Mapping mitochondrial respiratory chain deficiencies by respirometry: Beyond the Mito Stress Test - PubMed (original) (raw)

Mapping mitochondrial respiratory chain deficiencies by respirometry: Beyond the Mito Stress Test

Sausan M Jaber et al. Exp Neurol. 2020 Jun.

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 "map" 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.

Keywords: ABT-737; BCL-2; BH3; Bioenergetics; Cytochrome c; Oxygen; Pyruvate; Respiration; Seahorse; UK5099.

Copyright © 2020 Elsevier Inc. All rights reserved.

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Conflict of interest statement

Declaration of Competing Interest N.Y. receives royalties from Agilent Technologies for U.S. Patent US9915647B2. Agilent Technologies had no role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.

Figures

Fig. 1

Fig. 1. Comparison of cell respiratory capacities.

BCL-2 overexpressing MCF10A cells were compared with vector controls (CON) at the indicated plating cell densities (30,000 vs. 60,000 cells/well). Oxygen consumption rates (OCR) were measured following overnight cell proliferation. FCCP was added sequentially at 4 μM and 2 μM, respectively, followed by pyruvate (pyr, 10 mM) and antimycin A (Anti A, 1 μM). A & C) Absolute OCR values are shown (mean ± SD, n=3 wells). B & D) Baseline-normalized OCR values relative to the third rate, which was set at 100%, are displayed.

Fig. 2

Fig. 2. Identification of drugs that alter neuronal bioenergetics by the Mito Stress Test.

A & B) OCR of primary cortical neurons following the injection of 10 μM drug or vehicle control (CON), with ABT-737 added in A and UK5099 in B. The Mito Stress Test was conducted subsequent to the injection of the test drug, which consisted of the sequential addition of oligomycin (Oligo, 0.3 μg/ml), followed by FCCP (3 μM) or 2,4-dinitrophenol (DNP, 200 μM), and then antimycin A (Anti A, 1 μM). OCR values are means ± SD from three wells (with the exception of CON in B, which is the mean of two wells). OCR are baseline-normalized as in Fig. 1.

Fig. 3

Fig. 3. Respirometry test for a drug-induced cytochrome c deficit.

Cytochrome c (Cyt c) supplementation prevents the effect of ABT-737 (10 μM) on neuronal OCR (A) but does not rescue the UK5099 (10 μM)-induced respiratory deficit (B). To test for cytochrome _c_-limited respiration, neurons were permeabilized using saponin (SAP, 25 μg/ml), enabling delivery of purified Cyt c (100 μM), together with respiratory chain substrates, ADP, phosphate, and EGTA (see the Materials and Methods section for details). The substrate combinations were succinate and ADP in the presence of rotenone (S/R/A) in A and pyruvate, malate, and ADP (P/M/A) in B. To confirm that the OCR rescue by Cyt c was due to restored electron transfer to Complex IV, the Complex IV inhibitor azide (5 mM) was added. In B, antimycin A (Anti A, 1 μM) was also added subsequently to azide. CON, vehicle control. OCR values are means ± SD from three wells.

Fig. 4

Fig. 4. Respirometry test for Complex IV activity.

Complex IV functionality was assessed by monitoring azide-sensitive neuronal OCR supported by the exogenous electron donor TMPD in the presence of the TMPD-reducing agent ascorbate and the Complex III inhibitor antimycin A. UK5099 (10 μM) or vehicle (CON) was added first, followed by 200 μM DNP and 10 mM pyruvate (DNP+Pyr) to induce maximal respiration. Anti A (1 μM) was injected ± TMPD/ascorbate (TMPD/Asc, 0.4 mM/8 mM) as indicated. Finally, azide (5 mM) was added to confirm the specificity of the assay. OCR values are means ± SD from three wells.

Fig. 5

Fig. 5. Permeabilized cell assay to test for a Complex I- or II-linked respiration deficit.

Neurons were permeabilized by saponin (SAP) in the presence of either the Complex I-linked substrates pyruvate and malate, as well as ADP (P/M/A) (A), or the Complex II-linked substrate succinate, with the Complex I inhibitor rotenone and ADP included (S/R/A) (B). Subsequently UK5099 (10 μM) or vehicle (CON) was added to test for substrate-specific effects on respiration. See the Materials and Methods section for full details on the permeabilization mixture. OCR values are means from two wells.

Fig. 6

Fig. 6. Complex I-III-IV linked respirometry assay to evaluate the ability of Complex I to oxidize NADH.

Complex I activity was measured directly in permeabilized neurons by monitoring OCR supported by the substrate NADH. The neurons were permeabilized by saponin (25 μg/ml) and the pore-forming peptide alamethicin (40 μg/ml), which was used to deliver NADH across the mitochondrial inner membrane. Exogenous cytochrome c (100 μM) was added to compensate for the cytochrome c loss caused by alamethicin, allowing Complex I-III-IV electron transfer to occur so that OCR could be measured as a readout of Complex I activity. UK5099 (10 μM), vehicle (CON), or the positive control rotenone (Rot, 0.5 μM) was added together with NADH (2 mM) and the other permeabilization components (see Material and Methods section for full details). Subsequently, azide (5 mM) was added to block oxygen consumption by Complex IV. OCR values are means from two wells.

References

    1. Bordt EA, Clerc P, Roelofs BA et al. (2017) The Putative Drp1 Inhibitor mdivi-1 Is a Reversible Mitochondrial Complex I Inhibitor that Modulates Reactive Oxygen Species. Dev Cell, 40, 583–594 e586. - PMC - PubMed
    1. Brand MD and Nicholls DG (2011) Assessing mitochondrial dysfunction in cells. Biochem J, 435, 297–312. - PMC - PubMed
    1. Bricker DK, Taylor EB, Schell JC et al. (2012) A mitochondrial pyruvate carrier required for pyruvate uptake in yeast, Drosophila, and humans. Science, 337, 96–100. - PMC - PubMed
    1. Clerc P, Carey GB, Mehrabian Z, Wei M, Hwang H, Girnun GD, Chen H, Martin SS and Polster BM (2012) Rapid detection of an ABT-737-sensitive primed for death state in cells using microplate-based respirometry. PLoS One, 7, e42487. - PMC - PubMed
    1. Clerc P, Ge SX, Hwang H, Waddell J, Roelofs BA, Karbowski M, Sesaki H and Polster BM (2014) Drp1 is dispensable for apoptotic cytochrome c release in primed MCF10A and fibroblast cells but affects Bcl-2 antagonist-induced respiratory changes. Br. J. Pharmacol, 171, 1988–1999. - PMC - PubMed

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