AXON-TARGETED MICRODEVICES FOR CNS AXON TRANSPORT STUDIES

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、多发性硬化症以及神经退行性疾病，如阿尔茨海默病（AD）和帕金森病（PD）。本研究的总体目标是利用微制造技术开发分离培养轴突的隔间培养室，以促进轴突生长、功能和病理的研究。具体来说，微装置的设计和制造将允许有选择地定量递送药物到轴突，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，我们将获得神经退行性疾病和损伤的病理生理学机制，这反过来可以指导治疗退行性疾病或损伤的治疗方法的发展。该项目的重点是开发开放室微型装置，使轴突分离和药物/毒素的靶向应用成为可能。由于患者数据、遗传模型以及体内和体外毒素研究都支持PD中轴突功能障碍的重要作用，因此我们将使用一个具有良好特征的PD模型来研究这种疾病中轴突中的毒素作用。具体来说，在我们的微设备中，在酪氨酸羟化酶（TH）启动子下表达绿色荧光蛋白（GFP）的小鼠中脑神经元的培养将使我们能够实时测试帕金森模拟6-OHDA（6-羟多巴胺）触发轴突运输变化导致轴突功能丧失并最终导致细胞死亡的假设。这项建议的具体目的是：(1)确定开放室设计的设计限制，允许简单的细胞播种和轴突试验，可以很容易地扩展到中/高通量培养；(2)确定一种新型微设备的设计限制，允许将药物/毒素应用于与另一群神经元突触的轴突（而不是细胞体）；(3)利用新型微装置首次检测了pd -拟6-OHDA对多巴胺能（DA）轴突线粒体运输的影响。

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.