Aim1在视网膜感光细胞纤毛形成中的作用及机制研究

Retina dystrophy causes the majority of blindness worldwide. Defects in ciliogenesis and cilium function of retina photoreceptor lead to retina dystrophy. The molecular mechanism regarding to ciliogenesis of photoreceptor still under covered. In our previous studies, we identified the Aim1 gene which specifically high expressed in retina. In vitro studies suggested that Aim1 may act as a tumor suppressor. However, little is known about its function during organ development. Our data suggested that Aim1 predominantly expressed in OS of retina photoreceptor. In mammalian cells, knockdown of Aim1 by siRNA reduced the primary cilia length which induced by starvation. Aim1 interacted with α-tubulin and the interaction increased when cells were induced with starvation. We speculated that Aim1 is involved in protein traffic as cytoskeleton component during photoreceptor ciliogenesis. Meanwhile, we constructed Aim1 knockout mice using CRISPR-cas9 technology and identified Aim1-/- mice. In this project, with Aim1-/- mice and ARPE-19 cell line, we aim to study 1) the function of Aim1 in ciliogenesis and function maintenance of photoreceptor; 2) the molecular mechanism of microtubule-dependent cilia protein traffic regulated by Aim1. This project would explain the role of Aim1 in photoreceptor ciliogenesis and function, and shed some light on the understanding of retina dystrophy pathogenesis.

视网膜变性类疾病是世界范围内致盲的重要原因。感光细胞纤毛的生成和功能障碍是导致该疾病发生的主要原因。我们前期工作中发现一个视网膜特异性高表达的基因Aim1。目前对Aim1功能研究仅限于其具有抑癌作用，但Aim1在机体正常发育中的功能未知。我们前期工作发现Aim1在小鼠感光细胞内节中高表达，siRNA敲低Aim1导致细胞诱导的纤毛变短。Aim1与α-tubulin相互作用，并在纤毛诱导时结合增强。因此我们推测Aim1作为微管结合蛋白调控感光细胞纤毛的生成。本项目中我们利用CRISPR-Cas9技术构建Aim1敲除小鼠（已鉴定出纯合敲除鼠），并利用视网膜色素上皮细胞系（ARPE-19）分别从体内、体外探究：1) Aim1在视网膜感光细胞纤毛生成中的作用；2) Aim1与微管互作调控纤毛蛋白转运的分子机制。该项目将阐述Aim1在视网膜发育过程中的作用，为了解视网膜变性疾病的发病机制提供新的思路。

Aim1属于β/γ-crystallin结构域蛋白家族成员，目前已报道的研究较少。在前期工作基础中，我们检测发现Aim1在小鼠视网膜中高表达，因此构建Aim1敲除小鼠品系，研究Aim1在视网膜发育中的功能。后续分析中，我们发现Aim1敲除小鼠视网膜分层正常，光感受器无明显退行性病变发生。电镜结果提示视锥、视杆细胞外界膜盘发育正常。鉴于与小鼠相比，斑马鱼的视网膜感光细胞组成和结构与人更为相似，我们利用CRISPR-Cas9技术构建Aim1敲除斑马鱼品系。我们研究发现，和小鼠结果类似，Aim1敲除斑马鱼的视网膜发育正常，暂无退行性病变发生。在研究过程中，我们发现Aim1表达于斑马鱼侧线神经丘中。利用新霉素处理的毛细胞再生模型，我们确定Aim1可通过负调控细胞周期蛋白依赖性激酶抑制剂p27、p21和p57的表达，参与神经丘毛细胞的再生过程中。该发现为研究哺乳动物耳蜗毛细胞再生提供新的理论基础.