title: Spinal Fbxo3-Dependent Fbxl2 Ubiquitination of Active Zone Protein RIM1α Mediates Neuropathic Allodynia through CaV2.2 Activation.
abstract: Spinal plasticity, a key process mediating neuropathic pain development, requires ubiquitination-dependent protein turnover. Presynaptic active zone proteins have a crucial role in regulating vesicle exocytosis, which is essential for synaptic plasticity. Nevertheless, the mechanism for ubiquitination-regulated turnover of presynaptic active zone proteins in the progression of spinal plasticity-associated neuropathic pain remains unclear. Here, after research involving Sprague Dawley rats, we reported that spinal nerve ligation (SNL), in addition to causing allodynia, enhances the Rab3-interactive molecule-1α (RIM1α), a major active zone protein presumed to regulate neural plasticity, specifically in the synaptic plasma membranes (SPMs) of the ipsilateral dorsal horn. Spinal RIM1α-associated allodynia was mediated by Fbxo3, which abates Fbxl2-dependent RIM1α ubiquitination. Subsequently, following deubiquitination, enhanced RIM1α directly binds to CaV2.2, resulting in increased CaV2.2 expression in the SPMs of the dorsal horn. While exhibiting no effect on Fbxo3/Fbxl2 signaling, the focal knockdown of spinal RIM1α expression reversed the SNL-induced allodynia and increased spontaneous EPSC (sEPSC) frequency by suppressing RIM1α-facilitated CaV2.2 expression in the dorsal horn. Intrathecal applications of BC-1215 (a Fbxo3 activity inhibitor), Fbxl2 mRNA-targeting small-interfering RNA, and ω-conotoxin GVIA (a CaV2.2 blocker) attenuated RIM1α upregulation, enhanced RIM1α expression, and exhibited no effect on RIM1α expression, respectively. These results confirm the prediction that spinal presynaptic Fbxo3-dependent Fbxl2 ubiquitination promotes the subsequent RIM1α/CaV2.2 cascade in SNL-induced neuropathic pain. Our findings identify a role of the presynaptic active zone protein in pain-associated plasticity. That is, RIM1α-facilitated CaV2.2 expression plays a role in the downstream signaling of Fbxo3-dependent Fbxl2 ubiquitination/degradation to promote spinal plasticity underlying the progression of nociceptive hypersensitivity following neuropathic injury.
  
  
  
  Ubiquitination is a well known process required for protein degradation. Studies investigating pain pathology have demonstrated that ubiquitination contributes to chronic pain by regulating the turnover of synaptic proteins. Here, we found that the spinal presynaptic active zone protein Rab3-interactive molecule-1α (RIM1α) participates in neuropathic pain development by binding to and upregulating the expression of CaV2.2. In addition, Fbxo3 modifies this pathway by inhibiting Fbxl2-mediated RIM1α ubiquitination, suggesting that presynaptic protein ubiquitination makes a crucial contribution to the development of neuropathic pain. Research in this area, now in its infancy, could potentially provide a novel therapeutic strategy for pain relief.
authors.affiliation: Department of Veterinary Medicine, College of Veterinary Medicine, National Chung-Hsing University, Taichung 40227, Taiwan, Department of Medicine, Mackay Medical College, New Taipei 25160, Taiwan.
authors.forename: Cheng-Yuan
authors.identifier: 0000-0002-5732-9814
authors.initials: CY
authors.lastname: Lai
authors.affiliation: Department of Medicine, Mackay Medical College, New Taipei 25160, Taiwan.
authors.forename: Yu-Cheng
authors.initials: YC
authors.lastname: Ho
authors.affiliation: Department of Medicine, Mackay Medical College, New Taipei 25160, Taiwan, Department of Physiology, College of Medicine, National Taiwan University, Taipei 10051, Taiwan, and.
authors.forename: Ming-Chun
authors.initials: MC
authors.lastname: Hsieh
authors.affiliation: Department of Medicine, Mackay Medical College, New Taipei 25160, Taiwan.
authors.forename: Hsueh-Hsiao
authors.initials: HH
authors.lastname: Wang
authors.affiliation: Department of Medicine, Mackay Medical College, New Taipei 25160, Taiwan, Department of Anesthesiology, Mackay Memorial Hospital, Taipei 10449, Taiwan.
authors.forename: Jen-Kun
authors.identifier: 0000-0003-2384-7856
authors.initials: JK
authors.lastname: Cheng
authors.affiliation: Department of Medicine, Mackay Medical College, New Taipei 25160, Taiwan.
authors.forename: Yat-Pang
authors.initials: YP
authors.lastname: Chau
authors.affiliation: Department of Medicine, Mackay Medical College, New Taipei 25160, Taiwan, hsien.yu@gmail.com.
authors.forename: Hsien-Yu
authors.identifier: 0000-0002-9471-8130
authors.initials: HY
authors.lastname: Peng
mesh_terms: D000200:Action Potentials / Q000502:physiology; D000818:Animals; D001596:Benzylamines / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020864:Calcium Channels, N-Type / Q000378:metabolism*; D004195:Disease Models, Animal; D044783:F-Box Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006930:Hyperalgesia / Q000209:etiology / Q000378:metabolism*; D008297:Male; D009437:Neuralgia / Q000150:complications; D009474:Neurons / Q000502:physiology; D010147:Pain Measurement; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D066148:Spinal Cord Dorsal Horn / Q000187:drug effects / Q000378:metabolism; D013127:Spinal Nerves / Q000166:cytology / Q000293:injuries / Q000378:metabolism; D054875:Ubiquitination / Q000187:drug effects / Q000502:physiology; D020866:omega-Conotoxin GVIA / Q000494:pharmacology; D020692:rab3 GTP-Binding Proteins / Q000378:metabolism*
chemical_list: C585582:BC-1215; D001596:Benzylamines; C503279:Cacna1b protein, rat; D002121:Calcium Channel Blockers; D020864:Calcium Channels, N-Type; D044783:F-Box Proteins; C000606755:F-box protein 3, rat; D011725:Pyridines; D020866:omega-Conotoxin GVIA; D020692:rab3 GTP-Binding Proteins
keywords: Cav2.2; Fbxl2; Fbxo3; Rim1α; neuropathic pain