RND3在神经干细胞增殖调控中的作用及机制研究

Alzheimer's disease (AD) is the most common Dementia in humans. Motivation of endogenous neural stem cells (NSCs) into newly derived neurons provides new and potential for AD treatment. Understanding the fundamental mechanism of NSCs proliferation and differentiation becomes critical and challenging. In our preliminary study, we found that genetic deletion of a small GTPase RND3 gene in mice led to significant increases in NSC numbers at the subventricular zone (SVZ), suggesting that RND3 may be a new factor that regulates NSCs proliferation. Two aims are proposed: 1) To investigate the relationship between RND3 and NSCs proliferation. We have generated RND3 gene knockout mouse, and we propose to use this unique mouse model to study RND3 deficiency-medicated NSCs proliferation. First we will analyze the NSCs proliferation in vitro. Cell growth rate and molecular markers of proliferation will be assessed in RND3 knockout NSCs and the wild-type cells. Second, RND3 deficiency-mediated NSCs proliferation will be dissected in vivo as we conducted in our preliminary study. Finally, we will demonstrate that forcing RDN3 expression in NSCs will attenuate NSC proliferation. The outcome of the proposal will establish direct links between RND3 expression and NSCs cell proliferation. 2) To explore the molecular mechanism of RND3 deficiency-mediated NSCs cell proliferation. Our recent study found that RND3 is a new regulator of Notch1 signaling. RND3 deficiency results in decreased Notch intracellular domain (NICD) protein degradation through repressing NICD protein ubiquitination. This eventually enhances Notch signaling (PNAS). It is known that upregulation of Notch signaling facilitates NSCs proliferation. In this study, we propose that RND3 functions as a new factor that regulates Notch complex in NSCs cell proliferation. Using loss- and gain-of-function approaches, we will demonstrate that RND3 manipulates NSCs cell proliferation partially through Notch signaling. Our RND3 knockout mice are a unique mouse model for the proposed mechanistic study. The innovation of proposal includes 1) establishment of RND3 as a new factor in NSC proliferation regulation; 2) elucidation of RND3 downregulation→NICD→Notch signaling→NSCs proliferation; 3) implication of manipulating RND3 expression levels as a potential therapeutic target. Since RND3 regulates NSCs proliferation, and facilitating NSCs proliferation is a promising approach for AD treatment, the result from this project has clear basic and clinical significance, and will provide new information for AD treatment.

本课题将研究RND3基因在神经干细胞（NSCs）增殖中的作用和机制。我们前期工作发现RND3可以抑制神经系统室管膜细胞增殖，RND基因敲除小鼠大脑此区NSCs细胞多于对照组，提示RND3参与NSCs细胞的增殖调控。机制实验揭示RND3介导的抑制效应可能与Notch1信号通路有关。并证实，RND3可以与Notch1活性形式NICD结合进而降低细胞NICD蛋白水平。因此我们提出假说：RND3抑制NSCs细胞增殖， 其机制是通过降低细胞内NICD蛋白水平，进而抑制Notch1信号通路。具体实验包括：1）体外实验证实RND3上调或敲低可抑制或促进细胞增殖；2）利用RND3基因敲除小鼠体内实验探讨RND3与NSCs细胞增殖关系；3）体内外实验探讨RND3在NSCs细胞增殖及Notch1信号通路中作用与机制。

目的：探讨RND3在神经干细胞增殖的调控及其相关机制。.方法：我们首先进行免疫荧光染色检测RND3基因敲出小鼠(HOMO)及对照组小鼠(WT)脑组织SVZ区Histone H3磷酸化水平、nestin表达变化。构建高表达和低表达RND3的四个神经干细胞系：Myc、Myc-RND3、siCtrl和siRND3；免疫印记技术检测细胞内Histone H3磷酸化水平。免疫印记技术检测Myc、Myc-RND3、siCtrl、siRND3四种NSCs内Notch1、HES1蛋白表达情况；同时采用应用免疫荧光染色检测RND3基因敲出小鼠(HOMO)及对照组小鼠(WT)脑组织SVZ区Notch1、Hes1表达变化。最后应用免疫印迹技术和rt-PCR检测AD(APP/PS1转基因)小鼠及对照组小鼠（WT）脑组织RND3蛋白及mRNA水平的变化；免疫荧光染色检测AD小鼠脑组织RND3水平变化。.结果：免疫荧光染色提示HOMO组小鼠SVZ区Histone H3磷酸化水平、nestin表达较WT组明显升高（p<0.05）。Myc-RND3组Histone H3磷酸化水平及Notch1、Hes1蛋白水平明显降低，较对照组相比，差异有统计学意义，（p<0.05），而siRND3组Histone H3磷酸化水平及Notch1、Hes1蛋白水平明显升高，较sictrl组相比，差异有统计学意义（p<0.05），免疫荧光染色提示HOMO组小鼠SVZ区Notch1及HES1表达水平较WT组明显升高（p<0.05）。APP/PS1小鼠脑组织RND3蛋白表达较WT组增多，但mRNA水平无明显变化。免疫荧光染色提示AD小鼠脑组织RND3表达较对照组增多。.结论：RND3通过调节Notch1信号通路来调控神经干细胞增殖。