Role of Histamine H₃ Receptor Antagonist Pitolisant in Early Neural Differentiation of Mouse Embryonic Stem Cells

The histamine H₃ receptor, prominently expressed in neurons with a minor presence in glial cells, acts as both an autoreceptor and an alloreceptor, controlling the release of histamine and other neurotransmitters. The receptor impacts various essential physiological processes. Our team's initial investigations had demonstrated that the histamine H₃ receptor antagonists could facilitate nerve regeneration by promoting the histamine H₁ receptors on primary neural stem cells (NSCs) in the traumatic brain injury mouse, which suggested the potential of histamine H₃ receptor as a promising target for treating neurological disorders and promoting nerve regeneration. Pitolisant (PITO) is the only histamine H₃ receptor antagonist approved by the Food and Drug Administration (FDA) for treating narcolepsy. However, there is no report on Pitolisant in neural development or regeneration, and it is urgent to be further studied in strong biological activity models in vitro. The embryonic stem (ES) cells were differentiated into neural cells in vitro, which replicated the neurodevelopmental processes that occur in vivo. It also provided an alternative model for studying neurodevelopmental processes and testing drugs for neurological conditions. Therefore, we aimed to elucidate the regulatory role of Pitolisant in the early differentiation of ES cells into neural cells. Our results demonstrated that Pitolisant could promote the differentiation of ES cells toward NSCs and stimulated the formation of growth cones. Furthermore, Pitolisant was capable of inducing the polarization of NSCs through the cAMP-LKB1-SAD/MARK2 pathway, but had no significant effect on later neuronal maturation. Pitolisant altered mitochondrial morphology and upregulated the levels of mitochondrion-related proteins TOM20, Drp1, and p-Drp1, and reversed the inhibitory effect of Mdivi-1 on mitochondrial fission during the early neural differentiation of ES cells. In addition, Pitolisant induced the increase in cytosolic Ca²⁺. Our study provided an experimental foundation for the potential application of histamine H₃ receptor-targeted modulators in the field of neuroregeneration.