oligomycins and Retinitis-Pigmentosa

Mitochondrial (mt) DNA-associated NARP (neurogenic muscle weakness, ataxia, and retinitis pigmentosa) syndrome is due to mutation in the MT-ATP6 gene. We report the case of a 18-year-old man who presented with deafness, a myoclonic epilepsy, muscle weakness since the age of 10 and further developed a retinitis pigmentosa and ataxia. The whole mtDNA analysis by next-generation sequencing revealed the presence of the 2 bp microdeletion m.9127-9128 del AT in the ATP6 gene at 82% heteroplasmy in muscle and to a lower load in blood (10-20%) and fibroblasts (50%). Using the patient's fibroblasts, we demonstrated a 60% reduction of the oligomycin-sensitive ATPase hydrolytic activity, a 40% decrease in the ATP synthesis and determination of the mitochondrial membrane potential using the fluorescent probe tetramethylrhodamine, ethyl ester indicated a significant reduction in oligomycin sensitivity. In conclusion, we demonstrated that this novel AT deletion in the ATP6 gene is pathogenic and responsible for the NARP syndrome.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Base Sequence; Carrier Proteins; Cells, Cultured; DNA Mutational Analysis; DNA, Mitochondrial; High-Throughput Nucleotide Sequencing; Humans; Male; Membrane Potential, Mitochondrial; Membrane Proteins; Mitochondrial Myopathies; Mitochondrial Proton-Translocating ATPases; Oligomycins; Retinitis Pigmentosa; Sequence Deletion; Syndrome; Young Adult

To verify whether enhanced substrate-level phosphorylation increases viability and adenosine 5'-triphosphate (ATP) content of cells with neuropathy, ataxia, and retinitis pigmentosa/maternally inherited Leigh syndrome (NARP/MILS) mitochondrial DNA mutations and ATP synthase dysfunction.. We used cell lines "poisoned" with oligomycin, the specific inhibitor of ATP synthase, and "natural" models, including transmitochondrial human cell lines (cybrids) harboring 2 different pathogenic mutations associated with the NARP/MILS phenotypes.. Cell survival, morphology, and ATP content.. When normal human fibroblasts cultured in glucose-free medium were forced to increase energy consumption by exposure to the ionophore gramicidin or were energy challenged by oligomycin inhibition, their survival at 72 hours was 5%, but this increased to 70% when the medium was supplemented with alpha-ketoglutarate/aspartate to boost mitochondrial substrate-level phosphorylation. Homoplasmic cybrids harboring the 8993T-->G NARP mutation were also protected from death (75% vs 15% survival at 72 hours) by the supplemented medium and their ATP content was similar to controls.. These results show that ATP synthase-deficient cells can be rescued by increasing mitochondrial substrate-level phosphorylation and suggest potential dietary or pharmacological therapeutic approaches based on the supplementation of alpha-ketoglutarate/aspartate to patients with impaired ATP synthase activity.

Topics: Adenosine Triphosphate; Anti-Infective Agents, Local; Aspartic Acid; C-Reactive Protein; Cell Line; Cell Survival; DNA, Mitochondrial; Energy Metabolism; Gramicidin; Humans; Ketoglutaric Acids; Leigh Disease; Mitochondria; Mitochondrial Proton-Translocating ATPases; Mutation; Nerve Tissue Proteins; Oligomycins; Oxidative Phosphorylation; Retinitis Pigmentosa; Spinocerebellar Degenerations