Biology subjects

Tonello, R.

Publications and source records attributed to Tonello, R..

7 recordsLinked to original sources

bioRxiv2026

Endocytosis and Compartmentalized Intracellular Signaling of the Prostaglandin Receptor EP4 Mediate Pain

Prostaglandin E2 (PGE2) is recognized as a major mediator of inflammatory pain. However, the intracellular signaling mechanisms by which PGE2 mediates pain remain unclear. Here, we show that pain-like responses evoked by PGE2 in dorsal root ganglion (DRG) nociceptors are mediated by internalization and compartmentalized signaling of the EP4 receptor. The EP4-selective agonist L-902,688 induced immediate nocifensive behavior and prolonged mechanical allodynia in mice and sensitized isolated DRG nociceptors. Pharmacological inhibition of clathrin- and dynamin-mediated endocytosis (using pitstop2 and dyngo4a) and siRNA knockdown of Dnm1 prevented EP4-induced nociception and sensitization, implicating receptor trafficking in pain signaling. Using genetically-encoded biosensors, we monitored EP4 trafficking and downstream signaling within subcellular compartments of HEK293 cells. PGE2 stimulated dynamin- and {beta}-arrestin-dependent EP4 trafficking from the plasma membrane to early, late and recycling endosomes and Golgi apparatus, and mobilized intracellular EP4 pools from the endoplasmic reticulum. PGE2 induced the assembly of EP4, G proteins and {beta}-arrestin signaling complexes in endosomes and the Golgi apparatus. Inhibition of EP4 endocytosis suppressed intracellular cAMP production and ERK activation. Our findings reveal that EP4-mediated nociceptive signaling originates from intracellular compartments and suggest that modulating EP4 internalization and subcellular signaling dynamics could offer a novel strategy for inflammatory pain management.

cell biology↗

bioRxiv2026

Targeting Synaptic Vesicle Endocytosis in Nociceptors Provides Sustained Pain Relief

Endocytosis replenishes synaptic vesicle (SV) pools that are required for persistent transmission of chronic pain signals within nociceptive spinal circuits. The nociceptor-specific contribution of SV endocytosis to pain and the therapeutic potential of endocytosis inhibitors are unclear. We identified SV endocytosis in nociceptors as a critical driver of ongoing pain and developed a gene-based strategy to target this mechanism. Nociceptor-specific adeno-associated virus-mediated knockdown of adaptor-associated kinase 1 (AAK1) or dynamin 1 (Dnm1) in dorsal root ganglia Nav1.8-positive neurons inhibited postoperative and neuropathic hypersensitivity without affecting baseline mechanical or thermal sensitivity, locomotion or spontaneous behavior. Electrophysiological recordings from spinal neurons combined with optogenetic activation of nociceptor afferents showed that AAK1 or Dnm1 downregulation blocked the sustained synaptic transmission between nociceptors and dorsal horn neurons by disrupting SV recycling and reducing neurotransmitter release probability. Lipid nanoparticle (LNP)-encapsulated CRISPR/dCas9-repressor mRNA constructs (dCas9-R) were engineered to achieve sustained and reversible transcriptional and epigenetic repression of Aak1 or Dnm1 following intrathecal delivery. LNP-mediated gene modulation produced sustained downregulation of Aak1 or Dnm1 mRNA in sensory neurons and resulted in robust and long-lasting analgesia in preclinical models of postoperative, inflammatory, neuropathic and osteoarthritis pain without impairing acute nociception or locomotor activity. Mechanistically, targeting endocytic machinery disrupted SV recycling at nociceptor terminals, thereby reducing excitatory neurotransmission within spinal pain circuits. Together, these findings establish presynaptic endocytic regulation as a convergent mechanism underlying chronic pain and demonstrate the translational potential of LNP-delivered CRISPR/dCas9-R as a durable, non-opioid pain therapy that surmounts inherent redundancy of pain signaling mechanisms. One Sentence SummarySynaptic vesicle endocytosis in nociceptors is a critical mechanism driving ongoing pain and targeting this process with intrathecal LNP-delivered CRISPR/dCas9-mediated gene repression produces durable, non-opioid analgesia across multiple chronic pain models.

neuroscience↗

bioRxiv2025

Characterization and targeting of the endosomal signaling of the gastrin releasing peptide receptor in pruritus.

Chronic pruritus is a major unmet clinical problem affecting one in four adults. G protein-coupled receptors (GPCRs) are key receptors driving itch signaling and are a therapeutic target for itch relief. The endosomal signaling of GPCRs provides new challenges for understanding how GPCR signaling is regulated, how endosomal signaling of GPCRs contributes to disease states like chronic pruritus and opens new targets for therapeutic development. The Gastrin releasing peptide receptor (GRPR) is a key mediator of pruritus in the spinal cord. Yet, little is known about the molecular mechanisms regulating GRPR signaling in pruritus, if GRPR can signal from endosomes, or the role of endosomal GRPR in the development of pruritus. Here we show the importance of internalization and endosomal signaling of GRPR in pruritus. Agonist induced GRPR internalization and trafficking was quantified using BRET or microscopy while endosomal-mediated ERK signaling was measured using compartmentalized FRET biosensors. Recruitment of G proteins to endosomes was measured with NanoBit BRET. pH sensitive mesoporous silica nanoparticles (MSN) which accumulated in endosomes were used to deliver RC-3095, a GRPR specific antagonist, intracellularly to block endosomal signaling of GRPR. MSN-RC proved more effective than free RC-3095 at inhibiting chloroquine scratching in mice. Our results demonstrate a critical role for GRPR endosomal signaling in itch sensation. These results highlight the ability of endosomally targeted antagonist to inhibit GRPR signaling and provide a new target for developing therapeutics that block GRPR mediated pruritus. Significance StatementGPCRs are dynamic signaling receptors that can continue to signal following internalization and trafficking to endosomes. Using subcellular targeted BRET and FRET based biosensors we can quantify the recruitment of signaling partners like G proteins and arrestins to GRPR from the endosomal compartment. Inhibition of clathrin and dynamin mediated endocytosis allowed to differentiate plasma membrane vs endosomal signaling of GRPR. pH sensitive nanoparticles loaded with the GRPR antagonist RC-3095 are endocytosed to the endosomal network where they specifically target and block endosomal GRPR signaling. Intrathecal injection of RC-3095 loaded nanoparticles blocked chloroquine induced scratching behavior in mice. Thus, intracellular GRPR drives itch sensation and targeted inhibition of intracellular GRPR signaling is a more effective strategy to treat pruritus.

neuroscience↗

bioRxiv2024

Targeting the Schwann Cell EP2/cAMP Nanodomain to Block Pain but not Inflammation

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 EP2 receptor in Schwann cells via an adeno-associated viral vector 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 PG-mediated persistent inflammatory pain but not protective inflammation.

pharmacology and toxicology↗

bioRxiv2023

Neuropilin-1 is a co-receptor for NGF and TrkA-evoked pain

Nerve growth factor (NGF) monoclonal antibodies inhibit chronic pain yet, failed to gain approval due to worsened joint damage in osteoarthritis patients. We report that neuropilin-1 (NRP1) is a co-receptor for NGF and tropomyosin-related kinase A (TrkA) pain signaling. NRP1 is coexpressed with TrkA in human and mouse nociceptors. NRP1 inhibitors suppress NGF-stimulated excitation of human and mouse nociceptors and NGF-evoked nociception in mice. NRP1 knockdown inhibits NGF/TrkA signaling, whereas NRP1 overexpression enhances signaling. NGF binds NRP1 with high affinity and interacts with and chaperones TrkA from the biosynthetic pathway to the plasma membrane and endosomes, enhancing TrkA signaling. Molecular modeling suggests that C-terminal R/KXXR/K NGF motif interacts with extracellular "b" NRP1 domain within a plasma membrane NGF/TrkA/NRP1 of 2:2:2 stoichiometry. G Alpha Interacting Protein C-terminus 1 (GIPC1) scaffolds NRP1 and TrkA to myosin VI and colocalizes in nociceptors with NRP1/TrkA. GIPC1 knockdown abrogates NGF-evoked excitation of nociceptors and pain-like behavior. NRP1 is a nociceptor-enriched co-receptor that facilitates NGF/TrkA pain signaling. NRP binds NGF and chaperones TrkA to the plasma membrane and signaling endosomes via the GIPC1 adaptor. NRP1 and GIPC1 antagonism in nociceptors offers a long-awaited non-opioid alternative to systemic antibody NGF sequestration for the treatment of chronic pain. SummaryNeuropilin-1 and G Alpha Interacting Protein C-terminus 1 are necessary for nerve growth factor-evoked pain and are non-opioid therapeutic targets for chronic pain.

neuroscience↗

bioRxiv2023

Satellite glial GPR37L1 regulates maresin and potassium channel signaling for pain control

G protein coupled receptor 37-like 1 (GPR37L1) is an orphan GPCR and its function remains largely unknown. Here we report that GPR37L1 transcript is highly expressed compared to all known GPCRs in mouse and human dorsal root ganglia (DRGs) and selectively expressed in satellite glial cells (SGCs). Peripheral neuropathy following diabetes and chemotherapy by streptozotocin and paclitaxel resulted in downregulations of surface GPR37L1 in mouse and human DRGs. Transgenic mice with Gpr37l1 deficiency exhibited impaired resolution of neuropathic pain symptom (mechanical allodynia), whereas overexpression of Gpr37l1 in mouse DRGs can reverse neuropathic pain. Notably, GPR37L1 is co-expressed and coupled with potassium channels in SGCs. We found striking species differences in potassium channel expression in SGCs, with predominant expression of KCNJ10 and KCNJ3 in mouse and human SGCs, respectively. GPR37L1 regulates the surface expression and function of KCNJ10 and KCNJ3. We identified the pro-resolving lipid mediator maresin 1 (MaR1) as a GPR37L1 ligand. MaR1 increases KCNJ10/KCNJ3-mediated potassium influx in SGCs via GPR37L1. MaR1 protected chemotherapy-induced suppression of KCNJ13/KCNJ10 expression and function in SGCs. Finally, genetic analysis revealed that the GPR37L1-E296K variant is associated with increased chronic pain risk by destabilizing the protein. Thus, GPR37L1 in SGCs offers a new target for neuropathy protection and pain control.

neuroscience↗

bioRxiv2019

Resolvin D3 controls mouse and human nociceptive functions and preclinical progression of psoriasis

Psoriasis is a chronic inflammatory skin disease caused by a complex interplay between the immune and nervous systems. Here, we found that resolvin D3 (RvD3) significantly prevented both skin inflammation and itch in a preclinical animal model of psoriasis induced by repeated applications of imiquimod. We also found that RvD3 anti-psoriatic effects resulted from the inhibition of nociceptive functions, which inhibition was replicated in human nociceptive neurons. We conclude that RvD3 and targeting nociceptive functions are promising strategies to treat psoriasis.

neuroscience↗