Investigating endocannabinoid signalling using a novel cell reporter assay

• Main Author: Lu, Leanne
• Other Authors: Doherty, Patrick ; Williams, Gareth
• Published: King's College London (University of London) 2018
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.754967

The endocannabinoid (eCB) system consists of cannabinoid receptors 1 and 2 (CB1 and CB2) that are activated by two well characterised lipid ligands, namely 2-arachidonoylglycerol (2-AG) and N-arachidonyl ethanolamine (AEA). 2-AG is synthesised by the diacylglycerol lipases (DAGLα and β), however these enzymes also synthesise other related lipids including 2-linoleoylglycerol (2-LG). The synthetic pathway for AEA synthesis is less clear. N-acylphosphatidylethanolamine-specific phospholipase D (NAPEPLD) is the most studied enzyme that can make AEA, however genetic knockout of NAPEPLD from animal models has little impact on AEA levels. More recently, a calcium activated N-acyl transferase (CaNAT) called PLA2G4E has been identified as a key regulatory enzyme in the AEA pathway. In this thesis, three key questions have been addressed. Firstly, a detailed study using a CB1 reporter cell assay was undertaken to test whether 2-LG can signal via the CB1 receptor and/or modulate the activity of eCBs. The results clearly showed that 2-LG is a novel CB1 receptor partial agonist capable of signalling on its own, and also capable of modulating eCB signalling. Using the same human osteosarcoma reporter cell assay, the hypothesis that a nine amino acid peptide Hemopressin is an antagonist for the CB1 receptor was tested. Hemopressin failed to inhibit CB1 activation stimulated by a synthetic CB1 agonist, or the response stimulated by 2-AG and AEA. Finally, the CB1 reporter cell assay was utilised to measure activation of the receptor in response to stimulation by calcium or by direct activation of PKA or PKC, presumably as a consequence of the regulated synthesis of one or more eCBs. The CRISPR/Cas9 system was then used to systematically knockout the DAGLs that are involved in the generation of 2-AG. The results from this section confirmed that the DAGLs are responsible for over 90% of 2-AG synthesis in the cells and that in some circumstances they can contribute to eCB signalling. However robust eCB signalling is still found in their absence, possibly mediated by AEA. Interestingly, genetic deletion of NAPEPLD had no impact on AEA levels or eCB signalling. The cells do not express detectable levels of PLA2G4E suggesting that an as yet to be discovered enzyme is responsible for AEA synthesis in the cells. I identified PLA2G4B as a possible candidate, however genetic deletion of this also had no impact on AEA levels or eCB signalling.