Development of a neurotoxicity assay that is tuned to detect mitochondrial toxicants

Abstract

Many neurotoxicants affect energy metabolism in man, but currently available test methods may still fail to predict mito-and neurotoxicity. We addressed this issue using LUHMES cells, i.e., human neuronal precursors that easily differentiate into mature neurons. Within the NeuriTox assay, they have been used to screen for neurotoxicants. Our new approach is based on culturing the cells in either glucose or galactose (Glc-Gal-NeuriTox) as the main carbohydrate source during toxicity testing. Using this Glc-Gal-NeuriTox assay, 52 mitochondrial and non-mitochondrial toxicants were tested. The panel of chemicals comprised 11 inhibitors of mitochondrial respiratory chain complex I (cI), 4 inhibitors of cII, 8 of cIII, and 2 of cIV; 8 toxicants were included as they are assumed to be mitochondrial uncouplers. In galactose, cells became more dependent on mitochondrial function, which made them 2-3 orders of magnitude more sensitive to various mitotoxicants. Moreover, galactose enhanced the specific neurotoxicity (destruction of neurites) compared to a general cytotoxicity (plasma membrane lysis) of the toxicants. The Glc-Gal-NeuriTox assay worked particularly well for inhibitors of cI and cIII, while the toxicity of uncouplers and non-mitochondrial toxicants did not differ significantly upon glucose ↔ galactose exchange. As a secondary assay, we developed a method to quantify the inhibition of all mitochondrial respiratory chain functions/ complexes in LUHMES cells. The combination of the Glc-Gal-NeuriTox neurotoxicity screening assay with the mechanistic follow up of target site identification allowed both, a more sensitive detection of neurotoxicants and a sharper definition of the mode of action of mitochondrial toxicants. Keywords Neurotoxicity • Mitotoxicity • Metabolic reprogramming • High-throughput toxicity screening • High content imaging • Mechanistic safety assessment Abbreviations ADP Adenosine triphosphate AOP Adverse outcome pathway Asp l-Aspartate ATP Adenosine diphosphate cAMP N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate cI-V MRC complex I-V CNS Central nervous system Cyt c Cytochrome c FAD(H 2) Flavin adenine dinucleotide (FAD: oxidized, FADH2: reduced) FCCP Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone G6P Glucose-6-phosphate Gal1P Galactose-1-phosphate GALK Galactokinase GALT Galactose-1-phosphate uridylyltransferase GDNF Glial derived neurotrophic factor Glc1P Glucose-1-phosphate Hex Hexose, in this study either glucose or galactose HK Hexokinase Lac Lactate Mal Malate MoA Mode of action MPP 1-Methyl-4-phenylpyridinium Electronic supplementary material The online version of this article (

Time and space-resolved quantification of plasma membrane sialylation for measurements of cell function and neurotoxicity

“…This feature was used here to establish a particularly sensitive assay for toxicants disturbing mitochondrial function and glycoprotein synthesis. It allows especially the detection of functional deficits early after toxicant application and it complements another new assay for mitotoxicants that uses standard readouts, but alters the composition of the cell culture media by exchanging glucose for galactose (Delp et al 2019).…”

“…A large number of known neurotoxicants targets neurites (Delp et al 2018b(Delp et al , 2019Harrill 2018;Narro et al 2007;Stiegler et al 2011). Especially, growing neurites are more susceptible to the exposure of neurotoxicants (Krug et al 2013) (Suppl.…”

“…Here, we studied the metabolic incorporation of an azidetagged sugar precursor (Ac 4 ManNAz) as Az-Sia into the glycocalyx of mature human neurons (LUHMES). These cells (Scholz et al 2011) are a frequently used model for human neurotoxicity (Delp et al 2018b(Delp et al , 2019Efremova et al 2017;Ganjam et al 2019;Harris et al 2018;Krug et al 2013;Singh et al 2018;Skirzewski et al 2018;Stiegler et al 2011;Tong et al 2018). We asked the question on whether the sialylation capacity can be used as an early functional readout of neurotoxicity.…”

Thiazolides promote G1 cell cycle arrest in colorectal cancer cells by targeting the mitochondrial respiratory chain

“…Since NTZ has been described as mitochondrial uncoupler [30], we investigated this potential toxicity mechanism of thiazolides. Cells were permeabilized to directly probe their mitochondrial function by using an established protocol [49]. Speciଏc uncoupling effects were evaluated in the presence of oligomycin, an ATPase inhibitor.…”

“…For the quantiଏcation of mitochondrial respiration and glycolytic function, the OCR and the ECAR of intact LS174T and Caco-2 cells were analyzed on a Seahorse XFe24 analyzer (Agilent, Ratingen, Germany), as described in [58]. To assess the MRC complex activities, cells were permeabilized as described in [49]. To assess mitochondrial inner membrane integrity, assays were performed in the presence of oligomycin (1 µM).…”