Mitochondrial ND5 Gene Variation Associated with Encephalomyopathy and Mitochondrial ATP Consumption

Matthew Mckenzie,Danae Liolitsa,Natalya Akinshina,Michelangelo Campanella,Sanjay Sisodiya,Ian Hargreaves,Niranjanan Nirmalananthan,Mary G Sweeney,Patrick M Abou-Sleiman,Nicholas W Wood,Michael G Hanna,Michael R Duchen

doi:10.1074/jbc.m704158200

Matthew Mckenzie, Danae Liolitsa + Show 10 more

Open Access

https://doi.org/10.1074/jbc.m704158200

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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 13528A-->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 (Deltapsim) 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 13528A-->G mutation; Complex I-linked respiration and Deltapsim were both significantly reduced in patient mtDNA cybrids compared with controls. Inhibition of the adenine nucleotide translocase or the F1F0-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 Deltapsim. This was confirmed by inhibition of glycolysis with 2-deoxy-D-glucose, 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 Deltapsim, representing a potential pathophysiological mechanism in human mitochondrial disease.

Highlights

Defined cases of MELAS most commonly associate with heteroplasmic mitochondrial DNA (mtDNA) mutations in the tRNA leucine (UUR) gene [6]; the mitochondrial ND5 gene may represent a hotspot for MELAScausing mutations [7,8,9]
We have identified a patient with the mitochondrial disease MELAS who harbors two missense mutations at positions 13,528 and 13,565 in the mitochondrial ND5 gene
The 13,528 and 13,565 changes have been described previously on haplogroup U in the apparent absence of a disease phenotype [20]; the 13528A3 G mutation has been reported in association with Leber hereditary optic neuropathy (LHON) [19]

Summary

EXPERIMENTAL PROCEDURES

Case Report—The subject (referred to as Patient 1) presented with severe strokelike episodes and epilepsy in early adolescence. Oxygen Respiratory Activity—To measure respiration rate in intact cells ϳ1 ϫ 107 cells were suspended in respiration medium (5.37 mM KCl, 0.44 mM KH2PO4, 4.17 mM NaHCO3, 137 mM NaCl, 0.34 mM Na2HPO4, pH 7.2) with 5.6 mM D-glucose in a Clark-type oxygen electrode thermostatically maintained at 37 °C. Cells were incubated for 2 h in record solution (156 mM NaCl, 3 mM KCl, 2 mM MgSO4, 1.25 mM KH2PO4, 2 mM CaCl2, 20 mM HEPES, pH 7.35) with either 10 mM glucose, 10 mM glucose plus 2.5 ␮g/ml oligomycin (glycolytic ATP generation), or 5 mM 2-deoxy-Dglucose plus 5 mM pyruvate (oxidative ATP production). Fluorescent Imaging of Cells—All cells were loaded with fluorescent dyes in buffer containing 156 mM NaCl, 3 mM KCl, 2 mM MgSO4, 1.25 mM KH2PO4, 10 mM D-glucose, 2 mM CaCl2, 10 mM HEPES, pH 7.35 (record solution). The mean intensity of the TMRM signal per pixel was measured over mitochondrial structures only and was independent of mitochondrial mass or density

RESULTS

Respiration in intact cells

Ratio of

DISCUSSION