Final Report Summary - BRAINCANNABINOIDS (Understanding the molecular blueprint and functional complexity of the endocannabinoid metabolome in the brain)
Endocannabinoid signaling has a central function in the control of synaptic communication between nerve cells. However, the precise molecular architecture, the physiological regulatory principles and the pathological significance of the brain endocannabinoid system have largely remained enigmatic. Therefore, the overall aim of the BRAINCANNABINOIDS project was to advance our understanding on how these novel lipid signaling pathways are organized at the molecular level in chemical synapses; to uncover how they contribute to certain forms of synaptic plasticity; and finally, how endocannabinoid-mediated synaptic signaling is affected in certain brain disorders. With the support of the BRAINCANNABINOIDS ERC Starting Grant, experimental work at the Laboratory of Molecular Neurobiology at the Institute of Experimental Medicine of the Hungarian Academy of Sciences in Budapest has led to several important discoveries in endocannabinoid research. These findings were or will be soon published in ~13 scientific publications in top-tier neuroscience journals including Nature Neuroscience, Neuron, Nature Communications, Journal of Neuroscience, European Journal of Neuroscience or Annual Review of Neuroscience. The results uncovered that diacylglycerol lipase-alpha (DGL-alpha), the key synthesizing enzyme of the endocannabinoid molecule 2-arachidonoylglycerol is a core component of excitatory synapses throughout the central nervous system. The data showed that this enzyme plays a key role in several stress-related physiological processes including stress-induced analgesia in the midbrain and in multiple synaptic mechanisms involved in the stress response in central amygdala. Combined neuroanatomical and patch-clamp recordings demonstrated that DGL-alpha level varies in a cell-type- and synapse-specific manner, which results in different induction thresholds for long-term synaptic depression. Importantly, DGL-alpha triggered long-term depression was completely absent in the mouse model of Fragile X syndrome, a severe mental retardation disorder. Electron microscopic analysis revealed that the lack of endocannabinoid-mediated synaptic plasticity is due to the impaired nanoscale targeting of this endocannabinoid-synthesizing enzyme. To enable nanoscale molecular imaging in a cell type-specific manner and its combination with physiological and anatomical measurements, a new methodical approach based on super-resolution imaging was developed in the laboratory. This achievement is expected to have widespread impact on other neuroscience research fields and it required several technical innovations including the use of transgenic rabbits to produce more sensitive antibodies, the design of several new protocols for immunostaining and tissue handling as well as the generation of new algorithms and software tools to visualize and analyze confocal and super-resolution images in a correlated manner. These advances were instrumental to discover the cell type-specific principles of the nanoscale architecture of synaptic cannabinoid signaling as well as helped to reveal the molecular mechanisms of cannabis-induced functional tolerance and cognitive dysfunctions. Finally, an unexpected new functional interaction between the two endocannabinoid signaling pathways mediated either by 2-arachidonoylglycerol or by anandamide were also uncovered in the tonic synaptic regulation of neurotransmitter release. The molecular architecture mediating synaptic anandamide signaling were therefore characterized by anatomical and physiological approaches in details. In terms of these research efforts, a novel molecular player was also identified with a surprising new function in the regulation of cortical neurogenesis. This serendipitous latter finding also opened new unforeseen research horizons for the laboratory in the future.