MOLECULAR MECHANISMS OF β-AMYLOID-INDUCED INHIBITION OF GLUTATHIONE SYNTHESIS IN RATS

Abstract

Oxidative stress is one of the main pathogenic mechanisms of Alzheimer's disease (AD). Glutathione (GSH) is an endogenous antioxidant protecting cells from oxidative injury. However, it is reported that the content of GSH is lower both in the brain of AD patients and the AD animal models. The reason why GSH content is lower in such an oxidative environment of AD brain is not clear. Glutamatecysteine ligase (GCL), composed of a catalytic (GCLC) and a modifier (GCLM) subunit, is the rate-limiting enzyme in GSH synthesis. The transcription of both GCLC and GCLM are regulated by Keap1-Nrf2-ARE signal pathway. Meanwhile, as a chaperone protein of Nrf2, small Maf proteins form heterodimers with Nrf2 leading to activation or repression of ARE-mediated gene expression. The current study aimed at investigating the molecular mechanisms of regulation on GCL transcription by amyloid-β (Aβ) involving Keap1-Nrf2-ARE signal pathway. Aβ intrahippocampal injection rat model and human neuroblastoma SH-SY5Y cells treated with Aβ were used. The expressions of Nrf2, GCLC, GCLM and small Maf proteins as well as GSH level under Aβ insult at different time point were tested. Overexpression and knockdown of Maf proteins were used to explore the roles of Maf proteins on GCL expression. The banding of Keap1 and Nrf2 was examined by CoIP. The expressions of GCLC, GCLM and GSH level were increased in acute stage of Aβ treatment (within 6h), while the expressions of GCLC, GCLM and GSH level were decreased accompanying with activation of Keap1-Nrf2-ARE signal pathway after Aβ treatment for 6–24h. The nuclear level of Nrf2 restored to normal level accompanying with down regulation of GCL and low GSH in the late stage of Aβ treatment. The nuclear expressions of MafG and MafK were largely induced and displacement of Nrf2 nuclear binding to ARE in the late stage of Aβ treatment, while, MafG or MafK knockdown restored GCLC expression and GSH level. Meanwhile, the banding of Keap1 and Nrf2 were enhanced with decreased expression of p-Nrf2 Ser40. Aβ-mediated inhibition of Keap1-Nrf2-ARE signal pathway and induction of nuclear Maf proteins contributed to lowering GSH content due to inhibition of GCL expression.