Mitochondrial ND5 Gene Variation Associated with Encephalomyopathy and Mitochondrial ATP Consumption

Abstract

Mitochondrial encephalomyopathy and lactic acidosis with strokelike episodes (MELAS) is a severe young onset stroke disorder without effective treatment. We have identified a MELAS patient harboring a 13528A3 G mitochondrial DNA (mtDNA) mutation in the Complex I ND5 gene. This mutation was homoplasmic in mtDNA from patient muscle and nearly homoplasmic (99.9%) in blood. Fibroblasts from the patient exhibited decreased mitochondrial membrane potential (⌬ m ) and increased lactate production, consistent with impaired mitochondrial function. Transfer of patient mtDNA to a new nuclear background using transmitochondrial cybrid fusions confirmed the pathogenicity of the 13528A3 G mutation; Complex I-linked respiration and ⌬ m were both significantly reduced in patient mtDNA cybrids compared with controls. Inhibition of the adenine nucleotide translocase or the F 1 F 0 -ATPase with bongkrekic acid or oligomycin caused a loss of potential in patient mtDNA cybrid mitochondria, indicating a requirement for glycolytically generated ATP to maintain ⌬ m . This was confirmed by inhibition of glycolysis with 2-deoxy-Dglucose, which caused depletion of ATP and mitochondrial depolarization in patient mtDNA cybrids. These data suggest that in response to impaired respiration due to the mtDNA mutation, mitochondria consume ATP to maintain ⌬ m , representing a potential pathophysiological mechanism in human mitochondrial disease.Mitochondrial respiration is fundamental to the well being of most mammalian cells, since it provides the central mechanism that couples fuel and oxygen consumption to ATP synthesis. The mitochondrial respiratory chain resides at the inner membrane and consists of five multimeric enzyme complexes: I (NADH:ubiquinone oxidoreductase), II (succinate:ubiquinone oxidoreductase), III (ubiquinol:cytochrome c oxidoreductase), IV (cytochrome c oxidase), and V (F 1 F 0 -ATPase). Electrons donated from the tricarboxylic acid cycle to Complexes I and II are utilized by Complexes I, III, and IV to pump protons from the matrix to the intermembrane space. The resulting electrochemical potential gradient, normally expressed as a mitochondrial membrane potential (⌬ m ), 2 provides the energy to drive ATP synthesis by Complex V (1, 2).Mitochondria contain their own unique, double-stranded, circular genome, which encodes the 13 essential protein subunits of the respiratory complexes. It also encodes the 12 and 16 S rRNAs and the 22 tRNAs specific for mitochondrial protein synthesis. Mutations in mitochondrial DNA (mtDNA) are associated with a wide variety of multisystemic degenerative diseases (3). Aspects of central nervous system and muscle function are usually affected, whereas a number of mutations are also variably associated with deafness, diabetes, and optic nerve atrophy or retinal degeneration (2, 4). In many cases, the pathogenic mtDNA mutation associated with the respiratory defect has been assigned, but it is still unclear why different mtDNA mutations result in such a wide array of clinical phenotypes.Mitochondr...

TSPO interacts with VDAC1 and triggers a ROS-mediated inhibition of mitochondrial quality control

“…Cells cotransfected with YFP were loaded with 75 nM tetramethyl rhodamine methyl ester (TMRM; Sigma-Aldrich, T5428) and 10 mM verapamil HCl (Sigma-Aldrich, V4629; which is required to inhibit TMRM export from the cell via the multidrug transporter 82 ) in HEPES-buffered salt solution (156 mM NaCl, 3 mM KCl, 2 mM MgSO 4 [Sigma, M2643], 2 mM CaCl 2, 10 mM glucose; 10 mM HEPES [Sigma-Aldrich, H4034]). TMRM accumulates in mitochondria, and its signal intensity is a function of membrane potential.…”

Respiratory Complex I Dysfunction Due to Mitochondrial DNA Mutations Shifts the Voltage Threshold for Opening of the Permeability Transition Pore toward Resting Levels

“…The decreased ATP supply to the Ca 2ϩ pumps would then reduce Ca 2ϩ uptake in intracellular stores and cause increased cytosolic free [Ca 2ϩ ] followed by mitochondrial Ca 2ϩ and P i uptake, resulting in a shift of the PTP voltage threshold. The depolarizing effect of oligomycin, which was also observed in cybrids from a MELAS patient harboring a A13528G/ND5 mtDNA mutation (55) and in patients affected by Ullrich congenital muscular dystrophy (63,64), can therefore be explained on the basis of decreased ATP levels, eventually resulting in a shift of the PTP threshold above the resting membrane potential (25).…”

“…Once this occurs, repolarization cannot take place despite ATP hydrolysis. Based on the protective effect of the intracellular Ca 2ϩ chelator BAPTA-AM and of the antioxidant Trolox, we think that defective complex I sensitizes the PTP through Ca 2ϩ overload and increased production of reactive oxygen species (46), which have indeed been described in several mtDNA disease models (47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57).…”

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