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Research ArticleNeuroscience
Open Access | 
10.1172/JCI174847
1Department of Anesthesiology, State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
2Department of Physiology, Hebei Medical University, Shijiazhuang, Hebei, China.
3Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China.
Address correspondence to: Jingdun Xie, Department of Anesthesiology, Sun Yat-sen University Cancer Center, 651 Dongfeng East Road, Guangzhou 510060, China. Phone: 86.020.87343062; Email: xiejd6@mail.sysu.edu.cn. Or to: Huijie Ma, Department of Physiology, Hebei Medical University, 361 Eastern Zhongshan Road, Shijiazhuang 050017, China. Phone: 86.311.86261164; Email: huijiema@hebmu.edu.cn. Or to: Junting Huang, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan Second Road, Guangzhou 510060, China. Phone: 86.020.87330709; Email: huangjt56@mail.sysu.edu.cn.
Authorship note: WL, XY, W Zhong, and GC contributed equally to this work.
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1Department of Anesthesiology, State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
2Department of Physiology, Hebei Medical University, Shijiazhuang, Hebei, China.
3Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China.
Address correspondence to: Jingdun Xie, Department of Anesthesiology, Sun Yat-sen University Cancer Center, 651 Dongfeng East Road, Guangzhou 510060, China. Phone: 86.020.87343062; Email: xiejd6@mail.sysu.edu.cn. Or to: Huijie Ma, Department of Physiology, Hebei Medical University, 361 Eastern Zhongshan Road, Shijiazhuang 050017, China. Phone: 86.311.86261164; Email: huijiema@hebmu.edu.cn. Or to: Junting Huang, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan Second Road, Guangzhou 510060, China. Phone: 86.020.87330709; Email: huangjt56@mail.sysu.edu.cn.
Authorship note: WL, XY, W Zhong, and GC contributed equally to this work.
Find articles by Yang, X. in: JCI | PubMed | Google Scholar
1Department of Anesthesiology, State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
2Department of Physiology, Hebei Medical University, Shijiazhuang, Hebei, China.
3Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China.
Address correspondence to: Jingdun Xie, Department of Anesthesiology, Sun Yat-sen University Cancer Center, 651 Dongfeng East Road, Guangzhou 510060, China. Phone: 86.020.87343062; Email: xiejd6@mail.sysu.edu.cn. Or to: Huijie Ma, Department of Physiology, Hebei Medical University, 361 Eastern Zhongshan Road, Shijiazhuang 050017, China. Phone: 86.311.86261164; Email: huijiema@hebmu.edu.cn. Or to: Junting Huang, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan Second Road, Guangzhou 510060, China. Phone: 86.020.87330709; Email: huangjt56@mail.sysu.edu.cn.
Authorship note: WL, XY, W Zhong, and GC contributed equally to this work.
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1Department of Anesthesiology, State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
2Department of Physiology, Hebei Medical University, Shijiazhuang, Hebei, China.
3Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China.
Address correspondence to: Jingdun Xie, Department of Anesthesiology, Sun Yat-sen University Cancer Center, 651 Dongfeng East Road, Guangzhou 510060, China. Phone: 86.020.87343062; Email: xiejd6@mail.sysu.edu.cn. Or to: Huijie Ma, Department of Physiology, Hebei Medical University, 361 Eastern Zhongshan Road, Shijiazhuang 050017, China. Phone: 86.311.86261164; Email: huijiema@hebmu.edu.cn. Or to: Junting Huang, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan Second Road, Guangzhou 510060, China. Phone: 86.020.87330709; Email: huangjt56@mail.sysu.edu.cn.
Authorship note: WL, XY, W Zhong, and GC contributed equally to this work.
Find articles by Chen, G. in: JCI | PubMed | Google Scholar
1Department of Anesthesiology, State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
2Department of Physiology, Hebei Medical University, Shijiazhuang, Hebei, China.
3Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China.
Address correspondence to: Jingdun Xie, Department of Anesthesiology, Sun Yat-sen University Cancer Center, 651 Dongfeng East Road, Guangzhou 510060, China. Phone: 86.020.87343062; Email: xiejd6@mail.sysu.edu.cn. Or to: Huijie Ma, Department of Physiology, Hebei Medical University, 361 Eastern Zhongshan Road, Shijiazhuang 050017, China. Phone: 86.311.86261164; Email: huijiema@hebmu.edu.cn. Or to: Junting Huang, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan Second Road, Guangzhou 510060, China. Phone: 86.020.87330709; Email: huangjt56@mail.sysu.edu.cn.
Authorship note: WL, XY, W Zhong, and GC contributed equally to this work.
Find articles by Guo, X. in: JCI | PubMed | Google Scholar
1Department of Anesthesiology, State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
2Department of Physiology, Hebei Medical University, Shijiazhuang, Hebei, China.
3Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China.
Address correspondence to: Jingdun Xie, Department of Anesthesiology, Sun Yat-sen University Cancer Center, 651 Dongfeng East Road, Guangzhou 510060, China. Phone: 86.020.87343062; Email: xiejd6@mail.sysu.edu.cn. Or to: Huijie Ma, Department of Physiology, Hebei Medical University, 361 Eastern Zhongshan Road, Shijiazhuang 050017, China. Phone: 86.311.86261164; Email: huijiema@hebmu.edu.cn. Or to: Junting Huang, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan Second Road, Guangzhou 510060, China. Phone: 86.020.87330709; Email: huangjt56@mail.sysu.edu.cn.
Authorship note: WL, XY, W Zhong, and GC contributed equally to this work.
Find articles by Ye, Q. in: JCI | PubMed | Google Scholar
1Department of Anesthesiology, State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
2Department of Physiology, Hebei Medical University, Shijiazhuang, Hebei, China.
3Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China.
Address correspondence to: Jingdun Xie, Department of Anesthesiology, Sun Yat-sen University Cancer Center, 651 Dongfeng East Road, Guangzhou 510060, China. Phone: 86.020.87343062; Email: xiejd6@mail.sysu.edu.cn. Or to: Huijie Ma, Department of Physiology, Hebei Medical University, 361 Eastern Zhongshan Road, Shijiazhuang 050017, China. Phone: 86.311.86261164; Email: huijiema@hebmu.edu.cn. Or to: Junting Huang, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan Second Road, Guangzhou 510060, China. Phone: 86.020.87330709; Email: huangjt56@mail.sysu.edu.cn.
Authorship note: WL, XY, W Zhong, and GC contributed equally to this work.
Find articles by Xu, Y. in: JCI | PubMed | Google Scholar
1Department of Anesthesiology, State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
2Department of Physiology, Hebei Medical University, Shijiazhuang, Hebei, China.
3Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China.
Address correspondence to: Jingdun Xie, Department of Anesthesiology, Sun Yat-sen University Cancer Center, 651 Dongfeng East Road, Guangzhou 510060, China. Phone: 86.020.87343062; Email: xiejd6@mail.sysu.edu.cn. Or to: Huijie Ma, Department of Physiology, Hebei Medical University, 361 Eastern Zhongshan Road, Shijiazhuang 050017, China. Phone: 86.311.86261164; Email: huijiema@hebmu.edu.cn. Or to: Junting Huang, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan Second Road, Guangzhou 510060, China. Phone: 86.020.87330709; Email: huangjt56@mail.sysu.edu.cn.
Authorship note: WL, XY, W Zhong, and GC contributed equally to this work.
Find articles by Qi, Z. in: JCI | PubMed | Google Scholar
1Department of Anesthesiology, State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
2Department of Physiology, Hebei Medical University, Shijiazhuang, Hebei, China.
3Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China.
Address correspondence to: Jingdun Xie, Department of Anesthesiology, Sun Yat-sen University Cancer Center, 651 Dongfeng East Road, Guangzhou 510060, China. Phone: 86.020.87343062; Email: xiejd6@mail.sysu.edu.cn. Or to: Huijie Ma, Department of Physiology, Hebei Medical University, 361 Eastern Zhongshan Road, Shijiazhuang 050017, China. Phone: 86.311.86261164; Email: huijiema@hebmu.edu.cn. Or to: Junting Huang, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan Second Road, Guangzhou 510060, China. Phone: 86.020.87330709; Email: huangjt56@mail.sysu.edu.cn.
Authorship note: WL, XY, W Zhong, and GC contributed equally to this work.
Find articles by Ye, Y. in: JCI | PubMed | Google Scholar
1Department of Anesthesiology, State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
2Department of Physiology, Hebei Medical University, Shijiazhuang, Hebei, China.
3Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China.
Address correspondence to: Jingdun Xie, Department of Anesthesiology, Sun Yat-sen University Cancer Center, 651 Dongfeng East Road, Guangzhou 510060, China. Phone: 86.020.87343062; Email: xiejd6@mail.sysu.edu.cn. Or to: Huijie Ma, Department of Physiology, Hebei Medical University, 361 Eastern Zhongshan Road, Shijiazhuang 050017, China. Phone: 86.311.86261164; Email: huijiema@hebmu.edu.cn. Or to: Junting Huang, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan Second Road, Guangzhou 510060, China. Phone: 86.020.87330709; Email: huangjt56@mail.sysu.edu.cn.
Authorship note: WL, XY, W Zhong, and GC contributed equally to this work.
Find articles by Zhang, J. in: JCI | PubMed | Google Scholar
1Department of Anesthesiology, State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
2Department of Physiology, Hebei Medical University, Shijiazhuang, Hebei, China.
3Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China.
Address correspondence to: Jingdun Xie, Department of Anesthesiology, Sun Yat-sen University Cancer Center, 651 Dongfeng East Road, Guangzhou 510060, China. Phone: 86.020.87343062; Email: xiejd6@mail.sysu.edu.cn. Or to: Huijie Ma, Department of Physiology, Hebei Medical University, 361 Eastern Zhongshan Road, Shijiazhuang 050017, China. Phone: 86.311.86261164; Email: huijiema@hebmu.edu.cn. Or to: Junting Huang, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan Second Road, Guangzhou 510060, China. Phone: 86.020.87330709; Email: huangjt56@mail.sysu.edu.cn.
Authorship note: WL, XY, W Zhong, and GC contributed equally to this work.
Find articles by Wang, Y. in: JCI | PubMed | Google Scholar
1Department of Anesthesiology, State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
2Department of Physiology, Hebei Medical University, Shijiazhuang, Hebei, China.
3Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China.
Address correspondence to: Jingdun Xie, Department of Anesthesiology, Sun Yat-sen University Cancer Center, 651 Dongfeng East Road, Guangzhou 510060, China. Phone: 86.020.87343062; Email: xiejd6@mail.sysu.edu.cn. Or to: Huijie Ma, Department of Physiology, Hebei Medical University, 361 Eastern Zhongshan Road, Shijiazhuang 050017, China. Phone: 86.311.86261164; Email: huijiema@hebmu.edu.cn. Or to: Junting Huang, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan Second Road, Guangzhou 510060, China. Phone: 86.020.87330709; Email: huangjt56@mail.sysu.edu.cn.
Authorship note: WL, XY, W Zhong, and GC contributed equally to this work.
Find articles by Wang, X. in: JCI | PubMed | Google Scholar
1Department of Anesthesiology, State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
2Department of Physiology, Hebei Medical University, Shijiazhuang, Hebei, China.
3Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China.
Address correspondence to: Jingdun Xie, Department of Anesthesiology, Sun Yat-sen University Cancer Center, 651 Dongfeng East Road, Guangzhou 510060, China. Phone: 86.020.87343062; Email: xiejd6@mail.sysu.edu.cn. Or to: Huijie Ma, Department of Physiology, Hebei Medical University, 361 Eastern Zhongshan Road, Shijiazhuang 050017, China. Phone: 86.311.86261164; Email: huijiema@hebmu.edu.cn. Or to: Junting Huang, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan Second Road, Guangzhou 510060, China. Phone: 86.020.87330709; Email: huangjt56@mail.sysu.edu.cn.
Authorship note: WL, XY, W Zhong, and GC contributed equally to this work.
Find articles by Wang, S. in: JCI | PubMed | Google Scholar
1Department of Anesthesiology, State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
2Department of Physiology, Hebei Medical University, Shijiazhuang, Hebei, China.
3Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China.
Address correspondence to: Jingdun Xie, Department of Anesthesiology, Sun Yat-sen University Cancer Center, 651 Dongfeng East Road, Guangzhou 510060, China. Phone: 86.020.87343062; Email: xiejd6@mail.sysu.edu.cn. Or to: Huijie Ma, Department of Physiology, Hebei Medical University, 361 Eastern Zhongshan Road, Shijiazhuang 050017, China. Phone: 86.311.86261164; Email: huijiema@hebmu.edu.cn. Or to: Junting Huang, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan Second Road, Guangzhou 510060, China. Phone: 86.020.87330709; Email: huangjt56@mail.sysu.edu.cn.
Authorship note: WL, XY, W Zhong, and GC contributed equally to this work.
Find articles by Zhao, Q. in: JCI | PubMed | Google Scholar
1Department of Anesthesiology, State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
2Department of Physiology, Hebei Medical University, Shijiazhuang, Hebei, China.
3Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China.
Address correspondence to: Jingdun Xie, Department of Anesthesiology, Sun Yat-sen University Cancer Center, 651 Dongfeng East Road, Guangzhou 510060, China. Phone: 86.020.87343062; Email: xiejd6@mail.sysu.edu.cn. Or to: Huijie Ma, Department of Physiology, Hebei Medical University, 361 Eastern Zhongshan Road, Shijiazhuang 050017, China. Phone: 86.311.86261164; Email: huijiema@hebmu.edu.cn. Or to: Junting Huang, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan Second Road, Guangzhou 510060, China. Phone: 86.020.87330709; Email: huangjt56@mail.sysu.edu.cn.
Authorship note: WL, XY, W Zhong, and GC contributed equally to this work.
Find articles by Zeng, W. in: JCI | PubMed | Google Scholar
1Department of Anesthesiology, State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
2Department of Physiology, Hebei Medical University, Shijiazhuang, Hebei, China.
3Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China.
Address correspondence to: Jingdun Xie, Department of Anesthesiology, Sun Yat-sen University Cancer Center, 651 Dongfeng East Road, Guangzhou 510060, China. Phone: 86.020.87343062; Email: xiejd6@mail.sysu.edu.cn. Or to: Huijie Ma, Department of Physiology, Hebei Medical University, 361 Eastern Zhongshan Road, Shijiazhuang 050017, China. Phone: 86.311.86261164; Email: huijiema@hebmu.edu.cn. Or to: Junting Huang, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan Second Road, Guangzhou 510060, China. Phone: 86.020.87330709; Email: huangjt56@mail.sysu.edu.cn.
Authorship note: WL, XY, W Zhong, and GC contributed equally to this work.
Find articles by Huang, J. in: JCI | PubMed | Google Scholar
1Department of Anesthesiology, State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
2Department of Physiology, Hebei Medical University, Shijiazhuang, Hebei, China.
3Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China.
Address correspondence to: Jingdun Xie, Department of Anesthesiology, Sun Yat-sen University Cancer Center, 651 Dongfeng East Road, Guangzhou 510060, China. Phone: 86.020.87343062; Email: xiejd6@mail.sysu.edu.cn. Or to: Huijie Ma, Department of Physiology, Hebei Medical University, 361 Eastern Zhongshan Road, Shijiazhuang 050017, China. Phone: 86.311.86261164; Email: huijiema@hebmu.edu.cn. Or to: Junting Huang, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan Second Road, Guangzhou 510060, China. Phone: 86.020.87330709; Email: huangjt56@mail.sysu.edu.cn.
Authorship note: WL, XY, W Zhong, and GC contributed equally to this work.
Find articles by Ma, H. in: JCI | PubMed | Google Scholar
1Department of Anesthesiology, State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China.
2Department of Physiology, Hebei Medical University, Shijiazhuang, Hebei, China.
3Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China.
Address correspondence to: Jingdun Xie, Department of Anesthesiology, Sun Yat-sen University Cancer Center, 651 Dongfeng East Road, Guangzhou 510060, China. Phone: 86.020.87343062; Email: xiejd6@mail.sysu.edu.cn. Or to: Huijie Ma, Department of Physiology, Hebei Medical University, 361 Eastern Zhongshan Road, Shijiazhuang 050017, China. Phone: 86.311.86261164; Email: huijiema@hebmu.edu.cn. Or to: Junting Huang, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan Second Road, Guangzhou 510060, China. Phone: 86.020.87330709; Email: huangjt56@mail.sysu.edu.cn.
Authorship note: WL, XY, W Zhong, and GC contributed equally to this work.
Find articles by Xie, J. in: JCI | PubMed | Google Scholar
Authorship note: WL, XY, W Zhong, and GC contributed equally to this work.
Published February 6, 2024 - More info
Published in Volume 134, Issue 6 on March 15, 2024
AbstractEpigenetics 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.
Graphical Abstract
Introduction
Chemotherapy-induced neuropathic pain (CINP) is a debilitating complication caused by damage to the peripheral nervous system by chemotherapeutic agents, including paclitaxel (1). CINP occurs in approximately 50% to 90% of cancer survivors receiving chemotherapy, resulting in therapy discontinuation or even disability (2). Currently, effective management of CINP remains limited. Therefore, there is an urgent need to elucidate the underlying mechanisms of CINP and identify novel drug targets.
Epigenetics refers to long-lasting heritable changes in gene expression that do not affect the DNA sequences (3). N6-methyladenosine (m6A) is the most ubiquitous post-transcriptional modification in eukaryotes (4). Characterized by a consensus motif (RRACH), m6A is critical for gene expression, as it regulates RNA localization, processing, translation, and decay. It preferentially affects the sequence of the mRNA stop codon and 3′-untranslated region (3′-UTR) (5–7) and is mainly mediated by RNA methyltransferase complexes, including methyltransferase-like 14 (METTL14), methyltransferase-like 3 (METTL3), and Wilms’ tumor 1–associating protein (WTAP) (8, 9). It can be reversibly removed by demethylases, including fat mass/obesity–associated protein (FTO) and alkylation repair homolog protein (ALKBH5) (10, 11). Furthermore, transcripts with m6A modifications can be recognized and interpreted by multiple m6A-binding proteins, such as insulin-like growth factor 2 mRNA–binding proteins (IGF2BPs) (12). m6A dysregulation is associated with various physiological and pathological processes, including pain. FTO in dorsal root ganglion (DRG) neurons contributes to nerve injury–associated pain hypersensitivity (13). METTL3 facilitates complete Freund’s adjuvant–induced (CFA-induced) inflammatory hyperalgesia by regulating TET1 expression in the spinal cord (14). Targeting novel epitranscriptomic mechanisms appears to be a promising strategy for the treatment of nociceptive disorders. However, the specific role of m6A modifications in CINP has not been explicitly defined.
N-methyl-d-aspartate receptors (NMDARs) are ligand-gated ion channels that are essential for synaptic plasticity (15). Conventionally, NMDARs are predominantly localized to the postsynaptic membrane. However, NMDARs are also expressed and act presynaptically, particularly at the central terminals of primary sensory neurons (16). Enhanced presynaptic NMDAR activity contributes to glutamate release from primary afferent terminals to spinal dorsal horn neurons under painful conditions (17, 18). Upregulation of GluN2A, a key subunit of NMDARs, is indispensable for increased presynaptic NMDAR activity in CINP (19). In this regard, blocking GluN2A-containing NMDARs at the spinal cord level diminishes paclitaxel-induced hyperalgesia (20, 21). Nevertheless, the mechanism underlying the chemotherapy-induced GluN2A upregulation in primary sensory neurons remains unclear.
In this study, we aimed to determine the role of m6A modification in paclitaxel-induced CINP and to uncover the epigenetics of NMDARs in nociception. We demonstrated that paclitaxel treatment increased the m6A abundance in the DRG in CINP. The RNA methyltransferase METTL14 was highly expressed and contributed to CINP. GluN2A, but not GluN1 or GluN2B, in the DRG was a downstream target of METTL14. Mechanistically, METTL14-mediated m6A modification facilitated CINP by stabilizing Grin2a (coding for GluN2A) in an IGF2BP2-dependent manner, contributing to presynaptic NMDAR hyperactivity at the spinal dorsal horn level. Furthermore, the D-box–binding PAR BZIP transcription factor (DBP) contributed to the paclitaxel-induced increase in METTL14 expression by activating Mettl14 transcription in the DRG. Together, our findings provide new insight into the molecular pathogenesis of CINP.
ResultsPaclitaxel treatment increases m6A abundance and METTL14 expression in rat and human DRG. Following paclitaxel administration, the rats developed long-lasting exaggerated pain behaviors, as indicated by a marked and sustained decrease in the paw withdrawal threshold, a decrease in paw withdrawal latency, and an increase in acetone response scores (Figure 1, A–D). To systematically determine m6A abundance and associated enzyme alterations in the DRG of CINP, we conducted m6A dot blot and Western blot assays. A dot blot assay revealed that the total m6A levels in the DRG were notably upregulated by paclitaxel treatment (Figure 1E). Furthermore, the expression levels of the methyltransferases METTL3, METTL14, METTL16, and WTAP increased whereas the expression levels of the demethylase ALKBH5 decreased in the DRG after paclitaxel treatment (Figure 1F). As the elevation in METTL14 expression in the DRG caused by paclitaxel was most evident, we chose METTL14 for further studies.
Figure 1Paclitaxel treatment increases m6A levels and METTL14 expression in the DRG. (A) Timeline of paclitaxel treatment, behavioral assessment, and tissue collection in rats. (B–D) Paclitaxel induced long-lasting notable mechanical allodynia, thermal hyperalgesia, and cold pain in rats (at least 6 rats per group, 2-way ANOVA followed by Šidák’s post hoc test). PWT, paw withdrawal threshold; PWL, paw withdrawal latency. (E) Representative dot blot images and quantification showing the m6A level in the bilateral L4–L6 DRGs in rats 10 days after treatment with saline or paclitaxel (PCX) (n = 12 rats per group, Student’s t test). (F) Representative immunoblot images and quantification show the protein levels of FTO, METTL3, METTL14, METTL16, WTAP, and ALKBH5 in the bilateral L4–L6 DRGs in rats 10 days after treatment with saline or PCX (n = 6 rats per group, Student’s t test). (G and H) Quantification and representative immunoblot images show the mRNA and protein levels of METTL14 in the bilateral L4–L6 DRGs in rats 0–14 days after treatment with PCX (n = 4 rats per group, 1-way ANOVA followed by Dunnett’s post hoc test for mRNA and least significant difference post hoc test for protein). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
We investigated Mettl14 mRNA and protein levels using real-time quantitative PCR (qPCR) and Western blotting in rat DRG at different time points after paclitaxel treatment. A notable increase was observed from day 7, reaching the highest level on day 10 after paclitaxel treatment (Figure 1, G and H). Next, we characterized the METTL14 expression profile in DRG (Figure 2A). Double immunolabeling confirmed that METTL14 was expressed predominantly in DRG neurons (NeuN-positive cells) (Figure 2B), but less in satellite glial cells (GFAP-positive cells) and macrophages (IBA1-positive cells) (Supplemental Figure 1, A and B; supplemental material available online with this article; https://doi.org/10.1172/JCI174847DS1). The colocalization of METTL14 in DRG neurons consistently increased after paclitaxel treatment as previously indicated (Figure 2B). Moreover, double-labeling immunofluorescence showed that METTL14-positive cells were predominantly neurons positive for IB4 (a marker for DRG non-peptidergic neurons; 33.87% ± 3.007%) and calcitonin gene–related peptide (CGRP) (a marker for small DRG peptidergic neurons; 14.9% ± 2.123%), but fewer were neurons positive for neurofilament-200 (NF200) (a marker for medium/large DRG neurons with myelinated Aβ fibers; 9.587% ± 1.523%) (Figure 2C). Further results consistently indicated that human DRG METTL14 was also expressed mostly in neurons (NeuN-positive cells, 77.87% ± 4.198%) but less in glial cells (glutamine synthetase–positive [GS-positive] cells, 21.91% ± 4.188%) (Figure 2D). Increased METTL14 expression was confirmed in the DRG tissues of clinical patients with chemotherapeutic agents, but not in those without treatment, and was mainly located in the nucleus (costained with DAPI dihydrochloride, a nuclear staining marker) (Figure 2E). Taken together, these results indicate that paclitaxel treatment increases m6A abundance and METTL14 expression in rat and human DRG.
Figure 2Characterization of METTL14 expression profile in the rat and human DRG. (A) Experimental paradigm for METTL14 expression profile analysis. (B) Left: Representative immunofluorescence images show colabeling of METTL14 and NeuN in the rat DRG 10 days after treatment with saline or PCX. Right: The percentage of DRG neurons colabeled by METTL14 and NeuN in the L4 and L5 DRGs 10 days after treatment with saline or PCX (n = 9 DRGs from 3 rats per group, Student’s t test). Scale bars: 100 μm. (C) Representative double immunofluorescence staining and quantification showing the colocalization of METTL14 with IB4, CGRP, and NF200 in rat DRG (at least 9 DRG slices from 4 rats per group). Scale bars: 100 μm. (D) Representative double immunofluorescence staining and quantification showing the colocalization of METTL14 with NeuN and glutamine synthetase (GS) in human DRG (n = 10 DRG slices per group). Scale bars: 50 μm. (E) Representative immunofluorescence staining and quantification showing colabeling and alteration of METTL14 and DAPI in patients (n = 3 patients per group, Student’s t test). Scale bars: 100 μm. Data are shown as the mean ± SEM. *P < 0.05, ****P < 0.0001.
Paclitaxel increases m6A levels in the DRG via METTL14. Next, we investigated whether METTL14 regulates m6A levels in CINP DRG. Initially, we screened the siRNA sequences in PC12 cells for METTL14 ablation (Supplemental Figure 2A). Among the screened sequences, siRNA-Mettl14-03 (named siMettl14 hereafter) resulted in the most effective knockdown of Mettl14 mRNA. Mettl14 mRNA and protein expression in the DRG was decreased by approximately 50% following siMettl14 intrathecal injection in naive rats (Figure 3, A and B), compared with that of scrambled siRNA. Furthermore, treatment with siMettl14 in naive rats markedly decreased m6A levels in the L4–L6 DRGs (Figure 3C).
Figure 3Paclitaxel treatment increases the global m6A level in the DRG via METTL14. (A and B) Mettl14 mRNA (A) and protein (B) expression in the bilateral L4–L6 DRGs in naive rats after siMettl14 or scrambled siRNA intrathecal injection (n = 6 rats per group, 1-way ANOVA followed by Tukey’s post hoc test). (C) RNA m6A level in the bilateral L4–L6 DRGs in naive rats after siMettl14 or scrambled siRNA treatment (n = 5 rats per group, Student’s t test). (D and E) Mettl14 mRNA (D) and protein (E) expression in the bilateral L4–L6 DRGs in saline- or PCX-pretreated rats after siMettl14 intrathecal injection (n = 5 rats per group, 1-way ANOVA followed by Tukey’s post hoc test). (F) RNA m6A level in the bilateral L4–L6 DRGs in saline- or PCX-pretreated rats after siMettl14 or scrambled siRNA treatment (n = 10 rats per group, 1-way ANOVA followed by Tukey’s post hoc test). (G and H) Mettl14 mRNA (G) and protein (H) expression in the bilateral L4–L6 DRGs in naive rats after AAV-Mettl14 or AAV-Gfp intrathecal injection (at least 4 rats per group, Student’s t test). (I) RNA m6A level in the bilateral L4–L6 DRGs in naive rats after AAV-Mettl14 or AAV-Gfp injection (n = 5 rats per group, Student’s t test). Data are shown as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
We determined the role of METTL14 in regulating paclitaxel-induced m6A expression. METTL14 knockdown using a siMettl14 reduced the paclitaxel-induced increase in Mettl14 mRNA and protein levels in the DRG (Figure 3, D and E). Consistently, siMettl14 reduced m6A levels in the DRG potentiated by paclitaxel treatment (Figure 3F).
We also determined whether METTL14 overexpression affected m6A levels in the DRG. AAV9 expressing full-length Mettl14 (AAV-Mettl14) was injected intrathecally to overexpress METTL14 in the DRG of naive rats. The results showed that AAV-Mettl14 induced high Mettl14 mRNA and protein expression in the rat DRG (Figure 3, G and H), in which obvious FLAG-positive signal in neurons and upregulation of METTL14 immunofluorescence signaling indicated the high transfection efficiency of AAV-Mettl14 carrying FLAG label (Supplemental Figure 2, B and C). A dot blot assay showed that METTL14 overexpression in naive rats caused an increase in m6A levels in the DRG (Figure 3I). Collectively, our results suggest that METTL14 mediates paclitaxel-induced m6A upregulation in the DRG.
METTL14 contributes to pain hypersensitivity induced by paclitaxel treatment. To determine the role of METTL14 in the development of CINP, we intrathecally injected siMettl14 or scrambled control siRNA into rats treated with saline or paclitaxel (Figure 4A). Results showed that siMettl14 intrathecal injection notably attenuated paclitaxel-induced pain behaviors, including mechanical hypersensitivity (Figure 4B), thermal hyperalgesia (Figure 4C), and cold allodynia (Figure 4D). We also measured pain thresholds in female rats treated with siMettl14 and found that Mettl14 siRNA alleviated paclitaxel-induced mechanical, thermal, and cold allodynia in female rats, indicating no potential sex-dependent effects of siRNA in CINP (Supplemental Figure 3, A–C). Additionally, to determine whether METTL14 manipulation can serve as a potential treatment strategy for CINP, we applied siMettl14 after day 7, when the neuropathic pain was stably established. We found that siRNA administration for 4 consecutive days from day 8 attenuated paclitaxel-induced nociceptive behaviors (Figure 4, E–H), suggesting that targeting METTL14 is a promising treatment approach for CINP. These data suggest that METTL14 plays a crucial role in the development of CINP.
Figure 4Paclitaxel treatment induces tactile and thermal hypersensitivity and potentiates glutamate release from primary afferent nerve terminals via METTL14. (A) Illustration of the experimental design. (B–D) Mechanical allodynia, thermal hyperalgesia, and cold allodynia of saline- or PCX-treated rats after siMettl14 or scrambled siRNA treatment measured by the von Frey test (B), hot plate test (C), and acetone test (D), respectively (at least 7 rats per group; *PCX+siMettl14- vs. PCX+siNC-treated rats, #PCX+siNC- vs. SAL+siNC-treated rats, $PCX- vs. SAL-treated rats). siNC, negative control RNA. (E) Schematic of the METTL14 therapeutic role evaluation. (F–H) Mechanical allodynia, thermal hyperalgesia, and cold allodynia were measured following PCX treatment and siRNA injection (7 rats per group, 2-way ANOVA followed by Tukey’s post hoc test; *PCX+siMettl14 vs. PCX+siNC). (I and J) Representative recording traces and quantification show the baseline control and the effect of bath application of AP5 on monosynaptic EPSCs of a lamina II neuron evoked from the dorsal root from saline- or PCX-pretreated rats after siMettl14 or scrambled siRNA treatment. (K and L) Representative recording traces and quantification show the baseline control and the effect of bath application of AP5 on the EPSCs evoked by a pair of pulses from saline- or PCX-pretreated rats after siMettl14 or scrambled siRNA treatment (at least 3 rats, 8 neurons per group; *compared with baseline between each group, #compared with the respective baseline). *P < 0.05, **P < 0. 01, ***P < 0.001, ****P < 0. 0001; ##P < 0.01, ###P < 0.001, ####P < 0.0001; $P < 0.01, $$$P < 0.001, $$$$P < 0.0001. Two-way ANOVA followed by Tukey’s post hoc test.
METTL14 mediates paclitaxel treatment–induced presynaptic NMDAR hyperactivity in the spinal cord. We then determined whether METTL14 contributes to paclitaxel-induced presynaptic NMDAR hyperactivity at the spinal cord level. Double-labeling immunofluorescence was conducted and confirmed that METTL14 was also predominantly colocalized with NeuN-positive cells in lamina I–III of spinal dorsal horns (81.58% ± 2.82%) (Supplemental Figure 4A). To elucidate the potential role of METTL14 at the primary sensory neuron terminal and spinal cord levels, we performed whole-cell patch-clamp recordings of spinal cord slices with dorsal root input in lamina II neurons, which preferentially receive nociceptive input from primary afferent nerves (22). Whole-cell recordings showed
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