Physiologic Mechanisms of Action of APP and APLP2 in Axon Targeting

描述（由申请人提供）：淀粉样蛋白前体蛋白（APP）被认为在阿尔茨海默病的发病机制中起核心作用。在哺乳动物中，APP有两个淀粉样前体样蛋白（APLP）同源物，该蛋白家族的生理功能尚不清楚。APP的同源物在酵母中不存在，但从蠕虫到人类都是保守的，这表明它们在细胞间通讯中发挥了基本作用。在初步数据中，我们证明了APP或APLP2基因缺失的小鼠系表现出嗅觉感觉神经元（OSNs）轴突投射保真度的丧失，这表明这些蛋白在嗅觉神经回路精确接力图的建立和/或维持中发挥了重要作用。来自分离osn的下一代测序数据表明，APLP2和APP在osn中高表达（在27,390个注释转录本中分别排名第26位和第29位）。我们最近发表了BACE1蛋白酶（一种处理APP家族蛋白质以及许多其他轴突引导分子的关键蛋白酶）的功能丧失也导致小鼠嗅觉神经回路中的连接错误。在此，我们提出APP和APLP2在小鼠嗅觉神经回路的精确轴突投影图的形成和维持中具有生理功能。这一假设得到了其他人的几项观察结果的支持。APP与许多粘附分子以及在轴突引导中起既定作用的蛋白质（如netrin和contact蛋白）有关。事实上，正如我们的下一代测序数据所证明的那样，许多这些相关蛋白也在osn中表达。APP和APLP2的选择性剪接胞外结构域可在翻译后被硫酸软骨素修饰。这种选择性剪接的APLP2异构体在osn中高水平表达。此外，APP和APLP2的胞外结构域在体外与硫酸肝素结合。这两种蛋白多糖都参与排斥或吸引生长锥的环境信号，并存在于嵌入嗅觉神经回路的组织中。为了测试APP和APLP2在精确定位轴突投射中的作用，我们使用了一系列遗传工具来操纵小鼠嗅觉回路，以检查选择性缺失APP和/或APLP2在osn中的后果（目的1）。此外，我们利用APP和APLP2的高水平表达来纯化和鉴定来自小鼠osn的APP和/或APLP2相关蛋白（Aim 2）。总之，从这些研究中获得的知识将促进我们对这一重要蛋白质家族的生理功能的理解，并可能为阿尔茨海默病的发病机制以及阿尔茨海默病正在开发的改变APP及其切割产物水平的治疗方法的不良后果提供见解。

DESCRIPTION (provided by applicant): The amyloid precursor protein (APP) is thought to play a central role in the pathogenesis of Alzheimer's disease. In mammals, APP has two amyloid precursor like protein (APLP) homologs, and the physiological functions of this family of proteins remain poorly understood. Orthologs of APP are not present in yeast, but are conserved from worms to humans, suggesting they provide a fundamental role in intercellular communication. In preliminary data, we demonstrate that mouse lines with genetic deletion of either APP or APLP2 exhibit a loss of fidelity of axon projections of olfactory sensory neurons (OSNs), indicating an essential role for these proteins in the establishment and/or maintenance of the precise wiring diagram of the olfactory neural circuit. Next generation sequencing data derived from isolated OSNs indicates that APLP2 and APP are highly expressed in OSNs (ranked #26 and #29, respectively, of 27,390 annotated transcripts). We recently published that loss of function of the BACE1 protease, a key protease that processes proteins of the APP family as well as many other axon guidance molecules, also leads to connectivity errors in the mouse olfactory neural circuit. Here, we propose that APP and APLP2 function physiologically in the formation and maintenance of the precise axon projection map of the mouse olfactory neural circuit. This hypothesis is supported by several observations from others. APP associates with numerous adhesion molecules as well as proteins with established roles in axon guidance, such as netrin and contactin 4. Indeed, many of these associated proteins are expressed in OSNs, too, as evidenced by our next generation sequencing data. Alternatively spliced extracellular domains of APP and APLP2 can be modified posttranslationally by chondroitin sulfate. This alternatively spliced APLP2 isoform is expressed at high levels in OSNs. In addition, the extracellular domains of APP and APLP2 bind heparan sulfate in vitro. Both of these proteoglycans contribute to the environmental cues that repel or attract growth cones and are present in tissue embedding the olfactory neural circuit. To test the role of APP and APLP2 to precisely map axon projections, we deploy a combination of genetic tools developed to manipulate the mouse olfactory circuit to examine the consequences of selective deletion of APP and/or APLP2 exclusively in OSNs (Aim 1). Furthermore, we take advantage of the high levels of expression of APP and APLP2 to purify and identify proteins associated with APP and/or APLP2 derived from mouse OSNs (Aim 2). Together, the knowledge gained from these studies will advance our understanding of the physiologic function of this important protein family and may provide insight into the pathogenesis of Alzheimer's disease as well as into adverse consequences of the therapies under development for Alzheimer's disease to alter the levels of APP and its cleavage products.