A Screenable <em>In Vivo</em> Assay for Mitochondrial Modulators Using Transgenic Bioluminescent <em>Caenorhabditis elegans</em>

Abstract

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode's ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants. 4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

Cell Biology of the Mitochondrion

“…Lagido et al, in the Glover and Pettitt laboratories, developed a luciferase expressing strain that allowed in vivo assessment of relative ATP levels in C. elegans (Lagido et al 2008). They later described the use of this strain for monitoring toxicant effects on mitochondrial function (McLaggan et al 2012;Lagido et al 2015). Members from the Meyer laboratory extended the use of this strain to allow rapid evaluation of the effects of toxicants on mitochondrial function, particularly the electron transport chain, glycolysis and fatty acid oxidation (Luz et al 2016b).…”

Adverse Outcome Pathway on Inhibition of the mitochondrial complex I of nigro-striatal neurons leading to parkinsonian motor deficits

“…A sharp decline in bioluminescence (maximal inhibition) relative to controls occurred at the lowest rotenone concentration of 2.5 μM. This decline in bioluminescence was consistent with reduced cellular ATP (Lagido et al, 2015). The results obtained from C. elegans exposed to rotenone suggested that chronic exposure to low concentration (2 or 4 μM) caused mitochondrial damage through persistent suppression of mitochondrial biogenesis and mitochondrial gene expression leading to mitochondrial dysfunction that contributed to DA neuron degeneration (Zhou et al, 2013).…”

Effects of reduced mitochondrial DNA content on secondary mitochondrial toxicant exposure in Caenorhabditis elegans

“…As we were primarily interested in investigating the effects of mtDNA knockdown on somatic tissues, and development of the nematode germline during the L3/L4 transition is associated with large increases in mtDNA copy number [19, 38] which would confound the interpretation of our results, we used the germline-deficient, in vivo ATP reporter strain PE255 glp-4 for all experiments [22, 23]. EtBr, a well-known inhibitor of mtDNA replication [39–41] that is frequently used to generate rho 0 (mtDNA-deficient) cells, was used to reduce mtDNA copy number in nematodes.…”

“…In vivo steady-state ATP levels were determined as previously described [23], and as visualized in [22]. Briefly, 50 nematodes (suspended in K-medium) were loaded into each well of a white 96-well plate (four wells per treatment), and then GFP fluorescence was measured (emissions filter: 502nm; excitation filter: 485nm) using a FLUOstar Optima microplate reader (BMG Labtech, Germany).…”

“…As nematodes share highly conserved mitochondrial biology [19], energy metabolism pathways [20], and mtDNA [21] with humans, they represent an excellent in vivo model to test this hypothesis. Furthermore, availability of transgenic strains, such as the PE255 ATP-reporter strain [22, 23], allow for the rapid assessment of mitochondrial function in vivo . To test this hypothesis, we reduced mtDNA copy number 35-55% and then exposed nematodes to known and suspected mitochondrial toxicants (aflatoxin B 1 (AfB 1 ), arsenite, paraquat, rotenone, and ultraviolet C radiation (UVC)).…”