Distinguishing cell-intrinsic and cell-extrinsic effects of APP/Abeta in vivo

描述（由申请人提供）：早发性阿尔茨海默病的遗传学涉及淀粉样前体蛋白（APP）及其衍生物在该疾病的发病机制中，但我们尚未确定家族性APP变异导致神经元功能障碍和认知能力下降的机制。即使是基本的机制见解，如突变的APP是否主要损害其表达的细胞或通过分泌片段破坏邻近细胞，也一直难以捉摸。培养的神经元和器官型切片已被用于探索这一问题，但尚不清楚在体外测试的未成熟神经元中观察到的结果是否适用于成人大脑中的成熟神经元。此外，培养研究的持续时间有限，可能无法反映人类疾病长期暴露的后果。长时间的暴露可以在体内进行测试，然而，目前可用的大多数转基因模型都被设计为在整个大脑中提供无处不在的APP过表达，排除了细胞自主效应和细胞外源性效应的区分。我的实验室正在开发一种新的小鼠模型，该模型将提供分离突变APP的细胞内在和外在效应所需的空间分辨率，以及区分急性和慢性暴露后果的时间分辨率。我们的方法是基于病毒和标准转基因的结合来创建马赛克动物，其中突变APP的表达可以通过病毒表达的四环素-transactivator来控制。通过调节病毒滴度，我们可以控制转基因嵌合在脑内的程度，从而分离APP过表达的细胞内效应和细胞外效应。四环素反激活剂的结合提供了对转基因APP过表达的开始和持续时间的快速和可逆的时间控制。我们提供了显示新模型发展的初步数据，并描述了其优化的下一步。然后，我们提出使用新的马赛克测试APP小鼠进行实验，以解决在现有模型中无法测试的关键问题：APP过表达是否以细胞自主或细胞外在的方式改变神经元的结构和功能？我们将研究APP过表达对转基因神经元及其野生型邻居的结构和功能的影响。新的模型将允许我们把这个问题从培养皿转移到哺乳动物的大脑中，在那里它可以有更大的时间自由和更好的对人类疾病的保真度进行检查。

DESCRIPTION (provided by applicant): The genetics of early-onset Alzheimer's disease implicate the amyloid precursor protein (APP) and its derivatives in pathogenesis of the disease, yet we have not identified the mechanism through which familial APP variants lead to neuronal dysfunction and cognitive decline. Even fundamental mechanistic insight such as whether mutant APP primarily damages the cell in which it's expressed or neighboring cells through secreted fragments has been elusive. Cultured neurons and organotypic slices have been used to explore this issue, but it remains unclear whether observations made in immature neurons tested in vitro extend to mature neurons in the adult brain. Additionally, culture studies are limited in duration and may not reflect the consequences of chronic exposure present in the human disease. Longer exposures can be tested in vivo, however most transgenic models currently available were designed to provide ubiquitous APP over-expression throughout the brain, precluding distinction of cell-autonomous and cell-extrinsic effects. My laboratory is developing a novel mouse model that will provide the spatial resolution needed to separate cell-intrinsic and - extrinsic effects of mutant APP as well as the temporal resolution to distinguish the consequences of acute and chronic exposure. Our approach is based on a combination of viral and standard transgenesis to create mosaic animals in which the expression of mutant APP can be controlled through a virally-expressed tetracycline-transactivator. By adjusting the viral titer, we can control the degree of transgenic mosaicism in the brain to separate cell-intrinsic and cell-extrinsic effects of APP over-expression. Incorporation of the tetracycline transactivator provides rapid and reversible temporal control over the onset and duration of transgenic APP over-expression. We provide preliminary data showing the development of the new model and describe the next steps in its optimization. We then propose experiments using the new mosaic tet-off APP mice to address a critical question that has been un-testable in existing models: Does APP over- expression alter neuronal structure and function in a cell-autonomous or a cell-extrinsic manner? We will examine the impact of APP over-expression on the structure and function of transgenic neurons and their wild- type neighbors. The new model will allow us to move this question from the culture dish into the mammalian brain where it can be examined with greater temporal freedom and better fidelity to the human disease. PUBLIC HEALTH RELEVANCE: This study will create a new animal model for Alzheimer's disease that will help us understand how inherited mutations in familial forms of the disease cause neuronal dysfunction thought to underlie dementia. The new model will be used to test whether the Alzheimer's-related amyloid precursor protein damages the cells in which it's made or acts on nearby neighbors through secreted fragments, akin to asking whether neurons in the Alzheimer's brain die by suicide or murder. A better understanding of this fundamental issue will improve our ability to design and deliver effective therapeutics for the disease.