CYY-1/cyclin Y and CDK-5 differentially regulate synapse elimination and formation for rewiring neural circuits

The assembly and maturation of neural circuits require a delicate balance between synapse formation and elimination. The cellular and molecular mechanisms that coordinate synaptogenesis and synapse elimination are poorly understood. In C. elegans, DD motoneurons respecify their synaptic connectivity during development by completely eliminating existing synapses and forming new synapses without changing cell morphology. Using loss- and gain-of-function genetic approaches, we demonstrate that CYY-1, a cyclin box-containing protein, drives synapse removal in this process. In addition, cyclin-dependent kinase-5 (CDK-5) facilitates new synapse formation by regulating the transport of synaptic vesicles to the sites of synaptogenesis. Furthermore, we show that coordinated activation of UNC-104/Kinesin3 and Dynein is required for patterning newly formed synapses. During the remodeling process, presynaptic components from eliminated synapses are recycled to new synapses, suggesting that signaling mechanisms and molecular motors link the deconstruction of existing synapses and the assembly of new synapses during structural synaptic plasticity.     

Cadherin-mediated adhesion at the interneuronal synapse

One of the recent advances in the molecular definition of a synapse has been the identification of cadherins as major structural components. The presence of classic (N- and E-) cadherins in the synaptic complex is not surprising considering the ultrastructural similarities between interneuronal synapses and the adhesive junctions formed between epithelial cells. However, the role of these adhesion molecules and their junctions in this context is likely to encompass both developmental and physiological phenomena that are unique to the synapse. Moreover, the recent finding that a much broader family of cadherin-related receptors is also located at the synaptic complex has fuelled speculation that cadherins have a role in generation of specificity in synaptic connectivity as well as structure.     

PSD-95 promotes the stabilization of young synaptic contacts

Maintaining a population of stable synaptic connections is probably of critical importance for the preservation of memories and functional circuitry, but the molecular dynamics that underlie synapse stabilization is poorly understood. Here, we use simultaneous time-lapse imaging of post synaptic density-95 (PSD-95) and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) to investigate the dynamics of protein composition at axodendritic (AD) contacts. Our data reveal that this composition is highly dynamic, with both proteins moving into and out of the same synapse independently, so that synapses cycle rapidly between states in which they are enriched for none, one or both proteins. We assessed how PSD-95 and CaMKII interact at stable and transient AD sites and found that both phospho-CaMKII and PSD-95 are present more often at stable than labile contacts. Finally, we found that synaptic contacts are more stable in older neurons, and this process can be mimicked in younger neurons by overexpression of PSD-95. Taken together, these data show that synaptic protein composition is highly variable over a time-scale of hours, and that PSD-95 is probably a key synaptic protein that promotes synapse stability.     

The presynaptic active zone protein bassoon is essential for photoreceptor ribbon synapse formation in the retina

The photoreceptor ribbon synapse is a highly specialized glutamatergic synapse designed for the continuous flow of synaptic vesicles to the neurotransmitter release site. The molecular mechanisms underlying ribbon synapse formation are poorly understood. We have investigated the role of the presynaptic cytomatrix protein Bassoon, a major component of the photoreceptor ribbon, in a mouse retina deficient of functional Bassoon protein. Photoreceptor ribbons lacking Bassoon are not anchored to the presynaptic active zones. This results in an impaired photoreceptor synaptic transmission, an abnormal dendritic branching of neurons postsynaptic to photoreceptors, and the formation of ectopic synapses. These findings suggest a critical role of Bassoon in the formation and the function of photoreceptor ribbon synapses of the mammalian retina.     

Synaptic destabilization by neuronal Nogo-A

Formation and maintenance of a neuronal network is based on a balance between plasticity and stability of synaptic connections. Several molecules have been found to regulate the maintenance of excitatory synapses but nothing is known about the molecular mechanisms involved in synaptic stabilization versus disassembly at inhibitory synapses. Here, we demonstrate that Nogo-A, which is well known to be present in myelin and inhibit growth in the adult CNS, is present in inhibitory presynaptic terminals in cerebellar Purkinje cells at the time of Purkinje cell-Deep Cerebellar Nuclei (DCN) inhibitory synapse formation and is then downregulated during synapse maturation. We addressed the role of neuronal Nogo-A in synapse maturation by generating several mouse lines overexpressing Nogo-A, starting at postnatal ages and throughout adult life, specifically in cerebellar Purkinje cells and their terminals. The overexpression of Nogo-A induced a progressive disassembly, retraction and loss of the inhibitory Purkinje cell terminals. This led to deficits in motor learning and coordination in the transgenic mice. Prior to synapse disassembly, the overexpression of neuronal Nogo-A led to the downregulation of the synaptic scaffold proteins spectrin, spectrin-E and β-catenin in the postsynaptic neurons. Our data suggest that neuronal Nogo-A might play a role in the maintenance of inhibitory synapses by modulating the expression of synaptic anchoring molecules. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

LAR-RPTPs: synaptic adhesion molecules that shape synapse development

The synapse is the most elementary operating unit in neurons, creating neural circuits that underlie all brain functions. Synaptic adhesion molecules initiate neuronal synapse connections, promote their stabilization and refinement, and control long-term synaptic plasticity. Leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-RPTPs) have previously been implicated as essential elements in central nervous system (CNS) development. Recent studies have demonstrated that LAR-RPTP family members are also involved in diverse synaptic functions, playing a role in synaptic adhesion pathways together with a host of distinct transmembrane proteins and serving as major synaptic adhesion molecules in governing pre- and postsynaptic development, dysfunctions of which may underlie various disorders. This review highlights the emerging role of LAR-RPTPs as synapse organizers in orchestrating synapse development.     

alpha5 integrin signaling regulates the formation of spines and synapses in hippocampal neurons

The actin-based dynamics of dendritic spines play a key role in synaptic plasticity, which underlies learning and memory. Although it is becoming increasingly clear that modulation of actin is critical for spine dynamics, the upstream molecular signals that regulate the formation and plasticity of spines are poorly understood. In non-neuronal cells, integrins are critical modulators of the actin cytoskeleton, but their function in the nervous system is not well characterized. Here we show that alpha5 integrin regulates spine morphogenesis and synapse formation in hippocampal neurons. Knockdown of alpha5 integrin expression using small interfering RNA decreased the number of dendritic protrusions, spines, and synapses. Expression of constitutively active or dominant negative alpha5 integrin also resulted in alterations in the number of dendritic protrusions, spines, and synapses. alpha5 integrin signaling regulates spine morphogenesis and synapse formation by a mechanism that is dependent on Src kinase, Rac, and the signaling adaptor GIT1. Alterations in the activity or localization of these molecules result in a significant decrease in the number of spines and synapses. Thus, our results point to a critical role for integrin signaling in regulating the formation of dendritic spines and synapses in hippocampal neurons.     

Glycobiology of the synapse

Synapses are the fundamental units of connectivity that link together the nervous system. Lectin studies from 30 years ago suggested that specific glycans are concentrated at neuromuscular synapses in the peripheral nervous system and at excitatory synapses in the brain. Subsequent studies have confirmed that particular glycan structures are localized at these synapses, including polysialic acid, high mannose, the cytotoxic T cell antigen, and forms of heparan sulfate. Though the role of these molecules in synapse formation and function is still poorly understood, there is increasing evidence that the function of agrin, a synaptogenic factor in neuromuscular formation, is modulated by several glycans. In addition, the recent generation of ST8SiaIV null mice strongly suggests a role for polysialic acid in synaptic plasticity in the some regions of the central nervous system.     

Acetylcholinesterase mobility and stability at the neuromuscular junction of living mice

Acetylcholinesterase (AChE) is an enzyme that terminates acetylcholine neurotransmitter function at the synaptic cleft of cholinergic synapses. However, the mechanism by which AChE number and density are maintained at the synaptic cleft is poorly understood. In this work, we used fluorescence recovery after photobleaching, photo-unbinding, and quantitative fluorescence imaging to investigate the surface mobility and stability of AChE at the adult innervated neuromuscular junction of living mice. In wild-type synapses, we found that nonsynaptic (perisynaptic and extrasynaptic) AChEs are mobile and gradually recruited into synaptic sites and that most of the trapped AChEs come from the perijunctional pool. Selective labeling of a subset of synaptic AChEs within the synapse by using sequential unbinding and relabeling with different colors of streptavidin followed by time-lapse imaging showed that synaptic AChEs are nearly immobile. At neuromuscular junctions of mice deficient in alpha-dystrobrevin, a component of the dystrophin glycoprotein complex, we found that the density and distribution of synaptic AChEs are profoundly altered and that the loss rate of AChE significantly increased. These results demonstrate that nonsynaptic AChEs are mobile, whereas synaptic AChEs are more stable, and that alpha-dystrobrevin is important for controlling the density and stability of AChEs at neuromuscular synapses.     

The Neurexin/N-Ethylmaleimide-sensitive Factor (NSF) Interaction Regulates Short Term Synaptic Depression

Although Neurexins, which are cell adhesion molecules localized predominantly to the presynaptic terminals, are known to regulate synapse formation and synaptic transmission, their roles in the regulation of synaptic vesicle release during repetitive nerve stimulation are unknown. Here, we show that nrx mutant synapses exhibit rapid short term synaptic depression upon tetanic nerve stimulation. Moreover, we demonstrate that the intracellular region of NRX is essential for synaptic vesicle release upon tetanic nerve stimulation. Using a yeast two-hybrid screen, we find that the intracellular region of NRX interacts with N-ethylmaleimide-sensitive factor (NSF), an enzyme that mediates soluble NSF attachment protein receptor (SNARE) complex disassembly and plays an important role in synaptic vesicle release. We further map the binding sites of each molecule and demonstrate that the NRX/NSF interaction is critical for both the distribution of NSF at the presynaptic terminals and SNARE complex disassembly. Our results reveal a previously unknown role of NRX in the regulation of short term synaptic depression upon tetanic nerve stimulation and provide new mechanistic insights into the role of NRX in synaptic vesicle release.     

Assessing the Role of Cell-Surface Molecules in Central Synaptogenesis in the Drosophila Visual System

A hallmark of the central nervous system is its spatial and functional organization in synaptic layers. During neuronal development, axons form transient contacts with potential post-synaptic elements and establish synapses with appropriate partners at specific layers. These processes are regulated by synaptic cell-adhesion molecules. In the Drosophila visual system, R7 and R8 photoreceptor subtypes target distinct layers and form en passant pre-synaptic terminals at stereotypic loci of the axonal shaft. A leucine-rich repeat transmembrane protein, Capricious (Caps), is known to be selectively expressed in R8 axons and their recipient layer, which led to the attractive hypothesis that Caps mediates R8 synaptic specificity by homophilic adhesion. Contradicting this assumption, our results indicate that Caps does not have a prominent role in synaptic-layer targeting and synapse formation in Drosophila photoreceptors, and that the specific recognition of the R8 target layer does not involve Caps homophilic axon-target interactions. We generated flies that express a tagged synaptic marker to evaluate the presence and localization of synapses in R7 and R8 photoreceptors. These genetic tools were used to assess how the synaptic profile is affected when axons are forced to target abnormal layers by expressing axon guidance molecules. When R7 axons were mistargeted to the R8-recipient layer, R7s either maintained an R7-like synaptic profile or acquired a similar profile to r8s depending on the overexpressed protein. When R7 axons were redirected to a more superficial medulla layer, the number of presynaptic terminals was reduced. These results indicate that cell-surface molecules are able to dictate synapse loci by changing the axon terminal identity in a partially cell-autonomous manner, but that presynapse formation at specific sites also requires complex interactions between pre- and post-synaptic elements.     

Mechanisms of excitatory synapse maturation by trans-synaptic organizing complexes

Synapses are specialized cell-cell adhesion contacts that mediate communication within neural networks. During development, excitatory synapses are generated by step-wise recruitment of presynaptic and postsynaptic proteins to sites of contact. Several classes of synaptic organizing complexes have been identified that function during the initial stages of synapse formation. However, mechanisms underlying the later stages of synapse development are less well understood. In recent years, molecules have been discovered that appear to play a role in synapse maturation. In this review, we highlight recent findings that have provided key insights for understanding postsynaptic maturation of developing excitatory synapses with a focus on recruitment of AMPA receptors to developing synapses.     

Organization and regulation of proteins at synapses

The organization and regulation of synaptic connections in the mammalian nervous system entail complicated and co-ordinated molecular and cellular processes. The unveiling of various protein-protein interactions and their functional consequences at synapses have led to a greater understanding of the process of synapse formation and the modulation of synaptic transmission. Recent studies indicate that the major excitatory neurotransmitter receptors in the brain, the glutamate receptors, are associated with many different molecules that are involved in the formation of elaborate synaptic cytoskeletal networks and signal transduction cascades. These complex protein networks may play critical roles in the regulation of neurotransmitter receptor function and the efficacy of synaptic transmission.     

Organization of brain complexity--synapse proteome form and function.

Proteomic study of the synapse has generated an extensive list of molecular components, revealing one of the most complex functional systems currently known to cell biology. While fundamental to neural information processing, behaviour and disease, the molecular organisation of the synapse and its relation to higher-level function has yet to be clearly understood. Neurotransmitter receptor complexes, such as the N-methyl-D-aspartate receptor complex (NRC/MASC), are major components of the synaptic proteome. We have recently completed a detailed study of MASC, its functional organisation and involvement in behaviour and disease. This pointed to simple design principles underlying synaptic organisation. Drawing together the results of proteomic and analytical study, we sketch out a model for synaptic functional organisation.     

Synaptic Activity Regulates the Abundance and Binding of Complexin

Nervous system function relies on precise chemical communication between neurons at specialized junctions known as synapses. Complexin (CPX) is one of a small number of cytoplasmic proteins that are indispensable in controlling neurotransmitter release through SNARE and synaptic vesicle interactions. However, the mechanisms that recruit and stabilize CPX are poorly understood. The mobility of CPX tagged with photoactivatable green fluorescent protein (pGFP) was quantified in vivo using Caenorhabditis elegans. Although pGFP escaped the synapse within seconds, CPX-pGFP displayed both fast and slow decay components, requiring minutes for complete exchange of the synaptic pool. The longer synaptic residence time of CPX arose from both synaptic vesicle and SNARE interactions, and surprisingly, CPX mobility depended on synaptic activity. Moreover, mouse CPX-GFP reversibly dispersed out of hippocampal presynaptic terminals during stimulation, and blockade of vesicle fusion prevented CPX dispersion. Hence, synaptic CPX can rapidly redistribute and this exchange is influenced by neuronal activity, potentially contributing to use-dependent plasticity.     

MiR‐134‐dependent regulation of Pumilio‐2 is necessary for homeostatic synaptic depression

Neurons employ a set of homeostatic plasticity mechanisms to counterbalance altered levels of network activity. The molecular mechanisms underlying homeostatic plasticity in response to increased network excitability are still poorly understood. Here, we describe a sequential homeostatic synaptic depression mechanism in primary hippocampal neurons involving miRNA‐dependent translational regulation. This mechanism consists of an initial phase of synapse elimination followed by a reinforcing phase of synaptic downscaling. The activity‐regulated microRNA miR‐134 is necessary for both synapse elimination and the structural rearrangements leading to synaptic downscaling. Results from miR‐134 inhibition further uncover a differential requirement for GluA1/2 subunits for the functional expression of homeostatic synaptic depression. Downregulation of the miR‐134 target Pumilio‐2 in response to chronic activity, which selectively occurs in the synapto‐dendritic compartment, is required for miR‐134‐mediated homeostatic synaptic depression. We further identified polo‐like kinase 2 (Plk2) as a novel target of Pumilio‐2 involved in the control of GluA2 surface expression. In summary, we have described a novel pathway of homeostatic plasticity that stabilizes neuronal circuits in response to increased network activity.     

Altered Postsynaptic-Density-Levels of Caldendrin in the Para-Chloroamphetamine-Induced Serotonin Syndrome but not in the Rat Ketamine Model of Psychosis

Caldendrin is a synaptic calcium sensor protein that is tightly associated with the postsynaptic density (PSD). Previous work has shown that the association of the protein with the synapse is highly dynamic and is increased in an activity-dependent manner. In the present study the caldendrin-association with the postsynaptic cytomatrix was analyzed in animal models of psychosis and drug abuse induced neurotoxicity. Subchronic administration of the N-methyl-d-aspartate (NMDA)-receptor antagonist ketamine, serving as a model of NMDA-receptor hypofunction and schizophrenia showed no significant effect on the PSD-levels of caldendrin, indicating that NMDA-receptor activity is not required to keep caldendrin at the synapse. However, administration of high doses of the serotonergic neurotoxin p-chloroamphetamine (PCA) lead to significant changes in the association of caldendrin with the PSD. These results underscore the dynamic association of caldendrin with the PSD and suggest a role of this synaptic calcium sensor in the PCA-induced serotonin syndrome.     

The influence of limited presynaptic growth and synapse removal on adaptive synaptogenesis

This report continues our research into the effectiveness of adaptive synaptogenesis in constructing feed-forward networks which perform good transformations on their inputs. Good transformations are characterized by the maintenance of input information and the removal of statistical dependence. Adaptive synaptogenesis stochastically builds and sculpts a synaptic connectivity in initially unconnected networks using two mechanisms. The first, synaptogenesis, creates new, excitatory, feed-forward connections. The second, associative modification, adjusts the strength of existing synapses. Our previous implementations of synaptogenesis only incorporated a postsynaptic regulatory process, receptivity to new innervation (Adelsberger-Mangan and Levy 1993a, b). In the present study, a presynaptic regulatory process, presynaptic avidity, which regulates the tendency of a presynaptic neuron to participate in a new synaptic connection as a function of its total synaptic weight, is incorporated into the synaptogenesis process. In addition, we investigate a third mechanism, selective synapse removal. This process removes synapses between neurons whose firing is poorly correlated. Networks that are constructed with the presynaptic regulatory process maintain more information and remove more statistical dependence than networks constructed with postsynaptic receptivity and associative modification alone. Selective synapse removal also improves network performance, but only when implemented in conjunction with the presynaptic regulatory process. Received: 20 August 1993/Accepted in revised form: 16 April 1994     

Kinesin-3 mediated axonal delivery of presynaptic neurexin stabilizes dendritic spines and postsynaptic components

The functional properties of neural circuits are defined by the patterns of synaptic connections between their partnering neurons, but the mechanisms that stabilize circuit connectivity are poorly understood. We systemically examined this question at synapses onto newly characterized dendritic spines of C. elegans GABAergic motor neurons. We show that the presynaptic adhesion protein neurexin/NRX-1 is required for stabilization of postsynaptic structure. We find that early postsynaptic developmental events proceed without a strict requirement for synaptic activity and are not disrupted by deletion of neurexin/nrx-1. However, in the absence of presynaptic NRX-1, dendritic spines and receptor clusters become destabilized and collapse prior to adulthood. We demonstrate that NRX-1 delivery to presynaptic terminals is dependent on kinesin-3/UNC-104 and show that ongoing UNC-104 function is required for postsynaptic maintenance in mature animals. By defining the dynamics and temporal order of synapse formation and maintenance events in vivo, we describe a mechanism for stabilizing mature circuit connectivity through neurexin-based adhesion.