Heteromerization of G-protein-coupled receptors is an important event as they integrate the actions of extracellular signals to give heteromer-selective ligand binding and signaling, opening new ...avenues in the development of potential drug targets in pharmacotherapy. The aim of the present paper was to check for cannabinoid CB1–GPR55 receptor heteromers in the central nervous system (CNS), specifically in striatum. First, a direct interaction was demonstrated in cells transfected with the cDNA for the human version of the receptors, using bioluminescence resonance energy transfer and in situ proximity ligation assays (PLA). In the heterologous system, a biochemical fingerprint consisting on cross-antagonism in ERK1/2 phosphorylation was detected. The cross-antagonism was also observed on GPR55-mediated NFAT activation. Direct identification of GPR55 receptors in striatum is here demonstrated in rat brain slices using a specific agonist. Moreover, the heteromer fingerprint was identified in these rat slices by ERK1/2 phosphorylation assays whereas PLA assays showed results consistent with receptor–receptor interactions in both caudate and putamen nuclei of a non-human primate. The results indicate not only that GPR55 is expressed in striatum but also that CB1 and GPR55 receptors form heteromers in this specific CNS region.
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•GPR55 is expressed in the striatum.•Cannabinoid CB1 and GPR55 receptors form heteromers in the CNS.•Rat striatal slices display the CB1R–GPR55 heteromer fingerprint.•Non-human primate striatum displays CB1R–GPR55 heteromers.
Neuroprotective M2‐skewed microglia appear as promising to alter the course of neurodegenerative diseases and G protein‐coupled receptors (GPCRs) are potential targets to achieve such microglial ...polarization. A common feature of adenosine A2A (A2AR) and cannabinoid CB2 (CB2R) GPCRs in microglia is that their expression is upregulated in Alzheimer's disease (AD). On the one hand, CB2R seems a target for neuroprotection, delaying neurodegenerative processes like those associated to AD or Parkinson's diseases. A2AR antagonists reduce amyloid burden and improve cognitive performance and memory in AD animal models. We here show a close interrelationship between these two receptors in microglia; they are able to physically interact and affect the signaling of each other, likely due to conformational changes within the A2A‐CB2 receptor heteromer (A2A‐CB2Het). Particularly relevant is the upregulation of A2A‐CB2Het expression in samples from the APPSw,Ind AD transgenic mice model. The most relevant finding, confirmed in both heterologous cells and in primary cultures of microglia, was that blockade of A2A receptors results in increased CB2R‐mediated signaling. This heteromer‐specific feature suggests that A2AR antagonists would potentiate, via microglia, the neuroprotective action of endocannabinoids with implications for AD therapy.
Main Points
Adenosine A2A/cannabinoid CB2 receptor heteromers, expressed in microglia from Alzheimer's disease (AD) models, allow enhanced cannabinoid signaling upon A2A blockade.
A2AR antagonists are promising to combat AD and other neurodegenerative diseases.
•LPS plus interferon-γ upregulate CB2 receptors in microglia.•LPS plus interferon-γ upregulate CB1-CB2 receptor complexes in microglia.•ß amyloid and LPS plus interferon-γ lead to similar activation ...traits in microglia.•Activated microglia is identified in pups of an Alzheimer’s disease model.•Dyskinesia in Parkinson’s correlates with CB1-CB2 receptor complex upregulation.
Endocannabinoids are important regulators of neurotransmission and, acting on activated microglia, they are postulated as neuroprotective agents. Endocannabinoid action is mediated by CB1 and CB2 receptors, which may form heteromeric complexes (CB1-CB2Hets) with unknown function in microglia. We aimed at establishing the expression and signaling properties of cannabinoid receptors in resting and LPS/IFN-γ-activated microglia. In activated microglia mRNA transcripts increased (2 fold for CB1 and circa 20 fold for CB2), whereas receptor levels were similar for CB1 and markedly upregulated for CB2; CB1-CB2Hets were also upregulated. Unlike in resting cells, CB2 receptors became robustly coupled to Gi in activated cells, in which CB1-CB2Hets mediated a potentiation effect. Hence, resting cells were refractory while activated cells were highly responsive to cannabinoids. Interestingly, similar results were obtained in cultures treated with ß-amyloid (Aß1-42). Microglial activation markers were detected in the striatum of a Parkinson’s disease (PD) model and, remarkably, in primary microglia cultures from the hippocampus of mutant β-amyloid precursor protein (APPSw,Ind) mice, a transgenic Alzheimer’s disease (AD) model. Also of note was the similar cannabinoid receptor signaling found in primary cultures of microglia from APPSw,Ind and in cells from control animals activated using LPS plus IFN-γ. Expression of CB1-CB2Hets was increased in the striatum from rats rendered dyskinetic by chronic levodopa treatment. In summary, our results showed sensitivity of activated microglial cells to cannabinoids, increased CB1-CB2Het expression in activated microglia and in microglia from the hippocampus of an AD model, and a correlation between levodopa-induced dyskinesia and striatal microglial activation in a PD model. Cannabinoid receptors and the CB1-CB2 heteroreceptor complex in activated microglia have potential as targets in the treatment of neurodegenerative diseases.
Endocannabinoids act on G protein-coupled receptors that are considered potential targets for a variety of diseases. There are two different cannabinoid receptor types: ligands for cannabinoid type 2 ...receptors (CB2Rs) show more promise than those for cannabinoid type 1 receptors (CB1Rs) because they lack psychotropic actions. However, the complex pharmacology of these receptors, coupled with the lipophilic nature of ligands, is delaying the translational success of medications targeting the endocannabinoid system. We here report the discovery and synthesis of a fluorophore-conjugated CB2R-selective compound, CM-157 (3-4-2-tert-butyl-1-(tetrahydropyran-4-ylmethyl)benzimidazol-5-ylsulfonyl-2-pyridyloxypropan-1-amine), which was useful for pharmacological characterization of CB2R by using a time-resolved fluorescence resonance energy transfer assay. This methodology does not require radiolabeled compounds and may be undertaken in homogeneous conditions and in living cells (i.e., without the need to isolate receptor-containing membranes). The affinity of the labeled compound was similar to that of the unlabeled molecule. Time-resolved fluorescence resonance energy transfer assays disclosed a previously unreported second affinity site and showed conformational changes in CB2R forming receptor heteromers with G protein-coupled receptor GPR55, a receptor for l-α-lysophosphatidylinositol. The populations displaying subnanomolar and nanomolar affinities were undisclosed in competitive assays using a well known cannabinoid receptor ligand, AM630 (1-2-(morpholin-4-yl)ethyl-2-methyl-3-(4-methoxybenzoyl)-6-iodoindole), and TH-chrysenediol, not previously tested on binding to cannabinoid receptors. Variations in binding parameters upon formation of dimers with GPR55 may reflect decreases in binding sites or alterations of the quaternary structure of the macromolecular G protein-coupled receptor complexes. In summary, the homogeneous binding assay described here may serve to better characterize agonist binding to CB2R and to identify specific properties of CB2R on living cells.
Heteromerization of G-protein-coupled receptors is an important event as they integrate the actions of extracellular signals to give heteromer-selective ligand binding and signaling, opening new ...avenues in the development of potential drug targets in pharmacotherapy. The aim of the present paper was to check for cannabinoid CB sub(1)-GPR55 receptor heteromers in the central nervous system (CNS), specifically in striatum. First, a direct interaction was demonstrated in cells transfected with the cDNA for the human version of the receptors, using bioluminescence resonance energy transfer and in situ proximity ligation assays (PLA). In the heterologous system, a biochemical fingerprint consisting on cross-antagonism in ERK1/2 phosphorylation was detected. The cross-antagonism was also observed on GPR55-mediated NFAT activation. Direct identification of GPR55 receptors in striatum is here demonstrated in rat brain slices using a specific agonist. Moreover, the heteromer fingerprint was identified in these rat slices by ERK1/2 phosphorylation assays whereas PLA assays showed results consistent with receptor-receptor interactions in both caudate and putamen nuclei of a non-human primate. The results indicate not only that GPR55 is expressed in striatum but also that CB sub(1) and GPR55 receptors form heteromers in this specific CNS region.
Dopamine is derived from an amino acid, phenylalanine, which must be obtained through the diet. Dopamine, known primarily to be a neurotransmitter involved in almost any higher executive action, acts ...through five types of G-protein-coupled receptors. Dopamine has been studied extensively for its neuronal handling, synaptic actions, and in relation to Parkinson's disease. However, dopamine receptors can be found extra-synaptically and, in addition, they are not only expressed in neurons, but in many types of mammalian cells, inside and outside the central nervous system (CNS). Recent studies show a dopamine link between the gut and the CNS; the mechanisms are unknown, but they probably require cells to act as mediators and the involvement of the immune system. In fact, dopamine receptors are expressed in almost any cell of the immune system where dopamine regulates various processes, such as antigen presentation, T-cell activation, and inflammation. This likely immune cell-mediated linkage opens up a new perspective for the use of dopamine-related drugs, i.e., agonist-antagonist-allosteric modulators of dopamine receptors, in a variety of diseases.
The family of dopamine D2‐like receptors represents an interesting target for a variety of neurological diseases, e. g. Parkinson's disease (PD), addiction, or schizophrenia. In this study we ...describe the synthesis of a new set of fluorescent ligands as tools for visualization of dopamine D2‐like receptors. Pharmacological characterization in radioligand binding studies identified UR‐MN212 (20) as a high‐affinity ligand for D2‐like receptors (pKi (D2longR)=8.24, pKi (D3R)=8.58, pKi (D4R)=7.78) with decent selectivity towards D1‐like receptors. Compound 20 is a neutral antagonist in a Go1 activation assay at the D2longR, D3R, and D4R, which is an important feature for studies using whole cells. The neutral antagonist 20, equipped with a 5‐TAMRA dye, displayed rapid association to the D2longR in binding studies using confocal microscopy demonstrating its suitability for fluorescence microscopy. Furthermore, in molecular brightness studies, the ligand's binding affinity could be determined in a single‐digit nanomolar range that was in good agreement with radioligand binding data. Therefore, the fluorescent compound can be used for quantitative characterization of native D2‐like receptors in a broad variety of experimental setups.
In this study, we have identified UR‐MN212 (20), a spiperone‐based fluorescent ligand bearing a 5‐TAMRA dye, as suitable for fluorescence microscopy at the D2R. The binding affinity in the single‐digit nanomolar range could be determined in radioligand competition binding and molecular brightness studies at D2‐like receptors.
Dopamine D1‐like receptors are the most abundant type of dopamine receptors in the central nervous system and, even after decades of discovery, still highly interesting for the study of neurological ...diseases. We herein describe the synthesis of a new set of fluorescent ligands, structurally derived from D1R antagonist SCH‐23390 and labeled with two different fluorescent dyes, as tool compounds for the visualization of D1‐like receptors. Pharmacological characterization in radioligand binding studies identified UR‐NR435 (25) as a high‐affinity ligand for D1‐like receptors (pKi (D1R)=8.34, pKi (D5R)=7.62) with excellent selectivity towards D2‐like receptors. Compound 25 proved to be a neutral antagonist at the D1R and D5R in a Gs heterotrimer dissociation assay, an important feature to avoid receptor internalization and degradation when working with whole cells. The neutral antagonist 25 displayed rapid association and complete dissociation to the D1R in kinetic binding studies using confocal microscopy verifying its applicability for fluorescence microscopy. Moreover, molecular brightness studies determined a single‐digit nanomolar binding affinity of the ligand, which was in good agreement with radioligand binding data. For this reason, this fluorescent ligand is a useful tool for a sophisticated characterization of native D1 receptors in a variety of experimental setups.
We herein report the identification of UR‐NR435 (25), a SCH‐23390‐based fluorescent ligand bearing a 5‐TAMRA dye, as suitable for fluorescence microscopy at the D1R. Single‐digit nanomolar binding affinity could be determined in radioligand competition binding and molecular brightness studies at D1‐like receptors.
Synaptic dysfunction caused by soluble β‐amyloid peptide (Aβ) is a hallmark of early‐stage Alzheimer's disease (AD), and is tightly linked to cognitive decline. By yet unknown mechanisms, Aβ ...suppresses the transcriptional activity of cAMP‐responsive element‐binding protein (CREB), a master regulator of cell survival and plasticity‐related gene expression. Here, we report that Aβ elicits nucleocytoplasmic trafficking of Jacob, a protein that connects a NMDA‐receptor‐derived signalosome to CREB, in AD patient brains and mouse hippocampal neurons. Aβ‐regulated trafficking of Jacob induces transcriptional inactivation of CREB leading to impairment and loss of synapses in mouse models of AD. The small chemical compound Nitarsone selectively hinders the assembly of a Jacob/LIM‐only 4 (LMO4)/ Protein phosphatase 1 (PP1) signalosome and thereby restores CREB transcriptional activity. Nitarsone prevents impairment of synaptic plasticity as well as cognitive decline in mouse models of AD. Collectively, the data suggest targeting Jacob protein‐induced CREB shutoff as a therapeutic avenue against early synaptic dysfunction in AD.
Synopsis
The synapto‐nuclear shuttling protein Jacob assembles and docks a signalosome to the transcriptional regulator CREB to promote CREB de‐phosphorylation and neuronal cell death. Here, β‐amyloid peptide (Aβ) is found to cause Alzheimer‐associated synapse loss by regulating nuclear translocation of Jacob, providing a potential target for therapeutic intervention.
Aβ promotes nuclear translocation of Jacob in Alzheimer's disease (AD) patient brains and mouse hippocampal neurons.
Aβ‐regulated trafficking of Jacob induces transcriptional inactivation of cAMP‐responsive element‐binding protein (CREB), leading to synapse impairment and loss in an AD mouse model.
The small‐molecular compound Nitarsone selectively prevents the assembly of the Jacob/LMO4/PP1 signalosome to promote CREB transcriptional activity.
Nitarsone prevents synaptic plasticity impairment and cognitive decline in mouse models of AD.
A process by which β‐amyloid peptide regulates nuclear trafficking of the synapto‐nuclear shuttling protein Jacob to cause synapse impairment and loss in an AD mouse model can be inhibited by the small molecule Nitarsone.
The family of dopamine D
-like receptors represents an interesting target for a variety of neurological diseases, e. g. Parkinson's disease (PD), addiction, or schizophrenia. In this study we ...describe the synthesis of a new set of fluorescent ligands as tools for visualization of dopamine D
-like receptors. Pharmacological characterization in radioligand binding studies identified UR-MN212 (20) as a high-affinity ligand for D
-like receptors (pK
(D
R)=8.24, pK
(D
R)=8.58, pK
(D
R)=7.78) with decent selectivity towards D
-like receptors. Compound 20 is a neutral antagonist in a G
activation assay at the D
R, D
R, and D
R, which is an important feature for studies using whole cells. The neutral antagonist 20, equipped with a 5-TAMRA dye, displayed rapid association to the D
R in binding studies using confocal microscopy demonstrating its suitability for fluorescence microscopy. Furthermore, in molecular brightness studies, the ligand's binding affinity could be determined in a single-digit nanomolar range that was in good agreement with radioligand binding data. Therefore, the fluorescent compound can be used for quantitative characterization of native D
-like receptors in a broad variety of experimental setups.
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