Screening Of ChAT Gene Expression Modulators From Small-molecule Compounds And Study Of The Modulation Mechanisms

Currently, the induction of neural stem cells differentiation in the life scienceshas become the most hot spots. With the development of neural stem cell research,induction of neural stem cells differentiation and transplantation therapy is becomingpossible. Study of the differentiation mechanisms in vitro or in vivo, finding thetherapeutic targets to develop new treatments have become key issues of modernmedical and biological research.ACh has been shown to regulate many physiologic functions within the centralnervous system (CNS), including cognition, attention, and arousal. Cholinergicneuron dysfunction plays a role in age-related memory impairments and in the onsetof Alzheimer’s disease.The synthesis of acetylcholine (ACh) is catalyzed by cholineacetyltransferase, a rate-limiting enzyme that transfers an acetyl group from acetylcoenzyme A to choline. In view of ChAT is an important marker of nerve,we usedChAT promoter as the target for drug screening, in order to find compounds in thechemical compound library that can promote the ChAT promoter transcriptionactivity.In this study, we first constructed the pGL3-ChAT promoter luciferase reportervector and transfected into P19and HEK293T cells. The results of luciferase activityshown that the ChAT promoter had high activity. Then we screened the chamicalcompound library by using this luciferase reporter system, and found that TI5canpromote the activity of ChAT promoter. Further results revealed that TI5alsoincreased the expression of ChAT in both mRNA and protein levels. we thenresearched the molecular mechanisms of TI5promoting ChAT expression byapplication of the transcription factor responsive luciferase reporter assay and westernblot method, the result shown that TI5can significantly activated ERK/MAPKsignaling pathway.Overall, our results suggest that TI5can promote the expression of ChAT gene,and the ERK/MAPK pathway may involved in this process, and TI5may be anexcellent lead candidate for induction of cholinergic neuron differentiation.