Rab7在阿尔茨海默病神经元变性过程中的作用

A lot of studies have implied that dysfunction in intracellular substance transportation and clearing is closely associated with formation of senile plagues（SP）, a characteristic feature of Alzheimer's disease（AD），but the exact mechanism remains poorly elucidated. Rab7 is a key factor regulating late endosome trafficking and autophagy. The central hypothesis of this application is that failed activation of Rab7 in AD neurons, causes pathological deposits of amyloid beta by eliciting abnormality of maturation, transportation, and clearing of endosomes and autophagosomes. The approaches utilized here to test our hypothesis include immunohistochemistry, transmission electron microscopy, molecular biology, proteomics, 7.0 Tesla MRI scanning with use of lableled SP probe in SH-SY5Y cell line transfected with FAD gene and in transgenic mice. It is our expectation that activated GTP-Rab7 will ameliorate deposits of SP and degeneration of neurons in AD brain by improving maturation and function of late endosomes, competing with Rubicon to release UVRAG and stimulating binding of UVRAG to Beclin1 to induce autophagy, and accelarating RLIP and FYCO1-mediated transportation of endosomes and autophagic vacuoles. This research is significant because it will provide insights into mechanisms of pathogenesis of AD and identify a novel therapeutic target for AD.

研究显示细胞内物质转运及清除障碍与AD病理标志老年斑（SP）形成相关，但具体机制尚不明确。Rab7是调节晚期内体转运与自噬的重要蛋白。本项目将以FAD基因转染的SH-SY5Y细胞和转基因小鼠为对象，采用免疫组织/细胞化学、免疫电镜、分子生物学、蛋白组学、行为学和自主研发的SP分子探针标记后7.0T MRI扫描等方法验证我们的假说：在AD神经元中存在Rab7的活化障碍，导致内体和自噬小体的成熟、转运和清除异常，最终引起Aβ病理性沉积而致病。以慢病毒为载体过表达激活的GTP-Rab7将通过以下途径减少SP沉积和神经元变性死亡：1.促进内体成熟、改善晚期内体功能；2.与Rubicon竞争性结合释放UVRAG，使UVRAG与Beclin1结合促进自噬诱导；3.通过RILP和FYCO1加速晚期内体和自噬囊泡转运。本研究的完成有助于阐明AD的发病机制并为这一难治性疾病的治疗提供新靶标。

研究显示细胞内物质转运及清除障碍与AD病理标志老年斑（SP）形成相关，Rab7作为调节晚期内体转运与自噬的重要蛋白,其在AD病理形成中所扮演的角色和其具体作用机制目前尚不清楚。通过慢病毒介导的转基因技术构建AD细胞模型和AD转基因模型小鼠APP/PS1研究发现，A-beta与Rab7介导的内吞－自噬通路蛋白存在共定位关系，提示Rab7介导的内吞－自噬通路参与了A-beta的代谢过程。然而Rab7在皮层和海马中的表达存在差异，同时在这些部位中作为Rab7激活者的UVRAG-Beclin1共表达情况与Rab7的改变一致，因此我们推测Rab7及其介导的内吞－自噬通路相关蛋白的表达在AD病理的发生发展过程中可能是动态变化的，其状态与神经元所处的变性阶段有关：在AD病变早期Rab7介导的内吞－自噬通路激活，APP通过该通路代谢，使A-beta产生增加、沉积；到病程晚期，由于该通路的过度激活和自噬结构的堆积，Rab7耗竭而造成晚期内体、自噬体与溶酶体的融合障碍，出现A-beta清除障碍、神经元变性脱失。基于以上结果，我们采用携带活化Rab7的慢病毒侧脑室注射，使5xFAD小鼠脑中过表达活化Rab7，观察到了活化Rab7对AD小鼠行为学和脑组织糖代谢的改善，提示活化的Rab7可能通过促进内体成熟、改善晚期内体功能、自噬囊泡的转运和清除而改善AD的临床表型。Rab7有可能成为今后治疗AD的新靶点。本研究的完成有助于阐明AD及其相关神经系统变性的发病机制，为其治疗提供理论依据。