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.

Background: Experiments in the late nineties showed an inverse relationship in the eye levels of melatonin and dopamine, thereby constituting an example of eye parameters that are prone to circadian variations. The underlying mechanisms are not known but these relevant molecules act via specific cell surface dopamine and melatonin receptors. This study investigated whether these receptors formed heteromers whose function impact on eye physiology. We performed biophysical assays to identify interactions in heterologous systems. Particular heteromer functionality was detected using Gi coupling, MAPK activation, and label-free assays. The expression of the heteroreceptor complexes was assessed using proximity ligation assays in cells producing the aqueous humor and human eye samples. Dopamine D₃ receptors (D₃Rs) were identified in eye ciliary body epithelial cells. We discovered heteromers formed by D₃R and either MT₁ (MT₁R) or MT₂ (MT₂R) melatonin receptors. Heteromerization led to the blockade of D₃R-Gi coupling and regulation of signaling to the MAPK pathway. Heteromer expression was negatively correlated with intraocular hypertension. Conclusions: Heteromers likely mediate melatonin and dopamine actions in structures regulating intraocular pressure. Significant expression of D₃R–MT₁R and D₃R–MT₁R was associated with normotensive conditions, whereas expression diminished in a cell model of hypertension. A clear trend of expression reduction was observed in samples from glaucoma cases. The trend was marked but no statistical analysis was possible as the number of available eyes was 2.

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.

Objectives: Human tear analysis holds promise for biomarker discovery, but its clinical utility is hindered by the lack of standardized reference values, limiting interindividual comparisons. This study aimed at developing a protocol for normalizing metabolomic data from human tears, enhancing its potential for biomarker identification. Methods: Tear metabolomic profiling was conducted on 103 donors (64 females, 39 males, aged 18–82 years) without ocular pathology, using the AbsoluteIDQ™ p180 Kit for targeted metabolomics. A predictive normalization model incorporating age, sex, and fasting time was developed to correct for interindividual variability. Key metabolites from six compound families (amino acids, biogenic amines, acylcarnitines, lysophosphatidylcholines, phosphatidylcholines, and sphingomyelins) were identified as normalization references. The approach was validated using Linear Discriminant Analysis (LDA) to test its ability to classify donor sex based on metabolite concentrations. Results: Metabolite concentrations exhibited significant interindividual variability. The normalization model, which predicted metabolite concentrations based on a reference “concomitant” metabolite from each compound family, successfully reduced this variability. Using the ratio of observed-to-predicted concentrations, the model enabled robust comparisons across individuals. LDA classification of donor sex using acylcarnitine C4 achieved 78 % accuracy, correctly identifying 92 % of female donors. This approach outperformed traditional statistical and machine learning methods (Lasso logistic regression and Random Forest classification) in sex discrimination based on tear metabolomics. Conclusions: This novel normalization protocol significantly improves the reliability of tear metabolomics by enabling standardized interindividual comparisons. The approach facilitates biomarker discovery by mitigating variability in metabolite concentrations and may be extended to other biological fluids, enhancing its applicability in precision medicine.

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.