FoxO转录因子对神经软骨蛋白 (neurochondrin）的作用及机制研究

Forkhead box O (FoxO) transcription factors (FoxO1, FoxO3a, FoxO4 and FoxO6) are transcription factors playing important roles in cellular metabolism, proliferation, differentiation and survival. Our recent study has shown that FoxO3a inhibits neuronal differentiation stimulated by NGF in PC12 cells. Preliminary results from DNA microarray analysis and quantitative RT-PCR demonstrated that the overexpression of FoxO3a significantly attenuated the mRNA level of neurochoondrin (NCDN). Bioinformatic studies found that the regulatory region of the NCDN promoter contained 4 FoxO3a binding sites. Dual-Luciferase reporter assay with a reportor gene containing the NCDN promoter showed that FoxO3a significantly decreased the transcription activity of the NCDN promoter. These results suggest that NCDN may be a new direct target of FOXO3a. Knock-down of NCDN by siRNA blocked NGF-induced neuronal differentiation of PC12 cells while overexpression of NCDN had the opposite effect indicating that NCDN has an important role in neuronal differentiation. In this project, we will use transfection, PCR, siRNA, dual-Luciferase reporter assays, EMSA and ChIP in PC12 cells, primary neurons and transgene mice to study the effect and underlying mechanisms of FoxO transcription factor on neuronal differentiation genes like NCDN. The aims of this study(FoxO3a as example) are to investigate 1) the effect of FoxO3a on the expression of NCDN and its underlying machanism and to verify if NCDN is a new target of FoxO3a (ii) the role of NCDN in neuronal differentiation and its underlying mechanisms and (iii) to determine if NCDN is involved in the inhibitory effect of FoxO3a on the differentiation of PC12 induced by NGF. Our study will deepen the understanding of the mechanisms of neuronal differentiation and provide new evidence for FoxO and its downstream targets as new therapeutic targets for neurodegenative diseases.

FoxO转录因子广泛参与细胞的代谢、增殖、分化及凋亡等的调控，是当今生物科学的研究热点。我们最近研究发现FoxO3a可抑制神经细胞的分化但机制未明。初步结果显示FoxO3a抑制神经分化蛋白neurochondrin（NCDN）等基因的表达。NCDN启动子序列分析发现NCDN含有FoxO结合位点,报告基因分析也证明FOXO3a能抑制NCDN的转录活性提示NCDN可能是参与FOXO3a抑制分化的一个新下游靶标。SiRNA敲除NCDN抑制NGF诱导神经分化而过表达NCDN则促进PC12分化提示了它在分化中的重要作用。本项目拟采用电转染、PCR、siRNA、荧光报告基因、EMSA和ChIP分析等方法在PC12/RGC-5，神经元及转基因鼠研究FoxO对NCDN等基因的作用和机制及这些基因在神经分化中的作用。研究将为FoxO及其下游靶标作为神经退行性疾病治疗新靶点及神经细胞分化机制提供依据。

FoxO3a 是FoxO转录因子家族的成员之一，广泛参与细胞的增殖、分化、代谢及凋亡的调控。最近研究证明FoxO参与了多种非神经细胞分化的调控，但FoxO3a对神经细胞分化的作用及作用机制则不清楚。本项目研究了 FoxO转录因子对神经细胞分化, 神经软骨蛋白 (neurochondrin）的作用及机制。我们的结果证明 (1)FoxO3a抑制NGF诱导的神经分化和NCDN的表达; (2) NCDN是FoxO3a的直接下游靶基因; (3) NGF促进PC12细胞及原代培养神经元表达NCDN, 其作用是由TrkA/Akt/Foxo3a等信号通路调节的; (4) NCDN在NGF诱神经分化的作用中起重要的作用; (5) GAP-43, 一个和轴突生长相关的重要蛋白分子, 可能是Foxo3a的另一新靶标。Foxo3a 抑制NCDN 和GAP43的表达; NCDN则促进GAP-43的表达, 提示NCDN是GAP-43的上游调节蛋白, NCDN的作用可能通过/部分通过GAP-43起效。另外, 我们建立了Foxo3a及Foxo1的转基因鼠的动物模型，进一步证明了体内高表达Foxo3a能抑制NCDN的表达。行为分析初步结果证明高度表达Foxo3a转基因鼠能损害小鼠的学习记忆功能。这些发现将有助于为FoxO及其下游靶标作为神经退行性疾病治疗新靶点及神经细胞分化机制提供依据。.发表学术论文25篇，其中影响因子5分以上文章有10篇, 通讯作者文章24篇。获邀参编Encyclopedia of Signaling Molecules (2nd Edition)百科全书, 负责编写FoxO转录因子。在中山大学申请发明专利二个，获得授权一个。获邀在美国，加拿大，香港，澳洲和中国召开的国际会议上作口头演讲，分会主席及主题演讲20多次 (Foxo专题演讲>10次) 。积极参加国内外学术合作交流, 组织邀请国內外著名学者在中山大學，澳门大学学术讲座30多次。邀请外国著名学者为学生上课并组织学生和外国学者一起讨论。 毕业博士研究生2名，硕士研究生2名。