Endocannabinoids Released on Demand via Extracellular Microvesicles

Academic Background

Endocannabinoids (ECBs) are a class of lipid neurotransmitters that play a critical role in brain function by activating cannabinoid receptor CB1. Unlike classical neurotransmitters, the storage and release mechanisms of ECBs have remained elusive, leading to significant gaps in our understanding of how these signals are regulated. In particular, the molecular mechanism of release and transport of 2-arachidonoylglycerol (2-AG), one of the major ECBs, is still unclear. Previous studies proposed an “on-demand production” model, suggesting that 2-AG is synthesized and released by neurons upon specific stimuli. However, this model fails to fully explain how 2-AG is released from neurons and how it crosses the cell membrane to reach target cells.

To address this issue, researchers developed an experimental system combining genetically encoded fluorescent sensors, electrophysiology, and mathematical modeling to study endocannabinoid signaling with high temporal precision. Their findings propose a new “on-demand release” model, suggesting that the formation of microvesicles is a key step in 2-AG release. This model not only extends the “on-demand production” model but also reconciles the three previously proposed hypotheses of ECB trafficking, providing a new framework for understanding endocannabinoid signaling.

Source of the Paper

This paper was co-authored by Verena M. Straub, Benjamin Barti, Sebastian T. Tandar, and others from multiple research institutions including Leiden University, Indiana University, and Peking University. It was published in PNAS (Proceedings of the National Academy of Sciences) on February 20, 2025, titled The endocannabinoid 2-arachidonoylglycerol is released and transported on demand via extracellular microvesicles.

Research Workflow

1. Development of Experimental System

The researchers developed a dual-culture system based on the fluorescent sensor GRABECB2.0 to study the release and transport of 2-AG. The system consists of HEK293T cells expressing GRABECB2.0 and neuronal cells (Neuro2A). When Neuro2A cells are stimulated, the released 2-AG activates the GRABECB2.0 sensor on adjacent HEK293T cells, allowing real-time monitoring of 2-AG dynamics through changes in fluorescence signals.

2. Validation of Fluorescent Sensor

Using confocal microscopy and a fluorescence plate reader, the researchers validated the effectiveness of the GRABECB2.0 sensor in detecting 2-AG release. Experiments showed that when Neuro2A cells were stimulated with ATP, the fluorescence signal in HEK293T cells significantly increased, while the mutant GRABECB2.0 sensor showed no response. Additionally, by adding different inhibitors, the researchers further confirmed that 2-AG release is regulated by protein kinase C (PKC), diacylglycerol lipase (DAGL), and ADP-ribosylation factor 6 (ARF6).

3. Isolation and Characterization of Microvesicles

The researchers isolated extracellular vesicles (EVs) from the culture supernatant of Neuro2A cells and characterized them using nanoparticle tracking analysis (NTA) and proteomics. The experiments demonstrated that ATP stimulation significantly increased EV release, and these EVs contained 2-AG but not another ECB, anandamide. Each microvesicle contained approximately 2,000 2-AG molecules.

4. Electrophysiological Experiments

To verify the role of microvesicles in neuronal signaling, the researchers conducted electrophysiological experiments in acute hippocampal slices. The results showed that ARF6 and ECB transport inhibitors significantly affected 2-AG-mediated synaptic plasticity, further supporting the importance of microvesicles in 2-AG signaling.

5. Construction of Mathematical Model

To quantitatively describe the kinetics of 2-AG release, the researchers constructed a mathematical model. The model considered 2-AG production, metabolism, distribution, as well as the formation and release of microvesicles. The model fitting results indicated that the formation of microvesicles is the rate-limiting step in 2-AG release, rather than 2-AG production.

Main Results

1. Effectiveness of GRABECB2.0 Sensor: Experiments confirmed that the GRABECB2.0 sensor can monitor the release and transport of 2-AG in real time, with signal changes significantly correlated with ATP stimulation.
2. Release of Microvesicles and 2-AG Content: ATP stimulation significantly increased EV release, and these EVs contained 2-AG but not anandamide. Each microvesicle contained approximately 2,000 2-AG molecules.
3. Role of ARF6 and ECB Transport Inhibitors: ARF6 and ECB transport inhibitors significantly affected 2-AG-mediated synaptic plasticity, indicating the crucial role of microvesicles in 2-AG signaling.
4. Support from Mathematical Model: The mathematical model showed that the formation of microvesicles is the rate-limiting step in 2-AG release, while the absorption of 2-AG is the overall rate-limiting step in sensor activation.

Conclusion

This study proposes a new “on-demand release” model, suggesting that the formation and release of microvesicles are key steps in 2-AG signaling. This model not only extends the “on-demand production” model but also provides a new molecular mechanism framework for understanding endocannabinoid signaling. The results indicate that microvesicles carrying 2-AG play an important role in neuronal communication, working alongside classical synaptic vesicles to regulate neurotransmission.

Highlights of the Study

1. Proposal of a New Model: The study first proposed the “on-demand release” model, offering a new perspective on understanding endocannabinoid signaling.
2. Innovation of Experimental System: By combining genetically encoded fluorescent sensors, electrophysiology, and mathematical modeling, the researchers developed a high-temporal-precision experimental system capable of real-time monitoring of 2-AG dynamics.
3. Role of Microvesicles: The study found that microvesicles are important carriers of 2-AG release, with each microvesicle containing approximately 2,000 2-AG molecules.
4. Interdisciplinary Research: The study combined molecular biology, electrophysiology, and mathematical modeling, showcasing the advantages of interdisciplinary research in solving complex biological problems.

Significance and Value of the Study

This study not only deepens the understanding of the mechanisms of endocannabinoid signaling but also provides new insights for developing drugs targeting CB1 receptors. Moreover, the proposed “on-demand release” model may serve as a reference for studying other lipid signaling pathways. By revealing the role of microvesicles in neuronal communication, the study offers potential new targets for treating neurological diseases.

Other Valuable Information

1. Proteomic Analysis: Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), the researchers analyzed the proteins in EVs, identifying various proteins related to 2-AG release and transport, such as ARF6 and FABP5.
2. Detailed Parameters of Mathematical Model: The model estimated the distribution and release rate of 2-AG in microvesicles, providing a quantitative tool for further research.
3. Validation by Electrophysiological Experiments: The study verified the role of microvesicles in 2-AG signaling in hippocampal slices, indicating that this mechanism is applicable under physiological conditions.

This study holds significant scientific value and provides new directions for future drug development and the treatment of neurological diseases.