ENDOCANNABINOID SIGNALLING IN OTOTOXICITY (Uncovering novel biomolecular clues in in vitro and in vivo models)

Cisplatin-induced ototoxicity is a debilitating side effect of chemotherapy which represents a leading cause of sensorineural hearing loss (SNHL) and remains a significant clinical burden to date. Cisplatin is known to primarily target sensory hair cells (HCs) in cochlea resulting in HC damage/loss leading to hearing impairment. At present, no effective treatments exist to prevent/counteract these adverse effects. Therefore, the lack of viable pharmacological approaches necessitates the need for better understanding of the molecular mechanisms underlying cisplatin induced hearing loss to identify new potential targets. In this context, recent preliminary studies have pointed out promising therapeutic roles for bioactive lipids such as endocannabinoids (eCBs) in ototoxicity. However, despite its emerging interest, the endocannabinoid system (ECS) remains poorly investigated in the context of auditory health and disease. To this end, firstly, a systematic molecular characterization of the ECS in a cochlear HC-like cell line was performed. We demonstrated for the first time the presence of bioactive eCBs and eCB-like lipids (through targeted lipidomics) along with key receptor targets and metabolic enzymes at the protein level in the cell line. Afterwards, potential alterations of the ECS were explored in an in vitro model of cisplatin-induced ototoxicity. Interestingly, novel biochemical perturbations in the ECS were observed, such as decrease in the protein expressions of Cannabinoid receptor 2 (CB2R), Diacylglycerol lipase β (DAGLβ) and α/β Hydrolase Domain-Containing Protein 6 (ABHD6), revealing HC-related targets in the context of drug-induced hearing trauma. Following the characterization of the ECS and its modulation upon ototoxicity in vitro, we extended our investigation to an in vivo model for a more physiological and translational relevance. To this end, we investigated the ECS components that were found to be altered in vitro (CB2R, DAGLβ and ABHD6) within the organ of Corti (OC) of an in vivo model of cisplatin-induced ototoxicity. Intriguingly, the analysis revealed similar alterations in vivo thereby strengthening our findings. Furthermore, we demonstrated that antagonism of CB2R through SR144528 ameliorates cisplatin induced HC death through reduction of cleaved-caspase 3 levels, thereby providing mechanistic insights into the role of CB2R in ototoxicity. Taken together, the work described in the present thesis provided a comprehensive characterization of the ECS elements in cochlear physiology using HC-like cell line. Furthermore, the study uncovered novel biomolecular targets within the ECS that may hold potential therapeutic relevance for cisplatin-induced SNHL.