mutations in the TYMP gene, the pathological increase of thymidine caused by a defect of thymidine eliminating machinery might lead to secondary mtDNA depletion and/or mtDNA mutations. In order to improve the balance of the four nucleotides, we added a mixture of the three other nucleotides (dAMP/dGMP/dCMP) in two different concentrations (200 and 400 µ m ) to MNGIE cells. Significant changes in mtDNA copy number were not observed, and mtDNA deletions were not detected on long-range PCR (Fig.  3 ).

Figure 3.

Supplementation with a combination of three nucleotides (dAMP/dGMP/dCMP) at two different concentrations (200 and 400 µ m ) did not lead to a significant change ( P > 0.05) of mtDNA copy number in MNGIE ( TYMP ) cells (p4) and controls compared with unsupplemented cells. Error bars indicate standard deviation, n -values show the number of wells, investigated for each cell type.

Figure 3.

Supplementation with a combination of three nucleotides (dAMP/dGMP/dCMP) at two different concentrations (200 and 400 µ m ) did not lead to a significant change ( P > 0.05) of mtDNA copy number in MNGIE ( TYMP ) cells (p4) and controls compared with unsupplemented cells. Error bars indicate standard deviation, n -values show the number of wells, investigated for each cell type.

Biochemical and histochemical studies did not reveal a COX defect in patient myoblasts

To investigate whether the changes of mtDNA copy number were associated with a functional defect in these cells, biochemical and histochemical studies were performed. Biochemical activities of the RC enzymes in DGUOK and POLG1 deficient myoblasts showed normal results. Although mtDNA copy numbers decreased significantly in DGUOK and POLG1 defect myotubes, histochemical stains for cytochrome c oxidase (COX) were normal.

WST-1 test

The metabolic activity in fused DGUOK and POLG1 deficient cells as well as in two control cell lines with and without 200 and 400 µ m dAMP/dGMP supplementation was determined by treating cultures with WST-1 for 3 h (see Materials and Methods). The amount of dye formed correlates to the amount of mitochondrial dehydrogenases and overall mitochondrial activity. This test also represents mtDNA independent dehydrogenases and is generally used to assess cell viability/proliferation. It does not accurately measure mitochondrial RC function but rather the general mitochondrial content. However, it was shown that mitochondrial dehydrogenase activity is directly related to mitochondrial energy production and a fall in activity suggests a reduced production ( 29 ). Optical density was read on a spectrophotometer and percentage metabolic activity was compared with fused control cells from a healthy control incubated with WST-1 (100%). No difference was observed between patients and controls and supplementation did not change the result, implying normal growth and mitochondrial enzymatic function in all cell lines.

Triggering mtDNA depletion with EtBr

Administration of EtBr induced RC deficiency. After 16 days of supplementation with EtBr (50 ng/µl), severe mtDNA depletion was observed in both myotubes and myoblasts. The severe depletion caused by an additional EtBr supplementation in DGUOK deficient myotubes and also in control cells was not reversed by 200 or 400 µ m dAMP/dGMP (Fig.  4 ). Myotubes treated with EtBr also showed a decreased histochemical staining for COX, which did not improve for dAMP/dGMP supplementation. The level of mtDNA depletion was very severe (1000–2000 times lower compared with cells without EtBr), which is far bellow the biochemical threshold for expressing COX deficiency. Our results suggest that such low mtDNA copy numbers cannot be rescued in this in vitro model. Further experiments with different EtBr concentrations or shorter exposure periods may help to define whether there is a threshold for reversibility.

Figure 4.

Logarithmic scale: after 16 days of addition of EtBr (50 ng/µl) to the medium caused severe, highly significant 1000–2000-fold ( P < 0.01) mtDNA depletion both in myotubes (MT) and myoblasts (MB) with DGUOK deficiency (p1, p2) and also in controls (c1), and this was not reversed by 200 or 400 µ m dAMP/dGMP.

Figure 4.

Logarithmic scale: after 16 days of addition of EtBr (50 ng/µl) to the medium caused severe, highly significant 1000–2000-fold ( P < 0.01) mtDNA depletion both in myotubes (MT) and myoblasts (MB) with DGUOK deficiency (p1, p2) and also in controls (c1), and this was not reversed by 200 or 400 µ m dAMP/dGMP.

No rearrangements of mtDNA and nuclear DNA were observed on high-dose dAMP/dGMP

In order to test the possible toxic effect of nucleotides, we performed supplementation studies with dAMP/dGMP in increasing concentrations up to very high levels (1200 µ m ). We did not find any morphological evidence, decreased cell division rate or impaired fusion capacity as a toxic effect up to 800 µ m . Supplementation with 1200 µ m dAMP/dGMP resulted in a decreased fusion capacity, which was detected on a deficient staining with beta-dystroglycan (Fig.  5 ), while mtDNA copy numbers further increased (Fig.  2 A). In order to investigate the possible effect of high-dose dAMP/dGMP administration on DNA stability, long-range PCR analysis for the detection of multiple mtDNA deletions and CGH array for chromosomal instability were performed. Rearrangements of mtDNA and nuclear DNA were not detected in any of the treated cell cultures (data not shown).

Figure 5.

A negative staining of muscle cells with beta-dystroglycan suggests an adverse effect of 1200 µ m dAMP/dGMP on myotube differentiation of both DGUOK defect (p1) and control (c1) cells compared with untreated DGUOK (p1) and POLG1 (p3) deficient and control (c1) myotubes.

Figure 5.

A negative staining of muscle cells with beta-dystroglycan suggests an adverse effect of 1200 µ m dAMP/dGMP on myotube differentiation of both DGUOK defect (p1) and control (c1) cells compared with untreated DGUOK (p1) and POLG1 (p3) deficient and control (c1) myotubes.

DISCUSSION

MtDNA depletion disorders lead to clinical symptoms in a tissue specific manner caused by RC deficiency related to low mtDNA copy number. Therefore, mtDNA depletion and rearrangements might be detected in affected post-mitotic tissues more easily when compared with dividing, cultured cells. Studying these conditions was previously performed in cultured fibroblasts, which are dividing cells with glycolytic function, not depending on oxidative phosphorylation. However, mtDNA depletion is a tissue specific condition and may be better represented in muscle and liver.

We performed our studies on primary human muscle cell lines, which share more similarities with the affected tissue, skeletal muscle. Differentiation of myoblasts into myotubes is used in investigating muscle disorders. Several muscle specific proteins are expressed in myotubes, but not in the dividing myoblasts. Myotubes represent an excellent model system for post-mitotic tissues and may be more widely used in studying mitochondrial diseases. We aimed to utilize myotubes of patients with different mtDNA depletion syndromes in vitro to develop a cell culture model for mtDNA depletion. MtDNA depletion was previously triggered in dGK deficient fibroblasts by cell cycle inhibition with serum deprivation ( 27 , 28 ). On the basis of these results, we hypothesized that mtDNA copy number might decrease in patient myotubes, where differentiation is triggered by serum deprivation, which is the standard method for initiating myotube differentiation through cell cycle inhibition in human muscle cells. We detected significant mtDNA depletion in myotubes of patients with mtDNA depletion syndromes carrying pathogenic mutations in DGUOK and POLG1 , but not in controls, implying that the decreased mtDNA/nDNA ratio is not caused by an increase of nuclei. Multiple mtDNA deletions, another consequence of pathogenic POLG1 and TYMP mutations were not detected in the same samples. The appearance of multiple mtDNA deletions in human disease is usually in late adult life, which may imply that the formation of mtDNA deletions on a detectable amount is a long-term process and cannot be reliably studied in cell culture, as reported by others ( 3 ).

The detection of mtDNA depletion in myotubes is consistent with previous results, showing that nucleotides can enter the mitochondria from the cytosol in dividing cells. However, non-dividing cells rely only on the mitochondrially synthesized nucleotide supply synthesized by the mitochondrial enzymes DGUOK and TK2 ( 22 , 23 ). The significant mtDNA depletion in myotubes may serve as an in vitro model of mtDNA depletion and gives the possibility to further investigate the pathomechanism of mtDNA depletion and also be used for supplementation studies in order to develop possible future therapies.

It was previously shown that free nucleotides enter mitochondria ( 22 ) in the form of monophosphates. Therefore, we aimed to rescue the decrease in mtDNA copy number in patient myotubes by supplementation with different nucleotide monophosphates. We show that in myotubes with dGK defect, where the amount of available purine nucleotides for mtDNA synthesis is reduced, supplementation by dAMP/dGMP rescued mtDNA depletion and retained normal mtDNA copy numbers. These results extend the findings of previous studies ( 27 , 28 ) to a different cell model and suggest a possible avenue for future therapy. It would be interesting to measure dNTP pools in the myotubes, particularly in cells with DGUOK mutations with and without dAMP/dGMP supplementation, but this measurement is technically difficult to perform in the limited number of myotubes that can be generated in vitro .

Myotubes of a child with Alpers syndrome caused by POLG1 mutations developed mtDNA depletion and showed a milder, not significant increase in mtDNA copy numbers when supplemented. This may reflect a different pathomechanism of depletion in POLG1 mutated cells, caused by the dysfunction of the mitochondrial polymerase, when compared with the insufficient supply of nucleotides in dGK deficiency. Encouraged by the mild positive effect and based on the different pathomechanism, we suggest that supplementation with all four nucleotides should be studied in POLG1 deficiency.

In MNGIE, excessive levels of thymidine might lead to mtDNA depletion and/or mtDNA deletions or point mutations. Fibroblasts derived from MNGIE patients have been shown to excrete thymidine into the culture medium, whereas normal controls actively metabolize thymidine ( 30 ). The excess thymidine is generally thought to increase dTTP pools in MNGIE, and in fact, thymidine supplementation has been shown to increase dTTP in both mitochondria and cytosol ( 21 ) and to cause mtDNA depletion (50%), but not mtDNA deletions or point mutations in quiescent cells after 2 months ( 31 ). In order to trigger mtDNA depletion in MNGIE myotubes, supplementation with thymidine may be investigated in future studies. It was suggested that mtDNA mutations are generated in MNGIE as a result of an excess of dTTP in the mitochondria ( 32 ). Furthermore, thymidine excess is known to reduce the activity of TK ( 33 ); however, further studies failed to demonstrate the expected excess of intramitochondrial dTTP in MNGIE cells ( 34 ). Our results did not detect significant mtDNA depletion and/or mtDNA deletions in MNGIE myotubes. Adding the other three nucleotides dAMP/dGMP/dCMP did not lead to a change in mtDNA copy number. Our studies in MNGIE may have failed to have a beneficial effect for several reasons. First, the level of the three other nucleotides may not have reached sufficiently high concentrations within the cells to compete with the increased thymidine for mtDNA synthesis because of the intact excretion mechanisms leading to catabolism, while thymidine could not have been eliminated and the imbalance remained. Alternatively, the high levels might block mtDNA synthesis as a compensatory mechanism, however the absence of such an effect on high dose (1200 µ m ) dAMP/dGMP administration in other cell types makes this explanation unlikely. Our results suggest that supplementation with deoxy-mononucleotide phosphates in MNGIE do not show a significant positive effect in cell culture and does not provide support for a therapy in MNGIE.

To investigate the possible toxic effect of nucleotides, we supplemented the cells with very high dose of nucleotides (up to 1200 µ m ). Interestingly, a mild growth retardation and an insufficient differentiation were observed in cell culture without any decrease in mtDNA copy number. In order to test whether high dose of nucleotides might lead to the formation of mtDNA and/or nuclear DNA rearrangements ( 35 , 36 ), we performed CGH array and long-range PCR for mtDNA deletions in DNA samples extracted from cells treated with high doses of dAMP/dGMP. We did not detect any sign of chromosomal or mitochondrial rearrangements after 6 days. We cannot exclude that the duration of the nucleotide exposure was too short to develop such abnormalities, however previous studies showed that 3 days exposure with the nucleoside analogue zidovudine (AZT) lead to a dose-dependent increase of mutagenicity in different reporter genes ( 37 ).

We assume that if excretion mechanisms in nucleotide metabolism are intact, excess amount of physiological nucleotides can be eliminated and mtDNA maintenance can be preserved. Our results imply that the toxic effect of nucleotide supplementation might not disturb mtDNA maintenance significantly and this method might be considered in future therapy trials. Both uptake and efflux of nucleotides might occur at the monophosphate level ( 38 ). In contrast, imbalance of mtDNA synthesis might occur if these mechanisms are defect, like in MNGIE, or if non-physiological nucleoside analogues are used.

Ashley et al . ( 34 ) developed an in vitro system to measure the incorporation of radiolabelled dNTPs into mitochondria of saponin permeabilized cells. They observed reduced incorporation of exogeneous 32 P-dTTP in fibroblasts from a patient with Alpers syndrome, DGUOK mutations and mtDNA depletion of unknown origin compared with controls. However, incorporation of 32 P-dTTP relative to either cell doubling time or 32 P-dCTP incorporation was increased in patients with thymidine kinase deficiency or Twinkle mutations. Such DNA precursor asymmetry may cause pausing of the replication complex and exacerbate the propensity for age-related mtDNA mutations. Because deviations from the normal concentrations of dNTPs are known to be pathogenic, intramitochondrial imbalance may underlie the multiple mtDNA mutations observed in these patients.

Recent results showed that fibroblasts of children with Alpers syndrome caused by POLG1 mutations showed reduced incorporation of exogeneous 32 P-dTTP ( 34 ) and develop a mosaic cellular depletion, which may be a manifestation of severe replication stalling ( 30 ). Overall mtDNA depletion developed only if cells were cultured through several passages. However, there are only few control data to show whether this is specific for POLG1 deficient cell cultures.

MtDNA depletion needs to surpass a certain threshold to cause RC deficiency. It was shown in skeletal muscle fibres of patients with TK2 mutations that mtDNA copy numbers higher than 0.01 copies of mtDNA/µm 3 are sufficient to preserve COX activity ( 39 ). This defines the minimum amount of wild-type mtDNA molecules required to maintain COX activity in mature skeletal muscle and provides an explanation for the mosaic histochemical pattern seen in patients with mtDNA depletion syndrome. If mtDNA copy number increases above this level, the functional consequence of mtDNA depletion may be ameliorated. Even a small increase in mtDNA copy numbers might result in significant improvement of RC function and possibly improvement of the clinical phenotype, suggesting that changing mitochondrial nucleotide pools may be a possible way for future therapy. These findings may also explain, why we were unable to induce biochemical RC deficiency as a consequence of mtDNA depletion in our cellular model.

We showed an increase in mtDNA copy numbers in DGUOK deficient myotubes after nucleotide supplementation. However, biochemical measurement of untreated myotubes did not show a defect of RC activities despite significant mtDNA depletion. Probably, the decrease in mtDNA copy number did not exceed the threshold ( 39 ). In order to demonstrate functional benefit of nucleotide supplementation, we triggered more severe mtDNA depletion and RC deficiency.

Then, we applied EtBr to trigger mtDNA depletion. As it was shown previously ( 40 ), EtBr reduces mtDNA copy number in human cells. We observed very severe (1000–2000-fold), highly significant ( P < 0.01) mtDNA depletion and deficient COX staining in all cell types, both in myoblasts and myotubes of patients as well as in normal control myoblasts. However, supplementation with dAMP/dGMP did not lead to a significant change in any of the cell cultures investigated, implying that severe mtDNA depletion through this mechanism might not be reversible. More experiments are needed to titrate the amount and exposure time of EtBr in order to detect the condition where/if patients myotubes are more susceptible than control cells and to examine the effect of nucleotide supplementation. Previous studies with EtBr treated cells showed reversibility of mtDNA depletion in long-term culturing after EtBr withdrawal in controls ( 40 ). Unfortunately, myotubes cannot be maintained in culture for long enough to investigate mtDNA repopulation. Since myoblasts also showed severe mtDNA depletion on EtBr supplementation, long-term cell culture experiments with repopulation studies may provide another avenue for investigating the mtDNA depletion disorders.

Summarizing our results, we suggest that human primary human myotubes can be used as a cellular model for studying mtDNA depletion in vitro . Supplementation with dAMP/dGMP resulted in a significant increase of mtDNA copy number without evidence of toxicity on mtDNA copy number for DGUOK deficiency suggesting a possible therapeutic approach, which needs to be further investigated.

MATERIALS AND METHODS

Patients

Primary human myoblasts cultures of two patients (p1, p2) with DGUOK deficiency caused by homozygous mutations c.705+1_4delGTAA (P1) and p.52S>F (p2), respectively, of one patient (p3) with Alpers–Huttenlocher syndrome caused by the compound heterozygous p.Ala467Thr/p.Lys1191Asn mutations in the POLG1 gene and one MNGIE patient (p4) carrying a homozygous mutation p.Glu289Ala in the TYMP gene as well as two healthy controls (c1, c2) were obtained from the Muscle Tissue Culture Collection (Friedrich-Baur Institute, Munich, Germany). (Table  1 ).

Table 1.

Summary of the clinical and genetic data of the patients

Patient Clinical phenotype Defect gene Pathogenic mutations 
p1 Hepatoencephalopathy, muscular hypotonia DGUOK Homozygous c.705+1_4delGTAA 
p2 Hepatoencephalopathy, muscular hypotonia DGUOK Homozygous p.Ser52Phe 
p3 Alpers–Huttenlocher syndrome with liver failure POLG1 p.Ala467Thr/p.Lys1191Asn 
p4 MNGIE syndrome ECGF1 Homozygous p.Glu289Ala 
Patient Clinical phenotype Defect gene Pathogenic mutations 
p1 Hepatoencephalopathy, muscular hypotonia DGUOK Homozygous c.705+1_4delGTAA 
p2 Hepatoencephalopathy, muscular hypotonia DGUOK Homozygous p.Ser52Phe 
p3 Alpers–Huttenlocher syndrome with liver failure POLG1 p.Ala467Thr/p.Lys1191Asn 
p4 MNGIE syndrome ECGF1 Homozygous p.Glu289Ala 

Patient p1 was the first child of consanguineous Turkish parents (first-grade cousins). He developed an acute liver failure, muscular hypotonia and nystagmus between age 3 and 6 months of life and died of a severe liver failure at 9 months of age. Patient p4 is an adult German patient who presented in his teens with a severe demyelinating peripheral neuropathy and foot deformity resembling Charcot–Marie–Tooth disease, but no genetic defect on known CMT genes was identified. He developed severe gastrointestinal pseudo-obstruction with ophthalmoparesis and lactic acidosis at age 32 years. Brain MRI detected severe leukodystrophy, suggestive of MNGIE.

The case histories of p2 and p3 were reported previously ( 41 , 42 ). In brief, p2 presented with a severe hepatoencephalopathy and died at 18 months, p3 had Alpers syndrome and died at 16 months of age.

Cell cultures and growth conditions

Cells were grown in skeletal muscle growth medium (SGM) (PromoCell, Heidelberg, Germany) supplemented with 15% fetal calf serum (Invitrogen Life Technologies, Karlsruhe, Germany) and 2 m m glutamine (Invitrogen Life Technologies).

For differentiation into multinucleated myotubes, cells were grown in SGM in plastic dishes coated with laminin (Sigma). Differentiation and fusion into multinucleated myotubes were induced at 70% confluence by replacing SGM with serum-reduced fusion medium (Dulbecco’s modified Eagle’s medium supplemented with 2% horse serum and 2 m m glutamine) for 6 days. All cells were grown in a humidified atmosphere with 5% CO 2 at 37°C.

For supplementation studies, the cells were incubated with serum-reduced fusion medium with different concentrations (50, 100, 200, 400, 800, 1200 µ m ) of dAMP/dGMP and also of dAMP or dGMP or uridine alone during fusion. In order to improve the balance of the four nucleotides in MNGIE cells, a mixture of the three other nucleotides (dAMP/dGMP/dCMP) was added in two different concentrations (200 and 400 µ m ). The concentrations were calculated for each of the nucleotides separately in the mixture.

To induce mtDNA depletion, myoblasts were grown in SGM in the presence of 50 ng/ml ethidium bromide (EtBr) for 10 days as described ( 40 ). Cells were kept between 30 and 80% confluence with excess fresh medium to assure exponential growth. Afterwards, they were fused to myotubes in fusion medium with 50 ng/ml EtBr.

After 6 days of fusion (16 days total), all cells were harvested for DNA extraction using High Pure PCR Template Preperation Kit (Roche Diagnostics GmbH, Mannheim, Germany). All cell culture studies were done in triplicates and were repeated in at least two independent experiments.

Biochemical and histochemical measurement for COX

Biochemical measurement ( 43 ) and histochemical staining of cells for COX was performed as described ( 44 ).

WST-1 test

Myoblasts were plated at 1 × 10 4 cells/well into 96-well plates and allowed to adhere overnight before fusion was induced and medium changed to fusion medium. Cells were differentiated for 6 days under normal conditions or in fusion medium supplemented with dAMP/dGMP. The metabolic activity of the fused cells was determined by treating cultures with WST-1 from Roche Applied Science (Basel, Switzerland) for 3 h as described ( 45 ). The amount of dye formed correlates to the amount of mitochondrial dehydrogenases and overall mitochondrial activity. Optical density was read on a spectrophotometer and percentage metabolic activity was compared with fused control cells from a healthy donor incubated with WST-1 (100%).

Immunofluorescence

For immunofluorescence staining, myoblasts were grown in SGM on laminin-coated glass cover slips. Differentiation and fusion into multinucleated myotubes were induced at 70% confluence with serum-reduced fusion medium for 6 days.

Cover slips were briefly washed in phosphate-buffered saline (PBS) and fixed in 3.7% formaldehyde (freshly prepared from paraformaldehyde) in PBS for 15 min at room temperature. Permeabilization was in 0.1% Triton X-100 in PBS for 10 min. After three washes in PBS, blocking of unspecific binding sites with 5% horse serum in PBS for 1 h was followed by incubation with the primary antibody (1:20; beta-dystroglycan, mouse monoclonal; Novocastra Laboratories, Newcastle upon Tyne, UK) diluted in PBS containing 5% horse serum for 1 h in a humid chamber. After three washes in PBS, the cells were incubated with the secondary antibody conjugated to Cy3 (1:200; anti-mouse IgG; Sigma).

The nuclei were visualized using bis-benzimide H 33258 (40 µg/ml; Sigma) and finally, after three washes in PBS, the cover slips were mounted in DAKO fluorescent mounting medium (DAKO, Carpinteria, CA, USA) and sealed with nail polish.

Digital images were captured using a Zeiss Axiovert 200 M fluorescence microscope and a Zeiss AxioCam HR photo camera.

DNA analysis

Long-range PCR for mtDNA deletions was performed as described ( 46 ). MtDNA and nuclear DNA copy numbers and mtDNA/nDNA ratios in DNA samples extracted from 10 control myoblast cultures and from myoblasts and myotubes of p1–p4 (Table  1 ) with and without supplementation were determined by real-time PCR using a fluorescent temperature cycler (Light Cycler, Roche Molecular Biochemicals, Mannheim, Germany). The mitochondrial MTATP6 gene was amplified between nucleotide positions 8981 and 9061 with the forward primer, 5′-ACCAATAGCCCTGGCCGTAC-3′ and the reverse primer 5′-GGTGGCGCTTC-CAATTAGGT-3′. For the detection of nDNA, we selected exon number 8 of the GAPDH gene between nucleotide positions 4280–4342, using the forward primer 5′-CGGGGCTCTCCAGAACATC-3′ and the reverse primer 5′-ATGACCTTGCCCACAGCCT-3′. The extracted DNA was diluted to 10 ng/µl and 1 µl was added to the PCR mixture. The PCR amplification consisted of a single denaturation–enzyme activation step for 10 min at 95°C, followed by 45 amplification cycles of 5 s at 95°C, 9 s at 54°C and 30 s at 72°C. The ratio of mtDNA copy number to nuclear DNA was used as a measure of mtDNA content in each sample. All samples were run in triplicates. The control range for skeletal muscle was determined using 30 control muscle DNA samples. Sequencing of the nuclear-encoded DGUOK and POLG1 genes was performed as described ( 7 , 42 ).

Array CGH

Whole genomic DNA of myoblast cell cultures (extracted using High Pure PCR Template Preperation Kit) was labelled, sephadex G50 purified and hybridized together with human male reference DNA (400 ng each) on BAC arrays (CytoChip V2.1, BlueGnome, Cambridge) according to the manufacturers protocol. Briefly, after hybridisation for 21 h arrays were washed in three times in 1× PBS, 0.5% Tween (10 min each), 30 min in 50% formamide/2× SSC at 42°C followed by two washes in 1× PBS (5 min each) and dried in a swing out rotor 3 min at 200 g. Fluorescent scanning was performed on a PerkinElmer ProScan Array HT microarray scanner. Data were analysed using BlueFuse 3.5 array CGH software (BlueGnome, Cambridge). The statistical analysis was performed by Student’s t -test.

FUNDING

This work is supported by grants from Deutsche Forschungsgemeinschaft HO 2505/2-1 (R.H.). The Muscle Tissue Culture Collection is part of the German network on muscular dystrophies (MD-NET, service structure S1, 01GM0601) funded by the German ministry of education and research (BMBF, Bonn, Germany). The Muscle Tissue Culture Collection is a partner of EuroBioBank ( www.eurobiobank.org ) and TREAT-NMD (EC, 6th FP, proposal # 036825).

ACKNOWLEDGEMENTS

The excellent technical assistance of Ira Kaus and Eva Neugebauer is gratefully acknowledged.

Conflict of Interest statement . None declared.

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