Background & Aims

Nerve growth factor (NGF) is a neurotrophic protein essential for development that mediates multiple forms of pain. NGF blockade can reduce pain symptoms in rodents and patients (1). NGF interacts with Tropomyosin kinase receptor A (TrkA), which sensitizes transient receptor potential (TRP) ion channels, leading to hyperalgesia and allodynia (2). Neuropilin-1 (NRP1), a single transmembrane protein that lacks an intracellular signaling domain, is known to interact with growth factors to facilitate the activation of receptor tyrosine kinases (3). NRP1 also interacts with NGF and TrkA (4). NRP1 antagonism hinders NGF-triggered firing of rodent and human nociceptors and blunts NGF-evoked pain-like behavior in mice. G Alpha Interacting Protein C-terminus 1 (GIPC1) scaffolds NRP1/TrkA interactions and is necessary for NGF-induced nociceptors excitation and nociception (4). Herein we determined the mechanisms by which NGF, TrkA, NRP1 and GIPC1 interact to sensitize TRP channels and evoke pain

Methods

NGF-induced sensitization of TRPV1 in nociceptors was investigated by calcium imaging of mouse dorsal root ganglion (DRG) nociceptors. The effects of NRP1 antagonists on NGF-induced sensitization of calcium responses to the TRPV1 agonist capsaicin were determined. The contribution of NRP1 and GIPC1 to NGF-evoked nociception was studied in mice. NGF was injected into the right hindpaw together with NRP1 inhibitors or vehicle. GIPC1 or control siRNA was intrathecally injected 48 hours before NGF. Mechanical allodynia and thermal hyperalgesia were assessed by measuring paw withdrawal responses to stimulation with von Frey filaments and radiant heat, respectively. To examine the contribution of GIPC1 to the TrkA chaperone function of NRP1, bioluminescence resonance energy transfer (BRET) was used to measure the proximity between TrkA tagged with Renilla luciferase and plasma membrane marker tagged with Renilla GFP in HEK293T cells after GIPC1 siRNA knockdown.

Results

Preincubation of mouse DRG neurons with NGF augmented the calcium response to capsaicin, consistent with sensitization of TRPV1. NRP1 inhibitors suppressed NGF-induced sensitization of TRPV1. Intraplantar administration of NGF in mice decreased the withdrawal threshold to von Frey filament stimulation and reduced latency of withdrawal to thermal stimulation within 30 min, lasting for at least 4 hrs. NRP1 inhibitors fully mitigated NGF-induced mechanical allodynia and thermal hyperalgesia for 1-2 hrs. Intrathecal injection of GIPC1 siRNA abrogated NGF-induced pain-like behaviors. Therefore, NRP1 and GIPC1 are necessary for NGF-evoked nociception. BRET analysis revealed that GIPC1 siRNA reduced NRP1-induced plasma membrane expression of TrkA compared to control siRNA, indicating that GIPC1 is necessary for TrkA chaperone function of NRP1.

Conclusions

Despite extensive research, the effective treatment of pain continues to present significant challenges. High levels of NGF have been found in individuals with pain conditions and NGF monoclonal antibodies are effective analgesics (3). In this study, we focused on the mechanisms underlying NGF pain sensitization. We identified NRP1 as a co-receptor for NGF and TrkA that is required for NGF-triggered hyperexcitability of mouse and human nociceptors (4). NRP1 is involved in NGF-evoked TRPV1 sensitization of nociceptors, since NRP1 inhibitors abrogated capsaicin-dependent increase of intracellular calcium. Furthermore, NRP1 is necessary for the pronociceptive actions of exogenous NGF in mice. GIPC1 is a pivotal scaffold protein for TrkA and NRP1 endocytic cycle and in vivo studies reveal a critical role for GIPC1 in NGF-induced nociception. Overall, understanding the complex interplay of NRP1, NGF and GIPC1 provides valuable and potential therapeutic targets for pain management and relief

References

1. Mantyh PW, Koltzenburg M, Mendell LM, Tive L, Shelton DL. Antagonism of nerve growth factor-TrkA signaling and the relief of pain. Anesthesiology. 2011;115(1):189-204.
2. Shu X, Mendell LM. Nerve growth factor acutely sensitizes the response of adult rat sensory neurons to capsaicin. Neurosci Lett. 1999;274(3):159-62.
3. Barker PA, Mantyh P, Arendt-Nielsen L, Viktrup L, Tive L. Nerve Growth Factor Signaling and Its Contribution to Pain. J Pain Res. 2020;13:1223-41.
4. Peach CJ, Tonello R, Gomez K, Calderon-Rivera A, Sanchez MR, Maile L, et al. Neuropilin-1 is a co-receptor for NGF and TrkA-evoked pain. bioRxiv. 2023.
5. Bimonte S, Cascella M, Forte CA, Esposito G, Cuomo A. The Role of Anti-Nerve Growth Factor Monoclonal Antibodies in the Control of Chronic Cancer and Non-Cancer Pain. J Pain Res. 2021;14:1959-67.
6. Chang DS, Hsu E, Hottinger DG, Cohen SP. Anti-nerve growth factor in pain management: current evidence. J Pain Res. 2016;9:373-83.

Presenting Author

Elisa Damo

Poster Authors

Elisa Damo

PhD

New York University

Lead Author

E-mail

Raquel Tonello

New York University

Lead Author

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Chloe Peach

Lead Author

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Ana-Maria Manu

Lead Author

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Brian Schmidt

NYU Dentistry, Translational Research Center

Lead Author

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Nigel Bunnett

Department of Molecular Pathobiology, Pain Research Center, NYU

Lead Author

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Topics

  • Mechanisms: Biological-Molecular and Cell Biology