Resilience to Pain-Related Depression in σ1 Receptor Knockout Mice Is Associated with the Reversal of Pain-Induced Brain Changes in Affect-Related Genes.

Mice lacking the σ1 receptor chaperone (σ1R-/-) are resilient to depressive-like behaviors secondary to neuropathic pain. Examining the resilience's brain mechanisms could help develop conceptually novel therapeutic strategies. We explored the diminished motivation for a natural reinforcer (white chocolate) in the partial sciatic nerve ligation (PSNL) model in wild-type (WT) and σ1R-/- mice. In the same mice, we performed a comprehensive reverse transcription quantitative PCR (qPCR) analysis across ten brain regions of seven genes implicated in pain regulation and associated affective disorders, such as anxiety and depression. PSNL induced anhedonic-like behavior in WT but not in σ1R-/- mice. In WT mice, PSNL up-regulated dopamine transporter (DAT) and its rate-limiting enzyme, tyrosine hydroxylase (Th), in the ventral tegmental area (VTA) and periaqueductal gray (PAG) as well as the serotonin transporters (SERT) and its rate-limiting enzyme tryptophan hydroxylase 2 (Tph2) in VTA. In addition, µ-opioid receptor (MOR) and σ1R were up-regulated in PAG, and MOR was also elevated in the somatosensory cortex (SS) but down-regulated in the striatum (STR). Finally, increased BDNF was found in the medial prefrontal cortex (mPFC) and hypothalamus (HPT). Sham surgery also produced PSNL-like expression changes in VTA, HPT, and STR. Genetic deletion of the σ1R in mice submitted to PSNL or sham surgery prevented changes in the expression of most of these genes. σ1R is critically involved in the supraspinal gene expression changes produced by PSNL and sham surgery. The changes in gene expression observed in WT mice may be related to pain-related depression, and the absence of these changes observed in σ1R-/- mice may be related to resilience.

Discovery of WLB-89462, a New Drug-like and Highly Selective σ2 Receptor Ligand with Neuroprotective Properties.

The synthesis and pharmacological activity of a new series of isoxazolylpyrimidines as sigma-2 receptor (σ2R) ligands are reported. Modification of a new hit retrieved in an HTS campaign allowed the identification of the compound WLB-89462 (20c) with good σ2R affinity (Ki = 13 nM) and high selectivity vs both the σ1R (Ki = 1777 nM) and a general panel of 180 targets. It represents one of the first σ2R ligands with drug-like properties, linked to a good physicochemical and ADMET profile (good solubility, no CYP inhibition, good metabolic stability, high permeability, brain penetration, and high oral exposure in rodents). Compound 20c shows neuroprotective activity in vitro and improves short-term memory impairment induced by hippocampal injection of amyloid ß peptide in rats. Together with the promising effects in the chronic models where 20c is currently being evaluated, these results pave the way toward its clinical development as a neuroprotective agent.

Development of a Novel σ1 Receptor Biosensor Based on Its Heterodimerization with Binding Immunoglobulin Protein in Living Cells.

The σ1 receptor (S1R) is a ligand-regulated non-opioid intracellular receptor involved in several pathological conditions. The development of S1R-based drugs as therapeutic agents is a challenge due to the lack of simple functional assays to identify and classify S1R ligands. We have developed a novel nanoluciferase binary technology (NanoBiT) assay based on the ability of S1R to heteromerize with the binding immunoglobulin protein (BiP) in living cells. The S1R-BiP heterodimerization biosensor allows for rapid and accurate identification of S1R ligands by monitoring the dynamics of association-dissociation of S1R and BiP. Acute treatment of cells with the S1R agonist PRE-084 produced rapid and transient dissociation of the S1R-BiP heterodimer, which was blocked by haloperidol. The effect of PRE-084 was enhanced by calcium depletion, leading to a higher reduction in heterodimerization even in the presence of haloperidol. Prolonged incubation of cells with S1R antagonists (haloperidol, NE-100, BD-1047, and PD-144418) increased the formation of S1R-BiP heteromers, while agonists (PRE-084, 4-IBP, and pentazocine) did not alter heterodimerization under the same experimental conditions. The newly developed S1R-BiP biosensor is a simple and effective tool for exploring S1R pharmacology in an easy cellular setting. This biosensor is suitable for high-throughput applications and a valuable resource in the researcher's toolkit.

Bispecific sigma-1 receptor antagonism and mu-opioid receptor partial agonism: WLB-73502, an analgesic with improved efficacy and safety profile compared to strong opioids.

Opioids are the most effective painkillers, but their benefit-risk balance often hinder their therapeutic use. WLB-73502 is a dual, bispecific compound that binds sigma-1 (S1R) and mu-opioid (MOR) receptors. WLB-73502 is an antagonist at the S1R. It behaved as a partial MOR agonist at the G-protein pathway and produced no/unsignificant ß-arrestin-2 recruitment, thus demonstrating low intrinsic efficacy on MOR at both signalling pathways. Despite its partial MOR agonism, WLB-73502 exerted full antinociceptive efficacy, with potency superior to morphine and similar to oxycodone against nociceptive, inflammatory and osteoarthritis pain, and superior to both morphine and oxycodone against neuropathic pain. WLB-73502 crosses the blood-brain barrier and binds brain S1R and MOR to an extent consistent with its antinociceptive effect. Contrary to morphine and oxycodone, tolerance to its antinociceptive effect did not develop after repeated 4-week administration. Also, contrary to opioid comparators, WLB-73502 did not inhibit gastrointestinal transit or respiratory function in rats at doses inducing full efficacy, and it was devoid of proemetic effect (retching and vomiting) in ferrets at potentially effective doses. WLB-73502 benefits from its bivalent S1R antagonist and partial MOR agonist nature to provide an improved antinociceptive and safety profile respect to strong opioid therapy.

Long-Lasting Nociplastic Pain Modulation by Repeated Administration of Sigma-1 Receptor Antagonist BD1063 in Fibromyalgia-like Mouse Models.

Sigma-1 receptor (σ1R) ligands have been shown to be effective at relieving neuropathic and inflammatory pain, but have not yet been tested in experimental models of fibromyalgia. The objective of this study was to evaluate the effect of a σ1R antagonist (BD1063) compared to pregabalin. ICR-CD1 female mice were subjected to either six repeated injections of reserpine, to cause reserpine-induced myalgia (RIM6), or acidified saline intramuscular injections (ASI). In these two models, we evaluated the effect of BD1063 and pregabalin on thermal hypersensitivity, anxiety-like and depression-like behaviors, and on spinal cord gliosis. BD1063 exerted an antinociceptive effect on both reflexive (thermal hyperalgesia) and nonreflexive (anxiety- and depression-like) pain behaviors, and reduced spinal astroglial and microglial reactivity, following repeated treatment for 2 weeks. Interestingly, the effects of BD1063 were long-term, lasting several weeks after treatment discontinuation in both fibromyalgia-like models. Similar results were obtained with pregabalin, but the effects on pain behaviors lasted for a shorter length of time, and pregabalin did not significantly modulate spinal glial reactivity. The inhibitory and long-lasting effect of pharmacological blockade of σ1Rs on both sensory and affective dimensions of nociplastic-like pain and spinal cord gliosis in two experimental models of fibromyalgia support the application of this therapeutic strategy to treat fibromyalgia.

EST79232 and EST79376, Two Novel Sigma-1 Receptor Ligands, Exert Neuroprotection on Models of Motoneuron Degeneration.

Motor neuron diseases (MNDs) include sporadic and hereditary neurological disorders characterized by progressive degeneration of motor neurons (MNs). Sigma-1 receptor (Sig-1R) is a protein enriched in MNs, and mutations on its gene lead to various types of MND. Previous studies have suggested that Sig-1R is a target to prevent MN degeneration. In this study, two novel synthesized Sig-1R ligands, coded EST79232 and EST79376, from the same chemical series, with the same scaffold and similar physicochemical properties but opposite functionality on Sig-1R, were evaluated as neuroprotective compounds to prevent MN degeneration. We used an in vitro model of spinal cord organotypic cultures under chronic excitotoxicity and two in vivo models, the spinal nerve injury and the superoxide dismutase 1 (SOD1)G93A mice, to characterize the effects of these Sig-1R ligands on MN survival and modulation of glial reactivity. The antagonist EST79376 preserved MNs in vitro and after spinal nerve injury but was not able to improve MN death in SOD1G93A mice. In contrast, the agonist EST79232 significantly increased MN survival in the three models of MN degeneration evaluated and had a mild beneficial effect on motor function in SOD1G93A mice. In vivo, Sig-1R ligand EST79232 had a more potent effect on preventing MN degeneration than EST79376. These data further support the interest in Sig-1R as a therapeutic target for neurodegeneration.

Long-lasting reflexive and nonreflexive pain responses in two mouse models of fibromyalgia-like condition.

Nociplastic pain arises from altered nociception despite no clear evidence of tissue or somatosensory system damage, and fibromyalgia syndrome can be highlighted as a prototype of this chronic pain subtype. Currently, there is a lack of effective treatments to alleviate both reflexive and nonreflexive pain responses associated with fibromyalgia condition, and suitable preclinical models are needed to assess new pharmacological strategies. In this context, although in recent years some remarkable animal models have been developed to mimic the main characteristics of human fibromyalgia, most of them show pain responses in the short term. Considering the chronicity of this condition, the present work aimed to develop two mouse models showing long-lasting reflexive and nonreflexive pain responses after several reserpine (RIM) or intramuscular acid saline solution (ASI) injections. To our knowledge, this is the first study showing that RIM6 and ASI mouse models show reflexive and nonreflexive responses up to 5-6 weeks, accompanied by either astro- or microgliosis in the spinal cord as pivotal physiopathology processes related to such condition development. In addition, acute treatment with pregabalin resulted in reflexive pain response alleviation in both the RIM6 and ASI models. Consequently, both may be considered suitable experimental models of fibromyalgia-like condition, especially RIM6.

Comprehensive Preclinical Assessment of Sensory, Functional, Motivational-Affective, and Neurochemical Outcomes in Neuropathic Pain: The Case of the Sigma-1 Receptor.

Chronic pain remains a major health problem and is currently facing slow drug innovation. New drug treatments should address not only the sensory-discriminative but also functional and motivational-affective components of chronic pain. In a mouse model of neuropathic pain induced by partial sciatic nerve ligation (PSNL), we analyzed sensory and functional-like outcomes by hindpaw mechanical stimulation and automated gait analysis (CatWalk). We characterized over time a reward-seeking task based on diminished motivation for natural reinforcers (anhedonic-like behavior). To differentiate the appetitive ("wanting") and consummatory ("liking") aspects of motivational behavior, we quantified the latency and number of approaches to eat white chocolate, as well as the eating duration and amount consumed. We explored a putative chronic pain-induced dysregulation of monoamine function by measuring monoamine levels in the nucleus accumbens (NAc), a well-known brain reward area. Finally, we investigated the role of sigma-1 receptor (σ1R) modulation, a nonopioid target, in these multiple dimensions by genetic deletion and pharmacological dose-response studies. After 6 weeks, PSNL increased the approach latency and reduced the consumption of white chocolate in 20-25% of the mice, while around 50-60% had one or the other parameter affected independently. After 10 weeks, sham-operated mice also displayed anhedonic-like behavior. PSNL was associated with reduced extracellular baseline dopamine and increased norepinephrine in the NAc and with a suppression of increased dopamine and serotonin efflux in response to the rewarding stimulus. Genetic and pharmacological blockade of σ1R relieved these multiple alterations in nerve-injured mice. We comprehensively describe sensory, functional, and depression-like impairment of key components of motivated behavior associated with nerve injury. We provide a neurochemical substrate for the depressed mesocorticolimbic reward processing in chronic pain, with a potentially increased translational value. Our results also highlight σ1R for the therapeutic intervention of neuropathic pain.

Identification of Sodium Transients Through NaV1.5 Channels as Regulators of Differentiation in Immortalized Dorsal Root Ganglia Neurons.

Neuronal differentiation is a complex process through which newborn neurons acquire the morphology of mature neurons and become excitable. We employed a combination of functional and transcriptomic approaches to deconvolute and identify key regulators of the differentiation process of a DRG neuron-derived cell line, and we focused our study on the Na V 1.5 ion channel (encoded by Scn5a) as a channel involved in the acquisition of DRG neuronal features. Overexpression of Scn5a enhances the acquisition of neuronal phenotypic features and increases the KCl-elicited hyperexcitability response in a DRG-derived cell line. Moreover, pharmacologic inhibition of the Na V 1.5 channel during differentiation hinders the acquisition of phenotypic features of neuronal cells and the hyperexcitability increase in response to changes in the extracellular medium ionic composition. Taken together, these data highlight the relevance of sodium transients in regulating the neuronal differentiation process in a DRG neuron-derived cell line.

Neuroprotective Effects of Sigma 1 Receptor Ligands on Motoneuron Death after Spinal Root Injury in Mice.

Loss of motor neurons (MNs) after spinal root injury is a drawback limiting the recovery after palliative surgery by nerve or muscle transfers. Research based on preventing MN death is a hallmark to improve the perspectives of recovery following severe nerve injuries. Sigma-1 receptor (Sig-1R) is a protein highly expressed in MNs, proposed as neuroprotective target for ameliorating MN degenerative conditions. Here, we used a model of L4-L5 rhizotomy in adult mice to induce MN degeneration and to evaluate the neuroprotective role of Sig-1R ligands (PRE-084, SA4503 and BD1063). Lumbar spinal cord was collected at 7, 14, 28 and 42 days post-injury (dpi) for immunohistochemistry, immunofluorescence and Western blot analyses. This proximal axotomy at the immediate postganglionic level resulted in significant death, up to 40% of spinal MNs at 42 days after injury and showed markedly increased glial reactivity. Sig-1R ligands PRE-084, SA4503 and BD1063 reduced MN loss by about 20%, associated to modulation of endoplasmic reticulum stress markers IRE1α and XBP1. These pathways are Sig-1R specific since they were not produced in Sig-1R knockout mice. These findings suggest that Sig-1R is a promising target for the treatment of MN cell death after neural injuries.

Identification of Novel Regulators of Zalcitabine-Induced Neuropathic Pain.

Neuropathic pain is one of the foremost adverse effects that worsens quality of life for patients undergoing an antiretroviral treatment. Currently, there are no effective analgesics for relieving it; thus, there is an urgent need to develop novel treatments for neuropathic pain. Previously, we described and validated F11 cells as a model of DRG (dorsal root ganglia) neurons. In the current work, we employed F11 cells to identify regulators of antiretroviral-induced neuropathic pain combining functional and transcriptomic analysis. The antiretroviral zalcitabine (ddC) increased the excitability of differentiated F11 cells associated with calcium signaling without morphological changes in the neuronal phenotype, mimicking the observed increase of painful signaling in patients suffering from antiretroviral-induced neuropathic pain. Employing RNA sequencing, we observed that zalcitabine treatment upregulated genes related with oxidative stress and calcium homeostasis. The functional impact of the transcriptomic changes was explored, finding that the exposure to zalcitabine significantly increased intracellular oxidative stress and reduced store-operated calcium entry (SOCE). Because the functional and transcriptomic evidence points toward fundamental changes in calcium signaling and oxidative stress upon zalcitabine exposure, we identified that NAD(P)H quinone dehydrogenase and the sarcoplasmic/endoplasmic reticulum calcium ATPase 3 were involved in zalcitabine-induced hyperexcitability of F11 cells. Overexpression of those genes increases the calcium-elicited hyperexcitability response and reduces SOCE, as well as increases intracellular ROS levels. These data do not only mimic the effects of zalcitabine but also highlight the relevance of oxidative stress and of calcium-mediated signaling in antiretroviral-induced hyperexcitability of sensory neurons, shedding light on new therapeutic targets for antiviral-induced neuropathic pain.

Development of a novel in vitro assay to screen for neuroprotective drugs against iatrogenic neurite shortening.

This work tries to help overcome the lack of relevant translational screening assays, as a limitation for the identification of novel analgesics for neuropathic pain. Hyperexcitability and neurite shortening are common adverse effects of antiviral and antitumor drugs, leading to neuropathic pain. Now, as seen in the drug screening that we developed here, a high-content microscopy-based assay with immortalized dorsal root ganglia (DRG) neurons (differentiated F11 cells) allowed to identify drugs able to protect against the iatrogenic neurite shortening induced by the antitumor drug vincristine and the antiviral drug rilpivirine. We observed that vincristine and rilpivirine induced a significant reduction in the neurite length, which was reverted by α-lipoic acid. We had also evidenced protective effects of pregabalin and melatonin, acting through the α2δ-2 subunit of the voltage-dependent calcium channels and the MT1 receptor, respectively. Additionally, two hits originated from a previous primary screening aimed to detect inhibitors of hyperexcitability to inflammatory mediators in DRG neurons (nitrendipine and felodipine) also prevented neurite shortening in our model. In summary, in this work we developed a novel secondary assay for identifying hits with neuroprotective effect against iatrogenic neurite shortening, consistent with the anti-hyperexcitability action previously tested: highlighting nitrendipine and felodipine against iatrogenic damage in DRG neurons.

The Sigma 2 receptor promotes and the Sigma 1 receptor inhibits mu-opioid receptor-mediated antinociception.

The Sigma-1 receptor (σ1R) has emerged as an interesting pharmacological target because it inhibits analgesia mediated by mu-opioid receptors (MOR), and also facilitates the development of neuropathic pain. Based on these findings, the recent cloning of the Sigma-2 receptor (σ2R) led us to investigate its potential role as a regulator of opioid analgesia and of pain hypersensitivity in σ2R knockout mice. In contrast to σ1R deficient mice, σ2R knockout mice developed mechanical allodynia following establishment of chronic constriction injury-induced neuropathic pain, which was alleviated by the σ1R antagonist S1RA. The analgesic effects of morphine, [D-Ala, N-MePhe, Gly-ol]-encephalin (DAMGO) and ß-endorphin increased in σ1R-/- mice and diminished in σ2R-/- mice. The analgesic effect of morphine was increased in σ2R-/- mice by treatment with S1RA. However, σ2R-/- mice and wild-type mice exhibited comparable antinociceptive responses to the delta receptor agonist [D-Pen2,5]-encephalin (DPDPE), the cannabinoid type 1 receptor agonist WIN55,212-2 and the α2-adrenergic receptor agonist clonidine. Therefore, while σR1 inhibits and σ2R facilitates MOR-mediated analgesia these receptors exchange their roles when regulating neuropathic pain perception. Our study may help identify new pharmacological targets for diminishing pain perception and improving opioid detoxification therapies.

Discovery of EST73502, a Dual µ-Opioid Receptor Agonist and σ1 Receptor Antagonist Clinical Candidate for the Treatment of Pain.

The synthesis and pharmacological activity of a new series of 4-alkyl-1-oxa-4,9-diazaspiro[5.5]undecane derivatives as potent dual ligands for the σ1 receptor (σ1R) and the µ-opioid receptor (MOR) are reported. A lead optimization program over the initial 4-aryl analogues provided 4-alkyl derivatives with the desired functionality and good selectivity and ADME profiles. Compound 14u (EST73502) showed MOR agonism and σ1R antagonism and a potent analgesic activity, comparable to the MOR agonist oxycodone in animal models of acute and chronic pain after single and repeated administration. Contrary to oxycodone, 14u produces analgesic activity with reduced opioid-induced relevant adverse events, like intestinal transit inhibition and naloxone-precipitated behavioral signs of opiate withdrawal. These results provide evidence that dual MOR agonism and σ1R antagonism may be a useful strategy for obtaining potent and safer analgesics and were the basis for the selection of 14u as a clinical candidate for the treatment of pain.

Urinary bladder sigma-1 receptors: A new target for cystitis treatment.

No adequate treatment is available for painful urinary bladder disorders such as interstitial cystitis/bladder pain syndrome, and the identification of new urological therapeutic targets is an unmet need. The sigma-1 receptor (σ1-R) modulates somatic pain, but its role in painful urological disorders is unexplored. The urothelium expresses many receptors typical of primary sensory neurons (e.g. TRPV1, TRPA1 and P2X3) and high levels of σ1-R have been found in these neurons; we therefore hypothesized that σ1-R may also be expressed in the urothelium and may have functional relevance in this tissue. With western blotting and immunohistochemical methods, we detected σ1-R in the urinary bladder in wild-type (WT) but not in σ1-R-knockout (σ1-KO) mice. Interestingly, σ1-R was located in the bladder urothelium not only in mouse, but also in human bladder sections. The severity of histopathological (edema, hemorrhage and urothelial desquamation) and biochemical alterations (enhanced myeloperoxidase activity and phosphorylation of extracellular regulated kinases 1/2 [pERK1/2]) that characterize cyclophosphamide-induced cystitis was lower in σ1-KO than in WT mice. Moreover, cyclophosphamide-induced pain behaviors and referred mechanical hyperalgesia were dose-dependently reduced by σ1-R antagonists (BD-1063, NE-100 and S1RA) in WT but not in σ1-KO mice. In contrast, the analgesic effect of morphine was greater in σ1-KO than in WT mice. Together these findings suggest that σ1-R plays a functional role in the mechanisms underlying cyclophosphamide-induced cystitis, and modulates morphine analgesia against urological pain. Therefore, σ1-R may represent a new drug target for urinary bladder disorders.

Outside-in regulation of the readily releasable pool of synaptic vesicles by α2δ-1.

The readily releasable pool (RRP) of synaptic vesicles is a key determinant of phasic neurotransmission. Although the size of the RRP is tightly regulated by intracellular factors, there is little evidence for its modification by extracellular signals. By studying the homogeneous population of synapses present in autaptic microcultures, we show that pregabalin, a prototypical gabapentinoid, decreases the effective RRP size. Simultaneous imaging of presynaptic calcium influx and recording of postsynaptic responses shows that the effect is not related to a reduction of calcium entry. The main cause is the impairment of the functional coupling among N-type calcium channels and the RRP, resembling an increase of intracellular mobile calcium buffers. The ectodomain of neurexin-1α shows a similar action to pregabalin, acting as an endogenous ligand of α2δ-1 that reduces the RRP size without affecting presynaptic calcium influx. The regulatory actions described for pregabalin and the ectodomain of neurexin-1α are mutually exclusive. The overexpression of α2δ-1 enhances the effect of pregabalin and the ectodomain of neurexin-1α on neurotransmission by decreasing their effective concentration. In contrast, knockdown of α2δ-1 causes a profound inhibition of synaptic transmission. These observations prompt to consider α2δ-1 as an outside-in signaling platform that binds exogenous and endogenous cues for regulating the coupling of voltage-gated calcium channels to synaptic vesicles.

Sigma-1 receptor modulates neuroinflammation associated with mechanical hypersensitivity and opioid tolerance in a mouse model of osteoarthritis pain.

BACKGROUND AND PURPOSE: Osteoarthritic pain is a chronic disabling condition lacking effective treatment. Continuous use of opioid drugs during osteoarthritic pain induces tolerance and may result in dose escalation and abuse. Sigma-1 (σ1) receptors, a chaperone expressed in key areas for pain control, modulates µ-opioid receptor activity and represents a promising target to tackle these problems. The present study investigates the efficacy of the σ1 receptor antagonist E-52862 to inhibit pain sensitization, morphine tolerance, and associated electrophysiological and molecular changes in a murine model of osteoarthritic pain. EXPERIMENTAL APPROACH: Mice received an intra-knee injection of monoiodoacetate followed by 14-day treatment with E-52862, morphine, or vehicle, and mechanical sensitivity was assessed before and after the daily doses. KEY RESULTS: Monoiodoacetate-injected mice developed persistent mechanical hypersensitivity, which was dose-dependently inhibited by E-52862. Mechanical thresholds assessed before the daily E-52862 dose showed gradual recovery, reaching complete restoration by the end of the treatment. When repeated treatment started 15 days after knee injury, E-52862 produced enhanced short-term analgesia, but recovery to baseline threshold was slower. Both a σ1 receptor agonist and a µ receptor antagonist blocked the analgesic effects of E-52862. An acute, sub-effective dose of E-52862 restored morphine analgesia in opioid-tolerant mice. Moreover, E-52862 abolished spinal sensitization in osteoarthritic mice and inhibited pain-related molecular changes. CONCLUSION AND IMPLICATIONS: These findings show dual effects of σ1 receptor antagonism alleviating both short- and long-lasting antinociception during chronic osteoarthritis pain. They identify E-52862 as a promising pharmacological agent to treat chronic pain and avoid opioid tolerance.

Ligands Exert Biased Activity to Regulate Sigma 1 Receptor Interactions With Cationic TRPA1, TRPV1, and TRPM8 Channels.

The sigma 1 receptor (σ1R) and the mu-opioid receptor (MOR) regulate the transient receptor potential (TRP) V1 calcium channel. A series of proteins are involved in the cross-regulation between MORs and calcium channels like the glutamate N-methyl-D-aspartate receptor (NMDAR), including the histidine triad nucleotide-binding protein 1 (HINT1), calmodulin (CaM), and the σ1R. Thus, we assessed whether similar mechanisms also apply to the neural TRP ankyrin member 1 (TRPA1), TRP vanilloid member 1 (TRPV1), and TRP melastatin member 8 (TRPM8). Our results indicate that σ1R and CaM bound directly to cytosolic regions of these TRPs, and this binding increased in the presence of calcium. By contrast, the association of HINT1 with these TRPs was moderately dependent on calcium. The σ1R always competed with CaM for binding to the TRPs, except for its binding to the TRPA1 C-terminal where σ1R binding cooperated with that of CaM. However, σ1R dampened HINT1 binding to the TRPA1 N-terminal. When the effect of σ1R ligands was addressed, the σ1R agonists PRE084 and pregnenolone sulfate enhanced the association of the σ1R with the TRPM8 N-terminal and TRPV1 C-terminal in the presence of physiological calcium, as seen for the σ1R-NMDAR interactions. However, these agonists dampened σ1R binding to the TRPA1 and TRPV1 N-terminal domains, and also to the TRPA1 C-terminal, as seen for σ1R-binding immunoglobulin protein (BiP) interactions in the endoplasmic reticulum (ER). By contrast, the σ1R antagonists progesterone and S1RA reduced the association of σ1R with TRPA1 and TRPV1 C-terminal regions, as seen for the σ1R-NMDAR interactions. Conversely, they enhanced the σ1R interaction with the TRPA1 N-terminal, as seen for σ1R-BiP interactions, whereas they barely affected the association of σ1R with the TRPV1 N-terminal. Thus, depending on the calcium channel and the cytosolic region examined, the σ1R agonists pregnenolone sulfate and PRE084 opposed or collaborated with the σ1R antagonists progesterone and S1RA to disrupt or promote such interactions. Through the use of cloned cytosolic regions of selected TRP calcium channels, we were able to demonstrate that σ1R ligands exhibit biased activity to regulate particular σ1R interactions with other proteins. Since σ1Rs are implicated in essential physiological processes, exploiting such ligand biases may represent a means to develop more selective and efficacious pharmacological interventions.

Blockade of the Sigma-1 Receptor Relieves Cognitive and Emotional Impairments Associated to Chronic Osteoarthritis Pain.

Osteoarthritis is the most common musculoskeletal disease worldwide, often characterized by degradation of the articular cartilage, chronic joint pain and disability. Cognitive dysfunction, anxiety and depression are common comorbidities that impact the quality of life of these patients. In this study, we evaluated the involvement of sigma-1 receptor (σ1R) on the nociceptive, cognitive and emotional alterations associated with chronic osteoarthritis pain. Monosodium iodoacetate (MIA) was injected into the knee of Swiss-albino CD1 mice to induce osteoarthritis pain, which then received a repeated treatment with the σ1R antagonist E-52862 or its vehicle. Nociceptive responses and motor performance were assessed with the von Frey and the Catwalk gait tests. Cognitive alterations were evaluated using the novel object recognition task, anxiety-like behavior with the elevated plus maze and the zero-maze tests, whereas depressive-like responses were determined using the forced swimming test. We also studied the local effect of the σ1R antagonist on cartilage degradation, and its central effects on microglial reactivity in the medial prefrontal cortex. MIA induced mechanical allodynia and gait abnormalities that were prevented by the chronic treatment with the σ1R antagonist. E-52862 also reduced the memory impairment and the depressive-like behavior associated to osteoarthritis pain. Interestingly, the effect of E-52862 on depressive-like behavior was not accompanied by a modification of anxiety-like behavior. The pain-relieving effects of the σ1R antagonist were not due to a local effect on the articular cartilage, since E-52862 treatment did not modify the histological alterations of the knee joints. However, E-52862 induced central effects revealed by a reduction of the cortical microgliosis observed in mice with osteoarthritis pain. These findings show that σ1R antagonism inhibits mechanical hypersensitivity, cognitive deficits and depressive-like states associated with osteoarthritis pain in mice. These effects are associated with central modulation of glial activity but are unrelated to changes in cartilage degradation. Therefore, targeting the σ1R with E-52862 represents a promising pharmacological approach with effects on multiple aspects of chronic osteoarthritis pain that may go beyond the strict inhibition of nociception.

Supraspinal and Peripheral, but Not Intrathecal, σ1R Blockade by S1RA Enhances Morphine Antinociception.

Sigma-1 receptor (σ1R) antagonism increases the effects of morphine on acute nociceptive pain. S1RA (E-52862) is a selective σ1R antagonist widely used to study the role of σ1Rs. S1RA alone exerted antinociceptive effect in the formalin test in rats and increased noradrenaline levels in the spinal cord, thus accounting for its antinociceptive effect. Conversely, while systemic S1RA failed to elicit antinociceptive effect by itself in the tail-flick test in mice, it did potentiate the antinociceptive effect of opioids in this acute pain model. The present study aimed to investigate the site of action and the involvement of spinal noradrenaline on the potentiation of opioid antinociception by S1RA on acute thermal nociception using the tail-flick test in rats. Local administration was performed after intrathecal catheterization or intracerebroventricular and rostroventral medullar (RVM) cannulae implantation. Noradrenaline levels in the spinal cord were evaluated using the concentric microdialysis technique in awake, freely-moving rats. Systemic or supraspinal administration of S1RA alone, while having no effect on antinociception, enhanced the effect of morphine in rats. However, spinal S1RA administration did not potentiate the antinociceptive effect of morphine. Additionally, the peripherally restricted opioid agonist loperamide was devoid of antinociceptive effect but produced antinociception when combined with S1RA. Neurochemical studies revealed that noradrenaline levels in the dorsal horn of the spinal cord were not increased at doses exerting potentiation of the antinociceptive effect of the opioid. In conclusion, the site of action of σ1R for opioid modulation on acute thermal nociception is located at the peripheral and supraspinal levels, and the opioid-potentiating effect is independent of the spinal noradrenaline increase produced by S1RA.