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Treatment of Inflammatory and Neuropathic Pain by Uncoupling Src from the NMDA Receptor Complex
Abstract and Figures
Chronic pain hypersensitivity depends on N-methyl-D-aspartate receptors (NMDARs). However, clinical use of NMDAR blockers is limited by side effects resulting from suppression of the physiological functions of these receptors. Here we report a means to suppress pain hypersensitivity without blocking NMDARs, but rather by inhibiting the binding of a key enhancer of NMDAR function, the protein tyrosine kinase Src. We show that a peptide consisting of amino acids 40-49 of Src fused to the protein transduction domain of the HIV Tat protein (Src40-49Tat) prevented pain behaviors induced by intraplantar formalin and reversed pain hypersensitivity produced by intraplantar injection of complete Freund's adjuvant or by peripheral nerve injury. Src40-49Tat had no effect on basal sensory thresholds, acute nociceptive responses or cardiovascular, respiratory, locomotor or cognitive functions. Thus, through targeting of Src-mediated enhancement of NMDARs, inflammatory and neuropathic pain are suppressed without the deleterious consequences of directly blocking NMDARs, an approach that may be of broad relevance to managing chronic pain.
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... Intracellular stimulation of Src potentiates NMDAR activity and occludes NMDAR-dependent LTP [110], while blockade of endogenous Src activity suppresses NMDAR activity and induction of LTP in hippocampal neurons [109,111]. In addition, upregulation of NMDAR activity escalates the sensitivity of chronic pain [58], and this hyperfunction of NMDAR is thought to be regulated by both Src and Fyn [112][113][114]. However, our pharmacological experiments demonstrated that neither Src nor Fyn seem to be involved in the RGC death due to NMDAmediated excitotoxicity, although both Src and Fyn are expressed by RGCs. ...
Excessive levels of glutamate activity could potentially damage and kill neurons. Glutamate excitotoxicity is thought to play a critical role in many CNS and retinal diseases. Accordingly, glutamate excitotoxicity has been used as a model to study neuronal diseases. Immune proteins, such as major histocompatibility complex (MHC) class I molecules and their receptors, play important roles in many neuronal diseases, while T-cell receptors (TCR) are the primary receptors of MHCI. We previously showed that a critical component of TCR, CD3ζ, is expressed by mouse retinal ganglion cells (RGCs). The mutation of CD3ζ or MHCI molecules compromises the development of RGC structure and function. In this study, we investigated whether CD3ζ-mediated molecular signaling regulates RGC death in glutamate excitotoxicity. We show that mutation of CD3ζ significantly increased RGC survival in NMDA-induced excitotoxicity. In addition, we found that several downstream molecules of TCR, including Src (proto-oncogene tyrosine-protein kinase) family kinases (SFKs) and spleen tyrosine kinase (Syk), are expressed by RGCs. Selective inhibition of an SFK member, Hck, or Syk members, Syk or Zap70, significantly increased RGC survival in NMDA-induced excitotoxicity. These results provide direct evidence to reveal the underlying molecular mechanisms that control RGC death under disease conditions.
... NMDAR dysfunction contributes to a variety of neurological disorders and psychiatric disorders, such as cerebral ischemic injury, schizophrenia, depression, and cognitive impairment (Ferreira et al., 2017;Luo et al., 2018;Adell, 2020;Companys-Alemany et al., 2020). A great deal of evidence has demonstrated that NMDARs also play a significant role in nociceptive sensitization (Liu et al., 2008), particularly for NMDAR2B (NR2B), whose alterations include overexpression and altered phosphorylation status of GluN2B (Paoletti et al., 2013). Our previous study showed that a CM rat model involves changes in synaptic plasticity due to NR2B phosphorylation, which leads to hypersensitivity in the central nociceptive system (Wang et al., 2018). ...
Background
Central sensitization is one of the pivotal pathological mechanisms in chronic migraine (CM). Silent information regulator 1 (SIRT1) was shown to be involved in CM, but its specific mechanism is unclear. Reactive oxygen species (ROS) are increasingly regarded as important signaling molecules in several models of pain. However, studies about the role of ROS in the central sensitization of CM model are rare. We thus explored the specific process of SIRT1 involvement in the central sensitization of CM, focusing on the ROS pathway.
Methods
Inflammatory soup was repeatedly administered to male Sprague–Dawley rats to establish a CM model. The SIRT1 expression level in trigeminal nucleus caudalis (TNC) tissues was assessed by qRT–PCR and Western blotting analysis. The levels of ROS were detected by a Tissue Reactive Oxygen Detection Kit, DHE staining, and the fluorescence signal intensity of 8-OHdG. A ROS scavenger (tempol), a SIRT1 activator (SRT1720), a SIRT1 inhibitor (EX527), and a mitochondrial fission inhibitor (Mdivi-1) were used to investigate the specific molecular mechanisms involved. NMDAR2B, CGRP, ERK, and mitochondrial fission-related protein were evaluated by Western blotting, and the CGRP level in frozen sections of the TNC was detected via immunofluorescence staining.
Results
After repeated inflammatory soup infusion and successful establishment of the CM rat model, SIRT1 expression was found to be significantly reduced, accompanied by elevated ROS levels. Treatment with Tempol, SRT1720, or Mdivi-1 alleviated allodynia and reduced the increase in NMDAR2B phosphorylation and CGRP and ERK phosphorylation in the CM rat. In contrast, EX527 had the opposite effect in CM rat. SRT1720 and EX527 decreased and increased ROS levels, respectively, in CM rats, and tempol reversed the aggravating effect of EX527 in CM rats. Furthermore, the regulatory effect of SIRT1 on ROS may include the involvement of the mitochondrial fission protein DRP1.
Conclusion
The results indicate the importance of SIRT1 in CM may be due to its role in regulating the production of ROS, which are involved in modulating central sensitization in CM. These findings could lead to new ideas for CM treatment with the use of SIRT1 agonists and antioxidants.
... The mixed lineage kinase domain-like protein (MLKL) is the downstream mediator of the necroptosis process and also the molecular target for saracatinib that has shown promising results in the treatment of psoriasis, an autoimmune disease [66,67]. Additionally, the association of Src with the N-methyl-D-aspartate (NMDA) glutamate receptors, an important mediator of chronic pain hypersensitivity, with the activation of mast cells mediated by immunoglobulin E (IgE) makes saracatinib an interesting possible analgesic and anti-allergic drug, respectively [68,69]. ...
Saracatinib (AZD0530) is a dual Src/Abl inhibitor initially developed by AstraZeneca for cancer treatment; however, data from 2006 to 2024 reveal that this drug has been tested not only for cancer treatment, but also for the treatment of other diseases. Despite the promising pre-clinical results and the tolerability shown in phase I trials, where a maximum tolerated dose of 175 mg was defined, phase II clinical data demonstrated a low therapeutic action against several cancers and an elevated rate of adverse effects. Recently, pre-clinical research aimed at reducing the toxic effects and enhancing the therapeutic performance of saracatinib using nanoparticles and different pharmacological combinations has shown promising results. Concomitantly, saracatinib was repurposed to treat Alzheimer’s disease, targeting Fyn. It showed great clinical results and required a lower daily dose than that defined for cancer treatment, 125 mg and 175 mg, respectively. In addition to Alzheimer’s disease, this Src inhibitor has also been studied in relation to other health conditions such as pulmonary and liver fibrosis and even for analgesic and anti-allergic functions. Although saracatinib is still not approved by the Food and Drug Administration (FDA), the large number of alternative uses for saracatinib and the elevated number of pre-clinical and clinical trials performed suggest the huge potential of this drug for the treatment of different kinds of diseases.
... To investigate whether the effect of TID1S on frataxin is mediated by the last 6 amino acids of its C-terminus (448-453), we generated a competing peptide based on the amino acid sequence of TID1S448-453. To make the TID1S448-453 peptide permeable to cell membranes, it was coupled with a peptide from the transactivator of transcription (TAT) of human immunodeficiency virus (Liu et al., 2008). As a negative control, scrambled TID1S448-453 (sTID1S448-453) was also produced. ...
Friedreich’s ataxia (FRDA), the most common recessive inherited ataxia, results from homozygous guanine–adenine–adenine (GAA) repeat expansions in intron 1 of the FXN gene, which leads to the deficiency of frataxin, a mitochondrial protein essential for iron-sulphur cluster synthesis. The study of frataxin protein regulation might yield new approaches for FRDA treatment. Here, we report tumorous imaginal disc 1 (TID1), a mitochondrial J-protein cochaperone, as a binding partner of frataxin that negatively controls frataxin protein levels. TID1 interacts with frataxin both in vivo in mouse cortex and in vitro in cortical neurons. Acute and subacute depletion of frataxin using RNA interference markedly increases TID1 protein levels in multiple cell types. In addition, TID1 overexpression significantly increases frataxin precursor but decreases intermediate and mature frataxin levels in HEK293 cells. In primary cultured human skin fibroblasts, overexpression of TID1S results in decreased levels of mature frataxin and increased fragmentation of mitochondria. This effect is mediated by the last 6 amino acids of TID1S as a peptide made from this sequence rescues frataxin deficiency and mitochondrial defects in FRDA patient-derived cells. Our findings show that TID1 negatively modulates frataxin levels, and thereby suggests a novel therapeutic target for treating FRDA.
... IL-17, which originated from spinal astrocytes, was involved in pathological pain by promoting Ca 2+ /calmodulin-dependent protein kinase II (CaMKII)-mediated c-AMP-responsive elementbinding protein (CREB) phosphorylation in spinal neurons [24]. N-methyl-D-aspartate receptor (NMDAR) in spinal neurons is critical for the pathogenesis of pain [82]. IL-17 interacted with IL-17R and further promoted phosphorylating NR1 in the NMDAR of spinal neurons [83]. ...
Pathological pain imposes a huge burden on the economy and the lives of patients. At pre- sent, drugs used for the treatment of pathological pain have only modest efficacy and are also plagued by adverse effects and risk for misuse and abuse. Therefore, understanding the mechanisms of patho- logical pain is essential for the development of novel analgesics. Several lines of evidence indicate that interleukin-17 (IL-17) is upregulated in rodent models of pathological pain in the periphery and central nervous system. Besides, the administration of IL-17 antibody alleviated pathological pain. Moreover, IL-17 administration led to mechanical allodynia which was alleviated by the IL-17 antibody. In this review, we summarized and discussed the therapeutic potential of targeting IL-17 for pathological pain. The upregulation of IL-17 promoted the development of pathological pain by promoting neuroin- flammation, enhancing the excitability of dorsal root ganglion neurons, and promoting the communi- cation of glial cells and neurons in the spinal cord. In general, the existing research shows that IL-17 is an attractive therapeutic target for pathologic pain, but the underlying mechanisms still need to be in- vestigated.
Cannabinoids (the endocannabinoids, the synthetic cannabinoids, and the phytocannabinoids) are well known for their various pharmacological properties, including neuroprotective and anti-inflammatory features, which are fundamentally important for the treatment of neurodegenerative diseases. The aging of the global population is causing an increase in these diseases that require the development of effective drugs to be even more urgent. Taking into account the unavailability of effective drugs for neurodegenerative diseases, it seems appropriate to consider the role of cannabinoids in the treatment of these diseases. To our knowledge, few reviews are devoted to cannabinoids’ impact on modulating central and peripheral immunity in neurodegenerative diseases. The objective of this review is to provide the best possible information about the cannabinoid receptors and immuno-modulation features, peripheral immune modulation by cannabinoids, cannabinoid-based therapies for the treatment of neurological disorders, and the future development prospects of making cannabinoids versatile tools in the pursuit of effective drugs.
Inflammation is involved in the induction of chronic inflammatory and neuropathic pain. Moreover, the ketogenic diet, a high-fat, low-carbohydrate, and adequate protein diet, has an anti-inflammatory effect. Thus, we hypothesized that a ketogenic diet has a therapeutic effect on both types of chronic pain. In the present study, we investigated the effect of a ketogenic diet on mechanical allodynia, a chronic pain symptom, in formalin-induced chronic inflammatory pain and nerve injury-induced neuropathic pain models using adult male mice. Formalin injection into the hind paw induced mechanical allodynia in both the injected and intact hind paws, and the ketogenic diet alleviated mechanical allodynia in both hind paws. In addition, the ketogenic diet prevented formalin-induced edema. Furthermore, the diet alleviated mechanical allodynia induced by peripheral nerve injury. Thus, these findings indicate that a ketogenic diet has a therapeutic effect on chronic pain induced by inflammation and nerve injury.
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This study aimed to reduce cancer-associated pain by blocking pain signals through the intranasal administration of siRNA targeting the NMDA subunit NR2B (siNR2B).
Cancer pain models were established by injecting 3 × 105 B16F10 melanoma cells into the left hind paws of C57BL/6 mice. To evaluate pain reduction, 600 pmol of siNR2B was complexed with the RVG9R peptide at a 20:1 molar ratio, or 5 mg/kg NR2B receptor antagonist Ro25-6981 was used as a positive control. Melanoma-xenografted mice were intranasally administered the peptide/siRNA complex or intraperitoneally inoculated with Ro25-6981 three times a week for 3 weeks. The mechanical withdrawal threshold was determined using an electronic von Frey apparatus.
The therapeutic effect of intranasally administered siNR2B was observed 21 days after cancer cell implantation in a hind paw melanoma model. NR2B expression in the cancer model was approximately twice that in the normal animals. The groups treated with siNR2B or Ro25-6981 exhibited approximately 60 and 50% of NR2B expression in the thalamus, respectively. This reduced pain signaling in the thalamic region, as evidenced by a decrease in phosphorylated extracellular signal-regulated kinase. In addition, the siNR2B-treated group displayed significant behavioral improvements, a marked reduction in cancer-induced pain, compared with controls. siNR2B treatment in a cancer-induced murine model did not affect the general cognitive function.
This study demonstrated that the intranasal delivery of siNR2B in a murine model effectively reduced cancer-induced neuropathic pain by downregulating overexpressed NMDA receptor-mediated pain signaling in the thalamus.
The formalin test was introduced as a model of tonic pain in 1977, and has since been used extensively in rats and mice. In rats, formalin generates an initial phase of activity (510 min, phase 1), a quiescent interphase (510 min), and a second phase of activity (lasting 6090 min, phase 2), and this is seen with spontaneous behaviors, firing of afferent neurons, and activity in dorsal horn neurons. Both active phases involve ongoing peripheral afferent neural activity; inflammation contributes to phase 2 activity and the interphase results from active inhibition. Responses are concentration dependent between 0.25% and 2.5%, plateau from 2.5% to 5%, and can decline at higher concentrations. Formalin also results in tissue edema, and this is longer lasting. Responses to formalin up to 2.5% are predominantly neurogenic, while at higher concentrations, responses involve a further prominent inflammatory component. Within the spinal cord, formalin increases c-Fos expression in neurons and causes activation of microglia, and these may contribute more prominently to longer term changes. Acute responses (to 90 min) may represent a model of ongoing acute pain involving inflammation and aspects of central sensitization, while longer term responses (days, weeks) may represent a model of changes involved in persistent pathological pain.
Delivery of therapeutic proteins into tissues and across the blood-brain barrier is severely limited by the size and biochemical
properties of the proteins. Here it is shown that intraperitoneal injection of the 120-kilodalton β-galactosidase protein,
fused to the protein transduction domain from the human immunodeficiency virus TAT protein, results in delivery of the biologically
active fusion protein to all tissues in mice, including the brain. These results open new possibilities for direct delivery
of proteins into patients in the context of protein therapy, as well as for epigenetic experimentation with model organisms.
Long-term potentiation (LTP) is an activity-dependent enhancement of synaptic efficacy, considered a model of learning and memory. The biochemical cascade producing LTP requires activation of Src, which upregulates the function of NMDA receptors (NMDARs), but how Src becomes activated is unknown. Here, we show that the focal adhesion kinase CAKβ/Pyk2 upregulated NMDAR function by activating Src in CA1 hippocampal neurons. Induction of LTP was prevented by blocking CAKβ/Pyk2, and administering CAKβ/Pyk2 intracellularly mimicked and occluded LTP. Tyrosine phosphorylation of CAKβ/Pyk2 and its association with Src was increased by stimulation that produced LTP. Finally, CAKβ/Pyk2-stimulated enhancement of synaptic AMPA responses was prevented by blocking NMDARS, chelating intracellular Ca2+, or blocking Src. Thus, activating CAKβ/Pyk2 is required for inducing LTP and may depend upon downstream activation of Src to upregulate NMDA receptors.
Background: Spinal or systemic adenosine can alleviate neuropathic pain both in humans and in animal models. Peripheral analgesic effects of adenosine have been demonstrated in rodent inflammatory pain models.Purpose: To examine the effects of local peripheral administration of adenosine agonists and inhibitors of adenosine metabolism in a rat neuropathic pain model.Methods: Thermal hyperalgesia and mechanical allodynia were produced by tight ligation of the L5 and L6 spinal nerves in rats, and evaluated by radiant heat or von Frey hairs, respectively. Drugs were administered SC into the dorsal aspect of the rat hind paw.Results: The adenosine A1 receptor-selective agonists, N6-cyclopentyladenosine (CPA) and R-N6-(2-phenylisopropyl)-adenosine (L-PIA), the adenosine kinase inhibitor, 5-amino-5-deoxyadenosine (NH2dAD), and the adenosine deaminase inhibitor, 2-deoxycoformycin (DCF) reversed thermal hyperalgesia produced by nerve injury. This effect was peripherally mediated, as injection of active doses of these drugs into the nonligated paw produced no effect. The effect was due to an action on cell surface adenosine receptors, as the nonselective adenosine receptor antagonist caffeine completely blocked antihyperalgesic actions. CPA, L-PIA, NH2dAD, and DCF did not produce a peripheral antiallodynic effect, indicating the mechanisms involved in the peripheral maintenance of thermal hyperalgesia and mechanical allodynia are different.Conclusion: Activating peripheral adenosine receptors, both directly and indirectly, can reverse the thermal hyperalgesia produced by spinal nerve ligation in the rat.
A comparison was made of the spontaneous nociceptive behaviors elicited by s.c. injection into the rat hind paw of the following 8 irritants: acetic acid, carrageenan, formalin, kaolin, platelet-activating factor, mustard oil (given topically), serotonin, and yeast. Two distinct quantifiable behaviors indicative of pain were identified: flinching/shaking of the paw and hindquarters and licking/biting of the injected paw. These behaviors were prolonged and intense after formalin and acetic acid. Formalin-induced flinching was biphasic across time, a finding potentially useful for the study of both acute and tonic pain. Of the remaining test agents, only yeast caused significant spontaneous behavioral activity, which was of low intensity but long duration. Different time-courses for nociceptive behavior and development of edema were demonstrated for formalin, acetic acid and yeast. It is therefore unlikely that these endpoints are causally related. Overall, the present data strongly support the use of formalin as a noxious stimulus in tonic pain research.
A method for assessing pain and analgesia in rats and cats is described. The procedure involves subcutaneous injection of dilute formalin into the forepaw, after which the animal's responses are rated according to objective behavioral criteria. The formalin test is a statistically valid technique which has two advantages over other pain tests: (1) little or no restraint is necessary, permitting unhindered observation of the complete range of behavioral responses; and (2) the pain stimulus is continuous rather than transient, thus bearing greater resemblance to most clinical pain. The analgesic effects of morphine, meperidine, and stimulation of the periaqueductal grey matter are evaluated using this test.
Administration of 5% formalin into the rat or guinea pig hind paw evokes two spontaneous responses: flinching/shaking and licking/biting of the injected paw. The temporal and behavioral characteristics of these objective endpoints are described. Additionally, several practical suggestions aimed at standardizing this test for the evaluation of analgesics are presented. The early/acute and late/tonic (0-10 and 20-35 min post-formalin, respectively) phases of flinching were used to quantitate antinociception in the rat. PD 117302, the kappa selective agonist, was three times more potent than morphine against tonic flinching after SC administration. Formalin may therefore be a noxious stimulus of choice in the evaluation of kappa agonists. Morphine was only twice as potent against tonic flinching as against acute flinching or the tail-dip reflex to water (50 degrees C). In contrast, PD 117302 was 27 times less potent on early phase and was inactive in the tail-dip test. Thus, while morphine is essentially equipotent across tests, PD 117302 shows a spectrum of activity with impressive potency and efficacy being obtained against tonic pain. Kappa receptors may therefore be prominently involved in tonic pain states. Aspirin given orally was not consistently antinociceptive in either phase of the formalin test. Spinal transection completely abolished late phase responding but only partly attenuated flinching in the early phase. This suggests that the relative involvement of spinal (as opposed to supraspinal) processing of noxious inputs may, at least in part, be a function of stimulus intensity and underlie the differences in antinociceptive potency observed in this work.
To understand the normal, physiological role of the c-src proto-oncogene, a null mutation was introduced into the gene by homologous recombination in mouse embryonic stem cells. Two independent targeted clones were used to generate chimeras that transmitted the mutated allele to their offspring. Intercrossing of heterozygotes gave rise to live born homozygotes, but most of these mice died within the first few weeks of birth. Histological and hematological examination of the homozygous mutants did not reveal detectable abnormalities in the brain or platelets, where src is most highly expressed. However, these mutants were deficient in bone remodeling, indicating impaired osteoclast function, and developed osteopetrosis. These results demonstrate that src is not required for general cell viability (possibly because of functional overlap with other tyrosine kinases related to src) and uncover an essential role for src in bone formation.
A method to measure cutaneous hyperalgesia to thermal stimulation in unrestrained animals is described. The testing paradigm uses an automated detection of the behavioral end-point; repeated testing does not contribute to the development of the observed hyperalgesia. Carrageenan-induced inflammation resulted in significantly shorter paw withdrawal latencies as compared to saline-treated paws and these latency changes corresponded to a decreased thermal nociceptive threshold. Both the thermal method and the Randall-Selitto mechanical method detected dose-related hyperalgesia and its blockade by either morphine or indomethacin. However, the thermal method showed greater bioassay sensitivity and allowed for the measurement of other behavioral parameters in addition to the nociceptive threshold.