Transcriptional and metabolic adaptation of human neurons to the mitochondrial toxicant MPP+

Abstract

Assessment of the network of toxicity pathways by Omics technologies and bioinformatic data processing paves the road toward a new toxicology for the twenty-first century. Especially, the upstream network of responses, taking place in toxicant-treated cells before a point of no return is reached, is still little explored. We studied the effects of the model neurotoxicant 1-methyl-4-phenylpyridinium (MPP+) by a combined metabolomics (mass spectrometry) and transcriptomics (microarrays and deep sequencing) approach to provide unbiased data on earliest cellular adaptations to stress. Neural precursor cells (LUHMES) were differentiated to homogeneous cultures of fully postmitotic human dopaminergic neurons, and then exposed to the mitochondrial respiratory chain inhibitor MPP+ (5 μM). At 18–24 h after treatment, intracellular ATP and mitochondrial integrity were still close to control levels, but pronounced transcriptome and metabolome changes were seen. Data on altered glucose flux, depletion of phosphocreatine and oxidative stress (e.g., methionine sulfoxide formation) confirmed the validity of the approach. New findings were related to nuclear paraspeckle depletion, as well as an early activation of branches of the transsulfuration pathway to increase glutathione. Bioinformatic analysis of our data identified the transcription factor ATF-4 as an upstream regulator of early responses. Findings on this signaling pathway and on adaptive increases of glutathione production were confirmed biochemically. Metabolic and transcriptional profiling contributed complementary information on multiple primary and secondary changes that contribute to the cellular response to MPP+. Thus, combined ‘Omics' analysis is a new unbiased approach to unravel earliest metabolic changes, whose balance decides on the final cell fate.

Consensus report on the future of animal-free systemic toxicity testing

“…therefore, signaling pathway identification and analysis is a crucial research necessity in toxicology, and very detailed quantitative information needs to be derived (Fig. 12) to use such data for systems biology modeling (Jennings et al, 2013;Krug et al, 2014;. toxicogenomics technologies (Ramirez et al, 2013) are important tools that cover a multitude of cellular events.…”

Switching from astrocytic neuroprotection to neurodegeneration by cytokine stimulation

“…The percent LDH release was expressed as 100 × LDH (supernatant) /(LDH (supernatant) + LDH (lysate) ) (Krug et al 2014). Release of human-specific neuronal enolase into co-culture supernatants was analyzed using the 'Neuron-specific Enolase' Quantikine ELISA Kit (R&D, Wiesbaden) according to the manufacturer's protocol.…”

Reference compounds for alternative test methods to indicate developmental neurotoxicity (DNT) potential of chemicals: Example lists and criteria for their selection and use_suppl

“…Introduction of new endpoints or identification of biomarkers (Krug et al, 2014;Stiegler et al, 2011;Zimmer et al, 2012) requires the availability of a relevant set of test compounds that allows correlation studies from one system or from one endpoint to another. To an even greater extent, the same holds true for identification of general toxicity mechanisms (Fritsche et al, 2005;Gassmann et al, 2010Gassmann et al, , 2014Langeveld et al, 2012;Lein et al, 2007;Waldmann et al, 2014;Yang et al, 2014;Zimmer et al, 2011a) or for development of toxicant classifiers (Krug et al, 2013b;Rempel et al, 2015), as the selected compounds are the main anchoring point of such studies.…”

“…To assess the specificity of a test system for direct-acting DNT compounds, it is necessary to select a second group of negative control compounds, i.e., nonspecific controls known for their general cytotoxicity (Kadereit et al, 2012;Leist et al, 2010). The concentration ratio of these compounds concerning specific (e.g., neurite growth) and nonspecific (e.g., cytotoxicity) test endpoints can be used to define a prediction model for test specificity (Krug et al, 2014;Stiegler et al, 2011); (iii) The third problem is related to toxicokinetics (including drug metabolism). Several compounds would (based on their biochemical activity) affect fundamental neurodevelopmental/biological processes relevant to DNT, but they are not recognized as DNT compounds in the literature or by in vivo testing, as they do not reach the fetus or the central nervous system at the doses used.…”