Discovery of V-0219: A Small-Molecule Positive Allosteric Modulator of the Glucagon-Like Peptide-1 Receptor toward Oral Treatment for "Diabesity".

Peptidic agonists of the glucagon-like peptide-1 receptor (GLP-1R) have gained a prominent role in the therapy of type-2 diabetes and are being considered for reducing food intake in obesity. Potential advantages of small molecules acting as positive allosteric modulators (PAMs) of GLP-1R, including oral administration and reduced unwanted effects, could improve the utility of this class of drugs. Here, we describe the discovery of compound 9 (4-{[1-({3-[4-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5-yl}methyl)piperidin-3-yl]methyl}morpholine, V-0219) that exhibits enhanced efficacy of GLP-1R stimulation, subnanomolar potency in the potentiation of insulin secretion, and no significant off-target activities. The identified GLP-1R PAM shows a remarkable in vivo activity, reducing food intake and improving glucose handling in normal and diabetic rodents. Enantioselective synthesis revealed oral efficacy for (S)-9 in animal models. Compound 9 behavior bolsters the interest of a small-molecule PAM of GLP-1R as a promising therapeutic approach for the increasingly prevalent obesity-associated diabetes.

Involvement of the ghrelin system in the maintenance and reinstatement of cocaine-motivated behaviors: a role of adrenergic action at peripheral ß1 receptors.

Cocaine addiction is a significant medical and public concern. Despite decades of research effort, development of pharmacotherapy for cocaine use disorder remains largely unsuccessful. This may be partially due to insufficient understanding of the complex biological mechanisms involved in the pathophysiology of this disorder. In the present study, we show that: (1) elevation of ghrelin by cocaine plays a critical role in maintenance of cocaine self-administration and cocaine-seeking motivated by cocaine-conditioned stimuli; (2) acquisition of cocaine-taking behavior is associated with the acquisition of stimulatory effects of cocaine by cocaine-conditioned stimuli on ghrelin secretion, and with an upregulation of ghrelin receptor mRNA levels in the ventral tegmental area (VTA); (3) blockade of ghrelin signaling by pretreatment with JMV2959, a selective ghrelin receptor antagonist, dose-dependently inhibits reinstatement of cocaine-seeking triggered by either cocaine or yohimbine in behaviorally extinguished animals with a history of cocaine self-administration; (4) JMV2959 pretreatment also inhibits brain stimulation reward (BSR) and cocaine-potentiated BSR maintained by optogenetic stimulation of VTA dopamine neurons in DAT-Cre mice; (5) blockade of peripheral adrenergic ß1 receptors by atenolol potently attenuates the elevation in circulating ghrelin induced by cocaine and inhibits cocaine self-administration and cocaine reinstatement triggered by cocaine. These findings demonstrate that the endogenous ghrelin system plays an important role in cocaine-related addictive behaviors and suggest that manipulating and targeting this system may be viable for mitigating cocaine use disorder.

Sudden cessation of fluoxetine before alcohol drinking reinstatement alters microglial morphology and TLR4/inflammatory neuroadaptation in the rat brain.

Preclinical studies on the effects of abrupt cessation of selective serotonin reuptake inhibitors (SSRIs), a medication often prescribed in alcohol use disorder (AUD) patients with depression, results in alcohol consumption escalation after resuming drinking. However, a potential neuroinflammatory component on this escalation remains unexplored despite the immunomodulatory role of serotonin. Here, we utilized a rat model of 14-daily administration of the SSRI fluoxetine (10 mg/kg/day) along alcohol self-administration deprivation to study the effects of fluoxetine cessation on neuroinflammation after resuming alcohol drinking. Microglial morphology and inflammatory gene expression were analyzed in prelimbic cortex, striatum, basolateral amygdala and dorsal hippocampus. Results indicated that alcohol drinking reinstatement increased microglial IBA1 immunoreactivity and altered morphometric features of activated microglia (fractal dimension, lacunarity, density, roughness, and cell area, perimeter and circularity). Despite alcohol reinstatement, fluoxetine cessation modified microglial morphology in a brain region-specific manner, resulting in hyper-ramified (spatial complexity of branching), reactive (lower heterogeneity and circularity)-like microglia. We also found that microglial cell area correlated with changes in mRNA expression of chemokines (Cx3cl1/fractalkine, Cxcl12/SDF1α, Ccl2/MCP1), cytokines (IL1ß, IL6, IL10) and the innate immune toll-like receptor 4 (TLR4) in dorsal hippocampus. Specifically, TLR4 correlated with microglial spatial complexity assessed by fractal dimension in striatum, suggesting a role in process branching. These findings suggest that alcohol drinking reinstatement after fluoxetine treatment cessation disturbs microglial morphology and reactive phenotype associated with a TLR4/inflammatory response to alcohol in a brain region-specific manner, facts that might contribute to alcohol-induced damage through the promotion of escalation of alcohol drinking behavior.

Abrupt cessation of reboxetine along alcohol deprivation results in alcohol intake escalation after reinstatement of drinking.

Major depression (MD) is a frequent comorbidity in alcohol use disorder (AUD) patients. Antidepressant prescription is often limited by poor clinical outcomes or unwanted side effects in comorbid AUD-MD patients. Recent studies suggest that abrupt cessation of selective serotonin reuptake inhibitors antidepressant treatment increases alcohol consumption after an alcohol deprivation period in rats. However, the appearance of this effect after the treatment with selective noradrenaline reuptake inhibitors (SNRIs) is not known. Here, we report that interruption of subchronic (14 days) treatment with the SNRIs reboxetine (15 mg/kg/day intraperitoneally) resulted in escalation of ethanol intake when the animals resume alcohol self-administration. This effect of reboxetine treatment cessation was associated with a profound deactivation of the endocannabinoid/acylethanolamide signaling system in the prefrontal cortex but not in the dorsal hippocampus, as reflected by the decrease in the protein expression of the cannabinoid CB1 receptor, the PPARα receptor, the 2-arachidonoylglycerol synthesizing enzymes DAGLα and DGALß, and the endocanabinoid degrading enzyme MAGL. This was associated with dysregulation of the expression of glutamic acid receptors GluN1, GluA1, and mGlu5 in the medial prefrontal cortex and the dorsal hippocampus of the animals exposed to reboxetine. The present results further support the idea that abrupt cessation of antidepressant therapy along alcohol deprivation time can boost alcohol intake after relapse through mechanisms associated with endocannabinoid/glutamate signaling dysregulation. This finding might be relevant for patients suffering AUD/MD comorbidity where antidepressant therapy must be monitored with caution for avoiding unwanted side effects if adherence to the treatment is not fully achieved.

Sex-specific behavioral and neurogenic responses to cocaine in mice lacking and blocking dopamine D1 or dopamine D2 receptors.

Adult neurogenesis in rodents is modulated by dopaminergic signaling and inhibited by cocaine. However, the sex-specific role of dopamine D1 and D2 receptors (D1R, D2R) in the deleterious effect of cocaine on adult neurogenesis has not been described yet. Here, we explored sex differences in (a) cell proliferation (5'-bromo-2'-deoxyuridine [BrdU]), (b) neural precursor (nestin), (c) neuronal phenotype (BrdU/ß3-tubulin), and (d) neuronal maturity (NeuN) in the subventricular zone (SVZ) of the lateral ventricles and striatum of mice with genetic deletion (D1-/- , D2-/- ) or pharmacological blockage (SCH23390: 0.1 mg/kg/day/5 days; Raclopride: 0.3 mg/kg/day/5 days) of D1R and D2R, and treated (10 mg/kg/day/5 days) and then challenged (5 mg/kg, 48 hr later) with cocaine. Results indicated that hyperactivity responses to cocaine were absent in D1-/- mice and reduced in SCH23390-treated mice. Activity responses to cocaine were reduced in D2-/- males, but absent in D2-/- females and increased in Raclopride-treated females. D1R deletion blocked the deleterious effect of cocaine on SVZ cell proliferation in males. Cocaine-exposed D1-/- males also had reduced neuronal phenotype of SVZ newborn cells and increased striatal neuronal maturity. D2-/- mice had lower proliferative and neural precursor responses. Cocaine in D2-/- females or coadministered with Raclopride in wild-type females improved SVZ cell proliferation, an effect that positively correlated with plasma brain-derived neurotrophic factor (BDNF) concentrations. In conclusion, the sex-specific D1R and D2R signaling on SVZ cell proliferation, neural progenitor and neuronal maturity is differentially perturbed by cocaine, and BDNF may be required to link D2R to neuroplasticity in cocaine addiction in females.

Cerebellar and cortical TLR4 activation and behavioral impairments in Wernicke-Korsakoff Syndrome: Pharmacological effects of oleoylethanolamide.

Wernicke-Korsakoff Syndrome (WKS) is a neuropsychiatric disorder whose etiology is a thiamine deficiency (TD), with alcoholism being the main underlying cause. Previous evidence suggests the presence of initial neuroinflammation and oxidative/nitrosative stress in the physiopathology, although the specific molecular mechanisms underlying TD-induced brain damage and behavioral disabilities are unknown. We explored the specific role of the innate immune receptor TLR4 in three murine models of WKS, based on the combination of a thiamine-deficient diet and pyrithiamine injections (0.25 mg/kg, i.p.) over time. The Symptomatic Model (SM) allowed us to describe the complete neurological/neurobehavioral symptomatology over 16 days of TD. Animals showed an upregulation of the TLR4 signaling pathway both in the frontal cortex (FC) and cerebellum and clear motor impairments related with cerebellar dysfunction. However, in the Pre-Symptomatic Model (PSM), 12 days of TD induced the TLR4 pathway upregulation in the FC, which correlated with disinhibited-like behavior, but not in the cerebellum, and no motor impairments. In addition, we tested the effects of the biolipid oleoylethanolamide (OEA, 10 mg/kg, i.p., once daily, starting before any symptom of the pathology is manifested) through the Glucose-Precipitated Model (GPM), which was generated by glucose loading (5 g/kg, i.v., last day) in thiamine-deficient animals to accelerate damage. Pretreatment with OEA prevented the TLR4-induced signature in the FC, as well as an underlying incipient memory disability and disinhibited-like behavior. This study suggests a key role for TLR4 in TD-induced neuroinflammation in the FC and cerebellum, and it reveals different vulnerability of these brain regions in WKS over time. Pre-treatment with OEA counteracts TD-induced TLR4-associated neuroinflammation and may serve as co-adjuvant therapy to prevent WKS-induced neurobehavioral alterations.

Adaptive Stress Coping in Awake Bruxism.

Numerous studies have analyzed the relationship between psychological factors and bruxism. However, the data are often obscured by the lack of precise diagnostic criteria and the variety of the psychological questionnaires used. The purpose of this study is to determine the association between awake bruxism and psychological factors (anxiety, depression, sociability, stress coping, and personality traits). With this aim, 68 participants (13 males) completed a battery of psychological questionnaires, a self-reported bruxism questionnaire, and a clinical examination. Based on their scores on the bruxism questionnaire and the clinical examination, subjects were divided into two groups. Subjects who met the criteria for "probable awake bruxism" were assigned to the case group (n = 29, five males). The control group (n = 39, nine males) was composed of subjects who showed no signs or symptoms of bruxism in the examination nor in the questionnaire. The probable awake bruxism group presented significantly higher levels of trait and state anxiety, symptoms of somatization, and neuroticism than the control group. Despite this, and when their problem coping strategies were considered, awake bruxers showed higher levels in Positive Reappraisal (p < 0.05), a strategy generally considered as adaptive. In conclusion, although awake bruxers in our study showed larger levels of anxiety, somatization, and neuroticism, they also displayed more adapted coping strategies, while according to previous data TMD patients (which generally also present high levels of anxiety, somatization and neuroticism) might tend to present less adaptive coping styles. Thus, awake bruxism may play a positive role in stress coping, which would be compatible with the hypothesis of mastication as a means of relieving psychological tension. This finding should be further confirmed by future research comparing TMD patients with definitive awake bruxers and controls and using larger and more representative samples.

Antianhedonic and Antidepressant Effects of Affron®, a Standardized Saffron (Crocus Sativus L.) Extract.

Anxiety and depression have high prevalence in the general population, affecting millions of people worldwide, but there is still a need for effective and safe treatments. Nutritional supplements have recently received a lot of attention, particularly saffron. Thus, several pre-clinical studies support a beneficial role for bioactive compounds, such as saffron, in anxiety and depression. Here we used an animal model of depression based on social isolation to assess the effects of affron®, a standardized saffron extract containing ≥3.5% of total bioactive compounds safranal and crocin isomers. Affron® was administered both through the oral and the intraperitoneal routes, and several tasks related to anxiety and depression, such as the elevated plus maze, the forced swimming test or the sucrose preference test, were assessed. These tasks model key features of depressive states and anxious states relating to fear, behavioral despair or anhedonia, the lack of motivation and/or pleasure from everyday activities, respectively. Animals receiving oral affron® displayed behaviors congruent with improvements in their anxious/depressive state, showing the enhanced consumption of a sweet solution, as well as an increase in certain escape responses in the forced swimming test. Our data support a beneficial role for oral saffron in anxious/depressive states.

Cessation of fluoxetine treatment increases alcohol seeking during relapse and dysregulates endocannabinoid and glutamatergic signaling in the central amygdala.

Administration of selective serotonin reuptake inhibitors (SSRIs), typically used as antidepressants, induces long-lasting behavioral changes associated with alcohol use disorder (AUD). However, the contribution of SSRI (fluoxetine)-induced alterations in neurobiological processes underlying alcohol relapse such as endocannabinoid and glutamate signaling in the central amygdala (CeA) remains largely unknown. We utilized an integrative approach to study the effects of repeated fluoxetine administration during abstinence on ethanol drinking. Gene expression and biochemical and electrophysiological studies explored the hypothesis that dysregulation in glutamatergic and endocannabinoid mechanisms in the CeA underlie the susceptibility to alcohol relapse. Cessation of daily treatment with fluoxetine (10 mg/kg) during abstinence resulted in a marked increase in ethanol seeking during re-exposure periods. The increase in ethanol self-administration was associated with (a) reductions in levels of the endocannabinoids N-arachidonoylethanolomine and 2-arachidonoylglycerol in the CeA, (b) increased amygdalar gene expression of cannabinoid type-1 receptor (CB1), N-acyl phosphatidylethanolamine phospholipase D (Nape-pld), fatty acid amid hydrolase (Faah), (c) decreased amygdalar gene expression of ionotropic AMPA (GluA2 and GluA4) and metabotropic (mGlu3) glutamate receptors, and (d) increased glutamatergic receptor function. Overall, our data suggest that the administration of the antidepressant fluoxetine during abstinence dysregulates endocannabinoid signaling and glutamatergic receptor function in the amygdala, facts that likely facilitate alcohol drinking behavior during relapse.

Sex-Dimorphic Behavioral Alterations and Altered Neurogenesis in U12 Intron Splicing-Defective Zrsr1 Mutant Mice.

Mutant mice with respect to the splicing factor Zrsr1 present altered spermatogenesis and infertility. To investigate whether Zrsr1 is involved in the homeostatic control that the hypothalamus exerts over reproductive functions, we first analyzed both differential gene and isoform expression and alternative splicing alterations in Zrsr1 mutant (Zrsr1mu) hypothalamus; second, we analyzed the spontaneous and social behavior of Zrsr1mu mice; and third, we analyzed adult cell proliferation and survival in the Zrsr1mu hypothalamus. The Zrsr1mu hypothalamus showed altered expression of genes and isoforms related to the glutathione metabolic process, synaptonemal complex assembly, mRNA transport, and altered splicing events involving the enrichment of U12-type intron retention (IR). Furthermore, increased IR in U12-containing genes related with the prolactin, progesterone, and gonadotropin-releasing hormone (GnRH) reproductive signaling pathway was observed. This was associated with a hyperactive phenotype in both males and females, with an anxious phenotype in females, and with increased social interaction in males, instead of the classical aggressive behavior. In addition, Zrsr1mu females but not males exhibited reduced cell proliferation in both the hypothalamus and the subventricular zone. Overall, these results suggest that Zrsr1 expression and function are relevant to organization of the hypothalamic cell network controlling behavior.

Bupropion, a possible antidepressant without negative effects on alcohol relapse.

RATIONALE: the role that antidepressants play on alcohol consumption is not well understood. Previous studies have reported that treatment with a Selective Serotonin Reuptake Inhibitor (SSRIs) increases alcohol consumption in an animal model of relapse, however it is unknown whether this effect holds for other antidepressants such as the atypical dopamine/norepinephrine reuptake inhibitors (SNDRI). OBJECTIVES: the main goal of the present study was to compare the effects of two classes of antidepressants drugs, bupropion (SNDRI) and fluoxetine (SSRI), on alcohol consumption during relapse. Since glutamatergic and endocannabinoid signaling systems plays an important role in alcohol abuse and relapse, we also evaluated the effects of both antidepressants onthe expression of the main important genes and proteins of both systems in the prefrontal cortex, a critical brain region in alcohol relapse. METHODS: rats were trained to self-administered alcohol. During abstinence, rats received a 14d-treatment with vehicle, fluoxetine (10 mg/kg) or bupropion (20 mg/kg), and we evaluated alcohol consumption during relapse for 3 weeks. Samples of prefrontal cortex were taken to evaluate the mRNA and protein expression of the different components of glutamatergic and endocannabinoid signaling systems. RESULTS: fluoxetine treatment induced a long-lasting increase in alcohol consumption during relapse, an effect that was not observed in the case of bupropion treatment. The observed increases in alcohol consumption were accompanied by distinct alterations in the glutamate and endocannabinoid systems. CONCLUSIONS: our results suggest that SSRIs can negatively impact alcohol consumption in relapse while SNDRIs have no effects. The observed increase in alcohol consumption are accompanied by functional alterations in the glutamatergic and endocannabinoid systems. This finding could open new strategies for the treatment of depression in patients with alcohol use disorders.

Role of the galanin N-terminal fragment (1-15) in anhedonia: Involvement of the dopaminergic mesolimbic system.

BACKGROUND: Anhedonia is a core feature of depressive disorders. The galanin N-terminal fragment (1-15) plays a role in mood regulation since it induces depression and anxiogenic-like effects in rats. In this study, we analysed galanin N-terminal fragment (1-15) actions in anhedonic-like behaviours in rats using operant and non-operant tests and the areas involved with these effects. METHODS: Galanin N-terminal fragment (1-15) effects were analysed in saccharin self-administration, sucrose preference, novelty-suppressed feeding and female urine sniffing tests. The areas involved in galanin N-terminal fragment (1-15)-mediated effects were studied with positron emission tomography for in vivo imaging, and we analysed the ventral tegmental area and nucleus accumbens. Galanin N-terminal fragment (1-15) had effects on the mRNA expression of the dopamine transporters Dat and Vmat2; the C-Fos gene; the dopamine receptors D1, D2, D3, D5; and the galanin receptors 1 and 2. RESULTS: Galanin N-terminal fragment (1-15) at a concentration of 3 nmol induced a strong anhedonia-like phenotype in all tests. The involvement of galanin receptor 2 was demonstrated with the galanin receptor 2 antagonist M871 (3 nmol). The 18F-fluorodeoxyglucose positron emission tomography images indicated the action of galanin N-terminal fragment (1-15) over several nuclei of the limbic system. Galanin N-terminal fragment (1-15)-mediated effects also involved changes in the expression of Dat, Vmat2, D3 and galanin receptors in the ventral tegmental area as well as the expression of C-Fos, D1, D2 and D3 and TH immunoreactivity in the nucleus accumbens. CONCLUSIONS: Our results indicated that galanin N-terminal fragment (1-15) exerts strong anhedonic-like effects and that this effect was accompanied by changes in the dopaminergic mesolimbic system. These results may provide a basis for the development of novel therapeutic strategies using galanin N-terminal fragment (1-15) analogues for the treatment of depression and reward-related diseases.

Oleoylethanolamide Modulates BDNF-ERK Signaling and Neurogenesis in the Hippocampi of Rats Exposed to Δ9-THC and Ethanol Binge Drinking During Adolescence.

Oleoylethanolamide is an endogenous NAE that modulates ethanol-seeking behavior and ethanol-induced neuroinflammation. In the present study we further analyze the role of OEA in hippocampal neurogenesis, BDNF-ERK signaling, and spatial memory that are affected by alcohol. Additionally, we addressed the effects of OEA on the association of alcohol and cannabis, a frequent combination in human alcohol addicts, and whose long-term effects are far from being understood. To this end, OEA (10 mg/kg/day, i.p.) was pharmacologically administered for 5 days/week in a preclinical model of adolescent rats with binge-like consumption (1 day/week) of ethanol (3 g/kg, i.g.) combined or not with acute administrations of Δ9-THC (5 mg/kg, i.p.) for 5 weeks. OEA restored ethanol/THC-related decreases in both short-term spatial memory (spontaneous alternation by Y-maze) and circulating levels of BDNF, reduced cell proliferation (Mki67 and IdU+ cells) and maturation (Dcx, Calb1), and improved cell survival (Casp3 and BrdU+ cells) in the dorsal hippocampus. Interestingly, OEA alone or combined with THC also decreased the mRNA levels of neurotrophic factors (Bdnf, Ntf3) and the NT3 receptor TrkC, but increased the BDNF receptor TrkB in the hippocampus of ethanol-exposed rats. These effects were likely associated with a OEA-specific phosphorylation of AKT and ERK1, key signaling regulators of cell proliferation and survival. These results suggest a regulatory role of OEA in short-term spatial memory and hippocampal neurogenesis through BDNF/AKT/ERK1 signaling in response to acute THC in an alcoholic context during adolescence.

Plasma concentrations of oleoylethanolamide in a primary care sample of depressed patients are increased in those treated with selective serotonin reuptake inhibitor-type antidepressants.

Oleoylethanolamide (OEA) is a non-cannabinoid acylethanolamide with multiple physiological roles that has been proposed to have antidepressant-like activity in preclinical models. OEA shares biosynthetic pathways with anandamide (AEA) a transmitter involved in affective disorders and anxiety in humans. However, although the participation of OEA in depression has been proposed, both, the contribution of OEA to the depressive phenotype and the effect of antidepressant therapy on circulating levels of this and related non-cannabinoid acylethanolamides in humans are basically unknown. The main objective of this study is to compare the plasma concentrations of OEA and related acylethanolamides in a sample of primary care patients with depression (n = 69) with those of healthy non-depressed patients (n = 47). At the time of admission to the study, 22 patients were under selective serotonin reuptake inhibitor (SSRI) antidepressant treatment and 47 patients were not receiving any type of intervention. In addition, plasma concentrations of the endocannabinoid 2-AG and two related monoacylglycerols were monitored. Plasma OEA concentrations were found to be elevated in depressed patients and to correlate with somatic symptoms of depression. Plasma concentrations of both, AEA and 2-AG, were found to be elevated also in depressed patients. Further analysis demonstrated that the elevation observed in the plasma concentrations of both, OEA and 2-AG, was associated to SSRI antidepressant therapy at the time of recruitment. Further clinical research is needed to understand whether SSRI-induced elevations in OEA levels contribute to the response to SSRI in depressed patients as described in preclinical models.

Alcohol-induced cognitive deficits are associated with decreased circulating levels of the neurotrophin BDNF in humans and rats.

Chronic alcohol consumption is associated with neurocognitive and memory deficits, dramatically affecting plasticity and connectivity, with maximal expression as dementia. Neurotrophic factors may contribute to alcohol-related cognitive decline. For further investigation, a cross-sectional study was performed to evaluate the association of cognitive impairment, by using frontal assessment battery, and memory loss, using memory failures everyday, with the circulating levels of the neurotrophin brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3) in abstinent subjects with alcohol use disorders (AUDs, N = 58, average of 17.9 years of problematic use and 4.3 months of abstinence) compared with healthy control subjects (N = 22). This association was also explored in a pre-clinical model of adolescent rats chronically exposed to alcohol up to adulthood (~77 days old) in a three-bottle free-choice (5-10-20 percent), repeated abstinence and relapse paradigm. AUD subjects had low educational level and cognitive impairment associated with teenage consumption and lower circulating levels of BDNF and NT-3. Only BDNF concentration showed a positive correlation with frontal assessment battery in AUD patients. In the ethanol-exposed rats, the plasma levels of BDNF and NT-3 were also decreased, and a negative correlation between hippocampal Bdnf mRNA levels and recognition memory was found. The ethanol-exposed rat hippocampus showed a decrease in the mRNA levels of neurotrophic (Bdnf and Ntf-3) and neurogenic (Mki67, Sox2, Dcx, Ncam1 and Calb1) factors, associated to a deactivation of the neurogenic regulator mitogen-activated protein kinase extracellular signal-regulated kinase. Results suggest a relevant role of BDNF/extracellular signal-regulated kinase 2 signaling in alcohol-induced cognitive impairment and suggest that early alcohol exposure-derived effects on cognition are associated with neurotrophin signaling deficits.

PPARα/CB1 receptor dual ligands as a novel therapy for alcohol use disorder: Evaluation of a novel oleic acid conjugate in preclinical rat models.

Recent studies have demonstrated the utility of drugs modulating the endogenous cannabinoid system to control excessive alcohol intake. Among them, drugs interacting with acylethanolamide receptors including cannabinoid CB1 receptor antagonists/inverse agonists, peroxisome proliferator-activated receptor alpha (PPARα) agonists or peroxisome proliferator-activated receptor gamma (PPARγ) agonists have demonstrated utility in the reduction of alcohol intake in animal models. However, few studies have addressed the potential utility of combining these classes of drugs, especially because of expected safety problems. In the present work we took the advantage of the availability of two novel dual ligands for these receptors, to test the hypothesis that these types of drugs might reproduce and even improve the pharmacological profile of those drugs interacting with single targets. To this end we tested (R)-3-[(4-Benzyl-2-oxooxazolidin-3-yl)methyl]-N-[4-(dodecylcarbamoyl)phenyl]benzamide (NF 10-360), a dual PPARα/γ agonist, and N-[1-(3,4-dihydroxyphenyl)propan-2-yl]oleamide (OLHHA), a dual CB1 receptor antagonist/PPARα agonist, in animal models of alcohol consumption. Both drugs were effective in reducing alcohol intake and alcohol self-administration, being OLHHA a very potent alcohol intake inhibitor (EC50 0.2 mg/kg). OLHHA also reduced self-administration of the opioid oxycodone. OLHHA actions on alcohol self-administration were replicated in alcohol-preferring Marchigian-Sardinian msP rats. Repeated administration of OLHHA did result neither in tolerance nor in toxicological or deleterious metabolic changes in the liver of msP rats. These data support the feasibility of developing novel dual ligands interacting with cannabinoid targets to treat alcohol use disorder in humans.

Impaired Spermatogenesis, Muscle, and Erythrocyte Function in U12 Intron Splicing-Defective Zrsr1 Mutant Mice.

The U2AF35-like ZRSR1 has been implicated in the recognition of 3' splice site during spliceosome assembly, but ZRSR1 knockout mice do not show abnormal phenotypes. To analyze ZRSR1 function and its precise role in RNA splicing, we generated ZRSR1 mutant mice containing truncating mutations within its RNA-recognition motif. Homozygous mutant mice exhibited severe defects in erythrocytes, muscle stretch, and spermatogenesis, along with germ cell sloughing and apoptosis, ultimately leading to azoospermia and male sterility. Testis RNA sequencing (RNA-seq) analyses revealed increased intron retention of both U2- and U12-type introns, including U12-type intron events in genes with key functions in spermatogenesis and spermatid development. Affected U2 introns were commonly found flanking U12 introns, suggesting functional cross-talk between the two spliceosomes. The splicing and tissue defects observed in mutant mice attributed to ZRSR1 loss of function suggest a physiological role for this factor in U12 intron splicing.

Systemic blockade of LPA1/3 lysophosphatidic acid receptors by ki16425 modulates the effects of ethanol on the brain and behavior.

The systemic administration of lysophosphatidic acid (LPA) LPA1/3 receptor antagonists is a promising clinical tool for cancer, sclerosis and fibrosis-related diseases. Since LPA1 receptor-null mice engage in increased ethanol consumption, we evaluated the effects of systemic administration of an LPA1/3 receptor antagonist (intraperitoneal ki16425, 20 mg/kg) on ethanol-related behaviors as well as on brain and plasma correlates. Acute administration of ki16425 reduced motivation for ethanol but not for saccharine in ethanol self-administering Wistar rats. Mouse experiments were conducted in two different strains. In Swiss mice, ki16425 treatment reduced both ethanol-induced sedation (loss of righting reflex, LORR) and ethanol reward (escalation in ethanol consumption and ethanol-induced conditioned place preference, CPP). Furthermore, in the CPP-trained Swiss mice, ki16425 prevented the effects of ethanol on basal c-Fos expression in the medial prefrontal cortex and on adult neurogenesis in the hippocampus. In the c57BL6/J mouse strain, however, no effects of ki16425 on LORR or voluntary drinking were observed. The c57BL6/J mouse strain was then evaluated for ethanol withdrawal symptoms, which were attenuated when ethanol was preceded by ki16425 administration. In these animals, ki16425 modulated the expression of glutamate-related genes in brain limbic regions after ethanol exposure; and peripheral LPA signaling was dysregulated by either ki16425 or ethanol. Overall, these results suggest that LPA1/3 receptor antagonists might be a potential new class of drugs that are suitable for treating or preventing alcohol use disorders. A pharmacokinetic study revealed that systemic ki16425 showed poor brain penetration, suggesting the involvement of peripheral events to explain its effects.

Oleoylethanolamide, Neuroinflammation, and Alcohol Abuse.

Neuroinflammation is a complex process involved in the physiopathology of many central nervous system diseases, including addiction. Alcohol abuse is characterized by induction of peripheral inflammation and neuroinflammation, which hallmark is the activation of innate immunity toll-like receptors 4 (TLR4). In the last years, lipid transmitters have generated attention as modulators of parts of the addictive process. Specifically, the bioactive lipid oleoylethanolamide (OEA), which is an endogenous acylethanolamide, has shown a beneficial profile for alcohol abuse. Preclinical studies have shown that OEA is a potent anti-inflammatory and antioxidant compound that exerts neuroprotective effects in alcohol abuse. Exogenous administration of OEA blocks the alcohol-induced TLR4-mediated pro-inflammatory cascade, reducing the release of proinflammatory cytokines and chemokines, oxidative and nitrosative stress, and ultimately, preventing the neural damage in frontal cortex of rodents. The mechanisms of action of OEA are discussed in this review, including a protective action in the intestinal barrier. Additionally, OEA blocks cue-induced reinstatement of alcohol-seeking behavior and reduces the severity of withdrawal symptoms in animals, together with the modulation of alcohol-induced depression-like behavior and other negative motivational states associated with the abstinence, such as the anhedonia. Finally, exposure to alcohol induces OEA release in blood and brain of rodents. Clinical evidences will be highlighted, including the OEA release and the correlation of plasma OEA levels with TLR4-dependent peripheral inflammatory markers in alcohol abusers. In base of these evidences we hypothesize that the endogenous release of OEA could be a homeostatic signal to counteract the toxic action of alcohol and we propose the exploration of OEA-based pharmacotherapies to treat alcohol-use disorders.

Increased plasma oleoylethanolamide and palmitoleoylethanolamide levels correlate with inflammatory changes in alcohol binge drinkers: the case of HMGB1 in women.

Alcohol binge drinking is a heavy pattern of alcohol consumption increasingly used by young people. In a previous study, we reported that young drinkers with a 2-year history of binge alcohol consumption had an overactivation of the innate immune system and peripheral inflammation when compared with controls. In the present study, we measured several biolipids that are fatty acid derivatives belonging to the acylethanolamide or 2-acylglycerol families in the plasma of the same subjects (n = 42; 20 men and 22 women). We found that during abstinence, alcohol binge drinkers had elevated plasma levels of oleoylethanolamide, palmitoleoylethanolamide, arachidonoylethanolamide, dihomo-γ-linolenoyl ethanolamide and linoleoyl ethanolamide, which positively correlated with changes in the mRNA expression of key inflammatory markers in peripheral blood mononuclear cells, such as toll-like receptors (TLR4), pro-inflammatory cytokines/chemokines interleukin-1 beta, interleukin-6 and monocyte chemoattractant protein-1, and cyclooxygenase-2. Additionally, plasma oleoylethanolamide positively correlated with plasma levels of high mobility group box-1, which is a danger-associated molecular pattern and an endogenous TLR4 agonist, specifically in female alcohol binge drinkers. No changes were observed in 2-acylglycerols in alcohol binge drinkers, although sex-related differences in these bioactive lipids as well as in palmitoleoylethanolamide and docosatetraenoylethanolamide levels were detected. These results extend the previous clinical findings observed in patients diagnosed with long-term alcohol use disorder to young users and suggest a prominent role for these lipids in the response to acute alcohol exposure.