Midbrain dopaminergic (DAergic) neurotransmission plays a crucial role in regulating motor, cognitive, and emotional functions. The orphan nuclear receptor estrogen-related receptor gamma (ERRγ) is highly expressed in the adult brain and in the developing fetal brain. Our previous study showed the relevance of ERRγ in the regulation of the DAergic neuronal phenotype with the upregulation of dopamine synthesizing tyrosine hydroxylase (TH) and dopamine transporter (DAT) and the possibility that ERRγ could be a novel target for regulating DAergic neuronal differentiation. In this study, we examined whether ERRγ ligands could be small molecule regulators of DAergic phenotypes. The ERRγ agonist GSK4716 increased DAT and TH expression, and the ERRγ inverse agonist GSK5182 attenuated the retinoic acid-induced upregulation of DAT and TH in differentiated SH-SY5Y cells. We found that biphasic activation of the protein kinase A/cyclic AMP response element-binding (CREB) protein signaling pathway was involved in the GSK4716-induced increase in the DAergic phenotype in SH-SY5Y cells. CREB signaling activated as early as 3 h after GSK4716 treatment in an ERRγ-independent manner, but increased following ERRγ activation after 3 days. Protein kinase A inhibitor H-89 attenuated GSK4716-induced DAT and TH upregulation. In primary cultured DAergic neurons, GSK4716 increased neurite length and the number of DAT and TH-double-positive (DAT+TH+) neurons compared to that in control cells. These findings suggest that ERRγ ligands could serve as useful chemical tools for obtaining a better understanding of the regulation of DAergic phenotypes and might facilitate the development of small molecule therapeutics to treat DA-related neurological diseases.

中脑多巴胺能（DAergic）神经传递在调节运动，认知和情绪功能中起关键作用。孤儿核受体雌激素相关受体γ（ERRγ）在成年大脑和发育中的胎儿大脑中高度表达。我们先前的研究表明ERRγ与多巴胺合成酪氨酸羟化酶（TH）和多巴胺转运蛋白（DAT）上调的调控与DAergic神经元表型的相关性，以及ERRγ可能成为调节DAergic神经元分化的新靶标的可能性。在这项研究中，我们检查了ERRγ配体是否可能是DAergic表型的小分子调节剂。ERRγ激动剂GSK4716增加DAT和TH表达，ERRγ反向激动剂GSK5182减弱了视黄酸诱导分化的SH-SY5Y细胞中DAT和TH的上调。我们发现，蛋白激酶A /环AMP反应元件结合（CREB）蛋白信号通路的双相激活参与了GSK4716诱导的SH-SY5Y细胞DAergic表型的增加。CREB信号最早在GSK4716治疗后3小时以ERRγ无关的方式激活，但在ERRγ激活3天后增加。蛋白激酶A抑制剂H-89减弱了GSK4716-诱导的DAT和TH上调。与对照细胞相比，在原代培养的DAergic神经元中，GSK4716增加了神经突长度，并增加了DAT和TH双阳性（DAT + TH +）神经元的数量。