β-arrestin 2 and Epac2 cooperatively mediate DRD1-stimulated proliferation of human neural stem cells and growth of human cerebral organoids.

G protein coupled receptors (GPCRs) reportedly relay specific signals, such as dopamine and serotonin, to regulate neurogenic processes though the underlying signaling pathways are not fully elucidated. Based on our previous work which demonstrated Dopamine receptor D1 (DRD1) effectively induces the proliferation of human neural stem cells, here we continued to show the knockout of β-arrestin 2 by CRISPR/Cas9 technology significantly weakened the DRD1-induced proliferation and neurosphere growth. Furthermore, inhibition of the downstream p38 MAPK by its specific inhibitors or small hairpin RNA mimicked the weakening effect of β-arrestin 2 knockout. In addition, blocking of Epac2, a PKA independent signal pathway, by its specific inhibitors or small hairpin RNA also significantly reduced DRD1-induced effects. Simultaneous inhibition of β-arrestin 2/p38 MAPK and Epac2 pathways nearly abolished the DRD1-stimulated neurogenesis, indicating the cooperative contribution of both pathways. Consistently, the expansion and folding of human cerebral organoids as stimulated by DRD1 were also mediated cooperatively by both β-arrestin 2/p38 MAPK and Epac2 pathways. Taken together, our results reveal that GPCRs apply at least two different signal pathways to regulate neurogenic processes in a delicate and balanced manners.