Organization Of Active Zones Research Articles

Neuronal LRP4 directs the development, maturation and cytoskeletal organization of Drosophila peripheral synapses.

Synaptic development requires multiple signaling pathways to ensure successful connections. Transmembrane receptors are optimally positioned to connect the synapse and the rest of the neuron, often acting as synaptic organizers to synchronize downstream events. One such organizer, the LDL receptor-related protein LRP4, is a cell surface receptor that has been most well-studied postsynaptically at mammalian neuromuscular junctions. Recent work, however, identified emerging roles, but how LRP4 acts as a presynaptic organizer and the downstream mechanisms of LRP4 are not well understood. Here, we show that LRP4 functions presynaptically at Drosophila neuromuscular synapses, acting in motoneurons to instruct pre- and postsynaptic development. Loss of presynaptic LRP4 results in multiple defects, impairing active zone organization, synapse growth, physiological function, microtubule organization, synaptic ultrastructure and synapse maturation. We further demonstrate that LRP4 promotes most aspects of presynaptic development via a downstream SR-protein kinase, SRPK79D. These data demonstrate a function for presynaptic LRP4 as a peripheral synaptic organizer, highlight a downstream mechanism conserved with its CNS function in Drosophila, and underscore previously unappreciated but important developmental roles for LRP4 in cytoskeletal organization, synapse maturation and active zone organization.

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.

Protein phase separation hotspots at the presynapse.

Fundamental discoveries have shaped our molecular understanding of presynaptic processes, such as neurotransmitter release, active zone organization and mechanisms of synaptic vesicle (SV) recycling. However, certain regulatory steps still remain incompletely understood. Protein liquid-liquid phase separation (LLPS) and its role in SV clustering and active zone regulation now introduce a new perception of how the presynapse and its different compartments are organized. This article highlights the newly emerging concept of LLPS at the synapse, providing a systematic overview on LLPS tendencies of over 500 presynaptic proteins, spotlighting individual proteins and discussing recent progress in the field. Newly discovered LLPS systems like ELKS/liprin-alpha and Eps15/FCho are put into context, and further LLPS candidate proteins, including epsin1, dynamin, synaptojanin, complexin and rabphilin-3A, are highlighted.

RIM-BP2 primes synaptic vesicles via recruitment of Munc13-1 at hippocampal mossy fiber synapses.

All synapses require fusion-competent vesicles and coordinated Ca2+-secretion coupling for neurotransmission, yet functional and anatomical properties are diverse across different synapse types. We show that the presynaptic protein RIM-BP2 has diversified functions in neurotransmitter release at different central murine synapses and thus contributes to synaptic diversity. At hippocampal pyramidal CA3-CA1 synapses, RIM-BP2 loss has a mild effect on neurotransmitter release, by only regulating Ca2+-secretion coupling. However, at hippocampal mossy fiber synapses, RIM-BP2 has a substantial impact on neurotransmitter release by promoting vesicle docking/priming and vesicular release probability via stabilization of Munc13-1 at the active zone. We suggest that differences in the active zone organization may dictate the role a protein plays in synaptic transmission and that differences in active zone architecture is a major determinant factor in the functional diversity of synapses.

An Input-Specific Orphan Receptor GPR158-HSPG Interaction Organizes Hippocampal Mossy Fiber-CA3 Synapses

Pyramidal neuron dendrites integrate synaptic input from multiple partners. Different inputs converging on the same dendrite have distinct structural and functional features, but the molecular mechanisms organizing input-specific properties are poorly understood. We identify the orphan receptor GPR158 as a binding partner for the heparan sulfate proteoglycan (HSPG) glypican 4 (GPC4). GPC4 is enriched on hippocampal granule cell axons (mossy fibers), whereas postsynaptic GPR158 is restricted to the proximal segment of CA3 apical dendrites receiving mossy fiber input. GPR158-induced presynaptic differentiation in contacting axons requires cell-surface GPC4 and the co-receptor LAR. Loss of GPR158 increases mossy fiber synapse density but disrupts bouton morphology, impairs ultrastructural organization of active zone and postsynaptic density, and reduces synaptic strength of this connection, while adjacent inputs on the same dendrite are unaffected. Our work identifies an input-specific HSPG-GPR158 interaction that selectively organizes synaptic architecture and function of developing mossy fiber-CA3 synapses in the hippocampus.

Human APP Gene Expression Alters Active Zone Distribution and Spontaneous Neurotransmitter Release at the Drosophila Larval Neuromuscular Junction.

This study provides further insight into the molecular mechanisms that control neurotransmitter release. Experiments were performed on larval neuromuscular junctions of transgenic Drosophila melanogaster lines with different levels of human amyloid precursor protein (APP) production. To express human genes in motor neurons of Drosophila, the UAS-GAL4 system was used. Human APP gene expression increased the number of synaptic boutons per neuromuscular junction. The total number of active zones, detected by Bruchpilot protein puncta distribution, remained unchanged; however, the average number of active zones per bouton decreased. These disturbances were accompanied by a decrease in frequency of miniature excitatory junction potentials without alteration in random nature of spontaneous quantal release. Similar structural and functional changes were observed with co-overexpression of human APP and β-secretase genes. In Drosophila line with expression of human amyloid-β42 peptide itself, parameters analyzed did not differ from controls, suggesting the specificity of APP effects. These results confirm the involvement of APP in synaptogenesis and provide evidence to suggest that human APP overexpression specifically disturbs the structural and functional organization of active zone and results in altered Bruchpilot distribution and lowered probability of spontaneous neurotransmitter release.

Transmission, Development, and Plasticity of Synapses.

Chemical synapses are sites of contact and information transfer between a neuron and its partner cell. Each synapse is a specialized junction, where the presynaptic cell assembles machinery for the release of neurotransmitter, and the postsynaptic cell assembles components to receive and integrate this signal. Synapses also exhibit plasticity, during which synaptic function and/or structure are modified in response to activity. With a robust panel of genetic, imaging, and electrophysiology approaches, and strong evolutionary conservation of molecular components, Drosophila has emerged as an essential model system for investigating the mechanisms underlying synaptic assembly, function, and plasticity. We will discuss techniques for studying synapses in Drosophila, with a focus on the larval neuromuscular junction (NMJ), a well-established model glutamatergic synapse. Vesicle fusion, which underlies synaptic release of neurotransmitters, has been well characterized at this synapse. In addition, studies of synaptic assembly and organization of active zones and postsynaptic densities have revealed pathways that coordinate those events across the synaptic cleft. We will also review modes of synaptic growth and plasticity at the fly NMJ, and discuss how pre- and postsynaptic cells communicate to regulate plasticity in response to activity.

Presynaptic active zones in invertebrates and vertebrates.

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.

Postsynaptic actin regulates active zone spacing and glutamate receptor apposition at the Drosophila neuromuscular junction.

Synaptic communication requires precise alignment of presynaptic active zones with postsynaptic receptors to enable rapid and efficient neurotransmitter release. How transsynaptic signaling between connected partners organizes this synaptic apparatus is poorly understood. To further define the mechanisms that mediate synapse assembly, we carried out a chemical mutagenesis screen in Drosophila to identify mutants defective in the alignment of active zones with postsynaptic glutamate receptor fields at the larval neuromuscular junction. From this screen we identified a mutation in Actin 57B that disrupted synaptic morphology and presynaptic active zone organization. Actin 57B, one of six actin genes in Drosophila, is expressed within the postsynaptic bodywall musculature. The isolated allele, act(E84K), harbors a point mutation in a highly conserved glutamate residue in subdomain 1 that binds members of the Calponin Homology protein family, including spectrin. Homozygous act(E84K) mutants show impaired alignment and spacing of presynaptic active zones, as well as defects in apposition of active zones to postsynaptic glutamate receptor fields. act(E84K) mutants have disrupted postsynaptic actin networks surrounding presynaptic boutons, with the formation of aberrant actin swirls previously observed following disruption of postsynaptic spectrin. Consistent with a disruption of the postsynaptic actin cytoskeleton, spectrin, adducin and the PSD-95 homolog Discs-Large are all mislocalized in act(E84K) mutants. Genetic interactions between act(E84K) and neurexin mutants suggest that the postsynaptic actin cytoskeleton may function together with the Neurexin-Neuroligin transsynaptic signaling complex to mediate normal synapse development and presynaptic active zone organization.

Cryo–electron tomography reveals a critical role of RIM1α in synaptic vesicle tethering

Synaptic vesicles are embedded in a complex filamentous network at the presynaptic terminal. Before fusion, vesicles are linked to the active zone (AZ) by short filaments (tethers). The identity of the molecules that form and regulate tethers remains unknown, but Rab3-interacting molecule (RIM) is a prominent candidate, given its central role in AZ organization. In this paper, we analyzed presynaptic architecture of RIM1α knockout (KO) mice by cryo-electron tomography. In stark contrast to previous work on dehydrated, chemically fixed samples, our data show significant alterations in vesicle distribution and AZ tethering that could provide a structural basis for the functional deficits of RIM1α KO synapses. Proteasome inhibition reversed these structural defects, suggesting a functional recovery confirmed by electrophysiological recordings. Altogether, our results not only point to the ubiquitin-proteasome system as an important regulator of presynaptic architecture and function but also show that the tethering machinery plays a critical role in exocytosis, converging into a structural model of synaptic vesicle priming by RIM1α.

Active zones of mammalian neuromuscular junctions: formation, density, and aging

Presynaptic active zones are synaptic vesicle release sites that play essential roles in the function and pathology of mammalian neuromuscular junctions (NMJs). The molecular mechanisms of active zone organization use presynaptic voltage-dependent calcium channels (VDCCs) in NMJs as scaffolding proteins. VDCCs interact extracellularly with the muscle-derived synapse organizer, laminin β2 and interact intracellularly with active zone-specific proteins, such as Bassoon, CAST/Erc2/ELKS2alpha, ELKS, Piccolo, and RIMs. These molecular mechanisms are supported by studies in P/Q- and N-type VDCCs double-knockout mice, and they are consistent with the pathological conditions of Lambert-Eaton myasthenic syndrome and Pierson syndrome, which are caused by autoantibodies against VDCCs or by a laminin β2 mutation. During normal postnatal maturation, NMJs maintain the density of active zones, while NMJs triple their size. However, active zones become impaired during aging. Propitiously, muscle exercise ameliorates the active zone impairment in aged NMJs, which suggests the potential for therapeutic strategies.

Fife, aDrosophilaPiccolo-RIM Homolog, Promotes Active Zone Organization and Neurotransmitter Release

Neuronal communication depends on the precisely orchestrated release of neurotransmitter at specialized sites called active zones (AZs). A small number of scaffolding and cytoskeletal proteins comprising the cytomatrix of the active zone (CAZ) are thought to organize the architecture and functional properties of AZs. The majority of CAZ proteins are evolutionarily conserved, underscoring the fundamental similarities in neurotransmission at all synapses. However, core CAZ proteins Piccolo and Bassoon have long been believed exclusive to vertebrates, raising intriguing questions about the conservation of the molecular mechanisms that regulate presynaptic properties. Here, we present the identification of a piccolo-rim-related gene in invertebrates, together with molecular phylogenetic analyses that indicate the encoded proteins may represent Piccolo orthologs. In accordance, we find that the Drosophila homolog, Fife, is neuronal and localizes to presynaptic AZs. To investigate the in vivo function of Fife, we generated a deletion of the fife locus. We find that evoked neurotransmitter release is substantially decreased in fife mutants and loss of fife results in motor deficits. Through morphological analysis of fife synapses, we identify underlying AZ abnormalities including pervasive presynaptic membrane detachments and reduced synaptic vesicle clustering. Our data demonstrate the conservation of a Piccolo-related protein in invertebrates and identify critical roles for Fife in regulating AZ structure and function. These findings suggest the CAZ is more conserved than previously thought, and open the door to a more complete understanding of how CAZ proteins regulate presynaptic structure and function through genetic studies in simpler model systems.

Molecular Mechanism of Active Zone Organization at Vertebrate Neuromuscular Junctions

Organization of presynaptic active zones is essential for development, plasticity, and pathology of the nervous system. Recent studies indicate a trans-synaptic molecular mechanism that organizes the active zones by connecting the pre- and the postsynaptic specialization. The presynaptic component of this trans-synaptic mechanism is comprised of cytosolic active zone proteins bound to the cytosolic domains of voltage-dependent calcium channels (P/Q-, N-, and L-type) on the presynaptic membrane. The postsynaptic component of this mechanism is the synapse organizer (laminin β2) that is expressed by the postsynaptic cell and accumulates specifically on top of the postsynaptic specialization. The pre- and the postsynaptic components interact directly between the extracellular domains of calcium channels and laminin β2 to anchor the presynaptic protein complex in front of the postsynaptic specialization. Hence, the presynaptic calcium channel functions as a scaffolding protein for active zone organization and as an ion-conducting channel for synaptic transmission. In contrast to the requirement of calcium influx for synaptic transmission, the formation of the active zone does not require the calcium influx through the calcium channels. Importantly, the active zones of adult synapses are not stable structures and require maintenance for their integrity. Furthermore, aging or diseases of the central and peripheral nervous system impair the active zones. This review will focus on the molecular mechanisms that organize the presynaptic active zones and summarize recent findings at the neuromuscular junctions and other synapses.

Liprinα1 Degradation by Calcium/Calmodulin-Dependent Protein Kinase II Regulates LAR Receptor Tyrosine Phosphatase Distribution and Dendrite Development

Neural activity regulates dendrite and synapse development, but the underlying molecular mechanisms are unclear. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is an important sensor of synaptic activity, and the scaffold protein liprinalpha1 is involved in pre- and postsynaptic maturation. Here we show that synaptic activity can suppress liprinalpha1 protein level by two pathways: CaMKII-mediated degradation and the ubiquitin-proteasome system. In hippocampal neurons, liprinalpha1 mutants that are immune to CaMKII degradation impair dendrite arborization, reduce spine and synapse number, and inhibit dendritic targeting of receptor tyrosine phosphatase LAR, which is important for dendrite development. Thus, regulated degradation of liprinalpha1 is important for proper LAR receptor distribution, and could provide a mechanism for localized control of dendrite and synapse morphogenesis by activity and CaMKII.

Organization of the Presynaptic Active Zone by ERC2/CAST1-Dependent Clustering of the Tandem PDZ Protein Syntenin-1

Presynaptic active zones contain a cytoskeletal matrix called the CAZ, which is thought to play a critical role in the regulation of active zone formation and neurotransmitter release. Recent studies have identified several CAZ components, but little is known about how they contribute to the molecular organization of active zones. Here, we report a novel PDZ [postsynaptic density-95/Discs large/zona occludens-1] interaction between the CAZ protein ERC2/CAST1 and the tandem PDZ protein syntenin-1, which is known to associate with diverse synaptic proteins, including glutamate receptor subunits, SynCAM, and beta-neurexin. This interaction promotes the localization of syntenin-1 at presynaptic ERC2 clusters. In addition to the PDZ interaction, multimerization of both ERC2 and syntenin-1 mediates syntenin-1 clustering. These results suggest that ERC2 promotes presynaptic syntenin-1 clustering by two distinct mechanisms and that syntenin-1 may contribute to the molecular organization of active zones by linking ERC2 and other CAZ components to diverse syntenin-1-associated synaptic proteins.

The architecture of the active zone in the presynaptic nerve terminal.

Active zones are highly specialized sites for release of neurotransmitter from presynaptic nerve terminals. The architecture of the active zone is exquisitely designed to facilitate the regulated tethering, docking, and fusing of the synaptic vesicles with the plasma membrane. Here we present our view of the structural and molecular organization of active zones across species and propose that all active zones are organized according to a common principle in which the structural differences correlate with the kinetics of transmitter release.

Lambert-Eaton sera reduce low-voltage and high-voltage activated Ca2+ currents in murine dorsal root ganglion neurons.

Voltage-gated Ca2+ channels are categorized as either high-voltage activated (HVA) or low-voltage activated (LVA), and a subtype (or subtypes) of HVA Ca2+ channels link the presynaptic depolarization to rapid neuro-transmitter release. Reductions in transmitter release are characteristic of the autoimmune disorder, Lambert-Eaton syndrome (LES). Because antibodies from LES patients reduce Ca2+ influx in a variety of cell types and disrupt the intramembrane organization of active zones at neuromuscular synapses, specificity of LES antibodies for the Ca2+ channels that control transmitter release has been suggested as the mechanism for disease. We tested sera from four patients with LES. Serum samples from three of the four patients reduced both the maximal LVA and HVA Ca2+ conductances in murine dorsal root ganglion neurons. Thus, even though LES is expressed as a neuromuscular and autonomic disorder, our studies suggest that Ca2+ channels may be broadly affected in LES patients. To account for the specificity of disease expression, we suggest that incapacitation of only a fraction of the Ca2+ channels clustered at active zones would severely depress transmitter release. In particular, if several Ca2+ channels in a cluster are normally required to open simultaneously before transmitter release becomes likely, the loss of a few active zone Ca2+ channels would exponentially reduce the probability of transmitter release. This model may explain why LES is expressed as a neuromuscular disorder and can account for a clinical hallmark of LES, facilitation of neuromuscular transmission produced by vigorous voluntary effort.

Active zone organization and vesicle content scale with bouton size at a vertebrate central synapse

A common observation in studies of neuronal structure is that axons differ in the size of their synaptic boutons. The significance of this size variation is unclear, in part because we do not know how the size of synaptic boutons is related to their internal organization. The present study has addressed this issue by using three-dimensional reconstruction of serial thin sections to examine the ultrastructure of synaptic boutons that vary in size. Our observations are based on complete or near-complete reconstructions of 53 synaptic boutons contacting large neurons in the ventromedial gray matter of the upper cervical spinal cord (probable neck motor neurons). We characterized bouton size in terms of volume and total area of membrane apposed to the motor neuron surface (apposition area). Boutons vary in apposition area by a factor of 40, and there is a significant positive correlation between our two measures of bouton size. In addition, bouton size is systematically related to four ultrastructural variables: 1) total active zone area, 2) number of active zones, 3) individual active zone area, and 4) number of synaptic vesicles. The correlations between these variables and both of our measures of bouton size are positive and significant. These data suggest that bouton size may be an index of ultrastructural features that are thought to influence transmitter storage and release.

Disruption of active zones in frog neuromuscular junctions following treatment with proteolytic enzymes.

Frog neuromuscular junction treated with proteolytic enzymes to remove the basal lamina were studied with freeze-fracture techniques in order to examine the influence of the basal lamina in the maintenance of active zone ultrastructure. The active zone is believed to be the site of transmitter release and has a unique membrane organization and location in the neuromuscular junction. After removal of the basal lamina by successive treatment of 0.01% collagenase and 0.1% protease for 1 h each, active zone disruption was observed. Some active zones became segmented, and some were also randomly located and oriented, but they still had normal double-row particle organization. Others contained only clusters of large intramembrane particles. These disorganized active zones were still functional as indicated by the presence of vesicle openings. Some enzyme-treated junctions were also exposed to the membrane cholesterol probe, filipin, to examine the expression of membrane lipid heterogeneity in disrupted active zones. As in normal active zones, filipin-sterol complexes were absent. The densities of background particles in the presynaptic membranes and of large particles thought to be acetylcholine receptors were not significantly altered by the enzyme treatment. Although a direct effect of the enzymes on active zone ultrastructure can not be totally excluded, the present work is consistent with a maintenance role of the basal lamina in active zone organization and location.