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. 2010 Jan 15;19(2):374-86.
doi: 10.1093/hmg/ddp503. Epub 2009 Oct 29.

Mitochondrial DNA background modifies the bioenergetics of NARP/MILS ATP6 mutant cells

M D'Aurelio  1 , C Vives-Bauza, M M Davidson, G Manfredi
Affiliations

Mitochondrial DNA background modifies the bioenergetics of NARP/MILS ATP6 mutant cells

M D'Aurelio et al. Hum Mol Genet. .

Abstract

Mutations in the mitochondrial DNA (mtDNA) encoded subunit 6 of ATPase (ATP6) are associated with variable disease expression, ranging from adult onset neuropathy, ataxia and retinitis pigmentosa (NARP) to fatal childhood maternally inherited Leigh's syndrome (MILS). Phenotypical variations have largely been attributed to mtDNA heteroplasmy. However, there is often a discrepancy between the levels of mutant mtDNA and disease severity. Therefore, the correlation among genetic defect, bioenergetic impairment and clinical outcome in NARP/MILS remains to be elucidated. We investigated the bioenergetics of cybrids from five patients carrying different ATP6 mutations: three harboring the T8993G, one with the T8993C and one with the T9176G mutation. The bioenergetic defects varied dramatically, not only among different ATP6 mutants, but also among lines carrying the same T8993G mutation. Mutants with the most severe ATP synthesis impairment showed defective respiration and disassembly of respiratory chain complexes. This indicates that respiratory chain defects modulate the bioenergetic impairment in NARP/MILS cells. Sequencing of the entire mtDNA from the different mutant cell lines identified variations in structural genes, resulting in amino acid changes that destabilize the respiratory chain. Taken together, these results indicate that the mtDNA background plays an important role in modulating the biochemical defects and clinical outcome in NARP/MILS.

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Figures

Figure 1.
Figure 1.
Bioenergetic variations among the different homoplasmic ATP6 mutant cell lines. (A) ATP synthesis activities using pyruvate and malate as substrates. (B and C) Measurements of oxygen consumption in intact cells using pyruvate as substrate in the absence (coupled respiration) or in the presence of FCCP (uncoupled respiration). The values are average of at least three independent measurements and are expressed as a percentage of 143B cells. Statistically significant differences between 143B and mutant cell lines are indicated: *P < 0.05, **P < 0.005, *** < 0.0005 versus 143B.
Figure 2.
Figure 2.
RC enzymatic activities. Complex I activity in isolated mitochondria (A) and complex IV (COX) activity normalized by citrate synthase (CS) activity in cell lysates (B). The values are average of at least three independent measurements and are expressed as a percentage of 143B cells. *P < 0.05, **P < 0.005, ***P < 0.0005 versus 143B.
Figure 3.
Figure 3.
RC complexes assembly and mitochondrial protein levels. (A) BN-PAGE of RC complexes. CoI, complex I; CoII, complex II; CoIII, dimer of complex III; CoIV, complex IV; CoV, complex V; F1, F1 portion of complex V. The amount of RC complexes expressed as percentage of WT, estimated by densitometry of western blot bands from at least three independent experiments, are indicated below each lane. (B) RC complexes subunits from 143B cell line (left) and AT mutant cybrid (right) resolved by first dimension BN-PAGE (as in A), followed by separation by second dimension denaturing SDS–PAGE probed with antibodies against the 70 kDa complex II subunit, β subunit of ATPase, subunit I of COX (COX I), the inner membrane translocator TIM23 and subunit 6 of ATPase (ATP6). (C) Western blot of mitochondrial proteins solubilized as in (A) resolved by denaturing SDS–PAGE, blotted and detected with specific antibodies. VDAC is the voltage-dependent anion channel (porin). (D) Western blot of whole cell lysates separated by denaturing SDS–PAGE, blotted, and detected with specific antibodies. Cytc, cytochrome c.
Figure 4.
Figure 4.
Bioenergetic variations were preserved after ‘re-cybridization’. (A) BN-PAGE of RC complexes from cybrids obtained by ‘re-cybridization’. (B) Western blot of mitochondrial proteins from cybrids obtained by ‘re-cybridization’ solubilized as in (A), resolved by denaturing SDS–PAGE, and detected with specific antibodies. (C) ATP synthesis in cybrids obtained by ‘re-cybridization’ expressed as a percentage of 143B cells. (D) Coupled respiration (top) and ATP synthesis (bottom) of ATP6 mutant cybrids obtained by ‘re-cybridization’ in a d407 retinal pigmentary epithelium background expressed as a percentage of d407 wild-type control cells. In (C) and (D), values are average of at least three independent measurements. *P < 0.05, **P < 0.005 versus wild-type cells.
Figure 5.
Figure 5.
Bioenergetic variations in isogenic cybrids. (A) BN-PAGE of RC complexes from isogenic cybrids. (B) Western blot of mitochondrial proteins from isogenic control cybrid solubilized as in (A), resolved by denaturing SDS–PAGE and detected with specific antibodies. (C) ATP synthesis and (D) coupled respiration of isogenic cybrids. The values are average of at least three independent measurements and are expressed as percentage of 143B. *P < 0.05, **P < 0.005, ***P < 0.0005 versus 143B.
Figure 6.
Figure 6.
Assembly kinetics of RC complexes. (A) BN-PAGE of 143B (left) and JC (right) cells grown for 6 days in the presence of doxycicline followed by 6 days without the drug. Mitochondrial RC complexes were studied at different time points during the treatment and the recovery. Note that the JC blots are subjected to a longer exposure than 143B ones in order to obtain similar band intensities in the two lines. (B) Quantification of complexes I, III, IV and V band intensities in doxycline treated cells expressed as a percentage of the respective complexes from untreated cells (day 0). Each value is the average of two independent experiments.
Figure 7.
Figure 7.
Assembly kinetics of complex V. (A) BN-PAGE of complex V from 143B (left) and JC (right) cells grown and treated as in Figure 6. Samples were separated in parallel on two different gels, blotted and probed with antibodies against ATP6 subunit (top panels) and β-ATPase subunit (bottom panels). (B) Western blot of mitochondrial proteins solubilized as in (A), resolved by denaturing SDS–PAGE and detected with specific antibodies.
Figure 8.
Figure 8.
Cell replication in glucose and galactose medium. Equal numbers of cells were plated and the total cell count was obtained after 3 days of growth either in glucose (A) or galactose (B) medium. Cell growth is expressed as percentage of 143B. *P < 0.05, ***P < 0.0005.
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