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暗示淀粉样前体蛋白（APP）及其衍生物参与疾病的发病机制，但我们尚未确定家族性APP变体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APP是否主要损害细胞，它的表达或邻近细胞通过分泌片段一直难以捉摸。培养的神经元和器官型切片已被用于探索这个问题，但目前尚不清楚在体外测试的未成熟神经元中进行的观察是否延伸到成年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中存在的慢性暴露后果。可以在体内测试更长时间的暴露，然而，目前可用的大多数转基因模型被设计为在整个大脑中提供普遍存在的APP过表达，排除了细胞自主效应和细胞外源效应的区别。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组合，以创建嵌合体动物，其中突变APP的表达可以通过病毒表达的四环素反式激活因子来控制。通过调节病毒滴度，我们可以控制脑中转基因嵌合体的程度，以分离APP过表达的细胞内源性和细胞外源性效应。四环素反式激活因子的掺入提供了对转基因APP过度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，我们提出实验使用新的马赛克tet关闭APP小鼠，以解决一个关键的问题，已无法在现有的模型：APP过度表达改变神经元的结构和功能，在细胞自主或细胞外在的方式？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。新的模型将使我们能够将这个问题从培养皿转移到哺乳动物的大脑中，在那里它可以以更大的时间自由和更好的保真度来检查人类疾病。 公共卫生相关性：这项研究将为阿尔茨海默病创造一个新的动物模型，这将有助于我们了解该疾病家族形式中的遗传突变如何导致被认为是痴呆症基础的神经元功能障碍。新模型将用于测试阿尔茨海默氏症相关的淀粉样前体蛋白是否会损害其产生的细胞或通过分泌的片段作用于附近的邻居，类似于询问阿尔茨海默氏症大脑中的神经元是否死于自杀或谋杀。更好地理解这一基本问题将提高我们设计和提供有效治疗方法的能力。

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.