How does misregulation of PI3,5P2 signaling lead to neurodegeneration?

描述（由申请人提供）：低丰度信号脂质，磷脂酰肌醇(3,5)-二磷酸（pi3,5p2）存在于所有真核生物中，并被认为参与了来自晚期核内体的多种运输途径。合成这种脂质的机制包括PI3P 5'-激酶Fab1/PIKfyve和由Vac14和Fig4组成的调节复合体。在哺乳动物中，这些蛋白在所有组织中都有表达。我们最近发现，缺乏Vac14或Fig4的小鼠细胞中PI3、5P2水平降低，并过早死亡。出乎意料的是，主要的缺陷是大面积的神经变性。在大脑和周围神经系统中，受影响的神经元产生由核内体产生的大液泡。与PI3,5P2在神经系统中的重要性一致，我们确定了Charcot-Marie Tooth综合征（CMT4J）患者，其疾病对应于图4中的单点突变。人们对PI3、5P2知之甚少，对其在神经系统中的作用也几乎一无所知。本提案的总体目标是确定调节神经元中PI3,5P2水平的机制，并开展研究以确定为什么PI3,5P2的缺失会导致神经系统缺陷。我们的三个具体目标是：1)确定是否有任何或所有WIPI家族蛋白调节Fab1/PIKfyve活性。基于与酵母Atg18的同源性，我们预测WIPI家族的一个或多个成员负调控Fab1/PIKfyve。我们将直接验证这一假设，并将筛选Fab1/PIKfyve的其他阴性和阳性调节因子。2)确定神经元中的PI3、5P2是单独调节一般的膜转运，还是也调节神经元特异性的膜转运途径。为什么神经元特别受PI3,5P2缺失的影响？为了解决这个问题，将测试以下问题。神经元是否由于其漫长的过程而特别容易受到PI3,5P2调节的膜运输途径缺陷的影响？突触前和/或突触后末端是否存在需要PI3,5P2的神经元特异性细胞器？我们将测量PI3,5P2的损失对神经元特异性通路以及一般通路的影响。我们还将开发提高培养细胞中PI3、5P2水平的方法。基于酵母Fab1的显性活性突变体，其pi35,5p2的水平高出17倍，我们将测试候选的哺乳动物Fab1/PIKfyve突变体，我们预测这些突变体将具有显性活性。3)判断在没有Fab1/PIKfyve的情况下是否可以生成PI3、5P2。在酵母中，所有的PI3,5P2都是通过Fab1产生的。然而，哺乳动物的磷酸肌肽代谢更为复杂。我们将测试小鼠在缺乏Fab1/PIKfyve的情况下是否能产生PI3、5P2。这些目标的实现将为神经退行性疾病的病理生理学提供见解，并可能最终导致治疗各种神经退行性疾病的新方法。公共卫生相关性：常见的神经退行性疾病，如阿尔茨海默病和帕金森病，是由多种通路缺陷引起的复杂病症。我们的实验室发现了一种新的通路，当它被破坏时，会意外地导致神经变性。该应用程序的总体目标是确定该途径中的缺陷如何导致神经退行性变。

DESCRIPTION (provided by applicant): The low abundance signaling lipid, phosphatidylinositol (3,5)-bis phosphate (PI3,5P2) is present in all eukaryotes, and is postulated to be involved in multiple trafficking pathways from late endosomes. The machinery that synthesizes this lipid includes the PI3P 5'-kinase Fab1/PIKfyve and a regulatory complex composed of Vac14 and Fig4. In mammals, these proteins are expressed in all tissues. We recently discovered that mice that lack either Vac14 or Fig4 have reduced PI3,5P2 levels in their cells and die prematurely. Unexpectedly, the main defect is massive neurodegeneration. Affected neurons in both the brain and the peripheral nervous system develop large vacuoles that arise from endosomes. Consistent with the importance of PI3,5P2 in the nervous system, we identified patients with Charcot-Marie Tooth syndrome (CMT4J) whose disease corresponds to a single point mutation in Fig4. Little is known about PI3,5P2 and virtually nothing is known about its role in the nervous system. The overall goals of this proposal are to determine the mechanisms that regulate PI3,5P2 levels in neurons and to conduct studies to determine why loss of PI3,5P2 causes neurological defects. Our three specific aims are: 1) Determine whether any or all WIPI family proteins regulate Fab1/PIKfyve activity. Based on homology with yeast Atg18, we predict that one or more WIPI family members negatively regulate Fab1/PIKfyve. We will directly test this hypothesis and will also screen for additional negative and positive regulators of Fab1/PIKfyve. 2) Determine whether PI3,5P2 in neurons solely regulates general membrane trafficking, or whether it also regulates neuronal- specific membrane trafficking pathways. Why are neurons particularly affected by loss of PI3,5P2? To address this, the following questions will be tested. Are neurons, by virtue of their long processes especially vulnerable to defects in membrane trafficking pathways regulated by PI3,5P2? Are there neuronal-specific organelles either in presynaptic and/or postsynaptic termini that require PI3,5P2? We will measure the effects of loss of PI3,5P2 on pathways specific to neurons, as well as general pathways. We will also develop methods to elevate PI3,5P2 levels in cultured cells. Based on a dominant active yeast Fab1 mutant that produces 17-fold higher levels of PI3,5P2, we will test candidate mammalian Fab1/PIKfyve mutants that we predict will be dominant active. 3) Determine whether PI3,5P2 can be generated in the absence of Fab1/PIKfyve. In yeast, all PI3,5P2 is generated through Fab1. However, phosphoinositide metabolism in mammals is more complex. We will test whether PI3,5P2 in mice can be generated in the absence of Fab1/PIKfyve. Achievement of these aims will provide insights into the pathophysiology of neurodegenerative disorders and may ultimately lead to novel approaches for treatments for a variety of neurodegenerative diseases. PUBLIC HEALTH RELEVANCE: Common neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases, are complex conditions that arise from defects in a variety of pathways. Our laboratory has discovered a new pathway that when disrupted unexpectedly causes neurodegeneration. The overall goal of this application is to determine how defects in this pathway lead to neurodegeneration.