Four-octyl itaconate activates Keap1-Nrf2 signaling to protect neuronal cells from hydrogen peroxide

Hua Liu,Min Xu,Guangfu Di,Jian Yang,Yu Feng,Zhichun Wang

doi:10.1186/s12964-018-0294-2

Abstract

BackgroundFour-octyl itaconate (OI), the itaconate’s cell-permeable derivative, can activate Nrf2 signaling via alkylation of Keap1 at its cysteine residues. The current study tested the potential neuroprotective function of OI in hydrogen peroxide (H2O2)-treated neuronal cells.MethodsSH-SY5Y neuronal cells and epigenetically de-repressed (by TSA treatment) primary murine neurons were treated with OI and/or H2O2. Nrf2 pathway genes were examined by Western blotting assay and real-time quantitative PCR analysis. Neuronal cell death was tested by the LDH and trypan blue staining assays. Apoptosis was tested by TUNEL and Annexin V assays.ResultsIn SH-SY5Y neuronal cells and primary murine neurons, OI activated Nrf2 signaling, causing Keap1-Nrf2 disassociation, Nrf2 protein stabilization and nuclear translocation, as well as expression of Nrf2-regulated genes (HO1, NQO1 and GCLC) and ninjurin2 (Ninj2). Functional studies showed that OI attenuated H2O2-induced reactive oxygen species (ROS) production, lipid peroxidation and DNA damage as well as neuronal cell death and apoptosis. shRNA-mediated knockdown, or CRISPR/Cas9-induced knockout of Nrf2 almost abolished OI-induced neuroprotection against H2O2. Keap1 is the primary target of OI. Keap1 knockout by CRISPR/Cas9 method mimicked and abolished OI-induced actions in SH-SY5Y cells. Introduction of a Cys151S mutant Keap1 in SH-SY5Y cells reversed OI-induced Nrf2 activation and anti-H2O2 neuroprotection.ConclusionsOI activates Keap1-Nrf2 signaling to protect SH-SY5Y cells and epigenetically de-repressed primary neurons from H2O2 in vitro.

Highlights

IntroductionFour-octyl itaconate (OI), the itaconate’s cell-permeable derivative, can activate Nrf signaling via alkylation of Keap at its cysteine residues
Four-octyl itaconate (OI), the itaconate’s cell-permeable derivative, can activate neuronal cellNuclear factor E2-related factor 2 (Nrf2) signaling via alkylation of Kelch like ECH-associated protein 1 (Keap1) at its cysteine residues
By performing the Quantitative real-time PCR (qPCR) assay, we show that OI dose-dependently increased mRNA levels of the known Nrf2-dependent genes, including HO1, NAD(P) H quinone oxidoreductase-1 (NQO1) and glutamyl cysteine ligase catalytic subunit (GCLC) (Fig. 1a)

Summary

Introduction

Four-octyl itaconate (OI), the itaconate’s cell-permeable derivative, can activate Nrf signaling via alkylation of Keap at its cysteine residues. Excessive oxidative stress shall induce neuronal cell injury, which is implicated in the physiological process of aging and a variety of neurodegenerative disease, including Parkinson’s, Huntington’s and Alzheimer’s diseases [1,2,3,4]. Nuclear factor E2-related factor 2 (Nrf2) is a key transcription factor regulating expression of a number of antioxidant enzymes, offering significant cellular protection against oxidative stress [8,9,10]. Nrf translocates to cell nuclei, and binds to antioxidant response elements (ARE) in the promoter of multiple anti-oxidant and detoxifying enzymes, including heme oxygenase 1 (HO-1), NAD(P) H quinone oxidoreductase-1 (NQO1) and. Upregulation of Nrf2-driven antioxidant enzymes is beneficial in models of neurodegenerative diseases [11,12,13]

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