In Vivo Determination of Mitochondrial Function Using Luciferase‐Expressing Caenorhabditis elegans: Contribution of Oxidative Phosphorylation, Glycolysis, and Fatty Acid Oxidation to Toxicant‐Induced Dysfunction

Anthony L. Luz1, Cristina Lagido2, Matthew D. Hirschey3, Joel N. Meyer1

1 Nicholas School of the Environment, Duke University, Durham, North Carolina, 2 School of Medical Sciences, Institute of Medical Sciences, Aberdeen, 3 Department of Medicine, Duke University, Durham, North Carolina
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 25.8
DOI:  10.1002/cptx.10
Online Posting Date:  August, 2016
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Abstract

Mitochondria are a target of many drugs and environmental toxicants; however, how toxicant‐induced mitochondrial dysfunction contributes to the progression of human disease remains poorly understood. To address this issue, in vivo assays capable of rapidly assessing mitochondrial function need to be developed. Here, using the model organism Caenorhabditis elegans, we describe how to rapidly assess the in vivo role of the electron transport chain, glycolysis, or fatty acid oxidation in energy metabolism following toxicant exposure, using a luciferase‐expressing ATP reporter strain. Alterations in mitochondrial function subsequent to toxicant exposure are detected by depleting steady‐state ATP levels with inhibitors of the mitochondrial electron transport chain, glycolysis, or fatty acid oxidation. Differential changes in ATP following short‐term inhibitor exposure indicate toxicant‐induced alterations at the site of inhibition. Because a microplate reader is the only major piece of equipment required, this is a highly accessible method for studying toxicant‐induced mitochondrial dysfunction in vivo. © 2016 by John Wiley & Sons, Inc.

Keywords: Caenorhabditis elegans; fatty acid oxidation; glycolysis; mitochondrial toxicity; PE255; PE327; oxidative phosphorylation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Luciferase‐Based in Vivo Assessment of Mitochondrial Energy Metabolism in C. elegans
  • Support Protocol 1: Preparing OP50 Seeded K Agar Plates
  • Support Protocol 2: Age‐Synchronizing Nematodes Via Sodium Hypochlorite Treatment
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Luciferase‐Based in Vivo Assessment of Mitochondrial Energy Metabolism in C. elegans

  Materials
  • OP50‐seeded K agar plates (see protocol 2)
  • Synchronous populations of L1 PE255 nematodes (see protocol 3)
  • K medium (see recipe)
  • Inhibitor stocks (Table 25.8.1 outlines all required inhibitors, as well as storage conditions)
  • Unbuffered EPA H 2O (see recipe)
  • Dimethylsulfoxide (DMSO)
  • 0.1% (v/v) Triton X‐100 (diluted in ddH 2O; store at room temperature indefinitely)
  • Luminescence buffer (see recipe)
  • 70% ethanol
  • Incubator (capable of maintaining temperatures in the range of 15−25°C)
  • Centrifuge (e.g., Beckman Coulter equipped for 15‐ml tubes)
  • Dissecting light microscope
  • Glass microscope slides
  • 25‐ml disposable reagent reservoirs
  • Multichannel pipettor (capable of pipetting 20 to 200 µl)
  • White 96‐well plates without lids
  • Horizontal vortexer (e.g., Eppendorf MixMate PCR 96)
  • Orbital shaker
  • Microplate reader (FLUOstar OPTIMA, BMG Labtech) equipped with luminescence optic, 502‐nm emissions filter, and 485‐nm excitation filter
  • 50‐ml conical centrifuge tubes
  • Additional reagents and equipment for culturing nematodes (Stiernagle, )
Table 5.8.1   MaterialsPreparation of Inhibitors

Inhibitor (target) Stock concentration Working concentration (8×) Final concentration (1×) Incubation period (hr)
Rotenone (ETC complex I) 2 mM dissolved in 100% DMSO (store at −20°C in 30‐µl aliquots)
  • 160 µM dissolved in 8% DMSO
  • To make: Add 24 µl 2 mM rotenone (100% DMSO) to 276 µl unbuffered EPA H 2O
20 µM in 1% DMSO 1
TTFA (ETC complex II)a 100 mM dissolved in 100% DMSO (store at 4°C in 30‐µl aliquots)
  • 8mM dissolved in 8% DMSO
  • To make: Add 24 µl 100 mM TTFA to 276 µl unbuffered EPA H 2O
1 mM in 1% DMSO 1
  • Malonate
  • (ETC complex II) b
120 mM dissolved in 100% unbuffered EPA H 2O (store at 4°C in 1‐ml aliquots) 120 mM dissolved in 100% unbuffered EPA H 2O 15 mM in 100% unbuffered EPA H 2O 1
Antimycin A (ETC complex III) 15 mM dissolved in 100% DMSO (store at −20°C in 30‐µl aliquots)
  • 1.2 mM dissolved in 8% DMSO
  • To make: Add 24 µl 15 mM antimycin A (100% DMSO) to 276 µl unbuffered EPA H 2O
150 mM in 1% DMSO 1
Sodium azide (ETC complex IV) c 2 mM dissolved in 100% unbuffered EPA H 2O (store at 4°C in 1‐ml aliquots) 2 mM dissolved in 100% unbuffered EPA H 2O 250 µM in 100% unbuffered EPA H 2O 1
DCCD (ATP synthase) 2 mM dissolved in 100% DMSO (store at −20°C in 30‐µl aliquots)
  • 160 µM dissolved in 8% DMSO
  • To make: Add 24 µl 2 mM DCCD (100% DMSO) to 276 µl unbuffered EPA H 2O
20 µM in 1% DMSO 1
FCCP (mitochondrial uncoupler) 2.5 mM dissolved in 100% DMSO (store at −20°C in 30‐µl aliquots)
  • 200 µM dissolved in 8% DMSO
  • To make: Add 24 µl 2.5 mM FCCP (100% DMSO) to 276 µl unbuffered EPA H 2O
25 µM in 1% DMSO 1
Perhexiline (fatty acid oxidation) 10 mM dissolved in 100% DMSO (store at 4°C in 30‐µl aliquots)
  • 800 µM dissolved in 8% DMSO
  • To make: Add 24 µl 10 mM perhexiline (100% DMSO) to 276 µl unbuffered EPA H 2O
100 µM in 1% DMSO 1
2‐DG (glycolysis) 400 mM dissolved in unbuffered EPA H 2O (store at 4°C in 30‐µl aliquots) 400 mM dissolved in unbuffered EPA H 2O 50 mM in 100% unbuffered EPA H 2O 4.5

 a500 µM TTFA reduces luminescence approximately 50% in PE255 N2 nematodes (data not shown), while 1000 µM causes an 80% to 99% reduction in PE255 N2 bioluminescence (Fig.  ). Concentrations of ETC inhibitors listed in Table 25.8.1 caused roughly a 40% to 60% reduction in bioluminescence in young adult PE255 glp‐4‐deficient nematodes (see Supplemental Figures 2 to 7 at http://www.currentprotocols.com/protocol/tx2508), and with the exceptions of TTFA and sodium azide, cause similar reductions in both L4 and 8 day old PE255 N2 nematodes.
 bMalonate, a competitive inhibitor of ETC complex II, can be used in place of TTFA at the discretion of the experimenter. Pros and cons of this are discussed in Background Information.
 c250 µM sodium azide has no significant effect on PE255 N2 bioluminescence, while 500 µM azide reduces bioluminescence ∼50%.

Support Protocol 1: Preparing OP50 Seeded K Agar Plates

  Materials
  • Potassium chloride (KCl),
  • Sodium chloride (NaCl)
  • Bacto‐peptone (e.g., BD Difco)
  • Bacto‐agar (e.g., BD Difco)
  • 1 M calcium chloride (CaCl 2, dissolved in distilled deionized H 2O and autoclaved)
  • 1 M magnesium sulfate (MgSO 4, dissolved in distilled deionized H 2O and autoclaved)
  • 10 mg/ml cholesterol (dissolved in 100% ethanol and filter sterilized)
  • 5 ml 1.25 mg/ml nystatin (dissolved in 100% ethanol)
  • LB broth (see recipe)
  • E. coli OP50 (which can be purchased from the Caenorhabditis Genetics Center, University of Minnesota)
  • Erlenmeyer flask (2 liters or larger)
  • Magnetic stir bar
  • Magnetic stirrer/hot plate
  • Autoclave tape
  • Serological pipet (25 or 50 ml)
  • Petri dishes (100 × 50 mm)
  • Inoculating loop
  • 37°C shaking incubator
  • Repeating pipettor (e.g., Eppendorf) and 10‐ml displacement tips
  • Glass hockey stick spreaders
  • Rotating pedestal (turntable) for spreading bacteria on agar in Petri dish
  • Bunsen burner

Support Protocol 2: Age‐Synchronizing Nematodes Via Sodium Hypochlorite Treatment

  Materials
  • OP50‐seeded K agar plates (see protocol 2) containing gravid transgenic (PE255) adult nematodes
  • K medium (see recipe)
  • 70% ethanol
  • Sodium hydroxide/bleach solution (see recipe)
  • Complete K medium (see recipe)
  • OP50‐seeded K agar plates (see protocol 2)
  • 15‐ml centrifuge tubes (e.g., Corning Falcon)
  • Centrifuge (e.g., Beckman Coulter equipped for 15‐ml tubes)
  • Glass hockey stick spreader
  • Bunsen burner
  • Orbital shaker
  • 50‐ml cell culture flask
  • Dissecting light microscope
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Figures

Videos

Literature Cited

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