Physiologic Mechanisms of Action of APP and APLP2 in Axon Targeting

描述（由申请人提供）：淀粉样前体蛋白（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仍然知之甚少。APP的直系同源物不存在于酵母中，但从蠕虫到人类都是保守的，这表明它们在细胞间通讯中起着重要作用。在初步的数据中，我们证明，基因缺失APP或APLP 2的小鼠品系表现出嗅觉感觉神经元（OSN）轴突投射的保真度损失，表明这些蛋白质在嗅觉神经回路的精确接线图的建立和/或维护中的重要作用。源自分离的OSN的下一代测序数据表明APLP 2和APP在OSN中高度表达（在27，390个注释的转录物中分别排名#26和#29）。我们最近发表了BACE 1蛋白酶功能的丧失，这是一种处理APP家族蛋白质以及许多其他轴突导向分子的关键蛋白酶，也会导致小鼠嗅觉神经回路中的连接错误。在这里，我们提出，APP和APLP 2生理功能的形成和维护精确的轴突投影图的小鼠嗅觉神经回路。这一假设得到了其他人的一些观察结果的支持。APP与许多粘附分子以及在轴突引导中具有既定作用的蛋白质（如netrin和contactin 4）相关联。事实上，这些相关蛋白中的许多也在OSN中表达，正如我们的下一代测序数据所证明的那样。APP和APLP 2的选择性剪接的细胞外结构域可以通过硫酸软骨素进行后修饰。这种选择性剪接的APLP 2同种型在OSN中以高水平表达。此外，APP和APLP 2的胞外结构域在体外结合硫酸乙酰肝素。这两种蛋白聚糖都有助于排斥或吸引生长锥的环境线索，并存在于包埋嗅觉神经回路的组织中。为了测试APP和APLP 2的作用，以精确地映射轴突投射，我们部署了一个遗传工具的组合，开发来操纵小鼠嗅觉回路，以检查APP和/或APLP 2的选择性删除的后果，专门在OSN（目的1）。此外，我们利用APP和APLP 2的高水平表达来纯化和鉴定源自小鼠OSN的与APP和/或APLP 2相关的蛋白质（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，并可能深入了解阿尔茨海默病的发病机制以及正在开发的阿尔茨海默病治疗方法的不良后果，以改变APP及其裂解产物的水平。

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.