Protective effect of an Nrf2-ARE activator identified from a chemical library on 6-hydroxydopamine-induced dopaminergic neuronal death

Abstract

Oxidative stress, which is caused by reactive oxygen species, contributes to the pathogenesis of Parkinson's disease. Activation of nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway induces antioxidant enzymes in response to oxidative stress. We focused on the Nrf2-ARE activators as dopaminergic neuroprotective agents. Activators of the Nrf2-ARE pathway are often electrophilic compounds and they react with a specific thiol on Kelch-like ECH-associated protein 1 (Keap1), a suppressor of Nrf2. A general issue with electrophilic compounds is that they nonspecifically react with thiol groups, leading to cytotoxicity. In this study, we aimed to find novel compounds of Nrf2 activators with a wide safety margin and to demonstrate the protective effect of the activator against 6-hydroxydopamine (6-OHDA) -induced dopaminergic neuronal death.ARE-driven transcriptional activity was measured by luciferase reporter assay in rat adrenal pheochromocytoma PC12 cells. Cell viability of PC12 cells was determined by the MTT assay. Primary mesencephalic cultures were established from rat embryos. Viability of dopaminergic neurons was determined by counting tyrosine hydroxylase-positive cells in primary mesencephalic cultures.We identified TPNA10168 as a potent Nrf2-ARE activator from the chemical library containing 4776 low molecular weight compounds. TPNA10168 showed a higher efficacy in Nrf2 activation and a lower cytotoxicity as compared with sulforaphane, a well-known activator of the Nrf2-ARE pathway. In primary mesencephalic cultures, pretreatment of TPNA10168 suppressed 6-OHDA-induced dopaminergic neuronal death. Treatment of TPNA10168 increased the expression of heme oxygenese-1 (HO-1), which is a downstream target gene of the Nrf2-ARE pathway, in astrocytes. Carbon monoxide (CO) is one of the degradation products of heme by HO-1 and is reported to present anti-apoptotic effect via guanylate cyclase (GC)/ protein kinase G (PKG) signaling pathway. The protective effect of TPNA10168 was suppressed by each inhibitor of HO-1, GC and PKG and hemoglobin which has CO-scavenging activity.The present study suggests that TPNA10168, which was identified as a Nrf2-ARE activator from the chemical library, exhibits protective effect against 6-OHDA-induced dopaminergic neuronal death through upregulation of HO-1 in astrocytes.