Date of download: 5/28/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Human NARP Mitochondrial Mutation Metabolism Corrected With α-Ketoglutarate/Aspartate: A Potential New Therapy Arch Neurol. 2009;66(8): doi: /archneurol Substrates (5mM α-ketoglutarate + 5mM aspartate) rescue cell death in oligomycin-treated fibroblasts. Cells were incubated in Dulbecco modified Eagle medium–enriched medium (A), in the same medium plus 40 ng/mL of gramicidin (B), or in the same medium plus 40 ng/mL of gramicidin and 0.6nM oligomycin (C), as detailed in the “Methods” section. Dotted lines denote presence of the substrates in the culture medium. Values are given as mean (SD). Figure Legend:

Date of download: 5/28/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Human NARP Mitochondrial Mutation Metabolism Corrected With α-Ketoglutarate/Aspartate: A Potential New Therapy Arch Neurol. 2009;66(8): doi: /archneurol Microscopical evaluation of fibroblast viability. Cells were incubated for 72 hours in Dulbecco modified Eagle medium–enriched medium (A) or in the same medium plus 40 ng/mL of gramicidin (B) or 40 ng/mL of gramicidin and 0.6nM oligomycin (C), in the absence (A-C) or in the presence (D-F) of the substrates (5mM α-ketoglutarate + 5mM aspartate). Figure Legend:

Date of download: 5/28/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Human NARP Mitochondrial Mutation Metabolism Corrected With α-Ketoglutarate/Aspartate: A Potential New Therapy Arch Neurol. 2009;66(8): doi: /archneurol Substrates (5mM α-ketoglutarate + 5mM aspartate) contribute to preserve adenosine 5′-triphosphate (ATP) levels in resting oligomycin-treated fibroblasts. Cells were incubated in Dulbecco modified Eagle medium–enriched medium (A), in the same medium plus 40 ng/mL of gramicidin (B), or in the same medium plus 40 ng/mL of gramicidin and 0.6nM oligomycin (C). Total ATP levels were assayed every day by using a luminescent-based assay (see the “Methods” section). Dotted lines denote presence of the substrates in the medium. At zero time, the ATP content of the fibroblasts was mean (SD) (2.50) nmol/mg of protein. Values are given as mean (SD). Figure Legend:

Date of download: 5/28/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Human NARP Mitochondrial Mutation Metabolism Corrected With α-Ketoglutarate/Aspartate: A Potential New Therapy Arch Neurol. 2009;66(8): doi: /archneurol Effect of substrates (5mM α-ketoglutarate + 5mM aspartate) on viability and adenosine 5′-triphosphate (ATP) content of neuropathy, ataxia, and retinitis pigmentosa 8993T>G cybrid cell lines. Viability (A and B) and ATP content (C and D) in wild-type (A and C) and homoplasmic mutant (B and D) cell lines grown in Dulbecco modified Eagle medium–enriched medium. The dotted line indicates presence of the substrates. At zero time, the ATP content was mean (SD) 9.70 (0.99) nmol/mg of protein in wild-type cells and 8.07 (0.23) nmol/mg of protein in homoplasmic mutant cells. Values are given as mean (SD). Figure Legend:

Date of download: 5/28/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Human NARP Mitochondrial Mutation Metabolism Corrected With α-Ketoglutarate/Aspartate: A Potential New Therapy Arch Neurol. 2009;66(8): doi: /archneurol Effect of substrates (5mM α-ketoglutarate + 5mM aspartate) on viability and adenosine 5′-triphosphate (ATP) content of neuropathy, ataxia, and retinitis pigmentosa 8993T>C cybrid cell lines. Viability (A and B) and ATP content (C and D) of wild-type (A and C) and homoplasmic mutant (B and D) cell lines grew in Dulbecco modified Eagle medium–enriched medium. The dotted line indicates presence of the substrates. At zero time, the ATP content was mean (SD) 8.33 (0.64) mmol/mg of protein in wild-type cells and 8.25 (1.39) nmol/mg of protein in homoplasmic mutant cells. Values are given as mean (SD). Figure Legend:

Date of download: 5/28/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Human NARP Mitochondrial Mutation Metabolism Corrected With α-Ketoglutarate/Aspartate: A Potential New Therapy Arch Neurol. 2009;66(8): doi: /archneurol Metabolic pathways for anaerobic adenosine 5′-triphosphate (ATP) production by the supplemented substrates (5mM α- ketoglutarate [α-KG] + 5mM aspartate [Asp]). α-Ketoglutarate is transferred by the oxoglutarate carrier to the mitochondrial matrix, where it enters the tricarboxylic acid cycle. Here, it is converted first to succinyl coenzyme A (succinyl-CoA), then to succinate by the succinyl-CoA synthetase (A-SCS), with generation of ATP. Aspartate enters the mitochondria and is transaminated to oxaloacetate (OAA), which is reduced to malate (reducing equivalents as nicotinamide adenine dinucleotide [NADH] is removed from the matrix and nicotinamide adenine dinucleotide [NAD + ] is regenerated to support the reaction catalyzed by the α-KG dehydrogenase). After leaving the mitochondrial matrix, malate contributes to shuttling α-KG into the organelle. i.m.m. indicates inner mitochondrial membrane; ADP, adenosine diphosphate; and P i, inorganic phosphate. Figure Legend: