A method to identify and validate mitochondrial modulators using mammalian cells and the worm C. elegans

Abstract

Mitochondria are semi-autonomous organelles regulated by a complex network of proteins that are vital for many cellular functions. Because mitochondrial modulators can impact many aspects of cellular homeostasis, their identification and validation has proven challenging. It requires the measurement of multiple parameters in parallel to understand the exact nature of the changes induced by such compounds. We developed a platform of assays scoring for mitochondrial function in two complementary models systems, mammalian cells and C. elegans. We first optimized cell culture conditions and established the mitochondrial signature of 1,200 FDA-approved drugs in liver cells. Using cell-based and C. elegans assays, we further defined the metabolic effects of two pharmacological classes that emerged from our hit list, i.e. imidazoles and statins. We found that these two drug classes affect respiration through different and cholesterol-independent mechanisms in both models. Our screening strategy enabled us to unequivocally identify compounds that have toxic or beneficial effects on mitochondrial activity. Furthermore, the cross-species approach provided novel mechanistic insight and allowed early validation of hits that act on mitochondrial function.

Cellular Allometry of Mitochondrial Functionality Establishes the Optimal Cell Size

“…We therefore examined how the mevalonate pathway affects the cell size scaling of mitochondrial functionality. In Kc167 cells inhibition of the mevalonate pathway by pitavastatin resulted in increased rΔΨm in larger cells (Figure 6B), consistent with the statin-induced ΔΨm increase in other cell types (Andreux et al., 2014). These effects were rescued by mevalonate, the metabolite immediately downstream of the statin target HMG-CoA reductase (Figure 6B).…”

“…We previously identified the mevalonate pathway as a cell size regulator (Miettinen and Bjorklund, 2015, Miettinen et al., 2014), and this pathway has been shown to affect mitochondria (Andreux et al., 2014, Liu et al., 2014). We therefore examined how the mevalonate pathway affects the cell size scaling of mitochondrial functionality.…”

A screening-based platform for the assessment of cellular respiration in Caenorhabditis elegans

“…genome-wide screens to find genetic interactors affecting mitochondrial bioenergetics and compound screens for mitochondrial toxicity. Indeed, XF respirometry was already successfully applied to study the effects of compounds or gene knockdowns on mitochondrial function in C. elegans 15,26–27.…”

“…Numerous in vitro , in situ and in vivo methodologies have been developed to assess various aspects of mitochondrial function1,15–16. Assays include the estimation of mitochondrial membrane potential17, determining the production and/or concentration of ATP with bioluminescence18 and calcium retention19.…”

“…Nevertheless, the progress of the mitochondrial field in C. elegans was long delayed by the limited options to dedicatedly measure metabolism, in particular mitochondrial activity. Hence, we set out to develop a platform using Seahorse respirometry, which was successfully applied in several projects15,26–27. Here, we describe in detail how Seahorse XF96 respirometers can be used to study mitochondrial biology in C. elegans .…”