The Role of Mechanosensation Pathways in Osteoarthritis Joint Damage and Pain

项目摘要 膝骨性关节炎(OA)是一种痛苦的慢性疾病，在美国有2700万人受到影响。膝盖骨关节炎是 以所有关节组织的渐进性损伤和重塑为特征。骨性关节炎的主要特征是关节疼痛 X线片显示关节间隙变窄(软骨丢失)。生物力学因素在两个关节中起着重要作用。 疼痛和损伤，但究竟机械力是如何作用于感觉神经元和软骨来驱动 疾病是未知的。我们的长期目标是阐明机械负荷是如何通过关节组织和 对装载的反应如何影响办公自动化。细胞通过各种机制感知机械力， 包括机械敏感离子通道。近年来，感觉神经元的机械敏感离子通道 Piezo2被认为是炎症性和非炎症性小鼠机械性痛觉异常的关键因素。 神经病理性疼痛，但Piezo2在伤害性感受器表达和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还不是很清楚，但基于 之前的工作表明PIEZO1在软骨细胞对机械刺激的反应中发挥了作用，我们假设 PIEZO1在骨性关节炎的组织损伤中起作用。总体而言，尚不清楚压电离子通道信号是如何 导致骨性关节炎病理(疼痛和关节损伤)。我们的目标是通过使用小说来解决这个问题 我们开发的技术能够在执行任务时将机械刺激应用于完整的组织 钙离子或电压成像，实时评估细胞功能。这一建议解决了中心假说 伤害性感受器和软骨细胞中的压电通道信号在启动过程中驱动疼痛和关节损伤 和骨性关节炎的进展。中心假设将在两个具体目标中进行检验：1)定义 伤害性感受器表达的Piezo2离子通道介导外周敏化和持续性疼痛 DMM模型不同阶段的行为；2)研究软骨细胞PIEZO1离子 在DMM模型中，通道信号会促进关节损伤和疼痛。为了实现这些目标，我们有 产生了创新的技术，使机械刺激能够应用于完整的组织，同时执行 实时钙离子或电压成像。我们已经创造了表达荧光钙(GCaMP6s)或 在痛觉感觉神经元(伤害性感受器；NaV1.8cre)或 软骨细胞(COL2a1cre)。我们还有两种伤害性感受器特异的Piezo2条件性基因敲除小鼠 以及软骨细胞特异性Piezo1条件性基因敲除小鼠。这些目标的成功实现将 提高我们对伤害性感受器和软骨细胞如何使用压电通道做出反应的理解 骨关节炎中的机械负荷，这可能导致识别针对疼痛和关节的新途径 治疗上的损害。通过测量伤害性感受器Piezo2的疼痛相关行为和关节损伤 基因敲除和软骨细胞-Piezo1基因敲除小鼠之间的相互作用 这些过程，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.