Activity-dependent control of axonal amphisome trafficking governs norepinephrine release in vivo

Abstract

Amphisomes are autophagy-related organelles formed by the fusion of autophagosomes with late endosomes. In neurons, they contribute to both cargo degradation and signaling, yet their physiological roles in vivo remain unclear. Here, we show that axonal amphisome trafficking in highly branched locus coeruleus (LC) neurons is regulated by neuronal activity, novelty-associated behavioral state, and axonal norepinephrine (NE) signaling. Using fiber-mediated in vivo laser photoconversion and two-photon imaging, combined with chemogenetics and a genetically encoded norepinephrine sensor, we tracked amphisome transport in the intact brains. We show that organelles generated within distal LC axons projecting to the prefrontal and motor cortices (PFC/M1) undergo retrograde trafficking across the entire axonal length toward somatic compartments. Amphisome trafficking dynamics are bidirectionally regulated by presynaptic adrenergic signaling through opposing cAMP/PKA-dependent mechanisms. In vivo, elevated local autoreceptor-mediated norepinephrine signaling, involving association of activated {beta}2-adrenergic autoreceptors with SIPA1L2-positive amphisomes, constrains processive retrograde trafficking by promoting transient immobilization and localized, non-directional "jittery" motility states. Conversely, reduced local norepinephrine signaling promotes amphisome mobilization, and chemogenetic engagement of Gi-coupled signaling accelerated transport by reducing transient immobilization events and reinforcing directional processivity, thereby facilitating cargo delivery toward somatic degradative compartments. Together, these findings identify a local neuromodulatory mechanism linking norepinephrine signaling to axonal amphisome trafficking in vivo and suggest that neuromodulatory states, such as novelty- and wakefulness-associated LC activity, as well as sleep-associated silencing of LC activity, regulate neuronal proteostasis through local control of autophagic cargo transport.