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

ABSTRACT: 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 the rate-limiting step. A co31 culture model system that combines a reporter cell line expressing the fluorescent eCB sensor,GRABeCB2.0, and neuronal cells revealed that neurons release EVs containing 2-AG, but not anandamide, in a stimulus-dependent process regulated by PKC, DAGL, Arf6, and which was sensitive to inhibitors of eCB facilitated diffusion. A vesicle contained approximately 2000 2-AG molecules. Accordingly, hippocampal eCB-mediated synaptic plasticity was modulated by Arf6 and transport inhibitors. This “on demand release” model, supported by mathematical analysis, offers a cohesive framework for understanding eCB signaling at the molecular level and suggests that microvesicles carrying signaling lipids regulate neuronal functions in parallel to canonical synaptic vesicles. To show the identity and purity of the EVs isolated in this study, the proteome of the EVs and their parental cells were analyzed by LC-MS/MS

INSTRUMENT(S): timsTOF HT

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Verena Straub

LAB HEAD: Mario van der Stelt

PROVIDER: PXD056498 | Pride | 2025-02-27

REPOSITORIES: pride

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			Action	DRS
	155_1_EV1_dil._3_Slot1-19_1_1567.d.zip	Other
	155_2_EV2_dil._3_Slot1-20_1_1568.d.zip	Other
	155_3_EV3_dil._3_Slot1-21_1_1569.d.zip	Other
	155_4_N2A1_3_Slot1-22_1_1571.d.zip	Other
	155_5_N2A2_3_Slot1-23_1_1572.d.zip	Other

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Project description:Extracellular vesicles (EVs) are small membrane-derived vesicles that shuttle proteins or nucleic acids between glia and neurons, thereby promoting neuronal survival and plasticity in the CNS. Exosomes are small EVs (40-150 nm) that are derived from multi-vesicular bodies (MVBs) of the endo-lysosomal pathway, formed by inward budding of the limiting membrane into the MVB lumen and released into the extracellular space upon fusion of the MVB with the plasma membrane (PM). Previous research revealed certain effector molecules to be required for exosome release. For instance, RAB GTPases have been shown to control exosome release in a cell- and tissue-specific manner. In addition, exosome release appears to be evoked by membrane depolarization in conjunction with calcium influx and to depend on members of the SNARE family of proteins. However, the specific cellular and molecular factors that regulate neuronal exosome release and segregate it from the release of neurotransmitter vesicles are currently unknown. Here, we used a combination of molecular biology, patch-clamp electrophysiology and pH-sensitive dye imaging to examine the effect of the neuronal growth and differentiation factors basic fibroblast growth factor (bFGF), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) on neuronal EV release. We found that bFGF-treatment of cultured hippocampal neurons increased the abundance of EVs in the culture medium as measured by Western blot and nanoparticle tracking analysis (NTA), without affecting the number or size of neuronal MVBs. The effect of bFGF depended on calcium and on receptor tyrosine kinase (RTK) activity as the calcium chelating agent BAPTA and the tyrosine kinase inhibitor genistein both abolished the bFGF-induced increase in EV release. In accord with these results, untreated neurons had a low rate of spontaneous and stimulus-evoked MVB-PM fusion events as measured by pH-sensitive dye imaging in conjunction with patch-clamp electrophysiology, but treatment with bFGF significantly increased the number of neurons exhibiting MVB-PM fusion events in response to a high-frequency stimulus. Proteomic analysis of neuronal EVs by Liquid Chromatography Mass Spectrometry (LC-MS) demonstrated bFGF to increase the abundance of the v-SNARE vesicle-associated membrane protein 3 (VAMP3, cellubrevin) on EVs, whereas VAMP2 was decreased. Conversely, knocking-down VAMP3 in cultured neurons abolished the effect of bFGF on EV release. Similar to bFGF, the classical neurotrophins BDNF and NGF increased neuronal EV release in a VAMP3-dependent manner. In summary, our results thus reveal a new function for neurotrophic factor signalling in controlling neuronal exosome release and support the investigation of growth factor-mediated signal transduction via EVs in the healthy and diseased CNS.

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

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