| Objective: Neural progenitor cells(NPCs) are self-renewing and multipotent cells that generate neurons, astrocytes and oligodendrocytes in the central nervous system(CNS). NPCs proliferate, differentiate, migrate and eventually integrate into the neural network. The abnormalities in any of these processes will cause dysfunctions of the brain and leads to neurological diseases such as schizophrenia, autism, Alzheimer’s disease. Recent studies indicate patients with major depressive disorder(MDD) or bipolar affective disorder(BPAD) also exhibit an abnormal proliferation and differentiation of NPCs, indicating a function of NPCs in the pathogenesis of MDD and BPAD.G protein-coupled receptor 50(GPR50), which is also known as H9 or melatonin-related receptor, is an X-linked orphan G protein-coupled receptor. A recent study indicates that GPR50 is a genetic risk factor for MDD and BPAD which are associated with abnormality of cortical development in females. GPR50 is expressed in the pituitary, hypothalamus and hippocampus of adult mammalian brain. Moreover,expression of GPR50 in the developing mouse brain starts at embryonic day 13(E13),peaks at E18. These lines of evidence suggest a potential role of GPR50 in brain development. However, the function and mechanism of GPR50 in the brain remains unclear. In this study, we focus on the function and mechanism of GPR50 in proliferation and differentiation about neural progenitor cells.Methods: We investigated the expression of GPR50 in cultured NPCs. NPCs were isolated from the lateral ventricle walls of E14 C57/BL6 J mice and cultured 3-4 days for immunofluorescent staining, dissociated cells from primary neurospheres wereseeded into 24-well dishes and were cultured in DMEM-F12 culture medium containing B27, epidermal growth factor(EGF) and basic fibroblast growth factor(b FGF) for 3-4days, cultured NPCs were transfected with 200 n M si RNA per cuvette using nucleofector system(Lonza) according to the protocol provided in the Amaxa Nucleofector kit. For neurospheres formation, NPCs were cultured at a density of 500cells/well in 96-well plates for 8 days. The numbers of neurospheres per well were counted. For quantification of neurosphere size, NPCs were cultured at a density of2×104cells/well in 24-well plates for 8 days. For differentiation, transfected cells were seeded into 24-well dishes and were cultured in DMEM/F12 culture medium containing B27 and 0.5% fetal bovine serum(FBS, Gibco) for 5 days. We investigated the expression levels of gene related to proliferation and differentiation of neural stem cell on transcription level through real-time Quantitative polymerase chain reaction(q-PCR)and luciferase.Results:1) GPR50 is expressed in neural progenitor cells in the ventricular zone.2) Knockdown of GPR50 with si RNA decreased self-renewal of NPCs.3) Knockdown of GPR50 with si RNA showed no effect on astroglial differentiation of NPCs.4) Knockdown of GPR50 with si RNA increased hes1 expression level and deseased TCF7L2, Ngn1, Ngn2 and cyclin D1 espression levels.s5) The transcriptional activity of the intracellular domain of notch on hes1 gene was repressed by overexpression of GPR50.Conclusion: GPR50 is expressed in neural progenitor cells in the ventricular zone.It promotes self-renewal of NPCs, and shows no effect on astroglial differentiation of NPCs. The transcriptional activition of the intracellular domain of notch on hes1 gene was repressed by overexpression of GPR50. In addition, decreased levels of transcription factor 7-like 2(TCF7L2) m RNA was observed in GPR50 si RNA-transfected NPCs, suggesting that knockdown of GPR50 impairs wnt/β-catenin signaling. Moreover, the m RNA levels of neurogenin(Ngn) 1, Ngn2 and cyclin D1, the target genes of notch and wnt/β-catenin signalings, in NPCs were reduced byknockdown of GPR50. Therefore, GPR50 promotes self-renewal and neuronal differentiation of NPCs possibly through regulation of notch and wnt/β-catenin signalings. |
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