Assembly of Presynaptic Active Zones

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.

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.

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

Bassoon is a core scaffold protein of the presynaptic active zone. In brain synapses, the C-terminus of Bassoon is oriented toward the plasma membrane and its N-terminus is oriented toward synaptic vesicles. At the Golgi-apparatus, Bassoon is thought to assemble active zone precursor structures, but whether it is arranged in an orderly fashion is unknown. Understanding the topology of this large scaffold protein is important for models of active zone biogenesis. Using stimulated emission depletion nanoscopy in cultured hippocampal neurons, we found that an N-terminal intramolecular tag of recombinant Bassoon, but not C-terminal tag, colocalized with markers of the trans-Golgi network (TGN). The N-terminus of Bassoon was located between 48 and 69 nm away from TGN38, while its C-terminus was located between 100 and 115 nm away from TGN38. Sequences within the first 95 amino acids of Bassoon were required for this arrangement. Our results indicate that, at the Golgi-apparatus, Bassoon is oriented with its N-terminus toward and its C-terminus away from the trans Golgi network membrane. Moreover, they suggest that Bassoon is an extended molecule at the trans Golgi network with the distance between amino acids 97 and 3,938, estimated to be between 46 and 52 nm. Our data are consistent with a model, in which the N-terminus of Bassoon binds to the membranes of the trans-Golgi network, while the C-terminus associates with active zone components, thus reflecting the topographic arrangement characteristic of synapses also at the Golgi-apparatus.

The docking of synaptic vesicles on the presynaptic membrane and their priming for fusion with it to mediate synaptic transmission of nerve impulses typically occur at structurally specialized regions on the membrane called active zones. Stable components of active zones include aggregates of macromolecules, 'active zone material' (AZM), attached to the presynaptic membrane, and aggregates of Ca(2+)-channels in the membrane, through which Ca(2+) enters the cytosol to trigger impulse-evoked vesicle fusion with the presynaptic membrane by interacting with Ca(2+)-sensors on the vesicles. This laboratory has used electron tomography to study, at macromolecular spatial resolution, the structure and function of AZM at the simply arranged active zones of axon terminals at frog neuromuscular junctions. The results support the conclusion that AZM directs the docking and priming of synaptic vesicles and essential positioning of Ca(2+)-channels relative to the vesicles' Ca(2+)-sensors. Here we review the findings and comment on their applicability to understanding mechanisms of docking, priming and Ca(2+)-triggering at other synapses, where the arrangement of active zone components differs.

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

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.

Most interneuronal communication occurs with high spatiotemporal precision at structurally and molecularly specialized synaptic sites, where neurotransmitter-filled synaptic vesicles (SVs) fuse at dedicated presynaptic active zones (AZs) in response to an action potential (AP). The strength of synaptic communication is dynamically and purposefully regulated in an activity-dependent manner, known as synaptic plasticity, which manifests itself over a wide range of timescales. I investigated the mechanism underlying a form of synaptic plasticity that operates on a millisecond timescale, called short-term facilitation (STF), in which synaptic strength increases with repeated stimulation. In the mammalian brain, hippocampal mossy fiber bouton (hMFB) to CA3 pyramidal cell synapses exhibit a strong presynaptic STF. STF is crucial for the function of the hippocampal network by allowing hMFB-CA3 synapses to operate as conditional detonators (Henze et al., 2002b). Deciphering how STF works is key to understanding information processing in neuronal circuits associated with learning and memory processes. In recent years, considerable progress has been made with respect to the functional characterization of STF at hMFB-CA3 synapses, and several mechanisms have been proposed to contribute to STF, one of them involving the Ca2+ sensor synaptotagmin-7 (Syt-7) (Jackman et al., 2016). However, an ultrastructural characterization assessing the contribution of STF and Syt-7 to SV pool organization at individual hMFB AZs is still missing. This information is critical since SV organization is of fundamental relevance to the functional state of synapses, given that morphologically docked SVs serve as a proxy for molecularly primed SVs (Imig et al., 2014), and that synapse-specific levels of SV organization co-determine certain functional properties of neurotransmitter release at hippocampal synapses (Maus et al., 2020). Conventional electron microscopy (EM) techniques lack the temporal resolution and level of ultrastructural preservation required to monitor ultrastructural changes associated with STF. The development of light stimulation-coupled high-pressure freezing (HPF), also known as flash-and-freeze EM (Watanabe et al., 2013b) is a powerful method for investigating ultrastructure-function relationships associated with short-lived functional states. The goal of my PhD project was to employ optogenetics and flash-and-freeze EM to analyse the dynamic organization and activity-dependent remodelling of AZ-proximal SV pools during STF at hMFB-CA3 synapses, and to dissect the underlying molecular mechanisms. In order to do this, I used optogenetics as a tool to study synaptic structure in the context of different functional states of hMFB-CA3 synapses. To this end, I exploited mouse genetics to (i) selectively express channelrhodopsin 2 (ChR2) in granule cells (GCs) of the hippocampal dentate gyrus, and to (ii) delete Syt-7 protein expression to genetically isolate aspects of activity-dependent SV pool remodelling most relevant to the STF functional state. My data demonstrated that genetic deletion of Syt-7 at hMFB-CA3 synapses leads to an increased size of light stimulation-evoked EPSCs, and to a pronounced reduction in STF compared to Syt-7 WT synapses. Ultrastructural analysis of resting synapses revealed that Syt-7 KO AZs exhibited a SV docking deficit compared to Syt-7 WT AZs from hMFB-CA3 synapses, and that this SV docking deficit was exaggerated following stimulation, indicating a role for Syt-7 in regulating activity-dependent SV docking. This study advances our understanding of Syt-7-mediated STF, which is essential for information processing in hippocampal circuits, and lays the groundwork for a deeper understanding of mediated functions such as working memory and learning.

Determining the localization of proteins within a cell and their possible interactions is highly relevant to understand their functionality. Nevertheless, subcellular structures of interest in neurobiology, most importantly synapses with their pre- and postsynaptic compartments, are usually smaller than the resolution limit of conventional light microscopy. Indeed, diffraction of light limits to roughly half of the wavelength of light the resolution of a conventional light microscope. In this regard, super-resolution light microscopy (SRLM) techniques have emerged, achieving even more than ten times the resolution of conventional light microscopy, thus allowing to resolve subsynaptic structures also in situ. Importantly, stimulated emission depletion (STED) microscopy has been extensively used to image in situ the nanoscale organization of presynaptic compartments, such as the area of the presynaptic plasma membrane where synaptic vesicles fuse to release neurotransmitters, the so-called active zone. In this article, we outline a method to determine the localization of active zone scaffolding key players relative to voltage-gated calcium channels within the presynaptic active zone by time-gated STED (gSTED) microscopy in situ.

Synaptic vesicle (SV) release is spatially and temporally regulated by a network of proteins that form the presynaptic active zone (AZ). The hallmark of most AZs is an electron-dense projection (DP) surrounded by SVs. Despite their importance for our understanding of triggered SV release, high-resolution analyses of DP structures are limited. Using electron microscopy, we show that DPs at Caenorhabditis elegans neuromuscular junctions (NMJs) were highly structured, composed of building units forming bays in which SVs are docked to the AZ membrane. Furthermore, larger ribbonlike DPs that were multimers of the NMJ building unit are found at synapses between inter- and motoneurons. We also demonstrate that DP size is determined by the activity of the AZ protein SYD-2/Liprin-α. Whereas loss of syd-2 function led to smaller DPs, syd-2 gain-of-function mutants displayed larger ribbonlike DPs through increased recruitment of ELKS-1/ELKS. Therefore, our data suggest that a main role of SYD-2/Liprin-α in synaptogenesis is to regulate the polymerization of DPs.

Presynaptic Biogenesis Requires Axonal Transport of Lysosome-Related Vesicles

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.

Memories are assumed to be formed by sets of synapses changing their structural or functional performance. The efficacy of forming new memories declines with advancing age, but the synaptic changes underlying age-induced memory impairment remain poorly understood. Recently, we found spermidine feeding to specifically suppress age-dependent impairments in forming olfactory memories, providing a mean to search for synaptic changes involved in age-dependent memory impairment. Here, we show that a specific synaptic compartment, the presynaptic active zone (AZ), increases the size of its ultrastructural elaboration and releases significantly more synaptic vesicles with advancing age. These age-induced AZ changes, however, were fully suppressed by spermidine feeding. A genetically enforced enlargement of AZ scaffolds (four gene-copies of BRP) impaired memory formation in young animals. Thus, in the Drosophila nervous system, aging AZs seem to steer towards the upper limit of their operational range, limiting synaptic plasticity and contributing to impairment of memory formation. Spermidine feeding suppresses age-dependent memory impairment by counteracting these age-dependent changes directly at the synapse.

Synaptic vesicle exocytosis relies on the tethering of release ready vesicles close to voltage-gated Ca2+ channels and specific lipids at the future site of fusion. This enables rapid and efficient neurotransmitter secretion during presynaptic depolarization by an action potential. Extensive research has revealed that this tethering is mediated by an active zone, a protein dense structure that is attached to the presynaptic plasma membrane and opposed to postsynaptic receptors. Although roles of individual active zone proteins in exocytosis are in part understood, the molecular mechanisms that hold the protein scaffold at the active zone together and link it to the presynaptic plasma membrane have remained unknown. This is largely due to redundancy within and across scaffolding protein families at the active zone. Recent studies, however, have uncovered that ELKS proteins, also called ERC, Rab6IP2 or CAST, act as active zone scaffolds redundant with RIMs. This redundancy has led to diverse synaptic phenotypes in studies of ELKS knockout mice, perhaps because different synapses rely to a variable extent on scaffolding redundancy. In this review, we first evaluate the need for presynaptic scaffolding, and we then discuss how the diverse synaptic and non-synaptic functional roles of ELKS support the hypothesis that ELKS provides molecular scaffolding for organizing vesicle traffic at the presynaptic active zone and in other cellular compartments.

Neurotransmission at chemically transmitting synapses requires calcium-mediated fusion of synaptic vesicles with the presynaptic membrane. Utilizing ultrastructural information available for the crustacean excitatory neuromuscular junction, we developed a model that employs the Monte Carlo simulation technique to follow the entry and movement of Ca2+ ions at a presynaptic active zone, where synaptic vesicles are preferentially docked for release. The model includes interaction of Ca2+ with an intracellular buffer, and variable separation between calcium channels and vesicle-associated Ca(2+)-binding targets that react with Ca2+ to trigger vesicle fusion. The end point for vesicle recruitment for release was binding of four Ca2+ ions to the target controlling release. The results of the modeling experiments showed that intracellular structures that interfere with Ca2+ diffusion (in particular synaptic vesicles) influence recruitment or priming of vesicles for release. Vesicular recruitment is strongly influenced by the separation distance between an opened calcium channel and the target controlling release, and by the concentration and binding properties of the intracellular buffers, as in previous models. When a single opened calcium channel is very close to the target, a single synaptic vesicle can be recruited. However, many of the single-channel openings actuated by a nerve impulse are likely to be ineffective for release, although they contribute to the buildup of total intracellular Ca2+. Thus, the overall effectiveness of single calcium channels in causing vesicles to undergo exocytosis is likely quite low.