Mapping the joint-nerve interactome of the knee

Project Summary Our multidisciplinary team assembles basic and translational researchers with expertise in joint biology and neuroscience, proposing a holistic approach to mapping the sensory innervation of murine and human knee joints. We will use state-of-the-art imaging techniques, combined with transcriptomics to construct 3D models of

the sensory innervation of the knee, compose a cell atlas in which knee afferents are transcriptionally profiled at a single cell resolution, and document the nerve-joint cell interactome at the transcriptional level. Our overarching objective is to precisely describe the sensory innervation of the knee, and the dynamic changes occurring with

aging, joint injury, and osteoarthritis (OA). This will provide the Consortium with a rich anatomical and molecular resource to study mechanisms underlying joint pain and guide the development of novel analgesic strategies. Aim 1. Documenting the sensory innervation of the healthy and diseased mouse knee: Anatomical and molecular

perspectives. Using fluorescent reporter mice to label nociceptors, C-fiber subsets, and proprioceptors, we will map the anatomical innervation of the mouse knee in (a) naïve mice of different ages; (b) after joint injury; (c) in surgically induced OA. We will use ribbon scanning confocal and clearing-enabled lightsheet microscopy to

construct high-resolution 3-D anatomical models of joint innervation. We will backlabel knee-innervating afferents and use spatial transcriptomics to describe their molecular phenotypes compared to other non-knee innervating DRG neurons. Aim 2. Documenting the sensory innervation of the healthy and diseased human knee: Anatomical

and molecular perspectives. We will use a unique set of post mortem knee/DRG samples from (1) healthy knees, age 20-40 (n=15/sex); (2) knees from donors over 70 (n=15/sex), in which we anticipate 80-90% to exhibit OA pathology. Knee tissues will be collected in a standardized fashion, including synovium, osteochondral plugs

(medial tibial plateau), meniscus, ACL, fat pad, and quadriceps muscle. In each tissue, we will perform (1) histopathology; (2) IHC for sensory innervation; (3) bulk and scRNAseq; (4) spatial transcriptomics. Matched DRGs will be used for bulk RNAseq to identify differentially expressed genes (DEG) between the groups provide

information for ligand-receptor analysis. Aim 3. Identifying mediators in the knee synovium that drive disease- associated neuroplasticity. (1) We will reconstruct the cellular interactome between synovial cells and DRG neurons in mouse models of aging, joint injury, and OA using scRNAseq of matched synovium and DRG

samples. (2) We will compare patient reports of OA knee pain at the time of TKR to matched synovial histology, including extent of lining hyperplasia, single-cell transcriptional changes, and innervation. Overall, this project will provide the community with comprehensive databases of the neuro-articular environment, which can be

mined to (1) undertake mechanistic studies to inhibit pathological neuroplasticity and (2) identify and test new druggable targets. This strategy will pave the way for the development of novel, targeted, non-addictive, and safe analgesic therapeutics for the treatment of joint pain.