title: Fbxo3-Dependent Fbxl2 Ubiquitination Mediates Neuropathic Allodynia through the TRAF2/TNIK/GluR1 Cascade.
abstract: Emerging evidence has indicated that the pathogenesis of neuropathic pain is mediated by spinal neural plasticity in the dorsal horn, which provides insight for analgesic therapy. Here, we report that the abundance of tumor necrosis factor receptor-associated factor 2 and NcK-interacting kinase (TNIK), a kinase that is presumed to regulate neural plasticity, was specifically enhanced in ipsilateral dorsal horn neurons after spinal nerve ligation (SNL; left L5 and L6). Spinal TNIK-associated allodynia is mediated by downstream TNIK-GluR1 coupling and the subsequent phosphorylation-dependent trafficking of GluR1 toward the plasma membrane in dorsal horn neurons. Tumor necrosis factor receptor-associated factor 2 (TRAF2), which is regulated by spinal F-box protein 3 (Fbxo3)-dependent F-box and leucine-rich repeat protein 2 (Fbxl2) ubiquitination, contributes to SNL-induced allodynia by modifying TNIK/GluR1 phosphorylation-associated GluR1 trafficking. Although exhibiting no effect on Fbxo3/Fbxl2/TRAF2 signaling, focal knockdown of spinal TNIK expression prevented SNL-induced allodynia by attenuating TNIK/GluR1 phosphorylation-dependent subcellular GluR1 redistribution. In contrast, intrathecal administration of BC-1215 (N1,N2-Bis[[4-(2-pyridinyl)phenyl]methyl]-1,2-ethanediamine) (a novel Fbxo3 inhibitor) prevented SNL-induced Fbxl2 ubiquitination and subsequent TFAF2 de-ubiquitination to ameliorate behavioral allodynia via antagonizing TRAF2/TNIK/GluR1 signaling. By targeting spinal Fbxo3-dependent Fbxl2 ubiquitination and the subsequent TRAF2/TNIK/GluR1 cascade, spinal application of a TNF-α-neutralizing antibody ameliorated SNL-induced allodynia, and, conversely, intrathecal TNF-α injection into naive rats induced allodynia via a spinal Fbxo3/Fbxl2-dependent modification of the TRAF2/TNIK/GluR1 cascade. Together, our results suggest that spinal TNF-α contributes to the development of neuropathic pain by upregulating TRAF2/TNIK/GluR1 signaling via Fbxo3-dependent Fbxl2 ubiquitination and degradation. Thus, we propose a potential medical treatment strategy for neuropathic pain by targeting the F-box protein or TNIK.
  
  
  CONCLUSIONS
  TNF-α participates in neuropathic pain development by facilitating the spinal TRAF2-dependent TNIK-GluR1 association, which drives GluR1-containing AMPA receptor trafficking toward the plasma membrane. In addition, F-box protein 3 modifies this pathway by inhibiting F-box and leucine-rich repeat protein 2-mediated TRAF2 ubiquitination, suggesting that protein ubiquitination contributes crucially to the development of neuropathic pain. These results provide a novel therapeutic strategy for pain relief.
authors.affiliation: Department of Physiology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 11049, Taiwan, Graduate Institute of Basic Medical Science, China Medical University, Taichung 40402, Taiwan, Department of Biotechnology, Asia University, Taichung 41354, Taiwan.
authors.forename: Tzer-Bin
authors.initials: TB
authors.lastname: Lin
authors.affiliation: Department of Medicine, Mackay Medical College, New Taipei 25245, Taiwan, Department of Physiology, School of Medicine, College of Medicine, National Taiwan University, Taipei 10617, Taiwan.
authors.forename: Ming-Chun
authors.initials: MC
authors.lastname: Hsieh
authors.affiliation: Department of Medicine, Mackay Medical College, New Taipei 25245, Taiwan, Department of Veterinary Medicine, College of Veterinary Medicine, National Chung-Hsing University, Taichung 40254, Taiwan.
authors.forename: Cheng-Yuan
authors.identifier: http://orcid.org/0000-0002-5732-9814
authors.initials: CY
authors.lastname: Lai
authors.affiliation: Department of Medicine, Mackay Medical College, New Taipei 25245, Taiwan, Department of Anesthesiology, Mackay Memorial Hospital, New Taipei 10449, Taiwan.
authors.forename: Jen-Kun
authors.initials: JK
authors.lastname: Cheng
authors.affiliation: Department of Medicine, Mackay Medical College, New Taipei 25245, Taiwan.
authors.forename: Yat-Pang
authors.initials: YP
authors.lastname: Chau
authors.affiliation: School of Medicine, Fu-Jen Catholic University, New Taipei 24205, Taiwan, and.
authors.forename: Ting
authors.initials: T
authors.lastname: Ruan
authors.affiliation: Department of Obstetrics and Gynecology, Chung-Shan Medical University Hospital, Chung-Shan Medical University, Taichung 40201, Taiwan.
authors.forename: Gin-Den
authors.identifier: http://orcid.org/0000-0001-8613-5499
authors.initials: GD
authors.lastname: Chen
authors.affiliation: Department of Medicine, Mackay Medical College, New Taipei 25245, Taiwan, hsien.yu@gmail.com.
authors.forename: Hsien-Yu
authors.identifier: http://orcid.org/0000-0002-9471-8130
authors.initials: HY
authors.lastname: Peng
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000494:pharmacology; D001596:Benzylamines / Q000494:pharmacology; D044783:F-Box Proteins / Q000235:genetics* / Q000502:physiology*; D055785:Gene Knockdown Techniques; D006930:Hyperalgesia / Q000235:genetics* / Q000503:physiopathology*; D008297:Male; D010523:Peripheral Nervous System Diseases / Q000235:genetics* / Q000503:physiopathology*; D020671:Posterior Horn Cells / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics*; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D018091:Receptors, AMPA / Q000037:antagonists & inhibitors / Q000235:genetics*; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D013127:Spinal Nerves / Q000293:injuries; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000494:pharmacology; D054875:Ubiquitination / Q000187:drug effects / Q000235:genetics*
chemical_list: D057134:Antibodies, Neutralizing; D001596:Benzylamines; D044783:F-Box Proteins; D017346:Protein Serine-Threonine Kinases; D011725:Pyridines; D018091:Receptors, AMPA; D014409:Tumor Necrosis Factor-alpha; C585582:BC-1215; C000606755:F-box protein 3, rat; C000606756:Fbxl2 protein, rat; C553969:TNIK protein, rat
keywords: Fbxl2; Fbxo3; GluR1; TNIK; neuropathic; ubiquitination