Background & Aims

Neuropathic pain is evoked by an injury to the somatosensory system at the peripheral or central nervous system level leading to direct changes in neuronal activity (1). Moreover, there is a large body of evidence that changes occurring in primary peripheral sensory neurons also alter secondary central circuits thus causing hyperalgesia and allodynia. The latter are hallmarks of neuropathic pain and recent work suggests that both of those sensory abnormalities depend on alterations in spinal dorsal horn circuits likely caused by a loss of local inhibition(2)(3). However, the molecular mechanisms that lead to these circuit changes are still poorly understood. To redress this, we characterized gene expression changes occurring in different neuronal and non-neuronal spinal cell populations at different time points after peripheral nerve injury (PNI) in males and females employing the highly sensitive Translating Ribosome Affinity Purification (TRAP) method (4).

Methods

To characterize the translatomic changes occurring after peripheral nerve injury, we first induced neuropathic pain in mice by performing Spared Nerve Injury (SNI) surgery(5). Behavioral testing using Von Frey method is used to monitor mechanical sensitivity changes before and after surgical intervention. After 2 days and 7 days from SNI surgery, spinal cord polysomal RNA from different spinal cell populations is extracted employing highly sensitive Translating Ribosome Affinity Purification (TRAP) method. After RNA-bulk sequencing, Differential Gene Expression Analysis comparing SNI, Sham and Naïve conditions is employed to identify gene expression changes at different time points post-nerve injury. The resulting selected candidate genes are further mapped back into the spinal cord using Immunohistochemistry and in situ Hybridization methods. Ultimately, a spinal topographic map of acute and chronic pain state for males and females is obtained.

Results

We have meanwhile obtained results employing the TRAP method using two different TRAP lines (1. Ellacre:nuTRAP (all spinal cells) and 2. CCKcre:nuTRAP (CCK expressing spinal neurons). We have used the Ellacre:nuTRAP to compare the transcriptome (total RNA input) to the translatome (affinity purified RNA) of the mice in the different conditions. We found that approx. 30% of all differentially expressed genes (DEGs) found in the transcriptome were propagated to the translatome. Unexpectedly, approx. 45% of all differentially expressed genes (DEGs) were only detected in the translatome.
When analyzing the translatomes of CCKcre:nuTRAP mice we found more DEGs between SNI, Sham and Naïve conditions as in the first analysis. Employing pathway analysis tools, we found many components of the complement system to be upregulated 7 days after SNI in male and female mice (e.g. C1qa, C1qb, ccl7, ccl2). Furthermore. we found many transient changes occurring in Sham mice two days after surgery.

Conclusions

Based on our analysis so far, we conclude that several transcriptional changes in the spinal cord induced by injury are propagated to the translatome. Finding additional or more DEGs in the translatome than in the transcriptome may be due to technical issues or may hint at regulation of protein expression occurring at the translational and not transcriptional level.

In agreement with previous reports (6), analysis of the results from the CCKcre:nuTRAP sequencing suggests deregulation of the complement system as one potential underlying mechanism for the development of neuropathy.

Finally, finding many more DEGs in the analysis of CCKcre:nuTRAP than in the Ellacre:nuTRAP mice suggests that many of the expression changes occurring in different spinal cell population might shadow each other, preventing the detection of genes that selectively are up or down in regulated in smaller cell populations during chronic pain development.

References

1. Scholz J, Finnerup NB, Attal N, Aziz Q, Baron R, Bennett MI, et al. The IASP classification of chronic pain for ICD-11: Chronic neuropathic pain. Pain. 2019;160(1):53–9.

2. Basbaum AI, Bautista DM, Scherrer G, Julius D. Cellular and Molecular Mechanisms of Pain. Cell. 2009;139(2):267–84.

3. Peirs C, Williams SPG, Zhao X, Arokiaraj CM, Ferreira DW, Noh M chul, et al. Mechanical Allodynia Circuitry in the Dorsal Horn Is Defined by the Nature of the Injury. Neuron [Internet]. 2021;109(1):73-90.e7. Available from: https://doi.org/10.1016/j.neuron.2020.10.027

4. Heiman M, Kulicke R, Fenster RJ, Greengard P, Heintz N. Cell type-specific mRNA purification by translating ribosome affinity purification (TRAP). Nat Protoc [Internet]. 2014;9(6):1282–91. Available from: https://dx.doi.org/10.1038/nprot.2014.085

5. Cichon J, Sun L, Yang G. Spared Nerve Injury Model of Neuropathic Pain in Mice. Bio-Protocol. 2018;8(6):1–7.

6. Griffin RS, Costigan M, Brenner GJ, Ma CHE, Scholz J, Moss A, et al. Complement induction in spinal cord microglia results in anaphylatoxin C5a-mediated pain hypersensitivity. J Neurosci. 2007;27(32):8699–708.

7. Calvo M, Davies AJ, Hébert HL, Weir GA, Chesler EJ, Finnerup NB, et al. The Genetics of Neuropathic Pain from Model Organisms to Clinical Application. Neuron. 2019;104(4):637–53.

8. Foster E, Wildner H, Tudeau L, Haueter S, Ralvenius WT, Jegen M, et al. Targeted ablation, silencing, and activation establish glycinergic dorsal horn neurons as key components of a spinal gate for pain and itch. Neuron. 2015;85(6):1289–304.

9. Frezel N, Ranucci M, Foster E, Wende H, Pelczar P, Mendes R, et al. c-Maf-positive spinal cord neurons are critical elements of a dorsal horn circuit for mechanical hypersensitivity in neuropathy. Cell Rep. 2023;42(4).

Presenting Author

Camilla Beccarini

Poster Authors

Camilla Beccarini

MSc

University of Zurich

Lead Author

E-mail

Louis Scheurer

University of Zurich, Institute of Pharmacology and Toxicology

Lead Author

E-mail

Eva Roth

University of Zurich, Institute of Pharmacology and Toxicology

Lead Author

E-mail

Lennart Opitz

Functional Genomics Center Zurich

Lead Author

E-mail

Hendrik Wildner

PhD

University of Zurich, Institute of Pharmacology and Toxicology

Lead Author

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Topics

  • Mechanisms: Biological-Molecular and Cell Biology