Abstract
Neurotransmission is based on the exocytic fusion of synaptic vesicles (SVs) followed by endocytic membrane retrieval and the reformation of SVs. Conflicting models have been proposed regarding the mechanisms of SV endocytosis, most notably clathrin/adaptor protein complex 2 (AP-2)-mediated endocytosis and clathrin-independent ultrafast endocytosis. Partitioning between these pathways has been suggested to be controlled by temperature and stimulus paradigm. We report on the comprehensive survey of six major SV proteins to show that SV endocytosis in mouse hippocampal neurons at physiological temperature occurs independent of clathrin while the endocytic retrieval of a subset of SV proteins including the vesicular transporters for glutamate and GABA depend on sorting by the clathrin adaptor AP-2. Our findings highlight a clathrin-independent role of the clathrin adaptor AP-2 in the endocytic retrieval of select SV cargos from the presynaptic cell surface and suggest a revised model for the endocytosis of SV membranes at mammalian central synapses.
Highlights
Synaptic transmission relies on the release of neurotransmitters by calcium-triggered exocytic fusion of synaptic vesicles (SVs), tiny organelles (~40 nm in diameter) that store and secrete neurotransmitter molecules at specialized active zone (AZ) release sites within presynaptic nerve terminals (Südhof, 2004)
A second model supported by elegant high-p ressure freezing (Watanabe et al, 2014), electrophysiological (Delvendahl et al, 2016), and optical imaging (Kononenko et al, 2014; Soykan et al, 2017) experiments suggests that exocytosed SV proteins are internalized via clathrin- and action potential (AP)-2 -independent bulk endocytosis
Loss of clathrin or Adaptor protein complex 2 (AP-2) is predicted to result in distinct phenotypes: While endocytosis of SV proteins is unperturbed upon depletion of clathrin, loss of AP-2 would be expected to selectively affect the rate and efficacy of endocytosis of distinct SV cargos recognized by AP-2 (Figure 1C)
Summary
Synaptic transmission relies on the release of neurotransmitters by calcium-triggered exocytic fusion of synaptic vesicles (SVs), tiny organelles (~40 nm in diameter) that store and secrete neurotransmitter molecules at specialized active zone (AZ) release sites within presynaptic nerve terminals (Südhof, 2004). Temperature-sensitive, clathrin-independent SV endocytosis has been observed by presynaptic capacitance recordings at cerebellar mossy fiber boutons and by optical imaging at small hippocampal synapses (Delvendahl et al, 2016; Kononenko et al, 2014; Soykan et al, 2017) and is compatible with the accumulation of postendocytic presynaptic vacuoles upon acute or sustained genetic perturbation of clathrin at stimulated fly neuromuscular junctions (Kasprowicz et al, 2014; Kasprowicz et al, 2008; Heerssen et al, 2008) and at mammalian central synapses (Kononenko et al, 2014; Imig et al, 2020) These studies suggest that SV endocytosis under physiological conditions is primarily mediated by CIE (e.g., UFE), while the function of clathrin and clathrin adaptors such as AP-2 is limited to the reformation of functional SVs from internal ELVs rather than acting at the plasma membrane proper. Our findings highlight a clathrin-independent function of the clathrin adaptor AP-2 in the endocytic retrieval of select SV cargos from the presynaptic plasma membrane and suggest a revised model for SV endocytosis and recycling
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