INVESTIGATION OF A NOVEL 5-HT<SUB >2A</SUB> RECEPTOR-LINKED SIGNALLING PATHWAY INVOLVING NAADP-MEDIATED LYSOSOMAL CA<SUP>2+</SUP> RELEASE VIA TPC2

Psychedelic drugs such as psilocybin are currently under investigation for treatment of psychiatric disorders like major depression. These drugs exert their psychedelic, and possibly therapeutic, effects via the 5-HT_2A receptor, typically coupled to G_q-mediated Ca²⁺ release from endoplasmic reticulum (ER) stores. However, the possibility that Ca²⁺ could be released from additional stores which contribute to functional effects of these drugs, has not been considered. It has been reported that glutamate mGluR1, a G_q-coupled receptor, mobilises lysosomal Ca²⁺ stores through unknown mechanisms. Here we report a 5-HT_2A receptor signalling pathway involving Ca²⁺ release from lysosomes, mediated via nicotinic acid adenine dinucleotide phosphate (NAADP) acting on two-pore channels (TPC2). Ca²⁺ responses to 5-HT_2A receptor agonists were investigated using inhibitors of the NAADP pathway in C6 and SH-SY5Y cells expressing rat and human 5-HT_2A receptors, respectively. A genetically encoded TPC2-linked Ca²⁺ sensor (TPC2-GECO1.2) directly detected lysosomal Ca²⁺ release, and ELISA measured NAADP levels after 5-HT_2A receptor stimulation. Finally, head-twitch and plasticity-related gene expression responses to psilocin were measured in TPC2 KO mice compared to wildtypes. Results revealed that 5-HT_2A receptor agonists (5-HT, psilocin, DOI, 5-MeO-DMT and 25B-NBOMe) stimulated lysosomal Ca²⁺ release in C6 and SH-SY5Y cells. Additionally, 5-HT and 25B-NBOMe evoked increases in NAADP levels in C6 cells. Finally, TPC2 KO mice had reduced psilocin-evoked head-twitches and plasticity-related gene expression compared to wildtypes. Thus, this work revealed a novel 5-HT_2A receptor pathway involving NAADP-mediated Ca²⁺ release through lysosomal TPC2 channels. This pathway potentially contributes to driving psychedelic and neuroplastic effects of psychedelic drugs.