Presynaptic Active Zone Research Articles

Live-cell imaging of endocytosed synaptophysin around individual hippocampal presynaptic active zones.

In presynaptic terminals 4 types of endocytosis, kiss-and-run, clathrin-mediated, bulk and ultrafast endocytosis have been reported to maintain repetitive exocytosis of neurotransmitter. However, detailed characteristics and relative contribution of each type of endocytosis still need to be determined. Our previous live-cell imaging study demonstrated individual exocytosis events of synaptic vesicle within an active-zone-like membrane (AZLM) formed on glass using synaptophysin tagged with a pH-sensitive fluorescent protein. On the other hand, individual endocytosis events of postsynaptic receptors were recorded with a rapid extracellular pH exchange method. Combining these methods, here we live-cell imaged endocytosed synaptophysin with total internal reflection fluorescence microscopy in rat hippocampal culture preparations. Clathrin-dependent and -independent endocytosis, which was seemingly bulk endocytosis, occurred within several seconds after electrical stimulation at multiple locations around AZLM at room temperature, with the locations varying trial to trial. The contribution of clathrin-independent endocytosis was more prominent when the number of stimulation pulses was large. The skewness of synaptophysin distribution in intracellular vesicles became smaller after addition of a clathrin inhibitor, which suggests that clathrin-dependent endocytosis concentrates synaptophysin. Ultrafast endocytosis was evident immediately after stimulation only at near physiological temperature and was the predominant endocytosis when the number of stimulation pulses was small.

SynLight: a bicistronic strategy for simultaneous active zone and cell labeling in the Drosophila nervous system.

At synapses, chemical neurotransmission mediates the exchange of information between neurons, leading to complex movement, behaviors, and stimulus processing. The immense number and variety of neurons within the nervous system make discerning individual neuron populations difficult, necessitating the development of advanced neuronal labeling techniques. In Drosophila, Bruchpilot-Short and mCD8-GFP, which label presynaptic active zones and neuronal membranes, respectively, have been widely used to study synapse development and organization. This labeling is often achieved via the expression of 2 independent constructs by a single binary expression system, but expression can weaken when multiple transgenes are expressed by a single driver. Recent work has sought to circumvent these drawbacks by developing methods that encode multiple proteins from a single transcript. Self-cleaving peptides, specifically 2A peptides, have emerged as effective sequences for accomplishing this task. We leveraged 2A ribosomal skipping peptides to engineer a construct that produces both Bruchpilot-Short-mStraw and mCD8-GFP from the same mRNA, which we named SynLight. Using SynLight, we visualized the putative synaptic active zones and membranes of multiple classes of olfactory, visual, and motor neurons and observed the correct separation of signal, confirming that both proteins are being generated separately. Furthermore, we demonstrate proof of principle by quantifying synaptic puncta number and neurite volume in olfactory neurons and finding no difference between the synapse densities of neurons expressing SynLight or neurons expressing both transgenes separately. At the neuromuscular junction, we determined that the synaptic puncta number labeled by SynLight was comparable to the endogenous puncta labeled by antibody staining. Overall, SynLight is a versatile tool for examining synapse density in any nervous system region of interest and allows new questions to be answered about synaptic development and organization.

Real-time heterogeneity of supramolecular assembly of amyloid precursor protein is modulated by an endocytic risk factor PICALM.

Recently, the localization of amyloid precursor protein (APP) into reversible nanoscale supramolecular assembly or "nanodomains" has been highlighted as crucial towards understanding the onset of the molecular pathology of Alzheimer's disease (AD). Surface expression of APP is regulated by proteins interacting with it, controlling its retention and lateral trafficking on the synaptic membrane. Here, we evaluated the involvement of a key risk factor for AD, PICALM, as a critical regulator of nanoscale dynamics of APP. Although it was enriched in the postsynaptic density, PICALM was also localized to the presynaptic active zone and the endocytic zone. PICALM colocalized with APP and formed nanodomains with distinct morphological properties in different subsynaptic regions. Next, we evaluated if this localization to subsynaptic compartments was regulated by the C-terminal sequences of APP, namely, the "Y682ENPTY687" domain. Towards this, we found that deletion of C-terminal regions of APP with partial or complete deletion of Y682ENPTY687, namely, APP-Δ9 and APP-Δ14, affected the lateral diffusion and nanoscale segregation of APP. Lateral diffusion of APP mutant APP-Δ14 sequence mimicked that of a detrimental Swedish mutant of APP, namely, APP-SWE, while APP-Δ9 diffused similar to wild-type APP. Interestingly, elevated expression of PICALM differentially altered the lateral diffusion of the APP C-terminal deletion mutants. These observations confirm that the C-terminal sequence of APP regulates its lateral diffusion and the formation of reversible nanoscale domains. Thus, when combined with autosomal dominant mutations, it generates distinct molecular patterns leading to onset of Alzheimer's disease (AD).

Nanoscaled RIM clustering at presynaptic active zones revealed by endogenous tagging.

Chemical synaptic transmission involves neurotransmitter release from presynaptic active zones (AZs). The AZ protein Rab-3-interacting molecule (RIM) is important for normal Ca2+-triggered release. However, its precise localization within AZs of the glutamatergic neuromuscular junctions of Drosophila melanogaster remains elusive. We used CRISPR/Cas9-assisted genome engineering of the rim locus to incorporate small epitope tags for targeted super-resolution imaging. A V5-tag, derived from simian virus 5, and an HA-tag, derived from human influenza virus, were N-terminally fused to the RIM Zinc finger. Whereas both variants are expressed in co-localization with the core AZ scaffold Bruchpilot, electrophysiological characterization reveals that AP-evoked synaptic release is disturbed in rimV5-Znf but not in rimHA-Znf In addition, rimHA-Znf synapses show intact presynaptic homeostatic potentiation. Combining super-resolution localization microscopy and hierarchical clustering, we detect ∼10 RIMHA-Znf subclusters with ∼13 nm diameter per AZ that are compacted and increased in numbers in presynaptic homeostatic potentiation.

Age-dependent structural reorganization of utricular ribbon synapses.

In mammals, spatial orientation is synaptically-encoded by sensory hair cells of the vestibular labyrinth. Vestibular hair cells (VHCs) harbor synaptic ribbons at their presynaptic active zones (AZs), which play a critical role in molecular scaffolding and facilitate synaptic release and vesicular replenishment. With advancing age, the prevalence of vestibular deficits increases; yet, the underlying mechanisms are not well understood and the possible accompanying morphological changes in the VHC synapses have not yet been systematically examined. We investigated the effects of maturation and aging on the ultrastructure of the ribbon-type AZs in murine utricles using various electron microscopic techniques and combined them with confocal and super-resolution light microscopy as well as metabolic imaging up to 1year of age. In older animals, we detected predominantly in type I VHCs the formation of floating ribbon clusters, mostly consisting of newly synthesized ribbon material. Our findings suggest that VHC ribbon-type AZs undergo dramatic structural alterations upon aging.

AMPA and GABAA receptor nanodomains assemble in the absence of synaptic neurotransmitter release.

Postsynaptic neurotransmitter receptors and their associated scaffolding proteins assemble into discrete, nanometer-scale subsynaptic domains (SSDs) within the postsynaptic membrane at both excitatory and inhibitory synapses. Intriguingly, postsynaptic receptor SSDs are mirrored by closely apposed presynaptic active zones. These trans-synaptic molecular assemblies are thought to be important for efficient neurotransmission because they concentrate postsynaptic receptors near sites of presynaptic neurotransmitter release. While previous studies have characterized the role of synaptic activity in sculpting the number, size, and distribution of postsynaptic SSDs at established synapses, it remains unknown whether neurotransmitter signaling is required for their initial assembly during synapse development. Here, we evaluated synaptic nano-architecture under conditions where presynaptic neurotransmitter release was blocked prior to, and throughout synaptogenesis with tetanus neurotoxin (TeNT). In agreement with previous work, neurotransmitter release was not required for the formation of excitatory or inhibitory synapses. The overall size of the postsynaptic specialization at both excitatory and inhibitory synapses was reduced at chronically silenced synapses. However, both AMPARs and GABAARs still coalesced into SSDs, along with their respective scaffold proteins. Presynaptic active zone assemblies, defined by RIM1, were smaller and more numerous at silenced synapses, but maintained alignment with postsynaptic AMPAR SSDs. Thus, basic features of synaptic nano-architecture, including assembly of receptors and scaffolds into trans-synaptically aligned structures, are intrinsic properties that can be further regulated by subsequent activity-dependent mechanisms.

Insulin secretion hot spots in pancreatic β cells as secreting adhesions.

Pancreatic β cell secretion of insulin is crucial to the maintenance of glucose homeostasis and prevention of diseases related to glucose regulation, including diabetes. Pancreatic β cells accomplish efficient insulin secretion by clustering secretion events at the cell membrane facing the vasculature. Regions at the cell periphery characterized by clustered secretion are currently termed insulin secretion hot spots. Several proteins, many associated with the microtubule and actin cytoskeletons, are known to localize to and serve specific functions at hot spots. Among these proteins are the scaffolding protein ELKS, the membrane-associated proteins LL5β and liprins, the focal adhesion-associated protein KANK1, and other factors typically associated with the presynaptic active zone in neurons. These hot spot proteins have been shown to contribute to insulin secretion, but many questions remain regarding their organization and dynamics at hot spots. Current studies suggest microtubule- and F-actin are involved in regulation of hot spot proteins and their function in secretion. The hot spot protein association with the cytoskeleton networks also suggests a potential role for mechanical regulation of these proteins and hot spots in general. This perspective summarizes the existing knowledge of known hot spot proteins, their cytoskeletal-mediated regulation, and discuss questions remaining regarding mechanical regulation of pancreatic beta cell hot spots.

Visualizing the triheteromeric N-methyl-D-aspartate receptor subunit composition.

N-methyl-D-aspartate receptors (NMDARs) are one of three ligand-gated ionotropic channels that transduce the effects of neurotransmitter glutamate at excitatory synapses within the central nervous system. Their ability to influx Ca2+ into cells, unlike mature AMPA or kainate receptors, implicates them in a variety of processes ranging from synaptic plasticity to cell death. Many of the receptor's capabilities, including binding glutamate and regulating Ca2+ influx, have been attributed to their subunit composition, determined putatively using cell biology, electrophysiology and/or pharmacology. Here, we show that subunit composition of synaptic NMDARs can also be readily visualized in acute brain slices (rat) using highly specific antibodies directed against extracellular epitopes of the subunit proteins and high-resolution confocal microscopy. This has helped confirm the expression of triheteromeric t-NMDARs (containing GluN1, GluN2, and GluN3 subunits) at synapses for the first time and reconcile functional differences with diheteromeric d-NMDARs (containing GluN1 and GluN2 subunits) described previously. Even though structural information about individual receptors is still diffraction limited, fluorescently tagged receptor subunit puncta coalesce with precision at various magnifications and/or with the postsynaptic density (PSD-95) but not the presynaptic active zone marker Bassoon. These data are particularly relevant for identifying GluN3A-containing t-NMDARs that are highly Ca2+ permeable and whose expression at excitatory synapses renders neurons vulnerable to excitotoxicity and cell death. Imaging NMDAR subunit proteins at synapses not only offers firsthand insights into subunit composition to correlate function but may also help identify zones of vulnerability within brain structures underlying neurodegenerative diseases like Temporal Lobe Epilepsy.

Nanoscale Phosphoinositide Distribution on Cell Membranes of Mouse Cerebellar Neurons.

Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) plays an essential role in neuronal activities through interaction with various proteins involved in signaling at membranes. However, the distribution pattern of PI(4,5)P2 and the association with these proteins on the neuronal cell membranes remain elusive. In this study, we established a method for visualizing PI(4,5)P2 by SDS-digested freeze-fracture replica labeling (SDS-FRL) to investigate the quantitative nanoscale distribution of PI(4,5)P2 in cryo-fixed brain. We demonstrate that PI(4,5)P2 forms tiny clusters with a mean size of ∼1000 nm2 rather than randomly distributed in cerebellar neuronal membranes in male C57BL/6J mice. These clusters show preferential accumulation in specific membrane compartments of different cell types, in particular, in Purkinje cell (PC) spines and granule cell (GC) presynaptic active zones. Furthermore, we revealed extensive association of PI(4,5)P2 with CaV2.1 and GIRK3 across different membrane compartments, whereas its association with mGluR1α was compartment specific. These results suggest that our SDS-FRL method provides valuable insights into the physiological functions of PI(4,5)P2 in neurons.SIGNIFICANCE STATEMENT In this study, we established an electron microscopic method to visualize and analyze the quantitative distribution pattern of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) on cell membranes using cryo-fixed brain tissues and SDS-digested freeze-fracture replica labeling. PI(4,5)P2 interacts with various ion channels and receptors to regulate membrane signaling but its nanoscale distribution and association with these proteins remain elusive. This method revealed PI(4,5)P2 clusters preferentially accumulated in specific membrane compartments and its distinct associations with CaV2.1, GIRK3, and mGluR1α in the mouse cerebellum. These results demonstrate usefulness of the method for gaining insights into the physiological functions of PI(4,5)P2.

Active Zone Trafficking of CaV2/UNC-2 Channels Is Independent of β/CCB-1 and α2δ/UNC-36 Subunits.

The CaV2 voltage-gated calcium channel is the major conduit of calcium ions necessary for neurotransmitter release at presynaptic active zones (AZs). The CaV2 channel is a multimeric complex that consists of a pore-forming α1 subunit and two auxiliary β and α2δ subunits. Although auxiliary subunits are critical for channel function, whether they are required for α1 trafficking is unresolved. Using endogenously fluorescent protein-tagged CaV2 channel subunits in Caenorhabditis elegans, we show that UNC-2/α1 localizes to AZs even in the absence of CCB-1/β or UNC-36/α2δ, albeit at low levels. When UNC-2 is manipulated to be trapped in the endoplasmic reticulum (ER), CCB-1 and UNC-36 fail to colocalize with UNC-2 in the ER, indicating that they do not coassemble with UNC-2 in the ER. Moreover, blocking ER-associated degradation does not further increase presynaptic UNC-2 channels in ccb-1 or unc-36 mutants, indicating that UNC-2 levels are not regulated in the ER. An unc-2 mutant lacking C-terminal AZ protein interaction sites with intact auxiliary subunit binding sites displays persistent presynaptic UNC-2 localization and a prominent increase of UNC-2 channels in nonsynaptic axonal regions, underscoring a protective role of auxiliary subunits against UNC-2 degradation. In the absence of UNC-2, presynaptic CCB-1 and UNC-36 are profoundly diminished to barely detectable levels, indicating that UNC-2 is required for the presynaptic localization of CCB-1 and UNC-36. Together, our findings demonstrate that although the pore-forming subunit does not require auxiliary subunits for its trafficking and transport to AZs, it recruits auxiliary subunits to stabilize and expand calcium channel signalosomes.SIGNIFICANCE STATEMENT Synaptic transmission in the neuron hinges on the coupling of synaptic vesicle exocytosis with calcium influx. This calcium influx is mediated by CaV2 voltage-gated calcium channels. These channels consist of one pore-forming α1 subunit and two auxiliary β and α2δ subunits. The auxiliary subunits enhance channel function and regulate the overall level of channels at presynaptic terminals. However, it is not settled how these auxiliary subunits regulate the overall channel level. Our study in C. elegans finds that although the auxiliary subunits do not coassemble with α1 and aid trafficking, they are recruited to α1 and stabilize the channel complex at presynaptic terminals. Our study suggests that drugs that target the auxiliary subunits can directly destabilize and have an impact on CaV2 channels.

Simulations of active zone structure and function at mammalian NMJs predict that loss of calcium channels alone is not sufficient to replicate LEMS effects.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune-mediated neuromuscular disease thought to be caused by autoantibodies against P/Q-type voltage-gated calcium channels (VGCCs), which attack and reduce the number of VGCCs within transmitter release sites (active zones; AZs) at the neuromuscular junction (NMJ), resulting in neuromuscular weakness. However, patients with LEMS also have antibodies to other neuronal proteins, and about 15% of patients with LEMS are seronegative for antibodies against VGCCs. We hypothesized that a reduction in the number of P/Q-type VGCCs alone is not sufficient to explain LEMS effects on transmitter release. Here, we used a computational model to study a variety of LEMS-mediated effects on AZ organization and transmitter release constrained by electron microscopic, pharmacological, immunohistochemical, voltage imaging, and electrophysiological observations. We show that models of healthy AZs can be modified to predict the transmitter release and short-term facilitation characteristics of LEMS and that in addition to a decrease in the number of AZ VGCCs, disruption in the organization of AZ proteins, a reduction in AZ number, a reduction in the amount of synaptotagmin, and the compensatory expression of L-type channels outside the remaining AZs are important contributors to LEMS-mediated effects on transmitter release. Furthermore, our models predict that antibody-mediated removal of synaptotagmin in combination with disruption in AZ organization alone could mimic LEMS effects without the removal of VGCCs (a seronegative model). Overall, our results suggest that LEMS pathophysiology may be caused by a collection of pathological alterations to AZs at the NMJ, rather than by a simple loss of VGCCs.NEW & NOTEWORTHY We used a computational model of the active zone (AZ) in the mammalian neuromuscular junction to investigate Lambert-Eaton myasthenic syndrome (LEMS) pathophysiology. This model suggests that disruptions in presynaptic active zone organization and protein content (particularly synaptotagmin), beyond the simple removal of presynaptic calcium channels, play an important role in LEMS pathophysiology.

Critical Role of the Presynaptic Protein CAST in Maintaining the Photoreceptor Ribbon Synapse Triad.

The cytomatrix at the active zone-associated structural protein (CAST) and its homologue, named ELKS, being rich in glutamate (E), leucine (L), lysine (K), and serine (S), belong to a family of proteins that organize presynaptic active zones at nerve terminals. These proteins interact with other active zone proteins, including RIMs, Munc13s, Bassoon, and the β subunit of Ca2+ channels, and have various roles in neurotransmitter release. A previous study showed that depletion of CAST/ELKS in the retina causes morphological changes and functional impairment of this structure. In this study, we investigated the roles of CAST and ELKS in ectopic synapse localization. We found that the involvement of these proteins in ribbon synapse distribution is complex. Unexpectedly, CAST and ELKS, in photoreceptors or in horizontal cells, did not play a major role in ribbon synapse ectopic localization. However, depletion of CAST and ELKS in the mature retina resulted in degeneration of the photoreceptors. These findings suggest that CAST and ELKS play critical roles in maintaining neural signal transduction in the retina, but the regulation of photoreceptor triad synapse distribution is not solely dependent on their actions within photoreceptors and horizontal cells.

S-Afadin binds to MAGUIN/Cnksr2 and regulates the localization of the AMPA receptor and glutamatergic synaptic response in hippocampal neurons

A hippocampal mossy fiber synapse implicated in learning and memory is a complex structure in which a presynaptic bouton attaches to the dendritic trunk by puncta adherentia junctions (PAJs) and wraps multiply branched spines. The postsynaptic densities (PSDs) are localized at the heads of each of these spines and faces to the presynaptic active zones. We previously showed that the scaffolding protein afadin regulates the formation of the PAJs, PSDs, and active zones in the mossy fiber synapse. Afadin has two splice variants: l-afadin and s-afadin. l-Afadin, but not s-afadin, regulates the formation of the PAJs but the roles of s-afadin in synaptogenesis remain unknown. We found here that s-afadin more preferentially bound to MAGUIN (a product of the Cnksr2 gene) than l-afadin invivo and invitro. MAGUIN/CNKSR2 is one of the causative genes for nonsyndromic X-linked intellectual disability accompanied by epilepsy and aphasia. Genetic ablation of MAGUIN impaired PSD-95 localization and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptor surface accumulation in cultured hippocampal neurons. Our electrophysiological analysis revealed that the postsynaptic response to glutamate, but not its release from the presynapse, was impaired in the MAGUIN-deficient cultured hippocampal neurons. Furthermore, disruption of MAGUIN did not increase the seizure susceptibility to flurothyl, a GABAA receptor antagonist. These results indicate that s-afadin binds to MAGUIN and regulates the PSD-95-dependent cell surface localization of the AMPA receptor and glutamatergic synaptic responses in the hippocampal neurons and that MAGUIN is not involved in the induction of epileptic seizure by flurothyl in our mouse model.

Interactive nanocluster compaction of the ELKS scaffold and Cacophony Ca2+ channels drives sustained active zone potentiation.

At presynaptic active zones (AZs), conserved scaffold protein architectures control synaptic vesicle (SV) release by defining the nanoscale distribution and density of voltage-gated Ca2+ channels (VGCCs). While AZs can potentiate SV release in the minutes range, we lack an understanding of how AZ scaffold components and VGCCs engage into potentiation. We here establish dynamic, intravital single-molecule imaging of endogenously tagged proteins at Drosophila AZs undergoing presynaptic homeostatic potentiation. During potentiation, the numbers of α1 VGCC subunit Cacophony (Cac) increased per AZ, while their mobility decreased and nanoscale distribution compacted. These dynamic Cac changes depended on the interaction between Cac channel's intracellular carboxyl terminus and the membrane-close amino-terminal region of the ELKS-family protein Bruchpilot, whose distribution compacted drastically. The Cac-ELKS/Bruchpilot interaction was also needed for sustained AZ potentiation. Our single-molecule analysis illustrates how the AZ scaffold couples to VGCC nanoscale distribution and dynamics to establish a state of sustained potentiation.

Single-Molecule Localization Microscopy of Presynaptic Active Zones in Drosophila melanogaster after Rapid Cryofixation.

Single-molecule localization microscopy (SMLM) greatly advances structural studies of diverse biological tissues. For example, presynaptic active zone (AZ) nanotopology is resolved in increasing detail. Immunofluorescence imaging of AZ proteins usually relies on epitope preservation using aldehyde-based immunocompetent fixation. Cryofixation techniques, such as high-pressure freezing (HPF) and freeze substitution (FS), are widely used for ultrastructural studies of presynaptic architecture in electron microscopy (EM). HPF/FS demonstrated nearer-to-native preservation of AZ ultrastructure, e.g., by facilitating single filamentous structures. Here, we present a protocol combining the advantages of HPF/FS and direct stochastic optical reconstruction microscopy (dSTORM) to quantify nanotopology of the AZ scaffold protein Bruchpilot (Brp) at neuromuscular junctions (NMJs) of Drosophila melanogaster. Using this standardized model, we tested for preservation of Brp clusters in different FS protocols compared to classical aldehyde fixation. In HPF/FS samples, presynaptic boutons were structurally well preserved with ~22% smaller Brp clusters that allowed quantification of subcluster topology. In summary, we established a standardized near-to-native preparation and immunohistochemistry protocol for SMLM analyses of AZ protein clusters in a defined model synapse. Our protocol could be adapted to study protein arrangements at single-molecule resolution in other intact tissue preparations.

The Role of Tomosyn in the Regulation of Neurotransmitter Release.

Soluble NSF attachment protein receptor (SNARE) proteins play a central role in synaptic vesicle (SV) exocytosis. These proteins include the vesicle-associated SNARE protein (v-SNARE) synaptobrevin and the target membrane-associated SNARE proteins (t-SNAREs) syntaxin and SNAP-25. Together, these proteins drive membrane fusion between synaptic vesicles (SV) and the presynaptic plasma membrane to generate SV exocytosis. In the presynaptic active zone, various proteins may either enhance or inhibit SV exocytosis by acting on the SNAREs. Among the inhibitory proteins, tomosyn, a syntaxin-binding protein, is of particular importance because it plays a critical and evolutionarily conserved role in controlling synaptic transmission. In this chapter, we describe how tomosyn was discovered, how it interacts with SNAREs and other presynaptic regulatory proteins to regulate SV exocytosis and synaptic plasticity, and how its various domains contribute to its synaptic functions.

Complexins: Ubiquitously Expressed Presynaptic Regulators of SNARE-Mediated Synaptic Vesicle Fusion.

Neurotransmitter release is a spatially and temporally tightly regulated process, which requires assembly and disassembly of SNARE complexes to enable the exocytosis of transmitter-loaded synaptic vesicles (SVs) at presynaptic active zones (AZs). While the requirement for the core SNARE machinery is shared by most membrane fusion processes, SNARE-mediated fusion at AZs is uniquely regulated to allow very rapid Ca2+-triggered SV exocytosis following action potential (AP) arrival. To enable a sub-millisecond time course of AP-triggered SV fusion, synapse-specific accessory SNARE-binding proteins are required in addition to the core fusion machinery. Among the known SNARE regulators specific for Ca2+-triggered SV fusion are complexins, which are almost ubiquitously expressed in neurons. This chapter summarizes the structural features of complexins, models for their molecular interactions with SNAREs, and their roles in SV fusion.

Endogenous tagging of Unc-13 reveals nanoscale reorganization at active zones during presynaptic homeostatic potentiation.

Neurotransmitter release at presynaptic active zones (AZs) requires concerted protein interactions within a dense 3D nano-hemisphere. Among the complex protein meshwork the (M)unc-13 family member Unc-13 of Drosophila melanogaster is essential for docking of synaptic vesicles and transmitter release. We employ minos-mediated integration cassette (MiMIC)-based gene editing using GFSTF (EGFP-FlAsH-StrepII-TEV-3xFlag) to endogenously tag all annotated Drosophila Unc-13 isoforms enabling visualization of endogenous Unc-13 expression within the central and peripheral nervous system. Electrophysiological characterization using two-electrode voltage clamp (TEVC) reveals that evoked and spontaneous synaptic transmission remain unaffected in unc-13 GFSTF 3rd instar larvae and acute presynaptic homeostatic potentiation (PHP) can be induced at control levels. Furthermore, multi-color structured-illumination shows precise co-localization of Unc-13GFSTF, Bruchpilot, and GluRIIA-receptor subunits within the synaptic mesoscale. Localization microscopy in combination with HDBSCAN algorithms detect Unc-13GFSTF subclusters that move toward the AZ center during PHP with unaltered Unc-13GFSTF protein levels.

Diverse organization of voltage-gated calcium channels at presynaptic active zones.

Synapses are highly organized but are also highly diverse in their organization and properties to allow for optimizing the computing power of brain circuits. Along these lines, voltage-gated calcium (CaV) channels at the presynaptic active zone are heterogeneously organized, which creates a variety of calcium dynamics profiles that can shape neurotransmitter release properties of individual synapses. Extensive studies have revealed striking diversity in the subtype, number, and distribution of CaV channels, as well as the nanoscale topographic relationships to docked synaptic vesicles. Further, multi-protein complexes including RIMs, RIM-binding proteins, CAST/ELKS, and neurexins are required for coordinating the diverse organization of CaV channels at the presynaptic active zone. In this review, we highlight major advances in the studies of the functional organization of presynaptic CaV channels and discuss their physiological implications for synaptic transmission and short-term plasticity.

A brain-wide form of presynaptic active zone plasticity orchestrates resilience to brain aging in Drosophila.

The brain as a central regulator of stress integration determines what is threatening, stores memories, and regulates physiological adaptations across the aging trajectory. While sleep homeostasis seems to be linked to brain resilience, how age-associated changes intersect to adapt brain resilience to life history remains enigmatic. We here provide evidence that a brain-wide form of presynaptic active zone plasticity ("PreScale"), characterized by increases of active zone scaffold proteins and synaptic vesicle release factors, integrates resilience by coupling sleep, longevity, and memory during early aging of Drosophila. PreScale increased over the brain until mid-age, to then decreased again, and promoted the age-typical adaption of sleep patterns as well as extended longevity, while at the same time it reduced the ability of forming new memories. Genetic induction of PreScale also mimicked early aging-associated adaption of sleep patterns and the neuronal activity/excitability of sleep control neurons. Spermidine supplementation, previously shown to suppress early aging-associated PreScale, also attenuated the age-typical sleep pattern changes. Pharmacological induction of sleep for 2 days in mid-age flies also reset PreScale, restored memory formation, and rejuvenated sleep patterns. Our data suggest that early along the aging trajectory, PreScale acts as an acute, brain-wide form of presynaptic plasticity to steer trade-offs between longevity, sleep, and memory formation in a still plastic phase of early brain aging.