Safeguards of Neurotransmission: Endocytic Adaptors as Regulators of Synaptic Vesicle Composition and Function

Abstract

Communication between neurons relies on neurotransmitters which are released from synaptic vesicles (SVs) upon Ca²⁺ stimuli. To efficiently load neurotransmitters, sense the rise in intracellular Ca²⁺ and fuse with the presynaptic membrane, SVs need to be equipped with a stringently controlled set of transmembrane proteins. In fact, changes in SV protein composition quickly compromise neurotransmission and most prominently give rise to epileptic seizures. During exocytosis SVs fully collapse into the presynaptic membrane and consequently have to be replenished to sustain neurotransmission. Therefore, surface-stranded SV proteins have to be efficiently retrieved post-fusion to be used for the generation of a new set of fully functional SVs, a process in which dedicated endocytic sorting adaptors play a crucial role. The question of how the precise reformation of SVs is achieved is intimately linked to how SV membranes are retrieved. For a long time both processes were believed to be two sides of the same coin since Clathrin-mediated endocytosis (CME), the proposed predominant SV recycling mode, will jointly retrieve SV membranes and proteins. However, with the recent proposal of Clathrin-independent SV recycling pathways SV membrane retrieval and SV reformation turn into separable events. This review highlights the progress made in unraveling the molecular mechanisms mediating the high-fidelity retrieval of SV proteins and discusses how the gathered knowledge about SV protein recycling fits in with the new notions of SV membrane endocytosis.

Keywords: AP180; presynapse; recycling; release site clearance; sorting; stonin2.

Figure 1

Requirements for synaptic vesicle (SV) protein sorting in different endocytic modes. (A) In kiss-and-run endocytosis the SV only transiently fuses. Closure of the fusion pore restores the SV with unaltered protein composition eliminating the need for sorting. (B,C) After full collapse fusion SV proteins strand in the presynaptic membrane. There they can either stay associated and diffuse as cluster out of the active zone reducing the need for individual sorting adaptors (B) or they can first disperse and later recluster with the aid of self-aggregation mechanisms and specific sorting adaptors (C).

Figure 2

Model of SV protein reclustering and sorting at the presynapse. (A) After full collapse fusion freely diffusing SV proteins are confined and recaptured by endocytic sorting adaptors at the periactive zone to allow for release site clearance. At the plasma membrane SV proteins might either be clustered by AP-2 and additional cargo-specific adaptor proteins (I), they might interact with each other and thereby self-assemble into clusters (II) or form mixed clusters of self-assembled SV proteins together with sorting adaptors (III). These clusters can directly be endocytosed from the plasma membrane by Clathrin-mediated endocytosis (CME) to reform SVs. However, cargo-specific sorting proteins together with AP-2 and Clathrin can also operate on endosomal-like vacuoles (ELVs) after Clathrin-independent endocytosis (CIE) to recycle SVs with correct protein composition. (B–D) Sorting of individual SV proteins. (B) The precise sorting of Synaptotagmin1 can be accomplished by three possible mechanisms. (a) Synaptotagmin1 associates with AP-2μ2 via its C2B domain. However, this interaction does not suffice for efficient Synaptagmin1 retrieval. The specific adaptor protein Stonin2 is needed to strengthen the link between AP-2 and Synaptotagmin1 by interacting simultaneously with Synaptotagmin1’s C2A domain and the AP-2α ear. (b) Synaptotagmin1 can also be sorted by association with SV2, another SV protein. The N-terminus of SV2 binds to the C2B domain of Synaptotagmin1 and thereby facilitates correct Synaptotagmin1 sorting. (c) As Stonin2 and SV2 interact with distinct C2 domains of Synaptotagmin1, both proteins might bind at the same time to the Ca²⁺ sensor and link it to AP-2 to enable its precise retrieval. (C) Free Synaptobrevin2 can also be sorted by multiple mechanisms. (a) Synaptobrevin2 is recognized by the specific sorting adaptors AP180 and CALM via their ANTH domain that interacts with the SNARE domain of Synaptobrevin2 and with the plasma membrane. Via the unstructured C-terminus AP180 and CALM associate with AP-2 and Clathrin thereby linking Synaptobrevin2 to the CME machinery. (b) Synaptobrevin2 can also be sorted by association with the SV protein Synaptophysin1 via their transmembrane domains. (D) In inner hair cells Otoferlin is directly sorted by AP-2 via the interaction of di-leucine motifs within the linker regions between the C2A-C2B-C2C domains of Otoferlin with AP-2α/σ2. In addition, Otoferlin associates with AP-2μ2. However, the exact interaction sites are not clear yet.