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.

Anti-inflammatory and Anti-oxidant Activity of Hidrox® in Rotenone-Induced Parkinson’s Disease in Mice

“…Nrf2, one of the most important transcription factors that activates several genes with cytoprotective function, participates in antioxidant and anti-inflammatory reactions [ 72 , 73 , 74 , 75 , 76 , 77 ]. These cytoprotective genes encode a wide variety of phase II detoxification enzymes, such as NAD (P) H: quinone oxidoreductase 1 (NQO1), HO-1, γ-GCS, glutathione S-transferase (GST) and glutamate-cysteine ligase, thioredoxin, thioredoxin reductase, thermal shock proteins and many others [ 31 , 78 , 79 , 80 , 81 ]. Cell culture studies have shown that HT was the only olive oil phenol capable of increasing the transactivation of Nrf2, which suggests that this polyphenol could be responsible for the induction of Nrf2-dependent gene expression [ 82 ].…”

Assessment of developmental neurotoxicity induced by chemical mixtures using an adverse outcome pathway concept

“…In brief, NSCs obtained from neuroectodermal derivatives (rosettes) were passaged, plated onto reduced growth factor matrigel-coated 96 well plates (precoated with poly-D-lysine) at a density of 7000 cells/well (i.e., 21.000 cells/cm 2 ), and differentiated for either 21 or 28 days in vitro (DIV). At 21 DIV a mixed population of neurons (35-42% glutamatergic neurons, 15-20% GABAergic neurons, 13-20% dopaminergic neurons) and astrocytes (15-20%) was obtained [38,39].…”

Rotenone exerts developmental neurotoxicity in a human brain spheroid model

“…At certain concentrations, rotenone has been shown to produce progressive neurodegeneration of dopaminergic and non-do-paminergic neurons, and also of other brain cell populations such as astrocytes (Betarbet et al, 2000; Sindhu et al, 2005; Zagoura et al, 2017). Rotenone has been suggested as one of the most important environmental risk factor for Parkinson’s Disease (PD) (Betarbet et al, 2000; Perier et al, 2003; Spivey, 2011; Tanner et al, 2011; Nandipati and Litvan, 2016).…”

“…The major effect of rotenone is related to dopaminergic dysfunction, however, animal studies have found that rotenone may cause diffuse mitochondrial dysfunction in central non-dopaminergic and peripheral cells outside of the CNS (Fleming et al, 2004; Richter et al, 2007) and affect fish, chick and rat development (Khera et al, 1982; Spencer and Sing, 1982; Melo et al, 2015; Rao and Chauhan, 2004). In addition, in vitro studies on human iPSC-derived mixed neuronal and glial cultures have shown activation of the Keap1-Nrf2-ARE pathway after acute and chronical rotenone exposure, at non-cytotoxic concentrations (Pistollato et al, 2017; Zagoura et al, 2017). The Keap1-Nrf2 pathway is the major regulator of cytoprotective responses to oxidative and electrophilic stress (Kansanen et al, 2013).…”