Background & Aims
Neuropilin-1 (NRP1) is a membrane bound receptor that promotes and inhibits sensory neuron excitability in a ligand-specific manner. During development and healing, NRP1 has been implicated in the regulation of axon guidance, with elevated expression in primary afferents following nerve injury.1, 3 NRP1 is a co-receptor for receptor tyrosine kinases and can form homotypic intercellular interactions in the absence of RTKs.4 NRP1 implicated in Vegfa and NGF-dependent pain through RTK signaling.1, 2 However, NRP1 is expressed on a variety of cell types including leukocytes, endothelial cells, and neurons. This study was designed to investigate whether NRP1 expressed specifically on peptidergic sensory neurons regulates basal nociception.
Methods
RNAscope was used to determine the neurochemical phenotype of DRG neurons from male and female Spraque-Dawley rats. Basescope and fluorescence immunohistochemistry were used to validate deletion of NRP1 in CGRPcreER;NRP1-/- mice. Positive neurons and Intraepidermal nerve fibers (IENF) were quantified using Ilastik or Fiji software. The following behavioral assays were performed: Mechanical sensitivity (SUDO method), Heat sensitivity (Hargreaves’ apparatus), Cold plantar assay (dry ice method), and locomotor activity (Rotorod). Vegfa165 (4ng/20ul) or vehicle were injected intraplantar.
Results
NRP1 is widely expressed in Nefh, Calca, and Th DRG neurons. However, 74.37 ± 1.56% neurons in rat DRG exhibit expression of NRP1 while only 47.67 ± 4.94% of neurons in mouse expressed NRP1. In both rat and mouse, approximately two-thirds of NRP1 expressing neurons co-express CGRP or Calca mRNA. Nrp1 mRNA expression is significantly reduced in CGRP expressing neurons in DRG from CGRP-creER: NRP1fl/fl. NRP1 knockout mice exhibit normal numbers of CGRP positive DRG neurons as well as normal CGRP positive IENF density. NRP1 knockout mice show no deficits in locomotor activity or cold sensitivity. However, CGRP-creER: NRP1fl/fl mice exhibit significantly higher mechanical thresholds and paw withdrawal latencies to radiant heat. Importantly, intraplantar vegfa induces mechanical allodynia in control mice, but CGRP-creER: NRP1fl/fl mice have similar mechanical thresholds to vehicle treated mice.
Conclusions
Conditional deletion of NRP1 from peptidergic sensory afferents does not alter peptidergic innervation. However, loss of peptidergic NRP1 is sufficient to inhibit basal mechanical and heat sensitivity. Inhibition of basal nociception suggests that targeting NRP1 therapeutically may reduce pathological pain.
References
1.Gavazzi I, Stonehouse J, Sandvig A, Reza JN, Appiah-Kubi LS, Keynes R, Cohen J. Peripheral, but not central, axotomy induces neuropilin-1 mRNA expression in adult large diameter primary sensory neurons. J Comp Neurol. 423:492-499, 2000
2.Gomez K, Duran P, Tonello R, Allen HN, Boinon L, Calderon-Rivera A, Loya-Lopez S, Nelson TS, Ran D, Moutal A, Bunnett NW, Khanna R. Neuropilin-1 is essential for vascular endothelial growth factor A-mediated increase of sensory neuron activity and development of pain-like behaviors. Pain. 164:2696-2710, 2023
3.Lindholm T, Risling M, Carlstedt T, Hammarberg H, Wallquist W, Cullheim S, Skold MK. Expression of Semaphorins, Neuropilins, VEGF, and Tenascins in Rat and Human Primary Sensory Neurons after a Dorsal Root Injury. Front Neurol. 8:49, 2017
4.Sawma P, Roth L, Blanchard C, Bagnard D, Cremel G, Bouveret E, Duneau JP, Sturgis JN, Hubert P. Evidence for new homotypic and heterotypic interactions between transmembrane helices of proteins involved in receptor tyrosine kinase and neuropilin signaling. J Mol Biol. 426:4099-4111, 2014
Presenting Author
Jami Saloman
Poster Authors
Jami Saloman
PhD
University Of Pittsburgh
Lead Author
Shilang Xiao
Lead Author
Heather Allen
PhD
Lead Author
Stephanie Fulton
University of Pittsburgh
Lead Author
Olivia Babyok
University of Pittsburgh
Lead Author
Rajesh Khanna
PhD
University of Florida College of Medicine
Lead Author
Topics
- Mechanisms: Biological-Molecular and Cell Biology