Control of glutamate release by complexes of adenosine and cannabinoid receptors.

BACKGROUND: It has been hypothesized that heteromers of adenosine A2A receptors (A2AR) and cannabinoid CB1 receptors (CB1R) localized in glutamatergic nerve terminals mediate the integration of adenosine and endocannabinoid signaling involved in the modulation of striatal excitatory neurotransmission. Previous studies have demonstrated the existence of A2AR-CB1R heteromers in artificial cell systems. A dependence of A2AR signaling for the Gi protein-mediated CB1R signaling was described as one of its main biochemical characteristics. However, recent studies have questioned the localization of functionally significant A2AR-CB1R heteromers in striatal glutamatergic terminals. RESULTS: Using a peptide-interfering approach combined with biophysical and biochemical techniques in mammalian transfected cells and computational modeling, we could establish a tetrameric quaternary structure of the A2AR-CB1R heterotetramer. This quaternary structure was different to the also tetrameric structure of heteromers of A2AR with adenosine A1 receptors or dopamine D2 receptors, with different heteromeric or homomeric interfaces. The specific quaternary structure of the A2A-CB1R, which depended on intermolecular interactions involving the long C-terminus of the A2AR, determined a significant A2AR and Gs protein-mediated constitutive activation of adenylyl cyclase. Using heteromer-interfering peptides in experiments with striatal glutamatergic terminals, we could then demonstrate the presence of functionally significant A2AR-CB1R heteromers with the same biochemical characteristics of those studied in mammalian transfected cells. First, either an A2AR agonist or an A2AR antagonist allosterically counteracted Gi-mediated CB1R agonist-induced inhibition of depolarization-induced glutamate release. Second, co-application of both an A2AR agonist and an antagonist cancelled each other effects. Finally, a CB1R agonist inhibited glutamate release dependent on a constitutive activation of A2AR by a canonical Gs-Gi antagonistic interaction at the adenylyl cyclase level. CONCLUSIONS: We demonstrate that the well-established cannabinoid-induced inhibition of striatal glutamate release can mostly be explained by a CB1R-mediated counteraction of the A2AR-mediated constitutive activation of adenylyl cyclase in the A2AR-CB1R heteromer.

Downregulated Glia Interplay and Increased miRNA-155 as Promising Markers to Track ALS at an Early Stage.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of unknown cause. Absence of specific targets and biomarkers compromise the development of new therapeutic strategies and of innovative tools to stratify patients and assess their responses to treatment. Here, we investigate changes in neuroprotective-neuroinflammatory actions in the spinal cord of SOD1 G93A mice, at presymptomatic and symptomatic stages to identify stage-specific biomarkers and potential targets. Results showed that in the presymptomatic stage, there are alterations in both astrocytes and microglia, which comprise decreased expression of GFAP and S100B and upregulation of GLT-1, as well as reduced expression of CD11b, M2-phenotype markers, and a set of inflammatory mediators. Reduced levels of Connexin-43, Pannexin-1, CCL21, and CX3CL1 further indicate the existence of a compromised intercellular communication. In contrast, in the symptomatic stage, increased markers of inflammation became evident, such as NF-κB/Nlrp3-inflammasome, Iba1, pro-inflammatory cytokines, and M1-polarizion markers, together with a decreased expression of M2-phenotypic markers. We also observed upregulation of the CX3CL1-CX3CR1 axis, Connexin-43, Pannexin-1, and of microRNAs (miR)-124, miR-125b, miR-146a and miR-21. Reduced motor neuron number and presence of reactive astrocytes with decreased GFAP, GLT-1, and GLAST further characterized this inflammatory stage. Interestingly, upregulation of miR-155 and downregulation of MFG-E8 appear as consistent biomarkers of both presymptomatic and symptomatic stages. We hypothesize that downregulated cellular interplay at the early stages may represent neuroprotective mechanisms against inflammation, SOD1 aggregation, and ALS onset. The present study identified a set of inflamma-miRNAs, NLRP3-inflammasome, HMGB1, CX3CL1-CX3CR1, Connexin-43, and Pannexin-1 as emerging candidates and promising pharmacological targets that may represent potential neuroprotective strategies in ALS therapy.

Dissecting neurovascular coupling mechanisms: a role for adenosine A2A receptor: An Editorial highlight for 'Correlation of transient adenosine release and oxygen changes in the caudate-putamen'.

Read the highlighted article 'Correlation of transient adenosine release and oxygen changes in the caudate-putamen' on page 13.

Dissecting striatal adenosine-cannabinoid receptor interactions. New clues from rats over-expressing adenosine A2A receptors.

This Editorial highlights a study by Chiodi et al. () showing that the effects mediated by cannabinoid CB1 receptor (CB1R) activation in the striatum are significantly reduced in rats with neuronal over-expression of adenosine A2A receptors (A2AR). Two hypotheses are derived from that study. Hypothesis A: two subpopulations of pre-synaptic CB1R in corticostriatal glutamatergic terminals exist, one forming and another not forming heteromers with A2AR. Hypothesis B: CB1R are predominantly forming heteromers with A2AR. In the case of hypothesis A, the A2AR might be required for CB1R-A2AR heteromeric signaling, whereas non-heteromeric CB1R activity is inhibited by A2ARs. In the case of hypothesis B, up-regulation of A2ARs may perturb heteromeric stoichiometry, thus reducing CB1R functioning. In any case, pre-synaptic striatal A2AR-CB1R heteromers emerge as important targets of the effects of cannabinoids demonstrated at the neuronal and behavioral level. Read the highlighted article 'Striatal adenosine-cannabinoid receptor interactions in rats over-expressing adenosine A2A receptors' on page 907.

Adenosine A2A receptor activation is determinant for BDNF actions upon GABA and glutamate release from rat hippocampal synaptosomes.

Adenosine, through A(2A) receptor (A(2A)R) activation, can act as a metamodulator, controlling the actions of other modulators, as brain-derived neurotrophic factor (BDNF). Most of the metamodulatory actions of adenosine in the hippocampus have been evaluated in excitatory synapses. However, adenosine and BDNF can also influence GABAergic transmission. We thus evaluated the role of A(2A)R on the modulatory effect of BDNF upon glutamate and GABA release from isolated hippocampal nerve terminals (synaptosomes). BDNF (30 ng/ml) enhanced K(+)-evoked [(3)H]glutamate release and inhibited the K(+)-evoked [(3)H]GABA release from synaptosomes. The effect of BDNF on both glutamate and GABA release requires tonic activation of adenosine A(2A)R since for both neurotransmitters, the BDNF action was blocked by the A(2A)R antagonist SCH 58261 (50 nM). In the presence of the A(2A)R agonist, CGS21680 (30 nM), the effect of BDNF on either glutamate or GABA release was, however, not potentiated. It is concluded that both the inhibitory actions of BDNF on GABA release as well as the facilitatory action of the neurotrophin on glutamate release are dependent on the activation of adenosine A(2A)R by endogenous adenosine. However, these actions could not be further enhanced by exogenous activation of A(2A)R.

Brain-derived neurotrophic factor mediates neuroprotection against Aß-induced toxicity through a mechanism independent on adenosine 2A receptor activation.

Brain-derived neurotrophic factor (BDNF) promotes neuronal survival through TrkB-FL activation. The activation of adenosine A2A receptors (A2AR) is essential for most of BDNF-mediated synaptic actions, such as synaptic plasticity, transmission and neurotransmitter release. We now aimed at evaluating the A2AR influence upon BDNF-mediated neuroprotection against Aß25-35 toxicity in cultured neurons. Results showed that BDNF increases cell survival and reduces the caspase-3 and calpain activation induced by amyloid-ß (Aß) peptide, in a mechanism probably dependent on PLCγ pathway. This BDNF-mediated neuroprotection is not affected by A2AR activation or inhibition. Moreover neither activation nor inhibition of A2AR, per se, significantly influenced Aß-induced neuronal death on calpain-mediated cleavage of TrkB induced by Aß. In conclusion, these results suggest that, in opposition to the fast synaptic actions of BDNF, the neuroprotective actions of this neurotrophin against a strong Aß insult do not require the activation of A2AR.

Neuroinflammation after neonatal hypoxia-ischemia is associated with alterations in the purinergic system: adenosine deaminase 1 isoenzyme is the most predominant after insult.

Hypoxic-ischemic (HI) injury perinatal brain is a major contributor to morbidity and mortality to infants and children. Adenosine may play a role in the pathophysiology of HI, since it modulates the inflammatory process and the release of several neurotransmitters. Thus, the aim of this study was to identify the isoforms of adenosine deaminase (ADA) responsible for the enzymatic activity as well as the adenosine kinase (ADK) and A1 adenosine receptor (A1R) expression in the cerebral cortex eight days after HI. Myeloperoxidase (MPO) and N-acetyl-glucosaminidase (NAG) were assessed as inflammation markers. ADA activity was analyzed, in the presence or absence of a specific ADA1 inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine. The ADA1 activity (92.6%) was significantly higher than ADA2 (7.4%) activity in the cerebral cortex eight days after HI. A1Rs and ADK protein expression showed decreased 8 days after insult. Interestingly, the ADA1, MPO, and NAG activities were correlated positively. In view of this, we conclude that the inhibitor of ADA1, in in vitro conditions, was effective in decreasing the ADA activity, and that mainly ADA1 isoform is responsible for the increase in the ADA activity 8 days after HI insult. Therefore, HI neonatal was able to alter the ADK and A1R expression. Thus, due to the importance of adenosine signaling in the regulation of inflammatory and immune process and the crucial role of ADA in the postischemic homeostase of adenosine as well as during inflammatory process, we suggest that ADA1 inhibitors may play an important role in the regulation of events that follow the HI insult, favoring the increase in the adenosine in the sites of tissue injury. Together, these results highlight a role of the purinergic signaling cascade in the pathophysiology of HI neonatal.

Mechanisms of regulation of olfactory transduction and adaptation in the olfactory cilium.

Olfactory adaptation is a fundamental process for the functioning of the olfactory system, but the underlying mechanisms regulating its occurrence in intact olfactory sensory neurons (OSNs) are not fully understood. In this work, we have combined stochastic computational modeling and a systematic pharmacological study of different signaling pathways to investigate their impact during short-term adaptation (STA). We used odorant stimulation and electroolfactogram (EOG) recordings of the olfactory epithelium treated with pharmacological blockers to study the molecular mechanisms regulating the occurrence of adaptation in OSNs. EOG responses to paired-pulses of odorants showed that inhibition of phosphodiesterases (PDEs) and phosphatases enhanced the levels of STA in the olfactory epithelium, and this effect was mimicked by blocking vesicle exocytosis and reduced by blocking cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) and vesicle endocytosis. These results suggest that G-coupled receptors (GPCRs) cycling is involved with the occurrence of STA. To gain insights on the dynamical aspects of this process, we developed a stochastic computational model. The model consists of the olfactory transduction currents mediated by the cyclic nucleotide gated (CNG) channels and calcium ion (Ca(2+))-activated chloride (CAC) channels, and the dynamics of their respective ligands, cAMP and Ca(2+), and it simulates the EOG results obtained under different experimental conditions through changes in the amplitude and duration of cAMP and Ca(2+) response, two second messengers implicated with STA occurrence. The model reproduced the experimental data for each pharmacological treatment and provided a mechanistic explanation for the action of GPCR cycling in the levels of second messengers modulating the levels of STA. All together, these experimental and theoretical results indicate the existence of a mechanism of regulation of STA by signaling pathways that control GPCR cycling and tune the levels of second messengers in OSNs, and not only by CNG channel desensitization as previously thought.

Impact of in vivo chronic blockade of adenosine A2A receptors on the BDNF-mediated facilitation of LTP.

Brain-derived neurotrophic factor (BDNF) through the activation of its receptor (TrkB-FL) exert well-described neuroprotective effects playing a major role in hippocampal synaptic transmission and plasticity such as long-term potentiation (LTP), a molecular surrogate for learning and memory. Impairments in BDNF signalling have been associated to several neurodegenerative disorders such as Alzheimer's disease (AD). Therefore, the reestablishment of BDNF actions is considered a promising strategy for AD treatment. While, most of BDNF synaptic actions, namely on LTP, require the activation of adenosine A2A receptor (A2AR), the antagonists of A2AR have been proven to prevent AD induced deficits in different animal models. Therefore in this work we aimed to evaluate the impact of a chronic in vivo oral administration of an A2AR antagonist (KW-6002) in the BDNF actions upon hippocampal CA1 LTP. The results showed that chronic blockade of A2AR in male Wistar rats inhibits the facilitatory action of BDNF upon LTP on hippocampal CA1 area and decreases both mRNA and protein levels of the TrkB-FL receptor in hippocampus. These findings imply that BDNF signalling may be affected in chronic A2AR blocking conditions.

Hypoxia-ischemia alters nucleotide and nucleoside catabolism and Na+,K+-ATPase activity in the cerebral cortex of newborn rats.

It is well known that the levels of adenosine in the brain increase dramatically during cerebral hypoxic-ischemic (HI) insults. Its levels are tightly regulated by physiological and pathophysiological changes that occur during the injury acute phase. The aim of the present study was to examine the effects of the neonatal HI event on cytosolic and ecto-enzymes of purinergic system--NTPDase, 5'-nucleotidase (5'-NT) and adenosine deaminase (ADA)--in cerebral cortex of rats immediately post insult. Furthermore, the Na(+)/K(+)-ATPase activity, adenosine kinase (ADK) expression and thiobarbituric acid reactive species (TBARS) levels were assessed. Immediately after the HI event the cytosolic NTPDase and 5'-NT activities were increased in the cerebral cortex. In synaptosomes there was an increase in the ecto-ADA activity while the Na(+)/K(+) ATPase activity presented a decrease. The difference between ATP, ADP, AMP and adenosine degradation in synaptosomal and cytosolic fractions could indicate that NTPDase, 5'-NT and ADA were differently affected after insult. Interestingly, no alterations in the ADK expression were observed. Furthermore, the Na(+)/K(+)-ATPase activity was correlated negatively with the cytosolic NTPDase activity and TBARS content. The increased hydrolysis of nucleotides ATP, ADP and AMP in the cytosol could contribute to increased adenosine levels, which could be related to a possible innate neuroprotective mechanism aiming at potentiating the ambient levels of adenosine. Together, these results may help the understanding of the mechanism by which adenosine is produced following neonatal HI injury, therefore highlighting putative therapeutical targets to minimize ischemic injury and enhance recovery.

Interleukin-6-type cytokines in neuroprotection and neuromodulation: oncostatin M, but not leukemia inhibitory factor, requires neuronal adenosine A1 receptor function.

Neuroprotection is one of the prominent functions of the interleukin (IL)-6-type cytokine family, for which the underlying mechanism(s) are not fully understood. We have previously shown that neuroprotection and neuromodulation mediated by IL-6 require neuronal adenosine A(1) receptor (A(1) R) function. We now have investigated whether two other IL-6-type cytokines [oncostatin M (OSM) and leukemia inhibitory factor (LIF)] use a similar mechanism. It is presented here that OSM but not LIF, enhanced the expression of A(1) Rs (both mRNA and protein levels) in cultured neurons. Whereas the neuroprotective effect of LIF was unchanged in A(1) R deficient neurons, OSM failed to protect neurons in the absence of A(1) R. In addition, OSM pre-treatment for 4 h potentiated the A(1) R-mediated inhibition of electrically evoked excitatory post-synaptic currents recorded from hippocampal slices either under normal or hypoxic conditions. No such effect was observed after LIF pre-treatment. Our findings thus strongly suggest that, despite known structural and functional similarities, OSM and LIF use different mechanisms to achieve neuroprotection and neuromodulation.

Activation of adenosine A2A receptor facilitates brain-derived neurotrophic factor modulation of synaptic transmission in hippocampal slices.

Both brain-derived neurotrophic factor (BDNF) and adenosine influence neuronal plasticity. We now investigated how adenosine influences the action of BDNF on synaptic transmission in the CA1 area of the rat hippocampal slices. Alone, BDNF (20-100 ng/ml) did not significantly affect field EPSPs (fEPSPs). However, a 2 min pulse of high-K(+) (10 mm) 46 min before the application of BDNF (20 ng/ml) triggered an excitatory action, an effect blocked by the TrkB receptor inhibitor K252a (200 nm), by the adenosine A(2A) receptor antagonist ZM 241385 (50 nm), and by the protein kinase A inhibitor H-89 (1 microm). Presynaptic, rather than postsynaptic depolarization was required to trigger the BDNF action because after K(+) depolarization BDNF also increased EPSCs recorded from pyramidal neurons voltage-clamped at -60 mV, and transient postsynaptic depolarization was unable to unmask the BDNF action. A weak theta burst stimulation of the afferents could elicit potentiation of synaptic transmission only when applied in the presence of BDNF. Activation of adenosine A(2A) receptors with CGS 21680 (10 nm), or the increase in extracellular adenosine levels induced by 5-iodotubercidin (100 nm) triggered the excitatory action of BDNF, a process prevented by ZM 241385 and by H-89. In the presence of dibutyryl-cAMP (0.5 mm), BDNF also increased fEPSPs but postsynaptic cAMP (0.5 mm) was unable to trigger the BDNF action. It is concluded that presynaptic activity-dependent release of adenosine, through activation of A(2A) receptors, facilitates BDNF modulation of synaptic transmission at hippocampal synapses.