Mechanism of action and neuroplastic potential of the 5-HT2A serotonin receptor agonist psilocin

Major depressive disorder (MDD) is a debilitating illness characterized by depressed mood, anhedonia, and impaired cognitive function. In the last few years, several compounds from various pharmacological classes emerged as promising rapid-onset antidepressant agents, including serotoninergic agonists. These drugs, collectively known as fast-acting antidepressants, demonstrate clinical effectiveness within 24 hours from the first administration and their effects persist long after the compounds have been eliminated from the bloodstream. It is believed that this clinical effect is driven by the promotion of long-term structural and functional neuroplasticity in the prefrontal cortex (PFC), a brain region known to experience neuronal atrophy in patients with MDD. Among all these molecules, a great interest garnered around the therapeutic potential of serotonergic psychedelics, especially psilocybin and its active metabolite psilocin. Psilocin is a partial agonists of the serotonin receptor 5-HT2A (5-HT2AR). 5-HT2ARs are G-protein coupled receptors that are densely expressed by post-synaptic neurons of the PFC, where they promote the excitatory effects of serotonin via the activation of the Gq/11 signaling cascade. Despite the role of 5-HT2ARs in the hallucinogenic effect of these molecules is well documented, their involvement in the neuroplastic effect, and whether this action is mediated by the canonical Gq/11 signaling pathway originating at the plasma membrane or by different signal transduction routes, is still unclear. The work described in this thesis is conceived within a collaboration with MGGM LLC, a company interested in the clinical development of psilocybin and its derivates. Notably, MGGM LLC recently submitted a clinical trial application for a preliminary Phase 1 study of a new, extended-release psilocybin formulation. The aim of the project is to investigate the mechanism of action and neuroplastic potential of molecules targeting 5-HT2ARs. A recent study suggests that psilocin, after entering neurons, might promote neuroplasticity through the activation of intracellular 5-HT2ARs. An intriguing idea is that psilocin becomes protonated and trapped in acidic compartments, accumulating and explaining its prolonged effects. To investigate this possibility, we developed a fluorescent-labeled psilocin derivative that we used as a probe for exploring the intracellular distribution of psilocin. By testing the new probe on primary cortical neurons and neuronal cells, we demonstrated that it mirrors the biological activity of psilocin in terms of 5-HT2AR activation and dendritic spine plasticity, endorsing its use as a strategic tool to explore psilocin mechanism of action. Using various microscopy imaging techniques, results obtained with this probe allowed us to shed new light on the intracellular distribution of psilocin. Preliminary data, aimed at assessing the role of psilocin accumulation in the neuroplastic outcome, suggest that psilocin retention inside acidic compartments might indeed contribute to prolong its neuroplastic action even upon a brief, transient stimulation. If confirmed, these data would explain why psilocin and other protonable membrane-permeable psychedelics promote long-term clinical effect after a single dose. Our approach, that we are currently implementing with ex vivo and in vivo methodologies, represents a novel, powerful strategy to comprehend the contribution of intracellular acidic compartments in the pharmacology of other fast-acting antidepressants. This will also lay the groundwork for the rational design of new serotoninergic agonists that leverage drug retention within acidic compartments.