Signaling Mechanisms in Drosophila Neural Development

描述（由申请人提供）：本提案涉及痉挛素和蓝奶酪搁浅（bchs），这两个基因是我们在果蝇幼虫中进行的涉及运动轴突指导和突触发生的基因筛选中鉴定的。我们选择这些基因进行进一步研究，因为它们编码高度保守但知之甚少的蛋白质家族成员，这些蛋白质家族以前没有涉及神经发育。这两个基因彼此无关，但都编码可能参与神经元内蛋白质运输的蛋白质，并且可以使用类似的方法进行研究。这两个基因都有直系同源物或亲属，在人类遗传疾病中受到影响。(1)第一个基因，spastin，是一个人类基因的直系同源物影响常染色体显性遗传性痉挛性截瘫（ADSP）。spastin编码AAA ATP酶。这些ATP酶参与催化蛋白质复合物的组装和分解，所述蛋白质复合物参与囊泡运输、蛋白质降解、微管动力学和其他过程。然而，AAA ATP酶序列的分析不允许定义其参与的细胞过程，因此Spastin功能的靶点仍然未知。痉挛素神经元过表达会导致中枢神经系统（CNS）轴突会聚到中线，痉挛素功能丧失（LOF）无效突变幼虫在其神经肌肉接头（NMJ）处表现出突触形态的改变。他们也有减少诱发交界电位（EJP）幅度，表明他们的NMJ突触异常。存活到成年的无寄生虫的动物不能飞行，行走能力差，寿命缩短。为了进一步分析Spastin的功能，我们将完成幼虫NMJ的形态学和电生理分析。我们还将进行电生理测试，以检查为什么突变的成年人不能飞，并检查成年人的大脑结构缺陷和神经退行性变。我们将通过将导致人类痉挛的突变引入果蝇基因并确定这些突变是否在果蝇中作为显性负性来研究人类ADSP的机制。为了确定Spastin作用的途径的其他组分，我们将使用Spastin功能获得性（GOF）眼表型进行增强子/抑制子遗传筛选。将测试从眼筛选中出现的候选基因的痉挛蛋白GOF轴突表型的修饰以及与痉挛蛋白LOF突变的相互作用。(2)第二个基因bchs编码一种与海滩结构域蛋白家族的创始成员密切相关的蛋白质：这种蛋白质的缺失会导致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患者的细胞含有异常的巨大溶酶体，bchs在神经元中的过表达产生独特的表型，其中在运动轴突干和侧支之间的连接处形成隆起。bchs LOF突变导致大脑和眼睛中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，这表明单个轴突肿胀或额外的轴突加入了通路。Bchs含有FYVE结构域，其结合磷脂酰肌醇-3-磷酸（Ptdlns 3 P）。它是一种囊泡蛋白，偶尔与含Ptdlns 3 P的内体的荧光标记物（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隔室。我们将研究bchs LOF和获得功能（GOF）的表型在幼虫神经肌肉系统中使用抗体染色，电子显微镜和电生理学。我们还将寻找bchs GOF眼表型的增强子和抑制子。将测试来自眼部筛选的候选基因对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.