Proteolytic Processing of OPA1 Links Mitochondrial Dysfunction to Alterations in Mitochondrial Morphology

Many muscular and neurological disorders are associated with mitochondrial dysfunction and are often accompanied by changes in mitochondrial morphology. Mutations in the gene encoding OPA1, a protein required for fusion of mitochondria, are associated with hereditary autosomal dominant optic atrophy type I. Here we show that mitochondrial fragmentation correlates with processing of large isoforms of OPA1 in cybrid cells from a patient with myoclonus epilepsy and ragged-red fibers syndrome and in mouse embryonic fibroblasts harboring an error-prone mitochondrial mtDNA polymerase {gamma}. Furthermore, processed OPA1 was observed in heart tissue derived from heart-specific TFAM knock-out mice suffering from mitochondrial cardiomyopathy and in skeletal muscles from patients suffering from mitochondrial myopathies such as myopathy encephalopathy lactic acidosis and stroke-like episodes. Dissipation of the mitochondrial membrane potential leads to fast induction of proteolytic processing of OPA1 and concomitant fragmentation of mitochondria. Recovery of mitochondrial fusion depended on protein synthesis and was accompanied by resynthesis of large isoforms of OPA1. Fragmentation of mitochondria was prevented by overexpressing OPA1. Taken together, our data indicate that proteolytic processing of OPA1 has a key role in inducing fragmentation of energetically compromised mitochondria. We present the hypothesis that this pathway regulates mitochondrial morphology and serves as an early response to prevent fusion of dysfunctional mitochondria with the functional mitochondrial network

Mitochondrial DNA Mutations Provoke Dominant Inhibition of Mitochondrial Inner Membrane Fusion

<div><p>Mitochondria are highly dynamic organelles that continuously move, fuse and divide. Mitochondrial dynamics modulate overall mitochondrial morphology and are essential for the proper function, maintenance and transmission of mitochondria and mitochondrial DNA (mtDNA). We have investigated mitochondrial fusion in yeast cells with severe defects in oxidative phosphorylation (OXPHOS) due to removal or various specific mutations of mtDNA. We find that, under fermentative conditions, OXPHOS deficient cells maintain normal levels of cellular ATP and ADP but display a reduced mitochondrial inner membrane potential. We demonstrate that, despite metabolic compensation by glycolysis, OXPHOS defects are associated to a selective inhibition of inner but not outer membrane fusion. Fusion inhibition was dominant and hampered the fusion of mutant mitochondria with wild-type mitochondria. Inhibition of inner membrane fusion was not systematically associated to changes of mitochondrial distribution and morphology, nor to changes in the isoform pattern of Mgm1, the major fusion factor of the inner membrane. However, inhibition of inner membrane fusion correlated with specific alterations of mitochondrial ultrastructure, notably with the presence of aligned and unfused inner membranes that are connected to two mitochondrial boundaries. The fusion inhibition observed upon deletion of OXPHOS related genes or upon removal of the entire mtDNA was similar to that observed upon introduction of point mutations in the mitochondrial <em>ATP6</em> gene that are associated to neurogenic ataxia and retinitis pigmentosa (NARP) or to maternally inherited Leigh Syndrome (MILS) in humans. Our findings indicate that the consequences of mtDNA mutations may not be limited to OXPHOS defects but may also include alterations in mitochondrial fusion. Our results further imply that, in healthy cells, the dominant inhibition of fusion could mediate the exclusion of OXPHOS-deficient mitochondria from the network of functional, fusogenic mitochondria.</p> </div

Deletion or mutation of OXPHOS genes inhibits mitochondrial fusion.

<p>Cells expressing matrix-targeted mtGFP or mtRFP were conjugated and the proportion of zygotes with Total (T), Partial (P) or No fusion (N) was determined by fluorescence microscopy after the indicated times (<b>A–C</b>) or after 4 hours (<b>D</b>). <b>A</b>: Fusion in strains devoid of mitochondrial <i>COX2</i> (Δ<i>cox2</i>) or mitochondrial DNA (ρ⁰). <b>B</b>: Fusion in strains with defects in ATP-synthase genes (Δ<i>atp6, atp6-L183R, atp6-L247R,</i> Δ<i>atp12</i>). <b>C, D:</b> Comparison of total fusion as a function of time (<b>C</b>) or of Total, Partial and No fusion after 4 hours (<b>D</b>) in wild-type, Δ<i>mgm1</i> and OXPHOS-deficient cells. Dashed line: proportion of fusion in wild-type cells.</p

OXPHOS defects inhibit fusion with wild-type mitochondria in trans.

<p>Wild-type and mutant cells expressing matrix-targeted mtGFP or mtRFP were conjugated and mitochondrial fusion was analyzed by fluorescence microscopy after the indicated times (<b>A, B</b>) or after 4 hours (<b>C</b>). <b>A:</b> Kinetics of Total (T), Partial (P) and No fusion (N). <b>B, C:</b> Comparison of total fusion as a function of time (<b>B</b>) or of Total, Partial and No fusion after 4 hours (<b>C</b>). Dashed line: proportion of fusion in wild-type cells.</p

Properties of <i>ATP6</i> mutant strains.

<p>The <i>atp6</i>-L183R mutation is homologous to human T8993G/L156R, the most frequent mutation associated to neurogenic ataxia retinitis pigmentosa (NARP). The <i>atp6</i>-L247R mutation is homologous to human T9176G/L217R, which is associated to maternally-inherited Leigh syndrome (MILS), the most severe form of NARP. *COX activity was extimated by measuring oxygen consumption with ascorbate/TMPD (<i>N,N,N’,N’</i>-tetramethyl-<i>p</i>-phenylenediamine), that directly reduce cytochrome c.</p

OXPHOS deficient mitochondria display altered inner membrane structures.

<p>Yeast cells of the indicated genotypes were fixed and analyzed by electron microscopy. White arrowheads point to normal (short) cristae membranes. White arrows point to elongated and aligned inner membranes (septae) that connect two boundaries and separate matrix compartments. Bars 200 nm.</p

Outer membrane fusion is not affected by OXPHOS defects.

<p>Wild-type and mutant cells expressing fluorescent proteins targeted to the outer membrane (GFPOM, RFPOM) were conjugated and mitochondrial outer membrane fusion was analyzed by fluorescence microscopy after the indicated times (<b>A, B</b>) or after 4 hours (<b>C</b>). <b>A:</b> Kinetics of Total (T), Partial (P) and No fusion (N). <b>B, C:</b> Comparison of total fusion as a function of time (<b>B</b>) or of Total, Partial and No fusion after 4 hours (<b>C</b>). The dashed line indicates the proportion in wild-type cells. Dashed line: proportion of total fusion in wild-type cells.</p

Bioenergetic characterization of yeast strains.

<p>Yeast cells of the indicated genotypes were cultivated under the conditions of a mitochondrial fusion assay and analyzed for respiration (<b>A</b>) ATP and ADP content (<b>B</b>) mitochondrial inner membrane potential ΔΨ_m (<b>C</b>) or content or reactive oxygen species (<b>D</b>). <b>A:</b> Respiration rates in the presence of ethanol alone (all strains), after inhibition of ATP-synthase with tri-ethyl-tin (tet: <i>WT, atp6-L183R, atp6-L247R</i>) or after addition of the protonophore cccp (all strains). <b>B:</b> ATP-content, ADP-content and ATP/ADP ratio. <b>C, D:</b> Mean and median fluorescence of cells that were incubated with rh 123 (which accumulates in cells in a ΔΨ_m-dependent manner, <b>C</b>) and DHE (which is converted to fluorescent ethidium by superoxide, <b>D</b>) and analyzed by flow cytometry. The distributions of fluorescence in these cell populations are depicted in Supp. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049639#pone.0049639.s002" target="_blank">Fig. S2</a>.</p

Pattern of Mgm1-isoforms in yeast cells with different OXPHOS defects.

<p>Yeast cells of the indicated genotypes were maintained for 6 hours in glucose-containing medium (<b>A, </b><b>B</b>) or galactose-containing medium (<b>C</b>). In <b>A</b>, cells were treated, or not, with valinomycin (VM). Cells were then analyzed by Western-blot with Mgm1-antibodies and the relative amounts of l-Mgm1 and s-Mgm1 quantified by densitometry.</p