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

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.

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.

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.

Synaptogenesis has been studied in the electric organ of embryonic Torpedo marmorata by use of two antisera directed against components of synaptic vesicles (anti-SV) and presynaptic plasma membranes (ap-anti-TSM), respectively. The anti-SV serum was previously shown to recognize a proteoglycan specific for synaptic vesicles. The ap-anti-TSM serum was raised to plasma membranes of synaptosomes derived from the electromotor nerve terminals and affinity-purified on electric-organ gangliosides. The vesicular antigen was first detectable at the 81-mm stage of development, which is 1-2 weeks earlier than the formation of morphologically mature presynaptic terminals, but is coincident with a rise in choline acetyltransferase levels and the ability of the electric organ to generate discharges. The gangliosidic antigen recognized by the ap-anti-TSM was first detectable on the ventral electrocyte surface at the 93-mm stage of development. This indicates that specific carbohydrate epitopes, not present on the growth cones, are expressed during maturation of the nerve terminal. The nerve terminal components recognized by these sera arose pari passu with neurite coverage of the ventral surface of the electrocyte, reaching a maximum in the adult. In contrast, postsynaptic aggregates of acetylcholine receptor, rendered visible with rhodamine-labeled alpha-bungarotoxin, arose previous to the presynaptic antigens, reaching a maximum surface density at 110 mm and then declining in the adult.

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.

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.

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.

In vitro binding of isolated synaptic vesicles to presynaptic plasma membranes: Activation by Ca 2+ and protein kinase C

More than 300 new inborn errors or metabolism (IEM) have been described during the last 5 years and most of them have major neurological symptoms (Ferreira et al. 2018; Saudubray and Garcia-Cazorla 2018). These disorders have changed paradigms transforming the concept and classification of IEM and are contributing enormously to our understanding of mechanisms in neurological diseases. A pioneer initiative in this direction was the description of the vast new group of complex lipid synthesis and remodelling defects that stands at the frontier between classical IEM and cellular neurobiology (Lamari et al. 2015). More recently, many defects affecting systems involved in intracellular vesiculation, trafficking, processing and quality control of complex molecules, such as protein folding and autophagy (Ebrahimi-Fakhari et al. 2016), have described a number of diseases with important repercussion in the brain. The -omics era is currently transforming neurology, and in particular child neurology, with the description of an increasing amount of new monogenic diseases. However, the pathophysiological approach to these disorders is mostly based on neuroanatomy, neurophysiology and the description of individual proteins. The role of metabolism, which strongly orchestrates neuronal function, has been mostly neglected in the study of brain disorders. On the other hand, the description of biological mechanisms of disease in the field of IEM has not fully considered the particularities of the nervous system. The human brain is a unique system that exhibits a complex architecture and a great diversity of functions and cellular types. Neurons are large and highly polarised cells consisting of axons and dendritic arborisations that communicate with each other through synaptic contacts, which are essential to their function. To integrate information within and between compartments, neurons need sophisticated transfer mechanisms, ensuring that necessary molecules are available in the right place at the right time. Metabolism is regulating these functions and appears to have specific properties at every single neuronal compartment. Metabolism has also specific rules depending on the specific subtype of nervous cell (different populations of neurons, glia, oligodendrocytes, microglia…). An important model of compartmentalised signalling and metabolism occurs at the synapse, which is the main topic of this special issue. The synapse is a highly specialised cell junction that connects a presynaptic transmitting neuron with a postsynaptic receiving neuron. Synapses connect more than one hundred billion neurons present in the human brain, and wire-select neurons into functional circuits, enabling the brain to process and transfer information (Südhof 2013). The concept of “synaptic metabolism” is introduced in this special issue and is the conducting wire of most of its articles; it could be defined as the specific chemical composition and metabolic functions occurring at the synapse. The presynaptic and postsynaptic terminals have specific patterns of proteins and lipids; and even within each of these compartments, there are specialised areas. As an example, the active zone, a specialised region for neurotransmitter release at the presynaptic plasma membrane has a unique protein and lipid composition compared to other areas of the neuronal membrane (Cortès-Saladelafont et al. 2018; Mochel 2018). The same is true for the postsynaptic density, characteristic of the postsynaptic element of excitatory synapses (Bayés and Grant 2009). Mitochondria, for instance, are concentrated at the presynaptic terminal, where they have specific bioenergetic properties, yet they are absent from the postsynaptic compartment. Additionally, the synaptic vesicle (SV) cycle, crucial to neurotransmission, is a complex mechanism involving many different elements: the structure and composition of the SV itself, exocytic and endocytic processes and interactions between proteins and lipids among other biological functions. Since the SV is an independent organelle, such as mitochondria and lysosomes, diseases of the synaptic vesicle may encompass a category of disorders itself (Cortès-Saladelafont et al. 2018). These diseases present with the constant features, including neurodevelopmental delay or intellectual disability, and a spectrum of signs comprising epilepsy, behavioural problems and movement disorders. Interestingly, and as an innovative aspect in the field of IEM, our knowledge about synaptic metabolic diseases remains almost exclusive to the presynaptic and astrocytic level. In fact, little is known about the metabolic pathways operating at the postsynaptic terminal, although its complex signalling machinery has been strongly related with intellectual disability. This postsynaptic approach is described in this issue by Dr. Àlex Bayès (Bayés 2018; Bayés et al. 2011) as a global overview and by Drs. Abela and Kurian (Abela and Kurian 2018) in the case of movement disorders. In practical terms, deeping into the concept of synaptic metabolism will contribute to a better understanding of the biological basis of neurometabolic diseases as well as providing potential new categories of IEM (such as the diseases of the SV). Additionally, it represents a perfect example of the necessity of combining classical metabolism and cell biology in neurometabolic and neurogenetic diseases in general (García-Cazorla and Saudubray 2018). This integrative approach will probably redefine the current perspective about the pathophysiological categories of IEM and, most importantly, will provide new therapeutic options. The study of the cerebrospinal fluid by means of new -omic techniques (metabolomics, lipidomics and proteomics) seems a promising strategy to search for biomarkers leading to a better characterisation of these diseases. Finally, in order to face this extremely changing and complex field, clinical neurologist and metabolic physicians will need renewed and advance educational programmes able to efficiently combine the traditional clinical approach with a pathophysiological-based perspective. A.G.C. is funded by FIS: PI15/01082 (Instituto de Salud Carlos III: ISCIII and “Fondo Europeo de desarrollo regional” FEDER). A.G.C. has contributed the original idea and has written the editorial letter. R.A and A. B. have reviewed and contributed with several messages and new information to the original text. They have actively contributed their scientific knowledge throughout the elaboration of this special issue. Àngels García-Cazorla, Rafael Artuch and Àlex Bayès declare that they have no conflict of interest. Not applicable. Not applicable.

An action potential triggers neurotransmitter release from synaptic vesicles docking to a specialized release site of the presynaptic plasma membrane, the active zone. The active zone is a highly organized structure with proteins that serves as a platform for synaptic vesicle exocytosis, mediated by SNAREs complex and Ca2+ sensor proteins, within a sub-millisecond opening of nearby Ca2+ channels with the membrane depolarization. In response to incoming neuronal signals, each active zone protein plays a role in the release-ready site replenishment with synaptic vesicles for sustainable synaptic transmission. The active zone release apparatus provides a possible link between neuronal activity and plasticity. This review summarizes the mostly physiological role of active zone protein interactions that control synaptic strength, presynaptic short-term plasticity, and homeostatic synaptic plasticity.

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.

Calcium-Dependent Synaptic Vesicle Trafficking Underlies Indefatigable Release at the Hair Cell Afferent Fiber Synapse