Title: Loss of Skywalker reveals synaptic endosomes as sorting stations for synaptic vesicle proteins
Other Titles: Verlies van Skywalker onthult synaptische endosomen als sorteerstations voor synaptische vesikelproteïnen
Authors: Uytterhoeven, Valerie
Issue Date: 21-May-2012
Abstract: Maintenance of neurotransmission depends on an efficient vesicle fusion machinery but also on the continuous availability of vesicles at the synapse during stimulation. Given that neuronal cell bodies are often located far from nerve endings, synapses operate in part autonomously, and vesicles that are depleted during stimulation are rapidly and locally replenished for continued neurotransmission. Although, numerous proteins and lipids implicated in the regulation of presynaptic function have been characterized it is not known how synaptic terminals replace dysfunctional proteins and lipids and incorporate fresh components to protect against use dependent synaptic decline. To identify novel players in synaptic release, my collaborators and I have performed genome-wide mutagenesis screens that affect synaptic communication in Drosophila and isolated numerous genes. Upon genetic mapping our data indicate that one of the genes encodes a novel regulator of synaptic ageing and we named the protein Skywalker. Sky contains a TBC and TLDc domain, two evolutionary conserved domains commonly found in GTPase Activating Proteins (GAPs). GAPs accelerate the low endogenous GTPase activity of Rab GTPases which coordinate vesicle transport between organelles. The identification of Sky may thus suggest the existence of a GAP that may regulate synaptic vesicle trafficking.In this thesis I describe our investigation of Skywalker, a previously uncharacterized neuronal GAP protein that we showed to be localized at the synapse and that regulates Rab35 GTPase activity to control endosomal traffic of synaptic vesicles. Using a combination of 10 kDa dextran labeling, FM 1-43 labeling, photoconversion of FM 1-43 for electron microscopy, multiple time-point electron microscopy, electron tomography and electrophysiology, my data and the data of my collaborators indicate that in sky loss-of-function mutants or animals with over active Rab35, newly formed synaptic vesicles travel excessively via sorting endosomes marked by 2xFYVE-GFP and Rab5-GFP, and to a lesser extend via recycling endosomes marked by Rab4-GFP. Both biochemical and genetic interaction data indicate that Sky regulates Rab35 activity efficiently but not that of Rab5. Furthermore, animals expressing the constitutive active form of Rab35 as well as sky mutants show a dramatic increase in neurotransmitter release. While calcium influx upon stimulation is not affected in sky mutants, increased neurotransmitter release correlates with a larger size of the readily releasable synaptic vesicle pool. Consistent with our data, I propose that facilitation of endosomal trafficking in sky mutants mediates the exchange of inactive synaptic vesicle proteins for functional ones leading to a “cleaner” synaptic vesicle pool. Indeed, following FlAsH-FALI-mediated acute inactivation of the synaptic vesicle-associated protein Synaptotagmin in sky mutants and controls, I find much faster recovery of Synaptotagmin-function in sky mutants. In addition, loweringexpression of ESCRT complex components, involved in endo-lysosomal traffic of ubiquitinated proteins destined for degradation, dramatically suppresses increased neurotransmitter release in sky mutants. Furthermore, the synaptic levels of a chimeric Synaptobrevin-Ubiquitin protein that I expressed in sky mutants are much lower compared to controls and this can be rescued by removing an ESCRT-gene copy, suggesting that increased endosomal traffic of this chimeric synaptic vesicle protein results in its degradation. Taken together, these and other data suggest that endosomes mediate synaptic vesicle protein rejuvenation, and this function appears to be controlled by Skywalker and its main target Rab35, thus providing a novel mechanism by which neurons can regulate synaptic plasticity. These findings are not only of importance for the mechanisms of synaptic plasticity but have also implications for neuronal disease, as sky mutations in humans cause epilepsy.
ISBN: 978 94 6165 042 9
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Laboratory of Neuronal Communication
Department of Human Genetics - miscellaneous

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