Neuronal Polarity and Membrane Trafficking

神经元极性和膜运输

• 批准号: 7619105
• 负责人: GARY A BANKER
• 金额: $ 46.12万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-09 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7619105
• 关键词: Accounting; Acetylation; Affect; Animal Model; Award; Axon; Binding; Biochemical; Biological Assay; Carrier Proteins; Cell membrane; Cells; Color; Data; Defect; Dendrites; Development; Family member; Golgi Apparatus; Heart; Hippocampus (Brain); Human; Image; Individual; Kinesin; Label; Life; Mediating; Membrane Protein Traffic; Membrane Proteins; Membrane Transport Proteins; Methods; Microtubules; Modification; Molecular; Motor; Nerve Degeneration; Neurites; Neurodegenerative Disorders; Neuronal Dysfunction; Neurons; Organelles; Pathway interactions; Play; Population; Post-Translational Protein Processing; Proteins; RNA Interference; Role; Site; Tubulin; Variant; Work; anterograde transport; base; cellular imaging; knock-down; novel; preference; protein transport; public health relevance; trafficking

DESCRIPTION (provided by applicant): Nearly every aspect of neuronal function depends on the accurate polarization of membrane proteins to axons or dendrites. During the current award period we have identified the principal trafficking pathways that underlie the polarized localization of neuronal plasma membrane proteins in cultured hippocampal neurons and developed methods to image each of the different populations of the long-range carriers that transport proteins along these pathways. Our results show that the selectivity of anterograde, kinesin- mediated transport plays a central role in the targeting of polarized proteins. Carriers containing dendritic proteins are transported into dendrites but excluded from axons; carriers containing axonal proteins enter both dendrites and axons, but are transported preferentially into axons. We also developed an assay to assess the selective transport of constitutively active kinesin motor domains in the absence of cargo. We demonstrated that Kinesin-1 motor domains translocate preferentially into the axon, whereas a Kinesin-3 motor domain translocates with equal efficiency into both axons and dendrites. Recent evidence demonstrates that posttranslational modifications of tubulin (acetylation and glutamylation) regulate the efficiency of kinesin translocation and our preliminary data show that axonal and dendritic microtubules differ in both of these posttranslational modifications. Using the unique methods we have developed during the current award period, we now propose to identify the kinesins that transport each population of long-range carriers and to investigate the molecular features of kinesins and the molecular modifications of microtubules that determine the selectivity of carrier transport in hippocampal neurons. We will use two-color imaging to identify the carrier populations labeled by expressed kinesins. We will also use RNAi to inhibit the expression of individual kinesins and evaluate the populations of long-range carriers that are affected. We will compare the selectivity of motor domain translocation for all kinesin organelle motors expressed in hippocampal neurons and examine how posttranslational modifications of tubulin influence the selectivity of motor domain transport and the transport of long-range carriers. Defects in kinesin-mediated transport cause neuronal dysfunction in animal models and have been implicated in several human neurodegenerative diseases. Our results will identify novel targets for pharmacological manipulations that could compensate for transport defects and protect against neural degeneration. PUBLIC HEALTH RELEVANCE: Nearly every aspect of neuronal function depends on the accurate transport and trafficking of membrane proteins; defects in the long-range transport of membrane proteins are thought to underlie several neurodegenerative diseases. By elucidating the regulatory mechanisms that underlie the accuracy of kinesin-driven transport, our work may identify a novel set of targets for pharmacologic manipulations that could enhance long-range transport and perhaps ameliorate neural degeneration.

描述（由申请人提供）：神经元功能的几乎每个方面都取决于膜蛋白与轴突或树突的准确极化。在当前的奖励期间，我们确定了主要的运输途径，这些途径是神经元质膜蛋白在培养的海马神经元中的两极分化定位，并开发了方法来对沿这些途径运输蛋白质的远程载体的每个不同种群进行成像。我们的结果表明，动力素介导的转运的选择性在极化蛋白的靶向中起着核心作用。含有树突蛋白的载体被转运到树突中，但被排除在轴突之外。含有轴突蛋白的载体同时进入树突和轴突，但优先运输到轴突中。我们还开发了一种测定法，以评估在没有货物的情况下组成型活性运动蛋白运动结构域的选择性运输。我们证明了驱动蛋白-1电动机结构域优先转移到轴突中，而驱动蛋白3电动机结构域则以同等效率转移到轴突和树突上。最近的证据表明，小管蛋白的翻译后修饰（乙酰化和谷氨酸化）调节驱动蛋白易位的效率和我们的Preliriminal Data表明，这两种翻译后修饰中的轴突和树突小管都有所不同。使用我们在当前奖励期内开发的独特方法，我们现在建议鉴定运输每个远程载体的每个种群的动力素，并研究驱动蛋白的分子特征以及确定海马神经元中载体转运的微管的分子修饰。我们将使用两色成像来识别由表达的驱动蛋白标记的载体种群。我们还将使用RNAi抑制单个驱动蛋白的表达，并评估受影响的远程载体的种群。我们将比较在海马神经元中表达的所有驱动蛋白细胞器电动机的运动结构域易位的选择性，并检查微管蛋白的翻译后修饰如何影响运动结构域运输的选择性和远距离载体的运输。驱动蛋白介导的转运缺陷会导致动物模型的神经元功能障碍，并与几种人类神经退行性疾病有关。我们的结果将确定可以弥补运输缺陷并防止神经变性的药理操作的新颖靶标。公共卫生相关性：神经元功能的几乎每个方面都取决于膜蛋白的准确运输和运输；膜蛋白的远距离运输中的缺陷被认为是几种神经退行性疾病的基础。通过阐明动力蛋白驱动运输准确性的调节机制，我们的工作可以确定一组新型的药理学操纵目标，这些目标可以增强长期运输并可能改善神经变性。