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Purpose: This study elucidates the mechanism of the physiological effect of cannabidiol (CBD) by assessing its impact on lipopolysaccharide (LPS)-induced inflammation in RWPE-1 cells and prostatitis-induced by 17β-estradiol and dihydrotestosterone in a rat model, focusing on its therapeutic potential for chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). Materials and Methods: RWPE-1 cells were stratified in vitro into three groups: (1) controls, (2) cells with LPS-induced inflammation, and (3) cells with LPS-induced inflammation and treated with CBD. Enzyme-linked immunosorbent assays and western blots were performed on cellular components and supernatants after administration of CBD. Five groups of six Sprague– Dawley male rats were assigned: (1) control, (2) CP/CPPS, (3) CP/CPPS and treated with 50 mg/kg CBD, (4) CP/CPPS and treated with 100 mg/kg CBD, and (5) CP/CPPS and treated with 150 mg/kg CBD. Prostatitis was induced through administration of 17β-estradiol and dihydrotestosterone. After four weeks of CBD treatment, a pain index was evaluated, and prostate tissue was collected for subsequent histologic examination and western blot analysis. Results: CBD demonstrated efficacy in vivo for CP/CPPS and in vitro for inflammation. It inhibited the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) pathway by activating the CB2 receptor, reducing expression of interleukin-6, tumor necrosis factor-alpha, and cyclooxygenase-2 (COX2) (p<0.01). CBD exhibited analgesic effects by activating and desensitizing the TRPV1 receptor. Conclusions: CBD inhibits the TLR4/NF-κB pathway by activating the CB2 receptor, desensitizes the TRPV1 receptor, and decreases the release of COX2. This results in relief of inflammation and pain in patients with CP/CPPS, indicating CBD as a potential treatment for CP/CPPS. [ABSTRACT FROM AUTHOR]

Background and Purpose: Receptor activity‐modifying proteins (RAMPs) and melanocortin receptor accessory proteins (MRAPs) modulate expression and signalling of calcitonin and melanocortin GPCRs. Interactions with other GPCRs have also been reported. The cannabinoid receptors, CB1 and CB2, and two putative cannabinoid receptors, GPR18 and GPR55, exhibit substantial intracellular expression and there are discrepancies in ligand responsiveness between studies. We investigated whether interactions with RAMPs or MRAPs could explain these phenomena. Experimental Approach: Receptors and accessory proteins were co‐expressed in HEK‐293 cells. Selected receptors were studied at basal expression levels and also with enhanced expression produced by incorporation of a preprolactin signal sequence/peptide (pplss). Cell surface and total expression of receptors and accessory proteins were quantified using immunocytochemistry. Signalling was measured using cAMP (CAMYEL) and G protein dissociation (TRUPATH Gα13) biosensors. Key Results: MRAP2 enhanced surface and total expression of GPR18. Pplss‐GPR18 increased detection of cell surface MRAP2. MRAP1α and MRAP2 reduced GPR55 surface and total expression, correlating with reduced constitutive, but not agonist‐induced, signalling. GPR55, pplss‐CB1 and CB2 reduced detection of MRAP1α at the cell surface. Pplss‐CB1 agonist potency was reduced by MRAP2 in Gα13 but not cAMP assays, consistent with MRAP2 reducing pplss‐CB1 expression. Some cannabinoid receptors increased RAMP2 or RAMP3 total expression without influencing surface expression. Conclusions and Implications: Mutual influences on expression and/or function for specific accessory protein‐receptor pairings raises the strong potential for physiological and disease‐relevant consequences. Sequestration and/or hetero‐oligomerisation of cannabinoid receptors with accessory proteins is a possible novel mechanism for receptor crosstalk. LINKED ARTICLES: This article is part of a themed issue Therapeutic Targeting of G Protein‐Coupled Receptors: hot topics from the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists 2021 Virtual Annual Scientific Meeting. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.14/issuetoc [ABSTRACT FROM AUTHOR]

Taking into account homeostatic disorders resulting from arterial hypertension and the key importance of CacyBP/SIP, β‐catenin and endocannabinoids in the functioning of many organs, it was decided to assess the presence and distribution of CacyBP/SIP, β‐catenin, CB1 and CB2 in the adrenal glands of hypertensive rats of various aetiology. The study was conducted on the adrenal glands of rats with spontaneous and renovascular hypertension. The expression of CacyBP/SIP, β‐catenin, CB1 and CB2 was detected by immunohistochemistry and real‐time PCR method. The results of the present study revealed both lower gene expression and immunoreactivity of CacyBP/SIP in the adrenal glands of all hypertensive groups compared to the normotensive rats. This study demonstrated a reduction in the immunoreactivity and expression of the β‐catenin, CB1 and CB2 genes in the adrenals of 2K1C rats. While in SHR, the reaction showing β‐catenin and CB1 was very weak or negative, and the expression of CB2 in the adrenal glands of these rats increased. The results of this study show, for the first time, marked differences in the expression of CacyBP/SIP, β‐catenin and CB1 and CB2 cannabinoid receptors in the adrenal glands of rats with primary (SHR) and secondary hypertension (2K1C). [ABSTRACT FROM AUTHOR]

Abstract To test the hypothesis that genetic and pharmacological modulation of the classical cannabinoid type 1 (CB1) and 2 (CB2) receptors attenuate cancer-induced bone pain, we searched Medline, Web of Science and Scopus for relevant skeletal and non-skeletal cancer studies from inception to July 28, 2022. We identified 29 animal and 35 human studies. In mice, a meta-analysis of pooled studies showed that treatment of osteolysis-bearing males with the endocannabinoids AEA and 2-AG (mean difference [MD] − 24.83, 95% confidence interval [95%CI] − 34.89, − 14.76, p

Early adversity, the loss of the inhibitory GABAergic interneuron parvalbumin, and elevated neuroinflammation are associated with depression. Individuals with a maltreatment history initiate medicinal cannabis use earlier in life than non-maltreated individuals, suggesting self-medication. Female rats underwent maternal separation (MS) between 2 and 20 days of age to model early adversity or served as colony controls. The prelimbic cortex and behavior were examined to determine whether MS alters the cannabinoid receptor 2 (CB2), which has anti-inflammatory properties. A reduction in the CB2-associated regulatory enzyme MARCH7 leading to increased NLRP3 was observed with Western immunoblots in MS females. Immunohistochemistry with stereology quantified numbers of parvalbumin-immunoreactive cells and CB2 at 25, 40, and 100 days of age, revealing that the CB2 receptor associated with PV neurons initially increases at P25 and subsequently decreases by P40 in MS animals, with no change in controls. Confocal and triple-label microscopy suggest colocalization of these CB2 receptors to microglia wrapped around the parvalbumin neuron. Depressive-like behavior in MS animals was elevated at P40 and reduced with the CB2 agonist HU-308 or a CB2-overexpressing lentivirus microinjected into the prelimbic cortex. These results suggest that increasing CB2 expression by P40 in the prelimbic cortex prevents depressive behavior in MS female rats. [ABSTRACT FROM AUTHOR]

Endocannabinoid anandamide (AEA) and paracannabinoid lysophosphatidylinositol (LPI) play a significant role in cancer cell proliferation regulation. While anandamide inhibits the proliferation of cancer cells, LPI is known as a cancer stimulant. Despite the known endocannabinoid receptor crosstalk and simultaneous presence in the cancer microenvironment of both molecules, their combined activity has never been studied. We evaluated the effect of LPI on the AEA activity in six human breast cancer cell lines of different carcinogenicity (MCF-10A, MCF-7, BT-474, BT-20, SK-BR-3, MDA-MB-231) using resazurin and LDH tests after a 72 h incubation. AEA exerted both anti-proliferative and cytotoxic activity with EC50 in the range from 31 to 80 µM. LPI did not significantly affect the cell viability. Depending on the cell line, the response to the LPI–AEA combination varied from a decrease in AEA cytotoxicity to an increase in it. Based on the inhibitor analysis of the endocannabinoid receptor panel, we showed that for the former effect, an active GPR18 receptor was required and for the latter, an active CB2 receptor. The data obtained for the first time are important for the understanding the manner by which endocannabinoid receptor ligands acting simultaneously can modulate cancer growth at different stages. [ABSTRACT FROM AUTHOR]

The endocannabinoid system (ECS) exerts immunosuppressive effects, which are mostly mediated by cannabinoid receptor 2 (CBR2), whose expression on leukocytes is higher than CBR1, mainly localized in the brain. Targeted CBR2 activation could limit inflammation, avoiding CBR1-related psychoactive effects. Herein, we evaluated in vitro the biological activity of a novel, selective and high-affinity CBR2 agonist, called JT11, studying its potential CBR2-mediated anti-inflammatory effect. Trypan Blue and MTT assays were used to test the cytotoxic and anti-proliferative effect of JT11 in Jurkat cells. Its pro-apoptotic activity was investigated analyzing both cell cycle and poly PARP cleavage. Finally, we evaluated its impact on LPS-induced ERK1/2 and NF-kB-p65 activation, TNF-α, IL-1β, IL-6 and IL-8 release in peripheral blood mononuclear cells (PBMCs) from healthy donors. Selective CB2R antagonist SR144528 and CBR2 knockdown were used to further verify the selectivity of JT11. We confirmed selective CBR2 activation by JT11. JT11 regulated cell viability and proliferation through a CBR2-dependent mechanism in Jurkat cells, exhibiting a mild pro-apoptotic activity. Finally, it reduced LPS-induced ERK1/2 and NF-kB-p65 phosphorylation and pro-inflammatory cytokines release in human PBMCs, proving to possess in vitro anti-inflammatory properties. JT11 as CBR2 ligands could enhance ECS immunoregulatory activity and our results support the view that therapeutic strategies targeting CBR2 signaling could be promising for the treatment of chronic inflammatory diseases.

Copyright of Revista Foco (Interdisciplinary Studies Journal) is the property of Revista Foco and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Endometriosis, a chronic condition affecting around 10–14% of women, is challenging to manage, due to its complex pathogenesis and limited treatment options. Research has suggested a potential role of the gut microbiota and the endocannabinoid system in the development and progression of endometriosis. This narrative review aims to explore the role of, and any potential interactions between, the endocannabinoid system (ECS) and the gut microbiota in endometriosis. This review found that both the ECS and microbiota influence endometriosis, with the former regulating inflammation and pain perception and the latter influencing immune responses and hormonal balance. There is evidence that a dysregulation of the endocannabinoid system and the gut microbiota influence endometriosis symptoms and progression via changes in CB1 receptor expression and increased circulating levels of endocannabinoids. Microbial imbalances in the gut, such as increases in Prevotella, have been directly correlated to increased bloating, a common endometriosis symptom, while increases in E. coli have supported the bacterial contamination hypothesis as a potential pathway for endometriosis pathogenesis. These microbial imbalances have been correlated with increases in inflammatory markers such as TNF-α and IL-6, both often raised in those with endometriosis. Protective effects of the ECS on the gut were observed by increases in endocannabinoids, including 2-AG, resulting in decreased inflammation and improved gut permeability. Given these findings, both the ECS and the gut microbiota may be targets for therapeutic interventions for endometriosis; however, clinical studies are required to determine effectiveness. [ABSTRACT FROM AUTHOR]

Introduction: Over 1 billion humans carry infectious helminth parasites that can lead to chronic comorbidities such as anemia and growth retardation in children. Helminths induce a T-helper type 2 (Th2) immune response in the host and can cause severe tissue damage and fibrosis if chronic. We recently reported that mice infected with the soil-transmitted helminth, Nippostrongylus brasiliensis, displayed elevated levels of endocannabinoids (eCBs) in the lung and intestine. eCBs are lipid-signaling molecules that control inflammation; however, their function in infection is not well defined. Materials and Methods: A combination of pharmacological approaches and genetic mouse models was used to investigate roles for the eCB system in inflammatory responses and lung injury in mice during parasitic infection with N. brasiliensis. Results: Hemorrhaging of lung tissue in mice infected with N. brasiliensis was exacerbated by inhibiting peripheral cannabinoid receptor subtype-1 (CB1Rs) with the peripherally restricted CB1R antagonist, AM6545. In addition, these mice exhibited an increase in nonfunctional alveolar space and prolonged airway eosinophilia compared to vehicle-treated infected mice. In contrast to mice treated with AM6545, infected cannabinoid receptor subtype-2-null mice (Cnr2-/-) did not display any changes in these parameters compared to wild-type mice. Conclusions: Roles for the eCB system in Th2 immune responses are not well understood; however, increases in its activity in response to infection suggest an immunomodulatory role. Moreover, these findings suggest a role for eCB signaling at CB1Rs but not cannabinoid receptor subtypes-2 in the resolution of Th2 inflammatory responses, which become host destructive over time.

Abstract Introduction The cannabinoid receptor 2 (CB2) is mainly involved in the immune system. However, although CB2 has been reported to play an anti‐tumor function in breast cancer (BC), its specific mechanism in BC remains unclear. Methods We examined the expression and prognostic significance of CB2 in BC tissues by qPCR, second‐generation sequencing, western blot, and immunohistochemistry. We assessed the impacts of overexpression and a specific agonist of CB2 on the growth, proliferation, apoptosis, and drug resistance of BC cells in vitro and in vivo using CCK‐8, flow cytometry, TUNEL staining, immunofluorescence, tumor xenografts, western blot, and colony formation assays. Results CB2 expression was significantly lower in BC compared with paracancerous tissues. It was also highly expressed in benign tumors and ductal carcinoma in situ, and its expression was correlated with prognosis in BC patients. CB2 overexpression and treatment of BC cells with a CB2 agonist inhibited proliferation and promoted apoptosis, and these actions were achieved by suppressing the PI3K/Akt/mTOR signaling pathway. Moreover, CB2 expression was increased in MDA‐MB‐231 cell treated with cisplatin, doxorubicin, and docetaxel, and sensitivity to these anti‐tumor drugs was increased in BC cells overexpressing CB2. Conclusions These findings reveal that CB2 mediates BC via the PI3K/Akt/mTOR signaling pathway. CB2 could be a novel target for the diagnosis and treatment of BC.

Across many species, endocannabinoids play an important role in regulating social play, reward, and anxiety. These processes are mediated through at least two distinct cannabinoid receptors (CB), CB1 and CB2. CB1 expression is found in appreciable densities across regions of the brain that integrate memory with socio-spatial information; many of these regions have been directly linked to the neurobiology of pair bonding in monogamous species. Using receptor autoradiography, we provide the first distributional map of CB1 within the brains of closely related monogamous prairie voles and promiscuous meadow voles, and compare receptor densities across sexes and species in limbic regions. We observe CB1-specific signal using [3H] CP-55,940 and [3H] SR141716A, though the latter exhibited a lower signal to noise ratio. We confirmed the presence of CB2 in prairie vole spleen tissue using [3H] CP-55,940. However, we found no evidence of CB2 in the brain using either [3H] CP-55,940 or [3H] A-836,339. The overall distribution of putative CB1 in the brain was similar across vole species and followed the pattern of CB1 expression observed in other species-high intensity binding within the telencephalon, moderate binding within the diencephalon, and mild binding within the mesencephalon and metencephalon (aside from the cerebellar cortex). However, we found profound differences in CB1 densities across species, with prairie voles having higher CB1 binding in regions implicated in social attachment and spatial memory (e.g., periaqueductal gray, hippocampus). These findings suggest that CB1 densities, but not distribution, correlate with the social systems of vole species.

Copyright of Revista Foco (Interdisciplinary Studies Journal) is the property of Revista Foco and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The endocannabinoid system is a key regulator of the response to psychological stress. Inhibitors of monoacylglycerol lipase (MGL), the enzyme that deactivates the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG), exert anxiolytic-like effects in rodent models via 2-AG-dependent activation of CB1 cannabinoid receptors. In the present study, we examined whether the MGL inhibitor JZL184 might modulate persistent predator-induced fear in rats, a model that captures features of human post-traumatic stress disorder. Exposure to 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), a volatile chemical that is innately aversive to some rodent species, produced in male rats a long-lasting anxiety-like state that was measured 7 days later in the elevated plus maze test. Systemic administration of JZL184 [4, 8 and 16 mg/kg, intraperitoneal (IP)] 4 h before testing caused dose-dependent inhibition of MGL activity and elevation of 2-AG content in brain tissue. Concomitantly, the inhibitor suppressed TMT-induced fear behaviors with a median effective dose (ED50) of 4 mg/kg. A similar behavioral response was observed with another MGL inhibitor, KML29 (4 and 16 mg/kg, IP). Surprisingly, the effect of JZL184 was prevented by co-administration of the CB2 inverse agonist AM630 (5 mg/kg, IP), but not the CB1 inverse agonist rimonabant (1 mg/kg, IP). Supporting mediation of the response by CB2 receptors, the CB2 agonist JWH133 (0.3, 1 and 3 mg/kg, IP) also produced anxiolytic-like effects in TMT-stressed rats, which were suppressed by AM630. Notably, (i) JWH133 was behaviorally ineffective in animals that had no prior experience with TMT; and (ii) CB2 mRNA levels in rat prefrontal cortex were elevated 7 days after exposure to the aversive odorant. The results suggest that JZL184 attenuates the behavioral consequences of predator stress through a mechanism that requires 2-AG-mediated activation of CB2 receptors, whose transcription may be induced by the stress itself.

The number of functions controlled by the endocannabinoid system in health and disease continues growing over the years. In the brain, these include the modulation of harmful events such as glutamate excitotoxicity, oxidative stress, and inflammation, mainly regulated by activation/blockade of CB1/CB2 cannabinoid receptors. In the present work, we evaluated the capacity of the CB1 antagonist/CB2 agonist synthetic cannabinoid URB447 on reducing neurodegeneration after brain injury. By using a model of hypoxia-ischemia (HI) in neonatal rats, we found that URB447 strongly reduced brain injury when administered before HI. A comparable effect was observed with the CB1 antagonist SR141716A, whereas the CB1 agonist WIN-55,212-2 reduced the effect of URB447. When administered 3 h after HI, which is considered a clinically feasible therapeutic window to treat perinatal brain injury in humans, URB447 reduced neurodegeneration and white matter damage. Markers of astrogliosis and microglial activation also appeared reduced. These results confirm the important role played by the endocannabinoid system in the neurodegenerative process and strongly encourage further research into the mechanisms of URB447-induced neuroprotection.

Cannabinoid ligands are known to possess neuroprotective actions and may have utility in the treatment of neurodegeneration. The major targets for cannabinoids include the classical CB1 cannabinoid receptor, as well as the novel cannabinoid receptor GPR55 which binds to many synthetic cannabinoid ligands. In this study, novel thiopyranothiazoles 1, 3, 4, 6, and 7 were synthesized and their pharmacological activity as potential cannabinoid-like ligands was evaluated in glioblastoma cells, cultured cortical neurons, and cells of HEK293 line expressing GPR55. Stimulation of protein kinase ERK1/2, MAP-kinases and cAMP response element binding protein (CREB) was evaluated using Western-blot analysis. CREB activation was additionally monitored by means of confocal imaging of nuclear phospho-CREB labeling. Docking simulation confirmed the good affinity of the synthesized compounds to CB1 and CB2 receptors. Striking effects of the chromeno[4′,3′:4,5]thiopyrano[2,3-d][1,3]thiazol with ethylacetate moiety (3) and isothiochromeno[4a,4-d]thiazole with phenazone fragment (7) on pCREB activation as the indicator of stimulation of the pathway beneficial for neurons survival were observed.

Introducción: los cannabinoides son derivados de la planta de cannabis, y han captado la atención por sus efectos terapéuticos demostrados como antiinflamatorios, analgésicos, antieméticos y antitumorales. Su potencial radica en su interacción con los receptores endocannabinoides CB1 y CB2, confirmada por estudios in vitro e in vivo. Por ello, resulta interesante estudiar dicha interacción proteína-ligando mediante técnicas de acoplamiento molecular. Estos permiten describir las zonas de los cannabinoides responsables de su actividad biológica, posibilitando que estos compuestos den respuesta a patologías relacionadas con el sistema inmune. Objetivo: estudiar las interacciones proteína-ligando de 30 cannabinoides análogos del receptor CBN II. Métodos: el grupo de moléculas estudiadas en esta investigación incluyó cannabinoides endógenos, fitocannabinoides y cannabinoides sintéticos, con 30 análogos extraídos de PubChem. Se seleccionó la estructura 3D del receptor CB2 de Protein Data Bank con código 5ZTY. Se optimizó el receptor y ligandos para el acoplamiento molecular, validado con un RMSD de 1.76. Resultados: los ligandos SR141716, AM251 y JZL184 mostraron mejor afinidad por CB2. La fenilalanina fue el aminoácido más presente en la unión, con interacciones alquil y pi-alquil, sugiriendo la importancia de anillos aromáticos. Esto concuerda con otros estudios que indican que los grupos cetona en posición 3 del indol y grupos hidroxilo/metoxilo en posición 6 influyen en la afinidad de unión a CB2 mediante donación de hidrógeno al carbonilo del ligando. Conclusión: los análogos SR141716, AM251 y JZL184, relacionados con el THC y el CBD, se perfilan como moléculas prometedoras para evaluación experimental debido a su alta afinidad por el receptor CB2. Estos compuestos podrían presentar efectos beneficiosos mediados por CB2 en el tratamiento del dolor, inflamación, cáncer y trastornos inmunológicos.

Introduction: The cannabinoid receptor 2 (CB2) is mainly involved in the immune system. However, although CB2 has been reported to play an anti‐tumor function in breast cancer (BC), its specific mechanism in BC remains unclear. Methods: We examined the expression and prognostic significance of CB2 in BC tissues by qPCR, second‐generation sequencing, western blot, and immunohistochemistry. We assessed the impacts of overexpression and a specific agonist of CB2 on the growth, proliferation, apoptosis, and drug resistance of BC cells in vitro and in vivo using CCK‐8, flow cytometry, TUNEL staining, immunofluorescence, tumor xenografts, western blot, and colony formation assays. Results: CB2 expression was significantly lower in BC compared with paracancerous tissues. It was also highly expressed in benign tumors and ductal carcinoma in situ, and its expression was correlated with prognosis in BC patients. CB2 overexpression and treatment of BC cells with a CB2 agonist inhibited proliferation and promoted apoptosis, and these actions were achieved by suppressing the PI3K/Akt/mTOR signaling pathway. Moreover, CB2 expression was increased in MDA‐MB‐231 cell treated with cisplatin, doxorubicin, and docetaxel, and sensitivity to these anti‐tumor drugs was increased in BC cells overexpressing CB2. Conclusions: These findings reveal that CB2 mediates BC via the PI3K/Akt/mTOR signaling pathway. CB2 could be a novel target for the diagnosis and treatment of BC. [ABSTRACT FROM AUTHOR]

Simple Summary: Alzheimer's disease (AD) is a complex, multifactorial disease where numerous components, such as environment, lifestyle, comorbidities, and genetic predisposition, contribute to triggering the onset of the disease. Several neurobiological brain alterations have been reported during AD pathologies, including the endocannabinoid system (ECS) and associated lipid transmitter-based signaling systems. In this study, we have evaluated the expression levels of the cannabinoid receptors type 2 (CB2) and the novel cannabinoid/lysophospholipid G protein-coupled receptor 55 (GPR55) at different stages of AD. We deeply investigated CB2 and GPR55's close proximity with Aβ-plaque deposits, as well as neuronal and glial cells, in the AD AppNL-G-F knock-in mouse model. Additionally, we analyzed whether Aβ42 directly affects CB2 and GPR55 protein expression in neuronal and glial primary cell cultures. Our study shows that the ECS, specifically the CB2 and GPR55 receptors, are altered during AD pathology. Monitoring these receptors may provide new biomarkers for AD diagnosis. CB2 and GPR55 could be potential pharmacological targets for selective compounds to treat AD inflammation. Background: The endocannabinoid system (ECS) and associated lipid transmitter-based signaling systems play an important role in modulating brain neuroinflammation. ECS is affected in neurodegenerative disorders, such as Alzheimer's disease (AD). Here we have evaluated the non-psychotropic endocannabinoid receptor type 2 (CB2) and lysophosphatidylinositol G-protein-coupled receptor 55 (GPR55) localization and expression during Aβ-pathology progression. Methods: Hippocampal gene expression of CB2 and GPR55 was explored by qPCR analysis, and brain distribution was evaluated by immunofluorescence in the wild type (WT) and APP knock-in AppNL-G-F AD mouse model. Furthermore, the effects of Aβ42 on CB2 and GPR55 expression were assessed in primary cell cultures. Results: CB2 and GPR55 mRNA levels were significantly upregulated in AppNL-G-F mice at 6 and 12 months of age, compared to WT. CB2 was highly expressed in the microglia and astrocytes surrounding the Aβ plaques. Differently, GPR55 staining was mainly detected in neurons and microglia but not in astrocytes. In vitro, Aβ42 treatment enhanced CB2 receptor expression mainly in astrocytes and microglia cells, whereas GPR55 expression was enhanced primarily in neurons. Conclusions: These data show that Aβ pathology progression, particularly Aβ42, plays a crucial role in increasing the expression of CB2 and GPR55 receptors, supporting CB2 and GPR55 implications in AD. [ABSTRACT FROM AUTHOR]

Machaeriols and machaeridiols are unique hexahydrodibenzopyran-type aralkyl phytocannabinoids isolated from Machaerium Pers. Earlier studies of machaeriol A (1) and B (2) did not show any affinity for cannabinoid receptor 1 (CB1 or CNR1), although they are structural analogs of psychoactive hexahydrocannabinol. This study comprehensively reports on the affinities of isolated Machaerium Pers. compounds, namely machaeriol A–D (1–4) and machaeridiol A–C (5–7), against cannabinoid (CB1 and CB2) and opioid (κ, δ and µ) receptors. Among the isolated compounds, machaeriol D (4) and machaeridiol A–C (5–7) showed some selective binding affinity for the CB2 receptor, using a radioligand binding assay, with Ki values of >1.3, >1.77, >2.18 and >1.1 μM, respectively. On the other hand, none of the compounds showed any binding to the CB1 receptor. Due to recent reports on the anticancer potential of the endocannabinoid system, compounds 1–7 were tested against a battery of luciferase reporter gene vectors that assess the activity of many cancer-related signaling pathways, including Stat3, Smad2/3, AP-1, NF-κB, E2F, Myc, Ets, Notch, FoxO, Wnt, Hedgehog and pTK in HeLa and T98G glioblastoma cells. Complete dose–response curves have been determined for each compound in both of these cell lines, which revealed that machaeridiol 6 displayed activities (IC50 in µM in HeLa and T98G cells) towards Stat3 (4.7, 1.4), Smad2/3 (1.2, 3.0), AP-1 (5.9, 4.2), NF-κB (0.5, 4.0), E2F (5.7, 0.7), Myc (5.3, 2.0), ETS (inactive, 5.9), Notch (5.3, 4.6), Wnt (4.2, inactive) and Hedgehog (inactive, 5.0). Furthermore, a combination study between machaeriol C (3) and machaeridiol B (6) displayed additive effects for E2F, ETS, Wnt and Hedgehog pathways, where these compounds individually were either minimally active or inactive. None of the compounds inhibited luciferase expression driven by the minimal thymidine kinase promoter (pTK), indicating the lack of general cytotoxicity for luciferase enzyme inhibition at the 50 µM concentration in both of these cell lines. The significance of the inhibition of these signaling pathways via machaeridiol 5–7 and their cross-talk potential has been discussed. [ABSTRACT FROM AUTHOR]

Early adversity, the loss of the inhibitory GABAergic interneuron parvalbumin, and elevated neuroinflammation are associated with depression. Individuals with a maltreatment history initiate medicinal cannabis use earlier in life than non-maltreated individuals, suggesting self-medication. Female rats underwent maternal separation (MS) between 2 and 20 days of age to model early adversity or served as colony controls. The prelimbic cortex and behavior were examined to determine whether MS alters the cannabinoid receptor 2 (CB2), which has anti-inflammatory properties. A reduction in the CB2-associated regulatory enzyme MARCH7 leading to increased NLRP3 was observed with Western immunoblots in MS females. Immunohistochemistry with stereology quantified numbers of parvalbumin-immunoreactive cells and CB2 at 25, 40, and 100 days of age, revealing that the CB2 receptor associated with PV neurons initially increases at P25 and subsequently decreases by P40 in MS animals, with no change in controls. Confocal and triple-label microscopy suggest colocalization of these CB2 receptors to microglia wrapped around the parvalbumin neuron. Depressive-like behavior in MS animals was elevated at P40 and reduced with the CB2 agonist HU-308 or a CB2-overexpressing lentivirus microinjected into the prelimbic cortex. These results suggest that increasing CB2 expression by P40 in the prelimbic cortex prevents depressive behavior in MS female rats.

BACKGROUND AND AIMS: The gut immune, cannabinoid, and opioid systems constitute an integrated network contributing to visceral sensation and pain modulation. We aimed to assess the expression of the µ-opioid receptor (MOR), its ligand β-endorphin (β-END), and cannabinoid receptor-2 (CB2 ) in patients with irritable bowel syndrome (IBS) and asymptomatic controls (AC) and their correlation with sex and symptom perception. METHODS: Mucosal biopsies were obtained from the left colon of 31 IBS patients (45% women) with predominant constipation (IBS-C, 9) or diarrhea (IBS-D, 10) or with mixed bowel habits (IBS-M, 12) and 32 AC (44% women) and processed for qRT-PCR, Western blotting, and immunohistochemistry. KEY RESULTS: µ-opioid receptor and CB2 mRNA and protein expression and β-END protein levels were increased in patients with IBS compared to AC (all Ps=0.021). A significant sex by IBS interaction was found in relation to CB2 mRNA expression (P = .003) with women showing a markedly higher expression to men (P = .035). In contrast, in AC, men had higher expression than women (P = .033). β-END, MOR, and CB2 immunoreactivities (IR) were localized to CD4+T cells including EMR-1+ eosinophils and CD31+ T cells but not to mast cells. CONCLUSIONS: The increased expression of MOR, β-END, and CB2 in the mucosa of IBS patients, where they are localized to immune cells, suggests that opioid and cannabinoid systems play an immune-related compensatory role in visceral pain in IBS patients. Further work is necessary to support this hypothesis.

Abstract Background As effective immune modulators, Endocannabinoids may suppress the inflammatory responses in periodontitis. This study assessed the expression of cannabinoid receptors in gingiva and the impact on periodontitis. Methods A cross‐sectional study on 20 patients with more than stage II and Grade A periodontitis and a control group consisting of 19 healthy individuals was performed. The gingival biopsies were assessed for the expression of CB1 and CB2 using the quantitative reverse transcription polymerase chain reaction, TaqMan method. Results The study sample consisted of 39 subjects, 31 females (79.5%) and 8 males (20.5%), including 20 periodontitis subjects (80% female and 20% male), and control groups (78.9% female and 21.1% male). The mean ages of cases and controls were 33.3 ± 4.7 and 35.7 ± 5.1 years, respectively. The gene expression of CB2 in periodontitis was 27.62 ± 7.96 and in healthy subjects was 78.15 ± 23.07. The CB2 was significantly lower than the control group (p = .008). In comparison, the gene expression index of CB1 in the periodontal group (9.42 ± 3.03) was higher than the control group (6.62 ± 1.13) but did not meet a significant value (p = .671). Conclusion The lower expression of CB2 receptors in the periodontitis group may be due to the reduced protective effect of anti‐inflammatory agents. These elements include cannabinoids and the imbalance leading to the predominance of pro‐inflammatory effects. Therefore, the local effects of cannabinoids as an immunomodulator could be useful for oral inflammatory diseases such as periodontitis.

ABSTRACTBackground Preclinical and clinical evidence suggests that cannabis has potential analgesic properties. However, cannabinoid receptor expression and localization within spinal cord pain processing circuits remain to be characterized across sex and species.Aims We aimed to investigate the differential expression of the cannabinoid type 1 (CB1) receptor across dorsal horn laminae and cell populations in male and female adult rats and humans.Methods To investigate and quantify CB1 receptor expression in the spinal dorsal horn across species, we refined immunohistochemical procedures for successful rat and human fixed tissue staining and confocal imaging. Immunohistochemical results were complemented with analysis of CB1 gene (CNR1) expression within rodent and human dorsal horn using single-cell/nuclei RNA sequencing data sets.Results We found that CB1 was preferentially localized to the neuropil within the superficial dorsal horn of both rats and humans, with CB1 somatic staining across dorsal horn laminae. CB1 receptor immunoreactivity was significantly higher in the superficial dorsal horn compared to the deeper dorsal horn laminae for both rats and humans, which was conserved across sex. Interestingly, we found that CB1 immunoreactivity was not primarily localized to peptidergic afferents in rats and humans and that CNR1 (CB1) but not CNR2 (CB2) was robustly expressed in dorsal horn neuron subpopulations of both rodents and humans.Conclusions The conserved preferential expression of CB1 receptors in the superficial dorsal horn in male and female rodents and humans has significant implications for understanding the roles of this cannabinoid receptor in spinal mechanisms of nociception and analgesia.

Cannabinoid Receptor 2 (CB2) is a promising target for treating inflammatory diseases. We designed derivatives of 3-carbamoyl-2-pyridone and 1,8-naphthyridin-2(1H)-one-3-carboxamide CB2-selective agonists with reduced lipophilicity. The new compounds were measured for their affinity (radioligand binding) and ability to elicit cyclic adenosine monophosphate (cAMP) signalling and β-arrestin-2 translocation with temporal resolution (BRET-based biosensors). For the 3-carbamoyl-2-pyridone derivatives, we found that modifying the previously reported compound UOSS77 (also known as S-777469) by appending a PEG2-alcohol via a 3-carbomylcyclohexyl carboxamide (UOSS75) lowered lipophilicity, and preserved binding affinity and signalling profile. The 1,8-naphthyridin-2(1H)-one-3-carboxamide UOMM18, containing a cis configuration at the 3-carboxamide cyclohexyl and with an alcohol on the 4-position of the cyclohexyl, had lower lipophilicity but similar CB2 affinity and biological activity to previously reported compounds of this class. Relative to CP55,940, the new compounds acted as partial agonists and did not exhibit signalling bias. Interestingly, while all compounds shared similar temporal trajectories for maximal efficacy, differing temporal trajectories for potency were observed. Consequently, when applied at sub-maximal concentrations, CP55,940 tended to elicit sustained (cAMP) or increasing (arrestin) responses, whereas responses to the new compounds tended to be transient (cAMP) or sustained (arrestin). In future studies, the compounds characterised here may be useful in elucidating the consequences of differential temporal signalling profiles on CB2-mediated physiological responses. [ABSTRACT FROM AUTHOR]

Mast cells (MCs) are the main participants in the control of immune reactions associated with inflammation, allergies, defense against pathogens, and tumor growth. Bioactive lipids are lipophilic compounds able to modulate MC activation. Here, we explored some of the effects of the bioactive lipid lysophosphatidylinositol (LPI) on MCs. Utilizing murine bone marrow-derived mast cells (BMMCs), we found that LPI did not cause degranulation, but slightly increased FcεRI-dependent β-hexosaminidase release. However, LPI induced strong chemotaxis together with changes in LIM kinase (LIMK) and cofilin phosphorylation. LPI also promoted modifications to actin cytoskeleton dynamics that were detected by an increase in cell size and interruptions in the continuity of the cortical actin ring. The chemotaxis and cortical actin ring changes were dependent on GPR55 receptor activation, since the specific agonist O1602 mimicked the effects of LPI and the selective antagonist ML193 prevented them. The LPI and O1602-dependent stimulation of BMMC also led to VEGF, TNF, IL-1α, and IL-1β mRNA accumulation, but, in contrast with chemotaxis-related processes, the effects on cytokine transcription were dependent on GPR55 and cannabinoid (CB) 2 receptors, since they were sensitive to ML193 and to the specific CB2 receptor antagonist AM630. Remarkably, GPR55-dependent BMMC chemotaxis was observed towards conditioned media from distinct mouse and human cancer cells. Our data suggest that LPI induces the chemotaxis of MCs and leads to cytokine production in MC in vitro with the differential participation of GPR55 and CB2 receptors. These effects could play a significant role in the recruitment of MCs to tumors and the production of MC-derived pro-angiogenic factors in the tumor microenvironment. [ABSTRACT FROM AUTHOR]

Objective: The aim of the present study was to assess if the endocannabinoid system is involved differently in patients with recurrent and non-recurrent periodontal disease and if in sites that have a predisposition for reactivation, levels of anandamide (AEA) change after periodontal therapy. Background: Periodontal disease (PD) may be due to a dysregulation of the endocannabinoid system. Methods: Periodontal patients were recruited, treated for PD and monitored. Gingival samples from these patients with recurrent (n = 10) and non-recurrent (n = 10) periodontal disease were harvested before and after treatment and compared to those of periodontally healthy (n = 10) subjects. Levels of CB1 and CB2, AEA and CBs receptor activation were assessed in healthy and inflamed samples using immunohistochemistry, chromatography and autoradiography. In healed sites, AEA levels were also assessed. Results: The number of CBs in inflamed sites of recurrent patients was significantly higher than in those with non-recurrent disease and also higher than those in healthy subjects. Inflamed sites of recurrent patients had significantly lower CBs receptor activation than those of healthy subjects. Levels of AEA in inflamed sites of non-recurrent patients were significantly higher than those found both in inflamed recurrent sites and in healthy sites. Otherwise, the amount of AEA in healthy subjects and recurrent inflamed sites was similar. After periodontal therapy, levels of AEA were significantly lower in both periodontal groups. In recurrent sites, they resulted significantly lower than in non-recurrent and even in healthy subjects. Conclusions: The endocannabinoid system seems involved differently in subjects with recurrent and non-recurrent periodontal disease. [ABSTRACT FROM AUTHOR]

GPR55 is a non-canonical cannabinoid receptor, important for cancer proliferation. Depending on the ligand, it induces either cell proliferation or death. The objective of the study was to establish the mechanisms of this multidirectional signaling. Using the CRISPR-Cas9 system, the GPR55, CB1, CB2, and GPR18 receptor knockouts of the MDA-MB-231 line were obtained. After the CB2 receptor knockout, the pro-apoptotic activity of the pro-apoptotic ligand docosahexaenoyl dopamine (DHA-DA) slightly increased, while the pro-proliferative activity of the most active synthetic ligand of the GPR55 receptor (ML-184) completely disappeared. On the original cell line, the stimulatory effect of ML-184 was removed by the CB2 receptor blocker and by GPR55 receptor knockout. Thus, it can be confidently assumed that when proliferation is stimulated with the participation of the GPR55 receptor, a signal is transmitted from the CB2 receptor to the GPR55 receptor due to the formation of a heterodimer. GPR18 was additionally involved in the implementation of the pro-apoptotic effect of DHA-DA, while the CB1 receptor is not involved. In the implementation of the pro-apoptotic action of DHA-DA, the elimination of Gα13 led to a decrease in cytotoxicity. The obtained data provide novel details to the mechanism of the pro-proliferative action of GPR55. [ABSTRACT FROM AUTHOR]

Abstract Zebrafish is considered an unprecedented animal model in drug discovery. A review of the literature presents highlights and elucidates the biological effects of chemical components found in Cannabis sativa. Particular attention is paid to endocannabinoid system (eCB) and its main receptors (CB1 and CB2). The zebrafish model is a promising one for the study of cannabinoids because of the many similarities to the human system. Despite the recent advances on the eCB system, there is still the need to elucidate some of the interactions and, thus, the zebrafish model can be used for that purpose as it respects the 3Rs concept and reduced time and costs. In view of the relevance of cannabinoids in the treatment and prevention of diseases, as well as the importance of the zebrafish animal model in elucidating the biological effects of new drugs, the aim of this study was to bring to light information on the use of the zebrafish animal model in testing C. sativa-based medicines.

Chemotherapy-induced peripheral neuropathy (CIPN) is a severe and dose-limiting side effect of cancer treatment that affects millions of cancer survivors throughout the world and current treatment options are extremely limited by their side effects. Cannabinoids are highly effective in suppressing pain symptoms of chemotherapy-induced and other peripheral neuropathies but their widespread use is limited by central nervous system (CNS)-mediated side effects. Here, we tested one compound from a series of recently developed synthetic peripherally restricted cannabinoids (PRCBs) in a rat model of cisplatin-induced peripheral neuropathy. Results show that local or systemic administration of 4-{2-[-(1E)-1[(4-propylnaphthalen-1-yl)methylidene]-1H-inden-3-yl]ethyl}morpholine (PrNMI) dose-dependently suppressed CIPN mechanical and cold allodynia. Orally administered PrNMI also dose-dependently suppressed CIPN allodynia symptoms in both male and female rats without any CNS side effects. Co-administration with selective cannabinoid receptor subtype blockers revealed that PrNMI's anti-allodynic effects are mediated by CB1 receptor (CB1R) activation. Expression of CB2Rs was reduced in dorsal root ganglia from CIPN rats, whereas expression of CB1Rs and various endocannabinoid synthesizing and metabolizing enzymes was unaffected. Daily PrNMI treatment of CIPN rats for two weeks showed a lack of appreciable tolerance to PrNMI's anti-allodynic effects. In an operant task which reflects cerebral processing of pain, PrNMI also dose-dependently suppressed CIPN pain behaviors. Our results demonstrate that PRCBs exemplified by PrNMI may represent a viable option for the treatment of CIPN pain symptoms.

Background: Alcohol use disorder (AUD) commonly occurs in patients with chronic pain, and a major barrier to achieving abstinence and preventing relapse is the emergence of hyperalgesia during alcohol withdrawal. Elucidating novel therapeutic approaches to target hyperalgesia associated with alcohol withdrawal could have important implications for treating AUD. Here, we examined the role of 2‐arachidonoylglycerol (2‐AG)‐mediated endocannabinoid (eCB) signaling in the regulation of hyperalgesia associated with alcohol withdrawal in mice. We tested the hypothesis that pharmacological augmentation of 2‐AG signaling could reduce hyperalgesia during withdrawal. Methods: Male and female C57BL/6J mice were tested during withdrawal from a continuous access two‐bottle choice (2BC) paradigm to investigate how eCB signaling modulates mechanical and thermal sensitivity during withdrawal. Mice were pretreated with the monoacylglycerol lipase (MAGL) inhibitor JZL184 to elevate levels of 2‐AG. Rimonabant or AM630 were given to block CB1 and CB2 receptor activity, respectively. DO34 was given to reduce 2‐AG by inhibiting the 2‐AG synthetic enzyme diacylglycerol lipase (DAGL). Results: After 72 h of withdrawal, male and female mice exhibited increased mechanical, but not thermal, hypersensitivity, which normalized by 7 days. This effect was reversed by pretreatment with JZL184. The effects of JZL184 were prevented by coadministration of either the CB1 or the CB2 antagonist. DO34, Rimonabant, and AM630 exacerbated mechanical hypersensitivity during alcohol withdrawal, causing an earlier onset and persistent hypersensitivity even 1 week into withdrawal. Conclusions: Our findings demonstrate the critical role of 2‐AG signaling in the bidirectional regulation of mechanical sensitivity during alcohol withdrawal, with enhancement of 2‐AG levels reducing sensitivity, and inhibition of 2‐AG signaling exacerbating sensitivity. These data suggest that 2‐AG augmentation represents a novel approach to the treatment of alcohol withdrawal‐associated hyperalgesia and AUD in patients with comorbid pain disorders. [ABSTRACT FROM AUTHOR]

Cannabinoid (CB) receptors (CB1 and CB2) are expressed on cancer cells and their expression influences carcinogenesis in various tumor entities. Cells of the tumor microenvironment (TME) also express CB receptors, however, their role in tumor development is still unclear. We, therefore, investigated the role of TME-derived CB1 and CB2 receptors in a model of non-small cell lung cancer (NSCLC). Leukocytes in the TME of mouse and human NSCLC express CB receptors, with CB2 showing higher expression than CB1. In the tumor model, using CB1- (CB1-/-) and CB2-knockout (CB2-/-) mice, only deficiency of CB2, but not of CB1, resulted in reduction of tumor burden vs. wild type (WT) littermates. This was accompanied by increased accumulation and tumoricidal activity of CD8+ T and natural killer cells, as well as increased expression of programmed death-1 (PD-1) and its ligand on lymphoid and myeloid cells, respectively. CB2-/- mice responded significantly better to anti-PD-1 therapy than WT mice. The treatment further increased infiltration of cytotoxic lymphocytes into the TME of CB2-/- mice. Our findings demonstrate that TME-derived CB2 dictates the immune cell recruitment into tumors and the responsiveness to anti-PD-1 therapy in a model of NSCLC. CB2 could serve as an adjuvant target for immunotherapy.

Cannabinoid signaling, mainly via CB1 and CB2 receptors, plays an essential role in oligodendrocyte health and functions. However, the specific molecular signals associated with the activation or blockade of CB1 and CB2 receptors in this glial cell have yet to be elucidated. Mass spectrometry-based shotgun proteomics and in silico biology tools were used to determine which signaling pathways and molecular mechanisms are triggered in a human oligodendrocytic cell line (MO3.13) by several pharmacological stimuli: the phytocannabinoid cannabidiol (CBD); CB1 and CB2 agonists ACEA, HU308, and WIN55, 212–2; CB1 and CB2 antagonists AM251 and AM630; and endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG). The modulation of cannabinoid signaling in MO3.13 was found to affect pathways linked to cell proliferation, migration, and differentiation of oligodendrocyte progenitor cells. Additionally, we found that carbohydrate and lipid metabolism, as well as mitochondrial function, were modulated by these compounds. Comparing the proteome changes and upstream regulators among treatments, the highest overlap was between the CB1 and CB2 antagonists, followed by overlaps between AEA and 2-AG. Our study opens new windows of opportunities, suggesting that cannabinoid signaling in oligodendrocytes might be relevant in the context of demyelinating and neurodegenerative diseases. Proteomics data are available at ProteomeXchange (PXD031923). [ABSTRACT FROM AUTHOR]

Background: As effective immune modulators, Endocannabinoids may suppress the inflammatory responses in periodontitis. This study assessed the expression of cannabinoid receptors in gingiva and the impact on periodontitis. Methods: A cross‐sectional study on 20 patients with more than stage II and Grade A periodontitis and a control group consisting of 19 healthy individuals was performed. The gingival biopsies were assessed for the expression of CB1 and CB2 using the quantitative reverse transcription polymerase chain reaction, TaqMan method. Results: The study sample consisted of 39 subjects, 31 females (79.5%) and 8 males (20.5%), including 20 periodontitis subjects (80% female and 20% male), and control groups (78.9% female and 21.1% male). The mean ages of cases and controls were 33.3 ± 4.7 and 35.7 ± 5.1 years, respectively. The gene expression of CB2 in periodontitis was 27.62 ± 7.96 and in healthy subjects was 78.15 ± 23.07. The CB2 was significantly lower than the control group (p =.008). In comparison, the gene expression index of CB1 in the periodontal group (9.42 ± 3.03) was higher than the control group (6.62 ± 1.13) but did not meet a significant value (p =.671). Conclusion: The lower expression of CB2 receptors in the periodontitis group may be due to the reduced protective effect of anti‐inflammatory agents. These elements include cannabinoids and the imbalance leading to the predominance of pro‐inflammatory effects. Therefore, the local effects of cannabinoids as an immunomodulator could be useful for oral inflammatory diseases such as periodontitis. [ABSTRACT FROM AUTHOR]

Selective modulation of peripheral cannabinoid receptors (CBRs) has potential therapeutic applications in medical conditions, including obesity, diabetes, liver diseases, GI disorders and pain. While there have been considerable efforts to produce selective antagonists or full agonists of CBRs, there has been limited reports on the development of partial agonists. Partial agonists targeting peripheral CBRs may have desirable pharmacological profiles while not producing centrally mediated dissociative effects. Bayer reported that BAY 59-3074 is a CNS penetrant partial agonist of both CB1 and CB2 receptors with efficacy in rat models of neuropathic and inflammatory pain. In this report, we demonstrate our efforts to synthesize analogs that would favor peripheral selectivity, while maintaining partial agonism of CB1. Our efforts led to the identification of a novel compound, which is a partial agonist of the human CB1 (hCB1) receptor with vastly diminished brain exposure compared to BAY 59-3074. [ABSTRACT FROM AUTHOR]

Cannabinoid Receptor 2 (CB2) is a G protein-coupled receptor (GPCR) with considerable, though as yet unrealised, therapeutic potential. Promising preclinical data supports the applicability of CB2 activation in autoimmune and inflammatory diseases, pain, neurodegeneration, and osteoporosis. A diverse pharmacopoeia of cannabinoid ligands is available, which has led to considerable advancements in the understanding of CB2 function and extensive preclinical evaluation. However, until recently, most CB2 ligands were highly lipophilic and as such not optimal for clinical application due to unfavourable physicochemical properties. A number of strategies have been applied to develop CB2 ligands to achieve closer to 'drug-like' properties and a few such compounds have now undergone clinical trial. We review the current state of CB2 ligand development and progress in optimising physicochemical properties, understanding advanced molecular pharmacology such as functional selectivity, and clinical evaluation of CB2-targeting compounds. Cannabinoid Receptor 2 (CB2) is a promising therapeutic target, particularly for inflammatory disorders and pain; however, clinical trials to date have been unsuccessful. Medicinal chemistry efforts have produced selective ligands with a wide range of core scaffolds. Optimisation for drug-like properties and oral administration has been demonstrated to be feasible, but few such compounds have been tested clinically to date. Recent crystal and cryo-EM structures are expected to facilitate rational ligand development and further optimisation. Although CB2 ligands can produce functional selectivity (bias) of signalling responses, few new classes of ligand have been studied in this context. The physiological relevance of CB2 signalling bias, and subcellular spatial organisation of CB2 signalling, remain to be elucidated. [ABSTRACT FROM AUTHOR]

Cannabinoid receptor type 2 (CB2) is the primary receptor pathway mediating the immunologic consequences of cannabinoids. We recently reported that human peripheral blood B cells express CB2 on both the extracellular membrane and at intracellular sites, where-as monocytes and T cells only express intracellular CB2. To better understand the pattern of CB2 expression by human B cells, we examined CD20+ B cells from three tissue sources. Both surface and intracellular expression were present and uniform in cord blood B cells, where all cells exhibited a naïve mature phenotype (IgD+/CD38Dim). While naïve mature and quiescent memory B cells (IgD-/CD38-) from tonsils and peripheral blood exhibited a similar pattern, tonsillar activated B cells (IgD-/CD38+) expressed little to no surface CB2. We hypothesized that regulation of the surface CB2 receptor may occur during B cell activation. Consistent with this, a B cell lymphoma cell line known to exhibit an activated phenotype (SUDHL-4) was found to lack cell surface CB2 but express intracellular CB2. Furthermore, in vitro activation of human cord blood resulted in a down-regulation of surface CB2 on those B cells acquiring the activated phenotype but not on those retaining IgD expression. Using a CB2 expressing cell line (293 T/CB2-GFP), confocal microscopy confirmed the presence of both cell surface expression and multifocal intracellular expression, the latter of which co-localized with endoplasmic reticulum but not with mitochondria, lysosomes, or nucleus. Our findings suggest a dynamic multi-compartment expression pattern for CB2 in B cells that is specifically modulated during the course of B cell activation.

The hallmark of vaccines is their ability to prevent the spread of infectious pathogens and thereby serve as invaluable public health tool. Despite their medical relevance, there is a gap in our understanding of the physiological factors that mediate innate and adaptive immune response to vaccines. The endocannabinoid (eCB) system is a critical modulator of homeostasis in vertebrates. Our results indicate that macrophages and dendritic cells produce the endocannabinoid, 2-arachidonoyl-sn-glycerol (2-AG) upon antigen activation. We have also established that 2-AG levels are upregulated in the serum and in the lymph node of mice during vaccination. We hypothesized that the intrinsic release of eCBs from immune cells during activation by pathogenic antigens mitigate inflammation, but also suppress overall innate and adaptive immune response. Here we demonstrate, for the first time, that transient administration of the cannabinoid receptor 2 antagonist AM630 (10 mg/kg) or inverse agonist JTE907 (3 mg/kg) during immunization heightens the intensity and breadth of antigen-specific immune responses in young and aged mice through the upregulation of immunomodulatory genes in secondary lymphoid tissues.

B-lymphoid transcription factors, such as PAX5 and IKZF1, are critical for early B-cell development, yet lesions of the genes encoding these transcription factors occur in over 80% of cases of pre-B-cell acute lymphoblastic leukaemia (ALL). The importance of these lesions in ALL has, until now, remained unclear. Here, by combining studies using chromatin immunoprecipitation with sequencing and RNA sequencing, we identify a novel B-lymphoid program for transcriptional repression of glucose and energy supply. Our metabolic analyses revealed that PAX5 and IKZF1 enforce a state of chronic energy deprivation, resulting in constitutive activation of the energy-stress sensor AMPK. Dominant-negative mutants of PAX5 and IKZF1, however, relieved this glucose and energy restriction. In a transgenic pre-B ALL mouse model, the heterozygous deletion of Pax5 increased glucose uptake and ATP levels by more than 25-fold. Reconstitution of PAX5 and IKZF1 in samples from patients with pre-B ALL restored a non-permissive state and induced energy crisis and cell death. A CRISPR/Cas9-based screen of PAX5 and IKZF1 transcriptional targets identified the products of NR3C1 (encoding the glucocorticoid receptor), TXNIP (encoding a glucose-feedback sensor) and CNR2 (encoding a cannabinoid receptor) as central effectors of B-lymphoid restriction of glucose and energy supply. Notably, transport-independent lipophilic methyl-conjugates of pyruvate and tricarboxylic acid cycle metabolites bypassed the gatekeeper function of PAX5 and IKZF1 and readily enabled leukaemic transformation. Conversely, pharmacological TXNIP and CNR2 agonists and a small-molecule AMPK inhibitor strongly synergized with glucocorticoids, identifying TXNIP, CNR2 and AMPK as potential therapeutic targets. Furthermore, our results provide a mechanistic explanation for the empirical finding that glucocorticoids are effective in the treatment of B-lymphoid but not myeloid malignancies. Thus, B-lymphoid transcription factors function as metabolic gatekeepers by limiting the amount of cellular ATP to levels that are insufficient for malignant transformation.

Abstract Tauopathies are a group of neurodegenerative diseases characterized by the alteration/aggregation of TAU protein, for which there is still no effective treatment. Therefore, new pharmacological targets are being sought, such as elements of the endocannabinoid system (ECS). We analysed the occurrence of changes in the ECS in tauopathies and their implication in the pathogenesis. By integrating gene expression analysis, immunofluorescence, genetic and adeno-associated virus expressing TAU mouse models, we found a TAU-dependent increase in CB2 receptor expression in hippocampal neurons, that occurs as an early event in the pathology and was maintained until late stages. These changes were accompanied by alterations in the endocannabinoid metabolism. Remarkably, CB2 ablation in mice protects from neurodegeneration induced by hTAUP301L overexpression, corroborated at the level of cognitive behaviour, synaptic plasticity, and aggregates of insoluble TAU. At the level of neuroinflammation, the absence of CB2 did not produce significant changes in concordance with a possible neuronal location rather than its classic glial expression in these models. These findings were corroborated in post-mortem samples of patients with Alzheimer’s disease, the most common tauopathy. Our results show that neurons with accumulated TAU induce the expression of the CB2 receptor, which enhances neurodegeneration. These results are important for our understanding of disease mechanisms, providing a novel therapeutic strategy to be investigated in tauopathies.

Renal fibrosis is a common feature in various chronic kidney diseases (CKD). Tubular cell damage is a main characterization which results from dysregulated fatty acid oxidation (FAO) and lipid accumulation. Cannabinoid Receptor 2 (CB2) contributes to renal fibrosis, however, its role in FAO dysregulation in tubular cells is not clarified. In this study, we found CB2 plays a detrimental role in lipid metabolism in tubular cells. CB2 knockout mice were adopted to establish a folic acid-induced nephropathy (FAN) model. CB2-induced FAO dysfunction, lipid deposition, and fibrogenesis were assessed in vivo and vitro. To explore molecular mechanisms, β-catenin inhibitors and peroxisome proliferator-activated receptor alpha (PPARα) activators were also used in CB2-overexpressed cells. The mediative role of β-catenin in CB2-inhibited PPARα and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) activation was analyzed. CB2 activates β-catenin signaling, resulting in the suppression of PPARα/PGC-1α axis. This decreased FAO functions and led to lipid droplet formation in tubular cells. CB2 gene ablation effectively mitigated FAO dysfunction, lipid deposition and uremic toxins accumulation in FAN mice, consequently retarding renal fibrosis. Additionally, inhibition to β-catenin or PPARα activation could greatly inhibit lipid accumulation and fibrogenesis induced by CB2. This study highlights CB2 disrupts FAO in tubular cells through β-catenin activation and subsequent inhibition on PPARα/PGC-1α activity. Targeted inhibition on CB2 offers a perspective therapeutic strategy to fight against renal fibrosis. [Display omitted] • Impaired fatty acid oxidation linked to renal fibrosis in CKD • Cannabinoid receptor 2 disrupts FAO, promotes lipid deposit via β-catenin activation. • CB2/β-catenin inhibits renal FAO by suppressing PPARα/PGC-1α axis [ABSTRACT FROM AUTHOR]

Background: The endocannabinoid system (ECS) and associated lipid transmitter-based signaling systems play an important role in modulating brain neuroinflammation. ECS is affected in neurodegenerative disorders, such as Alzheimer’s disease (AD). Here we have evaluated the non-psychotropic endocannabinoid receptor type 2 (CB2) and lysophosphatidylinositol G-protein-coupled receptor 55 (GPR55) localization and expression during Aβ-pathology progression. Methods: Hippocampal gene expression of CB2 and GPR55 was explored by qPCR analysis, and brain distribution was evaluated by immunofluorescence in the wild type (WT) and APP knock-in AppNL-G-F AD mouse model. Furthermore, the effects of Aβ42 on CB2 and GPR55 expression were assessed in primary cell cultures. Results: CB2 and GPR55 mRNA levels were significantly upregulated in AppNL-G-F mice at 6 and 12 months of age, compared to WT. CB2 was highly expressed in the microglia and astrocytes surrounding the Aβ plaques. Differently, GPR55 staining was mainly detected in neurons and microglia but not in astrocytes. In vitro, Aβ42 treatment enhanced CB2 receptor expression mainly in astrocytes and microglia cells, whereas GPR55 expression was enhanced primarily in neurons. Conclusions: These data show that Aβ pathology progression, particularly Aβ42, plays a crucial role in increasing the expression of CB2 and GPR55 receptors, supporting CB2 and GPR55 implications in AD.

NMDA receptor blockade in rodents is commonly used to induce schizophrenia-like behavioral abnormalities, including cognitive deficits and social dysfunction. Aberrant glutamate and GABA transmission, particularly in adolescence, is implicated in these behavioral abnormalities. The endocannabinoid system modulates glutamate and GABA transmission, but the impact of endocannabinoid modulation on cognitive and social dysfunction is unclear. Here, we asked whether late-adolescence administration of the anandamide hydrolysis inhibitor URB597 can reverse behavioral deficits induced by early-adolescence administration of the NMDA receptor blocker MK-801. In parallel, we assessed the impact of MK-801 and URB597 on mRNA expression of glutamate and GABA markers. We found that URB597 prevented MK-801-induced novel object recognition deficits and social interaction abnormalities in adult rats, and reversed glutamate and GABA aberrations in the prelimbic PFC. URB597-mediated reversal of MK-801-induced social interaction deficits was mediated by the CB1 receptor, whereas the reversal of cognitive deficits was mediated by the CB2 receptor. This was paralleled by the reversal of CB1 and CB2 receptor expression abnormalities in the basolateral amygdala and prelimbic PFC, respectively. Together, our findings show that interfering with NMDA receptor function in early adolescence has a lasting impact on phenotypes resembling the negative symptoms and cognitive deficits of schizophrenia and on glutamate and GABA marker expression in the PFC. Prevention of behavioral and molecular abnormalities by late-adolescence URB597 via CB1 and CB2 receptors suggests that endocannabinoid stimulation may have therapeutic potential in addressing treatment-resistant symptoms. [ABSTRACT FROM AUTHOR]