The Role of Mechanosensation Pathways in Osteoarthritis Joint Damage and Pain

项目摘要 膝关节骨关节炎（OA）是一种痛苦的慢性疾病，影响着美国2700万人。膝关节OA是 其特征在于所有关节组织的进行性损伤和重塑。OA的主要特征是关节疼痛 X光片显示关节间隙变窄（软骨丧失）。生物力学因素在两关节中起重要作用 疼痛和损伤，但确切地说，机械力如何作用于感觉神经元和软骨，以驱动 疾病未知。我们的长期目标是阐明机械负荷是如何被关节组织感知的， 对负荷的反应如何影响OA。细胞通过多种机制感知机械力， 包括机械敏感离子通道。最近，感觉神经元机械敏感离子通道 PIEZO 2被鉴定为在炎性和慢性炎症的鼠模型中机械性异常性疼痛的关键贡献者。 PIEZO 2在神经病理性疼痛中的作用，但由伤害感受器表达的PIEZO 2和在持续性疼痛中的作用尚不清楚。 此外，软骨细胞中机械敏感离子通道的功能尚不清楚，但基于 先前的工作暗示了PIEZO 1在软骨细胞对机械刺激的反应中的作用，我们假设 PIEZO 1导致OA的组织损伤。总体而言，尚不清楚PIEZO离子通道信号传导如何 导致OA病理学（疼痛和关节损伤）。我们的目标是解决这个问题，通过使用新的 我们已经开发的技术，使机械刺激的应用，以完整的组织，同时执行 钙或电压成像，以真实的时间评估细胞功能。这一提议涉及中心假设 伤害感受器和软骨细胞中的PIEZO通道信号在开始时驱动疼痛和关节损伤。 和OA的进展。中心假设将在两个具体目标中进行检验：1）确定 伤害感受器表达的PIEZO 2离子通道介导外周敏化和持续痛 在OA DMM模型的不同阶段的行为;和2）研究软骨细胞PIEZO 1离子是否 通道信号传导促进DMM模型中的关节损伤和疼痛。为了实现这些目标，我们 产生的创新技术，使机械刺激的应用程序，以完整的组织，同时执行 实时钙或电压成像。我们创造了表达荧光钙（GCaMP 6）或 电压（ASAP 2s）指示蛋白在疼痛感觉神经元（伤害感受器; NaV1.8 cre）或 软骨细胞（Col 2a 1 cre）。我们也有两种类型的伤害感受器特异性Piezo 2条件性敲除小鼠 以及软骨细胞特异性Piezo 1条件性敲除小鼠。成功实现这些目标将 提高我们对伤害感受器和软骨细胞如何使用PIEZO通道来响应 OA中的机械负荷，这可能导致识别针对疼痛和关节的新途径 治疗上的伤害。通过测量伤害感受器-Piezo 2中的疼痛相关行为和关节损伤， 敲除和软骨细胞-Piezo 1敲除小鼠，这种方法将提供对 这些进程将为今后的工作奠定基础。

Project Summary Knee osteoarthritis (OA) is a painful chronic disease affecting 27 million people in the US. Knee OA is characterized by progressive damage and remodeling of all joint tissues. Major hallmarks of OA are joint pain and joint space narrowing on x-ray (cartilage loss). Biomechanical factors play an important role in both joint pain and damage, but exactly how mechanical forces act on sensory neurons and cartilage to drive the disease is unknown. Our long-term goal is to elucidate how mechanical loading is sensed by joint tissues and how responses to loading contribute to OA. Cells sense mechanical forces through a variety of mechanisms, including mechanosensitive ion channels. Recently, the sensory neuron mechanosensitive ion channel PIEZO2 was identified as a key contributor to mechanical allodynia in murine models of inflammatory and neuropathic pain, but the role of PIEZO2 expressed by nociceptors and in persistent pain is not clear. Furthermore, the function of mechanosensitive ion channels in chondrocytes is less clear, but based on previous work implicating a role for PIEZO1 in chondrocyte responses to mechanical stimuli, we hypothesize that PIEZO1 contributes to tissue damage in OA. Overall it is unknown how PIEZO ion channel signaling contributes to OA pathology (pain and joint damage). We aim to address this question by using novel techniques we have developed that enable application of mechanical stimuli to intact tissues while performing calcium or voltage imaging to assess cell function in real time. This proposal addresses the Central Hypothesis that: PIEZO channel signaling in nociceptors and chondrocytes drives pain and joint damage in the initiation and progression of OA. The central hypothesis will be tested in two specific aims: 1) To define the role of PIEZO2 ion channel expressed by nociceptors in mediating peripheral sensitization and persistent pain behaviors at different stages of the DMM model of OA; and 2) To investigate whether chondrocyte PIEZO1 ion channel signaling promotes joint damage and pain in the DMM model. To perform these aims, we have generated innovative techniques that enable application of mechanical stimuli to intact tissues while performing real-time calcium or voltage imaging. We have created mice that express fluorescent calcium (GCaMP6s) or voltage (ASAP2s) indicator proteins in either pain-sensing sensory neurons (nociceptors; NaV1.8 cre) or in chondrocytes (Col2a1 cre). We also have two types of nociceptor-specific Piezo2 conditional knock-out mice as well as chondrocyte-specific Piezo1 conditional knock-out mice. Successful completion of these aims will improve our understanding of how nociceptors and chondrocytes use PIEZO channels to respond to mechanical loading in OA, which may lead to the identification of novel pathways to target both pain and joint damage therapeutically. By measuring both pain-related behaviors and joint damage in nociceptor-Piezo2 knock-out and chondrocyte-Piezo1 knock-out mice, this approach will provide insight on interactions between these processes, which will set up future work.