Phosphorylation of α-synuclein directs trafficking of the v-SNARE synaptobrevin-2.

α-Synuclein has been extensively studied for its role in Parkinson's disease, however its native function remains unclear. Several lines of evidence suggest it modulates presynaptic function. As many presynaptic proteins have their functions regulated by phosphorylation, we sought to determine whether α-synuclein, a known phosphoprotein, might similarly have phospho-dependent roles. We identified two residues in α-synuclein, Ser42 and Thr81, that undergo activity-dependent phosphorylation in response to neuronal depolarisation. We employed a molecular replacement strategy, introducing α-synuclein phosphovariants into neuronal cultures from α-synuclein knockout (KO) mice (of either sex), and revealed that ablating phosphorylation at Thr81 altered the subcellular distribution of synaptobrevin-2/VAMP2, a crucial fusogenic vesicular SNARE protein and known binding partner of α-synuclein. Ablating phosphorylation of α-synuclein at Thr81 specifically accelerated endocytic retrieval of synaptobrevin-2 during stimulation without impacting global endocytosis, evoked exocytosis, or the size of the recycling pool. Conversely, mimicking phosphorylation at Thr81 augmented post-stimulation retrieval of synaptobrevin-2. This suggests that activity-dependent phosphorylation of Thr81 modulates the trafficking of synaptobrevin-2 to direct its retrieval via different endocytic pathways, thereby impacting presynaptic functionality and plasticity.Significance statement The presynaptic proteins α-synuclein has been extensively studied for its role in Parkinson's disease, however its native function remains unclear. As many presynaptic proteins have their functions regulated by phosphorylation, we sought to determine whether α-synuclein, a known phosphoprotein, might similarly have phospho-dependent roles. We identified two residues in α-synuclein, Ser42 and Thr81, that undergo activity-dependent phosphorylation Mimicking or ablating phosphorylation of α-synuclein at Thr81 specifically and differentially altered retrieval of synaptobrevin-2 without impacting global endocytosis, evoked exocytosis, or the size of the recycling pool. This suggests that activity-dependent phosphorylation of Thr81 modulates the trafficking of synaptobrevin-2 to direct its retrieval via different endocytic pathways, thereby impacting presynaptic functionality and plasticity.