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

项目总结/摘要 低丰度信号脂质磷脂酰肌醇（3，5）-二磷酸（PI 3，5 P2）存在于所有细胞中。 真核生物，并假定参与从晚期内体的多个运输途径。的 合成这种脂质的机制包括PI 3 P 5 '-激酶Fab 1/PIKfyve和调节复合物 由Vac 14和Fig 4组成。在哺乳动物中，这些蛋白质在所有组织中表达。我们最近 发现缺乏Vac 14或Fig 4的小鼠细胞中的PI 3，5 P2水平降低并死亡 过早地。出乎意料的是，主要的缺陷是大量的神经退行性变。两个大脑中受影响的神经元 周围神经系统产生大的空泡，这些空泡产生于核内体。符合 PI 3，5 P2在神经系统中的重要性，我们确定了Charcot-Marie Tooth综合征患者 （CMT 4J），其疾病对应于图4中的单点突变。关于PI 3，5 P2和 事实上，人们对它在神经系统中的作用一无所知。本提案的总体目标是 确定调节神经元中PI 3，5 P2水平的机制，并进行研究以确定为什么 PI 3、5 P2的缺失导致神经缺陷。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Fab 1/PIKfyve活性。基于与酵母Atg 18的同源性，我们预测， 一个或多个WIPI家族成员负调控Fab 1/PIKfyve。我们将直接测试这个假设， 还将筛选Fab 1/PIKfyve的其他阴性和阳性调节剂。2)确定PI 3、5 P2是否 在神经元中仅调节一般的膜运输，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PI 3，5 P2损失的影响？到 为了解决这个问题，将测试以下问题。是神经元，由于它们的长过程， 易受PI 3，5 P2调节的膜运输途径缺陷的影响？是否有神经特异性 细胞器无论是在突触前和/或突触后的终端，需要PI 3，5 P2？我们将测量 在神经元特异性通路以及一般通路上的PI 3、5 P2的丢失。我们还将开发 提高培养细胞中PI 3，5 P2水平的方法。基于显性活性酵母Fab 1突变体， 产生17倍高水平的PI 3，5 P2，我们将测试候选哺乳动物Fab 1/PIKfyve突变体，我们 预测将占主导地位。3)确定是否可以在不存在的情况下生成PI 3、5 P2 Fab1/PIKfyve。在酵母中，所有的PI 3，5 P2都是通过Fab 1产生的。然而，磷酸肌醇代谢， 哺乳动物更复杂。我们将测试小鼠中的PI 3，5 P2是否可以在缺乏 Fab1/PIKfyve。这些目标的实现将为神经退行性疾病的病理生理学提供深刻的见解。 并可能最终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.