The selective loss of cerebellar Purkinje cells (PCs) is a common hallmark of several neurodegenerative movement disorders. The mechanism underlying their selective vulnerability has not been yet identified. Here we show that endocytic adaptor AP-2 is essential to maintain the survival of PCs. We reveal that mice lacking the µ subunit of the AP-2 complex in cerebellar PCs develop severe gait abnormalities accompanied by PC progressive degeneration. Importantly, synaptic input dysfunction, characterized by a dominance of parallel fiber (PF) over climbing fiber (CF) synapses precedes the PC loss. We identified Delphilin as a novel AP-2 binding partner in the cerebellum. Delphilin is a postsynaptic scaffolding protein selectively expressed at the parallel fiber-PCs synapse. Delphilin interacts with the glutamate δ2 receptor (GRID2), a protein essential for cerebellar long-term depression, motor learning, and PF synapse formation, and its loss in humans causes spinocerebellar ataxia type 18. AP-2-deficient PCs reveal loss of synaptic Delphilin and accumulation of GRID2 to distal dendrites, a phenotype that results in PF/CF misbalance in the cerebellum of conditional AP-2 KO mice. Moreover, proteomics data obtained from the cerebellum of these mice show upregulation of proteins involved in processes such as synaptic pruning and downregulation of proteins related to long-term depression and spinocerebellar ataxia. Interestingly, the overrepresentation of PF leads to PC hyperexcitation that can be rescued by facilitating synaptic glutamate clearance. Our data suggest that AP2 prevents motor gait dysfunction by regulating dendritic levels of GRID2 in PCs, which is required to maintain synaptic connectivity in the cerebellum.

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Enteroendocrine cells (EECs) comprise a heterogenous group of chemo- and mechanosensitive cells in the gut epithelium that transmit information from the gut to the central nervous system via the release of peptide hormones and neurotransmitters. Recent studies indicate that distinct EEC subtypes regulate processes such as food reward and aversion (Bai et al., eLife, 2022), feeding behavior and gut motility (Hayashi et al., eLife, 2023), visceral pain and anxiety (Bayrer et al., Nature, 2023), or sodium appetite (Liu et al., Science Advances, 2023). EECs express a large number of proteins composing the neuronal presynaptic release machinery and may even form „axon-like“ processes or „synaptic-like“ contacts with vagal nerve endings. Despite their role in mediating important physiological processes and behaviors, little is known about the cell biological and molecular mechanisms underlying stimulus-induced vesicle fusion, transmitter and peptide release, and signaling to neurons in specific EEC subtypes. To gain a better understanding of the functional organization of secretory vesicle pools in genetically identified EEC subtypes, we established and characterized an in vitro experimental workflow combining mouse genetics, 2D-monolayer cultures of mouse gut epithelium, and single-cell electrophysiology and electrochemistry. We anticipate that our approach will make it possible to understand the molecular control underlying synaptic-like gut-brain-axis signaling by specific EEC subtypes in health and disease.