Force Clamp Systems for Evaluation of Mechanotransduction

描述（由申请人提供）：本研究的长期目标是发现负责触觉和本体感觉的力传递和力转导通路。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，从坐到站，再到跑；然而，它们的功能在遗传性和获得性疾病中都受到破坏，包括艾滋病毒/艾滋病和糖尿病。甚至感觉功能的部分丧失，如糖尿病周围神经病变（DPN），也有毁灭性的后果；DPN影响了大约1500万美国人，是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，触觉和本体感觉功能障碍的诊断工具和治疗方法仍然不发达，主要是因为对这些感觉的工作原理知之甚少。这种知识差距反映了缺乏足够的设备来提供受控的机械刺激和适合于触觉机械生物学分析的动物模型。这项研究的目的是通过开发新的控制力传递装置，改进动物模型来解剖力传递和转导途径，以及新的分析方法来研究这一基本生命过程的相关机制，从而弥补这一空白。这项拟议中的研究使用了简单的蛔虫秀丽隐杆线虫，因为人们对它的触觉的了解比其他任何动物都多。利用秀丽隐杆线虫的研究已经成功地揭示了几个基本的和保守的生物过程的机制方面，包括触摸感觉。例如，在秀丽隐杆线虫中，大约20年前首次发现了触觉所需的离子通道蛋白。因为类似的蛋白质在哺乳动物的触觉感受器神经元中也有表达，它们可能也对触觉有贡献。目前，秀丽隐杆线虫是唯一一种我们知道哪些蛋白质形成了触觉受体神经元中负责检测力的机电转导通道的动物。这些知识使我们能够进行目前在哺乳动物模型中无法获得的分析。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、神经元位置和细胞内细胞骨架结构的相互作用决定的。为了验证这一假设，我们将开发新的触摸灵敏度定量评估指标；新的微加工工具适合传递pN-5N力，新的触觉受体神经元体外模型，并建立新的力传递和力转导模型。具体目的是：1)验证皮肤力学、神经元位置和神经元细胞骨架在体内调节触摸敏感性的假说；2)评估体壁肌张力和内部静水压力对体内触觉的影响；3)确定机电转导通道的激活和适应机制。

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.