Non-amyloidogenic processing of APP

描述（由申请人提供）：阿尔茨海默病（AD）的神经病理学特征是细胞外淀粉样蛋白2 （A2）沉积和细胞内神经原纤维缠结的存在。淀粉样蛋白2来源于淀粉样蛋白2前体蛋白（A2PP）的淀粉样变性过程。在淀粉样变性途径中，A2PP被2-分泌酶（β -位点A2PP切割酶1；BACE1）和多亚基蛋白酶复合物（3-分泌酶）依次切割，产生可溶性外结构域片段（A2PPs2）和A2。在非淀粉样蛋白生成途径中，1-分泌酶切割A2序列中的A2PP，从而阻止A2肽的形成，并产生可溶的、神经保护的n端外畴A2PPs1。ADAM（一种崩解素和金属蛋白酶）家族的成员ADAM10 （EC 3.4.24.81）似乎负责1-分泌酶介导的A2PP的外结构域脱落。虽然AD的一些主要治疗途径涉及淀粉样蛋白A2PP加工抑制剂的开发，但ADAM10的激活和非淀粉样蛋白途径代表了一种合乎逻辑的替代策略。不幸的是，由于缺乏ADAM10敲除小鼠模型，涉及表征ADAM10作为1-分泌酶在A2PP加工和其他底物加工中的活性的研究受到阻碍。已经产生了ADAM10缺陷小鼠（Hartmann, 2002），但它们在E9.5天的早期致命性阻碍了对ADAM10体内1-分泌酶功能的可靠分析，特别是在神经元细胞中。本研究的目的是建立一种诱导条件敲除（cKO）动物模型，在体内研究ADAM10在神经元中的活性。我们认为，一个可诱导的cKO小鼠模型对于充分表征ADAM10 1-分泌酶活性的全部功能组合及其临床相关性至关重要，这可能远远超出了对AD的研究。我们的具体目标是：具体目标1：有条件地敲除（cKO）小鼠神经元中的Adam10基因。Adam10缺陷小鼠的早期死亡阻碍了对Adam10在神经元细胞体内功能的可靠分析。在这个目标中，我们将产生一个诱导的cKO小鼠模型，其中Adam10的功能丧失将针对表达CreERT2蛋白的神经元。在淀粉样蛋白沉积的动物模型中，我们将使用该模型来表征Adam10在A2PP和Notch代谢以及A2沉积中的功能丧失的后果。

DESCRIPTION (provided by applicant): Alzheimer disease (AD) is neuropathologically characterized by the presence of extracellular amyloid 2 (A2) deposits and intracellular neurofibrillary tangles. Amyloid 2 is derived from the amyloidogenic processing of the amyloid 2 precursor protein (A2PP). In the amyloidogenic pathway, A2PP is sequentially cleaved by 2-secretase (beta-site A2PP cleaving enzyme 1; BACE1) and a multi-subunit protease complex known as 3-secretase to produce a soluble ectodomain fragment (A2PPs2) and A2. In the non-amyloidogenic pathway, 1-secretase cleaves A2PP within the A2 sequence, thereby precluding the formation of A2-peptides and generating a soluble, neuroprotective, N-terminal ectodomain termed A2PPs1. A member of the ADAM (a disintegrin and metalloproteinase) family of enzymes, ADAM10 (EC 3.4.24.81), seems to be responsible for the 1-secretase-mediated ectodomain shedding of A2PP. Whilst some of the main therapeutic avenues for AD involve the development of inhibitors of amyloidogenic A2PP processing, the activation of ADAM10 and the non-amyloidogenic pathway represents a logical alternative strategy. Unfortunately, studies involving the characterization of ADAM10 activity as 1-secretase in A2PP processing and in the processing of other substrates have been obstructed by the lack of an Adam10 knock-out mouse model. Adam10-deficient mice have been generated (Hartmann, 2002), but their early lethality at day E9.5 prevents a reliable analysis of ADAM10's 1-secretase function in vivo, especially in neuronal cells. The aim of this study is to generate an inducible conditional knock-out (cKO) animal model in which investigate ADAM10 activity in neurons in vivo. We believe that an inducible cKO mouse model will be crucial to fully characterize both the full functional portfolio of ADAM10 1-secretase activity as well as its clinical relevance, which may go way beyond the study of AD. Our specific aims are: Specific Aim 1: To conditionally knock-out (cKO) the murine Adam10 gene in neurons. The early lethality of Adam10-deficient mice has prevented a reliable analysis of Adam10 function in vivo in neuronal cells. In this aim we will generate an inducible cKO mouse model in which loss of function of Adam10 will be targeted to neurons expressing the CreERT2 protein. We will use this model to characterize the consequences of Adam10 loss of function in the metabolism of A2PP and Notch, and in A2 deposition in an animal model of amyloid deposition. PUBLIC HEALTH RELEVANCE: The early lethality observed in Adam10-deficient mice generated by classical Cre recombination has prevented a reliable analysis of ADAM10's 1-secretase function in vivo, in particular in neuronal cells. The aim of this study is to generate an inducible conditional knock-out (cKO) animal model in which to investigate ADAM10's activity in neurons in vivo. We believe that an inducible cKO mouse model will be crucial to fully characterize both the full functional portfolio of ADAM10 1-secretase activity as well as its clinical relevance in Alzheimer disease, where the activation of ADAM10 and the non-amyloidogenic pathway represent a logical alternative strategy that has not been well investigated due to the lack of an Adam10 knock-out mouse model.