Iyer 2019 MiPschool Coimbra

From Bioblast
Shilpa Iyer
Characterizing metabolic and structural changes in Leigh’s Syndrome patient fibroblasts.

Link: MitoEAGLE

Iyer S, Bakare A, Kolenc O, Scheulin K, Daniel J, Stabach J, Lesnefsky EJ, West FD, Quinn KP (2019)

Event: MiPschool Coimbra 2019


Leigh’s Syndrome (LS), is a severe neuro-metabolic disorder and has no current cure or adequate cellular models to understand the rapid fatality associated with the disease. Other symptoms are widespread tissue malfunction in brain stem and muscle in LS patients. We hypothesize that altered mitochondrial structure and function caused by mitochondrial genome mutations in the electron transport chain (ETC) may lead to rapid fatality in LS. Critical studies to address this issue have been hampered by limitations in developing specific pluripotent stem cell models containing mitochondrial DNA (mtDNA) mutations in LS. We have recently reprogramed fibroblast cells containing mtDNA mutations in complex I and complex V of the electron transport chain to yield induced pluripotent stem cells (iPSC) by using a combination of mRNA and miRNA technology. These cell models can now be used to study the role of specific mtDNA mutation(s) capable of disrupting the balance between glycolysis and oxidative phosphorylation. Prior to reprogramming, we characterized the patient fibroblast cells to obtain a metabolic β€˜disease signature’ phenotype. A two-photon excited fluorescence microscopy approach relying entirely on endogenous fluorophores, was used to dynamically quantify functional metabolic readouts from individual cells. A ratio of FAD/(NADH+FAD) has been shown to be sensitive to the relative proportion of oxidative phosphorylation to glucose catabolism. We observed a higher optical redox ratio in cells with complex V (p=0.0006) mutations compared to normal fibroblast cells. This increase was likely due to increased ETC activity or possible proton leak associated with defective ATP synthase (CV). Using traditional assays, ETC enzyme activities was measured spectrophotometrically as specific donor–acceptor oxidoreductase activities in 0.1 M phosphate buffer. We observed an increase in NADH ferricyanide reductase (NFR) activity in Complex V mutants. NFR activity measured NADH dehydrogenase and mitochondrial outer membrane cytochrome b5 (rotenone-insensitive NADH-reductase). Using the Seahorse extracellular flux analyzer, we assessed mitochondrial respiration. Overall, results indicate that Basal Oxygen Consumption Rates and mitochondrial ATP production was compromised in cell lines exhibiting complex I and complex V mutations, while real time extracellular acidification rates (glycolysis) were elevated as cells had adapted to a compromised metabolic pathway. Using a specialized ImageJ macro mitochondrial network morphology analysis (MiNA) tool designed to analyze mitochondrial shape from live-cell images of MitoTracker-Red CM-H2Xros stained cells, we quantified mitochondrial morphology in healthy and diseased states. Our results indicate that some of the diseased cell lines exhibited mitochondrial morphologies associated with stressed conditions. Future experiments will determine whether mitochondrial morphology depend on mitochondrial DNA mutation load and whether they influence bioenergetics within a cell. Our ongoing studies are focused on evaluating mutation burden in hiPSCs, followed by bioenergetic analyses in differentiated neurons and muscle cells from LS-hiPSCs. Results from these studies will address the knowledge gaps that exist in the understanding of relationships among mtDNA mutations, morphology, function and cell fate that may ultimately contribute to devastating mitochondrial disorders.

β€’ Bioblast editor: Plangger M

Labels: MiParea: Respiration, mt-Structure;fission;fusion, mtDNA;mt-genetics  Pathology: Inherited 

Organism: Human  Tissue;cell: Fibroblast 


Iyer S(1), Bakare A(1), Kolenc O(2), Scheulin, K(3), Daniel J(1), Stabach J(1), Lesnefsky EJ(4), West FD(3), Quinn KP(2)
  1. Dept Biological Sciences, J William Fulbright College Arts Sciences, Univ Arkansas, Fayetteville, AR
  2. Dept Biomedical Engineering, College Engineering, Univ Arkansas, Fayetteville, AR
  3. Regenerative Bioscience Center, Univ Georgia, Athens, GA
  4. Hunter Holmes McGuire Veterans Affairs Medical Center, Richmond, VA, USA. - [email protected]


  1. Grace HE, Galdun P, Lesnefsky EJ, West FD, Iyer S (2019) mRNA reprogramming of T8993G Leigh's Syndrome fibroblast cells to create induced pluripotent stem cell models for mitochondrial disorders. Stem Cells Dev doi: 10.1089/scd.2019.0045.
  2. Valente, AJ, Maddalena LA, Robb EL, Moradi F, Stuart JA (2017) A simple ImageJ macro tool for analyzing mitochondrial network morphology in mammalian cell culture. Acta Histochem 119:315–26.
  3. Quinn KP, Bellas E, Fourligas N, Lee K, Kaplan DL, Georgakoudi I (2012) Characterization of metabolic changes associated with the functional development of 3D engineered tissues by non-invasive, dynamic measurement of individual cell redox ratios. Biomaterials 33:5341–48.
  4. Hoppel CL, Kerr DS, Dahms B, Roessmann U (1987) Deficiency of the reduced nicotinamide adenine dinucleotide dehydrogenase component of complex I of mitochondrial electron transport. Fatal infantile lactic acidosis and hypermetabolism with skeletal-cardiac myopathy and encephalopathy. J Clin Invest 80:71-77.
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