Tacrine‐6‐Ferulic Acid, a Novel Multifunctional Dimer Against Alzheimer's Disease, Prevents Oxidative Stress‐Induced Neuronal Death Through Activating Nrf2/ARE/HO‐1 Pathway in HT22 Cells

Abstract

Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder with progressive and devastating memory impairment 1. The majority of therapeutic strategies and drug development approaches for AD were based on dysfunction of acetylcholine to date, which mainly alleviate symptoms of AD 1. A great amount of researches proposed that oxidative stress (OS) be an important and early factor in the pathogenesis of AD and contribute to β-amyloid generation 2. Nuclear factor E2-related factor 2 (Nrf2) is a novel transcription factor known to induce expression of a variety of cytoprotective and detoxification genes. Increasing evidence demonstrated that Nrf2/ARE pathway may be considered a therapeutic target for neurodegenerative diseases, including AD 3. We have previously synthesized and proved that tacrine-6-ferulic acid (T6FA; Figure 1), a novel multifunctional dimer, can prevent amyloid-β peptide-induced AD-associated pathological changes, including anti-OS, in vitro and in vivo 4. But the molecular mechanism(s) underlying the neuroprotective effects of T6FA is still not fully elucidated. HT22 cells, an immortalized and ionotropic glutamate receptors-free mouse hippocampal cell line, have been widely used as an in vitro model for elucidating the mechanism of OS-induced neurotoxicity 5. In this study, HT22 cells were used to examined the potential effects of T6FA on the OS-induced cell death as well as the role of Nrf2/ARE pathway. HT22 cells were exposed to 5 mM glutamate in the absence or presence of T6FA (0- 30 μM). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to examine cell viability. H2-DCF-DA staining was used to monitor the ROS level. Immunocytofluorescence and confocol microscopy were used to monitor the Nrf2 translocation. Antioxidant response element (ARE)-luciferase reporter system was used to measure the ARE activity, and Eastern blot was used to assay the HO-1 protein level. Data were statistically analyzed by one-way ANOVA. Glutamate significantly induces cell death, and T6FA blocks the cell death in a concentration-dependent manner (Figure 1A). T6FA (30 μM) potently attenuate the ROS level induced by glutamte (Figure 1B). Immunocytofluorescence experiment showed that T6FA dramatically provoke the Nrf2 nuclear translocation (Figure 2A). In addition, T6FA activated the ARE-dependent transcription (Figure 2B) and also up-regulated the expression of heme oxygenase-1 (HO-1) in a time- and concentration-dependent manner (Figure 2C). The neuroprotective effects can be blocked by zinc protoporphyrin IX (ZnPP) (1 μM), an inhibitor of HO-1 (Figure 2D). This study showed that T6FA potently prevent OS-induced cell death in HT22 cells and activate Nrf2/ARE pathway and up-regulate the expression of HO-1. Modulation of Nrf2 has been shown in several neurodegenerative diseases, including AD 3, 6. Targeting Nrf2 pathway has become a potential therapeutic avenue for these diseases 6. The expression of phase II detoxification enzymes is governed by the cis-acting regulatory element known as ARE. The transcription factor Nrf2 binds to ARE thereby transcribing multitude of antioxidant genes to protect against OS in neurodegenerative diseases 6. Our findings support that T6FA confer neuroprotection through activating Nrf2/ARE pathway, suggesting that T6FA may be a promising multifunctional drug candidate for neurodegenerative diseases, not only AD. This research was supported by grants from Fundamental Research Funds for the Central Universities (No. 10ykpy23) to R. Pi and the Scientific and Technology Foundation of Guangdong Province (2011B061300049) to W. Huang. The authors declare no conflict of interests.