Force Clamp Systems for Evaluation of Mechanotransduction

描述(申请人提供)：这项研究的长期目标是发现负责触摸和本体感觉的力传递和力传递路径。这些感官对于社会交流和日常生活的方方面面都是必不可少的，从坐着、站着到跑步；然而，它们的功能在遗传性和获得性疾病中都被破坏了，包括艾滋病毒-艾滋病和糖尿病。即使是像糖尿病周围神经病变(DPN)那样的感觉功能部分丧失也会造成毁灭性的后果；据估计，DPN影响着1500万美国人，是导致下肢截肢的主要危险因素。因此，触觉和本体感觉的丧失是常见的，不仅与不适和疼痛有关，而且还与生活质量的下降有关。尽管如此，对于触觉和本体感觉功能障碍的诊断工具和治疗方法仍然很不发达，主要是因为人们对这些感觉是如何工作的知之甚少。这一认识差距反映了缺乏足够的设备来传递受控的机械刺激，以及缺乏能够分析触摸感觉的机械生物学的动物模型。拟议研究的目的是通过开发新的受控力量传递装置，改进解剖力量传递和转导途径的动物模型，以及基础研究这一基本生命过程的相关机制的新分析方法，来弥合这一差距。这项拟议的研究使用了简单的线虫--秀丽线虫，因为人们比任何其他动物都更了解它的触觉。对线虫的研究已经成功地揭示了几个基本和保守的生物过程的机械方面，包括触摸感觉。例如，大约20年前，在线虫中发现了第一个触觉所需的离子通道蛋白。由于类似的蛋白质在哺乳动物的触觉感受器神经元中表达，它们也可能对触觉起作用。目前，线虫是唯一一种我们知道哪些蛋白质形成机械-电转导通道的动物，该通道负责检测触摸感受器神经元中的力。这一知识使分析达到了目前在哺乳动物模型中无法获得的水平。我们正在测试的中心假设是，力敏感性和反应动力学都是由皮肤力学、神经元位置以及细胞内和细胞骨架结构的相互作用决定的。为了验证这一假设，我们将开发用于定量评估触摸敏感性的新指标、适用于传递PN-5N力的新微制造工具、新的触摸感受器神经元的体外模型，并建立新的力传递和力传导模型。具体目的是：1)在活体内验证皮肤力学、神经元位置和神经元细胞骨架调节触觉敏感性的假说；2)在活体内评估体壁肌张力和体内静水压力对触觉的影响；3)确定机电转导通道激活和适应的机制。 与公共健康相关：正常的触觉和本体感觉对日常生活至关重要，需要专门的机械感受器神经元激活。当这些神经元的功能因衰老、疾病(艾滋病毒-艾滋病、糖尿病)和医疗干预(化疗)而被破坏时，轻微的损伤往往会导致伤口无法愈合，只能通过截肢来治疗。这种疾病折磨着数以百万计的美国人，每年造成数百亿美元的医疗费用。通过揭示力量从皮肤传递到机械感受器神经元的机制，并开发用于研究的微型组装工具，这些研究可能为1)干预措施提供新的策略，这些干预措施可以恢复感觉神经病患者的敏感性，以及2)改进的诊断工具，可能有助于应用干预措施，将感觉功能的损失降至最低。

DESCRIPTION (provided by applicant): The long-term goal of this research is to discover the force transmission and force transduction pathways responsible for touch and proprioception. These senses are essential for social communication and every aspect of daily life from sitting, to standing, to running; however, their function is disrupted in both inherited and acquired diseases, including HIV-AIDS and diabetes. Even partial loss of sensory function as in diabetic peripheral neuropathy (DPN) has devastating consequences; DPN affects an estimated 15 million Americans and is the dominant risk factor in lower limb amputations. Thus, the loss of touch and proprioception is common, associated not only with discomfort and pain, but also with a decrease in the quality of life. Despite this, diagnostic tools and treatments for the dysfunction of touch and proprioception remain poorly developed, principally because little is known about how these senses work. This knowledge gap reflects a lack of adequate devices for delivering controlled mechanical stimuli and of animal models amenable to analysis of the mechanobiology of touch sensation. The objective of the proposed research is to bridge this gap by developing new devices for controlled force delivery, improved animal models for dissecting force transmission and transduction pathways, and new analytical methods for fundamental study of the relevant mechanics of this basic life process. The proposed research uses the simple roundworm, Caenorhabditis elegans, because more is understood about its sense of touch than that of any other animal. Research using C. elegans has successfully revealed mechanistic aspects of several fundamental and conserved biological processes, including touch sensation. It was in C. elegans, for instance, that the first ion channel proteins required for touch sensation were identified ~20 years ago. Because analogous proteins are expressed in mammalian touch receptor neurons, they may also contribute to touch sensation. At present, C. elegans is the only animal in which we know which proteins form the mechano-electrical transduction channels responsible for detecting force in touch receptor neurons. This knowledge enables a level of analysis that is not currently available in mammalian models. The central hypothesis we are testing is that both force-sensitivity and response dynamics are determined by the interplay of skin mechanics, neuron position, and intracellular, cytoskeletal structures. To test this hypothesis, we will develop new metrics for quantitative assessment of touch sensitivity; new microfabricated tools suitable for delivering pN-5N forces, new in vitro models of touch receptor neurons, and build new models of force transmission and force transduction. The specific aims are: 1) Test the hypothesis that skin mechanics, neuron position, and the neuronal cytoskeleton regulate touch sensitivity in vivo; 2) Assess the impact of body wall muscle tone and internal hydrostatic pressure on touch sensation in vivo; 3) Identify mechanisms of mechano- electrical transduction channel activation and adaptation. PUBLIC HEALTH RELEVANCE: Normal touch sensation and proprioception are essential for daily life and require the activation of specialized mechanoreceptor neurons. When the function of such neurons is disrupted by aging, disease (HIV-AIDS, diabetes), and medical interventions (chemotherapy), small injuries often lead to wounds that fail to heal and are treated only by limb amputation. Such pathologies afflict millions of Americans and account for tens of billions of dollars in health-care costs annually. By revealing the mechanisms by which force is transmitted from the skin to mechanoreceptor neurons and developing microfabricated tools for research, these studies may provide new strategies for 1) interventions that could restore sensitivity in individuals afflicted by sensory neuropathy and for 2) improved diagnostic tools that may aid in the application of interventions that minimize the loss of sensory function.