Genetic Analysis of the Roles and Regulation of Microtubule Severing in Neurons

描述（由申请人提供）：我们研究的长期目标是了解神经元发育和功能中细胞骨架的调节。特别是我们专注于微管细胞骨架，这是必不可少的神经元功能的许多方面，包括轴突生长，突触的形成和重塑，以及细胞体和远端隔间之间的细胞成分的运输。虽然动态不稳定性，即微管在其末端生长和收缩的过程，仍然是微管聚合物长度被认为是调节的主要机制，但称为微管切断的额外机制的重要性变得越来越明显。微管切割蛋白是ATP酶，其结合并切割聚合物中心能量更稳定区域的微管。它们存在于从植物到人类的各种门中，并已被证明在生长，细胞分裂和神经系统功能中发挥关键作用。在人类中，微管切断蛋白Spastin的突变是常染色体显性遗传性痉挛性截瘫的主要原因，这是一种使人衰弱的神经退行性疾病，其中中枢神经系统的最长轴突束退化。我们在果蝇中的工作表明，Spastin的缺失导致远端突触终扣中微管的缺乏，以及突触传递的减少。这些动物表现出严重受损的运动行为，让人想起人类疾病。本提案的目标是了解细胞生物学事件，其中微管切断在神经元中使用，以及这些过程的调节。在发育中的果蝇神经系统中表达三种主要蛋白质：spastin，katanin-60（kat 60）和kat-like。每一种都可能在不同的细胞类型、发育阶段或细胞区室中发挥作用;或者，它们可能是功能冗余的。基于它们的胚胎表达模式、突变表型和我们通过遗传筛选鉴定的调节蛋白的特异性，我们假设每种蛋白在神经元功能中具有独立的作用，但可能是部分冗余的。为了验证这一假设，我们将：1）表征这些基因中单个和组合功能丧失突变体的神经元形态和微管分布，2）阐明痉挛蛋白遗传调节因子DPak 3，Cdk 5和Pctaire的作用，以及3）对kat 60和kat样基因修饰剂进行体内筛选。我们将利用果蝇的优势来实现这些目标，重点是在实验室中建立良好的遗传和成像方法的组合。公共卫生相关性：该项目的目标是阐明预测在苍蝇中切断神经元微管的三种蛋白质的作用和调节：spastin，kat 60和kat样。微管的失调与许多人类疾病有关，包括遗传性痉挛性截瘫（由痉挛素突变引起），阿尔茨海默病和许多形式的癌症。因此，对这些蛋白质的理解将广泛有益于人类健康，揭示控制微管生长和/或损失的途径，从而为治疗干预提供额外的靶点。

DESCRIPTION (provided by applicant): The long-term goal of our research is to understand the regulation of the cytoskeleton in neuronal development and function. In particular we focus on the microtubule cytoskeleton, which is essential to many aspects of neuronal function, including neurite outgrowth, synapse formation and remodeling, and transport of cellular components between the cell body and distal compartments. Although dynamic instability, the process by which microtubules grow and shrink at their ends, remains the predominant mechanism by which microtubule polymer length is thought to be regulated, the importance of an additional mechanism known as microtubule severing is becoming increasingly apparent. Microtubule severing proteins are ATPases that bind to and cut the microtubule in the central, more energetically stable region of the polymer. They are found in phyla ranging from plants to humans, and have been shown to play critical roles in growth, cell division and nervous system function. In humans, mutations in the microtubule severing protein Spastin are the major cause of Autosomal Dominant Hereditary Spastic Paraplegia, a debilitating neurodegenerative disease in which the longest axon tracts of the central nervous system degenerate. Our work in Drosophila has shown that loss of Spastin leads to a paucity of microtubules in distal synaptic boutons, as well as reduced synaptic transmission. These animals exhibit severely compromised motor behavior, reminiscent of the human disease. The goal of this proposal is to understand the cell biological events in which microtubule severing is utilized in neurons, and the regulation of these processes. Three severing proteins are expressed in the developing fly nervous system: spastin, katanin-60 (kat60), and kat-like. Each may function in distinct cell types, developmental stages, or cell compartments; alternatively, they may be functionally redundant. Based on their embryonic expression patterns, mutant phenotypes, and specificity of regulatory proteins we have identified through genetic screens, we hypothesize that each protein has an independent role(s) in neuronal function, but may be partially redundant. To test this hypothesis we will: 1) characterize neuronal morphology and microtubule distribution in single and combinatorial loss-of- function mutants in these genes, 2) elucidate the roles of the spastin genetic regulators DPak3, Cdk5, and Pctaire, and 3) execute in vivo screens for genetic modifiers of kat60 and kat-like. We will exploit the strengths of Drosophila to achieve these goals, focusing on a combination of genetic and imaging approaches that are well-established in the lab. PUBLIC HEALTH RELEVANCE: The goals of this project are to elucidate the roles and regulation of the three proteins predicted to sever neuronal microtubules in the fly: spastin, kat60, and kat-like. Misregulation of microtubules is implicated in a host of human diseases, including Hereditary Spastic Paraplegia (caused by mutations in spastin), Alzheimer's Disease, and many forms of cancer. An understanding of these proteins will therefore be broadly beneficial to human health, revealing pathways controlling microtubule growth and/or loss, and thereby providing additional targets for therapeutic intervention.