Genetic Manipulation of sn-1-Diacylglycerol Lipase and CB1 Cannabinoid Receptor Gain-of-Function Uncover Neuronal 2-Linoleoyl Glycerol Signaling in Drosophila melanogaster

Giuseppe Tortoriello, Johannes Beiersdorf, Susana Romani, Gareth Williams, Gary Cameron, Ken Mackie, Michael Williams, Vincenzo Di Marzo, Erik Keimpema, Patrick Doherty, Tibor Harkany*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

7 Citations (Scopus)


Introduction: In mammals, sn-1-diacylglycerol lipases (DAGL) generate 2-arachidonoylglycerol (2-AG) that, as the major endocannabinoid, modulates synaptic neurotransmission by acting on CB1 cannabinoid receptors (CB1R). Even though the insect genome codes for inaE, which is a DAGL ortholog (dDAGL), its products and their functions remain unknown particularly because insects lack chordate-type cannabinoid receptors.

Materials and Methods: Gain-of-function and loss-of-function genetic manipulations were carried out in Drosophila melanogaster, including the generation of both dDAGL-deficient and mammalian CB1R-overexpressing flies. Neuroanatomy, dietary manipulations coupled with targeted mass spectrometry determination of arachidonic acid and 2-linoleoyl glycerol (2-LG) production, behavioral assays, and signal transduction profiling for Akt and Erk kinases were employed. Findings from Drosophilae were validated by a CB1R-binding assay for 2-LG in mammalian cortical homogenates with functionality confirmed in neurons using high-throughput real-time imaging in vitro.

Results: In this study, we show that dDAGL is primarily expressed in the brain and nerve cord of Drosophila during larval development and in adult with 2-LG being its chief product as defined by dietary precursor availability. Overexpression of the human CB1R in the ventral nerve cord compromised the mobility of adult Drosophilae. The causality of 2-LG signaling to CB1R-induced behavioral impairments was shown by inaE inactivation normalizing defunct motor coordination. The 2-LG-induced activation of transgenic CB1Rs affected both Akt and Erk kinase cascades by paradoxical signaling. Data from Drosophila models were substantiated by showing 2-LG-mediated displacement of [3H]CP 55,940 in mouse cortical homogenates and reduced neurite extension and growth cone collapsing responses in cultured mouse neurons.

Conclusions: Overall, these results suggest that 2-LG is an endocannabinoid-like signal lipid produced by dDAGL in Drosophila.
Original languageEnglish
Pages (from-to)119-136
Number of pages18
JournalCannabis and Cannabinoid Research
Issue number2
Early online date12 May 2020
Publication statusPublished - 15 Apr 2021

Bibliographical note

The authors thank O.K. Penz for her assistance with the maintenance of Drosophila stocks, M. Watanabe for anti-CB1 receptor antibodies, and T. Hummel for discussions and critical feedback on this article. GW Pharmaceuticals (United Kingdom) are acknowledged for providing access to an IncuCyte Zoom (Essen Bioscience) live-cell imaging platform.

Funding Information
This work was supported by the EMBO Young Investigator Program (T.H.), Swedish Research Council (T.H.); Novo Nordisk Foundation (T.H.); Hjärnfonden (T.H.); European Research Council (SECRET-CELLS, ERC-2015-AdG-695136; T.H.), intramural funds of the Medical University of Vienna (T.H.); and the Wellcome Trust (P.D.)


  • 2-monoacylglycerol
  • behavior
  • development
  • diacylglycerol lipase
  • inaE


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