Genetic Dissection of Synaptic Vesicle Endocytosis




Afuwape, Olusoji Adeyemi

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Synaptic transmission is mediated by the quantal release of neurotransmitters through the fusion of discrete synaptic vesicles with the presynaptic membrane. To maintain reliable transmission, synaptic vesicles and proteins must be recycled after release of neurotransmitters. A key protein in this recycling process is dynamin. Dynamin is a GTPase that catalyzes the scission of a budding endosome off its parent membrane. The mammalian brain expresses three isoforms of dynamin. Using genetically modified mouse hippocampal neurons, I analyzed the functional significance of dynamin in synaptic vesicle endocytosis. Specifically, I assessed dynamin 2 function in synaptic vesicle recycling and neurotransmission and investigated the role of dynamin independent endocytosis at the synapse. My data demonstrates that synaptic transmission after post-natal knockout of dynamin 2 remains intact and synaptic vesicle endocytosis, assessed by the trafficking of vesicular glutamate transporter fused to pHluorin, is unperturbed. Synaptic vesicle endocytosis in the absence of dynamin 2 was assessed at both room temperature and 32 oC. At both temperatures, my results reveal that synaptic vesicle recycling functions independent of dynamin 2 but also, the kinetics of single vesicle recycling is unaffected by changes in temperature suggesting that a single, temperature insensitive (within the limits of testing) form of endocytosis mediates single synaptic vesicle endocytosis. Further experiments reveal that the retrieval of single synaptic vesicles persists after the knockout of all dynamin isoforms. However, after multivesicular release, my results show an overall decrease in synaptic vesicle pool size and a retardation of subsequent vesicle endocytosis in neurons lacking all dynamin isoforms suggesting dynamin function at the synapse is activity dependent. This finding is consistent with prior reports showing dynamin function at the synapse is dependent on its dephosphorylation by the Ca2= dependent phosphatase, calcineurin. My results also demonstrate a dichotomy in the dependence of dynamin for synaptic neurotransmission. Whereas I observe a decrease in evoked amplitude, release probability and frequency of spontaneously released events in glutamatergic synapses, I observe no discernable defects in GABAergic neurotransmission. This result suggests inhibitory synapses are better equipped with compensatory mechanisms to deal with the loss of dynamins 1,2 and 3. Overall, my data demonstrate that dynamin is crucial but not essential for synaptic vesicle endocytosis. Dynamin is an activity dependent GTPase that is required for synaptic vesicle recycling after exocytosis of multiple vesicles. However, the underlying mechanism of single synaptic vesicle endocytosis is both dynamin and temperature independent.

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