METTL14-mediated m6A epitranscriptomic modification contributes to chemotherapy-induced neuropathic pain by stabilizing GluN2A expression via IGF2BP2
Weicheng Lu, … , Huijie Ma, Jingdun Xie
Weicheng Lu, … , Huijie Ma, Jingdun Xie
Published February 6, 2024
Citation Information: J Clin Invest. 2024;134(6):e174847. https://doi.org/10.1172/JCI174847.
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Research Article Neuroscience

METTL14-mediated m6A epitranscriptomic modification contributes to chemotherapy-induced neuropathic pain by stabilizing GluN2A expression via IGF2BP2

Abstract

Epigenetics is a biological process that modifies and regulates gene expression, affects neuronal function, and contributes to pain. However, the mechanism by which epigenetics facilitates and maintains chronic pain is poorly understood. We aimed to determine whether N6-methyladenosine (m6A) specifically modified by methyltransferase-like 14 (METTL14) alters neuronal activity and governs pain by sensitizing the GluN2A subunit of the N-methyl-d-aspartate receptor (NMDAR) in the dorsal root ganglion (DRG) neurons in a model of chemotherapy-induced neuropathic pain (CINP). Using dot blotting, immunofluorescence, gain/loss-of-function, and behavioral assays, we found that m6A levels were upregulated in L4–L6 DRG neurons in CINP in a DBP/METTL14-dependent manner, which was also confirmed in human DRGs. Blocking METTL14 reduced m6A methylation and attenuated pain hypersensitivity. Mechanistically, METTL14-mediated m6A modification facilitated the synaptic plasticity of DRG neurons by enhancing the GluN2A subunit of NMDAR, and inhibiting METTL14 blocked this effect. In contrast, overexpression of METTL14 upregulated m6A modifications, enhanced presynaptic NMDAR activity in DRG neurons, and facilitated pain sensation. Our findings reveal a previously unrecognized mechanism of METTL14-mediated m6A modification in DRG neurons to maintain neuropathic pain. Targeting these molecules may provide a new strategy for pain treatment.

Authors

Weicheng Lu, Xiaohua Yang, Weiqiang Zhong, Guojun Chen, Xinqi Guo, Qingqing Ye, Yixin Xu, Zhenhua Qi, Yaqi Ye, Jingyun Zhang, Yuge Wang, Xintong Wang, Shu Wang, Qiyue Zhao, Weian Zeng, Junting Huang, Huijie Ma, Jingdun Xie

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Figure 9

IGF2BP2 mediates the stability of Grin2a mRNA in an m6A-dependent manner and regulates pain hypersensitivity.

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IGF2BP2 mediates the stability of Grin2a mRNA in an m6A-dependent manner...
(A) RNA stability curves in vivo (at least 4 rats per group; *PCX vs. CON, #PCX+siMettl14 vs. PCX+siNC). (B) Double-labeled immunofluorescent images and quantification showing the colocalization and alteration of GluN2A in neurons. Bar plots show the relative GluN2A intensity in neurons and NeuN intensity (n = 5 rats per group). Scale bar: 50 μm. (C) Illustration of RNA stability assay in rat primary cultured DRG neurons. (D) RNA stability in cultured DRG neurons. (E) IGF2BP mRNA expression in DRGs in rats at day 10 (n = 6 rats per group, Student’s t test). (F) IGF2BP2 protein expression in DRGs at day 10 (n = 4, Student’s t test). (G) Western blotting results of IGF2BPs pulled down by different probes. (H) RIP-qPCR results for enriched Grin2a mRNA using an IGF2BP2 antibody compared with IgG control in saline- or PCX-pretreated rats (2-way ANOVA followed by Šidák’s post hoc test). (I) RNA stability curves in vivo (5 rats per group; *PCX vs. CON, #PCX+siMettl14 vs. PCX+siNC). (J) RNA stability in cultured DRG neurons. (K) RNA stability in ActD-incubated PC12 cells transfected with lectin-Mettl14 or lectin-Gfp combined with siIgf2bp2 or scrambled siRNA (*lectin-Mettl14 vs. the control, #lectin-Mettl14+siIgf2bp2 vs. lectin-Mettl14+siNC). (L–N) Mechanical allodynia (L), thermal hyperalgesia (M), and cold allodynia (N) of naive rats after AAV-Mettl14 or AAV-Gfp combined with siIgf2bp2 or scrambled siRNA intrathecal injection (*AAV-Mettl14+siIgf2bp2 vs. AAV-Mettl14+siNC). Two-way ANOVA followed by Tukey’s post hoc test was used for RNA stability, whole-cell patch-clamp recording, and behavior data analysis. Data are shown as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; #P < 0.05, ##P < 0.01, ###P < 0.001.