Abstract
Recycling of synaptic vesicles (SVs) is a fundamental step in the process of neurotransmission. Endocytosed SV can travel directly into the recycling pool or recycle through endosomes but little is known about the molecular actors regulating the switch between these SV recycling routes. ADP ribosylation factor 6 (Arf6) is a small GTPase known to participate in constitutive trafficking between plasma membrane and early endosomes. Here, we have morphologically and functionally investigated Arf6-silenced hippocampal synapses and found an activity dependent accumulation of synaptic endosome-like organelles and increased release-competent docked SVs. These features were phenocopied by pharmacological blockage of Arf6 activation. The data reveal an unexpected role for this small GTPase in reducing the size of the readily releasable pool of SVs and in channeling retrieved SVs toward direct recycling rather than endosomal sorting. We propose that Arf6 acts at the presynapse to define the fate of an endocytosed SV.
Highlights
Synaptic vesicles (SVs) are continuously recycled at the nerve terminal and this process is tightly controlled to ensure the fidelity of neurotransmission
We first investigated on the expression of the small GTPase ADP ribosylation factor 6 (Arf6) at synaptic level by biochemical experiments and revealed expression of Arf6 in isolated nerve terminal-extract; differential extraction of synaptosomal proteins (Phillips et al, 2001) revealed that Arf6 is not tightly associated with presynaptic or postsynaptic membranes, as it is mainly extracted at pH6 to the SV protein synaptophysin
Ultrastructural analysis revealed that Arf6-KD synapses were undistinguishable from control synapses in terms of synaptic area and active zone (AZ) length (Supplementary file 1), but were characterized by a decreased total number of SVs and a significantly increased number of SVs docked at the AZ (Figure 1A)
Summary
Synaptic vesicles (SVs) are continuously recycled at the nerve terminal and this process is tightly controlled to ensure the fidelity of neurotransmission. The existence of endosome-like compartments has been demonstrated at the synapse since the pioneering experiments on synapse ultrastructure (Heuser and Reese, 1973), but their functional role in SV cycling is highly debated and the molecular and signaling pathways that define the fate of an endocytosed SV are still largely unknown (see for recent reviews Morgan et al, 2013; Kokotos and Cousin, 2015; Jahne et al, 2015). At postsynaptic sites, accumulating evidence has recently shown that Arf plays a role in AMPA receptor trafficking and long-term synaptic plasticity
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