Constitutive activity of a G protein-coupled receptor, DRD1, contributes to human cerebral organoid formation

The intricate balance of neural stem cell (NSC) amplification and neurogenesis is central to nervous system development. Dopamine D1 receptor (DRD1) is a typical G protein-coupled receptor (GPCR) mainly expressed in neurogenic area, with high constitutive activity. The receptor appears in the embryonic period before the formation of mature synaptic contacts, which indicates that dopamine receptor and its constitutive activity play crucial roles in the embryonic brain development. Here, we found that DRD1 was enriched in human NSCs. Inhibition of the receptor activity by its inverse agonists promoted human NSCs proliferation and impeded its differentiation. These results were also mimicked by genetic knockdown of DRD1, which also blocked the effects of inverse agonists, suggesting a receptor-dependent manner. More interestingly, knock-in A229T mutant with reduced DRD1 constitutive activity by CRISPR-Cas9 genome editing technology resulted into increased endogenous human NSCs proliferation. These results were well reproduced in human cerebral organoids, and inhibition of the DRD1 constitutive activity by its inverse agonists induced the expansion and folding of human cerebral organoids. The anatomic analysis uncovered that decreasing the constitutive activity of DRD1 by its inverse agonists promoted the NSCs proliferation and maintenance that led to hindered cortical neurogenesis. Further mechanistic studies revealed that the PKC-CBP pathway was involved in the regulation by DRD1. Thus, our findings indicate that the constitutive activity of DRD1 and possibly other GPCRs plays an important role in the development of human nervous system.