The postsynaptic density

Glutamatergic synapses in the central nervous system are characterized by an electron-dense web underneath the postsynaptic membrane; this web is called the postsynaptic density (PSD). PSDs are composed of a dense network of several hundred proteins, creating a macromolecular complex that serves a wide range of functions. Prominent PSD proteins such as members of the MaGuk or ProSAP/Shank family build up a dense scaffold that creates an interface between clustered membrane-bound receptors, cell adhesion molecules and the actin-based cytoskeleton. Moreover, kinases, phosphatases and several proteins of different signalling pathways are specifically localized within the spine/PSD compartment. Small GTPases and regulating proteins are also enriched in PSDs being the molecular basis for regulated structural changes of cytoskeletal components within the synapse in response to external or internal stimuli, e.g. synaptic activation. This synaptic rearrangement (structural plasticity) is a rapid process and is believed to underlie learning and memory formation. The characterization of synapse/PSD proteins is especially important in the light of recent data suggesting that several mental disorders have their molecular defect at the synapse/PSD level.The work of former and current colleagues in my laboratory and the support with respect to research on components of the PSD network by the DFG (SFB497/B8, Bo1718/2-2) and by the Land Baden-Württemberg (1423/74) are gratefully acknowledged.     

Estimating the synaptic current in a multiconductance AMPA receptor model

Synaptic transmission starts after the presynaptic neuron has released diffusing neurotransmitters, leading to postsynaptic receptor activation and a postsynaptic current, mostly mediated by glutamatergic (AMPARs) receptors for excitatory neurons. Despite intense experimental and theoretical research, it is still unclear how factors such as the synaptic cleft geometry, the organization, the number and the multiconductance state of receptors, the geometry of postsynaptic density (PSD), and the neurotransmitter release location, shape the mean and the variance of the postsynaptic current and its plastic changes. To estimate the synaptic current amplitude and to account for the stochastic nature of synaptic transmission, we develop a semianalytical method in which we obtain a general expression for the coefficient of variation. The method uses the experimental data about the multiconductance channels. We find that PSD morphological changes can significantly modulate the synaptic current, which is maximally reliable (the coefficient of variation is minimal) for an optimal size of the PSD, that depends on the vesicular release active zone. We show that this optimal PSD size is due to nonlinear phenomena involving the receptor multibinding cooperativity. We conclude that changes in the PSD geometry can sustain a form of synaptic plasticity, independent of a change in the number of receptors.     

Mechanisms of synaptic plasticity

We have shown that the synapse maturation phase of synaptogenesis is a model for synaptic plasticity that can be particularly well-studied in chicken forebrain because for most forebrain synapses, the maturation changes occur slowly and are temporally well-separated from the synapse formation phase. We have used the synapse maturation phase of neuronal development in chicken forebrain to investigate the possible link between changes in the morphology and biochemical composition of the postsynaptic density (PSD) and the functional properties of glutamate receptors overlying the PSD. Morphometric studies of PSDs in forebrains and superior cervical ganglia of chickens and rats have shown that the morphological features of synapse maturation are characteristic of a synaptic type, but that the rate at which these changes occur can vary between types of synapses within one animal and between synapses of the same type in different species. We have investigated, during maturation in the chicken forebrain, the properties of the N-methyl-D-aspartate (NMDA) subtype of the glutamate receptors, which are concentrated in the junctional membranes overlying thick PSDs in the adult. There was no change in the number of NMDA receptors during maturation, but there was an increase in the rate of NMDA-stimulated uptake of 45Ca2+ into brain prisms. This functional change was not seen with the other ionotropic subtypes of the glutamate receptor and was NMDA receptor-mediated. The functional change also correlated with the increase in thickness of the PSD during maturation that has previously been shown to be due to an increase in the amount of PSD associated Ca(2+)-calmodulin stimulated protein kinase II (CaM-PK II). Our results provide strong circumstantial evidence for the regulation of NMDA receptors by the PSD and implicate changing local concentrations of CaM-PK II in this process. The results also indicate some of the ways in which properties of existing synapses can be modified by changes at the molecular level.     

Synapse geometry and receptor dynamics modulate synaptic strength

Synaptic transmission relies on several processes, such as the location of a released vesicle, the number and type of receptors, trafficking between the postsynaptic density (PSD) and extrasynaptic compartment, as well as the synapse organization. To study the impact of these parameters on excitatory synaptic transmission, we present a computational model for the fast AMPA-receptor mediated synaptic current. We show that in addition to the vesicular release probability, due to variations in their release locations and the AMPAR distribution, the postsynaptic current amplitude has a large variance, making a synapse an intrinsic unreliable device. We use our model to examine our experimental data recorded from CA1 mice hippocampal slices to study the differences between mEPSC and evoked EPSC variance. The synaptic current but not the coefficient of variation is maximal when the active zone where vesicles are released is apposed to the PSD. Moreover, we find that for certain type of synapses, receptor trafficking can affect the magnitude of synaptic depression. Finally, we demonstrate that perisynaptic microdomains located outside the PSD impacts synaptic transmission by regulating the number of desensitized receptors and their trafficking to the PSD. We conclude that geometrical modifications, reorganization of the PSD or perisynaptic microdomains modulate synaptic strength, as the mechanisms underlying long-term plasticity.     

Molecular mechanisms underlying specificity of excitotoxic signaling in neurons

The central role of glutamate receptors in mediating excitotoxic neuronal death in stroke, epilepsy and trauma has been well established. Glutamate is the major excitatory amino acid transmitter within the CNS and it's signaling is mediated by a number of postsynaptic ionotropic and metabotropic receptors. Although calcium ions are considered key regulators of excitotoxicity, new evidence suggests that specific second messenger pathways rather than total Ca(2+) load, are responsible for mediating neuronal degeneration. Glutamate receptors are found localized at the synapse within electron dense structures known as the postsynaptic density (PSD). Localization at the PSD is mediated by binding of glutamate receptors to submembrane proteins such as actin and PDZ containing proteins. PDZ domains are conserved motifs that mediate protein-protein interactions and self-association. In addition to glutamate receptors PDZ-containing proteins bind a multitude of intracellular signal molecules including nitric oxide synthase. In this way PDZ proteins provide a mechanism for clustering glutamate receptors at the synapse together with their corresponding signal transduction proteins. PSD organization may thus facilitate the individual neurotoxic signal mechanisms downstream of receptors during glutamate overactivity. Evidence exists showing that inhibiting signals downstream of glutamate receptors, such as nitric oxide and PARP-1 can reduce excitotoxic insult. Furthermore we have shown that uncoupling the interaction between specific glutamate receptors from their PDZ proteins protects neurons against glutamate-mediated excitotoxicity. These findings have significant implications for the treatment of neurodegenerative diseases using therapeutics that specifically target intracellular protein-protein interactions.     

Capping of the N‐terminus of PSD‐95 by calmodulin triggers its postsynaptic release

Postsynaptic density protein‐95 (PSD‐95) is a central element of the postsynaptic architecture of glutamatergic synapses. PSD‐95 mediates postsynaptic localization of AMPA receptors and NMDA receptors and plays an important role in synaptic plasticity. PSD‐95 is released from postsynaptic membranes in response to Ca²⁺ influx via NMDA receptors. Here, we show that Ca²⁺/calmodulin (CaM) binds at the N‐terminus of PSD‐95. Our NMR structure reveals that both lobes of CaM collapse onto a helical structure of PSD‐95 formed at its N‐terminus (residues 1–16). This N‐terminal capping of PSD‐95 by CaM blocks palmitoylation of C3 and C5, which is required for postsynaptic PSD‐95 targeting and the binding of CDKL5, a kinase important for synapse stability. CaM forms extensive hydrophobic contacts with Y12 of PSD‐95. The PSD‐95 mutant Y12E strongly impairs binding to CaM and Ca²⁺‐induced release of PSD‐95 from the postsynaptic membrane in dendritic spines. Our data indicate that CaM binding to PSD‐95 serves to block palmitoylation of PSD‐95, which in turn promotes Ca²⁺‐induced dissociation of PSD‐95 from the postsynaptic membrane.     

Modeling synaptic dynamics driven by receptor lateral diffusion

The synaptic weight between a pre- and a postsynaptic neuron depends in part on the number of postsynaptic receptors. On the surface of neurons, receptors traffic by random motion in and out from a microstructure called the postsynaptic density (PSD). In the PSD, receptors can be stabilized at the membrane when they bind to scaffolding proteins. We propose a mathematical model to compute the postsynaptic counterpart of the synaptic weight based on receptor trafficking. We take into account the receptor fluxes at the PSD, which can be regulated by neuronal activity, and the interactions of receptors with the scaffolding molecules. Using a Markovian approach, we estimate the mean and the fluctuations of the number of bound receptors. When the number of receptors is large, a deterministic system is also derived. Moreover, these equations can be used, for example, to fit fluorescence-recovery-after-photobleaching experiments to determine, in living neurons, the chemical binding constants for the receptors/scaffolding molecules interaction at synapses.     

Postsynaptic scaffold proteins at non-synaptic sites. The role of postsynaptic scaffold proteins in motor-protein-receptor complexes

Synapse-associated proteins that are located at the postsynaptic density (PSD) have recently been shown to have a structural role at non-synaptic locations. Here, they act as adaptor proteins between neurotransmitter receptors and the microtubule- or microfilament-based motor-protein complexes that are responsible for transport to the PSD. The use of a common set of proteins that contain multiple domains for protein-protein interactions as both intracellular transport adaptors and synaptic scaffold proteins might contribute to the transport specificity and postsynaptic integration of receptors that underlie synapse formation and plasticity.     

Posttranslational Modifications Regulate the Postsynaptic Localization of PSD-95

The postsynaptic density (PSD) consists of a lattice-like array of interacting proteins that organizes and stabilizes synaptic receptors, ion channels, structural proteins, and signaling molecules required for normal synaptic transmission and synaptic function. The scaffolding and hub protein postsynaptic density protein-95 (PSD-95) is a major element of central chemical synapses and interacts with glutamate receptors, cell adhesion molecules, and cytoskeletal elements. In fact, PSD-95 can regulate basal synaptic stability as well as the activity-dependent structural plasticity of the PSD and, therefore, of the excitatory chemical synapse. Several studies have shown that PSD-95 is highly enriched at excitatory synapses and have identified multiple protein structural domains and protein-protein interactions that mediate PSD-95 function and trafficking to the postsynaptic region. PSD-95 is also a target of several signaling pathways that induce posttranslational modifications, including palmitoylation, phosphorylation, ubiquitination, nitrosylation, and neddylation; these modifications determine the synaptic stability and function of PSD-95 and thus regulate the fates of individual dendritic spines in the nervous system. In the present work, we review the posttranslational modifications that regulate the synaptic localization of PSD-95 and describe their functional consequences. We also explore the signaling pathways that induce such changes.     

ProSAPiP2, a novel postsynaptic density protein that interacts with ProSAP2/Shank3

The postsynaptic density (PSD) is a highly specialized structure that is located juxtaposed to the presynaptic active zone of excitatory synapses. It is composed of a variety of proteins that include receptors, signaling molecules, cytoskeletal components and scaffolding proteins. ProSAP/Shank proteins are large multidomain proteins that facilitate multiple functions within the PSD. They build large scaffolds that are the structural basis for the direct and/or indirect connection between receptor proteins and the actin based cytoskeleton. Here, we characterize a novel interaction partner of ProSAP2/Shank3, named ProSAP interacting protein 2 (ProSAPiP2) that does not show any close homology to other known proteins. It binds to the PDZ domain of ProSAP2/Shank3 and is highly expressed in the neuronal system. ProSAPiP2 is located in dendrites and spines, is enriched in the PSD and interacts with actin. Therefore ProSAPiP2 could be involved in the linkage between molecules of the PSD and the cytoskeleton.     

Assembly and plasticity of the glutamatergic postsynaptic specialization

Glutamate mediates most excitatory synaptic transmission in the brain. Synaptic strength at glutamatergic synapses shows a remarkable degree of use-dependent plasticity and such modifications may represent a physiological correlate to learning and memory. Glutamate receptors and downstream enzymes are organized at synapses by cytoskeletal proteins containing multiple protein-interacting domains. Recent studies demonstrate that these 'scaffolding' proteins within the postsynaptic specialization have the capacity to promote synaptic maturation, influence synapse size, and modulate glutamate receptor function.     

Scaffold proteins (MAGUK, Shank and Homer) in postsynaptic density in the central nervous system

The postsynaptic density (PSD) is a dynamic multi-protein complex attached to the postsynaptic membrane composed of several hundred proteins such as receptors and channels, scaffolding and adaptor proteins, cell-adhesion proteins, cytoskeletal proteins, G-proteins and their modulators and signaling molecules including kinases and phosphtases. This review focuses on the prominent PSD scaffolds proteins such as members of the MAGUK (membrane-associated guanylyl kinase), Shank (SH3 domain and ankyrin repeat-containing protein) and Homer families. These molecules interact simultaneously with different kinds of receptors and modulate their function by linking the receptors to downstream signaling events. For example PSD 95, a main member of MAGUK family, interacts directly with carboxyl termini of NMDA receptor subunits and clusters them to the postsynaptic membrane. In addition, PSD 95 is involved in binding and organizing proteins connected with NMDAR signaling. Based on the modular character and ability to form multiproteins interactions, MAGUK, Shank and Homer are perfectly suited to act as a major scaffold in postsynaptic density.     

Aplysia synapse associated protein (APSAP): identification, characterization, and selective interactions with Shaker-type potassium channels

The vertebrate post-synaptic density (PSD) is a region of high molecular complexity in which dynamic protein interactions modulate receptor localization and synaptic function. Members of the membrane-associated guanylate kinase (MAGUK) family of proteins represent a major structural and functional component of the vertebrate PSD. In order to investigate the expression and significance of orthologous PSD components associated with the Aplysia sensory neuron-motor neuron synapse, we have cloned an Aplysia Dlg-MAGUK protein, which we identify as Aplysia synapse associated protein (ApSAP). As revealed by western blot, RT-PCR, and immunocytochemical analyses, ApSAP is predominantly expressed in the CNS and is located in both sensory neuron and motor neurons. The overall amino acid sequence of ApSAP is 55–61% identical to Drosophila Dlg and mammalian Dlg-MAGUK proteins, but is more highly conserved within L27, PDZ, SH3, and guanylate kinase domains. Because these conserved domains mediate salient interactions with receptors and other PSD components of the vertebrate synapse, we performed a series of GST pull-down assays using recombinant C-terminal tail proteins from various Aplysia receptors and channels containing C-terminal PDZ binding sequences. We have found that ApSAP selectively binds to an Aplysia Shaker-type channel AKv1.1, but not to (i) NMDA receptor subunit AcNR1-1, (ii) potassium channel AKv5.1, (iii) receptor tyrosine kinase ApTrkl, (iv) glutamate receptor ApGluR1/4, (v) glutamate receptor ApGluR2/3, or (vi) glutamate receptor ApGluR7. These findings provide preliminary information regarding the expression and interactions of Dlg-MAGUK proteins of the Aplysia CNS, and will inform questions aimed at a functional analysis of how interactions in a protein network such as the PSD may regulate synaptic strength.     

Morphine administration alters the profile of hippocampal postsynaptic density-associated proteins: a proteomics study focusing on endocytic proteins

Numerous studies have shown that drugs of abuse induce changes in protein expression in the brain that are thought to play a role in synaptic plasticity. Drug-induced plasticity can be mediated by changes at the synapse and more specifically at the postsynaptic density (PSD), which receives and transduces synaptic information. To date, the majority of studies examining synaptic protein profiles have focused on identifying the synaptic proteome. Only a handful of studies have examined the changes in synaptic profile by drug administration. We applied a quantitative proteomics analysis technique with the cleavable ICAT reagent to quantitate relative changes in protein levels of the hippocampal PSD in response to morphine administration. We identified a total of 102 proteins in the mouse hippocampal PSD. The majority of these were signaling, trafficking, and cytoskeletal proteins involved in synaptic plasticity, learning, and memory. Among the proteins whose levels were found to be altered by morphine administration, clathrin levels were increased to the largest extent. Immunoblotting and electron microscopy studies showed that this increase was localized to the PSD. Morphine treatment was also found to lead to a local increase in two other components of the endocytic machinery, dynamin and AP-2, suggesting a critical involvement of the endocytic machinery in the modulatory effects of morphine. Because alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are thought to undergo clathrin-mediated endocytosis, we examined the effect of morphine administration on the association of the AMPA receptor subunit, GluR1, with clathrin. We found a substantial decrease in the levels of GluR1 associated with clathrin. Taken together, these results suggest that, by causing a redistribution of endocytic proteins at the synapse, morphine modulates synaptic plasticity at hippocampal glutamatergic synapses.     

Genetic Deletion of NR3A Accelerates Glutamatergic Synapse Maturation

Glutamatergic synapse maturation is critically dependent upon activation of NMDA-type glutamate receptors (NMDARs); however, the contributions of NR3A subunit-containing NMDARs to this process have only begun to be considered. Here we characterized the expression of NR3A in the developing mouse forebrain and examined the consequences of NR3A deletion on excitatory synapse maturation. We found that NR3A is expressed in many subcellular compartments, and during early development, NR3A subunits are particularly concentrated in the postsynaptic density (PSD). NR3A levels dramatically decline with age and are no longer enriched at PSDs in juveniles and adults. Genetic deletion of NR3A accelerates glutamatergic synaptic transmission, as measured by AMPAR-mediated postsynaptic currents recorded in hippocampal CA1. Consistent with the functional observations, we observed that the deletion of NR3A accelerated the expression of the glutamate receptor subunits NR1, NR2A, and GluR1 in the PSD in postnatal day (P) 8 mice. These data support the idea that glutamate receptors concentrate at synapses earlier in NR3A-knockout (NR3A-KO) mice. The precocious maturation of both AMPAR function and glutamate receptor expression are transient in NR3A-KO mice, as AMPAR currents and glutamate receptor protein levels are similar in NR3A-KO and wildtype mice by P16, an age when endogenous NR3A levels are normally declining. Taken together, our data support a model whereby NR3A negatively regulates the developmental stabilization of glutamate receptors involved in excitatory neurotransmission, synaptogenesis, and spine growth.     

A Monte Carlo model reveals independent signaling at central glutamatergic synapses

We have developed a biophysically realistic model of receptor activation at an idealized central glutamatergic synapse that uses Monte Carlo techniques to simulate the stochastic nature of transmission following release of a single synaptic vesicle. For the a synapse with 80 AMPA and 20 NMDA receptors, a single quantum, with 3000 glutamate molecules, opened approximately 3 NMDARs and 20 AMPARs. The number of open receptors varied directly with the total number of receptors, and the fraction of open receptors did not depend on the ratio of co-localized AMPARs and NMDARs. Variability decreased with increases in either total receptor number or quantal size, and differences between the variability of AMPAR and NMDAR responses were due solely to unequal numbers of receptors at the synapse. Despite NMDARs having a much higher affinity for glutamate than AMPARs, quantal release resulted in similar occupancy levels in both receptor types. Receptor activation increased with number of transmitter molecules released or total receptor number, whereas occupancy levels were only dependent on quantal size. Tortuous diffusion spaces reduced the extent of spillover and the activation of extrasynaptic receptors. These results support the conclusion that signaling is spatially independent within and between central glutamatergic synapses.     

NCAM promotes assembly and activity-dependent remodeling of the postsynaptic signaling complex

The neural cell adhesion molecule (NCAM) regulates synapse formation and synaptic strength via mechanisms that have remained unknown. We show that NCAM associates with the postsynaptic spectrin-based scaffold, cross-linking NCAM with the N-methyl-d-aspartate (NMDA) receptor and Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIalpha) in a manner not firmly or directly linked to PSD95 and alpha-actinin. Clustering of NCAM promotes formation of detergent-insoluble complexes enriched in postsynaptic proteins and resembling postsynaptic densities. Disruption of the NCAM-spectrin complex decreases the size of postsynaptic densities and reduces synaptic targeting of NCAM-spectrin-associated postsynaptic proteins, including spectrin, NMDA receptors, and CaMKIIalpha. Degeneration of the spectrin scaffold in NCAM-deficient neurons results in an inability to recruit CaMKIIalpha to synapses after NMDA receptor activation, which is a critical process in NMDA receptor-dependent long-term potentiation. The combined observations indicate that NCAM promotes assembly of the spectrin-based postsynaptic signaling complex, which is required for activity-associated, long-lasting changes in synaptic strength. Its abnormal function may contribute to the etiology of neuropsychiatric disorders associated with mutations in or abnormal expression of NCAM.     

Clathrin-mediated endocytosis at synapses

Neurons are communication specialists that convert electrical into chemical signals at specialized cell-cell junctions termed synapses. Arrival of an action potential triggers calcium-regulated exocytosis of neurotransmitter (NT) from small synaptic vesicles (SVs), which then diffuses across the synaptic cleft and binds to postsynaptic receptors to elicit specific changes within the postsynaptic cell. Endocytosis of pre- and postsynaptic membrane proteins including SV components and postsynaptic NT receptors is essential for the proper functioning of the synapse. During the past several years, we have witnessed enormous progress in our understanding of the mechanics of clathrin-mediated endocytosis (CME) and its role in regulating exo-endocytic vesicle cycling at synapses. Here we summarize the molecular machinery used for recognition of synaptic membrane protein cargo and its clathrin-dependent internalization, and describe the inventory of tools that can be used to monitor vesicle cycling at synapses or to inhibit CME in a stage-specific manner.     

Towards a quantitative model of the post-synaptic proteome

The postsynaptic compartment of the excitatory glutamatergic synapse contains hundreds of distinct polypeptides with a wide range of functions (signalling, trafficking, cell-adhesion, etc.). Structural dynamics in the post-synaptic density (PSD) are believed to underpin cognitive processes. Although functionally and morphologically diverse, PSD proteins are generally enriched with specific domains, which precisely define the mode of clustering essential for signal processing. We applied a stochastic calculus of domain binding provided by a rule-based modelling approach to formalise the highly combinatorial signalling pathway in the PSD and perform the numerical analysis of the relative distribution of protein complexes and their sizes. We specified the combinatorics of protein interactions in the PSD by rules, taking into account protein domain structure, specific domain affinity and relative protein availability. With this model we interrogated the critical conditions for the protein aggregation into large complexes and distribution of both size and composition. The presented approach extends existing qualitative protein-protein interaction maps by considering the quantitative information for stoichiometry and binding properties for the elements of the network. This results in a more realistic view of the postsynaptic proteome at the molecular level.     

BDNF increases synapse density in dendrites of developing tectal neurons in vivo

Neuronal connections are established through a series of developmental events that involve close communication between pre- and postsynaptic neurons. In the visual system, BDNF modulates the development of neuronal connectivity by influencing presynaptic retinal ganglion cell (RGC) axons. Increasing BDNF levels in the optic tectum of Xenopus tadpoles significantly increases both axon arborization and synapse density per axon terminal within a few hours of treatment. Here, we have further explored the mechanisms by which BDNF shapes synaptic connectivity by imaging tectal neurons, the postsynaptic partners of RGCs. Individual neurons were co-labeled with DsRed2 and a GFP-tagged postsynaptic density protein (PSD95-GFP) to visualize dendritic morphology and postsynaptic specializations simultaneously in vivo. Immunoelectron microscopy confirmed that PSD95-GFP predominantly localized to ultrastructurally identified synapses. Time-lapse confocal microscopy of individual, double-labeled neurons revealed a coincident, activity-dependent mechanism of synaptogenesis and axon and dendritic arbor growth, which is differentially modulated by BDNF. Microinjection of BDNF into the optic tectum significantly increased synapse number in tectal neuron dendritic arbors within 24 hours, without significantly influencing arbor morphology. BDNF function-blocking antibodies had opposite effects. The BDNF-elicited increase in synapse number complements the previously observed increase in presynaptic sites on RGC axons. These results, together with the timescale of the response by tectal neurons, suggest that the effects of BDNF on dendritic synaptic connectivity are secondary to its effects on presynaptic RGCs. Thus, BDNF influences synaptic connectivity in multiple ways: it enhances axon arbor complexity expanding the synaptic territory of the axon, while simultaneously coordinating synapse formation and stabilization with individual postsynaptic cells.