AXON-TARGETED MICRODEVICES FOR CNS AXON TRANSPORT STUDIES

用于中枢神经系统轴突运输研究的轴突靶向微器件

• 批准号: 8048081
• 负责人: Shelly Elese Sakiyama-Elbert
• 金额: $ 19万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8048081
• 关键词: Affect; Alzheimer' s Disease; Area; Axon; Axonal Transport; Back; Biological Assay; Cell Adhesion; Cell Death; Cells; Cellular Stress; Controlled Environment; Data; Development; Devices; Dimensions; Disease; Disease model; Environment; Functional disorder; Genetic Models; Goals; Green Fluorescent Proteins; Growth; Image; Impairment; In Vitro; Injury; Knowledge; Label; Lead; Liquid substance; Microfabrication; Midbrain structure; Mitochondria; Molecular; Movement; Multiple Sclerosis; Mus; Nerve Degeneration; Neuraxis; Neurodegenerative Disorders; Neurons; Organelles; Oxidopamine; Parkinson Disease; Pathology; Patients; Pharmaceutical Preparations; Physiological; Play; Population; Process; Property; Public Health; Research; Research Personnel; Risk; Role; Sampling; Seeds; Side; Signal Transduction; Slide; Stroke; Synapses; System; Techniques; Testing; Time; Toxin; Transfection; Trauma; Tyrosine 3-Monooxygenase; Work; axon growth; axonal degeneration; design; disease mechanisms study; dopaminergic neuron; fluorophore; in vivo; insight; mimetics; nervous system disorder; neuronal cell body; novel; novel therapeutics; promoter; protein aggregation; prototype; public health relevance; research study; restraint; synaptic function; therapeutic development; tool; trafficking

DESCRIPTION (provided by applicant): Axonal injury is a central component in many neurological disorders including trauma, stroke, and multiple sclerosis as well as neurodegenerative disorders such as Alzheimer's (AD) and Parkinson's disease (PD). The overall goal of this research is to develop compartmentalized culture chambers using microfabrication techniques that isolate axons in culture to facilitate the study of axon growth, function and pathology. Specifically, the design and fabrication of microdevices will allow the quantitative delivery of pharmacological agents selectively to axons and allow their effects on axonal transport and growth to be examined in a controlled environment. By understanding the effects of toxins or drugs on axon growth and transport, we will gain mechanistic insight into the pathophysiology of neurodegenerative disorders and injury, which could in turn guide the development of therapeutics for treatment of degeneration or injury. The focus of this project is to develop open chamber microdevices that allow axonal isolation and the targeted application of drugs/toxins. Because patient data, genetic models, and in vivo and in vitro toxin studies all support a compelling role for axonal dysfunction in PD, we will use a well characterized PD model to study toxin effects in axons at risk in this disorder. Specifically, cultures of midbrain neurons from mice expressing green fluorescent protein (GFP) under the tyrosine hydroxylase (TH) promoter in our microdevices will allow us to test in real time the hypothesis that the Parkinson mimetic 6-OHDA (6-Hydroxydopamine) triggers changes in axonal transport resulting in the loss of axonal function and ultimately cell death. The specific aims of this proposal are to: (1) determine the design constraints for an open chamber design that allows easy cell seeding and axonal assays, which can be readily scaled for medium/high throughput cultures, (2) determine the design constraints for a novel microdevice that allows the application of a drug/toxin to axons (but not cell bodies) that are synapsing onto another population of neurons, and (3) use the new microdevices to test for the first time the effects of PD-mimetic 6-OHDA on mitochondrial trafficking in dopaminergic (DA) axons. PUBLIC HEALTH RELEVANCE: If the aims of this proposal are achieved, novel microdevices will become available to a large numbers of researchers trying to understand the role that axons play in neurological disease and injury. Establishing a causal role for axon dysfunction in neurodegenerative disorders such as Parkinson's and Alzheimer's disease is a challenging task given current methodological restraints. The tools proposed here will overcome current limitations in this field and further advance our knowledge in this area by providing unique platforms to study pharmacological and molecular mechanisms of disease and to screen for novel therapeutics.

描述（由申请人提供）：轴突损伤是许多神经系统疾病的核心组成部分，包括创伤、中风和多发性硬化症以及神经退行性疾病，例如阿尔茨海默病（AD）和帕金森病（PD）。 这项研究的总体目标是使用微加工技术开发分隔的培养室，分离培养物中的轴突，以促进轴突生长、功能和病理学的研究。 具体来说，微装置的设计和制造将允许选择性地将药物定量递送至轴突，并允许在受控环境中检查它们对轴突运输和生长的影响。 通过了解毒素或药物对轴突生长和运输的影响，我们将深入了解神经退行性疾病和损伤的病理生理学，这反过来可以指导退行性或损伤治疗方法的开发。 该项目的重点是开发允许轴突分离和药物/毒素的靶向应用的开放室微器件。 由于患者数据、遗传模型以及体内和体外毒素研究都支持轴突功能障碍在帕金森病中的重要作用，因此我们将使用特征明确的帕金森病模型来研究毒素对处于这种疾病风险中的轴突的影响。 具体来说，在我们的微型设备中，在酪氨酸羟化酶 (TH) 启动子的作用下表达绿色荧光蛋白 (GFP) 的小鼠的中脑神经元培养物将使我们能够实时测试以下假设：帕金森模拟物 6-OHDA（6-羟基多巴胺）会触发轴突运输的变化，导致轴突功能丧失并最终导致细胞死亡。 该提案的具体目标是：（1）确定开放室设计的设计约束，该设计允许轻松进行细胞接种和轴突测定，可以轻松地扩展到中/高通量培养，（2）确定新型微型设备的设计约束，该新型微型设备允许将药物/毒素应用于突触到另一神经元群体的轴突（但不是细胞体），以及（3）使用新的微型设备来测试 首次研究 PD 模拟 6-OHDA 对多巴胺能 (DA) 轴突线粒体运输的影响。 公共健康相关性：如果该提案的目标得以实现，大量研究人员将可以使用新型微型设备来了解轴突在神经系统疾病和损伤中所起的作用。 鉴于目前方法学的限制，确定轴突功能障碍在帕金森病和阿尔茨海默病等神经退行性疾病中的因果作用是一项具有挑战性的任务。 这里提出的工具将克服该领域当前的局限性，并通过提供独特的平台来研究疾病的药理学和分子机制以及筛选新的治疗方法，进一步推进我们在该领域的知识。