Martina Chieca

Nerve growth factor (NGF) is a key mediator of pain through activation of the high-affinity tropomyosin receptor kinase A (TrkA) and the low-affinity neurotrophin receptor (p75NTR). Although neuronal TrkA signaling is well established, the contribution of non-neuronal cells to NGF-dependent pain remains unclear. Here, we show that NGF and its precursor proNGF engage distinct cellular mechanisms. Intraplantar NGF induced acute nociception, heat hyperalgesia, mechanical allodynia, and cold hypersensitivity, whereas cleavage-resistant proNGF selectively evoked mechanical allodynia and cold hypersensitivity. Pharmacological and cell-specific genetic approaches demonstrated that acute nociception and heat hyperalgesia require neuronal TrkA, whereas mechanical and cold hypersensitivity depend on p75NTR activation in Schwann cells. In Schwann cells, NGF and proNGF induced p75NTR-dependent calcium release, followed by TRPA1 activation, mitochondrial ROS production, and NOX1-dependent oxidative amplification. Inhibition of ROS or TRPA1, or Schwann cell-specific Trpa1 deletion, markedly reduced mechanical allodynia and cold hypersensitivity without affecting acute nociception or heat hyperalgesia. These findings identify a Schwann cell p75NTR-ROS-TRPA1 pathway sustaining persistent pain and highlight non-neuronal p75NTR signaling as a potential therapeutic target.

Extracellular proteases are important signaling molecules in coagulation, inflammation, cell migration, and pain. Dysregulation of extracellular protease activity is common in diseases that perturb these critical functions. Engineering cells to sense and respond programmatically to protease activity has applications in biosensing, cell-based screening for protease activity, and therapeutics. Here we report synthetic protease-activated receptors ( SynPARs ) based on engineered, auto-inhibited G protein-coupled receptors (GPCRs). Relief of autoinhibition by proteolysis enables receptor activation by an exogenous or tethered agonist to generate transgene expression, real-time fluorescence, or endogenous G-protein signaling. We demonstrate SynPAR modularity with diverse secreted proteases, establish a cell-based SynPAR library selection to optimize protease recognition sequences, and control neuronal activity in response to protease activity. Finally, we use SynPARs in the dorsal root ganglion of mice to counteract hyperalgesia produced by trypsin activity, rewiring neurons to produce an analgesic response to a pain-inducing stimulus. Our study establishes SynPAR as a versatile and modular platform for recording, sensing, and responding to pericellular proteolysis. This fills a critical gap in protease-sensing tools and lays the groundwork for protease-activated genetic and cell-based medicines.

Analgesia by non-steroidal anti-inflammatory drugs (NSAIDs) is ascribed to inhibition of prostaglandin (PG) biosynthesis and ensuing inflammation. However, NSAIDs have life-threatening side effects, and inhibition of inflammation delays pain resolution. Decoupling the mechanisms underlying PG-evoked pain vs. protective inflammation would facilitate pain treatment. Herein, we reveal that selective silencing of the PGE₂ receptor 2 (EP2) in Schwann cells via adeno-associated viral vectors abrogates the indomethacin-sensitive component of pain-like responses in mice elicited by inflammatory stimuli without affecting inflammation. In human Schwann cells and in mice, EP2 activation and optogenetic stimulation of adenylyl cyclase evokes a plasma membrane-compartmentalized cyclic adenosine monophosphate (cAMP) signal that, via A-kinase anchor protein-associated protein kinase A, sustains inflammatory pain-like responses, but does not delay their resolution. Thus, an unforeseen and druggable EP2 receptor in Schwann cells, via specific cAMP nanodomains, encodes PGE₂-mediated persistent inflammatory pain but not PG-dependent protective inflammation.

Over 60% of women with endometriosis experience abdominopelvic pain and broader pain manifestations, including chronic back pain, fibromyalgia, chronic fatigue, vulvodynia, and migraine. Although the imbalance of proinflammatory mediators, including the complement component C5a, is associated with endometriosis-related pain, the mechanisms causing widespread pain and the C5a role remain unclear. Female mice and women with endometriosis exhibit increased plasma C5a levels and pain. We hypothesize the Schwann cells involvement in endometriotic pain. Here, we show that silencing the C5a receptor (C5aR1) in Schwann cells blocks the C5a-induced activation of the NLRP1 inflammasome and subsequent release of interleukin-1β (IL-1β). Macrophages, recruited to sciatic/trigeminal nerves by IL-1β from Schwann cells, increase oxidative stress, which activates the proalgesic TRPA1 pathway, resulting in widespread pain. These findings reveal a pathway involving Schwann cell C5aR1, NLRP1/IL-1β activation, macrophage recruitment, oxidative stress, and TRPA1 engagement, contributing to pain in a mouse model of endometriosis.

Differentiation of oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes (OLs) is a key event for axonal myelination in the brain; this process fails during demyelinating pathologies. Adenosine is emerging as an important player in oligodendrogliogenesis, by activating its metabotropic receptors (A₁R, A_2AR, A_2BR, and A₃R). We previously demonstrated that the Gs‐coupled A_2BR reduced differentiation of primary OPC cultures by inhibiting delayed rectifier (I_K) as well as transient (I_A) outward K⁺ currents. To deepen the unclear role of this receptor subtype in neuron‐OL interplay and in myelination process, we tested the effects of different A_2BR ligands in a dorsal root ganglion neuron (DRGN)/OPC cocultures, a corroborated in vitro myelination assay. The A_2BR agonist, BAY60‐6583, significantly reduced myelin basic protein levels but simultaneously increased myelination index in DRGN/OPC cocultures analyzed by confocal microscopy. The last effect was prevented by the selective A_2BR antagonists, PSB‐603 and MRS1706. To clarify this unexpected data, we wondered whether A_2BRs could play a functional role on DRGNs. We first demonstrated, by immunocytochemistry, that primary DRGN monoculture expressed A_2BRs. Their selective activation by BAY60‐6583 enhanced DRGN excitability, as demonstrated by increased action potential firing, decreased rheobase and depolarized resting membrane potential and were prevented by PSB‐603. Throughout this A_2BR‐dependent enhancement of neuronal activity, DRGNs could release factors to facilitate myelination processes. Finally, silencing A_2BR in DRGNs alone prevents the increased myelination induced by BAY60‐6583 in cocultures. In conclusion, our data suggest a different role of A_2BR during oligodendrogliogenesis and myelination, depending on their activation on neurons or oligodendroglial cells.

Over 60% of women with endometriosis experience abdominopelvic pain alongside pain in other areas of the body, such as chronic back pain, fibromyalgia and chronic fatigue, vulvodynia, and migraine. Although the imbalance of proinflammatory mediators, including the complement component C5a, has been implicated in endometriosis-associated pain symptoms, the mechanisms causing widespread pain and the role of C5a remain unclear. Female mice with endometriotic lesions displayed widespread pain and increased plasma C5a levels, similarly, observed in women with endometriosis. We hypothesized Schwann cells involvement in endometriotic pain. Silencing the C5a surface receptor (C5aR1) in mouse Schwann cells abolished C5a-induced activation of NLRP1 inflammasome and the ensuing interleukin-1β (IL-1β) release. IL-1β, from Schwann cells, recruited macrophages in sciatic/trigeminal nerve trunks. Macrophages induced oxidative stress, targeting proalgesic TRPA1, causing widespread mechanical allodynia. This pathway, initiated by C5aR1, engages Schwann cell signaling, including NLRP1/IL-1β/macrophages/oxidative stress/TRPA1, sustaining pain in an endometriosis mouse model.