Background: OnabotulinumtoxinA (onabotA) is approved globally for prevention of chronic migraine; however, the classical mechanism of action of onabotA in motor and autonomic neurons cannot fully explain the effectiveness of onabotulinumtoxinA in this sensory neurological disease. We sought to explore the direct effects of onabotulinumtoxinA on mouse trigeminal ganglion sensory neurons using an inflammatory soup-based model of sensitization. Methods: Primary cultured trigeminal ganglion neurons were pre-treated with inflammatory soup, then treated with onabotulinumtoxinA (2.75 pM). Treated neurons were used to examine transient receptor potential vanilloid subtype 1 and transient receptor potential ankyrin 1 cell-surface expression, calcium influx, and neuropeptide release. Results: We found that onabotulinumtoxinA cleaved synaptosomal-associated protein-25 kDa in cultured trigeminal ganglion neurons; synaptosomal-associated protein-25 kDa cleavage was enhanced by inflammatory soup pre-treatment, suggesting greater uptake of toxin under sensitized conditions. OnabotulinumtoxinA also prevented inflammatory soup-mediated increases in TRPV1 and TRPA1 cell-surface expression, without significantly altering TRPV1 or TRPA1 protein expression in unsensitized conditions. We observed similar inhibitory effects of onabotulinumtoxinA on TRP-mediated calcium influx and TRPV1- and TRPA1-mediated release of calcitonin gene-related peptide and prostaglandin 2 under sensitized, but not unsensitized control, conditions. Conclusions: Our data deepen the understanding of the sensory mechanism of action of onabotulinumtoxinA and support the notion that, once endocytosed, the cytosolic light chain of onabotulinumtoxinA cleaves synaptosomal-associated protein-25 kDa to prevent soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated processes more generally in motor, autonomic, and sensory neurons.

Transient receptor potential vanilloid 4 (TRPV4) is a polymodal cation channel that is widely expressed in sensory neurons, immune cells, and structural tissues, where it integrates mechanical, osmotic, and chemical stimuli to regulate both physiological responses and disease-associated signaling. Mast cells (MCs), key immune effector cells capable of rapid mediator release through degranulation, also express TRPV4. Increasing evidence supports TRPV4-MC signaling as an important neuroimmune interface, linking mechanical and inflammatory stimuli to tissue hypersensitivity and pain. In this review, we synthesize current evidence supporting a role for TRPV4 in MC-associated neuroimmune signaling across multiple disease contexts while distinguishing settings in which TRPV4 directly regulates MC activation from those in which MC responses arise through multicellular tissue interactions. Direct TRPV4-dependent MC activation has been described in conditions such as LL-37–driven rosacea and mechanically induced inflammation, whereas in disorders including asthma, visceral hypersensitivity, bladder pain syndromes, and osteoarthritis, TRPV4 activity in epithelial, neuronal, or stromal compartments more often influences MC function indirectly through ATP–purinergic signaling, cytokine release, and neuropeptide-mediated crosstalk. Across systems, TRPV4 emerges not as a single pathogenic switch but as part of a context-dependent signaling network whose functional consequences depend on cell type, tissue microenvironment, and disease stage. Altogether, these findings identify TRPV4 as a therapeutically actionable node within neuroimmune signaling pathways and support the development of tissue-specific and combination strategies targeting both TRPV4 activity and MC-mediated signaling in chronic inflammatory and pain disorders.

Skin inflammation is an evolutionary-honed protective mechanism that serves to clear noxious cues and irritants and initiate regeneration. Calcium-permeable transient-receptor-potential (TRP) ion channels have critical functions in sensory transduction which is sensitized in skin inflammation. Skin sensory transduction relies on skin-innervating sensory neurons in the dorsal root ganglion (DRG), but also on innervated keratinocytes (KC). The multimodally-activated TRPV4 is robustly expressed in KC, where it can readily be activated by Ultraviolet-B (UVB). Our goal was to deconstruct keratinocyte TRPV4-mediated signaling, specifically how TRPV4 can facilitate inflammatory injury, thus lowering pain thresholds and rendering KC into pain-generator cells. We wanted to uncover the effect of TRPV4-mediated signaling on UVB-induced inflammasome activation in KC given the powerful impact of the activated inflammasome on pro-inflammatory/pro-algesic secretory signaling, using mouse models and cultured human KC. In mice, our evidence suggests that TRPV4 functions as calcium-permeable channel upstream of the KC inflammasome. Furthermore, we found that UVB induced activation of TRPV4 caused rapid - within minutes - E xtracellular Signal R egulated K inase (ERK) phosphorylation, caspase-1 activation and Interleukin-1ß (IL-1ß) secretion. In human primary KC we demonstrated that UVB induced secretion of IL-1ß was dependent on the NLR family pyrin domain containing 1 (NLRP1) inflammasome. Direct chemical TRPV4 activation could also activate NLRP1 and to lesser extent NLPR3. Building on our previous work, we now define at increased resolution TPRV4-dependent forefront signaling mechanisms in KC in response to UVB, showing TRPV4 upstream of the NLRP1 inflammasome, subsequent rapid ERK activation and pro-inflammatory/pro-algesic secretory function.