Propofol impairs specification of retinal cell types in zebrafish by inhibiting Zisp-mediated Noggin-1 palmitoylation and trafficking

Background: Propofol can have adverse effects on developing neurons, leading to cognitive disorders, but the mechanism of such an effect remains elusive. Here, we aimed to investigate the effect of propofol on neuronal development in zebrafish and to identify the molecular mechanism(s) involved in this pathway.Methods: The effect of propofol on neuronal development was demonstrated by a series of in vitro and in vivo experiments. mRNA injections, whole-mount in situ hybridization and immunohistochemistry, quantitative real-time polymerase chain reaction, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, 5-ethynyl-2'-deoxyuridine labeling, co-immunoprecipitation, and acyl-biotin exchange labeling were used to identify the potential mechanisms of propofol-mediated zisp expression and determine its effect on the specification of retinal cell types.Results: Propofol impaired the specification of retinal cell types, thereby inhibiting neuronal and glial cell formation in retinas, mainly through the inhibition of Zisp expression. Furthermore, Zisp promoted the stabilization and secretion of a soluble form of the membrane-associated protein Noggin-1, a specific palmitoylation substrate.Conclusions: Propofol caused a severe phenotype during neuronal development in zebrafish. Our findings established a direct link between an anesthetic agent and protein palmitoylation in the regulation of neuronal development. This could be used to investigate the mechanisms via which the improper use of propofol might result in neuronal defects.

背景：异丙酚（Propofol）可对发育中的神经元产生不良影响，进而引发认知障碍，但其具体作用机制仍不明确。本研究旨在探究异丙酚对斑马鱼（zebrafish）神经元发育的影响，并阐明该通路中涉及的分子机制。方法：本研究通过一系列体外（in vitro）与体内（in vivo）实验验证了异丙酚对神经元发育的影响。采用mRNA显微注射、整体原位杂交、免疫组织化学、实时定量聚合酶链式反应（quantitative real-time polymerase chain reaction）、脱氧核糖核苷酸末端转移酶介导的dUTP缺口末端标记（terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling，TUNEL）、5-乙炔基-2'-脱氧尿苷（5-ethynyl-2'-deoxyuridine）标记、免疫共沉淀（co-immunoprecipitation）以及酰基生物素交换标记（acyl-biotin exchange labeling）等技术，阐明异丙酚介导Zisp表达的潜在机制，并明确其对视网膜细胞类型特化的调控作用。结果：异丙酚可损害视网膜细胞类型特化过程，进而抑制视网膜内神经元与神经胶质细胞的生成，该作用主要通过抑制Zisp表达实现。进一步研究发现，Zisp可促进膜相关蛋白Noggin-1（一种特异性棕榈酰化底物）的可溶性形式的稳定与分泌。结论：异丙酚可导致斑马鱼神经元发育出现严重异常表型。本研究结果证实，麻醉剂与蛋白质棕榈酰化（protein palmitoylation）在神经元发育调控中存在直接关联，该发现可为探究异丙酚不当使用引发神经元缺陷的潜在机制提供研究思路。