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

PI3、5P2 信号传导失调如何导致神经退行性变？

基本信息

批准号：
8197473
负责人：
Lois S Weisman
金额：
$ 33.12万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-01-01 至 2013-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8197473
关键词：
Achievement Address Affect Alzheimer&apos s Disease Axonal Transport Binding Binding Proteins Brain Candidate Disease Gene Cell Survival Cells Charcot-Marie-Tooth Disease Complex Cultured Cells Defect Dendritic Spines Disease Embryo Endosomes Enzymes Eukaryota Family member Fibroblasts Functional disorder Genes Goals Growth Introns Knock-out Laboratories Lead Lipids Mammals Mass Spectrum Analysis Measurement Measures Membrane Protein Traffic Metabolism Methods Missense Mutation Mus Nerve Degeneration Nerve Growth Factors Nervous system structure Neurodegenerative Disorders Neurologic Neurons Organelles Organism Parkinson Disease Pathway interactions Patients Peripheral Nervous System Phosphatidylinositols Phosphoric Monoester Hydrolases Phosphotransferases Point Mutation Process Protein Family Proteins Research Role Signal Transduction Syndrome Testing Tissues Transgenic Mice Vacuole Yeasts base embryonic stem cell inorganic phosphate insight knock-down late endosome member mutant novel strategies overexpression postsynaptic presynaptic protein complex protein function research study small hairpin RNA tool trafficking

项目摘要

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. PROJECT NARRATIVE 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.

项目概要/摘要低丰度信号脂质磷脂酰肌醇 (3,5)-二磷酸 (PI3,5P2) 存在于所有细胞中真核生物，并且被认为参与晚期内体的多种运输途径。这合成这种脂质的机制包括 PI3P 5'-激酶 Fab1/PIKfyve 和调节复合物由Vac14和Fig4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 Vac14 或 Fig4 的小鼠细胞中 PI3、5P2 水平降低并死亡过早地。出乎意料的是，主要缺陷是大规模的神经变性。两个大脑中的神经元都受到影响周围神经系统发育出由内体产生的大液泡。符合 PI3、5P2 在神经系统中的重要性，我们确定了夏科-玛丽图斯综合征患者（CMT4J），其疾病对应于图4中的单点突变。关于 PI3、5P2 和事实上，人们对它在神经系统中的作用一无所知。该提案的总体目标是确定神经元中调节 PI3、5P2 水平的机制并进行研究以确定其原因 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) 确定是否有任何或全部 WIPI 家族蛋白调节 Fab1/PIKfyve 活性。基于与酵母 Atg18 的同源性，我们预测一个或多个 WIPI 家族成员对 Fab1/PIKfyve 进行负调控。我们将直接检验这个假设还将筛选 Fab1/PIKfyve 的其他负调节因子和正调节因子。 2）判断是否PI3,5P2 在神经元中仅调节一般膜运输，或者它是否也调节神经元特定的膜运输途径。为什么神经元特别容易受到 PI3、5P2 缺失的影响？到为了解决这个问题，将测试以下问题。神经元，由于它们的长过程，特别是容易受到 PI3、5P2 调节的膜运输途径缺陷的影响吗？是否存在神经元特异性突触前和/或突触后末端的细胞器需要 PI3,5P2？我们将衡量效果神经元特异性通路以及一般通路上 PI3、5P2 的丢失。我们还将开发提高培养细胞中 PI3、5P2 水平的方法。基于显性活性酵母 Fab1 突变体产生 17 倍高水平的 PI3,5P2，我们将测试候选哺乳动物 Fab1/PIKfyve 突变体预测将主导主动。 3）判断在没有的情况下能否生成PI3,5P2 Fab1/PIKfyve。在酵母中，所有PI3、5P2都是通过Fab1产生的。然而，磷酸肌醇代谢哺乳动物则更为复杂。我们将测试小鼠体内PI3,5P2是否可以在缺乏的情况下产生 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入的见解疾病，并可能最终导致治疗各种神经退行性疾病的新方法疾病。项目叙述常见的神经退行性疾病，例如阿尔茨海默病和帕金森病，是复杂的病症由多种途径的缺陷引起的。我们的实验室发现了一条新途径意外破坏会导致神经退行性变。该应用程序的总体目标是确定如何该通路的缺陷会导致神经退行性变。