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.

Keywords: High-throughput screening, luciferase reporter assay, neurodegeneration, neuroglobin (Ngb), neuroprotection, Ngb promoter, primary neuron.

Graphical Abstract