Neurogenin2 (Ngn2), an activator-type bHLH transcriptional factor, promotes differentiation of neural stem cells into neurons by transcription of pro-neural genes. To find neural stem cell accelerators from the extract library of natural resources, we used a two-step screening including a Ngn2 promoter reporter gene screening and differentiation assay screening of neural stem cells. A reporter gene assay that can detect Ngn2 promoter activity by luciferase expression was constructed using C3H10T1/2 cells. Using this primary cell-based screening, Butea superba was found to include Ngn2 promoter activators from our tropical plant extract libraries. Bioassay-guided fractionation of this plant extract led to the isolation of 18 natural products, including pterocarpans and isoflavonoids. Dehydromaackiain (1), formononetin (6), (-)-variabilin (13), (-)-medicarpin (14), rothindin (17) and ononin (18) showed 1.8–2.8 times higher Ngn2 promoter activity at 5 mM compared with control. Of active natural compounds, 30-methoxydaidzein (3) showed promotion of neurite outgrowth of C17.2 in a secondary screen. 30-Methoxydaidzein (3) increased mRNA expression of pro-neural transcriptional factors (Ngn2, Ngn1, NeuroD2), a mature neuron-specific enzyme GAD1 and a pro-neural neurotrophic growth factor neurotrophin 3 (NT3) in C17.2 neural stem cells.
This study is the first example of a search for small molecules with focusing Ngn2 promoter activity. Considering the contribution and importance of natural pro-ducts for medicinal uses, investigating the naturally occurring modulators of bHLH transcriptional factors that control the fate of neural stem cells would be worthwhile for regenerative medicine.
Midori A. Arai, Kazune Koryudzu, Takashi Koyano, Thaworn Kowithayakornc and Masami Ishibashi
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