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Regulation of synaptic proteasome activity by the dynactin complex

dynactin 复合物对突触蛋白酶体活性的调节

基本信息

批准号：
8016005
负责人：
Benjamin Arthur Eaton
金额：
$ 31.83万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-02-01 至 2015-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8016005
关键词：
Affect Alzheimer&apos s Disease Amyotrophic Lateral Sclerosis Axonal Transport Binding Binding Proteins Biochemical Biological Assay Cell membrane Cellular Assay Cellular biology Complex Data Deposition Development Disease Drosophila genus Dynein ATPase Enhancers Event Family Fluorescence Frontotemporal Dementia Functional disorder Genes Genetic Genetic Screening Genetic Techniques Glues Goals Golgi Apparatus Growth Guanosine Triphosphate Phosphohydrolases Health Homologous Gene Human Human Pathology Image Impairment In Vitro Investigation Link Lipid Binding Maintenance Mediating Mediation Membrane Modeling Molecular Molecular Genetics Monomeric GTP-Binding Proteins Mus Mutation Nerve Nerve Degeneration Nervous System Physiology Nervous system structure Neurodegenerative Disorders Neurons Neurophysiology - biologic function Onset of illness Parkinson Disease Pathogenesis Pathway interactions Phenocopy Process Proteasome Inhibition Proteins Reagent Regulation Reporter Resolution Role Sequence Analysis Signal Transduction Structure Synapses System Tertiary Protein Structure Vesicle base design dynactin fly human disease in vivo interest multicatalytic endopeptidase complex mutant nervous system disorder neurodegenerative phenotype neurological pathology neurotransmission novel novel strategies overexpression presynaptic progressive neurodegeneration protein aggregate protein complex protein degradation response rho synaptic function tool trafficking

项目摘要

DESCRIPTION (provided by applicant): Pinpointing the underlying cause of many neurodegenerative diseases has been difficult due to the complexity of neuronal cellular biology and the lack of basic information about impaired mechanisms that contribute to disease. Mutations in the human DCTN1 gene, which encodes a component of the dynactin complex, have been strongly linked to both familial and sporadic cases of amyotrophic lateral sclerosis (ALS). Many of the neurodegenerative phenotypes associated with human disease have been recapitulated in flies and mice harboring dynactin complex mutations demonstrating a conserved pathogenesis in dynactin complex mutants. To clarify the mechanisms that contribute to the pathogenesis of neurodegeneration observed in dynactin complex mutants, especially the degeneration of synaptic contacts, we will use a combination of forward genetic screens and quantitative cellular assays to identify and characterize important modifiers of dynactin complex function at the synapse. We believe that this approach has the potential to identify and describe new genes and pathways required within the nervous system for normal synaptic growth, synapse stabilization, and function. A genetic screen designed to specifically identify genetic modifiers of the Drosophila DCTN1 homolog, glued, within the nervous system has identified the Drosophila homologue of the Arfaptin2 gene, Darfaptin2 (Darf2). We find that Darf2 is expressed in motorneurons and required for normal synaptic growth. The goal of this proposal is to investigate the hypothesis that Darfaptin2 (Darf2) represents a novel component of the dynactin complex required for normal synaptic growth, stabilization, and neurotransmission. Models of Arfaptin2 function include the mediation of cross-talk between Rho-like GTPases and Arf family GTPases during vesicle formation, and the regulation of proteasome activity within neurons. Using standard genetic techniques and synaptic analyses, we will first define the role of Darf2 in the regulation of synaptic growth and synapse stabilization. This will include the biochemical analysis of its association with the dynactin complex in the nervous system (Aim1). We will further investigate the role of Darf2 in the nervous system using a structure-function approach to define protein domains required for normal Darf2 activity. This will include determining the signaling context for Darf2 during synapse growth and stabilization (Aim2). Finally, we are developing reagents and assays to directly investigate the regulation of proteasome function in the nerve terminal by the dynactin complex, Darf2, and during synapse retraction (Aim3). It is expected that these studies will reveal a novel regulatory mechanism during synaptic growth, stabilization, and neurotransmission that will have important implications for the pathogenesis of late onset neurological diseases. PUBLIC HEALTH RELEVANCE: A hallmark of neurodegenerative disease is the early and prominent loss of synaptic contacts observed throughout the nervous system that tightly correlates with the decline in neural function. Recent data has demonstrated the importance synapse degeneration to the onset of disease but mechanisms involved in the maintenance of synaptic contacts remain unclear. We predict that studies aimed at elucidating the molecular mechanism associated with the regulation of synapse maintenance will have broad applications to neurological disease and provide the basis for novel strategies for treatment.

描述（由申请人提供）：由于神经元细胞生物学的复杂性和缺乏有关导致疾病的受损机制的基本信息，很难确定许多神经退行性疾病的根本原因。人类DCTN 1基因（编码动力蛋白复合物的一种成分）的突变与肌萎缩性侧索硬化症（ALS）的家族性和散发性病例密切相关。许多与人类疾病相关的神经退行性表型已在携带强肌动蛋白复合物突变的果蝇和小鼠中重现，证明了强肌动蛋白复合物突变体中保守的发病机制。为了阐明在动力肌动蛋白复合体突变体中观察到的神经变性的发病机制，特别是突触接触的变性，我们将使用正向遗传筛选和定量细胞测定的组合来识别和表征突触处动力肌动蛋白复合体功能的重要修饰剂。我们相信，这种方法有可能识别和描述神经系统内正常突触生长，突触稳定和功能所需的新基因和途径。一项旨在特异性鉴定果蝇DCTN 1同源物（胶合）在神经系统内的遗传修饰剂的遗传筛选已经鉴定出Arfaptin 2基因的果蝇同源物Darfaptin 2（Darf 2）。我们发现Darf 2在运动神经元中表达，并且是正常突触生长所必需的。该提案的目标是研究Darfaptin 2（Darf 2）代表正常突触生长，稳定和神经传递所需的dynactin复合物的新组分的假设。Arfaptin 2功能的模型包括在囊泡形成期间Rho样GTP酶和Arf家族GTP酶之间的串扰的介导，以及神经元内蛋白酶体活性的调节。使用标准的遗传技术和突触分析，我们将首先确定的作用，Darf 2的突触生长和突触稳定的调节。这将包括其与神经系统中的dynactin复合物（Aim 1）相关的生化分析。我们将使用结构-功能方法进一步研究Darf 2在神经系统中的作用，以确定正常Darf 2活性所需的蛋白质结构域。这将包括确定突触生长和稳定（Aim 2）期间Darf 2的信号背景。最后，我们正在开发试剂和检测方法，以直接研究在神经末梢的dynactin复合物，Darf 2的蛋白酶体功能的调节，并在突触回缩（AIM 3）。预计这些研究将揭示突触生长、稳定和神经传递过程中的一种新的调控机制，这将对晚发性神经系统疾病的发病机制产生重要影响。公共卫生关系：神经退行性疾病的一个标志是在整个神经系统中观察到的突触接触的早期和显著丧失，这与神经功能的下降密切相关。最近的数据已经证明了突触变性对疾病发作的重要性，但是涉及突触接触的维持的机制仍然不清楚。我们预测，旨在阐明与突触维持调节相关的分子机制的研究将对神经系统疾病具有广泛的应用，并为新的治疗策略提供基础。