Acute Cannabis Administration Transiently Reduces Mitochondrial DNA in Young Adults: Findings from a Secondary Analysis of a Double-Blind, Placebo-Controlled, Randomized Clinical Trial.

Background: Cannabis is one of the world's most commonly used substances; however, many questions remain unanswered as to how cannabis impacts the body. Recently, there has been a resurgence of research into the effects of plant-derived cannabinoids on mitochondrial health. In particular, a number of studies implicate mitochondrial-Δ9-tetrahydrocannabinol (Δ9-THC) interactions with altered memory, metabolism, and catalepsy in mice. Although the research in this field is expanding rapidly, there is little known about the effects of cannabis on mitochondria health in human subjects either in acute or chronic term use. Methods: Blood samples were obtained from a double-blind, placebo-controlled, parallel-group randomized clinical trial in which adults who regularly use cannabis (1-4 days/week) aged 19-25 years were randomized 2:1 to receive either an active (12.5% Δ9-THC) cigarette or placebo (<0.01% Δ9-THC) cigarette containing 750 mg of cannabis before driving simulator testing. DNA was extracted from whole blood using commercial spin columns, followed by measurement of mt-ND1, mt-ND4, and ß2M using quantitative polymerase chain reaction. One-way repeated measures analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test was used to observe changes in mitochondrial DNA (mtDNA) copy number over time. A two-tailed Pearsons R test was used to assess correlations between mtDNA copy number and cannabinoid levels (Δ9-THC and metabolites) in blood. Results: We found that exposure to active cannabis containing Δ9-THC, as opposed to placebo, was associated with an acute reduction in mitochondrial DNA copy number in whole blood at 15 min and 1 h after smoking. The observed decrease in mtDNA copy number negatively correlated with blood concentrations of 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and 11-Nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), the two primary metabolites of Δ9-THC, but not Δ9-THC itself. Further, the negative correlation between 11-OH THC and THC-COOH concentrations and mtDNA copy number was found in only a subgroup of participants who use cannabis infrequently, suggesting a tolerance effect. Conclusions: These results illuminate mitochondrial alterations attributed to Δ9-THC consumption, which may be mediated by metabolites. These results appear to suggest stronger effects in individuals who consume cannabis less frequently, suggesting some form of tolerance to the effects of Δ9-THC and its metabolites on mtDNA content in whole blood. Keywords: Mitochondria; mtDNA; cannabis; THC; THC metabolites; blood; THC-COOH; 11-OH-THC.

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

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: Bioluminescence resonance energy transfer (BRET) assays in HEK293 cells were performed to investigate G protein dissociation, ERK1/2 phosphorylation and ß-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. KEY RESULTS: 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. CONCLUSION AND IMPLICATIONS: 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 ß-arrestin 2 recruitment and internalisation. Thus, the spatiotemporal signalling of endogenous cannabinoids will not be retained in vivo.

In Vitro Characterization of 6-Methyl-3-(2-nitro-1-(thiophen-2-yl)ethyl)-2-phenyl-1H-indole (ZCZ011) at the Type 1 Cannabinoid Receptor: Allosteric Agonist or Allosteric Modulator?

Orthosteric activation of CB1 is known to cause a plethora of adverse side effects in vivo. Allosteric modulation is an exciting therapeutic approach and is hoped to offer improved therapeutic potential and a reduced on-target side effect profile compared to orthosteric agonists. This study aimed to systematically characterize the in vitro activity of the positive allosteric modulator ZCZ011, explicitly considering its effects on receptor regulation. HEK293 cells expressing hCB1 receptors were used to characterize ZCZ011 alone and in combination with orthosteric agonists. Real-time BRET approaches were employed for G protein dissociation, cAMP signaling, and ß-arrestin translocation. Characterization also included ERK1/2 phosphorylation (PerkinElmer AlphaLISA) and receptor internalization. ZCZ011 is an allosteric agonist of CB1 in all pathways tested, with a similar signaling profile to that of the partial orthosteric agonist Δ9-tetrahydrocannabinol. ZCZ011 also showed limited positive allosteric modulation in increasing the potency and efficacy of THC-induced ERK1/2 phosphorylation, ß-arrestin translocation, and receptor internalization. However, no positive allosteric modulation was observed for ZCZ011 in combination with either CP55940 or AMB-FUBINACA, in G protein dissociation, nor cAMP inhibition. Our study suggests that ZCZ011 is an allosteric agonist, with effects that are often difficult to differentiate from those of orthosteric agonists. Together with its pronounced agonist activity, the limited extent of ZCZ011 positive allosteric modulation suggests that further investigation into the differences between allosteric and orthosteric agonism is required, especially in receptor regulation end points.

Cannabidiol, but Not Δ9-Tetrahydrocannabinol, Has Strain- and Genotype-Specific Effects in Models of Psychosis.

Introduction: Cannabis use has been associated with an increased incidence of psychiatric disorders, yet the underlying neurobiological processes mediating these associations are poorly understood. Whereas exposure to Δ9-tetrahydrocannabinol (THC) has been associated with the development or exacerbation of psychosis, treatment with cannabidiol (CBD) has been associated with amelioration of psychosis. In this study, we demonstrate a complex effect of CBD in mouse models of psychosis, based on factors, including dose, strain, and genotype. Methods: Adult GluN1 knockdown (GluN1KD) and dopamine transporter knockout (DATKO) mice (almost equally balanced for male/female) were acutely treated with vehicle, THC (4 mg/kg), CBD (60, 120 mg/kg), or THC:CBD (1:15, 4:60 mg/kg) and tested in behavioral assays. Results: GluN1KD and DATKO mice displayed hyperactivity, impaired habituation, and sensorimotor gating, along with increased stereotypy and vertical activity. THC, alone and in combination with CBD, produced a robust "dampening" effect on the exploratory behavior regardless of strain or genotype. CBD exhibited a more complex profile. At 60 mg/kg, CBD had minimal effects on horizontal activity, but the effects varied in terms of directionality (increase vs. decrease) in other parameters; effects on stereotypic behaviors differ by genotype, while effects on vertical exploration differ by strain×genotype. CBD at 120 mg/kg had a "dampening" effect on exploration overall, except in GluN1KD mice, where no effect was observed. In terms of sensorimotor gating, both THC and CBD had minimal effects, except for 120 mg/kg CBD, which exacerbated the acoustic startle response. Conclusions: Here, we present a study that highlights the complex mechanism of phytocannabinoids, particularly CBD, in models of psychosis-like behavior. These data require careful interpretation, as agonism of the cannabinoid receptor 1 (CB1) resulting in a decrease in locomotion can be misinterpreted as "antipsychotic-like" activity in murine behavioral outputs of psychosis. Importantly, the THC-mediated decrease in hyperexploratory behavior observed in our models (alone or in combination) was not specific to the genetic mutants, but rather was observed regardless of strain or genotype. Furthermore, CBD treatment, when comparing mutants with their wild-type littermate controls, showed little to no "antipsychotic-like" activity in our models. Therefore, it is not only important to consider dose when designing/interpreting therapeutically driven phytocannabinoid studies, but also effects of strain or genetic vulnerability respective to the general population.

In vivo brain endocannabinoid metabolism is related to hippocampus glutamate and structure - a multimodal imaging study with PET, 1H-MRS, and MRI.

Dysregulation of hippocampus glutamatergic neurotransmission and reductions in hippocampal volume have been associated with psychiatric disorders. The endocannabinoid system modulates glutamate neurotransmission and brain development, including hippocampal remodeling. In humans, elevated levels of anandamide and lower activity of its catabolic enzyme fatty acid amide hydrolase (FAAH) are associated with schizophrenia diagnosis and psychotic symptom severity, respectively (Neuropsychopharmacol, 29(11), 2108-2114; Biol. Psychiatry 88 (9), 727-735). Although preclinical studies provide strong evidence linking anandamide and FAAH to hippocampus neurotransmission and structure, these relationships remain poorly understood in humans. We recruited young adults with and without psychotic disorders and measured FAAH activity, hippocampal glutamate and glutamine (Glx), and hippocampal volume using [11C]CURB positron emission tomography (PET), proton magnetic resonance spectroscopy (1H-MRS) and T1-weighted structural MRI, respectively. We hypothesized that higher FAAH activity would be associated with greater hippocampus Glx and lower hippocampus volume, and that these effects would differ in patients with psychotic disorders relative to healthy control participants. After attrition and quality control, a total of 37 participants (62% male) completed [11C]CURB PET and 1H-MRS of the left hippocampus, and 45 (69% male) completed [11C]CURB PET and hippocampal volumetry. Higher FAAH activity was associated with greater concentration of hippocampal Glx (F1,36.36 = 9.17, p = 0.0045; Cohen's f = 0.30, medium effect size) and smaller hippocampal volume (F1,44.70 = 5.94, p = 0.019, Cohen's f = 0.26, medium effect size). These effects did not differ between psychosis and healthy control groups (no group interaction). This multimodal imaging study provides the first in vivo evidence linking hippocampal Glx and hippocampus volume with endocannabinoid metabolism in the human brain.

Inhibition of a tonic inhibitory conductance in mouse hippocampal neurones by negative allosteric modulators of α5 subunit-containing γ-aminobutyric acid type A receptors: implications for treating cognitive deficits.

BACKGROUND: Multiple cognitive and psychiatric disorders are associated with an increased tonic inhibitory conductance that is generated by α5 subunit-containing γ-aminobutyric acid type A (α5 GABAA) receptors. Negative allosteric modulators that inhibit α5 GABAA receptors (α5-NAMs) are being developed as treatments for these disorders. The effects of α5-NAMs have been studied on recombinant GABAA receptors expressed in non-neuronal cells; however, no study has compared drug effects on the tonic conductance generated by native GABAA receptors in neurones, which was the goal of this study. METHODS: The effects of five α5-NAMs (basmisanil, Ono-160, L-655,708, α5IA, and MRK-016) on tonic current evoked by a low concentration of GABA were studied using whole-cell recordings in cultured mouse hippocampal neurones. Drug effects on current evoked by a saturating concentration of GABA and on miniature inhibitory postsynaptic currents (mIPSCs) were also examined. RESULTS: The α5-NAMs caused a concentration-dependent decrease in tonic current. The potencies varied as the inhibitory concentration for 50% inhibition (IC50) of basmisanil (127 nM) was significantly higher than those of the other compounds (0.4-0.8 nM). In contrast, the maximal efficacies of the drugs were similar (35.5-51.3% inhibition). The α5-NAMs did not modify current evoked by a saturating GABA concentration or mIPSCs. CONCLUSIONS: Basmisanil was markedly less potent than the other α5-NAMs, an unexpected result based on studies of recombinant α5 GABAA receptors. Studying the effects of α5 GABAA receptor-selective drugs on the tonic inhibitory current in neurones could inform the selection of compounds for future clinical trials.

Fatty acid amide hydrolase is lower in young cannabis users.

We have recently shown that levels of fatty acid amide hydrolase (FAAH), the enzyme that metabolizes the endocannabinoid anandamide, are lower in the brains of adult cannabis users (CUs) (34 ± 11 years of age), tested during early abstinence. Here, we examine replication of the lower FAAH levels in a separate, younger cohort (23 ± 5 years of age). Eighteen healthy volunteers (HVs) and fourteen CUs underwent a positron emission tomography scan using the FAAH radioligand [11 C]CURB. Regional [11 C]CURB binding was calculated using an irreversible two-tissue compartment model with a metabolite-corrected arterial plasma input function. The FAAH C385A genetic polymorphism (rs324420) was included as a covariate. All CUs underwent a urine screen to confirm recent cannabis use and had serum cannabinoids measured. One CU screened negative for cannabinoids via serum and was removed from analysis. All HVs reported less than five lifetime cannabis exposures more than a month prior to study initiation. There was a significant effect of group (F1,26 = 4.31; P = .048) when two A/A (rs324420) HVs were removed from analysis to match the genotype of the CU group (n = 16 HVs, n = 13 CUs). Overall, [11 C]CURB λk3 was 12% lower in CU compared with HV. Exploratory correlations showed that lower brain [11 C]CURB binding was related to greater use of cannabis throughout the past year. We confirmed our previous report and extended these findings by detecting lower [11 C]CURB binding in a younger cohort with less cumulative cannabis exposure.

CB1 allosteric modulators and their therapeutic potential in CNS disorders.

CB1 is the most abundant GPCR found in the mammalian brain. It has garnered considerable attention as a potential therapeutic drug target. CB1 is involved in a wide range of physiological and psychiatric processes and has the potential to be targeted in a wide range of disease states. However, most of the selective and non-selective synthetic CB1 agonists and antagonists/inverse agonists developed to date are primarily used as research tools. No novel synthetic cannabinoids are currently in the clinic for use in psychiatric illness; synthetic analogues of the phytocannabinoid THC are on the market to treat nausea and vomiting caused by cancer chemotherapy, along with off-label use for pain. Novel strategies are being explored to target CB1, but with emphasis on the elimination or mitigation of the potential psychiatric adverse effects that are observed by central agonism/antagonism of CB1. New pharmacological options are being pursued that may avoid these adverse effects while preserving the potential therapeutic benefits of CB1 modulation. Allosteric modulation of CB1 is one such approach. In this review, we will summarize and critically analyze both the in vitro characterization and in vivo validation of CB1 allosteric modulators developed to date, with a focus on CNS therapeutic effects.

A novel allosteric modulator of the cannabinoid CB1 receptor ameliorates hyperdopaminergia endophenotypes in rodent models.

The endocannabinoid system (eCBs) encompasses the endocannabinoids, their synthetic and degradative enzymes, and cannabinoid (CB) receptors. The eCBs mediates inhibition of neurotransmitter release and acts as a major homeostatic system. Many aspects of the eCBs are altered in a number of psychiatric disorders including schizophrenia, which is characterized by dysregulation of dopaminergic signaling. The GluN1-Knockdown (GluN1KD) and Dopamine Transporter Knockout (DATKO) mice are models of hyperdopaminergia, which display abnormal psychosis-related behaviors, including hyperlocomotion and changes in pre-pulse inhibition (PPI). Here, we investigate the ability of a novel CB1 receptor (CB1R) allosteric modulator, ABM300, to ameliorate these dysregulated behaviors. ABM300 was characterized in vitro (receptor binding, ß-arrestin2 recruitment, ERK1/2 phosphorylation, cAMP inhibition) and in vivo (anxiety-like behaviors, cannabimimetic effects, novel environment exploratory behavior, pre-pulse inhibition, conditioned avoidance response) to assess the effects of the compound in dysregulated behaviors within the transgenic models. In vitro, ABM300 increased CB1R agonist binding but acted as an inhibitor of CB1R agonist induced signaling, including ß-arrestin2 translocation, ERK phosphorylation and cAMP inhibition. In vivo, ABM300 did not elicit anxiogenic-like or cannabimimetic effects, but it decreased novelty-induced hyperactivity, exaggerated stereotypy, and vertical exploration in both transgenic models of hyperdopaminergia, as well as normalizing PPI in DATKO mice. The data demonstrate for the first time that a CB1R allosteric modulator ameliorates the behavioral deficits in two models of increased dopamine, warranting further investigation as a potential therapeutic target in psychiatry.

Consequences of NMDA receptor deficiency can be rescued in the adult brain.

N-methyl-D-aspartate receptors (NMDARs) are required to shape activity-dependent connections in the developing and adult brain. Impaired NMDAR signalling through genetic or environmental insults causes a constellation of neurodevelopmental disorders that manifest as intellectual disability, epilepsy, autism, or schizophrenia. It is not clear whether the developmental impacts of NMDAR dysfunction can be overcome by interventions in adulthood. This question is paramount for neurodevelopmental disorders arising from mutations that occur in the GRIN genes, which encode NMDAR subunits, and the broader set of mutations that disrupt NMDAR function. We developed a mouse model where a congenital loss-of-function allele of Grin1 can be restored to wild type by gene editing with Cre recombinase. Rescue of NMDARs in adult mice yields surprisingly robust improvements in cognitive functions, including those that are refractory to treatment with current medications. These results suggest that neurodevelopmental disorders arising from NMDAR deficiency can be effectively treated in adults.

Imaging Brain Fatty Acid Amide Hydrolase in Untreated Patients With Psychosis.

BACKGROUND: The brain's endocannabinoid system, the primary target of cannabis, has been implicated in psychosis. The endocannabinoid anandamide is elevated in cerebrospinal fluid of patients with schizophrenia. Fatty acid amide hydrolase (FAAH) controls brain anandamide levels; however, it is unknown if FAAH is altered in vivo in psychosis or related to positive psychotic symptoms. METHODS: Twenty-seven patients with schizophrenia spectrum disorders and 36 healthy control subjects completed high-resolution positron emission tomography scans with the novel FAAH radioligand [11C]CURB and structural magnetic resonance imaging. Data were analyzed using the validated irreversible 2-tissue compartment model with a metabolite-corrected arterial input function. RESULTS: FAAH did not differ significantly between patients with psychotic disorders and healthy control subjects (F1,62.85 = 0.48, p = .49). In contrast, lower FAAH predicted greater positive psychotic symptom severity, with the strongest effect observed for the positive symptom dimension, which includes suspiciousness, delusions, unusual thought content, and hallucinations (F1,26.69 = 12.42, p = .002; Cohen's f = 0.42, large effect). Shorter duration of illness (F1,26.95 = 13.78, p = .001; Cohen's f = 0.39, medium to large effect) and duration of untreated psychosis predicted lower FAAH (F1,26.95 = 6.03, p = .021, Cohen's f = 0.27, medium effect). These results were not explained by past cannabis exposure or current intake of antipsychotic medications. FAAH exhibited marked differences across brain regions (F7,112.62 = 175.85, p < 1 × 10-56; Cohen's f > 1). Overall, FAAH was higher in female subjects than in male subjects (F1,62.84 = 10.05, p = .002; Cohen's f = 0.37). CONCLUSIONS: This first study of brain FAAH in psychosis indicates that FAAH may represent a biomarker of disease state of potential utility for clinical studies targeting psychotic symptoms or as a novel target for interventions to treat psychotic symptoms.

The Trifluoromethyl Group as a Bioisosteric Replacement of the Aliphatic Nitro Group in CB1 Receptor Positive Allosteric Modulators.

The first generation of CB1 positive allosteric modulators (e.g., ZCZ011) featured a 3-nitroalkyl-2-phenyl-indole structure. Although a small number of drugs include the nitro group, it is generally not regarded as being "drug-like", and this is particularly true for aliphatic nitro groups. There are very few case studies where an appropriate bioisostere replaced a nitro group that had a direct role in binding. This may be indicative of the difficulty of replicating its binding interactions. Herein, we report the design and synthesis of ligands targeting the allosteric binding site on the CB1 cannabinoid receptor, in which a CF3 group successfully replaced the aliphatic NO2. In general, the CF3-bearing compounds were more potent than their NO2 equivalents and also showed improved in vitro metabolic stability. The CF3 analogue (1) with the best balance of properties was selected for further pharmacological evaluation. Pilot in vivo studies showed that (±)-1 has similar activity to (±)-ZCZ011, with both showing promising efficacy in a mouse model of neuropathic pain.

Serum Metabolomic Profiling Identifies Key Metabolic Signatures Associated With Pathogenesis of Alcoholic Liver Disease in Humans.

Alcoholic liver disease (ALD) develops in a subset of heavy drinkers (HDs). The goals of our study were to (1) characterize the global serum metabolomic changes in well-characterized cohorts of controls (Cs), HDs, and those with alcoholic cirrhosis (AC); (2) identify metabolomic signatures as potential diagnostic markers, and (3) determine the trajectory of serum metabolites in response to alcohol abstinence. Serum metabolic profiling was performed in 22 Cs, 147 HDs, and 33 patients with AC using ultraperformance liquid chromatography-tandem mass spectrometry. Hepatic gene expression was conducted in Cs (n = 16) and those with AC (n = 32). We found progressive changes in the quantities of metabolites from heavy drinking to AC. Taurine-conjugated bile acids (taurocholic acid [TCA], 127-fold; taurochenodeoxycholic acid [TCDCA], 131-fold; and tauroursodeoxycholic acid, 56-fold) showed more striking elevations than glycine-conjugated forms (glycocholic acid [GCA], 22-fold; glycochenodeoxycholic acid [GCDCA], 22-fold; and glycoursodeoxycholic acid [GUDCA], 11-fold). This was associated with increased liver cytochrome P450, family 7, subfamily B, member 1 and taurine content (more substrates); the latter was due to dysregulation of homocysteine metabolism. Increased levels of GCDCA, TCDCA, GCA, and TCA positively correlated with disease progression from Child-Pugh A to C and Model for End-Stage Liver Disease scores, whereas GCDCA, GCA, and GUDCA were better predictors of alcohol abstinence. The levels of glucagon-like peptide 1 (GLP-1) and fibroblast growth factor (FGF) 21 but not FGF19 were increased in HDs, and all three were further increased in those with AC. Conclusion: Serum taurine/glycine-conjugated bile acids could serve as noninvasive markers to predict the severity of AC, whereas GLP-1 and FGF21 may indicate a progression from heavy drinking to AC.

The F238L Point Mutation in the Cannabinoid Type 1 Receptor Enhances Basal Endocytosis via Lipid Rafts.

Defining functional domains and amino acid residues in G protein coupled receptors (GPCRs) represent an important way to improve rational drug design for this major class of drug targets. The cannabinoid type 1 (CB1) receptor is one of the most abundant GPCRs in the central nervous system and is involved in many physiological and pathophysiological processes. Interestingly, cannabinoid type 1 receptor with a phenylalanine 238 to leucine mutation (CB1F238L) has been already linked to a number of both in vitro and in vivo alterations. While CB1F238L causes significantly reduced presynaptic neurotransmitter release at the cellular level, behaviorally this mutation induces increased risk taking, social play behavior and reward sensitivity in rats. However, the molecular mechanisms underlying these changes are not fully understood. In this study, we tested whether the F238L mutation affects trafficking and axonal/presynaptic polarization of the CB1 receptor in vitro. Steady state or ligand modulated surface expression and lipid raft association was analyzed in human embryonic kidney 293 (HEK293) cells stably expressing either wild-type cannabinoid type 1 receptor (CB1wt) or CB1F238L receptor. Axonal/presynaptic polarization of the CB1F238L receptor was assessed in transfected primary hippocampal neurons. We show that in vitro the CB1F238L receptor displays increased association with lipid rafts, which coincides with increased lipid raft mediated constitutive endocytosis, leading to a reduction in steady state surface expression of the CB1F238L receptor. Furthermore, the CB1F238L receptor showed increased axonal polarization in primary hippocampal neurons. These data demonstrate that endocytosis of the CB1 receptor is an important mediator of axonal/presynaptic polarization and that phenylalanine 238 plays a key role in CB1 receptor trafficking and axonal polarization.

LncRNA AK054921 and AK128652 are potential serum biomarkers and predictors of patient survival with alcoholic cirrhosis.

BACKGROUND: Alcoholic liver disease (ALD) is one of the leading causes of chronic liver disease. Recent studies have demonstrated the roles of long noncoding RNAs (lncRNAs) in the pathogenesis of several disease processes. However, the roles of lncRNAs in patients with ALD remain unexplored. METHODS: Global profiling for human lncRNAs from peripheral blood RNA was performed in a well characterized cohort of healthy controls (HC, n=4), excessive drinkers without liver diseases (ED, n=4), and those with alcoholic cirrhosis with different severities (AC, n=12). The expression of unique lncRNA signatures were validated in a separate cohort of HC (n=17), ED (n=19), AC (n=48), and human liver tissues with ALD (n=19). RESULTS: Detailed analysis of plasma lncRNAs in AC subjects with different severities compared to HC identified 244 commonly up-regulated lncRNAs and 181 commonly down-regulated lncRNAs. We further validated top 20 most differentially up- and down-regulated lncRNAs in ED and AC as compared to HC and also determined the expression of selected lncRNAs in human liver tissues with or without AC. Among those lncRNAs, AK128652 and AK054921 were two of the most abundantly expressed lncRNAs in normal human plasma and liver, and their levels were significantly elevated in AC. The prognostic significance of AK128652 and AK054921 was determined in 48 subjects with AC; who were prospectively followed for 520 days. The expression of AK128652 and AK054921 was inversely associated with survival in patients with AC. CONCLUSIONS: LncRNAs AK054921 and AK128652 are potential biomarkers to predict the progression to ALD in those with excessive alcohol consumption and are predictors of survival with patients with alcoholic cirrhosis.

Quantity of alcohol drinking positively correlates with serum levels of endotoxin and markers of monocyte activation.

It is unknown if LPS (lipopolysaccharides) and markers of immune activation, soluble CD14 (sCD14) and CD163 (sCD163) are associated with the quantity of alcohol consumption. 148 subjects were enrolled (97 excessive drinkers (ED) and 51 controls). Time Line Follow-Back questionnaire was used to quantify the amount of alcohol consumed. Serum LPS, sCD14, and sCD163 were measured. Peripheral blood mononuclear cells (PBMCs) were also isolated. Compared to controls, ED had higher total drinks in the past 30 days, higher levels of LPS, sCD14 and sCD163. The levels of serum LPS, sCD14, and sCD163 were higher among ED with recent alcohol consumption (last drink <10 days before enrollment) compared to those without recent drinking. Similar bacterial genome copy numbers were detected in control and ED groups. We found that ethanol primed PBMCs for LPS-induced inflammatory responses. A positive correlation between serum LPS, sCD14, sCD163 and the quantity of alcohol drinking was observed after adjusting for covariates and that abstinence was associated with decline in the levels of LPS, sCD14 and sCd163. We found an increase in the levels of LPS and markers of monocyte activations in ED. Further studies are needed to determine whether these can be used as the biomarkers for excessive alcohol use.

Mitochondrial DNA-enriched microparticles promote acute-on-chronic alcoholic neutrophilia and hepatotoxicity.

Over the last several years, one of the major advances in the field of alcoholic liver disease research was the discovery that binge alcohol consumption induced neutrophilia and hepatic neutrophil infiltration in chronically ethanol-fed mice and human subjects with excessive alcohol use (EAU); however, the underlying mechanisms remain obscure. Here, we demonstrated that chronic EAU patients with a history of recent excessive drinking (EAU + RD) had higher serum levels of mitochondrial DNA (mtDNA)-enriched microparticles (MPs) than EAU without recent drinking (EAU - RD) and healthy controls, which correlated positively with circulating neutrophils. Similarly, mice with chronic-plus-binge (E10d + 1B) ethanol feeding also had markedly elevated serum levels of mtDNA-enriched MPs, with activation of hepatic ER stress and inflammatory responses. Inhibition of ER stress by gene KO or inhibitors attenuated ethanol-induced elevation of mtDNA-enriched MPs, neutrophilia, and liver injury. The data from the study of hepatocyte-specific deletion of the protein kinase RNA-like ER kinase (Perk) gene in mice and of cultured hepatocytes demonstrated that hepatocytes were the main source of mtDNA-enriched MPs after ethanol feeding. Finally, administration of mtDNA-enriched MPs isolated from E10d+1B-fed mice caused neutrophilia in mice. In conclusion, E10d + 1B ethanol consumption activates hepatic ER stress-dependent mtDNA-enriched MP release, leading to neutrophilia and liver injury.

MicroRNA-223 ameliorates alcoholic liver injury by inhibiting the IL-6-p47phox-oxidative stress pathway in neutrophils.

OBJECTIVES: Chronic-plus-binge ethanol feeding activates neutrophils and exacerbates liver injury in mice. This study investigates how recent excessive drinking affects peripheral neutrophils and liver injury in alcoholics, and how miR-223, one of the most abundant microRNAs (miRNAs) in neutrophils, modulates neutrophil function and liver injury in ethanol-fed mice. DESIGNS: Three hundred alcoholics with (n=140) or without (n=160) recent excessive drinking and 45 healthy controls were enrolled. Mice were fed an ethanol diet for 10 days followed by a single binge of ethanol. RESULTS: Compared with healthy controls or alcoholics without recent drinking, alcoholics with recent excessive drinking had higher levels of circulating neutrophils, which correlated with serum levels of alanine transaminase (ALT) and aspartate transaminase (AST). miRNA array analysis revealed that alcoholics had elevated serum miR-223 levels compared with healthy controls. In chronic-plus-binge ethanol feeding mouse model, the levels of miR-223 were increased in both serum and neutrophils. Genetic deletion of the miR-223 gene exacerbated ethanol-induced hepatic injury, neutrophil infiltration, reactive oxygen species (ROS) and upregulated hepatic expression of interleukin (IL)-6 and phagocytic oxidase (phox) p47phox. Mechanistic studies revealed that miR-223 directly inhibited IL-6 expression and subsequently inhibited p47phox expression in neutrophils. Deletion of the p47phox gene ameliorated ethanol-induced liver injury and ROS production by neutrophils. Finally, miR-223 expression was downregulated, while IL-6 and p47phox expression were upregulated in peripheral blood neutrophils from alcoholics compared with healthy controls. CONCLUSIONS: miR-223 is an important regulator to block neutrophil infiltration in alcoholic liver disease and could be a novel therapeutic target for the treatment of this malady.

Development of indole sulfonamides as cannabinoid receptor negative allosteric modulators.

Existing CB1 negative allosteric modulators (NAMs) fall into a limited range of structural classes. In spite of the theoretical potential of CB1 NAMs, published in vivo studies have generally not been able to demonstrate the expected therapeutically-relevant CB1-mediated effects. Thus, a greater range of molecular tools are required to allow definitive elucidation of the effects of CB1 allosteric modulation. In this study, we show a novel series of indole sulfonamides. Compounds 5e and 6c (ABD1075) had potencies of 4 and 3nM respectively, and showed good oral exposure and CNS penetration, making them highly versatile tools for investigating the therapeutic potential of allosteric modulation of the cannabinoid system.

Mechanical and material properties of cortical and trabecular bone from cannabinoid receptor-1-null (Cnr1(-/-)) mice.

The endocannabinoid system is known for its regulatory effects on bone metabolism through the cannabinoid receptors, Cnr1 and Cnr2. In this study we analysed the mechanical and material properties of long bones from Cnr1(-/-) mice on a C57BL/6 background. Tibiae and femora from 5- and 12-week-old mice were subjected to three-point bending to measure bending stiffness and yield strength. Elastic modulus, density and mineral content were measured in the diaphysis. Second moment of area (MOA2), inner and outer perimeters of the cortical shaft and trabecular fractional bone volume (BV/TV) were measured using micro-CT. In Cnr1(-/-) males and females at both ages the bending stiffness was reduced due to a smaller MOA2. Bone from Cnr1(-/-) females had a greater modulus than wild-type controls, although no differences were observed in males. BV/TV of 12-week-old Cnr1(-/-) females was greater than controls, although no difference was seen at 5-weeks. On the contrary, Cnr1(-/-) males had the same BV/TV as controls at 12-weeks while they had significantly lower values at 5-weeks. This study shows that deleting Cnr1 decreases the amount of cortical bone in both males and females at 12-weeks, but increases the amount of trabecular bone only in females.