Endocannabinoid Signaling Research Articles

Comparison of Agonist Activity between CB1 and CB2 Receptors with Orthosteric Site Mutations

Human endocannabinoid signaling is primarily mediated by the cannabinoid receptors, CB1 and CB2, which are G protein-coupled receptors (GPCRs). These receptors have been linked to a variety of physiological processes and are being pursued as prospective drug targets due to their potential in treating pain and inflammation. However, because of their homology and shared signaling mechanisms, investigating the individual physiological roles of these receptors and designing subtype-selective ligands has been challenging. Using active-state CB1 and CB2 structures as guides, homologous residues within the orthosteric pocket of each receptor were mutated to alanine to test whether they equally impair CB1 and CB2 activity in response to two high-affinity, nonselective agonists (CP55,940 and AM12033). Interestingly, mutating the Y5.39 position impairs CB1 but not CB2 function. Conversely, mutating residue C6.47 improves CB1 but impairs CB2 signaling. The F7.35A mutation leads to a decrease in CP55,940 potency at CB1 and impairs internalization; however, AM12033 gains potency and promotes CB1 internalization. In CB2, mutation of F7.35A decreases the potency of CP55,940 and neither agonist induces internalization. These observations provide some insight into functional sensitivity of CB1 and CB2 to different agonists when conserved residues are mutated in the orthosteric pocket.

From Classical to Alternative Pathways of 2-Arachidonoylglycerol Synthesis: AlterAGs at the Crossroad of Endocannabinoid and Lysophospholipid Signaling.

2-arachidonoylglycerol (2-AG) is the most abundant endocannabinoid (EC), acting as a full agonist at both CB1 and CB2 cannabinoid receptors. It is synthesized on demand in postsynaptic membranes through the sequential action of phosphoinositide-specific phospholipase Cβ1 (PLCβ1) and diacylglycerol lipase α (DAGLα), contributing to retrograde signaling upon interaction with presynaptic CB1. However, 2-AG production might also involve various combinations of PLC and DAGL isoforms, as well as additional intracellular pathways implying other enzymes and substrates. Three other alternative pathways of 2-AG synthesis rest on the extracellular cleavage of 2-arachidonoyl-lysophospholipids by three different hydrolases: glycerophosphodiesterase 3 (GDE3), lipid phosphate phosphatases (LPPs), and two members of ecto-nucleotide pyrophosphatase/phosphodiesterases (ENPP6-7). We propose the names of AlterAG-1, -2, and -3 for three pathways sharing an ectocellular localization, allowing them to convert extracellular lysophospholipid mediators into 2-AG, thus inducing typical signaling switches between various G-protein-coupled receptors (GPCRs). This implies the critical importance of the regioisomerism of both lysophospholipid (LPLs) and 2-AG, which is the object of deep analysis within this review. The precise functional roles of AlterAGs are still poorly understood and will require gene invalidation approaches, knowing that both 2-AG and its related lysophospholipids are involved in numerous aspects of physiology and pathology, including cancer, inflammation, immune defenses, obesity, bone development, neurodegeneration, or psychiatric disorders.

Lipolysis pathways modulate lipid mediator release and endocannabinoid system signaling in dairy cows’ adipocytes

BackgroundAs cows transition from pregnancy to lactation, free fatty acids (FFA) are mobilized from adipose tissues (AT) through lipolysis to counter energy deficits. In clinically healthy cows, lipolysis intensity is reduced throughout lactation; however, if FFA release exceeds tissue demands or the liver’s metabolic capacity, lipid byproducts accumulate, increasing cows’ risk of metabolic and infectious disease. Endocannabinoids (eCBs) and their congeners, N-acylethanolamines (NAEs), are lipid-based compounds that modulate metabolism and inflammation. Their synthesis and release depend upon the availability of FFA precursors and the abundance of synthesizing and degrading enzymes and transporters. Therefore, we hypothesized that eCB production and transcription of endocannabinoid system components are modulated by lipolysis pathways in adipocytes. To test this hypothesis, we stimulated canonical (isoproterenol, 1 µmol/L; ISO) and inflammatory (lipopolysaccharide, 1 µg/mL; LPS) lipolysis pathways in adipocytes isolated from the AT of 5 Holstein dairy cows. Following, we assessed lipolysis intensity, adipocytes’ release of eCBs, and transcription of endocannabinoid system components.ResultsWe found that ISO and LPS stimulated lipolysis at comparable intensities. Exposure to either treatment tended to elevate the release of eCBs and NAEs by cultured adipocytes; however, specific eCBs and NAEs and the transcriptional profiles differed by treatment. On one hand, ISO enhanced adipocytes’ release of 2-arachidonoylglycerol (2-AG) but reduced NAE production. Notably, ISO enhanced the cells’ expression of enzymes associated with 2-AG biosynthesis (INPP5F, GDPD5, GPAT4), transport (CD36), and adipogenesis (PPARG). Conversely, LPS enhanced adipocytes’ synthesis and release of N-arachidonoylethanolamide (AEA). This change coincided with enhanced transcription of the NAE-biosynthesizing enzyme, PTPN22, and adipocytes’ transcription of genes related to eCB degradation (PTGS2, MGLL, CYP27B1). Furthermore, LPS enhanced adipocytes’ transcription of eCB and NAE transporters (HSPA1A, SCP2) and the expression of the anti-adipogenic ion channel, TRPV3.ConclusionsOur data provide evidence for distinct modulatory roles of canonical and inflammatory lipolysis pathways over eCB release and transcriptional regulation of biosynthesis, degradation, transport, and ECS signaling in cows’ adipocytes. Based on our findings, we conclude that, within adipocytes, eCB production and ECS component expression are, at least in part, mediated by lipolysis in a pathway-dependent manner. These findings contribute to a deeper understanding of the molecular mechanisms underlying metabolic regulation in dairy cows’ AT, with potential implications for prevention and treatment of inflammatory and metabolic disorders.Graphical

Characterization of a Fatty Acid Amide Hydrolase (FAAH) in Hirudo Verbana

The endocannabinoid system plays a critical role in modulating both peripheral and central nervous system function. Despite being present throughout the animal kingdom, there has been relatively little investigation of the endocannabinoid system beyond traditional animal models. In this study, we report on the identification and characterization of a putative fatty acid amide hydrolase (FAAH) in the medicinal leech, Hirudo verbana. FAAH is the primary enzyme responsible for metabolizing the endocannabinoid signaling molecule arachidonoyl ethanolamide (anandamide or AEA) and therefore plays a critical role in regulating AEA levels in the nervous system. mRNA encoding Hirudo FAAH (HirFAAH) is expressed in the leech central nervous system (CNS) and sequence analysis suggests that this is an orthologue of FAAH-2 observed in vertebrates. Functionally, HirFAAH has serine hydrolase activity based on activity-based protein profiling (ABPP) studies using the fluorophosphonate probe TAMRA-FP. HirFAAH also hydrolyzes arachidonyl 7-amino, 4-methyl coumarin amide (AAMCA), a substrate specific to FAAH. Hydrolase activity during both the ABPP and AAMCA assays was eliminated by a mutation at a conserved catalytic serine. Activity was also blocked by the known FAAH inhibitor, URB597. Treatment of Hirudo ganglia with URB597 potentiated synapses made by the pressure-sensitive mechanosensory neuron (P cell), mimicking the effects of exogenously applied AEA. The Hirudo CNS has been a useful system in which to study properties of endocannabinoid modulation of nociception relevant to vertebrates. Therefore, this characterization of HirFAAH is an important contribution to comparative studies of the endocannabinoid system.

Exploring Intrinsic Disorder in Human Synucleins and Associated Proteins.

In this work, we explored the intrinsic disorder status of the three members of the synuclein family of proteins-α-, β-, and γ-synucleins-and showed that although all three human synucleins are highly disordered, the highest levels of disorder are observed in γ-synuclein. Our analysis of the peculiarities of the amino acid sequences and modeled 3D structures of the human synuclein family members revealed that the pathological mutations A30P, E46K, H50Q, A53T, and A53E associated with the early onset of Parkinson's disease caused some increase in the local disorder propensity of human α-synuclein. A comparative sequence-based analysis of the synuclein proteins from various evolutionary distant species and evaluation of their levels of intrinsic disorder using a set of commonly used bioinformatics tools revealed that, irrespective of their origin, all members of the synuclein family analyzed in this study were predicted to be highly disordered proteins, indicating that their intrinsically disordered nature represents an evolutionary conserved and therefore functionally important feature. A detailed functional disorder analysis of the proteins in the interactomes of the human synuclein family members utilizing a set of commonly used disorder analysis tools showed that the human α-synuclein interactome has relatively higher levels of intrinsic disorder as compared with the interactomes of human β- and γ- synucleins and revealed that, relative to the β- and γ-synuclein interactomes, α-synuclein interactors are involved in a much broader spectrum of highly diversified functional pathways. Although proteins interacting with three human synucleins were characterized by highly diversified functionalities, this analysis also revealed that the interactors of three human synucleins were involved in three common functional pathways, such as the synaptic vesicle cycle, serotonergic synapse, and retrograde endocannabinoid signaling. Taken together, these observations highlight the critical importance of the intrinsic disorder of human synucleins and their interactors in various neuronal processes.

Analgesic Properties of Next-Generation Modulators of Endocannabinoid Signaling: Leveraging Modern Tools for the Development of Novel Therapeutics.

Targeting the endocannabinoid (eCB) signaling system for pain relief is an important treatment option that is only now beginning to be mechanistically explored. In this review, we focus on two recently appreciated cannabinoid-based targeting strategies, treatments with cannabidiol (CBD) and α/β-hydrolase domain containing 6 (ABHD6) inhibitors, which have the exciting potential to produce pain relief through distinct mechanisms of action and without intoxication. We review evidence on plant-derived cannabinoids for pain, with an emphasis on CBD and its multiple molecular targets expressed in pain pathways. We also discuss the function of eCB signaling in regulating pain responses and the therapeutic promises of inhibitors targeting ABHD6, a 2-arachidonoylglycerol (2-AG)-hydrolyzing enzyme. Finally, we discuss how the novel cannabinoid biosensor GRABeCB2.0 may be leveraged to enable the discovery of targets modulated by cannabinoids at a circuit-specific level. SIGNIFICANCE STATEMENT: Cannabis has been used by humans as an effective medicine for millennia, including for pain management. Recent evidence emphasizes the therapeutic potential of compounds that modulate endocannabinoid signaling. Specifically, cannabidiol and inhibitors of the enzyme ABHD6 represent promising strategies to achieve pain relief by modulating endocannabinoid signaling in pain pathways via distinct, nonintoxicating mechanisms of action.

Endocannabinoid and neuroplasticity-related changes as susceptibility factors in a rat model of posttraumatic stress disorder

Traumatic experiences result in the development of posttraumatic stress disorder (PTSD) in 10–25% of exposed individuals. While human clinical studies suggest that susceptibility is potentially linked to endocannabinoid (eCB) signaling, neurobiological PTSD susceptibility factors are poorly understood. Employing a rat model of contextual conditioned fear, we characterized distinct resilient and susceptible subpopulations based on lasting generalized fear, a core symptom of PTSD. In these groups, we assessed i.) eCB levels by mass spectrometry and ii.) expression variations of eCB system- and iii.) neuroplasticity-related genes by real-time quantitative PCR in the circuitry relevant in trauma-induced changes. Furthermore, employing unsupervised and semi-supervised machine learning based statistical analytical models, we assessed iv.) gene expression patterns with the most robust predictive power regarding PTSD susceptibility. According to our findings, in our model, generalized fear responses occurred with sufficient variability to characterize distinct resilient and susceptible subpopulations. Resilient subjects showed elevated prelimbic and lower ventral hippocampal levels of eCB 2-arachidonoyl-glycerol (2-AG) compared to resilient and non-shocked control subjects. Ventral hippocampal 2-AG content positively correlated with the strength of fear generalization. Furthermore, susceptibility was associated with i.) prefrontal, hippocampal and amygdalar neuronal hypoactivity, ii.) marked decrease in the expression of genes of transcription factors modulating neuroplasticity and iii.) an altered expression pattern of eCB-related genes, including enzymes involved in eCB metabolism. Unsupervised and semi-supervised statistical approaches highlighted that hippocampal gene expression patterns possess strong predictive power regarding susceptibility. Taken together, the marked eCB and neuroplasticity changes in susceptible individuals associated with abnormal activity patterns in the fear circuitry possibly contribute to context coding deficits, resulting in generalized fear.

Interplay among Oxidative Stress, Autophagy, and the Endocannabinoid System in Neurodegenerative Diseases: Role of the Nrf2- p62/SQSTM1 Pathway and Nutraceutical Activation.

The onset of neurodegenerative diseases involves a complex interplay of pathological mechanisms, including protein aggregation, oxidative stress, and impaired autophagy. This review focuses on the intricate connection between oxidative stress and autophagy in neurodegenerative disorders, highlighting autophagy as pivotal in disease pathogenesis. Reactive oxygen species (ROS) play dual roles in cellular homeostasis and autophagy regulation, with disruptions of redox signaling contributing to neurodegeneration. The activation of the Nrf2 pathway represents a critical antioxidant mechanism, while autophagy maintains cellular homeostasis by degrading altered cell components. The interaction among p62/SQSTM1, Nrf2, and Keap1 forms a regulatory pathway essential for cellular stress response, whose dysregulation leads to impaired autophagy and aggregate accumulation. Targeting the Nrf2-p62/SQSTM1 pathway holds promise for therapeutic intervention, mitigating oxidative stress and preserving cellular functions. Additionally, this review explores the potential synergy between the endocannabinoid system and Nrf2 signaling for neuroprotection. Further research is needed to elucidate the involved molecular mechanisms and develop effective therapeutic strategies against neurodegeneration.

Neuroprotective Mechanism of MOTS-c in TBI Mice: Insights from Integrated Transcriptomic and Metabolomic Analyses.

Traumatic brain injury (TBI) is a condition characterized by structural and physiological disruptions in brain function caused by external forces. However, as the highly complex and heterogenous nature of TBI, effective treatments are currently lacking. Mitochondrial open reading frame of the 12S rRNA-c (MOTS-c) has shown notable antinociceptive and anti-inflammatory effects, yet its detailed neuroprotective effects and mode of action remain incompletely understood. This study investigated the neuroprotective effects and the underlying mechanisms of MOTS-c. Adult male C57BL/6 mice were randomly divided into three groups: control (CON) group, MOTS-c group and TBI group. Enzyme-linked immunosorbent assay (ELISA) kit method was used to measure the expression levels of MOTS-c in different groups. Behavioral tests were conducted to assess the effects of MOTS-c. Then, transcriptomics and metabolomics were performed to search Differentially Expressed Genes (DEGs) and Differentially Expressed Metabolites (DEMs), respectively. Moreover, the integrated transcriptomics and metabolomics analysis were employed using R packages and online Kyoto Encyclopedia of Genes and Genomes (KEGG) database. ELISA kit method showed that TBI resulted in a decrease in the expression of MOTS-c. and peripheral administration of MOTS-c could enter the brain tissue after TBI. Behavioral tests revealed that MOTS-c improved memory, learning, and motor function impairments in TBI mice. Additionally, transcriptomic analysis screened 159 differentially expressed genes. Metabolomic analysis identified 491 metabolites with significant differences. Integrated analysis found 14 KEGG pathways, primarily related to metabolic pathways. Besides, several signaling pathways were enriched, including neuroactive ligand-receptor interaction and retrograde endocannabinoid signaling. TBI reduced the expression of MOTS-c. MOTS-c reduced inflammatory responses, molecular damage, and cell death by down-regulating macrophage migration inhibitory factor (MIF) expression and activating the retrograde endocannabinoid signaling pathway. In addition, MOTS-c alleviated the response to hypoxic stress and enhanced lipid β-oxidation to provide energy for the body following TBI. Overall, our study offered new insights into the neuroprotective mechanisms of MOTS-c in TBI mice.

Acute and Long-Term Effects of App-Delivered Heartfulness Meditation on Psychological Outcomes and the Endocannabinoid Signaling System in Cyclic Vomiting Syndrome.

Cyclic vomiting syndrome (CVS) is a disorder of gut-brain interaction often triggered by stress. Interventions such as meditation may improve psychological outcomes and health-related quality of life (HRQoL), but their efficacy and the underlying mechanism are unknown. We conducted a 6-week single-arm pilot study to assess the effects of heartfulness meditation (HFM) in CVS using a custom-designed meditation app. Primary outcomes included state and trait anxiety and mood state changes pre vs post-meditation, and secondary outcomes were psychological distress, coping, sleep quality, and HRQoL at baseline and at weeks 3 and 6. Serum concentrations of endocannabinoids N -arachidonylethanolamine and 2-arachidonoylglycerol and related lipids were measured pre- and post-HFM at baseline and week 6. In 30 treatment completers, there was a significant improvement in state anxiety ( P < 0.001), total mood disturbance ( P < 0.001), and other mood states (all P values < 0.05) across the 3 time points. Trait anxiety was also improved at week 6. There was a significant improvement in psychological distress (Global Severity Index), sleep quality (daytime dysfunction), coping (using religion/spirituality), and HRQoL (mental and physical) across the 3 time points (all P < 0.05). Significant increases in N -arachidonylethanolamine and related lipids N -oleoylethanolamine and palmitoylethanolamide post vs pre-HFM were observed at week 6 ( P < 0.001, 0.002, 0.003, respectively). No adverse effects were noted. App-delivered HFM is feasible, safe, and effective and improves psychological outcomes and augments endocannabinoids. This provides insight into the mechanism underlying HFM and has potential for widespread use as a digital therapeutic in CVS and other disorder of gut-brain interaction.

O-demethyl galantamine alters protein expression in cerebellum of 5xFAD mice.

Alzheimer's disease (AD), one of the most common health issues, is characterized by memory loss, severe behavioral disorders, and eventually death. Despite many studies, there are still no drugs that can treat AD or stop it from progressing. Previous in vitro tests showed that O-demethyl galantamine (ODG) might have therapeutic potential owing to its 10 times higher acetylcholinesterase inhibitory activity than galantamine (GAL). We aimed to assess the effect of ODG at the molecular level in a 12-month-old 5xFAD Alzheimer's mouse model. To this end, following the administrations of ODG and GAL (used as a positive control), protein alterations were investigated in the cortex, hippocampus, and cerebellum regions of the brain. Surprisingly, GAL altered proteins prominently in the cortex, while ODG exclusively exerted its effect on the cerebellum. GNB1, GNB2, NDUFS6, PAK2, and RhoA proteins were identified as the top 5 hub proteins in the cerebellum of ODG-treated mice. Reregulation of these proteins through Ras signaling and retrograde endocannabinoid signaling pathways, which were found to be enriched, might contribute to reversing AD-induced molecular changes. We suggest that, since it targets specifically the cerebellum, ODG may be further evaluated for combination therapies for AD.

Transcriptomic Revealed That Selenium-Rich Lactobacillus plantarum Alleviated Cadmium-Induced Immune Responses in Bulatmai barbel Luciobarbus capito Kidneys

Cadmium (Cd) is a common environmental pollutant that accumulates mainly in the kidneys and thus endangers the physiological health of aquatic animals. Selenium (Se) is a natural antidote to heavy metals that antagonises heavy metal toxicity and enhances the antioxidant capacity of organisms. Lactobacillus plantarum (L. plantarum) can reduce the toxicity of heavy metals through adsorption, reduction and metabolism. Studies have confirmed that the biological synthesis of Se nanoparticles (Bio-SeNPs) using bacterial microorganisms is simple, safe and less toxic than the synthesis of inorganic and organic Se, but the effect on Cd-induced immunosuppression is un-known. One hundred and eighty Bulatmai barbel (Luciobarbus capito: L. capito) plants were randomly divided into control (C), Cd and Cd + Se-enriched L. plantarum groups (S1L1-Cd) and fed for 28 days. The analysis methods included histopathology, test kits, transcriptomics and real-time quantitative PCR. The addition of selenium-enriched L. plantarum significantly attenuated cadmium-induced pathological changes such as glomerular atrophy, detachment of renal tubular epithelial cells, mild swelling, and interstitial inflammatory cell infiltration. Cd stress can lead to significant decreases in RBC, HCT, WBC, LZM, C3, and IgM levels, and the addition of Se-enriched L. plantarum can significantly reverse the changes in these indicators. Transcriptomic analysis revealed 488 DEGs in the Cd groups, 301 of which were upregulated and 187 of which were downregulated. There were 1474 DEGs in the S1L1-Cd group, of which 720 were upregulated and 754 were downregulated. In addition, GO enrichment analysis revealed that the biological regulation of the most differentially expressed genes involved metal ion binding, ATP binding and nucleotide inclusion. KEGG enrichment analysis revealed six of the most enriched pathways: oxidative phosphorylation, Huntington disease, retrograde endocannabinoid signalling, natural killer cell-mediated cyto-toxicity, the IL-17 signalling pathway, and leukocyte transient migration. Moreover, we selected 12 DEGs for qRT-PCR, which showed that the qRT-PCR results were consistent with our RNA-Seq results. Our results suggest that Se-enriched L. plantarum can enhance immunity and alleviate Cd exposure-mediated immunosuppression in L. capito.

Integrated proteomics and metabolomics analysis of sclerosis-related proteins and femoral head necrosis following internal fixation of femoral neck fractures

Femoral head necrosis (FHN) is a serious complication after femoral neck fractures (FNF), often linked to sclerosis around screw paths. Our study aimed to uncover the proteomic and metabolomic underpinnings of FHN and sclerosis using integrated proteomics and metabolomics analyses. We identified differentially expressed proteins (DEPs) and metabolites (DEMs) among three groups: patients with FNF (Group A), sclerosis (Group B), and FHN (Group C). Using the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology enrichment analyses, we examined the roles of these proteins and metabolites. Our findings highlight the significant differences across the groups, with 218 DEPs and 44 DEMs identified between the sclerosis and FNF groups, 247 DEPs and 31 DEMs between the FHN and sclerosis groups, and a stark 682 DEPs and 94 DEMs between the FHN and FNF groups. Activities related to carbonate dehydratase and hydrolase were similar in the FHN and sclerosis groups, whereas extracellular region and lysosome were prevalent in the FHN and FNF groups. Our study also emphasized the involvement of the PI3K-Akt pathway in sclerosis and FHN. Moreover, the key metabolic pathways were implicated in glycerophospholipid metabolism and retrograde endocannabinoid signaling. Using western blotting, we confirmed the pivotal role of specific genes/proteins such as ITGB5, TNXB, CA II, and CA III in sclerosis and acid phosphatase 5 and cathepsin K in FHN. This comprehensive analyses elucidates the molecular mechanisms behind sclerosis and FHN and suggests potential biomarkers and therapeutic targets, paving the way for improved treatment strategies. Further validation of the findings is necessary to strengthen the robustness and reliability of the results.

Early life adversity promotes gastrointestinal dysfunction through a sex-dependent phenotypic switch in enteric glia.

Irritable bowel syndrome and related disorders of gut-brain interaction (DGBI) are common and exhibit a complex, poorly understood etiology that manifests as abnormal gut motility and pain. Risk factors such as biological sex, stressors during critical periods, and inflammation are thought to influence DGBI vulnerability by reprogramming gut-brain circuits, but the specific cells affected are unclear. Here, we used a model of early life stress to understand cellular mechanisms in the gut that produce DGBIs. Our findings identify enteric glia as a key cellular substrate in which stress and biological sex converge to dictate DGBI susceptibility. Enteric glia exhibit sexual dimorphism in genes and functions related to cellular communication, inflammation, and disease susceptibility. Experiencing early life stress has sex-specific effects on enteric glia that cause a phenotypic switch in male glia toward a phenotype normally observed in females. This phenotypic transformation is followed by physiological changes in the gut, mirroring those observed in DGBI in humans. These effects are mediated, in part, by alterations to glial prostaglandin and endocannabinoid signaling. Together, these data identify enteric glia as a cellular integration site through which DGBI risk factors produce changes in gut physiology and suggest that manipulating glial signaling may represent an attractive target for sex-specific therapeutic strategies in DGBIs.

Positive allosteric modulation of the cannabinoid CB1 receptor potentiates endocannabinoid signalling and changes ERK1/2 phosphorylation kinetics.

Activation of CB1 by exogenous agonists causes adverse effects in vivo. Positive allosteric modulation may offer improved therapeutic potential and a reduced on-target adverse effect profile compared with orthosteric agonists, due to reduced desensitisation/tolerance, but this has not been directly tested. This study investigated the ability of PAMs/ago-PAMs to induce receptor regulation pathways, including desensitisation and receptor internalisation. Bioluminescence resonance energy transfer (BRET) assays in HEK293 cells were performed to investigate G protein dissociation, ERK1/2 phosphorylation and β-arrestin 2 translocation, while immunocytochemistry was performed to measure internalisation of CB1 in response to the PAMs ZCZ011, GAT229 and ABD1236 alone and in combination with the orthosteric agonists AEA, 2-AG, and AMB-FUBINACA. ZCZ011, GAT229 and ABD1236 were allosteric agonists in all pathways tested. The ago-PAM ZCZ011 induced a biphasic ERK1/2 phosphorylation time course compared to transient activation by orthosteric agonists. In combination with 2-AG but not AEA or AMB-FUBINACA, ZCZ011 and ABD1236 caused the transient peak of ERK1/2 phosphorylation to become sustained. All PAMs increased the potency and efficacy of AEA-induced signalling in all pathways tested; however, no notable potentiation of 2-AG or AMB-FUBINACA was observed. Ago-PAMs can potentiate endocannabinoid CB1 agonism by AEA to a larger extent compared with 2-AG. However, all compounds were found to be allosteric agonists and induce activation of CB1 in the absence of endocannabinoid, including β-arrestin 2 recruitment and internalisation. Thus, the spatiotemporal signalling of endogenous cannabinoids will not be retained in vivo.

SGIP1 binding to the α-helical H9 domain of cannabinoid receptor 1 promotes axonal surface expression.

Endocannabinoid signalling mediated by cannabinoid receptor 1 (CB1R, also known as CNR1) is critical for homeostatic neuromodulation of both excitatory and inhibitory synapses. This requires highly polarised axonal surface expression of CB1R, but how this is achieved remains unclear. We previously reported that the α-helical H9 domain in the intracellular C terminus of CB1R contributes to axonal surface expression by an unknown mechanism. Here, we show in rat primary neuronal cultures that the H9 domain binds to the endocytic adaptor protein SGIP1 to promote CB1R expression in the axonal membrane. Overexpression of SGIP1 increases CB1R axonal surface localisation but has no effect on CB1R lacking the H9 domain (CB1RΔH9). Conversely, SGIP1 knockdown reduces axonal surface expression of CB1R but does not affect CB1RΔH9. Furthermore, SGIP1 knockdown diminishes CB1R-mediated inhibition of presynaptic Ca2+ influx in response to neuronal activity. Taken together, these data advance mechanistic understanding of endocannabinoid signalling by demonstrating that SGIP1 interaction with the H9 domain underpins axonal CB1R surface expression to regulate presynaptic responsiveness.

Mu-opioid receptor activation in the habenula modulates synaptic transmission and depression-like behaviors

Opioid system dysregulation in response to stress is known to lead to psychiatric disorders including major depression. Among three different types of opioid receptors, the mu-type receptors (mORs) are highly expressed in the habenula complex, however, the action of mORs in this area and its interaction with stress exposure is largely unknown. Therefore, we investigated the roles of mORs in the habenula using male rats of an acute learned helplessness (aLH) model. First, we found that mOR activation decreased both excitatory and inhibitory synaptic transmission onto the lateral habenula (LHb). Intriguingly, this mOR-induced synaptic depression was reduced in an animal model of depression compared to that of controls. In naïve animals, we found an unexpected interaction between mORs and the endocannabinoid (eCB) signaling occurring in the LHb, which mediates presynaptic alteration occurring with mOR activation. However, we did not observe presynaptic alteration by mOR activation after stress exposure. Moreover, selective mOR activation in the habenula before, but not after, stress exposure effectively reduced helpless behaviors compared to aLH animals. Our observations are consistent with clinical reports suggesting the involvement of mOR signaling in depression, and additionally reveal a critical time window of mOR action in the habenula for ameliorating helplessness symptoms.

Parvalbumin interneuron mGlu5 receptors govern sex differences in prefrontal cortex physiology and binge drinking.

Despite established sex differences in the prevalence and presentation of psychiatric disorders, little is known about the cellular and synaptic mechanisms that guide these differences under basal conditions. The proper function of the prefrontal cortex (PFC) is essential for the top-down regulation of motivated behaviors. The activity of the PFC is tightly controlled by parvalbumin-expressing interneurons (PV-INs), a key subpopulation of fast-spiking GABAergic cells that regulate cortical excitability through direct innervations onto the perisomatic regions of nearby pyramidal cells. Recent rodent studies have identified notable sex differences in PV-IN activity and adaptations to experiences such as binge drinking. Here, we investigated the cellular and molecular mechanisms that underlie sex-specific regulation of PFC PV-IN function. Using whole-cell patch-clamp electrophysiology and selective pharmacology, we report that PV-INs from female mice are more excitable than those from males. Moreover, we find that mGlu1 and mGlu5 metabotropic glutamate receptors regulate cell excitability, excitatory drive, and endocannabinoid signaling at PFC PV-INs in a sex-dependent manner. Genetic deletion of mGlu5 receptors from PV-expressing cells abrogates all sex differences observed in PV-IN membrane and synaptic physiology. Lastly, we report that female, but not male, PV-mGlu5-/- mice exhibit decreased voluntary drinking on an intermittent access schedule, which could be related to changes in ethanol's stimulant properties. Importantly, these studies identify mGlu1 and mGlu5 receptors as candidate signaling molecules involved in sex differences in PV-IN activity and behaviors relevant to alcohol use.

Transcriptomic signature, bioactivity and safety of a non-hepatotoxic analgesic generating AM404 in the midbrain PAG region

Safe and effective pain management is a critical healthcare and societal need. The potential for acute liver injury from paracetamol (ApAP) overdose; nephrotoxicity and gastrointestinal damage from chronic non-steroidal anti-inflammatory drug (NSAID) use; and opioids’ addiction are unresolved challenges. We developed SRP-001, a non-opioid and non-hepatotoxic small molecule that, unlike ApAP, does not produce the hepatotoxic metabolite N-acetyl-p-benzoquinone-imine (NAPQI) and preserves hepatic tight junction integrity at high doses. CD-1 mice exposed to SRP-001 showed no mortality, unlike a 70% mortality observed with increasing equimolar doses of ApAP within 72 h. SRP-001 and ApAP have comparable antinociceptive effects, including the complete Freund’s adjuvant-induced inflammatory von Frey model. Both induce analgesia via N-arachidonoylphenolamine (AM404) formation in the midbrain periaqueductal grey (PAG) nociception region, with SRP-001 generating higher amounts of AM404 than ApAP. Single-cell transcriptomics of PAG uncovered that SRP-001 and ApAP also share modulation of pain-related gene expression and cell signaling pathways/networks, including endocannabinoid signaling, genes pertaining to mechanical nociception, and fatty acid amide hydrolase (FAAH). Both regulate the expression of key genes encoding FAAH, 2-arachidonoylglycerol (2-AG), cannabinoid receptor 1 (CNR1), CNR2, transient receptor potential vanilloid type 4 (TRPV4), and voltage-gated Ca2+ channel. Phase 1 trial (NCT05484414) (02/08/2022) demonstrates SRP-001’s safety, tolerability, and favorable pharmacokinetics, including a half-life from 4.9 to 9.8 h. Given its non-hepatotoxicity and clinically validated analgesic mechanisms, SRP-001 offers a promising alternative to ApAP, NSAIDs, and opioids for safer pain treatment.

Interactions between physical exercise, associative memory, and genetic risk for Alzheimer's disease.

The ε4 allele of the APOE gene heightens the risk of late onset Alzheimer's disease. ε4 carriers, may exhibit cognitive and neural changes early on. Given the known memory-enhancing effects of physical exercise, particularly through hippocampal plasticity via endocannabinoid signaling, here we aimed to test whether a single session of physical exercise may benefit memory and underlying neurophysiological processes in young ε3 carriers (ε3/ε4 heterozygotes, risk group) compared with a matched control group (homozygotes for ε3). Participants underwent fMRI while learning picture sequences, followed by cycling or rest before a memory test. Blood samples measured endocannabinoid levels. At the behavioral level, the risk group exhibited poorer associative memory performance, regardless of the exercising condition. At the brain level, the risk group showed increased medial temporal lobe activity during memory retrieval irrespective of exercise (suggesting neural compensatory effects even at baseline), whereas, in the control group, such increase was only detectable after physical exercise. Critically, an exercise-related endocannabinoid increase correlated with task-related hippocampal activation in the control group only. In conclusion, healthy young individuals carrying the ε4 allele may present suboptimal associative memory performance (when compared with homozygote ε3 carriers), together with reduced plasticity (and functional over-compensation) within medial temporal structures.