Background & Aims

Chronic pain is common, poorly understood and difficult to treat. Existing therapeutics lack efficacy and have devastating side effects, as demonstrated by the opioid epidemic1. During chronic pain, synaptic transmission is sustained and depends on the balance between synaptic vesicle (SV) exocytosis, which releases neurotransmitters into the synaptic cleft, and SV endocytosis, which maintains a releasable pool of SVs in presynaptic nociceptor terminals2,3. Although endocytic mediators are known to control SV trafficking, their specific roles in synaptic transmission in nociceptive circuits are largely unknown. Whether therapeutic targeting of SV recycling ameliorates chronic pain remains to be determined. Here, we investigate if the disruption of endocytosis in nociceptors would provide sustained relief from chronic pain.

Methods

The endocytic mediators dynamin (Dnm) and adaptor protein-2-associated kinase 1 (AAK1) were localized in mouse and human dorsal root ganglia (DRG) using RNAScope®. To assess their role in pain, siRNA, shRNA or control was administered intrathecally (i.t.) to mice to knockdown endocytic proteins. The effects on nociception were evaluated in preclinical models of postoperative pain (paw incision), inflammatory pain (intraplantar complete Freund’s adjuvant), neuropathic pain (spared nerve injury), and cancer pain (melanoma cells intraplantar inoculation). As a therapeutic approach, lipid nanoparticles (LNP) encapsulating mRNA were administered to silence endocytic mediators by CRISPR interference (CRISPRi). Mechanical allodynia and thermal hyperalgesia were evaluated. To assess the impact of endocytic mediator knockdown on synaptic transmission in nociceptive circuits, cell-attached recordings were made from post-synaptic spinal neurons after stimulation of presynaptic neurons.

Results

Dnm 1 and AAK1 mRNA were expressed in DRG neurons of mouse and human. Intrathecal injection of Dnm 1 and AAK1 siRNA knocked down endocytosis mediators in DRG neurons and reversed mechanical allodynia and thermal hyperalgesia in preclinical models of postoperative, inflammatory, neuropathic and cancer pain. Endocytic mediator knockdown inhibited synaptic transmission between primary sensory neurons and neurons in lamina I/II of the spinal cord dorsal horn by suppressing SV release from presynaptic primary afferent neurons. A dCas9 mRNA CRISPRi construct with single-guide RNA targeting the promoter of the mouse AAK1 gene was encapsulated into LNPs. LNP-dCas9 AAK1 prevented both mechanical allodynia and thermal hyperalgesia in postoperative pain. Normal motor functions were unaffected by any treatment.

Conclusions

Our results reveal a major role for endocytic mediators in SV recycling in the central projections of nociceptors in the spinal cord. Disruption of synaptic transmission reverses chronic nociception without affecting normal motor functions. Moreover, intrathecal administration of LNP encapsulating CRISPRi formulations can be used to silence pain-associated genes, offering a non-opioid therapy for the relief of chronic pain.

References

  1. Shipton EA, Shipton EE, Shipton AJ. A Review of the opioid epidemic: what do we do about it? Pain Ther 2018;7:23–36
  2. Basbaum AI, Bautista DM, Scherrer G, Julius D. Cellular and molecular mechanisms of pain. Cell 139, 267-284 (2009).
  3. Chanaday NL, Cousin MA, Milosevic I, Watanabe S, Morgan JR. The Synaptic Vesicle Cycle Revisited: New Insights into the Modes and Mechanisms. The Journal of Neuroscience 39, 8209 (2019).
  4. Tonello R, Anderson WB, Davidson S, Escriou V, Yang L, Schmidt BL, Imlach WL, Bunnett NW. The contribution of endocytosis to sensitization of nociceptors and synaptic transmission in nociceptive circuits. Pain. 2023;164(6):1355-1374.

Presenting Author

Raquel Tonello

Poster Authors

Raquel Tonello

PhD

New York University

Lead Author

Maria Fialho

Federal University of Santa Maria, Santa Maria, RS, Brazil

Lead Author

Thomas Payne

Lead Author

Gokul Sriman Thanigai Arasu

Lead Author

Colin Pouton

PhD

Lead Author

Wendy Imlach

PhD

Lead Author

Nigel Bunnett

Department of Molecular Pathobiology, Pain Research Center, NYU

Lead Author

Topics

  • Mechanisms: Biological-Molecular and Cell Biology