Presynaptic trafficking of synaptotagmin I is regulated by protein palmitoylation

Protein palmitoylation plays a critical role in sorting and targeting of several proteins to pre- and postsynaptic sites. In this study, we have analyzed the role of palmitoylation in trafficking of synaptotagmin I and its modulation by synaptic activity. We found that palmitoylation of N-terminal cysteines contributed to sorting of synaptotagmin I to an intracellular vesicular compartment at the presynaptic terminal. Presynaptic targeting is a unique feature of N-terminal sequences of synaptotagmin I because the palmitoylated N terminus of synaptotagmin VII failed to localize to presynaptic sites. We also found that palmitate was stably associated with both synaptotagmin I and SNAP-25 and that rapid neuronal depolarization did not affect palmitate turnover on these proteins. However, long-term treatment with drugs that either block synaptic activity or disrupt SNARE complex assembly modulated palmitoylation and accumulation of synaptotagmin I at presynaptic sites. We conclude that palmitoylation is involved in trafficking of specific elements involved in transmitter release and that distinct mechanisms regulate addition and removal of palmitate on select neuronal proteins.     

Local presynaptic activity gates homeostatic changes in presynaptic function driven by dendritic BDNF synthesis

Homeostatic synaptic plasticity is important for maintaining stability of neuronal function, but heterogeneous expression mechanisms suggest that distinct facets of neuronal activity may shape the manner in which compensatory synaptic changes are implemented. Here, we demonstrate that local presynaptic activity gates a retrograde form of homeostatic plasticity induced by blockade of AMPA receptors (AMPARs) in cultured hippocampal neurons. We show that AMPAR blockade produces rapid (<3 hr) protein synthesis-dependent increases in both presynaptic and postsynaptic function and that the induction of presynaptic, but not postsynaptic, changes requires coincident local activity in presynaptic terminals. This "state-dependent" modulation of presynaptic function requires postsynaptic release of brain-derived neurotrophic factor (BDNF) as a retrograde messenger, which is locally synthesized in dendrites in response to AMPAR blockade. Taken together, our results reveal a local crosstalk between active presynaptic terminals and postsynaptic signaling that dictates the manner by which homeostatic plasticity is implemented at synapses.     

Central Presynaptic Terminals Are Enriched in ATP but the Majority Lack Mitochondria

Synaptic neurotransmission is known to be an energy demanding process. At the presynapse, ATP is required for loading neurotransmitters into synaptic vesicles, for priming synaptic vesicles before release, and as a substrate for various kinases and ATPases. Although it is assumed that presynaptic sites usually harbor local mitochondria, which may serve as energy powerhouse to generate ATP as well as a presynaptic calcium depot, a clear role of presynaptic mitochondria in biochemical functioning of the presynapse is not well-defined. Besides a few synaptic subtypes like the mossy fibers and the Calyx of Held, most central presynaptic sites are either en passant or tiny axonal terminals that have little space to accommodate a large mitochondrion. Here, we have used imaging studies to demonstrate that mitochondrial antigens poorly co-localize with the synaptic vesicle clusters and active zone marker in the cerebral cortex, hippocampus and the cerebellum. Confocal imaging analysis on neuronal cultures revealed that most neuronal mitochondria are either somatic or distributed in the proximal part of major dendrites. A large number of synapses in culture are devoid of any mitochondria. Electron micrographs from neuronal cultures further confirm our finding that the majority of presynapses may not harbor resident mitochondria. We corroborated our ultrastructural findings using serial block face scanning electron microscopy (SBFSEM) and found that more than 60% of the presynaptic terminals lacked discernible mitochondria in the wild-type mice hippocampus. Biochemical fractionation of crude synaptosomes into mitochondria and pure synaptosomes also revealed a sparse presence of mitochondrial antigen at the presynaptic boutons. Despite a low abundance of mitochondria, the synaptosomal membranes were found to be highly enriched in ATP suggesting that the presynapse may possess alternative mechanism/s for concentrating ATP for its function. The potential mechanisms including local glycolysis and the possible roles of ATP-binding synaptic proteins such as synapsins, are discussed.     

Constitutive Endocytosis and Turnover of the Neuronal Glycine Transporter GlyT2 Is Dependent on Ubiquitination of a C-Terminal Lysine Cluster

Inhibitory glycinergic neurotransmission is terminated by sodium and chloride-dependent plasma membrane glycine transporters (GlyTs). The mainly glial glycine transporter GlyT1 is primarily responsible for the completion of inhibitory neurotransmission and the neuronal glycine transporter GlyT2 mediates the reuptake of the neurotransmitter that is used to refill synaptic vesicles in the terminal, a fundamental role in the physiology and pathology of glycinergic neurotransmission. Indeed, inhibitory glycinergic neurotransmission is modulated by the exocytosis and endocytosis of GlyT2. We previously reported that constitutive and Protein Kinase C (PKC)-regulated endocytosis of GlyT2 is mediated by clathrin and that PKC accelerates GlyT2 endocytosis by increasing its ubiquitination. However, the role of ubiquitination in the constitutive endocytosis and turnover of this protein remains unexplored. Here, we show that ubiquitination of a C-terminus four lysine cluster of GlyT2 is required for constitutive endocytosis, sorting into the slow recycling pathway and turnover of the transporter. Ubiquitination negatively modulates the turnover of GlyT2, such that increased ubiquitination driven by PKC activation accelerates transporter degradation rate shortening its half-life while decreased ubiquitination increases transporter stability. Finally, ubiquitination of GlyT2 in neurons is highly responsive to the free pool of ubiquitin, suggesting that the deubiquitinating enzyme (DUB) ubiquitin C-terminal hydrolase-L1 (UCHL1), as the major regulator of neuronal ubiquitin homeostasis, indirectly modulates the turnover of GlyT2. Our results contribute to the elucidation of the mechanisms underlying the dynamic trafficking of this important neuronal protein which has pathological relevance since mutations in the GlyT2 gene (SLC6A5) are the second most common cause of human hyperekplexia.     

The Regulation of Synaptic Protein Turnover

Emerging evidence indicates that protein synthesis and degradation are necessary for the remodeling of synapses. These two processes govern cellular protein turnover, are tightly regulated, and are modulated by neuronal activity in time and space. The anisotropic anatomy of the neurons presents a challenge for the study of protein turnover, but the understanding of protein turnover in neurons and its modulation in response to activity can help us to unravel the fine-tuned changes that occur at synapses in response to activity. Here we review the key experimental evidence demonstrating the role of protein synthesis and degradation in synaptic plasticity, as well as the turnover rates of specific neuronal proteins.     

Neurochemical aspects of interneuronal communication

It is suggested that the term neurotransmission, which is used to designate neuronal communication at synaptic level, be associated to the less restrictive term neuromodulation. These two types of intercellular communication seem in fact to be two basically different mechanisms, both of which contribute to neuronal integration. The integration of neuronal information at cellular level appears to be more complex than the simple addition of excitatory plus inhibitory influences eliciting postsynaptic responses. Evidence has been obtained that non synaptic transmission can alter the capacity of a given synapse to transfer neuronal information from the presynaptic element to the postsynaptic neuron. For instance, presynaptic mechanisms provide evidence for the functional independence of the nerve terminals, since the release of neuromediators by the latter is sometimes independent of the axonal firing rate. Similarly, the somato-dendritic part of some neurons exhibits intrinsic functions, such as a dendritic release of neuromediator, suggesting that the control of the axonal firing rate takes place partly at this somato-dendritic level and does not depend for the totality on afferent axonic information. The intercellular operations which organize individual neurons into neuronal networks will also occur either at somato-dendritic level or at the level of specific nerve terminals selected as the result of presynaptic interactions. This integration of neuronal information also seems to take place at postsynaptic level, where cooperative interactions have been shown to occur between various receptors. These mechanisms will function at the level of a single nerve terminal containing more than one neuromediator. Neuromodulation can therefore be said to involve very efficient adaptive processes, which help to account for the fact that such large behavioral responses are expressed by such a small number of neuronal elements.     

Mechanisms and function of dendritic exocytosis

Dendritic exocytosis is required for a broad array of neuronal functions including retrograde signaling, neurotransmitter release, synaptic plasticity, and establishment of neuronal morphology. While the details of synaptic vesicle exocytosis from presynaptic terminals have been intensely studied for decades, the mechanisms of dendritic exocytosis are only now emerging. Here we review the molecules and mechanisms of dendritic exocytosis and discuss how exocytosis from dendrites influences neuronal function and circuit plasticity.     

The needs of a synapse—How local organelles serve synaptic proteostasis

Synaptic function crucially relies on the constant supply and removal of neuronal membranes. The morphological complexity of neurons poses a significant challenge for neuronal protein transport since the machineries for protein synthesis and degradation are mainly localized in the cell soma. In response to this unique challenge, local micro‐secretory systems have evolved that are adapted to the requirements of neuronal membrane protein proteostasis. However, our knowledge of how neuronal proteins are synthesized, trafficked to membranes, and eventually replaced and degraded remains scarce. Here, we review recent insights into membrane trafficking at synaptic sites and into the contribution of local organelles and micro‐secretory pathways to synaptic function. We describe the role of endoplasmic reticulum specializations in neurons, Golgi‐related organelles, and protein complexes like retromer in the synthesis and trafficking of synaptic transmembrane proteins. We discuss the contribution of autophagy and of proteasome‐mediated and endo‐lysosomal degradation to presynaptic proteostasis and synaptic function, as well as nondegradative roles of autophagosomes and lysosomes in signaling and synapse remodeling. We conclude that the complexity of neuronal cyto‐architecture necessitates long‐distance protein transport that combines degradation with signaling functions.     

Roles of ubiquitination at the synapse

The ubiquitin proteasome system (UPS) was first described as a mechanism for protein degradation more than three decades ago, but the critical roles of the UPS in regulating neuronal synapses have only recently begun to be revealed. Targeted ubiquitination of synaptic proteins affects multiple facets of the synapse throughout its life cycle; from synaptogenesis and synapse elimination to activity-dependent synaptic plasticity and remodeling. The recent identification of specific UPS molecular pathways that act locally at the synapse illustrates the exquisite specificity of ubiquitination in regulating synaptic protein trafficking and degradation events. Synaptic activity has also been shown to determine the subcellular distribution and composition of the proteasome, providing additional mechanisms for locally regulating synaptic protein degradation. Together these advances reveal that tight control of protein turnover plays a conserved, central role in establishing and modulating synapses in neural circuits.     

Rab2 regulates presynaptic precursor vesicle biogenesis at the trans-Golgi

Reliable delivery of presynaptic material, including active zone and synaptic vesicle proteins from neuronal somata to synaptic terminals, is prerequisite for successful synaptogenesis and neurotransmission. However, molecular mechanisms controlling the somatic assembly of presynaptic precursors remain insufficiently understood. We show here that in mutants of the small GTPase Rab2, both active zone and synaptic vesicle proteins accumulated in the neuronal cell body at the trans-Golgi and were, consequently, depleted at synaptic terminals, provoking neurotransmission deficits. Ectopic presynaptic material accumulations consisted of heterogeneous vesicles and short tubules of 40 × 60 nm, segregating in subfractions either positive for active zone or synaptic vesicle proteins and LAMP1, a lysosomal membrane protein. Genetically, Rab2 acts upstream of Arl8, a lysosomal adaptor controlling axonal export of precursors. Collectively, we identified a Golgi-associated assembly sequence of presynaptic precursor biogenesis dependent on a Rab2-regulated protein export and sorting step at the trans-Golgi.     

The ubiquitin proteasome system acutely regulates presynaptic protein turnover and synaptic efficacy

BACKGROUND: The ubiquitin proteasome system (UPS) mediates regulated protein degradation and provides a mechanism for closely controlling protein abundance in spatially restricted domains within cells. We hypothesized that the UPS may acutely determine the local concentration of key regulatory proteins at neuronal synapses as a means for locally modulating synaptic efficacy and the strength of neurotransmission communication. RESULTS: We investigated this hypothesis at the Drosophila neuromuscular synapse by using an array of genetic and pharmacological tools. This study demonstrates that UPS components are present in presynaptic boutons and that the UPS functions locally in the presynaptic compartment to rapidly eliminate a conditional transgenic reporter of proteasome activity. We assayed a panel of synaptic proteins to determine whether the UPS acutely regulates the local abundance of native synaptic targets. Both acute pharmacological inhibition of the proteasome (<1 hr) and targeted genetic perturbation of proteasome function in the presynaptic neuron cause the specific accumulation of the essential synaptic vesicle-priming protein DUNC-13. Most importantly, acute pharmacological inhibition of the proteasome (<1 hr) causes a rapid strengthening of neurotransmission (an approximately 50% increase in evoked amplitude) because of increased presynaptic efficacy. The proteasome-dependent regulation of presynaptic protein abundance, both of the exogenous reporter and native DUNC-13, and the modulation of presynaptic neurotransmitter release occur on an intermediate, rapid (tens of minutes) timescale. CONCLUSIONS: Taken together, these studies demonstrate that the UPS functions locally within synaptic boutons to acutely control levels of presynaptic protein and that the rate of UPS-dependent protein degradation is a primary determinant of neurotransmission strength.     

神经元突触前可塑性的结构及分子基础

突触可塑性是神经元间信息传递的重要生理调控机制,它包括突触前可塑性和突触后可塑性.突触前可塑性是指通过对神经递质释放过程的干预、修饰,调节突触强度的过程.突触强度的变化,是通过影响量子的大小,活动区的个数和囊泡释放概率来实现的.而突触前囊泡活动尤为重要：从转运、搭靠、融合至内吞进入下一轮循环,每一步都是由一群互相作用的蛋白质共同完成的.     

Miro1‐dependent mitochondrial positioning drives the rescaling of presynaptic Ca2+ signals during homeostatic plasticity

Mitochondrial trafficking is influenced by neuronal activity, but it remains unclear how mitochondrial positioning influences neuronal transmission and plasticity. Here, we use live cell imaging with the genetically encoded presynaptically targeted Ca²⁺ indicator, SyGCaMP5, to address whether presynaptic Ca²⁺ responses are altered by mitochondria in synaptic terminals. We find that presynaptic Ca²⁺ signals, as well as neurotransmitter release, are significantly decreased in terminals containing mitochondria. Moreover, the localisation of mitochondria at presynaptic sites can be altered during long‐term activity changes, dependent on the Ca²⁺‐sensing function of the mitochondrial trafficking protein, Miro1. In addition, we find that Miro1‐mediated activity‐dependent synaptic repositioning of mitochondria allows neurons to homeostatically alter the strength of presynaptic Ca²⁺ signals in response to prolonged changes in neuronal activity. Our results support a model in which mitochondria are recruited to presynaptic terminals during periods of raised neuronal activity and are involved in rescaling synaptic signals during homeostatic plasticity.     

Presynaptic N-type calcium channels regulate synaptic growth

Voltage-gated calcium channels couple changes in membrane potential to neuronal functions regulated by calcium, including neurotransmitter release. Here we report that presynaptic N-type calcium channels not only control neurotransmitter release but also regulate synaptic growth at Drosophila neuromuscular junctions. In a screen for behavioral mutants that disrupt synaptic transmission, an allele of the N-type calcium channel locus (Dmca1A) was identified that caused synaptic undergrowth. The underlying molecular defect was identified as a neutralization of a charged residue in the third S4 voltage sensor. RNA interference reduction of N-type calcium channel expression also reduced synaptic growth. Hypomorphic mutations in syntaxin-1A or n-synaptobrevin, which also disrupt neurotransmitter release, did not affect synapse proliferation at the neuromuscular junction, suggesting calcium entry through presynaptic N-type calcium channels, not neurotransmitter release per se, is important for synaptic growth. The reduced synapse proliferation in Dmca1A mutants is not due to increased synapse retraction but instead reflects a role for calcium influx in synaptic growth mechanisms. These results suggest N-type channels participate in synaptic growth through signaling pathways that are distinct from those that mediate neurotransmitter release. Linking presynaptic voltage-gated calcium entry to downstream calcium-sensitive synaptic growth regulators provides an efficient activity-dependent mechanism for modifying synaptic strength.     

Endosomal phosphatidylinositol 3‐phosphate controls synaptic vesicle cycling and neurotransmission

Neural circuit function requires mechanisms for controlling neurotransmitter release and the activity of neuronal networks, including modulation by synaptic contacts, synaptic plasticity, and homeostatic scaling. However, how neurons intrinsically monitor and feedback control presynaptic neurotransmitter release and synaptic vesicle (SV) recycling to restrict neuronal network activity remains poorly understood at the molecular level. Here, we investigated the reciprocal interplay between neuronal endosomes, organelles of central importance for the function of synapses, and synaptic activity. We show that elevated neuronal activity represses the synthesis of endosomal lipid phosphatidylinositol 3‐phosphate [PI(3)P] by the lipid kinase VPS34. Neuronal activity in turn is regulated by endosomal PI(3)P, the depletion of which reduces neurotransmission as a consequence of perturbed SV endocytosis. We find that this mechanism involves Calpain 2‐mediated hyperactivation of Cdk5 downstream of receptor‐ and activity‐dependent calcium influx. Our results unravel an unexpected function for PI(3)P‐containing neuronal endosomes in the control of presynaptic vesicle cycling and neurotransmission, which may explain the involvement of the PI(3)P‐producing VPS34 kinase in neurological disease and neurodegeneration.     

Regulation of presynaptic Ca(V)2.1 channels by Ca2+ sensor proteins mediates short-term synaptic plasticity

Short-term synaptic plasticity shapes the postsynaptic response to bursts of impulses and is crucial for encoding information in neurons, but the molecular mechanisms are unknown. Here we show that activity-dependent modulation of presynaptic Ca(V)2.1 channels mediated by neuronal Ca(2+) sensor proteins (CaS) induces synaptic plasticity in cultured superior cervical ganglion (SCG) neurons. A mutation of the IQ-like motif in the C terminus that blocks Ca(2+)/CaS-dependent facilitation of the P/Q-type Ca(2+) current markedly reduces facilitation of synaptic transmission. Deletion of the nearby calmodulin-binding domain, which inhibits CaS-dependent inactivation, substantially reduces depression of synaptic transmission. These results demonstrate that residual Ca(2+) in presynaptic terminals can act through CaS-dependent regulation of Ca(V)2.1 channels to induce short-term synaptic facilitation and rapid synaptic depression. Activity-dependent regulation of presynaptic Ca(V)2.1 channels by CaS proteins may therefore be a primary determinant of short-term synaptic plasticity and information-processing in the nervous system.     

Nerve growth factor collaborates with myocyte-derived factors to promote development of presynaptic sites in cultured sympathetic neurons

Nerve growth factor (NGF) acutely modulates synaptic transmission between sympathetic neurons and their cardiac myocyte targets. NGF also has developmental effects in establishing the level of synaptic transmission between sympathetic neurons and myocytes in culture, although little is known about the mechanisms by which NGF influences this synaptic connectivity. Here we report that NGF acts in conjunction with factors produced by cardiac myocytes to promote neuronal contact with the target and the extension of synaptic vesicle-containing growth cones. In conjunction with previously published results showing that NGF has long-term effects on synaptic transmission between sympathetic neurons and myocytes, this work suggests that NGF acts to promote sympathetic neurotransmission by increasing the number of sympathetic fibers establishing target contact. Further, we found that developmental changes in cardiac myocytes led to an increase in the density of synaptic vesicle-containing variocosities along sympathetic fibers, a process regulated by NGF. Thus, as myocytes mature they produce factors that promote the formation of sympathetic presynaptic structures. These results argue that multiple target interactions regulate the extent of synapse formation between sympathetic neurons and cardiac cells and suggest that NGF promotes presynaptic development by increasing neuronal contact with myocyte-derived cell surface or matrix-associated factors.     

BSN (bassoon) and PRKN/parkin in concert control presynaptic vesicle autophagy

ABSTRACT

Maintaining the integrity and function of the presynaptic neurotransmitter release apparatus is a demanding process for a post-mitotic neuron; the mechanisms behind it are still unclear. BSN (bassoon), an active zone scaffolding protein, has been implicated in the control of presynaptic macroautophagy/autophagy, a process we recently showed depends on poly-ubiquitination of synaptic proteins. Moreover, loss of BSN was found to lead to smaller synaptic vesicle (SV) pools and younger pools of the SV protein SV2. Of note, the E3 ligase PRKN/parkin appears to be involved in BSN deficiency-related changes in autophagy levels, as shRNA-mediated knockdown of PRKN counteracts BSN-deficiency and rescues decreased SV protein levels as well as impaired SV recycling in primary cultured neurons. These data imply that BSN and PRKN act in concert to control presynaptic autophagy and maintain presynaptic proteostasis and SV turnover at the physiologically required levels.     

Mapping molecular memory: navigating the cellular pathways of learning

A consolidated map of the signalling pathways that function in the formation of short- and long-term cellular memory could be considered the ultimate means of defining the molecular basis of learning. Research has established that experience-dependent activation of these complex cellular cascades leads to many changes in the composition and functioning of a neuron's proteome, resulting in the modulation of its synaptic strength and structure. However, although generally accepted that synaptic plasticity is the mechanism whereby memories are stored in the brain, there is much controversy over whether the site of this neuronal memory expression is predominantly pre- or postsynaptic. Much of the early research into the neuromolecular mechanisms of memory performed using the model organism, the marine snail Aplysia, has focused on the associated presynaptic events. Recently however, postsynaptic mechanisms have been shown to contribute definitively to long term memory processes, and are in fact critical for persistent learning-induced synaptic changes. In this review, in which we aimed to integrate many of the early and recent advances concerning coordinated neuronal signaling in both the pre- and postsynaptic neurons, we have provided a detailed account of the diverse cellular events that lead to modifications in synaptic strength. Thus, a comprehensive synaptic model is presented that could explain a few of the shortcomings that arise when the presynaptic and postsynaptic changes are considered separately. Although it is clear that there is still much to be learnt and that the exact nature of many of the signalling cascades and their components are yet to be fully understood, this still incomplete but integrated illustrative map of the cellular pathways involved provides an overview which expands understanding of the neuromolecular mechanisms of learning and memory.     

Coupling exo- and endocytosis: an essential role for PIP₂ at the synapse

Chemical synapses are specialist points of contact between two neurons, where information transfer takes place. Communication occurs through the release of neurotransmitter substances from small synaptic vesicles in the presynaptic terminal, which fuse with the presynaptic plasma membrane in response to neuronal stimulation. However, as neurons in the central nervous system typically only possess ~200 vesicles, high levels of release would quickly lead to a depletion in the number of vesicles, as well as leading to an increase in the area of the presynaptic plasma membrane (and possible misalignment with postsynaptic structures). Hence, synaptic vesicle fusion is tightly coupled to a local recycling of synaptic vesicles. For a long time, however, the exact molecular mechanisms coupling fusion and subsequent recycling remained unclear. Recent work now indicates a unique role for the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP(2)), acting together with the vesicular protein synaptotagmin, in coupling these two processes. In this work, we review the evidence for such a mechanism and discuss both the possible advantages and disadvantages for vesicle recycling (and hence signal transduction) in the nervous system. This article is part of a Special Issue entitled Lipids and Vesicular Transport.