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Sinomenine ameliorates fibroblast-like synoviocytes dysfunction by promoting phosphorylation and nuclear translocation of CRMP2

Yu, Jie; Wang, Song; Chen, Si-Jia; Zheng, Meng-Jia; Yuan, Cun-Rui; Lai, Wei-Dong; Wen, Jun-Jun; You, Wen-Ting; Liu, Pu-Qing; Khanna, Rajesh; Jin, Yan
ETHNOPHARMACOLOGICAL RELEVANCE/BACKGROUND:Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial inflammation and arthritic pain. Sinomenine (SIN), derived from the rhizome of Chinese medical herb Qing Teng (scientific name: Sinomenium acutum (Thunb.) Rehd. Et Wils), has a longstanding use in Chinese traditional medicine for treating rheumatoid arthritis. It has been shown to possess anti-inflammatory, analgesic, and immunosuppressive effects with minimal side-effects clinically. However, the mechanisms governing its effects in treatment of joint pathology, especially on fibroblast-like synoviocytes (FLSs) dysfunction, and arthritic pain remains unclear. AIM/OBJECTIVE:This study aimed to investigate the effect and underlying mechanism of SIN on arthritic joint inflammation and joint FLSs dysfunctions. MATERIALS AND METHODS/METHODS:Collagen-induced arthritis (CIA) was induced in rats and the therapeutic effects of SIN on joint pathology were evaluated histopathologically. Next, we conducted a series of experiments using LPS-induced FLSs, which were divided into five groups (Naïve, LPS, SIN 10, 20, 50 μg/ml). The expression of inflammatory factors was measured by qPCR and ELISA. The invasive ability of cells was detected by modified Transwell assay and qPCR. Transwell migration and cell scratch assays were used to assess the migration ability of cells. The distribution and content of relevant proteins were observed by immunofluorescence and laser confocal microscopy, as well as Western Blot and qPCR. FLSs were transfected with plasmids (CRMP2 T514A/D) to directly modulate the post-translational modification of CRMP2 protein and downstream effects on FLSs function was monitored. RESULTS:SIN alleviated joint inflammation in rats with CIA, as evidenced by improvement of synovial hyperplasia, inflammatory cell infiltration and cartilage damage, as well as inhibition of pro-inflammatory cytokines release from FLSs induced by LPS. In vitro studies revealed a concentration-dependent suppression of SIN on the invasion and migration of FLSs induced by LPS. In addition, SIN downregulated the expression of cellular CRMP2 that was induced by LPS in FLSs, but increased its phosphorylation at residue T514. Moreover, regulation of pCRMP2 T514 by plasmids transfection (CRMP2 T514A/D) significantly influenced the migration and invasion of FLSs. Finally, SIN promoted nuclear translocation of pCRMP2 T514 in FLSs. CONCLUSIONS:SIN may exert its anti-inflammatory and analgesic effects by modulating CRMP2 T514 phosphorylation and its nuclear translocation of FLSs, inhibiting pro-inflammatory cytokine release, and suppressing abnormal invasion and migration. Phosphorylation of CRMP2 at the T514 site in FLSs may present a new therapeutic target for treating inflammatory joint's destruction and arthritic pain in RA.
PMID: 38176664
ISSN: 1872-7573
CID: 5634942

Targeted transcriptional upregulation of SENP1 by CRISPR activation enhances deSUMOylation pathways to elicit antinociception in the spinal nerve ligation model of neuropathic pain

Gomez, Kimberly; Allen, Heather N; Duran, Paz; Loya-Lopez, Santiago; Calderon-Rivera, Aida; Moutal, Aubin; Tang, Cheng; Nelson, Tyler S; Perez-Miller, Samantha; Khanna, Rajesh
The voltage-gated sodium channel Na V 1.7 is an essential component of human pain signaling. Changes in Na V 1.7 trafficking are considered critical in the development of neuropathic pain. SUMOylation of collapsin response mediator protein 2 (CRMP2) regulates the membrane trafficking and function of Na V 1.7. Enhanced CRMP2 SUMOylation in neuropathic pain correlates with increased Na V 1.7 activity. Pharmacological and genetic interventions that interfere with CRMP2 SUMOylation in rodents with neuropathic pain have been shown to reverse mechanical allodynia. Sentrin or SUMO-specific proteases (SENPs) are vital for balancing SUMOylation and deSUMOylation of substrates. Overexpression of SENP1 and/or SENP2 in CRMP2-expressing cells results in increased deSUMOylation and decreased membrane expression and currents of Na V 1.7. Although SENP1 is present in the spinal cord and dorsal root ganglia, its role in regulating Na V 1.7 function and pain is not known. We hypothesized that favoring SENP1 expression can enhance CRMP2 deSUMOylation to modulate Na V 1.7 channels. In this study, we used a clustered regularly interspaced short palindromic repeats activation (CRISPRa) SENP1 lentivirus to overexpress SENP1 in dorsal root ganglia neurons. We found that SENP1 lentivirus reduced CRMP2 SUMOylation, Na V 1.7-CRMP2 interaction, and Na V 1.7 membrane expression. SENP1 overexpression decreased Na V 1.7 currents through clathrin-mediated endocytosis, directly linked to CRMP2 deSUMOylation. Moreover, enhancing SENP1 expression did not affect the activity of TRPV1 channels or voltage-gated calcium and potassium channels. Intrathecal injection of CRISPRa SENP1 lentivirus reversed mechanical allodynia in male and female rats with spinal nerve injury. These results provide evidence that the pain-regulating effects of SENP1 overexpression involve, in part, the modulation of Na V 1.7 channels through the indirect mechanism of CRMP2 deSUMOylation.
PMID: 37862053
ISSN: 1872-6623
CID: 5641182

Intranasal CRMP2-Ubc9 inhibitor regulates Na V 1.7 to alleviate trigeminal neuropathic pain

Loya-Lopez, Santiago I; Allen, Heather N; Duran, Paz; Calderon-Rivera, Aida; Gomez, Kimberly; Kumar, Upasana; Shields, Rory; Zeng, Rui; Dwivedi, Akshat; Saurabh, Saumya; Korczeniewska, Olga A; Khanna, Rajesh
Dysregulation of voltage-gated sodium Na V 1.7 channels in sensory neurons contributes to chronic pain conditions, including trigeminal neuropathic pain. We previously reported that chronic pain results in part from increased SUMOylation of collapsin response mediator protein 2 (CRMP2), leading to an increased CRMP2/Na V 1.7 interaction and increased functional activity of Na V 1.7. Targeting this feed-forward regulation, we developed compound 194 , which inhibits CRMP2 SUMOylation mediated by the SUMO-conjugating enzyme Ubc9. We further demonstrated that 194 effectively reduces the functional activity of Na V 1.7 channels in dorsal root ganglia neurons and alleviated inflammatory and neuropathic pain. Here, we used a comprehensive array of approaches, encompassing biochemical, pharmacological, genetic, electrophysiological, and behavioral analyses, to assess the functional implications of Na V 1.7 regulation by CRMP2 in trigeminal ganglia (TG) neurons. We confirmed the expression of Scn9a , Dpysl2 , and UBE2I within TG neurons. Furthermore, we found an interaction between CRMP2 and Na V 1.7, with CRMP2 being SUMOylated in these sensory ganglia. Disrupting CRMP2 SUMOylation with compound 194 uncoupled the CRMP2/Na V 1.7 interaction, impeded Na V 1.7 diffusion on the plasma membrane, and subsequently diminished Na V 1.7 activity. Compound 194 also led to a reduction in TG neuron excitability. Finally, when intranasally administered to rats with chronic constriction injury of the infraorbital nerve, 194 significantly decreased nociceptive behaviors. Collectively, our findings underscore the critical role of CRMP2 in regulating Na V 1.7 within TG neurons, emphasizing the importance of this indirect modulation in trigeminal neuropathic pain.
PMID: 37751532
ISSN: 1872-6623
CID: 5635032

Human Hsp70 Substrate-Binding Domains Recognize Distinct Client Proteins

Ambrose, Andrew J; Zerio, Christopher J; Sivinski, Jared; Zhu, Xiaoyi; Godek, Jack; Sanchez, Jonathan L; Khanna, May; Khanna, Rajesh; Lairson, Luke; Zhang, Donna D; Chapman, Eli
The 13 Hsp70 proteins in humans act on unique sets of substrates with diversity often being attributed to J-domain-containing protein (Hsp40 or JDP) cofactors. We were therefore surprised to find drastically different binding affinities for Hsp70-peptide substrates, leading us to probe substrate specificity among the 8 canonical Hsp70s from humans. We used peptide arrays to characterize Hsp70 binding and then mined these data using machine learning to develop an algorithm for isoform-specific prediction of Hsp70 binding sequences. The results of this algorithm revealed recognition patterns not predicted based on local sequence alignments. We then showed that none of the human isoforms can complement heat-shocked DnaK knockout Escherichia coli cells. However, chimeric Hsp70s consisting of the human nucleotide-binding domain and the substrate-binding domain of DnaK complement during heat shock, providing further evidence in vivo of the divergent function of the Hsp70 substrate-binding domains. We also demonstrated that the differences in heat shock complementation among the chimeras are not due to loss of DnaJ binding. Although we do not exclude JDPs as additional specificity factors, our data demonstrate substrate specificity among the Hsp70s, which has important implications for inhibitor development in cancer and neurodegeneration.
PMID: 38243804
ISSN: 1520-4995
CID: 5628862

Neuropilin-1 is essential for vascular endothelial growth factor A-mediated increase of sensory neuron activity and development of pain-like behaviors

Gomez, Kimberly; Duran, Paz; Tonello, Raquel; Allen, Heather N; Boinon, Lisa; Calderon-Rivera, Aida; Loya-López, Santiago; Nelson, Tyler S; Ran, Dongzhi; Moutal, Aubin; Bunnett, Nigel W; Khanna, Rajesh
Neuropilin-1 (NRP-1) is a transmembrane glycoprotein that binds numerous ligands including vascular endothelial growth factor A (VEGFA). Binding of this ligand to NRP-1 and the co-receptor, the tyrosine kinase receptor VEGFR2, elicits nociceptor sensitization resulting in pain through the enhancement of the activity of voltage-gated sodium and calcium channels. We previously reported that blocking the interaction between VEGFA and NRP-1 with the Spike protein of SARS-CoV-2 attenuates VEGFA-induced dorsal root ganglion (DRG) neuronal excitability and alleviates neuropathic pain, pointing to the VEGFA/NRP-1 signaling as a novel therapeutic target of pain. Here, we investigated whether peripheral sensory neurons and spinal cord hyperexcitability and pain behaviors were affected by the loss of NRP-1. Nrp-1 is expressed in both peptidergic and nonpeptidergic sensory neurons. A CRIPSR/Cas9 strategy targeting the second exon of nrp-1 gene was used to knockdown NRP-1. Neuropilin-1 editing in DRG neurons reduced VEGFA-mediated increases in CaV2.2 currents and sodium currents through NaV1.7. Neuropilin-1 editing had no impact on voltage-gated potassium channels. Following in vivo editing of NRP-1, lumbar dorsal horn slices showed a decrease in the frequency of VEGFA-mediated increases in spontaneous excitatory postsynaptic currents. Finally, intrathecal injection of a lentivirus packaged with an NRP-1 guide RNA and Cas9 enzyme prevented spinal nerve injury-induced mechanical allodynia and thermal hyperalgesia in both male and female rats. Collectively, our findings highlight a key role of NRP-1 in modulating pain pathways in the sensory nervous system.
PMID: 37366599
ISSN: 1872-6623
CID: 5540182

A peptidomimetic modulator of the CaV2.2 N-type calcium channel for chronic pain

Gomez, Kimberly; Santiago, Ulises; Nelson, Tyler S; Allen, Heather N; Calderon-Rivera, Aida; Hestehave, Sara; Rodríguez Palma, Erick J; Zhou, Yuan; Duran, Paz; Loya-Lopez, Santiago; Zhu, Elaine; Kumar, Upasana; Shields, Rory; Koseli, Eda; McKiver, Bryan; Giuvelis, Denise; Zuo, Wanhong; Inyang, Kufreobong E; Dorame, Angie; Chefdeville, Aude; Ran, Dongzhi; Perez-Miller, Samantha; Lu, Yi; Liu, Xia; Handoko,; Arora, Paramjit S; Patek, Marcel; Moutal, Aubin; Khanna, May; Hu, Huijuan; Laumet, Geoffroy; King, Tamara; Wang, Jing; Damaj, M Imad; Korczeniewska, Olga A; Camacho, Carlos J; Khanna, Rajesh
Transmembrane Cav2.2 (N-type) voltage-gated calcium channels are genetically and pharmacologically validated, clinically relevant pain targets. Clinical block of Cav2.2 (e.g., with Prialt/Ziconotide) or indirect modulation [e.g., with gabapentinoids such as Gabapentin (GBP)] mitigates chronic pain but is encumbered by side effects and abuse liability. The cytosolic auxiliary subunit collapsin response mediator protein 2 (CRMP2) targets Cav2.2 to the sensory neuron membrane and regulates their function via an intrinsically disordered motif. A CRMP2-derived peptide (CBD3) uncouples the Cav2.2-CRMP2 interaction to inhibit calcium influx, transmitter release, and pain. We developed and applied a molecular dynamics approach to identify the A1R2
PMID: 37972067
ISSN: 1091-6490
CID: 5608032

The Ca2+ channel ORAI1 is a regulator of oral cancer growth and nociceptive pain

Son, Ga-Yeon; Tu, Nguyen Huu; Santi, Maria Daniela; Loya Lopez, Santiago; Souza Bomfim, Guilherme H; Vinu, Manikandan; Zhou, Fang; Chaloemtoem, Ariya; Alhariri, Rama; Idaghdour, Youssef; Khanna, Rajesh; Ye, Yi; Lacruz, Rodrigo S
Oral cancer causes pain associated with cancer progression. We report here that the function of the Ca2+ channel ORAI1 is an important regulator of oral cancer pain. ORAI1 was highly expressed in tumor samples from patients with oral cancer, and ORAI1 activation caused sustained Ca2+ influx in human oral cancer cells. RNA-seq analysis showed that ORAI1 regulated many genes encoding oral cancer markers such as metalloproteases (MMPs) and pain modulators. Compared with control cells, oral cancer cells lacking ORAI1 formed smaller tumors that elicited decreased allodynia when inoculated into mouse paws. Exposure of trigeminal ganglia neurons to MMP1 evoked an increase in action potentials. These data demonstrate an important role of ORAI1 in oral cancer progression and pain, potentially by controlling MMP1 abundance.
PMID: 37669398
ISSN: 1937-9145
CID: 5617972

A new polymodal gating model of the proton-activated chloride channel

Zhao, Piao; Tang, Cheng; Yang, Yuqin; Xiao, Zhen; Perez-Miller, Samantha; Zhang, Heng; Luo, Guoqing; Liu, Hao; Li, Yaqi; Liao, Qingyi; Yang, Fan; Dong, Hao; Khanna, Rajesh; Liu, Zhonghua
The proton-activated chloride (PAC) channel plays critical roles in ischemic neuron death, but its activation mechanisms remain elusive. Here, we investigated the gating of PAC channels using its novel bifunctional modulator C77304. C77304 acted as a weak activator of the PAC channel, causing moderate activation by acting on its proton gating. However, at higher concentrations, C77304 acted as a weak inhibitor, suppressing channel activity. This dual function was achieved by interacting with 2 modulatory sites of the channel, each with different affinities and dependencies on the channel's state. Moreover, we discovered a protonation-independent voltage activation of the PAC channel that appears to operate through an ion-flux gating mechanism. Through scanning-mutagenesis and molecular dynamics simulation, we confirmed that E181, E257, and E261 in the human PAC channel serve as primary proton sensors, as their alanine mutations eliminated the channel's proton gating while sparing the voltage-dependent gating. This proton-sensing mechanism was conserved among orthologous PAC channels from different species. Collectively, our data unveils the polymodal gating and proton-sensing mechanisms in the PAC channel that may inspire potential drug development.
PMID: 37713449
ISSN: 1545-7885
CID: 5594392

Identification and targeting of a unique NaV1.7 domain driving chronic pain

Gomez, Kimberly; Stratton, Harrison J; Duran, Paz; Loya, Santiago; Tang, Cheng; Calderon-Rivera, Aida; François-Moutal, Liberty; Khanna, May; Madura, Cynthia L; Luo, Shizhen; McKiver, Bryan; Choi, Edward; Ran, Dongzhi; Boinon, Lisa; Perez-Miller, Samantha; Damaj, M Imad; Moutal, Aubin; Khanna, Rajesh
Small molecules directly targeting the voltage-gated sodium channel (VGSC) NaV1.7 have not been clinically successful. We reported that preventing the addition of a small ubiquitin-like modifier onto the NaV1.7-interacting cytosolic collapsin response mediator protein 2 (CRMP2) blocked NaV1.7 function and was antinociceptive in rodent models of neuropathic pain. Here, we discovered a CRMP2 regulatory sequence (CRS) unique to NaV1.7 that is essential for this regulatory coupling. CRMP2 preferentially bound to the NaV1.7 CRS over other NaV isoforms. Substitution of the NaV1.7 CRS with the homologous domains from the other eight VGSC isoforms decreased NaV1.7 currents. A cell-penetrant decoy peptide corresponding to the NaV1.7-CRS reduced NaV1.7 currents and trafficking, decreased presynaptic NaV1.7 expression, reduced spinal CGRP release, and reversed nerve injury-induced mechanical allodynia. Importantly, the NaV1.7-CRS peptide did not produce motor impairment, nor did it alter physiological pain sensation, which is essential for survival. As a proof-of-concept for a NaV1.7 -targeted gene therapy, we packaged a plasmid encoding the NaV1.7-CRS in an AAV virus. Treatment with this virus reduced NaV1.7 function in both rodent and rhesus macaque sensory neurons. This gene therapy reversed and prevented mechanical allodynia in a model of nerve injury and reversed mechanical and cold allodynia in a model of chemotherapy-induced peripheral neuropathy. These findings support the conclusion that the CRS domain is a targetable region for the treatment of chronic neuropathic pain.
PMID: 37498871
ISSN: 1091-6490
CID: 5592562

Targeting the vascular endothelial growth factor A/neuropilin 1 axis for relief of neuropathic pain

Stratton, Harrison J; Boinon, Lisa; Gomez, Kimberly; Martin, Laurent; Duran, Paz; Ran, Dongzhi; Zhou, Yuan; Luo, Shizhen; Perez-Miller, Samantha; Patek, Marcel; Ibrahim, Mohab M; Patwardhan, Amol; Moutal, Aubin; Khanna, Rajesh
Vascular endothelial growth factor A (VEGF-A) is a pronociceptive factor that causes neuronal sensitization and pain. We reported that blocking the interaction between the membrane receptor neuropilin 1 (NRP1) and VEGF-A-blocked VEGF-A-mediated sensory neuron hyperexcitability and reduced mechanical hypersensitivity in a rodent chronic neuropathic pain model. These findings identified the NRP1-VEGF-A signaling axis for therapeutic targeting of chronic pain. In an in-silico screening of approximately 480 K small molecules binding to the extracellular b1b2 pocket of NRP1, we identified 9 chemical series, with 6 compounds disrupting VEGF-A binding to NRP1. The small molecule with greatest efficacy, 4'-methyl-2'-morpholino-2-(phenylamino)-[4,5'-bipyrimidin]-6(1H)-one, designated NRP1-4, was selected for further evaluation. In cultured primary sensory neurons, VEGF-A enhanced excitability and decreased firing threshold, which was blocked by NRP1-4. In addition, NaV1.7 and CaV2.2 currents and membrane expression were potentiated by treatment with VEGF-A, and this potentiation was blocked by NRP1-4 cotreatment. Neuropilin 1-4 reduced VEGF-A-mediated increases in the frequency and amplitude of spontaneous excitatory postsynaptic currents in dorsal horn of the spinal cord. Neuropilin 1-4 did not bind to more than 300 G-protein-coupled receptors and receptors including human opioids receptors, indicating a favorable safety profile. In rats with spared nerve injury-induced neuropathic pain, intrathecal administration of NRP1-4 significantly attenuated mechanical allodynia. Intravenous treatment with NRP1-4 reversed both mechanical allodynia and thermal hyperalgesia in rats with L5/L6 spinal nerve ligation-induced neuropathic pain. Collectively, our findings show that NRP1-4 is a first-in-class compound targeting the NRP1-VEGF-A signaling axis to control voltage-gated ion channel function, neuronal excitability, and synaptic activity that curb chronic pain.
PMID: 36729125
ISSN: 1872-6623
CID: 5434812