Abstract：
DARPP-32 (dopamine and cAMP-regulated phosphoprotein Mr. 32 kDa) is a phosphoprotein that is modulated by multiple receptors integrating intracellular pathways and playing roles in various physiological functions. It is regulated by dopaminergic receptors through the cAMP/protein kinase A (PKA) pathway, which modulates the phosphorylation of threonine 34 (Thr34). When phosphorylated at Thr34, DARPP-32 becomes a potent protein phosphatase-1 (PP1) inhibitor. Since dopamine is involved in the development of GABAergic neurons and DARPP-32 is expressed in the developing brain, it is possible that DARPP-32 has a role in GABAergic neuronal development. We cloned the zebrafish darpp-32 gene (ppp1r1b) gene and observed that it is evolutionarily conserved in its inhibitory domain (Thr34 and surrounding residues) and the docking motif (residues 7-11 (KKIQF)). We also characterized darpp-32 protein expression throughout the 5 days post-fertilization (dpf) zebrafish larval brain by immunofluorescence and demonstrated that darpp-32 is mainly expressed in regions that receive dopaminergic projections (pallium, subpallium, preoptic region, and hypothalamus). We demonstrated that dopamine acutely suppressed darpp-32 activity by reducing the levels of p-darpp-32 in the 5dpf zebrafish larval brain. In addition, the knockdown of darpp-32 resulted in a decrease in the number of GABAergic neurons in the subpallium of the 5dpf larval brain, with a concomitant increase in the number of DAergic neurons. Finally, we demonstrated that darpp-32 downregulation during development reduced the motor behavior of 5dpf zebrafish larvae. Thus, our observations suggest that darpp-32 is an evolutionarily conserved regulator of dopamine receptor signaling and is required for the formation of GABAergic neurons in the developing telencephalon.
摘要：
DARPP-32（多巴胺和cAMP调节的磷蛋白Mr.32kDa）是一种磷蛋白，由整合细胞内途径并在各种生理功能中发挥作用的多种受体调节。它由多巴胺能受体通过cAMP/蛋白激酶A（PKA）途径调节，其调节苏氨酸34(Thr34)的磷酸化。当在Thr34磷酸化时，DARPP-32成为有效的蛋白磷酸酶-1（PP1）抑制剂。由于多巴胺参与GABA能神经元的发育，而DARPP-32在发育中的大脑中表达，DARPP-32可能在GABA能神经元发育中起作用。我们克隆了斑马鱼darpp-32基因（ppp1r1b）基因，并观察到它在其抑制域（Thr34及其周围残基）和对接基序（残基7-11（KKIQF））中在进化上是保守的。我们还通过免疫荧光对整个受精后5天（dpf）斑马鱼幼虫脑的darpp-32蛋白表达进行了表征，并证明darpp-32主要在接受多巴胺能投射的区域表达（pallium，亚镓,视前区,和下丘脑）。我们证明，多巴胺通过降低5dpf斑马鱼幼虫大脑中p-darpp-32的水平来急剧抑制darpp-32的活性。此外,darpp-32的击倒导致5dpf幼虫脑下皮层中GABA能神经元的数量减少，伴随着DAer能神经元数量的增加。最后,我们证明了darpp-32在发育过程中的下调降低了5dpf斑马鱼幼虫的运动行为。因此，我们的观察表明,darpp-32是一种进化上保守的多巴胺受体信号调节因子,是发育中的端脑GABA能神经元形成所必需的.
