The endocannabinoid 2-arachidonoylglycerol is released and transported on demand via extracellular microvesicles

Straub, Verena M. and Barti, Benjámin and Tandar, Sebastian T. and Stevens, A. Floor and Egmond, Noëlle van and Wel, Tom van der and Zhu, Na and Rüegger, Joel and Horst, Cas van der and Heitman, Laura H. and Li, Yulong and Stella, Nephi and Coen van Hasselt, J. G. and Katona, István and Stelt, Mario van der (2025) The endocannabinoid 2-arachidonoylglycerol is released and transported on demand via extracellular microvesicles. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 122 (8). No.-e2421717122. ISSN 0027-8424

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Abstract

Endocannabinoids (eCB) are lipid neurotransmitters that play a critical role in brain function by activating the cannabinoid CB1 receptor. Unlike classical neurotransmitters, their storage and release mechanisms have remained elusive, leading to major gaps in our understanding of how these signals are regulated. In this study, we developed an experimental system combining genetically encoded fluorescent sensors, electrophysiology, and mathematical modeling to study endocannabinoid signaling with temporal precision. Our findings led us to propose an ?on-demand release? model, where microvesicle formation governs endocannabinoid release. This comprehensive model extends the ?on-demand production? model and reconciliates the three previously proposed hypotheses for eCB trafficking. The model reshapes our understanding of endocannabinoid signaling and addresses key unanswered questions in the field. While it is known that endocannabinoids (eCB) modulate multiple neuronal functions, the molecular mechanism governing their release and transport remains elusive. Here, we propose an ?on-demand release? model, wherein the formation of microvesicles, a specific group of extracellular vesicles (EVs) containing the eCB, 2-arachidonoylglycerol (2-AG), is an important step. A coculture model system that combines a reporter cell line expressing the fluorescent eCB sensor, G protein-coupled receptor-based (GRAB)eCB2.0, and neuronal cells revealed that neurons release EVs containing 2-AG, but not anandamide, in a stimulus-dependent process regulated by protein kinase C, Diacylglycerol lipase, Adenosinediphosphate (ADP) ribosylation factor 6 (Arf6), and which was sensitive to inhibitors of eCB facilitated diffusion. A vesicle contained approximately 2,000 2-AG molecules. Accordingly, hippocampal eCB-mediated synaptic plasticity was modulated by Arf6 and transport inhibitors. The ?on-demand release? model, supported by mathematical analysis, offers a cohesive framework for understanding eCB trafficking at the molecular level and suggests that microvesicles carrying signaling lipids in their membrane regulate neuronal functions in parallel to canonical synaptic vesicles.

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