AP180 and CALM: Dedicated endocytic adaptors for the retrieval of synaptobrevin 2 at synapses

Abstract

Communication between neurons largely occurs at chemical synapses by conversion of electric to chemical signals. Chemical neurotransmission involves the action potential-driven release of neurotransmitters from synaptic vesicles (SVs) at presynaptic nerve terminals. Fusion of SVs is driven by SNARE complex formation comprising synaptobrevin 2 on the SV membrane and syntaxin 1A and SNAP-25 on the plasma membrane. In order to maintain neurotransmission during repetitive stimulation and to prevent expansion of the presynaptic plasma membrane, exocytic SV fusion needs to be balanced by compensatory retrieval of SV components to regenerate functional vesicles. Our recent work has unraveled a mechanism by which the R-SNARE synaptobrevin 2, the most abundant SV protein and an essential player for exocytic fusion, is recycled from the presynaptic membrane. The SNARE motif of synaptobrevin 2 is directly recognized by the ANTH domains of AP180 and CALM, monomeric endocytic adaptors for clathrin-mediated endocytosis. Given that key residues involved in synaptobrevin 2-ANTH domain complex formation are also essential for SNARE assembly, we propose that disassembly of SNARE complexes is a prerequisite for synaptobrevin 2 retrieval, thereby preventing endocytic mis-sorting of the plasma membrane Q-SNAREs syntaxin 1A and SNAP-25. It is tempting to speculate that perturbed synaptobrevin 2 recycling caused by reduction of CALM or AP180 levels may lead to disease as suggested by the genetic association of ANTH domain proteins with neurodegenerative disorders.

Figure 1

Representative electron micrographs of control and AP180-depleted synapses. Arrowheads in the enlarged insets illustrate tubular structures occasionally found in AP180-depleted nerve terminals. Such structures were not normally seen in synapses from control neurons. Morphology and SV density were unchanged but SVs appeared slightly larger and more heterogeneous in synapses depleted with AP180. Hippocampal neurons were cotransfected with AP180 sIRNA and an eGFP-encoding plasmid. Six to eight days after transfection neurons were fixed with 4% paraformaldehyde before permeabilization by freeze-cracking in liquid nitrogen. Samples were labeled with anti-GFP antibody and NANOGOLD^® particles, post-fixed with 2% glutaraldehyde, gold-enhanced and processed for electron microscopy. Images were taken using Zeiss 910 electron microscope. Neurites and terminals of control or KD neurons were identified by absence/presence of intense immunogold labeling scattered in the cytoplasm. Scale bar, 500 nm; 200 nm for the inset; KD, knockdown.

Figure 2

Hypothetical model detailing mechanisms involved in cargo-specific endocytic sorting of SV proteins. Upon the arrival of an action potential at the nerve terminal SVs fuse with the presynaptic membrane via a SNARE-dependent mechanism. As a result cis-SNARE complexes comprising synaptobrevin 2, syntaxin 1A and SNAP-25 remain on the plasma membrane. For new rounds of exocytosis, cis-SNARE complex are disassembled via NSF with its cofactor α-SNAP, releasing free synaptobrevin 2 molecules. Endocytic sorting of SV proteins on the plasma membrane requires dedicated endocytic adaptors: vGLUT1 recognition by AP2 and endophilin, synaptobrevin 2 binding to AP180 and CALM, and synaptotagmin 1 sorting by AP2 and stonin 2.– AP180 and CALM directly recognize the SNARE motif of synaptobrevin 2, perhaps to ensure that only free rather than SNARE-complexed synaptobrevin 2 is endocytically resorted to while syntaxin 1A and SNAP-25 are left behind. Recycling of synaptobrevin 2 has been shown to require synaptophysin although the molecular details of this mechanism remain unknown. SV cargo becomes concentrated in clathrin-coated pits containing clathrin, endocytic adaptors and accessory proteins resulting in SV reformation. SV, synaptic vesicle; SytI, Synaptotagmin 1; vGLUT 1, vesicular glutamate transporter 1; Syp, synaptophysin; Syb2, synaptobrevin 2; NSF, N-ethylmaleimide-sensitive factor; α-SNAP, soluble NSF attachment proteins; mHD, µ homology domain; SHD, stonin homology domain.