Evaluation of the rotenone-induced activation of the Nrf2 pathway in a neuronal model derived from human induced pluripotent stem cells

ABSTRACT Human induced pluripotent stem cells (hiPSCs) are considered as a powerful tool for drug and chemical screening and development of new in vitro testing strategies in the field of toxicology, including neurotoxicity evaluation. These cells are able to expand and efficiently differentiate into different types of neuronal and glial cells as well as peripheral neurons. These human cells‐based neuronal models serve as test systems for mechanistic studies on different pathways involved in neurotoxicity. One of the well‐known mechanisms that are activated by chemically‐induced oxidative stress is the Nrf2 signaling pathway. Therefore, in the current study, we evaluated whether Nrf2 signaling machinery is expressed in human induced pluripotent stem cells (hiPSCs)‐derived mixed neuronal/glial culture and if so whether it becomes activated by rotenone‐induced oxidative stress mediated by complex I inhibition of mitochondrial respiration. Rotenone was found to induce the activation of Nrf2 signaling particularly at the highest tested concentration (100 nM), as shown by Nrf2 nuclear translocation and the up‐regulation of the Nrf2‐downstream antioxidant enzymes, NQO1 and SRXN1. Interestingly, exposure to rotenone also increased the number of astroglial cells in which Nrf2 activation may play an important role in neuroprotection. Moreover, rotenone caused cell death of dopaminergic neurons since a decreased percentage of tyrosine hydroxylase (TH+) cells was observed. The obtained results suggest that hiPSC‐derived mixed neuronal/glial culture could be a valuable in vitro human model for the establishment of neuronal specific assays in order to link Nrf2 pathway activation (biomarker of oxidative stress) with additional neuronal specific readouts that could be applied to in vitro neurotoxicity evaluation. HIGHLIGHTSOxidative stress is a common key event in developmental and adult neurotoxicity.hiPSC‐derived mixed culture is a reliable model for neurotoxicity testing.Rotenone causes oxidative stress and leads to the activation of the Nrf2 pathway.Rotenone activates Nrf2 and increases astrocytes in hiPSC‐derived neurons and glia.Rotenone decreases the number of dopaminergic neurons derived from hiPSCs.

[1]  M. Rao,et al.  Using Human Pluripotent Stem Cell–Derived Dopaminergic Neurons to Evaluate Candidate Parkinson’s Disease Therapeutic Agents in MPP+ and Rotenone Models , 2013, Journal of biomolecular screening.

[2]  S. Biswal,et al.  Nrf2-regulated glutathione recycling independent of biosynthesis is critical for cell survival during oxidative stress. , 2009, Free radical biology & medicine.

[3]  Steven K. Gibb.  Toxicity testing in the 21st century: a vision and a strategy. , 2008, Reproductive toxicology.

[4]  G. Barreto,et al.  Mitochondrial functions in astrocytes: Neuroprotective implications from oxidative damage by rotenone , 2012, Neuroscience Research.

[5]  Donna D. Zhang,et al.  Keap1 Controls Postinduction Repression of the Nrf2-Mediated Antioxidant Response by Escorting Nuclear Export of Nrf2 , 2007, Molecular and Cellular Biology.

[6]  Martin Paparella,et al.  International STakeholder NETwork (ISTNET): creating a developmental neurotoxicity (DNT) testing road map for regulatory purposes , 2015, Archives of Toxicology.

[7]  Cheng He,et al.  Rotenone inhibits delayed rectifier K+ current via a protein kinase A-dependent mechanism , 2008, Neuroreport.

[8]  C. Tanner,et al.  Rotenone, Paraquat, and Parkinson’s Disease , 2011, Environmental health perspectives.

[9]  Jing Zhao,et al.  Sulfiredoxin-1 Attenuates Oxidative Stress via Nrf2/ARE Pathway and 2-Cys Prdxs After Oxygen-Glucose Deprivation in Astrocytes , 2014, Journal of Molecular Neuroscience.

[10]  L. Phebus,et al.  Measurement of striatal H2O2 by microdialysis following global forebrain ischemia and reperfusion in the rat: correlation with the cytotoxic potential of H2O2 in vitro , 1995, Brain Research.

[11]  K. Tufekci,et al.  The Nrf2/ARE Pathway: A Promising Target to Counteract Mitochondrial Dysfunction in Parkinson's Disease , 2011, Parkinson's disease.

[12]  Timothy H Murphy,et al.  Induction of the Nrf2-driven Antioxidant Response Confers Neuroprotection during Mitochondrial Stress in Vivo* , 2005, Journal of Biological Chemistry.

[13]  Q. Ma.  Role of nrf2 in oxidative stress and toxicity. , 2013, Annual review of pharmacology and toxicology.

[14]  B. Holzapfel,et al.  Hypoxic upregulation of tyrosine hydroxylase gene expression is paralleled, but not induced, by increased generation of reactive oxygen species in PC12 cells , 1999, FEBS letters.

[15]  Claudia M Testa,et al.  Rotenone induces oxidative stress and dopaminergic neuron damage in organotypic substantia nigra cultures. , 2005, Brain research. Molecular brain research.

[16]  S. Swarnkar,et al.  Astrocyte Activation: A Key Step in Rotenone Induced Cytotoxicity and DNA Damage , 2012, Neurochemical Research.

[17]  T. Murphy,et al.  NF-E2-related Factor-2 Mediates Neuroprotection against Mitochondrial Complex I Inhibitors and Increased Concentrations of Intracellular Calcium in Primary Cortical Neurons* , 2003, Journal of Biological Chemistry.

[18]  Mili B. Patel,et al.  Effect of Nrf2 activators on release of glutathione, cysteinylglycine and homocysteine by human U373 astroglial cells☆ , 2013, Redox biology.

[19]  Andrew Worth,et al.  Applying Adverse Outcome Pathways (AOPs) to support Integrated Approaches to Testing and Assessment (IATA). , 2014, Regulatory toxicology and pharmacology : RTP.

[20]  P. Stroman,et al.  The role(s) of astrocytes and astrocyte activity in neurometabolism, neurovascular coupling, and the production of functional neuroimaging signals , 2011, The European journal of neuroscience.

[21]  C. Glass,et al.  Expression of Nrf2 in Neurodegenerative Diseases , 2007, Journal of neuropathology and experimental neurology.

[22]  M. Osanai,et al.  Both Neurons and Astrocytes Exhibited Tetrodotoxin-Resistant Metabotropic Glutamate Receptor-Dependent Spontaneous Slow Ca2+ Oscillations in Striatum , 2014, PloS one.

[23]  Todd B. Sherer,et al.  Subcutaneous Rotenone Exposure Causes Highly Selective Dopaminergic Degeneration and α-Synuclein Aggregation , 2003, Experimental Neurology.

[24]  A. Odawara,et al.  Long-term electrophysiological activity and pharmacological response of a human induced pluripotent stem cell-derived neuron and astrocyte co-culture. , 2014, Biochemical and biophysical research communications.

[25]  E. Ullian,et al.  New roles for astrocytes in developing synaptic circuits , 2008, Communicative & integrative biology.

[26]  Iekhsan Othman,et al.  Neural Differentiation of Human Pluripotent Stem Cells for Nontherapeutic Applications: Toxicology, Pharmacology, and In Vitro Disease Modeling , 2015, Stem cells international.

[27]  Ying Zhang,et al.  Nrf2 regulates ROS production by mitochondria and NADPH oxidase , 2015, Biochimica et biophysica acta.

[28]  M. Dolan,et al.  Modeling Chemotherapeutic Neurotoxicity with Human Induced Pluripotent Stem Cell-Derived Neuronal Cells , 2015, PloS one.

[29]  W. Rausch,et al.  Dopaminergic neurons are preferentially sensitive to long-term rotenone toxicity in primary cell culture. , 2008, Toxicology in vitro : an international journal published in association with BIBRA.

[30]  Raymond R. Tice,et al.  Comparative neurotoxicity screening in human iPSC-derived neural stem cells, neurons and astrocytes , 2016, Brain Research.

[31]  K. Itoh,et al.  Keap1/Nrf2 system regulates neuronal survival as revealed through study of keap1 gene-knockout mice. , 2009, Biochemical and biophysical research communications.

[32]  C. Moraes,et al.  Titrating the Effects of Mitochondrial Complex I Impairment in the Cell Physiology* , 1999, The Journal of Biological Chemistry.

[33]  S. Budd,et al.  Mitochondria and neuronal survival. , 2000, Physiological reviews.

[34]  G. Daley,et al.  The promise of induced pluripotent stem cells in research and therapy , 2012, Nature.

[35]  H. Hogberg,et al.  Relevance of in vitro neurotoxicity testing for regulatory requirements: challenges to be considered. , 2010, Neurotoxicology and teratology.

[36]  P. Bernardi,et al.  High concordance of drug-induced human hepatotoxicity with in vitro cytotoxicity measured in a novel cell-based model using high content screening , 2006, Archives of Toxicology.

[37]  DelindaA . Johnson,et al.  The Nrf2–ARE Pathway , 2008, Annals of the New York Academy of Sciences.

[38]  Jaana M. Hartikainen,et al.  Genetic polymorphisms and protein expression of NRF2 and Sulfiredoxin predict survival outcomes in breast cancer. , 2012, Cancer research.

[39]  W. Krueger,et al.  Natural and artificial routes to pluripotency. , 2010, The International journal of developmental biology.

[40]  N. Benvenisty,et al.  Pluripotent stem cells in disease modelling and drug discovery , 2016, Nature Reviews Molecular Cell Biology.

[41]  L. Studer,et al.  Adapting human pluripotent stem cells to high-throughput and high-content screening , 2012, Nature Protocols.

[42]  M. Santello,et al.  Synaptic modulation by astrocytes via Ca2+-dependent glutamate release , 2009, Neuroscience.

[43]  T. Mak,et al.  Modulation of oxidative stress as an anticancer strategy , 2013, Nature Reviews Drug Discovery.

[44]  Donna D. Zhang,et al.  Molecular mechanisms of Nrf2 regulation and how these influence chemical modulation for disease intervention , 2015, Biochemical Society transactions.

[45]  Victor Tapias,et al.  A highly reproducible rotenone model of Parkinson's disease , 2009, Neurobiology of Disease.

[46]  Hui Dong,et al.  Nrf2/ARE pathway activation, HO-1 and NQO1 induction by polychlorinated biphenyl quinone is associated with reactive oxygen species and PI3K/AKT signaling. , 2014, Chemico-biological interactions.

[47]  Sergio Martinoia,et al.  A multi‐laboratory evaluation of microelectrode array‐based measurements of neural network activity for acute neurotoxicity testing , 2017, Neurotoxicology.

[48]  G. Basso,et al.  Development of a pluripotent stem cell derived neuronal model to identify chemically induced pathway perturbations in relation to neurotoxicity: effects of CREB pathway inhibition. , 2014, Toxicology and applied pharmacology.

[49]  A human pluripotent stem cell platform for assessing developmental neural toxicity screening , 2013, Stem Cell Research & Therapy.

[50]  Samik Ghosh,et al.  Integrating Pathways of Parkinson's Disease in a Molecular Interaction Map , 2013, Molecular Neurobiology.

[51]  S. Chandran,et al.  Mild oxidative stress activates Nrf2 in astrocytes, which contributes to neuroprotective ischemic preconditioning , 2010, Proceedings of the National Academy of Sciences.

[52]  K. Crofton,et al.  Putative adverse outcome pathways relevant to neurotoxicity , 2015, Critical reviews in toxicology.

[53]  B. K. Park,et al.  The Nrf2 cell defence pathway: Keap1-dependent and -independent mechanisms of regulation. , 2013, Biochemical pharmacology.

[54]  A. Levonen,et al.  The Keap1-Nrf2 pathway: Mechanisms of activation and dysregulation in cancer☆ , 2013, Redox biology.

[55]  Kevin K Kumar,et al.  The potential of induced pluripotent stem cells as a translational model for neurotoxicological risk. , 2012, Neurotoxicology.

[56]  J. Bailar,et al.  Toxicity Testing in the 21st Century: A Vision and a Strategy , 2010, Journal of toxicology and environmental health. Part B, Critical reviews.

[57]  K. Itoh,et al.  An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. , 1997, Biochemical and biophysical research communications.

[58]  F. Pistollato,et al.  Standardization of pluripotent stem cell cultures for toxicity testing , 2012, Expert opinion on drug metabolism & toxicology.