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

项目摘要/摘要 ALL中存在低丰度的信号脂质磷脂酰肌醇(3，5)-双磷酸(PI3，5P2) 真核生物，并被认为参与了晚期内小体的多种运输途径。这个 合成这种脂质的机制包括PI3P 5‘-激酶FAB1/PIKfyve和调节复合体 由Vac14和图4组成。在哺乳动物中，这些蛋白质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水平降低并死亡 为时过早。出乎意料的是，主要缺陷是大规模的神经变性。两个大脑中受影响的神经元 外周神经系统发育出由内小体形成的大空泡。与 PI3，5P2在神经系统中的重要性，我们确定了Charcot-Marie Tooth综合征的患者 (CMT4J)，其疾病对应于图4中的单点突变。关于PI3，5P2和 关于它在神经系统中的作用，人们几乎一无所知。这项提案的总体目标是 确定调节神经元中PI3，5P2水平的机制，并进行研究以确定原因 PI3，5P2的缺失会导致神经缺陷。我们的三个具体目标是：1)确定任何或所有 Wipi家族蛋白调节FAB1/PIKfyve活性。根据与酵母Atg18的同源性，我们预测 一个或多个Wipi家族成员负性调节FAB1/PIKfyve。我们将直接测试这一假设，并 还将筛选FAB1/PIKfyve的其他负调控因子和正调控因子。2)确定PI3，5P2是否 在神经元中只调节一般的膜运输，或者它是否也调节神经元- 特定的膜运输途径。为什么神经元特别受PI3，5P2缺失的影响？至 要解决这一问题，将测试以下问题。是神经元，由于它们的长过程，特别是 是否易受PI3，5P2调控的膜转运途径缺陷的影响？有没有神经元特异性的 突触前和/或突触后终末的细胞器需要PI3，5P2？我们将衡量其影响 PI3，5P2在神经元特有的通路和一般通路上的丢失。我们还将发展 方法提高培养细胞中PI3、5P2水平。基于显性活性酵母FAB1突变体 产生高出17倍的PI3，5P2，我们将测试候选哺乳动物FAB1/PIKfyve突变体，我们 预测将是主导活跃的。3)确定在没有PI3，5P2的情况下是否可以生成PI3，5P2 FAB1/PIKfyve。在酵母中，所有的PI3，5P2都是通过FAB1产生的。然而，磷脂酰肌醇在体内的代谢 哺乳动物要复杂得多。我们将测试在缺乏PI3，5P2的情况下是否能在小鼠中产生PI3，5P2 FAB1/PIKfyve。这些目标的实现将为神经退行性变的病理生理学提供洞察力 并最终可能导致治疗各种神经退行性疾病的新方法 疾病。

PROJECT SUMMARY/ABSTRACT 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.