AXON-TARGETED MICRODEVICES FOR CNS AXON TRANSPORT STUDIES

描述(申请人提供)：轴突损伤是许多神经疾病的中心组成部分，包括创伤、中风和多发性硬化症，以及神经退行性疾病，如阿尔茨海默氏症(AD)和帕金森氏病(PD)。本研究的总体目标是利用微细加工技术分离培养中的轴突，以便于研究轴突的生长、功能和病理。具体地说，微设备的设计和制造将允许有选择地将药物定量输送到轴突，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，我们将从机制上深入了解神经退行性疾病和损伤的病理生理学，进而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腔微设备，允许轴突分离和药物/毒素的靶向应用。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，我们将使用一个具有良好特征的帕金森病模型来研究这种疾病中处于危险状态的轴突中的毒素效应。具体地说，在我们的微型设备中，通过培养在酪氨酸羟基酶(TH)启动子下表达绿色荧光蛋白(GFP)的小鼠中脑神经元，我们可以实时测试以下假设：模拟帕金森症的6-OHDA(6-羟基多巴胺)会触发轴突运输的变化，从而导致轴突功能丧失，最终导致细胞死亡。这项建议的具体目的是：(1)确定能够轻松进行细胞播种和轴突分析的开放式小室设计的设计约束，这种设计可以方便地进行中高通量培养的轴突分析；(2)确定一种新型微设备的设计约束，该微设备允许将药物/毒素应用于突触到另一组神经元的轴突(但不是细胞体)，以及(3)使用新的微设备首次测试PD模拟6-OHDA对多巴胺能(DA)轴突中线粒体运输的影响。 与公共健康相关：如果这项提案的目标得以实现，大量试图了解轴突在神经疾病和损伤中所起作用的研究人员将可以使用新型微型设备。鉴于目前的方法限制，确定轴突功能障碍在帕金森氏症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目前这一领域的限制，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，进一步促进我们在这一领域的知识。

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.