阿尔茨海默病郎飞结区Aβ生成及调控的分子机制研究

Alzheimer's disease （AD） is a progressive, irreversible brain disorder. Abnormalities in APP processing have been linked to AD. Understanding the mechanisms regulating the amyloidogenic processing of APP is therefore a central area of investigation in AD research. Substantial controversy remains over the sites of APP processing and Aβ release. Some Aβ release occurs at synapses, but the release of Aβ in primary neuronal cultures that lack synapses and the occurrence of plaques in white-matter tracts that lack synaptic input suggest that Aβ might be released from more-proximal sites too. Recently, we have found APP is specifically located at the CNS nodes of Ranvier, implicating a potential role for NORs in the Aβ release. At nodes of Ranvier, there exists molecule complex forming intercellular or extracellular interactions between neurons and glia that result in profound molecular process.Presumably APP and other nodal molecules will constitute a protein complex to function in this region. To explore the relationship of NORs with Aβ release, we will be using an APP/PS1 transgenic mice to study the Aβ release and deposition along perforant pathway by microdialysis. We will uncover the interaction network of APP with some nodal molecules, such as tenascin-R, Nogo receptor and its ligands, MAG, Nogo-66 and OMgp. Further, we will try to study if these molecules could modulate APP processing and Aβ release. We will cross Tenascin-R knockout mice with APP/PS1 transgenic mice to reduce endogenous level of Tenascin-R, then examining Aβ release and deposition in APP-PS1/Tn-R-/- mice. We will interfere expression of nodal molecules such as MAG,Nogo-66,OMgp to APP/PS1 transgenic mice by RNAi technique, thus examining Aβ release and deposition. Also we will check the effect of these interfering in learning and memory of APP/PS1 mice. This project will help to understand the pathogenesis of AD and provide novel approach for treatment of Alzheimer's disease.

淀粉样前体蛋白(APP)代谢异常形成的β-淀粉样肽(Aβ)是阿尔茨海默病(AD)老年斑的主要成分，延缓和阻止Aβ的产生和沉积是控制AD的关键。然而APP剪切产生Aβ的部位仍存在争议。我们前期研究表明：APP特异汇聚中枢神经纤维郎飞氏结，和其周围髓鞘分子构成功能上的复合体（图4-7）。介于AD发病早期主要表现为髓鞘损害，且前穿质通路为Aβ沉积的主要部位，我们推测除突触之外，郎飞氏结亦可能是Aβ产生的另一位点,调控郎飞氏结区Aβ的生成将是治疗AD的新靶点。本研究是在前期研究基础上，从分子、形态和功能等多方面，进一步探讨AD发病期间郎飞氏结区APP代谢异常与Aβ沉积的时空关系；寻找APP与髓鞘分子Tn-R,Nogo,MAG,OMgp的关联性；筛选调控郎飞氏结区APP降解、Aβ产生的关键因子。通过上述研究，阐明郎飞氏结处调控Aβ生成的分子机制，明确干预Aβ生成的靶点，为AD治疗提供新的方法和策略。

阿尔茨海默病（AD）是一种渐进性不可逆转的神经退行性病，其病理特征为细胞外Aβ聚集形成的老年斑和细胞内神经纤维缠结。延缓和阻止Aβ的产生和沉积是控制AD的关键。然而Aβ产生部位仍存在争议, 经典的观点认为Aβ形成于细胞器，释放于突触处。但此观点不能解释AD病人白质亦有A的沉积、髓鞘的损害。我们的研究发现，A的代谢前体APP与Nav1.6特异汇集于郎飞氏结（NOR）,推测NOR是APP剪切产生A的另一靶点。本课题从整体和细胞水平探讨NOR区APP 代谢异常的可能性以及其调控机制，寻找治疗 AD的新靶点。.通过研究发现：AD模型鼠前穿质通路存在Aβ沉积。实时微透析前穿质通路及海马区局部间质液，发现前穿质通路间质液中存在Aβ，且电刺激内嗅皮层后Aβ水平升高，此效应能够被钠通道阻断剂TTX所压抑。尽管前穿质通路脑间质液中Aβ的水平仍较海马区低，但我们首次证实了前穿质通路这个轴突区有Aβ生成，也是对经典突触产生Aβ学说的补充。.鉴于电刺激内嗅皮层后Aβ水平升高，而Nav1.6与细胞电兴奋密切相关，既然APP与Nav1.6共聚于NOR, 那么二者之间的功能联系如何？通过研究我们发现，APP 能够增强Nav1.6电流，钠通道阻滞剂拉莫三嗪能够改善 AD小鼠学习记忆障碍。根据以上结果我们首次提出压抑钠通道是治疗AD的潜在靶点。.细胞外基质糖蛋白(Tn-R) 聚集于NOR, 有报道Tn-R可正性调控Na+通道，那么Tn-R是否也调控APP剪切？而这种剪切的调控是否是通过Nav1.6实现？我们研究发现，Tn-R 与APP形态共聚结构相连。过表达Tn-R后，小鼠和人细胞系中的APP表达均升高，且与Aβ生成相关的APP剪切酶 BACE1、剪切片段sAPPβ、β-CTF 蛋白表达水平也显著升高，细胞上清液中Aβ 含量增加。而这些改变同时伴行Nav1.6 蛋白表达水平的升高。与此结果相对应的是Tn-R 基因敲除鼠脑内 APP 及其剪切片段的表达与同窝孪生鼠对比水平降低，剪切酶 BACE1、剪切片段 sAPPβ以及 Nav1.6 蛋白表达水平均显著下降。提示Tn-R可通过Nav1.6调控APP的剪切。我们同时也发现，髓鞘分子NgR也有可能调控Aβ的产生。此课题的研究不仅对AD的发病机制进行了补充，并且为药物研发提供新靶点。