Synaptic vesicle endocytosis (SVE) is triggered by calcineurin-mediated dephosphorylation of the dephosphin proteins. SVE is maintained by the subsequent rephosphorylation of the dephosphins by unidentified protein kinases. Here, we show that cyclin-dependent kinase 5 (Cdk5) phosphorylates dynamin I on Ser 774 and Ser 778 in vitro, which are identical to its endogenous phosphorylation sites in vivo. Cdk5 antagonists and expression of dominant-negative Cdk5 block phosphorylation of dynamin I, but not of amphiphysin or AP180, in nerve terminals and inhibit SVE. Thus Cdk5 has an essential role in SVE and is the first dephosphin kinase identified in nerve terminals.

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Dynamin I is dephosphorylated at Ser-774 and Ser-778 during synaptic vesicle endocytosis (SVE) in nerve terminals. Phosphorylation was proposed to regulate assembly of an endocytic protein complex with amphiphysin or endophilin. Instead, we found it recruits syndapin I for SVE and does not control amphiphysin or endophilin binding in rat synaptosomes. After depolarisation, syndapin exhibited a calcineurin-mediated interaction with dynamin. A phosphorylation sitemimicking peptide disrupted the dynamin-syndapin complex, not the dynamin-endophilin complex, arrested SVE and produced glutamate release fatigue after repetitive stimulation. Pseudo-phosphorylation of Ser-774 or Ser-778 inhibited syndapin binding without affecting amphiphysin recruitment. Site mutagenesis to alanine arrested SVE in cultured neurons. The effects of the sites were additive for syndapin I binding and SVE. Thus syndapin I is a central component of the endocytic protein complex for SVE via stimulus-dependent recruitment to dynamin I and plays a key role in synaptic transmission. KeywordsDynamin; syndapin; protein phosphorylation; endocytosis; synaptosomes; neurons Neurons communicate via the release of neurotransmitter by exocytosis from nerve terminals. After exocytosis, synaptic vesicles (SV) are retrieved by endocytosis to accommodate multiple cycles of synaptic transmission. Synaptic vesicle endocytosis (SVE) is triggered by a coordinated calcineurin-dependent dephosphorylation of a group of at least eight proteins called the dephosphins. They are dynamin I, amphiphysin I/II, synaptojanin, epsin, eps15, AP180 and PIP kinase Iγ 1. The dephosphins are constitutively phosphorylated in nerve terminals and their collective rephosphorylation after SVE is necessary for maintaining the continuity of SV recycling and thus maintenance of synaptic transmission. To date only one dephosphin kinase has been identified, cyclin-dependent kinase 5 (Cdk5) 2. It phosphorylates dynamin I, synaptojanin I and PIP kinase Iγ in vivo 2-4 and other dephosphins such as amphiphysin I in vitro 5. Cdk5 activity is required for SVE 2, yet it remains unknown whether each phosphorylation site in these substrates is functionally important for the basic mechanism of SVE and what functional role they serve in the process.Correspondence should be addressed to PJR: Tel, +61-2-9687-2800; Fax, +61-2-9687-2120; E-mail: phrobins@mail.usyd.edu.au.. Europe PMC Funders GroupAuthor Manuscript Nat Neurosci. Author manuscript; available in PMC 2007 November 20. Published Dynamin I is a large GTPase enzyme, the activity of which is required for vesicle fission in SVE 6. The proline-rich domain (PRD) at the C-terminus contains numerous binding motifs for src-3-homology (SH3) domains, through which it interacts with proteins such as amphiphysin I 7, endophilin I 8, and syndapin I 9. The SH3-mediated dynamin I interactions of amphiphysin and endophilin are involved in SVE 10, 11. An emerging idea is that different synaptic proteins like endophilin and amphiphysin are involved in ...

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.

Synaptic vesicles (SVs) are retrieved by more than one mode in central nerve terminals. During mild stimulation, the dominant SV retrieval pathway is classical clathrin-mediated endocytosis (CME). During elevated neuronal activity, activity-dependent bulk endocytosis (ADBE) predominates, which requires activation of the calcium-dependent protein phosphatase calcineurin. We now report that calcineurin dephosphorylates dynamin I in nerve terminals only above the same activity threshold that triggers ADBE. ADBE was arrested when the two major phospho-sites on dynamin I were perturbed, suggesting that dynamin I dephosphorylation is a key step in its activation. Dynamin I dephosphorylation stimulates a specific dynamin I-syndapin I interaction. Inhibition of this interaction by competitive peptides or by site-directed mutagenesis exclusively inhibited ADBE but did not affect CME. The results reveal that the phospho-dependent dynamin-syndapin interaction recruits ADBE to massively increase SV endocytosis under conditions of elevated neuronal activity.