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-Pie...