Alzheimer’s disease (AD) is the main cause of dementia worldwide. Given that learning and memory are impaired in this pathology, NMDA receptors (NMDARs) appear as key players in the onset and progression of the disease. NMDARs are glutamate receptors, mainly located at the post-synapse, which regulate voltage-dependent influx of calcium into the neurons. They are heterotetramers, and there are different subunits that can be part of the receptors, which are usually composed of two obligatory GluN1 subunits plus either two NR2A or two NR2B subunits. NR2A are mostly located at the synapse, and their activation is involved in the expression of pro-survival genes. Conversely, NR2B are mainly extrasynaptic, and their activation has been related to cell death and neurodegeneration. Thus, activation of NR2A and/or inactivation of NR2B-containing NMDARS has been proposed as a therapeutic strategy to treat AD. Here, we wanted to investigate the main differences between both subunits signalling in neuronal primary cultures of the cortex and hippocampus. It has been observed that Aβ induces a significant increase in calcium release and also in MAPK phosphorylation signalling in NR2B-containing NMDAR in cortical and hippocampal neurons. However, while NR2A-containing NMDAR decreases neuronal death and favours cell viability after Aβ treatment, NR2B-containing NMDAR shows higher levels of cytotoxicity and low levels of neuronal survival. Finally, it has been detected that NMDAR has no effect on pTau axonal transport. The present results demonstrate a different role between GluNA and GluNB subunits in neurodegenerative diseases such as Alzheimer’s.

Microdialysis assays demonstrated a possible role of orexin in the regulation of amyloid beta peptide (Aß) levels in the hippocampal interstitial fluid in the APP transgenic model. CB₂R is overexpressed in activated microglia, showing a neuroprotective effect. These two receptors may interact, forming CB₂-OX₁-Hets and becoming a new target to combat Alzheimer’s disease. Aims: Demonstrate the potential role of CB₂-OX₁-Hets expression and function in microglia from animal models of Alzheimer’s disease. Receptor heteromer expression was detected by immunocytochemistry, bioluminescence resonance energy transfer (BRET) and proximity ligation assay (PLA) in transfected HEK-293T cells and microglia primary cultures. Quantitation of signal transduction events in a heterologous system and in microglia cells was performed using the AlphaScreen^® SureFire^® kit, western blot, the GCaMP6 calcium sensor and the Lance Ultra cAMP kit (PerkinElmer). The formation of CB₂-OX₁ receptor complexes in transfected HEK-293T cells has been demonstrated. The tetrameric complex is constituted by one CB₂R homodimer, one OX₁R homodimer and two G proteins, a G_i and a G_q. The use of TAT interfering peptides showed that the CB₂-OX₁ receptor complex interface is TM4-TM5. At the functional level it has been observed that the OX₁R antagonist, SB334867, potentiates the action induced by CB₂R agonist JWH133. This effect is observed in transfected HEK-293T cells and microglia, and it is stronger in the Alzheimer’s disease (AD) animal model APPSw/Ind where the expression of the complex assessed by the proximity ligation assay indicates an increase in the number of complexes compared to resting microglia. The CB₂-OX₁ receptor complex is overexpressed in microglia from AD animal models where OX₁R antagonists potentiate the neuroprotective actions of CB₂R activation. Taken together, these results point to OX₁R antagonists as drugs with therapeutic potential to combat AD. Data access statement: Raw data will be provided by the corresponding author upon reasonable requirement.

Background: Cannabidiol (CBD) is a phytocannabinoid with potential in one of the most prevalent syndromes occurring at birth, the hypoxia of the neonate. CBD targets a variety of proteins, cannabinoid CB₂ and serotonin 5HT_1A receptors included. These two receptors may interact to form heteromers (CB₂–5HT_1A-Hets) that are also a target of CBD. Aims: We aimed to assess whether the expression and function of CB₂–5HT_1A-Hets is affected by CBD in animal models of hypoxia of the neonate and in glucose- and oxygen-deprived neurons. Methods: We developed a quantitation of signal transduction events in a heterologous system and in glucose/oxygen-deprived neurons. The expression of receptors was assessed by immuno-cyto and -histochemistry and, also, by using the only existing technique to visualize CB₂–5HT_1A-Hets fixed cultured cells and tissue sections (in situ proximity ligation PLA assay). Results: CBD and cannabigerol, which were used for comparative purposes, affected the structure of the heteromer, but in a qualitatively different way; CBD but not CBG increased the affinity of the CB₂ and 5HT_1A receptor–receptor interaction. Both cannabinoids regulated the effects of CB₂ and 5HT_1A receptor agonists. CBD was able to revert the upregulation of heteromers occurring when neurons were deprived of oxygen and glucose. CBD significantly reduced the increased expression of the CB₂–5HT_1A-Het in glucose/oxygen-deprived neurons. Importantly, in brain sections of a hypoxia/ischemia animal model, administration of CBD led to a significant reduction in the expression of CB₂–5HT_1A-Hets. Conclusions: Benefits of CBD in the hypoxia of the neonate are mediated by acting on CB₂–5HT_1A-Hets and by reducing the aberrant expression of the receptor–receptor complex in hypoxic-ischemic conditions. These results reinforce the potential of CBD for the therapy of the hypoxia of the neonate.

Cannabidiol (CBD) is a phytocannabinoid with potential as a therapy for a variety of diseases. CBD may act via cannabinoid receptors but also via other G-protein-coupled receptors (GPCRs), including the adenosine A_2A receptor. Homogenous binding and signaling assays in Chinese hamster ovary (CHO) cells expressing the human version of the A_2A receptor were performed to address the effect of CBD on receptor functionality. CBD was not able to compete for the binding of a SCH 442416 derivative labeled with a red emitting fluorescent probe that is a selective antagonist that binds to the orthosteric site of the receptor. However, CBD reduced the effect of the selective A_2A receptor agonist, CGS 21680, on G_s-coupling and on the activation of the mitogen activated kinase signaling pathway. It is suggested that CBD is a negative allosteric modulator of the A_2A receptor.

The identification of new modulators for Cannabinoid Receptors (CBRs) has garnered significant attention in drug discovery over recent years, owing to their manifold pathophysiological implications. In the context of hit identification, the availability of robust and sensitive high-throughput screening assays is essential to enhance the likelihood of success. In this study, we present the development and validation of a Tag-lite^® binding assay designed for screening hCB₁/hCB₂ binding, employing a dual fluorescent ligand, CELT-335. Representative ligands for CBRs, exhibiting diverse affinity and functional profiles, were utilized as reference compounds to validate the robustness and efficiency of the newly developed Tag-lite^® binding assay protocol. The homogeneous format, coupled with the sensitivity and optimal performance of the fluorescent ligand CELT-335, establishes this assay as a viable and reliable method for screening in hit and lead identification campaigns.

Microglial activation often accompanies the plastic changes occurring in the brain of patients with neurodegenerative diseases. A_2A and A₃ adenosine receptors have been proposed as therapeutic targets to combat neurodegeneration. RNAseq was performed using samples isolated from lipopolysaccharide/interferon-γ activated microglia treated with SCH 58261, a selective A_2A receptor antagonist, and with both SCH 58261 and 2-Cl-IB-MECA, a selective A₃ receptor agonist. None of the treatments led to any clear microglial phenotype when gene expression for classical biomarkers of microglial polarization was assessed. However, many of the downregulated genes were directly or indirectly related to immune system-related events. Searching for genes whose expression was both significantly and synergistically affected when treated with the two adenosine receptor ligands, the AC122413.1 and Olfr56 were selected among those that were, respectively, upregulated and downregulated. We therefore propose that the products of these genes, olfactory receptor 56 and T-cell activation GTPase-activating protein 1, deserve attention as potential biomarkers of phenotypes that occur upon microglial activation.