Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Oct 25:20:554-566.
doi: 10.1016/j.isci.2019.09.020. Epub 2019 Sep 17.

RARβ Agonist Drug (C286) Demonstrates Efficacy in a Pre-clinical Neuropathic Pain Model Restoring Multiple Pathways via DNA Repair Mechanisms

Maria B Goncalves  1 , Julien Moehlin  2 , Earl Clarke  3 , John Grist  3 , Carl Hobbs  3 , Antony M Carr  4 , Julian Jack  3 , Marco Antonio Mendoza-Parra  5 , Jonathan P T Corcoran  6
Affiliations

RARβ Agonist Drug (C286) Demonstrates Efficacy in a Pre-clinical Neuropathic Pain Model Restoring Multiple Pathways via DNA Repair Mechanisms

Maria B Goncalves et al. iScience. .

Erratum in

Abstract

Neuropathic pain (NP) is associated with profound gene expression alterations within the nociceptive system. DNA mechanisms, such as epigenetic remodeling and repair pathways have been implicated in NP. Here we have used a rat model of peripheral nerve injury to study the effect of a recently developed RARβ agonist, C286, currently under clinical research, in NP. A 4-week treatment initiated 2 days after the injury normalized pain sensation. Genome-wide and pathway enrichment analysis showed that multiple mechanisms persistently altered in the spinal cord were restored to preinjury levels by the agonist. Concomitant upregulation of DNA repair proteins, ATM and BRCA1, the latter being required for C286-mediated pain modulation, suggests that early DNA repair may be important to prevent phenotypic epigenetic imprints in NP. Thus, C286 is a promising drug candidate for neuropathic pain and DNA repair mechanisms may be useful therapeutic targets to explore.

Keywords: Biological Sciences; Neuroscience; Transcriptomics.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing financial interests. C286 synthesis and use in nerve injuries and neuropathic pain is protected under patents (PCT/EP2015/08,002; PCT/EP2017/0604802; GB1907647).

Figures

None
Graphical abstract
Figure 1
Figure 1
C286 Modulates Multiple Pathways Chronically Altered after SNL (A–E) (A) Schematic of experimental paradigm. L5-SNL, spinal nerve ligation; SC L5 ipsi (spinal cord, L5 level ipsilateral to injury) indicates the area where tissue analysis was carried out and is delineated as a red square in subsequent figures. Measurement of mechanical and thermal sensitivities shown as paw withdrawal (PW) in grams (g) or seconds (sec), by (B) von Frey filaments, (C) hot plate, (D) cold plate, (E) and Randall Selitto test in vehicle (n = 8) or C286 (n = 8). Data shown as Mean ± SEM. Two-way ANOVA with Pairwise Multiple Comparison Procedures (Holm-Sidak method). **p ≤ 0.01, ***p ≤ 0.001. (F–H) Gene co-expression analysis assessed in the SC from L5-SNL rats and non-injured rats, treated with vehicle or C286. (F) Differential gene expression analysis relative to the sample non-injured + vehicle. (G) Differentially expressed genes are classified by their co-expression paths assessed after injury and injury + vehicle or C286 treatment. (H) Gene ontology analysis performed per co-expression path. The heatmap illustrates the GO enrichment confidence.
Figure 2
Figure 2
C286 Regulates Inflammatory and DNA Repair Pathways (A) Genes were classified on the basis of their co-expression behavior over the various conditions. C286 downregulated 165 genes that had been upregulated by the injury. (B) GO terms associated to the co-expressed genes are displayed on the basis of their confidence (−10*log10[p value]), red arrows highlight the MAPK and WNT pathways. (C) Diagram of experimental design. (D–G) (D and E) Representative images and quantification of FZD10 (scale bar, 100 μm and 20 μm for higher-magnification insets) and (F and G) of Daxx expression in microglia (Iba1) and neurons (βIII tubulin) in the SC at the end of the treatment period (scale bar, 50 μm). Two weeks after injury, a sub-set of vehicle- and C286-treated rats was used for immunohistological analysis (n = 3 per treatment group) and for RT-qPCR (n = 3 per treatment group). (H–K) (H and I) Levels of P2X4R+ microglia (highlighted by arrows in the merged upper panel, scale bar, 30 μm) and (J and K) BDNF (in neurons and microglia) in the SC (scale bar, 30 μm). (L and M) (L) Images showing BRCA1 expression in the same area (insets show higher magnification of BRCA1 in nuclei, scale bar, 100 μm) and (M) quantification by RT-qPCR. (N–P) Expression and quantification of BRCA1 and pATM in spinal microglia (scale bar, 20 μm). (Q and R) Expression and quantification of γH2AX (scale bar, 100 μm). In E, G, I, K, O, P, and R, data are shown as Mean ± SEM of fluorescence intensity (FI) in arbitrary units (a.u.). Student's t test, **p ≤ 0.01, ***p ≤ 0.001, n = 4 (E and G) or n = 3 (I, K, M, P, and R) per group, five sections per animal).
Figure 3
Figure 3
C286 Regulates DNA Damage in Microglia via BRCA1 and ATM Pathways (A) Diagram showing microglia culture conditions and markers assessed. (B–E) (B and C) BRCA1 and (D and E) γH2AX expression and quantification in microglia cultures. (F) Diagram showing the experimental design. (G and H) Expression and quantification of pATM in nuclei in the different culture conditions. Scale bars, 15 μm. Data show Mean FI ± SEM from three independent experiments (C and E), Student's t test, *p ≤ 0.05 and (H) one-way ANOVA with Pairwise Multiple Comparison Procedures (Tukey Test), ***p ≤ 0.001.
Figure 4
Figure 4
BRCA1 Is a Downstream Target of C286 in NP Modulation (A) Schematic of experimental paradigm. (B and C) (B) Measurement of thermal and mechanical sensitivities by von Frey filaments (C) and hot plate in vehicle (n = 6) or C286 (n = 6). Data shown as Mean ± SEM. Two-way ANOVA with Pairwise Multiple Comparison Procedures (Holm-Sidak method). ***p ≤ 0.001. (D) Co-labelling of GFP and Iba1 (scale bar, 20 μm), and GFP and BRCA1 in LVsc + C286- and LVBRCA1shRNA + C286-treated rats (scale bar, 100 μm). (E–H) (E and F) Western blots and quantification of BRCA1 in L5-SNL rats transduced with LV/SC + C286 (n = 3) or LV/BRCA1shRNA + C286 (n = 3). Student's t test. *p ≤ 0.05 (G) Immunohistological confirmation of LV transduction (scale bar, 20 μm), (H) higher-magnification inset shows BRCA1 in microglia in an LV/Sc + C286-treated rat (scale bar, 10 μm). (I) Quantification of BRCA1 in microglia. (J and K) Expression and quantification of γH2AX (scale bar, 20 μm), (L) higher-magnification inset shows γH2AX in microglia in an LV/BRCA1shRNA + C286-treated rat (scale bar, 10 μm). (M and N) Expression and quantification of CGRP in laminae I–III of the dorsal horn (scale bar, 100 μm). (O and P) Expression and quantification of P2X4R (scale bar, 100 μm). Inset shows higher-magnification image (scale bar, 20 μm) Data are shown as Mean +SEM of FI. Student's t test, **p ≤ 0.01, ***p ≤ 0.001 (n = 3 per group, 5 sections per animal).
Figure 5
Figure 5
BRCA1 Influences Inflammatory Mechanisms in NP (A) Diagram of the experimental design. (B–G) (B) Immunostaining of spinal microglia and NGF, (D) TNFα, and (F) TNFR1 in LV/sc + C286- and LV/BRCA1shRNA + C286treated rats (scale bars, 30 μm). Quantification of FI for (C) NGF, (E) TNFα, and (G) TNFR1. Data show Mean FI ± SEM, Student's t test, ***p ≤ 0.001 (n = 3 per group, 5 sections per animal).
Figure 6
Figure 6
BRCA1 Is Necessary for C286-Mediated Regulation of Pain (A) Diagram of the experimental design. (B–D) (B and C) Immunostaining of spinal microglia for BDNF (scale bar, 50 μm) and its quantification. (D) Western blots of spinal cords for BDNF. Student's t test, n = 3 per group, ***p ≤ 0.001. (E–H) (E and F) Expression and quantification of FZD10 (white arrows show colocalization with Iba1 and black arrows with βIII tubulin) and (G and H) Daxx in spinal neurons and microglia of LV/sc + C286- and LV/BRCA1shRNA + C286-treated rats (insets show its predominant nuclear localization). Scale bars, 100 μm for E and G. Data show Mean FI ± SEM, n = 3 per treatment group, 5 sections per animal. Student's t -test, **p ≤ 0.01, ***p ≤ 0.001.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Alt F.W., Schwer B. DNA double-strand breaks as drivers of neural genomic change, function, and disease. DNA Repair (Amst.) 2018;71:158–163. - PMC - PubMed
    1. Amaya F., Izumi Y., Matsuda M., Sasaki M. Tissue injury and related mediators of pain exacerbation. Curr. Neuropharmacol. 2013;11:592–597. - PMC - PubMed
    1. Bajpe P.K., Heynen G.J., Mittempergher L., Grernrum W., de Rink I.A., Nijkamp W., Beijersbergen R.L., Bernards R., Huang S. The corepressor CTBP2 is a coactivator of retinoic acid receptor/retinoid X receptor in retinoic acid signaling. Mol. Cell. Biol. 2013;33:3343–3353. - PMC - PubMed
    1. Bar-Shira A., Rashi-Elkeles S., Zlochover L., Moyal L., Smorodinsky N.I., Seger R., Shiloh Y. ATM-dependent activation of the gene encoding MAP kinase phosphatase 5 by radiomimetic DNA damage. Oncogene. 2002;21:849–855. - PubMed
    1. Beggs S., Trang T., Salter M.W. P2X4R+ microglia drive neuropathic pain. Nat. Neurosci. 2012;15:1068–1073. - PMC - PubMed