The structure and function of 'active zone material' at synapses.

The regulated release of neurotransmitter occurs via the fusion of synaptic vesicles (SVs) at specialized regions of the presynaptic membrane called active zones (AZs). These regions are defined by a cytoskeletal matrix assembled at AZs (CAZ), which functions to direct SVs toward docking and fusion sites and supports their maturation into the readily releasable pool. In addition, CAZ proteins localize voltage-gated Ca(2+) channels at SV release sites, bringing the fusion machinery in close proximity to the calcium source. Proteins of the CAZ therefore ensure that vesicle fusion is temporally and spatially organized, allowing for the precise and reliable release of neurotransmitter. Importantly, AZs are highly dynamic structures, supporting presynaptic remodeling, changes in neurotransmitter release efficacy, and thus presynaptic forms of plasticity. In this review, we discuss recent advances in the study of active zones, highlighting how the CAZ molecularly defines sites of neurotransmitter release, endocytic zones, and the integrity of synapses.

This report integrates knowledge of in situ macromolecular structures and synaptic protein biochemistry to propose a unified hypothesis for the regulation of certain vesicle trafficking events (i.e., docking, priming, Ca2+-triggering, and membrane fusion) that lead to neurotransmitter secretion from specialized “active zones” of presynaptic axon terminals. Advancements in electron tomography, to image tissue sections in 3D at nanometer scale resolution, have led to structural characterizations of a network of different classes of macromolecules at the active zone, called “Active Zone Material’. At frog neuromuscular junctions, the classes of Active Zone Material macromolecules “top-masts”, “booms”, “spars”, “ribs” and “pins” direct synaptic vesicle docking while “pins”, “ribs” and “pegs” regulate priming to influence Ca2+-triggering and membrane fusion. Other classes, “beams”, “steps”, “masts”, and “synaptic vesicle luminal filaments’ likely help organize and maintain the structural integrity of active zones. Extensive studies on the biochemistry that regulates secretion have led to comprehensive characterizations of the many conserved proteins universally involved in these trafficking events. Here, a hypothesis including a partial proteomic atlas of Active Zone Material is presented which considers the common roles, binding partners, physical features/structure, and relative positioning in the axon terminal of both the proteins and classes of macromolecules involved in the vesicle trafficking events. The hypothesis designates voltage-gated Ca2+ channels and Ca2+-gated K+ channels to ribs and pegs that are connected to macromolecules that span the presynaptic membrane at the active zone. SNARE proteins (Syntaxin, SNAP25, and Synaptobrevin), SNARE-interacting proteins Synaptotagmin, Munc13, Munc18, Complexin, and NSF are designated to ribs and/or pins. Rab3A and Rabphillin-3A are designated to top-masts and/or booms and/or spars. RIM, Bassoon, and Piccolo are designated to beams, steps, masts, ribs, spars, booms, and top-masts. Spectrin is designated to beams. Lastly, the luminal portions of SV2 are thought to form the bulk of the observed synaptic vesicle luminal filaments. The goal here is to help direct future studies that aim to bridge Active Zone Material structure, biochemistry, and function to ultimately determine how it regulates the trafficking events in vivo that lead to neurotransmitter secretion.

Regulated exocytosis requires the general membrane fusion machinery-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins. Using reconstituted giant unilamellar vesicles containing preassembled t-SNARE proteins (syntaxin 1·SNAP-25), we determined how Munc18-1 controls the docking, priming, and fusion of small unilamellar vesicles containing the v-SNARE VAMP2 and the Ca(2+) sensor synaptotagmin 1. In vitro assays allowed us to position Munc18-1 in the center of a sequential reaction cascade; vesicle docking by synaptotagmin 1 is a prerequisite for Munc18-1 to accelerate trans-SNARE complex (SNAREpin) assembly and membrane fusion. Complexin II stalls SNAREpin zippering at a late stage and, hence, contributes to synchronize membrane fusion in a Ca(2+)- and synaptotagmin 1-dependent manner. Thus, at the neuronal synapse, the priming factor Munc18-1 may accelerate the conversion of docked synaptic vesicles into a readily releasable pool by activating SNAREs for efficient membrane fusion.

Fusion Competent Synaptic Vesicles Persist upon Active Zone Disruption and Loss of Vesicle Docking

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The active zone defines the area of the presynaptic plasma membrane where synaptic vesicles (SV) dock, mature, and fuse with the cell membrane in a regulated manner. The active zone is defined by an electron-dense cytomatrix of specialized proteins, the cytomatrix assembled at the active zone (CAZ). Some of these proteins, e.g., RIMs, Munc13s, ERC/CASTs as well as Bassoon and Piccolo are exclusive components of the CAZ, and are thought to mediate specific functions, including the localization and regulation of the SV cycle. Recently, major progress has been achieved in studying the assembly of the active zone and the underlying cytoskeletal matrix, suggesting it is not assembled ‘brick-by-brick’ as originally assumed. Rather, mounting evidence suggests that active zone components are preassembled within the neuronal soma, presumably at a trans-Golgi compartment, and then transported on distinct precursor vesicles to primordial presynaptic sites where they fuse with the cell membrane. As Piccolo and Bassoon are unique marker proteins of these vesicles they are called Piccolo-Bassoon transport vesicles (PTVs). The present review summarizes evidence for this active zone precursor vesicle hypothesis, which predicts that presynaptic active zones are assembled in a more or less quantal manner.

Neurotransmitter release requires the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes by SNARE proteins syntaxin-1 (Stx1), synaptosomal-associated protein 25 (SNAP-25), and synaptobrevin-2 (Syb2). In mammalian systems, loss of SNAP-25 or Syb2 severely impairs neurotransmitter release; however, complete loss of function studies for Stx1 have been elusive due to the functional redundancy between Stx1 isoforms Stx1A and Stx1B and the embryonic lethality of Stx1A/1B double knock-out (DKO) mice. Here, we studied the roles of Stx1 in neuronal maintenance and neurotransmitter release in mice with constitutive or conditional deletion of Stx1B on an Stx1A-null background. Both constitutive and postnatal loss of Stx1 severely compromised neuronal viability in vivo and in vitro, indicating an obligatory role of Stx1 for maintenance of developing and mature neurons. Loss of Munc18-1, a high-affinity binding partner of Stx1, also showed severely impaired neuronal viability, but with a slower time course compared with Stx1A/1B DKO neurons, and exogenous Stx1A or Stx1B expression significantly delayed Munc18-1-dependent lethality. In addition, loss of Stx1 completely abolished fusion-competent vesicles and severely impaired vesicle docking, demonstrating its essential roles in neurotransmission. Putative partial SNARE complex assembly with the SNARE motif mutant Stx1A(AV) (A240V, V244A) was not sufficient to rescue neurotransmission despite full recovery of vesicle docking and neuronal survival. Together, these data suggest that Stx1 has independent functions in neuronal maintenance and neurotransmitter release and complete SNARE complex formation is required for vesicle fusion and priming, whereas partial SNARE complex formation is sufficient for vesicle docking and neuronal maintenance. Syntaxin-1 (Stx1) is a component of the synaptic vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex and is essential for neurotransmission. We present the first detailed loss-of-function characterization of the two Stx1 isoforms in central mammalian neurons. We show that Stx1 is fundamental for maintenance of developing and mature neurons and also for vesicle docking and neurotransmission. We also demonstrate that neuronal maintenance and neurotransmitter release are regulated by Stx1 through independent functions. Furthermore, we show that SNARE complex formation is required for vesicle fusion, whereas partial SNARE complex formation is sufficient for vesicle docking and neuronal maintenance. Therefore, our work provides insights into differential functions of Stx1 in neuronal maintenance and neurotransmission, with the latter explored further into its functions in vesicle docking and fusion.

Assembling the Presynaptic Active Zone: A Characterization of an Active Zone Precursor Vesicle

There are many special sites at the end of a synapse called active zones (AZs). Synaptic vesicles (SVs) fuse with presynaptic membranes at these sites, and this fusion is an important step in neurotransmitter release. The cytomatrix in the active zone (CAZ) is made up of proteins such as the regulating synaptic membrane exocytosis protein (RIM), RIM-binding proteins (RIM-BPs), ELKS/CAST, Bassoon/Piccolo, Liprin-α, and Munc13-1. RIM is a scaffold protein that interacts with CAZ proteins and presynaptic functional components to affect the docking, priming, and fusion of SVs. RIM is believed to play an important role in regulating the release of neurotransmitters (NTs). In addition, abnormal expression of RIM has been detected in many diseases, such as retinal diseases, Asperger's syndrome (AS), and degenerative scoliosis. Therefore, we believe that studying the molecular structure of RIM and its role in neurotransmitter release will help to clarify the molecular mechanism of neurotransmitter release and identify targets for the diagnosis and treatment of the aforementioned diseases.

Identification of a syntaxin-binding site on N-Type calcium channels

The animation shows calcium-stimulated exocytosis of synaptic vesicles followed by clathrin-mediated vesicle recycling. Many of the molecular components that are involved in synaptic vesicle priming, docking, fusion, and endocytosis are shown. Although the process in the animation describes synaptic vesicle cycling, similar cellular processes occur for most calcium-coupled secretory and clathrin-mediated endocytotic events. The animation could be used to help illustrate the sequence of events associated with both exocytosis and endocytosis, as well as aid in understanding the processes involved in neurotransmitter release in response to nerve stimulation. [ Resource Details ]

Expression Analysis and Protein Localization of the Human HPC-1/Syntaxin 1A, a Gene Deleted in Williams Syndrome

SummaryAction potentials trigger neurotransmitter release with minimal delay. Active zones mediate this temporal precision by co-organizing primed vesicles with CaV2 Ca2+ channels. The presumed model is that scaffolding proteins directly tether primed vesicles to CaV2s. We find that CaV2 clustering and vesicle priming are executed by separate machineries. At hippocampal synapses, CaV2 nanoclusters are positioned at variable distances from those of the priming protein Munc13. The active zone organizer RIM anchors both proteins, but distinct interaction motifs independently execute these functions. In heterologous cells, Liprin-α and RIM from co-assemblies that are separate from CaV2-organizing complexes upon co-transfection. At synapses, Liprin-α1–4 knockout impairs vesicle priming, but not CaV2 clustering. The cell adhesion protein PTPσ recruits Liprin-α, RIM and Munc13 into priming complexes without co-clustering of CaV2s. We conclude that active zones consist of distinct complexes to organize CaV2s and vesicle priming, and Liprin-α and PTPσ specifically support priming site assembly.

Brain-derived neurotrophic factor (BDNF) promotes long-term potentiation (LTP) at hippocampal CA1 synapses by a presynaptic enhancement of synaptic transmission during high-frequency stimulation (HFS). Here we have investigated the mechanisms of BDNF action using two lines of BDNF knockout mice. Among other presynaptic impairments, the mutant mice exhibited more pronounced synaptic fatigue at CA1 synapses during high-frequency stimulation, compared with wild-type animals. Quantitative analysis of CA1 synapses revealed a significant reduction in the number of vesicles docked at presynaptic active zones in the mutant mice. Synaptosomes prepared from the mutant hippocampus exhibited a marked decrease in the levels of synaptophysin as well as synaptobrevin [vesicle-associated membrane protein (VAMP-2)], a protein known to be involved in vesicle docking and fusion. Treatment of the mutant slices with BDNF reversed the electrophysiological and biochemical deficits in the hippocampal synapses. Taken together, these results suggest a novel role for BDNF in the mobilization and/or docking of synaptic vesicles to presynaptic active zones.

Synaptic vesicle docking and exocytosis require the specific interaction of synaptic vesicle proteins (such as VAMP/synaptobrevin) with presynaptic plasma membrane proteins (such as syntaxin and SNAP 25). These proteins form a stable, SDS-resistant, multimolecular complex, the SNARE complex. The subcellular distribution of VAMP and syntaxin within Torpedo electric organ nerve endings was studied by immunogoldlabeling of SDS-digested freeze-fracture replicas (Fujimoto, 1995). This technique allowed us to visualize large surface areas of the presynaptic plasma membrane and numerous synaptic vesicles from rapidly frozen nerve endings and synaptosomes. VAMP was found associated with synaptic vesicles, as also shown by conventional electron microscopy immunolabeling, and to the presynaptic plasma membrane (P leaflet). Syntaxin was also detected in the nerve ending plasma membrane, without gold labeling of synaptic vesicles. Comparison of gold particle densities suggests that the presynaptic plasma membrane contains 3 VAMP molecules per molecule of syntaxin. After biotinylation of intact synaptosomes, the synaptosomal plasma membrane was isolated on Streptavidin coated magnetic beads. Its antigenic content was compared to that of purified synaptic vesicles. VAMP was present in both membranes whereas syntaxin and SNAP 25 were highly enriched in the synaptosomal plasma membrane. This membrane has a low content of classical synaptic vesicle proteins (synaptophysin, SV2 and the vesicular acetylcholine transporter). The VAMP to syntaxin stoichiometry in the isolated synaptosomal membrane was estimated by comparison with purified antigens and close to 2, in accordance with morphological data. SDS-resistant SNARE complexes were detected in the isolated presynaptic membrane but absent in purified synaptic vesicles. Taken together, these results show that the presence of VAMP in the plasma membrane of nerve endings cannot result from exocytosis of synaptic vesicles, a process which could, as far as SNAREs are concerned, very much resemble homotypic fusion.