mTOR–neuropeptide Y signaling sensitizes nociceptors to drive neuropathic pain

Research ArticleNeuroscience Open Access | 10.1172/jci.insight.159247

Lunhao Chen,1 Yaling Hu,2,3,4 Siyuan Wang,1 Kelei Cao,2,3,4 Weihao Mai,2,3 Weilin Sha,5 Huan Ma,2,3,4 Ling-Hui Zeng,6 Zhen-Zhong Xu,2,3,4 Yong-Jing Gao,5 Shumin Duan,2,3,4 Yue Wang,1 and Zhihua Gao2,3,4

1Spine Lab, Department of Orthopedic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

2Department of Neurobiology and Department of Neurology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

3Liangzhu Laboratory, Zhejiang University Medical Center, MOE Frontier Science Center for Brain Science and Brain-machine Integration, State Key Laboratory of Brain-machine Intelligence, Zhejiang University, Hangzhou, China.

4NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University, Hangzhou, China.

5Institute of Pain Medicine and Special Environmental Medicine, Nantong University, Nantong, China.

6Key Laboratory of Novel Targets and Drug Study for Neural Repair of Zhejiang Province, School of Medicine, Zhejiang University City College, Hangzhou, China.

Address correspondence to: Zhihua Gao, 1369 West Wenyi Road, Hangzhou 311121, China. Phone: 86.571.87071107; Email: zhihuagao@zju.edu.cn. Or to: Yue Wang, 1367 West Wenyi Road, Hangzhou 311121, China. Phone: 86.571.87236128; Email: wangyuespine@zju.edu.cn.

Authorship note: LC, YH, and SW contributed equally to this work.

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1Spine Lab, Department of Orthopedic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

2Department of Neurobiology and Department of Neurology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

3Liangzhu Laboratory, Zhejiang University Medical Center, MOE Frontier Science Center for Brain Science and Brain-machine Integration, State Key Laboratory of Brain-machine Intelligence, Zhejiang University, Hangzhou, China.

4NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University, Hangzhou, China.

5Institute of Pain Medicine and Special Environmental Medicine, Nantong University, Nantong, China.

6Key Laboratory of Novel Targets and Drug Study for Neural Repair of Zhejiang Province, School of Medicine, Zhejiang University City College, Hangzhou, China.

Address correspondence to: Zhihua Gao, 1369 West Wenyi Road, Hangzhou 311121, China. Phone: 86.571.87071107; Email: zhihuagao@zju.edu.cn. Or to: Yue Wang, 1367 West Wenyi Road, Hangzhou 311121, China. Phone: 86.571.87236128; Email: wangyuespine@zju.edu.cn.

Authorship note: LC, YH, and SW contributed equally to this work.

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1Spine Lab, Department of Orthopedic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

2Department of Neurobiology and Department of Neurology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

3Liangzhu Laboratory, Zhejiang University Medical Center, MOE Frontier Science Center for Brain Science and Brain-machine Integration, State Key Laboratory of Brain-machine Intelligence, Zhejiang University, Hangzhou, China.

4NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University, Hangzhou, China.

5Institute of Pain Medicine and Special Environmental Medicine, Nantong University, Nantong, China.

6Key Laboratory of Novel Targets and Drug Study for Neural Repair of Zhejiang Province, School of Medicine, Zhejiang University City College, Hangzhou, China.

Address correspondence to: Zhihua Gao, 1369 West Wenyi Road, Hangzhou 311121, China. Phone: 86.571.87071107; Email: zhihuagao@zju.edu.cn. Or to: Yue Wang, 1367 West Wenyi Road, Hangzhou 311121, China. Phone: 86.571.87236128; Email: wangyuespine@zju.edu.cn.

Authorship note: LC, YH, and SW contributed equally to this work.

Find articles by Wang, S. in: JCI | PubMed | Google Scholar

1Spine Lab, Department of Orthopedic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

2Department of Neurobiology and Department of Neurology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

3Liangzhu Laboratory, Zhejiang University Medical Center, MOE Frontier Science Center for Brain Science and Brain-machine Integration, State Key Laboratory of Brain-machine Intelligence, Zhejiang University, Hangzhou, China.

4NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University, Hangzhou, China.

5Institute of Pain Medicine and Special Environmental Medicine, Nantong University, Nantong, China.

6Key Laboratory of Novel Targets and Drug Study for Neural Repair of Zhejiang Province, School of Medicine, Zhejiang University City College, Hangzhou, China.

Address correspondence to: Zhihua Gao, 1369 West Wenyi Road, Hangzhou 311121, China. Phone: 86.571.87071107; Email: zhihuagao@zju.edu.cn. Or to: Yue Wang, 1367 West Wenyi Road, Hangzhou 311121, China. Phone: 86.571.87236128; Email: wangyuespine@zju.edu.cn.

Authorship note: LC, YH, and SW contributed equally to this work.

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1Spine Lab, Department of Orthopedic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

2Department of Neurobiology and Department of Neurology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

3Liangzhu Laboratory, Zhejiang University Medical Center, MOE Frontier Science Center for Brain Science and Brain-machine Integration, State Key Laboratory of Brain-machine Intelligence, Zhejiang University, Hangzhou, China.

4NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University, Hangzhou, China.

5Institute of Pain Medicine and Special Environmental Medicine, Nantong University, Nantong, China.

6Key Laboratory of Novel Targets and Drug Study for Neural Repair of Zhejiang Province, School of Medicine, Zhejiang University City College, Hangzhou, China.

Address correspondence to: Zhihua Gao, 1369 West Wenyi Road, Hangzhou 311121, China. Phone: 86.571.87071107; Email: zhihuagao@zju.edu.cn. Or to: Yue Wang, 1367 West Wenyi Road, Hangzhou 311121, China. Phone: 86.571.87236128; Email: wangyuespine@zju.edu.cn.

Authorship note: LC, YH, and SW contributed equally to this work.

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1Spine Lab, Department of Orthopedic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

2Department of Neurobiology and Department of Neurology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

3Liangzhu Laboratory, Zhejiang University Medical Center, MOE Frontier Science Center for Brain Science and Brain-machine Integration, State Key Laboratory of Brain-machine Intelligence, Zhejiang University, Hangzhou, China.

4NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University, Hangzhou, China.

5Institute of Pain Medicine and Special Environmental Medicine, Nantong University, Nantong, China.

6Key Laboratory of Novel Targets and Drug Study for Neural Repair of Zhejiang Province, School of Medicine, Zhejiang University City College, Hangzhou, China.

Address correspondence to: Zhihua Gao, 1369 West Wenyi Road, Hangzhou 311121, China. Phone: 86.571.87071107; Email: zhihuagao@zju.edu.cn. Or to: Yue Wang, 1367 West Wenyi Road, Hangzhou 311121, China. Phone: 86.571.87236128; Email: wangyuespine@zju.edu.cn.

Authorship note: LC, YH, and SW contributed equally to this work.

Find articles by Sha, W. in: JCI | PubMed | Google Scholar

1Spine Lab, Department of Orthopedic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

2Department of Neurobiology and Department of Neurology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

3Liangzhu Laboratory, Zhejiang University Medical Center, MOE Frontier Science Center for Brain Science and Brain-machine Integration, State Key Laboratory of Brain-machine Intelligence, Zhejiang University, Hangzhou, China.

4NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University, Hangzhou, China.

5Institute of Pain Medicine and Special Environmental Medicine, Nantong University, Nantong, China.

6Key Laboratory of Novel Targets and Drug Study for Neural Repair of Zhejiang Province, School of Medicine, Zhejiang University City College, Hangzhou, China.

Address correspondence to: Zhihua Gao, 1369 West Wenyi Road, Hangzhou 311121, China. Phone: 86.571.87071107; Email: zhihuagao@zju.edu.cn. Or to: Yue Wang, 1367 West Wenyi Road, Hangzhou 311121, China. Phone: 86.571.87236128; Email: wangyuespine@zju.edu.cn.

Authorship note: LC, YH, and SW contributed equally to this work.

Find articles by Ma, H. in: JCI | PubMed | Google Scholar

1Spine Lab, Department of Orthopedic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

2Department of Neurobiology and Department of Neurology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

3Liangzhu Laboratory, Zhejiang University Medical Center, MOE Frontier Science Center for Brain Science and Brain-machine Integration, State Key Laboratory of Brain-machine Intelligence, Zhejiang University, Hangzhou, China.

4NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University, Hangzhou, China.

5Institute of Pain Medicine and Special Environmental Medicine, Nantong University, Nantong, China.

6Key Laboratory of Novel Targets and Drug Study for Neural Repair of Zhejiang Province, School of Medicine, Zhejiang University City College, Hangzhou, China.

Address correspondence to: Zhihua Gao, 1369 West Wenyi Road, Hangzhou 311121, China. Phone: 86.571.87071107; Email: zhihuagao@zju.edu.cn. Or to: Yue Wang, 1367 West Wenyi Road, Hangzhou 311121, China. Phone: 86.571.87236128; Email: wangyuespine@zju.edu.cn.

Authorship note: LC, YH, and SW contributed equally to this work.

Find articles by Zeng, L. in: JCI | PubMed | Google Scholar

1Spine Lab, Department of Orthopedic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

2Department of Neurobiology and Department of Neurology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

3Liangzhu Laboratory, Zhejiang University Medical Center, MOE Frontier Science Center for Brain Science and Brain-machine Integration, State Key Laboratory of Brain-machine Intelligence, Zhejiang University, Hangzhou, China.

4NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University, Hangzhou, China.

5Institute of Pain Medicine and Special Environmental Medicine, Nantong University, Nantong, China.

6Key Laboratory of Novel Targets and Drug Study for Neural Repair of Zhejiang Province, School of Medicine, Zhejiang University City College, Hangzhou, China.

Address correspondence to: Zhihua Gao, 1369 West Wenyi Road, Hangzhou 311121, China. Phone: 86.571.87071107; Email: zhihuagao@zju.edu.cn. Or to: Yue Wang, 1367 West Wenyi Road, Hangzhou 311121, China. Phone: 86.571.87236128; Email: wangyuespine@zju.edu.cn.

Authorship note: LC, YH, and SW contributed equally to this work.

Find articles by Xu, Z. in: JCI | PubMed | Google Scholar |

1Spine Lab, Department of Orthopedic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

2Department of Neurobiology and Department of Neurology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

3Liangzhu Laboratory, Zhejiang University Medical Center, MOE Frontier Science Center for Brain Science and Brain-machine Integration, State Key Laboratory of Brain-machine Intelligence, Zhejiang University, Hangzhou, China.

4NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University, Hangzhou, China.

5Institute of Pain Medicine and Special Environmental Medicine, Nantong University, Nantong, China.

6Key Laboratory of Novel Targets and Drug Study for Neural Repair of Zhejiang Province, School of Medicine, Zhejiang University City College, Hangzhou, China.

Address correspondence to: Zhihua Gao, 1369 West Wenyi Road, Hangzhou 311121, China. Phone: 86.571.87071107; Email: zhihuagao@zju.edu.cn. Or to: Yue Wang, 1367 West Wenyi Road, Hangzhou 311121, China. Phone: 86.571.87236128; Email: wangyuespine@zju.edu.cn.

Authorship note: LC, YH, and SW contributed equally to this work.

Find articles by Gao, Y. in: JCI | PubMed | Google Scholar |

1Spine Lab, Department of Orthopedic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

2Department of Neurobiology and Department of Neurology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

3Liangzhu Laboratory, Zhejiang University Medical Center, MOE Frontier Science Center for Brain Science and Brain-machine Integration, State Key Laboratory of Brain-machine Intelligence, Zhejiang University, Hangzhou, China.

4NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University, Hangzhou, China.

5Institute of Pain Medicine and Special Environmental Medicine, Nantong University, Nantong, China.

6Key Laboratory of Novel Targets and Drug Study for Neural Repair of Zhejiang Province, School of Medicine, Zhejiang University City College, Hangzhou, China.

Address correspondence to: Zhihua Gao, 1369 West Wenyi Road, Hangzhou 311121, China. Phone: 86.571.87071107; Email: zhihuagao@zju.edu.cn. Or to: Yue Wang, 1367 West Wenyi Road, Hangzhou 311121, China. Phone: 86.571.87236128; Email: wangyuespine@zju.edu.cn.

Authorship note: LC, YH, and SW contributed equally to this work.

Find articles by Duan, S. in: JCI | PubMed | Google Scholar

1Spine Lab, Department of Orthopedic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

2Department of Neurobiology and Department of Neurology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

3Liangzhu Laboratory, Zhejiang University Medical Center, MOE Frontier Science Center for Brain Science and Brain-machine Integration, State Key Laboratory of Brain-machine Intelligence, Zhejiang University, Hangzhou, China.

4NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University, Hangzhou, China.

5Institute of Pain Medicine and Special Environmental Medicine, Nantong University, Nantong, China.

6Key Laboratory of Novel Targets and Drug Study for Neural Repair of Zhejiang Province, School of Medicine, Zhejiang University City College, Hangzhou, China.

Address correspondence to: Zhihua Gao, 1369 West Wenyi Road, Hangzhou 311121, China. Phone: 86.571.87071107; Email: zhihuagao@zju.edu.cn. Or to: Yue Wang, 1367 West Wenyi Road, Hangzhou 311121, China. Phone: 86.571.87236128; Email: wangyuespine@zju.edu.cn.

Authorship note: LC, YH, and SW contributed equally to this work.

Find articles by Wang, Y. in: JCI | PubMed | Google Scholar |

1Spine Lab, Department of Orthopedic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

2Department of Neurobiology and Department of Neurology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

3Liangzhu Laboratory, Zhejiang University Medical Center, MOE Frontier Science Center for Brain Science and Brain-machine Integration, State Key Laboratory of Brain-machine Intelligence, Zhejiang University, Hangzhou, China.

4NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University, Hangzhou, China.

5Institute of Pain Medicine and Special Environmental Medicine, Nantong University, Nantong, China.

6Key Laboratory of Novel Targets and Drug Study for Neural Repair of Zhejiang Province, School of Medicine, Zhejiang University City College, Hangzhou, China.

Address correspondence to: Zhihua Gao, 1369 West Wenyi Road, Hangzhou 311121, China. Phone: 86.571.87071107; Email: zhihuagao@zju.edu.cn. Or to: Yue Wang, 1367 West Wenyi Road, Hangzhou 311121, China. Phone: 86.571.87236128; Email: wangyuespine@zju.edu.cn.

Authorship note: LC, YH, and SW contributed equally to this work.

Find articles by Gao, Z. in: JCI | PubMed | Google Scholar |

Authorship note: LC, YH, and SW contributed equally to this work.

Published October 4, 2022 - More info

Published in Volume 7, Issue 22 on November 22, 2022
JCI Insight. 2022;7(22):e159247. https://doi.org/10.1172/jci.insight.159247.
© 2022 Chen et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published October 4, 2022 - Version history
Received: February 9, 2022; Accepted: September 29, 2022 View PDF Abstract

Neuropathic pain is a refractory condition that involves de novo protein synthesis in the nociceptive pathway. The mTOR is a master regulator of protein translation; however, mechanisms underlying its role in neuropathic pain remain elusive. Using the spared nerve injury–induced neuropathic pain model, we found that mTOR was preferentially activated in large-diameter dorsal root ganglion (DRG) neurons and spinal microglia. However, selective ablation of mTOR in DRG neurons, rather than microglia, alleviated acute neuropathic pain in mice. We show that injury-induced mTOR activation promoted the transcriptional induction of neuropeptide Y (Npy), likely via signal transducer and activator of transcription 3 phosphorylation. NPY further acted primarily on Y2 receptors (Y2R) to enhance neuronal excitability. Peripheral replenishment of NPY reversed pain alleviation upon mTOR removal, whereas Y2R antagonists prevented pain restoration. Our findings reveal an unexpected link between mTOR and NPY/Y2R in promoting nociceptor sensitization and neuropathic pain.

Graphical Abstractgraphical abstract Introduction

Chronic pain, the leading cause of long-term human disability, poses a heavy health burden to society. Nerve injury–induced neuropathic pain accounts for approximately one-fifth of the chronic pain population (1). It is characterized by persistent hyperalgesia, allodynia, and spontaneous pain. Long-lasting sensitization of the nociceptive pathway, leading to a reduced pain threshold, has been considered a major mechanism mediating the persistent hypersensitivity in neuropathic pain (2).

Accumulating evidence has shown that nerve injury–induced de novo gene expression contributes to maladaptive responses in both peripheral and central nociceptive circuits, thereby promoting nociceptive sensitization and pain hypersensitivity (2, 3). Elevation of G protein–coupled receptors (GPCRs), such as GPR151, coupled with ion channels in the injured dorsal root ganglia (DRG), has been shown to facilitate the generation of ectopic action potentials (AP) in nociceptive neurons to promote pain (4, 5).

Other than ion channels and GPCRs, prominent induction of neuropeptides, including neuropeptide Y (NPY), galanin (Gal), neurotensin (NTS), and cholecystokinin (CCK), have also been observed in DRG neurons after nerve injury (68). The 36–amino acid peptide, NPY, is one of the most robustly upregulated neuropeptides in DRG neurons after nerve injury (9). However, mechanisms underlying its induction remain unclear. Conditional knockdown of spinal cord NPY has been shown to increase tactile and thermal hypersensitivity primarily through Y1 receptor (Y1R) in nerve injury–induced neuropathic pain models (10, 11), whereas s.c. injection of NPY or Y2R agonist appears to exacerbate pain after nerve injury, suggesting a biphasic role of NPY in neuropathic pain at different sites (1214). It remains to be elucidated how NPY is induced after injury and whether NPY plays opposing roles through different receptors in the nociceptive pathway.

The mTOR, a master regulator of protein translation, plays a pivotal role in regulating cell growth and metabolism. Deregulation of the mTOR signaling has been linked to various human diseases, including cancer, obesity, and neurodegeneration (1517). Activation of mTOR has been observed in the DRG and spinal cord in neuropathic pain models and in morphine-induced chronic pain (1821). Furthermore, pharmacologic blockade of mTOR activity has been demonstrated to reduce pain (2227). However, several studies also noted that inhibiting mTOR complex 1 (mTORC1) resulted in unexpected mechanical allodynia, likely associated with the negative feedback activation of extracellular signal–regulated kinase 1/2 (ERK 1/2) in primary sensory neurons (3, 28). The role of mTOR in pain remains to be clarified.

Combining genetic manipulation, transcriptomic profiling, and electrophysiological recording, we uncovered a previously unrecognized link between the nerve injury–triggered mTOR activation and NPY induction in DRG neurons. We further demonstrate that mTOR-mediated NPY production enhances nociceptor excitability and promotes pain hypersensitivity through Y2R in DRGs. Although mTOR-related signaling has been extensively studied, we present the first evidence to our knowledge for mTOR-regulated NPY signaling in driving neuropathic pain development.

Results

Nerve injury induces mTOR activation in subsets of DRG neurons and spinal cord microglia. To examine the status of mTOR activation after nerve injury, we carried out Western blot analysis of L4 and L5 DRGs and spinal dorsal horn (SDH) tissues from mice at different time points after the spared nerve injury (SNI) surgery (Figure 1A). The activity of mTOR was assessed by the levels of phosphorylated S6 protein (p-S6), a key downstream effector of mTOR. As shown in Figure 1, B and C, substantially upregulated p-S6 was found in the injured (ipsilateral) DRG at day 1 after SNI and lasted for at least 7 days (P < 0.05), consistent with elevated mTOR activity in DRGs after peripheral nerve injury (18).

Activation of the mTOR in subsets of DRG neurons and SDH microglia after spFigure 1

Activation of the mTOR in subsets of DRG neurons and SDH microglia after spared nerve injury (SNI). (A) A schematic diagram depicting the isolation of DRGs and SDH. (B) Representative blots indicating the upregulated p-S6 levels in the ipsilateral DRG after SNI at indicated time points. (C) Quantification of p-S6/S6 in the ipsilateral DRG at indicated time points after SNI (n = 4–7 mice per time point). (D) Coimmunostaining p-S6 with CGRP, NF160/200, or IB4 in DRGs after SNI (arrows indicating colabeled neurons). Scale bar: 50 μm. (EG) Quantification of p-S6+ neurons in different subpopulations of DRG neurons: CGRP (E), NF160/200 (F), and IB4 (G) (n = 4 mice). (H) Representative blots of p-S6 and S6 levels in SDH (L4 and L5) at days 3 and day 7 after SNI. (I) Quantification of p-S6/S6 in the ipsilateral and contralateral SDH (n = 5 and 3 for day 3 and day 7 after SNI, respectively). (J) Representative images of p-S6+ microglia (arrows) in the superficial contralateral and ipsilateral SDH (dotted lines) at indicated time points after SNI. Boxes show regions of higher magnification in the SDH. Scale bars: 100 and 20 μm for low- and high-magnification images, respectively. (K) Quantification of p-S6+ microglia in superficial SDH (n = 5–6 mice per time point). Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, 2-tailed paired Student’s t tests. BL, baseline; Ipsi, ipsilateral; Cont, contralateral; DRG: dorsal root ganglion; SDH, spinal dorsal horn.

To further determine the cellular identities with mTOR activation, we performed immunofluorescence analysis using the anti–p-S6 antibody along with different markers. Size frequency analysis showed that p-S6 was mainly present in medium and large neurons in DRG (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.159247DS1). In the contralateral DRG, positive p-S6 labeling, reflecting basal mTOR activity, was observed in a small subset of CGRP+ peptidergic neurons (9.7%) but a large fraction of NF160/200+ neurons (43.7%), reminiscent of large myelinated type A fiber mechanoreceptors. In the ipsilateral DRG, a substantial increase of p-S6 labeling in NF160/200+ large mechanoreceptors (from 43.7% to 71.2%, P < 0.01) and CGRP+ peptidergic neurons (from 9.7% to 18.7%, P < 0.05) was observed at 3 days after SNI (Figure 1, D–F). Notably, no elevation of mTOR activity was observed in Isolectin-B4+ (IB4+) nonpeptidergic small neurons (P > 0.05, Figure 1G).

By contrast, Western blot analysis of p-S6 from the SDH tissue extracts detected no difference between the contralateral and ipsilateral spinal cords following SNI (P > 0.05, Figure 1, H and I). Given that Western blot analysis detects the gross mTOR activity in the SDH, which may mask changes in sparsely distributed cells in the spinal cord, we carried out dual labeling of p-S6 with different cellular markers, including NeuN (neurons), GFAP (astrocytes), and Iba1 (microglia). No significant changes were observed in p-S6+ neurons or astrocytes between the contralateral and ipsilateral SDH within 1 week following the injury (Supplemental Figure 1, B–D). However, the number of p-S6+ microglia (GFP+) in the superficial layers of the ipsilateral SDH was robustly increased from day 3 to day 7 after SNI in Cx3cr1EGFP/+ mice (P < 0.05, Figure 1, J and K). Together, our results demonstrate that peripheral nerve injury induces mTOR activation mainly in large DRG mechanoreceptors and SDH microglia.

Blocking mTOR activity acutely alleviates pain. To further determine the contribution of mTOR signaling in neuropathic pain, we i.p. administered rapamycin, an mTORC1 inhibitor, to systematically block the mTORC1 activity; we also administered BrdU to label proliferating cells (Figure 2A). Daily administration of rapamycin from 1 day before to 7 days after SNI significantly inhibited mTOR activity in both DRG neurons and SDH microglia (Figure 2, B and C, and Supplemental Figure 2, A–C), and it suppressed mechanical allodynia and heat hyperalgesia for the first 3 days (P < 0.05; Figure 2, D and E), without affecting cold allodynia (Figure 2F). Rapamycin treatments also reduced the total number of microglia (vehicle, 839.9 ± 88.3 per mm2; rapamycin, 588.0 ± 27.8 per mm2; P < 0.05) and the percentage of proliferative microglia (BrdU+ Iba1+) (vehicle, 93.7% ± 0.1%; rapamycin, 86.1% ± 0.7%; P < 0.001) in the superficial layers of ipsilateral SDH at day 3 after SNI (Figure 2, G–J). These data demonstrate that blocking mTOR signaling inhibited pain development at the acute phase and suppressed nerve damage–induced microgliosis.

Rapamycin treatments inhibit mTOR activation and attenuate mechanical allodFigure 2

Rapamycin treatments inhibit mTOR activation and attenuate mechanical allodynia and heat hyperalgesia after SNI. (A) Experimental schedule for rapamycin or vehicle along with BrdU administration through intraperitoneal (i.p.) injection. (B) Representative blots indicating the decreased p-S6 levels in the ipsilateral DRG and SDH following 7-day continuous i.p. injection of rapamycin or vehicle in Mtorfl/fl mice after SNI. (C) Quantitation of p-S6/S6 in DRGs and SDH following rapamycin treatments (n = 3 mice per group). (DF) Measurements of mechanical allodynia (n = 12–13 per group) (D), heat hyperalgesia (n =7–8 per group) (E), and cold allodynia (n = 7–8 per group) (F) with daily i.p. injection of rapamycin or vehicle after SNI. (G and H) Representative images of Iba1 and BrdU immunolabeling in superficial SDH (dotted regions) after treated with Veh (G) or rapamycin (H) at day 3 after SNI. Boxes show regions of higher magnification in SDH, while arrowheads indicate Iba1+ BrdU+ mitotic microglia. Scale bars: 100 and 20 μm for low- and high-magnification images, respectively. (I and J) Quantitative analysis of microglia per square millimeter (I) and the percentage of mitotic microglia in total microglia (J) in both contralateral and ipsilateral SDH at day 3 after SNI (n = 5–7 mice per group). Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, using 2-way ANOVA followed by Bonferroni’s post hoc tests among group (DF), 2-tailed unpaired Student’s t tests (C), or 1-way ANOVA followed by Bonferroni’s post hoc tests (I and J). Rap, rapamycin; Veh, vehicle; BL, baseline; D, day; SDH, spinal dorsal horn; PWT, paw withdraw threshold.

Selective ablation of mTOR in DRG neurons, but not in microglia, alleviates neuropathic pain. To further discern the contributions of neuronal or microglial mTOR in neuropathic pain, we crossed specific Cre mouse lines Advcre or Cx3cr1creER with Mtorfl/fl mice to selectively delete the Mtor gene in primary sensory neurons or microglia, respectively. We observed complete elimination of p-S6 in DRG neurons and unchanged p-S6 levels in SDH in Advcre Mtorfl/fl (Mtor-cKOAdv) mice 7 days after SNI (Figure 3, A and B), demonstrating the selective ablation of mTOR in primary sensory neurons. Examination of sensory perception and motor activities found no significant differences between the control and Mtor-cKOAdv mice at basal states (Supplemental Figure 3, A–E). However, after SNI, Mtor-cKOAdv mice exhibited alleviated mechanical allodynia, heat hyperalgesia, and cold allodynia in both male and female mice for at least 14 days (Figure 3, C–E, and Supplemental Figure 3, F–H). Mtor-cKOAdv mice also had lower difference scores, representing the differences between post- and preconditioning time, in response to mechanical stimulation than the Mtorfl/fl mice in a 2-chamber conditioned place aversion (CPA) assay, which assesses the aversive responses to pain, suggesting that mTOR deletion in DRG neurons alleviated aversive responses to noxious stimuli (Figure 3F).

Ablation of Mtor in DRG neurons alleviates neuropathic pain.Figure 3

Ablation of Mtor in DRG neurons alleviates neuropathic pain. (A) Representative blots of p-S6 and S6 in the ipsilateral DRG and SDH from Mtorfl/fl and Mtor-cKOAdv mice at day 7 after SNI. (B) Representative images of p-S6 in the ipsilateral DRG at day 7 after SNI, indicating the ablation of mTOR in Mtor-cKOAdv mice rather than Mtorfl/fl mice after SNI. Scale bar: 100 μm. (CE) Measurements of mechanical allodynia (C), heat hyperalgesia (D), and cold allodynia (E) in male Mtorfl/fl and Mtor-cKOAdv mice before and after SNI (n = 6–8 mice per group). (F) Track plots of animal movements at pre- and postconditioning phases with a 2-chamber conditioned place aversion (CPA) test (n = 6–8 mice per group) in male Mtorfl/fl and Mtor-cKOAdv mice at day 15 after SNI. Difference scores = postconditioning time – preconditioning time spent in the stimulation chamber. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, by 2-way ANOVA followed by Bonferroni’s post hoc tests among groups (CE) or 2-tailed unpaired Student’s t tests (F). BL, baseline; PWT, paw withdraw threshold; PWL, paw withdraw latency.

To further examine whether microglial mTOR activation also contributes to neuropathic pain, we selectively deleted Mtor in microglia by gavaging tamoxifen into the Cx3cr1creER/+:Mtorfl/fl mice (Mtor-cKOMG mice) 4–6 weeks before the SNI surgery (Figure 4A and Supplemental Figure 4A). Cre-mediated recombination of Mtor gene in the CNS (brain and spinal cord) was detected by PCR analysis (Supplemental Figure 4B), and reduction of p-S6 levels in the SDH microglia was verified (Figure 4B). At day 7 after SNI, the total number of microglia (Figure 4, C and D) and mitotic microglia (BrdU+Iba1+) (Figure 4, E and F) was substantially reduced in the superficial layers of ipsilateral SDH in Mtor-cKOMG mice. However, no significant differences were observed in mechanical allodynia (Figure 4G), heat hyperalgesia (Figure 4H), or cold allodynia (Figure 4I) between the Mtor-cKOMG and control mice after SNI (from day 1 to day 7), suggesting that neuropathic pain is spared in the absence of microglial mTOR signaling.

Ablation of Mtor in microglia reduces microgliosis but does not affect neurFigure 4

Ablation of Mtor in microglia reduces microgliosis but does not affect neuropathic pain in male or female mice. (A) Experimental schedule showing the selected Mtor deletion in microglia and pain tests. (B) Representative images showing immunofluorescence labeling of Iba1 and p-S6 in ipsilateral SDH at day 7 after SNI in Cx3cr1CreER/+:Mtorfl/fl or control mice (Cx3cr1CreER/+ mice with TAM and Cx3cr1CreER/+:Mtorfl/fl mice with Veh). Arrows indicating Iba1+ p-S6+ microglia. Scale bar: 20 μm. (C) Representative images of bilateral SDH microglia (Iba1+) in Cx3cr1CreER/+:Mtorfl/fl mice with TAM or in control mice at day 7 after SNI. Scale bar: 100 μm. (D) Quantification of microglia in the ipsilateral and contralateral SDH in Cx3cr1CreER/+:Mtorfl/fl and control mice at day 7 after SNI (n = 5–7 per group). (E) Representative images of the ipsilateral SDH showing colocalization of Iba1 and BrdU (arrows) at day 7 after SNI. Boxes show regions of higher magnification in the SDH. Scale bars: 100 and 20 μm for low- and high-magnification images, respectively. (F) Quantitation of mitotic microglia (Iba1+BrdU+) in SDH in Cx3cr1CreER/+:Mtorfl/fl and control mice at day 7 after SNI (n = 5–7 mice per group). (GI) Measurements of mechanical allodynia (G), heat hyperalgesia (H), and cold allodynia (I) in Cx3cr1CreER/+:Mtorfl/fl and control mice before and after SNI (n = 10–13 mice per group; male and female mice were merged). Data are shown as mean ± SEM. **P < 0.01 and ***P < 0.001, by 1-way AVOVA (F) or 2-way ANOVA followed by Bonferroni’s post hoc tests among groups (D and GI). TAM, tamoxifen; Veh, vehicle; Cont, contralateral; Ipsi, ipsilateral; PWT, paw withdraw threshold; PWL, paw withdraw latency; D, day.

Mtor ablation in DRG neurons suppresses elevation of subsets of nerve injury–induced genes. To determine the downstream molecular targets of mTOR in DRG neurons involved in neuropathic pain, we performed RNA-Seq of DRGs from Mtorfl/fl and Mtor-cKOAdv mice before and 7 days after SNI surgery. In total, the expression levels of 189 genes (155 upregulated and 34 downregulated; Supplemental Table 2) were significantly changed (by at least 2 folds, q < 0.05) in the injured DRGs 7 days after SNI in Mtorfl/fl mice (Figure 5, A–C). Consistent with previous studies (6, 7, 29, 30), a large number of the upregulated genes, including those associated with injury (activating transcription factor 3 [Atf3] and small proline-rich protein 1A [Sprr1a]), GPCRs (including Gpr151 and Gpr119), neuropeptides (Npy, Gal, and Nts), cytokines (colony stimulating factor 1 [Csf1] and IL-1b [Il1b]), were identified in response to nerve injury (Figure 5B), verifying the reliability of the RNA-Seq data. Gene ontology (GO) analysis demonstrated that injury-affected genes were primarily enriched in 4 molecular functions (Figure 5D), including receptor ligand activity, hormone activity, and neuropeptide receptor binding and activity.

Ablation of Mtor in DRG neurons suppresses elevation of nerve injury–induceFigure 5

Ablation of Mtor in DRG neurons suppresses elevation of nerve injury–induced genes. (A) Venn diagram of DEGs identified in DRGs before and after SNI (day 7) in Mtorfl/fl mice (155 upregulated and 34 downregulated, n = 4–5 mice per group). (B) Heatmap of 189 DEGs by hierarchical clustering using z score values (n = 4–5 mice per group). (C) Volcano plots of DRG transcripts before and after SNI (day 7) in Mtorfl/fl mice. Red dots indicate 155 upregulated genes, and blue dots indicate 34 downregulated genes after SNI. (D) GO analysis of 155 upregulated genes after SNI and regroup into molecular function terms. All genes in each term are listed. (E) Pie chart of 155 injury-induced genes with 32 downregulated and 3 upregulated in Mtor-cKOAdv mice after SNI (n = 4–5 mice per group). (F) Heatmap of 35 DEGs in all samples using z score values. Only 3 (Inhbb, Lce6a, and Ucn) of the 155 injury-induced genes are upregulated upon deletion of Mtor in DRG neurons. (G) Volcano plots of 35 DEGs in control and Mtor-cKOAdv mice after SNI. Red dots indicate 3 upregulated genes, and blue dots indicate 32 downregulated genes after mTOR ablation. BL, baseline; D, day; DEGs, differentially expressed genes.

Importantly, approximately one-fifth (32 in 155 genes; Supplemental Table 3) of injury-induced genes were suppressed after mTOR ablation at day 7 after SNI (Figure 5E). In particular, the expression of 2 neuropeptide genes, Npy and Nts, induced by approximately 73.5 and 11.7 folds after injury, was strikingly reduced to 3.75 and 0.57 folds after ablation of Mtor in DRG neurons. By contrast, the expression of another 2 injury-induced neuropeptide genes, such as corticotropin releasing hormone (Crh) and Gal, remained largely unaffected, suggesting that mTOR specifically regulated the expression of subsets of injury-responsive genes (Figure 5, E–G). The reduced expression of Npy, Nts, and other genes (as indicated) in Mtor-cKOAdv mice was further verified by quantitative PCR (qPCR) analysis (Supplemental Figure 5). Notably, while mTOR was transiently activated during the first week after nerve injury, it may have long-term impacts through its downstream molecules. Collectively, these data demonstrate that mTOR regulates the transcription of subsets of injury-induced genes.

Injury-activated mTOR is required for NPY induction in DRG neurons. NPY is widely distributed in the CNS and peripheral nervous systems (31). It is absent in DRG neurons under homeostatic conditions but dramatically upregulated after peripheral nerve injury (8, 9). However, little is known about the molecular mechanisms regulating NPY induction aft

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