Ionotropic glutamate receptor trafficking--there and back again

Glutamate is a major excitatory neurotransmitter in brain. It engages mainly ionotropic glutamate receptors of AMPA and NMDA type. Thus, regulation of the number and properties of the receptors is crucial for correct neuronal communication, but also contributes to various forms of synaptic plasticity, namely neuronal development, learning and memory. Glutamate receptors are not static components of synapses. On the contrary, they are continuously delivered and removed from postsynaptic membranes and this process is regulated by synaptic activity, Receptor trafficking to synapses is a multi-step process, involving exit from endoplasmic reticulum, transport along dendrites, incorporation to postsynaptic membrane and finally removing them from synapses. The transport is regulated by numerous proteins, especially those bearing PDZ domains, or by receptors themselves.     

Dynamics of postsynaptic glutamate receptor targeting

Targeting of glutamate receptors to synapses is an important event in both developing and mature neurons. Glutamate receptors are delivered to nascent synapses during synaptogenesis and to existing synapses during activity-dependent synaptic strengthening. Increasing evidence suggests that glutamate receptors are inserted into the plasma membrane before they accumulate at the synapse. Lateral diffusion of receptors occurs at both synaptic and non-synaptic membranes, and glutamate receptors can exchange rapidly between synaptic and extrasynaptic sites. In addition, recent studies show that postsynaptic scaffold molecules can be highly mobile. The dynamic nature of the synapse suggests that many mechanisms might be involved in regulating synapse formation and synaptic plasticity.     

AMPA and NMDA glutamate receptor trafficking: multiple roads for reaching and leaving the synapse

Glutamate receptor trafficking in and out of synapses is one of the core mechanisms for rapid changes in the number of functional receptors during synaptic plasticity. Recent data have shown that the fast gain and loss of receptors from synaptic sites are accounted for by endocytic/exocytic processes and by their lateral diffusion in the plane of the membrane. These events are interdependent and regulated by neuronal activity and interactions with scaffolding proteins. We review here the main cellular steps for AMPA and NMDA receptor synthesis, traffic within intracellular organelles, membrane exocytosis/endocytosis and surface trafficking. We focus on new findings that shed light on the regulation of receptor cycling events and surface trafficking and the way that this might reshape our thinking about the specific regulation of receptor accumulation at synapses.     

Establishing and sculpting the synapse in Drosophila and C. elegans

Genetic approaches in flies and worms continue to dissect the intricate molecular machinery of chemical synapses. Investigations carried out in the last year provide important new insights into the development and modulation of the presynaptic active zones and postsynaptic receptor fields mediating synaptic function. Mutant screens have identified overlapping gene classes mediating synaptogenesis. The leucocyte common antigen-related receptor tyrosine phosphatase interacts with liprin in the formation of the active zone. Spectrins are essential for the spatial restriction of synaptic proteins to define active zones. Glutamate acts as a negative regulator of its cognate postsynaptic receptor to sculpt receptor field size. Finally, protein translation and degradation regulation emerge as possible key regulators of synaptic efficacy.     

CPG2: a brain- and synapse-specific protein that regulates the endocytosis of glutamate receptors

Long-term maintenance and modification of synaptic strength involve the turnover of neurotransmitter receptors. Glutamate receptors are constitutively and acutely internalized, presumptively through clathrin-mediated receptor endocytosis. Here, we show that cpg2 is a brain-specific splice variant of the syne-1 gene that encodes a protein specifically localized to a postsynaptic endocytotic zone of excitatory synapses. RNAi-mediated CPG2 knockdown increases the number of postsynaptic clathrin-coated vesicles, some of which traffic NMDA receptors, disrupts the constitutive internalization of glutamate receptors, and inhibits the activity-induced internalization of synaptic AMPA receptors. Manipulating CPG2 levels also affects dendritic spine size, further supporting a function in regulating membrane transport. Our results suggest that CPG2 is a key component of a specialized postsynaptic endocytic mechanism devoted to the internalization of synaptic proteins, including glutamate receptors. The activity dependence and distribution of cpg2 expression further suggest that it contributes to the capacity for postsynaptic plasticity inherent to excitatory synapses.     

受体、突触和记忆

大脑中神经元突触间的信号传递是由许多神经递质受体介导的。在过去,Richard L．Huganir实验室一直致力于神经递质受体功能调节的分子机制。而最近,该实验室又聚焦到大脑中一种最主要的兴奋性受体的研究——谷氨酸受体。谷氨酸受体主要可以分为两大类：AMPA受体和NMDA受体。AMPA受体主要介导了快速的兴奋性突触传递;而NMDA受体则在神经可塑性和发育中起到重要作用。实验发现,AMPA受体和NMDA受体都可以被一系列的蛋白激酶磷酸化,而磷酸化的水平则直接影响了这些受体的功能特性,包括通道电导和受体膜定位等。AMPA受体磷酸化的水平同时还在学习和记忆的细胞模型中发生改变,如长时程增强（LTP）和长时程抑制（LTD）。此外,AMPA受体中GluR1亚单位的磷酸化对于各种形式的可塑性以及空间记忆的维持有重要的作用。实验室主要研究突触部位谷氨酸受体在亚细胞水平的定位和聚集的分子机制。最近,一系列可以直接或间接与AMPA和NMDA受体相互作用的蛋白质得以发现,其中包括一个新发现的蛋白家族GRIPs（glutamate receptor interacting proteins）。GRIPs可以直接和AMPA受体的GluR2／3亚单位的C端结合。GRIPs包含7个PDZ结构域,可以介导蛋白与蛋白直接的相互连接,从而把各个AMPA受体交互连接在一起并与其他蛋白相连。另外,GluR2亚单位的c端还可以和兴奋性突触中的蛋白激酶C结合蛋白（PICK1）的PDZ结构域相互作用。另外,GluR2亚单位的C端也可以与一种参与膜融合的蛋白NSF相互作用。这些与AMPA受体相互作用的蛋白质对于受体在膜上的运输以及定位有至关重要的作用。同时,受体与PICK1和GRIP的结合对于小脑运动学习中的LTD有重要作用。总体上说,该实验室发现了一系列可以调节神经递质受体功能的分子机制,这些工作提示受体功能的调节可能是?     

Direct imaging of lateral movements of AMPA receptors inside synapses

Trafficking of AMPA receptors in and out of synapses is crucial for synaptic plasticity. Previous studies have focused on the role of endo/exocytosis processes or that of lateral diffusion of extra-synaptic receptors. We have now directly imaged AMPAR movements inside and outside synapses of live neurons using single-molecule fluorescence microscopy. Inside individual synapses, we found immobile and mobile receptors, which display restricted diffusion. Extra-synaptic receptors display free diffusion. Receptors could also exchange between these membrane compartments through lateral diffusion. Glutamate application increased both receptor mobility inside synapses and the fraction of mobile receptors present in a juxtasynaptic region. Block of inhibitory transmission to favor excitatory synaptic activity induced a transient increase in the fraction of mobile receptors and a decrease in the proportion of juxtasynaptic receptors. Altogether, our data show that rapid exchange of receptors between a synaptic and extra-synaptic localization occurs through regulation of receptor diffusion inside synapses.     

Mobile NMDA receptors at hippocampal synapses

Glutamate receptors are concentrated in the postsynaptic complex of central synapses. This implies a highly organized and stable postsynaptic membrane with tightly anchored receptors. Recent reports of rapid AMPA receptor insertion and removal at synapses have challenged this view. We examined the stability of synaptic NMDA receptors on cultured hippocampal neurons using the open-channel blockers (+)-MK-801 and ketamine to tag synaptic NMDA receptors. NMDA receptor-mediated EPSCs showed an anomalous recovery following "irreversible" MK-801 block. The recovery could not be attributed to MK-801 unbinding or insertion of new receptors, suggesting that membrane receptors had moved laterally into the synapse. At least 65% of synaptic NMDA receptors were mobile. Our results indicate that NMDA receptors can move laterally between synaptic and extrasynaptic pools, providing evidence for a dynamic organization of synaptic NMDA receptors in the postsynaptic complex.     

Glutamate transporters bring competition to the synapse

Glutamate transporters (GluTs) prevent the accumulation of glutamate and influence the occupancy of receptors at synapses. The ability of extrasynaptic NMDA receptors and metabotropic glutamate receptors to participate in signaling is tightly regulated by GluT activity. Astrocytes express the highest density of GluTs and dominate clearance away from these receptors; synapses that are not associated with astrocyte processes experience greater mGluR activation and can be exposed to glutamate released at adjacent synapses. Although less abundant, neuronal transporters residing in the postsynaptic membrane can also shield receptors from the glutamate that is released. The diversity in synaptic morphology suggests a correspondingly rich diversity of GluT function in excitatory transmission.     

Molecular dynamics of postsynaptic receptors and scaffold proteins

The activity of neurotransmitter receptors determines the strength of synaptic transmission. Therefore, the clustering of receptors at synapses is an important mechanism underlying synaptic plasticity. The dynamic exchange of receptors between synaptic and extrasynaptic membranes is dependent on their interaction with synaptic scaffold proteins. Here, we review the recent advances and emerging concepts related to the dynamics of synaptic proteins at inhibitory and excitatory synapses. These include the imaging techniques that enable the study of protein dynamics in cells, the differences and similarities of receptor dynamics at excitatory and inhibitory synapses, the relationship between the exchange of receptor and scaffold proteins, as well as the role of receptor fluxes in the modulation of synaptic strength.     

Contactin-associated protein 1 (Caspr1) regulates the traffic and synaptic content of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors

Glutamate receptors of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type mediate fast excitatory synaptic transmission in the CNS. Synaptic strength is modulated by AMPA receptor binding partners, which regulate receptor synaptic targeting and functional properties. We identify Contactin-associated protein 1 (Caspr1) as an AMPA receptor interactor. Caspr1 is present in synapses and interacts with AMPA receptors in brain synaptic fractions. Coexpression of Caspr1 with GluA1 increases the amplitude of glutamate-evoked currents. Caspr1 overexpression in hippocampal neurons increases the number and size of synaptic GluA1 clusters, whereas knockdown of Caspr1 decreases the intensity of synaptic GluA1 clusters. Hence, Caspr1 is a regulator of the trafficking of AMPA receptors to synapses.     

VGLUT1 and VGAT are sorted to the same population of synaptic vesicles in subsets of cortical axon terminals

Glutamate and GABA mediate most of the excitatory and inhibitory synaptic transmission; they are taken up and accumulated in synaptic vesicles by specific vesicular transporters named VGLUT1-3 and VGAT, respectively. Recent studies show that VGLUT2 and VGLUT3 are co-expressed with VGAT. Because of the relevance this information has for our understanding of synaptic physiology and plasticity, we investigated whether VGLUT1 and VGAT are co-expressed in rat cortical neurons. In cortical cultures and layer V cortical terminals we observed a population of terminals expressing VGLUT1 and VGAT. Post-embedding immunogold studies showed that VGLUT1+/VGAT+ terminals formed both symmetric and asymmetric synapses. Triple-labeling studies revealed GABAergic synapses expressing VGLUT1 and glutamatergic synapses expressing VGAT. Immunoisolation studies showed that anti-VGAT immunoisolated vesicles contained VGLUT1 and anti-VGLUT1 immunoisolated vesicles contained VGAT. Finally, vesicles containing VGAT resident in glutamatergic terminals undergo active recycling. In conclusion, we demonstrate that in neocortex VGLUT1 and VGAT are co-expressed in a subset of axon terminals forming both symmetric and asymmetric synapses, that VGLUT1 and VGAT are sorted to the same vesicles and that vesicles at synapses expressing the vesicular heterotransporter participate in the exo-endocytotic cycle.     

The expression pattern and assembly profile of synaptic membrane proteins in ribbon synapses of the developing mouse retina

In the present study, we generated a systematic overview of the expression pattern and assembly profile of synaptic membrane proteins in ribbon synapses of the developing mouse retina. Using indirect immunofluorescence microscopy, we analyzed the spatial and temporal distribution of 11 important membrane and membrane-associated synaptic proteins (syntaxin 1/3, SNAP-25, synaptobrevin 2, synaptogyrin, synaptotagmin I, SV2A, SV2B, Rab3A, clathrin light chains, CSP and neuroligin I) during synaptogenesis. The temporospatial distribution of these synaptic proteins was "normalized" by the simultaneous visualization of the synaptic vesicle protein synaptophysin, which served as an internal reference protein. We found that expression of various synaptic membrane proteins started at different time points and changed progressively during development. At early stages of development synaptic vesicle membrane proteins at extrasynaptic locations did not always colocalize with synaptophysin, indicating that these proteins probably do not reside in the same transport vesicles. Despite a non-synchronized onset of protein expression, clustering and colocalization of all synaptic membrane proteins at ribbon synapses roughly occurred in the same time window (between day 4 after birth, P4, and P5). Thus, the basic synaptic membrane machinery is already present in ribbon synapses before the well-known complete morphological maturation of ribbon synapses between P7 and P12. We conclude that ribbon synapse formation is a multistep process in which the concerted recruitment of synaptic membrane proteins is a relatively early event and clearly not the final step.     

Long-term potentiation of neuronal glutamate transporters

Persistent, use-dependent modulation of synaptic strength has been demonstrated for fast synaptic transmission mediated by glutamate and has been hypothesized to underlie persistent behavioral changes ranging from memory to addiction. Glutamate released at synapses is sequestered by the action of excitatory amino acid transporters (EAATs) in glia and postsynaptic neurons. So, the efficacy of glutamate transporter function is crucial for regulating glutamate spillover to adjacent presynaptic and postsynaptic receptors and the consequent induction of plastic or excitotoxic processes. Here, we report that tetanic stimulation of cerebellar climbing fiber-Purkinje cell synapses results in long-term potentiation (LTP) of a climbing fiber-evoked glutamate transporter current recorded in Purkinje cells. This LTP is postsynaptically expressed and requires activation of an mGluR1/PKC cascade. Together with a simultaneously induced long-term depression (LTD) of postsynaptic AMPA receptors, this might reflect an integrated antiexcitotoxic cellular response to strong climbing fiber synaptic activation, as occurs following an ischemic episode.     

The catenin/cadherin adhesion system is localized in synaptic junctions bordering transmitter release zones

Molecular mechanisms linking pre- and postsynaptic membranes at the interneuronal synapses are little known. We tested the cadherin adhesion system for its localization in synapses of mouse and chick brains. We found that two classes of cadherin-associated proteins, alpha N- and beta-catenin, are broadly distributed in adult brains, colocalizing with a synaptic marker, synaptophysin. At the ultrastructural level, these proteins were localized in synaptic junctions of various types, forming a symmetrical adhesion structure. These structures sharply bordered the transmitter release sites associated with synaptic vesicles, although their segregation was less clear in certain types of synapses. N-cadherin was also localized at a similar site of synaptic junctions but in restricted brain nuclei. In developing synapses, the catenin-bearing contacts dominated their junctional structures. These findings demonstrate that interneuronal synaptic junctions comprise two subdomains, transmitter release zone and catenin-based adherens junction. The catenins localized in these junctions are likely associated with certain cadherin molecules including N-cadherin, and the cadherin/ catenin complex may play a critical role in the formation or maintenance of synaptic junctions.     

Cell adhesion molecules: signalling functions at the synapse

Many cell adhesion molecules are localized at synaptic sites in neuronal axons and dendrites. These molecules bridge pre- and postsynaptic specializations but do far more than simply provide a mechanical link between cells. In this review, we will discuss the roles these proteins have during development and at mature synapses. Synaptic adhesion proteins participate in the formation, maturation, function and plasticity of synaptic connections. Together with conventional synaptic transmission mechanisms, these molecules are an important element in the trans-cellular communication mediated by synapses.     

Synapse-specific and developmentally regulated targeting of AMPA receptors by a family of MAGUK scaffolding proteins

Trafficking of AMPA receptors (AMPA-Rs) to and from synapses controls the strength of excitatory synaptic transmission. However, proteins that cluster AMPA-Rs at synapses remain poorly understood. Here we show that PSD-95-like membrane-associated guanylate kinases (PSD-MAGUKs) mediate this synaptic targeting, and we uncover a remarkable functional redundancy within this protein family. By manipulating endogenous neuronal PSD-MAGUK levels, we find that both PSD-95 and PSD-93 independently mediate AMPA-R targeting at mature synapses. We also reveal unanticipated synapse heterogeneity as loss of either PSD-95 or PSD-93 silences largely nonoverlapping populations of excitatory synapses. In adult PSD-95 and PSD-93 double knockout animals, SAP-102 is upregulated and compensates for the loss of synaptic AMPA-Rs. At immature synapses, PSD-95 and PSD-93 play little role in synaptic AMPA-R clustering; instead, SAP-102 dominates. These studies establish a PSD-MAGUK-specific regulation of AMPA-R synaptic expression that establishes and maintains glutamatergic synaptic transmission in the mammalian central nervous system.     

Ubiquitin and protein turnover in synapse function

Enduring modification of synapses is central to long-lasting neural circuit plasticity. Such adaptations include rapid posttranslational modification of existing synaptic proteins over periods of minutes and persisting changes in the abundance of synaptic proteins over hours to days. Recently, ubiquitination and protein degradation have emerged as additional mechanisms for modifying the function and molecular composition of synapses. These recent findings raise intriguing questions as to how enduring changes at synapses are accomplished in the face of robust, ongoing molecular turnover.     

A shift in protein S-palmitoylation, with persistence of growth-associated substrates, marks a critical period for synaptic plasticity in developing brain.

In the mammalian cortex, the initial formation of synaptic connections is followed by a prolonged period during which synaptic circuits are functional, but retain an elevated capacity for activity-dependent remodeling and functional plasticity. During this period, synaptic terminals appear fully mature, morphologically and physiologically. We show here, however, that synaptic terminals during this period are distinguished by their simultaneous accumulation of multiple growth-associated proteins at levels characteristic of axonal growth cones, and proteins involved in synaptic transmitter release at levels characteristic of adult synapses. We show further that newly formed synapses undergo a switch in the dynamic S-palmitoylation of proteins early in the critical period, which includes a large and specific decrease in the palmitoylation of GAP-43 and other major substrates characteristic of growth cones. Previous studies have shown that a similar reduction in ongoing palmitoylation of growth cone proteins is sufficient to stop advancing axons in vitro, suggesting that a developmental switch in protein S-palmitoylation serves to disengage the molecular machinery for axon extension in the absence of local triggers for remodeling during the critical period. Only much later does a decline in the availability of major growth cone components mark the molecular maturation of cortical synapses at the close of the critical period.     

AMPA receptor trafficking at excitatory synapses

Excitatory synapses in the CNS release glutamate, which acts primarily on two sides of ionotropic receptors: AMPA receptors and NMDA receptors. AMPA receptors mediate the postsynaptic depolarization that initiates neuronal firing, whereas NMDA receptors initiate synaptic plasticity. Recent studies have emphasized that distinct mechanisms control synaptic expression of these two receptor classes. Whereas NMDA receptor proteins are relatively fixed, AMPA receptors cycle synaptic membranes on and off. A large family of interacting proteins regulates AMPA receptor turnover at synapses and thereby influences synaptic strength. Furthermore, neuronal activity controls synaptic AMPA receptor trafficking, and this dynamic process plays a key role in the synaptic plasticity that is thought to underlie aspects of learning and memory.