Organization of Presynaptic Autophagy-Related Processes

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.

Polyhedral Protein Cages Encase Synaptic Vesicles and Participate in Their Attachment to the Active Zone

During neurotransmitter release, SVs (synaptic vesicles) fuse at the active zone and are recovered predominantly via clathrin-mediated endocytosis at the presynaptic compartment surrounding the site of release, referred to as the periactive zone. Exo- and endo-cytosis in synapses are tightly temporarily and spatially coupled to sustain synaptic transmission. The molecular mechanisms linking these two cellular events, which take place in separate compartments of the nerve terminal, remain largely enigmatic. Several lines of evidence indicate that multiple factors may be involved in exocytic-endocytic coupling including SV integral membrane proteins, SV membrane lipids and the membrane-associated actin cytoskeleton. A number of recent studies also indicate that multimodular adaptor proteins shuttling between the active and periactive zones aid the dynamic assembly of macromolecular protein complexes that execute the exo- and endo-cytic limbs of the SV cycle. Here, we discuss recent evidence implicating the multidomain scaffolding and adaptor protein ITSN1 (intersectin 1) as a central regulator of SV cycling.

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.

Synaptic vesicle protein trafficking at the glutamate synapse

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.

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.

Synaptic vesicles (SVs) transmit signals by releasing neurotransmitters from specialized synaptic regions of neurons. In the synaptic region, SVs are tightly clustered around small structures called active zones. The motor KIF1A transports SVs outward through axons until they are captured in the synaptic region. This transport must be guided in the forward direction because it is opposed by the dynein motor, which causes SVs to reverse direction multiple times en route. The core synapse stability (CSS) system contributes to both guided transport and capture of SVs. We identified Sentryn as a CSS protein that contributes to the synaptic localization of SVs in Caenorhabditis elegans. Like the CSS proteins SAD Kinase and SYD-2 (Liprin-α), Sentryn also prevents dynein-dependent accumulation of lysosomes in dendrites in strains lacking JIP3. Genetic analysis showed that Sentryn and SAD Kinase each have at least one nonoverlapping function for the stable accumulation of SVs at synapses that, when combined with their shared functions, enables most of the functions of SYD-2 (Liprin-α) for capturing SVs. Also like other CSS proteins, Sentryn appears enriched at active zones and contributes to active zone structure, suggesting that it is a novel, conserved active zone protein. Sentryn is recruited to active zones by a process dependent on the active zone-enriched CSS protein SYD-2 (Liprin-α). Our results define a specialized group of active zone enriched proteins that can affect motorized transport throughout the neuron and that have roles in both guided transport and capture of SVs.

Fine structure of the sensory epithelium of the guinea pig organ of corti: afferent and efferent synapses of hair cells

Filamentous (F) Actin is a known regulator of the synaptic vesicle (SV) cycle, with roles in SV mobilization, fusion, and endocytosis. However, the molecular pathways that regulate its dynamic assembly within presynaptic boutons remain unclear. In this study, we have used shRNA-mediated knockdown to demonstrate that Piccolo, a multi-domain protein of the active zone cytomatrix, is a key regulator of presynaptic F-Actin assembly. Boutons lacking Piccolo exhibit enhanced activity-dependent Synapsin1a dispersion and SV exocytosis, and reduced F-Actin polymerization and CaMKII recruitment. These phenotypes are rescued by stabilizing F-Actin filaments and mimicked by knocking down Profilin2, another regulator of presynaptic F-Actin assembly. Importantly, we find that mice with a targeted deletion of exon 14 from the PCLO gene, reported to lack >95% of Piccolo, continue to express multiple Piccolo isoforms. Furthermore, neurons cultured from these mice exhibit no defects in presynaptic F-Actin assembly due to the expression of these isoforms at presynaptic boutons. These data reveal that Piccolo regulates neurotransmitter release by facilitating activity-dependent F-Actin assembly and the dynamic recruitment of key signaling molecules into presynaptic boutons, and highlight the need for new genetic models with which to study Piccolo loss-of-function.

The synaptic cleft manifests enriched glycosylation, with structured glycans coordinating signaling between presynaptic and postsynaptic cells. Glycosylated signaling ligands orchestrating communication are tightly regulated by secreted glycan-binding lectins. Using the Drosophila neuromuscular junction (NMJ) as a model glutamatergic synapse, we identify a new Ca2+-binding (C-type) lectin, Lectin-galC1 (LGC1), which modulates presynaptic function and neurotransmission strength. We find that LGC1 is enriched in motoneuron presynaptic boutons and secreted into the NMJ extracellular synaptomatrix. We show that LGC1 limits locomotor peristalsis and coordinated movement speed, with a specific requirement for synaptic function, but not NMJ architecture. LGC1 controls neurotransmission strength by limiting presynaptic active zone (AZ) and postsynaptic glutamate receptor (GluR) aligned synapse number, reducing both spontaneous and stimulation-evoked synaptic vesicle (SV) release, and capping SV cycling rate. During high-frequency stimulation (HFS), mutants have faster synaptic depression and impaired recovery while replenishing depleted SV pools. Although LGC1 removal increases the number of glutamatergic synapses, we find that LGC1-null mutants exhibit decreased SV density within presynaptic boutons, particularly SV pools at presynaptic active zones. Thus, LGC1 regulates NMJ neurotransmission to modulate coordinated movement.

The fusion of synaptic vesicles (SVs) with the plasma membrane of the active zone (AZ) upon arrival of an action potential (AP) at the presynaptic compartment is a tightly regulated probabilistic process crucial for information transfer. The probability of a SV to release its transmitter content in response to an AP, termed release probability (Pr), is highly diverse both at the level of entire synapses and individual SVs at a given synapse. Differences in Pr exist between different types of synapses, between synapses of the same type, synapses originating from the same axon and even between different SV subpopulations within the same presynaptic terminal. The Pr of SVs at the AZ is set by a complex interplay of different presynaptic properties including the availability of release-ready SVs, the location of the SVs relative to the voltage-gated calcium channels (VGCCs) at the AZ, the magnitude of calcium influx upon arrival of the AP, the buffering of calcium ions as well as the identity and sensitivity of the calcium sensor. These properties are not only interconnected, but can also be regulated dynamically to match the requirements of activity patterns mediated by the synapse. Here, we review recent advances in identifying molecules and molecular machines taking part in the determination of vesicular Pr at the AZ.

With remarkably few exceptions, the molecules mediating synaptic vesicle exocytosis at active zones are structurally and functionally conserved between vertebrates and invertebrates. Mover was found in a yeast-2-hybrid assay using the vertebrate-specific active zone scaffolding protein bassoon as a bait. Peptides of Mover have been reported in proteomics screens for self-interacting proteins, phosphorylated proteins, and synaptic vesicle proteins, respectively. Here, we tested the predictions arising from these screens. Using flotation assays, carbonate stripping of peripheral membrane proteins, mass spectrometry, immunogold labelling of purified synaptic vesicles, and immuno-organelle isolation, we found that Mover is indeed a peripheral synaptic vesicle membrane protein. In addition, by generating an antibody against phosphorylated Mover and Western blot analysis of fractionated rat brain, we found that Mover is a bona fide phospho-protein. The localization of Mover to synaptic vesicles is phosphorylation dependent; treatment with a phosphatase caused Mover to dissociate from synaptic vesicles. A yeast-2-hybrid screen, co-immunoprecipitation and cell-based optical assays of homomerization revealed that Mover undergoes homophilic interaction, and regions within both the N- and C- terminus of the protein are required for this interaction. Deleting a region required for homomeric interaction abolished presynaptic targeting of recombinant Mover in cultured neurons. Together, these data prove that Mover is associated with synaptic vesicles, and implicate phosphorylation and multimerization in targeting of Mover to synaptic vesicles and presynaptic sites.

Frequency-Dependent Kinetics and Prevalence of Kiss-and-Run and Reuse at Hippocampal Synapses Studied with Novel Quenching Methods

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.