Role of the Amyloid Precursor Protein in the cellular phosphoinositide metabolism through its interaction with the PIKfyve complex

虽然淀粉样前体蛋白APP在阿尔茨海默病中的作用已经确定，但它在生物体中的正常作用还没有明确的定义。由于大多数完成复杂功能的蛋白质都是与它们物理结合的其他蛋白质(所谓的相互作用伙伴)协同工作的，了解结合伙伴可以为蛋白质功能提供有价值的见解。我们已经建立了大量的APP结合伙伴，其中包括由PIKfyve、Vac14和Fig4亚基组成的蛋白质复合体。众所周知，这三种蛋白质调节一种重要的信号脂质--磷脂酰肌醇-3，5-二磷酸(PI3，5P2)的水平。有趣的是，Vac14或图4的缺失会导致小鼠和人类的严重神经变性。APP和PIKfyve复合体都与神经退行性变有关，而且它们相互捆绑在一起，这一事实是新颖和令人惊讶的。这表明两者都参与了相同的细胞过程。我们认为，APP是通过与PIKfyve复合体的结合而调节PI3，5P2的。我们现在需要检验这一假设是否正确。我们将使用遗传模型生物秀丽线虫来测试这一想法。在这个简单的生物体中，有一些基因与人类APP、PIKfyve、Vac14和Fig4高度相似。使用线虫的强大优势是可以获得大量的突变体。我们已经获得了APP、PIKfyve和Vac14有缺陷的突变体。我们将从动物的外表、行为和神经系统的完整性等方面描述这些缺陷在动物身上的后果。在下一步，通过将APP突变体与PIKfyve或Vac14动物杂交，我们可以在一种动物身上结合突变。然后，这允许比较双突变和单突变的后果，并允许推断基因是否协同作用来实现生物功能(称为遗传相互作用)。我们预测，与单突变体相比，双突变体将具有更强的表型增强。这种类型的表型增强只有当几个基因在功能上相连时才能观察到。这种方法提供了任何其他方法无法获得的宝贵信息。我们还将分析信号脂质PI3，5P2的水平，这似乎对中枢神经系统的完整性至关重要。将这一水平与动物的特征(外表、行为等)联系起来。将让我们了解APP和PIKfyve复合体在其监管中扮演的角色。我们的工作表明，跨膜受体(APP)能够通过与PIKfyve复合体直接相互作用来模式化PI3，5P2的产生。众所周知，PIKfyve复合体功能的丧失会导致哺乳动物的神经退化。我们的工作首次将APP与PI3，5P2调控联系起来，从而可能提供一种全新的、意想不到的机制，即APP的丢失或异常处理如何引发神经退化。

While the role of the Amyloid Precursor Protein APP in Alzheimer's disease is well established, its normal role in organisms is ill defined. As most proteins that fulfill complex functions do so in concert with other proteins that they physically bind (so-called interaction partners), knowing binding partners can give valuable insights into protein function. We have established a large number of binding partners of APP, among them a protein complex consisting of the subunits PIKfyve, Vac14 and Fig4. These three proteins are well known to regulate the levels of an important signalling lipid, phosphatidylinositol-3,5-bisphosphate (PI3,5P2). Interestingly, loss of Vac14 or Fig4 lead to profound neurodegeneration in mice as well as humans. The fact that both APP and the PIKfyve complex are implicated in neurodegeneration and the fact that they bind each other is novel and surprising. This suggests that both participate in the same cellular process. We suggest that APP is a regulator of PI3,5P2 via its binding of the PIKfyve complex. We now need to test whether this hypothesis is correct. We will test this idea using the genetic model organism C. elegans. In this simple organism there are genes that are highly similar to human APP, PIKfyve, Vac14 and Fig4. The powerful advantage of using C. elegans is the availability of numerous mutants. We have obtained mutants in which APP, PIKfyve and Vac14 are defective. We will characterise the consequences of these defects in animals in terms of appearance of the animals, their behavior and the integrity of their neuronal system. In the next step, through crossing the APP mutants with PIKfyve or Vac14 animals we can combine the mutations in one animal. This then allows to compare the consequences of the double mutants with the single mutants and allows to deduce whether genes act in concert to fulfill a biological function (termed a genetic interaction). We predict that the double mutants will have a strongly enhanced phenotype compared to the single mutants. This type of phenotypic enhancement is only observed when several genes are functionally connected. This type of approach provides invaluable information that cannot be obtained by any other approach. We will also analyse the levels of the signalling lipid PI3,5P2 that appears to be of key importance for the integrity of the central nervous system. Correlating the levels of this with the characteristics of the animals (appearance, behavior etc.) will allow us to understand what role APP and the PIKfyve complex play in its regulation. Our work suggests that the transmembrane receptor (APP) is able to module the production of PI3,5P2 by a direct interaction with the PIKfyve complex. Loss of function of the PIKfyve complex is known to lead to neurodegeneration in mammals. Our work for the very first time links APP with PI3,5P2 regulation and thus may provide an entirely novel and unexpected mechanism how loss or aberrant processing of APP can instigate neurodegeneration.