Effect of morphine and ibuprofen on nociceptive behavior, preening and motor activity following tonic chemical pain in the Japanese quail (Coturnix japonica)
Khalilzadeh, Emad; Mousavi, Seyyedata; Dolatyarieslami, Mahdi; Bahadori, Reza; Khanna, Rajesh
OBJECTIVE:To establish a tonic chemical model of pain in quail and evaluate the efficacy of opioid and non-steroidal anti-inflammatory drugs. STUDY DESIGN/METHODS:A randomized, blinded, experimental study design. ANIMALS/METHODS:A total of 120 male Japanese quail, aged 7 weeks. METHODS:). The treatment effect was analyzed by one-way anova and the time course effect analyzed using repeated measures anova, both followed by Dunnett's post hoc test (p < 0.05). RESULTS:) significantly reduced foot lift responses. Preening activity was significantly decreased following injection of 0.6% and 0.9% formalin. Preening was normalized with ibuprofen, but not with morphine. Morphine, but not ibuprofen, reduced quail activity. CONCLUSION AND CLINICAL RELEVANCE/CONCLUSIONS:These results suggest that the formalin test was a reliable method for assessing tonic pain behavior in quail. The acute phase of the formalin test was not affected by morphine or ibuprofen. Although ibuprofen reduced the pain response in phase 2, the analgesic effects of morphine were not conclusive because morphine appeared to induce sedation.
Stereospecific Effects of Benzimidazolonepiperidine Compounds on T-Type Ca2+ Channels and Pain
Gomez, Kimberly; Tang, Cheng; Tan, Bin; Perez-Miller, Samantha; Ran, Dongzhi; Loya, Santiago; Calderon-Rivera, Aida; Stratton, Harrison J; Duran, Paz; Masterson, Kyleigh A; Gabrielsen, Anna T; Alsbiei, Omar; Dorame, Angie; Serafini, Maria; Moutal, Aubin; Wang, Jun; Khanna, Rajesh
T-type calcium channels activate in response to subthreshold membrane depolarizations and represent an important source of Ca2+ influx near the resting membrane potential. These channels regulate neuronal excitability and have been linked to pain. For this reason, T-type calcium channels are suitable molecular targets for the development of new non-opioid analgesics. Our previous work identified an analogue of benzimidazolonepiperidine, 5bk, that preferentially inhibited CaV3.2 channels and reversed mechanical allodynia. In this study, we synthesized and screened a small library of 47 compounds derived from 5bk. We found several compounds that inhibited the Ca2+ influx in DRG neurons of all sizes. After separating the enantiomers of each active compound, we found two compounds, 3-25-R and 3-14-3-S, that potently inhibited the Ca2+ influx. Whole-cell patch clamp recordings from small- to medium-sized DRG neurons revealed that both compounds decreased total Ca2+. Application of 3-14-3-S (but not 3-25-R) blocked transiently expressed CaV3.1-3.3 channels with a similar IC50 value. 3-14-3-S decreased T-type, but not N-type, Ca2+ currents in DRG neurons. Furthermore, intrathecal delivery of 3-14-3-S relieved tonic, neuropathic, and inflammatory pain in preclinical models. 3-14-3-S did not exhibit any activity against G protein-coupled opioid receptors. Preliminary docking studies also suggest that 3-14-3-S can bind to the central pore domain of T-type channels. Together, our chemical characterization and functional and behavioral data identify a novel T-type calcium channel blocker with in vivo efficacy in experimental models of tonic, neuropathic, and inflammatory pain.
Heat shock protein Grp78/BiP/HspA5 binds directly to TDP-43 and mitigates toxicity associated with disease pathology
FranÃ§ois-Moutal, Liberty; Scott, David Donald; Ambrose, Andrew J; Zerio, Christopher J; Rodriguez-Sanchez, Marina; Dissanayake, Kumara; May, Danielle G; Carlson, Jacob M; Barbieri, Edward; Moutal, Aubin; Roux, Kyle J; Shorter, James; Khanna, Rajesh; Barmada, Sami J; McGurk, Leeanne; Khanna, May
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no cure or effective treatment in which TAR DNA Binding Protein of 43Â kDa (TDP-43) abnormally accumulates into misfolded protein aggregates in affected neurons. It is widely accepted that protein misfolding and aggregation promotes proteotoxic stress. The molecular chaperones are a primary line of defense against proteotoxic stress, and there has been long-standing interest in understanding the relationship between chaperones and aggregated protein in ALS. Of particular interest are the heat shock protein of 70Â kDa (Hsp70) family of chaperones. However, defining which of the 13 human Hsp70 isoforms is critical for ALS has presented many challenges. To gain insight into the specific Hsp70 that modulates TDP-43, we investigated the relationship between TDP-43 and the Hsp70s using proximity-dependent biotin identification (BioID) and discovered several Hsp70 isoforms associated with TDP-43 in the nucleus, raising the possibility of an interaction with native TDP-43. We further found that HspA5 bound specifically to the RNA-binding domain of TDP-43 using recombinantly expressed proteins. Moreover, in a Drosophila strain that mimics ALS upon TDP-43 expression,Â the mRNA levels of the HspA5 homologue (Hsc70.3) were significantly increased. SimilarlyÂ we observed upregulationÂ of HspA5 in prefrontal cortex neurons from human ALS patients. Finally, overexpression of HspA5 in Drosophila rescued TDP-43-induced toxicity, suggesting that upregulation of HspA5 may have a compensatory role in ALS pathobiology.
Transmembrane protein TMEM184B is necessary for interleukin-31-induced itch
Larsen, Erik G; Cho, Tiffany S; McBride, Matthew L; Feng, Jing; Manivannan, Bhagyashree; Madura, Cynthia; Klein, Nathaniel E; Wright, Elizabeth B; Wickstead, Edward S; Garcia-Verdugo, Hector D; Jarvis, Chelsea; Khanna, Rajesh; Hu, Hongzhen; Largent-Milnes, Tally M; Bhattacharya, Martha R C
ABSTRACT/UNASSIGNED:Nociceptive and pruriceptive neurons in the dorsal root ganglia (DRG) convey sensations of pain and itch to the spinal cord, respectively. One subtype of mature DRG neurons, comprising 6% to 8% of neurons in the ganglia, is responsible for sensing mediators of acute itch and atopic dermatitis, including the cytokine IL-31. How itch-sensitive (pruriceptive) neurons are specified is unclear. Here, we show that transmembrane protein 184B (TMEM184B), a protein with roles in axon degeneration and nerve terminal maintenance, is required for the expression of a large cohort of itch receptors, including those for interleukin 31 (IL-31), leukotriene C4, and histamine. Male and female mice lacking TMEM184B show reduced responses to IL-31 but maintain normal responses to pain and mechanical force, indicating a specific behavioral defect in IL-31-induced pruriception. Calcium imaging experiments indicate that a reduction in IL-31-induced calcium entry is a likely contributor to this phenotype. We identified an early failure of proper Wnt-dependent transcriptional signatures and signaling components in Tmem184b mutant mice that may explain the improper DRG neuronal subtype specification. Accordingly, lentiviral re-expression of TMEM184B in mutant embryonic neurons restores Wnt signatures. Together, these data demonstrate that TMEM184B promotes adult somatosensation through developmental Wnt signaling and promotion of proper pruriceptive gene expression. Our data illuminate a new key regulatory step in the processes controlling the establishment of diversity in the somatosensory system.
The small molecule compound C65780 alleviates pain by stabilizing voltage-gated sodium channels in the inactivated and slowly-recovering state
Kong, Xiangjin; Li, Yinping; Perez-Miller, Samantha; Luo, Guoqing; Liao, Qingyi; Wu, Xiangyue; Liang, Songping; Tang, Cheng; Khanna, Rajesh; Liu, Zhonghua
Noxious pain signals are transduced in the peripheral nervous system as action potentials, which rely on the activities of voltage-gated sodium channels (NaVs). Blocking NaVs is thus a valuable strategy for pain treatment. Here, we report the characterization of a novel NaVs antagonist, 2-(2-(diethylamino)ethyl)indeno[1,2,3-de]phthalazin-3(2H)-one (C65780), and investigation of its action mechanisms. C65780 inhibited the resting NaV1.7, NaV1.8, and NaV1.9 channels with IC50s of 11.3Â Â±Â 0.4Â Î¼M, 2.7Â Â±Â 0.3Â Î¼M and 19.2Â Â±Â 2.3Â Î¼M, respectively. Mechanistic analysis revealed that C65780 quickly bound to its high-affinity receptor site in NaV1.7 as formed by the fast inactivation process and stabilized the channels in a slowly recovering state, for which it facilitated NaV1.7 channels' inactivation by shifting their inactivation-voltage relationship in the hyperpolarizing direction, increasing the plateau proportion of inactivated channels, and blunting their time-dependent recovery. The slow inactivation of NaV1.7, however, is not involved in the action of C65780. In DRG neurons, C65780 also inhibited activity of NaVs, thus dampening neuronal excitability. These effects parlayed into a broad efficacy of orally administrated C65780 in various models of pain, with an efficacy comparable to the antidepressant/neuropathic pain drug Amitriptyline. Excitingly, C65780 demonstrated weaker inactivated state inhibition of related NaV1.4 and NaV1.5 channels compared to amitriptyline, and no toxicity or inhibition of locomotion in a forced-swimming test was observed in mice at pain-relieving doses. These results demonstrate that C65780 acts by trapping NaVs in the inactivated and slowly-recovering state to produce pain relief and may represent an excellent starting compound for developing analgesics.
Neuronal allodynic mechanisms of Slc7a5 (LAT1) in the spared nerve injury rodent model of neuropathic pain
Goins, Aleyah E; Gomez, Kimberly; Ran, Dongzhi; Afaghpour-Becklund, Mitra; Khanna, Rajesh; Alles, Sascha R A
High-impact chronic pain is suffered by 1 in 5 patients in the USA and globally. Effective, non-addictive, non-opioid therapeutics are urgently needed for the treatment of chronic pain. Slc7a5 (Lat1), also known as system L-neutral amino acid transporter, is involved in a number of physiological processes related to inflammation. Transcriptomics studies have shown that Slc7a5 and its binding partner Slc3a2 are expressed in neurons of the dorsal root ganglia (DRG) and spinal dorsal horn, which are critical to the initiation and maintenance of nociception and pathophysiology of chronic pain. In addition, Slc7a5 is a transporter for the first-line anti-allodynic gabapentinoid drugs and binds to ion channels implicated in nociception and chronic pain including the voltage-gated sodium channel Nav1.7 and the voltage-gated potassium channels Kv1.1 and Kv1.2. We found that blocking Slc7a5 with intrathecal administration of the drug JPH203 alleviated allodynia in the spared nerve injury (SNI) rodent model of neuropathic pain. Western blot and immunohistochemistry studies revealed an increase in Slc7a5 protein levels in the spinal cord and DRGs of SNI mice compared to control mice. Using whole-cell current-clamp electrophysiology, we observed that JPH203 treatment reduced excitability of small-diameter (<â€‰30Â Âµm) DRG neurons from SNI mice, in agreement with its behavioral effects. Voltage-clamp recordings from JPH203-treated naÃ¯ve rat DRGs identified an effect on tetrodotoxin-resistant (TTX-R) sodium currents. Altogether, these results demonstrate that Slc7a5 is dysregulated in chronic neuropathic pain and can be targeted to provide relief of hypersensitivity.
Conditional knockout of CRMP2 in neurons, but not astrocytes, disrupts spinal nociceptive neurotransmission to control the initiation and maintenance of chronic neuropathic pain
Boinon, Lisa; Yu, Jie; Madura, Cynthia L; Chefdeville, Aude; Feinstein, Douglas L; Moutal, Aubin; Khanna, Rajesh
ABSTRACT/UNASSIGNED:Mechanistic studies principally focusing on primary afferent nociceptive neurons uncovered the upregulation of collapsin response mediator protein 2 (CRMP2)-a dual trafficking regulator of N-type voltage-gated calcium (Cav2.2) as well as Nav1.7 voltage-gated sodium channels-as a potential determinant of neuropathic pain. Whether CRMP2 contributes to aberrant excitatory synaptic transmission underlying neuropathic pain processing after peripheral nerve injury is unknown. Here, we interrogated CRMP2's role in synaptic transmission and in the initiation or maintenance of chronic pain. In rats, short-interfering RNA-mediated knockdown of CRMP2 in the spinal cord reduced the frequency and amplitude of spontaneous excitatory postsynaptic currents, but not spontaneous inhibitory postsynaptic currents, recorded from superficial dorsal horn neurons in acute spinal cord slices. No effect was observed on miniature excitatory postsynaptic currents and inhibitory postsynaptic currents. In a complementary targeted approach, conditional knockout of CRMP2 from mouse neurons using a calcium/calmodulin-dependent protein kinase II alpha promoter to drive Cre recombinase expression reduced the frequency and amplitude of spontaneous excitatory postsynaptic currents, but not miniature excitatory SCss. Conditional knockout of CRMP2 from mouse astrocytes using a glial fibrillary acidic protein promoter had no effect on synaptic transmission. Conditional knockout of CRMP2 in neurons reversed established mechanical allodynia induced by a spared nerve injury in both male and female mice. In addition, the development of spared nerve injury-induced allodynia was also prevented in these mice. Our data strongly suggest that CRMP2 is a key regulator of glutamatergic neurotransmission driving pain signaling and that it contributes to the transition of physiological pain into pathological pain.
Small molecule targeting NaV1.7 via inhibition of the CRMP2-Ubc9 interaction reduces and prevents pain chronification in a mouse model of oxaliplatin-induced neuropathic pain
Braden, Kathryn; Stratton, Harrison J; Salvemini, Daniela; Khanna, Rajesh
Treatment with anti-neoplastic agents can lead to the development of chemotherapy induced peripheral neuropathy (CIPN), which is long lasting and often refractory to treatment. This neuropathic pain develops along dermatomes innervated by peripheral nerves with cell bodies located in the dorsal root ganglia (DRG). The voltage-gated sodium channel NaV1.7 is expressed at high levels in peripheral nerve tissues and has been implicated in the development of CIPN. Efforts to develop novel analgesics directly inhibiting NaV1.7 have been unsuccessful, and our group has pioneered an alternative approach based on indirect modulation of channel trafficking by the accessory protein collapsin response mediator protein 2 (CRMP2). We have recently reported a small molecule, compound 194, that inhibits CRMP2 SUMOylation by the E2 SUMO-conjugating enzyme Ubc9 (Cai et al. , Sci. Transl. Med. 2021 13(6Â 1Â 9):eabh1314). Compound 194 is a potent and selective inhibitor of NaV1.7 currents in DRG neurons and reverses mechanical allodynia in models of surgical, inflammatory, and neuropathic pain, including spared nerve injury and paclitaxelinduced peripheral neuropathy. Here we report that, in addition to its reported effects in rats, 194 also reduces mechanical allodynia in male CD-1 mice treated with platinumcomplex agent oxaliplatin. Importantly, treatment with 194 prevented the development of mechanical allodynia when co-administered with oxaliplatin. No effects were observed on the body weight of animals treated with oxaliplatin or 194 throughout the study period. These findings support the notion that 194 is a robust inhibitor of CIPN that reduces established neuropathic pain and prevents the emergence of neuropathic pain during treatment with multiple anti-neoplastic agents in both mice and rats.
Cell specific regulation of NaV1.7 activity and trafficking in rat nodose ganglia neurons
Loya-López, Santiago I; Duran, Paz; Ran, Dongzhi; Calderon-Rivera, Aida; Gomez, Kimberly; Moutal, Aubin; Khanna, Rajesh
The voltage-gated sodium NaV1.7 channel sets the threshold for electrogenesis. Mutations in the gene encoding human NaV1.7 (SCN9A) cause painful neuropathies or pain insensitivity. In dorsal root ganglion (DRG) neurons, activity and trafficking of NaV1.7 are regulated by the auxiliary collapsin response mediator protein 2 (CRMP2). Specifically, preventing addition of a small ubiquitin-like modifier (SUMO), by the E2 SUMO-conjugating enzyme Ubc9, at lysine-374 (K374) of CRMP2 reduces NaV1.7 channel trafficking and activity. We previously identified a small molecule, designated 194, that prevented CRMP2 SUMOylation by Ubc9 to reduce NaV1.7 surface expression and currents, leading to a reduction in spinal nociceptive transmission, and culminating in normalization of mechanical allodynia in models of neuropathic pain. In this study, we investigated whether NaV1.7 control via CRMP2-SUMOylation is conserved in nodose ganglion (NG) neurons. This study was motivated by our desire to develop 194 as a safe, non-opioid substitute for persistent pain, which led us to wonder how 194 would impact NaV1.7 in NG neurons, which are responsible for driving the cough reflex. We found functioning NaV1.7 channels in NG neurons; however, they were resistant to downregulation via either CRMP2 knockdown or pharmacological inhibition of CRMP2 SUMOylation by 194. CRMP2 SUMOylation and interaction with NaV1.7 was consered in NG neurons but the endocytic machinery was deficient in the endocytic adaptor protein Numb. Overexpression of Numb rescued CRMP2-dependent regulation on NaV1.7, rendering NG neurons sensitive to 194. Altogether, these data point at the existence of cell-specific mechanisms regulating NaV1.7 trafficking.
Alternaria alternata-induced airway epithelial signaling and inflammatory responses via protease-activated receptor-2 expression
Rivas, Candy M; Schiff, Hillary V; Moutal, Aubin; Khanna, Rajesh; Kiela, Pawel R; Dussor, Gregory; Price, Theodore J; Vagner, Josef; DeFea, Kathryn A; Boitano, Scott
Inhalation of the fungus Alternaria alternata is associated with an increased risk of allergic asthma development and exacerbations. Recent work in acute exposure animal models suggests that A.Â alternata-induced asthma symptoms, which include inflammation, mucus overproduction and airway hyperresponsiveness, are due to A.Â alternata proteases that act via protease-activated receptor-2 (PAR2). However, because other active components present in A.Â alternata may be contributing to asthma pathophysiology through alternative signaling, the specific role PAR2 plays in asthma initiation and maintenance remains undefined. Airway epithelial cells provide the first encounter with A.Â alternata and are thought to play an important role in initiating the physiologic response. To better understand the role for PAR2 airway epithelial signaling we created a PAR2-deficient human bronchial epithelial cell line (16HBEPAR-