Signaling Mechanisms in Drosophila Neural Development

果蝇神经发育中的信号机制

• 批准号: 6777317
• 负责人: KAI G ZINN
• 金额: $ 29.97万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-15 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6777317
• 关键词: Drosophilidae; adenosinetriphosphatase; axon; biological signal transduction; developmental neurobiology; electron microscopy; electrophysiology; gene expression; genetic screening; immunocytochemistry; neural degeneration; neuromuscular junction; synaptogenesis; vesicle /vacuole

DESCRIPTION (provided by applicant): This proposal concerns spastin and blue cheese beached (bchs), two genes identified in screens we conducted for genes involved in motor axon guidance and synaptogenesis in Drosophila larvae. We selected these genes for further study because they encode members of highly conserved but poorly understood protein families that have not been previously implicated in neural development. The two genes are not related to each other, but both encode proteins likely to be involved in protein trafficking within neurons and can be studied using similar methods. Both genes have orthologs or relatives affected in human genetic diseases. (1) The first gene, spastin, is the ortholog of a human gene affected in autosomal dominant spastic paraplegia (ADSP). spastin encodes an AAA ATPase. These ATPases are involved in catalyzing assembly and disassembly of protein complexes involved in vesicle trafficking, protein degradation, microtubule dynamics, and other processes. Analysis of an AAA ATPase sequence does not allow definition of the cellular process(es) in which it participates, however, so the targets of Spastin function are still unknown. Neuronal overexpression of spastin causes convergence of central nervous system (CNS) axons onto the midline, spastin loss-of-function (LOF) null mutant larvae display altered synaptic morphologies at their neuromuscular junctions (NMJs). They also have reduced evoked junctional potential (EJP) amplitudes, indicating that their NMJ synapses are abnormal. Spastin-null animals that survive to adulthood are unable to fly, walk poorly, and have a shortened lifespan. To further analyze Spastin function, we will complete the morphological and electrophysiological analysis of larval NMJs. We will also perform electrophysiological tests to examine why the mutant adults cannot fly and examine the adult brain for structural defects and neurodegeneration. We will examine the mechanisms involved in human ADSP by introducing mutations that cause spasticity in humans into the fly gene and determining if these act as dominant negatives in Drosophila. To define other components of the pathways(s) in which Spastin acts, we will perform an enhancer/suppressor genetic screen using a spastin gain-of-function (GOF) eye phenotype. Candidate genes emerging from the eye screen will be tested for modification of the spastin GOF axonal phenotype and for interaction with spastin LOF mutations. (2) The second gene, bchs, encodes a protein closely related to the founding member of the BEACH domain protein family: the human protein whose loss causes Chediak-Higashi syndrome (CHS), a lethal genetic disease characterized by immunological and neurological defects. Cells from CHS patients contain abnormal giant lysosomes, bchs overexpression in neurons produces a unique phenotype in which bulges form at the junctions between motor axon trunks and side branches. bchs LOF mutations cause adult neurodegeneration phenotypes in the brain and eye, and bchs flies have short lifespans. In bchs larvae, some motor axon pathways are abnormally thickened, suggesting that individual axons are swollen or that additional axons have joined the pathways. Bchs contains a FYVE domain, which binds to phosphatidylinositol-3-phosphate (Ptdlns3P). It is a vesicular protein that occasionally colocalizes with a fluorescent marker (GFP-2XFYVE) for Ptdlns3P-containing endosomes; however, most Bchs vesicles are distinct from GFP-2XFYVE vesicles, suggesting that they represent different compartments. To study Bchs, we will analyze its subcellular localization and determine the vesicular compartment(s) in which it functions. We will examine bchs LOF and gain-of-function (GOF) phenotypes in the larval neuromuscular system using antibody staining, electron microscopy, and electrophysiology. We will also search for enhancers and suppressors of a bchs GOF eye phenotype. Candidate genes from the eye screen will be tested for modification of the bchs GOF neuromuscular phenotype and for interaction with bchs LOF mutations.

描述（由申请人提供）：该提案涉及Spastin和Blue Cheese Beached（BCHS），这是我们在屏幕上鉴定出的两个基因，我们针对参与果蝇幼虫的运动轴突引导和突触发生的基因进行了鉴定。我们选择了这些基因进行进一步研究，因为它们编码了以前没有与神经发育有关的高度保守但知之甚少的蛋白质家族的成员。这两个基因彼此无关，但是两种蛋白质都可能与神经元内的蛋白质运输有关，并且可以使用类似的方法进行研究。这两个基因都有在人类遗传疾病中受影响的直系同源物或亲戚。 （1）第一个基因Spastin是在常染色体显性痉挛性截瘫（ADSP）中受影响的人类基因的直系同源物。 Spastin编码AAA ATPase。这些ATPase参与了催化囊泡运输，蛋白质降解，微管动力学和其他过程的蛋白质复合物的催化组装和拆卸。但是，对AAA ATPase序列的分析不允许定义其参与的细胞过程（ES），因此Spastasin函数的目标仍然未知。痉挛的神经元过表达导致中枢神经系统（CNS）轴突在中线上收敛，Spastin功能丧失（LOF）NULL突变幼虫在其神经肌肉连接（NMJS）上显示出改变的突触形态。它们还降低了诱发的连接电位（EJP）幅度，表明其NMJ突触异常。生存到成年的痉挛无动物无法飞行，行走差，寿命缩短。为了进一步分析痉挛功能，我们将完成幼虫NMJ的形态和电生理分析。我们还将进行电生理测试，以检查为什么突变成年人无法飞行并检查成年大脑的结构缺陷和神经变性。我们将通过将引起人类痉挛的突变引入果蝇基因，并确定这些是否在果蝇中起主要负面因素来检查人类ADSP所涉及的机制。为了定义Spastin起作用的途径的其他组件，我们将使用Spastin功能获得（GOF）眼表型执行增强子/抑制器遗传筛选。将测试从眼屏中出现的候选基因，以修饰痉挛GOF轴突表型，并与Spastin Lof突变相互作用。 （2）第二个基因BCHS编码与海滩结构蛋白家族的创始成员密切相关的蛋白质：人类蛋白质的损失导致Chediak-Higashi综合征（CHS），这是一种由免疫学和神经学缺陷的致命遗传疾病。 CHS患者的细胞含有异常的巨型溶酶体，神经元中的BCHS过表达产生独特的表型，其中在运动轴突躯干和侧分支之间的连接处形成凸起。 BCHS LOF突变引起大脑和眼睛中的成年神经退行性表型，而BCHS苍蝇的寿命短。在BCHS幼虫中，某些运动轴突途径异常增厚，这表明单个轴突肿胀或其他轴突已连接了途径。 BCHS包含一个FYVE结构域，该结构域与磷脂酰肌醇-3-磷酸盐（PTDLNS3P）结合。这是一种囊泡蛋白，偶尔与含有PTDLNS3P的内体的荧光标记物（GFP-2XFYVE）共定位；但是，大多数BCH囊泡与GFP-2Xfyve囊泡不同，表明它们代表不同的隔室。为了研究BCHS，我们将分析其亚细胞定位，并确定其功能的囊泡室。我们将使用抗体染色，电子显微镜和电生理学研究BCHS LOF和幼体神经肌肉系统中的功能获得（GOF）表型。我们还将寻找BCHS GOF眼表型的增强剂和抑制器。将测试来自眼筛的候选基因，以修饰BCHS GOF神经肌肉表型以及与BCHS LOF突变的相互作用。