Jaber 2020 BM Exp Neurol

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Publications in the MiPMap
Jaber SM, Yadava N, Polster BM (2020) Mapping mitochondrial respiratory chain deficiencies by respirometry: beyond the Mito Stress Test. BM Exp Neurol 328:113282.

Β» PMC7202675 Open Access

Jaber SM, Yadava N, Polster BM (2020) BM Exp Neurol

Abstract: Cell-based respirometers, such as the Seahorse Extracellular Flux Analyzer, are valuable tools to assess the functionality of mitochondria within adherent neurons, as well as other cell types. The Mito Stress Test is the most frequently employed protocol of drug additions to evaluate mitochondrial bioenergetic function. Sequential exposure of cells to an ATP synthase inhibitor such as oligomycin and an uncoupler such as FCCP cause changes in oxygen consumption rate that allow estimation of the cellular efficiency and capacity for mitochondrial ATP synthesis. While a useful first step in assessing whether an experimental treatment or genetic manipulation affects mitochondrial energetics, the Mito Stress Test does not identify specific sites of altered respiratory chain function. This article discusses limitations of the Mito Stress Test, proposes a refined protocol for comparing cell populations that requires independent drug titrations at multiple cell densities, and describes a stepwise series of respirometry-based assays that β€œmap” locations of electron transport deficiency. These include strategies to test for cytochrome c release, to probe the functionality of specific electron transport chain complexes within intact or permeabilized cells, and to measure NADH oxidation by the linked activity of Complexes I, III, and IV. To illustrate utility, we show that although UK5099 and ABT-737 each decrease the spare respiratory capacity of cortical neurons, the stepwise assays reveal different underlying mechanisms consistent with their established drug targets: deficient Complex I substrate supply induced by the mitochondrial pyruvate carrier inhibitor UK5099 and cytochrome c release induced by the anti-apoptotic BCL-2 family protein inhibitor ABT-737.

β€’ Bioblast editor: Gnaiger E

Cited by

Gnaiger E (2021) Bioenergetic cluster analysis – mitochondrial respiratory control in human fibroblasts. MitoFit Preprints 2021.8.

Gnaiger E (2021) Bioenergetic cluster analysis – mitochondrial respiratory control in human fibroblasts. MitoFit Preprints 2021.8. https://doi.org/10.26124/mitofit:2021-0008

Labels: MiParea: Respiration 

Tissue;cell: Nervous system  Preparation: Intact cells 

Coupling state: LEAK, ROUTINE, ET  Pathway: ROX 

MitoFit 2021 BCA 

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