The multidomain scaffold protein intersectin 1 has been implicated in several cellular signaling pathways and processes. The best described function of intersectin 1 is its association with early proteins during clathrin-mediated endocytosis, a process with particular importance in the presynapse in neurons during the reformation of synaptic vesicles. However, intersectin’s numerous domains interact with proteins of diverse processes conceivably involving intersectin 1’s function in further pathways in the presynapse. In the present study we identified so far unknown functions of intersectin 1 in the synaptic vesicle cycle in neurons. Proper cycling of synaptic vesicles is essential for neurotransmission at synapses and includes several processes like clathrin-mediated reformation of synaptic vesicles. In a first project, we identified an accumulation of clathrin-coated vesicles in mice depleted of intersectin 1, a phenotype akin to loss of the endocytic protein endophilin and the phosphatase synaptojanin that is involved in uncoating of clathrin-coated vesicles. Furthermore, we found a direct interaction between intersectin 1 and endophilin in the brain which mediates the association of endophilin with the clathrin machinery. We argue that this association is involved in proper recruitment of the phosphatase synaptojanin to sites of clathrin coated vesicle formation to achieve efficient uncoating of vesicles and thereby reformation of synaptic vesicles. In a second project, we investigated the interaction between intersectin and synapsin, a protein implicated in clustering of reserve pool synaptic vesicles. During sustained neurotransmission synapsin’s dissociation from synaptic vesicles is crucial for replenishment of the recycling pool that comprises release-ready vesicles. We found that the interaction between intersectin and synapsin is regulated by a phosphorylation-dependent intramolecular lock within intersectin that enables binding to synapsin only during neuronal activity. Loss of intersectin in hippocampal neurons resulted in a mislocalization of synapsin in combination with a reduced recycling pool size. We rescued these phenotypes by reexpression of wild-type intersectin but not with a locked, synapsin-binding deficient intersectin mutant. We argue that intersectin associates with synapsin upon activity, prevents premature reclustering of synaptic vesicles and thereby enables replenishment of release-ready vesicles during sustained stimulation. In a third project, we identified an interaction between intersectin and the assembled SNARE complex. The assembly of SNARE complexes is essential for synaptic vesicle fusion at release sites within the active zone. Hippocampal neurons depleted of intersectin displayed exocytic depression in a frequency dependent manner, but also upon sustained stimulation. Additionally, neurons expressing an intersectin binding deficient mutant of the v-SNARE synaptobrevin 2 phenocopied this exocytic depression. We hypothesize that intersectin binds postfusion SNARE complexes and removes them from release sites to enable subsequent vesicle fusion and to prevent exocytic depression. In summary, we showed intersectin’s versatile functions within the synaptic vesicle cycle not only during clathrin-mediated endocytosis but also in synaptic vesicle clustering and exo-/endocytic coupling. Taken together, our data reveal intersectin’s importance for maintaining synaptic functionality especially during sustained neurotransmission.
