Signaling Mechanisms in Drosophila Neural Development

描述（由申请人提供）：该提案涉及spastin和blue cheese beached (bchs)，这两个基因是我们在果蝇幼虫中进行的涉及运动轴突引导和突触发生的基因筛选中发现的。我们选择这些基因进行进一步的研究，因为它们编码高度保守但鲜为人知的蛋白质家族成员，这些蛋白质家族以前没有涉及神经发育。这两个基因彼此没有关系，但都编码可能参与神经元内蛋白质运输的蛋白质，可以使用类似的方法进行研究。这两种基因在人类遗传疾病中都有同源物或亲属。(1)第一个基因spastin与常染色体显性痉挛性截瘫（ADSP）中受影响的人类基因同源。spastin编码一个AAA atp酶。这些atp酶参与催化蛋白质复合物的组装和拆卸，参与囊泡运输、蛋白质降解、微管动力学和其他过程。然而，对AAA atp酶序列的分析不能确定其参与的细胞过程，因此Spastin功能的靶标仍然未知。神经过度表达痉挛蛋白导致中枢神经系统（CNS）轴突向中线收敛，痉挛蛋白功能丧失（LOF）零突变幼虫在其神经肌肉连接处（NMJs）表现出突触形态的改变。它们还具有诱发连接电位（EJP）振幅降低，表明它们的NMJ突触异常。没有痉挛蛋白的动物存活到成年后不能飞行，行走能力差，寿命缩短。为了进一步分析Spastin的功能，我们将完成NMJs幼虫的形态和电生理分析。我们还将进行电生理测试，以检查突变成人不能飞行的原因，并检查成人大脑的结构缺陷和神经变性。我们将通过将导致人类痉挛的突变引入果蝇基因来研究人类ADSP的机制，并确定这些突变是否在果蝇中起显性阴性作用。为了确定Spastin作用途径的其他成分，我们将使用Spastin功能获得（GOF）眼表型进行增强子/抑制子遗传筛选。从眼屏幕中出现的候选基因将被测试用于spastin GOF轴突表型的修饰以及与spastin LOF突变的相互作用。(2)第二个基因bchs编码一种与BEACH结构域蛋白家族创始成员密切相关的蛋白，该蛋白的缺失导致Chediak-Higashi综合征（CHS），这是一种以免疫和神经缺陷为特征的致命遗传疾病。CHS患者的细胞中含有异常巨大的溶酶体，bchs在神经元中的过度表达产生一种独特的表型，在运动轴突干和侧分支之间的连接处形成凸起。bchs LOF突变导致大脑和眼睛的成年神经变性表型，并且bchs果蝇寿命短。在bchs幼虫中，一些运动轴突通路异常增厚，表明单个轴突肿胀或额外的轴突加入了通路。Bchs含有一个FYVE结构域，该结构域与磷酸磷脂酰肌醇-3-磷酸（Ptdlns3P）结合。它是一种囊泡蛋白，偶尔与含有ptdlns3p的核内体的荧光标记物（GFP-2XFYVE）共定位；然而，大多数Bchs囊泡与GFP-2XFYVE囊泡不同，表明它们代表不同的区室。为了研究Bchs，我们将分析其亚细胞定位并确定其起作用的囊室。我们将使用抗体染色、电子显微镜和电生理学检查幼虫神经肌肉系统中的LOF和功能获得（GOF）表型。我们还将寻找一种GOF眼表型的增强子和抑制子。将测试来自眼部筛查的候选基因对bchs GOF神经肌肉表型的修饰以及与bchs LOF突变的相互作用。

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.