O-Linked β-N-Acetylglucosamine (O-GlcNAc) Site Thr-87 Regulates Synapsin I Localization to Synapses and Size of the Reserve Pool of Synaptic Vesicles

Background: Synapsin I regulates synaptic plasticity and is modified by O-GlcNAc. Results: Mutation of O-GlcNAc site Thr-87 to alanine increases both localization of synapsin I to synapses and the reserve pool of synaptic vesicles. Conclusion:O-GlcNAcylation of Thr-87 may regulate synapsin I localization and function. Significance:O-GlcNAcylation of synapsin I may modulate synaptic plasticity. O-GlcNAc is a carbohydrate modification found on cytosolic and nuclear proteins. Our previous findings implicated O-GlcNAc in hippocampal presynaptic plasticity. An important mechanism in presynaptic plasticity is the establishment of the reserve pool of synaptic vesicles (RPSV). Dynamic association of synapsin I with synaptic vesicles (SVs) regulates the size and release of RPSV. Disruption of synapsin I function results in reduced size of the RPSV, increased synaptic depression, memory deficits, and epilepsy. Here, we investigate whether O-GlcNAc directly regulates synapsin I function in presynaptic plasticity. We found that synapsin I is modified by O-GlcNAc during hippocampal synaptogenesis in the rat. We identified three novel O-GlcNAc sites on synapsin I, two of which are known Ca2+/calmodulin-dependent protein kinase II phosphorylation sites. All O-GlcNAc sites mapped within the regulatory regions on synapsin I. Expression of synapsin I where a single O-GlcNAc site Thr-87 was mutated to alanine in primary hippocampal neurons dramatically increased localization of synapsin I to synapses, increased density of SV clusters along axons, and the size of the RPSV, suggesting that O-GlcNAcylation of synapsin I at Thr-87 may be a mechanism to modulate presynaptic plasticity. Thr-87 is located within an amphipathic lipid-packing sensor (ALPS) motif, which participates in targeting of synapsin I to synapses by contributing to the binding of synapsin I to SVs. We discuss the possibility that O-GlcNAcylation of Thr-87 interferes with folding of the ALPS motif, providing a means for regulating the association of synapsin I with SVs as a mechanism contributing to synapsin I localization and RPSV generation.