title: Establishment of Cell-Based Neuroglobin Promoter Reporter Assay for Neuroprotective Compounds Screening.
abstract: Neuroglobin (Ngb) has been demonstrated to be neuroprotective against stroke and neurodegenerative diseases, thus upregulating Ngb might be a novel approach for neuroprotection. In this study we aimed to establish cell-based Ngb reporter systems for screening neuroprotective compounds targeting Ngb upregulation. We developed both mouse and human stable Ngb reporter systems containing a luciferase reporter gene directed by mouse and human Ngb promoter, respectively. To validate these reporter systems, we used them to screen a pool of natural plant compounds. RT-PCR was used to verify the Ngb-upregulating effects of selected compounds, and neurotoxicity assay was used to test their neuroprotection effects in primary cultured neurons. We identified polydatin, genistein, daidzein, biochanin A and formononetin that can upregulate both mouse and human Ngb promoter activity. RT-PCR confirmed that polydatin, genistein and formononetin significantly increased Ngb mRNA expression in primary neurons. Furthermore, formononetin significantly decreased oxygen-glucose deprivation (OGD)-induced neurotoxicity. Moreover, inhibition of cAMP response element-binding protein (CREB) showed that CREB is required for formononetin-induced Ngb upregulation. These results suggest that these Ngb reporter systems are suitable for neuroprotective compound screening, which will be used to screen larger compound libraries for more potent neuroprotectants. This preliminary study will facilitate the development of Ngb-targeted therapeutics for stroke and neurodegenerative diseases.
authors.forename: Ning
authors.initials: N
authors.lastname: Liu
authors.forename: Zhanyang
authors.initials: Z
authors.lastname: Yu
authors.forename: Xiumei
authors.initials: X
authors.lastname: Gao
authors.forename: Yun S
authors.initials: YS
authors.lastname: Song
authors.forename: Jing
authors.initials: J
authors.lastname: Yuan
authors.forename: Yu
authors.initials: Y
authors.lastname: Xun
authors.forename: Tingting
authors.initials: T
authors.lastname: Wang
authors.forename: Feng
authors.initials: F
authors.lastname: Yan
authors.forename: Shishan
authors.initials: S
authors.lastname: Yuan
authors.forename: Jian
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Protein Chemistry and Development Biology of State Education Ministry of China, College of Life Sciences, Hunan Normal University, Changsha, 410081, China. xshlin@hunnu.edu.cn.
authors.forename: Shuanglin
authors.initials: S
authors.lastname: Xiang
authors.forename: Eng H
authors.initials: EH
authors.lastname: Lo
authors.affiliation: Neuroprotection Research Laboratory, Departments of Neurology and Radiology, Massachusetts General Hospital, 149 13th Street, Room 2411A, Charlestown, MA 02129, USA. wangxi@helix.mgh.harvard.edu.
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Wang
mesh_terms: D000818:Animals; D015687:Cell Hypoxia / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005914:Globins / Q000235:genetics* / Q000378:metabolism; D005947:Glucose / Q000172:deficiency; D006801:Humans; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D000078787:Neuroglobin; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology*; D064209:Phytochemicals / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
chemical_list: C115163:CREB1 protein, human; C492004:Creb1 protein, mouse; D017362:Cyclic AMP Response Element-Binding Protein; D009419:Nerve Tissue Proteins; D000078787:Neuroglobin; D018696:Neuroprotective Agents; D064209:Phytochemicals; D012333:RNA, Messenger; D005914:Globins; D005947:Glucose