Production of transgenic mice expressing familial AD relevant genes

本研究旨在建立一个实验动物模型，证明早老素1基因错配突变是大多数家族性阿尔茨海默病（FAD）的致病基因，导致AD病理病变之一的a β沉积表型的级联反应，并为分析级联反应相关蛋白的加工提供工具。为此，我们尝试构建表达FAD相关蛋白的转基因小鼠。我们制备了表达野生型人PS1（wtPS1）蛋白的Tg小鼠和表达错义PS1（密码子146 Met→Val）的Tg小鼠，其中一种错义突变被证明可以增加Aβ1-42的产生。此外，我们还培育了在密码子717（London type of FAD）表达突变APP的Tg小鼠。我们制备了针对n端1 ~ 81个氨基酸序列和c端299 ~ 407个氨基酸序列的多克隆抗体，两者均融合到GST中。利用这些工具，我们将分析Aβ沉积的机制。有报道显示突变体APP Tg小鼠随着年龄的增长（1.5岁后）在大脑中产生Aβ沉积，而突变体PS1 Tg小鼠在大脑中不能产生Aβ沉积。为了证明“Aβ沉积的播种假说”，我们对年轻的APP 717 Tg小鼠和M146V PS1 Tg小鼠进行了Aβ肽颅内注射。我们将观察在这些动物体内注射的物质是否作为加速内源性小鼠a β沉积的种子。

This study is dedicated to establish an experimental animal model which allows to demonstrate the cascade that missense mutation in presenilin 1 gene identified as causative gene for the majority of familial Alzheimer's disease (FAD) leads to the phenotype of Aβ deposition, one of the pathological lesions of AD, and to provide a tool for analysis of processing of proteins involved in the cascade. For this purpose we attempted to produce trasgenic mice which express FAD relevant proteins. We have produced Tg mice which express wild type human PS1(wtPS1) protein and those which express missense PS1 (codon 146 Met → Val), one of the missense mutations shown to increase the production of Aβ1-42. In addition, we have produced Tg mice which express mutant APP at codon 717(London type of FAD). We have produced polyclonal antibodies generated against N-terminal 1 to 81 amino acid sequence and C-terminal 299 to 407 amino acids sequence, both of which were fused to GST. Using these tools we will analyze the mechanism involved in the deposition of Aβ. There have been reports showing that mutant APP Tg mice generate Aβ deposits in the brain with aging (after 1.5years), whereas mutant PS1 Tg mice fail to generate Aβ deposits in the brain. To demonstrate "the seeding hypothesis for Aβ deposition", young APP 717 Tg mice and M146V PS1 Tg mice are subjected to intracranial administration of Aβ peptide. We will see whether the injected material in these animals serve as a seed for accelerated deposition of endogenous murine Aβ.