Mechanotransduction, the conversion of mechanical stimuli into electrical signals, is a fundamental process underlying essential physiological functions such as touch and pain sensing, hearing, and proprioception. Although the mechanisms for some of these functions have been identified, the molecules essential to the sense of pain have remained elusive. Here we report identification of TACAN (Tmem120A), an ion channel involved in sensing mechanical pain. TACAN is expressed in a subset of nociceptors, and its heterologous expression increases mechanically evoked currents in cell lines. Purification and reconstitution of TACAN in synthetic lipids generates a functional ion channel. Finally, a nociceptor-specific inducible knockout of TACAN decreases the mechanosensitivity of nociceptors and reduces behavioral responses to painful mechanical stimuli but not to thermal or touch stimuli. We propose that TACAN is an ion channel that contributes to sensing mechanical pain.

Curcumin and o-Vanillin cleared senescent intervertebral disc (IVD) cells and reduced the senescence-associated secretory phenotype (SASP) associated with inflammation and back pain. Cells from degenerate and non-mildly-degenerate human IVD were obtained from organ donors and from patients undergoing surgery for low back pain. Gene expression of senescence and SASP markers was evaluated by RT-qPCR in isolated cells, and protein expression of senescence, proliferation, and apoptotic markers was evaluated by immunocytochemistry (ICC). The expression levels of SASP factors were evaluated by enzyme-linked immunosorbent assay (ELISA). Matrix synthesis was verified with safranin-O staining and the Dimethyl-Methylene Blue Assay for proteoglycan content. Western blotting and ICC were used to determine the molecular pathways targeted by the drugs. We found a 40% higher level of senescent cells in degenerate compared to non-mildly-degenerate discs from unrelated individuals and a 10% higher level in degenerate compared to non-mildly-degenerate discs from the same individual. Higher levels of senescence were associated with increased SASP. Both drugs cleared senescent cells, and treatment increased the number of proliferating as well as apoptotic cells in cultures from degenerate IVDs. The expression of SASP factors was decreased, and matrix synthesis increased following treatment. These effects were mediated through the Nrf2 and NFkB pathways.

Toll-like receptors (TLR) are activated by endogenous alarmins such as fragmented extracellular matrix compounds found in the degenerating disc. TLRs regulate cytokine, neurotrophin, and protease expression in human disc cells in vitro, and thus control key factors in disc degeneration. However, whether TLR activation leads to degenerative changes in intact human discs is unclear. Nucleus pulposus (NP) cells isolated from non-degenerating discs increase IL-1β and nerve growth factor gene expression following treatment with Pam2CSK4 (TLR2/6 agonist) but not Pam3CSK4 (TLR1/2 agonist). Challenging NP cells with Pam2CSK4 or 30 kDa fibronectin fragments (FN-f, an endogenous TLR2 and TLR4 alarmin) increased secretion of proinflammatory cytokines. We then investigated the effect of TLR activation in intact, non-degenerate, ex vivo human discs. Discs were injected with PBS, Pam2CSK4 and FN-f, and cultured for 28 days. TLR activation increased proteoglycan and ECM protein release into the culture media and decreased proteoglycan content in the NP. Proteases, including MMP3, 13 and HTRA1, are secreted at higher levels following TLR activation. In addition, proinflammatory cytokine levels, including IL-6, TNFα and IFNγ, increased following TLR activation. These results indicate that TLR activation induces degeneration in human discs. Therefore, TLRs are potential disease-modifying therapeutic targets to slow disc degeneration.

Cellular senescence is a contributor to intervertebral disc (IVD) degeneration and low back pain. Here, we found that RG-7112, a potent mouse double-minute two protein inhibitor, selectively kills senescent IVD cells through apoptosis. Gene expression pathway analysis was used to compare the functional networks of genes affected by RG-7112, a pure synthetic senolytic with o-Vanillin a natural and anti-inflammatory senolytic. Both affected a functional gene network related to cell death and survival. O-Vanillin also affected networks related to cell cycle progression as well as connective tissue development and function. Both senolytics effectively decreased the senescence-associated secretory phenotype (SASP) of IVD cells. Furthermore, bioavailability and efficacy were verified ex vivo in the physiological environment of degenerating intact human discs where a single dose improved disc matrix homeostasis. Matrix improvement correlated with a reduction in senescent cells and SASP, supporting a translational potential of targeting senescent cells as a therapeutic intervention.