Dynamin GTPase activity increases when it oligomerizes either into helices in the presence of lipid templates or into rings in the presence of SH3 domain proteins. Dynasore is a dynamin inhibitor of moderate potency (IC₅₀ ˜ 15 μM in vitro). We show that dynasore binds stoichiometrically to detergents used for in vitro drug screening, drastically reducing its potency (IC₅₀ = 479 μM) and research tool utility. We synthesized a focused set of dihydroxyl and trihydroxyl dynasore analogs called the Dyngo™ compounds, five of which had improved potency, reduced detergent binding and reduced cytotoxicity, conferred by changes in the position and/or number of hydroxyl substituents. The Dyngo compound 4a was the most potent compound, exhibiting a 37‐fold improvement in potency over dynasore for liposome‐stimulated helical dynamin activity. In contrast, while dynasore about equally inhibited dynamin assembled in its helical or ring states, 4a and 6a exhibited >36‐fold reduced activity against rings, suggesting that they can discriminate between helical or ring oligomerization states. 4a and 6a inhibited dynamin‐dependent endocytosis of transferrin in multiple cell types (IC₅₀ of 5.7 and 5.8 μM, respectively), at least sixfold more potently than dynasore, but had no effect on dynamin‐independent endocytosis of cholera toxin. 4a also reduced synaptic vesicle endocytosis and activity‐dependent bulk endocytosis in cultured neurons and synaptosomes. Overall, 4a and 6a are improved and versatile helical dynamin and endocytosis inhibitors in terms of potency, non‐specific binding and cytotoxicity. The data further suggest that the ring oligomerization state of dynamin is not required for clathrin‐mediated endocytosis.

Neurotransmission is sustained by endocytosis and refilling of synaptic vesicles (SVs) locally within the presynapse. Until recently, a consensus formed that after exocytosis, SVs are recovered by either fusion pore closure (kiss-and-run) or clathrin-mediated endocytosis directly from the plasma membrane. However, recent data have revealed that SV formation is more complex than previously envisaged. For example, two additional recycling pathways have been discovered, ultrafast endocytosis and activity-dependent bulk endocytosis, in which SVs are regenerated from the internalized membrane and synaptic endosomes. Furthermore, these diverse modes of endocytosis appear to influence both the molecular composition and subsequent physiological role of individual SVs. In addition, previously unknown complexity in SV refilling and reclustering has been revealed. This review presents a modern view of the SV life cycle and discusses how neuronal subtype, physiological temperature, and individual activity patterns can recruit different endocytic modes to generate new SVs and sculpt subsequent presynaptic performance.

After neurotransmitter release evoked by intense stimulation, synaptic vesicles are retrieved either by clathrin-mediated endocytosis or by activity-dependent bulk endocytosis (ADBE), both of which require the GTPase dynamin I. Here the authors show that presynaptic glycogen synthase kinase-3 specifically regulates synaptic vesicle retrieval during ADBE via phosphorylation of dynamin I. Glycogen synthase kinase 3 (GSK3) is a critical enzyme in neuronal physiology; however, it is not yet known whether it has any specific role in presynaptic function. We found that GSK3 phosphorylates a residue on the large GTPase dynamin I (Ser-774) both in vitro and in primary rat neuronal cultures. This was dependent on prior phosphorylation of Ser-778 by cyclin-dependent kinase 5. Using both acute inhibition with pharmacological antagonists and silencing of expression with short hairpin RNA, we found that GSK3 was specifically required for activity-dependent bulk endocytosis (ADBE) but not clathrin-mediated endocytosis. Moreover we found that the specific phosphorylation of Ser-774 on dynamin I by GSK3 was both necessary and sufficient for ADBE. These results demonstrate a presynaptic role for GSK3 and they indicate that a protein kinase signaling cascade prepares synaptic vesicles for retrieval during elevated neuronal activity.