Project Summary Osteoarthritis (OA) is the most common form of arthritis, and a major source of chronic pain. No disease- modifying OA drugs are available to patients, and current analgesic approaches fall short of patients’ needs. Nerve growth factor (NGF) has emerged as a promising target for OA pain. Neutralizing antibodies that prevent NGF from binding to its receptor, tropomyosin receptor kinase A (TrkA), have shown strong analgesic effects in clinical trials for OA pain. However, up to 10% of patients treated with anti-NGF developed rapidly progressive OA, necessitating joint replacement. The mechanism of this deleterious effect of NGF blockade on joint integrity is unknown, exposing our ongoing lack of understanding the relationship between joint damage and pain in OA. During the previous funding cycles, we have developed assays, reporter mice, and neuroanatomical and biophysical techniques for monitoring neuronal activity in vivo to assess pain behaviors and underlying neurobiological mechanisms in mouse models of OA. We have uncovered that joint damage in OA is accompanied by extensive anatomical and functional neuronal plasticity of the nociceptive innervation of the knee. We will build on this discovery to test the central hypothesis that the NGF-TrkA axis is essential for the preservation of joint homeostasis in response to joint injury, through neuronal and non-neuronal mechanisms. We will (Aim 1) Determine the spatial and temporal NGF-TrkA “interactome” in the joint. Using reporter mice, single cell RNAseq of the dorsal root ganglia (DRG), RNAscope, and immunohistochemistry, we will detail which cells in the knee and DRG express Ngf and Ntrk1 during progressive experimental OA, in spatiotemporal relationship to neuronal plasticity and joint pathology. We will validate findings in human knee tissues; (Aim 2) Determine the role of the NGF-TrkA axis in promoting neuronal growth in the OA joint, and how this contributes to pain and joint integrity. We will assess if NGF causes neuroplasticity, by injecting it into knees of NaV1.8 reporter mice and assess pain behaviors, joint innervation, and joint integrity (histology and microCT), as well as functional effects on sensory neurons. Advillin-creERT2 mice will be used to delete Nrtrk1 from sensory neurons in adult mice subjected to DMM or PMX surgery; pain, neuroplasticity, and joint damage will be assessed up to 16 weeks; (Aim 3) Explore the role of the NGF-TrkA axis in OA through non-neuronal mechanisms. We will use Ngf-loxp and Ntrk1-loxp mice to conditionally delete Ngf or Ntrka from select non- neuronal cells (myeloid cells, osteoblasts, or chondrocytes). Mice will be subjected to surgery, and OA monitored up to week 8; (Aim 4) Assess the effect of anti-NGF Abs on the OA joint. The role of the NGF-TrkA axis in the whole joint as an organ will be determined by using neutralizing anti-NGF antibodies, either prophylactically (0-8 and 0-16 weeks) or therapeutically (8-16 weeks) after sur...