Background & Aims

Recent work has shown that intracellular metabotropic glutamate receptor 5 (mGluR5) mediates neuropathic and inflammatory pain (1, 2). This receptor is a G protein-coupled receptor expressed not only on the cell surface but also the intracellular membrane of spinal cord dorsal horn neurons (2). In these neurons, >80% of mGluR5 is localized within the inner and outer nuclear membrane (60%) and the endoplasmic reticulum. Scaffolding proteins, Homer 1a/b/c, control the trafficking of mGluR5 (3, 4). The interaction between mGluR5 and Homer 1b/c has been shown to promote a pain response that can be inhibited by Homer 1a (5). It is unknown whether inhibiting the interaction between nuclear or cell surface mGluR5 and Homer 1a/b/c can alter the pain response following injury. The aim of this study is to investigate whether using cell-penetrating peptides (CPP) that inhibit the mGluR5 and Homer 1b/c interaction, at the level of the cell membrane or nucleus, can induce an analgesic effect.

Methods

Previous work has described the design of a CPP capable of inhibiting the mGluR5 and Homer 1b/c interaction using a decoy Homer binding motif attached to a cell permeabilizing sequence referred to as TAT (6). In order to restrict this inhibition to the nuclear membrane, we added a nuclear localization sequence (NLS) to the previous CPP design (TAT-NLS-mGluR5ct). Consequently, rats with CFA-induced inflammatory pain were intrathecally injected with the CPP while control CFA rats received a scrambled peptide sequence. Mechanical sensitivity was evaluated by measuring paw withdrawal threshold via the up-down method. The 50% paw withdrawal threshold was assessed prior to the drug injection in addition to 0.5-, 1-, and 24-hours post-injection. The specificity and efficacy of the CPP was verified by measuring the levels of mGluR5 and Homer 1b/c in the nuclear and membrane fractions using subcellular fractionation and western blotting.

Results

The presence of Homer 1b/c within the nuclear fraction was confirmed by western blot and subcellular fractionation. Preliminary results showed that TAT-NLS-mGluR5ct induced an increase in paw withdrawal threshold compared to the control group. This was in agreement with the reduction in mGluR5 observed within the nuclear membrane of rats injected with the TAT-NLS-mGluR5 peptide. The membrane fraction of the peptide-treated group showed no change in mGluR5 levels compared to the control.

Conclusions

These preliminary findings highlight the potential analgesic effect of inhibiting the interaction between mGluR5 and Homer 1b/c, specifically at the level of the nuclear membrane in rats with inflammatory pain. Future studies will investigate the effect of using a CPP that targets mGluR5-Homer 1b/c interaction at the plasma membrane using a nuclear exclusion sequence (NES). Rodents with spared nerve injury can also be used to model neuropathic pain. Co-immunoprecipitation will be done to confirm the inhibition of binding between Homer 1b/c proteins and mGluR5 in the different fractions of the TAT-mGluR5ct treated groups.

References

1.Vincent K, Wang SF, Laferrière A, Kumar N, Coderre TJ. Spinal intracellular metabotropic glutamate receptor 5 (mGluR5) contributes to pain and c-fos expression in a rat model of inflammatory pain. Pain. 2017;158(4):705-16. 2.Vincent K, Cornea VM, Jong YI, Laferrière A, Kumar N, Mickeviciute A, et al. Intracellular mGluR5 plays a critical role in neuropathic pain. Nat Commun. 2016;7:10604. 3.Ango F, Pin JP, Tu JC, Xiao B, Worley PF, Bockaert J, et al. Dendritic and axonal targeting of type 5 metabotropic glutamate receptor is regulated by homer1 proteins and neuronal excitation. J Neurosci. 2000;20(23):8710-6. 4.Ribeiro FM, Paquet M, Cregan SP, Ferguson SS. Group I metabotropic glutamate receptor signalling and its implication in neurological disease. CNS Neurol Disord Drug Targets. 2010;9(5):574-95. 5.Obara I, Goulding SP, Hu JH, Klugmann M, Worley PF, Szumlinski KK. Nerve injury-induced changes in Homer/glutamate receptor signaling contribute to the development and maintenance of neuropathic pain. Pain. 2013;154(10):1932-45. 6.Mao L, Yang L, Tang Q, Samdani S, Zhang G, Wang JQ. The scaffold protein Homer1b/c links metabotropic glutamate receptor 5 to extracellular signal-regulated protein kinase cascades in neurons. J Neurosci. 2005;25(10):2741-52. 7.Viderman D, Tapinova K, Aubakirova M, Abdildin YG. The Prevalence of Pain in Chronic Diseases: An Umbrella Review of Systematic Reviews. J Clin Med. 2023;12(23). 8.Murray CB, de la Vega R, Murphy LK, Kashikar-Zuck S, Palermo TM. The prevalence of chronic pain in young adults: a systematic review and meta-analysis. Pain. 2022;163(9):e972-e84.

Presenting Author

Maya Jammoul

Poster Authors

Maya Jammoul

MS Neuroscience

McGill University

Lead Author

Nitasha Gill

MS Neuroscience

McGill University

Lead Author

Roseanna Rought

McGill University

Lead Author

Topics

  • Chronic Pain – Inflammatory
  • Models