Importance:Bipolar disorder (BD) and major depressive disorder (MDD) aggregate within families, with risk often first manifesting as early psychopathology, including attention-deficit/hyperactivity disorder (ADHD) and anxiety disorders. Objective:To determine whether polygenic scores (PGS) are associated with mood disorder onset independent of familial high risk for BD (FHR-BD) and early psychopathology. Design, Setting, and Participants:This cohort study used data from 7 prospective cohorts enriched in FHR-BD from Australia, Canada, the Netherlands, Spain, and the US. Participants with FHR-BD, defined as having at least 1 first-degree relative with BD, were compared with participants without FHR for any mood disorder. Participants were repeatedly assessed with variable follow-up intervals from July 1992 to July 2023. Data were analyzed from August 2023 to August 2024. Exposures:PGS indexed genetic liability for MDD, BD, anxiety, neuroticism, subjective well-being, ADHD, self-regulation, and addiction risk factor. Semistructured diagnostic interviews with relatives established FHR-BD. ADHD or anxiety disorder diagnoses before mood disorder onset constituted early psychopathology. Main Outcomes and Measures:The outcome of interest, mood disorder onset, was defined as a consensus-confirmed new diagnosis of MDD or BD. Cox regression examined associations of PGS, FHR-BD, ADHD, and anxiety with mood disorder onset. Kaplan-Meier curves and log-rank tests evaluated the probability of onset by PGS quartile and familial risk status. Results:A total of 1064 participants (546 [51.3%] female; mean [SD] age at last assessment, 21.7 [5.1] years), including 660 with FHR-BD and 404 without FHR for any mood disorder, were repeatedly assessed for mental disorders. A total of 399 mood disorder onsets occurred over a variable mean (SD) follow-up interval of 6.3 (5.7) years. Multiple PGS were associated with onset after correcting for FHR-BD and early psychopathology, including PGS for ADHD (hazard ratio [HR], 1.19; 95% CI, 1.06-1.34), self-regulation (HR, 1.19; 95% CI, 1.06-1.34), neuroticism (HR, 1.18; 95% CI, 1.06-1.32), MDD (HR, 1.17; 95% CI, 1.04-1.31), addiction risk factor (HR, 1.16; 95% CI, 1.04-1.30), anxiety (HR, 1.15; 95% CI, 1.02-1.28), BD (HR, 1.14; 95% CI, 1.02-1.28), and subjective well-being (HR, 0.89; 95% CI, 0.79-0.99). High PGS for addiction risk factor, anxiety, BD, and MDD were associated with increased probability of onset in the control group. High PGS for ADHD and self-regulation increased rates of onset among participants with FHR-BD. PGS for self-regulation, ADHD, and addiction risk factors showed stronger associations with onsets of BD than MDD. Conclusions and Relevance:In this cohort study, multiple PGS were associated with mood disorder onset independent of family history of BD and premorbid diagnoses of ADHD or anxiety. The association between PGS and mood disorder risk varied depending on family history status.

Microglia, the resident immune cells of the brain, regulate neuroinflammation which can lead to secondary neuronal damage and cognitive impairment under pathological conditions. Two of the many molecules that can elicit an inflammatory response from microglia are lipopolysaccharide (LPS), a component of gram-negative bacteria, and interferon gamma (IFNγ), an endogenous pro-inflammatory cytokine. We thoroughly examined the concentration-dependent relationship between LPS from multiple bacterial species and IFNγ in cultured microglia and macrophages. We measured the effects that these immunostimulatory molecules have on pro-inflammatory activity of microglia and used a battery of signaling inhibitors to identify the pathways that contribute to the microglial response. We found that LPS and IFNγ interacted synergistically to induce a pro-inflammatory phenotype in microglia, and that inhibition of JAK1/2 completely blunted the response. We determined that this synergistic action of LPS and IFNγ was likely dependent on JNK and Akt signaling rather than typical pro-inflammatory mediators such as NF-κB. Finally, we demonstrated that LPS derived from Escherichia coli, Klebsiella pneumoniae, and Akkermansia muciniphila can elicit different inflammatory responses from microglia and macrophages, but these responses could be consistently prevented using ruxolitinib, a JAK1/2 inhibitor. Collectively, this work reveals a mechanism by which microglia may become hyperactivated in response to the combination of LPS and IFNγ. Given that elevations in circulating LPS and IFNγ occur in a wide variety of pathological conditions, it is critical to understand the pharmacological interactions between these molecules to develop safe and effective treatments to suppress this process.

Microglia are resident immune cells of the central nervous system (CNS) and propagate inflammation following damage to the CNS, including the retina. Proliferative vitreoretinopathy (PVR) is a condition that can emerge following retinal detachment and is characterized by severe inflammation and microglial proliferation. The type 2 cannabinoid receptor (CB2) is an emerging pharmacological target to suppress microglial-mediated inflammation when the eyes or brain are damaged. CB2-knockout mice have exacerbated inflammation and retinal pathology during experimental PVR. We aimed to assess the anti-inflammatory effects of CB2 stimulation in the context of retinal damage and also explore the mechanistic roles of CB2 in microglia function. To target CB2, we used a highly selective agonist, HU-308, as well as its enantiomer, HU-433, which is a putative selective agonist. First, β-arrestin2 and Gαi recruitment was measured to compare activation of human CB2 in an in vitro heterologous expression system. Both agonists were then utilized in a mouse model of PVR, and the effects on retinal damage, inflammation, and cell death were assessed. Finally, we used an in vitro model of microglia to determine the effects of HU-308 and HU-433 on phagocytosis, cytokine release, migration, and intracellular signaling. We observed that HU-308 more strongly recruited both β-arrestin2 and Gαi compared to HU-433. Stimulation of CB2 with either drug effectively blunted LPS- and IFNγ-mediated signaling as well as NO and TNF release from microglia. Furthermore, both drugs reduced IL-6 accumulation, total caspase-3 cleavage, and retinal pathology following the induction of PVR. Ultimately, this work supports that CB2 is a valuable target for drugs to suppress inflammation and cell death associated with infection or sterile retinopathy, although the magnitude of effector recruitment may not be predictive of anti-inflammatory capacity.

Microglia, the resident immune cells of the brain, regulate the balance of inflammation in the central nervous system under healthy and pathogenic conditions. Huntington's disease (HD) is a chronic neurodegenerative disease characterized by activated microglia and elevated concentrations of pro-inflammatory cytokines within the brain. Chronic hyperactivation of microglia is associated with brain pathology and eventual neuron death. However, it is unclear which specific cytokines are required for neuron death and whether HD neurons may be hypersensitive to neuroinflammation. We assessed the profile of microglia-secreted proteins in response to LPS and IFNγ, and a conditioned media paradigm was used to examine the effects of these secreted proteins on cultured neuronal cells. STHdhQ7/Q7 and STHdhQ111/Q111 neuronal cells were used to model wild-type and HD neurons, respectively. We determined that STHdhQ111/Q111 cells were hypersensitive to pro-inflammatory factors secreted by microglia, and that TNF was required to induce neuronal death. Microglia-mediated neuronal death could be effectively halted through the use of JAK-STAT or TNF inhibitors which supported the requirement for TNF as well as IFNγ in the process of secondary neurotoxicity. Further data derived from human HD patients as well as HD mice were suggestive of enhanced receptor density for TNF (TNFR1) and IFNγ (IFNGR) which could sensitize the HD brain to these cytokines. This highlights several potential mechanisms by which microglia may induce neuronal death and suggests that these mechanisms may be upregulated in the brain of HD patients.

OBJECTIVE:Family history is an established risk factor for mental illness. The authors sought to investigate whether polygenic scores (PGSs) can complement family history to improve identification of risk for major mood and psychotic disorders. METHODS:Eight cohorts were combined to create a sample of 1,884 participants ages 2-36 years, including 1,339 offspring of parents with mood or psychotic disorders, who were prospectively assessed with diagnostic interviews over an average of 5.1 years. PGSs were constructed for depression, bipolar disorder, anxiety, attention deficit hyperactivity disorder (ADHD), schizophrenia, neuroticism, subjective well-being, p factor, and height (as a negative control). Cox regression was used to test associations between PGSs, family history of major mental illness, and onsets of major mood and psychotic disorders. RESULTS:There were 435 onsets of major mood and psychotic disorders across follow-up. PGSs for neuroticism (hazard ratio=1.23, 95% CI=1.12-1.36), schizophrenia (hazard ratio=1.15, 95% CI=1.04-1.26), depression (hazard ratio=1.11, 95% CI=1.01-1.22), ADHD (hazard ratio=1.10, 95% CI=1.00-1.21), subjective well-being (hazard ratio=0.90, 95% CI=0.82-0.99), and p factor (hazard ratio=1.14, 95% CI=1.04-1.26) were associated with onsets. After controlling for family history, neuroticism PGS remained significantly positively associated (hazard ratio=1.19, 95% CI=1.08-1.31) and subjective well-being PGS remained significantly negatively associated (hazard ratio=0.89, 95% CI=0.81-0.98) with onsets. CONCLUSIONS:Neuroticism and subjective well-being PGSs capture risk of major mood and psychotic disorders that is independent of family history, whereas PGSs for psychiatric illness provide limited predictive power when family history is known. Neuroticism and subjective well-being PGSs may complement family history in the early identification of persons at elevated risk.

Microglia are resident immune cells of the brain that survey the microenvironment, provide trophic support to neurons, and clear debris to maintain homeostasis and healthy brain function. Microglia are also drivers of neuroinflammation in several neurodegenerative diseases. Microglia produce endocannabinoids and express both cannabinoid receptor subtypes suggesting that this system is a target to suppress neuroinflammation. We tested whether cannabinoid type 1 (CB1) or type 2 (CB2) receptors could be targeted selectively or in combination to dampen the pro-inflammatory behavior of microglia, and whether this would have functional relevance to decrease secondary neuronal damage. We determined that components of the endocannabinoid system were altered when microglia are treated with lipopolysaccharide and interferon-gamma and shift to a pro-inflammatory phenotype. Furthermore, pro-inflammatory microglia released cytotoxic factors that induced cell death in cultured STHdhQ7/Q7 neurons. Treatment with synthetic cannabinoids that were selective for CB1 receptors (ACEA) or CB2 receptors (HU-308) dampened the release of nitric oxide (NO) and pro-inflammatory cytokines and decreased levels of mRNA for several pro-inflammatory markers. A nonselective agonist (CP 55,940) exhibited similar influence over NO release but to a lesser extent relative to ACEA or HU-308. All three classes of synthetic cannabinoids ultimately reduced the secondary damage to the cultured neurons. The mechanism for the observed neuroprotective effects appeared to be related to cannabinoid-mediated suppression of MAPK signaling in microglia. Taken together, the data indicate that activation of CB1 or CB2 receptors interfered with the pro-inflammatory activity of microglia in a manner that also reduced secondary damage to neurons.

Perturbation of the endocannabinoid system can have profound effects on immune function and synaptic plasticity. Microglia are one of few cell types with a self-contained endocannabinoid system and are positioned at the interface between the immune system and the central nervous system. Past work has produced conflicting results with respect to the effects of pro-inflammatory conditions on the microglial endocannabinoid system. Thus, we systematically investigated the relationship between the concentration of two distinct pro-inflammatory stimuli, lipopolysaccharide and interferon gamma, on the abundance of components of the endocannabinoid system within microglia. Here we show that lipopolysaccharide and interferon gamma influence messenger RNA abundances of the microglial endocannabinoid system in a concentration-dependent manner. Furthermore, we demonstrate that the efficacy of different synthetic cannabinoid treatments with respect to inhibition of microglia nitric oxide release is dependent on the concentration and type of pro-inflammatory stimuli presented to the microglia. This indicates that different pro-inflammatory stimuli influence the capacity of microglia to synthesize, degrade, and respond to cannabinoids which has implications for the development of cannabinoid-based treatments for neuroinflammation.

Microglia, the resident immune cells of the brain, can take on a range of pro- or anti-inflammatory phenotypes to maintain homeostasis. However, the sustained activation of pro-inflammatory microglia can lead to a state of chronic neuroinflammation characterized by high concentrations of neurotoxic soluble factors throughout the brain. In healthy brains, the inflammatory processes cease and microglia transition to an anti-inflammatory phenotype, but failure to halt the pro-inflammatory processes is a characteristic of many neurological disorders. The endocannabinoid system has been identified as a promising therapeutic target for chronic neuroinflammation as there is evidence that synthetic and endogenously produced cannabinoids temper the pro-inflammatory response of microglia and may encourage a switch to an anti-inflammatory phenotype. Activation of cannabinoid type 2 (CB2) receptors has been proposed as the mechanism of action responsible for these effects. The abundance of components of the endocannabinoid system in microglia also change dynamically in response to several brain pathologies. This can impact the ability of microglia to synthesize and degrade endocannabinoids or react to endogenous and exogenous cannabinoids. Cannabinoid receptors also participate in the formation of receptor heteromers which influences their function specifically in cells that express both receptors, such as microglia. This creates opportunities for drug-drug interactions between CB2 receptor-targeted therapies and other classes of drugs. In this article, we review the roles of pro- and anti-inflammatory microglia in the development and resolution of neuroinflammation. We also discuss the fluctuations observed in the components of the endocannabinoid in microglia and examine the potential of CB2 receptors as a therapeutic target in this context.

Type 1 and 2 cannabinoid receptors respond to endocannabinoids and contribute broadly to the function of the nervous system and immune system. Cannabinoid receptors participate in higher-order complex with other class A G protein-coupled receptors in both heterologous expression systems and native mammalian tissues. Receptor heteromers engage in protein-protein allosteric interactions that influence the conformation of receptor monomers within the complex. These altered conformations cause receptor heteromers to respond differently to cognate ligands compared to the individual receptors. This creates additional diversity in cell signaling that must be considered pharmacologically. In this chapter, we provide an overview of known cannabinoid receptor heteromers and signaling changes related to heteromer function. We also discuss the allosteric processes responsible for these effects and the therapeutic potential of this rapidly expanding group of receptor heteromers.

The human brain requires adequate cerebral blood flow to meet the high demand for nutrients and to clear waste products. With age, there is a chronic reduction in cerebral blood flow in small resistance arteries that can eventually limit proper brain function. The endothelin system is a key mediator in the regulation of cerebral blood flow, but the contributions of its constituent receptors in the endothelial and vascular smooth muscle layers of cerebral arteries have not been well defined in the context of aging. We isolated posterior cerebral arteries from young and aged Fischer 344 rats, as well as ETB receptor knock-out rats and mounted the vessels in plexiglass pressure myograph chambers to measure myogenic tone in response to increasing pressure and targeted pharmacological treatments. We used an ETA receptor antagonist (BQ-123), an ETB receptor antagonist (BQ-788), endothelin-1, an endothelin-1 synthesis inhibitor (phosphoramidon), and vessel denudation to dissect the roles of each receptor in aging vasculature. Aged rats exhibited a higher myogenic tone than young rats, and the tone was sensitive to the ETA antagonist, BQ-123, but insensitive to the ETB antagonist, BQ-788. By contrast, the tone in the vessels from young rats was raised by BQ-788 but unaffected by BQ-123. When the endothelial layer that is normally enriched with ETB1 receptors was removed from young vessels, myogenic tone increased. However, denudation of the endothelial layer did not influence vessels from aged animals. This indicated that endothelial ETB1 receptors were not functional in the vessels from aged rats. There was also an increase in ETA receptor expression with age, whereas ETB receptor expression remained constant between young and aged animals. These results demonstrate that in young vessels, ETB1 receptors maintain a lower myogenic tone, but in aged vessels, a loss of ETB receptor activity allows ETA receptors in vascular smooth muscle cells to raise myogenic tone. Our findings have potentially important clinical implications for treatments to improve cerebral perfusion in older adults with diseases characterized by reduced cerebral blood flow.

BACKGROUND: Methylenetetrahydrofolate reductase, the encoded by the MTHFR gene, plays a crucial role in converting the amino acid homocysteine to methionine. Two polymorphisms of the MTHFR gene, C677T and A1298C, reportedly reduce enzyme activity, resulting in hyperhomocysteinemia. Patients with C677T and A1298C polymorphisms may be at higher risk for developing abnormal hyperhomocysteinemia, which has been linked to catastrophic neurological including fatal outcomes. OBJECTIVE: Determine the prevalence of the MTHFR gene variants C677T and A1298C among pediatric dental patients treated at King Abdulaziz University Hospital. DESIGN: Cross-sectional. SETTING: Clinics of pediatric dentistry department. SUBJECTS AND METHODS: Healthy Saudi children 6–12 years old with no known allergies were screened for eligibility between May and December 2019. A single investigator collected saliva samples. The MTHFR C677T and A1298C polymorphisms were analyzed using polymerase chain reaction and restriction fragment length polymorphism. MAIN OUTCOME MEASURE: The prevalence of MTHFR gene variants (C677T and A1298C) among the subjects. SAMPLE SIZE: 138. RESULTS: MTHFR C677T polymorphism was present in 36.2% of the sample and 90.0% of children carrying this allele were heterozygotes. MTHFR A1298C polymorphism was present in 91.3% of the sample and 77.0% of the children carrying this allele were heterozygotes. No linkage disequilibrium between MTHFR C677T and MTHFR A1298C was observed within this sample. CONCLUSIONS: Our study found a high frequency of the MTHFR A1298C genotype, which was substantially more abundant than expected based on a Hardy-Weinberg distribution. Therefore, caution is advised in using N 2 O in Saudi children as the increased prevalence of this MTHFR allele may increase the incidence of serious adverse effects among these children. LIMITATIONS: Further studies are recommended with a larger sample size from randomly selected hospitals from different regions of Saudi Arabia. CONFLICT OF INTEREST: None.

Antipsychotics help alleviate the positive symptoms associated with schizophrenia; however, their debilitating side effects have spurred the search for better treatment options. Novel compounds can be screened for antipsychotic potential in neuronal cell cultures and following acute N-methyl-D-aspartate (NMDA) receptor blockade with non-competitive antagonists such as MK-801 in rodent behavioral models. Given the known interactions between NMDA receptors and type 1 cannabinoid receptors (CB1R), compounds that modulate CB1Rs may have therapeutic potential for schizophrenia. This study assessed whether the CB1R positive allosteric modulator GAT211, when compared to ∆9-tetrahydrocannabinol (THC), has potential to reduce psychiatric behavioral phenotypes following acute MK-801 treatment in rats, and block hyperdopaminergic signalling associated with those behaviors. The effects of GAT211 and THC on cellular signaling were compared in Neuro2a cells, and behavioral effects of GAT211 and THC on altered locomotor activity and prepulse inhibition of the acoustic startle response caused by acute MK-801 treatment were assessed in male, Long Evans rats. GAT211 limited dopamine D2 receptor-mediated extracellular regulated kinase (ERK) phosphorylation in Neuro2a cells, whereas THC did not. As expected, acute MK-801 (0.15 mg/kg) produced a significant increase in locomotor activity and impaired PPI. GAT211 treatment alone (0.3–3.0 mg/kg) dose-dependently reduced locomotor activity and the acoustic startle response. GAT211 (3.0 mg/kg) also prevented hyperlocomotion caused by MK-801 but did not significantly affect PPI impairments. Taken together, these findings support continued preclinical research regarding the usefulness of CB1R positive allosteric modulators as antipsychotics.

Attention-deficit/hyperactivity disorder (ADHD) and lower cognitive ability have been linked with increased likelihood of exposure to adversity. We hypothesized that these associations may be partly due to genetic factors. We calculated polygenic scores for ADHD and intelligence and assessed psychopathology and general cognitive ability in a sample of 297 youth aged 5–27 years enriched for offspring of parents with mood and psychotic disorders. We calculated an adversity score as a mean of 10 indicators, including socio-economic disadvantage, childhood maltreatment and bullying. We tested the effects of polygenic scores, externalizing symptoms and IQ on adversity scores using mixed-effects linear regression. Externalizing symptoms and general cognitive ability showed expected positive and negative relationships with adversity, respectively. Polygenic scores for intelligence were unrelated to adversity, but polygenic scores for ADHD were associated with adversity (β = 0.23, 95% CI 0.13 to 0.34, p  < .0001). ADHD polygenic scores uniquely explained 4.0% of variance in adversity score. The relationship between polygenic scores for ADHD and adversity was independently significant among individuals with (β = 0.49, 95% CI 0.25 to 0.75, p  < .0001) and without (β = 0.14, 95% CI 0.02 to 0.26, p  = .022) ADHD. A genetic score indexing liability to ADHD was associated with exposure to adversity in early life. Previously observed associations between externalizing symptoms, lower cognitive ability and adversity may be partially attributed to genetic liability to ADHD.

The endothelin receptor A (ETA) and endothelin receptor B (ETB) are G protein-coupled receptors that are co-expressed in vascular smooth muscle cells. Endothelin-1 (ET-1) activates endothelin receptors to cause microvascular vasoconstriction. Previous studies have shown that heteromerization between ETA and ETB prolongs Ca2+ transients, leading to prolongation of Gαq-dependent signaling and sustained vasoconstriction. We hypothesized that these effects are in part mediated by the resistance of ETA/ETB heteromers to β-arrestin recruitment and subsequent desensitization. Using bioluminescence resonance energy transfer 2 (BRET2), we found that ETB has a relatively equal affinity to form either homomers or heteromers with ETA when co-expressed in the human embryonic kidney 293 (HEK293) cells. When co-expressed, activation of ETA and ETB by ET-1 caused a heteromer-specific reduction and delay in β-arrestin-2 recruitment with a corresponding reduction and delay in ET-1-induced ETA/ETB co-internalization. Furthermore, the co-expression of ETA and ETB inhibited ET-1-induced β-arrestin-1-dependent extracellular signal-regulated kinase (ERK) phosphorylation while prolonging ET-1-induced Gαq-dependent ERK phosphorylation. ETA/ETB heteromerization mediates the long-lasting vasoconstrictor response to ET-1 by the prolongation of Gαq-dependent signaling and inhibition of β-arrestin function.

The cannabinoid type 1 (CB1) receptor and the dopamine type 2 (D2) receptor are co‐localized on medium spiny neuron terminals in the globus pallidus where they modulate neural circuits involved in voluntary movement. Physical interactions between the two receptors have been shown to alter receptor signaling in cell culture. The objectives of the current study were to identify the presence of CB1/D2 heteromers in the globus pallidus of C57BL/6J male mice, define how CB1/D2 heteromer levels are altered following treatment with cannabinoids and/or antipsychotics, and determine if fluctuating levels of CB1/D2 heteromers have functional consequences. Using in situ proximity ligation assays, we observed CB1/D2 heteromers in the globus pallidus of C57BL/6J mice. The abundance of the heteromers increased following treatment with the nonselective cannabinoid receptor agonist, CP55,940. In contrast, treatment with the typical antipsychotic haloperidol reduced the number of CB1/D2 heteromers, whereas the atypical antipsychotic olanzapine treatment had no effect. Co‐treatment with CP55,940 and haloperidol had similar effects to haloperidol alone, whereas co‐treatment with CP55,940 and olanzapine had similar effects to CP55,940. The observed changes were found to have functional consequences as the differential effects of haloperidol and olanzapine also applied to γ‐aminobutyric acid release in STHdhQ7/Q7 cells and motor function in C57BL/6J male mice. This work highlights the clinical relevance of co‐exposure to cannabinoids and different antipsychotics over acute and prolonged time periods.

BACKGROUND:Cannabis use is a risk factor for severe mental illness. However, cannabis does not affect everyone equally. Genetic information may help identify individuals who are more vulnerable to the harmful effects of cannabis on mental health. A common genetic variant within the AKT1 gene selectively increases risk of psychosis, only among those who use cannabis. Therapeutically oriented genetic counselling may enable us to reduce cannabis exposure among genetically sensitive individuals.METHODS:Using a trial-within-cohort design, we aim to test if genetic counselling, including the option to receive AKT1 rs2494732 genotype, reduces cannabis use. To this end, we have designed a genetic counselling intervention: Interdisciplinary approach to Maximize Adolescent potential: Genetic counselling Intervention to reduce Negative Environmental effects (IMAGINE).RESULTS:IMAGINE will be implemented in a cohort of children and youth enriched for familial risk for major mood and psychotic disorders. Approximately 110 eligible individuals aged 12-21 years will be randomized in a 1:1 ratio to be offered a single genetic counselling session with a board-certified genetic counsellor, or not. Allocated youth will also be invited to attend a follow-up session approximately 1 month following the intervention. The primary outcome will be cannabis use (measured by self-report or urine screen) at subsequent annual assessments as part of the larger cohort study. Secondary outcomes include intervention acceptability and psychopathology.CONCLUSION:This study represents the first translational application of a gene-environment interaction to improve mental health and test an intervention with potential public health benefits. This study is registered with clinicaltrials.gov (NCT03601026).

G protein-coupled receptors (GPCRs) are the target for many drugs. Evidence continues to accumulate demonstrating that multiple receptors form homo- and heteromeric complexes, which in turn dynamically couple with G proteins, and other interacting proteins. Here, we describe a method to simultaneously determine the identity of up to four distinct constituents of GPCR complexes using a combination of sequential bioluminescence resonance energy transfer 2-fluorescence resonance energy transfer (SRET2) with bimolecular fluorescence complementation (BiFC). The method is amenable to moderate throughput screening of changes in response to ligands and time-course analysis of protein-protein oligomerization.

GLT-1 is the major glutamate transporter in the brain, and is expressed in astrocytes and in axon terminals in the hippocampus, cortex, and striatum. Neuronal GLT-1 accounts for only 5-10% of total brain GLT-1 protein, and its function is uncertain. In HD, synaptic dysfunction of the corticostriate synapse is well-established. Transcriptional dysregulation is a key feature of HD. We hypothesized that deletion of neuronal GLT-1, because it is expressed in axon terminals in the striatum, might produce a synaptopathy similar to that present in HD. If true, then some of the gene expression changes observed in HD might also be observed in the neuronal GLT-1 knockout. In situ hybridization using 33P labeled oligonucleotide probes was carried out to assess localization and expression of a panel of genes known to be altered in expression in HD. We found changes in the expression of cannabinoid receptors 1 and 2, preproenkaphalin, and PDE10A in the striatum of mice in which the GLT-1 gene was inactivated in neurons by expression of synapsin-Cre, compared to wild-type littermates. These changes in expression were observed at 12 weeks of age but not at 6 weeks of age. No changes in DARPP-32, PDE1B, NGFIA, or β-actin expression were observed. In addition, we found widespread alteration in expression of the dynamin 1 gene. The changes in expression in the neuronal GLT-1 knockout of genes thought to exemplify HD transcriptional dysregulation suggest an overlap in the synaptopathy caused by neuronal GLT-1 deletion and HD. These data further suggest that specific changes in expression of cannabinoid receptors, preproenkephalin, and PDE10A, considered to be the hallmark of HD transcriptional dysregulation, may be produced by an abnormality of glutamate homeostasis under the regulation of neuronal GLT-1, or a synaptic disturbance caused by that abnormality, independently of mutation in huntingtin.

In addition to its known actions as a non-selective cyclooxygenase (COX) 1 and 2 inhibitor, we hypothesized that indomethacin can act as an allosteric modulator of the type 1 cannabinoid receptor (CB1R) because of its shared structural features with the known allosteric modulators of CB1R. Indomethacin enhanced the binding of [3H]CP55940 to hCB1R and enhanced AEA-dependent [35S]GTPγS binding to hCB1R in Chinese hamster ovary (CHO) cell membranes. Indomethacin (1 μM) also enhanced CP55940-dependent βarrestin1 recruitment, cAMP inhibition, ERK1/2 and PLCβ3 phosphorylation in HEK293A cells expressing hCB1R, but not in cells expressing hCB2R. Finally, indomethacin enhanced the magnitude and duration of CP55940-induced hypolocomotion, immobility, hypothermia, and anti-nociception in C57BL/6J mice. Together, these data support the hypothesis that indomethacin acted as a positive allosteric modulator of hCB1R. The identification of structural and functional features shared amongst allosteric modulators of CB1R may lead to the development of novel compounds designed for greater CB1R or COX selectivity and compounds designed to modulate both the prostaglandin and endocannabinoid systems.

Aggressive behavior exerts an enormous impact on society remaining among the main causes of worldwide premature death. Effective primary interventions, relying on predictive models of aggression that show adequate sensitivity and specificity are currently lacking. One strategy to increase the accuracy and precision of prediction would be to include biological data in the predictive models. Clearly, to be included in such models, biological markers should be reliably associated with the specific trait under study (i.e., diagnostic biomarkers). Aggression, however, is phenotypically highly heterogeneous, an element that has hindered the identification of reliable biomarkers. However, current research is trying to overcome these challenges by focusing on more homogenous aggression subtypes and/or by studying large sample size of aggressive individuals. Further advance is coming by bioinformatics approaches that are allowing the integration of inter-species biological data as well as the development of predictive algorithms able to discriminate subjects on the basis of the propensity toward aggressive behavior. In this review we first present a brief summary of the available evidence on neuroimaging of aggression. We will then treat extensively the data on genetic determinants, including those from hypothesis-free genome-wide association studies (GWAS) and candidate gene studies. Transcriptomic and neurochemical biomarkers will then be reviewed, and we will dedicate a section on the role of metabolomics in aggression. Finally, we will discuss how biomarkers can inform the development of new pharmacological tools as well as increase the efficacy of preventive strategies.