The contribution of endocytic mediators to itch transmission.

Chronic itch is a major burden, impacting the quality of life for one in four adults, and is closely associated with increased levels of anxiety, depression, and suicide. Despite its widespread burden, the precise mechanisms driving and sustaining chronic itch remain poorly understood. Non-histaminergic itch transmission in the spinal cord relies on the synaptic vesicle (SV) release of gastrin-releasing peptide (GRP) and the subsequent binding to the Gastrin-Releasing Peptide Receptor (GRPR). While SV exocytosis facilitates neurotransmitter release, such as GRP, SV endocytosis, mediated by key proteins including clathrin, adaptor associated kinase 1 (AAK1), and Dynamin (Dnm) are essential for retrieving and recycling SV from the presynaptic membrane to maintain signaling. Building upon evidence that AAK1- and Dnm-mediated endocytosis are viable targets to reverse pain, we characterized the role of these endocytic mediators in the regulation of synaptic transmission in itch pathways. We localized mRNA encoding AAK1, Dnm1 and Dnm3 within GRP positive neurons of the mouse dorsal root ganglia (DRG). Genetic and pharmacological disruption of AAK1, Dnm1 and Dnm3 were used to evaluate scratching behavior, locomotor activity, and anxiety-like behaviors. Finally, we assessed the impacts of these treatments on GRP release. Genetic and pharmacological disruption of AAK1 significantly reduced scratching behavior compared to control groups. This anti-pruritic effect correlated with confirmed Aak1 mRNA knockdown in both the DRG and spinal cord. Similarly, siRNA mediated knockdown of Dnm1 and Dnm3 in the DRGs also reduced scratching behavior. Crucially, these treatments decreased GRP release without altering locomotor activity or anxiety-like behaviors. Together, these findings suggest that the disruption of SV recycling reduces itch related signaling and behavior without affecting normal motor functions, providing a new approach for chronic pruritus.