Novel pyrrole based CB2 agonists: New insights on CB2 receptor role in regulating neurotransmitters' tone.

The cannabinoid system is one of the most investigated neuromodulatory systems because of its involvement in multiple pathologies such as cancer, inflammation, and psychiatric diseases. Recently, the CB2 receptor has gained increased attention considering its crucial role in modulating neuroinflammation in several pathological conditions like neurodegenerative diseases. Here we describe the rational design of pyrrole-based analogues, which led to a potent and pharmacokinetically suitable CB2 full agonist particularly effective in improving cognitive functions in a scopolamine-induced amnesia murine model. Therefore, we extended our study by investigating the interconnection between CB2 activation and neurotransmission in this experimental paradigm. To this purpose, we performed a MALDI imaging analysis on mice brains, observing that the administration of our lead compound was able to revert the effect of scopolamine on different neurotransmitter tones, such as acetylcholine, serotonin, and GABA, shedding light on important networks not fully explored, so far.

Cannabidiol negatively modulates adenosine A2A receptor functioning in living cells.

OBJECTIVES: Cannabidiol (CBD) is a phytocannabinoid with great potential in clinical applications. The mechanism(s) of action of CBD require further investigation. Previous studies suggested that adenosine A2A receptors (A2ARs) could play a role in CBD-induced effects. Here, we evaluated the ability of CBD to modify the function of A2AR. METHODS: We used HEK-293T cells transfected with the cDNA encoding the human A2AR and Gαs protein, both modified to perform bioluminescence-based assays. We first assessed the effect of CBD on A2AR ligand binding using an A2AR NanoLuciferase sensor. Next, we evaluated whether CBD modified A2AR coupling to mini-Gαs proteins using the NanoBiT™ assay. Finally, we further assessed CBD effects on A2AR intrinsic activity by recording agonist-induced cAMP accumulation. RESULTS: CBD did not bind orthosterically to A2AR but reduced the coupling of A2AR to Gαs protein and the subsequent generation of cAMP. CONCLUSION: CBD negatively modulates A2AR functioning.

Overcoming the Challenges of Detecting GPCR Oligomerization in the Brain.

G protein-coupled receptors (GPCRs) constitute the largest group of membrane receptor proteins controlling brain activity. Accordingly, GPCRs are the main target of commercial drugs for most neurological and neuropsychiatric disorders. One of the mechanisms by which GPCRs regulate neuronal function is by homo- and heteromerization, with the establishment of direct protein-protein interactions between the same and different GPCRs. The occurrence of GPCR homo- and heteromers in artificial systems is generally well accepted, but more specific methods are necessary to address GPCR oligomerization in the brain. Here, we revise some of the techniques that have mostly contributed to reveal GPCR oligomers in native tissue, which include immunogold electron microscopy, proximity ligation assay (PLA), resonance energy transfer (RET) between fluorescent ligands and the Amplified Luminescent Proximity Homogeneous Assay (ALPHA). Of note, we use the archetypical GPCR oligomer, the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromer as an example to illustrate the implementation of these techniques, which can allow visualizing GPCR oligomers in the human brain under normal and pathological conditions. Indeed, GPCR oligomerization may be involved in the pathophysiology of neurological and neuropsychiatric disorders.

In situ identification and G4-PPI-His-Mal-dendrimer-induced reduction of early-stage amyloid aggregates in Alzheimer's disease transgenic mice using synchrotron-based infrared imaging.

Amyloid plaques composed of Aß amyloid peptides and neurofibrillary tangles are a pathological hallmark of Alzheimer Disease. In situ identification of early-stage amyloid aggregates in Alzheimer's disease is relevant for their importance as potential targets for effective drugs. Synchrotron-based infrared imaging is here used to identify early-stage oligomeric/granular aggregated amyloid species in situ in the brain of APP/PS1 transgenic mice for the first time. Also, APP/PS1 mice show fibrillary aggregates at 6 and 12 months. A significant decreased burden of early-stage aggregates and fibrillary aggregates is obtained following treatment with poly(propylene imine) dendrimers with histidine-maltose shell (a neurodegenerative protector) in 6-month-old APP/PS1 mice, thus demonstrating their putative therapeutic properties of in AD models. Identification, localization, and characterization using infrared imaging of these non-fibrillary species in the cerebral cortex at early stages of AD progression in transgenic mice point to their relevance as putative pharmacological targets. No less important, early detection of these structures may be useful in the search for markers for non-invasive diagnostic techniques.

Decreased striatal adenosine A2A-dopamine D2 receptor heteromerization in schizophrenia.

According to the adenosine hypothesis of schizophrenia, the classically associated hyperdopaminergic state may be secondary to a loss of function of the adenosinergic system. Such a hypoadenosinergic state might either be due to a reduction of the extracellular levels of adenosine or alterations in the density of adenosine A2A receptors (A2ARs) or their degree of functional heteromerization with dopamine D2 receptors (D2R). In the present study, we provide preclinical and clinical evidences for this latter mechanism. Two animal models for the study of schizophrenia endophenotypes, namely the phencyclidine (PCP) mouse model and the A2AR knockout mice, were used to establish correlations between behavioural and molecular studies. In addition, a new AlphaLISA-based method was implemented to detect native A2AR-D2R heteromers in mouse and human brain. First, we observed a reduction of prepulse inhibition in A2AR knockout mice, similar to that observed in the PCP animal model of sensory gating impairment of schizophrenia, as well as a significant upregulation of striatal D2R without changes in A2AR expression in PCP-treated animals. In addition, PCP-treated animals showed a significant reduction of striatal A2AR-D2R heteromers, as demonstrated by the AlphaLISA-based method. A significant and pronounced reduction of A2AR-D2R heteromers was next demonstrated in postmortem caudate nucleus from schizophrenic subjects, even though both D2R and A2AR were upregulated. Finally, in PCP-treated animals, sub-chronic administration of haloperidol or clozapine counteracted the reduction of striatal A2AR-D2R heteromers. The degree of A2AR-D2R heteromer formation in schizophrenia might constitute a hallmark of the illness, which indeed should be further studied to establish possible correlations with chronic antipsychotic treatments.

Poly(propylene imine) dendrimers with histidine-maltose shell as novel type of nanoparticles for synapse and memory protection.

Poly(propylene imine) dendrimers have been shown to be promising 3-dimensional polymers for the use in the pharmaceutical and biomedical applications. Our aims of this study were first, to synthesize a novel type of dendrimer with poly(propylene imine) core and maltose-histidine shell (G4HisMal) assessing if maltose-histidine shell can improve the biocompatibility and the ability to cross the blood-brain barrier, and second, to investigate the potential of G4HisMal to protect Alzheimer disease transgenic mice from memory impairment. Our data demonstrate that G4HisMal has significantly improved biocompatibility and ability to cross the blood-brain barrier in vivo. Therefore, we suggest that a maltose-histidine shell can be used to improve biocompatibility and ability to cross the blood-brain barrier of dendrimers. Moreover, G4HisMal demonstrated properties for synapse and memory protection when administered to Alzheimer disease transgenic mice. Therefore, G4HisMal can be considered as a promising drug candidate to prevent Alzheimer disease via synapse protection.

Adenosine A2A-Cannabinoid CB1 Receptor Heteromers in the Hippocampus: Cannabidiol Blunts Δ9-Tetrahydrocannabinol-Induced Cognitive Impairment.

At present, clinical interest in the plant-derived cannabinoid compound cannabidiol (CBD) is rising exponentially, since it displays multiple therapeutic properties. In addition, CBD can counteract the undesirable effects of the psychoactive cannabinoid Δ9-tetrahydrocannabinol (Δ9-THC) that hinder clinical development of cannabis-based therapies. Despite this attention, the mechanisms of CBD action and its interaction with Δ9-THC are still not completely elucidated. Here, by combining in vivo and complementary molecular techniques, we demonstrate for the first time that CBD blunts the Δ9-THC-induced cognitive impairment in an adenosine A2A receptor (A2AR)-dependent manner. Furthermore, we reveal the existence of A2AR and cannabinoid CB1 receptor (CB1R) heteromers at the presynaptic level in CA1 neurons in the hippocampus. Interestingly, our findings support a brain region-dependent A2AR-CB1R functional interplay; indeed, CBD was not capable of modifying motor functions presumably regulated by striatal A2AR/CB1R complexes, nor anxiety responses related to other brain regions. Overall, these data provide new evidence regarding the mechanisms of action of CBD and the nature of A2AR-CB1R interactions in the brain.

Memory Improvement in the AßPP/PS1 Mouse Model of Familial Alzheimer's Disease Induced by Carbamylated-Erythropoietin is Accompanied by Modulation of Synaptic Genes.

Neuroprotection of erythropoietin (EPO) following long-term administration is hampered by the associated undesirable effects on hematopoiesis and body weight. For this reason, we tested carbamylated-EPO (CEPO), which has no effect on erythropoiesis, and compared it with EPO in the AßPP/PS1 mouse model of familial Alzheimer's disease. Groups of 5-month old wild type (WT) and transgenic mice received chronic treatment consisting of CEPO (2,500 or 5,000 UI/kg) or EPO (2,500 U I/kg) 3 days/week for 4 weeks. Memory at the end of treatment was assessed with the object recognition test. Microarray analysis and quantitative-PCR were used for gene expression studies. No alterations in erythropoiesis were observed in CEPO-treated WT and AßPP/PS1 transgenic mice. EPO and CEPO improved memory in AßPP/PS1 animals. However, only EPO decreased amyloid-ß (Aß)plaque burden and soluble Aß(40). Microarray analysis of gene expression revealed a limited number of common genes modulated by EPO and CEPO. CEPO but not EPO significantly increased gene expression of dopamine receptors 1 and 2, and adenosine receptor 2a, and significantly down-regulated adrenergic receptor 1D and gastrin releasing peptide. CEPO treatment resulted in higher protein levels of dopamine receptors 1 and 2 in WT and AßPP/PS1 animals, whereas the adenosine receptor 2a was reduced in WT animals. The present results suggest that the improved behavior observed in AßPP/PS1 transgenic mice after CEPO treatment may be mediated, at least in part, by the observed modulation of the expression of molecules involved in neurotransmission.

Baclofen and 2-hydroxysaclofen modify acute hypolocomotive and antinociceptive effects of nicotine.

The aim of the present study was to evaluate the possible involvement of GABAB receptors in nicotine-induced hypolocomotion and antinociceptive effects in mice. Animals were exposed to nicotine only once. Acute nicotine hydrogen tartrate salt (3mg/kg; subcutaneous, s.c.) administration induced hypolocomotion and antinociceptive responses in the tail-immersion and the hot-plate tests. The effects of pretreatment with either the GABAB receptor agonist baclofen (1, 2 and 3mg/kg; intraperitoneal, i.p.) or GABAB receptor antagonist 2-hydroxysaclofen (0.25, 0.5 and 1mg/kg; i.p.) were evaluated on these behavioral nicotine responses. The GABAB receptor agonist, baclofen (3mg/kg, i.p.) abolished nicotine-induced antinociceptive effects in the tail-immersion and the hot-plate tests, but did not modify nicotine-induced hypolocomotion. In addition, the GABAB receptor antagonist, 2-hydroxysaclofen (1mg/kg, i.p.) increased nicotine-induced antinociceptive effects in the tail-immersion and the hot-plate tests, and abolished nicotine-induced hypolocomotion. The present results shed light that the GABAB receptor has an important role in mediating specific acute nicotine responses such as hypolocomotion and antinociception in mice.

Attenuation by baclofen of nicotine rewarding properties and nicotine withdrawal manifestations.

RATIONALE: Nicotine is a major active ingredient in tobacco and plays a major role in tobacco addiction. In rodents, repeated nicotine administration produces behavioral responses related to its addictive properties, such as reinforcing effects and physical dependence. OBJECTIVES: The aim of the present study was to evaluate the possible role of GABAB receptor in responses induced by repeated nicotine administration in Swiss Webster mice. RESULTS: Nicotine hydrogen tartrate salt (0.5 mg/kg, s.c.) administration induced rewarding properties in the conditioning place preference test. The GABAB receptor agonist, baclofen (3 mg/kg, i.p.) abolished the rewarding properties induced by nicotine hydrogen tartrate salt (0.5 mg/kg, s.c.). In addition, naloxone-precipitated nicotine withdrawal induced somatic manifestations, anxiety-like effects in the elevated plus maze test and dysphoric manifestations in the conditioned place aversion paradigm. Baclofen (2 and 3 mg/kg, i.p.) prevented the somatic manifestations and the anxiety-like effects associated with naloxone-precipitated nicotine withdrawal but not the dysphoric manifestations. CONCLUSIONS: These results showed that nicotine rewarding properties and negative aspects of nicotine withdrawal, such as anxiety-like effects and somatic manifestations, can be modulated by the GABAB receptor activity. This study now reveals a novel possible application of baclofen to develop new therapeutic strategies to achieve smoking cessation.

Promoter hypermethylation of the phosphatase DUSP22 mediates PKA-dependent TAU phosphorylation and CREB activation in Alzheimer's disease.

Genetic screening in Alzheimer's disease (AD) has identified only a handful of genes that are mutated in the disorder. Thus, for a very large proportion of patients, the biology of their disease is poorly understood. Epigenetic alterations may provide an explanation in these cases. Using DNA methylation profiles of human hippocampus from controls and patients, we have identified the presence of promoter hypermethylation of the dual-specificity phosphatase 22 (DUSP22) gene in AD. DUSP22 is a likely candidate gene for involvement in the pathogenesis of the disorder since, as we demonstrate here, it inhibits PKA activity and thereby determines TAU phosphorylation status and CREB signaling.

Amyloid generation and dysfunctional immunoproteasome activation with disease progression in animal model of familial Alzheimer's disease.

Double-transgenic amyloid precursor protein/presenilin 1 (APP/PS1) mice express a chimeric mouse/human APP bearing the Swedish mutation (Mo/HuAPP695swe) and a mutant human PS1-dE9 both causative of familial Alzheimer's disease (FAD). Transgenic mice show impaired memory and learning performance from the age of 6 months onwards. Double-transgenic APP/PS1 mice express altered APP and PS1 mRNAs and proteins, reduced ß-secretase 1 (BACE1) mRNA and normal BACE1 protein, all of which suggest a particular mechanism of amyloidogenesis when compared with sporadic AD. The first ß-amyloid plaques in APP/PS1 mice appear at 3 months, and they increase in number and distribution with disease progression in parallel with increased levels of brain soluble ß-amyloid 1-42 and 1-40, but also with reduced 1-42/1-40 ratio with age. Amyloid deposition in plaques is accompanied by altered mitochondria and increased oxidative damage, post-translational modifications and accumulation of altered proteins at the dystrophic neurites surrounding plaques. Degradation pathways are also modified with disease progression including activation of the immunoproteasome together with variable alterations of the different protease activities of the ubiquitin-proteasome system. Present observations show modifications in the production of ß-amyloid and activation and malfunction of the subcellular degradation pathways that have general implications in the pathogenesis of AD and more particularly in specificities of FAD amyloidogenesis.

Role of the cannabinoid system in the effects induced by nicotine on anxiety-like behaviour in mice.

RATIONALE: Acute behavioural effects and motivational responses induced by nicotine can be modulated by the endocannabinoid system supporting the existence of a physiological interaction between these two systems. OBJECTIVES: The present study was designed to examine the possible involvement of the cannabinoid system in the anxiolytic- and anxiogenic-like responses induced by nicotine in mice. METHODS: Animals were only exposed once to nicotine. The acute administration of low (0.05) or high (0.8 mg/kg, s.c.) doses of nicotine produced opposite effects in the elevated plus-maze, i.e. anxiolytic- and anxiogenic-like responses, respectively. The effects of the pretreatment with the CB1 cannabinoid receptor antagonist, rimonabant (0.25, 0.5 and 1 mg/kg, i.p.), and the cannabinoid agonist, delta9-tetrahydrocannabinol (delta9-THC, 0.1 mg/kg, ip), were evaluated on the anxiolytic- and anxiogenic-like responses induced by nicotine. RESULTS: Rimonabant completely abolished nicotine-induced anxiolytic-like effects and increased the anxiogenic-like responses of nicotine, suggesting an involvement of CB1 receptors in these behavioural responses. On the other hand, delta9-THC failed to modify nicotine anxiolytic-like responses but attenuated its anxiogenic-like effects. In addition, the association of non-effective doses of delta9-THC and nicotine produced clear anxiolytic-like responses. CONCLUSIONS: These results demonstrate that the endogenous cannabinoid system is involved in the regulation of nicotine anxiety-like behaviour in mice and provide new findings to support the use of cannabinoid antagonists in the treatment of tobacco addiction.

Involvement of the opioid system in the effects induced by nicotine on anxiety-like behaviour in mice.

RATIONALE: Recent studies have revealed the participation of the endogenous opioid system in several behavioural responses induced by nicotine including antinociception, rewarding properties, and physical drug dependence. OBJECTIVES: The present study was designed to examine the possible involvement of the various opioid receptors in the anxiolytic- and anxiogenic-like responses induced by nicotine in mice. METHODS: The acute administration of low (0.05) or high (0.8 mg/kg) doses of nicotine subcutaneously produced opposite effects in the elevated plus maze, i.e. anxiolytic- and anxiogenic-like responses, respectively. Animals were only exposed once to nicotine. The effects of the pretreatment with the mu-opioid receptor antagonist, beta-funaltrexamine (5 mg/kg), the delta-opioid antagonist, naltrindole (2.5 mg/kg) and the kappa-opioid antagonist, nor-binaltorphimine (2.5 mg/kg) intraperitoneally were evaluated on the anxiolytic- and anxiogenic-like responses induced by nicotine. RESULTS: beta-funaltrexamine, but not nor-binaltorphimine or naltrindole, abolished nicotine-induced anxiolytic-like effects, suggesting an involvement of mu-opioid receptors in this behavioural response. On the other hand, naltrindole, but not nor-binaltorphimine or beta-funaltrexamine, increased the anxiogenic-like responses of nicotine, suggesting an involvement of delta-receptors in this behavioural effect. CONCLUSIONS: These results demonstrate that the endogenous opioid system is involved in the effects induced by nicotine on anxiety-like behaviour and provide new findings to further clarify the interaction between these two neurochemical systems.

Delta9-tetrahydrocannabinol decreases somatic and motivational manifestations of nicotine withdrawal in mice.

The possible interactions between Delta9-tetrahydrocannabinol (Delta9-THC) and nicotine remain unclear in spite of the current association of cannabis and tobacco in humans. The aim of the present study was to explore the interactions between these two drugs of abuse by evaluating the consequences of Delta9-THC administration on the somatic manifestations and the aversive motivational state associated with nicotine withdrawal in mice. Acute Delta9-THC administration significantly decreased the incidence of several nicotine withdrawal signs precipitated by mecamylamine or naloxone, such as wet-dog-shakes, paw tremor and scratches. In both experimental conditions, the global withdrawal score was also significantly attenuated by acute Delta9-THC administration. This effect of Delta9-THC was not due to possible adaptive changes induced by chronic nicotine on CB1 cannabinoid receptors, as the density and functional activity of these receptors were not modified by chronic nicotine administration in the different brain structures investigated. We also evaluated the consequences of Delta9-THC administration on c-Fos expression in several brain structures after chronic nicotine administration and withdrawal. c-Fos was decreased in the caudate putamen and the dentate gyrus after mecamylamine precipitated nicotine withdrawal. However, acute Delta9-THC administration did not modify c-Fos expression under these experimental conditions. Finally, Delta9-THC also reversed conditioned place aversion associated to naloxone precipitated nicotine withdrawal. Taken together, these results indicate that Delta9-THC administration attenuated somatic signs of nicotine withdrawal and this effect was not associated with compensatory changes on CB1 cannabinoid receptors during chronic nicotine administration. In addition, Delta9-THC also ameliorated the aversive motivational consequences of nicotine withdrawal.