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？

• 批准号: 7564524
• 负责人: Lois S Weisman
• 金额: $ 33.8万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 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/7564524
• 关键词: Achievement; Address; Affect; Alzheimer' 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; public health relevance; research study; small hairpin RNA; tool; trafficking

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.

描述(申请人提供)：低丰度的信号脂质，磷脂酰肌醇(3，5)-双磷酸(PI3，5P2)存在于所有真核生物中，并被认为参与了晚期内体的多种运输途径。合成这种脂质的机制包括PI3P5‘-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，并过早死亡。出乎意料的是，主要缺陷是大规模的神经变性。大脑和外周神经系统中受影响的神经元都会形成由内小体产生的大空泡。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)的患者，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，并进行研究以确定为什么PI3，5P2丢失会导致神经缺陷。我们的三个具体目标是：1)确定是否有任何或所有Wipi家族蛋白调控FAB1/PIKfyve活性。根据与酵母Atg18的同源性，我们预测一个或多个Wipi家族成员负调控FAB1/PIKfyve。我们将直接测试这一假说，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在神经元中是否只调节一般的膜转运，或者它是否也调节神经元特异性的膜转运通路。为什么神经元特别受PI3，5P2缺失的影响？为了解决这个问题，我们将测试以下问题。神经元，由于其长的突起，特别容易受到PI3，5P2调控的膜运输通路缺陷的影响？突触前和/或突触后终末是否存在需要PI3，5P2的神经元特异细胞器？我们将测量PI3，5P2缺失对神经元特有通路和一般通路的影响。我们还将开发提高培养细胞中PI3，5P2水平的方法。基于一个显性活性酵母FAB1突变体，它产生的PI3，5P2水平高出17倍，我们将测试我们预测将是显性活性的候选哺乳动物FAB1/PIKfyve突变体。3)确定在没有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，磷脂酰肌醇在哺乳动物中的代谢更为复杂。我们将测试在没有FAB1/PIKfyve的情况下，小鼠是否能产生PI3，5P2。这些目标的实现将为了解神经退行性疾病的病理生理学提供见解，并最终可能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，如阿尔茨海默氏症和帕金森氏病，是由多种途径缺陷引起的复杂疾病。我们的实验室发现了一种新的通路，当被干扰时，会意外地导致神经退化。这项应用的总体目标是确定该通路中的缺陷是如何导致神经退化的。