Physiologic Mechanisms of Action of APP and APLP2 in Axon Targeting

摘要 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着重要作用 疾病在哺乳动物中，APP具有两种淀粉样前体样蛋白（APLP）同源物，并且在生理学上， 该蛋白质家族的功能仍然知之甚少。APP的直系同源物不存在于酵母中，但存在于 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，我们证明了APP或APLP2基因缺失的小鼠品系表现出一种新的免疫应答。 嗅觉感觉神经元（OSNs）轴突投射的保真度丧失，表明这些神经元的重要作用。 蛋白质在建立和/或维持嗅觉神经回路的精确布线图中的作用。 来自分离的OSN的下一代测序数据表明APLP2和APP是高度同源的。 在OSN中表达（在27，390个注释的转录物中分别排名#26和#29）。我们最近发表 BACE1蛋白酶的功能丧失，BACE1蛋白酶是一种关键的蛋白酶，也可以处理APP家族的蛋白质， 和许多其他轴突导向分子一样，也会导致小鼠嗅觉神经回路的连接错误。 在这里，我们提出APP和APLP2在生理上的功能，在形成和维持的， 小鼠嗅觉神经回路的精确轴突投影图。这一假设得到了几个方面的支持。 其他人的观察。APP与许多粘附分子以及蛋白质结合， 在轴突引导中的既定作用，如netrin和contactin 4。事实上，许多相关蛋白质 也在OSN中表达，这一点已经被我们的下一代测序数据所证实。可变剪接 APP和APLP2的细胞外结构域可以被硫酸软骨素后修饰。这 选择性剪接的APLP2同种型在OSN中以高水平表达。此外，细胞外结构域 APP和APLP2在体外结合硫酸乙酰肝素。这两种蛋白聚糖都有助于环境 排斥或吸引生长锥的线索，存在于包埋嗅觉神经回路的组织中。测试 APP和APLP2的作用，以精确映射轴突投射，我们部署了一个组合的遗传工具， 开发来操纵小鼠嗅觉回路，以检查选择性删除APP的后果 和/或仅在OSN中的APLP2（目标1）。此外，我们利用了高水平的表达， APP和APLP2相关的蛋白质以纯化和鉴定源自小鼠OSN的APP和/或APLP2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生理学的理解。 这一重要的蛋白质家族的功能，并可能提供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， APP及其裂解产物的水平。

Abstract 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.