Phosphorylation of Synaptojanin Differentially Regulates Endocytosis of Functionally Distinct Synaptic Vesicle Pools

The rapid replenishment of synaptic vesiclesthrough endocytosisis crucial for sustaining synaptic transmission during intense neuronal activity. Synaptojanin(Synj), a phosphoinositide phosphatase, is known to play an important role in vesiclerecycling by promoting the uncoating of clathrinfollowing synaptic vesicleuptake. Synj has been shown to be a substrate of the minibrain (Mnb) kinase, a fly homolog of the dual-specificity tyrosine phosphorylation-regulated kinase 1A ( DYRK1A); however, the functional impacts of Synj phosphorylation by Mnb are not well understood. Here we identify that Mnb phosphorylates Synj at S1029 in Drosophila. We find that phosphorylation of Synj at S1029 enhances Synj phosphatase activity, alters interaction between Synj and endophilin, and promotes efficient endocytosisof the active cycling vesicle pool(also referred to as exo-endo cycling pool) at the expense of reserve pool vesicle endocytosis. DephosphorylatedSynj, on the other hand, is deficient in the endocytosisof the active recycling pool vesiclesbut maintains reserve pool vesicle endocytosisto restore total vesicle poolsize and sustain synaptic transmission. Together, our findings reveal a novel role for Synj in modulating reserve pool vesicle endocytosisand further indicate that dynamic phosphorylation and dephosphorylationof Synj differentially maintain endocytosisof distinct functional synaptic vesicle pools. SIGNIFICANCE STATEMENT Synaptic vesicle endocytosis sustains communicationbetween neurons during a wide range of neuronal activities by recycling used vesiclemembrane and protein components. Here we identify that Synaptojanin, a protein with a known role in synaptic vesicle endocytosis, is phosphorylated at S1029 in vivo by the Minibrain kinase. We further demonstrate that the phosphorylation status of Synaptojaninat S1029 differentially regulates its participation in the recycling of distinct synaptic vesicle pools. Our results reveal a new role for Synaptojaninin maintaining synaptic vesicle poolsize and in reserve vesicle endocytosis. As Synaptojaninand Minibrain perturbations are associated with various neurological disorders, such as Parkinson's, autism, and Down syndrome, understanding mechanisms modulating Synaptojaninfunction provides valuable insights into processes affecting neuronal communication.