Retrograde messengers adjust the precise timing of neurotransmitter release from the presynapse, thus modulating synaptic efficacy and neuronal activity. 2-Arachidonoyl glycerol, an endocannabinoid, is one such messenger produced in the postsynapse that inhibits neurotransmitter release upon activating presynaptic CB₁ cannabinoid receptors. Cognitive decline in Alzheimer’s disease is due to synaptic failure in hippocampal neuronal networks. We hypothesized that errant retrograde 2-arachidonoyl glycerol signalling impairs synaptic neurotransmission in Alzheimer’s disease. Comparative protein profiling and quantitative morphometry showed that overall CB₁ cannabinoid receptor protein levels in the hippocampi of patients with Alzheimer’s disease remain unchanged relative to age-matched controls, and CB₁ cannabinoid receptor-positive presynapses engulf amyloid-β-containing senile plaques. Hippocampal protein concentrations for the sn-1-diacylglycerol lipase α and β isoforms, synthesizing 2-arachidonoyl glycerol, significantly increased in definite Alzheimer’s (Braak stage VI), with ectopic sn-1-diacylglycerol lipase β expression found in microglia accumulating near senile plaques and apposing CB₁ cannabinoid receptor-positive presynapses. We found that microglia, expressing two 2-arachidonoyl glycerol-degrading enzymes, serine hydrolase α/β-hydrolase domain-containing 6 and monoacylglycerol lipase, begin to surround senile plaques in probable Alzheimer’s disease (Braak stage III). However, Alzheimer’s pathology differentially impacts serine hydrolase α/β-hydrolase domain-containing 6 and monoacylglycerol lipase in hippocampal neurons: serine hydrolase α/β-hydrolase domain-containing 6 expression ceases in neurofibrillary tangle-bearing pyramidal cells. In contrast, pyramidal cells containing hyperphosphorylated tau retain monoacylglycerol lipase expression, although at levels significantly lower than in neurons lacking neurofibrillary pathology. Here, monoacylglycerol lipase accumulates in CB₁ cannabinoid receptor-positive presynapses. Subcellular fractionation revealed impaired monoacylglycerol lipase recruitment to biological membranes in post-mortem Alzheimer’s tissues, suggesting that disease progression slows the termination of 2-arachidonoyl glycerol signalling. We have experimentally confirmed that altered 2-arachidonoyl glycerol signalling could contribute to synapse silencing in Alzheimer’s disease by demonstrating significantly prolonged depolarization-induced suppression of inhibition when superfusing mouse hippocampi with amyloid-β. We propose that the temporal dynamics and cellular specificity of molecular rearrangements impairing 2-arachidonoyl glycerol availability and actions may differ from those of anandamide. Thus, enhanced endocannabinoid signalling, particularly around senile plaques, can exacerbate synaptic failure in Alzheimer’s disease.

Adenosine A2A receptors (A2AR) overfunction causes synaptic and memory dysfunction in early Alzheimer's disease (AD). In a β-amyloid (Aβ1-42)-based model of early AD, we now unraveled that this involves an increased synaptic release of ATP coupled to an increased density and activity of ecto-5'-nucleotidase (CD73)-mediated formation of adenosine selectively activating A2AR. Thus, CD73 inhibition with α,β-methylene-ADP impaired long-term potentiation (LTP) in mouse hippocampal slices, which is occluded upon previous superfusion with the A2AR antagonist SCH58261. Furthermore, α,β-methylene-ADP did not alter LTP amplitude in global A2AR knockout (KO) and in forebrain neuron-selective A2AR-KO mice, but inhibited LTP amplitude in astrocyte-selective A2AR-KO mice; this shows that CD73-derived adenosine solely acts on neuronal A2AR. In agreement with the concept that ATP is a danger signal in the brain, ATP release from nerve terminals is increased after intracerebroventricular Aβ1-42 administration, together with CD73 and A2AR upregulation in hippocampal synapses. Importantly, this increased CD73 activity is critically required for Aβ1-42 to impair synaptic plasticity and memory since Aβ1-42-induced synaptic and memory deficits were eliminated in CD73-KO mice. These observations establish a key regulatory role of CD73 activity over neuronal A2AR and imply CD73 as a novel target for modulation of early AD.

Background: It has been hypothesized that heteromers of adenosine A_2A 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.